Molecularly imprinted electrochemical ratio sensor and preparation method thereof

Abstract

The invention discloses a molecularly imprinted electrochemical ratio sensor and a preparation method thereof, and belongs to the technical field of chemical detection. The molecularly imprinted electrochemical ratio sensor provided by the invention can be used for bFGF detection. The ratio sensing interface is prepared by electrically polymerizing FA and MB on the surface of ANME in sequence. Along with the increase of the concentration of the bFGF, the peak current of the pFA is gradually reduced, and the peak current of the pMB is kept relatively stable. The specific value of the pFA current and the pMB current is adopted as a signal for bFGF detection, and compared with a single-signal sensor, the molecularly imprinted electrochemical ratio sensor has higher sensitivity, accuracy and stability.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical detection, and particularly relates to a molecularly imprinted electrochemical ratio sensor and a preparation method thereof. Background Art

[0002] Research has shown that a variety of growth factors play important roles in the process of tumorigenesis and development, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and transforming growth factor β (TGF-β), etc. These factors promote tumor progression by regulating processes such as cell proliferation, angiogenesis, and metastasis. Here, we focus on bFGF. When secreted by tumor cells, it mediates several signal transduction pathways and exerts an influence in the tumor microenvironment. These mechanisms are the basis for the development of chemotherapy resistance and also indicate that bFGF has reliable prognostic value. Many evidences show that bFGF signaling is altered in the pathogenesis of many cancers that originate from different types of tissues in the human body. Abnormal FGF signaling can promote tumor development by directly promoting the proliferation of cancer cells and inhibiting their apoptosis, thereby inducing tumor angiogenesis, redefining the tumor microenvironment, and ultimately promoting the occurrence of metastasis. Among the currently identified 23 FGFs, acidic FGF (FGF-1) and basic FGF (bFGF) were the first to be isolated in the human body. bFGF was initially localized in the brain and pituitary gland. Physiologically, bFGF has been considered an important factor in the development and function of many body organ systems. bFGF is widely related to the biological functions of the hematopoietic system; it has also been proven to be an important neurotrophic factor in the central nervous system (CNS). bFGF exhibits unique functional diversity in different tissues: in the skin, it not only contributes to melanogenesis but also regulates the morphogenesis of basal upper keratinocytes; while in the eye, bFGF is not only crucial for maintaining the survival of photoreceptor cells but may also participate in its signal transduction process. Five bFGF isoforms (18, 22, 22.5, 24, and 34 kDa) have been identified in humans, and three bFGF isoforms (18, 21, and 23 kDa) have been identified in rodents and three bFGF isoforms (18.5, 20, and 21.5 kDa) have been identified in chickens. bFGF isoforms have different subcellular localizations and functions, and thus have a wide variety of effects on the cellular microenvironment in body regions.

[0003] bFGF has also been studied as a therapeutic agent for brain repair after traumatic events, such as brain and spinal cord injuries, and has been shown to have significant neuroprotective activity. However, in CNS diseases, the therapeutic use of bFGF is limited by its short half-life in the blood, low blood-brain barrier permeability, and several side effects, such as reduced blood pressure. Although bFGF administration has some potential human health benefits, its activity in different disease settings has been shown to be potentially harmful. In pathological conditions, bFGF, as a key regulator of tumor progression, is closely associated with poor patient prognosis and thus has become a highly promising anti-tumor therapeutic target.

[0004] Conventional analytical techniques such as chromatography, nuclear magnetic resonance (NMR), mass spectrometry (MS), etc. are usually complex to operate, with expensive and bulky equipment, and are usually difficult to achieve accurate detection and time-consuming; in addition, these methods usually also require professional operators, further limiting their wide application. To achieve rapid analytical detection, there is an urgent need to develop detection techniques with simple operation, low cost, and good selectivity, and at the same time, it is necessary to design portable detection devices suitable for on-site real-time monitoring. Among the devices that can meet this demand, sensors provide measurable signals and even the basic function of quantifying physical, biological, or even chemical phenomena, and can be directly used by humans. Electrochemical sensors show broad application prospects due to their significant technical advantages. This technology has the advantages of simple operation, high sensitivity, easy implementation, and low detection cost, and has application value in the fields of health, environment, or food quality control. Electrochemical sensors mainly adopt the following four electrochemical techniques: potentiometry, voltammetry, amperometry, and impedance spectroscopy. Biorecognition elements (such as antibodies, enzymes, microorganisms, DNA, etc.) can significantly improve the sensitivity and specificity of chemical sensors, compensating for the deficiencies of traditional chemical sensing materials. However, since they are biomolecules, they are extremely sensitive to environmental conditions such as pH value, temperature, and medium composition and need to be used under mild conditions to avoid protein denaturation. In addition, the use of these biological elements is not so simple. Professional operation techniques and experience accumulation are required from procurement to immobilization on the electrode surface. Due to the need to overcome these limitations, an alternative solution has been developed, including the use of molecularly imprinted polymers (MIPs). As a class of synthetic recognition materials, MIPs have binding sites that highly match the template molecule in terms of spatial structure and size, thus achieving specific recognition of target molecules. MIPs are characterized by their properties, such as physical resistance, robustness, resistance to high pressure and high temperature, and high inertness to various chemicals, and low production costs.

[0005] An electrochemical sensor is a detection device that converts chemical signals into quantifiable electrical signals through the specific interaction between the analyte and the recognition element on the electrode surface. Its working principle is based on the fact that the interaction between the analyte and the recognition element causes changes in electrical parameters such as electrode interface potential, current, conductivity or impedance. By measuring the changes in these parameters, qualitative and quantitative analysis of the target can be achieved. Due to the ease of preparation and modification of electrodes and their flexible design, electrochemical sensors have been used as a friendly platform for scientific research, including analysis and detection platforms. Traditional electrochemical sensors are generally identified as single-signal response technologies, which are sensitive to variable background noise in complex environments, resulting in relatively low detection accuracy and stability. Summary of the Invention

[0006] To solve the above problems, the present invention provides a molecular imprinting electrochemical ratio sensor and a preparation method thereof.

[0007] The novel electrochemical sensor presented in this paper combines the characteristics of ratiometric signaling and molecularly imprinted polymers (MIPs), creating a novel analytical technique. These electrochemical sensors, designated molecularly imprinted ratiometric electrochemical sensors (RMIECs), offer a novel analytical technique. The ratiometric detection mode of RMIECs not only allows for adjustment of the detection cycle by introducing a reference signal but also effectively utilizes dual signals, improving anti-interference capabilities. The MIPs enable specific target recognition and even simple sample pretreatment. Consequently, the newly developed RMIECs surpass conventional electrochemical sensors in stability, selectivity, and sensitivity.

[0008] To detect bFGF, the present invention proposes a dual-signal RMIECs combining polyferulic acid (pFA) and polymethylene blue (pMB), aiming to improve the sensitivity and selectivity of bFGF detection. The external reference signal is IpMB, which remains stable after the addition of the target analyte. At the same time, another signal IpMB is generated that changes proportionally with the concentration of the target analyte. pFA By calculating the signal response ratio (I pFA / I pMB ) to achieve accurate analysis of the target.

[0009] To achieve the above object, the present invention provides the following technical solutions:

[0010] One of the technical solutions of the present invention is to provide a method for preparing a molecularly imprinted electrochemical ratio sensor, comprising the following steps:

[0011] Step (1): depositing gold nanoparticles on the surface of the acupuncture needle microelectrode;

[0012] Step (2): continue to deposit a composite of carbon nanotubes and poly (3,4-ethylenedioxythiophene) (PEDOT);

[0013] Step (3): Further deposit polyferulic acid;

[0014] Step (4): Deposit gold nanoparticles again;

[0015] Step (5): Anchor the basic fibroblast growth factor protein template on the gold nanoparticles deposited in step (4);

[0016] Step (6): Continuously deposit the molecularly imprinted polymer film of polypyrrole and polymethylene blue;

[0017] Step (7): Finally, remove the basic fibroblast growth factor protein template to obtain the molecularly imprinted electrochemical ratio sensor;

[0018] The above operations are all carried out on the 0.1 mm - 10 mm near the tip of the acupuncture needle microelectrode.

[0019] Preferably, the acupuncture needle microelectrode further includes a surface treatment step before the first deposition of gold nanoparticles.

[0020] Preferably, the specific operation method for depositing gold nanoparticles in step (1) is: dipping the tip of the acupuncture needle microelectrode into the HAuCl4 solution and performing cyclic voltammetry cyclic scanning in the potential range of -1.5 V to +0.5 V.

[0021] Preferably, the specific operation method for depositing the composite of carbon nanotubes and poly(3,4-ethylenedioxythiophene) (PEDOT) in step (2) is: electrochemically polymerizing the composite of carbon nanotubes and poly(3,4-ethylenedioxythiophene) on the surface of the microelectrode treated in the previous step in a mixed solution containing carbon nanotubes and 3,4-ethylenedioxythiophene (EDOT).

[0022] Preferably, the specific operation method for depositing polyferulic acid in step (3) is: dipping the microelectrode treated in the previous step into the solution containing ferulic acid and performing cyclic voltammetry cyclic scanning in the potential range of -0.25 V to +0.75 V.

[0023] Preferably, the specific operation method for depositing gold nanoparticles in step (4) is: dipping the microelectrode treated in the previous step into the HAuCl4 solution and performing cyclic voltammetry cyclic scanning in the potential range of -1.5 V to +0.5 V.

[0024] Preferably, the specific operation method for anchoring the basic fibroblast growth factor protein template in step (5) is: dipping the microelectrode treated in the previous step into the solution containing the basic fibroblast growth factor protein and incubating at 4 °C to complete the anchoring.

[0025] Preferably, the specific operation method for depositing the molecularly imprinted polymer film of polypyrrole and polymethylene blue in step (6) is as follows: Immerse the microelectrode processed in the previous step into the solution containing pyrrole, and perform cyclic voltammetry cyclic scanning within the potential range of -0.3 V to +0.8 V. Then immerse it into the solution containing methylene blue, and perform cyclic voltammetry cyclic scanning within the potential range of -0.7 V to +0.4 V.

[0026] Preferably, the specific operation method for removing the basic fibroblast growth factor protein template in step (7) is as follows: Put the microelectrode processed in the previous step into an acetic acid aqueous solution containing sodium dodecyl sulfate (SDS) for elution.

[0027] Technical solution two of the present invention: Provide a molecularly imprinted electrochemical ratio sensor prepared according to the preparation method of the above-mentioned molecularly imprinted electrochemical ratio sensor.

[0028] The beneficial technical effects of the present invention are as follows:

[0029] The molecularly imprinted electrochemical ratio sensor provided by the present invention can be used for bFGF detection. By electro-polymerizing FA and MB on the ANME surface in sequence, a ratio sensing interface is prepared. As the concentration of bFGF increases, the peak current of pFA shows a gradually decreasing trend, while the peak current of pMB remains relatively stable. Using the ratio of the currents of pFA and pMB (I pFA / I pMB ) as the signal for bFGF detection, compared with single-signal sensors, the molecularly imprinted electrochemical ratio sensor has higher sensitivity, accuracy, and stability. In addition, the sensor exhibits excellent anti-interference performance. The simple and efficient molecularly imprinted electrochemical ratio sensor developed by the present invention provides an innovative solution for the highly sensitive and accurate detection of analytes. Description of the Drawings

[0030] Figure 1 It is the preparation process of the molecularly imprinted electrochemical ratio sensor and the schematic diagram of detecting bFGF protein in the embodiment of the present invention.

[0031] Figure 2 It is the electron transfer mechanism for the molecularly imprinted electrochemical ratio sensor prepared in the embodiment of the present invention to generate dual signals.

[0032] Figure 3Characterization results of the surface morphology and composition of the microelectrodes prepared in each step of Example 1. Among them, A is the SEM image of ANME after surface treatment, B is the SEM image of ANME / AuNPs, C is the SEM image of ANME / AuNPs / SWNTS, D is the SEM image of ANME / / pFA, E is the SEM image of ANME / / pFA / / pMB, F is the energy spectrum of ANME / AuNPs, and G is the element distribution map of ANME / AuNPs.

[0033] Figure 4 Electrochemical behavior study results of ANME / / pFA in Example 1. Among them, A is the CV curve of ANME / / pFA at different scanning rates, B is the relationship between I pa and I pc and the scanning rate, C is the CV curve of ANME / / pFA under different pH conditions, and D is the relationship diagram of pH value with peak potential and peak current.

[0034] Figure 5 Step-by-step modification results of ANME in Example 1 and Comparative Example 1 characterized by CVs. Among them, A is the CV curves of ANME (a), ANME / AuNPs (b), ANME / AuNPs / SWNTs (c), ANME / / pFA (d), ANME / / AuNPs (e), ANME / / AuNPs~bFGF (f), ANME / / pFA / / pMB (g), after elution (h), and after re-binding bFGF (i) in 0.1 M KCl containing 5.0 mM [Fe(CN)6] 3- / 4- ; B is the CV curves of ANME / / pFA (a), ANME / / pFA / / pPy (b), ANME / / pFA / / pMB (c), after elution (d), and after re-binding bFGF (e) in 0.1 M PBS solution (pH = 7.0) in Example 1, C is the DPV curves of ANME / / pFA (a), ANME / / pFA / / pPy (b), ANME / / pFA / / pMB (c), after elution (d), and after re-binding bFGF (e) in 0.1 M PBS solution (pH = 7.0) in Example 1; D is the DPV curves of ANME / / pFA (a), ANME / / pFA / AuNPs / pPy (b), ANME / / pFA / AuNPs / / pMB (c), after elution (d), and after re-binding bFGF (e) in 0.1 M PBS solution (pH = 7.0) in Comparative Example 1.

[0035] Figure 6The influence of the preparation parameters in the preparation method of Example 1 and the detection parameters of the detection method on the detection results, where A is the influence of the electrodeposition time of single-walled carbon nanotubes on the results, B is the influence of the number of cycles of polymerizing FA on the results, C is the influence of the scanning rate of polymerizing FA on the results, D is the influence of the incubation time of bFGF on the results, E is the influence of the number of cycles of polymerizing Py on the results, F is the influence of the scanning rate of polymerizing Py on the results, G is the influence of the elution time on the results, H is the influence of the rebinding time of bFGF on the results, and I is the influence of the pH value of the detection solution on the results.

[0036] Figure 7 The detection results of ANME / / pFA / / SMIpMB for bFGF prepared on the basis of optimized conditions, where A is the DPV curve of the prepared ANME / / pFA / / SMIpMB after incubating with different concentrations of bFGF for 60 min, B is the pFA I / I pMB linear relationship between the value and the logarithm of the bFGF concentration, and C is the pFA linear relationship between the value and the logarithm of the bFGF concentration.

[0037] Figure 8 The evaluation results of the selectivity (A), stability (B), and repeatability (C) of the ANME / / pFA / / SMIpMB prepared in Example 1. Detailed implementation manners

[0038] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention. It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention.

[0039] It should be noted that the aspects not described in detail in the present invention are all conventional operation means in the art and are not the focus of the present invention.

[0040] In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0041] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention.

[0042] Regarding the terms "comprising", "including", "having", "containing", etc. used in this text, they are all open-ended terms, meaning including but not limited to.

[0043] The preparation process of the molecularly imprinted electrochemical ratio sensor and the schematic diagram for detecting bFGF protein in the embodiments of the present invention are shown in Figure 1 .

[0044] The electron transfer mechanism for generating dual signals by the molecularly imprinted electrochemical ratio sensor prepared in the embodiments of the present invention is shown in Figure 2 .

[0045] Example 1

[0046] Preparation of the molecularly imprinted electrochemical ratio sensor (ANME / / pFA / / SMIpMB):

[0047] (1) Surface pretreatment of the acupuncture needle microelectrode (ANME):

[0048] First, polish with sandpaper for 3 min, then perform ultrasonic cleaning in ethanol and ultrapure water for 5 min respectively, and finally place it in the air to dry naturally to complete the surface pretreatment of ANME;

[0049] (2) Electrodeposition of gold nanoparticles (AuNPs):

[0050] Immerse the tip of 5 mm of the surface-pretreated ANME into a 2.5 mM HAuCl4 solution, and use cyclic voltammetry (CV) to scan 5 cycles at a scanning rate of 25 mV·s -1 within the potential range of -1.5 V to +0.5 V to obtain ANME modified with AuNPs, denoted as ANME / AuNPs;

[0051] (3) Co-deposition of PEDOT and carboxylated single-walled carbon nanotubes (SWNTs):

[0052] Immerse the tip of 5 mm of ANME / AuNPs in a mixed solution containing 2 mg·mL -1 SWNTs and 0.02 M 3,4-ethylenedioxythiophene (EDOT) for electropolymerization to prepare a stable interface. Ultrasonically treat the solution for 30 min before polymerization. The polymerization potential is set to 1.2 V, and the polymerization time is 110 s. After polymerization, thoroughly rinse the modified electrode with water and dry it at room temperature to obtain ANME / AuNPs co-deposited with PEDOT and carboxylated single-walled carbon nanotubes, denoted as ANME / AuNPs / SWNTs;

[0053] (4) Electropolymerization of ferulic acid (FA):

[0054] The tip of ANME / AuNPs / SWNTs with a length of 5 mm was subjected to 10 cycles of cyclic voltammetry (CV) scanning in a 0.1 M PBS electrolyte containing 0.4 mM FA (pH = 4.5) at a scanning rate of 60 mV·s -1 in the potential range of -0.25 V to +0.75 V to electro-polymerize FA (before the start of polymerization, the electro-polymerization solution was bubbled with nitrogen for 10 min to completely deoxygenate). The resulting microelectrode was denoted as ANME / AuNPs / SWNTs / pFA (abbreviated as ANME / / pFA);

[0055] (5) Further electrodeposit gold nanoparticles (AuNPs):

[0056] To anchor the protein template, a layer of AuNPs was continuously deposited. The tip of ANME / / pFA with a length of 5 mm was immersed in a 2.5 mM HAuCl4 solution, and cyclic voltammetry (CV) was used to scan 3 cycles in the potential range of -1.5 V to +0.5 V at a scanning rate of 25 mV·s -1 . The resulting microelectrode was denoted as ANME / AuNPs / SWNTs / pFA / AuNPs (abbreviated as ANME / / AuNPs);

[0057] (6) Anchor the protein template:

[0058] The tip of ANME / / AuNPs with a length of 5 mm was immersed in a bFGF protein solution (concentration: 10 μg·mL -1 ), and incubated at 4 °C for 10 h. The resulting microelectrode was denoted as ANME / / AuNPs~bFGF;

[0059] (7) Electrodeposit the molecularly imprinted polymer film:

[0060] The tip of ANME / / AuNPs~bFGF with a length of 5 mm was immersed in a 0.01 M PBS (pH = 7.0) solution containing 0.1 M pyrrole (Py). CV method was used to cycle 6 times in the voltage range of -0.3 V to +0.8 V, and the scanning rate was 100 mV·s -1 . ANME / / pFA / / pPy was obtained. Then, the microelectrode with electrodeposited polypyrrole was immersed in a 0.1 M PBS (pH = 7.0) solution containing 2.5 mM methylene blue (MB), and cycled 2 times in the voltage range of -0.7 V to +0.4 V by CV method with a scanning rate of 100 mV·s -1 . The free Py and MB were rinsed off with ultrapure water to complete the deposition of the molecularly imprinted polymer film. The resulting microelectrode was denoted as ANME / / pFA / / pMB;

[0061] (8) Removal of bFGF protein template:

[0062] The bFGF protein template in ANME / / pFA / / pMB was eluted with 10% (v / v) acetic acid aqueous solution containing 10 wt.% SDS to obtain the molecularly imprinted electrochemical ratio sensor ANME / AuNPs / SWNTs / pFA / AuNPs-bFGF@pPy@pMB (abbreviated as ANME / / pFA / / SMIpMB).

[0063] The surface morphology and composition of the electrodes prepared in each step of Example 1 were characterized using a scanning electron microscope (SEM) and an energy dispersive spectrometer (EDS). The results are shown in Figure 3 , where A is the SEM image of ANME after surface treatment, B is the SEM image of ANME / AuNPs, C is the SEM image of ANME / AuNPs / SWNTS, D is the SEM image of ANME / / pFA, E is the SEM image of ANME / / pFA / / pMB, F is the energy spectrum of ANME / AuNPs, and G is the elemental distribution map of ANME / AuNPs.

[0064] Figure 3 A in shows that the surface morphology of ANME presents longitudinal scratches, which are caused by polishing. The polishing treatment is beneficial for the subsequent modification of nanomaterials. Figure 3 B in shows that AuNPs are evenly distributed on the surface of ANME, and the diameter of AuNPs is about 60 nm. Figure 3 In F of , the characteristic peak of metallic Au can be clearly seen, confirming that AuNPs have been successfully modified onto ANME. Figure 3 G in is the elemental distribution map (mapping) of ANME / AuNPs, revealing the presence of Cr, Mn, Ni, Cl, Mo (from ANME itself) and Au elements. The appearance of Au elements further confirms the deposition of AuNPs on the surface of ANME, which provides a large surface area and high conductivity for subsequent modification. From Figure 3 C of , it can be clearly seen that an obvious network structure and a smooth surface appear on the surface, indicating that carbon nanotubes are successfully bonded to the surface of the microelectrode. From Figure 3 D of , pFA polymerized on the surface of carbon nanotubes can be seen. Figure 3 E of , further polymerized pPy and pMB can be seen.

[0065] Comparative Example 1

[0066] Preparation of non-molecularly imprinted electrochemical ratio sensor (compared with the method of Example 1, the difference is only that the step of anchoring protein is omitted):

[0067] (1)Surface pretreatment of acupuncture needle microelectrode (ANME):

[0068] First, polish with sandpaper for 3 min, then perform ultrasonic cleaning in ethanol and ultrapure water for 5 min respectively, and finally place it in the air to dry naturally to complete the surface pretreatment of ANME;

[0069] (2)Electrodeposition of gold nanoparticles (AuNPs):

[0070] Immerse the tip 5 mm of the surface-pretreated ANME into a 2.5 mM HAuCl4 solution, and use cyclic voltammetry (CV) to scan 5 cycles at a scan rate of 25 mV·s -1 in the potential range of -1.5 V to +0.5 V to obtain AuNPs-modified ANME, denoted as ANME / AuNPs;

[0071] (3)Co-deposition of PEDOT and carboxylated single-walled carbon nanotubes (SWNTs):

[0072] Immerse the tip 5 mm of ANME / AuNPs in a mixed solution containing 2 mg·mL -1 SWNTs and 0.02 M 3,4-ethylenedioxythiophene (EDOT) for electropolymerization to prepare a stable interface. Ultrasonically treat the solution for 30 min before polymerization. The polymerization potential is set to 1.2 V, and the polymerization time is 110 s. After polymerization, thoroughly rinse the modified electrode with water and dry it at room temperature to obtain ANME / AuNPs co-deposited with PEDOT and carboxylated single-walled carbon nanotubes, denoted as ANME / AuNPs / SWNTs;

[0073] (4)Electropolymerization of ferulic acid (FA):

[0074] Immerse the tip 5 mm of ANME / AuNPs / SWNTs in 0.1 M PBS electrolyte containing 0.4 mM FA (pH = 4.5) and perform 10 cycles of CV scanning at a scan rate of 60 mV·s -1 in the potential range of -0.25 V to +0.75 V for electropolymerization of FA (before starting polymerization, bubble nitrogen through the electropolymerization solution for 10 min to completely deoxygenate). The obtained microelectrode is denoted as ANME / AuNPs / SWNTs / pFA (abbreviated as ANME / / pFA);

[0075] (5)Further electrodeposition of gold nanoparticles (AuNPs):

[0076] To anchor the protein template, a layer of AuNPs was continuously deposited. The tip of 5 mm of ANME / / pFA was immersed in a 2.5 mM HAuCl4 solution, and cyclic voltammetry (CV) was used to scan 3 cycles in the potential range of -1.5 V to +0.5 V at a scanning rate of 25 mV·s -1 to obtain a microelectrode denoted as ANME / AuNPs / SWNTs / pFA / AuNPs (abbreviated as ANME / / pFA / AuNPs);

[0077] (6) Electrodeposition of molecularly imprinted polymer film:

[0078] The tip of 5 mm of ANME / / pFA / AuNPs was immersed in a 0.01 M PBS (pH = 7.0) solution containing 0.1 M pyrrole (Py), and CV was used to cycle 6 times in the voltage range of -0.3 V to +0.8 V at a scanning rate of 100 mV·s -1 to obtain ANME / / pFA / AuNPs / pPy; then, the microelectrode with electrodeposited polypyrrole was immersed in a 0.1 M PBS (pH = 7.0) solution containing 2.5 mM methylene blue (MB), and CV was used to cycle 2 times in the voltage range of -0.7 V to +0.4 V at a scanning rate of 100 mV·s -1 The free Py and MB were rinsed off with ultrapure water to complete the deposition of the molecularly imprinted polymer film. The obtained microelectrode was denoted as ANME / / pFA / AuNPs / / pMB;

[0079] (7) Removal of bFGF protein template:

[0080] The ANME / / pFA / AuNPs / / pMB was eluted with a 10% (v / v) acetic acid aqueous solution containing 10 wt.% SDS to obtain a non-molecularly imprinted electrochemical ratio sensor.

[0081] Method for detecting bFGF protein using the ANME / / pFA / / SMIpMB prepared in Example 1 or the non-molecularly imprinted electrochemical ratio sensor prepared in Comparative Example 1:

[0082] The prepared sensor was incubated in a 0.02 M PBS (pH = 7.4) solution containing different concentrations of bFGF at 4 °C for 60 min, and the unbound bFGF was washed with water. Then, in PBS of 0.1 M PBS (pH = 7.0), using FA and MB as dual-signal probes, the bound bFGF was detected by DPV.

[0083] Study on the electrochemical behavior of ANME / / pFA in Example 1:

[0084] The electrochemical behavior of electro - polymerized pFA adsorbed on the surface of a micro - electrode was studied in PBS under the conditions of scan rates set at 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, and 300 mV·s⁻¹ in 0.1 M PBS (pH = 7.0). -1 ; in 0.1 M PBS with pH values set at 5.0, 6.0, 7.0, 8.0, 9.0 and scan rate set at 100 mV·s⁻¹ -1 .

[0085] The research results are shown in Figure 4 , where A is the CV curve of ANME / / pFA at different scan rates, B is the relationship between I pa and I pc and the scan rate, C is the CV curve of ANME / / pFA under different pH conditions, and D is the relationship diagram of pH value with peak potential and peak current.

[0086] pFA shows a pair of redox peaks in the potential range from - 0.4 V to + 0.8 V. The oxidation peak potential (E pa ) is about 0.231 V, and the reduction peak potential (E pc ) is about 0.095 V. At a scan rate of 100 mV·s⁻¹ -1 , the difference (ΔE p ) is about 136 mV (A in Figure 4 ). E pa and E pc increase and decrease respectively with the increase of the scan rate, and ΔE p increases as the scan rate ranges from 10 to 300 mV·s⁻¹ -1 . The ratio of peak currents (I pa / I pc ) is about 0.93 - 1.31, indicating that the electrode reaction is reversible. In the range of 10 - 300 mV·s⁻¹ -1 , the peak current is proportional to the scan rate, and its linear equations are I pa (μA) = 0.9233v (mV·s⁻¹ -1 ) - 8.6726 (R 2 = 0.9932) and I pc (μA) = - 0.9337v (mV·s⁻¹ -1 ) + 10.2572 (R 2 = 0.9896). The anodic peak current is denoted as I pa , and the cathodic peak current is denoted as I pc. The results show that the electron transfer process of pFA on the modified electrode surface film is controlled by the surface process and is suitable for use as an electrochemical probe to provide current signals. The number of electrons gained or lost in the electrode reaction can be obtained by the following formula:

[0087]

[0088] In the above formula, Q = nFAΓ. Where Q is the electric charge (C), A is the electroactive surface area (cm 2 ), v is the scan rate, R is the gas constant [8.314 J / (K·mol) -1 , T is the temperature (298.15 K), F is the Faraday constant [96.487 kJ / (V·mol) -1 , Γ is the adsorption capacity (mol·cm -2 ), and in the present invention, n = 1.

[0089] The acidity and alkalinity of the solution have a great influence on the electrochemical performance of ANME / / pFA. Figure 4 Figure C in [reference] shows the CV curves of ANME / / pFA in PBS with different pH values. As the pH value increases, the oxidation peak potential and the reduction peak potential shift significantly negatively, indicating that H + participates in the electron transfer process. When the pH value is in the range of 5.0 - 9.0, E pa , E pc has a good linear relationship with the pH value, and the linear equations are as follows. E pa (V) = -0.0791pH + 0.8456 (R 2 = 0.9878) and E pc (V) = -0.0473pH + 0.4487 (R 2 = 0.9987) (curves a and b in Figure D in [reference]). As the pH increases from 5.0 to 7.0, the peak current of pFA gradually increases, reaches the maximum value at pH 7.0, and then decreases significantly between 7.0 and 9.0 (curve c in Figure D in [reference]). Therefore, pH 7.0 is selected as the optimal pH value in the experiment. Figure 4 Figure 4 Use CVs to further characterize the step-by-step modification results of ANME in Example 1 and Comparative Example 1:

[0090] Cyclic voltammetry (CV) scans were performed on the electrodes modified in each step in a solution containing 5.0 mM [Fe(CN)6]

[0091] and 0.1 M KCl solution, with a scan range of -0.2 V to +0.6 V and a scan rate of 100 mV s 3- / 4- . -1 .

[0092] The modified electrodes in each step were scanned by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) in 0.1 M PBS solution (pH = 7.0). The CV scanning range was -0.8 V to +0.8 V, and the scanning rate was 100 mV s -1 , and the DPV scanning range was -0.6 V to +0.4 V.

[0093] The characterization results are shown in Figure 5 , where A shows the CV curves of ANME (a), ANME / AuNPs (b), ANME / AuNPs / SWNTs (c), ANME / / pFA (d), ANME / / AuNPs (e), ANME / / AuNPs~bFGF (f), ANME / / pFA / / pMB (g), after elution (h), and after rebinding bFGF (i) in 0.1 M KCl containing 5.0 mM [Fe(CN)6] 3- / 4- ; B shows the CV curves of ANME / / pFA (a), ANME / / pFA / / pPy (b), ANME / / pFA / / pMB (c), after elution (d), and after rebinding bFGF (e) in 0.1 M PBS solution (pH = 7.0) in Comparative Example 1; C shows the DPV curves of ANME / / pFA (a), ANME / / pFA / / pPy (b), ANME / / pFA / / pMB (c), after elution (d), and after rebinding bFGF (e) in 0.1 M PBS solution (pH = 7.0) in Example 1; D shows the DPV curves of ANME / / pFA (a), ANME / / pFA / AuNPs / pPy (b), ANME / / pFA / AuNPs / / pMB (c), after elution (d), and after rebinding bFGF (e) in 0.1 M PBS solution (pH = 7.0) in Comparative Example 1.

[0094] Figure 5 In A, due to [Fe(CN)6] 3- / 4-The heterogeneous electron transfer process of the redox probe is severely inhibited by the stainless-steel material, and the ANME shows a rather low peak current. The peak current increases significantly at ANME / AuNPs, which is probably due to the enhanced heterogeneous electron transfer after AuNPs electrodeposition. The peak current of ANME / AuNPs / SWNTs increases significantly, which may be attributed to the presence of SWNTs on the electrode surface, which not only increases the specific surface area of the electrode and improves the electrode conductivity, but also can serve as an ideal carrier for MIP immobilization, providing a larger electroactive surface area and better opportunities for loading functional materials to construct an electrochemical microsensor with excellent sensing performance. The electrochemical properties were studied, and the electroactive surface areas of different electrodes were calculated according to the Randles-Sevcik equation:

[0095]

[0096] In the above formula, I p is the peak current of the anode (A), n is the number of electron transfers involved in the redox pair (= 1), A is the electroactive surface area (cm 2 ), C is the concentration of ferricyanide (5.0×10 -6 mol·cm -3 ), D is the diffusion coefficient of ferricyanide (7.6×10 -6 cm 2 ·s -1 ), and v is the scan rate (0.1 V·s -1 ).

[0097] After calculation, the electrochemically active surface area of ANME is 0.499×10 -3 cm 2 , while the electrochemically active specific surface area of ANME / AuNPs is 0.110 cm 2 . The active surface area of ANME / AuNPs / SWNTs is 0.192 cm 2 . The results show that compared with the case of ANME, the electrodeposition of AuNPs and SWNTs significantly increases the electroactive surface area and sites of the electrode. After removing the bFGF protein template, a significant increase in the peak current was observed. The reason is that cavities are formed on the MIP membrane. After the bFGF protein recombines with the cavities, the peak current decreases again.

[0098] Figure 5Figure B shows that a pair of clear and almost reversible redox peaks appear at +0.276 V and +0.081 V on the interface modified with pFA. When pPy is further modified onto the electrode surface, the sensor shows no obvious electrochemical response in PBS. When pMB is modified onto the electrode surface, a pair of peaks around -0.198 V and -0.416 V reappear in PBS, corresponding to the typical redox peaks of MB. After elution, the interface shows two relatively obvious electrochemical responses in PBS, indicating the formation of the imprinted cavity.

[0099] Figure 5 Figure C shows the DPV diagrams of different microelectrodes in 0.1 M PBS (pH = 7.0). Due to the oxidation of pFA, a huge oxidation peak is generated at +0.160 V. Then, after Py is modified on the electrode, an imprinted layer is formed, and due to the poor conductivity of pPy, the oxidation peak disappears. After electropolymerizing MB, a new signal peak appears at -0.284 V, corresponding to the oxidation peak of pMB. After the bFGF protein template is eluted, two non-interfering signal peaks are obtained at -0.284 V and +0.160 V respectively, which may be attributed to the charge transfer caused by the formation of the imprinted cavity. After the prepared electrochemical microsensor is incubated again in the bFGF protein solution, after the bFGF protein in the solution is captured by the imprinted cavity, the peak current of pFA at +0.160 V decreases again, while the peak current of pMB remains almost unchanged.

[0100] Figure 5 Figure D shows that for ANME / / pFA / AuNPs / / pMB, the peak current of pFA changes little before and after elution, which is due to the absence of an imprinted cavity in NIP.

[0101] Optimize the preparation parameters in the preparation method of Example 1 and the detection parameters of the detection method:

[0102] The optimization results are shown in Figure 6 , where A is the influence of the electrodeposition time of single-walled carbon nanotubes on the result, B is the influence of the number of cycles of polymerizing FA on the result, C is the influence of the scanning rate of polymerizing FA on the result, D is the influence of the incubation time of bFGF on the result, E is the influence of the number of cycles of polymerizing Py on the result, F is the influence of the scanning rate of polymerizing Py on the result, G is the influence of the elution time on the result, H is the influence of the rebinding time of bFGF on the result, and I is the influence of the pH value of the detection solution on the result.

[0103] For comparison, the peak currents of FA after ANME / / pFA, ANME / / pFA / / SMIpMB, and ANME / / pFA / / SMIpMB were incubated again in the FGF protein solution were defined as I1, I2, and I3, respectively.

[0104] By optimizing the deposition time of SWNTs preparation, a larger surface area can be obtained, thus obtaining more binding sites. As Figure 6 shown in A, a large number of SWNTs increases the conductivity and effective surface area, so the deposition time was selected as 110 s. Figure 6 B shows the relationship between I1 and the number of electropolymerization cycles of pFA. When the number of polymerization cycles is 6 - 10, the value of I1 gradually increases. When the polymerization proceeds to 10 cycles, the value of I1 reaches the maximum. When the number of polymerization cycles continues to increase, the change of I1 value is very small and almost unchanged, which is probably because after 10 cycles, the excessive thickness of pFA prevents the transfer of electrons. Since the sensitivity is closely related to the peak current of pFA, the optimal number of polymerization cycles was selected as 10 cycles. As Figure 6 shown in C, with the increase of the scanning rate, the maximum value of I1 appears at 60 mV·s -1 . During the electropolymerization process, a low scanning rate usually forms a dense film, while a high scanning rate usually forms a rough film, both of which limit the modification quality. Therefore, the optimal scanning rate was selected as 60 mV·s -1 . In the range of pH values of 5.0, 6.0, 7.0, 8.0, and 9.0, the influence of the pH value of the detection solution on the current response of FA was studied. As Figure 6 shown in I, during the process of pH rising from 5.0 to 7.0, the value of I1 gradually increases; during the process of pH rising from 7.0 to 9.0, the value of I1 gradually decreases. On this basis, the optimal pH value of the detection solution was determined to be 7.0.

[0105] In this experiment, the incubation time of ANME / / AuNPs in the bFGF protein solution was also optimized, and the results are as Figure 6 shown in D. The value of I2 reaches the maximum at 10 h, which may be due to the protein saturation in ANME / / AuNPs. That is to say, the immobilization of the bFGF protein template through the gold - amino bond interaction reaches saturation after 10 h of incubation. As Figure 6 shown in E and F, the value of I2 changes with the increase of the number of cycles and scanning rate of polyPy. Selecting the peak currents of these parameters, the optimal number of cycles was determined to be 6, and the scanning rate was 100 mV·s -1 . As Figure 6As shown in Fig. G, the DPV current signal intensity was positively correlated with the elution time. This phenomenon indicated that the bFGF protein template was gradually removed from the MIP membrane during the elution process, resulting in an increase in the imprinted cavities and thus enhancing the current response signal. The relationship diagram between the I2 value and the elution time showed a signal plateau from 2 to 3 h. Therefore, the optimal elution time for the FGF protein template was selected as 2 h. Figure 6 Fig. H shows the relationship between ΔI (ΔI = I2 - I3) and the rebinding time of the bFGF protein. At the rebinding time of 60 min, the ΔI value reached the maximum, indicating that the binding of the bFGF protein to the imprinted cavities reached saturation after 60 min.

[0106] On the basis of optimizing the detection conditions, a ratiometric electrochemical sensor for sensitive detection of bFGF was successfully constructed. Figure 7 Fig. 7 shows the detection results of ANME / / pFA / / SMIpMB for bFGF. Among them, A is the DPV curve of ANME / / pFA / / SMIpMB prepared after incubating with different concentrations of bFGF for 60 min, B is the pFA I pMB value and the linear relationship between the logarithm of the bFGF concentration, and C is the pFA value and the linear relationship between the logarithm of the bFGF concentration.

[0107] Figure 7 As shown in Fig. B, the pFA I pMB current ratio showed a good linear relationship with the bFGF concentration in the range of 0.001 - 3000 ng·mL -1 . The calibration equation pFA I pMB = -0.1074 lgC (ng mL -1 ) + 0.7838 (R 2 = 0.9969), and the detection limit (LOD) was 0.06 pg·mL −1 (S / N = 3). As Figure 7 shown in Fig. C, for comparison, the single current of pFA in Figure 7 Fig. A was used as the output signal to detect bFGF. Its linear range and calibration equation were 1 - 3000 ng·mL −1 and pFA I -1 (μA) = -3.8370 lgC (ng·mL 2 ) + 25.6358 (R −1(S / N = 3). Compared with single-signal output, the ratio signal has a wider linear range and a lower detection limit, demonstrating the superior performance of the ratio detection mode in bFGF detection. The results show that this method can effectively improve the sensitivity of bFGF detection.

[0108] Detection of the selectivity, stability, and repeatability of the prepared ANME / / pFA / / SMIpMB

[0109] Use [Fe(CN)6] 3- / 4- The probe was used to perform CV characterization on ANME / / pFA / / SMIpMB. CV or differential pulse voltammetry (DPV) was scanned in the potential range of -0.8 V to +0.8 V or -0.6 V to +0.4 V. Unless otherwise specified, the electrolyte solution was 0.1 M PBS (pH = 7.0), and DPV recorded the peak currents generated by the oxidation of pFA (I pFA ) and pMB (I pMB ), and calculated their current ratio (I pFA / I pMB ). ΔI was the difference in the peak current of pFA before and after SMIp adsorbed bFGF. VEGF, OPN, MSLN, UA, Mg 2+ , Na + Ca 2+ Cl - SO4 2- were selected as potential interferents (concentrations were 10 times that of bFGF-10 μg·mL -1 ) to evaluate the anti-interference ability of the prepared sensor.

[0110] The experimental results of the anti-interference ability of ANME / / pFA / / SMIpMB are as shown in Figure 8 A. Compared with the control group (a), the other groups (b ~ j) did not change much. This indicates that the sensor has outstanding affinity for bFGF and high anti-interference effect. The good selectivity of the obtained sensor should be attributed to the high recognition ability of the imprinted cavity. These data show that ANME / / pFA / / SMIpMB has high selectivity and affinity for bFGF.

[0111] Generally speaking, the ratio detection strategy can greatly improve stability and repeatability. In this case, to verify this, it was compared with non-ratio detection, and the detection method was the same as that in Figure 8 A. After storing for two weeks, I pFA was 87.2% of the initial value, while I pFA / I pMB retained 96.9% of the initial value (in Figure 8 B). As shown in Figure 8As shown in C, after adopting the ratio measurement strategy, the RSD decreased from 5.5% to 2.1%. The results indicate that the ratio measurement strategy effectively reduces the errors caused by internal and external environmental factors, suggesting that the ratio strategy is highly effective in improving sensing performance. These findings confirm that the ratio detection strategy has commendable stability and repeatability.

[0112] Detection of actual samples

[0113] To test the practical applicability of the proposed sensor, spiked samples (i.e., blood samples, saliva, and sweat) were analyzed. The detection method is described in the reference "Method for detecting bFGF protein using ANME / / pFA / / SMIpMB prepared in Example 1", and the detection results are shown in Table 1.

[0114] Table 1 Detection of bFGF in actual samples

[0115]

[0116] Table 1 shows that the recovery rate of bFGF is 95.80% - 104.70%, indicating that RMIECs have good application potential in detecting the content of bFGF in actual samples.

[0117] The above-described embodiments are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A preparation method of a molecularly imprinted electrochemical ratio sensor, characterized in that, It includes the following steps: Step (1): Deposit gold nanoparticles on the surface of the acupuncture needle microelectrode; Step (2): Continuously deposit a composite of carbon nanotubes and poly(3,4-ethylenedioxythiophene); Step (3): Further deposit polyferulic acid; Step (4): Deposit gold nanoparticles again; Step (5): Anchor the basic fibroblast growth factor protein template on the gold nanoparticles deposited in step (4); Step (6): Continuously deposit a molecularly imprinted polymer film of polypyrrole and polymethylene blue; Step (7): Finally, remove the basic fibroblast growth factor protein template to obtain the molecularly imprinted electrochemical ratio sensor; The operations of steps (1)-(7) are all carried out on the 0.1 mm - 10 mm near the tip of the acupuncture needle microelectrode.

2. The preparation method of the molecularly imprinted electrochemical ratio sensor according to claim 1, characterized in that, The acupuncture needle microelectrode also includes a surface treatment step before the first deposition of gold nanoparticles.

3. The preparation method of the molecularly imprinted electrochemical ratio sensor according to claim 1, characterized in that, The specific operation method for depositing gold nanoparticles in step (1) is: Immerse the tip of the acupuncture needle microelectrode in the HAuCl4 solution and perform cyclic voltammetry cyclic scanning in the potential range of -1.5 V to +0.5 V.

4. The preparation method of the molecularly imprinted electrochemical ratio sensor according to claim 1, wherein, The specific operation method for depositing the composite of carbon nanotubes and poly(3,4-ethylenedioxythiophene) in step (2) is: Electrochemically polymerize the composite of carbon nanotubes and poly(3,4-ethylenedioxythiophene) on the surface of the microelectrode treated in the previous step in a mixed solution containing carbon nanotubes and 3,4-ethylenedioxythiophene.

5. The preparation method of the molecularly imprinted electrochemical ratio sensor according to claim 1, characterized in that, The specific operation method for depositing polyferulic acid in step (3) is: Immerse the microelectrode treated in the previous step in a solution containing ferulic acid and perform cyclic voltammetry cyclic scanning in the potential range of -0.25 V to +0.75 V.

6. The preparation method of the molecularly imprinted electrochemical ratio sensor according to claim 1, characterized in that, The specific operation method for depositing gold nanoparticles in step (4) is: Immerse the microelectrode treated in the previous step in the HAuCl4 solution and perform cyclic voltammetry cyclic scanning in the potential range of -1.5 V to +0.5 V.

7. The preparation method of the molecularly imprinted electrochemical ratio sensor according to claim 1, characterized in that, The specific operation method for anchoring the basic fibroblast growth factor protein template in step (5) is: Immerse the microelectrode treated in the previous step in a solution containing the basic fibroblast growth factor protein and incubate at 4 °C to complete the anchoring.

8. The preparation method of the molecularly imprinted electrochemical ratio sensor according to claim 1, characterized in that, The specific operation method for depositing the molecularly imprinted polymer film of polypyrrole and polymethylene blue in step (6) is: Immerse the microelectrode treated in the previous step in a solution containing pyrrole and perform cyclic voltammetry cyclic scanning in the potential range of -0.3 V to +0.8 V, and then immerse it in a solution containing methylene blue and perform cyclic voltammetry cyclic scanning in the potential range of -0.7 V to +0.4 V.

9. The preparation method of the molecularly imprinted electrochemical ratio sensor according to claim 1, characterized in that, The specific operation method for removing the basic fibroblast growth factor protein template in step (7) is: Place the microelectrode treated in the previous step in an acetic acid aqueous solution containing sodium dodecyl sulfate for elution.

10. A molecularly imprinted electrochemical ratio sensor prepared by the preparation method of the molecularly imprinted electrochemical ratio sensor according to any one of claims 1-9.

Images