Elsevier

Talanta

Volume 139, 1 July 2015, Pages 35-39
Talanta

Coomassie brilliant blue R-250 as a new surface-enhanced Raman scattering probe for prion protein through a dual-aptamer mechanism

https://doi.org/10.1016/j.talanta.2014.12.050Get rights and content

Highlights

  • Coomassie brilliant dye was employed as Raman reporter and utilized to label the target protein.

  • Dual-aptamer was addressed, leading to high specificity towards PrP.

  • Silica shell of the nanoparticles served as versatile substrate for apamter immobilization.

  • The ensemble of Ag@Si NPs was utilized as the SERS substrate.

Abstract

Surface-enhanced Raman scattering (SERS) spectra, which can provide large information about trace amount of chemical and biological species have been widely performed as a well-established tool in complex biological system. In this work, coomassie brilliant blue (R-250) with high affinity to proteins and high Raman activity was employed as a Raman reporter to probe prion protein (PrP) through a dual-aptamer mechanism, and thus an original strategy for PrP determination was proposed, which showed great potential to turn on the SERS response through specific recognition of anti-prion aptamers towards the target protein. Aptamers (Apt1 and Apt 2) recognizing distinct epitopes of PrP with high affinity were first conjugated to Ag@Si NPs, and Ag@Si-PrP/R-250-Ag@Si conjugates were obtained in the presence of PrP/R-250, inducing dramatically enhanced Raman signal. SERS responses enhanced with increasing amount of PrP and a linear equation of ISERS=6729.7+3091.2 cPrP was obtained in the range of 3.0–12.0×10−9 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.

Graphical abstract

Coomassie Brilliant Blue R-250 was successfully applied as a new Surface-enhanced Raman scattering reporter for protein labeling through a dual-aptamer mechanism, and an original strategy for PrP determination was proposed, which is simple, rapid, and high specific.

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Introduction

Prion diseases or transmissible spongiform encephalopathies (TSEs), are a group of fatal neurodegenerative disorders that affect the nervous system in human and animals [1]. In the past decades, the emergence of variant Creutzfeldt–Jakob disease (vCJD), a kind of prion disease happened in human, has put prions in the spotlight due to its potential epidemic in human [2], [3]. The causative event of TSEs is believed to be the conformational conversion from cellular form (PrPC) to disease-causing isoform (PrPSc) [4], which can accumulate in the brain and lead to disease pathogenesis [5]. Although these rare but unique neurodegenerative disorders have attracted so much attention, a lot of questions remain far from clear, and a simple determination method of prions is meaningful and compulsory. Recently, Surface-enhanced Raman scattering (SERS) spectra, which can provide large information on the chemical structure of the probed substances, have been successfully performed as a well-established tool in complex biological system [6], [7], [8]. SERS-based method as a sensitive analytical technique has been used to investigate the prion protein expression and PrPC–Cu(II) interaction [9] and rapid detection of scrambled prions [10] in complex biological media. In addition, with its high sensitivity and selectivity, SERS spectroscopy can be applied for protein–ligand interactions without any label or fixation [11].

Since the discovery of aptamer, it has been widely used as an excellent ligand to recognize prions [12], and many high sensitive detection methods have been achieved [13], [14]. Aptamer is a type of DNA or RNA oligonucleotides that can bind to various molecular targets such as small molecules, proteins, nucleic acids even cells, tissues and organisms [15]. Compared to the traditional specific lock-key (antibody–antigen) recognition, aptamer has its own advantages, including easy preparation, good stability, reusability, high affinity and selectivity. As a result, aptamers have been widely applied in molecular recognition [16], cancer diagnosis [17], protein detection [18] and imaging in vivo [19]. PrP has been reported possessing two distinct binding epitopes for two aptamers. One aptamer (Apt1) recognizes epitope 23–90 of the N-terminal [20], while the other one (Apt2) specifically binds with the 90–231 of prion, which is corresponded to the β-sheet structure of PrP [21]. With the involvement of both of the anti-prion aptamers, which possess high affinity to the target, a high selective strategy was proposed to investigate the prions–aptamer interaction.

Coomassie dyes, which have been widely used to stain proteins [22], can bind to proteins with high affinity by physisorption to aromatic amino acids and other amino acids (e.g.: Arginine and Proline) [23], and it has been of quite importance for protein analysis due to its convenience and high sensitivity. Considering the properties of the dyes and their strong Raman activity, brilliant blue R-250 was employed to label the target proteins and applied as the Raman reporter. Recently, increasing experimental evidence demonstrates that metallic nanoparticles, especially gold or silver nanoparticles, were reported possessing resonant excitation of plasmons [24], [25], [26], while assembly of the nanoparticles has been applied as ideal SERS substrates due to their optical excitations known as plasmon resonance scattering (PRS) properties [27], [28], [29]. Considering the unique properties of silver nanoparticle and excellent biocompatibility of silica, core–shell nanoparticle architecture-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 brilliant blue (R-250) as Raman reporter and protein labeling.

Section snippets

Materials

Two aptamers of anti-prion protein, Apt1, NH2-CTT ACG GTG GGG CAA TT, and Apt2, GTT TTG TTA CAG TTC GTT TCT TTT CCC TGT CTT GTT TTG TTG TCT-NH2, were synthesized by Sangon Tech. Ltd. (Shanghai, China) without further purification. AgNO3 (99.8%, Tongbai Xinhong Silver Products Co. Ltd, Henan, China), (3-Aminopropyl) triethoxysilane (APTES, 98%, Sigma-Aldrich), Tetraethyl orthosilcate (TEOS, ≥99%, Fluka), and 1-ethyl-3-(3-(dimethylamino) propyl)-carbodiimide (EDC, Sigma) were used as received.

Characterization of aptamer-modifed Ag@Si NPs

Experimentally, AgNPs were prepared by the reduction of silver 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). Monodispersed Ag@Si NPs (Fig. 1a) were obtained according to the literature with little modification. Quantitative analysis of the thickness of silica shell was performed by measuring the shell from a large amount of individual particles by the software of Nano Measurer 1.2 software, which showed silica shell

Conclusions

In this communication, an original coomassie brilliant blue R-250-based labeling method for prion protein determination was proposed and employed to investigate protein–aptamer interaction. This method shows great potential to turn on the SERS response through specific recognition of aptamers towards the target protein, with the involvement of a common used cheap dye. On one hand, dual-aptamer strategy was addressed for its excellent recognition and selectivity to the target protein. Second,

Acknowledgment

This work was supported by the grants from the Ministry of Science and Technology of People׳s Republic of China (No. 2011CB933600) and the National Natural Science Foundation of China (NSFC, 21035005).

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