Multi-hotspot wearable dopamine detection SERS (Surface Enhanced Raman Scattering) sensor as well as preparation method and application thereof

Abstract

The invention discloses a multi-hot-spot wearable dopamine detection SERS sensor and a preparation method and application thereof.The sensor comprises a hydrogel substrate and an SERS chip loaded on the surface of the hydrogel substrate, the SERS chip comprises a solid PDMS substrate and a double-layer sandwich structure anchored through DNA hybridization, one single layer is of a multi-hot-spot structure, and the other single layer is of a multi-hot-spot structure. One single layer is a hydrophilic superlattice gold film which is an ordered array composed of double-layer gold and silver hollow nanoparticles modified with signal molecules and dopamine DNA aptamer chain segments, the thickness of the ordered array is 20-50 nm, the other single layer is a hydrophilic superlattice gold film composed of gold nanoparticles modified with dopamine DNA complementary chain segments, the thickness of the hydrophilic superlattice gold film is 15-50 nm, and the thickness of the hydrophilic superlattice gold film is 20-50 nm. The hydrophilic superlattice gold film modified with the dopamine DNA complementary chain segment is in direct contact with the solid PDMS substrate, and the solid PDMS substrate is in direct contact with the hydrogel substrate. According to the invention, high-sensitivity and high-stability detection of dopamine in sweat can be realized, and the device is convenient to wear.

Description

Technical Field

[0001] This invention relates to the fields of nanocomposite materials and biodetection technology, specifically to a multi-hotspot wearable dopamine detection SERS sensor, its preparation method, and its application. Background Technology

[0002] Dopamine (DA) is an important catecholamine neurotransmitter that plays a crucial role in physiological processes such as reward mechanisms, motor control, and mood regulation in the brain. Abnormalities in its concentration are closely related to various neuropsychiatric disorders, including Parkinson's disease, schizophrenia, and addictive disorders. Therefore, developing highly sensitive and selective dopamine detection technologies is of great significance for neuroscience research, disease diagnosis, and drug development. Traditional methods for dopamine detection include high-performance liquid chromatography, electrochemical methods, and fluorescence spectroscopy; however, these methods often suffer from drawbacks such as expensive equipment, complex operation, and susceptibility to interference from other electroactive substances.

[0003] Surface-enhanced Raman spectroscopy (SERS) is an enhancement technique based on the surface plasmon resonance effect of nanostructures, which can amplify the molecular Raman signal adsorbed on the surface of metal nanomaterials (such as gold and silver) by 10^6 times. 6 -10 14 For example, patent document CN106111974A discloses a gold-silver core-shell particle-gold nanorod self-assembly structure. This structure is formed by the self-assembly of nucleic acids from gold-silver core-shell nanoparticles and gold nanorods, and is modified with Raman beacons to enable precise quantification of dopamine.

[0004] For example, patent document CN105445252A discloses a dopamine detection method based on surface-enhanced resonance Raman spectroscopy. This method uses ferric iron as a central body to coordinate with citrate ions remaining on the surface of gold nanoparticles and dopamine molecules in the test solution. The resulting citrate-iron-dopamine complex resonates with Raman excitation light, generating a strong SERS signal, thereby achieving highly sensitive SERS detection of dopamine molecules.

[0005] SERS technology, with its advantages of high sensitivity, fingerprint specificity, and strong anti-interference ability, has become an important method for dopamine detection. However, dopamine molecules themselves have a small Raman scattering cross section and weak inherent signal, posing a serious challenge to direct detection in complex biological samples (such as sweat and cerebrospinal fluid). Although traditional rigid SERS substrates can provide strong signal enhancement, they cannot meet the requirements of wearable devices for flexibility, breathability, and long-term stability. Therefore, it is crucial to develop a wearable SERS sensor for dopamine detection that combines sensitivity, stability, and practicality. Summary of the Invention

[0006] To address the limitations of existing dopamine detection technologies in achieving a balance between sensitivity, stability, and wearability, this invention provides a multi-hotspot wearable dopamine detection SERS sensor. This sensor utilizes DNA-assembled gold and silver hollow nanoparticles combined with a hydrophilic superlattice gold film to form a multi-hotspot structure. Combined with a flexible solid PDMS substrate and a hydrogel substrate, it enables highly sensitive and stable dopamine detection while also providing convenient wearability.

[0007] The specific technical solution adopted is as follows: A wearable SERS sensor for dopamine detection with multiple hot spots includes a hydrogel substrate and a SERS chip loaded on its surface. The SERS chip includes a solid PDMS substrate and a double-layer sandwich structure anchored by DNA hybridization. One monolayer has a multiple hot spot structure and is an ordered array of double-layer gold and silver hollow nanoparticles modified with signal molecules and dopamine DNA aptamer segments, with a thickness of 20-50 nm. The other monolayer is a hydrophilic superlattice gold film modified with dopamine DNA complementary segments, with a thickness of 15-50 nm. The hydrophilic superlattice gold film is in direct contact with the solid PDMS substrate, and the solid PDMS substrate is in direct contact with the hydrogel substrate. Preferably, the particle size of the double-layer gold-silver hollow nanoparticles is 15-50 nm, and the structure includes gold nanoparticles and two layers of gold-silver alloy frameworks covering them. There are cavities between the gold nanoparticles and the first layer of gold-silver alloy frameworks, and between the first layer of gold-silver alloy frameworks and the second layer of gold-silver alloy frameworks. Preferably, the hydrophilic superlattice gold film is composed of gold nanoparticles with a particle size of 10-30 nm.

[0008] This invention constructs a SERS detection active substrate with high-density plasma hotspots by spatially and orderly assembling bilayer gold-silver hollow nanoparticles with a hydrophilic superlattice gold film, which can effectively improve the detection sensitivity. By loading a flexible solid PDMS substrate with a bilayer sandwich structure consisting of bilayer gold-silver hollow nanoparticles and a hydrophilic superlattice gold film, the stability and order of the hydrophilic superlattice gold film structure are effectively maintained, while direct contact between the bilayer sandwich structure and the uneven surface of the hydrogel substrate is avoided, thus improving the stability of the detection signal. By loading the SERS chip onto the hydrogel substrate, non-destructive adhesion between the SERS chip and the skin is achieved, enabling accurate detection of dopamine in skin sweat.

[0009] Preferably, the nucleotide sequence of the dopamine DNA aptamer segment is: 5'-GTCTCTGTGTGCGCCAGAGAACACTGGGGCAGATATGGGCCAGCACAGAATGAGGCCC-3', with a thiol group modified at the 3' end; the nucleotide sequence of the dopamine DNA complementary segment is: 5'-GGGCCTCATTCTGTGCTGGCCCATATCTGCCCCAGTGTTCTCTGGCGCACACAGAGAC-3', with a thiol group modified at the 5' end.

[0010] Preferably, cobalt chloride is coated on the hydrogel substrate near the SERS chip to detect whether a liquid sample has been collected for dopamine detection.

[0011] This invention also provides a method for preparing a multi-hotspot wearable dopamine detection SERS sensor, comprising the following steps: (1) Preparation of double-layered gold-silver hollow nanoparticles; (2) Prepare a gold nanoparticle solution, add toluene-oleylamine for phase inversion, and then induce self-assembly of the solution after phase inversion with diethylene glycol to form a superlattice gold film; (3) The superlattice gold film is attached to the solid PDMS substrate, and the attached superlattice gold film is subjected to hydrophilic treatment to obtain a hydrophilic superlattice gold film loaded on the solid PDMS substrate. (4) Modify the dopamine DNA aptamer strand and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) signal molecule on the double-layer gold-silver hollow nanoparticles prepared in step (1), and modify the dopamine DNA complementary strand on the hydrophilic superlattice gold film loaded on the solid PDMS substrate in step (3), and form a SERS chip by DNA hybridization anchoring; (5) Prepare hydrogel prepolymer, solidify and mold to obtain hydrogel substrate, and attach the SERS chip from step (4) directly onto the hydrogel substrate to obtain the multi-hotspot wearable dopamine detection SERS sensor.

[0012] Preferably, the preparation process of the double-layer gold-silver hollow nanoparticles in step (1) is as follows: First, a seed solution is prepared by CTAB, HAuCl4 and NaBH4. Then, a CTAC gold nanoparticle solution is prepared by CTAC, HAuCl4, ascorbic acid and the seed solution. Using the CTAC gold nanoparticle solution as raw material, a reaction system is constructed to reduce AgNO3 to form a silver shell on the CTAC gold nanoparticles, resulting in an Au@Ag solution. Au is partially replaced by Ag through an electro-displacement reaction to obtain an Au@Au-Ag solution. After removing the unreacted reducing agent in the system, a single-layer gold-silver hollow nanoparticle solution is obtained. The above process of reducing AgNO3-electro-displacement-removing reducing agent is repeated using the single-layer gold-silver hollow nanoparticle solution as raw material to obtain the double-layer gold-silver hollow nanoparticles.

[0013] Preferably, the gold nanoparticle solution described in step (2) is obtained by reducing chloroauric acid with trisodium citrate as a reducing agent under boiling conditions.

[0014] Preferably, the volume ratio of the gold nanoparticle solution to the toluene-oleylamine solution in step (2) is 2.5-5:1, the volume ratio of toluene to oleylamine is 1000-3400:1, and the volume ratio of the phase-inverted solution to diethylene glycol is 1:40-60.

[0015] Preferably, in step (3), a solid PDMS substrate is synthesized using a polyoxymethylene siloxane (PDMS) prepolymer and a crosslinking agent. The superlattice gold film after attachment is hydrophilized by plasma technology. The parameters of the plasma treatment are: power 50-150 W, time 30-90 s, and the reaction gas is oxygen.

[0016] Preferably, the hydrogel prepolymer liquid in step (5) is obtained by adding polyethylene glycol (PEG) to acrylic acid (HAc) and hydroxyethyl methacrylate (HEMA), and then adding a photoinitiator. The volume concentration of acrylic acid is 90-95%, the mass ratio of acrylic acid to hydroxyethyl methacrylate is 1-3:1, the mass ratio of the total amount of acrylic acid and hydroxyethyl methacrylate to polyethylene glycol is 1-3:1, the molecular weight of polyethylene glycol is 400-1000, the photoinitiator used is 2-hydroxy-2-methyl-1-phenyl-1-propanone, and the photoinitiator accounts for 0.25 wt%-0.5 wt% of the total mass of the prepolymer liquid. The curing process is completed by ultraviolet irradiation.

[0017] Furthermore, the present invention also provides a method for detecting dopamine in sweat, using the aforementioned multi-hotspot wearable dopamine detection SERS sensor.

[0018] The use of a multi-hotspot wearable dopamine detection SERS sensor to detect dopamine in sweat provides a new technical solution for dynamic monitoring of dopamine. Compared with the traditional blood sampling and irritating detection method, the non-invasive detection method of collecting sweat is gentler and more convenient to operate.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The present invention uses DNA-assembled gold and silver hollow nanoparticles and hydrophilic superlattice gold film to form a multi-hot spot structure, which enhances the initial intensity of signal molecules and achieves highly sensitive detection of low concentrations of dopamine.

[0020] (2) The present invention uses toluene phase inversion and oleylamine to modify gold nanoparticles. A dense hydrophilic superlattice gold film is formed by diethylene glycol induction, resulting in a more stable two-dimensional nanomaterial film. This film is then attached to a flat solid PDMS substrate, which greatly improves the stability of the detection results.

[0021] (3) The solid PDMS substrate and hydrogel substrate used in this invention have good flexibility, and the hydrogel can be directly adhered to human skin, making it convenient to wear directly.

[0022] (4) After coating with cobalt chloride, the successful collection of sweat samples can be directly observed. In addition, the present invention can also be equipped with drugs that promote sweat release, thereby realizing an integrated process from sampling to detection. Attached Figure Description

[0023] Figure 1 This is a TEM image of the monolayer gold-silver hollow nanoparticles in Example 1.

[0024] Figure 2 This is a TEM image of the double-layered gold-silver hollow nanoparticles in Example 1.

[0025] Figure 3 This is a TEM image of the gold nanoparticles in Example 2.

[0026] Figure 4 This is a TEM image of the hydrophilic superlattice gold film in Example 2.

[0027] Figure 5 This is a graph showing the SERS signal curve of the SERS chip in Example 4 as dopamine concentration changes.

[0028] Figure 6 This is a schematic diagram illustrating the working principle of the multi-hotspot wearable dopamine detection SERS sensor of the present invention.

[0029] Figure 7 This is a color change diagram of the multi-hotspot wearable dopamine detection SERS sensor coated with cobalt chloride in Example 6 before and after adding water.

[0030] Figure 8 This is a comparison chart of the dopamine detection signal stability between the hydrophilic superlattice gold film in Example 5 and the perfluorinated gold film in Comparative Example 1. In the figure, a represents the statistical result of the signal peak area of ​​the hydrophilic superlattice gold film in Example 5, and b represents the statistical result of the signal peak area of ​​the perfluorinated gold film in Comparative Example 1. Detailed Implementation

[0031] The present invention will be further illustrated below with reference to the embodiments and accompanying drawings. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Operating methods not specifically specified in the following embodiments are generally performed under conventional conditions or as recommended by the manufacturer.

[0032] Example 1 Preparation of double-layered gold-silver hollow nanoparticles S11. Two-step preparation of CTAC gold nanoparticles First, take 9.9 mL of CTAB (0.1 M 95% purity) and stir it appropriately with 100 μL of HAuCl4 solution with a concentration of 25 mM. After mixing thoroughly, add 450 μL of NaBH4 with a concentration of 0.01 mM (dissolved in ice water). Let it stand in a 30 ℃ water bath for three hours. During the standing process, seal it with sealing film to obtain the seed liquid.

[0033] The second step involves mixing 1.5 mL of AA (100 mM) solution and 2 mL of CTAC (200 mM), adding 50 μL of the seed solution prepared in the first step, and stirring at a constant speed for 10 min in a 50 mL beaker until the mixture is homogeneous. Then, immediately and quickly inject 2 mL of HAuCl4 (0.5 mM) solution and stir at a constant speed for 10 min. The solution changes from light brown to wine red, yielding a CTAC gold nanoparticle solution with a particle size of approximately 10 nm.

[0034] S12. Synthesis of monolayer gold-silver hollow nanoparticles Take 5 mL of the aqueous CTAC gold nanoparticle solution synthesized in step S11 and mix it with 5 mL of 0.1 M CTAC. Stir thoroughly in a 20 mL transparent glass bottle at approximately 600 rpm for 10 min in a 60 ℃ water bath. During this process, the solution gradually changes from pink to yellow. Add 1.25 mL of 10 mM AgNO3 and 1.25 mL of 100 mM AA to the solution and react for 4 h under stirring in a 60 ℃ water bath. The resulting solution is brownish-yellow. Centrifuge the reacted solution at 12000 rpm for 10 min using a pointed-bottom centrifuge tube, remove the supernatant, and redisperse the precipitate in 5 mL of deionized water to obtain the Au@Ag solution.

[0035] Take 5 mL of Au@Ag solution and mix it with 5 mL of 0.1 M CTAC in a 20 mL transparent glass bottle. The solution turns brownish-yellow. Under vigorous stirring at 1200 rpm, add 250 μL of 10 mM NaOH solution to adjust the pH of the system to meet the optimal conditions for the electrodisplacement reaction. At the same time, add 250 μL of 100 mM AA and 250 μL of 10 mM HAuCl4. The solution quickly changes from brownish-yellow to deep blue-purple. After adding the above reagents, resume slow stirring at 300 rpm and stir for 15 min at room temperature. After obtaining the solution, centrifuge at 11000 rpm for 10 min, remove the supernatant and redisperse it in 5 mL of deionized water to obtain Au@Au-Ag solution. Place it in a 50 mL centrifuge tube to provide sufficient reaction space.

[0036] Add 500 μL of 30% H2O2 solution and let stand at room temperature for 12 h to allow unreacted reducing agent AA to be completely consumed by H2O2. The final solution is a deep blue-purple color. Centrifuge at 12000 rpm for 10 min and redisperse in 5 mL of deionized water to obtain a monolayer gold-silver hollow nanoparticle solution. The TEM image of the monolayer gold-silver hollow nanoparticles is shown below. Figure 1 As shown, coating a single gold nanoparticle with a gold-silver alloy framework results in more signal hotspots compared to bare gold nanoparticles.

[0037] S13. Synthesis of bilayer gold-silver hollow nanoparticles The raw material aqueous CTAC gold nanoparticle solution in step S12 was replaced with the monolayer gold-silver hollow nanoparticle solution synthesized in step S12. Step S12 was repeated to obtain a bilayer gold-silver hollow nanoparticle solution. The TEM image of the bilayer gold-silver hollow nanoparticles is shown below. Figure 2As shown, its structure includes gold nanoparticles and a two-layer gold-silver alloy framework covering them. There are cavities between the gold nanoparticles and the first gold-silver alloy framework, and between the first gold-silver alloy framework and the second gold-silver alloy framework. The particle size range of the double-layer gold-silver hollow nanoparticles is 25-40 nm.

[0038] Example 2: Preparation of hydrophilic superlattice gold film S21. Preparation of gold nanoparticles Measure 90-110 mL of water into a three-necked flask, set up a condenser, and set the temperature to 110 °C. Stir slowly at 1200 rpm. Cover the top of the condenser with a piece of aluminum foil with a small opening to prevent dust from entering. After rapidly injecting 1 mL of HAuCl4·3H2O (25 mM), there was no obvious color change. Heat to boiling, then rapidly inject 1 mL of 0.03 g / mL trisodium citrate. Maintain the temperature for 30 min. The mixed solution changed color from colorless and transparent to deep purple, and finally to a wine-red liquid, yielding the gold nanoparticle solution. The TEM image of the gold nanoparticles is shown below. Figure 3 As shown, the synthesized particles have a diameter of 15-20 nm and are uniform in size.

[0039] S22. Preparation of superlattice gold films Take 5 mL of gold nanoparticle solution into a 15 mL centrifuge tube, add 1 mL of pre-prepared toluene-oleylamine solution (10 mL toluene corresponds to 3.3 μL oleylamine). The solution shows a clear, transparent upper layer and a pink lower layer. Stir thoroughly for 3 min on a vortex mixer. After stirring, the upper layer turns red and the lower layer becomes clear, indicating successful phase inversion. Take the upper phase-inverted solution and aliquot it into a new 15 mL centrifuge tube for sedimentation for 8 h to separate unmodified particles. Add approximately 45 mL of pre-diluted diethylene glycol solution (diethylene glycol to deionized water volume ratio 10:1) to a 50 mL plastic centrifuge tube, add 1.5 mL of the sedimented particles, and wait for the toluene to fully evaporate to obtain a dense superlattice gold film. Its TEM image is shown below. Figure 4 As shown, gold nanoparticles are tightly packed at the interface between water and diethylene glycol, forming a dense two-dimensional film. The thickness of the superlattice gold film is approximately 15 nm.

[0040] Example 3: Preparation of hydrophilic superlattice gold films supported on solid PDMS substrates S31. Preparation of solid PDMS substrate A polyoxymethylene siloxane (PDMS) prepolymer solution and crosslinking agent were mixed at a ratio of 10:1 and stirred thoroughly. The mixture was then poured into a large petri dish with a diameter of 21 cm and allowed to stand until there were almost no obvious bubbles. The solution was then placed in a 60 ℃ oven to cure for 2 h to form a solid. Solid PDMS substrates of 0.3 cm × 0.3 cm were cut out using a cutter.

[0041] S32. Combination and hydrophilic treatment The superlattice gold film synthesized in Example 2 was attached to the solid PDMS substrate prepared in S31. After rinsing the surface of the superlattice gold film with deionized water, the surface was treated with oxygen as the reaction gas in a plasma machine at a power of 100 W for 30 s to make it hydrophilic.

[0042] Figure 8 'a' represents the SERS detection result of the hydrophilic superlattice gold film supported on a solid PDMS substrate in this embodiment. Using 4-MBA signal molecules, detection was performed at different locations on the hydrophilic superlattice gold film at the same dopamine concentration, and corresponding 4-MBA characteristic signal peaks were obtained. By statistically integrating the characteristic peak areas, it was found that the signal distribution is uniform and the intensity is stable, proving that the hydrophilic superlattice gold film has high-density plasma hotspots and excellent uniformity.

[0043] Example 4: Fabrication of SERS Chip S41. The bilayer gold-silver hollow nanoparticle solution prepared in Example 1 was incubated with a dopamine DNA aptamer chain segment (SEQ ID NO.1: 5'-GTCTCTGTGTGCGCCAGAGAACACTGGGGCAGATATGGGCCAGCACAGAATGAGGCCC-3', with the 3' end modified with a thiol group) for 12 h. After incubation, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) signaling molecules were added and incubation continued for another 12 h.

[0044] S42. The hydrophilic superlattice gold film loaded on the solid PDMS substrate prepared in Example 2 was incubated with a dopamine DNA complementary strand (SEQ ID NO.2: 5'-GGGCCTCATTCTGTGCTGGCCCATATCTGCCCCAGTGTTCTCTGGCGCACACAGAGAC-3', with the 5' end modified with a thiol group) for 12 h.

[0045] S43. A solution of bilayer gold-silver hollow nanoparticles carrying DTNB signal molecules, incubated beforehand, was dropped onto a hydrophilic superlattice gold film supported on an incubated solid PDMS substrate. Both layers were incubated for 12 h to form a SERS chip containing the initial DTNB signal peak. After incubation, the thickness of the resulting bilayer gold-silver hollow nanoparticle array was approximately 45 nm. The SERS signal curve of the SERS chip as a function of dopamine concentration is shown below. Figure 5 As shown, when DTNB is used as a signaling molecule, and dopamine is introduced into the system, dopamine competes with the DTNB-containing double-layer gold-silver hollow nanoparticles for sites on the hydrophilic superlattice gold film, resulting in a decrease in signal intensity. By plotting curves for different concentrations, the relationship between concentration and signal intensity is obtained.

[0046] Example 5: Fabrication of a multi-hotspot wearable dopamine detection SERS sensor S51. Preparation of hydrogel prepolymer solution Acrylic acid (HAc) and hydroxyethyl methacrylate (HEMA) were mixed in a 1:1 mass ratio, resulting in an acrylic acid volume concentration of 95%. An equal mass of polyethylene glycol (molecular weight 400) solution was then added and thoroughly mixed. 0.5% by mass of the photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone (i1173) was added to the mixture to prepare a hydrogel prepolymer solution.

[0047] S52. Preparation of hydrogel substrate Commercially available 1.5 mm thick silicone was cut into square sheets measuring 6 cm long, 6 cm wide, and 1.5 mm thick, with a 5 cm long and 5 cm wide square section pre-cut out of each sheet. Two rectangular silicone strips, 3 cm long, 0.5 cm wide, and 0.5 mm thick, were used as pre-prepared constraint molds for the SERS chip and placed in the cut-out areas of the silicone. The cut-out silicone was then sandwiched between two square glass sheets, each 6 cm long and 6 cm wide, with a layer of silicone on their surface to prevent adhesion. The hydrogel prepolymer solution prepared in S51 was added to the mold, and the entire mold was secured using clamps. The mold was irradiated under a 20 W 352 nm UV lamp for 2 hours, after which the mold was removed to form the hydrogel substrate.

[0048] S53. Composition of a multi-hotspot wearable dopamine detection SERS sensor Two SERS chips prepared in Example 4 were taken as parallel groups and attached to the prepared hydrogel substrate to form a multi-hotspot wearable dopamine detection SERS sensor. Figure 6The diagram illustrates the working principle of the multi-hotspot wearable dopamine detection SERS sensor of this invention. Dopamine DNA aptamer segments and signal molecules are modified onto double-layered gold-silver hollow nanoparticles, forming a double-layered sandwich structure with a superlattice gold film modified with dopamine DNA complementary segments. Solid PDMS serves as the supporting substrate for this double-layered sandwich structure, which is then adhered to a hydrogel substrate to form the multi-hotspot wearable dopamine detection SERS sensor. The sensor works by allowing a small amount of dopamine in the analyte to compete with the double-layered gold-silver hollow nanoparticles carrying signal molecules for sites on the dopamine DNA complementary segments, causing the signal molecules to detach from the substrate, ultimately resulting in a decrease in the SERS signal.

[0049] Example 6: Fabrication of a wearable SERS sensor for dopamine detection with multiple hotspots and a sweat indicator area. Cobalt chloride (CoCl3) was prepared into a 100 g / L solution, which was pink in color. This cobalt chloride solution was then coated around the SERS chip on the hydrogel substrate of the multi-hotspot wearable dopamine detection SERS sensor prepared in Example 5. After the moisture evaporated completely, a sensor with an indicator block was obtained. Figure 8 As shown, a represents the dry state, where the color is dark blue, while b represents the state when there is sweat inside the chip, where the blue part has faded significantly.

[0050] Comparative Example 1 This comparative example uses a perfluorinated gold film instead of a superlattice gold film. A perfluorinated solution was prepared by adding perfluorodecyl mercaptan (50:1 ratio) to a mixed solution of ethanol and cyclohexane (ethanol to cyclohexane ratio 2:1). Due to the difference in the ethanol-cyclohexane interface and the reduction of the surface energy of the gold nanoparticles by the perfluorinated solution, the gold nanoparticles align at the interface between the two phases. The prepared perfluorinated gold film was attached to a solid PDMS substrate and subjected to the same hydrophilication treatment as the superlattice film before detection.

[0051] Figure 8 This is a comparison of the dopamine detection signal stability between the hydrophilic superlattice gold film in Example 5 and the perfluorinated gold film in Comparative Example 1. Figure b shows the statistical results of the signal peak area of ​​the perfluorinated gold film in Comparative Example 1. The results show that, compared with the statistical results of the signal peak area of ​​the hydrophilic superlattice gold film, its signal distribution is uneven and the signal intensity is lower. This indicates that the gold nanoparticles in the hydrophilic superlattice gold film are modified with spacing-regulating ligands, enabling the particles to form a uniform arrangement through stronger chemical bonding forces, thereby generating more uniformly distributed hot spots and improving the stability of the dopamine detection results.

[0052] The embodiments described above provide a detailed explanation of the technical solutions of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, additions, or similar substitutions made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A multi-hotspot wearable dopamine detection SERS sensor, characterized in that, The SERS chip includes a hydrogel substrate and its surface loading. The SERS chip includes a solid PDMS substrate and a bilayer sandwich structure anchored by DNA hybridization. One monolayer has a multi-hotspot structure and is an ordered array of bilayer gold and silver hollow nanoparticles modified with signal molecules and dopamine DNA aptamer segments, with a thickness of 20-50 nm. The other monolayer is a hydrophilic superlattice gold film modified with dopamine DNA complementary segments, with a thickness of 15-50 nm. The hydrophilic superlattice gold film is in direct contact with the solid PDMS substrate, and the solid PDMS substrate is in direct contact with the hydrogel substrate. The double-layer gold-silver hollow nanoparticles have a particle size of 15-50 nm and a structure consisting of gold nanoparticles and two layers of gold-silver alloy frameworks covering them. There are cavities between the gold nanoparticles and the first layer of gold-silver alloy frameworks, and between the first layer of gold-silver alloy frameworks and the second layer of gold-silver alloy frameworks. The hydrophilic superlattice gold film is composed of gold nanoparticles with a particle size of 10-30 nm.

2. The multi-hotspot wearable dopamine detection SERS sensor according to claim 1, characterized in that, The nucleotide sequence of the dopamine DNA aptamer segment is: 5'-GTCTCTGTGTGCGCCAGAGAACACTGGGGCAGATATGGGCCAGCACAGAATGAGGCCC-3', with a thiol group modified at the 3' end; the nucleotide sequence of the dopamine DNA complementary segment is: 5'-GGGCCTCATTCTGTGCTGGCCCATATCTGCCCCAGTGTTCTCTGGCGCACACAGAGAC-3', with a thiol group modified at the 5' end.

3. The multi-hotspot wearable dopamine detection SERS sensor according to claim 1, characterized in that, Cobalt chloride was coated onto the hydrogel substrate near the SERS chip to detect whether a liquid sample had been collected for dopamine detection.

4. The method for preparing a multi-hotspot wearable dopamine detection SERS sensor according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Preparation of double-layered gold-silver hollow nanoparticles; (2) Prepare a gold nanoparticle solution, add toluene-oleylamine for phase inversion, and then induce self-assembly of the solution after phase inversion with diethylene glycol to form a superlattice gold film; (3) The superlattice gold film is attached to the solid PDMS substrate, and the attached superlattice gold film is subjected to hydrophilic treatment to obtain a hydrophilic superlattice gold film loaded on the solid PDMS substrate. (4) Modify the dopamine DNA aptamer strand and 5,5'-dithiobis(2-nitrobenzoic acid) signal molecule on the double-layer gold-silver hollow nanoparticles prepared in step (1), and modify the dopamine DNA complementary strand on the hydrophilic superlattice gold film loaded on the solid PDMS substrate in step (3), and form a SERS chip by DNA hybridization anchoring. (5) Prepare hydrogel prepolymer, solidify and mold to obtain hydrogel substrate, and attach the SERS chip from step (4) directly onto the hydrogel substrate to obtain the multi-hotspot wearable dopamine detection SERS sensor.

5. The method for preparing a multi-hotspot wearable dopamine detection SERS sensor according to claim 4, characterized in that, The preparation process of the double-layer gold-silver hollow nanoparticles described in step (1) is as follows: First, a seed solution is prepared by CTAB, HAuCl4 and NaBH4. Then, a CTAC gold nanoparticle solution is prepared by CTAC, HAuCl4, ascorbic acid and the seed solution. Using the CTAC gold nanoparticle solution as raw material, a reaction system is constructed to reduce AgNO3 to form a silver shell on the CTAC gold nanoparticles, resulting in an Au@Ag solution. Au is partially replaced by Ag through an electro-displacement reaction to obtain an Au@Au-Ag solution. After removing the unreacted reducing agent in the system, a single-layer gold-silver hollow nanoparticle solution is obtained. The above process of reducing AgNO3-electro-displacement-removing reducing agent is repeated using the single-layer gold-silver hollow nanoparticle solution as raw material to obtain the double-layer gold-silver hollow nanoparticles.

6. The method for preparing a multi-hotspot wearable dopamine detection SERS sensor according to claim 4, characterized in that, The gold nanoparticle solution described in step (2) is obtained by reducing chloroauric acid with trisodium citrate as a reducing agent under boiling conditions.

7. The method for preparing a multi-hotspot wearable dopamine detection SERS sensor according to claim 4, characterized in that, In step (2), the volume ratio of gold nanoparticle solution to toluene-oleylamine solution is 2.5-5:1, and the volume ratio of toluene to oleylamine is 1000-3400:

1.

8. The method for preparing a multi-hotspot wearable dopamine detection SERS sensor according to claim 4, characterized in that, In step (3), a solid PDMS substrate is synthesized using polyoxysiloxane prepolymer and crosslinking agent. The superlattice gold film after attachment is hydrophilized by plasma technology. The parameters of the plasma treatment are: power 50-150 W, time 30-90s, and the reaction gas is oxygen.

9. The method for preparing a multi-hotspot wearable dopamine detection SERS sensor according to claim 4, characterized in that, The hydrogel prepolymer liquid described in step (5) is obtained by adding polyethylene glycol to acrylic acid and hydroxyethyl methacrylate, and then adding a photoinitiator. The volume concentration of acrylic acid is 90-95%, the mass ratio of acrylic acid to hydroxyethyl methacrylate is 1-3:1, the mass ratio of the total amount of acrylic acid and hydroxyethyl methacrylate to polyethylene glycol is 1-3:1, the molecular weight of polyethylene glycol is 400-1000, the photoinitiator used is 2-hydroxy-2-methyl-1-phenyl-1-propanone, and the photoinitiator accounts for 0.25 wt%-0.5 wt% of the total mass of the prepolymer liquid. The curing process is completed by ultraviolet irradiation.

10. A method for detecting dopamine in sweat, characterized in that, Use the multi-hotspot wearable dopamine detection SERS sensor as described in any one of claims 1-3.

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