Title Gold Nanoparticle Based Chemiluminescent Immunoassay and Sequence-Specific DNA Detection
Abstract

Immunoassay and DNA analysis have recently attracted much attention. Many different types of techniques, such as electrochemical, optical, fluorescent, radiochemical, chemiluminescent (CL) methods have been applied for immunoassay and DNA analysis. Each has its own pros and cons. Radiochemical method which is one of the commonly used methods suffers from the tedious labeling protocol, environmental pollution and the harmful effect of human Health. Hence, the nonradioactive technology including electrochemistry, enzymic method, and fluorescent method have been developing quickly in recent years.CL is the production of electromagnetic radiation by a chemical reaction. CL intensity is directly proportional to the concentration of a limiting reactant involved in the CL reaction. There is no optical source in the CL system, which avoids the interference of stray light and results in a high sensitivity. Due to its simple optical system and convenient manipulation, CL analysis has been widely applied in pharmaceutical, biological, molecular biological, clinical, and environmental fields and is becoming one of the most powerful techniques for use in analytical chemistry. Compared to the classical marker, gold nanoparticle is one of the ideal biological tags, because of the simple protocol, cheap price, stable character of the prepared nanoparticle tags, and retention of the biochemical activity of the labeled biomolecules. Hence, a series of CL immunoassays and sequence-specific DNA assays based on the colloidal gold as label have been developed. Description of research in my thesis is presented as follows:Chapter 1: A chemiluminescent metalloimmunoassay based on gold nanoparticlelabel1. Chemiluminescent metalloimmunoassay based on 96-well plateIt is well known that the sensitivity of the immunoassay could be greatly improved by attaching many labels into sqecondary antibody. However, this approach does not always work since the increased labeling level could decrease the biochemical activity of the labeled biomolecules. Each colloidal gold nanoparticle contains thousands of gold atoms (e.g., 2.3×105 gold atoms are theoretically contained in a 20-nm spherical gold particle). Consequently, a higher detection limit can be indirectly obtained by labeling colloidal gold label into secondary antibody, and then dissolving gold metal into many Au3+ ions for the immunoassay. Combining the advantage of gold nanoparticle as biological tag and the high sensitivity of CL detection of gold ions, gold nanoparticle is for the first time employed as a label in the CL metalloimmunoassay for the determination of biotinylated antibody and human IgG Three kinds of CL immunoassay based on the dissolution of gold nanoparticle have been developed. The experimental conditions such as gold dissolution and CL reaction were optimized. In the detection format of primary antibody-IgG- antibody modified gold (10 nm and 30 nm gold), the dynamic range of IgG extended between 1-75 ng and 0.5-25 ng, respectively. The detection limit was estimated to be 0.5 ng for 10 nm gold and 0.1 ng for 30 nm gold. In the detection format of primary antibody-IgG-biotinylated antibody-streptavidin gold (5 nm and 10 nm), the dynamic range of IgG extended between 10-250 ng and 1-250 ng respectively. The detection limit was estimated to be 5 ng for 5 nm gold and 1 ng for 10 nm gold.2. Chemiluminescent metalloimmunoassay based on magnetic separationImmunomagnetic bead is one of the new type materials which takes the advantage of magnetic separation technique and immunology. The targeting substrate in sample solution can be seprated in a magnetic field by the specific binding of antigen and antibody covered on the surface of magnetic baed. It realizes homogenous reaction and hetergenous separation. The immunomagnetic bead has lots of advantages, such as convenient separation, high separation efficiency, retention of the bioactivity of targeting substrat, high performance, low toxicity, and so on. Hence, it has been widely used in biotechnology, including cell segregation and purification, immunoassay, nucleic acid detection, gene Engineering, target releasing and so on. In this section, a sensitive CL immunoassay based on the magnetic separation and gold nanoparticle as label has been developed. In this protocol, the sandwich-type complex is first formed among the primary antibody immobilized on the surface of magnetic beads, the antigen in the sample and the second antibody labeled with colloidal gold. A large number of Au3+ ions from each gold particle anchored on the the surface of magnetic beads are released after oxidative gold metal dissolution in the HCl-NaCl-Br2 solution, and then indirectly determined by a simple and sensitive luminol CL reaction. The method is evaluated for a noncompetitive immunoassay of a human immunoglobulin G and a concentration as low as 3×10-12 mol/L is determined, which is competitive with colorimetric ELISA or with immunoassays based on electrochemical metalloimmunoassay based on a colloidal gold label. From the analytical chemistry point of view, the protocol can be readily extended to other metal nanoparticles-based immunoassays as well as a large variety of clinical bioaffinity assays of analytes and the detection of DNA hybridization.Chapter 2: Gold nanoparticle based chemiluminescent detection of sequence-specific DNA associated with the anthrax lethal factorsAnthrax, as a world-wide biowarfare agent, has attracted great attention of public and military agencies. The practical and effective methods are desired to identify and monitor the biowarfare agent, and control its infections in public. Herein, a sensitive CL detection of sequence-specific DNA associated with the anthrax lethal factors has been developed by taking advantage of a magnetic separation/mixing process and the amplification feature of colloidal gold label. First, the amine-modified capture DNA are immobilzed on the EDC actived carboxyl terminated magnetic beads. Then, the target oligonucleotides are hybridized with magnetic beads-linked capture probes, followed by the hybridization of the biotin-terminated reporter probe and the binding of streptavidin-coated gold nanoparticles. The nanometer-sized gold tags are then dissolved, the gold ions thus released in solution are quantitatively determined by the Au3+ catalyzed luminol CL reaction. The CL signal is proportional to the amount of target DNA in the standard or sample. The linear range extends from 0.02 pmol to 2.0 pmol. As low as 0.01 pmol of target DNA can be sensitively detected by using this method, which represents 150-fold increase in sensitivity over gold dissolution-based electrochemical format and 20 times higher than gold dissolution-based ICP-MS technique.Chapter 3: Polystyrene bead amplified chemiluminescent detection of sequence specific DNAAbout 80 biotinylated gold nanoparticles (5 nm) can be loaded on the surface of a 0.5-μm diameter polystyrene bead. Herein, a sensitive amplifying detection of sequence-specific DNA associated with the anthrax lethal factors by using streptavidin modified polystyrene beads instead of streptavidin modified gold nanoparticles as carrier has been developed. The assay relies on: (1) the immobilization of amine-modified capture DNA on the surface of carboxyl-terminated magnetic beads activated by EDC; (2) first hybridization event occurring between the bead-captured DNA probe and the target sequence; (3) second hybridization happening between the biotin modified reporter probe and the target sequence; (4) the reaction between the biotinylated reporter DNA and streptavidin modified polystyrene beads instead of streptavidin modified gold nanoparticles; (5) the binding of biotin modified gold nanoparticles onto the polystyrene beads; (6) dissolution of gold nanoparticles, and then direct detection of CL signal by a simple luminol reaction. The dynamic range for the assay extends between 0.5-200 fmol. The detection limit is estimated to be 1×10-12 mol/L (0.1 fmol), which is 100-fold improvement on non-amplified method. This new protocol also provids a good capability in discriminating target DNA sequence and mismatched sequences. Overall, this new CL protocol offers great promise for the determination of sequence-specific DNA associated with the anthrax lethal factors.Chapter 4: Hydroxylamine-amplified gold nanoparticles for the naked eye and chemiluminescent detection of sequence-specific DNA1. Hydroxylamine amplified visual colorimetic detection of sequence-specific DNA based on 96-well plate.Herein, we report a hydroxylamine-amplified gold nanoparticles-based assay with naked eye and CL detection of sequence-specific DNA associated with anthrax lethal factors. For the naked eye detection assay, the signal can be observed by naked eye directly, which provide a general way for other biological assays. In contrast, the CL detection method can improve the detection limit by two orders of magnitude as compared to the naked eye detection, and as low as 10 amol of target DNA can be sensitively detected. Most importantly, stringent control of either temperature or salt concentration are not needed during washing steps, this new methodology exhibits an excellent capability for differentiating a perfectly matched target oligonucleotide from 8 kinds of one-nucleotide mismatched oligonucleotides, and this detection specificity indicates that the present protocol could be applied to single-nucleotide polymorphism (SNP) analysis in many fields. In addition, the proposed CL and naked eye assay with simple instrumentation and shorter time consuming provided a value way for the determination of sequence-specific DNA.2. Homogenous visual colorimetric detection of sequence-specific DNAHerein, we present a magnetic bead-based hydroxylamine amplified homogenous visual colorimetric detection of sequence-specific DNA associated with anthrax factors. This method is composed of following steps: (1) the immobilization of amine-modified capture DNA on the surface of carboxyl-terminated magnetic beads activated by EDC; (2) first hybridization event occurring between the bead-captured DNA probe and the target sequence; (3) second hybridization happening between the target sequence and gold probe; (4) dissociation of gold nanoparticles from the surface of magnetic beads in 80℃of water bath and collection of the dissociated gold nanoparticles; (5) indirect quantitation of target DNA by measuring the absorbance at 630 nm after the gold staining. By employing this strategy, an excellent linearity is found within the range of 0.25-25 finol with the lowest detection amount of 0.1 fmol, which is 10-fold improvement on the 96-well based visual colorimetric method.Chapter 5: A simple way for direct colorimetric visualization of mercury (Hg2+) based on the formation of gold nanoparticlesHeavy metal contents of the human body, if exceeded, lead to chronic poisoning. Therefore, the impurity limit test of the heavy metal is very important in drug quality control. Mercury which is one kind of heavy metals has harmful effect on human body. Hence, it is very important to establish a rapid, sensitive method for the determination of Hg2+ ions. Herein, following on the work of Chapter four, a visual colorimetric method for the determination of Hg2+ ions based on the formation of gold nanoparticle by employing the hydroxylamine reduction reaction has been developed. The HAuCl4-NH2OH redox reaction can be accelerated by Hg2+ ions, and the speed of the formation of gold nanoparticle is increased with the increase of the concentration of Hg2+. The direct colorimetric visualization of Hg2+ ions based on the formation of gold nanoparticles is thus developed, which takes only 16 min to find out the concentration of Hg2+ ions in aqueous solution. The dynamic range of Hg2+ ions extends between 10-1000 nmol/L with the lowest detection concentration of 10 nmol/L. In addition, 12 kinds of metal ions would not interfere with the determination of Hg2+ ions even at a 100-fold higher concentration than that of Hg2+ ions, which indicates high selectivity of this method. In conclusion, our experimental results reported here open up a new possibility of rapid, easy, and reliable way for the determination of Hg2+ ions.Chapter 6: Review-chemiluminescence platforms in immunoassay and DNA analysesCL immunoassays and CL DNA detection techniques have become very popular in recent years. This review will discuss recent advances in CL immunoassay and CL DNA detection techniques that have occurred over the last few years (2000-2008). Both “monoplex” and “multiplex” assays are summarized. In the “monoplex” assay section, different classes of CL labels including nanoparticle, DNAzyme, acridinium ester, enzyme and luminol based CL immunoassay and DNA detection assay are reviewed. In the “multiplex” assay section, both spatial resolution and substrate zone resolution will be discussed.

Category Clinical
Keywords Chemiluminescence, chlorauric acid, gold nanoparticles, human IgG, hydroxylamine, magnetic beads, microwell palte, sequence-specific DNA, streptavidin modified polystyrene beads,
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Pages 158
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