IVDT_In Vitro Diagnostics Technology

IVD Technology, November/December 2012

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TMB Biotin Label HRP Wash Wash Figure 2. Portrait detection approach. Once bpHDA amplifi cation is complete, biotin-labeled amplicons are hybridized to target-specifi c probes immobilized on a modifi ed silicon surface. After an optional wash step, anti-biotin antibody/ HRP conjugate is incubated on the chip surface. After a required wash, the sur- face is incubated with a precipitable formulation of the substrate, TMB. After TMB incubation, the surface displays a visibly detectable signal, which can be imaged using CCD or CMOS cameras. for a low-cost process, and stability of probes on the surface has been mea- sured at greater than one year with no loss in activity. Th e tests are confi gured to perform in an ELISA format using reagents Introducing micropoc... that are well-characterized commer- cially and are very stable (Figure 2). To facilitate detection, bpHDA reactions use biotin-labeled primers. Biotin- labeled DNA targets are hybridized to probes arrayed on the chip surface. To detect target sequences, the array is exposed to an anti-biotin antibody/ HRP conjugate, washed, and then incubated with a precipitable formula- tion of the substrate TMB. Enzymatic product deposits on the chip surface and is observed as a blue-gray to sky- blue color. Th e LLOD is approximately 0.3 pM in this format for the detection of DNA duplexes. Because the quantity of bpHDA amplifi ed products (10-100 nM) is orders of magnitude above the limit of detection, short assay times can be used (5 minutes hybridize, 3 minutes conjugate, 2 minutes TMB). Combined with rapid amplifi cation, this assay approach has the potential for very rapid diagnostic test results. Th is is particularly signifi cant for test-and- treat indications, where the physician diagnostic solutions www.carclodiagnosticsolutions.com T: +44 (0)208 685 0500 ivdtechnology.com IVD TECHNOLOGY | NOVEMBER/DECEMBER 2012 19

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