A molecularly imprinted electrochemical ratio sensor and its preparation method

Abstract

The present invention discloses a molecularly imprinted electrochemical ratiometric sensor and a preparation method thereof, belonging to the field of chemical detection technology. The molecularly imprinted electrochemical ratiometric sensor provided by the present invention can be used for bFGF detection. FA and MB are sequentially electropolymerized on an anhydrous metalloproteinase (ANME) surface to prepare a ratiometric sensing interface. With increasing bFGF concentration, the peak current of pFA gradually decreases, while the peak current of pMB remains relatively stable. The ratio of the pFA to pMB currents is used as a signal for bFGF detection. Compared with single-signal sensors, the molecularly imprinted electrochemical ratiometric sensor has higher sensitivity, accuracy, and stability.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical detection, and in particular relates to a molecular imprinting electrochemical ratio sensor and a preparation method thereof. Background Art

[0002] Research has shown that multiple growth factors, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and transforming growth factor β (TGF-β), play important roles in tumor development and progression. These factors promote tumor progression by regulating processes such as cell proliferation, angiogenesis, and metastasis. Here, we focus on bFGF, which, when secreted by tumor cells, mediates several signaling pathways and exerts influence within the tumor environment. These mechanisms underlie the development of chemoresistance and suggest that bFGF has robust prognostic value. Numerous lines of evidence indicate that bFGF signaling is altered in the pathogenesis of numerous cancers originating from diverse tissue types. Aberrant FGF signaling can promote tumor progression by directly promoting cancer cell proliferation and inhibiting apoptosis, thereby inducing tumor angiogenesis, redefining the tumor microenvironment, and ultimately promoting metastasis. Of the 23 FGFs identified to date, acidic FGF (FGF-1) and basic FGF (bFGF) are the first to be isolated in humans. bFGF was originally localized to the brain and pituitary gland. Physiologically, bFGF has been recognized as a crucial factor in the development and function of numerous organ systems. bFGF is widely implicated in the biological functions of the hematopoietic system; it has also been shown to be an important neurotrophic factor in the central nervous system (CNS). bFGF exhibits unique functional diversity across different tissues: in the skin, it contributes to melanogenesis and regulates the morphogenesis of suprabasal keratinocytes; in the eye, bFGF is not only essential for maintaining photoreceptor cell survival but may also participate in their signaling processes. Five bFGF isoforms have been identified in humans (18, 22, 22.5, 24, and 34 kDa), and three bFGF isoforms have been identified in rodents (18, 21, and 23 kDa) and chickens (18.5, 20, and 21.5 kDa). These bFGF isoforms have distinct subcellular localizations and functions, resulting in a wide range of effects on the cellular microenvironment across various regions of the body.

[0003] bFGF has also been investigated as a therapeutic agent for brain repair after traumatic events, such as brain and spinal cord injury, and has demonstrated significant neuroprotective activity. However, the therapeutic use of bFGF in CNS diseases is limited by its short half-life in the blood, low blood-brain barrier permeability, and several side effects, such as decreased blood pressure. While bFGF administration has several potential human health benefits, its activity in various disease settings has been shown to be potentially detrimental. In pathological conditions, bFGF acts as a key regulator of tumor progression and is closely associated with poor patient prognosis, making it a promising target for anti-tumor therapy.

[0004] Conventional analytical techniques such as chromatography, nuclear magnetic resonance (NMR), and mass spectrometry (MS) are often complex, require expensive and bulky equipment, and often struggle to achieve accurate detection and are time-consuming. Furthermore, these methods often require specialized operators, further limiting their widespread application. To achieve rapid analytical detection, there is an urgent need to develop simple, low-cost, and highly selective detection technologies, along with portable detection devices suitable for on-site, real-time monitoring. Devices that address this need include sensors that provide measurable signals, or even quantify the fundamental functions of physical, biological, or even chemical phenomena, and can be directly used by humans. Electrochemical sensors, with their significant technological advantages, hold great promise for application. Their simplicity, high sensitivity, ease of implementation, and low cost make them valuable in areas such as health, environmental, and food quality control. Electrochemical sensors primarily utilize four electrochemical techniques: potentiometry, voltammetry, amperometry, and impedance spectroscopy. Biorecognition elements (such as antibodies, enzymes, microorganisms, and DNA) can significantly enhance the sensitivity and specificity of chemical sensors, complementing the shortcomings of traditional chemical sensing materials. However, since they are biological molecules, they are extremely sensitive to environmental conditions such as pH, temperature and medium composition, and need to be used under mild conditions to avoid protein denaturation. In addition, the use of these biological components is not so simple. From procurement to immobilization on the electrode surface, professional operating techniques and experience accumulation are required. 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 artificially synthesized recognition materials, MIPs have binding sites that have the characteristics of highly matching the spatial structure and size of the template molecule, thereby achieving specific recognition of the target molecule. 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 depositing polyferulic acid;

[0014] Step (4): re-depositing gold nanoparticles;

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

[0016] Step (6): continue to deposit molecularly imprinted polymer films of polypyrrole and polymethylene blue;

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

[0018] The above operations were all performed on the 0.1 mm to 10 mm portion of the acupuncture needle microelectrode close to the tip.

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

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

[0021] Preferably, the specific operation method of depositing the composite of carbon nanotubes and poly (3,4-ethylenedioxythiophene) (PEDOT) in step (2) is: electropolymerizing 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 of depositing polyferulic acid in step (3) is: immersing the microelectrode treated in the previous step in a solution containing ferulic acid, and performing cyclic scanning using cyclic voltammetry 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: immersing the microelectrode treated in the previous step in HAuCl4 solution, and performing cyclic scanning by cyclic voltammetry 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: immersing the microelectrode treated in the previous step in a solution containing basic fibroblast growth factor protein and incubating at 4°C to complete the anchoring.

[0025] Preferably, the specific operation method of depositing the molecularly imprinted polymer film of polypyrrole and polymethylene blue in step (6) is: immersing the microelectrode treated in the previous step in a solution containing pyrrole, and cyclically scanning the potential in the range of -0.3 V to +0.8 V using cyclic voltammetry, and then immersing the microelectrode in a solution containing methylene blue, and cyclically scanning the potential in the range of -0.7 V to +0.4 V using cyclic voltammetry.

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

[0027] The second technical solution of the present invention is to provide a molecular imprinted electrochemical ratio sensor prepared according to the preparation method of the molecular imprinted electrochemical ratio sensor.

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

[0029] The molecularly imprinted electrochemical ratiometric sensor provided by the present invention can be used for bFGF detection. A ratiometric sensing interface is prepared by sequentially electropolymerizing FA and MB onto the surface of an ANME. As the bFGF concentration increases, the peak current of pFA decreases, while the peak current of pMB remains relatively stable. The ratio of the pFA to pMB currents (I pFA / I pMB ) as a signal for bFGF detection. Compared with single-signal sensors, the molecularly imprinted electrochemical ratiometric sensor exhibits higher sensitivity, accuracy, and stability. Furthermore, the sensor exhibits excellent anti-interference performance. This simple and efficient molecularly imprinted electrochemical ratiometric sensor developed by the present invention provides an innovative solution for highly sensitive and accurate analyte detection. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0031] Figure 2 The electron transfer mechanism of the molecularly imprinted electrochemical ratio sensor produced by the embodiment of the present invention generates dual signals.

[0032] Figure 3The surface morphology and composition of the microelectrodes prepared in each step of Example 1 are characterized, wherein 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 The results of the electrochemical behavior study of ANME / / pFA in Example 1 are shown, where A is the CV curve of ANME / / pFA at different scan rates, B is the pa and I pc Figure 5 is the relationship between the pH value and the scan rate, C is the CV curve of ANME / / pFA under different pH conditions, and D is the relationship between pH value and peak potential and peak current.

[0034] Figure 5 The step-by-step modification results of ANME in Example 1 and Comparative Example 1 characterized by CVs, wherein A is 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 to bFGF (i) in Example 1 in the presence of 5.0 mM [Fe(CN)6] 3- / 4- A is the CV curve of ANME / / pFA (a), ANME / / pFA / / pPy (b), ANME / / pFA / / pMB (c), after elution (d), and after rebinding to bFGF (e) in 0.1 M PBS solution (pH = 7.0) in Example 1; C is the DPV curve of ANME / / pFA (a), ANME / / pFA / / pPy (b), ANME / / pFA / / pMB (c), after elution (d), and after rebinding to bFGF (e) in 0.1 M PBS solution (pH = 7.0) in Example 1; D is the DPV curve of ANME / / pFA (a), ANME / / pFA / AuNPs / pPy (b), ANME / / pFA / AuNPs / / pMB (c), after elution (d), and after rebinding to bFGF (e) in 0.1 M PBS solution (pH = 7.0).

[0035] Figure 6The figures show the influence of the preparation parameters in the preparation method of Example 1 and the detection parameters of the detection method on the detection results, wherein 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 polymerization FA on the results, C is the influence of the scanning rate of polymerization 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 polymerization Py on the results, F is the influence of the scanning rate of polymerization 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 results of the detection of bFGF by ANME / / pFA / / SMIpMB prepared based on the optimized conditions, where A is the DPV curve of ANME / / pFA / / SMIpMB prepared after incubation with different concentrations of bFGF for 60 min, B is the DPV curve of I pFA / I pMB The linear relationship between the value and the logarithm of bFGF concentration, C is I pFA There was a linear relationship between the values ​​and the logarithmic value of bFGF concentration.

[0037] Figure 8 Evaluation results of the selectivity (A), stability (B), and reproducibility (C) of ANME / / pFA / / SMIpMB prepared in Example 1. DETAILED DESCRIPTION

[0038] Various exemplary embodiments of the present invention are now 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, features, and embodiments of the present invention. It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention.

[0039] It should be pointed out that the matters not described in detail in the present invention are conventional operating means in this field and are not the focus of the present invention.

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

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

[0042] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0043] The preparation process of the molecular imprinting electrochemical ratio sensor and the principle diagram of detecting bFGF protein in the embodiment of the present invention are shown in FIG. Figure 1 .

[0044] The electron transfer mechanism of the molecularly imprinted electrochemical ratio sensor produced by the embodiment of the present invention to generate dual signals is shown in Figure 2 .

[0045] Example 1

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

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

[0048] First, the surface was polished with sandpaper for 3 minutes, then ultrasonically cleaned in ethanol and ultrapure water for 5 minutes respectively, and finally dried naturally in air to complete the surface pretreatment of ANME.

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

[0050] The tip of the ANME with a surface pretreatment of 5 mm was immersed in a 2.5 mM HAuCl4 solution and cyclic voltammetry (CV) was used in the potential range of -1.5 V to +0.5 V at a speed of 25 mV·s -1 The scanning rate was scanned for 5 cycles to obtain AuNPs-modified ANME, which was recorded as ANME / AuNPs;

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

[0052] The tip of ANME / AuNPs was 5 mm in a solution containing 2 mg·mL -1 A stable interface was prepared by electropolymerization in a mixed solution of SWNTs and 0.02 M 3,4-ethylenedioxythiophene (EDOT). The solution was sonicated for 30 minutes before polymerization. The polymerization potential was set at 1.2 V, and the polymerization time was 110 seconds. After polymerization, the modified electrode was thoroughly rinsed with water and dried 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 the ANME / AuNPs / SWNTs (5 mm) was placed in 0.1 M PBS electrolyte containing 0.4 mM FA (pH = 4.5) at 60 mV·s -1 10 cycles of CV scans were performed in the potential range of -0.25 V to +0.75 V at a scan rate to electropolymerize FA (before starting the polymerization, the electropolymerization solution was thoroughly deoxygenated by bubbling nitrogen for 10 min). The resulting microelectrode was designated ANME / AuNPs / SWNTs / pFA (abbreviated as ANME / / pFA).

[0055] (5) Further electrodeposition of gold nanoparticles (AuNPs):

[0056] To anchor the protein template, a layer of AuNPs was deposited. The tip of the ANME / / pFA was immersed in a 2.5 mM HAuCl4 solution for 5 mm. Cyclic voltammetry (CV) was performed in the potential range of -1.5 V to +0.5 V at a speed of 25 mV·s. -1 The scan rate was scanned for 3 cycles, and the obtained microelectrode was named ANME / AuNPs / SWNTs / pFA / AuNPs (abbreviated as ANME / / AuNPs);

[0057] (6) Anchor protein template:

[0058] The tip of the ANME / / AuNPs was immersed 5 mm into the bFGF protein solution (concentration 10 μg mL -1 ), incubated at 4 °C for 10 h, and the resulting microelectrode was named ANME / / AuNPs~bFGF;

[0059] (7) Electrodeposition of molecularly imprinted polymer films:

[0060] The tip of the ANME / / AuNPs~bFGF was immersed in 0.01 M PBS (pH = 7.0) solution containing 0.1 M pyrrole (Py) and the CV method was used to cycle 6 times in the voltage range of -0.3 V to +0.8 V at a scan rate of 100 mV·s. -1 , obtaining ANME / / pFA / / pPy; then, the microelectrode with electroplated polypyrrole was immersed in a 0.1 M PBS (pH = 7.0) solution containing 2.5 mM methylene blue (MB), and the CV method was used to cycle twice in the voltage range of -0.7 V to +0.4 V with a scan rate of 100 mV·s -1 , free Py and MB were washed away with ultrapure water to complete the deposition of molecularly imprinted polymer film, and the resulting microelectrode was designated as ANME / / pFA / / pMB;

[0061] (8) DebFGF protein template:

[0062] The bFGF protein template in ANME / / pFA / / pMB was eluted with a 10% (v / v) acetic acid aqueous solution containing 10 wt.% SDS to obtain the molecularly imprinted electrochemical ratiometric 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 scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS). Figure 3 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.

[0064] Figure 3 Middle A shows that the surface morphology of ANME presents longitudinal scratches, which are caused by polishing. Polishing treatment is beneficial to the subsequent modification of nanomaterials. Figure 3 Middle B shows that AuNPs are evenly distributed on the ANME surface, and the diameter of AuNPs is approximately 60 nm; Figure 3 The characteristic peaks of metallic Au can be clearly seen in F, confirming that AuNPs were successfully modified onto ANME; Figure 3 Figure G is the elemental distribution map of ANME / AuNPs, which reveals the presence of Cr, Mn, Ni, Cl, Mo (from ANME itself) and Au elements. The presence 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. Figure 3 It can be clearly seen from the C that the surface has a clear network structure and a smooth surface, indicating that the carbon nanotubes are successfully bonded to the surface of the microelectrode; Figure 3 In D, we can see the pFA polymerized on the surface of carbon nanotubes; Figure 3 Further polymerization of pPy and pMB can be seen in E.

[0065] Comparative Example 1

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

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

[0068] First, the surface was polished with sandpaper for 3 minutes, then ultrasonically cleaned in ethanol and ultrapure water for 5 minutes respectively, and finally dried naturally in air to complete the surface pretreatment of ANME.

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

[0070] The tip of the ANME with a surface pretreatment of 5 mm was immersed in a 2.5 mM HAuCl4 solution and cyclic voltammetry (CV) was used in the potential range of -1.5 V to +0.5 V at a speed of 25 mV·s -1 The scanning rate was scanned for 5 cycles to obtain AuNPs-modified ANME, which was recorded as ANME / AuNPs;

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

[0072] The tip of ANME / AuNPs was 5 mm in a solution containing 2 mg·mL -1 A stable interface was prepared by electropolymerization in a mixed solution of SWNTs and 0.02 M 3,4-ethylenedioxythiophene (EDOT). The solution was sonicated for 30 minutes before polymerization. The polymerization potential was set at 1.2 V, and the polymerization time was 110 seconds. After polymerization, the modified electrode was thoroughly rinsed with water and dried 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] The tip of the ANME / AuNPs / SWNTs (5 mm) was placed in 0.1 M PBS electrolyte containing 0.4 mM FA (pH = 4.5) at 60 mV·s -1 10 cycles of CV scans were performed in the potential range of -0.25 V to +0.75 V at a scan rate to electropolymerize FA (before starting the polymerization, the electropolymerization solution was thoroughly deoxygenated by bubbling nitrogen for 10 min). The resulting microelectrode was designated 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 deposited. The tip of the ANME / / pFA was immersed in a 2.5 mM HAuCl4 solution for 5 mm. Cyclic voltammetry (CV) was performed in the potential range of -1.5 V to +0.5 V at a speed of 25 mV·s. -1 The scan rate was scanned for 3 cycles, and the obtained microelectrode was named ANME / AuNPs / SWNTs / pFA / AuNPs (abbreviated as ANME / / pFA / AuNPs);

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

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

[0079] (7) DebFGF protein template:

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

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

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

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

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

[0085] The research results can be found in Figure 4 , where A is the CV curve of ANME / / pFA at different scan rates, B is I pa and I pc Figure 5 is the relationship between the pH value and the scan rate, C is the CV curve of ANME / / pFA under different pH conditions, and D is the relationship between pH value and peak potential and peak current.

[0086] pFA exhibits a pair of redox peaks in the potential range of -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 100 mV·s -1 At a scan rate of p ) is about 136 mV ( Figure 4 Middle A). E pa and E pc As the scan rate increases, ΔE increases and decreases, respectively. p As the scan rate increases from 10 to 300 mV·s -1 The peak current ratio (I pa / I pc ) is about 0.93 ~ 1.31, indicating that the electrode reaction is reversible. -1 In the range of , the peak current is proportional to the scan rate, and its linear equation is I pa (μA) =0.9233 v (mV·s -1 ) - 8.6726 (R 2 = 0.9932) and I pc (μA) = -0.9337 v (mV·s -1 ) +10.2572(R 2 = 0.9896). The anode peak current is expressed as I pa , the cathode peak current is expressed as I pcThe results show that the electron transfer process of pFA on the modified electrode surface film is controlled by surface processes and is suitable for use as an electrochemical probe to provide current signals. The number of electrons gained and lost in the electrode reaction can be obtained by the following formula:

[0087]

[0088] In the above formula, Q = nFAΓ. Where Q is the 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 ), in the present invention, n = 1.

[0089] The pH of the solution has a great influence on the electrochemical performance of ANME / / pFA. Figure 4 C 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 It has a good linear relationship with pH value, and the linear equation is as follows. pa (V) = -0.0791pH + 0.8456 (R 2 = 0.9878) and E pc (V) =-0.0473 pH +0.4487 (R 2 = 0.9987)( Figure 4 As the pH increases from 5.0 to 7.0, the peak current of pFA gradually increases, reaches a maximum at pH 7.0, and then decreases significantly between 7.0 and 9.0 ( Figure 4 Therefore, pH 7.0 was selected as the optimal pH value in the experiment.

[0090] CVs were used to further characterize the step-by-step modification results of ANME in Example 1 and Comparative Example 1:

[0091] The modified electrodes were placed in a 5.0 mM [Fe(CN)6] 3- / 4- Cyclic voltammetry (CV) was performed in 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 -1 .

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

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

[0094] Figure 5 A in the figure shows that 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 ANME shows a fairly low peak current. The peak current increases significantly at ANME / AuNPs, which is most likely due to the enhanced heterogeneous electron transfer after AuNPs electrodeposition. The peak current of ANME / AuNPs / SWNTs is significantly increased, 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, which provides a larger electroactive surface area and better opportunity to load functional materials to construct electrochemical microsensors with excellent sensing performance. The electrochemical performance was studied, and the electroactive surface area of ​​different electrodes was 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 couple (= 1), and 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 ferrocyanide (7.6×10 -6 cm 2 ·s -1 ), v is the scan rate (0.1 V·s -1 ).

[0097] The electrochemical active surface area of ​​ANME is calculated to be 0.499×10 -3 cm 2 , while the electrochemically active 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 showed that the electrodeposition of AuNPs and SWNTs significantly increased the electroactive surface area and sites of the electrode compared to the case of ANME. A significant increase in the peak current was observed after removal of the bFGF protein template. This was due to the formation of cavities in the MIP membrane. After the bFGF protein re-binded with the cavities, the peak current decreased again.

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

[0099] Figure 5 Center C shows DPV plots of different microelectrodes in 0.1 M PBS (pH = 7.0). A large oxidation peak at +0.160 V is generated due to the oxidation of pFA. Subsequently, after Py was modified on the electrode to form an imprinted layer, the oxidation peak disappeared due to the poor conductivity of pPy. After electropolymerization of MB, a new signal peak at -0.284 V was generated, corresponding to the oxidation peak of pMB. After the bFGF protein template was eluted, two non-interfering signal peaks were obtained at -0.284 V and +0.160 V, respectively, likely attributable to charge transfer caused by the formation of the imprinted cavity. Upon re-incubation of the prepared electrochemical microsensor in a bFGF protein solution, the bFGF protein in the solution was captured by the imprinted cavity, and the peak current of pFA at +0.160 V decreased again, while the peak current of pMB remained almost unchanged.

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

[0101] The preparation parameters in the preparation method of Example 1 and the detection parameters of the detection method were optimized:

[0102] Optimization results can be found in Figure 6 , where A is the effect of the electrodeposition time of single-walled carbon nanotubes on the results, B is the effect of the number of cycles of polymerization FA on the results, C is the effect of the scan rate of polymerization FA on the results, D is the effect of the incubation time of bFGF on the results, E is the effect of the number of cycles of polymerization Py on the results, F is the effect of the scan rate of polymerization Py on the results, G is the effect of the elution time on the results, H is the effect of the rebinding time of bFGF on the results, and I is the effect of the pH value of the detection solution on the results.

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

[0104] By optimizing the deposition time of SWNTs, a larger surface area can be obtained, thereby obtaining more binding sites. Figure 6 As shown in Figure 5A, a large number of SWNTs increases the conductivity and effective surface area, so the deposition time is selected as 110 s. Figure 6 Figure B shows the relationship between I1 and the number of pFA electropolymerization cycles. When the number of polymerization cycles is 6 to 10, the I1 value gradually increases, and when the polymerization reaches 10 cycles, the I1 value reaches the maximum. When the number of polymerization cycles continues to increase, the I1 value changes very little and remains almost unchanged. This is probably because after 10 cycles, the excessive pFA thickness prevents the transfer of electrons. Since the sensitivity is closely related to the peak current of pFA, the optimal number of polymerization cycles is selected as 10. Figure 6 As shown in C, with the increase of scanning rate, the maximum value of I1 appears at 60 mV·s -1 During the electron polymerization process, low scan rates usually form dense films, while high scan rates usually form rough films, both of which limit the modification quality. Therefore, the optimal scan rate is selected as 60 mV·s -1 The effect of the pH value of the detection solution on the FA current response was studied in the range of pH 5.0, 6.0, 7.0, 8.0, and 9.0. Figure 6 As shown in Figure 1, the I1 value gradually increases as the pH increases from 5.0 to 7.0, and gradually decreases as the pH increases from 7.0 to 9.0. Based on this, the optimal pH value of the test solution is determined to be 7.0.

[0105] This experiment also optimized the incubation time of ANME / / AuNPs in bFGF protein solution. Figure 6 As shown in Figure D. The I2 value reaches its maximum value at 10 h, which may be due to the saturation of the protein in ANME / / AuNPs. In other words, the immobilization of the bFGF protein template through the gold-amino bond interaction reaches saturation after incubation for 10 h. Figure 6 As shown in Figures E and F, the I2 value changes with the increase of the number of cycles and the scan rate of polyPy. The peak current of these parameters was selected and the optimal number of cycles was determined to be 6 and the scan rate was 100 mV·s -1 .like Figure 6As shown in Figure G, the DPV current signal intensity is positively correlated with elution time. This phenomenon indicates that the bFGF protein template is gradually removed from the MIP membrane during elution, resulting in an increase in the number of imprinted cavities and an enhancement of the current response signal. The plot of I² values ​​versus elution time shows a signal plateau between 2 and 3 hours, indicating that the optimal elution time for the FGF protein template is 2 hours. Figure 6 Figure H shows the relationship between ΔI (ΔI = I2-I3) and the rebinding time of bFGF protein. The ΔI value reaches a maximum at a rebinding time of 60 minutes, indicating that the binding of bFGF protein to the imprinted cavity reaches saturation after 60 minutes.

[0106] Based on the optimization of detection conditions, a ratiometric electrochemical sensor for sensitive detection of bFGF was successfully constructed. Figure 7 The results of the detection of bFGF by ANME / / pFA / / SMIpMB are shown in Figure 2, where A is the DPV curve of ANME / / pFA / / SMIpMB prepared after incubation with different concentrations of bFGF for 60 min, B is the DPV curve of I pFA / I pMB The linear relationship between the value and the logarithm of bFGF concentration, C is I pFA There was a linear relationship between the values ​​and the logarithmic value of bFGF concentration.

[0107] Figure 7 Middle B shows, I pFA / I pMB The current ratio and bFGF concentration ranged from 0.001 to 3000 ng·mL -1 There is a good linear relationship within the range. Calibration equation I pFA / I pMB = -0.1074 lgC (ng mL -1 ) + 0.7838 (R 2 = 0.9969), with a limit of detection (LOD) of 0.06 pg mL −1 (S / N = 3). Figure 7 As shown in C, for comparison, we use Figure 7 The single current of pFA in A was used as the output signal to detect bFGF, and its linear range and calibration equation were 1 ~ 3000 ng·mL −1 and I pFA (μA) = -3.8370 lgC (ng·mL -1 ) + 25.6358 (R 2 = 0.9936), LOD was 39.8 pg·mL −1(S / N = 3). Compared with single-signal output, the ratiometric signal exhibits a wider linear range and lower detection limit, demonstrating the superior performance of ratiometric detection in bFGF detection. The results demonstrate that this method can effectively improve the sensitivity of bFGF detection.

[0108] Detection of the selectivity, stability and reproducibility of ANME / / pFA / / SMIpMB prepared in Example 1

[0109] [Fe(CN)6] 3- / 4- CV characterization of ANME / / pFA / / SMIpMB was performed using the probe. CV or differential pulse voltammetry (DPV) was performed 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). DPV was used to record pFA (I pFA ) and pMB (I pMB ) and calculate the peak current generated by oxidation and the current ratio (I pFA / I pMB ΔI is the difference in pFA peak current before and after SMIP adsorbs bFGF. 2+ 、Na + , Ca 2+ 、Cl - 、SO4 2- As potential interfering substances (concentrations of bFGF-10 μg mL -1 10 times of that of the prepared sensor), and the anti-interference ability of the prepared sensor was evaluated.

[0110] The experimental results of ANME / / pFA / / SMIpMB's anti-interference ability are as follows: Figure 8 As shown in Figure A, compared to the control (a), the other groups (b–j) showed little change. This indicates that the sensor has outstanding affinity for bFGF and high anti-interference effectiveness. The excellent selectivity of the obtained sensor is attributed to the high recognition capacity of the imprinted cavity. These data demonstrate that ANME / / pFA / / SMIpMB has high selectivity and affinity for bFGF.

[0111] In general, a ratiometric detection strategy can greatly improve stability and reproducibility. In this case, to verify this, it was compared with a non-ratiometric detection strategy, using the same detection method. Figure 8 After two weeks of storage, I pFA is 87.2% of the initial value, and I pFA / I pMB Retained 96.9% of the initial Figure 8 (B). Figure 8As shown in Figure C, the RSD decreased from 5.5% to 2.1% after the ratiometric strategy was implemented. This result demonstrates that the ratiometric strategy effectively mitigates errors caused by internal and external environmental factors, indicating that the ratiometric strategy is highly effective in improving sensing performance. These findings confirm the commendable stability and reproducibility of the ratiometric detection strategy.

[0112] Actual sample testing

[0113] In order to test the practical applicability of the proposed sensor, spiked samples (i.e., blood, 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”. 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 rates of bFGF ranged from 95.80% to 104.70%, indicating that RMIECs have good application potential in detecting the content of bFGF in real samples.

[0117] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing a molecularly imprinted electrochemical ratio sensor, characterized in that: The following steps are involved: Step (1): depositing gold nanoparticles on the surface of the acupuncture needle microelectrode; Step (2): continuing to deposit a composite of carbon nanotubes and poly (3,4-ethylenedioxythiophene); Step (3): further depositing polyferulic acid; Step (4): re-depositing gold nanoparticles; Step (5): anchoring the basic fibroblast growth factor protein template on the gold nanoparticles deposited in step (4); Step (6): continue to deposit molecularly imprinted polymer films of polypyrrole and polymethylene blue; Step (7): finally removing the basic fibroblast growth factor protein template to obtain the molecularly imprinted electrochemical ratio sensor; The operations of steps (1) to (7) are all performed on the 0.1 mm to 10 mm portion of the acupuncture needle microelectrode close to the tip.

2. The method for preparing a molecularly imprinted electrochemical ratio sensor according to claim 1, wherein: The acupuncture needle microelectrode further comprises a surface treatment step before the first deposition of gold nanoparticles.

3. The method for preparing a molecularly imprinted electrochemical ratio sensor according to claim 1, wherein: The specific operation method for depositing gold nanoparticles in step (1) is as follows: immersing the tip of the acupuncture needle microelectrode in HAuCl4 solution, and performing cyclic scanning by cyclic voltammetry in the potential range of -1.5 V to +0.5 V.

4. The method for preparing a molecularly imprinted electrochemical ratio sensor according to claim 1, wherein: The specific operation method of depositing the composite of carbon nanotubes and poly (3,4-ethylenedioxythiophene) in step (2) is as follows: electropolymerizing 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 poly (3,4-ethylenedioxythiophene).

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

6. The method for preparing a molecularly imprinted electrochemical ratio sensor according to claim 1, wherein: The specific operation method for depositing gold nanoparticles in step (4) is as follows: immersing the microelectrode treated in the previous step in HAuCl4 solution, and performing cyclic scanning by cyclic voltammetry in the potential range of -1.5 V to +0.5 V.

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

8. The method for preparing a molecularly imprinted electrochemical ratio sensor according to claim 1, wherein: The specific operation method of depositing the molecularly imprinted polymer film of polypyrrole and polymethylene blue in step (6) is as follows: immersing the microelectrode treated in the previous step in a solution containing pyrrole, and cyclically scanning the potential in the range of -0.3 V to +0.8 V using cyclic voltammetry, and then immersing the microelectrode in a solution containing methylene blue, and cyclically scanning the potential in the range of -0.7 V to +0.4 V using cyclic voltammetry.

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

10. A molecularly imprinted electrochemical ratio sensor prepared according to the method for preparing a molecularly imprinted electrochemical ratio sensor according to any one of claims 1 to 9.

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