The work evaluated a series of approaches to optimise detection of polymerase chain reaction (PCR) amplified DNA samples by an optical sensor based on surface plasmon resonance (SPR) (BiacoreX™). The ...optimised procedure was based on an asymmetric PCR amplification system to amplify predominantly one DNA strand, containing the sequence complementary to a specific probe. The study moved into two directions, aiming to improve the analytical performance of SPR detection in PCR amplified products. One approach concerned the application of new strategies at the level of PCR, i.e. asymmetric PCR to obtain ssDNA amplified fragments containing the target capable of hybridisation with the immobilised complementary probe. The other strategy focused on the post-PCR amplification stage. Optimised denaturing conditions were applied to both symmetrically and asymmetrically amplified fragments. The effective combination of the two strategies allowed a rapid and specific hybridisation reaction. The developed method was successfully applied in the detection of genetically modified organisms.
An amperometric culture-based method was developed for rapid detection of viable
Escherichia
coli in water. The bacteria were recovered by filtration and incubated in a selective medium, lauryl ...sulphate broth (LSB) supplemented with the substrate 4-aminophenyl-β-
d-galactopyranoside (4-APGal) at 44.5°C. The electrochemically active molecule 4-aminophenol (4-AP) was produced after hydrolysis of 4-APGal by the enzyme β-galactosidase. 4-AP was measured by amperometry and was detected at a due concentration of
E.
coli. The time necessary for reaching that concentration was inversely related to the initial
E.
coli concentration of the sample. Environmental samples and suspensions of
E
coli IT1 were assayed. 4-AP was detected after 7.3 and 2.0
h in samples containing initial concentrations of
E.
coli IT1 of 4.5 and 4.5×10
6
cfu
ml
−1, respectively. For environmental samples with initial
E.
coli concentrations of 1.0 and 2.0×10
3
cfu
ml
−1, 4-AP were detected after 10 and 6.6
h, respectively.
Electrochemistry has superior properties respect to the other measurement systems because of the rapid, simple and sensitive characteristics. For all these reasons, electrochemical sensors are ...playing a key role in many scientific sectors.
Planar electrochemical sensors present many advantages respect to the three-dimensional devices, but this technology requires the use of specialty chemicals to meet the new functional requirements of planarization and miniaturization. In this paper, some production techniques and procedures to obtain planar electrochemical sensors are reported, together with their applications.
In the present work, electrochemical DNA biosensors are proposed as a screening device for the rapid bio-analysis of environmental pollution and DNA–drug interaction studies. The binding of small ...molecules to DNA immobilised on disposable screen-printed electrodes has been measured through the variation of the electrochemical signal of guanine by square wave voltammetric scans. These kinds of biosensors were used to evaluate the soil contamination level in an Italian polluted area and the results were compared with several methods for the DNA damage detection, as Comet genotoxicity effects, aberrant anatelophases and micronucleated cells frequency on plant roots, and with fixed wavelength fluorescence (FF) by using 2-aminoanthracene as standard compound. The results showed the ability of the biosensors to distinguish in 11
min low, medium and high contaminated soils with good correlation with well established techniques as well as FF, Comet and genotoxicity tests. The same kind of biosensors was also used to evaluate the interaction of DNA with some anti-proliferative metallo drugs, and the electrochemical responses reflected the kind of interaction. The reproducibility of the electrochemical measurements of DNA guanine peak was estimated as less than 10% of relative standard deviation (R.S.D.%).
An electrochemical DNA-based biosensor is proposed as a fast and easyscreening method for the detection of genotoxic compounds in soil samples. The biosensorwas assembled by immobilising double ...stranded Calf thymus DNA on screen-printedelectrodes. The interactions between DNA and environmental pollutants can causevariations of the electrochemical proprieties of DNA when they cause a DNA damage.Preliminary studies were performed using benzene, naphthalene and anthracene derivativesas model compounds. The effect of these compounds on the surface-confined DNA wasfound to be linearly related to their concentration in solution. On the other hand, theobjective was to optimise the ultrasonic extraction conditions of these compounds fromartificially spiked soil samples. Then, the applicability of such a biosensor was evaluated byanalysing soil samples from an Italian region with ecological risk (ACNA of Cengio, SV).DNA biosensor for qualitative analysis of soil presented a good correlation with a semiquantitativemethod for aromatic ring systems determination as fixed wavelengthfluorescence and interestingly, according results were found also with other bioassays.This kind of biosensors represent a new, easy and fast way of analysis of polluted sites, therefore they can be used as early warnings devices in areas with ecological risk as in situ measurement.
A novel low-cost platform to assess biomolecular interactions was investigated using surface plasmon resonance and an aptamer-based assay for thrombin detection. Gold SPR surface functionalized with ...a carboxylated cross-linked BSA film (cBSA) and commercially available carboxymethylated dextran chip (CM5) were used as immobilization platforms for the thrombin binding aptamer. The high end commercial instrument Biacore 3000 and a custom made FIA set-up involving TI Spreeta sensor (TSPR2K23) were used to assess different concentrations of thrombin within the range 0.1–150
nM both in buffer and in a complex matrix (plasma) using the obtained aptasensors. Based on data derived from both CM5 and cBSA platforms, the cBSA aptasensor exhibited good selectivity, stability and regeneration ability, both in buffer and in complex matrices (plasma), comparable with CM5.
The determination of organophosphate and carbamate pesticides was carried out using cobalt phthalocyanine-modified carbon epoxy composite electrodes coupled with acetylcholinesterase or ...butyrylcholinesterase. Covalent immobilization of enzymes on Immobilon membranes or nylon nets was examined; the highest sensitivity to inhibitors was found for the nylon net containing low enzyme loading and this was subsequently used for the construction of an amperometric biosensor for pesticides. Analyses were done using acetyl- or butyrylthiocholine as substrates; thiocholine produced by hydrolysis in the enzyme membrane was electrochemically oxidized at +300 mV (vs. Ag/AgCl reference). The decrease of substrate steady-state current caused by the addition of pesticide was used for evaluation. With this approach, 1.5 and 8.4 micrograms l-1 of paraoxon and heptenophos, respectively, can be detected in less than 3 min. These detection limits are similar as those obtained when analyses were performed using free cholinesterase and 10 min incubation with inhibitor.
Genosensor technology relying on the use of carbon and gold electrodes is reviewed. The key steps of each analytical procedure, namely DNA-probe immobilisation, hybridisation, labelling and ...electrochemical investigation of the surface, are discussed in detail with separate sections devoted to label-free and newly emerging magnetic assays. Special emphasis has been given to protocols that have been used with real DNA samples.
MicroRNAs (miRNAs, miRs) are naturally occurring small RNAs (approximately 22 nucleotides in length) that have critical functions in a variety of biological processes, including tumorigenesis. They ...are an important target for detection technology for future medical diagnostics. In this paper we report an electrochemical method for miRNA detection based on paramagnetic beads and enzyme amplification. In particular, miR 222 was chosen as model sequence, because of its involvement in brain, lung, and liver cancers. The proposed bioassay is based on biotinylated DNA capture probes immobilized on streptavidin-coated paramagnetic beads. Total RNA was extracted from the cell sample, enriched for small RNA, biotinylated, and then hybridized with the capture probe on the beads. The beads were then incubated with streptavidin–alkaline phosphatase and exposed to the appropriate enzymatic substrate. The product of the enzymatic reaction was electrochemically monitored. The assay was finally tested with a compact microfluidic device which enables multiplexed analysis of eight different samples with a detection limit of 7 pmol L
−1
and RSD = 15 %. RNA samples from non-small-cell lung cancer and glioblastoma cell lines were also analyzed.
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