Detection of Reactive Oxygen Species by a Carbon-Dot–Ascorbic Acid Hydrogel

Detection of reactive oxygen species (ROS) is important in varied biological processes, disease diagnostics, and chemotherapeutic drug screening. We constructed a ROS sensor comprising an ascorbic-acid-based hydrogel encapsulating luminescent amphiphilic carbon-dots (C-dots). The sensing mechanism is based upon ROS-induced oxidation of the ascorbic acid units within the hydrogel scaffold; as a consequence, the hydrogel framework collapses resulting in aggregation of the C-dots and quenching of their luminescence. The C-dot–hydrogel platform exhibits high sensitivity and detected ROS generated chemically in solution and in actual cell environments. We demonstrate application of the C-dot–hydrogel for evaluating the efficacy of a chemotherapeutic substance, underscoring the potential of the system for drug screening applications

Nitric Oxide Sensing through Azo-Dye Formation on Carbon Dots

Carbon dots (C-dots) prepared through heating of aminoguanidine and citric acid enable bimodal (colorimetric and fluorescence) detection of nitric oxide (NO) in aqueous solutions. The C-dots retained the functional units of aminoguanidine, which upon reaction with NO produced surface residues responsible for the color and fluorescence transformations. Notably, the aminoguanidine/citric acid C-dots were noncytotoxic, making possible real-time and high sensitivity detection of NO in cellular environments. Using multiprong spectroscopic and chromatography analyses we deciphered the molecular mechanism accounting for the NO-induced structural and photophysical transformations of the C-dots, demonstrating for the first time N₂ release and azo dye formation upon the C-dots’ surface

Qualitative and quantitative phytochemical analysis of PRME.

<p>“+” represents presence of the phytoconstituent; “−” represents absence of the phytoconstituent; “ND” represents “Not determined”. Phen- Phenol, Flav- Flavonoid, Carbo- Carbohydrate, Tan.- Tannin, Alka- Alkaloid, Asc- Ascorbic acid, Ter- Terpenoids, Triter- Triterpenoids, Anth-Anthraquinones, Sap- Saponin, Gly- Glycoside; Total phenolics (mg/100 mg extract gallic acid equivalent), Total flavonoids (mg/100 mg extract quercetin equivalent), Carbohydtrate (mg/100 mg extract glucose equivalent), Tannin (mg/100 mg extract catechin equivalent). Alkaloid (mg/100 mg extract reserpine equivalent), Ascorbic acid (mg/100 mg extract L-ascorbic acid equivalent) </p

Flow cytometric cell cycle distribution of PRME treated MCF-7 cells with increasing time.

<p>Sub-G1, G1, S, and G2/M phases of PRME (300 µg/ml) treated MCF-7 cells at (A) 0 hour, (B) 6 hours, (C) 12 hours, (D) 24 hours, (E) 36 hours, (F) 48 hours. (G) Graphical representation of % cell population in different phases.</p

Western blot analysis of apoptosis related proteins of MCF-7 cells treated with 300 µg/ml PRME.

<p>Graphs adjoining the blots represent the expression levels of corresponding proteins for indicated time intervals: (A) Pro and cleaved caspase-9, (B) Pro and cleaved caspase-3, (C) Native and cleaved PARP, (D) Bax and Bcl-2, (E) Pro and cleaved caspase-8, (F) Bid and t-Bid.</p

Western blot analysis of cell cycle related proteins of MCF-7 cells treated with 300 µg/ml PRME.

<p>Graphs adjoining the blots represent the expression levels of corresponding proteins for indicated time intervals: (A) Cyclin B1, (B) Cdk-2, (C) Cdc25c, (D) Cdk-1, (E) Cyclin A1, (F) p53, (G) p21.</p

Effect of various compounds on cell proliferation and viability of A549, MCF-7 and WI-38 cells.

<p>Cells were treated with increasing concentrations of compounds for 48 hours; cell proliferation and viability was determined with WST-1 cell proliferation reagent. Results were expressed as cell viability (% of control). (A) Tannic acid, (B) Catechin, (C) Purpurin, (D) Reserpine. All data is expressed as mean ± SD (n = 6). *p<0.05, **p<0.01 and ***p<0.001 vs. 0 µg/ml.</p

Trolox equivalent antioxidant capacity and IC₅₀ values of the lichen extract (PRME) and standard compounds for ROS and RNS scavenging.

<p>₅₀ values of all activities are determined in µg/ml. Data expressed as mean ± S.D (n = 6). EDTA represents Ethylenediamine tetraacetic acid.^# IC</p><p> p<0.001.</p

Proposed mechanisms of PRME-induced S and G2/M phase cell cycle arrest and apoptosis in MCF-7 cells.

<p>Proposed mechanisms of PRME-induced S and G2/M phase cell cycle arrest and apoptosis in MCF-7 cells.</p

HPLC chromatogram of PRME.

<p>Inset shows expanded region of the chromatogram with retention time of 4.5–10 minutes. Peaks marked signify the retention peak of purpurin (2.4 min), catechin (3.13 min), tannic acid (3.64 min) and reserpine (4.68 min).</p