Introduction

The ongoing revolution in biological sciences has generated high hopes for the advent of true personalized/preventive medicine. While the necessary biological tools are being developed at a fast pace, it has become clear that their cost, operation, and manufacturability are equally challenging issues that must be solved before the new methods can be widely accepted in medical practice. In the particular case of diagnostics, decentralized “near-patient” or “point-of-care” testing (1) has attempted to provide fast quantitative results at the bedside or in the clinic, thereby decreasing hospital stays, eliminating transportation and administrative expenses, and decreasing errors from mishandling and miscommunication. While a few single-analyte systems (1) have been developed (e.g., the now commonplace Glucometer®), the enormous potential for decentralized testing remains untapped because the vast majority of medical diagnostics is still conducted in clinical laboratories and with the use of large equipment.

A way for ubiquitous near-patient and point-of-care testing to reach fruition is for the current biological techniques to be reduced from the macroscale to the microscale, in a multi-analyte high-throughput format, preferably on handheld devices. In particular, reducing immunoassays to microfluidic scales has been extensively explored in recent years. Many approaches have been proposed, involving glass (2,3,4,5,6,7,8), TiO₂ (8), silicon (9,10), and silicone (9)(11,12,13,14,15,16,17) devices, but none possesses all of the desirable qualities: (i) capability to measure multiple antigens and samples per device, (ii) industrially feasible fabrication, (iii) parsimony of sample and reagents, (iv) adequate sensitivity and specificity, and (v) good reliability and reproducibility.

Here we report on a high-throughput multi-antigen high-specificity, high-sensitivity reproducible polydimethylsiloxane (PDMS) microfluidic system quantifying four representative blood analytes at the clinically relevant levels. An active microfluidic matrix (18) utilizes arrays of integrated micro-mechanical valves (19) to direct pressure-driven flow and multiplex analyte samples with immunoassay reagents. Enzyme-linked immuno-sorbent assay (ELISA)-like fluorescence immunostacks are formed in the microchambers at the intersections of sample and reagent channels. The fluorescence signals from these microchambers quantify the captured antigens. The 100-chamber prototype device can conduct 5 tests for each of 10 samples (with 2 replicates per sample-test combination). This test matrix could be expanded to significantly larger numbers (20).

We chose blood analytes for the initial validation of the system because blood tests represent an example of routine use of immunoassays. The current “gold standard” clinical technology is typically based on ELISA and requires 0.5–2 mL of sample per test per patient. High kit and instrumentation costs dictate centralization of measurements to large clinical or reference laboratories, resulting in transportation and batch delays of up to 14 days between the phlebotomist appointment and the final results. Such delays and the macroscale of samples and reagents increase costs in today's fast-paced, expensive healthcare environment. By contrast, the system described here uses only 100 nL of sample, while simultaneously measuring C-reactive protein (CRP), prostate-specific antigen (PSA), ferritin, and vascular endothelial growth factor (VEGF) within the clinically significant range. The system also uses only 300 nL of antibodies (as low as 0.8 ng) per assay to measure all 10 samples. Therefore, the microfluidic miniaturization of immunoassays described here may pave the way to efficient and portable handheld devices.

Materials and Methods

Reagents

Chip fabrication

Hexamethyldis-ilazane (HMDS) adhesion promoter was obtained from ShinEtsuMicroSi (Phoenix, AZ, USA); the photoresist Shipley SJR 5740 from MicroChem (Newton, MA, USA); tetramethyl-chlorosilane (TMCS) from Sigma (St. Louis, MO, USA); PDMS Sylgard 184 from Dow Corning (Midland, MI, USA); and Array It® SuperEpoxide SME slides from TeleChem International (Sunnyvale, CA, USA).

Antibodies and antigens

PSA antigen, monoclonal PSA antibody, ferritin antigen, monoclonal ferritin antibody, and monoclonal CRP antibody were procured from Fitzgerald Industries (Concord, MA, USA); VEGF antigen and antibodies and biotinylated CRP antibody from R&D Systems (Minneapolis, MN, USA); PSA biotinylated antibody from Lab Vision (Fremont, CA, USA); ferritin biotinylated antibody from U.S. Biological (Swampscott, MA, USA); and CRP antigen from EMD Biosciences (Calbiochem®; San Diego, CA, USA).