Microfluidic and electronic detection of protein biomarkers

Abstract

Proteins are essential components of our bodies and play critical roles in biological processes. Quantifying protein levels can help characterize these biological processes and detect malfunctions. Thus, protein testing is widely utilized in clinical diagnostics. The current workflow for protein testing involves the processing of patient samples that are collected at healthcare facilities. These samples are sent to centralized laboratories and analyzed using sophisticated instruments. As a result, it can take days to deliver results. However, to manage acute conditions such as sepsis, rapid results are desired to ensure timely diagnosis and treatment. In contrast, point-of-care testing has shown great promise for rapid results through the use of miniaturized and inexpensive devices in non-laboratory settings.

While there have been many advances in this field, only a small number of proteins (e.g., cardiac biomarkers) are covered by point-of-care devices, due to the profound challenge of maintaining sensitivity while miniaturizing testing system and reducing assay time. In this thesis, this technical challenge was tackled to develop a point-of-care system for the measurement of interleukin-6, which can be used as a biomarker for sepsis or cytokine release syndrome. Inspired by the success of commercial glucose meters, an all-electrical system was developed, due to easy miniaturization and low cost of electronics. First, surface chemistries were developed to coat antibodies onto electrodes and successful surface modification was validated via various techniques. Non-Faradaic impedimetric, Faradaic impedimetric and chemiresistive label-free electrical biosensors were developed and examined.

However, it was found that these biosensing platforms are susceptible to drifts due to the non-specific nature of the signal transduction. Then, building on the enzyme-linked immunosorbent assay (ELISA, the gold standard), a bead-based electronic ELISA was developed, where beads expedite the testing and electrical readout replaces the colorimetric readout in the gold standard ELISA. An integrated mathematical model was developed to comprehensively understand the assay and inform optimization. While providing comparable limit of detection with the gold standard ELISA (< 8 pg/ml), the bead-based electronic ELISA greatly reduces the assay time (40 min, as opposed to 5 hours in the conventional ELISA). A portable and multiplexed electrical readout system was then developed. In particular, a sequential multiplexing scheme was developed to incorporate multiplexing into the single-chip potentiostat.

Although simple, it was found that this multiplexing methodology may change the state of the biosensors by discharging the double layer capacitor and disrupting the mass transportation of redox species. With this regard, mathematical models were devised to analyze the sensor behavior and guide design. Finally, an integrated and automated system was obtained, by integrating the bead-based electronic ELISA on a microfluidic device and building an electronic interface to control the microfluidics. Validated with clinical patient samples, this system can provide clinically relevant limit of detection for interleukin-6 within 25 min and using less than 2.5 [mu]L sample.