02 Apr 2018
Shachaf C.M. et al A Novel Method for Detection of Phosphorylation in Single Cells by Surface Enhanced Raman Scattering (SERS) using Composite Organic-Inorganic Nanoparticles (COINs). Plos ONE. 4 1-12 (2009).
Kumar.S. et al Multiplex Flow Cytometry Barcoding and Antibody Arrays Identify Surface Antigen Profiles of Primary and Metastatic Colon Cancer Cell Lines. Plos ONE. 8 1-9 (2013).
On February 28, 1928, Chandrashekhara Venkat Raman addressed the South Indian Science Association and spoke about a "new radiation" that he had observed. Using some basic equipments like a telescope objective, a flask of benzene and a direct-vision spectroscope, C.V. Raman discovered a new light scattering phenomena that eventually won him the Nobel Prize in 1930. Normally when light interacts with matter it undergoes elastic scattering or Rayleigh scattering. This means that the scattered light maintains the same energy, frequency and wavelength as that of the incident light. Raman observed that a small fraction of the scattered light is scattered inelastically with a wavelength that is greater that the incident light. The change in wavelength of the scattered light depends in the molecular structure of the matter on which the light is incident. As a result, different objects will display a different Raman signature. This phenomena is termed as the Raman effect and has come a long way over the years to become the fundamental principle driving a wide range of diagnostic and detection techniques. One of the main drawbacks of the Raman effect is that the number of inelastically scattered photons could be as low as 1 for every 10 million elastically scattered photons. This drawback has been over come over the years by the usage of laser light sources which offer a high intensity monochromatic light source along with the discovery of the Surface Enhanced Raman Scattering (SERS) effect. The SERS effect is produced due to the plasmon resonance phenomena shown by small molecules that are adsorbed onto the surface of nano sized noble metals. This treatment increases the electromagnetic field of the incident light dramatically, which in turn produces a Raman effect that is several orders of magnitude higher that what would have been noticed in normal particles. By modifying the Raman active layer encompassing the nano particle, one can derive several different spectral signatures thereby giving us the freedom to investigate the presence of different molecules simultaneously. Along with advancements in instrumentation and image processing techniques, the Raman effect has become an invaluable tool for biomedical researchers.
Another technique that has evolved over the years and has been instrumental for biomedical researchers and diagnosticians to assess various cellular characteristics and functions is flow cytometry. The prefix cyto refers to cell and thhe suffix metry refers to counting. Together flow cytometry refers to a technique where cells are counted or scanned while being carried thorough a fluid stream. Another term that is commonly used for flow cytometry is Fluorescence activated cell scanning or sorting (FACS). As the name suggests, this technique uses fluorescence molecules or fluorochromes to recognize cells and intracellular organelles. An increased availability of monoclonal antibodies to which these fluorochromes can be conjugated has lead to rapid advancements and growth in the field of flow cytometry in recent years. The working principle of flow cytometry is different from that used in Raman spectroscopy. Rather than using the intrinsic property of the molecules under inspection that have a specific Raman signature, flow cytometry depends on the property of the antibody conjugated Fluorochromes. Fluorochromes absorb light at a characteristic wavelength which in turn excites its electrons to a higher energy state. When these electrons come back to their ground state, they release a photon of light that has a wavelength that is characteristic of the fluorochrome and not the cell or organelle under investigation.
This report will seek to compare the above mentioned novel cell and molecular detection techniques by reviewing two primary articles based on the same techniques.
Paper 1: Shachaf C.M. et al A Novel Method for Detection of Phosphorylation in Single Cells by Surface Enhanced Raman Scattering (SERS) using Composite Organic-Inorganic Nanoparticles (COINs). Plos ONE. 4 1-12 (2009).
The primary objective behind the research and experiments conducted by the authors of this article was to overcome some of the drawbacks and limitations of traditional flow cytometry detection techniques. Although fluorescence based techniques have been widely used for detection and diagnostic purposes, there has always been a limitation to the number of parameters that can be simultaneously studied using FACS. This is because of the overlapping spectral signals that is detected from the excited fluorophores. Therefore this paper investigates the utilization of the Raman effect as an alternative detection modality.
As mentioned earlier, native Raman scattered signals given out by objects or particle are extremely weak which eventually led to the discovery and utilization of the SERS effect. Berlin and colleagues (Intel Corporation) coalesced silver nano particles with Raman active organic layer and called them "Composite Organic-Inorganic Nanoparticles" (COINS). The same COIN clusters were found to enhance the Raman effect 104 to 105 fold. The authors further investigated the utility of these SERS based COINS to detect both cell surface epitopes as well as intracellular phospho-epitopes, thereby also investigating the efficacy of the COINS to be used as nano tags for immunophenotyping. Two different COINS were fabricated for the purpose of this experiment: the Acridine Orange (AOH COIN) and the Basic Fuchsin (BFU COIN). The peak Raman intensities for different sizes of the above mention COINs were measured and it was determined that optimal intensity of the Raman peaks were observed for COIN sizes of 60 for AOH COINS and 52 for BFU COINS. An automated Raman scanner sensitive enough to measure spectral shifts as little as 30nm was developed by Intel Corporation (Intel Raman BioAnalyser - IRBA) in order to facilitate the detection of the Raman signals for cellular analysis.
Detection of cell surface antigens using Raman Coins:
The efficacy of both AOH as well as BFU COINS to be conjugated to antibodies were first determined using an IL-8 ELISA sandwich assay and were subsequently deemed suitable for use in other biological assays. The U937 cell line which is a monocytic leukemia cell line with high ICAM-1 (CD54 adhesion molecule) was used to determine the utility of the COINS to detect cell surface protein expression. The COINs were conjugated with the CD54 antibodies and utilized to detect the CD54 antigen in the U937 cell line using ELISA. A linear regression analysis of the COIN signal against the antigen concentration revealed a correlation coefficients of 0.8-0.99 thereby ruling in favor of the COINs to be used for antigen detection on the cell surface. The optimal concentration of 0.25mM was determined at which both the AOH as well as the BFU COINs conjugated with αCD54 antibody exhibited high specific reactivity to the CD54 antigens on the U937 cell surface. The specificity of the binding of the COINs to CD54 antibodies were determined by using H82 small cell lung cancer (SCLC) cells since the SCLC do not express CD54. Similar results were obtained for both AOH and BFU COINs where specific binding was observed for the U937 cells but not the H82 cells. Scanning Electron Microscopy (SEM) images revealed that the COINs formed clusters on the apex of the U937 cells which also happens to be the characteristic location of the expression of CD54. Thus the localization of the COINs on the cell surface was also determined. Finally the immunostaining analysis was carried out using the IRBA. It was found that the peak height of the Raman intensity differed with respect to the type of COIN used, but the Raman peak height ratio for the antibody conjugated COIN to the non conjugated COIN was observed to be similar in case of both the AOH and the BFU COIN. Thus the utility of the SERS based COINs to carry out accurate immunostaining of a malignant cell line was demonstrated and established by the authors.
Paper 2: Kumar.S. et al Multiplex Flow Cytometry Barcoding and Antibody Arrays Identify Surface Antigen Profiles of Primary and Metastatic Colon Cancer Cell Lines. Plos ONE. 8 1-9 (2013).
The primary objective behind the research conducted by the authors of this paper is to identify cell surface antigen signatures in primary and metastatic colon cancer cell lines by combining fluorescent cell barcoding technique along with a high throughput flow cytometry profiling. The efficacy of Tumor-associated antigens (TAA) for the detection of Circulation tumor cells (CTC) as well as Disseminated tumor cells (DTC) has been well established. For the purpose of this research, two primary (SW480, HCT116) and one metastatic (SW620) colon cancer cells lines extracted from a single patient were investigated upon. The fluorescent barcoding of the three mentioned colon cancer cell lines was carried out using the VPD450 fluorochrome (SW480) and the CSFE fluorochrome (HCT116) dyes. The cell line SW620 was left unlabeled. The barcoded cells were then added into a pool of 242 antibodies and 9 isotype controls and distributed across three 96-wall plates for flow cytometry analysis. The high throughput flow cytometry analysis revealed that only 25 of these antibodies reacted with more that 50% of the three cell lines. These TAA signatures were cross compared to the Oncomine database of gene expression of these antibodies for colon cancer. This enabled the authors to further narrow their antigen list to over expressed genes CD44, integrin α6 (CD49f) and integrin β2 (CD49b). Further on, multi color flow cytometry using EpCAM, CD133 and CD44 were also performed and Cancer stem cell immunophenotypes with varied expressions were also detected. Therefore this multiplexed technique allowed for a much more comprehensive screening for Tumor associated antigens at a much faster rate and at lower cost.
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