Polymeric micelle-based bioassay with femtomolar sensitivity

Target-specific polymeric micelles loaded with fluorescence dye molecules in their hydrophobic cores were made from block copolymer of poly(caprolactones)23-b-poly(ethylene oxide)45. It was found that the micelles are stable against pH changes from pH 2 to 12 and temperature variation up to 65 degrees C. The dye molecules can be released to the solution on exposing the micelles to organic solvents or ultrasound. A rapid and highly sensitive immunoassay based on the above micelles was developed, and the assay can detect specific target proteins in the femtomolar range from complex biological samples such as serum mimics and cell lysate. For example, less than 0.15 U/ml of ovarian cancer-specific antigen 125, equivalent to 7.5 x 10(-15)M, can be reliably detected in solution. We also demonstrated that the assay can detect a cell surface biomarker, stage-specific embryonic antigen 4, from a single human embryonic stem cell.     

Porous silicon-based biosensor for pathogen detection

A porous silicon-based biosensor for rapid detection of bacteria was fabricated. Silicon (0.01 ohmcm, p-type) was anodized electrochemically in an electrochemical Teflon cell containing ethanoic hydrofluoric acid solution to produce sponge-like porous layer of silicon. Anodizing conditions of 5 mA/cm2 for 85 min proved best for biosensor fabrication. A single-tube chemiluminescence-based assay, previously developed, was adapted to the biosensor for detection of Escherichia coli. Porous silicon chips were functionalized with a dioxetane-Polymyxin B (cell wall permeabilizer) mixture by diffusion and adsorption on to the porous surface. The reaction of beta-galactosidase enzyme from E. coli with the dioxetane substrate generated light at 530 nm. Light emission for the porous silicon biosensor chip with E. coli was significantly greater than that of the control and planar silicon chip with E. coli (P<0.01). Sensitivity of the porous silicon biosensor was determined to be 101-102 colony forming units (CFU) of E. coli. The porous silicon-based biosensor was fabricated and functionalized to successfully detect E. coli and has potential applications in food and environmental testing.     

Development and comparison of whole-cell assay systems for quorum-sensing inhibitors based on TraR, LasR, and QscR

Quorum sensing (QS) is a cell density-dependent signaling system that is used by bacteria to coordinate gene expression within their population. In this study, the authors describe the development and characterization of various cell-based bioassay systems for detecting QS inhibitors based on three LuxR family proteins, TraR, LasR, and the recently identified QscR. Three different gram-negative bacteria, Escherichia coli, Agrobacterium tumefaciens, and Pseudomonas aeruginosa, were employed as reporter strains to overproduce one of the aforementioned QS activator proteins and respond to inhibitors. The nine different whole-cell assay systems (three reporter strains × three QS proteins) were evaluated for their applicability and reliability by studying quantitative responses to various furanones, which are potent inhibitors of the LuxR family proteins. These results demonstrate that the cell-based bioassay systems are sensitive and reliable tools for screening of QS activators and inhibitors. This study also suggests that furanones are potentially important QS inhibitors for many LuxR-type activator proteins.     

吡咯喹啉醌产生菌筛选方法建立及菌种筛选

吡咯喹啉醌(PQQ)是一种氧化还原酶的辅酶,具有多种生理功能。扩增得到大肠杆菌葡萄糖脱氢酶(GDH)基因,并利用表达载体pET28a在E.coli BL21(DE3)中进行了表达。纯化了可溶性表达产物,并建立了基于GDH的重组酶法分析PQQ的方法。确定了甲基营养菌筛选模型,从2000余份土样中分离得到一株PQQ高产生菌MP606,在未经培养条件优化及诱变选育的条件下PQQ产量达113mg/L。从该菌培养液中制备得到了产物的结晶,HPLC分析、特征光谱分析以及酶法分析均证实该产物为PQQ。扩增并分析了MP606的16S rDNA序列,结果显示该菌16S rDNA序列与12种甲基营养菌都具有95%以上同源性,其中与食甲基菌属两菌株的16S rDNA序列同源性达99%。     

Colistin-functionalised CdSe/ZnS quantum dots as fluorescent probe for the rapid detection of Escherichia coli

Intensely fluorescent, colistin-functionalised CdSe/ZnS QDs (Colis-QDs) nanoparticles, are synthesized and used as sensitive probes for the detection of Escherichia coli, a Gram-negative bacteria. Colistin molecules are attached to the terminal carboxyl of the mercaptoacetic acid-capped QDs in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) as amide bond promoters. The TEM analysis of bacteria treated with Colis-QDs conjugates showed the accumulation of Colis-QDs in the cell wall of E. coli. Under the recommended working conditions, the method provides a detection limit as few as 28 E. coli cells per mL, which is competitive which more elaborate detection systems. The simplicity of the method together with short analysis time (< 15 min, without including preparation and photoactivation of the Colis-QDs conjugate) make the proposed approach useful as quick bacteria screening system.     

Rapid and sensitive detection method of a bacterium by using a GFP reporter phage

A rapid, sensitive, and convenient method for detecting a specific bacterium was developed by using a GFP phage. Here we describe a model system that utilizes the temperate Escherichia coli-restricted bacteriophage lambda, which was genetically modified to express a reporter gene for GFP to identify the colon bacillus E. coli in the specimen. E. coli infected with GFP phage was detected by GFP fluorescence after 4-6 hr of incubation. The results show that a few bacteria in a specimen can be detected under fluorescence microscopy equipped with a sensitive cooled CCD camera. When E. coli and Mycobacterium smegmatis were mixed in a solution containing GFP phage, only E. coli was infected, indicating the specificity of this method. The method has the following advantages: 1) Bacteria from biological samples need not be purified unless they contain fluorescent impurities; 2) The infection of GFP phage to bacteria is specific; 3) The fluorescence of GFP within infected bacteria enables highly sensitive detection; 4) Exogenous substrates and cofactors are not required for fluorescence. Therefore this method is suitable for any phage-bacterium system when bacteria-specific phages are available.     

A bioassay based on recombinant DNA technology for determining selenium concentration.

The trace element selenium has recently attracted attention, particularly because (i) selenocysteine is involved in the active site of various prokaryotic and eukaryotic enzymes, some of which have a role in human health; (ii) selenocysteine incorporation into these proteins is coded by UGA codons; and (iii) as a result, selenocysteine is now considered to be the 21st amino acid in an expanded genetic code. Here, we built recombinant DNA constructs in which expression of the lac'Z gene is driven in Escherichia coli by UGA-directed selenocysteine incorporation. In this system, levels of beta-galactosidase activity are proportionally and specifically related to the presence and concentrations of several specific simple selenium derivatives. The system can thus be used as a sensitive bioassay for their determination. This bioassay is one of a few using recombinant DNA technology to provide a reporter for simple detection of a chemical trace element.     

Cellular injuries upon exposure of Escherichia coli and Lactobacillus rhamnosus to high-intensity ultrasound

AIMS: To examine cellular injuries occurring in cells of Escherichia coli (Gram-negative bacteria) and Lactobacillus rhamnosus (Gram-positive bacteria) in response to a high-intensity ultrasound treatment using classical plate count technique and flow cytometry. METHOD AND RESULTS: According to plate count results, E. coli (D-value 8.3 min) was far more sensitive than L. rhamnosus (D-value 18.1 min) in their response to the ultrasound intensity applied (20 kHz, 17.6 W). The dye precursor carboxyfluorescein diacetate (cFDA) could freely diffuse across the cytoplasmic membrane of intact cells of Gram-positive bacteria L. rhamnosus, resulting in its intracellular enzymatic conversion and emission of green fluorescence. In contrast, the presence of an outer membrane on E. coli, which represents the class of Gram-negative bacteria, apparently disabled the penetration of viability marker cFDA. Ultrasound application on E. coli yielded in an increasing population with disintegrated outer membrane, which allowed penetration of cFDA and its intracellular enzymatic conversion as well as accumulation. In both organisms evaluated only a small population was labelled by propidium iodide upon exposure to ultrasound for up to 20 min. Within the experimental conditions investigated ultrasound did not considerably affect the cytoplasmic membrane, although according to plate count results viability loss occurred. CONCLUSIONS: The results compiled suggest, that ultrasound induced cell death, which may not be related to membrane damage. SIGNIFICANCE AND IMPACT OF THE STUDY: Limitation on the use of bacteriocins, which are aimed on destabilization of cytoplasmic membrane but inhibited by the outer membrane, could be overcome by ultrasound-assisted physical disruption of the outer membrane.     

Rapid electrochemical detection and identification of catalase positive micro-organisms

The rapid detection and identification of bacteria has application in a number of fields, e.g. the food industry, environmental monitoring and biomedicine. While in biomedicine the number of organisms present during infection is multiples of millions in the other fields it is the detection of low numbers of organisms that is important, e.g. an infective dose of Escherichia coli O157:H7 from contaminated food is less than 100 organisms. A rapid and sensitive technique has been developed to detect low numbers of the model organism E. coli O55, combining Lateral Flow Immunoassay (LFI) for capture and amperometry for sensitive detection. Nitrocellulose membranes were used as the solid phase for selective capture of the bacteria using antibodies to E. coli O55. Different concentrations of E. coli O55 in Ringers solution were applied to LFI strips and allowed to flow through the membrane to an absorbent pad. The capture region of the LFI strip was placed in close contact with the electrodes of a Clarke cell poised at +0.7 V for the detection of hydrogen peroxide. Earlier research identified that the consumption of hydrogen peroxide by bacterial catalase provided a sensitive indicator of aerobic and facultative anaerobic microorganisms numbers. Modification and application of this technique to the LFI strips demonstrated that the consumption of 8 mM hydrogen peroxide was correlated with the number of microorganisms presented to the LFI strips in the range of 2 x 10(1)-2 x 10(7) colony forming units (cfu). Capture efficiency was dependent on the number of organisms applied and varied from 71% at 2 x 10(2) cfu to 25% at 2 x 10(7) cfu. The procedure was completed in less than 10 min and could detect less than 10 cfu captured from a 200 microl sample applied to the LFI strip. The approached adopted provides proof of principle for the basis of a new technological approach to the rapid, quantitative and sensitive detection of bacteria that express catalase activity.     

Fluorescent nanoparticles for multiplexed bacteria monitoring

Rapid, sensitive, and selective detection of pathogenic bacteria is extremely important for proper containment, diagnosis, and treatment of diseases like foodborne illness, sepsis, and bioterrorism. Most current bacterial detection methods are time-consuming and laborious and can detect only one bacterial pathogen at a time. We have developed a method for sensitive, multiplexed monitoring of bacterial pathogens within 30 min using multicolored FRET (fluorescence resonance energy transfer) silica NPs (nanoparticles). By varying the ratio of three tandem dyes coencapsulated into the NPs, we have synthesized NPs that emit unique colors upon excitation with a single wavelength. When these NPs were conjugated to monoclonal antibodies specific for the pathogenic bacteria species Escherichia coli, Salmonella typhimurium, and Staphylococcus aureus, and then incubated with small concentrations of the bacteria, simultaneous and sensitive detection of the multiple bacterial targets was achieved.     

Near-infrared fluorescent nanocapsules with reversible response to thermal/pH modulation for optical imaging

Polymeric near-infrared (NIR) fluorescent nanocapsules were developed, of which the fluorescence exhibited reversible response to local thermal/pH modulation. Our strategy was to use polymeric micelles made of temperature-sensitive Pluronic F-127 to encapsulate an amphiphilic NIR fluorescent dye-indocyanine green (ICG)-within the core and then cross-link the micelle corona by pH-sensitive poly(ethylenimine) (PEI). The size swelling/shrinking property of the micelles induced by temperature decrease/increase was used as a switch to control the fluorescence yield of the nanocapsules. It was found that the fluorescence yield significantly increased with the increase in temperature. The PEI cross-link made the fluorescence yield also sensitive to local pH change and enhanced intracellular delivery of the nanocapsules as well. Preliminary results suggest the NIR fluorescent probes could be potentially used as a contrast agent sensitive to local environment for translational optical imaging/sensing.     

Hydrotropic polymeric micelles for enhanced paclitaxel solubility: in vitro and in vivo characterization

The purpose of this investigation was to characterize the in vitro stability and in vivo disposition of paclitaxel in rats after solubilization of paclitaxel into hydrotropic polymeric micelles. The amphiphilic block copolymers consisted of a micellar shell-forming poly(ethylene glycol) (PEG) block and a core-forming poly(2-(4-vinylbenzyloxy)-N,N-diethylnicotinamide) (P(VBODENA)) block. N,N-Diethylnicotinamide (DENA) in the micellar inner core resulted in effective paclitaxel solubilization and stabilization. Solubilization of paclitaxel using polymeric micelles of poly(ethylene glycol)-b-P(D,L-lactide) (PEG-b-PLA) served as a control for the stability study. Up to 37.4 wt % paclitaxel could be loaded in PEG-b-P(VBODENA) micelles, whereas the maximum loading amount for PEG-b-PLA micelles was 27.6 wt %. Thermal analysis showed that paclitaxel in the polymeric micelles existed in the molecularly dispersed amorphous state even at loadings over 30 wt %. Paclitaxel-loaded hydrotropic polymeric micelles retained their stability in water for weeks, whereas paclitaxel-loaded PEG-b-PLA micelles precipitated in a few days. Hydrotropic polymer micelles were more effective than PEG-PLA micelle formulations in inhibiting the proliferation of human cancer cells. Paclitaxel in hydrotropic polymer micelles was administered orally (3.8 mg/kg), intravenously (2.5 mg/kg), or via the portal vein (2.5 mg/kg) to rats. The oral bioavailability was 12.4% of the intravenous administration. Our data suggest that polymeric micelles with a hydrotropic structure are superior as a carrier of paclitaxel due to a high solubilizing capacity combined with long-term stability, which has not been accomplished by other existing polymeric micelle systems.     

Multifunctional magnetic-plasmonic nanoparticles for fast concentration and sensitive detection of bacteria using SERS

Multifunctional magnetic-plasmonic Fe(3)O(4)-Au core-shell nanoparticles (Au-MNPs) were prepared for simultaneous fast concentration of bacterial cells by applying an external point magnetic field, and sensitive detection and identification of bacteria using surface-enhanced Raman spectroscopy (SERS). We demonstrated that a spread of a 10 μL drop of a mixture of 10(5) cfu/mL bacteria and 3 μg/mL Au-MNPs on a silicon surface can be effectively condensed into a highly compact dot within 5 min by applying an external point magnetic field, resulting in 60 times more concentrated bacteria in the dot area than on the spread area without concentration. Surrounded by dense uniformly packed Au-MNPs, bacteria can be sensitively and reproducibly detected directly using SERS. The principle component analysis (PCA) showed that three different Gram-negative bacterial strains can be clearly differentiated. We also demonstrated that the condensed multifunctional Au-MNPs dot can be used as a highly sensitive SERS-active substrate and a limit of detection better than 0.1 ppb was obtained in detection of small molecules such as 4-mercaptopyrine. This novel platform significantly simplifies the concentration and detection process, which holds great promise for applications in food safety, environmental monitoring, medical diagnoses, and chemical and biological threat detections.     

Determination of lipopolysaccharide by a bioluminescence technique.

The determination of the lipid A content of bacterial lipopolysaccharide by using a dim mutant of the luminous bacterium Beneckea harveyi is described. The luminous bacteria emitted light upon the addition of an acid hydrolysate of lipopolysaccharide which contained myristic acid, thus making it possible to detect as little as 1 ng of lipopolysaccharide. By converting the 3-OH-myristic acid to myristic acid, it was possible to further increase the detection sensitivity and to establish a basis for a specific and highly sensitive bioassay for the detection of lipopolysaccharide.     

Determination of lipopolysaccharide by a bioluminescence technique.

The determination of the lipid A content of bacterial lipopolysaccharide by using a dim mutant of the luminous bacterium Beneckea harveyi is described. The luminous bacteria emitted light upon the addition of an acid hydrolysate of lipopolysaccharide which contained myristic acid, thus making it possible to detect as little as 1 ng of lipopolysaccharide. By converting the 3-OH-myristic acid to myristic acid, it was possible to further increase the detection sensitivity and to establish a basis for a specific and highly sensitive bioassay for the detection of lipopolysaccharide.     

Conjugation of arginine-glycine-aspartic acid peptides to poly(ethylene oxide)-b-poly(epsilon-caprolactone) micelles for enhanced intracellular drug delivery to metastatic tumor cells

An arginine-glycine-aspartic acid (RGD) containing model peptide was conjugated to the surface of poly(ethylene oxide)-block-poly(epsilon-caprolactone) (PEO-b-PCL) micelles as a ligand that can recognize adhesion molecules overexpressed on the surface of metastatic cancer cells, that is, integrins, and that can enhance the micellar delivery of encapsulated hydrophobic drug into a tumor cell. Toward this goal, PEO-b-PCL copolymers bearing acetal groups on the PEO end were synthesized, characterized, and assembled to polymeric micelles. The acetal group on the surface of the PEO-b-PCL micelles was converted to reactive aldehyde under acidic condition at room temperature. An RGD-containing linear peptide, GRGDS, was conjugated on the surface of the aldehyde-decorated PEO-b-PCL micelles by incubation at room temperature. A hydrophobic fluorescent probe, that is, DiI, was physically loaded in prepared polymeric micelles to imitate hydrophobic drugs loaded in micellar carrier. The cellular uptake of DiI loaded GRGDS-modified micelles by melanoma B16-F10 cells was investigated at 4 and 37 degrees C by fluorescent spectroscopy and confocal microscopy techniques and was compared to the uptake of DiI loaded valine-PEO-b-PCL micelles (as the irrelevant ligand decorated micelles) and free DiI. GRGDS conjugation to polymeric micelles significantly facilitated the cellular uptake of encapsulated hydrophobic DiI most probably by intergrin-mediated cell attachment and endocytosis. The results indicate that acetal-terminated PEO-b-PCL micelles are amenable for introducing targeting moieties on the surface of polymeric micelles and that RGD-peptide conjugated PEO-b-PCL micelles are promising ligand-targeted carriers for enhanced drug delivery to metastatic tumor cells.     

Proving the antimicrobial spectrum of an amphoteric surfactant-sol-gel coating: a food-borne pathogen study

An antimicrobial coating was evaluated in this work for its antimicrobial efficacy against common food-borne pathogens. Dodecyl-di(aminoethyl)-glycine, an organic disinfectant, was immobilized in a silicon oxide matrix to generate thin films over surfaces by means of the sol-gel process. Tetraethoxysilane was used as the polymeric precursor. No alteration of optical transparency on the covered surfaces was observed. Topographic images obtained with atomic force microscopy showed a homogeneous film with no additional roughness added by the polymer to the surface. The attenuated total reflectance-Fourier transform infrared spectral data showed the presence of dodecyl-di(aminoethyl)-glycine in the silicon oxide network after a normal cleaning procedure. The antimicrobial efficacy test was performed by exposing coated slides to suspensions of common food-borne pathogens: Escherichia coli, Staphyloccocus aureus, E. coli O157:H7, Salmonella typhi, S. cholerasuiss, Listeria innocua and L. monocytogenes. The coating activity was not only bacteriostatic but also bactericidal. The percent reduction of viable microorganism exposure over 24 h to the coated surface ranged between 99.5%, for the more resistant gram-positive bacteria, and over 99.999%, for most gram-negative bacteria. The silicon matrix itself did not account for any reduction of viable microbial, even more an increase was observed.     

ENUMERATION OF IMMUNOMAGNETICALLY CAPTURED ESCHERICHIA COLI IN WATER SAMPLES USING QUANTUM DOT-LABELED ANTIBODIES

Immunomagnetic separation was coupled with quantum dot (QD) labeling for the rapid, selective and sensitive detection of Escherichia coli in water samples. The target bacteria were recovered from the solution by antibody-coated paramagnetic beads, and sandwich complexes were formed by using secondary antibodies labeled with QDs. The fluorescence intensities, as a result of the capturing of different concentrations of bacteria, were measured, and a linear correlation ( R ²  =  0.976) was obtained between log E. coli concentration ( x ) and the intensity ( y ) with a regression model of y =  26.9x  +  41.1 in a working range of 8.9  ×  10¹ and 1.9  ×  10⁶ cfu/mL. The selectivity of the developed sensor was examined with Enterobacter aerogenes and Enterobacter dissolvens, which did not produce any significant response. The ability of the immunoassay to detect E. coli in real water samples was investigated and the results were compared with the experimental results from plate-counting methods. A good agreement was observed between the QD-enhanced detection and plate counting.

PRACTICAL APPLICATIONS

In this study, a rapid, sensitive and convenient fluorometric assay based on the immunomagnetic separation (IMS) and quantum dot (QD) labeling was employed for the detection of Escherichia coli in water samples. The incorporation of QDs into fluorometric immunoassay techniques has various advantages over labeling with organic dyes and enzymes. In addition, the spectroscopic properties of QDs can allow multiplexed immunoassays coupled with IMS for bacteria detection, which will be investigated in further studies. Here we showed that QD labeling is a promising tool for the detection of E. coli in real water samples containing different components with a lower detection limit.     

An integrated, stacked microlaboratory for biological agent detection with DNA and immunoassays

An integrated, stacked microlaboratory for performing automated electric-field-driven immunoassays and DNA hybridization assays was developed. The stacked microlaboratory was fabricated by orderly laminating several different functional layers (all 76 x 76 mm(2)) including a patterned polyimide layer with a flip-chip bonded CMOS chip, a pressure sensitive acrylic adhesive (PSA) layer with a fluidic cutout, an optically transparent polymethyl methacrylate (PMMA) film, a PSA layer with a via, a patterned polyimide layer with a flip-chip bonded silicon chip, a PSA layer with a fluidic cutout, and a glass cover plate layer. Versatility of the stacked microlaboratory was demonstrated by various automated assays. Escherichia coli bacteria and Alexa-labeled protein toxin staphylococcal enterotoxin B (SEB) were detected by electric-field-driven immunoassays on a single chip with a specific-to-nonspecific signal ratios of 4.2:1 and 3.0:1, respectively. Furthermore, by integrating the microlaboratory with a module for strand displacement amplification (SDA), the identification of the Shiga-like toxin gene (SLT1) from E. coli was accomplished within 2.5 h starting from a dielectrophoretic concentration of intact E. coli bacteria and finishing with an electric-field-driven DNA hybridization assay, detected by fluorescently labeled DNA reporter probes. The integrated microlaboratory can be potentially used in a wide range of applications including detection of bacteria and biowarfare agents, and genetic identification.     

A novel bioassay for the detection of siderophores containing keto-hydroxy bidentate ligands

Abstract A convenient and sensitive pour-plate Petri dish bioassay for the detection of siderophores containing monoprotic keto-hydroxy bidentate ligands (KHBL) has been developed. The bioassay is based on the fact that bacteria of the Proteus-Providencia-Morganella group (Proteeae) utilize various ferric α-hydroxy- or α-ketocarboxylate complexes very efficiently. While P. vulgaris and P. rettgeri were able to utilize virtually all iron complexes supplied, Morganella morganii SBK3 was unable to utilize trihydroxamate type siderophores and was therefore selected as an indicator strain for iron complexes containing keto-hydroxy bidentate ligands (KHBL-siderophores). Filter paper disks containing the ferric complexes of siderophores were tested on tryptone or Luria broth agar, seeded with the indicator strains and supplemented with the ferrous iron chelator 2,2-dipyridyl (300 μM) to reduce the bioavailable iron. In the presence of siderophores, growth inhibition was reversed to provide a zone of growth stimulation. Ferric complexes of α-hydroxycarboxylates, α-ketocarboxylates, salicylic acid, tropolonederivatives, α-hydroxypyridinones, cepabactin, citrate, rhizoferrin and even epihydroxymugineic acid showed significant growth stimulation. From the results with the trihydroxamate-non-utilizing strain, M. morganii SBK3 , it may be inferred that the Proteeae prossess an iron transport system which recognizes ferric α-hydroxycarboxylates, α-ketocarboxylates as well as aromatic and heteroaromatic keto-hydroxy compounds, collectively named keto-hydroxy bidentate ligands. The bioassay is especially suited for detection of new siderophores from low-iron cultures of fungi and bacteria.