On-chip DNA analysis of Tuberculosis based on magnetic nanoparticle clustering induced by rolling circle amplification products

We present on-chip implementation of an isothermal nucleic acid amplification assay with optomagnetic detection. Isothermal rolling circle amplification (RCA) was designed for the highly specific detection of a single point mutation in the sub-sequence of the katG catalase peroxidase gene responsible for resistance against the antibiotic isoniazid in Mycobacterium tuberculosis. The assay was integrated using streptavidin coated magnetic microbeads (MMBs) as a movable substrate to transport the synthetic katG target and products between the sequential processing steps. RCA products were detected via optomagnetic measurements of the binding of functionalized magnetic nanoparticles (MNPs). Binding of MNPs to RCA products lead to an increase of their hydrodynamic size and consequently a shift of the optomagnetic signal to lower frequencies. We first analyzed the affinity of DNA probes used for target capture on MMBs and binding of MNPs to RCA products, respectively. This enabled us to choose conditions where amplification products were released from the MMBs and their binding to MNPs was optimal. The optimized assay was transferred to a polymer chip with three connected chambers filled with reaction buffers for capture, RCA and detection. The chip was operated in a setup integrating temperature control, transport of the DNA on magnetic carriers and dose-dependent detection of MNP clustering. For a total assay time of about 2 h, we demonstrated specific detection of the antibiotic resistance in synthetic Tuberculosis DNA target with a limit of detection of 4 pM.