Detection of viable Salmonella using microelectrode-based capacitance measurement coupled with immunomagnetic separation

In this study, we demonstrated the use of a general medium--brain heart infusion (BHI) broth that is not specifically formulated for impedance measurement, to achieve detectable impedance signals by using an interdigitated microelectrode (IME) with capacitance measurement at low frequencies. Anti-Salmonella antibody coated immunomagnetic beads were used to separate S. typhimurium from samples to provide the selectivity to this method. From analysis based on the equivalent circuit of the IME system, we found that the impedance change in BHI broth resulting from the growth of Salmonella was indeed the change in the double layer capacitance and could be monitored at 10 Hz using the IME. The results indicated that medium modification to improve impedance signal is not necessary with this IME system. However, effective immunological separation for the target organism is required for the selectivity when non-selective media are used. This finding provides a more flexible option of medium in impedance methods, which may provide opportunities to test those species of bacteria that have no suitable conductance growth medium. The detection time, t(d), was obtained from the impedance growth curve (impedance against bacterial growth time) at 10 Hz at the point where the impedance started to change. A linear relationship between the detection time and the logarithmic value of the initial cell number (N) was found in the Salmonella cell number ranging from 10(1) to 10(6) cfu/ml. The regression equation was t(d) = -1.22Log N + 8.90, with R2 = 0.95. The detection times for the initial cell number of 10(1) CFU/ml and 10(6) CFU/ml are 8 h and 1.5 h, respectively. This method is more sensitive than impedance methods using conventional electrodes.     

Rapid and sensitive detection of cancer cells by coupling with quantum dots and immunomagnetic separation at low concentrations

This work presents a rapid and sensitive method for detecting cancer cells at low concentration. In this method, two biomarkers of T-help cancer cells are detected simultaneously. One biomarker is conjugated with magnetic beads to separate T-help cell from the mixed cells and the other biomarker, associated with quantum dots, is used to detect fluorescence. The specific T-help cells can be quantified using the relationship between the QD fluorescence intensity and the cell frequency following magnetic separation. The intensity of fluorescence increases linearly with the frequency of T-help cells from 10(-7) to 10(-3), and neither B cells nor red blood cells interfere with the detection of T-help cells. Moreover, the total detection time is under 15 min, even though the frequency of specific T-help cells is as low as 5×10(-7). The numerous advantages of detecting specific cells at low concentration using the presented method include ease of preparation, low cost, fast detection, and high sensitivity.     

Spectroscopic characterization of streptavidin functionalized quantum dots

The spectroscopic properties of quantum dots can be strongly influenced by the conditions of their synthesis. In this work, we have characterized several spectroscopic properties of commercial, streptavidin functionalized quantum dots (QD525, lot 1005-0045, and QD585, lot 0905-0031, from Invitrogen). This is the first step in the development of calibration beads to be used in a generalizable quantification scheme of multiple fluorescent tags in flow cytometry or microscopy applications. We used light absorption, photoexcitation, and emission spectra, together with excited state lifetime measurements, to characterize their spectroscopic behavior, concentrating on the 400- to 500-nm wavelength ranges that are important in biological applications. Our data show an anomalous dependence of emission spectrum, lifetimes, and quantum yield (QY) on excitation wavelength that is particularly pronounced in the QD525. For QD525, QY values ranged from 0.2 at 480 nm excitation up to 0.4 at 450 nm and down again to 0.15 at 350 nm. For QD585, QY values were constant at 0.2 between 500 and 400 nm, but they dropped to 0.1 at 350 nm. We attribute the wavelength dependencies to heterogeneity in size and surface defects in the QD525, consistent with characteristics described previously in the chemistry literature. The results are discussed in the context of bridging the gap between what is currently known in the physical chemistry literature of quantum dots and the quantitative needs of assay development in biological applications.     

Detection of E. coli O157:H7 by immunomagnetic separation coupled with fluorescence immunoassay

Conventional culture-based methods for detection of E. coli O157:H7 in foods and water sources are time-consuming, and results can be ambiguous, requiring further confirmation by biochemical testing and PCR. A rapid immunoassay prior to cultivation to identify presumptive positive sample would save considerable time and resources. Immunomagnetic separation (IMS) techniques are routinely used for isolation of E. coli O157:H7 from enriched food and water samples, typically in conjunction with cultural detection followed by biochemical and serological confirmation. In this study, we developed a new method that combines IMS with fluorescence immunoassay, termed immunomagnetic fluorescence assay (IMFA), for the detection of E. coli O157:H7. E. coli O157:H7 cells were first captured by anti-O157 antibody-coated magnetic beads and then recognized by a fluorescent detector antibody, forming an immunosandwich complex. This complex was subsequently dissociated for measurement of fluorescence intensity with Signalyte™-II spectrofluorometer. Experiments were conducted to evaluate both linearity and sensitivity of the assay. Capture efficiencies were greater than 98%, as determined by cultural plating and quantitative real-time PCR, when cell concentrations were <10⁵ cells/mL. Capture efficiency decreased at higher cell concentrations, due to the limitation of bead binding capacity. At lower cell concentrations (10–10⁴ cells/mL), the fluorescence intensity of dissociated Cy5 solution was highly correlated with E. coli 157:H7 cell concentrations. The detection limit was 10 CFU per mL of water. The assay can be completed in less than 3 h since enrichment is not required, as compared to existing techniques that typically require a 24 h incubation for pre-enrichment, followed by confirmatory tests.     

Comparing recovering efficiency of immunomagnetic separation and centrifugation of mycobacteria in metalworking fluids

The accurate detection and enumeration of Mycobacterium immunogenum in metalworking fluids (MWFs) is imperative from an occupational health and industrial fluids management perspective. We report here a comparison of immunomagnetic separation (IMS) coupled to flow-cytometric enumeration, with traditional centrifugation techniques for mycobacteria in a semisynthetic MWF. This immunolabeling involves the coating of laboratory-synthesized nanometer-scale magnetic particles with protein A, to conjugate a primary antibody (Ab), specific to Mycobacterium spp. By using magnetic separation and flow-cytometric quantification, this approach enabled much higher recovery efficiency and fluorescent light intensities in comparison to the widely applied centrifugation technique. This IMS technique increased the cell recovery efficiency by one order of magnitude, and improved the fluorescence intensity of the secondary Ab conjugate by 2-fold, as compared with traditional techniques. By employing nanometer-scale magnetic particles, IMS was found to be compatible with flow cytometry (FCM), thereby increasing cell detection and enumeration speed by up to two orders of magnitude over microscopic techniques. Moreover, the use of primary Ab conjugated magnetic nanoparticles showed better correlation between epifluorescent microscopy counts and FCM analysis than that achieved using traditional centrifugation techniques. The results strongly support the applicability of the flow-cytometric IMS for microbial detection in complex matrices.

     

Detection of toxin B of Clostridium difficile based on immunomagnetic separation and aptamer-mediated colorimetric assay

Clostridium difficile can cause antibiotic-associated diarrhoea or pseudo-membranous colitis in humans and animals. Currently, the various methods such as microbiological culture, cytotoxic assay, ELISA and polymerase chain reaction have been used to detect Clostridium difficile infection (CDI). These conventional methods, however, require long detection time and professional staff. The paper is to describe a simple strategy which employs immunomagnetic separation and aptamer-mediated colorimetric assay for the detection of toxin B of C. difficile (TcdB) in the stool samples. HRP-labelled aptamer against TcdB selected by SELEX was firstly captured on the surface of magnetic beads (MB) by DNA hybridization with a complementary strand. In the presence of TcdB, aptamer specifically recognized and bound TcdB, disturbing the DNA hybridization and causing the release of HRP-aptamer from MB. This reduced the catalytic capacity of HRP and consequently the absorption intensity. As there was a relationship between the decrease in the absorption intensity and target concentration, a quantitative analysis of TcdB can be accomplished by the measurement of the absorption intensity. Under the optimal conditions, the assay system is able to detect TcdB at a concentration down to 5 ng ml⁻¹. Moreover the method had specificity of 97% and sensitivity of 66% and the system remained excellent stability within 4 weeks. The proposed method is a valuable screening procedure for CDI and can be extended readily to detection of other clinically important pathogens.     

Comparing recovering efficiency of immunomagnetic separation and centrifugation of mycobacteria in metalworking fluids

The accurate detection and enumeration of Mycobacterium immunogenum in metalworking fluids (MWFs) is imperative from an occupational health and industrial fluids management perspective. We report here a comparison of immunomagnetic separation (IMS) coupled to flow-cytometric enumeration, with traditional centrifugation techniques for mycobacteria in a semisynthetic MWF. This immunolabeling involves the coating of laboratory-synthesized nanometer-scale magnetic particles with protein A, to conjugate a primary antibody (Ab), specific to Mycobacterium spp. By using magnetic separation and flow-cytometric quantification, this approach enabled much higher recovery efficiency and fluorescent light intensities in comparison to the widely applied centrifugation technique. This IMS technique increased the cell recovery efficiency by one order of magnitude, and improved the fluorescence intensity of the secondary Ab conjugate by 2-fold, as compared with traditional techniques. By employing nanometer-scale magnetic particles, IMS was found to be compatible with flow cytometry (FCM), thereby increasing cell detection and enumeration speed by up to two orders of magnitude over microscopic techniques. Moreover, the use of primary Ab conjugated magnetic nanoparticles showed better correlation between epifluorescent microscopy counts and FCM analysis than that achieved using traditional centrifugation techniques. The results strongly support the applicability of the flow-cytometric IMS for microbial detection in complex matrices.     

Synthesis of functionalized amphiphilic polymers for coating quantum dots

Quantum dots (QDs) need to be attached to other chemical species if they are to be used as biomarkers, therapeutic agents or sensors. These materials also need to disperse well in water and have well-defined functional groups on their surfaces. QDs are most often synthesized in the presence of ligands such as trioctylphosphine oxide, which render the nanoparticle surfaces hydrophobic. We present a complete protocol for the synthesis and water solubilization of hydrophobic CdSe/ZnS QDs using designer amphiphilic polymeric coatings. The method is based on functionalization of an anhydride polymer backbone with nucleophilic agents. Small functional groups, bulky cyclic compounds and polymeric chains can be integrated into the coating prior to solubilization. We describe the preparation of acetylene- and azide-functionalized QDs for 'click' chemistry. The method is universal and applicable to any type of nanoparticle stabilized with hydrophobic ligands able to interact with the alkyl chains in the coating in water.     

Detection of Listeria monocytogenes in foods by immunomagnetic separation

Immunomagnetic separation with immunomagnetic beads was used to isolate strains of Listeria monocytogenes both from pure cultures and from heterogeneous suspensions. The monoclonal antibodies used recognized all six strains of serotype 4 but only one of three strains of serotype 1. Coating procedure, incubation time, and number of immunomagnetic beads influenced the sensitivity of the isolation method. Less than 1 x 10(2) bacteria per ml in pure cultures and less than 2 x 10(2) bacteria per ml in enriched foods could be detected. The method represents a new approach to extraction and isolation of pathogenic bacteria directly from foods, after resuscitation, or from enrichment broths.     

Detection of quercetin based on Al-amplified phosphorescence signals of manganese-doped ZnS quantum dots

A simple phosphorescence method is proposed for quercetin detection based on Al³⁺-amplified room-temperature phosphorescence (RTP) signals of 3-mercaptopropionic acid (MPA)-capped Mn-doped ZnS quantum dots (QDs). The sensor was established based on some properties as follows. Al³⁺ can interact with carboxyl groups on the surface of MPA-capped Mn-doped ZnS QDs via chelation, which will lead to the aggregation of QDs and amplification of RTP signals, After the addition of quercetin, it can form more stable complex with Al³⁺ in alkaline aqueous solution and dissociate Al³⁺ from the surface of Mn-doped ZnS QDs, which will result in significant recovery of RTP intensity of the MPA-capped Mn-doped ZnS–Al³⁺ system. Under the optimized conditions, the change of RTP intensity was proportional to the concentration of quercetin in the range from 0.1 to 6.0 mg L⁻¹, with a high correlation coefficient of 0.996 and a detection limit of 0.047 mg L⁻¹. The proposed method is potentially suitable for detection of quercetin in real samples without complicated pretreatment.     

Detection of Listeria monocytogenes in foods by immunomagnetic separation.

Immunomagnetic separation with immunomagnetic beads was used to isolate strains of Listeria monocytogenes both from pure cultures and from heterogeneous suspensions. The monoclonal antibodies used recognized all six strains of serotype 4 but only one of three strains of serotype 1. Coating procedure, incubation time, and number of immunomagnetic beads influenced the sensitivity of the isolation method. Less than 1 x 10(2) bacteria per ml in pure cultures and less than 2 x 10(2) bacteria per ml in enriched foods could be detected. The method represents a new approach to extraction and isolation of pathogenic bacteria directly from foods, after resuscitation, or from enrichment broths.     

Biomedical applications of glyconanoparticles based on quantum dots

Background

Quantum dots (QDs) are outstanding nanomaterials of great interest to life sciences. Their conjugation versatility added to unique optical properties, highlight these nanocrystals as very promising fluorescent probes. Among uncountable new nanosystems, in the last years, QDs conjugated to glycans or lectins have aroused a growing attention and their application as a tool to study biological and functional properties has increased.

Interactions of pathogenic mycobacteria with host macrophages

Tuberculosis, caused by the bacterium Mycobacterium tuberculosis, is one of the most deadly infectious diseases across the globe. The success of M. tuberculosis is related to its capacity to survive and replicate in macrophages, cells of the host innate immune system that are designed to detect and eliminate pathogens [1,2]. In this review, we will focus on the mechanisms used by the innate system of the host to detect and eliminate mycobacteria and the strategies used by M. tuberculosis to overcome host responses to establish a successful infection.     

Detection of single quantum dots in model organisms with sheet illumination microscopy

Single-molecule detection and tracking is important for observing biomolecule interactions in the microenvironment. Here we report selective plane illumination microscopy (SPIM) with single-molecule detection in living organisms, which enables fast imaging and single-molecule tracking and optical penetration beyond 300 μm. We detected single nanocrystals in Drosophila larvae and zebrafish embryo. We also report our first tracking of single quantum dots during zebrafish development, which displays a transition from flow to confined motion prior to the blastula stage. The new SPIM setup represents a new technique, which enables fast single-molecule imaging and tracking in living systems.     

Imaging Escherichia coli using functionalized core/shell CdSe/CdS quantum dots

The internalization of a series of water-soluble CdSe/CdS quantum dots (QDs) stabilized by citrate, isocitrate, succinate, and malate by Escherichia coli is established by epifluorescence and confocal fluorescence scanning microscopy, fluorimetry, and UV–vis spectroscopy on whole and lysed bacterial cells. The organic-acid-stabilized QDs span a range in size from 3.8±1.1 to 6.0±2.4 nm with emission wavelengths from 540 to 630 nm. QDs of different sizes (i.e., 3.8–6 nm) can enter the bacterium and be detected on different fluorescence channels with little interference from other QDs as a result of the distinct emission profiles (i.e., 540–630 nm, respectively). Costaining QD-labeled E. coli with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) demonstrates that the QDs and DAPI are colocalized within E. coli, whereas costaining QD-labeled E. coli with membrane dye FM4-64 shows that the FM4-64 is localized in the outer bacterial membrane and that the QDs are inside.Electronic Supplementary Material Supplementary material is available to authorized users in the online version of this article at .     

Detection of Clavibacter michiganensis subsp. michiganensis in tomato seeds using immunomagnetic separation

The use of pathogen-free plant material is the main strategy for controlling bacterial canker of tomato caused by Clavibacter michiganensis subsp. michiganensis. However, detection and isolation of this pathogen from seeds before field or greenhouse cultivation is difficult when the bacterium is at low concentration and associated microbiota are present. Immunomagnetic separation (IMS), based on the use of immunomagnetic beads (IMBs) coated with specific antibodies, was used to capture C. michiganensis subsp. michiganensis cells, allowing removal of non-target bacteria from samples before plating on non-selective medium. Different concentrations of IMBs and of two antisera were tested, showing that IMS with 10(6)IMBs/ml coated with a polyclonal antiserum at 1/3200 dilution recovered more than 50% of target cells from initial inocula of 10(3) to 10(0)CFU/ml. Threshold detection was lower than 10CFU/ml even in seed extracts containing seed debris and high populations of non-target bacteria. The IMS permitted C. michiganensis subsp. michiganensis isolation from naturally infected seeds with higher sensitivity and faster than direct isolation on the semiselective medium currently used and could become a simple viable system for routinely testing tomato seed lots in phytosanitary diagnostic laboratories.     

Synthesis and biological assay of GSH functionalized fluorescent quantum dots for staining Hydra vulgaris

Quantum dots (QDs) have been used extensively as fluorescent markers in several studies on living cells. Here, we report the synthesis of conjugates based on glutathione (GSH) and QDs (GSH-QDs) and we prove how these functionalized fluorescent probes can be used for staining a freshwater invertebrate called Hydra vulgaris. GSH is known to promote Hydra feeding response by inducing mouth opening. We demonstrate that GSH-QDs as well are able to elicit biological activity in such an animal, which results in the fluorescent staining of Hydra. GSH-QDs, once they reach the gastric region, are internalized by endodermal cells. The efficiency of GSH-QD internalization increases significantly when nanoparticles are coadministrated with free GSH. We also compared the behavior of bare QDs to that of GSH-QDs both in the presence and in the absence of free GSH. The conclusions from these series of experiments point to the presence of GSH binding proteins in the endodermal cell layer and uncover a novel role played by glutathione in this organism.     

Isolation of salmonellas by immunomagnetic separation

A.E.M. VERMUNT, A.A.J.M. FRANKEN AND R.R. BEUMER. 1992. Magnetisable particles, coated with anti-salmonella serum, were used to isolate Salmonella livingstone from pure cultures, mixed cultures and food samples. Beads (10⁷) were generally incubated with 10⁴ Salm. livingstone cells/ml for 60 min at room temperature. The incubation and washing medium (0.01 mol/l phosphate-buffered saline; PBS) contained 0.1% bovine serum albumin (BSA) and 0.1% Tween 20, respectively. This method gave a recovery for Salm. livingstone of 51.9±7.8%. However, other micro-organisms such as Aeromonas hydrophila interfered with this test because of non-specific reactions (recovery 50.9±12.7%). These non-specific reactions could be decreased by using 4% skim milk instead of 0.1% BSA in the incubation medium. The ratio of the recovery of Salim. livingstone relative to the recovery of Aer. hydrophila changed from 0.9 when PBS with 0.1% BSA was used, to 13.4 when PBS with 4% skim milk was used. Immunomagnetic separation of Salmonella spp. from food samples offers good prospects for concentrating salmonella cells from heterogeneous bacterial suspensions, such as enrichment broths.     

Interaction of pathogenic mycobacteria with the host immune system

Pathogenic mycobacteria, in particular Mycobacterium tuberculosis, the causative agent of tuberculosis, have the remarkable capacity to circumvent destruction within one of the most hostile cell types of a vertebrate host: the macrophage. The ability of pathogenic mycobacteria to survive inside macrophages has been known for more than 30 years; yet, only recently have advances in molecular genetics, biochemistry, immunology, as well as global analysis of gene expression, started to unravel the strategies utilized by these pathogens for intracellular persistence. In addition, the definition of key molecules that are important for intracellular survival opens the possibility to develop new drugs to combat mycobacterial diseases.