Monitoring Environmental WasteWater Using A Piezoelectric Impedance Microbial Sensing Technique

A new method was proposed for monitoring antibiotics and other pollutants based on the assessment of the growth of Staphylococcus aureus (S. aureus) with a piezoelectric impedance analysis technique. During the growth process of S. aureus, the motional resistance variation ( jR₁) increases and the frequency shift ( jf) of the piezoelectric quartz crystal (PQC) decreases correspondingly. The two responses can conveniently be used in process analysis. Under the given experimental conditions, a linear relationship between the resistance detection time (RDT) and logarithm value of the concentration of penicillin was obtained in the range of 0.01-10 µg/ml with a detection limit of 4 2 10^-3µg/ml. Other antibiotics and pollutants also have the similar linear relationships. The proposed method is simple, convenient and sensitive. Particularly, it does not need the immobilization of the microorganism. This method was used for monitoring of wastewater from a hospital.     

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 Approach to the Development of a Reusable Piezoelectric Crystal Biosensor

Abstract

A reusable piezoelectric crystal immunosensor for human albumin has been developed. The crystal was coated with protein a and then reacted with anti-human albumin antibody. Human albumin in the range 10^?4 mg/ml to 10^?1 mg/ml could be detected by the system. Crystals were regenerated by saturation with albumin and subsequent binding of a new anti-albumin antibody layer. The albumin assay could be repeated up to 5 times using the same crystal.     

Determination of Estradiol by Biotin‐Avidin‐Amplified Electrochemical Enzyme Immunoassay

Abstract

A simple, sensitive biotin‐avidin‐amplified electrochemical enzyme‐linked immunosorbent assay (ELISA) for the determination of estradiol (E₂) was proposed in this paper. The complex of biotinylated anti‐E₂ antibody and horseradish peroxidase‐labeled avidin (HRP‐avidin) were regarded as a probe in this system. The activity of labeled enzyme was measured with electrochemical methods using o‐phenylenediamine as substrate. Coupled with the plate‐coated antigen, indirect ELISA format using E₂‐ovalbumin, the electrochemical detection was performed for E₂ with the detection limit of 21.0 pg/ml, and the linear range of determination of 50.0–500.0 pg/ml. The proposed method has been used for the determination of E₂ in river water with satisfactory results. Compared with the traditionally spectrophotometric ELISA detection, this method shows greatly heightened sensitivity. The limit of detection improved by about two orders of magnitude, which is very suitable for the conditions with extremely low concentration of analyte or very small volumes of sample present.     

A New Bacteriophage-Modified Piezoelectric Sensor for Rapid and Specific detection of Mycobacterium

Detection of tuberculosis and related diseases caused by mycobacteria is costly, time-consuming, and labor-intensive. Here a new phage-modified piezoelectric system for rapid and specific detection of mycobacteria was developed. In this system, interdigital gold electrode immobilized with lytic phage was used as a probe in place of a steel electrode in the multi-channel series piezoelectric quartz crystal (MSPQC) system. The probe was directly connected to the piezoelectric detection system. Mycobacterium was specifically captured to the phage-modified electrode and then lysed by immobilized phage, which caused the electrode electrical properties change. This change can be sensitively monitored by the piezoelectric detection system. The detection time of Mycobacterium smegmatis was less than 2 hours and a detection limit of 10³cfu mL^?1 was obtained. Additionally, it was successfully used to detect Mycobacterium tuberculosis. The developed system using phage-modified interdigital electrode showed high specificity and reproducibility for mycobacterium detection. Compared with the MSPQC system, the proposed system was faster and more specific.     

A novel piezoelectric immunosensor for the detection of malarial Plasmodium falciparum histidine rich protein-2 antigen

A novel piezoelectric (PZ) immunosensor for the direct detection of malarial Plasmodium falciparum histidine rich protein-2 (PfHRP-2) antigen was developed. The mixed self-assembled monolayers (SAMs) of thioctic acid and 1-dodecanethiol were formed on gold surface of quartz crystal. Cyclic voltammetry, electrochemical impedance spectroscopy and surface Raman spectroscopy techniques were used to characterize the mixed SAMs. The rabbit anti-PfHRP-2 antibodies were coupled on mixed SAM modified gold surface of quartz crystal via NHS/EDC activation method. The PZ immunosensor was applied to detect PfHRP-2 in the linear range of 15-60 ng/ml with a detection limit of 12 ng/ml. It was also found that even after 14 days of storage, 50% of the activity still remained. Clinical human serum samples were tested with this method, and the results were in agreement with those obtained from commercially available ICT kit (NOW^® Malaria).     

Novel Reduction Kinetics Model of Dimethoate (DMA) on the Growth of Pseudomonas aeruginosa

Abstract

A new method for monitoring Pseudomonas aeruginosa growth with dimethoate (DMA) as sole carbon using the series piezoelectric quartz crystal (SPQC) and high-performance liquid chromatography (HPLC) was described. Compared with the general methods, SPQC gives real-time, multidimensional information. The determination limit of HPLC was 1.08 ng. A combined novel response model for Pseudomonas aeruginosa degrading DMA was derived. By fitting DMA reducing data, the value of the reduction rate constant (k) was obtained and in line with the actual trend. The proposed kinetic model was successfully applied to Pseudomonas aeruginosa degrading DMA.     

Flow Injection Piezoelectric Determination of Brix in Sugar Cane Juice and in the Alcoholic Fermentation Process

Abstract

A flow injection piezoelectric procedure is proposed for the determination of brix (total solids dissolved) in the alcoholic fermentation process. A piezoelectric Perspex cell was constructed to fit one of the gold electrodes of a 10 MHz crystal and this sensor was adapted to a flow injection system. The analytical curve is linear over the brix range from 0.5 to 20.0% with a detection limit of 0.3%. Seventy results can be obtained per hour and the relative standard deviation was less than 1.45% for a solution containing 6% of brix (n=15). Applications of this method for the determination of brix in sugar cane juice and in an alcoholic fermentation bench show satisfactory results in comparison with refractometric and densimetric methods, respectively.     

Study of the Fluorescence Enhancement of Eu-Y-DEM-Dibenzoylmethane-Ammonia System

Abstract

The fluorescence enhancement and solvent effect of Eu-dibenzoylmethane-ammonia system was studied. The fluorescence intensity of the system can be greatly increased by y³⁺. The maximum fluorescence intensity was obtained at pH 8.9 – 9.5. The fluorescence intensity is a linear function of Eu²⁺ in the range of 1.0x10^?9 M -4.0x10^?8 M. The detection limit is 1.1x10^?10 M. The optimized procedure is applied to the determination of europium in lanthanide oxide samples.     

The dual-modes of QCM immunosensing for estradiol analysis via dynamic or round-off calibration

In this work, a piezoelectric immunosensor is developed for the detection of estradiol (E₂). The gold-coated chip of quartz crystal microbalance was modified by a 3-mercaptopropionic acid self-assembly monolayer, activated by carbodiimide/n-hydroxylsuccinimide, and then to immobilise the E₂ antibody via the interaction with their amine groups. The resonance frequency change resulted by the binding of E₂ on immobilised antibody differs from that of a blank (ΔF), was correlated to the antigen concentration. The construction of the sensing interface was confirmed by electrochemical impedance spectroscopy. Both round-off or dynamic modes were explored as analytical detection strategy in this work. Using round-off mode, the output ΔF responds to the concentration of E₂ with a detection limit of 0.04 μg mL^?1 and a linear range from 0.1 to 10 μg mL^?1. Meanwhile, using a dynamic mode, the presence of E₂ induced in a real-time sensing output along with the incubation of antigen. The slope of sensing output is in linear relation with the concentration of E₂ with a detection limit of 0.06 μg mL^?1. Thus, a faster immunesensing technique is established for rapid analysis requiring only several seconds other than the round-off mode which needs a complete incubation period for at least 60 min. This finding will greatly promote the applicability of immunosensor for rapid, real-time or in-site assays.     

Piezoelectric Detection of Ricin and Affinity-Purified Goat Anti-ricin Antibody

Abstract

Detection of ricin, in a piezoelectric quartz crystal microbalance format, can be accomplished with the use of capture antibody techniques. These techniques allow for the specific attachment of ricin to immobilized capture antibodies on the quartz crystal transducer area. A reversed format can also be used to detect antibody in solution. In this case, the antigen is immobilized and antibody attaches specifically, thus increasing the overall mass and decreasing the resonant frequency. In this report we describe detection of both ricin and anti-ricin antibody using immunological piezoelectric quartz crystal microbalance techniques.

     

Rapid Detection of Escherichia Coliform Using A Series Electrode Piezoelectric Crystal Sensor

Abstract

A series electrode piezoelectric crystal sensor was used to determine Escherichia coliform (E. coli.). This method is simple and rapid, and requires only a single sample dilution. E. coli. in the range of 10 - 10⁶ cells per ml can be estimated by this method. The procedure is a frequency detection time (FDT) method. The factors which affect the determination are discussed. Thirty samples in pure culture were tested by this method and the results fell within the 95% confidence limits of the standard plate count values. A correlation coefficient of 0.93 was obtained between the frequency detection time and logarithm of initial bacterial concentration. This corresponds to the criteria used to evaluate E. coli. with the added advantage of single dilution and more rapid results.     

Novel potentiometric immunosensor for determination of diphtheria antigen based on compound nanoparticles and bilayer two-dimensional sol–gel as matrices

A novel method for fabrication of a diphtheria potentiometric immunosensor has been developed by means of self-assembling compound nanoparticles to a thiol-containing sol–gel network. A cleaned gold electrode was first immersed in a 3-mercaptopropyltrimethoxysilane (MPS) sol–gel solution to assemble a silica sol–gel monolayer. The silane entities were then polymerized into a two-dimensional sol–gel network (2D network) by dipping into aqueous NaOH. The second silane layer was formed by re-immersion in the MPS sol–gel solution overnight. The compound nanoparticles (nanocompounds) containing gold nanoparticles and silver nanoparticles were then chemisorbed on to the thiol groups of the second silane layer. Finally, diphtheria antibody (anti-Diph) was adsorbed on to the surface of the compound nanoparticles. The modified process was characterized by cyclic voltammetry (CV). Detection is based on the change in the potentiometric response before and after the antigen–antibody reaction. A direct potentiometric response to diphtheria antigen (Diph) was obtained from the immobilized diphtheria antibody. The potentiometric response of the resulting immunosensor was rapid and the linear range was from 22 to 800 ng mL^–1 with the linear regression equation E=–79.5+69.4 log [Diph] and a detection limit of 3.7 ng mL^–1 (at 3). Up to 19 successive assay cycles with retention of sensitivity were achieved for probes regenerated with 0.2 mol L^–1 glycine–hydrochloric acid (Gly–HCl) buffer solution. Moreover, analytical results from several serum samples obtained using the developed technique were in satisfactory agreement with those given by the ELISA method, implying a promising alternative approach for detecting diphtheria antigen in clinical diagnosis.     

Real-time immuno-PCR assay for detecting PCBs in soil samples

A fast and robust assay, based on immuno-polymerase chain reaction (IPCR) techniques, was developed for the detection of polychlorinated biphenyls (PCBs) in soil samples. Real-time IPCR (rt-IPCR) is a powerful technique that combines enzyme-linked immunosorbent assay (ELISA) with the specificity and sensitivity of PCR. In our assay, indirect ELISAs based on immobilization of PCB37 hapten–ovalbumin conjugates was used for evaluation of the immune response. The effect of optimal reagent concentrations to reduce background fluorescence was also investigated. Using the optimized assay, the linear sensitivity range of the assay covered more than six orders of magnitude, and the minimum detection limits reached 5 fg ml^–1 antigen. Rt-IPCR was tested for its cross-reactivity profiles using four selected congeners and four Aroclor products. The assays were highly specific for congeners but less specific for Aroclor1242. We took four soil samples to validate the method, and the results were confirmed by gas chromatography/mass spectrometry (GC/MS). The rt-IPCR results for soil samples correlated well with the concentrations of PCBs obtained by GC/MS (r = 0.99, n = 6). These data indicate that this highly specific, sensitive, and robust assay can be modified for detecting PCB compounds in the environment.     

A Piezoelectric Immunosensor for the Detection of Cortisol

Abstract

A piezoelectric crystal immunosensor has been developed for the detection and determination of cortisol. Cortisol antibody was layered onto the gold electrodes of a 10 MHz piezoelectric crystal which was precoated with either protein A or gluteraldehyde. Crystals pre-coated with protein A showed the best results with respect to stability and sensitivity.

The sensor was successfully used for the determination of cortisol in standard solutions from 36–3628 μg/L (part per billion). The advantages of the proposed sensor include simplicity, short analysis time, cost effectiveness and selectivity. The results demonstrate the feasibility of cortisol assay in clinical testing and in drug monitoring.     

Piezoelectric crystal for sensing bacteria by immobilizing antibodies on divinylsulphone activated poly-m-aminophenol film

A polymer sorbent formed at the surface of gold-plated piezoelectric crystal by anodic oxidation of m-aminophenol was investigated by voltammetric, FTIR techniques for antibody coupling after activation by divinylsulphone. A novel film with increased capacity for immobilizing antibodies was obtained using phloroglucinol to modify poly-m-aminophenol via divinylsulphone. Compared with the dip-coating methods using polyethylenimine and (gamma-aminopropyl)trimethoxysilane, this new technique gave more reproducible results for the immobilization of antibody from sample to sample due to the improved homogeneity and reproducibility of the coating. With present modification method, a piezoelectric immunosensor was developed for the detection of Salmonella enteritidis. A detection limit of 1x10(5) cells ml(-1) and an assay time of 25 min were achieved.     

Development of a liposome-based immunochromatographic strip assay for the detection of Salmonella

Salmonella species are ubiquitous human pathogens which pose a dangerous threat to the elderly and children worldwide. In this study, to develop a more efficient assay procedure for the rapid detection of Salmonella Typhimurium, an immunochromatographic strip assay was developed using immunoliposome (anti-Salmonella IgG-tagged) encapsulated with sulforhodamine B (SRB). The detection sensitivity of the developed immunochromatographic assay was compared with a commercial immunochromatographic test strip using colloidal gold nanoparticles. The liposomes were prepared through a reverse-phase evaporation method by using a lipid and phospholipid mixture and SRB, a fluorescence dye, which was encapsulated in the phospholipid bilayer. Furthermore, the outer surface of the SRB-encapsulated liposome was conjugated with antibody (affinity-purified polyclonal goat anti-Salmonella IgG) to form an immunoliposome (size, 223 nm), used as the analytical reagent in the developed immunoassay. For this strip assay, a plastic-backed nitrocellulose strip was immobilized with two antibody zones. The lower zone of the strip referred to Salmonella antigen capture zone (test line), while the other zone served as a positive control (control line). The lower zone was coated with affinity-purified polyclonal goat anti-Salmonella IgG, while the upper zone was coated with rabbit anti-goat IgG. During capillary migration of the wicking solution (diluted liposome and Salmonella culture, each 50 μl), Salmonella was captured with surface-bound immunoliposomes at the antigen capture zone, while the unbound liposomes migrated upward and bound to another zone. The color density of the antigen capture zone was directly proportional to the amount of S. Typhimurium in the test sample. As a result, the detection limit of the immunochromatographic strip assay developed in this study against S. Typhimurium was found to be 10² CFU/ml, which was significantly higher than the detection limit (10⁷ CFU/ml) of the commercial immunochromatographic test strip assay.     

A Multicommuted Flow‐based System for Hydrogen Peroxide Determination by Chemiluminescence Detection Using a Photodiode

Abstract

A simple, rapid, and automated assay for hydrogen peroxide in pharmaceutical samples was developed by combining the multicommutation system with a chemiluminescence (CL) detector. The detection was performed using a spiral flow‐cell reactor made from polyethylene tubing that was positioned in front of a photodiode. It allows the rapid mixing of CL reagent and analyte and simultaneous detection of the emitted light. The chemiluminescence was based on the reaction of luminol with hydrogen peroxide catalyzed by hexacyanoferrate(III).

The feasibility of the flow system was ascertained by analyzing a set of pharmaceutical samples. A linear response within the range of 2.2–210 µmol l^?1 H₂O₂ with a LD of 1.8 µmol l^?1 H₂O₂ and coefficient of variations smaller than 0.8% for 1.0×10^?5 mol l^?1 and 6.8×10^?5 mol l^?1 hydrogen peroxide solutions (n=10) were obtained. Reagents consumption of 90 µg of luminol and 0.7 mg of hexacyanoferrate(III) per determination and sampling rate of 200 samples per hour were also achieved.     

Immunochemical Detection of Microquantities of Bacterial Formate Dehydrogenase

Abstract

Two immunochemical methods were developed for detection of NAD⁺?dependent formate dehydrogenase (EC 1.2.1.2, FDH) isolated from the methylotrophic bacteria Pseudomonas sp. 101:1) the dot-blot analysis using rabbit polyclonal antibodies; and 2) the indirect competitive ELISA using poly- or monoclonal mouse antibodies. The first method was used for screening the bacterial gene bank, the sensitivity is 5 and 1 pg enzyme per sample using the anti-rabbit antibodies - horse radish peroxidase conjugate or the biotinylated anti-rabbit antibodies and avidin - peroxidase conjugate, respectively. The second method was applied for precise determination of FDH concentration in cell-free extracts of selected recombinant clones. Mouse polyclonal antibodies to bacterial FDH have exibited a rather high affinity binding also to FDH from the methylotrophic yeast Candida methylica. In the indirect competitive ELISA the sensitivity of bacterial FDH determination is 1 ng per sample.