The authors describe the fabrication of an electrochemical immunosensor for the determination of the activity of protein kinase A (PKA). The method involves (a) electrochemical deposition of gold nanoparticles (AuNPs) on a glassy carbon electrode, (b) PKA-induced catalytic phosphorylation of serine, and (c) the use of phosphoserine antibody and horseradish peroxidase conjugated to IgG on gold nanoparticles (HRP-IgG-AuNPs). The use of AuNPs and HRP-IgG-AuNPs results in large amplification so that the method, at a typical working potential as low as 0.08 V (vs. SCE), has a linear range that extends from 0.1 to 50 activity units per mL, and the detection limit is 0.026 units per mL (at an S/N ratio of 3). The assay is selective (not the least due to a rather low working potential) and well reproducible. It may also be applied to screening for PKA inhibitors and to quantify the PKA activity in human cell lysates.

It is extremely important for quantifying trace microRNAs in the biomedical applications. In this study, an ultrasensitive, rapid and efficient label-free fluorescence method was proposed and applied for detecting microRNA-21 in serum of gastric cancer patients based on DNA hybridization chain reaction (HCR). DNA H1 and DNA H2 were designed and used as hairpin probes, the HCR was proceeded in the presence of target microRNAs. Amounts of SYBR Green І dyes were used as signal molecules to intercalate long DNA concatemers from HCR, which guaranteed the model of label-free fluorescence and strong fluorescence density. The detection method showed a wide linear region from 1 fM to 105 fM, and the limit of detection was 0.2554 fM (at S/N = 3) for microRNAs. The results showed that this method had an excellent specificity and reproducibility. Furthermore, the label-free fluorescence strategy exhibited a sensitive response to microRNA-21 in real serum samples of gastric cancer patients and the results obtained were in accordance with reference method (R2 = 0.994). Overall, the proposed strategy could be satisfactory for rapid, ultrasensitive and efficient detection of microRNA-21, and held great potentials in clinic diagnosis of gastric cancer.Download high-res image (237KB)Download full-size image

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.

•A photoelectrochemical strategy is developed for methylated RNA detection.•The anti-m6A antibody is utilized to improve detection specificity.•Graphite-like C3N4/CdS quantum dots heterojunction was used as photoactive materials.•The photoelectrochemical immunosensor was validated for detecting m6A content in the blood serum of breast cancer patients.N6-methyladenosine (m6A) is an enigmatic and abundant internal modification in eukaryotic messenger RNA (mRNA), which could affect various aspects of RNA metabolism and mRNA translation. Herein, a novel photoelectrochemical (PEC) immunosensor was constructed for m6A detection based on the inhibition of Cu2+ to the photoactivity of g-C3N4/CdS quantum dots (g-C3N4/CdS) heterojunction, where g-C3N4/CdS heterojunction was used as photoactive material, anti-m6A antibody as recognition unit for m6A-containing RNA, Phos-tag-biotin as link unit and avidin functionalized CuO as PEC signal indicator. When CuO was captured on electrode through biotin-avidin affinity reaction and then treated with HCl, Cu2+ could be released and CuxS would be formed based on the selective interaction between CdS and Cu2+, leading the photocurrent obviously decreased. Under the optimal detection conditions, the PEC biosensor displayed a linear range of 0.01–10 nM and a low detection limit of 3.53 pM for methylated RNA determination. Furthermore, the developed method could also be used to detect the expression level of m6A methylated RNA in serum samples of breast cancer patient before and after operative treatment. The proposed assay strategy has a great potential for detecting the expression methylation level of RNA in real sample.Download high-res image (185KB)Download full-size image

•A novel photoelectrochemical biosensor is fabricated for kinase activity assay.•Phosphorylated g-C3N4 is used as the photoelectric conversion material with visible-light activity.•The high affinity between Zr4+ and the phosphate group causes the assembly of P-g-C3N4 on the substrate peptide.•The developed method can be applied to detect kinase in real samples.Protein kinases are general and significant regulators in the cell signaling pathway, and it is still greatly desired to achieve simple and quick kinase detection. Herein, we develop a simple and sensitive photoelectrochemical strategy for the detection of protein kinase activity based on the bond between phosphorylated peptide and phosphorylated graphite-like carbon nitride (P-g-C3N4) conjugates triggered by Zr4+ ion coordination. Under optimal conditions, the increased photocurrent is proportional to the protein kinase A (PKA) concentration ranging from 0.05 to 50 U/mL with a detection limit of 0.077 U/mL. Moreover, this photoelectrochemical assay can be also applied to quantitative analysis of kinase inhibition. The results indicated that the IC50 value (inhibitor concentration producing 50% inhibitor) for ellagic acid was 9.1 μM. Moreover, the developed method is further applied to detect PKA activity in real samples, which contains serum from healthy person and gastric cancer patients and breast tissue from healthy person and breast cancer patients. Therefore, the established protocol provides a new and simple tool for assay of kinase activity and its inhibitors with low cost and high sensitivity.

•A novel photoelectrochemical biosensor was developed for detection of 5-hydroxymethylcytosine.•The biosensor was based on in situ electron donor producing strategy.•The biosensor exhibited high sensitivity and excellent selectivity for 5-hydroxymethylcytosine.•The biosensor uses all wavelengths of light irradiation to increase the utilization rate of light.In this paper, a novel signal-on photoelectrochemical (PEC) biosensor was papered for detecting 5-hydroxymethylcytosine (5-hmC) based on in situ electron donor producing strategy and all wavelengths of light irradiation. Firstly, the amino modified double DNA strand containing 5-hmC was captured on the carboxyl functionalized magnetic bead surface via amide linkage. After treatment with cysteamine in the presence of M.HhaI, the dsDNA with 5-hmC could transformed to amino-derivatized DNA. Subsequently, NHS-biotin could be labeled on the amino-derivative dsDNA and further reacted with avidin conjugated alkaline phosphatase (avidin-ALP). Under the catalysis effect of ALP toward ascorbic acid 2-phosphatetrisodium salt (AAP), ascorbic acid (AA) was in situ produced as electron donor. After magnetic separation, the biosensor can achieved all wavelengths of light irritation and a strong PEC response was obtained using a CdS/ITO as working electrode. The prepared biosensor showed high detection sensitivity with low detection limit of 0.167 nM for 5-hmC.

•A simple electrochemical strategy was developed for detecting polynucleotide kinase activity.•The detection is based on phosphorylation triggered λ exonuclease and exonuclease I cleavage.•The proposed method showed wide linear range and low detection limit.•This method can be applied to screen PNK inhibitors.A simple and sensitive electrochemical strategy was developed for detecting T4 polynucleotide kinase (PNK) activity and screening inhibitors based on phosphorylation reaction triggered λ exonuclease and exonuclease I cleavage. Firstly, a double strand DNA was designed with 5′-SH modification of probe DNA and 5′-OH modification of target DNA. After assembly on the electrode surface, the double strand DNA can block the diffusion of the electrochemical redox probe of Fe(CN)63−, and lead to a weak electrochemical signal. However, the assembled target DNA can be phosphorylated at its 5′-OH terminal, which was catalyzed by PNK in the presence of ATP as phosphate group donor. Then, the phosphorylated target DNA can be digested by λ exonuclease and the probe DNA was remained on the electrode surface, which can be further digested by exonuclease I. After the above two kinds of digestion reactions, the interface transfer resistance of the electrode was decreased greatly and the electrochemical signal was increased significantly. The electrochemical signal intensity was proportional to the activity of PNK. The proposed method showed wide linear range of 0.005–5 unit/mL and low detection limit of 0.0018 unit/mL (S/N = 3). The inhibition activity of (NH4)2SO4, ADP, Na2HPO4 and EDTA on PNK was also verified using this method.

Nuclease S1 can catalyze the nonspecific endo- and exonucleolytic cleavage of single-stranded DNA and RNA to yield nucleoside 5′-phosphates and 5′-phosphooligonucleotides. However, it cannot hydrolyze double-stranded DNA, double-stranded RNA, or DNA-RNA hybrid. Inspired by this specific property, a simple electrochemical method was developed for microRNA detection based on hybridization protection against nuclease S1 digestion. In the absence of hybridization process, the assembled probe DNA on the electrode surface can be easily digested by nuclease S1 and a strong electrochemical signal can be generated due to the decreased repulsive force towards the redox probe. However, after hybridization with target microRNA, the digestion activity of nuclease S1 is inhibited, which can lead to a weak electrochemical signal. Based on the change of the electrochemical signal, the detection of target microRNA-319a can be achieved. Under optimal experiment conditions, the electrochemical signal was proportional to microRNA-319a concentration from 1000 to 5 pM and the detection limit was 1.8 pM (S/N = 3). The developed method also showed high detection selectivity and reproducibility. Furthermore, the proposed method was successfully applied to assay the expression level of microRNA-319a in the leaves of rice seedlings after being incubated with different concentrations of 6-benzylaminopurine.

•A sensitive electrochemical biosensor was fabricated for protein kinase A (PKA) detection.•The biosensor was based on biotinylated phos-tag and biotinylated β-galactosidase.•Multiple signal amplification technique was employed.•The biosensor can be applied to detect PKA in human cells.A sensitive and selective electrochemical biosensor was fabricated for protein kinase A (PKA) activity assay. Multiple signal amplification techniques were employed including the nanocomposite of gold nanoparticles and carbon nanospheres (Au@C), the biocomposite of SiO2 and streptavidin (SiO2-SA), the composite of AuNPs and biotinylated β-galactosidase (AuNPs-B-Gal) and in situ enzymatic generation of electrochemical activity molecule of p-aminophenol. After peptides were assembled on Au@C modified electrode surface, they were phosphorylated by PKA in the presence of ATP. Then, biotinylated Phos-tag was modified on electrode surface through the specific interaction between Phos-tag and phosphate group. Finally, SiO2-SA and AuNPs-B-Gal were captured through the specific interaction between biotin and streptavidin. Because the electrochemical response of p-aminophenol was directly related to PKA concentration, an innovative electrochemical assay could be realized for PKA detection. The detection limit was 0.014 unit/mL. The developed method showed high detection sensitivity and selectivity. In addition, the fabricated biosensor can be also applied to detect PKA in human normal gastricepithelial cell line and human gastric carcinoma cell line with satisfactory results.

•An electrochemical strategy is developed for detecting microRNA-21 in the blood serum of breast cancer patients.•The specificities of T7 Exonuclease and T4 RNA ligase 2 are utilized to improve detection specificity and sensitivity.•The cyclic enzymatic amplification method is used to improve the sensitivity of this biosensor.•The proposed method showed wide linear range and low detection limit.MicroRNA (miRNA) is riveting nowadays due to its close relevance to human malignancies. Exploiting a fast and convenient biosensor for sensitive and specific detecting miRNA is necessary and it has significant meaning for oncology. In this work, to understand the relationship between miRNA-21 and gastric cancer, we employ T4 RNA ligase 2 to initiate specific ligation reaction depending on the sequence of target RNA, and T7 exonuclease to catalyze the first stage cyclic enzymatic amplification method (CEAM) specifically, which also resting on the target RNA sequence, and the second stage CEAM to further amplify the response resulting from the initial target RNA. Our two-stage CEAM strategy can detect miRNA-21 as low as 0.36 fM with remarkable specificity, and most importantly, it can be used to inspect the expression level of miRNA-21 in blood serum of gastric cancer patients. This will offer new perspective for figuring out the pathways of miRNA-21 involving in cancer.

•A sensitive electrochemical biosensor was fabricated for microRNA-319a detection.•The strategy was based on poly(U) polymerase-mediated strand extension reaction.•Alkaline phosphatase catalytic signal amplification technique was used.•The developed method can detect as low as 1.7 fM microRNA-319a.•The effect of phytohormones on microRNA-319a in rice seedlings was investigated.MicroRNAs play crucial role in post-transcriptional regulation for gene expression in animals, plants, and viruses. For the better understanding of microRNA and its functions, it is very important to develop effectively analytical method for microRNA detection. Herein, a novel electrochemical biosensor was fabricated for sensitive and selective detection of microRNA based on poly(U) polymerase mediated isothermal signal amplification, where poly(U) polymerase can catalyze the template independent addition of UMP from UTP to the 3’ end of RNA. Using this activity, the target microRNA can be successfully labeled with biotin conjugated UMPs at its 3′-end using biotin conjugated UTP (biotin-UTP) as donor. Then, the avidin conjugated alkaline phosphatase can be further captured to the 3′-end of the target microRNA based on the specific interaction between biotin and avidin. Finally, under the catalytic activity of alkaline phosphatase, the substrate of p-nitrophenyl phosphate disodium salt hexahydrate can be hydrolyzed to produce 4-nitrophenol. According to the relationship between the electrochemical signal of p-nitrophenol and the concentration of microRNA-319a, the content of microRNA-319a can be detected. This signal amplification method is simple and sensitive. The developed method can detect as low as 1.7 fM microRNA and produce precise and accurate linear dynamic range from 10 to 1000 fM. The fabricated biosensor was further applied to detect the expression level change of microRNA-319a in rice seedlings after incubation with five kinds of different phytohormones.

A novel electrochemical immunosensor was fabricated for the quantitative detection of 5-hydroxymethylcytosine (5-hmC) in genomic DNA based on anti-5-hmC antibody, biotin functionalized phos-tag and avidin functionalized alkaline phosphatase. It is demonstrated that the levels of 5-hmC are dramatically reduced in human breast cancer tissue compared with those in normal tissue.

•A nano mesoporous silica-based biosensor was fabricated.•DNA methylation was detected using this biosensor.•G-quadruplex was used as a lock for methylene blue releasing.•Zebularine was used for the investigation of inhibitor on the activity of M. SssI MTase.The abnormal level of DNA methyltransferase (MTase) may cause the aberrant DNA methylation, which has been found being associated with a growing number of human diseases, so it is necessary to create a sensitive and selective method to detect DNA MTase activity. In this paper, a new type of DNA functionalized nano mesoporous silica (MSNs) was creatively introduced to the detection of DNA MTase activity with G-quadruplex as a lock for signal molecule to release. The method was carried out by designing a particular DNA which could fold into G-quadruplex and complement with probe DNA. Next, MSNs was prepared before blocking methylene blue (MB) by G-quadruplex. Probe DNA was then fixed on gold nanoparticles modified glass carbon electrode, and the material was able to be transferred to the surface of electrode by DNA hybridization. After methylation of DNA MTase and the cutting of restriction endonuclease, the electrode was transferred to phosphate buffer solution (pH 9.0) for the releasing of MB. The response of differential pulse voltammetry was obtained from the release of MB. Consequently, the difference of signals with or without methylation could prove the assay of M. SssI MTase activity. The results showed that the responses from MB increased linearly with the increasing of the M. SssI MTase concentrations from 0.28 to 50 U mL−1. The limit of detection was 0.28 U mL−1. In addition, Zebularine, a nucleoside analog of cytidine, was utilized for studying the inhibition activity of M. SssI MTase.

A sensitive and selective electrochemical biosensor was fabricated for polynucleotide kinase (PNK) activity assay and inhibitor screening based on phos-tag-biotin mediated double signal amplification, where streptavidin and alkaline phosphatase-conjugated biotin (biotin-ALP) were used as the signal amplification units. After the immobilization of probe DNA on the electrode surface, it was hybridized with complementary DNA containing the 5′-OH terminal, the OH can be phosphorylated under the catalytic effect of PNK in the presence of ATP. The generated phosphate group at the 5′-terminal can be further recognized by phos-tag-biotin, where phos-tag-biotin was not only used as the specific recognition reagent for the phosphate group, but also the capture reagent for streptavidin to induce the further assembly of biotin-ALP. Under the catalytic effect of ALP, the substrate of p-nitrophenyl phosphate disodium salt hexahydrate (PNPP) can be hydrolyzed to produce p-nitrophenol (PNP). According to the relationship between the electrochemical response of PNP and the logarithmic value of PNK concentration, a linear range of 0.01–5 units per mL and a low detection limit of 0.0027 unit per mL were achieved. The developed method showed high selectivity. The inhibition effect of (NH4)2SO4 and Na2HPO4 was also evaluated and the IC50 values were calculated.

•A novel signal-on photoelectrochemical immunosensor was fabricated.•M. SssI methyltransferase activity and inhibitor was detected using this immunosensor.•Anti-5-methylcytosine antibody was used to recognize methylated DNA.•In situ enzymatic ascorbic acid production as electron donor.•Immunogold labeled streptavidin amplification units were adopted to obtain high sensitivity.In this work, a novel signal-on photoelectrochemical (PEC) immunosensor was fabricated for M.SssI methyltransfease (MTase) activity analysis and inhibitor screening based on an in situ electron donor producing strategy, where the anti-5-methylcytosine antibody was selected as DNA CpG methylation recognition unit, gold nanoparticle labeled streptavidin (SA-AuNPs) as signal amplification unit and alkaline phosphatase conjugated biotin (ALP-Biotin) as enzymatic unit. In the presence of M.SssI MTase, hairpin DNA1 containing the palindromic sequences of 5′-CCGG-3′ in its stem was methylated. After hybridization with biotin-conjugated DNA2, the stem-loop structure of the hairpin DNA1 was unfolded and the duplex strand DNA (dsDNA) was formed. Then, the dsDNA was captured on the surface of anti-5-methylcytosine antibody modified electrode through the specific immuno-reaction. Afterwards, SA-AuNPs and ALP-Biotin was further captured on the electrode surface through the specific reaction between biotin and streptavidin. Under the catalysis effect of ALP towards ascorbic acid 2-phosphate trisodium salt (AAP), ascorbic acid (AA) was in situ produced as electron donor and a strong PEC response was obtained. The fabricated biosensor showed high detection sensitivity with low detection limit of 0.33 unit/mL for M.SssI MTase. Furthermore, the inhibition research suggested that RG108 could inhibit the M.SssI MTase activity with the IC50 value of 152.54 nM.

A selective and sensitive photoelectrochemical assay was proposed for microRNA detection based on the isothermal cycle hybridization amplification of microRNA-21 that originated from the specific cleavage activity of duplex-specific nuclease (DSN) toward a DNA probe in DNA-RNA double helix and in situ enzymatic production of electron donor of ascorbic acid. After the biotin-functionalized DNA probe hybridized with complementary target microRNA-21, the hybridized DNA probe could be cleaved by DSN and microRNA-21 was released back to the incubation solution. The released microRNA could be further hybridized with the remaining single-strand DNA probe; thus, the isothermal cycle hybridization amplification would be formed. As a result, little avidin-alkaline phosphatase conjugate could be captured due to the release of biotin originating from the DSN cleavage. The distinct photocurrent change between a control biosensor and the DSN cleavage biosensor achieved label-free microRNA-21 detection with the linear range from 1 to 500 fM. The fabricated biosensor showed high detection selectivity even for one-base mismatched sequence. The attempt was carried out to directly assay the expression level change of microRNA-21 in total RNA extracted from chicken fibroblast cells infected with subgroup J avian leukosis virus.

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.

•We developed a simple, sensitive, and label-free method for microRNAs biosensing.•This method was based on mimic enzyme catalysis signal amplification.•Carboxylic graphene–hemin hybrid nanosheets was synthesized and used as minic enzyme.•The biosensor showed high sensitivity with the detection limit of 0.17 pM.•Abscisic acid influences microRNA-159a expression in Arabidopsis thaliana seedlings.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.

•Signal "on" and visible light photoelectrochemical biosensor was fabricated.•MicroRNA was detected using this biosensor.•Bi2S3 showed high photoactivity and generated strong photocurrent.•In situ generation of ascorbic acid catalytic by enzyme was used as electron donor.•The expression of microRNA-21 in chicken cells infected with ALVs decreased.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.

•A visible light-activated photoelectrochemical biosensor based on BiOI was fabricated.•The biosensor was used to detect DNA methyltransferase activity.•Methyl binding domain (MBD1) protein was used to recognize methylated DNA.•This method showed high detection sensitivity with detection limit of 0.035 unit/mL.•The fabricated biosensor could be used to screen inhibitors.DNA methylation has important roles in gene regulation and relates to some diseases, especially cancers. Because DNA methylation is catalyzed by DNA methyltransferases (MTase), it is important to detect the activity of DNA MTase. In this work, we developed a novel visible light-activated photoelectrochemical (PEC) biosensor for DNA MTase activity assay, whereby bismuth oxyiodide (BiOI) nanoflake was synthesized as photoactive electrode material, M. SssI MTase as methylation reagent and methyl binding domain protein (MBD1 protein) as methylation recognition element. After cytosine methylation event occurred at the site of 5′-CG-3′, it could be probed by MBD1 protein and this protein could be combined tightly with methylated cytosine, which would lead to a decreased photocurrent due to the hindrance towards electron donor transferring to electrode surface by huge-volume protein. The decreased photocurrent was proportional to M. SssI MTase concentration from 0.1 to 50 unit/mL with the detection limit of 0.035 unit/mL (S/N=3). This detection limit was lower than that in some previous reports. This PEC biosensor showed high selectivity and good reproducibility for M. SssI MTase assay. Moreover, this method was successfully applied also to screen DNA MTase inhibitors, indicating that this PEC biosensor could be an alternative platform in anti-cancer pharmaceuticals discovery.

•A novel MPA-CdS/RGO nanocomposite was synthesized via a facile solverthermal method.•The MPA-CdS/RGO nanocomposite exhibited enhanced photocurrent.•A photoelectrochemical immunosensor was fabricated based on the MPA-CdS/RGO nanocomposites.•The strategy could be used for rapid detection of IAA with high sensitivity and specificity.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.

•DNA demethylase activity was assayed by electrochemical method.•Double enzyme system of DNA demethylase and BstUI endonuclease was applied.•The detection is based on alkaline phosphatase catalytic signal amplification.•This method showed high detection selectivity and good sensitivity.A novel electrochemical method is developed for detection of DNA demethylation and assay of DNA demethylase activity. This method is constructed by hybridizing the probe with biotin tagged hemi-methylated complementary DNA and further capturing streptavidin tagged alkaline phosphatase (SA-ALP) to catalyze the hydrolysis reaction of p-nitrophenyl phosphate. The hydrolysate of p-nitrophenol (PNP) is then used as electrochemical probe for detecting DNA demethylation and assaying the activity of DNA demethylase. Demethylation of target DNA initiates a degradation reaction of the double-stranded DNA (dsDNA) by restriction endonuclease of BstUI. It makes the failed immobilization of ALP, resulting in a decreased electrochemical oxidation signal of PNP. Through the change of this electrochemical signal, the DNA demethylation is identified and the activity of DNA demethylase is analyzed with low detection limit of 1.3 ng mL−1. This method shows the advantages of simple operation, cheap and miniaturized instrument, high selectivity. Thus, it provides a useful platform for detecting DNA demethylation, analyzing demethylase activity and screening inhibited drug.

•We presented an electrochemical method for assay of DNA MTase activity.•It was based on DNA methylation-sensitive cleavage method.•It was used DNA-Au bio bar code and HRP to amplify signals.•Four DNA MTase inhibitors were investigated to study on the assay.A sensitive electrochemical method for detection of DNA methylation and assay of DNA methyltransferase (Dam) was investigated based on methylation-sensitive restriction endonuclease, DNA-Au bio bar code and enzymatic signal amplification. After the DNA probe was hybridized with complementary DNA sequence, and the obtained double-strand DNA (dsDNA) was further methylated by Dam MTase, it could be successively digested by Dpn I at the methylated sites. As a result, the bio bar code and streptavidin-horseradish peroxidase (S-HRP) could not be conjugated on the electrode surface through the specific interaction between biotin and S-HRP, where biotin was labeled at the DNA-Au bio bar code. Thus, only a low electrochemical reduction signal was obtained. However, the hybridized probe could hybrid with DNA-Au bio bar code, and then S-HRP could be conjugated on the electrode surface in the absence of Dam MTase and Dpn I, so a larger reduction current of enzymatic product was obtained. The change of the reduction current of enzymatic product was used to detect DNA methylation and Dam MTase activity. The detection limit of Dam MTase was 0.02 unit/mL (S/N = 3). Moreover, the inhibitions of mitomycin C, paclitaxel, chlorogenic acid and epicatechin were also investigated. Therefore, this method provided a sensitive platform for monitoring DNA methylation and DNA MTase activity.

A label-free electrochemical immunosensor for sensitive detection of indole-3-acetic acid (IAA) was developed using gold nanoparticles (AuNPs) functionalized with horseradish peroxidase labeled immunoglobulins G (AuNPs-HRP-IgG) as signal amplification probe and rat monoclonal antibody against IAA as capture probe. AuNPs-HRP-IgG could be immobilized and through the interaction between 4-aminophenylboronic acid and a single N-glycosylation site, which exist in the CH2 domain of IgG. The electrochemical immunosensor was characterized by differential pulse voltammetry (DPV) in 0.1 M PBS (pH 7.4) containing Fe(CN)63−/4− as redox probe. The decreased oxidation peak current of Fe(CN)63−/4− was used to monitor the antibody–antigen interaction. The electrochemical immunosensor exhibited a linear range from 1 × 10−9 to 5 × 10−6 M with a limit of detection of 5.5 × 10−10 M (S/N = 3). The proposed electrochemical immunosensor could well discriminate IAA from other phytohormones, such as gibberellin, abscisic acid and salicylic acid. In addition, IAA extracted from leaf of mung bean sprouts was detected using the developed immunosensor for evaluating its applicability, and the recovery was from 93.5% to 106.75%.Graphical abstractHighlights► An electrochemical immunosensor was fabricated for indole-3-acetic acid detection. ► The proposed method showed high sensitivity and selectivity for indole-3-acetic acid detection. ► AuNPs-HRP-IgG can improve immobilization amount of anti-IAA antibody. ► IAA extracted from leaf of mung bean sprouts was detected using this method.

A simple and novel microRNA (miRNA) biosensor was developed using DNA-Au bio bar code (DNA-Au) and G-quadruplex-based DNAenzyme. DNA-Au increased the amount of miRNA-21 participating in hybridization. Hemin/G-quadruplex DNAenzyme significantly improved the catalysis of H2O2 by oxidation of hydroquinone, resulting in an obvious reduction current of benzoquinone for miRNA-21 indirect detection. Under optimum conditions, the linear relationship between miRNA-21 concentration and reduction response was obtained with the detection limit of 0.006 pM, which showed a good sensitivity. Besides, selectivity of the biosensor was investigated by detecting the base mismatched miRNAs. This proposed method was further applied to detect miRNA-21 extracted from human hepatocarcinoma BEL-7402 cells and human mastocarcinoma MCF-7 cells. The influence of bisphenol A (BPA) on the expression of miRNA-21 in cells was also investigated. The biosensor performs well in practical applications, which suggests it may provide a new platform for gene diagnosis.

In this paper, we developed a novel electrochemical method to quantify DNA methyltransferase (MTase) and analyze DNA MTase activity. After the double DNA helix structure was assembled on the surface of gold nanoparticle modified glassy carbon electrode, it was first methylated by M. SssI MTase and then digested by restriction endonuclease HpaII, which could not recognize the methylated CpG site. Successively, anti-5-methylcytosine antibody was specifically conjugated on the CpG methylation site and horseradish peroxidase labeled goat antimouse IgG (HRP-IgG) was conjugated on anti-5-methylcytosine antibody. In the detection buffer solution containing H2O2 and hydroquinone, HRP-IgG can catalyze hydroquinone oxidation by H2O2 to generate benzoquinone, resulting in a highly electrochemical reduction signal. Consequently, the activity of M. SssI MTase was assayed, and DNA methylation was detected using the signal change with and without methylation. Furthermore, the inhibition investigation demonstrated that, in the presence of 160 μM S-adenosyl-l-methionine as methyl donor, 5-aza-2′-deoxycytidine, procaine, epicatechin, and caffeic acid could inhibit the M. SssI MTase activity with the IC50 values of 45.77, 410.3, 129.03, and 124.2 μM, respectively. Therefore, this study may provide a sensitive platform for screening DNA MTase inhibitors.

A sensitive and selective electrochemical method for microRNA-21 (miRNA-21) detection was developed. This detection tactic was based on graphene, gold nanoparticles (AuNPs), locked nucleic acid (LNA) incorporated hairpin DNA switching and enzymatic signal amplification. Graphene and AuNPs can improve the electrode conductivity and increase the electrode active surface. The LNA incorporated hairpin DNA probe was modified with biotin at its 5′-end and –SH at its 3′-end. After the probe hybridized with target miRNA-21, its loop-and-stem structure was unfolded to force the biotin away from the electrode surface. Through the specific interaction between biotin and streptavidin-horseradish peroxidase (streptavidin-HRP), miRNA-21 can be quantitatively realized by electrochemical detection of the enzymatic product of benzoquinone in the presence of hydrogen peroxide and hydroquinone. The determination conditions, such as probe concentration, probe immobilization time, hybridization time and applied potential, were optimized. This assay allowed the detection of miRNA-21 in the range of 1–5000 pM with a detection limit of 0.4 pM (S/N = 3). Based on the locked nucleic acid incorporated probe with hairpin structure, the sensor can well discriminated complementary and base mismatched sequences. Successful attempts were made in miRNA-21 expression analysis of human HeLa cells and normal human hepatic L02 cells.

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%.

A novel electrochemical immunosensor was fabricated for the quantitative detection of 5-hydroxymethylcytosine (5-hmC) in genomic DNA based on anti-5-hmC antibody, biotin functionalized phos-tag and avidin functionalized alkaline phosphatase. It is demonstrated that the levels of 5-hmC are dramatically reduced in human breast cancer tissue compared with those in normal tissue.

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%.

A sensitive and selective electrochemical biosensor was fabricated for polynucleotide kinase (PNK) activity assay and inhibitor screening based on phos-tag-biotin mediated double signal amplification, where streptavidin and alkaline phosphatase-conjugated biotin (biotin-ALP) were used as the signal amplification units. After the immobilization of probe DNA on the electrode surface, it was hybridized with complementary DNA containing the 5′-OH terminal, the OH can be phosphorylated under the catalytic effect of PNK in the presence of ATP. The generated phosphate group at the 5′-terminal can be further recognized by phos-tag-biotin, where phos-tag-biotin was not only used as the specific recognition reagent for the phosphate group, but also the capture reagent for streptavidin to induce the further assembly of biotin-ALP. Under the catalytic effect of ALP, the substrate of p-nitrophenyl phosphate disodium salt hexahydrate (PNPP) can be hydrolyzed to produce p-nitrophenol (PNP). According to the relationship between the electrochemical response of PNP and the logarithmic value of PNK concentration, a linear range of 0.01–5 units per mL and a low detection limit of 0.0027 unit per mL were achieved. The developed method showed high selectivity. The inhibition effect of (NH4)2SO4 and Na2HPO4 was also evaluated and the IC50 values were calculated.