Fluorescence detection of total count of Escherichia coli and Staphylococcus aureus on water-soluble CdSe quantum dots coupled with bacteria

The aim of this paper was to demonstrate a fluorescence measurement method for rapid detection of two bacterial count by using water-soluble quantum dots (QDs) as a fluorescence marker, and spectrofluorometer acted as detection apparatus, while Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were as detection target bacteria. Highly luminescent water-soluble CdSe QDs were first prepared by using thioglycolic acid (TGA) as a ligand, and were then covalently coupled with target bacteria. The bacterial cell images were obtained using fluorescence microscopy. Our results showed that CdSe QDs prepared in water phase were highly luminescent, stable, and successfully conjugated with E. coli and S. aureus. The fluorescence method could detect 10²-10⁷ CFU/mL total count of E. coli and S. aureus in 1-2 h and the low detection limit is 10² CFU/mL. A linear relationship of the fluorescence peak intensity and log total count of E. coli and S. aureus have been established using the equation Y = 118.68X − 141.75 (r = 0.9907).     

Immunomagnetic separation and rapid detection of bacteria using bioluminescence and microfluidics

This paper describes an immunomagnetic separation of target bacterial cells from others by using magnetic bead. The surface of bead was coated with antibodies which can capture specific organism. The binding efficiency of immunomagnetic bead (IMB) capturing target bacterial cells was higher than 98% when the concentrations of target and interferent bacterial cells were at the same level. The concentration of bacteria was determined indirectly by detecting adenosine 5′-triphosphate (ATP) employing bioluminescence (BL) reaction of firefly luciferin-ATP. Benzalkonium chloride (BAC) was used as an ATP extractant from living bacterial cells. We found that BAC could enhance the light emission when the concentration of BAC was less than 5.3 × 10⁻²% (w/v) and the BL intensity reached its maximum at the concentration of BAC was 2.7 × 10⁻²%, which was 10-fold stronger than that without BAC. Based on the principle of the IMB, a microfluidic chip combined with immunofluorescence assay for separating and detecting bacteria simultaneously was also developed. The IMBs were magnetically fixed in the bead-beds of chip channels with a 3-mm diameter of NdFeB permanent magnet. The target bacterial cells can be captured magnetically and observed by a fluorescent microscope.     

Dual-color upconversion fluorescence and aptamer-functionalized magnetic nanoparticles-based bioassay for the simultaneous detection of Salmonella Typhimurium and Staphylococcus aureus

A sensitive luminescent bioassay for the simultaneous detection of Salmonella Typhimurium and Staphylococcus aureus was developed using aptamer-conjugated magnetic nanoparticles (MNPs) for both recognition and concentration elements and using upconversion nanoparticles (UCNPs) as highly sensitive dual-color labels. The bioassay system was fabricated by immobilizing aptamer 1 and aptamer 2 onto the surface of MNPs, which were employed to capture and concentrate S. Typhimurium and S. aureus. NaY_0.78F₄:Yb_0.2,Tm_0.02 UCNPs modified aptamer 1 and NaY_0.28F₄:Yb_0.70,Er_0.02 UCNPs modified aptamer 2 further were bond onto the captured bacteria surface to form sandwich-type complexes. Under optimal conditions, the correlation between the concentration of S. Typhimurium and the luminescent signal was found to be linear within the range of 10¹–10⁵ cfu mL⁻¹ (R² = 0.9964), and the signal was in the range of 10¹–10⁵ cfu mL⁻¹ (R² = 0.9936) for S. aureus. The limits of detection of the developed method were found to be 5 and 8 cfu mL⁻¹ for S. Typhimurium and S. aureus, respectively. The ability of the bioassay to detect S. Typhimurium and S. aureus in real water samples was also investigated, and the results were compared to the experimental results from the plate-counting methods. Improved by the magnetic separation and concentration effect of MNPs, the high sensitivity of UCNPs, and the different emission lines of Yb/Er- and Yb/Tm-doped NaYF₄ UCNPs excited by a 980 nm laser, the present method performs with both high sensitivity and selectivity for the two different types of bacteria.     

Light scattering sensing detection of pathogens based on the molecular recognition of immunoglobulin with cell wall-associated protein A

In this contribution, we report a rapid optical detection method of pathogens using Staphylococcus aureus (S. aureus) as the model analyte based on the molecular recognition of immunoglobulin with cell wall-associated Protein A (SpA). It was found that the molecular recognition of human immunoglobulin (IgG) with protein A on the cell wall of S. aureus on glass slide sensing area could result in strong surface enhanced light scattering (SELS) signals, and the SELS intensity (ΔI) increases proportionally with the concentration of S. aureus over the range of 2.5 × 10⁵-1.0 × 10⁸ CFU mL⁻¹ with right angle light scattering (RALS) signals detection mode. In order to identify the solid support based molecular recognition between IgG with SpA, we also employed water-soluble CdS quantum dots (CdS-QDs) as a fluorescent marker for IgG by immobilizing the IgG onto the surfaces of CdS-QDs through covalent binding in order to generate recognition probes for SpA on the cell wall of S. aureus. Consequently, the fluorescent method also showed that the detection for pathogens with solid supports is reliable based on the molecular recognition of IgG with SpA.     

量子点在法庭科学手印显现中的研究进展

随着纳米技术的进步,纳米颗粒正在被逐步应用到法庭科学领域的手印检验之中。近年来,半导体量子点因其良好的荧光特性而备受国内外法庭科学家的推崇,但大多数半导体量子点具有毒性,且会对环境造成污染,这些问题制约了半导体量子点在法庭科学领域中的应用。与传统有机染料和金属内核的半导体量子点相比,碳量子点具有毒性低、污染小、生物相容性优异的特点,现已应用于医学、生物、化学等多个领域。本文综述了半导体量子点在手印显现中的应用,介绍了碳量子点的研究进展,并指出碳量子点显现手印是今后法庭科学领域的重要研究方向。     

A novel fluorescence immunoassay for the sensitive detection of Escherichia coli O157:H7 in milk based on catalase-mediated fluorescence quenching of CdTe quantum dots

Immunoassay is a powerful tool for rapid detection of food borne pathogens in food safety monitoring. However, conventional immunoassay always suffers from low sensitivity when it employs enzyme-catalyzing chromogenic substrates to generate colored molecules as signal outputs. In the present study, we report a novel fluorescence immunoassay for the sensitive detection of E. coli O157:H7 through combination of the ultrahigh bioactivity of catalase to hydrogen peroxide (H₂O₂) and H₂O₂-sensitive mercaptopropionic acid modified CdTe QDs (MPA-QDs) as a signal transduction. Various parameters, including the concentrations of anti-E. coli O157:H7 polyclonal antibody and biotinylated monoclonal antibody, the amounts of H₂O₂ and streptavidin labeled catalase (CAT), the hydrolysis temperature and time of CAT to H₂O₂, as well as the incubation time between H₂O₂ and MPA-QDs, were systematically investigated and optimized. With optimal conditions, the catalase-mediated fluorescence quenching immunoassay exhibits an excellent sensitivity for E. coli O157:H7 with a detection limit of 5 × 10² CFU/mL, which was approximately 140 times lower than that of horseradish peroxidase-based colorimetric immunoassay. The reliability of the proposed method was further evaluated using E. coli O157:H7 spiked milk samples. The average recoveries of E. coli O157:H7 concentrations from 1.18 × 10³ CFU/mL to 1.18 × 10⁶ CFU/mL were in the range of 65.88%–105.6%. In brief, the proposed immunoassay offers a great potential for rapid and sensitive detection of other pathogens in food quality control.     

Use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide for rapid detection of methicillin-resistant Staphylococcus aureus by resonance light scattering

A rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) using resonance light scattering (RLS) on an ordinary fluorescence spectrometer was developed. The viable MRSA reduced 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to produce insoluble particles which displayed intense resonance scattering light. It showed a linear relationship between the number of viable MRSA and the RLS intensity. Dead MRSA were unable to reduce MTT. MRSA exposed to flavonoids extracted from Marchantia convoluta (MCF) showed a MCF concentration-dependent inhibition of the ability to reduce MTT. The RLS could, in combination with the MTT assay, be a rapid and sensitive detection method for vitro-cultured MRSA.     

氮掺杂鸡毛碳点荧光关开探针用于测定百草枯

以鸡毛和乙二胺为碳源和氮源,通过一步水热法合成强荧光性能的氮掺杂碳量子点(N-CQDs),并优化其制备和掺杂条件.该碳量子点具有良好的光学、结构性质和稳定性,平均粒径7.89 nm,荧光量子产率为14％.最大激发波长为320 nm,最大发射波长为386 nm.Hg2+存在条件下N-CQDs溶液的荧光被碎灭(关),添加百...     

Synthesis of chloroquinocin, a pyranonaphthoquinone antibiotic against Gram-positive bacteria

Chloroquinocin 1 is an antimicrobial agent against Gram-positive bacteria, including MRSA (methicillin-resistant Staphylococcus aureus). A successful synthesis of 1 was attained through a unique chlorination of the corresponding naphthoquinone derivative 12 as a key step.     

A dual-color flow cytometry protocol for the simultaneous detection of Vibrio parahaemolyticus and Salmonella typhimurium using aptamer conjugated quantum dots as labels

A sensitive, specific method for the collection and detection of pathogenic bacteria was demonstrated using quantum dots (QDs) as a fluorescence marker coupled with aptamers as the molecular recognition element by flow cytometry. The aptamer sequences were selected using a bacterium-based SELEX strategy in our laboratory for Vibrio parahaemolyticus and Salmonella typhimurium that, when applied in this method, allows for the specific recognition of the bacteria from complex mixtures including shrimp samples. Aptamer-modified QDs (QD-apt) were employed to selectively capture and simultaneously detect the target bacteria with high sensitivity using the fluorescence of the labeled QDs. The signal intensity is amplified due to the high photostability of QDs nanoparticles, resulting in improved sensitivity over methods using individual dye-labeled probes. This proposed method is promising for the sensitive detection of other pathogenic bacteria in food stuff if suitable aptamers are chosen. The method may also provide another potential platform for the application of aptamer-conjugated QDs in flow cytometry.     

Rapid detection of Escherichia coli contamination in packaged fresh spinach using hyperspectral imaging

A rapid method based on hyperspectral imaging for detection of Escherichia coli contamination in fresh vegetable was developed. E. coli K12 was inoculated into spinach with different initial concentrations. Samples were analyzed using a colony count and a hyperspectroscopic technique. A hyperspectral camera of 400-1000 nm, with a spectral resolution of 5 nm was employed to acquire hyperspectral images of packaged spinach. Reflectance spectra were obtained from various positions on the sample surface and pretreated using Sawitzky-Golay. Chemometrics including principal component analysis (PCA) and artificial neural network (ANN) were then used to analyze the pre-processed data. The PCA was implemented to remove redundant information of the hyperspectral data. The ANN was trained using Bayesian regularization and was capable of correlating hyperspectral data with number of E. coli. Once trained, the ANN was also used to construct a prediction map of all pixel spectra of an image to display the number of E. coli in the sample. The prediction map allowed a rapid and easy interpretation of the hyperspectral data. The results suggested that incorporation of hyperspectral imaging with chemometrics provided a rapid and innovative approach for the detection of E. coli contamination in packaged fresh spinach.     

Combining biofunctional magnetic nanoparticles and ATP bioluminescence for rapid detection of Escherichia coli

A rapid, specific and sensitive method for assay of Escherichia coli (E. coli) using biofunctional magnetic nanoparticles (BMNPs) in combination with adenosine triphosphate (ATP) bioluminescence was proposed. The BMNPs were fabricated by immobilizing a specific anti-E. coli antibody on the surface of amine-functionalized magnetic nanoparticles (about 20 nm in diameter), and then was applied to capture the target bacteria E. coli from samples. The BMNPs exhibited high capture efficiency to E. coli. Transmission electron microscope (TEM) images showed that the BMNPs were bound to the surface of entire E. coli cells. The target bacteria became magnetic so that could be isolated easily from the sample solution by employing an external magnetic field. The concentration of E. coli captured by the BMNPs was then detected by an ATP bioluminescence method. The optimization of ATP measurement was carried out to improve the detection sensitivity. The proposed method was applied to detect the E. coli inoculated into pasteurized milk with low detection limit (20 cfu/mL) and short detection time (about 1 h).     

氮掺杂荧光碳点用于Co2+的超灵敏检测及细胞成像

首次以金银花和乙二胺为原料,通过水热法合成出性能优异的氮掺杂碳点(N-CDs)。制备的N-CDs具有丰富的官能团、良好的水溶性、低细胞毒性、高的荧光稳定性和良好的生物相容性。在最佳条件下,N-CDs能够高选择性地检出Co²⁺,N-CDs的荧光强度在0.5～3.6 nmol·L^-1范围内被Co²⁺线性猝灭,检出限低至1.38 nmol·L^-1,其猝灭机制属于内滤效应和静态猝灭。该方法也已成功应用于实际样品的精确分析。此外,N-CDs还可用于细胞成像及细胞内Co²⁺传感。     

荧光磁性碳点对卵巢癌细胞的靶向成像与检测

利用高温碳化法制备了荧光磁性钆掺杂碳点(Gd-CDs),其具有明亮的绿色荧光发射,荧光量子产率为32.6%,T1弛豫效率高达 9.02 L·mmol^-1·s^-1。将 Gd-CDs与卵巢癌特异性抗体 Anti-HE4连接,得到的 Anti-HE4/Gd-CDs纳米探针能对 SKOV-3卵巢癌细胞进行特异性的荧光成像和 T1加权成像,其荧光信号强度与 SKOV-3的细胞浓度成正比,对 SKOV-3细胞的检测限为658 cell·mL^-1。以该方法检测了血液样品中的SKOV-3细胞。     

Total internal reflection imaging of microorganism adhesion using an oil immersion objective

In this paper, we report the results of total internal reflection microscopy investigations of the interaction of two types of microorganisms: Saccharomyces cerevisiae and Escherichia coli with substrates. It is shown that with this method qualitative and quantitative information about cells-substrate interaction can be obtained. One can easily make a difference between attached and non-attached as well as between dead and alive cells, and more generally can follow the dynamics of the process of cells' attachment to substrates. Quantitative information about the cell size and cell-substrate distance is obtained by using a model in which yeast cells and bacteria are approximated by ellipsoids, and multiple reflections of the evanescent waves between the cells and the substrate are neglected.     

荧光磁性碳点对卵巢癌细胞的靶向成像与检测

利用高温碳化法制备了荧光磁性钆掺杂碳点(Gd-CDs),其具有明亮的绿色荧光发射,荧光量子产率为32.6%,T1弛豫效率高达 9.02 L·mmol^-1·s^-1。将 Gd-CDs与卵巢癌特异性抗体 Anti-HE4连接,得到的 Anti-HE4/Gd-CDs纳米探针能对 SKOV-3卵巢癌细胞进行特异性的荧光成像和 T1加权成像,其荧光信号强度与 SKOV-3的细胞浓度成正比,对 SKOV-3细胞的检测限为658 cell·mL^-1。以该方法检测了血液样品中的SKOV-3细胞。     

Synthesis of magnetic framework composites for the discrimination of Escherichia coli at the strain level

Rapid and efficient characterization and identification of pathogens at the strain level is of key importance for epidemiologic investigations, which still remains a challenge. In this work, solvothermically Fe₃O₄-COOH@MIL-101 composites were fabricated by in situ crystallization approach. The composites combine the excellent properties of both chromium (III) terephthalate (MIL-101) and carboxylic-functionalized magnetite (Fe₃O₄-COOH) particles and possess the efficient peptides/proteins enrichment properties and magnetic responsiveness. Fe₃O₄-COOH@MIL-101 composites as magnetic solid phase extraction materials were used to increase the discriminatory power of MALDI-TOF MS profiles. BSA tryptic peptides at a low concentration of 0.25 fmol μL⁻¹ could be detected by MALDI-TOF MS. In addition, Fe₃O₄-COOH@MIL-101 composites were successfully applied in the selective enrichment of the protein biomarkers from bacterial cell lysates and discrimination of Escherichia coli at the strain level. This work provides the possibility for wide applications of magnetic MOFs to discriminate pathogens below the species level.     

Fluorescence detection of adenosine-5′-triphosphate and alkaline phosphatase based on the generation of CdS quantum dots

We have developed an analytical method to detect adenosine-5′-triphosphate (ATP) and alkaline phosphatase (ALP) based on the generation of CdS quantum dots (QDs). We demonstrated that Cd²⁺ cation reacts with S²⁻ anion to generate fluorescent CdS QDs in the presence of some certain amount of ATP. With increase in the ATP concentration, the fluorescence intensity of CdS QDs was also enhanced. ATP can be converted into adenosine by the dephosphorylation of ALP, so that the generation of CdS QDs would be inhibited in the presence of ALP. Therefore, this novel analysis system could be applied to assay ATP and ALP based on the growth of fluorescent CdS QDs.     

水溶性CdSe量子点的合成及其作为荧光探针对大肠杆菌的快速检测

采用水相法以谷胱甘肽为稳定剂合成高稳定性的CdSe量子点,利用化学偶联剂的作用使得量子点表面基团与菌体之间的成功结合,对偶联的条件进行了优化.基于荧光分析法建立了一种快速简便的大肠杆菌检测定量分析方法.研究结果表明:合成的量子点具有稳定、荧光性能良好等突出优点.通过偶联剂量子点能与大肠杆菌结合,其荧光强度与大肠杆菌浓度...     

Colorimetric detection of pathogenic bacteria using platinum-coated magnetic nanoparticle clusters and magnetophoretic chromatography

A colorimetric method that uses platinum-coated magnetic nanoparticle clusters (Pt/MNCs) and magnetophoretic chromatography is developed to detect pathogenic bacteria. Half-fragments of monoclonal Escherichia coli O157:H7 (EC) antibodies were functionalized to Pt/MNCs and used to capture E. coli bacteria in milk. After magnetic separation of free Pt/MNCs and Pt/MNC-EC complexes from the milk, a precision pipette was used to imbibe the E. coli-containing solution, then a viscous polyethylene glycol solution. Due to difference in viscosities, the solutions separate into two liquid layers inside the pipette tip. The Pt/MNC-EC complexes were separated from the free Pt/MNCs by applying an external magnetic field, then added to a tetramethylbenzidine (TMB) solution. Catalytic oxidation of TMB by Pt produced color changes of the solution, which enabled identification of the presence of 10 cfu mL⁻¹E. coli bacteria with the naked eye. The total assay time including separation, binding and detection was 30 min.