Effect of molecular crowding on the response of an electrochemical DNA sensor

E-DNA sensors, the electrochemical equivalent of molecular beacons, appear to be a promising means of detecting oligonucleotides. E-DNA sensors are comprised of a redox-modified (here, methylene blue or ferrocene) DNA stem-loop covalently attached to an interrogating electrode. Because E-DNA signaling arises due to binding-induced changes in the conformation of the stem-loop probe, it is likely sensitive to the nature of the molecular packing on the electrode surface. Here we detail the effects of probe density, target length, and other aspects of molecular crowding on the signaling properties, specificity, and response time of a model E-DNA sensor. We find that the highest signal suppression is obtained at the highest probe densities investigated, and that greater suppression is observed with longer and bulkier targets. In contrast, sensor equilibration time slows monotonically with increasing probe density, and the specificity of hybridization is not significantly affected. In addition to providing insight into the optimization of electrochemical DNA sensors, these results suggest that E-DNA signaling arises due to hybridization-linked changes in the rate, and thus efficiency, with which the redox moiety collides with the electrode and transfers electrons.     

Surface chemistry effects on the performance of an electrochemical DNA sensor

E-DNA sensors are a reagentless, electrochemical oligonucleotide sensing platform based on a redox-tag modified, electrode-bound probe DNA. Because E-DNA signaling is linked to hybridization-linked changes in the dynamics of this probe, sensor performance is likely dependent on the nature of the self-assembled monolayer coating the electrode. We have investigated this question by characterizing the gain, specificity, response time and shelf-life of E-DNA sensors fabricated using a range of co-adsorbates, including both charged and neutral alkane thiols. We find that, among the thiols tested, the positively charged cysteamine gives rise to the largest and most rapid response to target and leads to significantly improved storage stability. The best mismatch specificity, however, is achieved with mercaptoethanesulfonic and mercaptoundecanol, presumably due to the destabilizing effects of, respectively, the negative charge and steric bulk of these co-adsorbates. These results demonstrate that a careful choice of co-adsorbate chemistry can lead to significant improvements in the performance of this broad class of electrochemical DNA sensors.     

An enzyme-based E-DNA sensor for sequence-specific detection of femtomolar DNA targets

In this work, we report an enzyme-based E-DNA sensor for the sequence-specific detection of nucleic acids. This DNA sensor employs a "stem-loop" DNA probe dually labeled with biotin and digoxigenin (DIG). The probe is immobilized at an avidin-modified electrode surface via the biotin-avidin bridge, and the DIG serves as an affinity tag for the enzyme binding. In the initial state of the sensor, the probe adopts the stem-loop structure, which shields DIG from being approached by a bulky horseradish peroxidase-linked-anti-DIG antibody (anti-DIG-HRP) due to the steric effect. After hybridization, the probe undergoes a significant conformational change, forcing DIG away from the electrode. As a result, the DIG label becomes accessible by the anti-DIG-HRP, and the target hybridization event can be sensitively transduced via the enzymatically amplified electrochemical current signal. By using this new strategy, we demonstrate that the prototype E-DNA sensor has been able to detect as low as femtomolar DNA targets with excellent differentiation ability for even single mismatches.     

DNA-PEG-DNA triblock macromolecules for reagentless DNA detection

The sandwich assay is the most common design for electrochemical DNA sensors. This assay consists of three individual DNA components: an immobilized capture strand, a target strand, and a probe strand containing a redox-active reporter group. We report a simplified DNA assay where two strands of ssDNA, the capture and probe strands, are linked together via a flexible poly(ethylene glycol) (PEG) spacer forming an ABA triblock macromolecule. We have developed an electrochemical assay where the detection signal arises as a consequence of a large structural change induced upon hybridization with target DNA. In this system, the DNA-PEG-DNA macromolecule folds or wraps around the target DNA, bringing the ferrocene probe in close proximity to the electrode, affording an electrochemical response.     

Probe accessibility effects on the performance of electrochemical biosensors employing DNA monolayers

Surface-confined DNA probes are increasingly used as recognition elements (or presentation scaffolds) for detection of proteins, enzymes, and other macromolecules. Here we demonstrate that the density of the DNA probe monolayer on the gold electrode is a crucial determinant of the final signalling of such devices. We do so using redox modified single-stranded and double-stranded DNA probes attached to the surface of a gold electrode and measuring the rate of digestion in the presence of a non-specific nuclease enzyme. We demonstrate that accessibility of DNA probes for binding to their macromolecular target is, as expected, improved at lower probe densities. However, with double-stranded DNA probes, even at the lowest densities investigated, a significant fraction of the immobilized probe is inaccessible to nuclease digestion. These results stress the importance of the accessibility issue and of probe density effects when DNA-based sensors are used for detection of macromolecular targets.     

Signal enhancement for gene detection based on a redox reaction of [Fe(CN)(6)](4-) mediated by ferrocene at the terminal of a peptide nucleic acid as a probe with hybridization-amenable conformational flexibility

Electrochemically enhanced DNA detection was demonstrated by utilizing the couple of a synthesized ferrocene-terminated peptide nucleic acid (PNA) with a cysteine anchor and a sacrificial electron donor [Fe(CN)(6)](4-). DNA detection sensors were prepared by modifying a gold electrode surface with a mixed monolayer of the probe PNA and 11-hydroxy-1-undecanethiol (11-HUT), protecting [Fe(CN)(6)](4-) from any unexpected redox reaction. Before hybridization, the terminal ferrocene moiety of the probe was subject to a redox reaction due to the flexible probe structure and, in the presence of [Fe(CN)(6)](4-), the observed current was amplified based on regeneration of the ferrocene moiety. Hybridization decreased the redox current of the ferrocene. This occurred because hybridization rigidified the probe structure: the ferrocene moiety was then removed from the electrode surface, and the redox reaction of [Fe(CN)(6)](4-) was again prevented. The change in the anodic current before and after hybridization was enhanced 1.75-fold by using the electron donor [Fe(CN)(6)](4-). Sequence-specific detection of the complementary target DNA was also demonstrated.     

A regenerated electrochemical biosensor for label-free detection of glucose and urea based on conformational switch of i-motif oligonucleotide probe

Improving the reproducibility of electrochemical signal remains a great challenge over the past decades. In this work, i-motif oligonucleotide probe-based electrochemical DNA (E-DNA) sensor is introduced for the first time as a regenerated sensing platform, which enhances the reproducibility of electrochemical signal, for label-free detection of glucose and urea. The addition of glucose or urea is able to activate glucose oxidase-catalyzed or urease-catalyzed reaction, inducing or destroying the formation of i-motif oligonucleotide probe. The conformational switch of oligonucleotide probe can be recorded by electrochemical impedance spectroscopy. Thus, the difference of electron transfer resistance is utilized for the quantitative determination of glucose and urea. We further demonstrate that the E-DNA sensor exhibits high selectivity, excellent stability, and remarkable regenerated ability. The human serum analysis indicates that this simple and regenerated strategy holds promising potential in future biosensing applications.     

Designs of Enterobacteriaceae Lac Z Gene DNA Gold Screen Printed Biosensors

This paper describes specific electrochemical enterobacteriaceae lac Z gene DNA sensors based on immobilization of a thiolated 25 base single stranded probe onto disposable screen printed gold electrodes (gold SPEs). Two configurations have been evaluated. In the first one, the capture probe was attached to the electrode surface through its ? SH moiety, while mercaptohexanol (MCH) was used as spacer for the displacement of nonspecifically adsorbed oligonucleotide molecules. The hybridization event between the probe and target DNA sequences was detected at ?0.20 V by square‐wave voltammetry (SWV), using methylene blue (MB) as electrochemical indicator. The second genosensor configuration involved modification of gold high temperature SPEs with a 3,3′‐dithiodipropionic acid di(N‐succinimidyl ester) (DTSP) self‐assembled monolayer (SAM). Moreover, 2‐aminoethanol was used as blocking agent, and further modification with avidin allowed binding of the biotinylated enterobacteriaceae lac Z gene DNA probe. An enzyme amplified detection scheme was applied, based on the coupling of streptavidin‐peroxidase to the biotinylated complementary target, after the hybridization process, and immobilization of tetrathiafulvalene (TTF) as redox mediator atop the modified electrode. The amperometric response obtained at ?0.15 V after the addition of hydrogen peroxide was used to detect the hybridization process. Experimental variables concerning sensors composition and electrochemical transduction were evaluated in both cases. A better precision and reproducibility in the fabrication process, as well as a higher sensitivity were achieved using the biotinylated probe‐based sensor configuration. A limit of detection of 0.002 ng/μL was obtained without any preconcentration step.     

Effect of diluent chain length on the performance of the electrochemical DNA sensor at elevated temperature

Here we report the effect of passivating diluent chain length and sensor interrogation temperature on the electrochemical DNA (E-DNA) sensor's mismatch discrimination capability. Both stem-loop and linear probe-based E-DNA sensors were constructed with various diluents, including 6-mercapto-1-hexanol and longer chain hydroxyl-terminated alkanethiols. Contrary to previously reported results, we find that the E-DNA sensors work optimally in the presence of the longer chain diluents, signified by the enhanced % signal suppression observed upon target hybridization. Of note, the sensors' signaling efficiency maintains even when interrogated at an elevated temperature, permitting the use of stringent temperature conditions to improve sensor specificity. For example, a stem-loop E-DNA sensor fabricated with 8-mercapto-1-octanol, when employed at 47 °C, produces signal suppression of 79%, 35% and 1.6% for the perfect match, single-base mismatch, and 2-base mismatch DNA targets, respectively. In addition to the significant enhancement in sensor discrimination capacity, high temperature operation also improves hybridization kinetics. Our results also suggest that the stem-loop E-DNA sensors demonstrate better mismatch discrimination capability when compared to the linear probe system under the same experimental condition.     

Aptamer-based electrochemical sensors that are not based on the target binding-induced conformational change of aptamers

This study describes a new kind of aptamer-based electrochemical sensor that is not based on the target binding-induced conformational change of the aptamers by using a 15-mer thrombin-binding aptamer (5'-GGTTGGTGTGGTTGG-3') as the model oligonucleotide. The sensors are developed by first self-assembling the aptamer (i.e. a thrombin-binding aptamer) onto an Au electrode and then hybridizing the assembled aptamer with a ferrocene (Fc)-labeled short aptamer-complementary DNA oligonucleotide to form an electroactive double-stranded DNA (ds-DNA) oligonucleotide onto the Au electrode. The binding of the target (i.e. thrombin) towards the aptamer essentially destroys the Watson-Crick helix structure of the ds-DNA oligonucleotide assembled onto the electrode and leads to the dissociation of the Fc-labeled short complementary DNA oligonucleotide from the electrode surface to the solution, resulting in a decrease in the current signal obtained at the electrode, which can be used for the determination of the target. With the thrombin-binding aptamer as the model oligonucleotide, the current decrease obtained with the aptamer-based electrochemical sensors is linear with the concentration of thrombin within the concentration range from 0 to 10 nM (DeltaI/nA = 6.7C(thrombin)/nM + 2.8, gamma = 0.975). Unlike most kinds of existing aptamer-based electrochemical sensor, the electrochemical aptasensors demonstrated here are not based on the conformational change of the aptamers induced by the specific target binding. Moreover, the aptasensors are essentially label-free and are very responsive toward the targets. This study may pave a facile and general way to the development of aptamer-based electrochemical sensors.     

Effect of redox label tether length and flexibility on sensor performance of displacement-based electrochemical DNA sensors

This article summarizes the sensor performance of four electrochemical DNA sensors that exploit the recently developed displacement-replacement sensing motif. In the absence of the target, the capture probe is partially hybridized to the signaling probe at the distal end, positioning the redox label, methylene blue (MB), away from the electrode. In the presence of the target, the MB-modified signaling probe is released; one type of probe is capable of assuming a stem-loop probe (SLP) conformation, whereas the other type adopts a linear probe (LP) conformation. Independent of the sensor architecture, all four sensors showed “signal-on” sensor behavior. Unlike the previous report, here we focused on elucidating the effect of the redox label tether length and flexibility on sensor sensitivity, specificity, selectivity, and reusability. For both SLP and LP sensors, the limit of detection was 10 pM for sensors fabricated using a signaling probe with three extra thymine (T3) bases linked to the MB label. A limit of detection of 100 pM was determined for sensors fabricated using a signaling probe with five extra thymine (T5) bases. The linear dynamic range was between 10 pM and 100 nM for the T3 sensors, and between 100 pM and 100 nM for the T5 sensors. When compared to the LP sensors, the SLP sensors showed higher signal enhancement in the presence of the full-complement target. More importantly, the SLP-T5 sensor was found to be highly specific; it is capable of discriminating between the full complement and single-base mismatch targets even when employed in undiluted blood serum. Overall, these results highlight the advantages of using oligo-T(s) as a tunable linker to control flexibility of the tethered redox label, so as to achieve the desired sensor response.     

DNA hybridization at microbeads with cathodic stripping voltammetric detection

In electrochemical DNA hybridization sensors generally a single-stranded probe DNA was immobilized at the electrode followed by hybridization with the target DNA and electrochemical detection of the hybridization event at the same electrode. In this type of experiments nonspecific adsorption of DNA at the electrode caused serious difficulties especially in the case of the analysis of long target DNAs. We propose a new technology in which DNA is hybridized at a surface H and the hybridization is detected at the detection electrode (DE). This technology significantly extends the choice of hybridization surfaces and DEs. Here we use paramagnetic Dynabeads Oligo(dT)(25) (DBT) as a transportable reactive surface H and a hanging mercury drop electrode as DE. We describe a label-free detection of DNA and RNA (selectively captured at DBT) based on the determination of adenines (at ppb levels, by cathodic stripping voltammetry) released from the nucleic acids by acid treatment. The DNA and RNA nonspecific adsorption at DBT is negligible, making thus possible to detect the hybridization event with a great specificity and sensitivity. Specific detection of the hybridization of polyribonucleotides, mRNA, oligodeoxynucleotides, and a DNA PCR product (226 base pairs) is demonstrated. New possibilities in the development of the DNA hybridization sensors opened by the proposed technology, including utilization of catalytic signals in nucleic acid determination at mercury (e.g. signals of osmium complexes covalently bound to DNA) and solid DEs (e.g. using enzyme-labeled antibodies against chemically modified DNAs) are discussed.     

Label- and marker-free gene detection based on hybridization-induced conformational flexibility changes in a ferrocene-PNA conjugate probe

We report a strategy for label-free and marker-free gene detection transducing the hybridization event to an electrochemical signal based on the hybridization-induced conformational flexibility change in probe structure. The probe structure was designed to possess a ferrocene moiety as a reporter part and a cysteine moiety as an anchor part at each end of a peptide nucleic acid (PNA) as a recognition part. Electrochemical examination of probe-modified gold electrodes revealed that the ferrocene moiety was placed at the flexible end of the linear probe chain. Upon hybridization with a complementary target DNA, the resultant rigid duplex restricted the ferrocene motion to the electrode surface, causing a decrease in the observed current. The target DNA was detected with the detection limit of 1.44 x 10(-11) M. Thus the probe functioned as a 'self-reporting probe' and detection of the target DNA was demonstrated without the need for external indicators. Moreover, the sensor electrode was able repeatedly to detect the target DNA by the process of regeneration and could discriminate a mismatched DNA.     

Electrochemical Label‐Free Nucleotide Sensors

Numerous researchers have devoted a great deal of effort over the last few decades to the development of electrochemical oligonucleotide detection techniques, owing to their advantages of simple design, inherently small dimensions, and low power requirements. Their simplicity and rapidity of detection makes label‐free oligonucleotide sensors of great potential use as first‐aid screening tools in the analytical field of environmental measurements and healthcare management. This review article covers label‐free oligonucleotide sensors, focusing specifically on topical electrochemical techniques, including intrinsic redox reaction of bases, conductive polymers, the use of electrochemical indicators, and highly ordered probe structures.     

A sensitive and label-free impedimetric biosensor based on an adjunct probe

A highly sensitive and label-free impedimetric biosensor is achieved based on an adjunct probe attached nearby the capture probe. In this work, the adjunct probe was co-assembled on the surface of gold electrode with the capture probe hybridized with the reporter probe, and then 6-mercapto-1-hexanol was employed to block the nonspecific binding sites. When target DNA was added, the adjunct probe functioned as a fixer to immobilize the element of reporter probe displaced by the target DNA sequences and made the reporter probe approach the electrode surface, leading to effective inhibition of charge transfer. The increase in charge transfer resistance is related to the quantity of the target DNA in a wide range. The linear range for target DNA with specific sequences was from 0.1 nM to 0.5 μM with a good linearity (R = 0.9988) and a low detection limit of 6.3 pM. This impedimetric biosensor has the advantages of simplicity, sensitivity, good selectivity, and large dynamic range.     

Electrochemical detection of E. coli 16S rDNA sequence using air-plasma-activated fullerene-impregnated screen printed electrodes

A new DNA modified electrode for the electrochemical detection of 16S rDNA extracted from Escherichia coli (JCM1649) is proposed. The electrodes were fabricated by screen printing a fullerene-impregnated carbon ink onto a poly(methylmethacrylate) substrate and immobilizing a probe DNA on the surface after activating the electrode with air plasma. The results indicated a dramatic improvement in the surface coverage of the immobilized probe DNA, and of the reduction peak of the redox indicator (Co(phen)(3)(3+)) due to the incorporation of fullerene. By immobilizing the probe onto the fullerene-impregnated screen-printed electrodes, the PCR product of the 16S rDNA extracted from E. coli was directly detected without any pretreatment. A well defined signal difference was observed between the perfectly matching oligonucleotide and the mismatching one, and it was possible to detect the target at the modified electrode. This method enabled us to clearly detect the two base mismatches in the ca. 1500-bases long 16S rDNA sequence.     

Selective DNA detection at Zeptomole level based on coulometric measurement of gold nanoparticle-mediated electron transfer across a self-assembled monolayer

A selective DNA sensing with zeptomole detection level is developed based on coulometric measurement of gold nanoparticle (AuNPs)-mediated electron transfer (ET) across a self-assembled monolayer on the gold electrode. After immobilization of a thiolated hairpin-structured DNA probe, an alkanethiol monolayer was self-assembled on the resultant electrode to block [Fe(CN)6 ]-3-/4in a solution from accessing the electrode. In the presence of DNA target, hybridization between the DNA probe and the DNA target breaks the stem duplex of DNA probe. Consequently, stem moiety at the 3′-end of the DNA probes was removed from the electrode surface and made available for hybridization with the reporter DNA-AuNPs conjugates (reporter DNA-AuNPs). The thiolated reporter DNA matches the stem moiety at the 3′-end of the DNA probe. AuNPs were then enlarged by immersing the electrode in a growth solution containing HAuCl 4 and H2O2 after the reporter DNA-AuNPs bound onto the electrode surface. The enlarged AuNPs on the electrode restored the ET between the electrode and the [Fe(CN)6]3 -/4- , as a result, amplified signals were achieved for DNA target detection using the coulometric measurement of Fe(CN)6 3- electro-reduction by prolonging the electrolysis time. The quantities of ET on the DNA sensor increased with the increase in DNA target concentration through a linear range of 3.0 fM to 1.0 pM when electrolysis time was set to 300 s, and the detection limit was 1.0 fM. Correspondingly, thousands of DNA (zeptomole) copies were detected in 10L samples. Furthermore, the DNA sensor showed excellent differentiation ability for single-base mismatch.     

A nanocomposite consisting of plasma-polymerized propargylamine and graphene for use in DNA sensing

We report on a novel graphene-based nanoarchitecture modified with plasma-polymerized propargylamine (G-PpPG) and its application in electrochemical sensors for DNA. Films of G-PpPG were characterized by X-ray photoelectron spectroscopy and electrochemical impedance spectroscopy. The presence of graphene enhances the electrochemical activity of the films, and the high density of amino groups (deposited at a low plasma input power) on their surface assists in the immobilization of probe DNA on the water-swollen polymeric network. By contrast, the degree of hybridization of the total complementary target DNA to the probe DNA remains unchanged when G-PpPG nanofilms prepared at higher input power. No substantial non-specific adsorption of totally mismatched target DNA on the polymer films is observed because of the complete coverage of the probe DNA. The detection limit for total complementary target DNA is approximately 1.84 nmol?·?L^?1. The dynamic range extends from 0.1 to 1,000 nmol?·?L^?1. The new nanocomposite may also be used to immobilize other probe DNA sequences, and this makes the approach potentially applicable to the detection of other oligomers. Figure

Preparing the DNA sensor made from the graphene-based nanoarchitecture modified by using PpPG (G-PpPG) includes the following processes: (a) Modifying the Au electrode with the graphene nanosheet, (b) depositing the PpPG film onto the Au electrode coated with graphene, (c) immobilizing the probe DNA onto the G-PpPG film, and (d) hybridizing the MM0 target with the G-PpPG film immobilized with P1     

A novel label-free electrochemical aptasensor for thrombin based on the {nano-Au/thionine}n multilayer films as redox probes

Herein, a novel label-free electrochemical aptasensor based on direct immobilization of the redox probes on an electrode surface was reported. Gold electrode coated Nafion was firstly modified with redox probe-thionine (Thi) through ion exchange adsorption. Then, with the help of chemisorption and electrostatic adsorption, negatively charged nano-Au and positively charged Thi were layer-by-layer (LBL) self-assembled onto the modified electrode surface, which formed {nano-Au/Thi⁺}_n multilayer films for improving the amount of redox probes and immobilizing thiolated thrombin aptamers (TBA). In the presence of target thrombin (TB), the TBA on the multilayer film could catch the TB onto the electrode surface, which resulted in a barrier for electro-transfer, leading to decrease of the current. The proposed method avoided the cubsome redox probe labeling process, increased the amount of redox probe and reduced the distance between the redox probe and electrode surface. Thus, the approach showed a high sensitivity and a wider linearity to TB in the range from 0.12 nM to 46 nM with a detection limit of 40 pM.