Modified cfme, dopamine sensor, and method for detecting dopamine in vitro

Abstract

The application belongs to the technical field of dopamine sensor, and particularly relates to a modified CFME, a dopamine sensor and a method for detecting dopamine in vitro. The preparation method of the modified CFME is as follows: gold nanoparticles are modified on the surface of pretreated carbon fibers through sputtering to obtain AuNPs / CF; gold nanocoral is grown on the surface of the AuNPs / CF electrode by using an electrodeposition method to obtain AuNCs / CF; then the AuNCs / CF is modified by using egg white-based EWCDs to obtain the modified CFME. The dopamine sensor developed by using the same shows high selectivity and high sensitivity in real-time tracking of DA, has a good linear relationship in the range of 0.001-20 muM, and the detection limit is 0.765 nM. The dopamine sensor provided by the application can be implanted in the brain, and through the verification of a pathophysiological model, the sensor can reliably track the complex dynamic changes of DA in the living brain.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of dopamine sensor, and particularly relates to a modified CFME, a dopamine sensor and a method for detecting dopamine in vitro. BACKGROUND

[0002] Neurotransmitters are important molecules for maintaining brain activity and homeostasis, and their relative and basal levels in the brain are directly related to specific disease states. Dopamine (DA) is an endogenous nitrogen-containing organic substance, which is an intermediate product of tyrosine metabolism to dihydroxyphenylalanine. As one of the main neurotransmitters in the central nervous system (CNS) and peripheral nervous system, it plays an important role in the human central nervous system, regulating various physiological functions, including movement, cognition, memory, learning and reward. Abnormal levels of DA are closely related to the occurrence of neurological diseases such as addiction, drug abuse, Parkinson's disease, schizophrenia, etc. Therefore, obtaining chemical information of dopamine in situ in vivo is crucial for understanding the molecular mechanisms of DA in brain function.

[0003] The electrochemical platform based on microelectrode, especially carbon fiber microelectrode (CFME), has excellent temporal and spatial resolution. Its diameter is only a few microns, and it causes less damage to the brain neural tissue, and has become the main method for detecting neurochemical substances in vivo. Although many electrochemical sensors have been reported for DA detection, there are two major challenges that hinder the application of DA electrochemical sensors in vivo. On the one hand, the redox process of DA is severely interfered by the coexisting electroactive substances in the brain, such as ascorbic acid (AA) and uric acid (UA), which have highly similar oxidation potentials on bare electrodes. On the other hand, the content of DA is as low as nanomolar level, which is 2-4 orders of magnitude lower than the content of electroactive interferents in the actual brain.

[0004] In order to solve these problems, the skilled person in the art has developed fast scan cyclic voltammetry (FSCV), electrode chemical modification and electrochemical microtransistor devices to improve the selectivity and sensitivity of DA in vivo detection. FSCV pioneered by Wightman et al. is used for high time resolution DA detection. However, this method requires substrate oxidation to collect signals, so it cannot effectively distinguish DA from other structurally similar neurotransmitters. In addition, the skilled person in the art has also developed a fast scan potential gated organic electrochemical transistor (OECT) for sensitive detection of DA. Unfortunately, there are still a series of problems with OECT materials, which limit their wide application. In contrast, the chemical modification strategy of using polymers or molecular recognition ligands on the electrode surface seems to have more potential. However, how to modify the electrode to meet the requirements of antifouling, biocompatibility and long-term stability for in vivo DA detection while ensuring high sensitivity and selectivity is still a challenging task.

[0005] Biomass-derived carbon dots (BDCDs) have attracted increasing attention due to their large surface area, optical properties, excellent electrical conductivity and high chemical stability, and have been applied in bioimaging, sensors, drug carriers and other fields. Compared with other electrode modification materials, BDCDs exhibit excellent electrical conductivity, high biocompatibility and hydrophilicity. SUMMARY

[0006] Therefore, one of the purposes of the present application is to provide a modified CFME (EWCDs@Nafion / AuNCs / CFME) for detecting dopamine in the brain and cells, wherein the microelectrode is modified by a negatively charged biocompatible material, the prepared modified microelectrode has high sensitivity, high selectivity and biocompatibility, and provides technical support for real-time in-situ detection of dopamine in cells and living brains.

[0007] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0008] The preparation method of the modified CFME for detecting dopamine in the brain and cells comprises the following steps: modifying gold nanoparticles on the surface of the pretreated carbon fiber by sputtering to obtain AuNPs / CF; growing gold nanocoral on the surface of the AuNPs / CF electrode by electrodeposition to obtain AuNCs / CF; and then modifying the AuNCs / CF with EWCDs based on egg white to obtain the modified CFME.

[0009] The present application proposes a new in-vivo DA sensing strategy, which is designed and optimized by modifying the microelectrode with a negatively charged biocompatible material. First, the biomass-derived carbon dots based on egg white are used for electrode surface modification to provide rich active sites for the electrochemical catalysis of DA. At the same time, the negatively charged characteristics make EWCDs a good material for resisting negatively charged neurochemicals (AA, UA), thereby improving the selectivity. In addition, the safe and traceable food source endows EWCDs with excellent biocompatibility, which greatly improves the reliability of in-vivo DA detection.

[0010] Further, the pretreatment comprises the following steps: sequentially subjecting the CF to ultrasonic treatment in acetone, HNO3, KOH and distilled water; and then electrochemically activating the CF.

[0011] Further, the concentration of the HNO3 is 3M, the concentration of the KOH is 1M, and the ultrasonic treatment time is 5 minutes.

[0012] Further, the electrochemical activation comprises the following steps: first, performing potential control amperometry at a potential of +1.5V in 0.5M H2SO4 for 80s, and then performing cyclic voltammetry in a potential range of-1.0V to 1.0V at a scanning rate of 50mV·s-1.-1 The scan rate was cycled voltammetry until a stable cyclic voltammogram was obtained.

[0013] Further, the sputtering time is 70s.

[0014] Further, the EWCDs are immobilized by Nafion, and the concentration of the EWCDs is 0.01g / L.

[0015] Further, the preparation method of the EWCDs is: 200mL egg white is heated by microwave for 25 minutes under 750W, and the egg white changes from light yellow transparent liquid to yellow-brown solid; the yellow-brown solid is ground by a mortar to obtain a powder, which is added into 100mL ultrapure water and stirred thoroughly for a night to obtain a mixture; the mixture is centrifuged at 12000r / min for 20 minutes to remove large-size particles. The supernatant is vacuum filtered by using a 0.22μm membrane, and further dialyzed in ultrapure water by using a dialysis bag (MWCO=1.0kD) for 72 hours, and the dialysate is replaced every 4 hours to obtain a suspension of the EWCDs; the powder of the EWCDs is obtained by freeze-drying.

[0016] Further, the preparation method of the EWCDs / Nafion composite solution is: 5mL of the EWCDs (0.01mg / mL) is dispersed in 20mL of the Nafion solution (0.5wt.%), to obtain the EWCDs / Nafion composite solution. Then, the mixed solution is subjected to ultrasonic treatment to produce a uniformly dispersed solution.

[0017] Preparation of the EWCDs@Nafion / AuNCs / CFME: the AuNCs / CF electrode is immersed in the EWCDs / Nafion composite film solution for five times, and dried for 5 minutes between each time, to obtain the modified CFME.

[0018] Further, the electro-deposition method is: taking the AuNPs / CF as a working electrode, Ag / AgCl as a reference electrode, and Pt wire as a counter electrode, electrochemical deposition is carried out in the mixed solution; the electro-deposition time is 12h.

[0019] Further, the control voltage is-0.7V.

[0020] Further, the mixed solution is a mixed solution of AuCl3, CTAB, NaNO3 and ultrapure water, the concentration of the AuCl3 is 5mM, the concentration of the CTAB is 9mM, and the concentration of the NaNO3 is 20mM.

[0021] Further, the XRD pattern of the modified CFME has diffraction peaks at 2 theta values of 38.27, 44.44, 64.70 and 77.702, corresponding to the 111, 200, 220 and 311 crystal planes of gold, respectively; the O1s spectrum of the modified CFME has two characteristic peaks at 535.5 eV and 532.1 eV, corresponding to the O elements in Nafion and EWCDs, respectively; the C1s spectrum of the modified CFME has three characteristic peaks at 287.4 eV, 289.2 eV and 291.8 eV, corresponding to the C element; the Au 4f spectrum has two characteristic peaks at 88.1 eV and 84.4 eV, corresponding to the atomic orbitals of Au 4f 5 / 2 and Au 4f 7 / 2 .

[0022] The second object of the present application is to provide a dopamine sensor containing the modified CFME.

[0023] The dopamine sensor is based on CFME and gold nanostructures, and uses egg white-based biomass-derived carbon dots for electrode surface modification. Based on the negative charge and biocompatibility of EWCDs, the present application provides an optimized brain-implantable dopamine sensor, which realizes reliable tracking of the complex dynamic changes of DA in the living brain through the verification of pathophysiological models.

[0024] As a preferred, the dopamine sensor is a three-electrode system, the modified CFME is used as the working electrode, the platinum wire is used as the counter electrode, and the Ag / AgCl electrode is used as the reference electrode.

[0025] Selectivity and sensitivity are two major obstacles for the in vivo application of dopamine sensors. How to design and construct electrodes to meet the requirements of anti-fouling, stability and biocompatibility for DA detection in complex biological fluids while ensuring high sensitivity and selectivity is still a great challenge. The present application improves the selectivity of the dopamine sensor by modifying EWCDs on the electrode, while sensitively measuring DA in the living brain. On this basis, the modified CFME (EWCDs@Nafion / AuNCs / CFME) shows high selectivity for real-time tracking of DA, and is successfully applied to real-time monitoring of DA in cells and rat brains. The results show that EWCDs@Nafion / AuNCs / CFME can be used as a new reliable in vivo DA tracking platform, which helps to understand the physiological and pathological functions of DA involved in the nervous system.

[0026] The third object of the present application is to provide a method for detecting dopamine in vitro using the dopamine sensor, which has a good linear relationship in the range of 0.001 μM-20 μM, and the detection limit is 0.765 nM.

[0027] To achieve the above object, the application adopts the following technical solutions:

[0028] The method for detecting dopamine in vitro by using the dopamine sensor comprises the following steps:

[0029] (1) preparing a sample to be detected;

[0030] (2) electrochemically detecting the sample to be detected by using the dopamine sensor according to claim 2 to obtain a current response;

[0031] (3) qualitatively and / or quantitatively analyzing dopamine in the sample to be detected according to the current response.

[0032] Further, the sample to be detected is an ex vivo living cell and / or a brain slice.

[0033] Further, the detection potential is 0.25 V.

[0034] Further, the content of dopamine is calculated by substituting the measured current response into a standard curve equation; when the concentration of dopamine is 0.001 μM-20 μM, the standard curve equation is Y=0.489X-9.741, R 2 =0.996, wherein Y is the Y axis, representing a current signal, and the unit is nA; X is the X axis, representing the concentration of dopamine, and the unit is μM.

[0035] The application has the following beneficial effects:

[0036] (1) The application develops a DA selective sensing strategy by fixing EWCDs on Nafion. The addition of EWCDs not only improves the biocompatibility, but also changes the surface charge of the electrode, thereby improving the selectivity of the electrode to DA. The modified CFME (EWCDs@Nafion / AuNCs / CFME) developed by the application can realize in-situ real-time detection of DA and accurately track the concentration change of DA at a low level of nanomolar in a complex brain.

[0037] (2) The dopamine sensor prepared by the application is used to detect DA in PC12 cell groups and brain slices, and it is found that the release processes of dopamine in PC12 cell groups and brain slices are different. The basic release level of DA in brain slices is high, and is accompanied by reabsorption of DA. The application proves the potential of the charge-carrying biological derivative material in neurotransmitter sensing, and the DA selective sensing strategy provided by the application is expected to promote the further development of a new, simple in-vivo measurement platform for brain neurochemical substances.

[0038] (3) The dopamine sensor of the application is not interfered by coexisting electroactive substances such as ascorbic acid and uric acid in the brain, and can realize effective detection of dopamine at a low level of nanomolar. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 SEM image of bare CFME;

[0040] Figure 2 SEM image of EWCDs@Nafion / AuNCs / CFME;

[0041] Figure 3 EDX image, scale bar: 5 pm;

[0042] Figure 4 XRD image;

[0043] Figure 5 XPS survey scan image;

[0044] Figures 6-9 Spectra of O 1s, Au 4f, C 1s, F 1s, respectively;

[0045] Figure 10 Result plot of electrodeposition time optimization;

[0046] Figure 11 Result plot of EWCDs concentration optimization;

[0047] Figure 12 Result plot of potential optimization;

[0048] Figure 13 Result plot of potential optimization by selection;

[0049] Figure 14 CV plot of EWCDs@Nafion / AuNCs / CFME after adding 10 pM, 20 pM and 50 pM DA in aCSF;

[0050] Figure 15 Result plot of CV response of EWCDs@Nafion / AuNCs / CFME to 20 pM DA at different scan rates;

[0051] Figure 16 Linear relationship plot of square root of scan rate and peak current;

[0052] Figure 17 Amperometric response of EWCDs@Nafion / AuNCs / CFME to DA from 1 nM to 20 pM in 10 mL aCSF, inset is the fitted plot of amperometric response;

[0053] Figure 18 Result plot of selectivity of EWCDs@Nafion / AuNCs / CFME to DA in the presence of interfering substances at physiological concentration;

[0054] Figure 19 For the selective statistical results chart, the insert is a chart of the surface zeta potential of EWCDs@Nafion / AuNCs / CC and Nafion / AuNCs / CC;

[0055] Figure 20 For the amperometric response results chart of EWCDs@Nafion / AuNCs / CFME before and after soaking when 5 μM DA is continuously added in aCSF;

[0056] Figure 21 For Figure 20 The chart of the corresponding fitting curve;

[0057] Figure 22 For the amperometric response results chart of bare CFME before and after soaking when 5 μM DA is continuously added in aCSF;

[0058] Figure 23 For Figure 22 The chart of the corresponding fitting curve;

[0059] Figure 24 For the ratio of the post-calibration sensitivity to the pre-calibration sensitivity of EWCDs@Nafion / AuNCs / CFME and bare CFME;

[0060] Figure 25 For the images of EWCDs@Nafion / AuNCs / CFME before and after bending;

[0061] Figure 26 For Figure 25 The corresponding CV curve chart of the results;

[0062] Figure 27 For the results chart of the stability comparison of long-term monitoring for 1 h in aCSF containing 10 μM DA;

[0063] Figure 28 For the CCK8 cell viability comparison chart of EWCDs@Nafion / AuNCs / CC and bare CC;

[0064] Figure 29 For the fluorescence images of cell growth on EWCDs@Nafion / AuNCs / CC and bare CC;

[0065] Figure 30 For K + The mechanism chart of DA release stimulation and nifedipine inhibition;

[0066] Figure 31Amperometric response curves of DA release from PC12 cells monitored by EWCDs@Nafion / AuNCs / CFME at different treatments;

[0067] Figure 32 For Figure 31 The corresponding data statistical chart;

[0068] Figure 33 Amperometric response curves of DA release from rat brain tissue slices monitored by EWCDs@Nafion / AuNCs / CFME at different treatments;

[0069] Figure 34 For Figure 33 The corresponding data statistical chart. DETAILED DESCRIPTION

[0070] The technical solutions of the present application will be described further and more clearly in combination with specific examples. Obviously, the described examples are only some of the examples of the present application, but not all the examples. Therefore, all the other examples obtained by those skilled in the art based on the examples in the present application without creative labor fall within the protection scope of the present application.

[0071] In the embodiments of the present application, AuCl3 is purchased from Shandong Xiya Chemical Service Co., Ltd. (Shandong, China); cetyltrimethylammonium chloride (CTAC) is provided by Adamas beta (Shanghai, China); glucose is purchased from Jiangsu Johnson Functional Chemical Co., Ltd. (Jiangsu, China); Nafion 117 solution, dopamine (DA), uric acid (UA), ascorbic acid (AA), 3,4-dihydroxyphenylacetic acid (DOPAC), L-glutamic acid and nifedipine are purchased from Sigma-Aldrich and can be used without further purification; RPMI Medium 1640 culture medium is purchased from Gibco; fetal bovine serum (FBS) is provided by Lonsera; CCK-8 and Calcein / PI cell viability / cytotoxicity assay kit are purchased from Biyun Tian Biotechnology Co., Ltd.

[0072] In the embodiments of the present application, all the water (≥18M) used in the experiments is purified by a Millipore system.

[0073] In the embodiments of the present application, the preparation method of artificial cerebrospinal fluid (aCSF) is as follows: NaCl (126 mM), KCl (2.4 mM), KH2PO4 (0.5 mM), MgCl2 (0.85 mM), NaHCO3 (27.5 mM) and Na2SO4 (0.5 mM) and CaCl2 (1.1 mM) are mixed into ultrapure water (pH is adjusted to 7.4) to prepare artificial cerebrospinal fluid.

[0074] In the embodiments of the present application, phosphate buffered solution (PBS, 0.1 M, pH 7.4) is prepared by mixing NaH2PO4(0.1 M) and Na2HPO4(0.1 M).

[0075] In the embodiments of the present application, potassium ferricyanide solution ([Fe(CN)6] 3- / 4- : 5 mM K3[Fe(CN)6], 5 mM K4[Fe(CN)6]) is prepared using PBS containing 0.1 M KCl.

[0076] In the embodiments of the present application, fresh eggs are obtained from the local market in Chongqing, China.

[0077] Example 1

[0078] 1. Carbon fiber (CF) pretreatment: The CFs were sequentially treated in acetone, 3 M HNO3, 1 M KOH and distilled water for 5 min under ultrasonication. Before modification, the CFs were electrochemically activated by first performing a potential-controlled amperometry at +1.5 V for 80 s in 0.5 M H2SO4, and then by cyclic voltammetry at a scan rate of 50 mV·s -1 -1.0 V to 1.0 V until a stable cyclic voltammogram was obtained. After drying, the CFs were sputtered in a SEM gold sputter coater for 70 s to obtain gold nanoparticle-modified CFs (AuNPs / CFs).

[0079] 2. Electrode preparation: synthesis of gold nanocoral, AuCl3, CTAB and NaNO3 were added to ultrapure water to form a mixed solution containing 5 mM AuCl3, 9 mM CTAB and 20 mM NaNO3. After ultrasonic treatment, the mixture was placed in an electrolytic cup and deposited using a simple three-electrode electrochemical deposition system. The AuNPs / CFs obtained in step 1 were used as the working electrode, Ag / AgCl as the reference electrode, and Pt wire as the counter electrode. After connecting the three-electrode system, a control voltage of -0.7 V was applied to the electrode. After 12 hours, the growth of gold nanocoral (AuNCs) on the surface of the working electrode was observed, and the electrode (AuNCs / CF) was obtained. Finally, the electrode (AuNCs / CF) was ultrasonically cleaned in alcohol for 3 minutes and rinsed with ultrapure water before further study.

[0080] 3. Preparation of EWCDs powder and EWCDs / Nafion composite solution: 200 mL of egg white was obtained using egg white (EW) separator, which was placed in a beaker and microwaved at 750 W for 25 minutes using a microwave oven. The EW changed from light yellow transparent liquid to yellow-brown solid. The yellow-brown solid was ground using a mortar to obtain a powder, which was added to 100 mL of ultrapure water and stirred thoroughly overnight, and the mixture was centrifuged at 12000 rpm for 20 minutes to remove large size particles. The supernatant was vacuum filtered using a 0.22 pm membrane, and further dialyzed in ultrapure water using a dialysis bag (MWCO = 1.0 kD) for 72 hours, with the dialysate being changed every 4 hours. A suspension of CDs was finally obtained, and the EWCDs powder was obtained by freeze-drying. The EWCDs / Nafion composite solution was prepared by dispersing 5 mL of EWCDs (0.01 mg / mL) in 20 mL of Nafion solution (0.5 wt.%). Then, the mixed solution was subjected to ultrasonic treatment to produce a uniformly dispersed solution. The prepared EWCDs powder and EWCDs / Nafion composite solution were stored at 4 °C for further characterization and application.

[0081] 4. Preparation of EWCDs@Nafion / AuNCs / CFME (modified CFME): The AuNCs / CF electrode was immersed in the EWCDs / Nafion composite membrane solution for five times, with a 5-minute delay between each immersion for drying, to prepare the EWCDs@Nafion / AuNCs / CFME. The resulting EWCD / Nafion composite membrane-coated biosensor (EWCDs@Nafion / AuNCs / CFME) was stored at room temperature (at least 15 minutes at room temperature before use).

[0082] 5. Measurement: Morphology analysis and elemental distribution characteristic analysis of the modified CFME were performed on a field emission Hitachi S-8010 scanning electron microscope (Hitachi, Tokyo, Japan) and X-Max N (Oxford Instruments, UK) respectively, and energy dispersive X-ray spectroscopy (EDX) images were obtained. X-ray photoelectron spectroscopy (XPS) measurements were performed using a photoelectron spectrometer (K-Alpha, Thermo Fisher Scientific). X-ray diffraction (XRD) images were measured on an X-ray diffractometer (XRD-6100, Shimadzu). Fluorescence imaging was performed using an inverted fluorescence microscope (ECLIPSE Ti2, Nikon). The electrode was operated to penetrate and withdraw the brain slice using a micro manipulator MM-500 (RWD Life Science Co., Ltd., Shenzhen, China).

[0083] 6. Electrochemical measurements: Electrochemical measurements were performed using a computer-controlled electrochemical analyzer (CHI 630e; 660e; 440c, Shanghai, China). Modified CFME was used as the working electrode, platinum wire as the counter electrode, and Ag / AgCl electrode as the reference electrode.

[0084] Example 2. Characterization of EWCDs@Nafion / AuNCs / CFME

[0085] The present application used scanning electron microscopy and energy dispersive spectrometer to characterize the EWCDs@Nafion / AuNCs / CFME. Figure 1 、 Figure 2 The surface morphology of bare carbon fiber and EWCDs@Nafion / AuNCs / CFME is shown. It can be seen that, compared with the smooth unmodified bare CFME, the modified CF surface forms a coral-like gold nanostructure. This relatively uniform AuNCs is considered to be more conducive to electrocatalytic applications due to its loose structure, rough texture and large surface area. In order to further prove the successful modification of the EWCDs@Nafion selective membrane, the present application studied the elemental mapping of the electrode surface, as shown in Figure 3 , it can be seen that Au (characteristic element of AuNCs), C (characteristic element of EWCDs) and F (characteristic element of Nafion). These results show that the AuNCs / CFME surface is uniformly covered by the EWCDs@Nafion coating, which is crucial for the sensor to resist interference from other substances.

[0086] The XRD pattern of AuNCs / CFME is shown in Figure 4 . The diffraction peaks of Au are concentrated at 2θ values of 38.27, 44.44, 64.70 and 77.70, which are perfectly matched with (111), (200), (220) and (311) crystal planes, respectively, corresponding to the face-centered cubic crystal structure of gold. In addition, the EWCDs@Nafion / AuNCs / CFME was studied by full XPS spectrum, and the results are shown in Figures 5-9 . The O 1s spectrum Figure 6 shows the characteristic peaks of spin-orbit O 1s at 535.5 eV (-CF2-O-CF2-) and 532.1 eV (C-O-C) from O elements in Nafion and EWCDs, respectively. Figure 8 The C 1s characteristic peak shown in Figure 7 shows the Au 4f 5 / 2 at 88.1 eV and Au 4f 7 / 2A peak at 84.4 eV indicates the presence of Au(0) state. The above results confirm the successful synthesis of EWCDs@Nafion / AuNCs / CFME.

[0087] Example 3. Optimization of experimental parameters

[0088] The growth of gold nanostructures is largely influenced by the combination of reaction parameters. These parameters include reaction temperature, applied potential, concentration of metal precursor solution and other reagents. By controlling other parameters during the deposition process, this example evaluates the effect of different deposition times on electrode morphology and detection performance. As shown in Figure 10 , the increase of deposition time results in the current response of the electrode to 5 mM DA rising to the first peak at 12 hours, then decreasing, and finally reaching the maximum at 24 hours. Combining the SEM images of the evolution of gold nanostructure morphology under time control, the electrode diameter has exceeded 20 pm after 24 hours of deposition, which severely limits the application of the electrode in the detection of DA in vitro and in vivo. Therefore, 12 hours is chosen as the optimal time for electrodeposition. As shown in Figure 11 , the resistance percentage of the electrode to AA reaches the maximum when the concentration of EWCDs is 0.01 g / L, under which condition, the composite film has the strongest resistance to AA. However, with the further increase of EWCDs concentration, the resistance of the electrode to AA gradually decreases, which can be attributed to the destruction of the structure and proton exchange ability of Nafion by high EWCDs content. Even so, the ability of EWCDs@Nafion to resist AA is still higher than that of pure Nafion, proving that EWCDs can indeed improve the selectivity of the electrode to AA through its own charge. In addition, the amperometric determination of DA is also affected by the applied potential. Figure 12 shows that the current response obtained on EWCDs@Nafion / AuNCs / CFME increases with the increase of potential at different potentials between 0.2 V and 0.4 V in aCSF (pH 7.4) with the continuous addition of 5 mM DA. In order to more intuitively evaluate the effect of potential on the selectivity of the electrode, the current ratio of DA to AA at different potentials is compared by the present application, as shown in Figure 13 , the selectivity of the electrode to DA changes with the change of potential. At 0.25 V, the selectivity reaches the peak, the signal is sensitive and stable, and the noise value is relatively small. Therefore, in the subsequent experiments, the present application selects 0.25 V as the detection potential.

[0089] Example 4. In vitro detection

[0090] Prior to in vivo detection, the response of the DA sensor was examined in an in vitro system. To investigate the feasibility of using EWCDs@Nafion / AuNCs / CFME for DA detection, a simplified analysis was performed within a potential range of -0.2V to +0.6V, similar to analyses observed in previously reported DA sensors fabricated using different materials. Figure 14 As shown, when the DA concentration increases from 0 to 50 μM, the corresponding anolyte current increases significantly. For comparison ( Figure 14 (Illustration) Under the same conditions, the increase in current was smaller when 50 μM DA was added to bare CFME. The anolyte current of EWCDs@Nafion / AuNCs / CFME was significantly higher than that of bare CFME. First, EWCDs are negatively charged, which may be beneficial for improving the sensitivity of DA determination by electrostatic attraction. In addition, the microstructure of the electrode surface provides a larger active surface area, which is beneficial for improving electrocatalytic activity. To further investigate the kinetics of the electrocatalytic reaction, the effect of scan rate on the peak DA current was investigated as follows. Figure 15 As shown, the CV response of EWCDs@Nafion / AuNCs / CFME in aCSF (pH 7.4) with 20 μM DA is displayed at different scan rates. It can be observed that the peak current value increases with increasing scan rate. The oxidation and reduction peak currents increase with v. 1 / 2 Linear change ( Figure 16 The results indicate that the electrochemical surface interaction is a diffusion-controlled process. These results demonstrate that EWCDs@Nafion / AuNCs / CFME is effective for the electrocatalytic oxidation of DA.

[0091] Real-time current sensing of DA by EWCDs@Nafion / AuNCs / CFME was performed by continuously injecting DA (1 nM to 20 μM) into the aCSF solution at an applied potential of +0.25 V. For example... Figure 17 As shown, once the standard solution is added to aCSF, the catalytic oxidation current rapidly rises to a stable value. The constructed sensor exhibits a good linear relationship with DA in the range of 0.001–20 μM. Figure 17 (Illustration), this range covers normal DA levels in the striatum (2.5–15 nM). The linear correlation regression equation for DA concentration is I / nA = 0.489C. DA / μM-9.741(R 2 =0.996). The detection limit of EWCDs@Nafion / AuNCs / CFME is estimated to be 0.765 nM based on the signal-to-noise ratio (S / N=3) obtained from the current value of 1 nM DA, which is equal to or lower than other DA electrochemical biosensors reported in the literature, as detailed in Table 1.

[0092] Table 1. Comparison of the performance of DA detection in aCSF with that of reported sensors

[0093]

[0094] In vivo DA analysis, negatively charged neurochemicals such as ascorbic acid (AA) in the brain can seriously interfere with the detection of DA. To achieve selective detection of DA, researchers usually modify the electrode surface to be negatively charged. In this study, we evaluated the effect of EWCDs on the electrode charge, and the zeta potential results showed that the electrode surface was negatively charged in the presence of EWCDs Figure 19 The inset), which is likely to be the main reason for the improved selectivity of the electrode. By measuring the current response of AA, glutamate, DOPAC, UA, glucose and DA at physiological concentrations in aCSF, the selectivity of EWCDs@Nafion / AuNCs / CFME for DA under physiological conditions was studied. As shown in 18 and 19, other species showed little significant amperometric response compared to DA, indicating that the presence of these species does not affect the detection of DA, and this high selectivity provides strong support for the in vivo application of the sensor.

[0095] This example also evaluated the anti-fouling properties of EWCDs@Nafion / AuNCs / CFME. To this end, the electrode was immersed in cell-cultured medium for several hours (usually 2 hours), and then its amperometric response to DA was evaluated. As shown in Figure 20 , Figure 21 The sensitivity and response to DA of EWCDs@Nafion / AuNCs / CFME after immersion were similar to those before immersion. The ratio of the sensitivity after post-calibration (Spost) to that of pre-calibration (Spre) was calculated to be 0.83, as detailed in Figure 24 . In contrast, a much greater decrease in the sensitivity of bare CFE was observed after immersion in culture medium due to non-specific adsorption of proteins on the CFE surface, see Figures 22-23 This result indicates that the presence of proteins does not significantly affect the performance of EWCDs@Nafion / AuNCs / CFME in DA sensing, thus ensuring the reliability of EWCDs@Nafion / AuNCs / CFME for cell and brain slice DA monitoring. Cyclic voltammetry was used to evaluate the mechanical stability of the sensor. Figures 25-26 CV images before and after bending the electrode by 45° are shown, and the DA peak current changed little after 50 bends, which proves that mechanical deformation of the electrode during application does not significantly affect its electrocatalytic activity. In addition, as shown in Figure 27As shown, the current recorded by EWCDs@Nafion / AuNCs / CFME is more stable compared to bare CFE, indicating that the electrode does not adsorb DA oxidation products.

[0096] Example 5. Real-time monitoring of live cell DA

[0097] To verify the feasibility of the electrode to monitor the release of live cell DA, the following experiment was conducted: First, the cytotoxicity of EWCDs@Nafion / AuNCs / CFME was studied by CCK-8 experiment, and the results are shown in FIG. 6A. Figure 28 As shown, the viability of PC12 cells after material modification increased, indicating that the electrode material has low toxicity to cells. Second, the biocompatibility of the electrode was further studied using live / dead cell staining, and the results are shown in FIG. 6B. Figure 29 As shown, after 24 hours, PC12 cells on the same modified carbon cloth proliferated well and maintained a high viability, with significantly fewer dead cells than bare carbon cloth, proving that EWCDs@Nafion / AuNCs / CFME has good biocompatibility.

[0098] Figure 30 The molecular mechanism of potassium ion stimulation and nifedipine inhibition of dopamine release is shown, and this model is used to evaluate the feasibility of EWCDs@Nafion / AuNCs / CFME to detect cell and brain slice DA release. The ease of use of EWCDs@Nafion / AuNCs / CFME was detected by real-time monitoring of DA release from PC12 cells. High concentrations of KCl were chosen as the drug to stimulate live cells to release DA. Figure 31 The current response of the EWCDs@Nafion / AuNCs / CFME electrode under different conditions at a potential of 0.25 V is shown. After the addition of 100 mM KCl, a clear response of DA release from the cells and a stable current response on the EWCDs@Nafion / AuNCs / CFME were observed. In this case, the current first rapidly increased to a maximum value and then gradually decreased, which means that KCl stimulates the release of DA in PC12 cells through a rapid process. In contrast, when KCl and nifedipine were simultaneously added to the above PC12 cell system, the current only increased slightly compared to the potassium ion stimulation, indicating that the calcium channel inducing DA release in the cells has been blocked by nifedipine. Figure 32 The DA response data with and without nifedipine are shown. The data show that the response current of DA secreted by PC12 cells treated with nifedipine decreases, but does not decay to the initial level, indicating that there is still a small amount of DA release, which is consistent with the results observed in previous studies. These results confirm that the sensor of the present application has a full response to DA with high sensitivity in the extracellular environment.

[0099] Example 6. Detection of DA in rat brain slices

[0100] The present application further verified the applicability of the electrode of the present application in the brain of a living rat. The results are shown in Figure 33 Figure 33 The results show the amperometric response of the brain slice after local injection of 100 mM KCl to stimulate the electrode inserted into the striatum of the rat. Consistent with previous reports, a steady increase in current response was observed, which is due to K + stimulation-induced cell membrane depolarization, inducing Ca 2+ influx and leading to the release of DA in vesicles. At the same time, in the inhibition experiment, the incubation buffer without brain slices was used as a blank control to exclude the current signal of K + , and nifedipine was used as a calcium channel blocker to evaluate the performance of the electrode in in vivo detection. The statistical data are similar to the results of the PC12 cell experiment Figure 34 ), except that the basal level of DA in the brain slice is relatively high, but the electrode of the present application can still accurately capture the subtle current changes caused by a small amount of dopamine. The above results reveal the great application prospect of the sensor in brain slice and even in vivo detection and the study of the mechanism of nervous system diseases.​

Claims

1. A modified CFME for detecting dopamine in the brain or cells, characterized in that, The modified CFME is prepared by: sputtering gold nanoparticles onto the surface of pretreated carbon fibers to obtain AuNPs / CF; growing gold nanocoral on the surface of the AuNPs / CF electrode by electrodeposition to obtain AuNCs / CF; and then modifying the AuNCs / CF with egg white-based EWCDs to obtain the modified CFME; the EWCDs are immobilized using Nafion.

2. The modified CFME according to claim 1, characterized in that, The pretreatment involves first subjecting CF to ultrasonic treatment in acetone, HNO3, KOH, and distilled water sequentially; then electrochemically activating CF.

3. The modified CFME according to claim 1, characterized in that, The concentration of the EWCDs was 0.01 g / L.

4. The modified CFME according to claim 1, characterized in that, The electrodeposition method is as follows: using the AuNPs / CF as the working electrode, Ag / AgCl as the reference electrode, and Pt wire as the counter electrode, electrochemical deposition is carried out in a mixed solution; the electrodeposition time is 12 h; the mixed solution is a mixture of AuCl3, CTAB, NaNO3 and ultrapure water.

5. The modified CFME according to claim 1, characterized in that, The XRD pattern of the modified CFME shows diffraction peaks at 2θ values ​​of 38.27, 44.44, 64.70, and 77.702, corresponding to the 111, 200, 220, and 311 crystal planes of gold, respectively. The O 1s spectrum of the modified CFME has two characteristic peaks at 535.5 eV and 532.1 eV, corresponding to the O element in Nafion and EWCDs, respectively. The C 1s spectrum of the modified CFME has three characteristic peaks at 287.4 eV, 289.2 eV, and 291.8 eV, corresponding to the C element. The Au 4f spectrum has two characteristic peaks at 88.1 eV and 84.4 eV, corresponding to Au 4f. 5 / 2 and Au 4f 7 / 2 Atomic orbitals.

6. A dopamine sensor containing the modified CFME according to any one of claims 1-5.

7. A method for in vitro detection of dopamine using the dopamine sensor according to claim 6, characterized in that, Includes the following steps: (1) Prepare the sample to be tested; (2) The dopamine sensor described in claim 6 is used to perform electrochemical detection on the sample to be tested to obtain the current response; (3) Perform qualitative and / or quantitative analysis of dopamine in the test sample based on the current response.

8. The method according to claim 7, characterized in that, The samples to be tested are isolated live cells and / or brain slices.

9. The method according to claim 7, characterized in that, The detection potential is 0.25V.

10. The method according to claim 7, characterized in that, The measured current response was substituted into the standard curve equation to calculate the dopamine content; when the dopamine concentration was between 0.001 μM and 20 μM, the standard curve equation was: Y = 0.489X - 9.741, R 2 =0.996, where Y is the Y-axis, representing the current signal, and X is the X-axis, representing the dopamine concentration.

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