Ata Tuna Ciftlik

Microfluidic processor allows rapid HER2 immunohistochemistry of breast carcinomas and significantly reduces ambiguous (2+) read-outs

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.

High throughput-per-footprint inertial focusing.

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.

A low-temperature parylene-to-silicon dioxide bonding technique for high-pressure microfluidics

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.

Continuous quantification of HER2 expression by microfluidic precision immunofluorescence estimates HER2 gene amplification in breast cancer

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.

Low temperature Pyrex/silicon wafer bonding via a single intermediate parylene layer

We introduce a new low temperature (280 °C) parylene-C wafer bonding technique, where parylene-C bonds directly a Pyrex wafer to a silicon wafer with either a Si, SiO2 or Si3N4 surface with a bonding strength up to 23 MPa. The technique uses a single layer of parylene-C deposited only on the Pyrex wafer. Moreover, the process is compatible for bonding any type of wafer with small-sized micropatterned features, or containing microfluidic channels and electrodes. This technique can be an alternative for conventional bonding methods like anodic bonding in applications requiring a low temperature and diverse bonding interfaces.

A MEMS based gravimetric resonator for mass sensing applications

This paper presents the design and implementation of a resonator-type high-resolution gravimetric sensor, which allows detection in liquid media without severe degradation of the quality factor and the resolution. The proposed system eliminates the drawbacks of traditional cantilever-based detectors by lateral motion of the mechanical structure, which significantly decreases squeeze-film damping. It has been experimentally verified that the system is capable of achieving 5.91 fg/Hz mass sensitivity by measuring the mass of a single 3 µm diameter polystyrene bead bound on the proof mass surface.

Programmable parylene-C bonding layer fluorescence for storing information on microfluidic chips

We demonstrate data storage on glass/silicon microfluidic devices fabricated using parylene-C as a bonding layer. In particular, we report intermediate parylene-C bonding layer fluorescence (iPBLF) and its use as an on-chip medium for data storage by dynamic programming of iPBLF intensity, using alternating exposure of parylene-C to UV and Green light. This technique allows data on the microfluidic chip to be read, written and erased by a common fluorescent microscope. Until now, no studies have focused on storing data like expiry date, protocol or operational parameters on a chip. However, this can be useful to overcome certain automation challenges in industrial applications for which communication of information is required, like needed during operation of remote microfluidic platforms. Finally, we also demonstrate the application of iPBLF for detecting channel dimensions and positions, and for marking on-chip zones of particular interest.

A MEMS-based spiral channel dielectrophoretic chromatography system for cytometry applications

In this paper design, fabrication, and evaluation of an easy-to-use and low cost dielectrophoretic quantizer are introduced. The device works with standard tools in a biomedical laboratory: a stereo microscope with CCD camera and a voltage supply. A novel spiral microchannel geometry together with the coaxial electrode configuration is established. The device works with a droplet of sample, eliminating microfluidic connections, and external syringes. The proposed geometry decreases the footprint, therefore reduces the device cost, without compromizing the separation and quantization performances. Coaxial electrode geometry enables continuous electric-field application with simple voltage supplies. The devices are fabricated using a simple 3-mask process, and experiments are realized with 1 and 10 μm polystyrene beads. The results show that 1 μm particles have an average speed of 4.57 μm/s with 1.06 μm/s SD, and 10 μm particles have an average speed of 544 μm/s with 105 μm/s SD. The speed variation coefficient for 1 and 10 μm beads can be calculated as 23 and 19%, respectively. The size accuracy of the device is ± 10%, while the resolution is 20%, i.e., particles with radii different from each other by 20% can be separated. Hence, moderate separation performance with minimized cost and standard laboratory equipment is enabled.

A dielectrophoretic cell/particle separator fabricated by spiral channels and concentric gold electrodes

This paper presents the design and implementation of a novel dielectrophoresis (DEP) system with spiral channels and concentric electrodes for high resolution cell separation applications. The device is fabricated with a 4 mask parylene process and the design is optimized in MATLAB Simulink® to confine the operation. Tests with micro particles of different sizes are performed to show size-based separation by dielectrophoresis. Proposed device is also tested with K562 leukemia cell lines to prove that they can be separated from healthy leukocytes due to the difference in their electrical properties.

A direct injection method for blood cells into microchannels from pure blood droplets with switchable in-situ distillation of erythrocytes

This paper represents a direct injection method for leukocytes into microfluidic channels using negative dielectrophoresis. The blood sample is a heparinated pure blood droplet and injection is realized on-chip without any external microfluidic connections to the microchip. During injection erythrocytes are immobilized in-situ at injection reservoir electrodes. The method is easily applicable to most BioMEMS applications requiring direct injection of cells from pure blood and to eliminate pressure driven control.