Talanta 139 (2015) 35–39

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Talanta
journal homepage: www.elsevier.com/locate/talanta

Coomassie brilliant blue R-250 as a new surface-enhanced Raman

scattering probe for prion protein through a dual-aptamer mechanism
Ping Ping Hu a, Hui Liu a, Lei Zhan b, Lin Ling Zheng b, Cheng Zhi Huang a,b,n
a
Education Ministry Key Laboratory on Luminescence and Real-Time Analysis, College of Pharmaceutical Sciences, Southwest University,
Chongqing 400715, China
b
College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, China

art ic l e i nf o a b s t r a c t

Article history: Surface-enhanced Raman scattering (SERS) spectra, which can provide large information about trace
Received 29 October 2014 amount of chemical and biological species have been widely performed as a well-established tool in
Received in revised form complex biological system. In this work, coomassie brilliant blue (R-250) with high affinity to proteins
27 December 2014
and high Raman activity was employed as a Raman reporter to probe prion protein (PrP) through a dual-
Accepted 30 December 2014
aptamer mechanism, and thus an original strategy for PrP determination was proposed, which showed
Available online 8 January 2015
great potential to turn on the SERS response through specific recognition of anti-prion aptamers towards
Keywords: the target protein. Aptamers (Apt1 and Apt 2) recognizing distinct epitopes of PrP with high affinity were
Prion protein first conjugated to Ag@Si NPs, and Ag@Si-PrP/R-250-Ag@Si conjugates were obtained in the presence of
Aptamer
PrP/R-250, inducing dramatically enhanced Raman signal. SERS responses enhanced with increasing
Surface-enhanced Raman scattering
amount of PrP and a linear equation of ISERS ¼ 6729.7 þ3091.2 cPrP was obtained in the range of 3.0–
Ag@Si NPs
Protein labeling 12.0  10  9 M with the determination coefficient of 0.988. The proposed strategy is simple, rapid, and
high specificity to probe protein–aptamer recognition in the solution.
& 2015 Elsevier B.V. All rights reserved.

1. Introduction scrambled prions [10] in complex biological media. In addition,

with its high sensitivity and selectivity, SERS spectroscopy can be
Prion diseases or transmissible spongiform encephalopathies applied for protein–ligand interactions without any label or fixa-
(TSEs), are a group of fatal neurodegenerative disorders that affect tion [11].
the nervous system in human and animals [1]. In the past decades, Since the discovery of aptamer, it has been widely used as an
the emergence of variant Creutzfeldt–Jakob disease (vCJD), a kind excellent ligand to recognize prions [12], and many high sensitive
of prion disease happened in human, has put prions in the detection methods have been achieved [13–14]. Aptamer is a type
spotlight due to its potential epidemic in human [2–3]. The of DNA or RNA oligonucleotides that can bind to various molecular
causative event of TSEs is believed to be the conformational targets such as small molecules, proteins, nucleic acids even cells,
conversion from cellular form (PrPC) to disease-causing isoform tissues and organisms [15]. Compared to the traditional specific
(PrPSc) [4], which can accumulate in the brain and lead to disease lock-key (antibody–antigen) recognition, aptamer has its own
pathogenesis [5]. Although these rare but unique neurodegenera- advantages, including easy preparation, good stability, reusability,
tive disorders have attracted so much attention, a lot of questions high affinity and selectivity. As a result, aptamers have been
remain far from clear, and a simple determination method of widely applied in molecular recognition [16], cancer diagnosis
prions is meaningful and compulsory. Recently, Surface-enhanced [17], protein detection [18] and imaging in vivo [19]. PrP has been
Raman scattering (SERS) spectra, which can provide large informa- reported possessing two distinct binding epitopes for two apta-
tion on the chemical structure of the probed substances, have been mers. One aptamer (Apt1) recognizes epitope 23–90 of the N-
successfully performed as a well-established tool in complex terminal [20], while the other one (Apt2) specifically binds with
biological system [6–8]. SERS-based method as a sensitive analy- the 90–231 of prion, which is corresponded to the β-sheet
tical technique has been used to investigate the prion protein structure of PrP [21]. With the involvement of both of the anti-
expression and PrPC–Cu(II) interaction [9] and rapid detection of prion aptamers, which possess high affinity to the target, a high
selective strategy was proposed to investigate the prions–aptamer
n
interaction.
Corresponding author at: Education Ministry Key Laboratory on Luminescence
and Real-Time Analysis, College of Pharmaceutical Sciences, Southwest University,
Coomassie dyes, which have been widely used to stain proteins
Chongqing 400715, China. Tel.: þ 86 23 68254659; fax: þ86 23 688866796. [22], can bind to proteins with high affinity by physisorption to
E-mail address: [email protected] (C.Z. Huang). aromatic amino acids and other amino acids (e.g.: Arginine and

http://dx.doi.org/10.1016/j.talanta.2014.12.050
0039-9140/& 2015 Elsevier B.V. All rights reserved.
36 P.P. Hu et al. / Talanta 139 (2015) 35–39

Proline) [23], and it has been of quite importance for protein HR800 Spectrometer (HORIBA Jobin Yvon, France) with 532 nm
analysis due to its convenience and high sensitivity. Considering wavelength incident laser light.
the properties of the dyes and their strong Raman activity, brilliant
blue R-250 was employed to label the target proteins and applied as 2.3. Expression and purification of recombinant human prion protein
the Raman reporter. Recently, increasing experimental evidence
demonstrates that metallic nanoparticles, especially gold or silver The plasmid rhPrP23-231 was transformed to competent bacteria of
nanoparticles, were reported possessing resonant excitation of strain Escherichia coli BL21-DE3 and induced by isopropyl-D-
plasmons [24–26], while assembly of the nanoparticles has been thiogalactopyranoside (IPTG). The cells were harvested and then
applied as ideal SERS substrates due to their optical excitations purified according to the method developed by Xiao's group with
known as plasmon resonance scattering (PRS) properties [27–29]. some modifications [13–14,30]. Protein concentration was determined
Considering the unique properties of silver nanoparticle and excel- using the molar extinction coefficients ε280 ¼56,590 M  1 cm  1 for
lent biocompatibility of silica, core–shell nanoparticle architecture- rhPrP (23–231) [31].
silica coated silver (Ag@Si) NPs were utilized in this work for the
immobilization of target proteins as well as the SERS enhancement
substrate and an original PrP quantitative employing coomassie 2.4. Synthesis and functionalization of Ag@Si NPs
brilliant blue (R-250) as Raman reporter and protein labeling.
Silver nanoparticles (AgNPs) were prepared according to the
reference with small modification [32]. In short, 1.5 mL of 2% (w/w)
2. Material and methods trisodium citrate was added into a boiling 50.0 mL solution contain-
ing 1.0 mM AgNO3 in a conical flask, followed by continuous stirring
2.1. Materials and boiling for about 20 min. For the synthesis and DNA-modified
of Ag@Si NPs [33–35], 10 mL of the as-prepared silver colloid, which
Two aptamers of anti-prion protein, Apt1, NH2-CTT ACG GTG GGG was then centrifuged at 500 rpm for 1 h to remove larger NPs, was
CAA TT, and Apt2, GTT TTG TTA CAG TTC GTT TCT TTT CCC TGT CTT transferred into a conical glass flask containing 40 mL of ethanol,
GTT TTG TTG TCT-NH2, were synthesized by Sangon Tech. Ltd. followed by the addition of 0.8 mL of 25% ammonia and 1.5 mL of
(Shanghai, China) without further purification. AgNO3 (99.8%, Tongbai 10 mM TEOS ethanol solution under vigorous shaking, respectively,
Xinhong Silver Products Co. Ltd, Henan, China), (3-Aminopropyl) and the resulting solution further react for 24 h at 30 1C. Ag@Si NPs
triethoxysilane (APTES, 98%, Sigma-Aldrich), Tetraethyl orthosilcate were collected by centrifugation and further washed with ethanol
(TEOS, Z99%, Fluka), and 1-ethyl-3-(3-(dimethylamino) propyl)-car- three times. Subsequently, 0.2 mL of APTES was added to 4.8 mL of
bodiimide (EDC, Sigma) were used as received. Coomassie brilliant the as-obtained Ag@Si NPs and stirring at room temperature for 1 h,
blue R-250 was purchased from Beijing dingguo biotech CO. LTD. the reaction mixture was centrifuged to remove the unreacted
Other commercial reagents were analytical reagent grade without APTES, and then heated to 120 1C for 1 h. Lastly, the aminated Ag@Si
further purification. The solutions were prepared using ultrapure NPs were modified with NH2-Apt according to the literature [33].
water, which was obtained through a Milli-Q Integral-5 water pur-
ification system (Millipore, U.S.A.) and had an electric resistance of 2.5. Protein labeling
18.2 MΩ.
A certain amount of PrP was mixed with 50 μL of 0.5 mg mL  1
2.2. Apparatus R-250 (dissolved in PBS) and incubated for 10 min, and then, 50 μL
of 1% BSA was added for blocking isolated R-250 molecules. After
The plasmon resonance absorption (PRA) of AgNPs and Ag@SiO2 these additions, the solutions were well mixed.
was measured with a Hitachi U-3010 spectrophotometer (Tokyo,
Japan). SEM observations were carried out on a Hitachi S-4800 2.6. General procedure
scanning electron microscopy (Tokyo, Japan). A Zetasizer Nano-ZS
System (Malvern Inc.) was used to detect the size of particles in For the detection of PrP, Ag@Si-Apt1 were added to a certain
solution based on photo correlation spectroscopy. The morphology of amount of PrP/R-250 conjugates at 37 1C for 30 min, followed by
Ag@Si NPs in the absence and presence of prion protein was the addition of Ag@Si-Apt2 and incubating at 37 1C for 1 h more in
observed on Nanoscope Quadrex atom force microscope (AFM) the presence of 20 mmol/L MES buffer (pH 6.25) and 0.25 mol/L
(Veeco, USA), and Raman spectra were obtained on a LabRan NaCl. Ag@Si aggregates were collected and washed by

Fig. 1. Features of Si@Ag NPs during the modification process with aptamer. SEM images of Ag@Si NPs (a), scale bar, 400 nm. (b) Size information of silica shell. (c) Extinction
spectra of AgNPs (black), Ag@Si NPs (red) and Ag@Si-Apt (cyan), respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the
web version of this article.)
 P.P. Hu et al. / Talanta 139 (2015) 35–39 37

centrifugation and resuspension. The SRES response was mea- NPs. On the other hand, Sybr Green I, a double-strand specific dye
sured with laser excitation at 532 nm the excitation at 490 nm. [36], was employed to confirm the conjugation of aptamer with the
nanoparticles. After being washed three times more, the vast majority
of the free aptamers in the solution were removed. The complex of
3. Results and discussion Ag@Si-Apt1 was mixed with the corresponding complementary DNA
(cDNA), resulting in a significant fluorescence enhancement of Sybr
3.1. Characterization of aptamer-modifed Ag@Si NPs Green I, as observed from Fig. A1, and similar results were observed
with Ag@Si-Apt2 (Data was not shown). All of these results proved
Experimentally, AgNPs were prepared by the reduction of silver the efficient conjugation of aptamers to the nanoparticles.
nitrate in water [32] with an average diameter of about 57.1 nm,
and the metal plasmon absorbance at 418 nm in water (Fig. 1). 3.2. State investigation of the designed dual-aptamer–PrP
Monodispersed Ag@Si NPs (Fig. 1a) were obtained according to the interaction assay
literature with little modification. Quantitative analysis of the
thickness of silica shell was performed by measuring the shell As shown in Scheme 1, anti-prion aptamers (Apt1 and Apt 2)
from a large amount of individual particles by the software of were first chemically coated on the surface of Ag@Si NPs. Then, a
Nano Measurer 1.2 software, which showed silica shell of the certain amount of PrP was mixed with R-250, and PrP/R-250
core–shell nanoparticle is about 9.4 nm (Fig. 1b), while the average bioconjugates were obtained, followed by the addition of Ag@Si-
diameter changed to 75.9 nm. Then aldehyde-functionalized Apt1 and Ag@Si-Apt2, which can bind to PrP through the specific
Ag@Si NPs were obtained via silica surface chemistry for biocon- recognition of the aptamers with their corresponding epitopes of
jugation with aminoterminated oligonucleotides, which was PrP, and Ag@Si-PrP/R-250-Ag@Si sandwiches were formed, lead-
applied as bioprobe for subsequent experiments. The character- ing to the aggregation of nanoparticles, sequentially inducing
istic absorption peaks of silver red shifted approximate 30 nm enhanced Raman scattering.
with the modifications (Fig. 1c). Dynamic light scattering (DLS) DLS was used to characterize the formation of the dispersed
measurements also revealed that the hydrodynamic diameters of assemblies (Table 1), and the corresponding hydrodynamic dia-
Ag@Si NPs increased evidently after the modification of aptamers meters of the sandwich product were found increased with the
(Data were not shown), and the corresponding ξ-potential increasing amount of protein, which correlates well with the
decreased significantly due to the conjugation of DNA, which plasmon resonance scattering (PRS) signals of silver nanoparticles.
possesses more negative charges, suggesting the modification of As demonstrated in Fig. A2, the PRS signal of the PrP-Ag@Si-Apt
aptamers on the surface of nanoparticles is successful and do not system increased significant in the presence of PrP, which further
induce apparent aggregation or significantly alter the morphology proved the recognition between PrP and the Apt, suggesting the
of the particles during the process of functional modification. present method could be successfully applied to the prion protein–
The hybridization efficiency was determined in a strong basic ligand interaction investigation. In order to confirm the
solution (pH 12) according to Ref. [33]. In detail, 0.2 mL of 0.25 M
NaOH solution was added into the suspension of Ag@Si-Apt and
further shaked for 4 h. After centrifugation and washing to remove Table 1
the free Ag@Si NPs particles, supernatant containing aptamers were DLS aggregate size comparisons for Ag@Si NPs at the present of PrP.
collected, and the corresponding concentration of oligonucleotides
was determined by its strong absorption at 260 nm (the molar Concentration of PrP/mM 0 0.17 0. 35 0.52

extinction coefficient of the aptamer is about 5.2  103 M  1 cm  1 Size(nm) a

571 1240 1472 1740
and 3.9  105 M  1 cm  1 for Apt1 and Apt2, respectively). And the
result showed the concentration of aptamers coupled with Ag@Si NPs a
Represents the size of hydrated species in solution, either nanorods or
is around 0.3 nM, which is nearly 10 times higher than that of Ag@Si aggregates, as determined by DLS.

Scheme 1. Schematic diagram shows the design and the formation of Ag@Si assemblies. (A), Ag@Si NPs were chemically modified with Anti-prion aptamers (Apt1 and Apt 2),
while PrP was mixed with R-250, and the PrP/R-250 conjugates were obtained. (B), Ag@Si-PrP-Ag@Si assembly formed in the presence of target protein leading to enhanced
Raman scattering, while quite low Raman signal was observed without PrP.
38 P.P. Hu et al. / Talanta 139 (2015) 35–39

conjunction of the assembly, atomic force microscopic (AFM) enhancement of the SERS response with the increasing concentra-
imaging was also achieved (Fig. 2), in which thickness of the tion of PrP was observed due to the biological recognition and the
aggregates increased significantly to 245.7 nm, and it was realized induced Ag@Si NPs assembly. It was found that the increased SERS
that not only two Ag@Si particles are involved in a Ag@Si-PrP– intensity at 1626 cm  1 followed a linear relationship with con-
Ag@Si complex, providing a convincing evidence for the assembly centration of the target protein, which could be expressed as
of Ag@Si NPs, which will also induce the high SERS effect. ISERS ¼6729.7 þ3091.2 cPrP in the range of 3.0–12.0  10  9 M with
the determination coefficient (r2) of 0.988.
3.3. SERS spectral feature of the sample For the specificity study, the SERS changes brought by other
coexisting proteins including α-amylase, lysozyme, ovalbumin,
As known, the target proteins are able to be labled rapidly by papain, pepsin, trypsin, BSA and proteinase K (PK) were compared
Raman dye (R-250), and SERS-based detection of prion–aptamer with that in the presence of PrP, and the results were showed in
interaction was carried out. Since the high-affinity of R-250 to Fig. 4. It was clear that PrP caused a significant enhanced Raman
proteins is nonspecific, BSA was involved in this experiment to change, while other coexisting proteins which has different iso-
block the free excessive dyes. As shown in Fig. A3, the absorption electric points (pI), even if at much higher concentrations, influ-
maximum of R-250 solution was located at 556 nm. While a red- enced the Raman signal slightly, suggesting that biomolecules
shift occurred with the increasing amount of proteins and the mentioned above could not interact with the aptamers to form the
absorption peak remained at around 562 nm until excess BSA was sandwich structure. Thus, the present method could be success-
available. And then, with the addition of Ag@Si-Apt1 and Ag@Si- fully applied for the selective detection of PrP, due to the inherent
Apt2, the isolated dyes reacted with BSA can be removed easily by specificity of aptamers to PrP. The proposed strategy, which is
centrifugation and resuspension. addressing two aptamers recognizing distinct epitopes of the
Fig. 3 demonstrated SERS intensity of the Ag@Si-PrP/R-250- target, is simple, rapid, and high specificity to probe protein–
Ag@Si system with the laser excitation at 532 nm, and dramatic aptamer recognition in the solution.

Fig. 2. The morphology of Ag@Si NP-Apt (a) and Ag@Si-PrP-Ag@Si assembly (b) was observed by tapping mode AFM images (6 nm  6 nm). Scale bar, 20 nm. 3.25  10  12 M
Ag@Si-Apt1 and Ag@Si-Apt2 were incubated with 0.52 mM PrP.

Fig. 3. The SERS response of the system with increasing amount of PrP. a, SERS spectra of R-250 probing Ag@Si aggregate with increasing PrP. b, PrP concentration-
dependent SERS intensity at 1626 cm  1 of R-250 from the R-250-PrP-Apt-Ag@Si system, and PrP concentrations from down to up were 0, 0, 3.0, 4.5, 6.0, 7.5, 9.0, 10.5 and
12.0  10  9 M. Concentrations of Ag@Si-Apt1 and Ag@Si-Apt2 are both 3.25  10  12 mol L  1.
 P.P. Hu et al. / Talanta 139 (2015) 35–39 39

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Appendix A. Supporting information

Supplementary data associated with this article can be found in

the online version at http://dx.doi.org/10.1016/j.talanta.2014.12.050.