Studies Of Novel Methods For Homogeneous Immunoassay Based On Single Molecule/Particle Detection Technique

Immunoassay is based on highly selective immune reaction of antigen with antibody, and can be used to determine antigen or antibody. Currently, immunoassay becomes one of very widely used analytical technologies in clinical diagnosis, food and environmental analyses and biological and biomedical studies. The conventional heterogeneous immunoassays mainly use a microwell plate as an assay platform, and this method is considered to be labour intensive and time-consuming due to the use of tedious separation and washing steps before signal measurement. Compared to heterogeneous assays, homogeneous assay is to directly determine analytes in immune reaction mixture. This method is usually fast and amenable to miniaturization and automation. To date, several analytical methods have been used in homogeneous immunoassays, which mainly include fluorescence polarization, fluorescence resonance energy transfer, bioluminescence resonance energy transfer, surface plasmon resonance, and chemiluminescence. However, most of the homogeneous immunoassays do not have enough sensitivity, and they have currently been not satisfied for certain requirements in clinical diagnosis.In this dissertation, on the basis of single molecule/nanoparticle detection techniques, we develop highly sensitive homogeneous immunoassays for cancer biomarkers and biological macromolecules. The main contributions are as follows:1) We develop homogeneous immunoassay methods based on fluorescence correlation spectroscopy (FCS) and fluorescence cross correlation spectroscopy (FCCS). To distinguish the two components in FCS analysis, their diffusion coefficients must differ by a factor of at least 1.6, which corresponds to a molecular weight ratio of 4. In order to overcome the limitation, we use two strategies that are using fluorescence cross-correlation spectroscopy (FCCS) model and decreasing the antigen molecular weight. Firstly, we established a FCCS setup with single wavelength laser. Using quantum dots as labeling probes, the homebuilt setup was successfully applied for the study of the homogeneous immunoassay reaction. We used the immune reaction of human immunoglobulin G with goat antihuman immunoglobulin G as a reaction model. The molecule numbers in a highly focused volume, the concentration, and the diffusion time and hydrodynamic radii of the reaction products can be determined by FCCS system. The results demonstrated that the single wavelength FCCS system has simple instrument structure and operation process. The cross-talk effect was almost completely suppressed. This FCCS system was adapted to homogeneous immunoassay. Secondly, we develop a homogeneous competitive immunoassay method based on FCS using synthetic peptides as a tracer antigen. Synthetic peptides were peptide-mimic of the native antigen, and usually are short protein sequences, which can be recognized by corresponding antibodies. Synthetic peptides represent ideal antigenic targets for immunoassays since they can be cheaply and easily produced in large scale and in a reproducible manner. The most important thing is that synthetic peptides antigens have low molecule weight, which satisfied the requirement of FCS for molecule weight. We systematically investigated the immune reaction of CA125 (cancer biomarker) peptide antigen and its antibody, and obtained the dissociation constants and dissociation rates. Furthermore, we evaluated the sensitivity, specificity, reversibility, dissociation constant and dissociation rate of this competitive immunoassay. Our data illustrated that the detection limit of homogeneous immunoassay method based on FCS was about 0.1 nM. This method possesses potential applications.2) We present for first time an ultrasensitive method for detection of small gold nanoparticles, call as single gold nanoparticle counter (SGNPC). Its principle is based on the photon burst counting in a highly focused laser beam due to Brownian motion and strong resonance Rayleigh scattering of GNPs. We systematically investigated the effects of certain factors on the photon burst intensity. The photon burst intensity markedly depended on the laser wavelengths and power and GNPs sizes. The results demonstrated that the relationship between the photon burst counts and GNPs concentration showed an excellent linearity (R=0.992). The linear range was over four orders of magnitude, and the detection limit was 17 fM for GNPs (36 nm).The reproducibility of the photon burst counting was quite satisfactory and the relative standard deviations (RSDs) for intra-day and inter-day were 4.1 % (n=11) and 3.6 % (n=7), respectively. Moreover, our method was successfully applied for single gold nanoparticle detection in micro-fluidic chip channel. The results showed that there was the excellent linearity relationship between the photon burst counts and GNPs concentration under a given pressure. In addition, lower detection limit was obtained by increasing the velocity of GNPs flow. We believe the combination of SGNPC with a micro-fluidic chip have great potential applications in homogeneous bioassays.3) On the basis of SGNPC technique, we developed an ultrasensitive detection platform for homogeneous immunoassays for cancer biomarkers. A sandwich format is used in homogeneous immunoassays. We conjugated two antibodies to GNPs based on the strong adsorption of GNPs to antibodies. When GNPs-antibody conjugates are mixed in a sample containing antigens, the binding of antigens will cause GNPs to form dimmers (or oligomers). The number of GNPs decreased with an increase of antigens in solution, and SGNPC sensitively detected the change in the number of GNPs according to the photon burst counts. We used this technology to construct homogeneous immunoassays for AFP and CEA. The detection limits were 130 fM for CEA, 714 fM for AFP. Our method was successfully applied for direct determination of CEA and AFP levels in sera from healthy subjects and cancer patients. The results were in good agreement with ELISA assays Compared to current methods, our method can be characterized as extremely high sensitivity, good selectivity, simplicity and short analysis time. Furthermore, SGNPC can be used for real-time monitoring of certain biochemical processes in vitro and vivo due to its high spatial and good time resolutions. More importantly, our detection volume is less than 1 fL, and the sample requirement can be reduced to nano liter level. Therefore, our method has the potential to become a high-throughput detection platform for homogeneous immunoassay.4) Gold Nanoparticles (GNPs) possasse very strong absorption and scattering properties and currently, are extensively used in several fields of biological labeling, gene carrier and photothermal therapy sensing. The mean size and the size distribution of GNPs play a paramount important role in these applications. So far, we lack effective methods for characterization of GNPs in aqueous solution. Herein, we presented a novel method to determine the size and size distribution of GNPs in solution at single particle level, called as resonance light scattering correlation spectroscopy (RLSCS). We presented a data-fitting algorithm for RLSCS based on Genetic Algorithms to invert particle-size distribution from RLSCS curves. According to FCS method, we established a theoretical model of GNPs size distribution GNPs using RLSCS technique. We performed computer simulations to verify the validity of the method by using different size distributions modes. In this study, we firstly suppose certain size distributions modes, and then got the correlation curves by calculation, and finally Genetic Algorithms (GA) was used to invert the size distribution from autocorrelation curve of GNPs. GA program was written in Matlab language. The results showed that GA-RLSCS could retrieve initial values exactly. This result demonstrated that theoretically, GA-RLSCS could be used to measure size and size distribution. Variable sizes of GNPs were measured by this method. The results agree reasonably with that obtained by TEM. Compared with current methods, our method is simple, reliable, and may become a useful tool for characterization of GNPs and other nanoparticles. We believe that RLSCS may become an effective method for immunoassay, nucleic acid hybridize and aptamer recognition.