Sensitive and rapid assay of BNP in patient blood by micro-ELISA
Microfluidic immunoassay is expected to be a next-generation diagnostic method due to the small size and high performance. Various tests will be performed in clinics or for home medical care. However, the analytical markers are restricted in comparison to the clinical tests performed in medical facilities. We have developed a microfluidic ELISA (micro-ELISA) system which utilized a microfluidic device for reactions and a thermal lens microscope (TLM) for detection. This system allowed very sensitive and rapid assay due to the small size of the reaction space. In this paper, in order to apply this system for clinic uses, we investigated to detect unstable and low concentration (pg/mL) marker in patient plasma. Among various markers, brain natriuretic peptide (BNP), a heart failure marker, was chosen, because BNP is one of the difficult marker to handle in clinics and home due to its instability (biological half-life: ~20 minutes), its activity in patient sample declines within several hours. In order to overcome these problems, we investigated the chemical procedure. Streptavidin-beads/biotin-antibody reaction has been applied because of high association constant and, one-step pre-mixed method was developed for easier process and higher sensitivity. However, commercial standard BNP with sodium azide cannot be used in this pre-mixed method, because sodium azide would inhibit the acitivity of HRP. Therefore, preparation and preservation stability of standard sample was evaluated. To keep the stability of BNP, the buffer solution containing aprotinin used for dilution was investigated. In addition, in order to reduce the influences of the viscosity change of patient bloods, dilution procedure was also considered. Based on these investigations, quantitative analysis of BNP in only 10 L of patient plasma has been realized successfully, whereas the conventional 96-well microtiter plates method required about 500 μL. Also, superior performances were shown in the quantitation limit (3.61 pg/mL), short assay time (20 min. for 1 sample and 70 min. for 5 samples). The excellent correlation was shown between the conventional method used in The University of Tokyo Hospital and micro-ELISA method.