CombiMatrix oligonucleotide arrays: genotyping and gene expression assays employing electrochemical detection.

Electrochemical detection has been developed and assay performances studied for the CombiMatrix oligonucleotide microarray platform that contains 12,544 individually addressable microelectrodes (features) in a semiconductor matrix. The approach is based on the detection of redox active chemistries (such as horseradish peroxidase (HRP) and the associated substrate TMB) proximal to specific microarray electrodes. First, microarray probes are hybridized to biotin-labeled targets, second, the HRP-streptavidin conjugate binds to biotin, and enzymatic oxidation of the electron donor substrate then occurs. The detection current is generated due to electro-reduction of the HRP reaction product, and it is measured with the CombiMatrix ElectraSense Reader. Performance of the ElectraSense platform has been characterized using gene expression and genotyping assays to analyze: (i) signal to concentration dependence, (ii) assay resolution, (iii) coefficients of variation, (CV) and (iv) array-to-array reproducibility and data correlation. The ElectraSense platform was also compared to the standard fluorescent detection, and good consistency was observed between these two different detection techniques. A lower detection limit of 0.75 pM was obtained for ElectraSense as compared to the detection limit of 1.5 pM obtained for fluorescent detection. Thus, the ElectraSense platform has been used to develop nucleic acid assays for highly accurate genotyping of a variety of pathogens including bio-threat agents (such as Bacillus anthracis, Yersinia pestis, and other microorganisms including Escherichia coli, Bacillus subtilis, etc.) and common pathogens of the respiratory tract (e.g. influenza A virus).

Multiplexed analyte and oligonucleotide detection on microarrays using several redox enzymes in conjunction with electrochemical detection.

We show that multiple enzyme tags may be used in an immunoassay format or for the detection of sequence-specific DNA on microarrays. The assays may be multiplexed and monitored under separate solution and voltage differences. Thus, the detection method relies on an electrochemical detection format, whereby multiple enzymes can be sensed. In our case we utilize horseradish peroxidase, laccase, and glucose dehydrogenase as enzymes attached to specific antibodies or to streptavidin.

Recent advances in microarrays.

An overview is presented for the current DNA-based microarray market, including applications for microarrays in areas such as gene expression, single-nucleotide polymorphism, strain differentiation, de novo DNA synthesis, aptamers and advances in 'in situ' synthesis technology. The development of new detection methods, simplified methodologies and broad application to molecular diagnostics are rapidly migrating microarray technologies into the arena of diagnostics and personalized medicine. Comparisons of microarray technologies from various manufacturers are presented.:

Immunoassays and sequence-specific DNA detection on a microchip using enzyme amplified electrochemical detection.

A CMOS fabricated silicon microchip was used as a platform for immunoassays and DNA synthesis and hybridization. The chip is covered with a biofriendly matrix wherein the chemistries occur. The active silicon chip has over 1000 active electrodes that can be individually addressed for both synthesis of DNA and protein attachment to a membrane on the chip surface. Additionally, the active chip can be further used for the detection of various analytes at the chip surface via digital read out resulting from the redox enzymes on the captured oligonucleotide or antibody.

Integrated microfluidic biochips for DNA microarray analysis.

A fully integrated and self-contained microfluidic biochip device has been developed to automate the fluidic handling steps required to perform a gene expression study of the human leukemia cell line (K-562). The device consists of a DNA microarray semiconductor chip with 12,000 features and a microfluidic cartridge that consists of microfluidic pumps, mixers, valves, fluid channels and reagent storage chambers. Microarray hybridization and subsequent fluidic handling and reactions (including a number of washing and labeling steps) were performed in this fully automated and miniature device before fluorescent image scanning of the microarray chip. Electrochemical micropumps were integrated in the cartridge to provide pumping of liquid solutions. A micromixing technique based on gas bubbling generated by electrochemical micropumps was developed. Low-cost check valves were implemented in the cartridge to prevent cross-talk of the stored reagents. A single-color transcriptional analysis of K-562 cells with a series of calibration controls (spiked-in controls) was performed to characterize this new platform with regard to sensitivity, specificity and dynamic range. The device detected sample RNAs with a concentration as low as 0.375 pM. Detection was quantitative over more than 3 orders of magnitude. Experiments also demonstrated that chip-to-chip variability was low, indicating that the integrated microfluidic devices eliminate manual fluidic handling steps that can be a significant source of variability in genomic analysis.

Electrochemically generated acid and its containment to 100 micron reaction areas for the production of DNA microarrays.

An addressable electrode array was used for the production of acid at sufficient concentration to allow deprotection of the dimethoxytrityl (DMT) protecting group from an overlaying substrate bound to a porous reaction layer. Containment of the generated acid to an active electrode of 100 micron diameter was achieved by the presence of an organic base. This procedure was then used for the production of a DNA array, in which synthesis was directed by the electrochemical removal of the DMT group during synthesis. The product array was found to have a detection sensitivity to as low as 0.5 pM DNA in a complex background sample.

Self-assembling protein arrays using electronic semiconductor microchips and in vitro translation.

Protein arrays will greatly accelerate research and development in medical and biological sciences. We have used cell-free protein biosynthesis and a parallel immobilization strategy for producing protein biochips. We demonstrate a model two-protein microarray using luciferase and green fluorescent protein, both expressed in a cell-free system and specifically immobilized on CombiMatrix semiconductor oligonucleotide microarrays. This demonstration provides evidence for the appropriate folding, activity, robust presentation, and efficient flexible detection of proteins on the microscale.