DNA compaction enhances the sensitivity of fluorescence-based nucleic acid assays: a game changer in point of care sensors?

Fluorescence-based nucleic acid assays frequently exhibit a feeble signal at low analyte concentrations, necessitating complex, expensive methods such as the development of sequence-specific oligo tags, molecular beacons, and chemical modifications to maintain high detection sensitivity. Hence, there is growing interest in accomplishing fluorescence enhancement in nucleic acid assays using robust and cost-effective strategies. The study exploits the use of two compaction agents, PEG 8000 and CTAB, to compact the ITS-2 amplicon of the fungus Candida albicans and evaluates the effect of both of these agents on the fluorescence intensity of SYTO-9 labelled nucleic acids. Conventional fluorometric measurements showed that both CTAB and PEG 8000 enhanced the emission intensity by ∼1.2- and 2-fold, respectively. Furthermore, we leveraged paper-based spot tests and distance-based assays to validate the effect of DNA compaction for enhancing sensitivity in the point-of-care context. The spot assay performed on paper with compacted samples showed an increase in the emission intensity of SYTO-9 and this was manifested by an elevated G channel intensity in the order of PEG 8000 compacted > CTAB compacted > amplified. Moreover, in the distance-based assay, the PEG 8000 compacted sample was found to migrate farther compared to CTAB compacted and amplified DNA samples at amplicon concentrations, 15 µg ml-1 and 39.65 µg ml-1. The limit of detection (LOD) for PEG 8000 and CTAB compacted samples on both paper-spot and distance-based assays were found to be 0.4 µg ml-1 and 0.5 µg ml-1, respectively. Hence our work provides an overview of employing DNA compaction as an approach for enhancing the sensitivity of fluorescence-based point-of-care nucleic acid assays without the need for cumbersome sensitivity enhancement methods.

Paper and thread as media for the frugal detection of urinary tract infections (UTIs).

Urinary tract infections (UTIs) make up a significant proportion of the global burden of disease in vulnerable groups and tend to substantially impair the quality of life of those affected, making timely detection of UTIs a priority for public health. However, economic and societal barriers drastically reduce accessibility of traditional lab-based testing methods for critical patient groups in low-resource areas, negatively affecting their overall healthcare outcomes. As a result, cellulose-based materials such as paper and thread have garnered significant interest among researchers as substrates for so-called frugal analytical devices which leverage the material's portability and adaptability for facile and reproducible diagnoses of UTIs. Although the field may be only in its infancy, strategies aimed at commercial penetration can appreciably increase access to more healthcare options for at-risk people. In this review, we catalogue recent advances in devices that use cellulose-based materials as the primary housing or medium for UTI detection and chart out trends in the field. We also explore different modalities employed for detection, with particular emphasis on their ability to be ported onto discreet casings such as sanitary products.

Medical students' perception of changes in assessments implemented during the COVID-19 pandemic.

BACKGROUND: COVID-19 posed many challenges to medical education in the United Kingdom (UK). This includes implementing assessments during 4 months of national lockdowns within a 2-year period, where in-person education was prohibited. This study aimed to identify medical school assessment formats emerging during COVID-19 restrictions, investigate medical students' perspectives on these and identify influencing factors. METHODS: The study consisted of two phases: a questionnaire asking medical students about assessment changes they experienced, satisfaction with these changes and preference regarding different assessments that emerged. The second phase involved semi-structured interviews with medical students across the UK to provide a deeper contextualized understanding of the complex factors influencing their perspectives. RESULTS: In the questionnaire responses, open-book assessments had the highest satisfaction, and were the preferred option indicated. Furthermore, in the case of assessment cancellation, an increase in weighting of future assessments was preferred over increase in weighting of past assessments. Students were also satisfied with formative or pass-fail assessments. Interview analyses indicate that although cancellation or replacement of summative assessments with formative assessments reduced heightened anxiety from additional COVID-19 stressors, students worried about possible future knowledge gaps resulting from reduced motivation for assessment-related study. Students' satisfaction level was also affected by timeliness of communication from universities regarding changes, and student involvement in the decision-making processes. Perceived fairness and standardisation of test-taking conditions were ranked as the most important factors influencing student satisfaction, followed closely by familiarity with the format. In contrast, technical issues, lack of transparency about changes, perceived unfairness around invigilation, and uncertainty around changes in assessment format and weighting contributed to dissatisfaction. CONCLUSIONS: Online open-book assessments were seen as the most ideal amongst all participants, and students who experienced these were the most satisfied with their assessment change. They were perceived as most fair and authentic compared to real-life medical training. We seek to inform educators about student perceptions of successful assessment strategies under COVID-19 restrictions and provide evidence to allow debate on ongoing assessment reform and innovation. While this work looks specifically at assessment changes during COVID-19, understanding factors affecting student perception of assessment is applicable to examinations beyond COVID-19.

Methylglyoxal Induced Modifications to Stabilize Therapeutic Proteins: A Review.

Therapeutic proteins are potent, fast-acting drugs that are highly effective in treating various conditions. Medicinal protein usage has increased in the past 10 years, and it will evolve further as we better understand disease molecular pathways. However, it is associated with high processing costs, limited stability, difficulty in being administered as an oral medication, and the inability of large proteins to penetrate tissue and reach their target locations. Many methods have been developed to overcome the problems with the stability and chaperone activity of therapeutic proteins, viz., the addition of external agents (changing the properties of the surrounding solvent by using stabilizing excipients, e.g., amino acids, sugars, polyols) and internal agents (chemical modifications that influence its structural properties, e.g., mutations, glycosylation). However, these methods must completely clear protein instability and chaperone issues. There is still much work to be done on finetuning chaperone proteins to increase their biological efficacy and stability. Methylglyoxal (MGO), a potent dicarbonyl compound, reacts with proteins and forms covalent cross-links. Much research on MGO scavengers has been conducted since they are known to alter protein structure, which may result in alterations in biological activity and stability. MGO is naturally produced within our body, however, its impact on chaperones and protein stability needs to be better understood and seems to vary based on concentration. This review highlights the efforts of several research groups on the effect of MGO on various proteins. It also addresses the impact of MGO on a client protein, α-crystallin, to understand the potential solutions to the protein's chaperone and stability problems.

Paper-based dots and smartphone for detecting counterfeit country eggs.

Herein, we present for the first time, the employment of paper-based devices for rapidly differentiating original country eggs from the plain broiler eggs that have been coated with tea to appear as the former. The devices leverage two types of phenomena involving the phenols present in tea in precisely 5 min, namely precipitation, which produces a well-defined dark bluish precipitate on the surface of the counterfeit country eggs or tea-coated broiler eggs and de-coloration, wherein the dried layer of tea coating present on the surface of the dummy country eggs get dissolved, thereby revealing the white colour of the plain broiler egg shell. To reduce the subjectivity, a smartphone application 'Eggo' has been developed which is capable of detecting the spots produced by both the methods using mobile's camera. Fourier Transform Infrared Spectroscopy (FTIR) analysis was performed to study the changes occurring on the shell surface. Such sophisticated yet simple technologies will revolutionize food fraud analysis.

Embellishing 2-D MoS2 Nanosheets on Lotus Thread Devices for Enhanced Hydrophobicity and Antimicrobial Activity.

Herein, we report cellulose-based threads from Indian sacred Lotus (Nelumbo nucifera) of the Nymphaceae family embellished with MoS2 nanosheets for its enhanced hydrophobic and antimicrobial properties. MoS2 nanosheets synthesized by a coprecipitation method using sodium molybdate dihydrate (Na2MoO4·2H2O) and thioacetamide (CH3CSNH2) were used as a sourse for MoS2 particle growth with cellulose threads extracted from lotus peduncles. The size, crystallinity, and morphology of pure and MoS2-coated fibers were studied using X-ray diffractometry (XRD) and scanning electron microscopy (SEM). the XRD pattern of pure lotus threads showed a semicrystalline nature, and the threads@MoS2 composite showed more crystallinity than the pure threads. SEM depicts that pure lotus threads possess a smooth surface, and the MoS2 nanosheets growth can be easily identified on the threads@MoS2. Further, the presence of MoS2 nanosheets on threads was confirmed with EDX elemental analysis. Antimicrobial studies with Escherichia coli and Candida albicans reveal that threads@MoS2 have better resistance than its counterpart, i.e., pure threads. MoS2 sheets play a predominant role in restricting the wicking capability of the pure threads due to their enhanced hydrophobic property. The water absorbency assay denotes the absorption rate of threads@MoS2 to 80%, and threads@MoS2 shows no penetration for the observed 60 min, thus confirming its wicking restriction. The contact angle for threads@MoS2 is 128°, indicating its improved hydrophobicity.

Paper-based microfluidic devices for food adulterants: Cost-effective technological monitoring systems.

Analytical sciences have witnessed emergent techniques for efficient clinical and industrial food adulterants detection. In this review, the contributions made by the paper-based devices are highlighted for efficient and rapid detection of food adulterants and additives, which is the need of the hour and how different categories of techniques have been developed in the past decade for upgrading the performance for point-of-care testing. A simple strategy with an arrangement for detecting specific adulterants followed by the addition of samples to obtain well-defined qualitative or quantitative signals for confirming the presence of target species. The paper-based microfluidics-based technology advances and prospects for food adulterant detection are discussed given the high-demand from the food sectors and serve as a valued technology for food researchers working in interdisciplinary technological frontiers.

"Scratch it out": carbon copy based paper devices for microbial assays and liver disease diagnosis.

We present a facile paper-based microfluidic device fabrication technique leveraging off-the-shelf carbon paper for the deposition of hydrophobic barriers using a novel "stencil scratching" method. This exceedingly frugal approach (0.05$) requires practically no technical training to employ. Hydrophobic barriers fabricated using this approach offer a width of 3 mm and a hydrophilic channel width of 849 µm, with an ability to confine major aqueous solvents without leakage. The utility of the device is demonstrated by porting a cell viability assay showing a limit-of-detection (LOD) of 0.6 × 108 CFU mL-1 and bilirubin assay with human serum showing a detection range of 1.76-6.9 mg dL-1 and a limit-of-detection (LOD) of 1.76 mg dL-1. The intuitiveness and economic viability of the fabrication method afford it great potential in the field of point-of-care diagnostics geared towards providing testing infrastructure in resource-scarce regions globally.

Knitting Thread Devices: Detecting Candida albicans Using Napkins and Tampons.

Reproducible and in situ microbial detection, particularly of microbes significant in urinary tract infections (UTIs) such as Candida albicans, provides a unique opportunity to bring equity in the healthcare outcomes of disenfranchised groups like women in low-resource settings. Here, we demonstrate a system to potentially detect vulvovaginal candidiasis by leveraging the properties of multifilament cotton threads in the form of microfluidic-thread-based analytical devices (µTADs) to develop a frugal microbial identification assay. A facile mercerization method using heptane wash to boost reagent absorption and penetration is also performed and is shown to be robust compared to other existing conventional mercerization methods. Furthermore, the twisted mercerized fibers are drop-cast with media consisting of l-proline ß-naphthylamide, which undergoes hydrolysis by the enzyme l-proline aminopeptidase secreted by C. albicans, hence signaling the presence of the pathogen via simple color change with a limit of detection of 0.58 × 106 cfu/mL. The flexible and easily disposable thread-based detection device when integrated with menstrual hygiene products showed a detection time of 10 min using spiked vaginal discharge. The developed method boasts a long shelf life and high stability, making it a discreet detection device for testing, which provides new vistas for self-testing multiple diseases that are considered taboo in certain societies.

Thread integrated smart-phone imaging facilitates early turning point colorimetric assay for microbes.

This study employs a commercial multifilament cotton thread as a low-cost microbial identification assay integrated with smartphone-based imaging for high throughput and rapid detection of pathogens. The thread device with inter-twined fibers was drop-cast with test media and a pH indicator. The target pathogens scavenge the media components with different sugars and release acidic by-products, which in turn act as markers for pH-based color change. The developed thread-based proof-of-concept was demonstrated for the visual color detection (red to yellow) of Candida albicans (≈16 hours) and Escherichia coli (≈5 hours). Besides that, using a smart-phone to capture images of the thread-based colorimetric assay facilitates early detection of turning point of the pH-based color change and further reduces the detection time of pathogens viz. Candida albicans (≈10 hours) and Escherichia coli (≈1.5 hours). The reported thread and smartphone integrated image analysis works towards identifying the turning point of the colorimetric change rather than the end-point analysis. Using this approach, the interpretation time can be significantly reduced compared to the existing conventional microbial methods (≈24 hours). The thread-based colorimetric microbial assay represents a ready-to-use, low-cost and straightforward technology with applicability in resource-constrained environments, surpassing the need for frequent fresh media preparation, expensive instrumentation, complex fabrication techniques and expert intervention. The proposed method possesses high scalability and reproducibility, which can be further extended to bio(chemical) assays.

Fabricating Paper Based Devices Using Correction Pens.

We present a rapid (<10 s), cost-effective, unique single-step method for fabricating paper-based devices without necessitating any expensive instrumentation, simply by deploying correction pens that are otherwise commonly used for masking typos in printed or written matters. The marked regions formed by deposits from the correction pen demonstrate ubiquitous flow resistances to typical aqueous solutions and organic solvents in the transverse direction, resulting in a preferential bulk flow along the axial direction of the paper channels 'fabricated' in the process. Considering the simplicity and cost-effectiveness of this platform, it is deemed to be ideal for (bio) chemical sensing and point-of-care diagnostics in resource-limited settings.