A Novel QCM-based Biosensor for Detection of Microorganisms Producing Hydrogen Sulfide

Abstract

A novel piezoelectric quartz crystal microbalance (QCM) device with gas permeable membrane is proposed for the detection of microorganisms producing hydrogen sulfide (H₂S). The detection theory is based on the adsorption of hydrogen sulfide onto the silver electrode of the piezoelectric crystal sensor, which causes a dramatic decrease in the resonant frequency of QCM. A 100 Hz frequency shift is chosen as the criteria value to judge the presence of microorganisms producing H₂S. Factors affecting detection were investigated. Desiccant is of great practical importance in sensor response. This new biosensor can be a potential candidate for detecting bacteria which produce hydrogen sulfide.     

A reusable piezo-immunosensor with amplified sensitivity for ceruloplasmin based on plasma-polymerized film

A reusable piezoelectric immunosensor with amplified sensitivity has been developed for the detection of ceruloplasmin (CP) in human serum. The quartz crystal microbalance (QCM) was deposited with plasma-polymerized n-butyl amine film with the surface topology further characterized by using atomic force microscopy (AFM). Anti-ceruloplasmin antibody (CP-Ab) was electrostatically adsorbed on the PPF-modified crystal via an oppositely charged polyelectrolyte layer of alginate. It was found that the alginate-mediated immobilization interface could allow for antibodies to be largely immobilized with well-retained immunoactivity. In particular, a simple regeneration process for the sensor produced, i.e. by shifting the pH, can also be realized. Moreover, an optimized assay medium containing polyethylene glycol (PEG) was tested with enhanced immunosensing response (sensitivity). A dynamic concentration range of two orders of magnitude and a detection limit of 0.15 μg ml⁻¹ of CP were observed. Analytical results of clinical samples show that the developed immunoassay is comparable with the enzyme-linked immunosorbent assay (ELISA) method. However, it presents some superior advantages over the traditional sandwich format in that the analyzing performances are direct, rapid and simple without multiple separation and labeling steps.     

QCM‐Based Immunosensor for the Determination of Ochratoxin A

Abstract

A piezoelectric immunosensor based on a competitive format was developed for determination of ochratoxin A (OTA) concentration. Surface modifications via two self‐assembled monolayers (SAMs) were investigated respectively and a better result was obtained with the SAM of 16‐mercaptohexadecanoic acid (16‐MHDA). The quartz crystal microbalance (QCM)‐based immunosensor was fabricated by immobilizing anti‐OTA antibodies onto the surface of the 16‐MHDA‐modified electrode, and allowing competition between free OTA and that conjugated with BSA to occur. The assay exhibited a working range of 50–1000 ng/mL and a detection limit of 16.1 ng/mL. Studies of interference and matrix effects were performed to evaluate the feasibility of the developed immunosensor for the direct analysis of OTA in real samples. Recoveries were conducted at 50, 200, and 1000 ng/g and were determined to be in the range of 142%–76%. The OTA assay is specific. No cross‐reactivates were observed with citrinin.     

QCM-DNA biosensor based on plasma modified PT/TiO2 nanocomposites

A quartz crystal microbalance DNA biosensor based on plasma prepared polythiophene /titanium dioxide (PT/TiO₂) nanocomposite was developed for the detection of genetically modified organisms (GMOs). DNA hybridization was studied by quartz crystal microbalance (QCM) and cyclic voltammetry (CV) measurements. Single stranded DNA probes were immobilized on the PT/TiO₂ coated quartz crystal electrode and the hybridization between the immobilized probe and the target complementary sequence in solution was monitored. The developed QCM-DNA biosensor represented promising results for a real-time, label-free, direct detection of DNA samples for the screening of genetically modified organisms.     

A Piezoelectric Quartz Crystal Biosensor as a Direct Affinity Sensor

Abstract

A microprocessor controlled piezoelectric detector as sensor was employed to monitor in real time protein adsorption and immunoreactions using piezoelectric quartz crystals (AT-cut) with a basic resonant frequency of 10 MHz. The adsorbed protein was an immunoglobulin (h-lg G); in the immunosensing a covalent immobilized molecule (the pesticide 2,4-D) formed the receptor for the immobilized ligand sample (Mab anti-2,4-D) in a competitive assay.     

A Piezoelectric Crystal Detector for Acetoin

Abstract

A piezoelectric quartz crystal coated with tetrabutyphosphoniun chloride was found to be a good detector for acetoin. The coating is sensitive in the ppb range and completely reversible. The response curve is linear over a concentration range of 8-120 ppb. The detector can be tuned to give a satisfactory response in 30 seconds.     

The Detection of Pseudomonas aeruginosa Using the Quartz Crystal Microbalance

Abstract

The development of a piezoelectric immunosensor for the detection of Pseudomonas aeruginosa in milk and dairy samples was undertaken here. This was achieved primarily by optimising the system using ELISA, investigating capture, competitive and displacement assays. Results from ELISA supplied information on detection limits and linear ranges obtained with each assay. A displacement assay was chosen to be transferred to the piezoelectric system and the reduction in mass on the surface of the crystal due to antigen displacement was measured by recording the frequency changes of the quartz crystal microbalance. The linear range obtained was from 2x10⁶ cell/ml to 1x10⁸ cell/ml and the limit of detection was 100,000 cells. The system was also tested for cross reactivity with a non-specific antigen, Pseudomonas fluorescens.     

Piezoelectric crystal/surface acoustic wave biosensors based on fullerene C60 and enzymes/antibodies/proteins

The development of piezoelectric (PZ) quartz crystal and surface acoustic wave (SAW) biosensors based on fullerene C60 and immobilized C60-enzymes/antibodies/proteins for the detection of various biological species are reported. The C60 coated piezoelectric crystal sensors can be applied to the study of interactions between fullerene C60 and some biological species, such as enzymes, antibodies, proteins and heparin. The partial irreversible responses for some biospecies from C60 molecules were observed by the desorption study which implied that C60 could chemically react with these biological species. Thus, immobilized biological species, e.g. C60-GOD, C60-catalase, C60-urease, C60-lipase, C60-anti IgG, C60-heparin, C60-Hb, C60-Mb and C60-anti-Hb were successfully prepared. The immobilized C60-GOD, C60-catalase, C60-urease, C60-anti-IgG and C60-anti-Hb were employed as adsorbents onto quartz crystal of various piezoelectric biosensors to detect glucose, H₂O₂, urea, IgG, and hemoglobin respectively. The immobilized C60-lipase was applied to distinguishably catalyze the hydrolysis of some optical isomers such as L- and D-phenyalanine methyl ester and to determine these optical isomers. The immobilized C60-heparin was employed as a good inhibitor for blood clotting like solvated heparin. The H₂O₂ bio-sensor was set up with the immobilized C60-catalase to detect oxygen, the product of the hydrolysis of H₂O₂ by C60-catalase. The immobilized C60-GOD enzyme piezoelectric glucose sensor exhibited a good sensitivity and a good lower limit for glucose. A piezoelectric crystal urea biosensor based on immobilized C60-urease was also prepared to detect urea. Comparison between solvated and immobilized enzymes used for biosensors was also made. The C60-anti IgG or C60-anti-Hb coated IgG piezoelectric crystal sensors exhibited good sensitivity, selectivity and repeatability for IgG or hemoglobin. Fullerene C60-Hb and C60-myoglobin (C60-Mb) coated surface acoustic wave (SAW) immunosensors were prepared to detect the anti-hemoglobin (anti-Hb) and anti-myoglobin (anti-Mb) antibody, respectively. An electrochemical SAW (ESAW) detection system was also developed to detect glucose in aqueous solutions.     

Electrochemical Immunosensing Strategies Based on Immobilization of Anti-IgC on Mixed Self-Assembly Monolayers Carrying Surface Amide or Carboxyl Groups

Abstract

In the development of electrochemical immunosensing strategies, stability or activity of the immobilized biocomponents and signal amplification of the immunoconjugates are two key factors. In this study, a comparative study of immunoglobulin G antibody (anti‐IgG) immobilization, as a model, was performed on cysteine (Cys), 2‐aminoethane thiol (AET), and 11‐mercaptoundecanoic acid (MUA) monolayers. The change of anti‐IgG layer formation on the three base layers as a function of the anti‐IgG concentration was investigated in parallel via electrochemical impedance spectroscopy, cyclic voltammetry, surface plasmon resonance, and quartz crystal microbalance. Through the parallel measurements, we demonstrate that the Cys‐modified layer is more suitable for the immobilization of the anti‐IgG molecules than the MUA or AET‐modified layer. Based on the CV and EIS analyses, it was determined that the current responses decreased with the increment of anti‐IgG concentration, while the resistance responses increased with the concentration of anti‐IgG increased. Moreover, the current and resistance shifts were more remarkable on the Cys layer than that of the other two layers. In the SPR and QCM measurements, the SPR and QCM response signals were similar in shape but differing in time scales, reflecting differences in detection mechanisms. With regard to the fundamental problem of comparing different measurement principles, the mechanism of the IgG immobilized on the three layers was proposed. Consequently, the surface concentration of anti‐IgG immobilized on the electrode should be optimized to improve the sensitivity of the immunosensors.     

Studying the interaction of alpha-gal carbohydrate antigen and proteins by quartz-crystal microbalance

The interaction of alpha-galacosyl antigen and its binding protein (anti-Gal antibody) has been studied by quartz crystal microbalance through a SAM immobilization technique which overcomes the nonspecific interactions and molecule trapping for current piezoelectric biosensors.     

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 piezoelectric immunosensor for <Emphasis Type="Italic">Leishmania chagasi</Emphasis> antibodies in canine serum

The American visceral leishmaniasis is an important cause of morbidity and mortality in Brazil for both humans and dogs. Attempts to make a diagnosis of this disease need to be improved, especially in endemic areas, and in the tracking and screening of asymptomatic dogs, which are their main host in urban areas. A quartz crystal microbalance immunosensor for the diagnosis of the canine visceral leishmaniasis using a recombinant antigen of Leishmania chagasi (rLci2B-NH6) was developed. The rLci2B-NH6 was tightly immobilized on a quartz crystal gold electrode by self-assembled monolayer based on short-chain length thiol. The strategy was the use of the antigen-histidine tail covalently linked to glutaraldehyde performing a Schift base which permits a major exposure of epitopes and a reduced steric hindrance. The immunosensor showed good results regarding sensitivity and reproducibility, being able to distinguish positive and negative canine serum for L. chagasi. Furthermore, the immunosensor can be reused through exposure to sodium dodecyl sulfate solution, which promotes the dissociation of antigen–antibody binding, restoring the sensor surface with immobilized biologically active antigens for further analysis.     

A novel piezoelectric biosensor for the detection of phytohormone beta-indole acetic acid.

A novel piezoelectric immunosensor has been developed for the determination of beta-indole acetic acid (IAA) in dilute solutions. The detection is based on competitive immunoreaction between a hapten (IAA) and an antigen (IAA-BSA, hapten-protein conjugation) bound to an anti-IAA antibody, immobilized on a quartz crystal microbalance (QCM). The frequency change (y) of the sensor caused by antigen is linearly related to the logarithm of the concentration of IAA (x) in the range of 0.5 ng/ml - 5 microg/ml with a regression equation of the form y = -23x + 151 (r = 0.9937).     

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.     

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.     

丙肝压电免疫传感器的研制

研制了一种用于丙肝抗原检测的新型传感器——压电免疫传感器。采用AT切型、基频为10MHz伍英晶体作为换能器,先在晶体表面崮定巯基丙酸（MPA）,经过EDC和NHS活化后固定丙肝抗原（HCVAg）、最后检测不吲浓度的标准抗体溶液？分别研究了它的重复性和灵敏度,并通过临床验证了它的准确性此传感器其有灵敏度高、特异性好、不需标记、操作简单、能实时在线检测和重复使用等优点,能用于临床实验诊断、具有临床推广应用价值。     

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 Coated Piezoelectric Crystal Sensor for the Determination of 2, 4-Toluene Diisocyanate in Air

Abstract

2, 4-toluene diisocyanate (TDI) in air was detected and determined by a piezoelectric quartz crystal sensor coated with tetrakis(hydroxyethyl)ethylenediamine (THEED)/dithizone(diphenylthiocarbazone) solution (l:3v/v in acetone). The response curve is linear over the concentration range 3–24 ppb of TDI. The sensor can be used for more than 2 months without loss in sensitivity and presented good reversibility and reproducibility. Of the different possible interferents tested, only SO₂ caused a frequency change.     

Piezoelectric immuno sensor for the detection of candida albicans microbes

The system consists of an AT-cut quartz piezoelectric crystal, oscillator and frequency counter. The surface of the palladium-plated electrodes is oxidized anodically, and anti-Candida antibody is immobilized onto the surface. The crystal sensor is dipped into Candida suspension and the surface mass increase, caused by immuno-adsorption of Candida, is measured by the decrease in the resonant frequency of the crystal. The frequency shift is correlated with C. albicans concentrations in the range 10⁶?5 × 10⁸ cells cm^?3. The crystal sensor showed no response to Saccharomyces cerevisiae.     

Direct electrochemistry and superficial characterization of DNA-cytochrome c-MUA films on chemically modified gold surface

Cytochrome c (Cyt. c) was immobilized on the 11-mercaptohendecanoic acid (MUA)-modified gold electrode. The electrode was stable and sensitive to Cyt. c. Later, DNA was also immobilized on the two-layer modified electrode. Cyclic voltammetry studies show that Cyt. c can interact with dsDNA and ssDNA. The binding site sizes were determined to be 15 base pairs per Cyt. c molecule with dsDNA and 30 nucleotides binding 1 Cyt. c molecule with ssDNA. The modified electrodes were characterized by quartz crystal microbalance (QCM), impedance spectroscopy and atomic force microscope (AFM). The modified electrode can be used for determining DNA.