Single-cell immunoassay method based on quenched aptamer probe and application thereof

Abstract

The application discloses a single-cell immunoblotting method based on quenchable aptamer probes and relates to the technical field of single-cell protein detection. The method comprises the following steps: S1, a single-cell suspension sample is settled in a microwell array on a composite hydrogel chip; S2, the composite hydrogel chip is placed in an electrophoresis tank, then a lysis solution is slowly poured into the tank, and incubation lysis is carried out; S3, after cell lysis, an electric field is applied to both ends of the chip, and proteins enter a gel coating layer on the surface of the chip under the action of the electric field and start electrophoretic separation; S4, after gel electrophoresis is completed, the chip is placed under an ultraviolet lamp for exposure, and the gel is taken out after exposure is completed; S5, the gel is placed in an aptamer probe solution which is targeted to target proteins, and is soaked and dyed, and the fluorescence signal intensity of the target protein molecules is measured under a laser confocal fluorescence microscope. The application can improve the detection sensitivity of target proteins and has fast imaging speed.

Description

Technical Field

[0001] This invention relates to the field of single-cell protein detection technology, specifically to the field of single-cell protein detection technology for multi-target protein detection, and particularly to a single-cell immunoblotting method based on quenchable aptamer probes and its application. Background Technology

[0002] Single-cell immunoblotting is a single-cell protein detection technique proposed by Professor Amy Herr of UC Berkeley. The specific process is as follows: (1) Single-cell capture: The single-cell suspension settles into a single-cell capture unit with a diameter of tens of micrometers under gravity; (2) In-situ chemical lysis of cells: The cells are lysed in situ for 10 seconds using RIPA-like lysis electrophoresis buffer preheated to 55℃; (3) Gel electrophoresis: After cell lysis, an electric field of 40V / cm is applied to both ends of the microchip, and the proteins are separated by gel electrophoresis under the action of the electric field; (4) Protein fixation: After gel electrophoresis, the surface of the microchip is excited and irradiated, so that the protein molecules in the hydrogel coating protein bands and the monomer molecules in the hydrogel undergo an in-situ polymerization reaction; (5) Immunoblot protein analysis: The fixed target protein molecules are bound by a specific primary antibody, eluted, and then bound by a secondary antibody labeled with a fluorescent or luminescent group, and eluted; (6) Fluorescence imaging: The fluorescence signal intensity of the target protein molecules is measured under a laser confocal fluorescence microscope. Antibody probes with specificity to the target protein are used to detect single cells in the cells. Because of the integration of protein gel electrophoresis and antibody recognition, single-cell immunoblotting is less affected by antibody cross-reactivity. Therefore, single-cell immunoblotting can specifically detect proteins of different molecular weights in single cells. Furthermore, since the hydrogel chip contains thousands of cell traps, single-cell immunoblotting can simultaneously measure different expression levels in thousands of single cells on a single hydrogel microchip, enabling the study of cellular heterogeneity at both the population and individual cell scales.

[0003] For antibody-based single-cell immunoblotting, during antibody incubation, the large molecular weight of the antibody (typically 150-1000 kDa) creates significant hydrogel steric hindrance within the gel matrix, limiting primary antibody recognition and secondary antibody signal amplification. This restricts the detection of multiple target proteins in single-cell immunoblotting.

[0004] For antibody-based single-cell immunoblotting, the limitation of protein loss due to the unavoidable repeated antibody stripping steps in multi-target protein detection remains, making it difficult to detect medium- and low-abundance proteins at single-cell resolution. Therefore, the sensitivity of antibody probe-based single-cell immunoblotting is limited for the detection of multi-target proteins.

[0005] The detection of multi-target proteins in cells has been validated to play a crucial role in human pathological research. This presents new requirements for detection probes capable of detecting low-abundance multi-target proteins. Therefore, employing detection probes suitable for multi-target protein detection, achieving high resolution in multi-target protein separation, is of significant practical importance for the biological applications of single-cell immunoblotting technology.

[0006] Amy Herr et al. [Nature Methods 2014, 11(7), 749-755] reported a single-cell multi-target protein immunoblotting technique based on antibody stripping to enable the detection of multiple target proteins within a single cell. However, Amy Herr et al. [Scientific Reports 2019, 9(1)] demonstrated that up to 40% of the protein is lost during each round of antibody stripping in in situ immunoblotting, which significantly hinders the quantification and sensitivity of single-cell immunoblotting. This protein loss affects protein detection at single-cell resolution, especially for low-abundance proteins.

[0007] Amy Herr et al. [Analytical Chemistry 2014, 86(20), 10429-10436] reported an improved single-cell immunoblotting technique based on metal antibody probes. By using metal-labeled primary antibody probes for imaging instead of fluorescently labeled secondary antibodies, they were able to effectively detect low molecular weight proteins. This method eliminates the need for secondary antibody probe imaging, reducing the molecular weight of the imaging probe and minimizing the hydrogel steric hindrance effect of the primary and secondary antibody probes, thus improving the detection quality of antibody probes for low molecular weight proteins. This technique is expected to be used for the detection of multi-target proteins in the future. However, metal-labeled primary antibody probe imaging requires IMC technology, resulting in a complex detection process, slow detection speed, and complicated preparation methods and procedures for metal-labeled primary antibody probes.

[0008] Alex et al. [Journal of the American Chemical Society 2022, 144(4), 1572-1579.] reported a color-changing fluorescent barcode technique based on a chain displacement reaction, achieving multiple labeling through a simple and small nucleic acid structure design. The color-changing fluorescent barcode complex consists of several oligonucleotides, and its color sequence can be easily further extended. The color-changing fluorescent barcode technique is simple and time-saving to operate because an irreversible color-changing reaction occurs simply by adding complementary oligonucleotides. This method can label multiple targets by attaching color-changing fluorescent barcode tags to polystyrene beads. In addition, when the color-changing fluorescent barcode is attached to an antibody, it can be used to simultaneously detect intracellular proteins. However, the preparation method and process of the color-changing fluorescent barcode tags are complex, and the color-changing fluorescent barcode tags need to be labeled on the detection probe to achieve the color-changing reaction.

[0009] Therefore, those skilled in the art are dedicated to developing a single-cell immunoblotting method and its applications that can improve the detection sensitivity of target proteins, accelerate imaging, save experimental time, and reduce manpower and time costs. Summary of the Invention

[0010] In view of the above-mentioned deficiencies of the prior art, the technical problem to be solved by the present invention is to provide a single-cell immunoblotting method based on quenchable aptamer probes and its application, thereby solving the problems of low detection sensitivity, slow imaging speed and long experimental time in the prior art.

[0011] To achieve the above technical objectives, the present invention provides a single-cell immunoblotting method based on quenchable aptamer probes, comprising the following steps:

[0012] S1 settles single-cell suspension samples into a microporous array on a composite hydrogel chip;

[0013] S2. Place the composite hydrogel chip into the electrophoresis tank, then slowly pour in the lysis buffer and incubate for lysis.

[0014] After S3 cells were lysed, an electric field was applied to both ends of the chip, and the proteins entered the gel coating on the chip surface under the action of the electric field and began to be separated by electrophoresis.

[0015] After S4 gel electrophoresis, the chip is exposed to ultraviolet light. After exposure, the gel is removed.

[0016] S5 involves placing the gel in an aptamer probe solution that targets and binds to the target protein, immersing and staining it, and then measuring the fluorescence signal intensity of the target protein molecule under a laser confocal fluorescence microscope.

[0017] In a preferred embodiment of the present invention, the single-cell immunoblotting method based on quenchable aptamer probes further includes step S6: placing the gel in a quenching probe solution for quenching, and measuring the fluorescence signal intensity of the target protein molecule under a laser confocal fluorescence microscope, wherein the quenching probe is the complementary strand of the aptamer probe.

[0018] In another preferred embodiment of the present invention, the lysis buffer is a RIPA-like lysis buffer, wherein the final concentration of the RIPA-like lysis buffer is a mixture of 0.5% SDS, 0.1% v / v Triton X-100, 0.25% sodium deoxycholate, 12.5 mM Tris, and 96 mM glycine, with a pH of 8.3.

[0019] In a preferred embodiment of the present invention, in step S2, the pyrolysis solution is preheated to 50-55°C in a water bath.

[0020] In a preferred embodiment of the present invention, in step S3, the protein is separated by electrophoresis for 30 seconds under a voltage of 200V.

[0021] In a preferred embodiment of the present invention, in step S4, the chip is exposed to ultraviolet light for 45 seconds.

[0022] In a preferred embodiment of the present invention, in step S5, the aptamer probe is selected from any one of DNA or RNA labeled with a coupled fluorescent or luminescent group, a metal group, a quantum dot group, an enzyme, colloidal gold, or superparamagnetic microspheres. The molecular weight of the aptamer probe is 1-15 kDa, the incubation concentration of the aptamer probe is 2-100 nM, and the soaking and staining time is 5-120 min.

[0023] In a preferred embodiment of the present invention, in step S6, the quenching probe is selected from DNA or RNA of any one of coupled halide ions, heavy metal ions, oxidizing organic compounds, and oxygen quenching agents. The quenching probe has a length of 7 bp, a concentration of 20-1000 nM, and a quenching time of 1-120 min.

[0024] Among them, the oxidizing organic compounds are selected from any one or more of nitro compounds, diazo compounds, carbonyl compounds and hydroxy compounds.

[0025] This invention also discloses the application of the single-cell immunoblotting method based on quenchable aptamer probes in microfluidic protein immunoblotting, single-cell protein immunoblotting, capillary electrophoresis protein immunoblotting, DNA-protein immunoblotting, and RNA-protein immunoblotting.

[0026] The present invention also discloses the application of the single-cell immunoblotting method based on quenchable aptamer probes in the detection or determination of single-cell or multi-cell proteins or extracted proteins or purified protein solutions or one or more protein analytes.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1. This invention uses aptamer probes to detect target proteins, which have a good ability to specifically identify target proteins and have higher epitope binding density and better epitope recognition ability, thereby improving the detection sensitivity of target proteins.

[0029] 2. Small-volume aptamer probes facilitate diffusion within hydrogel systems. Compared to antibody probes with a molecular weight of 150-1000 kDa, aptamer probes typically have a molecular weight of 1-15 kDa. Therefore, aptamer probes exhibit less steric hindrance in hydrogel systems. Furthermore, probe incubation times are shorter, imaging speed is faster, experimental time is saved, and labor costs are reduced.

[0030] 3. Small-volume aptamer probes are easier to wash away excess probes, reducing background fluorescence.

[0031] 4. Aptamer probes are easy to synthesize and inexpensive to synthesize; they are easy to modify; and they have low immunogenicity.

[0032] 5. The quenching probe has a fast quenching speed. The complementary chain of the detection probe is only 7bp in length, which results in a shorter probe quenching time, faster imaging speed, saves experimental time, and reduces labor and time costs.

[0033] 6. Quenching probes do not damage the protein stripping process, are suitable for multiple rounds of staining, and are easy to synthesize and modify, and are inexpensive.

[0034] 7. The small pore size of the gel at room temperature ensures the separation resolution of low molecular weight proteins.

[0035] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0036] Figure 1 This is a schematic diagram illustrating the principle of aptamer probe binding to protein in this application.

[0037] Figure 2 This is a schematic diagram illustrating the fluorescence quenching principle of the aptamer probe in this application.

[0038] Figure 3 A schematic diagram illustrating the principle of single-cell immunoblotting technology based on quenchable aptamer probes provided in this application.

[0039] Figure 4 This describes the fluorescence quenching kinetics of the quenchable aptamer probe in this application.

[0040] Figure 5 This is a comparison of the results of detecting CA125 protein in tumor cells using single-cell immunoblotting technology based on quenchable aptamer probes in this application. Detailed Implementation

[0041] The following description, with reference to the accompanying drawings, illustrates several preferred embodiments of the present invention to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0042] In the accompanying drawings, components with the same structure are indicated by the same numerical designation, and components with similar structures or functions are indicated by similar numerical designations. The dimensions and thicknesses of each component shown in the drawings are arbitrary, and the present invention does not limit the dimensions and thicknesses of each component. To make the illustrations clearer, the thickness of some components has been appropriately exaggerated in the drawings.

[0043] Example 1

[0044] Detection of fluorescence quenching kinetics of fluorescent aptamer probes in solution.

[0045] like Figure 1 The diagram shown illustrates the principle of aptamer probe binding to proteins in this application. A functional aptamer with a specific spatial conformation is formed through three-dimensional structural transformation of single-stranded DNA or RNA structures, enabling it to specifically bind to biomarkers.

[0046] like Figure 2 The diagram shown illustrates the fluorescence quenching principle of the aptamer probe in this application. An aptamer that specifically binds to a biomarker is coupled to a substance with a fluorescent group. Then, a quenching probe complementary to the aptamer is added. At this point, the quenching probe and the aptamer sequence are complementary, forming a double-stranded structure. The fluorescent group reacts with the quenching group, resulting in fluorescence quenching.

[0047] like Figure 4 As shown, the fluorescence kinetics after the binding of the quenching probe and the fluorescent aptamer probe were detected using a fluorescence spectrophotometer. Quenching of the fluorophore was observed after quenching the chain in the aptamer probe solution. This indicates that the desired partial hybridization double strands were formed between the quenching probe and the fluorescent aptamer probe.

[0048] Example 2

[0049] This invention provides a single-cell immunoblotting method based on quenchable aptamer probes, such as... Figure 3As shown, it includes the following steps:

[0050] S1 settles single-cell suspension samples into a microporous array on a composite hydrogel chip;

[0051] S2 The composite hydrogel chip was placed in the electrophoresis tank, and then RIPA-like lysis buffer was slowly poured in and incubated for lysis. The final concentration of the RIPA-like lysis buffer was a mixture of 0.5% SDS, 0.1% v / v Triton X-100, 0.25% sodium deoxycholate, 12.5 mM Tris, and 96 mM glycine, with a pH of 8.3.

[0052] After S3 cells were lysed, an electric field was applied to both ends of the chip, and the proteins entered the gel coating on the chip surface under the action of the electric field and began to be separated by electrophoresis.

[0053] After S4 gel electrophoresis, the chip is exposed to ultraviolet light. After exposure, the gel is removed.

[0054] S5 involves placing the gel in an aptamer probe solution that targets and binds to the target protein, immersing and staining it, and then measuring the fluorescence signal intensity of the target protein molecule under a laser confocal fluorescence microscope.

[0055] S6 places the gel in a quenching probe solution for quenching, and then measures the fluorescence signal intensity of the target protein molecule under a laser confocal fluorescence microscope. The quenching probe is the complementary strand of the aptamer probe.

[0056] In some embodiments, the aptamer probe is selected from any one of the following DNA or RNA labeled with a fluorescent or luminescent group, a metal group, a quantum dot group, an enzyme, colloidal gold, or a superparamagnetic microsphere. The molecular weight of the aptamer probe is 1-15 kDa, the incubation concentration of the aptamer probe is 2-100 nM, and the soaking and staining time is 5-120 min.

[0057] In some embodiments, the quenching probe is selected from DNA or RNA that is coupled with any one of halide ions, heavy metal ions, oxidizing organic compounds, or oxygen quenchers. The quenching probe has a length of 7 bp, a concentration of 20-1000 nM, and a quenching time of 1-120 min.

[0058] Among them, the oxidizing organic compounds are selected from any one or more of nitro compounds, diazo compounds, carbonyl compounds and hydroxy compounds.

[0059] Test case

[0060] The following section details the detection of CA125 protein in tumor cells using single-cell immunoblotting based on quenchable aptamer probes. The results are as follows: Figure 5 As shown, the main steps include the following:

[0061] 1. Preparation of composite hydrogels.

[0062] 2. Prepare RIPA-like lysis buffer (Radio immune-precipitation assay lysis buffer, also known as electrophoresis buffer). 0.5% SDS, 0.1% v / v Triton X-100, 0.25% sodium deoxycholate, 12.5 mM Tris, 96 mM glycine, pH 8.3. Store at 4°C.

[0063] 3. Heat the lysis buffer / electrophoresis buffer to 50-55 degrees Celsius in a water bath. Turn on the UV light in advance to stabilize the light source.

[0064] 4. Place the chip in a new dish with the gel side facing up, add HeLa cells, and wait for the cells to settle and fall into the cell trap. Let stand for 10 minutes.

[0065] 5. Place the gel in the electrophoresis tank and gently and quickly pour in 10 ml of RIPA-like lysis buffer preheated to 55 degrees Celsius from one corner of the tank.

[0066] 6. Immediately turn on the power supply, 200V (E=40v / cm2), and separate the proteins by electrophoresis for 30s.

[0067] 7. Immediately stop the voltage and begin UV exposure. Set the exposure time to 45 seconds.

[0068] 8. Once exposure is complete, remove the adhesive.

[0069] 9. Place the gel in TBST and shake for 30 minutes, changing the solution every 10 minutes.

[0070] 10. Soak and stain in aptamer probe solution (20 nM) for 5 min.

[0071] 11. Observe the glue under a confocal microscope, and the results are shown in the attached figure. Figure 5 As shown, this demonstrates that single-cell immunoblotting based on quenchable aptamer probes can achieve strong detection of proteins within a relatively short time (5 minutes).

[0072] 12. Immerse the probe in quenching solution (200 nM) for 10 min to quench it.

[0073] 13. Observe the glue under a confocal microscope, and the results are shown in the attached figure. Figure 5As shown, this demonstrates that single-cell immunoblotting based on quenchable aptamer probes can quench intragel fluorescence in a relatively short time (10 min).

[0074] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. The application of a single-cell immunoblotting method based on quenchable aptamer probes in the preparation of reagents for detecting or determining one or more protein analytes, wherein the single-cell immunoblotting method based on quenchable aptamer probes includes the following steps: S1 settles single-cell suspension samples into a microporous array on a composite hydrogel chip; S2. Place the composite hydrogel chip into the electrophoresis tank, then slowly pour in the lysis buffer and incubate for lysis. After S3 cells were lysed, an electric field was applied to both ends of the chip, and the proteins entered the gel coating on the chip surface under the action of the electric field and began to be separated by electrophoresis. After S4 gel electrophoresis, the chip is exposed to ultraviolet light. After exposure, the gel is removed. S5 involves placing the gel in an aptamer probe solution that targets and binds to the target protein, immersing and staining it, and then measuring the fluorescence signal intensity of the target protein molecule under a laser confocal fluorescence microscope. S6. The gel is placed in the quenching probe solution and immersed to quench the probe. The fluorescence signal intensity of the target protein molecule is measured under a laser confocal fluorescence microscope. The quenching probe is the complementary strand of the aptamer probe. In step S5, the aptamer probe is selected from DNA or RNA with any one of the following: coupled fluorescent or luminescent groups or quantum dot groups. The molecular weight of the aptamer probe is 1-15 kDa, the incubation concentration of the aptamer probe is 2-100 nM, and the soaking and staining time is 5-120 min. In step S6, the quenching probe is selected from DNA or RNA that is coupled with any one of the following: halide ions, heavy metal ions, oxidizing organic compounds, or oxygen molecule quenchers. The quenching probe is 7 bp in length, has a concentration of 20-1000 nM, and quenches for 1-120 min.

2. The application as described in claim 1, characterized in that: In step S2, the lysis buffer is a RIPA-like lysis buffer, and the final concentration of the RIPA-like lysis buffer is a mixture of 0.5% SDS, 0.1% v / v Triton X-100, 0.25% sodium deoxycholate, 12.5 mM Tris, and 96 mM glycine, with a pH of 8.

3.

3. The application as described in claim 1, characterized in that: In step S2, the lysis solution is preheated to 50-55°C in a water bath.

4. The application as described in claim 1, characterized in that: In step S3, the proteins are separated by electrophoresis at 200V for 30s.

5. The application as described in claim 1, characterized in that: In step S4, the chip is exposed to ultraviolet light for 45 seconds.

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