A self-assembled monolayer-based piezoelectric immunosensor for rapid detection of Escherichia coli O157:H7

A piezoelectric immunosensor was developed for rapid detection of Escherichia coli O157:H7. It was based on the immobilization of affinity-purified antibodies onto a monolayer of 16-mercaptohexadecanoic acid (MHDA), a long-chain carboxylic acid-terminating alkanethiol, self-assembled on an AT-cut quartz crystal's Au electrode surface with N-hydroxysuccinimide (NHS) ester as a reactive intermediate. The binding of target bacteria onto the immobilized antibodies decreased the sensor's resonant frequency, and the frequency shift was correlated to the bacterial concentration. The stepwise assembly of the immunosensor was characterized by means of both quartz crystal microbalance (QCM) and cyclic voltammetry techniques. Three analytical procedures, namely immersion, dip-and-dry and flow-through methods, were investigated. The immunosensor could detect the target bacteria in a range of 10(3)-10(8)CFU/ml within 30-50 min, and the sensor-to-sensor reproducibility obtained at 10(3) and 10(5) colony-forming units (CFU)/ml was 18 and 11% R.S.D., respectively. The proposed sensor was comparable to Protein A-based piezoelectric immunosensor in terms of the amount of immobilized antibodies and detection sensitivity.     

Liposome-based microcapillary immunosensor for detection of Escherichia coli O157:H7

Our group has previously reported a sandwich-based strip immunoassay for rapid detection of Escherichia coli O157:H7 [Anal. Chem. 75 (2003) 4330]. In the present study, a microcapillary flow injection liposome immunoanalysis (mFILIA) system was developed for the detection of heat-killed E. coli O157:H7. A fused-silica microcapillary with anti-E. coli O157:H7 antibodies chemically immobilized on the internal surface via protein A served as an immunoreactor/immunoseparator for the mFILIA system. Liposomes tagged with anti-E. coli O157:H7 and encapsulating a fluorescent dye were used as the detectable label. In the presence of E. coli O157:H7, sandwich complexes were formed between the immobilized antibodies in the column, the sample of E. coli O157:H7 and the antibody-tagged sulforhodamine-dye-loaded liposomes. Signals generated by lysing the bound liposomes with 30 mM n-octyl-beta-D-glucopyranoside were measured by a fluorometer. The detected signal was directly proportional to the amount of E. coli O157:H7 in the test sample. The mFILIA system successfully detected as low as 360 cells/mL (equivalent to 53 heat-killed bacteria in the 150 microL of the sample solution injected). MeOH (30%) was used for the regeneration of antibody binding sites in the capillary after each measurement, which allowed the immunoreactor/immunoseparator to be used for at least 50 repeated assays. The calibration curve for heat-killed E. coli O157:H7 has a working range of 6 x 10(3)-6 x 10(7)cells, and the total assay time was less than 45 min. A coefficient of variation for triplicate measurements was < or =8.9%, which indicates an acceptable level of reproducibility for this newly developed method.     

Amperometric immunosensor for the detection of Escherichia coli O157:H7 in food specimens

A novel, label-free amperometric immunosensor has been developed for the rapid detection of heat-killed Escherichia coli O157:H7 (E. coli O157:H7). This immunosensor was prepared as follows. First, the long-chain, amine-terminated alkanethiol 11-amino-1-undecanethiol hydrochloride (AUT) was self-assembled onto a gold electrode surface to form an ordered, oriented, compact, and stable monolayer possessing -NH(2) functional groups that could immobilize massive gold nanoparticles (GNPs). Next, chitosan-multiwalled carbon nanotubes-SiO(2)/thionine (CHIT-MWNTs-SiO(2)@THI) nanocomposites and GNPs multilayer films were prepared via layer-by-layer (LBL) assembly. The surface area enhancement from the LBL assembly of the multilayer films improves the stability of the immobilized CHIT-MWNTs-SiO(2)@THI. More important, the sensitivity and stability of the immunosensor can be enhanced proportionally to the quantity of the THI mediator immobilized on the electrode surface. Finally, the E. coli O157:H7 antibody (anti-E. coli O157:H7) was covalently bound to the GNP monolayer and its bioactivity was measured by enzyme-linked immunosorbent assay (ELISA). Transmission electron microscopy (TEM) was employed to characterize the morphology of the MWNTs, CHIT-MWNTs, and CHIT-MWNTs-SiO(2)@THI. Under optimal conditions, the calibration curve for heat-killed E. coli O157:H7 has a working range of 4.12×10(2)-4.12×10(5) colony-forming units (CFU)/ml, and the total assay time was less than 45 min.     

A high density microelectrode array biosensor for detection of E. coli O157:H7

A high density microelectrode array biosensor was developed for the detection of Escherichia coli O157:H7. The biosensor was fabricated from (100) silicon with a 2 microm layer of thermal oxide as an insulating layer, an active area of 9.6 mm2 and consists of an interdigitated gold electrode array. The sensor surface was functionalised for bacterial detection using heterobifunctional crosslinkers and immobilised polyclonal antibodies to create a biological sensing surface. Bacteria suspended in solution became attached to the immobilised antibodies when the biosensor was tested in liquid samples. The change in impedance caused by the bacteria was measured over a frequency range of 100 Hz-10 M Hz. The biosensor was evaluated for E. coli O157:H7 detection in pure culture and inoculated food samples. The biosensor was able to discriminate between cellular concentrations of 10(4)-10(7)CFU/mL and has applications in detecting pathogens in food samples.     

Magnetoresistive immunosensor for the detection of Escherichia coli O157:H7 including a microfluidic network

A hand held device has been designed for the immunomagnetic detection and quantification of the pathogen Escherichia coli O157:H7 in food and clinical samples. In this work, a technology to manufacture a Lab on a Chip that integrates a 3D microfluidic network with a microfabricated biosensor has been developed. With this aim, the sensing film optimization, the design of the microfluidic circuitry, the development of the biological protocols involved in the measurements and, finally, the packaging needed to carry out the assays in a safe and straightforward way have been completed. The biosensor is designed to be capable to detect and quantify small magnetic field variations caused by the presence of superparamagnetic beads bound to the antigens previously immobilized on the sensor surface via an antibody-antigen reaction. The giant magnetoresistive multilayer structure implemented as sensing film consists of 20[Cu(5.10nm)/Co(2.47 nm)] with a magnetoresistance of 3.20% at 235Oe and a sensitivity up to 0.06 Omega/Oe between 150Oe and 230Oe. Silicon nitride has been selected as optimum sensor surface coating due to its suitability for antibody immobilization. In order to guide the biological samples towards the sensing area, a microfluidic network made of SU-8 photoresist has been included. Finally, a novel packaging design has been fabricated employing 3D stereolithographic techniques. The microchannels are connected to the outside using standard tubing. Hence, this packaging allows an easy replacement of the used devices.     

Flow-through immunofiltration assay system for rapid detection of E. coli O157:H7

A flow-through amperometric immunofiltration assay system based on disposable porous filter-membranes for rapid detection of Escherichia coli O157:H7 has been developed. The analytical system utilizes flow-through, immunofiltration and enzyme immunoassay techniques in conjunction with an amperometric sensor. The parameters affecting the immunoassay such as selection of appropriate filter membranes, membrane pore size, antibody binding capacity and the concentrations of immunoreagents were investigated and optimized. Non-specific adsorption of the enzyme conjugate was investigated and minimized. A sandwich scheme of immunoassay was employed and the immunofiltration system allows to specifically and directly detect E. coli cells with a lower detection limit of 100 cells/ml. The working range is from 100 to 600 cells/ml with an overall analysis time of 30 min. No pre-enrichment was needed. This immunosensor can be easily adapted for assay of other microorganisms and may be a basis for a new class of highly sensitive bioanalytical devices for rapid quantitative detection of bacteria.     

Evaluation of different micro/nanobeads used as amplifiers in QCM immunosensor for more sensitive detection of E. coli O157:H7

Micro/nanobeads with different materials (magnetic, silica and polymer) and different sizes (diameters from 30nm to 970nm) were investigated for their use as amplifiers in a quartz crystal microbalance (QCM) immunosensor for more sensitive detection of Escherichia coli O157:H7. The micro/nanobeads were conjugated with anti-E. coli antibodies. E. coli O157:H7 cells were first captured by the first antibody immobilized on the electrode surface, and then micro/nanobeads labeled secondary antibodies attached to the cells, and finally the complexes of antibody-E. coli-antibody modified beads were formed. The results showed that antibody-labeled beads lead to signal amplification in both the change in frequency (ΔF) and the change in resistance (ΔR). Since the penetration depth of the oscillation-induced shear-waves for a ～8MHz crystal is limited to 200nm, the interpretation of how the signal is amplified by the adsorbed particles was represented in terms of the coupled-oscillator theory. The amplification is not sensed in terms of increase in mass on the sensor surface. Amplification is sensed as a change in bacterial resonance frequency when the spheres adsorb to the bacteria. The change in the values of ΔF caused by different micro/nanobeads (amplifiers) attaching on target bacterial cells is indicative of the ratio between the resonance frequency of the absorbed bacterial-particle complex (ω(s)), and the resonance frequency of the crystal (ω).     

Protein array consisting of sol-gel bioactive platform for detection of E. coli O157:H7

Sol-gel-derived bioactive platform was fabricated for detection of pathogenic microbes, E. coli O157:H7. Design flexibility of sol-gel technique and ease of fabrication can fulfill to create the surfaces with structural and chemical features that are compatible with biomaterials such as antibody, enzymes, etc. In this study, the bioactive platform was prepared based on the silica gels, which were produced by hydrolyzing tetraethylorthosilane (TEOS) in ethanol. The mercaptopropyl triethoxysilane (MPTS) was mixed with the TEOS solution for the surface functionalization of bioactive platform. During TEOS hydrolysis, the modified thin film was prepared by sol-gel dip coating. Antibody against E. coli O157:H7 was immobilized with a configuration of protein array using piezo-type dispensing system. Surface morphology of the prepared bioactive platform was analyzed using atomic force microscopy (AFM). The antibody-antigen interaction was investigated with fluorescence microscopy and sandwich type immunoassay using fluorescein isothiocyanate (FITC)-labeled antibody. The results showed that antibody was sequestered within the sol-gel-derived bio-gel due to physical adsorption. The measurement of E. coli O157:H7 was done using the fabricated antibody surface. The fluorescence intensity was proportional to the concentration of E. coli O157:H7, of which the detection limit was 10(2)CFU/ml.     

Direct PCR detection of Escherichia coli O157:H7

AIMS: This paper reports a simple, rapid approach for the detection of Shiga toxin (Stx)-producing Escherichia coli (STEC). METHODS AND RESULTS: Direct PCR (DPCR) obviates the need for the recovery of cells from the sample or DNA extraction prior to PCR. Primers specific for Stx-encoding genes stx1 and stx2 were used in DPCR for the detection of E. coli O157:H7 added to environmental water samples and milk. CONCLUSIONS: PCR reactions containing one cell yielded a DPCR product. SIGNIFICANCE AND IMPACT OF THE STUDY: This should provide an improved method to assess contamination of environmental and other samples by STEC and other pathogens.     

肠出血性大肠杆菌O157：H7监测及分析

为了了解长春地区动物和人感染肠出血性大肠杆菌O157H7状况,建立流行病学监测网.采集长春市动物养殖场动物粪便和腹泻病人便样进行监测.结果在牛粪和鸡粪中共检出2株O157H7大肠杆菌.可见,在长春地区存在肠出血性大肠杆菌O157H7菌潜在污染的威胁,需要加强监测力度.     

Rapid electrochemical detection of polyaniline-labeled Escherichia coli O157:H7

There is a high demand for rapid, sensitive, and field-ready detection methods for Escherichia coli O157:H7, a highly infectious and potentially fatal food and water borne pathogen. In this study, E. coli O157:H7 cells are isolated via immunomagnetic separation (IMS) and labeled with biofunctionalized electroactive polyaniline (immuno-PANI). Labeled cell complexes are deposited onto a disposable screen-printed carbon electrode (SPCE) sensor and pulled to the electrode surface by an external magnetic field, to amplify the electrochemical signal generated by the polyaniline. Cyclic voltammetry is used to detect polyaniline and signal magnitude indicates the presence or absence of E. coli O157:H7. As few as 7CFU of E. coli O157:H7 (corresponding to an original concentration of 70 CFU/ml) were successfully detected on the SPCE sensor. The assay requires 70 min from sampling to detection, giving it a major advantage over standard culture methods in applications requiring high-throughput screening of samples and rapid results. The method can be performed with portable, handheld instrumentation and no biological modification of the sensor surface is required. Potential applications include field-based pathogen detection for food and water safety, environmental monitoring, healthcare, and biodefense.     

The lipopolysaccharide core type of Escherichia coli O157:H7 and other non-O157 verotoxin-producing E. coli

A mouse monoclonal antibody specific for the R3 lipopolysaccharide core type of Escherichia coli was used to determine the core type of E. coli O157:H7 and other non-O157 verotoxin-producing E. coli strains. Lipopolysaccharide extracts from 28 clinical isolates were examined by sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting and all were found to have the R3 core. None of the core lipopolysaccharide from the strains tested reacted with the control R1 and R2 specific monoclonal antibodies. A common core type between all the verotoxin-producing E. coli strains tested may be significant when considering the immune response to these bacteria, and to the receptor for the VT bacteriophage.     

Development of surface plasmon resonance immunosensor through metal ion affinity and mixed self-assembled monolayer

An immunosensor based on surface plasmon resonance (SPR) with enhanced performance was developed through a mixed self-assembled monolayer. A mixture of 16- mercaptohexadecanic acid (16-MHA) and 1-undecanethiol with various molar ratios was self-assembled on gold (Au) surface and the carboxylic acid groups of 16-MHA were then coordinated to Zn ions by exposing the substrate to an ethanolic solution of Zn(NO(3))(2)d6H2O. The antibody was immobilized on the SPR surface by exposing the functionalized substrate to the desired solution of antibody in phosphatebuffered saline (PBS) molecules. The film formation in series was confirmed by SPR and atomic force microscopy (AFM). The functionalized surface was applied to develop an SPR immunosensor for detecting human serum albumin (HSA) and the estimated detection limit (DL) was 4.27 nM. The limit value concentration can be well measured between ill and healthy conditions.     

Immunoliposome sandwich assay for the detection of Escherichia coli O157:H7

We describe the development of a field-portable colorimetric immunoassay for the detection of Escherichia coli O157:H7, using antibody-directed liposomes (immunoliposomes) encapsulating dye as an analytical reagent. Antibodies (anti-E. coli O157:H7) thiolated by 2-iminothiolane were coupled to malemide-tagged liposomes encapsulating the marker dye, sulforhodamine B. Transmission electron microscopy showed that the immunoliposomes bound only to the serotype without any cross-reactivity with tested negative controls. A wicking reagent containing immunoliposomes and the test sample and a plastic-backed nitrocellulose strip with a measurement zone were used in a sandwich (noncompetitive) assay format. During the capillary migration of the wicking reagent, E. coli, with surface-bound immunoliposomes, was captured at the measurement zone on which antibodies to E. coli O157:H7 were immobilized. The color density of the measurement zone was directly proportional to the amount of E. coli O157:H7 in the sample. The detection limit of the current assay with pure cultures of the serotype was ca. 10(4) colony-forming units (CFU)/mL. The assay, which does not need washing and incubation steps, can be completed in 8 min. These results demonstrate the feasibility of using dye-encapsulating immunoliposomes in microporous membranes for the rapid detection of molecules with multivalent antigenic sites.     

Multiplex PCR for detection of Escherichia coli O157:H7 in foods

Escherichia coli O157:H7 is well known enterohemorrhagic pathogen responsible for infections among animals including a man. The main source of this bacterium is cattle, that is mostly asymptomatic and through that E. coli O157:H7 can simple transfer to food products. Therefore, there is a need for rapid, sensitive and specific detection method. The present work is focused on its detection by a heptaplex polymerase chain reaction, which targets genes from known virulent regions of E. coli O157:H7. According to obtained results this approach is able to reach the detection sensitivity of 4 colony-forming units (CFU) from a culture and 6 and 8 CFU from milk and meat samples, respectively, independently of tested sample volume.     

Rapid PCR confirmation of E. coli O157:H7 after evanescent wave fiber optic biosensor detection

Escherichia coli O157:H7 is an enteric pathogen of public health importance, which is monitored by several government agencies. Many rapid detection tests have been developed to identify foodstuff and water supplies contaminated by E. coli O157:H7. However, these methods can be time consuming (24-48 h) due to the need to culture the bacteria to confirm detection results. Fiber optic biosensors can rapidly detect pathogens from complex matrices, yet confirmation tests can take up to 10h to complete. In addition, fiber optic biosensors can also be used to reduce the impact of PCR inhibitors present in complex matrices such as food and water. This paper presents methodologies to reduce the time necessary for confirmation from 10 to about 2 h, by direct PCR of bacteria from the fiber optic waveguides without the need for culture or enrichment steps.     

Confirmation of viable E. coli O157:H7 by enrichment and PCR after rapid biosensor detection

Many rapid tests have been developed for the detection of Escherichia coli O157:H7 from complex matrices such as food and water. However, many of these methods rely on traditional culture steps for confirmation, which can take an extra 24-48 h. The fiber optic biosensor has been used to rapidly detect pathogens from complex matrices. In this paper, we demonstrate a method using a rapid biosensor assay, recovery through a short enrichment, and PCR to detect and confirm the presence of at least 10(3) CFU/ml of E. coli O157:H7 in a sample in less than 10 h.