Opportunities for high-pressure microfluidics in biomedical applications

Pressure lies at the basis of operation of most microfluidic systems, and is a determinant factor in the extent of miniaturization and in limiting the throughput and time constant of the microfluidic assays. Despite the apparent importance given to fluidic and reagent exchange control in the reported biomedical assays in microfluidic systems, high-pressure enabled systems are not studied in depth until now. Therefore, this thesis deals with realization of high-pressure microfluidics, microfabrication methods that are at the basis of high pressure-resistant devices, and their potential to address and solve biologically and clinically relevant questions. We first develop a microfabrication technology that helps realization of high pressure-resistant microfluidic devices for diverse applications. This requires a bonding technology that can be performed using multiple bonding interfaces and at low temperature to enable integration with other technologies required for realizing a complete diagnostic assay. In a first study, we introduce a new low temperature (280°C) parylene-C wafer bonding technique, where parylene-C deposited on a Pyrex wafer bonds directly to a silicon wafer with either a Si, SiO2 or Si3N4 surface, with a bonding strength up to 23 MPa, and this by using a single layer of parylene-C. Moreover, the process is compatible for bonding any type of wafer with small-sized micropatterned features, or containing microfluidic channels and electrodes. This technique presents an alternative for conventional bonding methods like anodic bonding in applications requiring a low temperature and diverse bonding interfaces. This is an important point when integrating high-pressure microfluidic devices with additional electrical and biological functionalities. Following this, we use the bonding technique to fabricate standard packaged microfluidic devices and also develop a high-pressure microfluidic and electrical integration technology, eliminating special fluidic interconnections and wire-bonding steps. Finally, we introduce an easy, low-cost and efficient method for realizing high-pressure microfluidics-to-CMOS integrated devices. Exploiting parylene-C-to-SiN bonding technology, we demonstrate a microfluidic chip burst pressure as high as 16 MPa, while metalelectrodestructures on the CMOS wafer remain functional after bonding. Using the developed technologies, we immediately show that such integration can be key to realize CMOS-integrated flow focusing devices for monolithic cytometer applications, since such integration is appealing only if such down-scaling does not compromise the fluidic throughput. By assuming throughput-per-footprint (TPFP) as the main parameter determining the performance and unit assay cost for this kind of applications, we explore the scaling limits of TPFP in inertial focusing, by studying the interplay between theory, the effect of channel Reynolds numbers up to 1500 on focusing, the entry length for the laminar flow to develop, and pressure resistance of the microchannels. We experimentally demonstrate inertial particle focusing with a TPFP up to 0.3 L/(min cm2) in high aspect-ratio rectangular microfluidic channels that are readily fabricated with a post-CMOS integrable process, suggesting at least a 100-fold improvement, when compared to demonstrated techniques. Later, we turn to applying the technique to biomedically and clinically relevant questions. In this context, two aspects were studied: 1) improving the throughput and 2) decreasing the time constants of biomedical assays to improve accuracy of such applications. In particular, our study focuses on establishing quantitative diagnostic immunohistochemistry for breast cancer diagnosis. Quantitative assessment of immunohistochemical biomarker expression is of clinical relevance when deciding targeted treatments for certain cancer patients, as is the case for human epidermal growth factor receptor 2 (HER2) expression in invasive breast carcinoma. However conventional immunohistochemistry often produces ambiguous results, which requires complementary time-consuming and expensive reflex hybridization testing. We therefore introduce a Microfluidic Tissue Processor that permits the rapid (< 5 min) quantification by immunofluorescence of the expression of biomarkers on human tissues. Using the device, we performe tests on 76 formalin-fixed paraffin-embedded invasive breast carcinomas expressing various levels of the HER2 biomaker and score the cases, combining information obtained from both pathological cell topologies and quantitative immunofluorescent signal amplitude. We show that the Microfluidic Tissue Processor eliminates more than 90% of the ambiguous results, correctly assigning cases to the amplification status as assessed by in situ hybridization, while the concordance for HER2-negative and -positive cases is 100%. As a consequence, thanks to the higher fluidic exchange rates enabled by our high-pressure microfluidic circuits, both the throughput and the accuracy in cancer diagnosis are shown to be improved by our study. Other factors contributing to the success of our approach are the engineered reagent distribution uniformity and the increased effective processing area on the tissue sections.