Dark Field Microscopic Imaging Analysis At Single Nanoparticle Level:Sensitivity And Precision Improvement And The Application At Multi-target Detection

Owing to the unique oblique illumination,there are many advantages of the dark-field microscopic imaging technique,including the dark background,the high signal-to-noise and high spatial resolution.As an important branch of plasmonic nanoprobes,noble nanoparticles have some excellent optical properties,such as strong,stable,and tunable light signals and the convenience to modification.The combination of single noble nanoparticles and dark-field microscopic imaging technique results in dark-field microscopic imaging analysis at single nanoparticle level.Compared with traditional technique,dark-field microscopic imaging analysis at single nanoparticle level has some advantages,including high sensitivity,high spatial resolution and accurate localized information,enabling its applications in monitoring some reactions and spatial resolution at single nanoparticle level.However,there are still some limitations,such as the insufficient study on the localized surface plasmon resonance of single nanoparticles,the low accuracy of the dark-field microscopic imaging and the deficiency of multi-target determination.To address these limitations,we carried out researches to improve the imaging performance,which are summarized as follows:1.The influence of aspect ratios of single gold nanorods for the sensitivity of light scattering analysisFirst,gold nanoparticles(AuNPs)with different multiple shapes were synthesized.Dark-field microscope and scanning electron microscope were combined to determine the relationship between the shape and the scattering light color.The results displayed that gold nanospheres or nanocubes scattered green light,while gold ellipsoids scattered yellow light.Gold nanorods(Au NRs)scattered red light and the scattering spectra of single Au NRs shifted towards to longer wavelength as the aspect ratio increased.When these nanoparticles were put into solvent with different refractive index,it was found that the refractive index sensitivities of single Au NRs with different aspect ratios were different,and the refractive index sensitivities got higher with increasing aspect ratio.These results are important for further designing highly sensitive nanobiosensors and improving the detection sensitivity.2.Precision improvement in dark-field microscopic imaging by using gold nanoparticles as internal referenceThere are some large deviations caused by manual manipulation in dark-field microscopic imaging.To calibrate these deviations,internal reference(IR)was introduced,and the large deviations in the achieved scattered light signals from the probes can be regulated by the average value of the scattered light signals from the IR.The bare AuNPs were used as the IR,and silver nanoparticles(AgNPs)and the functionalized AuNPs were used as nanoprobes.First,we investigate the feasibility for introducing AuNPs as the IR.The results showed that the ratio of the scattering light intensity of blue light to that of green light obtained from the same region at different captures was constant without chemical reactions or physical changes occur and factors,indicating that the introduction of the bare AuNPs as the IR in dark-field microscopic imaging technique is feasible and reliable.Next,the improved precision by using AuNPs as the IR has been identified by three experiments: monitoring the oxidation process of AgNPs in air at room temperature,quantifying the level of glucose based on the hydrogen peroxide(H2O2)induced reduced light intensity of AgNPs,and quantifying the change in the light intensity of AuNPs after the plasmon resonance enery transfer(PRET)between AuNPs and TAMRA in the presence of the unavoidable errors.It can be concluded that the precision of the scattered light signals of nanoprobes under dark-field microscope can be greatly improved by utilizing the bare AuNPs as the IR.3.The influence of the distance between gold nanoparticles and dye to the efficiency of the plasmon resonance enery transferThe IR was further applied in the study of the PRET.The light scattering peak of AuNPs with the diameter of 50 nm is located at 550 nm,and TAMRA or Cy3 has strong absorbance at about 550 nm.When the distance between AuNPs and the dye is close enough,PRET between them will occur during which AuNPs are the donor and TAMRA or Cy3 is the acceptor.In our work,AuNPs and the dye were linked by the double stranded DNA(dsDNA),and the distance between the donor and the acceptor can be tuned by altering the length of dsDNA.The efficiency of PRET between AuNPs and the dye with different distance can be achieved by comparing the scattering light intensity of AuNPs before and after the occurrence of PRET.The finite difference time domain(FDTD)and the experimental results showed that the distance between the donor and the acceptor had important influences on the efficiency of PRET,and the smaller distance had the higher efficiency.4.Dual probes with different scattering light colors for the simultaneous quantification of dual cancer biomarkersThe coating-anti-AFP and coating-anti-CEA were mixed at first and immobilized on the glass slides.After the binding of AFP and CEA to these antibodies,the slides would specifically attach labeling-anti-AFP-modified AuNPs and labeling-anti-CEA-modified AgNPs by forming the sandwich-type structure.Since the anti-AFP-modified spherical single AuNPs and anti-CEA-modified spherical single AgNPs scatter green and blue light under dark-field microscopic system,respectively,the level of AFP and CEA can be easily quantified by counting the green and blue light spots separately,and the linearship was in the range of 0.5-100 ng/m L.The mutual interference between the detection process of AFP and CEA in the dual detection was investigated,and the negligible interference was found when the concentration of the antigen was in the range of 0.5-10 ng/m L.Furthermore,we believe this simple strategy can be applied for simultaneous detection of multiple targets with high sensitivity by using multiple probes with different scattering light colors.In summary,this thesis mainly focused on the improvement of dark-field microscopic imaging analysis from the sensitivity,the precision,and the single target determination,which is significant to develop more simple and sensitive analytical detecting methods.In addition,the theory related to PRET,which is the important branch of dark-field microscopic imaging analysis,has also been investigated.