Express test for NT-proBNP competitive detection based on lateral flow immunoassay using silanized fluorescent quantum dots

Abstract

This paper describes the step-by-step development and the application of a lateral flow immunoassay (LFIA) for the detection of the N-terminal pro-brain natriuretic peptide (NT-proBNP). NT-proBNP is widely used as a biomarker for diagnosing heart failure and other cardiac dysfunctions, distinguishing patients with heart failure from patients without heart failure, and for risk assessment. CdSe/CdS fluorescent quantum dots (QDs) were synthesized as immunoassay labels, carefully coated with a silicon dioxide shell with a high fluorescence quantum yield of 51% for silanized QDs, and finally functionalized with carboxy- or epoxy groups. The conjugation pathways of the resulting silanized QDs with antibodies specific to amino acid residues 61-76 in NT-proBNP were optimized. The LFIA uses the principle of competitive detection with the test line containing proBNP precursor molecule as the cover antigen and the control line containing rabbit anti-mouse immunoglobulins. All components of the LFIA strip, including membrane type, blocking buffer composition, and QD-antibody conjugate composition, were optimized to reduce nonspecific interaction. The LFIA test was applied to determine NT-proBNP in human plasma. The optimal dilution of plasma for application of the test was found to be 10-fold. Under optimized conditions, the LFIA cut-off level was set as 0.5 ng/mL NT-proBNP in human plasma. The LFIA design ensures that the test line fluorescence disappears at the cut-off concentration.