Carbohydrate-based label-free detection of Escherichia coli ORN 178 using electrochemical impedance spectroscopy

A label-free biosensor for Escherichia coli (E. coli) ORN 178 based on faradaic electrochemical impedance spectroscopy (EIS) was developed. α-Mannoside or β-galactoside was immobilized on a gold disk electrode using a self-assembled monolayer (SAM) via a spacer terminated in a thiol functionality. Impedance measurements (Nyquist plot) showed shifts due to the binding of E. coli ORN 178, which is specific for α-mannoside. No significant change in impedance was observed for E. coli ORN 208, which does not bind to α-mannoside. With increasing concentrations of E. coli ORN 178, electron-transfer resistance (R(et)) increases before the sensor is saturated. After the Nyquist plot of E. coli/mixed SAM/gold electrode was modeled, a linear relationship between normalized R(et) and the logarithmic value of E. coli concentrations was found in a range of bacterial concentration from 10(2) to 10(3) CFU/mL. The combination of robust carbohydrate ligands with EIS provides a label-free, sensitive, specific, user-friendly, robust, and portable biosensing system that could potentially be used in a point-of-care or continuous environmental monitoring setting.     

Interdigitated Array microelectrode-based electrochemical impedance immunosensor for detection of Escherichia coli O157:H7

A label-free electrochemical impedance immunosensor for rapid detection of Escherichia coli O157:H7 was developed by immobilizing anti-E. coli antibodies onto an indium-tin oxide interdigitated array (IDA) microelectrode. Based on the general electronic equivalent model of an electrochemical cell and the behavior of the IDA microelectrode, an equivalent circuit, consisting of an ohmic resistor of the electrolyte between two electrodes and a double layer capacitor, an electron-transfer resistor, and a Warburg impedance around each electrode, was introduced for interpretation of the impedance components of the IDA microelectrode system. The results showed that the immobilization of antibodies and the binding of E. coli cells to the IDA microelectrode surface increased the electron-transfer resistance, which was directly measured with electrochemical impedance spectroscopy in the presence of [Fe(CN)(6)](3-/4-) as a redox probe. The electron-transfer resistance was correlated with the concentration of E. coli cells in a range from 4.36 x 10(5) to 4.36 x 10(8) cfu/mL with the detection limit of 10(6) cfu/mL.     

Design and fabrication of a microimpedance biosensor for bacterial detection

A biosensor for bacterial detection was developed based on microelectromechanical systems, heterobifunctional crosslinkers and immobilized antibodies. The sensor detected the change in impedance caused by the presence of bacteria immobilized on interdigitated gold electrodes and was fabricated from (100) silicon with a 2-/spl mu/m layer of thermal oxide as an insulating layer. The sensor active area is 9.6 mm/sup 2/ and consists of two interdigital gold electrode arrays measuring 0.8 /spl times/ 6 mm. Escherichia coli specific antibodies were immobilized to the oxide between the electrodes to create a biological sensing surface. The impedance across the interdigital electrodes was measured after immersing the biosensor in solution. Bacteria cells present in the sample solution attached to the antibodies and became tethered to the electrode array, thereby causing a change in measured impedance. The biosensor was able to discriminate between different cellular concentrations from 10/sup 5/ to 10/sup 7/ CFU/mL in pure culture. The sample testing process, including data acquisition, required 5 min. The design, fabrication, and testing of the biosensor is discussed along with the implications of these findings toward further biosensor development.     

Immunobiosensor chips for detection of Escherichia coil O157:H7 using electrochemical impedance spectroscopy

Impedance biosensor chips were developed for detection of Escherichia coli O157:H7 based on the surface immobilization of affinity-purified antibodies onto indium tin oxide (ITO) electrode chips. The immobilization of antibodies onto ITO chips was carried out using an epoxysilane monolayer to serve as a template for chemical anchoring of antibodies. The surface characteristics of chips before and after the binding reaction between the antibodies and antigens were characterized by atomic force microscopy (AFM). The patterns of the epoxysilanes monolayer, antibodies, and E. coli cells were clearly observed from the AFM images. Alkaline phosphatase as the labeled enzyme to anti-E. coli O157:H7 antibody was used to amplify the binding reaction of antibody-antigen on the chips. The biocatalyzed precipitation of 5-bromo-4-chloro-3-indolyl phosphate by alkaline phosphatase on the chips in pH 10 PBS buffer containing 0.1 M MgCl2 increased the electron-transfer resistance for a redox probe of Fe(CN)6(3-/4-) at the electrode-solution interface or the electrode resistance itself. Electrochemical impedance spectroscopy and cyclic voltammetric method were employed to follow the stepwise assembly of the systems and the electronic transduction for the detection of E. coli. The biosensor could detect the target bacteria with a detection limit of 6 x 10(3) cells/mL. A linear response in the electron-transfer resistance for the concentration of E. coli cells was found between 6 x 10(4) and 6 x 10(7) cells/mL.     

Rapid detection of urinary tract infections using isotachophoresis and molecular beacons

We present a novel assay for rapid detection and identification of bacterial urinary tract infections using isotachophoresis (ITP) and molecular beacons. We applied on-chip ITP to extract and focus 16S rRNA directly from bacterial lysate and used molecular beacons to achieve detection of bacteria specific sequences. We demonstrated detection of E. coli in bacteria cultures as well as in patient urine samples in the clinically relevant range 1E6-1E8 cfu/mL. For bacterial cultures we further demonstrate quantification in this range. The assay requires minimal sample preparation (a single centrifugation and dilution), and can be completed, from beginning of lysing to detection, in under 15 min. We believe that the principles presented here can be used for design of other rapid diagnostics or detection methods for pathogenic diseases.     

Surface plasmon resonance and impedance spectroscopy on gold electrode for biosensor application

This paper describes new-label free immunoassays for the detection of atrazine using optical Surface Plasmon Resonance (SPR) and Electrochemical Impedance Spectroscopy (EIS) by employing gold functionalised with self-assembled mixed monolayer. The mixed monolayer, formed by 16-mercaptohexadecanoic acid (MHDA) and biotinyl–PE, is used as the basic layer in the fabrication of the immunoprobe. The interfacial interaction of a biotinyl–Fab fragment K47 antibody with the mixed monolayer is based on a biotin–avidin system using the neutravidin molecule. Thus, in this study the multilayer engineering is mentored by SPR and EIS methods. In addition, the specificity and sensitivity of this self-assembled multilayer system to the presence of atrazine are investigated. The limit detection of Atrazine obtained with EIS technique is equal to 20 ng/ml and with SPR technique is equal to 50 ng/ml.     

Electrochemical phagemid assay for the specific detection of bacteria using Escherichia coli TG-1 and the M13KO7 phagemid in a model system

We describe a reporter phagemid system for the specific amperometric detection of bacteria. We constructed a phagemid a bacteriophage containing a bacterial plasmid using the M13KO7 helper phage and a commercial plasmid, pFLAG-ATS-BAP, which contains a gene encoding for a reporter enzyme, alkaline phosphatase. In the bacteria, the enzyme reacts with the substrate, p-aminophenyl phosphate, in the periplamic space that separates the outer plasma membrane from the cell wall. Thus, the activity of the reporter enzyme can be measured directly in an electrochemical cell without further treatment. The product of the enzymatic activity, p-aminophenol, diffuses out and is oxidized at the working electrode with an applied potential of 220 mV vs the reference electrode Ag/AgCl. The lower detection limit was 1 cfu/mL E. coli TG1 in less than 3 h in a very specific manner. The use of plasmid alkaline phosphatase as the reporter increased the sensitivity by 10-fold over our earlier electrochemical lytic phage method. Such a system can be used for the rapid detection of any strain of bacteria using the appropriate bacteriophage and reporter gene.     

Quantum dot biolabeling coupled with immunomagnetic separation for detection of Escherichia coli O157:H7

A sensitive, specific, and rapid method for the detection of E. coli O157:H7 was demonstrated using quantum dots (QDs) as a fluorescence marker coupled with immunomagnetic separation. Magnetic beads coated with anti-E. coli O157 antibodies were employed to selectively capture the target bacteria, and biotin-conjugated anti-E. coli antibodies were added to form sandwich immuno complexes. After magnetic separation, the immuno complexes were labeled with QDs via biotin-streptavidin conjugation. This was followed by a fluorescence measurement using a laptop-controlled portable device, which consisted of a blue LED and a CCD-array spectrometer. The peak intensity of the fluorescence emission was proportional to the initial cell concentration of E. coli O157:H7 in the range of 10(3)-10(7) CFU/mL with a detection limit at least 100 times lower than that of the FITC-based method. The total detection time was less than 2 h. Neither E. coli K12 nor Salmonella typhimurium interfered with the detection of E. coli O157:H7.     

Label-free detection of antibody-antigen interactions on (R)-lipo-diaza-18-crown-6 self-assembled monolayer modified gold electrodes

Novel (R)-diaza-18-crown-6 has been prepared by a simple two-step synthetic method and characterized for its ability to form a uniform self-assembled monolayer (SAM) on gold as well as to immobilize proteins using atomic force microscopy, quartz crystal microbalance, and electrochemical impedance spectroscopy (EIS) experiments. The (R)-lipo-diaza-18-crown-6 was shown to form a well-defined SAM on gold, which subsequently captures the antibody (Ab) molecules that in turn capture the antigen (Ag) molecules. The Ab molecules studied include antibody C-reactive protein (Ab-CRP) and antibody ferritin (Ab-ferritin) along with their Ag's, i.e., CRP and ferritin. Quantitative detection of the Ab-Ag interactions was accomplished by EIS experiments with a Fe(CN)6(3-/4-) redox probe present. The ratios of the charge-transfer resistances for the redox probe on the SAM-antibody-covered electrode to those with the antigen molecules attached show an excellent linearity for log[Ag] with lower detection limits than those of other SAMs for the electrochemical sensing of proteins.     

Deposition behavior of self-assembled monolayers and bacteria on metallic surfaces using an electrochemical quartz crystal nanobalance

This paper presents an investigation on the deposition behavior of self-assembled monolayers (SAMs) and bacteria on titanium and gold surfaces using an electrochemical quartz crystal nanobalance (EQCN). The tests included alkanethiol and alkanesilane and three bacteria, namely: E. coli, P. fluorescens and K. aerogenes. The mass change with respect to immersion time was measured by EQCN. The results showed SAM formation on both titanium and gold surfaces, but SAM formation on gold was generally higher by 26-74% as compared to that of titanium. On the other hand, bacteria also adhered well on the two metal surfaces. E. coli and P. fluorescens had high affinity on gold and titanium surfaces, respectively, while K. aerogenes was more adherent to titanium. The results showed that titanium and gold are good metals for biomaterials yet at the same time, their bioinert property provide excellent condition for bacterial adhesion. Therefore, there is a need for proper surface preparation to optimize the use of titanium and gold as biomaterials.     

Mid-IR biosensor: detection and fingerprinting of pathogens on gold island functionalized chalcogenide films

Antibody (human IgG, anti-E. coli O157:H7, and anti-Salmonella) complexes on the surface of IR-transparent Ge-containing chalcogenide glass films were formed via thiol chemistry on 20-nm-thick gold islands. As a first step, the protocol was validated by monitoring fluorescently tagged targets to validate binding. FT-IR spectroscopy confirmed that the coating of the films with 20-nm gold did not have a significant effect on the propagation and penetration of IR evanescent waves through the film. The films functionalized with anti-E. coli O157:H7 and anti-Salmonella antibodies were used to detect E. coli O157:H7 and S. enteriditis through label-free IR fingerprinting. Highly selective detection of bacterial targets was achieved at both the species (E. coli vs. S. enteriditis) and strain level (E. coli O157:H7 vs E. coli K12). A mid-infrared approach could thus be used as a biosensor as well as a molecular fingerprinting tool.     

In-a-day electrochemical detection of coliforms in drinking water using a tyrosinase composite biosensor

A rapid method for the detection of fecal contamination in water based on the use of a tyrosinase composite biosensor for improved amperometric detection of beta-galactosidase activity is reported. The method relies on the detection of phenol released after the hydrolysis of phenyl beta-D-galactopyranoside (PG) by beta-galactosidase. Under the optimized PG concentration and pH (4.0) values, a detection limit of 1.2x10(-3) unit of beta-galactosidase/mL-1 was obtained. The capability of the sensor for the detection of Escherichia coli was evaluated using polymyxin B sulfate to allow permeabilization of the bacteria membrane. A detection limit of 1x10(6) cfu of E. coli mL-1 was obtained with no preconcentration or pre-enrichment steps. To improve the analytical characteristics for bacteria detection, the processes involving galactosidase induction during incubation and membrane permeabilization were optimized. Using 0.25 mM isopropyl beta-D-thiogalactopyranoside for the enzyme activity induction, and 10 microg mL-1 polymyxin B sulfate as permeabilizer agent, it was possible to detect bacteria concentrations as low as 10 cfu mL-1 after 5 h of enrichment. The possibility of detecting E. coli at the required levels for drinking water quality assessment (1 cfu/100 mL) is demonstrated, the time of analysis being shorter than 6.5 h and involving a simple methodology.     

A microfabricated biosensor for detecting foodborne bioterrorism agents

A biosensor for the detection of pathogenic bacteria was developed for biosecurity applications. The sensor was fabricated using photolithography and incorporates heterobifunctional crosslinkers and immobilized antibodies. The sensor detected the change in impedance caused by the presence of bacteria immobilized on interdigitated gold electrodes and was fabricated from (100) silicon with a 2-/spl mu/m layer of thermal oxide as an insulating layer. The sensor has a large active area of 9.6 mm/sup 2/ and consists of two interdigital gold electrode arrays each measuring 0.8 /spl times/ 6 mm. Pathogenic Escherichia coli and Salmonella infantis were tested in serially diluted pure culture. Analyte specific antibodies were immobilized to the oxide between the electrodes to create a biological sensing surface. After immersing the biosensor in solution, the impedance across the interdigital electrodes was measured. Bacteria cells present in the sample solution attached to the antibodies and became tethered to the electrode array thereby causing a change in measured impedance. The biosensor was able to discriminate between different cellular concentrations from 10/sup 4/ - 10/sup 7/ CFU/mL (colony-forming units per milliliter) in solution. The sample testing process, including data acquisition, required 5 min. The design, fabrication, and testing of the biosensor is discussed along with the implications of these findings toward further biosensor development.     

A new method of immobilization of proteins on activated ester terminated alkanethiol monolayers towards the label free impedancemetric detection

This paper investigates a new immobilization procedure for biological molecules that is based on the formation of reactive ω-functionalized-self-assembled thiol monolayers onto a gold electrode. The homogeneous self-assembled monolayer was characterized by X-ray photoelectron spectroscopy (XPS), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The SAM modified gold electrode showed a clear peak corresponding to S_2p that characterized the Au-thiolate bond, while cyclic voltammetry and electrochemical impedance spectroscopy measurements, in 10 mM phosphate buffer pH 7, in the presence of Fe(CN)₆^{− 3/− 4} as redox probe, showed that these monolayers were densely packed and prevented electron transfer towards the gold surface. These homogeneous SAMs were used to immobilize biotin hydrazide by covalent attachment, after the nucleophilic attack of the amino group of biotin hydrazide on the ω-activated ester function of thiols. The biotin–avidin interaction was then examined as a model for an affinity biosensor with electrochemical impedance spectroscopy. A Randles equivalent circuit was used for the interpretation of impedance data and the change in the interfacial properties at the modified-electrode/electrolyte interface were monitored through charge-transfer resistance variation. The proposed affinity biosensor showed a detection range that was linear between 200 and 800 ng/ml for avidin. In order to improve the sensitivity the technique of mixed self-assembled monolayers was adopted. Mixed SAMs were elaborated by co-adsorption of two differently substituted thiols, one was substituted by a reactive group that was used to react with the amino group of biotin hydrazide, whereas the other was substituted by an hydroxyl group that was chosen to mimic protein resistance. In this study, we started with a 1:3 activated ester:hydroxyl-terminated alkanethiol ratio. The results obtained with the mixed SAMs appeared to be better than those obtained with the homogeneous SAMs, and the corresponding affinity biosensor presented two detection ranges that were linear between 20 and 100 ng/ml and between 100 and 1200 ng/ml, respectively, with two different slopes.     

Selective electrochemical sensing of glycated hemoglobin (HbA1c) on thiophene-3-boronic acid self-assembled monolayer covered gold electrodes

We report a novel concept of sensing glycated hemoglobin, HbA 1c, which is now the most important index for a long-term average blood glucose level, by first selectively immobilizing it on the thiophene-3-boronic acid (T3BA) self-assembled monolayer (SAM)-covered gold electrode by a selective chemical reaction with boronic acid. HbA 1c thus immobilized is then detected by the label-free electrochemical impedance spectroscopic (EIS) measurements with a redox probe, an equimolar mixture of K 3Fe(CN) 6 and K 4Fe(CN) 6, present. The rate of charge transfer between the electrode and the redox probe is shown to be modulated by the amount of HbA 1c in the matrix hemoglobin solution due to the blocking effect caused by the binding of HbA 1c with boronic acid. Both the formation of a well-defined T3BA-SAM on the gold surface and the chemical binding of its boronic acid with HbA 1c in solution were confirmed by quartz crystal microbalance, atomic force microscopy, and EIS experiments.     

An antibody-immobilized capillary column as a bioseparator/bioreactor for detection of Escherichia coil O157:H7 with absorbance measurement.

A capillary-column-based bioseparator/bioreactor was developed for detection of Escherichia coli O157:H7 by chemically immobilizing anti-E. coli O157:H7 antibodies onto the inner wall of the column, forming the "sandwich" immunocomplexes (immobilized antibody-E. coli O157: H7-enzyme-labeled antibody) after the sample and the enzyme-labeled antibody passed through the column and detecting the absorbance of the product in the bioreactor with an optical detector. The effects of the blocking agent, flow rate of samples and substrates, buffer, MgCl2, and pH on the detection of E. coli O157:H7 were investigated. The parameters, 2% BSA in 1.0 x 10-2 M, pH 7.4, PBS as the blocking agent, 0.5 mL/h as the sample flow rate, 1.0 x 10(-2) M MgCl2, and 2.0 x 10(-4) M p-nitrophenyl phosphate in 1.0 M, pH 9.0 Tris buffer as the substrate for the enzymatic reaction, and 1.0 mL/h as the substrate flow rate, were used in the bioseparator/bioreactor system for detection of E. coli O157:H7. The selectivity of the system was checked, and other pathogens, including Salmonella typhimurium, Campylobacterjejuni, and Listeria monocytogenes, had no interference with the detection of E. coli O157:H7. Its working range was from 5.0 x 10(2) to 5.0 x 10(6) cfu/mL, and the total assay time was < 1.5 h without any enrichment. The relative standard deviation was approximately 2.0-7.3%.     

Electrochemical impedance immunosensor based on three-dimensionally ordered macroporous gold film

A novel label-free immunosensor for the detection of C-reactive protein (CRP) was developed based on a three-dimensional ordered macroporous (3DOM) gold film modified electrode by using the electrochemical impedance spectroscopy (EIS) technique. The electrode was electrochemically fabricated with an inverted opal template, making the surface area of the 3DOM gold film up to 14.4 times higher than that of a classical bare flat one, characterized by the cyclic voltammetric (CV) technique. The 3DOM gold film which was composed of interconnected gold nanoparticles not only has a good biocompatible microenvironment but also promotes the increase of conductivity and stability. The CRP immunosensor was developed by covalently conjugating CRP antibodies with 3-mercaptopropionic acid (MPA) on the 3DOM gold film electrode. The CRP concentration was measured through the increase of impedance values in the corresponding specific binding of CRP antigen and CRP antibody. The increased electron-transfer resistance (R(et)) values were proportional to the logarithmic value of CRP concentrations in the range of 0.1 to 20 ng mL(-1). The detection of CRP levels in three sera obtained from hospital showed acceptable accuracy.     

Nanostructured disposable impedimetric sensors as tools for specific biomolecular interactions: sensitive recognition of concanavalin A

The development of sensors to detect specific weak biological interactions is still today a challenging topic. Characteristics of carbohydrate-protein (lectin) interactions include high specificity and low affinity. This work describes the development of nanostructured impedimetric sensors for the detection of concanavalin A (Con A) binding to immobilized thiolated carbohydrate derivatives (D-mannose or D-glucose) onto screen-printed carbon electrodes (SPCEs) modified with gold nanoparticles. Thiolated D-galactose derivative was employed as negative control to evaluate the selectivity of the proposed methodology. After binding the thiolated carbohydrate to the nanostructured SPCEs, different functionalized thiols were employed to form mixed self-assembled monolayers (SAM). Electrochemical impedance spectroscopy (EIS) was employed as a technique to evaluate the binding of Con A to selected carbohydrates through the increase of electron transfer resistance of the ferri/ferrocyanide redox probe at the differently SAM modified electrodes. Different variables of the assay protocol were studied in order to optimize the sensor performance. Selective Con A determinations were only achieved by the formation of mixed SAMs with adequate functionalized thiols. Important differences were obtained depending on the chain lengths and functional groups of these thiols. For the 3-mercapto-1-propanesulfonate mixed SAMs, the electron transfer resistance varied linearly with the Con A concentration in the 2.2-40.0 μg mL(-1) range for D-mannose and D-glucose modified sensors. Low detection limits (0.099 and 0.078 pmol) and good reproducibility (6.9 and 6.1%, n=10) were obtained for the D-glucose and D-mannose modified sensors, respectively, without any amplification strategy.     

Electron permeable self-assembled monolayers of dithiolated aromatic scaffolds on gold for biosensor applications

Self-assembled monolayers (SAMs) of thiolated compounds are formed by the spontaneous chemisorption of thiolate groups on metal surfaces. In biosensors, they are most commonly used to covalently immobilize a biorecognition molecule onto the surface of the transducer, thus offering the possibility of controlling the orientation, distribution, and spacing of the sensing element while reducing nonspecific interactions. In this paper, self-assembled monolayers of dithiolated derivatives of 3,5-dihydroxybenzyl alcohol containing carboxyl and hydroxyl end groups have been prepared on gold surfaces and characterized by cyclic voltammetry and electrochemical impedance spectroscopy. Impedance measurements revealed that SAM formation is essentially completed after 3-5 h of exposure by observing the successive blocking of the faradic response of ferricyanide anion due to the adsorption of the dithiol molecules. The surface coverage of these molecules, estimated by reductive desorption experiments, was in the range of (1.1-2.8) x 10-10 mol/cm2. To demonstrate the potential of the dithiol SAM, a model system for detection of a tumor marker, prostate-specific antigen (PSA), was developed. The carboxyl groups of the SAM were succinimide-activated, and an anti-PSA antibody was covalently immobilized via amide bonds. The modified SAM was used for the label-free detection of prostate-specific antigen using EIS with a detection limit of 9 ng/mL. The results described here demonstrate that this kind of dithiol-modified SAM can be used as supports in electrochemical biosensors and the results are explained in terms of the structural features of these dithiols.     

Magnetoelastic immunosensors: amplified mass immunosorbent assay for detection of Escherichia coli O157:H7

A mass-sensitive magnetoelastic immunosensor for detection of Escherichia coli O157:H7 is described, based on immobilization of affinity-purified antibodies attached to the surface of a micrometer-scale magnetoelastic cantilever. Alkaline phosphatase is used as a labeled enzyme to the anti-E. coli O157:H7 antibody, amplifying the mass change associated with the antibody-antigen binding reaction by biocatalytic precipitation of 5-bromo-4-chloro-3-indolyl phosphate in a pH 10.0 PBS solution. The detection limit of the biosensor is 10(2) E. coli O157:H7 cells/mL. A linear change in the resonance frequency of the biosensor was found to E. coli O157:H7 concentrations ranging from 10(2) to 10(6) cells/mL.