Development of a Flexible Sensor-Integrated Tissue Patch to Monitor Early Organ Rejection Processes Using Impedance Spectroscopy.

Heart failure represents a primary cause of hospitalization and mortality in both developed and developing countries, often necessitating heart transplantation as the only viable recovery path. Despite advances in transplantation medicine, organ rejection remains a significant post-operative challenge, traditionally monitored through invasive endomyocardial biopsies (EMB). This study introduces a rapid prototyping approach to organ rejection monitoring via a sensor-integrated flexible patch, employing electrical impedance spectroscopy (EIS) for the non-invasive, continuous assessment of resistive and capacitive changes indicative of tissue rejection processes. Utilizing titanium-dioxide-coated electrodes for contactless impedance sensing, this method aims to mitigate the limitations associated with EMB, including procedural risks and the psychological burden on patients. The biosensor's design features, including electrode passivation and three-dimensional microelectrode protrusions, facilitate effective monitoring of cardiac rejection by aligning with the heart's curvature and responding to muscle contractions. Evaluation of sensor performance utilized SPICE simulations, scanning electron microscopy, and cyclic voltammetry, alongside experimental validation using chicken heart tissue to simulate healthy and rejected states. The study highlights the potential of EIS in reducing the need for invasive biopsy procedures and offering a promising avenue for early detection and monitoring of organ rejection, with implications for patient care and healthcare resource utilization.

Thiol-ene-based microfluidic chips for glycopeptide enrichment and online digestion of inflammation-related proteins osteopontin and immunoglobulin G.

Proteins, and more specifically glycoproteins, have been widely used as biomarkers, e.g., to monitor disease states. Bottom-up approaches based on mass spectrometry (MS) are techniques commonly utilized in glycoproteomics, involving protein digestion and glycopeptide enrichment. Here, a dual function polymeric thiol-ene-based microfluidic chip (TE microchip) was applied for the analysis of the proteins osteopontin (OPN) and immunoglobulin G (IgG), which have important roles in autoimmune diseases, in inflammatory diseases, and in coronavirus disease 2019 (COVID-19). TE microchips with larger internal surface features immobilized with trypsin were successfully utilized for OPN digestion, providing rapid and efficient digestion with a residence time of a few seconds. Furthermore, TE microchips surface-modified with ascorbic acid linker (TEA microchip) have been successfully utilized for IgG glycopeptide enrichment. To illustrate the use of the chips for more complex samples, they were applied to enrich IgG glycopeptides from human serum samples with antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The dual functional TE microchips could provide high throughput for online protein digestion and glycopeptide enrichment, showing great promise for future extended applications in proteomics and the study of related diseases.

A microfluidic microparticle-labeled impedance sensor array for enhancing immunoassay sensitivity.

An impedimetric biosensor is used to measure electrical impedance changes in the presence of biomolecules from sinusoidal input voltages. In this paper, we present a new portable impedance-based biosensor platform to improve the sensitivity of immunoassays with microparticles as a label. Using a 2 × 4 interdigitated electrode array with a 10/10 µm electrode/gap and a miniaturized impedance analyzer, we performed immunoassays with microparticles by integrating a microfluidic channel to evaluate signal enhancement. First, to understand the material dependency of microparticles on the sensor array, magnetic, silica, and polystyrene microparticles were tested. Among these microparticles, magnetic microparticles presented a high signal enhancement with relevant stability from the sensor array. With the magnetic microparticles, we demonstrate a series of immunoassays to detect human tumor necrosis factor (TNF-α) and compare the level of signal enhancement by measuring the limit of detection (LOD). With the microparticles, we achieved over ten times improvement of LOD from sandwich immunoassays. By incorporating with sample preparation and flow manipulation systems, this impedance sensor array can be utilized for digital diagnostics for a real sample-in answer-out system.

Thiol-ene microfluidic chip for fast on-chip sample clean-up, separation and ESI mass spectrometry of peptides and proteins.

Mass spectrometry (MS) is a key technology for sensitive and high-resolution mass analysis of peptides and proteins. Sample clean-up and chromatographic separation is typically performed prior to MS analysis to limit adduct formation and ionization suppression. Usually, this requires a high-pressure LC pump system equipped with expensive metal chromatographic columns placed in-line of an electrospray ionization (ESI) source. Microfluidic devices coupled to MS have gained considerable attention, due to the promise of low manufacturing costs, low sample consumption and channels with a high surface area to volume ratio and tailorable functional groups. Here, we describe a thiol-ene microfluidic chip capable of fast chromatographic sample clean-up, concentration, and separation of complex protein and peptide mixtures with direct on-chip ESI. On-chip reversed-phase chromatography (RPC) was performed through an in-situ polymerized monolith frit for retaining inexpensive commercially available reversed-phase (RP) spherical particles, while on-chip ESI is achieved through an emitter monolithically implemented by precision micro milling. The on-chip integration of both RPC and ESI emitter allowed for a minimization of dead-volumes and enables very fast sample clean-up, efficient ionization, and mass analysis of peptides and proteins from complex matrices.

A thiol-ene microfluidic device enabling continuous enzymatic digestion and electrophoretic separation as front-end to mass spectrometric peptide analysis.

One of the most attractive aspects of microfluidic chips is their capability of integrating several functional units into one single platform. In particular, enzymatic digestion and chemical separation are important steps in processing samples for many biochemical assays. This study presents the development and application of a free-flow electrophoresis microfluidic chip, and its upstream combination with an enzyme microreactor with immobilized pepsin in the same miniaturized platform. The whole microfluidic chip was fabricated by making use of thiol-ene click chemistry. As a proof of concept, different fluorescent dyes and labeled amino acids were continuously separated in the 2D electrophoretic channel. The protease pepsin was immobilized using a covalent linkage with ascorbic acid onto a high-surface monolithic support, also made of thiol-ene. To show the potential of the microfluidic chip for continuous sample preparation and analysis, an oligopeptide was enzymatically digested, and the resulting fragments were separated and collected in a single step (prior to mass spectrometric detection), without the need of further time-consuming liquid handling steps.

Thiol-Ene Based Polymers as Versatile Materials for Microfluidic Devices for Life Sciences Applications.

While there is a steady growth in the number of microfluidics applications, the search for an optimal material that delivers the diverse characteristics needed for the numerous tasks is still nowhere close to being settled. Often overlooked and still underrepresented, the thiol-ene family of polymer materials has an enormous potential for applications in organs-on-a-chip, droplet productions, microanalytics, and point of care testing. In this review, the main characteristics of the thiol-ene materials are given, and advantages and drawbacks with respect to their potential in microfluidic chip fabrication are critically assessed. Select applications, which exploit the versatility of the thiol-ene polymers, are presented and discussed. It is concluded that, in particular, the rapid prototyping possibility combined with the material's resulting mechanical strength, solvent resistance, and biocompatibility, as well as the inherently easy surface functionalization, are strong factors to make thiol-ene polymers strong contenders for promising future materials for many biological, clinical, and technical lab-on-a-chip applications.

Monitoring transient cell-to-cell interactions in a multi-layered and multi-functional allergy-on-a-chip system.

We have developed a highly integrated lab-on-a-chip containing embedded electrical microsensors, µdegassers and pneumatically-actuated micropumps to monitor allergic hypersensitivity. Rapid antigen-mediated histamine release (e.g. s to min) and resulting muscle contraction (<30 min) is detected by connecting an immune compartment containing sensitized basophile cells to a vascular co-culture model.

Oxygen Management at the Microscale: A Functional Biochip Material with Long-Lasting and Tunable Oxygen Scavenging Properties for Cell Culture Applications.

Oxygen plays a pivotal role in cellular homeostasis, and its partial pressure determines cellular function and fate. Consequently, the ability to control oxygen tension is a critical parameter for recreating physiologically relevant in vitro culture conditions for mammalian cells and microorganisms. Despite its importance, most microdevices and organ-on-a-chip systems to date overlook oxygen gradient parameters because controlling oxygen often requires bulky and expensive external instrumental setups. To overcome this limitation, we have adapted an off-stoichiometric thiol-ene-epoxy polymer to efficiently remove dissolved oxygen to below 1 hPa and also integrated this modified polymer into a functional biochip material. The relevance of using an oxygen scavenging material in microfluidics is that it makes it feasible to readily control oxygen depletion rates inside the biochip by simply changing the surface-to-volume aspect ratio of the microfluidic channel network as well as by changing the temperature and curing times during the fabrication process.

Chloroform compatible, thiol-ene based replica molded micro chemical devices as an alternative to glass microfluidic chips.

Polymeric microfluidic chips offer a number of benefits compared to their glass equivalents, including lower material costs and ease and flexibility of fabrication. However, the main drawback of polymeric materials is often their limited resistance to (organic) solvents. Previously, thiol-ene materials were shown to be more solvent resistant than most other commonly used polymers; however, they still fall short in "harsh" chemical environments, such as when chlorinated solvents are present. Here, we show that a simple yet effective treatment of thiol-ene materials results in exceptional solvent compatibility, even for very challenging chemical environments. Our approach, based on a temperature treatment, results in a 50-fold increase in the chloroform compatibility of thiol-enes (in terms of longevity). We show that prolonged heat exposure allows for the operation of the microfluidic chips in chloroform for several days with no discernable deformation or solvent-induced swelling. The method is applicable to many different thiol-ene-based materials, including commercially available formulations, and also when using other commonly considered "harsh" solvents. To demonstrate the utility of the solvent compatible thiol-enes for applications where chloroform is frequently employed, we show the continuous and uniform production of polymeric microspheres for drug delivery purposes over a period of 8 hours. The material thus holds great promise as an alternative choice for microfluidic applications requiring harsh chemical environments, a domain so far mainly restricted to glass chips.

Thiol-ene Microfluidic Chip for Performing Hydrogen/Deuterium Exchange of Proteins at Subsecond Time Scales.

Hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) has become a routine approach for sensitive analysis of the dynamic structure and interactions of proteins. However, transient conformational changes and weak affinity interactions found in many biological systems typically only perturb fast-exchanging amides in proteins. Detection of HDX changes for such amides require shorter deuterium labeling times (subsecond) than can be performed reproducibly by manual sample handling. Here, we describe the development and validation of a microfluidic chip capable of rapid on-chip protein labeling and reaction quenching. The fastHDX thiol-ene microchip is fabricated entirely using thiol-ene photochemistry. The chip has a three-channel design for introduction of protein sample, deuterated buffer, and quench buffer. Thiol-ene based monolith plugs (i.e., polymerized thiol-ene emulsions) situated within microchannels are generated in situ using a 3D-printed photolithography mask. We show that efficient on-chip mixing can be achieved at channel junctions by spatially confined in-channel monolith mixers. Using human hemoglobin (Hb), we demonstrate the ability of the chip to perform highly reproducible HDX in the 0.14-1.1 s time frame. The HDX of Hb at 0.14-1.1 s, resolved to peptide segments, correlates closely with structural features of the crystal structure of the Hb tetramer, with helices exhibiting no or minor HDX and loops undergoing pronounced HDX even at subsecond time scales. On-chip HDX of Hb at time points ranging from 0.14-1.1 s demonstrates the ability to distinguish fast exchanging amides and thus provides enhanced detection of transient structure and interactions in dynamic or exposed regions of proteins in solution.

Nanoliter-Scale Electromembrane Extraction and Enrichment in a Microfluidic Chip.

This paper reports for the first time nanoliter-scale electromembrane extraction (nanoliter-scale EME) in a microfluidic device. Six basic drug substances (model analytes) were extracted from 70 µL samples of human whole blood, plasma, or urine through a supported liquid membrane (SLM) of 2-nitrophenyl octyl ether (NPOE) and into 6 nL of 10 mM formic acid as an acceptor solution. A DC potential of 15 V was applied across the SLM and served as the driving force for the extraction. The cathode was located in the acceptor solution. Because of the small area of the SLM (0.06 mm2), the system provided soft extraction with recoveries <1% for the 70 µL samples. Because of the large sample-to-acceptor-volume ratio, analytes were enriched in the acceptor solution. The enrichment capacity was 6-7-fold per minute, and after 60 min of operation, most of the model analytes were enriched by a factor of approximately 400. Because of the SLM and the direction of the applied electrical field, substantial sample cleanup was obtained. The chips were based on thiol-ene polymers, and the soft-lithography-fabrication procedure and the materials were selected in such a way that future mass production should be feasible. The chip-to-chip variability was within 23% RSD (and less than 10% in most cases) with respect to extraction recovery. Our findings have verified that nanoliter-scale EME is highly feasible and provides reliable data, and for future studies, the concept should be tested for applicability in connection with in vitro microphysiological systems, organ-on-a-chip systems, and point-of-care diagnostics. These are potential areas where the combination of soft extraction and high enrichment from limited sample volumes is required for reliable analytical measurements.

Microfluidic Migration and Wound Healing Assay Based on Mechanically Induced Injuries of Defined and Highly Reproducible Areas.

All cell migration and wound healing assays are based on the inherent ability of adherent cells to move into adjacent cell-free areas, thus providing information on cell culture viability, cellular mechanisms and multicellular movements. Despite their widespread use for toxicological screening, biomedical research and pharmaceutical studies, to date no satisfactory technological solutions are available for the automated, miniaturized and integrated induction of defined wound areas. To bridge this technological gap, we have developed a lab-on-a-chip capable of mechanically inducing circular cell-free areas within confluent cell layers. The microdevices were fabricated using off-stoichiometric thiol-ene-epoxy (OSTEMER) polymer resulting in hard-polymer devices that are robust, cost-effective and disposable. We show that the pneumatically controlled membrane deflection/compression method not only generates highly reproducible (RSD 4%) injuries but also allows for repeated wounding in microfluidic environments. Performance analysis demonstrated that applied surface coating remains intact even after multiple wounding, while cell debris is simultaneously removed using laminar flow conditions. Furthermore, only a few injured cells were found along the edge of the circular cell-free areas, thus allowing reliable and reproducible cell migration of a wide range of surface sensitive anchorage dependent cell types. Practical application is demonstrated by investigating healing progression and endothelial cell migration in the absence and presence of an inflammatory cytokine (TNF-α) and a well-known cell proliferation inhibitor (mitomycin-C).

Multi-layered, membrane-integrated microfluidics based on replica molding of a thiol-ene epoxy thermoset for organ-on-a-chip applications.

In this study we have investigated a photosensitive thermoset (OSTEMER 322-40) as a complementary material to readily fabricate complex multi-layered microdevices for applications in life science. Simple, versatile and robust fabrication of multifunctional microfluidics is becoming increasingly important for the development of customized tissue-, organ- and body-on-a-chip systems capable of mimicking tissue interfaces and biological barriers. In the present work key material properties including optical properties, vapor permeability, hydrophilicity and biocompatibility are evaluated for cell-based assays using fibroblasts, endothelial cells and mesenchymal stem cells. The excellent bonding strength of the OSTEMER thermoset to flexible fluoropolymer (FEP) sheets and poly(dimethylsiloxane) (PDMS) membranes further allows for the fabrication of integrated microfluidic components such as membrane-based microdegassers, microvalves and micropumps. We demonstrate the application of multi-layered, membrane-integrated microdevices that consist of up to seven layers and three membranes that specially confine and separate vascular cells from the epithelial barrier and 3D tissue structures.

Monitoring cellular stress responses using integrated high-frequency impedance spectroscopy and time-resolved ELISA.

We have developed a lab-on-a-chip system for continuous and non-invasive monitoring of microfluidic cell cultures using integrated high-frequency contactless impedance spectroscopy. Electrically insulated microfabricated interdigitated electrode structures were embedded into four individually addressable microchambers to reliably and reproducibly detect cell-substrate interactions, cell viability and metabolic activity. While silicon nitride passivated sensor substrates provided a homogeneous cell culture surface that minimized cell orientation along interdigitated electrode structures, the application of high-frequency AC fields reduced the impact of the 300 nm thick passivation layer on sensor sensitivity. The additional implementation of multivariate data analysis methods such as partial least square (PLS) for high-frequency impedance spectra provided unambiguous information on intracellular pathway activation, up and down-regulation of protein synthesis as well as global cellular stress responses. A comparative cell analysis using connective tissue fibroblasts showed that high-frequency contactless impedance spectroscopy and time-resolved quantification of IL-6 secretion using ELISA provided similar results following stimulation with circulating pro-inflammatory cytokines IL-1ß and TNFα. The combination of microfluidics with contactless impedance sensing and time-resolved quantification of stress factor release will provide biologist with a new tool to (a) establish a variety of uniform cell culture surfaces that feature complex biochemistries, micro- and nanopatterns; and (b) to simultaneously characterize cell responses under physiologically relevant conditions using a complementary non-invasive cell analysis method.

Lab-on-a-chip technologies for stem cell analysis.

The combination of microfabrication-based technologies with cell biology has laid the foundation for the development of advanced in vitro diagnostic systems capable of analyzing cell cultures under physiologically relevant conditions. In the present review, we address recent lab-on-a-chip developments for stem cell analysis. We highlight in particular the tangible advantages of microfluidic devices to overcome most of the challenges associated with stem cell identification, expansion and differentiation, with the greatest advantage being that lab-on-a-chip technology allows for the precise regulation of culturing conditions, while simultaneously monitoring relevant parameters using embedded sensory systems. State-of-the-art lab-on-a-chip platforms for in vitro assessment of stem cell cultures are presented and their potential future applications discussed.

Exploitation of S-layer anisotropy: pH-dependent nanolayer orientation for cellular micropatterning.

We have developed a tunable, facile, and reliable cell patterning method using a self-assembled crystalline protein monolayer that, depending on its orientation, can exhibit either cell adhesive (cytophilic) or cell repulsive (cytophobic) surface properties. Our technique exploits, for the first time, the inherent biological anisotropy of the bacterial cell wall protein SbpA capable of interacting with its cytophilic inner side with components of the cell wall, while its outer cytophobic side interacts with the environment. By simply altering the recrystallization protocol from a basic to an acidic condition, the SbpA-protein layer orientation and function can be switched from preventing unspecific protein adsorption and cell adhesion to effectively promote cell attachment, spreading, and proliferation. As a result, the same protein solution can be used to form cell adhesive and repulsive regions over large areas on a single substrate using a simple pH-dependent self-assembly procedure. The reliable establishment of cytophobic and cytophilic SbpA layers allows the generation of well-defined surface patterns that exhibit uniform height (9-10 nm), p4 lattice symmetry with center-to-center spacing of the morphological units of 12 nm, as well as similar surface potential and charge distributions under cell culture conditions. The pH-dependent "orientation switch" of the SbpA protein nanolayer was integrated with micromolding in capillaries (MIMIC) technology to demonstrate its application for cell patterning using a variety of cell lines including epithelial, fibroblast and endothelial cells.