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.