Aptamer-based Electrochemical Biosensor Research Articles

Evaluation of Transducer Elements Based on Different Material Configurations for Aptamer-Based Electrochemical Biosensors.

The selection of an appropriate transducer is a key element in biosensor development. Currently, a wide variety of substrates and working electrode materials utilizing different fabrication techniques are used in the field of biosensors. In the frame of this study, the following three specific material configurations with gold-finish layers were investigated regarding their efficacy to be used as electrochemical (EC) biosensors: (I) a silicone-based sensor substrate with a layer configuration of 50 nm SiO/50 nm SiN/100 nm Au/30-50 nm WTi/140 nm SiO/bulk Si); (II) polyethylene naphthalate (PEN) with a gold inkjet-printed layer; and (III) polyethylene terephthalate (PET) with a screen-printed gold layer. Electrodes were characterized using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) to evaluate their performance as electrochemical transducers in an aptamer-based biosensor for the detection of cardiac troponin I using the redox molecule hexacyanoferrade/hexacyaniferrade (K3[Fe (CN)6]/K4[Fe (CN)6]. Baseline signals were obtained from clean electrodes after a specific cleaning procedure and after functionalization with the thiolate cardiac troponin I aptamers "Tro4" and "Tro6". With the goal of improving the PEN-based and PET-based performance, sintered PEN-based samples and PET-based samples with a carbon or silver layer under the gold were studied. The effect of a high number of immobilized aptamers will be tested in further work using the PEN-based sample. In this study, the charge-transfer resistance (Rct), anodic peak height (Ipa), cathodic peak height (Ipc) and peak separation (∆E) were determined. The PEN-based electrodes demonstrated better biosensor properties such as lower initial Rct values, a greater change in Rct after the immobilization of the Tro4 aptamer on its surface, higher Ipc and Ipa values and lower ∆E, which correlated with a higher number of immobilized aptamers compared with the other two types of samples functionalized using the same procedure.

P-696 Continuous monitoring of female hormones as a tool to improve IVF treatment’s efficacy

Abstract Study question Is it possible to employ an unobtrusive and injectable biosensor for real-time remote monitoring of patient’s hormone levels (LH, P4, E2) during an ART cycle? Summary answer In-situ implantable biosensors can effectively monitor clinically relevant hormone levels in real-time to improve ART cycle. What is known already Worldwide 48.5 million couples suffer from infertility. Hormonal imbalances can impede pregnancy by compromising the intricate processes of the reproductive system, affecting ovulation, egg quality, fallopian tube function, and endometrial receptivity. In the UK, assisted reproductive treatments (ART) have surged ten-fold in the last 20 years. ART success tremendously depends on the accurate measurement of hormone levels, namely Luteinizing Hormone (LH), oestradiol (E2), and progesterone (P4). These hormone levels change and pulsate during each patient cycle, requiring women to undergo a blood draw and endocrinology analysis every 2-3 days, making ART treatment a highly invasive and time consuming method. Study design, size, duration In order to determine the feasibility of the study question, Impli designed a minimally invasive implantable biosensor to continuously monitor essential hormones for women’s fertility such as LH, P4 and E2 in order to enhance the quality of treatment and improve the ART experience. An implantable electrochemical aptamer-based biosensors has been developed using cutting-edge electronics and electrochemical technologies. This minimally invasive biosensor allows patients to monitor their hormone levels in real-time during ART without venipuncture. Participants/materials, setting, methods The implantable biosensor contains the biorecognition interface, designed using the DNA-based aptamers coupled to the detection gold working electrodes. When the biosensor is in touch with the sample, hormones bind to the respective aptamers, which leads to changes in the electrical properties of the gold electrode. The changes are then monitored and calibrated through sensitive electrochemical techniques such as differential pulse voltammetry (DPV), using CHI potentiostat. Main results and the role of chance The biosensor was tested in vitro in artificial interstitial fluid (ISF) containing the range of 1 pg/ml to 1 µg/ml of hormones yielding a limit of detection (LOD) of 0.46 pg/ml, 1.15 pg/ml, and 0.58 pg/ml for E2, P4 and LH, respectively. Our data was validated using ELISA assays. A notably high linear correlation coefficient (R2) of approximately 0.9 was observed between the signals obtained from the ELISA and the biosensors, confirming the reliability of the aptamer biosensors for hormone analysis. The stability of the biosensor was also investigated over one week. The DPV technique was employed to assess the cross-reactivity of potential interferences with each biosensor. The results revealed that each biosensor exhibited notable selectivity towards its respective analyte over other interferences, at significantly higher concentrations. The biosensor was also characterised by microscopy techniques such as SEM and Raman analysis. Limitations, reasons for caution This novel implantable biosensor technology requires further upcoming validation in animal models to assess its ability for widespread use. Once validated, the appropriate human clinical trials will be undertaken. Wider implications of the findings The development and implementation of an unobtrusive implantable remote and continuous monitoring system, capturing hormone concentrations that are vital for success in an ART procedure, could transform the industry and positively influence patient’s access to care and the patient journey. Trial registration number No clinical trial

Ultrasensitive Aptamer-Based Electrochemical Nanobiosensor in Diagnosis of Prostate Cancer Using 2D:2D Reduced Graphene Oxide/graphitic Carbon Nitride decorated with Au Nanoparticles

Prostate-specific antigen (PSA) remains the cornerstone for prostate cancer diagnosis. This study presents a highly sensitive aptamer-based electrochemical biosensor for PSA detection utilizing a novel two-dimensional (2D):2D reduced graphene oxide (rGO)/graphitic carbon nitride (g-C3N4) composite decorated with gold nanoparticles (Au NPs). The aptamer chains were immobilized on a glassy carbon electrode (GCE) modified with the rGO/g-C3N4/Au NPs composite. The successful synthesis of the nanomaterial and electrode modification were confirmed using X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and transmission electron microscopy (TEM). The electrochemical properties and selectivity of the modified GCE were characterized by cyclic voltammetry (CV), square wave voltammetry (SQW), and electrochemical impedance spectroscopy (EIS). The biosensor exhibited high selectivity towards PSA compared to potential interferents like carbohydrate antigen 15-3 (CA 15-3), bovine serum albumin (BSA), fetal bovine serum (FBS), and glucose (C6H12O6). Under optimized conditions, the biosensor achieved a rapid detection time of 30 minutes for PSA and a remarkable limit of detection (LOD) of 0.44 fM (femtomolar) using methylene blue (MB) as the redox mediator. The limit of quantification (LOQ) was also exceptionally low at 2.5 fM. The applicability of the developed biosensor was further validated by analyzing real serum samples, demonstrating its potential for clinical use.

Cell-SELEX for aptamer discovery and its utilization in constructing electrochemical biosensor for rapid and highly sensitive detection of Legionella pneumophila serogroup 1

This study introduces an innovative electrochemical aptasensor designed for the highly sensitive and rapid detection of Legionella pneumophila serogroup 1 (L. pneumophila SG1), a particularly virulent strain associated with Legionellosis. Employing a rigorous selection process utilizing cell-based systematic evolution of ligands by exponential enrichment (cell-SELEX), we identified new high-affinity aptamers specifically tailored for L. pneumophila SG1. The selection process encompassed ten rounds of cell-SELEX cycles with live L. pneumophila, including multiple counter-selection steps against the closely related Legionella sub-species. The dissociation constant (Kd) of the highest affinity sequence to L. pneumophila SG1 was measured at 14.2 nM, representing a ten-fold increase in affinity in comparison with the previously reported aptamers. For the development of electrochemical aptasensor, a gold electrode was modified with the selected aptamer through the formation of self-assembled monolayers (SAMs). The newly developed aptasensor exhibited exceptional sensitivity, and specificity in detecting and differentiating various Legionella sp., with a detection limit of 5 colony forming units (CFU)/mL and an insignificant/negligible cross-reactivity with closely related sub-species. Furthermore, the aptasensor effectively detected L. pneumophila SG1 in spiked water samples, demonstrating an appreciable recovery percentage. This study shows the potential of our aptamer-based electrochemical biosensor as a promising approach for detecting L. pneumophila SG1 in diverse environments.

Rapid nanomolar detection of Δ9-tetrahydrocannabinol in biofluids via electrochemical aptamer-based biosensor

BackgroundThe fabrication of sensors capable of achieving rapid, sensitive, and highly selective detection of target molecules in complex fluids is key to realizing their real-world applications. For example, there is an urgent need in drugged driving roadside screening scenarios to develop a method that can be used for rapid drug detection and that avoids interference from the matrix in the sample. How to minimize the interference of complex matrices in biofluids at the electrode interface is the key to improve the sensitivity of the sensor. ResultsThis work develops a facile and green method to prepare rough electrodes with a porous structure for constructing electrochemical aptamer-based (EAB) sensors for rapid, sensitive and accurate detection of Δ9-tetrahydrocannabinol (THC) in biofluids. The electroactive area of the rough electrode was 21 times of smooth electrode. And the antifouling performance of the rough electrode was much better than that of smooth electrode. Based on the unique advantages of the rough electrode, the developed EAB sensor achieves rapid nanomolar detection of THC in undiluted serum, undiluted urine and 50 % saliva with the detection limit of 5.0 nM, 10 nM and 10 nM, respectively. Moreover, our method possesses good reproducibility, accuracy and specificity. SignificanceThe porous structure can effectively reduce the non-specific adsorption and enhance the stability of the signal, while the larger active area can modify more aptamers, thus improving the sensitivity. The detection limits of the EAB sensor were lower than the cutoff concentration of THC in drugged driving and the measuring process was completed within 60 s after target addition, which makes the present sensors capable for real-world applications.

Electrochemical aptamer-based biosensing of glycan-conjugated targets

As the most complex post-translational modification, glycosylation involves the covalent linking of glycan(s) to a biomolecule. The glycosylation of proteins, peptides, lipids, and nucleic acids results in the formation of diverse glycoconjugates, like glycoproteins, peptidoglycans, glycopeptides, glycolipids, lipopolysaccharides, and glycoRNAs, and glycoconjugates are also ubiquitous on the surface of extracellular vesicles (EVs), cells, bacteria, and viruses. The conjugated glycans are integral to cell recognition (e.g., adhesion and homing), cell trafficking, immune recognition, hormone-receptor binding, protein folding and stabilization, and many other biological processes. Currently, the glycan-conjugated biological objects have found widespread applications in disease diagnosis (e.g., glycoproteins, exosomes, and circulating tumor cells) and therapy (e.g., therapeutic monoclonal antibodies). Over the past decades, a variety of methods have been established for the quantitative assay of the glycan-conjugated biological objects, in light of their significant diagnostic and therapeutic values, using antibodies, aptamers, or affinity peptides as recognition ligands. Relative to other methods, the aptamer-based electrochemical biosensors (i.e., electrochemical aptasensors) have attracted much attention in recent years in the detection of the glycan-conjugated targets, by virtue of their intrinsic advantages such as low cost, good portability, simple instrument, superior stability and anti-interference capability, and high sensitivity and selectivity. This review summarizes the biological, diagnostic, and therapeutic significance of the glycan-conjugated biological objects, and then presents the progress in the electrochemical aptasensing of the glycan-conjugated biological objects, including glycoproteins, lipopolysaccharides (LPSs), exosomes, circulating tumor cells (CTCs), and pathogenic microbes (e.g., bacteria and viruses). Moreover, the challenges and future prospects of the electrochemical aptasensing of the glycan-conjugated biological objects are discussed.

Whole blood multiplex measurements using electrochemical aptamer-based biosensors.

Simultaneous measurements of various molecules ("multiplex") using electrochemical biosensors typically require multiple electrode implementations, which for neonates, hemophiliacs, etc. is problematic. Here, we introduce the oxazine ATTO 700 into electrochemical aptamer-based biosensors to achieve "true" multiplex, continuous and real-time measurements of two different molecules in undiluted whole blood using a single electrode.

Electrochemical Aptamer-Based Biosensor Developed to Monitor PCA3 and PSA Released by Prostate Cancer Cells

1. Introduction Detection of prostate cancer (PCa) in the early stage of the disease is fundamental for the prevention of metastases and for the initiation of treatment, which may bring greater chance of patient cured; so several research groups have sought the manufacture of a simple and non-invasive methodology [1]. The early diagnosis relies on prostate specific antigen (PSA) serum test, even if it showed clear limits [2]. Recently, prostate cancer gene 3 (PCA3), a non-coding mRNA which is overexpressed in tumor cells when compared with noncancerous prostate tissue [3]. Here, we report a new method for detection of prostate cancer-specific antigen 3 (PCA3) and prostate specific antigen (PSA) released by cancer cells using an aptasensor. Electrochemical impedance spectroscopy (EIS), SEM and EDS were used to evaluate biosensor surface. 2. Experimental Part The aptamers were modified through Biosearch Technologies at 5′-terminus in C6-disulfide linker to have it bond to the gold electrode; and at 3′-end in methylene blue (MB) (as redox probe) to perform the electrochemical analysis, which was followed by the HPLC purification process to remove truncated-synthesis products and to enrich the purity of the aptamer sequences. Aptamer immobilization was done by incubation of 1 μM thiolated aptamer on the top of the gold-coated silicon electrode overnight, in the dark, at 4°C. Subsequently, the electrodes were washed with PBS and a backfilling consisted of 3 mM of MCH aqueous solution for 30 minutes was done to displace the nonspecifically adsorbed aptamer molecules and to passivate the electrode surface [26]. Finally, the electrodes were once more rinsed with PBS to remove MCH excesses. The electrochemical characterization was done in a potentiostat μAutolabIII (Metrohn, NL), using a three-electrode system, which consisted of Ag/AgCl (3M KCl), as reference electrode; Pt wire, as a counter electrode; and gold-coated silicon wafer, as a working electrode. The prostate cell lines used were normal human prostate cells (RWPE-1; ATCC® CRL-11™), androgen-sensitive human prostate cancer cells (LNCaP; ATCC® CRL-1740™), and androgen-independent human prostate cancer cells (PC-3; ATCC® CRL-1435™). The three lineages were chosen to better understand PCA3 and PSA production patterns by different cell types. 3. Results and Discussion The gold-coated silicon wafe electrode was analyzed by SEM and EDS, which acts as working electrode and substrate to anchor the thiolated aptamer on the surface. By using square wave voltammetry, the present aptasensor was able of monitoring the PCA3 and PSA released from prostate cells (normal and cancerous). The PSA and PCA3 detection limits were 1 ng/mL and 9.2 ng/mL, respectively, at linear range up to 150 ng/mL, and it was also able to detect both biomarker even in presence of several interfering compounds (UA, AA, BSA and MUC1). In addition, the herein developed aptasensor was able to verify, at accurate and robust response, the patterns of the PCA3 and PSA released by different cell lines (RWPE-1, LNCaP and PC3). The detection of PSA and PCA3 by aptasensor agree with the cell lineage type, the LNCaP is an androgen-dependent lineage. In this way, an overexpressed both biomarkres was release was verified [49,50] and it can be compared to early-stage patients. On the other hand, the PC3 cells PSA and PCA3 releasing was nearly background signal, demonstrating almost absent production of studied targets, the PC3 lineage is androgen-independent which can be compared to advanced-disease patients. Funding This work has been supported by a grant from FAPESP-Brazil, CNPq and Capes.

Fast Fourier-Transform Impedance Spectroscopy Applied to Electrochemical Sensors

We show that using fast Fourier transform electrochemical impedance spectroscopy (FFT-EIS) to interrogate electrochemical aptamer-based biosensors leads to improved (here better than 2 s) time resolution and calibration-free operation, even when such sensors are deployed in vivo. Because this approach uses the heterogeneous electron transfer rate constant as a means of monitoring target concentration, rather than absolute current, it is independent of both sensor-to-sensor fabrication variation and the drift arising due to fouling in biological fluids, rendering the technique suitable for performing calibration-free in vivo measurements. To showcase these benefits, we demonstrate the approach’s ability to perform real-time molecular measurements in the veins of living rats.

Aptamer-based electrochemical biosensor with S protein binding affinity for COVID-19 detection: Integrating computational design with experimental validation of S protein binding affinity

Aptamer-based biosensors capable of rapid one-step detection of viruses provide new capabilities for large-scale detection of COVID-19. Here we report a combined aptamer-based electrochemical and computational approach to characterize the interaction between a 60 base single stranded (ss)DNA aptamer and the viral S protein using molecular docking and bioinformatics tools. To construct the biosensor, the DNA aptamer was immobilized to the surface of a gold nanoparticles (AuNPs) modified electrode and methylene blue (MB) was used as redox indicator. Based on thermodynamic calculations for the aptamer sequence as well as several runs in APBS (Adaptive Poisson-Boltzmann Equation), we found that S protein GLN104-ASP115 pocket is the candidate for DNA 30–39(GGGAGTGGTT) sequence interaction. In the presence of S-protein, the target induced conformational changes in the aptamer’s structure, generating an increase in the MB redox signal, with a linear range between 10 pM to 6 nM and a limit of detection of 91.1 pM using differential pulse voltammetry (DPV). The aptasensor was investigated in spiked human saliva and serum samples, demonstrating performance in both media. This study demonstrates the potential of the MB-labeled electrochemical aptasensor for direct one-step detection of viral proteins, COVID-19 and potentially other viral agents.

A capacitive laser-induced graphene based aptasensor for SARS-CoV-2 detection in human saliva.

SARS-CoV-2 virus induced CoVID-19 pandemic has accelerated the development of diagnostic tools. Devices integrated with electrochemical biosensors may be an interesting alternative to respond to the high demand for testing, particularly in contexts where access to standard detection technologies is lacking. Aptamers as recognition elements are useful due to their stability, specificity, and sensitivity to binding target molecules. We have developed a non-invasive electrochemical aptamer-based biosensor targeting SARS-CoV-2 in human saliva. The aptamer is expected to detect the Spike protein of SARS-CoV-2 wildtype and its variants. Laser-induced graphene (LIG) electrodes coated with platinum nanoparticles were biofunctionalized with a biotin-tagged aptamer. Electrochemical Impedance Spectroscopy (EIS) for BA.1 sensing was conducted in sodium chloride/sodium bicarbonate solution supplemented with pooled saliva. To estimate sensing performance, the aptasensor was tested with contrived samples of UV-attenuated virions from 10 to 10,000 copies/ml. Selectivity was assessed by exposing the aptasensor to non-targeted viruses (hCoV-OC43, Influenza A, and RSV-A). EIS data outputs were further used to select a suitable response variable and cutoff frequency. Capacitance increases in response to the gradual loading of the attenuated BA.1. The aptasensor was sensitive and specific for BA.1 at a lower viral load (10-100 copies/ml) and was capable of discriminating between negative and positive contrived samples (with strain specificity against other viruses: OC43, Influenza A, and RSV-A). The aptasensor detected SARS-CoV-2 with an estimated LOD of 1790 copies/ml in contrived samples. In human clinical samples, the aptasensor presents an accuracy of 72%, with 75% of positive percent of agreement and 67% of negative percent of agreement. Our results show that the aptasensor is a promising candidate to detect SARS-CoV-2 during early stages of infection when virion concentrations are low, which may be useful for preventing the asymptomatic spread of CoVID-19.

The Ultrasensitive Detection of Aflatoxin M1 Using Gold Nanoparticles Modified Electrode with Fe3+ as a Probe.

The increasing incidence of diseases caused by highly carcinogenic aflatoxin M1 (AFM1) in food demands a simple, fast, and cost-effective detection technique capable of sensitively monitoring AFM1. Recent works predominantly focus on the electrochemical aptamer-based biosensor, which still faces challenges and high costs in experimentally identifying an efficient candidate aptamer. However, the direct electrochemical detection of AFM1 has been scarcely reported thus far. In this study, we observed a significant influence on the electrochemical signals of ferric ions at a gold nanoparticle-modified glassy carbon electrode (AuNPs/GCE) by adding varying amounts of AFM1. Utilizing ferricyanide as a sensitive indicator of AFM1, we have introduced a novel approach for detecting AFM1, achieving an unprecedentedly low detection limit of 1.6 × 10-21 g/L. Through monitoring the fluorescence quenching of AFM1 with Fe3+ addition, the interaction between them has been identified at a ratio of 1:936. Transient fluorescence analysis reveals that the fluorescence quenching process is predominantly static. It is interesting that the application of iron chelator diethylenetriaminepentaacetic acid (DTPA) cannot prevent the interaction between AFM1 and Fe3+. With a particle size distribution analysis, it is suggested that a combination of AFM1 and Fe3+ occurs and forms a polymer-like aggregate. Nonetheless, the mutual reaction mechanism between AFM1 and Fe3+ remains unexplained and urgently necessitates unveiling. Finally, the developed sensor is successfully applied for the AFM1 test in real samples, fully meeting the detection requirements for milk.

Aptamer-based carbohydrate antigen 125 sensor with molybdenum disulfide functional hybrid materials

Epithelial ovarian cancer is a malignant tumor of the female reproductive system with insidious symptoms, aggressiveness, risk of metastasis, and high mortality. Carbohydrate antigen 125 (CA125), a standard biomarker for screening epithelial ovarian cancer, can be applied to track cancer progression and treatment response. Here, we constructed an aptamer-based electrochemical biosensor to achieve sensitive detection of CA125. Molybdenum disulfide (MoS2) was used as the stable layered substrate, combined with the irregular branched structure of gold nanoflowers (AuNFs) to provide the sensing interface with a large specific surface area by one-step electrodeposition AuNFs@MoS2. The simplified electrode modification step increased the stability of the electrode while ensuring excellent electrochemical performance and providing many sulfhydryl binding sites. Then, AuNFs@MoS2/CA125 aptamer/MCH sensor was designed for CA125 detection. Based on AuNFs@MoS2 electrode, CA125 aptamer with sulfhydryl as the sensitive layer was fixed on the electrode by gold sulfur bonds. 6-Mercapto-1-hexanol (MCH) was used to block the electrode and reduce the non-specific adsorption. Finally, DPV analysis was applied for CA125 detection with the range of 0.0001 U/mL to 500 U/mL. Our designed aptamer sensor showed reasonable specificity, reproducibility, and stability. Clinical sample testing also proved the consistency of our sensor with the gold standard in negative/positive judgment. This work demonstrated a novel strategy for integrating nanostructures and biocompatibility to build advanced cancer biomarker sensors with promising applications.

Redox Reporter - Ligand Competition to Support Signaling in the Cocaine-Binding Electrochemical Aptamer-Based Biosensor.

Electrochemical aptamer-based (E-AB) biosensors have demonstrated capabilities in monitoring molecules directly in undiluted complex matrices and in the body with the hopes of addressing personalized medicine challenges. This sensing platform relies on an electrode-bound, redox-reporter-modified aptamer. The electrochemical signal is thought to originate from the aptamer undergoing a binding-induced conformational change capable of moving the redox reporter closer to the electrode surface. While this is the generally accepted mechanism, it is notable that there is limited evidence demonstrating conformational change or distance-dependent change in electron transfer rates in E-AB sensors. In response, we investigate here the signal transduction of the well-studied cocaine-binding aptamer with different analytical methods and found that this sensor relies on a redox-reporter - ligand competition mechanism rather than a ligand-induced structure formation mechanism. Our results show that the covalently bound redox reporter, methylene blue, binds at or near the ligand binding site on the aptamer resulting in a folded conformation of the cocaine-binding aptamer. Addition of ligand then competes with the redox reporter for binding, altering its electron transfer rate. While we show this for the cocaine-binding aptamer, given the prevalence of methylene blue in E-AB sensors, a similar competition-based may occur in other systems.

A Simple Label-Free Aptamer-Based Electrochemical Biosensor for the Sensitive Detection of C-Reactive Proteins

The level of C-reactive protein (CRP) in the human body is closely associated with cardiovascular diseases and inflammation. In this study, a label-free functionalized aptamer sensor was attached to an electrode trimmed with in-gold nanoparticles and carboxylated graphene oxide (AuNPs/GO-COOH) to achieve sensitive measurements relative to CRP. Gold nanoparticles were selected for this study due to super stability, remarkably high electrical conductivity, and biocompatibility. In addition, carboxylated graphene oxide was utilized to promote the anchorage of inducer molecules and to increase detection accuracies. The sensing signal was recorded using differential pulse voltammetry (DPV), and it produced a conspicuous peak current obtained at approximately -0.4 V. Furthermore, the adapted sensor manifested a broad linear span from 0.001 ng/mL to 100 ng/mL. The results also demonstrated that this aptamer sensor had superior stability, specificity, and reproducibility. This aptamer-based electrochemical sensor has enormous potential in complex application situations with interfering substances.

A Novel Approach for Designing Electrochemical Aptamer-Based Biosensor for Ultrasensitive Detection of Zearalenone as a Prevalent Estrogenic Mycotoxin.

Zearalenone is a well-known estrogenic mycotoxin produced by Fusarium species, a serious threat to the agricultural and food industries worldwide. Zearalenone, with its known metabolites, is a biomarker of exposure to certain fungi, primarily through food. It has considerable toxic effects on biological systems due to its carcinogenicity, mutagenicity, renal toxicity, teratogenicity, and immunotoxicity. This study aims to design a simple, quick, precise, and cost-effective method on a biosensor platform to evaluate the low levels of this toxin in foodstuffs and agricultural products. An aptamer-based electrochemical biosensor was introduced that utilizes screen-printed gold electrodes instead of conventional electrodes. The electrodeposition process was employed to develop a gold nanoparticle-modified surface to enhance the electroactive surface area. Thiolated aptamers were immobilized on the surface of gold nanoparticles, and subsequently, the blocker and analyte were added to the modified surface. In the presence of a redox probe, electrochemical characterization of differential pulse voltammetry, cyclic voltammetry, and electrochemical impedance spectroscopy were used to investigate the various stages of aptasensor fabrication. The proposed aptasensor for zearalenone concentration had a wide linear dynamic range covering the 0.5 pg/mL to 100 ng/mL with a 0.14 pg/mL detection limit. Moreover, this aptasensor had high specificity so that a non-specific analyte cannot negatively affect the selectivity of the aptasensor. Overall, due to its simple design, high sensitivity, and fast performance, this aptasensor showed a high potential for assessing zearalenone in real samples, providing a clear perspective for designing a portable and cost-effective device.

Optimization of Aptamer-Based Electrochemical Biosensor for ATP Detection Using Screen-Printed Carbon Electrode/Gold Nanoparticles (SPCE/AuNP)

Electrochemical biosensors are used to detect adenosine triphosphate (ATP) levels, which are involved in a variety of biological processes, such as regulating cellular metabolism and biochemical pathways. Therefore, this research aims to develop an aptamer-based electrochemical biosensor with Screen Printed Carbon Electrode/gold nanoparticles (SPCE/AuNP) and collect data as well as information related to ATP detection. The modification of SPCE with AuNP increased the analyte’s binding sensitivity and biocompatibility. The aptamer was selected based on its excellent bioreceptor characteristics. Furthermore, aptamer–SH (F1) and aptamer-NH2 (F2) were immobilized on the SPCE/AuNP surface, which had been characterized using SEM, EIS, and DPV. Also, the ATP-binding aptamers were electrochemically characterized using the K3[Fe(CN)6] redox system and Differential Pulse Voltammetry (DPV). According to the optimization results using the Box-Behnken experimental design, the ideal conditions obtained from the factors influencing the experiment were the F1 concentration and incubation time of 4 µM and 24 h, respectively, as well as F1/F2/ATP incubation time of 7.5 min. Meanwhile, for the range of 0.1 to 100 µM, the detection (LoD) and quantification (LoQ) limits were 7.43 and 24.78 µM, respectively. Therefore, this aptasensor method can be used to measure ATP levels in real samples.

Synthetic antibodies for methamphetamine analysis: Design of high affinity aptamers and their use in electrochemical biosensors

An aptamer-based electrochemical biosensor was designed and fabricated for methamphetamine (METH) analysis. Several aptamer sequences developed by the GO-SELEX method were immobilized onto gold electrodes (GE) via gold-thiol affinity. Surface characterizations were performed by electrochemical methods including cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectrometry (EIS). GE/Apta-4/METH aptasensor was selected for further studies since the best results were obtained with the Apta-4 sequence in terms of analytical parameters. Accordingly, it exhibited good selectivity for METH analysis with a wide linear range of 0.1–50 ng/mL and a low limit of detection (LOD) which was found to be 0.467 ng/mL. The proposed aptasensor platform displayed good selectivity for the detection of METH in synthetic urine samples spiked with METH.

Detection of Oxytetracycline Using an Electrochemical Label-Free Aptamer-Based Biosensor.

One of the most effective ways to detect and measure antibiotics is to detect their biomarkers. The best biomarker for the control and detection of oxytetracycline (OTC) is the OTC-specific aptamer. In this study, a novel, rapid, and label-free aptamer-based electrochemical biosensor (electrochemical aptasensor) was designed for OTC determination based on a newly synthesized nanocomposite including multi-walled carbon nanotubes (MWCNTs), gold nanoparticles (AuNPs), reduced graphene oxide (rGO), and chitosan (CS), as well as nanosheets to modify a glassy carbon electrode, which extremely enhanced electrical conductivity and increased the electrode surface to bind well with the amine-terminated OTC-specific aptamer through self-assembly. The (MWCNTs-AuNPs/CS-AuNPs/rGO-AuNPs) nanocomposite modified electrode was synthesized using a layer- by-layer modification method which had the highest efficiency for better aptamer stabilization. Differential pulse voltammetry (DPV), cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and scanning electron microscopy (SEM) techniques were used to investigate and evaluate the electrochemical properties and importance of the synthesized nanocomposite in different steps. The designed aptasensor was very sensitive for measuring the OTC content of milk samples, and the results were compared with those of our previously published paper. Based on the calibration curve, the detection limit was 30.0 pM, and the linear range was 1.00–540 nM for OTC. The repeatability and reproducibility of the aptasensor were obtained for 10.0 nM of OTC with a relative standard deviation (RSD%) of 2.39% and 4.01%, respectively, which were not affected by the coexistence of similar derivatives. The measurement in real samples with the recovery range of 93.5% to 98.76% shows that this aptasensor with a low detection limit and wide linear range can be a good tool for detecting OTC.

Application of Nanomaterial Modified Aptamer-Based Electrochemical Sensor in Detection of Heavy Metal Ions.

Heavy metal pollution resulting from significant heavy metal waste discharge is increasingly serious. Traditional methods for the detection of heavy metal ions have high requirements on external conditions, so developing a sensitive, simple, and reproducible detection method is becoming an urgent need. The aptamer, as a new kind of artificial probe, has received more attention in recent years for its high sensitivity, easy acquisition, wide target range, and wide use in the detection of various harmful substances. The detection platform that an aptamer-based electrochemical biosensor (E-apt sensor) provides is a new approach for the detection of heavy metal ions. Nanomaterials are particularly important in the construction of E-apt sensors, as they can be used as aptamer carriers or sensitizers to stimulate or inhibit electrochemical signals, thus significantly improving the detection sensitivity. This review summarizes the application of different types of nanomaterials in E-apt sensors. The construction methods and research progress of the E-apt sensor based on different working principles are systematically introduced. Moreover, the advantages and challenges of the E-apt sensor in heavy metal ion detection are summarized.