Sustainable agricultural production has the aim to maximise the yield and quality of harvests. For that, farmers need to monitor nutrient levels within soils to apply fertilizers accordingly and to prevent land degradation and soil exhaustion. Current methods involve time-consuming sampling and transport to a laboratory; and most farmers do not have the resources to measure frequently at multiple locations, limiting their sustainable production. In order to address this challenge, a paper-based analytical device (PAD) for the multiplexed detection of soil nutrients, i.e. phosphate, nitrate and pH, extracted with a cafetière-based method, is developed in this work. The compact and easy-to-use platform enables an accurate soil analysis in less than 20 min; 3 min for extraction and 15 min for readout. Two detection channels for nitrate and phosphate analysis, and one circular detection area for pH, including the selective reagents to each analyte, are defined within the PAD. Upon contact with the specific nutrients, the platform produces a colorimetric response that is easily readable by naked eye and smartphone camera. The detection reactions were optimized in the range 1 − 22.5 mg L⁻¹, 10 −100 mg L⁻¹ and 5.0 – 8.5 for phosphate, nitrate and pH, respectively, in agreement with the relevant levels in soils. The volume of water, the number of pushes of the plunger, the extraction time and the mass of soil were also optimized for the nutrient extraction method with the cafetière. The optimal workflow was validated with commercial soils and compared with the conventional UV-Vis method and the labels from the soil package, showing excellent agreement. This paper-based platform provides the possibility of a cheap, sensitive and specific monitoring of soil nutrients by minimally trained operators, such as farmers, enabling in-the-field analyses of multiple key soil nutrients in resource-limited settings and therefore addressing the challenge of routine monitoring for sustainable agriculture.

Introduction Annually, approximately 174 million people globally are affected by Trichomonas vaginalis (T. vaginalis) infection. Half of these infections occur in resource-limited regions. Untreated T. vaginalis infections are associated with complications such as pelvic inflammatory disease and adverse pregnancy outcomes mostly seen in women. In resource-limited regions, the World Health Organization (WHO) advocates for syndromic case management. However, this can lead to unnecessary treatment. Accurate diagnosis of T. vaginalis is required for effective and prompt treatment. Molecular tests such as Polymerase Chain Reaction (PCR) have the advantage of having a short turn-around time and allow the use of non-invasive specimens such as urine and vaginal swabs. However, these diagnostic techniques have numerous disadvantages such as high infrastructure costs, false negative and positive results, and interstrain variation among others. This study aimed to evaluate the use of identical multi-repeat sequences (IMRS) as amplification primers for developing ultrasensitive diagnostic for T. vaginalis. Methods We used genome-mining approaches based on identical multi-repeat sequences (IMRS) algorithm to identify sequences distributed on the T. vaginalis genome to design a primer pair that targets a total of 69 repeat sequences. Genomic T. vaginalis DNA was diluted from 5.8×102 to 5.8×10−4 genome copies/μl and used as a template in the IMRS-based amplification assay. For performance comparison, 18S rRNA PCR assay was employed. Results The T. vaginalis -IMRS primers offered a higher test sensitivity of 0.03 fg/μL compared to the 18S rRNA PCR (0.714 pg/μL). The limit of detection for the Isothermal assay was 0.58 genome copies/mL. Using real-time PCR, the analytical sensitivity of the T. vaginalis -IMRS primers was <0.01 pg/μL, equivalent to less than one genome copy/μL. Conclusion De novo genome mining of T. vaginalis IMRS as amplification primers serves as a platform for developing ultrasensitive diagnostics for Trichomoniasis and a wide range of infectious pathogens.

Clinical diagnostics of infectious diseases via nucleic acid amplification tests (NAATs) depend on a separate step of isolation of nucleic acids from cells/viruses embedded in complex biological matrices. The most recent example has been reverse transcription polymerase chain reaction (RT-PCR) for amplification and detection of SARS-CoV-2 RNA for COVID-19 diagnostics. Kits for RNA extraction and purification are commercially available; however, their integration with amplification systems is generally lacking, resulting in two separate steps, i.e., sample preparation and amplification. This makes NAATs more time-consuming, requiring skilled personnel, and can increase the likelihood of contamination. Here, we describe a setup and methodology to perform the quick extraction and detection of nucleic acids in an integrated manner. In particular, we focus on the use of an immiscible filtration device for capture, isolation, concentration, amplification, and colorimetric detection of SARS-CoV-2 RNA.

Microfluidics – as most of us call their research field today; micro total analysis systems as it was named at the beginning of the 1990s, was heavily influenced by contributions from Andreas Manz. The first inventions and publications in the field of analytical chemistry, such as electrophoresis-on-chip and chromatography-on-chip, showed the amazing potential of miniaturized systems. The pioneering work for separation methods was followed by further seminal studies on bioanalytical and biological applications of microfluidic chips, such as PCR-on-chip, adaptations to various analytical detection methods, chemical reactors; and numerous more fancy ideas, such as powder-on-chip, leaves as molds, or the miniaturized London city map, with shortest travel distances visualized by a gas plasma. The world of microfluidics has very creative aspects and Andreas was one of the catalysts for the next generation of researchers.

There is a growing need for easy-to-use, low cost and portable quantitative assays to determine active pharmaceutical ingredients in the pharmaceutical industry. Here, we developed a batch spectrophotometric method and a method employing a paper-based microfluidic device for the estimation of Amoxicillin (AMX) in pure solution and pharmaceutical preparations. The detection depends on the coupling reaction of Amoxicillin with diazotized sulfadimidine (DSDM) in an alkaline medium. The yellow azo dye reaction product was measured at λ_max 425 nm and linearity was observed from 2 to 30 mg L⁻¹ with a detection limit of 0.32 mg L⁻¹ and a quantification limit of 1.2 mg L⁻¹ was found. The reaction was then transferred onto the paper-based microfluidic device and a plateau change in color intensity was found above 10 mg L⁻¹. Thus, the paper-based microfluidic device can be applied for the semi-quantitative determination of Amoxicillin in pure solution and commercial pharmaceutical products for rapid screening.

Soil nutrition is a key pillar in agricultural productivity. However, point-of-need testing for soil nutrition is not readily available in resource-limited settings such as Kenya. We set out to study the perceived need for soil testing among farmers in this country. A group of 547 farmers from Murang'a and Kiambu counties in central Kenya were recruited through multi-stage sampling to help assess the perceptions and willingness to pay (WTP) toward a prototype technology for surveillance of in-situ soil nutrition. The technology is based on a cafetière-style filter system for extraction and a microfluidic paper-based analytical device (μPAD) for nutrient readout. We employed the double bounded choice contingent valuation method (CVM) to analyze the willingness of farmers to accept and pay for the prototype if the technology was available on the market. It was found that currently, only 1.5 % of farmers carry out soil testing. The high costs of analysis at testing centers, which are often far from the farmers, are among the main reasons contributing to the majority of farmers not testing their soils. The farmers surveyed were generally willing to make their soil data publicly accessible, especially to extension officers. CVM showed that uncontrolled WTP had a 94.24 % premium above KSh1,000 ($6.60) incurred by using the existing rapid testing method. Factoring the control variables and disaggregating the model into gender categories, the findings showed that youth, women, and men had WTP values of KSh1,612.53 ($10.75), KSh1,558.68 ($10.39), and KSh1,504.83 ($10.03), respectively, indicating that farmers can indeed pay for the convenience to test their soils in situ. Through the democratization of soil nutrition data, extension agents can enhance the improvement of agricultural productivity, which implies that farmers can commercialize their agricultural activities.

Background: Curable sexually transmitted infections (STIs), such as Neisseria gonorrhoeae (N. gonorrhoeae), are a major cause of poor pregnancy outcomes. The infection is often asymptomatic in pregnant women, and a syndrome-based approach of testing leads to a missed diagnosis. Culture followed by microscopy is inadequate and time-consuming. The gold standard nucleic acid amplification tests require advanced infrastructure settings, whereas point-of-care tests are limited to immunoassays with sensitivities and specificities insufficient to accurately diagnose asymptomatic cases. This necessitates the development and validation of assays that are fit for purpose. Methods: We identified new diagnostic target biomarker regions for N. gonorrhoeae using an algorithm for genome mining of identical multi-repeat sequences (IMRS). These were then developed as DNA amplification primers to design better diagnostic assays. To test the primer pair, genomic DNA was 10-fold serially diluted (100 pg/μL to 1 × 10−3 pg/μL) and used as DNA template for PCR reactions. The gold standard PCR using 16S rRNA primers was also run as a comparative test, and both assay products were resolved on 1% agarose gel. Results: Our newly developed N. gonorrhoeae IMRS-PCR assay had an analytical sensitivity of 6 fg/μL representing better sensitivity than the 16S rRNA PCR assay with an analytical sensitivity of 4.3096 pg/μL. The assay was also successfully validated using clinical urethral swab samples. We further advanced this technique by developing an isothermal IMRS, which was both reliable and sensitive for detecting cultured N. gonorrhoeae isolates at a concentration of 38 ng/μL. Combining isothermal IMRS with a low-cost lateral flow assay, we were able to detect N. gonorrhoeae amplicons at a starting concentration of 100 pg/μL. Conclusion: Therefore, there is a potential to implement this concept within miniaturized, isothermal, microfluidic platforms, and laboratory-on-a-chip diagnostic devices for highly reliable point-of-care testing.

Agroathelia rolfsii (A. rolfsii) is a fungal infection and poses a significant threat to over 500 plant species worldwide. It can reduce crop yields drastically resulting in substantial economic losses. While conventional detection methods like PCR offer high sensitivity and specificity, they require specialized and expensive equipment, limiting their applicability in resource -limited settings and in the field. Herein, we present an integrated workflow with nucleic acid extraction and isothermal amplification in a lab -on -a -chip cartridge based on immiscible filtration assisted by surface tension (IFAST) to detect A. rolfsii fungi in soil for point -of -need application. Our approach enabled both DNA extraction of A. rolfsii from soil and subsequent colorimetric loop -mediated isothermal amplification (LAMP) to be completed on a single chip, termed IFAST-LAMP. LAMP primers targeting ITS region of A. rolfsii were newly designed and tested. Two DNA extraction methods based on silica paramagnetic particles (PMPs) and three LAMP assays were compared. The best -performing assay was selected for on -chip extraction and detection of A. rolfsii from soil samples inoculated with concentrations of 3.75, 0.375 and 0.0375 mg fresh weight per 100-g soil (%FW). The full on -chip workflow was achieved within a 1-h turnaround time. The platform was capable of detecting as low as 3.75 %FW at 2 days after inoculation and down to 0.0375 %FW at 3 days after inoculation. The IFAST-LAMP could be suitable for field -applicability for A. rolfsii detection in low -resource settings.

Solid tumours are complex and heterogeneous systems, which exist in a dynamic biophysical microenvironment. Conventional cancer research methods have long relied on two-dimensional (2D) static cultures which neglect the dynamic, three-dimensional (3D) nature of the biophysical tumour microenvironment (TME), especially the role and impact of interstitial fluid flow (IFF). To address this, we undertook a transcriptome-wide analysis of the impact of IFF-like perfusion flow using a spheroid-on-chip microfluidic platform, which allows 3D cancer spheroids to be integrated into extracellular matrices (ECM)-like hydrogels and exposed to continuous perfusion, to mimic IFF in the TME. Importantly, we have performed these studies both in experimental (normoxia) and pathophysiological (hypoxia) oxygen conditions. Our data indicated that gene expression was altered by flow when compared to static conditions, and for the first time showed that these gene expression patterns differed in different oxygen tensions, reflecting a differential role of spheroid perfusion in IFF-like flow in tumour-relevant hypoxic conditions in the biophysical TME. We were also able to identify factors primarily linked with IFF-like conditions which are linked with prognostic value in cancer patients and therefore could correspond to a potential novel biomarker of IFF in cancer. This study therefore highlights the need to consider relevant oxygen conditions when studying the impact of flow in cancer biology, as well as demonstrating the potential of microfluidic models of flow to identify IFF-relevant tumour biomarkers.