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E-mail: mohammad. Limited quantitative and equipment-free POC assays have been reported. This study aims to develop, validate, and apply a simple, quantitative, paper-based POC assay. Methods : wax-channeled paper treated with specific anti- Brucella and anti- Salmonella antibodies was used for distance-based chromatographic elution of stained bacterial cell agglutinations. The optimization of the test for paper type, microfluidic channel design, antibody and bacterial cell concentrations, and elution methods was carried out. Quantitative assay transformation was successfully developed using the color intensity of the original reaction zone, intensity of elution tail, and distance-based migration that correspond to bacterial agglutination size. The migration distance of eluted bacterial agglutination bands corresponds to the target concentration with good linearity and minimal variability. Reporting of colored band migration with numbers using microfluidic patterns was used to enhance non-technical end-user applications. A distance-based POC assay prototype was then successfully used for the accurate detection of known and unknown samples in comparison with standard assays. Conclusions : the migration distance of an eluted stained bacterial agglutination correlated with anti-bacterial antibody concentrations. A simple, cheap, quantitative, and equipment-free paper-based POC assay of bacterial cell agglutination was developed. The assay has extended applications to different human disease biomarkers. Quantitative, equipment-free, distance-based paper POCs that rely on reading a visual signal length that corresponds to target concentration in a thermometer-like approach have been developed. Some of these POC devices require the presence of a smart phone for accurate detection. Distance-based paper POCs were used for the measurement of cholesterol, high-density lipoprotein, glucose, bilirubin, nickel, glutathione, immunoglobulin, aerosol oxidative activity, cocaine, lactoferrin, theophylline, and metals. Serological tests for the detection of anti- Brucella or anti- Salmonella antibodies are commonly used for establishing the diagnosis using slide agglutination test or ELISA methods. Furthermore, converting distance to text numbers is a new and simple approach that shows results directly with no need for further interpretation. The Salmonella slide test applies a similar principle and procedure for the qualitative and semi-quantitative detection of anti- Salmonella antibodies using visible agglutination. The test was performed according to manufacturer instructions Arcomex, Jordan. Rose Bengal reagent was concentrated using a centrifuge Sigma, USA , followed by discarding the required volume of the supernatant. Pictures of the reactions were taken by a Canon EOS D digital camera Canon, Japan and the washed tail length was measured using a ruler. Kleenex had the highest differences between the two concentrations absorbances but failed to leave a washing tail, which could be possibly used as a concentration measurement tool. Filter paper grade and chromatography paper were tested for the possibility of using direct washing of positive and negative agglutination reactions. Filter paper grade had a nearly completely washed negative reaction zone and a non-washed positive agglutination. The washed tails lengths were measured for each concentration on each paper type too. Grade showed tail lengths positively correlated with the expected wash level according to each concentration. However, glass fiber MGC paper showed no tail lengths according to the concentrations Fig. Filter paper grade was used for a semi-quantitative agglutination reaction using a direct wash of normal saline. In this study, we combined color intensity, chromatographic migration distance, and text reporting by ruler or numbers using patterned paper to generate a new quantitative and equipment-free prototype for antibody detection using stained bacterial agents. The assay prototype was optimized, validated and successfully detected anti- Brucella and anti- Salmonella antibodies in samples. The color can be provided using naturally colored cells e. Limited assays have reported equipment-free, text-based signal readout of paper-based POCs. The agglutination test depends on the ability of antibodies to react with antigens on cells or particles to form clumps. Agglutination can be visualized by the naked eye using slide, card, and tube methods or through automated machines. The test has a wide range of applications including blood grouping, detection of infections, and autoimmunity. The assay is simple, fast, reliable, uses cheap materials in an innovative design, and can have extended applications of agglutination assays. The advantage of using the paper-based agglutination assay developed in this study includes open reading time as the results are stable for days in the paper while standard slide agglutination had a very short reading window of a few minutes before the reaction fades due to dryness and disabled agglutination. The paper-based assay used the chromatographic elution of colored agglutination clumps based on size within a porous paper matrix to provide a distance-based migration that corresponds to the target concentration. The mechanism has been described previously for the qualitative detection of blood groups or plasma and serum separation. Implications of POC tests for infectious diseases diagnosis including Brucella and Salmonella are huge, especially in endemic low resources countries. DOI: Received 22nd April , Accepted 6th June Paper type. Different paper types were considered for test optimization. Filter paper grade and glass fiber MGC paper showed poor agglutination color variation among the positive and negative results after washing with the three washing types. Filter paper grade and chromatography paper had better color intensity variations using normal saline as a washing buffer compared to the other two wash types. Kleenex displayed a clear color intensity difference between positive and negative results when normal saline was used as a wash. Target concentration. However, filter paper grade and glass fiber MGC paper had the least semi-quantitative results. Rose Bengal reagent concentration. Positive control concentrations were used for this purpose on chromatography paper and filter paper grade Channels shape. Hydrophobic waxed channels were printed on chromatography paper to control the direction of the reaction zone for a more accurate semi-quantification of the reaction. The measured tail lengths increased when the concentration decreased. The average tail lengths were 1. B : a table of tail length readings for the four repeated channeled reactions. B : Head and tail absorbance readings as well as tail lengths in cm. The average absorbance OD of the same 4 trails was calculated with different concentrations B. Immobilized specific antibody, enzyme reagent glucose oxidase and horseradish peroxidase and a color-developer solution containing substrates for both enzymes. Simple, rapid, stable, reliable, with no need for external calibration, or blood separation. Immobilization of glucose oxidase and monoclonal antibody to theophylline on chromatographic paper followed by the addition of enzyme reagents glucose, dicarboxidine, ascorbate, and theophylline-labeled horseradish peroxidase HRP. Require 20 minutes with multiple reagents and antibodies and applications limited to therapeutic drug monitoring. Enzymatic reaction with cholesterol to produce hydrogen peroxide and quantification using redox-coupled indicator. Serum separation and erythrocyte agglutination followed by lipoprotein separation, the first reaction chamber contains chromatography paper with immobilized HRP, the second chamber contains immobilized cholesterol esterase, the last chamber contains immobilized cholesterol oxidase. Aptamer cross-linked hydrogel for target recognition, cascade enzymatic reactions for signal amplification, within microfluidic paper-based device. Cost-effective, disposable, easy to fabricate, sensitive, selective, detection of a variety of targets. Detection by a cellular phone camera after 30 minutes, requires controlled conditions and multiple washing steps. Aptamer and invertase-DNA conjugate functionalized sepharose beads, and cascade enzymatic reactions, within a 3D microfluidic paper-based device. One step, low-cost, small volume, disposable, applicable to many targets, and simple. Invasive fungi Candida and Aspergillus. CRISPR Cas12a for target recognition, a DNA hydrogel coupled with a cascade of enzymatic reactions for signal amplification and transduction, within a paper-based microfluidic chips. Immobilizing 4-cyanopentylbiphenyl 5CB liquid crystalline followed by ultrasound-assisted decomposition by hydroxyl radical generated from the oxidase enzymatic reaction of the analyte. Simple, accurate with low detection limit, applicable to many biomarkers and reproducible. Elman's reagent with chemical oxidation of dithiothreitol on silver nanoparticle aggregation coupled to glutathione oxidation in a paper-based device. Utilizes the movement of immunoassay complexes with magnetic beads using a permanent magnet in PMMA. Amphiphilic biosample surfactants, DNA, bovine serum albumin, human albumin, nitrite, glucose, and low-density lipoprotein. The flow length decreased with increasing concentration of an amphiphilic sample because of adsorption of the sample on the hydrophobic barrier.

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