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CRISPR-based diagnostics (CRISPR/Dx) have revolutionized the field of molecular diagnostics. It enables home self-test, field-deployable, and point-of-care testing (POCT). Despite the great potential of CRISPR/Dx in diagnoses of biologically complex diseases, preamplification of the template often is required for the sensitive detection of low-abundance nucleic acids. Various amplification-free CRISPR/Dx systems were recently developed to enhance signal detection at sufficient sensitivity. Broadly, these amplification-free CRISPR/Dx systems are classified into five groups depending on the signal enhancement strategies employed: CRISPR/Cas12a and/or CRISPR/Cas13a are integrated with: (1) other catalytic enzymes (Cas14a, Csm6, Argonaute, duplex-specific nuclease, nanozyme, or T7 exonuclease), (2) rational-designed oligonucleotides (multivalent aptamer, tetrahedral DNA framework, RNA G-quadruplexes, DNA roller machine, switchable-caged guide RNA, hybrid locked RNA/DNA probe, hybridized cascade probe, or "U" rich stem-loop RNA), (3) nanomaterials (nanophotonic structure, gold nanoparticle, micromotor, or microbeads), (4) electrochemical and piezoelectric plate biosensors (SERS nanoprobes, graphene field-effect transistor, redox probe, or primer exchange reaction), or (5) cutting-edge detection technology platforms (digital bioanalysis, droplet microfluidic, smartphone camera, or single nanoparticle counting). Herein, we critically discuss the advances, pitfalls and future perspectives for these amplification-free CRISPR/Dx systems in nucleic acids detection. The continued refinement of these CRISPR/Dx systems will pave the road for rapid, cost-effective, ultrasensitive, and ultraspecific on-site detection without resorting to target amplification, with the ultimate goal of establishing CRISPR/Dx as the paragon of diagnostics.

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A laboratory-based lateral flow (LF) test that utilizes up-converting reporter particles (UCP) for ultrasensitive quantification of Schistosoma circulating anodic antigen (CAA) in urine is a well-accepted test to identify active infection. However, this UCP-LF CAA test requires sample pre-treatment steps not compatible with field applications. Flow, a new low-cost disposable, allows integration of large-volume pre-concentration of urine analytes and LF detection into a single field-deployable device. We assessed a prototype Flow-Schistosoma (Flow-S) device with an integrated UCP-LF CAA test strip, omitting all laboratory-based steps, to enable diagnosis of active Schistosoma infection in the field using urine. Flow-S is designed for large-volume (5-20 mL) urine, applying passive paper-based filtration and antibody-based CAA concentration. Samples tested for schistosome infection were collected from women of reproductive age living in a Tanzania region where S. haematobium infection is endemic. Fifteen negative and fifteen positive urine samples, selected based on CAA levels quantified in paired serum, were analyzed with the prototype Flow-S. The current Flow-S prototype, with an analytical lower detection limit of 1 pg CAA/mL, produced results correlated with the laboratory-based UCP-LF CAA test. Urine precipitates occurred in frozen banked samples and affected accurate quantification; however, this should not occur in fresh urine. Based on the findings of this study, Flow-S appears suitable to replace the urine pre-treatment required for the laboratory-based UCP-LF CAA test, thus allowing true field-based applications with fresh urine samples. The urine precipitates observed with frozen samples, though less important given the goal of testing fresh urines, warrant additional investigation to evaluate methods for mitigation. Flow-S devices permit testing of pooled urine samples with applications for population stratified testing. A field test with fresh urine samples, a further optimized Flow-S device, and larger statistical power has been scheduled.

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The mpox outbreak has subdued with fewer reported cases at the present in high-income countries. It is known that mpox virus (MPXV) infection has been epidemic for more than 50 years in African countries. The ancestral MPXV strain has changed into multiple clades, indicating the ongoing evolution of MPXV, which reflects the historical neglect of mpox in Africa, especially after smallpox eradication, and bestows the danger of more severe mpox epidemics in the future. It is thus imperative to continue the development of mpox diagnostics and treatments so we can be prepared in the event of a new mpox epidemic. In this study, we have developed an MPXV detection tool that leverages the recombinase-aid amplification assay by integrating lateral flow strips (RAA-LF) and one-step sample DNA preparation, with visible readout, no need of laboratory instrument, and ready for field deployment. The detection limit reaches 10 copies per reaction. The performance of our RAA-FL assay in diagnosing mpox clinical samples is on par with that of the quantitative polymerase chain reaction (PCR) assay. Taken together, we have developed a point-of-care RAA-LF method of high accuracy and sensitivity, readily deployable for field detection of MPXV. This diagnostic tool is expected to improve and accelerate field- and self-diagnosis, allow timely isolation and treatment, reduce the spread of MPXV, thus effectively mitigate MPXV outbreak in the future.

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Here, we developed a field-deployable molecular diagnostic kit for the detection of RNA viruses that operates in a pipette-free manner. The kit is composed of acrylic sticks, PCR tubes, and palm-sized three-dimensional(3D)-printed heaters operated by batteries. The kit performs RNA extraction, reverse transcriptase loop-mediated isothermal amplification (RT-LAMP), and visual detection in one kit. An acrylic stick was engraved with one shallow and one deep cylindrical chamber at each end for the insertion of an FTA card and ethidium homodimer-1 (EthD-1), respectively, to perform RNA extraction/purification and bimodal visual detection of the target amplicons. First, an intercalation of EthD-1 into the target DNA initially produces fluorescence upon UV illumination. Next, the addition of a strong oxidant, in this case sodium (meta) periodate (NaIO4 ), produces intense aggregates in the presence of EthD-1-intercalated DNA, realized by electrostatic interaction. In the absence of the target amplicon, no fluorescence or aggregates are observed. Using this kit, two major infectious viruses-severe fever with thrombocytopenia syndrome virus (SFTSV) and severe acute respiratory syndrome coronavirus (SARS-CoV-2)-were successfully detected in 1 h, and the limits of detection (LOD) were approximately 1 virus µL-1 for SFTSV and 103 copies µL-1 for SARS-CoV-2 RNA. The introduced kit is portable, end-user-friendly, and can be operated in a pipette-free manner, paving the way for simple and convenient virus detection in resource-limited settings.

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Leishmaniasis is a neglected tropical disease endemic primarily to low- and middle-income countries, for which there has been inadequate development of affordable, safe, and efficacious therapies. Clinical manifestations of leishmaniasis range from self-healing skin lesions to lethal visceral infection with chances of relapse. Although treatments are available, secondary effects limit their use outside the clinic and negatively impact the quality of life of patients in endemic areas. Other non-medicinal treatments, such as thermotherapies, are limited to use in patients with cutaneous leishmaniasis but not with visceral infection. Recent studies shed light to mechanisms through which Leishmania can persist by hiding in cellular safe havens, even after chemotherapies. This review focuses on exploring the cellular niches that Leishmania parasites may be leveraging to persist within the host. Also, the cellular, metabolic, and molecular implications of Leishmania infection and how those could be targeted for therapeutic purposes are discussed. Other therapies, such as those developed against cancer or for manipulation of the ferroptosis pathway, are proposed as possible treatments against leishmaniasis due to their mechanisms of action. In particular, treatments that target hematopoietic stem cells and monocytes, which have recently been found to be necessary components to sustain the infection and provide a safe niche for the parasites are discussed in this review as potential field-deployable treatments against leishmaniasis.

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CRISPR-based nucleic acid detection is easy to implement, field deployable, and always coupled with isothermal amplification to improve the sensitivity. However, the conventional detection requires two separate steps, which can cause long-lasting amplicon aerosol contaminants, hence leading to false-positive results. To address this problem, we proposed a one-tube assay based on CRISPR-Cas13a coupled with reverse transcription-recombinase polymerase amplification to avoid aerosol pollution. The one-tube assay could be completed within 40 min with a sensitivity of up to 180 copies of RNA per reaction, and exhibited no cross reactivity with two related coronaviruses. Our technology showed reproducibility with relative standard deviation of 4.6% responding to 1 fM nucleic acid for three times. It could be used to detect SARS-CoV-2 nucleic acids in raw wastewater with a limit of detection of 103 copies/mL. We also validated the practicability of this technique for viral detection in environmental water samples by detecting SARS-CoV-2 in wastewater, which were not detectable by RT-qPCR technology, showing resistance of this technology to wastewater matrix. It is anticipated that the robustness and high sensitivity will significantly promote the development of a point-of-care method in environmental virus monitoring.

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Soft robotics technology can aid in achieving United Nations' Sustainable Development Goals (SDGs) and the Paris Climate Agreement through development of autonomous, environmentally responsible machines powered by renewable energy. By utilizing soft robotics, we can mitigate the detrimental effects of climate change on human society and the natural world through fostering adaptation, restoration, and remediation. Moreover, the implementation of soft robotics can lead to groundbreaking discoveries in material science, biology, control systems, energy efficiency, and sustainable manufacturing processes. However, to achieve these goals, we need further improvements in understanding biological principles at the basis of embodied and physical intelligence, environment-friendly materials, and energy-saving strategies to design and manufacture self-piloting and field-ready soft robots. This paper provides insights on how soft robotics can address the pressing issue of environmental sustainability. Sustainable manufacturing of soft robots at a large scale, exploring the potential of biodegradable and bioinspired materials, and integrating onboard renewable energy sources to promote autonomy and intelligence are some of the urgent challenges of this field that we discuss in this paper. Specifically, we will present field-ready soft robots that address targeted productive applications in urban farming, healthcare, land and ocean preservation, disaster remediation, and clean and affordable energy, thus supporting some of the SDGs. By embracing soft robotics as a solution, we can concretely support economic growth and sustainable industry, drive solutions for environment protection and clean energy, and improve overall health and well-being.

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Point-of-care testing (POCT) can be the method of choice for detecting infectious pathogens; these pathogens are responsible for not only infectious diseases such as COVID-19, but also for certain types of cancers. For example, infections by human papillomavirus (HPV) or Helicobacter pylori (H. pylori) are the main cause of cervical and stomach cancers, respectively. COVID-19 and many cancers are treatable with early diagnoses using POCT. A variety of nucleic acid testing have been developed for use in resource-limited environments. However, questions like unintegrated nucleic acid extraction, open detection systems increase the risk of cross-contamination, and dependence on expensive equipment and alternating current (AC) power supply, significantly limit the application of POCT, especially for on-site testing. In this paper, a simple portable platform is reported capable of rapid sample-to-answer testing within 30 min based on recombinase polymerase amplification (RPA) at a lower temperature, to detect SARS-CoV-2 virus and H. pylori bacteria with a limit of detection as low as 4 × 102 copies mL-1 . The platform used a battery-powered portable reader for on-chip one-pot amplification and fluorescence detection, and can test for multiple (up to four) infectious pathogens simultaneously. This platform can provide an alternative method for fast and reliable on-site diagnostic testing.

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Acute Hepatopancreatic Necrosis Disease (AHPND), caused by bacterial isolates expressing PirAB binary toxins, represents the severest and most economically destructive disease affecting penaeid shrimp. Its rapid disease progression and associated massive mortalities call for vigilant monitoring and early diagnosis, but molecular detection methods that simultaneously satisfy the requirements of sensitivity, specificity, and portability are still scarce. In this work, the CRISPR-Cas12a technology was harnessed for the development of two fluorescent assays compatible with naked-eye visualization. The first assay, AP4-Cas12a, was based on the OIE-recommended AP4 two-tubed nested PCR method and was designed to bypass the time-consuming and potentially hazardous agarose gel electrophoresis step. Using AP4-Cas12a, the detection limit of 10 copies per reaction could be achieved within less than 30 minutes post-PCR. The second assay, RPA-Cas12a, utilized recombinase polymerase amplification (RPA) to rapidly and isothermally amplify the target DNA, followed by amplicon detection by Cas12a, resulting in a protocol that can be completed in less than an hour at a constant temperature of 37°C. The detection limit of RPA-Cas12a is 100 copies of plasmid DNA or 100 fg of bacterial genomic DNA per reaction. Importantly, we validated that both assays are compatible with a previously reported smartphone-based device for facile visualization of fluorescence, thereby providing an affordable option that requires less consumables than lateral flow detection. Using this portable device for readouts, the AP4-Cas12a and RPA-Cas12a methods showed excellent concordance with the AP4-agarose gel electrophoresis approach in the evaluation of clinical samples. Therefore, the developed Cas12a assays have the potential to streamline both in-laboratory and onsite diagnosis of AHPND.

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Using pesticides is a common agricultural and horticultural practice to serve as a control against weeds, fungi, and insects in plant systems. The application of these chemical agents is usually by spraying them on the crop or plant. However, this methodology is not highly directional, and so only a fraction of the pesticide actually adsorbs onto the plant, and the rest seeps through into the soil base contaminating its composition and eventually leaching into groundwater sources. Electrochemical sensors which are more practical for in situ analysis used for pesticide detection in soil runoff systems are still in dearth, while the ones published in the literature are attributed with complex sensor modification/functionalization and preprocessing of samples. Hence, in this work, we present a highly intuitive electroanalytical sensor approach toward rapid (10 min), on-demand screening of commonly used pesticides-glyphosate and atrazine-in soil runoff. The proposed sensor functions based on the affinity biosensing mechanism driven via thiol cross-linker and antibody receptors that holistically behaves as a recognition immunoassay stack that is specific and sensitive to track test pesticide analytes. Then, this developed sensor is integrated further to create a pesticide-sensing ecosystem using a front-end field-deployable smart device. The method put forward in this work is compared and validated against a standard laboratory potentiostat instrument to determine efficacy, feasibility, and robustness for a point-of-use (PoU) setting yielding LoD levels of 0.001 ng/ml for atrazine and 1 ng/ml for glyphosate. Also, the ML model integration resulted in an accurate prediction rate of ≈80% in real soil samples. Therefore, a universal pesticide screening analytical device is designed, fabricated, and tested for pesticide assessment in real soil runoff samples.

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The global equine industry provides significant economic contributions worldwide, producing approximately USD $300 billion annually. However, with the continuous national and international movement and importation of horses, there is an ongoing threat of a viral outbreak causing large epidemics and subsequent significant economic losses. Additionally, horses serve as a host for several zoonotic diseases that could cause significant human health problems. The ability to rapidly diagnose equine viral diseases early could lead to better management, treatment, and biosecurity strategies. Current serological and molecular methods cannot be field-deployable and are not suitable for resource-poor laboratories due to the requirement of expensive equipment and trained personnel. Recently, isothermal nucleic acid amplification technologies, such as loop-mediated isothermal amplification (LAMP) and insulated isothermal polymerase chain reaction (iiPCR), have been developed to be utilized in-field, and provide rapid results within an hour. We will review current isothermal diagnostic techniques available to diagnose equine viruses of biosecurity and zoonotic concern and provide insight into their potential for in-field deployment.

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Rathayibacter toxicus is a toxigenic bacterial pathogen of several grass species and is responsible for massive livestock deaths in Australia and South Africa. Due to concern for animal health and livestock industries, it was designated a U.S. Select Agent. A rapid, accurate, and sensitive in-field detection method was designed to assist biosecurity surveillance surveys and to support export certification of annual ryegrass hay and seed. Complete genomes from all known R. toxicus populations were explored, unique diagnostic sequences identified, and target-specific primers and a probe for recombinase polymerase amplification (RPA) and endpoint PCR were designed. The RPA reaction ran at 37 °C and a lateral flow device (LFD) was used to visualize the amplified products. To enhance reliability and accuracy, primers and probes were also designed to detect portions of host ITS regions. RPA assay specificity and sensitivity were compared to endpoint PCR using appropriate inclusivity and exclusivity panels. The RPA assay sensitivity (10 fg) was 10 times more sensitive than endpoint PCR with and without a host DNA background. In comparative tests, the RPA assay was unaffected by plant-derived amplification inhibitors, unlike the LAMP and end-point PCR assays. In-field validation of the RPA assay at multiple sites in South Australia confirmed the efficiency, specificity, and applicability of the RPA assay. The RPA assay will support disease management and evidence-based in-field biosecurity decisions.

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Recent advances in smartphone technologies have opened the door to the development of accessible, highly portable sensing tools capable of accurate and reliable data collection in a range of environmental settings. In this article, we introduce a low-cost smartphone-based hyperspectral imaging system that can convert a standard smartphone camera into a visible wavelength hyperspectral sensor for ca. £100. To the best of our knowledge, this represents the first smartphone capable of hyperspectral data collection without the need for extensive post processing. The Hyperspectral Smartphone's abilities are tested in a variety of environmental applications and its capabilities directly compared to the laboratory-based analogue from our previous research, as well as the wider existing literature. The Hyperspectral Smartphone is capable of accurate, laboratory- and field-based hyperspectral data collection, demonstrating the significant promise of both this device and smartphone-based hyperspectral imaging as a whole.

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For the past two decades, microbial monitoring of the International Space Station (ISS) has relied on culture-dependent methods that require return to Earth for analysis. This has a number of limitations, with the most significant being bias towards the detection of culturable organisms and the inherent delay between sample collection and ground-based analysis. In recent years, portable and easy-to-use molecular-based tools, such as Oxford Nanopore Technologies' MinION™ sequencer and miniPCR bio's miniPCR™ thermal cycler, have been validated onboard the ISS. Here, we report on the development, validation, and implementation of a swab-to-sequencer method that provides a culture-independent solution to real-time microbial profiling onboard the ISS. Method development focused on analysis of swabs collected in a low-biomass environment with limited facility resources and stringent controls on allowed processes and reagents. ISS-optimized procedures included enzymatic DNA extraction from a swab tip, bead-based purifications, altered buffers, and the use of miniPCR and the MinION. Validation was conducted through extensive ground-based assessments comparing current standard culture-dependent and newly developed culture-independent methods. Similar microbial distributions were observed between the two methods; however, as expected, the culture-independent data revealed microbial profiles with greater diversity. Protocol optimization and verification was established during NASA Extreme Environment Mission Operations (NEEMO) analog missions 21 and 22, respectively. Unique microbial profiles obtained from analog testing validated the swab-to-sequencer method in an extreme environment. Finally, four independent swab-to-sequencer experiments were conducted onboard the ISS by two crewmembers. Microorganisms identified from ISS swabs were consistent with historical culture-based data, and primarily consisted of commonly observed human-associated microbes. This simplified method has been streamlined for high ease-of-use for a non-trained crew to complete in an extreme environment, thereby enabling environmental and human health diagnostics in real-time as future missions take us beyond low-Earth orbit.

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Management of the coronavirus disease 2019 (COVID-19) pandemic requires widespread testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A main limitation for widespread SARS-CoV-2 testing is the global shortage of essential supplies, among them RNA extraction kits. The need for commercial RNA extraction kits places a bottleneck on tests that detect SARS-CoV-2 genetic material, including PCR-based reference tests. Here, we propose an alternative method we call PEARL (precipitation-enhanced analyte retrieval) that addresses this limitation. PEARL uses a lysis solution that disrupts cell membranes and viral envelopes while simultaneously providing conditions suitable for alcohol-based precipitation of RNA, DNA, and proteins. PEARL is a fast, low-cost, and simple method that uses common laboratory reagents and offers performance comparable to that of commercial RNA extraction kits. PEARL offers an alternative method to isolate host and pathogen nucleic acids and proteins to streamline the detection of DNA and RNA viruses, including SARS-CoV-2.

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Driven by complex and interconnected factors, including population growth, climate change, and geopolitics, infectious diseases represent one of the greatest healthcare challenges of the 21st century. Diagnostic technologies are the first line of defense in the fight against infectious disease, providing critical information to inform epidemiological models, track diseases, decide treatment choices, and ultimately prevent epidemics. The diagnosis of infectious disease at the genomic level using nucleic acid disease biomarkers has proven to be the most effective approach to date. Such methods rely heavily on enzymes to specifically amplify or detect nucleic acids in complex samples, and significant effort has been exerted to harness the power of enzymes for in vitro nucleic acid diagnostics. Unfortunately, significant challenges limit the potential of enzyme-assisted nucleic acid diagnostics, particularly when translating diagnostic technologies from the lab toward the point-of-use or point-of-care. Herein, we discuss the current state of the field and highlight cross-disciplinary efforts to solve the challenges associated with the successful deployment of this important class of diagnostics at or near the point-of-care.

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Dickeya spp. cause blackleg and soft rot diseases of potato and several other plant species worldwide, resulting in high economic losses. Rapid detection and identification of the pathogen is essential for facilitating efficient disease management. Our aim in this research was to develop a rapid and field-deployable recombinase polymerase amplification (RPA) assay coupled with a lateral flow device (LFD) that will accurately detect Dickeya spp. in infected plant tissues without the need for DNA isolation. A unique genomic region (mglA/mglC genes) conserved among Dickeya spp. was used to design highly specific robust primers and probes for an RPA assay. Assay specificity was validated with 34 representative strains from all Dickeya spp. and 24 strains from other genera and species; no false positives or negatives were detected. An RPA assay targeting the internal transcribed spacer region of the host genome was included to enhance the reliability and accuracy of the Dickeya assay. The detection limit of 1 fg was determined by both sensitivity and spiked sensitivity assays; no inhibitory effects were observed when 1 µl of host sap, macerated in Tris-EDTA buffer, was added to each reaction in the sensitivity tests. The developed RPA assay is rapid, highly accurate, sensitive, and fully field deployable. It has numerous applications in routine diagnostics, surveillance, biosecurity, and disease management.

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Fusarium circinatum is the causal agent of pitch canker, a lethal disease of pine and other conifers. Since F. circinatum is a quarantine organism, its timely detection could efficiently prevent its introduction into new areas or facilitate spread management in already infected sites. In this study, we developed a sequence-specific probe loop-mediated isothermal amplification (LAMP) assay for F. circinatum using a field-deployable portable instrument. The assay was able to recognize the pathogen in host tissues in just 30 min, and the sensitivity of the assay made it possible to detect even small amounts of F. circinatum DNA (as low as 0.5 pg/µl). The high efficiency of this method suggests its use as a standard diagnostic tool during phytosanitary controls.

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This paper presents a capacitive differential bridge structure with both AC and DC excitation and balancing capability for low cost electrode-solution interfacial capacitance biosensing applications. The proposed series RC balancing structure offers higher sensitivity, lower susceptibility to common-mode interferences, and drift control. To evaluate the bridge performance in practice, possible effects of initial bridge imbalance due to component mismatches are investigated considering the required resolution of the balancing networks, sensitivity, and linearity. This evaluation is also a guideline to designing the balancing networks, balancing algorithm and the proceeding readout interface circuitry. The proposed series RC bridge structure is implemented along with a custom single frequency real-time amplification/filtering readout board with real-time data acquisition and sine fitting. The main specifications for the implemented structure are 8-bit detection resolution if the total expected fractional capacitance change at the interface is roughly 1%. The characterization and measurement results show the effectiveness of the proposed structure in achieving the design target. The implemented structure successfully achieves distinct detection levels for tiny total capacitance change at the electrode-solution interface, utilizing Microcystin-(Leucine-Arginine) toxin dilutions as a proof of concept.

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One of the major challenges for scientists and engineers today is to develop technologies for the improvement of human health in both developed and developing countries. However, the need for cost-effective, high-performance diagnostic techniques is very crucial for providing accessible, affordable, and high-quality healthcare devices. In this context, microfluidic-based devices (MFDs) offer powerful platforms for automation and integration of complex tasks onto a single chip. The distinct advantage of MFDs lies in precise control of the sample quantities and flow rate of samples and reagents that enable quantification and detection of analytes with high resolution and sensitivity. With these excellent properties, microfluidics (MFs) have been used for various applications in healthcare, along with other biological and medical areas. This review focuses on the emerging demands of MFs in different fields such as biomedical diagnostics, environmental analysis, food and agriculture research, etc., in the last three or so years. It also aims to reveal new opportunities in these areas and future prospects of commercial MFDs.

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