Fig 1.
PCR reaction with sPIN particle and multiplex qPCR.
Fig 2.
(a) Effective capture of primer on supplimer of sPIN particle. In order to see the capturing efficiency, sPIN particles were prepared with matched (red) or mismatched (black) supplimer to primer. After incubation with FAM-modified primer, both of them showed high intensity depending on the injected FAM-primer concentration (filled red and black circle). After rinsing, mismatched sPIN particle lost the fluorescence (empty black circle), meanwhile matched sPIN particle showed consistently remained fluorescence (empty red circle). It was caused by the stable binding between the primer and corresponding supplimer. (b) Positive qPCR graph of each sPIN particle using supplimers with different Tm. (c) Negative qPCR graph of each sPIN particle with supplimers of different Tm. (d) Fluorescent signal subtracted No Template Control (NTC) signal from Positive Template Control (PTC) showing regular S-shaped curves.
Fig 3.
Fluorescent snapshots of sPIN particle at each cycle of qPCR (a) and its graphs (b) according to serial dilution of template. In particular, the graphs consist of triplicate sets for each concentration. (c) Calibration curve showing the Ct values of sPIN qPCR with serial dilution. This shows a constant interval of 3.5 for the Ct value, indicating that sPIN qPCR is stable and reliable.
Fig 4.
(a) Fluorescent image sequence of multiplex qPCR with a synthetic template. Six different sPIN particles (P. f, P. m, P. o, P. v, PAN, and β-actin) were located in single channel. During qPCR, sPIN particles targeting PAN, P. v, and β-actin selectively showed fluorescent signals. Although there were air bubbles in the channel which were inevitable when the plastic chip was used, they did not interfere fluorescent signal of sPIN particles. That’s because the fluorescent signal was measured in the sPIN particles only. (b) qPCR graph analyzed with DNA sample obtained from P. v patients. Solid lines are results of multiplex assay and dotted lines are for the single-plex assay. They showed no significant difference on amplification curves and no other cross-talk. (c) The result results of eight different clinical tests. The Y-axis was set to ‘40-Ct value’ to emphasize that the high height of a column corresponds to a high concentration of the target. A value of one on the y-axis means that there was no signal at all for 50 PCR cycles.