Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 237 (2020) 118364

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Spectrochimica Acta Part A: Molecular and Biomolecular

Spectroscopy
journal homepage: www.elsevier.com/locate/saa

SERS studies on normal epithelial and cancer cells derived from clinical
breast cancer specimens
LiShengNan Shen a, Ye Du a, Na Wei b, Qian Li a, SiMin Li a, TianMeng Sun c,d,e, Shuping Xu f, Han Wang g,h,
XiaXia Man i, Bing Han a,⁎
a
Department of Breast Surgery, The First Hospital, Jilin University, Changchun 130000, Jilin, China
b
Third Operating Room, The First Hospital, Jilin University, Changchun 13000, Jilin, China
c
Key Laboratory of Organ Regeneration and Transplantation of Ministry of Education, Institute of Immunology, The First Hospital, Jilin University, Changchun 130000, Jilin, China
d
International Center of Future Science, Jilin University, Changchun 130000, Jilin, China
e
National-local Joint Engineering Laboratory of Animal Models for Human Diseases, Changchun 130000, Jilin, China
f
State Key Laboratory of Supramolecular Structure and Materials, Institute of Theoretical Chemistry, Jilin University, Changchun, Jilin 130012, China
g
College of Information Science and Technology, Northeast Normal University, Changchun 130117, China
h
Institution of Computational Biology, Northeast Normal University, Changchun 130117, China
i
Department of Gynaecology, The First Hospital, Jilin University, Changchun 130000, Jilin, China

a r t i c l e i n f o a b s t r a c t

Article history: Surface-enhanced Raman scattering (SERS) spectroscopy of single-cell suspensions obtained from fresh speci-
Received 30 November 2019 mens of breast cancer tissue and normal breast tissue by mechanical enzymatic digestion was obtained and
Received in revised form 9 April 2020 analysed, which is different from most Raman studies using breast cancer cell lines. Random forest classification
Accepted 10 April 2020
was implemented to develop effective diagnostic algorithms for the classification of SERS of different typed cells.
Available online 12 April 2020
We first examined the SERS spectra of the primary breast cancer single cell and normal epithelial single cell ob-
Keywords:
tained by flow sorting cytometry due to their biomarkers of CD326+/CD45−. Comparison analyses on their SERS
Surface-enhanced Raman spectroscopy spectra disclose that the nucleic acid and protein levels of the primary breast cancer single cell are higher than
Primary breast cancer cell those of the normal epithelial single cell, while the lipids are at a relatively lower level. An important finding is
Flow cytometry that the cholesterol, palmitic acid, and sphingomyelin in the cancer cell profiles exhibit stronger than those of
Random forest normal cells, while the glycans are at a relatively lower level. Furthermore, the standard deviation (SD) of the
normal epithelial single cell is larger than that of the breast cancer cell, and the SD of the primary breast cancer
single cell is more obvious than that of the normal epithelial cells. In addition, the prospective application of an
algorithm to the dataset results in an accuracy of 78.2%, a precision of 75.5%, and a recall of 66.7%. The breast can-
cer diagnostic model laid a solid foundation for judgment of breast-conserving surgical margins and early diag-
nosis of breast cancer.
© 2020 Elsevier B.V. All rights reserved.

1. Introduction
In 2018, the World Health Organization reported that 627,000
women died of breast cancer [1]. Needle biopsy, as the gold standard
method of breast cancer diagnosis, is invasive, inconvenient, and ineffi-
cient. The pathologic result of needle biopsy is also time-consuming,
which is an urgent problem to be solved especially during breast cancer
surgery.
Raman spectroscopy can provide fingerprint information of analytes
and owns many merits in bio-applications. It allows simple sample
preparation and can achieve noncontact detections, giving almost no
damage to sample structures. Raman analysis has the characteristics of
⁎ Corresponding author.
E-mail address: han_bing@jlu.edu.cn (B. Han).
fast analysis, simplicity and high resolution, and has broad application
prospects in aqueous phase systems. Surface-enhanced Raman spec-
troscopy (SERS) is a sensitive and powerful tool for chemical composi-
tion identification by virtue of the enhancement effect from metal
nanoparticles on the spectral signals [2]. In recent years, many studies
utilized SERS for deep learning of breast cancers, such as the diagnosis
of breast cancer tissues[36] [3], the study of breast cancer with specific
antibody-related functionalized nanoparticles (NPs) [4], and the differ-
ential diagnosis of breast cancer cell lines [5]. However, due to the com-
plex compositions of breast tissues and a large amount of adipose tissue,
SERS analysis of cancerous tissues is complex and may be overwhelmed
by the Raman signal of the adipose tissue [6]. Moreover, most of the re-
search subjects use breast cancer cell lines as models. Very few studies
referred to primary epithelial single cell. In González-Solís's study,
they explored the Raman spectra of primary breast cancer cells,

https://doi.org/10.1016/j.saa.2020.118364
1386-1425/© 2020 Elsevier B.V. All rights reserved.
2 L. Shen et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 237 (2020) 118364
however, the primary breast cancer cell acquisition methods require
culture for several generations in their work [7]. Although in vitro cul-
tured cell lines and primary cells reflect most of the characteristics of
breast cancer, primary single cell isolated from human tissue samples
retain more cell functions, growth characteristics, cell markers and sig-
nalling pathways [8]. Therefore, it is of great significance for achieving
primary breast cancer single cell and their SERS should be rebuilt to bet-
ter reflect their intrinsic characteristics.
Our study obtained single-cell suspensions from breast cancer and
normal breast tissue samples from the same patients by a mechanical
enzymatic digestion method, and the CD326+/CD45− cells in the sus-
pensions were selected by flow cytometry. Then, the SERS spectra of the
cells were recorded by mixing them with Ag nanoparticles (AgNPs). For
the first time to our knowledge, with these data, a supervised learning
algorithm based on the ensemble method “Random forest” as a classi-
fier was trained and used to simultaneously and automatically identify
primary breast cancer single cell and normal epithelial single cell. The
novelties and achievements of this study can be summarized as three
aspects: (1) SERS of single cell show more uniform spectral behaviours
rather than the tissues, which allows for more stable and producible
Raman spectra and solve the spectral interference from a large amount
of adipose tissue. (2) SERS of the primary single cell isolated from
human tissue samples were built, and for the first time compared
with cancer cell lines. (3) The assessments on nucleic acid, protein and
lipid levels of the primary breast cancer single cell and normal epithelial
single cell were achieved, which provides a deeper understanding from
the view of molecular profiles of breast cancers.
2. Materials and methods
2.1. Chemicals and antibodies
Phosphate-buffered saline (PBS), foetal bovine serum (FBS), penicil-
lin/streptomycin, and L-glutamine were all purchased from Invitrogen
(Carlsbad, CA, USA). Fluorophore-labelled anti-human CD326 and
anti-human CD45 monoclonal antibodies (mAbs) were obtained from
BioLegend (San Diego, CA, USA). DNase and collagenase type III were
purchased from Worthington (Lakewood, NJ, USA). Silver nitrate
(AgNO3) and sodium citrate were purchased from Shanghai Chemical
Company.
2.2. Patients and samples
Matched fresh specimens of breast cancer tissue and normal breast
tissue from the same patient (the normal tissue sample was taken
more than 5 cm away from the cancerous lesion) were collected from
15 patients who underwent surgical resection at the Department of
Breast Surgery, the First Hospital of Jilin University. Table S1 shows
age and pathological parameters of 15 patients. All the patients agreed
to participate in this research project through Institutional Review
Board (IRB) protocol (2017-111). The specimens were divided into
two parts, one for pathological examination and the other for cryopres-
ervation in the laboratory.
2.3. Flow cytometry sorting
Each specimen was washed twice with PBS containing penicillin and
streptomycin. Then the fat and blood vessels were removed and each
tissue specimen was cut into small pieces by eye scissors. The small
pieces were digested into single cell using a 0.01% DNase and collage-
nase type III solution at 37 °C for 40 min. Then, the dissociated specimen
was filtered through 40-μm mesh filters to remove debris and cell seg-
ments. The solution was centrifuged at 1650 rpm for 5 min at room tem-
perature (RT, 21 °C). Then, the pellet was re-suspended in PBS. Then, the
cells were stained with appropriate dilutions of anti-CD45 and anti-
CD326 antibodies at RT. The CD326+CD45− cells were collected using
BD influx cell sorter at RT (BD Bioscience), and the data were analysed
using FlowJo10 software.
2.4. Synthesis of silver nanoparticles (AgNPs)
In total, 0.018 g of AgNO3 was mixed with 100 mL of deionized water
in a three-necked flask, heated and stirred until slightly boiling. Then,
2.0 mL of 1% w sodium citrate was quickly added, and we waited for
the colour change from light yellow to dark yellow-grey-green. The
mixture was kept stirring at 90 °C for 40 min. Fig. S2 shows the trans-
mission electron micrographs of the AgNPs and UV–visible light absorp-
tion spectra of the Ag colloids.
2.5. Raman spectroscopy
Fifty microliters (about 5 × 103/ml) cell solution were mixed with
10 μL of 4 pM AgNPs, and the mixture was then dripped onto a glass
slide. Under the microscope, we can observe some of AgNPs were inter-
nalized by the cells. A confocal Raman system (LabRAM Aramis, Horiba
Jobin Yvon) with a ~0.7-mm spatial resolution and a 5 mW, 633 nm
HeNe laser as the excitation source was used for the collection of
Raman spectra. The Raman signals were detected with a Synapse Ther-
moelectric cooled charge-coupled device (CCD) camera (Horiba Jobin
Yvon). Raman scattering light was collected with a 50× microscope ob-
jective lens (0.50 NA, LMPLFLN, Olympus) that was also used to focus
the excitation laser light. The laser beam was focused on the AgNPs lo-
cated in cells. Strong Rayleigh scattering light was then blocked using
a four-notch filter (Horiba Jobin Yvon). Extended scan spectra with a
spectral range of 400–1800 cm-1 were acquired using an integration
time of 10 s and two accumulations. The wavenumber calibration was
set by reference to the 520.7 cm−1 vibrational band of a silicon wafer.
These settings were kept constant for all spectral measurements. The
Raman scattering light collection was performed at RT and lasted for
an hour for each sample. Approximately 5 spectra were collected from
15 patients who underwent surgical resection. As the fresh specimens
of breast cancer tissue from Patients No.5, 6, 10 too small to get the pri-
mary breast cancer cells by the mechanical enzymatic digestion, and a
total of 135 SERS spectra were obtained from different samples
(Table 1). S2 shows the SERS spectra of the primary breast cancer single
cell and the normal epithelial single cell from 15 patients.
2.6. Statistical analyses
All the spectra were baseline corrected by fitting and subtracting a
third-order polynomial (NGSLabSpec, Horiba Jobin Yvon). Then, the
spectra were smoothed and normalized using Origin 8.0 software. The
final data used to build the model are shown in Table 1. Finally, variance
values were obtained with respect to the mean spectrum. The spectral
data were automatically classified by the random forest (RF) method.
3. Result and discussion
Primary epithelial single cell was sorted from breast tissue samples
by flow cytometry according to the expression of different cell surface
markers to obtain primary breast cancer single cell and normal epithe-
lial single cell. Fig. 1 shows the results of flow cytometry sorting using
CD45 and CD326 as cell surface markers. The CD326+/CD45− cell is a
Table 1
The numbers of collected spectra from 15 patients.
Patients no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Sum
Primary breast 5 5 5 5 0 0 5 5 5 0 5 5 5 5 5 60
cancer cells
Normal epithelial 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 75
cells
 L. Shen et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 237 (2020) 118364
Fig. 1. The flow cytometric isolation the normal (A) and cancerous epithelial cells (B) by CD45 and CD326.
primary epithelial single cell and accounts for about 5.78% and 11.2% of
the total number of nuclear cells respectively in cancer and normal tis-
sues. The normal cytological components of fine needle aspiration cytol-
ogy specimens of the breast include epithelium (catheter, lobule,
apocrine gland, squamous component, etc.), myoepithelial cells, macro-
phages, vascular endothelium, fat, stroma, and other mesenchymal tis-
sues. Epithelial cells are characteristic cells in breast tissue, and CD326
is a transmembrane glycoprotein composed of 314 amino acids that
play a function of an adhesion molecule between epithelial cells ex-
pressing the same molecule. Therefore, high activity epithelial cells in
a tissue can be selected by their CD326+/CD45− phenotype, and the
difference between the SERS spectra of primary cancer single cell and
those of normal single cell can be determined to characterize the differ-
ence between cancerous tissues and normal tissues, which can avoid
many difficulties in the spectral analysis of tissues. This approach can
also avoid the interference of adipose tissue on the Raman signal of can-
cerous tissue and preserve the specificity of the enhanced spectra of
cancer cells in different patients, which lays a foundation for the devel-
opment of a Raman spectroscopy-based diagnostic model.
3
Raman and SERS spectra of primary breast cancer single cell and nor-
mal epithelial single cell were collected under the same conditions. The
collected Raman spectra are shown in Fig. 2. Notably, the intensity of
Raman bands was weak. However, the SERS spectral bands at
400–1800 cm−1 are greatly enhanced[10]. This phenomenon is caused
by the plasmonic effect of AgNPs, especially in a collective form [9]. A
comparison of a SERS spectrum and regular Raman spectrum indicate
that the intensities of many predominant vibration bands were remark-
ably increased by the SERS-active AgNPs. In contrast, only a few weak
Raman bands could be observed in breast cancer cells without Ag
colloids.
After the initial SERS spectra were baseline corrected and smoothed,
all the full SERS spectra were normalized to the integrated area from
400 cm−1 to 1800 cm−1 to improve the comparison of the SERS spectral
shape in the analysis. Fig. 3 shows the mean normalized SERS spectra of
the primary breast cancer single cell (red), and normal epithelial single
cell (violet), with standard deviations (SDs) overlaid as shaded colour
fills. Fig. 3 shows that the yellow shaded area is larger than the green
shaded area, that is, the standard deviation of normal epithelial cells is
4 L. Shen et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 237 (2020) 118364
Fig. 2. Comparison of the SERS spectrum of breast cancer cell–AgNP mixtures (red), the
normal Raman spectrum of breast cancer cell without AgNPs solution (blue) and the
blank spectrum of Ag NPs (black).
larger than that of primary breast cancer cells. The SERS of the two pri-
mary cell types show multiple bands, and both cell types contain lipids,
proteins, sugars, and DNA. The SD of the primary breast single cell is
more obvious than that of the breast cancer cell lines, and the SD of
the normal epithelial cells is larger than that of the breast cancer cells.
The first reason may be differences among different patients, which
would also provide a basis for subgroup analysis when the data are
more variable. The second reason may be that the primary cells were
not in the same proliferative state, but the breast cancer cell lines
were in the same proliferative state undergoing exponential growth or
plated cancer cells. Short reported that the Raman spectra of cells in dif-
ferent proliferative phases were different [11]. An index of the prolifer-
ative period of the cancer cells shows that the relative levels of nucleic
acids and proteins increase while the plateau cell lipid content also in-
creases, indicating that when studying cell populations including cells
in different phases of cell proliferation, the SD of Raman spectra is larger.
It is possible that we could determine the state of breast cancer cell
Fig. 3. Intensity standard deviations of the primary breast cancer single cell (green-shaded
areas) and normal epithelial single cell (yellow-shaded areas).
proliferation by Raman spectra. The third reason may be that normal
breast tissue has more lipids than cancerous tissue, causing the SD of
the normal breast tissue is more distinct than that of cancerous breast
tissue [12].
Prominent SERS bands were observed in both normal and breast tu-
mour cells at 568, 628, 712, 761, 822, 885, 957, 1004, 1063, 1090, 1222,
1271, 1308, 1327, 1337, 1345, 1374, 1445, 1471, 1575, and 1610 cm-1.
The spectrum (Fig. 4) is obtained by subtracting the average spectrum
of the normal epithelial single cell from the average spectrum of the
breast cancer single cell, and it shows the difference between the SERS
spectrum of primary breast cancer single cell and that of normal epithe-
lial single cell relatively clearly. The difference spectrum in Fig. 4 shows
both positive and negative bands, suggesting that their spectral intensi-
ties are comparable. The bands at 1173 and 1456 cm−1 are associated
with nucleic acids, and the bands appearing at 853, 991, 1026, 1239,
1491, 1617, and 1649 cm−1[22–26] could be attributed to proteins,
which might be related to the occurrence of profibrotic proliferative re-
sponses in breast cancer tissue[21][21–26] [13]. Therefore, the levels of
nucleic acids and proteins in the breast cells become increasing [14]. In
addition to these bands, several predominant bands at 418, 608, 704,
1135, and 1295 cm−1 in the cancer cells profiles exhibit stronger than
those of normal cells. Bands near 418, 608, and 704 cm−1 are due to
cholesterol. The cholesterol level in the primary breast cancer single
cell is significantly higher than that in the normal epithelial cells,
which might be attributed to rapid proliferation and the need for large
amounts of cholesterol to synthesize new cell membranes. The bands
at 1135 and 1295 cm−1 are associated with palmitic acid and
sphingomyelin. These results were consistent with Abramczyk's sug-
gestion that significantly increased levels of saturated fatty acids and
sphingomyelin are associated with metastasis and invasiveness in tu-
mour cells [15]. The troughs at 719 and 1090 cm−1 were attributed to
the C\\N, C\\C stretch of lipids. Therefore, normal breast cells contain
excessive lipids. Many studies have shown that the levels of monoun-
saturated fatty acids and triglycerides in cancer cells are significantly
lower than those in normal cells. Louw found that the oleic acid content
in cervical cancer tissue was higher than that in normal tissue [16].
Mamalakis reported similar findings for prostate cancer [17]. In addi-
tion, researchers have suggested that amino acid metabolism may be
closely related to carcinogenesis. Su found that the amino acid content
in malignant cells was reduced [18]. The Raman bands at 1342 and
1547 cm−1 attributed to amino acids are stronger in the normal epithe-
lial cells than in the breast cancer cells, suggesting that amino acid me-
tabolism may be related to tissue carcinogenesis. In summary, the
assignments of the bands and troughs reveal that nucleic acid and
Fig. 4. Difference spectra of the normal to the breast cancer cell.
 L. Shen et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 237 (2020) 118364
Fig. 5. The mean SERS spectra of primary breast cancer single cell and normal epithelial
single cell of patients.
protein levels in malignant cells increase while lipid levels decrease
[12]. The difference in SERS was consistent with the pathological fea-
tures of breast cancer cell proliferation, large nuclei, and a small
nucleus-to-cytoplasm ratio. These features may provide important
clues on understanding differences within the biochemical composition
of tissues.
Besides the intensity changes, the SERS band shifts of two kinds of
single cell were analysed (Fig. 5). The bands of the normal cell associ-
ated with Amide III in proteins previously laid at 1228 and
1275 cm−1, blueshift to 1222 and 1271 cm−1 in the cancer cells,
which indicates a 3-strand or random-coil increase in malignant tu-
mours [27]. The bands attributed to the C\\N or C\\H vibration mode
of lipids also shift from 714 and 1450 cm−1 in the normal cells to 709
and 1448 cm−1 in the cancer cells, and become lower, indicating that
the stability of lipid chains has been reduced in tumour tissue [28]. For
the nucleic acids of the cells, SERS of the cancer cells produced bands lo-
cated at 1147, 1337, 1575, and 1610 cm−1, while the bands of the nor-
mal epithelial cells shift to 1036, 1327, 1577, and 1609 cm−1. The bands
of the cancer cell get into wider and larger than those of the normal ep-
ithelial single cell, indicating that the nucleic acid content in the tumour
Fig. 6. Schematic diagram of Random Forest.
5
tissue has greatly increased. These results are consistent with the con-
clusion that the ratio of nucleic acid to cytoplasm in malignant tissue
is elevated with pathological progression [29]. The DNA band of the nor-
mal epithelial single cell was located at 1090 cm−1, but that of the can-
cer single cell redshifts to 1095 cm−1. Single-strand breaks produce
DNA fragments in cancerous tissues [30]. These breaks have been re-
ported in other organs (the larynx, cervix, colon, and bladder)
[31–34]. In addition, the normal epithelial single cell contains more gly-
cans (855 cm−1) than the cancer single cell. Since many tumour-specific
antigens are carbohydrates, a lot of cancer markers are also in the glyco-
protein family. Therefore, the difference in the glycan Raman spectral
shift between cancer cells and normal glandular epithelial cells is critical
for breast cancer diagnosis [35].
RF is a machine learning model. In this model, multiple samples are
extracted from pre-processed datasets by bootstrap aggregating. Each
bootstrap sample is modelled by a decision tree, and then the predic-
tions of multiple randomly selected decision trees are combined. The
final prediction results are obtained by voting. The specific process is
shown in Fig. 6.
135 spectra were automatically classified by the RF method. We
used the ten-fold cross-validation algorithm to randomly divide the
dataset into 10 parts, and nine of which were used for training and
the other was used for testing. Each of the 10 parts was used as a test
set. Prospective application of the algorithm to the dataset resulted in
an accuracy of 78.2%, a precision of 75.5%, and a recall of 66.7%.
4. Conclusion
We aimed to distinguish the SERS spectral characteristics of primary
breast cancer single cell from those of primary normal epithelial single
cell and to construct diagnostic models with the SERS spectra of primary
single cell. The SD of the primary breast cells is more obvious than that
of the breast cancer cell lines，the SD of the normal epithelial cells is
larger than that of the breast cancer cells. The primary cell model con-
structed herein can be used to establish a foundation for the study of dif-
ferent molecular types, genotypes of breast cancer cells, determining
the degree of malignancy of breast cancer and monitoring tumour
growth, response to treatment and provide valuable information for
predicting the occurrence and metastasis of breast cancer. The diagnos-
tic model created by machine learning can identify subtle changes in
SERS. The results of this experiment will help us improve the diagnostic
technology for breast cancer using SERS spectroscopy to improve the
rate of breast conservation and the survival rate of breast cancer pa-
tients and reduce the rate of secondary surgery.
6 L. Shen et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 237 (2020) 118364
Ethical statement
The authors are accountable for all aspects of the work in ensuring
that questions related to the accuracy or integrity of any part of the
work are appropriately investigated and resolved. This study was ap-
proved by an institutional ethics committee and followed the tenets of
the Declaration of Helsinki.
CRediT authorship contribution statement
Shen LiShengNan:Methodology, Writing - original draft.Du Ye:Val-
idation.Na Wei:Formal analysis.Li Qian:Investigation.Li SiMin:Data
curation.Sun TianMeng:Resources, Supervision, Writing - review &
editing.Xu Shuping:Methodology, Supervision.Wang Han:Software.
Man XiaXia:Investigation.Bing Han:Conceptualization, Supervision,
Writing - review & editing.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
This work was supported by the Natural Science Foundation of China
(81773171) and the Education Department of Jilin Province
(JJKH20180218KJ).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.saa.2020.118364.
References
[1] The Global Cancer Observatory, Breast Cancer Fact Sheet: Globocan 2018, Available
from: http://gco.iarc.fr/today/data/factsheets/cancers/20-Breast-fact-sheet.pdf
2018.
[2] D. Cialla, A. März, R. Böhme, F. Theil, K. Weber, M. Schmitt, et al., Surface-enhanced
Raman spectroscopy (SERS): progress and trends, Anal. Bioanal. Chem. 403 (1)
(2012) 27–54.
[3] E. Cepeda-Pérez, T. López-Luke, P. Salas, G. Plascencia-Villa, A. Ponce, J. Vivero-
Escoto, et al., SERS-active Au/SiO2 clouds in powder for rapid ex vivo breast adeno-
carcinoma diagnosis, Biomed. Opt. Express 7 (6) (2016) 2407–2418.
[4] R.M. Davis, J.L. Campbell, S. Burkitt, Z. Qiu, S. Kang, M. Mehraein, et al., A Raman im-
aging approach using CD47 antibody-labeled SERS nanoparticles for identifying
breast cancer and its potential to guide surgical resection, Nanomaterials 8 (11)
(2018) 953.
[5] M. Yarbakht, M. Nikkhah, A. Moshaii, K. Weber, C. Matthäus, D. Cialla-May, et al., Si-
multaneous isolation and detection of single breast cancer cells using surface-
enhanced Raman spectroscopy, Talanta 186 (2018) 44–52.
[6] N. Huang, M. Short, J. Zhao, H. Wang, H. Lui, M. Korbelik, et al., Full range character-
ization of the Raman spectra of organs in a murine model, Opt. Express 19 (23)
(2011) 22892–22909.
[7] J. González-Solís, G. Luévano-Colmenero, J. Vargas-Mancilla, Surface enhanced
Raman spectroscopy in breast cancer cells, Laser Ther. 22 (1) (2013) 37–42.
[8] N. Faridi, S.Z. Bathaie, S. Abroun, P. Farzaneh, H. Karbasian, F. Tamanoi, et al., Isola-
tion and characterization of the primary epithelial breast cancer cells and the adja-
cent normal epithelial cells from Iranian women’s breast cancer tumors, 70 (2)
(2018) 625–639.
[9] E. Dulkeith, A.C. Morteani, T. Niedereichholz, T.A. Klar, J. Feldman, S. Levi, et al., Fluo-
rescence quenching of dye molecules near gold nanoparticles: radiative and
nonradiative effects, Phys. Rev. Lett. 89 (20) (2002) 2030021–2030024.
[10] aacute Gonz, S. lez, s iacute, Lu JL, et al., Surface enhanced Raman spectroscopy in
breast cancer cells, Laser Ther. 22 (1) (2013) 37–42.
[11] K.W. Short, S. Carpenter, J.P. Freyer, J.R. Mourant, Raman spectroscopy detects bio-
chemical changes due to proliferation in mammalian cell cultures, Biophys. J. 88
(6) (2005) 4274–4288.
[12] N. Stone, C. Kendall, J. Smith, P. Crow, H. Barr, Raman spectroscopy for identification
of epithelial cancers, Faraday Discuss. 126 (2004) 141–157 (discussion 69-83).
[13] S. Feng, S. Huang, D. Lin, G. Chen, Y. Xu, Y. Li, et al., Surface-enhanced Raman spec-
troscopy of saliva proteins for the noninvasive differentiation of benign and malig-
nant breast tumors, Int. J. Nanomedicine 10 (2015) 537–547.
[14] A.S. Haka, K.E. Shafer-Peltier, M. Fitzmaurice, J. Crowe, R.R. Dasari, M.S. Feld, Identi-
fying microcalcifications in benign and malignant breast lesions by probing differ-
ences in their chemical composition using Raman spectroscopy, Cancer Res. 62
(18) (2002) 5375–5380.
[15] H. Abramczyk, B. Brozek-Pluska, New look inside human breast ducts with Raman
imaging. Raman candidates as diagnostic markers for breast cancer prognosis:
mammaglobin, palmitic acid and sphingomyelin, Anal. Chim. Acta 909 (2016)
91–100.
[16] L. Louw, A.M. Engelbrecht, F. Cloete, Comparison of the fatty acid compositions in
intraepithelial and infiltrating lesions of the cervix: part I, total fatty acid profiles,
Prostaglandins Leukot. Essent. Fat. Acids 59 (4) (1998) 247–251.
[17] G. Mamalakis, A. Kafatos, N. Kalogeropoulos, N. Andrikopoulos, G. Daskalopulos, A.
Kranidis, Prostate cancer vs hyperplasia: relationships with prostatic and adipose
tissue fatty acid composition, Prostaglandins Leukot. Essent. Fat. Acids 66 (5–6)
(2002) 467–477.
[18] L. Su, Y.F. Sun, Y. Chen, P. Chen, A.G. Shen, X.H. Wang, et al., Raman spectral proper-
ties of squamous cell carcinoma of oral tissues and cells, 22 (1) (2012) 311–316.
[21] A.J. Ruiz-Chica, M.A. Medina, F. Sanchez-Jimenez, F.J. Ramirez, Characterization by
Raman spectroscopy of conformational changes on guanine-cytosine and adenine-
thymine oligonucleotides induced by aminooxy analogues of spermidine, J. Raman
Spectrosc. 35 (2) (2004) 93–100.
[22] E. O Faolain, M.B. Hunter, J.M. Byrne, P. Kelehan, M. McNamara, H.J. Byrne, et al., A
study examining the effects of tissue processing on human tissue sections using vi-
brational spectroscopy, Vib. Spectrosc. 38 (1–2) (2005) 121–127.
[23] Z. Liu, C. Davis, W.B. Cai, L. He, X.Y. Chen, H.J. Dai, Circulation and long-term fate of
functionalized, biocompatible single-walled carbon nanotubes in mice probed by
Raman spectroscopy, Proc. Natl. Acad. Sci. U. S. A. 105 (5) (2008) 1410–1415.
[24] N. Stone, C. Kendall, J. Smith, P. Crow, H. Barr, Raman spectroscopy for identification
of epithelial cancers, Faraday Discuss. 126 (2004) 141–157.
[25] C.H. Liu, Y. Zhou, Y. Sun, J.Y. Li, L.X. Zhou, S. Boydston-White, et al., Resonance Raman
and Raman spectroscopy for breast cancer detection, Technol. Cancer Res. Treat. 12
(4) (2013) 371–382.
[26] M.V. Chowdary, K.K. Kumar, J. Kurien, S. Mathew, C.M. Krishna, Discrimination of
normal, benign, and malignant breast tissues by Raman spectroscopy, Biopolymers
83 (5) (2006) 556–569.
[27] A. Mahadevan-Jansen, R.R. Richards-Kortum, Raman spectroscopy for the detection
of cancers and precancers, J. Biomed. Opt. 1 (1) (1996) 31–70.
[28] Z. Huang, A. McWilliams, H. Lui, D.I. McLean, S. Lam, H. Zeng, Near-infrared Raman
spectroscopy for optical diagnosis of lung cancer, 107 (6) (2003) 1047–1052.
[29] T.A. Steitz, Structural studies of protein-nucleic acid interaction: the sources of
sequence-specific binding, Q. Rev. Biophys. 23 (3) (1990) 205–280.
[30] B. Han, Y. Du, T. Fu, Z. Fan, S. Xu, C. Hu, et al., Differences and relationships between
normal and atypical ductal hyperplasia, ductal carcinoma in situ, and invasive ductal
carcinoma tissues in the breast based on Raman spectroscopy, Appl. Spectrosc. 71
(2) (2017) 300–307.
[31] M.G. Shim, B.C. Wilson, The effects of ex vivo handling procedures on the near-
infrared Raman spectra of normal mammalian tissues, Photochem. Photobiol. 63
(5) (1996) 662–671.
[32] N. Stone, P. Stavroulaki, C. Kendall, M. Birchall, H. Barr, Raman spectroscopy for early
detection of laryngeal malignancy: preliminary results, Laryngoscope 110 (10)
(2000) 1756–1763 Pt 1.
[33] A. Mahadevan-Jansen, M.F. Mitchell, N. Ramanujam, A. Malpica, S. Thomsen, U.
Utzinger, et al., Near-infrared Raman spectroscopy for in vitro detection of cervical
precancers, Photochem. Photobiol. 68 (1) (1998) 123–132.
[34] C.H. Liu, B.B. Das, W.L. Sha Glassman, G.C. Tang, K.M. Yoo, H.R. Zhu, et al., Raman,
fluorescence, and time-resolved light scattering as optical diagnostic techniques to
separate diseased and normal biomedical media, J. Photochem. Photobiol. B 16 (2)
(1992) 187–209.
[35] Hakomori S-i, Tumor-associated carbohydrate antigens defining tumor malignancy:
basis for development of anti-cancer vaccines, in: A.M. Wu (Ed.), The Molecular Im-
munology of Complex Carbohydrates—2, Springer US, Boston, MA 2001,
pp. 369–402.
[36] A. Pallaoro, M.R. Hoonejani, G.B. Braun, C.D. Meinhart, M. Moskovits, Rapid identifi-
cation by surface-enhanced Raman spectroscopy of cancer cells at low concentra-
tions flowing in a microfluidic channel, ACS Nano 9 (4) (2015) 4328–4336.