Electrochemical enzyme biosensor based on gold nanoparticles/polyaniline composites for highly specific rapidly detection of chlorpyrifos residues in traditional Chinese medicines

Electrochemical enzyme biosensor based on gold nanoparticles/polyaniline composites for highly specific rapidly detection of chlorpyrifos residues in traditional Chinese medicines | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Electrochemical enzyme biosensor based on gold nanoparticles/polyaniline composites for highly specific rapidly detection of chlorpyrifos residues in traditional Chinese medicines Bolu Sun, Ying Lv, Quhuan Ma, Hongxia Shi, Qiaoning Dang, Xinlan Wang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5165698/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Dec, 2024 Read the published version in Journal of Applied Electrochemistry → Version 1 posted 12 You are reading this latest preprint version Abstract Chlorpyrifos (CPF) is an insecticide and acaricide that interrupts nerve conduction by reducing the activity of the enzyme acetylcholinesterase. It can enter the human body through a variety of channels and produce serious physiological effects. However, excessive use of CPF in herbal production produces excessive pesticide residues and reduces the yield of high-quality herbs. Therefore, the development of effective and convenient CPF residue detection technology is critical for pesticide residue monitoring as well as danger avoidance. Based on this, an electrochemical enzyme biosensor using acetylcholinesterase (AChE) as the recognition element was constructed for highly sensitive and rapid detection of chlorpyrifos in traditional Chinese medicine. The electrode substrate modification material was a polyaniline/gold nanoparticles (PANI/AuNPs) composite material prepared by PANI functional modification of AuNPs with high conductivity and large specific surface area, which has excellent electrical conductivity and biocompatibility. Specifically, the PANI/AuNPs offered an exceptional active site for AChE immobilization, greatly enhancing the electrical signals resulting from AChE-catalyzed iodination of acetylthiocholine and accelerating electron transfer. In contrast, AChE's better identification of the target analyte CPF meant that the sensor was highly selective for organophosphorus pesticide residues. Under optimized conditions, the sensor showed good linearity in the range of 1.00 × 10 − 3 ~ 1.00 × 10 1 ppm with a detection limit of 7.90 × 10 − 5 ppm. During practical sample testing, the sensor exhibited remarkable stability, reproducibility, and sensitivity in detecting CPF pesticide residues in Chinese herbal medicines. This offers a reliable tool for precise monitoring and propels the progress of enzyme-based biosensors, both in fostering the cultivation and production of superior-quality herbal medicines, as well as in enabling rapid on-site pesticide residue detection. Chinese herbal medicine pesticide residues chlorpyrifos enzyme biosensor trace fast detection Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Chinese medications (CM) are natural preventives and treatments crafted based on traditional Chinese medicine (TCM) principles. TCM, known for its natural ingredients and therapeutic efficacy with minimal side effects, has surged in popularity globally, boosting China's industry growth and exports. According to the World Health Organization (WHO), TCM is used by 80% of the world's population [ 1 ]. The scarcity of wild Chinese herbal medicines (CHM), due to rising demand, has led to cultivation becoming crucial for meeting market needs. However, pesticides are often used by herbalists as chemicals to regulate plant growth and protect against pests and diseases. These pesticides can not only increase the yield of Chinese herbal medicines, but also cause pesticide residues in them, affecting the quality of Chinese herbal medicines, turning them into "poisoned medicine", endangering human health, and seriously affecting the export of Chinese herbal medicines and the international reputation of China [ 2 ]. Organophosphorus pesticides (OPs) are effective in controlling pests, diseases, and weeds in Chinese herbal medicines due to their potent insecticidal, fungicidal, and herbicidal properties. Their widespread utilization has contributed positively to ensuring the yield and quality of Chinese herbal medicines [ 3 , 4 ]. CPF (C 9 H 11 Cl 3 NO 3 PS) is a widely used organophosphorus insecticide [ 5 , 6 ]. It has the effect of disrupting normal nerve impulse conduction by combating AChE or cholinesterase ChE activity in vivo. CPF's low water solubility and high adsorption coefficient allow it to remain on the surface of Chinese herbal medicines for an extended period of time [ 7 – 9 ], and then enter the human body through the gastrointestinal tract, skin, and respiratory tract, leading neurological deficiencies, developmental and autoimmune diseases [ 10 ], posing a serious threat to human health. Consequently, ensuring the safety of Chinese herbal medicines and their global market growth hinges on an accurate and efficient method to detect CPF residues, which is vital for both public health and the industry's sustainability. Currently, the traditional methods for the detection of chlorpyrifos include gas chromatography (GC) [ 11 ], gas chromatography-mass spectrometry (GC-MS) [ 12 ], liquid chromatography (HPLC) [ 13 ], liquid chromatography-mass spectrometry (LC-MS) [ 14 ], and so on, which have highly sensitive and accurate, yet demanding in time, labor, and user expertise. In addition, the conventional analytical approach is cumbersome and prone to sample decomposition, unsuitable for rapid field analysis. There's an urgent demand for a simplified, rapid, portable detection technology to efficiently screen large sample sizes for organophosphorus pesticide residues in agricultural products before confirming positives according to national standards. As an enzyme-based biosensor, electrochemical enzyme biosensors have the advantages of small detection equipment, low cost, short time to result, ease of operation, and high accuracy, making them promising in the fields of clinical diagnosis, environmental monitoring, and food safety [ 15 , 16 ]. Enzymes' unique 3D nanostructure enables biorecognition and signal amplification, enhancing biosensor performance through their high catalytic specificity and activity. Recent reports highlight enzyme-based biosensors that detect OPs residues by inhibiting AChE, reducing electrically active substances from AChE-catalyzed substrates [ 17 , 18 ]. Researchers can quantitatively analyze trace OP pesticide residues by assessing the electrochemical sensor's response signal strength to electroactive chemicals. Bo Wang et al [ 19 ]. developed a graphene and transition metal carbide-modified acetylcholinesterase biosensor for the detection of organophosphorus pesticides. The combined modified electrode of graphene and transition metal carbide demonstrated excellent electrical properties, biocompatibility, and high specific surface area, resulting in highly sensitive detection of the organophosphorus pesticide dichlorvos with a linear range of 1.31 × 10 − 2 mmol L − 1 to 2.26 × 10 − 5 mmol L − 1 and a limit of detection of 1.45 × 10 − 8 mmol L − 1 . The biosensor showed promise for real-sample detection with its stability, sensitivity, and resistance to interference, which are vital for monitoring pesticide residues. However, the immobilization of bioactive enzymes, being a crucial step in enzyme sensor preparation, faces significant challenges due to the inherent fragility of enzymes. Enzymes are sensitive to denaturation from high temperatures, solvents, and inhibitors, complicating biosensor industrialization. Enzyme immobilization technology is key to enhancing enzyme sensor performance [ 20 ]. Therefore, the enzyme immobilization step is crucial for maintaining biocatalytic activity and substrate selectivity; it must not interfere with the rapid transfer of electrons in order to ensure a sensitive sensor response. Based on this, researchers have optimized conventional electrodes with nanomaterials to maintain enzyme bioactivity and facilitate direct electron transfer [ 21 , 22 ]. AuNPs, renowned for their vast specific surface area and high surface energy, efficiently augment electrode active sites, enhancing their interaction with electrolytes. Their quick and efficient synthesis, uniform size, biocompatibility, and easy bioconjugation make them ideal host nanostructures for enzymes [ 23 ]. It has also been discovered that metal particles, particularly AuNPs, greatly facilitate direct electron transport to oxidoreductase. B. Perk et al [ 24 ], investigation confirmed that AuNP-modified electrodes increased surface area and enhanced HA electrochemical signals by facilitating direct electron transfer between Hb and the electrodes. In addition, when AuNPs are combined with PANI, they may significantly increase the sensitivity and selectivity of electrochemical sensors sensitivity and selectivity. For example, PANI, a conductive polymer renowned for its superior conductivity and environmental stability, is ideal for enhancing electrode conductivity. Its surface modification facilitates ion exchange, enabling the detection of potential changes in solutions [ 25 – 27 ]. Combining AuNPs may significantly increase the sensitivity and selectivity of electrochemical sensors. Moreover, this composite material, which is easy to fabricate, not only features low resistance and rapid response, but it is also cost-effective, displaying remarkable electrical conductivity, environmental stability, and biocompatibility. It enhances sensor sensitivity by amplifying electrochemical signals on the electrode surface, widely used in modified electrode applications. For example, in a study by V. Gupta et al [ 28 ], PANI-doped flexible electrochemical sensors enable swift and sensitive bisphenol A detection, maintaining performance for 20.00 weeks with superior flexibility, highlighting PANI's potential for sensor enhancement. Using PANI to modify electrode substrates notably enhances electrochemical sensor response, offering significant performance benefits. Based on this, PANI/AuNPs composites were created by modifying PANI with AuNPs, which are recognized for their excellent conductivity and surface area. These composites have great electrical conductivity and biocompatibility, making them ideal for electrode substrate modification materials. An electrochemical enzyme biosensor, utilizing AChE, was developed for the rapid and sensitive detection of CPF in TCM. Firstly, PANI/AuNPs/GCE electrodes were prepared by cyclic voltammetry (CV), involving sequential deposition of AuNPs and aniline on glassy carbon substrates. The Au-NH 2 can form a gold-ammonia bond with the amino group on AChE, effectively anchoring it to the electrode. Since, AChE catalyzes the generation of thiocholine from acetylthiocholine iodide (ATCHI), whereas PANI/AuNPs catalyze the redox reaction of thiocholine to produce electrochemical signals. Chlorpyrifos inhibits AChE activity, blocking thiocholine formation from ATCHI, which in turn diminishes the electrochemical response signal, thereby allowing indirect chlorpyrifos detection in samples. On the contrary, PANI/AuNPs' high surface area and conductivity make them ideal for loading AChE and facilitating thiocholine transfer, boosting catalytic efficiency and electron generation. This research project will provide an efficient and convenient detection method for the highly sensitive identification of trace chlorpyrifos pesticide residues in Chinese herbal medicines. It will also serve as a reference and technical support for the development of new, portable, and on-site pesticide residue law enforcement detection techniques. 2. Experimental section 2.1. Materials and reagents Chlorpyrifos (National Standard Material Website www.bzwz.com ), chloroauric acid HAuCl 4 (Tianjin Komio Chemical Reagent Co., Ltd.); aniline (Beijing Enocai Science and Technology Co., Ltd.). CHI 760E electrochemical workstation (Shanghai Chenhua Instrument Co., Ltd.); JSM-6701F cold field emission scanning electron microscope (Japan Electro-Optics Corporation). ( Please see the supplementary material for details ). 2.2. Preparation of AChE/PANI/AuNPs/GCE 2.2.1 Preparation of AuNPs/GCE modified electrodes Before modification, the glassy carbon electrode (GCE) was polished using 0.30 µm and 0.05 µm alumina powders on smooth chamois leather. It was then ultrasonically cleaned with methanol and distilled water. The glassy carbon electrode was placed in a 5.00 × 10 2 mmol L − 1 H 2 SO 4 solution containing 5.00 mmol L − 1 HAuCl 4 , and the AuNPs were deposited by scanning 15.00 cycles with the CV method at a rate of 50.00 mV s − 1 in the potential range of − 0.40 ~ 0.80 V. The AuNPs/GCE modified electrode was obtained. 2.2.2 Preparation of PANI/AuNPs/GCE modified electrodes The AuNPs/GCE-modifid electrode was immersed in a solution containing 1.00 mol L − 1 hydrochloric acid and 0.10 mol L − 1 aniline, and electrochemical deposition was achieved by scanning 30.00 cycles using the CV method at a rate of 50.00 mV s − 1 in the potential range of − 0.40 ~ 0.80 V. The aniline was deposited on the AuNPs/GCE-modified electrode, and the PANI/AuNPs/GCE-modified electrode was formed. 2.2.3 Preparation of AChE/PANI/AuNPs/GCE modified electrodes Apply 5.00 µL of AChE (10.00 U mL − 1 ) solution to the surface of the AChE/PANI/AuNPs/GCE electrode. The acetylcholinesterase sensor (AChE/PANI/AuNPs/GCE) was dried at ambient temperature and then refrigerated at 4.00 ℃. 2.3. Sample standard preparation An appropriate quantity of CPF was weighed and dissolved in anhydrous methanol to produce an anhydrous methanol solution of CPF. To conduct quantitative measurement, a volume of CPF solution was mixed with PBS and CPF standards of 1.00 × 10 1 , 0.50 × 10 1 , 0.10 × 10 1 , 5.00 × 10 − 2 , 1.00 × 10 − 2 , 5.00 × 10 − 3 , 1.00 × 10 − 3 , 5.00 × 10 − 3 , and 1.00 × 10 − 3 ppm were obtained. 2.4. Electrochemical measurements and enzymatic inhibition of reaction processes 2.4.1 Electrochemical characterization of basic electrodes In this work, CV and EIS methods were used to characterize the electrochemical behaviors of GCE, AuNPs/GCE, and PANI/AuNPs/GCE. The electrolyte was a 4.00 mmol L − 1 solution of K 3 [Fe(CN) 6 ]/K 4 [Fe(CN) 6 ] with 10.00 mmol L − 1 KCl. The following measurement conditions were employed to characterize the basic electrodes using the CV method: The sweep rate was 50.00 mV s − 1 and the voltage range was − 0.40 V to 0.80 V. The EIS method's test setup covered a frequency range of 1 Hz to 106 Hz, with sinusoidal waveform signals with an amplitude of 5 mV at a voltage of 0.22 V. 2.4.2 Cyclic voltammetric characterization of electrochemical enzyme biosensors The AChE/PANI/AuNPs/GCE electrochemical enzyme biosensors were evaluated using the CV technique in a buffer solution of 10.00 mL of PBS (pH = 7.60) containing 1.00 mmol L − 1 ATCHI at a sweep rate of 50 mV s − 1 over the voltage range of − 0.40 V to 0.80 V. The CV technique was used on the electrochemical enzymes of the AChE/PANI/AuNPs/GCE biosensors at voltages ranging from − 0.40 V to 0.80 V. The CV method is based on the application of the CV approach. 2.4.3Measurement process of CPF AChE/PANI/AuNPs/GCE modified electrodes had been produced using the drop coating and electrodeposition procedure. Using differential pulse voltammetry (DPV) [ 29 ] in PBS (pH = 7.60) buffer containing 1.00 mmol L − 1 ATCHI, the electrochemical response of AChE/PANI/AuNPs/GCE modified electrodes was examined, and the initial current I 0 was observed. The modified electrode was removed, rinsed with PBS (pH = 7.60) buffer, and then immersed in different concentrations of CPF standards for 10 min of inhibition, followed by a DPV scan to measure the currentin. The inhibition rate was calculated according to inhibition rate = (I 0 - In) / I 0 × 100%. The DPV was measured using the following conditions: voltage range of 0 to 0.416; potential increment of 0.004 V; amplitude of 0.05 V; pulse width of 0.05 s; sampling width of 0.02 s; pulse period of 0.50 s; and resting duration of 2.00 s. Finally, mathematical modeling revealed a linear functional link between inhibition rate and electrochemical response value. 3. Results and discussion 3.1Characterization of modified materials The morphology and microstructure of AuNPs and PANI/AuNPs were characterized using scanning electron microscopy (SEM) [ 30 ]. As shown in Fig. 1 a, the synthesized AuNPs exhibit a uniform spherical morphology, smooth surfaces, and consistent particle sizes, exhibiting excellent dispersion, enabling them to serve as host nanostructures for enzyme molecules, and enhance electron transfer in oxidoreductase; Fig. 1 b shows that the overall PANI/AuNPs surface is relatively smooth, and the AuNPs surface follows the uniformly stacked PANI particles, forming a uniformly dense and flat PANI layer. The interface between the PANI and the AuNPs is very clear, indicating that the two are tightly bonded. This tight bonding helps to realize efficient electron transport between PANI and AuNPs, which improves the electrical conductivity and electrochemical properties of the composites. This indicates that PANI was successfully deposited on top of AuNPs by electrodeposition. The signals of gold (Au) elements are clearly apparent in the spectra, showing that AuNPs were effectively loaded onto the surface of GCE, as shown in Fig. 1 c. AuNPs are distributed very homogeneously, with no evident agglomeration phenomena, which contributes to their high catalytic activity and stability. In addition, the higher signal intensities of carbon (C) and nitrogen (N) components are caused by the fact that PANI is mostly made of these two elements, implying that PANI creates a continuous and uniform covering on the surface of AuNPs. The nitrogen signal, on the other hand, follows closely behind the carbon signal, indicating the existence of PANI. The distribution of elements such as C, N, and Au can be observed clearly in Fig. 1 (d-g) . The C and N elements are mostly obtained from PANI, and the graphic shows a distribution pattern that matches the PANI structure. In contrast, the Au element is mostly concentrated in the presence of nanoparticles, and its distribution density is directly related to AuNP loading. The mapped images confirmed the uniform distribution of each element in the PANI/AuNPs composites. Saberi et al. [ 31 ] prepared an amplified electrochemical DNA sensor based on a PANI membrane and AuNPs for sensitive detection. The sensors were also characterized by scanning electron microscopy (SEM), cyclic voltammetry and impedance testing, which demonstrated the successful preparation of AuNPs and PANI composites. The chemical composition of AuNPs/PANI nanocomposites was confirmed by energy dispersive X-ray spectroscopy (EDX). The effective synthesis of PANI/AuNPs composites was verified by the gold (Au), nitrogen (N), carbon (C), and oxygen (O) peaks in Fig. 1 c, which correspond to the composition of AuNPs and PANI. Table 1 shows the composition of AuNPs and PANI composites. Table I EDX compositional analysis of AuNPs and PANI composites elemental wt% wt%Sigma Atomic percentage C 61.39 2.11 90.39 N 1.71 2.94 2.16 O 4.08 0.64 4.51 Au 32.83 1.36 2.95 overall amount: 100.00 100.00 3.2. Electrochemical characterization of basic electrodes The characterization of the preparation process of electrochemical sensors was evaluated using CV and EIS techniques (Fig. 2 A and 2 B), and Fig. 2 A reveals that the current responsiveness of the AuNPs (curve b) is enhanced and the peak current is raised as compared to the naked GCE (curve a); when PANI was further deposited, the peak currents of AuNPs/GCE (curve b) and PANI/AuNPs/GCE (curve c) increased, indicating that the high specific surface area and affinity of PANI and AuNPs were combined with the former's high electrical conductivity performance. EIS was employed to describe the progressive alteration of PANI/AuNPs/GCE composites, further demonstrating their electrical conductivity. The Nyquist plot in Fig. 2 B shows regular semicircles in the high-frequency region with a diameter equal to the electron transfer resistance (Ret), which effectively reflects the conductivity of the base electrode-electrolyte interface. As shown in Fig. 2 B, comparing the size of the semicircle diameters of the Nyquist plots of GCE (curve a), AuNPs/GCE (curve b), and PANI/AuNPs/GCE (curve c), the results showed that the semicircle diameters of PANI/AuNPs/GCE (curve c) were the smallest, indicating that the modification of GCE by PANI/AuNPs dramatically reduced the Ret and effectively promoted rapid electron transfer on the electrode surface. The results are congruent with those obtained by CV measures. Figure 2 C shows the CV curves of composite electrodes at different scan speeds (10.00 mV s − 1 ~ 200.00 mV s − 1 ). As the scan rate increases, so does the peak current of the modified electrode, and the peak point location shifts gradually, implying that the adsorption affects the redox reaction on the constructed electrochemical sensors and tends to a linear equation. Figure 2 D indicates that the logarithm of the redox peak current has a strong linear connection with the logarithm of the scan rate. This provides more evidence that the electrode is responsive to adsorption-diffusion regulation. The size of the effective specific surface area of the sensor is directly related to the strength of its electrochemical response signal. In this work, the electrochemically effective surface areas of GCE (curve a), AuNPs/GCE (curve b), and PANI/AuNPs/GCE (curve c) electrodes at the optimal degree of modification were evaluated and compared using the chronocoulometric technique in a 5.00 mmol L − 1 K 3 [Fe(CN) 6 ]/K 4 [Fe(CN) 6 ] solution (including 1.00 mmol L − 1 KCl). The relevant experimental settings were set: beginning potential = 0 V, termination potential = 0.30 V, number of steps = 1.00, pulse width = 0.25 s, settling time = 2.00 s, and sampling interval = 2.50 × 10 − 4 s. The corresponding Q-t 1/2 curve is shown in Fig. 2 F, according to the Anson equation [ 32 ]: $$\:Q=\frac{2nFAc\left(Dt\right)1/2}{\varPi\:1/2}+Qdl+Qads$$ Where A is the electrode's surface area, c is the substance concentration, F is the Faraday electrolysis constant, D is the diffusion coefficient, Qdl is the double layer charge, Qads is the Faraday charge, and n is the number of electron transfers. The A-values of GCE (curve a), AuNPs/GCE (curve b), and PANI/AuNPs/GCE (curve c) were 0.28 cm − 1 , 0.79 cm − 1 , and 1.26 cm − 1 , respectively, and the results showed that, compared with other materials, the surface area of the modified PANI/AuNPs/GCE electrode was clearly increased and the electrochemical response signal is clearly enhanced. 3.3 Electrochemical characterization of CPF AChE catalyzes the generation of redox-active thiocholine from the substrate ATCHI, and electrode surface-modified PANI/AuNPs catalyze the thiocholine's redox reaction, generating a detectable electrochemical signal. If chlorpyrifos is present in the system to be tested, the activity of AChE is inhibited, resulting in the inability of acetylthiocholine iodide to generate thiocholine, which eventually leads to a weakening or even disappearance of the electrochemical response signal, and this principle can be used to indirectly detect organophosphorus pesticide residues-chlorpyrifos in the samples. Figure 3 G shows the DPV plots of AChE/PANI/AuNPs/GCE modified electrodes in PBS (pH = 7.60) buffer containing 1.00 mmol L − 1 ATCHI before and after chlorpyrifos inhibition. Curve a shows the DPV plot of the AChE/PANI/AuNPs/GCE modified electrode in 1.00 mmol L − 1 ATCHI in PBS (pH = 7.60) buffer before inhibition, and curve b shows the DPV plot of the sensor in 1.00 mmol L − 1 ATCHI in PBS (pH = 7.60) buffer after inhibition by chlorpyrifos. The figures show that the sensor's response current was drastically lowered following inhibition with chlorpyrifos. 3.4 Optimization of experimental conditions 3.4.1 Effect of the number of polymerization cycles of AuNPs on modified sensors As shown in Fig. 3 A, the peak current of AuNPs/GCE reached its maximum after AuNPs were deposited for 15.00 cycles, showing that AuNPs' high specific surface area and affinity boosted sensor sensitivity and signal response intensity. And it served as the modification quantity for further studies. 3.4.2 Effect of the number of PANI polymerization cycles on modified sensors As shown in Fig. 3 B, increasing the number of aniline polymerization cycles improved the current response and gradually increased the peak current; when the number of aniline polymerization cycles was 30.00 cycles, the peak current reached its maximum, and as the number of polymerization cycles increased, the peak current gradually decreased, indicating that 30.00 cycles was the optimal number of cycles for aniline polymerization. 3.4.3 Effect of solution ambient pH on sensor performance The pH of the test substrate, phosphate buffer solution (PBS), affects the activity of AChE, which influences the response current produced by the enzyme sensor catalyzing ATCHI. The catalytic activity of AChE was higher in the pH = 7.00 ~ 8.00 range. As shown in Fig. 3 C, peak current rose as the pH climbed from 7.00 to 7.60, but the rise decreased when the pH went to 7.80 vs. 8.00, indicating that too high a pH might lead to CPF hydrolysis. Finally, a suitable pH = 7.60 PBS buffer was selected as the test substrate, and the electrochemical sensor performed best at pH = 7.60. 3.4.4 Effect of substrate (ATCHI) concentration on the sensor A change in substrate concentration has a significant effect on the rate of the enzyme reaction. AChE/PANI/AuNPs/GCE-modified electrodes were used to measure the concentration of iodinated acetylthiocholine in 10.00 mmol L − 1 pH 7.06 PBS buffer at a constant potential of + 0.20 V. Figure 3 D shows that when the quantity of iodinated acetylthiocholine rose from 0.25 to 0.75 mmol L − 1 , the catalytic current increased quickly. The catalytic current remained steady when the content of chlorinated acetylthiocholine fluctuated between 0.75 and 1.25 mmol L − 1 . Acetylthiocholine's catalytic current rose fast as the substrate concentration increased. Therefore, 1.00 mmol L − 1 of iodinated acetylthiocholine was chosen as the optimal substrate concentration. 3.4.5 Effect of AChE modification amount on sensor performance Drops of material of 4.00 µL, 5.00 µL, 6.00 µL, 7.00 µL, and 8.00 µL of PBS (pH = 7.60) containing 10.00 U mL -1 AChE were applied to the surface of PANI/AuNPs/GCE-modified electrodes and dried. The modified electrodes were examined by the DPV method, and the results are shown in Fig. 3 E. AChE modification at 5.00 µL had the highest reaction value. As the quantity of modification increased, the enzyme layer thickened, preventing the diffusion of enzymatic reaction products to the electrode surface, limiting sensor sensitivity, and eventually decreasing the response value. Therefore, the optimal amount of AChE modification was 5.00 µL and was used as the modification amount for subsequent experiments. 3.5. Performance of electrochemical enzyme biosensors The CPF pesticide residue standard solutions at various concentrations (1.00 × 10 − 3 ~ 1.00 × 10 1 ppm) were determined under optimum experimental circumstances utilizing the. DPV method. Figure 4 A shows the connection between the DPV response values obtained when the electrochemical enzyme biosensor was tested for various CPF standard concentrations. The calibration curve for CPF concentration and inhibition rate is shown in Fig. 4 B. The logarithm of CPF concentration shows a good linear relationship with its corresponding enzyme inhibition rate in the CPF concentration range of 1.00 × 10 − 3 ~ 1.00 × 10 1 ppm, with the linear equation Y = 3.7708 LgC CPF + 37.133 (R 2 = 0.9948) and the detection limit of 7.90 × 10 − 5 ppm (S/N = 3). Compared to previous approaches reported in the literature, this method produced superior results and had high selectivity for determining CPF content. Table II Comparison of different analytical methods for CPF detection Detection methods Detection limit Linear range References Electropolymerization photoelectric chemical sensor 3.60× 10 2 ppm 1.00 ~ 218.92 ppm [ 33 ] Microfluidic electrochemical sensor 2.00× 10 − 1 ppm 1.00 ~ 1.50×10 2 ppm [ 34 ] MC 1.20 × 10 5 ppm 8.00 × 10 1 ~ 1.50×10 1 ppm [ 35 ] HPLC 2.50 × 10 6 ppm 0.050 ~ 5.00 ppm [ 36 ] ELISA 6.00× 10 2 ppm 2.00 ~ 1.00 × 10 3 ppm [ 37 ] Electrochemical enzyme biosensor 7.90 × 10 − 5 ppm 1.00 × 10 − 3 ~ 1.00 × 10 1 ppm This work To further demonstrate the good performance of the sensor, we compared the performance of the AChE/PANI/AuNPs/GCE electrochemical enzyme biosensor with the reported CPF assays ( Table II ), and the method constructed in this study showed superior analytical performance with a wide linear range and low detection limit, which was superior to the other reported methods. 3.6 Selectivity, Repeatability, and Stability The specificity of the electrochemical enzyme sensor was evaluated by subjecting it to various compositional interfering terms that may interfere with the detection of CPF pesticide residues, such as glycine, glyphosate, soluble starch, urea, and ascorbic acid, and comparing the signal changes of these interferences to the presence of CPF pesticide residue. Figure 5 A shows that the incubated interference term group had essentially no change in DPV response signals to the sensors, with an RSD of less than 2.85% when compared to the blank control group. It indicates that AChE/PANI/AuNPs/GCE, based on the principle of enzyme inhibition, has great specificity for CPF pesticide residue detection. We constructed five modified electrodes in parallel and tested solutions containing CPF (1.00 × 10 − 1 ppm) using the DPV technique. As depicted in Fig. 5 C, the electrochemical platform constructed with AChE/PANI/AuNPs composites exhibits remarkable reproducibility, as evidenced by an average relative standard deviation (RSD) of merely 1.73% across five parallelly fabricated electrodes. This figure underscores the consistency and reliability of the platform for electrochemical applications.The electrodes were sealed and stored at room temperature (25.00℃) for 5, 10, 15, 20 and 25 days. As depicted in Fig. 5 B, the electrochemical sensor demonstrated remarkable stability, maintaining a detection rate ranging from 88.37–97.84% of its initial performance when tested in a solution containing CPF at a concentration of 1.00 × 10 − 1 ppm, as determined by the differential DPV method. 3.7. Analysis of actual samples In order to evaluate the viability of the sensing platform created based on AChE/PANI/AuNPs composites for the sensitive detection of CPF in actual samples, in this investigation, 10.00 mL of the actual sample solution was chosen for a10.00 minute incubation, crushed astragalus was placed in 100.00 mL volumetric flasks, and PBS solution with a pH of 7.60 was added to the scale. This solution was divided into three 10.00 mL aliquots, and a methanol solution of CPF was added at concentrations of 25.00, 50.00, and 250.00 ppm. The DPV test was carried out with a combination of PBS (pH = 7.60) and 1.00 mmol L − 1 ATCHI as the supporting electrolyte, and the recoveries achieved in the three analyses utilizing AChE/PANI/AuNPs/GCE varied from 96.68–102.32%, with relative standard deviations of 1.97–2.41% ( Table III ). This indicates that the sensor is sensitive and convenient to use for instantaneous, fast detection of CPF in real samples. Table III Electrochemical enzyme biosensor determination of Astragalus samples SaCPFle Added (ppm) Found (ppm) a Recovery (%) RSD (%) 1 25.00 24.17 96.68% 1.97 2 50.00 51.16 102.32% 2.15 3 250.00 248.13 99.25% 2.41 a The average of three measurements 4. Conclusion In this study, PANI/AuNPs composites were prepared from PANI functionally modified with AuNPs, which have excellent conductivity and biocompatibility, to be used as electrode substrate modification materials, and an electrochemical enzyme biosensor was constructed for the highly sensitive and rapid detection of CPF in TCM, with the use of AChE as the recognition element. Cyclic voltammetry was used to deposit AuNPs and aniline onto the glassy carbon electrode, resulting in a PANI/AuNPs/GCE-modified electrode. Then AChE was immobilized using the gold-ammonia bonding of AuNPs to AChE. AChE catalyzes the production of thiocholine by ATCHI. With PANI's stronger catalytic ability, it can catalyze the redox reaction of thiocholine in a timely and efficient manner while also greatly promoting electron transmission, significantly amplifying the detection signal. Under optimized conditions, the sensor showed good linearity in the concentration range of 1.00 × 10 − 3 ~ 1.00 × 10 1 ppm with a detection limit of 7.90 × 10 − 5 ppm (S/N = 3). Meanwhile, the electrochemical enzyme biosensor has remarkable performance, including high selectivity, sensitivity, a wide linear range, an ultra-low detection limit, and strong anti-interference capability. The findings of this study not only provide an effective method for the highly sensitive identification and detection of trace CPF pesticide residues in Chinese herbal medicines, but they also provide critical technical support for on-site law enforcement and supervision of new and portable organophosphorus pesticide residues in agriculture, as well as ensuring the production guarantee of high-quality agricultural products. Declarations Author Contribution B.Sun, L. Yang put forward the idea of the experiment and gave guidance to the experiment.S. Yu, Q. Ma and H. Shi prepared PANI/AuNPs composites with excellent electroactivity, prepared electrochemical enzyme biosensors and wrote the manuscript. B. Sun,Y. Lv, Q. Ma, H. Shi, Q. Dang, X. Wang, M. Zhou, X. Da, L. Yang, X. Shi helped with the experimental design, analyzed the data, interpreted the results, and revised the manuscript. All authors read and approved the completed manuscript. Acknowledgments The authors acknowledge science and Technology Plan of Gansu Province -Major Science and Technology Project-Social Development Category (No. 23ZDFA013-1); Science and Technology Plan of Gansu Province-Innovation Fund for Technology Based Firms (No. 23CXGA0097); Gansu Province 2024 Drug Safety Supervision Research Project (No. 2024GSMPA070); the Education Science and Technology Innovation Project of Gansu Provincial Department of Education 2025-Young PhD Support Project (2025QB-000); the Postdoctoral Research Workstation Project of Lanzhou Foci Pharmaceutical Co., Ltd.; the fifth batch of Hongliu outstanding young talents support program of Lanzhou University of Technology; the 2020 PhD Research Start-up Fee of Lanzhou University of Technology and the 2024 Lanzhou University of Technology Innovation and Entrepreneurship Training Program for college students (No. DC20240753; DC20240287). Data Availability The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors alone are responsible for the content and writing of this article. References Xiao JJ, Duan JS, Xu X, Li SN, Wang F, Fang QK, Liao M, Cao HQ (2018) Behavior of pesticides and their metabolites in traditional Chinese medicine PANIeoniae Radix Alba during processing and associated health risk. J Pharm Biomed Anal 16120. https://doi:10.1016/j.jpba.2018.08.029 Liu Y, Xiao Y, Zhang Y, Gao X, Wang H, Niu B, Li W (2024) ZnO-rGO-based electrochemical biosensor for the detection of organophosphorus pesticides. Bioelectrochemistry 156:108599. https://doi:10.1016/j.bioelechem.2023.108599 Ghorbani M, Mohammadi P, Keshavarzi M, Hossien Saghi M, Mohammadi M, Shams A, Aghamohammadhasan M (2021) Simultaneous determination of organophosphorus pesticides residues in vegetable, fruit juice, and milk samples with magnetic dispersive micro solid-phase extraction and chromatographic method; recruitment of simplex lattice mixture design for optimization of novel sorbent composites. Anal Chim Acta 1183:338802. https://doi:10.1016/j.aca.2021.338802 Pundir CS, Malik A, Preety (2019) Bio-sensing of organophosphorus pesticides:A review. Biosens Bioelectron. Sep 1; 140:111348. https://doi:10.1016/j.bios.2019.111348 Fu HY, Liu HZ, Yao G, Chen YF, Tan P, Bai J, Dai ZL, Yang Y Z.L. Wu,Chitosan oligosaccharide alleviates and removes the toxicological effects of organophosphorus pesticide chlorpyrifos residues. J Hazard Mater (2023) Mar 15;446:130669. https://doi:10.1016/j.jhazmat.2022.130669 Sradha SA, George L, Varghese PKA (2022) Recent advances in electrochemical and optical sensing of the organophosphate chlorpyrifos: a review. Crit Rev Toxicol 52(6):431–448. https://doi:10.1080/10408444.2022.2122770 Nagabooshanam S, Roy S, Deshmukh S, Wadhwa S, Sulania I, Mathur A, Krishnamurthy S, Bharadwaj LM, Roy SS (2020) Microfluidic Affinity Sensor Based on a Molecularly Imprinted Polymer for Ultrasensitive Detection of Chlorpyrifos. ACS Omega 2;5(49):31765–31773. https://doi:10.1021/acsomega.0c04436 Shi QP, Guo W, Shen QC, Han J, Lei L, Chen LG, Feng CL, Yang LH, Zhou BS (2021) In vitro biolayer interferometry analysis of acetylcholinesterase as a potential target of aryl-organophosphorus flame-retardants. J Hazard Mater 409:124999. https://doi:10.1016/j.jhazmat.2020.124999 Hamadeen HM, Elkhatib EA (2022) Nanostructured modified biochar for effective elimination of chlorpyrifos from wastewater: Enhancement, mechanisms and performance. J Water Process Eng 47:102703. https://doi:10.1016/j.jwpe.2022.102703 Liu HF, Ku CH, Chang SS, Chang CM, Wang IK, Yang HY, Weng CH, Huang WH, Hsu CW, Yen TH (2020) Outcome of patients with chlorpyrifos intoxication. Hum Exp Toxicol 39(10):1291–1300. https://doi:10.1177/0960327120920911 Gaba Y, Vashishat N (2023) Pesticide residue anlysis in excreta of spotted owlet Athene brama and barn owl Tyto alba. Indian J Entomol 205–208. https://doi:10.55446/ije.2022.498 Tay BYP, Wai WH (2021) A gas chromatography–mass spectrometry method for the detection of chlorpyrifos contamination in palm-based fatty acids. 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Bioelectrochemistry 161:108806. https://doi:10.1016/j.bioelechem.2024.108806 Singh AP, Balayan S, Hooda V, Sarin RK (2020) Nidhi. Chauhan, Nano-interface driven electrochemical sensor for pesticides detection based on the acetylcholinesterase enzyme inhibition. Int J Biol Macromol 164:3943–3952. https://doi:10.1016/j.ijbiomac.2020.08.215 Arkhypova V, Soldatkin O, Soldatkin A, Dzyadevych S (2024) Electrochemical biosensors based on enzyme inhibition effect. Chem Rec 24(2):e202300214. https://doi:10.1002/tcr.202300214 Wang B, Li Y, Hu H, Shu W, Yang L, Zhang J Acetylcholinesterase electrochemical biosensors with graphene-transition metal carbides nanocomposites modified for detection of organophosphate pesticides. Plos One (2020) Apr 29;15(4): e0231981. https://doi:10.1371/journal.pone.0231981 Rajagopalan V, Venkataraman S, Rajendran DS, Vinoth Kumar V, Kumar VV, Rangasamy G Acetylcholinesterase biosensors for electrochemical detection of neurotoxic pesticides and acetylcholine neurotransmitterA literature review. Environ Res (2023) Jun 15;227:115724. https://doi:10.1016/j.envres.2023.115724 Gao Y, Yang F, Yu Q, Fan R, Yang M, Rao S, Lan Q, Yang Z, Yang Z (2019) Three-dimensional porous Cu@ Cu 2 O aerogels for direct voltammetric sensing of glucose. Microchim Acta 186(3):192. https://doi:10.1007/s00604-019-3263-6 Nemiwal M, Zhang TC, Kumar D (2022) Enzyme immobilized nanomaterials as electrochemical biosensors for detection of biomolecules. Enzyme Microb Tech 156:110006. https://doi:10.1016/j.enzmictec.2022.110006 Lipińska W, Grochowska K, Siuzdak K (2021) Enzyme immobilization on gold nanoparticles for electrochemical glucose biosensors. Nanomaterials-Basel 11(5):1156. https://doi:10.3390/nano11051156 Perk B, Büyüksünetçi YT, Anık Ü (2023) Gold nanoparticle deposited electrochemical sensor for hyaluronic acid detection. Chem Pap 77(8):4319–4329. https://doi:10.1007/s11696-023-02781-9 Uzunçar S, Meng L, Turner APF, Mak WC (2021) Processable and nanofibrous polyaniline:polystyrene-sulphonate (nano-PANI:PSS) for the fabrication of catalyst-free ammonium sensors and enzyme-coupled urea biosensors. Biosens Bioelectron 171:112725. https://doi:10.1016/j.bios.2020.112725 Chen HH, Xiang YH, Cai RF, Zhang L, Zhang YT, Zhou ND (2021) An ultrasensitive biosensor for dual-specific DNA based on deposition of polyaniline on a self-assembled multi-functional DNA hexahedral-nanostructure. Biosens Bioelectron 179113066. https://doi:10.1016/j.bios.2021.113066 Saravanakumar K, Balakumar V, Govindan K, Jang A, Lee G, Muthuraj V (2020) Polyaniline intercalated with Ag1. 2V 3 O 8 nanorods based electrochemical sensor. J Ind Eng Chem 91:93–101. https://doi:10.1016/j.jiec.2020.07.036 Cheema S, Vashishth L, Kumar (2024) Highly efficient polyaniline based flexible electrochemical sensor for bisphenol a detection. Microchem J 197:109914. https://doi:10.1016/j.microc.2024.109914 Thakur A, Kumar A (2023) Recent trends in nanostructured carbon-based electrochemical sensors for the detection and remediation of persistent toxic substances in real-time analysis. Mater Res Express 10(3):034001. https://doi:10.1088/2053-1591/acbd1a Sapurina I, Bubulinca C, Trchová M, Prokeš J, Stejskal J (2022) Solid manganese dioxide as heterogeneous oxidant of aniline in the preparation of conducting polyaniline or polyaniline/manganese dioxide coCPFosites. Colloid Surf A 638:128298. https://doi:org/10.1016/j.colsurfa.2022.128298 del Campo FJ (2023) Self-powered electrochemical sensors. Curr Opin Electroche 101356. https://doi:10.1016/j.coelec.2023.101356 Kataria N, Garg VK (2018) Green synthesis of Fe 3 O 4 nanoparticles loaded sawdust carbon for cadmium (II) removal from water. Chemosphere 208:818–828. https://doi.org/10.1016/j.chemosphere.2018.06.022 Xu X, Zhou H, Zhang J, Li Y, Yang Y, Fang Y, Wu Z, Cui B, Hu Q (2022) One-Step Electropolymerization of Polythiophene Derivative Film for Photoelectrochemical Detection of Chlorpyrifos. J Electrochem Soc 169(10):106502. https://doi:10.1149/1945-7111/ac8fbe McClain ES, Miller DR, Cliffel DE (2019) Communication—microfluidic electrochemical acetylcholine detection in the presence of chlorpyrifos. J Electrochem Soc 166(16):G178. https://doi:10.1149/2.0711916jes Gao N, Guo X, Zhang K et al (2014) High-Performance Liquid Chromatography and Gas Chromatography—Mass Spectrometry Methods for the Determination of Imidacloprid, Chlorpyrifos, and Bifenthrin Residues in Tea Leaves[J]. Instrum Sci Technol 42(3):267–277. https://doi:10.1080/10739149.2013.862629 Alkan C, Çabuk H (2022) Matrix-induced sugaring‐out liquid‐liquid microextraction coupled with high‐performance liquid chromatography for the determination of organophosphorus pesticides in fruit jams. Sep Sci Plus 5(8):416–423. https://doi:10.1002/sscp.202200039 Chen ZJ, Zhao LY, Zhang ZT, Wu J, Zhang LX, Jing X, Wang XW (2023) Dispersive liquid–liquid microextraction combined with enzyme-linked immunosorbent assay for the analysis of chlorpyrifos in cereal samples. Talanta 265:124802. https://doi:10.1016/j.talanta.2023.124802 Scheme 1 Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Scheme1.png Scheme 1. Schematic diagram of CPF pesticide residue electrochemical sensor platform based on glassy carbon electrode. SupplementaryMaterials.doc Cite Share Download PDF Status: Published Journal Publication published 26 Dec, 2024 Read the published version in Journal of Applied Electrochemistry → Version 1 posted Editorial decision: Revision requested 28 Nov, 2024 Reviews received at journal 20 Nov, 2024 Reviews received at journal 06 Nov, 2024 Reviews received at journal 05 Nov, 2024 Reviewers agreed at journal 05 Nov, 2024 Reviewers agreed at journal 05 Nov, 2024 Reviewers agreed at journal 03 Nov, 2024 Reviewers agreed at journal 03 Nov, 2024 Reviewers invited by journal 03 Nov, 2024 Editor assigned by journal 28 Sep, 2024 Submission checks completed at journal 27 Sep, 2024 First submitted to journal 27 Sep, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5165698","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":383987697,"identity":"f2adf8d6-8413-45e9-8823-ca7f33ab7e92","order_by":0,"name":"Bolu Sun","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAx0lEQVRIiWNgGAWjYHACNoYHBmxyfFA2kVoSCviM2UjU8kEusY1oLfIzco89SDAwS2/jP2PA8KHsMAP/7Ab8Wgxu5KUbJBik5bYxnDFgnHHuMIPEnQMEtEjkmEkkGBzLbWPsMWDmbTsMFEkg5DCwlv/pbMw8Bsx/idHCcAOshS2BjQ2ohZEYLQZn3pgD/cJm2MbDVnCw51w6j8QNQg5rzzF78OEPmzw//+GND36UWcvxzyDkMGRwAIh5SFA/CkbBKBgFowAXAADN6joqKQjzcgAAAABJRU5ErkJggg==","orcid":"","institution":"Lanzhou University of Technology","correspondingAuthor":true,"prefix":"","firstName":"Bolu","middleName":"","lastName":"Sun","suffix":""},{"id":383987698,"identity":"fe3e693b-ceba-47a9-849e-b89633a9e0e7","order_by":1,"name":"Ying Lv","email":"","orcid":"","institution":"Lanzhou University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Lv","suffix":""},{"id":383987699,"identity":"907180b9-3234-4306-9ae4-de0065a9de01","order_by":2,"name":"Quhuan Ma","email":"","orcid":"","institution":"Gansu Academy of Medical Science, Lanzhou 730050, Gansu, China.","correspondingAuthor":false,"prefix":"","firstName":"Quhuan","middleName":"","lastName":"Ma","suffix":""},{"id":383987700,"identity":"405a65af-7dc2-4ba2-9f44-05fb1b22e7cb","order_by":3,"name":"Hongxia Shi","email":"","orcid":"","institution":"Lanzhou CAIQTEST Co., Ltd, Lanzhou 730000, Gansu.","correspondingAuthor":false,"prefix":"","firstName":"Hongxia","middleName":"","lastName":"Shi","suffix":""},{"id":383987701,"identity":"8f05fe81-5408-4ae0-afbc-2c6f85dd185a","order_by":4,"name":"Qiaoning Dang","email":"","orcid":"","institution":"Lanzhou University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Qiaoning","middleName":"","lastName":"Dang","suffix":""},{"id":383987702,"identity":"14278048-5b67-4e78-b7b8-3a1ad28413b0","order_by":5,"name":"Xinlan Wang","email":"","orcid":"","institution":"Lanzhou University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Xinlan","middleName":"","lastName":"Wang","suffix":""},{"id":383987703,"identity":"c71af8bb-c58a-4890-9125-714f1c99bcad","order_by":6,"name":"Miao Zhou","email":"","orcid":"","institution":"Lanzhou University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Miao","middleName":"","lastName":"Zhou","suffix":""},{"id":383987704,"identity":"050734d2-606c-4775-b35d-1e6a05680af3","order_by":7,"name":"Xuanxiu Da","email":"","orcid":"","institution":"Lanzhou University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Xuanxiu","middleName":"","lastName":"Da","suffix":""},{"id":383987705,"identity":"f26c93eb-d0fc-42bd-ae4d-182168967fcd","order_by":8,"name":"Lin Yang","email":"","orcid":"","institution":"Lanzhou University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Lin","middleName":"","lastName":"Yang","suffix":""},{"id":383987706,"identity":"61f30948-0370-486c-950f-0ce55f8ec9f3","order_by":9,"name":"Xiaofeng Shi","email":"","orcid":"","institution":"Gansu Academy of Medical Science, Lanzhou 730050, Gansu, China.","correspondingAuthor":false,"prefix":"","firstName":"Xiaofeng","middleName":"","lastName":"Shi","suffix":""}],"badges":[],"createdAt":"2024-09-27 14:08:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5165698/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5165698/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10800-024-02244-3","type":"published","date":"2024-12-26T15:56:56+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":71017170,"identity":"f69f6d07-518b-4350-984d-a795aa122b80","added_by":"auto","created_at":"2024-12-10 08:52:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":502026,"visible":true,"origin":"","legend":"\u003cp\u003e(a) SEM images of AuNPs, (b) PANI/AuNPs; (c) EDX spectra of PANI/AuNPs; (d-g) corresponding elemental uptake maps of PANI AuNPs\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-5165698/v1/eadd215bd5985dba72fe5195.png"},{"id":71017167,"identity":"66d94930-b4c5-4222-8a86-ffa5768a4622","added_by":"auto","created_at":"2024-12-10 08:52:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":292358,"visible":true,"origin":"","legend":"\u003cp\u003eGCE (a), AuNPs/GCE (b), and PANI/AuNPs/GCE (c) of (A) CV curves and (B) EIS curves of GCE (a), AuNPs/GCE (b), and PANI/AuNPs/GCE (c) in K\u003csub\u003e3\u003c/sub\u003e[Fe(CN)\u003csub\u003e6\u003c/sub\u003e]/K\u003csub\u003e4\u003c/sub\u003e[Fe(CN)\u003csub\u003e6\u003c/sub\u003e] solutions containing 0.01 mmol L\u003csup\u003e-1\u003c/sup\u003e KCl at 4.00 mmol L\u003csup\u003e-1\u003c/sup\u003e; (C) CV curves of PANI/AuNPs/GCE at different scanning rates (10 mV s\u003csup\u003e-1 \u003c/sup\u003e~ 200 mV s\u003csup\u003e-1\u003c/sup\u003e) CV curves of PANI/AuNPs/GCE; (D) logarithm of the peak current versus the logarithm of the scan rate; in the presence of 1.00 × 10\u003csup\u003e2\u003c/sup\u003e mmol L\u003csup\u003e-1\u003c/sup\u003e KCl and 4.00 mmol L\u003csup\u003e-1\u003c/sup\u003e [Fe(CN)\u003csub\u003e6\u003c/sub\u003e]\u003csup\u003e3-/4-\u003c/sup\u003eprobe solution (E) Timed coulometric curves of GCE (a), AuNPs/GCE (b), and PANI/AuNPs/GCE (c); Figure F shows the Q-t\u003csup\u003e1/2\u003c/sup\u003e curves of GCE (a), AuNPs/GCE (b), and PANI/AuNPs/GCE (c).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-5165698/v1/3a04d281c778ea8b9a5d6017.png"},{"id":71018141,"identity":"eddb9a0f-ca2e-4f63-9500-2d83c2aeeb51","added_by":"auto","created_at":"2024-12-10 09:00:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":288394,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Effect of the number of polymerization cycles of AuNPs on modified sensors; (B) Effect of the number of PANI polymerization cycles on modified sensors; (C) Effect of solution ambient pH on sensor performance; (D) Effect of substrate (ATCHI) concentration on the sensor; (E) Effect of AChE modification amount on sensor performance; (F) Effect of the CPF inhibition time on the sensor sensitivity; (G) Sensor current response before and after inhibition.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-5165698/v1/d5f97b6c89df168ce40e5865.png"},{"id":71017172,"identity":"01e266e9-635d-45e7-906d-fc3eab81c190","added_by":"auto","created_at":"2024-12-10 08:52:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":110274,"visible":true,"origin":"","legend":"\u003cp\u003e(A) DPV curves of various CPF concentrations on PBS (pH = 7.60) buffer AChE/PANI/AuNPs/GCE containing 1.00 mmol L\u003csup\u003e-1\u003c/sup\u003e ATCHI; (B) linear connection between inhibition rate of the produced sensor and logarithm of CPF concentration.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-5165698/v1/8d723fc95bb02421d9fcc672.png"},{"id":71017171,"identity":"c86e3fb4-e8d7-4480-a878-25dd14400ee0","added_by":"auto","created_at":"2024-12-10 08:52:04","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":170636,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Tests were performed in the presence of different interfering substances, where chlorpyrifos was at a concentration of 1.00 × 10\u003csup\u003e-1\u003c/sup\u003e ppm and each interfering term was at a concentration of 1.00 × 10\u003csup\u003e2 \u003c/sup\u003eppm; (B) The modified electrode was prepared and stored in a sealed container, and the solution containing CPF (1.00 × 10\u003csup\u003e-1\u003c/sup\u003e ppm) solution. The test solution was PBS buffer (pH = 7.60) with 1.00 mmol L\u003csup\u003e-1\u003c/sup\u003e ATCHI; (C) The same electrode was subjected to five DPV tests in the test solution.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-5165698/v1/3e29b42fce7c82fdeeeef0b0.png"},{"id":72640359,"identity":"cf82b9cb-e5a0-4b8f-b016-02ee3e5ab914","added_by":"auto","created_at":"2024-12-30 16:04:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2246811,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5165698/v1/ba58ce2e-84d0-4814-b0e0-951b88023c32.pdf"},{"id":71018271,"identity":"64f1531d-2a8d-4c59-864e-39bfa3a62793","added_by":"auto","created_at":"2024-12-10 09:08:04","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":317121,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1.\u003c/strong\u003e Schematic diagram of CPF pesticide residue electrochemical sensor platform based on glassy carbon electrode.\u003c/p\u003e","description":"","filename":"Scheme1.png","url":"https://assets-eu.researchsquare.com/files/rs-5165698/v1/ceb210e00b11c8788975244a.png"},{"id":71018143,"identity":"38ae5fcd-89bb-4626-955d-f318f028d56b","added_by":"auto","created_at":"2024-12-10 09:00:04","extension":"doc","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1086464,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials.doc","url":"https://assets-eu.researchsquare.com/files/rs-5165698/v1/9110752616279759344ba6c3.doc"}],"financialInterests":"No competing interests reported.","formattedTitle":"Electrochemical enzyme biosensor based on gold nanoparticles/polyaniline composites for highly specific rapidly detection of chlorpyrifos residues in traditional Chinese medicines","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eChinese medications (CM) are natural preventives and treatments crafted based on traditional Chinese medicine (TCM) principles. TCM, known for its natural ingredients and therapeutic efficacy with minimal side effects, has surged in popularity globally, boosting China's industry growth and exports. According to the World Health Organization (WHO), TCM is used by 80% of the world's population [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The scarcity of wild Chinese herbal medicines (CHM), due to rising demand, has led to cultivation becoming crucial for meeting market needs. However, pesticides are often used by herbalists as chemicals to regulate plant growth and protect against pests and diseases. These pesticides can not only increase the yield of Chinese herbal medicines, but also cause pesticide residues in them, affecting the quality of Chinese herbal medicines, turning them into \"poisoned medicine\", endangering human health, and seriously affecting the export of Chinese herbal medicines and the international reputation of China [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOrganophosphorus pesticides (OPs) are effective in controlling pests, diseases, and weeds in Chinese herbal medicines due to their potent insecticidal, fungicidal, and herbicidal properties. Their widespread utilization has contributed positively to ensuring the yield and quality of Chinese herbal medicines [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. CPF (C\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e11\u003c/sub\u003eCl\u003csub\u003e3\u003c/sub\u003eNO\u003csub\u003e3\u003c/sub\u003ePS) is a widely used organophosphorus insecticide [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. It has the effect of disrupting normal nerve impulse conduction by combating AChE or cholinesterase ChE activity in vivo. CPF's low water solubility and high adsorption coefficient allow it to remain on the surface of Chinese herbal medicines for an extended period of time [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], and then enter the human body through the gastrointestinal tract, skin, and respiratory tract, leading neurological deficiencies, developmental and autoimmune diseases [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], posing a serious threat to human health. Consequently, ensuring the safety of Chinese herbal medicines and their global market growth hinges on an accurate and efficient method to detect CPF residues, which is vital for both public health and the industry's sustainability.\u003c/p\u003e \u003cp\u003eCurrently, the traditional methods for the detection of chlorpyrifos include gas chromatography (GC) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], gas chromatography-mass spectrometry (GC-MS) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], liquid chromatography (HPLC) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], liquid chromatography-mass spectrometry (LC-MS) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], and so on, which have highly sensitive and accurate, yet demanding in time, labor, and user expertise. In addition, the conventional analytical approach is cumbersome and prone to sample decomposition, unsuitable for rapid field analysis. There's an urgent demand for a simplified, rapid, portable detection technology to efficiently screen large sample sizes for organophosphorus pesticide residues in agricultural products before confirming positives according to national standards.\u003c/p\u003e \u003cp\u003eAs an enzyme-based biosensor, electrochemical enzyme biosensors have the advantages of small detection equipment, low cost, short time to result, ease of operation, and high accuracy, making them promising in the fields of clinical diagnosis, environmental monitoring, and food safety [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Enzymes' unique 3D nanostructure enables biorecognition and signal amplification, enhancing biosensor performance through their high catalytic specificity and activity. Recent reports highlight enzyme-based biosensors that detect OPs residues by inhibiting AChE, reducing electrically active substances from AChE-catalyzed substrates [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Researchers can quantitatively analyze trace OP pesticide residues by assessing the electrochemical sensor's response signal strength to electroactive chemicals. Bo Wang et al [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. developed a graphene and transition metal carbide-modified acetylcholinesterase biosensor for the detection of organophosphorus pesticides. The combined modified electrode of graphene and transition metal carbide demonstrated excellent electrical properties, biocompatibility, and high specific surface area, resulting in highly sensitive detection of the organophosphorus pesticide dichlorvos with a linear range of 1.31 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e mmol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 2.26 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e mmol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and a limit of detection of 1.45 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e mmol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The biosensor showed promise for real-sample detection with its stability, sensitivity, and resistance to interference, which are vital for monitoring pesticide residues.\u003c/p\u003e \u003cp\u003eHowever, the immobilization of bioactive enzymes, being a crucial step in enzyme sensor preparation, faces significant challenges due to the inherent fragility of enzymes. Enzymes are sensitive to denaturation from high temperatures, solvents, and inhibitors, complicating biosensor industrialization. Enzyme immobilization technology is key to enhancing enzyme sensor performance [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Therefore, the enzyme immobilization step is crucial for maintaining biocatalytic activity and substrate selectivity; it must not interfere with the rapid transfer of electrons in order to ensure a sensitive sensor response. Based on this, researchers have optimized conventional electrodes with nanomaterials to maintain enzyme bioactivity and facilitate direct electron transfer [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. AuNPs, renowned for their vast specific surface area and high surface energy, efficiently augment electrode active sites, enhancing their interaction with electrolytes. Their quick and efficient synthesis, uniform size, biocompatibility, and easy bioconjugation make them ideal host nanostructures for enzymes [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. It has also been discovered that metal particles, particularly AuNPs, greatly facilitate direct electron transport to oxidoreductase. B. Perk et al [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], investigation confirmed that AuNP-modified electrodes increased surface area and enhanced HA electrochemical signals by facilitating direct electron transfer between Hb and the electrodes.\u003c/p\u003e \u003cp\u003eIn addition, when AuNPs are combined with PANI, they may significantly increase the sensitivity and selectivity of electrochemical sensors sensitivity and selectivity. For example, PANI, a conductive polymer renowned for its superior conductivity and environmental stability, is ideal for enhancing electrode conductivity. Its surface modification facilitates ion exchange, enabling the detection of potential changes in solutions [\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Combining AuNPs may significantly increase the sensitivity and selectivity of electrochemical sensors. Moreover, this composite material, which is easy to fabricate, not only features low resistance and rapid response, but it is also cost-effective, displaying remarkable electrical conductivity, environmental stability, and biocompatibility. It enhances sensor sensitivity by amplifying electrochemical signals on the electrode surface, widely used in modified electrode applications. For example, in a study by V. Gupta et al [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], PANI-doped flexible electrochemical sensors enable swift and sensitive bisphenol A detection, maintaining performance for 20.00 weeks with superior flexibility, highlighting PANI's potential for sensor enhancement. Using PANI to modify electrode substrates notably enhances electrochemical sensor response, offering significant performance benefits.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBased on this, PANI/AuNPs composites were created by modifying PANI with AuNPs, which are recognized for their excellent conductivity and surface area. These composites have great electrical conductivity and biocompatibility, making them ideal for electrode substrate modification materials. An electrochemical enzyme biosensor, utilizing AChE, was developed for the rapid and sensitive detection of CPF in TCM. Firstly, PANI/AuNPs/GCE electrodes were prepared by cyclic voltammetry (CV), involving sequential deposition of AuNPs and aniline on glassy carbon substrates. The Au-NH\u003csub\u003e2\u003c/sub\u003e can form a gold-ammonia bond with the amino group on AChE, effectively anchoring it to the electrode. Since, AChE catalyzes the generation of thiocholine from acetylthiocholine iodide (ATCHI), whereas PANI/AuNPs catalyze the redox reaction of thiocholine to produce electrochemical signals. Chlorpyrifos inhibits AChE activity, blocking thiocholine formation from ATCHI, which in turn diminishes the electrochemical response signal, thereby allowing indirect chlorpyrifos detection in samples. On the contrary, PANI/AuNPs' high surface area and conductivity make them ideal for loading AChE and facilitating thiocholine transfer, boosting catalytic efficiency and electron generation. This research project will provide an efficient and convenient detection method for the highly sensitive identification of trace chlorpyrifos pesticide residues in Chinese herbal medicines. It will also serve as a reference and technical support for the development of new, portable, and on-site pesticide residue law enforcement detection techniques.\u003c/p\u003e"},{"header":"2. Experimental section","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials and reagents\u003c/h2\u003e \u003cp\u003eChlorpyrifos (National Standard Material Website \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://www.bzwz.com\" target=\"_blank\"\u003ewww.bzwz.com\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.bzwz.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), chloroauric acid HAuCl\u003csub\u003e4\u003c/sub\u003e (Tianjin Komio Chemical Reagent Co., Ltd.); aniline (Beijing Enocai Science and Technology Co., Ltd.).\u003c/p\u003e \u003cp\u003eCHI 760E electrochemical workstation (Shanghai Chenhua Instrument Co., Ltd.); JSM-6701F cold field emission scanning electron microscope (Japan Electro-Optics Corporation). (\u003cb\u003ePlease see the supplementary material for details\u003c/b\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Preparation of AChE/PANI/AuNPs/GCE\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1 Preparation of AuNPs/GCE modified electrodes\u003c/h2\u003e \u003cp\u003eBefore modification, the glassy carbon electrode (GCE) was polished using 0.30 \u0026micro;m and 0.05 \u0026micro;m alumina powders on smooth chamois leather. It was then ultrasonically cleaned with methanol and distilled water. The glassy carbon electrode was placed in a 5.00 \u0026times; 10\u003csup\u003e2\u003c/sup\u003e mmol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e solution containing 5.00 mmol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e HAuCl\u003csub\u003e4\u003c/sub\u003e, and the AuNPs were deposited by scanning 15.00 cycles with the CV method at a rate of 50.00 mV s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in the potential range of \u0026minus;\u0026thinsp;0.40\u0026thinsp;~\u0026thinsp;0.80 V. The AuNPs/GCE modified electrode was obtained.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2 Preparation of PANI/AuNPs/GCE modified electrodes\u003c/h2\u003e \u003cp\u003eThe AuNPs/GCE-modifid electrode was immersed in a solution containing 1.00 mol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e hydrochloric acid and 0.10 mol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e aniline, and electrochemical deposition was achieved by scanning 30.00 cycles using the CV method at a rate of 50.00 mV s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in the potential range of \u0026minus;\u0026thinsp;0.40\u0026thinsp;~\u0026thinsp;0.80 V. The aniline was deposited on the AuNPs/GCE-modified electrode, and the PANI/AuNPs/GCE-modified electrode was formed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e\u003cb\u003e2.2.3 Preparation of AChE/PANI/AuNPs/GCE modified electrodes\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eApply 5.00 \u0026micro;L of AChE (10.00 U mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) solution to the surface of the AChE/PANI/AuNPs/GCE electrode. The acetylcholinesterase sensor (AChE/PANI/AuNPs/GCE) was dried at ambient temperature and then refrigerated at 4.00 ℃.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Sample standard preparation\u003c/h2\u003e \u003cp\u003eAn appropriate quantity of CPF was weighed and dissolved in anhydrous methanol to produce an anhydrous methanol solution of CPF. To conduct quantitative measurement, a volume of CPF solution was mixed with PBS and CPF standards of 1.00 \u0026times; 10\u003csup\u003e1\u003c/sup\u003e, 0.50 \u0026times; 10\u003csup\u003e1\u003c/sup\u003e, 0.10 \u0026times; 10\u003csup\u003e1\u003c/sup\u003e, 5.00 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, 1.00 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, 5.00 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e, 1.00 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e, 5.00 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e, and 1.00 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e ppm were obtained.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Electrochemical measurements and enzymatic inhibition of reaction processes\u003c/h2\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e\u003cb\u003e2.4.1 Electrochemical characterization of basic electrodes\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eIn this work, CV and EIS methods were used to characterize the electrochemical behaviors of GCE, AuNPs/GCE, and PANI/AuNPs/GCE. The electrolyte was a 4.00 mmol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e solution of K\u003csub\u003e3\u003c/sub\u003e[Fe(CN)\u003csub\u003e6\u003c/sub\u003e]/K\u003csub\u003e4\u003c/sub\u003e[Fe(CN)\u003csub\u003e6\u003c/sub\u003e] with 10.00 mmol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e KCl. The following measurement conditions were employed to characterize the basic electrodes using the CV method: The sweep rate was 50.00 mV s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and the voltage range was \u0026minus;\u0026thinsp;0.40 V to 0.80 V. The EIS method's test setup covered a frequency range of 1 Hz to 106 Hz, with sinusoidal waveform signals with an amplitude of 5 mV at a voltage of 0.22 V.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e\u003cb\u003e2.4.2 Cyclic voltammetric characterization of electrochemical enzyme biosensors\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eThe AChE/PANI/AuNPs/GCE electrochemical enzyme biosensors were evaluated using the CV technique in a buffer solution of 10.00 mL of PBS (pH\u0026thinsp;=\u0026thinsp;7.60) containing 1.00 mmol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e ATCHI at a sweep rate of 50 mV s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e over the voltage range of \u0026minus;\u0026thinsp;0.40 V to 0.80 V. The CV technique was used on the electrochemical enzymes of the AChE/PANI/AuNPs/GCE biosensors at voltages ranging from \u0026minus;\u0026thinsp;0.40 V to 0.80 V. The CV method is based on the application of the CV approach.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.4.3Measurement process of CPF\u003c/h2\u003e \u003cp\u003eAChE/PANI/AuNPs/GCE modified electrodes had been produced using the drop coating and electrodeposition procedure. Using differential pulse voltammetry (DPV) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] in PBS (pH\u0026thinsp;=\u0026thinsp;7.60) buffer containing 1.00 mmol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e ATCHI, the electrochemical response of AChE/PANI/AuNPs/GCE modified electrodes was examined, and the initial current I\u003csub\u003e0\u003c/sub\u003e was observed. The modified electrode was removed, rinsed with PBS (pH\u0026thinsp;=\u0026thinsp;7.60) buffer, and then immersed in different concentrations of CPF standards for 10 min of inhibition, followed by a DPV scan to measure the currentin. The inhibition rate was calculated according to inhibition rate = (I\u003csub\u003e0\u003c/sub\u003e - In) / I\u003csub\u003e0\u003c/sub\u003e \u0026times; 100%. The DPV was measured using the following conditions: voltage range of 0 to 0.416; potential increment of 0.004 V; amplitude of 0.05 V; pulse width of 0.05 s; sampling width of 0.02 s; pulse period of 0.50 s; and resting duration of 2.00 s. Finally, mathematical modeling revealed a linear functional link between inhibition rate and electrochemical response value.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1Characterization of modified materials\u003c/h2\u003e \u003cp\u003eThe morphology and microstructure of AuNPs and PANI/AuNPs were characterized using scanning electron microscopy (SEM) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, the synthesized AuNPs exhibit a uniform spherical morphology, smooth surfaces, and consistent particle sizes, exhibiting excellent dispersion, enabling them to serve as host nanostructures for enzyme molecules, and enhance electron transfer in oxidoreductase; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb shows that the overall PANI/AuNPs surface is relatively smooth, and the AuNPs surface follows the uniformly stacked PANI particles, forming a uniformly dense and flat PANI layer. The interface between the PANI and the AuNPs is very clear, indicating that the two are tightly bonded. This tight bonding helps to realize efficient electron transport between PANI and AuNPs, which improves the electrical conductivity and electrochemical properties of the composites. This indicates that PANI was successfully deposited on top of AuNPs by electrodeposition. The signals of gold (Au) elements are clearly apparent in the spectra, showing that AuNPs were effectively loaded onto the surface of GCE, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec. AuNPs are distributed very homogeneously, with no evident agglomeration phenomena, which contributes to their high catalytic activity and stability.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition, the higher signal intensities of carbon (C) and nitrogen (N) components are caused by the fact that PANI is mostly made of these two elements, implying that PANI creates a continuous and uniform covering on the surface of AuNPs. The nitrogen signal, on the other hand, follows closely behind the carbon signal, indicating the existence of PANI. The distribution of elements such as C, N, and Au can be observed clearly in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e \u003cb\u003e(d-g)\u003c/b\u003e. The C and N elements are mostly obtained from PANI, and the graphic shows a distribution pattern that matches the PANI structure. In contrast, the Au element is mostly concentrated in the presence of nanoparticles, and its distribution density is directly related to AuNP loading. The mapped images confirmed the uniform distribution of each element in the PANI/AuNPs composites. Saberi et al. [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] prepared an amplified electrochemical DNA sensor based on a PANI membrane and AuNPs for sensitive detection. The sensors were also characterized by scanning electron microscopy (SEM), cyclic voltammetry and impedance testing, which demonstrated the successful preparation of AuNPs and PANI composites.\u003c/p\u003e \u003cp\u003eThe chemical composition of AuNPs/PANI nanocomposites was confirmed by energy dispersive X-ray spectroscopy (EDX). The effective synthesis of PANI/AuNPs composites was verified by the gold (Au), nitrogen (N), carbon (C), and oxygen (O) peaks in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, which correspond to the composition of AuNPs and PANI. Table\u0026nbsp;1 shows the composition of AuNPs and PANI composites.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTable I\u003c/b\u003e EDX compositional analysis of AuNPs and PANI composites\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eelemental\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ewt%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ewt%Sigma\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAtomic percentage\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e61.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e90.39\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.51\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e32.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.95\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eoverall amount:\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Electrochemical characterization of basic electrodes\u003c/h2\u003e \u003cp\u003eThe characterization of the preparation process of electrochemical sensors was evaluated using CV and EIS techniques (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA reveals that the current responsiveness of the AuNPs (curve b) is enhanced and the peak current is raised as compared to the naked GCE (curve a); when PANI was further deposited, the peak currents of AuNPs/GCE (curve b) and PANI/AuNPs/GCE (curve c) increased, indicating that the high specific surface area and affinity of PANI and AuNPs were combined with the former's high electrical conductivity performance.\u003c/p\u003e \u003cp\u003eEIS was employed to describe the progressive alteration of PANI/AuNPs/GCE composites, further demonstrating their electrical conductivity. The Nyquist plot in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB shows regular semicircles in the high-frequency region with a diameter equal to the electron transfer resistance (Ret), which effectively reflects the conductivity of the base electrode-electrolyte interface. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, comparing the size of the semicircle diameters of the Nyquist plots of GCE (curve a), AuNPs/GCE (curve b), and PANI/AuNPs/GCE (curve c), the results showed that the semicircle diameters of PANI/AuNPs/GCE (curve c) were the smallest, indicating that the modification of GCE by PANI/AuNPs dramatically reduced the Ret and effectively promoted rapid electron transfer on the electrode surface. The results are congruent with those obtained by CV measures.\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC shows the CV curves of composite electrodes at different scan speeds (10.00 mV s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e ~ 200.00 mV s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). As the scan rate increases, so does the peak current of the modified electrode, and the peak point location shifts gradually, implying that the adsorption affects the redox reaction on the constructed electrochemical sensors and tends to a linear equation. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD indicates that the logarithm of the redox peak current has a strong linear connection with the logarithm of the scan rate. This provides more evidence that the electrode is responsive to adsorption-diffusion regulation.\u003c/p\u003e \u003cp\u003eThe size of the effective specific surface area of the sensor is directly related to the strength of its electrochemical response signal. In this work, the electrochemically effective surface areas of GCE (curve a), AuNPs/GCE (curve b), and PANI/AuNPs/GCE (curve c) electrodes at the optimal degree of modification were evaluated and compared using the chronocoulometric technique in a 5.00 mmol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e K\u003csub\u003e3\u003c/sub\u003e[Fe(CN)\u003csub\u003e6\u003c/sub\u003e]/K\u003csub\u003e4\u003c/sub\u003e[Fe(CN)\u003csub\u003e6\u003c/sub\u003e] solution (including 1.00 mmol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e KCl). The relevant experimental settings were set: beginning potential\u0026thinsp;=\u0026thinsp;0 V, termination potential\u0026thinsp;=\u0026thinsp;0.30 V, number of steps\u0026thinsp;=\u0026thinsp;1.00, pulse width\u0026thinsp;=\u0026thinsp;0.25 s, settling time\u0026thinsp;=\u0026thinsp;2.00 s, and sampling interval\u0026thinsp;=\u0026thinsp;2.50 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e s. The corresponding Q-t\u003csup\u003e1/2\u003c/sup\u003e curve is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, according to the Anson equation [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:Q=\\frac{2nFAc\\left(Dt\\right)1/2}{\\varPi\\:1/2}+Qdl+Qads$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eWhere A is the electrode's surface area, c is the substance concentration, F is the Faraday electrolysis constant, D is the diffusion coefficient, Qdl is the double layer charge, Qads is the Faraday charge, and n is the number of electron transfers. The A-values of GCE (curve a), AuNPs/GCE (curve b), and PANI/AuNPs/GCE (curve c) were 0.28 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 0.79 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and 1.26 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively, and the results showed that, compared with other materials, the surface area of the modified PANI/AuNPs/GCE electrode was clearly increased and the electrochemical response signal is clearly enhanced.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Electrochemical characterization of CPF\u003c/h2\u003e \u003cp\u003eAChE catalyzes the generation of redox-active thiocholine from the substrate ATCHI, and electrode surface-modified PANI/AuNPs catalyze the thiocholine's redox reaction, generating a detectable electrochemical signal. If chlorpyrifos is present in the system to be tested, the activity of AChE is inhibited, resulting in the inability of acetylthiocholine iodide to generate thiocholine, which eventually leads to a weakening or even disappearance of the electrochemical response signal, and this principle can be used to indirectly detect organophosphorus pesticide residues-chlorpyrifos in the samples. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG shows the DPV plots of AChE/PANI/AuNPs/GCE modified electrodes in PBS (pH\u0026thinsp;=\u0026thinsp;7.60) buffer containing 1.00 mmol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e ATCHI before and after chlorpyrifos inhibition. Curve a shows the DPV plot of the AChE/PANI/AuNPs/GCE modified electrode in 1.00 mmol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e ATCHI in PBS (pH\u0026thinsp;=\u0026thinsp;7.60) buffer before inhibition, and curve b shows the DPV plot of the sensor in 1.00 mmol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e ATCHI in PBS (pH\u0026thinsp;=\u0026thinsp;7.60) buffer after inhibition by chlorpyrifos. The figures show that the sensor's response current was drastically lowered following inhibition with chlorpyrifos.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Optimization of experimental conditions\u003c/h2\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e\u003cb\u003e3.4.1 Effect of the number of polymerization cycles of AuNPs on modified sensors\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, the peak current of AuNPs/GCE reached its maximum after AuNPs were deposited for 15.00 cycles, showing that AuNPs' high specific surface area and affinity boosted sensor sensitivity and signal response intensity. And it served as the modification quantity for further studies.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e3.4.2 Effect of the number of PANI polymerization cycles on modified sensors\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, increasing the number of aniline polymerization cycles improved the current response and gradually increased the peak current; when the number of aniline polymerization cycles was 30.00 cycles, the peak current reached its maximum, and as the number of polymerization cycles increased, the peak current gradually decreased, indicating that 30.00 cycles was the optimal number of cycles for aniline polymerization.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e3.4.3 Effect of solution ambient pH on sensor performance\u003c/h2\u003e \u003cp\u003eThe pH of the test substrate, phosphate buffer solution (PBS), affects the activity of AChE, which influences the response current produced by the enzyme sensor catalyzing ATCHI. The catalytic activity of AChE was higher in the pH\u0026thinsp;=\u0026thinsp;7.00\u0026thinsp;~\u0026thinsp;8.00 range. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, peak current rose as the pH climbed from 7.00 to 7.60, but the rise decreased when the pH went to 7.80 vs. 8.00, indicating that too high a pH might lead to CPF hydrolysis. Finally, a suitable pH\u0026thinsp;=\u0026thinsp;7.60 PBS buffer was selected as the test substrate, and the electrochemical sensor performed best at pH\u0026thinsp;=\u0026thinsp;7.60.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e \u003ch2\u003e3.4.4 Effect of substrate (ATCHI) concentration on the sensor\u003c/h2\u003e \u003cp\u003eA change in substrate concentration has a significant effect on the rate of the enzyme reaction. AChE/PANI/AuNPs/GCE-modified electrodes were used to measure the concentration of iodinated acetylthiocholine in 10.00 mmol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e pH 7.06 PBS buffer at a constant potential of +\u0026thinsp;0.20 V. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD shows that when the quantity of iodinated acetylthiocholine rose from 0.25 to 0.75 mmol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, the catalytic current increased quickly. The catalytic current remained steady when the content of chlorinated acetylthiocholine fluctuated between 0.75 and 1.25 mmol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Acetylthiocholine's catalytic current rose fast as the substrate concentration increased. Therefore, 1.00 mmol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of iodinated acetylthiocholine was chosen as the optimal substrate concentration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e \u003ch2\u003e3.4.5 Effect of AChE modification amount on sensor performance\u003c/h2\u003e \u003cp\u003eDrops of material of 4.00 \u0026micro;L, 5.00 \u0026micro;L, 6.00 \u0026micro;L, 7.00 \u0026micro;L, and 8.00 \u0026micro;L of PBS (pH\u0026thinsp;=\u0026thinsp;7.60) containing 10.00 U mL\u003csup\u003e-1\u003c/sup\u003e AChE were applied to the surface of PANI/AuNPs/GCE-modified electrodes and dried. The modified electrodes were examined by the DPV method, and the results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE. AChE modification at 5.00 \u0026micro;L had the highest reaction value. As the quantity of modification increased, the enzyme layer thickened, preventing the diffusion of enzymatic reaction products to the electrode surface, limiting sensor sensitivity, and eventually decreasing the response value. Therefore, the optimal amount of AChE modification was 5.00 \u0026micro;L and was used as the modification amount for subsequent experiments.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Performance of electrochemical enzyme biosensors\u003c/h2\u003e \u003cp\u003eThe CPF pesticide residue standard solutions at various concentrations (1.00 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e ~ 1.00 \u0026times; 10\u003csup\u003e1\u003c/sup\u003e ppm) were determined under optimum experimental circumstances utilizing the.\u003c/p\u003e \u003cp\u003eDPV method. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA shows the connection between the DPV response values obtained when the electrochemical enzyme biosensor was tested for various CPF standard concentrations. The calibration curve for CPF concentration and inhibition rate is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB. The logarithm of CPF concentration shows a good linear relationship with its corresponding enzyme inhibition rate in the CPF concentration range of 1.00 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e ~ 1.00 \u0026times; 10\u003csup\u003e1\u003c/sup\u003e ppm, with the linear equation Y\u0026thinsp;=\u0026thinsp;3.7708 LgC\u003csub\u003eCPF\u003c/sub\u003e + 37.133 (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9948) and the detection limit of 7.90 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e ppm (S/N\u0026thinsp;=\u0026thinsp;3). Compared to previous approaches reported in the literature, this method produced superior results and had high selectivity for determining CPF content.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eTable II\u003c/b\u003e Comparison of different analytical methods for CPF detection\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabb\" border=\"1\"\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDetection methods\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDetection limit\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLinear range\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eReferences\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eElectropolymerization photoelectric chemical sensor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.60\u0026times; 10\u003csup\u003e2\u003c/sup\u003e ppm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.00\u0026thinsp;~\u0026thinsp;218.92 ppm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMicrofluidic electrochemical sensor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.00\u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e ppm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.00\u0026thinsp;~\u0026thinsp;1.50\u0026times;10\u003csup\u003e2\u003c/sup\u003e ppm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.20 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e ppm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.00 \u0026times; 10\u003csup\u003e1\u003c/sup\u003e ~ 1.50\u0026times;10\u003csup\u003e1\u003c/sup\u003e ppm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHPLC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.50 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e ppm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.050\u0026thinsp;~\u0026thinsp;5.00 ppm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eELISA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.00\u0026times; 10\u003csup\u003e2\u003c/sup\u003e ppm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.00\u0026thinsp;~\u0026thinsp;1.00 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e ppm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eElectrochemical enzyme biosensor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.90 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e ppm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.00 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e ~ 1.00 \u0026times; 10\u003csup\u003e1\u003c/sup\u003e ppm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eThis work\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTo further demonstrate the good performance of the sensor, we compared the performance of the AChE/PANI/AuNPs/GCE electrochemical enzyme biosensor with the reported CPF assays (\u003cb\u003eTable II\u003c/b\u003e), and the method constructed in this study showed superior analytical performance with a wide linear range and low detection limit, which was superior to the other reported methods.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Selectivity, Repeatability, and Stability\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe specificity of the electrochemical enzyme sensor was evaluated by subjecting it to various compositional interfering terms that may interfere with the detection of CPF pesticide residues, such as glycine, glyphosate, soluble starch, urea, and ascorbic acid, and comparing the signal changes of these interferences to the presence of CPF pesticide residue. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eA shows that the incubated interference term group had essentially no change in DPV response signals to the sensors, with an RSD of less than 2.85% when compared to the blank control group. It indicates that AChE/PANI/AuNPs/GCE, based on the principle of enzyme inhibition, has great specificity for CPF pesticide residue detection.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe constructed five modified electrodes in parallel and tested solutions containing CPF (1.00 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e ppm) using the DPV technique. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, the electrochemical platform constructed with AChE/PANI/AuNPs composites exhibits remarkable reproducibility, as evidenced by an average relative standard deviation (RSD) of merely 1.73% across five parallelly fabricated electrodes. This figure underscores the consistency and reliability of the platform for electrochemical applications.The electrodes were sealed and stored at room temperature (25.00℃) for 5, 10, 15, 20 and 25 days. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, the electrochemical sensor demonstrated remarkable stability, maintaining a detection rate ranging from 88.37\u0026ndash;97.84% of its initial performance when tested in a solution containing CPF at a concentration of 1.00 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e ppm, as determined by the differential DPV method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Analysis of actual samples\u003c/h2\u003e \u003cp\u003eIn order to evaluate the viability of the sensing platform created based on AChE/PANI/AuNPs composites for the sensitive detection of CPF in actual samples, in this investigation, 10.00 mL of the actual sample solution was chosen for a10.00 minute incubation, crushed astragalus was placed in 100.00 mL volumetric flasks, and PBS solution with a pH of 7.60 was added to the scale. This solution was divided into three 10.00 mL aliquots, and a methanol solution of CPF was added at concentrations of 25.00, 50.00, and 250.00 ppm. The DPV test was carried out with a combination of PBS (pH\u0026thinsp;=\u0026thinsp;7.60) and 1.00 mmol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e ATCHI as the supporting electrolyte, and the recoveries achieved in the three analyses utilizing AChE/PANI/AuNPs/GCE varied from 96.68\u0026ndash;102.32%, with relative standard deviations of 1.97\u0026ndash;2.41% (\u003cb\u003eTable III\u003c/b\u003e). This indicates that the sensor is sensitive and convenient to use for instantaneous, fast detection of CPF in real samples.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTable III\u003c/b\u003e Electrochemical enzyme biosensor determination of Astragalus samples\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabc\" border=\"1\"\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSaCPFle\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAdded (ppm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFound (ppm) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRecovery (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRSD (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e25.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e24.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e96.68%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.97\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e51.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e102.32%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e250.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e248.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e99.25%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.41\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003csup\u003ea\u003c/sup\u003eThe average of three measurements\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn this study, PANI/AuNPs composites were prepared from PANI functionally modified with AuNPs, which have excellent conductivity and biocompatibility, to be used as electrode substrate modification materials, and an electrochemical enzyme biosensor was constructed for the highly sensitive and rapid detection of CPF in TCM, with the use of AChE as the recognition element. Cyclic voltammetry was used to deposit AuNPs and aniline onto the glassy carbon electrode, resulting in a PANI/AuNPs/GCE-modified electrode. Then AChE was immobilized using the gold-ammonia bonding of AuNPs to AChE. AChE catalyzes the production of thiocholine by ATCHI. With PANI's stronger catalytic ability, it can catalyze the redox reaction of thiocholine in a timely and efficient manner while also greatly promoting electron transmission, significantly amplifying the detection signal. Under optimized conditions, the sensor showed good linearity in the concentration range of 1.00 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e ~ 1.00 \u0026times; 10\u003csup\u003e1\u003c/sup\u003e ppm with a detection limit of 7.90 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e ppm (S/N\u0026thinsp;=\u0026thinsp;3). Meanwhile, the electrochemical enzyme biosensor has remarkable performance, including high selectivity, sensitivity, a wide linear range, an ultra-low detection limit, and strong anti-interference capability. The findings of this study not only provide an effective method for the highly sensitive identification and detection of trace CPF pesticide residues in Chinese herbal medicines, but they also provide critical technical support for on-site law enforcement and supervision of new and portable organophosphorus pesticide residues in agriculture, as well as ensuring the production guarantee of high-quality agricultural products.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eB.Sun, L. Yang put forward the idea of the experiment and gave guidance to the experiment.S. Yu, Q. Ma and H. Shi prepared PANI/AuNPs composites with excellent electroactivity, prepared electrochemical enzyme biosensors and wrote the manuscript. B. Sun,Y. Lv, Q. Ma, H. Shi, Q. Dang, X. Wang, M. Zhou, X. Da, L. Yang, X. Shi helped with the experimental design, analyzed the data, interpreted the results, and revised the manuscript. All authors read and approved the completed manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThe authors acknowledge science and Technology Plan of Gansu Province -Major Science and Technology Project-Social Development Category (No. 23ZDFA013-1); Science and Technology Plan of Gansu Province-Innovation Fund for Technology Based Firms (No. 23CXGA0097); Gansu Province 2024 Drug Safety Supervision Research Project (No. 2024GSMPA070); the Education Science and Technology Innovation Project of Gansu Provincial Department of Education 2025-Young PhD Support Project (2025QB-000); the Postdoctoral Research Workstation Project of Lanzhou Foci Pharmaceutical Co., Ltd.; the fifth batch of Hongliu outstanding young talents support program of Lanzhou University of Technology; the 2020 PhD Research Start-up Fee of Lanzhou University of Technology and the 2024 Lanzhou University of Technology Innovation and Entrepreneurship Training Program for college students (No. DC20240753; DC20240287).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors alone are responsible for the content and writing of this article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eXiao JJ, Duan JS, Xu X, Li SN, Wang F, Fang QK, Liao M, Cao HQ (2018) Behavior of pesticides and their metabolites in traditional Chinese medicine PANIeoniae Radix Alba during processing and associated health risk. J Pharm Biomed Anal 16120. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.1016/j.jpba.2018.08.029\u003c/span\u003e\u003cspan address=\"https://doi:10.1016/j.jpba.2018.08.029\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu Y, Xiao Y, Zhang Y, Gao X, Wang H, Niu B, Li W (2024) ZnO-rGO-based electrochemical biosensor for the detection of organophosphorus pesticides. 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Talanta 265:124802. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.1016/j.talanta.2023.124802\u003c/span\u003e\u003cspan address=\"https://doi:10.1016/j.talanta.2023.124802\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-applied-electrochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jach","sideBox":"Learn more about [Journal of Applied Electrochemistry](http://link.springer.com/journal/10800)","snPcode":"10800","submissionUrl":"https://submission.nature.com/new-submission/10800/3","title":"Journal of Applied Electrochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Chinese herbal medicine pesticide residues, chlorpyrifos, enzyme biosensor, trace fast detection","lastPublishedDoi":"10.21203/rs.3.rs-5165698/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5165698/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChlorpyrifos (CPF) is an insecticide and acaricide that interrupts nerve conduction by reducing the activity of the enzyme acetylcholinesterase. It can enter the human body through a variety of channels and produce serious physiological effects. However, excessive use of CPF in herbal production produces excessive pesticide residues and reduces the yield of high-quality herbs. Therefore, the development of effective and convenient CPF residue detection technology is critical for pesticide residue monitoring as well as danger avoidance. Based on this, an electrochemical enzyme biosensor using acetylcholinesterase (AChE) as the recognition element was constructed for highly sensitive and rapid detection of chlorpyrifos in traditional Chinese medicine. The electrode substrate modification material was a polyaniline/gold nanoparticles (PANI/AuNPs) composite material prepared by PANI functional modification of AuNPs with high conductivity and large specific surface area, which has excellent electrical conductivity and biocompatibility. Specifically, the PANI/AuNPs offered an exceptional active site for AChE immobilization, greatly enhancing the electrical signals resulting from AChE-catalyzed iodination of acetylthiocholine and accelerating electron transfer. In contrast, AChE's better identification of the target analyte CPF meant that the sensor was highly selective for organophosphorus pesticide residues. Under optimized conditions, the sensor showed good linearity in the range of 1.00 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e ~ 1.00 \u0026times; 10\u003csup\u003e1\u003c/sup\u003e ppm with a detection limit of 7.90 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e ppm. During practical sample testing, the sensor exhibited remarkable stability, reproducibility, and sensitivity in detecting CPF pesticide residues in Chinese herbal medicines. This offers a reliable tool for precise monitoring and propels the progress of enzyme-based biosensors, both in fostering the cultivation and production of superior-quality herbal medicines, as well as in enabling rapid on-site pesticide residue detection.\u003c/p\u003e","manuscriptTitle":"Electrochemical enzyme biosensor based on gold nanoparticles/polyaniline composites for highly specific rapidly detection of chlorpyrifos residues in traditional Chinese medicines","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-10 08:51:59","doi":"10.21203/rs.3.rs-5165698/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-11-28T18:25:30+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-20T09:15:55+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-07T02:36:15+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-06T03:03:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"90681894259519719520910909599242867765","date":"2024-11-06T00:13:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"305288210023017654857763614612370367631","date":"2024-11-05T21:55:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"60327914426355471179023132075085181167","date":"2024-11-04T04:50:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"96051133032586435319730540745765019107","date":"2024-11-03T22:10:28+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-03T21:45:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-09-28T16:01:55+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-09-28T02:00:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Applied Electrochemistry","date":"2024-09-27T14:03:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-applied-electrochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jach","sideBox":"Learn more about [Journal of Applied Electrochemistry](http://link.springer.com/journal/10800)","snPcode":"10800","submissionUrl":"https://submission.nature.com/new-submission/10800/3","title":"Journal of Applied Electrochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"011cf8f0-794d-42b5-b25d-a07471ee8b20","owner":[],"postedDate":"December 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-30T15:58:28+00:00","versionOfRecord":{"articleIdentity":"rs-5165698","link":"https://doi.org/10.1007/s10800-024-02244-3","journal":{"identity":"journal-of-applied-electrochemistry","isVorOnly":false,"title":"Journal of Applied Electrochemistry"},"publishedOn":"2024-12-26 15:56:56","publishedOnDateReadable":"December 26th, 2024"},"versionCreatedAt":"2024-12-10 08:51:59","video":"","vorDoi":"10.1007/s10800-024-02244-3","vorDoiUrl":"https://doi.org/10.1007/s10800-024-02244-3","workflowStages":[]},"version":"v1","identity":"rs-5165698","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5165698","identity":"rs-5165698","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}