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George Orwell

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ELECTROCHEMICAL MICROARRAY FOR IDENTIFICATION PATHOGENS: A REVIEW

Combimatrix oligonucleotide microarray platform
Overview of electrochemical biosensors for identification of pathogenic agents
The methods of enzyme-linked immunoSorbent assay, reverse transcription-polymerase chain reaction (RT-PCR) or PCR are commonly used techniques for diagnosing of viruses or bacteria [1-6].
Despite the relatively high cost of equipment, microarray-based detection methods are highly attractive due to their high sensitivity and rapidity, because these DNA/RNA microarrays are an important tool in gene expression studies, genotyping, pharmacogenomics, pathogen classification, drug discovery, sequencing and molecular diagnostics. Application of oligonucleotide microarrays in different areas of molecular clinical diagnostic and infectious disease monitoring has been rapidly growing during the last years [7-10]. Electrochemical biosensor arrays have been described by different authors [11-13]. The impedance-based horseradish peroxidase (HRP)-labelled immunosensor Ag-PSA-based DNA sensors was developed for identification Hepatitis B (HBsAg), as a model sample, and confirmed the feasibility of applying electrochemical impedance spectroscopy to the electrode array [14]. Magnetically-assisted impedimetric with phage-modified carbon microarrays method was described for more specific detection of bacteria in milk [15]. We can highlight different methods based microarrays sensor as interdigitated array microelectrodes (IDA) amplication-based RNA/DNA microarray sensor [16-18], screen-printed electrode (SPE) amplication-based RNA/DNA microarray sensor [19,20], esterase 2 amplication-based DNA array sensor [21], Hoechst 33258 amplication-based DNA array sensor [22] and HRP amplication-based DNA microarray sensor (CombiMatrix ElectroSenseTM. In this review, we attempted to summarize the utilization of CombiMatrix for detection or/and identification pathogenic agents in biological samples.

Component of CombiMatrix ElectroSense TM CombiMatrix core technology is based on a specially modified semiconductor adapted for biological applications, which contains arrays of platinum microelectrodes. The CombiMatrix CustomArray™ and CatalogArray microarrays are currently used with fluorescent detection [23,24]. However, the underlying electronics employed for the oligonucleotide synthesis can also be utilized for electrochemical detection (ElectraSense) of target molecules bound to the microarray. CombiMatrix has developed a commercial system that is based on this approach and unique semiconductor-based microelectrode arrays. The CombiMatrix system has the ability to address each electrode individually and measure the signal present at that electrode site. The two main components are ElectraSenseTM Reader and ElectraSenseTM microarray with hybridization cap (Fig. 1). The pre-hybridization, hybridization and washing solutions are easily loaded through the portals in each chamber. Preparation of the hybridized ElectraSense™ microarray for electrochemical detection involves a post-hybridization labelling step that is performed using the CombiMatrix ElectraSense™ Detection Kit (Fig. 1A). The ElectraSense reader is able to detect electric current values for an array of 12,544 electrodes in approximately 25 s, with each electrode being “read” for less than 2 ms. The fast reading time ensures that horseradish peroxidase (HRP) retains a high level of reactivity throughout reading of the whole microarray (Fig. 1B). The range of the highest sensitivity is from 0.1 to 200 nA, and it can be configured to detect higher signals [25]. The ElectraSenseTM microarray reader electrochemically measures thousands of probes in less than one minute (Fig. 1C). The ElectraSenseTM reader electronically reads the data by performing amperometric detection of the current flux for each individual spot through the underlying platinum microelectrode. The resulting data provides direct numeric quantification of the hybridization signals for all spots. The ElectraSenseTM Software takes as input a chip design file, which gives detailed information about the placement of probes on the microarray and data from the ElectraSenseTM reader (Fig. 1D). This system provides an optimized and completely automated data extraction, background subtraction and data normalization solution. Results can be further visualized in Excel for species identification. Another major advantage of is the ability to successfully strip and re-use each array (up to 4 times). The microarray re-using procedure is performed using the CombiMatrix CustomArrayTM Stripping Kit. Component of CombiMatrix ElectroSenseTM. (A) ElectraSenseTM 12K microarray, hybridization cap and hybridization chamber. (B) Assembly of the ElectraSenseTM 12K microarray in hybridization chamber. (C) Load samples into the hybridization cap. (D) Different part of The ElectraSense™ Reader.

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