| In this dissertation, nucleic acid probe, nucleic acid probe based biosensors, nanomaterial and nucleic acid probe involved ECL biosensors were reviewed. Recently, nanomaterial related ECL detection has become an important subject in bioassays. Among them, the idea of enrichment of multiple chemiluminescent signal molecules on the nanoparticles, i.e chemiluminescent functionalized nanomaterial (CF NPs) is the new trend in the development of functional nanomaterials and has drawn much attention. Up to now, the reported CF NPs can be divided into two types: encapsulated or doped CF NPs and "bridging" dendritic CF NPs. The synthesis of these CF NPs requires multi-step reaction and the functional reagents are limited to Ru complexes. These CF NPs have already found their applications in nucleic-acid probe based bioassays, which improved the sensitivity, stability, selectivity of these biosensors. Our previous work has demonstrated the direct synthesis of chemiluminescent functionalized gold nanopaticles (CF-AuNPs) using luminol and its analogues as reductant and stabilizing reagent. This new type of CF NPs exhibited great ECL properties. However, few works has been reported on their applications in nucleic acid probe based biosensors. In the dissertation, we explored the applications of luminol and N-(aminobutyl)-N-ethylisoluminol (ABEI) functionalized gold nanoparticles in nucleic-acid probe based biosensors. We studied the assembly of these CF-AuNPs with DNA and aptamer molecules to build nucleic acid gold nanoprobes. On the basis, three novel analysis strategies were proposed for the development of ECL biosensors against DNA, protein and small molecule. Moreover, the direct synthesis strategy of CF-AuNPs was extended to the synthesis of biological functional nanomaterial, the biotinylated gold nanoparitcles. The main results are as follows:1. A novel ECL strategy for the detection of specific DNA sequences was developed based on luminol functionalized gold nanoparticles (luminol-AuNPs) labeling and the amplification of gold nanoparticles (AuNPs) as well as streptavdin-biotin system. The luminol-AuNPs labeled single stranded DNA was employed as signal probe and thousands of luminol molecules bounded on luminol-AuNPs acted as signal reporters, which greatly amplified the ECL signal. In this strategy, streptavidin coated AuNPs were self-assembled on a1,3-propanedithiol modified gold electrode, the biotinylated DNA capture probes were then linked on electrode through streptavidin-biotin biological system. The target ss-DNA and luminol-AuNPs labeled signal probe were subsequently assembled on the electrode through hybridization to form a "sandwich" DNA conjugate modified electrode, i.e. DNA sensor. Extremely high sensitivity for detecting sequence-specific DNA was achieved with a detection limit of0.19fM, which was more sensitive than previously reported methods except magnetically amplified DNA assay. The assay exhibited good stability and high specificity to discriminate one-base mismatch. The fabricated DNA sensor was also proved to be unaffected by complex sample matrix blood serum. It is of great application potential in the fields such as clinic diagnosis, food safety and environmental monitoring.2. A novel ECL aptasensor for platelet-derived growth factor B chain (PDGF-BB) was developed by assembling N-(aminobutyl)-N-ethylisoluminol functionalized gold nanoparticles (ABEI-AuNPs) with aptamers as nanoprobes. In the protocol, the biotinylated aptamer capture probes were first immobilized on a streptavidin coated AuNPs modified electrode, afterwards, the target PDGF-BB and the ABEI-AuNPs tagged aptamer signal probe were successively attached to the modified electrode by virtue of the dirner structure of PDGF-BB to fabricate a "sandwich" conjugate modified electrode, i.e. an aptasensor. ECL measurement was carried out with a double-step potential in carbonate buffer solution containing H2O2. The aptasensor showed high sensitivity and selectivity toward PDGF-BB and specificity toward PDGF-BB aptamer. The detection limit was as low as27fmol/L. The aptasensor was also applied for the detection of PDGF-BB in human serum samples, showing great application potential. Given these advantages, the ECL aptasensor is well suited for the direct, sensitive and rapid detection of protein in complex clinical samples.3. An ECL biosensor for simultaneous detection of adenosine and thrombin in one sample based on bifunctional aptamer and ABEI-AuNPs was developed. A streptavidin coated gold nanoparticles modified electrode was utilized to immobilize biotinylated bifunctional aptamer (ATA), which consisted of adenosine and thrombin aptamer. The ATA performed as recognition element of capture probe. For adenosine detection, ABEI-AuNPs labeled hybridization probe with a partial complementary sequence of ATA reacted with ATA, leading to a strong ECL response of ABEI enriched on ABEI-AuNPs. After recognition of adenosine, the hybridization probe was displaced by adenosine and ECL signal declined. The decrease of ECL signal was in proportion to the concentration of adenosine over the range of5.0×10-12～5.0×10-9mol/L with a detection limit of2.2×10～12mol/L. For thrombin detection, thrombin was assembled on ATA modified electrode via aptamer-target recognition, another aptamer of thrombin tagged with ABEI-AuNPs was bounded to another reactive site of thrombin, producing ECL signals. The ECL intensity was linearly with the concentration of thrombin from5.0×10-14～5.0×10-10mol/L with a detection limit of1.2×10-14mol/L. In the ECL biosensor, adenosine and thrombin can be detected when they coexisted in one sample and a multi-analytes assay was established. The biosensor also showed good selectivity towards the targets. Being challenged in real plasma sample, the biosensor was confirmed to be a good prospect for multi-analytes assay of small molecules and proteins in biological samples.4. It was found that the biotinylated AuNPs could be prepared by reducing HAuCl4; with biotin in aqueous solution at room temperature through a one-pot method. The reaction conditions such as concentration of HAuCl4and biotin, volume of biotin solution and pH played critical roles in the size, morphology and synthesis rate of biotinylated AuNPs. The characterizations of transmission electron microscopy, UV-visible spectroscopy showed that the obtained AuNPs were spherical gold nanoparticles with diameter of1.8～7.2nm. X-ray photoelectron spectroscopy, mass spectroscopy,1H nuclear magnetic resonance and fluorescence spectrum were used to characterize the surface component of AuNPs, showing that biotin and its oxidation product biotin sulfoxide were coexisted on the surface of AuNPs as protective reagents and the AuNPs was biotinylated AuNPs. The biotinylated AuNPs exhibited similar fluorescence properties with biotin. The maximum excitation was at378nm and the maximum emission was at434nm. Static injection chemiluminescence (CL) studies showed that the biotinylated AuNPs could remarkably enhance the CL of luminol-H2O2by one order of magnitude and its CL kinetics behaved as a sustainable platform. Moreover, the biotinylated AuNPs could be assembled on streptavidin coated AuNPs to form AuNPs-AuNPs assembly. The biotinylated AuNPs will find application prospect in the study of CL, nano-sensors, nanomaterials assembly and other fields. |
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