WO2017117113A1

WO2017117113A1 - Single molecule counting for analysis from dried blood spots

Info

Publication number: WO2017117113A1
Application number: PCT/US2016/068685
Authority: WO
Inventors: Jeffrey Bishop; Ali Mukherjee; Tam DANG; Heather MORRELL
Original assignee: Singulex Inc
Current assignee: Singulex Inc
Priority date: 2015-12-30
Filing date: 2016-12-27
Publication date: 2017-07-06
Anticipated expiration: 2018-06-30
Also published as: WO2017117113A8

Abstract

The disclosure is directed to the ultra-sensitive detection of analytes obtained from blood samples collected on blood collection cards. Disclosed herein is a method for analyzing a blood sample for the presence or amount of an analyte. The method includes extracting dried blood representing a whole blood sample volume from a blood collection card; forming a complex between analyte in the extracted sample and a labeled binding partner for the analyte comprising a fluorescent moiety; and determining the amount of binding partner from the complex by analyzing a processing sample comprising the fluorescent moiety from the complex with an analyzer capable of the ultra-sensitive detection of the moiety.

Description

SINGLE MOLECULE COUNTING FOR ANALYSIS OF

ANALYTES FROM DRIED BLOOD SPOTS

BACKGROUND

Related Application

[0001] This application claims the benefit of U.S. Provisional Patent Application

Serial No. 62/273,400, filed December 30, 2015, which is incorporated by reference herein in its entirety.

Field

[0002] This disclosure is directed to the analysis of dried blood spot samples for the presence or amount of analytes. The analysis is conducted using single molecule counting technology.

Related Art

[0003] The concept of a dried blood spot (DBS) for blood sampling and analysis was first introduced in 1963 in the context of neonatal screening of phenylketonuria. DBS testing is now utilized in toxicology, pharmacologic, and biomarker testing.

[0004] DBS testing is advantageous for applications that require low blood volume testing, such as the critically ill or pediatric populations. Additionally, DBS testing offers several logistical advantages over venous blood draws, including the avoidance of centrifuges for plasma preparation, freezers for storage prior to shipping, and the ability to ship samples at ambient conditions in an envelope with desiccants.

SUMMARY

[0005] In various aspects, the disclosure is directed to a method for analyzing a blood sample for the presence or amount of an analyte. The method includes extracting dried blood representing a whole blood sample volume from a blood collection card; forming a complex between analyte in the extracted sample and a labeled binding partner for the analyte comprising a fluorescent moiety; and determining the amount of binding partner from the complex by analyzing a processing sample comprising the fluorescent moiety from the complex with an analyzer. The Analyzer includes an electromagnetic radiation source; an objective that directs electromagnetic radiation from the electromagnetic radiation source to an interrogation space in a processing sample; a detector that detects electromagnetic radiation emitted from a photon emitting moiety in the interrogation space if the moiety is present, and a processor operatively connected to the detector. The processor is configured to execute instructions stored on a non-transitory computer-readable medium, and wherein the instructions, when executed by the processor, cause the processor to: determine a threshold photon value corresponding to a background signal in the processing sample, determine the presence of a fluorescent moiety in the interrogation space in each of a plurality of bins by identifying bins having a photon value greater than the threshold value, and determining the presence or amount of the analyte in the blood sample by comparing the number of bins having a photon value greater than the threshold value to a standard curve

[0006] In various embodiments of the aspects of the invention, the blood sample is extracted with an extraction buffer. The dried blood spot may represent a whole blood sample of less than 10 μί. In other embodiments, the method includes determining the volume of the whole blood that represents the dried extracted blood.

[0007] Still further, the instructions may cause the processor to determine the threshold photon value as a function of the background photon level. For example, the threshold photon value may be a fixed number of standard deviations above the background photon level. The instructions may cause the processor to determine detected events representing photon bin counts above the threshold photon value as single molecules of the fluorescent moiety. For example, the instructions may cause the processor to analyze each bin as a "yes" or "no" for the presence of the fluorescent moiety.

[0008] In other embodiments, the analyzer may include a translating system that moves the interrogation space through at least a portion of the processing sample, or the analyzer may include a capillary flow cell for moving at least a portion of the sample through the interrogation space.

[0009] Still further, the analyzer may include an attenuator operatively connected between either the interrogation space and the detector or between the electromagnetic radiation source and the interrogation space and configured to receive electromagnetic radiation emitted from the interrogation space, wherein the instructions cause the processor to instruct the attenuator to attenuate the electromagnetic radiation when the number of photons detected in one or more bins exceeds a saturation threshold. In this aspect, the instructions cause the processor to determine the presence or amount of a photon emitting moiety by measuring the total number of photons per bin.

[0010] The translating system of the analyzer may be configured such that the bins are longer or shorter than the time that the fluorescent moiety is present in the interrogation space during each bin. For example, the translating system may be configured such that the bins are one-half to two times longer than the time that the fluorescent moiety is present in the interrogation space during each bin. Or, the translating system may configured such that bins are the same as the time that the fluorescent moiety is present in the interrogation space during each bin.

[0011] The method of the disclosure includes detection of various analytes. For example wherein the analyte is cardiac troponin I (cTnl) and the concentration of cardiac troponin I in the original whole blood sample is less than or equal to 10 pg/ml; wherein the analyte is interleukin 6 (IL-6) and the concentration of IL-6 in the original whole blood sample is less than or equal to 10 pg/ml; wherein the analyte is endothelin-1 (ET-1) and the concentration of ET-1 in the original whole blood sample is less than or equal to 10 pg/ml; wherein the analyte is Interleukin 17A (IL-17A) and the concentration of IL-17A in the original whole blood sample is less than or equal to 10 pg/ml; wherein the analyte is B-type natriuretic peptide (BNP) and the concentration of BNP in the original whole blood sample is less than or equal to 10 pg/ml; wherein the analyte is vascular endothelial growth factor (VEGF) and the concentration of VEGF in the original whole blood sample is less than or equal to 10 pg/ml; wherein the analyte is tumor necrosis factor alpha (TNF-a) and the concentration of TNF-a in the original whole blood sample is less than or equal to 10 pg/ml; wherein the analyte is leptin and the concentration of leptin in the original whole blood sample is less than or equal to 10 pg/ml; wherein the analyte is thyroid stimulating hormone (TSH) and the concentration of TSH in the original whole blood sample is less than or equal to 10 pg/ml; wherein the analyte is prostate specific antigen (PSA) and the concentration of PSA in the original whole blood sample is less than or equal to 10 pg/ml; and/or the analyte is estradiol and the concentration of estradiol in the original whole blood sample is less than or equal to 10 pg/ml.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The following description can be best understood when read in conjunction with the following drawings, where like structure is indicated with like reference numerals and in which: [0013] Figure 1A shows the range of blood volumes and dried blood spot (DBS) sizes utilized for volume calculation validation, as described in Example 2.

[0014] Figure IB shows the correlation of the calculated DBS volume to the actual pipetted blood volume, as described in Example 2.

[0015] Figure 2A shows the scanning single molecule analyzer as viewed from the top.

[0016] Figure 2B shows the scanning single molecule analyzer as viewed from the side.

[0017] Figure 3 shows a graph of the diffusion time for a 155 KDa molecular weight molecule as a function of the diffusion radius of the molecule.

[0018] Figure 4 shows the DE, EP, and TP signal standard curves for the

SINGULEX^® Clinical Laboratory (SCL) data (left curves (squares)) and DBS data as (right curves (diamonds) described in Example 4.

[0019] Figure 5 shows the correlation of cardiac troponin I (cTnl) concentration data obtained by DBS assay to SCL data for the same donors, as described in Example 4.

[0020] Figure 6 shows the coefficient of variation (CV) between data obtained by

DBS assay and SCL data for six samples spanning the low end of the DBS analyte measuring range, as described in Example 4.

[0021] Figure 7 shows the correlation of prostate specific antigen (PSA)

concentration data obtained by DBS assay to SCL data for the same donor, as described in Example 5.

[0022] Figure 8 shows the correlation of thyroid stimulating hormone (TSH) concentration data obtained by DBS assay to SCL data for the same donors, as described in Example 6.

[0023] Figure 9 shows the correlation of C-reactive protein (CRP) concentration data obtained by DBS assay to SCL data for the same donors, as described in Example 7.

[0024] Figure 10 shows the distribution of differences between cTnl concentration data obtained by finger prick DBS assay and venous ethylenediaminetetraacetic acid (EDTA) plasma assay, as described in Example 8.

[0025] Figure 11 shows cTnl concentration data obtained by DBS assay of samples from control donors and pre-race marathon runners, as described in Example 9.

[0026] Figure 12A shows the change in cTnl concentration between samples of marathon runners pre-race and post-race, obtained by DBS assay, as described in Example 10.

[0027] Figure 12B shows the change in cTnl concentration between samples of control donors before and after 24 hours of avoiding extreme exercise, as described in Example 10.

[0028] Figure 13 shows the distribution of differences between cTnl concentration data obtained by venous DBS assay and venous EDTA plasma assay, as described in Example 11.

[0029] Figure 14 shows the distribution of percent differences between cTnl concentration data obtained by finger prick DBS assay and venous DBS assay, as described in Example 12.

DESCRIPTION

[0030] The disclosure is directed to a method for testing analytes in a dried blood spot sample that have been collected on blood collection cards. In light of the small volume of sample that can be collected and extracted from a blood collection card and the dilution of the sample that results from the extraction of the dried blood from the cards, it is advantageous to be able to detect analytes in low concentrations, especially for analytes having a normal blood concentration range that is already below the sensitivity of most standard analyzers and assay methods. Accordingly, the disclosure describes the ultra-sensitive detection of analytes obtained from blood samples collected on blood collection cards.

[0031] Blood from human subjects is typically collected on blood collection cards with between 10 and 100 of blood obtained from a finger or heal prick by a lancet.

Samples are typically allowed to dry and may be shipped to a reference laboratory at room temperature using commercially available transportation and delivery services (e.g., U.S. Mail, FEDEX®, UPS®) in standard delivery envelopes without refrigeration.

Blood samples collected and dried on the cards are generally stable for up to 7 days at room temperature and longer at colder temperatures. Use of a desiccant in the shipping container helps to avoid degradation.

[0032] Blood collection cards are commercially available from a variety of sources and formats that may be selected based upon the analyte(s) to be determined. For example, WHATMAN^® FTA^® DMPK-A and FTA^® DMPK-B cards lyse cells and denature proteins on contact. A WHATMAN ^® FTA^® DMPK-C card is not impregnated with chemicals.

PerkinElmer 226 Sample Collection cards are 100% cotton linter fiber filter paper designed to hold bio-samples. Long-term stability has been demonstrated on these cards for analytes and metabolites sensitive to plasma enzymes.

[0033] Analytes that can be determined from dried blood spots include, for example, nucleic acids and polypeptides. Using a high sensitivity analyzer, examples of analytes that can be determined in low concentrations are show in Table 1. The approximate volume of reconstituted blood (the diluted blood/buffer mixture which results from re-hydrating the dried blood spot and extracting it from the card with a reconstitution buffer) which is needed by the presently disclosed method is also shown.

Table 1

Creatinine 1 μΐ,

[0034] The analytes in Table 1 are only examples. Most blood-based analytes that can form a complex with a suitable binding partner (i.e. antibody, oligonucleotide, or synthetic binder) can be detected. More examples of analytes can be found, for example, in U. S. Patent No. 8,264,684, which is incorporated by reference herein in its entirety. Using a high sensitivity analyzer as described herein, the analytes can be detected individually or in combination in a manner that is described, for example, in U.S. Patent Application

Publication No. US 20160178520, and Intemational Patent Publication No. WO20091 17033, each of which is incorporated by reference herein its entirety.

[0035] The blood volume necessary for the detection of any particular analyte depends on the analyte and its concentration in whole blood. Using the high sensitivity analyzer and assays as described further herein, the volume of blood necessary for any blood- based analyte is significantly lower than necessary for traditional assay analyzers and procedures. Typically, an original blood sample of less than 80 is used. Once dried, the blood sample representing the original sample volume is extracted from the card with an extraction buffer. The buffer is processed to provide a processing sample that is analyzed by the high sensitivity analyzer as more fully described herein.

[0036] The disclosure provides systems and methods for highly sensitive detection and quantitation of one or more target molecules, such as markers for biological states. Such systems, which may include instruments, kits, and compositions, may be referred to as "single molecule detectors," "single particle detectors," "single molecule analyzers," "single particle analyzers," "single molecule readers," or "single particle readers." Compositions and methods for diagnosis, prognosis, and/or determination of treatment based on such highly sensitive detection and quantization are also described.

[0037] The volume of whole blood dried in a blood collection spot on a blood collection card can be determined by optically scanning the spot. Figure 1 A shows blood spots from known volumes of whole blood. The area of each spot was determined by scanning the spots with an Epson scanner. Using this area in conjunction with the known thickness of the card, as well as calibrating the area calculation with standard spots created from known blood volumes, allows for the accurate determination of blood volume in the test sample. The volume of blood identified by the scanner closely correlates to the known volumes as show in Figure IB.

[0038] Accordingly, in one aspect, the disclosure is directed to a method for determining the presence or amount of an analyte using dried blood spot technology. The method includes obtaining a blood sample on a collection card. Optionally, the volume of original blood sample that dries to form the dried blood spot can be calculated by the methods described herein or other known methods. Dried blood is extracted from the card with an elution buffer. The method also includes forming a complex between analyte in the sample and a labeled binding partner for the analyte comprising a fluorescent moiety. The amount of binding in the complex is determined by analyzing a solution comprising the fluorescent moiety from the complex with a single molecule analyzer. A single molecule analyzer in accordance with the disclosure includes an electromagnetic radiation source, an objective that directs electromagnetic radiation from the electromagnetic radiation source to an

interrogation space in a processing sample, a detector that detects electromagnetic radiation emitted from a photon emitting moiety in the interrogation space if the moiety is present, and a processor operatively connected to the detector. In various aspects, the processor is configured to execute instructions stored on a non-transitory computer-readable medium, and wherein the instructions, when executed by the processor, cause the processor to determine a threshold photon value corresponding to a background signal in the interrogation space. The processor may also determine the presence of a fluorescent moiety in the interrogation space in each of a plurality of bins by identifying bins having a photon value greater than the threshold value. The processor can determine the presence or amount of the analyte in the sample by comparing the number of bins having a photon value greater than the threshold value to a standard curve.

[0039] In various embodiments of the disclosure, the analyte(s) is(are) present in patient blood, serum, or plasma at concentrations of less than 10 pg/mL. In particular, one or more of these analytes that can be detected according the disclosure herein include cardiac troponin I (cTnl), Interleukin-6 (IL-6), endothelin-1 (ET-1), Interleukin 17A (IL-17A), B- type natriuretic peptide (BNP) (all forms), vascular endothelial growth factor (VEGF), tumor necrosis factor alpha (TNF-a), thyroid stimulating hormone (TSH), prostate specific antigen (PSA), leptin, and estradiol. Depending on how many of these or other analytes are determined from a dried blood spot, the extracted blood may be further diluted in buffer to make a working volume of up to about 50 to 300 total volume.

[0040] Example Scanning Single Molecule Analyzer [0041] An example of a high sensitivity analyzer is shown in Figures 2A and 2B. The analyzer system 100 includes electromagnetic radiation source 110, a first alignment mirror 112, a second alignment mirror 114, a dichroic mirror 160, and a rotating scan mirror 122 mounted to the shaft 124 of a scan motor 120. As shown in Figure 2B, the rotating scan mirror 122 deflects the electromagnetic radiation source through a first scan lens 130, through a second scan lens 132, and through a microscope objective lens 140, to a sample plate 170. The fluorescence associated with the single molecules contained on or in the sample plate 170 is detected using a tube lens 180, an aperture 182, a detector filter 188, a detector lens 186, and a detector 184. The signal is then processed by a processor (not shown) operatively coupled to the detector 184. In some embodiments, the entire scanning analyzer system 100 is mounted to a baseboard 190.

[0042] In operation, the electromagnetic radiation source 110 is aligned so that its output 126, e.g., a beam, is reflected off the front surface 111 of a first alignment

mirror 112 to the front surface 113 of a second alignment mirror 114 to the dichroic mirror 160 mounted to a dichroic mirror mount 162. The dichroic mirror 160 then reflects the electromagnetic radiation 126 to the front surface of a scan mirror 122 located at the tip of the shaft 124 of the scan motor 120. The electromagnetic radiation 126 then passes through a first scan lens 130 and a second scan lens 132 to the microscope objective lens 140. The objective lens 140 focuses the beam 126 through the base 172 of the sample plate 170 and directs the beam 126 to an interrogation space located on the opposite side of the sample plate 170 from which the beam 126 entered. Passing the electromagnetic radiation beam 126 through a first scan lens 130 and a second scan lens 132 ensures all light to the objective lens 140 is coupled efficiently. The beam 126 excites the label attached to the single molecule of interest contained on or in the sample plate 170. The label emits radiation that is collected by the objective 140. The electromagnetic radiation is then passed back through the scan lenses 130, 132, which then ensure coupling efficiency of the radiation from the objective 140. The detected radiation is reflected off of the front surface of the scan mirror 122 to the dichroic mirror 160. Because the wavelength of the fluorescent light detected is different than the wavelength emitted by the electromagnetic radiation source 110, the fluorescent light passes through the dichroic mirror 160. The fluorescent light then passes through a tube lens 180, an aperture 182, a detector filter 188, and a detector lens 186 to a detector 184. The detector filter 188 minimizes aberrant noise signals due to light scatter or ambient light while maximizing the signal emitted by the excited fluorescent moiety bound to the particle. A processor processes the light signal from the particle according to the methods described herein.

[0043] In one embodiment, the microscope objective 140 has a numerical aperture.

As used herein, "high numerical aperture lens" includes a lens with a numerical aperture of equal to or greater than 0.6. The numerical aperture is a measure of the number of highly diffracted image-forming light rays captured by the objective. A higher numerical aperture allows increasingly oblique rays to enter the objective lens and thereby produce a higher resolution image. The brightness of an image also increases with higher numerical aperture. High numerical aperture lenses are commercially available from a variety of vendors, and any one lens having a numerical aperture of equal to or greater than approximately 0.6 can be used in the analyzer system. In some examples, the lens may have a numerical aperture falling within the range of 0.6 to about 1.3, in particular, 0.6 to about 1.0, 0.7 to about 1.2, 0.7 to about 1.0, 0.7 to about 0.9, 0.8 to about 1.3, 0.8 to about 1.2, or 0.8 to about 1.0. In some embodiments, the lens has a numerical aperture of at least about 0.6, for example, at least about 0.7, at least about 0.8, at least about 0.9, or at least about 1.0. In some embodiments, the numerical aperture of the microscope objective lens 140 is approximately 1.25.

[0044] The high numerical aperture (NA) microscope objective, used when performing single molecule detection through the walls or the base of the sample plate 170, has short working distances. The working distance is the distance from the front of the lens to the object in focus. The objective in some embodiments can be within 350 microns of the object. In some embodiments, where a microscope objective lens 140 with NA of 0.8 is used, an Olympus 40*/0.8 NA water immersion objective (Olympus America, Inc., USA) can be used. This objective has a 3.3 mm working distance. In some embodiments, an Olympus 60x/0.9 NA water immersion objective with a 2 mm working distance can be used. Because the later lens is a water immersion lens, the space 142 between the objective and the sample can be filled with water. This can be accomplished using a water bubbler (not shown) or some other suitable plumbing for depositing water between the objective and the base of the sample plate.

[0045] The electromagnetic radiation source 110 is set so that the wavelength of the laser is sufficient to excite the fluorescent label attached to the particle. In some

embodiments, the electromagnetic radiation source 110 is a laser that emits light in the visible spectrum. In some embodiments, the laser is a continuous-wave laser with a wavelength of 639 nm, 532 nm, 488 nm, 422 nm, or 405 nm. Any continuous-wave laser with a wavelength suitable for exciting a fluorescent moiety as used in the methods and compositions of the disclosure can be used without departing from the scope of the disclosure.

[0046] As the interrogation space in the single molecule analyzer system 100 passes over the labeled single molecule, the beam 126 of the electromagnetic radiation source directed into the interrogation space causes the label to enter an excited state. When the particle relaxes from its excited state, a detectable burst of light is emitted. In the length of time it takes for the interrogation space to pass over the particle, the excitation-emission cycle is repeated many times by each particle. This allows the analyzer system 100 to detect tens to thousands of photons for each particle as the interrogation space passes over the particle. Photons emitted by the fluorescent particles are registered by the detector 184 with a time delay indicative of the time for the interrogation space to pass over the labeled particle. The photon intensity is recorded by the detector 184 and the sampling time is divided into bins, wherein the bins are uniform, arbitrary time segments with freely selectable time channel widths. The number of signals contained in each bin is evaluated. One or more of several statistical analytical methods are used to determine when a particle is present. As will be discussed further below, these methods include determining the baseline noise of the analyzer system 100 and determining signal strength for the fluorescent label at a statistical level above baseline noise to mitigate false positive signals from the detector 184.

[0047] Electromagnetic Radiation Source

[0048] Some embodiments of the analyzer system use a chemiluminescent label.

These embodiments may not require an electromagnetic radiation source for particle detection. In other embodiments, the extrinsic label or intrinsic characteristic of the particle is light-interacting, such as a fluorescent label or light-scattering label. In such an embodiment, a source of EM radiation is used to illuminate the label and/or the particle. EM radiation sources for excitation of fluorescent labels are preferred.

[0049] In some embodiments, the analyzer system 100 consists of an electromagnetic radiation source 110. Any number of radiation sources can be used in a scanning analyzer system 100 without departing from the scope of the disclosure. For example, the

electromagnetic radiation source 110 can be a continuous-wave laser producing wavelengths of between 200 nm and 1000 nm. Continuous-wave lasers provide continuous illumination without accessory electronic or mechanical devices, such as shutters, to interrupt their illumination. Such electromagnetic radiation sources have the advantage of being small, durable, and relatively inexpensive. In addition, they generally have the capacity to generate larger fluorescent signals than other light sources. Specific examples of suitable continuous- wave electromagnetic radiation sources include, but are not limited to: lasers of the argon, krypton, helium-neon, helium-cadmium types, as well as, diode lasers (red to infrared regions), each with the possibility of frequency doubling. In an embodiment where a continuous-wave laser is used, an electromagnetic radiation source of less than 3 mW, for example 2 mW and 1 mW, may have sufficient energy to excite a fluorescent label depending on the label selected. A beam of such energy output can be between 2 to 5 μιτι in diameter. When exposed at 3 mW, a labeled particle can be exposed to the laser beam for about 1 msec, equal to or less than about 500 μββΰ, equal to or less than about 100 μββΰ, equal to or less than about 50 μββΰ, or equal to or less than about 10 μββα

[0050] Light-emitting diodes (LEDs) are another low-cost, highly reliable illumination source. Advances in ultra-bright LEDs and dyes with high absorption cross- section and quantum yield have made LEDs applicable for single molecule detection. Such LED light can be used for particle detection alone or in combination with other light sources such as mercury arc lamps, elemental arc lamps, halogen lamps, arc discharges, plasma discharges, and any combination of these.

[0051] The electromagnetic radiation source can also comprise a pulsed laser. In such an embodiment, the pulse width, pulse delay, beam cross-section, focus spot size, instantaneous power, and average power emitted by the laser may be sufficient to excite the fluorescent label. In some embodiments, a laser pulse width of less than 1 nanosecond can be used. A pulse of this duration can be preferable in some pulsed laser applications. In other embodiments, a laser pulse width of 1, 2, 3, 4, or 5 nanoseconds can be used. In still other embodiments, a pulse width of between 2 to 5 nanoseconds can be used. In other embodiments, a pulse width of longer duration can be used.

[0052] The optimal laser intensity depends on the photo bleaching characteristics of the single dyes and the length of time required to traverse the interrogation space (including the speed of the particle, the distance between interrogation spaces, if more than one is used, and the size of the interrogation space(s)). To obtain a maximal signal, the sample can be illuminated at the highest intensity that will not photo bleach a high percentage of the dyes. The preferred intensity is such that no more that 5% of the dyes are bleached by the time the particle has traversed the interrogation space. [0053] The power of the laser is set depending on the type of dye molecules and the length of time the dye molecules are stimulated. The power can also depend on the speed that the interrogation space passes through the sample. Laser power is defined as the rate at which energy is delivered by the beam and is measured in units of Joules/second, or Watts. To provide a constant amount of energy to the interrogation space as the particle passes through, the less time the laser can illuminate the particle as the power output of the laser is increased. In some embodiments, the combination of laser power and illumination time is such that the total energy received by the interrogation space during the time of illumination is more than about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or 110 microJoules. In some embodiments, the combination of laser power and illumination time is such that the total energy received by the interrogation space during the time of illumination is less than about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or 110 microJoules. In some embodiments, the combination of laser power and illumination time is such that the total energy received by the interrogation space during the time of illumination is between about 0.1 and 100 microJoules, for example, between about 1 and 100 microJoules, between about 1 and 50 microJoules, between about 2 and 50 microJoules, between about 3 and 60 microJoules, between about 3 and 50 microJoules, between about 3 and 40 microJoules, or between about 3 and 30 microJoules. In some embodiments, the combination of laser power and illumination time is such that the total energy received by the interrogation space during the time of illumination is about 1 microJoule, about 3 microJoules, about 5 microJoules, about 10 microJoules, about 15 microJoules, about 20 microJoules, about 30 microJoules, about 40 microJoules, about 50 microJoules, about 60 microJoules, about 70 microJoules, about 80 microJoules, about 90 microJoules, or about 100 microJoules.

[0054] In some embodiments, the laser power output is set to at least about 1 mW, 2 mW, 3 mW, 4 mW, 5 mW, 6 mW, 7 mW, 8 mW, 9 mW, 10 mW, 13 mW, 15 mW, 20 mW, 25 mW, 30 mW, 40 mW, 50 mW, 60 mW, 70 mW, 80 mW, 90 mW, 100 mW, or more than 100 mW. In some embodiments, the laser power output is set to at least about 1 mW, at least about 3 mW, at least about 5 mW, at least about 10 mW, at least about 15 mW, at least about 20 mW, at least about 30 mW, at least about 40 mW, at least about 50 mW, at least about 60 mW, or at least about 90 mW.

[0055] The time that the laser illuminates the interrogation space can be set to no less than about 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1500, or 2000 microseconds. The time that the laser illuminates the interrogation space can be set to no more than about 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1500, or 2000 microseconds. The time that the laser illuminates the interrogation space can be set between about 1 and 1000 microseconds. For example, the time that the laser illuminates the interrogation space can be set between about 5 and 500 microseconds, between about 5 and 100 microseconds, between about 10 and 100 microseconds, between about 10 and 50 microseconds, between about 10 and 20 microseconds, between about 5 and 50 microseconds, or between about 1 and 100 microseconds. In some embodiments, the time that the laser illuminates the interrogation space is about 1 microsecond, about 5

microseconds, about 10 microseconds, about 25 microseconds, about 50 microseconds, about 100 microseconds, about 250 microseconds, about 500 microseconds, or about 1000 microseconds.

[0056] In some embodiments, the laser illuminates the interrogation space for 1 millisecond, 250 microseconds, 100 microseconds, 50 microseconds, 25 microseconds, or 10 microseconds with a laser that provides a power output of ImW, 2mW, 3 mW, 4 mW, 5 mW, or more than 5 mW. In some embodiments, a label is illuminated with a laser that provides a power output of 3 mW and illuminates the label for about 1000 microseconds. In other embodiments, a label is illuminated for less than 1000 milliseconds with a laser providing a power output of not more than about 20 mW. In other embodiments, the label is illuminated with a laser power output of 20 mW for less than or equal to about 250 microseconds. In some embodiments, the label is illuminated with a laser power output of about 5 mW for less than or equal to about 1000 microseconds.

[0057] Optical Scanning System

[0058] The scanning analyzer system described herein is, in some embodiments, different than traditional single molecule analyzers previously described elsewhere. In flow cytometry and other methods of fluorescence spectroscopy, a sample flows through an interrogation space. In contrast, the interrogation space in one embodiment of the analyzer provided herein is moved relative to the sample. This can be done by fixing the sample container relative to the instrument and moving the electromagnetic radiation beam.

Alternatively, the electromagnetic radiation beam can be fixed and the sample plate moved relative to the beam. In some embodiments, a combination of both can be used. In an embodiment wherein the sample plate is translated to create the moveable interrogation space, the limiting factor is the ability to move the plate smoothly enough so that the sample located on the sample plate is not jarred and the interrogation space is in the desired location.

[0059] In one embodiment, the electromagnetic radiation source 110 is focused onto a sample plate 170 of the analyzer systemlOO. The beam 126 from the continuous-wave electromagnetic radiation source 110 is optically focused through the base of the sample plate to a specified depth plane within the sample contained on or in the sample plate 170. Optical scanning of the sample can be accomplished using mirrors or lenses. In some embodiments, a mirror 122 is mounted on the end of a scan motor shaft 124 of the scan motor 120 but is tilted at a slight angle relative to the shaft 124. In some embodiments, as the

mirror 122 turns, it can deflect the electromagnetic radiation beam 126 thereby creating a small circle. By placing the mirror 122 between the objective 140 and the dichroic mirror 160, the spot at the focus of the objective can move around the sample. In some embodiments, the sample is scanned in a circular partem. In such an embodiment, a scan circle with a diameter of between about 500 μιτι and about 750 μιτι can be formed. In some embodiments, a scan circle with a diameter of between about 550 μιτι and 700 μιτι can be formed. In some embodiments, a scan circle with a diameter of between about 600 μιτι and 650 μιτι can be formed. In some embodiments a scan circle with a diameter of about 630 μιτι can be formed. In some embodiments, when a scan circle with a diameter of 630 μιτι is used, the scan circle can be traversed at about 8 revolutions per second (or about 500 RPM), equivalent to pumping the sample through a flow source at a rate of about 5 μΐ/min.

[0060] In some embodiments, the scan speed of the interrogation space is more than

100 RPM, is more than 300 RPM, is more than 500 RPM, is more than 700 RPM, or is more than 900 RPM. In some embodiments, the scan speed of the interrogation space is less than 1000 RPM, is less than 800 RPM, is less than 600 RPM, is less than 400 RPM, of is less than 200 RPM. In some embodiments, the scan speed of the interrogation space is between about 100 RPM and about 1000 RPM, between about 200 RPM and about 900 RPM, between about 300 RPM and about 800 RPM, between about 400 RPM and about 700 RPM, between about 450 RPM and about 600 RPM, or between about 450 RPM and about 550 RPM. With the development of improved electronics and optics, scanning in the z-axis may be required in addition to scanning in a two-dimensional pattern to avoid duplicate scanning of the same molecule. In some of the embodiments previously mentioned, the optical scanning pattern allows the scanning of a substantially different volume each time a portion of the sample is scanned. [0061] Scan speeds can range from about 10 cm/min to about 1000 cm/min. For example, depending on the scan pattern, scan speed can be about 10, 20, 40, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 cm/min.

[0062] The sample is scanned by an electromagnetic radiation source that interrogates a portion of the sample. A single molecule of interest may or may not be present in the interrogation space. In some embodiments, a portion of the sample is scanned a first time and then subsequently scanned a second time. In some embodiments the same portion of sample is scanned multiple times. In some embodiments, the sample is scanned such that the detection spot returns to a portion of sample a second time after sufficient time has passed so that the molecules detected in the first pass have drifted or diffused out of the portion, and other molecules have drifted or diffused into the portion. When the same portion of sample is scanned at least one or more times, the scanning speed can be slow enough to allow molecules to diffuse into, and out of, the space being interrogated. In some embodiments, the interrogation space is translated through a same portion of sample a first time and a second time at a sufficiently slow speed as to allow a molecule of interest that is detected the first time the interrogation space is translated through the portion of sample to substantially diffuse out of the portion of sample after the first time the portion of sample is interrogated by the interrogation space, and to further allow a subsequent molecule of interest, if present, to substantially diffuse into the portion of sample the second time the portion of sample is interrogated by the interrogation space. Figure 3 shows a graph of the diffusion time versus corresponding diffusion radius for molecules with a 155 KDa molecular weight. As used herein, "diffusion radius" refers to the standard deviation of the distance from the starting point that the molecule will most likely diffuse in the time indicated on the X-axis.

[0063] In some embodiments an alternative scan pattern is used. In some

embodiments, the scan partem can approximate an arc. In some embodiments, the scan pattern comprises at least one 90 degree angle. In some embodiments, the scan pattern comprises at least one angle less than 90 degrees. In some embodiments, the scan pattern comprises at least one angle that is more than 90 degrees. In some embodiments, the scan partem is substantially sinusoidal. In some embodiments, the optical scanning can be done with one mirror as previously described. In an alternative embodiment, the optical scanning can be done with at least two mirrors. Multiple mirrors allow scanning in a straight line, as well as allowing the system to scan back and forth, so that a serpentine partem is created. Alternatively, a multiple mirror optical scanning configuration allows for scanning in a raster pattern.

[0064] In an alternative embodiment, optical scanning can be done using an optical wedge. A wedge scanner provides a circular scan pattern and shortens the optical path because scan lenses are not required. An optical wedge approximates a prism with a very small angle. The optical wedge can be mounted to the shaft of the electromagnetic radiation source. The optical wedge rotates to create an optical scan partem. In an alternative embodiment, the scan mirror can be mounted using an electro-mechanical mount. In such an embodiment, the electro-mechanical mount would have two voice coils. One voice coil would cause displacement of the mirror in a vertical direction. The other voice coil would cause displacement of the mirror in a horizontal direction. Using this embodiment, any scan pattern desired can be created.

[0065] The scanning particle analyzer can scan the sample located in the sample plate in a two-dimensional orientation, e.g., following the x-y plane of the sample. In some embodiments, the sample can be scanned in a three-dimensional orientation consisting of scanning in an x-y plane and z direction. In some embodiments, the sample can be scanned along the x-y and z directions simultaneously. For example, the sample can be scanned in a helical pattern. In some embodiments, the sample can be scanned in the z direction only.

[0066] In some embodiments, a scan lens (130 as shown in Figures 2A and 2B) can re-direct the scanning optical path to the pupil of the objective. The scan lens focuses the image of the optical axis on the scan mirror to the exit pupil of the objective. The scan lens ensures that the scanning beam remains centered on the obj ective, despite the distance between the scan mirror and the microscope objective, thus improving the image and light collection efficiency of the scanning beam.

[0067] Interrogation Space

[0068] An interrogation space can be thought of as an effective volume of sample in which a single molecule of interest can be detected when present. Although there are various ways to calculate the interrogation space of the sample, the simplest method for determining the effective volume (V) of the interrogation space is to calculate the effective cross section of the detection volume. Because the detection volume is typically swept through the sample by translating the detection volume through the stationary sample, the volume is typically the result of the cross sectional area of the detection volume being swept through some distance during the time of measurement. If the sample concentration (C) is known and the number of molecules detected (N) during a period of time is known, then the sample volume consists of the number of molecules detected divided by the concentration of the sample, or V=N/C (where the sample concentration has units of molecules per unit volume).

[0069] For example, in some embodiments of the system described herein, all photons detected are counted and added up in 1 msec segments (photon counting bins). If a molecule of interest is present in the 1 msec segment, the count of photons detected is typically significantly higher than background. Therefore, the distance the detection volume has moved with respect to the sample is the appropriate distance to use to calculate the volume sampled in a single segment, i.e., the interrogation space. In this example, if the sample is analyzed for 60 seconds, then effectively 60,000 segments are scanned. If the effective volume is divided by the number of segments, the resulting volume is in essence the volume of a single segment, i.e., the interrogation space. Mathematically, the volume of the single segment, i.e., the interrogation space volume (Vs), equals the number of molecules detected (N) divided by the concentration of the sample multiplied by the number of segment bins (C n— where n represents the number of segment bins during the time the N number of molecules were counted). For exemplary purposes only, consider that a known standard of one femtomolar concentration is run through 60,000 segments, and 20 molecules of the standard are detected. Accordingly, the interrogation space volume, Vs, equals N/(C n) or 20/(6.02214E8- 6E4), or 553.513 μπι³. Thus, in this example, the interrogation space volume, which is the effective volume for one sample corresponding to one photon counting bin, is 553.513 μιη³.

[0070] In addition, from the interrogation volume described previously, the cross sectional area of the sample segment can be approximated using a capillary flow system with similar optics to the disclosure described herein. The cross section area (A) is approximated by dividing the interrogation volume (Vs) by the distance (t) the detection segment moves. The distance (t) the detection segment moves is given by 2-r-s/x, where t a function of the flow rate (r), the segment bin time (s), and the cross section of the capillary (x). For exemplary purposes only, consider a bin time (s) of 1 msec, a flow rate (r) of 0.08 μί/ββΰ, and a capillary cross sectional area (x) of 10,000 μιτι². Accordingly, the distance the interrogation space moves (t) is given by 2-rs/x, or (2 .08 μΐνββΰ- Ι msec)/(10,000 μπι²), or 16.0 μιτι. The effective cross sectional area (A) of the detector spot can further be calculated as Vs/t, or (553.513 μιη )/(16.7 μιτι), or 33 μιτι². Note that both the value of the interrogation volume, Vs, and the cross sectional area of the interrogation volume depend on the binning time.

[0071] The lower limit on the size of the interrogation space is bounded by the wavelengths of excitation energy currently available. The upper limit of the interrogation space size is determined by the desired signal-to-noise ratios— the larger the interrogation space, the greater the noise from, e.g., Raman scattering. In some embodiments, the volume of the interrogation space is more than about 1 μιη³, more than about 2 μηι³, more than about 3 μηι³, more than about 4 μηι³, more than about 5 μηι³, more than about 10 μηι³, more than about 15 μηι³, more than about 30 μηι³, more than about 50 μηι³, more than about 75 μηι³, more than about 100 μηι³, more than about 150 μηι³, more than about 200 μηι³, more than about 250 μηι³, more than about 300 μηι³, more than about 400 μηι³, more than about 500 μηι³, more than about 550 μηι³, more than about 600 μηι³, more than about 750 μηι³, more than about 1000 μηι³, more than about 2000 μηι³, more than about 4000 μηι³, more than about 6000 μηι³, more than about 8000 μηι³, more than about 10000 μηι³, more than about 12000 μιτι³, more than about 13000 μηι³, more than about 14000 μηι³, more than about 15000 μηι³, more than about 20000 μηι³, more than about 30000 μηι³, more than about 40000 μηι³, or more than about 50000 μηι³. In some embodiments, the interrogation space is of a volume less than about 50000 μηι³, less than about 40000 μηι³, less than about 30000 μηι³, less than about 20000 μηι³, less than about 15000 μηι³, less than about 14000 μηι³, less than about 13000 μηι³, less than about 12000 μηι³, less than about 11000 μηι³, less than about 9500 μηι³, less than about 8000 μηι³, less than about 6500 μηι³, less than about 6000 μηι³, less than about 5000 μηι³, less than about 4000 μηι³, less than about 3000 μηι³, less than about 2500 μιη³, less than about 2000 μηι³, less than about 1500 μηι³, less than about 1000 μηι³, less than about 800 μηι³, less than about 600 μηι³, less than about 400 μηι³, less than about 200 μηι³, less than about 100 μηι³, less than about 75 μηι³, less than about 50 μηι³, less than about 25 μιη³, less than about 20 μηι³, less than about 15 μηι³, less than about 14 μηι³, less than about 13 μηι³, less than about 12 μηι³, less than about 11 μιη³, less than about 10 μηι³, less than about 5 μηι³, less than about 4 μηι³, less than about 3 μηι³, less than about 2 μηι³, or less than about 1 μιη³. In some embodiments, the volume of the interrogation space is between about 1 μιη³ and about 10000 μηι³. In some embodiments, the interrogation space is between about 1 μιη³ and about 1000 μηι³. In some embodiments, the interrogation space is between about 1 μιη³ and about 100 μηι³. In some embodiments, the interrogation space is between about 1 μιη³ and about 50 μηι³. In some embodiments, the interrogation space is between about 1 μηι³ and about 10 μηι³. In some embodiments, the interrogation space is between about 2 μιη³ and about 10 μηι³. In some embodiments, the interrogation space is between about 3 μιη³ and about 7 μηι³.

[0072] Sample Plate

[0073] Some embodiments of the disclosure described herein use a sample plate 170 to hold the sample being detected for a single molecule of interest. The sample plate in some embodiments is a microtiter plate. The microtiter plate consists of a base 172 and a top surface 174. The top surface 174 of the microtiter plate in some embodiments consists of at least one well for containing a sample of interest. In some embodiments, the microtiter plate consists of a plurality of wells to contain a plurality of samples. The system described herein is sensitive enough so that only a small sample size is needed. In some embodiments the sample size can be less than approximately 100, 10, 1 , 0.1 , 0.01 , or 0.001 μΐ. The microtiter plate in some embodiments can be one constructed using microfabrication techniques. In some embodiments, the top surface 174 of the plate can be smooth. The sample can be sized so that the sample is self-contained by the surface tension of the sample itself. In such an embodiment, the sample forms a droplet on the surface of the plate. In some embodiments, the sample can then be scanned for a molecule of interest.

[0074] The sample is scanned through the sample plate material, e.g., through the walls of the microwells. In some embodiments, the sample is scanned through the base 172 of the sample plate. In some embodiments, the base 172 of the sample plate is made of a material that is transparent to light. In some embodiments, the base 172 of the sample plate is made of a material that is transparent to electromagnetic radiation. The sample plate 170 is transparent to an excitation wavelength of interest. Using a transparent material allows the wavelength of the excitation beam to pass through the sample plate 170 and excite the molecule of interest or the fluorescent label conjugated to the molecule of interest. The transparency of the plate 170 further allows the detector 184 to detect the emissions from the excited molecules of interest. In some embodiments, the base material is substantially transparent to light of wavelengths for all the wavelength associated with each of the electromagnetic radiation sources and each of the emission spectra of the labels used in multiplex single molecule analysis.

[0075] The thickness of the sample plate 170 is also considered. The sample is scanned by electromagnetic radiation that passes through a portion of the material of the plate 170. The thickness of the plate 170 allows an image to be formed on a first side of the portion of the plate 170 that is scanned by a high numerical aperture lens that is positioned on a second side of the portion of the plate 170 that is scanned. Such an embodiment facilitates the formation of an image within the sample and not within the base 172. The image formed corresponds to the interrogation space of the system 100. The image should be formed at the depth of the single molecule of interest. As previously mentioned, the thickness of the plate 170 depends on the working distance and depth of field of the lens that is used. Commercial plates available are typically 650 microns thick.

[0076] The plate 170 can be made out of any suitable material that allows the excitation energy to pass through the plate. In some embodiments, the plate 170 is made of polycarbonate. In some embodiments, the plate 170 is made of polyethylene. In some embodiments, a commercially available plate can be used, such as a NUNC™ brand plate. Any plate made of a suitable material and of a suitable thickness can be used. In preferred embodiments, the plate 170 is made out of a material with low fluorescence, thereby reducing background fluorescence. Background fluorescence resulting from the plate 170 material can be further avoided by minimizing the thickness of the plate 170.

[0077] In some embodiments, the sample consists of a small volume of fluid that can contain a particular type of molecule. In such an embodiment, the single molecule of interest, if present, can be detected and counted in a location anywhere in the fluid volume. In some embodiments, scanning the sample comprises scanning a smaller concentrated sample. In such an embodiment, the optical scanning can occur at the surface 174 of the sample plate 170, for example, if the highest concentration of molecules is located at the surface 174 of the sample plate 170. This can occur if the single molecules are adsorbed to the surface 174 of the plate 170 or if they are bound to antibodies or other binding molecules adhered to the surface 174 of the plate 170. When antibodies are used to capture a single molecule of interest, the antibodies can be applied to the surface 174 of the sample plate 170, e.g., to the bottom of a microwell(s). The single molecule of interest then binds to the antibodies located within the microwell. In some embodiments, an elution step is done to remove the bound single molecule of interest. The presence or absence of the unbound molecules can then be detected in a smaller sample volume. In some embodiments wherein the elution step is done, the single molecules may or may not be attached to paramagnetic beads. If no beads are used, the elution buffer can be added to the sample well and the presence or absence of the single molecule of interest can be detected. In some embodiments, a paramagnetic bead is used as a capture bead to capture the single molecule of interest.

[0078] In some embodiments of the scanning single molecule analyzer described herein, the electromagnetic (EM) radiation source is directed to the sample interrogation space without passing through the material of the sample plate 170. Image formation occurs in the sample on the same side as the beam 126 directed to the sample. In such an embodiment, a water immersion lens can be used but is not required to image the sample through the air-liquid interface. In zero carryover systems wherein the objective does not come in contact with the sample, sample carryover between samples does not occur.

[0079] In some embodiments, the sample container is associated with a microfluidic cell or chip that includes appropriate sample processing reagents and binding surfaces on the chip. In some aspects, some, or all, of the sample processing occurs on the chip, which may be accompanied by an apparatus to mobilize the sample on reagents throughout the chip (e.g., electromagnetic, pneumatic, and/or centrifugal). A sample container, well, chamber or surface that is transparent to electromagnetic radiation as described above for the plate 170 allows for the analysis of the processed sample as described herein.

[0080] Detectors

[0081] In one embodiment, light emitted by a fluorescent label after exposure to electromagnetic radiation is detected. The emitted light can be, e.g., ultra-violet, visible or infrared. Referring to Figures 2A and 2B, the detector 184 (or other embodiments) can capture the amplitude and duration of photon bursts from a fluorescent moiety, and convert the amplitude and duration of the photon bursts to electrical signals. Detection devices such as CCD cameras, video input module cameras, and streak cameras can be used to produce images with contiguous signals. Other embodiments use devices such as a bolometer, a photodiode, a photodiode array, avalanche photodiodes, and photomultipliers, which produce sequential signals. Any combination of the aforementioned detectors can be used.

[0082] Several distinct characteristics of the emitted electromagnetic radiation 126 between an interrogation space and its corresponding detector 184, can be detected including: emission wavelength, emission intensity, burst size, burst duration, and fluorescence polarization. In some embodiments, the detector 184 is a photodiode used in reverse bias. Such a photodiode usually has an extremely high resistance. This resistance is reduced when light of an appropriate frequency shines on the P/N junction. Hence, a reverse-biased diode can be used as a detector by monitoring the current running through it. Circuits based on this effect are more sensitive to light than circuits based on zero bias.

[0083] The photodiode can be provided as an avalanche photodiode. These photodiodes can be operated with much higher reverse bias than conventional photodiodes, thus allowing each photo-generated carrier to be multiplied by avalanche breakdown. This results in internal gain within the photodiode, thereby increasing the effective responsiveness and sensitivity of the device. The choice of photodiode is determined by the energy or emission wavelength emitted by the fluorescently labeled particle. In some embodiments, the detector is an avalanche photodiode detector that detects energy between 300 nm and 1700 nm. In another embodiment, silicon avalanche photodiodes can be used to detect

wavelengths between 300 nm and 1100 nm. In another embodiment, the photodiode is an indium gallium arsenide photodiode that detects energy in the range of 800-2600 nm. In another embodiment, indium gallium arsenide photodiodes can be used to detect wavelengths between 900 nm and 1700 nm. In some embodiments, the photodiode is a silicon photodiode that detects energy in the range of 190-1 100 nm. In another embodiment, the photodiode is a germanium photodiode that detects energy in the range of 800-1700 nm. In yet other embodiments, the photodiode is a lead sulfide photodiode that detects energy in the range of between less than 1000 nm to 3500 nm. In some embodiments, the avalanche photodiode is a single-photon detector designed to detect energy in the 400 nm to 1 100 nm wavelength range. Single photon detectors are commercially available (for example Perkin Elmer and

Hamamatsu).

[0084] Once a particle is labeled to render it detectable (or if the particle possesses an intrinsic characteristic rendering it detectable), any suitable detection mechanism known in the art can be used without departing from the scope of the disclosure, for example a CCD camera, a video input module camera, a streak camera, a bolometer, a photodiode, a photodiode array, avalanche photodiodes, and photomultipliers producing sequential signals, and combinations thereof. Different characteristics of the electromagnetic radiation can be detected including: emission wavelength, emission intensity, burst size, burst duration, fluorescence polarization, and any combination thereof.

[0085] Binding Partners

[0086] Any suitable binding partner with the requisite specificity for the form of molecule, e.g., a marker, to be detected can be used. If the molecule, e.g., a marker, has several different forms, various specificities of binding partners are possible. Suitable binding partners are known in the art and include antibodies, aptamers, lectins, and receptors. A useful and versatile type of binding partner is an antibody.

[0087] In some embodiments, the binding partner is an antibody specific for a molecule to be detected. The term "antibody," as used herein, is a broad term and is used in its ordinary sense, including, without limitation, to refer to naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, single chain antibodies, chimeric, bifunctional, and humanized antibodies, as well as antigen-binding fragments thereof. It will be appreciated that the choice of epitope or region of the molecule to which the antibody is raised will determine its specificity, e.g., for various forms of the molecule, if present, or for total (e.g., all, or substantially all, of the molecule).

[0088] Methods for producing antibodies are well-established. One skilled in the art will recognize that many procedures are available for the production of antibodies, for example, as described in Antibodies, A Laboratory Manual, Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988), Cold Spring Harbor, N.Y. One skilled in the art will also appreciate that binding fragments or Fab fragments that mimic antibodies can be prepared from genetic information by various procedures (Antibody Engineering: A Practical Approach (Borrebaeck, C, ed.), 1995, Oxford University Press, Oxford; J. Immunol. 149, 3914-3920 (1992)). Monoclonal and polyclonal antibodies to molecules, e.g., proteins, and markers are also commercially available (R and D Systems, Minneapolis, Minn.; HyTest, HyTest Ltd., Turku Finland; Abeam Inc., Cambridge, Mass., USA, Life Diagnostics, Inc., West Chester, Pa., USA; Fitzgerald Industries International, Inc., Concord, Mass. 01742- 3049 USA; BiosPacific, Emeryville, Calif). The antibody may be a monoclonal or a polyclonal antibody.

[0089] Capture binding partners and detection binding partner pairs, e.g., capture and detection antibody pairs, can be used in embodiments of the disclosure. Thus, in some embodiments, a heterogeneous assay protocol is used in which, typically, two binding partners, e.g., two antibodies, are used. One binding partner is a capture partner, usually immobilized on a solid support, and the other binding partner is a detection binding partner, typically with a detectable label attached. Such antibody pairs are available from the sources described above, e.g., BiosPacific, Emeryville, Calif. Antibody pairs can also be designed and prepared by methods well-known in the art. Compositions of the disclosure include antibody pairs wherein one member of the antibody pair is a label as described herein, and the other member is a capture antibody. [0090] In some embodiments it is useful to use an antibody that cross-reacts with a variety of species, either as a capture antibody, a detection antibody, or both. Such embodiments include the measurement of drug toxicity by determining, e.g., release of cardiac troponin into the blood as a marker of cardiac damage. A cross-reacting antibody allows studies of toxicity to be done in one species, e.g. a non-human species, and direct transfer of the results to studies or clinical observations of another species, e.g., humans, using the same antibody or antibody pair in the reagents of the assays, thus decreasing variability between assays. Thus, in some embodiments, one or more of the antibodies for use as a binding partner to the marker of the molecule of interest, e.g., cardiac troponin, such as cardiac troponin I, can be a cross-reacting antibody. In some embodiments, the antibody cross-reacts with the marker, e.g. cardiac troponin, from at least two species selected from the group consisting of human, monkey, dog, and mouse. In some embodiments, the antibody cross-reacts with the marker, e.g., cardiac troponin, from the entire group consisting of human, monkey, dog, and mouse.

[0091] Labels

[0092] Many strategies can be used for labeling target molecules to enable their detection or discrimination in a mixture of particles. The labels can be attached by any known means, including methods that utilize non-specific or specific interactions of label and target molecule. Labels can provide a detectable signal or affect the mobility of the particle in an electric field. Labeling can be accomplished directly or through binding partners.

[0093] In some embodiments, the label comprises a binding partner to the molecule of interest, where the binding partner is attached to a fluorescent moiety. The compositions and methods of the disclosure can use highly fluorescent moieties. Moieties suitable for the compositions and methods of the disclosure are described in more detail below. Fluorescent molecules may be attached to binding partners by any known means such as direct conjugation or indirectly (e.g., biotin/streptavidin).

[0094] In some embodiments, the disclosure provides a label for detecting a biological molecule comprising a binding partner for the biological molecule that is attached to a fluorescent moiety, wherein the fluorescent moiety is capable of emitting at least about 200 photons when simulated by a laser emitting light at the excitation wavelength of the moiety, wherein the laser is focused on a spot not less than about 5 microns in diameter that contains the moiety, and wherein the total energy directed at the spot by the laser is no more than about 3 microJoules. In some embodiments, the moiety comprises a plurality of fluorescent entities, e.g., about 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, or about 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, or 3 to 10 fluorescent entities. In some embodiments, the moiety comprises about 2 to 4 fluorescent entities.

[0095] In some embodiments, the binding partner comprises an antibody. In some embodiments, the antibody is a monoclonal antibody. In other embodiments, the antibody is a polyclonal antibody. The antibody can be specific to any suitable marker. In some embodiments, the antibody is specific to a marker that is selected from the group consisting of cytokines, growth factors, oncology markers, markers of inflammation, endocrine markers, autoimmune markers, thyroid markers, cardiovascular markers, markers of diabetes, markers of infectious disease, neurological markers, respiratory markers, gastrointestinal markers, musculoskeletal markers, dermatological disorders, and metabolic markers.

[0096] A "fluorescent moiety," as that term is used herein, includes one or more fluorescent entities whose total fluorescence is such that the moiety can be detected in the single molecule detectors described herein. Thus, a fluorescent moiety can comprise a single entity (e.g., a Quantum Dot or fluorescent molecule) or a plurality of entities (e.g., a plurality of fluorescent molecules). It will be appreciated that when "moiety," as that term is used herein, refers to a group of fluorescent entities, e.g., a plurality of fluorescent dye molecules, each individual entity can be attached to the binding partner separately or the entities can be attached together, as long as the entities as a group provide sufficient fluorescence to be detected.

[0097] Typically, the fluorescence of the moiety involves a combination of quantum efficiency and lack of photobleaching sufficient that the moiety is detectable above background levels in a single molecule detector, with the consistency necessary for the desired limit of detection, accuracy, and precision of the assay. For example, in some embodiments, the fluorescence of the fluorescent moiety is such that it allows detection and/or quantitation of a molecule, e.g., a marker, at a limit of detection of less than about 10, 5, 4, 3, 2, 1, 0.1, 0.01, 0.001 , 0.0001, 0.00001 , or 0.000001 pg/ml and with a coefficient of variation of less than about 20, 15, 14, 13, 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 % or less, e.g., about 10% or less, in the instruments described herein. In some embodiments, the fluorescence of the fluorescent moiety is such that it allows detection and/or quantitation of a molecule, e.g., a marker, at a limit of detection of less than about 5, 1 , 0.5, 0.1 , 0.05, 0.01 , 0.005, 0.001 pg/ml and with a coefficient of variation of less than about 10%, in the instruments described herein.

[0098] "Limit of detection," as that term is used herein, includes the lowest concentration at which one can identify a sample as containing a molecule of the substance of interest, e.g., the first non-zero value. It can be defined by the variability of zeros and the slope of the standard curve. For example, the limit of detection of an assay can be determined by running a standard curve, determining the standard curve zero value, and adding two standard deviations to that value. A concentration of the substance of interest that produces a signal equal to this value is the "lower limit of detection" concentration.

[0099] Furthermore, the moiety has properties that are consistent with its use in the assay of choice. In some embodiments, the assay is an immunoassay, where the fluorescent moiety is attached to an antibody; the moiety must not aggregate with other antibodies or proteins, or must not undergo any more aggregation than is consistent with the required accuracy and precision of the assay. In some embodiments, fluorescent moieties that are preferred are fluorescent moieties, e.g., dye molecules, that have a combination of: 1) high absorption coefficient; 2) high quantum yield; 3) high photostability (low photobleaching); and 4) compatibility with labeling the molecule of interest (e.g., protein) so that it can be analyzed using the analyzers and systems of the disclosure (e.g., does not cause precipitation of the protein of interest, or precipitation of a protein to which the moiety has been attached).

[00100] Fluorescent moieties, e.g., a single fluorescent dye molecule or a plurality of fluorescent dye molecules, which are useful in some embodiments of the disclosure, can be defined in terms of their photon emission characteristics when stimulated by EM radiation. For example, in some embodiments, the disclosure utilizes a fluorescent moiety, e.g., a moiety comprising a single fluorescent dye molecule or a plurality of fluorescent dye molecules, that is capable of emitting an average of at least about 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 350, 400, 500, 600, 700, 800, 900, or 1000 photons when simulated by a laser emitting light at the excitation wavelength of the moiety, where the laser is focused on a spot of not less than about 5 microns in diameter that contains the moiety, and where the total energy directed at the spot by the laser is no more than about 3 micro Joules. It will be appreciated that the total energy can be achieved by many different combinations of power output of the laser and length of time of exposure of the dye moiety. E.g., a laser of a power output of 1 mW can be used for 3 ms, 3 mW for 1 ms, 6 mW for 0.5 ms, 12 mW for 0.25 ms, and so on. [00101] In some embodiments, the fluorescent moiety comprises an average of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 fluorescent entities, e.g., fluorescent molecules. In some embodiments, the fluorescent moiety comprises an average of no more than about 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 fluorescent entities, e.g., fluorescent molecules. In some embodiments, the fluorescent moiety comprises an average of about 1 to 11 fluorescent entities. By "average" it is meant that, in a given sample that is representative of a group of labels of the disclosure, where the sample contains a plurality of the binding partner-fluorescent moiety units, the molar ratio of the particular fluorescent entity to the binding partner, as determined by standard analytical methods, corresponds to the number or range of numbers specified. For example, in embodiments wherein the label comprises a binding partner that is an antibody and a fluorescent moiety that comprises a plurality of fluorescent dye molecules of a specific absorbance, a spectrophotometric assay can be used in which a solution of the label is diluted to an appropriate level and the absorbance at 280 nm is taken to determine the molarity of the protein (antibody) and an absorbance at, e.g., 650 nm (for ALEXA FLUOR^® 647), is taken to determine the molarity of the fluorescent dye molecule. The ratio of the latter molarity to the former represents the average number of fluorescent entities (dye molecules) in the fluorescent moiety attached to each antibody.

[00102] In some embodiments, the disclosure uses fluorescent moieties that comprise fluorescent dye molecules. The dye should emit sufficient photons when stimulated by an excitation source such that it is useful in the measurement of analytes as described herein.

[00103] A non-inclusive list of useful fluorescent entities for use in the fluorescent moieties of the disclosure is given in Table 2.

TABLE 2

FLUORESCENT ENTITIES

Dye E Ex (nm) E (M)-l Em (nm) MMw

Bimane 380 5,700 458 282.31

Dapoxyl 373 22,000 551 362.83

Dimethylamino coumarin-4-

375 22,000 470 344.32 acetic acid

Marina blue 365 19,000 460 367.26

8-Anilino naphthalene- 1 -sulfonic

372 480

acid

Cascade blue 376 23,000 420 607.42

ALEXA FLUOR^® 405 402 35,000 421 1028.26 Cascade blue 400 29.000 420 607.42

Cascade yellow 402 24,000 545 563.54

BD Horizon Brilliant™BV510 405 510

BD Horizon Brilliant™BV421 407 2,500,000 421 70,000

Pacific blue 410 46,000 455 339.21

PyMPO 415 26.000 570 582.41

ALEXA FLUOR^® 430 433 15,000 539 701.75

ATTO-425 438 486

NBD 465 22,000 535 391.34

ALEXA FLUOR^® 488 495 73.000 519 643.41

Fluorescein 494 79,000 518 376.32

Oregon Green 488 496 76,000 524 509.38

Atto 495 495 522

Cy2 489 150,000 506 713.78

DY-480-XL 500 40.000 630 514.60

DY-485-XL 485 20,000 560 502.59

DY-490-XL 486 27,000 532 536.58

BD Horizon Brilliant™BB515 490 515

DY-500-XL 505 90.000 555 596.68

DY-520-XL 520 40,000 664 514.60

ALEXA FLUOR^® 532 531 81,000 554 723.77

BODIPY 530/550 534 77,000 554 513.31

6-HEX 535 98.000 556 680.07

6-JOE 522 75,000 550 602.34

Rhodamine 6G 525 108,000 555 555.59

Atto-520 520 542

Cy3B 558 130,000 572 658.00

ALEXA FLUOR^® 610 612 138.000 628

ALEXA FLUOR^® 633 632 159,000 647 ca. 1200

ALEXA FLUOR^® 647 650 250,000 668 ca. 1250

BODIPY 630/650 625 101,000 640 660.50

Cy5 649 250.000 670 791.99

ALEXA FLUOR^® 660 663 110,000 690

ALEXA FLUOR^® 680 679 184,000 702

ALEXA FLUOR^® 700 702 192,000 723

ALEXA FLUOR^® 750 749 240,000 782

B-phycoerythrin 546. 565 2,410.000 575 240,000

R-phycoerythrin 480, 546, 565 1,960,000 578 240,000 Allophycocyanin 650 700,000 660 700,000

PBXL-1 545 666

PBXL-3 614 662

Atto-tec dyes

Name Ex (nm) Em (nm) QY □ (ns)

ATTO 425 436 486 0.9 3.5

ATTO 495 495 522 0.45 2.4

ATTO 520 520 542 0.9 3.6

ATTO 532 532 553 0.9 3.8

ATTO 560 561 585 0.92 3.4

ATTO 590 598 634 0.8 3.7

ATTO 610 605 630 0.7 3.3

ATTO 655 665 690 0.3 1.9

ATTO 680 680 702 0.3 1.8

Dyomics Fluors

Molar absorbance* Molecular weight label Ex (nm) Em (nm)

[1-mol - 1-cm - 1] #[g mol - 1]

DY-495/5 495 70,000 520 489.47

DY-495/6 495 70,000 520 489.47

DY-495X/5 495 70,000 520 525.95

DY-495X/6 495 70,000 520 525.95

DY-505/5 505 85,000 530 485.49

DY-505/6 505 85,000 530 485.49

DY-505X/5 505 85,000 530 523.97

DY-505X/6 505 85,000 530 523.97

DY-550 553 122,000 578 667.76

DY-555 555 100.000 580 636.18

DY-610 609 81.000 629 667.75

DY-615 621 200.000 641 578.73

DY-630 636 200.000 657 634.84

DY-631 637 185.000 658 736.88

DY-633 637 180.000 657 751.92

DY-635 647 175.000 671 658.86

DY-636 645 190.000 671 760.91

DY-650 653 170.000 674 686.92

DY-651 653 160.000 678 888.96

DYQ-660 660 117,000 — 668.86

DYQ-661 661 116,000 — 770.90 DY-675 674 110.000 699 706.91

DY-676 674 145.000 699 807.95

DY-680 690 125.000 709 634.84

DY-681 691 125.000 708 736.88

DY-700 702 96.000 723 668.86

DY-701 706 115.000 731 770.90

DY-730 734 185.000 750 660.88

DY-731 736 225.000 759 762.92

DY-750 747 240.000 776 712.96

DY-751 751 220.000 779 814.99

DY-776 771 147.000 801 834.98

DY-780-OH 770 70.000 810 757.34

DY-780-P 770 70.000 810 957.55

DY-781 783 98.000 800 762.92

DY-782 782 102.000 800 660.88

EVOblue-10 651 101.440 664 389.88

EVOblue-30 652 102.000 672 447.51

Quantum Dots: Qdot 525, QD 565, QD 585, QD 605, QD 655, QD 705, QD 800

[00104] Suitable dyes for use in the disclosure include modified carbocyanine dyes. Such modification comprises modification of an indolium ring of the carbocyanine dye to permit a reactive group or conjugated substance at the number three position. The modification of the indolium ring provides dye conjugates that are uniformly and substantially more fluorescent on proteins, nucleic acids and other biopolymers, than conjugates labeled with structurally similar carbocyanine dyes bound through the nitrogen atom at the number one position. In addition to having more intense fluorescence emission than structurally similar dyes at virtually identical wavelengths, and decreased artifacts in their absorption spectra upon conjugation to biopolymers, the modified carbocyanine dyes have greater photostability and higher absorbance (extinction coefficients) at the wavelengths of peak absorbance than the structurally similar dyes. Thus, the modified carbocyanine dyes result in greater sensitivity in assays using the modified dyes and their conjugates. Preferred modified dyes include compounds that have at least one substituted indolium ring system in which the substituent on the 3-carbon of the indolium ring contains a chemically reactive group or a conjugated substance. Other dye compounds include compounds that incorporate an azabenzazolium ring moiety and at least one sulfonate moiety. The modified

carbocyanine dyes that can be used to detect individual molecules in various embodiments of the disclosure are described in U. S. Pat. No. 6,977,305, which is herein incorporated by reference in its entirety. Thus, in some embodiments the labels of the disclosure utilize a fluorescent dye that includes a substituted indolium ring system in which the substituent on the 3-carbon of the indolium ring contains a chemically reactive group or a conjugated substance group.

[00105] Currently available organic fluors can be improved by rendering them less hydrophobic by adding hydrophilic groups such as polyethylene. Alternatively, currently sulfonated organic fluors such as the ALEXA FLUOR® 647 dye can be rendered less acidic by making them zwitterionic. Particles such as antibodies that are labeled with the modified fluors are less likely to bind non-specifically to surfaces and proteins in immunoassays, and thus enable assays that have greater sensitivity and lower backgrounds. Methods for modifying and improving the properties of fluorescent dyes for the purpose of increasing the sensitivity of a system that detects single molecules are known in the art. Preferably, the modification improves the Stokes shift while maintaining a high quantum yield.

[00106] Quantum Dots

[00107] In some embodiments, the fluorescent label moiety that is used to detect a molecule in a sample using the analyzer systems of the disclosure is a quantum dot.

Quantum dots (QDs), also known as semiconductor nanocrystals or artificial atoms, are semiconductor crystals that contain anywhere between 100 to 1 ,000 electrons and range from 2-10 nm. Some QDs can be between 10-20 nm in diameter. QDs have high quantum yields, which makes them particularly useful for optical applications. QDs are fluorophores that fluoresce by forming excitons, which are similar to the excited state of traditional fluorophores, but have much longer lifetimes of up to 200 nanoseconds. This property provides QDs with low photobleaching. The energy level of QDs can be controlled by changing the size and shape of the QD, and the depth of the QDs' potential wells. One optical feature of small excitonic QDs is coloration, which is determined by the size of the dot. The larger the dot, the redder, or more towards the red end of the spectrum, the fluorescence. The smaller the dot, the bluer, or more towards the blue end, it is. The bandgap energy that determines the energy and hence the color of the fluoresced light is inversely proportional to the square of the size of the QD. Larger QDs have more energy levels which are more closely spaced, thus allowing the QD to absorb photons containing less energy, i.e., those closer to the red end of the spectrum. Because the emission frequency of a dot is dependent on the bandgap, it is possible to control the output wavelength of a dot with extreme precision. In some embodiments the protein that is detected with the single molecule analyzer system is labeled with a QD. In some embodiments, the single molecule analyzer is used to detect a protein labeled with one QD and uses a filter to allow for the detection of different proteins at different wavelengths.

[00108] QDs have broad excitation and narrow emission properties that, when used with color filtering, require only a single electromagnetic source to resolve individual signals during multiplex analysis of multiple targets in a single sample. Thus, in some embodiments, the analyzer system comprises one continuous-wave laser and particles that are each labeled with one QD. Colloidally prepared QDs are free floating and can be attached to a variety of molecules via metal-coordinating functional groups. These groups include, but are not limited to, thiol, amine, nitrile, phosphine, phosphine oxide, phosphonic acid, carboxylic acids or other ligands. By bonding appropriate molecules to the surface, the quantum dots can be dispersed or dissolved in nearly any solvent or incorporated into a variety of inorganic and organic films. Quantum dots (QDs) can be coupled to streptavidin directly through a maleimide ester coupling reaction or to antibodies through a meleimide-thiol coupling reaction. This yields a material with a biomolecule covalently attached on the surface, which produces conjugates with high specific activity. In some embodiments, the protein that is detected with the single molecule analyzer is labeled with one quantum dot. In some embodiments, the quantum dot is between 10 and 20 nm in diameter. In other embodiments, the quantum dot is between 2 and 10 nm in diameter. In other embodiments, the quantum dot is about 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, or 20 nm in diameter. Useful Quantum Dots comprise QD 605, QD 610, QD 655, and QD 705. A preferred Quantum Dot is QD 605.

[00109] Polymeric Fluorophores

[00110] In some embodiments, the fluorescent label moiety is a polymeric fluorophore.

Polymeric fluorophores are designed to have greater absorption of excitation light and brighter emission fluorescence than convention fluorophores. Polymeric fluorophores work as molecular antennae and gather higher levels of excitation energy. This energy can either be emitted by the polymer itself as fluorescence or can be transferred to a covalently linked tandem fluorescent dye through a fluorescence resonance energy transfer (FRET) process.

[00111] Polymeric fluorophores can be designed such that they have a polymeric backbone that has intrinsic absorption and fluorescence at a specific wavelength, such as BD Horizon™ BV421 that is excited at 407 nm and maximally fluoresces at 421 nm and BD Horizon™ BB515 that is excited at 490 nm and maximally fluoresces at 515 nm. Tandem dyes can also be created where acceptor dyes are covalently linked to the polymeric backbone to allow multiple emission spectra besides the intrinsic polymeric one. This allows for a family of fluorophores that can all be excited at the same wavelength but emit at different wavelengths. In some embodiments the protein that is detected with the single molecule analyzer system is labeled with a polymeric fluorophore. In some embodiments, the protein that is detected with the single molecule analyzer system is labeled with biotin and a streptavidin molecule covalently bound to a polymeric fluorophore is added, which binds to the biotin. In some embodiments, the single molecule analyzer is used to detect a protein labeled with a polymeric fluorophore.

[00112] Some polymeric fluorophores have been designed to have a narrow excitation and multiple potential emission properties that depend on covalently linked fluorophores. These fluorophores, when used with color filtering, require only a single electromagnetic excitation source to resolve individual signals during multiplex analysis of multiple targets in a single sample. Thus, in some embodiments, the analyzer system comprises one continuous- wave laser and particles that are each labeled with a different polymeric fluorophore, which may be detected by multiple detectors. Polymeric fluorophores can be coupled to antibodies directly or to antibodies indirectly through a coupling reaction (e.g., biotin/streptavin). This yields a material with a biomolecule covalently attached on the surface, which produces conjugates with high specific activity. In some embodiments, the protein that is detected with the single molecule analyzer is labeled with one polymeric fluorophore. Useful polymeric fluorophores comprise BV421, BV510, and BB515.

[00113] Binding Partner-Fluorescent Moiety Compositions

[00114] The labels of the disclosure generally contain a binding partner, e.g., an antibody, bound to a fluorescent moiety to provide the requisite fluorescence for detection and quantitation in the instruments described herein. Any suitable combination of binding partner and fluorescent moiety for detection in the single molecule detectors described herein can be used as a label in the disclosure. In some embodiments, the disclosure provides a label for a marker of a biological state, where the label includes an antibody to the marker and a fluorescent moiety. The marker can be any of the markers described above. The antibody can be any antibody as described above. A fluorescent moiety can be attached such that the label is capable of emitting an average of at least about 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 350, 400, 500, 600, 700, 800, 900, or 1000 photons when stimulated by a laser emitting light at the excitation wavelength of the moiety, where the laser is focused on a spot of not less than about 5 microns in diameter that contains the label, and wherein the total energy directed at the spot by the laser is no more than about 3 microJoules.

[00115] Attachment of the fluorescent moiety, or fluorescent entities that make up the fluorescent moiety, to the binding partner, e.g., an antibody, can be by any suitable means; such methods are well-known in the art and exemplary methods are given in the Examples. In some embodiments, after attachment of the fluorescent moiety to the binding partner to form a label for use in the methods of the disclosure, and prior to the use of the label for labeling the marker of interest, it is useful to perform a filtration step. E.g., an antibody-dye label can be filtered prior to use, e.g., through a 0.2 micron filter, or any suitable filter, for removing aggregates. Other reagents for use in the assays of the disclosure can also be filtered, e.g., through a 0.2 micron filter, or any suitable filter. Without being bound by theory, it is thought that such filtration removes a portion of the aggregates of the, e.g., antibody-dye labels. Such aggregates can bind as a unit to the protein of interest, but, upon release in elution buffer, the aggregates are likely to disaggregate. Therefore, false positives can result when several labels are detected from an aggregate that has bound to only a single protein molecule of interest. Regardless of theory, filtration has been found to reduce false positives in the subsequent assay and to improve accuracy and precision.

[00116] It will be appreciated that immunoassays often employ a sandwich format in which binding partner pairs, e.g. antibodies, to the same molecule, e.g., a marker, are used. The disclosure also encompasses binding partner pairs, e.g., antibodies, wherein both antibodies are specific to the same molecule, e.g., the same marker, and wherein at least one member of the pair is a label as described herein. Thus, for any label that includes a binding- partner and a fluorescent moiety, the disclosure also encompasses a pair of binding partners wherein the first binding partner, e.g., an antibody, is part of the label, and the second binding partner, e.g., an antibody, is, typically, unlabeled and serves as a capture binding partner. In addition, binding partner pairs are frequently used in FRET assays. FRET assays useful in the disclosure are disclosed in U. S. Patent Application Publication No. US2006/0078998, and the disclosure also encompasses binding partner pairs, each of which includes a FRET label.

[00117] Highly Sensitive Analysis of Molecules

[00118] In one aspect, the disclosure provides a method for determining the presence or absence of a single molecule, e.g., a molecule of a marker, in a sample, by: i) labeling the molecule if present, with a label; and ii) detecting the presence or absence of the label, wherein the detection of the presence of the label indicates the presence of the single molecule in the sample. In some embodiments, the method is capable of detecting the molecule at a limit of detection of less than about 100, 80, 60, 50, 40, 30, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01 , 0.005, or 0.001 femtomolar. Detection limits can be determined by use of an appropriate standard, e.g., National Institute of Standards and Technology reference standard material.

[00119] The methods also provide methods of determining a concentration of a molecule, e.g., a marker indicative of a biological state, in a sample by detecting single molecules of the molecule in the sample. The "detecting" of a single molecule includes detecting the molecule directly or indirectly. In the case of indirect detection, labels that correspond to single molecules, e.g., labels attached to the single molecules, can be detected.

[00120] In some embodiments, the disclosure provides a method for determining the presence or absence of a single molecule of a protein in a biological sample, comprising labeling the molecule with a label and detecting the presence or absence of the label in a single molecule detector, wherein the label comprises a fluorescent moiety that is capable of emitting at least about 200 photons when simulated by a laser emitting light at the excitation wavelength of the moiety, wherein the laser is focused on a spot not less than about 5 microns in diameter that contains the moiety, and wherein the total energy directed at the spot by the laser is no more than about 3 microJoules. The single molecule detector may, in some embodiments, comprise not more than one interrogation space. The limit of detection of the single molecule in the sample can be less than about 10, 1, 0.1 , 0.01 , or 0.001 femtomolar. In some embodiments, the limit of detection is less than about 1 femtomolar. The detecting can comprise detecting electromagnetic radiation emitted by the fluorescent moiety. The method can further comprise exposing the fluorescent moiety to electromagnetic radiation, e.g., electromagnetic radiation provided by a laser, such as a laser with a power output of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 40, 60 80, 100, 120, 140, 160, 180 or 200 mW. In some embodiments, the laser stimulus provides light to the interrogation space for between about 10 to 1000 microseconds, or about 1000, 250, 100, 50, 25 or 10 microseconds. In some embodiments, the label further comprises a binding partner specific for binding the molecule, such as an antibody.

[00121] In some embodiments, detecting the presence or absence of the label comprises: (i) directing electromagnetic radiation from an electromagnetic radiation source to an interrogation space; (ii) providing electromagnetic radiation that is sufficient to stimulate the label, such as a fluorescent moiety, to emit photons if the label is present in the interrogation space; (iii) translating the interrogation space through the sample thereby moving the interrogation space to detect the presence or absence of other single molecules; and (iv) detecting photons emitted during the exposure of step (ii). The method can further comprise determining a background photon level in the interrogation space, wherein the background level represents the average photon emission of the interrogation space when it is subjected to electromagnetic radiation in the same manner as in step (ii), but without label in the interrogation space. The method can further comprise comparing the amount of photons detected in step (iv) to a threshold photon level, wherein the threshold photon level is a function of the background photon level, wherein an amount of photons detected in step (iv) greater than the threshold level indicates the presence of the label, and an amount of photons detected in step (iv) equal to or less than the threshold level indicates the absence of the label.

[00122] Sample Preparation

[00123] In general, any method of sample preparation can be used that produces a label corresponding to a molecule of interest, e.g., a marker of a biological state to be measured, where the label is detectable in the instruments described herein. As is known in the art, sample preparation in which a label is added to one or more molecules can be performed in a homogeneous or heterogeneous format. In some embodiments, the sample preparation is formed in a homogenous format. In analyzer systems employing a homogenous format, unbound label is not removed from the sample. See, e.g. , US 2006/0078998. In some embodiments, the particle or particles of interest are labeled by addition of labeled antibody or antibodies that bind to the particle or particles of interest.

[00124] In some embodiments, a heterogeneous assay format is used, wherein, typically, a step is employed for removing unbound label. Such assay formats are well- known in the art. One particularly useful assay format is a sandwich assay, e.g., a sandwich immunoassay. In this format, the molecule of interest, e.g., a marker of a biological state, is captured, e.g., on a solid support, using a capture binding partner. Unwanted molecules and other substances can then optionally be washed away, followed by binding of a label comprising a detection binding partner and a detectable label, e.g., a fluorescent moiety. Further washes remove unbound label, then the detectable label is released, usually, though not necessarily, still attached to the detection binding partner. In alternative embodiments, sample and label are added to the capture binding partner without a wash in between, e.g., at the same time. Other variations will be apparent to one of skill in the art.

[00125] In some embodiments, the method for detecting the molecule of interest, e.g., a marker of a biological state, uses a sandwich assay with antibodies, e.g., monoclonal antibodies, as capture binding partners. The method comprises binding molecules in a sample to a capture antibody that is immobilized on a binding surface, and binding the label comprising a detection antibody to the molecule to form a "sandwich" complex. The label comprises the detection antibody and a fluorescent moiety, as described herein, which is detected, e.g., using the single molecule analyzers of the disclosure. Both the capture and detection antibodies specifically bind the molecule. Many examples of sandwich

immunoassays are known, and some are described in U. S. Pat. No. 4,168,146 to Grubb et al. and U. S. Pat. No. 4,366,241 to Tom et al, both of which are incorporated herein by reference. Further examples specific to specific markers are described in the Examples.

[00126] The capture binding partner can be attached to a solid support, e.g., a microtiter plate or paramagnetic beads. In some embodiments, the disclosure provides a binding partner for a molecule of interest, e.g., a marker of a biological state, attached to a paramagnetic bead. Any suitable binding partner that is specific for the molecule that it is wished to capture can be used. The binding partner can be an antibody, e.g., a monoclonal antibody. Production and sources of antibodies are described elsewhere herein. It will be appreciated that antibodies identified herein as useful as a capture antibody can also be useful as detection antibodies, and vice versa.

[00127] The attachment of the binding partner, e.g., an antibody, to the solid support can be covalent or noncovalent. In some embodiments, the attachment is noncovalent. An example of a noncovalent attachment well-known in the art is that between biotin-avidin and streptavidin. Thus, in some embodiments, a solid support, e.g., a microtiter plate or a paramagnetic bead, is attached to the capture binding partner, e.g., an antibody, through noncovalent attachment, e.g., biotin-avidin/streptavidin interactions. In some embodiments, the attachment is covalent. Thus, in some embodiments, a solid support, e.g., a microtiter plate or a paramagnetic bead, is attached to the capture binding partner, e.g., an antibody, through covalent attachment.

[00128] The capture antibody can be covalently attached in an orientation that optimizes the capture of the molecule of interest. For example, in some embodiments, a binding partner, e.g., an antibody, is attached in a orientated manner to a solid support, e.g., a microtiter plate or a paramagnetic microparticle.

[00129] In some embodiments, the solid support is a microtiter plate. In some embodiments, the solid support is a paramagnetic bead. An exemplary paramagnetic bead is Streptavidin C l(Dynal, 650.01 -03). Other suitable beads will be apparent to those of skill in the art. Methods for attachment of antibodies to paramagnetic beads are well-known in the art. One example is given in Example 4.

[00130] The molecule of interest is contacted with the capture binding partner, e.g., capture antibody immobilized on a solid support. Some sample preparation can be used, e.g., preparation of serum from blood samples or concentration procedures before the sample is contacted with the capture antibody. Protocols for binding of proteins in immunoassays are well-known in the art.

[00131] The time allowed for binding will vary depending on the conditions; it will be apparent that shorter binding times are desirable in some settings, especially in a clinical setting. The use of, e.g., paramagnetic beads can reduce the time required for binding. In some embodiments, the time allowed for binding of the molecule of interest to the capture binding partner, e.g., an antibody, is less that about 12, 10, 8, 6, 4, 3, 2, or 1 hours, or less than about 60, 50, 40, 30, 25, 20, 15, 10, or 5 minutes.

[00132] In some embodiments, following the binding of particles of the molecule of interest to the capture binding partner, e.g., a capture antibody, particles that bound nonspecifically, as well as other unwanted substances in the sample, are washed away leaving, substantially, only specifically bound particles of the molecule of interest. In other embodiments, no wash is used between additions of sample and label, which can reduce sample preparation time. Thus, in some embodiments, the time allowed for both binding of the molecule of interest to the capture binding partner, e.g., an antibody, and binding of the label to the molecule of interest, is less that about 12, 10, 8, 6, 4, 3, 2, or 1 hours, or less than about 60, 50, 40, 30, 25, 20, 15, 10, or 5 minutes.

[00133] Label is added either with or following the addition of sample and washing.

Protocols for binding antibodies and other immunolabels to proteins and other molecules are well-known in the art. If the label binding step is separate from that of capture binding, the time allowed for label binding can be important, e.g., in clinical applications or other time sensitive settings. In some embodiments, the time allowed for binding of the molecule of interest to the label, e.g., an antibody-dye, is less than about 12, 10, 8, 6, 4, 3, 2, or 1 hours, or less than about 60, 50, 40, 30, 25, 20, 15, 10, or 5 minutes. Excess label is removed by washing.

[00134] In some embodiments, the label is not eluted from the protein of interest. In other embodiments, the label is eluted from the protein of interest. Preferred elution buffers are effective in releasing the label without generating significant background. It is useful if the elution buffer is bacteriostatic. Elution buffers used in the disclosure can comprise a chaotrope, a buffer, an albumin to coat the surface of the microtiter plate, and a surfactant, selected so as to produce a relatively low background. The chaotrope can comprise urea, a guanidinium compound, or other useful chaotropes. The buffer can comprise borate buffered saline, or other useful buffers. The protein carrier can comprise, e.g., an albumin, such as human, bovine, or fish albumin, an IgG, or other useful carriers. The surfactant can comprise an ionic or nonionic detergent including Tween 20, Triton X-100, sodium dodecyl sulfate (SDS), and others.

[00135] In another embodiment, the solid phase binding assay can be a competitive binding assay. One such method is as follows. First, a capture antibody immobilized on a binding surface is competitively bound by i) a molecule of interest, e.g., marker of a biological state, in a sample, and ii) a labeled analog of the molecule comprising a detectable label (the detection reagent). Second, the amount of the label using a single molecule analyzer is measured. Another such method is as follows. First, an antibody having a detectable label (the detection reagent) is competitively bound to i) a molecule of interest, e.g., marker of a biological state in a sample, and ii) an analog of the molecule that is immobilized on a binding surface (the capture reagent). Second, the amount of the label using a single molecule analyzer is measured. An "analog of a molecule" refers, herein, to a species that competes with a molecule for binding to a capture antibody. Examples of competitive immunoassays are disclosed in U.S. Pat. No. 4,235,601 to Deutsch et al., U.S. Pat. No. 4,442,204 to Liotta, and U.S. Pat. No. 5,208,535 to Buechler et al, all of which are incorporated herein by reference.

[00136] Detection of Molecule of Interest and Determination of Concentration

[00137] Following elution, the presence or absence of the label in the sample is detected using a single molecule detector. A sample can contain no label, a single label, or a plurality of labels. The number of labels corresponds to or is proportional to (if dilutions or fractions of samples are used) the number of molecules of the molecule of interest, e.g., a marker of a biological state captured during the capture step.

[00138] Any suitable single molecule detector capable of detecting the label used with the molecule of interest can be used, including scanning analyzer system 100. Suitable single molecule detectors are described herein. Typically the detector is part of a system that includes an automatic sampler for sampling prepared samples, and, optionally, a recovery system to recover samples.

[00139] In some embodiments, the sample is analyzed in a single molecule analyzer that uses a laser to illuminate an interrogation space containing a sample, a detector to detect radiation emitted from the interrogation space, and a scan motor and mirror attached to the motor to translate the interrogation space through the sample. In some embodiments, the single molecule analyzer further comprises a microscope objective lens that collects light emitted from the sample as the interrogation space is translated through the sample, e.g., a high numerical aperture microscope objective. In some embodiments, the laser and detector are configured in a confocal arrangement. In some embodiments, the laser is a continuous- wave laser. In some embodiments, the detector is an avalanche photodiode detector. In some embodiments, the interrogation space is translated through the sample using a mirror attached to the scan motor. In some embodiments, the interrogation space is translated through the sample using multiple mirrors or a prism attached to the scan motor. In some embodiments, the disclosure provides an analyzer system that includes a sampling system capable of automatically sampling a plurality of samples with zero carryover between subsequently measured samples.

[00140] In some embodiments, the single molecule detector used in the methods of the disclosure uses a sample plate, a continuous-wave laser directed toward a sample plate in which the sample is contained, a high numerical aperture microscope objective lens that collects light emitted from the sample as interrogation space is translated through the sample, wherein the lens has a numerical aperture of at least about 0.8, an avalanche photodiode detector to detect radiation emitted from the interrogation space, and a scan motor with a moveable mirror to translate the interrogation space through the sample wherein the interrogation space is between about 1 μιτι³ and about 10000 μιτι³.

[00141] In some embodiments, the single molecule detector is capable of determining a concentration for a molecule of interest in a sample wherein the sample can range in concentration over a range of at least about 100-fold, 1000-fold, 10,000-fold, 100,000-fold, 300,000-fold, 1,000,000-fold, 10,000,000-fold, or 30,000,000-fold. In some embodiments, the methods of the disclosure use a single molecule detector capable detecting a difference of less than about 50%, 40%, 30%, 20%, 15%, or 10% in concentration of an analyte between a first sample and a second sample contained in a sample plate, wherein the volume of the first sample and the second sample introduced into the analyzer is less than about 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 10, 5, 4, 3, 2, or 1 μΐ, and wherein the analyte is present at a concentration of less than about 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 10, 5, 4, 3, 2, or 1 femtomolar.

[00142] A feature that contributes to the extremely high sensitivity of the instruments and methods of the disclosure is the method of detecting and counting molecules. Briefly, the sample contained in the sample plate is effectively divided into a series of detection events, by translating an interrogation space through the sample plate wherein EM radiation from a laser for a predetermined period of time is directed to the wavelength, and photons emitted during that time are detected. In some embodiments where labels are used, the wavelength of the EM radiation may be chosen as an appropriate excitation wavelength for the fluorescent moiety used in the label. Each predetermined period of time is a "bin." In certain embodiments, each bin is consecutive without overlap. If the total number of photons detected in a given bin exceeds a predetermined threshold level, a detection event ("DE") is registered for that bin, i.e., a label has been detected. A detection event can also be thought of as each "flash" of light that is brighter than the threshold. If the total number of photons is not at the predetermined threshold level, no detection event is registered. In certain embodiments, no pattern of fluorescence is determined for each bin or any number of bins. The only data recorded is whether the photon value for a bin is greater than a threshold level.

[00143] In some embodiments, the processing sample concentration is dilute enough that, for a large percentage of detection events, the detection event represents only one label passing through the window, which corresponds to a single molecule of interest in the original sample. Accordingly, few detection events represent more than one label in a single bin. However, as the concentration goes up, the probability that two molecules will transit the detector at the same time (in the same counting bin) becomes significant. In this case, one flash of light represents two (or more) molecules. In some embodiments, further refinements are applied to allow greater concentrations of label in the processing sample to be detected accurately, i.e., concentrations at which the probability of two or more labels being detected as a single detection event is no longer insignificant. To detect single molecules at greater concentrations, the number of photons detected over a threshold level is counted. In other words, the brightness of each flash is measured. The sum of the photon counts is called event photons ("EP").

[00144] Although other bin times can be used without departing from the scope of the disclosure, in some embodiments the bin times are selected in the range of about 1 microsecond to about 5 ms. In some embodiments, the bin time is more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900, 1000, 2000, 3000, 4000, or 5000 microseconds. In some embodiments, the bin time is less than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900, 1000, 2000, 3000, 4000, or 5000 microseconds. In some embodiments, the bin time is about 1 to 1000 microseconds. In some embodiments, the bin time is about 1 to 750 microseconds. In some embodiments, the bin time is about 1 to 500 microseconds. In some embodiments, the bin time is about 1 to 250 microseconds. In some embodiments, the bin time is about 1 to 100 microseconds. In some embodiments, the bin time is about 1 to 50 microseconds. In some embodiments, the bin time is about 1 to 40 microseconds. In some embodiments, the bin time is about 1 to 30 microseconds. In some embodiments, the bin time is about 1 to 25 microseconds. In some embodiments, the bin time is about 1 to 20 microseconds. In some embodiments, the bin time is about 1 to 10 microseconds. In some embodiments, the bin time is about 1 to 7.5 microseconds. In some embodiments, the bin time is about 1 to 5 microseconds. In some embodiments, the bin time is about 5 to 500 microseconds. In some embodiments, the bin time is about 5 to 250 microseconds. In some embodiments, the bin time is about 5 to 100 microseconds. In some embodiments, the bin time is about 5 to 50 microseconds. In some embodiments, the bin time is about 5 to 20 microseconds. In some embodiments, the bin time is about 5 to 10 microseconds. In some embodiments, the bin time is about 10 to 500 microseconds. In some embodiments, the bin time is about 10 to 250 microseconds. In some embodiments, the bin time is about 10 to 100 microseconds. In some embodiments, the bin time is about 10 to 50 microseconds. In some embodiments, the bin time is about 10 to 30 microseconds. In some embodiments, the bin time is about 10 to 20 microseconds. In some embodiments, the bin time is about 1 microsecond. In some embodiments, the bin time is about 2 microseconds. In some embodiments, the bin time is about 3 microseconds. In some embodiments, the bin time is about 4 microseconds. In some embodiments, the bin time is about 5 microseconds. In some embodiments, the bin time is about 6 microseconds. In some embodiments, the bin time is about 7 microseconds. In some embodiments, the bin time is about 8 microseconds. In some embodiments, the bin time is about 9 microseconds. In some embodiments, the bin time is about 10 microseconds. In some embodiments, the bin time is about 11

microseconds. In some embodiments, the bin time is about 12 microseconds. In some embodiments, the bin time is about 13 microseconds. In some embodiments, the bin time is about 14 microseconds. In some embodiments, the bin time is about 15 microseconds. In some embodiments, the bin time is about 16 microseconds. In some embodiments, the bin time is about 17 microseconds. In some embodiments, the bin time is about 18

microseconds. In some embodiments, the bin time is about 19 microseconds. In some embodiments, the bin time is about 20 microseconds. In some embodiments, the bin time is about 25 microseconds. In some embodiments, the bin time is about 30 microseconds. In some embodiments, the bin time is about 40 microseconds. In some embodiments, the bin time is about 50 microseconds. In some embodiments, the bin time is about 100

microseconds. In some embodiments, the bin time is about 250 microseconds. In some embodiments, the bin time is about 500 microseconds. In some embodiments, the bin time is about 750 microseconds. In some embodiments, the bin time is about 1000 microseconds.

[00145] In some embodiments, determining the concentration of a particle-label complex in a sample comprises determining the background noise level. In some

embodiments, the background noise level is determined from the mean noise level, or the root-mean-square noise. In other embodiments, a typical noise value or a statistical value is chosen. Often, the noise is expected to follow a Poisson distribution. The background signal can be determined in a reagent sample in the absence of the analyte or labels corresponding to the analyte.

[00146] As the interrogation space is translated through the sample, the laser beam directed to the interrogation space generates a burst of photons when a label is encountered. The photons emitted by the label are discriminated from background light or background noise emission by considering only the bursts of photons with energy above a predetermined threshold energy level, thereby accounting for the amount of background noise present in the sample. Background noise typically comprises low frequency emission produced, e.g., by the intrinsic fluorescence of non-labeled particles that are present in the sample, the buffer, or diluent used in preparing the sample for analysis; Raman scattering; and electronic noise. In some embodiments, the value assigned to the background noise is calculated as the average background signal noise detected in a plurality of bins, which are measurements of photon signals that are detected in an interrogation space during a predetermined length of time. In some embodiments, background noise is calculated for each sample as a number specific to that sample.

[00147] The background signal can also be determined during a scan of the processing sample. For example, during a scan of sample including a plurality of bins, a selected number of bins can be used to determine the background noise level. In various

embodiments, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% or 25% of the bins having the lowest photon count are used in the calculation of the background noise level. Therefore, the mean photon value from the selected amount (e.g., 10%) of the bins having the lowest readings is used as the background noise level from which a threshold photon can be determined.

[00148] Given the value for the background noise, a threshold energy level can be assigned. As discussed above, the threshold value is determined to discriminate true signals resulting from the fluorescence of a label from the background noise. A threshold value can be chosen such that the number of false positive signals from random noise is minimized while the number of true signals which are rejected is also minimized. Methods for choosing a threshold value include determining a fixed value above the noise level and calculating a threshold value based on the distribution of the noise signal. In one embodiment, the threshold is set at a fixed number of standard deviations above the background level.

Assuming a Poisson distribution of the noise, using this method one can estimate the number of false positive signals over the time course of the experiment. In some embodiments, the threshold level is calculated as a value of four standard deviations (σ) above the background noise. For example, given an average background noise level of 200 photons, the analyzer system establishes a threshold level of 4V200 above the average background/noise level of 200 photons to be 256 photons. Thus, in some embodiments, determining the concentration of a label in a sample includes establishing the threshold level above which photon signals represent the presence of a label. Conversely, the absence of photon signals with an energy level greater than the threshold level indicates the absence of a label.

[00149] Many bin measurements are taken to determine the concentration of a sample, and the absence or presence of a label is ascertained for each bin measurement. Typically, 60,000 measurements or more can be made in 1 min. 60,000 measurements are made in 1 min when the bin size is 1 ms. For smaller bin sizes, the number of measurements is correspondingly larger, e.g., 6,000,000 measurements per minute equates to a bin size of 10 microseconds. Because so many measurements are taken, no single measurement is crucial, thus providing for a high margin of error. Bins that are determined not to contain a label ("no" bins) are discounted and only the measurements made in the bins that are determined to contain label ("yes" bins) are accounted in determining the concentration of the label in the processing sample. Discounting measurements made in the "no" bins or bins that are devoid of label increases the signal to noise ratio and the accuracy of the measurements. Thus, in some embodiments, determining the concentration of a label in a sample comprises detecting the bin measurements that reflect the presence of a label.

[00150] The signal to noise ratio or the sensitivity of the analyzer system can be increased by minimizing the time that background noise is detected during a bin

measurement in which a particle-label complex is detected. For example, consider a bin measurement lasting 1 millisecond during which one particle-label complex is detected as it passes across an interrogation space in 250 microseconds. Under these conditions, 750 microseconds of the 1 millisecond are spent detecting background noise emission. The signal to noise ratio can be improved by decreasing the bin time. In some embodiments, the bin time is 1 millisecond. In other embodiments, the bin time is 750 microseconds, 500 microseconds, 250 microseconds, 100 microseconds, 50 microseconds, 25 microseconds, or 10 microseconds. Other bin times are as described herein.

[00151] Other factors that affect measurements are the brightness or dimness of the fluorescent moiety, size of the aperture image or lateral extent of the laser beam, the rate at which the interrogation space is translated through the sample, and the power of the laser. Various combinations of the relevant factors that allow for detection of label will be apparent to those of skill in the art. In some embodiments, the bin time is adjusted without changing the scan speed. It will be appreciated by those of skill in the art that as bin time decreases, laser power output directed at the interrogation space must increase to maintain a constant total energy applied to the interrogation space during the bin time. For example, if bin time is decreased from 1000 microseconds to 250 microseconds, as a first approximation, laser power output must be increased approximately four-fold. These settings allow for the detection of the same number of photons in a 250 microseconds as the number of photons counted during the 1000 microseconds given the previous settings, and allow for faster analysis of sample with lower backgrounds and greater sensitivity. In addition, the speed at which the interrogation space is translated through the sample can be adjusted in order to speed processing of sample. For instance, the bin time can be about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 250% or 300% of the time that it takes a fluorescent moiety to pass through an interrogation space. These numbers are merely exemplary, and the skilled practitioner can adjust the parameters as necessary to achieve the desired result.

[00152] In some embodiments, the interrogation space is smaller than the volume of sample when, for example, the interrogation space is defined by the size of the spot illuminated by the laser beam. In some embodiments, the interrogation space can be defined by adjusting the apertures 182 (Figures 2A and 2B) of the analyzer and reducing the illuminated volume that is imaged by the objective lens to the detector. In embodiments wherein the interrogation space is defined to be smaller than the cross-sectional area of the sample, the concentration of the label can be determined by interpolation of the signal emitted by the complex from a standard curve that is generated using one or more samples of known standard concentrations. In other embodiments, the concentration of the label can be determined by comparing the measured particles to an internal label standard. In

embodiments wherein a diluted sample is analyzed, the dilution factor is accounted for when calculating the concentration of the molecule of interest in the starting sample.

[00153] To determine the concentration of labels in the processing sample, the total number of labels contained in the "yes" bins is determined relative to the sample volume represented by the total number of bins. Thus, in one embodiment, determining the concentration of a label in a processing sample comprises determining the total number of labels detected "yes" and relating the total number of detected labels to the total sample volume that was analyzed. The total sample volume that is analyzed is the sample volume through which the interrogation space is translated in a specified time interval. Alternatively, the concentration of the label complex in a sample is determined by interpolation of the signal emitted by the label in a number of bins from a standard curve that is generated by determining the signal emitted by labels in the same number of bins by standard samples containing known concentrations of the label.

[00154] In some embodiments, the number of individual labels detected in a bin is related to the relative concentration of the particle in the processing sample. At relatively low concentrations, e.g., at concentrations below about 10^~16 M, the number of labels is proportional to the photon signal detected in a bin. Thus, at low concentrations of label, the photon signal is provided as a digital signal. At relatively higher concentrations, for example at concentrations greater than about lCf ¹⁶ M, the proportionality of photon signal to a label is lost as the likelihood of two or more labels crossing the interrogation space at about the same time and being counted as one becomes significant. Thus, in some embodiments, individual particles in a sample of a concentration greater than about lCf ¹⁶ M are resolved by decreasing the length of time of the bin measurement.

[00155] In other embodiments, the total photon signal that is emitted by a plurality of particles that are present in any one bin is detected. These embodiments allow for single molecule detectors of the disclosure wherein the dynamic range is at least 3, 3.5, 4, 4.5, 5.5, 6, 6.5, 7, 7.5, 8, or more than 8 logs.

[00156] "Dynamic range," as that term is used herein, refers to the range of sample concentrations that can be quantitated by the instrument without need for dilution or other treatment to alter the concentration of successive samples of differing concentrations, where concentrations are determined with accuracy appropriate for the intended use. For example, if a microtiter plate contains a sample of 1 femtomolar concentration for an analyte of interest in one well, a sample of 10,000 femtomolar concentration for an analyte of interest in another well, and a sample of 100 femtomolar concentration for the analyte in a third well, an instrument with a dynamic range of at least 4 logs and a lower limit of quantitation of 1 femtomolar can accurately quantitate the concentration of all the samples without further treatment to adjust concentration, e.g., dilution. Accuracy can be determined by standard methods, e.g., measuring a series of standards with concentrations spanning the dynamic range and constructing a standard curve. Standard measures of fit of the resulting standard curve can be used as a measure of accuracy, e.g., an r² greater than about 0.7, 0.75, 0.8, 0.85, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, or 0.99.

[00157] Dynamic range can be increased by altering how data from the detector is analyzed, and perhaps using an attenuator between the detector and the interrogation space. At the low end of the range, the processing sample is sufficiently dilute that each detection event, i.e., each burst of photons above a threshold level in a bin (the "event photons"), likely represents only one label. Under these conditions, the data is analyzed to count detection events as single molecules so that each bin is analyzed as a simple "yes" or "no" for the presence of label, as described above. For a more concentrated processing sample, where the likelihood of two or more labels occupying a single bin becomes significant, the number of event photons in a significant number of bins is substantially greater than the number expected for a single label. For example, the number of event photons in a significant number of bins corresponds to two-fold, three-fold, or more than the number of event photons expected for a single label. For these samples, the instrument changes its method of data analysis to integrate the total number of event photons for the bins of the processing sample. This total is proportional to the total number of labels in all the bins. For an even more concentrated processing sample, where many labels are present in most bins, background noise becomes an insignificant portion of the total signal from each bin, and the instrument changes its method of data analysis to count total photons per bin (including background). An even further increase in dynamic range can be achieved by the use of an attenuator, between the sample plate and the detector, when concentrations are such that the intensity of light reaching the detector would otherwise exceed the capacity of the detector for accurately counting photons, i.e., saturate the detector.

[00158] The instrument can include a data analysis system that receives input from the detector and determines the appropriate analysis method for the sample being run, and outputs values based on such analysis. The data analysis system can further output instructions to use or not use an attenuator, if an attenuator is included in the instrument. For instance, the data processing system includes a processor operatively connected to the detector, wherein the processor is configured to execute instructions stored on a non- transitory computer-readable medium, and wherein the instructions, when executed by the processor, cause the processor to operate in any one of the following manners: determine a threshold photon value corresponding to a background signal in the interrogation space, determine the presence of a photon emitting moiety in the interrogation space in each of a plurality of bins by identifying bins having a photon value greater than the threshold value, and compare the number of bins having a photon value greater than the threshold value to a standard curve.

[00159] By utilizing such methods, the dynamic range of the instrument can be dramatically increased. In some embodiments, the instrument is capable of measuring concentrations of samples over a dynamic range of more than about 1000 (3 log), 10,000 (4 log), 100,000 (5 log), 350,000 (5.5 log), 1,000,000 (6 log), 3,500,000 (6.5 log), 10,000,000 (7 log), 35,000,000 (7.5 log), or 100,000,000 (8 log). In some embodiments, the instrument is capable of measuring the concentrations of samples over a dynamic range of from about 1 to 10 femtomolar to at least about 1000, 10,000, 100,000, 350,000, 1,000,000, 3,500,000, 10,000,000, or 35,000,000 femtomolar. [00160] In some embodiments, an analyzer or analyzer system of the disclosure is capable of detecting an analyte, e.g., a biomarker, at a limit of detection of less than about 1 nanomolar, or 1 picomolar, or 1 femtomolar, or 1 attomolar, or 1 zeptomolar. In some embodiments, the analyzer or analyzer system is capable of detecting a change in

concentration of the analyte, or of multiple analytes, e.g., a biomarker or biomarkers, from one sample to another sample of less than about 0.1 %, 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, or 80% when the biomarker is present at a concentration of less than about 1 nanomolar, or 1 picomolar, or 1 femtomolar, or 1 attomolar, or 1 zeptomolar, in the samples, and when the size of each of the sample is less than about 100, 50, 40, 30, 20, 10, 5, 2, 1, 0.1 , 0.01 , 0.001, or 0.0001 μΐ.

[00161] Methods of Use of Single Molecule Analyzer

[00162] Further provided herein is a method for detecting the presence or absence of a single molecule in a sample comprising: (a) directing electromagnetic radiation from an electromagnetic radiation source to an interrogation space in the sample; (b) detecting the presence or absence of a first single molecule in the interrogation space located at a first position in the sample; (c) translating the interrogation space through the sample to a subsequent position in the sample; (d) detecting the presence or absence of a subsequent single molecule in the subsequent position in the sample; and (e) repeating steps (c) and (d) as required to detect the presence or absence of a single molecule in more than one position of the sample.

[00163] Further provided herein is a method for detecting the presence or absence of a single molecule wherein the interrogation space is translated in a non-linear path. In a further embodiment, the non-linear path comprises a substantially circular path. In another embodiment, the non-linear path comprises a helical pattern. The disclosure provides for a method of detecting the presence or absence of a single molecule in an interrogation space, wherein the interrogation space is translated through the sample. In some embodiments, the method provides for the sample to remain substantially stationary relative to the

instrumentation. In some embodiments, the method provides that the sample is translated with respect to the instrumentation. In some embodiments, both the sample and the electromagnetic radiation are translated with respect to one another. For instance, the sample container can be moved in a linear pattern to minimize movement of the container while the electromagnetic radiation is moved in a non-linear partem or a linear pattern that bisects, but does substantially overlap, the linear partem of movement of the sample. In an embodiment where the sample is translated with respect to the instrumentation, the sample can remain stationary within its container, e.g., a microwell. While single molecules can diffuse in and out of an interrogation space or a series of interrogations spaces, the medium in which the single molecules are present remains stationary. Therefore, this system allows for single molecule detection without the need for flowing fluid.

EXAMPLES

[00164] Example 1 : Preparation of cTnl Standard Curves

[00165] Dried blood spot (DBS) standard curves were prepared by centrifuging and washing whole blood with lx phosphate buffered saline (PBS) three times. The final whole blood was adjusted to 50% hematocrit before separating the whole blood into 12 samples and spiking each with a different level of cardiac troponin I (cTnl) analyte, as shown in Table 5 (below). A 70 aliquot of each sample was pipetted to the center of a spot on a

PerkinElmer 226 sample collection device and allowed to dry overnight at ambient temperature.

[00166] Example 2: DBS Sample Collection and Validation

[00167] DBS samples were collected from donors finger pricked with

SURGILANCE™ Safety Lancets. Finger prick samples were collected on PerkinElmer 226 sample collection devices and dried overnight at ambient temperature. After drying overnight, DBS samples were stored at -80°C in bags containing desiccant until needed.

[00168] DBS samples were scanned using an Epson scanner and images were processed using ImageJ version 1.48. The average of two measurements of the area of the DBS samples (in mm²) was multiplied by the paper thickness to calculate the blood volume (in μί), which was used to determine the correction factor for each DBS sample. The sample collection method and calculation were validated by using an autopipette to deposit blood spot volumes ranging from 10-100 μΚ See, Figures 1A and IB and Table 3.

Table 3

DBS Volume Validation

40 69.83 70.10 69.96 0% 32.88

50 94.96 103.26 99.11 6% 46.58

60 121.72 118.30 120.01 2% 56.41

70 134.23 132.83 133.53 1% 62.76

80 147.48 140.19 143.83 4% 67.60

90 162.66 177.37 170.02 6% 79.91

100 179.28 189.63 184.45 4% 86.69

[00169] Example 3: DBS Sample Extraction

[00170] Whole spots or one or more 6 mm punches of DBS (depending upon the amount of blood obtained and the volume needed to obtain quantitative results for the analyte to be tested, based on the expected or known endogenous concentration) were removed and placed into wells on a VWR^® 96 deep well plate. 600 μΐ. extraction buffer (IX PBS, 0.1% BSA, 0.1% Triton-X100) was added to each well and samples were extracted on a shaker at room temperature. After 2 hours, DBS extracts were removed from the extraction plate and transferred to a new 96 deep well plate for testing.

[00171] Example 4: cTnl Assay

[00172] DBS samples were analyzed using the SINGULEX^® SMC™ cTnl assay.

Volume-normalized extracts of the standards described in Example 1 and the DBS samples described in Examples 2-3 were incubated overnight at 4°C with i) DYNABEADS^®

MY ONE™ Streptavidin CI paramagnetic microparticles (Cat. # 65001, Invitrogen) coated with SDIX cTnl antibodies (Cat. # B9085MA07-MA, SDIX and Cat. # 4T21 clone 19C7cc, Hytest) in a ratio of 25 μg Ig per mg MP (cTnl capture reagent) and ii) Hytest cTnl antibodies (Cat. # 4T21 clone 16A12cc, Cat. # 4T21 clone MF4cc) labelled with ALEXA FLUOR^® 647 dye (Cat. # A-20106, Invitrogen) (detection reagent).

[00173] After incubation, samples were washed 3 times with 200 borate buffered saline with pH 8.3 TRITON™ X-100 ("BBST") wash buffer prepared according to Table 4, transferred to a new 96 deep well plate, and washed again 3 times with 200 μΐ. BBST wash buffer.

Table 4

BBST Wash Buffer Composition

Borate Buffer 77 mmol/L

77 mmol Boric Acid + 7.5

mM Na₂B₄0₇ (Borax)

NaCl pH 8.3 1.5 mol/L

(0.5 PROCLIN™ optional)

[00174] After washing, the detection reagent was eluted with 20 of pH 2.8 elution buffer (100 mmol/L glycine, 0.02% TRITON™ X-100) and neutralized with 4 of buffer C (1 mol/L Tris). The neutralized sample was read on a SINGULEX^® ERENNA^® instrument (which is described in U.S. Patent No. 7,838,250, which is incorporated by reference herein in its entirety). Standard curve analysis was performed using SGX LINK™ software (See, Figure 4 (SCL data left curves (squaresz); DBS data, right curves (diamonds)). Table 5 shows the standard results are compared to data obtained from an in-house clinical laboratory (Singulex Clinical Laboratory ("SLC")). DBS sample signal data was converted to concentrations by interpolation from the DBS standard curve according to normal methods.

Table 5

Standard Curve Validation

* Values obtained from the SCL

[00175] A total of 36 DBS samples were assayed for cTnl concentration. cTnl data was also obtained from the SCL using plasma samples and compared to the DBS assay results in Table 6 and Figure 5.

Table 6

cTnl Assay Results and Validation

30 3.30 2 2.94 1

[00176] The Pearson correlation coefficient between the DBS assay and SCL data was 0.98, and only 5 of the 30 DBS assay results fell into a different risk category than that of the SCL results (The risk categories are based on the distribution of cTnl values in a normal population, with all cTnl values greater than the 99^th percentile are considered high risk (Red), values between the 95^th and the 99^th percentile are considered moderate risk (yellow), and values lower than the 95^th percentile are on target (green)).

[00177] In addition, data was obtained from the SCL for five replicates of six assayed DBS samples spanning the low end of the DBS analyte measuring range (100, 50, 25, 12.5, 6.25, and 3.13 pg/mL) and compared with the DBS assay data. The coefficient of variation (CV) between each DBS assay and SCL result is shown in Figure 6.

[00178] Example 5: PSA Assay

[00179] A total of 36 DBS samples were assayed for PSA concentration. The SMC

PSA assay was performed by adding 100 of PSA standard (90: 10 complexed and free PSA) or DBS extract (samples) onto a 96 well assay plate. Assay reagents (100 volume, PSA capture antibody; Cat. # 4P33, clone 5A6, Hytest, Finland; coated onto microparticles and ALEXA FLUOR^® 647 labeled PSA detection antibody; Cat. # A45110136P, clone 8301, BiosPacific) were added to the standard and sample wells. The reaction was incubated for one hour at 25°C on a Jitterbug. The assay was completed by washing the beads with borate buffered saline with Triton X-100 using a Hydroflex microplate washer. The detection reagent was eluted with 20 elution buffer B (Tris-Glycine, pH 2.8) after washing, and the eluate neutralized with 4 μΐ. buffer C (Tris 1 mol/L, pH 8.2) before reading on the Singulex ERENNA^® instrument. Standard curve analysis and sample interpolation was performed using SGX LINK software (Singulex). Because analyte concentrations are typically reported as the concentration in serum or plasma (i.e. after separation and removal of red blood cells), a hematocrit correction factor is applied to the DBS data to obtain the final concentrations of the analytes in DBS samples by correcting the concentrations assuming a hematocrit (defined as the percentage of the total blood volume that is taken up by red blood cells) of 50%.

[00180] PSA data was also obtained from the SCL using plasma samples and compared to the DBS assay results in Table 7 and Figure 7.

Table 7

Assay Results and Validation SCL Risk DBS Assay DBS Risk

SCL Results Category Results Category

PSA PSA

Sample

(pg/mL) (pg/mL)

1 702.0 3 764.8 3

2 2.2 1 4.1 1

3 3.7 1 7.5 1

4 704.6 3 883.0 3

5 3.1 1 8.2 1

6 1.6 1 2.7 1

7 1.1 1 3.9 1

8 781.4 3 889.3 3

9 361.1 3 494.3 3

10 63.4 2 154.6 2

11 749.7 3 752.9 3

12 870.8 3 930.2 3

13 2.0 1 8.4 1

14 5.5 1 10.3 1

15 75.0 2 99.6

16 0.4 1 0.5 1

17 0.3 1 0.3 1

18 62.6 2 81.9

19 0.5 1 5.3 1

20 0.5 1 25.3 1

21 707.2 3 692.9

22 1.1 1 4.4 1

23 0.1 1 1.0 1

24 401.8 3 451.1 3

25 62.7 2 74.9 2

26 1.2 1 0.4 1

27 356.6 3 347.1 3

28 686.5 3 589.0 3

29 599.2 3 562.8 3

30 2.5 1 2.0 2

31 596.0 3 585.5 3

32 938.7 3 841.9 3

33 933.1 3 723.3 3

34 131.2 2 149.8 2 35 360.5 3 264.8 3

36 332.2 3 329.5 3

[00181] The Pearson correlation coefficient between the DBS assay and SCL data was 0.98, and none of the 36 DBS assay results fell into a different risk category than that of the SCL results (risk categories defined as explained above based on percentile analysis of a normal population).

[00182] Example 6: TSH Assay

[00183] A total of 40 DBS samples were assayed for TSH concentration. The SMC^®

TSH assay was performed by adding 50 of TSH standard or DBS extract (samples) onto a 96 well assay plate. Assay reagents (50 volume, TSH capture antibody coated onto microparticles and Alexa 647 labeled TSH detection antibody) were added to the standard and sample wells. The reaction was incubated for 15 min at 37°C on Jitterbug. The assay was completed by washing the beads with borate buffered saline with Triton X-100 using a Hydroflex microplate washer. The detection reagent was eluted with 20 elution buffer B (Tris-Glycine, pH 2.8) after washing, and the eluate neutralized with 4 μΐ. buffer C (Tris 1 mol/L, pH 8.2) before reading on the Singulex Erenna instrument. Standard curve analysis and sample interpolation was performed using Sgx Link software (Singulex). A hematocrit correction factor was applied to the DBS data. TSH data was also obtained from the SCL using plasma samples and compared to the DBS assay results in Table 8 and Figure 8.

Table 8

Assay Results and Validation

10 3.07 1 3.61 1

11 1.27 1 0.97 1

12 1.98 1 1.66 1

13 2.49 1 2.39 1

14 0.72 1 0.74 1

15 2.07 1 2.22 1

16 1.45 1 1.79 1

17 1.94 1 2.23 1

18 1.26 1 1.63 1

19 1.93 1 2.15 1

20 2.14 1 2.00 1

21 2.92 1 3.14 1

22 1.53 1 1.76 1

23 1.68 1 2.00 1

24 1.65 1 1.89 1

25 1.40 1 1.48 1

26 2.24 1 2.76 1

27 4.51 3 4.72 3

28 5.12 3 5.56 3

29 2.63 1 2.13 1

30 1.94 1 1.79 1

31 1.87 1 1.76 1

32 3.17 1 2.69 1

33 1.76 1 1.24 1

34 4.33 4.71

35 0.53 1 0.48 1

36 2.95 1 3.24 1

37 0.97 1 0.93 1

38 1.39 1 1.47 1

39 6.82 3 6.3 3

40 3.10 1 2.7 1

[00184] The Pearson correlation coefficient between the DBS assay and TSH data was 0.96, and none of the 40 DBS assay results fell into a different risk category than that of the SCL results (risk categories defined as explained above based on a percentile analysis of a normal population).

[00185] Example 7: CRP Assay [00186] A total of 40 DBS samples were assayed for hs-CRP concentration. The SMC

CRP assay was performed by adding 50 of CRP standard or DBS extract diluted 1: 10,000 (samples) onto a 96 well assay plate. Assay reagents (50 volume, CRP capture antibody coated onto microparticles and Alexa 647 labeled CRP detection antibody) were added to the standard and sample wells. The reaction was incubated for 30 min at 25°C on Jitterbug. The assay was completed by washing the beads with borate buffered saline with Triton X-100 using a Hydroflex microplate washer. The detection reagent was eluted with 20 elution buffer B (Tris-Glycine, pH 2.8) after washing, and the eluate neutralized with 4 buffer C (Tris 1 mol/L, pH 8.2) before reading on the Singulex Erenna instrument. Standard curve analysis and sample interpolation was performed using Sgx Link software (Singulex). A hematocrit correction factor was applied to the DBS data. TSH data was also obtained from the SCL using plasma samples and compared to the DBS assay results in Table 9 and Figure 9.

Table 9

Assay Results and Validation

18 1.0 2 0.9 1

19 2.5 2 1.6 2

20 0.3 1 0.2 1

21 1.1 2 1.0 2

22 5.3 3 4.5 3

23 1.5 2 1.3 2

24 0.3 1 0.3 1

25 0.9 1 0.9 1

26 - - 0.2 1

27 1.4 3 1.7 2

28 5.7 4 6.5 3

29 12.6 4 14.0 3

30 9 4 8.5 3

31 0.5 2 0.5 1

32 0.6 2 0.8 1

33 0.3 2 0.2 1

34 0.3 2 0.2 1

35 10.1 4 10.7 3

36 0.8 2 0.8 1

37 0.7 2 0.6 1

38 1 3 0.9 1

39 1.7 3 1.5 2

40 0.8 2 0.6 1

[00187] The Pearson correlation coefficient between the DBS assay and TSH data was 0.99, and only 4 of the 40 DBS assay results fell into a different risk category than that of the SCL results (risk categories defined as explained above based on a percentile analysis of a normal population).

[00188] Example 8: Comparison of Venous EDTA Plasma and Finger Prick DBS cTnl Measurements

[00189] 22 donors provided both venous EDTA plasma and finger prick DBS samples.

The EDTA plasma and DBS sample (extracted according to Example 3) were both subjected to the cTnl assay as described in Example 4. The results of the assays are shown in Table 10, and the difference between the venous EDTA plasma and DBS assays is shown in Figure 10. A sign rank test was performed, yielding P = 0.5905. Table 10

Plasma and DBS Assay Results

[00190] Example 9. Marathon Runner Baseline cTnl Measurements

[00191] Before running, 42 marathon participants provided finger prick DBS samples.

Samples were assayed for cTnl concentration as described in Example 4, and compared to the results of the 22 control donors of Example 8. The results of the assay, listed in Table 11 and depicted in Figure 11, showed that marathon runners have a higher baseline (pre-race) cTnl concentration than the control donors. A Mann-Whitney test was preformed, yielding P <

Table 11

Pre-Race Runner vs Control cTnl Results

[00192] Example 10. Marathon Running Post-Race cTnl Measurements

[00193] After running, the 42 marathon participants of Example 9 provided finger prick DBS samples. The 22 control donors of Example 8 avoided extreme exercise for 1 day and provided a second finger prick DBS sample. Samples were assayed for cTnl concentration as described in Example 4. Comparison of the results, listed in Table 12 and depicted in Figures 12A and 12B, showed no change in cTnl concentration for the control group and a significant increase in cTnl concentration for the marathon runners post-race. The cTnl concentration in 40 of 42 marathon participant DBS samples increased by at least 10 pg/mL post-race.

Table 12

Post-Race Runner vs Control cTnl Change

[00194] Example 11. Comparison of Venous EDTA Plasma and Venous DBS cTnl

Measurements

[00195] 22 donors provided both venous EDTA plasma and venous DBS samples.

The EDTA plasma and DBS sample (extracted according to Example 3) were both subjected to the cTnl assay as described in Example 4. The results of the assays are shown in Table 13, and the difference between the venous EDTA plasma and DBS assays is shown in Figure 13. A sign rank test was performed, yielding P = 0.6866.

Table 13

Plasma and DBS Assay Results

[00196] Example 12. Comparison of Venous DBS and Finger Prick DBS cTnl

Measurements

[00197] The cTnl assay results of the DBS samples collected in Example 8 (finger prick) and Example 11 (venous) were compared. The percent difference between the two collection methods median + IQR was 0.0 (-30,0.0). The distribution of percent differences is shown in Figure 14. A sign rank test was performed, yielding P = 0.5078.

[00198] Although preferred embodiments of the disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein can be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS What is claimed is:

1. A method for analyzing a blood sample for the presence or amount of an analyte,

comprising,

(a) extracting dried blood representing a whole blood sample volume from a blood collection card;

(b) forming a complex between analyte in the extracted sample and a labeled binding partner for the analyte comprising a fluorescent moiety;

(c) determining the amount of binding partner from the complex by analyzing a processing sample comprising the fluorescent moiety from the complex with an analyzer comprising:

i. an electromagnetic radiation source;

ii. an objective that directs electromagnetic radiation from the

electromagnetic radiation source to an interrogation space in a processing sample;

iii. a detector that detects electromagnetic radiation emitted from a photon emitting moiety in the interrogation space if the moiety is present, and

iv. a processor operatively connected to the detector, wherein the processor is configured to execute instructions stored on a non- transitory computer-readable medium, and wherein the instructions, when executed by the processor, cause the processor to: determine a threshold photon value corresponding to a background signal in the processing sample, determine the presence of a fluorescent moiety in the interrogation space in each of a plurality of bins by identifying bins having a photon value greater than the threshold value, and determining the presence or amount of the analyte in the blood sample by comparing the number of bins having a photon value greater than the threshold value to a standard curve

2. The method claim 1, wherein the blood sample is extracted with an extraction buffer.

3. The method of claim 1, wherein the dried blood spot represents a whole blood sample of less than 10 μί.

4. The method of claim 1 , further comprising determining the volume of the whole blood that represents the dried extracted blood.

5. The method of claim 1, wherein the instructions cause the processor to determine the threshold photon value as a function of the background photon level.

6. The method of claim 5, wherein the threshold photon value is a fixed number of standard deviations above the background photon level.

7. The method of claim 1, wherein the instructions cause the processor to determine

detection events representing photon bin counts above the threshold photon value as single molecule of the fluorescent moiety.

8. The analyzer of claim 7, wherein the instructions cause the processor to analyze each bin as a "yes" or "no" for the presence of the fluorescent moiety.

9. The method of claim 1, wherein the analyzer further comprises a translating system that moves the interrogation space through at least a portion of the processing sample.

10. The method of claim 1, wherein the analyzer comprises a capillary flow cell for moving at least a portion of the sample through the interrogation space.

11. The method of claim 1, further comprising an attenuator operatively connected between the interrogation space and the detector and configured to receive electromagnetic radiation emitted from the interrogation space, wherein the instructions cause the processor to instruct the attenuator to attenuate the electromagnetic radiation from the interrogation space when number of photons detected in one or more bins exceeds a saturation threshold.

12. The method of claim 11, wherein the instructions cause the processor to determine the presence or amount of a photon emitting moiety by measuring a total number of photons per bin.

13. The method of claim 1 , wherein the translating system is configured such that the bins are longer than the time that the fluorescent moiety is present in the interrogation space during each bin.

14. The method of claim 1 , wherein the translating system is configured such that the bins are one-half to two times longer than the time that the fluorescent moiety is present in the interrogation space during each bin.

15. The method of claim 1 , wherein the translating system is configured such that bins are the same as the time that the fluorescent moiety is present in the interrogation space during each bin.

16. The method of claim 1 , wherein the analyte is cardiac troponin I and the concentration of cardiac troponin I in the original whole blood sample is less than or equal to 10 pg/ml.

17. The method of claim 1 , wherein the analyte is interleukin 6 (IL-6) and the concentration of IL-6 in the original whole blood sample is less than or equal to 10 pg/ml.

18. The method of claim 1, wherein the analyte is endothelin-1 (ET-1) and the concentration of ET-1 in the original whole blood sample is less than or equal to 10 pg/ml.

19. The method of claim 1 , wherein the analyte is interleukin 17A (IL-17A) and the

concentration of IL-17A in the original whole blood sample is less than or equal to 10 pg/ml.

20. The method of claim 1, wherein the analyte is B-type natriuretic peptide (BNP), and the concentration of BNP in the original whole blood sample is less than or equal to 10 pg/ml.

21. The method of claim 1, wherein the analyte is vascular endothelial growth factor (VEGF) and the concentration of VEGF in the original whole blood sample is less than or equal to 10 pg/ml.

22. The method of claim 1, wherein the analyte is Tumor Necrosis Factor alpha (TNF-a) and the concentration of TNF-a in the original whole blood sample is less than or equal to 10 pg/ml.

23. The method of claim 1 , wherein the analyte is leptin and the concentration of leptin in the original whole blood sample is less than or equal to 10 pg/ml.

24. The method of claim 1 , wherein the analyte is thyroid stimulating hormone (TSH) and the concentration of TSH in the original whole blood sample is less than or equal to 10 pg/ml.

25. The method of claim 1 , wherein the analyte is prostate specific antigen (PSA) and the concentration of PSA in the original whole blood sample is less than or equal to 10 pg/ml.

26. The method of claim 1, wherein the analyte is estradiol and the concentration of estradiol in the original whole blood sample is less than or equal to 10 pg/ml.