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Formalin has been reported to be added to milk to prolong the shelf life of milk. The present study was conducted to develop and validate liquid-phase and paper-based strip methods for the rapid detection of formalin in milk using four different procedures i.e., A, B, C and D. Among all the procedures, procedure A worked successfully both in the liquid-phase and paper-based assays. The developed strip-based method involved the dipping of the strips in milk followed by visualization of change in colour. Formalin adulterated milk gave yellow colour to the strip, while for normal milk, it remained colourless. Limit of detection of developed strips was 0.05% with a response time of 7–8 min. Shelf life of the strips was a minimum of 6 months at both room (30–37 °C) and refrigeration (4 ℃) temperature. The strips can be used both at field as well as household levels for rapid detection of formalin presence in milk with greater sensitivity and hence can contribute in supplying authentic milk to the consumers. [ABSTRACT FROM AUTHOR]

The development of fluorometric paper-based analytical devices (fPADs) integrated with smartphone for fluorometric quantification of ethanol in an instrument-free and portable setup is described. The NAD+-dependent alcohol dehydrogenase immobilized within chitosan modified paper substate was utilized as a bio-recognition element and enzymatically generated NADH was used as a fluorescent probe. 3D-printed imaging setup which houses a paper chip holder and UV-light emitting device (LED) was developed for rapid, accurate capture of the fluorescent images. The biocompatible chitosan layer covering the paper provides a feasible environment for enzyme immobilization and enhances the fluorescence signal. The developed fPADs exhibited high sensitivity for ethanol detection and has a linear range for ethanol detection from 17 µM to 8.75 mM (R2 =0.99). Additionally, the fPADs were applied to quantify ethanol in four different wine samples including red, white, rose, and sparkling wines successfully. Moreover, the fPADs produce reproducible signals without loss of enzyme activity for at least 14 days and ~80% activity remained till 28 days. Thus, the proposed approach can provide a facile, affordable, portable, and instrument-free tool for the onsite quantification of ethanol in real samples and is applicable for food quality controls. [ABSTRACT FROM AUTHOR]

Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are important liver enzymes in clinical settings. Their levels are known to be elevated in individuals with underlying liver diseases and those consuming hepatotoxic drugs. Serum ALT and AST levels are crucial for diagnosing and assessing liver diseases. Serum ALT is considered the most reliable and specific candidate as a disease biomarker for liver diseases. ALT and AST levels are routinely analyzed in high-risk individuals for the bioanalysis of both liver function and complications associated with drug-induced liver injury. Typically, ALT and AST require blood sampling, serum separation, and testing. Traditional methods require expensive or sophisticated equipment and trained specialists, which is often time-consuming. Therefore, developing countries have limited or no access to these methods. To address the above issues, we hypothesize that low-cost biosensing methods (paper-based assays) can be applied to the analysis of ALT and AST levels in biological fluids. The paper-based biodetection technique can semi-quantitatively measure ALT and AST from capillary finger sticks, and it will pave the way for the development of an inexpensive and rapid alternative method for the early detection and diagnosis of liver diseases. This method is expected to significantly reduce the economic burden and aid routine clinical analysis in both developed and underdeveloped countries. The development of low-cost testing platforms and their diagnostic utility will be extremely beneficial in helping millions of patients with liver disorders. [ABSTRACT FROM AUTHOR]

Point-of-care tests play an important role in serological diagnostics of infectious diseases and post-vaccination immunity monitoring, including in COVID-19. Currently, lateral flow tests dominate in this area and show good analytical performance. However, studies to improve the effectiveness of such tests remain important. In comparison with lateral flow tests, vertical flow immunoassays allow for a reduction in assay duration and the influence of the hook effect. Additionally, the use of carbon black nanoparticles (CNPs) as a color label can provide a lower detection limit (LOD) compared to conventional colloidal gold. Therefore, we have developed a vertical flow immunoassay for the detection of IgG against SARS-CoV-2 spike protein in human serum samples by applying a conjugate of CNPs with anti-human IgG mouse monoclonal antibodies (CNP@MAb). The vertical flow assay device consists of a plastic cassette with a hole on its top containing a nitrocellulose membrane coated with spike protein and an absorbent pad. The serum sample, washing buffer, and CNP@MAb flow vertically through the nitrocellulose membrane and absorbent pads, reducing assay time and simplifying the procedure. In positive samples, the interaction of CNP@MAb with anti-spike antibodies leads to the appearance of black spots, which can be visually detected. The developed method allows for rapid visual detection (5–7 min) of IgG vs. spike protein, with a LOD of 7.81 BAU/mL. It has been shown that an untrained operator can perform the assay and visually evaluate its results. Thus, the presented assay can be used in the further development of test systems for the serological diagnostics of COVID-19 or post-vaccination immunity monitoring. [ABSTRACT FROM AUTHOR]

Neopterin (NPT) is a Guanosine Tri Phosphate (GTP) metabolite produced by activated macrophages and is considered a pro‐inflammatory and atherosclerotic agent. Beyond this NPT is a multi‐utility biomarker for assessing the status of various diseases including cardiovascular disease, depression, and malignant tumors as well as infections including viral and bacterial origin. Recent clinical investigations suggest the use NPT as a prognostic marker for the early prediction of the severity of Covid‐19, which is the prime concern of global public health. One of the main immunopathological characteristics of Covid‐19 is an excessive inflammatory response, and the severity of the infection is linked with macrophage activation syndrome (MAS). Hence the assessment of neopterin serves as an effective diagnostic tool for the risk prediction of Covid‐19. We present a fluorescence turn‐on sensor for neopterin using ferric ion‐modulated sulfur and nitrogen co‐doped carbon quantum dots. The linear detection of NPT was achieved with a limit of detection of 3.11 nM. [ABSTRACT FROM AUTHOR]

Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are important liver enzymes in clinical settings. Their levels are known to be elevated in individuals with underlying liver diseases and those consuming hepatotoxic drugs. Serum ALT and AST levels are crucial for diagnosing and assessing liver diseases. Serum ALT is considered the most reliable and specific candidate as a disease biomarker for liver diseases. ALT and AST levels are routinely analyzed in high-risk individuals for the bioanalysis of both liver function and complications associated with drug-induced liver injury. Typically, ALT and AST require blood sampling, serum separation, and testing. Traditional methods require expensive or sophisticated equipment and trained specialists, which is often time-consuming. Therefore, developing countries have limited or no access to these methods. To address the above issues, we hypothesize that low-cost biosensing methods (paper-based assays) can be applied to the analysis of ALT and AST levels in biological fluids. The paper-based biodetection technique can semi-quantitatively measure ALT and AST from capillary finger sticks, and it will pave the way for the development of an inexpensive and rapid alternative method for the early detection and diagnosis of liver diseases. This method is expected to significantly reduce the economic burden and aid routine clinical analysis in both developed and underdeveloped countries. The development of low-cost testing platforms and their diagnostic utility will be extremely beneficial in helping millions of patients with liver disorders.

Point-of-care tests play an important role in serological diagnostics of infectious diseases and post-vaccination immunity monitoring, including in COVID-19. Currently, lateral flow tests dominate in this area and show good analytical performance. However, studies to improve the effectiveness of such tests remain important. In comparison with lateral flow tests, vertical flow immunoassays allow for a reduction in assay duration and the influence of the hook effect. Additionally, the use of carbon black nanoparticles (CNPs) as a color label can provide a lower detection limit (LOD) compared to conventional colloidal gold. Therefore, we have developed a vertical flow immunoassay for the detection of IgG against SARS-CoV-2 spike protein in human serum samples by applying a conjugate of CNPs with anti-human IgG mouse monoclonal antibodies (CNP@MAb). The vertical flow assay device consists of a plastic cassette with a hole on its top containing a nitrocellulose membrane coated with spike protein and an absorbent pad. The serum sample, washing buffer, and CNP@MAb flow vertically through the nitrocellulose membrane and absorbent pads, reducing assay time and simplifying the procedure. In positive samples, the interaction of CNP@MAb with anti-spike antibodies leads to the appearance of black spots, which can be visually detected. The developed method allows for rapid visual detection (5–7 min) of IgG vs. spike protein, with a LOD of 7.81 BAU/mL. It has been shown that an untrained operator can perform the assay and visually evaluate its results. Thus, the presented assay can be used in the further development of test systems for the serological diagnostics of COVID-19 or post-vaccination immunity monitoring.

Nucleic acid-based molecular detection of mitochondrial DNA (mtDNA) has been used for the detection of eukaryotic pathogens, genetic markers, and biomedical studies in humans and animals, forensic investigation, conservation biology, and environmental monitoring. Currently available molecular mtDNA testing is predominately dependent on relatively expensive laboratory equipment, which limits test applicability for on-site field testing. Recent technical advances in molecular testing of mtDNA using microfluidic chip technology and paper-based methods, and assays based on isothermal nucleic acid amplification show promise for field assay development. Microfluidic and paper-based components for mtDNA sample preparation and isothermal amplification establish the groundwork for targeted detection. The goal is on-site field detection mtDNA assays that combine sample processing, assay setup, and visual representation of the presence of mtDNA. This review examines advantages and disadvantages of alternative field-compatible tests for on-site detection of mtDNA. Despite challenges, developing an integrated "sample-through detection" field test platform is the future direction for mtDNA molecular detection in environmental samples. [ABSTRACT FROM AUTHOR]

Paper-based assays have revolutionized POC diagnostics, offering significant values across diverse sectors due to their simplicity, cost-effectiveness, and portability. Detecting E. coli is critical for public health but faces challenges in traditional methods, prompting ongoing exploration of alternative approaches. To address the need for heightened sensitivity, this study introduces lithium acetate as a novel element in a paper-based E. coli detection assay, accompanying absorbance analysis using microplate readers. Utilization of lithium acetate as a lysis reagent and carrying out absorbance measurements in paper microtiter plates provide a significant stride in improving sensitivity and assay efficiency. The assay substantially reduced the analysis time to 4 hours with a lower quantification limit of 103 cfu/ml for detecting E. coli in pure cultures. The results suggest that the assay could determine 10 cfu/ml of E. coli in a contaminated milk sample with 4 hours of enrichment time. Thus, the study contributes towards improving E. coli detection with lower quantification limits and shorter analysis times. It is anticipated that the diagnostic methodology would circumvent the challenges in conventional E. coli detection methods, placing emphasis on absorbance analysis to achieve a streamlined, accurate, and rapid paper-based detection technique. [Display omitted] • Colorimetric detection of E. coli using a novel paper-based assay. • An improved assay using lithium acetate for cell lysis. • Absorbance analyses using microplate readers for analyte quantification and assessment. • Obtained LOD is 10 cfu/ml in contaminated milk samples. [ABSTRACT FROM AUTHOR]

The overutilization of dimethoate may lead to its deposition in both the environment and crops, which potentially induces neurological, respiratory, and hepatic abnormalities upon absorption by organisms. Colorimetry can accurately determine dimethoate, but there are still many challenges in the rapid, convenient, and point-of-care detection. Herein, based on the inhibitory effect of dimethoate on the laccase activity of silver citrate (AgCit), a smartphone-assisted colorimetric sensor and rapid detection enzyme sheets were constructed. It was demonstrated that dimethoate could adsorb on the surface of AgCit and inhibit the production of semiquinone radicals in the reaction system. Then, the AgCit laccase activity was inhibited, causing the colorimetry sensor and the enzyme sheets turned light red or even colorless. The constructed sensor has higher sensitivity and the limit of detection for dimethoate is 1.0 μg⋅L−1. Moreover, the developed enzyme sheets have good selectivity and specificity, and display the detection results within 5 min. Thus, the colorimetric sensor and the constructed enzyme sheets are expected to be used for large-scale preliminary detection of dimethoate residues in actual agricultural products. • The laccase-like activity of enzyme sheet for dimethoate was first developed. • The inhibition mechanism of AgCit laccase-like activity by dimethoate was studied. • The AgCit was first used as a chromogenic agent for enzyme sheets. • The enzyme sheets can directly distinguish dimethoate with the naked eye in 5 min. [ABSTRACT FROM AUTHOR]

Chemiluminescence (CL) reagent luminol was loaded into the porous structure of cobalt-imidazole metal-organic framework (MOF) ZIF-67 to obtain luminol-functionalized ZIF-67 (luminol@ZIF-67) with CL property. The morphology, composition, CL property, and CL mechanism of luminol@ZIF-67 were carefully investigated. The obtained luminol@ZIF-67 exhibited strong, stable, and visible CL emission that reacted with H2O2, attributed to the strong catalytic effect of ZIF-67 combined with the shortened diffusion distance between luminol and the catalytic center. The CL intensity of luminol@ZIF-67 was more than 550 times higher than that of luminol. Catechol can effectively quench the CL emission of luminol@ZIF-67 that reacted with H2O2. Then, a simple paper-based CL imaging detection method was developed for the detection of catechol by using a smartphone as a portable detector. The linear calibration curve of the developed CL assay for catechol ranged from 5 to 100 mg/L with detection limit of 1.1 mg/L (S/N = 3δ). The strong CL emission of luminol@ZIF-67 combined with the effective quench ability of catechol guaranteed high sensitivity of the detection method. The practical application ability of the developed CL assay was tested by the determination of catechol in tea and tap water samples, resulting in acceptable results. This work provides an effective paper-based CL detection method for catechol and enriches the species of the chemiluminescent MOF material. [ABSTRACT FROM AUTHOR]

Food fortification in low-income settings is limited due to the lack of simple quality control sensing tools. In this study, we field validated a paper-based, smartphone-assisted colorimetric assay (Nu3Px) for the determination of iron in fortified flours against the gold standard method, atomic emission spectrometry (AES). Samples from commercial brands (n = 6) were collected from supermarkets, convenience stores, and directly from companies in Mexico and characterized using both Nu3Px and AES. Nu3Px’s final error parameters were quantified (n = 45) via method validation final experiments (replication and comparison of methods experiment). Qualitative pilot testing was conducted, assessing Nu3Px’s accept/reject batch decision making (accept ≥ 40 μg Fe/g flour; reject < 40 μg Fe/g flour) against Mexico’s fortification policy. A modified user-centered design process was followed to develop and evaluate an alternative sampling procedure using affordable tools. Variation of iron content in Mexican corn flours ranged from 23% to 39%. Nu3Px’s random error was 12%, and its bias was 1.79 ± 9.99 μg Fe/g flour. Nu3Px had a true mean difference from AES equal to 0 and similar variances. AES and Nu3Px made similar classifications based on Mexico’s policy. Using simple, affordable tools for sampling resulted in similar output to the traditional sampling preparation (r = 0.952, p = 0.01). The affordable sample preparation kit has similar precision to using analytical tools. The sample preparation kit coupled with the smartphone app and paper-based assay measure iron within the performance parameters required for the application to corn flour fortification programs, such as in the case of Mexico.

Directed evolution has become an important method to unleash the latent potential of enzymes to make them uniquely suited for human purposes. However, the need for a large reagent volume and sophisticated instrumentation hampers its broad implementation. In an attempt to address this problem, here we report a paper-based high-throughput screening approach that should find broad application in generating desired enzymes. As an example case, the dehalogenation reaction of the halohydrin dehalogenase was adopted for assay development. In addition to visual detection, quantitative measurements were performed by measuring the color intensity of an image that was photographed by a smartphone and processed using ImageJ free software. The proposed method was first validated using a gold standard method and then applied to mutagenesis library screening with reduced consumption of reagents (i.e., ≤ 10 μl per assay) and a shorter assay time. We identified two active mutants (P135A and G137A) with improved activities toward four tested substrates. The assay not only consumes less reagents but also eliminates the need for expensive instrumentation. The proposed method demonstrates the potential of paper-based whole-cell screening coupled with digital image colorimetry as a promising approach for the discovery of industrially important enzymes. Key Points • A frugal method was developed for directed enzyme evolution. • Mutagenesis libraries were successfully screened on a paper platform. • Smartphone imaging was efficiently used to measure enzyme activities. [ABSTRACT FROM AUTHOR]

This study unveils the electrochemically-enhanced nanozymatic activity exhibited by borophene during the reaction of 3,3′,5,5′-tetramethylbenzidine (TMB) and H 2 O 2. Herein, the surface of the pristine borophene was first modified with the addition of thiocyanate groups to improve hydroxyl radical (•OH) scavenging activity. Then, the oxidation reaction of TMB was accelerated under applied electrochemical potential. Both factors significantly improved the detection limit and drastically decreased the detection time. DPPH testing revealed that the radical scavenging nature of borophene was more than 70%, boosting its catalytic activity. In the presence of H 2 O 2 , borophene catalyzed the oxidation of TMB and produced a blue-colored solution that was linearly correlated with the concentration of H 2 O 2 and allowed for the detection of H 2 O 2 up to 38 nM. The present finding was further extended to nanozymatic detection of tetracyclines (TCs) using a target-specific aptamer, and the results were colorimetrically quantifiable up to 1 μM with a LOD value of 150 nM. Moreover, transferring the principles of the discussed detection method to form a portable and disposable paper-based system enabled the quantification of TCs up to 0.2 μM. All the sensing experiments in this study indicate that the nanozymatic activity of borophene has significantly improved under electrochemical potential compared to conventional nanozyme-based colorimetric detection. Hence, the present discovery of electrochemically-enhanced nanozymatic activity would be promising for various sensitive and time-dependent colorimetric sensor development initiatives in the future. [ABSTRACT FROM AUTHOR]

In the last decades, there is a growing search for analytical strategies to ensure clinical analysis without the need of laboratory set-up and skilled personnel. Indeed, user-friendly and low-cost devices are highly valued in the era of sustainability for their capability to be applied in low-resource contexts, such as developing countries. To address this issue, herein we report a 96-well paper-based and laboratory setup-free optical platform for the detection of butyrylcholinesterase enzyme (BChE) activity in human serum. We used chromatographic paper to realize a novel analytical tool exploiting its porous structure for reagentless synthesize Prussian Blue Nanoparticles (the sensing element), as well to load all the reagents required for the measurement. The principle of BChE activity detection relies on the reaction between the enzymatic product thiocholine and Prussian Blue, giving the Prussian White with subsequently Prussian Blue's fading, detected by a common office scanner supported by ImageJ software. Using this novel paper-based optical platform, BChE activity was linearly detected in the 2–15 U/mL range with a detection limit down to 0.8 U/mL. The accuracy was successfully demonstrated by recovery study with spiked serum and by comparing the data with the gold standard method. • 96-wells paper-based platform for BChE activity quantification in serum. • Colorimetric detection by following the Prussian Blue reduction by thiocholine. • The measurement requires only some µL of diluted human serum. • Color reading by using a common office scanner. • BChE activity is linearly detected up to 15 U/mL with LOD equal to 0.8 U/mL. [ABSTRACT FROM AUTHOR]

Bioassays based on cellulose paper substrates are gaining increasing popularity for the development of field portable and low-cost diagnostic applications. Herein, we report a paper-based nucleic acid hybridization assay using immobilized upconversion nanoparticles (UCNPs) as donors in luminescence resonance energy transfer (LRET). UCNPs with intense green emission served as donors with Cy3 dye as the acceptor. The avidin functionalized UCNPs were immobilized on cellulose paper and subsequently bioconjugated to biotinylated oligonucleotide probes. Introduction of unlabeled oligonucleotide targets resulted in a formation of probe-target duplexes. A subsequent hybridization of Cy3 labeled reporter with the remaining single stranded portion of target brought the Cy3 dye in close proximity to the UCNPs to trigger a LRET-sensitized emission from the acceptor dye. The hybridization assays provided a limit of detection (LOD) of 146.0 fmol and exhibited selectivity for one base pair mismatch discrimination. The assay was functional even in undiluted serum samples. This work embodies important progress in developing DNA hybridization assays on paper. Detection of unlabeled targets is achieved using UCNPs as LRET donors, with minimization of background signal from paper substrates owing to the implementation of low energy near-infrared (NIR) excitation.

An integrated microfluidic platform comprising a microfluidic paper-based analytical device (µPAD) and a portable detection system is proposed for the concentration detection of benzoic acid via Janovsky reaction theory. In the proposed approach, the reaction zone of the µPAD is implanted with 5 N sodium hydroxide and dried at 30 °C for 20 min. The benzoic acid sample is derived to 3,5-Dinitrobenzoic acid using KNO 3 and H 2 SO 4 at 40 °C for 40 min and is then dripped on the reaction zone of the µPAD. Finally, the µPAD is transferred to the portable detection system and heated at a temperature of 45 °C for 20 min on a hot plate to prompt a Janovsky reaction. The resulting color change of the detection zone is observed using a CMOS camera. The reaction color image is delivered to a smartphone via a connector and the benzoic acid concentration is determined using self-written RGB analysis software. The experimental results obtained using control samples with known benzoic acid concentrations in the range of 500–4000 ppm show that the R(ed) + B(lue) intensity (Y) and benzoic acid concentration (X) are related as Y =  −0.0264 X + 408.79. Moreover, the correlation coefficient is equal to R 2  = 0.9953. The proposed detection platform is used to measure the benzoic acid concentrations of twenty-one commercial food samples. It is shown that the concentration measurements deviate by no more than 6.6% from those obtained using a standard HPLC macroscale method. Overall, the results presented in this study show that the proposed integrated microfluidic paper-based chip platform provides a compact and reliable tool for benzoic acid concentration measurement purposes. [ABSTRACT FROM AUTHOR]

Porcine sperm motility was assessed via resazurin reduction color change in sperm cells using a novel paper-based assay of our own design. We applied mixtures of resazurin solution and porcine semen onto hydrophilic test circles on our paper-based device and investigated the resulting reduction reaction expressed as red and blue color intensity (RBCI). We quantified this reaction using a blue/pink color ratio from our 8 × 3 = 24 bit RGB color image. To examine enzymatic reactivity in sperm cells, we used two inhibitors: 3-Nitropropanoic acid (3-NPA) and 3-Bromopyruvic acid (3-BP). 3-NPA inhibits the citric acid cycle and electron transfer reaction in mitochondria, but did not strongly reduce sperm motility in our tests. 3-BP decreases reactivity of both mitochondrial electron transfer and glycolytic enzymes in cytosol, which significantly lowers porcine sperm motility. RBCIs of 3-NPA- and 3-BP-treated samples were significantly lower compared to our untreated control (p < 0.025). Based on these results, we feel that resazurin can be used to estimate the amount of reductants with and without inhibitor treatment. For continued research assessing the molecular mechanisms of resazurin reduction in porcine sperm, a combination assay using two or more redox indicators (e.g., resazurin and Thiazolyl Blue Tetrazolium Bromide (MTT)) embedded into our paper-based device could further our understanding of sperm cell bioenergetics.

The diagnosis of periprosthetic joint infection (PJI) remains a labor-intensive and challenging issue, with life-threatening complications associated with misdiagnoses. Superior diagnostic approaches are therefore urgently needed, and synovial biomarkers are gaining substantial attention in this capacity. A new aptamer-based sandwich assay was developed where the aptamer probes specific to one such biomarker, alpha-defensin human neutrophil protein 1 (HNP 1), was integrated herein into a new microfluidic platform. The magnetic beads coated with the primary aptamer probe were able to bind the target protein with high affinity and high specificity in synovial fluid and a fluorescent-labelled secondary aptamer were further used to quantify HNP 1 in a sandwich approach. Up to four clinical samples with low volume (∼50 μL each) in a much faster assay including detection within <60 min with 100% accuracy (with totally 13 clinical samples without the need of sample pretreatment) through the use of the aptamer-based sandwich assay were automatically detected on a single chip. The wide dynamic range of this compact device, 0.5–100 mg/L, highlights its utility for future PJI diagnostics in the clinic. [ABSTRACT FROM AUTHOR]

In this work, a facial ratiometric fluorescence nanorpobe (RFP) based on gadolinium and nitrogen co-doped CDs (Gd , N -CDs) was for the first time proposed for visual detection of folic acid (FA). The Gd , N -CDs were prepared by hydrothermal heating of Gd(NO 3) 3 ⋅6H 2 O, o-phenylenediamine (OPD), 4-aminobenzoic acid (PABA). The as-prepared Gd , N -CDs had an average particle size of 10.27 ± 0.35 nm and exhibited two emission bands at 396 and 548 nm, respectively upon excitation at 340 nm. In the presence of FA, the 560 nm emission of Gd , N -CDs was reduced while the fluorescence signal at 457 nm was enhanced, and the quantitatively determination of FA was based on the fluorescence intensity ratio (F 457 /F 560). The as-constructed RFP achieved a linear range of 0.0–30.0 μM and a low detection limit (LOD) of 3.43 nM. In particular, a distinguishable change in fluorescence colour from bright orange to blue enabled the development of a smartphone-assisted visual sensing platform and a paper-based analytical device for FA detection with LODs of 3.06 μM and 0.83 μM, respectively. The proposed sensing platform was successfully utilised for onsite detection of FA in vegetable, fruit and yolk samples with satisfactory results were achieved. This work highlights the application potential of smartphone-integrated CDs-based RFP as a promising portable method for on-site analysis of FA. [Display omitted] • A Gd , N -CDs-based ratiometric fluorescence method was proposed for ultrasensitive sensing of folic acid. • The proposed method realized folic acid visualization by integrating with a smartphone and a paper device. • The proposed fluorescence methods were successfully used for folic acid detection in food samples. [ABSTRACT FROM AUTHOR]

Malaria is responsible for over 435,000 deaths annually, mostly occurring in sub-Saharan Africa. Detecting Plasmodium spp. sporozoites (spzs) in the salivary glands of Anopheles (Diptera: Culicidae) vectors with circumsporozoite enzyme-linked immunosorbent assay (csELISA) is an important surveillance method. However, current technological advances are intellectual property and often require of distribution and highly trained users. The transition into paper-based rapid plataforms would allow for decentralization of survillance, especially in areas where it was virtually eliminated. The addition of bio-based materials have shown the potential to improve binding of target antigens, while being widely available. Here, we evaluate the use of chitosan and cellulose nanocrystals (CNC) as antibody carriers and substrate coatings on 96-well plates and on wax hydrophobized paper plates for the detection of Plasmodium falciparum (Pf), P. vivax VK210 (Pv210), and P. vivax VK247 (Pv247). To further improve the user-friendliness of the paper plates a quantitative photograph image-based color analysis was done. Interactions between the materials and the assay antibodies were studied by quartz crystal microbalance with dissipation monitoring (QCM-D). Overall, the addition of chitosan increased the interaction with antibodies and enhanced signaling in all tests. This work demonstrated that the adaptation of a PcsELISA shows potential as a cost-effective alternative assay platform easily adaptable in deployable testing sites that also showed reduction in reagent volumes by 80% and assay run time by seventh. While dipstick assays were previously developed, paper-based assays are a cost-effective and field-deployable alternative, reducing volumes of reagents that could be used in malaria control and elimination settings., (Published by Oxford University Press on behalf of Entomological Society of America 2023.)

This article describes a paper-based low cost single cell HaloChip assay that can be used to assess drug- and radiation-induced DNA damage at point-of-care. Printing ink on paper effectively blocks fluorescence of paper materials, provides high affinity to charged polyelectrolytes, and prevents penetration of water in paper. After exposure to drug or ionizing radiation, cells are patterned on paper to create discrete and ordered single cell arrays, embedded inside an agarose gel, lysed with alkaline solution to allow damaged DNA fragments to diffuse out of nucleus cores, and form diffusing halos in the gel matrix. After staining DNA with a fluorescent dye, characteristic halos formed around cells, and the level of DNA damage can be quantified by determining sizes of halos and nucleus with an image processing program based on MATLAB. With its low fabrication cost and easy operation, this HaloChip on paper platform will be attractive to rapidly and accurately determine DNA damage for point-of-care evaluation of drug efficacy and radiation condition. [ABSTRACT FROM AUTHOR]

The article describes a simple optical assay for glucose. It is based on the use of paper spots loaded with colloidal CdSe/ZnS quantum dots (Q-dots) and the enzyme glucose oxidase (GOx). Circular paper sheets were uniformly loaded with Q-dots and then displayed strong fluorescence under a UV lamp (365 nm exCitation). The action of GOx causes the production of HO which, after a typical exposure time of 20 min, causes fluorescence intensity to be quenched. To obtain a reading, the paper sheets were photographed under 365 nm excitation using a digital camera. Several parameters, including the amount of Q-dots, sample pH, and amount of GOx were optimized to maximize the response to glucose. The paper-based assay showed a sigmoidal-shaped response with respect to the glucose concentration in the 5-200 mg·dL range (limit of detection of 5 μg·dL), demonstrating their potential use for biomedical applications. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Food fortification in low-income settings is limited due to the lack of simple quality control sensing tools. In this study, we field validated a paper-based, smartphone-assisted colorimetric assay (Nu3Px) for the determination of iron in fortified flours against the gold standard method, atomic emission spectrometry (AES). Samples from commercial brands (n = 6) were collected from supermarkets, convenience stores, and directly from companies in Mexico and characterized using both Nu3Px and AES. Nu3Px's final error parameters were quantified (n = 45) via method validation final experiments (replication and comparison of methods experiment). Qualitative pilot testing was conducted, assessing Nu3Px's accept/reject batch decision making (accept ≥ 40 μg Fe/g flour; reject < 40 μg Fe/g flour) against Mexico's fortification policy. A modified user-centered design process was followed to develop and evaluate an alternative sampling procedure using affordable tools. Variation of iron content in Mexican corn flours ranged from 23% to 39%. Nu3Px's random error was 12%, and its bias was 1.79 ± 9.99 μg Fe/g flour. Nu3Px had a true mean difference from AES equal to 0 and similar variances. AES and Nu3Px made similar classifications based on Mexico's policy. Using simple, affordable tools for sampling resulted in similar output to the traditional sampling preparation (r = 0.952, p = 0.01). The affordable sample preparation kit has similar precision to using analytical tools. The sample preparation kit coupled with the smartphone app and paper-based assay measure iron within the performance parameters required for the application to corn flour fortification programs, such as in the case of Mexico. [ABSTRACT FROM AUTHOR]

Smartphone based devices (SBDs) have the potential to revolutionize food safety control by empowering citizens to perform screening tests. To achieve this, it is of paramount importance to understand current research efforts and identify key technology gaps. Therefore, a systematic review of optical SBDs in the food safety sector was performed. An overview of reviewed SBDs is given focusing on performance characteristics as well as image analysis procedures. The state-of-the-art on commercially available SBDs is also provided. This analysis revealed several important technology gaps, the most prominent of which are: (i) the need to reach a consensus regarding optimal image analysis, (ii) the need to assess the effect of measurement variation caused by using different smartphones and (iii) the need to standardize validation procedures to obtain robust data. Addressing these issues will drive the development of SBDs and potentially unlock their massive potential for citizen-based food control. • Optical smartphone based sensors in the food safety field are systematically reviewed. • Recommendations on image analysis optimization are given. • The analytical performance of smartphone based sensors is discussed. • Available commercial devises are critically compared. [ABSTRACT FROM AUTHOR]

Biosensor development exploiting various transduction principles is characterized by a strong competition to reach high detectability, portability and robustness. Nevertheless, a literature-based comparison is not possible, as different conditions are employed in each paper. Herein, we aim at evaluating which measurement, photons or electrons, yields better biosensor performance. Upon outlining an update in recent achievements to boost analytical performance, amperometry and chemiluminescence (CL)-based biosensors are directly compared employing the same biospecific reagents and analytical formats. Horseradish peroxidase (HRP) and hydrogen peroxide concentrations were directly measured, while glucose and mouse IgG were detected employing an enzyme paper-based biosensor and an immunosensor, respectively. Detectability was down to picomoles of hydrogen peroxide (4 pmol for CL and 210 pmol for amperometry) and zeptomoles of HRP (45 zmol for CL and 20 zmol for amperometry); IgG was detected down to 12 fM (CL) and 120 fM (amperometry), while glucose down to 17 μM (CL) and 40 μM (amperometry). Results showed that amperometric and CL biosensors offered similar detectability and analytical performance, with some peculiarities that suggest complementary application fields. As they generally provided slightly higher detectability and wider dynamic ranges, CL-based biosensors appear more suitable for point-of-care testing of clinical biomarkers, where detectability is crucial. Nevertheless, as high detectability in CL biosensors usually requires longer acquisition times, their rapidity will allocate electrochemical biosensors in real-time monitoring and wearable biosensors. The analytical challenge demonstrated that these biosensors have competitive and similar performance, and between photons and electrons the competition is still open. Image 1 • Challenge photon vs electron, which measurement yields better biosensor performance? • An update on electrochemical and chemiluminescent biosensor development is reported. • Amperometric and CL biosensors are challenged employing the same protocols. [ABSTRACT FROM AUTHOR]

The ability to identify single-nucleotide mutations is critical for probing cell biology and for precise detection of disease. However, the small differences in hybridization energy provided by single-base changes makes identification of these mutations challenging in living cells and complex reaction environments. Here, we report a class of de novo -designed prokaryotic riboregulators that provide ultraspecific RNA detection capabilities in vivo and in cell-free transcription-translation reactions. These single-nucleotide-specific programmable riboregulators (SNIPRs) provide over 100-fold differences in gene expression in response to target RNAs differing by a single nucleotide in E. coli and resolve single epitranscriptomic marks in vitro. By exploiting the programmable SNIPR design, we implement an automated design algorithm to develop riboregulators for a range of mutations associated with cancer, drug resistance, and genetic disorders. Integrating SNIPRs with portable paper-based cell-free reactions enables convenient isothermal detection of cancer-associated mutations from clinical samples and identification of Zika strains through unambiguous colorimetric reactions. • SNIPRs are riboregulators with single-nucleotide-specific RNA detection capabilities • They operate in live cells and in paper-based cell-free reactions • Automated in silico design allows SNIPRs to detect many different harmful mutations • Isothermal SNIPR tests enable portable human genotyping and viral strain detection The development of high-specificity RNA toehold-based sensors allows for the discrimination of point mutations and RNA base modifications and enables genotypic diagnosis for E. coli , human samples, and Zika virus strains [ABSTRACT FROM AUTHOR]

Rapid and inexpensive serological tests for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antibodies are essential to conduct large-scale seroprevalence surveys and can potentially complement nucleic acid or antigen tests at the point of care. During the COVID-19 pandemic, extreme demand for traditional lateral flow tests has stressed manufacturing capacity and supply chains. Motivated by this limitation, we developed a SARS-CoV-2 antibody test using cellulose, an alternative membrane material, and a double-antigen sandwich format. Functionalized SARS-CoV-2 antigens were used as both capture and reporter binders, replacing the anti-human antibodies currently used in lateral flow tests. The test could provide enhanced sensitivity because it labels only antibodies against SARS-CoV-2 and the signal intensity is not diminished due to other human antibodies in serum. Three-dimensional channels in the assay were designed to have consistent flow rates and be easily manufactured by folding wax-printed paper. We demonstrated that this simple, vertical flow, cellulose-based assay could detect SARS-CoV-2 antibodies in clinical samples within 15 min, and the results were consistent with those from a laboratory, bead-based chemiluminescence immunoassay that was granted emergency use approval by the US FDA.

Porcine sperm motility was assessed via resazurin reduction color change in sperm cells using a novel paper-based assay of our own design. We applied mixtures of resazurin solution and porcine semen onto hydrophilic test circles on our paper-based device and investigated the resulting reduction reaction expressed as red and blue color intensity (RBCI). We quantified this reaction using a blue/pink color ratio from our 8 × 3 = 24 bit RGB color image. To examine enzymatic reactivity in sperm cells, we used two inhibitors: 3-Nitropropanoic acid (3-NPA) and 3-Bromopyruvic acid (3-BP). 3-NPA inhibits the citric acid cycle and electron transfer reaction in mitochondria, but did not strongly reduce sperm motility in our tests. 3-BP decreases reactivity of both mitochondrial electron transfer and glycolytic enzymes in cytosol, which significantly lowers porcine sperm motility. RBCIs of 3-NPA- and 3-BP-treated samples were significantly lower compared to our untreated control (p < 0.025). Based on these results, we feel that resazurin can be used to estimate the amount of reductants with and without inhibitor treatment. For continued research assessing the molecular mechanisms of resazurin reduction in porcine sperm, a combination assay using two or more redox indicators (e.g., resazurin and Thiazolyl Blue Tetrazolium Bromide (MTT)) embedded into our paper-based device could further our understanding of sperm cell bioenergetics. [ABSTRACT FROM AUTHOR]

A lack of quality control tools limits the enforcement of fortification policies. In alignment with the World Health Organization's ASSURED criteria (affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free, and deliverable), a paper-based assay that interfaces with a smartphone application for the quantification of iron fortificants is presented. The assay is based on the Ferrozine colorimetric method. The reaction started after deposition of the 5 µL aqueous sample and drying. After developing color, pixel intensity values were obtained using a smartphone camera and image processing software or a mobile application, Nu3px. From these values, the actual iron concentration from ferrous sulfate and ferrous fumarate was calculated. The limits of detection, quantification, linearity, range, and errors (systematic and random) were ascertained. The paper-based values from real samples (wheat flour, nixtamalized corn flour, and infant formula) were compared against atomic emission spectroscopy. The comparison of several concentrations of atomic iron between the spectrophotometric and paper-based assays showed a strong positive linear correlation (y = 47.01x + 126.18; R2 = 0.9932). The dynamic range (5.0–100 µg/mL) and limit of detection (3.691 µg/mL) of the paper-based assay are relevant for fortified food matrices. Random and systematic errors were 15.9% and + 8.65 µg/g food, respectively. The concept can be applied to limited-resource settings to measure iron in fortified foods. [ABSTRACT FROM AUTHOR]

Bioassays based on cellulose paper substrates are gaining increasing popularity for the development of field portable and low-cost diagnostic applications. Herein, we report a paper-based nucleic acid hybridization assay using immobilized upconversion nanoparticles (UCNPs) as donors in luminescence resonance energy transfer (LRET). UCNPs with intense green emission served as donors with Cy3 dye as the acceptor. The avidin functionalized UCNPs were immobilized on cellulose paper and subsequently bioconjugated to biotinylated oligonucleotide probes. Introduction of unlabeled oligonucleotide targets resulted in a formation of probe-target duplexes. A subsequent hybridization of Cy3 labeled reporter with the remaining single stranded portion of target brought the Cy3 dye in close proximity to the UCNPs to trigger a LRET-sensitized emission from the acceptor dye. The hybridization assays provided a limit of detection (LOD) of 146.0 fmol and exhibited selectivity for one base pair mismatch discrimination. The assay was functional even in undiluted serum samples. This work embodies important progress in developing DNA hybridization assays on paper. Detection of unlabeled targets is achieved using UCNPs as LRET donors, with minimization of background signal from paper substrates owing to the implementation of low energy near-infrared (NIR) excitation.