Single-molecule immunodetection method and single-molecule chip

Abstract

The invention relates to a single-molecule immunodetection method and a single-molecule chip, and the single-molecule immunodetection method comprises the following steps: slowly adding a microsphere reagent mixed with different codes into the chip, capturing and fixing microspheres by a microsphere clamping groove at the side edge of a main flow channel in the flowing process of the microspheres in the chip, the residual reagent passes through the main flow channel and the secondary flow channel and then is collected to a waste liquid collecting area; the method comprises the following steps: introducing an antigen to be detected and a mixed fluorescent substance with different detection items into a chip, and then introducing a cleaning solution to wash the redundant antigen and the free mixed fluorescent substance; counting the number of the luminous microspheres and the fluorescence brightness through a Poisson distribution probability formula, and establishing a standard curve to obtain the concentration of the to-be-detected sample. The microspheres are captured and fixed by the microsphere clamping grooves to the maximum extent, the loss amount of the microspheres is low, an antigen to be detected is introduced into the chip, the immunoreaction is fully carried out, the detection sensitivity is high, the detection operation process is highly simplified, the detection cost is reduced, the detection efficiency is improved, and multiple target molecule detection can be realized at the same time.

Description

Technical Field

[0001] The present application relates to the field of single molecule detection technology, and in particular to a single molecule immunoassay method and a single molecule chip. Background Art

[0002] Single-molecule detection technology is a biomedical detection technology that has developed rapidly in recent years. It can realize high-sensitivity and high-resolution detection and analysis of single molecules of biological or chemical molecules.

[0003] In the single-molecule detection method in the prior art, the method is: the washed microspheres, the target molecules to be detected and the signal molecules are mixed, loaded into a detection chip containing a microwell array, and then digitally detected and measured. For example, the product of simoa requires an external automated reaction instrument, which is bulky, expensive to build and maintain, and the detection operation process is too cumbersome and complicated. In the process of repeatedly washing the excess unreacted fluorescent signal molecules, 90% of the microspheres and the samples to be tested are lost, the reagent sample utilization rate is low, which is not conducive to the detection of ultra-low abundance proteins, and in order to compensate for the loss of microspheres in the washing process, the amount of reagents and samples needs to be increased, which is not conducive to reducing costs.

[0004] Therefore, the prior art still needs to be improved and developed. Summary of the invention

[0005] Based on this, the present application provides a single-molecule immunoassay method and a single-molecule chip, which have high detection sensitivity, effectively simplify the detection process, reduce detection costs, improve detection efficiency, and can simultaneously realize multiple target molecule detection.

[0006] In order to achieve the above objectives, the present invention provides a single molecule immunoassay method, which includes:

[0007] The microsphere reagents mixed with different codes are slowly added into the chip. The microspheres are captured and fixed by the microsphere slots on the side of the main channel during the flow in the chip. The remaining reagents are collected into the waste liquid collection area after passing through the main channel and the secondary channel.

[0008] The antigen to be tested and mixed fluorescent substances with different detection items are introduced into the chip, and then a cleaning solution is introduced to wash away the excess antigen and free mixed fluorescent substances;

[0009] The number of luminescent microspheres and the fluorescence brightness were counted using the Poisson distribution probability formula, and a standard curve was established to obtain the concentration of the sample to be tested.

[0010] Preferably, the step of introducing the antigen to be detected and the mixed fluorescent substance with different detection items into the chip, and then introducing a cleaning solution to wash away the excess antigen and the free mixed fluorescent substance comprises:

[0011] After incubating and combining 10-50ul of the antigen to be tested and 5-20ul of a mixed fluorescent substance with a concentration of 0.001-0.5mg / ml containing different detection items, the mixture is passed into the chip at a flow rate of 3-15ul / min, and then 20-100ul of cleaning solution is passed to wash away the excess antigen and free mixed fluorescent substance.

[0012] Preferably, the step of introducing the antigen to be detected and the mixed fluorescent substance with different detection items into the chip, and then introducing a cleaning solution to wash away the excess antigen and the free mixed fluorescent substance comprises:

[0013] 10-50ul of the antigen to be tested is introduced into the chip at a flow rate of 3-15ul / min, followed by 5-20ul of a mixed fluorescent substance with a concentration of 0.01-0.5mg / ml containing different test items, and finally 20-100ul of cleaning solution is introduced to wash away excess antigen and free mixed fluorescent substance.

[0014] Preferably, the incubation temperature of the chip is 35-39°C.

[0015] Preferably, the microsphere reagent is added into the chip via a syringe pump or a constant pressure pump;

[0016] Or it can be driven into the chip by itself due to the super hydrophilic surface of the chip.

[0017] Preferably, the chip is surface treated by physical adsorption or hydrophilic reagent spraying or coating or chemical vapor deposition.

[0018] Preferably, the preparation method of the mixed microsphere reagents with different codes is:

[0019] The same volume of the first encoded microsphere reagent labeled with IL-6 antibody at a concentration of 0.05 mg / ml and the second encoded microsphere reagent labeled with cTnI antibody at a concentration of 0.05 mg / ml were added to the oscillator, mixed in the oscillator for 30 seconds, and then sonicated in a water bath for 30 seconds.

[0020] Preferably, the average diameter of the microspheres in the microsphere reagent mixed with different codes is 1-5 um.

[0021] The present application also provides a single molecule chip for use in the above-mentioned single molecule immunoassay method, which comprises:

[0022] A sample adding area, which is used to add microsphere reagents mixed with different codes into the single molecule chip and then enter the microsphere capture area;

[0023] Microsphere capture area: After the microsphere reagents mixed with different codes enter the microsphere capture area, the microspheres are captured and fixed by the microsphere slots on the side of the main flow channel during the flow process, and the remaining reagents flow into the waste liquid collection area through the main flow channel and the secondary flow channel;

[0024] The waste liquid collection area is used to collect the liquid flowing out of the microsphere capture area.

[0025] Preferably, the microsphere capture zone is provided with a plurality of main flow channels and secondary flow channels connected to the main flow channels;

[0026] The height of the main channel is 2 to 3 times the diameter of the microsphere, and the width of the main channel is 4 to 5 times the diameter of the microsphere;

[0027] At least one of the width or height of the secondary flow channel is smaller than the diameter of the microsphere, thereby ensuring that the microsphere cannot pass through the secondary flow channel;

[0028] At least one microball groove is provided at one end of the secondary flow channel, and the diameter of the microball groove is close to or slightly larger than the diameter of the microball, so that when the reagent flows along the secondary flow channel, the microball can enter the microball groove at a certain speed and block the microball groove.

[0029] The single molecule immunoassay method provided by the present invention has the following advantages and beneficial effects:

[0030] A mixture of microsphere reagents with different codes is slowly added to the chip. The microspheres are captured and fixed by the microsphere slots to the maximum extent, with low microsphere loss. The antigen to be tested is passed into the chip for immune reaction to proceed fully, especially for low-abundance samples, which can be increased to fg level with high sensitivity.

[0031] The detection operation process is highly simplified, large-scale automated reaction equipment is abandoned, and the detection cost is reduced. The entire test process is less than 20 minutes, and the detection efficiency is greatly improved.

[0032] Based on the imaging system of multi-color upconversion fluorescence-encoded microspheres and multiple detection methods, multiple target molecules can be detected simultaneously on the chip. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a schematic diagram of the process of the single molecule immunoassay method embodiment of the present application.

[0034] Figure 2 A microscopic image of a chip capturing immobilized microspheres in the single-molecule immunoassay method of this application.

[0035] Figure 3 This is a graph of the test data for different antigen concentration gradients for the two projects IL-6 and cTnI.

[0036] Figure 4This is a module schematic diagram of a single-molecule chip embodiment of the present application. DETAILED DESCRIPTION

[0037] In order to facilitate understanding of the present application, the present application will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present application are given in the drawings. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive.

[0038] It should be noted that when an element is considered to be "connected" to another element, it may be directly connected to the other element and integrated therewith, or there may be an intermediate element at the same time. The terms "mounted" and similar expressions used herein are for illustrative purposes only.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0040] In the first embodiment, Figure 1 As shown, a single molecule immunoassay method is provided, which comprises the following steps:

[0041] S100, slowly adding microsphere reagents mixed with different codes into the chip, the microspheres are captured and fixed by the microsphere slots on the side of the main channel during the flow of the microspheres in the chip, and the remaining reagents are collected into the waste liquid collection area after passing through the main channel and the secondary channel;

[0042] S200, introducing the antigen to be tested and mixed fluorescent substances with different detection items into the chip, and then introducing a cleaning solution to wash away excess antigen and free mixed fluorescent substances;

[0043] S300, counting the number of luminescent microspheres and the fluorescence brightness using the Poisson distribution probability formula, and establishing a standard curve to obtain the concentration of the sample to be tested.

[0044] Through the image recognition algorithm, the number of luminescent microspheres in low-abundance samples is counted using the Poisson distribution probability formula, and the brightness of fluorescence is tested for high-concentration samples. The concentration of the indicator to be tested is obtained by establishing a standard curve.

[0045] The microspheres that can be captured include: various fluorescent microspheres used for detecting reactions, microspheres, cells, and other spherical or other similarly shaped tiny structures with a specific diameter.

[0046] In specific implementation, there are two ways to add antigens and fluorescent substances:

[0047] One-step method: 10-50ul of the antigen to be tested and 5-20ul of a mixed fluorescent substance with a concentration of 0.001-0.5mg / ml containing different detection items are incubated and combined, and then passed into the chip at a flow rate of 3-15ul / min, and then 20-100ul of cleaning solution is passed to wash away excess antigen and free mixed fluorescent substance.

[0048] Two-step method: 10-50ul of the antigen to be tested is introduced into the chip at a flow rate of 3-15ul / min, followed by 5-20ul of a mixed fluorescent substance with a concentration of 0.01-0.5mg / ml containing different test items, and finally 20-100ul of cleaning solution is introduced to wash away excess antigen and free mixed fluorescent substance.

[0049] In specific implementation, the microsphere reagent is added into the chip through a syringe pump or a constant pressure pump or other sample addition power source; or is self-driven into the chip by relying on the super-hydrophilic surface of the chip.

[0050] In situ reactions can be carried out with the aid of a sample addition power source or chips with different hydrophilicities after surface treatment, and different flow rates can be designed to ensure that the fluid speed can be accurately controlled at each step. The immune response time can be accurately regulated, and the incubation temperature of the test (preferably 35-39°C) can be set as needed, so that the test results are more accurate and more repeatable.

[0051] In specific implementation, physical adsorption (such as titanium dioxide, surfactants, biological molecules), hydrophilic reagent spraying, coating or chemical vapor deposition (APTES) is used to treat the surface of the microsphere capture area of ​​the chip, so that the contact angle of the microchannel is 5 to 70 degrees, and the hydrophobic sites of the channel are blocked by passivation reagents. The modified surface reduces nonspecific protein adsorption and reduces background noise.

[0052] In specific implementation, the preparation method of the mixed microsphere reagent with different codes is:

[0053] The same volume of the first encoded microsphere reagent labeled with IL-6 antibody at a concentration of 0.05 mg / ml and the second encoded microsphere reagent labeled with cTnI antibody at a concentration of 0.05 mg / ml were added to the oscillator, mixed in the oscillator for 30 seconds, and then sonicated in a water bath for 30 seconds.

[0054] In a specific implementation, the average diameter of the microspheres in the microsphere reagent mixed with different codes is 1-5 um.

[0055] Use a syringe pump or super-hydrophilic surface self-drive to inject 10-100ul of microsphere mixture and slowly inject it into the chip at a flow rate of 2-10ul / min. The microspheres enter the capture area and are fixed by specific microgrooves to form a well-dispersed array arrangement. Figure 2 shown.

[0056] Set the chamber temperature to 35-39°C for incubation, select the two-step sample addition method, pass 10-50ul of the mixed antigen to be tested at a flow rate of 2-10ul / min, and then pass the fluorescent substance labeled with IL-6 antibody and cTnI antibody at an equal volume concentration of 0.01-0.5mg / ml at a speed of 2-10ul / min. Pass 20-100ul of cleaning solution at a speed of 4-15ul / min to wash away unreacted antigens and free fluorescent substances to reduce background signals.

[0057] The quantitative and qualitative images are processed to obtain the number and type of biological molecules in the biological sample to be tested. The different analytes in the biological sample to be tested are qualitatively and quantitatively detected by the color type of the fluorescence signal under near-infrared laser excitation and the fluorescence quantity and brightness value under visible light, and finally statistically analyzed by software. The number of luminescent microspheres is counted for low-abundance samples through the Poisson distribution probability formula, and the brightness of fluorescence is tested for high-concentration samples. The concentration of the indicator to be tested is obtained by establishing a standard curve.

[0058] from Figure 3 It can be seen that for the IL-6 project, the signal of 0pg / ml is less than 100, the lowest sensitivity is 100fg / ml, the detection range is 0-1000pg / ml, and it has good gradient responsiveness.

[0059] For the cTnI project, the 0pg / ml signal is less than 100, the lowest sensitivity is 500fg / ml, the detection range is 0-1000pg / ml, and it has good gradient responsiveness.

[0060] According to experiments, in-situ reactions can detect multiple indicators, and each item has good gradient reactivity and fg-level sensitivity.

[0061] This application can be designed for different usage scenarios according to different immune projects and surface treatment methods, and can adjust the immune response time and control the temperature to allow the immune response to proceed fully, reduce background signals, and improve detection responsiveness.

[0062] Based on the imaging system of multi-color upconversion fluorescence-encoded microspheres and multiple detection methods, 5-7 target molecule detections can be achieved simultaneously on the chip.

[0063] The equipment required for this application occupies a small area and is easy to move and carry. Chips, liquid inlet devices, and fluorescence microscopes can be selected as integrated machines for automated clinical testing, or split machines can be used for scientific research.

[0064] The present application also provides a single molecule chip for use in the above-mentioned single molecule immunoassay method, which comprises:

[0065] A sample adding area, which is used to add microsphere reagents mixed with different codes into the single molecule chip and then enter the microsphere capture area;

[0066] Microsphere capture area: After the microsphere reagents mixed with different codes enter the microsphere capture area, the microspheres are captured and fixed by the microsphere slots on the side of the main flow channel during the flow process, and the remaining reagents flow into the waste liquid collection area through the main flow channel and the secondary flow channel;

[0067] The waste liquid collection area is used to collect the liquid flowing out of the microsphere capture area.

[0068] In a specific implementation, the microsphere capture area is provided with a plurality of main flow channels and secondary flow channels connected to the main flow channels;

[0069] The height of the main channel is 2 to 3 times the diameter of the microsphere, and the width of the main channel is 4 to 5 times the diameter of the microsphere;

[0070] At least one of the width or height of the secondary flow channel is smaller than the diameter of the microsphere, thereby ensuring that the microsphere cannot pass through the secondary flow channel;

[0071] At least one microball groove is provided at one end of the secondary flow channel, and the diameter of the microball groove is close to or slightly larger than the diameter of the microball, so that when the reagent flows along the secondary flow channel, the microball can enter the microball groove at a certain speed and block the microball groove.

[0072] The height of the main channel can be 4-10um, and the optimal value is 5um; the width of the main channel can be 5-30um, and the optimal value is 13um; the width of the secondary channel is 1-3um, and the optimal value is 2um; the diameter of the microball groove at one end of the secondary channel is 3-5um, and the optimal value is 4um.

[0073] The single-molecule chip is divided into a cover plate and a base plate. The cover plate is processed with sample addition holes, and the base plate is processed with sample addition area, microsphere capture area, waste liquid collection area and other structures. Of course, a liquid distribution area can also be set. The diameter of the sample addition hole can be 0.5-2mm, and the optimal is 1mm; the size of the microsphere capture area of ​​the chip is 5.5mm*6.5mm; there are 220,000 microsphere slots in the entire area; the waste liquid area can accommodate 200-500ul of waste liquid.

[0074] The cover plate and the base plate are sealed together by using methods such as heat pressing, ultrasonic bonding, gluing, laser bonding, and plasma bonding to form a well-sealed microfluidic structure.

[0075] The cover and base can be processed into PDMS, PS, PMMA, glass, quartz, or other materials by photolithography, etching, nanoimprinting, injection molding, mold replication, vacuum hot pressing, etc. Figure 4 The overall structure of the chip is shown.

[0076] The microsphere capture area can be processed into PDMS, PS, PMMA, glass, quartz, or other materials by photolithography, etching, nanoimprinting, injection molding, overmolding, vacuum hot pressing, etc.

[0077] The single-molecule chip is based on a microsphere array capture chip. By introducing secondary flow / lateral flow factors into the microchannel, the microspheres are fixed in a specific arrayed microgroove structure, thereby improving the microsphere capture rate and capture density.

[0078] Appropriate surface treatment of the chip flow channel provides interfaces with different hydrophilicities, which can achieve self-driven detection with the help of capillary action; at the same time, it reduces nonspecific adsorption and drops the background value to below 100, further improving sensitivity.

[0079] This application uses MEMS micromachining technology and a matching surface treatment process to process a microfluidic chip with a specific structure. During the detection process, microspheres (slightly larger than the capture amount) are added to the chip in sequence, so that the microspheres are distributed into the array microwells of the microfluidic channel, and then the antigens to be tested and fluorescent molecules are added to achieve a single-molecule detection sensitivity of fg / ml level.

[0080] In summary, the present application provides a single-molecule immunoassay method and a single-molecule chip, which include the following steps: slowly adding a microsphere reagent mixed with different codes into the chip, the microspheres are captured and fixed by the microsphere slots on the side of the main channel during the flow of the microspheres in the chip, and the remaining reagents are collected into the waste liquid collection area after passing through the main channel and the secondary channel; the antigen to be tested and the mixed fluorescent substance with different detection items are introduced into the chip, and then the cleaning solution is introduced to wash away the excess antigen and the free mixed fluorescent substance; the number of luminescent microspheres and the fluorescence brightness are counted by the Poisson distribution probability formula, and a standard curve is established to obtain the concentration of the sample to be tested, so that the microspheres are captured and fixed by the microsphere slots to the maximum extent, the loss of microspheres is low, the immune reaction of the antigen to be tested is fully carried out when it is introduced into the chip, the detection sensitivity is high, the detection operation process is highly simplified, the detection cost is reduced, the detection efficiency is improved, and multiple target molecule detection can be achieved simultaneously.

[0081] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0082] The above-described embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be construed as limiting the scope of the patent application. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent application shall be subject to the attached claims.

Claims

1. A single molecule immunoassay method, characterized in that: The steps include: The microsphere reagents mixed with different codes are slowly added into the chip. The microspheres are captured and fixed by the microsphere slots on the side of the main channel during the flow in the chip. The remaining reagents are collected into the waste liquid collection area after passing through the main channel and the secondary channel. The antigen to be tested and mixed fluorescent substances with different detection items are introduced into the chip, and then a cleaning solution is introduced to wash away the excess antigen and free mixed fluorescent substances; The number of luminescent microspheres and the fluorescence brightness were counted using the Poisson distribution probability formula, and a standard curve was established to obtain the concentration of the sample to be tested.

2. The single molecule immunoassay method according to claim 1, characterized in that: The method of introducing the antigen to be tested and the mixed fluorescent substances with different detection items into the chip, and then introducing a cleaning solution to wash away the excess antigen and the free mixed fluorescent substances comprises: After incubating and combining 10-50ul of the antigen to be tested and 5-20ul of a mixed fluorescent substance with a concentration of 0.001-0.5mg / ml containing different detection items, the mixture is passed into the chip at a flow rate of 3-15ul / min, and then 20-100ul of cleaning solution is passed to wash away the excess antigen and free mixed fluorescent substance.

3. The single molecule immunoassay method according to claim 1, characterized in that: The method of introducing the antigen to be tested and the mixed fluorescent substances with different detection items into the chip, and then introducing a cleaning solution to wash away the excess antigen and the free mixed fluorescent substances comprises: 10-50ul of the antigen to be tested is introduced into the chip at a flow rate of 3-15ul / min, followed by 5-20ul of a mixed fluorescent substance with a concentration of 0.01-0.5mg / ml containing different test items, and finally 20-100ul of cleaning solution is introduced to wash away excess antigen and free mixed fluorescent substance.

4. The single molecule immunoassay method according to claim 1, characterized in that: The incubation temperature of the chip is 35-39°C.

5. The single molecule immunoassay method according to claim 1, characterized in that: The microsphere reagent is added into the chip via a syringe pump or a constant pressure pump; Or it can be driven into the chip by itself due to the super hydrophilic surface of the chip.

6. The single molecule immunoassay method according to claim 5, characterized in that: The chip is surface treated by physical adsorption or hydrophilic reagent spraying or coating or chemical vapor deposition.

7. The single molecule immunoassay method according to claim 1, characterized in that: The preparation method of the microsphere reagent mixed with different codes is: The same volume of the first encoded microsphere reagent labeled with IL-6 antibody at a concentration of 0.05 mg / ml and the second encoded microsphere reagent labeled with cTnI antibody at a concentration of 0.05 mg / ml were added to the oscillator, mixed in the oscillator for 30 seconds, and then sonicated in a water bath for 30 seconds.

8. The single molecule immunoassay method according to claim 7, characterized in that: The average diameter of the microspheres in the microsphere reagent mixed with different codes is 1-5 um.

9. A single molecule chip, used in the single molecule immunoassay method according to any one of claims 1 to 8, characterized in that: include: A sample adding area, which is used to add microsphere reagents mixed with different codes into the single molecule chip and then enter the microsphere capture area; Microsphere capture area: After the microsphere reagents mixed with different codes enter the microsphere capture area, the microspheres are captured and fixed by the microsphere slots on the side of the main flow channel during the flow process, and the remaining reagents flow into the waste liquid collection area through the main flow channel and the secondary flow channel; The waste liquid collection area is used to collect the liquid flowing out of the microsphere capture area.

10. The single-molecule chip according to claim 9, characterized in that: The microsphere capture area is provided with a plurality of main flow channels and secondary flow channels connected to the main flow channels; The height of the main channel is 2-3 times the diameter of the microsphere, and the width of the main channel is 4-5 times the diameter of the microsphere; At least one of the width or height of the secondary flow channel is smaller than the diameter of the microsphere, thereby ensuring that the microsphere cannot pass through the secondary flow channel; At least one microball groove is provided at one end of the secondary flow channel, and the diameter of the microball groove is close to or slightly larger than the diameter of the microball, so that when the reagent flows along the secondary flow channel, the microball can enter the microball groove at a certain speed and block the microball groove.

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