A review on the electrochemical biosensors for determination of microRNAs.

MicroRNAs (miRNAs) are a family of non-protein-coding, endogenous, small RNAs. They are a group of gene regulators that function mainly by binding the 3' untranslated regions of specific target messenger RNA (mRNA) leading to gene inactivation by repression of mRNA transcription or induction of mRNA. Mature miRNAs are short molecules approximately 22 nucleotides in length. They regulate a wide range of biological functions from cell proliferation and death to cancer progression. Cellular miRNA expression levels can be used as biomarkers for the onset of disease states and in gene therapy for genetic disorders. Methods for detection of miRNA mainly include northern blotting, microarray, polymerase chain reaction (PCR). This review focuses on the use of electrochemical biosensors for the detection of microRNA.

Yeast surface displaying glucose oxidase as whole-cell biocatalyst: construction, characterization, and its electrochemical glucose sensing application.

The display of glucose oxidase (GOx) on yeast cell surface using a-agglutinin as an anchor motif was successfully developed. Both the immunochemical analysis and enzymatic assay showed that active GOx was efficiently expressed and translocated on the cell surface. Compared with conventional GOx, the yeast cell surface that displayed GOx (GOx-yeast) demonstrated excellent enzyme properties, such as good stability within a wide pH range (pH 3.5-11.5), good thermostability (retaining over 94.8% enzyme activity at 52 °C and 84.2% enzyme activity at 56 °C), and high d-glucose specificity. In addition, direct electrochemistry was achieved at a GOx-yeast/multiwalled-carbon-nanotube modified electrode, suggesting that the host cell of yeast did not have any adverse effect on the electrocatalytic property of the recombinant GOx. Thus, a novel electrochemical glucose biosensor based on this GOx-yeast was developed. The as-prepared biosensor was linear with the concentration of d-glucose within the range of 0.1-14 mM and a low detection limit of 0.05 mM (signal-to-noise ratio of S/N = 3). Moreover, the as-prepared biosensor is stable, specific, reproducible, simple, and cost-effective, which can be applicable for real sample detection. The proposed strategy to construct robust GOx-yeast may be applied to explore other oxidase-displaying-system-based whole-cell biocatalysts, which can find broad potential application in biosensors, bioenergy, and industrial catalysis.

Microbial surface display of glucose dehydrogenase for amperometric glucose biosensor.

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 50-800 µM and a low detection limit of 4 µM 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.

Direct energy conversion from xylose using xylose dehydrogenase surface displayed bacteria based enzymatic biofuel cell.

Xylose is an important and major monosaccharide that extensively exists in the cellulose fermentation industry. Here we present the first report on the direct energy conversion from xylose achieved by using novel xylose dehydrogenase (XDH) surface displayed bacteria (XDH-bacteria) based enzymatic biofuel cell. The maximum power density and the open-circuit potential of the cell are 63 µWcm(-2) and 0.58 V, respectively. The as-prepared BFC holds great potential to make use of biomass from lignocellulose degradation as source energy, which avoids the bottleneck in conversion of xylose to ethanol met by conventional fermentation method.

Electrochemical detection of miRNA-222 by use of a magnetic bead-based bioassay.

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.

Co-immobilization of glucose oxidase and xylose dehydrogenase displayed whole cell on multiwalled carbon nanotube nanocomposite films modified electrode for simultaneous voltammetric detection of D-glucose and D-xylose.

In this paper, we first report the construction of Nafion/glucose oxidase (GOD)/xylose dehydrogenase displayed bacteria (XDH-bacteria)/multiwalled carbon nanotubes (MWNTs) modified electrode for simultaneous voltammetric determination of D-glucose and D-xylose. The optimal conditions for the immobilized enzymes were established. Both enzymes retained their good stability and activities. In the mixture solution of D-glucose and D-xylose containing coenzyme NAD⁺ (the oxidized form of nicotinamide adenine dinucleotide), the Nafion/GOD/XDH-bacteria/MWNTs modified electrode exhibited quasi-reversible oxidation-reduction peak at -0.5 V (vs. saturated calomel electrode, SCE) originating from the catalytic oxidation of D-glucose, and oxidation peak at +0.55 V(vs. SCE) responding to the oxidation of NADH (the reduced form of nicotinamide adenine dinucleotide) by the carbon nanotubes, where NADH is the resultant product of coenzyme NAD⁺ involved in the catalysis of D-xylose by XDH-displayed bacteria. For the proposed biosensor, cathodic peak current at -0.5 V was linear with the concentration of D-glucose within the range of 0.25-6 mM with a low detection limit of 0.1 mM D-glucose (S/N=3), and the anodic peak current at +0.55 V was linear with the concentration of d-xylose in the range of 0.25∼4 mM with a low detection limit of 0.1 mM D-xylose (S/N=3). Further, D-xylose and D-glucose did not interfere with each other. 300-fold excess saccharides including D-maltose, D-galactose, D-mannose, D-sucrose, D-fructose, D-cellobiose, and 60-fold excess L-arabinose, and common interfering substances (100-fold excess ascorbic acid, dopamine, uric acid) as well as 300-fold excess D-xylitol did not affect the detection of D-glucose and D-xylose (both 1 mM). Therefore, the proposed biosensor is stable, specific, reproducible, simple, rapid and cost-effective, which holds great potential in real applications.

Electrochemical bioassay for the detection of TNF-α using magnetic beads and disposable screen-printed array of electrodes.

BACKGROUND: In this study we have developed an electrochemical bioassay for the analysis of TNF-α, coupling magnetic beads with disposable electrochemical platforms. TNF-α is a pro inflammatory cytokine that participates in the regulation of immune defense against various pathogens and the recovery from injury. It plays a central role in the development of many inflammatory diseases. The bioassay was based on a sandwich format using alkaline phosphatase as an enzymatic label and an eight-sensor screen-printed array as an electrochemical transducer. RESULTS: The modified magnetic beads were captured by a magnet on the surface of each graphite working electrode of the array and the electrochemical detection was thus achieved through the addition of the alkaline phosphatase substrate (1-naphthylphosphate); 1-naphthol produced during the enzymatic reaction was detected using differential pulse voltammetry. The parameters influencing the different steps of the assay were optimized in order to reach the best sensitivity and specificity. CONCLUSION: The proposed strategy offers great promise for analysis of clinical diagnostics, considering also that arrays allow the simultaneous analysis of different samples.

Electrochemical nanomaterial-based nucleic acid aptasensors.

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.

An ELIME assay for the rapid diagnosis of coeliac disease.

Coeliac disease (CD) is a gluten-induced autoimmune enteropathy found in genetically susceptible subjects. Because of the high number of undetected cases, rapid and cheaper screening methods are needed. Currently, the CD diagnosis involves the detection of anti-transglutaminase IgA antibodies (anti-tTG IgA) in blood serum through the use of ELISA systems with confirmation by histology of the intestinal mucosa. A new, rapid magneto-electrochemical immunosensor for CD diagnosis has been developed and applied to serum sample analysis. The system uses magnetic beads coated with tTG antigen to detect anti-tTG antibodies in positive serum samples and an alkaline phosphatase-conjugated anti-human IgA as label. An electrochemical readout, using magnetized screen-printed electrodes coupled with a portable instrument, is made after the addition of α-naphtyl phosphate, which is enzymatically converted into the electrochemically active α-naphthol product. The work involved the following considerations: (1) optimization of analytical parameters; (2) recovery evaluation, adding known concentrations of anti-tTG IgA to "blank" sera; (3) analysis of 107 blood serum samples; (4) calculation of the ROC curve, resulting in a cut-off of 1.0 U/ml, 100% of clinical sensitivity and 98.36% of clinical specificity; evaluation of the agreement between electrochemical and ELISA kit values (r (2) of 0.943). The system developed could be an useful tool for a correct and rapid CD diagnosis. This method is simple, cheap, rapid, and suitable for screening analyses performed outside of the classical diagnostic laboratory.

A selective and sensitive D-xylose electrochemical biosensor based on xylose dehydrogenase displayed on the surface of bacteria and multi-walled carbon nanotubes modified electrode.

A novel Nafion/bacteria-displaying xylose dehydrogenase (XDH)/multi-walled carbon nanotubes (MWNTs) composite film-modified electrode was fabricated and applied for the sensitive and selective determination of d-xylose (INS 967), where the XDH-displayed bacteria (XDH-bacteria) was prepared using a newly identified ice nucleation protein from Pseudomonas borealis DL7 as an anchoring motif. The XDH-displayed bacteria can be used directly, eliminating further enzyme-extraction and purification, thus greatly improved the stability of the enzyme. The optimal conditions for the construction of biosensor were established: homogeneous Nafion-MWNTs composite dispersion (10 µL) was cast onto the inverted glassy carbon electrode, followed by casting 10-µL of XDH-bacteria aqueous solution to stand overnight to dry, then a 5-µL of Nafion solution (0.05 wt%) is syringed to the electrode surface. The bacteria-displaying XDH could catalyze the oxidization of xylose to xylonolactone with coenzyme NAD(+) in 0.1M PBS buffer (pH7.4), where NAD(+) (nicotinamide adenine dinucleotide) is reduced to NADH (the reduced form of nicotinamide adenine dinucleotide). The resultant NADH is further electrocatalytically oxidized by MWNTs on the electrode, resulting in an obvious oxidation peak around 0.50 V (vs. Ag/AgCl). In contrast, the bacteria-XDH-only modified electrode showed oxidation peak at higher potential of 0.7 V and less sensitivity. Therefore, the electrode/MWNTs/bacteria-XDH/Nafion exhibited good analytical performance such as long-term stability, a wide dynamic range of 0.6-100 µM and a low detection limit of 0.5 µM D-xylose (S/N=3). No interference was observed in the presence of 300-fold excess of other saccharides including D-glucose, D-fructose, D-maltose, D-galactose, D-mannose, D-sucrose, and D-cellbiose as well as 60-fold excess of L-arabinose. The proposed microbial biosensor is stable, specific, sensitive, reproducible, simple, rapid and cost-effective, which holds great potential in real applications.

Nucleic acid and peptide aptamers: fundamentals and bioanalytical aspects.

In recent years new nucleic acid and protein-based combinatorial molecules have attracted the attention of researchers working in various areas of science, ranging from medicine to analytical chemistry. These molecules, called aptamers, have been proposed as alternatives to antibodies in many different applications. The aim of this Review is to illustrate the peculiarities of these combinatorial molecules which have initially been explored for their importance in molecular medicine, but have enormous potential in other biotechnological fields historically dominated by antibodies, such as bioassays. A description of these molecules is given, and the methods for their selection and production are also summarized. Moreover, critical aspects related to these molecules are discussed.

Simultaneous detection of transgenic DNA by surface plasmon resonance imaging with potential application to gene doping detection.

Surface plasmon resonance imaging (SPRi) was used as the transduction principle for the development of optical-based sensing for transgenes detection in human cell lines. The objective was to develop a multianalyte, label-free, and real-time approach for DNA sequences that are identified as markers of transgenosis events. The strategy exploits SPRi sensing to detect the transgenic event by targeting selected marker sequences, which are present on shuttle vector backbone used to carry out the transfection of human embryonic kidney (HEK) cell lines. Here, we identified DNA sequences belonging to the Cytomegalovirus promoter and the Enhanced Green Fluorescent Protein gene. System development is discussed in terms of probe efficiency and influence of secondary structures on biorecognition reaction on sensor; moreover, optimization of PCR samples pretreatment was carried out to allow hybridization on biosensor, together with an approach to increase SPRi signals by in situ mass enhancement. Real-time PCR was also employed as reference technique for marker sequences detection on human HEK cells. We can foresee that the developed system may have potential applications in the field of antidoping research focused on the so-called gene doping.

Selection of thrombin-binding aptamers by using computational approach for aptasensor application.

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, 1mM MgCl2, 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 (R²) and reproducibility (CVav %). The experimental results were in agreement with the simulation findings.

Conference report: 9th International Symposium on Drug Analysis.

The successful International Symposium series on Drug Analysis started in Brussels in 1983. So far it has been followed by a further eight editions. Presently, this meeting series is organized on a once every 4-years basis. The 9th edition was held in Antwerp (Belgium) from 21 to 24 September 2010.

Surface plasmon resonance imaging (SPRi)-based sensing: a new approach in signal sampling and management.

Surface plasmon resonance imaging (SPRi) is at the forefront of optical sensing, allowing real-time and label free simultaneous multi-analyte measurements. It represents an interesting technology for studying a broad variety of affinity interactions with impact in chemistry, both in fundamental and applied research. Signal sampling and management is a key step in SPRi measurements to achieve successful performances. This work aims to develop a strategy for selecting the sensing areas, called Regions of Interest (ROIs), to be sampled for recording SPRi signals that could results in improved sensor performances. The approach has been evaluated using antigen-antibody interaction: anti-human IgGs are immobilized on the chip surface in an array format, while the specific ligand (hIgG antigen) is in solution. This approach has general applicability and demonstrates that rational selection of sensitive areas and standard management of SPRi data has dramatic impact on sensor behaviour. The criteria of the method are: (a) creation of high density maps of ROIs, (b) evaluation of the SPRi binding signals on all the ROIs during a pre-analysis step, (c) 3D elaboration of the results, and (d) ranking of the ROIs for their final selection in further biosensor analysis. Using standard solution of antigen, three different ROIs selection approaches have been compared for their analytical performances. The proposed innovative method results to be the best one for SPRi-based sensing applications.

Aptamers biosensors for pharmaceutical compounds.

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.

A novel low-cost and easy to develop functionalization platform. Case study: aptamer-based detection of thrombin by surface plasmon resonance.

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.

Effects of formamide on the thermal stability of DNA duplexes on biochips.

In molecular biology, formamide (FA) is a commonly used denaturing agent for DNA. Although its influence on DNA duplex stability in solution is well established, little is known about immobilized DNA on microarrays. We measured thermal denaturation curves for oligonucleotides immobilized by two standard protocols: thiol self-assembling and pyrrole electrospotting. A decrease of the DNA denaturation temperature with increasing FA fraction of the solvent was observed on sequences with mutations for both surface chemistries. The average dissociation temperature decrease was found to be -0.58+/-0.05 degrees C/% FA (v/v) independently of grafting chemistry and probe sequence.

Surface plasmon resonance imaging for affinity-based biosensors.

SPR imaging (SPRi) is at the forefront of optical label-free and real-time detection. It offers the possibility of monitoring hundreds of biological interactions simultaneously and from the binding profiles, allows the estimation of the kinetic parameters of the interactions between the immobilised probes and the ligands in solution. We review the current state of development of SPRi technology and its application including commercially available SPRi instruments. Attention is also given to surface chemistries for biochip functionalisation and suitable approaches to improve sensitivity.

Affinity sensing for transgenes detection in antidoping control.

Sports authorities fear that a new form of doping called gene doping, based on the misuse of gene therapy, represents an emerging important problem and so far no methods are available for detecting it. The World Anti-Doping Agency (WADA) has included since 2003 for the first time gene doping methods in the "Prohibited List of Substances and Methods", thus detection of this new form of doping is challenging for analytical chemists. In this work, we apply affinity-based biosensors (ABBs), in particular DNA piezoelectric sensing, for detection of target DNA sequences selected as transgenosis markers. In this work, two sequences widely used in transgenosis experiments have been identified as markers: the enhanced green fluorescence protein (EGFP) gene and the promoter of Cytomegalovirus (CMV). The biosensors are characterized in their analytical performances using synthetic oligonucleotides and amplified DNA obtained from purified plasmid used as a template. Finally they have been applied to transgenic human cell cultures (human embryonic kidney HEK-EGFP), transformed with the same plasmid and carrying the target markers. This represents the closest human real matrix available for our transgenes.