Preparation And Application Of Piezoelectric Biosensor Based On Signal Amplification Technology

With the continuous progress of the analytical science, a variety of analysis and testing process in life sciences, more and more with the aid of bio-sensing technology to obtain the required information. After 40 years the continuous rapid development and mutual integrationin modern measurement techniques, molecular biology, bio-electronics and bionics, biosensing technology has made remarkable progress in basic research, applied research, new product development as well as in commercialization. The current biosensors have been reported in hundreds of species and new types of biosensors are emerging, equipment performance continuously improving, have made a wide range of applications in health care, food hygiene, environmental monitoring, defense and security. This research paper concerns on a number of bottleneck problems for the current development of sensor technology, focusing on how to improve the sensor signals and so on. Combination of the advantages of simple, rapid, real-time output data with piezoelectric immunosensor sensing analysis and high sensitivity, characteristics of electrochemical sensing technology, using superior performance of nano-materials, and the enzyme-catalyzed signal amplification technology, the several new biological sensors for aflatoxin B1, human IgG,α-thalassemia mutations, the quantitative simulation of DNA sequence determination were developed and the results were compared with conventional methods, the detection sensitivity, linear range has improved and verified the practicality of the techniques.Piezoelectric immunosensor combines the high sensitivity of the piezoelectric effect and high specificity of immune response as a biological sensor, has the characteristics of simple, rapid, sensitive, low cost, to respond in a broad spectrum, real-time data output, etc., with wide application prospects in the biological technology, clinical diagnostics, environmental monitoring, food industry, medicine and military fields. We use enzymes and nano-materials in the biological sensor applications, combined with the effective fixed method of bio-active component in analysis using signal amplification technology to improve the signal, lower detection limit, has developed three new types of piezoelectric immunosensor. At the same time, try to use quartz crystal microbalance as a sensor on its surface fixed with a hairpin DNA probes used to identify and target DNA to produce a signal to build a simple and efficient DNA detection method. Also proposed a new method of electrochemical detection of DNA in order to ferrocene labeled oligonucleotide construct a simple general-purpose the type of signal opening molecular switch, realized its reagent-free detection of DNA sequences. The main contents are 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 0.10 ~ 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 (Chapter 2).(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 (Chapter 3). (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 (Chapter 4).(4) Try to use quartz crystal microbalance as a sensor on its surface fixed with a hairpin DNA probes used to identify and target DNA to produce a signal to build a simple and efficient DNA detection method. It features combined with restriction endonuclease ECoR I and nano-gold-labeled detection probe that can reduce the background of a very good value and effective way to boost the signal. The program are as follows: first, the quartz crystal surface through the thiol-based self-assembly of a fixed period with a hairpin DNA probes can be part of its hairpin endonuclease ECoR I specifically recognize and cut. Mercapto-hexanol then closed, then the role of the target DNA. If the system under test contains a target DNA, the DNA probe of the hairpin turn and the subsequent enzyme is not their role, and can be combined with nano-gold-labeled detection probe making signal amplification. Otherwise, in the absence of target DNA, the hairpin DNA probe can be digested, and nano-Au labeled probes role can not produce any signal. The experimental results show that this method is a simple and practical, high sensitivity analytics adapting the detection of DNA (Chapter 5).(5) In the present approach, we revealed a new scheme for electrochemical detection the target DNA sequence using Fc-labeled oligonucleotide as a molecular switch, which is a signal-on sensor featuring both generalizability and simplicity in design toward reagentless detection of DNA. The present approach has been demonstrated with the identification of a single-base mutation in Hb Constant Spring codon 142 (terminating codon TAA→CAA) that is one of the major types ofα-thalassemia point mutations for clinical diagnosis. After reaction with various concentration of target DNA under the optimum experimental conditions, the calibration curve was plotted. The results showed that the current intensity was linear to the logarithm of the target concentration in the range from 0.01 to 100 pM with a detection limit 0.01 pM. The experiment found that the DNA sensor could be reused via immersed into NaOH solution to be easily and successfully regenerated. All these features revealed that the system is a promising candidate for single-base mutation discrimination, owing the advantages of both generalizability and simplicity toward reagentless detection of DNA with sensitivity and selectivity (Chapter 6).