Opening the black box of traumatic brain injury: a holistic approach combining human 3D neural tissue and an in vitro traumatic brain injury induction device.

Traumatic brain injury (TBI) is caused by a wide range of physical events and can induce an even larger spectrum of short- to long-term pathophysiologies. Neuroscientists have relied on animal models to understand the relationship between mechanical damages and functional alterations of neural cells. These in vivo and animal-based in vitro models represent important approaches to mimic traumas on whole brains or organized brain structures but are not fully representative of pathologies occurring after traumas on human brain parenchyma. To overcome these limitations and to establish a more accurate and comprehensive model of human TBI, we engineered an in vitro platform to induce injuries via the controlled projection of a small drop of liquid onto a 3D neural tissue engineered from human iPS cells. With this platform, biological mechanisms involved in neural cellular injury are recorded through electrophysiology measurements, quantification of biomarkers released, and two imaging methods [confocal laser scanning microscope (CLSM) and optical projection tomography (OPT)]. The results showed drastic changes in tissue electrophysiological activities and significant releases of glial and neuronal biomarkers. Tissue imaging allowed us to reconstruct the injured area spatially in 3D after staining it with specific nuclear dyes and to determine TBI resulting in cell death. In future experiments, we seek to monitor the effects of TBI-induced injuries over a prolonged time and at a higher temporal resolution to better understand the subtleties of the biomarker release kinetics and the cell recovery phases.

A novel point-of-care diagnostic prototype system for the simultaneous electrochemiluminescent sensing of multiple traumatic brain injury biomarkers.

Traumatic brain injuries (TBI) are typically acquired when a sudden violent event causes damage to the brain tissue. A high percentage (70-85%) of all TBI patients are suffering from mild TBI (mTBI), which is often difficult to detect and diagnose with standard imaging tools (MRI, CT scan) due to the absence of significant lesions and specific symptoms. Recent studies suggest that a screening test based on the measurement of a protein biomarker panel directly from a patient's blood can facilitate mTBI diagnosis. Herein, we report a novel prototype system designed as a precursor of a future hand-held point-of-care (POC) diagnostic device for the simultaneous multi-biomarker sensing, employing a microarray-type spatially resolved electrochemiluminescence immunoassay (SR-ECLIA). The small tabletop prototype consists of a screen-printed electrode compartment to conduct multi-analyte ECL sandwich assays, a potentiostat module and a light collection module, all integrated into a compact 3D-printed housing (18.2 × 16.5 × 5.0 cm), as well as an sCMOS detector. Based on this design concept, further miniaturization, system integration, performance optimization and clinical evaluation shall pave the way towards the development of a portable instrument for use at the site of accident and healthcare. To demonstrate the system's feasibility, current performance and efficiency, the simultaneous detection of three mTBI biomarkers (GFAP, h-FABP, S100ß) in 50% serum was achieved in the upper pg mL-1 range. The proposed device is amenable to the detection of other biomarker panels and thus could open new medical diagnostic avenues for sensitive multi-analyte measurements with low-volume biological sample requirements.

Manufacturing of Peptide Microarrays Based on Catalyst-Free Click Chemistry.

Immobilization of peptides to a solid surface is frequently an important first step before they can be probed with a variety of biological samples in a heterogeneous assay format for research and clinical diagnostic purposes. Peptides can be derivatized in many ways to subsequently covalently attach them to an activated solid surface such as, for instance, epoxy-functionalized glass slides. Here, we describe a clean, efficient, and reproducible fabrication process based on catalyst-free click chemistry compatible with the construction of low- to high-density peptide microarrays.

Fluorescence-polarization immunoassays within glass fiber micro-chambers enable tobramycin quantification in whole blood for therapeutic drug monitoring at the point of care.

Many therapeutic drugs require monitoring of their concentration in blood followed by dose adjustments in order to ensure efficacy while minimizing adverse effects. It would be highly desirable to perform such measurements rapidly and with reduced sample volumes to support point-of-care testing. Here, we demonstrate that the concentration of small therapeutics can be determined in whole blood within paper-like membranes using Fluorescence Polarization Immunoassay (FPIA). Different types of paper-like materials such as glass microfibers, cellulose and filter paper were investigated for artefacts such as scattering or autofluorescence. Accurate determination of the fluorescence polarization of red-emitting fluorophores at sub-nanomolar concentrations was feasible within glass fiber membranes. This enabled the development of a competitive immunoassay for the quantification of the antibiotic tobramycin using only 1 µL of plasma in glass fiber micro-chambers. Furthermore, the same membrane was used for transversal separation of blood cells followed by accurate FPIA read-out at the bottom part of the micro-chamber. For quantification of tobramycin, 1 µL of whole blood was incubated with the immunoassay reagents during only 3 min before deposition in the micro-chamber and analysis. Within the therapeutic window, coefficients of variation were around 20% and recoveries between 80 and 105%. Owing to the simplified procedure requiring no centrifugation, the reduced blood sample volume and the rapid analysis time, we envision that this novel method supports the performance of therapeutic drug monitoring directly at the point of care.

Towards a Point-of-Care (POC) Diagnostic Platform for the Multiplex Electrochemiluminescent (ECL) Sensing of Mild Traumatic Brain Injury (mTBI) Biomarkers.

Globally, 70 million people are annually affected by TBI. A significant proportion of all TBI cases are actually mild TBI (concussion, 70-85%), which is considerably more difficult to diagnose due to the absence of apparent symptoms. Current clinical practice of diagnosing mTBI largely resides on the patients' history, clinical aspects, and CT and MRI neuroimaging observations. The latter methods are costly, time-consuming, and not amenable for decentralized or accident site measurements. As an alternative (and/or complementary), mTBI diagnostics can be performed by detection of mTBI biomarkers from patients' blood. Herein, we proposed two strategies for the detection of three mTBI-relevant biomarkers (GFAP, h-FABP, and S100ß), in standard solutions and in human serum samples by using an electrochemiluminescence (ECL) immunoassay on (i) a commercial ECL platform in 96-well plate format, and (ii) a "POC-friendly" platform with disposable screen-printed carbon electrodes (SPCE) and a portable ECL reader. We further demonstrated a proof-of-concept for integrating three individually developed mTBI assays ("singleplex") into a three-plex ("multiplex") assay on a single SPCE using a spatially resolved ECL approach. The presented methodology demonstrates feasibility and a first step towards the development of a rapid POC multiplex diagnostic system for the detection of a mTBI biomarker panel on a single SPCE.

Analytical Platforms at Swiss Universities of Applied Sciences.

Numerous projects and industrial and academic collaborations benefit from state-of-the-art facilities and expertise in analytical chemistry available at the Swiss Universities of Applied Sciences. This review summarizes areas of expertise in analytical sciences at the University of Applied Sciences and Arts Northwestern Switzerland (FHNW), the University of Applied Sciences and Arts Western Switzerland (HES-SO), and the Zurich University of Applied Sciences (ZHAW). We briefly discuss selected projects in different fields of analytical sciences.

Point-of-Care Therapeutic Drug Monitoring for Precision Dosing of Immunosuppressive Drugs.

BACKGROUND: Immunosuppressive drugs (ISD) are an essential tool in the treatment of transplant rejection and immune-mediated diseases. Therapeutic drug monitoring (TDM) for determination of ISD concentrations in biological samples is an important instrument for dose personalization for improving efficacy while reducing side effects. While currently ISD concentration measurements are performed at specialized, centralized facilities, making the process complex and laborious for the patient, various innovative technical solutions have recently been proposed for bringing TDM to the point-of-care (POC). CONTENT: In this review, we evaluate current ISD-TDM and its value, limitations, and proposed implementations. Then, we discuss the potential of POC-TDM in the era of personalized medicine, and provide an updated review on the unmet needs and available technological solutions for the development of POC-TDM devices for ISD monitoring. Finally, we provide concrete suggestions for the generation of a meaningful and more patient-centric process for ISD monitoring. SUMMARY: POC-based ISD monitoring may improve clinical care by reducing turnaround time, by enabling more frequent measurements in order to obtain meaningful pharmacokinetic data (i.e., area under the curve) faster reaction in case of problems and by increasing patient convenience and compliance. The analysis of the ISD-TDM field prompts the evolution of POC testing toward the development of fully integrated platforms able to support clinical decision-making. We identify 4 major areas requiring careful combined implementation: patient usability, data meaningfulness, clinicians' acceptance, and cost-effectiveness.

Manufacturing of Peptide Microarrays Based on Catalyst-Free Click Chemistry.

Immobilization of peptides to a solid surface is frequently an important first step before they can be probed with a variety of biological samples in a heterogeneous assay format for research and clinical diagnostic purposes. Peptides can be derivatized in many ways to subsequently covalently attach them to an activated solid surface such as epoxy-functionalized glass slides. Here, we describe a clean, efficient, and reproducible fabrication process based on catalyst-free click chemistry compatible with the construction of low- to high-density peptide microarrays.

Identification of bioaccessible and uptaken phenolic compounds from strawberry fruits in in vitro digestion/Caco-2 absorption model.

Strawberry fruits are highly valued for their taste and nutritional value. However, results describing the bioaccessibility and intestinal absorption of phenolic compounds from strawberries are still scarce. In our study, a combined in vitro digestion/Caco-2 absorption model was used to mimic physiological conditions in the gastrointestinal track and identify compounds transported across intestinal epithelium. In the course of digestion, the loss of anthocyanins was noted whilst pelargonidin-3-glucoside remained the most abundant compound, amounting to nearly 12 mg per 100 g of digested strawberries. Digestion increased the amount of ellagic acid available by nearly 50%, probably due to decomposition of ellagitannins. Only trace amounts of pelargonidin-3-glucoside were found to be absorbed in the intestine model. Dihydrocoumaric acid sulphate and p-coumaric acid were identified as metabolites formed in enterocytes and released at the serosal side of the model.

ADIBO-based "click" chemistry for diagnostic peptide micro-array fabrication: physicochemical and assay characteristics.

Several azide-derivatized and fluorescently-labeled peptides were immobilized on azadibenzocyclooctyne (ADIBO)-activated slide surfaces via a strain-promoted alkyne-azide cycloaddition (SPAAC) reaction revealing excellent immobilization kinetics, good spot homogeneities and reproducible fluorescence signal intensities. A myc-peptide micro-array immunoassay showed an antibody limit-of-detection (LOD) superior to a microtiter plate-based ELISA. Bovine serum albumin (BSA) and dextran covalently attached via "click" chemistry more efficiently reduced non-specific binding (NSB) of fluorescently-labeled IgG to the microarray surface in comparison to immobilized hexanoic acid and various types of polyethylene glycol (PEG) derivatives. Confirmation of these findings via further studies with other proteins and serum components could open up new possibilities for human sample and microarray platform-based molecular diagnostic tests.

Construction of a peptide microarray for auto-anti- body detection.

Peptide and protein microarrays provide a multiplex approach to identification and quantification of protein-protein interactions (PPI), useful to study for instance antigen-antibody properties. Multivariate serology assays detecting multiple tumor auto-antibodies (TAA) is an emerging class of blood tests for cancer detection. Here we describe the efficient coupling of peptide baits derived from the BRCA1-associated RING domain protein 1 (BARD1) to a solid surface and detection of a commercially available anti-BARD1 antibody with this newly designed peptide microarray. Analytical sensitivity and specificity were shown to be comparable to a microtiter plate based enzyme-linked immunosorbent assay (ELISA).

Coupling of a microfluidic mixer to a Fourier-transform infrared spectrometer for protein-conformation studies.

The biological properties of a protein critically depend on its conformation, which can vary as a result of changes in conditions such as pH or following the addition of various substances. Being able to reliably assess the quality of protein structures under various conditions is therefore of crucial importance. Infrared (IR) spectroscopy of the Amide I band of proteins is a powerful method for the determination of protein conformations and further allows the analysis of continuously flowing solutions of the target molecule. Here, a commercial Fourier-transform infrared spectrometer was coupled to a microfluidic mixer to allow the on-line monitoring of protein conformation under varying conditions. The validity of the concept was demonstrated by continuously recording the variations of the IR spectrum of poly-L-lysine resulting from repetitive, pH-induced conformational changes.

Fluorometric Method to Assess Lipase Inhibition Activity.

Obesity and excess weight have become serious health problems in our developed societies today. Increased blood pressure, blood glucose levels and abnormal blood lipids are frequent consequences. Inhibition of digestive enzymes by pharmacological or nutritional intervention are one avenue to be considered to treat this population. In the present study a robust assay to screen biologically active materials for their ability to inhibit pancreatic lipase, the most important enzyme in fat digestion, has been developed. Methyl-umbelliferyl butyrate was used as an artificial substrate, enabling assessment of lipase activity via specific fluorescence emission. Applicability of the assay was shown by assessment of lipase inhibition activity of wild plants from Switzerland and France. Testing showed some plants to have a high inhibition rate of about 70%. In further projects, this lipase inhibition assay could be used for a scientific proof of biological activity of raw materials with the intention to develop functional foods for weight reduction.