The development of a metalloimmunoassay for the detection of NT-proBNP
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The purpose of this doctoral research is to develop a biosensor for the monitoring of heart failure (HF) in humans. Currently, there is no quantitative patient-facilitated method to monitor HF, and the physical symptoms that result are a poor representation of the acute state of the disease. The biomarker of interest is N-terminal prohormone brain natriuretic peptide (NT-proBNP) which is secreted from the cardiac muscle tissue when the heart is experiencing decompensation. The concentration of NT-proBNP has a direct correlation to the severity of HF, and it is used as the antigen in a metalloimmunoassay, where two monoclonal antibodies are used to sandwich NT-proBNP. One is conjugated to a magnetic microbead via a streptavidin-biotin interaction, and the other is conjugated to a 20 nm-diameter silver nanoparticle (AgNP) using a heterobifunctional cross-linker. The fully formed metalloimmunoassay is placed on a carbon screen-printed and Au electrodeposited sensing electrode to detect AgNP labels electrochemically. Ag charge collected from the assay is representative of the concentration of NT-proBNP in the sample. A phenomenon known as galvanic exchange (GE) is utilized in the detection of Ag. GE is a process that occurs when a zerovalent metal is immersed in a solution containing the oxidized form of a more noble metal. In this specific case, the exchange occurs between AgNP in the metalloimmunoassay and Au³⁺ generated on the sensing electrode. GE occurs because the standard reduction potential of Ag⁺ is slightly lower than Au³⁺. Significant findings of this project reveal that GE between AgNP and Au³⁺ is a process that results in only a partial exchange of AgNP with the Au³⁺ under physiologic conditions. It has also been found that, implementing two subsequent Au³⁺ electrogeneration steps improve the Ag collection efficiency and the reproducibility. Additionally, using heterobifunctional cross-linkers to covalently attach antibodies to AgNP and silver nanocubes (AgNCs) results in a lower limit of detection. These findings have led to the detection of NT-proBNP in buffer within clinically relevant ranges of 0.06-3.49 nM.