Enhancing the Efficiency of Conventional Surface Immunoassays Within Standard Labware Using Microscale Flows.

In this chapter, we present the design and fabrication of a device and implementation of a protocol to realize increased efficiency of immunoassays within microtiter plates. The device, WellProbe, is a 3D-structured probe that can be used to deliver precise flows at the bottom of standard well plates to establish concentric areas of shear stress intensities using hydrodynamically confined flows. The protocols involve both operation and data analysis.

Space in cancer biology: its role and implications.

Tumor cells present complex behaviors in their interactions with other cells. This intricate behavior is driving the need to develop new tools to understand these ecosystems. The surge of spatial technologies allows evaluation of the complexity of relationships between cells present in a tumor, giving insights about tumor heterogeneity and the tumor microenvironment while providing clinically relevant metrics for tumor classification. In this review, we describe key results obtained using spatial techniques, present recent advances in methods to uncover spatially relevant biological significance, and summarize their main characteristics. We expect spatial technologies to significantly broaden our understanding of tumor biology and to generate clinically relevant tools that will ultimately impact personalized medicine.

Quantifying Antibody Binding Kinetics on Fixed Cells and Tissues via Fluorescence Lifetime Imaging.

We present a method for monitoring spatially localized antigen-antibody binding events on physiologically relevant substrates (cell and tissue sections) using fluorescence lifetime imaging. Specifically, we use the difference between the fluorescence decay times of fluorescently tagged antibodies in free solution and in the bound state to track the bound fraction over time and hence deduce the binding kinetics. We make use of a microfluidic probe format to minimize the mass transport effects and localize the analysis to specific regions of interest on the biological substrates. This enables measurement of binding constants (kon) on surface-bound antigens and on cell blocks using model biomarkers. Finally, we directly measure p53 kinetics with differential biomarker expression in ovarian cancer tissue sections, observing that the degree of expression corresponds to the changes in kon, with values of 3.27-3.50 × 103 M-1 s-1 for high biomarker expression and 2.27-2.79 × 103 M-1 s-1 for low biomarker expression.

Quantification of tumor heterogeneity: from data acquisition to metric generation.

Tumors are unique and complex ecosystems, in which heterogeneous cell subpopulations with variable molecular profiles, aggressiveness, and proliferation potential coexist and interact. Understanding how heterogeneity influences tumor progression has important clinical implications for improving diagnosis, prognosis, and treatment response prediction. Several recent innovations in data acquisition methods and computational metrics have enabled the quantification of spatiotemporal heterogeneity across different scales of tumor organization. Here, we summarize the most promising efforts from a common experimental and computational perspective, discussing their advantages, shortcomings, and challenges. With personalized medicine entering a new era of unprecedented opportunities, our vision is that of future workflows integrating across modalities, scales, and dimensions to capture intricate aspects of the tumor ecosystem and to open new avenues for improved patient care.

Spatial protein heterogeneity analysis in frozen tissues to evaluate tumor heterogeneity.

A new workflow for protein-based tumor heterogeneity probing in tissues is here presented. Tumor heterogeneity is believed to be key for therapy failure and differences in prognosis in cancer patients. Comprehending tumor heterogeneity, especially at the protein level, is critical for tracking tumor evolution, and showing the presence of different phenotypical variants and their location with respect to tissue architecture. Although a variety of techniques is available for quantifying protein expression, the heterogeneity observed in the tissue is rarely addressed. The proposed method is validated in breast cancer fresh-frozen tissues derived from five patients. Protein expression is quantified on the tissue regions of interest (ROI) with a resolution of up to 100 µm in diameter. High heterogeneity values across the analyzed patients in proteins such as cytokeratin 7, ß-actin and epidermal growth factor receptor (EGFR) using a Shannon entropy analysis are observed. Additionally, ROIs are clustered according to their expression levels, showing their location in the tissue section, and highlighting that similar phenotypical variants are not always located in neighboring regions. Interestingly, a patient with a phenotype related to increased aggressiveness of the tumor presents a unique protein expression pattern. In summary, a workflow for the localized extraction and protein analysis of regions of interest from frozen tissues, enabling the evaluation of tumor heterogeneity at the protein level is presented.

Advection-Enhanced Kinetics in Microtiter Plates for Improved Surface Assay Quantitation and Multiplexing Capabilities.

Surface assays such as ELISA are pervasive in clinics and research and predominantly standardized in microtiter plates (MTP). MTPs provide many advantages but are often detrimental to surface assay efficiency due to inherent mass transport limitations. Microscale flows can overcome these and largely improve assay kinetics. However, the disruptive nature of microfluidics with existing labware and protocols has narrowed its transformative potential. We present WellProbe, a novel microfluidic concept compatible with MTPs. With it, we show that immunoassays become more sensitive at low concentrations (up to 9× signal improvement in 12× less time), richer in information with 3-4 different kinetic conditions, and can be used to estimate kinetic parameters, minimize washing steps and non-specific binding, and identify compromised results. We further multiplex single-well assays combining WellProbe's kinetic regions with tailored microarrays. Finally, we demonstrate our system in a context of immunoglobulin subclass evaluation, increasingly regarded as clinically relevant.

Open Space Diffusive Filter for Simultaneous Species Retrieval and Separation.

We present a novel method for the local retrieval of surface bound species and their rapid in-line separation using an open space microfluidic device. Separation can be performed in less than 30 s using the difference in diffusivities within parallel microfluidic flows. As a proof-of-principle, we report the rapid and efficient filtration of polystyrene beads from small molecules and surface bound red blood cells from dimethyl sulfoxide for antigen typing.

Shake It or Shrink It: Mass Transport and Kinetics in Surface Bioassays Using Agitation and Microfluidics.

Surface assays, such as ELISA and immunofluorescence, are nothing short of ubiquitous in biotechnology and medical diagnostics today. The development and optimization of these assays generally focuses on three aspects: immobilization chemistry, ligand-receptor interaction, and concentrations of ligands, buffers, and sample. A fourth aspect, the transport of the analyte to the surface, is more rarely delved into during assay design and analysis. Improving transport is generally limited to the agitation of reagents, a mode of flow generation inherently difficult to control, often resulting in inconsistent reaction kinetics. However, with assay optimization reaching theoretical limits, the role of transport becomes decisive. This perspective develops an intuitive and practical understanding of transport in conventional agitation systems and in microfluidics, the latter underpinning many new life science technologies. We give rules of thumb to guide the user on system behavior, such as advection regimes and shear stress, and derive estimates for relevant quantities that delimit assay parameters. Illustrative cases with examples of experimental results are used to clarify the role of fundamental concepts such as boundary and depletion layers, mass diffusivity, or surface tension.

Spatially multiplexed RNA in situ hybridization to reveal tumor heterogeneity.

Multiplexed RNA in situ hybridization for the analysis of gene expression patterns plays an important role in investigating development and disease. Here, we present a method for multiplexed RNA-ISH to detect spatial tumor heterogeneity in tissue sections. We made use of a microfluidic chip to deliver ISH-probes locally to regions of a few hundred micrometers over time periods of tens of minutes. This spatial multiplexing method can be combined with ISH-approaches based on signal amplification, with bright field detection and with the commonly used format of formalin-fixed paraffin-embedded tissue sections. By using this method, we analyzed the expression of HER2 with internal positive and negative controls (ActB, dapB) as well as predictive biomarker panels (ER, PgR, HER2) in a spatially multiplexed manner on single mammary carcinoma sections. We further demonstrated the applicability of the technique for subtype differentiation in breast cancer. Local analysis of HER2 revealed medium to high spatial heterogeneity of gene expression (Cohen effect size r = 0.4) in equivocally tested tumor tissues. Thereby, we exemplify the importance of using such a complementary approach for the analysis of spatial heterogeneity, in particular for equivocally tested tumor samples. As the method is compatible with a range of ISH approaches and tissue samples, it has the potential to find broad applicability in the context of molecular analysis of human diseases.

Nip the bubble in the bud: a guide to avoid gas nucleation in microfluidics.

Gas bubbles are almost a routine occurrence encountered by researchers working in the field of microfluidics. The spontaneous and unexpected nature of gas bubbles represents a major challenge for experimentalists and a stumbling block for the translation of microfluidic concepts to commercial products. This is a startling example of successful scientific results in the field overshadowing the practical hurdles of day-to-day usage. We however believe such hurdles can be overcome with a sound understanding of the underlying conditions that lead to bubble formation. In this tutorial, we focus on the two main conditions that result in bubble nucleation: surface nuclei and gas supersaturation in liquids. Key theoretical concepts such as Henry's law, Laplace pressure, the role of surface properties, nanobubbles and surfactants are presented along with a view of practical implementations that serve as preventive and curative measures. These considerations include not only microfluidic chip design and bubble traps but also often-overlooked conditions that regulate bubble formation, such as gas saturation under pressure or temperature gradients. Scenarios involving electrolysis, laser and acoustic cavitation or T-junction/co-flow geometries are also explored to provide the reader with a broader understanding on the topic. Interestingly, despite their often-disruptive nature, gas bubbles have also been cleverly utilized for certain practical applications, which we briefly review. We hope this tutorial will provide a reference guide in helping to deal with a familiar foe, the "bubble".

Quantitative microimmunohistochemistry for the grading of immunostains on tumour tissues.

Immunohistochemistry is the gold-standard method for cancer-biomarker identification and patient stratification. Yet, owing to signal saturation, its use as a quantitative assay is limited as it cannot distinguish tumours with similar biomarker-expression levels. Here, we introduce a quantitative microimmunochemistry assay that enables the acquisition of dynamic information, via a metric of the evolution of the immunohistochemistry signal during tissue staining, for the quantification of relative antigen density on tissue surfaces. We used the assay to stratify 30 patient-derived breast-cancer samples into conventional classes and to determine the proximity of each sample to the other classes. We also show that the assay enables the quantification of multiple biomarkers (human epidermal growth factor receptor, oestrogen receptor and progesterone receptor) in a standard breast-cancer panel. The integration of quantitative microimmunohistochemistry into current pathology workflows may lead to improvements in the precision of biomarker quantification.

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 ß-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.