Chemiluminescence Functionalized Nanomaterials And Their Applications In Label-free Chemiluminescence Bioassays

In this dissertation, the state of arts on the development of chemiluminescence (CL), chemiluminescent functionalized nanomaterials (CL-NMs) and label-free CL bioassays were reviewed. Recently, lots of attention has been paid to CL-NMs for bioassays because their promising application potential in clinical diagnosis, environmental monitoring and food safety, etc. However, CL-NMs were mainly used as analytical probe for label-related bioassays. Recently, label-free bioassays have attracted great interests due to that they do not need complicated labeling and operation procedures and thus are simple, fast and low-cost. On the other hand, the CL efficiencies of the previous synthesized CL-NMs are limited and great challenges remains for the determination of trace substance. The aim of this dissertation is to explore the application of CL-NMs in label-free bioassays and the synthesis of CL-NMs with high CL activity. Luminol functionalized gold nanoparticles (luminol-AuNPs) and ruthenium(II) complex functionalized graphene oxide (Ru-GO) were used as nanointerfaces to develop label-free electrochemiluminescence (ECL) sensors for the sensitive and fast detection of thrombin, cardiac troponin I (cTnl) and rpoB genes of Mycobacterium tuberculosis. N-(aminobytyl)-N-(ethylisoluminol)(ABEI)/catalyst metal complexes bifunctionalized gold nanoparticles and ABEI-gold nanodots/Cu2+bifunctionalized chitosan-grafted carbon nanotubes (ABEI-Au/Cu2+-cs-CNTs) with excellent CL properties were successfully synthesized and used as sensing platform to label-free CL method for the sensitive and selective detection of pyrophosphate ion and pyrophosphatase. The main results are as follows:1. A label-free ECL aptasensor for the sensitive and selective detection of thrombin was constructed based on target-induced direct ECL signal change by virtue of a novel assembly strategy of oligonucleotide and luminol-AuNPs. Streptavidin AuNPs coated with biotinylated DNA capture probe1(AuNPs-probe1) were firstly assembled onto an gold electrode through1,3-propanedithiol. Then luminol-AuNPs co-loaded with thiolated DNA capture probe2and thiolated thrombin binding aptamer (TBA)(luminol-AuNPs-probe2/TBA) were assembled onto AuNPs-probe1modified electrode through the hybridization between capture probe1and capture probe2. The luminol-AuNPs-probe2/TBA acted as both molecule recognition probe and sensing interface. An Au/AuNPs/ds-DNA/luminol-AuNPs/TBA multilayer architecture was obtained. In the presence of target thrombin, TBA on the luminol-AuNPs could capture the thrombin onto the electrode surface, which produced a barrier for electro-transfer and influenced the electro-oxidation reaction of luminol, leading to a decrease in ECL intensity. The change of ECL intensity indirectly reflected the concentration of thrombin. The approach showed a a wide linearity for the detection of thrombin in the range of5.0pmol/L-50nmol/L with a detection limit of1.7pmol/L. The developed aptasensor is selective for the detection of thrombin. It is promising to use it for the detection of thrombin in real samples such as human serum since sample matrix could be separated from the target analyte during the measurement. As the aptamers for various proteins become available, this aptasensor model might be used for the detection of other proteins in clinical analysis.2. A simple and sensitive label-free ECL immunosensor based on the use of luminol-AuNPs as antibody carriers and sensing platform is fabricated for detecting the acute myocardial infarction biomarker cTnI. The ECL immunosensor was fabricated by the assembly of luminol-AuNPs conjugated with biotinylated antibodies against cTnI (biotin-anti-cTnI-luminol-AuNPs) with the streptavidin coated AuNPs (SA-AuNPs) modified Au electrode directly by virtue of the biotin-SA system. The fabricated sensing platform exhibited stable and strong ECL intensity and could be used for the recognition of target antigen. In the presence of cTnI, a decrease in the ECL intensity was observed. Direct detection of the ECL signal changes during antigen-antibody immunoreactions can be used for the quantification of cTnI. The ECL response exhibited a quite wide dynamic range (0.1-1000ng/mL). The proposed method has been successfully applied in the detection of cTnI in real plasma samples. This protocol is simple, fast, sensitive, specific, stable and reliable. This work reveals that the luminol-AuNPs are excellent sensing platforms for the fabrication of simple and sensitive immunosensors. Moreover, the proposed strategy may also be extended to detect other biomarkers, which is of great application potential.3. Tuberculosis (TB) remains one of the leading causes of morbidity and mortality all over the world and multidrug resistance TB (MDR-TB) pose a serious threat to the TB control and represent an increasing public health problem. A homogeneous signal-on ECL DNA sensor for the sensitive and specific detection of rpoB genes of MDR-TB by using Ru-GO as suspension sensing interface and ferrocene modified ssDNA (Fc-ssDNA) as ECL intensity controller. The ECL of Ru-GO could be effectively quenched by Fc-ssDNA absorbed on the Ru-GO nanosheets. The Ru-GO has good discrimination ability over ssDNA and dsDNA. Mutant ssDNA target responsible for the drug resistant tuberculosis can hybridize with Fc-ssDNA and release Fc-ssDNA from Ru-GO surface, leading to the recovery of ECL. Mutant ssDNA target can be detected in a range from0.1-100nmol/L with a detection limit of0.04nmol/L. The proposed protocol is sensitive, specific, simple, time-saving and polymerase chain reaction free without complicated immobilization, separation and washing steps, which creates a simple but valuable tool for facilitating fast and accurate detection of disease related specific sequences or gene mutations.4. CL reagent and catalyst metal ion complexes bifunctionalized gold nanoparticles (BF-AuNPs) with high CL efficiency were synthesized via an improved synthesis strategy. Biothiols, such as cysteine (Cys), cysteinyl-glycine (Cys-Gly), homocysteine (Hcy) and glutathione (GSH) were used as new chelators. ABEI was used as a model of CL reagents and Cu2+as a model of metal ion. In this strategy, biothiols were first grafted on the surface of ABEI-AuNPs by Au-S bond. Then Cu2+was captured onto the surface of ABEI-AuNPs by the coordination reaction to form BF-AuNPs. BF-AuNPs synthesized by using Cys as chelators exhibited much higher CL intensity than Hcy and GSH. The CL intensity of Cu2+-Cys/ABEI-AuNPs was1order of magnitude higher than that of DTDTPA/Cu2+-ABEI-AuNPs synthesized by the previous work. Moreover, strong CL emission of Cu2+-Cys/ABEI-AuNPs was also observed in neutral pH conditions. Finally, by virtue of Cu2+-Cys/ABEI-AuNPs as a platform, a simple CL chemosensor for the sensitive and selective detection of pyrophosphate ion (PPi) was established based on the competitive coordination interactions of Cu2+between Cys and PPi. The method exhibited a wide detection range from10-100μmol/L, with a low detection limit of3.6nmol/L. The chemosensor was successfully applied to the detection of PPi in human plasma samples. This work reveals that BF-AuNPs could be used as ideal nano-interface for the development of novel analytical methods.5. A simple and effective strategy for the synthesis of CL reagent and catalyst metal ion bifunctionalized carbon nanotubes (BF-CNTs) with excellent CL activity was presented for the first time. ABEI was used as a model of CL reagents. In this strategy, firstly, ABEI-Au functionalized chitosan-grated carbon nanotubes (ABEI-Au-cs-CNTs) were synthesized. Then, catalyst Cu2+was further assembled onto the surface of ABEI-Au-cs-CNTs via the affinity interactions between Cu2+and cs-CNTs to form ABEI-Au/Cu2+-cs-CNTs with highly enhanced CL intensity. The CL intensity of as-prepared ABEI-Au/Cu2+-cs-CNTs was almost2orders of magnitude higher than that of ABEI-Au-cs-CNTs and the ECL intensity of ABEI-Au/Cu2+-cs-CNTs was also about2times higher than that of ABEI-Au-cs-CNTs. The CL and ECL catalysis mechanism of ABEI-Au/Cu2+-cs-CNTs was proposed. PPi can coordinate with Cu2+to form stable PPi-Cu2+coordination complex, hampering the synthesis of ABEI-Au/Cu2+-cs-CNTs. The addition of PPase could catalyze the hydrolysis of PPi into Pi and release Cu2+from PPi-Cu2+. The released free Cu2+could then be selectively and effectively captured onto the surface of ABEI-Au-cs-CNTs to form ABEI-Au/Cu2+-cs-CNTs with enhanced CL intensity. Then, by virtue of ABEI-Au-cs-CNTs as sensing platform, Cu2+as signal transition medium, a simple, sensitive and convenient chemiluminescent assay for PPase activity was developed. The enhanced CL intensity has a linear relationship with the activity units of PPase from0.025U to0.5U with a detection limit down to9mU. This method is simple, sensitive and convenient without any labels or complicated operations. Moreover, the method is also employed to monitor PPase inhibitors efficiently.