Studies On Piezoelectric Immunosensors Based On Signal Amplification Technique For Quantification Of Aflatoxin B₁ And Other Analytes

Piezoelectric immunosensors, which incorporate high sensitivity of piezoelectric response and high specificity of antibody-antigen immunoreaction, have some outstanding advantages including desirable simplicity, rapid response, high sensitivity, effective cost, wide detection range, and real-time output etc. They have become increasingly practical and useful tools in biotechnology, clinical diagnostics, environmental monitoring, food industry, medicine and military affairs. In recent years, nanostructured materials have been widely researched and used. The performances of the resulting biosensors will be greatly improved when the nanomaterials are applied in the fabrication. Moreover, enzyme and nanoparticles labels are attractive due to their advantage properties and have been extensively applied in bioassay. In this paper,we developed three novel piezoelectric immunosensors and a H2O2 sensor by use of enzyme and nanoparticles based on efficient immobilization of biomolecule and signal-amplified methods to improve detection sensitivity and decrease the detection limit. The detailed contents are shown as follows:(1) A simple, rapid and highly sensitive piezoelectric immunosensor has been proposed and applied to detect aflatoxin B₁ (AFB₁). It is unlikely that direct binding of small molecules such like AFB₁ to the piezoelectric sensor surface could result in a satisfactory detection limit and sensitivity. Thus, indirect competitive immunoassay technique had been used for the detection of the target and gold nanoparticles (GNP) been employed as a'weight label'to the secondary antibody for amplifying the response. This method is proven in its ability to detect AFB₁ down to a level of 0.01 ng mL^-1 in artificially contaminated milk, which is comparable to or even exceeding the sensitivity of microtitre plate ELISA. Furthermore, the frequency responses of the immunoassay are linearly correlated to the logarithm of AFB₁ concentration in the range of 0.1 100 ng mL^-1. The sensor could be regenerated under very mild conditions simply by immersing the sensor into glycine buffer solution to desorb the combined antibody. It is found that the as-renewed sensor could be reused at least 9 runs without obvious loss of sensing sensitivity.(2) An ultrasensitive piezoelectric method for the detection of the aflatoxin B₁ based on the indirect competitive immunoassay and the biocatalyzed deposition amplification has been developed. In this method, the quartz crystal surface was coated with a self-assembled monolayer of 3-mercaptopropionic acid (MPA) for covalently immobilization of the BSA-AFB₁ conjugate, which could compete with the free AFB₁ for binding to the anti-AFB₁ antibody (MsIgG). After the competitive immunoreaction, the horseradish peroxidase (HRP) labeled goat anti-mouse IgG (G-Anti-MsIgG) was introduced into the detection cell to combine with the anti-AFB₁ antibody on the crystal surface. The enzyme labeled G-Anti-MsIgG as a biocatalyst could accelerate the oxidation of 4-chloro-1-naphthol by H₂O₂ to yield the insoluble product benzo-4-chlorohexadienone on the surface of quartz crystal microbalance (QCM), resulting in a mass increase that was reflected by a decrease in the resonance frequency of the QCM. The proposed approach could allow for the determination of AFB₁ in the concentration range of 0.01 10.0 ng mL^-1. Furthermore, several artificially contaminated milk samples were analyzed with good recoveries obtained, which demonstrated the suitability of the proposed method for detecting AFB₁.(3) A simple piezoelectric immunoagglutination assay technique with antibody-modified nanoparticles has been developed for direct quantitative detection of protein. The proposed technique is based on the specific agglutination of goat anti-hIgG-coated silica nanoparticles in the presence of human immunoglobulin G (hIgG),which causes a frequency change and is monitored by a piezoelectric device. The antibody modified on the probe surface would combine with antibody-coated nanoparticles in the presence of antigen (hIgG) when the surface agglutination reaction took place, which couples both the mass effect and viscoelastic effect acting on the probe. The results indicate that the background interference can be substantially minimized and the probe signal can be observably multiplied. In addition, the surfaces of the modified probe and that after combining the complex of immunoagglutination were imaged by scanning electronic microscopy (SEM). Moreover, an optimization of assay medium composition with the addition of poly(ethylene glycol) (PEG) serving as immunoagglutination enhancer and sodium chloride to control the ion-strength was investigated. The frequency responses of the immunoagglutination assay were found to correlate well with the hIgG concentration with a detection limit of 0.084μg mL^-1.(4) In this paper, we prepared a kind of inorganic-organic composite nanoparticles, poly(vinylpyrrolidone) (PVP)-Prussian blue (PB), adopting PVP as a protecting polymer to protect PB. A series of PVP-PB particles with different diameters were obtained via mixing various proportions of PVP and Fe²⁺. Transmission electron microscopy (TEM) indicated that this particle was of the same shape and the diameter was about 100nm when the proportion of PVP and Fe²⁺ was 1:1. Amperometric response also showed that particle was much active with this proportion. We constructed a new kind of electrochemical sensor for effective modification glassy carbon electrode with this nanoparticle and MWCNT. The sensor with this complex film was demonstrated to possess high catalytic activity for the electrochemical reduction of hydrogen peroxide in neutral condition, which not only owned the advantages of inorganic nanoparticles, such as easy synthesis, great catalytic property and high selectivity, but also had great stability and reproduction. Cyclic voltammetry (CV) was employed to investigate the ability of oxidation and reduction. Two pairs of redox peaks arising from the reversible transformation of Fe³⁺/²⁺ and Fe(CN)₆^3-/4- was shown clearly in the CV. The amount of PVP-PB particles and MWCNT also had great influence on the catalytic activity of the sensor. We thoroughly investigated these two factors and chose 1 mg mL^-1 for each particle at last. Under the optimized condition, we obtained great amperometric response to H₂O₂ with this complex film at -0.2 V. The linear range of this sensor is 5×10^-7 - 7.5×10^-3 M and detecting limit is 8×10^-8 M. It also provides a new platform for sensor of enzyme which can produce H2O2 as a substrate.