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In this study, we developed a microfluidic chip integrated with on-chip valves for quantitative detection of biomarkers. The sandwich-structured microfluidic chip consists of a top fluidic layer embedded with a zigzag channel, a middle tinfoil layer pre-patterned with protein stripes, and a bottom substrate layer with two waste chambers. A set of customized on-chip valves was introduced for precise control of fluid flow. To improve the production volume and uniformity among chips, we selected an injection molding method for mass-production of the main components. We standardized the process of sealing chip layers and assembling the valve holder to improve the effectiveness of cooperation with the instrument. To demonstrate the versatility of the microfluidic chip for quantitative detection of biomarkers, we used it to automatically detect C-reactive proteins (CRP), interleukin-6 (IL-6), insulin, and osteocalcin with high sensitivity and excellent reproducibility inside a customized and portable instrument. We further conducted the automated chip-based chemiluminescence immunoassay to test CRP in serum samples for demonstration of potential clinical applications, assess the activity of capture antibodies of CRP (CRP-Ab1) on the tinfoil layer for preliminary assessment of storage stability, and simultaneously detect CRP and IL-6 for confirmation of scalable multiplexation. This microfluidic chip for quantitative, automated, and portable immunoassay provides a promising choice for clinical practices.

Microfluidic platforms capable of automated, rapid, sensitive, and quantitative detection of biomarkers from patient samples could make a major impact on clinical or point-of-care (POC) diagnosis. In this work, we realize an automated diagnostic platform composed of two main components: (1) a disposable, self-contained, and integrated microfluidic chip and (2) a portable instrument that carries out completely automated operations. To demonstrate its potential for real-world application, we use injection molding for mass fabrication of the main components of disposable microfluidic chips. The assembled three-layered chip with on-chip mechanical valves for fluid control consists of (1) a top silicone fluidic layer with embedded zigzag microchannels, reagent reservoirs and a negative pressure port, (2) a middle tinfoil layer with patterned antibody/antigen stripes, and (3) a bottom silicone substrate layer with waste reservoirs. The versatility of the microfluidics-based system is demonstrated by implementation of a chemiluminescence immunoassay for quantitative detection of C-reactive protein (CRP) and testosterone in real clinical samples. This lab-on-a-chip platform with features of quantitation, portability and automation provides a promising strategy for POC diagnosis.