Ultra-low fouling and functionalizable zwitterionic coatings grafted onto SiO2 via a biomimetic adhesive group for sensing and detection in complex media

doi:10.1016/j.bios.2010.03.012

Biosensors and Bioelectronics

Volume 25, Issue 10, 15 June 2010, Pages 2276-2282

https://doi.org/10.1016/j.bios.2010.03.012 Get rights and content

Abstract

Non-specific protein binding from human plasma and serum has severely hindered the full capabilities of biosensors concerned with cancer biomarker detection. Currently, there is a strong desire for developing new materials which allow for the convenient attachment of an ultra-low fouling and functionalizable surface coating which can be used for highly sensitive and label-free detection of target analytes directly from complex media. In this work, a short 20 min in situ “graft to” protocol using Tris pH 8.5 buffer was developed for zwitterionic carboxybetaine methacrylate (CBMA) polymer conjugates containing the adhesive biomimetic moiety, 3,4-dihydroxy-l-phenylalanine (DOPA), on SiO₂ substrates. Using a surface plasmon resonance (SPR) biosensor, different buffers, pH values, salt concentrations, and temperatures were investigated for determining the “graft to” conditions that yield dense polymer films which both minimize non-specific protein adsorption and maximize antibody immobilization. The optimized surface coatings were shown to be highly protein resistant to 100% human blood plasma and serum. Subsequent antibody functionalized surfaces without any blocking agents enabled the specific detection of the cancer biomarker ALCAM directly from undiluted human serum down to 64 ng/mL. The successful use of this zwitterionic surface coating for detection from complex media on SiO₂ surfaces indicates its potential for broad impacts in the development of implantable medical devices, in vivo diagnostics, and nano-scale biosensors.

Introduction

The inability to identify and verify suitable biomarkers using various sensing devices has been the major shortcoming in cancer diagnostics (Benson et al., 2006, Hanash et al., 2008, Sawyers, 2008). As many of these low concentration proteins are found in complex bodily fluids (e.g. plasma, serum, urine) (Feng et al., 2009, Radpour et al., 2009) the culprit underlying detection using biosensors is the overwhelming background noise as a result of non-specific protein adsorption (Cheng et al., 2006, Ferrari, 2005, Paulovich et al., 2008, Ullah and Aatif, 2009).

Recent studies have shown the ability of polymers containing the carboxybetaine (CB) moiety, formed via surface initiated atom transfer radical polymerization (SI-ATRP), to outperform commonly used protein resistant ethylene glycol (EG) based coatings. These polyCB (pCB) films were shown to be ultra-low fouling to 100% human plasma and serum (Ladd et al., 2008, Vaisocherova et al., 2008, Yang et al., 2009). It has been hypothesized that the surface hydration formed via ionic solvation due to the zwitterionic groups is primarily responsible for the ability of CB polymers to prevent protein adsorption (Chen et al., 2005). Subsequent functionalization with antibodies using the abundant carboxylic acid groups and convenient N-hydroxysuccinimide (NHS)/N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide (EDC) chemistry enabled sensitive and specific detection of analytes directly from undiluted human plasma (Vaisocherova et al., 2008).

However, many biosensors today incorporate the use of a variety of nanomaterials with differing geometries into their design (Chen et al., 2004, Zhang et al., 2009). The dimensions of the sensing surface have also been decreasing to the nanometer length scale (Kim et al., 2009). Therefore, other methods for creating a bifunctional coating on such devices, capable of highly sensitive detection from complex media, need to be developed.

As an alternative to SI-ATRP, there has been much attention focusing on developing “graft to” technologies due to their simplicity and convenience (Ryu et al., 2005). However, this technique requires a method for chemically attaching the non-fouling material “to” the substrate via some form of an adhesive moiety. During a study of surface modification strategies which could be applied to a variety of materials (Lee et al., 2007) it was found that the compound 3,4-dihydroxy-l-phenylalanine (DOPA) could strongly adhere to numerous substrates (noble metals, native oxides, ceramics, etc.).

Several studies on the ability of PEG polymers containing DOPA anchors for forming protein resistant coatings on oxide surfaces have been performed (Dalsin et al., 2005). It was found that the number of residues in the DOPA anchor significantly affected the surface density of the grafted polymer and its subsequent ability to resist protein adsorption. In previous work (Li et al., 2008), a single DOPA anchor conjugated to polymers composed of zwitterionic sulfobetaine methacrylate (SBMA) was shown to form protein resistant coatings on various surfaces. To increase the surface density of the grafted zwitterionic polymers and provide for functionalization capabilities, two linked DOPA–poly(carboxybetaine methacrylate) (DOPA₂–pCBMA₂) polymer conjugates was synthesized and grafted to gold substrates (Gao et al., 2010). Following antibody immobilization, the detection of a target protein directly from undiluted human plasma was demonstrated using a SPR biosensor.

Conventional SPR sensor chips are composed of a top layer of gold (∼48 nm) for which many surface chemistries have been developed. However, many other materials are incorporated into the design of biosensors today which include silica, titanium, platinum, among others (Archakov and Ivanov, 2007, Chen et al., 2004, Homola, 2008, Zhang et al., 2009). Thus, there has been a push to use available sensing platforms as tools for developing new surface chemistries for these materials. Silica is an attractive material for many biosensor platforms due to the widely established use of silicon (from which SiO₂ is formed) in the semiconductor industry which has enabled the development of convenient micro-fabrication methods. Silicon is currently used in many microelectromechanical system (MEMS) devices (Zhu et al., 2005) and has since been applied to developing nanoelectromechanical system (NEMS) based biosensors with recent advances in nanotechnology (Burg et al., 2007). Hence, new surface chemistries for this material need to be developed.

In this work, the “graft to” behavior of novel DOPA₂–pCBMA₂ polymer conjugates onto silica substrates was studied using a SPR sensor. The grafting conditions were investigated in order to both minimize biofouling and maximize protein immobilization efficiency. The ability of the DOPA₂–pCBMA₂ surface functionalized with antibodies to detect analytes directly from undiluted human blood serum was demonstrated using the cancer biomarker, activated leukocyte cell adhesion molecule (ALCAM, CD 166). Using a simple 20 min in situ attachment protocol, the detection of ALCAM at concentrations down to ∼64 ng/mL was enabled.

Section snippets

Materials

DOPA₂–pCBMA₂ polymer conjugates were synthesized as described previously (Gao et al., 2010). Sodium hydroxide (99.998%), tetrahydrofuran (THF, HPLC grade), N-hydroxysuccinimide (NHS), and N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were purchased from Sigma–Aldrich (Milwaukee, WI). Tris, crystallized free base (molecular biology grade) was purchased from Fisher Scientific. Fibrinogen (fraction I from bovine plasma), lysozyme (from chicken egg white), and 3-(N

SiO₂ coated SPR chips

In order for the SiO₂ thin film coated onto the SPR chips to serve as a model system for developing new surface chemistries for biosensing applications, it must be compatible to a variety of conditions (i.e. various temperatures, pHs, solvents, etc.) and maintain its stability during detections. Prior to grafting DOPA₂–pCBMA₂ onto the substrates, the stability of the SiO₂ layer was tested under a variety of pH values (1–10) and solutions/organic solvents (e.g. 3 M NaCl in PBS, DMF/water, THF,

Conclusions

A novel polymer conjugate containing ultra-low fouling and functionalizable zwitterionic pCB polymers and two DOPA moieties (for increased polymer surface coverage) was successfully grafted to SiO₂ coated SPR chips. SiO₂ is being increasingly incorporated into the design of novel NEMS devices which emphasize the importance of developing new surface chemistries for this material. Subsequent antibody functionalization enabled the detection of a target analyte directly from undiluted human serum

Acknowledgements

This work is funded by the University of Washington and the National Cancer Institute via SAIC-Frederick (28XS119). N.D.B. was supported by the Center for Nanotechnology at the University of Washington with funding from an IGERT Fellowship Award NSF #DGE-0504573.

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Ultra-low fouling and functionalizable zwitterionic coatings grafted onto SiO2 via a biomimetic adhesive group for sensing and detection in complex media

Abstract

Introduction

Section snippets

Materials

SiO2 coated SPR chips

Conclusions

Acknowledgements

J. Colloid Interface Sci.

Curr. Opin. Chem. Biol.

Biomaterials

J. Colloid Interface Sci.

Biomaterials

Cancer Treat. Rev.

Biosens. Bioelectron.

Int. J. Adhes. Adhes.

Eur. Polym. J.

J. Electrochem. Soc.

Mol. Biosyst.

Nature

Nature

J. Electrochem. Soc.

Biotechnol. Adv.

J. Am. Chem. Soc.

Langmuir

Plos Med.

Ultra-low fouling and functionalizable zwitterionic coatings grafted onto SiO₂ via a biomimetic adhesive group for sensing and detection in complex media

SiO₂ coated SPR chips