New Gold Nanoparticles-based Chemiluminescent Technologies For The Detection Of Biomolecules

The unique electric, optical, thermodynamic and catalytic properties of gold nanoparticles (AuNPs) have stimulated the increasing interest in the application of AuNPs in physics, chemistry, biology, materials science and cross subjects. Due to their less toxicity, small size and ease of cellular uptake, AuNPs have been an obvious choice in gene delivery, drug delivery, bioimaging and other therapeutic and diagnostic applications. The excellent conductivity, surface plasmon resonance (SPR) and high catalytic properties of AuNPs facilitate extensive application of AuNPs in construction of optical, electrochemical and piezoelectric biosensors with enhanced analytical performance.Chemiluminescence (CL) is the production of electromagnetic radiation by a chemical reaction. The CL intensity is directly proportional to the concentration of a limiting reactant involved in the CL reaction. CL has been combined with other technologies for a wide range of applications in various fields owing to its extremely hypersensitivity, simple instrumentation, wide calibration ranges and suitability for miniaturization in analytical chemistry. Due to their ease of modification, functionalized AuNPs are suitable for the carriers of CL reagents, such as AuNPs functionalized with luminol can effectively improve the CL sensitivity of analyte. Owing to their small size and prominent surface effect, AuNPs have been used as CL enhancer. For instance, AuNPs as nucleation centers catalyzed the reduction of AgN03to Ag atoms by luminol to yield Au/Ag core/shell nanoparticles. Meanwhile, luminol was oxidized to luminol radical, which further reacted with the dissolved oxygen, giving rise to light emission. The luminol-AgNO3-AuNPs CL system has the advantages of low background and good stability, which may be of great potential for the CL immunoassay, DNA and small molecules analysis. Hence, combining with other technologies, a series of CL analysis methods based on AuNPs have been developed to detect human plasma proteins, adenosine and telomerase activity. Description of research in the thesis is presented as follows:Chapter1:IntroductionPrecious metal nanoparticles, especially AuNPs are being one of the hot research areas due to their unique physical and chemical properties. This chapter includes the following parts:the synthesis methods and excellent properties of AuNPs; the applications of AuNPs in bioimaging, diagnostics, biosensors, CL catalyst, drug and gene delivery; the important application of AuNPs in luminol-AgNO3CL system.Chapter2:Aptameric system for detection of IgE based on gold nanoparticlesAptamers are small oligonucleotides that bind with affinity and specificity to a large number of target molecules, which are selected from a large combinatorial oligonucleotides library through an in vitro evolution process termed SELEX (systematic evolution of ligands by exponential enrichment). Aptamers have possessed a broader range of applications in therapeutics, diagnostics and biotechnology. IgE is one of the five classes of immunoglobulins, which plays a key role in allergic responses. Unlike other immunoglobulins, the circulating concentration of IgE is very low, however, elevated in patients afflicted with allergic asthma. A novel approach is proposed in this chapter for the development of an aptameric assay system for protein based on non-stripping gold nanoparticles-triggered CL upon target binding. The strategy chiefly depends on the formation of a sandwich-type immunocomplex among the capture antibody immobilized on the polystyrene microwells, target protein and aptamer-functionalized AuNPs. Introduction of target protein into the assay system leads to the attachment of AuNPs onto the surface of the microwells and thus the assembled AuNPs could trigger the reaction between luminol and AgNO3with a CL emission. Further signal amplification was achieved by a simple gold metal catalytic deposition onto the AuNPs. Such an amplified CL transduction allowed for the detection of model target IgE down to50fM, which is better than most existing aptameric methods for IgE detection. This new protocol also provided a good capability in discriminating IgE from nontarget proteins such as IgG, IgA, IgM and interferon. The practical application of the proposed AuNPs-based immunoassay was successfully carried out for the determination of IgE in35human serum samples. Overall, the proposed assay system exhibits excellent analytical characteristics (e.g., a detection limit on the attomolar scale and a linear dynamic range of4orders of magnitude), and it is also straightforward to adapt this strategy to detect a spectrum of other proteins by using different aptamers. This new CL strategy might create a novel technology for developing simple biosensors in the sensitive and selective detection of target protein in a variety of clinical, environmental and biodefense applications. Chapter3:Aptamer-based biosensor for chemiluminescence immunoassay of platelet-derived growth factor using gold nanoparticlesPlatelet-derived growth factor (PDGF) is a growth factor protein that has growth-promoting activity to stimulate the division and proliferation of the cells through binding its receptors on the cell membrane surface. Therefore, PDGF plays an important role in angiogenesis and is linked to cell transformation and tumor growth and progression. PDGF-BB, one of the important isoforms of PDGF, is expressed at low level or undetectable in normal cells, but is found to be overexpressed in some human tumors, including gliboblastomas and sarcomas. Detection of PDGF is significant for its potential use as a protein marker in cancer diagnosis. In this chapter, a novel aptamer-based CL immunoassay coupling with AuNPs as the tag was developed for the rapid, sensitive detection of PDGF-BB. Typically, PDGF-BB antibodies were immobilized on the surface of96-well plate to capture target PDGF-BB, and then sandwiched with the aptamer conjugates which were prepared by assembling AuNPs with PDGF-BB aptamer. The captured AuNPs on the96-well plate was further enlarged in the presence of hydroxylamine and chlorauric acid. Thus the enlarged AuNPs triggered the reaction between luminol and silver nitrate for the generation of a CL signal. Under the optimal conditions, a good linear relation was achieved in the range of0.1-1000pM PDGF-BB with a detection limit down to10fM. Other PDGF isoforms (PDGF-AA, PDGF-AB), IgG, IgA, IgM and IFN-α2b showed no obvious interference for the determination of PDGF-BB. Therefore, our strategy provided a simple and sensitive detection of PDGF-BB, and with high specificity, which could find wide applications in protein assay.Chapter4:Ultrasensitive chemiluminescence aptasensor based on Exonuclease-assisted recycling amplificationAdenosine is a nucleoside with value role in biochemistry, including the energy translation in the form of adenosine triphosphate or adenosine diphosphate, and the signal transmission in adenosine monophasphate form. Adenosine has received much attention due to its crucial signaling functions in both the peripheral and central nervous system. Elevated levels of adenosine in the brain appear to promote sleep. The monitor of adenosine under physiological conditions therefore is of great value. Exonuclease-III (Exo-III) could catalyze the stepwise removal of mononucleotides from3’-OH termini of double-stranded DNA when the substrates are blunt or recessed3’-terminus. However, its activity on single-strand DNA and protruding3’-terminus of double-stranded DNA is limited. In this chapter, we develop an Exo Ⅲ-assisted aptamer-based target recycling amplification strategy for sensitive and selective CL determination of small molecules, employing adenosine as the model target analyte. The system contained an amino DNA sequence as capture probe, aptamer A as detection probe, AuNPs-linked DNA sequence as reporter probe. Aptamer A consisted of the whole adenosine aptamer and an extension sequence which hybridized with capture probe immobilized on the wells, whereas12bases of the adenosine aptamer on the3’-terminal hybridized to the reporter probe functionalized with AuNPs. When adenosine was added, aptamer A turned into a duplex DNA with3’-hydroxyl termini relied on the structure-switching properties of aptamers binding to their target molecules. Therefore, adenosine could reduce the gold probes hybridized with aptamer A, then minus CL signal could be detected as a readout signal for the quantitative detection of adenosine. As the Exo Ⅲ catalyzed the stepwise removal of mononucleotides from3’-hydroxyl termini of double stranded DNA when the substrates are recessed3’-terminus or blunt, adenosine was released and hybridized with the second aptamer A. Thus Exo Ⅲ could catalyze a new cycle of probe transformation and led to enlarged unusual high sensitivity. Experimental results revealed that the enzymatic-assisted recycling strategy enabled the monitoring of adenosine with low detection limits of0.5nM, which is much lower than other analysis methods previously reported. This unique property of Exo Ⅲ makes the proposed sensor hold great potential for highly sensitive, selective and simple detection of a wide range of target molecules. The approach thus provides a versatile platform for clinical diagnosis, environmental monitoring, pharmaceutical and biomedical analysis.Chapter5:Amplified detection of telomerase activity using chemiluminescence analysis based on molecular beacon-gold nanoparticlesTelomerase has been regarded as an important sensitive and specific cancers marker, which is closely linked to malignancy and tumor progression. Rapid and ultrasensitive detection of telomerase activity plays a vital role in measure of malignancy and evaluation of treatment plans. In this chapter, a CL methods based on molecular beacon-gold nanoparticles (MB-AuNPs) for amplified detection telomerase activity originating from non-small-cell lung cancer A549cell is described. This strategy involves the telomerization of primer, in the presence of telomerase from A549cell extract and dNTP, followed linked to molecular beacon functionalized AuNPs by hybridization. MB could be open to linear structure and hybridize with the capture probe immobilized on the surface of DNA-binding96-well plate. As a consequence, AuNPs were captured on the plate-surface, which were further enlarged by HAuCl4-NH2OH. Then CL signals were considerably enhanced. The methods enable the detection telomerase activity that was extracted from200A549cancer cells, and good linear relationship between CL intensity and the concentrations of synthetic targeted DNA and human cancer cells were obtained. These easily fabricated MB-AuNPs biosensors show excellent promise for simple, sensitive and visible detection of telomerase activity in cancer cells.