Immobilization Of Probe DNA Research Articles

A novel DNA based bioassay toward ultrasensitive detection of Brucella using gold nanoparticles supported histidine: A new platform for the assay of bacteria in the cultured and human biofluids with and without polymerase chain reactions (PCR)

Brucella organisms, which are small aerobic intracellular coccobacilli, localize in the reproductive organs of host animals, causing abortions and sterility. In this work, we used a novel method to preparation of excellent genosensor on the surface of low-toxic substrate (gold nanoparticles) supported histidine prepared by fully electrodeposition method. The results of the present work show that the nano-Au-Hist provide suitable active sites for the DNA probe immobilization. The fabricated DNA genosensor employs cyclic voltammetry (CV), and square wave voltammetry (SWV) techniques for monitoring the behavior of the redox probe. To survey the morphological pattern and surface structural characterizations, the Field Emission-Scanning Electron Microscopy (FE-SEM) has been applied. In summary, the gold nanoparticles supported by histidine was checked for immobilization of a Brucella-specific probe and detection of hybridization with a variety of sequences with a high sensitivity. The high sensitivity would be related to more favorable conformation and deflection angle of the probe for an efficient hybridization, higher surface concentration of the probe, and/or enhanced diffusion regime. These lead to better display of the entangled target sequences arising from the nanobiotechnology. The proposed genosensor showed a perfect distinction between complementary, non-complementary and mismatched DNA sequences. The engineered genosensor for detection of the complementary/non-complementary sequences were assayed. The fabricated genosensor was evaluated for the assay of the bacteria in the cultured and human samples with and without polymerase chain reactions (PCR). The genosensor could detect the complementary sequence in linear concentration range of 1 × 10−1 to 1 × 10−10 μM, and a low limit of quantification 0.1 pM.

SPRi-Based Biosensing Platforms for Detection of Specific DNA Sequences Using Thiolate and Dithiocarbamate Assemblies.

The framework of presented study covers the development and examination of the analytical performance of surface plasmon resonance-based (SPR) DNA biosensors dedicated for a detection of model target oligonucleotide sequence. For this aim, various strategies of immobilization of DNA probes on gold transducers were tested. Besides the typical approaches: chemisorption of thiolated ssDNA (DNA-thiol) and physisorption of non-functionalized oligonucleotides, relatively new method based on chemisorption of dithiocarbamate-functionalized ssDNA (DNA-DTC) was applied for the first time for preparation of DNA-based SPR biosensor. The special emphasis was put on the correlation between the method of DNA immobilization and the composition of obtained receptor layer. The carried out studies focused on the examination of the capability of developed receptors layers to interact with both target DNA and DNA-functionalized AuNPs. It was found, that the detection limit of target DNA sequence (27 nb length) depends on the strategy of probe immobilization and backfilling method, and in the best case it amounted to 0.66 nM. Moreover, the application of ssDNA-functionalized gold nanoparticles (AuNPs) as plasmonic labels for secondary enhancement of SPR response is presented. The influence of spatial organization and surface density of a receptor layer on the ability to interact with DNA-functionalized AuNPs is discussed. Due to the best compatibility of receptors immobilized via DTC chemisorption: 1.47 ± 0.4 · 1012 molecules · cm−2 (with the calculated area occupied by single nanoparticle label of ~132.7 nm2), DNA chemisorption based on DTCs is pointed as especially promising for DNA biosensors utilizing indirect detection in competitive assays.

Electrically Guided DNA Immobilization and Multiplexed DNA Detection with Nanoporous Gold Electrodes.

Molecular diagnostics have significantly advanced the early detection of diseases, where the electrochemical sensing of biomarkers (e.g., DNA, RNA, proteins) using multiple electrode arrays (MEAs) has shown considerable promise. Nanostructuring the electrode surface results in higher surface coverage of capture probes and more favorable orientation, as well as transport phenomena unique to nanoscale, ultimately leading to enhanced sensor performance. The central goal of this study is to investigate the influence of electrode nanostructure on electrically-guided immobilization of DNA probes for nucleic acid detection in a multiplexed format. To that end, we used nanoporous gold (np-Au) electrodes that reduced the limit of detection (LOD) for DNA targets by two orders of magnitude compared to their planar counterparts, where the LOD was further improved by an additional order of magnitude after reducing the electrode diameter. The reduced electrode diameter also made it possible to create a np-Au MEA encapsulated in a microfluidic channel. The electro-grafting reduced the necessary incubation time to immobilize DNA probes into the porous electrodes down to 10 min (25-fold reduction compared to passive immobilization) and allowed for grafting a different DNA probe sequence onto each electrode in the array. The resulting platform was successfully used for the multiplexed detection of three different biomarker genes relevant to breast cancer diagnosis.

Ultrasensitive detection of cancer biomarkers using conducting polymer/electrochemically reduced graphene oxide-based biosensor: Application toward BRCA1 sensing

Breast Cancer (BRCA) is the most common threat in women worldwide. Increasing death rate of diagnosed cases is the main leading cause of designing specific genosensors for BRCA − related cancer detection. In the present study, an ultrasensitive label − free electrochemical DNA (E − DNA) sensor based on conducting polymer/reduced graphene − oxide platform has been developed for the detection of BRCA1 gene. An electrochemical method was applied as a simple and controllable technique for the electrochemical reduction of graphene oxide and also, electro − polymerization of pyrrole − 3 − carboxylic acid monomer. The results of the present work show that the polymer − coated reduced graphene − oxide provide more active sites compare to polymer − coated glassy carbon electrode for the DNA probe immobilization. The fabricated signal − off E − DNA sensor employs Cyclic Voltammetry (CV), Differential Pulse Voltammetry (DPV) and Electrochemical Impedance Spectroscopy (EIS) techniques for monitoring the electrochemical behavior of the redox probe. To survey the morphological pattern and surface structural characterizations, the Atomic Force Microscopy (AFM) and Scanning Electron Microscopy (SEM) have been applied, respectively. The applicability of the modified surface layer toward DNA sensing was evaluated by both DPV and EIS measurements. This platform permits quantitative determination of BRCA1 in the range of 10 fM–0.1 μM with a limit of detection as low as 3 fM. The proposed genosensor showed a perfect discriminatory power between complementary, non − complementary and mismatched DNA sequences. The described method combined the outstanding features in terms of selectivity, sensitivity, repeatability, reproducibility and remarkable reusability, without demanding for labor − intensive labeling steps. Moreover, the modified electrode was successfully used for accurate determination of trace amounts of DNA target in blood plasma samples.

Ultrasensitive photoelectrochemical aptasensor for lead ion detection based on sensitization effect of CdTe QDs on MoS2-CdS:Mn nanocomposites by the formation of G-quadruplex structure

An ultrasensitive photoelectrochemical (PEC) aptasensor for lead ion (Pb2+) detection was fabricated based on MoS2-CdS:Mn nanocomposites and sensitization effect of CdTe quantum dots (QDs). MoS2-CdS:Mn modified electrode was used as the PEC matrix for the immobilization of probe DNA (pDNA) labeled with CdTe QDs. Target DNA (tDNA) were hybridized with pDNA to made the QDs locate away from the electrode surface by the rod-like double helix. The detection of Pb2+ was based on the conformational change of the pDNA to G-quadruplex structure in the presence of Pb2+, which made the labeled QDs move close to the electrode surface, leading to the generation of sensitization effect and evident increase of the photocurrent intensity. The linear range was 50 fM to 100 nM with a detection limit of 16.7 fM. The recoveries of the determination of Pb2+ in real samples were in the range of 102.5–108.0%. This proposed PEC aptasensor provides a new sensing strategy for various heavy metal ions at ultralow levels.

Monitoring Potential‐Induced DNA Dehybridization Kinetics for Single Nucleotide Polymorphism Detection by using In Situ Surface Enhanced Raman Scattering

AbstractChanges in temperature, ionic strength or electrical field are generally employed to dehybridize double‐stranded DNA (dsDNA). In contrast, we propose potential‐induced dsDNA dehybridization to distinguish fully matched target DNA (tDNA) from tDNA with a single nucleotide polymorphism (SNP) by following their dehybridization kinetics through in situ surface enhanced Raman scattering (SERS). The determination of the potential that evokes notable dehybridization of dsDNA without causing any desorption of the immobilized probe DNA (pDNA) from the electrode surface was performed by investigating the desorption kinetics of labelled single‐stranded DNA (ssDNA) and dehybridization kinetics of dsDNA with labelled tDNA. This comparatively simple approach allows for SNP detection within minutes.

Substrate Properties of New Fluorescently Labeled Deoxycytidine Triphosphates in Enzymatic Synthesis of DNA with Polymerases of Families A and B

The efficiency of the incorporation of fluorescently labeled derivatives of 2'-deoxycytidine in DNA synthesized de novo has been studied using PCR with Taq and Tth polymerases of family A and Vent (exo-) and Deep Vent (exo-) polymerases of family B. Four derivatives of 5'-triphosphate-2'-deoxycytidine (dCTP) have different chemical structures of the indodicarbocyanine dye and Cy5 analogue attached to position 5 of cytosine. The kinetics of the accumulation of the PCR products and the intensity of the fluorescent signals in the hybridization analysis with immobilized DNA probes depend on the modification of the fluorescently labeled dCTP counterpart, its concentration, and the type of DNA polymerase. All labeled triphosphates showed some inhibitory effects on PCR. The best balance between the efficiency of incorporating labeled cytidine derivatives and the negative effect on the PCR kinetics has been shown in the case of Hot Taq polymerase in combination with the Cy5-dCTP analogue, which contains containing electrically neutral chro-mophore, the axis of which is a continuation of the linker between the chromophore and the pyrimidine base.

Photonic crystals on copolymer film for label-free detection of DNA hybridization

The presence of a single-nucleotide polymorphism in Apolipoprotein E4 gene is implicated with the increased risk of developing Alzheimer's disease (AD). In this study, detection of AD-related DNA oligonucleotide sequence associated with Apolipoprotein E4 gene sequence was achieved using localized-surface plasmon resonance (LSPR) on 2D-Photonic crystal (2D-PC) and Au-coated 2D-PC surfaces. 2D-PC surfaces were fabricated on a flexible copolymer film using nano-imprint lithography (NIL). The film surface was then coated with a dual-functionalized polymer to react with surface immobilized DNA probe. DNA hybridization was detected by monitoring the optical responses of either a Fresnel decrease in reflectance on 2D-PC surfaces or an increase in LSPR on Au-coated 2D-PC surfaces. The change in response due to DNA hybridization on the modified surfaces was also investigated using mismatched and non-complementary oligonucleotides sequences. The proof-of-concept results are promising towards the development of 2D-PC on copolymer film surfaces as miniaturized and wearable biosensors for various diagnostic and defense applications.

Synthesis and Characterization of Polyaniline/Graphene Composite Nanofiber and Its Application as an Electrochemical DNA Biosensor for the Detection of Mycobacterium tuberculosis.

This article describes chemically modified polyaniline and graphene (PANI/GP) composite nanofibers prepared by self-assembly process using oxidative polymerization of aniline monomer and graphene in the presence of a solution containing poly(methyl vinyl ether-alt-maleic acid) (PMVEA). Characterization of the composite nanofibers was carried out by Fourier transform infrared (FTIR) and Raman spectroscopy, transmission electron microscopy (TEM) and scanning electron microscopy (SEM). SEM images revealed the size of the PANI nanofibers ranged from 90 to 360 nm in diameter and was greatly influenced by the proportion of PMVEA and graphene. The composite nanofibers with an immobilized DNA probe were used for the detection of Mycobacterium tuberculosis by using an electrochemical technique. A photochemical indicator, methylene blue (MB) was used to monitor the hybridization of target DNA by using differential pulse voltammetry (DPV) method. The detection range of DNA biosensor was obtained from of 10−6–10−9 M with the detection limit of 7.853 × 10−7 M under optimum conditions. The results show that the composite nanofibers have a great potential in a range of applications for DNA sensors.

Impedimetric DNA biosensor based on polyurethane/poly(m-anthranilic acid) nanofibers

An impedimetric DNA biosensor based on nanofiber mats were fabricated for the detection of Salmonella spp. Polyurethane/Poly(m-anthranilic acid) (PU/P3ANA) nanofibers were obtained by electrospinning. Capture DNA probe was covalently immobilized onto PU/P3ANA nanofibers by activation of carboxyl groups on the nanofibers. The hybridization was carried out with fully complementary and mutant target DNA with a single or double-base mismatch. The immobilization and hybridization process was investigated by electrochemical impedance spectroscopy (EIS) and the data were fitted to equivalent electrical circuit model (ECM). The hybridization of complementary DNA with the immobilized probe DNA increased the charge transfer resistance of the PU/P3ANA nanofibers. The fabricated DNA biosensor was stable up to one month and displayed linear response (0.1μM − 10μM), high selectivity (8.17 kΩ/μM) and single-base mismatch selectivity. This study represents a sensitive and advanced nanofiber based DNA biosensor for the detection of Salmonella spp. by EIS.

Sensitive detection of microRNAs based on the conversion of colorimetric assay into electrochemical analysis with duplex-specific nuclease-assisted signal amplification.

miRNAs have emerged as new biomarkers for the detection of a wide variety of cancers. By employing duplex-specific nuclease for signal amplification and gold nanoparticles (AuNPs) as the carriers of detection probes, a novel electrochemical assay of miRNAs was performed. The method is based on conversion of the well-known colorimetric assay into electrochemical analysis with enhanced sensitivity. DNA capture probes immobilized on the electrode surface and ferrocene (Fc)-labeled DNA detection probes (denoted “Fc-DNA-Fc”) presented in the solution induced the assembly of positively charged AuNPs on the electrode surface through the electrostatic interaction. As a result, a large number of Fc-DNA-Fc molecules were attached on the electrode surface, thus amplifying the electrochemical signal. When duplex-specific nuclease was added to recycle the process of miRNA-initiated digestion of the immobilized DNA probes, Fc-DNA-Fc-induced assembly of AuNPs on the electrode surface could not occur. This resulted in a significant fall in the oxidation current of Fc. The current was found to be inversely proportional to the concentration of miRNAs in the range of 0–25 fM, and a detection limit of 0.1 fM was achieved. Moreover, this work presents a new method for converting colorimetric assays into sensitive electrochemical analyses, and thus would be valuable for design of novel chemical/biosensors.

A needle-like Cu2CdSnS4 alloy nanostructure-based integrated electrochemical biosensor for detecting the DNA of Dengue serotype 2

The authors describe an integrated biosensor for amperometric DNA detection of Dengue virus in real time. Cu2CdSnS4 (CCTS) quaternary alloy nanostructures were successfully synthesized, deposited on an oxygen-etched silicon substrate (O2/Si) via spin coating, and annealed at 400 °C. The nanostructures were investigated by using UV-vis spectroscopy, X-ray diffraction, atomic force microscopy and scanning electron microscopy. Interdigitated electrodes were fabricated using silver as a metal contact deposited on the CCTS/O2/Si substrate using a thermal evaporator vacuum coater and a hard mask. The quaternary alloy acts as a support for immobilization of a Dengue-specific DNA probe that is employed as the recognition element. Single-stranded DNA in concentrations from 100 f. to 10 nM were successfully recognized via amperometry, typically at a working voltage of 1.5 V. The lower detection limit is 17 nM. Sensitivity is found to be 24.2 μA nM−1 cm−2. The biosensor is inexpensive, fast, highly sensitive, and has low power consumption.

Rational Design of Electrochemical DNA Biosensors for Point‐of‐Care Applications

AbstractElectrochemical DNA biosensors show great potential for the sensitive and sequence‐specific detection of disease pathogens. To date, although there are a large number of published articles showing various sensing strategies of an electrochemical biosensor, only a few of those studies actually reach the commercialization phase, because addressing one technical issue would usually be at the expense of another. Moreover, there are various adoptable formats in electrochemical biosensors. Current approaches may or may not use enzymes, immobilize DNA probes, label the sensing substrates with an electroactive reporter molecule, or any combination of these. Although much has been paid to individual advantages and disadvantages, there are a very limited number Review articles studying and analyzing the synergistic aspects of various detection strategies when combined with each other. It is the aim of this Review to highlight the hotspots for innovation when these strategies are used in tandem, in order to create more streamlined improvements in making a versatile biosensor platform that can be administered at point of care.

Preparation of a Surface-functionalized Power-free PDMS Microchip for MicroRNA Detection Utilizing Electron Beam-induced Graft Polymerization.

We propose an easy microchannel surface functionalization method for a poly(dimethylsiloxane) (PDMS) microchip that utilizes electron beam-induced graft polymerization (EIGP) as a platform for microchip-based biomarker analysis. Unlike other grafting techniques, EIGP enables rapid surface modification of PDMS without initiator immobilization. The grafted microchip is preservable, and can be easily functionalized for versatile applications. In this study, the surface-functionalized power-free microchip (SF-PF microchip) was used for the detection of microRNA (miRNA), which is a biomarker for many serious diseases. The EIGP enables high-density three-dimensional probe DNA immobilization, resulting in rapid and sensitive miRNA detection on the portable SF-PF microchip. The limit of detection was 0.8 pM, the required sample volume was 0.5 μL, and the analysis time was 15 min. The SF-PF microchip will be a versatile platform for microchip-based point-of-care diagnosis.

High-Curvature Nanostructuring Enhances Probe Display for Biomolecular Detection.

High-curvature electrodes facilitate rapid and sensitive detection of a broad class of molecular analytes. These sensors have reached detection limits not attained using bulk macroscale materials. It has been proposed that immobilized DNA probes are displayed at a high deflection angle on the sensor surface, which allows greater accessibility and more efficient hybridization. Here we report the first use of all-atom molecular dynamics simulations coupled with electrochemical experiments to explore the dynamics of single-stranded DNA immobilized on high-curvature versus flat surfaces. We find that high-curvature structures suppress DNA probe aggregation among adjacent probes. This results in conformations that are more freely accessed by target molecules. The effect observed is amplified in the presence of highly charged cations commonly used in electrochemical biosensing. The results of the simulations agree with experiments that measure the degree of hybridization in the presence of mono-, di-, and trivalent cations. On high-curvature structures, hybridization current density increases as positive charge increases, whereas on flat electrodes, the trivalent cations cause aggregation due to electrostatic overscreening, which leads to decreased current density and less sensitive detection.

Simplified detection of the hybridized DNA using a graphene field effect transistor

Detection of disease-related gene expression by DNA hybridization is a useful diagnostic method. In this study a monolayer graphene field effect transistor (GFET) was fabricated for the detection of a particular single-stranded DNA (target DNA). The probe DNA, which is a single-stranded DNA with a complementary nucleotide sequence, was directly immobilized onto the graphene surface without any linker. The VDirac was shifted to the negative direction in the probe DNA immobilization. A further shift of VDirac in the negative direction was observed when the target DNA was applied to GFET, but no shift was observed upon the application of non-complementary mismatched DNA. Direct immobilization of double-stranded DNA onto the graphene surface also shifted the VDirac in the negative direction to the same extent as that of the shift induced by the immobilization of probe DNA and following target DNA application. These results suggest that the further shift of VDirac after application of the target DNA to the GFET was caused by the hybridization between the probe DNA and target DNA.

Voltammetric DNA Biosensor using Gold Electrode Modified by Self Assembled Monolayer of Thiol for Detection of Mycobacterium Tuberculosis

Mycobacterium tuberculosis is an infectious agent that causes tuberculosis (TB). TB is one of the major causes of death worldwide, mainly in the developing country. Early and rapid diagnosis of TB will be of great help to isolate the patients and control the disease. The aim of this research is to detect Mycobacterium tuberculosis using voltammetric DNA biosensor by using gold electrode modified by self assembled monolayer with thiol. Single-stranded probe DNA was immobilized on the surface of self assembled monolayer gold electrode with the assistance of cysteamine and glutaraldehyde, which was further used to hybridize with the target DNA sequence and non-complementary target sequence. Differential Pulse Voltammetry (DPV) was used to study the immobilization of DNA probe and hybridization with the target DNA. The hybridization reaction on the gold electrode surface was detected by monitoring a guanine oxidation signal at +0.2 Volt. Voltammetric DNA biosensor using gold electrode modified with thiol can be used to determine hybridization between probe DNA and target DNA sequence of M. tuberculosis with sensitivity value is 0.5175 for target DNA in concentration range 0- 30 μg/mL; detection limit is 2.7046 μg/mL and quantification limit is 9.0155 μg/mL, accuracy is 99.22%, precision 99.86%

All-Electrical Graphene DNA Sensor Array.

Electrical sensing of biomolecules has been an important pursuit due to the label-free operation and chip-scale construct such sensing modality can enable. In particular, electrical biomolecular sensors based on nanomaterials such as semiconductor nanowires, carbon nanotubes, and graphene have demonstrated high sensitivity, which in the case of nanowires and carbon nanotubes can surpass typical optical detection sensitivity. Among these nanomaterials, graphene is well suited for a practical candidate for implementing a large-scale array of biomolecular sensors, as its two-dimensional morphology is readily compatible with industry standard top-down fabrication techniques. In our recent work published in 2014 Nature Communications, we demonstrated these benefits by creating DNA sensor arrays from chemical vapor deposition (CVD) graphene. The present chapter, which is a review of this recent work, outlines procedures demonstrating the use of individual graphene sites of the array in dual roles--electrophoretic electrodes for site specific probe DNA immobilization and field effect transistor (FET) sensors for detection of target DNA hybridization. The 100fM detection sensitivity achieved in 7 out of 8 graphene FET sensors in the array combined with the alternative use of the graphene channels as electrophoretic electrodes for probe deposition represent steps toward creating an all-electrical multiplexed DNA array.

Impedance sensing of DNA hybridization onto nanostructured phthalocyanine-modified electrodes

DNA detection is still undergoing major innovations in pursuit of low-cost and simple approaches for decentralized applications. Label-free sensing of DNA hybridization via impedance measurements is a popular strategy to fulfil the goals of cost-efficiency and simplicity. Several materials are often reported for electrode modification to improve the sensitivity of impedance-based sensors. Herein we evaluate the electronic properties of copper phthalocyanine tetrasulfonate (CuPcTs) in Layer-by-Layer (LbL) films for impedimetric sensing of DNA hybridization using silanized Fluorine-doped Tin Oxide (FTO) electrodes. 1 to 5 bilayers were prepared by alternate immersion of the substrate in CuPcTs and poly(allylamine hydrochloride) (PAH). DNA probe immobilization was carried out electrostatically onto the last PAH layer, followed by hybridization with the target sequence leading to the formation of a partial double stranded (pds) structure onto the films. Impedance decreased after hybridization proportionally with the concentration of the target sequence at picomolar levels. Not only are these findings useful as a potential biosensing strategy, but also leave an open question about the electronic and synergistic properties of DNA interacting with different materials and surfaces.

Aptamer Lateral Flow Assays for Ultrasensitive Detection of β-Conglutin Combining Recombinase Polymerase Amplification and Tailed Primers.

In this work, different methodologies were evaluated in search of robust, simple, rapid, ultrasensitive, and user-friendly lateral flow aptamer assays. In one approach, we developed a competitive based lateral flow aptamer assay, in which β-conglutin immobilized on the test line of a nitrocellulose membrane and β-conglutin in the test sample compete for binding to AuNP labeled aptamer. The control line exploits an immobilized DNA probe complementary to the labeled aptamer, forcing displacement of the aptamer from the β-conglutin-aptamer complex. In a second approach, the competition for aptamer binding takes place off-strip, and following competition, aptamer bound to the immobilized β-conglutin is eluted and used as a template for isothermal recombinase polymerase amplification, exploiting tailed primers, resulting in an amplicon of a duplex flanked by single stranded DNA tails. The amplicon is rapidly and quantitatively detected using a nucleic acid lateral flow with an immobilized capture probe and a gold nanoparticle labeled reporter probe. The competitive lateral flow is completed in just 5 min, achieving a detection limit of 55 pM (1.1 fmol), and the combined competitive-amplification lateral flow requires just 30 min, with a detection limit of 9 fM (0.17 amol).