A DNA Aptasensor for Electrochemical Detection of Vascular Endothelial Growth Factor.

Electrochemical aptasensors can detect different cancer biomarkers to provide point-of-care diagnosis that is low cost, rapid, specific and sensitive. In this work, we described the development of an electrochemical single-use aptasensor for detection and analysis of vascular endothelial growth factor (VEGF). Gold nanostructured graphite screen-printed electrodes were firstly modified with a mixed monolayer of a primary thiolated DNA aptamer and a spacer thiol, 6-mercapto-1-hexanol. VEGF protein was then incubated with the aptasensor. An enzyme-amplified detection scheme, based on the coupling of a streptavidin-alkaline phosphatase conjugate and secondary biotinylated aptamer was then applied. The enzyme catalyzed the hydrolysis of the electroinactive 1 -naphthyl-phosphate to 1 -naphthol; this product is electroactive and has been detected by means of differential pulse voltammetry (DPV). The aptasensor response was found to be linearly related to the target concentration between 0 and 250 nmol L(-1); the detection limit was 30 nmol L(-1). The performance of the immunoassay in terms of reproducibility and selectivity has been also studied.

Disposable electrochemical genosensor for the simultaneous analysis of different bacterial food contaminants.

This paper deals with the use of an electrochemical genosensor array for the rapid and simultaneous detection of different food-contaminating pathogenic bacteria. The method includes PCR amplification followed by analysis of the amplicons by hybridisation with toxin-specific oligonucleotide probes. A screen-printed array of four gold electrodes, modified using thiol-tethered oligonucleotide probes, was used. Unmodified PCR products were captured at the sensor interface via sandwich hybridisation with surface-tethered probes and biotinylated signaling probes. The resulting biotinylated hybrids were coupled with a streptavidin-alkaline phosphatase conjugate and then exposed to an alpha-naphthyl phosphate solution. Differential pulse voltammetry was finally used to detect the alpha-naphthol oxidation signal. Mixtures of DNA samples from different bacteria were detected at the nanomolar level without any cross-interference. The selectivity of the assay was also confirmed by the analysis of PCR products unrelated to the immobilised probes.

Electrochemical aptasensors for contaminants detection in food and environment: Recent advances.

The growing number of contaminants requires the development of new analytical tools to meet the increasing demand for legislative actions on food safety and environmental pollution control. In this context, electrochemical aptamer-based sensors appear promising among all biosensors because they permit multiplexed analysis and provide fast response, sensitivity, specificity and low cost. The aim of this review is to give the readers an overview of recent important achievements in the development of electrochemical aptamer-based biosensors for contaminant detection over the last two years. Special emphasis is placed on aptasensors based on screen-printed electrodes which show a substantial improvement of analytical performances.

Acetamiprid multidetection by disposable electrochemical DNA aptasensor.

In this work, we propose an electrochemical DNA aptasensor for sensitive multidetection of acetamiprid based on a competitive format and disposable screen-printed arrays. To improve the sensitivity of the aptasensor, polyaniline film and gold nanoparticles were progressively electrodeposited on the graphite screen-printed electrode surface by cyclic voltammetry. Gold nanoparticles were then employed as platform for thiol-tethered DNA aptamer immobilization. Different acetamiprid solutions containing a fixed amount of biotinylated complementary oligonucleotide sequence by DNA aptasensor arrays were analyzed. Streptavidin-alkaline phosphatase conjugate was then added to trace the affinity reaction. The enzyme catalyzed the hydrolysis of 1-naphthyl phosphate to 1-naphthol. The enzymatic product was detected by differential pulse voltammetry. A decrease of the signal was obtained when the pesticide concentration was increased, making the sensor work as signal off sensor. Under optimized conditions by testing key experimental parameters, a dose-response curve was constructed between 0.25 and 2.0µM acetamiprid concentration range and a limit of detection of 0.086µM was calculated. The selectivity of the aptasensor was also confirmed by the analysis of atrazine pesticide. Finally, preliminary experiments in fruit juice samples spiked with acetamiprid were also performed.

Measurement of volatile organic compounds (VOCs) in libraries and archives in Florence (Italy).

Indoor air samples from libraries and archives in Florence, Italy, were collected and analysed for a variety of volatile organic compounds. The aim was to perform a characterisation of the indoor air quality, and try to elucidate if there are VOCs that may cause or result from the determination of the cultural heritage institutions. All compounds of interest were regularly detected, with BTEXs (Benzene, Toluene, Ethylbenzene, Xylenes) being the most abundant and followed by cyclic volatile methylsiloxanes, aldehydes, terpenes and organic acids. The prevalence and qualitative characteristics, such as concentrations, profiles and indoor/outdoor ratios of BTEXs underline the important influence of the outdoor air infiltrations on the indoor air concentrations. Acetic acid that is a substance that can oxidise books and other exposed objects was detected at concentrations ranging between 1.04 and 18.9µgm-3, while furfural, that is a known marker of paper degradation, was constantly present at concentrations that ranged between 5.26 and 32.6µgm-3. This work shows the importance that indoor air quality monitoring campaigns can have in order to give early warning to cultural heritage institution managers about the impact that indoor air quality can have on exposed and/or preserved objects.

Disposable DNA electrochemical sensor for hybridization detection.

A disposable electrochemical sensor for the detection of short DNA sequences is described. Synthetic single-stranded oligonucleotides have been immobilized onto graphite screen printed electrodes with two procedures, the first involving the binding of avidinbiotinylated oligonucleotide and the second adsorption at a controlled potential. The probes were hybridized with different concentrations of complementary sequences. The formed hybrids on the electrode surface were evaluated by differential pulse voltammetry and chronopotentiometric stripping analysis using daunomycin hydrochloride as indicator of hybridization reaction. The probe immobilization step, the hybridization event and the indicator detection, have been optimized. The DNA sensor obtained by adsorption at a controlled potential was able to detect 1 microgram/ml of target sequence in the buffer solution using chronopotentiometric stripping analysis.

Detection of human apolipoprotein E genotypes by DNA biosensors coupled with PCR.

Apolipoprotein E (apoE) is an important constituent of several plasma lipoproteins and has been associated with the risk of developing cardiovascular diseases and in familiar type III hyperlipoproteinemia. We developed new procedures for the detection of apolipoprotein E polymorphism in human blood based on coupling DNA electrochemical or piezoelectric sensors with polymerase chain reaction (PCR). The electrochemical sensor was obtained by immobilizing single-stranded oligonucleotides onto graphite screen-printed electrodes by adsorption at controlled potential. The hybridization reaction that occurred on the electrode surface was evidenced by chronopotentiometric stripping analysis-using daunomycin as indicator. In the piezoelectric sensor, biotinylated 23-mer probes were immobilized on the streptavidin-coated gold surface of a quartz crystal; streptavidin was covalently bound to the thiol/dextran modified gold surface. The hybridization of the immobilized probes with complementary and mismatched DNA was investigated. With the use of two different probes, it was possible to investigate both positions in which apoE polymorphism takes place and consequently, to distinguish the different genotypes. The procedure was validated with both kinds of biosensor with a reference method based on polyacrilamide gel electrophoresis.

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.

l- and d-Lactate assay in real milk samples with immobilized enzyme reactors and graphite electrode.

A sensitive flow system for the determination of l- and d-lactate in milk samples is described. l- and d-Lactate dehydrogenase, LDH, were immobilized on aminopropyl-controlled pore glass beads. l- and d-Lactate are oxidized to pyruvate in the presence of NAD(+) and NADH is produced. The electrochemical determination of NADH allows the measurement of the substrate involved in the reaction. We used a graphite-based anode sensor without any mediator at +500 mV vs. Ag/AgCl. The analytes were measured, in standard solutions, in the concentration range from 1 x 10(-6) to 4 x 10(-4)M using 1 mM NAD(+) concentration and 0.1M Tris buffer pH 9. Experiments with real milk samples showed large values of currents probably due to electroactive substances usually contained in milk. To eliminate interfering compounds a microdialysis probe coupled with a pre-oxidizing cell was used. This method of pre-treatment removes the interfering substances, but leaves the analytes under study unaffected. The procedure allows the determination of l- and d-lactate in milk samples in the concentration range from 1 x 10(-5) to 5 x 10(-4)M. The assay was applied to monitor continuously the bacterial fermentation of Staphylococcus aureus in UHT milk as an example of possible contamination detection in the manufacturing process.

DNA electrochemical biosensors.

Disposable electrochemical DNA-based biosensors are reviewed; they have been used for the determination of low-molecular weight compounds with affinity for nucleic acids and for the detection of the hybridisation reaction. The first application is related to the molecular interaction between surface-linked DNA and the target pollutants or drugs, in order to develop a simple device for rapid screening of toxic or similar compounds. The determination of such compounds was measured by their effect on the oxidation signal of the guanine peak of calf thymus DNA immobilised on the electrode surface and investigated by chronopotentiometric analysis. The DNA biosensor is able to detect known intercalating compounds, such as daunomycin, polychlorinated biphenyls (PCBs), aflatoxin B1, and aromatic amines. Applicability to river and waste water samples is also demonstrated. Disposable electrochemical sensors for the detection of a specific sequence of DNA were realised by immobilising synthetic single-stranded oligonucleotides onto a graphite screen-printed electrode. The probes became hybridised with different concentrations of complementary sequences present in the sample. The hybrids formed on the electrode surface were evaluated by chronopotentiometric analysis using daunomycin as indicator of the hybridisation reaction. The hybridisation was also performed using real samples. Application to apolipoprotein E (ApoE) is described, in this case samples have to be amplified by PCR and then analysed by DNA biosensor. The extension of such procedures to samples of environmental interest or to contamination of food is discussed.

Electrochemical DNA biosensor as a screening tool for the detection of toxicants in water and wastewater samples.

Applications of a disposable electrochemical DNA biosensor to standard solutions and to real samples are reported. The DNA biosensor is assembled by immobilising the double stranded calf thymus DNA on the surface of a disposable carbon screen-printed electrode. The immobilised ds-DNA interacts with the sample for 2 min; then is washed and immersed in a clean buffer where the analytical signal (the oxidation peak area of the guanine base) is obtained by a square-wave voltammetric scan. The results were compared with some currently used toxicity tests and in particular with a commercial luminescent bacteria test, Toxalert(R)100.

Detection of human apolipoprotein E genotypes by DNA electrochemical biosensor coupled with PCR.

BACKGROUND: Apolipoprotein E (apoE) is an important constituent of several plasma lipoproteins, mainly VLDL, HDL, and chylomicrons. It is involved in the redistribution of lipids in the liver and is implicated in growth and repair of injured neurons in the nervous system. apoE has also been associated with the risk of developing cardiovascular diseases and in familial type III hyperlipoproteinemia. METHODS: We developed a new procedure for detecting genetic polymorphisms of apoE in human blood samples. The procedure is based on coupling of DNA electrochemical sensors with PCR-amplified DNA extracted from human blood. The DNA electrochemical sensor incorporated single-stranded oligonucleotides immobilized on graphite screen-printed electrodes (SPEs) by adsorption at controlled potential. The hybridization reaction on the electrode surface was monitored by chronopotentiometric stripping analysis (PSA), using daunomycin as indicator. RESULTS: With use of two different probes, it was possible to investigate both DNA positions in which the apoE polymorphism takes place and thus to distinguish different genotypes. Real samples containing only complementary sequences gave a good increase in the area of the daunomycin peak ( approximately 600 ms) compared with the peak observed with the buffer. Samples containing 50% complementary sequences gave a much lower increase, and samples containing only mismatch sequences gave a decrease in the daunomycin area. The procedure was validated by comparison with a method based on polyacrylamide gel electrophoresis. CONCLUSION: The coupling of DNA electrochemical sensors with PCR allowed quick discrimination between the different genotypes of apoE.