A genetically engineered Escherichia coli (E. coli) strain displaying glucose dehydrogenase (GDH) with ice-nucleation protein (INP) as the anchoring motif was first constructed. The surface ...localization and functionality of the fusion protein containing GDH were verified by SDS-PAGE, Western blotting and enzymatic activity assay. The fusion of INP had no effects on the functionality of GDH cofactor binding domain. The activity assay showed that 74.6% of the cell lysate GDH activity was detected in the outer membrane fractions. Compared with the crude enzyme solution from E. coli expressing intracellular GDH, the GDH-displaying bacteria (GDH-bacteria) was stable within pH 6–10 below 40°C. Further, a novel electrochemical glucose biosensor was developed by construction of Nafion/GDH-bacteria/multiwalled-carbon-nanotube modified electrode. The as-prepared biosensor is linear with the concentration of d-glucose within the range of 50–800μM and a low detection limit of 4μM d-glucose (S/N=3). Excess saccharides including d-galactose, d-fructose, d-cellbiose, l-arabinose and d-sucrose, d-maltose, d-mannose and d-xylose as well as common interfering substances (acetaminophen, ascorbic acid and uric acid) did not affect the detection of d-glucose (0.1mM). The proposed biosensor is stable, specific, reproducible, simple, rapid and cost-effective, which can be used for detection of real samples. It is envisioned that this GDH-bacteria will be found promising applications in biofuel cell, glucose detection and cofactor reproduction system.
► First construction of genetically engineered bacteria displaying glucose dehydrogenase. ► Development of a novel electrochemical glucose biosensor. ► Without interference from other saccharides and common interfering substances to the detection of glucose. ► Capable of glucose measurement in real samples.
A disposable electrochemical assay involving magnetic particles and carbon‐based screen‐printed electrodes (SPCEs) was developed for the detection of C Reactive Protein (CRP). CRP is a plasma protein ...and is among the most expressed proteins in acute phase inflammation cases, being a known biomarker for inflammatory states. The assay was based on a sandwich format in which a RNA aptamer was coupled to a monoclonal antibody and alkaline phosphatase (AP) was used as enzymatic label. After the sandwich assay, the modified magnetic beads were captured by a magnet on the surface of a graphite working electrode and the electrochemical detection was thus achieved through the addition of the AP substrate (α‐naphthyl‐phosphate) and α‐naphthol produced during the enzymatic reaction was detected using differential pulse voltammetry (DPV). The parameters influencing the different steps of the assay were optimized in order to reach the best sensitivity and specificity. With the optimized conditions, the assay was applied to the analysis of CRP free serum and serum samples.
The development of an electrochemical genosensor involving DNA biotinylated capture probe immobilized on streptavidin coated paramagnetic beads and microfluidic based platform for the detection of ...P53 gene PCR product is reported. The novelty of this work is the combination of a sensitive electrochemical platform and a proper microfluidic system with a simple and effective enzyme signal amplification technology, ELISA, for detection of target DNA sequence and single nucleotide mutation in p53 tumor suppressor gene sequence. The biosensor has been applied to detect the PCR amplified samples and the results shows that it can discriminate successfully perfect matched DNA from mutant form.
The possibility of introducing a computationally assisted method to study aptamer–protein interaction was evaluated with the aim of streamlining the screening and selection of new aptamers. Starting ...from information on the 15-mer (5′-GGTTGGTGTGGTTGG-3′) thrombin binding aptamer (TBA), a library of mutated DNA sequences (994 elements) was generated and screened using shapegauss a shape-based scoring function from openeye software to generate computationally derived binding scores. The TBA and three other mutated oligonucleotides, selected on the basis of their binding score (best, medium, worst), were incorporated into surface plasmon resonance (SPR) biosensors. By reducing the ionic strength (binding buffer, 50
mM TrisHCl pH 7.4, 140
mM NaCl, 1
mM MgCl
2, diluted 1:50) in order to match the simulated condition, the analytical performances of the four oligonucleotide sequences were compared using signal amplitude, sensitivity (slope), linearity (
R
2) and reproducibility (CVav %). The experimental results were in agreement with the simulation findings.
A DNA piezoelectric sensor has been developed for the detection of genetically modified organisms (GMOs). Single stranded DNA (ssDNA) probes were immobilised on the sensor surface of a quartz crystal ...microbalance (QCM) device and the hybridisation between the immobilised probe and the target complementary sequence in solution was monitored. The probe sequences were internal to the sequence of the 35S promoter (P) and Nos terminator (T), which are inserted sequences in the genome of GMOs regulating the transgene expression. Two different probe immobilisation procedures were applied: (a) a thiol–dextran procedure and (b) a thiol-derivatised probe and blocking thiol procedure. The system has been optimised using synthetic oligonucleotides, which were then applied to samples of plasmidic and genomic DNA isolated from the pBI121 plasmid, certified reference materials (CRM), and real samples amplified by the polymerase chain reaction (PCR). The analytical parameters of the sensor have been investigated (sensitivity, reproducibility, lifetime etc.). The results obtained showed that both immobilisation procedures enabled sensitive and specific detection of GMOs, providing a useful tool for screening analysis in food samples.
This paper describes the optimisation and the analytical performances of an enzyme-based electrochemical genosensor, developed using disposable oligonucleotide-modified screen-printed gold ...electrodes.
The immobilisation of a thiol-tethered probe was qualitatively investigated by means of faradic impedance spectroscopy. Impedance spectra confirmed that the thiol moiety unambiguously drives the immobilisation of the oligonucleotide probe. Furthermore, both probe surface densities and hybridisation efficiencies were quantified through chronocoulometric measurements.
Electrochemical transduction of the hybridisation process was also performed by means of faradic impedance spectroscopy, after coupling of a streptavidin–alkaline phosphatase conjugate and bio-catalysed precipitation of an insoluble and insulating product onto the sensing interface. Chronocoulometric results allowed discussion of the magnitude of hybridisation signals in terms of probe surface densities and their corresponding hybridisation efficiency. The genosensor response varied linearly (
r
2 = 0.9998) with the oligonucleotide target concentration over three orders of magnitude, between 12
pmol/L and 12
nmol/L. The estimated detection limit was 1.2
pmol/L (i.e., 7.2 × 10
6 target molecules in 10
μL of sample solution).
The analytical usefulness of the impedimetric genosensor was finally demonstrated analysing amplified samples obtained from the pBI121 plasmid and soy and maize powders containing 1 and 5% of genetically modified product. Sensing of such unmodified amplicons was achieved via sandwich hybridisation with a biotinylated signaling probe. The electrochemical enzyme-amplified assay allowed unambiguous identification of all genetically modified samples, while no significant non-specific signal was detected in the case of all negative controls.
Recent progress in the development of electrochemical nanomaterial–aptamer-based biosensors is summarized. Aptamers are nucleic acid ligands that can be generated against amino acids, drugs, ...proteins, and other molecules. They are isolated from a large random library of synthetic nucleic acids by an iterative process of binding, separation, and amplification, called systematic evolution of ligands by exponential enrichment (SELEX). In this review, different methods of integrating aptamers with different nanomaterials and nanoparticles for electrochemical biosensing application are described.
The detection and identification of foodborne pathogens continue to rely on conventional culturing techniques. These are very elaborate, time-consuming, and have to be completed in a microbiology ...laboratory and are therefore not suitable for on-site monitoring. The need for a more rapid, reliable, specific, and sensitive method of detecting a target analyte, at low cost, is the focus of a great deal of research. Biosensor technology has the potential to speed up the detection, increase specificity and sensitivity, enable high-throughput analysis, and to be used for monitoring of critical control points in food production. This article reviews food pathogen detection methods based on electrochemical biosensors, specifically amperometric, potentiometric, and impedimetric biosensors. The underlying principles and application of these biosensors are discussed with special emphasis on new biorecognition elements, nanomaterials, and lab on a chip technology.
The development of an acetylcholinesterase (AChE) based biosensor is described. As transducers cobalt(II) phthalocyanine (CoPC)-modified screen-printed carbon electrodes (SPCEs) were used. Through ...the study of the AChE catalytic activity and by using acetylthiocholine (ATCh) as enzyme substrate, it was possible to detect pesticides, such as Carbofuran, by means these modified SPCEs under optimised chronoamperometric conditions. The method was based on the incubation of the AChE-based biosensor with the pesticide. The inhibitory effect of the pesticide determined a decrease of the catalytic activity of AChE; as a consequence, less thiocholine (TCh) was produced from ATCh. Therefore, the current value, due to the oxidation of TCh at the modified SPCEs, was lower than that recorded in a blank solution. This current decrease was correlated with the pesticide concentration. A detection limit of 4.9
×
10
−10
M for Carbofuran was found with an analysis time of 15
min. The study of the immobilisation layer composition (enzyme units and cross-linker amounts) was emphasised. A comparison of the inhibitory effect of pesticides among AChE in solution as well as immobilised was also reported.
Aptamers are single stranded DNA or RNA ligands which can be selected for different targets starting from a huge library of molecules containing randomly created sequences. Aptamers have been ...selected to bind very different targets, from proteins to small organic dyes. In the last years great progress has been accomplished in the development of aptamer-based bioanalytical assays with different detection techniques. This review will describe some recent aptamer-based biosensors which have been developed for the detection of small molecules that could be interesting in the pharmaceutical field. The use of aptamers to develop assays for small molecules has not been extensively studied as for protein targets. This is mainly due to difficulties in selecting aptamers for small molecules which present fewer binding possibilities for the aptamers with respect to proteins. Despite these difficulties, a few works aiming at developing aptamer-based biosensor for small molecules have been reported which take advantage of the versatility and the flexibility of aptamers.
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