Article pubs.acs.org/JPCC
Toward Plasmonic Biosensors Functionalized by a Photoinduced Surface Reaction Tina A. Gschneidtner,† Si Chen,‡ Jørn B. Christensen,§ Mikael Kal̈ l,‡ and Kasper Moth-Poulsen*,† †
Department of Chemical and Biological Engineering, Chalmers University of Technology, SE-412 96 Göteborg, Sweden Department of Applied Physics, Chalmers University of Technology, SE-412 96 Göteborg, Sweden § Department of Chemistry, University of Copenhagen, DK-2100 Copenhagen, Denmark ‡
S Supporting Information *
ABSTRACT: We present a method for efficient coupling of amine nucleophilic molecules of choice to a nanostructured gold surface via photoinduced surface chemistry. The method is based on photoactive self-assembled monolayers and can be used to functionalize localized surface plasmon resonance (LSPR) based biosensors with biorecognition motifs while reducing nonspecific binding via introduction of hydrophilic units. The photoactive linker molecule, 5-bromo-7-nitroindoline, couples nucleophilic molecules such as biotin ethylenediamine to a surface when exposed to UV-light. The specific, noncovalent recognition between biotin and streptavidin is used for demonstration of a simple biorecognition assay based on the LSPR sensing principle. By doing so, one can envision that the binding of any streptavidin fusion protein, being attached to specific spots at the gold surface, is monitored by an LSPR peak shift. Since the surface functionalization is based on a photoinduced reaction, this method can be used to functionalize the surface in a local and site-specific way, and biomedical applications such as drug-screening platforms, microarrays, solid support protein synthesis, and even single molecule experiments can be envisioned.
1. INTRODUCTION Functionalization of surfaces with active (bio)molecules is becoming increasingly important for several applications, including protein synthesis,1,2 oligonucleotide synthesis for DNA chips,3−7 drug-screening platforms, antifouling and antiseptic surfaces, and biosensors.8 In this context, photoinduced surface reactions are particularly interesting since they offer a simple strategy to functionalize surfaces in a local and site-specific way using spatially structured illumination. During recent years, several photoactive chemical groups have been employed in surface modification schemes, including onitrobenzyl (2-nitrobenzyl (NB),9−11 1-(2-nitrophenyl)ethyl (NPE)12), (coumarin-4-yl)methyl (7-dialkyamino group (e.g., diethylaminocoumarine (DEACM))),13,14 7-alkoxy group (e.g., methoxycoumarine (MCM), 7-carboxymethoxycoumarine (CMCM)),15−17 6,7-dialkoxy group (e.g., dimethoxycoumarin (DMCM)), 6-bromo-7-alkoxy group (Bhc),18,19 and nitroindoline (Bni).20−23 These examples include photocleavable protecting groups for amines,24 alcohols, sugars,10,25−27 thiols,28,29 and phosphoric acids for ATP or cAMP, for example,14,15,26,30−34 carboxylic acids,20,21 sulfates,35 peptides, and proteins.16,36 These caged molecules can be photoactivated to expose their relevant functional groups by irradiation with UV-light to induce the attachment of wanted compounds to the surface. Thereby, an illumination-controlled formation of spatial patterns of chemical reactivity is viable.37 Surface plasmon resonance (SPR) phenomena and their applications, in particular, those based on noble metal © 2013 American Chemical Society
nanostructures, have become one of the most dynamic research areas in contemporary nanoscience. The decay length of the evanescent light field generated at resonance can range from about half the free-space wavelength in the case of propagating plasmons down to a few nanometers in the case of hot spots associated with localized surface plasmon resonances (LSPRs) in nanostructures.38 The short LSPR decay length often results in extremely high local enhancement of the electromagnetic field, which can be utilized for surface-enhanced Raman scattering39 and fluorescence,40 plasmon-assisted photolithography,41 and many other applications. Both propagating and localized plasmons are extremely sensitive to refractive index changes that occur within the evanescent field region near the metal surface, a phenomenon that has been utilized for constructing ultrasensitive refractometric biosensors. Gold is typically the metal of choice for this purpose because of its chemical inertness and excellent plasmonic properties. Virtually all SPR biosensing applications are based on surface modification of the metal surface with some kind of target-specific capture molecule. Biorecognition between capture and target molecules then results in a refractive index increase within the plasmon-induced evanescent field region that in turn leads to a shift in the SPR condition. In the case of plasmonic nanoparticles, the typical Received: February 26, 2013 Revised: June 19, 2013 Published: June 19, 2013 14751
dx.doi.org/10.1021/jp402002n | J. Phys. Chem. C 2013, 117, 14751−14758
The Journal of Physical Chemistry C
Article
sensitivity and signal when only a small amount of protein binds to the surface of plasmonic nanoparticles, an enzymatic enhancement strategy is utilized.2 The concept we use is similar to the well-established enzyme-linked immune assay (ELISA). Here, a streptavidin-functionalized horse radish peroxidase (HRP) enzyme is utilized to catalyze a polymer precipitation reaction of 3,3-diaminobenzidine (DAB) (Figure 1). The precipitated polymer of DAB has a high optical mass, which greatly increases the signal from the protein on the surface. Additional experiments are carried out to probe the effective surface functionalization, including grazing incidence reflection−absorption Fourier transform infrared spectroscopy (IRRA), X-ray photoelectron spectroscopy (XPS/ESCA), contact angle measurements, and quartz crystal microbalance (QCM) experiments.
observable is a red shift of the LSPR wavelength peak position, which can be measured using optical extinction or scattering techniques down to the level of the single nanoparticle. Although LSPR sensors typically have a much lower macroscopic refractive index sensitivity compared to thin film SPR sensors, the highly confined LSPR field increases the response from biomolecular attachment close to the metal surface resulting in similar macroscopic detection efficiencies.42 While classical SPR sensing based on flat gold films is a mature technology used in a large number of applications, including drug-screening platforms,43 biophysical research,44 and in-situ monitoring of catalytic processes,45 LSPR sensing is the focus of intense recent research efforts.46 The main motivation behind this interest is the many new developments, solutions, and applications made possible by sophisticated nanotechnology; examples range from sensor solutions based on simple transmission measurements on colloidal particles, single particle sensing with single molecule sensitivity, and multiplexing and measurements within extremely confined volumes.47−50 However, many of these developments are challenging from a surface functionalization point of view because of metal surface roughness or high surface curvatures associated with nanofabrication and because the sensor surface often exposes several different materials to the target, for example, glass and gold. New techniques that facilitate simple and spatially localized surface functionalization and yield specific binding with high affinity to the metal surface while minimizing nonspecific binding to other areas of the sensor surface are therefore highly desirable. In this study, we addressed some of the challenges associated with LSPR sensor functionalization by using a photoactive linker molecule based on the 5-bromo-7-nitroindolinyl (Bni) functional group.20,23,51 The Bni group is activated by UV photons (hν < 365 nm) and reacts once activated with nucleophiles such as primary amines to transfer the linker moiety of the molecule from the Bni group to the nucleophile (Figure 1). The result is an effective covalent linkage between
2. MATERIALS AND METHODS 2.1. Materials. Triethylene glycol mono-11-mercaptoundecyl ether, streptavidin from Streptomyces avidinii, hydrogenperoxide, phosphate-buffered saline, and biotin ethylendiamine hydrobromide (biotin-NH2) were purchased from Sigma-Aldrich, and 4-(trifluoromethyl)benzylamine and 4(aminomethyl)benzonitrile hydrochloride were purchased from TCI. Ethanol (99.5%) was purchased from Solveco, and 25% ammonia solution was purchased from Fisher Scientific. Amino-poly(ethylene glycol) (PEG) (OH-PEG-NH2, MW 3400) was purchased from Laysan Bio Inc. 2.2. Methods. The quartz crystals for quartz crystal microbalance measurements (QCM) are covered with a 100 nm thick gold layer onto a 50 nm chromium adhesive layer with a fundamental frequency of 4.95 MHz obtained from QSense AB, Sweden. The QCM-D instrument model E4 (QSense, Sweden) was used for all measurements. UV/vis extinction spectra were measured using a Varian Cary 5000 spectrophotometer. Nuclear magnetic resonance (NMR) data were recorded by an automated Agilent (Varian) MR 400 MHz spectrometer equipped with one-probe, 1H frequency 399.95 MHz and 13C frequency 100.58 MHz. UV irradiation of the self-assembled monolayer on a gold surface was performed using a mercury lamp and a 40 nm bandpass filter centered at 365 nm. The UV light irradiance at the sample surface was approximately 200 μW/cm2. IRRA, contact angle, and XPSM/ESCA measurements (Supporting Information (SI) 1−3) were performed on 200 nm thick gold films on glass slides. The films were evaporated by electron beam assisted thermoheating using 1 nm thin Cr adhesion layer. IRRA, XPS, and contact angle measurements and protocols are described in the Supporting Information (SI 1−3). 2.3. Experimental Protocols. 2.3.1. Synthesis of N-(11Mercaptoundecanoyl)-5-bromo-7-nitroindoline 1. 11-Bromoundecanoic acid was prepared according to previously reported procedures.23 2.3.2. Self-Assembly of 1 on a Gold-Surface Followed by a Photoinduced Reaction. Prior to the self-assembly of 1 onto gold substrates, the gold substrates were cleaned thoroughly by a 10 min treatment in a UV/ozone chamber followed by immersion in a 5:1:1 solution of ultrapure water (Milli-Q, resistivity > 18 MΩ cm), 30% hydrogen peroxide (H2O2), and 25% ammonia (NH3(aq)) at 80 °C for 10 min. The surface was rinsed in pure water and ethanol (99.5%) and was dried under nitrogen gas. The formation of self-assembled monolayers of
Figure 1. Photoinduced reaction on a nanostructured gold biosensor surface (orange). The photoinduced reaction of nitroindoline (1) with biotin (2) forms 4 with the release of the indoline moiety 3 (step A). The biotin-functionalized surfaces enable specific biorecognition of streptavidin (B). This can be monitored as a peak shift of the LSPR of the gold biosensor. The LSPR peak shift can be further enhanced by an enzymatic polymerization of 3,3-diaminobenzidine catalyzed by horse radish peroxidase enzyme (HRP) conjugated to the streptavidin (C).
the nucleophile and the surface. One advantage of this method is that both nucleophile and light has to be present for reaction. Therefore, accidental exposure of the surface to light will only cleave the molecule to a lesser extent. The synthesis of the Bni linker molecule and its basic function has been reported previously.23 The focus here is to present work on LSPR biosensor functionalization and methods to reduce nonspecific binding. To increase the 14752
dx.doi.org/10.1021/jp402002n | J. Phys. Chem. C 2013, 117, 14751−14758
The Journal of Physical Chemistry C
Article
nitroindoline (1) occurred during a period of
