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.     

基于纳米ZnO/壳聚糖复合膜DNA电化学传感器用于HIV基因的免标记检测

以室温固相合成法制备纳米ZnO,通过壳聚糖(CHIT)的成膜效应将纳米ZnO固定在玻碳电极(GCE)表面,制得的ZnO/CHIT/GCE电极成为DNA固定和杂交的良好平台。DNA的固定和杂交通过电化学交流阻抗进行表征。以电化学交流阻抗免标记法检测目标DNA,固定于电极表面的DNA探针与目标DNA杂交后使电极表面的电子传递电阻增大,以此作为检测信号可以高灵敏度地测定目标DNA。电化学阻抗谱检测人类免疫缺陷病毒(HIV)基因片段的线性范围为2.0×10-11~2.0×10-6mol/L,检出限为2.0×10-12mol/L。     

以乙二胺为手臂分子制备的DNA修饰电极及其伏安性能

Carboxyl was formed on the surface of glassy carbon electrode(GCE) by electrochemical oxidation. Ethylenediamine(En) was used as the arm molecule to link carboxyl with dsDNA using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N- hydroxysuccinimide (NHS) as the activators to prepare dsDNA modified electrode(dsDNA/En/GCE). It was shown that dsDNA couM be covalently immobilized on the surface of GCE. ssDNA modified electrode(ssDNA/En/GCE) was obtained via the thermal denaturation of dsDNA/En/GCE. The dsDNA/En/GCE and ssDNA/En/GCE were characterized by voltammetry with methylene blue(MB) as the indicator. The results indicated that the currents of the redox peaks of MB at ssDNA/En/GCE were larger than those at dsDNA/En/GCE, and the currents of the redox peaks at En/GCE were the smallest. The peak-currents of MB at the DNA modified electrode had good reproducibility after multi-denaturation and hybridization cycles.     

氧化锌纳米簇-金纳米颗粒-壳聚糖复合膜修饰电极用于示差脉冲伏安法测定吗啡

将制备的氧化锌纳米簇和金纳米颗粒分散在壳聚糖中并滴涂在玻碳电极表面,制备了氧化锌纳米簇-金纳米颗粒-壳聚糖复合膜修饰电极(Au-ZnO-CHIT/GCE)。采用循环伏安法研究了吗啡在修饰电极上的电化学行为。结果表明:吗啡在该修饰电极上出现了一个氧化峰,提出了用示差脉冲伏安法测定吗啡的方法。吗啡浓度在5.3×10-6~6.5×10-4mol.L-1范围内与氧化峰电流呈线性关系,检出限(3S/N)为1.8×10-6mol.L-1。修饰电极用于尿液中吗啡的测定,回收率在80.0%~99.6%之间。     

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.     

聚苯胺纳米管/壳聚糖修饰玻碳电极循环伏安法测定阿米卡星

将1.0g·L~(-1)聚苯胺纳米管-N,N-二甲基甲酰胺(DMF)溶液和1.0g·L~(-1)壳聚糖乙酸溶液以1比50的比例混合,并超声处理1h后滴涂在玻碳电极表面,制得聚苯胺纳米管/壳聚糖修饰电极(记为PANT′s/CTS/GCE)。采用循环伏安法研究了阿米卡星在聚苯胺纳米管/壳聚糖修饰电极上的电化学行为。试验结果表明:在pH5.0磷酸盐缓冲溶液中,阿米卡星在聚苯胺纳米管/壳聚糖修饰电极于-0.2V处出现一个不可逆的还原峰,且还原峰电流与阿米卡星的质量浓度在10.0～80.0mg·L~(-1)范围内呈线性关系,检出限(3S/N)为8.0mg·L~(-1)。应用此法测定注射液中阿米卡星的含量,测定结果与分光光度法测定值相一致。     

Nano‐Gold Modified Genosensor for Direct Detection of DNA Hybridization

In this paper, nano‐gold modified carbon paste electrode (NGMCPE) was employed to develop an electrochemical DNA hybridization biosensor. The proposed sensor was made up by immobilization of 15‐mer single stranded oligonucleotide probe for detection of target DNA. Hybridization detection relies on the alternation in guanine oxidation signal following hybridization of the probe with complementary genomic DNA. The guanine oxidation was monitored using differential pulse voltammetry (DPV). Different factors such as activation potential, activation time and probe immobilization conditions were optimized. The selectivity of the sensor was investigated by non‐complementary oligonucleotides. Diagnostic performance of the biosensor was described and the detection limit was found 1.9 × 10^?13 M at the NGMCPE surface. All of the investigations were performed in both CPE and NGMCPE and finally their results were compared.     

Polyaniline／Prussian Blue Composite Film Electrochemical Biosensors for Cholesterol Detection

ＩｎｔｒｏｄｕｃｔｉｏｎＣｈｏｌｅｓｔｅｒｏｌｉｓａｖｅｒｙｉｍｐｏｒｔａｎｔｂｉｏａｃｔｉｖｅｃｏｍｐｏｕｎｄ .Ｎｕｍｅｒｏｕｓａｔｔｅｍｐｔｓｈａｖｅｂｅｅｎｍａｄｅｔｏｃｒｅａｔｅｓｅｎｓｉｔｉｖｅ ,ｓｅｌｅｃｔｉｖｅ ,ｒｅｌｉａｂｌｅａｎｄｌｏｗｃｏｓｔｃｈｏｌｅｓｔｅｒｏｌｓｅｎｓｏｒｓｄｕｒｉｎｇｔｈｅｌａｓｔｄｅｃａｄｅｂｅｃａｕｓｅｏｆｔｈｅｓｉｇｎｉｆｉｃａｎｃｅｉｎｃｌｉｎｉｃａｌｄｉ ａｇｎｏｓｉｓｏｆｃｏｒｏｎａｒｙｈｅａｒｔｄｉｓｅａｓｅｓ ,ａｒｔｅｒｉｏｓｃｌｅｒｏｓ…     

A Novel DNA Probe Based on Molecularly Imprinted Polymer Modified Electrode for the Electrochemical Monitoring of DNA

A DNA probe that was based on methylene blue (MB) imprinted polyvinyl pyridine polymer (MIP) modified carbon paste electrodes were developed for the first time for electrochemical monitoring of DNA. Probes were built up by adsorbing MB onto modified electrodes prior to DNA immobilization. It was shown that DNA strongly immobilizes on MIP modified electrodes when MB was adsorbed in advance of DNA immobilization. The performance of the MB imprinted polymer modified carbon paste electrodes (MIP‐CPE) to rebind the template molecule (MB) were compared to those of control polymer modified (non‐imprinted polymer NIP‐CPE) and bare (CPE) electrodes. Electrochemical signal resulting from the oxidation of guanine moiety of the immobilized probe DNA was high enough on the constructed platform, implicating that probes of this kind could be favorably used for DNA analysis. These probes exhibited high selectivity for its complementary DNA sequences (target). HBV‐DNA hybridization was studied to evaluate the selectivity of the probes for complementary, non‐complementary and mismatch sequences. The detection limit of the probe for the target DNA was 8.72 µg/mL (1.38 µM), which was better than those attained by some earlier DNA sensor studies.     

A Selective Cholesterol Biosensor Based on Composite Film Modified Electrode for Amperometric Detection

An amperometric cholesterol biosensor based on immobilization of cholesterol oxidase in a Prussian blue (PB)/polypyrrole (PPy) composite film on the surface of a glassy carbon electrode was fabricated. Hydrogen peroxide produced by the enzymatic reaction was catalytically reduced on the PB film electrode at 0 V with a sensitivity of 39 μA (mol/L)^?1. Cholesterol in the concentration range of 10^?5 ? 10^?4 mol/L was determined with a detection limit of 6 × 10^?7 mol/L by amperometric method. Normal coexisting compounds in the bio‐samples such as ascorbic acid and uric acid do not interfere with the determination. The excellent properties of the sensor in sensitivity and selectivity are attributed to the PB/PPy layer modified on the sensor.     

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。     

Voltammetric Determination of Folic Acid Using Adsorption of Methylene Blue onto Electrodeposited of Reduced Graphene Oxide Film Modified Glassy Carbon Electrode

This work presents a sensitive voltammetric method for determination of folic acid by adsorbing methylene blue onto electrodeposited reduced graphene oxide film modified glassy carbon electrode (MB/ERGO/GCE) in 100 mM KCl‐10 mM sodium phosphate buffer solution (pH 7.40). The surface morphology of the MB/ERGO/GCE modified electrode was characterized using scanning electron microscopy, displays that both MB and ERGO distributed homogeneously on the surface of GCE. The MB/ERGO/GCE modified electrode shows more favorable electron transfer kinetics for potassium ferricyanide and potassium ferrocyanide probe molecules, which are important electroactive compounds, compared with bare GCE, MB/GCE, and ERGO/GCE. The electrochemical behaviors of folic acid at MB/ERGO/GCE were investigated by cyclic voltammetry, suggesting that the modified electrode exhibited excellent electrocatalytic activity towards folic acid compared with other electrodes. Under physiological condition, the MB/ERGO/GCE modified electrode showed a linear voltammetric response from 4.0 μM to 167 μM for folic acid, and with the detection limit of 0.5 μM (S/N=3). The stability, reproducibility and anti‐interference ability of the modified electrode were examined. The developed method has been successfully applied to determination of FA in tablets with a satisfactory recovery from 96 % to 100 %. The work demonstrated that the electroactive MB adsorbing onto graphene modified electrode showed an enhanced electron transfer property and a high resolution capacity to FA.     

Parallel Detection of Different DNA Sequences on One Gold Electrode

Motivated by the potential of electrochemical techniques to analyze hybridization events fast and in a simple and cost‐effective way we present here a detection system allowing a parallel electrochemical DNA analysis. For this purpose different probe DNA strands have been immobilized on one electrode. By the use of two different target DNA sequences, both marked with the redox active methylene blue, we can show that hybridization with the complementary probe sh“NA strands can occur without steric hindrance. Each target has been recognized down to 3nM with a very high specificity of the sensor. In addition, we can detect two different ssDNA targets labeled with different redox active molecules, methylene blue and ferrocene, on one sensor surface simultaneously.     

L-半胱氨酸/纳米金/DNA/壳聚糖修饰金电极测定布洛芬

用电沉积和滴涂的方法依次将L-半胱氨酸、纳米金、DNA、壳聚糖修饰在金电极表面,制备了稳定性、响应性良好的修饰电极(L-Cys/Au colloid/DNA/CS/Au/CME)。考察了影响实验结果的关键因素,确定了最佳实验条件,进一步采用循环伏安法和差分脉冲伏安法研究了布洛芬在L-Cys/Au colloid/DNA/CS/Au/CME上的电化学行为。在最佳实验条件下,当布洛芬浓度在1.0×10~(-7)~1.0×10~(-4) mol/L范围内时相应的氧化还原峰电流与浓度呈现良好的线性关系,检出限(S/N=3)达2.3×10~(-8) mol/L。结果表明,该修饰电极对布洛芬有良好的响应性能,据此建立了一种测定布洛芬的新方法。     

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.     

Electrochemical DNA Biosensor Based on Graphene and TiO2 Nanorods Composite Film for the Detection of Transgenic Soybean Gene Sequence of MON89788

Based on graphene (GR), TiO₂ nanorods, and chitosan (CTS) nanocomposite modified carbon ionic liquid electrode (CILE) as substrate electrode, a new electrochemical DNA biosensor was effectively fabricated for the detection of the transgenic soybean sequence of MON89788. By using methylene blue (MB) as hybridization indicator for monitoring the hybridization with different ssDNA sequences, the differential pulse voltammetric response of MB on DNA modified electrodes were recorded and compared. Due to the synergistic effects of TiO₂ nanorods and GR on the electrode surface, the electrochemical responses of MB were greatly increased. Under optimal conditions the differential pulse voltammetric response of the target ssDNA sequence could be detected in the range from 1.0×10^?12 to 1.0×10^?6 mol/L with a detection limit of 7.21×10^?13 mol/L (3σ). This electrochemical DNA biosensor was further applied to the polymerase chain reaction (PCR) product of transgenic soybeans with satisfactory results.     

普鲁士蓝膜修饰电极循环伏安行为的研究

本文研究普鲁士蓝(PB)膜修饰电极的循环伏安行为,结果表明,膜中电活性中心间的相互作用导致PB膜电极的伏安行为偏离理想表面波.用有限扩散边界模型解释了高电位扫速下膜电极的伏安行为.在含钾离子的电解质溶液中测得PB膜中电荷传输扩散系数为10~(-9)～10~(-11)cm~2/s.     

Cationic Dye Modified Sawdust as Electrode Modifier for Electrochemical Detection of Anions

Because of its chemical properties, sawdust displays poor anionic exchange capacity. Here we demonstrate that sawdust modification with methylene blue (MB) dye represents an interesting and facile alternative to render this natural biomaterial capable to accumulate anionic species. MB adsorption onto sawdust was monitored by cyclic voltammetry and experimental parameters carefully optimized. Under the ideal experimental conditions (composition of accumulation and desorption solution, accumulation and desorption time and the nature of the electrolytic solution), the adsorbed MB showed poor mobility, which results in the absence of the characteristic electrochemical signal of MB. The ability of the material to accumulate anionic species was thus evaluated using Fe(CN)₆^3? as a model anions. The slow Fe(CN)₆^3?/4? system recorded onto the electrode modified by pristine sawdust (P/SFE) become fast and reversible after immobilization of MB onto P/SFE (MB/SFE). Electrochemical impedance spectroscopy confirms this result through the spectacular decrease of charge transfer resistance after MB adsorption (from 83 kΩ on P/SFE to 637 Ω on MB/SFE). MB/SFE was applied to the electroanalysis of nitrites and a sensitivity of 7.4 μA mM^?1 was obtained. Although this sensitivity was less important compared to that obtained on glassy carbon electrode (9.4 μA mM^?1), the dye modified electrode displays by far the best reproducibility even at higher nitrite concentration.     

维生素C在多壁碳纳米管/壳聚糖复合膜修饰玻碳电极上的电化学行为及测定

研究了维生素C在多壁碳纳米管/壳聚糖复合膜修饰玻碳电极上的电化学行为及测定。实验结果表明,在0.2 mol/L PBS(pH6.0)缓冲溶液中,修饰电极对抗坏血酸的氧化具有明显的催化和增敏效应,其氧化峰电位由 0.5 V负移至 0.1 V(vs.AgCl/Ag)。对修饰剂碳纳米管的用量、支持电解质、富集电位和富集时间等进行了优化。采用半微分伏安法进行定量测定,其线性范围为4.0×10-6~2.0×10-3mol/L,r=-0.998 3,检出限为1.0μmol/L。对抗坏血酸在修饰电极上的电化学行为进行了探讨,其电极反应为具有吸附特性和不可逆的电极过程,测得参加反应的质子数为2,电极反应的电子转移系数为0.59。测定了维生素C药片中抗坏血酸的含量,回收率在93%~105%。     

ssDNA/十八酸修饰碳糊电极的制备及伏安法表征

将石墨粉与十八酸在80 ℃下混合制成表面富含—COOH的基底碳糊电极(SA/CPE), 然后在活化剂N-羟基琥珀酰亚胺(NHS)和1-乙基-3-(3-二甲基氨丙基)碳二亚胺盐酸盐(EDC)存在下将ssDNA固定到电极表面制备ssDNA修饰电极(ssDNA/SA/CPE). 以亚甲基蓝(MB)为指示剂, 用循环伏安法对SA/CPE和ssDNA/SA/CPE进行电化学表征, 发现其在ssDNA/SA/CPE上较在SA/CPE上的氧化峰电流(i_pa)和还原峰电流(i_pc)分别增大1.9倍和1.7倍, 式电势(E_f)负移8 mV. 把ssDNA/SA/CPE放在互补ssDNA溶液中杂交后, MB的i_pa 和i_pc较在SA/CPE上分别增大1.0倍和0.8倍, E_f负移18 mV. 用0.5 mol/L 的NaOH溶液冲洗使电极表面杂交而成的dsDNA变性洗脱, MB的伏安信号几乎与在ssDNA/SA/CPE上一样. i_pc与SA/CPE上固定的ssDNA质量在1.0×10^－7～5.0×10^－6 g范围内成线性关系, 检测限为2.0×10^－9 g (S/N＝3). 这种既廉价又灵敏的电化学生物传感器有望在转基因植物产品检测研究中得到应用.