Fabrication Of Sers Probes Based On Gold Nanostars And The Application In Cell Detection

Raman spectroscopy is a non-invasive technology with fingerprint identification, which can be able to obtain rich structural information of samples at the molecular level. Surface enhanced Raman scattering (SERS) is a unique optical enhancement based principally on the rough surface in nanoscale. Compared with conventional Raman scattering, SERS can enhance the Raman signal of molecules up to 102～106 orders of magnitude, which has higher sensitivity for detection. SERS has many significant advantages over traditional technologies, such as its high stability, its resistance to photo bleaching and weak Raman signal of water. So it is suitable for studying biological sample that contains water. Au nanostars (AuNSs) are a novel nanomaterial, containing a small core size and multiple long thin branches. AuNSs not only have plasmon bands that are tunable in the NIR tissue optical window but also have multiple sharp branches acting as "hot-spots" for localized electromagnetic field enhancement. In this paper, SERS tags and label-free SERS probes based on AuNSs were prepared for cellular detection. The details are as follows:1. AuNSs were synthesized with a seed-mediated growth approach. The length and size of the branches of AuNSs can be controlled by adjusting the ratio of HAuCl4, hydroquinone and gold seeds in the reaction system. The morphology, optical properties and SERS effect of AuNSs were characterized using scanning electron microscope (SEM), UV-vis-NIR spectrophotometer and confocal Raman spectrometer, respectively. AuNSs with well monodispersity, homogeneous and excellent SERS enhancement effect were synthesized by the optimized reaction conditions.2. The SERS-active tags based on AuNSs were constructed for cellular imaging and sensing. Nile blue A (NBA) and bovine serum albumin (BSA) were used as Raman reporter molecules and capping materials, respectively. The reliable aggregation test and CCK8 assay indicated a high chemical stability and a low cytotoxicity for the SERS tags, respectively. The prepared SERS tags were used for SERS imaging of human lung adenocarcinoma cell line (A549) and human alveolar type Ⅱ epithelial cell line (ATII). The majority of SERS tags appeared to be localized in the cytoplasm, and few SERS tags were observed inside the nucleus. The cells loaded SERS tags exhibited much stronger SERS signals than the cells unloaded SERS tags. The dark-field imaging and TEM of cells incubated with SERS tags were consistent with the previous results of SERS measurement.3. This work presents novel SERS tags for targeting cancer cells overexpressing epidermal growth factor receptor (EGFR). In such a targeting SERS tag,4-mercaptobenzoic acid (4-MBA) was served as the Raman reporter to generate SERS signals as well as the conjugation agent for attaching anti-EGFR molecules to AuNSs. The targeting SERS tags were used for distinguishing human lung cancer cells (A549, H1229) from human bronchial epithelial cell (BEAS-2B). A549 cells and H1229 cells incubated with targeting SERS tags exhibited much stronger SERS signals than those of BEAS-2B cells, indicating that more SERS tags were targeted to lung cancer cells. The dark-field imaging and energy-dispersed spectrum (EDS) further confirmed that anti-EGFR conjugated AuNSs could be used as an efficient cancer cell targeting agent.4. The transactivator of transcription (TAT) peptide-conjugated AuNSs were used as label-free SERS probes for distinguishing the differentiation of lung resident mesenchymal stem cells (LR-MSCs). The stability, biocompatibility and cellular uptake of TAT-functionalized SERS probes were studied. The average SERS spectra of LR-MSCs during their differentiation into alveolar type Ⅱ epithelial cell line (ATII) and fibroblasts (3T3) were measured, respectively. Finally, SERS spectra were analyzed using principal component analysis (PCA), which had been demonstrated to be a highly sensitive method for distinguishing and identifying the different subtypes of LR-MSCs.5. By using TAT-conjugated AuNSs as label-free SERS probes, the bleomycin (BLM)-induced epithelial-mesenchymal transition (EMT) process in ATII cells was characterized by SERS spectroscopy. The expression of epithelial and fibroblastic markers on the cells was detected by immunofluorescence and protein immunoblot, which demonstrated that ATII cells incubated with BLM, could differentiate into 3T3 cells. SERS spectra of the cells at different stages of EMT could be acquired. The differences in SERS spectra among different stages revealed the modulation of cellular biochemical components during the EMT process. PCA plots indicated that the SERS spectra of ATII cells at different stages of EMT were distinguished into four disparate groups. The different types of cells were well separated from each other based on their SERS spectra.