We investigated the biocompatibility, specificity, and activity of a ligand-receptor-protein system covalently bound to oxidized single-walled carbon nanotubes (SWNTs) as a model proof-of-concept for employing such SWNTs as biosensors. SWNTs were functionalized under ambient conditions with either the Knob protein domain from adenovirus serotype 12 (Ad 12 Knob) or its human cellular receptor, the CAR protein, via diimide-activated amidation. We confirmed the biological activity of Knob protein immobilized on the nanotube surfaces by using its labeled conjugate antibody and evaluated the activity and specificity of bound CAR on SWNTs, first, in the presence of fluorescently labeled Knob, which interacts specifically with CAR, and second, with a negative control protein, YieF, which is not recognized by biologically active CAR proteins. In addition, current-gate voltage (I-V(g)) measurements on a dozen nanotube devices explored the effect of protein binding on the intrinsic electronic properties of the SWNTs, and also demonstrated the devices' high sensitivity in detecting protein activity. All data showed that both Knob and CAR immobilized on SWNT surfaces fully retained their biological activities, suggesting that SWNT-CAR complexes can serve as biosensors for detecting environmental adenoviruses.

Figure S1, schematic concept of the three-dimensional dark-field microscopy imaging setup; Figure S2, dynamic light scattering measurement of anti-CA125 and anti-Biotin antibody-conjugated 80 nm spherical gold plasmonic nanoparticles (PNPs); Figure S3, dynamic light scattering measurement and UV-visible absorption spectra of anti-CA125 and anti-Biotin antibody-conjugated PNPs treated with various concentrations of CA125 antigen; Figure S4, color quantization for monomer, dimer, and trimer based on the red/ green intensity ratios (R/G ratios); Figure S5, mathematical model for evaluating the systemic error of the color quantization method for bound PNPs in MUC16 binding on the surface of the cell; Figure S6, PNP-based digital cytometric assay on ovarian cancer cells (OVCAR3) with and without centrifugation; Figure S7, optimization of incubation conditions for the ratio of treated PNPs to cells in the PNP-based digital cytometric assay on ovarian cancer cells (OVCAR3); Figure S8, cell membrane mask generated by a deep convolutional neural network (U-Net) to exclude unbound PNPs nearby the cells in the enumeration of bound PNPs on the surface of cells; Figure S9, longitudinal study of bound MUC16/CA125 on the surface of EOC patient's PBMCs over a 17 month period at 1 month intervals; Figure S10, evaluation of the specific binding ability of anti-CA125 PNPs toward its targets on the patient's and healthy subject's PBMCs with various PBMC to PNP ratios; Figure S11, dark-field microscopy image montages of anti-CA125 PNPs bound to individual PBMCs in samples from five healthy donors and five serous invasive EOC patients; Figure S12, flow cytometric analysis for the evaluation of bound MUC16 on the surface of PBMCs from five healthy donors and five serous invasive ovarian cancer patients; Figure S13, scanning electron microscopy images of OVCAR3 clone treated with antibody-conjugated PNPs; and Table S1, ages and the CA125 levels in the serum of healthy donors and ovarian cancer patients (PDF)