Huanshun Yin

Electrochemical determination of microRNA-21 based on graphene, LNA integrated molecular beacon, AuNPs and biotin multifunctional bio bar codes and enzymatic assay system

MicroRNAs (miRNAs), a kind of small, endogenous, noncoding RNAs (∼22 nucleotides), might play a crucial role in early cancer diagnose due to its abnormal expression in many solid tumors. As a result, label-free and PCR-amplification-free assay for miRNAs is of great significance. In this work, a highly sensitive biosensor for sequence specific miRNA-21 detection without miRNA-21 labeling and enrichment was constructed based on the substrate electrode of dendritic gold nanostructure (DenAu) and graphene nanosheets modified glassy carbon electrode. Sulfydryl functionalized locked nucleic acid (LNA) integrated hairpin molecule beacon (MB) probe was used as miRNA-21 capture probe. After hybridized with miRNA-21 and reported DNA loading in gold nanoparticles (AuNPs) and biotin multi-functionalized bio bar codes, streptavidin-HRP was brought to the electrode through the specific interaction with biotin to catalyze the chemical oxidation of hydroquinone by H(2)O(2) to form benzoquinone. The electrochemical reduction signal of benzoquinone was utilized to monitor the miRNA-21 hybridization event. The effect of experimental variables on the amperometric response was investigated and optimized. Based on the specific confirmation of probe and signal amplification, the biosensor showed excellent selectivity and high sensitivity with low detection limit of 0.06 pM. Successful attempts are made in miRNA-21 expression analysis of human hepatocarcinoma BEL-7402 cells and normal human hepatic L02 cells.

Amperometric biosensor based on tyrosinase immobilized onto multiwalled carbon nanotubes-cobalt phthalocyanine-silk fibroin film and its application to determine bisphenol A.

An amperometric bisphenol A (BPA) biosensor was fabricated by immobilizing tyrosinase on multiwalled carbon nanotubes (MWNTs)-cobalt phthalocyanine (CoPc)-silk fibroin (SF) composite modified glassy carbon electrode (GCE). In MWNTs-CoPc-SF composite film, SF provided a biocompatible microenvironment for the tyrosinase to retain its bioactivity, MWNTs possessed excellent inherent conductivity to enhance the electron transfer rate and CoPc showed good electrocatalytic activity to electrooxidation of BPA. The cyclic voltammogram of BPA at this biosensor exhibited a well defined anodic peak at 0.625 V. Compared with bare GCE, the oxidation signal of BPA significantly increased; therefore, this oxidation signal was used to determine BPA. The effect factors were optimized and the electrochemical parameters were calculated. The possible oxidation mechanism was also discussed. Under optimum conditions, the oxidation current was proportional to BPA concentration in the range from 5.0 x 10(-8) to 3.0 x 10(-6) M with correlation coefficient of 0.9979 and detection limit of 3.0 x 10(-8) M (S/N=3). The proposed method was successfully applied to determine BPA in plastic products and the recovery was in the range from 95.36% to 104.39%.

Electrocatalytic oxidation behavior of guanosine at graphene, chitosan and Fe3O4 nanoparticles modified glassy carbon electrode and its determination

A graphene, chitosan and Fe(3)O(4) nanoparticles (nano-Fe(3)O(4)) modified glassy carbon electrode (graphene-chitosan/nano-Fe(3)O(4)/GCE) was fabricated. The modified electrode was characterized by scanning electron microscope and electrochemical impedance spectroscopy. The electrochemical oxidation behavior of guanosine was investigated in pH 7.0 phosphate buffer solution by cyclic voltammetry and differential pulse voltammetry. The experimental results indicated that the modified electrode exhibited an electrocatalytic and adsorptive activities towards the oxidation of guanosine. The transfer electron number (n), transfer proton number (m) and electrochemically effective surface area (A) were calculated. Under the optimized conditions, the oxidation peak current was proportional to guanosine concentration in the range of 2.0 x 10(-6) to 3.5 x 10(-4) mol L(-1) with the correlation coefficient of 0.9939 and the detection limit of 7.5 x 10(-7) mol L(-1) (S/N=3). Moreover, the modified electrode showed good ability to discriminate the electrochemical oxidation response of guanosine, guanine and adenosine. The proposed method was further applied to determine guanosine in spiked urine samples and traditional Chinese medicines with satisfactory results.

A nitrite biosensor based on the immobilization of Cytochrome c on multi-walled carbon nanotubes–PAMAM–chitosan nanocomposite modified glass carbon electrode

A novel nitrite biosensor was successfully prepared via immobilizing Cytochrome c (Cyt c) onto the multi-walled carbon nanotubes-poly(amidoamine) (PAMAM)-chitosan (MWNT-PAMAM-Chit) nanocomposite modified glass carbon electrode (GCE). Ultraviolet and visible (UV-vis) absorption spectrum, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to examine the native conformation and bioactivity of the immobilized Cyt c, and the electrochemical properties of the modified electrodes, respectively. The results indicate that the immobilized Cyt c retained its native characters, and the MWNT-PAMAM-Chit nanocomposite is a good platform for the immobilization of Cyt c as well as an excellent promoter for the electron transfer between Cyt c and electrode. The high reactive Cyt c pi-cation, which can oxidize NO(2)(-) into NO(3)(-) in the solution, is generated at higher potential (>0.7 V) based on the further oxidation of Cyt c. The nitrite biosensor showed a fast response to nitrite (about 5 s) in two concentration intervals, one was from 0.1 to 29 microM, and the other from 29 to 254 microM. The low detection limit of 0.01 microM was obtained.

Sensitivity and selectivity determination of BPA in real water samples using PAMAM dendrimer and CoTe quantum dots modified glassy carbon electrode

Bisphenol A (BPA) is an environmental pollutant to disrupt endocrine system or cause cancer, thus the detection of BPA is very important. Herein, an amperometric sensor was fabricated based on immobilized CoTe quantum dots (CoTe QDs) and PAMAM dendrimer (PAMAM) onto glassy carbon electrode (GCE) surface. The cyclic voltammogram of BPA on the sensor exhibited a well-defined anodic peak at 0.490V in 0.1M pH 8.0 PBS. The determination conditions were optimized and the kinetic parameters were calculated. The linear range was 1.3 x 10(-8) to 9.89 x 10(-6)M with the correlation coefficient of 0.9999. The limit of detection was estimated to be 1 x 10(-9)M. The current reached the steady-state current within about 5s. Furthermore, the fabricated sensor was successfully applied to determine BPA in real water samples.

Electrochemical behaviour of Sudan I at Fe3O4 nanoparticles modified glassy carbon electrode and its determination in food samples

In this work, a simple and sensitive electrochemical method was developed to determine Sudan I based on magnetic Fe3O4 nanoparticles modified glassy carbon electrode using cyclic voltammetry and differential pulse voltammetry. The sensor exhibited an obviously electrocatalytic activity towards the oxidation of Sudan I, which can be confirmed by the increased oxidation peak current and the decreased oxidation peak potential when compared with the bare GCE. The determination conditions, such as pH, modifier amount, accumulation time and accumulation potential, were optimised. And some kinetic parameters were calculated. Under the optimum experimental conditions, the oxidation current of Sudan I was proportional to its concentration from 0.01 to 1μM and 1 to 20μM. The detection limit was estimated to be 0.001μM (S/N=3). The developed method was successfully applied to determine Sudan I content in food samples with satisfactory results.

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.

Ultrasensitive photoelectrochemical immunoassay of indole-3-acetic acid based on the MPA modified CdS/RGO nanocomposites decorated ITO electrode

A novel ultrasensitive photoelectrochemical immunosensor was fabricated based on 3-mercaptopropionic acid stabilized CdS/reduced graphene oxide (MPA-CdS/RGO) nanocomposites for indole-3-acetic acid (IAA) detection. The MPA-CdS/RGO nanocomposites were synthesized by in situ solvothermal growth of triangulated pyramidal CdS nanoparticles on the RGO sheet. 3-Mercaptopropionic acid (MPA) was employed as the modifier and bridge to immobilize the antibody. The nanocomposites were characterized by X-ray diffraction, transmission electron microscopy, scanning electron microscopy and UV/vis spectra. The results showed that the MPA-CdS/RGO nanocomposites revealed enhanced photocurrent response due to excellent electron transport properties of RGO and the improved assembly of CdS nanoparticles onto RGO sheet with the introduction of MPA. Based on the dependence of the photocurrent decline on the concentration of IAA, the proposed photoelectrochemical immunosensor for IAA depicted a linear range from 0.1 to 1000 ng/mL with a lower detection limit (0.05 ng/mL). The high sensitivity, reproducibility and specificity of the method permitted the method suitable to be used in real samples.