Sensitive detection of dengue virus NS1 by highly stable affibody-functionalized gold nanoparticles

Infection with dengue virus (DENV) is a serious health issue that causes severe dengue fever and occasionally lethal complications, such as dengue hemorrhagic fever. Therefore, rapid and sensitive DENV detection is important to reduce morbidity and mortality. Here, we screened dengue NS1-specific affibodies and developed highly stable anti-NS1 affibody-functionalized AuNPs to improve the detection sensitivity of the NS1 antigen in ELISA. First, we screened NS1 antigen-specific affibody molecules (Z(Ns1)12, Z(Ns1)16, and Z(Ns1) 46) from an affibody phage Library. The affibodies were then expressed and purified from Escherichia coli. Among them, the Z(Ns1)12 affibody showed the highest equilibrium binding constant (K-d) of 1 mu.M and was sufficiently stable at high temperatures. This affibody was functionalized on AuNPs measuring 20 nm in diameter, which were used as carriers to achieve amplification of the signal in ELISA. The developed anti-NS1 affibody-functionalized AuNPs ((Z(Ns)12)(2) -AuNP) showed good properties, such as easy synthesis, a high number of affibody conjugations on the AuNPs, and excellent stability under harsh conditions with high salt concentrations and temperature. In addition, this nanoparticle-based enhanced ELISA resulted in a 14.2-fold signal amplification performance for dengue NS1 detection in comparison with the affibody-based ELISA. This novel and sensitive method using (Z(Ns)12)(2) -AuNPs may have applications in the detection of DENV in infected patients at an early stage and for the detection of other pathogens in clinical diagnostics.