Synthesis Of Novel Surface-enhanced Raman Scattering (SERS) Tags And Their Application In Single-Cell Imaging

Surface-enhanced Raman scattering (SERS) probes have attracted considerable attention as an emerging class of biolabels in cellular imaging. One reason for this is that SERS provides a 106-1014 fold enhancement of the Raman signal intensity, which is sufficient even for single molecule detection. Secondly, the extremely short scattering times of the Raman process prevent photo-bleaching, energy transfer, or quenching of reporters in the excited state, resulting in a high photostability of the SERS probes. Thirdly, an optimal contrast can be achieved by using red to near-infrared (NIR) excitation to minimize the disturbing autofluorescence of cells and tissues, making SERS an important tool for noninvasive imaging in living subjects. And finally, SERS tags yield much narrower peaks (typically< 2 nm) in the resulting spectral response and thus offer the potential to access an increased number (about 10-100) of unique optical'signatures'by varying the Raman reporter molecules, which make them ideally suited for multiplex detection or multicolour imaging. Except for label imaging, a potentially label-free imaging method based on strong SERS signals from the native chemical constituents of a cell was developed by incorporating colloidal noble metal (Au or Ag) NPs into a cell, localizing them on subcellular organelles or bio-macromolecules. Therefore, unlike fluorescence microscopy, this type of SERS microscopy provides a tool for the sensitive and structurally selective detection of native chemicals inside a cell and their intracellular distribution, suggesting many applications in biomedical resea.Based on the above advantage of SERS probes, we carried out the following reaearch work used laser confocal Raman microscopy in the field of single-cell imaging:1. The preparation of multi-targeting SERS tags and their application for triplex three-dimensional cellular imagingWe proposed a simple and wide applicability SERS tags synthesis method, due to the effect of encapsulation of the polypropylene amine (PAH), the choice of SERS signal molecules will have great randomness. Furthermore, a large number of -NH2 group of PAH is advantageous to generate stable amide bond with the carboxyl group by EDC reacts. In this way, three kinds of organelles targeting labelled surface-enhanced Raman scattering (SERS) tags were prepared and applied for simultaneous and three-dimensional imaging of double organelles (nucleus and membrane) in single HeLa cells. The multiplex three-dimensional SERS imaging technique allows for both temporal (real time) and spatial (multiple organelles and molecules in three-dimensional space) live-cell imaging and therefore provides a new and attractive 2D/3D tracing method in biomedicine on subcellular level.2. Multi-targeting membrane/nucleus SERS tags for direct and indirect detection research during cell apoptosisTo in vivo, in situ and real time obtain the complex information including the compositions and structures of cells, multi-molecular location and their interaction during the physical or pathological processof cells play an important role on the explanation of several subjects in cell biology and the investigation of the early diagnosis and pathogenesis of many serious diseases. Here, we demonstrate that the combination of labelled and label-free SERS tags enable us to capture the dynamic chemical information of nuclei and to display the real-time location of the FA receptors and the receptors for LHRH on the membrane with very good height resolution. Our results demonstrate the novel SERS-based single cell analysis approach offers powerful strategies for analyzing the complex physiological.3. Alkynyl-coded SERS tags for interference-free triplex cellular imagingThe alkyne moiety is an excellent Raman tag for bioimaging due to its unique interference-free Raman signals in a cellular Raman-silent region (1800-2800 cm-1). Unfortunately, the extremely weak spontaneous Raman scattering of alkyne-tag hinders its further application in the field of biochemical labels. Herein, we developed a narrow emission, band shift-tunable and ultra-sensitive alkynl-modulated SERS palette through combining the advantages of alkyne-tags and SERS effect, which was successfully applied to triplex-targeted and simultaneous cellular imaging without interference. Due to its super sensitivity and minimal perturbation, the alkynyl-modulated SERS-palette is of great significance for multiplex sensing and multi-color biological imaging when the hyperspectral and fairly intense optical noises originating from lower wave number region (<1800 cm-1) are inevitable under complex ambient conditions.4. The application of SERS tags in the study of plant cell imagingAs a supplementary application of SERS tags in the field of plant cell imaging, the SERS tags modified with CALNNR8 peptide chain was preparaed and applied for bioimaging study of BY2 tobacco cells and lily pollen cells. The results confirmed that there is a big difference in morphology struture of cell walls between BY2 tobacco cells and lily pollen cells. After studying the composotion of pollen wall utilizing resonance Raman spectrum, we found the reticalar rigid structure composed of flavonoids compound have the effect of the resistane to environmental stress;and the carotenoids filled the gap in the reticalar struture maybe help pollen against microbial attack and cell recognition during pollination.