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5-Formylcytosine (5fC) is a rare base found in mammalian DNA, which is thought to be involved in the demethylation of DNA. As a stable epigenetic modification, 5fC participates in gene regulation and cell differentiation, and plays an important role in the growth and development of plants. However, the abundance of 5fC is only as low as 0.002-0.02% of cytosine. Therefore, to further understand the functions of 5fC, a rapid, highly sensitive, and efficient method is needed for detecting 5fC. Herein, a novel photoelectrochemical (PEC) biosensor was constructed for 5fC detection, where a MoS2/WS2 nanosheet heterojunction was employed as a photoactive material, amino-functionalized Fe3O4 and SMCC were used as a linker, 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole was adopted as 5fC recognition reagent, and black TiO2 (B-TiO2) was used as a signal amplification unit. Under the optimal experimental conditions, this PEC biosensor showed a wide linear range of 0.01-200 nM and a low detection limit of 2.7 pM (S/N = 3). Due to the specific covalent reaction between -NH2 and -CHO, the biosensor presented high detection sensitivity, even discriminating 5fC with 5-methylcytosine and 5-hydroxymethylcytosine. The biosensor was then applied to investigate the effect of heavy metal Cd2+ on 5fC content in the root, stem, and leaves of maize seedlings.

Herein, a novel photoelectrochemical (PEC) biosensor was developed for the ultrasensitive detection of dual microRNAs (miRNAs), with the detection being based on energy transfer (ET) between carbon quantum dots (CQDs) and gold nanoparticles (AuNPs). The PEC platform consisted of a CQDs@Mo2C nanotube modified ITO electrode. Two hairpin probes (H1 and H2) carrying the Au NPs were used “switch off” and “switch on” the PEC signal of the CQDs, with a close approach of the tagged AuNPs to the CQDs quenching the PEC signal. The introduction of different miRNAs (miRNA-159b and miRNA-166a) altered the interparticle distance between the AuNPs and CQDs, thereby affecting the intensity of the PEC response. This approach allowed the highly sensitive detection of both miRNA-159b and miRNA-166a. The linear range of the biosensor for miRNA-159b and miRNA-166a detection were 0.5–5000 fM, with low detection limits of 0.15 fM and 0.21 fM, respectively. To our knowledge, this is the first reported CQDs-based ET biosensor for the PEC detection of dual miRNAs. Results suggest that this approach offers a promising platform for the ultrasensitive detection of multiple miRNAs.

In this work, a novel and sensitive photoelectrochemical (PEC) strategy was designed for protein kinase A (PKA) detection, comprising carbon microsphere (CMS) modified ITO electrode, TiO2 as the phosphate group recognition material and graphite-carbon nitride (g-C3N4) as photoactive material. For the first time, gold nanoparticle decorated zeolitic imidazolate frameworks (Au-ZIF-8) was employed to fabricate biosensor for PKA activity assay with the function of substrate peptide immobilization and signal amplification. Firstly, substrate peptides were assembled on the Au-ZIF-8/CMS/ITO surface through the covalent bonding between the gold nanoparticles (AuNPs) and sulfydryl groups of the peptides. Then, in the presence of ATP, phosphorylation of the substrate peptide was achieved under PKA catalysis. Finally, TiO2-g-C3N4 composites were further modified on the electrode surface based on bonding between TiO2 and phosphate groups created via phosphorylation of the peptide (yielding TiO2-g-C3N4/P-peptide/Au-ZIF-8/CMS/ITO), which is different with our previous work by directly immobilizing g-C3N4 composite on electrode surface. The developed method showed a wide linear range from 0.05–50 U mL−1. The detection limit was 0.02 U mL−1 (S/N = 3). The constructed biosensor exhibited high detection specificity for PKA. In addition, the wide applicability of this biosensor was demonstrated by evaluating the inhibition ability of ellagic acid towards PKA.

Herein, a novel photoelectrochemical (PEC) biosensor was developed for the ultrasensitive detection of microRNA-396a based on a MoS2/g-C3N4/black TiO2 heterojunction as the photoactive material and gold nanoparticles carrying Histostar antibodies (Histostar@AuNPs) for signal amplification. Briefly, MoS2/g-C3N4/black TiO2 was deposited on an indium tin oxide (ITO) electrode surface, after which gold nanoparticles (AuNPs) and probe DNA were assembled on the modified electrode. Hybridization with miRNA-396a resulted in a rigid DNA: RNA hybrid being formed, which was recognized by the S9.6 antibody. The captured antibody can further conjugate with the secondary IgG antibodies of Histostar@AuNPs, thereby leading to the immobilization of horse radish peroxidase (HRP). In the presence of HRP, the oxidation of 4-chloro-1-naphthol (4-CN) by H2O2 was accelerated, producing the insoluble product benzo-4-chlorohexadienone on the electrode surface and causing a significant decrease in the photocurrent. The developed biosensor could detect miRNA-396a at concentrations from 0.5 fM to 5000 fM, with a detection limit of 0.13 fM. Further, the proposed method can also be used to investigate the effect of heavy metal ions on the expression level of miRNAs. Results suggest that the biosensor developed herein offers a promising platform for the ultrasensitive detection of miRNA.

A simple and selective photoelectrochemical method was reported for 5-hydroxymethylcytosine (5hmC) detection in genomic DNA based on DNA methyltransferase (MTase) catalytic covalent bonding of –CH2OH of 5hmC with thiol compounds, where WS2 nanomaterial was used as a photoactive material, polydopamine (PDA) was employed as a solid-phase electron donor, phos-tag-biotin was used as bridge to link 5hmC and streptavidin. PDA was attached to 4-mercaptophenulboronic acid (MPBA) via the covalent bonding between its vicinal diol and the boracic acid of MPBA. Then, under the catalytic effect of M. HhaI MTase, 5hmC deoxyribonucleotide was captured on electrode surface through the covalent bonding between –CH2OH and –SH. Afterwards, phos-tag-biotin, a kind of specific reagent for phosphate group, was further captured through the specific bonding between the phosphate group of 5hmC deoxyribonucleotide and phos-tag-biotin. Subsequently, based on the “bridge” role of phos-tag-biotin, streptavidin was further modified on electrode surface to further block the transfer of photogenerated-electron to electrode surface. The photocurrent shows linear relationship with the logarithm value of 5hmC concentration in the range from 0.01 to 100 nM, and the detection limit of 4.12 pM (3σ). The developed method presents high detection specificity, which can discriminate 5-methylcytosine and 5hmC. Moreover, the applicability of this photoelectrochemical method was evaluated by detecting 5hmC content change in maize seedlings leaves and chicken embryo fibroblast cell.

5-Hydroxymethylcytosine (5hmC) plays an important role in switching genes on and off in mammals, and it is implicated in both embryonic development and cancer progression. Herein, a novel photoelectrochemical (PEC) biosensor was developed for 5hmC detection based on WS2 nanosheets as the photoactive material and boronic acid functionalized carbon dots (B-CDs) for signal amplification unit. This biosensor can also be used for T4-β-glucosyltransferase (β-GT) activity assessment. Firstly, WS2 nanosheets and gold nanoparticles (AuNPs) were immobilized on an ITO electrode surface. Then probe DNA was immobilized on this electrode surface via Au-S bond. Afterwards, the complementary DNA containing 5hmC was then captured on the modified electrode surface by hybridization. Subsequently, β-GT transferred glucose from uridine diphosphoglucose to the hydroxyl groups of the 5hmC residues. After glycosylation, B-CDs could further be immobilized on the modified electrode surface resulting in a strong photocurrent. The PEC biosensor afforded high selectivity, excellent sensitivity and good reproducibility, with detection limits of 0.0034 nM and 0.028 unit/mL for 5hmC and β-GT, respectively. Results demonstrate that the photoelectrochemical strategy introduced here based on WS2 nanosheets and B-CDs offers a versatile platform for hydroxymethylated DNA detection, β-GT activity assessment and β-GT inhibitor screening.

A novel photoelectrochemical immunosensor has been constructed for the determination of methylated RNA. MoS2 nanosheets with large specific area were employed as photoactive material, gold nanoparticles were used as signal amplification unit and immobilization matrix of 4-mercaptophenylboronic acid, anti-m6A antibody was adopted as methylated RNA recognition reagent, and poly(U) polymerase–mediated RNA chain extension and Ru(NH3)63+ were used as assisted signal amplification unit. With the sensitization effect of Ru(NH3)63+, the photoactivity of WS2 nanosheets was improved greatly, which also improved the sensitivity. Using visible-light excitation and ascorbic acid as electron donor, the sensitive determination of methylated RNA was achieved by monitoring the photocurrent change with different concentrations of methylated RNA. This photoelectrochemical immunosensor has a wide linear relationship with methylated RNA concentration from 0.05 to 35 nM under optimal experimental conditions. The low detection limit of 14.5 pM was realized based on 3σ criterion. In addition to the good selectivity, this sensor also presents high reproducibility with a relative standard deviation of 1.4% for the photocurrent of seven electrodes. The applicability of the developed method was also investigated by detecting the level of methylated RNA in corn seedling leaves with and without sulfadiazine treatment.

A photoelectrochemical (PEC) biosensor was constructed for 5-formylcytosine (5fC) nucleotide detection based on Ag2S@WS2 photoactive material and FeVO4 catalytic signal quenching. After Ag2S@WS2 was modified onto the ITO substrate surface, 5fC recognition reagent of Au@4-amino3hydrazino5mercapto-1,2,4-triazol (Au@AHMT) was further modified through electrostatic adsorption. Afterwards, based on the specific chemical reaction between –NH2 and –CHO, 5fC can be selectively recognized and captured. Subsequently, the nanoenzyme of FeVO4 was recognized based on the specific reaction between the phosphate group of 5fC nucleotide and Fe3+. Under the catalysis of FeVO4, the 4-chloro-1-naphthol in the detection solution can be oxidized to generate a precipitate, which will be retained on the electrode surface to inhibit the PEC signal. The developed method presented a widely dynamic range from 0.1 to 400 nM. The detection limit was 0.062 nM (3σ). This method also showed good detection selectivity, reproducibility and stability. The applicability was verified by investigating 5fC content change in genomic DNA of rice tissues after incubated with heavy metals. Moreover, the inhibited influence of hydrochar towards heavy metals was also assessed.

Graphite-like carbon nitride (g-C3N4) nanosheet is a new type of photoactive semiconducting material with promising application in photoelectrochemical (PEC) biosensor. In the present work, a simple and selective PEC biosensor was fabricated using g-C3N4 nanosheets as photoactive material coupled with capture-release strategy. DNA aptamer labeled with ferrocene (DNA-Fc) was served as both recognition reagent of antibiotic chloramphenicol and electron donor of PEC biosensor. Based on the decreased affinity of aptamer to g-C3N4 nanosheets causing by the formation of aptamer-chloramphenicol complex, DNA-Fc could be released from g-C3N4 nanosheets modified ITO surface, resulting a decreased PEC response. The developed method showed a wide linear range from 1 pM to 100 nM and a low detection limit of 0.22 pM (3σ). The fabricated PEC biosensor also illustrated good detection specificity and can be applied to detect chloramphenicol residue in water samples with recoveries ranging from 94.5% to 107.3%.

Herein, a novel dual-signal amplified photoelectrochemical (PEC) biosensor was successfully developed for the highly selective detection of N6-methyladenosine (m6A) methylated RNA. The PEC biosensor comprised BiVO4-110-TiO2 heterojunction and gold nanoparticle decorated MoS2 (MoS2-AuNPs) as the photoactive materials, horseradish peroxidase conjugated biotin (HRP-Biotin) as the enzymatic unit, Ag+-mediated cytosine pairs (C-Ag+-C) as the signal amplification unit, and the anti-m6A antibody as the m6A methylated RNA recognition unit. Following immunoreaction between m6A and the anti-m6A antibody, the C-Ag+-C structure of the hairpin DNA unfolded, yielding the duplex strand DNA (dsDNA) and releasing Ag+ ions. Superoxide ions (O2-) generated by the action of HRP on H2O2 then served as an electron donor, resulting in the deposition of Ag on AuNPs surface and resulting in an increased photocurrent. Based on this change f the photocurrent, m6A could be accurately assayed using this dual-signal amplified PEC biosensor. The biosensor showed high selectivity and a very low detection limit of 1.665 pM for m6A, and was successfully applied to evaluate the content change of m6A in leaves of maize seedling and chicken fetal hepatocytes samples after treatment with chemical mutagen of ethylmethane sulfonate and hormone of insulin, respectively.

Zeatin is a kind of typical cytokinin, which plays important roles in regulating the proliferation and differentiation of plant cells, and widely distributed in various plant tissues. Herein, a simple and sensitive photoelectrochemical method was developed for zeatin detection using graphite-like carbon nitride (g-C3N4) as photoactive material, graphene quantum dots as photoactivity improvement reagent, gold nanoparticles as immobilization substrate for DNA probe, DNA biotin labeled aptamer as zeatin recognition and streptavidin capture element, and streptavidin as PEC response inhibitor factor. In the presence of zeatin, DNA aptamer can bind with zeatin to form conjugate, which make aptamer releasing from electrode surface and decrease the capture amount of streptavidin, causing the increase of the PEC response. Under optimal conditions, the biosensor showed wide linear range from 0.1 to 100 nM and low detection limit of 0.031 nM (3σ). The developed method also presented good selectivity, even discriminating zeatin analogues. In addition, the proposed detection strategy can be further applied to detect zeatin in complex biological matrix, revealing great potential in practical applicability. This work might provide an excellent platform for zeatin or other phytohormone detection using aptamer as recognition reagent.

MicroRNAs play a crucial role in regulating plant growth, such as involving in the pathways of phytohormons signaling, which function in broad range of important aspects in many stages of plant development and evolution. To figure out the components part of microRNA in phytohormone signaling network, a sensitive photoelectrochemical biosensor was developed to detect miRNA-319a specifically using CuO-CuWO 4 as photoactive material and in situ generation electron donor technique. Taking the advantage of rolling circle amplification, nicking endonuclease triggered exponential amplification of target molecule, specific interaction between phosphate group and phos-tag and between biotin and avidin, the developed strategy can greatly amplify the detection signal only using trace amount of target miRNA-319a. The wide linear range was obtained from 1 fM to 0.1 nM and the detection limit was estimated to be 0.47 fM (S/N = 3). The developed method also showed good detection specificity. The effect of five phytohormones on the expression level of miRNA-319a in rice leaves was also investigated. This will be a basal principle of investigating the signaling network of phytohormones.

As an important epigenetic modification, 5-hydroxymethylcytosine (5hmC) aroused wide concern about the distribution and the function. Due to the necessity of 5hmC detection, a novel photoelectrochemical (PEC) biosensor was established based on the in-situ generated heterojunction of Bi4NbO8Cl@Bi2S3, which was employed as the substrate material with excellent photoelectric property. The specific recognition of 5hmC relied on the covalent reaction between –CH2OH of 5hmC and –SH on the substrate electrode under the catalysis of M.HhaI methyltransferase. Afterwards, ZrO2 was used as signal amplification unit capturing by the specific reaction of Zr with the phosphate group of 5hmC. The experimental results demonstrated well specificity and sensitivity of this biosensor. Under optimal conditions, the linear relationship between the photocurrent and the logarithm value of 5hmC concentration was constructed with the range from 0.3 to 300 nM and the detection limit of 0.0779 nM (S/N = 3). The procedures of constructing this biosensor were compact and convenient, and this biosensor realized actual detection of 5hmC level in wheat sample. Significantly, this biosensor was applied to a preliminary study that the heavy metal Pb2+ and the perfluorooctanoic acid influence the expression of 5hmC in the genomic DNA of wheat seedling roots and leaves.

The endogenous epigenetic marker 5-formylcytosine (5 fC) is introduced by 5-methylcytosine (5 mC) oxidation under action of enzyme oxidation, and plays an important role in many life activities. Since the content of 5 fC in mammalian tissues and cells is very low, it is necessary to exploit a sensitive and specific detection method to further understand the function of 5 fC. In this work, a sensitively and selectively photoelectrochemical (PEC) biosensor was developed for 5-formyl-2′-deoxycytidine (5fdC) detection. CuBi2O4/ZnPc@WS2 was used as photoactive material, where the formed ternary heterojunction structure greatly enhanced the PEC response and increased the detection sensitivity. Positively charged polyethyleneimine (PEI) was employed as 5fdC recognition and capture unit, where the amine group on PEI specifically reacted with aldehyde group of 5fdC to form stable amide bond. 4-Carboxyphenylboronic acid (4-CPBA) was adopted as crosslinker for 5fdC and amino functionalized CuBi2O4 based on the covalent interaction between 1,3-diol bond on 5fdC and boric acid structure on 4-CPBA, and the covalent interaction between –COOH on 4-CPBA and –NH2 on amino functionalized CuBi2O4. On the basis of the positive synergistic effect of ZnPc and CuBi2O4 on improving the photoelectric performance of WS2, the separation of photo-generated electron-hole pairs in semiconductors were promoted, and the examination range was expanded from 0.1 to 500 nM, and the detection limit was 0.0483 nM (3σ). Based on the unique covalent reaction between –NH2 and –CHO, the PEC biosensor has excellent detection sensitivity, and can even separate 5fdC from 5-methylcytosine deoxyribonucleoside and 5-hydroxymethylcytosine deoxyribonucleoside. The effect of antibiotics and heavy metals on the 5fdC content in wheat tissue genome has also been further investigated using this sensor.

A simple and sensitive electrochemical aptasensor was constructed for zeatin detection, where MoS2 nanosheets were used as the immobilization matrix for gold nanoparticles (AuNPs), and AuNPs were employed as the immobilization matrix to probe DNA. After the aptamer DNA and assist DNA hybridized with probe DNA, Y-type DNA can be formed with two biotins at the terminals of aptamer DNA. Then, avidin modified alkaline phosphatase (Avidin-ALP) can be further modified on the electrode surface through the biotin and avidin interaction. Under the catalytic effect of ALP, p-nitrophenylphosphate disodium (PNPP) can be hydrolyzed to produce p-nitrophenol (PNP). However, in the presence of zeatin, the formed Y-type DNA can be destroyed due to the formation of the zeatin–aptamer conjugate, which further reduces the amount of PNP and leads to the decrease of the oxidation signal of PNP. Under the optimum conditions, the change of the oxidation peak current of PNP was inversely proportional to the logarithm value of zeatin concentration in the range of 50 pM–50 nM. The detection limit was calculated to be 16.6 pM. This electrochemical method also showed good detection selectivity and stability. The potential applicability of this method was proved by detecting zeatin in real samples.

A novel and selective photoelectrochemical immunosensor was successfully prepared for N6-methyladenosine (m6A) detection based on the in-situ production of electron donor strategy. Black titanium dioxide (TiO2-x) and molybdenum sulfide (MoS2) heterojunction (TiO2-x-MoS2) was employed as photoactive material, m6A antibody was used as target molecule recognition reagent, biotin-functionalized phos-tag (Phos-tag-biotin) was adopted as bridging reagent to link m6A and avidin-functionalized alkaline phosphatase (avidin-ALP). With the catalytic effect of ALP, its substrate of l -ascorbic acid 2-phosphate trisodium salt in detection buffer can be hydrolyzed to produce ascorbic acid (AA), which will offer electron to capture the hole of photoactive material, improve the photoelectrochemical response and increase the detection sensitivity. Under the optimal conditions, the detection range for m6A is ranged from 0.005 − 35 nM, and the detection limit is 2.57 pM (S/N = 3). The biosensor has good selectivity, reproducibility and stability. Moreover, the applicability of this biosensor was also assessed by investigating the effect of antibiotics on the content of m6A in root, stem and leaf of rice seedlings.

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.

MicroRNAs have been involved into many biological processes and are regarded as disease biomarkers. Simple, rapid, sensitive and selective method for microRNA detection is crucial for early diagnosis and therapy of diseases. In this work, sensitive fluorescence assay was developed for microRNA-21 detection based on DNA polymerase induced strand displacement amplification reaction, Mg2+-dependent DNAzyme catalysis reaction, and magnetic separation. In the presence of target microRNA-21, amounts of trigger DNA could be produced with DNA polymerase induced strand displacement amplification reaction, and the trigger DNA could be further hybridized with signal DNA, which was labeled with biotin and AMCA dye. After introduction of Mg2+, trigger DNA could form DNAzyme to cleave signal DNA. After magnetic separation, the DNA fragment with AMCA dye could give fluorescence signal, which was related to microRNA-21 concentration. Based on the two efficient signal amplifications, the developed method showed high detection sensitivity with low detection limit of 0.27 fM (3σ). In addition, this fluorescence strategy also possessed excellent detection specificity, and could be applied to analyze microRNA-21 expression level in serum of cancer patient. According to the obtained results, the developed fluorescence method might be a promising detection platform for microRNA-21 quantitative analysis in biomedical research and clinical diagnosis.

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.

A simple and sensitive electrochemical biosensor was fabricated for hydroxymethylated DNA detection and T4 β-glucosyltransferase activity assay based on the glycosylation of hydroxymethylated DNA and HpaII restriction endonuclease system triggering signal amplification. After immobilizing double-stranded DNA (dsDNA) on the surface of carboxyl functionalized Fe3O4 nanoparticle, the glycosylation reaction of hydroxymethylated cytosine can be performed with the catalytic effect of T4 β-glucosyltransferase using uridine diphosphoglucose as glucose donor. Then, 4-mercaptophenylboronic acid (4-MPBA) can be further reacted with glucose and captured on Fe3O4 nanoparticle surface. After the dsDNA was digested by HpaII, the fragment with 4-MPBA can be released from Fe3O4 nanoparticle surface. After magnetic separation, this fragment can be further modified on gold electrode surface through the interaction between Au and −SH, which can result in a decreased electrochemical signal of [Fe(CN)6]3−. According to this detection strategy, the electrochemical biosensor showed high sensitivity with the low detection limits of 0.14 nM and 0.27 unit/mL for hydroxymethylated DNA and T4 β-glucosyltransferase. The fabricated biosensor also presented high selectivity and acceptable reproducibility. In addition, the inhibition activity of 4-phenylimidazole towards T4 β-glucosyltransferase was also investigated. This work provides a novel platform for hydroxymethylated DNA detection, T4 β-glucosyltransferase activity assay and its inhibition screening.

N6-methyladenosine (m6A), a kind of RNA methylation form and important epigenetic event, plays crucial roles in many biological progresses. Thus it is essential to quantitatively detect m6A in complicated biological samples. Herein, a simple and sensitive electrochemical method was developed for m6A detection using N6-methyladenosine-5′-triphosphate (m6ATP) as detection target molecule. In this detection strategy, anti-m6A antibody was selected as m6A recognition and capture reagent, silver nanoparticles and amine-PEG3-biotin functionalized SiO2 nanospheres (Ag@SiO2) was prepared and used as signal amplification label, and phos-tag-biotin played a vital role of “bridge” to link m6ATP and Ag@SiO2 through the two forms of specific interaction between phosphate group of m6ATP and phos-tag, biotin and streptavidin, respectively. Under the optimal experimental conditions, the immunosensor presented a wide linear range from 0.2 to 500 nM and a low detection limit of 0.078 nM (S/N=3). The reproducibility and specificity were acceptable. Moreover, the developed method was also validated for detect m6A content in human cell lines. Importantly, this detection strategy provides a promising immunodetection platform for ribonucleotides and deoxyribonucleotides with the advantages of simplicity, low-costing, specificity and sensitivity.

Herein, a simple and selective electrochemical method was developed for sulfadimethoxine detection based on the triggered cleavage activity of nuclease P1 by the formation of aptamer and sulfadimethoxine conjugate. After probe DNA was immobilized on gold electrode surface, aptamer DNA labeled with biotin at its 5′-terminal was then captured on electrode surface through the hybridization reaction between probe DNA and aptamer DNA. The formed double-stranded DNA (dsDNA) can block the digestion activity of Nuclease P1 towards the single-stranded probe DNA. Then, the anti-dsDNA antibody was further modified on electrode surface based on the specific interaction between dsDNA and antibody. Due to the electrostatic repulsion effect and steric-hindrance effect, a weak electrochemical signal was obtained at this electrode. However, in the presence of sulfadimethoxine, it can interact with aptamer DNA, and then the formation of dsDNA can be blocked. As a result, the probe DNA at its single-strand state can be digested by Nuclease P1, which leads to the failure of the immobilization of anti-dsDNA antibody. At this state, a strong electrochemical signal was obtained. Based on the change of the electrochemical signal, sulfadimethoxine can be detected with linear range of 0.1–500 nmol/L. The detection limit was 0.038 nmol/L. The developed method possesses high detection selectivity and sensitivity. The applicability of this method was also proved by detecting sulfadimethoxine in veterinary drug and milk with satisfactory results.

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 SiO 2 and streptavidin (SiO 2 -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, SiO 2 -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.

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 Zr(4+) 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 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) 6 3− , 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 (NH 4 ) 2 SO 4 , ADP, Na 2 HPO 4 and EDTA on PNK was also verified using this method.

As a significant potential biomarker for early disease diagnostics, the detection of 5-hydroxymethylcytosine (5hmC) has garnered widespread attention. In this work, a simple and sensitive electrochemiluminescence (ECL) was developed for 5hmC detection. After substrate electrode preparation, anti-5hmC antibody was captured on electrode surface by the formation of amide bond. Then, through immunoreaction, 5hmC was specifically recognized and captured. Utilizing the crosslink effect of Zr4+ with phosphoric acid recognition, aminomethyl phosphate was modified on electrode to further capture PAMAM-silver nanoclusters/Nitrogen-doped graphene nanocomposite (PAMAM-Ag-NG), where this nanocomposite was employed as ECL signal probe. The designed immunosensor showed a linear concentration range from 10 pM to 30 nM with a low detection limit of 2.47 pM (S/N = 3). This method presented good selectivity and reproducibility. To prove the applicability, this method was further used to study the influence of phytohormone and avian leukemia virus on 5hmC content in maize seedling leaves and chicken fibroblast cells, respectively.

A sensitively homogeneous electrochemiluminescence (ECL) method was developed for 5-hydroxymethylcytosine (5hmC) detection using TiO2/MoS2/g-C3N4/GCE as substrate electrode, where g-C3N4 was employed as the ECL active material, the MoS2 nanosheets were used as co-catalyst, and TiO2 was adopted as phosphate group capture reagent. To achieve the specific recognition and capture of 5hmC, the covalent reaction between –CH2OH and –SH was employed under the catalysis of HhaI methyltransferase, in which, –SH functionalized ferrocenedicarboxylic acid polymer (PFc-SH) was prepared as 5hmC capture reagent and ECL signal quencher. Then, based on the interaction between TiO2 and phosphate group of 5hmC, the target was recognized and captured on electrode, resulting in a decreased ECL response due to the quenching effect of PFc-SH. Under optimal conditions, the biosensor presented the linear range from 0.01 to 500 nM with the detection limit of 3.21 pM (S/N = 3). The steric effect on electrode surface is a bottle-neck issue restricting devised biosensors advancement. In this work, the reaction between 5hmC and PFc was carried out in the solution, which can avoid steric effect on electrode surface to keep the high activity of enzyme. In addition, the biosensor was successfully applied to detect 5hmC in genomic DNA of chicken embryo fibroblast cells and different tissues of rice seedlings.

A nanocomposite was prepared from β-cyclodextrin (β-CD) functionalized graphene oxide and magnetic nanoparticles (GO/Fe3O4/β-CD). In parallel, a polyamidoamine dendrimer conjugated to avidinylated alkaline phosphatase (PAMAM-avidin-ALP) was prepared and exploited as a signal amplification unit in a voltammetric immunoassay for 5-methylcytosine (5mC) in genomic DNA. The GO/Fe3O4/β-CD as a substrate material exhibited good solubility, electrical conductivity and large surface. This is beneficial for the further modification of antibodies (Ab) by host-guest interaction and amide bonds. By taking advantage of three-dimensional structure to capture avidin-ALP by amide linkages, PAMAM was used as a catalytic signal amplification element in this assay. Under the optimized condition and at a typical working potential of 0.94 V, the response to 5mC is linear in the 0.01–50 nM concentration range with a detection limit of 3.2 pM (at S/N = 3). The method is stable, selective and reproducible. It was applied to the determination of 5mC in genomic DNA of human tissue.

Carbon nitride and WS2 nanocomposite (CN-WS2) with excellent photoelectric activity was prepared by one-step thermal polymerization. By the new strategy of simultaneously loading C and N elements on pure WS2, the formation of CN-WS2 brings high mobility, ultra-fast charge carrier separation and transport, and slows down the recombination rate of electrons and holes. Then, a novel photoelectrochemical biosensor was constructed for 5-formylcytosine detection based on CN-WS2 composite, aldehyde reaction probe (ARP) and ZnFe2O4, where CN-WS2 composite was employed as photoactive material, ARP was used as 5-formylcytosine capture reagent, and ZnFe2O4 was adopted as artificial mimic enzyme. Under the catalysis effect of ZnFe2O4, 4-chloro-1-naphthol was quickly oxidized by H2O2 to produce the insoluble substance of benzo-4-chlorohexadienone, which would be deposited on the electrode surface and caused a decreased PEC response. Under optimal experimental conditions, the biosensor showed low detection limit of 3 pM. This method can also discriminate 5-formylcytosine with other cytosine derivatives. More importantly, the applicability of this method was demonstrated by assessing the effect of antibiotics on the content of 5-formylcytosine in the root, stem and leave of maize seedlings.

5-Hydroxymethylcytosine (5 hmC) is a novel epigenetic modification that plays an important role in mammalian nuclear reprogramming, regulation of gene activity, and initiation of DNA demethylation. In this paper, an electrochemiluminescence sensor was constructed for 5 hmC detection based on thiol functional Fe3O4 magnetic beads and covalent chemical reaction of –CH2OH in 5 hmC. First, Fe3O4 magnetic beads were prepared and modified with thiol. Then, 5 hmC was captured on the surface of the magnetic beads by the reaction between –CH2OH of 5 hmC and –SH of the thiol-functionalized Fe3O4 under the catalysis of DNA methyltransferase (M. HhaI). After that, through a series of reactions, phos-tag-biotin, avidin, and bis(hexafluorophosphate) (Ru (bpy)2 (phen-5-NH2) (PF6)2) (Ru) were further successively immobilized on the surface of the magnetic beads. More importantly, these reactions were carried out in a solution to ensure the activity of the biomolecules, and further to ensure that the reaction proceeded sufficiently. Finally, an ECL signal was generated by the introduction of Ru. The concentration of 5 hmC presented a good linear relationship with the ECL signal intensity in the range of 0.01–500 nM, and the detection limit was 2.86 pM. Moreover, we also used this method to study the 5 hmC content change in rice seedlings treated with antibiotics and heavy metal composite pollutants, and in chicken embryo fibroblast cell infected with and without avian leukosis virus subgroup J.

A sensitive and selective photoelectrochemical (PEC) method is described for the detection of protein kinase A (PKA) activity based on the use of graphite-like carbon nitride (g-C3N4) and the CdS quantum dots (QDs). Firstly, a complex was synthesized from g-C3N4 and gold nanoparticles (AuNPs). It was employed as both the PEC-active material and as a support for immobilization of peptides. The latter were assembled on an ITO electrode modified with g-C3N4-AuNPs and subsequently phosphorylated by PKA in the presence of adenosine 5′-[γ-thio]triphosphate (ATP-S). Finally, CdS quantum dots (QDs) were introduced on the ITO in order to increase the PEC response of g-C3N4 based on the Cd-S binding between the QDs and thiol groups. Under the optimal conditions and a typical working voltage of −0.3 V, the method has a dynamic range that extends from 0.05 to 50 unit·mL−1, with a 0.017 unit·mL−1 lower detection limit. The method was successfully applied to the quantification of the inhibitory effect of ellagic acid on the activity of PKA, and to monitor enzyme activity in cell lysates.

A method is described for photoelectrochemical determination of chloramphenicol (CLOA). It is based on the use of (a) aptamers protected with photoactive WS2 nanosheets, and (b) DNase I-assisted target recycling. The DNA aptamer without label was employed for recognition of CLOA. In the absence of CLOA, the aptamer is adsorbed on the surface of WS2. This leads to a decrease of photocurrent due to the steric-hindrance effect of aptamer DNA. The adsorption of WS2 also protects the aptamer from digestion by DNase. In the presence of CLOA, the aptamer will be desorbed from the WS2 surface due to formation of an aptamer/CLOA conjugate. This results in an increased photocurrent due to a decreased amount of aptamer DNA on the electrode surface. The increase of photocurrent can be further improved by applying DNase triggered catalytic recycling of CLOA. Under optimal experimental conditions, the response is linear 10 pM – 10 nM CLOA concentration range, with a 3.6 pM lower detection limit (at 3σ). This method is acceptably selective, accurate and stable. It was applied to the determination of CLOA in spiked milk samples and gave satisfactory results.

The DNA isothermal amplification cycle is an effective expansion strategy, which can overcome the inherent limitation of the target to signal ratio of 1 : 1. In this work, we fabricated an electrochemical biosensor for the rapid detection of Dam methyltransferase activity based on the exonuclease III (Exo III) assisted isothermal amplification cycle. First, double stranded DNA (ds-DNA) containing the symmetric sequence of 5′-GATC-3′ was immobilized on the magnetic bead surface. After the ds DNA was methylated by Dam MTase, it could be further cleaved by DpnI and formed a blunt end, which could lead to the digestion of a DNA strand by Exo III and release of a target single stranded DNA (ts-DNA). Then, the ts-DNA can further hybridize with DNA probes (DNA3) immobilized on the AuNPs/Au electrode surface and form a blunt end DNA–DNA duplex. Subsequently, the DNA3 in the DNA–DNA duplex can be digested by Exo III and release ts-DNA. The released ts-DNA can further hybridize with the unhybridized single stranded DNA (ss-DNA) and an isothermal amplification cycle is achieved. The method showed high sensitivity with a low detection limit of 0.031 unit per mL. Moreover, inhibition investigation indicates that Dam MTase activity can be inhibited by using 5-fluorouracil with an IC50 value of 131.22 μM.

Herein, a sensitive and selective photoelectrochemical (PEC) immunoassay is developed for microRNA, where the graphite-like C3N4 (g-C3N4) is used as photoactive material and anti-DNA:RNA antibody is used as target microRNA recognition unit. With the doping of gold nanoparticles (AuNPs), the photoactivity of g-C3N4 is greatly improved. The capture probe for microRNA is assembled on the AuNPs-g-C3N4 modified ITO surface through the Au S bond. After hybridizing with target microRNA, the DNA:RNA hybrids can be recognized by anti-DNA:RNA antibody and this antibody can be further captured on the electrode surface. Through the specific interaction between antibody and alkaline phosphatase labeled IgG (ALP-IgG), ALP-IgG can be captured, which can catalyze the hydrolysis of ascorbic acid-2-phosphate (AAP) to produce ascorbic acid as electron donor. As a result, a strong photocurrent is obtained and the expression level of microRNA can be detected based the change of photocurrent. The fabricated biosensor presents good detection sensitivity with the linear range of 5–3000 fM and the detection limit of 2.26 fM. The developed biosensor also shows accepted detection selectivity with identifying even single-base mismatched microRNA. The applicability of the biosensor is further evaluated by assaying the expression level change of microRNA in rice leaves with and without phytohormone incubation. This work is not only providing a new detection platform for microRNA assay, but also affording some information on the role of microRNA in phytohormone signaling.

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.

In this work, we proposed a selective and sensitive biosensor for microRNA detection based on the high specificity and affinity of anti-DNA:RNA hybrids antibody (S9.6 antibody) and alkaline phosphatase catalytic signal amplification. Briefly, after the hybridization of probe DNA and the target microRNA, the S9.6 antibody can be captured on the electrode surface through antigen-antibody immunoreaction. Then, alkaline phosphatase labeled goat anti-mouse IgG (ALP-IgG) was further captured on the electrode surface through the specific recognition effect between the primary antibody and the secondary antibody. Finally, ALP catalyzed the hydrolysis reaction of p -nitrophenyl phosphate to generate p -nitrophenol, resulting in a electrochemical oxidation signal. The simple signal amplification assay performed a successful linear range from 0.5-500 fM with a detection limit of 0.40 fM. Moreover, this biosensor exhibited high selectivity with discriminating only single-base mismatched microRNA sequence. Additionally, the simplicity of this method hold a great promise for further investigation of the role of microRNAs in phytohormone signaling transduction.

A simple, highly sensitive and selective electrochemical assay is developed for the detection of protein kinase A (PKA) activity based on the specific recognition utility of Phos-tag for kinase-induced phosphopeptides and enzymatic signal amplification. When the substrate peptide was phosphorylated by PKA reaction, they could specifically bind with Phos-tag-biotin in the presence of Zn(2+) through the formation of a specific noncovalent complex with the phosphomonoester dianion in phosphorylated peptides. Through the further specific interaction between biotin and avidin, avidin functionalized horseradish peroxidase (HRP) can be captured on the electrode surface. Under the catalytic effect of HRP, a sensitive electrochemical signal for benzoquinone was obtained, which was related to PKA activity. Under the optimal experiment conditions, the proposed electrochemical method presented dynamic range from 0.5 to 25 unit/mL with low detection limit of 0.15 unit/mL. This new detection strategy was also successfully applied to analyze the inhibition effect of inhibitors (ellagic acid and H-89) on PKA activity and monitored the PKA activity in cell lysates. Therefore, this Phos-tag-based electrochemical assay offers an alternative platform for PKA activity assay and inhibitor screening, and thus it might be a valuable tool for development of targeted therapy and clinical diagnosis.

MicroRNAs (miRNAs), a class of small endogenous nonprotein-coding RNAs, regulate a wide range of biological processes, and their abnormal expressions are related to the growth and development of plants. Thus, a simple, rapid, and highly sensitive assay for miRNA detection is of great significance. In this work, a label-free and ultrasensitive assay for miRNA detection using protein cage nanoparticles has been developed. Apoferritin-encapsulated Cu nanoparticles (Cu-apoferritin) could be immobilized on the electrode through special reaction between amino and carboxyl. Hybridization event between the probe DNA and the target miRNA-159a is confirmed by electrochemical oxidation signal after Cu released into the detection buffer by adjusting the pH. This assay is highly selective and sensitive with a low detection limit of 3.5 fM. Moreover, the developed method can even discriminate single-base mismatched strand between the complementary targets. The effect of abscisic acid on the expression level of miRNA-159a in Arabidopsis thaliana seeds was also investigated.

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.

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 H 2 O 2 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.

A selective and sensitive electrochemical aptasensing strategy was developed for kanamycin detection based on target-triggered single-strand DNA adsorption on MoS2 nanosheets and enzymatic signal amplification. Firstly, a Y-shape double-stranded DNA (dsDNA) was designed containing probe DNA, assist DNA (labeled with biotin at its 3′-terminal) and aptamer DNA. Then, in the presence of kanamycin, aptamer DNA can be released from the Y-shape dsDNA structure due to its interaction with kanamycin to form stable conjugate, which will destroy the Y-shape dsDNA structure and lead to the presence of two-fragment of single-stranded DNA (ssDNA) on the remained half-hybrid DNA structure (containing probe DNA and assist DNA). Based on the strong interaction between ssDNA and MoS2 nanosheets, the half-hybrid DNA structure can be captured on MoS2 nanosheets modified electrode surface, which triggers the subsequent immobilization of streptavidin-alkaline phosphatase (SA-ALP) compound. Under the catalysis of ALP, p-nitrophenol phosphate (PNPP) is hydrolyzed to produce p-nitrophenol (PNP). Based on the linear relationship between the electrochemical oxidation signal of PNP and kanamycin concentration, kanamycin can be detected with the detection limit of 0.029 nM (S/N = 3). The aptamer-MoS2 nanosheets based electrochemical biosensor shows good selectivity, reproducibility for kanamycin detection.

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) 6 3−/4− as redox probe. The decreased oxidation peak current of Fe(CN) 6 3−/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%.

Cu2O nanoparticles (nano-Cu2O) modified glassy carbon electrode (GCE) was fabricated and used to investigate the electrochemical behaviour of 4-nitrophenol (4-NP) by cyclic voltammetry (CV), chronoamperometry (CA), chronocoulometry (CC) and differential pulse voltammetry (DPV). Compared with GCE, a remarkable increase in oxidation peak current was observed. It indicates that nano-Cu2O exhibits remarkable enhancement effect on the electrochemical oxidation of 4-NP. Under the optimised experimental conditions, the oxidation peak currents were propotional to 4-NP concentration in the range from 1.0 × 10−6 to 4.0 × 10−4 mol L−1 with a detection limit of 5.0 × 10−7 mol L−1 (S/N = 3). The fabricated electrode presented good repeatability, stability and anti-interference. Finally, the proposed method was applied to determine 4-NP in water samples. The recoveries for these samples were from 94.60% to 105.5%.

In this work, a simple and sensitive electrochemical method was developed to determine Sudan I based on magnetic Fe 3 O 4 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 1 μM and 1 to 20 μM. The detection limit was estimated to be 0.001 μM ( S / N = 3). The developed method was successfully applied to determine Sudan I content in food samples with satisfactory results.

In this work, 3-aminopropyltriethoxysilane modified Fe3O4 nanoparticles (ATPS-Fe3O4) were used to modify glassy carbon electrode for aminopyrine determination. ATPS-Fe3O4 showed obviously catalytic activity and adsorptivity towards aminopyrine oxidation proven by the increased oxidation peak current and the decreased oxidation peak potential. The best analytical response was obtained by immobilizing 8 μL 3 mg/mL APTS-Fe3O4 dispersion with an accumulation time of 200 s at −0.2 V in 0.1 M phosphate buffer solution (pH 9.0). The oxidation peak current of aminopyrine showed linear relationship with its concentration in the range from 0.5 to 100 and 100 to 1600 μM. The detection limit was 0.1 μM (S/N = 3). The proposed method showed satisfactory repeatability and anti-interference ability. The fabricated electrode was successfully applied to determine aminopyrine in pharmaceutical formulations.

The electrochemical behavior of guanine and adenine on the graphene and Nafion composite film modified glassy carbon electrode was investigated by differential pulse voltammetry (DPV). The results indicated that the modified electrode exhibited an excellent electrocatalytic activity towards the oxidation of guanine and adenine, testified by the increased oxidation peak current and decreased oxidation potential. The experimental conditions were optimized. The separation of the two oxidation peaks was 0.364 V in 0.1 M pH 4.4 acetate buffer solution (ABS). Based on this, a novel electrochemical method was proposed to simultaneously determine guanine and adenine with the detection limit of 0.58 (guanine) and 0.75 (adenine) μM (S/N = 3). The proposed method was applied to determine guanine and adenine in milk powder, urine and herring sperm DNA samples with satisfactory results. The value of (G + C)/(A + T) in herring sperm DNA was calculated to be 0.8065. The fabricated electrode showed excellent reproducibility, stability and anti-interference.

Electrochemical behavior of bisphenol A (BPA) at glassy carbon electrode-modified with layered double hydroxide (LDH) and anionic surfactant (sodium dodecyl sulfate) is investigated by electrochemical techniques. Compared with the bare electrode and LDH-modified electrode, the oxidation peak potential of BPA shifted negatively and the peak current increased significantly due to the enhanced accumulation of BPA via electrostatic interaction with LDH at the hydrophobic electrode surface. Some determination conditions such as LDH loading, pH, scan rate, accumulation potential, and accumulation time on the oxidation of BPA were optimized. And some kinetic parameters were investigated. Under the optimized conditions, the oxidation current was proportional to BPA concentration in the range of 8 × 10−9 to 2.808 × 10−6 M with the detection limit of 2.0 × 10−9 M by amperometry. The fabricated electrode showed good reproducibility, stability, and anti-interference. The proposed method was successfully applied to determine BPA in water samples, and the results were satisfactory.

A glassy carbon electrode (GCE) modified with Mg-Al-SDS hydrotalcite-like clay (SDS-HTLC) was used for the sensitive voltammetric determination of 2-nitrophenol (2-NP) utilizing the oxidation process. The results indicate the prepared modified electrode has an excellent electrocatalytic activity toward 2-NP oxidation, lowering the oxidation overpotential and increasing the oxidation current. Under optimal conditions, the oxidation current was proportional to 2-NP concentration in the range from 1.0×10−6 to 6.0×10−4 M with the detection limit of 5.0×10−7 M by DPV (S/N=3). The fabricated electrode was applied for 2-NP determination in water samples and the recovery for these samples was from 95.6 to 103.5%.

A glassy carbon electrode was modified with hydroxyapatite nanopowder (HA-NP) and characterized in terms of electrochemical oxidation of 4-nitrophenol (4-NP) via cyclic voltammetry, differential pulse voltammetry, chronoamperometry, and chronocoulumetry. The oxidation peak current of 4-NP at the modified electrode was increased (compared to the bare GCE), thus indicating that the HA-NP exhibits a remarkable enhancement effect on the electrochemical oxidation of 4-NP. The effects of loading with HA-NP, pH value, scan rate and accumulation time were examined. The oxidation peak current of 4-NP is proportional to its concentration in the range from 1.0 μM to 300 μM, with a correlation coefficient of 0.9996. The detection limit is 0.6 μM (at an S/N = 3). The method is simple, selective and sensitive. It was successfully applied to the determination of 4-NP in water samples, with recoveries ranging from 96% to 104%.

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 × 10 −8 to 3.0 × 10 −6 M with correlation coefficient of 0.9979 and detection limit of 3.0 × 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%.