Elsevier

Analytica Chimica Acta

Volume 689, Issue 2, 18 March 2011, Pages 243-249
Analytica Chimica Acta

Magnetic relaxation switch and colorimetric detection of thrombin using aptamer-functionalized gold-coated iron oxide nanoparticles

https://doi.org/10.1016/j.aca.2011.01.046Get rights and content

Abstract

We describe a sensitive biosensing system combining magnetic relaxation switch diagnosis and colorimetric detection of human α-thrombin, based on the aptamer–protein interaction induced aggregation of Fe3O4@Au nanoparticles. To demonstrate the concept, gold-coated iron oxide nanoparticle was synthesized by iterative reduction of HAuCl4 onto the dextran-coated Fe3O4 nanoparticles. The resulting core–shell structure had a flowerlike shape with pretty narrow size distribution (referred to as “nanorose”). The two aptamers corresponding to human α-thrombin were conjugated separately to two distinct nanorose populations. Once a solution containing human α-thrombin was introduced, the nanoroses switched from a well dispersed state to an aggregated one, leading to a change in the spin–spin relaxation time (T2) as well as the UV–Vis absorption spectra of the solution. Thus the qualitative and quantitative detection method for human α-thrombin was established. The dual-mode detection is clearly advantageous in obtaining a more reliable result; the detection range is widened as well. By using the dual-mode detection method, a detectable T2 change is observed with 1.0 nM human α-thrombin, and the detection range is from 1.6 nM to 30.4 nM.

Introduction

Due to the remarkable chemical and physical properties on the nanometer scale, various types of nanoparticles have been extensively studied for numerous biotechnological applications [1]. For example, magnetic nanoparticles are widely used in tissue imaging, drug delivery, molecular sensing and bio-separation. Since the first magnetic resonance based assay was developed by Weissleder and co-workers [2], superparamagnetic nanoparticle based biosensors, also known as magnetic relaxation switches (MRSw), have been designed to identify and quantify a wide variety of target analytes [3], [4], [5], [6], [7], [8], [9]. The potential to probe nonoptical samples may eliminate time-consuming and complex processing steps, which makes MRSw assay an attractive biosensing approach. The principle underlying the detection mechanism of MRSw assay is based on the switch of the magnetic nanoparticles between a dispersed and aggregated state upon target interaction, with a concomitant change in the spin–spin relaxation time (T2) of the solution's water protons around the magnetic nanoparticles [10]. On the basis of changes in T2 (δT2) the analyte concentration can be determined. Thus the size distribution and the stability in solution are important factors in selecting superparamagnetic nanoparticles for use in MRSw assay application. Cross-linked iron oxide (CLIO) is the most usually used superparamagnetic nanoparticle, owing to its narrow size distribution and excellent stability in a variety of fluids. Now, a number of CLIO-based MRSw sensors have been developed [11]. For example, Westmeyer et al. [12] reported an enzyme reporter system using CLIO as a turn-on sensor. Osborne et al. [13] covalently attached spiropyran derivative to CLIO, which was used as a “smart” T2 agent that was reversibly activated by a visible light stimulus. However, most of these sensors have linear T2 response over a comparatively narrow concentration of analytes. Overtitration results in erratic T2 values, which arise from the CLIO clusters becoming unstable in solution [5], [10]. On the other hand, gold nanoparticle-based colorimetric biosensor has been increasingly applied for the detection of a large variety of targets. Sharing similar dispersion-to-aggregation process with MRSw assay, the gold nanoparticle-based colorimetric biosensor has a broader detection range, and typically the detection limit is in the range of nM to μM without signal amplification steps [14].

As we all know, the multifunctional gold-coated iron oxide nanoparticles (Fe3O4@Au) integrate excellent surface chemistry, special optical properties, and superparamagnetic properties in a core–shell structure, which makes them extremely interesting for optical, magnetic, and biomedical applications. So far, many research works on synthesis of Fe3O4@Au nanoparticles have been reported, and Fe3O4@Au nanoparticles with controlled plasmonic and magnetic properties can be successfully synthesized by various methods [15], [16], [17], [18]. We herein propose that the core–shell structures with combined magnetic and optical properties can provide a novel platform for MRSw assay, using the magnetic property, and colorimetric sensing, using the optical property. Thus the potential of such multifunctional nanoparticle is enhanced, and the application is broadened as well. The Fe3O4@Au nanoparticles can be excellent multifunctional sensors especially in aptamer-based protein detection. For one thing the Au shell provides enhanced stability and allows for convenient surface-modification with aptamer, for another we can get a δT2 as well as a colorimetric change, by comparing the colorimetric result with the MRSw one, we are more confident to quantify the analytes. To the best of our knowledge there is no published paper using Fe3O4@Au nanoparticles for MRSw assay or colorimetric detection application.

Based on these principles, we herein report the application of Fe3O4@Au nanoparticles as double-functional sensors for the detection of protein. Flowerlike Fe3O4@Au nanoparticles reported by Ma et al. [15] were chosen as they could stay monodispersed in solution for a long time, also they were easy to synthesize. Human α-thrombin (TB) with its two binding aptamers was chosen as the target analyte of interest. TB is a common but critical protein that naturally functions as a blood clotting factor to convert fibrinogen to firin. Excessive TB will induce thrombosis while low content of TB will induce an excessive bleeding. Two aptamers have been identified to bind with TB: a 29-mer aptamer (Apt29), which binds to the heparin-binding site of TB (Kd  0.5 nM), and a 15-mer aptamer (Apt15), which binds to the fibrinogen-binding site of TB (Kd  26 nM) [19]. Fig. 1 schematically shows the procedure of the aptamer sandwich assay that is generally applicable to the analysis of proteins. To assay for TB, the thiolated aptamers were conjugated to nanoroses seperately through the strong Au–S bond. In the absence of TB, the aptamer-functionalized nanoroses (Apt29-nanoroses and Apt15-nanoroses) dispersed well in the solution. However, Once a solution containing TB was introduced, the nanoroses switched from a well dispersed state to an aggregated one, leading to a change in the spin–spin relaxation time (T2) as well as the UV–Vis absorption spectra of the solution. Thus two distinct signals were obtained and a comparison between them could be made.

Section snippets

Reagents

Chloroauri acid (HAuCl4·4H2O), ferric chloride (FeCl3·6H2O, 99.0%), ferrous chloride (FeCl2·6H2O, 99.0%) and dextran (MW = 20,000 g mol−1) were purchased from Shanghai Chem. Corp. Human α-thrombin and bovine serum albumin (BSA) were purchased from Aldrich. The aptamers of TB used in this study had the following sequences: Apt29, 5′-SH-(CH2)6-T8-AGT CCG TGG TAG GGC AGG TTG GGG TGA CT-3′; Apt15, 5′-SH-(CH2)6-T8-GGT TGG TGT GGT TGG-3′. They were synthesized and purified by Sangon Corp., Shanghai. DNA,

Preparation and characterization of nanoroses

The iron oxide nanoparticle cores were synthesized in the presence of dextran, and then the nanoroses were formed by the reduction of HAuCl4 onto the surfaces of iron oxide nanoparticles with hydroxylamine as a seeding agent and dextrose as a mild reducing agent. Transmission electron microscopy (TEM) and energy-dispersive spectroscopy (EDS) were used to study the particle structure. The TEM images show that the nanoroses have flowerlike cluster shape, the diameter ranged from 50 to 70 nm with a

Conclusions

Aptamer-functionalized Fe3O4@Au nanoparticles were synthesized and employed as magnetic relaxation and colorimetric switches for the first time. The method developed here is promising in the detection of TB with a high sensitivity and specificity. Similar approaches can also be applied to other aptamer-based protein assay. The combination of MRSw diagnostics and colorimetric detection utilizes the special properties of both Au and Fe3O4 with a good result, which can be a successful attempt to

Acknowledgments

This work was supported by Shanghai Nano Project (0852nm03800), Shanghai Leading Academic Discipline Project (B109), Shanghai Academic Leader Project (08XD14009), National Natural Science Foundation of China (Nos. 20805009 and 20890022) and Natural Science Foundation of Fujian Province of China (Grant no. 2007J0009).

References (39)

  • D.M. Tasset et al.

    J. Mol. Biol.

    (1997)
  • H. Yang et al.

    Electrochem. Commun.

    (2009)
  • P. Alivisatos

    Nat. Biotechnol.

    (2004)
  • L. Josephson et al.

    Angew. Chem. Int. Ed.

    (2001)
  • T. Atanasijevic et al.

    Nat. Protocols

    (2007)
  • J.M. Perez et al.

    Nat. Biotechnol.

    (2002)
  • I. Koh et al.

    Anal. Chem.

    (2009)
  • M.V. Yigit et al.

    Bioconjugate Chem.

    (2008)
  • J.M. Perez et al.

    J. Am. Chem. Soc.

    (2003)
  • H. Lee et al.

    Nat. Med.

    (2008)
  • H. Lee et al.

    Proc. Natl. Acad. Sci. U. S. A.

    (2009)
  • T.J. Lowery et al.

    Anal. Chem.

    (2008)
  • I. Koh et al.

    Sensors

    (2009)
  • G.G. Westmeyer et al.

    Angew. Chem. Int. Ed.

    (2010)
  • E.A. Osborne et al.

    J. Am. Chem. Soc.

    (2010)
  • W. Zhao et al.

    Chembiochem

    (2008)
  • L.L. Ma et al.

    ACS Nano

    (2009)
  • J.L. Lyon et al.

    Nano Lett.

    (2004)
  • I.Y. Goon et al.

    Chem. Mater.

    (2009)
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