Aptamer-based Electrochemical Biosensor Research Articles

Aptamer-based electrochemical biosensor for detection of adenosine triphosphate using a nanoporous gold platform

In spite of the promising applications of aptamers in the bioassays, the development of aptamer-based electrochemical biosensors with the improved limit of detection has remained a great challenge. A strategy for the amplification of signal, based on application of nanostructures as platforms for the construction of an electrochemical adenosine triphosphate (ATP) aptasensor, is introduced in the present manuscript. A sandwich assay is designed by immobilizing a fragment of aptamer on a nanoporous gold electrode (NPGE) and its association to second fragment in the presence of ATP. Consequently, 3, 4-diaminobenzoic acid (DABA), as a molecular reporter, is covalently attached to the amine-label of the second fragment, and the direct oxidation signal of DABA is followed as the analytical signal. The sensor can detect the concentrations of ATP as low as submicromolar scales. Furthermore, 3.2% decrease in signal is observed by keeping the aptasensor at 4°C for a week in buffer solution, implying a desirable stability. Moreover, analog nucleotides, including GTP, UTP and CTP, do not show serious interferences and this sensor easily detects its target in deproteinized human blood plasma.

Immobilization of redox-labeled hairpin DNA aptamers on gold: Electrochemical quantitation of epithelial tumor marker mucin 1

We report herein an aptamer-based electrochemical biosensor for the quantitative determination of mucin 1 (MUC1), a glycoprotein expressed on most epithelial cell surfaces, based on the analyte-binding induced, global-scale conformational change of electrode-bound anti-MUC1 DNA aptamers. The modified electrode was readily prepared by directly immobilizing thiolated, single-stranded anti-MUC1 DNA aptamers on gold; they were also tethered at the distal end with methylene blue (MB) for electrochemical measurements. In the absence of MUC1, these DNA single strands fold into a thermodynamically stable “hairpin” conformation, which facilities direct electron transfer between MB and the gold electrode. Upon MUC1 binding, the aptamer no longer retains its inherent hairpin conformation, which relocates the redox center (MB) further away from the electrode surface. The surface density of the DNA aptamer probes on gold measured by MB voltammetric response is consistent with that of electrostatically bound [Ru(NH3)6]3+, indicating that every MB molecule is electroactive at low scan rates; yet, the electroactivity of MB decreases remarkably upon binding MUC1. The detection limit (50nM) and dynamic response range (up to 1.5μM) are superior to the commercially available enzyme-linked immunosorbent assay (ELISA) kits and our previously developed quantum dot-based fluorescence protocol. The sensing principle pertaining to the specific folding conformation described in this paper has the potential to serve as a general approach of using hairpin DNA aptamers to construct biosensors for other molecular analytes.

RNA Aptamer-Based Electrochemical Biosensor for Selective and Label-Free Analysis of Dopamine

The inherent redox activity of dopamine enables its direct electrochemical in vivo analysis ( Venton , B. J.; Wightman, M. R. Anal. Chem. 2003, 75, 414A). However, dopamine analysis is complicated by the interference from other electrochemically active endogenous compounds present in the brain, including dopamine precursors and metabolites and other neurotransmitters (NT). Here we report an electrochemical RNA aptamer-based biosensor for analysis of dopamine in the presence of other NT. The biosensor exploits a specific binding of dopamine by the RNA aptamer, immobilized at a cysteamine-modified Au electrode, and further electrochemical oxidation of dopamine. Specific recognition of dopamine by the aptamer allowed a selective amperometric detection of dopamine within the physiologically relevant 100 nM to 5 μM range in the presence of competitive concentrations of catechol, epinephrine, norepinephrine, 3,4-dihydroxy-phenylalanine (L-DOPA), 3,4-dihydroxyphenylacetic acid (DOPAC), methyldopamine, and tyramine, which gave negligible signals under conditions of experiments (electroanalysis at 0.185 V vs Ag/AgCl). The interference from ascorbic and uric acids was eliminated by application of a Nafion-coated membrane. The aptasensor response time was <1 s, and the sensitivity of analysis was 62 nA μM(-1) cm(-2). The proposed design of the aptasensor, based on electrostatic interactions between the positively charged cysteamine-modified electrode and the negatively charged aptamer, may be used as a general strategy not to restrict the conformational freedom and binding properties of surface-bound aptamers and, thus, be applicable for the development of other aptasensors.

Analyte-Driven Switching of DNA Charge Transport: De Novo Creation of Electronic Sensors for an Early Lung Cancer Biomarker

A general approach is described for the de novo design and construction of aptamer-based electrochemical biosensors, for potentially any analyte of interest (ranging from small ligands to biological macromolecules). As a demonstration of the approach, we report the rapid development of a made-to-order electronic sensor for a newly reported early biomarker for lung cancer (CTAP III/NAP2). The steps include the in vitro selection and characterization of DNA aptamer sequences, design and biochemical testing of wholly DNA sensor constructs, and translation to a functional electrode-bound sensor format. The working principle of this distinct class of electronic biosensors is the enhancement of DNA-mediated charge transport in response to analyte binding. We first verify such analyte-responsive charge transport switching in solution, using biochemical methods; successful sensor variants were then immobilized on gold electrodes. We show that using these sensor-modified electrodes, CTAP III/NAP2 can be detected with both high specificity and sensitivity (K(d) ~1 nM) through a direct electrochemical reading. To investigate the underlying basis of analyte binding-induced conductivity switching, we carried out Förster Resonance Energy Transfer (FRET) experiments. The FRET data establish that analyte binding-induced conductivity switching in these sensors results from very subtle structural/conformational changes, rather than large scale, global folding events. The implications of this finding are discussed with respect to possible charge transport switching mechanisms in electrode-bound sensors. Overall, the approach we describe here represents a unique design principle for aptamer-based electrochemical sensors; its application should enable rapid, on-demand access to a class of portable biosensors that offer robust, inexpensive, and operationally simplified alternatives to conventional antibody-based immunoassays.

Sensitive label-free electrochemical analysis of human IgE using an aptasensor with cDNA amplification

In this study, we developed an ultrasensitive label-free aptamer-based electrochemical biosensor, featuring a highly specific anti-human immunoglobulin E (IgE) aptamer as a capture probe, for human IgE detection. Construction of the aptasensor began with the electrodeposition of gold nanoparticles (AuNPs) onto a graphite-based screen-printed electrode (SPE). After immobilizing the thiol-capped anti-human IgE aptamer onto the AuNPs through self-assembly, we treated the electrode with mercaptohexanol (MCH) to ensure that the remaining unoccupied surfaces of the AuNPs would not undergo nonspecific binding. We employed a designed complementary DNA featuring a guanine-rich section in its sequence (cDNA G1) as a detection probe to bind with the unbound anti-human IgE aptamer. We measured the redox current of methylene blue (MB) to determine the concentration of human IgE in the sample. When the aptamer captured human IgE, the binding of cDNA G1 to the aptamer was inhibited. Using cDNA G1 in the assay greatly amplified the redox signal of MB bound to the detection probe. Accordingly, this approach allowed the linear range (coefficient of determination: 0.996) for the analysis of human IgE to extend from 1 to 100,000pM; the limit of detection was 0.16pM. The fabricated aptasensor exhibited good selectivity toward human IgE even when human IgG, thrombin, and human serum albumin were present at 100-fold concentrations. This method should be readily applicable to the detection of other analytes, merely by replacing the anti-human IgE aptamer/cDNA G1 pair with a suitable anti-target molecule aptamer and cDNA.

Aptasensing of Chloramphenicol in the Presence of Its Analogues: Reaching the Maximum Residue Limit

A novel, label-free folding induced aptamer-based electrochemical biosensor for the detection of chloramphenicol (CAP) in the presence of its analogues has been developed. CAP is a broad-spectrum antibiotic that has lost its favor due to its serious adverse toxic effects on human health. Aptamers are artificial nucleic acid ligands (ssDNA or RNA) able to specifically recognize a target such as CAP. In this article, the aptamers are fixed onto a gold electrode surface by a self-assembly approach. In the presence of CAP, the unfolded ssDNA on the electrode surface changes to a hairpin structure, bringing the target molecules close to the surface and triggering electron transfer. Detection limits were determined to be 1.6 × 10(-9) mol L(-1). In addition, thiamphenicol (TAP) and florfenicol (FF), antibiotics with a structure similar to CAP, did not influence the performance of the aptasensor, suggesting a good selectivity of the CAP-aptasensor. Its simplicity and low detection limit (because of the home-selected aptamers) suggest that the electrochemical aptasensor is suitable for practical use in the detection of CAP in milk samples.

Aptamer based electrochemical sensor for detection of human lung adenocarcinoma A549 cells

We report results of the studies relating to development of an aptamer-based electrochemical biosensor for detection of human lung adenocarcinoma A549 cells. The aminated 85-mer DNA aptamer probe specific for the A549 cells has been covalently immobilized onto silane self assembled monolayer (SAM) onto ITO surface using glutaraldehyde as the crosslinker. The results of cyclic voltammetry and differential pulse voltammetry studies reveal that the aptamer functionalized bioelectrode can specifically detect lung cancer cells in the concentration range of 103 to 107 cells/ml with detection limit of 103 cells/ml within 60 s. The specificity studies of the bioelectrode have been carried out with control KB cells. No significant change in response is observed for control KB cells as compared to that of the A549 target cells.

Aptamer-based electrochemical biosensor for label-free voltammetric detection of thrombin and adenosine

A bifunctional aptamer-based electrochemical biosensor for label-free detection of thrombin and adenosine has been developed. It was based on the principle of switching structures of aptamers from DNA/DNA duplex to DNA/target complex upon target addition. Thrombin aptamer was firstly immobilized on gold electrode surface by self-assembly, and then it was hybridized with adenosine aptamer. Methylene blue (MB), as an electrochemical indicator, was abundantly adsorbed on the aptamers via specific interaction of MB with guanine base. In the presence of thrombin or adenosine, the aptamer part could bind with thrombin or adenosine instead of MB. The decreased peak current of MB was intimately related to the concentration of thrombin or adenosine. In present work, the assay was linear in the ranges from 6 to 60 nM for thrombin and from 10 to 1000 nM for adenosine. Detection limits were determined to be 3 nM for thrombin and 10 nM for adenosine, respectively. Moreover, bovine plasma albumin (BSA) and lysozyme, or uridine and guanosine, did not influence performance of the biosensor, indicating good selectivity of this sensor for thrombin or adenosine detection. Finally, this sensor was successfully applied to thrombin and adenosine analysis in human plasma samples.

Recent Advances in Electrochemical Aptamer-Based Sensors

Aptamers are with their selectivity and affinity for specific target ligands ideal biorecognition units for any biosensor development. The development of improved techniques for in vitro selection of aptamers with high affinities (comparable to antibodies) for target molecules has transformed aptamers from costly and restrictedly accessible species into readily synthetically available biorecognition material for almost all possible targets - from small molecules to proteins and whole cells. Unsurprisingly, the new potential of aptamers has also been applied for development of novel aptamer-based electrochemical biosensors. In this paper we overview general tendencies and recent advances in this field, exemplified by impedimetric and electrochemical conformational aptamer-based biosensors, electrochemical aptamer-based biosensors exploiting redox indicators, catalytic and quantum dot labels, and inherent redox activity of DNA, and aptamer-modified field-effect transistors. Keywords: Apatmers, DNA, RNA, electrochemical aptamer-based biosensors, aptasensors, SELEX, adenosine monophosphate (AMP), neomycin-modified electrode, neomycin B, methylene blue (MB), aptamer-thrombin, ferrocene (Fc) redox, platelet-derived growth factor (PGDF), cocaine biosensor

Electrochemical Aptamer-Based Biosensors: Recent Advances and Perspectives

This paper reviews the advancements of a wide range of electrochemical aptamer-based biosensors, electrochemical aptasensors, for target analytes monitoring. Methods for immobilizing aptamers onto an electrode surface are discussed. Aptasensors are presented according to their detection strategies. Many of these are simply electrochemical, aptamer-based equivalents of traditional immunochemical approaches, sandwich and competition assays employing electroactive signaling moieties. Others, exploiting the unusual physical properties of aptamers, are signal-on (positive readout signal) and signal-off (negative readout signal) aptasensors based on target binding-induced conformational change of aptamers. Aptamer label-free devices are also discussed.

Rational design and performance testing of aptamer-based electrochemical biosensors for adenosine

This work emphasizes on the design criteria of aptamer-based biosensors for adenosine and the comparison of their performances by using both electrochemical and biochemical methods. The adenosine biosensors have been typically prepared by hybridizing a short bridging DNA (modified with a thiol for the attachment to gold electrode) to the aptamer strand that releases upon adenosine binding. We have carefully compared the two “isomeric” designs, one with the thiol-linker tethered to the 3′-hydroxyl and the other to the 5′-phosphate end of the bridging DNA. It has been demonstrated through electrochemical and gel electrophoresis experiments that the 3′-thiol bridged sensor shows much better performance than its 5′-counterpart on surface, whereas in solution phase the two sensor systems are equally sensitive. We believe that the “transient” secondary structure of the aptamer sequence restricts the freedom of the strand displacement upon analyte binding, leading to a sluggish release of the aptamer strand from the 5′-thiol-bridging DNA.

Electrochemical Aptasensors – Recent Achievements and Perspectives

AbstractThis article reviews recent achievements in developing aptamer‐based electrochemical biosensors (electrochemical aptasensors). Aptamers are single stranded DNA or RNA molecules with high specificity to various ligands. Their specificity is comparable and in certain cases even higher than those of antibodies. In contrast to antibodies, aptamers are prepared by an in vitro selection procedure developed simultaneously in the early 1990s by L. Gold and A. Ellington. Due to their stability and the possibility of chemical modification aptamers can be immobilized on various supports and serve as artificial receptors in biosensors. The first aptasensors developed in the second half of 1990s were based on optical detection. However, in early 2000 substantial interest arose to the development of electrochemical aptasensors. It has been shown that due to their simplicity and fast response they represent an excellent tool in practical applications. The main focus of this review is to discuss the configuration of aptamers and electrochemical methods for detecting aptamers–analyte interactions. We will also provide a brief history of aptamer development, along with molecular structure and methods of aptamer engineering. Methods for immobilizing aptamers onto a solid support are also discussed.

Design and testing of aptamer-based electrochemical biosensors for proteins and small molecules

The fabrication of aptamer-based electrochemical biosensors as an emerging technology has made the detection of small and macromolecular analytes easier, faster, and more suited for the ongoing transition from fundamental analytical science to the early detection of protein biomarkers. Aptamers are synthetic oligonucleotides that have undergone iterative rounds of in vitro selection for binding with high affinity to specific analytes of choice; a sensitive yet simple method to utilize aptamers as recognition entities for the development of biosensors is to transduce the signal electrochemically. In this review article, we attempt to summarize the state-of-the-art research progresses that have been published in recent years; in particular, we focus on the electrochemical biosensors that incorporate aptamers for sensing small organic molecules and proteins. Based on differences in the design of the DNA/RNA-modified electrodes, we classify aptamer-based electrochemical sensors into three categories, for which the analyte detection relies on: (a) configurational change, i.e., the analyte binding induces either an assembly or dissociation of the sensor construct; (b) conformational change, i.e., the analyte binding induces an alteration in the conformation (folding) of the surface immobilized aptamer strands; and (c) conductivity change, i.e., the analyte binding “switches on” the conductivity of the surface-bound aptamer–DNA constructs. In each section, we will discuss the performance of these novel biosensors with representative examples reported in recent literature.

Target induced dissociation (TID) strategy for the development of electrochemical aptamer-based biosensor

In this paper, we report a novel and more general signal-on strategy for the fabrication of electrochemical aptamer-based (E-AB) biosensor. The principle is that the interaction between the target and the aptamer strand may induce the formation and subsequent dissociation of target–aptamer complex from an electrode surface, and consequently, the remaining DNA strand on the electrode surface can hybridize again with a ssDNA containing an electrochemical probe. Differential pulse voltammetric studies have revealed that this target induced disassociation (TID) strategy is an effective signal-on method for the detection of ATP molecules with good selectivity. The TID strategy may also have several advantages, such as independence on the specific structure of either the aptamers or their complementary sequences and promotion of the generalization of E-AB sensors, the more convincible results due to the signal-on model, and the unnecessity to label the aptamers, which provides the optimized status for the reaction with the targets, etc.

Aptamer-based Electrochemical Biosensors for Highly Selective and Quantitative Detection of Adenosine

Abstract A new adenosine biosensor based on aptamer probe is introduced in this article. An amino-labeled aptamer probe was immobilized on the gold electrode modified with an o -phenylenediamine electropolymerized film. When adenosine is bound specifically to the aptamer probe, the interface of the biosensor is changed, resulting in the decrement of the peak current. The response current is proportional to the amount of adenosine in sample. The used electrode can be easily regenerated in hot water. The proposed biosensor represents a linear response to adenosine over a concentration range of 1.0x10 −7 —1.0x10 −4 mol/L with a detection limit of 1.0x10 −8 mol/L. The presented biosensor exhibits a nice specificity towards adenosine. It offers a promising approach for adenosine assay due to its excellent electrochemical properties that are believed to be very attractive for electrochemical studies and electroanalytical applications.