Detection of Dengue Fever Nonstructural Protein 1 Antigen by Proteolytic Peptide Imprinting Technology and UHPLC–MS/MS

Dengue fever is caused by mosquito-transmitted dengue virus infection and continues to increase worldwide, threatening public health in tropical and subtropical regions. The primary difficulties in preventing a reduction of the medical burden of dengue fever lies in the lack of effective mosquito control, preventive dengue vaccines, and clinically effective antiviral drugs to treat dengue infections. Rapid and accurate diagnosis is crucial for proper patient care and effective control of epidemics. The present work proposes an alternative strategy for detecting the dengue virus nonstructural protein 1 (NS1) antigen in clinical serum samples by using ultrahigh-performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) in combination with the molecularly imprinted polymers. Rather than the whole protein, the NS1 signature peptide is selected as a template for molecular imprinting and quantified as a stoichiometric readout of NS1. Three functional monomers with hydrophobic, positively charged, and negatively charged groups were synthesized by click reactions in terms of the signature peptide. These three functional monomers provide abundant recognition sites for the peptide, allowing the peptide template to be effectively imprinted during polymerization. The imprinting conditions were optimized, and the molecularly imprinted polymers were characterized and used for enriching the signature peptide from digested serum samples by solid-phase extraction and then detected by UHPLC–MS/MS. The proposed method is used to detect the dengue virus NS1 in clinical samples and holds significant promise for early confirmation of dengue virus infection.