[go: up one dir, main page]

WO2017205868A1 - Lecteur de molécule unique basé sur un appareil photo - Google Patents

Lecteur de molécule unique basé sur un appareil photo Download PDF

Info

Publication number
WO2017205868A1
WO2017205868A1 PCT/US2017/034988 US2017034988W WO2017205868A1 WO 2017205868 A1 WO2017205868 A1 WO 2017205868A1 US 2017034988 W US2017034988 W US 2017034988W WO 2017205868 A1 WO2017205868 A1 WO 2017205868A1
Authority
WO
WIPO (PCT)
Prior art keywords
pixels
target molecules
image
subset
electromagnetic radiation
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2017/034988
Other languages
English (en)
Inventor
Richard Livingston
Robert ONORATO
Danni WANG
Michele Gilbert
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Singulex Inc
Original Assignee
Singulex Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Singulex Inc filed Critical Singulex Inc
Publication of WO2017205868A1 publication Critical patent/WO2017205868A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G06—COMPUTING OR CALCULATING; COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T7/00—Image analysis
    • G06T7/10—Segmentation; Edge detection
    • G06T7/11—Region-based segmentation
    • G—PHYSICS
    • G06—COMPUTING OR CALCULATING; COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T7/00—Image analysis
    • G06T7/10—Segmentation; Edge detection
    • G06T7/136—Segmentation; Edge detection involving thresholding
    • H—ELECTRICITY
    • H04—ELECTRIC COMMUNICATION TECHNIQUE
    • H04N—PICTORIAL COMMUNICATION, e.g. TELEVISION
    • H04N25/00—Circuitry of solid-state image sensors [SSIS]; Control thereof
    • H04N25/60—Noise processing, e.g. detecting, correcting, reducing or removing noise
    • H04N25/68—Noise processing, e.g. detecting, correcting, reducing or removing noise applied to defects

Definitions

  • This disclosure is directed to the detection and quantification of molecules of interest using camera-based imaging technology.
  • single-molecule counting instruments count molecules free in a solution by scanning a small detection zone (e.g., approximately 3 ⁇ in diameter) through the solution. Because the location of the molecules is not known, the molecules can encounter the detection volume with some unknown offset from the optimal position in the center of the detection zone. As a result, many molecules may be detected weakly or not at all; it would be advantageous, however, to detect a larger fraction of the molecules.
  • An additional disadvantage of this scheme is that binary detection of molecules in the detection zone is done serially, one small volume after another. If more volumes were detected simultaneously, the reader could count molecules more quickly and with higher throughput.
  • systems and methods provide highly sensitive detection and quantitation of one or more target molecules using camera- based imaging technology.
  • a system in an example embodiment, includes an electromagnetic radiation source.
  • the system includes one or more optical elements configured to direct a first electromagnetic radiation from the electromagnetic radiation source to a binding surface.
  • the first electromagnetic radiation is received by labels associated with a plurality of target molecules bound to the binding surface.
  • the labels emit a second electromagnetic radiation in response to receiving the first electromagnetic radiation.
  • the system includes a detector configured to capture an image of the binding surface in response to the one or more optical elements directing the first electromagnetic radiation to the binding surface.
  • the image is defined by a plurality of pixels. Each pixel provides a signal.
  • the signals provided by a subset of the pixels result at least in part from the second electromagnetic radiation emitted by the target molecules, whereby the subset of pixels indicates a presence of the target molecules in corresponding areas of the binding surface.
  • the system includes a processor operatively connected to the detector and configured to execute instructions stored on a non- transitory computer-readable medium. The instructions, when executed by the processor, cause the processor to perform the steps of: receiving the image from the detector;
  • determining a noise signal threshold based on an average background noise signal for the plurality of pixels in the image identifying the subset of pixels indicating the presence of the target molecules by identifying the pixels having a signal greater than the noise signal threshold, and determining a count of the target molecules based on the identified subset of pixels.
  • a method in another example embodiment, includes receiving, by a processor, an image of a binding surface captured by a detector.
  • the detector captures the image in response to one or more optical elements directing a first electromagnetic radiation from a source to the binding surface.
  • the image is defined by a plurality of pixels. Each pixel provides a signal.
  • the signals provided by a subset of the pixels results at least in part from a second electromagnetic radiation emitted by target molecules bound to the binding surface.
  • the target molecules emit the second electromagnetic radiation in response to receiving the first electromagnetic radiation, whereby the subset of pixels indicates a presence of the target molecules in corresponding areas of the binding surface.
  • the method includes determining, by the processor, a noise signal threshold based on an average background noise signal for the plurality of pixels in the image.
  • the method includes identifying, by the processor, the subset of pixels indicating the presence of the target molecules by identifying the pixels having a signal greater than the noise signal threshold.
  • the method includes determining, by the processor, a count of the target molecules based on the identified subset of pixels.
  • FIG. 1 illustrates an example camera-based single molecule reader system, according to aspects of the present disclosure.
  • FIG. 2 illustrates an example method for detecting labeled target molecules, according to aspects of the present invention.
  • FIG. 3 illustrates an example image captured with a camera-based single molecule reader system, according to aspects of the present disclosure.
  • FIG. 4 illustrates an example method for identifying and excising defects in an image captured with a camera-based single molecule reader system, according to aspects of the present disclosure.
  • FIG. 5 illustrates a table of the raw data gathered from tests conducted with a camera-based single molecule reader system, according to aspects of the present disclosure.
  • FIG. 6A illustrates a table of the averaged data in FIG. 5.
  • FIG. 6B illustrates a graph of the averaged data in FIG. 5.
  • FIG. 7 illustrates graph of data for an assay conducted with a camera-based single molecule reader system, according to aspects of the present disclosure.
  • the present 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 molecule analyzers,” or “single molecule readers.”
  • Compositions and methods for diagnosis, prognosis, and/or determination of treatment based on such highly sensitive detection and quantization are also described.
  • FIG. 1 illustrates an example of a camera-based single-molecule reader system
  • target molecules may be bound to a surface to: (1) immobilize the molecules, and (2) confine the molecules to a single focal plane where they can be in focus at the same time.
  • the target molecules may be bound to a surface in a relatively high-density, uniform manner. In some cases, it may be impractical to illuminate the entire object area for a camera sensor with the intensity of illumination employed in the single scanned detection zone of current systems. Accordingly, immobilization of the target molecules may be particularly advantageous.
  • Some current single molecule detector systems use a 5 mW laser focused on the detection zone. However, multiplying this laser power for a million pixels or so in an entire object area would be sufficient to almost instantly destroy the sample.
  • a lower intensity light source can therefore be used in the present system.
  • the ability to detect millions of pixels simultaneously allows the use of lower intensity light source, such as an unfocused laser or a low-cost LED.
  • using a lower intensity source permits photons to be collected from the pixels over a longer period of time (e.g., 1 second versus 100 for the scanning systems), while still allowing for high- throughput analysis.
  • a sample can be read in 1 second, versus 15 seconds as is currently achieved with the scanning readers, thereby increasing the throughput by more than 15 times.
  • the optical system of reader system 100 may include an electromagnetic radiation source 110, such as a LED 1 12, a detector 120, such as a CCD or CMOS camera, and a wider field of view in the sample.
  • an electromagnetic radiation source 110 such as a LED 1 12
  • a detector 120 such as a CCD or CMOS camera
  • FIG. 1 shows an example schematic of the reader system 100.
  • the LED light source 1 12 may be selected to provide the required intensity of illumination at a wavelength appropriate to excite the chosen fluorophour or other electromagnetic radiation emitting label associated with the target molecules.
  • a LED lens 114 collimates the light from the LED 1 12 to most efficiently couple the light into an obj ective 130 and provide the highest practical intensity at a molecule binding surface 140, and over the entire field of view of a camera sensor 126 of the detector 120.
  • a LED cleanup filter 116 rejects wavelengths from the LED light source 1 12 that overlap a camera emission filter 122 pass band.
  • a dichroic mirror 118 reflects the LED light into the objective 130, which then focuses an image of the LED light source onto the molecule binding surface 140.
  • the LED light source 1 12 it is not necessary, and in some cases may not be desirable, for the LED light source 1 12 to be in sharp focus at the molecule binding surface 140, only that the light intensity is as high as practical and the illumination is as uniform as practical over the entire field of view of the camera sensor 126.
  • the entire reader system 100 is mounted to a baseboard (not shown).
  • a baseboard not shown.
  • embodiments may employ LED illumination, other embodiments may employ other types of illumination, e.g., a continuous wave (CW) laser.
  • CW continuous wave
  • embodiments may employ any low intensity light source with a wavelength suitable for exciting a fluorescent moiety.
  • the label on any target molecules bound to the surface will be excited by the LED illumination.
  • the labels may emit photons at a slightly longer wavelength than the illumination, i.e., at wavelengths characteristic of the label used.
  • the objective 130 collects these photons from across the detection area and directs them towards the camera sensor 126.
  • the dichroic mirror 118 passes them through instead of reflecting them back to the LED.
  • the camera emission filter 122 only allows photons associated with the label to pass through. This filter 122, along with the dichroic mirror 1 18, prevent excessive background light from Raman scattering, scattered LED light or ambient light from reaching the camera sensor 126.
  • a camera lens 124 focuses an image of the molecule binding area onto the camera sensor array. Executing instructions stored on a non-transitory computer-readable storage medium 160, a processor 150 can processes the light signal from the labels according to the methods described herein.
  • the LED emission area and the camera can be mapped to the same area on the binding surface 140.
  • the focal length of the camera lens 124 can be chosen such that the area of each pixel mapped to the binding surface 140 is optimal for the intended sample concentration and the required signal to noise ratio for the detection of the labels.
  • the camera 120 provides the most uniform pixel response available and minimizes variations in pixel quantum efficiency and noise.
  • the microscope objective 130 may have a high numerical aperture.
  • a "high numerical aperture objective” or “high NA objective” includes a lens with a numerical aperture of equal to or greater than approximately 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 more highly resolved image.
  • the brightness of an image also increases with higher numerical aperture.
  • the objective 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.
  • 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.
  • the numerical aperture of the microscope objective lens 130 is approximately 0.7.
  • the high numerical aperture (NA) microscope obj ective 130 used when performing single molecule detection through the walls or the base of the binding plate 140, 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.
  • a microscope objective lens 130 with a NA of 0.7 such as an Olympus 20x/0.7NA (Olympus America, Inc., USA), can be used.
  • an Olympus 40*/0.8 NA water immersion objective Olympus America, Inc., USA
  • This objective has a 3.3 mm working distance.
  • an Olympus 60*/0.9 NA water immersion objective with a 2 mm working distance can be used. Because the lens is a water immersion lens, the space 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 binding plate.
  • FIG. 2 illustrates an example method 200 for reliably detecting the labels associated with target molecules bound to a binding surface.
  • the example method 200 may be implemented with the camera-based single-molecule reader system 100 above.
  • one or more optical elements e.g., the objective 130
  • a first electromagnetic radiation e.g., LED illumination
  • a source e.g., the LED light source 112
  • the first electromagnetic radiation is received by a plurality of labels associated with the target molecules of a sample bound to the binding surface.
  • Labels, such as fluorescent moieties, on the target molecules may be excited by the first electromagnetic radiation and emit a second electromagnetic radiation (i.e., photons at a characteristic wavelength).
  • a detector e.g., the camera 120
  • the captured image is defined by a plurality of pixels, where each pixel corresponds to a respective area of the binding surface.
  • the captured image may be defined by about 262,000 pixels to about 5 megapixels.
  • Each pixel may provide a signal.
  • the signal may be a brightness or intensity associated with the pixel.
  • the signal may indicate a count of photons captured from the corresponding area of the binding surface.
  • the signals provided by a subset of the pixels results at least in part from the second electromagnetic radiation emitted by the labels associated with target molecules in the corresponding areas of the binding surface. As such, the subset of pixels indicates a presence of the target molecules in those corresponding areas.
  • An example of a captured image 300 is shown in FIG. 3 where an example pixel 302 (white dot) indicates the presence of target molecule(s).
  • a processor e.g., the processor 150
  • the example method 200 processes each pixel of the captured image to determine if one or more labeled target molecules are present in the corresponding area of the binding surface.
  • pixels in an image of the binding surface may provide a signal due to noise associated with various aspects of the binding surface and reader system.
  • the signal from such pixels can be characterized by an average background noise signal.
  • the example method 200 can distinguish pixels with signals indicating the presence of target molecules from pixels with signals resulting merely from background noise.
  • the example method 200 includes step 202 which determines a noise signal threshold based on an average background noise signal for the pixels in the image.
  • the average background noise signal can be calculated by delivering the first electromagnetic radiation to a blank sample (without target molecules) and capturing an image for the blank sample as described above (e.g., with the reader system 100).
  • the average background noise signal can be determined by measuring the signal for each pixel in the image. Because there are non-uniformities based on pixel quantum efficiency and noise, it may be advantageous to use the measurements of background noise for each pixel to calibrate the detector and minimize the non-uniformities. In particular, such calibration permits the effective use of lower-cost cameras for the methods described herein.
  • a single spot scanning reader can measure a blank sample, whereby the effect of the buffer solution in particular can be taken into account.
  • the example method 200 can identify the subset of pixels indicating the presence of the target molecules. Additionally, in step 206, the example method 200 can determine a number of target molecules in the sample based on this subset of pixels.
  • the number of pixels having a signal greater than the noise signal threshold is used as an estimate of the number of target molecules in the sample.
  • each pixel exceeding the noise signal threshold is associated with detection of a single target molecule, i.e., a single detection event (DE).
  • DE detection event
  • the number of target molecules can be compared with the number of target molecules in samples of known concentration.
  • FIG. 5, for instance, illustrates estimated values for concentration based on measured detection events taken from samples of known concentration.
  • FIG. 7 further illustrates a graph of data for an IL-4 assay, showing pixel counts (i.e., DE) as a function of concentration.
  • pixels having a signal greater than the noise signal threshold may correspond to an area with more than one target molecule.
  • pixels exceeding the noise signal threshold may be associated with more than one DE.
  • other embodiments of the apparatus and method described herein may count multiple detection events within a single pixel. By determining a generally regular signal associated with a detection event, multiple detection events within single pixels can be identified and counted to enhance sensitivity and accuracy. Additionally or alternatively, embodiments may be employ statistical methods to predict the frequency of pixels with multiple molecules.
  • the example method 200 may determine that the concentration of the sample is very high, where a large percentage, e.g., most or all, of the pixels indicate the presence of target molecules. For instance, in decision 208, the example method 200 may determine whether this subset of pixels exceeds a percentage threshold of the total pixels. In some cases, the percentage threshold may range between approximately 10% to approximately 50% of the total pixels.
  • the example method 200 may alternatively measure the total signal from the target molecules to estimate molecule count. In particular, in step 210, the example method 200 determines a sum of the signals (event photons or an analog measurement thereof) from the subset of pixels indicating the presence of the target molecules.
  • the example method 200 can determine, in step 212, the number of the target molecules in the sample by comparing the sum of the signals from the subset of pixels to another sum of signals produced from a sample having a known number of target molecules.
  • the count of target molecules may be affected by defects in the captured image.
  • FIG. 3 illustrates an example defect 304 in the image 300.
  • the example method 200 can correct for such defects in step 201. Defects may be caused for instance by paramagnetic beads, which are often part of the sample prepeparation process. Although not intended, paramagnetic beads may sometimes be present in the sample processed by the reader system. Their presence may appear in the captured image with the size of a typical pixel and a very strong signal.
  • the signal associated with paramagnetic beads may be stronger than the signals associated with the labeled target molecules. As such, the presence of the paramagnetic beads may be confused with the presence of target molecules. To correct defects in the image caused by paramagnetic beads and avoid such confusion, the corresponding pixels can be removed and calculations by the example method 200 can be adjusted to account for the removal of the pixels.
  • Other defects may also include bright or blank areas in the captured image caused by scratches and/or other surface abnormalities in the binding surface.
  • the example method 200 can detect such defects by identifying adjacent pixels that are either anomalously bright or dim. To correct defects in the image caused by abnormalities in the binding surface, the corresponding pixels can be removed and calculations by the example method 200 can be adjusted to account for the removal of the pixels.
  • Further defects may be associated with irregularities related to the use of plastic windows in the reader system. These irregularities may be glowing specks inside the window material, or fluorescent dust or lint on the interior or exterior surfaces of the window. The corresponding defects in the image may appear as very bright areas.
  • the example method 200 can determine a brightness threshold for such defects and identify pixels that meet or exceed this brightness threshold. The example method 200 can remove the corresponding pixels and adjust calculations to account for the removal of the pixels.
  • FIG. 4 illustrates an example method 400 for addressing defects in a captured image.
  • the example method 400 excises defects based on a number of configurable parameters.
  • the example method 400 processes each pixel of an image, where each pixel has a respective signal 402.
  • decision 410 the example method 400 determines whether the signal 402 for the pixel is anomalously high relative to the mean signal for all pixels in the image, e.g., global mean plus 20 times the standard deviation ⁇ of the average background noise signal. If the answer in decision 410 is affirmative ("yes"), the pixel is excised.
  • decision 410 in FIG. 4 may indicate a value of global mean plus the standard deviation multiplied by a factor of 20, other factors may be employed in other implementations.
  • the factor may be in a range of approximately 10 to approximately 15, or approximately 15 to approximately 20.
  • the factor is based on an analysis of the background signal, molecule label brightness, and their statistics.
  • the threshold for excising anomalously bright pixels is typically 25 to 50 times the standard deviation above the average background noise signal.
  • the actual factor is determined from the actual average background noise signal, the standard deviation of the average background noise signal, as well as the mean signal and standard deviation for pixels indicating the presence of the target molecules.
  • a system with a very low background noise level may permit much higher factor than a similar system with a higher background noise level.
  • the example method 400 in decision 420 determines if the signal 402 of the pixel is greater than the global mean plus a multiple of the standard deviation, e.g., global mean + 2 ⁇ . If the answer in decision 420 is negative, the pixel is not excised.
  • the example method 400 in decision 430 determines if the pixel is adjacent to a flagged pixel.
  • a pixel is flagged if the signal 402 is greater than the mean signal for a local area plus a given multiple of the standard deviation ⁇ , e.g., local mean + 2 ⁇ .
  • the local area of pixels may be defined by a rectangle with sides of two times a radius plus one, and the local mean and ⁇ may be calculated disregarding the points outside one sigma on the high side. For instance, the radius may be equal to five so the local area is 11 pixels x 1 1 pixels. If the answer in decision 430 is affirmative, the pixel is flagged.
  • the example method 400 in decision 440 determines whether the signal 402 is greater than local mean plus the given multiple of ⁇ . If the answer in decision 440 is affirmative, the pixel is flagged. If the answer in decision 440 is negative, the pixel is not excised.
  • the example method 400 completes the determination of decision 440 both from the lower left (LL) and upper right (UR) of the image to remove bias. These LL and UR flag matrices are kept separate for comparison. For both LL and UR flag matrices, if the example method 400 determines in decision 450 that the size of an object of adjacent flagged pixels is greater than or equal to an object threshold defined as a number X of pixels, the pixels remain flagged. In one scenario, for instance, the object threshold may be 300 pixels; in another scenario, however, the object threshold may be three pixels. If the answer in decision 450 is negative, the flags are removed and the pixels are not excised.
  • the example method 400 compares the LL and UR flag matrices in decision 460. According to decision 460, any pixels flagged in both matrices are excised. In another scenario, any pixels flagged in one matrix and within a radius defined by a number of pixels of a flagged pixel in the other matrix may be excised.
  • a guardband may be added in step 470 around any excised pixels.
  • the guardband may be zero pixels; in another scenario, however, the guardband may be one, two, three, four, or five or even more pixels.
  • the image 300 of FIG. 3 illustrates the effects of some non-uniform illumination.
  • image-flattening may be applied to the image.
  • the background of the image is flattened according to a comparison between the local mean pixel intensity and the global mean pixel intensity.
  • the local area for instance, may be an 1 lxl 1 box around a pixel of interest.
  • the mean intensity is calculated, any pixel with intensity greater than one standard deviation from the mean is excluded, and the mean intensity is recalculated.
  • the signal is then set as the recalculated mean intensity plus the global mean. In the case that all pixels in the 1 lxl 1 box are excised, the pixel of interest is also excised.
  • the steps for processing each raster scan image may include: (1) parsing the raw data to remove the "dead-time" between each line scan; (2) excising any defects with the function input parameters optimized for large defects; (3) flattening the image to remove baseline signal variations along scan lines; (4) excising defects with the function input parameters optimized for small defects; and (5) reshaping the image matrix into a ID array and feeding this into a function to extract detection events (DE 1 ) and event photons ( ⁇ ').
  • FIGS. 6A-B illustrate a table and a graph of the averaged data, respectively.
  • embodiments utilize optimized optics, allowing quantification of the concentration of the sample in a relatively short time.
  • the optimized optics allow the system to count as many molecules of interest as possible, by a combination of choice of NA of the light collecting objective and the field of view.
  • the optics are optimized to achieve maximum signal to noise ratio, based on the expected number of photons to be emitted by a labeled molecule (signal), and the expected background photon signal detected from the interrogation window as emitted by the substrate and buffer solution, without molecules of interest (noise), when illuminated by the chosen light source through the same optics.
  • signal labeled molecule
  • noise molecules of interest
  • the present system optimizes the equivalent pixel size in the sample, resulting in the optimum signal with minimum noise.
  • the signal to noise ratio is optimized often by using a fast pulsed laser and using high speed electronics to separate prompt Raman emissions from the slightly delayed fluorescence emissions.
  • the cost of these lasers and electronics may be avoided by designing the optics so that adequate signal to noise ratio is achieved, while using lower cost light sources (e.g., LED, continuous -wave laser) and photon counting detectors. Additional features of the present disclosure help to achieve a high signal-to-noise ratio.
  • the present systems utilize a low-cost plastic substrate that minimizes noise due to autofluorescence.
  • Electromagnetic Radiation Source provides for chemistry that binds molecules of interest to the binding surface with high density and uniformity, this does not appreciably increase to the noise background. Further, where naturally-occurring defects occur, they are detected and corrected with the approaches utilized in the systems and methods described herein.
  • Some embodiments employ a chemiluminescent label. These embodiments might not require an electromagnetic radiation source for particle detection. In other embodiments, however, 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 may be preferred.
  • the example reader system 100 includes an
  • electromagnetic radiation source 110 Any number of radiation sources can be used in a reader system 100 without departing from the scope of the present disclosure.
  • Light-emitting diodes provide a 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.
  • the optimal light intensity depends on the photo bleaching characteristics of the single dyes and the camera exposure time. 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 within the camera exposure time.
  • the power of the electromagnetic radiation source 110 is set depending on the type of dye molecules and the length of time the dye molecules are stimulated.
  • 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.
  • the detector 120 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.
  • 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 CMOS 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
  • target molecules of interest are bound on a top surface of the binding surface and both illumination and detection occur from the opposite (e.g., bottom) surface of the binding surface.
  • molecules of interest are bound to top surface of the binding surface, with illumination and detection occurring from the same surface (e.g., top).
  • the binding surface may be a clear or opaque material.
  • molecules of interest are bound to top surface of the binding surface, with illumination coming from below the binding surface and detection occurring from above.
  • the binding surface in this example may be made of a clear material.
  • molecules of interest are bound to a top surface the preferably clear binding surface, with illumination coming from above the binding surface and detection occurring below.
  • molecules of interest in a sample are immobilized on the binding surface 140.
  • the sample is interrogated through the binding surface material.
  • the sample is interrogated through the base of the binding surface.
  • the base of the binding surface is made of a material that is transparent to light.
  • the base of the binding surface is made of a material that is transparent to electromagnetic radiation.
  • the binding surface is transparent to an excitation wavelength of interest. Using a transparent material allows the wavelength of the excitation beam to pass through the binding surface and excite the molecule of interest or the label associated with the molecule of interest. The transparency of the binding surface further allows the camera to detect the emissions from the excited labels.
  • the binding surface material is substantially transparent to light of wavelengths for all of the wavelengths associated with each of the electromagnetic radiation sources and each of the emission spectra of the labels used in multiplex single molecule analysis.
  • the binding surface is a slide, such as a Nexterion-E slide.
  • the binding surface is coated with an epoxy.
  • the binding surface may also be metallic, or coated with a metallic material, thereby effectively intensifying the light at the molecules by the surface plasmon effect.
  • the thickness of binding surface is also considered.
  • the sample is interrogated by electromagnetic radiation that passes through a portion of the material of the slide.
  • the thickness of the binding surface allows an image to be formed on a first side of the portion of the binding surface that is imaged by a high numerical aperture lens that is positioned on a second side of the portion of the binding surface that is imaged.
  • Such an embodiment facilitates the formation of an image within the sample and not within the base.
  • the image formed corresponds to the field of view the system.
  • the image should be formed at the depth of the single molecule of interest.
  • the thickness of the binding surface depends on the working distance and depth of field of the lens that is used.
  • the binding surface can be made out of any suitable material that allows the excitation energy to pass through the surface.
  • the binding surface is made of cyclo-olefin polymer (COP) or cyclic-olefin co-polymer (COC).
  • the binding surface is made of fused silica. Nexterion glass, which is similar to COP in autofluorescence, but may offer desirable properties for the binding chemistry, may also be used for the binding surface.
  • a commercially available slide can be used. Any binding surface made of a suitable material and of a suitable thickness can be used.
  • the binding surface is made out of a material with low fluorescence, thereby reducing background fluorescence. Background fluorescence resulting from the binding surface material can be further avoided by minimizing the thickness of the binding surface.
  • the antibodies When antibodies are used to capture a single molecule of interest, the antibodies can be applied to the surface of the binding surface. The single molecule of interest then binds to the antibodies immobilized on the binding surface. In some
  • a paramagnetic bead is used as a capture bead during the assay process to capture the single molecule of interest. The bead is then removed before surface attachment.
  • 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.
  • the binding partner is an antibody specific for a molecule to be detected.
  • antibody 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).
  • Monoclonal and polyclonal antibodies to molecules e.g., proteins, and markers 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.
  • Capture binding partners and detection binding partner pairs can be used in embodiments of the disclosure.
  • 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.
  • 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.
  • the label comprises a binding partner to the molecule of interest, where the binding partner is attached to a photon emitting species such as, for example, a flourescent moiety. Moieties suitable for the compositions and methods of the disclosure are described in more detail below.
  • Signal producing molecules may be attached to binding partners by any known means such as direct conjugation or indirectly (e.g., biotin/streptavidin).
  • 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 stimulated by an illumination source emitting light at the excitation wavelength of the moiety, wherein the illumination area corresponds to the field of view of the system .
  • 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.
  • the moiety comprises about 2 to 4 fluorescent entities.
  • 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.
  • 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).
  • a fluorescent moiety 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.
  • 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.
  • 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, 0.00001 , 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.
  • a molecule e.g., a marker
  • 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.
  • a molecule e.g., a marker
  • “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.
  • the moiety has properties that are consistent with its use in the assay of choice.
  • 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.
  • 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).
  • 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).
  • 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.
  • 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 stimulated by an illumination source emitting light at the excitation wavelength of the moiety, where the light illuminates the plane that contains the moiety, and where the total energy directed at each pixel area is on the order of 0.1 to 10 microJoules.
  • 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
  • the total energy can be achieved by many different combinations of power output of the light source, number of camera pixels, and length of time of exposure of the dye moiety, e.g. an LED with a power output of 0.5 W, a 1 megapixel camera, and a two second illumination time and an LED power output of 1 W, a 4 megapixel camera, and a four second illumination time would both achieve a total energy of 1 microJoule per pixel area and so on.
  • 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.
  • 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.
  • 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.
  • 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.
  • ALEXA FLUOR® 750 749 240,000 782 B-phycoerythrin 546, 565 2,410,000 575 240,000
  • Quantum Dots Qdot 525, QD 565, QD 585, QD 605, QD 655, QD 705, QD 800
  • Suitable dyes for use in the disclosure include modified carbocyanine dyes.
  • 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.
  • the modified carbocyanine dyes 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.
  • 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.
  • organic fluors can be improved by rendering them less hydrophobic by adding hydrophilic groups such as polyethylene.
  • 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.
  • the modification improves the Stokes shift while maintaining a high quantum yield.
  • 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 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.
  • One optical features 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.
  • the protein that is detected with the single molecule analyzer system is labeled with a QD.
  • the single molecule analyzer is used to detect a protein labeled with one QD and using a filter to allow for the detection of different proteins at different wavelengths.
  • the analyzer system comprises one illumination source 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 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.
  • the protein that is detected with the single molecule analyzer is labeled with one quantum dot.
  • 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.
  • 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.
  • 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.
  • FRET fluorescence resonance energy transfer
  • 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 HorizonTM BV421 that is excited at 407 nm and maximally fluoresces at 421 nm and BD HorizonTM 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.
  • the protein that is detected with the single molecule analyzer system is labeled with a polymeric fluorophore.
  • 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.
  • the single molecule analyzer is used to detect a protein labeled with a polymeric fluorophore.
  • the analyzer system comprises one illumination source 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.
  • the protein that is detected with the single molecule analyzer is labeled with one polymeric fluorophore.
  • Useful polymeric fluorophores comprise BV421 , BV510, and BB515.
  • 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.
  • a binding partner e.g., an antibody
  • 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.
  • 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 light source emitting light at the excitation wavelength of the moiety.
  • Attachment of the fluorescent moiety, or fluorescent entities that make up the fluorescent moiety, to the binding partner can be by any suitable means; such methods are well-known in the art and exemplary methods are given in the Examples.
  • the binding partner e.g., an antibody
  • 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.
  • 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.
  • immunoassays often employ a sandwich format in which binding partner pairs, e.g. antibodies, to the same molecule, e.g., a marker, are used.
  • binding partner pairs e.g. antibodies
  • 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.
  • 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.
  • first binding partner e.g., an antibody
  • second binding partner e.g., an antibody
  • 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.
  • a single molecule e.g., a molecule of a marker
  • 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.
  • 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.
  • labels that correspond to single molecules e.g., labels attached to the single molecules, can be detected.
  • 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 light source at the excitation wavelength of the moiety.
  • 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
  • the method can further comprise exposing the fluorescent moiety to electromagnetic radiation, e.g., electromagnetic radiation provided by an LED.
  • electromagnetic radiation e.g., electromagnetic radiation provided by an LED.
  • the LED stimulus provides light to the binding surface window for a range of about 0.5 to about 5 seconds.
  • the label further comprises a binding partner specific for binding the molecule, such as an antibody.
  • detecting the presence or absence of the label comprises: (i) directing electromagnetic radiation from an electromagnetic radiation source to a window of a binding surface; (ii) providing electromagnetic radiation that is sufficient to stimulate the label, such as a fluorescent moiety, to emit photons if the label is present on the surface; and (iii) detecting photons emitted during the exposure of step (ii).
  • the method can further comprise determining a background photon level in the window of the binding surface, wherein the background level represents the average photon emission of the binding surface when it is subjected to electromagnetic radiation in the same manner as in step (ii), but without label present.
  • the method can further comprise comparing the amount of photons detected in step (iii) 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 (iii) greater that the threshold level indicates the presence of the label, and an amount of photons detected in step (iii) equal to or less than the threshold level indicates the absence of the label.
  • sample preparation in which a label is added to one or more molecules can be performed in a homogeneous or heterogeneous format.
  • the sample preparation is formed in a homogenous format.
  • unbound label is not removed from the sample.
  • a heterogeneous assay format is used, wherein, typically, a step is employed for removing unbound label.
  • assay formats are well-known in the art.
  • One particularly useful assay format is a sandwich assay, e.g., a sandwich immunoassay.
  • the molecule of interest e.g., a marker of a biological state
  • 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 antibody conjugated to a detectable moiety. Further washes remove unbound label.
  • 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.
  • the method for detecting the molecule of interest 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 detectable 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.
  • the capture antibody can be covalently attached to the binding surface in an orientation that optimizes the capture of the molecule of interest.
  • 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 time allowed for binding of the molecule of interest to the capture binding partner e.g., an antibody
  • the capture binding partner 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.
  • the capture binding partner e.g., a capture antibody
  • 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.
  • Label is added either with or following the addition of sample and washing.
  • the time allowed for label binding can be important, e.g., in clinical applications or other time sensitive settings.
  • 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.
  • the solid phase binding assay can be a competitive binding assay.
  • 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).
  • the amount of the label using a single molecule analyzer is measured. Another such method is as follows.
  • 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).
  • 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.

Landscapes

  • Engineering & Computer Science (AREA)
  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Computer Vision & Pattern Recognition (AREA)
  • Theoretical Computer Science (AREA)
  • Multimedia (AREA)
  • Signal Processing (AREA)
  • Health & Medical Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

La présente invention a trait à un lecteur de molécule unique basé sur un appareil photo, lequel lecteur comprend un détecteur qui capture une image en réponse à un ou plusieurs éléments optiques dirigeant un premier rayonnement électromagnétique vers une surface de liaison. L'image est définie par une pluralité de pixels. Chaque pixel produit un signal. Les signaux produits par un sous-ensemble des pixels résultent au moins en partie d'un second rayonnement électromagnétique émis par des molécules cibles liées à la surface de liaison, en réponse au premier rayonnement électromagnétique. Le sous-ensemble de pixels indique la présence des molécules cibles dans des zones correspondantes de la surface de liaison. Un processeur détermine un seuil de signal de bruit sur la base d'un signal de bruit de fond moyen pour la pluralité de pixels. Le processeur identifie le sous-ensemble de pixels indiquant la présence des molécules cibles en identifiant les pixels présentant un signal supérieur au seuil de signal de bruit. Le processeur détermine un comptage des molécules cibles sur la base du sous-ensemble de pixels.
PCT/US2017/034988 2016-05-27 2017-05-30 Lecteur de molécule unique basé sur un appareil photo Ceased WO2017205868A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201662342567P 2016-05-27 2016-05-27
US62/342,567 2016-05-27

Publications (1)

Publication Number Publication Date
WO2017205868A1 true WO2017205868A1 (fr) 2017-11-30

Family

ID=60412637

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2017/034988 Ceased WO2017205868A1 (fr) 2016-05-27 2017-05-30 Lecteur de molécule unique basé sur un appareil photo

Country Status (1)

Country Link
WO (1) WO2017205868A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008032096A2 (fr) * 2006-09-14 2008-03-20 Oxford Gene Technology Ip Limited Appareil permettant de former l'image de molécules simples
WO2009001276A1 (fr) * 2007-06-25 2008-12-31 Koninklijke Philips Electronics N.V. Dispositif de détection microélectronique destiné à la détection de particules de marquage
CA2930836A1 (fr) * 2013-11-17 2015-05-21 Quantum-Si Incorporated Pixel a source active, dispositif integre pour analyse rapide d'eprouvettes biologiques et chimiques

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008032096A2 (fr) * 2006-09-14 2008-03-20 Oxford Gene Technology Ip Limited Appareil permettant de former l'image de molécules simples
WO2009001276A1 (fr) * 2007-06-25 2008-12-31 Koninklijke Philips Electronics N.V. Dispositif de détection microélectronique destiné à la détection de particules de marquage
CA2930836A1 (fr) * 2013-11-17 2015-05-21 Quantum-Si Incorporated Pixel a source active, dispositif integre pour analyse rapide d'eprouvettes biologiques et chimiques

Similar Documents

Publication Publication Date Title
US20200249164A1 (en) Multiplexed Single Molecule Analyzer
US9977031B2 (en) Highly sensitive system and method for analysis of troponin
EP2696193A1 (fr) Systèmes et procédés de détection de dosage
US20130171623A1 (en) Binding Assays Utilizing Time-Resolved Up-Converting Luminescence Detection
US20070254376A1 (en) Method and apparatus for the detection of labeling elements in a sample
CN112964688B (zh) 全内反射检测基因芯片杂交结果的方法
US10564156B2 (en) Use of a colorant in order to improve signal detection in an analysis method
US10054593B2 (en) Multiplexed spectral lifetime detection of phosphors
WO2017205868A1 (fr) Lecteur de molécule unique basé sur un appareil photo
WO2002073198A2 (fr) Essai biologique
CN103353531A (zh) 用于肌钙蛋白分析的高灵敏系统和方法
US20220041928A1 (en) Multiplexed spectral lifetime detection of phosphors
WO2017117113A1 (fr) Comptage de molécule unique pour une analyse à partir de taches de sang séché
JPH03221837A (ja) 検体測定方法及び検体測定装置
AU2014200749B2 (en) Highly sensitive system and methods for analysis of troponin
US20130316468A1 (en) Biomarkers for Alzheimer's Disease
CN118980671A (zh) 溶液中目标物的检测方法及装置、电子设备
JPH04340448A (ja) 表面状態の検査方法及び検査装置
AU2016204877A1 (en) Highly sensitive system and methods for analysis of troponin
HK1235464A (en) Highly sensitive methods for analysis of troponin
HK1189657A (en) Highly sensitive system and methods for analysis of troponin
HK1163818B (en) Highly sensitive system and methods for analysis of troponin

Legal Events

Date Code Title Description
NENP Non-entry into the national phase

Ref country code: DE

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17803750

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 17803750

Country of ref document: EP

Kind code of ref document: A1