Novel Biosensing Technology Based On Gold Nanoparticle Signal Transduction And Signal Amplification

Gold nanoparticles (GNPs) possess the distance-dependent optical properties and high extinction coefficients. The wavelength at which they absorb light and the absorption value depend on their size and shape, the dielectric properties of the surrounding medium and their interactions with neighbouring particles. For GNPs-based colorimetric assays, the color changes of GNPs indicating molecular recognition events can be directly observed by the naked eyes or monitored with simple UV-VIS spectrophotometer. In addition, the unique catalytic properties of gold nanoparticles stimulate their enlargement by reducing the same or another metal ion to metal and depositing on the surface of the GNPs, which leads to the changes of physical or chemical parameters of the analytical system. Therefore, GNPs have been widely used as versatile and sensitive tracers for the signal transduction of different biomolecular recognition events.Nanotechnology offers unique opportunities for creating highly sensitive innovative biosensing devices and ultrasensitive bioassays. Immobilization of biomolecules with GNPs plays an important role in the construction of biosensors. Due to their large specific surface area and high surface free energy, easy conjugation chemistry, good conductivity and biocompatibility, using GNPs as substrates for biomolecule immobilization can effectively retain the activity of biomolecules, enhance immobilization results, improve the stability of biosensors and facilitate the electron transfer efficiency. Furthermore, enormous signal amplification can also be achieved by linking the biorecognition unit to nanoparticle carrying multiple signal tracers.This dissertation focuses on developing a series of biosensing techniques based on signal transduction (in chapter 2, 3, 4) and signal amplification (in chapter 5, 6, 7) of GNPs for the detection of ever-increasing amounts of analytes in the clinical diagnosis, environmental monitoring and food ananlysis including human IgE,?-fetoprotein, ochratoxin A and Hg2+. The detailed materials are shown as follows:(1) A simple and rapid colorimetric approach for the determination of adenosine has been developed via target inducing aptamer structure switching, thus leading to GNP aggregation (in chapter 2). In the absence of the analytes, the aptamer/gold nanoparticle (GNP) solution remained well dispersed under a given high ionic strength condition in that the random-coil aptamer was readily wrapped on the surface of the GNPs, which resulted in the enhancement of the repulsive force between the nanoparticles due to the high negative charge density of DNA molecules. While in the presence of adenosine, target-aptamer complexes were formed and the conformation of the aptamer was changed to a folded structure which disfavored its adsorption on the GNP surface. As a result, the aggregation of the GNP solution occurred. The changes of the absorption spectrum could be easily monitored by a UV-Vis spectrophotometer. A linear correlation exists between the ratio of the absorbance of the system at 522 to 700 nm (A522 nm/A700 nm) and the logarithm of adenosine concentration between 100 nM and 10?M, with a detection limit of 51.5 nM.(2) In chapter 3, a new working mechanism, hairpin end pairing-induced GNP assembly, is introduced on the basis of the unique instability of aptamer-modified nanoparticles. The salt-induced aggregation of oligonucleotide-modified GNPs can readily occur while addition of target molecules favors the formation of hairpin structure and inhibits substantially the nanoparticle assembly. Along this line, we developed a proof-of-concept colorimetric homogeneous assay using immunoglobulin E (IgE) as analyte model via transforming commonly designed“light-down”colorimetric biosensor into“light-up”one. From the points of view of both conformational transition of aptamer and enhancement of steric effect, oligonucleotide-GNPs display an additional stability upon binding to target molecules. The assay showed an excellent sensitivity with both the naked eye and absorbance measurements. Compared with almost all existing IgE sensing strategies, the proposed colorimetric system possesses a greatly improved analytical performance. Success in this biosensing protocol indicates that investigating the assembly behavior of hairpin aptamer-modified GNPs would offer new insight into the dependence of GNP property on the structure switching and open a new way to design signaling probes and develop colorimetric assay schemes.(3) In chapter 4, using adenosine as model analyte, a novel homogeneous fluorescent spectrometry with high sensitivity and specificity was proposed. It involves two kinds of DNA probe conjugated nanoparticles-aptamer modified magnetic nanoparticles and capture DNA probe derivatized GNPs, which were firstly connected together via hybridization. The conformational transition of aptamer induced by adenosine-aptamer complex contributes to the displacement of probe conjugated GNP, which further catalyzes the reduction of Cu2+ by ascorbic acid, thus enlarged by the deposition of Cu atom on the surface of GNP. The fluorescence quenching of calcein was restored as the consumption of Cu2+. The experimental results show that the dynamic range for adenosine detection was from 100 pM to 10 nM, with a detection limit of 80 pM.(4) A sensitive and specific electrochemical immunosensor by using GNP label for enzymatic catalytic amplification was developed with?-fetoprotein (AFP) as the model analyte (in chapter 5). A self-assembled monolayer membrane of mercaptopropionic acid (MPA) was firstly formed on the electrode surface through gold-sulfur interaction. Monoclonal mouse anti-human AFP was covalently immobilized to serve as the capture antibody. In the presence of the target human AFP, GNPs coated with polycolonal rabbit anti-human AFP were bound to the electrode via the formation of a sandwiched complex. With the introduction of goat anti-rabbit IgG conjugated with alkaline phosphatase, dentritical enzyme complex was formed through selective interaction of the secondary antibodies with the GNP-based primary antibody at the electrode, thus affording the possibility of signal amplification for AFP detection. Current response arising from the oxidation of enzymatic product was significantly amplified by the dentritical enzyme complex. The current signal was proportional to the logarithm of AFP concentration from 1.0 ng/mL to 500 ng/mL with a detection limit of 0.8 ng/mL. This system could be extended to detect other target molecules with the corresponding antibody pairs.(5) A convenient, specific, and highly sensitive electrochemical immunosensor based on an indirect competitive assay format was developed for the determination of ochratoxin A (OTA), a common toxic contaminant in various kinds of agricultural products (in chapter 6). The sensing substrate was prepared using a gold electrode modified with a self-assembled monolayer of 1,6-hexanedithiol that mediated the assembly of a GNP layer, which could enhance the surface loading of OTA–ovalbumin conjugates and improve the sensitivity in electrochemical readouts. After competition of the limited anti-OTA mouse monoclonal antibody between immobilized hapten and OTA analyte in sample solution, alkaline phosphatase (ALP)-labeled horse anti-mouse immunoglobulin G (IgG) antibody was selectively bound onto the surface of the electrode, affording an indicator for OTA concentration in the sample. Electrochemical response arising from the oxidation of enzymatic product of 1-naphthyl phosphate was observed to be inversely proportional to the logarithm of OTA concentration in the range from 10 pg/ml to 100 ng/ml with a detection limit as low as 8.2 pg/ml. Furthermore, a negligible matrix effect and good recoveries were obtained in the determination of corn samples, evidencing the feasibility of the proposed method for accurate determination of OTA in corn samples.(6) A novel electrochemical biosensor for the determination of mercury ions in aqueous solution has been reported (in chapter 7). The sensing substrate with a layer of GNPs on the gold electrode surface was formed through 1,6-hexanedithiol connection, which could enhance the surface loading of capture probe and improve the electron transfer performance. Another DNA probe used as mercury ion specific binding (MSB) probe hybridized with capture probe. In the presence of mercury ions, the easy formation of thymine-Hg(?)-thymine structure destabilized the hybrid complex and caused MSB probe released from the interface. As a result, the amount of electroactive indicator methylene blue (MB) adsorbed by the remained DNA on the electrode surface decreased, leading to the decrement of the current signal. Therefore, the change of the redox current could reflect the concentration of the analyte. A linear relationship between the current signal and the logarithm of the target concentration up to 500 nM was obtained, with a detection limit of 0.32 nM. The fabricated sensor is shown to exhibit high sensitivity, desirable selectivity and excellent application in real sample analysis.