A ratio-type acupuncture needle sensor for detecting growth hormone releasing peptide and a method of manufacturing the same

Abstract

The application discloses a ratio type acupuncture needle sensor for detecting growth hormone releasing peptide and a preparation method thereof. The sensor is based on a microelectrode of an acupuncture needle, gold nanoparticles, single-wall carbon nanotubes are sequentially electrodeposited, an internal signal layer of methylene blue is layer-by-layer electropolymerized, an anchor layer of beta-cyclodextrin, a molecular imprinting recognition layer of growth hormone releasing peptide and a reference signal layer of ferulic acid are sequentially electrodeposited, and the sensor is obtained by eluting templates to obtain a double-signal ratio type molecular imprinting sensor. The sensor relies on the peak current ratio of methylene blue and ferulic acid to quantitatively correct the environment and non-specific adsorption interference. The nanocomposite modification improves the conductivity of the electrode, and the surface imprinting shortens the diffusion path of the polypeptide. The linear range of the sensor is 0.001-5000 ng / mL, the detection limit is as low as 0.92 pg / mL, the anti-interference, repeatability and long-term stability are excellent. The sensor is simple to prepare, low in cost, suitable for on-site rapid detection of trace biological samples, and suitable for primary medical institutions and large-scale screening of clinical diseases.

Description

Technical Field

[0001] This invention relates to a sensor and its preparation method, and more specifically, to a ratiometric acupuncture needle sensor for detecting growth hormone-releasing peptide and its preparation method, belonging to the field of electrochemical analysis and detection technology. Background Technology

[0002] Growth hormone-releasing peptide (GHRL) is a 28-amino acid bioactive polypeptide secreted by gastric mucosal cells. It plays a crucial role in regulating energy homeostasis and appetite, and also participates in the physiological regulation of gastrointestinal secretion, intestinal motility, and pancreatic endocrine function. Emerging clinical and biological evidence suggests that abnormal circulating GHRL levels are closely associated with the progression of various diseases, including Parkinson's disease, Alzheimer's disease, end-stage renal disease, and gastrointestinal malignancies. Therefore, GHRL has been identified as a promising peripheral biomarker for early pathological assessment and prognostic evaluation of these diseases.

[0003] Currently, conventional quantitative detection methods for GHRL mainly include radioimmunoassay, liquid chromatography-tandem mass spectrometry, and enzyme-linked immunosorbent assay (ELISA). However, these methods are highly dependent on sophisticated analytical instruments, expensive experimental consumables, and specialized operating skills, while also posing potential occupational hazards and being difficult to adapt to resource-constrained clinical settings. Therefore, developing a portable, low-cost GHRL micro-electrochemical sensor with dual-signal self-calibration, fast recognition response, and high specificity is an inevitable trend. Summary of the Invention

[0004] To address the technical problems of existing methods for detecting growth hormone-releasing peptide (GHRP), such as expensive instruments, complex operation, susceptibility to environmental interference in single-signal electrochemical detection, difficulty in eluting traditional molecularly imprinted polymer templates, and slow target response, this invention provides a ratiometric acupuncture needle sensor for detecting GHRP and its preparation method.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solution: This invention discloses a method for preparing a ratiometric acupuncture needle sensor for detecting growth hormone-releasing peptide, the method comprising the following steps: Step 1) Pretreatment of acupuncture needles: Grind and polish the acupuncture needles, ultrasonically clean them in ethanol and deionized water for 5 minutes each, and dry them with nitrogen gas for later use. Step 2) Modification of gold nanoparticles: The acupuncture needles pretreated in Step 1) are used as working electrodes. The needle tips are immersed in a HAuCl4 solution containing KCl and cyclic voltammetry is performed. The resulting electrodes are denoted as ANME / AuNPs. Step 3) Modification of single-walled carbon nanotubes: The ANME / AuNPs electrode obtained in step 2) is used as the working electrode and electropolymerized in an aqueous solution containing 3,4-ethylenedioxythiophene and single-walled carbon nanotubes. After polymerization, the modified electrode is obtained, washed with water and dried to obtain electrode ANME / AuNPs / SWCNTs. Step 4) Modification of built-in signal: Immerse the ANME / AuNPs / SWCNTs electrode obtained in step 3) in menaquinone solution and perform cyclic voltammetry scan to obtain the electrode ANME / AuNPs / SWCNTs / pMNQ; Step 5) Modification of β-cyclodextrin: The ANME / AuNPs / SWCNTs / pMNQ electrode obtained in step 4) was immersed in a β-cyclodextrin solution and cyclic voltammetry was performed to obtain the electrode ANME / AuNPs / SWCNTs / pMNQ / pβ-CD. Step 6) Binding of template molecules: Immerse the ANME / AuNPs / SWCNTs / pMNQ / pβ-CD electrode obtained in step 5) in GHRL (growth hormone releasing peptide solution) solution and incubate at 0℃ to 4℃ for 10h to obtain the electrode ANME / AuNPs / SWCNTs / pMNQ / pβ-CD~GHRL. Step 7) Modification of the imprinted polymer film: Using the electrode obtained in step 6) as the working electrode, electropolymerization of acid chrome blue K was carried out by cyclic voltammetry to obtain the electrode ANME / AuNPs / SWCNTs / pMNQ / pβ-CD~GHRL / pACBK. Step 8) Modification of reference signal: Immerse the portion of the electrode obtained in step 7) 5 mm from the needle tip in ferulic acid solution for cyclic voltammetry scanning to obtain the electrode ANME / AuNPs / SWCNTs / pMNQ / pβ-CD~GHRL / pACBK / pFA; Step 9) Elution of template molecules: Elute the GHRL (growth hormone-releasing peptide) template wrapped on the electrode in step 8) to obtain the surface molecularly imprinted microsensor ANME / AuNPs / SWCNTs / pMNQ / pβ-CD~GHRL@SMIP~pFA.

[0006] Preferably, in step 2), the cyclic voltammetry scan rate is 25 mV / s, the potential range is -1.5 V to 0.5 V, and 5 scan cycles are performed; the HAuCl4 solution concentration is 2.5 mmol / L, and the 10 mm tip of the acupuncture needle is immersed in the HAuCl4 solution containing KCl.

[0007] Preferably, in step 3), the electropolymerization potential is 1.2 V, the polymerization time is 110 s, the concentration of 3,4-ethylenedioxythiophene solution is 0.02 mol / L, and the concentration of single-walled carbon nanotube solution is 2 mg / ml.

[0008] Preferably, the menadione solution in step 4) is 0.1 M PBS (a solution of menadione dissolved in 0.1 M phosphate buffer), with a pH of 7.0 and a concentration of 1 mM; the electropolymerization potential range is -0.6 V to 0 V, the scan rate is 50 mV / s, and the cycle scan is 10 times.

[0009] Preferably, in step 5), the β-cyclodextrin solution is 0.1 M PBS (β-cyclodextrin dissolved in 0.1 M phosphate buffer), with a pH of 7.5 and a concentration of 10 mM; the electropolymerization potential range is -1 V to 1.0 V, the scan rate is 70 mV / s, and the cycle is 4 times.

[0010] Preferably, in step 6), the GHRL solution is 0.01 M PBS (the growth hormone-releasing peptide is dissolved in 0.1 M phosphate buffer to obtain a solution), with a pH of 7.4, a concentration of 5 μg / ml, and an incubation time of 10 h.

[0011] Preferably, in step 7), the solution of Acid Chrome Blue K is 0.05 M PBS (Acid Chrome Blue K is dissolved in 0.05 M phosphate buffer to obtain a solution), with a pH of 7.0 and a concentration of 0.5 mM; the electropolymerization potential range is -0.4 V to 1.5 V, the scan rate is 70 mV / s, and the cycle scan is 45 times.

[0012] Preferably, in step 8), the ferulic acid solution is 0.1 M PBS (ferulic acid is dissolved in 0.1 M phosphate buffer to obtain a solution), with a pH of 4.5 and a concentration of 0.4 mM; the electropolymerization potential range is -0.25 V to 0.75 V, the scan rate is 50 mV / s, and the cycle scan is 10 times; nitrogen gas is introduced into the electrode for 10 min before use.

[0013] Preferably, in step 9), the elution agent used is 0.1 M PBS containing 5% methanol (5% methanol dissolved in 0.1 M phosphate buffer solution), with a pH of 5.0, and the elution time is 60 min.

[0014] This invention relates to a ratiometric acupuncture needle sensor for detecting growth hormone-releasing peptide, prepared using the method described above.

[0015] Compared with the prior art, this application has the following advantages: 1) This invention constructs a dual-output mode of built-in signal (MNQ) and reference signal (FA), and uses the ratio of their peak currents for quantitative calibration. This effectively offsets background interference caused by factors such as ambient temperature, solution pH, electrode surface state fluctuations, and non-specific adsorption, significantly reducing the risk of false positives or false negatives that are easily generated by single-signal output mode, and significantly improving the detection accuracy and reliability of GHRL in complex biological samples.

[0016] 2) This invention uses acupuncture needle microelectrodes (ANMEs) as a substrate and performs dual functionalization modification by electrodepositing gold nanoparticles (AuNPs) and single-walled carbon nanotubes (SWCNTs). By utilizing the high conductivity and biocompatibility of AuNPs and the large specific surface area and excellent electron transport capability of SWCNTs, the effective specific surface area of ​​the electrode is synergistically increased and the interfacial electron transfer rate is accelerated, thereby significantly amplifying the electrochemical response signal and realizing trace-level high-sensitivity detection of GHRL.

[0017] 3) This invention utilizes electropolymerization technology to sequentially and controllably construct an MNQ built-in signal layer, a β-cyclodextrin anchoring layer, an ACBK imprint recognition layer, and an FA reference signal layer. The thickness and modification amount of each functional layer can be precisely controlled through electropolymerization parameters (potential, time, number of scans), exhibiting excellent batch-to-batch repeatability. System optimization and verification have shown that the resulting ratiometric electrochemical sensor demonstrates a wide linear detection range, ultra-low detection limit, excellent repeatability, long-term stability, and outstanding anti-interference capability.

[0018] 4) This invention uses surface molecularly imprinted polymers (SMIPs) to construct the recognition layer, directly forming the specific recognition cavity of GHRL on the surface of the electrode material. This effectively shortens the diffusion path of the target molecule and reduces the interfacial mass transfer resistance. While maintaining excellent specific binding ability, it achieves rapid response equilibrium and overcomes the technical defects of traditional bulk imprinted polymer templates, such as excessively deep embedding, difficult elution, and slow binding kinetics.

[0019] 5) The ratiometric electrochemical sensor constructed in this invention has low manufacturing cost, and the detection process does not require complex sample pretreatment and expensive markers. It can achieve rapid detection with the help of a portable electrochemical workstation. It is easy to operate and requires low professional skills from operators. Moreover, the microelectrode of acupuncture needle is small in size and suitable for the detection of small samples. It is particularly suitable for resource-limited primary medical institutions, point-of-care testing (POCT), and large-scale clinical screening scenarios, and has good prospects for industrial application. Attached Figure Description

[0020] Figure 1 This is a surface morphology diagram of the electrode layer-by-layer modification of the present invention.

[0021] Figure 2These are the cyclic voltammetry curves of the electrodes at each modification stage of this invention.

[0022] Figure 3 These are differential pulse voltammetry curves of electrodes at each modification stage of this invention.

[0023] Figure 4 This invention provides differential pulse voltammetry results for detecting GHRL solutions containing different concentrations.

[0024] Figure 5 This is a comparison chart of linear fitting between the ratio type and single-signal sensor of the present invention.

[0025] Figure 6 This is a comparison chart of the anti-interference performance test of the sensor of this invention.

[0026] Figure 7 This is the differential pulse voltammetry curve of the non-imprinted control electrode of the present invention.

[0027] Figure 8 This is a comparison diagram of the menadione signal with and without SWCNTs modification according to the present invention. Detailed Implementation

[0028] The following will refer to the accompanying drawings in the embodiments of the present invention. Figures 1 to 8 The technical solutions in the embodiments of the present invention are clearly and completely described herein. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0029] I. Technical Solution / Principle: This application employs surface molecular imprinting to optimize the spatial distribution of recognition sites, shorten the diffusion path of target molecules, and accelerate the recognition response dynamics. It also constructs a ratiometric dual-signal output mode based on an electrochemical detection system and uses a stable internal reference signal to eliminate background interference caused by environmental fluctuations and non-specific adsorption, thereby significantly improving the accuracy and reliability of GHRL detection results and demonstrating good potential for clinical biomarker detection applications.

[0030] The complete solution is divided into two parts: preparation method and sensor. 1.1 Complete preparation method of a ratiometric electrochemical sensor for detecting growth hormone-releasing peptide A method for preparing a ratiometric electrochemical sensor for detecting growth hormone-releasing peptide, the method comprising the following steps: Step 1) Pretreatment of acupuncture needles: Grind and polish the acupuncture needles, ultrasonically clean them in ethanol and deionized water for 5 minutes each, and dry them with nitrogen gas for later use. Step 2) Modification of gold nanoparticles: The acupuncture needles pretreated in Step 1) were used as working electrodes. The needle tips were immersed in a KCl-containing HAuCl4 solution, and cyclic voltammetry was performed. The resulting electrodes were denoted as ANME / AuNPs. The cyclic voltammetry scan rate was 25 mV / s, the potential range was -1.5 V to 0.5 V, and 5 scan cycles were performed. The HAuCl4 solution concentration was 2.5 mmol / L, and the 10 mm needle tip was immersed in the KCl-containing HAuCl4 solution. Step 3) Modification of single-walled carbon nanotubes: Using the ANME / AuNPs electrode obtained in Step 2) as the working electrode, electropolymerization was carried out in an aqueous solution containing 3,4-ethylenedioxythiophene and single-walled carbon nanotubes. After polymerization, the modified electrode was obtained, washed with water and dried to obtain the electrode ANME / AuNPs / SWCNTs. The electropolymerization potential in this step was 1.2 V, and the polymerization time was 110 s. The concentration of 3,4-ethylenedioxythiophene solution was 0.02 mol / L, and the concentration of single-walled carbon nanotube solution was 2 mg / ml. Step 4) Modification of the built-in signal: The ANME / AuNPs / SWCNTs electrode obtained in Step 3) was immersed in a menadione solution and subjected to cyclic voltammetry scanning to obtain the electrode ANME / AuNPs / SWCNTs / pMNQ; the menadione solution was prepared with 0.1 M PBS, with a pH of 7.0 and a concentration of 1 mM; the electropolymerization potential range was -0.6 V to 0 V, the scan rate was 50 mV / s, and the cycle was 10 times; Step 5) β-Cyclodextrin modification: The ANME / AuNPs / SWCNTs / pMNQ electrode obtained in Step 4) was immersed in a β-cyclodextrin solution and cyclic voltammetry was performed to obtain the electrode ANME / AuNPs / SWCNTs / pMNQ / pβ-CD; the β-cyclodextrin solution was prepared with 0.1 M PBS, with a pH of 7.5 and a concentration of 10 mM; the electropolymerization potential range was -1 V to 1.0 V, the scan rate was 70 mV / s, and the cycle was repeated 4 times; Step 6) Template molecule binding: The ANME / AuNPs / SWCNTs / pMNQ / pβ-CD electrode obtained in step 5) was immersed in GHRL (growth hormone releasing peptide) solution and incubated at 0℃ to 4℃ for 10h to obtain the electrode ANME / AuNPs / SWCNTs / pMNQ / pβ-CD~GHRL; the GHRL solution was prepared with 0.01 M PBS, pH 7.4, concentration 5 μg / ml, and incubation time 10h; Step 7) Modification of the imprinted polymerized film: Using the electrode obtained in step 6) as the working electrode, electropolymerization of Acid Chrome Blue K was performed by cyclic voltammetry to obtain the electrode ANME / AuNPs / SWCNTs / pMNQ / pβ-CD~GHRL / pACBK; the solution of Acid Chrome Blue K was prepared with 0.05 M PBS, with a pH of 7.0 and a concentration of 0.5 mM; the electropolymerization potential range was -0.4 V to 1.5 V, the scan rate was 70 mV / s, and the cyclic scan was performed 45 times; Step 8) Modification of the reference signal: The portion of the electrode obtained in Step 7) 5 mm from the needle tip was immersed in ferulic acid solution for cyclic voltammetry scanning to obtain the electrode ANME / AuNPs / SWCNTs / pMNQ / pβ-CD~GHRL / pACBK / pFA; the ferulic acid solution was prepared with 0.1 M PBS, pH 4.5, and concentration 0.4 mM; the electropolymerization potential range was -0.25 V to 0.75 V, the scan rate was 50 mV / s, and the cyclic scan was performed 10 times; nitrogen gas was introduced into the electrode for 10 min before use; Step 9) Elution of template molecules: Elute the GHRL (growth hormone-releasing peptide) template wrapped on the electrode in step 8) to obtain the surface molecularly imprinted microsensor ANME / AuNPs / SWCNTs / pMNQ / pβ-CD~GHRL@SMIP~pFA; The elution agent used is a 0.1 M PBS solution containing 5% methanol with a pH of 5.0, and the elution time is 60 min.

[0031] 1.2 Ratio-modulated electrochemical sensor for detecting growth hormone-releasing peptide A ratiometric electrochemical sensor for detecting growth hormone-releasing peptide (GHRP) is disclosed. The sensor is fabricated using the method described above. The substrate is an acupuncture needle microelectrode. The electrode surface is sequentially modified from the inside out with an electrodeposited gold nanoparticle layer, a single-walled carbon nanotube conductive layer, an electropolymerized menadione embedded signal layer, an electropolymerized β-cyclodextrin anchoring layer, and a GHRP surface molecular imprint recognition layer formed by electropolymerization of acid chrome blue K. An additional electropolymerized ferulic acid reference signal layer is modified in a 5 mm region of the electrode tip. A specific recognition cavity is left on the electrode surface after the template molecules are eluted. The GHRP is quantitatively detected by relying on the ratio of the two electrochemical signals of menadione and ferulic acid.

[0032] II. The following specific embodiments are used to describe the technical solution in detail. The specific descriptions of each parameter should not be construed as limitations on the technical solution. Electrochemical tests uniformly adopt a three-electrode system: the acupuncture needle is the working electrode, the saturated calomel electrode is the reference electrode, and the platinum wire electrode is the counter electrode; unless otherwise specified, all reagents / solutions are commercially available analytical grade.

[0033] 2.1 Example 1: A method for preparing a ratiometric electrochemical sensor for detecting GHRL includes the following steps: Step 1) Pretreatment of acupuncture needles: Use metallographic sandpaper to thoroughly polish the acupuncture needles for 3-5 minutes, and then immerse them in ethanol and deionized water for five minutes respectively to obtain a clean electrode substrate.

[0034] Step 2) Modification of gold nanoparticles: The pretreated acupuncture needle was used as the working electrode, a saturated calomel electrode as the reference electrode, and a platinum wire electrode as the counter electrode. The 10 mm tip of the acupuncture needle electrode was immersed in a solution containing 2.5 mM HAuCl4 and 0.1 M KCl, and five cycles were performed at a scan rate of 25 mV / s, with the potential alternating between -1.5 V and 0.5 V. The resulting electrode is denoted as ANME / AuNPs. This step electrochemically deposits a uniformly distributed layer of gold nanoparticles with similar particle sizes onto the needle tip surface.

[0035] Step 3) Modification of Single-Walled Carbon Nanotubes: The ANME / AuNPs electrode obtained in Step 2) was used as the working electrode for electropolymerization in an aqueous solution containing 0.02 mol / L 3,4-ethylenedioxythiophene and 2 mg / ml single-walled carbon nanotubes (SWCNTs). The polymerization potential was set to 1.2 V, and the polymerization time was 110 s. After polymerization, the modified electrode was thoroughly washed with water and dried. The prepared electrode was labeled ANME / AuNPs / SWCNTs. This process constructed a more conductive three-dimensional network structure, further amplifying the electrical signal.

[0036] Step 4) Modification of built-in signal menadione: The electrode ANME / AuNPs / SWCNTs prepared in step 3) is immersed in a solution of 0.1 M PBS (pH = 7.0) containing 1 mM menadione. Cyclic voltammetry is performed 10 times at a scan rate of 50 mV / s between -0.6 and 0 V to obtain the electrode ANME / AuNPs / SWCNTs / pMNQ (abbreviated as ANME / / pMNQ).

[0037] Step 5) Modification with β-cyclodextrin: The electrode ANME / / pMNQ prepared in step 4) was immersed in a solution of 0.1 M PBS (pH = 7.5) containing 10 mM β-cyclodextrin. Four cyclic voltammetric scans were performed between -1 V and 1 V at a scan rate of 70 mV / s to obtain the electrode ANME / AuNPs / SWCNTs / pMNQ / pβ-CD.

[0038] Step 6) Binding of template molecules: The electrode prepared in step 5) is immersed in a solution of 0.01M PBS (pH = 7.4) containing 5 mg / mL GHRL and incubated at 4°C for 10 h to obtain the electrode ANME / AuNPs / SWCNTs / pMNQ / pβ-CD~GHRL.

[0039] Step 7) Modification of the imprinted polymer: The electrode prepared in step 6) is immersed in a solution of 0.05 M PBS (pH = 7.0) containing 0.5 mM ACBK. Cyclic voltammetry is performed for 45 cycles at a scan rate of 70 mV / s between -0.4 and 1.5 V to obtain the electrode ANME / AuNPs / SWCNTs / pMNQ / pβ-CD~GHRL / pACBK.

[0040] Step 8) Modification of the reference signal ferulic acid: The electrode prepared in step 7) is immersed in a 0.1 M PBS (pH = 4.5) solution containing 0.4 mM FA (nitrogen gas needs to be purged for 10 min before use). Cyclic voltammetry is performed 10 times at a scan rate of 50 mV / s between -0.25 V and 0.75 V to obtain the electrode ANME / AuNPs / SWCNTs / pMNQ / pβ-CD~GHRL / pACBK / pFA.

[0041] Step 9) Elution of template molecules: The electrode prepared in step 8) is immersed in a 0.1 MPBS (pH = 5.0) solution containing 5% methanol and eluted at 4°C for 60 min. The electrode ANME / AuNPs / SWCNTs / pMNQ / pβ-CD~GHRL@SMIP~pFA (abbreviated as ANME / / pMNQ / / SMIP-pFA) was obtained.

[0042] Figure 1 These are surface morphology images of the electrode layer-by-layer modification of the present invention (surface morphology characterization of the step-by-step modification process of the electrochemical sensor using an inverted fluorescence microscope and a scanning electron microscope (SEM). Figure 1 A and B are bare acupuncture needle microelectrodes (ANMEs), with smooth and flat surfaces and intact needle tip structures; Figure 1 C is a SEM image of ANME after metallographic sandpaper polishing. The longitudinal scratches on its surface are conducive to the subsequent firm deposition of nanomaterials and provide a good substrate for the electrodeposition of AuNPs. Figure 1The image in D is a SEM image of gold nanoparticles (AuNPs) electrodeposited on the surface of ANME. It can be seen that the AuNPs are uniformly distributed, have uniform particle size, and are completely covered, which is beneficial for further modification of the functional layer. Compared with the bare electrode, the surface roughness is significantly increased, which confirms that AuNPs effectively increase the specific surface area of ​​the electrode. Figure 1 Image E in the middle is an image of ANME / AuNPs / SWCNTs, which clearly shows the single-walled carbon nanotubes (SWCNTs) with a fibrous network structure deposited on the AuNPs layer. The roughness is further increased compared to the previous step, indicating that the introduction of SWCNTs further increases the effective area and roughness of the electrode surface. Figure 1 Image F shows the result of electropolymerization of menaquinone (MNQ). It can be seen that a uniform polyMNQ (pMNQ) film is covered on the surface of the SWCNT network structure, and the network skeleton of SWCNTs remains intact and undamaged, indicating that pMNQ has been successfully attached to the carbon nanotube surface. Figure 1 Image G is an image of the Acid Chrome Blue K (ACBK) imprinted layer and ferulic acid (FA) reference layer after electropolymerization, i.e. before template molecules are eluted. Because the GHRL template molecules fill the imprinted cavity, the electrode surface presents a relatively uniform and smooth coating layer. Figure 1 Image H shows the eluted template molecules. The electrode surface exhibits a rough and uneven morphology, indicating that the GHRL template molecules were successfully eluted, forming a large number of nanoscale imprinted cavities on the electrode surface, which can specifically recognize and capture the target GHRL molecules.

[0043] Figure 2This is a cyclic voltammetry curve of the electrodes at each modification stage of the present invention (cyclic voltammetry characterization of the GHRL sensor during the layer-by-layer modification process). Due to the poor conductivity of the stainless steel substrate, the bare acupuncture needle microelectrode exhibits low peak current and weak electrochemical response, hindering interfacial electron transfer. After electrodeposition of gold nanoparticles, significant redox peaks appear, and the peak current is greatly enhanced, with the oxidation-to-reduction peak current ratio approximately 1.0, indicating that gold nanoparticles can effectively promote heterogeneous electron transfer and achieve a reversible electrode reaction process. Further composite with single-walled carbon nanotubes results in a continuous increase in the redox peak current, attributed to their large specific surface area and porous structure providing abundant active sites and continuous electron transport pathways, laying the structural foundation for subsequent functional material loading. After electropolymerization of menadione, the peak current decreases slightly because the polymer film partially covers the carbon nanotube channels and active sites, leading to a reduction in the electron transfer rate. Subsequently, the introduction of GHRL template molecules further reduces the peak current, indicating that the template occupies recognition sites and hinders electron transport. After being covered with an Acid Chrome Blue K imprinted layer, the redox peak current significantly decreased, confirming the shielding effect of the dense imprinted film on interfacial electron transfer. After template elution, the peak current rebounded significantly, attributed to the formation of molecularly imprinted cavities on the electrode surface that allow probe diffusion, thus restoring electron transport channels. When the GHRL target molecules recombine, the peak current decreased again, confirming that the imprinted cavity is highly matched to the target in size and spatial configuration; after specific recognition, the cavity is occupied, and the electron transport pathway is blocked. The above-described layer-by-layer response CV variation systematically verifies the successful assembly of the functional interfaces of this invention and its specific recognition performance for GHRL.

[0044] Figure 3 This is a differential pulse voltammogram of the electrodes at each modification stage of this invention (differential pulse voltammograms of electrodes at different modification stages in 0.1 MPBS at pH 7.0). After modification with menadione, the electrode exhibits a strong oxidation peak at -0.28 V. After electropolymerization of β-cyclodextrin and incubation with the GHRL template, this oxidation peak significantly decreases, attributed to the formation of a poorly electroactive β-cyclodextrin polymer film on the electrode surface and the connection of GHRL, which jointly hinder electron transfer. Further construction of an acid chrome blue K imprint layer and polymerization of ferulic acid results in a new oxidation peak at 0.16 V, corresponding to the anodic oxidation current of the ferulic acid polymer, which can serve as a stable reference signal. After eluting the template, nanocavities capable of electron transfer are formed on the imprint surface, and the menadione oxidation peak significantly rebounds. When GHRL is re-incubated, the target molecules again occupy the imprint cavities, hindering electron transfer, and the peak current decreases accordingly. During this process, the ferulic acid reference signal remains almost unchanged, confirming its good signal stability.

[0045] 2.2 Application Example 1: The prepared ratiometric electrochemical sensor was incubated for 30 minutes in solutions containing different concentrations of GHRL (0.001 ng / ml to 5000 ng / ml). Figure 4 The image shows differential pulse voltammetry plots of the fabricated sensor detecting solutions containing different concentrations of GHRL. As the GHRL concentration increases, the peak current of the built-in menadione gradually decreases. This decrease in current is because the imprinted nanocavities are occupied by structurally complementary GHRL molecules, thus hindering electron transfer within the built-in menadione.

[0046] 2.3 Application Example 2 like Figure 5 Specifically, the ratiometric and built-in electrochemical sensors prepared for GHRL detection are shown, with D(I) MNQ / I FA ) and I MNQ The linear relationship between D(I) and the logarithm of GHRL concentration (1 gC) shows that when the prepared ratiometric electrochemical sensor detects GHRL, D(I) MNQ / I FA There is a strong linear relationship between the value of D(I) and the logarithm of GHRL concentration, and the linear equation is D(I) = ... NQ / I FA ) = 0.39093 lgC + 1.48818 (R 2 = 0.9939), with a linear range of 0.001 ~ 5000 ng / mL and a limit of detection (LOD) of 0.92 pg / mL (S / N=3). In contrast, the built-in electrochemical sensor showed better performance for GHRL detection. MNQ There is a weak linear relationship between the value and the logarithm of GHRL concentration, DI NQ (mA) = 7.92927 lgC + 14.77554 (R 2 =0.9739), with a linear range of 0.01 ~ 5000 ng / mL and a LOD of 16.2 pg / mL (S / N=3). These results demonstrate that the ratiometric electrochemical sensor can be successfully applied to the sensitive detection of GHRL.

[0047] 2.4 Application Example 3: By detecting GHRL and separately mixed β-amyloid protein 1-40 (Aβ) 1-40 ), β-amyloid 1-42 (Aβ) 1-42 ), fibroblast growth factor (bFGF), uric acid (UA), dopamine (DA), Cl - Na + Ca + To study the specificity of micro electrochemical sensors, such as Figure 6The diagram shows the response results of the prepared ratiometric and built-in electrochemical sensors after combining GHRL and non-specific molecules. The signals generated by the ratiometric and built-in sensors after adding other molecules are similar to the signals in the presence of GHRL alone. Compared with the non-ratiometric type, the ratiometric sensor of the present invention shows stronger stability and selectivity in the presence of interfering substances, which indicates that the sensor has good specificity, and the specificity of the ratiometric sensor is slightly better than that of the built-in sensor.

[0048] 2.5 Comparative Example 1: To further demonstrate the successful fabrication of the imprinted electrochemical sensor, a non-imprinted electrochemical sensor was also fabricated for comparison. Figure 7 The figure shows the differential pulse voltammogram of the non-imprinted sensor without the GHRL template in 0.1 M PBS (pH 7.0) during the modification process. Unlike the imprinted sensor, the oxidation peak current of menadione showed almost no change before and after elution, indicating that the sensor did not form a recognition cavity that matches GHRL, and therefore could not produce a specific response to GHRL. This further confirms the key role of the molecular imprinted cavity in the sensor recognition mechanism.

[0049] 2.6 Comparative Example 2: In this invention, MNQ was electropolymerized on the surfaces of ANME / AuNPs and ANME / AuNPs / SWCNTs electrodes to investigate the effect of different modified substrates on the peak current response of pMNQ differential pulse voltammetry. After electropolymerization, the two modified electrodes were characterized by differential pulse voltammetry in 0.1 M PBS (pH = 7.0). Figure 8 The results showed that the ANME / AuNPs / SWCNTs / pMNQ electrode exhibited a distinct and sharp oxidation peak at -0.28 V, which is attributed to the characteristic response of the quinone / hydroquinone redox pair in MNQ. However, no obvious pMNQ oxidation peak was observed at this potential on the ANME / AuNPs / pMNQ electrode. These results indicate that MNQ is stably anchored to the SWCNTs surface mainly through π-π stacking and hydrophobic interactions, thus forming a pMNQ film with good electrochemical activity. AuNPs modification alone is insufficient to provide enough binding sites and a suitable interfacial environment for effective MNQ immobilization. Therefore, SWCNTs are the decisive factor in enhancing the electrochemical signal intensity of pMNQ, playing a crucial structural and electronic conduction role in the composite modification layer.

[0050] 2.7 Example 2: In this embodiment, all solution concentrations, incubation / elution temperatures and durations, and potential parameters remained consistent with those in Example 1. Only the number of electropolymerization cycle scans was adjusted to verify the tolerance of process parameters: Step 4) 8 cycles of menadione electropolymerization; Step 5) 6 cycles of β-cyclodextrin electropolymerization; Step 7) 40 cycles of Acid Chrome Blue K electropolymerization; Step 8) 12 cycles of ferulic acid electropolymerization. The resulting sensor exhibited a linear correlation coefficient R² = 0.9912, a detection limit of 1.15 pg / mL, and an unchanged linear range. The detection performance was close to that of the standard embodiment, demonstrating that the number of electropolymerization cycle scans in this invention has an adjustable range and good process tolerance.

[0051] 2.8 Example 3: All other operating steps and parameters in this embodiment are exactly the same as in Example 1, except that the concentration of the nano-modified solution is adjusted: in step 3), the concentration of the single-walled carbon nanotube solution is adjusted to 1.5 mg / mL; in step 2), the concentration of the HAuCl4 solution is adjusted to 2.0 mmol / L. The resulting sensor has a slightly lower electrochemical peak current than that in Example 1, a linear correlation coefficient R² = 0.9905, and a detection limit LOD = 1.31 pg / mL, which still meets the requirements for GHRL trace detection. The concentration of the nanomaterial can be fine-tuned according to the actual detection sensitivity requirements.

[0052] Finally, it should be noted that the present invention is not limited to the above embodiments, and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a ratiometric acupuncture needle sensor for detecting growth hormone-releasing peptide, characterized in that... The preparation method includes the following steps: Step 1) Pretreatment of acupuncture needles: Grind and polish the acupuncture needles, ultrasonically clean them in ethanol and deionized water for 5 minutes each, and dry them with nitrogen gas for later use. Step 2) Modification of gold nanoparticles: The acupuncture needles pretreated in Step 1) are used as working electrodes. The needle tips are immersed in a HAuCl4 solution containing KCl and cyclic voltammetry is performed. The resulting electrodes are denoted as ANME / AuNPs. Step 3) Modification of single-walled carbon nanotubes: The ANME / AuNPs electrode obtained in step 2) is used as the working electrode and electropolymerized in an aqueous solution containing 3,4-ethylenedioxythiophene and single-walled carbon nanotubes. After polymerization, the modified electrode is obtained, washed with water and dried to obtain electrode ANME / AuNPs / SWCNTs. Step 4) Modification of built-in signal: Immerse the ANME / AuNPs / SWCNTs electrode obtained in step 3) in menaquinone solution and perform cyclic voltammetry scan to obtain the electrode ANME / AuNPs / SWCNTs / pMNQ; Step 5) Modification of β-cyclodextrin: The ANME / AuNPs / SWCNTs / pMNQ electrode obtained in step 4) was immersed in a β-cyclodextrin solution and cyclic voltammetry was performed to obtain the electrode ANME / AuNPs / SWCNTs / pMNQ / pβ-CD. Step 6) Binding of template molecules: Immerse the ANME / AuNPs / SWCNTs / pMNQ / pb-CD electrode obtained in step 5) in GHRL solution and incubate at 0℃ to 4℃ for 10h to obtain the electrode ANME / AuNPs / SWCNTs / pMNQ / pβ-CD~GHRL. Step 7) Modification of the imprinted polymer film: Using the electrode obtained in step 6) as the working electrode, electropolymerization of acid chrome blue K was carried out by cyclic voltammetry to obtain the electrode ANME / AuNPs / SWCNTs / pMNQ / pβ-CD~GHRL / pACBK. Step 8) Modification of reference signal: Immerse the portion of the electrode obtained in step 7) 5 mm from the needle tip in ferulic acid solution and perform cyclic voltammetry scan to obtain the electrode ANME / AuNPs / SWCNTs / pMNQ / pβ-CD~GHRL / pACBK / pFA; Step 9) Elution of template molecules: Elute the GHRL template wrapped on the electrode from step 8) to obtain the surface molecular imprinted microsensor ANME / AuNPs / SWCNTs / pMNQ / pβ-CD~GHRL@SMIP~pFA.

2. The method for preparing a ratiometric acupuncture needle sensor for detecting growth hormone-releasing peptide according to claim 1, characterized in that, Step 2) Cyclic voltammetry scan rate 25 mV / s, potential range -1.5 V to 0.5 V, 5 scan cycles; HAuCl4 solution concentration 2.5 mmol / L, acupuncture needle tip 10 mm is immersed in HAuCl4 solution containing KCl.

3. The method for preparing a ratiometric acupuncture needle sensor for detecting growth hormone-releasing peptide according to claim 1, characterized in that, Step 3) Electropolymerization potential 1.2 V, polymerization time 110 s; 3,4-ethylenedioxythiophene solution concentration 0.02 mol / L, single-walled carbon nanotube solution concentration 2 mg / ml.

4. The method for preparing a ratiometric acupuncture needle sensor for detecting growth hormone-releasing peptide according to claim 1, characterized in that, The menadione solution mentioned in step 4) is 0.1 M PBS with a pH of 7.0 and a concentration of 1 mM; the electropolymerization potential range is -0.6 V to 0 V, the scan rate is 50 mV / s, and the scan is performed 10 times.

5. The method for preparing a ratiometric acupuncture needle sensor for detecting growth hormone-releasing peptide according to claim 1, characterized in that, Step 5) The β-cyclodextrin solution was 0.1 M PBS with a pH of 7.5 and a concentration of 10 mM; the electropolymerization potential range was -1 V to 1.0 V, the scan rate was 70 mV / s, and the scan was performed 4 times.

6. The method for preparing a ratiometric acupuncture needle sensor for detecting growth hormone-releasing peptide according to claim 1, characterized in that, Step 6) The GHRL solution is 0.01 M PBS with a pH of 7.4 and a concentration of 5 μg / ml. The incubation time is 10 h.

7. The method for preparing a ratiometric acupuncture needle sensor for detecting growth hormone-releasing peptide according to claim 1, characterized in that, Step 7) The solution of Acid Chrome Blue K is 0.05 M PBS with a pH of 7.0 and a concentration of 0.5 mM; the electropolymerization potential range is -0.4 V to 1.5 V, the scan rate is 70 mV / s, and the cycle scan is 45 times.

8. The method for preparing a ratiometric acupuncture needle sensor for detecting growth hormone-releasing peptide according to claim 1, characterized in that, Step 8) The ferulic acid solution is 0.1 M PBS with a pH of 4.5 and a concentration of 0.4 mM; the electropolymerization potential range is -0.25 V to 0.75 V, the scan rate is 50 mV / s, and the cycle scan is 10 times; nitrogen gas is introduced into the electrode for 10 min before use.

9. The method for preparing a ratiometric acupuncture needle sensor for detecting growth hormone-releasing peptide according to claim 1, characterized in that, Step 9) The elution agent used is 0.1 M PBS containing 5% methanol, with a pH of 5.0, and the elution time is 60 min.

10. A ratiometric acupuncture needle sensor for detecting growth hormone-releasing peptide prepared by the method described in any one of claims 1-9.

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