SERS Based Analysis Of Cell Viability

Cell viability analysis plays an essential role in the research fields of cytology and biomedicine.Cell viability analysis is not only useful for effectively evaluating basic survival capacity of biological organisms and metabolic activities,but also reflect the effect of the environmental variations on cells,which provides meaningful references for the study of complicated diseases in the real life,especially in the case of tumor diseases.Among the approaches for cell viability analysis,immunohistochemical analytical method due to its advantage of specificity,has been widely used in the fields of both fundamental studies and clinical medicine.It shows great potential in a variety of applications including clinical diagnosis and treatment of tumors,auxiliary examination of immunological diseases,and examination of pathogenic microorganism.Surface-enhanced Raman scattering(SERS)can provide abundant fingerprint information of target molecules with high selectivity and sensitivity,which can avoid false-negative and false-positive results caused by environment or other factors like color.In this thesis,based on the major problems in the current analytical methods for cell viability,we developed SERS-based analytical approaches for cell viability.The main contents are as follows: 1.SERS-based 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)assay for cell viabilityOn the basis of the traditional MTT assay,we developed a SERS-based MTT assay for cell viability by employing SERS detection instead of the colorimetric analysis,which was used for viability analysis of MDA-MB231 cells from human breast cancer.According to the experimental procedure,MTT molecule was firstly added into the 96-well plates coated by the target cells,resulting in the reduction of MTT to formazan catalyzed by the dehydrogenase in active cells.After dissolved in DMSO,formazan was mixed with gold(Au)colloid and the SERS spectra were collected.We found that the SERS intensities of formazan increased with the increase of the living cell numbers,which demonstrated the possibility of SERS for cell viability analysis.Compared to the traditional methods for cell viability analysis,the SERS-based MTT analytical method displayed a 1000-fold higher sensitivity for formazan,and 5-fold higher sensitivity for human breast cancer cells(MDA-MB231).Moreover,the SERS intensities of the probe molecule had a linear relationship with the cell numbers(Y=1.66X+0.01 R2=0.99),which showed the potential application of the present method for viability analysis of single cell.Furthermore,we shortened the detection time from 4 hour to 0.5 hour,and simplified the detection steps,which significantly increased the detection efficiency.Thus,the proposed SERS-based approach has combined rapidness and sensitivity,providing a new idea for cell viability analysis.2.SERS-based 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4- disulfophenyl)-2H-tetrazolium,monosodium salt(WST-8)assay for cell viabilityMTT and formazan molecules are insoluble in water,and the organic solvent DMSO will inactivate the cells,leading to the difficulty in monitoring the changes of its biological function,which thus limits the application of this method.In this part,we chose a highly water-soluble molecule WST-8 as a probe instead of MTT for cell viability analysis.WST-8 shows the same property as MTT and the reduced product WST-8 formazan molecule is also water-soluble.After the WST-8 was catalyzed by the dehydrogenase,the product WST-8 formazan was mixed with silver(Ag)nanoparticles,SERS spectra of the formazan molecules was observed and their intensities displayed a good positive correlation with cell numbers(Y=2.34X+1151.4 R2=0.998).This method exhibits obvious advantages: the WST-8 and WST-8 formazan is water-soluble molecule,which avoid the negative effect of the organic solvent on cells.Thus the cells after SERS spectrum collection can be re-cultured for other related experiments.In addition,compared to the traditional WST-8 analytical method,the SERS-based approach can effectively avoid the false-positive and false-negative results caused by visual errors.We found that the detection limits of this method for the WST-8 formazan molecule can also reach 0.1m M,and for the number of MDA-MB231 cells are 50.3.SERS-based immunohistochemical analysisWe combined the basic theory of SERS and immunohistochemistry,and developed a SERS-based immunohistochemical method for location and quantitatively analysis of the targets in rat's neuron cells and histological sections of rat's brain tissues.We prepared an immune SERS-active probe with the MBA molecules and goat anti rabbit immunoglobulin adsorbed on the surface of Au nanoparticles.The immune SERS-active probe was used to recognize the rabbit immunoglobulin assembled on the glass substrate through the immune interaction between antigens and antibodies.The quantitative analysis of rabbit immunoglobulin was realized by the SERS intensity of the MBA molecules.This result proved the feasibility of the SERS-based immune detection.Subsequently,this method was combined with immunohistochemical ABC analysis method and a SERS-active probe was fabricated with MBA molecules and NF-200 antibodies adsorbed on the Au nanoparticle surface.This immune SERS-active probe was utilized in the analysis of NF-200 protein in rat's neuron cells and histological sections of rat's brain tissues.We found that the immune SERS-active probe can recognize the NF-200 protein through the immune interaction.After the SERS spectrum collection,the neuron cell viability and the quantitative analysis of the NF-200 protein can be realized by building the relationship between the SERS intensities of the MBA molecules and the NF-200 concentrations.Compared with the traditional methods,the SERS-based detection method increased the detection sensitivity(about 2-fold higher than the traditional method)and analysis efficiency(omitting the procedure of second antibody addition).Moreover,the SERS probe MBA molecules had nonspecific affinity to biomolecules in cells or tissues,avoiding false-positive results,and SERS measurements instead of visual examination by naked eyes and microscopy allowed quantitative analysis of cell viability.4.Hollow Au-Ag hybrid core-shell SERS substrate for the immunohistochemical analysisWe synthesized hollow Au-Ag hybrid core-shell nanoparticles with different Ag shell thickness as SERS substrates,and utilized them in immunohistochemical analysis.The enhancement ability of the as prepared hollow Au-Ag hybrid core-shell substrate is 5 times higher than the traditional Ag nanoparticles and 20 times higher than the Au nanoparticles.The human immunoglobulin antibodies and the MBA molecules were labeled on the metal shell surface and they were then used to recognize the human immunoglobulin.After collecting the SERS spectra of the MBA adsorbed on the shell surface,the quantitative analysis of was realized by the SERS intensities of the MBA molecules with the change of the protein concentration.The detection limit can reach 50 ng/ml.Furthermore,we combined this detection method with immunohistochemical analysis.We prepared a 3,3'-diethylthiatricarbocyanine perchlorate(DTTC)labeled hollow Au-Ag hybrid core-shell SERS-active probe and used this probe in the detection of insulin in histological sections of a rat's islet tissue.The SERS spectra of DTTC can also probe the location of the ? cells in the rat's islet tissue.