Electrochemical DNA biosensor for human papillomavirus 16 detection in real samples

An electrochemical DNA biosensor for human papillomavirus (HPV) 16 detection has been developed. For this proposed biosensor, l-cysteine was first electrodeposited on the gold electrode surface to form l-cysteine film (CYSFILM). Subsequently, HPV16-specific probe was immobilized on the electrode surface with CYSFILM. Electrochemistry measurement was studied by differential pulse voltammetry method (DPV). The measurement was based on the reduction signals of methylene blue (MB) before and after hybridization either between probe and synthetic target or extracted DNA from clinical samples. The effect of probe concentration was analyzed and the best results were seen at 1000 nM. The hybridization detection presented high sensitivity and broad linear response to the synthetic-target concentration comprised between 18.75 nM and 250 nM as well as to a detection limit of 18.13 nM. The performance of this biosensor was also investigated by checking probe-modified electrode hybridization with extracted DNA from samples. The results showed that the biosensor was successfully developed and exhibited high sensitivity and satisfactory selectivity to HPV16. These results allow for the possibility of developing a new portable detection system for HPVs and for providing help in making an effective diagnosis in the early stages of infection.     

Voltammetric determination of DNA hybridization using methylene blue and self-assembled alkanethiol monolayer on gold electrodes

An electrochemical DNA biosensor based on the recognition of single stranded DNA (ssDNA) by hybridization detection with immobilized complementary DNA oligonucleotides is presented. DNA and oligonucleotides were covalently attached through free amines on the DNA bases using N-hydroxysulfosuccinimide (NHS) and N-(3-dimethylamino)propyl-N′-ethylcarbodiimide hydrochloride (EDC) onto a carboxylate terminated alkanethiol self-assembled monolayers (SAM) preformed on a gold electrode (AuE). Differential pulse voltammetry (DPV) was used to investigate the surface coverage and molecular orientation of the immobilized DNA molecules. The covalently immobilized probe could selectively hybridize with the target DNA to form a hybrid on the surface despite the bases being attached to the SAM. The changes in the peak currents of methylene blue (MB), an electroactive label, were observed upon hybridization of probe with the target. Peak currents were found to increase in the following order: hybrid-modified AuE, mismatched hybrid-modified AuE, and the probe-modified AuE which indicates the MB signal is determined by the extent of exposed bases. Control experiments were performed using a non-complementary DNA sequence. The effect of the DNA target concentration on the hybridization signal was also studied. The interaction of MB with inosine substituted probes was investigated. Performance characteristics of the sensor are described.     

A novel electrochemical biosensor based on the hemin-graphene nano-sheets and gold nano-particles hybrid film for the analysis of hydrogen peroxide

Hydrogen peroxide is an important analyte in biochemical, industrial and environmental systems. Therefore, development of novel rapid and sensitive analytical methods is useful. In this work, a hemin-graphene nano-sheets (H-GNs)/gold nano-particles (AuNPs) electrochemical biosensor for the detection of hydrogen peroxide (H₂O₂) was researched and developed; it was constructed by consecutive, selective modification of the GCE electrode. Performance of the H-GNs/AuNPs/GCE was investigated by chronoamperometry, and AFM measurements suggested that the graphene flakes thickness was ∼1.3 nm and that of H-GNs was ∼1.8 nm, which ultimately indicated that each hemin layer was ∼0.25 nm. This biosensor exhibited significantly better electrocatalytic activity for the reduction of hydrogen peroxide in comparison with the simpler AuNPs/GCE and H-GNs/GCE; it also displayed a linear response for the reduction of H₂O₂ in the range of 0.3 μM to 1.8 mM with a detection limit of 0.11 μM (S N⁻¹ = 3), high sensitivity of 2774.8 μA mM⁻¹ cm⁻², and a rapid response, which reached 95% of the steady state condition within 5 s. In addition, the biosensor was unaffected by many interfering substances, and was stable over time. Thus, it was demonstrated that this biosensor was potentially suitable for H₂O₂ analysis in many types of sample.     

基于聚苯胺掺杂乙醇胺固定DNA电化学传感器的研究

本研究以电化学聚合法制备了聚苯胺掺杂乙醇胺修饰电极,并成功固定了DNA探针。文中对修饰电极的制备和DNA的固定杂交条件进行了探讨,并利用循环伏安法测定嵌入双链DNA(dsDNA)分子碱基对中的亚甲基蓝的氧化还原峰电流,识别和测定溶液中互补的单链DNA(ssDNA)片段,从而实现对溶液中不同基因片段的检测。     

Selective detection and estimation of C-reactive protein in serum using surface-functionalized gold nano-particles

A new method for the detection of C-reactive protein (CRP) in serum using functionalized gold nano-particles (GNP) is reported. The affinity towards CRP is imparted to GNP by tethering O-phosphorylethanolamine (PEA) onto their surface. GNP and modified GNP were characterized using TEM, particle size analysis, zeta potential measurements, absorption spectroscopy and FT-IR techniques. The event of binding of CRP onto the PEA-GNP is followed by visibly observable colour change. We observed a red shift as well as a decrease in absorption in the plasmon peak of the modified GNP with the concentration of CRP. When the concentration of CRP exceeded 450 ng mL⁻¹, particles were aggregated and the solution became turbid. The method exhibited a linear range for CRP from 50 to 450 ng mL⁻¹ with a detection limit of 50 ng mL⁻¹. The colour change and the variation in absorption of the GNP were highly specific to CRP even in the presence of albumin. We estimated CRP in blood serum collected from patients and the results obtained compared well with the estimation using the technique of nephelometry based on the antibody-antigen interaction.     

Enzyme-based electrochemical biosensor for sensitive detection of DNA demethylation and the activity of DNA demethylase

A novel electrochemical method is developed for detection of DNA demethylation and assay of DNA demethylase activity. This method is constructed by hybridizing the probe with biotin tagged hemi-methylated complementary DNA and further capturing streptavidin tagged alkaline phosphatase (SA-ALP) to catalyze the hydrolysis reaction of p-nitrophenyl phosphate. The hydrolysate of p-nitrophenol (PNP) is then used as electrochemical probe for detecting DNA demethylation and assaying the activity of DNA demethylase. Demethylation of target DNA initiates a degradation reaction of the double-stranded DNA (dsDNA) by restriction endonuclease of BstUI. It makes the failed immobilization of ALP, resulting in a decreased electrochemical oxidation signal of PNP. Through the change of this electrochemical signal, the DNA demethylation is identified and the activity of DNA demethylase is analyzed with low detection limit of 1.3 ng mL⁻¹. This method shows the advantages of simple operation, cheap and miniaturized instrument, high selectivity. Thus, it provides a useful platform for detecting DNA demethylation, analyzing demethylase activity and screening inhibited drug.     

Nano-ZnO/Chitosan Composite Film Modified Electrode for Voltammetric Detection of DNA Hybridization

Abstract

A sensitive electrochemical DNA biosensor based on nano-ZnO/chitosan composite matrix for DNA hybridization detection was developed. The Nano-ZnO was synthesized by the hydrothermal method and dispersed in chitosan, which was used to fabricate the modification of the glassy carbon electrode (GCE) surface. The ZnO/chitosan-modified electrode exhibited good biocompatibility and excellent electrochemical conductivity. The hybridization detection was monitored with differential pulse voltammetry (DPV) measurement using methylene blue (MB) as an indicator. The established biosensor can effectively discriminate complementary target sequence and two-base-mismatched sequence, with a detection limit of 1.09 × 10^?11 mol L^?1 of complementary target.     

Fabrication and characterization of gold nano particles for DNA biosensor applications

This research involves the preparation of a biosensor using silicon oxide for biomedical applications, and its effective use for the detection of target DNA hybridization. An electrochemical DNA biosensor was successfully fabricated by using(3-aminopropyl) tri-ethoxysilane(APTES) as a linker molecule combined with gold nanoparticles(GNPs) on a thermally oxidized SiO_2 thin film. The size of the GNPs was calculated by utilizing UV–vis data with an average calculated particle size within the range of 30±5 nm, and characterization by transmission electron microscopy(TEM) and atomic force microscopy(AFM). The GNP-modified SiO_2 thin films were electrically characterized through the measurement of capacitance, permittivity and conductivity using a low-cost dielectric analyzer. The capacitance, permittivity and conductivity profiles of the fabricated sensor clearly differentiated DNA immobilization and hybridization.     

Electrochemical DNA biosensor for the detection of short DNA species of Chronic Myelogenous Leukemia by using methylene blue

A novel assay for the voltammetric detection of 18-bases DNA sequences relating to Chronic Myelogenous Leukemia (CML, Type b3a2) using methylene blue (MB) as the hybridization indicator was reported. DNA was covalently attached onto a glassy carbon electrode (GCE) through amines of the DNA bases using N-hydroxysulfosuccinimide (NHS) and N-(3-dimethylamion)propyl-N′-ethyl carbodiimidehydrochloride (EDC). The covalently immobilized single-stranded DNA (ssDNA) could selectively hybridize with its complementary DNA (cDNA) in solution to form double-stranded DNA (dsDNA) on the surface. A significant increase of the peak current for methylene blue upon the hybridization of immobilized ssDNA with cDNA in the solution was observed. This peak current change was used to monitor the recognition of CML DNA sequence. This electrochemical approach is sequence specific as indicated by the control experiments in which no peak current change was observed if a non-complementary DNA sequence was used. Factors, such as DNA target concentration and hybridization conditions determining the sensitivity of the electrochemical assay were investigated. Under optimal conditions, this sensor has a good calibration range between 1.25 × 10⁻⁷ and 6.75 × 10⁻⁷ M, with CML DNA sequence detection limit of 5.9 × 10⁻⁸ M.     

Gold nanoparticle aggregation-based highly sensitive DNA detection using atomic force microscopy

The potential ability of atomic force microscopy (AFM) as a quantitative bioanalysis tool is demonstrated by using gold nanoparticles as a size enhancer in a DNA hybridization reaction. Two sets of probe DNA were functionalized on gold nanoparticles and sandwich hybridization occurred between two probe DNAs and target DNA, resulting in aggregation of the nanoparticles. At high concentrations of target DNA in the range from 100 nM to 10 μM, the aggregation of gold nanoparticles was determined by monitoring the color change with UV-vis spectroscopy. The absorption spectra broadened after the exposure of DNA–gold nanoparticles to target DNA and a new absorption band at wavelengths >600 nm was observed. However, no differences were observed in the absorption spectra of the gold nanoparticles at low concentrations of target DNA (10 pM to 10 nM) due to insufficient aggregation. AFM was used as a biosensing tool over this range of target DNA concentrations in order to monitor the aggregation of gold nanoparticles and to quantify the concentration of target DNA. Based on the AFM images, we successfully evaluated particle number and size at low concentrations of target DNA. The calibration curve obtained when mean particle aggregate diameter was plotted against concentration of target DNA showed good linearity over the range 10 pM to 10 nM, the working range for quantitative target DNA analysis. This AFM-based DNA detection technique was three orders of magnitude more sensitive than a DNA detection method based on UV-vis spectroscopy.     

4‐Aminothiophenol Self‐Assembled Monolayer for the Development of a DNA Biosensor Aiming the Detection of Cylindrospermopsin Producing Cyanobacteria

The development of a DNA biosensor for the detection of cylindrospermopsin, based on self‐assembled monolayers (SAMs) of 4‐aminothiophenol, is investigated. SAMs were characterized by electrochemical reductive desorption. Detection of probe immobilization and hybridization has been achieved by cyclic and square‐wave voltammetry (SWV), using methylene blue (MB) as electroactive indicator. The SWV data obtained in phosphate buffer, with and without NaCl, after MB accumulation, revealed an increase of the redox indicator current peaks after the hybridization step. This behavior is consistent with MB intercalation into DNA, for high ionic strength media and attributed to electrostatic interactions in the absence of salt. Evidence for surface modification is also provided by atomic force microscopy and ellipsometry.     

以高氯酸·三-2,2′-联吡啶合钴(Ⅲ)作为电子媒介体金纳米颗粒增强的葡萄糖生物传感器

用纳米金溶胶与聚乙烯醇缩丁醛(PVB)构成复合固酶基质,采用溶胶-凝胶法固定葡萄糖氧化酶(GOx)于铂电极表面,并在葡萄糖溶液中加入高氯酸·三-2,2′-联吡啶合钴(Ⅲ)作为电子媒介体,制成了高灵敏的葡萄糖生物传感器.葡萄糖氧化酶吸附在纳米金颗粒表面上稳定且保持其生物活性;而电子媒介体的存在,显著提高了传感器的响应灵敏度.该传感器对葡萄糖响应的线性范围为1.2×10-8～6.2×10-6 mol/L,检出限6.2×10-9 mol/L(S/N=3).该生物传感器有效消除了抗坏血酸、尿酸的干扰,可用于人体血清中葡萄糖的测定.     

纳米金在DNA生物传感器和基因芯片研究中的应用

综述了近年来纳米金在DNA生物传感器及基因芯片中的研究、应用和发展,并对其在生物科技方面的发展趋势进行了展望。参考文献31篇。     

Electrochemical DNA Biosensors Based on Palladium Nanoparticles Combined with Carbon Nanotubes

Palladium nanoparticles, in combination with multi‐walled carbon nanotubes (MWCNTs), were used to fabricate a sensitivity‐enhanced electrochemical DNA biosensor. MWCNTs and palladium nanoparticles were dispersed in Nafion, which were used to modify a glassy carbon electrode (GCE). Oligonucleotides with amino groups at the 5′ end were covalently linked onto carboxylic groups of MWCNTs on the electrode. The hybridization events were monitored by differential pulse voltammetry (DPV) measurement using methylene blue (MB) as an indicator. Due to the ability of carbon nanotubes to promote electron‐transfer and the high catalytic activities of palladium nanoparticles for electrochemical reaction of MB, the sensitivity of presented electrochemical DNA biosensors was remarkably improved. The detection limit of the method for target DNA was 1.2×10^?13 M.     

A sensitive DNA biosensor based on a facile sulfamide coupling reaction for capture probe immobilization

A novel DNA biosensor was fabricated through a facile sulfamide coupling reaction. First, the versatile sulfonic dye molecule of 1-amino-2-naphthol-4-sulfonate (AN-SO₃⁻) was electrodeposited on the surface of a glassy carbon electrode (GCE) to form a steady and ordered AN-SO₃⁻ layer. Then the amino-terminated capture probe was covalently grafted to the surface of SO₃⁻-AN deposited GCE through the sulfamide coupling reaction between the amino groups in the probe DNA and the sulfonic groups in the AN-SO₃⁻. The step-by-step modification process was characterized by electrochemistry and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. Using Ru(NH₃)₆³⁺ as probe, the probe density and the hybridization efficiency of the biosensor were determined to be 3.18 × 10¹³ strands cm⁻² and 86.5%, respectively. The hybridization performance of the biosensor was examined by differential pulse voltammetry using Co(phen)₃^3+/2+ (phen = 1,10-phenanthroline) as the indicator. The selectivity experiments showed that the biosensor presented distinguishable response after hybridization with the three-base mismatched, non-complementary and complementary sequences. Under the optimal conditions, the oxidation peak currents of Co(phen)₃^3+/2+ increased linearly with the logarithm values of the concentration of the complementary sequences in the range from 1.0 × 10⁻¹³ M to 1.0 × 10⁻⁸ M with a regression coefficient of 0.9961. The detection limit was estimated to be 7.2 × 10⁻¹⁴ M based on 3σ.     

Developing an electrochemical deoxyribonucleic acid (DNA) biosensor on the basis of human interleukine-2 gene using an electroactive label

Development of an electrochemical DNA biosensor based on a human interleukine-2 (IL-2) gene probe, using a pencil graphite electrode (PGE) as transducer and methylene blue (MB) as electroactive label is described. The sensor relies on the immobilization of a 20-mer single stranded oligonucleotide probe (hIL-2) related to the IL-2 gene on the electrode. The hybridization between the probe and its complementary sequence (chIL-2) as the target was studied by square wave voltammetry (SWV) of MB accumulated on the PGE. In this approach the extent of hybridization is evaluated on the basis of the difference between SWV signals of MB accumulated on the probe-PGE and MB accumulated on the probe-target-PGE. Some hybridization experiments with non-complementary oligonucleotides were carried out to assess whether the suggested DNA sensor responds selectively to the target. Some experimental variables affecting the performance of the biosensor including: polishing of PGE, its electrochemical activation conditions (i.e., activation potential and activation time) and probe immobilization conditions on the electrodes (i.e., immobilization potential and time) were investigated and the optimum values of 1.80 V and 300 s for PGE activation, and −0.5 V and 400 s for the probe immobilization on the electrode were suggested.     

Towards a fast-responding,label-free electrochemical DNA biosensor

DNA sensors and sensor arrays (biochips) have become an important tool in molecular biology and biotechnology in recent years. For low-throughput, easy-to-use devices it is desirable that they be of low cost, reagentless, and label-free. Displacement sensors with electrochemical detection offer these advantages, and therefore the development of such a detection principle is show in this work. An HRP-labeled oligonucleotide was sub-optimally pre-hybridized with a capture probe and was displaced upon introduction of the fully complementary probe target, producing a decrease in signal that was proportional to the sample concentration. This detection scheme has been demonstrated colorimetrically and electrochemically, obtaining a total signal displacement of 55% only 5 min after introduction of the sample.     

Label‐Free and Label Based Electrochemical Detection of Hybridization by Using Methylene Blue and Peptide Nucleic Acid Probes at Chitosan Modified Carbon Paste Electrodes

A chitosan modified carbon paste electrode (ChiCPE) based DNA biosensor for the recognition of calf thymus double stranded DNA (dsDNA), single stranded DNA (ssDNA) and hybridization detection between complementary DNA oligonucleotides is presented. DNA and oligonucleotides were electrostatically attached by using chitosan onto CPE. The amino groups of chitosan formed a strong complex with the phosphate backbone of DNA. The immobilized probe could selectively hybridize with the target DNA to form hybrid on the CPE surface. The detection of hybridization was observed by using the label‐free and label based protocols. The oxidation signals of guanine and adenine greatly decreased when a hybrid was formed on the ChiCPE surface. The changes in the peak currents of methylene blue (MB), an electroactive label, were observed upon hybridization of probe with target. The signals of MB were investigated at dsDNA modified ChiCPE and ssDNA modified ChiCPE and the increased peak currents were observed, in respect to the order of electrodes. The hybridization of peptide nucleic acid (PNA) probes with the DNA target sequences at ChiCPE was also investigated. Performance characteristics of the sensor were described, along with future prospects.     

巯基乙酸自组装膜DNA电化学传感器对转基因NOS的定量检测

以转基因植物中常用的根癌农杆菌终止子(NOS)为检测对象, 将巯基乙酸自组装于金电极表面形成巯基乙酸自组装单分子膜, 再利用乙基-(3-二甲基氨丙基)碳二亚胺盐酸盐(EDC)和N-羟基琥珀酰亚胺(NHS)的活化作用将NOS探针ssDNA序列固定于金电极表面形成NOS电化学生物传感器, 以亚甲基蓝(MB)为杂交指示剂, 对NOS靶基因相关序列进行了定量检测.