Integrating machine learning and biosensors in microfluidic devices: A review.

Microfluidic devices are increasingly widespread in the literature, being applied to numerous exciting applications, from chemical research to Point-of-Care devices, passing through drug development and clinical scenarios. Setting up these microenvironments, however, introduces the necessity of locally controlling the variables involved in the phenomena under investigation. For this reason, the literature has deeply explored the possibility of introducing sensing elements to investigate the physical quantities and the biochemical concentration inside microfluidic devices. Biosensors, particularly, are well known for their high accuracy, selectivity, and responsiveness. However, their signals could be challenging to interpret and must be carefully analysed to carry out the correct information. In addition, proper data analysis has been demonstrated even to increase biosensors' mentioned qualities. To this regard, machine learning algorithms are undoubtedly among the most suitable approaches to undertake this job, automatically learning from data and highlighting biosensor signals' characteristics at best. Interestingly, it was also demonstrated to benefit microfluidic devices themselves, in a new paradigm that the literature is starting to name "intelligent microfluidics", ideally closing this benefic interaction among these disciplines. This review aims to demonstrate the advantages of the triad paradigm microfluidics-biosensors-machine learning, which is still little used but has a great perspective. After briefly describing the single entities, the different sections will demonstrate the benefits of the dual interactions, highlighting the applications where the reviewed triad paradigm was employed.

A microfluidic impedance cytometry device for robust identification of H. pluvialis.

H. pluvialis contains rich oleic acid and astaxanthin, which have important applications in the fields of biodiesel and biomedicine. Detection of live H. pluvialis is the prerequisite to obtaining oleic acid and astaxanthin. For this purpose, we successfully developed a reliable microfluidic impedance cytometry for the identification of live H. pluvialis. Firstly, we established a simulation model for detecting H. pluvialis based on their morphology and studied the effect of medium conductivity on the impedance of H. pluvialis at different frequencies. From the simulations, we determined that the optimal solution conductivity for the detection of H. pluvialis was 1500 µS cm-1 and studied the frequency responses of the impedance of H. pluvialis. Secondly, we fabricated the microchannels and stainless-steel detection electrodes and assembled them into microfluidic impedance cytometry. The frequency dependence of live and dead H. pluvialis was explored under different frequencies, and live and dead H. pluvialis were distinguished at a frequency of 1 MHz. The impedance of live H. pluvialis at the frequency of 1 MHz ranges from 33.73 to 52.23 Ω, while that of dead ones ranges from 13.05 to 19.59 Ω. Based on these findings, we accomplished the identification and counting of live H. pluvialis in the live and dead sample solutions. Furthermore, we accomplished the identification and counting of live H. pluvialis in the mixed samples containing Euglena and H. pluvialis. This approach possesses the promising capacity to serve as a robust tool in the identification of target microalgae, addressing a challenge in the fields of biodiesel and biomedicine.

Quantitatively Evaluating Interactions between Patient-Derived Organoids and Autologous Immune Cells by Microfluidic Chip.

The coculture of patient-derived tumor organoids (PDOs) and autologous immune cells has been considered as a useful ex vivo surrogate of in vivo tumor-immune environment. However, the immune interactions between PDOs and autologous immune cells, including immune-mediated killing behaviors and immune-related cytokine variations, have yet to be quantitatively evaluated. This study presents a microfluidic chip for quantifying interactions between PDOs and autologous immune cells (IOI-Chip). A baffle-well structure is designed to ensure efficient trapping, long-term coculturing, and in situ fluorescent observation of a limited amount of precious PDOS and autologous immune cells, while a microbeads-based immunofluorescence assay is designed to simultaneously quantify multiple kinds of immune-related cytokines in situ. The PDO apoptosis and 2 main immune-related cytokines, TNF-α and IFN-γ, are simultaneously quantified using samples from a lung cancer patient. This study provides, for the first time, a capability to quantify interactions between PDOs and autologous immune cells at 2 levels, the immune-mediated killing behavior, and multiple immune-related cytokines, laying the technical foundation of ex vivo assessment of patient immune response.

Coupling Microdroplet-Based Sample Preparation, Multiplexed Isobaric Labeling, and Nanoflow Peptide Fractionation for Deep Proteome Profiling of the Tissue Microenvironment.

There is increasing interest in developing in-depth proteomic approaches for mapping tissue heterogeneity in a cell-type-specific manner to better understand and predict the function of complex biological systems such as human organs. Existing spatially resolved proteomics technologies cannot provide deep proteome coverage due to limited sensitivity and poor sample recovery. Herein, we seamlessly combined laser capture microdissection with a low-volume sample processing technology that includes a microfluidic device named microPOTS (microdroplet processing in one pot for trace samples), multiplexed isobaric labeling, and a nanoflow peptide fractionation approach. The integrated workflow allowed us to maximize proteome coverage of laser-isolated tissue samples containing nanogram levels of proteins. We demonstrated that the deep spatial proteomics platform can quantify more than 5000 unique proteins from a small-sized human pancreatic tissue pixel (∼60,000 µm2) and differentiate unique protein abundance patterns in pancreas. Furthermore, the use of the microPOTS chip eliminated the requirement for advanced microfabrication capabilities and specialized nanoliter liquid handling equipment, making it more accessible to proteomic laboratories.

Portable Microfluidic Viscometer for Formulation Development and in Situ Quality Control of Protein and Antibody Solutions.

Viscosity of protein solutions is a critical product quality attribute for protein therapeutics such as monoclonal antibodies. Here we introduce a portable single-use analytical chip-based viscometer for determining the viscosity of protein solutions using low sample volumes of 10 µL. Through the combined use of a microfluidic viscometer, a smartphone camera for image capture, and an automated data processing algorithm for the calculation of the viscosity of fluids, we enable measurement of viscosity of multiple samples in parallel. We first validate the viscometer using glycerol-water mixtures and subsequently demonstrate the ability to perform rapid characterization of viscosity in four different monoclonal antibody formulations in a broad concentration (1 to 320 mg/mL) and viscosity (1 to 600 cP) range, showing excellent agreement with values obtained by a conventional cone-plate rheometer. Not only does the platform offer benefits of viscosity measurements using minimal sample volumes, but enables higher throughput compared to gold-standard methodologies owing to multiplexing of the measurement and single-use characteristics of the viscometer, thus showing great promise in developability studies. Additionally, as our platform has the capability of performing viscosity measurements at the point of sample collection, it offers the opportunity to employ viscosity measurement as an in situ quality control of therapeutic proteins and antibodies.

Micro blood analysis technology (µBAT): multiplexed analysis of neutrophil phenotype and function from microliter whole blood samples.

There is an ongoing need to do more with less and provide highly multiplexed analysis from limited sample volumes. Improved "sample sparing" assays would have a broad impact across pediatric and other rare sample type studies in addition to enabling sequential sampling. This capability would advance both clinical and basic research applications. Here we report the micro blood analysis technology (µBAT), a microfluidic platform that supports multiplexed analysis of neutrophils from a single drop of blood. We demonstrate the multiplexed orthogonal capabilities of µBAT including functional assays (phagocytosis, neutrophil extracellular traps, optical metabolic imaging) and molecular assays (gene expression, cytokine secretion). Importantly we validate our microscale platform using a macroscale benchmark assay. µBAT is compatible with lancet puncture or microdraw devices, and its design facilitates rapid operations without the need for specialized equipment. µBAT offers a new method for investigating neutrophil function in populations with restricted sample amounts.

Integration of 3D printed Mg2+ potentiometric sensors into microfluidic devices for bioanalysis.

Electrochemical sensors provide an affordable and reliable approach towards the detection and monitoring of important biological species ranging from simple ions to complex biomolecules. The ability to miniaturize electrochemical sensors, coupled with their affordability and simple equipment requirements for signal readout, permits the use of these sensors at the point-of-care where analysis using non-invasively obtainable biofluids is receiving growing interest by the research community. This paper describes the design, fabrication, and integration of a 3D printed Mg2+ potentiometric sensor into a 3D printed microfluidic device for the quantification of Mg2+ in low-sample volume biological fluids. The sensor employs a functionalized 3D printable photocurable methacrylate-based ion-selective membrane affixed to a carbon-mesh/epoxy solid-contact transducer for the selective determination of Mg2+ in sweat, saliva and urine. The 3D printed Mg2+ ion-selective electrode (3Dp-Mg2+-ISE) provided a Nernstian response of 27.5 mV per decade with a linear range of 10 mM to 39 µM, covering the normal physiological and clinically relevant levels of Mg2+ in biofluids. 3Dp-Mg2+-ISEs selectively measure Mg2+ over other biologically present cations - sodium, potassium, calcium, ammonium - as well as provide high stability in the analytical signal with a drift of just 13 µV h-1 over 10 hours. Comparison with poly(vinylchloride)-based Mg2+-ISEs showed distinct advantages to the use of 3Dp-Mg2+-ISEs, with respect to stability, resilience towards biofouling and importantly providing a streamlined and rapid approach towards mass production of selective and reliable sensors. The miniaturization capabilities of 3D printing coupled with the benefits of microfluidic analysis (i.e., low sample volumes, minimal reagent consumption, automation of multiple assays, etc.), provides exciting opportunities for the realization of the next-generation of point-of-care diagnostic devices.

Microfluidics for macrofluidics: addressing marine-ecosystem challenges in an era of climate change.

Climate change presents a mounting challenge with profound impacts on ocean and marine ecosystems, leading to significant environmental, health, and economic consequences. Microfluidic technologies, with their unique capabilities, play a crucial role in understanding and addressing the marine aspects of the climate crisis. These technologies leverage quantitative, precise, and miniaturized formats that enhance the capabilities of sensing, imaging, and molecular tools. Such advancements are critical for monitoring marine systems under the stress of climate change and elucidating their response mechanisms. This review explores microfluidic technologies employed both in laboratory settings for testing and in the field for monitoring purposes. We delve into the application of miniaturized tools in evaluating ocean-based solutions to climate change, thus offering fresh perspectives from the solution-oriented end of the spectrum. We further aim to synthesize recent developments in technology around critical questions concerning the ocean environment and marine ecosystems, while discussing the potential for future innovations in microfluidic technology. The purpose of this review is to enhance understanding of current capabilities and assist researchers interested in mitigating the effects of climate change to identify new avenues for tackling the pressing issues posed by climate change in marine ecosystems.

An all-in-one microfluidic SlipChip for power-free and rapid biosensing of pathogenic bacteria.

Point-of-care testing of pathogens is becoming more and more important for the prevention and control of food poisoning. Herein, a power-free colorimetric biosensor was presented for rapid detection of Salmonella using a microfluidic SlipChip for fluidic control and Au@PtPd nanocatalysts for signal amplification. All the procedures, including solution mixing, immune reaction, magnetic separation, residual washing, mimicking catalysis and colorimetric detection, were integrated on this SlipChip. First, the mixture of the bacterial sample, immune magnetic nanobeads (IMBs) and immune Au@PtPd nanocatalysts (INCs), washing buffer and H2O2-TMB chromogenic substrate were preloaded into the sample, washing and catalysis chambers, respectively. After the top layer of this SlipChip was slid to connect the sample chamber with the separation chamber, the mixture was moved back and forth through the asymmetrical split-and-recombine micromixer by using a disposable syringe to form the IMB-Salmonella-INC sandwich conjugates. Then, the conjugates were captured in the separation chamber using a magnetic field, and the top layer was slid to connect the washing chamber with the separation chamber for washing away excessive INCs. Finally, the top layer was slid to connect the catalysis chamber with the separation chamber, and the colorless substrate was catalyzed by the INCs with peroxidase-mimic activity to generate color change, followed by using a smartphone app to collect and analyze the image to determine the bacterial concentration. This all-in-one microfluidic biosensor enabled simple detection of Salmonella as low as 101.2 CFU mL-1 within 30 min and was featured with low cost, straightforward operation, and compact design.

Extending the shelf life of HLM chips through freeze-drying of human liver microsomes immobilized onto thiol-ene micropillar arrays.

Microfluidic flow reactors functionalized with immobilized human liver microsomes (HLM chips) represent a powerful tool for drug discovery and development by enabling mechanism-based enzyme inhibition studies under flow-through conditions. Additionally, HLM chips may be exploited in streamlined production of human drug metabolites for subsequent microfluidic in vitro organ models or as metabolite standards for drug safety assessment. However, the limited shelf life of the biofunctionalized microreactors generally poses a major barrier to their commercial adaptation in terms of both storage and shipping. The shelf life of the HLM chips in the wetted state is ca. 2-3 weeks only and requires cold storage at 4 °C. In this study, we developed a freeze-drying method for lyophilization of HLMs that are readily immobilized inside microfluidic pillar arrays made from off-stoichiometric thiol-ene polymer. The success of lyophilization was evaluated by monitoring the cytochrome P450 and UDP-glucuronosyltransferase enzyme activities of rehydrated HLMs for several months post-freeze-drying. By adapting the freeze-drying protocol, the HLM chips could be stored at room temperature (protected from light and moisture) for at least 9 months (n = 2 independent batches) and up to 16 months at best, with recovered enzyme activities within 60-120% of the non-freeze-dried control chips. This is a major improvement over the cold-storage requirement and the limited shelf life of the non-freeze-dried HLM chips, which can significantly ease the design of experiments, decrease energy consumption during storage, and reduce the shipping costs with a view to commercial adaptation.

Label-Free Imaging of Mesenchymal Stem Cell Spheroid Differentiation with Flexible-Probe SECM and a Microfluidic Device.

Mesenchymal stem cells (MSCs) have emerged as an indispensable source for stem cell research and preclinical studies due to their capacity for in vitro proliferation and their potential to differentiate into mesodermal lineages, particularly into osteoblasts. This capability has propelled their application in the fields of bone regeneration and osteochondral repair. Traditional methodologies for assessing the differentiation status of MSCs necessitate invasive procedures such as cell lysis or fixation. In this study, we introduce a nondestructive technique that utilizes an integrated label-free approach to evaluate the osteogenic maturation of MSC spheroid aggregates. This method employs scanning electrochemical microscopy (SECM) with a flexible probe in conjunction with a top-removable microfluidic device designed for easy SECM access. By tracking the production rate of p-aminophenol (PAP) in the generation/collection mode and assessing morphological changes via the negative feedback mode using [Ru(NH3)6]Cl3 (Ruhex), we can discern variations in the alkaline phosphatase (ALP) activity indicative of osteogenic differentiation. This innovative strategy enables the direct evaluation of osteogenic differentiation in MSC spheroids cultured within microwell arrays without necessitating any labeling procedures. The utilization of a flexible microelectrode as the probe that scans in contact mode (with probe-substrate distances potentially as minimal as 0 µm) affords enhanced resolution compared to the traditional stiff-probe technique. Furthermore, this method is compatible with subsequent molecular biology assays, including gene expression analysis and immunofluorescence, thereby confirming the electrochemical findings and establishing the validity of this integrative approach.

Multi-dimensional microfluidic paper-based analytical devices (µPADs) for noninvasive testing: A review of structural design and applications.

The rapid emergence of microfluidic paper-based devices as point-of-care testing (POCT) tools for early disease diagnosis and health monitoring, particularly in resource-limited areas, holds immense potential for enhancing healthcare accessibility. Leveraging the numerous advantages of paper, such as capillary-driven flow, porous structure, hydrophilic functional groups, biodegradability, cost-effectiveness, and flexibility, it has become a pivotal choice for microfluidic substrates. The repertoire of microfluidic paper-based devices includes one-dimensional lateral flow assays (1D LFAs), two-dimensional microfluidic paper-based analytical devices (2D µPADs), and three-dimensional (3D) µPADs. In this comprehensive review, we provide and examine crucial information related to paper substrates, design strategies, and detection methods in multi-dimensional microfluidic paper-based devices. We also investigate potential applications of microfluidic paper-based devices for detecting viruses, metabolites and hormones in non-invasive samples such as human saliva, sweat and urine. Additionally, we delve into capillary-driven flow alternative theoretical models of fluids within the paper to provide guidance. Finally, we critically examine the potential for future developments and address challenges for multi-dimensional microfluidic paper-based devices in advancing noninvasive early diagnosis and health monitoring. This article showcases their transformative impact on healthcare, paving the way for enhanced medical services worldwide.

A rapid and facile immunoassay for C-reactive protein using PDMS-based digital magnetofluidics.

BACKGROUND: C-reactive protein has been reported as a biomarker of inflammation caused by acute injury, infection or tissue damage and also a prediction marker of cardiovascular diseases. Commonly, the gold standard for the detection of CRP is enzyme-linked immunosorbent assays (ELISAs). Normally, traditional immunoassays in multiwell plates typically suffer from prolonged assay time due to slow mass transport controlled by diffusion. Herein, a PDMS based magnetofluidic approach has been applied for a rapid and facile immunoassay using a sandwich enzyme-linked immunosorbent assay (ELISA) for the analysis of CRP. RESULTS: Due to the superhydrophobic PDMS, droplets of reagent and sample solutions were obtained when pipetting all solutions onto the PDMS substrate. These droplets were individually controlled by an external magnet to perform the assays. Magnetic beads immobilized with a capture antibody were not only used for immunomagnetic separation (IMS) of the captured CRP from the sample matrix, but also used as a carrier for droplet movement on the magnetofluidic device, expediting the immunoassay procedure, especially washing steps. The immunoassay of CRP was successfully performed within 1 h with a limit of detection of 0.015 mg L-1 in the concentration range of 0.1-10 mg L-1. The recovery percentages of CRP spiked in human serum were found in the range of 90-114 % with %RSD of less than 5 %, indicating acceptable accuracy and precision. SIGNIFICANCE: By individually controlling the droplet movement using an external magnet, all steps of immunoassays were simply and rapidly performed. In addition, the microfluidic format allows for small volumes of reagents and samples and rapid assay kinetics. Therefore, the proposed magnetofluidic approach has shown its potential of becoming a rapid, facile and cost-effective method to perform traditional immunoassays in a variety of applications. In addition, the proposed approach is also particularly well-suited for analyses/reactions with multiple steps.

1-Octanol-assisted ultra-small volume droplet microfluidics with nanoelectrospray ionization mass spectrometry.

BACKGROUND: Droplet microfluidics with push-pull and microdialysis sampling from brain slices, cultured cells and engineered tissues produce low volume mass limited samples containing analytes sampled from the extracellular space. This sampling approach coupled to mass spectrometry (MS) detection allows evaluation of time-dependent chemical changes. Our goal is an approach for continuous sampling and segregation of extracellular samples into picoliter droplets followed by the characterization of the droplets using nanoelectrospray ionization (nESI) MS. The main focus here is the optimization of the carrier oil for the microfluidic device that neither affects the stability of picoliter droplets nor compatibility with MS detection of a range of analytes. RESULTS: We developed and characterized a 1-octanol-assisted ultra-small volume droplet microfluidic nESI MS system for the analysis of neurotransmitters in distinct samples including cerebrospinal fluid (CSF). The use of a 1-octanol oil phase was effective for generation of aqueous droplets as small as 65 pL and enabled detection of acetylcholine (ACh) and gamma-aminobutyric acid (GABA) in water and artificial CSF. Continuous MS analysis of droplets for extended periods up to 220 min validated the long-term stability of droplet generation and analyte detection by nESI-MS. As an example, ACh response demonstrated a linear working range (R2 = 0.99) between 0.4 µM and 25 µM with a limit of detection of 370 nM (24 amol), enabling its quantitation in rodent CSF. SIGNIFICANCE: The established droplet microfluidics - nESI MS approach allows the analysis of microenvironments at high spatiotemporal resolution. The approach may allow microsampling and monitoring of spatiotemporal dynamics of neurochemicals and drugs in the brain and spinal cord of live animals.

Detachable acoustofluidic droplet-sorter.

BACKGROUND: Cell sorting is crucial in isolating specific cell populations. It enables detailed analysis of their functions and characteristics and plays a vital role in disease diagnosis, drug discovery, and regenerative medicine. Fluorescence-activated cell sorting (FACS) is considered the gold standard for high-speed single-cell sorting. However, its high cost, complex instrumentation, and lack of portability are significant limitations. Additionally, the high pressure and electric fields used in FACS can harm cell integrity. In this work, an acoustofluidic device was developed in combination with surface acoustic wave (SAW) and droplet microfluidics to isolate single-cell droplets with high purity while maintaining high cell viability. RESULT: Human embryonic kidney cells, transfected with fluorescent reporter plasmids, were used to demonstrate the targeted droplet sorting containing single cells. The acoustofluidic sorter achieved a recovery rate of 81 % and an accuracy rate higher than 97 %. The device maintained a cell viability rate of 95 % and demonstrated repeatability over 20 consecutive trials without compromising efficiency, thus underscoring its reliability. Thermal image analysis revealed that the temperature of the interdigital transducer (IDT) during SAW operation remained within the permissible range for maintaining cell viability. SIGNIFICANCE: The findings highlighted the sensitivity and effectiveness of the developed acoustofluidic device as a tool for single-cell sorting. The detachable microfluidic chip design enables the reusability of the expensive IDT, making it cost-effective and reducing the risk of cross-contamination between different biological samples. The results underscore its capability to accurately isolate individual cells on the basis of specific criteria, showcasing its potential to advance research and clinical applications requiring precise cell sorting methodologies.

Microfluidic Lab-on-CMOS Packaging Using Wafer-Level Molding and 3D-Printed Interconnects.

Lab-on-a-chip (LoC) technologies continue to promise lower cost and more accessible platforms for performing biomedical testing in low-cost and disposable form factors. Lab-on-CMOS or lab-on-microchip methods extend this paradigm by merging passive LoC systems with active complementary metal-oxide semiconductor (CMOS) integrated circuits (IC) to enable front-end signal conditioning and digitization immediately next to sensors in fluid channels. However, integrating ICs with microfluidics remains a challenge due to size mismatch and geometric constraints, such as non-planar wirebonds or flip-chip approaches in conflict with planar microfluidics. In this work, we present a hybrid packaging solution for IC-enabled microfluidic sensor systems. Our approach uses a combination of wafer-level molding and direct-write 3D printed interconnects, which are compatible with post-fabrication of planar dielectric and microfluidic layers. In addition, high-resolution direct-write printing can be used to rapidly fabricate electrical interconnects at a scale compatible with IC packaging without the need for fixed tooling. Two demonstration sensor-in-package systems with integrated microfluidics are shown, including measurement of electrical impedance and optical scattering to detect and size particles flowing through microfluidic channels over or adjacent to CMOS sensor and read-out ICs. The approach enables fabrication of impedance measurement electrodes less than 1 mm from the readout IC, directly on package surface. As shown, direct fluid contact with the IC surface is prevented by passivation, but long-term this approach can also enable fluid access to IC-integrated electrodes or other top-level IC features, making it broadly enabling for lab-on-CMOS applications.

Application of Microfluidic Devices for Automated Two-Step Radiolabeling of Antibodies.

Radioimmunoconjugates (RICs) composed of tumor-targeting monoclonal antibodies and radionuclides have been developed for diagnostic and therapeutic application. A new radiolabeling method using microfluidic devices is expected to facilitate simpler and more rapid synthesis of RICs. In the microfluidic method, microfluidic chips can promote the reaction between reactants by mixing them efficiently, and pumping systems enable automated synthesis. In this study, we synthesized RICs by the pre-labeling method, in which the radiometal is coordinated to the chelator and then the radiolabeled chelator is incorporated into the antibodies, using microfluidic devices for the first time. As a result of examining the reaction parameters including the material of mixing units, reaction temperature, and flow rate, RICs with radiochemical purity (RCP) exceeding 90% were obtained. These high-purity RICs were successfully synthesized without any purification simply by pumping three solutions of a chelating agent, radiometal, and antibody into microfluidic devices. Under the same conditions, the RCP of RICs labeled by conventional methods was below 50%. These findings indicate the utility of microfluidic devices for automatic and rapid synthesis of high-quality RICs.

Live Imaging Analysis of Axonal Regeneration in Human iPSC-Derived Motor Neurons Using a Microfluidic System.

Axonal damage is a common feature of traumatic injury and neurodegenerative disease. The capacity for axons to regenerate and to recover functionality after injury is a phenomenon that is seen readily in the peripheral nervous system, especially in rodent models, but human axonal regeneration is limited and does not lead to full functional recovery. Here we describe a system where dynamics of human axonal outgrowth and regeneration can be evaluated via live imaging of human-induced pluripotent stem cell (hiPSC)-derived neurons cultured in microfluidic systems, in which cell bodies are isolated from their axons. This system could aid in studying axonal outgrowth dynamics and could be useful for testing potential drugs that encourage regeneration and repair of the nervous system.

The sensing of circRNA with tetrahedral DNA nanostructure modified microfluidic chip.

BACKGROUND: Circular ribonucleic acids (circRNAs) are a type of covalently closed noncoding RNA with disease-relevant expressions, making them promising biomarkers for diagnosis and prognosis. Accurate quantification of circRNA in biological samples is a necessity for their clinical application. So far, methods developed for detecting circRNAs include northern blotting, reverse transcription quantitative polymerase chain reaction (RT-qPCR), microarray analysis, and RNA sequencing. These methods generally suffer from disadvantages such as large sample consumption, cumbersome process, low selectivity, leading to inaccurate quantification of circRNA. It was thought that the above drawbacks could be eliminated by the construction of a microfluidic sensor. RESULTS: Herein, for the first time, a microfluidic sensor was constructed for circRNA analysis by using tetrahedral DNA nanostructure (TDN) as the skeleton for recognition probes and target-initiated hybridization chain reaction (HCR) as the signal amplification strategy. In the presence of circRNA, the recognition probe targets the circRNA-specific backsplice junction (BSJ). The captured circRNA then triggers the HCR by reacting with two hairpin species whose ends were labeled with 6-FAM, producing long DNA strands with abundant fluorescent labels. By using circ_0061276 as a model circRNA, this method has proven to be able to detect circRNA of attomolar concentration. It also eliminated the interference of linear RNA counterpart, showing high selectivity towards circRNA. The detection process can be implemented isothermally and does not require expensive complicated instruments. Moreover, this biosensor exhibited good performance in analyzing circRNA targets in total RNA extracted from cancer cells. SIGNIFICANCE: This represents the first microfluidic system for detection of circRNA. The biosensor showed merits such as ease of use, low-cost, small sample consumption, high sensitivity and specificity, and good reliability in complex biological matrix, providing a facile tool for circRNA analysis and related disease diagnosis in point-of care application scenes.

Paper-Based Microfluidic Analytical Device Patterned by Label Printer for Point-of-Care Blood Glucose and Hematocrit Detection Using 3D-Printed Smartphone Cassette.

This study presents a portable, low-cost, point-of-care (POC) system for the simultaneous detection of blood glucose and hematocrit. The system consists of a disposable origami microfluidic paper-based analytical device (µPAD) for plasma separation, filtration, and reaction functions and a 3D-printed cassette for hematocrit and blood glucose detection using a smartphone. The origami µPAD is patterned using a cost-effective label printing technique instead of the conventional wax printing method. The 3D-printed cassette incorporates an array of LED lights, which mitigates the effects of intensity variations in the ambient light and hence improves the accuracy of the blood glucose and hematocrit concentration measurements. The hematocrit concentration is determined quantitatively by measuring the distance of plasma wicking along the upper layer of the origami µPAD, which is pretreated with sodium chloride and Tween 20 to induce dehydration and aggregation of the red blood cells. The filtered plasma also penetrates to the lower layer of the origami µPAD, where it reacts with embedded colorimetric assay reagents to produce a yellowish-brown complex. A color image of the reaction complex is captured using a smartphone inserted into the 3D-printed cassette. The image is analyzed using self-written RGB software to quantify the blood glucose concentration. The calibration results indicate that the proposed detection platform provides an accurate assessment of the blood glucose level over the range of 45-630 mg/dL (R2 = 0.9958). The practical feasibility of the proposed platform is demonstrated by measuring the blood glucose and hematocrit concentrations in 13 human whole blood samples. Taking the measurements obtained from commercial glucose and hematocrit meters as a benchmark, the proposed system has a differential of no more than 6.4% for blood glucose detection and 9.1% for hematocrit detection. Overall, the results confirm that the proposed µPAD is a promising solution for cost-effective and reliable POC health monitoring.