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WO2009046251A2 - Amelioration de la specificite et de la sensibilite d'une detection en porte-a-faux - Google Patents

Amelioration de la specificite et de la sensibilite d'une detection en porte-a-faux Download PDF

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Publication number
WO2009046251A2
WO2009046251A2 PCT/US2008/078675 US2008078675W WO2009046251A2 WO 2009046251 A2 WO2009046251 A2 WO 2009046251A2 US 2008078675 W US2008078675 W US 2008078675W WO 2009046251 A2 WO2009046251 A2 WO 2009046251A2
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Prior art keywords
sensor
target
compound
sensor system
substrates
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WO2009046251A3 (fr
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Wei-Heng Shih
Wan Y. Shih
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Drexel University
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Drexel University
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Priority claimed from US11/943,790 external-priority patent/US8491818B2/en
Application filed by Drexel University filed Critical Drexel University
Priority to US12/677,613 priority Critical patent/US8481335B2/en
Publication of WO2009046251A2 publication Critical patent/WO2009046251A2/fr
Publication of WO2009046251A3 publication Critical patent/WO2009046251A3/fr
Anticipated expiration legal-status Critical
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings

Definitions

  • the invention is directed to a sensor system and a method for providing a higher degree of specificity and sensitivity in cantilever sensing. More specifically, the invention relates to the binding of substrates to target specific receptors to improve detection specificity and sensitivity in cantilever sensors.
  • the quartz crystal microbalance uses thickness-mode resonance detection.
  • the detection sensitivity of a QCM is related to the resonance frequency and the thickness of the quartz membrane.
  • Sensitivity is therefore generally limited to a range of about 10 ⁇ 8 g/Hz.
  • the QCM has a relatively low detection sensitivity in the ng/ml range.
  • protein detection for diagnostic purposes generally requires a sensitivity in the range of pg/ml to fg/ml.
  • ELISA Another popular sensor system, ELISA, for example, has a detection sensitivity of 0.01-50 ng/ml for proteins in solution, depending on the affinity of antibodies. Detection using the ELISA system, however, is time intensive and requires labeling. Recently, H. Zhang, et al, "A Sensitive and High-Throughput Assay to Detect Low-Abundance Proteins in Serum," Nature Medicine 12(4) 473-477 (2006) showed that ELISA detection can be enhanced 10 5 fold (5 pg/ml) by replacing enzymes with RNA polymerase in HER-2 detection. Even with this improvement, processing speed and expense remain a significant concern.
  • the present invention is directed to a sensor system and a method for its use.
  • the system uses substrates provided with target specific receptors to enhance detection sensitivity.
  • the invention relates to a sensor system including at least one sensor including a plurality of target specific receptors capable of binding a specific target molecule or compound.
  • a detector is operatively associated with said sensor and is capable of detecting a change in at least one property of said sensor.
  • Substrates are positioned to contact said at least one sensor, each said substrate having at least one target specific receptor directly or indirectly bound to said substrate, said target specific receptor being capable of binding the target molecule or compound.
  • the target specific receptors bound to said substrates do not bind to the same binding site on said target molecule or compound as said target specific receptors of said sensor.
  • Another aspect of the invention involves a method for using the sensor system comprising exposing a sensor system to a test sample and determining a presence or concentration of a target molecule or compound.
  • Fig. l(a) is a schematic representation of compound detection using the sensor system in accordance with one embodiment of the present invention.
  • Fig. l(b) is a cross-sectional view of a first embodiment of a sensor in accordance with the present invention.
  • Fig. l(c) is a cross-sectional view of a second embodiment of a sensor in accordance with the present invention.
  • Fig. 2(a) is a scanning electron micrograph of a 40 ⁇ m long PZTVSiO 2 piezoelectric microcantilever sensor.
  • Fig. 2(b) is a graph of the resonance frequency shift versus relative humidity of the sensor of Fig. 2(a).
  • the insert of Fig. 2(b) shows the resonance spectra at various levels of relative humidity.
  • Fig. 3(b) shows an absorption and photoluminescent emission spectra of the ZnS quantum dots of Fig. 3(a).
  • Fig. 3(c) shows aqueous quantum dots lighting up Salmonella t cells in the optical images of quantum dots-labeled salmonella cells under noraml light.
  • Fig. 3(d) shows aqueous quantum dots lighting up Salmonella t cells in the optical images of quantum dots-labeled salmonella cells under ultraviolet light.
  • Fig. 4 shows a schematic representation of a target molecule or compound detection using a sensor coupled with target specific receptor bound substrates having quantum dots.
  • Fig. 5(a) depicts a flow cell system which can be used in conjunction with the sensor system of the present invention.
  • Fig. 5(b) depicts a 3.5 in by 7.5 in construct of a portable sensor system construct capable of working with 8 sensors and powered by a 9- V battery.
  • Fig. 6 is a graph of resonance frequency shift versus time of a piezoelectric microcantilever sensor in a biotinylated polystyrene suspension.
  • the insert is a photograph of the PZT/stainless steel sensor coated with avidin.
  • Fig. 7 is a transmission electron microscope image of ZnS quantum dots synthesized with MPS capping molecules.
  • Fig. 8(a) is an optical micrograph of CdS quantum dots attached to microbeads in normal light.
  • Fig. 8(b) is an optical micrograph of CdS quantum dots attached to microbeads in ultraviolet light.
  • Figs. 9(a)-(i) depict a schematic representation of a method for fabricating PZT/SiO2 piezoelectric microcantilever sensors.
  • Fig. 10(a) is a graph of resonance frequency shift as a function of time of a PMN-PT PEMS in 1 ng/ml of HER-2.
  • Fig. 10(b) is a graph of resonance frequency shift as a function of time of a
  • Fig. 1 l(a) is a graph of resonance frequency shift as a function of time for a cantilever coated with an scFv selective for HER-2 (a) and a cantilever coated with an antibody for E. coli (b) when exposed to a 0.86 mg/ml solution of HER-2, a rinse in PBS and herceptin coated polystyrene beads.
  • Fig. 1 l(b) is a graph of resonance frequency shift as a function of time for a cantilever coated with an scFv selective for HER-2 when exposed to HER-2, a PBS rinse and herceptin coated polystyrene beads.
  • the invention is directed to a sensor system 1 and method for enhanced detection using a substrate bound to a target specific receptor.
  • the system enables rapid real time label free highly specific and sensitive detection and, optionally, may further enable a means for rapid independent verification of the detection results.
  • the sensor system 1 may be used for the detection of any molecule or compound in any sample medium for which a receptor and target pair is available.
  • the sensor system 1 includes at least one sensor 2 provided with receptors attached to at least a surface of sensor 2.
  • Receptors 6 are preferably selected for their ability to bind to a particular target molecule or compound or a class of target molecules or compounds. Receptors 6 may be bound to the surface of sensor 2 in any suitable conventional manner.
  • sensor 2 may be rendered capable of detecting the presence of a target molecule or compound 5 since target 5 will bind to receptors 6 on the surface of sensor 2.
  • the resultant shift in the mechanical resonance frequency of the sensor 2 due to the binding of target 5 may be monitored to indicate the presence of target 5 in the media to which the sensor 2 and receptors 6 have been exposed.
  • Sensor 2 may have any suitable structural configuration and may be constructed from any suitable material that enables detection.
  • Sensor 2 may be configured as a cantilever sensor in which binding stresses due to the bound target 5 can be detected. Detection can be facilitated, for example, by the provision of a piezoelectric material in association with sensor 2 whereby the resonance frequency of sensor 2 can be measured by measuring the output from the piezoelectric material and relating that resonance frequency shift to the binding of target 5 and the associated binding stress exerted on sensor 2.
  • One particularly useful embodiment of sensor 2 is in the form of a piezoelectric microcantilever since this embodiment facilitates sensitivity at very low concentrations of target 5.
  • Exemplary piezoelectric microcantilevers may have detection sensitivities in the range of about fg/ml to about pg/ml, depending on the specific sensing application.
  • Figs. 2(a) and 2(b) show one preferred embodiment wherein the sensor 2 is a piezoelectric microcantilever that can detect proteins in solution.
  • Sensor 2 of Figs. 2(a)-2(b) has been demonstrated to have a sensitivity of at least 10 " g/Hz Z. Shen, W. Y. Shih, and W.-H. Shih, "Self-Exciting, Self-Sensing PZlVSiO 2 Piezoelectric Microcantilever Sensors with Femtogram/Hz Sensitivity," Appl Phys Lett 89, 023506 (2006).
  • Fig. 1 (b) is another preferred embodiment wherein the sensor is a piezoelectric microcantilever comprising conductive elements 7 operatively associated with electrical leads 8, an electrically insulating layer 9, a receptor immobilization layer 10, target specific receptors 6, at least one non-piezoelectric layer 11 and at least one piezoelectric layer 12.
  • the non- piezoelectric layer 11 may also function as a conductive layer 7, wherein an insulation layer 9 and an immobilization layer 10 may also be present on the surface of the non- piezoelectric layer.
  • the piezoelectric microcantilever sensor detects the presence of a target molecule or compound by monitoring a shift in the mechanical resonance frequency of the sensor due to a change in mass or elastic modulus of the cantilever portion when target molecules or compounds bind to receptors 6.
  • Target specific receptor antibodies 6, for example, may be immobilized on at least a surface of the piezoelectric microcantilever sensor enabling binding of a target protein 5. This binding causes the resonance frequency of the piezoelectric microcantilever to shift and generates a real-time label-free detection signal which corresponds to the amount of a target proteins 5 bound to the sensor, thus allowing determination of the concentration of a target protein 5.
  • the sensor system 1 of the present invention provides binding substrates 3 in a sample material which is to be sensed.
  • Each binding substrate 3 may be provided with target specific receptors 4 bound to binding substrate 3.
  • Target specific receptors 4 include binding sites which are specific for binding target molecules or compounds 5.
  • substrates 3 are provided with at least one, preferably target specific receptors 4 bound to each substrate 3.
  • each target specific receptor 4 includes one binding site which is specific for binding target molecule or compound 5.
  • target specific receptors 4 are employed to indirectly bind substrate 3 to sensor 2 via binding of target specific receptors 4 to target molecules or compounds 5 that are also bound to target specific receptors 6 bound to a surface of sensor 2, as shown in Fig. 1.
  • the resonance frequency shift can be mathematically related to the concentration of target 5 in the sample by calibration of the sensor system.
  • target specific receptors 4 In an embodiment of the invention where it is desirable to measure the concentration of target molecules or compounds 5, a key aspect of target specific receptors 4 is their ability to selectively bind a specific target molecule or compound 5 at a different binding site on target 5 than is used to form the bond between target 5 and target specific receptors 6. Thus, target specific receptors 4 do not compete with target specific receptors 6 immobilized on a surface of sensor 2 for binding sites on target molecules or compounds 5.
  • each substrate 3 includes one target specific receptor 4, in which case target specific receptors 4 function to bind substrates 3 to targets 5 which targets 5 will also be bound to target specific receptors 6 on the surface of sensor 2.
  • the weight of target specific receptors 4 and substrate 3 is added to the weight of the bound target molecules or compounds 5 thereby enhancing the detection sensitivity of sensor 2.
  • Detection sensitivity is enhanced since lower concentrations of target 5 can be employed to generate a larger signal from sensor 2 due to the added binding stress providing by the additional binding of target specific receptors 4 and substrates 3 to the surface of sensor 2.
  • selecting for micron sized or larger substrates 3, the large mass of the bound substrates and stress generated by the bound substrates will dramatically enhance the detection signal even when there are only few target molecules on the sensor surface.
  • the micron-size target specific receptors 4 bound to substrate 3 are estimated to be able to enhance detection sensitivity by a factor of approximately 10 6 .
  • the target specific receptors 4 may be any receptor such as specially synthesized cavitants, DNA oligonucleotides, proteins, single chain variable fragments (scFvs), enzymes, antibodies, etc. which selectively bind a particular cell, protein, antigen, pathogen, etc.
  • scFvs single chain variable fragments
  • enzymes enzymes
  • antibodies etc. which selectively bind a particular cell, protein, antigen, pathogen, etc.
  • monomeric and dimeric anti-tumor scFv molecules composed of variable light and heavy chains of antibody molecule anti-ECD scFV, which react to cancer markers are useful target specific receptors.
  • BA Bacillus anthracis
  • antibodies specific to BA spore surface antigens may be used.
  • the target specific receptors 4 are high affinity, high specificity non-competing secondary antibodies which target a specific antigen; a primary antibody located on the surface of a sensor may be used to capture the antigens and subsequently capture any secondary antibody specific receptors which binds to a non-competiting epitope on the antigen.
  • Secondary antibodies that do not compete with the primary antibodies may be identified from panels of single-chain variable fragment (scFv) antibodies isolated from combinatorial naive phage display libraries or from commercial sources. Additionally, the secondary antibodies may be formulated from new scFv antibodies that are isolated from other scFv phage display libraries in the presence of high concentrations of the primary antibodies to promote the isolation of non-competing clones.
  • scFv single-chain variable fragment
  • Combinatorial naive phage display libraries are another source for non- competing secondary antibodies. These libraries are typically created through the random combination of human variable light and variable heavy chain domains, resulting in the creation of antibodies that are specific for regions, i.e. epitopes, on target antigens that are not normally immunogenic. The use of phage display therefore significantly increases the areas on the antigen that can be bound by a secondary antibody.
  • Substrate 3 may be any microparticle, more preferably, the substrate is a microsphere, microrod, microplate and most preferably the substrate is a microsphere, microrod or microplate having a diameter of about 0.1 microns to about 100 microns.
  • the microspheres may function like cells or spores that can be captured by a target molecule or compound attached to a sensor.
  • substrate 3 may be populated with one or more quantum dots 7 to further enable visualization and imaging of the captured target molecules or compounds 5.
  • Quantum dots 7 fluoresce under excitation light providing visual or fluorescent verification that the target molecules or compounds 5 are captured on the sensor 2 surface and thus confirming the presence of the target molecule or compound 5 in a sample. Therefore, it is possible to view a sample under a fluorescent microscope and determine the concentration of target molecules or compounds 5 based on the photoluminescence of the quantum dot 7 populated substrates 3.
  • Quantum dots 7 are particularly useful in imaging proteins and cells in biological systems due to their stability against photo-bleaching and their ability to be conjugated to target proteins such as antibodies.
  • Figs. 3(c)-3(d) demonstrate the imagining of bacteria, Salmonella t cells, using quantum dots directly conjugated with target specific receptor antibodies under normal light and under ultraviolet light, respectively.
  • Fig. 3(b) shows the absorption and photo luminescent emission spectra of the suspension.
  • Quantum dots 7 may be synthesized using any standard fabrication techniques and may be of any suitable size.
  • microsphere substrates having a diameter of about 0.1 micron- 100 microns may be coated with quantum dots having a size ranging from 3 nm-100 nm.
  • the sensor system 1 of the present invention may be used to detect and verify the presence of a select molecule or compound 5 by exposing target specific receptor 4 bound substrates 3 to a testing environment containing the sensor 2 and sample to be tested.
  • the target specific receptor 4 bound substrates 3 may be introduced to the testing environment or sample before, at the same time as or after the sensor 2 is exposed to the sample.
  • the sample and sensor 2 are first allowed to react for a defined period of time before the target specific receptor 4 bound substrates 3 are introduced to allow the target molecules or compounds 5 to first bind to the sensor 2. This reaction time can vary depending upon the sample size, target molecules or compounds 5 and target specific receptors 4.
  • the sample and target specific receptors 4 are first mixed to together and then exposed to the sensor 2.
  • Sensor system 1 functions by binding target molecules or compounds 5 that react to a first set of target specific receptors 6 immobilized on a conductive element.
  • the binding of a target molecule or compound 5 causes a subsequent change in mass and a change in the spring constant of the sensor 2 which correspondingly shifts the mechanical resonance frequency of the sensor 2.
  • the sensor 2 detects these shifts in resonance frequency ⁇ f, expressed in Equation 1, which models the functionality of the sensor:
  • the sensor 2 may be immersed in a flowing solution for in-solution detection.
  • the sensors are preferably situated in a flow cell system 8 to enable tailored, rapid and simultaneous detection and quantification of multiple compounds or molecules.
  • Fig. 5(a) shows a flow cell system 8, with a sensor holder/measuring unit 9, having a total volume of less than 0.03-30 ml, pump 10, and a mechanism for controlling temperature and humidity (not shown).
  • the flow cell 8 may attain flow rates of up to 0.1-100 ml/min.
  • the total volume of the flow cell, number of channels and flow rate may vary depending upon the number of compounds to be measured.
  • the flow cell 8 may cooperate with a portable sensor unit, shown in Fig. 5(b), which has multiple channels for the simultaneous quantification of multiple receptor specific molecules.
  • the portable sensor unit is inexpensive and capable of obtaining quick measurements.
  • the sensor system 1 of the present application may be used in various sensing applications such as solid-liquid transition detectors, liquid viscosity and density sensors, mass sensors for in situ and in-solution detection.
  • the sensor 2 may generally be used for detection of any molecule or compound in any sample medium but is particularly effective for in-solution detection and detection in biological samples.
  • the sensor system 1 may be particularly promising as a diagnostic instrument. It may be useful as a means for early detection for various forms of cancers, such as breast cancer, ovarian cancer, prostate cancer and/or other diseases, and the incorporation of quantum dots may minimize the possibility of false-positive and false-negative results. It may also be used to monitor the progress of the disease throughout treatment, and the sensor system 1 may even be incorporated in a portable device and used as a noninvasive means for testing blood and other biological samples for various pathogens, infectious agents and other markers indicative of disease in a highly sensitive and verifiable manner. For example, the sensor system 1 would enable real time protein or DNA detection with sensitivities in the range of fg/ml.
  • the system can be formulated as a cost-efficient and portable device, such a diagnostic tool may be easily incorporated in a patient's annual physical.
  • a rapid direct label free detection system for pathogens or indicators such as cancer markers can be used to screen a patients' blood.
  • no additional chemical analysis is required, so patients will be able to rapidly obtain their results at the point of care.
  • the sensor system 1 may be useful for the detection of bioterrorism agents.
  • Primary antibody receptors specific to at least one bioterrorism agent may be bound to an electrode and secondary antibody receptors may be bound to a substrate for use in detecting the presence of bioterrorism antigens.
  • bioterrorism antigens In addition to identifying the existence of a bioterrorism agent, it may also be used to quantify the concentration of the agent.
  • the sensor system 1 may also be applicable for the food science and food manufacturing industry.
  • the sensor system may be used as a diagnostic instrument for detecting pathogens or other disease agents present in food supplies and prepared or processed foods. Additionally, it may also be useful in manufacturing plants and food service industries as a means of intermittently checking food products during different phases of food preparations thereby preventing contamination and the spread of bacterial or viral diseases such as salmonella and E cob.
  • Sensor system 1 may also be applicable for detecting and/or measuring water- borne pathogens in water treatment plants.
  • the sensor system may be used as an evaluation tool for monitoring the purity of drinking water or waste water.
  • Antigens attached to substrates are easier to detect in comparison to sensors without substrates because the additional mass of the substrates affects the detection signal of the sensor.
  • Previous studies have established that using biotinylated polystyrene spheres to bind biotin on immobilized avidin can be easily detected whereas the same binding event using molecular biotin is more difficult.
  • Using a PZT/stainless steel piezoelectric microcantilever having a PZT layer 1.3 mm long and 3.35 mm wide and having a 2.87 mm long gold-coated stainless steel tip coated with avidin it was possible to bind biotin in a biotinylated polystyrene suspension.
  • the biotinylated polystyrene spheres were approximately 2 ⁇ m in diameter and produced a resonance frequency shift of about ⁇ f ⁇ 250 Hz, as shown in Fig. 6.
  • a 1- ⁇ m size microsphere has roughly 4x10 4 quantum dots on its surface and can amplify the optical signal of QD-marker protein binding 4x10 4 fold. Moreover, since a 1- ⁇ m size microsphere has a mass 8x10 6 times that of a quantum dot, the binding of a microsphere can enhance the piezoelectric microcantilever sensor resonance frequency shift by 8x10 6 times that which is achievable with a single quantum dot. As a result, quantum dot coated-microspheres, not only can image but also enhance detection sensitivity.
  • the sensor system comprises antigen-coated microspheres decorated with metal sulfide (MS) quantum dots.
  • MS quantum dots may be fabricated from various materials using various different fabrication means. MS quantum dots were be synthesized using 3-mercaptopropionic acid (MPA), metal nitrate (M(NOs) 2 ) and sodium sulfide (Na 2 S). The well-dispersed transparent and highly photo luminescent MS quantum dots, which may be fabricated in 30 minutes at room temperature, produced a MPA/M ratio of about 8:2.
  • MPA-capped quantum dots were previously used to bind amine-modif ⁇ ed 1 ⁇ m in diameter polystyrene spheres through peptide bonds.
  • the carboxyl group of MPA on the CdS quantum dots surface was first activated by l-ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDC) and N-hydrocylsulfo-succinimide (sulfo- NHS).
  • EDC l-ethyl-3-(3- dimethylaminopropyl) carbodiimide
  • sulfo- NHS N-hydrocylsulfo-succinimide
  • Figs. 8(a)-8(b) is an optical micrograph of CdS quantum dots attached to microbeads in normal light.
  • Fig. 8(b) is an optical micrograph of CdS quantum dots attached to microbeads in UV light. Clusters of microbeads contain aggregates of quantum dots giving brighter image under UV light. For more stable MPS-capped quantum dots, there are several approaches to fabricate the quantum dots decorated and antibody coated microspheres.
  • avidin-conjugated quantum dots were first synthesized using SMCC (sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane- 1 -carboxylate) as linker to bind MPS-capped quantum dots to avidin.
  • the avidin-conjugated quantum dots were then coated on biotinylated polystyrene spheres. The number ratio between the quantum dots and polystyrene and their concentrations were varied in order to achieve dispersed antibody coated quantum dot microsphere suspensions.
  • Another approach involved spheres with amine surface groups where SMCC was added to the polystyrene suspension to facilitate the NHS ester to react with primary amines.
  • the precipitated polystyrene spheres were collected and mixed with MPS-capped quantum dots.
  • a third approach involves spheres with carboxyl groups on their surface wherein sodium silicate was added to the MPS-capped quantum dot suspension to form a silica layer.
  • the silica-coated quantum dots were then reacted with aminopropylsilane (APS) to modify the surface with primary amine groups.
  • APS aminopropylsilane
  • the reaction between amine and carboxyl groups resulted in the formation of quantum dot decorated and antibody coated microspheres.
  • the conjugation of secondary antibody to microspheres utilizes the same surface chemistry employed in the immobilization of the primary antibody on the surface of sensor as described in Example 4.
  • Example 4 It is envisioned that the sensor system of the present invention may be particularly useful for diagnosing breast cancer from a patient's blood sample. Because the sensor system of the present invention improves sensitivity by several orders of magnitude and is capable of verifying the detection results, it may be possible to more successfully and more accurately detect breast cancer at an early stage when treatment is most effective.
  • a sensor system for breast cancer detection is envisioned to comprise a piezoelectric microcantilever sensor (PEMS) having a primary antibody coating and polystyrene spheres decorated with quantum dots (Q- spheres) Secondary antibodies will then be immobilized on the surface of the Q- spheres to bind to the breast cancer markers.
  • PEMS piezoelectric microcantilever sensor
  • Q- spheres quantum dots
  • HER-2 a member of the epidermal growth factor receptor family. 20-40% of breast cancers are HER-2 positive and over-expression of this receptor is correlated with a poor prognosis.
  • a panel of antibodies will be used which is expected to achieve at least a 95% detection rate for breast cancer.
  • the panel of anti-HER-2 scFv molecules will be isolated from two na ⁇ ve human scFv phage display libraries. These scFv molecules are composed of variable light and variable heavy chains of antibody molecules, thereby duplicating an antibody's antigen binding pocket.
  • scFv molecules are readily expressed from E. coli and can be modified e.g., with caiboxy-terminal residues to facilitate site-specific coupling to a piezoelectric microcantilever sensor.
  • the relatively small size of the secondary antibody coated microspheres about 25 kDa, facilitates a dense distribution on a piezoelectric microcantilever surface.
  • Panels of scFv molecules specific for a variety of human breast cancer antigens including, HER-2, HER3, HER4, EGFR and the Mullerian Inhibiting Substance Type II receptor (MISIIR) have already been isolated and thus may provide suitable candidates for the present invention. Additional panels of scFv specific for additional breast cancer antigens, e.g.
  • CA-125, CEA, CEA15.3 etc. may be isolated as the experiment progresses.
  • Milligram quantities of recombinant HER-2 extracellular domain (ECD) for use in the selection of scFv molecules and in the development of the assay system may also be expressed.
  • H3 anti-HER-2 scFv molecules
  • This scFv has been previously successful in detecting nanogram quantities of recombinant HER-2 from solutions containing one milligram per milliliter of serum albumin (from 1/40 serum).
  • Secondary scFv and IgG candidates for conjugation to the Q-spheres that do not block binding of H3 to HER-2 will then be identified.
  • the secondary antibody candidates will be conjugated to the Q-spheres and then re-assayed so as to enable binding to the H3-HER-2 ECD complex.
  • the candidate antibody that generates the greatest signal will be selected as the secondary antibody.
  • the effectiveness of the sensor system to detect HER-2 will be tested by comparing the detection sensitivity of a PEMS to that of a system including a PEMS and secondary antibody bound Q-spheres.
  • the PEMS of the sensor system will be fabricated by two approaches.
  • a PZT/SiCh system is fabricated according to the method shown in Fig. 9.
  • Another approach involves the formation of lead magnesium niobate-lead titanate (PMN-PT) freestanding films followed by electroplating and wire-saw cutting.
  • PMN-PT lead magnesium niobate-lead titanate
  • the sensor system of the present invention including the antibody coated Q-spheres, will be used to detect the presence and concentration of HER-2 in serum.
  • a fluorescence microscope will be used to image the photoluminescence of the Q-spheres.
  • Q-spheres that have been conjugated with secondary antibody will be first mixed with a serum that contains the HER-2 antigen.
  • PEMS will then be immersed in the Q-spheres-antigen serum.
  • the resonance frequency of PEMS will be monitored to study the enhancement effect due to the Q- spheres. While not wishing to be bound by theory, it is expected that the antigens will bind with the Q-spheres in solution and then the antigen Q-sphere complex will bind to the PEMS.
  • Comparative Example A & Example 5 The ability of a PEMS coated with microspheres and a PEMS having no microspheres to detect HER-2 in low concentration solutions was investigated.
  • a PEMS was coated with scFv specific for HER-2 was tested.
  • Sulfo-SMCC was used as a linker for antibody immobilization.
  • 5 mM SMCC was mixed with 400 nM scFv for 2 hours.
  • the unreacted SMCC was removed by 4 repetitions of microcentrifugation with a 10 kDa filter.
  • the MPS-coated PEMS was then soaked in the scFv-bound SMCC solution.
  • the sulfhydryl of the MPS on the sensor surface reacted with the maleimide of the scFv-bound SMCC to immobilize the scFv.
  • the MPS-coated PEMS was soaked in a 5 mM (2.6 mg/ml) maleimide activated biotin solution in PBS for 2 hours and rinsed well with PBS to remove any excess biotin.
  • the cantilever was then soaked in a 4 mg/ml avidin solution.
  • the scFv was then biotinylated with NHS-Biotin (Pierce).
  • a 15 molar fold excess of the scFv (1 ⁇ M) was prepared and allowed to react at 4 0 C for 3 hr.
  • the excess biotin was then removed by microcentrifugation using a 10 k filter (Millipore) at 4000 RPM for 10 min.
  • the retentate was then mixed with PBS and microcentrifuged again. The process was repeated 3 times. After the second spin the centrifuge began to warm up. The tubes were placed in the refrigerator and the final centrifugation was performed when the machine cooled to room temperature.
  • the scFv was then immobilized on the PEMS surface by dipping the avidin-coated PEMS in the biotinylated scFv solution. To block nonspecific binding, the scFv-immobilized PEMS was soaked in a 3% Bovine Serum Albumin (BSA) solution prepared in PBS. After blocking, the PEMS was rinsed in a solution with 1% BSA and 0.1% TWEEN20. After rinsing and in between trials the PEMS was submerged in a diluted fetal bovine serum (serum/PBS 1/40).
  • BSA Bovine Serum Albumin
  • Figs. 10(a)-(b) show the detection results of HER-2 in the 1/40 diluted fetal bovine serum using a PMN-PT PEMS.
  • the resonance frequency shift versus time in 1 ng/ml and 100 pg/ml of HER-2 in 1/40 diluted fetal bovine serum are shown in Fig.lO(a) and Fig.lO(b), respectively.
  • the detection in 1 ng/ml of HER-2 yielded -2000 Hz frequency shift and that in 100 pg/ml HER-2 in diluted serum yielded -700 Hz frequency shift.
  • the PMN-PT PEMS was 600 ⁇ m long, 370 ⁇ m wide consisting of an 8 ⁇ m thick PMN-PT film and a 3 ⁇ m thick copper layer and exhibited 4xlO " ⁇ g/Hz mass detection sensitivity.
  • concentration sensitivity of 100 pg/ml was outstanding because of the better immobilization and better blocking scheme.
  • the biotin-avidin-biotin immobilization scheme packed more than twice the number of scFv on the sensor surface and consequently bound more than twice as many HER-2 as other schemes that we have tried.
  • the better scFv immobilization also made blocking of the nonspecific binding more effective, thus enabling the detection of HER-2 at a lower concentration.
  • Example 5 Two PZT/glass PEMS are used to demonstrate the ability of selectively binding polystyrene microspheres which have been coated with an antibody specific for HER-2.
  • the PEMS was first insulated with MPS (Sigma, St. Louis, MO) using a solution method. First, the cantilevers' glass tip was submerged in a piranha solution (two parts of 98% sulfuric acid (Fisher, Fair Lawn, NJ) with one part of 30% hydrogen peroxide (FisherBiotech, Fair Lawn, NJ)) at 20 0 C for 20 minutes to clean the glass surface. The cantilever tip was rinsed with deionized water and then with ethanol. The cantilevers were then totally submerged in a 1% MPS solution in ethanol titrated to a pH of 4.5 using acetic acid. The cantilevers were allowed to soak for a total of twelve hours.
  • MPS Sigma, St. Louis, MO
  • the glass tips were then immobilized with different receptors.
  • One cantilever was coated with anti-£ coli (Kirkegaard & Perry Laboratory, Gaithersburg, MD) and the other was coated with an anti-HER-2 single chain variable fragment (scFv).
  • the immobilization of these receptors was carried out under identical conditions using a heterobifunctional cross linker Sulfosuccinimidyl-4-N-maleimidomethyl cyclohexane- 1-carboxylate sulfo-SMCC (Pierce).
  • a 2.8 ⁇ M solution of receptor was activated with 50 molar fold excess SMCC for 1.5 hours at 4°C. Next the excess ⁇ unreacted SMCC was removed through centrifugation using a 1OK filtered centrifuge tube.
  • the filtered retentate was then added to a conjugation buffer containing 5 mM EDTA.
  • the MPS coated cantilever was first soaked in 5 mM EDTA in DI water for 20 minutes, and then it was soaked in the activated receptor solution. EDTA functioned to chelate divalent metals, thereby reducing disulfide formation.
  • Carboxylic acid terminated polystyrene beads were conjugated with Herceptin using carbodiimide chemistry. A stock solution containing 10 9 beads/ml was used.
  • Each bead has an approximate area of 5x10 "11 m 2 .
  • the approximate area of each NHS molecule is 8xlO '19 m 2 , as a result, 6 x 10 15 molecules were needed. This requirement was sufficiently met by using 3 mM NHS and 5 mM EDC in an MES buffer for 25 minutes at room temperature. The excess/unreacted NHS-EDC was removed using a 300 K centrifugation filter. After activation the beads were mixed with Herceptin. The approximate area of each Herceptin molecule is 8 x 10 " m . As a result, 6x10 antibodies were needed to coat the surface of all the beads. A 100-fold excess of Herceptin was used. The activated bead-Hercetpin solution was allowed to react for 2 hours at 4 0 C. Afterward the unbound Herceptin was removed using a 300 K centrifuge filter.
  • the cantilevers were rinsed with PBS and then placed in the same 3.5 ml home built flow cell containing an 86 ⁇ g/ml solution of HER-2 ECD. The solution was flowed parallel to the face of the cantilever at a rate of 0.7 ml/minute. The results of this experiment are depicted in Fig. 1 l(a). After 85 minutes the cantilever with the anti-HER-2 scFv responded by shifting 300 Hz, and the control cantilever did not change from its resonant frequency. The control cantilever was coated with an anti-Salmonella antibody. Following the exposure to HER-2 the flow cell was rinsed with PBS, which was circulated through the flow cell for 20 minutes to rinse the cantilever surface.
  • Herceptin beads detection was carried out using sensors with HER-2 immobilized on the surface.
  • HER-2 was first activated in PBS with sulfo-SMCC.
  • An 88 ⁇ g/ml solution of HER-2 was activated with a 50 fold molar excess of SMCC for 1.5 hours at 4 0 C.
  • the excess ⁇ unreacted SMCC was removed through centrifugation using a 10 K filtered centrifuge tube.
  • the filtered retentate was then added to a conjugation buffer containing 5 mM EDTA.
  • the MPS coated cantilever was first soaked in 5 mM EDTA in DI water for 20 minutes, and then it was soaked in the activated HER-2 solution for immobilization.
  • Fig. 1 l(b) shows the resonance frequency shift due to binding of the HER-2 to the cantilever surface and then that from the binding of Heiceptin beads to the HER-2 coated surface.
  • the resonance frequency shift from binding of the HER-2 to the sensor surface resulted in about a 250 Hz shift.
  • the resonance frequency shift due to the binding of the Herceptin beads was about 900 Hz, about twice what was observed in Fig. 1 l(a), supporting the notion that it may be the cross reactivity that limited the binding of the beads in the previous example.

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Abstract

L'invention concerne un système de détection comprenant au moins un capteur et des récepteurs spécifiques de cibles liés à des substrats permettant d'améliorer la sensibilité de détection. Le système de détection selon l'invention comprend facultativement des points quantiques permettant de vérifier indépendamment la présence d'une molécule ou d'un composé cible. Ledit système peut être particulièrement utile dans le diagnostic médical, la bio-défense, la préservation de la salubrité des aliments et de l'eau et la détection chimique en général.
PCT/US2008/078675 2006-11-27 2008-10-03 Amelioration de la specificite et de la sensibilite d'une detection en porte-a-faux Ceased WO2009046251A2 (fr)

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US7288404B2 (en) * 2002-04-29 2007-10-30 Regents Of The University Of California Microcantilevers for biological and chemical assays and methods of making and using thereof
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