Rapid field testing of mercury pollution by designed fluorescent biosensor and its cells-alginate hydrogel-based paper assay

With increasing industrial activities,mercury has been largely discharged into environment and caused serious environmental problems.The growing level of mercury pollution has become a huge threat to human health due to its significant biotoxicity.Therefore,the simple and fast means for on-site monitoring discharged mercury pollution are highly necessary to protect human beings from its pernicious effects in time.Herein,a"turn off"fluorescent biosensor(mCherry L199 C)for sensing Hg2+was successfully designed based on direct modification of the chromophore environment of fluorescent protein mCherry.For rapid screening and characterization,the designed variant of mCherry(mCherry L199 C)was directly expressed on outer-membrane of Escherichia coli cells by cell surface display technique.The fluorescent biosensor was characterized to have favorable response to Hg2+at micromole level among other metal ions and over a broad pH range.Further,the cells of the fluorescent biosensor were encapsulated in alginate hydrogel to develop the cells-alginate hydrogel-based paper.The cells-alginate hydrogel-based paper could detect mercury pollution in 5 min with simple operation process and inexpensive equipment,and it could keep fluorescence and activity stable at 4℃ for 24 hr,which would be a high-throughput screening tool in preliminarily reporting the presence of mercury pollution in natural setting.

Self-assembled Nanosheets of Perylene Monoamide Derivative as Sensitive Fluorescent Biosensor for Exonuclease Ⅲ Activity

Enzymes containing 3′→5′exonuclease activities play an important role in various key cellular and physiological processes.The development of fluorescence biosensor is an efficient method to detecting enzyme activity.Herein,a fluorescence resonance energy transfer(FRET)“on”and“off”strategy for detecting exonuclease III(Exo III)activity has been developed.We report here that the double-stranded DNA(dsDNA)enables to bind tightly to self-assembled nanosheets of cationic perylene monoimide derivative(PMI-O7)through electrostatic interaction,and the 6-carboxyfluorescein(FAM)-modified dsDNA could be efficiently quenched via FRET between FAM and PMI-O7.Upon the addition of Exo III,the dsDNA will be digested and the FAM fluorophore will be released,resulting in the fluorescence recovery of FAM.This method provides a simple and sensitive biosensor platform with a low detection limit of 0.077 U/mL for Exo III.Importantly,this method exhibits similar and calibration curves for the detection of Exo III in both buffer and fetal bovine serum samples,indicating that this platform has potential to detect Exo III activity in complex samples.

Chondroitin Sulfate Fluorescence Biosensor Based on Graphere Quantum Dots Aggregating on CMC/CS Polyelectrolyte Microspheres

Here, we report an efficient fluorescence biosensor for chondroitin sulfate（CHS） based on polyelectrolyte microspheres of carboxymethyl cellulose（CMC） and chitosan（CS） composites inducing the aggregation of graphene quantum dots（GQDs）, calling CMC/CS-GQDs. The polyelectrolyte microspheres（CMC/CS microspheres） were fabricated by using anioniccationic electrostatic attraction between CMC and CS by high voltage electrostatic spray technology. The aggregating process of GQDs was based on the anionic-cationic electrostatic attraction as well. After combing with the polyelectrolyte microspheres, the fluorescence of GQDs disappeared. CHS, which widely consists in the cell surface of human beings and animals, carries a large number of negative charges on the surface. The addition of CHS enabled CHS and GQDs to compete with each other to composite with the CMC/CS microshpheres. As a result of the higher surface charge density of CHS, CMC/CS-CHS formed accompanied by the release of GQDs, and the fluorescence of the system recovered. The CHS content was detected by analyzing the system＇s fluorescence recovery, which suggested that the obtained fluorescence biosensor can accurately detect the concentration of CHS. The test results showed that the linear range of the fluorescence recovery for this biosensor with respect to CHS was 0~12.00 mg/mL, and the detection limit was 10-8 M. Besides, to test the stability of the biosensor, the CMC/CS-GQDs micropsheres persisted for one month, with a low fluorescence quenching of 9.48%. These results suggested that CMC/CS-GQDs can be utilized as efficient fluorescence biosensor for the detection of CHS. Moreover, the detection method was simple and efficient, and could be widely popularized.

Magnetic covalent organic framework nanospheres-based miRNA biosensor for sensitive glioma detection

Sensitive detection and accurate diagnosis/prognosis of glioma remain urgent challenges.Herein,dispersed magnetic covalent organic framework nanospheres(MCOF)with uniformed Fe3O4 nano-assembly as cores and high-crystalline COF as shells were prepared by monomer-mediated in-situ interface growth strategy.Based on the unique interaction between MCOF and hairpin DNA,a fluorescent signal amplified miRNA biosensor was constructed.It could realize the sensitive detection of miRNA-182 in different matrixes,where the detection limit,linearity range and determination coefficient(R^(2))in real blood samples reached 20 fM,0.1 pM-10 pM and 0.991,respectively.Also,it possessed good stability and precision as observed from the low intra-day/inter-day RSD and high extraction recovery.As a result,it could quantify miRNA-182 in serum of glioma patients,the concentration of which was significantly higher than that of healthy people and obviously decreased after surgery.Finally,a proof-of-concept capillary chip system using this biosensor was proposed to realize the visualized detection of miRNA-182 in microsample.These findings suggest a robust way for sensitive detection and accurate diagnosis/prognosis of glioma.

Construction of a dual fluorescence whole-cell biosensor to detect N-acyl homoserine lactones

Detection of N-acyl homoserine lactones （AHLs） is useful for understanding quorum sensing （QS） behaviors, including biofilm formation, virulence and metabolism. For detecting AHLs and indicating the host cells in situ, we constructed the plasmid pUCGMA2T1-4 to make a dual fluorescent whole- cell biosensor based on the AhlI/R AHL system of Pseudomonas syringae pv. syringae B728a. The plasmid contains three components： constitutively expressed enptll：：gfP for indicating host cells, Pahll：：mcherry that produces red fluorescence in response to AHL, and the ahIR gene that encodes an AHL regulatory protein. Meanwhile, two copies of T1-4 （four tandem copies of a transcriptional terminator） were added into the plasmid to reduce background. The results showed that when the plasmid was placed into Escherichia coli, the dual fluorescence whole-cell biosensor was able to respond with red fluorescence within 6 hr to 5 × 10^-8-1 × 10^-5 mol/L of 3OC6-HSL. Bright green fluorescence indicated the host cells. Furthermore, when the plasmid was transferred to wild- type Pseudomonas PhTA125 （an AHL-producing bacterium）, it also showed both green and red fluorescence. This result demonstrates that this plasmid can be used to construct whole-cell indicators that can indicate the AHL response and spatial behaviors of microbes in a mi tal niche

Cross-regulation between CDK and MAPK control cellular fate

Background:Commitment to a new cell cycle is controlled by a number of cellular signals.Mitogen-activated protein kinase(MAPK)pathways,which transduce multiple extracellular cues,have been shown to be interconnected with the cell cycle and can modulate its progression.Methods:In budding yeast,we have introduced fluorescent biosensors that monitor in real time the signaling activity of the MAPKs Fus3 and Kssl and the cyclin-dependent kinase(CDK)in individual cells.We have quantified in hundreds of live single cells the interplay between the MAPKs regulating the mating response and the CDK controlling cell cycle progression.Results:Different patterns of MAPK activity dynamics could be identified by clustering cells based on their CDK activity,denoting the tight relationship between these two cellular signals.Our data suggest that beyond the already well-established mechanisms of regulation between the MAPK and the CDK,additional mechanisms remain to be identified.Conclusion:A tight interplay between MAPK pathways and the cell cycle is essential to control cellular proliferation and cell fate decisions.

Two-dimensional coordination polymer-based nanosensor for sensitive and reliable nucleic acids detection in living cells

A reliable and sensitive strategy which can assess nucleic acid levels in living cells would be essential for fundamental research of biomedical applications. Some nanomaterial-based fluorescence biosensors recently developed for detecting nucleic acids, however, are often with expensive, complicated and timeconsuming preparation process. Here, by using a facile bottom-up synthesis method, a two-dimensional(2 D) coordination polymer(CP) nanosheet, [Cu(tz)](Htz = 1,2,4-triazole), was successfully prepared after optimizing reaction conditions. These ultrathin CP nanosheets with thickness of 4.7 ± 1.1 nm could readily form nanosensors by assembly with DNA probes, which exhibited a low limit of detection(LOD)for p53 DNA fragment as 144 pmol/L. Furthermore, by integrating [Cu(tz)] nanosheets with hybridization chain reaction(HCR) probes, mi R-21, one kind of micro RNA upregulated in many cancer cells, can be sensitively detected with a LOD of 100 pmol/L and monitored in living cells, giving consistent results with those obtained by quantitative reverse-transcription polymerase chain reaction(q RT-PCR) analysis.Thus [Cu(tz)] nanosheets, which not only possess much better nucleic acids sensing performance than bulk cystals, but also exhibit nucleic acid delivery functions, could be used as a novel nanoplatform in biomedical imaging and sensing applications.