Surface-enhanced Raman scattering (SERS) continues to strive to gather molecular level information from dynamic biological systems. It is our ongoing effort to utilize the technique for understanding of the biomolecular processes in living systems such as eukaryotic and prokaryotic cells. In this study, the technique is investigated to identify cell death mechanisms in 2D and 3D in vitro cell culture models, which is a very important process in tissue engineering and pharmaceutical applications. Second, in situ biofilm formation monitoring is investigated to understand how microorganisms respond to the environmental stimuli, which inferred information can be used to interfere with biofilm formation and fight against their pathogenic activity.
There is an ongoing effort to obtain molecular level information from living cells using surface-enhanced Raman scattering (SERS) not only to understand changes of cellular processes upon exposure to external stimuli but also to decide the status of cells; whether they are healthy or abnormal. In our research effort, we investigate how much information can be obtained from living cells to use for decision making about the cellular processes using SERS. The undertaken studies include cytotoxicity assessment of the nanomaterials and differentiation of the healthy and cancer cells. In the first case, A549 (lung cancer) and HDF (human dermal fibroblast) cells were incubated with 50 nm gold nanoparticles (AuNP) and exposed to three different nanoparticles (Zinc oxide nanoparticles (ZnO NPs), titanium dioxide nanoparticles (TiO2) and single walled carbon nanotubes (SWCNTs)) to perform SERS analysis and track the cellular response to these nanomaterials (NMs). After the principal component analysis on the spectral data, it was shown that the NPs exposed samples could be differentiated through SERS. In the second case, SERS spectra obtained from human kidney adenocarcinoma (ACHN), human kidney carcinoma (A-498) and non-cancerous human kidney embryonic cells (HEK 293) were used to diagnose metastatic, primary and non-cancerous cell lines. Linear discriminant analysis (LDA) based on principal component analysis (PCA) was applied to collected multidimensional SERS spectral data set to differentiate three different cell lines.
The detection of kidney cancers at an early stage is critical for diagnosis and therapy. Surface-enhanced Raman scattering (SERS) is investigated for early detection of cancer cases from biopsy samples. The colloidal silver nanoparticles as the SERS-active nanostructures are directly mixed with homogenized tissue samples. The SERS spectra from the normal and abnormal tissue samples collected from 40 cancer patients, 28 of them at T1 stage and 12 of them at T2–T3 stages, are analyzed using principal component analysis combined linear discriminant analysis with leave-one-out cross-validation method. It is found that the diagnosis sensitivity, specificity, and total accuracy of the approach can be as high as 100%. The results suggest that SERS can be used as a potential technique for the identification of the different tumor stages.
The multiplex detection of biologically important molecules such as proteins in complex mixtures has critical importance not only in disease diagnosis but also in other fields such as proteomics and biotechnology. Surface-enhanced Raman scattering (SERS) is a powerful technique for multiplex identification of molecular components in a mixture. We combined the multiplexing power of SERS and heat denaturation of proteins to identify proteins in ternary protein mixtures. The heat denaturation profiles of four model blood proteins, transferrin, human serum albumin, fibrinogen, and hemoglobin, were studied with SERS. Then, two ternary mixtures of these four proteins were used to test the feasibility of the approach. It was demonstrated that unique denaturation profiles of each protein could be used for their identification in the mixture.
Surface-enhanced Raman scattering (SERS) is a powerful technique to study the biological molecules and structures.
SERS of proteins is always difficult due to their complex, flexible and diverse structures. This difficulty is one of the
major obstacles hindering the applicability of SERS for the label-free detection and identification. In this study, we have
employed several sample preparation approaches involving the packing AgNPs with protein molecules in a proper
manner to allow the polarization of the electron system of proteins in coherence with the nanostructured noble metal
system. The applicability of heat denaturation kinetics is perused for the detection and identification of proteins in
model protein mixtures. Human serum albumin, transferrin, hemoglobin and the binary mixtures of these proteins are
used as models. We have found that the SERS spectrum of each protein in the protein mixture is rather different at an
increased temperature, which could be used to distinguish a protein in the protein mixture.
Detection and identification of biomacromolecules is of critical importance in many fields ranging from biotechnology
to medicine. Surface-enhanced Raman scattering (SERS) is an emerging technique for the label-free detection and
identification of biological molecules and structures with its fingerprinting properties and high sensitivity. However,
there are a number of obstacles for its applications for biological macromolecules due to their complexity. In this report,
manipulation of microscopic processes in play during the drying of a sessile droplet as a tool to influence the
nanoparticle-macromolecule packing, which has dramatic effect on SERS performance, before the SERS acquisition is
demonstrated. A process known as the coffee ring phenomenon jams all particles and molecular species to the edges of
the droplet during drying. This uncontrolled process has dramatic effects on a SERS experiment, using colloidal metal
nanoparticles as substrates, by sweeping everything to the edges and influencing the packing of nanoparticles in the
droplet area. A plastic tip was dipped into a drying sample droplet to influence the uncontrolled piling up. A negatively-charged
protein, BSA, a positively-charged protein, cytochrom c, and a 20-base long oligonucleotide, were used as
model biomacromolecules in this study. While a minimum of one order of magnitude lower concentration improvement
in detection limit was observed with negatively-charged biomacromolecules, no significant improvement was observed
with positively-charged ones compared to a sample droplet left on the surface without any interference. With the
demonstrated approach, picomolar-level biomolecular detection using SERS is possible.
The development of an assay for the detection of gene mutations has been attempted based on surface-enhanced Raman
scattering (SERS). Using multiplexing property and high sensitivity of SERS technique, the detection of all mutation
possibilities on one given spot is achievable. To test the feasibility of approach, SNPs and other types of mutations such
as insertion and deletion are investigated. The PCR amplified and isolated genomic DNA without PCR amplification is
immobilized on poly-L/D-lysine coated glass surface after denaturing with heating. The SERS probes are prepared by
simultaneous attachment of oligonucleotides complementary to the target mutation regions and Raman active dyes to 13
nm gold nanoparticles (GNPs). After the hybridization of SERS probes on the poly-L/D-lysine surfaces, it was stained
with silver colloidal nanoparticles for further enhancement of Raman scattering. In the second approach, Raman active
dyes are chemically attached on gold nanoparticles and a thin layer of silver film is deposited on top of it to prepare core-shell
nanoparticles. The complementary oligonucleotides to the target regions of the gene are chemically attached to
silver surfaces of the nanoparticles. The promising results indicate that it is possible to detect certain mutation types
without PCR amplification using the approach.
Surface-enhanced Raman scattering (SERS) is a powerful technique for the analysis of a variety of molecules and molecular structures. Due to its great complexity, the acquisition of detailed molecular information from biological organizations such as bacteria is still a challenging task. SERS can provide valuable information once silver or gold surfaces can be brought in close contact with the biological organization. Because several experimental parameters can affect SERS spectra of bacteria, the experimental conditions must be well defined for comparable and reproducible results. The influence of experimental parameters, such as the type of noble metal, size, and aggregation properties of nanoparticles, and the wavelength of the laser light on the SERS of E. coli and B. megaterium are examined. It is demonstrated that the impact of these parameters could be enormous and a standard protocol must be developed depending on the goal of the study.
We describe the use of a biochip based on complementary metal oxide semiconductor (CMOS) technology for detection of specific genetic sequences using molecular beacons (MB) immobilized on solid surfaces as probes. The applicability of this miniature detection system for screening for the BRCA1 gene is evaluated using MB probes, designed especially for the BRCA1 gene. MB probes are immobilized on a zeta-probe membrane by biotin-streptavidin immobilization. Two immobilization strategies are investigated to obtain optimal assay sensitivity. The MB is immobilized by manual spotting on zeta-probe membrane surfaces with the use of a custom-made stamping system. The detection of the BRCA1 gene using an MB probe is successfully demonstrated and expands the use of the CMOS biochip for medical applications.
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