Simple Electrochemical Aptasensor Research Articles

Magneto-assisted enzymatic DNA walkers for simultaneous electrochemical detection of amyloid-beta oligomers and Tau.

DNA walkers have been widely explored and applied as biosensor elements to detect disease-related biomarkers. Traditional interface-anchored DNA walkers typically have a fixed swing arm range and an orientation of the preset track, which might complicate the design of a sensor system and limit its application in more scenes. We propose a simple electrochemical aptasensor to accurately detect Alzheimer's disease (AD) based on a nicking enzyme-powered DNA walker. In this method, bifunctional magnetic nanoparticles are used to identify and capture Aβ oligomers (AβO) and Tau and release the DNA walker. As the DNA walker moves freely on the surface of the electrode, the nicking enzymes circularly cleave and release the two signal substrate chains, significantly amplifying the signal. It has been demonstrated that the constructed sensor can sensitively detect AβO and Tau, and the combined analysis of dual markers improves the accuracy of AD diagnosis. Furthermore, this method can distinguish normal individuals from AD patients in real cerebrospinal fluid samples. The excellent performance of this biosensor makes it promising for clinical applications in diagnosing AD patients and prognosis assessment.

A highly sensitive electrochemical aptasensor for cocaine detection based on CRISPR-Cas12a and terminal deoxynucleotidyl transferase as signal amplifiers.

Cocaine is one of the mainly used illegal drugs in the world. Using the signal amplification elements of terminal deoxynucleotidyl transferase (TdT) and CRISPR-Cas12a, a highly sensitive and simple electrochemical aptasensor was introduced for cocaine quantification. When, no cocaine existed in the sample, the 3'-end of complementary strand of aptamer (CS) was extended by TdT, leading to the activation of CRISPR-Cas12a and remaining of very short oligonucleotides on the working electrode. So, the current signal was remarkably promoted. With the presence of cocaine, CS left the electrode surface. Thus, nothing changed following the incubation of TdT and CRISPR-Cas12a and the Aptamer/Cocaine complex presented on the electrode. Consequently, the [Fe(CN)6]3-/4- could not freely reach the electrode surface and the signal response was weak. Under optimal situations, the biosensor revealed a wide linear relation from 40 pM to 150nM with detection limit of 15 pM for cocaine. The sensitivity of the analytical system was comparable and even better than other reported methods for cocaine detection. The designed method displayed excellent cocaine selectivity. The aptasensor could work well for cocaine assay in serum samples. So, the aptasensor is expected to be an efficient analytical method with broad applications in the determination of diverse analytes.

A simple electrochemical aptasensor for saxitoxin detection.

In this study, a simple electrochemical aptasensor for saxitoxin (STX) detection was developed based on a short anti-STX aptamer with a methylene blue (MB) incorporated at the 3′-end. The aptamer was immobilized on the surface of gold electrode and served as the recognition element. The aptasensor was successfully used as a highly selective and sensitive biosensor for STX detection because of the selection of aptamer along with the excellent electrocatalytical properties of MB. With the increase of the dynamic concentration of STX from 1 nM to 1 μM, the aptasensor exhibited a sensitivity with a peak current change of 2.92 μA per decade and a low detection limit of 1 nM. In addition, the aptasensor demonstrated excellent selectivity to STX, which prevented the interferences from neo-STX, okadaic acid and common metal ions. Using the proposed principle, several other aptasensors with improved performance can be constructed via the modification of the electrochemical sensor with appropriate aptamers.

Highly sensitive electrochemical detection of circulating EpCAM-positive tumor cells using a dual signal amplification strategy

Circulating tumor cells (CTCs) play an important role in diagnosing cancer and metastasis; thus, appropriate sensors are required for analyzing CTCs in patient samples. Here, we developed a simple electrochemical aptasensor for highly sensitive and selective detection of CTCs based on rolling circle amplification (RCA) coupled with the catalytic ability of the hemin/G-quadruplex complex. In detail, we first modified the surface of a screen-printed carbon electrode using a biocompatible mixture of thiol functionalized mesoporous silica nanoparticles and ionic liquid. Next, gold nanostar structures were prepared by electrodeposition to increase the electrode surface area and electrochemical signal, followed by EpCAM aptamer immobilization. When EpCAM-positive cancer cells are present in the sample, they bind to the EpCAM aptamer on the modified electrode, and a number of cholesterol-labeled DNA probes that act as initiators for RCA are inserted into the cell membrane. Consequently, RCA is initiated to generate multiple tandem repeats of the G-quadruplex DNA structure, which is complexed with hemin to catalyze the H2O2-mediated oxidation of 3,3’,5,5’-tetramethyl benzidine and produces strong electrochemical signals. Using this strategy, we could sensitively determine target cancer cells down to a single cell with high selectivity even in human serum. The proposed strategy with the dual signal amplification can thus be a promising platform for simple and ultrasensitive detection of CTCs, and be used for the detection of other cancer cells and biomarkers in different clinical and biological samples.

An electrochemical sensing method based on an oligonucleotide structure for ultrasensitive detection of malachite green

Herein, a simple electrochemical aptasensor with the implementation of an oligonucleotide structure is introduced for determination of malachite green (MG) as a fungicide. The oligonucleotide structure is composed of MG aptamer (Apt) and its two complementary strands (CSs). Benefiting from physical barrier and high negatively charged of the oligonucleotide structure, the proposed sensing strategy indicated improved selectivity and sensitivity for MG detection with a detection limit of 0.15 nM. Addition of MG leads to conformational change of Apt sequence, its release from the electrode surface and dismantlement of the oligonucleotide structure. So, [Fe(CN)6]3−/4− as redox marker has high accessibility to the gold electrode surface and the peak current remarkably increases. Also, the aptasensor exhibited great potential for MG detection in tap water. The designed sensing method opens a simple way for ultrasensitive detection of MG in a relatively short time.

A signal-on electrochemical aptasensor for rapid detection of aflatoxin B1 based on competition with complementary DNA

Aflatoxin B1 (AFB1) is the most toxic mycotoxin, causing harmful effects on human and animal health, and the rapid and sensitive detection of AFB1 is highly demanded. We developed a simple electrochemical aptasensor achieving rapid detection of aflatoxin B1 (AFB1). A short anti-AFB1 aptamer having a methylene blue (MB) redox tag at the 3′-end was immobilized on the surface of a gold electrode. In the absence of AFB1, a complementary DNA (cDNA) strand hybridized with the MB-labeled aptamer, causing MB apart from the electrode surface and low current of MB. In the presence of AFB1, AFB1 competed with the cDNA in the binding to the MB-labeled aptamer, and the aptamer-AFB1 binding caused formation of a hairpin structure, making the MB close to the electrode surface and current of MB increase. Under optimized conditions, we achieved detection of AFB1 over dynamic concentration range of 2 nM–4 μM by using this signal-on electrochemical aptasensor. This method only required a simple 5-min incubation of sample solution prior to rapid electrochemical sensing, more rapid than other electrochemical aptasensors. The sensor could be well regenerated and reused. This sensor allowed to detect AFB1 spiked in 20-fold diluted beer and 50-fold diluted white wine, respectively. It shows potential for detection of AFB1 in wide applications.

Electrochemical aptasensor for activated protein C using a gold nanoparticle – Chitosan/graphene paste modified carbon paste electrode

In this work, a selective and simple electrochemical aptasensor was developed for the detection of activated protein C by employing methylene blue (MB) as a redox indicator. An activated protein C aptamer (APC-apt) was covalently immobilized on the surface of a carbon paste electrode modified with gold nanoparticle – chitosan /graphene paste (AuNPs-Chi/Gr). The AuNPs-Chi/Gr paste increased electrochemical peak current and immobilized the aptamer on the electrode surface. The process of aptasensor construction and successful immobilization of the aptamer on the electrode surface was confirmed by electrochemical cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Differential pulse voltammetry (DPV) was used to determine the methylene blue peak current. By replacing APC instead of MB at the electrode surface, the cathodic current of the MB decreases, and this decrease corresponds to the APC concentration. Under optimum conditions, the APC concentration was detected in the range from of 0.1 ng·mL−1 to 40 μg·mL−1 with a relatively low detection limit of 0.073 ng·mL−1. This method was then applied to the determination of APC in human serum samples. The results revealed that this strategy can be used to measure other proteins in biological samples.

Electrochemical aptasensor for aflatoxin B1 based on smart host-guest recognition of β-cyclodextrin polymer

Developing a simple and reliable method for the detection of the highly concerning mycotoxin, aflatoxin B1 (AFB1), is of great importance to food safety monitoring. In this study, a simple electrochemical aptasensor was presented for the detection of AFB1 based on the host-guest recognition between ferrocene and β-cyclodextrin (β-CD). Fc-labeled aptamer of AFB1 first hybridized with its complementary Fc-cDNA. Two ferrocene molecules were brought closely together and couldn’t enter into the cavity of β-CD modified on the electrode. Negligible signal could be observed. Once AFB1 captured the aptamer from the AFB1-sensitive dsDNA, Fc-cDNA was released and subsequently entered into the cavity of β-CD to form inclusion complexes, giving rise to an distinct increase of Ret and peak current because of the molecular recognition of β-CD. AC impedance method is more sensitive than DPV method. The electrochemical aptasensor displayed a sensitive response to AFB1 in a wide linear range of 0.1 pg/mL to 10 ng/mL, with a low detection limit of 0.049 pg/mL (0.147 pmol/mL) by AC impedance detection, which is 10–100 lower than previously reported methods. The aptasensor has good selectivity and reliability, which has been successfully applied to the determination of AFB1 in real peanut oil samples with recoveries ranging from 94.5% to 106.7% and inter-assay RSD lower than 11.51%.

Glycated Albumin Detection Using Electrochemical Aptasensor for Screening and Monitoring of Diabetes Mellitus

A simple electrochemical aptasensor was developed for the detection of glycated albumin (GHSA) using ssDNA aptamer that selectively binds to GHSA as a recognition element. The biotinylated ssDNA aptamer was immobilized on a streptavidin-modified screen-printed carbon electrode (SPCE). The changes of interfacial features of the electrode surface, which were based on the aptamer-GHSA interaction, were probed in the presence of the redox Fe(CN)6 3- using square wave voltammetry (SWV) measurements (Fig 1). The results showed that limit of detection of our sensor was 3 µg/ml, with a calibration curve to the range of 16 mg/ml. The aptasensor showed high selectivity for GHSA over other molecules that is usually available in the blood. Our aptasensor was successfully applied to detect GHSA in serum samples, which demonstrated the higher levels of GHSA concentrations in diabetes than normal persons. These indicate that our electrochemical aptasensor has a potential for diagnosis and monitoring of diabetes mellitus. Figure 1

Sensitive Electrochemical Aptasensor by Coupling “Signal-on’’ and “Signal-off’’ Strategies

A simple electrochemical aptasensor for sensitive and selective determination of adenosine triphosphate (ATP) has been developed on the basis of a new dual-signaling amplification strategy. This aptasensor features both ''signal-on'' and ''signal-off'' elements. The ferrocene (Fc)-labeled aptamer probe (Fc-P) is designed to hybridize with the thiolated methylene blue (MB)-modified DNA probe (MB-P) on gold electrode to form rigid duplex DNA. In the presence of ATP, the interaction between ATP and the aptamer leads to the dissociation of the duplex DNA structure and thereby the release of the Fc-P from the sensing interface. The single-stranded MB-P could thus tend to form a hairpin structure through the hybridization of the complementary sequences at both its ends. Such conformational changes result in the oxidation peak current of Fc decreases and that of MB increases, and the changes of dual signals are linear with the concentration of ATP. When "ΔI = ΔI(MB) + |ΔI(Fc)|" (ΔI(MB) and ΔI(Fc) are the change values of the oxidation peak currents of MB and Fc, respectively.) is used as the response signal for quantitative determination of ATP, the detection limit (1.9 nM) is much lower than that by using either MB-P or Fc-P alone. This new dual-signaling aptasensor is readily regenerated and shows good response toward the target. It will have important applications in the sensitive and selective electrochemical determination of other small molecules and proteins.

A simple electrochemical aptasensor for ultrasensitive protein detection using cyclic target-induced primer extension

A simple electrochemical aptasensor was developed for ultrasensitive protein detection by combining a novel strategy of cyclic target-induced primer extension (CTIPE) with an aptamer-hairpin probe and enzyme-amplified electrochemical readout. In the presence of protein target, the immobilized aptamer-hairpin probe recognized the protein to trigger primer extension reaction by target-induced conformational transition, which released the protein from replicated DNA duplex. The released target could cyclically bind with other aptamer-hairpin probes and trigger new primer extension, leading to formation of numerous biotin-tagged DNA duplex, which significantly amplified the protein recognition event and facilitated the subsequent enzymatic signal enhancement, leading to an ultrasensitive electrochemical aptasensor. Using human vascular endothelial growth factor as a model protein, the designed aptasensor could detect protein down to 0.82pgmL−1 with a linear range from 1pgmL−1 to 1ngmL−1. The proposed aptasensor was amenable to quantification of protein in complex biological matrixes, and would become a simple and powerful tool for bioanalysis and clinic diagnostic application.

Electrochemical aptasensor based on the dual-amplification of G-quadruplex horseradish peroxidase-mimicking DNAzyme and blocking reagent-horseradish peroxidase

A simple electrochemical aptasensor for sensitive detection of thrombin was fabricated with G-quadruplex horseradish peroxidase-mimicking DNAzyme (hemin/G-quadruplex system) and blocking reagent-horseradish peroxidase as dual signal-amplification scheme. Gold nanoparticles (nano-Au) were firstly electrodeposited onto single wall nanotube (SWNT)-graphene modified electrode surface for the immobilization of electrochemical probe of nickel hexacyanoferrates nanoparticles (NiHCFNPs). Subsequently, another nano-Au layer was electrodeposited for further immobilization of thrombin aptamer (TBA), which later formed hemin/G-quadruplex system with hemin. Horseradish peroxidases (HRP) then served as blocking reagent to block possible remaining active sites and avoided the non-specific adsorption. In the presence of thrombin, the TBA binded to thrombin and the hemin released from the hemin/G-quadruplex electrocatalytic structure, increasing steric hindrance of the aptasensor and decomposing hemin/G-quadruplex electrocatalytic structure, which finally decreased the electrocatalytic efficiency of aptasensor toward H 2O 2 in the presence of NiHCFNPs with a decreased electrochemical signal. On the basis of the synergistic amplifying action, a detection limit as low as 2 pM for thrombin was obtained.