Preparation method of novel suspension array bar code microspheres of mass spectrometry flow cytometry

Abstract

The invention discloses a preparation method of novel suspension array bar code microspheres for a mass spectrometry flow cytometry, and relates to the technical field of nano biology. Various metal nanoparticles (such as gold, silver, platinum and rare earth metal nanoparticles) are controllably loaded on the surface of the magnetic microsphere through a streptavidin-biotin specific coupling technology, so that the mass spectrum coding magnetic microsphere with high resolution and high specificity in a mass spectrum flow cytometry is prepared. According to the method, the coding capacity of the suspension array is remarkably expanded by regulating and controlling the types and the load concentration of the metal nanoparticles, generation of more than millions of barcodes can be supported, and the upper limit of the capacity of the barcodes of the suspension array is broken through. The bar code microsphere prepared by the invention has the characteristics of high throughput, high resolution, high specificity and the like, provides an innovative technical support for high-throughput screening of complex biomarkers and multi-component analysis in precision medicine, and has a wide clinical application prospect.

Description

Technical Field

[0001] The present invention relates to the field of nanobiotechnology, and in particular to a method for encoding suspended array microspheres for mass spectrometry flow cytometers. Mass spectrometry encoding is achieved by controllable loading of metal nanoparticles on the surface of magnetic microspheres, and the obtained barcode magnetic microspheres have broad application prospects in high-throughput detection fields such as clinical analysis. Background Art

[0002] Suspension array is an efficient micro-quantitative multifunctional analysis platform with the advantages of high detection density and high throughput. It is widely used in genomics, proteomics and other fields. The core technology of suspension array is to achieve highly selective signal decoding by combining coded microcarriers with multiple signal detection platforms. However, traditional optical encoding strategies are limited by problems such as spectral overlap and energy transfer of luminophores, and it is difficult to break through the barcode capacity (the current reported barcode capacity is limited to about 1000 types). In recent years, mass spectrometry encoding methods based on inorganic metal elements have become a new direction for suspension array research due to their high resolution and high selectivity in mass spectrometers (ICP-TOF-MS). Mass spectrometry flow cytometers can use more than 34 metal elements in the periodic table as coding tags, which greatly improves the flexibility and diversity of coding selection. However, the existing microsphere encoding method based on metal ion doping is limited by the volume of microspheres, and the number of barcode types is still limited. Therefore, developing breakthrough encoding technologies and improving the barcode capacity of suspension arrays is of great significance to meet the increasingly complex needs of biomarker screening and high-throughput detection. Summary of the invention

[0003] The present invention provides a metal nanoparticle loading technology based on streptavidin-biotin specific coupling for preparing suspended array barcode magnetic microspheres for mass spectrometry flow cytometers. By regulating the loading concentration and type of metal nanoparticles, this method can use all metal elements that can synthesize metal nanoparticles in the periodic table. In theory, a mass spectrometry flow coding library with a capacity of more than one million barcode microspheres can be constructed, significantly expanding the coding capacity of the suspended array, and providing new coding technology support for high-throughput screening and precise diagnosis of multi-component biomarkers.

[0004] The present invention provides a method for preparing a suspension array barcode microsphere for a mass spectrometry flow cytometer, the steps of which are as follows: (1) Synthesis and functional modification of metal nanoparticles: preparation of gold (AuNPs), silver (AgNPs), platinum (PtNPs) and rare earth metal nanoparticles and surface modification; (2) Biotinylation and streptavidin modification of magnetic microspheres: Optimizing the surface properties of magnetic microspheres and improving the binding efficiency with metal nanoparticles; (3) Load coding by specific binding of streptavidin-biotin: by controlling the type and loading concentration of metal nanoparticles, biofunctionalized barcoded magnetic microspheres are prepared; (4) Bioanalytical applications of encoded magnetic microspheres: High-throughput detection of multi-component biomarkers can be achieved using encoded microspheres.

[0005] This method achieves a significant expansion of barcode encoding through the flexible combination and controllable loading of multi-metal nanoparticles, and has the advantages of high throughput, high resolution, and high specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Figure 1 :Encoding principle of metal nanoparticles loaded magnetic microspheres;

[0007] Figure 2 :Characterization of encoding channels and mass spectrometry signal distribution of AuNPs and AgNPs loaded magnetic microspheres;

[0008] Figure 3 :TEM characterization and coding diagram of rare earth metal nanoparticle-loaded magnetic microspheres;

[0009] Figure 4 :Coding schematic and signal distribution of dual loading of AuNPs and AgNPs;

[0010] Figure 5 : Hierarchical loading coding results of metal nanoparticles;

[0011] Figure 6 :Application results of encoded magnetic microspheres in high-throughput immunoassay of 9 antigen markers. Specific embodiments

[0012] The present invention will be described in more detail below with the help of examples, the specific contents of which are as follows: The analysis method of the present invention is not limited to the following examples.

[0013] Synthesis of AuNPs, AgNPs, PtNPs and rare earth metal nanoparticles: (1) Synthesis of AuNPs: HAuCl4 (0.01%, w / v) and ultrapure water were mixed in a three-necked flask with vigorous magnetic stirring and kept boiling for 10 min. An appropriate volume of sodium citrate solution was added as a reducing agent to reduce HAuCl4 and prepare AuNPs of the target particle size. After continuous heating and reflux for about 20 min, the solution was slowly stirred and cooled to room temperature. The newly prepared 15 nm AuNPs were filtered through polyethersulfone (PES) and stored at 4 °C for future use. (2) Synthesis of AgNPs: (A) Stir a citrate (about 0.2%, w / v) aqueous solution in a three-necked flask at 70°C for 15 minutes; (B) Prepare a seed solution, add 1.7 mL of 1% (w / v) AgNO3 and 140.3 μL of 1% KBH4 and store the solution at 70°C for about 1 hour. After cooling, remove it for use; (C) Add 75 mL of ultrapure water and 2 mL of 1% citrate solution to a three-necked flask, heat to boiling and reflux for 15 minutes. Further add 10 mL of starting seeds and 1.7 mL of 1% (w / v) AgNO3, keep boiling and reflux for 1 hour, cool it, and remove it for use; (D) The required 34 nm AgNPs were prepared through the cyclic step (E). The freshly prepared AgNPs were filtered through polyethersulfone (PES) and stored at 4°C for use; (3) Synthesis of PtNPs: (A) Add appropriate amount of H2PtCl6 and ultrapure water to a three-necked flask, heat to boiling and reflux for 1 minute, and add citrate solution and potassium borate solution in turn for reduction; (B) After 10 minutes, cool to room temperature, remove the seed solution and set aside; (C) Add appropriate amount of ultrapure water, PtNPs seed solution, 1% H2PtCl6 and a mixed solution of citric acid and ascorbic acid to a three-necked flask, slowly heat to boiling while stirring, and keep reflux for 15 minutes; (D) After cooling to room temperature, remove the PtNPs and store at 4°C for later use; (4) Synthesis of rare earth metal nanoparticles: Taking the synthesis of NaYF4 nanoparticles as an example: (A) Mix an appropriate amount of 1 mmol of yttrium chloride hydrate and methanol solution in a three-necked flask, then add an appropriate amount of 90% oleic acid and 90% 1-octadecene, and stir evenly at room temperature; (B) Introduce argon (Ar) to exhaust the air in the system to form an anaerobic environment; (C) Slowly heat the mixed solution to 170°C with a heating jacket while stirring. After all the solvents evaporate, stabilize the temperature at about 160°C and keep it stable for 60 minutes; (D) Turn off the heating and slowly reduce the solution temperature to 50°C, while maintaining the Ar atmosphere; (E) Slowly add 4 mmol of ammonium fluoride, 2.5 mmol sodium hydroxide in methanol mixture, keep stirring; (F) slowly raise the temperature to 160℃ and keep it for 30 minutes, keep the Ar atmosphere continuously, until there is no obvious precipitation; (G) quickly raise the temperature to about 305℃ at a rate of 10℃ / min and keep it at this temperature for 90 minutes, keep the Ar atmosphere continuously; (H) turn off the heating and slowly reduce the temperature to room temperature, keep the Ar atmosphere continuously; (I) after the solution has cooled to room temperature, pour the product into a centrifuge tube; (J) centrifuge the solution for 10 minutes, remove the NaY F4 nanoparticles are completely precipitated to the bottom of the tube, the supernatant is discarded, and cyclohexane is added to re-dissolve; (K) The solution is centrifuged for 10 minutes to completely precipitate the NaYF4 nanoparticles to the bottom of the tube, the supernatant is discarded, and cyclohexane is added to re-dissolve; (L) The purification steps are repeated 2 to 3 times to ensure effective washing of the NaYF4 nanoparticles; (M) After the NaYF4 nanoparticles are precipitated to the bottom of the tube for the last time and the supernatant is discarded, an appropriate amount of cyclohexane is added to re-dissolve and concentrate the precipitate, and the sample is transferred to a brown glass bottle and stored at room temperature. The synthesis steps for the other seven rare earth nanoparticles are similar to the synthesis steps of the above-mentioned NaYF4 nanoparticles.

[0014] Biofunctionalization of AuNPs, AgNPs and rare earth metal nanoparticles: (1) Streptavidin functionalization of AuNPs: Take 1 mL of freshly prepared AuNPs, add an appropriate amount of boric acid buffer (pH 9.0) to adjust the pH of the solution to a weak alkaline state (pH about 8.4); add 20 μg of streptavidin by pipetting, and incubate at room temperature with slow shaking for 1 hour; further add PBS solution containing BSA to block the unoccupied sites on the surface of AuNPs; the streptavidin-functionalized AuNPs probe is subjected to refrigerated centrifugation at 9000 rpm, and the streptavidin-modified AuNPs colloid at the bottom is finally redissolved in PBS buffer containing 1% BSA and dispersed to 1 mL, and stored at 4°C for later use; (2) Streptavidin functionalization of AgNPs: Take 1 mL of freshly prepared AgNPs, remove the suspension by refrigerated centrifugation at an appropriate speed, and finally redissolve the bottom AgNPs colloid in 0.01 M boric acid buffered ultrapure water solution; add 20 μg of streptavidin under pipetting, and incubate slowly with shaking at room temperature for 1 hour; further add PBS solution containing BSA to block the unoccupied sites on the AgNPs surface; the streptavidin-functionalized AgNPs probe is subjected to refrigerated centrifugation at an appropriate speed, and the bottom colloid is finally redissolved in PBS buffer containing BSA and dispersed to 1 mL, and stored at 4°C for future use; (3) Biotin functionalization of rare earth metal nanoparticles: Take one equivalent of (4-amino-1-hydroxybutane-1,1-diyl) diphosphate in a 1 mL centrifuge tube and dissolve it in a weak alkaline buffer solution. Then add two equivalents of Biotin-PEG4-NHS ester for coupling to first prepare the biotin-diphosphate coupling agent (linker); pipette the linker solution into 10 mg of freshly weighed rare earth nanoparticles and incubate overnight at room temperature; the biotin-functionalized rare earth nanoparticle probe is purified by refrigerated centrifugation at an appropriate speed, and the bottom colloid is finally redissolved in MES buffer solution.

[0015] Biotinylation and streptavidin modification of magnetic microsphere surface: (1) Biotinylation modification of the surface of magnetic microspheres for surface loading of streptavidin-functionalized metal nanoparticles: The biotinylation modification scheme for the surface of carboxyl magnetic microspheres with a particle size of 1 μm is as follows: 200 μg of 1 μm magnetic microspheres were added to a 200 μL centrifuge tube; the carboxyl groups on the surface of the magnetic microspheres were activated with 150 mg / mL EDC and 25 mg / mL sulfo-NHS under weak acid conditions of pH 6.5; 10% BSA dissolved in MES buffer solution was fully mixed with the activated magnetic microspheres and coupled at room temperature for 12 hours; the BSA-labeled magnetic microspheres were blocked with 10% BSA for 30 minutes, and finally washed three times with 0.01 M boric acid buffer ultrapure water solution for use; an appropriate amount of Biotin-PEG4-NHS ester was added under the blowing of a pipette to biotinylate the amino groups in the BSA on the magnetic microspheres, and incubated slowly with shaking at room temperature for 6 hours; the biotinylated 1 μm magnetic microspheres were stained with PBS solution containing 1% BSA. The 1 μm magnetic microspheres were washed four times, and then the biotinylated 1 μm magnetic microspheres were washed four times with PBS solution containing 0.01% Tween 20 (0.01% PBST), and redissolved in PBS solution for later use; The biotinylation modification scheme for the surface of amino-modified magnetic microspheres with a particle size of 2.8 μm is as follows: add 200 μg of 2.8 μm magnetic microspheres to a 200 μL centrifuge tube; add an appropriate amount of Biotin-PEG4-NHS ester under the blowing of a pipette to biotinylate the amino groups on the magnetic microspheres, and incubate slowly with shaking at room temperature for 6 hours; wash the biotinylated 2.8 μm magnetic microspheres four times with a PBS solution containing 1% BSA, and then wash the biotinylated 2.8 μm magnetic microspheres four times with a PBS solution containing 0.01% Tween 20 (0.01% PBST), and redissolve in a PBS solution for use; The biotinylation modification scheme for the surface of M-450 epoxy resin-modified magnetic microspheres with a particle size of 4.5 μm is as follows: 400 μg of 4.5 μm magnetic microspheres are added to a 200 μL centrifuge tube; an appropriate amount of 10% BSA dissolved in 0.01 M boric acid ultrapure aqueous solution is added under the blowing of a pipette to couple BSA to the surface of the 4.5 μm magnetic microspheres, and this process is incubated with slow shaking at room temperature for 12 hours; the BSA-functionalized 4.5 μm magnetic microspheres are washed four times with a PBS solution containing 1% BSA, and then washed four times with a 0.01 M boric acid ultrapure aqueous solution for use; an appropriate amount of Biotin-PEG4-NHS ester is added under the blowing of a pipette to biotinylate the amino groups in the BSA on the magnetic microspheres, and incubated with slow shaking at room temperature for 6 hours; the biotinylated 4.5 μm magnetic microspheres are washed with a PBS solution containing 1% BSA The 4.5 μm magnetic microspheres were washed four times, and then the biotinylated 4.5 μm magnetic microspheres were washed four times with PBS solution containing 0.01% Tween 20 (0.01% PBST), and redissolved in PBS solution for later use; (2) Streptavidin modification of the surface of magnetic microspheres for surface loading of biotin-functionalized metal nanoparticles: The streptavidin modification scheme for the surface of carboxyl magnetic microspheres with a particle size of 1 μm was as follows: 200 μg of 1 μm magnetic microspheres were added to a 200 μL centrifuge tube; the carboxyl groups on the surface of the magnetic microspheres were activated with 150 mg / mL EDC and 25 mg / mL sulfo-NHS under weak acid conditions of pH 6.5; 20 μg of streptavidin dissolved in MES buffer solution was fully mixed with the activated magnetic microspheres and coupled at room temperature for 12 hours; the magnetic microspheres were blocked with 5 μg of streptavidin for 1 hour, and finally washed three times with MES buffer solution to complete the streptavidinization of the 1 μm magnetic microspheres; Streptavidin-conjugated magnetic microspheres with a particle size of 2.8 μm were purchased directly from commercial sources; The streptavidin modification scheme for the surface of M-450 epoxy resin-coated magnetic microspheres with a particle size of 4.5 μm is as follows: add 400 μg of 4.5 μm magnetic microspheres to a 200 μL centrifuge tube; add 40 μg of streptavidin dissolved in an appropriate amount of 0.01 M boric acid ultrapure aqueous solution under pipetting, and incubate slowly with shaking at room temperature for 12 hours; block the magnetic microspheres with 5 μg of streptavidin for 1 hour, and finally wash three times with MES buffer solution to complete the streptavidinization of the 4.5 μm magnetic microspheres;

[0016] Load encoding using streptavidin-functionalized AuNPs or AgNPs on biotinylated magnetic microspheres: (1) Use a pipette to draw an appropriate amount of biotinylated magnetic microsphere solution into a 200 μL centrifuge tube, wash twice with PBS buffer solution, add an appropriate amount of streptavidin-ylated AuNPs, and incubate at 37°C with shaking for 1 hour to achieve controllable loading of AuNPs on the surface of the magnetic microspheres. By controlling the loading concentration of AuNPs, barcode magnetic microspheres with different loading amounts can be prepared on the surface of biotinylated magnetic microspheres of different particle sizes. Wash the nanoparticle-encoded magnetic microspheres four times with a MES solution containing 1% BSA, then wash them four times with a MES solution containing 0.01% Tween 20, and finally dissolve the prepared barcode microspheres in a MES solution containing 1% BSA for later use; (2) The scheme of using AgNPs to load and encode on the surface of biotinylated magnetic microspheres is similar to the AuNPs scheme. By controlling the loading concentration of AgNPs on the surface of magnetic microspheres, barcode magnetic microspheres with different loading amounts can be prepared on the surface of biotinylated magnetic microspheres with different particle sizes. The magnetic microspheres encoded with nanoparticles were washed four times with MES solution containing 1% BSA, and then washed four times with MES solution containing 0.01% Tween 20. Finally, the prepared barcode microspheres were re-dissolved in MES solution containing 1% BSA for use.

[0017] Use biotin-functionalized rare earth nanoparticles (synthesized NaYF4, LaF3, NaSmF4, NaEuF4, NaTbF4, NaDyF4, NaHoF4, NaTmF4) to load encoding on the surface of streptavidin-labeled magnetic microspheres: Use a pipette to draw an appropriate amount of streptavidin-labeled magnetic microsphere solution into a 200 μL centrifuge tube, wash three times with MES buffer solution, add an appropriate amount of biotin-functionalized rare earth nanoparticles, and incubate at 37°C for 1 hour under shaking. By controlling the loading concentration, the method can achieve the loading encoding of magnetic microspheres by rare earth nanoparticles. The magnetic microspheres encoded with rare earth nanoparticles were washed 4 times with MES buffer solution containing 0.01% Tween 20, and an appropriate amount of streptavidin was added to block the biotin on the surface of the magnetic microspheres, and finally redissolved in PBS solution containing 1% BSA for use.

[0018] Application of new mass spectrometry coded microspheres in multi-component immunoassays: In order to further verify the practical application effect of metal nanoparticle-encoded microspheres, a multi-component immunoassay of suspended array microspheres based on mass spectrometry was carried out; First, AuNPs / AgNPs, two metal nanoparticles-encoded microspheres functionalized with antibodies, were prepared for antigen capture in immunoassays: Use a pipette to draw an appropriate amount of metal nanoparticle-encoded magnetic microsphere solution with rich streptavidin on the surface into a 200 μL centrifuge tube, add biotinylated mouse anti-human monoclonal capture antibody, incubate at 37°C with shaking for 1 hour to complete antibody functionalization of the encoded magnetic microspheres, and redissolve in PBS solution containing 1% BSA for later use; Secondly, antibody-functionalized PtNPs probes were prepared for signal output: Take 1 mL of pre-centrifuged purified PtNPs, add an appropriate amount of boric acid buffer (pH 9.0) to adjust the pH of the solution to weak alkalinity (pH about 8.4); add an appropriate amount of mouse anti-human monoclonal reporter antibody and mix well, incubate at 25°C overnight for coupling by electrostatic adsorption; the reporter antibody-functionalized PtNPs probe is centrifuged at 9000 rpm to remove the suspension, and finally the colloidal solution at the bottom is redissolved and mixed in PBS buffer containing 1% BSA for dispersion, and stored at 4°C for use.

[0019] High-throughput multi-component immunoassay using antibody-functionalized barcoded microspheres and antibody-functionalized PtNPs probes: Prepare 9 batches of 200 μL centrifuge tubes, corresponding to 9 types of antigen marker analysis. For a single batch of centrifuge tubes, use a pipette to draw an appropriate amount of antibody-functionalized encoded magnetic microspheres into the centrifuge tube, add an appropriate amount of standard concentration antigen solution, and add antibody-functionalized PtNPs probes, incubate at 37°C with shaking for 2 hours to complete the specific recognition and capture of antigens and PtNPs probes by the encoded magnetic microspheres. After the immune reaction, the encoded magnetic microspheres were separated by a magnetic solution and washed 8 times with PBS containing 0.01% Tween 20 to remove excess unbound PtNPs probes. After the immune reaction of 9 batches was carried out simultaneously and the washing was completed, an appropriate amount of 9 types of immune encoded magnetic microspheres from 9 batches were respectively drawn into a 4 mL centrifuge tube, and an appropriate amount of ultrapure water was added to dilute the mixed solution of the 9 types of encoded microspheres to meet the injection concentration and detection requirements of the mass spectrometry flow cytometer. The samples were sent into the ICP-TOF-MS through a pneumatic injection device to achieve simultaneous detection of multiple barcoded microsphere signals in the samples. 57 Fe + and 54 Fe + The signal positioning magnetic microspheres are used to load the surface of the microspheres. 197 Au + and 194 Pt + The signals distinguish different barcoded microspheres and capture the 194 Pt + The signal construction has a direct linear relationship with the corresponding antigen standard concentration, realizing the simultaneous analysis of the metabolic levels of 9 antigen markers.

[0020] Example 1: Metal nanoparticle encoding behavior and barcode microsphere preparation effect based on mass spectrometry flow cytometry: The establishment of an ideal barcode microsphere library based on mass spectrometry flow cytometry suspension array technology depends on two basic prerequisites: first, the barcode unit (the metal nanoparticle encoding unit used in the encoding of this patent) must have high resolution and no mutual interference to achieve extensive barcode compilation; second, the barcode compiled based on these high-resolution encoding units must also have high resolution; Figure 1 As shown, the principle and process of preparing barcode microspheres for mass spectrometry flow cytometry based on metal nanoparticle surface loading driven by streptavidin-biotin binding presented in the patent of the present invention are demonstrated; In order to verify the signal resolution of the barcoded magnetic microspheres prepared based on metal nanoparticle loading encoding in this patent, the prepared barcoded microspheres were detected by mass spectrometry flow cytometry. The full spectrum of ions from 6-280 m / z in the wide scan of ICP-TOF-MS was simultaneously collected. 54 Fe+ , 57 Fe + , 89 Y + , 139 La + , 152 Sm + , 153 Eu + , 159 Tb + , 164 Dy + , 165 Ho + , 169 Tm + , 107 Ag + , 197 Au + The signals can respectively realize the accurate positioning of magnetic microspheres and the precise distinction of barcodes. Figure 2 As shown in (a), the signal peaks of the 12 metal ions used for barcode microsphere positioning in ICP-TOF-MS have excellent resolution, and there is no peak overlap or interference, which meets the first premise. AuNPs with a particle size of 14 nm (AuNPs morphology and maximum absorption wavelength are characterized as follows Figure 2 (b) is used as an example to encode the load on the surface of magnetic microspheres. By decoding the barcode microspheres, Figure 2 As shown in (c), a total of 9 signal-resolved barcode channels were prepared on the surfaces of magnetic microspheres with three sizes (named #101, #102, #103; #201, #202, #203; #301, #302, #303; the first digit represents the particle size of the magnetic microspheres, "1" for 1 μm magnetic microspheres, "2" for 2.8 μm magnetic microspheres, and "3" for 4.5 μm magnetic microspheres; the second digit represents the relative size of the loading amount of AgNPs, "1" for low concentration loading, "2" for medium concentration loading, and "3" for high concentration loading), and the third digit represents the relative size of the loading amount of AuNPs, "1" for low concentration loading, "2" for medium concentration loading, and "3" for high concentration loading). The signal distribution of the channels is excellent, and there is no overlap, which meets the second premise mentioned above; AgNPs with a particle size of 34 nm (AgNPs morphology and maximum absorption wavelength are characterized as follows Figure 2 (d) is used as an example to encode the load on the surface of magnetic microspheres. By decoding the barcode microspheres, Figure 2 As shown in (e), a total of 9 signal-resolved barcode channels were prepared on the surfaces of magnetic microspheres of three sizes, and the signal distribution of the channels was excellent without any overlap. To further verify the universality of this loading coding strategy, the loading coding effects of 8 kinds of rare earth nanoparticles on the surface of magnetic microspheres were investigated. Figure 3 TEM characterization) to encode the load on the surface of the magnetic microspheres, and then decode the barcoded microspheres, as shown in Figure 3 As shown, 8 signal-resolved barcode channels were prepared on the surface of magnetic microspheres of three sizes respectively, and 64 barcode channels were prepared by encoding 8 kinds of rare earth nanoparticles in total. The signal distribution of the channels was excellent and there was no overlap. In addition to the above-mentioned study of the loading encoding of a single nanoparticle on the surface of the magnetic microspheres (such as Figure 4 (c)), further explored the dual loading encoding effect of AuNPs and AgNPs on the surface of magnetic microspheres. Figure 4 As shown in (a) and (b), two types of AuNPs-AgNPs dual-loaded magnetic microspheres named #213 and #231 were characterized by TEM and STEM elements. From the data results, it can be seen that the Fe content of the two barcoded magnetic microspheres #213 and #231 is the same, and the distribution and content of Au and Ag elements are significantly different with the difference in the relative loading ratio of AuNPs and AgNPs. By integrating the loading coding of AuNPs and AgNPs on the surface of magnetic microspheres with three particle sizes, a total of 11 barcoded microspheres were prepared, and their signal distribution is shown in Figure 1. Figure 4 (d) shown; Figure 4 (e) Magnetic microspheres with three different particle sizes 54 Fe + , 57 Fe + Signal distribution; Figure 4 (f) for #111, #131, #113 107 Ag + , 197 Au + Signal distribution; Figure 4 (g) for #211, #231, #213, #222 107 Ag + , 197 Au + Signal distribution; Figure 4 (h) for #311, #331, #313, #322 107 Ag + , 197 Au + Signal distribution; In addition to the above-mentioned single-layer loading of metal nanoparticles on the surface of magnetic microspheres, we also explored the possibility of achieving a breakthrough in the number of codes by loading metal nanoparticles in layers on the surface of magnetic microspheres. Taking the layered loading of AuNPs as an example, Figure 5 As shown in (a), a layer of streptavidin-functionalized AuNPs is first loaded on the surface of the magnetic microspheres with the maximum loading amount, followed by a layer of biotin-functionalized AuNPs, and then a layer of streptavidin-functionalized AuNPs, to achieve a reciprocating hierarchical loading of metal nanoparticles on the surface of the magnetic microspheres. Since the hierarchical loading introduces more AuNPs, after a total of three layers of AuNPs are loaded, as shown in Figure 5 As shown in (b), a new barcode magnetic microsphere #204 was successfully prepared; similarly, after loading a total of three layers of AgNPs, as shown in Figure 5 As shown in (c), a new barcoded magnetic microsphere #240 was successfully prepared, completing the further expansion of the barcoded microsphere types.

[0021] Example 2: Application of suspension array multi-component immunoassay based on mass spectrometry flow cytometry barcoded microspheres: The patent studies the application of the prepared metal nanoparticle-encoded magnetic microspheres in the detection of various cancer protein markers. Figure 6 As shown in (a), the capture antibody-functionalized metal nanoparticle-encoded magnetic microspheres in a uniform solution will capture the corresponding antigen markers and the corresponding reporter antibody-functionalized PtNPs probes based on the antigen-antibody recognition principle; the formed sandwich structure microspheres are separated from the unbound PtNPs probes by magnetic separation, and after 8 rounds of magnetic microsphere cleaning and appropriate dilution, they are introduced into the sample through a single particle injection system (such as Figure 6 (b)) is introduced into ICP-TOF-MS for high-throughput detection. In multi-component detection, such as Figure 6 (c) shows that the 54 Fe + , 57 Fe + Signal to realize the positioning of magnetic microspheres ( Figure 6 (d1)), using 107 Ag + , 197 Au + Signal to decode the barcode ( Figure 6 (d2)), plotting the different concentrations of cancer markers 194 Pt + The signal distribution data of the signal is calculated based on the average value ( Figure 6 (d3)), combined with the distribution mean to construct a linear relationship with the antigen concentration ( Figure 6(d4)), solve for the concentration of the unknown sample. Figure 6 As shown in (e) and (f), the constructed metal nanoparticle-encoded magnetic microsphere suspension array multi-component immunoassay strategy showed a good linear relationship for the detection of 9 antigen markers (as shown in Figure 7). The selectivity of 9 capture antibody-functionalized PtNPs probes in mixed antigen marker samples was also studied. Figure 6 As shown in (g), a single PtNP only recognizes and binds to the corresponding antigen marker and produces significant differences. 94 Pt + The high signal value, excellent selectivity and no obvious cross-interference verified the feasibility of the mass spectrometry flow cytometry suspension array.

Claims

1. A method for preparing a novel suspension array barcode microsphere for mass spectrometry flow cytometer, characterized in that: Microspheres with particle sizes ranging from 1 to 5 μm are used as encoding objects, and metal nanoparticles with particle sizes ranging from 10 to 100 nm are used as encoding units. The controllable loading of metal nanoparticles on the surface of microspheres is achieved through the specific binding between the functional groups on the surface of microspheres and the functional groups on the surface of metal nanoparticles. By regulating the relative loading concentration and loading ratio of the same metal nanoparticles and different metal nanoparticles on the surface of microspheres, barcoded microspheres with good signal differentiation and resolution in mass spectrometry flow cytometer are prepared. By modifying the surface of magnetic microspheres encoded by metal nanoparticles with active molecules used for biological analysis, such as antibodies, nucleic acids, peptides, etc., a suspension array high-throughput detection technology for the corresponding types of biomarkers can be constructed.

2. The method according to claim 1, characterized in that: The mass spectrometry flow cytometer microsphere encoding object used is not limited to magnetic microspheres, but can also be polystyrene microspheres, silica microspheres, etc. with a particle size ranging from 1 to 5 μm, which can provide a certain loading area for the loading of metal nanoparticles.

3. The method according to claim 1, characterized in that: The coding units used are not limited to the 10 metal nanoparticles described in the patent, as all elements in the periodic table can be prepared into metal nanoparticles with particle sizes ranging from 10 to 100 nm, because it is foreseeable that barcoded microspheres for mass spectrometry flow cytometry can be prepared by loading other types of metal nanoparticles on the surface of the microspheres.

4. The method according to claim 1, characterized in that: The means used for loading between microspheres and metal nanoparticles are not limited to the specific binding between streptavidin-biotin, but should be all chemical and biological coupling means that can be used to achieve loading between metal nanoparticles and microspheres; the decoding platform used is mass spectrometry flow cytometer, which is also called inductively coupled plasma time-of-flight mass spectrometry (ICP-TOF-MS).

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