Characterization and Visualization of Vesicles in the Endo-Lysosomal Pathway with Surface-Enhanced Raman Spectroscopy and Chemometrics

Abstract

Surface-enhanced Raman spectroscopy (SERS) is an ultra-sensitive vibrational fingerprinting technique widely used in analytical and biosensing applications. For intracellular sensing, typically gold nanoparticles (AuNPs) are employed as transducers to enhance the otherwise weak Raman spectroscopy signals. Thus the signature patterns of the molecular nanoenvironment around intracellular unlabelled AuNPs can be monitored in a reporter-free manner by SERS. The challenge of selectively identifying molecular changes resulting from cellular processes in large and multidimensional data sets and the lack of simple tools for extracting this information has resulted in limited characterization of fundamental cellular processes by SERS. Here, this shortcoming in analysis of SERS data sets is tackled by developing a suitable methodology of reference-based PCA-LDA (principal component analysis-linear discriminant analysis). This method is validated and exemplarily used to extract spectral features characteristic of the endocytic compartment inside cells. The voluntary uptake through vesicular endocytosis is widely used for the internalisation of AuNPs into cells but the characterization of the individual stages of this pathway has not been carried out. Herein, we use reporter-free SERS to identify the stages of endocytosis of AuNPs in cells, visualize them and map the molecular changes via the adaptation and advantageous use of chemometric methods in combination with tailored sample preparation. Thus our study demonstrates the capabilities of reporter-free SERS for intracellular analysis and its ability to provide a way of characterizing intracellular composition. The developed analytical approach is generic and enables the application of reporter-free SERS to identify unknown components in different biological matrices and materials.3 During the last decade, nanoparticle-based surface-enhanced Raman spectroscopy (SERS) has been extensively employed to study biological systems such as cells and tissues. [1][2][3] There are primarily two approaches: The SERS reporter approach and the label-free (reporter-free) SERS approach. In the former, a molecule is functionalized as a self-assembled monolayer on the nanoparticle and the SERS detection of its characteristic spectrum serves to visualize the location of the nanoparticle(s) inside the cell or tissue. 1, 2 This SERS reporter approach has been used in a variety of applications including detection of pathologic cells and tissues such as in cancer 4-6 or cancer markers in biofluids such as blood. 7,8 The reporters can also serve as pH sensors by an appropriate choice of the functionalized molecule with an exchangeable H + . 9 On the other hand, reporter-free SERS samples the direct environment in the vicinity of the nanoparticles and makes the spectral features contributed by the multiple molecular components available for the identification and understanding of the molecular changes around them. The nanoparticle probes can be tailored, such as by sparse functionalizatio...

Development of graphene oxide-wrapped gold nanorods as robust nanoplatform for ultrafast near-infrared SERS bioimaging

“…Notes: (A) UV-Vis absorption spectra of gNrs (solid lines) and gO@gNrs (dotted lines) with different aspect ratios. (B) raman spectra obtained from the gO@gNrs with different lsPr peaks (680 nm [1], 700 nm [2], 760 nm [3], 780 nm [4], and 810 nm [5]). Inset shows the sers intensities of 1,207 cm -1 .…”

“…[1][2][3][4][5][6] The SERS substrate can also be designed to serve as a sensitive Raman probe for bioimaging under in vitro and in vivo environments. [7][8][9][10][11] Despite the remarkable progress in SERS, there are many bottlenecks hindering its development.…”

What do we actually see in intracellular SERS? Investigating nanosensor-induced variation

“…Correlation of ROS activity with internalisation of NPs allows identification of one of the effects in the complex cellular events which unfold during NP-cell interaction and affect SERS spectra. Principal component analysis (PCA) and linear discriminant analysis (PCA-LDA) have previously been applied to SERS datasets by our group to achieve characterisation of the endolysosomal pathway 7 and distinguishing cell phenotypes 43 . In this application, we carefully apply PCA to observe alterations of cellular SERS spectra induced following various AuNP treatments to provide molecular-level detail of the changing cellular environment.…”

“…Varying media-serum content has been reported to alter intracellular localisation of AuNPs as a result of varying protein corona make-up, with decreased enrichment of protein corona found to increase the likelihood of NPs evading endolysosomal vesicles to achieve free cytosolic residence 23 . Due to the sensitivity of intracellular SERS measurements, sampling from different cellular locations can give drastically different spectral signatures and has been used as such for cellular characterisation and mapping 7,[47][48][49] . Figure 3 Mean SERS spectra of SY-SY5Y cells following 24 h incubation with AuNPs at 10, 100 and 250 µM in 1 % serum media (A, red) and 100 and 250 µM in 10 % serum media (C, blue).…”

“…Employed as SERS probes, their applications include monitoring of cellular functions 3,4 , dynamics [5][6][7] , enzyme kinetics 8,9 , stress response [10][11][12][13] , apoptosis and cell death 11,14,15 along with probing specific compartments such as the mitochondria 16 and tracking of drugs released into the cytoplasm by NP carriers [17][18][19][20] . Cellular uptake is achieved through voluntary incorporation into living cells by endocytotic mechanisms, following a simple incubation period with NP-doped cell culture media.…”