Le Deng

Fluorescent aptasensor for the determination of Salmonella typhimurium based on a graphene oxide platform

AbstractWe report on an aptamer with high affinity against Salmonella typhimurium (S. typhimurium) and selected from an enriched oligonucleotide pool by a whole-cell SELEX process in a method for the fluorimetric determination of S. typhimurium using a graphene oxide platform. In the absence of target, the fluorescence was fairly weak as result of the FAM-labeled aptamer adjacent to graphene oxide. If, however, the fluorophore is released from the graphene oxide due to the formation of the target/aptamer complexes, fluorescence intensity is substantially increased. Under the optimum conditions, the assay displays a linear response to bacteria in the concentration range from 1u2009×u2009103 to 1u2009×u2009108xa0CFU·mL−1, with a detection limit of 100xa0CFU·mL−1. The method is selective in that fluorescence is not much enhanced in case of other bacteria. This aptasensor displays higher sensitivity and selectivity than others and is believed to possess a large potential with respect to the rapid detection of bacteria.n FigureA useful fluorescence aptasensor based on a graphene oxide platform was constructed for Salmonella typhimurium detection, which has a great potential application in rapid detection of pathogen as it has high sensitivity and selectivity.

Nicking enzyme-assisted biosensor for Salmonella enteritidis detection based on fluorescence resonance energy transfer

Salmonella enteritidis (S. enteritidis) outbreaks continue to occur, and have increased public awareness of this pathogen. Nicking endonuclease Nb.BbvC I is widely used for the detection of biomolecules and displays activity for specific double-stranded DNA (dsDNA). In this study, we developed a biosensor to detect S. enteritidis based on fluorescence resonance energy transfer (FRET) using nicking enzyme and carbon nanoparticles (CNPs). Because of the quenching effect of black hole quencher 1 (BHQ 1), the CNPs do not fluoresce in the reaction system. When the target bacteria are added, the nicking enzyme recognizes and cleaves the dsDNA fabricated by the interaction between probe and target. As a result, the CNPs dissociate from BHQ 1 and emit strong fluorescence. Using the nicking enzyme, the fluorescence signals of the biosensor are greatly amplified. The biosensor exhibited a linear relationship with the concentration of S. enteritidis ranging from 10(2) to 3 × 10(3)CFU/mL in water and from 1.5 × 10(2) to 3 × 10(3)CFU/mL in milk. The present results indicate that our FRET-based detection system can be widely employed for the effective detection of pathogens.

Aptamer-based detection of Salmonella enteritidis using double signal amplification by Klenow fragment and dual fluorescence

AbstractThis article describes a sensitive and selective fluorometric method for the determination of Salmonella enteritidis by exploiting the polymerase activity of the Klenow fragment and dual fluorescence. First, one end of a target-selective aptamer was labeled with the fluorophore 6-carboxyfluorescein (FAM). Once the labeled aptamer binds to graphene oxide (GO) via π-stacking interaction, the fluorescence of FAM is quenched. However, the addition of target (16S rRNA) leads to the restoration of fluorescence due to the binding of probe and target which shifts the FAM fluorophore away from the quenching GO. By using the Klenow fragment and by exploiting the synergistic effect of FAM and the DNA probe SYBR Green I (which is strongly fluorescent in presence of dsDNA only), fluorescence is strongly amplified and sensitivity improved. The analyte 16SrRNA can be determined by this method in the 60 pM to 100xa0nM concentration range, and the detection limit is 60 pM. It is also shown that Salmonella enteritidis can be determined in milk samples by this method in concentrations between 102 to 105xa0cfu⋅mL‾1, with a detection limit of 300xa0cfu⋅mL‾1. This assay displays high sensitivity and selectivity and may possess wide applications in pathogen detection.n Graphical Abstract:If labeled aptamer against Salmonella enteritidis 16S rRNA binds to graphene oxide GO) by π-interaction, the fluorescence of the label is quenched. The addition of target rRNA leads to the restoration of fluorescence, and this effect can be used to quantify Salmonella enteritidis.

High Specific DNAzyme-Aptamer Sensor for Salmonella paratyphi A Using Single-Walled Nanotubes–Based Dual Fluorescence-Spectrophotometric Methods:

In this work, single-stranded DNA aptamers that are highly specific to enterotoxigenic Salmonella paratyphi A were obtained from an enriched oligonucleotide pool using Systematic Evolution of Ligands by Exponential Enrichment (SELEX) to target the flagellin protein. The selected aptamers were confirmed to have high sensitivity and specificity to the flagellin. In addition, a probe (P0) containing the DNAzyme and fluorescein isothiocyanate–labeled aptamer3 sequences was employed as a dual probe for observing fluorescence and absorbance changes. The flagellin demonstrated low detection limits of 5 ng/mL by fluorescence and 20 ng/mL by spectrophotometry. Moreover, milk samples spiked with Salmonella paratyphi A were also detected, with the low detection limits increasing to 105 CFU/mL by fluorescence and 106 CFU/mL by spectrophotometry. The combination of fluorescence and spectrophotometry offers a specific, rapid, and sensitive way for detecting Salmonella paratyphi A and has potential for detecting other pathogens in food.

Label-Free Fluorescent Aptasensor Based on a Graphene Oxide Self-Assembled Probe for the Determination of Adenosine Triphosphate

A sensitive and selective fluorescent aptasensor for adenosine triphosphate (ATP) was fabricated, composed of unbound SYBR Green I, graphene oxide, and a label-free detection probe. When ATP and complementary DNA of a signal probe were introduced, π-stacking interactions repelled the probe from the graphene oxide and formed a DNA-SYBR Green I duplex structure, triggering an increase in the fluorescence. ATP was determined over a linear range of 10 to 700 nM with a detection limit of 1 nM. The method displayed good selectivity, and is currently the most sensitive ATP fluorescence method. Furthermore, prominent fluorescence signals were also obtained in cellular assays. Consequently, the biosensor may have significant applications in protein, pathogenic microorganisms, and small molecule detection.

Highly sensitive fluorescent aptasensor for Salmonella paratyphi A via DNase I-mediated cyclic signal amplification

Outbreaks of Salmonella paratyphi A (S. paratyphi A) infection continue to occur worldwide and have drawn close attention. A useful practical detection platform is essential to the early rapid diagnosis of the infection. In this study, a simple and cost-effective DNA aptasensor was constructed, which was composed of a designed aptamer (DA) and two short carboxyfluorescein (FAM)-modified sequences (probe 1 and probe 2) for fluorimetric determination of S. paratyphi A. In the absence of a target, the two-FAM aptasensor (the aptasensor) was bound to graphene oxide (GO) and the fluorescence of FAM was quenched. In the presence of a target, however, the aptasensor was released from the surface of GO due to specific binding of the aptasensor to the target and a strong fluorescence signal could subsequently be detected. More importantly, the fluorescence signal could be substantially amplified by a DNase I-mediated target recycling process. Under the optimized conditions, the fluorescence intensity increased linearly with the target concentrations ranging from 1 × 102 to 1 × 1011 cells per mL with a detection limit of 1 × 102 cells per mL. These results demonstrated that this detection platform exhibited high sensitivity and specificity for the detection of S. paratyphi A, and it might even be a potential alternative approach for the detection of other bacteria.

Determination of Shigella flexneri by a Novel Fluorescent Aptasensor

A simple, sensitive, selective, and homogeneous fluorescent aptasensor was constructed for Shigella flexneri by using a dye-labeled aptamer and graphene oxide using target recycling amplification. Unique recognition sequences of restriction endonuclease ApaI from E. coli that carried the cloned apaIR gene from Acetobacter pasteurianus were incorporated into a single-stranded signal probe labeled with carboxyfluorescein at the 5′ and 3′ ends. In the absence of target bacteria, probes were adsorbed and quenched by graphene oxide. After the addition of the analyte, an iterative process called target recycling amplification was triggered. Specific binding of the aptamer to the target liberated probes from graphene oxide. The associated probes were recognized and cleaved by the enzyme. In addition, the released targets were reemployed in subsequent measurements. Under the optimum conditions, a linear calibration relationship was displayed from 500 to 109 CFU/mL with a limit of detection of 100 CFU/mL and high selectivity. Consequently, this aptasensor is an attractive alternative to conventional methodologies.

A Novel Biosensor for Detection of Salmonella typhimurium Carrying SSeC Gene Based on the Secondary Quenching Effect of Carbon Nanotubes

A Novel Biosensor for Detection of Salmonella typhimurium Carrying SSeC Gene Based on the Secondary Quenching Effect of Carbon Nanotubes nIn this paper, a sensitive and selective biosensor was constructed for detection of Salmonella typhimurium carrying SSeC gene, based on covalently coupling of molecular beacons (MBs) stained with daunorubicins (DNR) to single-walled carbon nanotubes (SWNTs) through EDC/Sulfo-NHS chemistry. In the absence of target, the fluorescence of daunorubicin was fairly week as result of dual fluorescence quenching. On the contrary, the daunorubicin was competed from the beacon due to the target-induced formation of rigid structure between the loop structure of the MB and the target sequence, which resulted in a decrease in the effect of dual fluorescence quenching, thereby the fluorescence intensity increased substantially. The target quantum was achieved by fluorescence increment. The experimental results showed that the recovery of fluorescence of daunorubicin is proportion to theconcentration of the target DNA with the range 0.2-0.7 μM and the low detection limit is 50 nM. The fluorescence intensity did not augment considerably when other Salmonella sps. were detected via the same method, which clearly displayed a high selectivity and specificity for the biosensor. Additionally, the real samples were also detected and their low detection limits were up to 105 CFU/mL. Consequently, the biosensor should be a potential alternative to the conventional detection ones and has great prospect in pathogenic microorganisms’ detection, clinical diagnosis and treatment.

Combat biofilm by bacteriostatic aptamer‐functionalized graphene oxide

Biofilms are the main reason for a large number deaths and high health costs. Their better protection compared to planktonic form against conventional antibiotics leads to poor treatment efficiency. Nanoagent‐targeted delivery is a promising avenue for disease therapeutic, but its application targeting biofilms has not been reported currently. The roles, if any, of aptamers acting as delivery carrier and targeting factor, the graphene oxide (GO), and GO modified with aptamers against biofilms were then systematically evaluated. Here, we successfully developed an aptamer‐targeted GO strategy against biofilms. We investigated the efficacy of aptamer–GO conjugates by UV spectrophotometer, inverted microscopy, and atomic force microscopy; 93.5 ± 3.4% Salmonella typhimurium biofilms were inhibited and 84.6 ± 5.1% of biofilms were dispersed by a ST‐3‐GO conjugate. More importantly, this conjugate represented distinctively toxicity to S. typhimurium. Thus, this strategy significantly displays excellent antibiofilm properties and may serve as a long‐term solution for biofilm control.

Application of Activated Biomaterial in the Rapid Start-up and Stable Operation of Biological Processes for Removal Cadmium from Effluent

With the aim of treating effluent containing Cd2+, a low-cost and efficient technique has been established in this work. By a combination of sulfate-reducing bacteria (SRB), carboxymethyl konjac glucomannan (CMKGM), and nickel–iron bimetallic (Ni/Fe) nanoparticles, we greatly enhanced Cd2+ removal and bacteria resistance to metals toxicity. Furthermore, it had much higher removal efficiencies (99.72%) than SRB (57.38%), CMKGM (52.46%), and Ni/Fe (58.91%) systems after 48xa0h in the treat processes. The parameters effecting Cd2+ removal of this system were obtained: the initial Cd2+ concentrations 150xa0mg/L, optimum pH 7.0, optimum temperature 37xa0°C, optimum time 48xa0h, respectively. CMKGM and SRB played significant roles in Cd2+ adsorption as they contained many functional groups on their surfaces. In addition, SRB promoted the degeneration of inorganic contaminants. The mechanism of adsorption was clarified by a serious of analysis. Overall, this study provided a highly efficient activated biomaterial for the practical treatment of Cd2+ in wastewater.