We have developed a simple and direct method to fabricate paper-based microfluidic devices that can be used for a wide range of colorimetric assay applications. With these devices, assays can be performed within minutes to allow for quantitative colorimetric analysis by use of a widely accessible iPhone camera and an RGB color reader application (app) to measure color intensity. In the described laboratory experiment, students design and create their own microfluidic devices with common laboratory supplies such as Kimwipes, Parafilm, and a thermal laminator, and gain hands-on experience in the analysis of Fe 2+ and Cu 2+ by colorimetric determination.

A sensitive, selective, environmentally friendly, high-throughput, well-plate-based immunosorbent assay was developed to detect human α-fetoprotein (AFP) using carbon dots (C-Dots). Highly fluorescent C-Dots were synthesized using a one-step hydrothermal reaction, with citric acid serving as the carbon source and ethylene diamine acting as the nitrogen source. The reaction conditions were optimized to obtain the desired surface functionality. Then, the C-Dots were used to label one member of the anti-AFP pair (Ab2) via amine-amine coupling using glutaraldehyde. The capture anti-AFP (Ab1) was coated onto polystyrene well plates and bovine serum albumin (BSA) was used to block unsaturated binding sites. AFP was incubated in Ab1-coated wells; unbound AFP was then washed away with Tween-20. Next, the C-Dot-labeled Ab2 was added to form a sandwich immunocomplex with the AFP bound to the Ab1-coated wells. The fluorescence intensities detected from the C-Dots on these sandwich immunocomplexes were positively correlated to the concentrations of AFP antigen. A five-parameter logistic regression calibration curve was established between fluorescence and clinically important AFP concentrations (range: 0-350 ng/mL with a correlation coefficient of R(2) = 0.995). The results from the C-Dot-based immunoassay were in agreement with results from traditional immunoassays, which used horseradish peroxidase (HRP, R(2) = 0.964) and fluorescein isothiocyanate (FITC, R(2) = 0.973). These results indicated that C-Dots have great potential to be applied as biolabels for high-throughput well-plate-based immunoassays.

The objective of this study was to investigate the relationship of acute pesticide exposures and acute changes in thyroid hormones among Thai farmers. We recruited 78 farmers, who were scheduled to spray insecticides (chlorpyrifos and/or cypermethrin) or herbicides (paraquat and/or glyphosate). On the day before spraying, farmers collected their first morning void urine and went for blood collection. On the spray day, urine samples were collected at end of the spraying event and they were interviewed with questionnaires. The next morning, the first morning void urine and blood samples were collected. Blood samples were analyzed for thyroid hormones. Urine samples were analyzed for the metabolites of the pesticide sprayed. The results showed that the thyroid hormones, free triiodothyronine (FT3) and total triiodothyronine (T3) were significantly reduced as urinary chlorpyrifos metabolite increased the day after spraying. Total thyroxine (T4) significantly increased as cypermethrin metabolites increased the day after spraying. T4 significantly increased as urinary glyphosate levels increased; however, FT3 and T3 decreased significantly as urinary paraquat levels increased the day after spraying. These findings suggest that acute exposures to the pesticides chlorpyrifos, cypermethrin, paraquat and glyphosate can produce acute effects on the hypothalamic–pituitary–thyroid (HPT) axis, acutely altering thyroid hormone levels.

The application of fluorescent carbon nanodots (C-dots or CD), non-toxic particulate organic labels, to disease biomarker detection is still in its earliest stage of development. In the effort described here, a novel ratiometric immunoassay was developed to target a model protein disease biomarker, alpha-fetoprotein (AFP), using C-dot doped silica nanoparticles (CD-SNPs) and fluorescein isothiocyanate (FITC) as signaling agents. Highly fluorescent C-dots were hydrothermally synthesized from citric acid and ethylene diamine. The C-dots were then encapsulated in silicate shells to yield 45 nm nanoparticles using a novel reverse microemulsion method, enabling convenient handling (centrifugation and washing) and straightforward surface chemistry modification to facilitate development of bioassays. Capture antibody capped CD-SNPs (Ab1-CD-SNPs), together with FITC labeled antibodies (Ab2-FITC) constituted a new ratiometric immunosensor for AFP, in which CD-SNPs functioned as both solid supports for washing and separation and as built-in reference signaling agents to correct for inconsistent environmental effects. A calibration curve was established between the ratiometric signal (the ratio of fluorescence signals of FITC and C-dots, F/C) and AFP concentration in the broad range of 0.317-280 μg/dL, exhibiting a useful linear range (0.317-35 μg/dL, R 2 =0.9977), low detection limit (0.317 μg/dL) and acceptable recovery (105-120%). This assay format can be applied to a wide range of immunoassay targets. Our demonstration of encapsulating low-cost, easily synthesized, highly-fluorescent C-dots into silica nanoparticles for use in immunoassays will be useful in expanding future applications of these carbon nanomaterials to areas such as in-vivo cellular imaging, drug delivery, and in-vitro cell labeling and biomolecule sensing.