Study On New Bioanalysis Approaches In Electrochemiluminescence Of Semiconductor Nanocrystals

Electrogenerated chemilumiescence (ECL), the generation of an optical signal triggered by an electrochemical reaction, has become a very powerful analytical technique extensively used in many domains. Semiconductor nanocrystals (S-NCs)with excellent luminescent properties and good biocompatabilities have extensively applied in biolabel and biosensing fields. The ECL analysis has many advantages over photoluminescence, such as the absence of background from unselective photoexcitation,the potential control over the ECL reaction,the higher sensitivity, the wider linear range and better selectivity, which has shown great application potentials in bioanalytical chemistry. However, in order to achieve the goal of the detection for the more biological samples in the nanocrystalline film, the new detection methods,new principles and new light-emitting materials would be urgently needed. The success of these could present novel and general routes for future ECL bioanalysis.Based on the current developments of S-NCs ECL and biosensors in our group,in this thesis, through the design and fabrication of innovative ECL system and the exploitation of ingenious signaling mechanism and development of novel emitters, we established some advanced methods in ECL bioanalysis. The major contents were described as follows:1. Highly efficient quenching of electrochemiluminescence from CdS nanocrystal film based on biocatalytic depositionAiming to find an alternative electrochemiluminescence (ECL) quenching route of high efficiency, biocatalytic precipitation (BCP) was firstly coupled with ECL for investigating its insulating effect on ECL. An insulating layer could be formed via BCP onto the electrode surface, inhibiting the reaction between the coreactant and luminophore and thereby impairing the ECL emission dramatically. Since the extent of insulating effect associated strongly with analyte concentration, a new ECL biosensor was successfully realized. In a model horseradish peroxidase (HRP)-based system, the fabricated biosensor possessed high sensitivity and wide linear range from 1.0×10-10 M to 1.0×10-6 M for H2O2 determination with a detection limit of 4× 10-11M(S/N=3). This method has great potential in extending the application of ECL biosensor for various bioanalytes.2. Potassium-doped graphene enhanced electrochemiluminescence of SiO2@CdS nanocomposites for sensitive detection of TATA-binding proteinThe K-doped graphene as an enhanced electron transfer substrate combined with CdS NCs coated SiO2 nanoparticles as an ECL emitter for double signal amplification,which provides a feasible route for improving the sensitivity of ECL biosensor. And the DNA-binding protein was realized sensitive detection by the ECL analytical techniques for the first time. Alkali metal K doped graphene could not only well maintain the two-dimensional structure of graphene morphology with the high surface/volume ratio but also enhance the rate of electron transfer. Meanwhile, SiO2 nanopraticals was employed to load large number of CdS NCs with the aim to amplify the ECL signal. However, the enhanced ECL could be greatly suppressed by the binding of target protein. The prepared biosensor possesses excellent analytical performance. Sensitive detection of the sequence-specific DNA-binding proteins are essential to elucidate gene expression mechanisms and the development of new diagnostics of disease states.3. Gold nanoparticle enhanced electrochemiluminescence of CdS thin films for ultrasensitive thrombin detectionThe ECL of CdS thin films had been greatly enhanced by gold nanoparticles (Au NPs) for ultrasensitive detection of thrombin. The system was composed of a CdS nanocrystals (NCs) film on glassy carbon electrode (GCE) as ECL emitter attached an aptamer of thrombin. Then ssDNA-AuNP conjugates hybridized with the aptamer to form a separation length of ca. 12 nm between CdS NCs and Au NPs. The system showed 5-fold enhancement of ECL intensity as compared to that without Au NPs,which might be attributed to the long-distance interaction between the S-NCs and surface plasmons resonance (SPR)-field of noble metal nanoparticles (MNPs).We also found that the enhanced ECL could be influenced by the involving factors such as the separation distance, spectral overlap and magnetic field. Such enhancement in combination with smart recognition of aptamer and target protein allowed us to construct an ultrasensitive aptasensor for attomolar detection of thrombin. The presence of target protein was reflected by the ECL signal decrease caused by the target-induced removal of ssDNA-AuNP conjugates. The principle described in this work could be also applied to many other bioassays.4. Amplified electrochemiluminescence detection of DNA-binding protein based on the synergy effect of electron and energy transfer between CdS nanocrystals and gold nanoparticlesIn an electrochemiluminescence (ECL) system involving the CdS NCs and Au NPs, we firstly enunciated the presence of synergy effect between the electron and energy transfer,based on which an amplified ECL biosensor was constructed for sensitive detection of DNA-protein interactions. Specifically, Au NPs modified on electrode, following by assembled probe DNA. Then the modified GCE was hybridized with SiO2@CdS/DNA conjugates. With DNA duplex as a rigid spacer, Au NPs could not only accelerate the electron transfer but also produce the surface plasma resonance upon excited ECL of CdS NCs. However, the enhanced ECL could be greatly suppressed by the binding of target protein. The prepared biosensor possesses excellent analytical performance with the linear range of target protein from 0.015 to 150 nM.5. Study on the anodic ECL properties of CdSe:Co NCs and their application in detection of alkaline phosphataseCo2+ doped CdSe:Co NCs with a series of different doping amount of Co2+ were synthesized via co-precipitation method in water. XRD, TEM, HRTEM and EDS were used to characterize the prepared NCs materials, and UV-vis, PL and ECL were also use to characterize the optical properties of CdSe:Co NCs. Specifically, the anodic ECL emission of CdSe:Co were discussed, the result showed that in the presence of co-reactant TPrA, the ECL intensity reached the maximum when the doping percent was 2%, resulting in a 2.8-fold ECL enhancement compared to pure CdSe NCs.Hence, this work could provide new NCs opportunities in anodic ECL bioanalysis and biosensing applications. Alkaline phosphatase (ALP) triggered hydrolyzation of phenyl phosphate disodium (PPNa) to phenol would induce the formation of phenolic polymer and the subsequent ECL inhibition,based on which the sensitive detection of ALP was realized, and the linear range was from 0.5 nM to 10 nM.