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.     

A fiber optic biosensor for fluorimetric detection of triple-helical DNA.

A fiber optic biosensor was used for the fluorimetric detection of T/AT triple-helical DNA formation. The surfaces of two sets of fused silica optical fibers were functionalized with hexaethylene oxide linkers from which decaadenylic acid oligonucleotides were grown in the 3'to 5'and 5'to 3'direction, respectively, using a DNA synthesizer. Fluorescence studies of hybridization showed unequivocal hybridization between oligomers immobilized on the fibers and complementary oligonucleotides from the solution phase, as detected by fluorescence from intercalated ethidium bromide. The complementary oligonucleotide, dT10, which was expected to Watson-Crick hybridize upon cooling the system below the duplex melting temperature ( T m), provided a fluorescence intensity with a negative temperature coefficient. Upon further cooling, to the point where the pyrimidine motif T*AT triple-helix formation occurred, a fluorescence intensity change with a positive temperature coefficient was observed. The reverse-Hoogsteen T.AT triplex, which is known to form with branched nucleic acids, provided a corresponding decrease in fluorescence intensity with decreasing temperature. Full analytical signal evolution was attainable in minutes.     

An absolute method for protein determination based on difference in absorbance at 235 and 280 nm

Because of the importance of quantitative determination of protein in the research laboratory as well as in the food and feed industries (1), search for the ideal method continues unabated after many years. Methods available include nitrogen determination (Kjeldahl (2) and Dumas (3)), hydrolysis of the protein, derivatization of the amino acids with phthalaldehyde and fluorescence determination (4), determination of bound or free lysine (5) or glutamate (4), and the Lowry (6), biuret (7) dye-binding (8–11) turbidity (12) and spectral methods (13). With the exception of the spectral methods, the methods involve destruction of the sample.In this paper we report the use of difference in absorbance between 235 and 280 nm for determination of protein concentration.     

Rapid label-free detection of E. coli using a novel SPR biosensor containing a fragment of tail protein from phage lambda

Abstract

In efforts to speed up the assessment of microorganisms, researchers have sought to use bacteriophages as a biosensing tool, due to their host-specificity, wide abundance, and safety. However, the lytic cycle of the phage has limited its efficacy as a biosensor. Here, we cloned a fragment of tail protein J from phage lambda and characterized its binding with the host, E. coli K-12, and other microorganism. The N-terminus of J was fused with a His-tag (6HN-J), overexpressed, purified, and characterized using anti-His monoclonal antibodies. The purified protein demonstrated a size of ～38?kDa upon SDS-PAGE and bound with the anti-His monoclonal antibodies. ELISA, dot blot, and TEM data revealed that it specifically bound to E. coli K-12, but not to Pseudomonas aeruginosa. The observed protein binding occurred over a concentration range of 0.01–5?μg/ml and was found to inhibit the in vivo adsorption of phage to host cells. This specific binding was exploited by surface plasmon resonance (SPR) to generate a novel 6HN-J-functionalized SPR biosensor. This biosensor showed rapid label-free detection of E. coli K-12 in the range of 2?×?10⁴ ?2?×?10⁹ CFU/ml, and exhibited a lower detection limit of 2?×?10⁴ CFU/ml.     

Aptamer biosensor for label-free square-wave voltammetry detection of angiogenin

Angiogenin (Ang), one of the most potent angiogenic factor, is related with the growth and metastasis of numerous tumors. This paper presents a very simple and label-free square-wave voltammetry (SWV) aptasensor to detect angiogenin, in which an anti-angiogenin-aptamer was used as a molecular recognition element, and the couple ferro/ferricyanide as a redox probe. At the bare gold electrode, the redox couple (K₄[Fe(CN)₆]/K₃[Fe(CN)₆]) can be very easily accessed to the electrode surface to give a very strong SWV signal. At the anti-angiogenin/Au electrode surface, when angiogenin was added to the electrochemical cell, the binding of the analyte results in less availability for a redox reaction, which led to smaller SWV current. To quantify the amount of angiogenin, current suppressions of SWV peak were monitored using the redox couple of an [Fe(CN)₆]^4−/3− probe. The plot of signal suppression against the logarithm of angiogenin concentration is linear with over the range from 0.01 nM to 30 nM with a detection limit of 1 pM. The aptasensor also showed very good selectivity for angiogenin without being affected by the presence of other proteins in serum. It is the first time to use a very simple method to detect the cancer marker. Such an aptasensor opens a rapid, selective and sensitive route for angiogenin detection and provides a promising strategy for other protein detections.     

A biosensor platform for rapid detection of E. coli in drinking water

There remains a need for rapid, specific and sensitive assays for the detection of bacterial indicators for water quality monitoring. In this study, a strategy for rapid detection of Escherichia coli in drinking water has been developed. This strategy is based on the use of the substrate 4-methylumbelliferyl-β-d-glucuronide (MUG), which is hydrolyzed rapidly by the action of E. coli β-d-glucuronidase (GUD) enzyme to yield a fluorogenic 4-methylumbelliferone (4-MU) product that can be quantified and related to the number of E. coli cells present in water samples. In this study, the detection time required for the biosensor response ranged between 20 and 120 min, depending on the number of bacteria in the sample. This approach does not need extensive sample processing with a rapid detection capability. The specificity of the MUG substrate was examined in both, pure cultures of non-target bacterial genera such as Klebsiella, Salmonella, Enterobacter and Bacillus. Non-target substrates that included 4-methylumbelliferyl-β-d-galactopyranoside (MUGal) and l-leucine β-naphthylamide aminopeptidase (LLβ-N) were also investigated to identify nonspecific patterns of enzymatic activities in E. coli. GUD activity was found to be specific for E. coli and no further enzymatic activity was detected by other species. In addition, fluorescence assays were performed for the detection of E. coli to generate standard curves; and the sensitivity of the GUD enzymatic response was measured and repeatedly determined to be less than 10 E. coli cells in a reaction vial. The applicability of the method was tested by performing multiple fluorescence assays under pure and mixed bacterial flora in environmental samples. The results of this study showed that the fluorescence signals generated in samples using specific substrate molecules can be utilized to develop a bio-sensing platform for the detection of E. coli in drinking water. Furthermore, this system can be applied independently or in conjunction with other methods as a part of an array of biochemical assays in order to reliably detect E. coli in water.     

Preliminary study of real-time fiber optic based protein C biosensor

Deficiency of protein C (PC), one of the human body's key anticoagulants, can lead to massive thrombotic complications. There is a diagnostic need to perform real-time assays, in order to quickly identify and treat this disease. An immuno-optical biosensor for the diagnosing of PC deficiencies and monitoring of PC concentrations is being developed for this purpose. Monoclonal antibody against PC (anti-PC) is immobilized on the surface of a tapered quartz fiber that is enclosed in a glass tube (capacity approximately 200 microL). Following sample injection, PC within a sample binds to the anti-PC in a highly specific reaction. The system is then probed with a fluorophore-tagged secondary antibody against PC. Excitation light is applied through the fiber, and the fluorescence intensity is correlated with the PC concentration in the sample. This study presents (1) a feasibility, direct binding assay, (2) a comparison of methods to immobilize anti-PC upon the fiber (direct immobilization vs an avidin-biotin bridge), and (3) effectiveness of an elution step to regenerate the fiber. PC-deficient patients typically have a concentration range less than 2.5 microg/mL. It was found that the sensor could detect PC levels down to 0.1 microg/mL in pure buffer with minimal optimization. Avidin-biotin immobilization of the primary antibody produced enhanced signals, up to 470% of the original intensities. Preliminary fiber regeneration tests achieved nearly a 50% increase in fiber lifetime with the use of a CaCl(2) elution step. Ultimately, further development may lead to automation and the use of the system as a multi-blood factor analyzer.     

A proteomic biosensor for enteropathogenic E. coli

The study of proteins and the molecules with which they interact on an organismwide scale is critical to understanding basic biology, and understanding and improving human health. New platform technologies allowing label-free, quantitative array-based analysis of proteins are particularly desirable. We have developed an analytical technology, reflective interferometry (RI), which provides specific, rapid, and label-free optical detection of biomolecules in complex mixtures. In order to evaluate the suitability of RI for proteomics, we have prepared a series of arrays bearing the extracellular domain of the secreted enteropathogenic Escherichia coli (EPEC) protein Translocated Intimin Receptor (Tir). These arrays are able to selectively detect the extracellular domain of the protein Intimin, Tir's natural binding partner. Furthermore, we demonstrate the use of RI and Tir-functionalized arrays for the selective detection of EPEC directly from culture.     

A DNA sequence-specific electrochemical biosensor based on alginic acid-coated cobalt magnetic beads for the detection of E. coli

A new type of DNA sequence-specific electrochemical biosensor based on magnetic beads for the detection of Escherichia coli is reported in the present work. Alginic acid-coated cobalt magnetic beads, capped with 5'-(NH(2)) oligonucleotide and employed not only for magnetic separation but also as the solid adsorbent, were used as DNA probes to hybridize with the target E. coli DNA sequence. This assay was specific for E. coli detection depending on the uid A gene, which encodes for the enzyme β-d-glucuronidase produced by E. coli strains. When daunomycin (DNR) was used as DNA hybridization indicator, the target sequences of E. coli hybridized with the probes resulted in the decrease of DNR reduction peak current, which was proportional to the E. coli concentration. The optimization of the hybridization detection was carried out and the specificity of the probes was also demonstrated. This DNA biosensor can be employed to detect a complementary target sequence for 3.0×10(-10) mol/L and denatured PCR products for 0.5 ng/μL. The linear range of the developed biosensor for the detection of E. coli cells was from 1.0×10(2) to 2.0×10(3) cells/mL with a detection limit of 50 cells/mL. After a brief enrichment process, a concentration of 10 cells/mL E. coli in real water samples was detected by the electrochemical biosensor.     

Interferometric biosensor based on planar optical waveguide sensor chips for label-free detection of surface bound bioreactions

A label free optical biosensor based on a free-space Young interferometer configuration is presented. Commercial planar Ta(2) O(5) waveguides are used as sensing elements and allow the investigation of surface bound bioreactions like immunoreactions or biological affinity systems. Design criteria are discussed and a detailed characterization of the sensor performance is presented. The developed interferometer yields an effective refractive index resolution of 9 x 10(-9), corresponding to a surface coverage of approximately 13 fg/mm(2). The performance of the system is characterized by two different affinity systems: the antibody-antigen complex protein G-immunoglobulin G is used as a model system for monitoring reaction kinetics. Further measurements on a silanized surface show the formation of a streptavidin monolayer on a biotinylated surface.     

Development of a label-free impedance biosensor for detection of antibody-antigen interactions based on a novel conductive linker

We developed a label-free impedance biosensor based on an innovative conductive linker for detecting antibody-antigen interactions. As the often used conventional long chain thiol is a poor conductor, it is not a suitable material for use in a faradaic biosensor. In this study, we adopted a thiophene-based conductive bio-linker to form a self-assembled monolayer and to immobilize the bio-molecules. We used cyclic voltammetry and impedance spectroscopy to verify the enhanced conductivity properties. Results showed that the electron transfer resistance of this new conductive linker was 3 orders of a magnitude lower than for a case using a conventional long chain thiol linker. With the decreased impedance (i.e. increased faradaic current), we can obtain a higher signal/noise ratio such that the detection limit is improved. Using fluorescence microscopy, we verified that our new conductive linker has a protein immobilization capability similar to a conventional long chain thiol linker. Also, using S100 proteins, we verified the protein interaction detection capability of our system. Our obtained results showed a linear dynamic range from 10 ng/ml to 10 μg/ml and a detection limit of 10 ng/ml. With our new conductive linker, an electrochemical impedance biosensor shows great potential to be used for point-of-care applications.     

Long period grating based biosensor for the detection of Escherichia coli bacteria

In this paper we report a stable, label-free, bacteriophage-based detection of Escherichia coli (E. coli) using ultra sensitive long-period fiber gratings (LPFGs). Bacteriophage T4 was covalently immobilized on optical fiber surface and the E. coli binding was investigated using the highly accurate spectral interrogation mechanism. In contrast to the widely used surface plasmon resonance (SPR) based sensors, no moving part or metal deposition is required in our sensor, making the present sensor extremely accurate, very compact and cost effective. We demonstrated that our detection mechanism is capable of reliable detection of E. coli concentrations as low as 10(3)cfu/ml with an experimental accuracy greater than 99%.     

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.     

A fluorescence-based fiber optic biosensor for the flow-injection analysis of penicillin

A penicillin fiber optic sensor is described. The sensor is based on co-immobilization of a pH indicator, fluorescein isothiocyanate (FITC), and penicillinase on a preactivated biodyne B membrane attached to the end of a bifurcated optical fiber. The characteristics of the sensor are investigated in conjunction with a flow injection analysis system. The proposed sensor is reversible and responds to penicillin in the concentration range of 1 x 10(-4) to 5 x 10(-2) mol/L. The application of this sensor to penicillin analysis in some pharmaceutical samples is demonstrated.     

An optimised electrochemical biosensor for the label-free detection of C-reactive protein in blood

C-reactive protein (CRP) is an acute phase protein whose levels are increased in many disorders. There exists, in particular, a great deal of interest in the correlation between blood serum levels and the severity of risk for cardiovascular disease. A sensitive, label-free, non-amplified and reusable electrochemical impedimetric biosensor for the detection of CRP in blood serum was developed herein based on controlled and coverage optimised antibody immobilization on standard polycrystalline gold electrodes. Charge transfer resistance changes were highly target specific, linear with logCRPconcentration across a 0.5-50nM range and associated with a limit of detection of 176pM. Significantly, the detection limits are better than those of current CRP clinical methods and the assays are potentially cheap, relatively automated, reusable, multiplexed and highly portable. The generated interfaces were capable not only of comfortably quantifying CRP across a clinically relevant range of concentrations but also of doing this in whole blood serum with interfaces that were, subsequently, reusable. The importance of optimising receptor layer resistance in maximising assay sensitivity is also detailed.     

Potentiometric analysis of Escherichia coli cytochromes in the optical absorbance range of 500 nm to 700 nm.

The oxidation-reduction potentials of Escherichia coli cytochromes have been studied by a recently described technique for automated electrodic potentiometry (Hendler, R.W., Songco, D., and Clem, T.R. (1977) Anal. Chem. 49, 1908-1913; Hendler, R.W. (1977) Anal. Chem. 49, 1914-1918), where entire spectra are recorded at a series of solution potentials. New techniques for resolution of the spectra versus voltage data have been applied. The results indicate that a 1-electron transport chain conducts electrons from substrate to cytochrome d, which is the cytochrome oxidase. Cytochrome d contains several components which appear to increase electron transfer first to a 2-electron stage and then to a 4-electron stage for the final reduction of a molecule of oxygen to 2 molecules of water.     

A label-free photonic crystal biosensor imaging method for detection of cancer cell cytotoxicity and proliferation

A label-free method for detecting the attachment of human cancer cells to a biosensor surface for rapid screening for biological activity is described, in which attachment of a cell results in highly localized increase of the resonant reflected wavelength of a photonic crystal narrowband reflectance filter incorporated into a standard 96-well microplate. An imaging detection instrument is used to determine the spatial distribution of attached cells by mapping the shift in reflected resonant wavelength as a function of position. The method enables monitoring of cancer cell attachment, cell proliferation, and cell detachment that is induced by exposure of the cells to drug compounds. We demonstrate the efficacy of this method as an early screening technique for the rapid quantification of the rate of cancer cell proliferation on the sensor surface, and subsequently as a means for quantifying cell detachment resulting from apoptosis that is induced by exposure of the cells to cytotoxic chemicals.     

Rapid biosensor for detection of antibiotic-selective growth of Escherichia coli

A rapid biosensor for the detection of bacterial growth was developed using micromechanical oscillators coated in common nutritive layers. The change in resonance frequency as a function of the increasing mass on a cantilever array forms the basis of the detection scheme. The calculated mass sensitivity according to the mechanical properties of the cantilever sensor is approximately 50 pg/Hz; this mass corresponds to an approximate sensitivity of approximately 100 Escherichia coli cells. The sensor is able to detect active growth of E. coli cells within 1 h. The starting number of E. coli cells initially attached to the sensor cantilever was, on average, approximately 1,000 cells. Furthermore, this method allows the detection of selective growth of E. coli within only 2 h by adding antibiotics to the nutritive layers. The growth of E. coli was confirmed by scanning electron microscopy. This new sensing method for the detection of selective bacterial growth allows future applications in, e.g., rapid antibiotic susceptibility testing.     

A Love wave immunosensor for whole E. coli bacteria detection using an innovative two-step immobilisation approach

The efficiency of a monomolecular film of (3-glycidoxypropyl) trimethoxysilane (GPTS) on a shear horizontal guided (Love) acoustic wave immunosensor to detect whole Escherichia coli (E. coli) bacteria is demonstrated. Direct anti-E. coli antibodies grafting onto the sensor surface did not lead to a significant bacteria immobilisation, partially attributed to the SiO2 sensor surface roughness. An innovative method has been set up to get around this difficulty and to detect whole bacteria. It consists in grafting goat anti-mouse antibodies (GAM) onto the sensor surface in a first step and introducing E. coli bacteria mixed with anti-E. coli antibodies onto the sensor in a second step. We describe the characteristics of such a technique like sample preparation time (lower than 30 min) and temperature improvements. A 37 degrees C experimental temperature led to the fastest bacteria binding kinetic, reducing the total analysis time. This method enables to keep the specificity of the antibody/antigen interaction and provides significant results in less than 1h. This leads to a detection threshold of 10(6) bacteria/ml in a 500 microl chamber.     

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.