Electrode for identifying and detecting cysteine and application

Abstract

The invention belongs to the technical field of amino acid detection, and relates to an electrode for identifying and detecting cysteine and application, the electrode is a glassy carbon electrode modified by vitamin B12 and subjected to electrochemical activation treatment; the electrochemical activation treatment comprises oxidation treatment and reduction treatment; the vitamin B12 modification method is an impregnation method and / or the electrolyte in the reduction treatment process contains vitamin B12, and modification is carried out in the electrochemical activation process. According to the invention, direct electric polarization of the molecular catalyst is realized, Cys can be effectively distinguished from sulfydryl of other similar structures, and interference of common electroactive compounds coexisting in a biological sample is eliminated; the super-hydrophilic surface endows the sensor with excellent biological pollution resistance; a new example is provided for electrochemical sulfydryl analysis, an important theoretical basis is provided for bionic interface engineering, and the method is expected to be applied to clinical diagnosis and on-site rapid detection; and a thought is provided for modification of other types of molecular catalysts such as Co-N4.

Description

Technical Field

[0001] The invention belongs to the technical field of amino acid detection, and relates to an electrode for identifying and detecting cysteine ​​and an application thereof. Background Art

[0002] L-Cysteine ​​(Cys), also known as L-2-amino-3-mercaptopropionic acid, is a sulfur-containing α-amino acid that is considered a conditionally essential amino acid. Cys is converted from methionine in vivo through a series of reactions involving the conversion of methionine to homocysteine ​​and further transsulfuration. In biological fluids and tissues, L-Cys exists in three forms: free reduced, free oxidized (cystine), and protein-bound via S-cystylation. Cys plays a key role in a variety of biological processes. It is a subunit of protein synthesis and contributes to protein folding and stability, thereby supporting its biological activity. It also plays an important role in development, signal transduction, biological defense, aging, and disease. Cys is considered a potential marker or mediator of oxidative stress.

[0003] The concentration of Cys in tissues is tightly regulated through controlled production and efficient clearance mechanisms. As a precursor amino acid, Cys plays a key role in the synthesis of proteins, coenzyme A, and glutathione (GSH), and is also involved in the production of their metabolites such as taurine and inorganic sulfur. To support these biosynthetic processes, the concentration of Cys in tissues must be sufficient. However, the human body can still function effectively at relatively low Cys concentrations because it can store Cys in the form of GSH. When needed, GSH can be hydrolyzed to release Cys, thereby ensuring essential metabolic functions. The concentration of Cys in the liver is maintained at approximately 0.01 to 0.1 mmol / L. In human plasma, the concentration of Cys ranges from 240 to 360 µM. Low levels of Cys are associated with a variety of diseases, including cardiovascular disease (CVD), ischemic stroke, neurological diseases, diabetes, cancers (such as lung and colorectal cancer), diseases related to renal dysfunction, and vitiligo.

[0004] Over the past few decades, a variety of techniques have been developed for Cys detection, including HPLC-UV, capillary electrophoresis, and mass spectrometry, while recent advances in fluorescence methods have further expanded the detection capabilities. Electrochemical methods have general advantages such as simplicity, high stability, high sensitivity, low equipment cost, and on-site supervision. In addition, they have shown significant potential in in vivo analysis and wearable applications, making them very promising for real-time health monitoring and personalized diagnosis. However, the electrochemical analysis of thiol groups remains extremely challenging. For example, conventional electrodes (such as glassy carbon and gold electrodes) generally exhibit poor electrochemical responses due to sluggish electron transfer kinetics, high overpotentials, and strong adsorption of thiol groups on the electrode surface (gold electrodes). In order to increase the electron transfer rate and reduce the overpotential, a variety of catalysts have been used for the electrochemical oxidation and detection of Cys, including graphene nanoribbons, boron-doped carbon nanotube-modified electrodes, ordered mesoporous carbon, metal-organic frameworks, cycloterphenyls, catechol, BiPr composite nanosheets, multi-walled carbon nanotubes / gold nanorods, Cu x S electrodes for recognition and detection of cysteine ​​SOD zeolite and Ni2P nanocrystals anchored on black phosphorus nanosheets.

[0005] However, these methods usually involve the use of two or more layers of modification, meaning that these catalysts or layers are usually fixed on the surface of traditional electrodes. In contrast, single-layer modification for the construction of simpler biothiol electrochemical sensors has also been reported. For example, screen-printed diamond electrodes for Cys detection. There is a unique class of catalysts that not only show excellent catalytic oxidation ability for biothiols, but also reduce the oxidation product of biothiols, disulfides. This allows the simultaneous detection of biothiols and disulfides. Such catalysts include Co-N4 catalysts, such as natural vitamin B12 (VB12), cobalt phthalocyanine (CoPc) and its derivatives. CoPc has extremely low solubility in water and organic solvents, which usually leads to aggregation and reduced electroactivity. In contrast, VB12 shows excellent water solubility and good compatibility in the modification process. It has been verified that VB12 can be effectively adsorbed on the surfaces of carbon nanotubes (CNTs), graphene oxide (GO) and graphite. However, the adsorption of VB12 is unstable on GCE, which is the most commonly used in electrochemistry and electroanalysis. After several scans, VB12 molecules tend to fall off the GCE surface. Therefore, it is very important to directly immobilize VB12 on GCE without the aid of other support materials and study its catalytic activity in the conversion of biothiols and disulfides. Summary of the invention

[0006] In view of the shortcomings of the above-mentioned prior art, the present invention innovatively proposes a construction strategy of "direct electric polarization of molecular catalysts". Without the need for other carrier materials, VB12 is directly modified on the surface of GCE, thereby proposing an electrode and application for identifying and detecting cysteine. The specific technical solution is as follows: The first object of the present invention is to provide an electrode for identifying and detecting cysteine, which is a glassy carbon electrode (GCE) modified with electrochemical activation by vitamin B12; The electrochemical activation treatment includes oxidation treatment and reduction treatment; The method for modifying the vitamin B12 is an immersion method; and / or the electrolyte in the reduction treatment process contains vitamin B12, which is modified simultaneously during the electrochemical activation process.

[0007] The present invention has developed an electrode for Cys recognition and detection. By co-modifying VB12 with electrochemically activated GCE, direct electropolarization of VB12 is achieved, and dual catalytic functions are exhibited: excellent electrocatalytic oxidation of Cys and efficient reduction of its oxidation products. This unique interface simulates enzyme behavior, in which VB12 acts as a redox mediator, reduces the reaction overpotential and enhances the electron transfer kinetics, similar to natural metalloenzymes. In addition, its superhydrophilic surface gives the sensor excellent anti-biofouling performance.

[0008] Furthermore, the solvent of the immersion method is deionized water or pH 7.0 PBS solution (phosphate buffer solution), that is, VB12 is dissolved in deionized water or pH 7.0 PBS.

[0009] Furthermore, in the immersion method, the VB12 concentration of the solution is 0.3-0.7 mM, and the immersion time is 20-40 minutes; preferably, the VB12 concentration is 0.5 mM, and the immersion time is 30 minutes.

[0010] Furthermore, the pH of the electrolyte subjected to the oxidation treatment is 5.5-13, preferably 7-12, and the optimal pH is 7.

[0011] Furthermore, the electrolyte for the oxidation treatment is one or more of acetate solution, phosphate solution, carbonate solution, potassium chloride solution, sodium hydroxide solution, and HCl-Tris solution, the applied potential is 1.3~2V (relative to Ag / AgCl saturated potassium chloride reference electrode), and the treatment time is 400~600s; preferably, the electrolyte is phosphate solution, carbonate solution, and potassium chloride solution, and the applied potential is 1.65~1.85V (relative to Ag / AgCl saturated potassium chloride reference electrode); more preferably, the electrolyte is phosphate and carbonate solution, the applied potential is 1.75V, and the treatment time is 500s.

[0012] Furthermore, the electrolyte for the reduction treatment is one or more of sodium hydroxide solution, carbonate solution, HCl-Tris solution, phosphate solution or acetate solution, with a pH of 5.5-13, an applied potential of -0.3-0.9V (relative to Ag / AgCl saturated potassium chloride reference electrode), and a treatment time of 400-600s; preferably, the electrolyte is phosphate, Tris-HCl solution, sodium hydroxide solution, with a pH of 7.0-13, an applied potential of -0.6-0.9 (relative to Ag / AgCl saturated potassium chloride reference electrode); more preferably, the electrolyte is pH7.0PBS, the applied potential is -0.7V, and the treatment time is 500s.

[0013] Furthermore, the VB12 concentration of the reduction-treated electrolyte is 0.3-0.7 mM, preferably 0.5 mM.

[0014] Co-N4 type molecular catalysts such as VB12 are usually loaded on the surface of porous carbon-based carriers such as graphene and carbon nanotubes through π-π stacking or covalent bonding, and then modified on the GCE substrate through a drop coating film formation process; although this multi-level modification strategy can increase the density of catalytic sites, the complex preparation process may lead to active site embedding and reduced interfacial charge transfer efficiency. The present invention breaks through the traditional loading mode and innovatively proposes a "molecular catalyst direct electric polarization" construction strategy to directly modify VB12 on GCE. The method is simple, fast and green. This method can also be used to modify other molecular catalysts other than VB12, such as TEMPO, cobalt phthalocyanine, etc. The "molecular catalyst direct electric polarization" method provides a new method for modifying other Co-N4 type molecular catalysts.

[0015] The second object of the present invention is to provide the application of the above-mentioned electrode in the identification and detection of electroactive amino acids; the electroactive amino acids are cysteine, glutathione, cystine or other electroactive amino acids; by changing the pH value of the detection solution, it is used for the identification and detection of cysteine, glutathione, and cystine; it can also be used for the detection of other electroactive substances, such as the identification and detection of dopamine, etc.

[0016] Furthermore, the application achieves multi-peak and multi-mode (potential and current) spectral identification through negative differential pulse voltammetry, which satisfies dynamic detection and identification in a wide concentration range.

[0017] The electrode interface for identifying and detecting cysteine ​​of the present invention realizes the ultra-low potential oxidation of Cys and the reversible reduction of its oxidation products. The synergistic effect between the nanostructured surface of GCE (rich in oxygen-containing functional groups) and the redox-active cobalt center of VB12 makes multipath interaction with Cys possible, thereby producing extremely low oxidation potential and spectral-like DPV fingerprints. In addition to low-potential, high-sensitivity IT detection, the interface also realizes multi-mode spectral-like differentiation through negative differential pulse voltammetry (DPV) fingerprinting, which can effectively distinguish Cys from other thiol groups with similar structures. It is a multi-mode spectral-like electrochemical fingerprint platform for identifying and detecting cysteine ​​(Cys), which can simulate the reversible transformation cycle of Cys / cystine.

[0018] The synergistic effect between the nanostructured surface of GCE (rich in oxygen-containing functional groups) and the redox-active cobalt center of VB12 enables multipath interactions with Cys, resulting in extremely low oxidation potentials and spectroscopic DPV-like fingerprints. The unique advantage of this platform lies in its "spectro-electrochemical" duality: the DPV curve is similar to a spectroscopic fingerprint, enabling the selective identification of Cys in complex matrices through machine learning-assisted pattern recognition, while retaining the simplicity, cost-effectiveness and portability of electroanalysis.

[0019] Compared with the prior art, the present invention has the following beneficial effects: The present invention has developed an electrode and a multi-mode spectroelectrochemical fingerprint platform for identifying and detecting cysteine, which can effectively distinguish Cys from other thiol groups with similar structures, and can also eliminate the interference of common electroactive compounds coexisting in biological samples; in addition, its super-hydrophilic surface gives the sensor excellent anti-biological contamination performance. The present invention not only provides a new paradigm for electrochemical thiol analysis, but also provides an important theoretical basis for biomimetic interface engineering, and is expected to be applied to clinical diagnosis and on-site rapid detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 For the present invention GCE, R VB12- GCE and GCE-D VB12 Cyclic voltammograms in 0.1 mol / L, pH 7.0 PBS (short dot line), 5 mM Cys (red solid line) and 5 mM GSH (short dash line), respectively, with a scan rate of 0.05 V / s; Figure 2 Cyclic voltammetry curves (CVs) of O-GCE and OR-GCE of the present invention in 0.1 M PBS, pH 7.0 (short dotted line), 5 mM Cys (red solid line) and 5 mM GSH (blue solid line) at a scan rate of 0.05 V / s; Figure 3Cyclic voltammetry curves of OR-GCE of the present invention at different final potentials in PBS, all scans started from 0.6 V, and the scan rate was 0.05 V / s; Figure 4 Thirty consecutive cyclic voltammograms of O-GCE in 5 mM Cys of the present invention, with a scan rate of 0.05 V / s; the initial potential was set to 0 V, the highest potential reached 0.6 V, and the lowest potential dropped to -1.05 V; the scan direction was forward; Figure 5 For the present invention VB12 -GCE and O VB12 -GCE cyclic voltammetry curve; a:O VB12 Cyclic voltammetry curves of -GCE in 0.1 M PBS, pH 7.0 (short dotted line) and 5 mM Cys (solid line), with the potential cycle range from -0.2 V to 0.6 V, and then from -0.2 V to -1.05 V, with a scan rate of 0.05 V / s; b:O VB12 Cyclic voltammograms of -GCE in 0.1 M PBS, pH 7.0 (short dotted line) and 5 mM Cys (solid line), with the potential cycled from -0.2 V to -1.05 V, then from -0.2 V to 0.6 V, with a scan rate of 0.05 V / s; Figure 6 CV curves of VB12 modified electrodes prepared by different modification methods of the present invention; Figure 7 The cyclic voltammograms of the glassy carbon electrodes treated differently in the present invention in 0.5 mM vitamin B12 (VB12) in 0.1 M, pH 7.0 phosphate buffer solution (PBS): The short dotted line represents 0.5 mM VVB12, the red solid line represents 5 mM cysteine ​​(Cys), and the blue solid line represents 5 mM glutathione (GSH); the potential scanned from -1.05 V to 0.6 V in the positive direction, and the scan rate was 0.05 V / s; Figure 8 For the present invention, in 5mMCys, OR VB12 -30 consecutive cyclic voltammograms of GCE, scan rate 0.05 V / s; initial potential: -0.4 V, highest potential: 0.6 V, lowest potential: -1.05 V, scan direction: negative; Fig. 9 The cyclic voltammetry curve of GCE in 0.5 mM VVB12 of the present invention, the scanning rate is 0.05 V / s; the initial potential is 0 V, the highest potential is 1.75 V, the lowest potential is -0.7 V, and the scanning direction is forward; Fig.10Cyclic voltammetry curves of OR-VB12-GCE in 0.1 M PBS, pH 7.0 (short dot line) and 5 mM Cys (solid line) according to the present invention, with a scan rate of 0.05 V / s; initial potential: -1.05 V; scan direction: forward; Fig.11 Cyclic voltammetry curves of O-GCE-DVB12 in 0.1 M PBS, pH 7.0, in the absence (short dotted line) and presence (solid line) of 5 mM Cys, at a scan rate of 0.05 V / s; Fig.12 For the present invention GCE, O-GCE, OR VB12 -Nyquist diagrams and corresponding Bode diagrams of GCE and OR-GCE; (a) In 0.1M KCl solution, containing 5mMFe(CN)6 3- / Fe(CN)6 4- When, GCE, O-GCE, OR VB12 -Nyquist plots of GCE and OR-GCE; Inset: Enlarged Nyquist plot; (b) Corresponding Bode plot; Inset: equivalent circuit; Symbols and solid lines represent experimental data and fitting data, respectively; the frequency range of EIS was 0.1 MHz to 0.1 Hz and was performed at 0.24 V; Fig.13 For the present invention VB12 -GCE, O VB12 R-GCE, OV B12 C1s X-ray photoelectron spectroscopy (XPS) spectra of -GCE, OR-GCE, O-GCE and GCE: (a)OR VB12 -GCE, (b)O VB12 R-GCE, (c)OV B12 -GCE, (d)OR-GCE, (e)O-GCE, (f)GCE; Fig.14 For the present invention VB12 -GCE, O VB12 R-GCE, O VB12 -O1sX-ray photoelectron spectroscopy (XPS) spectra of GCE, OR-GCE, O-GCE and GCE; (a)OR VB12 -GCE, (b)O VB12 R-GCE, (c)O VB12 -GCE, (d)OR-GCE, (e)O-GCE, (f)GCE; Fig.15 For the present invention VB12 -GCE, O VB12R -GCE, O VB12 -N1sXPS spectra of GCE, OR-GCE, O-GCE and GCE; (a)OR VB12 -GCE; (b)O VB12R -GCE; (c)O VB12 -GCE; (d)OR-GCE; (e)O-GCE; (f)GCE; Fig.16 For the present invention VB12 -GCE, O VB12R -GCE and OR VB12 -Co2pXPS spectrum of GCE; (a)O VB12 -GCE, (b)O VB12R -GCE and (c)OR VB12 -GCE; Fig.17 For the present invention (a) OR VB12 -GCE, (b)O VB12 R-GCE, (c)O VB12 -GCE, (d) OR-GCE, (e) O-GCE, and (f) GCE P2pXPS spectra; Fig.18 For the present invention VB12 - Raman spectra of GCE, OR-GCE, O-GCE and GCE; Fig.19 Cyclic voltammetry curves of the electrode treated in different electrolytes (containing 0.5 mM VVB12) in 0.1 M phosphate buffer solution (PBS) with a pH of 7.0 (short dotted line), 5 mM cysteine ​​(Cys) (red solid line) and 5 mM glutathione (GSH) (blue solid line) during the oxidation stage of the OR-GCE of the present invention; the scan rate is 0.05 V / s; Fig. 20 For the present invention VB12 -GCE cyclic voltammetry curves (CVs) at different oxidation potentials during the oxidation stage, measured in 0.1 M phosphate buffer solution (PBS), pH 7.0 (short dotted line), 5 mM cysteine ​​(Cys) (red solid line) and 5 mM glutathione (GSH) (blue solid line), with a scan rate of 0.05 V / s; Fig.21 The bar graph of the present invention shows the effect of different oxidation potentials on the reduction peak currents, which are observed at 0V and -0.65V respectively; Fig. 22 Cyclic voltammetry curves of the OR-GCE of the present invention using different electrolytes (0.5 mM vitamin B12 as solute) in the reduction stage, the measurements were carried out in 0.1 M, pH 7.0 phosphate buffer solution (PBS) (short dotted line), 5 mM cysteine ​​(Cys) (red solid line) and 5 mM glutathione (GSH) (blue solid line), respectively, with a scan rate of 0.05 V / s; Fig.23 For the present invention VB12 -Cyclic voltammetry curves of GCE at different reduction potentials during the reduction stage, in 0.1 M PBS, pH 7.0 (short dotted line), and 5 mM Cys (solid line); the potential was scanned from -0.4 V to 0.6 V, and then reversely scanned to -1.05 V, with a scan rate of 0.05 V / s; Fig.24 For the present invention VB12 - Cyclic voltammetry curves of GCE in different concentrations of Cys; the inset shows the linear relationship between the peak current (labeled as 'a' and 'b') and the Cys concentration; Fig.25 For the present invention VB12 -Amperometric response of GCE to the continuous addition of cysteine ​​(Cys) in a stirred 0.1 M phosphate buffer solution (PBS, pH 7.0) with an applied potential of 0 V; The inset (A) shows the calibration curves between the steady-state current and different concentrations of Cys; the inset (B) shows the amperometric response at lower concentrations of Cys; Fig.26 For the present invention VB12 -Amperometric response of GCE to the continuous addition of cysteine ​​(Cys) in a stirred 0.1 M phosphate buffer solution (PBS, pH 7.0) with an applied potential of 0.15 V; The inset (A) shows the calibration curves between the steady-state current and different concentrations of Cys; the inset (B) shows the amperometric response at lower concentrations of Cys; Fig. 27 In the present invention, in a stirred 0.1 M PBS (pH 7.0), OR VB12 -Amperometric response of GCE upon continuous addition of 5 μM (A) and 20 μM (B) Cys, with a constant potential of 0.15 V applied during the measurement; Fig.28 For the present invention VB12 - Long-term amperometric response of the GCE electrode to 5 μM (A) and 20 μM (B) Cys in 0.1 M PBS (pH 7.0), with a constant potential of 0.15 V applied during recording; Fig.29In the present invention, in 0.1M PBS (pH=7.0), OR VB12 -Amperometric responses of GCE to single and continuous addition of 20 μM Cys, measured at 0 V potential; Fig.30 For the present invention VB12 Negative scanning differential pulse voltammetry (DPVs) of -GCE in 0.1 M phosphate buffer solution (PBS, pH 7.0) with cysteine ​​(Cys) concentration gradually increasing from 1 μM to 10 mM; the inset shows the current response calibration curves of peaks a, b, d, and e at different Cys concentrations, and the potential shift of peak c; Fig.31 For the present invention VB12 - Negative scan differential pulse voltammograms (DPVs) of GCE in solutions containing 0.1 M PBS (pH 7.0), 500 μM Cys, 200 μM AA, and 200 μM UA; Fig.32 In the present invention, in 0.1M PBS and 5mM Cys solution, OR VB12 Cyclic voltammetry curves of -GCE were recorded before and after the addition of 20 mg / mL BSA. DETAILED DESCRIPTION

[0021] The principles and features of the present invention are described below in conjunction with embodiments. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0022] Chemical reagents and solutions: Cysteine ​​(Cys), glutathione (GSH), ascorbic acid (AA), uric acid (UA), tris(hydroxymethyl)aminomethane, Fe(CN)6³ - and Fe(CN)6 4- All chemical reagents were purchased from Sigma-Aldrich.

[0023] VB 12 Reagents such as NaH2PO4, Na2HPO4, acetic acid (HAc), NaAc, NaHCO3, KCl, HCl, and Na2CO3 were purchased from AlfaAesar.

[0024] All supplementary reagents were of analytical grade and all solutions were prepared with double distilled water.

[0025] Instruments and equipment: XPS analyses were performed using a Thermo ESCALAB 250 Xi spectrometer equipped with a monochromatic Al Kα X-ray source.

[0026] Raman spectroscopy measurements were performed using an inVia Qontor Raman spectrometer from Renishaw using a 633 nm laser wavelength.

[0027] The electrochemical experiments were carried out using a CHI 842C electrochemical workstation and an ASL RRDE-3A instrument.

[0028] The experiments were performed using a conventional three-electrode configuration, including GCE or its modified electrode as the working electrode, a platinum wire as the auxiliary electrode, and a KCl-saturated Ag / AgCl electrode as the reference electrode.

[0029] EIS was performed in 0.1M KCl solution containing 5mM Fe(CN)3 - / 4 - , the frequency range is 1MHz to 0.1Hz, and the applied potential is 0.25V.

[0030] The above chemical reagents and solutions, instruments and equipment are used in the following examples, comparative examples or tests.

[0031] Embodiment 1: An electrode for identifying and detecting cysteine ​​comprises the following steps: Step 1: Anodize the glassy carbon electrode: The glassy carbon electrode has a diameter of 3 mm and is polished to a mirror finish using 1 µm, 0.3 µm and 0.05 µm alumina slurries in sequence; after polishing, the glassy carbon electrode is carefully rinsed with deionized water and ultrasonically cleaned in distilled water to ensure absolute cleanliness; The electrolyte for the anodizing treatment was a 0.1 M pH 7.0 PBS solution; a potential of 1.75 V was applied to the electrode and the it curve was used for 500 seconds; Step 2: Connect the glassy carbon electrode to the cathode for reduction treatment: The electrolyte for the cathode reduction treatment was a pH 7.0 PBS solution containing 0.5 mM VB12; a potential of -0.7 V was applied to the electrode and the it curve was used for 500 seconds.

[0032] The obtained electrode was used for OR VB12 -GCE means.

[0033] Embodiment 2: An electrode for identifying and detecting cysteine ​​comprises the following steps: Step 1: Anodize the glassy carbon electrode: The glassy carbon electrode has a diameter of 3 mm and is polished to a mirror finish using 1 µm, 0.3 µm and 0.05 µm alumina slurries in sequence; after polishing, the glassy carbon electrode is carefully rinsed with deionized water and ultrasonically cleaned in distilled water to ensure absolute cleanliness; The electrolyte for the anodization treatment was a pH 7.0 PBS solution containing 0.5 mM VB12; a potential of 1.75 V was applied to the electrode, and the it curve was used for 500 seconds; Step 2: Connect the glassy carbon electrode to the cathode for reduction treatment: The electrolyte for the cathode reduction treatment was a pH 7.0 PBS solution containing 0.5 mM VB12; a potential of -0.7 V was applied to the electrode and the it curve was used for 500 seconds.

[0034] The obtained electrode was treated with O VB12 R VB12 -GCE means.

[0035] Embodiment 3: An electrode for identifying and detecting cysteine ​​comprises the following steps: Step 1: Anodize the glassy carbon electrode: The glassy carbon electrode has a diameter of 3 mm and is polished to a mirror finish using 1 µm, 0.3 µm and 0.05 µm alumina slurries in sequence; after polishing, the glassy carbon electrode is carefully rinsed with deionized water and ultrasonically cleaned in distilled water to ensure absolute cleanliness; The electrolyte for the anodizing treatment was a 0.1 M pH 7.0 PBS solution; a potential of 1.75 V was applied to the electrode and the it curve was used for 500 seconds; Step 2: Connect the glassy carbon electrode to the cathode for reduction treatment: The electrolyte for the cathode reduction treatment was a 0.1 M pH 7.0 PBS solution; a potential of -0.7 V was applied to the electrode and the it curve was used for 500 seconds.

[0036] Step 3: Dipping treatment: The electrochemically activated glassy carbon electrode was immersed in an ultrapure deionized water solution containing 0.5 mM VB12 for 30 min.

[0037] The obtained electrode was used for OR-GCE-D VB12 express.

[0038] Comparative Example 1: Step 1: Anodize the glassy carbon electrode: This step is the same as step 1 of Example 1 and will not be described again; Step 2: Dipping treatment: The electrochemically activated glassy carbon electrode was immersed in a pH 7.0 PBS solution containing 0.5 mM VB12 for 30 min.

[0039] The obtained electrode was used for O-GCE-D VB12(pH7.0PBS) express.

[0040] Comparative Example 2: An electrode for identifying and detecting cysteine ​​comprises the following steps: The glassy carbon electrode is anodized: this step is the same as step 1 of Example 2 and will not be repeated here; the obtained electrode is anodic oxidized. VB12 -GCE means.

[0041] Comparative Example 3: An electrode for identifying and detecting cysteine ​​comprises the following steps: Step 1: Anodize the glassy carbon electrode: This step is the same as step 1 of Example 2 and will not be described again; Step 2: Connecting the glassy carbon electrode to the cathode for reduction treatment: This step is the same as step 2 of Example 3 and will not be described again here; The obtained electrode was treated with O VB12 R-GCE indicates.

[0042] Comparative Example 4: An electrode for identifying and detecting cysteine ​​comprises the following steps: Step 1: Anodize the glassy carbon electrode: This step is the same as step 1 of Example 1 and will not be described again; Step 2: Immersion treatment: This step is the same as step 3 of Example 3 and will not be described again; The obtained electrode was used for O-GCE-D VB12 express.

[0043] Comparative Example 5: Connecting the glassy carbon electrode to the cathode for reduction treatment: This step is the same as step 2 of Example 1 and will not be described again here; The obtained electrode is R VB12 -GCE means.

[0044] Comparative Example 6: Immersion treatment: This step is the same as step 3 of Example 3 and will not be described in detail here; The obtained electrode was used for GCE-D VB12 express.

[0045] Comparative Example 7: Step 1: Anodize the glassy carbon electrode: This step is the same as step 1 of Example 1 and will not be described again; Step 2: Connecting the glassy carbon electrode to the cathode for reduction treatment: This step is the same as step 2 of Example 3 and will not be described again here; The obtained electrode is denoted as OR-GCE.

[0046] Comparative Example 8: The glassy carbon electrode is anodized: this step is the same as step 1 of Example 1 and will not be repeated here; the obtained electrode is denoted by O-GCE.

[0047] Comparative Example 9: Cyclic voltammetry (CV) preparation method: OR-VB12-GCE was prepared by cyclic voltammetry in 0.5 mM vitamin B12 (VB12) solution. The parameters were set as: starting potential 0 V, maximum potential 1.75 V, minimum potential -0.7 V, and scan rate 0.05 V / s.

[0048] The obtained electrode is denoted by OR-VB12-GCE.

[0049] Table 1 lists various electrodes using PBS as the electrolyte and their specific preparation parameters.

[0050] Table 1. Comparison of preparation parameters of different electrodes

[0051] Test results and analysis: 1. Immobilization of VB12 and electrocatalysis of thiol groups from Figure 1 Displayed GCE, R VB12 -GCE and GCE-D VB12 From the cyclic voltammetry (CV) curves in 0.1M pH 7.0 PBS (short dashed line), 5mM Cys (red solid line) and 5mM GSH (short dashed line), it can be seen that the CV curves of these three electrodes in the background electrolyte are basically the same, without obvious redox peaks. This shows that the electrode treated with electrochemical reduction and immersed in VB12 is no different from the bare GCE, which means that VB12 cannot be adsorbed on the bare GCE by the impregnation method. Both the anodized GCE (O-GCE) and the oxidized and then reduced GCE (OR-GCE) show a pair of redox peaks in PBS, with a potential range of -0.3 to 0.1 V (see Figure 2 ). It is noteworthy that this pair of redox peaks remains unchanged regardless of the scan range (see Figure 3 ). This is due to the redox reaction of the quinone groups on the electrode surface.

[0052] After the addition of Cys, an oxidation peak appeared at 0.3 V, but no reduction peak was observed, indicating that the activated GCE was able to catalyze the oxidation of Cys but could not further catalyze the reduction of the oxidation product. However, since the CV scan started from a more negative potential, it cannot be ruled out that the reduction reaction may occur at a more negative potential. Therefore, we performed a reproducible CV scan of O-GCE, starting from an initial potential of 0 V and scanning in the positive potential direction (see Figure 4 and Figure 5 ). The results show that in the first cycle, the oxidation curve is Figure 2 Although there is an oxidation current, no oxidation peak appears, and the reduction curve is also significantly different: in the first cycle, there is a large reduction peak current, which is due to the reduction of oxygen-containing functional groups on the surface of O-GCE. After two cycles, the CV curve is basically the same as Figure 4 This observation is also confirmed by CV experiments with anodic and cathodic scans in different directions. Figure 2 The O-GCE in represents the characteristics of OR-GCE because the O-GCE is immediately reduced once the CV scan potential is applied.

[0053] Figure 6 The CV curves of VB12 modified electrodes prepared by different modification methods are shown (in 0.1 M pH 7.0 phosphate buffer solution (PBS), the potential scan range is 0.6 V to -1.05 V, and the scan rate is 0.05 V / s). All electrodes show a pair of redox peaks corresponding to the quinone group. Compared with O-GCE and OR-GCE, VB12 can be successfully modified on other electrodes by different methods, although significant differences in electrochemical activity are detected. The differences in electrochemical performance between these electrodes may be attributed to the presence of different oxygen-containing functional groups, which may affect the binding mode of VB12 or its intrinsic activity. After multiple CV scans until stability, in the cathode direction, O-GCE-D VB12 , O VB12 R-GCE and O VB12 R-GCE only exhibits an obvious reduction peak at about -0.7 V. In contrast, O VB12 R VB12 -GCE, OR VB12 -GCE and OR-GCE-D VB12Shows more complex electrochemical behavior, characterized by multiple redox peaks. In other words, GCE must be treated with oxidation and reduction before it can interact with VB12 to show enhanced electrochemical activity. Electrochemical activation of GCE can form oxygen-functionalized graphene microdomains on its surface, as well as numerous oxygen-containing functional groups. This structure leads to a non-uniform interaction with VB12, which may involve multiple interactions such as π-π stacking, coordination bonds, hydrophobic / hydrophilic interactions, and hydrogen bonds. Compared with O-GCE, OR-GCE has a different interaction mode with VB12. The electrochemical oxidation process introduces oxygen-containing functional groups (such as carboxyl and hydroxyl groups) on the electrode surface, which enhances the hydrophilicity of the surface but destroys the carbon conjugated structure, thereby reducing the π-π interaction. The reduction process can partially remove these oxygen-containing functional groups, restore the conjugated structure, and increase the hydrophobicity and π electron density of the surface. This makes the π-π interaction with VB12 more favorable. In addition, the reduction process also facilitates electron transfer. OR-GCE-D VB12 The successive CVs show that the reduction peak in the first cycle is different from the subsequent CV peaks. In addition, the reduction peak of VB12 (such as Co³ + →Co² + ) overlaps with the reduction peak of the quinone group (C=O→C-OH), indicating that there is an electronic interaction between them. This phenomenon can be attributed to the ligand displacement under reducing conditions, where the cyanide (CN - ) ligand is hydroxyl group (OH - ) or hydrated (H2O) ligand displacement. During the reduction process, cyanocobalamin (vitamin B12) undergoes a structural transformation to hydroxocobalamin. Here, we assume that there are quinone groups on the electrode surface, which may coordinate competitively with the cobalt center. We speculate that VB12 can pull the cobalt center closer to the electrode surface through π-π interactions (e.g., with the benzimidazole group attached to the corrin ring) and interact with oxygen-containing functional groups.

[0054] from Figure 7 The electrocatalytic performance of VB12 modified electrodes prepared by different modification methods towards glutathione (GSH) and cysteine ​​(Cys) is shown. It is worth noting that the electrocatalytic activity of all VB12 modified electrodes towards Cys is higher than that of GSH, which may be due to the differences in the dissociation behavior of biothiols and their different interfacial interactions with the electrode surface. The VB12 functionalized electrodes exhibited obvious electrochemical oxidation activity towards Cys, and at the same time had electrocatalytic reduction ability towards the oxidation products of Cys. In Cys, when scanning from a starting potential of -0.4 V, only a background reduction peak was observed during the negative scan. An obvious reduction peak appeared after the second cycle, indicating that the reduction peak originated from the oxidation process (see Figure 8). For the electrode that was pre-treated with reduction before VB12 immobilization, two obvious oxidation peaks were observed during the Cys oxidation process with abnormally low potentials - one around 0 V and the other around 0.4 V (relative to Ag / AgCl). In the reverse scan, two reduction peaks were also accompanied, including a smaller shoulder peak. This multi-peak behavior indicates the presence of multiple catalytic active sites on the electrode surface. The oxidation onset potential was approximately -0.2 V, and there was a significant reduction peak at -0.67 V. In contrast, the electrode exposed to VB12 during the oxidation process showed a more positive oxidation onset potential (approximately -0.15 V) and a cathodic peak shifted to -0.78 V. These electrochemical changes were attributed to the different binding configurations of VB12 molecules on the electrode surface through different modification methods. However, the VB12 electrode modified by cyclic voltammetry (CV) exhibited very poor catalytic activity (see Fig. 9 and Fig.10 ). In addition, it is quite interesting that the catalytic activity of the electrode modified with VB12 dissolved in pure water is higher than that of the electrode modified with VB12 dissolved in PBS. This may be due to the fact that the presence of the electrolyte affects the interaction between VB12 and the electrode surface (see Fig.11 ).

[0055] 2. Electrode Characterization 2.1 Electrochemical impedance spectroscopy (EIS) characterization The interfacial charge transfer characteristics and surface modification of the modified electrode were analyzed by EIS. - / Fe(CN)6 4- 0.1M KCl solution (see Table 2 and Fig.12 ). The order of charge transfer resistance (R1) is: O-GCE (2744Ω) >> GCE (776.7Ω) > OR VB12 -GCE (9.025Ω) > OR -GCE (6.082Ω). CPE1-T (amplitude) values ​​are as follows: OR VB12 -GCE(0.00042)≈OR-GCE(0.00041)>>O-GCE(7.48×10 -6 )≈GCE(3.73×10 -6 ). The electrochemical anodic activation of GCE (forming O-GCE) introduced a large number of oxygen-containing functional groups on the electrode surface, resulting in a higher charge transfer resistance. After the subsequent cathodic reduction (forming OR-GCE), some of the oxygen-containing functional groups were removed, enhancing the conductivity of the electrode and thus reducing the charge transfer resistance. The slight resistance increase after VB12 modification suggests that the presence of VB12 may have introduced some additional resistance. OR VB12The higher CPE1-T values ​​of -GCE and OR-GCE indicate that their effective surface area or surface roughness increased due to the modification. This enhanced surface morphology may improve the catalytic activity of electrochemical reactions by providing more active sites.

[0056] Table 2. Fitting parameters of EIS of different electrodes

[0057] 2.2 X-ray photoelectron spectroscopy (XPS) characterization XPS spectroscopy provides valuable insights into the surface chemistry of VB12 modified electrodes. The C1s spectrum is decomposed into several peaks corresponding to different carbon environments, including CC / C=C, CO, OC=O, C=O, and possibly others such as C-OH (see Fig.13 , Table 3). The O1s spectrum is usually decomposed into peaks corresponding to different oxygen environments, such as C=O, CO, and C-OH (see Fig.14 , Table 3). GCE exhibited fewer oxygen-containing functional groups, lacking CO and OH. After electrochemical anodic oxidation (O-GCE), the contents of CO and OH increased significantly. After subsequent electrochemical cathodic reduction (OR-GCE), the content of CO bonds decreased, while the content of C=O increased. The application of cathodic potential induced a complex transformation of surface oxygen-containing functional groups. Compared with O-GCE, OVB12-GCE had a significantly higher OC=O content, which may be attributed to the amide groups introduced by VB12. In addition, significant changes in the peak positions of CO, OC=O, and C=O were observed, which may be due to the interaction between VB12 and these functional groups. In contrast, O VB12 R-GCE showed a decrease in CO and OC=O contents, while an increase in C=O content, and no significant change in the peak position. VB12 -GCE showed a slight change in the C=O peak position, and no significant change in the OC=O content. Obviously, the three VB12 modified electrodes showed different interaction mechanisms between VB12 and the underlying electrode substrate.

[0058] Table 3. Fitting parameters of C1s and O1s X-ray photoelectron spectroscopy (XPS) spectra

[0059] The presence of nitrogen was detected on the bare glassy carbon electrode (GCE), redox-treated GCE (OR-GCE), and oxidative-treated GCE (O-GCE), which may be due to the adsorption of nitrogen from the surrounding atmosphere (see Fig.15, Table 4). VB12 contains a cobalt-centered cobalt ring rich in nitrogen ligands, including pyridinic, pyrrolic, and amine groups. The deconvolution of X-ray photoelectron spectroscopy (XPS) N1s confirmed the presence of these nitrogen species in the VB12 modified electrodes. The differences in the content of each nitrogen component in the three VB12 modified electrodes can be attributed to the different amounts of VB12 fixed on the electrode surface and the different interaction modes between VB12 and the surface. The changes in these nitrogen species reflect the unique chemical environment created by the VB12 functional groups, which may affect the overall electrochemical behavior of the electrode.

[0060] All three VB12-modified electrodes showed XPS cobalt peaks (see Fig.16 ), which originates from the cobalt center in VB12. This also indicates that VB12 has been successfully immobilized on the electrode surface. No phosphorus peak was detected on the bare GCE (see Fig.17 , Table 4). However, after anodic oxidation, an obvious phosphorus peak appeared, which originated from the phosphate groups in the PBS solution. During the subsequent cathodic reduction process, the phosphorus peak weakened. All three VB12-modified electrodes showed significant phosphorus peaks, which may be derived from both PBS and the phosphate groups present in the VB12 molecules.

[0061] Table 4. Fitting parameters of N1s and P2p XPS spectra

[0062] 2.3 Raman spectroscopy characterization Raman spectroscopy data (see Fig.18 ) revealed that in the process of gradual electrode modification, the oxidation-treated glassy carbon electrode (O-GCE), the redox-treated glassy carbon electrode (OR-GCE), and the VB12-modified redox-treated glassy carbon electrode (OR VB12 -GCE) and the pristine glassy carbon electrode (pristine GCE) showed significant changes in structure and composition. The pristine glassy carbon electrode (pristine GCE) showed the highest ID / IG ratio (2.1), which indicates that there are inherent defects or disordered sp² domains on the untreated carbon surface. Electrochemical oxidation treatment (O-GCE) reduced the ID / IG ratio to 1.21, which is consistent with literature reports that the introduction of oxygen-containing functional groups (such as carboxyl, epoxy) during the oxidation process will partially passivate defects or destroy the graphitized sp² network, which is reflected by the upward shift of the G band (1591.8 cm - ¹) and an increase in the G-band full width at half maximum (FWHM) (68.5 cm -¹). This trend is consistent with the conversion of sp² carbon to sp³ carbon during oxidation, which has been described in studies on the structural evolution of graphene oxide. The subsequent electrochemical reduction treatment (OR-GCE) slightly increased the ID / IG ratio to 1.27, indicating partial recovery of the sp² domains or defect regeneration, possibly due to the removal of unstable oxygen-containing groups, while the G band position (1594.1 cm - ¹) and FWHM (62.5cm - ¹) reflects the disordered nature of the residues. Modification with VB12 (OR VB12 -GCE) further increased the ID / IG ratio to 1.28, which may be due to the surface heterogeneity caused by VB12 adsorption, such as local electron transfer or steric hindrance, which shifted the G band slightly (1595.6 cm - ¹) and a reduction in FWHM (58.4cm - These observations are consistent with the literature, where oxidation primarily passivates defects, reduction partially reverses structural disorder, and biomolecular functionalization introduces subtle interface modifications.

[0063] 3. Optimization of key factors to achieve excellent electrocatalytic activity Given the choice OR VB12 -GCE is used as a modified electrode in subsequent processes, and it is crucial to optimize the key parameters of the oxidation and reduction stages, especially the electrolyte composition and applied potential.

[0064] In pursuit of OR VB12 To achieve the best performance of -GCE, the parameters used in the oxidation and reduction processes were optimized in detail, including the electrolyte type, pH value, and applied potential. Unless otherwise stated, the electrolyte used in both the oxidation and reduction processes was pH 7.0 PBS. When focusing on optimizing the reduction process, the oxidation process always used pH 7.0 PBS as the electrolyte; conversely, when optimizing the oxidation process, the reduction process always used an electrolyte of pH 7.0 PBS containing 0.5 mM V VB12. Unless otherwise stated, the oxidation potential was generally set to 1.75 V, the reduction potential to -0.7 V, and the oxidation and reduction times to 500 s.

[0065] Fig.19The electrocatalytic activity of VB12 modified electrodes prepared using different electrolyte types and pH values ​​during the oxidation stage towards Cys and GSH is demonstrated. Notably, the electrodes oxidized using pH 5.5 ABS buffer and 0.1 M Tris-HCl (pH = 7.0) showed no detectable electrocatalytic activity towards both biothiols. Similarly, the electrodes oxidized using 0.1 M NaOH as the oxidation electrolyte and in pH 5.5 PBS solution also showed unsatisfactory performance. In contrast, the modified electrodes prepared by oxidation in an electrolyte of pH ≥ 7 showed excellent electrocatalytic activity towards both target biothiols. PO4³ - and CO3² - It may participate in the formation of functional groups on the electrode surface during electrochemical oxidation, promoting the formation of an oxide layer rich in carboxyl or hydroxyl groups. These oxygen-containing functional groups can enhance the chemical adsorption or electrostatic binding of VB12.

[0066] Fig. 20 and Fig.21 The effect of oxidation potential on the electrode catalytic activity was investigated. The results showed that when the oxidation potential was low, GCE failed to be effectively oxidized, resulting in the failure of VB12 to be successfully modified on the electrode surface, thereby reducing the catalytic activity. As the electrode potential increased, the electrocatalytic activity gradually increased. When the oxidation potential reached 1.75V, the oxidation peak current of Cys reached its maximum value. When the potential exceeded 1.75V, although the reduction peak current increased, the oxidation peak current gradually decreased. Therefore, the optimal oxidation potential was selected as 1.75V.

[0067] Different from the effect in the oxidation stage, in the reduction stage, the electrode treated with 0.1 M NaOH as the electrolyte also showed good electrocatalytic activity (see Fig. 22 ). When carbonate and acetate were used as reducing electrolytes, although the oxidation activity for thiol groups was poor, good catalytic activity was shown for the reduction of oxidation products. When pH 7.0 PBS was used as the reducing electrolyte, the electrode showed the best performance. In general, the effect of the electrolyte during the reduction process was not as significant as that during the oxidation process. Regarding the effect of the reduction potential on the reduction process, the electrocatalytic activity gradually increased with the negative shift of the potential and reached the best at -0.7 V (see Fig.23 ).

[0068] 4. Electrochemical Detection of Cysteine We used a variety of techniques including cyclic voltammetry (CV), chronoamperometry (IT), and differential pulse voltammetry (DPV) for the detection of Cys. Initially, CV showed low sensitivity with a linear range of 0.5–10 mM (see Fig.24), which is mainly due to the high background current of the electrode. Taking advantage of the high catalytic activity of the VB12 modified electrode, the electrode is capable of detection at extremely low oxidation potentials, so we tested two oxidation potentials. At 0 V, the linear range is divided into two segments: 8.8–688.8µM and 688.8–1738.8µM, and the detection limit is 5µM (see Fig.25 At 0.15 V, the linear ranges improved to 1.0–588.8 µM and 588.8–1788.8 µM, and the detection limit was significantly reduced to 0.1 µM (see Fig.26 The modified electrode showed excellent stability and reproducibility in long-term operation in it mode and in repeated measurements at a fixed Cys concentration (see Fig. 27 – Fig.29 ).

[0069] In addition, cathodic DPV detection revealed that the DPV curve showed multi-peak and multi-mode changes with the change of Cys concentration (see Fig.30 ). These changes include shifts in peak potential (e.g., peak c) as well as changes in current response. Some peaks (a and d) are positively correlated with Cys concentration, while others (b and e) are negatively correlated. These quasi-spectral DPV multimodal curves enable wide range detection from 1µM to 10mM, with a calculated detection limit of 0.5µM. The cathodic DPV-based Cys detection method, whose curves are similar to spectral curves, uses multi-peak electrochemical features to achieve high sensitivity, selectivity, and dynamic kinetic resolution. The DPV curves exhibit different peaks (a–e), corresponding to multiple interaction pathways between Cys and the electrode interface, such as redox reactions, adsorption, or complexation at specific functional groups (-SH, -NH2). These multimodal signals provide multidimensional insights, where opposite trends in peak currents (e.g., peak a / d increases while peak b / e decreases) can be cross-validated by ratio or differential signal normalization (e.g., Ia / Ib or Ia-Ib), effectively reducing background noise and misinterpretation. The spectral profile further allows the use of advanced pattern recognition algorithms (e.g. PCA, machine learning) to extract high-dimensional features (peak position, area, symmetry) to enhance specificity in complex biological matrices (such as serum or urine). Compared with literature reports, this method not only has the lowest oxidation potential and lower detection limit, but also exhibits a wider linear range and higher recognition ability, highlighting its excellent sensing performance.

[0070] Enhanced selectivity through multi-signal synergy, where interferents (e.g., GSH, Hcy) affect specific peaks differently, allows for robust differentiation through multivariate analysis. Unlike optical methods that require derivatization, DPV directly exploits the intrinsic electroactivity of Cys, simplifying the workflow and reducing sample loss. In addition, the platform's compatibility with miniaturized systems (e.g., handheld potentiometers, screen-printed electrodes) supports rapid on-site detection, while engineered electrodes (e.g., nanocomposites) enable simultaneous detection of Cys with other biothiols or metal ions. By integrating multimodal fingerprint analysis with scalable hardware, this approach bridges the gap between laboratory-grade sensitivity and practical application, providing a versatile tool for precision medicine and clinical diagnosis.

[0071] To evaluate potential interferences, the effects of UA and AA (common electrochemically oxidizable components in biological samples) on Cys detection were investigated. Neither UA nor AA caused significant interference in the detection of Cys in cathodic DPV mode (see Fig.31 ). In addition, since adsorption of biomacromolecules on the electrode surface usually impairs activity, sensitivity, and accuracy, we systematically tested the impact of biofouling. Notably, the electrode response in blank electrolyte and Cys-containing solution remained stable even after exposure to high concentrations of BSA, a model foulant (see Fig.32 ). This excellent anti-biofouling ability can be attributed to the hydrophilicity of VB12 and active GCE, which may prevent nonspecific protein adsorption. The analytical potential of the developed sensor was verified by detecting Cys in adulterated human serum samples. As shown in Table 5, the sensor achieved a recovery rate of 92% to 106% in the tested concentration range, indicating that the method can be applied to real sample analysis.

[0072] Table 5. Serum spiked Cys detection (n=3)

[0073] In summary, the present invention has developed a novel multimodal electrochemical sensing platform for the identification and detection of Cys. By co-modifying VB12 with electrochemically activated GCE to achieve "direct electropolarization of molecular catalysts", an electroactive interface with dual catalytic functions was designed: excellent electrocatalytic oxidation of Cys and efficient reduction of its oxidized products. This unique interface mimics enzyme behavior, in which VB12 acts as a redox mediator, reduces the reaction overpotential and enhances the electron transfer kinetics, similar to natural metalloenzymes. The formation mechanism of the biomimetic catalytic interface was systematically revealed using techniques such as electrochemical impedance spectroscopy (EIS), X-ray photoelectron spectroscopy (XPS) and Raman spectroscopy.

[0074] The synergistic effect between the nanostructured surface of GCE (rich in oxygen-containing functional groups) and the redox-active cobalt center of VB12 enables multipath interactions with Cys, resulting in extremely low oxidation potentials and spectroscopic-like DPV fingerprints. The unique advantage of this platform lies in its “spectro-electrochemical-like” duality: the DPV curves resemble spectroscopic fingerprints, enabling the selective identification of Cys in complex matrices through machine learning-assisted pattern recognition, while retaining the simplicity, cost-effectiveness, and portability of electroanalysis. The dynamic peak potential changes further provide real-time insights into reaction kinetics and interfacial phenomena, such as the formation and reduction of Cys oxidation intermediates, highlighting the biomimetic catalytic multifunctionality of the interface. By coordinating multimodal signal analysis, enzyme-like interface design, and compatibility with miniaturized devices, this work bridges the gap between laboratory-grade precision and applicability for rapid on-site detection. Future studies could extend this paradigm to multiplexed detection of biothiols or integrate with wearable sensors to achieve real-time monitoring of redox biomarkers using the dual catalytic properties of the engineered interface.

[0075] This invention not only advances the development of Cys sensing technology, but also establishes a generalizable framework for designing enzyme electrochemical interfaces, transforming traditional voltammetry into a high-information-density differentiation tool. Detailed exploration of activation and modification protocols provides a blueprint for tailoring catalytic surfaces for specific analytes, paving the way for smart electrochemical systems in precision medicine and disease monitoring.

[0076] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An electrode for identifying and detecting cysteine, characterized in that: Glassy carbon electrode modified with electrochemical activation for vitamin B12; The electrochemical activation treatment includes oxidation treatment and reduction treatment; The method for modifying the vitamin B12 is an immersion method; and / or the electrolyte in the reduction treatment process contains vitamin B12, which is modified simultaneously during the electrochemical activation process.

2. The electrode for identifying and detecting cysteine ​​according to claim 1, characterized in that: The solvent of the immersion method is deionized water or pH 7.0 PBS solution.

3. The electrode for identifying and detecting cysteine ​​according to claim 2, characterized in that: In the immersion method, the vitamin B12 concentration of the solution is 0.3-0.7 mM, and the immersion time is 20-40 minutes.

4. The electrode for identifying and detecting cysteine ​​according to claim 1, characterized in that: The pH of the electrolyte for the oxidation treatment is 5.5-13, the applied potential is 1.3-2V, and the treatment time is 400-600s.

5. The electrode for identifying and detecting cysteine ​​according to claim 1, characterized in that: The electrolyte for the oxidation treatment is one or more of acetate solution, phosphate solution, carbonate solution, potassium chloride solution, sodium hydroxide solution, and HCl-Tris solution.

6. The electrode for identifying and detecting cysteine ​​according to claim 1, characterized in that: The pH of the electrolyte for the reduction treatment is 5.5-13, the applied potential is -0.3-0.9V, and the treatment time is 400-600s.

7. The electrode for identifying and detecting cysteine ​​according to claim 1, characterized in that: The electrolyte for the reduction treatment is one or more of a sodium hydroxide solution, a carbonate solution, a HCl-Tris solution, a phosphate solution or an acetate solution.

8. The electrode for identifying and detecting cysteine ​​according to claim 1, characterized in that: The vitamin B12 concentration of the reduced electrolyte is 0.3-0.7 mM.

9. Use of the electrode for identifying and detecting cysteine ​​as claimed in any one of claims 1 to 8 in identifying and detecting electroactive amino acids or dopamine.

10. Use of the electrode for identifying and detecting cysteine ​​according to claim 9 in identifying and detecting electroactive amino acids or dopamine, characterized in that: Multi-peak and multi-mode spectral identification was achieved by negative differential pulse voltammetry.

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