Scanning electron microscopy coupled with energydispersive X-ray spectrometry for quick detection of sulfuroxidizing bacteria in environmental water samples

Detection of sulfur-oxidizing bacteria has largely been dependent on targeted gene sequencing technology or traditional cell cultivation, which usually takes from days to months to carry out. This clearly does not meet the requirements of analysis for time-sensitive samples and/or complicated environmental samples. Since energy-dispersive X-ray spectrometry(EDS) can be used to simultaneously detect multiple elements in a sample, including sulfur, with minimal sample treatment, this technology was applied to detect sulfur-oxidizing bacteria using their high sulfur content within the cell. This article describes the application of scanning electron microscopy imaging coupled with EDS mapping for quick detection of sulfur oxidizers in contaminated environmental water samples, with minimal sample handling. Scanning electron microscopy imaging revealed the existence of dense granules within the bacterial cells, while EDS identified large amounts of sulfur within them. EDS mapping localized the sulfur to these granules. Subsequent 16S rRNA gene sequencing showed that the bacteria detected in our samples belonged to the genus Chromatium, which are sulfur oxidizers. Thus, EDS mapping made it possible to identify sulfur oxidizers in environmental samples based on localized sulfur within their cells, within a short time(within 24 h of sampling). This technique has wide ranging applications for detection of sulfur bacteria in environmental water samples.

Comparative quantification of human intestinal bacteria based on cPCR and LDR/LCR

AIM: To establish a multiple detection method based on comparative polymerase chain reaction (cPCR) and ligase detection reaction (LDR)/ligase chain reaction (LCR) to quantify the intestinal bacterial components. METHODS: Comparative quantification of 16S rDNAs from different intestinal bacterial components was used to quantify multiple intestinal bacteria. The 16S rDNAs of different bacteria were amplified simultaneously by cPCR. The LDR/LCR was examined to actualize the genotyping and quantification. Two beneficial (Bifidobacterium , Lactobacillus ) and three conditionally pathogenic bacteria (Enterococcus , Enterobacterium and Eubacterium ) were used in this detection. With cloned standard bacterial 16S rDNAs, standard curves were prepared to validate the quantitative relations between the ratio of original concentrations of two templates and the ratio ofthe fluorescence signals of their final ligation products. The internal controls were added to monitor the whole detection flow. The quantity ratio between two bacteria was tested. RESULTS: cPCR and LDR revealed obvious linear correlations with standard DNAs, but cPCR and LCR did not. In the sample test, the distributions of the quantity ratio between each two bacterial species were obtained. There were significant differences among these distributions in the total samples. But these distributions of quantity ratio of each two bacteria remained stable among groups divided by age or sex. CONCLUSION: The detection method in this study can be used to conduct multiple intestinal bacteria genotyping and quantification, and to monitor the human intestinal health status as well.

A lab-on-a-disc platform based on nickel nanowire net and smartphone imaging for rapid and automatic detection of foodborne bacteria

Foodborne pathogenic bacteria have been considered as a major risk factor for food safety. It is of great significance to carry out in-field screening of pathogenic bacteria to prevent the outbreaks of foodborne diseases. In this study, a portable lab-on-a-disc platform with a microfluidic disc was developed for rapid and automatic detection of Salmonella typhimurium using a nickel nanowire(Ni NW) net for effective separation of target bacteria, horseradish peroxidase nanoflowers(HRP NFs) for efficient amplification of biological signals, and a self-developed smartphone APP for accurate analysis of colorimetric images. First,the microfluidic disc was preloaded with reagents and samples and centrifuged to form one bacterial sample column, one immune Ni NW column, one HRP NF column, two washing buffer columns and one tetramethylbenzidine(TMB) column, which were separated by air gaps. Then, a rotatable magnetic field was specifically developed to assemble the Ni NWs into a net, which was automatically controlled by a stepped motor to successively pass through the sample column for specific capture of target bacteria, the HRP NF column for specific label of target bacteria, the washing columns for effective removal of sample background and non-specific binding NFs, and the TMB column for colorimetric determination of target bacteria. The color change of TMB from colorless to blue was finally analyzed using the smartphone APP to quantitatively determine the target bacteria. This lab-on-a-disc platform could detect Salmonella typhimurium from 5.6 × 10^(1) CFU/20 μL to 5.6 × 10^(5) CFU/20 μL in 1 h with a lower detection limit of 56 CFU/20 μL. The recovery of target bacteria in spiked chicken samples ranged from 97.5% to 101.8%. This portable platform integrating separation, labeling, washing, catalysis and detection onto a single disc is featured with automatic operation, fast reaction, and small size and has shown its potential for in-field detection of foodborne pathogens.

Multiplexed detection and differentiation of bacterial enzymes and bacteria by color-encoded sensor hydrogels

We report on the fabrication and characterization of color-encoded chitosan hydrogels for the rapid,sensitive and specific detection of bacterial enzymes as well as the selective detection of a set of tested bacteria through characteristic enzyme reactions.These patterned sensor hydrogels are functionalized with three different colorimetric enzyme substrates affording the multiplexed detection and differentiation ofα-glucosidase,β-galactosidase andβ-glucuronidase.The limits of detection of the hydrogels for an observation time of 60 min using a conventional microplate reader correspond to concentrations of 0.2,3.4 and 4.5 nM of these enzymes,respectively.Based on their different enzyme expression patterns,Staphylococcus aureus strain RN4220,methicillin-resistant S.aureus(MRSA)strain N315,both producingα-glucosidase,but notβ-glucuronidase andβ-galactosidase,Escherichia coli strain DH5α,producingβ-glucuronidase andα-glucosidase,but notβ-galactosidase,and the enterohemorrhagic E.coli(EHEC)strain E32511,producingβ-galactosidase,but none of the other two enzymes,can be reliably and rapidly distinguished from each other.These results confirm the applicability of enzyme sensing hydrogels for the detection and discrimination of specific enzymes to facilitate differentiation of bacterial strains.Patterned hydrogels thus possess the potential to be further refined as detection units of a multiplexed format to identify certain bacteria for future application in point-of-care microbiological diagnostics in food safety and medical settings.

β-Cyclodextrin-modified AuBi metallic aerogels enable efficient peroxidase mimicking for colorimetric sensing of urease-positive pathogenic bacteria

The detection of pathogenic bacteria with improved accessibility,reduced analysis time,and increased sensitivity is of great importance for diagnosing the infected disease.Nanozymes have attracted rising attention in the bioassay field.Designing a model nanozyme needs the combined merit of sensible nanostructures and a large specific surface area to guarantee exceptional enzyme-mimic activity.Herein,aβ-cyclodextrin modified AuBi aerogel is prepared by a one-pot reduction strategy.The introduction ofβ-cyclodextrin(featured with a hydrophobic cavity and hydrophilic surface)enhances the catalytic activity of AuBi aerogels by engendering host-guest complex and improving dispersity/stability.Based on the specific urea hydrolysis,which could produce NH_(3)to raise pH by urease,the pH up-regulation would inhibit the peroxidase-mimicking performances ofβ-cyclodextrin/AuBi aerogels.Therefore,the sensitive colorimetric detection platform for urease activity could be constructed.Moreover,the sensing platform can detect straightforwardly urease-positive Proteus mirabilis in urine circumstances with a wide detection range and a low limit of detection(LOD)of 4 colony-forming unit(CFU)·mL^(-1).The reproducibility,stability,and specificity of this approach are verified to be satisfactory.Also,as an inhibitor of urease activity,the fluoride ion could be detected by the constructed sensing platform sensitively and specifically.Overall,this work provides a blueprint for designing an ideal nanozyme and paves a new roadway for detecting pathogenic bacteria.

Specific detection and effective inhibition of a single bacterial species in situ using peptide mineralized Au cluster probes

Increasingly serious microbial infections call for the development of new simpler methods for the precise diagnosis and specific inhibition of such pathogens. In this work, a peptide mineralized Au cluster probe was applied as a new simplified strategy to both recognize and inhibit a single bacteria species of Staphylococcus aureus(S. aureus) simultaneously. The probes are composed of peptides and Au clusters. Moreover, the peptides specifically target S. aureus cells and the Au clusters provide fluorescent imaging and have an antibacterial effect. These new probes enable the simultaneous specific detection and effective destruction S. aureus cells in situ.