Biomarker analysis is playing an essential role in cancer diagnosis, prognosis, and prediction. Quantitative assessment of immunohistochemical biomarker expression on tumor tissues is of clinical relevance when deciding targeted treatments for cancer patients. Here, we report a microfluidic tissue processor that permits accurate quantification of the expression of biomarkers on tissue sections, enabled by the ultra-rapid and uniform fluidic exchange of the device. An important clinical biomarker for invasive breast cancer is human epidermal growth factor receptor 2 [(HER2), also known as neu], a transmembrane tyrosine kinase that connotes adverse prognostic information for the patients concerned and serves as a target for personalized treatment using the humanized antibody trastuzumab. Unfortunately, when using state-of-the-art methods, the intensity of an immunohistochemical signal is not proportional to the extent of biomarker expression, causing ambiguous outcomes. Using our device, we performed tests on 76 invasive breast carcinoma cases expressing various levels of HER2. We eliminated more than 90% of the ambiguous results (n = 27), correctly assigning cases to the amplification status as assessed by in situ hybridization controls, whereas the concordance for HER2-negative (n = 31) and -positive (n = 18) cases was 100%. Our results demonstrate the clinical potential of microfluidics for accurate biomarker expression analysis. We anticipate our technique will be a diagnostic tool that will provide better and more reliable data, onto which future treatment regimes can be based.

Matching the scale of microfluidic flow systems with that of microelectronic chips for realizing monolithically integrated systems still needs to be accomplished. However, this is appealing only if such re-scaling does not compromise the fluidic throughput. This is related to the fact that the cost of microelectronic circuits primarily depends on the layout footprint, while the performance of many microfluidic systems, like flow cytometers, is measured by the throughput. The simple operation of inertial particle focusing makes it a promising technique for use in such integrated flow cytometer applications, however, microfluidic footprints demonstrated so far preclude monolithic integration. Here, the scaling limits of throughput-per-footprint (TPFP) in using inertial focusing are explored 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. Inertial particle focusing is demonstrated 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 integratable process, suggesting at least a 100-fold improvement compared to previously demonstrated techniques. Not only can this be an enabling technology for realizing cost-effective monolithically integrated flow cytometry devices, but the methodology represented here can also open perspectives for miniaturization of many biomedical microfluidic applications requiring monolithic integration with microelectronics without compromising the throughput.

Chromogenic immunohistochemistry (IHC) is omnipresent in cancer diagnosis, but has also been criticized for its technical limit in quantifying the level of protein expression on tissue sections, thus potentially masking clinically relevant data. Shifting from qualitative to quantitative, immunofluorescence (IF) has recently gained attention, yet the question of how precisely IF can quantify antigen expression remains unanswered, regarding in particular its technical limitations and applicability to multiple markers. Here we introduce microfluidic precision IF, which accurately quantifies the target expression level in a continuous scale based on microfluidic IF staining of standard tissue sections and low-complexity automated image analysis. We show that the level of HER2 protein expression, as continuously quantified using microfluidic precision IF in 25 breast cancer cases, including several cases with equivocal IHC result, can predict the number of HER2 gene copies as assessed by fluorescence in situ hybridization (FISH). Finally, we demonstrate that the working principle of this technology is not restricted to HER2 but can be extended to other biomarkers. We anticipate that our method has the potential of providing automated, fast and high-quality quantitative in situ biomarker data using low-cost immunofluorescence assays, as increasingly required in the era of individually tailored cancer therapy.

We introduce a new low-temperature (280 °C) parylene-to-SiO2 bonding process with high device yield (>90%) for the fabrication and integration of high-pressure-rated microfluidic chips. Pull tests demonstrate a parylene-to-SiO2 bonding strength of 10 ± 3 MPa. We apply this technique for bonding Pyrex and silicon wafers having multiple metal layers to fabricate standard packaged microfluidic devices. By performing electrochemical impedance spectroscopy of electrolyte solutions in such devices, we demonstrate that electrodes remain functional after the etching, bonding and dicing steps. We also develop a high-pressure microfluidic and electrical integration technology, eliminating special fluidic interconnections and wire-bonding steps. The burst pressure of the integrated system is statistically shown to be 7.6 ± 1.3 MPa, with a maximum achieved burst pressure of 11.1 MPa, opening perspectives for high-pressure applications of these types of microfluidic devices.

High pressure-rated channels allow microfluidic assays to be performed on a smaller footprint while keeping the throughput, thanks to the higher enabled flow rates, opening up perspectives for cost-effective integration of CMOS chips to microfluidic circuits. Accordingly, this study introduces an easy, low-cost and efficient method for realizing high pressure microfluidics-to-CMOS integration. First, we report a new low temperature (280 °C) Parylene-C wafer bonding technique, where O(2) plasma-treated Parylene-C bonds directly to Si(3)N(4) with an average bonding strength of 23 MPa. The technique works for silicon wafers with a nitride surface and uses a single layer of Parylene-C deposited only on one wafer, and allows microfluidic structures to be easily formed by directly bonding to the nitride passivation layer of the CMOS devices. Exploiting this technology, we demonstrated a microfluidic chip burst pressure as high as 16 MPa, while metal electrode structures on the silicon wafer remained functional after bonding.