Distance-based paper device using combined SYBR safe and gold nanoparticle probe LAMP assay to detect Leishmania among patients with HIV.

Asymptomatic visceral leishmaniasis cases increase continuously, particularly among patients with HIV who are at risk to develop further symptoms of leishmaniasis. A simple, sensitive and reliable diagnosis is crucially needed due to risk populations mostly residing in rural communities with limited resources of laboratory equipment. In this study, a highly sensitive and selective determination of Leishmania among asymptomatic patients with Leishmania/HIV co-infection was achieved to simultaneously interpret and semi-quantify using colorimetric precipitates (gold-nanoparticle probe; AuNP-probe) and fluorescence (SYBR safe dye and distance-based paper device; dPAD) in one-step loop-mediated isothermal amplification (LAMP) assay. The sensitivities and specificities of 3 detection methods were equivalent and had reliable performances achieving as high as 95.5%. Detection limits were 102 parasites/mL (0.0147 ng/µL) which were 10 times more sensitive than other related studies. To empower leishmaniasis surveillance as well as prevention and control, this dPAD combined with SYBR safe and gold nanoparticle probe LAMP assay is reliably fast, simple, inexpensive and practical for field diagnostics to point-of-care settings in resource-limited areas which can be set up in all levels of healthcare facilities, especially in low to middle income countries.

Distance-Based Paper Device for a Naked-Eye Albumin-to-Alkaline Phosphatase Ratio Assay.

The albumin-to-alkaline phosphatase ratio (AAPR) has been a cancer prognostic indicator. This paper presents the concept of a dual-color change distance-based paper device (dPAD) for albumin (Alb) and alkaline phosphatase (ALP) detection to evaluate this cancer prognostic index. Whereas Alb interacts with the bromocresol green (BCG) indicator to form a bluish-green complex, ALP hydrolyzes l-ascorbic acid-2-phosphate (AAP) to produce ascorbic acid (AA), which reacts with KIO3 to generate I2 and I-. I2/I- reacts with silver hexagonal nanoprisms (purple color) in the presence of Cu2+, resulting in a color change from purple to colorless. The distance of the color change from yellow to the bluish-green and purple to colorless correlates to Alb and ALP concentration, respectively. The angle index for the AAPR is then defined by drawing a straight line that connects the tops of the two changed band lengths in the detection area. The highest bluish-green color band length on the Alb region is the midpoint, which is the position set of the protractor at 0°, and the angle is measured using a simple protractor. The results indicate that an AAPR below 0.57 will have an angle greater than 40° and correlates with a risk factor for lung cancer. The naked-eye detection limits for Alb and ALP were found to be 0.8 g/L and 5 U/L (n = 10), respectively. The practical application of the developed dPAD was successfully demonstrated by Alb and ALP analysis in human serum and validated against standard methods. The proposed method does not require incubation conditions for the ALP assay, which strongly reduces the overall analysis steps and time. Moreover, our device provides a low-cost, simple, sensitive, selective, accurate, and precise determination of the AAPR.

Dual Sample Preconcentration for Simultaneous Quantification of Metal Ions Using Electrochemical and Colorimetric Assays.

A paper-based method for heating preconcentration (PAD-HP) has been developed for the determination of Pb2+, Cd2+, Fe3+, and Ni2+. The design of our heating system was evaluated for dual quantification of ions using electrochemical and colorimetric methods simultaneously. The PAD-HP was used to detect Pb2+ and Cd2+ by anodic stripping voltammetry and to detect Fe3+ and Ni2+ by colorimetric reactions. Assay conditions were optimized by evaluating performance when changing the concentration of the colorimetric reagent, eluent volume, electrolyte concentration, and electrochemical parameters. Limits of detection (LOD) were determined to be 0.97 and 2.33 µg L-1 for Pb2+ and Cd2+ (via voltammetry) and 0.03 and 0.04 mg L-1 for Fe3+ and Ni2+ (via colorimetric assay), respectively. The relative standard deviations for assays were in the range of 5.76 to 10.12%. We observed that the PAD-HP method significantly enhanced the signal of all metals ions (14-100-fold, depending on the metal) in comparison to paper-based devices that did not use a heating preconcentration system. This PAD-HP method was successfully applied to the determination of metals ions in samples of drinking water, tap water, pond water, and wastewater. These results suggest that our approach can provide a convenient strategy to monitor aqueous samples for heavy metals with high sensitivity and selectivity.

A paper-based conductive immunosensor for the determination of Salmonella Typhimurium.

We report for the first time a highly sensitive and rapid quantitative method for the detection of Salmonella Typhimurium (S. Typhimurium) using a conductive immunosensor on a paper-based device (PAD). S. Typhimurium monoclonal antibodies (MA) were first immobilized on a paper-based device and then captured by S. Typhimurium. After an immunoreaction on the device, the polyclonal antibody-colloidal gold conjugate (PA-AuNPs) was dropped to bind with S. Typhimurium. After a complete sandwich reaction, a dark red color appeared on the paper-based device, which can be observed by the naked eye for a rapid screening test. The electrical conductivity of PA-AuNPs between the screen-printed electrodes on the paper-based device was also measured for an accurate quantitative analysis. The electrical conductivity correlated well with the concentration of S. Typhimurium, which was controlled by the amount of S. Typhimurium attached to the polyclonal antibody-colloidal gold conjugate. The device showed a linear correlation for the concentration of the S. Typhimurium in the range of 10-108 CFU mL-1 in a logarithmic plot, with an R2 value of 0.9882 and a limit of detection (LOD) as low as 10 CFU mL-1. This simple, highly sensitive, and rapid method for the S. Typhimurium detection was successfully performed within 30 min, and it can be developed into small portable measuring devices in order to facilitate preliminary screening tests.

Sensitive distance-based paper-based quantification of mercury ions using carbon nanodots and heating-based preconcentration.

This article reports the first fluorescent distance-based paper device coupled with an evaporating preconcentration system for determining trace mercury ions (Hg2+) in water. The fluorescent nitrogen-doped carbon dots (NCDs) were synthesized by a one-step microwave method using citric acid and ethylenediamine. The fluorescence turn-off of the NCDs in the presence of Hg2+ was visualized with a common black light, and the distance of the quenched fluorescence correlated to Hg2+ concentration. The optimal conditions for pH, NCD concentration, sample volume, and reaction time were investigated. Heating preconcentration was used to improve the detection limits of the fluorescent distance-based paper device by a factor of 100. Under the optimal conditions, the naked eye limit of detection (LOD) was 5 µg L-1 Hg2+. This LOD is sufficient for monitoring drinking water where the maximum allowable mercury level is 6 µg L-1 as established by the World Health Organization (WHO). The fluorescent distance-based paper device was successfully applied for Hg2+ quantification in water samples without interference from other cations. The proposed method provides several advantages over atomic absorption spectroscopy including ease of use, inexpensive material and fabrication, and portability. In addition, the devices are simple to fabricate and have a long shelf-life (>5 months).

Distance-Based Paper Device Combined with Headspace Extraction for Determination of Cyanide.

We report for the first time a distance-based paper device based on gold/silver core shell nanoparticles (Au@Ag NPs) for a simple, inexpensive, instrument-free, and portable determination of cyanide by the naked eye. Au@Ag NPs immobilized on a paper channel were etched by cyanide ions so that a yellow color band length of Au@Ag NPs is proportional to a decrease in the cyanide concentration. Quantification is achieved by measuring color length, thus eliminating the need to differentiate hues and intensities by the user, and the processing data of each imaging device. Moreover, the paper-based headspace extraction was combined with the distance-based paper device to improve the sensitivity. The enrichment factor was found to be 30-fold and the linearity was found in the range 0.05-1 mg L-1. The naked eye detection limit was 10 µg L-1 where the World Health Organization (WHO) have regulated the maximum level of cyanide in drinking water as 70 µg L-1. Our proposed device also showed no interference from common cations and anions presenting in seawater and waste water including thiocyanate, chloride. Finally, our device has been successfully applied to determine cyanide ions in seawater, drinking water, tap water and wastewater providing satisfactory precision and accuracy.

Cotton fiber-based assay with time-based microfluidic absorption sampling for point-of-care applications.

Aim: Time-based microfluidic absorption sampling was proposed using cotton fiber-based device made in swab stick. The assay was optimized and compared with conventional pipetted drop sampling using the same device. Materials & methods: Reagents were integrated into cotton fiber device for assessing concentration of analytes by the colorimetric detection method through time-based absorption sampling microfluidic system. All assay parameters were first optimized using conventional pipette-based drop sampling. Results: The color intensity is linear in the relevant concentration range of the analytes. The LOD are 0.189 mM for glucose and 6.56 µM for nitrite, respectively. These values are better than conventional drop sampling. The fiber-containing swab itself functions as sampling, assay and calibration device. Conclusion: Microfluidic cotton fiber-based assay device was fabricated and can determine analyte concentration in artificial salivary samples, colorimetrically, by time-based absorption sampling without the need of complex equipments.

Disposable Nonenzymatic Uric Acid and Creatinine Sensors Using µPAD Coupled with Screen-Printed Reduced Graphene Oxide-Gold Nanocomposites.

Uric acid (UA) and creatinine are the imperative biological substance for clinical monitoring and diagnosis. Measuring the ratio between uric acid and creatinine in urine helps differentiate acute uric acid nephropathy from the hyperuricemia that secondarily occurs to renal failure. In general, the ratio is greater than 0.9 in acute uric acid nephropathy and less than 0.7 in hyperuricemia. In this work, disposable nonenzymatic screen-printed reduced graphene oxide-gold nanocomposites electrodes were firstly developed for the quantitative analysis of uric acid. Our sensors were also coupled with the paper-based colorimetric sensor of the determination of creatinine. Hence, an alternative high-throughput screening test for the uric acid to creatinine ratio with high sensitivity, specificity, simplicity, and rapidity was developed. Under the optimum conditions, our disposable nonenzymatic screen-printed electrode for the determination of uric acid shows the acceptable analytical performance in a wide range of linearity (2.5-1,000 µM) with a low detection limit of 0.74 µM. Our electrodes also showed no interference from common physiologic compound in urine. The determination of creatinine has been developed using Jaffé reaction between the creatinine and picric acid in alkaline condition. The alkaline picrate color on µPAD changed from yellow to orange in the presence of creatinine and the orange intensity is directly proportional to the creatinine amount in a linearity range of 0.20-6.0 mM as a detection limit of 180 µM. Finally, our device has been utilized to determine uric acid and creatinine simultaneously in control urine samples with acceptable result.

A distance-based paper sensor for the determination of chloride ions using silver nanoparticles.

We report for the first time the development of a distance-based paper sensor for a simple, inexpensive, instrument-free, and portable determination of chloride ions. Our analysis reaction is based on the oxidative etching of silver nanoparticles (AgNPs) to form AgCl in the presence of Cl- and H2O2. H2O2 reacts with AgNPs in the channel of the paper device and Cl- in the sample forming a white precipitate (AgCl) where the white color band length is proportional to the Cl- concentration. Quantification of Cl- is achieved by measuring the length of the white color band using a ruler printed on the side of the channel. Under optimal conditions, the distance-based paper sensor was characterized by a working range of 25-1000 mg L-1 (R2 = 0.9954) and the naked eye detection limit (LOD) was 2 mg L-1 (0.08 µg). Our sensor was found to be reproducible with a relative standard deviation of less than 4.51% (n = 10). The levels of Cl- in real water samples measured using our proposed device were within the error of the values measured using traditional tests but without the need for any external instrumentation. Therefore, our proposed method presents acceptable accuracy, precision, and high selectivity for point of need monitoring of Cl- in real water samples.

A Simple Paper-based Colorimetric Device for Rapid and Sensitive Urinary Oxalate Determinations.

In this work, a simple and inexpensive paper-based colorimetric device (cPAD) was developed for oxalate measurements. The colorimetric assay is based on the formation of formazan via the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by oxalate decarboxylase and formate dehydrogenase on the paper device. After the sample was spotted on the device, MTT changed color from yellow to purple, and the purple color intensity correlated with the oxalate concentration. The quantitative detection of oxalate ranged from 10 - 1000 µM, with a linear equation, y = 0.0086x + 34.978, and a correlation coefficient (R2) = 0.9994. The detection limit was 10 µM by the naked eye. The recoveries of oxalate spiked in artificial urine samples evaluated by our cPADs were in the range of 81 - 92%. This simple cPAD for rapid and sensitive oxalate determination should be useful for diagnostic urinary oxalate measurements for point-of-care monitoring.

Highly sensitive colorimetric detection of lead using maleic acid functionalized gold nanoparticles.

Highly sensitive colorimetric detection for Pb(2+) has been developed using maleic acid (MA) functionalized GNP. The -COOH on MA was used to modify GNP surface whereas the other -COOH functional group have strong affinity to coordination behavior of Pb(2+) allowing the selective formation more than other ions. MA-GNPs solution changed from red to blue color after the addition of Pb(2+) due to nanoparticle aggregation. The different optical absorption and discriminate of particle size between the MA-GNPs solution with and without Pb(2+) were characterized by UV-visible spectroscopy and transmission electron microscopy (TEM), respectively. The color intensity as a function of Pb(2+) concentration gave a linear response in the range of 0.0-10.0 µg L(-1) (R(2)=0.990). The detection limit was found at 0.5 µg L(-1) by naked eye and can be completed the analysis within 15 min. The MA-GNPs aggregated with Pb(2+) showed high selectivity when was compared to other metal ions (As(3+), Ca(2+), Cd(2+), Co(2+), Cu(2+), Fe(3+), Hg(2+), Mg(2+), Mn(2+), Ni(2+), Pb(2+) and Zn(2+)) and anions (Cl(-), NO3(-) and SO4(2-)). Our proposed method was also applied for the determination of Pb(2+) in real drinking water samples from 5 sources. The result of real water samples were not statistically significant different from the standard methods at the 95% confidence level (pair t-test method). Moreover, we evaluated our proposed method for the determination of trace Pb(2+) concentration in real breast milk samples. The recoveries were acceptable and ranged from 101 to 104% for spiked Pb(2+) in real breast milk samples. Thus, MA-GNP colorimetric sensing provides a simple, rapid, sensitive, easy-to-use, inexpensive and low detection limit for the monitoring of Pb(2+).

Simple silver nanoparticle colorimetric sensing for copper by paper-based devices.

The first investigation of silver nanoparticle (AgNP) colorimetric sensing of Cu(2+) by paper-based analytical devices (PADs) is reported here. AgNP colorimetric sensing for the detection of Cu(2+) was first characterized by UV-visible spectroscopy. The -SH groups on homocysteine (Hcy) and dithiothreitol (DTT) were used to modify the AgNP surface whereas the -COOH and -NH(2) functional groups have strong affinity to Cu(2+) relative to other ions in solution. The plasmon resonance absorption peak intensity at 404 nm decreased and a new red-shifted band at 502 nm occurred in the presence of Cu(2+). Paper devices coated with the modified AgNP solution changed from yellow to orange and green-brown color after the addition of Cu(2+) due to nanoparticle aggregation. The color intensity change as a function of Cu(2+) concentration gave a linear response in the range of 7.8-62.8 µM (R(2)=0.992). The limit of naked-eye detection is 7.8 nM or 0.5 µg L(-1). A color change observed by the naked eye with the addition of Cu(2+) can be clearly differentiated from the other metals (As(3+), Cd(2+), Co(2+), Hg(2+), Ni(2+), Pb(2+), Zn(2+), Mg(2+), Mn(2+), Ca(2+), Fe(3+), Na(+), and K(+)) at 15.7 µM. The use of different flow directions in the PADs and µPADs for Cu(2+) detection was also demonstrated. Levels of Cu(2+) in real water samples were measured using the paper devices to be 2.9±0.24 µM (tap water) and 3.2±0.30 µM (pond water), respectively, and were within error of the values measured using an atomic absorption spectrometer (2.8±0.08 µM in tap water, and 3.4±0.04 µM in pond water). Thus, this work shows the successful integration of paper devices and AgNP colorimetric sensing as a simple, rapid, easy-to-use, inexpensive and portable alternative point-of-measurement monitoring.