High sensitivity biolayer interferometry with enzymatic amplification

Abstract

The present invention provides a high sensitivity immunoassay method for determining the concentration of an analyte in a sample based on interferometry. The present invention provides a high molecular weight conjugate comprising a plurality of streptavidin and horse radish peroxidase (HRP) and using the conjugate to increase the sensitivity of biochemical assays. The present invention relates to a method for preparing a high molecular weight conjugate comprising at least 2 HRP, preferably 4 HRP, and binding a precipitated HRP substrate to a solid phase to increase the signal of a biochemical assay.

Description

Technical Field

[0001] The present invention relates to highly sensitive biochemical assays based on interferometry. The present invention provides a high molecular weight conjugate comprising streptavidin and horseradish peroxidase and uses the conjugate to increase the sensitivity of biolayer interferometry assays. Background Art

[0002] Biolayer interferometry (BLI) is a commonly used analytical method in life science research. The method detects the shift of the optical interference pattern caused by the change in the thickness of the optical layer at the tip of the glass probe. When biomolecules bind to the surface of the optical layer, the thickness of the optical layer can be changed. The main applications of this method are the characterization of ligand / receptor binding kinetics and biomolecule quantification. However, quantitative BLI assays have lower sensitivity compared to other methods (such as ELISA). BLI assays are also susceptible to interference from crude biological samples such as cell lysates and blood, because any sample non-specific binding to the optical layer will produce changes in the BLI interference signal. Therefore, the overall application of BLI is limited by the lack of high-sensitivity quantitative assays and tolerance to crude samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0003] Figure 1A to Figure 1B An example of a biosensor interferometer system is illustrated.

[0004] Figure 2A A spectral interference pattern generated by two light signals reflected from a boundary between a first refractive surface and a second refractive surface is shown. Figure 2B The spectral interference pattern is shown shifting from T0 to T1 when the analyte molecules bind to the analyte binding molecules on the distal surface of the interference layer.

[0005] Figure 3 One embodiment of a high-sensitivity BLI immunoassay for procalcitonin (PCT) is depicted.

[0006] Figure 4 Describes the preparation of cross-linked (Copolymer of sucrose and epichlorohydrin).

[0007] Figure 5 Depicts the preparation of streptavidin / HRP cross-linked Flowchart of the process.

[0008] Figure 6 Describes the preparation of anti-fluorescein cross-linked Flowchart of the process.

[0009] Figure 7 A table presents the protocol, reagents, timing steps, volumes and orbital rpm for performing the procalcitonin (PCT) assay.

[0010] Figure 8 Shown are the results of BLI measurements of step 13 monitoring probe-bound HRP enzyme activity when the probe tip is immersed in a substrate reagent.

[0011] Fig. 9 Shown is the use of streptavidin / HRP cross-linking (Cx) PCT assay results for the conjugate and streptavidin / HRP conjugate (200,000 Daltons).

[0012] Fig.10 The results show that in a PCT assay at 2 ng / ml PCT, streptavidin / HR-Cx Comparison results with two commercially available streptavidin-HRP reagents.

[0013] Fig.11 The correlation of clinical PCT samples analyzed by the high-sensitivity BLI assay of the present invention with those analyzed by fluorescence assay using the Pylon 3D instrument (ET Healthcare Inc) is shown. DETAILED DESCRIPTION

[0014] definition

[0015] The terms used in the claims and specification should be interpreted according to their ordinary meanings as understood by those skilled in the art, except for the definitions set forth below.

[0016] As used herein, "about" means within ±10% of the stated value.

[0017] As used herein, an "analyte binding" molecule refers to any molecule that is capable of participating in a specific binding reaction with an analyte molecule. Examples include, but are not limited to: (i) an antigen molecule for detecting the presence of an antibody specific for the antigen; (ii) an antibody molecule for detecting the presence of an antigen; (iii) a protein molecule for detecting the presence of a binding partner for the protein; (iv) a ligand for detecting the presence of a binding partner; or (v) a single-stranded nucleic acid molecule for detecting the presence of a nucleic acid binding molecule.

[0018] The "aspect ratio" of a shape is the ratio of its longer dimension to its shorter dimension.

[0019] "Binding molecule" refers to a molecule that is capable of binding to another molecule of interest.

[0020] As used herein, "binding pair" refers to two molecules that attract each other and specifically bind to each other. Examples of binding pairs include, but are not limited to, antigens and antibodies to antigens, ligands and receptors thereof, complementary strands of nucleic acids, biotin and avidin, biotin and streptavidin, lectins and carbohydrates. Preferred binding pairs are biotin and streptavidin, biotin and avidin, fluorescein and anti-fluorescein, digoxin / anti-digoxin.

[0021] As used herein, "immobilization" refers to an agent being fixed to a solid surface. When an agent is immobilized to a solid surface, it is either non-covalently bound or covalently bound to the surface.

[0022] As used herein, "monolithic substrate" refers to a single piece of solid material having one refractive index, such as glass, quartz, or plastic.

[0023] As used herein, "probe" refers to a monolithic substrate having an aspect ratio (length to width) of at least 2 to 1 and coated with a thin film layer on the sensing side. The probe has a distal end and a proximal end. The proximal end (also referred to as the probe tip in this application) has a sensing surface coated with a thin layer of analyte binding molecules.

[0024] "Waveguide" refers to a device (eg, a conduit, coaxial cable, or optical fiber) designed to confine and direct the propagation of electromagnetic waves (eg, light).

[0025] The present invention uses a high molecular weight conjugate comprising multiple HRP molecules to increase the number of enzyme molecules bound to the tip of a BLI biosensor as part of the signal generation step in a sandwich immunoassay format. Horseradish peroxidase (HRP) is commonly used to increase the sensitivity of immunoassays in BLI. HRP enzyme activity generates free radical products that, when reacting with certain substrates, can deposit the substrate on the surface of a solid phase matrix used in the assay. In the case of BLI, certain HRP products bind to the surface of an optical layer, increasing the thickness of the optical layer and causing a shift in the interference pattern. Compared to conventional HRP reagents, the present invention provides a high molecular weight conjugate comprising multiple HRP molecules that increases the sensitivity of an immunoassay. The present invention also provides the unexpected result that the high molecular weight HRP conjugate of the present invention significantly increases the immunospecific signal without significantly increasing the background caused by nonspecific binding, so that the signal-to-noise ratio is improved.

[0026] Since the present method measures HRP enzyme activity by BLI, any non-specific sample binding that does not increase HRP conjugate binding will not cause non-specific signals, which circumvents one of the limitations of BLI. Therefore, the present invention can be applied to crude biological samples that require high-sensitivity detection of analytes. Therefore, the present invention extends the sensitivity of immunoassays using BLI.

[0027] Biosensor Interferometer System

[0028] The present invention is applicable to several biosensor interferometer systems. Figures 1A to 1B An example of such a system is illustrated. Figure 1A Depicted is a biosensor interferometer 100 (or simply "interferometer") that includes a light source 102, a detector 104, a waveguide 106, and an optical assembly 108 (also referred to as a "probe"). The probe 108 may be connected to the waveguide 106 via a coupling medium.

[0029] The light source 102 can emit white light that is guided by the waveguide 106 toward the probe 108. For example, the light source 102 can be a light emitting diode (LED) configured to generate light within a range of at least 50 nanometers (nm), 100 nm, or 150 nm within a specified spectrum (e.g., 400 nm or less to 700 nm or greater). Alternatively, the interferometer 100 can employ multiple light sources with different characteristic wavelengths, such as LEDs designed to emit light of different wavelengths in the visible range. The same functionality can be achieved with a single light source having appropriate filters for directing light of different wavelengths onto the probe 108.

[0030] The detector 104 is preferably a spectrometer, such as the Ocean Optics USB4000, which is capable of recording the spectrum of the interfering light received from the probe 108. Alternatively, if the light source 102 operates to direct different wavelengths onto the probe 108, the detector 104 can be a simple photodetector capable of recording the intensity at each wavelength. In another embodiment, the detector 104 can include multiple filters that allow detection of the intensity at each of the multiple wavelengths.

[0031] The waveguide 106 can be configured to transmit light emitted by the light source 102 to the probe 108, and then transmit light reflected by surfaces within the probe 108 to the detector 104. In some embodiments, the waveguide 106 is a bundle of optical fibers (e.g., a single-mode fiber optic cable), while in other embodiments, the waveguide 106 is a multi-mode fiber optic cable.

[0032] like Figure 1B As shown, the probe 108 includes a monolithic substrate 114, a thin film layer (also referred to as an "interference layer"), and a biomolecule layer (also referred to as a "biolayer") composed of analyte molecules 122, which are bound to analyte binding molecules 120. The monolithic substrate 114 is composed of a transparent material through which light can pass. The interference layer is also composed of a transparent material. When light is irradiated on the probe 108, the proximal surface of the interference layer can act as a first reflective surface, and the biolayer can act as a second reflective surface. As further described below, the light reflected by the first reflective surface and the second reflective surface can form an interference pattern that can be monitored by the interferometer 100.

[0033] The interference layer typically includes multiple layers combined in a manner that improves the detectability of the interference pattern. For example, the interference layer is composed of a tantalum pentoxide (Ta2O5) layer 116 and a silicon dioxide (SiO2) layer 118. The tantalum pentoxide layer 116 can be thin (e.g., about 10nm to 40nm) because its main purpose is to improve the reflectivity at the proximal surface of the interference layer. At the same time, the silicon dioxide layer 118 can be relatively thick (e.g., about 650nm to 900nm) because its main purpose is to increase the distance between the first and second reflective surfaces.

[0034] To illustrate a simple interferometry test, a probe 108 may be suspended in a well 110 containing a sample 112. During a diagnostic test, analyte molecules 122 will bind to analyte binding molecules 120 along the distal end of the probe 108, and these binding events will result in an interference pattern that can be observed by the detector 104. The interferometer 100 can monitor the thickness of the biological layer formed along the distal end of the probe 108 by detecting shifts in the phase characteristics of the interference pattern.

[0035] Figure 1C Another biosensor interferometer probe is shown. The probe includes a monolithic substrate, an interference layer coated on the second surface of the monolithic substrate, and an analyte binding molecule layer coated on the interference layer, wherein the monolithic substrate has a first surface and a second surface arranged substantially parallel to each other at opposite ends of the monolithic substrate. The interference layer is generally composed of magnesium fluoride (MgF2). When light is irradiated on the interference measurement sensor, the first interface between the monolithic substrate and the interference layer acts as a first reflection surface, and when light is irradiated on the probe, the second interface between the biolayer formed by the analyte molecules in the sample combined with the analyte binding molecules and the solution containing the sample acts as a second reflection surface. As described above, the thickness of the biolayer can be estimated based on the interference pattern of the light reflected by the first and second reflection surfaces.

[0036] The probe 200 includes an interference layer 204 fixed along the distal end of a monolithic substrate 202. Analyte binding molecules 206 may be deposited along the distal surface of the interference layer 204. During a biochemical test, as analyte molecules 208 in a sample bind to the analyte binding molecules 206, a biolayer will be formed.

[0037] like Figure 1CAs shown, the monolithic substrate 202 has a proximal surface (also referred to as the "coupling side") that can be coupled to a waveguide of, for example, an interferometer, and a distal surface (also referred to as the "sensing side") on which additional layers are deposited. Typically, the monolithic substrate 202 has a length of at least 3 millimeters (mm), 5 mm, 10 mm, or 15 mm. In a preferred embodiment, the aspect ratio (length to width) of the monolithic substrate 202 is at least 5 to 1. In such an embodiment, the monolithic substrate 202 can be said to have a cylindrical form. The cross-section of the monolithic substrate 202 can be circular, elliptical, square, rectangular, triangular, pentagonal, etc. The monolithic substrate 202 preferably has a refractive index that is substantially higher than the refractive index of the interference layer 204, so that the proximal surface of the interference layer 204 effectively reflects light directed to the probe 200. The preferred refractive index of the monolithic substrate can be higher than 1.5, 1.8, or 2.0. Thus, the monolithic substrate 202 may be composed of a high refractive index material such as glass (refractive index 2.0) rather than a low refractive index material such as quartz (refractive index 1.46) or plastic (refractive index 1.32 to 1.49).

[0038] Interference layer 204 is composed of at least one transparent material, which is coated on the distal surface of monolithic substrate 202. These transparent materials are deposited on the distal surface of monolithic substrate 202 in the form of a thin film with a thickness ranging from a fraction of a nanometer (e.g., a monolayer) to a few microns. Interference layer 204 can have a thickness of at least 500nm, 700nm, or 900nm. Exemplary thicknesses are between 500nm and 5,000nm (and preferably 800nm ​​to 1,200nm). In this article, for example, interference layer 204 has a thickness of approximately 900nm to 1,000nm or 940nm.

[0039] Compared to conventional probes, the interference layer 204 has a refractive index substantially similar to that of the biological layer. This ensures that reflections from the distal end of the probe 200 are primarily due to the analyte molecules 208 rather than the interface between the interference layer 204 and the analyte binding molecules 206. In some embodiments, the interference layer 204 is composed of magnesium fluoride (MgF2), while in other embodiments, the interference layer 204 is composed of potassium fluoride (KF), lithium fluoride (LiF), sodium fluoride (NaF), lithium calcium aluminum fluoride (LiCaAlF6), sodium aluminum fluoride (Na3AlF6), strontium fluoride (SrF2), aluminum fluoride (AlF3), sulfur hexafluoride (SF6), etc. Magnesium fluoride has a refractive index of 1.38, which is substantially the same as the refractive index of the biological layer formed along the distal end of the probe 200. For comparison, the interference layer of a conventional probe is typically composed of silicon dioxide, and the refractive index of silicon dioxide is approximately 1.4 to 1.5 in the visible light range. Because the interference layer 204 and the biological layer have similar refractive indices, the light will experience minimal scattering as it travels from the interference layer 204 into the biological layer and then from the biological layer back into the interference layer 204 .

[0040] In one embodiment, the probe 200 includes an adhesion layer that is deposited along the distal surface of the interference layer 204 fixed to the monolithic substrate 202. The adhesion layer can include a material that promotes the adhesion of the analyte binding molecules 206. An example of such a material is silicon dioxide. Compared to the interference layer 204, the adhesion layer is usually very thin, so its effect on light traveling toward or returning from the biological layer will be minimal. For example, the adhesion layer 310 can have a thickness of about 3nm to 10nm, while the interference layer 304 can have a thickness of about 800nm ​​to 1,000nm. The biological layer formed by the analyte binding molecules 306 and the analyte molecules 308 will usually have a thickness of several nm.

[0041] When light is irradiated on probe 200, the proximal surface of interference layer 204 can serve as the first reflective surface, and the distal surface of the biolayer can serve as the second reflective surface. The presence, concentration or binding rate of analyte molecule 208 and probe 200 can be estimated based on the interference of the light beams reflected by these two reflective surfaces. When analyte molecule 208 is attached to (or detached from) analyte binding molecule 206, the distance between the first and second reflective surfaces will change. Because the size of all other components in probe 200 remains the same, the interference pattern formed by the light reflected by the first and second reflective surfaces is phase-shifted according to the change in the biolayer thickness caused by the binding event.

[0042] In operation, an incident light signal 210 emitted by a light source is transmitted toward the biological layer through the monolithic substrate 202. Within the probe 200, the light will be reflected at the first reflective surface, thereby generating a first reflected light signal 212. The light will also be reflected at the second reflective surface, thereby generating a second reflected light signal 214. The second reflective surface initially corresponds to the interface between the analyte binding molecules 206 and the sample in which the probe 200 is immersed. When binding occurs during a biochemical test, the second reflective surface becomes the interface between the analyte molecules 208 and the sample.

[0043] The two optical signals reflected from the boundary between the first and second refractive surfaces generate a spectral interference pattern, such as Figure 2A When the analyte molecules bind to the analyte binding molecules on the distal surface of the interference layer, the optical path of the second reflected light signal becomes longer. As a result, Figure 2B As shown, the spectral interference pattern shifts from T0 to T1. By measuring the phase shift after a period of time, the association of the analyte molecule with the analyte binding molecule fixed on the distal surface of the interference layer 204 can be used to calculate the analyte concentration in the sample. By continuously measuring the phase shift in real time, a kinetic binding curve can be drawn with the offset versus time. The association rate of the analyte molecule with the analyte binding molecule fixed on the distal surface of the interference layer can be used to calculate the analyte concentration in the sample. Therefore, the measurement of the phase shift is the detection principle of the thin film interferometer.

[0044] Methods used to determine analyte concentration

[0045] The present invention relates to a method for determining the concentration of an analyte in a sample. The method comprises the following steps: (a) obtaining a probe having a first antibody against the analyte fixed on the tip of the probe, wherein the diameter of the tip surface is ≤5 mm; (b) immersing the tip of the probe in a sample solution containing the analyte to capture the analyte at the tip of the probe; (c) immersing the probe in a reagent solution containing a second antibody against the analyte, wherein the second antibody is labeled with biotin; (d) immersing the probe in a conjugate solution having a conjugate to bind the conjugate to the probe, wherein the conjugate comprises (i) streptavidin, HRP and a polymer, or (ii) streptavidin and cross-linked HRP, or (iii) cross-linked streptavidin and HRP, wherein the conjugate has a molecular weight of at least 400,000 Daltons and comprises at least 4 HRPs; (e) immersing the probe in an HRP substrate solution to bind the substrate to the HRP in the conjugate for a period of time; and (f) determining the concentration of an analyte in the sample by measuring the wavelength shift due to light interference and quantifying the wavelength shift relative to a calibration curve to determine the analyte concentration.

[0046] Figure 3An embodiment of a highly sensitive BLI immunoassay for procalcitonin (PCT) performed in a sandwich assay format is depicted. A BLI probe tipped with an antibody against PCT is immersed in a sample containing PCT. After a wash sequence, the probe is transferred to a biotin-labeled anti-PCT reagent to form a sandwich immune complex with the PCT captured on the probe. After another wash sequence, the probe is transferred to a streptavidin / HRP- (copolymer of sucrose and epichlorohydrin) reagent. After a wash sequence, the probe is placed in an HRP substrate solution and the enzyme activity bound to the probe is monitored by the BLI instrument for a period of time, such as about 30 seconds. The HRP substrate is selected based on the binding of the enzyme product of the HRP substrate to the probe surface to increase the thickness of the biolayer, which then produces a detectable shift in the BLI interference pattern.

[0047] In step (a) of the present method, a probe with a small tip for binding an analyte is obtained. The tip has a relatively small surface area, with a diameter of ≤5 mm, preferably ≤2 mm or ≤1 mm. The small surface of the probe tip makes it have several advantages. In solid phase immunoassays, a probe with a small surface area is advantageous because it has less non-specific binding and therefore produces a lower background signal. In addition, due to the small surface area of ​​the tip, the reagent or sample carried on the probe tip is extremely small. This feature makes the probe tip easy to clean, and due to the large volume of the washing solution, the contamination in the washing solution is negligible. Another aspect of the small surface area of ​​the probe tip is that it has a small binding capacity. Therefore, when the probe tip is immersed in the reagent solution, the combination of the reagent does not consume a large amount of reagent. The reagent concentration is actually unchanged. The negligible contamination of the washing solution and the small amount of consumption of the reagent enable the reagent and the washing solution to be reused multiple times, for example, 3 to 10 times, 3 to 15 times or 3 to 20 times.

[0048] Methods for fixing proteins such as antibodies to a solid phase (the sensing surface of the probe tip) are common in immunochemistry. The antibody can be directly bound to the solid phase by adsorption, or it can be indirectly bound to the solid phase by a binding pair. For example, the probe surface can be coated with a first member of the binding pair (e.g., an anti-hapten), and an antibody labeled with a second member of the binding pair (e.g., a hapten) can be fixed to the probe by biotin-streptavidin binding.

[0049] In the step (b) of the method, the probe tip is immersed in a sample solution containing the analyte for a period of time to capture the analyte on the probe under the defined binding conditions. In one embodiment, the binding time is from 1 minute or 2 minutes to 30 minutes or 1 hour. For example, the binding time is from 2 minutes to 10 minutes.

[0050] After the binding step, the probe is optionally immersed in a washing solution comprising an aqueous solution having a pH preferably of 6.0-8.5 for a period of time (e.g., 5 seconds to 5 minutes, 10 seconds to 2 minutes, or 15 seconds to 1 minute). The aqueous solution can be water or a buffer having a pH between 6.0 and 8.5. Preferably, the aqueous solution contains 1-10 mM or 1-100 mM phosphate buffer, tris buffer, citrate buffer or other buffer suitable for pH 6.0 to 8.5. The probe is washed 1 to 3 times.

[0051] In step (c), the probe is immersed in a reagent solution containing a biotin-labeled secondary antibody against the analyte for a period of time (eg, 30 seconds to 5 minutes), wherein the secondary antibody binds to the analyte.

[0052] The probe is then optionally washed in a wash solution as described in step (b).

[0053] In step (d), the probe is immersed in a conjugate solution containing a high molecular weight conjugate for a period of time (e.g., 10 seconds to 5 minutes, 15 seconds to 1 minute, or 20 seconds to 2 minutes) to bind the conjugate to the probe, wherein the high molecular weight conjugate contains streptavidin and horseradish peroxidase (HRP).

[0054] Typically, the high molecular weight conjugate has a molecular weight of at least 200,000 Daltons, or at least 400,000 Daltons, or at least 500,000 Daltons, at least 1 million Daltons, or at least 2 million Daltons. For example, the conjugate has a molecular weight of 200,000 Daltons to 10,000,000 Daltons, 500,000 Daltons to 10 million Daltons, 1 million Daltons to 10 million Daltons, or 2 million Daltons to 10 million Daltons.

[0055] In one embodiment, the high molecular weight conjugate comprises streptavidin and cross-linked HRP, wherein the conjugate comprises at least 2, or at least 4, or at least 5, or at least 10 HRPs.

[0056] In one embodiment, the high molecular weight conjugate comprises streptavidin and HRP, wherein the streptavidin and HRP are cross-linked together. The conjugate comprises at least 2, or at least 4, at least 5, or at least 10 HRPs, and the molar ratio of streptavidin to HRP is between about 1:2 and about 2:1, or between about 1:1.5 and about 1.5:1, for example about 1:1.

[0057] In a preferred embodiment, the high molecular weight conjugate comprises streptavidin, HRP and a polymer. In the conjugate, streptavidin and HRP may be bound to the polymer. The polymer typically has a molecular weight of 200,000 Daltons to 10 million Daltons, or 500,000 Daltons to 10 million Daltons, or 1 million Daltons to 10 million Daltons. The polymer may be a polysaccharide (e.g., dextran, amylose, polysucrose), a dendrimer, or a polyethylene glycol. In a preferred embodiment, the polymer is (copolymer of sucrose and epichlorohydrin). The conjugate comprises at least 2, or at least 4, at least 5, or at least 10 HRPs, and the molar ratio of streptavidin to HRP is between about 1:2 to about 2:1, or between about 1:1.5 to about 1.5:1, such as about 1:1.

[0058] After binding, the probe is then washed one to three times in washing solution as described in step (c).

[0059] In step (e), the probe is immersed in an HRP substrate solution to bind the substrate to the HRP in the conjugate for a period of time (eg, 30 seconds to 5 minutes).

[0060] When the product of the HRP / substrate reaction is bound to the surface of the optical layer, the optical layer increases its thickness and produces a nanometer wavelength shift of light interference, and the wavelength shift is proportional to the amount of HRP bound to the probe. Any HRP substrate whose product is bound to the solid surface is suitable for the present invention. For BLI determination, precipitated substrates are preferred. Suitable substrates include 3,3'-diaminobenzidine tetrahydrochloride (DAB), 3,3',5,5'-tetramethylbenzidine (TMB), benzamidine, chloronaphthol, nitro tetrazolium chloride and 5-bromo-4-chloro-3'-indolyl phosphate p-toluidine salt. Preferred substrates are chloronaphthol, benzamidine and tetramethylbenzidine, and chloronaphthol is more preferred.

[0061] In step (f), the analyte concentration in the sample is determined by measuring the wavelength shift of the light interference and quantifying the wavelength shift against a calibration curve having the wavelength shift plotted against the analyte concentration. The phase shift can be monitored dynamically or determined by the difference between the start time point (T0) and the end time point (T1) (see Figure 2B ).

[0062] In the above-mentioned binding and washing steps, the reaction is optionally accelerated by stirring or mixing the solution in the container. For example, the solution can be induced to flow across the probe tip in one or more reaction containers (including sample containers, reagent containers, washing containers and conjugate containers), such as lateral flow or orbital flow, to accelerate the binding reaction and dissociation. For example, the reaction container can be mounted on an orbital oscillator, and the orbital oscillator rotates at a speed of at least 50rpm, preferably at least 200rpm or at least 500rpm, such as 50rpm-200rpm or 500rpm-1,500rpm. In addition, the probe tip can move up and down and perpendicular to the plane of the orbital flow at a speed of 0.01mm / sec to 10mm / sec to cause additional mixing of the solution above and below the probe tip.

[0063] like Figure 3 The assay protocol shown in is for a sandwich assay format. High molecular weight streptavidin / Conjugates to design competition assay formats.

[0064] High molecular weight conjugate

[0065] The present invention also relates to a method comprising streptavidin, HRP and cross-linked Conjugates. Cross-linked The conjugate has a molecular weight of at least 500,000 Daltons, preferably at least 1 million Daltons, or 2 million Daltons, or 5 million Daltons, or 10 million Daltons. The conjugate has at least 2 streptavidins and at least 2 HRPs, and preferably has at least 4 streptavidins and at least 4 HRPs. In one embodiment, the cross-linked having a molecular weight of 500,000 Daltons to 10 million Daltons or 1 million Daltons to 10 million Daltons, and each cross-linked In one embodiment, the cross-linked having a molecular weight of at least 2 million Daltons (e.g., 1 million Daltons to 10 million Daltons), and each cross-linked In one embodiment, the cross-linked having a molecular weight of at least 2 million Daltons or at least 5 million Daltons (e.g., 2 million Daltons to 10 million Daltons or 5 million Daltons to 10 million Daltons), and each cross-linked Combine at least 10 HRP and at least 10 streptavidin.

[0066] In one embodiment, the conjugate The molar ratio of streptavidin:HRP is 1:2-10:2-10. In another embodiment, the conjugate The molar ratio of streptavidin:HRP is 1:3-7:3-7. For example, each cross-linked The molecule binds at least 4 to 10 HRP and 4 to 10 streptavidin.

[0067] Probe

[0068] The present invention also relates to a probe comprising a single-chip substrate, a thin film layer and a biomolecule layer fixed on the tip of the probe, wherein the biomolecule layer comprises analyte molecules, analyte binding molecules, HRP and chloronaphthol, wherein the diameter of the tip surface is ≤5 mm.

[0069] The present invention is further illustrated by the following examples, which are not to be construed as limiting the scope of the invention to the specific procedures described therein.

[0070] Example

[0071] Material

[0072] Recombinant streptavidin was obtained from IBA. Horseradish peroxidase (HRP) RZ=1 was obtained from Santa Cruz BioTech. SMCC, 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimidyl ester, DMF, anhydrous dimethylformamide were obtained from Thermo Scientific and used without further purification. Cy5-NHS, cyanine 5-monosuccinimidyl ester was ordered from ATT Bioquest. N-acetyl-L-cysteine ​​and n-ethylmaleimide were purchased from MP Biomedicals and Thermo Scientific.

[0073] instrument

[0074] UV / VIS measurements were performed by spectrophotometer (Thermo BioMate 3S). Centrifugation was performed by centrifuge (Beckman Allegra 6R). Proteins were desalted by Zebra spin columns 2 ml size, 7K MWCO (Thermo Scientific). The bioconjugates were purified with Cytiva CL-4B Sepharose medium custom packaged in an XK 16×400 column connected to a Cytiva AKTA Go purification system.

[0075] Will The Prime system (Gator Bio, Inc.) was used for BLI measurements.

[0076] Example 1. Cross-linking Preparation of 400-SPDP

[0077] Cross-linked-FICOLL 400-SPDP (6-[3-[2-pyridyldithio]-propionylamino]hexanoic acid succinimidyl ester, Invitrogen) was prepared according to Example 1 of US2011 / 0312105. Figure 4 A flow chart for its preparation is shown.

[0078] Example 2. Preparation of cyanine 5 and maleimide-labeled streptavidin

[0079] 10 mg of streptavidin (molecular weight of about 60,000 Daltons) was dissolved in 500 μl of PBS buffer to 20 mg / ml. 50 μl of 0.5 M carbonate buffer was added to the streptavidin solution. 30 μl (MCR = 1) of a 5 mg / ml solution of cyanine 5 (Cy5) monosuccinimidyl ester in anhydrous dimethylformamide (DMF) was added to the streptavidin solution and allowed to react at room temperature for 1 hour. In this case, Cy5 acts as a tracer, thereby enabling the quantification of the Cy5-streptavidin / HRP cross-linked prepared subsequently. Streptavidin content in the conjugate. 10 mg / ml SMCC, 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimidyl ester in anhydrous dimethylformamide (DMF) was freshly prepared, and 52 μl (MCR=8.7) thereof was added to the above Cy5-labeled streptavidin. The SMCC labeling reaction was allowed to proceed for another hour at room temperature. A 2 ml size Zeba spin desalting column (7KMWCO) was prepared according to the Thermo Scientific instruction manual. The Cy5 / SMCC dual-labeled streptavidin reaction mixture was loaded onto the column and centrifuged at 1000×g for 2 minutes, and the effluent containing the sample was collected. The concentration of streptavidin and Cy5 labeling ratio was estimated by the absorbance at 280 nm and 655 nm of the spectrophotometer. Streptavidin was labeled with Cy5 so that the labeled streptavidin could be monitored throughout the conjugation step.

[0080] Example 3. Preparation of maleimide-labeled horseradish peroxidase

[0081] 12 mg of horseradish peroxidase (HRP) with a molecular weight of about 40,000 Daltons was dissolved in 600 μl of 1×PBS buffer, and then 60 μl of 0.5 M carbonate buffer was added. 72 μl of freshly prepared 10 mg / ml SMCC, 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester in anhydrous dimethylformamide (DMF) was added to the HRP solution to make the molar ratio of SMCC to HRP MCR=7.2. After 1 hour of reaction at room temperature, a 2 ml Zeba spin desalting column (7K MWCO) was prepared according to the Thermo Scientific instruction manual. The reaction mixture of maleimide-labeled HRP was loaded on the top of the spin column and centrifuged at 1000×g for 2 minutes. The purified samples were collected and the amount of maleimide-labeled HRP was estimated by the 280 nm absorbance of the spectrophotometer.

[0082] Example 4. Cy5-streptavidin, horseradish peroxidase, cross-linked Preparation of conjugates

[0083] Preparation of streptavidin / HRP cross-linked The flow chart is as follows Figure 5 Cross-linked -SPDP (3-(2-pyridyldithio) propionic acid succinimidyl ester) was reacted with 38 mg / ml DTT (dithiothreitol) at a 1:500 molar excess at room temperature for 1 hour. At the end of the reaction, the mixture was loaded onto a 2 ml size Zebra spin desalting column (7K MWCO) prepared according to the Thermo Scientific instruction manual. The thiol-labeled cross-linked The sample was reacted with cyanine 5 / maleimide-labeled streptavidin (Example 2) and maleimide-labeled horseradish peroxidase (Example 3) in Table 1 as the effluent from the Zeba spin column.

[0084] Table 1. Starting ratios of reagents in the reaction mixture

[0085]

[0086] Cross-linked The coupling of thiol (1 mg) with maleimide-streptavidin (1.1 mg) and maleimide-horseradish peroxidase (0.8 mg) was carried out at room temperature for 4 hours and then quenched with n-acetyl-l-cysteine ​​16 mg / ml in 1×PBS (6.25 ul per mg streptavidin) and n-ethylmaleimide 32 mg / ml in 1×PBS (10 u1 per mg streptavidin). The quenched reaction mixture was purified using a CL-4B column with an AKTA Go purification system at a flow rate of 1 ml / min (1×PBS mobile phase). The first eluting peak was the desired product. About 90% of the streptavidin and HRP were cross-linked. Conjugation.

[0087] Example 5. Anti-fluorescein cross-linking Preparation of conjugates

[0088] Figure 6 Presents a method for preparing anti-fluorescein cross-linked 1.5 mg / ml anti-fluorescein (Jackson ImmunoResearch) in 1 ml PBS was mixed with 5 mg / ml 1.9 μl SMCC in DMF and reacted at room temperature for 1 hour, followed by purification on a PD 10 column.

[0089] Cross-linking The thiol on the SPDP was deprotected by adding 30 ul of 38 mg / ml DTT to 0.7 mg of the cross-linked 400-SPDP in 1 ml PBS solution and reacted at room temperature for 1 hour, and then the cross-linked Cross-linking of anti-fluorescein-SMCC -SH were mixed and reacted at room temperature overnight. Then 10 μl of 12.5 mg / ml NEM (Aldrich) was added and reacted at room temperature for 1 / 2 hour. The conjugate was then purified on a Sepharose 4BCL column.

[0090] Example 6: Preparation of Streptavidin-HRP Conjugate (for comparison purposes)

[0091] 6.3 mg of streptavidin (IBA 2-0203-100) in 1.5 ml PBS pH 7.4 was reacted with 28 ul of SPDP (3-(2-pyridyldithio) propionic acid succinimidyl ester, Invitrogen 51531) in 10 mg / ml DMF and reacted for 1 hour at room temperature. The material was then purified on a Sephadex G25 column. To generate free thiols, 4.5 mg of streptavidin-SPDP in 1.5 ml of PBD S was reacted with 30 μl of dithiothreitol 38 mg / ml PBS and reacted for 30 minutes at room temperature and then purified on a Sephadex G25 column. 3.7 mg of HRP (Santa Cruz Bio H0621) in 1 ml of PBS was reacted with 21 ul of SMCC 10 mg / ml DMF and then purified on a Sephadex G25 column. Streptavidin-SH and HRP-SMCC were mixed and reacted overnight at 4°C. The conjugate was then purified on a Sepharose 6B CL column. The column was pre-calibrated with molecular weight markers, streptavidin, IgG, and β-galactosidase. The streptavidin-HRP conjugate with an estimated molecular weight of 200,000 Daltons was collected.

[0092] Example 7. Preparation of reagents and anti-PCT probes for PCT assay

[0093] A quartz probe with a diameter of 1 mm and a length of 2 cm, with a BLI optical layer at the distal tip, was coated with aminopropylsilane using a chemical vapor deposition method (Yield Engineering Systems, 1224P) according to the manufacturer's protocol. The probe tip was then immersed in 10 μg / ml mouse monoclonal anti-fluorescein-conjugated (from Example 5) in solution in PBS at pH 7.4. After allowing the antibody to adsorb to the probe for 15 minutes, the probe tip was washed in PBS.

[0094] The probe tip was then immersed in a solution containing 10 μg / ml fluorescently labeled mouse monoclonal anti-procalcitonin PCT (Hytest). The antibody was fluorescein labeled by standard methods. After 10 minutes, the probe was washed in PBS. The probe was then immersed in a 15% sucrose solution and then dried at 37°C for 20 minutes. The polyclonal anti-PCT (Hytest) was biotinylated by standard methods.

[0095] Example 8: Highly sensitive BLI assay for PCT

[0096] Figure 7 A table of protocols, reagents, timing steps, volumes and orbital rpm for performing the PCT assay is presented. Figure 8The results of BLI measurements of step 13 are shown, which monitor the activity of the HRP enzyme bound to the probe when the probe tip is immersed in a substrate reagent. Suitable substrates include 3,3'-diaminobenzidine tetrahydrochloride (DAB), 3,3',5,5'-tetramethylbenzidine (TMB), benzamidine, chloronaphthol, nitro blue tetrazolium chloride, and 5-bromo-4-chloro-3'-indolyl phosphate p-toluidine salt. Preferred substrates are chloronaphthol, benzamidine, and tetramethylbenzidine, and chloronaphthol is more preferred. PCT samples at 0pg / ml, 50pg / ml, 100pg / ml, and 200pg / ml produce dose responses with different nano-shifts.

[0097] Example 9: Streptavidin-HRP and Streptavidin / HRP cross-linking Comparison

[0098] Comparisons with different streptavidin-HRP conjugates were performed in a high sensitivity BLI format. A conjugate with a molecular weight of approximately 200,000 Daltons with a direct linkage between streptavidin and HRP as described in Example 6 and a streptavidin / HRP cross-linked conjugate with a molecular weight estimated to be several million Daltons as described in Example 4 were used. The conjugates were used in the PCT assay as described in Example 8. Both conjugates were loaded at 10 μg / ml in the assay in step 9. Fig. 9 PCT curves using two conjugates are presented, in which streptavidin / HRP cross-linked (Cx) The nano-shift signal generated is much higher. The sensitivity of the PCT assay using Streptavidin / HRP-CxFicoll is about 500 times that of Streptavidin-HRP, with a minimum detection limit (MDL) of 4.6 pg / ml compared to 2500 pg / ml, respectively. The MDL is defined as the PCT concentration that is 2 times the standard deviation of the negative samples. Streptavidin / HRP-Cx The reagent increased the specific PCT signal without a significant increase in the background of negative samples.

[0099] Example 10: Comparison of different streptavidin-HRP conjugates in PCT assay

[0100] exist Fig.10 In step 9, a different HRP reagent was used, and the protocol of Example 8 was used to add streptavidin / HR-Cx in the PCT assay at 2 ng / ml PCT. Results compared to two commercial streptavidin-HRP reagents. All three HRP reagents produced some wavelength (nm) shift in the BLI assay, with SA / HRP cross-linked FICOLL producing the highest wavelength (nm) shift (5.83 nm compared to 1.28 nm or 0.79 nm).

[0101] Example 11: Correlation with clinical samples

[0102] Plasma clinical PCT samples were analyzed by high sensitivity BLI and Pylon 3D instrument (ET Healthcare Inc, a commercial clinical immunoanalyzer using fluorescence detection). Samples were analyzed by BLI according to the protocol in Example 8 and by Pylon 3D according to the manufacturer's recommended protocol. Fig.11 A high correlation (R = 0.98) between the two methods was shown, indicating that high-sensitivity BLI can be used in applications of crude biological samples requiring high sensitivity and accurate quantification.

[0103] The invention and the manner and process of making and using the same are now described in full, clear, concise and precise terms to enable any person skilled in the art to make and use the invention. It should be understood that the foregoing describes preferred embodiments of the invention and that modifications may be made therein without departing from the scope of the invention as set forth in the claims. In order to particularly point out and distinctly claim the subject matter regarded as inventive, the specification ends with the following claims.

Claims

1. A method for detecting an analyte in a sample, the method comprising the following steps: (a) obtaining a probe having a first antibody against the analyte immobilized on the tip of the probe, wherein the diameter of the tip surface is ≤5 mm; (b) immersing the probe tip into a sample solution containing the analyte to capture the analyte at the probe tip; (c) immersing the probe in a reagent solution containing a second antibody against the analyte, wherein the second antibody is labeled with biotin; (d) immersing the probe in a conjugate solution having a conjugate to bind the conjugate to the probe, the conjugate comprising (i) streptavidin, HRP, and a polymer, or (ii) streptavidin and cross-linked HRP, or (iii) cross-linked streptavidin and HRP, wherein the conjugate has a molecular weight of at least 400,000 Daltons and comprises at least 4 HRPs; (e) immersing the probe in an HRP substrate solution to bind the substrate to the HRP in the conjugate for a period of time; as well as (f) determining the concentration of an analyte in the sample by measuring a wavelength shift due to light interference and quantifying the wavelength shift relative to a calibration curve to determine the analyte concentration.

2. The method of claim 1, wherein the conjugate comprises (i) streptavidin, HRP, and the polymer, and the polymer has a molecular weight of at least 1 million Daltons.

3. The method of claim 1, wherein the conjugate comprises (i) streptavidin, HRP, and the polymer, and the polymer has a molecular weight of at least 2 million Daltons.

4. The method of claim 1, wherein the conjugate comprises (i) streptavidin, HRP and the polymer, and the polymer is a cross-linked copolymer of sucrose and epichlorohydrin.

5. The method of claim 4, wherein the polymer has a molecular weight in excess of 1 million Daltons.

6. The method of claim 4, wherein the polymer has a molecular weight in excess of 2 million Daltons.

7. The method according to any one of claims 1 to 6, wherein the substrate is chloronaphthol, benzamidine and tetramethylbenzidine.

8. The method of claim 7, wherein the substrate is chloronaphthol.

9. A conjugate comprising streptavidin, HRP, and a cross-linked copolymer of sucrose and epichlorohydrin, wherein the cross-linked copolymer of sucrose and epichlorohydrin has a molecular weight of at least 1 million Daltons, and each cross-linked copolymer of sucrose and epichlorohydrin binds at least 2 HRPs and at least 2 streptavidins.

10. The conjugate of claim 9, wherein the cross-linked copolymer of sucrose and epichlorohydrin has a molecular weight of at least 2 million Daltons, and each cross-linked copolymer binds at least 4 HRPs and at least 4 streptavidins.

11. The conjugate according to claim 9, wherein the conjugate has a ratio of 1:3-7:3-7 Molar ratio of streptavidin:HRP.

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