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Both the ABO and Rhesus (Rh) blood groups play crucial roles in blood transfusion medicine. Herein, we report a simple and low-cost paper-based analytical device (PAD) for phenotyping red blood cell (RBC) antigens. Using this Rh typing format, 5 Rh antigens on RBCs can be simultaneously detected and macroscopically visualized within 12 min. The proposed Rh phenotyping relies on the presence or absence of hemagglutination in the sample zones after immobilizing the antibodies targeting each Rh antigen. The PAD was optimized in terms of filter paper type, antibodies, and distance of the visualization zone. In this study, the optimal conditions were Whatman filter paper Grade 4; anti-D, -C, -E, -c, and -e antibodies; RBC suspension of 30%; and a visualization zone of 1 cm above the sample zone. The accuracy of simultaneously phenotyping the five Rh RBC antigens in the blood samples (n = 4692) was 99.19%, comparable with the accuracy of the gold-standard tube method used by blood bank laboratories in several regions of Thailand. Furthermore, decision making based on this method can be assisted by deep learning. After implementing a two-stage objective detection algorithm (YOLO v4-tiny) and classification model (DenseNet-201), the ambiguous images (n = 48) were interpreted with 100% accuracy. The PAD integrated with customized-region convolutional neural networks can reduce the interpretation discrepancies in RBC antigen phenotyping in any laboratory., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Caenorhabditis elegans is an in vivo model known for its easy handling and maintenance and lack of associated ethical issues. The release of chitinase can be used to monitor the egg-laying stage in C. elegans . The aim of this study was to develop a simple and cost-effective device to monitor the activity of chitinase in embryos of C. elegans . Colloid chitin azure (CCA), a substrate for chitinase, was preimmobilized on the detection area of paper, forming a purple region, to generate a CCA paper-based analytical device (CCA-PAD). The degradation of CCA by chitinase could be observed as the purple color became faint and the filter paper eventually became colorless. Under the optimum conditions, the proposed device quantified the chitinase enzyme in the range of 15.625-125 mU/mL within 48 h ( R 2 = 0.993). In this work, 10 young adult-staged wild-type C. elegans (Bristol N2) worms were analyzed on the CCA-PAD, which was supplemented with the laboratory food source E. coli OP50 on a gauze layer. The same strain treated with 5-fluoro-2'-deoxyuridine was used to prevent egg production in C. elegans . A significant difference in the color intensity was observed between these two groups at the end of the experiment ( P = <0.001, independent t -test, n = 3). We successfully developed a simple and effective method for monitoring chitinase activity. The device may have potential applications in drug-screening studies as it efficiently distinguishes drugs that can impact egg laying.

Point-of-care testing (POCT) for uropathogen detection and chemical screening has great benefits for the diagnosis of urinary tract infections (UTIs). The goal of this study was to develop a portable and inexpensive paper-based analytical device (PAD) for cultivating bacteria in situ and rapidly testing for nitrite on the same device. The PAD was fabricated using a wax printing technique to create a pattern on Whatman No. 1 filter paper, which was then combined with a cotton sheet to support bacterial growth. Nitrite detection was based on the principle of the Griess reaction, and a linear detection range of 0-1.6 mg/dL (R 2  = 0.989) was obtained. Scanning electron microscopy (SEM) analysis demonstrated that the bacteria were able to grow and formed a cluster on the cellulose fibres within 2 hours. The enzyme β-glucuronidase, which is specifically produced by Escherichia coli, was able to convert the pre-immobilized 5-bromo-4-chloro-3-indolyl-β-D-glucuronide sodium salt (X-GlcA), a colourless substrate, generating a blue colour. Under optimum conditions, the proposed device allowed bacterial concentrations in the range of 10 4 -10 7 colony forming units (CFU)/mL to be quantified within 6 hours. Moreover, the use of this device enables the identification of E. coli pathogens with selectivity in real urine samples. In conclusion, the PAD developed in this study for UTI screening provides a rapid, cost-effective diagnostic method for use in remote areas.

Rationale: Saliva as a sample matrix is rapidly gaining interest for disease diagnosis and point-of-care assays because it is easy to collect (non-invasive) and contains many health-related biomarkers. However, saliva poses particular problems relative to more common urine and blood matrices, which includes low analyte concentrations, lack of understanding of biomolecule transportation and inherent viscosity variability in human samples. While several studies have sought to improve assay sensitivity, few have addressed sample viscosity specifically. The goal of this study is to minimize the effect of sample viscosity on paper-based analytical devices (PADs) for the measurement of pH and nitrite in human saliva. Methods: PADs were used to measure salivary pH from 5.0 to 10.0 with a universal indicator consisting of chlorophenol red, phenol red and phenolphthalein. Nitrite determination was performed using the Griess reaction. Artificial saliva with viscosity values between 1.54 and 5.10 mPa∙s was tested on the proposed PAD. To ensure the proposed PADs can be tailored for use in-field analysis, the devices were shipped to Australia and tested with human specimens. Results: Initial experiments showed that viscosity had a significant impact on the calibration curve for nitrite; however, a more consistent curve could be generated when buffer was added after the sample, irrespective of sample viscosity. The linear range for nitrite detection was 0.1 to 2.4 mg/dL using the improved method. The nitrite measurement in artificial saliva also showed a good correlation with the standard spectrophotometry method ( p =0.8484, paired sample t -test, n=20). Measured pH values from samples with varying viscosities correlated well with the results from our pH meter. Conclusions: The inherent variation of salivary viscosity that impacts nitrite and pH results can be addressed using a simple washing step on the PAD without the need for complex procedures., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Creatinine is one of many markers used to investigate kidney function. This paper describes a low-cost enzymatic paper-based analytical device (enz-PAD) for determining urine creatinine. The disposable dead volumes of creatinine enzyme reagents from an automatic analyser cassette were utilised. Whatman No. 3 paper was cut into long rectangular shapes (4×40 mm(2)) on which the enzyme reagents, R1 and R2, were adsorbed in two consecutive regions. The assay was performed by immersing test strips into urine samples contained in microwells to allow creatinine in the sample to react with immobilised active ingredients and, then, traverse via capillary action to the detection area where chromogen products accumulated. The method is based on hydrogen peroxide (H2O2) formation via creatinine conversion using creatininase, creatinase, and sarcosine oxidase. The liberated H2O2 reacts with 4-aminophenazone and 2,4,6-triiodo-3-hydroxybenzoic acid to form quinoneimine with a pink-red colour at the detection zone. The linear range of the creatinine assay was 2.5-25 mg dL(-1) (r(2)=0.983), and the detection limit was 2.0 mg dL(-1). The colorimetric enz-PAD for the creatinine assay was highly correlated with a conventional alkaline picrate method when real urine samples were evaluated (r(2)=0.977; n=40). This simple and nearly zero-cost paper-based device provides a novel alternative method for screening urinary creatinine and will be highly beneficial for developing countries., (Copyright © 2015 Elsevier B.V. All rights reserved.)

We propose a new, paper-based analytical device (PAD) for blood typing that allows for the simultaneous determination of ABO and Rh blood groups on the same device. The device was successfully fabricated by using a combination of wax printing and wax dipping methods. A 1:2 blood dilution was used for forward grouping, whereas whole blood could be used for reverse grouping. A 30% cell suspension of A-cells or B-cells was used for haemagglutination on the reverse grouping side. The total assay time was 10 min. The ratio between the distance of red blood cell movement and plasma separation is the criterion for agglutination and indicates the presence of the corresponding antigen or antibody. The proposed PAD has excellent reproducibility in that the same blood groups, namely A, AB, and O, were reported by using different PADs that were fabricated on the same day (n=10). The accuracy for detecting blood group A (n=12), B (n=13), AB (n=9), O (n=14), and Rh (n=48) typing were 92%, 85%, 89%, 93%, and 96%, respectively, in comparison with the conventional slide test method. The haematocrit of the sample affects the accuracy of the results, and appropriate dilution is suggested before typing. In conclusion, this study proposes a novel method that is straightforward, time-saving, and inexpensive for the simultaneous determination of ABO and Rh blood groups, which is promising for use in developing countries., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Electrochemical paper-based analytical devices (ePADs) with integrated plasma isolation for determination of glucose from whole blood samples have been developed. A dumbbell shaped ePAD containing two blood separation zones (VF2 membranes) with a middle detection zone was fabricated using the wax dipping method. The dumbbell shaped device was designed to separate plasma while generating homogeneous flow to the middle detection zone of the ePAD. The proposed ePADs work with whole blood samples with 24-60% hematocrit without dilution, and the plasma was completely separated within 4 min. Glucose in isolated plasma separated was detected using glucose oxidase immobilized on the middle of the paper device. The hydrogen peroxide generated from the reaction between glucose and the enzyme pass through to a Prussian blue modified screen printed electrode (PB-SPEs). The currents measured using chronoamperometry at the optimal detection potential for H2O2 (-0.1 V versus Ag/AgCl reference electrode) were proportional to glucose concentrations in the whole blood. The linear range for glucose assay was in the range 0-33.1 mM (r(2)=0.987). The coefficients of variation (CVs) of currents were 6.5%, 9.0% and 8.0% when assay whole blood sample containing glucose concentration at 3.4, 6.3, and 15.6mM, respectively. Because each sample displayed intra-individual variation of electrochemical signal, glucose assay in whole blood samples were measured using the standard addition method. Results demonstrate that the ePAD glucose assay was not significantly different from the spectrophotometric method (p=0.376, paired sample t-test, n=10)., (Copyright © 2013 Elsevier B.V. All rights reserved.)