Antibody-Conjugated Nanodiamonds as Dual-Functional Immunosensors for In Vitro Diagnostics

Nanodiamonds (NDs) are carbon nanoparticles with a large refractive index, a high density, and exceptional chemical stability. When excited by green light, they can emit bright red fluorescence from implanted nitrogen-vacancy (NV) centers. Taking advantage of these properties, we have developed antibody-conjugated NDs as in vitro diagnostic sensors for two complementary assays: particle-enhanced turbidimetric immunoassay (PETIA) and spin-enhanced lateral flow immunoassay (SELFIA). To achieve this goal, monocrystalline diamond powders (∼100 nm in diameter) with or without NV implantation were first treated in molten KNO3 to reduce their size and shape inhomogeneity, followed by surface carboxylation in strong oxidative acids and non-covalent conjugation with antibodies in water. PETIA and SELFIA were carried out separately with a microplate reader and a magnetically modulated fluorescence analyzer. Using C-reactive protein (CRP) as the target antigen, we found that anti-CRP-conjugated NDs exhibited high colloidal stability over 1 month at 4 °C in buffer solution. The limits of detection for 3 μL of CRP sample solution were 0.06 μg/mL and 1 ng/mL with variation coefficients of less than 10 and 15% for PETIA and SELFIA, respectively. These two methods together provide a detection range of 1 ng/mL–10 μg/mL, potentially useful for clinical applications. This work represents the first practical use of rounded monocrystalline NDs as in vitro diagnostic reagents.