Detection of sub-microscopic blood levels of Plasmodium falciparum using Tandem Oligonucleotide Repeat Cascade Amplification (TORCA) assay with an attomolar detection limit.

Tandem Oligonucleotide Repeat Cascade Amplification (TORCA) based on signal rather than target amplification under isothermal conditions was developed for nucleic acid assays. The initial signal was generated by hybridization of single stranded DNA targets to immobilized recognition probes followed by hybrid cleavage with specific restriction endonuclease (REase), and release of trigger oligonucleotides (Tr1). The signal amplification chamber contained two bead types carrying single-stranded amplification probes and two amplification REases. The probes consisted of multiple tandem repeats of either Tr1 or another trigger Tr2, with the tandem-Tr1 anchored to the beads through the antisense Tr2 linker and vice versa. Addition of the recognition reaction solution and Tr1 hybridization to the anti-Tr1 linkers started cleavage and release of additional Tr1 and Tr2, resulting in exponential signal amplification. The cleavage cascade also released horseradish peroxidase (HRP) pre-attached to the amplification probes, and the resultant signal was measured colorimetrically. A TORCA assay was developed for detection of Plasmodium falciparum parasites in blood. It had the detection limit in the attomolar concentration range, successfully detecting sub-microscopic P. falciparum infections at less than 0.75 infected erythrocytes per microliter. Further TORCA optimization will likely produce the quantitative isothermal alternative to PCR at a fraction of its cost.

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).

Restriction Cascade Exponential Amplification (RCEA) assay with an attomolar detection limit: a novel, highly specific, isothermal alternative to qPCR.

An alternative to qPCR was developed for nucleic acid assays, involving signal rather than target amplification. The new technology, Restriction Cascade Exponential Amplification (RCEA), relies on specific cleavage of probe-target hybrids by restriction endonucleases (REase). Two mutant REases for amplification (Ramp), S17C BamHI and K249C EcoRI, were conjugated to oligonucleotides, and immobilized on a solid surface. The signal generation was based on: (i) hybridization of a target DNA to a Ramp-oligonucleotide probe conjugate, followed by (ii) specific cleavage of the probe-target hybrid using a non-immobilized recognition REase. The amount of Ramp released into solution upon cleavage was proportionate to the DNA target amount. Signal amplification was achieved through catalysis, by the free Ramp, of a restriction cascade containing additional oligonucleotide-conjugated Ramp and horseradish peroxidase (HRP). Colorimetric quantification of free HRP indicated that the RCEA achieved a detection limit of 10 aM (10(-17) M) target concentration, or approximately 200 molecules, comparable to the sensitivity of qPCR-based assays. The RCEA assay had high specificity, it was insensitive to non-specific binding, and detected target sequences in the presence of foreign DNA. RCEA is an inexpensive isothermal assay that allows coupling of the restriction cascade signal amplification with any DNA target of interest.

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.

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.