New MSPQC Method for Rapid Identification and Quantification of Pseudomonas aeruginosa

Abstract

A new method for the rapid identification and quantification of Pseudomonas aeruginosa using multichannel series piezoelectric quartz crystal (MSPQC) was proposed. The identification of P. aeruginosa was based on the development of acetamide broth, which can selective culture P. aeruginosa and performed perfectly in MSPQC. The quantitative detection of P. aeruginosa was based on the fact that the frequency detection time (FDT) detected by MSPQC in developed medium had a linear relationship with the logarithm of its initial concentration in the range of 10–10⁸ colony ? forming units (cfu) ml^–1 (R=?0.984). The detection limit was 10 cfu ml^–1.     

Piezoelectric Crystal Biosensor System for Detection of Escherichia Coli

Abstract

A piezoelectric crystal biosensor system was applied to the detection of Escherichia coli. the system consists of an oscillator, a frequency counter, a flow cell and a modified piezoelectric crystal. Anti-E. coli antibody is immobilized on the surface of the crystal. It is used as an E. coli detection by measuring its resonant frequency shift due to a mass change caused by specific binding of the micro organisms to the surface. the frequency shift correlates with an E. coli concentration in the range of 10⁶?10⁸ cells·cm^?3. the resonant frequency shift is increased by further treatment to bind micro-particles modified with anti-E. coli antibody. This method allows us to improve the determination limit to 10⁵ cells · cm^?3.     

A New Sensor Method for Studying the Effect of Gentamicin and Cefotaxime Combination Against Escherichia coli

Abstract

A new sensor method based on a multi-channel series piezoelectric quartz crystal (MSPQC) was proposed for studying the effect of gentamicin and cefotaxime combination against Escherichia coli ATCC 25922. The frequency curves under different combination regimens were obtained and compared with each other. When antibiotic had an inhibitory effect to the growth of bacteria, frequency detection time (FDT) would be prolonged. The FDT was used to assess the effect of an antibiotic combination. By using the proposed method, simultaneous administration, different order, and time interval of nonsimultaneous administration were investigated in detail. The MSPQC method can provide the process information in real time. It is simple, rapid, and easy to perform.     

A Rapid and Sensitive Aptamer‐Based Electrochemical Biosensor for Direct Detection of Escherichia Coli O111

A sensitive and specific electrochemical biosensor based on target‐induced aptamer displacement was developed for direct detection of Escherichia coli O111. The aptamer for Escherichia coli O111 was immobilized on a gold electrode by hybridization with the capture probe anchored on the electrode surface through Au‐thiol binding. In the presence of Escherichia coli O111, the aptamer was dissociated from the capture probe‐aptamer duplex due to the stronger interaction between the aptamer and the Escherichia coli O111. The consequent single‐strand capture probe could be hybridized with biotinylated detection probe and tagged with streptavidin‐alkaline phosphatase, producing sensitive enzyme‐catalyzed electrochemical response to Escherichia coli O111. The designed biosensor showed weak electrochemical signal to Salmonella typhimurium, Staphylococcus aureus and common non‐pathogenic Escherichia coli, indicating high specificity for Escherichia coli O111. Under the optimal conditions, the proposed strategy could directly detect Escherichia coli O111 with the detection limit of 112 CFU mL^?1 in phosphate buffer saline and 305 CFU mL^?1 in milk within 3.5 h, demonstrated the sensitive and accurate quantification of target pathogenic bacteria. The designed biosensor could become a powerful tool for pathogenic microorganisms screening in clinical diagnostics, food safety, biothreat detection and environmental monitoring.     

Rapid detection of trace Cu2+ using an l‐cysteine based interdigitated electrode sensor integrated with AC electrokinetic enrichment

Copper is an indispensable trace element for human health. Too much or too little intake of copper ion (Cu²⁺) can lead to its own adverse health conditions. Therefore, detection of Cu²⁺ is always of vital importance. In this work, a simple sensor was developed for rapid detection of trace Cu²⁺ in water, in which L‐cysteine (Cys) as a molecular probe was self‐assembled on a gold interdigital electrode to form a monolayer for specific capture of Cu²⁺. The interfacial capacitance of interdigital electrode was detected to indicate the target adsorption level under an AC signal working as the excitation to induce directed movement and enrichment of Cu²⁺ to the electrode surface. This sensor reached a limit of detection of 4.14 fM and a satisfactory selectivity against eight other ions (Zn²⁺, Hg²⁺, Pb²⁺, Cd²⁺, Mg²⁺, Fe²⁺, As³⁺, and As⁵⁺). Testing of spiked tap water was also performed, demonstrating the sensor's usability. This sensor as well as the detection method shows a great application potential in fields such as environmental monitoring and medical diagnosis.     

Fabrication of a Sensitive Impedance Biosensor of DNA Hybridization Based on Gold Nanoparticles Modified Gold Electrode

In this work, we report on the preparation of a simple, sensitive DNA impedance sensor. Firstly gold nanoparticles were electrodeposited on the surface of a gold electrode, and then probe DNA was immobilized on the surface of gold nanoparticles through a 5′‐thiol‐linker. Electrochemical impedance spectroscopy (EIS) was used to investigate probe DNA immobilization and hybridization. Compared to the bare gold electrode, the gold nanoparticles modified electrode could improve the density of probe DNA attachment and the sensitivity of DNA sensor greatly. The difference of electron transfer resistance (ΔR_et) was linear with the logarithm of complementary oligonucleotides sequence concentrations in the range of 2.0×10^?12 to 9.0×10^?8 M, and the detection limit was 6.7×10^?13 M. In addition, the DNA sensor showed a fairly good reproducibility and stability during repeated regeneration and hybridization cycles.     

Electrochemical Evaluation of the Interaction between Avidin and Biotin at Biotinylated Polypyrrole Electrode Using a Redox Marker

The interaction between avidin and biotin was evaluated electrochemically by monitoring the change in the electrode response of redox markers. Biotin was immobilized on the electrode surface by means of the electrochemical polymerization of biotinylated pyrrole and pyrrole. When avidin was introduced onto the biotinylated polypyrrole electrode surface, the large change in the electrode response of the redox marker was detected. The fact that the change in the electrode response of a marker ion could be attributed to the electrostatic interaction between avidin on the electrode surface and the redox marker ion present in a solution was verified by replacing avidin with NutrAvidin. At a pH lower than the isoelectric point of avidin, the electrode response of ferrocyanide as an anionic marker ion increased linearly within the range of 5.0×10^?9 ?3.0×10^?8 M avidin. The relative standard deviation at 1.5×10^?8 M avidin was about 5.4% (n=5). The detection of biotin was also performed using a competitive reaction between biotin in solution and biotin that had been immobilized on the electrode surface in the form of the biotinylated polypyrrole.     

Studies on biotin–avidin indirect conjugated technology for a piezoelectric DNA sensor

Because of their high sensitivity, piezoelectric sensor techniques are extremely useful for environmental or clinical analysis. We developed a piezoelectric crystal DNA biosensor for the detection of the hybridization reaction based on the self-assembled monolayer technology and biotin–avidin system. 3,3′-Dithiopropionic acid was applied to form a self-assembled monolayer (SAM) on the gold surface of the quartz crystal. Avidin was coated on the gold electrode conjugated with 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide (EDC) and N-hydroxysuccinimide (NHS), and then biotinylated nucleotide acids were immobilized on the gold electrode surface through the specific interaction of biotin and avidin. Our results indicated that, using this immobilization method, the piezoelectric DNA sensor shows a higher sensitivity and specificity in detecting the hybridization reaction. The sensor can be used repeatedly by electrode regeneration.     

Label-free capacitive immunosensor based on quartz crystal Au electrode for rapid and sensitive detection of Escherichia coli O157:H7

A label-free capacitive immunosensor based on quartz crystal Au electrode was developed for rapid and sensitive detection of Escherichia coli O157:H7. The immunosensor was fabricated by immobilizing affinity-purified anti-E. coli O157:H7 antibodies onto self-assembled monolayers (SAMs) of 3-mercaptopropionic acid (MPA) on the surface of a quartz crystal Au electrode. Bacteria suspended in solution became attached to the immobilized antibodies when the immunosensor was tested in liquid samples. The change in capacitance caused by the bacteria was directly measured by an electrochemical detector. An equivalent circuit was introduced to simulate the capacitive immunosensor. The immunosensor was evaluated for E. coli O157:H7 detection in pure culture and inoculated food samples. The experimental results indicated that the capacitance change was linearly correlated with the cell concentration of E. coli O157:H7. The immunosensor was able to discriminate between cellular concentrations of 10²–10⁵ cfu mL⁻¹ and has applications in detecting pathogens in food samples. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were also employed to characterize the stepwise assembly of the immunosensor.     

An Electrochemical Aptasensor for Detection of Thrombin based on Target Protein‐induced Strand Displacement

A sensitive electrochemical aptasensor for detection of thrombin based on target protein‐induced strand displacement is presented. For this proposed aptasensor, dsDNA which was prepared by the hybridization reaction of the immobilized probe ssDNA (IP) containing thiol group and thrombin aptamer base sequence was initially immobilized on the Au electrode by self‐assembling via Au? S bind, and a single DNA labeled with CdS nanoparticles (DP‐CdS) was used as a detection probe. When the so prepared dsDNA modified Au electrode was immersed into a solution containing target protein and DP‐CdS, the aptamer in the dsDNA preferred to form G‐quarter structure with the present target protein resulting that the dsDNA sequence released one single strand and returned to IP strand which consequently hybridized with DP‐CdS. After dissolving the captured CdS particles from the electrode, a mercury‐film electrode was used for electrochemical detection of these Cd²⁺ ions which offered sensitive electrochemical signal transduction. The peak current of Cd²⁺ ions had a good linear relationship with the thrombin concentration in the range of 2.3×10^?9–2.3×10^?12 mol/L and the detection limit was 4.3×10^?13 mol/L of thrombin. The detection was also specific for thrombin without being affected by the coexistence of other proteins, such as BSA and lysozyme.     

Highly Sensitive in vitro Biosensor for Enterotoxigenic Escherichia coli Detection Based on ssDNA Anchored on PtNPs‐Chitosan Nanocomposite

Detection of Enterotoxigenic Escherichia coli in various biological samples has tremendous importance in human health. In this direction, we have designed a label free electrochemical biosensor for highly selective detection of Escherichia coli through detecting ST gene. The ability of sensor probe to detect ST_G was confirmed using polymerase chain reaction. The biosensor was fabricated based on ST_G specific probes immobilized on platinum nanoparticles chitosan nanocomposite on screen printed carbon electrode, which was characterized by cyclic voltammetry, transmission electron microscopy, and fourier transform infrared spectroscopy. A highly sensitive label free sensing was achieved by analyzing ST_G hybridization using electrochemical impedance spectroscopy (EIS) technique. The EIS analysis showed a significant increase in charge transfer resistance after ST_G interaction with the highly selective ssDNA probe immobilized on the nanocomposite film. The increase in charge transfer resistance was evaluated for varying concentrations of ST_G, which shows a dynamic range between 1.0×10⁻¹² and 1.0×10⁻⁴ with the detection limit of 3.6×10⁻¹⁴ M (RSD<4.5 %). The regeneration of sensor probe was also studied and interference due to non‐target sequences was evaluated to ensure the selectivity of the designed sensor. The practical applicability of sensor probe was also analyzed by detecting the ST_G from the bacteria present in surface water.     

Potentiometric Determination of L-Asparagine with an Enzymatic Electrode

A new enzyme electrode for the determination of L-asparagine is constructed by chemical immobilization of L-aspartase [E.C. 4.3.1.1] on an ammonia-gas-sensing probe. L-Asparagine was determined over a concentration range of 1.6 × 10^?5 to 1.5 × 10^?3 M with a subnerstian response of -51 mV/decade. The electrode was stable for more than 30 days. The effect of pH, substrate and metal activator concentrations, amount of immobilized enzyme, and interferents on the electrode response are reported.     

Redox cycling amplified electrochemical detection of DNA hybridization: application to pathogen E. coli bacterial RNA

An electrochemical genosensor in which signal amplification is achieved using p-aminophenol (p-AP) redox cycling by nicotinamide adenine dinucleotide (NADH) is presented. An immobilized thiolated capture probe is combined with a sandwich-type hybridization assay, using biotin as a tracer in the detection probe, and streptavidin-alkaline phosphatase as reporter enzyme. The phosphatase liberates the electrochemical mediator p-AP from its electrically inactive phosphate derivative. This generated p-AP is electrooxidized at an Au electrode modified self-assembled monolayer to p-quinone imine (p-QI). In the presence of NADH, p-QI is reduced back to p-AP, which can be re-oxidized on the electrode and produce amplified signal. A detection limit of 1 pM DNA target is offered by this simple one-electrode, one-enzyme format redox cycling strategy. The redox cycling design is applied successfully to the monitoring of the 16S rRNA of E. coli pathogenic bacteria, and provides a detection limit of 250 CFU μL⁻¹.     

Electrochemical Investigation of Cytochrome c Immobilized onto Self‐Assembled Monolayer of Captopril

The electrochemical behavior of cytochrome c (cyt‐c) that was electrostatically immobilized onto a self‐assembled monolayer (SAM) of captopril (capt) on a gold electrode has been investigated. Cyclic voltammetry, scanning electrochemical microscopy (SECM) and electrochemical impedance spectroscopy were employed to evaluate the blocking property of the capt SAM. SECM was used to measure the bimolecular electron transfer (ET) kinetics (k_BI) between a solution‐based redox probe and the immobilized protein. In addition, the tunneling ET between the immobilized protein and the underlying gold electrode was calculated. A k_BI value of (5.0±0.6)×10⁸ mol^?1 cm³ s^?1 for the bimolecular ET and a standard tunneling rate constant (k⁰) of 46.4±0.2 s^?1 for the tunneling ET have been obtained.     

Application of Impedimetric and Voltammetric Genosensor for Detection of a Biological Warfare: Anthrax

A strategy for the detection of anthrax, which is a potential biological weapon by using an electrochemical genosensing technology, is investigated. An alkanathiol‐linked or unlabeled capture probe related to B. anthracis is immobilized onto gold or graphite electrode surface. A 101‐mer anthrax target is used for hybridization. The extent of hybridization between probe and target sequences is determined by using differential pulse voltammetry (DPV) and electrochemical impedance spectrometry (EIS). EIS analysis are based on electron transfer resistance (R_ct) in the presence of [Fe(CN)₆]^3?/4? and DPV measurements are based on transduction of both guanine oxidation and Meldola's blue (MDB) reduction signal as hybridization indicator. The response of the probe‐modified electrodes which was interacted with a noncomplementary sequence was the same as the responses of probe‐modified surface and proved the specifity of the hybridization with the target. According to these results the developed genosensors based on EIS and DPV techniques can be employed for rapid and selective detection of B. anthracis.     

Detection of Listeria Monocytogenes by Electrochemical Impedance Spectroscopy

Listeria monocytogenes is detected by electrochemical impedance spectroscopy using a mouse monoclonal antibody immobilized onto an Au electrode. This yields sensitivities of 0.825 kΩ cm²/(CFU/mL) and 1.129 kΩ cm²/(CFU/mL) and detection limits of 5 CFU/mL and 4 CFU/mL for ideal solutions and filtered tomato extract, respectively. Control experiments with an Au electrode onto which a mouse monoclonal antibody to GAPDH is immobilized demonstrate that non‐specific adsorption is insignificant for the system and methodology studied here. Control experiments with Salmonella enterica demonstrate no cross‐reactivity to this food pathogen. Potential technological hurdles to development of a multiplexed impedance biosensor for food pathogens are also discussed.     

Development of an Amperometric Immunosensor for the Quantification of Staphylococcus aureus Using Self‐Assembled Monolayer‐Modified Electrodes as Immobilization Platforms

Amperometric immunosensors for the detection and quantification of S. aureus using MPA self‐assembled monolayer modified electrodes for the immobilization of the immunoreagents are reported. Two different immunosensor configurations were compared. A competitive mode, in which protein A‐bearing S. aureus cells and antiRbIgG labeled with horseradish peroxidase (HRP) compete for the binding sites of RbIgG immobilized onto the 3‐mercaptopropionic acid (MPA) modified electrode, was evaluated. Moreover, a sandwich configuration in which S. aureus cells were immobilized onto the MPA SAM, and RbIgG and antiRbIgG labeled with HRP were further linked to the electrode surface, was also tested. In both cases, TTF was used as the redox mediator of the HRP reaction with H₂O₂, and it was co‐immobilized onto the MPA‐modified gold electrode. After optimization of the working variables for both configurations, the analytical performance of the amperometric measurements carried out at 0.00 V (vs. Ag/AgCl) showed that the competitive immunosensor exhibited a lower limit of detection (1.6×10⁵ S. aureus cells mL^?1), as well as a better repeatability and reproducibility of the measurements.     

Amperometric Choline Sensor with Enzyme Immobilized by Gamma-Irradiation in a Biocompatible Membrane

Abstract

An amperometric choline biosensor was constructed using choline oxidase immobilized on poly(2-hydroxyethylmethacrylate) membranes obtained by gamma radiation-induced polymerization at low temperature. The measurements were carried out by Clark-type oxygen or hydrogen peroxide electrodes. Calibration curves were linear in the 10-200 umol · 1^?1 range for the oxygen probe and 5-250 umol · 1^?1 for the H₂O₂-based probe. Temperature and pH effects on the activity of immobilized enzyme are described and the response characteristics of the sensor are summarized. The immobilized enzyme membranes stored in glycine buffer or in a dry state were very stable and no significant decrease in the electrode response was observed after three months. The biosensor was employed also to analyse a choline-containing pharmaceutical product and the results were compared to those obtained by enzymatic-spectrophotometric detection.     

Label-Free Detection of DNA Hybridization Based on MnO2 Nanoparticles

Abstract

Nano-MnO₂/chitosan composite film modified glassy carbon electrode (MnO₂/CHIT/GCE) was fabricated and a DNA probe was immobilized on the electrode surface. The immobilization and hybridization events of DNA were characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The EIS was applied to the label-free detection of the target DNA. The human immunodeficiency virus (HIV) gene fragment was successfully detected by this DNA electrochemical sensor. The dynamic detection range was from 2.0 × 10^?11 to 2.0 × 10^?6 mol/L, with a detection limit of 1.0 × 10^?12 mol/L.     

Determination of phenol and catechol concentrations with oxygen probes coated with immobilized enzymes or immobilized cells

The enzyme phenol 2-hydroxylase was immobilized on Sepharose and used in conjunction with an O₂ electrode for quantitating phenol. Similarly, catechol 1,2-oxygenase was used for quantitating catechol. A third probe was prepared by immobilization ofTrichosporon cutaneum cells rather than purified phenol 2-hydroxylase for phenol quantitation. The whole cell system gave results comparable to the immobilized enzyme system.