Carbon Nanodots as Biolabels for Fluorescence Immunoassays Public Deposited

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  • There has been a tremendous growth in interest in carbon nanodots (C-dots) in the past several years. As a nascent nanomaterial, C-dots have shown great promise in applications that benefit from their superior water dispersibility, low toxicity, non-blinking fluorescent output, chemical and biological compatibility, ease of functionalization and resistance to photobleaching. With this positive outlook, however, challenges remain for the practical application of these fluorophores in specific biolabeling processes and bioassays. The goal of this effort has been to combine C-dots with biorecognition units for quantitative biomolecule determinations; specifically, the development of novel fluorescent immunoassays is presented here. These studies largely focused on the use of C-dot-labeled antibodies to target a protein disease biomarker, human alpha-fetoprotein (AFP). The research effort began with C-dot synthesis, followed by the application of capillary electrophoresis (CE) as an analysis tool for C-dot characterization and synthesis optimization. In addition, the CE work facilitated optimization of ensuing C-dot biolabeling reactions. C-dots were hydrothermally synthesized from citric acid (CA) and ethylene diamine (EDA) with a quantum yield as high as 99%. A novel CE method utilizing an alkaline working buffer was developed for rapid and reliable analysis of C-dots. A calibration curve was established over a broad concentration range (0.5-10 mg/mL) with excellent linearity (R²=0.9989). Then, the C-dots were used to label antibodies via amine-amine coupling using glutaraldehyde. All of the components in the conjugation reaction were baseline separated using the newly developed CE method. One peak in the electropherogram, having a migration time between that of the unlabeled antibody and that for the unmodified C-dots, was identified as representing the C-dot labeled antibodies. In summary, a novel, simple and rapid CE method was developed for: (1) quantitation of C-dots and (2) analysis of a C-dot-antibody bioconjugate, thus providing a method for optimization of conditions for acquiring the desired C-dot bioconjugate. The research continued with the development of a sensitive, selective, environmentally-friendly, high throughput, well plate based immunosorbent assay for human AFP using C-dots. The capture anti-AFP (Ab₁) was coated onto polystyrene well plates and bovine serum albumin (BSA) was used to block unsaturated binding sites. The C-dots were used to label the other member of the anti-AFP pair (Ab₂) via amine-amine coupling using glutaraldehyde. AFP was incubated to form a sandwich immunocomplex between Ab₁ and Ab₂ in the well plates, with unbound AFP and Ab₂ washed away with Tween-20. The fluorescence intensities detected from the C-dots in these immunocomplexes positively correlated to the concentrations of AFP antigen. A 5-parameter logistic regression curve was established between fluorescence and clinically important AFP concentrations (range: 0-350 ng/mL with an R-squared value of 0.995). The results were in agreement with those from two more traditional immunoassays which used horseradish peroxidase (HRP, R²=0.964) and fluorescein isothiocyanate (FITC, R²=0.973) as biolabels. This demonstration was the first example of a C-dot linked immunosorbent (solid phase) assay. To enable convenient separation by centrifugation and straightforward surface chemistry, in the next stage of the research described here, C-dots were encapsulated into a stable suspension of 45 nm silica nanoparticles through a reverse microemulsion method. A novel ratiometric immunoassay was developed to target human AFP, based on C-dot doped silica nanoparticles (CD-SNPs) and fluorescein isothiocyanate (FITC). CD-SNPs (Ab₁-CD-SNPs) capped with capture antibodies together with FITC labeled secondary antibodies (Ab₂-FITC) constituted a ratiometric immunoassay pair, in which CD-SNPs functioned as both a solid support enabling separation and washing, and as a built-in source of correction to account for inconsistent environmental effects and experimental errors. A linear calibration curve was established between ratio of FITC to C-dot fluorescent signals ("F/C") and a broad range of AFP concentrations (0-280 μg/dL with an R-squared value of 0.9977) with a low detection limit (0.317 μg/dL or 3.17 ng/mL) and acceptable recoveries. This is the first application of carbon nanodot doped silica nanoparticles to quantitative immunoassays for protein disease biomarkers. Moreover, the demonstration that these low-cost, simply-obtained and highly- fluorescent C-dots can be simply encapsulated into silica nanoparticles for specific biolabeling may expand future applications of C-dots into areas of in-vivo cellular imaging, drug delivery, and in-vitro cell labeling and biomolecule sensing. In the last part of the research effort described here, an alternative colorimetric detection platform, where an Apple iPhone 4 camera equipped with a color analysis application (ColorAssist) was combined with Vitros® blood urea nitrogen (BUN) colorimetric assays, was examined as a model for rapid and inexpensive clinical diagnostic testing. Color images of assay slides at various concentrations of urea were collected with the smartphone camera and quantified in three spectral ranges (red/green/blue or RGB) using the color analysis application. Absorbance values were converted from these diffuse reflectance data to quantitate BUN over its clinically important concentration range (2-190 mg/dL) with good linearity (R² = 0.9996 [n = 5]). This method was also applied to canine serum samples, the urea concentrations obtained were in good agreement with those from the instrumental "gold standard" (Beckman Coulter AU480) and a commercial colorimetric dry slide analyzer (HeskaTM Element DC)). This effort demonstrated that smart phones have the potential to be used as simple, effective colorimetric detectors for quantitative diagnostic tests. Furthermore, many additional demonstrations of the use of C-dots for sensitive detection of ions showed discernible color changes as well. This in turn suggests a valuable direction for future research: to explore combinations of these simple and effective colorimetric C-dot assays for both point-of-care applications in the developed world and field deployment in developing nations.
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