Zhong Feng Gao

Ultrasensitive Label-Free Resonance Rayleigh Scattering Aptasensor for Hg2+ Using Hg2+-Triggered Exonuclease III-Assisted Target Recycling and Growth of G-Wires for Signal Amplification

A novel signal-on and label-free resonance Rayleigh scattering (RRS) aptasensor was constructed for detection of Hg(2+) based on Hg(2+)-triggered Exonuclease III-assisted target recycling and growth of G-quadruplex nanowires (G-wires) for signal amplification. The hairpin DNA (H-DNA) was wisely designed with thymine-rich recognition termini and a G-quadruplex sequence in the loop and employed as a signal probe for specially recognizing trace Hg(2+) by a stable T-Hg(2+)-T structure, which automatically triggered Exonuclease III (Exo-III) digestion to recycle Hg(2+) and liberate the G-quadruplex sequence. The free G-quadruplex sequences were self-assembled into guanine nanowire (G-wire) superstructure in the presence of Mg(2+) and demonstrated by gel electrophoresis. The RRS intensity was dramatically amplified by the resultant G-wires, and the maximum RRS signal at 370 nm was linear with the logarithm of Hg(2+) concentration in the range of 50.0 pM to 500.0 nM (R = 0.9957). Selectivity experiments revealed that the as-prepared RRS sensor was specific for Hg(2+), even coexisting with high concentrations of other metal ions. This optical aptasensor was successfully applied to identify Hg(2+) in laboratory tap water and river water samples. With excellent sensitivity and selectivity, the proposed RRS aptasensor was potentially suitable for not only routine detection of Hg(2+) in environmental monitoring but also various target detection just by changing the recognition sequence of the H-DNA probe.

Fluorescent detection of hydrogen peroxide and glucose with polyethyleneimine-templated Cu nanoclusters.

An interesting, simple, and label-free strategy for the detection of hydrogen peroxide and glucose has been developed with polyethyleneimine (PEI)-capped copper nanoclusters as a fluorescence probe in aqueous solution. The PEI-templated Cu nanoclusters which we have synthesized have an average diameter of 1.8 nm and show a blue emission at 480 nm. In the presence of hydrogen peroxide, the fluorescence of the Cu nanoclusters is quenched. Similarly, glucose oxidase catalyzes the oxidation of glucose to gluconic acid and H2O2, so we can also use this probe to detect glucose. Because of the high zymolyte specificity of glucose oxidase, the detection of glucose has good selectivity. Under the optimized experimental conditions, the linear ranges for H2O2 and glucose are 0.5-10 μM and 10-100 μM, respectively. And the detection limits for H2O2 and glucose are 0.4 and 8 μM, respectively. Furthermore, we discussed the mechanism of fluorescence quenching which is caused by the interaction between H2O2 and Cu nanoclusters. This sensing system has been applied successfully to the detection of glucose in human serum samples.

Highly selective and sensitive electrochemical biosensor for ATP based on the dual strategy integrating the cofactor-dependent enzymatic ligation reaction with self-cleaving DNAzyme-amplified electrochemical detection.

A dual strategy that combines the adenosine triphosphate (ATP)-dependent enzymatic ligation reaction with self-cleaving DNAzyme-amplified electrochemical detection is employed to construct the biosensor. In this design, the methylene blue-labeled hairpin-structured DNA was self-assembled onto a gold electrode surface to prepare the modified electrode through the interaction of Au-S bond. In the procedure of ATP-dependent ligation reaction, when the specific cofactor ATP was added, the two split oligonucleotide fragments of 8-17 DNAzyme were linked by T4 DNA ligase and then released to hybridize with the labeled hairpin-structured DNA substrate. The linked 8-17 DNAzyme catalyzes the cleavage of the hairpin-structured substrate by the addition of Zn(2+), causing the methylene blue which contains high electrochemical activity to leave the surface of the gold electrode, therefore generating a dramatic decrease of electrochemical signal. The decrease of peak current was readily measured by square wave voltammetry and a relatively low detection limit (0.05 nM) was obtained with a linear response range from 0.1 to 1000 nM. By taking advantage of the highly specific cofactor dependence of the DNA ligation reaction, the proposed ligation-induced DNAzyme cascades demonstrate ultrahigh selectivity toward the target cofactor ATP. A catalytic and molecular beacons strategy is further adopted to amplify the electrochemical signal detection achieved by cycling and regenerating the 8-17 DNAzyme to realize enzymatic multiple turnover, thus one DNAzyme can catalyze the cleavage of several hairpin-structured substrates, which improves the sensitivity of the newly designed electrochemical sensing system.

Multidimensional optical sensing platform for detection of heparin and reversible molecular logic gate operation based on the phloxine B/polyethyleneimine system.

A multidimensional optical sensing platform which combines the advantages of resonance Rayleigh scattering (RRS), fluorescence, and colorimetry has been designed for detection of heparin. Phloxine B, a fluorescein derivative showing the special RRS spectrum in the long wavelength region, was selected to develop an easy-to-get system which can achieve switch-on sensing to obtain high sensitivity. The noise level of RRS in the long wavelength region is much weaker, and the reproducibility is much better; in this way, the sensitivity and selectivity can be improved. In the absence of heparin, the phloxine B and polyethyleneimine (PEI) form a complex through electrostatic interaction. Thus, the RRS signal at 554 nm is low; the phloxine B fluorescence is quenched, and the absorption signal is low. In the presence of heparin, competitive binding occurred between phloxine B and heparin toward PEI; then, phloxine B is gradually released from the phloxine B/PEI complex, causing obvious enhancement of the RRS, fluorescence, and absorption signals. Besides, the desorption of phloxine B is less effective for the heparin analogues, such as hyaluronic acid and chondroitin sulfate. In addition, the system presents a low detection limit of heparin to 5.0 × 10(-4) U mL(-1) and can also be applied to the detection of heparin in heparin sodium injection and 50% human serum samples with satisfactory results. Finally, the potential application of this method in reversible on-off molecular logic gate fabrication was discussed using the triple-channel optical signals as outputs.

Detection of mercury ions (II) based on non-cross-linking aggregation of double-stranded DNA modified gold nanoparticles by resonance Rayleigh scattering method

This work describes a sensitive approach utilizing non-cross-linking aggregation of double-stranded DNA modified gold nanoparticles (dsDNA-AuNPs) for the detection of mercury ions (Hg(2+)) by resonance Rayleigh scattering (RRS) method for the first time. The double-stranded DNA contains a mismatched T-T base pair in the chain terminus, resulting in a flexible DNA tail and preventing the AuNPs from aggregation. Thus, a low RRS signal is obtained. However, in the presence of Hg(2+), the non-cross-linking aggregation of dsDNA-AuNPs occurs, due to the Hg(2+)-mediated coordination of T-Hg(2+)-T base pair. The aggregation of nanoparticles generates a high RRS value. Particularly, the solution color and ultraviolet-visible absorption barely changed under the same conditions, while it is capable of detecting by RRS method with a low detection limit (0.4nM), which is 1000-fold lower than that of the colorimetric method. The proposed method was successfully applied to the detection of Hg(2+) in real samples. The sensitive and selective assay might be inspiring for the development of new detectors for other metal ions or biomolecules.

Detection of single-nucleotide polymorphisms using an ON-OFF switching of regenerated biosensor based on a locked nucleic acid-integrated and toehold-mediated strand displacement reaction.

Although various strategies have been reported for single-nucleotide polymorphisms (SNPs) detection, development of a time-saving, specific, and regenerated electrochemical sensing platform still remains a realistic goal. In this study, an ON-OFF switching of a regenerated biosensor based on a locked nucleic acid (LNA)-integrated and toehold-mediated strand displacement reaction technique is constructed for detection of SNPs. The LNA-integrated and methylene blue-labeled capture probe with an external toehold is designed to switch on the sensing system. The mutant-type DNA probe completes complementary with the capture probe to trigger the strand displacement reaction, which switches off the sensing system. However, when the single-base mismatched wild-type DNA probe is presented, the strand displacement reaction cannot be achieved; therefore, the sensing system still keeps the ON state. This DNA sensor is stable over five reuses. We further testify that the LNA-integrated sequence has better recognition ability for SNPs detection compared to the DNA-integrated sequence. Moreover, this DNA senor exhibits a remarkable discrimination capability of SNPs among abundant wild-type targets and 6000-fold (m/m) excess of genomic DNA. In addition, it is selective enough in complex and contaminant-ridden samples, such as human urine, soil, saliva, and beer. Overall, these results demonstrate that this reliable DNA sensor is easy to be fabricated, simple to operate, and stable enough to be readily regenerated.

A potential fluorescent probe: Maillard reaction product from glutathione and ascorbic acid for rapid and label-free dual detection of Hg2+ and biothiols

Maillard reactions and their fluorescent products have drawn much attention in the fields of food and life science, however, the application of fluorescent products separated from the reaction as an indicator for detection of certain substances in sensor field has not been mentioned. In this article, we report on an easy-to-synthesize and water-soluble fluorescent probe separated from the typical Maillard reaction products of glutathione and ascorbic acid, with excellent stability and high quantum yield (18.2%). The further application of the probe has been explored for dual detection of Hg(2+) and biothiols including cysteine, homocysteine, and glutathione, which is based on Hg(2+)-induced fluorescence quenching of the Maillard reaction fluorescent products (MRFPs) and the fluorescence recovery as the introduction of biothiols. This sensing system exhibits a good selectivity and sensitivity, and the linear ranges for Hg(2+), cysteine, homocysteine, and glutathione are 0.05-12, 0.5-10, 0.3-20, and 0.3-20μM, respectively. The detection limits for Hg(2+), cysteine, homocysteine, and glutathione are 22, 47, 96, and 30nM at a signal-to-noise ratio of 3, respectively. Furthermore, the practical applications of this sensor for Hg(2+) and biothiols determination in water samples and human plasma sample have been demonstrated with satisfactory results.

Green light-emitting polyepinephrine-based fluorescent organic dots and its application in intracellular metal ions sensing.

In this paper, we present a class of bio-dots, polyepinephrine (PEP)-based fluorescent organic dots (PEP-FODs) for selective and sensitive detection of Fe(2+), Fe(3+), and Cu(2+). The PEP-FODs were derived from epinephrine via self-polymerization at relatively low temperature down to 60°C with low cytotoxicity and relative long lifetime (7.24ns). The surface morphology and optical properties of the synthesized PEP-FODs were characterized. We found that the diameters of PEP-FODs were mainly distributed in the narrow range of 2-4nm with an average diameter of 2.9nm. An optimal emission peak located at 490nm was observed when the green light-emitting PEP-FODs were excited at 400nm. It is discovered that Fe(2+), Fe(3+), and Cu(2+)can strongly quench the fluorescence of PEP-FODs through the nonradiative electron-transfer. The detection limit of 0.16, 0.67, and 0.15μM was obtained for Fe(2+), Fe(3+), and Cu(2+), respectively. The independent sensing platform of Fe(2+), Fe(3+), and Cu(2+)could be established by using NaF as a complexing agent and by regulating the reaction time between NaF and metal ions. Cell viability studies reveal that the as-prepared PEP-FODs possess good solubility and biocompatibility, making it as excellent imaging nanoprobes for intracellular Fe(2+), Fe(3+), and Cu(2+)sensing. The developed PEP-FODs might hold great promise to broaden applications in nanotechnology and bioanalysis.

Diverse States and Properties of Polymer Nanoparticles and Gel Formed by Polyethyleneimine and Aldehydes and Analytical Applications

Multicolor polymer nanoparticles (or dots) were prepared via the reaction between hyperbranched polyethyleneimine (PEI) and aldehydes, and when the concentration of aldehydes was lower, the final mixture displayed gelation behavior. This phenomenon can be applied to visual detection of aldehydes. Moreover, the colors of the polymer dots and gel are varied by using different kinds of aldehydes, which can be utilized for visual discrimination of aldehydes. For simplicity, we focus our attention on the interaction between PEI and formaldehyde. The nanoparticles show an average diameter of 42 nm, emit bright cyan fluorescence with high quantum yield, and exhibit high water dispersibility and excellent photostability. Due to the advantages, our polymer nanoparticles (PNPs) are utilized as a fluorescent probe for imaging in living SK-N-SH cells. Furthermore, valuable explorations have been carried out on the fundamental properties of PNPs, such as concentration-dependent fluorescence, pH-dependent fluorescence, and solvent effect.

Guanine nanowire based amplification strategy: Enzyme-free biosensing of nucleic acids and proteins

Sensitive and specific detection of nucleic acids and proteins plays a vital role in food, forensic screening, clinical and environmental monitoring. There remains a great challenge in the development of signal amplification method for biomolecules detection. Herein, we describe a novel signal amplification strategy based on the formation of guanine nanowire for quantitative detection of nucleic acids and proteins (thrombin) at room temperature. In the presence of analytes and magnesium ions, the guanine nanowire could be formed within 10 min. Compared to the widely used single G-quadruplex biocatalytic label unit, the detection limits are improved by two orders of magnitude in our assay. The proposed enzyme-free method avoids fussy chemical label-ling process, complex programming task, and sophisticated equipment, which might provide an ideal candidate for the fabrication of selective and sensitive biosensing platform.