Microscale Surface-based Bioanalytical Applications Using Microfluidic Probe Technologies

Compartmentalization is central to the study of heterogeneity in biological substrates in several areas of research in the life-sciences. For this, it is important to localize chemical reactions at the micrometer length-scale in immersed environments with conservative use of reagents and samples. Such controlled localization of chemicals on surfaces has been demonstrated in microfluidic systems wherein the small scale provides unique advantages in terms of transport and reaction kinetics resulting in lower reagent and sample consumption as well as reduced time for testing. Most current microfluidic systems are “closed”, i.e. they are sealed within four walls. This limits their interactivity, versatility and precision in use with a diverse set of biological samples such as tissue sections and adherent cells. To address this large diversity of samples, and enable interaction with standard biological substrates (e.g. microscope slide, Petri dishes, microtiter plates), the Microfluidic Probe (MFP) was developed. In this thesis, technological advancements in the instrumentation, including the design and microfabrication of new MFP heads along with numerical and analytical models of the MFP and their applications to selected problems in the bioanalytical sciences are developed. The MFP is a scanning, non-contact microfluidic technology, which enables local (bio)chemical reactions on surfaces in the “open-space” by hydrodynamically confining nanoliter volumes of liquids in a wet environment (water, buffer solutions, culture media). Within the framework of this dissertation, a compact MFP (cMFP) system that can be used on standard inverted microscopes, that assists in local processing of tissue sections and biological specimens was developed. The cMFP has a footprint of 175 × 100 × 140 mm3 and can scan an area of 45 × 45 mm2 on a surface with an accuracy of ±15 μm. The applicability of the cMFP to tissue staining, exemplified by melanoma cell blocks was shown using hematoxylin to create contours, lines, spots and gradients. Further, a new methodology was introduced for efficient and high-quality patterning of biological reagents for surface-based biological assays. The interplay between diffusion, advection, and surface chemistry is studied. As compared to standard deposition techniques, a 2- to 5-fold increase in deposition rate is demonstrated together with a 10- vi fold reduction in analyte consumption with unprecedented level of deposition homogeneity. In order to expand the applicability of the MFP to a larger set of bioassays, a new concept was developed termed Hydrodynamic Thermal Confinement (HTC), that can be generally applicable, along with its implementation, for the creation of microscale dynamic thermochemical microenvironments on biological surfaces. HTC is based on an MFP and operates under physiological conditions. The temperature can be regulated between 30° and 80° C with ±2° C precision and temperature ramps of 5° C/s over a footprint of ~50 μm × 80 μm in a volume of ~50 × 80 × 15 μm3. Finally, the concept of Tissue Lithography (TL), which enables retrospective studies on formalin-fixed paraffin-embedded tissue sections was described and implemented. TL uses a microfluidic probe to remove microscale areas of the paraffin on formalin-fixed paraffin-embedded biopsy samples. Stain patterns as small as 100 μm × 50 μm as well as multiplexed immunostaining within a single tissue microarray core are achieved with a 20-fold time reduction for local dewaxing as compared to standard protocols. It is very likely that such a bioanalytical tool and the associated analytical models that we developed in this thesis will be a facilitator towards the development of quantitative surface-based biological assays and will bring a new level of versatility in biological sample processing and budgeting

Tissue lithography: Microscale dewaxing to enable retrospective studies on formalin-fixed paraffin-embedded (FFPE) tissue sections

We present a new concept, termed tissue lithography (TL), and its implementation which enables retrospective studies on formalin-fixed paraffin-embedded tissue sections. Tissue lithography uses a microfluidic probe to remove microscale areas of the paraffin layer on formalin-fixed paraffin-embedded biopsy samples. Current practices in sample utilization for research and diagnostics require complete deparaffinization of the sample prior to molecular testing. This imposes strong limitations in terms of the number of tests as well as the time when they can be performed on a single sample. Microscale dewaxing lifts these constraints by permitting deprotection of a fraction of a tissue for testing while keeping the remaining of the sample intact for future analysis. After testing, the sample can be sent back to storage instead of being discarded, as is done in standard workflows. We achieve this microscale dewaxing by hydrodynamically confining nanoliter volumes of xylene on top of the sample with a probe head. We demonstrate micrometer-scale, chromogenic and fluorescence-based immunohistochemistry against multiple biomarkers (p53, CD45, HER2 and β-actin) on tonsil and breast tissue sections and microarrays. We achieve stain patterns as small as 100 μm × 50 μm as well as multiplexed immunostaining within a single tissue microarray core with a 20-fold time reduction for local dewaxing as compared to standard protocols. We also demonstrate a 10-fold reduction in the rehydration time, leading to lower processing times between different stains. We further show the potential of TL for retrospective studies by sequentially dewaxing and staining four individual cores within the same tissue microarray over four consecutive days. By combining tissue lithography with the concept of micro-immunohistochemistry, we implement each step of the IHC protocol-dewaxing, rehydration and staining-with the same microfluidic probe head. Tissue lithography brings a new level of versatility and flexibility in sample processing and budgeting in biobanks, which may alleviate current sample limitations for retrospective studies in biomarker discovery and drug screening

Hydrodynamic thermal confinement: creating thermo-chemical microenvironments on surfaces

We present a new, general concept termed Hydrodynamic Thermal Confinement (HTC), and its implementation for the creation of microscale dynamic thermo-chemical microenvironments on biological surfaces. HTC is based on a scanning probe and operates under physiological conditions. The temperature can be regulated between 30° and 80 °C with ±0.2 °C precision and temperature ramps of 5 °C s−1 over a footprint of ∼50 μm × 80 μm in a volume of ∼50 × 80 × 15 μm3 (∼50 pl).ISSN:1359-7345ISSN:1364-548

Optionen der Lebensgestaltung junger Ehen und Kinderwunsch (Verbundstudie) Endbericht

StBA Wiesbaden(282) / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekSIGLEDEGerman

Tissue lithography: Microscale dewaxing to enable retrospective studies on formalin-fixed paraffin-embedded (FFPE) tissue sections

<div><p>We present a new concept, termed tissue lithography (TL), and its implementation which enables retrospective studies on formalin-fixed paraffin-embedded tissue sections. Tissue lithography uses a microfluidic probe to remove microscale areas of the paraffin layer on formalin-fixed paraffin-embedded biopsy samples. Current practices in sample utilization for research and diagnostics require complete deparaffinization of the sample prior to molecular testing. This imposes strong limitations in terms of the number of tests as well as the time when they can be performed on a single sample. Microscale dewaxing lifts these constraints by permitting deprotection of a fraction of a tissue for testing while keeping the remaining of the sample intact for future analysis. After testing, the sample can be sent back to storage instead of being discarded, as is done in standard workflows. We achieve this microscale dewaxing by hydrodynamically confining nanoliter volumes of xylene on top of the sample with a probe head. We demonstrate micrometer-scale, chromogenic and fluorescence-based immunohistochemistry against multiple biomarkers (p53, CD45, HER2 and β-actin) on tonsil and breast tissue sections and microarrays. We achieve stain patterns as small as 100 μm × 50 μm as well as multiplexed immunostaining within a single tissue microarray core with a 20-fold time reduction for local dewaxing as compared to standard protocols. We also demonstrate a 10-fold reduction in the rehydration time, leading to lower processing times between different stains. We further show the potential of TL for retrospective studies by sequentially dewaxing and staining four individual cores within the same tissue microarray over four consecutive days. By combining tissue lithography with the concept of micro-immunohistochemistry, we implement each step of the IHC protocol—dewaxing, rehydration and staining—with the same microfluidic probe head. Tissue lithography brings a new level of versatility and flexibility in sample processing and budgeting in biobanks, which may alleviate current sample limitations for retrospective studies in biomarker discovery and drug screening.</p></div