Quantifying non-transferrin-bound iron (NTBI) in human plasma: incorporating BODIPY-pyridylhydrazone (BODIPY-PH) within a thin green film linked to a portable fluorescence-based device.

Free iron in human serum or non-transferrin-bound iron (NTBI) can generate free radicals and lead to oxidative damage. Moreover, it is highly toxic to various tissues and a vital biomarker related to the iron-loading status of thalassemia and Alzheimer's patients. In NTBI in healthy individuals, NTBI levels are typically less than 1 µM; current NTBI analysis usually requires advanced instrumentation and many-step sample pretreatment. To address this issue, we employed our invented BODIPY derivative, BODIPY-PH, as a fluorescence probe and trapped it onto the microcentrifuge tube lid using tapioca starch. The fluorescence intensity of BODIPY-PH increased with increasing NTBI concentration (turn-on). The developed portable reaction chamber facilitates rapid analysis (∼5 min) using small sample volumes (10 µL sample in a total volume of 600 µL). Under optimum conditions, using the sample-developed portable fluorescence device and fluorescence spectrometer, we achieved impressive limits of detection (LOD) of 0.003 and 0.0015 µM, respectively. Furthermore, the developed sensors show relatively high selectivity toward Fe3+ over other metal ions and biomolecules (i.e., Fe2+, Cr3+, Cu2+, and glucose). The sensor performance in serum samples of thalassemia patients exhibited no significant difference compared to the labeled value (obtained from standard methods). Overall, the developed fluorescence sensor is suitable for determining NTBI and offers high sensitivity, high selectivity, and a short incubation time (5 min). Moreover, the method requires a limited number of reagents, is simple to use, and uses low-cost equipment to determine NTBI in human serum samples.

Point-of-care blood tests using a smartphone-based colorimetric analyzer for health check-up.

A microscale colorimetric assay was designed and implemented for the simultaneous determination of clinical chemistry tests measuring six parameters, including glucose (GLU), total protein (TP), human serum albumin (HSA), uric acid (UA), total cholesterol (TC), and triglycerides (TGs) in plasma samples. The test kit was fabricated using chromogenic reagents, comprising specific enzymes and binding dyes. Multiple colors that appeared on the reaction well when it was exposed to each analyte were captured by a smartphone and processed by the homemade Check6 application, which was designed as a colorimetric analyzer and simultaneously generated a report that assessed test results against gender-dependent reference ranges. Six blood checkup parameters for four plasma samples were conducted within 12 min on one capture picture. The assay achieved wide working concentration ranges of 10.45-600 mg dL-1 GLU, 1.39-10.0 g dL-1 TP, 1.85-8.0 g dL-1 HSA, 0.86-40.0 mg dL-1 UA, 11.28-600 mg dL-1 TC, and 11.93-400 mg dL-1 TGs. The smartphone-based assay was accurate with recoveries of 93-108% GLU, 93-107% TP, 92-107% HSA, 93-107% UA, 92-107% TC, and 99-113% TGs. The coefficient of variation for intra-assay and inter-assay precision ranged from 3.2-5.2% GLU, 4.6-5.3% TP, 4.3-5.3% HSA, 2.8-6.6% UA, 2.7-6.5% TC, and 1.1-3.9% TGs. This assay demonstrated remarkable accuracy in quantifying the concentration-dependent color intensity of the plasma, even in the presence of other suspected interferences commonly present in serum. The results of the proposed method correlated well with results determined by the microplate spectrophotometer (R2 > 0.95). Measurement of these six clinical chemistry parameters in plasma using a microscale colorimetric test kit coupled with the Check6 smartphone application showed potential for real-time point-of-care analysis, providing cost-effective and rapid assays for health checkup testing.

Development of lectin-based lateral flow assay for fucosylated alpha-fetoprotein.

Fucosylated alpha-fetoprotein (AFP-L3) is a more specific and sensitive biomarker for early diagnosis of hepatocellular carcinoma (HCC) than only the alpha-fetoprotein (AFP) level. Rapid and simple detection of AFP-L3 level greatly facilitates the early detection as well as the treatment of HCC, resulting in the reduction of mortality. Here, we developed a rapid and sensitive lateral flow assay (LFA) using lectin Lens culinaris agglutinin (LCA), which has a specific affinity to AFP-L3 fraction of AFP, as a biorecognition element for determination of the fucosylation of AFP. The assay is based on a sandwich format performed on a lateral flow test strip. LCA was immobilized on the membrane as a test line (T). Quantitative detection of AFP-L3 was achieved by measuring the green color intensity of captured gold nanoparticle conjugates on the T and control line (C) utilizing an in-house test strip reader. The calculated absorbance obtained by the green color intensity signals proportionally increased with AFP concentrations. The developed lectin-based LFA provided a detection limit of 0.8 ng/mL for AFP with a linear range between 1.5 and 160.0 ng/mL within an assay time of 10 min. Recoveries between 74.5% and 113.2% with relative standard deviations of 5.2%-8.7% for measuring spiked human serum were also achieved. The results reveal that the proposed assay offers a rapid, sensitive, and specific method, which is useful for development in point-of-care testing for early detection and treatment of HCC.

Sensitive detection of trace level Cd (II) triggered by chelation enhanced fluorescence (CHEF) "turn on": Nitrogen-doped graphene quantum dots (N-GQDs) as fluorometric paper-based sensor.

Cadmium ion (Cd (II)) is a highly toxic heavy metal usually found in natural water. Exposure to Cd (II) can produce serious effects in human organs such as Itai-Itai disease. Therefore, the maximum allowance levels of Cd (II) in drinking water and herbal medicines imposed by the World Health Organization (WHO) are 3 µg L-1 and 300 µg kg-1, respectively. In this work, nitrogen-doped graphene quantum dots (N-GQDs) as a fluorescent sensor for Cd (II) determination was developed in both solution-based and paper-based systems. N-GQDs were synthesized from citric acid (CA) and ethylenediamine (EDA) via the hydrothermal method. The synthesized N-GQDs emitted intense blue fluorescence with a quantum yield (QY) of up to 80%. The functional groups on the surface of N-GQDs measured by FTIR were carboxyl (COO-), hydroxyl (OH-), and amine (NH2) groups, suggesting that they could be bound to Cd (II) for complexation. The fluorescence intensity of N-GQDs was gradually enhanced with the increase of Cd (II) concentration. This phenomenon was proved to result from the fluorescence enhancement (turn-on) based on the chelation enhanced fluorescence (CHEF) mechanism. Under the optimum conditions in the solution-based and paper-based systems, the limits of detection (LODs) were found to be 1.09 and 0.59 µg L-1, respectively. Furthermore, the developed sensors showed relatively high selectivity toward Cd (II) over ten other metal cations and six other anions of different charges. The performance of the sensor in real water and herbal medicine samples exhibited no significant difference as compared to the results of the validation method (ICP-OES). Therefore, the developed sensors can be used as fluorescent sensors for Cd (II) determination with high sensitivity, high selectivity, short incubation time (5 min). As such, the paper-based strategy has excellent promising potential for practical analysis of Cd (II) in water and herbal medicine samples with a trace level of Cd (II) concentrations.

Use of a Smartphone as a Colorimetric Analyzer in Paper-based Devices for Sensitive and Selective Determination of Mercury in Water Samples.

A smartphone application, called CAnal, was developed as a colorimetric analyzer in paper-based devices for sensitive and selective determination of mercury(II) in water samples. Measurement on the double layer of a microfluidic paper-based analytical device (µPAD) fabricated by alkyl ketene dimer (AKD)-inkjet printing technique with special design doped with unmodified silver nanoparticles (AgNPs) onto the detection zones was performed by monitoring the gray intensity in the blue channel of AgNPs, which disintegrated when exposed to mercury(II) on µPAD. Under the optimized conditions, the developed approach showed high sensitivity, low limit of detection (0.003 mg L-1, 3SD blank/slope of the calibration curve), small sample volume uptake (two times of 2 µL), and short analysis time. The linearity range of this technique ranged from 0.01 to 10 mg L-1 (r2 = 0.993). Furthermore, practical analysis of various water samples was also demonstrated to have acceptable performance that was in agreement with the data from cold vapor atomic absorption spectrophotometry (CV-AAS), a conventional method. The proposed technique allows for a rapid, simple (instant report of the final mercury(II) concentration in water samples via smartphone display), sensitive, selective, and on-site analysis with high sample throughput (48 samples h-1, n = 3) of trace mercury(II) in water samples, which is suitable for end users who are unskilled in analyzing mercury(II) in water samples.

Exploiting an automated microfluidic hydrodynamic sequential injection system for determination of phosphate.

A microfluidic hydrodynamic sequential injection (µHSI) spectrophotometric system was designed and fabricated. The system was built by laser engraving a manifold pattern on an acrylic block and sealing with another flat acrylic plate to form a microfluidic channel platform. The platform was incorporated with small solenoid valves to obtain a portable setup for programmable control of the liquid flow into the channel according to the HSI principle. The system was demonstrated for the determination of phosphate using a molybdenum blue method. An ascorbic acid, standard or sample, and acidic molybdate solutions were sequentially aspirated to fill the channel forming a stack zone before flowing to the detector. Under the optimum condition, a linear calibration graph in the range of 0.1-6mg P L-1 was obtained. The detection limit was 0.1mgL-1. The system is compact (5.0mm thick, 80mm wide × 140mm long), durable, portable, cost-effective, and consumes little amount of chemicals (83µL each of molybdate and ascorbic acid, 133µL of the sample solution and 1.7mL of water carrier/run). It was applied for the determination of phosphate content in extracted soil samples. The percent recoveries of the analysis were obtained in the range of 91.2-107.3. The results obtained agreed well with those of the batch spectrophotometric method.

Colorimetric analyzer based on mobile phone camera for determination of available phosphorus in soil.

A field deployable colorimetric analyzer based on an "Android mobile phone" was developed for the determination of available phosphorus content in soil. An inexpensive mobile phone embedded with digital camera was used for taking photograph of the chemical solution under test. The method involved a reaction of the phosphorus (orthophosphate form), ammonium molybdate and potassium antimonyl tartrate to form phosphomolybdic acid which was reduced by ascorbic acid to produce the intense colored molybdenum blue. The software program was developed to use with the phone for recording and analyzing RGB color of the picture. A light tight box with LED light to control illumination was fabricated to improve precision and accuracy of the measurement. Under the optimum conditions, the calibration graph was created by measuring blue color intensity of a series of standard phosphorus solution (0.0-1.0mgPL(-1)), then, the calibration equation obtained was retained by the program for the analysis of sample solution. The results obtained from the proposed method agreed well with the spectrophotometric method, with a detection limit of 0.01mgPL(-1) and a sample throughput about 40h(-1) was achieved. The developed system provided good accuracy (RE<5%) and precision (RSD<2%, intra- and inter-day), fast and cheap analysis, and especially convenient to use in crop field for soil analysis of phosphorus nutrient.

Sequential injection monosegmented flow voltammetric determination of cadmium and lead using a bismuth film working electrode.

A cost-effective sequential injection monosegmented flow analysis (SI-MSFA) with anodic stripping voltammetric (ASV) detection has been developed for determination of Cd(II) and Pb(II). The bismuth film working electrode (BiFE) was employed for accumulative preconcentration of the metals by applying a fixed potential of -1.10 V versus Ag/AgCl electrode for 90 s. The SI-MSFA provides a convenient means for preparation of a homogeneous solution zone containing sample in an acetate buffer electrolyte solution and Bi(III) solution for in situ plating of BiFE, ready for ASV measurement at a flow through thin layer electrochemical cell. Under the optimum conditions, linear calibration graphs in range of 10-100 microg L(-1) of both Cd(II) and Pb(II) were obtained with detection limits of 1.4 and 6.9 microg L(-1) of Cd(II) and Pb(II), respectively. Relative standard deviations were 2.7 and 3.1%, for 11 replicate analyses of 25 microg L(-1) Cd(II) and 25 microg L(-1) Pb(II), respectively. A sample throughput of 12h(-1) was achieved with low consumption of reagent and sample solutions. The system was successfully applied for analysis of water samples collected from a draining pond of zinc mining, validating by inductively coupled plasma-optical emission spectroscopy (ICP-OES) method.