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A sensitive and portable microfluidic electrochemical array device (μFED) was developed for the immunoassay of trace amounts of human cardiac troponin I (cTnI), which is an attractive biomarker for acute myocardial infarction (AMI). The classical "sandwich" method was adopted for the immunoassay. The capture antibody was immobilized using a self-assembled monolayer (SAM) technique, and the process was reorganized to be compatible with the bonding process. The detection antibody was labeled with alkaline phosphatase (AP) for signal amplification. The performance of the μFED was improved by eliminating the shielding effect of the microelectrode array (MEA) integrated in the μFED. The effects of the interstice and the width of the MEA on the response peak current were analyzed and simulated. The concentration gradient, about 3% of the gradient at the surface, was considered as the criterion for estimation of the optimal interstice between electrodes, and its effectiveness was proved. A stable and miniaturized reference electrode was integrated in the μFED, and its potential deviation was less than 5 mV in 15 min. These efforts resulted in the enhanced immunoassay performance of the μFED. A low limit of detection of about 5 pg/mL was obtained in serum samples, and the response current was proportional to the logarithm of concentration from 50 pg/mL to 1 μg/mL. The immunoassay process was accomplished in 15 min. The μFED was thus qualified and is a promising candidate for point-of-care immunoassay of cTnI. Graphical abstract.

To set up a point-of-care whole-blood immunoassay system, sample preparation and on-chip storage of conjugate reagents are indispensable functional units. Here, we merge these functions into a deposited microbead plug (DMBP) to simultaneously play the roles of a blood filter and a conjugate reagent carrier. The DMBP was easily fabricated by the use of natural deposition of beads without the need of weirs. Conjugate reagents (FITC labeled antibodies used here) were incorporated into the DMBP during the assembly of the DMBP. To demonstrate the ability of the DMBP, we constructed a DMBP-based microfluidic chip and used it for the detection of human IgG (hIgG). The DMBP enabled to remove blood cells from whole blood and provide the pure plasma for the downstream on-chip immunoreactions. The release of reconstituted FITC labeled antibodies from the DMBP was controlled in a passive fashion. Dry FITC labeled antibodies retained at least 81% of their activity after 60 days of storage at the room temperature. The DMBP presented here makes an important step towards the development of the self-contained, integrated, sample-to-answer microfluidic chips for point-of-care diagnostics., (Copyright © 2012 Elsevier B.V. All rights reserved.)