Reagent storage and delivery on integrated microfluidic chips for point-of-care diagnostics.

Microfluidic-based point-of-care diagnostics offer several unique advantages over existing bioanalytical solutions, such as automation, miniaturisation, and integration of sensors to rapidly detect on-site specific biomarkers. It is important to highlight that a microfluidic POC system needs to perform a number of steps, including sample preparation, nucleic acid extraction, amplification, and detection. Each of these stages involves mixing and elution to go from sample to result. To address these complex sample preparation procedures, a vast number of different approaches have been developed to solve the problem of reagent storage and delivery. However, to date, no universal method has been proposed that can be applied as a working solution for all cases. Herein, both current self-contained (stored within the chip) and off-chip (stored in a separate device and brought together at the point of use) are reviewed, and their merits and limitations are discussed. This review focuses on reagent storage devices that could be integrated with microfluidic devices, discussing further issues or merits of these storage solutions in two different sections: direct on-chip storage and external storage with their application devices. Furthermore, the different microvalves and micropumps are considered to provide guidelines for designing appropriate integrated microfluidic point-of-care devices.

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.

Sensor-integrated brain-on-a-chip platforms: Improving the predictive validity in neurodegenerative research.

Affecting millions of individuals worldwide, neurodegenerative diseases (NDDs) pose a significant and growing health concern in people over the age of 60 years. Contributing to this trend are the steady increase in the aging population coupled with a persistent lack of disease-altering treatment strategies targeting NDDs. The absence of efficient therapeutics can be attributed to high failure rates in clinical trials and the ineptness of animal models in preceding preclinical studies. To that end, in recent years, significant research effort has been dedicated to the development of human cell-based preclinical disease models characterized by a higher degree of predictive validity. However, a key requirement of any in vitro model constitutes the precise knowledge and replication of the target tissues' (patho-)physiological microenvironment. Herein, microphysiological systems have demonstrated superiority over conventional static 2D/3D in vitro cell culture systems, as they allow for the emulation and continuous monitoring of the onset, progression, and remission of disease-associated phenotypes. This review provides an overview of recent advances in the field of NDD research using organ-on-a-chip platforms. Specific focus is directed toward non-invasive sensing strategies encompassing electrical, electrochemical, and optical sensors. Additionally, promising on- and integrable off-chip sensing strategies targeting key analytes in NDDs will be presented and discussed in detail.

A multi-channel microfluidic platform based on human flavin-containing monooxygenase 3 for personalised medicine.

Human flavin-containing monooxygenase 3 (FMO3) is a drug-metabolizing enzyme (DME) which is known to be highly polymorphic. Some of its polymorphic variants are associated with inter-individual differences that contribute to drug response. In order to measure these differences, the implementation of a quick and efficient in vitro assay is highly desirable. To this end, in this work a microfluidic immobilized enzyme reactor (µ-IMER) was developed with four separate serpentines where FMO3 and its two common polymorphic variants (V257M and E158K) were covalently immobilized via glutaraldehyde cross-linking in the presence of a polylysine coating. Computational fluid dynamics simulations were performed to calculate the selected substrate retention time in serpentines with different surface areas at various flow rates. The oxidation of tamoxifen, an anti-breast cancer drug, was used as a model reaction to characterize the new device in terms of available surface area for immobilization, channel coating, and applied flow rate. The highest amount of product was obtained when applying a 10 µL min-1 flow rate on polylysine-coated serpentines with a surface area of 90 mm2 each. Moreover, these conditions were used to test the device as a multi-enzymatic platform by simultaneously assessing the conversion of tamoxifen by FMO3 and its two polymorphic variants immobilized on different serpentines of the same chip. The results obtained demonstrate that the differences observed in the conversion of tamoxifen within the chip are similar to those already published (E158K > WT > V257M). Therefore, this microfluidic platform provides a feasible option for fabricating devices for personalised medicine.

Direct electrochemical reduction of graphene oxide thin film for aptamer-based selective and highly sensitive detection of matrix metalloproteinase 2.

Simple and low-cost biosensing solutions are suitable for point-of-care applications aiming to overcome the gap between scientific concepts and technological production. To compete with sensitivity and selectivity of golden standards, such as liquid chromatography, the functionalization of biosensors is continuously optimized to enhance the signal and improve their performance, often leading to complex chemical assay development. In this research, the efforts are made on optimizing the methodology for electrochemical reduction of graphene oxide to produce thin film-modified gold electrodes. Under the employed specific conditions, 20 cycles of cyclic voltammetry (CV) are shown to be optimal for superior electrical activation of graphene oxide into electrochemically reduced graphene oxide (ERGO). This platform is further used to develop a matrix metalloproteinase 2 (MMP-2) biosensor, where specific anti-MMP2 aptamers are utilized as a biorecognition element. MMP-2 is a protein which is typically overexpressed in tumor tissues, with important roles in tumor invasion, metastasis as well as in tumor angiogenesis. Based on impedimetric measurements, we were able to detect as low as 3.32 pg mL-1 of MMP-2 in PBS with a dynamic range of 10 pg mL-1 - 10 ng mL-1. Further experiments with real blood samples revealed a promising potential of the developed sensor for direct measurement of MMP-2 in complex media. High specificity of detection is demonstrated - even to the closely related enzyme MMP-9. Finally, the potential of reuse was demonstrated by signal restoration after experimental detection of MMP-2.

Archaeal ether lipids improve internalization and transfection with mRNA lipid nanoparticles.

Neutral and positively charged archaeal ether lipids (AEL) have been studied for their utilization as novel delivery systems for pDNA, showing efficient immune response with a strong memory effect while lacking noticeable toxicity. Recent technological advances placed mRNA lipid nanoparticles (LNPs) at the forefront of next-generation delivery systems; however, no study has examined AELs in mRNA delivery yet. In this study, we investigated either a crude lipid extract or the purified tetraether lipid caldarchaeol from Sulfolobus acidocaldarius as potential novel excipients for mRNA LNPs. Depending on their molar share in the respective LNP, particle uptake, and mRNA expression levels could be increased by up to 10-fold in in vitro transfection experiments using both primary cell sources (HSMM) and established cell lines (Caco-2, C2C12) compared to a well-known reference formulation. This increased efficiency might be linked to a substantial effect on endosomal escape, indicating fusogenic and lyotropic features of AELs. This study shows the high value of archaeal ether lipids for mRNA delivery and provides a solid foundation for future in vivo experiments and further research.

Development of a Paper-based Hematocrit Test and a Lateral Flow Assay to Detect Critical Fibrinogen Concentrations Using a Bottom-Up Pyramid Workflow Approach.

Fibrinogen is a coagulation factor in human blood and the first one to reach critical levels in major bleeding. Hypofibrinogenemia (a too low fibrinogen concentration in blood) poses great challenges to first responders, clinicians, and healthcare providers since it represents a risk factor for exsanguination and massive transfusion requirements. Thus, the rapid assessment of the fibrinogen concentration at the point of care has gained considerable importance in preventing and managing major blood loss. However, in whole blood measurements, hematocrit variations affect the amount (volume fraction) of plasma that passes the detection zone. In an attempt to accurately determine realistic critical levels of fibrinogen (<1.5 mg/mL) in patients needing immediate treatment and medical interventions, we have developed novel diagnostic systems capable of estimating hematocrit and critical fibrinogen concentrations. A lateral flow assay (LFA) for the detection of fibrinogen has been developed by establishing a workflow employing rapid characterization methods to streamline LFA development. The integration of two detection lines enables (i) the identification of fibrinogen (first line) present in the sample and (ii) the determination of the clinically critical fibrinogen concentrations below 1.5 mg/mL (second line). Furthermore, the paper-based separation of blood cells from plasma provides a semiquantitative estimate of the hematocrit by analyzing the fractions. Initial validation of the point-of-care (PoC) hematocrit test revealed good comparability to a standard laboratory method. The developed diagnostic systems have the ability to accelerate decision-making in cases with major bleeding.

Application of a Biomimetic Nanoparticle-Based Mock Virus to Determine SARS-CoV-2 Neutralizing Antibody Levels in Blood Samples Using a Lateral Flow Assay.

The presence of neutralizing antibodies against SARS-CoV-2 in blood, acquired through previous infection or vaccination, is known to prevent the (re)occurrence of outbreaks unless the virus mutates. Therefore, the measurement of neutralizing antibodies constitutes an indispensable tool in assessing an individual's and a population's immunity against SARS-CoV-2. For this reason, we have developed an innovative lateral flow assay (LFA) capable of detecting blood-derived neutralizing antibodies using a biomimetic SARS-CoV-2 mock virus system. Here, functionalized gold nanoparticles (AuNPs) featuring the trimeric spike (S) protein at its surface imitate the virus's structure and are applied to monitor the presence and efficacy of neutralizing antibodies in blood samples. The detection principle relies on the interaction between mock virus and the immobilized angiotensin-converting enzyme 2 (ACE2) receptor, which is inhibited when neutralizing antibodies are present. To further enhance the sensitivity of our competitive assay and identify low titers of neutralizing antibodies, an additional mixing pad is embedded into the device to increase the interaction time between mock virus and neutralizing antibodies. The developed LFA is benchmarked against the WHO International Standard (21/338) and demonstrated reliable quantification of neutralizing antibodies that inhibit ACE2 binding events down to a detection limit of an antibody titer of 59 IU/mL. Additional validation using whole blood and plasma samples showed reproducible results and good comparability to a laboratory-based reference test, thus highlighting its applicability for point-of-care testing.

Using Rapid Prototyping to Develop a Cell-Based Platform with Electrical Impedance Sensor Membranes for In Vitro RPMI2650 Nasal Nanotoxicology Monitoring.

Due to advances in additive manufacturing and prototyping, affordable and rapid microfluidic sensor-integrated assays can be fabricated using additive manufacturing, xurography and electrode shadow masking to create versatile platform technologies aimed toward qualitative assessment of acute cytotoxic or cytolytic events using stand-alone biochip platforms in the context of environmental risk assessment. In the current study, we established a nasal mucosa biosensing platform using RPMI2650 mucosa cells inside a membrane-integrated impedance-sensing biochip using exclusively rapid prototyping technologies. In a final proof-of-concept, we applied this biosensing platform to create human cell models of nasal mucosa for monitoring the acute cytotoxic effect of zinc oxide reference nanoparticles. Our data generated with the biochip platform successfully monitored the acute toxicity and cytolytic activity of 6 mM zinc oxide nanoparticles, which was non-invasively monitored as a negative impedance slope on nasal epithelial models, demonstrating the feasibility of rapid prototyping technologies such as additive manufacturing and xurography for cell-based platform development.

Database of 4 Million Medicinal Chemistry-Relevant Ring Systems.

Central ring systems are the most important part of bioactive molecules. They determine molecule shape, keep substituents in their proper positions, and also influence global molecular properties. In the present study, a database of 4 million medicinal chemistry-relevant ring systems has been created, not by crude random enumeration but by applying a set of rules derived by analyzing rings present in bioactive molecules. The aromatic properties and tautomer stability of generated rings have also been considered to ensure that the rings in the database are stable and chemically reasonable. 99.2% of these rings are novel and not included in molecules in the ChEMBL or PubChem databases. This large database of ring systems has been created with the goal to provide support for bioisosteric design and scaffold hopping as well as to be used in generative chemistry applications. The complete set of created rings is available for download in the SMILES format from https://peter-ertl.com/molecular/data/.

MicroCycle: An Integrated and Automated Platform to Accelerate Drug Discovery.

We herein describe the development and application of a modular technology platform which incorporates recent advances in plate-based microscale chemistry, automated purification, in situ quantification, and robotic liquid handling to enable rapid access to high-quality chemical matter already formatted for assays. In using microscale chemistry and thus consuming minimal chemical matter, the platform is not only efficient but also follows green chemistry principles. By reorienting existing high-throughput assay technology, the platform can generate a full package of relevant data on each set of compounds in every learning cycle. The multiparameter exploration of chemical and property space is hereby driven by active learning models. The enhanced compound optimization process is generating knowledge for drug discovery projects in a time frame never before possible.

MELLODDY: Cross-pharma Federated Learning at Unprecedented Scale Unlocks Benefits in QSAR without Compromising Proprietary Information.

Federated multipartner machine learning has been touted as an appealing and efficient method to increase the effective training data volume and thereby the predictivity of models, particularly when the generation of training data is resource-intensive. In the landmark MELLODDY project, indeed, each of ten pharmaceutical companies realized aggregated improvements on its own classification or regression models through federated learning. To this end, they leveraged a novel implementation extending multitask learning across partners, on a platform audited for privacy and security. The experiments involved an unprecedented cross-pharma data set of 2.6+ billion confidential experimental activity data points, documenting 21+ million physical small molecules and 40+ thousand assays in on-target and secondary pharmacodynamics and pharmacokinetics. Appropriate complementary metrics were developed to evaluate the predictive performance in the federated setting. In addition to predictive performance increases in labeled space, the results point toward an extended applicability domain in federated learning. Increases in collective training data volume, including by means of auxiliary data resulting from single concentration high-throughput and imaging assays, continued to boost predictive performance, albeit with a saturating return. Markedly higher improvements were observed for the pharmacokinetics and safety panel assay-based task subsets.

Optical glucose sensor for microfluidic cell culture systems.

Glucose is the primary energy source of human cells. Therefore, monitoring glucose inside microphysiological systems (MPS) provides valuable information on the viability and metabolic state of the cultured cells. However, continuous glucose monitoring inside MPS is challenging due to a lack of suitable miniaturized sensors. Here we present an enzymatic, optical glucose sensor element for measurement inside microfluidic systems. The miniaturized glucose sensor (Ø 1 mm) is fabricated together with a reference oxygen sensor onto biocompatible, pressure-sensitive adhesive tape for easy integration inside microfluidic systems. Furthermore, the proposed microfluidic system can be used as plug and play sensor system with existing MPS. It was characterized under cell culture conditions (37 °C and pH 7.4) for five days, exhibiting minor drift (3% day-1). The influence of further cell culture parameters like oxygen concentration, pH, flow rate, and sterilization methods was investigated. The plug-and-play system was used for at-line measurements of glucose levels in (static) cell culture and achieved good agreement with a commercially available glucose sensor. In conclusion, we developed an optical glucose sensor element that can be easily integrated in microfluidic systems and is able to perform stable glucose measurements under cell culture conditions.

Towards a microfluidic H295R steroidogenesis assay-biocompatibility study and steroid detection on a thiol-ene-based chip.

The development of cell-based microfluidic assays offers exciting new opportunities in toxicity testing, allowing for integration of new functionalities, automation, and high throughput in comparison to traditional well-plate assays. As endocrine disruption caused by environmental chemicals and pharmaceuticals represents a growing global health burden, the purpose of the current study was to contribute towards the miniaturization of the H295R steroidogenesis assay, from the well-plate to the microfluidic format. Microfluidic chip fabrication with the established well-plate material polystyrene (PS) is expensive and complicated; PDMS and thiol-ene were therefore tested as potential chip materials for microfluidic H295R cell culture, and evaluated in terms of cell attachment, cell viability, and steroid synthesis in the absence and presence of collagen surface modification. Additionally, spike-recovery experiments were performed, to investigate potential steroid adsorption to chip materials. Cell aggregation with poor steroid recoveries was observed for PDMS, while cells formed monolayer cultures on the thiol-ene chip material, with cell viability and steroid synthesis comparable to cells grown on a PS surface. As thiol-ene overall displayed more favorable properties for H295R cell culture, a microfluidic chip design and corresponding cell seeding procedure were successfully developed, achieving repeatable and uniform cell distribution in microfluidic channels. Finally, H295R perfusion culture on thiol-ene chips was investigated at different flow rates (20, 10, and 2.5 µL/min), and 13 steroids were detected in eluting cell medium over 48 h at the lowest flow rate. The presented work and results pave the way for a time-resolved microfluidic H295R steroidogenesis assay.

Which boronic acids are used most frequently for synthesis of bioactive molecules?

Boronic acids are essential building blocks used for the synthesis of bioactive molecules, the generation of chemical libraries and the exploration of structure-activity relationships. As a result, more than ten thousand boronic acids are commercially available. Medicinal chemists are therefore facing a challenge; which of them should they select to maximize information obtained by the synthesis of new target molecules. The present article aims to help them to make the right choices. The boronic acids used frequently in the synthesis of bioactive molecules were identified by mining several large molecular and reaction databases and their properties were analyzed. Based on the results a diverse set of boronic acids covering well the bioactive chemical space was selected and is suggested as a basis for library design for the efficient exploration of structure-activity relationships. A Boronic Acid Navigator web tool which helps chemists to make their own selection is also made available at https://bit.ly/boronics.

Rapid identification of in vitro cell toxicity using an electrochemical membrane screening platform.

This study compares the performance and output of an electrochemical phospholipid membrane platform against respective in vitro cell-based toxicity testing methods using three toxicants of different biological action (chlorpromazine (CPZ), colchicine (COL) and methyl methanesulphonate (MMS)). Human cell lines from seven different tissues (lung, liver, kidney, placenta, intestine, immune system) were used to validate this physicochemical testing system. For the cell-based systems, the effective concentration at 50 % cell death (EC50) values are calculated. For the membrane sensor, a limit of detection (LoD) value was extracted as a quantitative parameter describing the minimum concentration of toxicant which significantly affects the structure of the phospholipid sensor membrane layer. LoD values were found to align well with the EC50 values when acute cell viability was used as an end-point and showed a similar toxicity ranking of the tested toxicants. Using the colony forming efficiency (CFE) or DNA damage as end-point, a different order of toxicity ranking was observed. The results of this study showed that the electrochemical membrane sensor generates a parameter relating to biomembrane damage, which is the predominant factor in decreasing cell viability when in vitro models are acutely exposed to toxicants. These results lead the way to using electrochemical membrane-based sensors for rapid relevant preliminary toxicity screens.

The Cultivation Modality and Barrier Maturity Modulate the Toxicity of Industrial Zinc Oxide and Titanium Dioxide Nanoparticles on Nasal, Buccal, Bronchial, and Alveolar Mucosa Cell-Derived Barrier Models.

As common industrial by-products, airborne engineered nanomaterials are considered important environmental toxins to monitor due to their potential health risks to humans and animals. The main uptake routes of airborne nanoparticles are nasal and/or oral inhalation, which are known to enable the transfer of nanomaterials into the bloodstream resulting in the rapid distribution throughout the human body. Consequently, mucosal barriers present in the nose, buccal, and lung have been identified and intensively studied as the key tissue barrier to nanoparticle translocation. Despite decades of research, surprisingly little is known about the differences among various mucosa tissue types to tolerate nanoparticle exposures. One limitation in comparing nanotoxicological data sets can be linked to a lack of harmonization and standardization of cell-based assays, where (a) different cultivation conditions such as an air-liquid interface or submerged cultures, (b) varying barrier maturity, and (c) diverse media substitutes have been used. The current comparative nanotoxicological study, therefore, aims at analyzing the toxic effects of nanomaterials on four human mucosa barrier models including nasal (RPMI2650), buccal (TR146), alveolar (A549), and bronchial (Calu-3) mucosal cell lines to better understand the modulating effects of tissue maturity, cultivation conditions, and tissue type using standard transwell cultivations at liquid-liquid and air-liquid interfaces. Overall, cell size, confluency, tight junction localization, and cell viability as well as barrier formation using 50% and 100% confluency was monitored using trans-epithelial-electrical resistance (TEER) measurements and resazurin-based Presto Blue assays of immature (e.g., 5 days) and mature (e.g., 22 days) cultures in the presence and absence of corticosteroids such as hydrocortisone. Results of our study show that cellular viability in response to increasing nanoparticle exposure scenarios is highly compound and cell-type specific (TR146 6 ± 0.7% at 2 mM ZnO (ZnO) vs. ~90% at 2 mM TiO2 (TiO2) for 24 h; Calu3 93.9 ± 4.21% at 2 mM ZnO vs. ~100% at 2 mM TiO2). Nanoparticle-induced cytotoxic effects under air-liquid cultivation conditions declined in RPMI2650, A549, TR146, and Calu-3 cells (~0.7 to ~0.2-fold), with increasing 50 to 100% barrier maturity under the influence of ZnO (2 mM). Cell viability in early and late mucosa barriers where hardly influenced by TiO2 as well as most cell types did not fall below 77% viability when added to Individual ALI cultures. Fully maturated bronchial mucosal cell barrier models cultivated under ALI conditions showed less tolerance to acute ZnO nanoparticle exposures (~50% remaining viability at 2 mM ZnO for 24 h) than the similarly treated but more robust nasal (~74%), buccal (~73%), and alveolar (~82%) cell-based models.

Design and Biochemical Characterization of Peptidic Inhibitors of the Myb/p300 Interaction.

The Myb transcription factor is involved in the proliferation of hematopoietic cells, and deregulation of its expression can lead to cancers such as leukemia. Myb interacts with various proteins, including the histone acetyltransferases p300 and CBP. Myb binds to a small domain of p300, the KIX domain (p300KIX), and inhibiting this interaction is a potential new drug discovery strategy in oncology. The available structures show that Myb binds to a very shallow pocket of the KIX domain, indicating that it might be challenging to identify inhibitors of this interaction. Here, we report the design of Myb-derived peptides which interact with p300KIX. We show that by mutating only two Myb residues that bind in or near a hotspot at the surface of p300KIX, it is possible to obtain single-digit nanomolar peptidic inhibitors of the Myb/p300KIX interaction that bind 400-fold tighter to p300KIX than wildtype Myb. These findings suggest that it might also be possible to design potent low molecular-weight compounds to disrupt the Myb/p300KIX interaction.

The most common linkers in bioactive molecules and their bioisosteric replacement network.

Structures of the large majority of bioactive molecules are composed of several rings that are decorated by substituents and connected by linkers. While numerous cheminformatics studies focusing on rings and substituents are available, practically nothing has been published about the third important structural constituent of bioactive molecules - the linkers. The current study attempts to fill this gap. The most common linkers present in bioactive molecules are identified, their properties analyzed and a method for linker similarity search introduced. The bioisosteric replacement network of linkers is generated based on a large corpus of structure-activity data from medicinal chemistry literature. The results are presented in a graphical form and the underlying data are also made available for download. This analysis is intended to help medicinal chemists to better understand the role of linkers, particularly heterocyclic rings in bioactive molecules and to select an optimal set of linkers in their future project.