Recent Progresses in Electrochemical DNA Biosensors for MicroRNA Detection

MicroRNAs(miRNAs),as the small,non-coding,evolutionary conserved,and post-transcriptional gene regulators of the genome,have been highly associated with various diseases such as cancers,viral infections,and cardiovascular diseases.Several techniques have been established to detect miRNAs,including northern blotting,real-time polymerase chain reaction(RT-PCR),and fluorescent microarray platform.However,it remains a significant challenge to develop sensitive,accurate,rapid,and cost-effective methods to detect miRNAs due to their short size,high similarity,and low abundance.The electro-chemical biosensors exhibit tremendous potential in miRNA detection because they satisfy feature integration,portability,mass production,short response time,and minimal sample consumption.This article reviewed the working principles and signal amplification strategies of electrochemical DNA biosensors summarized the recent improvements.With the develop-ment of DNA nanotechnology,nanomaterials and biotechnology,electrochemical DNA biosensors of high sensitivity and specificity for microRNA detection will shortly be commercially accessible.

Celestine blue as a new indicator in electrochemical DNA biosensors

Celestine blue(CB)was introduced as a new electroactive indicator in DNA biosensors.The interaction of CB with DNA was investigated by electrochemical and spectroscopic methods.The effect of buffer kind and p H on the electrochemical behavior of CB was studied.The peak currents of CB were linearly related to DNA concentration in the range of 5.0×10^(-9) to 1.0×10^(-7)mol/L.The detection limit of this approach was 4.76×10^(-10) mol/L.Based on spectrometry data a hypochromic effect was observed in UV-Vis spectra of CB with increasing DNA concentration.The results illustrate the possible interaction mode between CB and DNA is electrostatic binding.

Tapered optical fiber DNA biosensor for detecting Leptospira DNA

Objective: To establish a DNA detection platform based on a tapered optical fiber to detect Leptospira DNA by targeting the leptospiral secY gene.Methods: The biosensor works on the principle of light propagating in the special geometry of the optical fiber tapered from a waist diameter of 125 to 12 μm. The fiber surface was functionalized through a cascade of chemical treatments and the immobilization of a DNA capture probe targeting the secY gene. The presence of the target DNA was determined from the wavelength shift in the optical transmission spectrum.Results: The biosensor demonstrated good sensitivity, detecting Leptospira DNA at 0.001 ng/μL, and was selective for Leptospira DNA without cross-reactivity with non-leptospiral microorganisms. The biosensor specifically detected DNA that was specifically amplified through the loop-mediated isothermal amplification approach.Conclusions: These findings warrant the potential of this platform to be developed as a novel alternative approach to diagnose leptospirosis.

Amperometric detection of gold by differential pulse voltammetry using a DNA biosensor

A DNA biosensor with [Ru（DA-bpy）3]Cl2（DA-bpy：4,4＇-diamino-2,2＇-bipyridine） （RuL） as the electrochemical probe was prepared on pyrolytic graphite electrode （PGE） through the supramolecular interaction between RuL complex and DNA template. Cyclic voltammetry of RuL-DNA film showed a pair of stable and reversible peaks corresponding to the Ru（Ⅲ）/Ru（Ⅲ） redox potential of-0.165 V versus AglAgCl in pa 7.4 0.1 mol·L^-1Tris-HCl. The electron transfer was expected across the double-strand DNA by an ＂electron tunneling＂ mechanism. When the DNA biosensor was immerged in gold （Ⅲ） buffer solution, the current peak signal （I） of the RuL-DNA supramolecular depressed and △I was linear in the concentration range of Au ion from 1×10^-7 to 2×10^-5 mol·L^-1 with a regression coefficient of 0.9879. The detection limit was 5×10^-8 mol·L^-1. The developed procedures were applied to the analysis of synthetic samples of real materials with good sensitivity and selectivity.

Detection of Breast Cancer 1 (BRCA1) Gene Using an Electrochemical DNA Biosensor Based on Immobilized ZnO Nanowires

Herein we report an electrochemical DNA biosensor for the rapid detection of sequence (5’ AAT GGA TTT ATC TGC TCT TCG 3’) specific for the breast cancer 1 (BRCA1) gene. The proposed electrochemical genosensor is based on short oligonucleotide DNA probe immobilized onto zinc oxide nanowires (ZnONWs) chemically synthesized onto gold electrode via hydrothermal technique. The morphology studies of the ZnONWs, performed by field emission scanning electron microscopy (FESEM), showed that the ZnO nanowires are uniform, highly dense and oriented perpendicularly to the substrate. Recognition event between the DNA probe and the target was investigated by differential pulse voltammetry (DPV) in 0.1 M acetate buffer solution (ABS), pH 7.00;as a result of the hybridization, an oxidation signal was observed at +0.8 V. The influences of pH, target concentration, and non-complimentary DNA on biosensor performance were examined. The proposed DNA biosensor has the ability to detect the target sequence in the range of concentration between 10.0 and 100.0 μM with a detection limit of 3.32 μM. The experimental results demonstrated that the prepared ZnONWs/Au electrodes are suitable platform for the immobilization of DNA.

Application of diazo-thiourea and gold nano-particles in the design of a highly sensitive and selective DNA biosensor

An effective procedure for constructing a DNA biosensor is developed based on covalent immobilization of NH2 labeled,single strand DNA（NH2-ssDNA） onto a self-assembled diazo-thiourea and gold nanoparticles modified Au electrode（diazo-thiourea/GNM/Au）.Gold nano-particles expand the electrode surface area and increase the amount of immobilized thiourea and single stranded DNA（ssDNA） onto the electrode surface.Diazo-thiourea film provides a surface with high conductibility for electron transfer and a bed for the covalent coupling of NH2-ssDNA onto the electrode surface.The immobilization and hybridization of the probe DNA on the modified electrode is studied by differential pulse voltammetry（DPV） using methylene blue（MB） as a well-known electrochemical hybridization indicator.The linear range for the determination of complementary target ssDNA is from 9.5（±0.1） × 10^-13 mol/L to1.2（±0.2） x 10^-9 mol/L with a detection limit of 1.2（±0.1） 〉 10^-13 mol/L.

Synthesis of core-shell structured Au@Bi2S3 nanorod and its application as DNA immobilization matrix for electrochemical biosensor construction

The core-shell structured Au@Bi2S3 nanorods have been prepared through direct in-situ growth of Bi2S3 at the surface of pre-synthesized gold nanorods.The product was characterized by X-ray diffraction,transmission electron microscopy and energy-dispersive X-ray spectroscopy.Then the obtained Au@Bi2S3 nanorods were coated onto glassy carbon electrode to act as a scaffold for fabrication of electrochemical DNA biosensor on the basis of the coordination of-NH2 modified on 5’-end of probe DNA and Au@Bi2S3.Electrochemical characterization assays demonstrate that the Au@Bi2S3 nanorods behave as an excellent electronic transport channel to promote the electron transfer kinetics and increase the effective surface area by their nanosize effect.The hybridization experiments reveal that the Au@Bi2S3 matrix-based DNA biosensor is capable of recognizing complementary DNA over a wide concentration ranging from 10 fmol/L to 1 nmol/L.The limit of detection was estimated to be 2 fmol/L(S/N=3).The biosensor also presents remarkable selectivity to distinguish fully complementa ry sequences from basemismatched and non-complementary ones,showing great promising in practical application.

Nano Au/TiO_2 hollow microsphere membranes for the improved sensitivity of detecting specific DNA sequences related to transgenes in transgenic plants

Gold nanoparticles (nano Au)/titanium dioxide (TiO2) hollow microsphere membranes were prepared on the carbon paste electrode (CPE) for enhancing the sensitivity of DNA hybridization detection. The immobilization of nano Au and TiO2 microsphere was investigated with cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The hybridization events were monitored with EIS us-ing [Fe(CN)6]3-/4- as indicator. The sequence-specific DNA of the 35S promoter from cauliflower mosaic virus (CaMV35S) gene was detected with this DNA electrochemical sensor. The dynamic detection range was from 1.0×10-12 to 1.0×10-8 mol/L DNA and a detection limit of 2.3×10-13 mol/L could be ob-tained. The polymerase chain reaction (PCR) amplification of the terminator of nopaline synthase (NOS) gene from the real sample of a kind of transgenic soybean was also satisfactorily detected.

A non-labeled DNA biosensor based on light addressable potentiometric sensor modified with TiO_2 thin film

Titanium dioxide (TiO2) thin film was deposited on the surface of the light addressable potentiometric sensor (LAPS) to modify the sensor surface for the non-labeled detection of DNA molecules. To evaluate the effect of ultraviolet (UV) treatment on the silanization level of TiO2 thin film by 3-aminopropyltriethoxysilane (APTS),fluorescein isothiocyanate (FITC) was used to label the amine group on the end of APTS immobilized onto the TiO2 thin film. We found that,with UV irradiation,the silanization level of the irradiated area of the TiO2 film was improved compared with the non-irradiated area under well-controlled conditions. This result indicates that TiO2 can act as a coating material on the biosensor surface to improve the effect and efficiency of the covalent immobilization of biomolecules on the sensor surface. The artificially synthesized probe DNA molecules were covalently linked onto the surface of TiO2 film. The hybridization of probe DNA and target DNA was monitored by the recording of I-V curves that shift along the voltage axis during the process of reaction. A significant LAPS signal can be detected at 10 μmol/L of target DNA sample.

Electrochemical DNA nano-biosensor for the detection of genotoxins in water samples

In the present study, a disposable electrochemical DNA nano-biosensor is proposed for the rapid detection of genotoxic compounds and bio-analysis of water pollution. The DNA nano-biosensor is prepared by immobilizing DNA on Au nanoparticles and a self-assembled monolayer of cysteamine modified Au electrode. The assembly processes of cysteamine, Au nanoparticles and DNA were characterized by cyclic voltammetry （CV）. The Au nanoparticles enhanced DNA immobilization resulting in an increased guanine signal. The interaction of the analyte with the immobilized DNA was measured through the variation of the electrochemical signal of guanine by square wave voltammetry （SWV）. The biosensor was able to detect the known genotoxic compounds： 2-anthramine, acridine orange and 2- naphthylamine with detection limits of 2, 3 and 50 nmol/L, respectively. The biosensor was also used to test actual water samples to evaluate the contamination level. Additionally, the comparison of results from the classical genotoxiciw bioassay has confirmed the applicability of the method for real samoles.

Probing the dynamic structural changes of DNA using ultrafast laser pulse in graphene-based optofluidic device

The ultrafast monitoring of deoxyribonucleic acid(DNA)dynamic structural changes is an emerging and rapidly growing research topic in biotechnology.The existing optical spectroscopy used to identify different dynamical DNA structures lacks quick response while requiring large consumption of samples and bulky instrumental facilities.It is highly demanded to develop an ultrafast technique that monitors DNA structural changes with the external stimulus or cancer-related disease scenarios.Here,we demonstrate a novel photonic integrated graphene-optofluidic device to monitor DNA structural changes with the ultrafast response time.Our approach is featured with an effective and straightforward design of decoding the electronic structure change of graphene induced by its interactions with DNAs in different conformations using ultrafast nanosecond pulse laser and achieving refractive index sensitivity of~3×10^(−5) RIU.This innovative technique for the first time allows us to perform ultrafast monitoring of the conformational changes of special DNA molecules structures,including G-quadruplex formation by K+ions and i-motif formation by the low pH stimulus.The graphene-optofluidic device as presented here provides a new class of label-free,ultrafast,ultrasensitive,compact,and cost-effective optical biosensors for medical and healthcare applications.

A new method of preparing fiber-optic DNA biosensor and its array for gene detection

A new method of preparing fiber-optic DNA biosensor and its arrayfor the simultaneous detection of multiple genes is described. The optical fibers were first treated with poly-l-lysine, and then were made into fiber-optic DNA biosensors by adsorbing and immobilizing the oligonucleotide probe on its end. By assembling the fiber-optic DNA biosensors in a bundle in which each fiber carried a different DNA probe, the fiber-optic DNA biosensor array was well prepared. Hybridization of fluorescent- labeled cDNA of p53 gene, N-ras gene and Rb1 gene to the DNA array was monitored by CCD camera. A good result was achieved.

A localized surface plasmon resonance DNA biosensor based on gold nanospheres coated on the tip of the fiber

A localized surface plasmon resonance(LSPR) biosensor was prepared with gold nanospheres(Au NSs) coated on the tip face of the optical silica fiber. Au NSs with the sizes of 20 nm and 80 nm were used. The sensitivities of Au NS_(20 nm) and Au NS_(80 nm) modified sensors to bulk refractive index(RI) variation are 82.86 nm/RIU and 218.98 nm/RIU, respectively. The Au NS_(80 nm) modified sensor was used for the detection of 40 bases DNA hybridization and the limit of detection is 50 nmol/L, where the 40-bases DNA probe was covalently linked with Au NS_(80 nm). The complementary DNA sequence in tris-acetate-EDTA(TAE) buffer solution was detected as the target DNA. This fiber sensor has the advantages of small sample consumption, easy fabrication and high sensitivity.

Simple Enzyme-Free Biosensor for Highly Sensitive and Selective Detection of miR-21 Based on Multiple Signal Amplification Strategy

Due to the fact that most microRNAs are small in size,low abundance in biological samples,homologous sequence among family members,and protein enzymes-based strategies display limited practical applications,therefore,we reported a simple enzyme-free DNA sensor for microRNA detection utilizing a multiple signal amplification strategy.The sensing system termed as C-CHA-HCR includes six hairpin DNA reactants that are metastable on account of intramolecular hybridization.The DNA hairpin reactants are opened and hybridized with the corresponding complementary DNA strand in the presence of miR-21 via toehold-mediated CHA,HCR reaction,and circulation between CHA and HCR,resulting in a hugely amplifying signal output.Without introducing external protein enzymes,this sensing system showed highly sensitive and selective on the detection of miR-21.A linear response range of miR-21 from 25 pmol/L to 1 nmol/L with a limit of detection(LOD)of 1.8 pmol/L was obtained.This promising biosensor was successfully applied to the detection of microRNA in human serum samples with acceptable recovery rates,suggesting the potential applications in disease diagnosis,treatment,and prognosis.