A digital biosensing method based on particle floatation and sinking mechanism

Abstract

The application discloses a digital biosensing method based on a particle floatation and sinking mechanism and belongs to the technical field of biomarker detection. The method comprises the following steps: loading specific capture elements of a to-be-detected biomarker on the surface of microspheres to obtain microspheres loaded with the capture elements; mixing the microspheres loaded with the capture elements, the to-be-detected biomarker and nano-sedimentation particles, and then performing reaction, so that each microsphere surface is loaded with at most one to-be-detected biomarker, and nano-sedimentation particles are combined to the surface of the microspheres to obtain the microspheres after reaction; adjusting the density of a sedimentation solution, mixing the microspheres after reaction with the sedimentation solution and then standing, so that the microspheres loaded with the to-be-detected biomarker are sedimented to the bottom of the solution, and the microspheres without the to-be-detected biomarker float on the uppermost layer of the liquid surface; and counting the microspheres sedimented to the bottom of the solution, so that digital analysis of the to-be-detected biomarker is realized. The method can realize rapid bedside diagnosis and on-site instant detection.

Description

Technical Field

[0001] This invention belongs to the field of biomarker detection technology, specifically relating to a digital biosensing method based on particle buoyancy and sedimentation mechanism. Background Technology

[0002] The accurate assessment of biomarkers in body fluids (such as proteins, nucleic acids, enzymes, extracellular vesicles, and small biological molecules) has become a key approach for disease diagnosis, prognosis, and personalized treatment in the field of liquid biopsy. However, in the early stages of disease development, the concentration of relevant biomarkers in body fluids is as low as aM to fM, while the detection limits of existing conventional analytical methods are usually at the pM level. Moreover, the weak signals induced by target biomarker molecules in conventional detection methods are dispersed and diluted in the overall solution, which greatly limits the sensitivity and accuracy of detecting ultra-low abundance biomarkers. Therefore, developing sensitive and efficient new sensing strategies for ultra-high sensitivity or even single-molecule level biomarker detection remains an urgent need in the field of early disease diagnosis based on liquid biopsy.

[0003] Unlike traditional analytical methods that rely on averaging the overall sample signal, digital analytical methods offer unique advantages and are the most effective way to solve the aforementioned problems. Based on the Poisson distribution, digital analytical methods confine the reaction initiated by a single target analyte to tiny reaction units ranging from fL to nL. By analyzing the negative and positive signals (0 or 1) of each independent unit using binary counting, digital detection of the target analyte is achieved. Therefore, digital analytical methods can concentrate and enrich the signal initiated by the target analyte molecule within tiny, independent units, enabling highly sensitive analysis of the target analyte at the single-molecule level. Currently, microemulsions and micropores are the most commonly used independent reaction units in digital analytical methods; however, they rely on sophisticated microfabrication techniques or complex microfluidic equipment for large-scale preparation, significantly increasing operational difficulty and cost. Furthermore, conventional digital analytical methods require the introduction of enzyme catalysis or nucleic acid amplification reactions to achieve clear distinction between negative and positive signals, making it difficult to extend to rapid point-of-care diagnostics and real-time on-site detection. Summary of the Invention

[0004] The purpose of this invention is to provide a digital biosensing method based on particle buoyancy and sedimentation mechanism to solve the technical problem that existing detection methods are difficult to extend to rapid bedside diagnosis and on-site real-time detection.

[0005] To achieve the above objectives, the present invention employs the following technical solution: This invention discloses a digital biosensing method based on particle buoyancy and sinking mechanism, comprising the following steps: Step 1: Load specific capture elements of the biomarker to be tested onto the surface of the microspheres to obtain microspheres loaded with capture elements; Step 2: Mix the microspheres loaded with the capture element, the biomarker to be detected, and the nano-precipitation particles and then react them so that each microsphere surface is loaded with a maximum of one biomarker to be detected, and induce the nano-precipitation particles to bind to the surface of the microspheres to obtain the reacted microspheres. Step 3: Adjust the density of the sedimentation solution. After mixing the reacted microspheres with the sedimentation solution and letting them stand, the microspheres loaded with the biomarkers to be tested will settle to the bottom of the solution, while the microspheres without the biomarkers to be tested will float on the top layer of the liquid. Step 4: Count the microspheres that have settled to the bottom of the solution to achieve digital analysis of the biomarker to be tested.

[0006] Furthermore, in step 1, the density of the microsphere material ranges from 1.0 to 1.2 g / cm³. 3 .

[0007] Further, in step 1, the microsphere material is any one of polystyrene, polyethylene, polypropylene, polyvinyl alcohol, polyvinyl chloride, polylactic acid, polymethyl methacrylate, polyethylene terephthalate, and sodium alginate.

[0008] Further, in step 1, the specific capture element is any one of the following: a specific antibody and aptamer against an antigen, a specific antibody and aptamer against extracellular vesicles, a specific antibody and aptamer against small biological molecules, a specific complementary nucleic acid probe against nucleic acids, and a specific catalytic substrate against enzymes.

[0009] Furthermore, in step 1, a specific trapping element is added so that the surface of the microsphere is fully loaded with the specific trapping element, thus obtaining a microsphere loaded with the trapping element.

[0010] Furthermore, in step 2, the nano-sedimented particles are either magnetic nanoparticles or high-density heavy nanoparticles; the density of the high-density heavy nanoparticles is ≥ 1.2 g / cm³. 3 .

[0011] In step 2, the ratio of the number of microspheres carrying the capture element to the number of biomarkers to be detected is ≥ 1:1, and the ratio of the number of nano-precipitation particles to the number of microspheres carrying the capture element is ≥ 5:1. Further, in step 3, the density of the sedimentation solution is 1.0~1.4 g / cm³. 3 .

[0012] Further, in step 3, the sedimentation solution is any one of iodixanol solution, iohexol solution, meglumine methylglucamine solution, sucrose solution, and cesium chloride solution.

[0013] Furthermore, in step 4, the method for counting the microspheres that have settled to the bottom of the solution is any one of flow cytometry, bright-field microscopy, and mobile phone photography.

[0014] Compared with the prior art, the present invention has the following beneficial effects: This invention discloses a digital biosensing method based on particle buoyancy and sedimentation. This method uses microspheres instead of micropores and microemulsions as a single reaction unit. Compared with micropores that require precision microfabrication technology and microemulsions that are prone to demulsification, various surface-functionalized polymer microspheres are easy to synthesize or commercially available, have high stability, and enhance the universality of the method. Furthermore, by adjusting the density of the sedimentation solution, a critical buoyancy and sedimentation system of microspheres is constructed, eliminating the need for any complex signal amplification methods, greatly simplifying the experimental steps, and enabling rapid point-of-care diagnosis and on-site instant detection in a minimally simplistic "mix and test" mode. This solves the technical problem that existing digital detection methods are difficult to extend to rapid point-of-care diagnosis and on-site instant detection.

[0015] Furthermore, the method of the present invention uses conventional flow cytometers, bright-field microscopes, and more portable and convenient mobile phone photography as counting means, so the invention can be openly expanded and applied in clinical diagnosis and general biochemical laboratories.

[0016] Furthermore, the method of this invention is compatible with a variety of biomarker affinity systems (immune response, nucleic acid hybridization, enzyme catalysis), and can be extended to the analysis of various biomarkers such as antigens, extracellular vesicles, small biological molecules, nucleic acids, and enzymes, thus possessing high versatility. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the digital immunoassay based on the particle float-sinking differentiation mechanism in Embodiment 1 of the present invention; Wherein: a- Schematic diagram of digital immunoassay based on particle floating and settling mechanism; b- Bright field microscopy image of microspheres settling at the bottom of the solution in the presence of different concentrations of prostate-specific antigen (PSA) molecules. Figure 2 This is a linear relationship graph between the concentration of antigen molecules and the number of microspheres that settle at the bottom of the solution in Example 1 of the present invention; Figure 3 This is a schematic diagram of digital enzyme analysis based on the particle float-sinking differentiation mechanism in Embodiment 2 of the present invention; Wherein: a- Schematic diagram of digital enzyme analysis based on particle floating and settling mechanism; b- Bright field microscopy image of microspheres settling at the bottom of the solution in the presence of different concentrations of T4 polynucleotide kinase phosphatase (T4 PNK) molecules. Figure 4 This is a linear relationship graph between the concentration of T4 PNK molecules and the number of microspheres that settle at the bottom of the solution in Example 2 of the present invention. Detailed Implementation

[0018] To enable those skilled in the art to understand the features and effects of the present invention, the terms and expressions used in the specification and claims are explained and defined in general below. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in case of conflict, the definitions in this specification shall prevail.

[0019] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0020] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0021] In this article, unless otherwise specified, “contains,” “includes,” “containing,” “has,” or similar terms cover the meanings of “composed of” and “mainly composed of,” for example, “A contains a” covers the meanings of “A contains a and others” and “A contains only a.”

[0022] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0023] This invention innovatively proposes a novel digital biosensor system based on a single-molecule binding-driven, density-mediated particle buoyancy differentiation mechanism, which is revolutionary compared to conventional digital detection technologies. It overcomes the technical barriers of existing digital detection platforms that rely on complex operating systems and expensive consumables, such as closed microchamber processing and isolation, fluorescence signal amplification and readout. This system boasts significant advantages, including extremely simple operation and high-throughput detection capabilities in signal output mode, completely open instrumentation and reagents, low cost, and the ability to be fully expanded in ordinary clinical / biochemical laboratories. It provides a simple, flexible, and high-throughput digital single-molecule detection technology platform for early disease screening.

[0024] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0025] The following examples use instruments and equipment conventional in the art. Experimental methods in the following examples, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. All raw materials used in the following examples are conventional commercially available products with specifications conventional in the art. In this specification and the following examples, unless otherwise specified, "%" refers to weight percentage, "parts" refers to parts by weight, and "ratio" refers to weight proportion.

[0026] This invention establishes a novel digital analysis method based on particle buoyancy and sedimentation mechanisms, achieving ultra-high sensitivity and instantaneous detection of multiple biomarkers. The technical solution of this patent is described in detail below with reference to specific embodiments and accompanying drawings. These embodiments do not constitute a limitation of this patent.

[0027] The method of this invention constructs a critical floating and sinking state of microspheres by controlling the solution density. That is, blank polymer microspheres float on the uppermost liquid surface of the dense solution, but the combination of nano-sedimentation particles triggered by a single biomarker molecule is sufficient to make the polymer microspheres sink to the bottom of the liquid surface. The polymer microspheres that have settled to the bottom of the liquid surface are counted using a simplified "reaction-on-demand" operation mode and conventional equipment, thereby realizing digital sensing of biomarker molecules.

[0028] Example 1 This embodiment is a digital immunoassay based on the particle buoyancy mechanism. Taking the detection of PSA molecules as an example, the digital analysis method is as follows: As attached Figure 1As shown in Figure a, PSA-specific capture antibodies and detection antibodies are loaded onto microspheres and magnetic nanoparticles, respectively. When the target PSA molecule is present in the system, a sandwich-type immune complex of "capture antibody - antigen molecule - magnetic nanoparticles coupled with detection antibody" will form on the surface of the microspheres. During this process, by controlling the number of PSA molecules to be much less than the number of microspheres, the surface of most microspheres carries 0 or only 1 immune complex. After the reacted microspheres are mixed with a density solution, microspheres that have not undergone an immune reaction will float completely on the surface of the liquid (negative), while microspheres loaded with a single immune complex will quickly settle to the bottom of the density solution under the action of a magnetic field (positive). Since each settled microsphere corresponds to a single-molecule immune binding event, when the total amount of microspheres used initially is known, the precise count of microspheres at the bottom of the density solution can be obtained by taking a wide-field bright-field photograph, thus obtaining quantitative information on the target PSA molecule. Specifically, it includes the following steps: Step 1: Take approximately 7.8 × 10 4 Carboxyl-modified polystyrene microspheres (ρ ~1.059 g / cm³) 3 After washing three times with 10 μL of 2-morpholine ethanesulfonic acid buffer (MES), 10 μL of a mixture containing 25 mg / mL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxythiosuccinimide (sulfo-NHS) was added to activate the carboxyl groups on the microspheres. After centrifugation, 100 ng of antigen-specific capture antibody was added to obtain capture antibody-loaded microspheres, and the unreacted activated carboxyl groups on the surface of the microspheres were blocked with tris(hydroxymethyl)aminomethane (Tris) or ethanolamine; simultaneously, approximately 3.9 × 10 7 Magnetic nanoparticles loaded with streptavidin (STV) are mixed with 100 ng of biotin-modified detection antibody to form detection antibody-loaded magnetic nanoparticles. Step 2: The antibody-loaded microspheres and the antibody-loaded magnetic nanoparticles from Step 1 were reacted with a series of PSA molecules in 10 μL of 1 × PBS buffer solution containing 1% BSA, so that an immune response triggered by a maximum of one PSA molecule occurred on each microsphere. Step 3: Dilute a 50% (mass-volume ratio) cesium chloride solution (ρ ~1.30 g / cm³) 3 ), to obtain cesium chloride with a critical density (ρ ~1.059 g / cm³), 3 ) sedimentation solution; Step 4: Mix the microspheres obtained in Step 2 with the cesium chloride solution obtained in Step 3, place the mixture in a 96-well plate, and place it on a magnet for 10 minutes. Then, use a bright-field microscope to count the microspheres that have settled to the bottom of the solution, thereby achieving digital quantitative analysis of PSA molecules in the sample. (See attached image) Figure 1 As shown in b, with the target PSA concentration (C) PSA With the increase of ), the number of microspheres (N) that settle to the bottom of the solution. observed The number of cases increases, and there is a good linear relationship (see appendix). Figure 2 ).

[0029] Example 2 This embodiment uses digital enzyme analysis based on particle buoyancy and sedimentation mechanisms, taking the detection of T4 PNK enzyme molecules as an example. The digital analysis method is as follows: T4 PNK exhibits unique single-particle surface-confined catalytic behavior for DNA substrates loaded on the surface of microspheres. That is, a single T4 PNK molecule can be spatially self-confined on the surface of a single microsphere, thereby stepwise catalyzing the substrate reaction without physically binding to the microsphere. Based on this, we applied the particle floating and settling mechanism to the digital analysis of T4 PNK molecules. Appendix Figure 3 This paper illustrates the basic principle of the digital enzyme analysis method based on particle floating and settling mechanism proposed in this invention. In the presence of biotin-modified adenosine triphosphate (ATP), a single T4 PNK molecule catalyzes the loading of γ-phosphate-biotin at the γ-position of ATP onto the 5' hydroxyl terminus of a nucleic acid probe on the microsphere surface, phosphorylating it and introducing a biotin group. Subsequently, it catalyzes the anchoring of adjacent nucleic acid probes on the same microsphere surface until all nucleic acid probes on the microsphere are consumed. In this way, a single T4 PNK molecule can induce the enrichment of a large number of biotin molecules on a single microsphere, which can then be recognized by STV-modified magnetic nanoparticles. Therefore, microspheres without T4 PNK molecules float on the top layer of the liquid, while microspheres that have undergone the T4 PNK catalytic reaction settle to the bottom. The counting of the settled microspheres using a bright-field microscope enables digital analysis of T4 PNK molecules. Specifically, it includes the following steps: Step 1: Take approximately 7.8 × 10 4 Carboxyl-modified polystyrene microspheres (ρ ~1.059 g / cm³) 3 After washing three times with 10 μL LMES buffer, 10 μL of a mixture containing 25 mg / mL EDC and sulfo-NHS was added to activate the carboxyl groups on the microspheres; after centrifugation, 10 pmol of a nucleic acid probe labeled with NH2 at the 3' end and OH at the 5' end was added to obtain microspheres loaded with 5'-OH, and the unreacted activated carboxyl groups on the surface of the microspheres were blocked. Step 2: The microspheres prepared in Step 1 were mixed with 0.2 mM biotin-modified ATP and a series of concentrations of T4 PNK molecules, and then phosphorylated at 37 °C to load a large number of biotin molecules onto the microspheres. Step 3: After centrifuging the biotin-loaded microspheres, add approximately 3.9 × 10⁻⁶ ppm. 7 Magnetic nanoparticles loaded with STV were reacted in 10 μL of 1 × PBS buffer at room temperature for 30 minutes. Step 4: Dilute a 50% cesium chloride solution (ρ ~ 1.30 g / cm³) 3 ), to obtain cesium chloride with a critical density (ρ ~1.059 g / cm³), 3 ) sedimentation solution; Step 5: Mix the microspheres obtained in Step 3 with the cesium chloride solution obtained in Step 4, place the mixture in a 96-well plate, and place it on a magnet for 10 minutes. Then, use a bright-field microscope to count the microspheres that have settled to the bottom of the solution, thereby achieving digital quantitative analysis of T4 PNK molecules in the sample. (See attached image) Figure 3 As shown in b, with the target T4 PNK concentration (C T4 PNK With the increase of ), the number of microspheres (N) that settle to the bottom of the solution. observed The number of cases increases, and there is a good linear relationship (see appendix). Figure 4 ).

[0030] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A digital biosensing method based on particle buoyancy and sinking mechanism, characterized in that, Includes the following steps: Step 1: Load specific capture elements of the biomarker to be tested onto the surface of the microspheres to obtain microspheres loaded with capture elements; Step 2: Mix the microspheres loaded with the capture element, the biomarker to be detected, and the nano-precipitation particles and then react them so that each microsphere surface is loaded with a maximum of one biomarker to be detected, and induce the nano-precipitation particles to bind to the surface of the microspheres to obtain the reacted microspheres. Step 3: Adjust the density of the sedimentation solution. After mixing the reacted microspheres with the sedimentation solution and letting them stand, the microspheres loaded with the biomarkers to be tested will settle to the bottom of the solution, while the microspheres without the biomarkers to be tested will float on the top layer of the liquid. Step 4: Count the microspheres that have settled to the bottom of the solution to achieve digital analysis of the biomarker to be tested.

2. The digital biosensing method based on particle buoyancy and sinking mechanism according to claim 1, characterized in that, In step 1, the density of the microsphere material ranges from 1.0 to 1.2 g / cm³. 3 .

3. The digital biosensing method based on particle buoyancy and sinking mechanism according to claim 1, characterized in that, In step 1, the microsphere material is any one of polystyrene, polyethylene, polypropylene, polyvinyl alcohol, polyvinyl chloride, polylactic acid, polymethyl methacrylate, polyethylene terephthalate, and sodium alginate.

4. The digital biosensing method based on particle buoyancy and sinking mechanism according to claim 1, characterized in that, In step 1, the specific capture element is any one of the following: a specific antibody and aptamer against an antigen, a specific antibody and aptamer against extracellular vesicles, a specific antibody and aptamer against small biological molecules, a specific complementary nucleic acid probe against nucleic acids, and a specific catalytic substrate against an enzyme.

5. The digital biosensing method based on particle buoyancy and sinking mechanism according to claim 1, characterized in that, In step 1, a specific trapping element is added so that the surface of the microsphere is fully loaded with the specific trapping element, thus obtaining a microsphere loaded with the trapping element.

6. A digital biosensing method based on particle buoyancy and sinking mechanism according to claim 1, characterized in that, In step 2, the nano-sedimented particles are either magnetic nanoparticles or high-density heavy nanoparticles; the density of the high-density heavy nanoparticles is ≥ 1.2 g / cm³. 3 .

7. A digital biosensing method based on particle buoyancy and sinking mechanism according to claim 1, characterized in that, In step 2, the ratio of the number of microspheres of the load-capturing element to the number of biomarkers to be detected is ≥ 1:1, and the ratio of the number of nano-precipitated particles to the number of microspheres of the load-capturing element is ≥ 5:

1.

8. A digital biosensing method based on particle buoyancy and sinking mechanism according to claim 1, characterized in that, In step 3, the density of the sedimentation solution is 1.0~1.4 g / cm³. 3 .

9. A digital biosensing method based on particle buoyancy and sinking mechanism according to claim 1, characterized in that, In step 3, the sedimentation solution is any one of iodixanol solution, iohexol solution, meglumine methyl methacrylate solution, sucrose solution, and cesium chloride solution.

10. A digital biosensing method based on particle buoyancy and sinking mechanism according to claim 1, characterized in that, In step 4, the method for counting the microspheres that have settled to the bottom of the solution is any one of flow cytometry, bright-field microscopy, and mobile phone photography.

Images