Extended Data Fig. 1: Constructing a DNA microarray on a modified pGOLD substrate.
a, Schematic of surface chemical modification on pGOLD substrate and construction of FEMMAN chip with N501N-probe (Supplementary Table 2). b, Detailed molecular schematic of DNA detection without amplification. c, IRDye800 fluorescence image of a typical titration curve for N501N-target-20 nt (Supplementary Table 4) detection from 2 nM to 2 pM on bare pGOLD substrate, cysteamine modified pGOLD substrate, PEG (Mw = 1,000) modified pGOLD substrate, PEG (Mw = 5,000) modified pGOLD substrate, BSA modified pGOLD substrate and glass substrate. d, Fluorescence quantification of DNA titration on the six substrates. e, Optimization of binding buffer in amplicon capturing. f, Schematic and corresponding IRDye800 fluorescence images of 20 nt DNA target (N501N-target-20 nt in Supplementary Table 4) detection on FEMMAN chip. DNA probes in Type 1 (Type 1-probe in Supplementary Table 2) and Type 2 (N501N-probe in Supplementary Table 2) had reverse direction for DNA target hybridization. The spacer containing 30 Adenosine bases (dA30) was inserted between DNA probe and thiol group in Type 3 (Type 3-probe in Supplementary Table 2) and dA10 in Type 4 (N501N-probe in Supplementary Table 2). g, Schematic and corresponding IRDye800 fluorescence images of amplicon detection on FEMMAN chip. The forward primer used for Type 5 and Type 8 was N501-RPA-PF1, for Type 6 and Type 7 was N501-RPA-PF2; The reverse primer used for Type 6 and Type 8 was N501-RPA-PR1, for Type 5 and Type 7 was N501-RPA-PR2 (primer sequence in Supplementary Table 1, target sequence in Supplementary Table 4). h, Fluorescence quantification comparison between Type1 & Type 2, and Type 3 & Type 4. i, Fluorescence quantification of Type 5, Type 6, Type 7, and Type 8. Probe concentration optimization for Asy-RT-RPA in FEMMAN assay for j, images and k, statistical results. N = 3 technical replicates, bars represent mean ± s.d.
