SERS (Surface Enhanced Raman Scattering) component for capturing marker in blood fluid by utilizing antigen-antibody reaction and preparation method of SERS component

Abstract

The invention relates to an SERS (Surface Enhanced Raman Scattering) component for capturing markers in blood fluid by utilizing antigen-antibody reaction and a preparation method of the SERS component. The preparation method comprises the following steps: preparing a PDA (Personal Digital Assistant) chip aiming at a target marker, preparing a micro-fluidic chip and assembling the SERS assembly. The obtained SERS assembly can be applied to sudden cardiac death early screening. The manufacturing cost and the process difficulty of the micro-fluidic chip can be reduced, and the micro-fluidic chip can be used in cooperation with a portable Raman spectrometer for detection and can be thrown for use.

Description

Technical Field

[0001] The invention relates to a SERS component for capturing markers in blood fluid by using antigen-antibody reaction and a preparation method thereof, and belongs to the intersection of biotechnology and fluid mechanics, and the field of SERS technology. Background Art

[0002] Microfluidics is the science and technology involved in systems that use microchannels (with a size of tens to hundreds of microns) to process or manipulate tiny fluids (volumes of picoliters to nanoliters). It is an emerging interdisciplinary subject involving chemistry, fluid physics, microelectronics, new materials, biology, and biomedical engineering. At present, microfluidic chip products are mainly used for gene identification with integrated sample pretreatment, such as chain polymerization reaction (PCR) based on chips. The material of the chip determines the performance of the chip. To make a microfluidic chip, the chip material must follow the following principles: good biocompatibility, good electrical insulation and heat dissipation, good optical properties, good surface modifiability, simple manufacturing process and low manufacturing cost. At present, the commonly used materials for making microfluidic chips are: single crystal silicon wafers, quartz, glass, organic polymers (PMMA, PDMS, PC, COP), etc., among which polymers are the most common. The production techniques of polymers mainly include hot pressing, molding, injection molding, laser ablation, LIGA, soft etching, etc.

[0003] The microfluidic technology chips and ELISA methods in the prior art have the following problems: (1) The manufacturing process of silicon microfluidic chips is complicated and requires professional personnel to operate. (2) Silicon microfluidic chips have high production costs and are expensive. (3) When using microfluidic chips alone to achieve the same separation effect, the structure is more complicated and the analysis time is longer, making it difficult to achieve rapid detection. (4) Although the simple ELISA method has a better separation effect, its detection time is longer and the operation is more difficult. (5) The ELISA method has high instrument requirements and the raw material cost is expensive. It is urgent to develop a disposable SERS component that can reduce the production cost and can capture markers in the blood fluid using antigen-antibody reactions. Summary of the invention

[0004] The main purpose of the present invention is to overcome the problems existing in the prior art and provide a SERS component and a preparation method thereof for capturing markers in blood fluid using antigen-antibody reaction, which can reduce the cost and process difficulty of microfluidic chip production, can be used in conjunction with a portable Raman spectrometer for detection, and can be disposable.

[0005] The technical solution of the present invention to solve the technical problem is as follows:

[0006] A method for preparing a SERS component for capturing a marker in a blood fluid using an antigen-antibody reaction comprises the following steps:

[0007] Step 1: Prepare PDA chip for target markers:

[0008] The silicon wafer is soaked in an alkaline tank and then washed with deionized water, then soaked in piranha solution and washed with deionized water; the silicon wafer is incubated with a specific antibody solution containing PDA, then washed with deionized water to obtain a PDA chip, which is stored for future use; the specific antibody is one of a high-sensitivity troponin T antibody, a creatine kinase isoenzyme antibody, an aspartate aminotransferase antibody, a lactate dehydrogenase B antibody, a pyruvate antibody, and a spermine antibody;

[0009] Step 2: Preparation of microfluidic chip:

[0010] Using a prefabricated composite template, polycarbonate is molded into a microfluidic chip by casting.

[0011] Step 3: Assemble SERS components:

[0012] A hole sample is opened at a preset position on the tube wall of the microfluidic chip, and the PDA chip is installed at the hole sample, thus completing the assembly and obtaining a finished SERS component.

[0013] The technical solution further improved by the present invention is as follows:

[0014] Preferably, in the first step, soaking in an alkaline tank for 2 to 3 hours; soaking in a piranha solution for 2 to 3 hours; the concentration of PDA in the specific antibody solution containing PDA is 33±5mM; the incubation time is 1 to 2 hours; and storage is performed at 0°C to 4°C; the solution in the alkaline tank is a piranha solution; the piranha solution is prepared by mixing 98% concentrated sulfuric acid and 30% hydrogen peroxide solution in a ratio of 7±1:3.

[0015] Preferably, in the first step, the silicon wafer is a single crystal silicon wafer.

[0016] Preferably, in the second step, the preparation process of the complex template is: on the template substrate, a chemically heterogeneous surface is constructed by planar patterning of a hydrogel, and three-dimensional liquid micro-stripes are prepared on the surface to obtain the complex template.

[0017] Preferably, in the second step, the ratio of the lumen diameter of the microfluidic chip to the diameter of the PDA chip is 3.0-3.25:1.

[0018] More preferably, the ratio of the lumen diameter of the microfluidic chip to the diameter of the PDA chip is 3.231:1.

[0019] More preferably, the lumen diameter of the microfluidic chip is 323.1 microns and the length is 6.9 cm; the diameter of the PDA chip is 100 microns; in the third step, the preset positions are 3.0 cm, 4.0 cm, and 5.0 cm in length of the microfluidic chip respectively; the open hole sample refers to a circular hole with a diameter of 100 microns.

[0020] Preferably, in the third step, a fixing rod is used to fix the PDA chip; one end of the fixing rod is fixedly connected to the outside of the PDA chip, and the other end of the fixing rod is fixedly connected to the tube wall of the microfluidic chip.

[0021] The present invention also provides:

[0022] The SERS component prepared by the preparation method described above utilizes antigen-antibody reaction to capture markers in blood fluid.

[0023] Preferably, the SERS component is a detection component used for early screening of sudden cardiac death; the SERS component is installed with at least: a PDA chip for capturing high-sensitivity troponin T, a PDA chip for capturing creatine kinase isoenzymes, a PDA chip for capturing aspartate aminotransferase, a PDA chip for capturing lactate dehydrogenase B, a PDA chip for capturing pyruvate, and a PDA chip for capturing spermine.

[0024] The present invention can reduce the cost and process difficulty of microfluidic chip production, can be used in conjunction with a portable Raman spectrometer for detection, and can be disposable. The present invention uses microfluidic technology to control the flow and expansion of blood so that the antigen-antibody reaction can be fully carried out. The cost of each chip to complete a day's experimental operation is about US$5, while the cost of high-throughput applications is hundreds to thousands of dollars, so the product of the present invention has obvious cost advantages. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the basic preparation process of the PDA chip in Example 1 of the present invention.

[0026] Figure 2 Schematic diagram of characteristic peaks of the spectrum at different ratios in Example 1 of the present invention.

[0027] Figure 3 This is a schematic diagram of assembling a SERS component in Example 1 of the present invention.

[0028] Figure 4 Schematic diagram of the overall structure of the kit of Example 2 of the present invention.

[0029] Figure 5 Schematic diagram of the top view of the kit of Example 2 of the present invention.

[0030] Figures 6 to 11They are respectively the test result diagrams of Example 2 of the present invention. Among them, Figure 6 This is the test result of high-sensitivity troponin T. Figure 7 This is the test result of creatine kinase isoenzyme. Figure 8 This is the test result of aspartate aminotransferase. Fig. 9 This is the test result of pyruvic acid. Fig.10 This is the test result of lactate dehydrogenase B. Fig.11 This is the detection result of spermine.

[0031] Fig.12 This is a Raman spectrum diagram obtained from 40 blood samples in Example 2 of the present invention. DETAILED DESCRIPTION

[0032] Terminology explanation:

[0033] Surface-enhanced Raman spectroscopy (SERS) is a method that increases the surface roughness of precious metals, adsorbs the object to be tested onto the metal surface, and makes the scattered laser intensity 10 times stronger than ordinary Raman. 6 -10 11 The SERS sensor is mainly composed of a carrier, an enhanced substrate, etc. It can be directly used for non-destructive testing or in-situ analysis of analytical materials without complicated pretreatment.

[0034] PDA chip: Polydopamine (PDA) chip uses polymerized dopamine (PDA) to synthesize nanomaterials because it has good biocompatibility and can stabilize SERS signals. By enriching marker-specific antibodies embedded in PDA on the surface of Si substrate, the markers in the blood sample are captured using antigen-antibody reaction.

[0035] The present invention will be described in further detail below with reference to the accompanying drawings and in combination with embodiments, but the present invention is not limited to the examples given.

[0036] Example 1

[0037] This example is to prepare a SERS component that uses antigen-antibody reaction to capture markers in blood fluid.

[0038] The specific contents of this embodiment are as follows:

[0039] 1. Raw materials:

[0040] Polycarbonate (PC), Polydopamine (PDA), Piranha solution (RCA), Single crystal silicon chip (Si), High-sensitivity troponin T antibody (Anti-Hs-cTnT), Lactate dehydrogenase B antibody (Anti-LDHB), Pyruvate antibody (Anti-2-oxo-propanoicaci, Anti-Pyruvicacid), Spermine antibody (Anti-N,N'-Bis(3-aminopropyl)-1,4-diaminobutane, Anti-Spermine), Creatine Kinase Isoenzymes antibodies (Anti-CK-MB), Aspartate Aminotransferase antibodies (Anti-AST) are all commodities purchased from the market.

[0041] 2. Process steps:

[0042] (I) Preparation of PDA chip for capturing various myocardial ischemia markers:

[0043] The single crystal silicon wafer was soaked in an alkaline tank and then washed with deionized water, then soaked in piranha solution and washed with deionized water. In order to increase the density of captured antibodies and maintain their biological activity, a self-assembled dopamine layer was used for wrapping: the silicon wafer was incubated with a specific antibody solution containing PDA, then washed with deionized water to obtain a PDA chip, which was stored for later use (such as Figure 1 As shown). Among them, soaking in an alkaline tank for 2 hours; soaking in a piranha solution for 2 hours. The concentration of PDA in the specific antibody solution containing PDA is 33mM; the incubation time is 1 hour; and the storage is below 4°C. The silicon wafer is a single crystal silicon wafer. The solution in the alkaline tank is a piranha solution. The piranha solution is a mixture of 98% concentrated sulfuric acid and 30% hydrogen peroxide solution in a ratio of 7:3.

[0044] Specific to each PDA chip:

[0045] (1) A PDA chip for capturing high-sensitivity troponin T is prepared using a high-sensitivity troponin T antibody in the above basic preparation process.

[0046] (2) A PDA chip for capturing creatine kinase isoenzymes is prepared using creatine kinase isoenzyme antibodies in the above basic preparation process.

[0047] (3) Preparation of a PDA chip for capturing aspartate aminotransferase, using aspartate aminotransferase antibodies in the above basic preparation process.

[0048] (4) Preparation of a PDA chip for capturing lactate dehydrogenase B, using lactate dehydrogenase B antibody in the above basic preparation process.

[0049] (5) Preparation of a PDA chip for capturing pyruvate, using pyruvate antibodies in the above basic preparation process.

[0050] (6) Preparation of a PDA chip for capturing spermine, using spermine antibodies in the above basic preparation process.

[0051] (II) Preparation of microfluidic chip:

[0052] The microfluidic chip is made of polycarbonate by casting method using a prefabricated composite template.

[0053] The preparation process of the complex template is as follows: on the template substrate, a chemically heterogeneous surface is constructed by planar patterning of hydrogel, and three-dimensional liquid micro-stripes are prepared on the surface to obtain the complex template.

[0054] This embodiment optimizes the capture effect by screening the optimal ratio of the microfluidic chip lumen diameter: the PDA chip diameter under turbulent flow conditions of the fluid (blood).

[0055] First, the ratios of the microfluidic chip lumen diameter and the PDA chip diameter were set to the following ratios: 0.50:1, 1.0:1, 2.0:1, 2.25:1, 2.5:1, 2.75:1, 3.0:1, and 3.25:1. Then, the markers in the same test samples were captured, and then the Raman spectra were obtained by detection with a portable Raman spectrometer. It was found that the PDA chips in the 3.0:1 and 3.25:1 groups had the best capture effects.

[0056] Afterwards, a similar method was used to set a more detailed ratio in the range of 3.0-3.25:1 for screening, and the final conclusion was: through measurement, the SERS signal was strongest when the microfluidic chip lumen diameter: PDA chip diameter was 3.231:1. The summary results are as follows Figure 2 shown.

[0057] Therefore, in this embodiment, the microfluidic chip lumen diameter is set to 323.1 microns, the length is set to 6.9 cm, and the PDA chip diameter is set to 100 microns to obtain the best capture effect.

[0058] (III) Assembling SERS components:

[0059] like Figure 3As shown, holes (i.e., circular holes with a diameter of 100 microns) are made at preset positions (i.e., at lengths of 3.0 cm, 4.0 cm, and 5.0 cm) on the tube wall of the microfluidic chip, and the PDA chip is installed at the hole, and the assembly is completed. Among them, a fixing rod 31 is used to fix and install the PDA chip 32; one end of the fixing rod 31 is fixedly connected to the outside of the PDA chip 32, and the other end is fixedly connected to the tube wall of the microfluidic chip.

[0060] Example 2

[0061] This embodiment is a specific application example based on Embodiment 1. This embodiment adopts the SERS component of Embodiment 1.

[0062] This embodiment uses a disposable test kit that cooperates with a portable Raman spectrometer for detection as a tool for testing the implementation effect of the SERS component.

[0063] like Figure 4 , Figure 5 As shown, the kit includes a box body 01, and a group of SERS scanning holes 02, a rod hole 03 and a sample injection port 04 are arranged on the top surface of the box body 01; a rod 05 is arranged in the rod hole 03; a sample tube 06 is arranged in the box body 01, one end of the sample tube 06 is connected to the sample injection port 04, and the other end is connected to the side wall of the box body; a group of capture devices 07 are arranged in the box body 01, and each capture device 07 is transmission-connected to the rod 05 through a transmission mechanism; each capture device 07 has a vertically arranged capillary bundle, and the end of each capillary bundle extends into the sample tube 06; a group of SERS detection substrates 08 using a microfluidic chip are arranged above the capture device 07 in the box body 01, each SERS detection substrate 08 corresponds to the capture device 07 one by one, each SERS detection substrate 08 corresponds to the SERS scanning hole 02 one by one, and each SERS detection substrate 08 is located directly below the corresponding SERS scanning hole 02; a substrate protection device 09 for locking the rod 05 is arranged in the box body 01.

[0064] The specific states of the reagent box are: (1) the substrate protection device 09 locks the pull rod 05, and each SERS detection substrate 08 is in the initial state separated from the corresponding capture device 07. (2) The substrate protection device 09 is disengaged from the pull rod 05 to unlock it, and the pull rod 05 is pulled out of the box body 01 under the action of external force, and the pull rod 05 moves each capture device 07 to a processing state combined with the corresponding SERS detection substrate 08 through the transmission mechanism. In this processing state, the capture device 07 and the SERS detection substrate 08 are combined to form a sandwich structure, and at this time, the portable Raman spectrometer can be used to align the SERS scanning hole 02 to detect each SERS detection substrate 08.

[0065] The sample tube 06 is formed by connecting a vertical tube and a horizontal tube, the top of the vertical tube is connected to the sample adding port 04, and the end of the horizontal tube away from the vertical tube is connected to the side wall of the box body. The sample tube 06 is "L" shaped.

[0066] The SERS component of Example 1 is used as the SERS detection substrate of the above-mentioned kit.

[0067] The detection process is as follows: add the sample to be tested to the sample injection port, the sample to be tested enters the sample tube, contacts and enters the capillary bundle of each capture device; operate the substrate protection device to unlock the pull rod, and use the pull rod to move each capture device to combine with the corresponding SERS detection substrate to form a sandwich structure; then, use a portable Raman spectrometer to detect each SERS detection substrate through the SERS scanning hole and obtain the test results. Afterwards, analyze and judge based on the test results. If the characteristic peak meets the preset value, it can be highly suspected that the corresponding patient of the sample to be tested has a higher risk of sudden cardiac death.

[0068] The SERS assembly of Example 1 was used in combination with the above kit to test sensitivity and repeatability:

[0069] The results of testing different concentrations of markers: standard solutions of high-sensitivity troponin T, creatine kinase isoenzyme, aspartate aminotransferase, lactate dehydrogenase B, pyruvate, and spermine were as follows: Figures 6 to 11 As shown. The Raman spectra of the standard solutions each have an obvious characteristic peak, while the blank control has no characteristic peak. In the range of 1 to 5000 ng / mL concentration of each marker, the SERS signal intensity gradually increases with the increase of concentration. The Raman signal intensity is positively correlated with the logarithm of the concentration of each marker in a piecewise manner. The results of further curve fitting show that the SERS component of Example 1 has a high sensitivity, and there is a good linear relationship between the SERS signal intensity and the logarithm of the concentration of each marker, which provides application prospects for the quantitative analysis of each marker in actual clinical blood samples.

[0070] Test specificity: Other molecules that may exist in clinical blood samples were selected for detection. After comparison, it was found that the signal intensity detected at the characteristic peak of the above six marker groups was significantly higher than that of other molecule groups, which indicates that the SERS component of Example 1 has a significantly higher detection ability for the above markers than other antigens.

[0071] The above results indicate that the SERS component of Example 1 has good specificity and selectivity, further demonstrating its excellent performance and can be used for actual clinical analysis.

[0072] Next, the SERS component of Example 1 was used in combination with the above kit to test the blood of 40 patients with myocardial ischemia. Each sample was tested at least 3 times to obtain the Raman spectra as shown in FIG. Fig.12As shown. The characteristic peaks of myocardial ischemia markers (high-sensitivity troponin T, creatine kinase isoenzymes, aspartate aminotransferase, lactate dehydrogenase B, pyruvate, spermine) were selected as markers, and the concentrations of 40 blood samples were calculated according to the Raman spectrum. The standard content of each marker was then compared, for example, myocardial enzyme spectrum: CK 405.3U / L, CK-MB 196.94U / L, LDH 896.65U / L, AST 91.9U / L; troponin 0.238ng / mL. By comparison, the degree of myocardial ischemia in each patient can be obtained: if it is within the normal range, although the patient has symptoms of myocardial ischemia, the risk of sudden cardiac death is still low, and continued maintenance treatment can be considered. If it exceeds the normal range, it can be highly suspected that the patient has a high risk of sudden cardiac death, and active means should be used to treat the patient. In this way, the kit of Example 2 can be used to quickly screen for sudden cardiac death and assist in the discovery of heart disease.

[0073] In summary, the present invention has relatively low cost, and the use of antigen-antibody reaction capture has reduced requirements for the microstructure manufacturing process of the microfluidic chip, which can greatly reduce the technical cost and labor cost of production. If a silicon microfluidic chip is used, the process is complicated and requires professional personnel to operate. Silicon microfluidic chips have high production costs and are expensive. When using a microfluidic chip alone to achieve the same separation effect, the structure is more complicated, the analysis time is longer, and it is difficult to achieve the effect of rapid detection. Although the simple ELISA method has a good separation effect, its detection time is long and the operation difficulty is high. The ELISA method has high instrument requirements and the raw material cost is expensive. The present invention combines a microfluidic chip with an antigen-antibody reaction, specifically detects markers in the blood, and adjusts the microfluidic chip lumen diameter and the PDA chip diameter according to fluid mechanics to obtain a diameter ratio with high capture sensitivity and strong specificity, thereby achieving the best capture effect.

[0074] In addition to the above embodiments, the present invention may also have other implementation modes. Any technical solution formed by equivalent replacement or equivalent transformation falls within the protection scope required by the present invention.

Claims

1. A method for preparing a SERS component for capturing markers in blood fluid using antigen-antibody reaction, characterized in that: The following steps are involved: Step 1: Prepare PDA chip for target markers: The silicon wafer is soaked in an alkaline tank and then washed with deionized water, then soaked in piranha solution and washed with deionized water; the silicon wafer is incubated with a specific antibody solution containing PDA, then washed with deionized water to obtain a PDA chip, which is stored for future use; the specific antibody is one of a high-sensitivity troponin T antibody, a creatine kinase isoenzyme antibody, an aspartate aminotransferase antibody, a lactate dehydrogenase B antibody, a pyruvate antibody, and a spermine antibody; Step 2: Preparation of microfluidic chip: Using a prefabricated composite template, polycarbonate is molded into a microfluidic chip by casting. Step 3: Assemble SERS components: A hole sample is opened at a preset position on the tube wall of the microfluidic chip, and the PDA chip is installed at the hole sample, thus completing the assembly and obtaining a finished SERS component.

2. According to claim 1, a method for preparing a SERS component for capturing markers in blood fluid using antigen-antibody reaction is characterized in that, in the first step, the sample is immersed in an alkaline tank for 2 to 3 hours; immersed in a piranha solution for 2 to 3 hours; the concentration of PDA in the specific antibody solution containing PDA is 33±5mM; the incubation time is 1 to 2 hours; and stored at 0°C to 4°C; the solution in the alkaline tank is a piranha solution; and the piranha solution is a mixture of 98% concentrated sulfuric acid and 30% hydrogen peroxide solution in a ratio of 7±1:

3.

3. The method for preparing a SERS component for capturing markers in blood fluid using antigen-antibody reaction according to claim 1, characterized in that: In the first step, the silicon wafer is a single crystal silicon wafer.

4. A method for preparing a SERS component for capturing markers in blood fluid using antigen-antibody reaction according to any one of claims 1 to 3, characterized in that: In the second step, the preparation process of the complex template is as follows: on the template substrate, a chemically heterogeneous surface is constructed by planar patterning of a hydrogel, and three-dimensional liquid micro-stripes are prepared on the surface to obtain a complex template.

5. A method for preparing a SERS component for capturing markers in blood fluid using antigen-antibody reaction according to any one of claims 1 to 3, characterized in that: In the second step, the ratio of the lumen diameter of the microfluidic chip: the diameter of the PDA chip was 3.0-3.25:

1.

6. The method for preparing a SERS component for capturing markers in blood fluid using antigen-antibody reaction according to claim 5, characterized in that: The ratio of the lumen diameter of the microfluidic chip to the diameter of the PDA chip is 3.231:

1.

7. The method for preparing a SERS component for capturing markers in blood fluid by antigen-antibody reaction according to claim 6, characterized in that: The lumen diameter of the microfluidic chip is 323.1 microns and the length is 6.9 cm; the diameter of the PDA chip is 100 microns; in the third step, the preset positions are respectively at 3.0 cm, 4.0 cm, and 5.0 cm in length of the microfluidic chip; the open hole sample refers to a circular hole with a diameter of 100 microns.

8. A method for preparing a SERS component for capturing markers in blood fluid using antigen-antibody reaction according to any one of claims 1 to 3, characterized in that: In the third step, the PDA chip is fixedly installed using a fixing rod; one end of the fixing rod is fixedly connected to the outer side of the PDA chip, and the other end of the fixing rod is fixedly connected to the tube wall of the microfluidic chip.

9. A SERS component for capturing markers in blood fluid by antigen-antibody reaction, prepared by the SERS component preparation method according to any one of claims 1 to 8.

10. The SERS component according to claim 9, characterized in that: The SERS component is a detection component used for early screening of sudden cardiac death; the SERS component is at least installed with: a PDA chip for capturing high-sensitivity troponin T, a PDA chip for capturing creatine kinase isoenzymes, a PDA chip for capturing aspartate aminotransferase, a PDA chip for capturing lactate dehydrogenase B, a PDA chip for capturing pyruvate, and a PDA chip for capturing spermine.

Images