Yunlei Zhou

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.

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.

One-Step, Ultrasensitive, and Electrochemical Assay of microRNAs Based on T7 Exonuclease Assisted Cyclic Enzymatic Amplification

Taking advantage of the special exodeoxyribonuclease activity of T7 exonuclease, a simple, sensitive, selective, and label-free microRNA biosensor based on the cyclic enzymatic amplification method (CEAM) has been proposed. First, thiol functionalized DNA probes were assembled onto a gold nanoparticles modified gold electrode surface through a Au-S bond, followed by hybridizing with target miRNA. Subsequently, DNA in RNA/DNA duplexes was digested by T7 exonuclease, which can release the microRNA molecules from the electrode surface and return into the buffer solution. Meanwhile, the released microRNA can further hybridize with the unhybridized DNA probes on the modified electrode surface. On the basis of it, an isothermal amplification cycle is realized. The T7 exonuclease-assisted CEAM achieved a low detection limit of 0.17 fM. Moreover, this assay presents excellent specificity with discriminating only a single-base mismatched microRNA sequence. Furthermore, this work can also be applied to detect avian leukemia based on the decreased expression level of microRNA-21.

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.

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.

A signal “on” photoelectrochemical biosensor for assay of protein kinase activity and its inhibitor based on graphite-like carbon nitride, Phos-tag and alkaline phosphatase

A highly sensitive and selective photoelectrochemical (PEC) biosensor is fabricated for the detection of protein kinase activity based on visible-light active graphite-like carbon nitride (g-C3N4) and the specific recognition utility of Phos-tag for protein kinase A (PKA)-induced phosphopeptides. For assembling the substrate peptides, g-C3N4 and gold nanoparticles (g-C3N4-AuNPs) complex is synthesized and characterized. When the immobilized peptides on g-C3N4-AuNPs modified ITO electrode are phosphorylated under PKA catalysis, they can be specifically identified and binded with biotin functionalized Phos-tag (Phos-tag-biotin) in the presence of Zn(2+). Then, through the specific interaction between biotin and avidin, avidin functionalized alkaline phosphatase (avidin-ALP) is further assembled to catalyze its substrate of l-ascorbic acid-2-phosphate trisodium salt (AAP) to produce electron donor of ascorbic acid (AA), resulting an increased photocurrent compared with the absence of phosphorylation event. Based on the specific identification effect of Phos-tag, the fabricated biosensor presents excellent selectivity for capturing the phosphorylated serine residues in the substrate peptides. With the good photoactivity of g-C3N4 and ALP-catalyzed signal amplification, the fabricated biosensor presents high sensitivity and low detection limit (0.015 unit/mL, S/N = 3) for PKA. The applicability of this PEC biosensor is further testified by the evaluation of PKA inhibition by HA-1077 with the IC50 value of 1.18μM. This new strategy is also successfully applied to detect the change of PKA activity in cancer cell lysate with and without drug stimulation. Therefore, the developed PEC method has great potential in screening of kinase inhibitors and highly sensitive detection of kinase activity.

Enhanced Photoelectrochemical Method for Sensitive Detection of Protein Kinase A Activity Using TiO2/g-C3N4, PAMAM Dendrimer, and Alkaline Phosphatase

A novel photoelectrochemical (PEC) assay is developed for sensitive detection of protein kinase A (PKA) activity based on PKA-catalyzed phosphorylation reaction in solution and signal amplification strategy triggered by PAMAM dendrimer and alkaline phosphatase (ALP). In this strategy, it is noteworthy at this point that PKA phosphorylation was achieved in solution instead of on the surface of the electrode, which has advantages of the good contact in reactants and simple experimental procedure. For immobilizing the phosphorylated peptide (P-peptide) on electrode surface, graphite-like carbon nitride (g-C3N4) and titanium dioxide (TiO2) complex is synthesized and characterized, which plays a significant role for TiO2 conjugating phosphate groups and g-C3N4 providing PEC signal. Subsequently, PAMAM dendrimer and ALP can be captured on P-peptide and TiO2/g-C3N4 modified ITO electrode via interaction between the -COOH groups on the surface of PAMAM dendrimer and the -NH2 groups of peptide and ALP, which can lead to the increase of ALP amount on the modified electrode surface assisted with the PAMAM dendrimer. As a result, the amount of ALP catalyzes of L-ascorbic acid 2-phosphate trisodium salt (AAP) to produce electron donor of ascorbic acid (AA), resulting in an increased photocurrent. The proposed detection assay displays high selectivity and low detection limit of 0.048 U/mL (S/N = 3) for PKA activity. This biosensor can also be applied for the evaluation of PKA inhibition and PKA activity assay in cell samples. Therefore, the fabricated PEC biosensor is potentionally well in PKA activity detection and inhibitor screening.