Electrochemical DNA biosensor for analysis of wastewater samples

The application of a disposable electrochemical DNA biosensor to wastewater samples is reported. The DNA biosensor is assembled by immobilising double-stranded calf thymus DNA on the surface of a disposable, carbon screen-printed electrode (SPE). The oxidation signal of the guanine base, obtained by a square wave voltammetric scan, is used as analytical signal. The presence of compounds with affinity for DNA is measured by their effect on the guanine oxidation. The comparison of the results with a toxicity test based on bioluminescent bacteria has confirmed the applicability of the method to real samples.     

A comparison study between a disposable electrochemical DNA biosensor and a Vibrio fischeri-based luminescent sensor for the detection of toxicants in water samples

In the present study, a comparison between a disposable electrochemical DNA biosensor and a Vibrio fischeri-based luminescent sensor for the detection of toxicants in water samples was made.In order to realize this study, a disposable electrochemical DNA biosensor has been reported. The DNA biosensor is assembled by immobilizing double stranded Calf Thymus DNA onto the surface of a disposable carbon screen-printed electrode. The oxidation signal of the guanine base, obtained by a square wave voltammetric scan, is used as analytical signal to detect the DNA damage; the presence of low molecular weight compounds with affinity for nucleic acids is measured by their effect on the guanine oxidation peak.Wastewater samples provided during First European Interlaboratory Exercise on water toxicity in the course of the project SWIFT-WFD were analyzed, and biosensor results were compared with a currently used toxicity test ToxAlert^®100 based on the bioluminescence inhibition of Vibrio fischeri. This test have been used because is rapid, easy handling and cost effectively responses for the toxicity assessment in real water samples.The results showed a promising correlation between two tests used for the detection of toxic compounds in water samples.     

Chitosan/Nitrogen Doped Reduced Graphene Oxide Modified Biosensor for Impedimetric Detection of microRNA

The development of a low‐cost and disposable biosensor platform for the sensitive and rapid detection of microRNAs (miRNAs) is of great interest for healthcare, pharmaceuticals, and medical science. We designed an impedimetric biosensing platform using Chitosan (CHIT)/nitrogen doped reduced graphene oxide (NRGO) conductive composite to modify the surface of pencil graphite electrodes (PGE) for the sensitive detection of miRNAs. An initial optimisation protocol involved investigation of the effect of NRGO concentration and miR 660 DNA probe concentration on the response of the modified electrode. After the optimization protocol, the sequence‐selective hybridization between miR 660 DNA probe and its RNA target was evaluated by measuring changes on charge transfer resistance, R_ct values. Moreover, the selectivity of impedimetric biosensor was tested in the presence of non‐complementary miRNA (NC) sequences, such as miR 34a and miR 16. The hybridization process was examined both in phosphate buffer (PBS) and in PBS diluted fetal bovine serum (FBS:PBS) solutions. The biosensor demonstrated a detection limit of 1.72 μg/mL in PBS and 1.65 μg/mL in FBS:PBS diluted solution. Given the easy, quick and disposable attributes, the proposed conductive nanocomposite biosensor platform shows great promise as a low‐cost sensor kit for healthcare monitoring, clinical diagnostics, and biomedical devices.     

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

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.     

DNA-based biosensor for the detection of strong damage to DNA by the quinazoline derivative as a potential anticancer agent

A disposable electrochemical DNA-based biosensor was developed and applied as a screening device to detect an effect of a synthetically prepared quinazoline derivative on the surface-attached double stranded calf thymus DNA. Screen-printed carbon electrodes without and with multi-walled carbon nanotubes interface served as the signal transducer. The quinazoline interaction with DNA was investigated voltammetrically using DNA-bound electrochemical indicators such as [Co(phen)₃]³⁺, [Ru(bpy)₃]²⁺, methylene blue, the K₃[Fe(CN)₆] complex present in the solution phase as well as by electrochemical impedance spectroscopy. A severe damage to DNA at the incubation of the biosensor in quinazoline solution was found which leads to the loss of DNA from the electrode surface. Agarose gel electrophoresis was used to verify the results.     

Prostate Cancer Biomarker Detection with Carbon Nanotubes Modified Screen Printed Electrodes

DNA hypermethylation is an epigenetic alteration and a promising biomarker for early prostate cancer detection. Simple, sensitive, easy to handle and rapid detection methodologies are imperative for point of care diagnostics especially for cancer. Herein, we describe for the first time a regenerable and compatible electrochemical biosensor for detection of Glutathione S‐Transferase P‐1 (GSTP‐1) gene hypermethylation related to prostate cancer via DNA hybridization onto the disposable Carbon and Multi Walled Carbon Nanotubes (MWCNT) Screen Printed Electrodes (SPEs). In the study, capture probes were adsorbed onto the SPEs by simple passive adsorption and then hybridization was achieved by sending the complementary target onto the probe‐modified electrodes. The selectivity of the biosensor was proved by control studies. Differential Pulse Voltammetry (DPV) technique was used to detect hybridization via guanine oxidation signals changes. The total time of the optimized method was nearly 1h, measurements took for less than 1 min, and the biosensor response was stable up to 40 days of storage period at 4 °C. The main advantages of the biosensor are very low detection limit (picomolar range) and capability of reusing the biosensor for at least 3 times after very simple regeneration process that is a unique property to reduce the cost of the assay. In addition, this is the first study that demonstrates the detection of GSTP‐1 hypermethylation electrochemically by using SPEs in order to create point of care diagnostics. The optimum parameters for the biosensor, as well as its future prospects to enhance the performance of DNA biosensors were also presented.     

Coupling of an indicator-free electrochemical DNA biosensor with polymerase chain reaction for the detection of DNA sequences related to the apolipoprotein E

This paper describes a disposable indicator-free electrochemical DNA biosensor applied to the detection of apolipoprotein E (apoE) sequences in PCR samples. In the indicator-free assays, the duplex formation was detected by measuring the electrochemical signal of the guanine base of nucleic acids. The biosensor format involved the immobilisation of an inosine-modified (guanine-free) probe onto a screen-printed electrode (SPE) transducer and the detection of the duplex formation in connection with the square-wave voltammetric measurement of the oxidation peak of the guanine of the target sequence.The indicator-free scheme has been characterised using 23-mer oligonucleotides as model: parameters affecting the hybridisation assay such as probe immobilisation conditions, hybridisation time, use of hybridisation accelerators were examined and optimised.The analysis of PCR samples (244 bp DNA fragments, obtained by amplification of DNA extracted from human blood) required a further optimisation of the experimental procedure. In particular, a lower steric hyndrance of the probe modified surface was essential to allow an efficient hybridisation of the target DNA fragment. Negative controls have been performed using the PCR blank and amplicons unrelated to the immobilised probe. A 10 min hybridisation time allowed a full characterisation of each sample.     

Disposable electrochemiluminescent biosensor using bidentate-chelated CdTe quantum dots as emitters for sensitive detection of glucose

A novel disposable solid-state electrochemiluminescent (ECL) biosensor was fabricated by immobilizing glucose oxidase and surface-unpassivated CdTe quantum dots (QDs) on a screen-printed carbon electrode (SPCE). The surface morphology of the biosensor was characterized with scanning electron microscopy and atomic force microscopy. With dissolved O(2) as an endogenous coreactant, QDs/SPCE showed strong ECL emission in pH 9.0 HCl-Tris buffer solution with low ECL peak potential at -0.89 V. The ECL intensity was twice that with hydrogen peroxide as coreactant at the same concentration. This phenomenon meant the ECL decreased upon consumption of dissolved O(2) and thus could be applied to the construction of oxidase-based ECL biosensors. With glucose oxidase as a model enzyme, the biosensor showed rapid response to glucose with a linear range of 0.8 to 100 μM and a detection limit of 0.3 μM. Further detection of glucose contained in human serum samples showed acceptable sensitivity and selectivity. This work provided a promising application of QDs in ECL-based disposable biosensors.     

Highly sensitive hybridization assay using the electrochemiluminescence of an ITO electrode, CdTe quantum dots functionalized with hierarchical nanoporous PtFe nanoparticles, and magnetic graphene nanosheets

We report on a disposable microdevice suitable for sandwich-type electrochemiluminescence (ECL) detection of DNA. The method is making use of CdTe quantum dots functionalized with hierarchical nanoporous PtFe (CdTe@PtFe) nanoparticles and with magnetic graphene nanosheets. The latter were selected as carriers for the capture DNA due to their excellent biomagnetic separation capability and electrical properties. The CdTe@PtFe nanoparticles were used to label the signal DNA which resulted in distinctly enhanced ECL owing to the large specific surface area and good electrical conductivity of the PtFe alloy. A DNA sensor was constructed on a disk-shaped indium tin oxide electrode that was fabricated via etching. Under optimal conditions, the biosensor responds linearly to DNA in the 0.02 fM to 5000 fM concentration range, with a detection limit as low as 15 aM. The electrode is regenerable. The method displays excellent specificity, extremely good sensitivity, and is highly reproducible.

Detection of damage to DNA and antioxidative activity of yeast polysaccharides at the DNA-modified screen-printed electrode

A simple procedure for the voltammetric detection of the DNA damage and antioxidants protecting DNA from its damage using a disposable electrochemical DNA biosensor is reported. The carbon-based screen-printed electrode (SPE) modified by a surface layer of the calf thymus double stranded (ds) DNA was used as a working electrode in combination with a silver/silver chloride reference electrode and a separate platinum auxiliary electrode. The [Co(phen)(3)](3+) ion served as the dsDNA redox marker and the [Cu(phen)(2)](2+) and [Fe(EDTA)](-) complex compounds were used as the DNA cleavage agents under the reduction by a chemical reductant (ascorbic acid). Four yeast polysaccharides with different chemical structure were investigated as the antioxidants within the concentration range of 0.05-4 mg ml(-1) in the cleavage mixture. A remarkable antioxidative activity of polysaccharides in order mannan (Candida krusei)>extracellular glucomannan (Candida utilis)>mannan (Candida albicans)>glucomannan (C. utilis) was found which is in agreement with that refered to trolox (a structural derivative of alpha-tocopherol) and determined by photochemiluminescent method.     

Rapid Electrochemical Assessment of Tumor Suppressor Gene Methylations in Raw Human Serum and Tumor Cells and Tissues Using Immunomagnetic Beads and Selective DNA Hybridization

We report a rapid and sensitive electrochemical strategy for the detection of gene‐specific 5‐methylcytosine DNA methylation. Magnetic beads (MBs) modified with an antibody for 5‐methylcytosines (5‐mC) are used for the capture of any 5‐mC methylated single‐stranded (ss)DNA sequence. A flanking region next to the 5‐mCs of the captured methylated ssDNA is recognized by hybridization with a synthetic biotinylated DNA sequence. Amperometric transduction at disposable screen‐printed carbon electrodes (SPCEs) is employed. The developed biosensor has a dynamic range from 3.9 to 500 pm and a limit of detection of 1.2 pm for the methylated synthetic sequence of the tumor suppressor gene O‐6‐methylguanine‐DNA methyltransferase (MGMT) promoter region. The method is applied in the 45‐min analysis of specific methylation in the MGMT promoter region directly in raw spiked human serum samples and in genomic DNA extracted from U‐87 glioblastoma cells and paraffin‐embedded brain tumor tissues without any amplification and pretreatment step.     

Structure simulation into a lamellar supramolecular network and calculation of the metal ions/ligands ratio

Background

Biosensors have attracted increasing attention as reliable analytical instruments in in situ monitoring of public health and environmental pollution. For enzyme-based biosensors, the stabilization of enzymatic activity on the biological recognition element is of great importance. It is generally acknowledged that an effective immobilization technique is a key step to achieve the construction quality of biosensors.

Results

A novel disposable biosensor was constructed by immobilizing laccase (Lac) with silica spheres on the surface of multi-walled carbon nanotubes (MWCNTs)-doped screen-printed electrode (SPE). Then, it was characterized in morphology and electrochemical properties by scanning electron microscopy (SEM) and cyclic voltammetry (CV). The characterization results indicated that a high loading of Lac and a good electrocatalytic activity could be obtained, attributing to the porous structure, large specific area and good biocompatibility of silica spheres and MWCNTs. Furthermore, the electrochemical sensing properties of the constructed biosensor were investigated by choosing dopamine (DA) as the typical model of phenolic compounds. It was shown that the biosensor displays a good linearity in the range from 1.3 to 85.5 ??M with a detection limit of 0.42 ??M (S/N = 3), and the Michaelis-Menten constant (K_m ^app) was calculated to be 3.78 ??M.

Conclusion

The immobilization of Lac was successfully achieved with silica spheres to construct a disposable biosensor on the MWCNTs-doped SPE (MWCNTs/SPE). This biosensor could determine DA based on a non-oxidative mechanism in a rapid, selective and sensitive way. Besides, the developed biosensor could retain high enzymatic activity and possess good stability without cross-linking reagents. The proposed immobilization approach and the constructed biosensor offer a great potential for the fabrication of the enzyme-based biosensors and the analysis of phenolic compounds.     

Electrochemical Biosensor Based on Bienzyme and Carbon Nanotubes Incorporated into an Os‐complex Thin Film for Continuous Glucose Detection in Human Saliva

Saliva opens a door for noninvasive and painless glucose testing since it reflects changes in the body physiology of diabetic individuals as compared to healthy ones. In this paper, a unique, disposable saliva biosensor has been developed for accurate, low cost, and continuous glucose monitoring. The biosensor exhibits linear dependence of the catalytic current upon glucose bulk concentration over the 0.05–1.5 mM range (R=0.998). A detection limit of 0.003 mM can be calculated considering three times the standard deviation of the blank signal divided by the sensitivity of the sensor. The selectivity of the biosensor was evaluated by adding the interferent species of lactate, ascorbic acid and uric acid into in 0.5 mM glucose; the nearly negligible interference current indicates its good selectivity. The operational stability of the biosensor was measured in 1 mM glucose over a 2 h period (RSD=3.27 %). A clinical trial on real‐time noninvasive salivary glucose monitoring was carried out on 30 individuals by measuring subjects’ salivary glucose and blood glucose in parallel. The results show that there is a good correlation of glucose levels in saliva and in blood 2 h after breakfast. Thus, the disposable biosensor would be a potential alternative for continuous glucose detection in human saliva.     

基于纳米铂黑修饰的快速检测用乳酸生物传感器研究

制备了一种可用于运动员血清样品乳酸快速检测的L-乳酸传感器.这种便携式平面电化学生物传感器采用金薄膜两电极系统;先后修饰纳米铂黑粒子层和铁氰化钾媒介体.铂黑纳米粒子沉积于金电极表面以提高传感器的灵敏度和稳定性,然后将乳酸氧化酶(LOD, E.C.1.1.3.2)和相关试剂固定在工作电极表面,铁氰化钾作为媒介体用以提高电极表面电子传递能力,并将工作电压降低为0.2 V.通过优化铂黑颗粒的沉积、乳酸氧化酶的浓度、铁氰化钾的浓度、添加剂的成分和浓度等条件,将传感器的检测范围扩展至1～20 mmol/L乳酸,检测灵敏度提高到1.43 μA·L/mmol,检测时间为50 s.生物传感器的批间r为0.0549;生物传感器经室温储存1年后仍可保持90%的活性.这种传感器成功地用于无稀释乳酸血清样品的快速检测,结合便携式检测仪(YT 2005-1 乳酸测试仪)将在快速诊断领域具有很好的应用前景.     

一次性唾液α-淀粉酶生物传感器的研制

基于丝网印刷技术在PVC薄膜上制备了一次性碳电极,用Nafion固定二茂铁(Fc)作为电子介体,将α-糖苷酶和葡萄糖氧化酶(GOD)滴加于二茂铁修饰的电极上,滴加明胶晾干,戊二醛间接交联固定制成一次性唾液α-淀粉酶生物传感器,用计时电流法测定对α-淀粉酶的响应.实验结果表明,该传感器响应电流与α-淀粉酶活性在60～840 U/L之间呈现良好的线性关系,检出限为17 U/L.该生物传感器响应时间短,达到95%稳态响应时间不超过30 s,具有良好的一致性、准确性和稳定性.探讨了pH、缓冲液、温度及其它干扰物质等对该传感器的影响.该传感器可用于唾液α-淀粉酶浓度的快速、准确检测.     

A novel electrochemical biosensor for detection of cholesterol

We report on a highly sensitive electrochemical biosensor for determination of cholesterol. The biosensor was fabricated by co-immobilizing bi-enzymes, cholesterol oxidase (ChOx), and horseradish peroxidase (HRP). Voltammetric technique such as cyclic voltammetry and impedance experiment were used to study the characterization of modified electrode step by step. The developed sensor is cheap, disposable, portable and exhibits higher sensitivity. The biosensor expressed a wide linear range up to 300 mg dL^–1 in a physiological condition (pH 7.0), with a correlation coefficient of 0.9969. A sensitivity of 13.28 μA mg^–1 dL cm^?2 which makes it very promising for the clinical determination of cholesterol.     

Electrochemical DNA biosensor using a disposable electrochemical printed (DEP) chip for the detection of SNPs from unpurified PCR amplicons

In this study, we are reporting for the first time the elucidation of single nucleotide polymorphisms (SNPs) of clinically important alleles from consenting human subjects using a disposable electrochemical printed (DEP) chip in connection with differential pulse voltammetry (DPV) and a redox active molecule Hoechst 33258 [H33258, 2'-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5'-bi(1H-benzimidazole)]. Post-PCR products were analyzed directly without any purification process. The aggregation of the DNA-H33258 complex causes a significant drop in the peak current intensity of H33258 oxidation. The phenomenon of DNA aggregation induced by H33258 in addition to changes in anodic current peak are used to detect SNPs. Since laborious probe immobilization was not required, our biosensor offers several benefits due to its simplicity and rapid response as a promising device for genetic analysis.     

Disposable Biosensor Based on Amidase/CeO2/GNR Modified Inkjet‐printed CNT Electrodes‐droplet Based Paracetamol Detection in Biological Fluids for “Point‐of‐care” Applications

A disposable acetaminophen biosensor based on inkjet‐printed CNT electrodes (IJPCNT) modified with amidase/cerium dioxide@graphene nanoribbons composite was developed (ACeO2@GNR/IJPCNT). The enzyme amidase A was used for the first time as a recognition element. Inkjet‐printed CNT electrodes served as a basis for the construction of a biosensor that enables droplet detection using 5 μL sample volume. The biosensor showed high selectivity, sensitivity, a low detection limit of 0.18 μM and a wide working linear range from 1 to 100 μM. The proposed approach allows fast and reliable detection of acetaminophen in biological fluids with negligible matrix effect and remarkable reproducibility.     

基于多壁碳纳米管的三电极血乙醇生物传感器的研究

采用丝网印刷技术在PVC基板上印制三电极,将多壁碳纳米管、麦尔多拉蓝、乙醇脱氢酶(ADH)以及氧化型辅酶Ⅰ(NAD+)依次修饰在工作电极表面,然后在该三电极表面贴一层亲水膜,形成一个5μL反应池,制成血乙醇检测新型生物传感器检测条。结果表明,此生物传感器具有良好的准确性和稳定性;检测线性范围为0.5～20mmol/L,r=0.99493;检出限为0.22mmol/L;电流达到95%稳态时间小于15s。考察了pH值、温度及干扰物对生物传感器的影响。用此生物传感器和顶空气相色谱法对10份全血标本乙醇浓度平行测试,两者相关性良好r=0.97583。利用虹吸现象吸取微量全血直接定量测定乙醇浓度是此生物传感器的特点。     

A disposable biosensor for the determination of alpha-amylase in human saliva

A disposable tri-enzymatic biosensor is presented for the determination of α-amylase in human saliva. It is based on the quantity of maltose generated by hydrolysis of maltopentose in the presence of salivary α-amylase. The biosensor is fabricated by co-immobilization of the enzymes α-glucosidase, glucose oxidase, and mutarotase on screen-printed electrodes modified with Prussian Blue. The assay can be performed with a “drop” of sample, this allowing for ease and simplicity. A linear relationship is found for the range from 5 to 250 units per mL, with an LOD of 5 units per mL. The biosensor is stable for at least one month and over this time retains 80% of its original activity. The system was then evaluated for matrix effects of human saliva and compared to a spectrometric method using a commercially available kit.