Organ-on-a-Chip Approach for Accelerating Blood-Brain Barrier Nanoshuttle Discovery.

Organ-on-a-chip, which recapitulates the dynamics of in vivo vasculature, has emerged as a promising platform for studying organ-specific vascular beds. However, its practical advantages in identifying vascular-targeted drug delivery systems (DDS) over traditional in vitro models remain underexplored. This study demonstrates the reliability and efficacy of the organ-on-a-chip in screening efficient DDS by comparing its performance with that of a conventional transwell, both designed to simulate the blood-brain barrier (BBB). The BBB nanoshuttles discovered through BBB Chip-based screening demonstrated superior functionality in vivo compared to those identified using transwell methods. This enhanced effectiveness is attributed to the BBB Chip's accurate replication of the structure and dynamics of the endothelial glycocalyx, a crucial protective layer within blood vessels, especially under shear stress. This capability of the BBB Chip has enabled the identification of molecular shuttles that efficiently exploit the endothelial glycocalyx, thereby enhancing transendothelial transport efficacy. Our findings suggest that organ-on-a-chip technology holds considerable promise for advancing research in vascular-targeted DDS due to its accurate simulation of molecular transport within endothelial systems.

Recent advances in micro-physiological systems for investigating tumor metastasis and organotropism.

Tumor metastasis involves complex processes that traditional 2D cultures and animal models struggle to fully replicate. Metastatic tumors undergo a multitude of transformations, including genetic diversification, adaptation to diverse microenvironments, and modified drug responses, contributing significantly to cancer-related mortality. Micro-physiological systems (MPS) technology emerges as a promising approach to emulate the metastatic process by integrating critical biochemical, biomechanical, and geometrical cues at a microscale. These systems are particularly advantageous simulating metastasis organotropism, the phenomenon where tumors exhibit a preference for metastasizing to particular organs. Organotropism is influenced by various factors, such as tumor cell characteristics, unique organ microenvironments, and organ-specific vascular conditions, all of which can be effectively examined using MPS. This review surveys the recent developments in MPS research from the past five years, with a specific focus on their applications in replicating tumor metastasis and organotropism. Furthermore, we discuss the current limitations in MPS-based studies of organotropism and propose strategies for more accurately replicating and analyzing the intricate aspects of organ-specific metastasis, which is pivotal in the development of targeted therapeutic approaches against metastatic cancers.

Organoid-Based Human Stomach Micro-Physiological System to Recapitulate the Dynamic Mucosal Defense Mechanism.

Several stomach diseases are attributed to the dysregulation of physiological function of gastric mucosal barrier by pathogens. Gastric organoids are a promising tool to develop treatment strategies for gastric infections. However, their functional features of in vivo gastric mucosal barrier and host-microbe interactions are limited due to the lack of physiological stimuli. Herein, a human stomach micro-physiological system (hsMPS) with physiologically relevant gastric mucosal defense system is described based on the combination of organoid and MPS technology. A fluid flow enhanced epithelial-mesenchymal interaction in the hsMPS enables functional maturation of gastric epithelial cells, which allows for the recreation of mesh-like mucus layer containing high level of mucus protective peptides and well-developed epithelial junctional complexes. Furthermore, gastroprotection mechanisms against Helicobacter pylori (H. pylori) are successfully demonstrated in this system. Therefore, hsMPS represents a new in vitro tool for research where gastric mucosal defense mechanism is pivotal for developing therapeutic strategies.

Integrated technologies for continuous monitoring of organs-on-chips: Current challenges and potential solutions.

Organs-on-chips (OoCs) are biomimetic in vitro systems based on microfluidic cell cultures that recapitulate the in vivo physicochemical microenvironments and the physiologies and key functional units of specific human organs. These systems are versatile and can be customized to investigate organ-specific physiology, pathology, or pharmacology. They are more physiologically relevant than traditional two-dimensional cultures, can potentially replace the animal models or reduce the use of these models, and represent a unique opportunity for the development of personalized medicine when combined with human induced pluripotent stem cells. Continuous monitoring of important quality parameters of OoCs via a label-free, non-destructive, reliable, high-throughput, and multiplex method is critical for assessing the conditions of these systems and generating relevant analytical data; moreover, elaboration of quality predictive models is required for clinical trials of OoCs. Presently, these analytical data are obtained by manual or automatic sampling and analyzed using single-point, off-chip traditional methods. In this review, we describe recent efforts to integrate biosensing technologies into OoCs for monitoring the physiologies, functions, and physicochemical microenvironments of OoCs. Furthermore, we present potential alternative solutions to current challenges and future directions for the application of artificial intelligence in the development of OoCs and cyber-physical systems. These "smart" OoCs can learn and make autonomous decisions for process optimization, self-regulation, and data analysis.

SEEDING to Enable Sensitive Electrochemical Detection of Biomarkers in Undiluted Biological Samples.

Electrochemical biosensors have shown great potential for simple, fast, and cost-effective point-of-care diagnostic tools. However, direct analysis of complex biological fluids such as plasma has been limited by the loss of sensitivity caused by biofouling. By increasing the surface area, the nanostructured electrode can improve detection sensitivity. However, like a double-edged sword, a large surface area increases the nonspecific adsorption of contaminating proteins. The use of nanoporous structures may prevent fouling proteins. However, there is no straightforward approach for creating nanostructured and nanoporous surfaces compatible with microfabricated thin-film electrodes. Herein, the preferential etching of chloride and surfactant-assisted anisotropic gold reduction to create homogeneous, nanostructured, and nanoporous gold electrodes is demonstrated, yielding a 190 ± 20 times larger surface area within a minute without using templates. This process, "surfactant-based electrochemical etch-deposit interplay for nanostructure/nanopore growth" (SEEDING), on electrodes enhances the sensitivity and antibiofouling capabilities of amperometric biosensors, enabling direct analysis of tumor-derived extracellular vesicles (tEVs) in complex biofluids with a limit of detection of 300 tEVs µL-1  from undiluted plasma and good discrimination between patients with prostate cancer from healthy ones with an area under the curve of 0.91 in urine and 0.90 in plasma samples.

Fully automated light transmission aggregometry on a disc for platelet function tests.

Platelet function tests, a group of assays that measure the ability of platelets to aggregate and promote clotting in a sample of blood, are performed in various medical fields to assess inherited platelet function disorders and monitor antiplatelet therapies. Light transmission aggregometry (LTA) is considered the gold standard for platelet function assessment. However, the lack of a standardized protocol is a major drawback when applied at the point of care. Moreover, it is a time-consuming and labor-intensive assay that requires a large volume of blood. Here, we describe the design, fabrication, and operation of a centrifugal microfluidic disc that can perform a fully automated LTA assay from a small volume of a whole blood sample (<1 mL), achieving highly reproducible results (3.2% coefficient of variation) within a short period (<25 min). The assays performed with this device yield more precise and accurate results than traditional LTA because of the automation of the reaction steps, minimal human operation, robust detection strategy via the distinctive structure of the microfluidic chamber, and quick analysis that minimizes the adverse effects of platelet instability.

Lab-on-a-Disc for Point-of-Care Infection Diagnostics.

Reliable, inexpensive, and rapid diagnostic tools are essential to control and prevent the spread of infectious diseases. Many commercial kits for coronavirus disease 2019 (COVID-19) diagnostics have played a crucial role in the fight against the COVID-19 pandemic. Most current standard in vitro diagnostic (IVD) protocols for infectious diseases are sensitive but time-consuming and require sophisticated laboratory equipment and specially trained personnel. Recent advances in biosensor technology suggest the potential to deliver point-of-care (POC) diagnostics that are affordable and provide accurate results in a short time. The ideal "sample-in-answer-out" type fully integrated POC infection diagnostic platforms are expected to be autonomous or easy-to-operate, equipment-free or infrastructure-independent, and high-throughput or easy to upscale. In this Account, we detail the recent progress made by our group and others in the development of centrifugal microfluidic devices or lab-on-a-disc (LOAD) systems. Unlike conventional pump-based fluid actuation, the centrifugal force generated by spinning the disc induces liquid pumping and no external fluidic interconnects are required. This allows a total fluidic network required for multiple steps of biological assays to be integrated on a disc, enabling fully automated POC diagnostics. Various applications have been demonstrated, including liquid biopsy for personalized cancer management, food applications, and environmental monitoring; here, we focus on IVD for infectious disease. First, we introduce various on-disc unit operation technologies, including reagent storage, sedimentation, filtration, valving, decanting, aliquoting, mixing, separation, serial dilution, washing, and calibration. Such centrifugal microfluidic technologies have already proved promising for micro-total-analysis systems for automated IVD ranging from molecular detection of pathogens to multiplexed enzyme-linked immunosorbent assays (ELISAs) that use raw samples such as whole blood or saliva. Some recent examples of LOAD systems for molecular diagnostics in which some or all steps of the assays are integrated on a disc, including pathogen enrichment, nucleic acid extraction, amplification, and detection, are discussed in detail. We then introduce fully automated ELISA systems with enhanced sensitivity. Furthermore, we demonstrate a toy-inspired fidget spinner that enables electricity-free and rapid analysis of pathogens from undiluted urine samples of patients with urinary tract infection symptoms and a phenotypic antimicrobial susceptibility test for an extreme POC diagnostics application. Considering the urgent need for cost-effective and reliable POC infection diagnostic tools, especially in the current pandemic crisis, the current limitations and future directions of fast and broad adaptation in real-world settings are also discussed. With proper attention to key challenges and leverage with recent advances in bio-sensing technologies, molecular biology, nanomaterials, analytical chemistry, miniaturization, system integration, and data management, LOAD systems hold the potential to deliver POC infection diagnostic tools with unprecedented performance regarding time, accuracy, and cost. We hope the new insight and promise of LOAD systems for POC infection diagnostics presented in this Account can spark new ideas and inspire further research and development to create better healthcare systems for current and future pandemics.

Highly sensitive detection of hydrazine by a disposable, Poly(Tannic Acid)-Coated carbon electrode.

We propose an electrochemical sensor based on the enhanced electrocatalytic oxidation exhibited on a functionalized poly(tannic acid) coating to detect hydrazine. Tannic acid, a naturally abundant and low-cost polyphenol, was enzymatically polymerized with horseradish peroxidase and subsequently adsorbed on a disposable screen-printed carbon electrode with a short incubation time (30 min). The fabrication method proved to be reproducible (4.2 % relative standard deviation), with the sensors displaying high sensitivity (7 × 10-3 µA mm-2 µM-1) and selectivity even in the presence of various common interfering agents. The low detection limit (100 nM) and robustness of the sensor demonstrated its suitability for environmental applications. It can be used to quantify hydrazine in tap and river water samples.

An antifouling coating that enables affinity-based electrochemical biosensing in complex biological fluids.

Affinity-based electrochemical detection in complex biological fluids could enable multiplexed point-of-care diagnostics for home healthcare; however, commercialization of point-of-care devices has been limited by the rapid loss of sensitivity caused by electrode surface inactivation and biofouling. Here, we describe a simple and robust antifouling coating for electrodes consisting of a three-dimensional porous matrix of cross-linked bovine serum albumin supported by a network of conductive nanomaterials composed of either gold nanowires, gold nanoparticles or carbon nanotubes. These nanocomposites prevent non-specific interactions while enhancing electron transfer to the electrode surface, preserving 88% of the original signal after 1 month of exposure to unprocessed human plasma, and functionalization with specific antibodies enables quantification of anti-interleukin 6 in plasma with high sensitivity. The easy preparation, stability and simplicity of this nanocomposite allow the generation of electrochemical biosensors that can operate in complex biological fluids such as blood plasma or serum.

Universal method for direct bioconjugation of electrode surfaces by fast enzymatic polymerization.

We report HRP-catalyzed polymerization of Tannic acid (TA) and application of the poly (Tannic acid) (p(TA)) as a versatile platform for covalent immobilization of biomolecules on various electrode surfaces based on electrochemical oxidation of the p(TA) and subsequent oxidative coupling reactions with the biomolecules. We also used this method for capturing cancer cells through a linker molecule, folic acid (FA). Furthermore, we have demonstrated that enhanced electrocatalytic activity of the p(TA)-modified surface could be used for simultaneous electrochemical determination of biologically important electroactive molecules such as ascorbic acid (AA), dopamine (DA), and uric acid (UA). This HRP-catalyzed polymerization of TA and p(TA)-mediated surface modification method can provide a simple and new framework to construct multifunctional platforms for covalent attachment of biomolecules and development of sensitive electrochemical sensing devices.

Ultrasensitive and rapid detection of ß-conglutin combining aptamers and isothermal recombinase polymerase amplification.

Lupin is increasingly being used in a variety of food products due to its nutritional, functional and nutraceutical properties. However, several examples of severe and even fatal food-associated anaphylaxis due to lupin inhalation or ingestion have been reported, resulting in the lupin subunit ß-conglutin, being defined as the Lup an 1 allergen by the International Union of Immunological Societies (IUIS) in 2008. Here, we report an innovative method termed aptamer-recombinase polymerase amplification (Apta-RPA) exploiting the affinity and specificity of a DNA aptamer selected against the anaphylactic ß-conglutin allergen termed ß-conglutin binding aptamer II (ß-CBA II), facilitating ultrasensitive detection via isothermal amplification. Combining magnetic beads as the solid phase with Apta-RPA detection, the total assay time was reduced from 210 min to just 25 min, with a limit of detection of 3.5 × 10-11 M, demonstrating a rapid and ultrasensitive generic methodology that can be used with any aptamer. Future work will focus on further simplification of the assay to a lateral flow format. Graphical Abstract Schematic representation of the rapid and novel bead-based Apta-RPA assay.

Isothermal solid-phase amplification system for detection of Yersinia pestis.

DNA amplification is required for most molecular diagnostic applications, but conventional polymerase chain reaction (PCR) has disadvantages for field testing. Isothermal amplification techniques are being developed to respond to this problem. One of them is the recombinase polymerase amplification (RPA) that operates at isothermal conditions without sacrificing specificity and sensitivity in easy-to-use formats. In this work, RPA was used for the optical detection of solid-phase amplification of the potential biowarfare agent Yersinia pestis. Thiolated forward primers were immobilized on the surface of maleimide-activated microtitre plates for the quantitative detection of synthetic and genomic DNA, with elongation occurring only in the presence of the specific template DNA and solution phase reverse primers. Quantitative detection was achieved via the use of biotinylated reverse primers and post-amplification addition of streptavidin-HRP conjugate. The overall time of amplification and detection was less than 1 h at a constant temperature of 37 °C. Single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) sequences were detected, achieving detection limits of 4.04*10(-13) and 3.14*10(-16) M, respectively. The system demonstrated high specificity with negligible responses to non-specific targets.

Real-time and label-free ring-resonator monitoring of solid-phase recombinase polymerase amplification.

In this work we present the use of a silicon-on-insulator (SOI) chip featuring an array of 64 optical ring resonators used as refractive index sensors for real-time and label-free DNA detection. Single ring functionalisation was achieved using a click reaction after precise nanolitre spotting of specific hexynyl-terminated DNA capture probes to link to an azido-silanised chip surface. To demonstrate detectability using the ring resonators and to optimise conditions for solid-phase amplification, hybridisation between short 25-mer single stranded DNA (ssDNA) fragments and a complementary capture probe immobilised on the surface of the ring resonators was carried out and detected through the shift in the resonant wavelength. Using the optimised conditions demonstrated via the solid-phase hybridisation, a 144-bp double stranded DNA (dsDNA) was then detected directly using recombinase and polymerase proteins through on-chip target amplification and solid-phase elongation of immobilised forward primers on specific rings, at a constant temperature of 37°C and in less than 60min, achieving a limit of detection of 7.8·10(-13)M (6·10(5) copies in 50µL). The use of an automatic liquid handler injection instrument connected to an integrated resealable chip interface (RCI) allowed programmable multiple injection protocols. Air plugs between different solutions were introduced to prevent intermixing and a proportional-integral-derivative (PID) temperature controller minimised temperature based drifts.