Dual-Responsive Carbon Quantum Dots for the Simultaneous Detection of Cytosine and 5-Methylcytosine Interpreted by a Machine Learning-Assisted Smartphone.

DNA methylation is an epigenetic alteration that results in 5-methylcytosine (5-mC) through the addition of a methyl group to the fifth carbon of a cytosine (C) residue. The methylation level, the ratio of 5-mC to C, in urine might be related to the whole-body epigenetic status and the occurrence of common cancers. To date, never before have any nanomaterials been developed to simultaneously determine C and 5-mC in urine samples. Herein, a dual-responsive fluorescent sensor for the urinary detection of C and 5-mC has been developed. This assay relied on changes in the optical properties of nitrogen-doped carbon quantum dots (CQDs) prepared by microwave-assisted pyrolysis. In the presence of C, the blue-shifted fluorescence intensity of the CQDs increased. However, fluorescence quenching was observed upon the addition of 5-mC. This was primarily due to photoinduced electron transfer as confirmed by the density functional theory calculation. In urine samples, our sensitive fluorescent sensor had detection limits for C and 5-mC of 43.4 and 74.4 µM, respectively, and achieved satisfactory recoveries ranging from 103.5 to 115.8%. The simultaneous detection of C and 5-mC leads to effective methylation level detection, achieving recoveries in the range of 104.6-109.5%. Besides, a machine learning-enabled smartphone was also developed, which can be effectively applied to the determination of methylation levels (0-100%). These results demonstrate a simple but very effective approach for detecting the methylation level in urine, which could have significant implications for predicting the clinical prognosis.

Rapid detection of cancer DNA in human blood using cysteamine-capped AuNPs and a machine learning-enabled smartphone.

DNA methylation occurs when a methyl group is added to a cytosine (C) residue's fifth carbon atom, forming 5-methylcytosine (5-mC). Cancer genomes have a distinct methylation landscape (Methylscape), which could be used as a universal cancer biomarker. This study developed a simple, low-cost, and straightforward Methylscape sensing platform using cysteamine-decorated gold nanoparticles (Cyst/AuNPs), in which the sensing principle is based on methylation-dependent DNA solvation. Normal and cancer DNAs have distinct methylation profiles; thus, they can be distinguished by observing the dispersion of Cyst/AuNPs adsorbed on these DNA aggregates in MgCl2 solution. After optimising the MgCl2, Cyst/AuNPs, DNA concentration, and incubation time, the optimised conditions were used for leukemia screening, by comparing the relative absorbance (ΔA 650/525). Following the DNA extraction from actual blood samples, this sensor demonstrated effective leukemia screening in 15 minutes with high sensitivity, achieving 95.3% accuracy based on the measurement by an optical spectrophotometer. To further develop for practical realisation, a smartphone assisted by machine learning was used to screen cancer patients, achieving 90.0% accuracy in leukemia screening. This sensing platform can be applied not only for leukemia screening but also for other cancers associated with epigenetic modification.

Development of sensitive colorimetric aptasensor based on short DNA aptamer and its application to screening for paraquat residues in agricultural soil.

Paraquat is a widely used herbicide for controlling weeds and grasses in agriculture, and its contaminated residues in agricultural areas are of increasing concern. This work reports the development of the sensitive and easy-to-use colorimetric aptasensor for screening paraquat residues in agricultural soil. The short DNA fragments derived from the original aptamer were analyzed for their capability to interact with paraquat by molecular dynamic simulation. The paraquat-aptasensor was developed using the selected DNA fragment and gold nanoparticles. Its limit of detection (LOD) for paraquat is 2.76 nM, which is more sensitive than the aptasensor with long-length aptamer (LOD = 12.98 nM). The developed aptasensor shows the selectivity to paraquat, but not to other tested herbicides; ametryn, atrazine, difenzoquat, 2,4-D-dimethyl ammonium, and glufosinate. The recovery rates of paraquat detection in the spiked soil samples were in a range of 99.5%-105.1%, with relative standard deviation values of <4%. The developed aptasensor was used to screen for paraquat residues in agricultural soils, and three out of 23 soil samples were tested positive for paraquat, which was confirmed by a high-performance liquid chromatography analysis. These results suggested the potential application of the developed aptasensor to detect paraquat residues in agricultural sites.

Yellow Twig (Nauclea orientalis) from Thailand: Strictosamide as the Key Alkaloid of This Plant Species

Comprehensive phytochemical examination from different perspectives using preparative and analytical chromatographic techniques combined with spectroscopic/spectrometric methods of the so-called "yellow twig" Nauclea orientalis (L.) L. (Rubiaceae) led to the identification of 13 tryptamine-derived (=monoterpene-indole) alkaloids. The identified alkaloids comprise strictosamide and four of its glucosidic derivatives, three oxindole derivatives, and five yellow-colored angustine-type aglycones. Qualitative and quantitative HPLC analyses showed the enrichment of strictosamide in all studied organs. Based on these results, we performed metabolomic analyses of monoterpene-indole alkaloids and made a 1H NMR in vitro monitoring of enzymatic deglucosylation of strictosamide. A comparison of the stability of strictosamide and its enantiomer vincoside lactam by theoretical calculations was also performed revealing a slightly higher stability of vincoside lactam. Additionally, we conducted two different anti-feedant assays of strictosamide using larvae of the polyphageous moth Spodoptera littoralis Boisduval. The obtained results indicate that generally two different biosynthetic pathways are most likely responsible for the overall alkaloid composition in this plant. Strictosamide is the key compound in the broader pathway and most likely the source of the identified angustine-type aglycones, which may contribute significantly to the yellow color of the wood. Its cross-organ accumulation makes it likely that strictosamide is not only important as a reservoir for the further biosynthesis, but also acts in the plants' defense strategy.

pH modulated luminescent switching and discriminative detection of amino acid based on metal-organic framework.

The detection of glutamic (Glu) or aspartic (Asp) acids is vital for human nutrition and diagnosis of disease. Herein, the dht ligand containing hydroxy group (-OH) is used to design and synthesize a 2D luminescent [Cd2(idc)(dht)(H2O)4] (1); H2idc = 4,5-imidazoledicarboxylic acid and H2dht = 2,5-dihydroxyterephthalic acid for sensing amino acids. The compound 1 can discriminatively detect Asp and Glu among other amino acids through blue-shifted emission (yellow → green). The dual sensing mechanism may be attributed to the intermolecular excited-state proton transfer between MOF and water to produce keto form along with the subsequent switching of keto form to enol form by protonation causing the increased band gap energy. This material can serve several benefits in terms of high selectivity, fast response (30s), good reproducibility and low LOD value of 11.34 µM which is less than the harmful concentration of Glu for human health (>400 µM). In addition, 1 shows the broad range detection of Glu covering in safe and unsafe levels. For on-site detection of Glu, MOF-based paper is devised and can be applied through color-scanning application in smartphone. Besides, this sensor can serve to detect Glu in real samples with good recovery.

Computational Insights into Molecular Adsorption Characteristics of Methylated DNA on Graphene Oxide for Multicancer Early Detection.

DNA methylation is an epigenetic modification involving the transfer of a methyl group to cytosine residues of a DNA molecule. Altered DNA methylation of certain genes is associated with several diseases including cancer. Nanomaterials, such as graphene oxide (GO), offer great potential as sensing elements for methylated DNA (mDNA) detection due to their distinct properties. Understanding molecular interactions between mDNA and GO can make provision for developing a universal cancer screening test. Molecular dynamics (MD) simulation and density functional theory (DFT) calculation have been employed for investigating their detailed macro- and microscale interactions. Based upon the MD simulation, different adsorption levels of methylated and unmethylated DNAs on GO were represented by a contacting surface area (CSA), which depends on surrounding conditions (in water or a MgCl2 solution). In water, the CSAs of the methylated and unmethylated single-stranded DNA (ssDNA) were ≈13 and ≈5 nm2, respectively, representing more preferable adsorption on GO for the methylated ssDNA. In the presence of divalent ions (Mg2+), the CSAs of both methylated and unmethylated DNA molecules were ≈8 nm2, suggesting that there was no significant difference in adsorption in a saline solution. To reveal the electrical property of GO covered by either methylated or unmethylated DNA, its electronic structure was investigated by the DFT calculation. The energy gaps of pristine graphene (pG) and GO adsorbed by 5-methylcytosine (5mC) were 1.6 and 12.9 meV, respectively, while cytosine adsorption resulted in lower energy gaps (1.2 meV for pG and 9.5 meV for GO). When comparing methylated DNA-covered GO with that covered with unmethylated DNA, remarkable differences in electrical conductivity, which were caused by the electronic structure of GO, were observed. These findings will provide a new route for an efficient detection method of DNA methylation, which can further be used to develop a universal cancer test.

Highly Aromatic Flavan-3-ol Derivatives from Palaeotropical Artocarpus lacucha Buch.-Ham Possess Radical Scavenging and Antiproliferative Properties.

Phytochemical investigation of leaves and stembark of Artocarpus lacucha collected in Thailand resulted in three yet undescribed isomeric flavan-3-ol derivatives (1-3), the four known compounds gambircatechol (4), (+)-catechin (5), (+)-afzelechin (6) and the stilbene oxyresveratrol (7). Compounds 1 to 3 feature 6/6/5/6/5/6 core structures. All structures were deduced by NMR and MS, while density functional theory (DFT) calculations on B3LYP theory level were performed of compounds 1 to 3 to support the stereochemistry in positions 2 and 3 in the C-ring. Possible biosynthetic pathways leading to 4 are discussed. The DPPH assay revealed high radical scavenging activities for 1 (EC50 = 9.4 ± 1.0 µmol mL-1), 2 (12.2 ± 1.1), 3 (10.0 ± 1.5) and 4 (19.0 ± 2.6), remarkably lower than ascorbic acid (EC50 = 34.9) and α-tocopherol (EC50 = 48.6). A cytotoxicity assay revealed moderate but consistent antiproliferative properties of 1 in CH1/PA-1 (ovarian teratocarcinoma) and SW480 (colon carcinoma) cells, with IC50 values of 25 ± 6 and 34 ± 4 µM, respectively, whereas effects in A549 (non-small cell lung cancer) cells were rather negligible. The performed DCFH-DA assay of 1 in the former cell lines confirmed potent antioxidative effects even in the cellular environment.

Designing an Aptasensor Based on Cysteamine-Capped AuNPs for 8-Oxo-dG Detection: A Molecular Dynamics Approach and Experimental Validation.

The concentration of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) in urine or serum is associated with the degree of oxidative damage of DNA and broadly used as a sensitive biomarker for various diseases. However, determination of a low concentration of 8-oxo-dG in biosamples is not an easy task owing to the complexity of coexisting substances. Herein, we design an aptasensor based on aptamer-mediated aggregation of cysteamine-capped gold nanoparticles (Cyst/AuNPs) for the detection of 8-oxo-dG by molecular dynamics simulation. Our simulations reveal that a positively charged Cyst modified onto the surfaces of AuNP exists in two conformers including gauche and trans. The trans conformer was prevalent on the AuNP surfaces and can stabilize AuNPs in the aqueous solution, even in the presence of 8-oxo-dG. Molecular recognition between 8-oxo-dG and the aptamer was demonstrated and bonding between these biomolecules was thoroughly elucidated. During the complex formation, van der Waals stacking interactions between 8-oxo-dG molecules were observed and found to play a significant role in the binding stability. The sensing mechanism of the colorimetric aptasensor was studied and the feasibility study of the proposed aptasensor was assessed by experimental validation. The experimental results are in good agreement with the computational study. Our in silico design can pave the way for, but is not limited to, a highly sensitive aptasensor for the naked-eye detection of 8-oxo-dG.

Combined in silico and in vitro study of an aptasensor based on citrate-capped AuNPs for naked-eye detection of a critical biomarker of oxidative stress.

An elevated level of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) in biosamples has been found to correlate to oxidative stress, and it has been assigned as a critical biomarker of various diseases. Herein, insights into the mechanisms of an aptasensor, based on citrate-capped gold nanoparticles (AuNPs), for 8-oxo-dG detection were elucidated using molecular dynamics (MD) simulations and validated experimentally. We found that the binding mechanism for binding between the anti-8-oxo-dG aptamer and 8-oxo-dG has the following characteristic stages: (i) adsorption stage, (ii) binding stage, and (iii) complex stabilization stage. Our simulations also reveal the binding sites between the anti-8-oxo-dG aptamer and 8-oxo-dG formed through hydrogen bonding during complex formation. A shortened anti-8-oxo-dG-aptamer was also engineered using in silico design, which was expected to improve the analytical performance of the colorimetric aptasensor. The mechanisms of the colorimetric aptasensor in the presence and absence of 8-oxo-dG were also investigated, and found to be in good agreement with the experiments. Complete understanding of the mechanism of the colorimetric aptasensor would open the door for development of novel naked-eye aptasensors.

Insight into the Molecular Mechanisms of AuNP-Based Aptasensor for Colorimetric Detection: A Molecular Dynamics Approach.

Colorimetric aptasensor based on assembly of salt-induced gold nanoparticles (AuNPs) is a promising biosensor. However, the molecular mechanism of the aptasensor is far from being fully understood. Herein, molecular dynamics (MD) simulation was used to investigate molecular interactions in the detection of ochratoxin A (OTA) including the following: (i) the molecular recognition of the anti-OTA aptamer, (ii) OTA-aptamer interactions in monovalent (Na+) and divalent (Mg2+) electrolytes, (iii) the binding mode of citrate on the AuNP surface, (iv) interactions of the aptamer with citrate-capped AuNPs, and (v) a detailed mechanism of the aptasensor. Our MD simulations revealed a specific binding of the OTA-aptamer complex, compared with OTB and warfarin. Compared with Na+, Mg2+ ions exerted a more effective attractive force between OTA and anti-OTA aptamer. Three different binding modes of citrate on AuNP surfaces were found. The kinetics of the adsorption of unfolded aptamers onto the citrate-capped AuNP was also elucidated. Most importantly, our MD simulation revealed an insightful analysis of the molecular mechanisms in the AuNP-based aptasensor and paved the way for the design of a novel colorimetric aptasensor for other target molecules, which is not limited to OTA detection.