A simple smartphone-assisted paper-based colorimetric biosensor for the detection of urea adulteration in milk based on an environment-friendly pH-sensitive nanocomposite.

Urea is a common milk adulterant that falsely increases its protein content. Excessive consumption of urea is harmful to the kidney, liver, and gastrointestinal system. The conventional methods for urea detection in milk are time-consuming, costly, and require highly skilled operators. So, there is an increasing demand for the development of rapid, convenient, and cost-efficient methods for the detection of urea adulteration in milk. Herein, we report a novel colorimetric paper-based urea biosensor, consisting of a novel environment-friendly nanocomposite of halloysite nanotubes (HNT), that urease enzyme and an anthocyanin-rich extract, as a natural pH indicator are simultaneously immobilized into its internal and external surfaces. The biosensing mechanism of this biosensor is based on anthocyanin color change, which occurs due to urease-mediated hydrolysis of urea and pH increment of the environment. The colorimetric signal of this biosensor is measured through smartphone-assisted analysis of the mean RGB (Red-Green-Blue) intensity of samples and is capable of detecting urea with a detection limit of 0.2 mM, and a linear range from 0.5 to 100 mM. This biosensor has demonstrated promising results for the detection of urea in milk samples, in the presence of other milk adulterants and interferents.

Paper-based colorimetric detection of COVID-19 using aptasenor based on biomimetic peroxidase like activity of ChF/ZnO/CNT nano-hybrid.

Corona Virus Disease 2019 (COVID-19) as the infectious disease caused the pandemic disease around the world through infection by SARS-CoV-2 virus. The common diagnosis approach is Quantitative RT-PCR (qRT-PCR) which is time consuming and labor intensive. In the present study a novel colorimetric aptasensor was developed based on intrinsic catalytic activity of chitosan film embedded with ZnO/CNT (ChF/ZnO/CNT) on 3,3',5,5'-tetramethylbenzidine (TMB) substrate. The main nanocomposite platform was constructed and functionalized with specific COVID-19 aptamer. The construction subjected with TMB substrate and H2O2 in the presence of different concentration of COVID-19 virus. Separation of aptamer after binding with virus particles declined the nanozyme activity. Upon addition of virus concentration, the peroxidase like activity of developed platform and colorimetric signals of oxidized TMB decreased gradually. Under optimal conditions the nanozyme could detect the virus in the linear range of 1-500 pg mL and LOD of 0.05 pg mL. Also, a paper-based platform was used for set up the strategy on applicable device. The paper-based strategy showed a linear range between 50 and 500 pg mL with LOD of 8 pg mL. The applied paper based colorimetric strategy showed reliable results for sensitive and selective detection of COVID-19 virus with the cost-effective approach.

A stem-loop like aptasensor for sensitive detection of aflatoxin based on graphene oxide/AuNPs nanocomposite platform.

Aflatoxin as the potent carcinogenic mycotoxin had been received great attention for detection in food industry and safety. Due to low quantity of aflatoxin in food samples, there is a need to develop a sensitive method toward its detection. In the present study, an aflatoxin B1 (AFB1) specific aptasensor with internal complementary sequence was developed for detection of AFB1. The FAM-functionalized aptamer was deposited on the surface of graphene oxide decorated with gold nanoparticles (GO/AuNPs) and following formation of heteroduplex stem-loop structure led to fluorescence quenching of FAM-labeled aptamer. After incubation of proposed aptasensor with AFB1, the aptamer-AFB1 complex resulted to denaturation in stem-loop structure of aptamer which caused restoration of single strand aptamer and recovery of fluorescence. The assay showed a convenient detection response in the range of 0.5-20 pg/mL with a very low detection limit (0.1 pg/mL) and it was highly selective for AFB1 over other mycotoxins.

Dual mode fluorometric and colorimetric detection of matrix metalloproteinase MMP-9 as a cancer biomarker based on AuNPs@gelatin/AuNCs nanocomposite.

Matrix metalloproteinase (MMPs) are proteolytic enzymes which considered as important enzymes and their higher expression is associated with some malignant progression. In the present study, a novel and sensitive method was developed for detection of MMP-9 based on its gelatinase activity. Green emitting gold nanoclusters (AuNCs) has been synthesized by gelatin substrate and then AuNPs@gelatin/AuNCs nanocomposite structure were prepared through coating of gold nanoparticles (AuNPs) with gelatin/AuNCs. Characterization and morphology of synthesized nanocomposite showed successful formation of AuNPs@gelatin/AuNCs. Constructed nanocomposite exhibited internal FRET occurrence and fluorescence quenching of AuNCs due to proximity of AuNCs/AuNPs structures. Hydrolysis effect of MMPs enzyme on AuNPs@gelatin/AuNCs in saline buffer induced changes in surface plasmon resonance (SPR) of gold nanoparticles and also enhanced the emitted fluorescence by AuNCs due to inhibition of internal FRET process. The proposed platform served as the efficient approach for semi-quantitative detection of MMP-9 enzyme by naked eye and precise activity detection with LOD of 2 ng/mL calorimetrically and 0.25 ng/mL fluorometrically. The approach was also showed convenient recovery for detection of MMP-9 enzyme in human serum matrix. Our work provides an efficient, convenient and practical tool for simple identification and precise detection of MMP-9 enzyme.

Fabrication of Graphene-Based TiO2@CeO2 and CeO2@TiO2 Core-Shell Heterostructures for Enhanced Photocatalytic Activity and Cytotoxicity.

Development of light-harvesting properties and inhibition of photogenerated charge carrier recombination are of paramount significance in the photocatalytic process. In the present work, we described the synthesis of core-shell heterostructures, which are composed of titanium oxide (TiO2) and cerium oxide (CeO2) deposited on a reduced graphene oxide (rGO) surface as a conductive substrate. Following the synthesis of ternary rGO-CeO2@TiO2 and rGO-TiO2@CeO2 nanostructures, their photocatalytic activity was investigated toward the degradation of rhodamine B dye as an organic pollutant under UV light irradiation. The obtained structures were characterized with high-resolution transmission electron microscopy, field-emission scanning electron microscopy, energy-dispersive X-ray spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction, Brunauer-Emmett-Teller, X-ray photoelectron spectroscopy surface analysis, and UV-Vis spectroscopy. Various parameters including pH, catalyst dosage, temperature, and contact time were studied for photocatalysis optimization. Heterostructures showed considerable advantages because of their high surface area and superior photocatalytic performance. In contrast, rGO-CeO2@TiO2 showed the highest photocatalytic activity, which is attributed to the more effective electron-hole separation and quick suppression of charge recombination at core-shell phases. A biological assay of the prepared heterostructure was performed to determine the cytotoxicity against breast cancer cells (MCF-7) and demonstrated a very low survival rate at 7.65% of cells at the 17.5 mg mL-1 concentration of applied photocatalyst.

A robust electrochemical sensing platform for the detection of erlotinib based on nitrogen-doped graphene quantum dots/copper nanoparticles-polyaniline-graphene oxide nanohybrid.

Erlotinib is a potent and highly specific tyrosine kinase inhibitor with the hindering effects on the growth of cancer cells. An electrochemical sensor with the great sensitivity and selectivity was fabricated for determining erlotinib by using a graphite rod electrode modified by the nitrogen-doped graphene quantum dots (N-GQDs) and a ternary nanohybrid comprising copper nanoparticles, polyaniline, along with graphene oxide (N-GQDs/CuNPs-PANI@GO) for the first time. The establishment of PANI and CuNPs was done simultaneously on the GO surface by thein situoxidative polymerization method. The morphological characteristics and elemental structure of the synthesized nanoparticles were examined by some microscopy techniques and x-ray energy/diffraction methods. The fabricated sensor represented the electrocatalytic activity towards erlotinib with a linear detection range from 1.0 nM to 35.0µM, a detection limit of 0.712 nM, and a sensitivity of 1.3604µAµM-1. Moreover, the N-GQDs/CuNPs-PANI@GO sensor showed acceptable stability up to 30 d (94.82%), reproducibility (RSD values of 3.19% intraday and 3.52% interday), and repeatability (RSD value of 3.65%) as a novel and powerful electrochemical sensor. It was successfully applied to monitor erlotinib in the drug-injected aqueous solution, serum, and urine samples that proved the capability of the sensor for the erlotinib monitoring in the biological samples.

Fluorometric detection of phytase enzyme activity and phosphate ion based on gelatin supported silver nanoclusters.

Phytase is the commercial enzyme for bioconversion of phytate substrate to digestible phosphate ions. Recently silver nanoclusters (AgNCs) have received great attention as the optical transducer nanoparticles in biosensors structure. The novel detection platform was developed to detect the phytase enzyme activity and phosphate ions based on fluorescence quenching of AgNCs. The AgNCs were synthesized through gelatin supported reaction and characterized by TEM, FTIR and XRD analysis. The hydrolytic effect of phytase enzyme and subsequent phosphate release led to suppression of AgNCs fluorescence. The linear range was observed for enzyme in the range of 0.5-5 U/mL with the detection limit of 0.2 U/mL. Also, the same fluorescence quenching effect was observed in the presence of phosphate ion in the linear range of 1 to 16 µM with a detection limit of 0.5 µM. The proposed mechanism showed effectiveness of detection strategy for detection of phytase enzyme and phosphate ion.

Photocatalytic activity of green synthesized cadmium sulfide quantum dots on the removal of RhB dye and its cytotoxicity and antibacterial studies.

Presence of inorganic pollutants in water reservoirs is the treating factor for human health and environment. Semiconductor quantum dots (QDs) has been regarded as one of the most efficient nanoparticles for their enhanced photocatalytic activity. Medicinal plants are the safe sources to provide green template for biosynthesis of inorganic nanoparticles such as quantum dots. In order to determine the photocatalytic and biological application of cadmium sulfide quantum dots (CdS QDs), a biosynthesis approach was employed using saffron (Crocus sativus L.) stigma extract as the green reaction substrate. The biosynthesis process was evaluated at different pH condition to obtain the most efficient CdS QDs. Characterization of prepared CdS QDs were determined through UV-vis and fluorescence spectroscopy, FTIR and TEM analysis. The obtained results showed well dsispersed and uniform QDs during green synthesis at the optimum condition. The absorption and electrical properties of green synthesized CdS QDs showed the lowest energy bandgap of 2.4 at pH 11. Photocatalytic activity of CdS QDs on Rhodamine B degradation showed 92% degradation after 80 min under UV light irradiation. The antibacterial and cell cytotoxicity of green synthesized CdS QDs were assayed by disk diffusion and MTT assays respectively. Obtained results showed significant antibacterial effect of CdS QDs against gram-positive and gram-negative bacteria includingB. subtilis(90%) andE. coli(96%) respectively. Moreover, cytotoxicity of prepared CdS Qds through MTT assay indicated 79% apoptosis induction on MCF-7 breast cancer cells.

Facile fabrication of ternary MWCNTs/ZnO/Chitosan nanocomposite for enhanced photocatalytic degradation of methylene blue and antibacterial activity.

Developing a cheap, stable and effective photocatalyst is necessary for remediation of persistent organic pollutants. To address this challenge, we proposed a unique interfacial engineering technique and proper bandgap matching strategy to synthesize MWCNTs/ZnO/Chitosan ternary nanocomposite for effective photocatalytic application. The features of the prepared samples were determined by FESEM, TEM, EDX, elemental mapping, AFM, FT-IR, XRD, UV-Vis spectroscopy and BET surface analysis. The obtained results showed successful fabrication of synthesized nanocomposites with enhanced surface area. Degradation effect of nanostructures on methylene blue (MB) and antibacterial activity against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Bacillus subtilis (B. subtilis) pathogenic strains were investigated. The proposed photocatalytic mechanism illustrated the electron transfer facilitated by MWCNTs/ZnO/Chitosan structure which results in spatial separation of electron-hole pairs. Compared with ZnO and ZnO/Chitosan, the prepared MWCNTs/ZnO/Chitosan ternary nanocomposite showed high usage of UV illumination and superior separation of photogenerated electron-hole pairs. MWCNTs/ZnO/Chitosan illustrated 86.26% adsorption rate and outstanding increased photocatalytic activity on MB degradation efficiency of 98.76% after 20 min. Stability of photocatalyst reached from 98.76% initial decolorization to 85% at the fourth cycle. In addition, the ternary nanocomposite also exhibited remarkable bactericidal activity against gram-positive (S. aureus) and (B. subtilis) and gram-negative (E. coli) bacteria strains. Due to the obtained results, the prepared nanocomposite would be an efficient candidate photocatalyst with antibacterial properties.

Colorimetric and label free detection of gelatinase positive bacteria and gelatinase activity based on aggregation and dissolution of gold nanoparticles.

A simple and sensitive method was developed for the detection of bacteria gelatinase activity based on their enzymatic hydrolysis effect on the surface plasmon resonance (SPR) of gelatin functionalized gold nanoparticles (Au@gelatin NPs) in bacteria supernatant. Characterization of synthesized NPs showed a very thin gelatin layer on the surface of about 20 nm AuNPs which modified the intrinsic SPR property of AuNPs. The extracted supernatants of applied bacteria were incubated with Au@gelatin NPs. Gelatinase activity of bacteria resulted in gradual gelatin shell removal and subsequent dissolution of bare AuNPs. The presence of inducer agents such as NaCl as the common ingredient in the bacterial medium led to the aggregation process of AuNPs and further bacterial activity resulted in AuNPs dissolution. AuNPs colloid solution color was changed from red to purple after addition of bacteria supernatants with gelatinase activity to the reaction. Also, the spectroscopic studies showed that the gelatinase activity of bacteria resulted in the gradual decrease of absorbance at 529 nm and subsequently led to extinction of SPR characteristics. So, the observed absorbance decrease in UV-Vis spectra at 529 nm was indicated as the gelatinase activity of applied bacteria. Different strains of gelatinase positive Bacillus strains were used as the real sample and their gelatinase activity was determined in the present study. Also, sensitivity analysis of the applied method was determined through this method and the obtained results showed Bacillus subtilis gelatinase activity in the linear range of 0-120 U/mL and detection limit of 0.5 U/mL. This method introduced label free, facile and sensitive assay of the bacterial gelatinase activity without any complicated instrument, affording convenience and simplicity.

A novel colorimetric biosensor for sensitive detection of aflatoxin mediated by bacterial enzymatic reaction in saffron samples.

Aflatoxin is regarded as the potent carcinogenic agent which is secreted from fungi and present in some food products. So far, many detection methods have been developed to determine the trace amounts of aflatoxin in foods. In the present study a colorimetric competitive assay for detection of aflatoxin B1 (AFB1) has been developed based on interaction of gelatin functionalized gold nanoparticles (AuNPs@gelatin) in specific enzymatic reaction. Bacterial supernatant containing gelatinase enzyme were used as the substrate that could digest the coated gelatin on the surface of AuNPs and following in the presence of NaCl medium ingredient resulted to color change of AuNPs colloidal solution from red to purple. It was observed that with addition of aflatoxin to the bacterial supernatant, aflatoxin could interfere in aggregation of AuNPs and inhibited the process which subsequently prevent the expected color change induced by AuNPs aggregation. The supernatant containing AuNPs were investigated to analyze their induced surface plasmon resonance spectra through UV-visible spectroscopy. The absorption values were directly proportional with the applied AFB1 concentration. The experiment conditions including incubation time, AuNPs concentration and pH were investigated. The obtained results showed that through this approach we could detect the AFB1 in a linear range from 10 to 140 pg ml-1, with detection limit of 4 pg ml-1. Real sample assay in saffron samples showed recoveries percentage of 92.4%-95.3%. The applied approach proposed simple, cost effective and specific method for detection of AFB1 toxin in food samples.

Fluorimetric detection of methylated DNA of Sept9 promoter by silver nanoclusters at intrastrand 6C-loop.

Methylation of DNA at carbon 5 of cytosines is the most common epigenetic modification of human genome. Due to its critical role in many normal cell processes such as growth and development, any aberrant methylation pattern in a particular locus may lead to abnormal functions and diseases such as cancer. Development of methods to detect methylation state of DNA which may eliminate labor-intensive chemical or enzymatic treatments has received considerable attention in recent years. Herein, we report a DNA methylation detection procedure based on fluorescence turn-on strategy. Target sequence was selected from Sept9 promoter region that has been reported as one of the most frequently methylated sites in colorectal cancer. Probe DNA was designed to be complementary to this sequence with an additional six cytosines in the middle to form an internal loop to host silver nanoclusters. The fluorescence intensity of the synthesized silver nanoclusters with the duplexes of probe-non-methylated target was significantly different from that of probe-methylated target. The fluorescence enhanced with increasing the methylated DNA concentration with a linear relation in the range of 1.0 × 10-8 M to 5.0 × 10-7 M with the detection limit of 8.2 × 10-9 M, and quenched with non-methylated ones. The method was very specific in the presence of non-complementary sequences with maximum similarity of 40%. Circular dichroism spectra indicated that silver ions significantly affected the structure of methylated and non-methylated DNA into different extents which could further influence the nanocluster fluorescence. Finally, a method was introduced to meet the concerns in the applicability of the proposed method in real situation.

A signal-on fluorescence based biosensing platform for highly sensitive detection of DNA methyltransferase enzyme activity and inhibition.

DNA methylation mediated by DNA methyltransferase (MTase) enzyme is internal cell mechanism which regulate the expression or suppression of crucial genes involve in cancer early diagnosis. Herein, highly sensitive fluorescence biosensing platform was developed for monitoring of DNA Dam MTase enzyme activity and inhibition based on fluorescence signal on mechanism. The specific Au NP functionalized oligonucleotide probe with overhang end as a template for the synthesis of fluorescent silver nanoclusters (Ag NCs) was designed to provide the FRET occurrence. Following, methylation and cleavage processes by Dam MTAse and DpnI enzymes respectively at specific probe recognition site could resulted to release of AgNCs synthesizer DNA fragment and returned the platform to fluorescence signal-on state through interrupting in FRET. Subsequently, amplified fluorescence emission signals of Ag NCs showed increasing linear relationship with amount of Dam MTase enzyme at the range of 0.1-20 U/mL and the detection limit was estimated at 0.05 U/mL. Superior selectivity of experiment was illustrated among other tested MTase and restriction enzymes due to the specific recognition of MTase toward its substrate. Furthermore, the inhibition effect of applied Dam MTase drug inhibitors screened and evaluated with satisfactory results which would be helpful for discovery of antimicrobial drugs. The real sample assay also showed the applicability of proposed method in human serum condition. This novel strategy presented an efficient and cost effective platform for sensitive monitoring of DNA MTase activity and inhibition which illustrated its great potential for further application in medical diagnosis and drug discovery.

Paper based colorimetric detection of miRNA-21 using Ag/Pt nanoclusters.

Abnormal expression of MicroRNA-21 (miRNA-21) is considered to be a reliable biomarker for the early diagnosis of cancer. In this work, a novel paper based biosensor was fabricated to detect sub-micro molar concentrations of miRNA-21 based on peroxidase mimetic activity of DNA-templated Ag/Pt nanoclusters (DNA-Ag/Pt NCs), which could catalyze the reaction of hydrogen peroxide and 3,3',5,5' tetramethylbenzidine (TMB), to produce a blue color. The Mechanism of reaction was based on the inhibition effect of miRNA-21 on peroxidase-like activity of nanosensor which resulted to quantitative determination of miRNA-21 concentration. It was found that miRNA-21 could be linearly detected in the range from 1-700 pM (A652 = 0.16x-0.96, R2 = 0.99; x = -log [miRNA-21]) with a detection limit of 0.6 pM. Moreover, a paper assay was carried out on a Y-shaped paper-based microfluidic device in order to use the distinctive features of micro-channels such as short response time, very low reagent volume, low fabrication cost, etc. After performing paper based assay, a good linear range was observed between 10-1000 pM (y = 0.06x+147.48, R2 = 0.99; x = [miRNA-21]) with detection limit of 4.1 pM. The practical application of proposed method for detection of miRNA-21 in real sample was assayed in the human urine sample and indicated the colorimetric method had acceptable accuracy.

Density Functional Theory Study of Antioxidant Adsorption onto Single-Wall Boron Nitride Nanotubes: Design of New Antioxidant Delivery Systems.

BACKGROUND: Boron Nitride Nanotubes (BNNTs) have recently emerged as an interesting field of study, because they could be used for the realization of developed, integrated and compact nanostructures to be formulated. BNNTs with similar surface morphology, alternating B and N atoms completely substitute for C atoms in a graphitic-like sheet with nearly no alterations in atomic spacing, with uniformity in dispersion in the solution, and readily applicable in biomedical applications with no obvious toxicity. Also demonstrating a good cell interaction and cell targeting. AIM AND OBJECTIVE: With a purpose of increasing the field of BNNT for drug delivery, a theoretical investigation of the interaction of Melatonin, Vitamin C, Glutathione and lipoic acid antioxidants using (9, 0) zigzag BNNTs is shown using density functional theory. METHODS: The geometries corresponding to Melatonin, Vitamin C, Glutathione and lipoic acid and BNNT with different lengths were individually optimized with the DMOL3 program at the LDA/ DNP (fine) level of theory. RESULTS: In the presence of external electric field Melatonin, Vitamin C, Glutathione and lipoic acid could be absorbed considerably on BNNT with lengths 22 and 29 Å, as the adsorption energy values in the presence of external electric field are considerably increased. CONCLUSION: The external electric field is an appropriate technique for adsorbing and storing antioxidants on BNNTs. Moreover, it is believed that applying the external electric field may be a proper method for controlling release rate of drugs.

A fluorometric study on the effect of DNA methylation on DNA interaction with graphene quantum dots.

DNA methylation plays an important role in development process which contributes to genome stability and also regulates gene expression and gene silencing. Detection of genome regions with altered 5-methylcytosine distribution at a genome-wide scale is very important for early detection of gene silencing related diseases. In the present study as a continuation of studies on DNA methylation, the interactions between graphene quantum dots (GQDs) and unmethylated and methylated deoxyribonucleic acid (DNA) fragment were investigated. Based on above interaction a novel GQDs-DNA nanoassembly was developed. Two types of DNA including unmethylated and methylated sequences were interacted with GQDs and contributed to the formation of unmethylated and methylated nanoassemlies. Analysis of the interaction indicated that the GQDs could bind to DNA fragments and led to different fluorescence pattern in two different mechanisms and could provide an efficient biosensing platform for label free and sensitive fluorescent assay of DNA. The excitation and emission wavelengths of experiment were 380 and 480 nm respectively. Fluorescence intensity of unmethylated DNA concentration were detectable from methylated DNA in linear range from 10.0-10M to 10.0-6M and the detection limit was estimated at 7.3 × 10-11 M. Above interaction was not observed in methylated DNA, indicated of distinguished interaction effect. Herein we further showed that GQDs could induce B-DNA to A-DNA form in methylated structure of DNA. The methylation sensitivity of the experiment was also testified by methylation sensitive restriction process. It was assumed that the involvement of methylation alteration in DNA structure could alter not only mechanism of DNA/GDQs interaction but also helical structure of DNA.

Sensitive detection of methylated DNA and methyltransferase activity based on the lighting up of FAM-labeled DNA quenched fluorescence by gold nanoparticles.

DNA methylation of cytosine bases, which is catalyzed by methyltransferase enzymes, involve biochemical processes that contribute to gene expression and gene regulation in cells. Detection of abnormal patterns of both methylated DNA and methyltransferase enzyme activity at early stages could be considered as promising targets for early cancer diagnosis. In the present study, a novel and facile method is introduced for the sensitive detection of the M.SssI methyltransferase (M.SssI MTase) enzyme and methylated DNA based on the fluorescence recovery of FAM-labeled DNA coupled with gold nanoparticles (AuNPs). Thiol-modified probes were functionalized with AuNPs, which brought the FAM fluorophore into the close proximity of the AuNPs. This led to the overlap between the FAM fluorescence emission and AuNPs absorption spectra, introducing a FRET occurrence and causing fluorescence quenching. The hybridization of the probe and its complementary target provided specific CpG sites for M.SssI MTase enzyme activity. The methylation process gradually converted the quenched FAM fluorophore into an emissive fluorophore upon the addition of the MTase enzyme, and the observed fluorescence recovery proved the efficiency of the assay for the detection of MTase enzyme. The fluorescence intensity showed an increasing trend with M.SssI MTase enzyme activity in the range of 1-8 U mL-1 with a detection limit of 0.14 U mL-1. The addition of methylated ssDNA targets to a ssDNA FAM-labeled probe resulted in a DNA duplex formation, leading to a strong fluorescence signal emission due to the recovery of the fluorophore signal. Conversely, the unmethylated ssDNA target caused no changes in the fluorescence signal. In the presence of methylated DNA targets, the biosensor could specifically recognize it and accordingly trigger the methylated targets through a fluorescence enhancement in the range of 5-100 pM by monitoring the increase in the fluorescence intensity with a detection limit of 2.2 pM. The obtained results showed that the assay could realize the detection of M.SssI MTase and methylated DNA effectively in diluted human serum samples. Human serum conditions showed no significant interference with the assay performance, indicating that the present method has great potential for further application in real samples.

A colorimetric assay of DNA methyltransferase activity based on peroxidase mimicking of DNA template Ag/Pt bimetallic nanoclusters.

DNA methylation catalyzed by DNA methyl transferase (MTase) is a significant epigenetic process for modulating gene expression. Abnormal levels of DNA MTase enzyme have been regarded as a cancer biomarker or a sign of bacterial diseases. We developed a novel colorimetric method to assay M.SssI MTase activity employing peroxidase-like activity of DNA template Ag/Pt NCs without using restriction enzymes. Based on inhibiting the peroxidase reaction that occurred in the TMB-H2O2 system, in the presence of MTase, a highly sensitive and selective colorimetric biosensor was fabricated with a detection limit (LOD) of 0.05 U/mL and a linear range from 0.5 to 10 U/mL. The changes in absorption intensity were monitored to quantify the M.SssI activity. This strategy had a high selectivity over other proteins. Furthermore, it is also demonstrated that this method can be used for the evaluation and screening of inhibitors for DNA MTase.

DNA-Templated Silver Nanoclusters for DNA Methylation Detection.

DNA methylation entails the covalent addition of a methyl group to C-5 position of cytosine by a family of DNA methyltransferase enzymes and has a significant role in gene regulation. Epigenetic changes such as DNA methylation of CpG islands located in the promoter region of some tumor suppressor genes are very common in human diseases such as cancer. Detection of aberrant methylation pattern could serve as an excellent diagnostic approach. It is key to develop methods for the direct and simple detection of methylated DNA or of methyltransferase activity without using antibodies, chemical modification, labeling and enzymatic treatments. In this study, we employ DNA-templated silver nanoclusters for detection of DNA methylation. This method entails use of cytosine rich DNA sequence as an effective template. By monitoring changes in fluorescence intensity, DNA methylation and DNA methyltransferase activity is detected. Upon DNA methylation, the fluorescence intensity of DNA templated Ag/NCs is decreased in a linear range when the concentration of methylated DNA is increased.

Study on the Interaction of the CpG Alternating DNA with CdTe Quantum Dots.

A novel sensitive method for detection of DNA methylation was developed with thioglycollic acid (TGA)-capped CdTe quantum dots (QDs) as fluorescence probes. Recognition of methylated DNA sites would be useful strategy due to the important roles of methylation in disease occurrence and developmental processes. DNA methylation occurs most often at cytosine-guanine sites (CpG dinucleotides) of gene promoters. The QDs significantly interacted with hybridized unmethylated and methylated DNA. The interaction of CpG rich methylated and unmethylated DNA hybrid with quantum dots as an optical probe has been investigated by fluorescence spectroscopy and electrophoresis assay. The fluorescence intensity of QDs was highly dependent to unmethylated and methylated DNA. Specific site of CpG islands of Adenomatous polyposis coli (APC), a well-studied tumor suppressor gene, was used as the detection target. Under optimum conditions, upon the addition of unmethylated dsDNA, the fluorescence intensity increased in linear range from 1.0 × 10- 10 to 1.0 × 10- 6M with detection limit of 6.2 × 10- 11 M and on the other hand, the intensity of QDs showed no changes with addition of methylated dsDNA. We also demonstrated that the unmethylated and methylated DNA and QDs complexes showed different mobility in electrophoresis assay. This easy and reliable method could distinguish between methylated and unmethylated DNA sequences.