Immobilization Of Probe DNA Research Articles

Silver Nanoparticles Decorated Polyaniline Nanowires-Based Electrochemical DNA Sensor: Two-step Electrochemical Synthesis

In this work, silver nanoparticles decorated onto polyaniline nanowires were synthesized site-specifically onto Pt working electrodes (with only 0.8 mm2 area) using a novel, rapid, and effective electrochemical procedure: electropolymerization of PANi NWs and electrodeposition of AgNPs. The complementary properties of PANi NWs and AgNPs were obtained, including high surface area, high electrochemical activity, high biocompatibility, high chemical stability and good adhesion with the electrode surface. Obtained results showed advantages of the electrosynthesis method compared to traditional methods, which have difficulties in binding between modifying materials with electrode surfaces. The sensor surface modification with the PANi NWs/AgNPs material facilitated the probe DNA immobilization and improved the electrochemical signal of the DNA sensors. The detection limit of the sensors was 2.80 × 10−15 M. The DNA sensors exhibited advantages including direct detection, high sensitivity, good specificity, and potential miniaturization for development of lab-on-a-chip systems.

A highly sensitive electrochemical biosensor for protein based on a tetrahedral DNA probe, N- and P-co-doped graphene, and rolling circle amplification

A tetrahedral DNA probe can effectively overcome the steric effects of a single-stranded probe to obtain well-controlled density and minimize nonspecific adsorption. Herein, a highly sensitive electrochemical biosensor is fabricated for determination of protein using a tetrahedral DNA probe and rolling circle amplification (RCA). N- and P-co-doped graphene (NP-rGO) is prepared, and AuNPs are then electrodeposited on it for DNA probe immobilization. Benefitting from the synergistic effects of the excellent electrical conductivity of NP-rGO, the stability of the tetrahedral DNA probe and the signal amplification of RCA, the biosensor achieves a low limit of 3.53 × 10-14M for thrombin and a wide linear range from 1 × 10-13 to 1 × 10-7M. This study provides a sensitive and effective method for the detection of protein in peripheral biofluids, and paves the way for future clinical diagnostics and treatment of disease. Graphical abstract.

Immobilization of DNA probes on a high frequency piezoelectric biosensor

In recent years, researchers have taken to biosensors as effective tools for detection due to their portability, low-cost, fast response, and practicality. Piezoelectricity gave way to quartz crystal microbalances (QCM), of which high-frequency QCMs (HFF-QCM 100MHz) are still being researched. In this paper, we use DNA immobilization on a HFF-QCM via self-assembled monolayers (SAM) technique. Immobilization was initially verified with ATR-FTIR. Then, DNA was immobilized in real time on the HFF-QCM crystals. A variation in the phase of the signal suggests fixation of DNA to the surface, in accordance with ATR-FTIR results. A density of 629 ng/cm2 was computed. Also, a positive correlation between immobilized DNA and DNA concentration, and the appearance of a saturation point between 1 and 5 μM were shown after analysis of different DNA concentrations.

Super-Resolution Imaging of Competitive Unlabeled DNA Hybridization Reveals the Influence of Fluorescent Labels on Duplex Formation and Dissociation Kinetics.

Single-molecule fluorescence imaging is a powerful method to measure reversible reaction kinetics, allowing one to monitor the bound state of individual probe molecules with fluorescently labeled targets. In the case of DNA hybridization, previous studies have shown that the presence of a fluorescent label on a target strand can exhibit significant influence on the stability of a DNA duplex that is formed. In this work, we have developed a super-resolution imaging method to measure the hybridization kinetics of unlabeled target DNA that compete with a fluorescently labeled tracer DNA strand to hybridize with an unlabeled probe DNA immobilized at a surface. The hybridization of an unlabeled DNA target cannot be detected directly, but its presence blocks the immobilized probe DNA, influencing the measured time intervals between labeled DNA hybridization events. We derive a model for competitive hybridization kinetics to extract the association and dissociation rate constants of the unlabeled species from the distribution of time intervals between hybridization events of the labeled tracer DNA at individual localized DNA probe sites. We use this methodology to determine the hybridization kinetics of a model 11-mer unlabeled target DNA strand and then determine how five different fluorescent labels attached to the same target DNA strand impact the hybridization kinetics. Compared to the unlabeled target, these labels can slow the association and dissociation rates by as much as a factor of 5. The super-resolution time-interval methodology provides a unique approach to determining fundamental (label-free) rates of DNA hybridization, revealing the significant influence of fluorescent labels on these kinetics. This measurement concept can be extended to studies of other reversible reaction systems, where kinetics of unlabeled species can be determined from their influence on the reaction of a labeled species with localized probe molecules on a surface.

Electrochemical Detection of Micro-RNAs As Potential Cancer Biomarker

MicroRNAs (miRNAs) are single-stranded RNA transcripts, typically of 19–23 nucleotides in length. Recently, miRNAs have been identified as important biomarkers that hold promise in early cancer diagnosis. Accumulative evidences have proved that abnormal expression of miRNAs is closely related with the occurrence, diagnosis and treatment of cancer [1]. Conventional methods of miRNA detection have some intrinsic drawbacks including the need for tedious and complex procedures, requirement of large amount of samples, highly skilled experts and high-end equipment. To address these shortcomings as well as to provide multiplexing capability, different types of electrochemical cancer biosensors have been developed. Electrochemical biosensors have been widely paid attention due to their merits including high sensitivity, specificity, low-cost, and multiplexing [2]. Herein, a label-free and sensitive biosensor was developed for miRNA-21 detection at femtomolar concentrations. A glassy carbon electrode (GCE) was first modified with carbon nanotube (CNT) which serves as platform for immobilization of probe DNA as well as providing higher surface area. Capture DNA probes were immobilized onto the surface of CNT modified electrode using an established molecular tethering method [3,4]. Electrochemical analyses confirmed the surface modification, immobilization, and hybridization between probe DNA and the target miRNA. Besides, different variables such as probe concentration and hybridization time were investigated and optimized to find the optimal experimental conditions for miRNA detection. The obtained results demonstrated that the proposed biosensor exhibits excellent analytical properties including high sensitivity, low detection limit, selectivity, and reproducibility for miRNA detection. References Lan H, Lu H, Wang X, Jin H (2015) MicroRNAs as potential biomarkers in cancer: opportunities and challenges. BioMed research international 2015Kilic T, Erdem A, Ozsoz M, Carrara S (2018) MicroRNA biosensors: Opportunities and challenges among conventional and commercially available techniques. Biosensors and Bioelectronics 99:525-546Ramasamy RP, Luckarift HR, Ivnitski DM, Atanassov PB, Johnson GR (2010) High electrocatalytic activity of tethered multicopper oxidase–carbon nanotube conjugates. Chemical Communications 46 (33):6045-6047Zhou Y, Fang Y, Ramasamy RP (2019) Non-covalent functionalization of carbon nanotubes for electrochemical biosensor development. Sensors 19 (2):392

Fe3O4@Au nanoparticles-based magnetoplatform for the HMGA maize endogenous gene electrochemical genosensing

This work addresses a technological advance applied to the construction of a magnetogenoassay with electrochemical transduction for the maize taxon-specific (HMGA gene) detection using gold-coated magnetic nanoparticles as nanosized platform.Superparamagnetic core-shell Fe3O4@Au nanoparticles (10.4 ± 1.7 nm) were used to assemble the genoassay through the covalent immobilization of HMGA DNA probes onto carboxylated self-assembled monolayers at the nanoparticles surface.A hybridization reaction using sandwich format was selected to prevent inefficient hybridization connected with stable secondary DNA structures using also fluorescein isothiocyanate as DNA signaling tag. The labelling of the hybridization reaction with enzymes allowed the chronoamperometric measurement of the peroxidase activity linked to the nanoplatform located on gold surface.Using this electrochemical magnetogenoassay a linear concentration range from 0.5 to 5 nM and a LOD of 90 pM with a RSD <1.2% was calculated. Certified maize was evaluated without further purification after PCR amplification. This work highlights the efficacy of the electrochemical magnetogenoassay for the HMGA detection, showing its potential as alternative procedure for the verification of the compliance of the legislation.

CIN2+ detection of the HPV DNA Array genotyping assay in comparison with the Cobas 4800 HPV test and cytology

BackgroundHPV DNA Array is an E1-targeting PCR genotyping test, with capability of distinguishing 18 high-risk (16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, 82) and 11 low-risk HPV types (6, 11, 40, 42, 44, 54, 67, 69, 70, 85, 97). HPV DNA Array uses multiplex PCR for E1-gene sequence amplification. The amplicons are detected and genotyped by reverse hybridization to immobilized DNA probes spotted as triplets in single 96 well-plate wells and read by AID ELISPOT reader.MethodsAim of the study was to evaluate the clinical performance of the assay against internationally accepted and FDA approved Cobas 4800 HPV test (Roche Diagnostics). Study population comprised of 500 cervical samples.ResultsHPV DNA Array demonstrated a very high sensitivity of 100% for CIN2+ and 100% for CIN3+ detection, same as Cobas 4800. HPV DNA Array showed greater sensitivity for CIN2+ detection than cytology (100% vs. 13.6%). The agreement to Cobas 4800 for HPV detection, irrespective of type, was 81.4% with κ = 0.613. The agreement for HPV 16 was 92.8% (κ = 0.929), and for HPV 18 54.2% (κ = 0.681).ConclusionHPV DNA Array demonstrated good clinical performance for detection of high-grade lesions, and may be considered for usage in a screening setting.

Novel 3D Printed Device for Dual-Signaling Ratiometric Photoelectrochemical Readout of Biomarker Using λ-Exonuclease-Assisted Recycling Amplification.

A ratiometric photoelectrochemical (PEC) sensing strategy was proposed for monitoring of carcinoembryonic antigen (CEA) based on a homemade 3D printing device with dual-working photoelectrodes (PE1 and PE2), coupling λ-exonuclease (λ-Exo)-assisted recycling amplification with CdS quantum dots. Gold nanoparticles-functionalized ZnO nanorods were utilized as PEC substrate for generating initial photocurrent and immobilizing DNA probe. Upon incubation of target with DNA trigger/CEA aptamer-modified magnetic bead (tri/apt-MB), DNA trigger dissociated from magnetic bead and then hybridized with capture probe (cp) on PE1 or opened hairpin probe (hp) on PE2 to form double-stranded DNA (dsDNA). The exonuclease could recognize and cleave two newly generated dsDNA, leading to the release of trigger. The free trigger strand continued to hybridize with the remaining cp/hp, which were cleaved by λ-Exo, and then trigger was released again and restarted next recycle with the λ-Exo. After digestion of λ-Exo, the number of capture probes on PE1 was reduced, and many short DNA fragments were produced on PE2, thereby resulting in the decreasing CdS QDs on PE1 and the increasing CdS QDs on PE2. As a result, it was observed that the ratio value of photocurrents (PE1/PE2) significantly decreased with the increasing CEA. Under optimum conditions, the sensing method showed a good linear relationship toward CEA within the dynamic range of 0.02-10 ng mL-1 and a detection limit of 6.0 pg mL-1. Moreover, the ratiometric PEC sensor exhibited good reproducibility, satisfying stability, and remarkable anti-interference performance, which suggests its promising application prospect to detect target CEA.

Influence of Oligonucleotide Grafting Density on Surface-Mediated DNA Transport and Hybridization.

Adsorption of soluble DNA to surfaces decorated with complementary DNA plays an important role in many bionanotechnology applications, and previous studies have reported complex dependencies of the surface density of immobilized DNA on hybridization. While these effects have been speculatively ascribed to steric or electrostatic effects, the influence of surface-mediated molecular transport (i.e., intermittent "hopping diffusion") has not been fully appreciated. Here, single-molecule tracking and Förster resonance energy transfer (FRET) were employed to characterize the mobility and the hybridization efficiency of adsorbed ssDNA oligonucleotides ("target") at solid-liquid interfaces exhibiting surface-immobilized ssDNA ("probe") over a wide range of surface grafting densities. Two distinct regimes were observed, with qualitatively different transport and hybridization behaviors. At dilute grafting density, only 1-3% of target molecules were observed to associate with probes (i.e., to hybridize). Adsorbing target molecules often searched unsuccessfully and "flew", via desorption-mediated diffusion, to secondary locations before hybridizing. In contrast, at high probe grafting density, approximately 20% of target DNA hybridized to immobilized probes, and almost always in the vicinity of initial adsorption. Moreover, following a dehybridization event, target molecules rehybridized at high probe density, but rehybridization was infrequent in the dilute density regime. Interestingly, the intermittent interfacial transport of mobile target molecules was suppressed by the presence of immobilized probe DNA, presumably due to an increased probability of readsorption following each "hop". Together, these findings suggested that many salient effects of grafting density on surface-mediated DNA hybridization can be directly related to the mechanisms of surface-mediated intermittent diffusion.

Electrochemical detection of DNA hybridization based on three-dimensional ZnO nanowires/graphite hybrid microfiber structure

In the present work, zinc oxide (ZnO) nanowire (NWs)/graphite microfiber hybrid electrodes were used for the detection of DNA hybridization. ZnO NWs were in-situ synthesized onto graphite fibers using the hydrothermal technique, and the result was characterized by X-ray diffraction (XRD), scanning electron microscope (SEM) as well as transmission electron microscope (TEM). An electrochemical sensor for the analysis of DNA hybridization was developed based on immobilizing DNA probes onto the ZnO NWs/graphite microfiber electrodes surface via electrostatic interaction. Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and X-ray photoelectron spectroscopy (XPS) were used to corroborate the fabrication of the sensor. Based on the results, ZnO NWs/graphite microfiber hybrid electrodes showed a great hybridization capacity for the determination of target DNA. A wide dynamic detection range from 1.0 × 10−14 M to 1.0 × 10−7 M with an ultralow detection limit of 3.3 × 10−15 M was achieved for detecting the target DNA. Moreover, it showed good selectivity and stability during the detection process.

Investigation of Electrical Stability and Sensitivity of Electric Double Layer Gated Field-Effect Transistors (FETs) for miRNA Detection.

In this research, we developed a miRNA sensor using an electrical double layer (EDL) gated field-effect transistor (FET)-based biosensor with enhanced sensitivity and stability. We conducted an in-depth investigation of the mechanisms that give rise to fluctuations in the electrical signal, affecting the stability and sensitivity of the miRNA sensor. Firstly, surface characteristics were studied by examining the metal electrodes deposited using different metal deposition techniques. The lower surface roughness of the gold electrode improved the electrical current stability. The temperature and viscosity of the sample solution were proven to affect the electrical stability, which was attributed to reducing the effect of Brownian motion. Therefore, by controlling the test conditions, such as temperature and sample viscosity, and the surface characteristics of the metal electrodes, we can enhance the stability of the sensor. Metal electrodes deposited via sputtering and e-beam evaporator yielded the lowest signal fluctuation. When ambient temperature was reduced to 3 °C, the sensor had better noise characteristics compared to room temperature testing. Higher viscosity of samples resulted in lower signal fluctuations. Lastly, surface functionalization was demonstrated to be a critical factor in enhancing the stability and sensitivity. MiRNA sensors with higher surface ratios of immobilized DNA probes performed with higher sensitivity and stability. This study reveals methods to improve the characteristics of EDL FET biosensors to facilitate practical implementation in clinical applications.

Construction of an electrochemical genosensor based on screen-printed gold electrodes (SPGE) for detection of a mutation in the adenomatous polyposis coli gene

Studies have well documented that mutations in the adenomatous polyposis coli gene (APC) are involved in the development of different types of cancer. Therefore, the objective of the present report was to design and construct a sensitive nanostructured genosensor able to detect specific sequences of the APC gene in order to be applied in early carcinogenesis diagnosis. We used screen-printed gold electrodes (SPGE) modified with DNA probes and mercaptoundecanoic acid chemically immobilized on a gold sensor surface. APC complementary DNA sequences were hybridized on the previously immobilized DNA probes. Besides, we tested effects of different intercalating agents, and various temperatures on the hybridization reaction to increase the sensitivity and selectivity of our device. The evaluation of these processes was carried out by means of the cyclic voltammetry technique (CV) to monitor the redox reaction of [Fe(CN)6]3−/4− on the SPGE-s. The constructed genosensor exhibited an excellent response to identify the APC gene in a concentration range from 100 pM to 100μM, with 43.92 pM as the limit of detection in the presence of doxorubicin as DNA intercalating agent. The selectivity of the device was tested by using a DNA sequence different from the APC gene, which shows negative response. Considering the importance of APC mutations in the development of tumors in the digestive, urogenital, and mammary tissues, here developed electrochemical genosensor, due to its easy management, short time of response, and high performance, may be useful for early cancer detection.

Biomimetic Nanochannel-Ionchannel Hybrid for Ultrasensitive and Label-Free Detection of MicroRNA in Cells.

A biomimetic nanochannel-ionchannel hybrid coupled with electrochemical detector was developed for label-free and ultrasensitive detection of microRNA (miRNA) in cells. Probe single stranded DNA (ssDNA) was first immobilized on the outer surface of the nanochannel-ionchannel hybrid membrane, which can hybridize with the target miRNA in cells. Due to the unique mass transfer property of the hybrid, the DNA-miRNA hybridization kinetics can be sensitively monitored in real-time using the electrochemical technique. More importantly, due to the super small size of the ionchannels, the DNA probe immobilization and hybridization process can be carried out on the outer surface of the ionchannel side, which can effectively avoid the blockage and damage of channels and thus considerably enhance the reproducibility and accuracy of the method. Using this strategy, the miRNA ranging from 0.1 fM to 0.1 μM can be facilely detected with a low detection limit of 15.4 aM, which is much lower than most reported work. The present strategy provides a sensitive and label-free miRNA detection platform, which will be of great significance in biomedical research and clinical diagnosis.

Development of a rapid bead-based microfluidic platform for DNA hybridization using single- and multi-mode interactions for probe immobilization

The demand for portable, rapid, sensitive and low-cost DNA detection systems that could be used for rapid diagnostic of disease and infection has greatly motivated the development of new DNA detection platforms in recent years. However, these platforms are often highly complex, requiring an excessively long time of analysis and/or suffer from inadequate sensitivity. Here we report a rapid and simple bead-based microfluidic platform to detect a specific 22-mer DNA sequence via hybridization, serving as a model of a miRNA biomarker for tumor and cardiovascular diseases. Commercial nanoporous chromatography beads were used as solid support for probe DNA immobilization, using either a single-mode electrostatic interaction or multi-mode interactions (electrostatic and hydrophobic). Using a mass balance approach, a probe density of 2.4 × 1013±18% (± RSD%) molecules/cm2 was quantified in optimized conditions, which was found to provide hybridization efficiencies above 95% and a hybridization dissociation constant below 1 nM. Comparing targets with different optical labels, namely Atto 430LS, quantum dots and horseradish peroxidase, the lowest limit of detection of 9.5 ±1.1 pM was achieved with an assay time of 10 min using a quantum dot label coupled with a multi-mode immobilization. These results highlight the potential of the system to be applied as a simple and highly sensitive DNA hybridization platform, achieving low pM sensitivities without the need of a DNA amplification procedure.

Attomolar Label-Free Detection of DNA Hybridization with Electrolyte-Gated Graphene Field-Effect Transistors.

In this work, we develop a field-effect transistor with a two-dimensional channel made of a single graphene layer to achieve label-free detection of DNA hybridization down to attomolar concentration, while being able to discriminate a single nucleotide polymorphism (SNP). The SNP-level target specificity is achieved by immobilization of probe DNA on the graphene surface through a pyrene-derivative heterobifunctional linker. Biorecognition events result in a positive gate voltage shift of the graphene charge neutrality point. The graphene transistor biosensor displays a sensitivity of 24 mV/dec with a detection limit of 25 aM: the lowest target DNA concentration for which the sensor can discriminate between a perfect-match target sequence and SNP-containing one.

A versatile method for the UVA-induced cross-linking of acetophenone- or benzophenone-functionalized DNA

Bioconjugation, biosensing, bioimaging, bionanomaterials, etc., are only a few examples of application of functionalized DNA. Since base-modified nucleic acids contribute not only to a broad range of biotechnological fields but also to the understanding of various cellular processes, it is crucial to design novel modifications with unique properties. Here, we demonstrate the utilization of N4-cytidine modified oligonucleotides, which contain reactive acetophenone (AP) or benzophenone (BP) groups, for the UV-induced cross-linking. We find that terminal deoxynucleotidyl transferase-mediated 3′-tailing using AP/BP-containing modified nucleotides generates photoactive DNA, suitable for a straightforward covalent cross-linking with both interacting proteins and a variety of well-known solid polymeric supports. Moreover, we show that AP/BP-functionalization of nucleic acid molecules induces an efficient cross-linking upon exposure to UVA light. Our findings reveal that 3′-tailed single-stranded DNA bearing AP/BP-moieties is easily photoimmobilized onto untreated polystyrene, polypropylene, polylactate, polydimethylsiloxane, sol-gel and borosilicate glass substrates. Furthermore, we demonstrate that such immobilized DNA probes can be further used for successful hybridization of complementary DNA targets. Our results establish novel N4-cytosine nucleobase modifications as photoreactive labels and suggest an effortless approach for photoimmobilization of nucleic acids.

Electrochemical functionalization of polypyrrole nanowires for the development of ultrasensitive biosensors for detecting microRNA

A simple electrochemical patterning strategy has been developed for the construction of ultrasensitive biosensors based on polyethylene glycol (PEG)-polypyrrole (PPy) nanowires substrate in order to supply improved antifouling performances. PPy nanowires array was produced through electrochemical polymerization of pyrrole at constant potential onto glassy carbon electrode (GCE) surface. PPy nanowires surfaces were decorated with 4-armed PEG molecules by electrochemical oxidation of amine groups offered by PEG end chains. The prepared PEG/PPy nanowires have integrated the excellent electrical conductivity of conducting polymer PPy nanowires with the good antifouling property of PEG. MicroRNAs (miRNAs) play very important roles in cancer development and a variety of diseases, which make them become promising biomarkers of the onset, prognosis and risk of diseases. Therefore the establishment of miRNA profiles for diseases and the detection of miRNAs in biological samples are critical milestones in diagnostics. An ultrasensitive electrochemical biosensor for miRNA can be effortlessly developed by the immobilization of DNA probes onto PEG/PPy nanowires. DNA/RNA hybridization was monitored by changes of methylene blue (MB) redox signal using differential pulse voltammetry (DPV) method. A wide linear range (0.10 pM ∼ 1.0 nM) to target miRNA was obtained by the fabricated biosensor, and miRNA mismatches can also be easily identified with satisfactory. Furthermore, the biosensor based on PEG/PPy nanowires can be extended to the development of any type of DNA-based biosensor.

Increasing target hybridization kinetics by immobilizing DNA probes at Au nanoflower ultramicroelectrode

Although different electrochemical DNA sensors have been widely developed for analytical purpose, the effective way to immobilize DNA probes on basic electrode surface for increasing their binding rate with target is rarely reported. Herein, a new scheme for increasing DNA probes-target hybridization kinetics using gold nanoflower ultramicroelectrode (UME) as DNA probe immobilization surface was developed. In details, the ATP aptamer was selected as the model to demonstrate this E-DNA sensor’s properties. Our results showed that the DNA aptamer/ATP hybridization rate obtained at gold nanoflower UME was about 10-fold and 4-fold greater than that obtained at the macroelectrode surface, and even in the homogeneous solution, respectively. This increase of DNA aptamer hybridization rate was confirmed to be related with the smaller size of UME and the specific structure of gold nanoflower.

Electrochemical detection of plant virus using gold nanoparticle-modified electrodes

Tristeza is one of the destructive diseases of citrus causing by citrus tristeza virus (CTV). Historically, CTV has been associated with serious outbreaks of quick decline of citrus, therefore CTV monitoring is important aspect for avoiding such re-emerging epidemics, which would threat citrus production through the world. In this context, we have designed for the first time a label-free impedimetric biosensor for the detection of nucleic acid of CTV. The sensing platform based on a screen-printed carbon electrode (SPCE) was modified by electrodeposited gold nanoparticles (AuNPs), which allowed to efficiently immobilizing thiolated ssDNA probes as well to enhance the electrode conductivity. The growth of AuNPs was optimized and characterized using scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). We investigated the behavior of thiolated ssDNA probe layer and its hybridization with target DNA onto AuNP surfaces by EIS measurements in Fe(CN6)4-/Fe(CN6)3- red-ox system. The main sensor design aspects such as AuNPs size, probe DNA concentration and immobilization time together with DNA hybridization time were optimized so as to achieve the best performance. Impedance values of DNA hybridization increased with Citrus tristeza-related synthetic DNA concentration, showing a logarithmic relation in the range of 0.1–10 μM. The results also indicate that the biosensor was able to selectively detect CTV nucleic acids in the presence of other non-specific DNAs. Moreover, we have demonstrated the good performance of the system in a real plant sample matrix. In addition, the sensor reproducibility enhanced after the hybridization onto MCH/poly (AT) thiolated DNA probes which was confirmed by intra- and inter-day variability assays.

Application of FRET Microscopy to the Study of the Local Environment and Dynamics of DNA SAMs on Au Electrodes.

Immobilized DNA probe strands self-assembled on an electrode surface are the bases of many electrochemically based biosensors. Control or measurement of the local environment around each DNA molecule tethered to the electrode surface is needed because the local environment can influence the binding or hybridization efficiency of the target in solution. Measurement of this local environment in buffer or under electrochemical control can be challenging. Here we demonstrate the use of fluorescence microscopy and a Förster resonance energy transfer (FRET) methodology to characterize multicomponent DNA SAMs. The DNA SAMs that were studied were composed of a series of mole fraction ratios of alkylthiol-modified DNA which was labeled with either AlexaFluor488 or AlexaFluor647, a FRET donor and acceptor, respectively. The DNA SAMs were hybridized before assembly onto the electrode surface. Wide-field filter-based FRET microscopy was used to study the assembly of DNA SAMs onto gold bead electrodes. These single-crystal gold bead electrodes contain many surface crystallographic regions which enable the comparison of the adsorbed DNA local environment. These surfaces show that most surface modifications are uniformly prepared, and the FRET efficiency can be explained through simple surface density considerations. The FRET efficiency for different compositions of the donor and acceptor for these regions is also explained through 2D FRET modeling. Not all surfaces were similar to the (111) and (110) regions showing deviations from the expected FRET behavior. Also demonstrated is FRET imaging using a confocal microscope. This approach proves useful in the analysis of a more dynamic system, such as the analysis of reductive desorption of the mixed-component DNA SAM. FRET microscopy is useful for surface analysis of the DNA local environment, enabling a measure of the surface modification, local density, and clustering and eventually a new detection modality.