A lysozyme and magnetic bead based method of separating intact bacteria

As a response to environmental stress, bacterial cells can enter a physiological state called viable but noncultivable (VBNC). In this state, bacteria fail to grow on routine bacteriological media. Consequently, standard methods of contamination detection based on bacteria cultivation fail. Although they are not growing, the cells are still alive and are able to reactivate their metabolism. The VBNC state and low bacterial densities are big challenges for cultivation-based pathogen detection in drinking water and the food industry, for example. In this context, a new molecular-biological separation method for bacteria using point-mutated lysozymes immobilised on magnetic beads for separating bacteria is described. The immobilised mutated lysozymes on magnetic beads serve as bait for the specific capture of bacteria from complex matrices or water due to their remaining affinity for bacterial cell wall components. Beads with bacteria can be separated using magnetic racks. To avoid bacterial cell lysis by the lysozymes, the protein was mutated at amino acid position 35, leading to the exchange of the catalytic glutamate for alanine (LysE35A) and glutamine (LysE35Q). As proved by turbidity assay with reference bacteria, the muramidase activity was knocked out. The mutated constructs were expressed by the yeast Pichia pastoris and secreted into expression medium. Protein enrichment and purification were carried out by SO₃-functionalised nanoscale cationic exchanger particles. For a proof of principle, the proteins were biotinylated and immobilised on streptavidin-functionalised, fluorescence dye-labelled magnetic beads. These constructs were used for the successful capture of Syto9-marked Microccocus luteus cells from cell suspension, as visualised by fluorescence microscopy, which confirmed the success of the strategy.     

Human pathogenic Cryptosporidium species bioanalytical detection method with single oocyst detection capability

A bioanalytical detection method for specific detection of viable human pathogenic Cryptosporidium species, C. parvum, C. hominis, and C. meleagridis is described. Oocysts were isolated from water samples via immunomagnetic separation, and mRNA was extracted with oligo-dT magnetic beads, amplified using nucleic acid sequence-based amplification (NASBA), and then detected in a nucleic acid hybridization lateral flow assay. The amplified target sequence employed was hsp70 mRNA, production of which is stimulated via a brief heat shock. The described method was capable of detecting one oocyst in 10 μL using flow-cytometer-counted samples. Only viable oocysts were detected, as confirmed using 4′,6-diamidino-2-phenylindole and propidium iodide (DAPI/PI) staining. The detection system was challenged by detecting oocysts in the presence of large numbers of common waterborne microorganisms and packed pellet material filtered from environmental water samples. When the method was compared with EPA Method 1622 for C. parvum detection, highly comparable results were obtained. Since the described detection system yields unambiguous results within 4.5 h, it is an ideal method for monitoring the safety of drinking water.     

Rapid preparation of methyltrimethoxy‐modified magnetic mesoporous silica as an effective solid‐phase extraction adsorbent

A time‐saving method was applied to synthesize methyltrimethoxy‐modified magnetic mesoporous silica with or without p‐toluenesulfonic acid as the catalyst for magnetic solid‐phase extraction. The synthesized materials were systematically characterized. Results demonstrated that methyltrimethoxy modified magnetic mesoporous silica with p‐toluenesulfonic acid as the catalyst has a relatively smaller aperture and extreme hydrophobicity (water contact angle of 135°). To evaluate the feasibility of these prepared materials as effective adsorbents, it was combined with gas chromatography and electron capture detection to determine 26 polychlorinated biphenyls in environmental water. The result revealed that methyltrimethoxy modified magnetic mesoporous silica with p‐toluenesulfonic acid as the catalyst had the best extraction efficiency and recovery. Under the optimized extracted conditions, the proposed method showed good linearity within the concentration range of 5 to 200 ng/L with correlation coefficients of 0.9969 to 0.9999. The limits of detection and quantification based on signal‐to‐noise ratios of 3 and 10 were in the range of 0.16 to 0.91 and 0.52 to 3.0 ng/L, respectively. The polychlorinated biphenyl concentrations in environmental water samples were successfully determined using the developed method. PCB008 and PCB110 were 4.05 and 8.52 ng/L in Red‐Star lake water (Hubei Province, China), respectively.     

Immunomagnetic nanoparticle-based sandwich chemiluminescence-ELISA for the enrichment and quantification of E. coli

Rapid methods for the quantification of Escherichia coli are required for the monitoring of faecal contamination in water to secure public health. The immunomagnetic separation (IMS) offers rapid enrichment and purification of bacteria in complex matrices and is compatible with immunoassays. By means of this technique, non-target cells and matrix components which might interfere with subsequent analytical methods are removed. We present the synthesis of magnetic nanoparticles (MNPs) and covalent coupling to antibodies against the enterobacterial common antigen (ECA) for use with IMS. Quantification was carried out with a chemiluminescence-based sandwich enzyme-linked immunosorbent assay (ELISA). Our anti-ECA-MNPs allow for a group-specific enrichment of bacterial cells, which can be combined with a species-specific analytical method. The particles were used along with commercially available magnetic columns for the selective enrichment of E. coli from 10-mL water samples. The volumetric enrichment factor was 9. For enriched samples, the limit of detection was reduced from 5.0?×?10⁶ cells·mL^-1 to 2.6?×?10⁵ cells·mL^-1. Using 200 µL anti-ECA-MNPs, we determined a recovery of 97?±?6% for a sample containing 10⁶ cells·mL^-1 and 89?±?2% for a sample containing 10⁷ cells·mL^-1. The overall time for cell enrichment and detection was 3 h 45 min.     

Fast detection of methyl tert-butyl ether from water using solid phase microextraction and ion mobility spectrometry

Methyl tert-butyl ether (MTBE) is commonly used as chemical additive to increase oxygen content and octane rating of reformulated gasoline. Despite its impact on enhancing cleaner combustion of gasoline, MTBE poses a threat to surface and ground water when gasoline is released into the environment. Methods for onsite analysis of MTBE in water samples are also needed. A less common technique for MTBE detection from water is ion mobility spectrometry (IMS). We describe a method for fast sampling and screening of MTBE from water by solid phase microextraction (SPME) and IMS. MTBE is adsorbed from the head space of a sample to the coating of SPME fiber. The interface containing a heated sample chamber, which couples SPME and IMS, was constructed and the SPME fiber was introduced into the sample chamber for thermal desorption and IMS detection of MTBE vapors. The demonstrated SPME-IMS method proved to be a straightforward method for the detection of trace quantities of MTBE from waters including surface and ground water. We determined the relative standard deviation of 8.3% and detection limit of 5 mg L⁻¹ for MTBE. Because of short sampling, desorption, and detection times, the described configuration of combined SPME and IMS is a feasible method for the detection of hazardous substances from environmental matrices.     

Aptamer based strategy for cytosensing and evaluation of HER-3 on the surface of MCF-7 cells by using the signal amplification of nucleic acid-functionalized nanocrystals

The electrochemical detection of cell lines of MCF-7 (human breast cancer) has been reported, using magnetic beads for the separation tool and high-affinity DNA aptamers for signal recognition. The high specificity was obtained by using the magnetic beads and aptamers, and the good sensitivity was realized with the signal amplification of DNA capped CdS or PbS nanocrystals. The ASV (anodic stripping voltammetry) technology was employed for the detection of cadmic cation and lead ions, for electrochemical assay of the amount of the target cells and biomarkers on the membrane of target cells, respectively. This electrochemical method could respond to as low as 100 cells mL⁻¹ of cancer cells with a linear calibration range from 1.0 × 10² to 1.0 × 10⁶ cells mL⁻¹, showing very high sensitivity. Moreover, the amounts of HER-3 which were overexpressed on MCF-7 cells were calculated correspond to be 3.56 × 10⁴ anti-HER-3 antibody molecules. In addition, the assay was able to differentiate between different types of target and control cells based on the aptamers and magnetic beads used in the assay, indicating the wide applicability of the assay for early and accurate diagnose of cancers.     

Application of flow cytometry for rapid detection ofLactococcus garvieae

Flow cytometry (FCM) technique was applied to rapid determination of cell number ofLactococcus garvieae. An antiserum againstL. garvieae was prepared and its immunological property was examined. The present antibody would recognize some epitopes ofL. garvieae with a high specificity. The optimum conditions for the FCM assay were as follows: discriminate value, 60; dilution ratio of the antiserum, 1.0 X 10⁴. Calibration curve forL. garvieae cells was linear, in the range of 2.4 X 10⁴-1.5 X 10⁷ cells/mL. The detection ofL. garvieae in cell suspensions contaminated withEscherichia coli was carried out. A good correlation was observed in the range of 20–90% for the mixing ratio ofL. garvieae. One FCM assay could be completed within 2 min, and the total assay time, including the preparation of bacterial sample, was within 3 h.     

An Optimized Bioassay for Mucin1 Detection in Serum Samples

Two simple and sensitive electrochemical approaches for Mucin1 (MUC1) tumor marker using magnetic beads coupling screen‐printed arrays were developed. The single‐use bioassays are based on a sandwich format in which aptamers or antibodies were coupled respectively to Streptavidin or Protein G‐modified magnetic beads. The bioreceptor‐modified beads are used to capture the MUC1 protein from the sample and sandwich assay is performed by the addition of a labeled secondary aptamer or antibody. The enzyme alkaline phosphatase and its substrate (1‐naphthyl phosphate) are then used for the electrochemical detection by differential pulse voltammetry (DPV). The analytical performances of the designed bioassays were compared in terms of sensitivity, selectivity and reproducibility. Using the optimized conditions, a linear range from 0 to 0.28 nM was obtained, with 0.19 nM LOD using antibody‐based and 0.07 nM LOD using aptamer‐based sandwich assay in MUC1 buffered solutions. The results also showed that the aptamer‐based approach exhibited higher selectivity for MUC1, allowing the detection of the protein in complex matrices. The developed aptasensor for MUC1 detection was applied on serum samples obtained from cancer patients, providing promising perspectives for clinical applications.     

Affinity capture using chimeric membrane proteins bound to magnetic beads for rapid ligand screening by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

The rapid and specific detection of therapeutically important ligands in complex mixtures, that may bind to membrane proteins, remains challenging for many research laboratories and pharmaceutical industries. Through its use in the development of screening assays, mass spectrometry (MS) is currently experiencing a period of tremendous expansion. In the study presented here, we took advantage of the remarkable stability properties of a bacterial membrane protein, the KcsA K⁺ channel, produced in E. coli and purified as a tetrameric protein in the presence of a detergent. This membrane protein can subserve as a molecular template to display the pore‐forming region of human K⁺ channels, which are considered as targets in the search for inhibitory ligands. The engineered chimeric proteins were linked to metal‐bound magnetic beads, for the screening of complex peptide mixtures, such as that of scorpion venoms. The affinity‐captured scorpion toxins were eluted prior to matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOFMS), and to nano‐electrospray ionization tandem mass QqTOF mass spectrometry (MS/MS) analysis. The de novo sequence of the toxins was deduced by combining the MS/MS fragmentation of the reduced form (up to the 33 first residues) and the trypsin digest peptides of the native toxins. This affinity‐capture screening assay led to the isolation and characterization of potent and specific ligands of the human K⁺ channel, Kv1.3. The affinity‐capture procedure is fast and reproducible. When linked to magnetic beads, the chimeric membrane protein can be re‐used several times without losing any of its selectivity or specificity. This assay also benefits from the fact that it requires minimal amounts of animal venoms or complex mixtures, which can be expensive or difficult to procure. Copyright © 2009 John Wiley & Sons, Ltd.     

Fully automated immunoassay for detection of prostate-specific antigen using nano-magnetic beads and micro-polystyrene bead composites, 'Beads on Beads'

Magnetic beads have served as a conventional bioassay platform in biotechnology. In this study, a fully automated immunoassay was performed using novel nano- and microbead-composites constructed by assembling nano-magnetic beads onto polystyrene microbeads, designated ‘Beads on Beads’. Nano-sized bacterial magnetic particles (BacMPs) displaying the immunoglobulin G (IgG)-binding domain of protein A (ZZ domain) were used for the construction of ‘Beads on Beads’ via the interaction of biotin-streptavidin. The efficient assembly of ‘Beads on Beads’ was performed by gradual addition of biotin-labeled BacMPs onto streptavidin-coated polystyrene microbeads. Approximately 2000 BacMPs were uniformly assembled on a single microbead without aggregation. The constructed ‘Beads on Beads’ were magnetized and separated from the suspension by using an automated magnetic separation system with a higher efficiency than BacMPs alone. Furthermore, fully automated detection of prostate-specific antigens was performed with the detection limit of 1.48 ng mL⁻¹. From this preliminary assay, it can be seen that ‘Beads on Beads’ could be a powerful tool in the development of high-throughput, fully automated multiplexed bioassays.     

Screening inhibitors of xanthine oxidase from natural products using enzyme immobilized magnetic beads by high‐performance liquid chromatography coupled with tandem mass spectrometry

In this study, high‐performance liquid chromatography coupled with tandem mass spectrometry was used to assess the results of bioactive compound screening from natural products using immobilized enzyme magnetic beads. We compared three commercial magnetic beads with modified amino, carboxy, and N‐hydroxysuccinimide groups, respectively. Amino magnetic beads performed best for immobilization and were selected for further experiments. Xanthine oxidase was immobilized on amino magnetic beads and applied to screen potential inhibitors in fresh Zingiber officinale Roscoe, extracts of Scutellaria baicalensis Georgi, and Pueraria lobata Ohwi. In total, 12 potential xanthine oxidase ligands were identified from fresh Zingiber root and Scutellaria root extracts, of which eight were characterized and the concentration required for 50% inhibition was determined. Preliminary structure–function relationships were discussed based on these results. A convenient and effective method was therefore developed for the identification of active compounds from complex natural product mixtures.     

Label-free aptamer-based chemiluminescence detection of adenosine

We have developed a novel sensitive chemiluminescence (CL) aptasensor for the target assay as exemplified by using adenosine as a model target. In this work, we have demonstrated the signaling mechanism to make detection based on magnetic separation and 3,4,5-trimethoxyl-phenylglyoxal (TMPG), a special CL reagent as the signaling molecule, which reacts instantaneously with guanine nucleobases (G) of adenosine-binding aptamer strands. Briefly, amino-functioned capture DNA sequences are immobilized on the surface of carboxyl-modified magnetic beads, and then hybridized with label-free G-rich (including 15 guanine nucleobases) adenosine-binding aptamer strands to form our CL aptasensor. Upon the introduction of adenosine, the aptamer on the surface of magnetic beads is triggered to make structure switching to the formation of the adenosine/aptamer complex. Consequently, G-rich aptamer strands are forced to dissociate from magnetic beads sensing interface, resulting in a decrease of CL signal. The decrement of peak signal is proportional to the amount of adenosine. The effects of the amounts of capture DNA, aptamer, magnetic beads are investigated and optimized. It was found that the CL intensity had a linear dependency on the concentration of adenosine in the range of 4 × 10⁻⁷ to 1 × 10⁻⁵ M. With a low detection limit of 8 × 10⁻⁸ M and simplicity in CL detection, this novel technique will offer a great promise for future target/aptamer analysis.     

Activation of Nanoparticles by Biosorption for <Emphasis Type="Italic">E. coli</Emphasis> Detection in Milk and Apple Juice

Two types of silver nanoparticles were activated by specific sorption of biomolecules for the detection of Escherichia coli. The capture of this bacterium was performed using polyclonal antibodies (anti-E. coli) biosorbed onto nanospheres or nanorice through a protein-A layer. The bacterial detection was achieved using surface enhancement Raman scattering in order to compare the performance of these two nanoparticles. The activated silver nanospheres showed a better performance mainly due to the dimension of these nanoparticles. The detection limit has been established using the automated Raman mapping system. The technique was capable of detecting 10³ cells/mL in milk and apple juice without any pre-enrichment. With an overall assay time less than 1 h, the process could be easily adapted to detect other pathogens by selecting the pertinent antibody. Furthermore, PCR was used for the DNA verification to assess whether the selected bacterial strain was identical before and after detection.     

Specific peptides as alternative to antibody ligands for biomagnetic separation of Clostridium tyrobutyricum spores

Nowadays, the reference method for the detection of Clostridium tyrobutyricum in milk is the most-probable-number method, a very time-consuming and non-specific method. In this work, the suitability of the use of superparamagnetic beads coated with specific antibodies and peptides for bioseparation and concentration of spores of C. tyrobutyricum has been assessed. Peptide or antibody functionalized nanoparticles were able to specifically bind C. tyrobutyricum spores and concentrate them up to detectable levels. Moreover, several factors, such as particle size (200 nm and 1 μm), particle derivatization (aminated and carboxylated beads), coating method, and type of ligand have been studied in order to establish the most appropriate conditions for spore separation. Results show that concentration of spore is favored by a smaller bead size due to the wider surface of interaction in relation to particle volume. Antibody orientation, related to the binding method, is also critical in spore recovery. However, specific peptides seem to be a better ligand than antibodies, not only due to the higher recovery ratio of spores obtained but also due to the prolonged stability over time, allowing an optimal recovery of spores up to 3 weeks after bead coating. These results demonstrate that specific peptides bound to magnetic nanoparticles can be used instead of traditional antibodies to specifically bind C. tyrobutyricum spores being a potential basis for a rapid method to detect this bacterial target.     

Continuous-flow separation and pre-concentration coupled on-line to solid-surface fluorescence spectroscopy for the simultaneous determination of<Emphasis Type="Italic"> o</Emphasis>-phenylphenol and thiabendazole

A novel and single flow-injection system combined with solid-surface fluorescence detection is proposed in this work for the resolution of a mixture of two widely used pesticides (o-phenylphenol and thiabendazole). The continuous-flow methodology is based on the implementation of on-line pre-concentration and separation of both analytes on the surface of C₁₈ silica gel beads placed just inside the flow cell, implemented with gel-phase fluorimetric multi-wavelength detection (using 305/358 and 250/345 nm as excitation/emission wavelengths for thiabendazole and o-phenylphenol, respectively). The separation of the pesticides was possible owing to the different retention/desorption kinetics of their interactions with the solid support in the zone where the stream impinges on the solid material. No previous separation of the analytes before they reach the flow cell is needed thereby simplifying substantially both the procedure and the manifold. By using a sample volume of 2,600 L, the system was calibrated in the range 0.5–16 and 5–120 ng mL^–1 with detection limits of 0.09 and 0.60 ng mL^–1 for thiabendazole and o-phenylphenol, respectively. The RSD values (n=10) were about 1% for both analytes. The proposed methodology was applied to environmental water samples and also to various commercial pesticide formulations containing both analytes. Recovery percentages were 97–103% and 98–102% for thiabendazole and o-phenylphenol, respectively.     

A new strategy for the development of monoclonal antibodies for the determination of human procalcitonin in serum samples

Procalcitonin (PCT)—a diagnostic serum parameter for bacterial infection and sepsis—is of great interest in the field of biosensors for point-of-care testing. Its detection needs specific biological recognition elements, such as antibodies. Herein, we describe the development and characterization of rat monoclonal antibodies (mAbs) for PCT, and their application in enzyme-linked immunosorbent assays (ELISAs) for the determination of PCT in patient serum samples. From about 50 mAbs, two mAbs, CALCA 2F3 and CALCA 4A6, were selected as a pair with high affinity for PCT in sandwich immunoassays. Both mAbs could be used either as capture or as detection mAb. They were Protein G-purified and biotinylated when used as detection mAb. The setup of two sandwich ELISAs with standards of human recombinant (hr) PCT, using either CALCA 2F3 (assay A) or CALCA 4A6 (assay B) as capture mAbs and the biotinylated mAbs CALCA 4A6 or CALCA 2F3, respectively, as detection mAbs, led to highly specific determinations of PCT without cross-reactivity to calcitonin and katacalcin. Test midpoints (IC₅₀) of both assays were determined for hrPCT standards in 4% (w/v) human serum albumin and found with 2.5 (assay A) and 2.7 μg L⁻¹ (assay B). With both sandwich ELISAs a collection of eight patient serum samples have been determined in comparison to the determination by the Elecsys BRAHMS PCT assay. Good correlations between our prototype ELISAs and the BRAHMS assay could be demonstrated (R ²: assay A, 0.996 and assay B, 0.990). The use of these newly developed anti-PCT mAbs should find broad applications in immunosensors for point-of-care diagnostics of sepsis and systemic inflammation processes.     

Detection of microbial volatile organic compounds (MVOCs) by ion-mobility spectrometry

Traces of microbial volatile organic compounds (MVOCs) in air can indicate the presence of growth of moulds in the indoor environment. Ion-mobility spectrometry is a very promising method for detection of these MVOCs, because of its high sensitivity. For development of an in-situ method for detection of MVOCs, a portable ion-mobility spectrometer (IMS) was used and test gases of 14 MVOCs and their respective mixtures were investigated. IMS spectra were recorded as a function of concentration of MVOCs in air. Drift time and mobility of reactant ions formed in positive polarity mode were determined and correlated with the mass-to-charge ratio (m/z) of the MVOCs investigated. The estimated detection limit has a specific value for each MVOC and is in the range 3 to 96 μg m⁻³ (1 to 52 ppb_V). Indoor trials show that IMS can indicate hidden mould growth.     

Electrochemical Immunoassay for Free Prostate Specific Antigen (f‐PSA) Using Magnetic Beads

Prostate specific antigen (PSA) is a prominent marker for the prostate carcinoma. It is found in human blood in free (f‐PSA) and complex forms. These two forms together are called total PSA (t‐PSA). Estimation of both forms is essential to predict malignancy. In this study we report a unique and effective technique of electrochemical detection of f‐PSA using magnetic beads on a three‐electrode screen‐printed sensor. A magnetic bead enzyme linked immunosorbent assay (ELISA) was performed in a cuvette. Following the immunoassay, magnetic beads were recovered by a magnetic concentrator and transferred on the working electrode of the 3‐electrode assembly. The amperometric response, a measure of the amount of residual enzyme activity on the beads and hence the concentration of analyte in solution, was determined by addition of enzyme substrate. The device has a detection limit of <0.1 ng mL^?1 f‐PSA and a linear range of 0 to 1 ng mL^?1 f‐PSA.     

Effect of some physico-chemical conditions on an immunoassay for viable Escherichia coli

Viable Escherichia coli can be detected by an immunoassay in which live bacteria captured on antibody-coated paramagnetic beads are induced to synthesize the enzyme β-galactosidase, which catalyzes the hydrolysis of the slightly fluorescent substrate 4-methyl umbelliferyl-β-d-galactoside to the highly fluorescent product 7-hydroxy-4-methylcoumarin for detection. The effects of bacterial strain, presence of dead bacteria, and some environmental stresses on assay performance were evaluated.