Yuna Guo

Label-free and highly sensitive electrochemical detection of E. coli based on rolling circle amplifications coupled peroxidase-mimicking DNAzyme amplification

In this work, a simple, label-free, low cost electrochemical biosensor for highly sensitive and selective detection of Escherichia coli has been developed on the basis of rolling circle amplification (RCA) coupled peroxidase-mimicking DNAzyme amplification. A aptamer-primer probe (APP) containing anti-E. coli aptamer and a primer sequence complementary to a circular probe, which includes two G-quadruplex units, is used for recognizing target and triggering RCA-based polymerase elongation. Due to RCA coupled DNAzyme amplification strategy, the presence of target E. coli leads to the formation of numerous G-quadruplex oligomers on electrode, which folds into G-quadruplex/hemin complexs with the help of K(+) and hemin, thus generating extremely strong catalytic activity toward H2O2 and giving a remarkably strong electrochemical response. As far as we know, this work is the first time that RCA coupled peroxidase-mimicking DNAzyme amplification technique have been integrated into electrochemical assay for detecting pathogenic bacteria. Under optimal conditions, the proposed biosensor exhibits ultrahigh sensitivity toward E. coli with detection limits of 8cfumL(-1) and a detection range of 5 orders of magnitude. Besides, our biosensor also shows high selectivity toward target E. coli and has the advantages in its rapidness, low cost, simplified operations without the need of electrochemical labeling steps and additional labile reagents. Hence, the RCA coupled peroxidase-mimicking DNAzyme amplification-based electrochemical method might create a useful and practical platform for detecting E. coli and related food safety analysis and clinical diagnosis.

A novel sandwich-type electrochemical aptasensor based on GR-3D Au and aptamer-AuNPs-HRP for sensitive detection of oxytetracycline

In this paper, a novel sandwich-type electrochemical aptasensor has been fabricated and applied for sensitive and selective detection of antibiotic oxytetracycline (OTC). This sensor was based on graphene-three dimensional nanostructure gold nanocomposite (GR-3D Au) and aptamer-AuNPs-horseradish peroxidase (aptamer-AuNPs-HRP) nanoprobes as signal amplification. Firstly, GR-3D Au film was modified on glassy carbon electrode only by one-step electrochemical coreduction with graphite oxide (GO) and HAuCl4 at cathodic potentials, which enhanced the electron transfer and loading capacity of biomolecules. Then the aptamer and HRP modified Au nanoparticles provide high affinity and ultrasensitive electrochemical probe with excellent specificity for OTC. Under the optimized conditions, the peak current was linearly proportional to the concentration of OTC in the range of 5×10-10-2×10-3gL-1, with a detection limit of 4.98×10-10gL-1. Additionally, this aptasensor had the advantages in high sensitivity, superb specificity and showed good recovery in synthetic samples. Hence, the developed sandwich-type electrochemical aptasensor might provide a useful and practical tool for OTC determination and related food safety analysis and clinical diagnosis.

Ultrasensitive and rapid detection of miRNA with three-way junction structure-based trigger-assisted exponential enzymatic amplification.

Aberrant expression of micro RNA (miRNA) is associated with development of cancers and diseases, so miRNA has become a tissue-based biomarker for cancer prognosis and diagnosis. Herein, a novel trigger-assisted exponential enzymatic amplification (T-EXPEA) method for ultrasensitive miRNA detection based on three-way junction (3-WJ) structure driven has been reported, which can be used in potential applications in cancer prognosis and diagnoses. In this assay, target miRNA can unfold hairpin probe and start the reaction, and thus specifically form stable 3-WJ structure with helper. Then it can produce triggers under the synergetic polymerase and restriction endonucleases amplification. The produced triggers could be used to unfold molecular beacon (MB) and initiate T-EXPEA process. In the EXPEA part, the exponential triggers were generated to initiate new T-EXPEA and high enhancement fluorescence amplification efficiency was obtained. The feature of our strategy lies in the T-EXPEA combining with 3-WJ structure has been utilized for fluorescence miRNA detection. It is worth noting that the sequence of the triggers in T-EXPEA part is the same to that of triggers generated from the 3-WJ part. In addition, the design of restriction enzyme cutting sites using the same restriction enzyme (Nt.BbvCI) in hairpin probe and MB respectively, improved reaction efficiency cost-efficiently. This method can quantitatively detect sequence-specific miRNA in a dynamic range from 10 aM to 10 pM with a detection limit as low as 7.8 aM.

An ultrasensitive HRP labeled competitive aptasensor for oxytetracycline detection based on grapheme oxide–polyaniline composites as the signal amplifiers

In this work, we explored an amplification strategy which was based on graphene oxide–polyaniline (GO–PANI) and horseradish peroxidase (HRP) to construct the competitive aptasensor for ultrasensitive detection of oxytetracycline (OTC). In the protocol, the GO–PANI film was immobilized on the surface of the electrodes. Then, gold nanoparticles (AuNPs) were electrodeposited on the electrode surface using a constant potential stripping technique. The selected aptamer which had high affinity and specificity for OTC was used as a capture probe and labeled with HRP. The linear response to OTC concentration of the developed aptasensor was in the range of 4.0 × 10−6 mg L−1 to 1.0 mg L−1. The detection limit (LOD) of 2.3 × 10−6 mg L−1 was obtained (S/N = 3). In addition to good repeatability and stability, the proposed aptamer sensor also showed the advantages of low background current and high sensitivity to examine OTC in real samples.

Label-free, homogeneous, and ultrasensitive detection of pathogenic bacteria based on target-triggered isothermally exponential amplification

In this work, a simple, isothermal, and ultrasensitive homogeneous colorimetric sensor for pathogenic bacteria detection has been developed on the basis of target-triggered exponential amplification reaction (EXPAR). An aptamer–primer probe (arched probe) containing anti-target aptamer and a primer sequence, which is released under the challenging of target, is used for recognizing target and triggering EXPAR-based polymerase elongation. Due to EXPAR coupled DNAzyme amplification strategy, the presence of target pathogenic bacteria leads to the formation of numerous G-quadruplex oligomers in solution, which folds into G-quadruplex/hemin complexes with the help of K+ and hemin, thus generating extremely strong catalytic activity toward H2O2 and giving a remarkably strong UV-vis absorption. This work is a novel design that EXPAR coupled DNAzyme amplification technique has been integrated into colorimetric assay for detecting pathogenic bacteria. Under optimal conditions, the proposed biosensor exhibits ultrahigh sensitivity toward target pathogenic bacteria with detection limits of 80 cfu mL−1. Besides, our biosensor also shows high selectivity toward target pathogenic bacteria and has the advantages in its low cost, simplified operations without the need of labeling steps and additional labile reagents. Hence, the EXPAR coupled DNAzyme amplification-based colorimetric method might create a useful and practical platform for detecting pathogenic bacteria and related food safety analysis and environmental monitoring.

An RNA aptamer-based electrochemical biosensor for sensitive detection of malachite green

An RNA aptamer-based electrochemical biosensing strategy has been developed for sensitive and selective detection of malachite green (MG). This biosensor is fabricated by the self-assembly of a thiolated MG aptamer (MGA) on AuNPs/graphene–chitosan nanocomposite modified glass carbon electrode. In addition, a short alkanethiol is further assembled on the AuNPs surface to generate uniform packing and reduce nonspecific adsorption. When the modified electrode is incubated in the presence of MG, MGA combines specifically with MG, which causes the horseradish peroxidase (HRP)-labelled MG antibody close to the electrode surfaces. As a result, MG detection is realized by outputting a redox current from electro-reduction of the hydrogen peroxide reaction catalyzed by HRP. Differential pulse voltammetry (DPV) is performed to record the signal responses. The results reveal the biosensor displays a very low detection limit as low as 16.3 pg mL−1 and a wide linear range from 1 × 10−4 to 10 μg mL−1 of MG. Hence, this proposed RNA aptamer-based electrochemical strategy may offer a simple, rapid, cost-effective, highly selective and sensitive method for the quantification of MG.

Signal-on electrochemical detection of antibiotics based on exonuclease III-assisted autocatalytic DNA biosensing platform

In this work, a novel electrochemical DNA sensor based on exonuclease III (Exo III)-assisted autocatalytic DNA biosensing platform for ultrasensitive detection of antibiotics has been reported. When the arched probe was challenged with target, the released primer caused by the specific recognition of the aptamer and target antibiotics hybridized with the 3′-protruding terminus in the HP1. Then, Exo III could catalyze the stepwise removal of mononucleotides from this flat terminus, resulting in the release of the primer and trigger. As a secondary target, trigger could replace Helper from the 5′-MB labeled HP2, and HP2 formed a “close” probe structure, confining MB close to the electrode surface for efficient electron transfer. To our best knowledge, such work is the first report about Exo III-assisted autocatalytic DNA biosensing platform combing with signal-on sensing strategy, which has been utilized for quantitative determination of antibiotics. It would be further used as a general strategy associated with more analytical techniques toward the detection of a wide spectrum of analytes. Thus, it holds great potential for the development of ultrasensitive biosensing platform for the applications in bioanalysis, disease diagnostics, and clinical biomedicine.

A functional oligonucleotide probe from an encapsulated silver nanocluster assembled by rolling circle amplification and its application in label-free sensors

A novel label-free, low cost electrochemical biosensor for highly sensitive and selective detection of E. coli has been developed based on rolling circle amplification (RCA) coupled silver nanoclusters (AgNCs) as an effective electrochemical probe. This strategy is to our knowledge the first approach where the combination of RCA with the efficient catalytic property of AgNCs has been applied in bioanalysis. Our biosensor relies on sandwich type immuno-recognition and an in situ RCA reaction, which results in the enrichment of functional oligonucleotide probe (FOP) encapsulated AgNCs. Due to the high electrocatalytic activity of AgNCs toward H2O2 reduction, stable and sensitive electrochemical signals can be achieved from the biological recognition events. The results reveal the calibration plot obtained for E. coli O157:H7 is approximately linear from 3.7 × 10 to 3.7 × 106 cfu mL−1 with a limit of detection of 31 cfu mL−1. This label-free biosensor has advantages over conventional electrochemical assays in its significantly improved sensitivity, remarkably widened dynamic range, and substantially lowered background signal. Moreover, the proposed biosensor has been demonstrated to allow accurate quantitative assay of E. coli O157:H7 in milk samples. By virtue of these merits, our RCA coupled AgNCs-based electrochemical biosensor might create a useful and valuable tool for the detection of E. coli O157:H7 and related molecular diagnostics in food safety analysis.