Ting Hou

Ultrasensitive homogeneous electrochemical strategy for DNA methyltransferase activity assay based on autonomous exonuclease III-assisted isothermal cycling signal amplification.

DNA methylation catalyzed by methyltransferase (MTase) plays an important role in many biological processes, including gene transcription, genomic imprinting and cellular differentiation. Herein, a simple and novel homogeneous electrochemical strategy for ultrasensitive DNA MTase activity assay has been successfully developed, which is based on methylation-triggered exonuclease (Exo) III-assisted autonomous isothermal cycling signal amplification. A duplex DNA (P1-P2 hybrid) containing the methylation-responsive sequence is ingeniously designed. In the presence of DNA adenine methylation (Dam) methyltransferase (MTase), P1-P2 hybrid is methylated and subsequently recognized and cleaved by Dpn I endonuclease, which triggers the Exo III-catalyzed autonomous cycling cleavage processes. Therefore, a large amount of methylene blue-labeled mononucleotides are released, generating a significantly amplified electrochemical signal toward the Dam MTase activity assay. The directly measured detection limit down to 0.004 U/mL is obtained, which is one or two orders magnitude lower than that of the approaches reported in literature. Since this assay is carried out in homogeneous solution phase under isothermal condition and sophisticated probe immobilization processes are avoided, it is very simple and easy to implement. Due to its advantages of ultrahigh sensitivity, excellent selectivity and simple operation, the as-proposed strategy has great potential in the applications in DNA methylation related clinical practices and biochemical researches.

Affinity-Mediated Homogeneous Electrochemical Aptasensor on a Graphene Platform for Ultrasensitive Biomolecule Detection via Exonuclease-Assisted Target-Analog Recycling Amplification

As is well-known, graphene shows a remarkable difference in affinity toward nonstructured single-stranded (ss) DNA and double-stranded (ds) DNA. This property makes it popular to prepare DNA-based optical sensors. In this work, taking this unique property of graphene in combination with the sensitive electrochemical transducer, we report a novel affinity-mediated homogeneous electrochemical aptasensor using graphene modified glassy carbon electrode (GCE) as the sensing platform. In this approach, the specific aptamer-target recognition is converted into an ultrasensitive electrochemical signal output with the aid of a novel T7 exonuclease (T7Exo)-assisted target-analog recycling amplification strategy, in which the ingeniously designed methylene blue (MB)-labeled hairpin DNA reporters are digested in the presence of target and, then, converted to numerous MB-labeled long ssDNAs. The distinct difference in differential pulse voltammetry response between the designed hairpin reporters and the generated long ssDNAs on the graphene/GCE allows ultrasensitive detection of target biomolecules. Herein, the design and working principle of this homogeneous electrochemical aptasensor were elucidated, and the working conditions were optimized. The gel electrophoresis results further demonstrate that the designed T7Exo-assisted target-analog recycling amplification strategy can work well. This electrochemical aptasensor realizes the detection of biomolecule in a homogeneous solution without immobilization of any bioprobe on electrode surface. Moreover, this versatile homogeneous electrochemical sensing system was used for the determination of biomolecules in real serum samples with satisfying results.

Enzyme-free and label-free fluorescence aptasensing strategy for highly sensitive detection of protein based on target-triggered hybridization chain reaction amplification.

Proteins are of great importance in medical and biological fields. In this paper, a novel fluorescent aptasensing strategy for protein assay has been developed based on target-triggered hybridization chain reaction (HCR) and graphene oxide (GO)-based selective fluorescence quenching. Three DNA probes, a helper DNA probe (HP), hairpin probe 1 (H1) and hairpin probe 2 (H2) are ingeniously designed. In the presence of the target, the aptamer sequences in HP recognize the target to form a target-aptamer complex, which causes the HP conformation change, and then triggers the chain-like assembly of H1 and H2 through the hybridization chain reaction, generating a long chain of HP leading complex of H1 and H2. At last the fluorescence indicator SYBR Green I (SG) binds with the long double strands of the HCR product through both intercalation and minor groove binding. When GO was added into the solutions after HCR, the free H1, H2 and SG would be closely adsorbed onto GO surface via π-π stacking. However, the HCR product cannot be adsorbed on GO surface, thereby the SG bound to HCR product gives a strong fluorescence signal dependent on the concentration of the target. With the use of platelet-derived growth factor BB (PDGF-BB) as the model analyte, this newly designed protocol provides a highly sensitive fluorescence detection of PDGF-BB with a limit of detection down to 1.25 pM, and also exhibit good selectivity and applicability in complex matrixes. Therefore, the proposed aptasensing strategy based on target-triggered hybridization chain reaction amplification should have wide applications in the diagnosis of genetic diseases due to its simplicity, low cost, and high sensitivity at extremely low target concentrations.

A versatile immobilization-free photoelectrochemical biosensor for ultrasensitive detection of cancer biomarker based on enzyme-free cascaded quadratic amplification strategy.

In this work, an ultrasensitive immobilization-free photoelectrochemical (PEC) biosensor was successfully developed for the first time based on a novel enzyme-free cascaded quadratic signal amplification strategy. This rationally designed homogeneous dual amplification strategy consists of a target-analog recycling circuit based on catalytic hairpin assembly (CHA) and a hybridization chain reaction (HCR) mediated amplification circuit. In the presence of carcinoembryonic antigen (CEA), a proof-of-concept target, target-analog is released to trigger the upstream CHA recycling circuit. The generated dsDNA complexes from CHA recycling could further induce the downstream HCR amplification, leading to the formation of numerous hemin/G-quadruplex DNAzymes. This would accordingly stimulate the biocatalytic precipitation of 4-chloro-1-naphthol, inducing a distinct decrease in the photocurrent signal due to the formed insoluble/insulating products on electrode surface. Under the optimal conditions, this PEC biosensor achieved ultrasensitive detection of CEA down to the atto-gram level. The introduction of this aptamer-based cascaded quadratic amplification strategy not only remarkably improves the selectivity and sensitivity of CEA assay, but also allows the ultrasensitive detection of other proteins by designing specific aptamers, providing a universal, isothermal and label-free PEC biosensing platform for ultrasensitive detection of different kinds of cancer biomarkers and holding a great potential for early-diagnosis of cancer.

Autonomous Exonuclease III-Assisted Isothermal Cycling Signal Amplification: A Facile and Highly Sensitive Fluorescence DNA Glycosylase Activity Assay

One common form of DNA damage is the oxidation of guanine to 8-oxo-7,8-dihydroguanine (8-oxoG), which can be carcinogenic. Human 8-oxoguanine DNA glycosylase (hOGG1) is a key base excision repair (BER) enzyme that repairs 8-oxoG, and the expression level of hOGG1 is closely related to many types of human cancers. Herein, a novel and highly sensitive fluorescence biosensing platform for hOGG1 activity detection has been constructed based on autonomous exonuclease III (Exo III)-assisted signal amplification. Two hairpin probes (HP1 and HP2) are ingeniously designed. In the presence of hOGG1, HP1 is cleaved at the 8-oxoG site, and the stem is subsequently digested by Exo III, releasing the trigger DNA fragment (tDNA1). Successively, tDNA1 partially hybridizes with HP2 to initiate the Exo III-assisted cycling cleavage to release another trigger DNA fragment (tDNA2), which in turn triggers the cycling cleavage of DNA fluorescence probe (FP). Therefore, large amount of fluorophore fragments are released, leading to a significantly amplified fluorescence signal toward hOGG1 activity detection. A directly measured detection limit down to 0.001 U/mL is obtained, which is much lower than that of the approaches reported in literature. In addition to high sensitivity and good selectivity, the as-proposed strategy also exhibits the advantages of isothermal experimental condition, simplicity, and convenience. Furthermore, the Exo III-assisted autonomous cycling cleavage approach we proposed here is a universal sensing strategy and has great potential in assays of many other biological analytes.

Paper-based fluorescent sensor for rapid naked-eye detection of acetylcholinesterase activity and organophosphorus pesticides with high sensitivity and selectivity

Various strategies have been proposed for the sensing of acetylcholinesterase (AChE) activity and organophosphorus pesticides (OPs). However, the practical application of most methods is restricted by their intrinsic drawbacks such as complexity, long analysis time, and high cost. Thus, it is highly desirable to develop simple, fast and sensitive approaches for AChE activity and OPs detection. Herein, we reported a simple paper-based fluorescent sensor (PFS) based on the aggregation induced emission (AIE) effect of tetraphenylethylene (TPE) and the addition reaction capability of maleimide, which has been used as a powerful tool for rapid naked-eye detection of AChE activity and OPs. The introduction of TPE provides the probe with unique fluorescence property in solid state and is of great importance for improving the sensitivity of PFS. The hydrolysis product of acetylthiocholine catalyzed by AChE induced the maleimide ring destruction and activated the fluorescence performance of TPE. Given that AChE activity can be specifically inhibited by OPs, the as-proposed PFS can also be utilized for sensitive detection of OPs. Meanwhile, the variation of fluorescence signal can be readily detected by naked eyes, and low detection limits of 2.5mUmL(-1) and 0.5ngmL(-1) for AChE activity and OPs are obtained, respectively. Moreover, it has been successfully applied for AChE activity and OPs detection in diluted human serum samples, showing its great potential to be applied in real samples. Thus, this strategy possesses considerable advantages of simplicity, rapid detection, portability, cost efficiency and visualization.

Label-free fluorescence strategy for sensitive microRNA detection based on isothermal exponential amplification and graphene oxide.

MicroRNAs (miRNAs) play an important role in many biological processes, and have been regarded as potential targets and biomarkers in cancer diagnosis and therapy. Also, to meet the big challenge imposed by the characteristics of miRNAs, such as small size and vulnerability to enzymatic digestion, it is of great importance to develop accurate, sensitive and simple miRNA assays. Herein, we developed a label-free fluorescence strategy for sensitive miRNA detection by combining isothermal exponential amplification and the unique features of SYBR Green I (SG) and graphene oxide (GO), in which SG gives significantly enhanced fluorescence upon intercalation into double-stranded DNAs (dsDNAs), and GO selectively adsorbs miRNA, single-stranded DNA and SG, to protect miRNA from enzymatic digestion, and to quench the fluorescence of the adsorbed SG. In the presence of the target miRNA, the ingeniously designed hairpin probe (HP) is unfolded and the subsequent polymerization and strand displacement reaction takes place to initiate the target recycling process. The newly formed dsDNAs are then recognized and cleaved by the nicking enzyme, generating new DNA triggers with the same sequence as the target miRNA, which hybridize with intact HPs to initiate new extension reactions. As a result, the circular exponential amplification for target miRNA is achieved and large amount of dsDNAs are formed to generate significantly enhanced fluorescence upon the intercalation of SG. Thus sensitive and selective fluorescence miRNA detection is realized, and the detection limit of 3 fM is obtained. Besides, this method exhibits additional advantages of simplicity and low cost, since expensive and tedious labeling process is avoided. Therefore, the as-proposed label-free fluorescence strategy has great potential in the applications in miRNA-related clinical practices and biochemical researches.

Biphasic photoelectrochemical sensing strategy based on in situ formation of CdS quantum dots for highly sensitive detection of acetylcholinesterase activity and inhibition

Herein, we reported a facile and highly sensitive biphasic photoelectrochemical (PEC) sensing strategy based on enzymatic product-mediated in situ formation of CdS quantum dots (QDs), and assayed the activity and inhibition of acetylcholinesterase (AChE) in its optimal state. Upon the hydrolysis of acetylthiocholine catalyzed by AChE, the product thiocholine stabilizes the in situ formation of CdS QDs in homogenous solution. Due to the electrostatic attraction, the resulting tertiary amino group-functionalized CdS QDs are attached to the surface of the negatively charged indium tin oxide (ITO) electrode, generating significant PEC response upon illumination in the presence of electron donors. By taking full advantage of the in situ formation of CdS QDs in homogenous solution, this strategy is capable of detecting AChE activity and inhibition in its optimal state. A directly measured detection limit of 0.01mU/mL for AChE activity is obtained, which is superior to those obtained by some fluorescence methods. The inhibition of AChE activity by aldicarb is successfully detected, and the corresponding IC50 is determined to be 13μg/L. In addition to high sensitivity and good selectivity, this strategy also exhibits additional advantages of simplicity, low cost and easy operation. To the best of our knowledge, the as-proposed strategy is the first example demonstrating the application of CdS QDs formed in situ for biphasic PEC detection of enzyme activity and inhibition. More significantly, it opens up a new horizon for the development of homogenous PEC sensing platforms, and has great potential in probing many other analytes.

Graphene-Assisted Label-Free Homogeneous Electrochemical Biosensing Strategy based on Aptamer-Switched Bidirectional DNA Polymerization

In this contribution, taking the discrimination ability of graphene over single-stranded (ss) DNA/double-stranded (ds) DNA in combination with the electrochemical impedance transducer, we developed a novel label-free homogeneous electrochemical biosensor using graphene-modified glassy carbon electrode (GCE) as the sensing platform. To convert the specific aptamer-target recognition into ultrasensitive electrochemical signal output, a novel aptamer-switched bidirectional DNA polymerization (BDP) strategy, capable of both target recycling and exponential signal amplification, was compatibly developed in this study. In this strategy, all the designed DNA structures could be adsorbed on the graphene/GCE and, thus, serve as the electrochemical impedance signal reporter, while the target acts as a trigger of this BDP reaction, in which these designed DNA structures are bound together and, then, converted to long dsDNA duplex. The distinct difference in electrochemical impedance spectroscopy between the designed structures and generated long dsDNA duplex on the graphene/GCE allows label-free and homogeneous detection of target down to femto-gram level. The target can be displaced from aptamer through the polymerization to initiate the next recognition-polymerization cycle. Herein, the design and signaling principle of aptamer-switched BDP amplification system were elucidated, and the working conditions were optimized. This method not only provides a universal platform for electrochemical biosensing but also shows great potential in biological process researches and clinic diagnostics.

Ultrasensitive Self-Powered Aptasensor Based on Enzyme Biofuel Cell and DNA Bioconjugate: A Facile and Powerful Tool for Antibiotic Residue Detection

Herein, we reported a novel ultrasensitive one-compartment enzyme biofuel cells (EBFCs)-based self-powered aptasensing platform for antibiotic residue detection. By taking full advantage of the unique features of both EBFCs-based self-powered sensors and aptamers, the as-proposed aptasensing platform has the merits of simple instrumentation, anti-interference ability, high selectivity, and low cost. In this study, DNA bioconjugate, i.e., SiO2@gold nanoparticles-complementary strand of aptamer (SiO2@AuNPs-csDNA), was elaborately designed and played a key role in blocking the mass transport of glucose to the bioanode. While in the presence of the target antibiotic, SiO2@AuNPs-csDNA bioconjugate broke away from the bioanode due to the aptamer recognition of the target. Without the blocking of glucose by the DNA bioconjugate, a significantly elevated open circuit voltage of the EBFCs-based aptasensor was obtained, whose amplitude was dependent on the antibiotic concentration. In addition, this proposed aptasensor was the first reported self-powered aptasensing platform for antibiotic determination and featured high sensitivity owing to the elaborate design of the DNA bioconjugate modified bioanode of EBFC, which was superior to those previously reported in the literature. Furthermore, due to the anti-interference ability and the excellent selectivity of the aptasensor, no special sample pretreatment was needed for the detection of antibiotics in milk samples. Therefore, the proposed EBFCs-based self-powered aptasensor has a great promise to be applied as a powerful tool for on-site assay in the field of food safety.