(a) Schematic displaying the structures of five different fIXa aptamer variants (R_A–R_E) used to create a single-stranded RNA microarray. Both R_A and R_E share a conserved AUA sequence that is boxed in Loop 1 (L1), but RE differs significantly elsewhere. The sequences R_B, R_C and R_D are modified R_A aptamers with single or double base mutations indicated using the dark circles. (b) A representative SPRI difference image obtained for the differential binding of 100 nM fIXa onto the five-component array, whose pattern is shown in (c). A prominent increase in percent reflectivity was observed at the R_A and R_E array elements: the R_A aptamer has a very strong binding affinity (2.9% increase in ΔR), while the R_E aptamer has a weaker affinity for fIXa (1.1% increase in ΔR). The R_B, R_C and R_D aptamers do not have a quantifiable binding affinity to fIXa. Adapted from ref. 63.

(a) Schematic of the enzymatically amplified detection of VEGF using an aptamer– protein–antibody sandwich assay. A three-component aptamer microarray is exposed sequentially to i) 1 pM VEGF, ii) 10 nM biotinylated VEGF antibody, and iii) 50 nM anti-biotinconjugated HRP. (b) The SPRI difference image shows that an increase in Δ%R was observed only at the R_V array elements, which is a known RNA aptamer specific to VEGF. R₂ and R_C are negative control sequences. (c) Pattern of the three-component RNA microarray. Adapted with permission from ref. 62.

Simplified scheme for the detection of biomarker binding onto an antibody microarray through (a) a fluorescently labeled secondary antibody in a sandwich assay, (b) a fluorescence label (F) directly attached to the biomarker, and (c) label-free measurements on a thin gold film using SPRI. Here, the output from a collimated white light source is passed through a polarizer (P), onto a prism/microarray assembly and collected through a filter (F) onto a CCD camera. On the bottom left is a representative fluorescence image based on a two-color analysis of directly labeled sera from cystic fibrosis patients. This image is compared to images acquired from normal patient samples: proteins that are elevated in one source compared to the other either fluoresce green or red, while proteins with similar levels in both samples appear in yellow. Adapted with permission from ref. 36. The bottom right is an SPRI difference image obtained by subtracting images acquired before and after 10 nM β₂-microglobulin binding onto a three-component antibody array composed of anti-cysC (square), anti-β₂m (circle) and anti-SEB (triangle). Adapted with permission from ref. 39.

(a) Schematic showing enzymatic amplification of biomarker detection signals via RCA. First, target proteins are labeled with biotin in serum solution and incubated on the antibody array. Primer DNA-conjugated antibodies (i.e. anti-biotin) are then bound to the surface-immobilized biotinylated protein target. Following the binding of circular DNA, a polymerase reaction significantly extends the primer sequence using the circle as a template. This enables the hybridization of multiple labeled oligonucleotides resulting in an amplified fluorescent signal. Adapted from ref. 85. (b) Fluorescence image of an antibody microarray analyzing sera from lung cancer and healthy samples using two-color RCA. Adapted from ref. 29.

Schematic of the biobarcode assay for the multiplexed detection of protein biomarkers. For each of the three protein targets, a corresponding set of Au-NP and MMP probes are prepared. The Au-NPs are conjugated to both antibodies that recognize the target protein and ‘reporter’ barcode oligonucleotides, while the MMPs are functionalized with antibodies specific to a different epitope on the target. Each MMP and Au-NP bind in a sandwich assay with the resulting complexes isolated under an applied magnetic field and the barcode strands released by a ligand exchange process. Half of each released barcode sequence is unique to a particular target while the remaining half is universal to all the barcodes. The released DNA is quantified via hybridization onto a complementary microarray element followed by hybridization of a second Au-NP probe that is modified with DNA complementary to the universal component of each barcode. A silver deposition step is then used to further amplify the light scattering detection signal. Adapted from ref. 94.