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Rapid diagnostic systems are essential in controlling the spread of viral pathogens and efficient patient management. The available technologies for low-cost viral antigen testing have several limitations, including a lack of accuracy and sensitivity. Here, we introduce a platform based on cellulose II nanoparticles (oppositely charged NPan and NPcat) for effective control of surface protein interactions, leading to rapid and sensitive antigen tests. Passivation against non-specific adsorption and augmented immobilization of sensing antibodies is achieved by adjusting the electrostatic charge of the nanoparticles. The interactions affecting the performance of the system are investigated by microgravimetry and confocal imaging. As a proof-of-concept test, SARS-CoV-2 nucleocapsid sensing was carried out by using saliva-wicking by channels that were stencil-printed on paper. We conclude that inkjet-printed NPcat elicits strong optical signals, visible after a few minutes, opening the opportunity for cost-effective and rapid diagnostic. [ABSTRACT FROM AUTHOR]

Paper-based diagnostics offer a promising alternative to traditional diagnostic methods for point-of-care use due to their low cost, ease of use, portability, rapid results, versatility, and low environmental impact. While paper-based serology tests in the form of lateral flow assays can provide rapid test results for past pathogen exposure, they currently lack the accuracy and sensitivity offered by molecular diagnostic tests such as the polymerase chain reaction (PCR). Loop-mediated isothermal amplification (LAMP)—an isothermal nucleic acid amplification test (NAAT)—provides PCR-like performance while simultaneously reducing the instrumentation and assay complexity associated with PCR. In this review, we discuss a newly emerging class of paper-based LAMP platforms that integrates the versatility of paper microfluidics with the accuracy of NAATs. Since its first adoption in 2015, we have discussed all paper-based LAMP platforms in terms of the paper substrates, reagent incorporation techniques, paper platform design, heating hardware, detection methods, and sensitivity and specificity of paper-based LAMP assays. We conclude by identifying the current challenges and future prospects of paper-based NAATs. [ABSTRACT FROM AUTHOR]

Laboratory-based nucleic acid amplification tests (NAATs) are highly sensitive and specific, but they require the transportation of samples to centralized testing facilities and have long turnaround times. During the Coronavirus Disease 2019 (COVID-19) pandemic, substantial advancement has been achieved with the development of paper-based point-of-care (POC) NAATs, offering features such as low cost, being easy to use, and providing rapid sample-to-answer times. Although most of the POC NAATs innovations are towards clinical settings, we have developed a portable, paper-based loop-mediated isothermal amplification (LAMP) testing platform for on-farm applications, capable of detecting Bacteroidales as a fecal contamination biomarker. Our integrated platform includes a drop generator, a heating and imaging unit, and paper-based biosensors, providing sensitive results (limit of detection 3 copies of Bacteroidales per cm2) within an hour of sample collection. We evaluated this integrated platform on a commercial lettuce farm with a concordance of 100% when compared to lab-based tests. Our integrated paper-based LAMP testing platform holds great promise as a reliable and convenient tool for on-site NAATs. We expect that this innovation will encourage the fresh produce industry to adopt NAATs as a complementary tool for decision-making in growing and harvesting. We also hope that our work can stimulate further research in the development of on-farm diagnostic tools for other agricultural applications, leading to improved food safety and technology innovation. [Display omitted] [ABSTRACT FROM AUTHOR]

We report the development and initial validation of a paper-based nucleic acid testing platform that integrates Loop-mediated isothermal amplification (LAMP) with clustered regularly interspaced short palindromic repeats (CRISPR) technology, referred to as PLACID (Paper-based LAMP-CRISPR Integrated Diagnostics). LAMP eliminates the need for thermal cycling, resulting in simplified instrumentation, and the CRISPR-associated protein (Cas 12a) system eliminates false positive signals from LAMP products, resulting in highly selective and sensitive assays. We optimized the assay to perform both amplification and detection entirely on paper, eliminating the need for complex fluid handling steps and lateral flow assay transfers. Additionally, we engineered a smartphone-operated system that includes a low-powered, non-contact IR heating chamber to actuate paper-based LAMP and CRISPR reactions and enable the detection of fluorescent signals from the paper. The platform demonstrates high specificity and sensitivity in detecting nucleic acid targets with a limit of detection of 50 copies/μL. We integrate an equipment-free sample preparation separation technology designed to streamline the preparation of crude samples prior to nucleic acid testing. The practical utility of our platform is demonstrated by the successful detection of spiked SARS-CoV-2 RNA fragments in saliva, E. Coli in soil, and pathogenic E. Coli in clinically fecal samples of infected patients. Furthermore, we demonstrate that the paper-based LAMP CRISPR chips employed in our assays possess a shelf life of several weeks, establishing them as viable candidates for on-site diagnostics. [ABSTRACT FROM AUTHOR]

Paper-based diagnostics offer a promising alternative to traditional diagnostic methods for point-of-care use due to their low cost, ease of use, portability, rapid results, versatility, and low environmental impact. While paper-based serology tests in the form of lateral flow assays can provide rapid test results for past pathogen exposure, they currently lack the accuracy and sensitivity offered by molecular diagnostic tests such as the polymerase chain reaction (PCR). Loop-mediated isothermal amplification (LAMP)—an isothermal nucleic acid amplification test (NAAT)—provides PCR-like performance while simultaneously reducing the instrumentation and assay complexity associated with PCR. In this review, we discuss a newly emerging class of paper-based LAMP platforms that integrates the versatility of paper microfluidics with the accuracy of NAATs. Since its first adoption in 2015, we have discussed all paper-based LAMP platforms in terms of the paper substrates, reagent incorporation techniques, paper platform design, heating hardware, detection methods, and sensitivity and specificity of paper-based LAMP assays. We conclude by identifying the current challenges and future prospects of paper-based NAATs.

Salmonella, the most prevalent food-borne pathogen, poses significant medical and economic threats. Swift and accurate on-site identification and serotyping of Salmonella is crucial to curb its spread and contamination. Here, a synthetic biology cascade reaction is presented on a paper substrate using CRISPR-Cas12a and recombinase polymerase amplification (RPA), enabling the programming of a standard toehold RNA switch for a genome of choice. This approach employs just one toehold RNA switch design to differentiate between two different Salmonella serotypes, i.e., S. Typhimurium and S. Enteritidis, without the need for reengineering the toehold RNA switch. The sensor exhibits high sensitivity, capable of visually detecting as few as 100 copies of the whole genome from a model Salmonella pathogen on a paper substrate. Furthermore, this robust assay is successfully applied to detect whole genomes in contaminated milk and lettuce samples, demonstrating its potential in real sample analysis. Due to its versatility and practical features, genomes from different organisms can be detected by merely changing a single RNA element in this universal cell-free cascade reaction., (© 2024 Wiley‐VCH GmbH.)

Herein, we describe the development of a paper-based device to detect nucleic acids of pathogens of interest in complex samples using loop-mediated isothermal amplification (LAMP) by producing a colorimetric response visible to the human eye. To demonstrate the utility of this device in emerging public health emergencies, we developed and optimized our device to detect SARS-CoV-2 in human saliva without preprocessing. The resulting device was capable of detecting the virus within 60 min and had an analytical sensitivity of 97% and a specificity of 100% with a limit of detection of 200 genomic copies/μL of patient saliva using image analysis. The device consists of a configurable number of reaction zones constructed of Grade 222 chromatography paper separated by 20 mil polystyrene spacers attached to a Melinex® backing via an ARclean® double-sided adhesive. The resulting device is easily configurable to detect multiple targets and has the potential to detect a variety of pathogens simply by changing the LAMP primer sets.

Equipment‐free paper‐based devices with quantitative readout have gained considerable interests in recent years, due to the features of remarkable cheapness, prominent portability, and excellent user friendliness. These equipment‐free strategies have been demonstrated to be able to detect a variety of analytes with good performance, such as metal ions, nucleic acids, and cells, thus showing great potential for point‐of‐care testing in resource‐limited settings. In this review, the latest advances of equipment‐free quantitative readout strategies in paper‐based point‐of‐care testing are summarized, including distance‐based strategy, counting‐based strategy, time‐based strategy, and text‐based strategy. Each type of equipment‐free strategy is sequentially introduced and discussed. Finally, conclusions and perspectives are discussed. [ABSTRACT FROM AUTHOR]

(1) Background: The complexity, amount of time, and the large amount of resource required to perform gold-standard bacteria culture procedures makes it difficult to perform timely pathogenic analyses, especially in areas where such resources are not readily available. A paper-based biochemical analytical tool can potentially tackle problems economically in terms of time and convenience, potentially finding utility in applications where simple and timely detection of bacteria is necessary; (2) Methods: The utility of paper-based MTT-PMS strips was tested using a simple colorimetric analytical methodology; (3) Results: Sufficient evidence was obtained to suggest that the strips can potentially be used as a rapid and convenient early, alternative bacteria screening tool for a variety of applications; (4) Conclusions: The potential of strips for the rapid detection of bacteria compared to standard bacteria culture is a key advantage in certain clinical, agricultural, and environmental applications.

Due to its unique material properties, paper offers many practical advantages as a viable platform for sensing devices. In view of paper-based microfluidic biosensing applications, the covalent immobilization of enzymes with preserved functional activity is highly desirable and ultimately challenging. In the present manuscript, we report an efficient approach to achieving the covalent attachment of certain enzymes on paper fibers via a surface-bound network of hydrophilic polymers bearing protein-modifiable sites. This tailor-made macromolecular system consisting of polar, highly swellable copolymers is anchored to the paper exterior upon light-induced crosslinking of engineered benzophenone motifs. On the other hand, this framework contains active esters that can be efficiently modified by the nucleophiles of biomolecules. This strategy allowed the covalent immobilization of glucose oxidase and horseradish peroxidase onto cotton linters without sacrificing their bioactivities and performance upon surface binding. As a proof-of-concept application, a microfluidic chromatic paper-based glucose sensor was developed and achieved successful glucose detection in a simple yet efficient cascade reaction.

In this work, we report the development of a general strategy for enhancing the efficiency of target capture in immunoassays, using a bifunctional fusion protein construct which incorporates a substrate-anchoring moiety for the high-abundance immobilization of an antigen-binding domain. This approach was informed by the development of a pseudo first-order rate constant model, and tested in a paper-based assay format using a fusion construct consisting of an rcSso7d binding module and a cellulose-binding domain. These rcSso7d-CBD fusion proteins were solubly expressed and purified from bacteria in high molar yields, and enable oriented, high-density adsorption of the rcSso7d binding species to unmodified cellulose within a 30-second incubation period. These findings were validated using two distinct, antigen-specific rcSso7d variants, which were isolated from a yeast surface display library via flow cytometry. Up to 1.6 micromoles of rcSso7d-CBD was found to adsorb per gram of cellulose, yielding a volume-averaged binder concentration of up to 760 μM within the resulting active material. At this molar abundance, the target antigen is captured from solution with nearly 100% efficiency, maximizing the attainable sensitivity for any given diagnostic system. [ABSTRACT FROM AUTHOR]

Helicobacter pylori is a species of bacteria that can colonize the human stomach mucosa. It is closely associated with gastric diseases. The restrictions of traditional methods have encouraged the development of innovative methods for rapid, reliable, and cost-effective diagnosis of H. pylori infection. In recent years, the concept of biosensor and microfluidic-based devices has opened new horizons in high-precision detection. Once combined with nanomaterials, nano-scale biosensors and microfluidic systems provide powerful analytical platforms for point of care (POC) diagnosing of H. pylori . In this article, a brief overview of general aspects of H. pylori infection and current diagnostic methods are firstly discussed. In addition, a clear and concise review of recent advances of biosensors, paper-based and microfluidic systems based on nanomaterials for the detection of H. pylori are discussed herein. Subsequently, the latest development of integrated and miniaturized microfluidic biosensing technologies for POC detection of H. pylori is explained. [ABSTRACT FROM AUTHOR]

Sickle cell anemia (SCA) is a genetic blood disorder that is particularly lethal in early childhood. Universal newborn screening programs and subsequent early treatment are known to drastically reduce under-five SCA mortality. However, in resource-limited settings, cost and infrastructure constraints limit the effectiveness of laboratory-based SCA screening programs. To address this limitation our laboratory previously developed a low-cost, equipment-free, point-of-care, paper-based SCA test. Here, we improved the stability and performance of the test by replacing sodium hydrosulfite (HS), a key reducing agent in the hemoglobin solubility buffer which is not stable in aqueous solutions, with sodium metabisulfite (MS). The MS formulation of the test was compared to the HS formulation in a laboratory setting by inexperienced users (n = 3), to determine visual limit of detection (LOD), readout time, diagnostic accuracy, intra- and inter-observer agreement, and shelf life. The MS test was found to have a 10% sickle hemoglobin LOD, 21-min readout time, 97.3% sensitivity and 99.5% specificity for SCA, almost perfect intra- and inter-observer agreement, at least 24 weeks of shelf stability at room temperature, and could be packaged into a self-contained, distributable test kits comprised of off-the-shelf disposable components and food-grade reagents with a total cost of only $0.21 (USD). [ABSTRACT FROM AUTHOR]

Water-associated or water-related infectious disease outbreaks are caused by pathogens such as bacteria, viruses, and protozoa, which can be transmitted through contaminated water sources, poor sanitation practices, or insect vectors. Low- and middle-income countries bear the major burden of these infections due to inadequate hygiene and subpar laboratory facilities, making it challenging to monitor and detect infections in a timely manner. However, even developed countries are not immune to these diseases, as inadequate wastewater management and contaminated drinking water supplies can also contribute to disease outbreaks. Nucleic acid amplification tests have proven to be effective for early disease intervention and surveillance of both new and existing diseases. In recent years, paper-based diagnostic devices have made significant progress and become an essential tool in detecting and managing water-associated infectious diseases. In this review, we have highlighted the importance of paper and its variants as a diagnostic tool and discussed the properties, designs, modifications, and various paper-based device formats developed and used for detecting water-associated pathogens. [Display omitted] [ABSTRACT FROM AUTHOR]

Monitoring blood sugar level is highly important for managing diabetes and preventing complications. Herein, we describe a paper-based portable assay system for highly accurate monitoring of glucose levels in tears. The system combines enzyme reactions, the perturbation of fluorescence properties, automated image analysis, and pattern recognition powered by a machine-learning algorithms in a single assay without extra manual steps. A small size (20 × 20 mm) assay system was prepared simply by printing black wax crosswise on cellulose paper, melting the wax with heat, and depositing four different fluorescent compounds and glucose oxidase at the test zones. Applying a small drop of sample in the center zone of the system evenly distributes the samples to the test zone via the paper capillary process and eventually induces a generation of unique fluorescence pattern that is specific to glucose. Two models are used to analyze image data collected from the fluorescent compounds array to make accurate estimations on glucose concentration: a random forest model that discretely classifies the concentration with respect to the training data granularity and an SVM regression model that estimates glucose concentration at interpolated finer granularity. These models are combined with feature engineering techniques for efficient sensor data processing on resource limited mobile platforms. As a result, using the random forest approach, glucose concentration in artificial tears can be accurately classified with 96.7% accuracy (0.2 mM intervals) (MSE of 0.060 mM) and the SVM regression model results in an MSE of 0.026 mM. • A fluorescent compounds array system enables noninvasive glucose monitoring. • Photophysical properties of the compounds are altered by an enzyme reaction. • Smartphone-based system enables automated image analysis and pattern recognition. • We achieved 96.7% detection accuracy in two steps using the system. [ABSTRACT FROM AUTHOR]

The growing prevalence of antibiotic resistance calls for new approaches in the development of antimicrobial therapeutics. Likewise, improved diagnostic measures are essential in guiding the application of targeted therapies and preventing the evolution of therapeutic resistance. Discovery platforms are also needed to form new treatment strategies and identify novel antimicrobial agents. By applying engineering principles to molecular biology, synthetic biologists have developed platforms that improve upon, supplement, and will perhaps supplant traditional broad-spectrum antibiotics. Efforts in engineering bacteriophages and synthetic probiotics demonstrate targeted antimicrobial approaches that can be fine-tuned using synthetic biology-derived principles. Further, the development of paper-based, cell-free expression systems holds promise in promoting the clinical translation of molecular biology tools for diagnostic purposes. In this review, we highlight emerging synthetic biology platform technologies that are geared toward the generation of new antimicrobial therapies, diagnostics, and discovery channels. [ABSTRACT FROM AUTHOR]

Lateral-flow tests were first launched commercially in 1984, as a simple urine-based pregnancy test for home use. The simplicity of the visual readout delivered by the basic lateral-flow format proved to be a very popular. However, the recent apparently unstoppable trend towards portable and wearable technology is driving the lateral-flow strip towards an industrial interface that will enable it to interface with big data and expert systems, and where ready transmission of data is essential. In this review, we chart the inevitable evolution of the visually-read lateral-flow strip to more advanced instrumented versions and consider the future of this very flexible approach to delivering simple affinity assays. We examine recent labelling strategies, the relative merits of optical and electrochemical transducers and explore the evolution of recognition elements that are now being incorporated into these systems. [ABSTRACT FROM AUTHOR]

Cardiovascular diseases (CVDs) are the main causes of morbidity and mortality in the world where about 4 in every 5 CVD deaths happen in low- and middle-income countries (LMICs). Most CVDs are preventable and curable, which is largely dependent on timely and effective interventions, including diagnosis, prognosis and therapeutic monitoring. However, these interventions are high-cost in high income countries and are usually lacking in LMICs. Thanks to the rapid development of microfluidics and nanotechnology, lots of portable analytical devices are developed for detection of CVDs at the point-of-care (POC). In the meantime, smartphone, as a versatile and powerful handheld tool, has been employed not only as a reader for microfluidic assays, but also as an analyzer for physiological indexes. In this review, we present a comprehensive introduction of the current status and potential development direction on POC diagnostics for CVDs. First of all, we introduce some main facts about CVDs and their standard diagnostic procedures and methods. Second, we discuss about both commercially available POC devices and developed prototypes for detection of CVDs via immunoassays. Subsequently, we report the advances in smartphone-based readout for microfluidic assays. Finally, we present some examples using smartphone, individually or combined with other components or devices, for CVD monitoring. We envision an integrated smartphone-based system capable of functioning blood tests, disease examination, and imaging will come in the future. [ABSTRACT FROM AUTHOR]

Sickle cell anemia (SCA) is a genetic blood disorder that is particularly lethal in early childhood. Universal newborn screening programs and subsequent early treatment are known to drastically reduce under-five SCA mortality. However, in resource-limited settings, cost and infrastructure constraints limit the effectiveness of laboratory-based SCA screening programs. To address this limitation our laboratory previously developed a low-cost, equipment-free, point-of-care, paper-based SCA test. Here, we improved the stability and performance of the test by replacing sodium hydrosulfite (HS), a key reducing agent in the hemoglobin solubility buffer which is not stable in aqueous solutions, with sodium metabisulfite (MS). The MS formulation of the test was compared to the HS formulation in a laboratory setting by inexperienced users (n = 3), to determine visual limit of detection (LOD), readout time, diagnostic accuracy, intra- and inter-observer agreement, and shelf life. The MS test was found to have a 10% sickle hemoglobin LOD, 21-min readout time, 97.3% sensitivity and 99.5% specificity for SCA, almost perfect intra- and inter-observer agreement, at least 24 weeks of shelf stability at room temperature, and could be packaged into a self-contained, distributable test kits comprised of off-the-shelf disposable components and food-grade reagents with a total cost of only $0.21 (USD).

Abstract: Advanced diagnostic technologies, such as polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), have been widely used in well-equipped laboratories. However, they are not affordable or accessible in resource-limited settings due to the lack of basic infrastructure and/or trained operators. Paper-based diagnostic technologies are affordable, user-friendly, rapid, robust, and scalable for manufacturing, thus holding great potential to deliver point-of-care (POC) diagnostics to resource-limited settings. In this review, we present the working principles and reaction mechanism of paper-based diagnostics, including dipstick assays, lateral flow assays (LFAs), and microfluidic paper-based analytical devices (μPADs), as well as the selection of substrates and fabrication methods. Further, we report the advances in improving detection sensitivity, quantification readout, procedure simplification and multi-functionalization of paper-based diagnostics, and discuss the disadvantages of paper-based diagnostics. We envision that miniaturized and integrated paper-based diagnostic devices with the sample-in-answer-out capability will meet the diverse requirements for diagnosis and treatment monitoring at the POC. [Copyright &y& Elsevier]

(1) Background: The complexity, amount of time, and the large amount of resource required to perform gold-standard bacteria culture procedures makes it difficult to perform timely pathogenic analyses, especially in areas where such resources are not readily available. A paper-based biochemical analytical tool can potentially tackle problems economically in terms of time and convenience, potentially finding utility in applications where simple and timely detection of bacteria is necessary; (2) Methods: The utility of paper-based MTT-PMS strips was tested using a simple colorimetric analytical methodology; (3) Results: Sufficient evidence was obtained to suggest that the strips can potentially be used as a rapid and convenient early, alternative bacteria screening tool for a variety of applications; (4) Conclusions: The potential of strips for the rapid detection of bacteria compared to standard bacteria culture is a key advantage in certain clinical, agricultural, and environmental applications. [ABSTRACT FROM AUTHOR]

Many point-of-care diagnostic tests rely on a pair of monoclonal antibodies that bind to two distinct epitopes of a molecule of interest. This protocol describes the identification and generation of such affinity pairs based on an easily produced small protein scaffold rcSso7d which can substitute monoclonal antibodies. These strong binding variants are identified from a large yeast display library. The approach described can be significantly faster than antibody generation and epitope binning, yielding affinity pairs synthesized in common bacterial protein synthesis strains, enabling the rapid generation of novel diagnostic tools., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Norovirus infections are the leading cause of foodborne illness and human gastroenteritis, afflicting hundreds of millions of people each year. Molecular assays with the capacity to detect norovirus without expensive equipment and with high sensitivity and specificity represent useful tools to track and contain future outbreaks. Here we describe how norovirus can be detected in low-cost paper-based cell-free reactions. These assays combine freeze-dried, thermostable cell-free transcription-translation reactions with toehold switch riboregulators designed to target the norovirus genome, enabling convenient colorimetric assay readouts. Coupling cell-free reactions with synbody-based viral enrichment and isothermal amplification enables detection of norovirus from clinical samples down to concentrations as low as 270 zM. These diagnostic tests are promising assays for confronting norovirus outbreaks and can be adapted to a variety of other human pathogens., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The gut microbiome and its interactions with the host have been shown to affect several aspects of human health and disease. Investigations to elucidate these mechanisms typically involve sequence analysis of fecal samples. To support these studies, we present methods to design RNA toehold switch sensors to detect microbial and host transcripts. The sensors are embedded in paper-based, cell-free reactions that enable affordable and rapid analysis of microbiome samples., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

As new infectious disease outbreaks become more likely, it is important to be able to develop and deploy appropriate testing in time. Paper-based immunoassays are rapid, cheap, and easy to produce at scale and relatively user friendly but often suffer from low selectivity and sensitivity. Understanding the molecular mechanisms of paper immunoassays may help improve and hasten development and therefore production and market availability. Here, we study how the behavior of nanoparticle-antibody immunoprobes in paper dipstick immunoassays is impacted by synthesis strategy and surface chemistry architecture. We conjugate gold nanoparticles to polyclonal anti-immunoglobulin G (IgG) and anti-zika NS1 antibodies by electrostatic adsorption and N -hydroxysuccinimide (NHS) and hydrazide (Hz) chemistries. The immunoprobes were used in paper immunoassays and the effective affinity for the antigen was quantified from the test line intensities, as well as the distribution of the immunoprobes throughout the strips. The results show that nanoparticle colloidal stability, both post synthesis and during antigen binding, is a key factor and affects immunoassay results and performance, often through reduction or loss of signal.