Surface-Enhanced Raman Scattering Surface Selection Rules for the Proteomic Liquid Biopsy in Real Samples: Efficient Detection of the Oncoprotein c-MYC

Article abstract of DOI:10.1021/jacs.6b08957

Blood-based biomarkers (liquid biopsy) offer extremely valuable tools for the noninvasive diagnosis and monitoring of tumors. The protein c-MYC, a transcription factor that has been shown to be deregulated in up to 70% of human cancers, can be used as a r

Full text of DOI:10.1021/jacs.6b08957

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Communication
SERS Surface Selection Rules for the Proteomic Liquid Biopsy
in Real Samples: Efficient Detection of the Oncoprotein c-MYC
Elena Pazos, Manuel Garcia Algar, Cristina Penas, Moritz Nazarenus,
Arnau Torruella, Nicolas Pazos-Perez, Luca Guerrini, M. Eugenio Vázquez,
Eduardo Garcia-Rico, José L. Mascareñas, and Ramon A. Alvarez-Puebla
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b08957 • Publication Date (Web): 12 Oct 2016
Downloaded from http://pubs.acs.org on October 12, 2016
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SERS Surface Selection Rules for the Proteomic Liquid Biopsy in
Real Samples: Efficient Detection of the Oncoprotein c-MYC
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Elena Pazos, Manuel Garcia-Algar, Cristina Penas, Moritz Nazarenus, Arnau Torruella, Ni-
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colas Pazos-Perez, Luca Guerrini, M. Eugenio Vázquez, Eduardo Garcia-Rico, * José L. Masca-
reñas * and Ramon A. Alvarez-Puebla *
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1,5
1
Universitat Rovira i Virgili and Centro de Tecnología Química de Catalunya, Carrer de Marcellí Domingo s/n,
43007 Tarragona, Spain.
2
Medcom Advance S.A., Av. Roma, 08840 Barcelona, Spain.
3
Centro Singular de Investigación en Química Biolóxica e Materiais Moleculares (CIQUS) and Departamento de
Química Orgánica, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain.
4
Department of Clinical Oncology, Hospital Universitario Hm Madrid-Torrelodones, 28250 Madrid, Spain
5
ICREA, Passeig Lluís Companys 23, 08010 Barcelona, Spain.
Supporting Information Placeholder
c-myc gene that plays a major role in coordinating cellular
ABSTRACT: Blood-based biomarkers (liquid biopsy) offer
extremely valuable tools for the non-invasive diagnosis and
metabolism with cell cycle and it has been shown to be ele-
4
vated or deregulated in up to 70% of human cancers. Unlike
other proto-oncogenes whose activity is dependent on muta-
tions, truncations or gene fusions, the oncogenicity of c-myc
gene is usually secondary to loss of transcriptional control
leading to over-expression and accumulation of normal pro-
monitoring of tumors. The protein c-MYC, a transcription
factor that has been shown to be deregulated in up to 70% of
human cancers, can be used as a robust proteomic signature
for cancer. Herein, we developed a rapid, highly specific and
sensitive SERS assay for the quantification of c-MYC in real
blood samples. The sensing scheme relies on the use of spe-
cifically designed hybrid plasmonic materials and their bio-
derivatization with a selective peptidic receptor modified
with a SERS transducer. Peptide/c-MYC recognition events
translate into measurable alterations of the SERS spectra
associated with a molecular re-orientation of the transducer,
in agreement with the surface selection rules. The efficiency
of the sensor is demonstrated in cellular lines, healthy do-
nors and a cancer patient.
5
tein. Furthermore, the c-MYC protein is not associated with
any specific transcriptional program, but it appears to be a
universal amplifier of gene expression, increasing the tran-
6
scription at all active promoters. Thus, the evaluation of the
concentration of this protein in blood can be used as a crite-
ria for the diagnosis and monitoring of tumors.
Identification and quantification of proteins in blood is
normally performed by using standard immunological tech-
niques such as Western Blot (WB) or ELISA. Although ro-
bust, these methods are tedious, lengthy, expensive and, in
the case of WB, have low sensitivity. In recent years, surface-
7
enhanced Raman scattering (SERS) spectroscopy has
The molecular characterization of tumors is consistently
applied in clinical oncology to guide treatment decisions.
These analyses are typically performed on solid tissues ac-
quired through an invasive biopsy at diagnosis. However,
tissue biopsies are challenging as they are costly, painful, or
potentially risky for the patient. Thus, the development of
non-invasive methods to detect and monitor tumors is a
major need in oncology. Blood-based biomarkers (liquid
emerged as an excellent alternative for the detection, quanti-
fication and characterization of biopolymers. SERS has been
employed directly for ultrasensitive quantification of prion-
8
9
10
11
ic, green or yellow fluorescent proteins, and others. Most
of these determinations were carried out in lab solutions
since the analysis of protein markers in real samples is ex-
tremely challenging due to both the intrinsic complexity of
these samples and the usually low concentration (~pg/mL)
1
12
biopsy) have a considerable potential for the diagnosis and
and poor SERS cross-section of the targets. A possible solu-
2
monitoring of cancer. Circulating tumor cells (CTC) and
tion relies on the functionalization of the plasmonic materi-
als with chemical species (chemoreceptors) with high SERS
cross-section and displaying high binding affinity and speci-
3
circulating tumor nucleic acids are all options, alongside
1
protein markers. However, the protein expression patterns
1
2
in plasma circulating tumor cells as well as in peripheral
blood mononuclear cells fractions remain a largely unex-
ficity for the target protein. In this approach, detection is
determined by the spectroscopic changes in the chemorecep-
1
13
plored aspect.
tor SERS spectrum upon binding with the target. Species
The protein c-MYC is a helix-loop-helix leucine zipper
bHLHZ) transcription factor encoded by the corresponding
such as antibodies, aptamers or peptides can be used as
chemoreceptors. However, these molecules are often char-
1
4
(
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Figure 1. (A) UV-Vis spectra of the AgNPs in solution (red) and
aggregated either in cast film (yellow) or on the silica beads (blue).
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(
B) SERS spectra and (C) comparison of the intensities (1075 cm ) of
Figure 2. (A) Optical images at low (×5) and high (×50) magnifica-
MB-H1 on a spin-coated film of non-aggregated AgNPs (sp, red), a
film of aggregated AgNPs produced by drop casting and air-drying
tion of beads from a 100 µL aliquot of suspensions with concentra-
tions of 2400 (upper panels) and 240 (bottom panels) beads/mL. (B)
SERS spectra of MB-H1 at different concentration on SiO2@Ag
5
2
0 µL of solution in a glass slide (a, yellow), and SiO @Ag (b, blue).
(
2400 beads/mL and, highlighted, 240 beads/mL), and (C) their
-1
acterized by a remarkable small SERS activity, which severely
limits their application for direct ultradetection. This issue
can be addressed by derivatizing the macromolecular recep-
tor with a terminal moiety with high SERS cross-section and
affinity for the plasmonic surface to mediate the interaction
corresponding intensity at 1075 cm .
no appreciable SERS signal is observed for the non-
aggregated AgNPs slides, while the aggregated AgNPs layer
and SiO @Ag display similar SERS intensities but with small-
2
1
4a
er standard deviation for the case of beads. The large size of
these beads allows their easy localization with a low magnifi-
cation objective (e.g., ×5) for the later scanning with a higher
resolution objective (e.g., ×50 or ×100) (Figure 2A). This
paves the way for a drastic reduction of the amount of plas-
monic material required for sensing, which in turn improves
the sensitivity by increasing the number of molecules per
bead ratio. This is exemplified in Figures 2B and C, which
show that SERS of MB-H1 is distinguishable down to 10 pM
for a particle concentration of 2400 beads/mL, while lower
between the receptor and the metallic substrate. In this
communication, we designed and prepared a SERS-active
peptide-conjugate (MB-H1) that binds c-MYC with high
affinity and selectivity. Silica microbeads rather homogene-
ously coated with silver nanoparticles (SiO @Ag) were ap-
2
propriately biofunctionalized with the chemoreceptor
(
SiO @Ag@MB-H1) and successfully used for the quantifica-
2
tion of c-MYC protein on laboratory and real samples from
cancer patients and healthy donors.
The chemoreceptor is based on a double mutant peptide
520
522
SiO @Ag amounts (240 beads/mL) allows for detection down
(
Ser → Ala, Phe → Ala) derived from the helix 1 of the
2
515
528
to 1 pM.
helix-loop-helix region of c-MYC ( NELKRAFAALRDQI
,
Capturing of the c-MYC target by MB-H1 bound to
H1), which is known to interact with high affinity and selec-
15
SiO @Ag beads could result in a large set of different pertur-
tivity with c-MYC. In order to build the SERS probe, we
2
bations on the SERS spectra of the mercaptobenzoyl moiety,
ranging from subtle shifts of the vibrational features to the
appearance of new bands. For interpreting such variations,
we acquired the Raman and SERS spectra of the SERS-active
terminal moiety (4-mercapto-N-methylbenzamide, MMB),
and those of MB-H1 before and after reaction with c-MYC
orthogonally attached a 4-mercaptobenzoyl unit to the
sidechain amino group of a Lys residue appended at the C-
terminus of the H1 peptide. Such modification should not
alter the intrinsic helical propensity of the peptide, or its c-
MYC-binding properties. The synthesis of the MB-H1 pep-
tide-conjugate is described in the supporting information
(S.I). Citrate-capped silver nanoparticles (AgNPs) of ca. 65
nm diameter (Figures S5A and B) were electrostatically de-
(
Figure 3A). The SERS spectra of MMB and MB-H1 display
similar vibrational patterns but with some differences in
relative intensities. This result points toward a slight change
of the molecular orientation of the mercaptobenzene group
on the silver surface when is coupled with a larger molecule
such as H1, in full agreement with the surface selection
posited onto 8 µm diameter silica beads (SiO @Ag). Previ-
2
ously, the negatively charged silica microparticles were se-
quentially coated with polyelectrolytes (polyethyleneimine,
PEI, positive), polyacrylic acid (PAA, negative), and again
PEI, using the layer-by-layer (LbL) assembly protocol (Fig-
17
rules. Such perturbation is exacerbated upon binding with
16
the very large c-MYC protein.
ures S5C-E). The UV-Vis spectrum of the prepared AgNPs
In order to identify the vibrational modes of the mercap-
tobenzene moiety which are more sensitive to its surface re-
orientation, we carried out DFT calculations on MMB at the
(
Figure 1A) shows a defined localized surface plasmon reso-
nance centered at 449 nm. Nanoparticle deposition onto a
solid support results in the appearance of an intense and
broad feature at longer wavelength, which is an indicator of
the interparticle coupling. The SERS fingerprint of MB-H1 on
1
8
RB3LYP-6311++G(d,p) theory level (Figure 3A). Results indi-
cate that MMB belongs to the C1 point group of symmetry,
implying that its structure is not planar. However, due to the
SiO @Ag is shown in Figure 1B (blue curve). Remarkably, the
2
1
9
strong interaction silver-thiol, we can assume a quasi-
perpendicular orientation of the phenyl ring onto the met-
SiO @Ag offer large surface area, close adsorbent-adsorbate
2
interaction and high density of electromagnetic hot spots,
thus combining the benefits of colloids and films, while
overcoming important limitations of both. In this regard, we
also produced silver-coated glass slides via spin-coating of
either non-aggregated (Figure S6B) or aggregated MB-H1-
modified AgNPs to compare and evaluate the SERS efficiency
20
ʌľ
al. On the other hand, the surface electric field, ̿, effective-
21
ly has only a normal component (Z direction in Figure 3B).
Based on these considerations, the intensity of a vibrational
mode results proportional to the square of the scalar product
of the electric field and the dipole moment derivative of the
22
of SiO @Ag@MB-H1. Results (Figures 1B and C) indicate that
mode. This scenario defines two degrees of freedom for the
2
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Figure 3. (A) Theoretical and experimental Raman spectrum of MMB and SERS spectra of MMB, MB-H1, and MB-H1 in presence of c-MYC, on
SiO @Ag. Magnification of the spectral windows between 730-800 and 990-1050 are also shown. (B) Model used in the estimation of the molecu-
2
lar orientation. Absolute orientation of the molecule on the surface and relative orientation of the ring over the surface are represented by XYZ
and xyz axes, respectively. (C) Vibrational mode assignment based on DFT calculations (theoretical values are reported into brackets).
phenyl ring to change its orientation upon interaction with
other substances: and χ (Figure 3B). is the tilt angle of the
z axis of the mercaptobenzene unit with the surface normal
Z). While, χ is the twist angle of the molecular plane around
the z axis (which is 0 degrees when y is parallel to the sur-
face).
following 24 h after extraction to obtain the mononuclear
cells by a Ficoll process. The mononuclear cells collected
from the blood samples as well as those from an epithelial
tumor cell line (MCF7) were lysed and centrifuged. Superna-
tants were then extracted and, for ELISA, the total protein
amount was quantified by the Bradford protein assay. Levels
of c-MYC were then determined with a total-human c-MYC
θ
θ
(
The theoretical Raman MMB spectrum shows good corre-
lation with the experimental observations, which allows the
safe assignation of the vibrational modes. The SERS spec-
trum of MBB displays weak out-of-plane modes, related to
ELISA kit. In the case of SERS, SiO @Ag@MB-H1 (100 µL of a
2
2400 beads/mL solution, which is equivalent to a concentra-
6
tion of 2.5 × 10 molecules of MB-H1 per bead) were directly
-1
the ring plane, at 756 and 997 cm , confirming a perpendicu-
added to each of the supernatants up to a final concentration
of ~218 beads/mL. Then, 100 µL aliquots were cast on glass
slides, and beads were analyzed by SERS before the evapora-
tion of the solvent. Figure 4B shows the SERS spectra for
both c-MYC standards (in dotted thin lines), the same as
used for calibration in ELISA, and the real human samples
(in solid thick lines). The corresponding calibrations curves
obtained for each method are presented in Figure 4C. Nota-
bly, both ELISA and SERS show adequate correlation coeffi-
cients with analogous c-MYC detection limits, albeit with a
slightly larger standard deviation for the optical method.
However, SERS results are obtained within minutes, while
ELISA assays require lengthy 4-5 h preparation procedures.
Similar results are obtained for real samples (Figure 4D).
Finally, MCF7 yields to much higher values than real sample,
probably because in that case all cells in the culture are can-
cerous, where in the real samples just a small fraction are
tumor cells.
In summary, we have developed a rapid, highly specific
and sensitive SERS assay for the determination of c-MYC in
real blood samples. The sensing scheme relies on the use of
specifically designed hybrid materials consisting of silica
microparticles rather homogenously coated with interacting
silver nanoparticles. The metallic surfaces are bioderivatized
with a c-MYC binding peptide modified with a 4-mercapto-
N-methylbenzamide antenna (MB-H1) that transduces the
specific c-MYC recognition events into a measurable altera-
tion of the SERS signal from MB-H1. Deconvolution of the
chemoreceptor vibrational fingerprint allowed us to identify
characteristic vibrational modes affected by c-MYC complex-
ation. The extent of such spectral changes was quantitatively
lar orientation of the phenyl group over the surface. When
MMB is included in the MB-H1 structure, we observe a slight
intensity increase of these features. More importantly, this
effect becomes remarkable when MB-H1 interacts with the
oncoprotein due to the c-MYC massive size (57 kDa). In fact,
the rise of the out-of-plane bands intensities, up to the same
-
1
level of the ring breathing at 1075 cm , indicates the adoption
of a flatter orientation of the ring onto the surface. This is
further supported by the drastic intensity increase of the N-C
-
1
stretching mode (1022 cm ) upon conjugation with c-MYC.
Selection of the spectral markers for c-MYC sensing should
take into account that the acquired SERS spectra result from
the contributions of both interacting and non-interacting
MB-H1 molecules. Thus, rather than monitoring the absolute
intensity of a specific feature, c-MYC quantification was
performed by recording the ratiometric intensities I756/I
1075
–1
(
ratio between the out-of-plane CCH deformation at 756 cm
–1
and the in-plane ring breathing at 1075 cm ).
The efficiency of our sensor was demonstrated with both
tumor cell lines and real human samples, while ELISA was
performed to validate the results. The study has received
ethical approval by the Ethics and Clinical Research Commit-
tee of the HM Hospitales Group, and all participants signed a
written informed consent form. Figure 4A shows a scheme of
the protocol followed for the quantification of c-MYC by
ELISA and SERS. Blood samples were obtained from two
healthy donors and a patient diagnosed of ovarian adenocar-
cinoma (stage IV). The patient was in progression at the time
of sample collection. The presence of the tumor was con-
firmed histologically in compliance with common standards.
Each sample of peripheral blood (8 mL) was processed in the
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Figure 4. (A) Protocol for the isolation of mononuclear cells in human blood samples from a cancer patient and several healthy donors. (B)
SERS spectra of SiO @Ag@MB-H1 in the presence of decreasing concentrations of c-MYC (thin dotted lines) and of real samples (thick solid
lines). (C) Calibration curves for ELISA (red) and our optical sensor (blue). (D) Results obtained for an epithelial tumor cell line (MCF7), and
samples extracted from a cancer patient (CP) and two healthy donors (HD1 and HD2). ELISA in red, and optical sensor in blue. Concentration of
c-MYC per million of cells.
2
correlated with the c-MYC content in complex biological
media of real samples.
(3) Crowley, E.; Di Nicolantonio, F.; Loupakis, F.; Bardelli, A. Nat. Rev.
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ASSOCIATED CONTENT
Supporting Information
Mautner, J.; Bornkamm, G. W.; Gerbitz, A. PLoS ONE 2013, 8, e77375.
(6) Nie, Z.; Hu, G.; Wei, G.; Cui, K.; Yamane, A.; Resch, W.; Wang, R.;
Green, Douglas R.; Tessarollo, L.; Casellas, R.; Zhao, K.; Levens, D. Cell
2012, 151, 68.
Experimental section; general peptide synthesis procedures;
synthesis, characterization and functionalization of SiO2@Ag
beads; cell and human samples preparation. Additional
SEM/TEM and SERS spectra. This material is available free of
charge via the Internet at http://pubs.acs.org.
(7) Schlücker, S. Angew. Chem. Int. Ed. 2014, 53, 4756.
(8) AlvarezꢀPuebla, R. A.; Agarwal, A.; Manna, P.; Khanal, B. P.; Aldeaꢀ
nuevaꢀPotel, P.; CarbóꢀArgibay, E.; PazosꢀPérez, N.; Vigderman, L.;
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USA 2011, 108, 8157.
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AUTHOR INFORMATION
Corresponding Authors
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8446.
(10) Singhal, K.; Kalkan, A. K. J. Am. Chem. Soc. 2010, 132, 429.
(11) (a) Lin, L.; Tian, X.; Hong, S.; Dai, P.; You, Q.; Wang, R.; Feng, L.;
E-mails: ramon.alvarez@urv.cat; joseluis.mascarenas@usc.es;
egarcia@hmhospitales.com
Xie, C.; Tian, Z.ꢀQ.; Chen, X. Angew. Chem. Int. Ed. 2013, 52, 7266; (b)
Feng, M.; Tachikawa, H. J. Am. Chem. Soc. 2008, 130, 7443.
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(12) AlvarezꢀPuebla, R. A.; LizꢀMarzan, L. M. Chem. Soc. Rev. 2012, 41,
4
3.
The authors declare no competing financial interests.
(13) Guerrini, L.; Arenal, R.; Mannini, B.; Chiti, F.; Pini, R.; Matteini, P.;
AlvarezꢀPuebla, R. A. ACS Appl. Mater. Interfaces 2015, 7, 9420.
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ACKNOWLEDGMENT
This work was funded by Marie Curie Actions (FP7/2007-
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No. 340055) the Spanish MINECO (CTQ2014-59808R,
SAF2013-41943-R, CTQ2015-70698-R, CTQ2013-49317-EXP
and the orfeo-cinqa network), the Generalitat of Catalonia
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