This paper reports a miniaturized immunosensor designed to determine a trace level cardiac marker, B-type
natriuretic peptide (BNP), using a microfluidic device
combined with a portable surface plasmon resonance
(SPR) sensor system. Sample BNP solution was introduced into the microchannel after an immunoreaction
with acetylcholine esterase-labeled antibody (conjugate),
and only unbound conjugate was trapped on the BNP-immobilized surface in the flow channel. Then, the thiol
compound generated by the enzymatic reaction with the
trapped conjugate was accumulated on a gold thin film
located downstream in the microchannel to monitor the
real-time SPR angle shift. We achieved a detectable
concentration range of 5 pg/mL−100 ng/mL by monitoring the SPR angle shift, which covers the required detection range for the BNP concentrations found in blood. This
success resulted from the use of a T-shaped microfluidic
device structure, which prevents the sample solution from
flowing over the gold film used for SPR detection. We were
able to measure trace levels of BNP peptide (15 fg) within
30 min since the procedure with our immunosensor is
simpler than a multistep immunoassay through the simultaneous use of a labeled enzymatic reaction and the
real-time monitoring of enzymatic product accumulation
in the microfluidic device. We employed the procedure
to detect serum BNP by using spiked samples in human
serum and achieved satisfactory recovery for heat-treated
samples to denature the esterase in the serum before the
immunoreaction.
We developed an electrochemical surface plasmon resonance flow cell for the simultaneous measurement of the binding affinity and catalytic activity of bifunctional biomolecules. These measurements will be useful for evaluating the performance of such biomolecules as ribozyme and abzyme. The simultaneous measurements were performed on a gold surface modified with a multilayer consisting of poly-l-lysine and poly(styrene sulfonate) assembled with the layer-by-layer method using an enzyme-labeled monoclonal antibody as a model compound. We obtained the amount of immunocomplex formation from the surface plasmon resonance angle shift value by injecting the compound into the flow cell containing the multilayer modified with tumor necrosis factor-alpha. Then we compared this surface plasmon resonance result with that for the in situ electrochemical oxidation of p-aminophenol formed by the catalytic reaction of labeled enzyme on the same gold film. We were able to obtain a high correlation coefficient of 0.999 between the two responses. This is because the compound could be captured with high stability with a less than 3% coulometric response decrease in the catalyzed product in the multilayer whose thickness was easily controllable. In addition, we were able to measure the catalytic activity by coulometry and thus avoid the effect of peak broadening. We also report that the dephosphorylation activity of a bound compound could be estimated from the measurement results and an equation.
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