Zhenning Xu

A new strategy for methylated DNA detection based on photoelectrochemical immunosensor using Bi2S3 nanorods, methyl bonding domain protein and anti-his tag antibody.

In this work, we fabricated a novel photoelectrochemical immunosensor for assay of DNA methylation, where Bi2S3 nanorods were used as photoelectric conversion material, MBD1 protein (a kind of methyl bonding domain protein) was used as DNA methylation recognizing unit, anti-his tag antibody was used to further inhibit the photocurrent and increase the detection sensitivity. The results demonstrated that Bi2S3 possessed excellent photoelectron property. The detection conditions, such as Bi2S3 concentration, MBD1 protein concentration, incubation time of MBD1 protein, antibody concentration and antibody incubation time, were optimized. Under optimal experimental conditions, the photocurrent variation was proportional to the logarithm of methylated target DNA concentration from 10(-9) to 10(-13) M with detection limit of 3.5×10(-14) M (S/N=3). Moreover, the immunosensor presented high detection specificity, even distinguishing single-base mismatched sequence.

Signal-on photoelectrochemical biosensor for microRNA detection based on Bi2S3 nanorods and enzymatic amplification

In this work, a photoelectrochemical (PEC) biosensor was fabricated for sensitive and specific detection of microRNA based on Bi2S3 nanorods and enzymatic signal amplification. Using the catalytic effect of alkaline phosphatase on l-ascorbic acid 2-phosphate trisodium salt (AAP), ascorbic acid (AA) was in situ generated and used as electron donor. Based on this, a signal-on protocol was successively achieved for microRNAs detection due to the dependence of photocurrent response on the concentration of electron donor of AA. The results demonstrated that the photocurrent response enhanced with increasing the hybridized concentration of microRNA. Under the amplification of the immunogold labeled streptavidin (SA-AuNPs), a low detection limit of 1.67 fM was obtained. The fabricated biosensor showed good detection stability and specificity, and it could discriminate only one-base mismatched microRNA sequence. Moreover, the down-regulated expression of microRNA-21 in DF-1 chicken fibroblast cells infected with subgroup J avian leukemia virus (ALVs) was confirmed by the developed method, indicating that microRNA-21 might be a new biomarker for avian leukemia. This work opens a different perspective for microRNAs detection and early diagnose of avian leukemia.

An electrochemical assay for DNA methylation, methyltransferase activity and inhibitor screening based on methyl binding domain protein

DNA methylation is one of important epigenetics events, and responsible to transcription, genomic imprinting and cellular differentiation. Aberrant DNA methylation is always contacted with various diseases. Methyl binding domain (MBD) proteins can specifically bind to the methylated CpG dinucleotides. Conventional assay for DNA methylation normally need bisulfide treatment, methylated nucleotide labeling or PCR amplification. Here, we fabricated a novel electrochemical biosensor for detection of DNA methylation, assay of DNA methyltransferase (MTase) activity and screening of MTase inhibitor based on MBD protein and coomassie brilliant blue G250 (CBB-G250), where the electrochemical signal of CBB-G250 was used to monitor the methylation event. After the hybrids of DNA S1 and DNA S2 were treated with M. SssI MTase in the presence of S-adenosylmethionine, the MBD proteins were specifically conjugated to the methylation site of CpG dinucleotides, and then, the MBD proteins were stained with CBB-G250. The electrochemical signal of CBB-G250 increased linearly with increasing M. SssI MTase concentration in the range from 0.1 to 40 unit/mL. Furthermore, the inhibition investigation demonstrates that fisetin and chlorogenic acid can inhibit the M. SssI MTase activity with the IC(50) value of 153.12 and 137.07 μM, respectively. Therefore, we think that this study may provide a sensitive platform for screening of DNA MTase inhibitors.

Colorimetric sensing of dopamine based on the aggregation of gold nanoparticles induced by copper ions

A facile, economic and eco-friendly colorimetric detection method for dopamine has been well-established in this paper. It is based on the interaction between Cu2+ ions with amino group and hydroxyl groups of the dopamine adsorbed on the surface of gold nanoparticles (GNPs). Cu2+ ions serve as the selective “discriminator and linker” for dopamine detection. At high ionic strength, introduction of Cu2+ ions to GNPs solution including dopamine arouses the aggregation of GNPs, the color of the solution changes from wine red to blue, and the red shift of ultraviolet absorption peaks. The concentration of dopamine can be determined by monitoring with the naked eye or a UV-vis spectrometer. The calibration curve showed that the absorption ratio value at 650 and 525 nm (A650/A525) increased linearly over the dopamine concentration range of 5 × 10−7 M to 1 × 10−5 M with a limit of detection of 2 × 10−7 M (3σ). This method exhibits excellent selectivity for dopamine over other α-amino acids, glutathione, glucose, uric acid and ascorbic acid, and the process of the experiment, including the preparation of GNPs, detection of dopamine, pretreatment of sample, is finished within 30 min at room temperature. On the other hand, this probe was successfully applied to detect dopamine in human serum with high sensitivity, and this strategy may provide a selective sensing approach for measuring dopamine under physiological conditions.

Investigation of the effect of phytohormone on the expression of microRNA-159a in Arabidopsis thaliana seedlings based on mimic enzyme catalysis systematic electrochemical biosensor

MicroRNAs (miRNAs) play very important roles in plant growth and development as well as phytohormones. More importantly, microRNAs were recently found to be a new growth regulator involved in plant hormone signaling. Therefore, for investigating the expression change of microRNAs in plants exposed to phytohormones and understanding the effect of phytohormones on microRNAs expression, we developed a simple, sensitive, and label-free method for microRNAs biosensing based on mimic enzyme catalysis signal amplification, where carboxylic graphene-hemin hybrid nanosheets was synthesized and used to catalyze the oxidation reaction of hydroquinone in the presence of H2O2 due to the intrinsic peroxidase-like activity of hemin on the carboxylic graphene surface. The electrochemical reduction current of the oxidative product of benzoquinone was depended on the hybridization amount of microRNAs and used to monitor the microRNAs hybridization event. Under optimal detection conditions, the current response was proportional to the logarithm concentration of microRNA-159a from 0.5 pM to 1.0 nM with the detection limit of 0.17 pM (S/N=3). The fabricated biosensor showed highly reproducible (Relative standard deviation (RSD) was 3.53% for 10 biosensors fabricated independently) and detection selectivity (Even discriminating single-base mismatched microRNA sequence). We also found that abscisic acid, a kind of phytohormone, had greatly influence on microRNA-159a expression in Arabidopsis thaliana seedlings. With increasing abscisic acid concentration and prolonging incubation time, both the expression level of microRNA-159a increased. This graphene-hemin-based approach provides a novel avenue to detect microRNA with high sensitivity and selectivity while avoiding laborious label, disadvantages of bio-enzymes and complex operations for microRNAs separation and enrichment, which might be attractive for genetic analysis and clinic biomedical application.

An ultrasensitive electrochemical immunosensor platform with double signal amplification for indole-3-acetic acid determinations in plant seeds

A label-free electrochemical immunosensor for ultra-sensitive detection of indole-3-acetic acid (IAA), a very important phytohormone, has been developed in this work. The detection strategy was mainly based on 4-aminophenylboronic acid, magnetic nanoparticles functionalized with horseradish peroxidase-conjugated goat anti-rat immunoglobulin G (HRP-IgG-Fe(3)O(4)) and rat monoclonal antibody against IAA-modified gold nanoparticles (anti-IAA-AuNPs). HRP-IgG-AuNPs was covalently assembled on the electrode surface through the specific chemical reaction between boronic acid and the vicinal diol in HRP-IgG. Then, anti-IAA-AuNPs was further assembled on the electrode via the interaction between IgG and antibody. Through the dual amplification of HRP-IgG-Fe(3)O(4) and anti-IAA-AuNPs, the trapping capacity of the immunosensor for IAA was significantly enhanced based on the promotion of the immunoreaction between antibody and antigen, which resulted in a large decrease of the electrochemical response of the redox probe, Fe(CN)(6)(3-), and an increase in sensitivity. The developed electrochemical immunosensor exhibited a wide linear range from 0.02 to 500 ng mL(-1) with a low detection limit of 0.018 ng mL(-1) (S/N = 3). Moreover, the proposed immunosensor showed acceptable selectivity, reproducibility, accuracy and stability. The IAA extracted from various seeds was successfully detected using the developed immunosensor. This assay method might provide an alternative strategy for the detection of various phytohormones.

Electrochemical behavior of phenacetin on CdSe microspheres modified glassy carbon electrode and its simultaneous determination with paracetamol and 4-aminophenol

A selective and sensitive electrochemistry method was developed for the determination of phenacetin on CdSe microspheres modified glassy carbon electrode (GCE). The electrode exhibited an effectively catalytic response to the oxidation of phenacetin, which was testified by the increased oxidation peak current and the decreased oxidation peak potential compared with the bare GCE. The scan rate investigation demonstrated that the electrochemical oxidation was an adsorption-controlled process in the range from 20 to 500 mV s−1. Under optimal determination conditions, the oxidation peak current of phenacetin was proportional to its concentration in the range of 0.5 to 800 μM. The limit of detection was estimated to be 0.1 μM (S/N = 3). The developed method showed good reproducibility, acceptable stability and excellent anti-interference performance. The fabricated electrode was successfully used to determine phenacetin in pharmaceutical formulation samples.

DNA-based hybridization chain reaction amplification for assaying the effect of environmental phenolic hormone on DNA methyltransferase activity.

In this work, a novel electrochemical protocol with signal amplification for determination of DNA methylation and methyltransferase activity using DNA-based hybridization chain reaction (HCR) was proposed. After the gold electrode was modified with dsDNA, it was treated with M.SssI MTase, HpaII endonuclease, respectively. And then the HCR was initiated by the target DNA and two hairpin helper DNAs, which lead to the formation of extended dsDNA polymers on the electrode surface. The signal was amplified by the labeled biotin on the hairpin probes. As a result, the streptavidin-alkaline phosphatase (S-ALP) conjugated on the electrode surface through the specific interaction between biotin and S-ALP. ALP could convert 1-naphthyl phosphate into 1-naphthol and the latter could be electrochemically oxidized, which was used to monitor the methylation event and MTase activity. The HCR assay presents good electrochemical responses for the determination of M.SssI MTase at a concentration as low as 0.0067 uni tmL(-1). Moreover, the effects of anti-cancer drug and environmental phenolic hormone on M.SssI MTase activity were also investigated. The results indicated that 5-fluorouracil and daunorubicin hydrochloride could inhibit the activity, and the opposite results were obtained with bisphenol A and nonylphenol. Therefore, this method can not only provide a platform to screen the inhibitors of DNA MTase and develop new anticancer drugs, but also offer a novel technique to investigate the possible carcinogenesis mechanism.

Electrochemical behavior of antipyrine at a Bi2S3 modified glassy carbon electrode and its determination in pharmaceutical formulations

A simple electrochemical method based on a Bi2S3 modified glassy carbon electrode (GCE) was developed to determine antipyrine using cyclic voltammetry and differential pulse voltammetry. Antipyrine shows a well-defined oxidation peak at the fabricated electrode in phosphate buffer solution and the oxidation peak current is much higher than that at the bare GCE, indicating that Bi2S3 can effectively improve the oxidation of antipyrine. Several effect factors on antipyrine determination were optimized, such as Bi2S3 amount, solution pH, scan rate and accumulation time. Under the optimal conditions, the oxidation peak current of antipyrine was proportional to its concentration in the range of 2.0 to 100 μM and 100 to 800 μM with a correlation coefficient of 0.9974 and 0.9956, respectively. The limit of detection was estimated to be 0.7 μM (S/N = 3). The developed method showed good reproducibility and excellent anti-interference performance. The fabricated electrode was successfully used to determine antipyrine in pharmaceutical formulations with recovery from 96% to 103.5%.