Integrated Amplification Microarrays for Infectious Disease Diagnostics

Abstract

This overview describes microarray-based tests that combine solution-phase amplification chemistry and microarray hybridization within a single microfluidic chamber. The integrated biochemical approach improves microarray workflow for diagnostic applications by reducing the number of steps and minimizing the potential for sample or amplicon cross-contamination. Examples described herein illustrate a basic, integrated approach for DNA and RNA genomes, and a simple consumable architecture for incorporating wash steps while retaining an entirely closed system. It is anticipated that integrated microarray biochemistry will provide an opportunity to significantly reduce the complexity and cost of microarray consumables, equipment, and workflow, which in turn will enable a broader spectrum of users to exploit the intrinsic multiplexing power of microarrays for infectious disease diagnostics.

Keywords: RT-PCR; asymmetric PCR; diagnostics; gel element arrays; integrated microarrays; microfluidics; multiplex; reverse transcriptase.

Figure 1

Integrating amplification and hybridization chemistry within an amplification microarray and single microfluidic chamber. (A) Gene-specific reverse amplification primers (pA-R and pB-R) are labeled with a fluorophore and provided in excess relative to the forward primers (pA-F and pB-F). The microarray may contain one or more probes for each target gene (GeneA and Gene B) and resulting amplicon. Two probes are shown for Gene A and one probe is shown for Gene B. (B) The initial rounds of the amplification create both double-stranded and single-stranded amplicon, but hybridization to the microarray is kinetically limited because single stranded amplicon has not yet accumulated. (C) Towards the final rounds of amplification, single stranded amplicons abound, so hybridization to the microarray is kinetically favorable. Hybridization times can be extended beyond the amplification reaction to achieve thermodynamic equilibrium, if desired.

Figure 2

Sensitivity and specificity of a prototype influenza RT-amplification microarray. Results are the average from two technical replicates, where positive detection is defined as a signal to noise threshold ≥3.

Figure 3

(A) Closed-amplicon amplification microarray with integrated waste chamber. (B) Amplification microarray flow cells in a Quanta flat block thermocycler. (C) Akonni portable microarray analyzer used to image amplification microarrays. (D) Image of a prototype MDR-TB array following a 50-cycle closed-amplicon asymmetric PCR protocol.