Aptamer-based Electrochemical Biosensor Research Articles

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.

Cancer Diagnostics and Early Detection Using Electrochemical Aptasensors.

The detection of early-stage cancer offers patients the best chance of treatment and could help reduce cancer mortality rates. However, cancer cells or biomarkers are present in extremely small amounts in the early stages of cancer, requiring high-precision quantitative approaches with high sensitivity for accurate detection. With the advantages of simplicity, rapid response, reusability, and a low cost, aptamer-based electrochemical biosensors have received considerable attention as a promising approach for the clinical diagnosis of early-stage cancer. Various methods for developing highly sensitive aptasensors for the early detection of cancers in clinical samples are in progress. In this article, we discuss recent advances in the development of electrochemical aptasensors for the early detection of different cancer biomarkers and cells based on different detection strategies. Clinical applications of the aptasensors and future perspectives are also discussed.

Rapid nanomolar detection of methamphetamine in biofluids via a reagentless electrochemical aptamer-based biosensor

The availability of sensing platforms able to rapidly measure abused drugs directly in biological fluids in a single step would allow performing drugged driving screening on the site. The achievement of this goal is extremely important for preventing and controlling drug abuse and crime incidence. Motived by this, we constructed a simple, cost-effective and reagentless electrochemical aptamer-based (EAB) sensor with methamphetamine (MAMP) as the target molecule. This EAB sensor produced a nanomolar level of detection accuracy in unprocessed or minimally processed bio-samples. Specifically, circular dichroic spectrum was used to confirm that the truncated aptamer from the original sequence would undergo large binding-induced conformational changes. We then engineered the aptamer to work in the EAB platform and the resulting sensor enabled sensitive and specific detection of MAMP with the detection limit of 30 nM in undiluted serum, 50 nM in undiluted urine and 20 nM in 50% saliva. The sensor has good recovery rate, implying this method has good reliability and repeatability. The detection limit is far below the clinical detection threshold, it would be hopefully used for preliminary screening of drugged driving in real world.

An Amine-Reactive Phenazine Ethosulfate (arPES)-A Novel Redox Probe for Electrochemical Aptamer-Based Sensor.

Electrochemical aptamer-based biosensors (E-ABs) are attractive candidates for use in biomarker detection systems due to their sensitivity, rapid response, and design flexibility. There are only several redox probes that were employed previously for this application, and a combination of redox probes affords some advantages in target detection. Thus, it would be advantageous to study new redox probes in an E-AB system. In this study, we report the use of amine-reactive phenazine ethosulfate (arPES) for E-AB through its conjugation to the terminus of thrombin-binding aptamer. The constructed E-AB can detect thrombin by square-wave voltammetry (SWV), showing peak current at −0.15 V vs. Ag/AgCl at pH 7, which differs from redox probes used previously for E-ABs. We also compared the characteristics of PES as a redox probe for E-AB to methylene blue (MB), which is widely used. arPES showed stable signal at physiological pH. Moreover, the pH profile of arPES modified thrombin-binding aptamer revealed the potential application of arPES for a simultaneous multianalyte detection system. This could be achieved using different aptamers with several redox probes in tandem that harbor various electrochemical peak potentials. Our findings present a great opportunity to improve the current standard of biological fluid monitoring using E-AB.

Electrochemical aptamer-based biosensors for <italic>in vivo</italic> analysis

<p indent="0mm">Chemistry is the molecular basis of life activities. Accurate acquisition of the distribution and composition variation of physiologically important molecules <italic>in vivo</italic> is of great significance for understanding sophisticated physiological and pathological processes of the human body. Electrochemical approaches, especially electrochemical aptamer-based biosensors, have the excellent characteristics of high specificity, a wide range of target molecules, and easy miniaturization, providing an effective tool for rapid, sensitive, and highly selective detection in complex physiological environments. In this review, we briefly summarize the design of the nucleic-acid-aptamer-based electrochemical biosensor and its application in <italic>in vivo</italic> analysis, and its future prospects and challenges were also discussed.

An electrochemical aptasensor for detection of prostate-specific antigen using reduced graphene gold nanocomposite and Cu/carbon quantum dots.

We report a label-free electrochemical aptamer-based biosensor for the detection of human prostate-specific antigen (PSA). The thiolate DNA aptamer against PSA was conjugated to the reduced graphene oxide/Au (RGO-Au) nanocomposite through the self-assembly of Au-S groups. Owing to the large volume to surface ratio, the RGO-Au nanocomposite provides a large surface for aptamer loading. The RGO-Au/aptamer was combined with a Nafion polymer and immobilized on a glassy carbon electrode. The interaction of aptamer with PSA was studied by cyclic voltammetry, square wave voltammetry, and electrochemical impedance spectroscopy. The detection of limit for prepared electrode was obtained about 50 pg/mL at the potential of 0.4 V in potassium hexacyanoferrate [K4 Fe(CN)6 ] medium. To decrease the limit of detection (LOD) and applied potential of the prepared nanoprobe Cu/carbon quantum dots (CuCQD) is introduced as a new redox. The results show that this new electrochemical medium provides better conditions for the detection of PSA. LOD of a nanoprobe in CuCQD media was obtained as 40 pg/mL at the potential of -0.2 V. Under optimal conditions, the aptasensor exhibits a linear response to PSA with a LOD as small as 3 pg/mL. The present aptasensor is highly selective and sensitive and shows satisfactory stability and repeatability.

Aptamer-based electrochemical biosensor for rapid detection of SARS-CoV-2: Nanoscale electrode-aptamer-SARS-CoV-2 imaging by photo-induced force microscopy

Rapid, mass diagnosis of the coronavirus disease 2019 (COVID-19) is critical to stop the ongoing infection spread. The two standard screening methods to confirm the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are polymerase chain reaction (PCR), through the RNA of the virus, and serology by detecting antibodies produced as a response to the viral infection. However, given the detection complexity, cost and relatively long analysis times of these techniques, novel technologies are urgently needed. Here, we report an aptamer-based biosensor developed on a screen-printed carbon electrode platform for rapid, sensitive, and user-friendly detection of SARS-CoV-2. The aptasensor relies on an aptamer targeting the receptor-binding domain (RBD) in the spike protein (S-protein) of the SARS-CoV-2. The aptamer immobilization on gold nanoparticles, and the presence of S-protein in the aptamer-target complex, investigated for the first time by photo-induced force microscopy mapping between 770 and 1910 cm−1 of the electromagnetic spectrum, revealed abundant S-protein homogeneously distributed on the sensing probe. The detection of SARS-CoV-2 S-protein was achieved by electrochemical impedance spectroscopy after 40 min incubation with several analyte concentrations, yielding a limit of detection of 1.30 pM (66 pg/mL). Moreover, the aptasensor was successfully applied for the detection of a SARS-CoV-2 pseudovirus, thus suggesting it is a promising tool for the diagnosis of COVID-19.

Advances in Electrochemical and Acoustic Aptamer-Based Biosensors and Immunosensors in Diagnostics of Leukemia.

Early diagnostics of leukemia is crucial for successful therapy of this disease. Therefore, development of rapid, sensitive, and easy-to-use methods for detection of this disease is of increased interest. Biosensor technology is challenged for this purpose. This review includes a brief description of the methods used in current clinical diagnostics of leukemia and provides recent achievements in sensor technology based on immuno- and DNA aptamer-based electrochemical and acoustic biosensors. The comparative analysis of immuno- and aptamer-based sensors shows a significant advantage of DNA aptasensors over immunosensors in the detection of cancer cells. The acoustic technique is of comparable sensitivity with those based on electrochemical methods; moreover, it is label-free and provides straightforward evaluation of the signal. Several examples of sensor development are provided and discussed.

A multivalent aptamer-based electrochemical biosensor for biomarker detection in urinary tract infection

Lactoferrin is a multifunctional protein of the transferrin family and is known as a biomarker for various clinical diseases including urinary tract infection (UTI). However, wide concentration range of lactoferrin in urine samples due to the high interpatient variations, requires a more practical biosensor. In this article, we used a novel multivalent aptamer immobilized on the surface of screen-printed gold electrode (aptamer/SPGE) to develop the first electrochemical aptasensor for label-free detection of lactoferrin with wide dynamic detection range and high sensitivity. The performance of the fabricated biosensor was tested using electrochemical impedance spectroscopy and differential pulse voltammetry. The multivalent aptamer as the bioreceptor with high affinity and good specificity against human lactoferrin, acts to enhance the electrochemical signals and widen the working window. The aptamer/SPGE demonstrates superior sensing performances for lactoferrin in buffer solution, with a wide linear range of 10 to 1300 ng/mL with LOD of 0.9 ng/mL, as well as high selectivity, and excellent reproducibility. Besides, the constructed aptasensor was successfully applied to quantify lactoferrin concentrations in spiked urine solutions. Owing to excellent sensitivity, ease of miniaturization, simple sensing procedure, low-cost, and fast response, the proposed electrochemical aptasensor indicates a great potential towards the development of lactoferrin analysis systems, which would be helpful in the early diagnosis of UTI.

Magnetic and gold nanocomposite as a novel aptasensor for early detection of tetracycline residues

Tetracycline (TET) is one of the most conventional antibiotics with a widespread application in veterinary medicine and livestock like mastitis. Detection of this antibiotic in milk is far important because its presence reduces the dairy products and also can increase the antibiotic resistance in human body. In current study, an electrochemical aptamer-based biosensor was designed for TET residues detection as a sensitive, selective, and rapid way based on a pencil graphite electrode (PGE) using magnetic nanoparticles (MNP) and gold nanoparticles (GNP) composite. The linear concentration range and detection limit values of the aptasensor was obtained as 1.0 pM to 1.0 μM, and 0.03 pM toward TET, which is lower than the value determined as allowable by the European Union (225 nM). A high selectivity for TET was shown by aptasensor over the other antibiotics including Sulfadiazine, Ampicillin, Penicillin G, and Streptomycin. The results of the TET-spiked in milk samples suggested reasonable recoveries between 92 and 102%. As a result, 90 min was the optimization time for aptasensor response. The relative standard deviation (RSD) was obtained as 5.9%, indicating an acceptable reproducibility for the aptasensor. The obtained results showed that the fabricated aptasensor is an effective, selective, and rapid assay for TET detection in milk and food quality control.

Design Strategies for Electrochemical Aptasensors for Cancer Diagnostic Devices

Improved outcomes for many types of cancer achieved during recent years is due, among other factors, to the earlier detection of tumours and the greater availability of screening tests. With this, non-invasive, fast and accurate diagnostic devices for cancer diagnosis strongly improve the quality of healthcare by delivering screening results in the most cost-effective and safe way. Biosensors for cancer diagnostics exploiting aptamers offer several important advantages over traditional antibodies-based assays, such as the in-vitro aptamer production, their inexpensive and easy chemical synthesis and modification, and excellent thermal stability. On the other hand, electrochemical biosensing approaches allow sensitive, accurate and inexpensive way of sensing, due to the rapid detection with lower costs, smaller equipment size and lower power requirements. This review presents an up-to-date assessment of the recent design strategies and analytical performance of the electrochemical aptamer-based biosensors for cancer diagnosis and their future perspectives in cancer diagnostics.

Current signal amplification strategies in aptamer-based electrochemical biosensor: A review

Due to their high specificity and affinity towards various targets, along with other unique advantages such as stability and low cost, aptamers are widely applied in analytical techniques. A typical aptamer-based electrochemical biosensor is composed of a aptamer as the biological recognition element and transducer converting the biologic interaction into electrical signals for the quantitative measurement of targets. Improvement of the sensitivity of a biosensor is significantly important in order to achieve the detection of biomolecules with low abundance, and different amplification strategies have been explored. The strategies either employ nanomaterials such as gold nanoparticles to construct electrodes which can transfer the biological reactions more efficiently, or attempt to obtain enhanced signal through multi-labeled carriers or utilize enzyme mimics to catalyze redox cycling. This review discusses recent advances in signal amplification methods and their applications. Critical assessment of each method is also considered.

A wrinkled structure of gold film greatly improves the signaling of electrochemical aptamer-based biosensors.

Electrochemical aptamer-based (E-AB) sensors provide a great opportunity towards the goal of point-of-care and wearable sensing technologies due to their good sensitivity and selectivity. Nevertheless, the output signals from this sensor class remain low when sensors are interrogated via square-wave voltammetry. This low signaling limits the sensor's precision for its capability to detect small changes in target concentrations. To circumvent this, we proposed here the use of a readily shrink-induced, wrinkled Au-coating polyolefin film to immobilize a greater number of DNA probes and thus improve the signaling. Specifically, wrinkled gold film exhibits a 5.5-fold increase of surface area in comparison to the unwrinkled ones. Using these substrates we fabricated a set of E-AB sensors of three biological molecules, including kanamycin, doxorubicin and ATP. We achieved up to 10-fold increase in its current and also good accuracies within ±20% error in the target concentration range across 2 orders of magnitude.

Re-engineering Electrochemical Aptamer-Based Biosensors to Tune Their Useful Dynamic Range via Distal-Site Mutation and Allosteric Inhibition.

Electrochemical aptamer-based (E-AB) sensors, exploiting binding-induced changes in biomolecular conformation, are rapid, specific, and selective and perform well even in a complex matrix, such as directly in whole blood and even in vivo. However, like all sensors employing biomolecular recognitions, E-AB sensors suffer from an inherent limitation of single-site binding, i.e., its fixed dose-response curve. To circumvent this, we employ here distal-site mutation and allosteric inhibition to rationally tune the dynamic range of E-AB sensors, achieving sets of sensors with a significantly varied target affinity (∼3 orders of magnitude). Using their combination, we recreate several approaches to narrow (down to 5-fold) or extend (up to 2000-fold) the dynamic range of biological receptors. The thermodynamic consequences of aptamer-surface interactions are estimated via the free-energy difference in solution-phase and surface-bound biosensors employing the same aptamer as a recognition element, revealing that an allostery strategy provides a more predictable and efficient means to finely control the target affinity and dynamic range. Such an ability to rationally modulate the affinity of biomolecule receptors would open the door to applications including cancer therapy, bioelectronics, and many other fields employing biomolecule recognition.

Employing an Intercalated Redox Reporter in Electrochemical Aptamer-Based Biosensors to Enable Calibration-Free Molecular Measurements in Undiluted Serum.

Electrochemical aptamer-based (E-AB) biosensors suffer from sensor-to-sensor signal variations due to the variation of the total number and the heterogeneity of probes immobilized on the electrode surface, with the former attracting more attention. As such, a calibration process to correct for such variations is required for this type of sensor, causing inconvenience and inaccessibility in harsh sensing environments such as blood samples, which has dramatically limited the widespread clinical use of biosensors. In response, here, we have adopted E-AB sensors to achieve calibration-free measurements of small biological/drug molecules. Specifically, we employ one probe-attached redox reporter and a second intercalated redox reporter to generate two signals, achieving good sensor-to-sensor reproducibility and thus obviating the need for calibration. We first demonstrated the capability of E-AB sensors for the accurate measurement of kanamycin, tobramycin, and adenosine triphosphate (ATP) in phosphate-buffered saline (PBS) buffer, achieving concentration ranges of approximately 4.7 × 103-, 2.0 × 103-, and 12.7-fold, respectively. Then, we applied this calibration-free approach to the measurement of these three target molecules directly in undiluted serum, achieving a concentration precision of a few micromolars.

Recent Advances in the Aptamer-Based Electrochemical Biosensors for Detecting Aflatoxin B1 and Its Pertinent Metabolite Aflatoxin M1.

The notable toxicological impacts of aflatoxin B1 (AFB1) and its main metabolite, aflatoxin M1 (AFM1), on human being health make the evaluation of food quality highly significant. Due to the toxicity of those metabolites—even very low content in foodstuffs—it is crucial to design a sensitive and reliable procedure for their detection. Electrochemical aptamer-based biosensors are considered the most encouraging option, based on multi-placed analysis, rapid response, high sensitivity and specificity. The present review specifically emphasizes the potential utilization of the electrochemical aptasensors for determining the AFM1 and AFB1 with different electrodes.