Electrochemical sensor based on nanogold enhanced molecular imprinting as well as preparation method and application of electrochemical sensor

Abstract

The invention discloses an electrochemical sensor based on nanogold enhanced molecular imprinting and a preparation method and application thereof. Ti / Au is sputtered on a PET substrate to form a conductive layer; the preparation method comprises the following steps: electrically depositing gold nanoparticles on a conductive layer to form a fern-shaped structure, electrically depositing a molecularly imprinted polymer film on the surface of the fern-shaped structure, and then eluting, removing template molecules and forming a specific cavity. A fern-shaped structure composed of nano-gold particles is electrically deposited on the gold substrate, the specific surface area is large, active catalytic sites on the surface of a working electrode are increased, more attachment points are provided for a molecularly imprinted polymer film, and the sensitivity and stability of the electrochemical sensor are improved; according to the invention, the wearable flexible electrochemical sensor with three different working electrodes is prepared on the same substrate through a screen printing technology, so that simultaneous detection of three depression biomarkers in artificial sweat is realized, and the pressure condition of a depression patient is reflected more accurately.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of electrochemical biosensors, and particularly relates to a gold nanoparticle-enhanced molecular imprinting-based electrochemical sensor and a preparation method and application thereof. TECHNICAL BACKGROUND

[0002] With the development of society and the progress of technology, the pace of life of people is getting faster and faster today, which has brought many psychological problems such as depression, anxiety and the like. Depression, as a common mental illness, has plagued hundreds of millions of patients, causing a very serious hindrance to their life and work. The content of depression biomarkers in the human body can provide effective help for the diagnosis of depression and the stress monitoring of depression patients. For example, glutamine can be used to improve brain function, anti-depression, etc., and can be converted into gamma-aminobutyric acid (GABA). GABA, as an inhibitory neurotransmitter, promotes the relaxation and calmness of the nervous system, and relieves anxiety, fear, etc. Tryptophan can be converted into serotonin, melatonin and vitamin b3, etc. in the human body. When the content of tryptophan in the human body is lower than the normal value, it may affect sleep and cause anxiety or depression. Therefore, the content of these substances related to depression in the human body can well reflect the physical condition and psychological state of an individual. However, the traditional detection often uses invasive methods such as blood sampling. This traditional detection method relies on professional and large medical equipment, and may cause other infections to the patient. Wearable devices give a new answer to solve these problems, and it is crucial for personal stress management to wear a wearable flexible electrochemical sensor to detect these biomarkers in real time and non-invasively.

[0003] Chinese invention patent application with publication number CN119688799A discloses a preparation method and application of a MoS2-Ti / Au modified PET film electrode molecular imprinting electrochemical sensor. The preparation method is to sputter Ti and Au metals on the surface of the PET film, coat a MoS2 film on the surface of the electrode, and then electrodeposit a molecular imprinting film on the surface of the MoS2-Ti / Au electrode material. However, the method for increasing the active sites on the surface of the electrode in this scheme is to coat a MoS2 film. This film formed by coating is not stable and will fall off when the electrode is bent. Moreover, the film often falls off when tested in an electrolyte, so that the sensor cannot be used continuously, affecting the service life of the electrode. SUMMARY

[0004] In order to overcome the above-mentioned defects in the prior art, the purpose of the present application is to provide a kind of electrochemical sensor based on nano gold enhanced molecular imprinting and its preparation method and application, by the fern structure of nano gold particle on gold substrate (PET / Ti / Au) Electrodeposition, with larger specific surface area, can increase active catalytic site on the surface of working electrode, provide more attachment point for molecular imprinting polymer film, broaden the detection range of electrochemical sensor and the minimum detection limit is as low as 0.612 μM, with higher detection sensitivity.In addition, the lattice matching degree of the nano gold particles prepared in the present application and the gold substrate is high, and the connection between them is also very firm.The nano gold structure formed in the present application is relatively stable, and the electrode can still maintain integrity after bending or long-term immersion in liquid environment.The electrochemical sensor prepared in the present application has a detection range of tryptophan of 1 μM-100 μM, and the minimum detection limit is as low as 0.612 μM;In addition, the electrochemical sensors of two other biomarkers related to depression, glutamine (10 μM-80 μM) and GABA (1 μM-10 μM) can be prepared under the same technical system, and have excellent sensing characteristics and anti-interference ability.The electrochemical sensor with three different working electrodes can be prepared on the same substrate by screen printing technology, and the simultaneous detection of three biomarkers of depression in artificial sweat can be realized.Compared with detecting only a single substance, multi-substance combined detection can more accurately reflect the stress condition of patients with depression.

[0005] To achieve the above object, the technical scheme adopted by the present application is as follows:

[0006] A preparation method of an electrochemical sensor based on nano gold enhanced molecular imprinting, comprising the following steps:

[0007] Step 1: sputter Ti and Au on the surface of the cleaned PET substrate in sequence to obtain a PET / Ti / Au composite electrode;

[0008] Step 2: using PET / Ti / Au as working electrode, using a mixed solution of HAuCl4 with a molar concentration of 0.5-1.5 mmol / L and glycine with a concentration of 5-25 mmol / L as deposition solution, depositing nano gold particles on the surface of PET / Ti / Au composite electrode by constant potential method to obtain a PET / Ti / Au / AuNHS composite electrode with fern structure; by volume ratio, HAuCl4: glycine = 1: (25-35);

[0009] Step 3: using PET / Ti / Au / AuNHS composite electrode as working electrode, using molecular imprinting polymerization solution as electrolyte, depositing molecular imprinting polymer film on the surface of PET / Ti / Au / AuNHS composite electrode by cyclic voltammetry to obtain a PET / Ti / Au / AuNHS / MIP composite electrode;

[0010] Step 4: elution is carried out on the PET / Ti / Au / AuNHS / MIP composite electrode to remove the template molecules on the molecularly imprinted polymer film, so as to form specific cavities, and an electrochemical sensor based on the nano-gold enhanced molecular imprinting is obtained.

[0011] In step 1, a Ti layer with a thickness of 25 nm and an Au layer with a thickness of 50 nm are sequentially deposited on the surface of the cleaned PET substrate by a magnetron sputtering process to form a conductive layer.

[0012] In step 2, the deposition potential is -0.15 to -0.05 v, and the deposition time is 3000-4000 s.

[0013] In step 3, the preparation process of the molecular imprinting polymer solution is as follows: the template molecules are ultrasonically decomposed in a PBS solution to obtain a template molecule solution; pyrrole is dissolved in ethanol to obtain a pyrrole solution with a molar concentration of 50-100 mM; the template molecule solution and the pyrrole solution are stirred for 30-60 min to obtain the molecular imprinting polymer solution; the volume ratio of the template molecule solution to the pyrrole solution is (10-20):1; and the template molecules in the molecular imprinting polymer solution are one of tryptophan with a molar concentration of 10-20 mM, glutamine with a molar concentration of 10-20 mM and GABA with a molar concentration of 20-40 mM.

[0014] In step 3, the scanning potential is -0.3 v to 0.9 v, the scanning speed is 50-150 mv / s, and the scanning number is 5-15.

[0015] In step 4, the elution process is as follows: the PET / Ti / Au / AuNHS / MIP composite electrode is placed in a 1×PBS solution with a pH of 7.2-7.6, and elution is carried out at a potential of 0.8-1.2 v for 300-600 s.

[0016] The electrochemical sensor comprises a PET substrate, a Ti / Au layer is arranged on the surface of the PET substrate, a fern-shaped structure composed of nano-gold particles is arranged on the Ti / Au layer, and a molecularly imprinted polymer film with specific cavities is arranged on the surface of the fern-shaped structure; the molecularly imprinted polymer film is a polymer film of pyrrole and a template molecule, and the template molecule is one of tryptophan, glutamine and GABA.

[0017] The application further provides the electrochemical sensor prepared by the preparation method of the electrochemical sensor based on the nano-gold enhanced molecular imprinting, and the application of the electrochemical sensor in the detection of biomarkers of depression.

[0018] The application further provides a preparation method of a wearable flexible electrochemical sensor integrated with three working electrodes, which comprises the following steps:

[0019] S1: Cover the mask plate of the working electrode on the cleaned PET substrate, and sequentially deposit Ti and Au by using a magnetron sputtering process, and repeat the operation three times by changing the sputtering position to obtain three PET / Ti / Au composite electrodes;

[0020] S2: A connecting wire, a counter electrode, a reference electrode and an insulating layer are sequentially prepared on the PET substrate by using a screen printing process.

[0021] S3: Three PET / Ti / Au / AuNHS / MIP / Elu composite electrodes for specifically recognizing tryptophan, glutamine and GABA are prepared on the surfaces of the three PET / Ti / Au composite electrodes obtained in step S1 by using the preparation method of the electrochemical sensor based on the nanogold-enhanced molecular imprinting, and a wearable flexible electrochemical sensor integrated with three working electrodes is obtained; the template molecule solutions in the three molecular imprinting polymerization solutions are respectively 10-20 mM of tryptophan, 10-20 mM of glutamine and 20-40 mM of GABA.

[0022] The application further provides the wearable flexible electrochemical sensor prepared by the preparation method of the wearable flexible electrochemical sensor integrated with three working electrodes, and the wearable flexible electrochemical sensor is applied to the detection of biomarkers of depression.

[0023] Compared with the prior art, the application has the following advantages:

[0024] 1. The fern-shaped structure composed of nanogold particles is electrodeposited on a gold substrate, the gold substrate itself has high conductivity, can provide uniform current distribution for the electrodeposition process, promote uniform nucleation and growth of nanogold particles, and form a fern-shaped structure. In addition, the gold substrate has high lattice matching degree with nanogold particles, can reduce interface defects, and improves the stability of the electrode structure. In addition, the gold substrate is not easy to oxidize or corrode in an electrochemical environment, and is suitable for long-term stable device applications (such as sensors).

[0025] 2. This invention forms a fern-like structure by electrodepositing gold nanoparticles on the working electrode surface, followed by electrodepositing a molecularly imprinted polymer (MIP) film on the fern-like structure. The hyperbranched three-dimensional dendritic structure of the fern-like structure provides an exponentially increased specific surface area compared to spherical nanoparticles or flat electrode surfaces. This increased surface area supports more pyrrole monomers for electropolymerization, resulting in a higher density of imprinted cavities complementary to the template molecules within the MIP matrix, leading to a lower detection limit for the sensor. The complex texture of the fern-like structure provides more adhesion points for the MIP film during electropolymerization, preventing delamination and peeling of the fragile film during repeated measurements, template elution, or washing, significantly improving sensor operational stability and result reproducibility. The fern-like structure composed of gold nanoparticles forms a highly conductive interconnect network, significantly improving the electron transfer efficiency between the detection solution and the working electrode. This invention can be directly adapted to the detection of other biomarkers (such as glutamine and GABA) by replacing the template molecules in the MIP, without requiring a redesign of the underlying nanotransduction layer.

[0026] 3. By electrodepositing a fern-like structure composed of gold nanoparticles on the surface of the working electrode, the response current of the electrochemical sensor is significantly increased, the detection range is widened, and the lowest detection limit is as low as 0.612 μM. Compared with other types of nanoparticles (such as MoS2, TiO2), gold nanoparticles have better stability.

[0027] 4. This invention utilizes screen printing technology to fabricate a wearable flexible electrochemical sensor with three different working electrodes on a single substrate. This sensor simultaneously detects three biomarkers of depression in artificial sweat, providing a more accurate reflection of the stress status of patients with depression compared to traditional independent three-electrode systems. Furthermore, the screen printing process enables the electrochemical sensor to be miniaturized and wearable, achieving non-invasive, real-time human stress monitoring. This wearable flexible electrochemical sensor holds promise for detecting changes in the concentration of depression biomarkers in human sweat, providing real-time and accurate stress monitoring for patients with depression.

[0028] In summary, this invention provides an electrochemical sensor based on gold nanoparticle-enhanced molecular imprinting, obtained by electrodepositing a fern-like structure composed of gold nanoparticles on a gold substrate, electrodepositing a molecularly imprinted polymer film on the surface of the fern-like structure, and eluting the resulting film. This sensor can specifically identify three biomarkers of depression: tryptophan, glutamine, and GABA, exhibiting good reproducibility, high sensitivity, and stability. Furthermore, this invention uses screen printing technology to fabricate a wearable flexible electrochemical sensor with three different working electrodes on the same substrate, enabling simultaneous detection of the three biomarkers of depression in artificial sweat, thus more accurately reflecting the stress status of patients with depression. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 is a preparation flow chart of the gold nanoparticle-based molecular imprinting electrochemical sensor of the present application.

[0030] Figure 2 is a structural schematic diagram of the wearable flexible electrochemical sensor integrating three working electrodes provided by the present application.

[0031] Figure 3 is a physical diagram of different bending directions of the wearable flexible electrochemical sensor integrating three working electrodes provided by the present application; wherein, Figure 3 A is outward bending, Figure 3 B is inward bending.

[0032] Figure 4 is a SEM diagram of different modified electrodes provided by Example 1; wherein, Figure 4 A is a PET / Ti / Au sample; Figure 4 B is a PET / Ti / Au / AuNHS sample; Figure 4 C is a PET / Ti / Au / AuNHS / MIP sample; Figure 4 D is a PET / Ti / Au / AuNHS / MIP / ELU sample.

[0033] Figure 5 is a FTIR spectrum diagram of different modified electrodes provided by Example 1.

[0034] Figure 6 is a XRD spectrum diagram of different modified electrodes provided by Example 1.

[0035] Figure 7 is a XPS spectrum of N1s of different modified electrodes provided by Example 1; wherein, Figure 7 A is a PET / Ti / Au / AuNHS sample; Figure 7 B is a PET / Ti / Au / AuNHS / MIP sample; Figure 7 C is a PET / Ti / Au / AuNHS / MIP / ELU.

[0036] Figure 8 is a CV curve of different modified electrodes provided by Example 1 and Comparative Example 1.

[0037] Figure 9 is an EIS curve of different modified electrodes provided by Example 1 and Comparative Example 1.

[0038] Figure 10 is a SWV current response and corresponding calibration curve of different to-be-detected substances detected by the wearable flexible electrochemical sensor integrating three working electrodes in a PBS solution; wherein, Figure 10A is the SWV current response of tryptophan in the range of 1-100 μM; Figure 10 B is the SWV current response of Glu in the range of 10-80 μM; Figure 10 C is the SWV current response of GABA in the range of 1-10 μM; Figure 10 D is the calibration curve of tryptophan; Figure 10 E is the calibration curve of Glu; Figure 10 F is the calibration curve of GABA.

[0039] Figure 11 is the specificity of the electrochemical sensor provided in Examples 1-3.

[0040] Figure 12 is the reproducibility of the electrochemical sensor provided in Example 1.

[0041] Figure 13 is the stability of the electrochemical sensor provided in Example 1.

[0042] Figure 14 is the SWV current response of different analytes in artificial sweat and the corresponding calibration curves detected by a wearable flexible electrochemical sensor integrating three working electrodes; wherein, Figure 14 A is the SWV current response of tryptophan in the range of 5-100 μM; Figure 14 B is the SWV current response of Gln in the range of 10-80 μM; Figure 14 C is the SWV current response of GABA in the range of 1-8 μM; Figure 14 D is the calibration curve of tryptophan; Figure 14 E is the calibration curve of Gln; Figure 14 F is the calibration curve of GABA. DETAILED DESCRIPTION

[0043] The technical solutions of the present application will be further described below in combination with the drawings and examples.

[0044] An electrochemical sensor based on nano-gold enhanced molecular imprinting is used for specific identification of one of three depression biomarkers of tryptophan (L-TRP), glutamine (Gln), and GABA; comprising a substrate, the surface of the substrate is provided with a conductive layer, the conductive layer is provided with a fern-shaped structure composed of nano-gold particles, and the surface of the fern-shaped structure is modified with a molecular imprinting polymer film having a specific cavity; the substrate is a flexible PET film; the conductive layer is a Ti / Au layer deposited by a magnetron sputtering process; the molecular imprinting polymer film is a polymer film of pyrrole and a template molecule, and the template molecule is one of tryptophan, glutamine, and GABA.

[0045] As Figure 1As shown, a preparation method of an electrochemical sensor based on gold nanoparticle enhanced molecular imprinting, comprising the following steps:

[0046] Step 1: sequentially perform ultrasonic cleaning and oxygen plasma cleaning on the flexible PET substrate;

[0047] Specifically, the flexible PET substrate is cut into a shape of 8cm*8cm, then immersed in ethanol, cleaned using an ultrasonic cleaning machine at 80% power for 20min, then immersed in deionized water, cleaned using an ultrasonic cleaning machine at 80% power for 20min, after ultrasonic cleaning, dried using an N2 gun, and then oxygen plasma cleaned at a power of 30w for 30s;

[0048] Step 2: sequentially deposit a Ti layer with a thickness of 25nm and an Au layer with a thickness of 50nm on the surface of the cleaned PET substrate by a magnetron sputtering process to form a conductive layer, thereby obtaining a PET / Ti / Au composite electrode; the Ti layer with a thickness of 25nm is an adhesion layer between the PET substrate and the Au layer, which can promote the firm combination between the Au layer and the substrate and prevent the Au layer from falling off;

[0049] The process parameters of the magnetron sputtering are as follows: the chamber pressure for sputtering Ti is 1.6pa, the radio frequency power is 95w, and the sputtering time is 55s; the chamber pressure for sputtering Au is 0.3pa, the radio frequency power is 90w, and the sputtering time is 50s;

[0050] Step 3: in a three-electrode system, take the PET / Ti / Au as a working electrode, take a platinum sheet as a counter electrode, take Ag / AgCl as a reference electrode, take a mixed solution of HAuCl4 with a molar concentration of 0.5-1.5mmol / L and Gly with a concentration of 5-25mmol / L as a deposition liquid, and deposit gold nanoparticles on the surface of the PET / Ti / Au composite electrode by a constant potential method to obtain a PET / Ti / Au / AuNHS composite electrode; the deposition potential is-0.15 to-0.05v, and the deposition time is 3000-4000s; after the gold nanoparticles are deposited on the surface of the PET / Ti / Au composite electrode, a fern-shaped nano dendrite is formed, which can increase the specific surface area of the working electrode; the ratio of HAuCl4 to Gly is 1:(25-35) by volume;

[0051] Step 4: taking PET / Ti / Au / AuNHS composite electrode as working electrode, Ag / AgCl as reference electrode, platinum sheet as counter electrode, and taking molecular imprinting polymerization solution as electrolyte, a PET / Ti / Au / AuNHS / MIP composite electrode is obtained by depositing a molecular imprinting polymer film on the surface of the PET / Ti / Au / AuNHS composite electrode by cyclic voltammetry, wherein the scanning potential range of the cyclic voltammetry is -0.3v-0.9v, the scanning speed is 50-150mv / s, and the scanning number is 5-15;

[0052] The molecular imprinting polymer film is a polymer film of pyrrole and template molecules; the preparation process of the molecular imprinting polymerization solution is as follows: the template molecules are dissolved in PBS solution and fully dissolved by ultrasonic treatment to obtain a template molecule solution; pyrrole is dissolved in ethanol to obtain a pyrrole solution with a molar concentration of 50-100mM; the template molecule solution and the pyrrole solution are stirred by a magnetic stirrer for 30-60min to obtain the molecular imprinting polymerization solution; the template molecule solution:pyrrole solution is (10-20):1 by volume ratio; the template molecules in the molecular imprinting polymerization solution are one of tryptophan with a molar concentration of 10-20mM, glutamine with a molar concentration of 10-20mM, and GABA with a molar concentration of 20-40mM;

[0053] Step 5: the PET / Ti / Au / AuNHS / MIP composite electrode is placed in PBS solution and eluted by constant potential method to oxidize the molecular imprinting polymer film, remove the template molecules on the molecular imprinting polymer film, form specific cavities, and obtain an electrochemical sensor based on nano-gold enhanced molecular imprinting, which is recorded as PET / Ti / Au / AuNHS / MIP / Elu; the oxidation potential is 0.8-1.2v, and the time is 300-600s.

[0054] It should be noted that the PBS solution used in the present application is 1x PBS buffer with pH of 7.2-7.6. The working mechanism of the electrochemical sensor for detection is as follows: taking tryptophan as an example, the molecularly imprinted polymer film generated by electro-polymerization is embedded with tryptophan molecules in a polypyrrole network, and the isoelectric point of tryptophan is pH = 5.89; when the pH of the PBS solution is higher than this value, tryptophan exists in the form of anion in the PBS solution, and the positive charge on the main chain formed during the pyrrole polymerization process is combined with the tryptophan anion to form a pyrrole and tryptophan polymer film. The peroxidized polypyrrole shows higher selectivity, which is attributed to the introduction of oxygen-containing functional groups such as carbonyl groups, and the positive charge on the polypyrrole (PPy) main chain is removed, and the negatively charged tryptophan molecules are excluded, leaving a three-dimensional cavity matching the shape and size of the tryptophan molecule, i.e. a specific cavity, thereby realizing specific recognition of the tryptophan molecule. When the working electrode is scanned in the PBS solution by square wave voltammetry (SWV), the current peak value of SWV is affected by the electron transfer ability between the working electrode and the PBS solution; when there is a tryptophan molecule in the PBS solution, the three-dimensional cavity matching the shape and size of the tryptophan molecule will capture the tryptophan molecule, which hinders the electron transfer, so the current peak value of SWV decreases. The degree of this current reduction reflects the concentration change of tryptophan, so it can be used as an analysis signal for concentration detection.

[0055] Example 1

[0056] Step 1: cut the flexible PET substrate into a shape of 8cm*8cm, then immerse it in ethanol, and clean it using an ultrasonic cleaning machine at 80% power for 20min; then immerse it in deionized water, and clean it using an ultrasonic cleaning machine at 80% power for 20min; after the ultrasonic cleaning is completed, dry it using an N2 gun, and then perform oxygen plasma cleaning at a power of 30w for 30s;

[0057] Step 2: deposit a Ti layer with a thickness of 25nm and an Au layer with a thickness of 50nm on the surface of the cleaned PET substrate in sequence by a magnetron sputtering process to form a conductive layer, and obtain a PET / Ti / Au composite electrode; the process parameters of the magnetron sputtering are as follows: the chamber pressure for sputtering Ti is 1.6pa, the radio frequency power is 95w, and the sputtering time is 55s; the chamber pressure for sputtering Au is 0.3pa, the radio frequency power is 90w, and the sputtering time is 50s;

[0058] Step 3: PET / Ti / Au is used as the working electrode, platinum sheet is used as the counter electrode, Ag / AgCl is used as the reference electrode, a mixed solution of 1 mmol / L of HAuCl4 and 15 mmol / L of Gly is used as the deposition solution, the deposition solution is deposited on the conductive layer by using the constant potential method, nano-gold particles are generated, and a PET / Ti / Au / AuNHS composite electrode is obtained; the deposition potential is -0.1 V, the deposition time is 3500 s, and the volume ratio of the HAuCl4 to the Gly is 1:30;

[0059] Step 4: PET / Ti / Au / AuNHS composite electrode is used as the working electrode, platinum sheet is used as the counter electrode, Ag / AgCl is used as the reference electrode, a molecular imprinting polymerization solution is used as the electrolyte, and a molecular imprinting polymer thin film is deposited on the surface of the PET / Ti / Au / AuNHS composite electrode by using the cyclic voltammetry method, so as to obtain a PET / Ti / Au / AuNHS / MIP composite electrode; the scanning potential range is -0.3 V to 0.9 V, the scanning speed is 100 mv / s, and the scanning number is 5 times;

[0060] Trp is ultrasonically decomposed in a PBS solution to obtain a 10 mM Trp solution; pyrrole is dissolved in ethanol to obtain a 50 mM pyrrole solution; the volume ratio of the Trp solution to the pyrrole solution is 15:1, and the mixture is stirred for 60 min by using a magnetic stirrer to obtain a molecular imprinting polymerization solution;

[0061] Step 5: the PET / Ti / Au / AuNHS / MIP composite electrode is placed in a PBS solution and eluted by using the constant potential method, so as to obtain an electrochemical sensor based on nano-gold enhanced molecular imprinting, which is denoted as PET / Ti / Au / AuNHS / MIP / Elu; the elution potential is 0.8 V, and the time is 600 s.

[0062] Example 2

[0063] The process and raw materials of this example are the same as those of Example 1, and the difference lies in that:

[0064] Step 3: PET / Ti / Au is used as the working electrode, a mixed solution of 0.5 mmol / L of HAuCl4 and 5 mmol / L of Gly is used as the deposition solution, the deposition solution is deposited on the conductive layer by using the constant potential method, nano-gold particles are generated, and a PET / Ti / Au / AuNHS composite electrode is obtained; the deposition potential is -0.15 V, the deposition time is 4000 s, and the volume ratio of the HAuCl4 to the Gly is 1:25;

[0065] Step 4: taking the PET / Ti / Au / AuNHS composite electrode as a working electrode, taking the molecular imprinting polymerization solution as an electrolyte, and adopting a cyclic voltammetry method to deposit a molecular imprinting polymer film on the surface of the PET / Ti / Au / AuNHS composite electrode, so as to obtain a PET / Ti / Au / AuNHS / MIP composite electrode; the scanning potential range of the cyclic voltammetry method is-0.3v-0.9v, the scanning speed is 150mv / s, and the scanning number of circles is 10;

[0066] The glutamine was ultrasonically decomposed in a PBS solution to obtain a 15mM glutamine solution; the pyrrole was dissolved in ethanol to obtain a 75mM pyrrole solution; the glutamine solution and the pyrrole solution in a volume ratio of 10:1 were stirred for 30min by using a magnetic stirrer to obtain a molecular imprinting polymerization solution;

[0067] Step 5: the PET / Ti / Au / AuNHS / MIP composite electrode was placed in a PBS solution to adopt a constant potential method for elution, so as to obtain an electrochemical sensor based on a gold nanoparticle enhanced molecular imprinting, which is recorded as PET / Ti / Au / AuNHS / MIP / Elu; the potential of the peroxidation is 1.0v, and the time is 500s.

[0068] Example 3

[0069] The process and raw materials of the embodiment are the same as those of example 1, and the difference lies in that:

[0070] Step 3: taking the PET / Ti / Au as a working electrode, taking a mixed solution of 1.5mmol / L of HAuCl4 and 25mmol / L of Gly as a deposition solution, and adopting a constant potential method to deposit the deposition solution on the conductive layer to generate gold nanoparticles, so as to obtain a PET / Ti / Au / AuNHS composite electrode; the deposition potential is-0.05v, and the deposition time is 3000s; according to the volume ratio, the HAuCl4:Gly is 1:35;

[0071] Step 4: taking the PET / Ti / Au / AuNHS composite electrode as a working electrode, taking the molecular imprinting polymerization solution as an electrolyte, and adopting a cyclic voltammetry method to deposit a molecular imprinting polymer film on the surface of the PET / Ti / Au / AuNHS composite electrode, so as to obtain a PET / Ti / Au / AuNHS / MIP composite electrode; the scanning potential range of the cyclic voltammetry method is-0.3v-0.9v, the scanning speed is 150mv / s, and the scanning number of circles is 10;

[0072] The GABA is ultrasonically decomposed in a PBS solution to obtain a 30 mM GABA solution; the pyrrole is dissolved in ethanol to obtain a 100 mM pyrrole solution; the GABA solution and the pyrrole solution in a volume ratio of 20:1 are stirred by using a magnetic stirrer for 45 min to obtain a molecularly imprinted polymerization solution;

[0073] Step 5: The PET / Ti / Au / AuNHS / MIP composite electrode is placed in a PBS solution to perform elution by using a constant potential method, to obtain an electrochemical sensor based on a gold nanoparticle enhanced molecular imprinting, which is denoted as PET / Ti / Au / AuNHS / MIP / Elu; the peroxidation potential is 1.2 V, and the time is 300 s.

[0074] Comparative Example 1

[0075] The steps and raw materials of the present comparative example 1 are the same as those of Example 1, and the difference lies in that:

[0076] The electrolyte used in Step 4 is a non-molecularly imprinted polymerization solution, to obtain a PET / Ti / Au / AuNHS / NIP composite electrode; the non-molecularly imprinted polymer thin film is a polymer thin film only containing pyrrole; the preparation process of the non-molecularly imprinted polymerization solution is the same as that of the molecularly imprinted polymerization solution, and the difference lies in that: no tryptophan is added in the non-molecularly imprinted polymerization solution.

[0077] Step 5: The PET / Ti / Au / AuNHS / NIP composite electrode is placed in a PBS buffer to perform peroxidation of the non-molecularly imprinted polymer thin film by using a constant potential method, to obtain a PET / Ti / Au / AuNHS / NIP / Elu composite electrode.

[0078] In view of the actual application scene of the electrochemical sensor in the detection of depression, the present application further provides a wearable flexible electrochemical sensor integrating three working electrodes, which can simultaneously detect three depression biomarkers of tryptophan, glutamine and GABA, and the three working electrodes are integrated on the same flexible PET substrate by using a magnetron sputtering technology and a screen printing technology, and the structure is as shown in Figure 2 The specific steps are as follows:

[0079] S1: AutoCAD software is used to draw the shapes of the working electrode, the counter electrode, the reference electrode, the connecting wire and the insulating layer, to prepare corresponding mask plates;

[0080] S2: The mask plate of the working electrode is covered on the cleaned PET substrate, and Ti and Au are sequentially deposited in a circular shape with a diameter of 4 mm by using a magnetron sputtering process, and the sputtering position is replaced to repeat the operation for three times, to obtain three PET / Ti / Au composite electrodes;

[0081] S3: Covering the mask plate of the connecting wire, printing the conductive silver paste by screen printing process, then annealing at 100℃ for 20min to fix, obtaining the connecting wire; the resistance of the conductive silver paste is ≤0.02-0.01Ω / cm 2 ;

[0082] S4: Covering the mask plate of the counter electrode, printing the conductive carbon paste by screen printing process, then annealing at 100℃ for 20min to fix, obtaining the counter electrode; the resistance of the conductive carbon paste is 10.0ohm / sq;

[0083] S5: Covering the mask plate of the reference electrode, printing the Ag / AgCl paste by screen printing process, then annealing at 100℃ for 20min to fix, obtaining the reference electrode; the resistance of the Ag / AgCl paste is ≤30mΩ / sq;

[0084] S6: Covering the mask plate of the insulating layer, printing the semi-transparent insulating ink by screen printing process, then annealing at 80℃ for 20min to fix, forming the insulating layer, preliminarily obtaining the electrochemical sensor integrating three working electrodes; the resistance of the semi-transparent insulating ink (purchased from Shenzhen Laiyuan Science and Technology Development Co., Ltd.) is >200MΩ;

[0085] S7: Modifying the electrochemical sensor obtained in step S6, depositing the nano-gold particles on the surface of the three PET / Ti / Au composite electrodes by constant potential method; the process of depositing the nano-gold particles is the same as that in step 3 of Example 1;

[0086] S8: After modifying the nano-gold particles, in a three-electrode system, three PET / Ti / Au / AuNHS / MIP / Elu composite electrodes for specifically recognizing tryptophan, glutamine and GABA are prepared respectively; the process of depositing the molecularly imprinted polymer film is the same as that in step 4 of Example 1, except that the template molecule solution in the two other molecularly imprinted polymer solutions is 15mM glutamine solution and 30mM GABA solution respectively, in addition to the 10mM tryptophan solution prepared in Example 1; finally, elution is performed, and the elution process is the same as that in step 5 of Example 1, finally obtaining the wearable flexible electrochemical sensor integrating three working electrodes.

[0087] It should be noted that the polymerization of the molecularly imprinted film and the elution process of the electrochemical sensor integrating three working electrodes are performed separately for each working electrode, i.e. each time only a single working electrode is powered on with the connecting wire, and the other two working electrodes are not powered on. In this way, the three working electrodes are modified in three different molecularly imprinted solutions respectively, and three working electrodes for specifically recognizing tryptophan, glutamine and GABA are integrated on one electrochemical sensor.

[0088] As Figure 3 shown in the integrated three working electrode wearable flexible electrochemical sensor, can be bent inward, outward, has very good flexibility, can be worn on the human body to detect the stress condition of depression patients.

[0089] The wearable flexible electrochemical sensor integrated with three working electrodes provided by the application can simultaneously detect solutions of tryptophan, glutamine, GABA and other depression biomarkers; specifically, the tryptophan, glutamine and GABA solutions are prepared by using PBS solution, and the ultrasonic method is used to fully dissolve the test substances, and 10mM of tryptophan, 10mM of glutamine and 1mM of GABA solutions are obtained. The PBS solution is used as an electrolyte, and the test substance solutions are added dropwise into the electrolyte, and the three test substances are detected respectively. The electrochemical detection method is square wave voltammetry (SWV), and the decrease of the peak current of square wave voltammetry reflects the increase of the concentration of the test substance.

[0090] As Figure 4 A shown, after magnetron sputtering Ti / Au on the surface of the PET substrate, a conductive layer with a relatively uniform and flat surface is formed. As Figure 4 B shown, after electrodeposition of nano-gold particles, three-dimensional fern-like nanocrystals are formed on the surface of the conductive layer, each fern-like nanocrystal is composed of a main stem crystal, a secondary crystal grown along the main stem crystal and a tertiary crystal grown along the secondary crystal, and the angles between each level of crystal are the same; the lengths of the secondary and tertiary crystals are both less than the length of the main stem crystal. As Figure 4 C shown, after electrodeposition of the molecularly imprinted polymer film, a layer of material, i.e. the polymer film of pyrrole and tryptophan, is obviously covered on the surface of the three-dimensional fern-like nanocrystals. As Figure 4 D shown, after peroxidation of the molecularly imprinted polymer film, it can be seen that with the separation of tryptophan molecules, the molecularly imprinted polymer film becomes no longer dense, and forms an obvious specific cavity.

[0091] As Figure 5 shown in the FTIR spectrum, it can be seen that: at 1095cm -1 , C-O absorption peak appears, at 1259cm -1 , C-O-C stretching vibration peak appears, at 1700cm -1 , C=O stretching vibration peak appears, which is consistent with the typical absorption of PET. After electrodeposition of nano-gold particles, there is no obvious change in the FTIR spectrum, indicating that no other groups are introduced. After electrodeposition of the molecularly imprinted polymer film, the C-O-C belonging to PET is reduced, and the 900-1200cm -1The absorption peak in the range of 1700-1600 cm-1 is enhanced, which may be caused by the overlapping of the C-C skeleton vibration of the indole ring of tryptophan and the C-O peak of PET; after the MIP film is oxidized and the tryptophan molecules are removed, the C=O stretching vibration (1700 cm -1 ) is enhanced, and the oxidation treatment introduces more oxygen-containing groups into the polypyrrole network, which causes the overlapping of the ester and carbonyl peaks to be strengthened, indicating that the tryptophan molecules are successfully removed.

[0092] As shown in the XRD spectrum of Figure 6 , for the PET / Ti / Au sample, strong diffraction peaks appear at 38.1° (Au (111) ), 44.3° (Au (200) ), 64.4° (Au (220) ), 77.5° (Au (311) ) and 81.71° (Au (222) ), which are consistent with the face-centered cubic structure of metal Au; for the PET / Ti / Au / AuNHS sample, the width of the diffraction peak of Au at (111) does not change significantly, and the average particle size corresponding to the (111) peak of the four samples is 26.9 nm, 29.1 nm, 25.6 nm and 28.4 nm respectively, indicating that the introduction of gold nanoparticles does not significantly change the crystalline state of Au, but the intensity of the peak is significantly increased, indicating that the gold nanoparticles grow along the (111) direction. In the PET / Ti / Au / AuNHS / MIP sample, after the electrodeposited MIP film is covered on the Au surface, the diffraction signal at (111) is weakened. In the PET / Ti / Au / AuNHS / MIP / Elu sample, after elution by applying voltage, the diffraction signal intensity at (111) is restored due to the formation of specific cavities.

[0093] As shown in the XPS spectrum of Figure 7 , in the N1s spectrum of the PET / Ti / Au / AuNHS sample, no N-related bonding is observed; while Figure 7 in the N1s spectrum of the PET / Ti / Au / AuNHS / MIP sample in B, a C-NH-C characteristic peak appears at 399.6 eV, indicating that the pyrrole introduces N-containing substances into the MIP film, indicating that the MIP film is successfully prepared; while Figure 7 in the N1s spectrum of the PET / Ti / Au / AuNHS / MIP / ELU sample in C, the C-NH-C characteristic peak is reduced, indicating that the tryptophan molecules are successfully removed and specific cavities are formed.

[0094] As shown in the XPS spectrum of Figure 8As shown in the CV scan chart, it can be seen that a pair of redox peaks appears on the bare gold electrode (PET / Ti / Au), and the redox peaks are significantly increased after the modification of the gold nanoparticles, which indicates that the modified gold nanoparticles increase the specific surface area, conductivity and electron transfer capacity of the working electrode; after the electrodeposition of the molecularly imprinted polymer film, the redox peak current value sharply decreases due to the block of the electron transfer between the PBS buffer and the electrode by the molecularly imprinted polymer film; after the elution of the tryptophan template molecule, the specific cavity appears due to the shedding of the tryptophan template molecule, which provides a path for the electron transfer between the working electrode and the PBS buffer, and the redox current is increased, while the SWV method can more obviously reflect the change; the PET / Ti / Au / AuNHS / NIP / Elu sample prepared in the comparative example 1 does not have the specific cavity, and the pyrrole loses the conductivity due to the peroxidation, which is consistent with the CV chart.

[0095] As shown in the AC impedance test result, Figure 9 it can be seen that the electron transfer resistance Rct of the bare gold electrode (PET / Ti / Au) is 160.9 Ω, and the Rct is reduced to 50.2 Ω after the electrodeposition of the gold nanoparticles, which indicates that the surface modified gold nanoparticles enhance the electron transfer capacity of the working electrode; after the electrodeposition of the working electrode in the molecularly imprinted polymer solution, the pyrrole and tryptophan polymer network is formed on the surface of the working electrode, and thus the electron transfer resistance of the working electrode is sharply increased; after the voltage elution of the PET / Ti / Au / AuNHS / MIP sample, the tryptophan molecules leave the polymer network and leave the specific cavity, so that the resistance between the working electrode and the PBS buffer is reduced, and the transfer resistance Rct is 120.4 Ω; while for the PET / Ti / Au / AuNHS / NIP / Elu sample prepared in the comparative example 1, the transfer resistance Rct is further increased after the voltage elution due to the absence of the tryptophan template molecule. The electrochemical impedance spectrum of the working electrode is basically consistent with the result of the electrochemical cyclic voltammetry, which indicates that the PET / Ti / Au / AuNHS / MIP electrochemical sensor is successfully prepared by the preparation method of the application.

[0096] As shown in the AC impedance test result, Figure 10 A, the detection range of the electrochemical sensor for detecting tryptophan is 1-100 μM, and the lowest detection limit is as low as 0.612 μM. As shown in the CV scan chart, Figure 10 D, in the range of 0-10 μM, the corresponding relationship between the current peak value and the concentration of the measured substance is Ip=-3.92C+505.56, and the correlation coefficient R 2 =0.935; in the range of 10-50 μM, Ip=-1.17C+479.98, and the correlation coefficient R 2= 0.998; Ip = -0.67C + 456.04, correlation coefficient R = 0.999 in the range of 50-100 μM 2 = 0.997, respectively, showing a good linear relationship, and showing better stability in a larger concentration range.

[0097] As shown in Figure 10 B and Figure 10 E, the linear detection range of the electrochemical sensor for glutamine (Gln) is 10-80 μM, Ip = -0.503C + 416.1, correlation coefficient R = 0.996. 2 = 0.996. As shown in Figure 10 C and Figure 10 F, the linear detection range of the electrochemical sensor for GABA is 1-10 μM, Ip = -3.16C + 312.21, correlation coefficient R = 0.99. 2

[0098] In order to evaluate the anti-interference ability of the electrochemical sensor prepared in the present application in the human body or sweat, some biological small molecules commonly found in the human body or sweat, such as glucose (Glucose), urea (Urea), sucrose (Sucrose), and glycine involved in the preparation process of the fern-like structure, and three test substances, i.e., L-Trp, Gln, and GABA, were selected. In a PBS solution with PH = 7.4, glucose (100 μM), glycine (50 μM), sucrose (25 μM), urea (50 μM), and Gln (50 μM), GABA (20 μM), and L-Trp (50 μM) were added, respectively, and two of the three test substances were also used as interference substances. As shown in Figure 11 compared with the test substance corresponding to each sensor, the sensor showed no obvious current response to the added interference substances, indicating that the specific cavity formed could not recognize and capture these molecules with unmatched shapes and structures. Therefore, it is shown that the prepared electrochemical sensor has good selectivity. Under the same conditions, the working electrodes of five electrochemical sensors were prepared, as shown in Figure 12 for the same concentration (10 μM) of tryptophan, the current response difference is not large, and the RSD is 2.42%, indicating that the prepared electrochemical sensor has good reproducibility. As shown in Figure 13 An electrochemical sensor was prepared, and the peak current slightly decreased after being placed at room temperature for 5 days, but still maintained 98.4% of the initial value, indicating that the prepared electrochemical sensor has good stability.

[0099] As shown in Figure 14 A, L-Trp was detected in the range of 5-100 μM in artificial sweat, as shown in Figure 14 D, in the range of 5-10 μM, Ip = -0.67C + 456.04, correlation coefficient R = 0.999 in the range of 50-100 μM p ​= -0.61C + 221.33, correlation coefficient R 2 = 0.85; 10-100 μM range, I p = -0.36C + 215.63, correlation coefficient R 2 = 0.98. As Figure 14 B and Figure 14 E shows, in artificial sweat, Gln is detected in the range of 10-80 μM, and there is a good linear relationship in the range of 10-80 μM, I p = -0.19C + 301.07, correlation coefficient R 2 = 0.99. As Figure 14 C and Figure 14 F shows, in artificial sweat, GABA is detected in the range of 1-8 μM, and there is a good linear relationship in the range of 1-8 μM, I p = -1.18C + 267.42, correlation coefficient R 2 = 0.98. The above data show that the nano-gold enhanced molecular imprinting-based electrochemical sensor prepared in the application can detect L-Trp, Gln and GABA, three biomarkers of depression, under artificial sweat conditions, and the detection range covers the physiological concentration range of these substances, which is of key significance to promote the clinical practicality of the sensor.

[0100] In summary, the PET / Ti / Au / AuNHS / MIP / Elu flexible electrochemical sensor of the application is used for the combined detection of biomarkers of depression for the first time. The fern-like structure composed of nano-gold particles is modified on the surface of the working electrode by direct synthesis, which enhances the specific surface area and surface roughness of the working electrode, widens the detection limit of the electrochemical sensor, and the minimum detection limit changes from 2.694 μM to 0.612 μM. The nano-gold structure enhances the electrochemical sensor of the molecular imprinting, which has a good linear relationship with tryptophan (1-100 μM), glutamine (10-80 μM) and GABA (1-10 μM), and the sensor has good selectivity, reproducibility and stability. Based on the sensor, a wearable flexible electrochemical sensor integrating three working electrodes is prepared, which has good sensing performance for the three biomarkers of depression in artificial sweat. This real-time and non-invasive detection method further improves the possibility of practical application of the flexible wearable electrochemical sensor for stress monitoring of patients with depression.

[0101] The above description is only several specific examples of the present application, describes the basic principles, main features and advantages of the present application, and does not constitute any limitation on the present application. After understanding the content and principles of the present application, those skilled in the art can make various modifications and changes in form and details without departing from the structure and method of the present application, but these should fall within the scope of the present application.

Claims

1. A method for preparing an electrochemical sensor based on gold nanoparticle-enhanced molecular imprinting, characterized in that, Includes the following steps: Step 1: Ti and Au are sputtered sequentially on the cleaned PET substrate surface to obtain a PET / Ti / Au composite electrode; Step 2: Using PET / Ti / Au as the working electrode, and a mixed solution of HAuCl4 with a molar concentration of 0.5-1.5 mmol / L and glycine with a molar concentration of 5-25 mmol / L as the deposition solution, gold nanoparticles are deposited on the surface of the PET / Ti / Au composite electrode using a potentiostatic method to obtain a PET / Ti / Au / AuNHS composite electrode with a fern-like structure; the volume ratio of HAuCl4 to glycine is 1:(25-35). Step 3: Using the PET / Ti / Au / AuNHS composite electrode as the working electrode and the molecularly imprinted polymerization solution as the electrolyte, a molecularly imprinted polymer film is deposited on the surface of the PET / Ti / Au / AuNHS composite electrode by cyclic voltammetry to obtain the PET / Ti / Au / AuNHS / MIP composite electrode. Step 4: Elute the PET / Ti / Au / AuNHS / MIP composite electrode to remove the template molecules on the molecularly imprinted polymer film, forming a specific cavity, and obtain an electrochemical sensor based on nano-gold-enhanced molecular imprinting.

2. The method for preparing an electrochemical sensor based on gold nanoparticle-enhanced molecular imprinting according to claim 1, characterized in that: In step 1, a 25nm thick Ti layer and a 50nm thick Au layer are sequentially deposited on the surface of the cleaned PET substrate using a magnetron sputtering process to form a conductive layer.

3. The method for preparing an electrochemical sensor based on gold nanoparticle-enhanced molecular imprinting according to claim 1, characterized in that: In step 2, the deposition potential is -0.15 to -0.05V, and the deposition time is 3000-4000s.

4. The method for preparing an electrochemical sensor based on gold nanoparticle-enhanced molecular imprinting according to claim 1, characterized in that, In step 3, the preparation process of the molecularly imprinted polymerization solution is as follows: the template molecule is ultrasonically decomposed in PBS solution to obtain a template molecule solution; pyrrole is dissolved in ethanol to obtain a pyrrole solution with a molar concentration of 50-100 mM; the template molecule solution and the pyrrole solution are stirred for 30-60 min to obtain the final product; the template molecule solution: pyrrole solution = (10-20): 1 by volume; the template molecule solution in the molecularly imprinted polymerization solution is one of tryptophan, glutamine, or GABA with a molar concentration of 10-20 mM, 10-20 mM, or 20-40 mM.

5. The method for preparing an electrochemical sensor based on gold nanoparticle-enhanced molecular imprinting according to claim 1, characterized in that: In step 3, the scanning potential is -0.3V to 0.9V, the scanning speed is 50-150mV / s, and the number of scanning revolutions is 5-15 revolutions.

6. The method for preparing an electrochemical sensor based on gold nanoparticle-enhanced molecular imprinting according to claim 1, characterized in that: In step 4, the elution process is as follows: the PET / Ti / Au / AuNHS / MIP composite electrode is placed in a 1×PBS solution with a pH of 7.2-7.6 and eluted for 300-600 seconds at a potential of 0.8-1.2V.

7. The electrochemical sensor based on gold nanoparticle-enhanced molecular imprints prepared by the preparation method according to any one of claims 1-6, characterized in that: The electrochemical sensor includes a PET substrate, on which a Ti / Au layer is disposed. A fern-like structure composed of gold nanoparticles is disposed on the Ti / Au layer. The surface of the fern-like structure is modified with a molecularly imprinted polymer film with specific cavities. The molecularly imprinted polymer film is a polymer film of pyrrole and template molecules, wherein the template molecule is one of tryptophan, glutamine, and GABA.

8. The application of the electrochemical sensor based on nano-gold-enhanced molecular imprinting prepared by the preparation method according to claim 7 in the detection of biomarkers for depression.

9. A method for fabricating a wearable flexible electrochemical sensor integrating three working electrodes, characterized in that, Includes the following steps: S1: Cover the working electrode mask on the cleaned PET substrate, and deposit Ti and Au sequentially using magnetron sputtering. Repeat the operation three times by changing the sputtering position to obtain three PET / Ti / Au composite electrodes. S2: Connecting wires, counter electrode, reference electrode, and insulating layer are sequentially prepared on a PET substrate using screen printing. S3: Using the preparation method described in claim 1, three PET / Ti / Au / AuNHS / MIP / Elu composite electrodes for specific recognition of tryptophan, glutamine, and GABA are respectively prepared on the surface of the three PET / Ti / Au composite electrodes obtained in step S1, to obtain a wearable flexible electrochemical sensor integrating three working electrodes; the template molecule solutions in the three molecular imprinting polymerization solutions are tryptophan with a molar concentration of 10-20 mM, glutamine with a molar concentration of 10-20 mM, and GABA with a molar concentration of 20-40 mM.

10. The wearable flexible electrochemical sensor with three integrated working electrodes prepared by the preparation method according to claim 9, and its application in the detection of biomarkers of depression.

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