Surface passivation using oligo(ethylene glycol) in ATRP-assisted DNA detection

https://doi.org/10.1016/j.snb.2007.07.130Get rights and content

Abstract

We recently reported a DNA sensing method using polymerization to amplify signal outputs [X. Lou, M.S. Lewis, C.B. Gorman, L. He, Anal. Chem. 77 (2005) 4698–4705]. In the current report an optimization of sensor surface chemistry is conducted in which (S–CH2CH2–OEG6–OMe)2 is used as the preferred surface passivation agent to reduce nonspecific adsorption experienced during ATRP-assisted DNA detection. The presence of oligo(ethylene glycol) (OEG) moieties on the sensor surface significantly reduces background adsorption of proteins, polymers, and reaction monomers used during DNA detection. The level of reduction in background nonspecific adsorption closely depends on the incubation solvent and the incubation time used, as evidenced by the ellipsometric and surface plasmon resonance (SPR) results. Reflectance FTIR results suggest that the surfaces with moderately ordered SAMs exhibit better protein resistance, consistent to the previous observations. Subsequent DNA binding experiments show no apparent decrease in DNA hybridization and ligation efficiencies when OEGylated self-assembled monolayers are used as the passivation layer. Improved DNA detection sensitivity is achieved from reduced background noises.

Introduction

Analytical limit of detection (LOD) is, by definition, decided by analyte-specific signal output and the background signal level. Consequently, reduction of background noise from nonspecific adsorption is an important strategy to improve bioassay sensitivity. Mercaptohexanol (MCH) is one of the most popular passivation molecules used in DNA biosensors for its ability to remove loosely adsorbed DNA molecules and to align attached DNA molecules to a more accessible orientation [1]. It has also been used in our recently demonstrated DNA sensing platform where atom transfer radical polymerization (ATRP) was used as an effective means to amplify DNA sensing signal outputs [2]. While direct visualization of 1 nM DNA target molecules was achieved using this ATRP-assisted DNA detection approach, detection of lower concentrations of DNA molecules was hindered by the small but nevertheless discernable nonspecific background. To meet the modern genetic assays’ demand of detecting <10 copies of DNA in complex biological matrices, much improved detection sensitivity with lower background is needed [3].

A number of self-assembled monolayers (SAMs) with oligo(ethylene glycol) (OEG) motifs have been found to be more effective in resisting nonspecific adsorption of biomolecules on Au surfaces than simple thiolated alkanes. Extensive theoretical and experimental studies have been conducted in the past to understand the passivation mechanism of OEG-containing alkyl thiol SAMs [4], [5], [6]. It has been suggested that the ability of water molecules to penetrate surface layers determines the interaction between SAMs and biomolecules at the solid–liquid interface; hence the level of nonspecific adsorption on a solid surface [7], [8]. For example, Vanderah et al. have found that BSA adsorbs atop highly ordered helical OEG-SAMs, but not much on the disordered ones because of the greater chain solvation and superior conformation flexibility of the latter [9]. An optimal surface coverage of thiolated OEG molecules at 60–80% has been found to be the most effective in inhibiting protein adsorption [4], [10]. Deviation from this surface density range in either direction results in increased nonspecific adsorption. The importance of OEG chain hydration in surface passivation has been well-explained using both thermodynamics theories and molecular simulations [7], [11], [12].

In this report, we used a disulfide molecule with the OEG moiety, i.e. [S(CH2)2(OCH2CH2)6OCH3]2 (OEG6–OMe), as the passivation agent in the ATRP-based DNA sensing process. Different SAM immobilization conditions were examined to optimize surface anti-fouling capability. The amount of nonspecifically adsorbed materials was characterized using surface plasmon resonance (SPR), ellipsometry, and reflectance FTIR spectroscopy. The efficiency of DNA hybridization and detection when OEG6–OMe was used as the passivation layer was compared to MCH-protected substrates.

Section snippets

Materials

DNA molecules used in this study were purchased from Integrated DNA Technologies, Inc. (Coralville, IA). 2-Hydroxyethyl methacrylate (HEMA, 98%) and the inhibitor remover (to remove hydroquinone and monomethyl ether hydroquinone-based inhibitors in HEMA) were purchased from Sigma–Aldrich. An inhibitor-removal column was packed in house and used to purify HEMA. Dioxane, dithiothreitol (DTT), triethylamine (TEA), 6-mercapto-1-hexanol (MCH), copper(I) chloride (CuCl), copper(II) bromide (CuBr2),

Results and discussion

Symmetric disulfide SAM molecules with the OEG6–OMe moieties, i.e. [S–CH2CH2–OEG6–OMe]2 (OEG6–OMe), were chosen in our study because they are commercially available, relatively stable in comparison to the corresponding free thiolated PEG molecules, and capable of resisting protein nonspecific adsorption [7], [16]. These disulfide molecules also have a relatively slow immobilization rate that allows fine-tuning final surface densities by simply varying incubation times [17].

The benefit of using

Conclusions

Dialkyl disulfide molecules with oligo(ethylene oxide) moieties were used to self-assemble on Au substrates in place of MCH to prevent nonspecific absorption of proteins (BSA), synthetic polymers (PEG 8000) and small organic molecules (HEMA). Different incubation solvents and incubation times were studied to achieve the optimal blocking efficiency. Our results show that longer incubation in OEG6–OMe/H2O resulted in better prevention of protein adsorption when the SAM layer was close to but not

Acknowledgments

We thank Dr. Jan Genzer and Rajendra Bhat for the use of their ellipsometer in the measurement of film thickness. We also thank Dr. Franzen and his group for the help on reflectance FTIR measurements. NCSU FPRD Award is thanked for the financial support. Partial support from National Science Foundation (CHE-0644865) is acknowledged.

Dr. Xinhui Lou received her PhD degree under the direction of Prof. Lin He at North Carolina State University, and she is currently working as a postdoctoral associate at UC Santa Barbara under the guidance of Profs. Hyongsok (Tom) Soh and Alan J. Heeger.

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Dr. Xinhui Lou received her PhD degree under the direction of Prof. Lin He at North Carolina State University, and she is currently working as a postdoctoral associate at UC Santa Barbara under the guidance of Profs. Hyongsok (Tom) Soh and Alan J. Heeger.

Prof. Lin He is a chemistry faculty at North Carolina State University. Her research interests include development of radical polymerization-assisted biosensing and ordered nanoarray-assisted laser desorption/ionization mass spectrometry (NALDI-MS)-based metabolite profiling and chemical imaging.

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Current address: Department of Materials, University of California, Santa Barbara, Santa Barbara, CA 93106, United States.

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