Entropy spontaneous ratiometric electrochemical aptasensor based on polymerization and AuNPs signal amplification for Acetamiprid residue analysis

Entropy spontaneous ratiometric electrochemical aptasensor based on polymerization and AuNPs signal amplification for Acetamiprid residue analysis | 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 Entropy spontaneous ratiometric electrochemical aptasensor based on polymerization and AuNPs signal amplification for Acetamiprid residue analysis Weiming Li, Yuzhen Jia, Kunyilan Chen, Huifang Li, Huaixia Yang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6299477/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Jun, 2025 Read the published version in Microchimica Acta → Version 1 posted 14 You are reading this latest preprint version Abstract Acetamiprid (ACE), a next-generation chlorinated neonicotinoid insecticide, has been extensively employed for pest control. However, its excessive residues in food and the environment have raised significant concerns regarding human health. To address the need for a simple, accurate, and efficient ACE detection method, this study developed a ratiometric electrochemical aptasensor utilizing a dual signal amplification strategy involving atom transfer radical polymerization (ATRP) and gold nanoparticles (AuNPs). Methylene blue (MB) served as the internal reference signal, which was attached to the ACE aptamer-DNA 1 (dsDNA) through electrostatic adsorption and intercalation. Ferrocenyl methyl methacrylate (FMMA) was polymerized into long chains via ATRP, generating a significantly amplified electrical signal compared to that of monomeric FMMA. AuNPs, known for their bioconjugability, were linked to DNA 2 (AuNPs-DNA 2 ), enhancing the DNA 2 loading capacity and expanding the ATRP reaction sites. In the presence of ACE, the MB signal decreased while the FMMA polymer signal increased, achieving a ratiometric detection strategy with a limit of detection (LOD) of 19.26 pg/mL. This ratiometric aptasensor not only enhances selectivity but also mitigates the influence of background currents, offering a novel and effective approach for ACE detection in real-world sample analysis. Acetamidine ratiometric aptasensor electrochemical ATRP AuNPs Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1 Introduction Acidamidine (ACE), a selective agonist of type II nicotinic acetylcholine receptors (nAChRs), targets nAChRs in the central nervous system of insects and is one of the most extensively utilized neonicotinoid insecticides [ 1 , 2 ]. However, the excessive and improper use of ACE has led to significant concerns regarding pesticide residues. In recent years, ACE residues have been detected in various common foods and medicinal herbs [ 3 , 4 ]. Studies have shown that the long-term accumulation of ACE and its toxic metabolites in mammals can alter hematological, biochemical and structural characteristics, ultimately leading to neurological, hepatorenal, immune, reproductive effects and genotoxicity [ 5 , 6 , 7 ]. Given its potential risks to human health and the environment, the development of simple and efficient ACE detection technologies is crucial for monitoring and assessing ACE contamination levels, as well as ensuring the safety of food and pharmaceutical products. Electrochemical aptasensors represent a well-established and advanced method for trace detection of small molecules, having been extensively developed in recent years [ 8 ]. Owing to their high sensitivity, rapid analysis, cost-effectiveness and operational simplicity, they have gained widespread application in various fields, such as food safety, environmental monitoring, clinical detection [ 9 , 10 , 11 ]. Whereas, conventional electrochemical sensors typically rely on a single signal output for target molecule detection. This single-signal approach is prone to aptasensor false signal interference due to unavoidable instrumental variations or external environmental factors, particularly when detecting trace amounts of small molecules in complex sample matrices [ 12 , 13 ]. To address these limitations, the integration of ratiometric sensing with dual or multiple signal responses has been proposed [ 14 , 15 ]. By utilizing the ratio of two distinct signals as the output, this approach can effectively compensate for response deviations caused by internal or external factors, providing inherent built-in correction for system or background electrical signals [ 16 ]. Consequently, ratiometric aptasensors based on dual-signal responses demonstrate superior accuracy and reliability, offering significant potential for practical applications in complex sample analysis. Currently, ratiometric electrochemical strategies are widely employed in aptasensor construction, utilizing redox tags such as methylene blue (MB), ferrocene (Fc) and thionin (Thi) [ 17 , 18 ]. Notably, MB exhibits versatile binding interactions with DNA, enabling it to attach to phosphate groups on single-stranded DNA (ssDNA) through electrostatic adsorption or intercalate into the double-helix structure of double-stranded DNA (dsDNA) [ 19 , 20 , 21 ]. Leveraging this principle, MB can serve as a label-free binding probe to enhance the response signal strength in electrochemical aptasensors. Zhu et al. [ 22 ] designed a ratiometric electrochemical aptasensor for ultrasensitive detection of Ochratoxin A, employing a dual signal amplification strategy. This approach utilized the binding of MB to DNA, combined with the introduction of hybrid DNA (hDNA) and MB post-OTA recognition, to amplify the response signal. The target concentration was accurately quantified by measuring the signal ratio of I Fc /I MB . Similarly, Ding et al. [ 23 ] developed an antifouling electrochemical biosensor based on a Y-shaped peptide and MXene loaded with Au@ZIF-67 and MB. In this system, electrochemical signal molecules (Fc and MB) were modified at distinct locations (on the peptide and within the MXene-Au@ZIF-67 composite, respectively) to generate a ratiometric electrochemical signal. In addition, to further enhance the sensitivity of ratiometric electrochemical aptasensor, signal amplification strategies involving polymerization reactions and nanomaterials are often employed. For example, atom transfer radical polymerization (ATRP) [ 24 , 25 , 26 ], a prominent controlled/living radical polymerization technique, offers advantages such as wide range of monomers and narrow molecular distribution. ATRP is widely recognized as an effective for synthesizing grafted polymer copolymers. Additionally, gold nanoparticles (AuNPs), known for their excellent biocompatibility, easy of surface modification, and superior electronic conductivity, are frequently employed as carriers to load signaling probes [ 27 , 28 ]. The loading process typically involves the formation of covalent bonds between functional groups (e.g., -SH, -NH 2 ) on DNA and the surface of AuNPs. Due to its superior characteristics, AuNPs are widely used in electrochemical aptasensor. Therefore, this study has introduced the two signal amplification strategies of ATRP and AuNPs into the development of ratiometric electrochemical aptasensor for the detection of ACE. In this work, a ratiometric electrochemical aptasensor was developed for the detection of ACE, incorporating a dual-signal amplification strategy based on ATRP and AuNPs aptasensor. MB was employed as an internal reference signal, while the ATRP reaction was utilized to synthesize polymers using ferrocenyl methyl methacrylate (FMMA) as the signal unit. The polymer serves as the amplification signal for the proposed aptasensor. Additionally, AuNPs were functionalized with multiple DNA 2 probes to increase the active site of ATRP reaction. The aptasensor was constructed by self-assembling thiolated DNA 1 -aptamer (SH-DNA 1 -Apt) aptasensor onto the gold electrode via an Au-S bond, with MB bound to the double-stranded DNA through intercalation and weak electrostatic interactions. In the presence of ACE, the electrical signal of MB was attenuated, exposing the DNA 1 site, which then hybridized with the AuNPs-DNA 2 probes through complementary base pairing. Subsequently, upon activation of the ATRP initiator, the signal unit FMMA was grafted onto the electrode surface, forming a conductive polymer. This strategy achieved a ratiometric signal response characterized by one signal decreasing while the other increased, demonstrating high sensitivity and specificity. The proposed aptasensor provides a novel and effective method for the analysis of pesticide residues in agricultural products and environmental samples, offering significant potential for practical applications. 2 Experimental 2.1 Reagents and materials ACE standard, gold perchlorate (HAuCl 4 ), α-bromoisobutyric acid (BMP), n-hydroxysuccinimide (NHS), N-(3-dimethylaminopropyl)-N'-ethyl diimide hydrochloride (EDC), Ferrocenyl methyl methacrylate (FMMA) and 6-mercaptohexyl alcohol (MCH) were derived from Shanghai Aladdin Reagent Co., Ltd (Shanghai, China). Ascorbic acid (AA) and Trisodium citrate dihydrate (C 6 H 5 Na 3 O 7 ·2H 2 O) were bought by Balinway Technology Co., Ltd (Shanghai, China). Copper bromide (CuBr 2 ), K 3 [Fe(CN) 6 ] and K 4 [Fe(CN) 6 ]·3H 2 O were purchased from Sinopharmate Chemical Reagent Co., Ltd. 0.1 M PBS (pH = 7.4) was the buffer system and electrochemical measurement solution of the whole experiment. Enzyme linked immunosorbent assay (ELISA) of ACE was purchased from Jiangsu Enzyme Free Industrial Co., Ltd (Jiangsu, China). The DA used in all experiments was treated with the Millipore system (≥ 18.25 MΩ cm). All the HPLC-purified DNA sequences were synthesized and purified by Wuhan Cloud Clone Technology Co., Ltd (Wuhan, China). Detailed sequences are listed as follows: Aptamer of ACE: 5'-SH-(CH 2 ) 6 -CTG ACA CCA TAT TAT GAA GA-3' DNA 1 : 5'-SH-(CH 2 ) 6 -TCT TCA TAA TAT GGT GTC AG-3' DNA 2 : 5'-SH-(CH 2 ) 6 -CTG ACA CCA TAT TAT-(CH 2 ) 6 -NH 2 -3' 2.2 Apparatus The standard three-electrode system (reference electrode: saturated calomel, counter electrode: Pt filament) was used for electrochemical tests in this experiment. Square wave voltammetry (SWV) was performed on the CHI 760E Electrochemical Workstation (Shanghai Chenhua Instrument Co., Ltd, China). Characterization of electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) was performed at the Autolab PGSTAT204 Electrochemical workstation (Eco Chemie, Netherlands). Ultraviolet-visible spectroscopy (Shimadzu UV-3600 Plus) measures the absorbance of AuNPs-DNA 2 . The modified gold electrode morphology was characterized by Dimension Icon Atomic force microscope (AFM, Bruker, USA) and Sigma HD field emission scanning electron microscope (TEM, Zeiss, Germany), respectively. 2.3 Synthesis of AuNPs-DNA 2 Firstly, the synthesis method of AuNPs was slightly modified based on previously reported procedure [ 29 ]. Briefly, 50 mL 0.01% HAuCl 4 was added to the 100 mL conical flask and heated to boiling on a magnetic stirrer at 1000 r/min at 120°C. Afterwards, 1.75 mL 1%C 6 H 5 Na 3 O 7 ·2H 2 O was quickly added in the above liquid stirring state, the liquid color gradually changed from colorless to wine red, and continued to boil for 10 mins. Finally, the heating was stopped and cooled to room temperature at 1000 r/min. The obtained AuNPs were placed in the refrigerator at 4°C for storage. During the preparation of the AuNPs-DNA 2 , 500 µL of the above AuNPs was taken and mixed with 10 µL 100 µM DNA 2 and shaken well, and incubated in a shaker for 3 h. 2.4 Construction of this ratiometric aptasensor The gold electrodes were polished with 0.05 µM Al 2 O 3 powder on a chamois leather clockwise for 3 mins to remove surface impurities before used. Afterwards, the treated gold electrode was ultrasounded with DW for 1 min and immersed in a mixture of 98% H 2 SO 4 and 30% H 2 O 2 (v/v = 3:1) for 15 min. Eventually, CV scanning was performed in 0.5 mM H 2 SO 4 solution to remove impurities on the electrode surface (potential range: -0.3 V − 1.5 V. Scan rate: 0.2 V s − 1 ). The mixture of 2 µM DNA 1 and 3 µM Apt in equal volume was shaken well and incubated in the shaker overnight at room temperature, so that DNA 1 and Apt reacted as completely as possible to obtain 1 µM DNA 1 -Apt. Then, 10 µL 1 µM DNA 1 -Apt was added to the surface of the gold electrode and reacted at room temperature overnight. The modified electrode was immersed in 2 mM MCH solution (solvent 70% ethanol) and reacted at 37 ℃ for 1 h to close the remaining active site on the electrode surface. 10 µL 0.2 mM MB (internal reference signal) reacted with the modified electrode at 37℃ for 1 h, which bound to DNA 1 -Apt by electrostatic adsorption and insertion. Subsequently, 10 µL ACE with different concentrations was incubated on the electrode at 37℃ for 50 mins. Subsequently, 10 µL 1 µM AuNPs-DNA 2 was added and incubated at 37℃ for 70 mins. The initiator BMP was mixed with EDC/NHS (20 mM:5 mM) in equal volume to activate the carboxyl group on the surface. 10 µL 3 mM activated BMP was reacted on the modified electrode at 37℃ for 60 min. Finally, the modified electrode was immersed in 200 µL ATRP reaction solution (20 µL 10 mM FMMA, 20 µL 2 mM AA, 20 µL 2mM CuBr 2 /Me 6 TREN + and 140 µL PBS) and reacted at 37℃ for 2 h. 2.5 Detection of the aptasensor The constructed aptasensor was measured by square wave voltammetry (SWV) on CHI760E electrochemical work using 0.1 M pH = 7.4 PBS buffer as the electrolyte solution (the sweep potential: -0.6 V − 0.6 V, the step potential: 4 mV, the frequency: 25 Hz, the amplitude: 25 mV). The signal variation trend between MB and FMMA was observed under different concentrations of ACE. 3 Results and discussion 3.1 The principle of the aptasensor This study proposed the construction of an FMMA/MB ratiometric electrochemical aptasensor based on ATRP amplification strategy to detect ACE (Scheme 1 ). DNA 1 -Apt was immobilized on the electrode surface by Au-S bond formed by sulfhydryl group (-SH) on DNA 1 . MCH was employed to block the remaining active sites on the electrode surface, thereby preventing non-specific binding interference. Furthermore, MB, serving as an internal reference signal, was attached to the electrode surface via intercalation or electrostatic interactions with the phosphate group on DNA 1 -Apt. Upon the introduction of varying concentrations of ACE, the Apt containing MB specifically captured ACE and was subsequently released from the gold electrode surface, leading to a reduction in the MB electrical signal. Following this, the AuNPs-DNA 2 probe hybridized with the single-stranded DNA 1 through complementary base pairing. The initiator BMP was then conjugated to the electrode surface via an amide bond (-CO-NH-) formed between the activated carboxyl group (-COOH) on BMP and the amino group (-NH 2 ) on DNA 2 . Finally, the signal unit FMMA was grafted onto the electrode surface through an in situ ATRP reaction, utilizing BMP as the initiator, AA as the reducing agent, and CuBr 2 /Me 6 TREN + as the catalyst. This study achieved ratiometric electrochemical detection of ACE by monitoring the ratio of the MB signal to the FMMA signal. The reaction mechanism of ATRP was illustrated in Scheme 1 B. The Br-Cu II /Me 6 TREN + complex was reduced to the catalytically active Cu I /Me 6 TREN by the reducing agent AA. The Cu I /Me 6 TREN then reacted with the initiator (Gn-Br) to generate free radicals (Gn•) and the oxidation state complex dormant substance Br-Cu II /Me 6 TREN + . The free radicals (Gn•) and the monomer (FMMA) initiated the polymerization reaction through double bond addition, ultimately forming a polymer (GnGm). Simultaneously, excessive AA reduced Br-Cu II /Me 6 TREN + , initiating a new cycle of polymerization. According to Eq. 1[ 30 ], the rate of ATRP (R p ) was determined by the propagation rate constant ( k p ) and the concentration of monomer (M) and free radicals (Gn•). In turn, the concentration of Gn• depended on the ATRP equilibrium constant ( K ATRP ), which was defined by the concentrations of the Gn-X, Cu I L and X-Cu II L. 3.2 Characterization of AuNPs-DNA 2 The morphology of the synthesized AuNPs-DNA 2 probe was characterized by TEM. As shown in Fig. 1 A and Fig. 1 B, the synthesized AuNPs exhibited a spherical shape with uniformly dispersion and an average diameter of approximately 21.80 nm. Following the covalent incubation of DNA 2 with AuNPs, the diameter of the nanoparticles increased to 27.54 nm, attributed to the presence of residual salt residues surrounding the DNA 2 . This result confirmed the successful conjugation of DNA 2 onto the AuNPs. In addition, the UV-Vis spectroscopy was employed to assess the modification efficiency of DNA 2 on AuNPs (Fig. 1 C). The UV-Vis spectrum of AuNPs displayed a characteristic absorption peak at 518.78 nm (curve a), while DNA 2 exhibited a characteristic peak at 258.02 nm (curve b). After the modification of AuNPs with DNA 2 , the resulting spectrum showed the presence of both DNA 2 and AuNPs characteristic peaks. Notably, the AuNPs peak exhibited a redshift from 518.78 nm to 521.86 nm (curve c), further confirming the successful functionalization of AuNPs with DNA 2 . 3.3 Feasibility and characterization of the aptasensor In order to analyze the feasibility of the ratiometric electrochemical aptasensor, this section examined and compared the changes in the electrical signal intensities of MB and FMMA under conditions where one of the key modifications was systematically omitted. As shown in Fig. 2 A, in the fully modified experimental group (curve f), two distinct signal peaks were observed at the potential − 0.288 V and + 0.265 V, corresponding to the signals of MB and FMMA, respectively. In contrast, when the aptasensor lacked the carrier DNA 1 -Apt (curve a), no significant signal peaks for either MB or FMMA were detected. In the absence of ACE (curve c), the current intensity of MB remained unchanged, and no noticeable FMMA signal was observed. This was attributed to the failure of the aptamer to recognize ACE, preventing the probe DNA 2 -AuNPs from hybridizing with DNA 1 and consequently hindering the grafting of FMMA onto the electrode surface. Similarly, when the DNA 2 -AuNPs/BMP (curve d) modification was omitted, the results mirrored those of curve c. Additionally, in the absence of MB (curve b) and FMMA (curve e) modifications, no corresponding current signal peaks were observed. Based on the above analysis, it could be concluded that the construction of the aptasensor is feasible. To further investigate the construction of the aptasensor, the EIS was employed to characterize the layer-by-layer assembly process. The measurements were conducted in a frequency range of aptasensor 0.1 MHz to 0.1 Hz with a sine wave potential amplitude of 5 mV. The electrodes, under different modification conditions, were immersed in a 5 mM [Fe (CN) 6 ] 3−/4− electrolyte solution containing 0.1 M KNO 3 . As shown in Fig. 2 B, the bare gold electrode exhibited the lowest charge transfer resistance ( Rct ) of -197.55 Ω (curve a). Upon modification with DNA 1 -Apt and MCH, the Rct value increased to -651.69 Ω (curve b) due to the strong electrostatic repulsion between the phosphate group of DNA 1 -Apt and [Fe (CN) 6 ] 3−/4− , as well as the blocking effect of MCH, which hindered the electron transfer at the electrode surface. When ACE specifically recognized by the aptamer, the detachment of the MB-containing Apt from the electrode surface led to a decrease in Rct to -476.93 Ω (curve d). Subsequent modification with AuNPs-DNA 2 resulted in a further increase in Rct to -1147.2Ω (curve e), attributed to the poor conductivity and high steric hindrance of the AuNPs. The introduction of the initiator BMP via amide bonding further impeded electron transfer, raising the Rct to -1355.93 Ω (curve f). Finally, the formation of a large amount of FMMA polymer on the electrode surface significantly increased steric hindrance, causing a substantial rise in Rct to -2182.90Ω (curve g). In addition, the progressive modification of the electrode was characterized using CV. As depicted in Fig. 2 C, the current intensity decreased progressively with each modification step, following the order: curve a > curve c > curve b > curve d > curve f > curve g. This trend in current intensity was consistent with the EIS results, confirming the successful layer-by-layer assembly of the aptasensor. Furthermore, AFM was utilized to compare the height changes of the aptasensor surface before and after the modifications involving AuNPs-DNA 2 modification and ATRP reaction. Following the self-assembly of DNA 1 -Apt/MB/ACE on the gold substrate, the surface height was measured at 34.9 nm (Fig. 3 A). Upon modification with AuNPs-DNA 2 , the height increased to 58.7 nm, reflecting a rise of 23.8 nm, which confirmed the successful assembly of AuNPs-DNA 2 (Fig. 3 B). After the introduction of the initiator BMP, the surface height further increased to 65.1 nm (Fig. 3 C). During the ATRP reaction, the surface height of gold substrate reached 105.1 nm, marking an additional increase of 40.0 nm, indicating the successful grafting of FMMA onto the aptasensor surface (Fig. 3 D). These results collectively demonstrated the effective step-by-step modification and functionalization of the aptasensor. 3.4 Optimization of experimental parameters To improve the ACE detection performance of the ratio aptasensor, several experimental parameters of the aptasensor were optimized, such as the incubation time of ACE, the volume and reaction time of AuNPs-DNA 2 , the reaction time of BMP and the incubation time of ATRP reaction. The specific recognition of ACE by the Apt exposed the DNA 1 site, enabling subsequent hybridization with DNA 2 and the grafting of electrochemical signals. Therefore, optimizing the reaction time between ACE and aptamer was crucial for achieving optimal aptasensor performance. In Fig. 4 A, the I FMMA /I MB signal ratio increased with the ACE reaction time within the range of 20 to 50 mins. However, after 50 mins, the reaction between aptamer and ACE reached saturation, and the I FMMA /I MB signal ratio stabilized, indicating no further significant changes. Thus, 50 mins was determined to be the optimal reaction time for ACE and Apt. The amount of AuNPs-DNA 2 modification directly affected the quantity of FMMA grafted onto the electrode. Consequently, the effects of both the volume and reaction time of AuNPs-DNA 2 on aptasensor performance were studied. As shown in Fig. 4 B, the I FMMA /I MB signal ratio increased with the volume of AuNPs-DNA 2 in the range of 3 to 15 µL. When the volume of AuNPs-DNA 2 reached 7 µL, the I FMMA /I MB ratio reached its maximum and subsequently plateaued, indicating that the electrode surface was saturated with AuNPs-DNA 2 at this volume. Therefore, 7 µL was identified as the optimal incubation volume for AuNPs-DNA 2 . Similarly, as shown in Fig. 4 C, the I FMMA /I MB ratio increased with the reaction time of AuNPs-DNA 2 within the range of 20 to 120 mins. However, after 80 mins, the I FMMA /I MB ratio reached a maximum and showed no significant further changes, suggesting that the electrode surface was saturated with AuNPs-DNA 2 at this time point. Thus, 80 mins was determined to be the optimal reaction time for AuNPs-DNA 2 . BMP, as the initiator of the ATRP reaction, played a crucial role in determining whether FMMA could be successfully polymerized and grafted onto the electrode surface. Since the concentration of BMP was maintained in excess, only its reaction time needed to be optimized. As illustrated in Fig. 4 D, within the range of 20 to 120 mins, the I FMMA /I MB signal ratio reached a maximum when the BMP incubation time was 60 mins. Beyond this point, the ratio showed no significant fluctuations, indicating that BMP had achieved maximum modification on the electrode. Therefore, 60 mins was identified as the optimal reaction time for BMP. 3.5 Detection performance analysis Under optimal conditions, the aptasensor's electrical signal response was evaluated across varying ACE concentrations to assess its detection performance. In Fig. 5 A, the electrical signal intensity of MB decreased with increasing ACE concentration, while the signal intensity of the FMMA polymer exhibited a corresponding increase. This behavior aligns with the aptasensor's conceptual design aptasensor. Within the concentration range of 70 pg/mL to 300 ng/mL, the logarithm of ACE concentration presented a good linear relationship with the I FMMA /I MB current intensity signal ratio (Fig. 5 B). The linear regression equation was as follows: I FMMA /I MB =1.71365 lgC ACE + 3.03591 ( R 2 = 0.998), and the limit of detection (LOD) was 19.26 pg/mL (S/N = 3). Compared with other aptasensor detection methods (Table 1 ), the dual-signal ratiometric aptasensor based on I FMMA /I MB exhibited superior detection and analytical performance over single-signal aptasensor relying solely on I FMMA or I MB aptasensor. Table 1 Comparison of the ratiometric aptasensor with other detection methods Methods The LOD (pg/mL) The detection range (ng/mL) Ref EC aptasensor 1.6×10 5 4.45×10 5 − 4.01×10 7 [ 31 ] ECL aptasensor 6.68×10 1 2.23×10 1 − 2.23×10 3 [ 32 ] FL aptasensor 28.5 1×10 2 − 3×10 3 [ 33 ] EC-SERS aptasensor 4.45×10 3 1.11×10 4 − 1.11×10 7 [ 34 ] ratiometric EC aptasensor 19.26 7×10 − 2 − 3×10 2 In the work 3.6 Selectivity, stability and reproducibility Under optimal conditions, the specificity of the aptasensor for ACE was evaluated by testing structurally similar pesticides, including imidacloprid (IMI), methyl parathion (MP) and chlorpyrifos (CPF). The aptasensor's response to above pesticides, ACE, and a mixed solution containing all four pesticides (each at a concentration of 1 ng/mL) was analyzed. As shown in Fig. 6 A, the I FMMA /I MB electrical signal strength ratios for IMI, MP and CPF were relatively weak and similar to each other. In contrast, the I FMMA /I MB signal intensity of mixed group was comparable to that of ACE alone, indicating that the presence of structurally similar pesticides did not significantly interfere with ACE detection. To assess the stability of the aptasensor system, the current response of freshly prepared electrodes was compared with that of electrodes stored at 4°C for 15 days. As depicted Fig. 6 B, the current response of the stored electrode retained 91.69% of its initial value, demonstrating the favorable stability of the aptasensor. In addition, the reproducibility of the aptasensor was investigated through parallel experiments (n = 5) conducted under identical conditions, both within and between groups. As illustrated in Fig. 6 C and Fig. 6 D, the relative standard deviation (RSD) within the group was 4.05%, while the RSD between groups was 4.70%. These results confirmed that the aptasensor system exhibited excellent reproducibility for ACE detection. 3.7 Application of real samples To further evaluate the practical application of the aptasensor for ACE detection in real samples, three different concentrations of ACE (0.5 ng/mL, 1 ng/mL and 5 ng/mL) were spiked into honeysuckle, wolfberry and almond samples. As shown in Table 2 , the recoveries from these samples ranged from 83.20–102.72%, with an average recovery of 93.10%. The relative standard deviations (RSDs) were between 0.75% and 4.89%, with an average RSD of 2.68%. To validate the reliability of the aptasensor, the results were compared with those obtained using an enzyme-linked immunosorbent assay (ELISA). The ACE concentration determined by ELISA were based on the percentage absorbance values, yielding recoveries of 89.70–100.20%, with an average recovery of 95.15%, and RSDs ranging from 0.47–4.63%, with an average RSD of 1.87%. The analysis results from the proposed aptasensor were consistent with those from ELISA, demonstrating the reliability of the aptasensor for real sample detection. These findings indicate that the ratiometric aptasensor was highly suitable for the detection of ACE in real samples. Table 2 ACE detection and recovery of this aptasensor in real samples The aptasensor ELISA Sample Added (ng/mL) Mean measured (ng/mL) Recovery (%) RSD (%) Mean measured (ng/mL) Recovery (%) RSD (%) Honeysuckle 0.50 0.4679 93.59 1.57 0.4896 97.91 3.37 1.00 0.8680 86.80 4.89 0.8969 89.69 1.77 5.00 4.1597 83.19 2.05 4.7910 95.82 1.03 Wolfberry 0.50 0.4751 95.03 1.87 0.4664 93.28 4.63 1.00 0.9703 97.03 2.06 1.0021 100.21 2.42 5.00 4.5727 91.45 4.70 4.8293 96.59 0.95 Almond 0.50 0.4907 98.13 0.75 0.4693 93.87 1.22 1.00 0.8992 89.92 2.81 0.8989 89.89 0.96 5.00 5.1356 102.71 3.39 4.9574 99.15 0.47 4 Conclusion In summary, a ratiometric electrochemical aptasensor based on ATRP reaction and AuNPs dual signal amplification strategy for the detection of ACE is developed for the first time. MB, serving as an internal reference signal, is bound to DNA 1 -Apt by electrostatic adsorption and insertion, which is stronger than the labelling signal 1:1 that can only be modified at the end of the sequence The ATRP reaction enables the in situ grafting of the signal unit FMMA onto the electrode, acting as an amplified growth signal. Meanwhile, AuNPs facilitate the loading of multiple DNA 2 probes, thereby expanding the reaction sites for polymerization. These dual signal amplification strategies significantly improve the sensitivity of the aptasensor. Furthermore, the aptasensor achieves a wide dynamic range (0.07 to 300 ng/mL), a low LOD of 19.26 pg/mL, and excellent interference resistance in complex environments. Notably, this versatile strategy can be adapted for the detection of other pesticide residues by simply replacing the target-specific aptamer, highlighting its potential for broad applications in monitoring and evaluating pesticide residue levels in food and medicinal products. Declarations Author Contribution Contributions All authors contributed to the study conception and design. Weiming Li: Investigation, Methodology, Validation, Data curation, Writing – original draft. Yuzhen Jia: Data curation, Resources. Kunyilan Chen: Validation. Huifang Li: Resources. Huaixia Yang: Conceptualization, Supervision, Writing – review & editing. Liang Guo: Data curation, Methodology, Funding acquisition. Mingsan Miao: Formal analysis, Investigation, Resources, Writing – review & editing. All authors reviewed the manuscript. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 51903248). Data availability The data supporting the findings of this study are available upon reasonable request. References Qin N, Liu J, Li F, Liu J (2024) Recent Advances in Aptasensors for Rapid Pesticide Residues Detection. Crit Rev Anal Chem 54(8):3592–3613. https://doi.org/10.1080/10408347.2023.2257795 Ouyang M, Liu T, Yuan X, Xie C, Luo K, Zhou L (2025) Nanomaterials-based aptasensors for rapid detection and early warning of key food contaminants: A review. Food Chem 462:140990. https://doi.org/10.1016/j.foodchem.2024.140990 Li D, Dong H, Li Z, Wang H, Sun J, Huang J, Li P, Zhou S, Zhai S, Zhao M, Sun X, Guo Y (2024) Novel cross-linkable fluorescent probe with oriented antibody to enhance lateral immunoassay strip for the detection of acetamiprid. J Hazard Mater 476:134935. https://doi.org/10.1016/j.jhazmat.2024.134935 Xi S, Wang L, Cheng M, Hu M, Liu R, Dong Y (2022) Developing a DNA logic gate nanosensing platform for the detection of acetamiprid. 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EFSA J 22(5):e8759. https://doi.org/10.2903/j.efsa.2024.8759 Zhang HX, Yu D, Sun JF, Zeng L, Wang CY, Bai LP, Zhu GY, Jiang ZH, Zhang W (2023) An integrated approach to evaluate acetamiprid-induced oxidative damage to tRNA in human cells based on oxidized nucleotide and tRNA profiling. Environ Int 178:108038. https://doi.org/10.1016/j.envint.2023.108038 Guo L, Zhou S, Xue J, Liu Z, Xu S, He Z, Yang H (2024) Signal-enhanced electrochemical sensor employing MWCNTs/CMK-3/AuNPs and Au@Pd core-shell structure for sensitive determination of AFB1 in complex matrix. Microchim Acta 191(10):594. https://doi.org/10.1007/s00604-024-06665-x Si F, Cui X, Zhang Y, Li Y, Yang H, Liu Y (2024) A novel electrochemical biosensor based on dual signal amplification of CMK-3@AuNPs and ATRP for DR1 detection. Sens Actuator B-Chem 403:135080. https://doi.org/10.1016/j.snb.2023.135080 He X, Zhang W, Wang Y, Wang Z, Wang J, Li X, Yang H, Liu Y, Miao M (2024) Synergistic signal amplification for HER2 biosensing using tetrahedral DNA nanostructures and 2D transition metal carbon/nitride@Au composites via ARGET ATRP. Microchem J 207:112029. https://doi.org/10.1016/j.microc.2024.112029 Yang J, Chen J, Li Y, Wang H, Yang H, Li X, Liu Y (2025) High-Sensitivity Detection of Tobacco Mosaic Virus RNA Based on a Ring-Opening Polymerization Fluorescent Biosensor. J Anal Test press. https://doi.org/10.1007/s41664-025-00364-z Dong H, Zhao L, Zhou Y, Wei X, Xu C, Zhang Y, Xu M (2021) Novel Self-Calibrating Amperometric and Ratiometric Electrochemical Nanotip Microsensor for pH Measurement in Rat Brain. Anal Chem 93(41):13815–13822. https://doi.org/10.1021/acs.analchem.1c02436 Yang H, Wang W, Zeng Y, Tang R, Yan H, Yang Y, Wang H, Wang J, Guo L, Xu J, Li L (2025) A Novel Composite of Aptamer-Based Ti 3 C 2 T x and Molecularly Imprinted Polymer with Double Recognition Property for Sensitive Electrochemical Detection of Ofloxacin. J Anal Test 9:109–117. https://doi.org/10.1007/s41664-024-00335-w Feng A, Li L, He N, Li D, Zheng D, Liu Y, Yang H (2024) A ratiometric electrochemical biosensor based on ARGET ATRP for detection of HER2 gene. Talanta 275:126130. https://doi.org/10.1016/j.talanta.2024.126130 Kunpatee K, Khantasup K, Komolpis K, Yakoh A, Nuanualsuwan S, Sain MM, Chaiyo S (2023) Ratiometric electrochemical lateral flow immunoassay for the detection of Streptococcus suis serotype 2. Biosens Bioelectron 242:115742. https://doi.org/10.1016/j.bios.2023.115742 Li Y, Qin H, Hu C, Sun M, Li P, Liu H, Li J, Li Z, Wu L, Zhu J (2022) Research Progress of Nanomaterials-Based Sensors for Food Safety. J Anal Test 6:431–440. https://doi.org/10.3390/molecules26082130 Ciobanu D, Hosu-Stancioiu O, Melinte G, Ognean F, Simon I, Cristea C (2023) Recent Progress of Electrochemical Aptasensors toward AFB1 Detection (2018–2023). Biosensors 14(1):7. https://doi.org/10.3390/bios14010007 Grabowska I, Hepel M, Kurzątkowska-Adaszyńska K (2021) Advances in Design Strategies of Multiplex Electrochemical Aptasensors. Sensors 22(1):161. https://doi.org/10.3390/s22010161 Ding Z, Yang S, Wang J, Zhao Z, Xu H, Chen Z, Liu Z, Wang Y, Bao J, Chang K, Chen M (2023) Rolling Circle Amplification/G-Quadruplex‐Based Dual‐Signal Ratiometric Electrochemical Aptasensor for Ultrasensitive Detection of Pathogenic Bacteria. ChemElectroChem 10(17):e202300257. https://doi.org/10.1002/celc.202300257 Lu N, Pei H, Ge Z, Simmons C, Yan H, Fan C (2012) Charge Transport within a Three-Dimensional DNA Nanostructure Framework. J Am Chem Soc 134(32):13148–13151. https://doi.org/10.1021/ja302447r Zhang Z, Ou X, Ma L, Li C, Yang Z, Duan J (2024) A double methylene blue labeled single-stranded DNA and hairpin DNA coupling biosensor for the detection of Fusarium oxysporum f. sp. cubense race 4. Bioelectrochemistry 156:108612. https://doi.org/10.1016/j.bioelechem.2023.108612 Zhu C, Liu D, Li Y, Shen X, Ma S, Liu Y, You T (2020) Ratiometric electrochemical aptasensor for ultrasensitive detection of Ochratoxin A based on a dual signal amplification strategy: Engineering the binding of methylene blue to DNA. Biosens Bioelectron 150:111814. https://doi.org/10.1016/j.bios.2019.111814 Ding Y, Zhang S, Zang X, Ding M, Ding C (2024) Ratiometric antifouling electrochemical biosensors based on designed Y-shaped peptide and MXene loaded with Au@ZIF-67 and methylene blue. Microchim Acta 191(1):5. https://doi.org/10.1007/s00604-023-06079-1 Ma N, Qiu W, Wei G, Zhang J, Yu H, Kong J, Zhang X (2024) GOX mediated oxygen tolerance ATRP for detection of esophageal cancer biomarker miRNA-144. Chem Eng J 495:153543. https://doi.org/10.1016/j.cej.2024.153543 Yazdi MK, Zarrintaj P, Saeb MR, Mozafari M, Bencherif SA (2024) Progress in ATRP-derived materials for biomedical applications. Prog Mater Sci 143:101248. https://doi.org/10.1016/j.pmatsci.2024.101248 Coskun HI, Bossa FDL, Hu XL, Jockusch S, Sobieski J, Yilmaz G, Matyjaszewski K (2024) ATRP with ppb Concentrations of Photocatalysts. J Am Chem Soc 146(12):28994–29005. https://doi.org/10.1021/jacs.4c09927 Pei X, Liu J, Zhang Y, Huang Y, Li Z, Niu X, Zhang W, Sun W (2024) Tetrahedral DNA-linked aptamer-antibody-based sandwich-type electrochemical sensor with Ag@Au core-shell nanoparticles as a signal amplifier for highly sensitive detection of α-fetoprotein. Microchim Acta 191:414. https://doi.org/10.1007/s00604-024-06485-z Zhong Y, Li Z, Zhang A, Peng Y, Zhou H, Guo Y, Lu D, Xie L, Shi S (2024) Gold nanoparticle-mediated molecularly imprinted electrochemical sensor MIP/AuNPs/GCE for highly sensitive and selective detection of neutral phosmet residues in fruits and vegetables. Microchem J 201:110728. https://doi.org/10.1016/j.microc.2024.110728 Guo L, Zhou S, Liu Y, Yang H, Miao M, Gao W (2024) Facile and controllable hybrid-nanoengineering of MWCNTs/Au@ZIF-8 and AuPt@CeO 2 based sandwich electrochemical aptasensor for AFB1 determination in foods and herbs. J Saudi Chem Soc 28(6):101946. https://doi.org/10.1016/j.jscs.2024.101946 Chmielarz P, Fantin M, Park S, Isse AA, Gennaro A, Magenau AJD, Sobkowiak A, Matyjaszewski K (2017) Electrochemically mediated atom transfer radical polymerization (eATRP). Prog Polym Sci 69:47–78. https://doi.org/10.1016/j.progpolymsci.2017.02.005 Ai J, Wang X, Zhang Y, Hu H, Zhou H, Duan Y, Wang D, Wang H, Du H, Yang Y (2022) A sensitive electrochemical sensor for nitenpyram detection based on CeO 2 /MWCNTs nanocomposite. Appl Phys A 128(9):831. https://doi.org/10.1007/s00339-022-05952-9 Sun J, Wang H, Li P, Li C, Li D, Dong H, Guo Z, Geng L, Zhang X, Fang M, Xu Y, Ahmed MBM, Guo Y, Sun X (2024) Metal-organic framework-based aptasensor utilizing a novel electrochemiluminescence system for detecting acetamiprid residues in vegetables. Biosens Bioelectron 259:116371. https://doi.org/10.1016/j.bios.2024.116371 Yu Y, Ye S, Sun Z, You J, Li W, Song Y, Zhang H (2022) A fluorescent aptasensor based on gold nanoparticles quenching the fluorescence of rhodamine B to detect acetamiprid. RSC Adv 12(54):35260–35269. https://doi.org/10.1039/D2RA05037D Wu T, Tang X, Zeng W, Han Y, Zhang S, Wei J, Wu L (2024) Potential powered EC-SERS for sensitive detection of acetamiprid. J Food Eng 378:112109. https://doi.org/10.1016/j.jfoodeng.2024.112109 Scheme 1 Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Scheme1.png Scheme 1. (A) ACE ratiometric electrochemical aptasensor construction detection diagram. (B) ATRP response mechanism. Cite Share Download PDF Status: Published Journal Publication published 23 Jun, 2025 Read the published version in Microchimica Acta → Version 1 posted Editorial decision: Revision requested 07 Apr, 2025 Reviews received at journal 06 Apr, 2025 Reviews received at journal 05 Apr, 2025 Reviewers agreed at journal 31 Mar, 2025 Reviewers agreed at journal 30 Mar, 2025 Reviews received at journal 29 Mar, 2025 Reviewers agreed at journal 29 Mar, 2025 Reviewers agreed at journal 28 Mar, 2025 Reviews received at journal 28 Mar, 2025 Reviewers agreed at journal 28 Mar, 2025 Reviewers invited by journal 28 Mar, 2025 Editor assigned by journal 26 Mar, 2025 Submission checks completed at journal 26 Mar, 2025 First submitted to journal 24 Mar, 2025 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-6299477","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":433483120,"identity":"ee8523dc-b82e-4788-93fb-f054b43840db","order_by":0,"name":"Weiming Li","email":"","orcid":"","institution":"Henan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Weiming","middleName":"","lastName":"Li","suffix":""},{"id":433483121,"identity":"dabb8f7a-8733-42e1-8b05-411e77ef5573","order_by":1,"name":"Yuzhen Jia","email":"","orcid":"","institution":"Henan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yuzhen","middleName":"","lastName":"Jia","suffix":""},{"id":433483122,"identity":"2f593405-8f47-41e7-b01c-49e9b70cd63e","order_by":2,"name":"Kunyilan Chen","email":"","orcid":"","institution":"Henan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Kunyilan","middleName":"","lastName":"Chen","suffix":""},{"id":433483123,"identity":"6c95fbd9-1c47-49d2-86d9-35bd43b52dfa","order_by":3,"name":"Huifang Li","email":"","orcid":"","institution":"Henan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Huifang","middleName":"","lastName":"Li","suffix":""},{"id":433483124,"identity":"54b4f12c-b22f-4d00-9b1d-ac5abea436fd","order_by":4,"name":"Huaixia Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/klEQVRIiWNgGAWjYJACZiDmAZKMDz5U2PDw8zcQr4XZcMaZNBnJGQeI0wICbNK8LYdtDBoS8Cs3OH728OvCNjsZg+PMjw1nNpznMWA4wPjhYw4eLWfy0qxntiXzSDazGT74uOM2jzlzA7PkzG14tBzIMTPmbWPm4WdmMDaceeY2j2XDATZmXnxazr8BaannYWNm/ybN23aOx+BAAgEtN3KMH/O2HQbawmMG1HKAsBbJG2/MmHnOHQf6hacYGMhAT8042IzXL3znc4w/85RV2xucP74RGJV29vz8zQc/fMSjReEAA5sEmhhjA271QCDfwMD8Aa+KUTAKRsEoGAUAaQxRNwjFSLEAAAAASUVORK5CYII=","orcid":"","institution":"Henan University of Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Huaixia","middleName":"","lastName":"Yang","suffix":""},{"id":433483126,"identity":"501684af-17ce-4b3f-aa48-dc3abcf3f9b9","order_by":5,"name":"Liang Guo","email":"","orcid":"","institution":"Henan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Liang","middleName":"","lastName":"Guo","suffix":""},{"id":433483127,"identity":"6ce9459a-21fe-411f-ae0d-8cd62601064a","order_by":6,"name":"Mingsan Miao","email":"","orcid":"","institution":"Henan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Mingsan","middleName":"","lastName":"Miao","suffix":""}],"badges":[],"createdAt":"2025-03-25 03:08:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6299477/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6299477/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00604-025-07217-7","type":"published","date":"2025-06-23T15:57:17+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79237085,"identity":"cb889ef7-bc49-4fc3-828b-4fa2f5185d18","added_by":"auto","created_at":"2025-03-26 04:25:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1338010,"visible":true,"origin":"","legend":"\u003cp\u003eThe TEM images of AuNPs (A), AuNP-DNA\u003csub\u003e2\u003c/sub\u003e (B), inserting the respective diameter dispersion respectively. (C) the UV-vis spectra of AuNPs (curve a), DNA\u003csub\u003e2 \u003c/sub\u003e(curve b) and AuNP-DNA\u003csub\u003e2\u003c/sub\u003e (curve c).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6299477/v1/b8be6ffa639390431594efea.png"},{"id":79236402,"identity":"e5de892d-fd9a-4642-bab4-d4d6f94ac478","added_by":"auto","created_at":"2025-03-26 04:17:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2234711,"visible":true,"origin":"","legend":"\u003cp\u003e(A) The SWV current signal intensity: control without Apt-DNA\u003csub\u003e1\u003c/sub\u003e (a), without MB (b), without ACE (c), without DNA\u003csub\u003e2\u003c/sub\u003e-AuNPs/BMP (d), without FMMA (e) and experimental (d). (B) and (C) Electrochemical impedance (EIS) and cyclic voltammetry (CV), respectively, for bare Au (a), DNA\u003csub\u003e1\u003c/sub\u003e-Apt (b), MB (c), ACE (d), Au-DNA\u003csub\u003e2\u003c/sub\u003e (e), BMP (f) and FMMA (g).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6299477/v1/6e1a6a3ac2b33e24c44e4e63.png"},{"id":79237086,"identity":"907e70a3-f73b-4200-8d3a-15709dc0ad87","added_by":"auto","created_at":"2025-03-26 04:25:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1932614,"visible":true,"origin":"","legend":"\u003cp\u003eThe AFM images of the DNA\u003csub\u003e1\u003c/sub\u003e-Apt/MB/ACE (A), DNA\u003csub\u003e1\u003c/sub\u003e-Apt/MB/ACE/AuNPs-DNA\u003csub\u003e2\u003c/sub\u003e (B), DNA\u003csub\u003e1\u003c/sub\u003e-Apt/MB/ACE/AuNPs-DNA\u003csub\u003e2\u003c/sub\u003e/BMP (C), and DNA\u003csub\u003e1\u003c/sub\u003e-Apt/MB/ACE/AuNPs-DNA\u003csub\u003e2\u003c/sub\u003e/BMP/FMMA(D), with the corresponding cross section.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6299477/v1/59380cea232bc8e1ec21e52b.png"},{"id":79236411,"identity":"27dd0d76-5769-48b0-8745-6f5858c2ba46","added_by":"auto","created_at":"2025-03-26 04:17:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2889913,"visible":true,"origin":"","legend":"\u003cp\u003e(A) ACE time optimization. (B) and (C) The volume optimization and time optimization of AuNPs-DNA\u003csub\u003e2\u003c/sub\u003e, respectively. (D) BMP time optimization.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6299477/v1/6b9f131cb4dc16bf5c3a7733.png"},{"id":79237089,"identity":"9c52ba69-5b63-4a7c-bff9-0dd60b69b6aa","added_by":"auto","created_at":"2025-03-26 04:25:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":709114,"visible":true,"origin":"","legend":"\u003cp\u003e(A) The SWV electrical signal response corresponding to the range of varying concentrations of ACE. (B) The linear relationship between ACE concentration and I\u003csub\u003eFMMA\u003c/sub\u003e/I\u003csub\u003eMB\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6299477/v1/246647052fa5703e46af2a75.png"},{"id":79236408,"identity":"6c05a96b-4be7-4ca7-8122-512a46008add","added_by":"auto","created_at":"2025-03-26 04:17:39","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":663512,"visible":true,"origin":"","legend":"\u003cp\u003e(A)The responses of the developed aptasensor towards different analytes: IMI, MP, CPF, ACE and Mix. (B) The sensing current changes after 15 days. (C) Interclass (1 - 5) and interblock (6 - 10) signal responses of the sensing system, respectively.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6299477/v1/5f0cf666dc5bc4bfe196090b.png"},{"id":85686212,"identity":"cbcd2449-3124-42a9-81b4-1b546a7ff07e","added_by":"auto","created_at":"2025-06-30 16:04:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8834084,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6299477/v1/5e15b258-7d52-4793-ae12-e3de569ed575.pdf"},{"id":79236403,"identity":"9be42031-aee6-4e89-8969-830ed0b98e74","added_by":"auto","created_at":"2025-03-26 04:17:39","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1284455,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1. \u003c/strong\u003e(A) ACE ratiometric electrochemical aptasensor construction detection diagram. (B) ATRP response mechanism.\u003c/p\u003e","description":"","filename":"Scheme1.png","url":"https://assets-eu.researchsquare.com/files/rs-6299477/v1/272ddf877319a4362c851bb0.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Entropy spontaneous ratiometric electrochemical aptasensor based on polymerization and AuNPs signal amplification for Acetamiprid residue analysis","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eAcidamidine (ACE), a selective agonist of type II nicotinic acetylcholine receptors (nAChRs), targets nAChRs in the central nervous system of insects and is one of the most extensively utilized neonicotinoid insecticides [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, the excessive and improper use of ACE has led to significant concerns regarding pesticide residues. In recent years, ACE residues have been detected in various common foods and medicinal herbs [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Studies have shown that the long-term accumulation of ACE and its toxic metabolites in mammals can alter hematological, biochemical and structural characteristics, ultimately leading to neurological, hepatorenal, immune, reproductive effects and genotoxicity [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Given its potential risks to human health and the environment, the development of simple and efficient ACE detection technologies is crucial for monitoring and assessing ACE contamination levels, as well as ensuring the safety of food and pharmaceutical products.\u003c/p\u003e \u003cp\u003eElectrochemical aptasensors represent a well-established and advanced method for trace detection of small molecules, having been extensively developed in recent years [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Owing to their high sensitivity, rapid analysis, cost-effectiveness and operational simplicity, they have gained widespread application in various fields, such as food safety, environmental monitoring, clinical detection [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Whereas, conventional electrochemical sensors typically rely on a single signal output for target molecule detection. This single-signal approach is prone to aptasensor false signal interference due to unavoidable instrumental variations or external environmental factors, particularly when detecting trace amounts of small molecules in complex sample matrices [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. To address these limitations, the integration of ratiometric sensing with dual or multiple signal responses has been proposed [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. By utilizing the ratio of two distinct signals as the output, this approach can effectively compensate for response deviations caused by internal or external factors, providing inherent built-in correction for system or background electrical signals [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Consequently, ratiometric aptasensors based on dual-signal responses demonstrate superior accuracy and reliability, offering significant potential for practical applications in complex sample analysis.\u003c/p\u003e \u003cp\u003eCurrently, ratiometric electrochemical strategies are widely employed in aptasensor construction, utilizing redox tags such as methylene blue (MB), ferrocene (Fc) and thionin (Thi) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Notably, MB exhibits versatile binding interactions with DNA, enabling it to attach to phosphate groups on single-stranded DNA (ssDNA) through electrostatic adsorption or intercalate into the double-helix structure of double-stranded DNA (dsDNA) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Leveraging this principle, MB can serve as a label-free binding probe to enhance the response signal strength in electrochemical aptasensors. Zhu et al. [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] designed a ratiometric electrochemical aptasensor for ultrasensitive detection of Ochratoxin A, employing a dual signal amplification strategy. This approach utilized the binding of MB to DNA, combined with the introduction of hybrid DNA (hDNA) and MB post-OTA recognition, to amplify the response signal. The target concentration was accurately quantified by measuring the signal ratio of I\u003csub\u003eFc\u003c/sub\u003e/I\u003csub\u003eMB\u003c/sub\u003e. Similarly, Ding et al. [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] developed an antifouling electrochemical biosensor based on a Y-shaped peptide and MXene loaded with Au@ZIF-67 and MB. In this system, electrochemical signal molecules (Fc and MB) were modified at distinct locations (on the peptide and within the MXene-Au@ZIF-67 composite, respectively) to generate a ratiometric electrochemical signal.\u003c/p\u003e \u003cp\u003eIn addition, to further enhance the sensitivity of ratiometric electrochemical aptasensor, signal amplification strategies involving polymerization reactions and nanomaterials are often employed. For example, atom transfer radical polymerization (ATRP) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], a prominent controlled/living radical polymerization technique, offers advantages such as wide range of monomers and narrow molecular distribution. ATRP is widely recognized as an effective for synthesizing grafted polymer copolymers. Additionally, gold nanoparticles (AuNPs), known for their excellent biocompatibility, easy of surface modification, and superior electronic conductivity, are frequently employed as carriers to load signaling probes [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The loading process typically involves the formation of covalent bonds between functional groups (e.g., -SH, -NH\u003csub\u003e2\u003c/sub\u003e) on DNA and the surface of AuNPs. Due to its superior characteristics, AuNPs are widely used in electrochemical aptasensor. Therefore, this study has introduced the two signal amplification strategies of ATRP and AuNPs into the development of ratiometric electrochemical aptasensor for the detection of ACE.\u003c/p\u003e \u003cp\u003eIn this work, a ratiometric electrochemical aptasensor was developed for the detection of ACE, incorporating a dual-signal amplification strategy based on ATRP and AuNPs aptasensor. MB was employed as an internal reference signal, while the ATRP reaction was utilized to synthesize polymers using ferrocenyl methyl methacrylate (FMMA) as the signal unit. The polymer serves as the amplification signal for the proposed aptasensor. Additionally, AuNPs were functionalized with multiple DNA\u003csub\u003e2\u003c/sub\u003e probes to increase the active site of ATRP reaction. The aptasensor was constructed by self-assembling thiolated DNA\u003csub\u003e1\u003c/sub\u003e-aptamer (SH-DNA\u003csub\u003e1\u003c/sub\u003e-Apt) aptasensor onto the gold electrode via an Au-S bond, with MB bound to the double-stranded DNA through intercalation and weak electrostatic interactions. In the presence of ACE, the electrical signal of MB was attenuated, exposing the DNA\u003csub\u003e1\u003c/sub\u003e site, which then hybridized with the AuNPs-DNA\u003csub\u003e2\u003c/sub\u003e probes through complementary base pairing. Subsequently, upon activation of the ATRP initiator, the signal unit FMMA was grafted onto the electrode surface, forming a conductive polymer. This strategy achieved a ratiometric signal response characterized by one signal decreasing while the other increased, demonstrating high sensitivity and specificity. The proposed aptasensor provides a novel and effective method for the analysis of pesticide residues in agricultural products and environmental samples, offering significant potential for practical applications.\u003c/p\u003e"},{"header":"2 Experimental","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Reagents and materials\u003c/h2\u003e \u003cp\u003eACE standard, gold perchlorate (HAuCl\u003csub\u003e4\u003c/sub\u003e), α-bromoisobutyric acid (BMP), n-hydroxysuccinimide (NHS), N-(3-dimethylaminopropyl)-N'-ethyl diimide hydrochloride (EDC), Ferrocenyl methyl methacrylate (FMMA) and 6-mercaptohexyl alcohol (MCH) were derived from Shanghai Aladdin Reagent Co., Ltd (Shanghai, China). Ascorbic acid (AA) and Trisodium citrate dihydrate (C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e5\u003c/sub\u003eNa\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e\u0026middot;2H\u003csub\u003e2\u003c/sub\u003eO) were bought by Balinway Technology Co., Ltd (Shanghai, China). Copper bromide (CuBr\u003csub\u003e2\u003c/sub\u003e), K\u003csub\u003e3\u003c/sub\u003e[Fe(CN)\u003csub\u003e6\u003c/sub\u003e] and K\u003csub\u003e4\u003c/sub\u003e[Fe(CN)\u003csub\u003e6\u003c/sub\u003e]\u0026middot;3H\u003csub\u003e2\u003c/sub\u003eO were purchased from Sinopharmate Chemical Reagent Co., Ltd. 0.1 M PBS (pH\u0026thinsp;=\u0026thinsp;7.4) was the buffer system and electrochemical measurement solution of the whole experiment. Enzyme linked immunosorbent assay (ELISA) of ACE was purchased from Jiangsu Enzyme Free Industrial Co., Ltd (Jiangsu, China). The DA used in all experiments was treated with the Millipore system (\u0026ge;\u0026thinsp;18.25 MΩ cm). All the HPLC-purified DNA sequences were synthesized and purified by Wuhan Cloud Clone Technology Co., Ltd (Wuhan, China). Detailed sequences are listed as follows:\u003c/p\u003e \u003cp\u003eAptamer of ACE: 5'-SH-(CH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e6\u003c/sub\u003e-CTG ACA CCA TAT TAT GAA GA-3'\u003c/p\u003e \u003cp\u003eDNA\u003csub\u003e1\u003c/sub\u003e: 5'-SH-(CH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e6\u003c/sub\u003e-TCT TCA TAA TAT GGT GTC AG-3'\u003c/p\u003e \u003cp\u003eDNA\u003csub\u003e2\u003c/sub\u003e: 5'-SH-(CH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e6\u003c/sub\u003e-CTG ACA CCA TAT TAT-(CH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e6\u003c/sub\u003e-NH\u003csub\u003e2\u003c/sub\u003e-3'\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Apparatus\u003c/h2\u003e \u003cp\u003eThe standard three-electrode system (reference electrode: saturated calomel, counter electrode: Pt filament) was used for electrochemical tests in this experiment. Square wave voltammetry (SWV) was performed on the CHI 760E Electrochemical Workstation (Shanghai Chenhua Instrument Co., Ltd, China). Characterization of electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) was performed at the Autolab PGSTAT204 Electrochemical workstation (Eco Chemie, Netherlands). Ultraviolet-visible spectroscopy (Shimadzu UV-3600 Plus) measures the absorbance of AuNPs-DNA\u003csub\u003e2\u003c/sub\u003e. The modified gold electrode morphology was characterized by Dimension Icon Atomic force microscope (AFM, Bruker, USA) and Sigma HD field emission scanning electron microscope (TEM, Zeiss, Germany), respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Synthesis of AuNPs-DNA\u003csub\u003e2\u003c/sub\u003e\u003c/h2\u003e \u003cp\u003eFirstly, the synthesis method of AuNPs was slightly modified based on previously reported procedure [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Briefly, 50 mL 0.01% HAuCl\u003csub\u003e4\u003c/sub\u003e was added to the 100 mL conical flask and heated to boiling on a magnetic stirrer at 1000 r/min at 120\u0026deg;C. Afterwards, 1.75 mL 1%C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e5\u003c/sub\u003eNa\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e\u0026middot;2H\u003csub\u003e2\u003c/sub\u003eO was quickly added in the above liquid stirring state, the liquid color gradually changed from colorless to wine red, and continued to boil for 10 mins. Finally, the heating was stopped and cooled to room temperature at 1000 r/min. The obtained AuNPs were placed in the refrigerator at 4\u0026deg;C for storage. During the preparation of the AuNPs-DNA\u003csub\u003e2\u003c/sub\u003e, 500 \u0026micro;L of the above AuNPs was taken and mixed with 10 \u0026micro;L 100 \u0026micro;M DNA\u003csub\u003e2\u003c/sub\u003e and shaken well, and incubated in a shaker for 3 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Construction of this ratiometric aptasensor\u003c/h2\u003e \u003cp\u003eThe gold electrodes were polished with 0.05 \u0026micro;M Al\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e powder on a chamois leather clockwise for 3 mins to remove surface impurities before used. Afterwards, the treated gold electrode was ultrasounded with DW for 1 min and immersed in a mixture of 98% H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e and 30% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (v/v\u0026thinsp;=\u0026thinsp;3:1) for 15 min. Eventually, CV scanning was performed in 0.5 mM H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e solution to remove impurities on the electrode surface (potential range: -0.3 V \u0026minus;\u0026thinsp;1.5 V. Scan rate: 0.2 V s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e).\u003c/p\u003e \u003cp\u003eThe mixture of 2 \u0026micro;M DNA\u003csub\u003e1\u003c/sub\u003e and 3 \u0026micro;M Apt in equal volume was shaken well and incubated in the shaker overnight at room temperature, so that DNA\u003csub\u003e1\u003c/sub\u003e and Apt reacted as completely as possible to obtain 1 \u0026micro;M DNA\u003csub\u003e1\u003c/sub\u003e-Apt. Then, 10 \u0026micro;L 1 \u0026micro;M DNA\u003csub\u003e1\u003c/sub\u003e-Apt was added to the surface of the gold electrode and reacted at room temperature overnight. The modified electrode was immersed in 2 mM MCH solution (solvent 70% ethanol) and reacted at 37 ℃ for 1 h to close the remaining active site on the electrode surface. 10 \u0026micro;L 0.2 mM MB (internal reference signal) reacted with the modified electrode at 37℃ for 1 h, which bound to DNA\u003csub\u003e1\u003c/sub\u003e-Apt by electrostatic adsorption and insertion. Subsequently, 10 \u0026micro;L ACE with different concentrations was incubated on the electrode at 37℃ for 50 mins. Subsequently, 10 \u0026micro;L 1 \u0026micro;M AuNPs-DNA\u003csub\u003e2\u003c/sub\u003e was added and incubated at 37℃ for 70 mins. The initiator BMP was mixed with EDC/NHS (20 mM:5 mM) in equal volume to activate the carboxyl group on the surface. 10 \u0026micro;L 3 mM activated BMP was reacted on the modified electrode at 37℃ for 60 min. Finally, the modified electrode was immersed in 200 \u0026micro;L ATRP reaction solution (20 \u0026micro;L 10 mM FMMA, 20 \u0026micro;L 2 mM AA, 20 \u0026micro;L 2mM CuBr\u003csub\u003e2\u003c/sub\u003e/Me\u003csub\u003e6\u003c/sub\u003eTREN\u003csup\u003e+\u003c/sup\u003e and 140 \u0026micro;L PBS) and reacted at 37℃ for 2 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Detection of the aptasensor\u003c/h2\u003e \u003cp\u003eThe constructed aptasensor was measured by square wave voltammetry (SWV) on CHI760E electrochemical work using 0.1 M pH\u0026thinsp;=\u0026thinsp;7.4 PBS buffer as the electrolyte solution (the sweep potential: -0.6 V \u0026minus;\u0026thinsp;0.6 V, the step potential: 4 mV, the frequency: 25 Hz, the amplitude: 25 mV). The signal variation trend between MB and FMMA was observed under different concentrations of ACE.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results and discussion","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 The principle of the aptasensor\u003c/h2\u003e\n \u003cp\u003eThis study proposed the construction of an FMMA/MB ratiometric electrochemical aptasensor based on ATRP amplification strategy to detect ACE (Scheme\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). DNA\u003csub\u003e1\u003c/sub\u003e-Apt was immobilized on the electrode surface by Au-S bond formed by sulfhydryl group (-SH) on DNA\u003csub\u003e1\u003c/sub\u003e. MCH was employed to block the remaining active sites on the electrode surface, thereby preventing non-specific binding interference. Furthermore, MB, serving as an internal reference signal, was attached to the electrode surface via intercalation or electrostatic interactions with the phosphate group on DNA\u003csub\u003e1\u003c/sub\u003e-Apt. Upon the introduction of varying concentrations of ACE, the Apt containing MB specifically captured ACE and was subsequently released from the gold electrode surface, leading to a reduction in the MB electrical signal. Following this, the AuNPs-DNA\u003csub\u003e2\u003c/sub\u003e probe hybridized with the single-stranded DNA\u003csub\u003e1\u003c/sub\u003e through complementary base pairing. The initiator BMP was then conjugated to the electrode surface via an amide bond (-CO-NH-) formed between the activated carboxyl group (-COOH) on BMP and the amino group (-NH\u003csub\u003e2\u003c/sub\u003e) on DNA\u003csub\u003e2\u003c/sub\u003e. Finally, the signal unit FMMA was grafted onto the electrode surface through an in situ ATRP reaction, utilizing BMP as the initiator, AA as the reducing agent, and CuBr\u003csub\u003e2\u003c/sub\u003e/Me\u003csub\u003e6\u003c/sub\u003eTREN\u003csup\u003e+\u003c/sup\u003e as the catalyst. This study achieved ratiometric electrochemical detection of ACE by monitoring the ratio of the MB signal to the FMMA signal.\u003c/p\u003e\n \u003cp\u003eThe reaction mechanism of ATRP was illustrated in Scheme \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB. The Br-Cu\u003csup\u003eII\u003c/sup\u003e/Me\u003csub\u003e6\u003c/sub\u003eTREN\u003csup\u003e+\u003c/sup\u003e complex was reduced to the catalytically active Cu\u003csup\u003eI\u003c/sup\u003e/Me\u003csub\u003e6\u003c/sub\u003eTREN by the reducing agent AA. The Cu\u003csup\u003eI\u003c/sup\u003e/Me\u003csub\u003e6\u003c/sub\u003eTREN then reacted with the initiator (Gn-Br) to generate free radicals (Gn\u0026bull;) and the oxidation state complex dormant substance Br-Cu\u003csup\u003eII\u003c/sup\u003e /Me\u003csub\u003e6\u003c/sub\u003eTREN\u003csup\u003e+\u003c/sup\u003e. The free radicals (Gn\u0026bull;) and the monomer (FMMA) initiated the polymerization reaction through double bond addition, ultimately forming a polymer (GnGm). Simultaneously, excessive AA reduced Br-Cu\u003csup\u003eII\u003c/sup\u003e /Me\u003csub\u003e6\u003c/sub\u003eTREN\u003csup\u003e+\u003c/sup\u003e, initiating a new cycle of polymerization. According to Eq.\u0026nbsp;1[\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e], the rate of ATRP (R\u003csub\u003ep\u003c/sub\u003e) was determined by the propagation rate constant (\u003cem\u003ek\u003c/em\u003e\u003csub\u003ep\u003c/sub\u003e) and the concentration of monomer (M) and free radicals (Gn\u0026bull;). In turn, the concentration of Gn\u0026bull; depended on the ATRP equilibrium constant (\u003cem\u003eK\u003c/em\u003e\u003csub\u003eATRP\u003c/sub\u003e), which was defined by the concentrations of the Gn-X, Cu\u003csup\u003eI\u003c/sup\u003eL and X-Cu\u003csup\u003eII\u003c/sup\u003eL.\u003c/p\u003e\n \u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" style=\"width: 501px;\"\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Characterization of AuNPs-DNA\u003csub\u003e2\u003c/sub\u003e\u003c/h2\u003e\n \u003cp\u003eThe morphology of the synthesized AuNPs-DNA\u003csub\u003e2\u003c/sub\u003e probe was characterized by TEM. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA and Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB, the synthesized AuNPs exhibited a spherical shape with uniformly dispersion and an average diameter of approximately 21.80 nm. Following the covalent incubation of DNA\u003csub\u003e2\u003c/sub\u003e with AuNPs, the diameter of the nanoparticles increased to 27.54 nm, attributed to the presence of residual salt residues surrounding the DNA\u003csub\u003e2\u003c/sub\u003e. This result confirmed the successful conjugation of DNA\u003csub\u003e2\u003c/sub\u003e onto the AuNPs. In addition, the UV-Vis spectroscopy was employed to assess the modification efficiency of DNA\u003csub\u003e2\u003c/sub\u003e on AuNPs (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC). The UV-Vis spectrum of AuNPs displayed a characteristic absorption peak at 518.78 nm (curve a), while DNA\u003csub\u003e2\u003c/sub\u003e exhibited a characteristic peak at 258.02 nm (curve b). After the modification of AuNPs with DNA\u003csub\u003e2\u003c/sub\u003e, the resulting spectrum showed the presence of both DNA\u003csub\u003e2\u003c/sub\u003e and AuNPs characteristic peaks. Notably, the AuNPs peak exhibited a redshift from 518.78 nm to 521.86 nm (curve c), further confirming the successful functionalization of AuNPs with DNA\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 Feasibility and characterization of the aptasensor\u003c/h2\u003e\n \u003cp\u003eIn order to analyze the feasibility of the ratiometric electrochemical aptasensor, this section examined and compared the changes in the electrical signal intensities of MB and FMMA under conditions where one of the key modifications was systematically omitted. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA, in the fully modified experimental group (curve f), two distinct signal peaks were observed at the potential \u0026minus;\u0026thinsp;0.288 V and +\u0026thinsp;0.265 V, corresponding to the signals of MB and FMMA, respectively. In contrast, when the aptasensor lacked the carrier DNA\u003csub\u003e1\u003c/sub\u003e-Apt (curve a), no significant signal peaks for either MB or FMMA were detected. In the absence of ACE (curve c), the current intensity of MB remained unchanged, and no noticeable FMMA signal was observed. This was attributed to the failure of the aptamer to recognize ACE, preventing the probe DNA\u003csub\u003e2\u003c/sub\u003e-AuNPs from hybridizing with DNA\u003csub\u003e1\u003c/sub\u003e and consequently hindering the grafting of FMMA onto the electrode surface. Similarly, when the DNA\u003csub\u003e2\u003c/sub\u003e-AuNPs/BMP (curve d) modification was omitted, the results mirrored those of curve c. Additionally, in the absence of MB (curve b) and FMMA (curve e) modifications, no corresponding current signal peaks were observed. Based on the above analysis, it could be concluded that the construction of the aptasensor is feasible.\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003eTo further investigate the construction of the aptasensor, the EIS was employed to characterize the layer-by-layer assembly process. The measurements were conducted in a frequency range of aptasensor 0.1 MHz to 0.1 Hz with a sine wave potential amplitude of 5 mV. The electrodes, under different modification conditions, were immersed in a 5 mM [Fe (CN)\u003csub\u003e6\u003c/sub\u003e]\u003csup\u003e3\u0026minus;/4\u0026minus;\u003c/sup\u003e electrolyte solution containing 0.1 M KNO\u003csub\u003e3\u003c/sub\u003e. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB, the bare gold electrode exhibited the lowest charge transfer resistance (\u003cem\u003eRct\u003c/em\u003e) of -197.55 Ω (curve a). Upon modification with DNA\u003csub\u003e1\u003c/sub\u003e-Apt and MCH, the \u003cem\u003eRct\u003c/em\u003e value increased to -651.69 Ω (curve b) due to the strong electrostatic repulsion between the phosphate group of DNA\u003csub\u003e1\u003c/sub\u003e-Apt and [Fe (CN)\u003csub\u003e6\u003c/sub\u003e]\u003csup\u003e3\u0026minus;/4\u0026minus;\u003c/sup\u003e, as well as the blocking effect of MCH, which hindered the electron transfer at the electrode surface. When ACE specifically recognized by the aptamer, the detachment of the MB-containing Apt from the electrode surface led to a decrease in \u003cem\u003eRct\u003c/em\u003e to -476.93 Ω (curve d). Subsequent modification with AuNPs-DNA\u003csub\u003e2\u003c/sub\u003e resulted in a further increase in \u003cem\u003eRct\u003c/em\u003e to -1147.2Ω (curve e), attributed to the poor conductivity and high steric hindrance of the AuNPs. The introduction of the initiator BMP via amide bonding further impeded electron transfer, raising the \u003cem\u003eRct\u003c/em\u003e to -1355.93 Ω (curve f). Finally, the formation of a large amount of FMMA polymer on the electrode surface significantly increased steric hindrance, causing a substantial rise in \u003cem\u003eRct\u003c/em\u003e to -2182.90Ω (curve g). In addition, the progressive modification of the electrode was characterized using CV. As depicted in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC, the current intensity decreased progressively with each modification step, following the order: curve a\u0026thinsp;\u0026gt;\u0026thinsp;curve c\u0026thinsp;\u0026gt;\u0026thinsp;curve b\u0026thinsp;\u0026gt;\u0026thinsp;curve d\u0026thinsp;\u0026gt;\u0026thinsp;curve f\u0026thinsp;\u0026gt;\u0026thinsp;curve g. This trend in current intensity was consistent with the EIS results, confirming the successful layer-by-layer assembly of the aptasensor.\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003eFurthermore, AFM was utilized to compare the height changes of the aptasensor surface before and after the modifications involving AuNPs-DNA\u003csub\u003e2\u003c/sub\u003e modification and ATRP reaction. Following the self-assembly of DNA\u003csub\u003e1\u003c/sub\u003e-Apt/MB/ACE on the gold substrate, the surface height was measured at 34.9 nm (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). Upon modification with AuNPs-DNA\u003csub\u003e2\u003c/sub\u003e, the height increased to 58.7 nm, reflecting a rise of 23.8 nm, which confirmed the successful assembly of AuNPs-DNA\u003csub\u003e2\u003c/sub\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). After the introduction of the initiator BMP, the surface height further increased to 65.1 nm (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC). During the ATRP reaction, the surface height of gold substrate reached 105.1 nm, marking an additional increase of 40.0 nm, indicating the successful grafting of FMMA onto the aptasensor surface (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD). These results collectively demonstrated the effective step-by-step modification and functionalization of the aptasensor.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4 Optimization of experimental parameters\u003c/h2\u003e\n \u003cp\u003eTo improve the ACE detection performance of the ratio aptasensor, several experimental parameters of the aptasensor were optimized, such as the incubation time of ACE, the volume and reaction time of AuNPs-DNA\u003csub\u003e2\u003c/sub\u003e, the reaction time of BMP and the incubation time of ATRP reaction.\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003eThe specific recognition of ACE by the Apt exposed the DNA\u003csub\u003e1\u003c/sub\u003e site, enabling subsequent hybridization with DNA\u003csub\u003e2\u003c/sub\u003e and the grafting of electrochemical signals. Therefore, optimizing the reaction time between ACE and aptamer was crucial for achieving optimal aptasensor performance. In Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA, the I\u003csub\u003eFMMA\u003c/sub\u003e/I\u003csub\u003eMB\u003c/sub\u003e signal ratio increased with the ACE reaction time within the range of 20 to 50 mins. However, after 50 mins, the reaction between aptamer and ACE reached saturation, and the I\u003csub\u003eFMMA\u003c/sub\u003e/I\u003csub\u003eMB\u003c/sub\u003e signal ratio stabilized, indicating no further significant changes. Thus, 50 mins was determined to be the optimal reaction time for ACE and Apt.\u003c/p\u003e\n \u003cp\u003eThe amount of AuNPs-DNA\u003csub\u003e2\u003c/sub\u003e modification directly affected the quantity of FMMA grafted onto the electrode. Consequently, the effects of both the volume and reaction time of AuNPs-DNA\u003csub\u003e2\u003c/sub\u003e on aptasensor performance were studied. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB, the I\u003csub\u003eFMMA\u003c/sub\u003e/I\u003csub\u003eMB\u003c/sub\u003e signal ratio increased with the volume of AuNPs-DNA\u003csub\u003e2\u003c/sub\u003e in the range of 3 to 15 \u0026micro;L. When the volume of AuNPs-DNA\u003csub\u003e2\u003c/sub\u003e reached 7 \u0026micro;L, the I\u003csub\u003eFMMA\u003c/sub\u003e/I\u003csub\u003eMB\u003c/sub\u003e ratio reached its maximum and subsequently plateaued, indicating that the electrode surface was saturated with AuNPs-DNA\u003csub\u003e2\u003c/sub\u003e at this volume. Therefore, 7 \u0026micro;L was identified as the optimal incubation volume for AuNPs-DNA\u003csub\u003e2\u003c/sub\u003e. Similarly, as shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC, the I\u003csub\u003eFMMA\u003c/sub\u003e/I\u003csub\u003eMB\u003c/sub\u003e ratio increased with the reaction time of AuNPs-DNA\u003csub\u003e2\u003c/sub\u003e within the range of 20 to 120 mins. However, after 80 mins, the I\u003csub\u003eFMMA\u003c/sub\u003e/I\u003csub\u003eMB\u003c/sub\u003e ratio reached a maximum and showed no significant further changes, suggesting that the electrode surface was saturated with AuNPs-DNA\u003csub\u003e2\u003c/sub\u003e at this time point. Thus, 80 mins was determined to be the optimal reaction time for AuNPs-DNA\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n \u003cp\u003eBMP, as the initiator of the ATRP reaction, played a crucial role in determining whether FMMA could be successfully polymerized and grafted onto the electrode surface. Since the concentration of BMP was maintained in excess, only its reaction time needed to be optimized. As illustrated in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD, within the range of 20 to 120 mins, the I\u003csub\u003eFMMA\u003c/sub\u003e/I\u003csub\u003eMB\u003c/sub\u003e signal ratio reached a maximum when the BMP incubation time was 60 mins. Beyond this point, the ratio showed no significant fluctuations, indicating that BMP had achieved maximum modification on the electrode. Therefore, 60 mins was identified as the optimal reaction time for BMP.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5 Detection performance analysis\u003c/h2\u003e\n \u003cp\u003eUnder optimal conditions, the aptasensor\u0026apos;s electrical signal response was evaluated across varying ACE concentrations to assess its detection performance. In Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA, the electrical signal intensity of MB decreased with increasing ACE concentration, while the signal intensity of the FMMA polymer exhibited a corresponding increase. This behavior aligns with the aptasensor\u0026apos;s conceptual design aptasensor. Within the concentration range of 70 pg/mL to 300 ng/mL, the logarithm of ACE concentration presented a good linear relationship with the I\u003csub\u003eFMMA\u003c/sub\u003e/I\u003csub\u003eMB\u003c/sub\u003e current intensity signal ratio (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB). The linear regression equation was as follows: I\u003csub\u003eFMMA\u003c/sub\u003e/I\u003csub\u003eMB\u003c/sub\u003e=1.71365 lgC\u003csub\u003eACE\u003c/sub\u003e + 3.03591 (\u003cem\u003eR\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.998), and the limit of detection (LOD) was 19.26 pg/mL (S/N\u0026thinsp;=\u0026thinsp;3). Compared with other aptasensor detection methods (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e), the dual-signal ratiometric aptasensor based on I\u003csub\u003eFMMA\u003c/sub\u003e/I\u003csub\u003eMB\u003c/sub\u003e exhibited superior detection and analytical performance over single-signal aptasensor relying solely on I\u003csub\u003eFMMA\u003c/sub\u003e or I\u003csub\u003eMB\u003c/sub\u003e aptasensor.\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eComparison of the ratiometric aptasensor with other detection methods\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMethods\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eThe LOD (pg/mL)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eThe detection range (ng/mL)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRef\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEC aptasensor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.6\u0026times;10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.45\u0026times;10\u003csup\u003e5\u003c/sup\u003e \u0026minus;\u0026thinsp;4.01\u0026times;10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eECL aptasensor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.68\u0026times;10\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.23\u0026times;10\u003csup\u003e1\u003c/sup\u003e \u0026minus;\u0026thinsp;2.23\u0026times;10\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFL aptasensor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u0026times;10\u003csup\u003e2\u003c/sup\u003e \u0026minus;\u0026thinsp;3\u0026times;10\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEC-SERS aptasensor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.45\u0026times;10\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.11\u0026times;10\u003csup\u003e4\u003c/sup\u003e \u0026minus;\u0026thinsp;1.11\u0026times;10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eratiometric EC aptasensor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e \u0026minus;\u0026thinsp;3\u0026times;10\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIn the work\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e3.6 Selectivity, stability and reproducibility\u003c/h2\u003e\n \u003cp\u003eUnder optimal conditions, the specificity of the aptasensor for ACE was evaluated by testing structurally similar pesticides, including imidacloprid (IMI), methyl parathion (MP) and chlorpyrifos (CPF). The aptasensor\u0026apos;s response to above pesticides, ACE, and a mixed solution containing all four pesticides (each at a concentration of 1 ng/mL) was analyzed. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA, the I\u003csub\u003eFMMA\u003c/sub\u003e/I\u003csub\u003eMB\u003c/sub\u003e electrical signal strength ratios for IMI, MP and CPF were relatively weak and similar to each other. In contrast, the I\u003csub\u003eFMMA\u003c/sub\u003e/I\u003csub\u003eMB\u003c/sub\u003e signal intensity of mixed group was comparable to that of ACE alone, indicating that the presence of structurally similar pesticides did not significantly interfere with ACE detection.\u003c/p\u003e\n \u003cp\u003eTo assess the stability of the aptasensor system, the current response of freshly prepared electrodes was compared with that of electrodes stored at 4\u0026deg;C for 15 days. As depicted Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB, the current response of the stored electrode retained 91.69% of its initial value, demonstrating the favorable stability of the aptasensor. In addition, the reproducibility of the aptasensor was investigated through parallel experiments (n\u0026thinsp;=\u0026thinsp;5) conducted under identical conditions, both within and between groups. As illustrated in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC and Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eD, the relative standard deviation (RSD) within the group was 4.05%, while the RSD between groups was 4.70%. These results confirmed that the aptasensor system exhibited excellent reproducibility for ACE detection.\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003e3.7 Application of real samples\u003c/h2\u003e\n \u003cp\u003eTo further evaluate the practical application of the aptasensor for ACE detection in real samples, three different concentrations of ACE (0.5 ng/mL, 1 ng/mL and 5 ng/mL) were spiked into honeysuckle, wolfberry and almond samples. As shown in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, the recoveries from these samples ranged from 83.20\u0026ndash;102.72%, with an average recovery of 93.10%. The relative standard deviations (RSDs) were between 0.75% and 4.89%, with an average RSD of 2.68%. To validate the reliability of the aptasensor, the results were compared with those obtained using an enzyme-linked immunosorbent assay (ELISA). The ACE concentration determined by ELISA were based on the percentage absorbance values, yielding recoveries of 89.70\u0026ndash;100.20%, with an average recovery of 95.15%, and RSDs ranging from 0.47\u0026ndash;4.63%, with an average RSD of 1.87%. The analysis results from the proposed aptasensor were consistent with those from ELISA, demonstrating the reliability of the aptasensor for real sample detection. These findings indicate that the ratiometric aptasensor was highly suitable for the detection of ACE in real samples.\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eACE detection and recovery of this aptasensor in real samples\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eThe aptasensor\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eELISA\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eSample\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAdded (ng/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMean measured (ng/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRecovery (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRSD (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMean measured (ng/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRecovery (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRSD (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHoneysuckle\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.4679\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e93.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.4896\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e97.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.37\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.8680\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e86.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.8969\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e89.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.77\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.1597\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e83.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.7910\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e95.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eWolfberry\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.4751\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e95.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.4664\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e93.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.63\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.9703\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e97.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.0021\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.42\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.5727\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e91.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.8293\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e96.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.95\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eAlmond\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.4907\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e98.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.4693\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e93.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.22\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.8992\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e89.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.8989\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e89.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.96\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.1356\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e102.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.9574\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e99.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.47\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4 Conclusion","content":"\u003cp\u003eIn summary, a ratiometric electrochemical aptasensor based on ATRP reaction and AuNPs dual signal amplification strategy for the detection of ACE is developed for the first time. MB, serving as an internal reference signal, is bound to DNA\u003csub\u003e1\u003c/sub\u003e-Apt by electrostatic adsorption and insertion, which is stronger than the labelling signal 1:1 that can only be modified at the end of the sequence The ATRP reaction enables the in situ grafting of the signal unit FMMA onto the electrode, acting as an amplified growth signal. Meanwhile, AuNPs facilitate the loading of multiple DNA\u003csub\u003e2\u003c/sub\u003e probes, thereby expanding the reaction sites for polymerization. These dual signal amplification strategies significantly improve the sensitivity of the aptasensor. Furthermore, the aptasensor achieves a wide dynamic range (0.07 to 300 ng/mL), a low LOD of 19.26 pg/mL, and excellent interference resistance in complex environments. Notably, this versatile strategy can be adapted for the detection of other pesticide residues by simply replacing the target-specific aptamer, highlighting its potential for broad applications in monitoring and evaluating pesticide residue levels in food and medicinal products.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eContributions All authors contributed to the study conception and design. Weiming Li: Investigation, Methodology, Validation, Data curation, Writing \u0026ndash; original draft. Yuzhen Jia: Data curation, Resources. Kunyilan Chen: Validation. Huifang Li: Resources. Huaixia Yang: Conceptualization, Supervision, Writing \u0026ndash; review \u0026amp; editing. Liang Guo: Data curation, Methodology, Funding acquisition. Mingsan Miao: Formal analysis, Investigation, Resources, Writing \u0026ndash; review \u0026amp; editing. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThis work was supported by the National Natural Science Foundation of China (No. 51903248).\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe data supporting the findings of this study are available upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eQin N, Liu J, Li F, Liu J (2024) Recent Advances in Aptasensors for Rapid Pesticide Residues Detection. 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J Food Eng 378:112109. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jfoodeng.2024.112109\u003c/span\u003e\u003cspan address=\"10.1016/j.jfoodeng.2024.112109\" 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":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"microchimica-acta","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"miac","sideBox":"Learn more about [Microchimica Acta](https://link.springer.com/journal/604)","snPcode":"604","submissionUrl":"https://submission.springernature.com/new-submission/604/3","title":"Microchimica Acta","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Acetamidine, ratiometric aptasensor, electrochemical, ATRP, AuNPs","lastPublishedDoi":"10.21203/rs.3.rs-6299477/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6299477/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAcetamiprid (ACE), a next-generation chlorinated neonicotinoid insecticide, has been extensively employed for pest control. However, its excessive residues in food and the environment have raised significant concerns regarding human health. To address the need for a simple, accurate, and efficient ACE detection method, this study developed a ratiometric electrochemical aptasensor utilizing a dual signal amplification strategy involving atom transfer radical polymerization (ATRP) and gold nanoparticles (AuNPs). Methylene blue (MB) served as the internal reference signal, which was attached to the ACE aptamer-DNA\u003csub\u003e1\u003c/sub\u003e (dsDNA) through electrostatic adsorption and intercalation. Ferrocenyl methyl methacrylate (FMMA) was polymerized into long chains via ATRP, generating a significantly amplified electrical signal compared to that of monomeric FMMA. AuNPs, known for their bioconjugability, were linked to DNA\u003csub\u003e2\u003c/sub\u003e (AuNPs-DNA\u003csub\u003e2\u003c/sub\u003e), enhancing the DNA\u003csub\u003e2\u003c/sub\u003e loading capacity and expanding the ATRP reaction sites. In the presence of ACE, the MB signal decreased while the FMMA polymer signal increased, achieving a ratiometric detection strategy with a limit of detection (LOD) of 19.26 pg/mL. This ratiometric aptasensor not only enhances selectivity but also mitigates the influence of background currents, offering a novel and effective approach for ACE detection in real-world sample analysis.\u003c/p\u003e","manuscriptTitle":"Entropy spontaneous ratiometric electrochemical aptasensor based on polymerization and AuNPs signal amplification for Acetamiprid residue analysis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-26 04:17:34","doi":"10.21203/rs.3.rs-6299477/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-07T14:07:26+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-07T01:01:58+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-05T13:41:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"169670728688956365008918306726285055224","date":"2025-03-31T11:15:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"121418020360686425857417438357739542286","date":"2025-03-30T14:43:53+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-30T02:59:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"252829796156869641310371832841444767554","date":"2025-03-29T06:52:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"240543869623870025919202559177655354334","date":"2025-03-29T00:51:42+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-28T15:42:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"273352393521542118282770648463006993890","date":"2025-03-28T15:17:51+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-28T14:31:52+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-27T00:54:51+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-27T00:53:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microchimica Acta","date":"2025-03-25T03:06:28+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microchimica-acta","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"miac","sideBox":"Learn more about [Microchimica Acta](https://link.springer.com/journal/604)","snPcode":"604","submissionUrl":"https://submission.springernature.com/new-submission/604/3","title":"Microchimica Acta","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"1ce7b055-e87a-4910-91af-6064e43fe7c4","owner":[],"postedDate":"March 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-30T16:02:07+00:00","versionOfRecord":{"articleIdentity":"rs-6299477","link":"https://doi.org/10.1007/s00604-025-07217-7","journal":{"identity":"microchimica-acta","isVorOnly":false,"title":"Microchimica Acta"},"publishedOn":"2025-06-23 15:57:17","publishedOnDateReadable":"June 23rd, 2025"},"versionCreatedAt":"2025-03-26 04:17:34","video":"","vorDoi":"10.1007/s00604-025-07217-7","vorDoiUrl":"https://doi.org/10.1007/s00604-025-07217-7","workflowStages":[]},"version":"v1","identity":"rs-6299477","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6299477","identity":"rs-6299477","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}