Humanized brain organoids-on-chip integrated with sensors for screening neuronal activity and neurotoxicity.

The complex structure and function of the human central nervous system that develops from the neural tube made in vitro modeling quite challenging until the discovery of brain organoids. Human-induced pluripotent stem cells-derived brain organoids offer recapitulation of the features of early human neurodevelopment in vitro, including the generation, proliferation, and differentiation into mature neurons and micro-macroglial cells, as well as the complex interactions among these diverse cell types of the developing brain. Recent advancements in brain organoids, microfluidic systems, real-time sensing technologies, and their cutting-edge integrated use provide excellent models and tools for emulation of fundamental neurodevelopmental processes, the pathology of neurological disorders, personalized transplantation therapy, and high-throughput neurotoxicity testing by bridging the gap between two-dimensional models and the complex three-dimensional environment in vivo. In this review, we summarize how bioengineering approaches are applied to mitigate the limitations of brain organoids for biomedical and clinical research. We further provide an extensive overview and future perspectives of the humanized brain organoids-on-chip platforms with integrated sensors toward brain organoid intelligence and biocomputing studies. Such approaches might pave the way for increasing approvable clinical applications by solving their current limitations.

Smart Wearable Nanopaper Patch for Continuous Multiplexed Optical Monitoring of Sweat Parameters.

Notwithstanding the substantial progress in optical wearable sensing devices, developing wearable optical sensors for simultaneous, real-time, and continuous monitoring of multiple biomarkers is still an important, yet unmet, demand. Aiming to address this need, we introduced for the first time a smart wearable optical sensor (SWOS) platform combining a multiplexed sweat sensor sticker with its IoT-enabled readout module. We employed our SWOS system for on-body continuous, real-time, and simultaneous fluorimetric monitoring of sweat volume (physical parameter) and pH (chemical marker). Herein, a variation in moisture (5-45 µL) or pH (4.0-7.0) causes a color/fluorescence change in the copper chloride/fluorescein immobilized within a transparent chitin nanopaper (ChNP) in a selective and reversible manner. Human experiments conducted on athletic volunteers during exercise confirm that our developed SWOS platform can be efficiently exploited for smart perspiration analysis toward personalized health monitoring. Moreover, our system can be further extended for the continuous and real-time multiplexed monitoring of various biomarkers (metabolites, proteins, or drugs) of sweat or other biofluids (for example, analyzing exhaled breath by integrating onto a facemask).

A three-level model for therapeutic drug monitoring of antimicrobials at the site of infection.

The silent pandemic of bacterial antimicrobial resistance is a leading cause of death worldwide, prolonging hospital stays and raising health-care costs. Poor incentives to develop novel pharmacological compounds and the misuse of antibiotics contribute to the bacterial antimicrobial resistance crisis. Therapeutic drug monitoring (TDM) based on blood analysis can help alleviate the emergence of bacterial antimicrobial resistance and effectively decreases the risk of toxic drug concentrations in patients' blood. Antibiotic tissue penetration can vary in patients who are critically or chronically ill and can potentially lead to treatment failure. Antibiotics such as ß-lactams and glycopeptides are detectable in non-invasively collectable biofluids, such as sweat and exhaled breath. The emergence of wearable sensors enables easy access to these non-invasive biofluids, and thus a laboratory-independent analysis of various disease-associated biomarkers and drugs. In this Personal View, we introduce a three-level model for TDM of antibiotics to describe concentrations at the site of infection (SOI) by use of wearable sensors. Our model links blood-based drug measurement with the analysis of drug concentrations in non-invasively collectable biofluids stemming from the SOI to characterise drug concentrations at the SOI. Finally, we outline the necessary clinical and technical steps for the development of wearable sensing platforms for SOI applications.

Wearable sweat analysis to determine biological age.

A real-time, noninvasive, and clinically applicable aging test in humans has yet to be established. Herein we propose a sweat- and wearable-based test to determine biological age. This test would empower users to monitor their aging process and take an active role in managing their lifestyle and health.

Multiplexed biosensor for point-of-care COVID-19 monitoring: CRISPR-powered unamplified RNA diagnostics and protein-based therapeutic drug management.

In late 2019 SARS-CoV-2 rapidly spread to become a global pandemic, therefore, measures to attenuate chains of infection, such as high-throughput screenings and isolation of carriers were taken. Prerequisite for a reasonable and democratic implementation of such measures, however, is the availability of sufficient testing opportunities (beyond reverse transcription PCR, the current gold standard). We, therefore, propose an electrochemical, microfluidic multiplexed polymer-based biosensor in combination with CRISPR/Cas-powered assays for low-cost and accessible point-of-care nucleic acid testing. In this study, we simultaneously screen for and identify SARS-CoV-2 infections (Omicron-variant) in clinical specimens (Sample-to-result time: ∼30 min), employing LbuCas13a, whilst bypassing reverse transcription as well as target amplification of the viral RNA (LODs of 2,000 and 7,520 copies/µl for the E and RdRP genes, respectively, and 50 copies/ml for combined targets), both of which are necessary for detection via PCR and other isothermal methods. In addition, we demonstrate the feasibility of combining synthetic biology-driven assays based on different classes of biomolecules, in this case protein-based ß-lactam antibiotic detection, on the same device. The programmability of the effector and multiplexing capacity (up to six analytes) of our platform, in combination with a miniaturized measurement setup, including a credit card sized near field communication (NFC) potentiostat and a microperistaltic pump, provide a promising on-site tool for identifying individuals infected with variants of concern and monitoring their disease progression alongside other potential biomarkers or medication clearance.

Challenges and Opportunities for Printed Electrical Gas Sensors.

Printed electrical gas sensors are a low-cost, lightweight, low-power, and potentially disposable alternative to gas sensors manufactured using conventional methods such as photolithography, etching, and chemical vapor deposition. The growing interest in Internet-of-Things, smart homes, wearable devices, and point-of-need sensors has been the main driver fueling the development of new classes of printed electrical gas sensors. In this Perspective, we provide an insight into the current research related to printed electrical gas sensors including materials, methods of fabrication, and applications in monitoring food quality, air quality, diagnosis of diseases, and detection of hazardous gases. We further describe the challenges and future opportunities for this emerging technology.

End-to-end design of wearable sensors.

Wearable devices provide an alternative pathway to clinical diagnostics by exploiting various physical, chemical and biological sensors to mine physiological (biophysical and/or biochemical) information in real time (preferably, continuously) and in a non-invasive or minimally invasive manner. These sensors can be worn in the form of glasses, jewellery, face masks, wristwatches, fitness bands, tattoo-like devices, bandages or other patches, and textiles. Wearables such as smartwatches have already proved their capability for the early detection and monitoring of the progression and treatment of various diseases, such as COVID-19 and Parkinson disease, through biophysical signals. Next-generation wearable sensors that enable the multimodal and/or multiplexed measurement of physical parameters and biochemical markers in real time and continuously could be a transformative technology for diagnostics, allowing for high-resolution and time-resolved historical recording of the health status of an individual. In this Review, we examine the building blocks of such wearable sensors, including the substrate materials, sensing mechanisms, power modules and decision-making units, by reflecting on the recent developments in the materials, engineering and data science of these components. Finally, we synthesize current trends in the field to provide predictions for the future trajectory of wearable sensors.

Designing electrochemical microfluidic multiplexed biosensors for on-site applications.

Clinical assessment based on a single biomarker is in many circumstances not sufficient for adequate diagnosis of a disease or for monitoring its therapy. Multiplexing, the measurement of multiple analytes from one sample and/or of the same target from different samples simultaneously, could enhance the accuracy of the diagnosis of diseases and their therapy success. Thus, there is a great and urgent demand for multiplexed biosensors allowing a low-cost, easy-to-use, and rapid on-site testing. In this work, we present a simple, flexible, and highly scalable strategy for implementing microfluidic multiplexed electrochemical biosensors (BiosensorX). Our technology is able to detect 4, 6, or 8 (different) analytes or samples simultaneously using a sequential design concept: multiple immobilization areas, where the assay components are adsorbed, followed by their individual electrochemical cells, where the amperometric signal readout takes place, within a single microfluidic channel. Here, first we compare vertical and horizontal designs of BiosensorX chips using a model assay. Owing to its easier handling and superior fluidic behavior, the vertical format is chosen as the final multiplexed chip design. Consequently, the feasibility of the BiosensorX for multiplexed on-site testing is successfully demonstrated by measuring meropenem antibiotics via an antibody-free ß-lactam assay. The multiplexed biosensor platform introduced can be further extended for the simultaneous detection of other anti-infective agents and/or biomarkers (such as renal or inflammation biomarkers) as well as different (invasive and non-invasive) sample types, which would be a major step towards sepsis management and beyond.

Emerging Biosensing Technologies for the Diagnostics of Viral Infectious Diseases.

Several viral infectious diseases appear limitless since the beginning of the 21st century, expanding into pandemic lengths. Thus, there are extensive efforts to provide more efficient means of diagnosis, a better understanding of acquired immunity, and improved monitoring of inflammatory biomarkers, as these are all crucial for controlling the spread of infection while aiding in vaccine development and improving patient outcomes. In this regard, various biosensors have been developed recently to streamline pathogen and immune response detection by addressing the limitations of traditional methods, including isothermal amplification-based systems and lateral flow assays. This review explores state-of-the-art biosensors for detecting viral pathogens, serological assays, and inflammatory biomarkers from the material perspective, by discussing their advantages, limitations, and further potential regarding their analytical performance, clinical utility, and point-of-care adaptability. Additionally, next-generation biosensing technologies that offer better sensitivity and selectivity, and easy handling for end-users are highlighted. An emerging example of these next-generation biosensors are those powered by novel synthetic biology tools, such as clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated proteins (Cas), in combination with integrated point-of-care devices. Lastly, the current challenges are discussed and a roadmap for furthering these advanced biosensing technologies to manage future pandemics is provided.

Sensors in blockchain.

Blockchain, the technology behind bitcoin, has stimulated global conversation around digital decentralization to connect societies and economies. Connected low-cost sensors within Internet of Things (IoT) networks may soon perform measurements, exchange data, and make decisions automatically over decentralized networks; these systems could improve healthcare, the quality of goods, and supply chain efficiency.

CRISPR/Cas-powered nanobiosensors for diagnostics.

CRISPR diagnostics (CRISPR-Dx) offer a wide range of enhancements compared to traditional nanobiosensors by taking advantage of the excellent trans-cleavage activity of the CRISPR/Cas systems. However, the single-stranded DNA/RNA reporters of the current CRISPR-Dx suffer from poor stability and limited sensitivity, which make their application in complex biological environments difficult. In comparison, nanomaterials, especially metal nanoparticles, exhibits robust stability and desirable optical and electrocatalytical properties, which make them ideal as reporter molecules. Therefore, biosensing research is moving towards the use of the trans-cleavage activity of CRISPR/Cas effectors on metal nanoparticles and apply the new phenomenon to develop novel nanobiosensors to target various targets such as viral infections, genetic mutations and tumor biomarkers, by using different sensing methods, including, but not limited to fluorescence, luminescence resonance, colorimetric and electrochemical signal readout. In this review, we explore some of the most recent advances in the field of CRISPR-powered nanotechnological biosensors. Demonstrating high accuracy, sensitivity, selectivity and versatility, nanobiosensors along with CRISPR/Cas technology offer tremendous potential for next-generation diagnostics of multiple targets, especially at the point of care and without any target amplification.

Biosensor-Enabled Multiplexed On-Site Therapeutic Drug Monitoring of Antibiotics.

Personalized antibiotherapy ensures that the antibiotic concentration remains in the optimal therapeutic window to maximize efficacy, minimize side effects, and avoid the emergence of drug resistance due to insufficient dosing. However, such individualized schemes need frequent sampling to tailor the blood antibiotic concentrations. To optimally integrate therapeutic drug monitoring (TDM) into the clinical workflow, antibiotic levels can either be measured in blood using point-of-care testing (POCT), or can rely on noninvasive sampling. Here, a versatile biosensor with an antibody-free assay for on-site TDM is presented. The platform is evaluated with an animal study, where antibiotic concentrations are quantified in different matrices including whole blood, plasma, urine, saliva, and exhaled breath condensate (EBC). The clearance and the temporal evaluation of antibiotic levels in EBC and plasma are demonstrated. Influence of matrix effects on measured drug concentrations is determined by comparing the plasma levels with those in noninvasive samples. The system's potential for blood-based POCT is further illustrated by tracking ß-lactam concentrations in untreated blood samples. Finally, multiplexing capabilities are explored successfully for multianalyte/sample analysis. By enabling a rapid, low-cost, sample-independent, and multiplexed on-site TDM, this system can shift the paradigm of "one-size-fits-all" strategy.

Microfluidic Roadmap for Translational Nanotheranostics.

Nanotheranostic materials (NTMs) shed light on the mechanisms responsible for complex diseases such as cancer because they enable making a diagnosis, monitoring the disease progression, and applying a targeted therapy simultaneously. However, several issues such as the reproducibility and mass production of NTMs hamper their application for clinical practice. To address these issues and facilitate the clinical application of NTMs, microfluidic systems have been increasingly used. This perspective provides a glimpse into the current state-of-art of NTM research, emphasizing the methods currently employed at each development stage of NTMs and the related open problems. This work reviews microfluidic technologies used to develop NTMs, ranging from the fabrication and testing of a single NTM up to their manufacturing on a large scale. Ultimately, a step-by-step vision on the future development of NTMs for clinical practice enabled by microfluidics techniques is provided.

Current Technologies for RNA-Directed Liquid Diagnostics.

There is unequivocal acceptance of the variety of enormous potential liquid nucleic acid-based diagnostics seems to offer. However, the existing controversies and the increased awareness of RNA-based techniques in society during the current global COVID-19 pandemic have made the readiness of liquid nucleic acid-based diagnostics for routine use a matter of concern. In this regard-and in the context of oncology-our review presented and discussed the status quo of RNA-based liquid diagnostics. We summarized the technical background of the available assays and benchmarked their applicability against each other. Herein, we compared the technology readiness level in the clinical context, economic aspects, implementation as part of routine point-of-care testing as well as performance power. Since the preventive care market is the most promising application sector, we also investigated whether the developments predominantly occur in the context of early disease detection or surveillance of therapy success. In addition, we provided a careful view on the current biotechnology investment activities in this sector to indicate the most attractive strategies for future economic success. Taken together, our review shall serve as a current reference, at the interplay of technology, clinical use and economic potential, to guide the interested readers in this rapid developing sector of precision medicine.

Low-Cost Optical Assays for Point-of-Care Diagnosis in Resource-Limited Settings.

Readily deployable, low-cost point-of-care medical devices such as lateral flow assays (LFAs), microfluidic paper-based analytical devices (µPADs), and microfluidic thread-based analytical devices (µTADs) are urgently needed in resource-poor settings. Governed by the ASSURED criteria (affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free, and deliverability) set by the World Health Organization, these reliable platforms can screen a myriad of chemical and biological analytes including viruses, bacteria, proteins, electrolytes, and narcotics. The Ebola epidemic in 2014 and the ongoing pandemic of SARS-CoV-2 have exemplified the ever-increasing importance of timely diagnostics to limit the spread of diseases. This review provides a comprehensive survey of LFAs, µPADs, and µTADs that can be deployed in resource-limited settings. The subsequent commercialization of these technologies will benefit the public health, especially in areas where access to healthcare is limited.

A CRISPR/Cas13a-powered catalytic electrochemical biosensor for successive and highly sensitive RNA diagnostics.

Rapid and specific quantitation of a variety of RNAs with low expression levels in early-stage cancer is highly desirable but remains a challenge. Here, we present a dual signal amplification strategy consisting of the CRISPR/Cas13a system and a catalytic hairpin DNA circuit (CHDC), integrated on a reusable electrochemical biosensor for rapid and accurate detection of RNAs. Signal amplification is accomplished through the unique combination of the CRISPR/Cas13a system with CHDC, achieving a limit of detection of 50 aM within a readout time of 6 min and an overall process time of 36 min, using a measuring volume of 10 µL. Enzymatic regeneration of the sensor surface and ratiometric correction of background signal allow up to 37 sequential RNA quantifications by square-wave voltammetry on a single biosensor chip without loss of sensitivity. The reusable biosensor platform could selectively (specificity = 0.952) and sensitively (sensitivity = 0.900) identify low expression RNA targets in human serum, distinguishing early-stage patients (n = 20) suffering from non-small-cell lung carcinoma (NSCLC) from healthy subjects (n = 30) and patients with benign lung disease (n = 12). Measurement of six NSCLC-related RNAs (miR-17, miR-155, TTF-1 mRNA, miR-19b, miR-210 and EGFR mRNA) shows the ability of the electrochemical CRISPR/CHDC system to be a fast, low-cost and highly accurate tool for early cancer diagnostics.

CRISPR-powered electrochemical microfluidic multiplexed biosensor for target amplification-free miRNA diagnostics.

Recently the use of microRNAs (miRNAs) as biomarkers for a multitude of diseases has gained substantial significance for clinical as well as point-of-care diagnostics. Amongst other challenges, however, it holds the central requirement that the concentration of a given miRNA must be evaluated within the context of other factors in order to unambiguously diagnose one specific disease. In terms of the development of diagnostic methods and devices, this implies an inevitable demand for multiplexing in order to be able to gauge the abundance of several components of interest in a patient's sample in parallel. In this study, we design and implement different multiplexed versions of our electrochemical microfluidic biosensor by dividing its channel into subsections, creating four novel chip designs for the amplification-free and simultaneous quantification of up to eight miRNAs on the CRISPR-Biosensor X ('X' highlighting the multiplexing aspect of the device). We then use a one-step model assay followed by amperometric readout in combination with a 2-min-stop-flow-protocol to explore the fluidic and mechanical characteristics and limitations of the different versions of the device. The sensor showing the best performance, is subsequently used for the Cas13a-powered proof-of-concept measurement of two miRNAs (miRNA-19b and miRNA-20a) from the miRNA-17-92 cluster, which is dysregulated in the blood of pediatric medulloblastoma patients. Quantification of the latter, alongside simultaneous negative control measurements are accomplished on the same device. We thereby confirm the applicability of our platform to the challenge of amplification-free, parallel detection of multiple nucleic acids.