Metal Nanoparticles Initiated Chemiluminescence Reactions And Their Applications In Immunoassays

In this dissertation,the state of arts in the field of chemiluminescence(CL), chemiluminescence immunoassays,the synthesis and properties of metal nano-materials and their applications in immunoassays were reviewed.Although the theory of CL and its application in immunoassay have been investigated for many years,the study of CL was limited to molecular and ion systems.Recently,metal nanoparticle-involved CL has attracted great attentions,in which metal nanoparticles can participate in CL reactions as catalyst,reductant,naonosized platform and energy acceptor.However,most of the reported CL systems involving nanoparticles were of low quantum yield,limiting there applications in analysis.Meanwhile,the study of nanoparticle-controlled CL system has been rarely reported.On the other hand,with the advance of nanoparticle-involved CL,chemiluminescence immunoassays based on metal nanoparticle labels were developed.Most of these reported chemiluminescence immunoassays contained "stripping" process,in which strict conditions were required to dissolve metal nanoparticles,making the method complicated,limiting there practical application.There were few reports about non-stripping chemiluminescence immunoassays based on metal nanoparticle-catalyzed CL reactions.Therefore,in the present dissertation,gold and platinum nanoparticles were utilized to design new CL systems.The nanoparticle-dependent or-controlled CL behaviors,rules and mechanisms of these new CL systems were investigated.Moreover,based on the CL activity of gold nanoparticles,new methods for non-stripping chemiluminescence immunoassays were developed.The main results are as follows:1.It was found that gold nanoparticles of small size(＜5 nm) could inhibit the chemiluminescence(CL) of the luminol-ferricyanide system,whereas gold nanoparticles of large size(＞10 nm) could enhance this CL,and the most intensive CL signals were obtained with 25-nm-diameter gold nanoparticles.The luminophor was identified as the excited-state 3-aminophthalate anion(Ap^2-).The studies of UV-visible spectra,CL spectra,X-ray photoelectron spectra(XPS),effects of concentrations of luminol and ferricyanide solution,and fluorescence quenching efficiency of gold colloids were carried out to explore the CL inhibition and enhancement mechanism.The CL inhibition by gold nanoparticles of small size was supposed to originate from the competitive consumption of ferricyanide by gold nanoparticles and the relatively high quenching efficiency of the luminophor by gold nanoparticles.In contrast,the CL enhancement by gold nanoparticles of large size was ascribed to the catalysis of gold nanoparticles in the electron-transfer process during the luminol CL reaction and the relatively low quenching efficiency of the luminophor by gold nanoparticles.This work demonstrates that gold nanoparticles have the size-dependent inhibition and enhancement in the CL reaction,proposing a perspective for the investigation of new and efficient nanosized inhibitors and enhancers in CL reactions for analytical purposes.2.An intensive and delayed CL could be observed from the alkaline lucigenin solution in the presence of ethanol and Pt nanopartieles simultaneously.The "induction" period of the CL was largely dependent on the concentration of Pt nanoparticles.The time from injection to the occurrence of the maximum CL intensity, which defined as T_p here,was exponentially related to the concentration of Pt nanoparticles;while the maximum CL intensity was linear with Pt nanoparticles concentration.The products and luminophor of lucigenin CL reaction were characterized by UV-visible spectra,fluorescence spectra and CL spectra.The luminophor of this system was identified as the excited-state N-methylacridone (NMA).The variation of the concentration of lucigenin and its CL product NMA during the process was monitored in situ.The results showed that the reaction of lucigenin and NaOH involving Pt nanoparticles and ethanol was autocatalytic.The studies of transmission electron microscope(TEM),XPS and nuclear magnetic resonance(NMR) were carried out to explore the mechanism of the autocatalytic CL reaction.The results suggested that Pt nanoparticles acted as a "general" catalyst in the CL system that not only catalyzed the oxidation of ethanol to acetaldehyde,but also catalyzed the CL enhancement by acetaldehyde.Ethanol in this system was the initiator for catalysis.Similar to ethanol,other alcohols such as methanol,ethylene glycol and n-propanol could also initiate autocatalytic lucigenin CL in the presence of Pt nanoparticles.This is the first time autocatalytic light emission has been obtained from nanoparticles-involved CL reactions.The strong light emission in this system may find future applications in bioanalysis and fabrication of"cold light sources".3.A novel microplate-compatible chemiluminescence immunoassay has been developed for the determination of human immunoglobulin G(IgG) based on the luminol-AgNO₃-gold nanoparticles CL system.Polystyrene microtiter plates were used for both immunoreactions and CL measurements.The primary antibody, goat-anti-human IgG,was first immobilized on polystyrene microwells.Then the antigen(human IgG) and the gold-labeled second antibody were connected to the microwells successively to form a sandwich-type immunocomplex.The gold label could trigger the reaction between luminol and AgNO₃,accompanied by light emission.A flocculation test was carried out to determine the minimum amount of goat-anti-human IgG required to form stable nanoparticle-protein bioconjugate.Some experimental parameters influencing the CL reaction,including luminol pH and the concentration of luminol and AgNO₃,were optimized.Under the optimized conditions, the CL intensity of the system was linear with the logarithm of the concentration of human IgG in the range from 25 to 5000 ng/mL,with a detection limit of 12.8 ng/mL (～80 pM) at a signal to noise ratio of three(S/N = 3).Compared with other reported CL immunoassay method based on gold labels,the proposed CL protocol avoids a strict stripping procedure or difficult to control synthesis processes,making the method more simple,time-saving and easily automated.The present CL method is promising for the determination of clinically important bioactive analytes.4.A new non-stripping and microplate-compatible chemiluminescence immunoassay protocol based on gold label with silver staining has been developed for the determination of human IgG.The primary antibody,goat-anti-human IgG,was first immobilized on polystyrene microwells,and then the antigen(human IgG) and the gold-labeled second antibody were captured onto the microwells successively to form a sandwich-type immunocomplex.The gold label could catalyze the reduction of silver acetate by hydroquinone,leading to the deposition of silver on the gold nanoparticles,followed by direct CL detection using luminol-H₂O₂ system.The effect of staining time on the CL response was carefully studied.With different staining time, both enhanced and inhibited CL detention modes were established for the CL immunoassay.Some CL conditions in both modes,including the buffer media of luminol,luminol pH and the concentration of luminol and H₂O₂,were optimized. Under the optimized conditions,the detection limit of this CL method(S/N = 3) was 0.9 ng/mL for enhanced mode,and 5.6 ng/mL for inhibited mode,respectively.The sensitivity of the enhanced mode was higher than other reported non-stripping CL immunoassay methods.This work revealed that the staining of silver on the gold nanoparticles was useful to improve the sensitivity.This is the first time to imply gold-label-silver-staining technology to develop non-stripping CL immunoassays.The proposed CL protocol avoids strict stripping procedure or the synthesis of irregular gold nanoparticles.However,there are still some disadvantages,such as the narrow linear range,and the further improvement required for the sensitivity and the stability.