Extra short incubation microfluidic assisted – fluorescence in situ hybridization (ESIMA-FISH)

Fluorescence in situ hybridization (FISH) is a powerful technique for evaluating the HER2 gene status in breast cancer specimen (1). However, most of FISH assays currently used in clinical laboratories are expensive and require long experimental time, up to two days with an overnight incubation (2). Indeed, despite the development of faster FISH probes (HER2 IQFISH pharmDxTM from DAKO, Denmark) cutting the assay time to one day (3), the cost of these new probes is still high (more than 200$/test) and impedes the dissemination of this technique. In this study, we present the extra short incubation microfluidic assisted- fluorescence in situ hybridization (ESIMA-FISH) technique that uses microfluidics to improve FISH for HER2 assessment in breast cancer samples. ESIMA-FISH requires a very short incubation time (35 minutes) and uses 4-fold less probe solution per test. The system is based on a microfluidic chip, developed in our laboratory (4), that is clamped against a microscope slide containing a breast cancer tissue specimen (figure 1). A fluorescent DNA probe solution, specific to the target DNA, is then applied to the tissue section within a thin chamber using the microfluidic system. The probe solution used is obtained by diluting 4 times the standard HER2 IQFISH pharmDxTM probe solution (DAKO, Denmark). Oscillating flows can then be implemented using syringe pumps to improve the delivery of the probe to the tissue. Thanks to this hydrodynamic enhancement of mass transport, the probe-target hybridization efficiency is increased, resulting in overall reductions in the cost and duration of the assay. To validate the ESIMA-FISH technique, several tissue specimens were blindly tested with ESIMA-FISH and standard IQFISH. The results from these two techniques were comparable (figure 2, 3), supporting the possibility of a future clinical use of ESIMA-FISH.

Microfluidics-assisted multiplexed biomarker detection for in situ mapping of immune cells in tumor sections

Diego Gabriel Dupouy, Martinus Gijs, Daniel Migliozzi, Benjamin Pelz

Because of the close interaction between tumors and the immune system, immunotherapies are nowadays considered as the most promising treatment against cancer. In order to define the diagnosis and the subsequent therapy, crucial information about the immune cells at the tumor site is needed. Indeed, different types or activation status of cells may be indicative for specific and personalized treatments. Here, we present a quantitative method to identify ten different immuno-markers in the same tumor cut section, thereby saving precious samples and enabling correlative analysis on several cell families and their activation status in a tumor microenvironment context. We designed and fabricated a microfluidic chip with optimal thermomechanical and optical properties for fast delivery of reagents on tissue slides and for fully automatic imaging by integration with an optical microscope. The multiplexing capability of the system is enabled by an optimized cyclic immunofluorescence protocol, with which we demonstrated quantitative sequential immunostaining of up to ten biomarkers on the same tissue section. Furthermore, we developed high-quality image-processing algorithms to map each cell in the entire tissue. As proofof-concept analyses, we identified coexpression and colocalization patterns of biomarkers to classify the immune cells and their activation status. Thanks to the quantitativeness and the automation of both the experimental and analytical methods, we believe that this multiplexing approach will meet the increasing clinical need of personalized diagnostics and therapy in cancer pathology.

2019

Microfluidics-assisted fluorescence in situ hybridization for advantageous human epidermal growth factor receptor 2 assessment in breast cancer

Maryse Fiche, Martinus Gijs, Seiya Matsuoka, Huu Tuan Nguyen, Raphaël Etienne Jean Trouillon

Fluorescence in situ hybridization (FISH) is one of the recommended techniques for human epidermal growth factor receptor 2 (HER2) status assessment on cancer tissues. Here we develop microfluidics-assisted FISH (MA-FISH), in which hybridization of the FISH probes with their target DNA strands is obtained by applying square-wave oscillatory flows of diluted probe solutions in a thin microfluidic chamber of 5 mu l volume. By optimizing the experimental parameters, MA-FISH decreases the consumption of the expensive probe solution by a factor 5 with respect to the standard technique, and reduces the hybridization time to 4 h, which is four times faster than in the standard protocol. To validate the method, we blindly conducted HER2 MA-FISH on 51 formalin-fixed paraffin-embedded tissue slides of 17 breast cancer samples, and compared the results with standard HER2 FISH testing. HER2 status classification was determined according to published guidelines, based on average number of HER2 copies per cell and average HER2/CEP17 ratio. Excellent agreement was observed between the two methods, supporting the validity of MA-FISH and further promoting its short hybridization time and reduced reagent consumption.

Nature Publishing Group2017

Microelectronics Meets Biology

Emile Dupont

Microfluidics plays a key role in the design of automated platforms for realizing biological assays in a miniaturized format. Several advantages are offered by a microfluidic system, namely, low sample and reagent consumption (typically a few microliters), the possibility to mass-produce and to integrate several analysis modules enabling portable automated applications, fast analysis and low cost. Furthermore, the combination of advanced Complementary Metal-Oxide-Semiconductor (CMOS) technology and microfluidics can result in flexible and easy-to-use hybrid systems that hold large potential for application in an analytical laboratory or at the point-of-care. In this Thesis, two different monolithic silicon CMOS chips were designed, fabricated and tested, demonstrating state-of-the-art performance utilising a novel fluorescence detection principle. They allowed the detection of monoclonal antibodies in a solution or the precise counting of specifically labelled fluorescent cells in a mixed-cell sample. The proposed detection method does not require the utilization of a fluorescence camera and filter set. The first part of this Thesis presents different methods, fabrication processes and materials for microfluidic cartridges that can be integrated in a hybrid way with silicon CMOS chips. Glass and SU-8 capillaries are tested, a novel material (NOA 63) is proposed for fabricating a natively hydrophilic microfluidic cartridge, and the fabrication of custom-designed polydimethyl(-siloxane) (PDMS) cartridges for easy integration on top of a CMOS chip is introduced. The second part of this Thesis presents the combination of microfluidics with a monolithic and fully-integrated CMOS chip for the manipulation and detection of fluorescent magnetic beads, inside a PDMS microchannel that is loosely positioned on top of the chip. Magnetic manipulation is done by current actuation of microcoils on the chip; detection is achieved using single photon avalanche diodes (SPADs), located in the centre of each microcoil, that count the fluorescent photons originating from a single labeled magnetic bead. This approach permits in principle microscope-less fluorescence detection with high sensitivity. Using sandwich immunoassays on the beads' surfaces, the selective detection of the cancer biomarker 5D10 monoclonal antibody (mAb) from a non-purified hybridoma cell-culture medium is demonstrated. In the third part, a CMOS chip comprising a matrix of SPADs is designed, fabricated and tested. The recognition of single fluorescently-labelled cells from the breast cancer cell line MCF-7 in a test solution of mixed MCF-7 and non-fluorescent Jurkat cells is demonstrated, without the need of a bulky and expensive fluorescent microscope setup. Moreover, due to the use of a SPAD matrix spanning the complete width of the microchannel, the system does not require any cell focusing mechanism, needed when using a single detector. This allowed the use of a very simple, easily replicable and disposable plastic microfluidic cartridge.

EPFL2010

Microelectronics Meets Biology

Emile Dupont

EPFL2010