Bingchen Li

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

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

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

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

A sensitive electrochemical biosensor for detection of protein kinase A activity and inhibitors based on Phos-tag and enzymatic signal amplification.

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.

Two-stage cyclic enzymatic amplification method for ultrasensitive electrochemical assay of microRNA-21 in the blood serum of gastric cancer patients

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 0.36fM 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.

Signal-on fluorescence biosensor for microRNA-21 detection based on DNA strand displacement reaction and Mg2+-dependent DNAzyme cleavage

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 0.27fM (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.

Enzyme-based electrochemical biosensor for sensitive detection of DNA demethylation and the activity of DNA demethylase

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.

Photoelectrochemical biosensor for highly sensitive detection of microRNA based on duplex-specific nuclease-triggered signal amplification

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.

Electrochemical biosensor for DNA demethylase detection based on demethylation triggered endonuclease BstUI and Exonuclease III digestion

Herein, an electrochemical biosensor was fabricated for DNA demethylase detection based on DNA demethylation triggered endonuclease BstUI and Exonuclease III digestion. After the double-strand DNA was demethylated, it can be further digested by BstUI and formed a blunt end at the electrode surface. Then, the remained fragment of DNA-DNA duplex was further cleaved by exonuclease III and led to increased electrochemical signal. Based on this detection strategy, the biosensor showed high sensitivity with low detection limit of 0.15ng/mL. Moreover, the developed method also presented high selectivity and acceptable reproducibility. This work provides a novel detection platform for DNA demethylase detection.

An electrochemical biosensor for the activity assay of polynucleotide kinase and inhibitor screening

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.