Surekha Barkur

A Micro-Raman Study of Live, Single Red Blood Cells (RBCs) Treated with AgNO3 Nanoparticles

Silver nanoparticles (Ag NPs) are known to exhibit broad antimicrobial activity. However, such activity continues to raise concerns in the context of the interaction of such NPs with biomolecules. In a physiological environment NPs interact with individual biological cells either by penetrating through the cell membrane or by adhering to the membrane. We have explored the interaction of Ag NPs with single optically-trapped, live erythrocytes (red blood cells, RBCs) using Raman Tweezers spectroscopy. Our experiments reveal that Ag NPs induce modifications within an RBC that appear to be irreversible. In particular we are able to identify that the heme conformation in an RBC transforms from the usual R-state (oxy-state) to the T-state (deoxy-state). We rationalize our observations by proposing a model for the nanoparticle cytotoxicity pathway when the NP size is larger than the membrane pore size. We propose that the interaction of Ag NPs with the cell surface induces damage brought about by alteration of intracellular pH caused by the blockage of the cell membrane transport.

Probing differentiation in cancer cell lines by single-cell micro-Raman spectroscopy

Abstract. Single-cell micro-Raman spectroscopy has been applied to explore cell differentiation in single, live, and malignant cells from two tumor cell lines. The spectra of differentiated cells exhibit substantial enhancement primarily in the intensities of protein peaks with concomitant decrease in intensities of O─P─O asymmetric stretching peaks in DNA/RNA. Principal component analyses show that the spectral score of differentiated cells tends to asymptotically approach that of spectra obtained from normal neural stem cells/progenitors. This lends credence to the notion that the observed spectral changes are specific to differentiation, since upon differentiation, malignant cells become less malignant and tend toward benignity.

Effect of infrared light on live blood cells: Role of β-carotene

We have utilized Raman tweezers to measure and assign micro-Raman spectra of optically trapped, live red blood cells (RBCs), white blood cells (WBCs) and platelets. Various types of WBCs- both granulocytes, lymphocytes, and their different types have been studied. The Raman bands are assigned to different biomolecules of blood cells. The Raman spectra thus obtained has been enabled detection of β-carotene in these blood cells, the spectral features of which act as a signature that facilitates experimental probing of the effect of 785nm laser light on different blood cells as a function of incident laser power in the mW range. The spectral changes that we obtain upon laser irradiation indicate that, both haemoglobin as well as the cell membrane sustains damage. In case of lymphocytes and platelets the peaks corresponding to β-carotene showed drastic changes. Thorough analysis of the spectral changes indicates possibility of free radical induced damage of β-carotene in lymphocytes and platelets. Among different blood cells, RBCs have a power threshold of only 10mW. The power threshold for other types of blood cells is somewhat higher, but always below about 30mW. These values are likely to serve as useful guides for Raman tweezers based experiments on live cells.

Surface Enhanced Raman Spectroscopy Study of Red Blood Cells and Platelets

Surface-enhanced Raman spectroscopy (SERS) is an emerging technique finding applications in different areas. SERS can yield signal enhancement of several orders of magnitude compared to conventional Raman spectroscopy. While SERS bears the advantages of Raman spectroscopy such as minimal interference from water and can give molecular fingerprint, at the same time the technique overcomes the disadvantage of feeble Raman spectral signal. This makes the SERS technique an attractive candidate for studying samples with low concentrations opening up applications in biology, forensic science, food industry etc. SERS is based on the inelastic photon scattering of molecules positioned in the proximity of a nanostructured metal surface. In the visible and near-infrared frequency range, silver and gold nanostructures can give enhancement (Kumar, 2012). In biological and chemical applications, the key factor being sensitivity and reliability, it is vital to fabricate highly sensitive, uniform, and reproducible SERS substrates. In spite of the advantages that SERS has, it is challenging in real-world applications because of the difficulty in getting highly reproducible SERS signals. Different types of nanostructures prepared using different methods have been employed in SERS (Kumar, 2012). Nanocubes, nanorods, nanowires, pyramid structures, shell-isolated nanoparticles etc. structures were used in preparing SERS substrate to achieve high enhancement factor (Kumar, 2012). SERS is advantageous for studying biological samples since components with less quantity can be detected (Premasiri, Lee, & Ziegler, 2012). The SERS can be of much importance in several biomedical applications (Huh, Chung, & Erickson, 2009). Silver colloid has been used to study the SERS of RNA. Highly reproducible SERS spectra from singleand double-stranded thiolated DNA oligomers have been recorded using nanoshell substrate. SERS of amino acids has been studied using silver SERS substrate (Stewart & Fredericks, 1999). SERS using vertically aligned silicon nanowire arrays was also used for label free DNA detection. SERS was used to study the interaction between hypocrelin A (an antiviral and antitumor photoactive drug) and human serum albumin (Kočišová et al., 1999). SERS can be performed on single cells (Kneipp et al., 2002). There are reports on SERS study of single optically trapped bacterial spores. This method is based on the simultaneous use of the optical trapping and SERS phenomena. SERS of human whole blood, red blood cells, and blood plasma has been reported by Premasiri et al. (2012). Oxy and deoxy state of hemoglobin was investigated and the two states could be spectrally differentiated. SERS of whole blood done using visible and near infra-red laser excitations enabled recognition of hemoglobin and β-carotene fingerprints. SERS of blood plasma and serum has been reported which is done using colloidal silver and gold nanoparticles. The nanoparticle and hemoglobin interaction was studied by Drescher, Büchner, McNaughton, and Kneipp (2013). In this report, the SERS of RBCs and platelets has been discussed. The capability of SERS to give enhanced Raman signal makes it possible to gain the information more rapidly. The spectra were recorded with minimum laser power (6 mW) and few seconds of acquisition time. The Raman frequency bands were assigned based on the literature data. Extremely good Raman bands were obtained from the blood cells even with few seconds of exposure time. Present study shows that, enough care must be taken while recording the SERS spectra of cells since the heat induced due to metal nanostructures while exposing the probe laser radiation may damage the cells.

Raman tweezers spectroscopy study of free radical induced oxidative stress leading to eryptosis

Raman tweezers spectroscopy study of effect of free radicals was carried out on erythrocytes. We prepared hydroxyl radicals using Fenton reaction (which yields hydroxyl radicals). Raman spectra were acquired from single, trapped erythrocytes after supplementing with these free radicals. The changes in the Raman bands such as 1211 cm-1, 1224 cm-1, 1375 cm-1 indicate deoxygenation of red blood cells (RBCs). Our study shows that free radicals can induce oxidative stress on erythrocytes. The changes in the Raman spectra as well as shape of erythrocytes indicate that oxidative stress can trigger eryptosis in erythrocytes.