Narayana M. S. Sirimuthu

Quantitative surface-enhanced Raman spectroscopy

The purpose of this tutorial review is to show how surface-enhanced Raman (SERS) and resonance Raman (SERRS) spectroscopy have evolved to the stage where they can be used as a quantitative analytical technique. SER(R)S has enormous potential for a range of applications where high sensitivity needs to be combined with good discrimination between molecular targets, particularly since low cost, compact spectrometers can read the high signal levels that SER(R)S typically provides. These advantages over conventional Raman measurements come at the cost of increased complexity and this review discusses the factors that need to be controlled to generate stable and reproducible SER(R)S calibrations.

Rapid characterization and quality control of complex cell culture media solutions using raman spectroscopy and chemometrics

The use of Raman spectroscopy coupled with chemometrics for the rapid identification, characterization, and quality assessment of complex cell culture media components used for industrial mammalian cell culture was investigated. Raman spectroscopy offers significant advantages for the analysis of complex, aqueous‐based materials used in biotechnology because there is no need for sample preparation and water is a weak Raman scatterer. We demonstrate the efficacy of the method for the routine analysis of dilute aqueous solution of five different chemically defined (CD) commercial media components used in a Chinese Hamster Ovary (CHO) cell manufacturing process for recombinant proteins.The chemometric processing of the Raman spectral data is the key factor in developing robust methods. Here, we discuss the optimum methods for eliminating baseline drift, background fluctuations, and other instrumentation artifacts to generate reproducible spectral data. Principal component analysis (PCA) and soft independent modeling of class analogy (SIMCA) were then employed in the development of a robust routine for both identification and quality evaluation of the five different media components. These methods have the potential to be extremely useful in an industrial context for “in‐house” sample handling, tracking, and quality control. Biotechnol. Bioeng. 2010;107: 290–301.

Monitoring the Uptake and Redistribution of Metal Nanoparticles during Cell Culture Using Surface-Enhanced Raman Scattering Spectroscopy

We describe the uptake of silver nanoparticles by CHO (Chinese hamster ovary) cells and their subsequent fate as a result of cell division during culture, as monitored by surface-enhanced Raman scattering (SERS) spectroscopy. Mapping of populations of cells containing both labeled and native nanoparticles by SERS spectroscopy imaging provided a quantitative method by which the number of intracellular nanoparticles could be monitored. Initially, for a given amount of nanoparticles, the relationship between the number taken up into the cell and the time of incubation was explored. Subsequently, the redistribution of intracellular nanoparticles upon multiple rounds of cell division was investigated. Intracellular SERS signatures remained detectable in the cells for up to four generations, although the abundance and intensity of the signals declined rapidly as nanoparticles were shared with daughter cells. The intensity of the SERS signal was dependent both on stability of the label and their abundance (nanoparticle aggregation increases the extent of the SERS enhancement). The data show that while the labeled nanoparticles remain stable for prolonged periods, during cell division, the changes in signal could be attributed both to a decrease in abundance and distribution (and hence aggregation).

Screening Tablets for DOB Using Surface‐Enhanced Raman Spectroscopy*

Abstract:  2,5,‐Dimethoxy‐4‐bromoamphetamine (DOB) is of particular interest among the various “ecstasy” variants because there is an unusually long delay between consumption and effect, which dramatically increases the danger of accidental overdose in users. Screening for DOB in tablets is problematic because it is pharmacologically active at 0.2–3 mg, which is c. 50 times less than 3,4‐methylenedioxy‐N‐methylamphetamine (MDMA) and makes it more difficult to detect in seized tablets using conventional spot tests. The normal Raman spectra of seized DOB tablets are dominated by the bands of the excipient with no evidence of the drug component. Here we report the first use of on‐tablet surface‐enhanced Raman spectroscopy (SERS) to enhance the signal from a low concentration drug. Raman studies (785‐nm excitation) were carried on series of model DOB/lactose tablets (total mass c. 400 mg) containing between 1 mg and 15 μg of DOB and on seized DOB tablets. To generate surface‐enhanced spectra, 5 μL of centrifuged silver colloid was dispensed onto the upper surface of the tablets, followed by 5 μL of 1.0 mol/dm3 NaCl. The probe laser was directed onto the treated area and spectra accumulated for c. 20 sec (10 sec × 2). It was found that the enhancement of the DOB component in the model tablets containing 1 mg DOB/tablet and in the seized tablets tested was so large that their spectra were completely dominated by the vibrational bands of DOB with little or no contribution from the unenhanced lactose excipient. Indeed, the most intense DOB band was visible even in tablets containing just 15 μg of the drug. On‐tablet surface‐enhanced Raman spectroscopy is a simple method to distinguish between low dose DOB tablets and those with no active constituent. The fact that unique spectra are obtained allows identification of the drug while the lack of sample preparation and short signal accumulation times mean that the entire test can be carried out in <1 min.

Characterization of silicone elastomer vaginal rings containing HIV microbicide TMC120 by Raman spectroscopy.

Silicone elastomer vaginal rings are currently being pursued as a controlled‐release strategy for delivering microbicidal substances for the prevention of heterosexual transmission of HIV. Although it is well established that the distribution of drugs in delivery systems influences the release characteristics, in practice the distribution is often difficult to quantify in‐situ. Therefore, the aim of this work was to determine whether Raman spectroscopy might provide a rapid, non‐contact means of measuring the concentrations of the lead candidate HIV microbicide TMC120 in a silicone elastomer reservoir‐type vaginal ring. Vaginal rings loaded with TMC120 were manufactured and sectioned before either Raman mapping an entire ring cross‐section (100 μm resolution) or running line scans at appropriate time intervals up to 30 h after manufacture. The results demonstrated that detectable amounts of TMC120, above the silicone elastomer saturation concentration, could be detected up to 1 mm into the sheath, presumably as a consequence of permeation and subsequent reprecipitation. The extent of permeation was found to be similar in rings manufactured at 25 and 80°C.

Quantitative Characterization of Individual Microdroplets using Surface-Enhanced Resonance Raman Scattering Spectroscopy

Surface-enhanced resonance Raman scattering (SERRS) spectroscopy is a highly sensitive optical technique capable of detecting multiple analytes rapidly and simultaneously. There is significant interest in SERRS detection in micro- and nanotechnologies, as it can be used to detect extremely low analyte concentrations in small volumes of fluids, particularly in microfluidic systems. There is also rapidly growing interest in the field of microdroplets, which promises to offer the analyst many potential advantages over existing technologies for both design and control of microfluidic assays. While there have been rapid advances in both fields in recent years, the literature on SERRS-based detection of individual microdroplets remains lacking. In this paper, we demonstrate the ability to quantitatively detect multiple variable analyte concentrations from within individual microdroplets in real time using SERRS spectroscopy. We also demonstrate the use of a programmable pump control algorithm to generate concentration gradients across a chain of droplets.

Investigation of the stability of labelled nanoparticles for SE(R)RS measurements in cells

We explore the long-term stability of two different classes of labelled nanoparticles as intracellular SE(R)RS probes. Whilst chemisorbed labels gave stable responses inside cells for extended periods of time, signals from physisorbed labels could only be measured for short periods of time. These results help inform strategies for cellular imaging using vibrational spectroscopies.

Intracellular multiplex detection and imaging of stable chemisorbed labels by SERS spectroscopy

SERS spectroscopy is currently gaining wider acceptance in biological research due to its ability to obtain signals from very low quantities of material, and to obtain information from within live cells. SERS spectroscopy yields very narrow bands (10-100 times narrower than typical fluorescence bands) and spectra suffer from minimal interference from aqueous media, making SERS spectroscopy ideal for multiplex detection of intracellular components. Typically for sensing, nanoparticles are labelled with suitable sensing molecules such as a dye or thiol. Nanoparticle labelling involves two different types of interaction between the label and the enhancing surface, chemisorption and physisorption. The former is considerably stronger and more stable than the latter and hence chemisorbed labels are more appropriate for intracellular nanosensor design. In this paper, we demonstrate the difference in stability of both types of Raman label inside live cells over periods of time. Chinese hamster ovary (CHO) cells were infused with a mixture of differently labelled stable nanosensors and were imaged using SERS microspectroscopy. We also demonstrate the applicability of SERS mapping for high-throughput multiplex detection using micropatterned cell arrays.

Characterization of individual microdroplets by SERRS spectroscopy

Raman spectroscopy and its various derivatives continue to offer the analyst fast, powerful, non-invasive and nondestructive means by which to identify multiple analytes simultaneously and in real time. By virtue of the huge enhancements possible in Raman scattering, generated by both surface enhancement and the resonance Raman effect, or when coupled with other techniques such as confocal microscopy, Raman spectroscopy is becoming more and more applicable to the types of assay being conducted in lab-on-a-chip applications, such as flow cytometry, cell patterning and trapping, and microarrays, all of which often involve the detection of extremely low quantities of analyte. Surface enhanced Raman scattering (SERS, or when coupled with the resonance Raman phenomenon, SERRS) spectroscopy has proven to be of particular use as a robust optical detection method in microfluidic environments. In this paper, we demonstrate the use of SERRS multiplex detection to quantitatively characterize individual microdroplets in a continuous stream whose contents are gradually varied using a bespoke pump control algorithm.