SERS chip for blood neurotransmitter detection and preparation and detection method

Abstract

The present invention discloses a SERS chip for detecting blood neurotransmitters and a preparation and detection method. The chip includes a PDMS gel composite mold, a gel layer and a chip detection substrate. The overall structure from bottom to top is a glass substrate, a gel filtration membrane and a PDMS mold; a plurality of parallel rows of detection points are arranged on the glass substrate, each detection point is covered with a layer of hydrogel with filtering properties, and a blood sample to be tested is passed into each detection point and left to stand. Small molecules in the sample can be quantitatively analyzed by collecting SERS spectra of each detection area. The present invention can detect the level of specific neurotransmitter molecules in a blood sample without pretreatment, improves the convenience of detection, shortens the detection time, and has broad application prospects in the field of rapid disease diagnosis and analysis.

Description

Technical Field

[0001] The invention relates to a surface enhanced Raman scattering chip detection technology, belongs to the field of spectroscopy biological applications, and specifically relates to a preparation and detection method of a SERS chip for blood neurotransmitter detection. Background Art

[0002] Neurotransmitters are chemical molecules in the body that are responsible for transmitting or regulating specific signals between neurons and other cell types. They play a vital role in brain development, behavior, emotions, and life maintenance. There are many types of neurotransmitters and their quantities are extremely small. In recent years, the level of neurotransmitters in the blood has become an important indicator for clinical medical testing. How to meet the needs of convenient, rapid, and high-throughput analysis and detection of neurotransmitters in clinical testing is an urgent problem to be solved.

[0003] At present, the commonly used methods for detecting neurotransmitters in the blood include enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography (HPLC). Among them, although the ELISA has good specificity and sensitivity, it consumes a large amount of biological reagents and has complex operations and a long detection cycle; HPLC can accurately detect various neurotransmitters, but requires expensive instruments and time-consuming pretreatment steps. Therefore, the development of a fast, accurate and convenient neurotransmitter detection technology is an urgent problem to be solved.

[0004] Surface Enhanced Raman Scattering (SERS) is an ultra-sensitive analytical technique. When the analyte molecules approach or adsorb on the surface of precious metal (such as gold, silver, etc.) nanoparticles under the irradiation of incident laser, their Raman scattering signals will increase by 6-14 orders of magnitude. SERS spectroscopy retains many advantages of Raman spectroscopy, such as strong molecular fingerprint characteristics and non-destructive in-situ analysis. It also has the advantages of short analysis time, high sensitivity, and wide detection range. In addition, the instrument is portable, easy to operate, and does not require complicated sample pretreatment steps. It has very good application value for the detection of trace small molecules in blood. Summary of the invention

[0005] Technical problem: The purpose of the present invention is to overcome the shortcomings of the prior art and provide a preparation and detection method based on SERS chip to achieve rapid, highly sensitive and accurate quantitative analysis and detection of multiple different components in whole blood samples.

[0006] Technical solution: To achieve the above purpose, the technical solution of the present invention is as follows:

[0007] A SERS chip for blood neurotransmitter detection includes a PDMS gel composite mold and a chip detection substrate. The overall structure from bottom to top is a glass substrate, a gel filtration membrane and a PDMS mold. Multiple rows of parallel detection points are arranged on the glass substrate, and each detection channel is covered with a layer of hydrogel with filtering properties. A sample to be tested is passed into each detection channel and left to stand. By collecting the SERS spectrum of each detection area, the neurotransmitter content in the sample can be quantitatively analyzed.

[0008] A preparation and detection method of a SERS chip for blood neurotransmitter detection, the specific steps are as follows:

[0009] (1) Using metal nanoparticles as a substrate, a PDMS mask with a microarray structure is covered on the substrate to form a two-dimensional array detection substrate;

[0010] (2) preparing a gel filtration membrane in each detection channel in step (1), so that the small molecules to be detected in the blood sample to be detected can be filtered by the gel and contact the surface of the metal nanoparticles on the glass substrate, thereby preparing a gel microarray detection channel;

[0011] (3) The blood sample to be tested is passed through the microarray detection channel and the residue is removed. The SERS spectra of the microarray detection channel are collected in sequence using a Raman spectrometer to obtain the SERS signals of the neurotransmitters in the blood. The SERS signals are processed to obtain the types of neurotransmitters contained in the blood sample to be tested and to measure their concentrations.

[0012] Furthermore, the SERS chip for blood neurotransmitter detection and the preparation and detection method are characterized in that: the metal nanoparticles are gold core silver shell nanorods, the PDMS mask is obtained by 3D printing template inversion, and the gel filtration membrane covering each channel can allow smaller molecules in the blood to pass through the mesh of the gel and non-specifically bind to the surface of the gold core silver shell nanorods.

[0013] Beneficial effects: The advantages of the present invention are as follows:

[0014] 1. The present invention utilizes the hydrogel filtration method to remove the complicated pretreatment steps of blood, shorten the detection time, and is low-cost and environmentally friendly;

[0015] 2. The chip can detect multiple targets at the same time, improving the convenience and accuracy of detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Attached Figure 1 The schematic diagram is a structural diagram of the SERS gel composite microarray chip for blood neurotransmitter detection of the present invention.

[0017] Attached Figure 2This is the SERS spectrum of the neurotransmitter in the blood sample to be tested. DETAILED DESCRIPTION

[0018] The present invention will be further described below in conjunction with the accompanying drawings.

[0019] As attached Figure 1 As shown, a SERS chip for blood neurotransmitter detection includes a PDMS gel composite mold and a chip detection substrate. The overall structure from bottom to top is a glass substrate, a gel filtration membrane and a PDMS mold; multiple rows of parallel detection points are arranged on the glass substrate, and each detection channel is covered with a layer of hydrogel with filtering properties. A sample to be tested is passed into each detection channel and left to stand, and the neurotransmitter content in the sample can be quantitatively analyzed by collecting the SERS spectrum of each detection area.

[0020] A preparation and detection method of a SERS chip for blood neurotransmitter detection, the specific steps are as follows:

[0021] (1) Using metal nanoparticles as a substrate, a PDMS mask with a microarray structure is covered on the substrate to form a two-dimensional array detection substrate;

[0022] (2) preparing a gel filtration membrane in each detection channel in step (1), so that the small molecules to be detected in the blood sample to be detected can be filtered by the gel and contact the surface of the metal nanoparticles on the glass substrate, thereby preparing a gel microarray detection channel;

[0023] (3) The blood sample to be tested is passed through the microarray detection channel and the residue is removed. The SERS spectra of the microarray detection channel are collected in sequence using a Raman spectrometer to obtain the SERS signals of the neurotransmitters in the blood. The SERS signals are processed to obtain the types of neurotransmitters contained in the blood sample to be tested and to measure their concentrations.

[0024] Furthermore, the SERS chip for blood neurotransmitter detection and the preparation and detection method are characterized in that: the metal nanoparticles are gold core silver shell nanorods, the PDMS mask is obtained by 3D printing template inversion, and the gel filtration membrane covering each channel can allow smaller molecules in the blood to pass through the mesh of the gel and non-specifically bind to the surface of the gold core silver shell nanorods.

[0025] The present utility model is further described below in conjunction with specific embodiments, but the examples are only exemplary and do not constitute any limitation on the scope of the present invention. It should be understood that the details and forms of the technical solution can be improved and modified without departing from the scope of the present embodiment, but these improvements and modifications do not deviate from the protection scope of the present utility model.

[0026] Specific examples: Example 1: A SERS detection substrate is prepared with gold core silver shell nanorods, and the filtering effect of gel is used to achieve small molecule screening. Example 2: A SERS detection substrate is prepared with gold core silver shell nanorods, and the filtering effect of gel is used to achieve high-throughput detection of blood neurotransmitters.

[0027] Example 1: A SERS detection substrate was prepared using gold core-silver shell nanorods, and the filtering effect of gel was used to achieve blood small molecule screening.

[0028] 1) Preparation of gold core-silver shell nanorods.

[0029] First, prepare gold seeds. Add 200μl 0.05M tetrachloroauric acid solution and 100μl ascorbic acid solution to 20ml 50mM hexadecyltrimethylammonium bromide (CTAB) and 13.5mM n-decanol solution at 25-27°C and stir to obtain a colorless solution. After 1-2 minutes, add 0.8ml 0.02M iced sodium borohydride solution and stir vigorously to obtain a yellow-brown seed solution. Then prepare small gold nanorods. Add the following reagents in 30ml 50mM CTAB and 13.5mM n-decanol solution at room temperature: 240μl 0.01M silver nitrate solution, 2.1ml 1M hydrochloric acid solution, 300μl 0.05M tetrachloroauric acid solution and 390μl 0.1M ascorbic acid solution and stir slowly. Then add 1.8ml gold seed solution and stir evenly. Let stand at room temperature for at least 4h to obtain a small gold nanorod solution. The size of the obtained small gold nanorods is about 7.5nm×21nm. Finally, the gold nanorods are synthesized, 2ml of the small gold nanorod solution is centrifuged and washed twice to remove the CTAB on the surface, and dispersed into 4ml of 1mM CTAB solution. At room temperature, the following reagents are added to 10ml of 50mM CTAB and 11mM n-decanol solution: 200μl 0.01M silver nitrate solution, 100μl 0.05M tetrachloroauric acid solution, 80μl 0.1M ascorbic acid solution, 500μl 1M hydrochloric acid solution and 250μl small gold nanorod solution, stirred until colorless and then allowed to stand for 4h to obtain gold nanorods. The size of the obtained gold nanorods is about 100nmx20nm.

[0030] Take 2 ml of the above solution and wash it twice by centrifugation to remove CTAB on the surface, disperse it back into 2 ml of water, stir and add 0.0728 g of CTAB, 130 μl of 0.1 M ascorbic acid, 50 μl of 15 mM silver nitrate solution, and 240 μl of 0.1 M sodium hydroxide in sequence to obtain a deep red solution, centrifuge it once, remove the supernatant and disperse it again in 2 ml of water.

[0031] 2) The surface of the glass sheet is modified with nanoparticles.

[0032] The glass slide was placed in a freshly prepared piranha solution (98% concentrated sulfuric acid and 30% aqueous hydrogen peroxide solution mixed in a volume ratio of 3:1), and then ultrasonicated for 1 hour and washed with deionized water, followed by drying with argon gas three times and placed in a 60°C oven for 1 hour. In order to modify the surface of the substrate with sulfur bonds, the glass slide was soaked in a 10% mercaptopropyltrimethoxysilane solution for 4 hours, then washed with ethanol and dried. At the same time, a polydimethylsiloxane (PDMS) mask was attached to the glass slide, and then the gold core silver shell nanorods were added to the microarray channel, washed with deionized water and dried overnight to obtain a microarray SERS substrate.

[0033] 3) Gel microarray composite substrate

[0034] At room temperature, add 0.04g of photoinitiator 2595 and 0.25g of polyethylene glycol (diol) diacrylate to 5ml of deionized water and stir until completely dissolved to obtain a gel precursor solution. Take another clean PDMS mask and attach it to the microarray SERS substrate obtained in step 2) (the same position as the PDMS mask in step 2)). Add 200μl of gel precursor solution to each detection channel and expose it to ultraviolet light for cross-linking polymerization. After 2 minutes, the gel microarray composite substrate is obtained.

[0035] 4) SERS signal acquisition

[0036] After the detection channel is cleaned with deionized water, the SERS spectra of each unit of the substrate are collected in sequence using a Raman spectrometer. The content of small molecules in the sample to be tested can be measured by interpreting the SERS signal.

[0037] Example 2: A SERS probe was prepared using gold core silver shell nanorods as a substrate, and the specific reaction between antibodies and antigens was used to detect neurotransmitters in the blood.

[0038] 1) Preparation of gold core-silver shell nanorods.

[0039] First, prepare gold seeds. Add 200μl 0.05M tetrachloroauric acid solution and 100μl ascorbic acid solution to 20ml 50mM hexadecyltrimethylammonium bromide (CTAB) and 13.5mM n-decanol solution at 25-27°C and stir to obtain a colorless solution. After 1-2 minutes, add 0.8ml 0.02M iced sodium borohydride solution and stir vigorously to obtain a yellow-brown seed solution. Then prepare small gold nanorods. Add the following reagents in 30ml 50mM CTAB and 13.5mM n-decanol solution at room temperature: 240μl 0.01M silver nitrate solution, 2.1ml 1M hydrochloric acid solution, 300μl 0.05M tetrachloroauric acid solution and 390μl 0.1M ascorbic acid solution and stir slowly. Then add 1.8ml gold seed solution and stir evenly. Let stand at room temperature for at least 4h to obtain a small gold nanorod solution. The size of the obtained small gold nanorods is about 7.5nm×21nm. Finally, the gold nanorods are synthesized, 2ml of the small gold nanorod solution is centrifuged and washed twice to remove the CTAB on the surface, and dispersed into 4ml of 1mM CTAB solution. At room temperature, the following reagents are added to 10ml of 50mM CTAB and 11mM n-decanol solution: 200μl 0.01M silver nitrate solution, 100μl 0.05M tetrachloroauric acid solution, 80μl 0.1M ascorbic acid solution, 500μl 1M hydrochloric acid solution and 250μl small gold nanorod solution, stirred until colorless and then allowed to stand for 4h to obtain gold nanorods. The size of the obtained gold nanorods is about 100nmx20nm.

[0040] Take 2 ml of the above solution and wash it twice by centrifugation to remove CTAB on the surface, disperse it back into 2 ml of water, stir and add 0.0728 g of CTAB, 130 μl of 0.1 M ascorbic acid, 50 μl of 15 mM silver nitrate solution, and 240 μl of 0.1 M sodium hydroxide in sequence to obtain a deep red solution, centrifuge it once, remove the supernatant and disperse it again in 2 ml of water.

[0041] 2) The surface of the glass sheet is modified with nanoparticles.

[0042] The glass slide was placed in a freshly prepared piranha solution (98% concentrated sulfuric acid and 30% aqueous hydrogen peroxide solution mixed in a volume ratio of 3:1), and then ultrasonicated for 1 hour and washed with deionized water, followed by drying with argon gas three times and placed in a 60°C oven for 1 hour. In order to modify the surface of the substrate with sulfur bonds, the glass slide was soaked in a 10% mercaptopropyltrimethoxysilane solution for 4 hours, then washed with ethanol and dried. At the same time, a polydimethylsiloxane (PDMS) mask was attached to the glass slide, and then the gold core silver shell nanorods were added to the microarray channel, washed with deionized water and dried overnight to obtain a microarray SERS substrate.

[0043] 3) Gel microarray composite substrate

[0044] At room temperature, add 0.04g of photoinitiator 2595 and 0.25g of polyethylene glycol (diol) diacrylate to 5ml of deionized water and stir until completely dissolved. Take another clean PDMS mask and attach it to the microarray SERS substrate obtained in step (2) (the same position as the PDMS mask in step (2)). Add 200μl of gel precursor solution to each detection channel and expose it to ultraviolet light for cross-linking polymerization. After 2 minutes, the gel microarray composite substrate is obtained.

[0045] 4) SERS signal acquisition

[0046] After the detection channel is cleaned with deionized water, the SERS spectra of each unit of the substrate are collected in sequence using a Raman spectrometer. The content of small molecules in the sample to be tested can be measured by interpreting the SERS signal.

[0047] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A SERS chip for blood neurotransmitter detection, characterized in that: The overall structure of the chip is a chip detection substrate and a PDMS mask from bottom to top; a microarray mechanism is arranged on the chip detection substrate, and metal nanoparticles are arranged in the microarray channel; a microarray structure consistent with the chip detection substrate is arranged on the PDMS mask; a gel filtration membrane with filtering properties is covered on the detection channel formed between the chip detection substrate and the PDMS mask, and the gel filtration membrane can allow smaller molecules in the blood to be tested to pass through the mesh of the gel and non-specifically bind to the surface of the metal nanoparticles; the blood to be tested is passed into each detection channel and left to stand, and the neurotransmitter content in the blood to be tested can be quantitatively analyzed by collecting the SERS spectrum of each detection area; The preparation method of the SERS chip comprises the following specific steps: preparing a microarray SERS substrate, i.e. a chip detection substrate; A PDMS mask with a microarray structure is covered on the microarray SERS substrate to form a detection channel; Adding a gel precursor solution into each detection channel and exposing it to ultraviolet light for cross-linking polymerization to form a gel filtration membrane to prepare a SERS gel composite microarray chip; The preparation of the microarray SERS substrate specifically includes: Preparation of metal nanoparticles; The PDMS mask with the microarray structure is attached to a glass sheet with sulfur bonds on the surface, and metal nanoparticles are added into the microarray channel to obtain a microarray SERS substrate. The metal nanoparticles are gold core and silver shell nanorods.

2. A SERS chip for blood neurotransmitter detection according to claim 1, characterized in that: The PDMS mask is obtained by inverting.

3. The detection method based on the SERS chip according to claim 1, characterized in that: The specific steps include: passing the blood sample to be tested into the detection channel to remove blood cells, proteins and DNA through the gel mesh structure, using a Raman spectrometer to collect the SERS spectra of the detection channel in sequence, obtaining the SERS signals of the neurotransmitters in the blood, and processing the SERS signals to obtain the types of neurotransmitters contained in the blood sample to be tested and measure their concentrations.

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