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Surface functionalization chemistries on highly sensitive silica-based sensorchips†

Subash C. B. Gopinath,*a Koichi Awazu,a Makoto Fujimaki,*a Kazufumi Shimizu,b Wataru Mizutanic

and Kiyomi Tsukagoshic

Received 5th December 2011, Accepted 6th May 2012

DOI: 10.1039/c2an35159e

The surfaces of silica-based sensor chips, designed for evanescent-field-coupled waveguide-mode

sensors, were functionalized using various surface chemistries. The immobilization of molecular entities

on the functionalized silica surfaces was monitored using various microscopic techniques (scanning

electron, fluorescence, and atomic force microscopies). Further, gold nanoparticle-based signal

enhancement analyses were performed with protein conjugation on different functionalized surfaces

using a waveguide-mode sensor. Based on these analyses, the sensor surfaces modified with

glutaraldehyde (Glu) and carbonyldiimidazole were found to be good for molecules of different sizes.

In addition, it can be inferred that the Glu-modified surface may be suitable for small molecules with

diameters around 5 nm owing to its surface roughness. The modified surface with carbonyldiimidazole

is suitable for the direct immobilization of larger molecules especially for biomolecular assemblies

without intermediate chemical modifications.

Introduction

Surface modification is an approach for modifying the surface of

a material by physical, chemical, or biological means to produce

characteristics that are different from those of the original

surface. These surface modifications are highly dependent on

various factors such as roughness, hydrophilicity, surface charge,

surface energy, biocompatibility, and reactivity. On the other

hand, surface functionalization is a process of introducing

chemical functional groups on the surface to capture the mole-

cules to be analyzed, with a view to several applications, in

various fields involving biochemical, biophysical, biomedical and

molecular biological reactions for specific assays. High-density

microarrays on a solid glass support, known as protein chips or

DNA chips, are highly dependent on chemical modification in

order to accommodate higher numbers of molecules, and they

are powerful tools for gene discovery, expression, and mapping

analyses.1–4 The development of analytical devices requires the

formatting of various substrates in order to couple them with

the molecules of interest for various read-out formats of the

aElectronics and Photonics Research Institute, National Institute ofAdvanced Industrial Science and Technology (AIST), Central 5, 1-1-1Higashi, Tsukuba, Ibaraki, 305-8565, Japan. E-mail:gopi-subashchandrabose@aist.go.jp; m-fujimaki@aist.go.jpbOpen Research Center for Genome and Infectious Diseases, NihonUniversity School of Medicine, 30-1 Oyaguchikami-chou, Itabashi-ku,Tokyo 173-8610, JapancNanosystem Research Institute, AIST, Central 4, 1-1-1 Higashi, Tsukuba,Ibaraki, 305-8562, Japan

† Electronic supplementary information (ESI) available. See DOI:10.1039/c2an35159e

3520 | Analyst, 2012, 137, 3520–3527

molecular assembly or application area, such as surface plasmon

resonance, quartz crystal microbalance, microscopic methods,

cantilever-based methods, affinity chromatography, and elec-

trochemical and fluorescence methods.5

Among various metallic and non-metallic substrates, silica-

derived substrates are considered as one of the most versatile

materials with the potential for several chemical modifications.

They have been used to develop relatively cheap sensor surfaces

for analytical systems.6–13 Silica-based substrates provide a vari-

ation in the degree of packing densities, thicknesses, and

morphologies on the deposited surfaces.14,15 Silica-based nano-

particles are potential candidates for the development of nano-

scale composites with optical and chemical properties on a single

structure.16 In order to attach biomolecules to the silica surfaces,

various surface modifications have been proposed in previous

studies, including the attachment of modified biotin through

amino-coupling,15,17,18 a biotin–streptavidin–biotin sandwich on

an amino surface,10,19 the use of N-(2-trifluoroethane-

sulfonatoethyl)-N-(methyl)-triethoxysilylpropyl-3-amine-linked

oligonucleotides for duplex formation,6,10 the attachment of

proteins to amino couplings through glutaraldehyde (Glu)

molecules,11,19,20 an antibody–protein–antibody sandwich on

amino-coupled Glu,11 and thiol-coupling.8,19 By using a similar

strategy, aptamers have been attached to silica-based nano-

particles for cancer cell detection.21 Amino-coupling based

attachments on the silica surface have been quite common for

several years, and it is recognized that the high-density immo-

bilization of proteins on silica or other surfaces is necessary to

allow the use of low sample volumes.3,21,22 Parameters such as

chemistry, incubation time, reaction temperature, homogeneity,

This journal is ª The Royal Society of Chemistry 2012

Fig. 1 Spectral readout system of the EFC-WM sensor. The light from

the tungsten halogen lamp was guided to a collimator lens and the

collimated light was irradiated to a prism. The monolithic sensing plate

placed on the bottom of a prism was illuminated and a spectrum of the

reflected light was observed by a spectrophotometer. The prism was made

of SiO2 glass, and the bottom angle of the prism was 38�.

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spacing between molecules, conformation of captures, and

buffering condition influence the attachment of biomolecules.23

Further, improper immobilization may lead to loss of protein

activity partially or completely with wrong orientation of mole-

cules and structural deformation. Therefore, the choice of

immobilization chemistry is important before attaching the

ligand and analyte.3 Availing of these surface properties,

biosensor developments were pursued to analyze various sizes of

biomolecules and biomolecule–nanoparticle conjugates. Among

various proteins and oligonucleotides, there will be size varia-

tions and whole cell analyses will have larger molecules, neces-

sary to immobilize on the sensing surface with the suitable linker.

Recent interest in the sensor developments is to create a system to

analyze and screen small molecules, especially for the drug-

discovery processes. Previous studies carried out with different

surface modifications are mainly focused to study the property of

molecular interactions.7,10,11,13,15,24 Chemical and physical

properties of size-dependent nanoparticles for the sensitive

detections were overviewed by Wittenberg and Haynes.25

However, there are lacunae in the study of surface modifica-

tions for the immobilization of molecules based on molecular

sizes. To fill these lacunae, the present study investigates the use

of different surface functionalization chemistry, using carbonyl-

diimidazole (CDI), glycidoxypropyltrimethoxysilane (GOPTS),

and Glu for molecular size selection. All these molecules were

intended to be used with the sensor chip surfaces that are

routinely used in our laboratory to monitor various biomolecular

interactions, for an evanescent-field-coupled waveguide-mode

sensor (EFC-WM sensor) operating on the Kretschmann

principle.10,11,13,15,17–19,26

Experimental

Chemicals and biomolecules

3-(Triethoxysilyl)propan-1-amine (3APT) was purchased from

Sigma-Aldrich (Tokyo, Japan). N,N0-Carbonyldiimidazole

(CDI) and Glu were procured from Wako Chemicals (Osaka,

Japan); 3-glycidoxypropyltrimethoxysilane (GOPTS), from

Shin-Etsu, Silicon Chemicals, Japan; Alexa Fluor 555-labeled

goat anti-rabbit IgG (H+L), from Invitrogen, USA; gold nano-

particle (GNP) conjugated streptavidin (5 nm of 3 O.D. and

20 nm of 4 O.D.), from BBInternational, UK; 40 nm GNP–

streptavidin conjugates (15 O.D.), from BioAssay Works, MD,

USA; and anti-HA serum for H1N1 (Brisbane/59/2007) from

Denka Seiken, Tokyo, Japan.

Setup of the EFC-WM sensor

The EFC-WM sensor utilizes a sensing plate having a multilayer

structure consisting of a dielectric waveguide, a high-refractive

index layer, and a glass substrate.18 The sensing plate illuminated

under the Kretschmann configuration operates as a sensor that is

capable of detecting modifications in the dielectric environment

near the waveguide surface by measuring change in reflectivity.

In the present research, we applied a spectral readout system to

the EFC-WM sensor, which had been reported as a compact

optical system of SPR sensors.27 The optical setup is shown in

Fig. 1. In the system, a tungsten halogen lamp was used as a light

source. The light from the lamp was guided to a collimator lens

This journal is ª The Royal Society of Chemistry 2012

and the collimated light was irradiated to a prism, where the

incident angle was parallel to the bottom face of the prism. Then,

the monolithic sensing plate placed on the bottom of a prism is

illuminated and a spectrum of the reflected light is observed by

a spectrophotometer. The prism was made of SiO2 glass, and the

bottom angle of the prism was 38�. As the sensing plate,

a monolithic sensing plate that we previously developed was

applied.18 The monolithic sensing plate consists of a SiO2 glass

substrate, a single crystalline Si layer, and a thermally grown

SiO2 waveguide. In the present experiment, the thicknesses of the

SiO2 glass waveguide and the single crystalline Si layer were set to

be 45 and 360 nm, respectively. The optical system was designed

to show a dip in reflectance at around 520 nm, which corresponds

to the optical absorption of GNPs. If GNPs are attached on the

waveguide surface, the dip will be deepened by the optical

absorption of the GNPs.15

Reactions for the functionalization of the silica surface

Three different chemical strategies (CDI, GOPTS and Glu) were

employed for attaching the biomolecules to the silica-based

sensor chips. Before performing the chemical modifications, the

chips were treated with alkali solution for 30 min, washed

thoroughly with water, and dried to attach OH groups to the

surface of the silica. Free hydroxide ions were removed by

washing several times with phosphate buffer (pH 7.4). For the

CDI modification, the alkali-treated sensor chip was soaked in

a 0.5 M solution of CDI in dioxane. The reaction was carried out

at 37 �C for 2 h, after which the chip was rinsed with acetone

followed by distilled water and dried. For GOPTS modification,

GOPTS reactions on the sensor surface were performed by

immersion in anhydrous toluene containing 2% GOPTS for 4 h

at 50 �C, followed by washing with toluene and water to remove

adsorbed silane. For the Glu modification, silane coupling using

3APT on the chip followed by immobilization with Glu was

performed as previously described.10,19 In brief, the alkali

modified surface of the waveguide substrate was further modified

by immersion in a 0.5% (v/v) ethanolic solution of 3APT for 24 h.

The 3APT reacted with surface hydroxyl groups of the SiO2

waveguide to give an amine group-functionalized substrate that

was rinsed with ethanol and dried in a stream of nitrogen gas.

The amine-functionalized SiO2 surface was treated with a 2.5%

solution of aqueous Glu for 3 h at room temperature to form an

aldehyde-activated surface. After incubation, the surface was

washed thoroughly with the phosphate buffer. Unless otherwise

stated, surface modified glass substrates were stored in glass Petri

dishes until further analysis.

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On each surface modified sensing plate, experiments were

performed with 1 optical density of the GNP–streptavidin

conjugate, as this concentration did not give non-specificity on

the unmodified surfaces under optimal room temperature. The

dilutions for 1 optical density were made from the original stock

with phosphate buffer (pH 7.4). Reactions were performed for 20

min on the shell of the waveguide sensor and after each step the

sensing surface was thoroughly washed three times with 300 ml of

phosphate buffer.

Microscopic analyses

All microscopic observations were performed with 1 optical

density of GNP–streptavidin conjugates, as described above. The

GNP–streptavidin conjugate attached surfaces were examined

using field emission scanning electron microscopy (SEM; JEOL,

JSM-6340F). The surface functionalized with different chemis-

tries was observed by atomic force microscopy (AFM; Digital

Instruments Nanoscope). The AFM measurements were carried

out with a 1 mm scan size at a scan rate of 0.5003 Hz. Fluores-

cence images of Alexa Fluor 555-labeled goat anti-rabbit IgG

(H+L) attached surfaces were visualized using a BIOREVO

Keyence BZ-9000 instrument. 50 nM of a fluorescent-labeled

antibody was attached on each chemically modified surface and

incubated for 30 min at room temperature, washed, and dried

before observations were carried out.

Results and discussion

The sensor chips used for surface functionalization are correctly

designed for the EFC-WM sensors, and are stable against

chemical and physical changes. The principles and design of the

waveguide sensor system that we used were similar to that of

a surface plasmon resonance (SPR) system, the only difference

being that the mode used for measurement was not a surface

mode but a waveguide mode. The advantage of the waveguide

sensor over the SPR sensor is that higher sensitivity is easily

obtained by perforating the waveguide layer and/or by using dyes

or metal nanoparticles. In the case of the SPR sensor, the

wavelength of an incident light is restricted by the material used

in order to induce the surface plasmon, whereas there is no such

restriction in the case of the waveguide sensor. In addition,

amorphous SiO2, one of the most popular materials of the

waveguide layer of the waveguide sensor, is physically and

chemically more stable than typical materials (Au or Ag)

generally used for SPR.10,15,18,19 Moreover, silica materials are

inexpensive and have a relatively homogeneous chemical surface,

which offers the added advantage of easier scanning for visual-

ization of the spots for chemical and biochemical analyses.6,28

Silicates and silicon oxide surfaces require surface modifications

before attachment of molecules of interest.5 Attempts were made

to control the surface chemistry of these chips because surface

control is crucial for efficient immobilization of proteins in the

preparation of micro-nanodevices. There are two in vogue

approaches to the surface immobilization and patterning of solid

surfaces, namely, in situ surface synthesis and preparation of

prefabricated molecules.29Generally, in order to retain biological

activities on the sensor-surfaces, modifications of physico-

chemical and chemical properties are the pertinent options.

3522 | Analyst, 2012, 137, 3520–3527

Surface functionalization chemistry on waveguide sensor chips

The sensor surface was activated by alkali treatment and the

resulting surface has Si–OH groups. On these Si–OH surfaces,

the functionalization chemistry was incorporated with three

independent appropriate surface modifications, namely CDI,

GOPTS and Glu. CDI is an organic compound that forms

a white crystalline solid. It is often used for the coupling of amino

acids for peptide synthesis and as a reagent in organic synthesis.

CDI is very often used for coupling carboxylic acids with

aliphatic or aromatic amines to form amides for both small- and

large-scale applications.30 It can be dissolved in the solvent and

directly attached to the silica surfaces without the need for an

intermediate step, to produce silane residues,9 and thus, modifi-

cation with this reagent reduces the number of experimental

steps. A CDI functionalized surface will possess an imidazole

carbamate functionality and it can form a stable carbamate

linkage with amine groups. The epoxy-silane in GOPTS allows

for the formation of a covalent linkage to tertiary amine groups

on amino acids.20 Epoxy activated surfaces are considered to be

a universal type of support, on which nucleophilic and electro-

philic groups can be attached with high efficiency.31However, the

reaction time for this covalent reaction with proteins is higher

than that of the other modifications discussed;3,6 also, GOPTS

polymerizes easily in the presence of water to form multilayer

assemblies.32 This compound was dissolved in anhydrous toluene

to create an epoxylated surface and it forms a secondary amine

bond upon reacting with amines. GOPTS is stable at neutral pH,

wettable and reactive with several nucleophilic groups to form

strong bonds with protein.3 Glu needs an additional modification

step to immobilize the molecules on the sensor chip by first

modifying the chip with silane groups and then attaching Glu. A

glass substrate derived from organosilanes can be functionalized

for –OH, –NH2, –SH, –COOH, –CHO and so on, to capture the

subsequent molecules of interest (Todt and Blohm, 2009). In this

study, 3-(triethoxysilyl)propan-1-amine (in 95% ethanol) is used,

as this compound is considered as one of the potent functiona-

lization chemistries for glass substrates that can introduce reac-

tive amino groups on the surface. This surface activated with

amines was reacted with aldehyde groups of Glu. Glu is

a homobifunctional cross-linker, frequently used in biochemistry

applications as an amine-reactive group, and the oligomeric state

of proteins can be examined through this application. Glu has

two aldehyde groups at the ends and both ends can be coupled

with amine groups to form a Schiff’s base. As reported in the

literature, the Glu coupling reaction on chip was performed at

a pH of 7.4, a slightly alkaline environment, as a lower pH than

this would reduce the efficiency of the coupling reaction.33

Attachment of gold nanoparticle conjugated-streptavidin on

sensor chips: SEM and waveguide sensor analyses

Nanomaterials can facilitate signal transduction just like fluo-

rophores or electroactive tags and be useful for imaging studies.

GNP is the ideal nanomaterial commonly used in the sensor

developments, has unique characteristics, such as easy to disperse

in water, compatible with surface functionalization to link

biomolecules and can be tailored with the desired nano-sizes.34,35

In this study, the activity of the modified waveguide sensing

This journal is ª The Royal Society of Chemistry 2012

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surfaces was evaluated using GNPs of three different sizes. As

mentioned above, the surface chemistry modifications were

carried out using CDI, GOPTS, and Glu as modifiers (Fig. 2a).

Then, GNP–streptavidin conjugates were immobilized on the

modified waveguide-mode sensor chips, using GNPs of three

different sizes (Fig. 2b). Streptavidin is a tetravalent protein

molecule with a diameter of approximately 5 nm.10 Upon coating

the 40 nm sized gold nanoparticles with this molecule, the

conjugates may have a total diameter of approximately 50 nm,

whereas the 20 and 5 nm GNPs will produce conjugates with

sizes of 30 and 10 or 15 nm, respectively (Fig. 2b). Streptavidin

was chosen as the model protein for conjugation with GNP

because it finds widespread use in molecular biology owing to its

extraordinarily strong affinity for the vitamin biotin. The disso-

ciation constant (Kd) of the biotin–streptavidin complex is of the

order of 4 � 10�14 mol l�1, ranking it as one of the strongest

known non-covalent interactions in molecular, immunological

and cellular assays.36

SEM observation of the 40 nm GNP–streptavidin conjugates

showed clear attachment on all three chemically modified silica

surfaces used in the present study (Fig. 3a–c). As mentioned,

streptavidin has a size of about 5 nm, and the size of the GNP

conjugates using a 40 nm GNP is increased to roughly 50 nm

upon conjugation, in a non-aggregation situation (Fig. 2b). It is

hard to predict the number of streptavidin molecules attached on

GNP. To minimize the formation of aggregated forms of GNP,

the usage of NaCl was avoided. The addition of salt (NaCl) will

cap the repulsion among the unmodified negatively charged

GNPs, inducing the aggregation of these particles and failure to

stabilize.37,38 As mentioned in our previous report, the waveguide

system detects less than one GNP adsorbed per square

micrometer. According to our theoretical calculation based on

the experimental data the reflectance is expected to decrease by

0.01 (reflectivity in the vertical direction) by the adsorption of

0.75 GNP per square micrometer.15 When sensing surfaces are

analyzed with the EFC-WM sensor before and after attaching

the GNP–streptavidin conjugates, prominent changes in the dip

of the resonance were observed in the cases of CDI and Glu

Fig. 2 (a) Schematic surface functionalization on the sensor chip. CDI,

GOPTS, and Glu modifications are shown. (b) Diagrammatic represen-

tation of the sizes of the GNP–streptavidin conjugates. Three different

sizes (40, 20, and 5 nm) of GNP were used. The size of streptavidin is

considered to be 5 nm. The expected total sizes of GNP-conjugated

streptavidin are shown.

This journal is ª The Royal Society of Chemistry 2012

modification (Fig. 3d and f). In both cases, the reflection

changes in the dip were with values of 18.7 and 21.4, respectively.

In contrast, epoxy activation by surface modification with

GOPTS produced a reflection change with nearly half the value

(11.7) of those observed in the other two cases, indicating

stronger molecular attachments with CDI and Glu molecules

(Fig. 3d–f).

Further evaluation was performed by similar studies on all

three surfaces using 20 nm GNPs–streptavidin conjugates. These

GNP conjugates may have an estimated size of approximately

30 nm. Direct observation with SEM using the three chemically

modified surfaces shows the attachment of a larger number of

molecules of this size on the CDI modified surfaces than on the

GOPTS and Glu modified surfaces (Fig. 4a–c). Confirmatory

evidence was provided in the observation of the reflectance

changes using a EFC-WM sensor, similar to that presented

above. The observations with the sensor also support the

conclusion that the CDI-modified surface exhibited preferential

attachment of these particles, as more prominent reflectance

changes in the resonance dip were observed in the case of the

CDI modified surface, compared with the other two. With CDI-

modification the reflectance changes were about 18.7 which is

similar to 40 nm GNP–streptavidin attachment, whereas in the

case of Glu the reflectance change was roughly one fourth of the

reflectance observed for the CDI case. In the case of GOPTS

modification, no significant changes were observed in the dip in

the resonance (Fig. 4d–f).

Because the two studies presented above indicated clear

molecular-size discrimination, similar analyses were extended to

include 5 nmGNPs conjugated with streptavidin. The 5 nmGNP

can accommodate fewer streptavidin molecules, potentially

accommodating one or two streptavidin molecules per particle

on either side, to give conjugates with a size of 10 nm or 15 nm in

total (Fig. 2b). The 5 nm GNP was too small for the SEM system

that we used and we could not observe it using the system.

Waveguide-mode reflectance analysis of the 5 nm GNP–strep-

tavidin conjugates showed a reflectance change of 7.7 in the case

of the Glu-modified surface, which is higher than the value

observed for this surface using the 20 nm GNP–streptavidin

conjugates. The reflectance differences (with 20 nm and 5 nm

GNP–streptavidin) might be attributed to molecular accommo-

dation fitness on the Glu-modified surface in the case of the 5 nm

conjugates, relative to the 20 nm GNP–streptavidin conjugates.

The reflectance difference is, thus, due to a higher molecular

density of the 5 nm molecules attached to the Glu-immobilized

surface and a relatively lower density in the 20 nm case. However,

in the case of CDI-modification, there was a marked decrease in

the changes of the resonance dip, due to fewer molecular

attachments of the 5 nm conjugates on this surface compared to

the case of the 20 nm GNP–streptavidin conjugates (Fig. 5a and

b). As observed in the case of the 20 nm GNP–streptavidin

conjugates, there were no visible changes with the 5 nm GNP–

streptavidin conjugates for the GOPTS modifications (Fig. 5c).

The comprehensive analyses with three sizes of GNP–streptavi-

din conjugates and three chemical modifications are shown in

Fig. 5d. The non-specific attachments of all of the GNPs used in

this study were monitored on chemically un-modified surfaces,

and no significant non-specific attachments were observed (ESI,

Fig. 1a–c†).

Analyst, 2012, 137, 3520–3527 | 3523

Fig. 3 SEM images of 40 nmGNP–streptavidin conjugates on the surfaces modified with (a) CDI, (b) GOPTS and (c) Glu. Magnified SEM images are

shown as figure inset. Reflectivity measurements were carried out with the EFC-WM sensor using these surfaces on which the 40 nm GNP–streptavidin

conjugate was immobilized. (d), (e), and (f) illustrate the immobilization of the particles on CDI, GOPTS and Glu, respectively. The black and the red

curves indicate the spectra before and after the immobilization, respectively. Higher changes in reflectivity were observed with CDI and Glu, and lower

one was observed with GOPTS. Vertical arrows on the figures indicate the direction of the changes.

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Based on this observation, it can be deduced that particles

having larger sizes, i.e., �50 nm, are best suited for the surfaces

chemically modified with CDI and Glu, from which it can also be

extrapolated that these surfaces might be well suited for the

larger biomolecules such as nanoparticles and viruses. In one of

our recent studies to observe the interactions of antibody and

whole viral particles, a Glu-modified surface was used and good

sensitivity was achieved.19 In addition, the results indicate that

there is a significant dependence of the molecular attachments on

these surfaces, upon the molecular sizes below 50 nm. This might

be attributed to the high chemical molecular crowdedness in the

case of Glu modification, thereby restricting molecular sizes

around 30 nm, as the surface roughness forms ridges. Ultimately,

Fig. 4 SEM images of 20 nmGNP–streptavidin conjugates on the surfaces m

shown as figure inset. The particle immobilized surfaces were monitored by th

the particles on CDI, GOPTS and Glu, respectively. The black and the red cur

Vertical arrows on the figures indicate the direction of the changes.

3524 | Analyst, 2012, 137, 3520–3527

there was the possibility for the formation of ordered, arranged

molecular attachments on the CDI modified surfaces, but in the

case of 50 nm particle sizes used in the above study, the reflec-

tance was high due to larger particle sizes. These studies clearly

illustrate that the fit on the modified surfaces is based on the

chemical molecular crowding and molecular accommodation

operates within size-limitations.

Fluorescence microscopy analyses

Based on the above studies, there were expectations that there

would be differences in the surfaces of the sensors with the

various modifications. Consequently, the studies were further

odified with (a) CDI, (b) GOPTS, and (c) Glu. Magnified SEM images are

e waveguide-mode sensor. (d), (e), and (f) illustrate the immobilization of

ves indicate the spectra before and after the immobilization, respectively.

This journal is ª The Royal Society of Chemistry 2012

Fig. 5 Reflectivity measurements with the EFC-WM sensor using the

surfaces modified with (a) CDI, (b) GOPTS and (c) Glu, on which the

5 nm GNP–streptavidin conjugate was immobilized. The black and

the red curves indicate the spectra before and after the immobilization,

respectively. The changes in reflectivity observed by the immobilization

of the three GNP–streptavidin conjugates with different sizes on the three

chemical modifications are summarized in (d). The error bars indicate the

averages of three values.

Fig. 6 Fluorescence microscopy images with fluorescent (Alexa Fluor

555) labeled antibody immobilized on the surfaces modified with (a) CDI,

(b) GOPTS, (c) Glu, and (d) the unmodified surface.

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extended to other microscopic approaches. Fluorescence

microscopy analyses were performed on the three chemically

modified surfaces using Alexa Fluor 555 labeled goat anti-rabbit

IgG. Alexa Fluor is a family of fluorescent dyes, a unique dye

obtained from Invitrogen, typically used for fluorescence

microscopy observations. The excitation and emission spectra of

Alexa Fluor dyes covers the visible spectrum and extends into the

infrared; and these dyes are generally more stable, brighter, and

less pH-sensitive than other common dyes. The results of the

fluorescence microscopy studies showed the presence of fewer

antibody molecules on the GOPTS-modified surface compared

with the CDI-modified surface (Fig. 6a and b). Several tens of

molecules could be clearly observed on the CDI surface. In the

case of the Glu-modified surface, a totally different behavior was

observed compared with the CDI and GOPTS surfaces, in which

there was a dense image as seen in the fluorescence microscopy

studies (Fig. 6c). The immobilizations of the fluorescent-labeled

antibody on the unmodified surfaces were also visualized as

blanks (Fig. 6d). The high-background caused by Glu has the

potential for creating cross-linkages between two protein mole-

cules via homobifunctional aldehyde groups leading to undefined

complex formation on the immobilized surface. Glu is also

reported to polymerize in aqueous solution to give a number of

molecular structures, which may cause immobilization of

a branched polyaldehyde rather than the native bifunctional

molecule.9 Another possibility could be the higher number of

molecular attachments of the amine and the Glu functionalized

surface as reported by Goddard and Erickson,9 resulting in

conjugation of Glu with higher hybridization densities. This clear

discrimination on the Glu-coated surface evidenced by the

indistinct image exemplified the correlation with the GNP–

streptavidin studies regarding the molecular size requirement for

attachment. Usually antibodies have a molecular weight in the

region of 150 000 and they may be similar in size to the 5 nm

This journal is ª The Royal Society of Chemistry 2012

GNP–streptavidin conjugated particles. In both cases, there

might be plenty of molecular attachments. Further, the fluores-

cence microscopy analyses allow us to conclude that Glu-modi-

fication may allow for the attachment of several proteins, as they

are the right-sized molecules. Even though our studies indicate

that the GOPTS-modified surface cannot accommodate many

molecules, the report of Goddard and Erickson9 indicates that

GOPTS and CDI behave similarly in their studies. As proposed

before, because GOPTS requires a longer reaction time, the

reaction efficiency may be lower than that of the other two

chemistries used.6

Validation of surface roughness by AFM on the CDI and Glu

surfaces

The immobilization of proteins via chemical reactions of the

amino acid side-chains are generally considered as random due

to the presence of different residues on different proteins, thereby

forming a heterogenic population on the sensor chip. Attach-

ments of the proteins through chemical bonds may be guided by

ordered processes to give proper orientation.3 However, this is

dependent on the chemical processes involved in the modification

procedure. The AFM approach was chosen for direct visualiza-

tion of the surface ordered-ness on the present sensor chip.

Based on the above experiments using protein conjugated

GNP and un-conjugated protein molecules, it was concluded

that there is a drastic change in the surface with both CDI and

Glu. To prove this, we monitored and measured the surface

roughness of both CDI and Glu modified surfaces after immo-

bilization of these chemical compounds. In the case of CDI, the

modification proceeds by a single step; therefore, the surface

roughness was observed directly for CDI using AFM, whereas in

the case of Glu the modification involves two steps, and surface

observations were made after each step. When the 3APT-coupled

surface, which is first step for Glu modification, was analyzed,

slight changes were observed in the roughness of the surface

(Fig. 7a). The surface roughness was increased drastically upon

Analyst, 2012, 137, 3520–3527 | 3525

Fig. 7 AFM images of the surfaces modified with (a) 3-APT, (b) Glu, and (c) CDI. The scanned images are also given at the bottom of each figure.

Graphical representation of surface roughness is shown in (d). The root-mean-square (RMS) roughnesses were measured at three different positions. (e)

An AFM image of antibodies attached on the CDI-modified surface.

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immobilization of the Glu molecules on the 3APT-coupled

surface (Fig. 7b), whereas, in the case of CDI, the roughness was

not as high compared with the Glu modified surface (Fig. 7c).

The overall comparison of these three surface roughnesses indi-

cates that they were different in each case as shown in Fig. 7d.

Antibody attachment on the CDI surface was monitored using

anti-Brisbane influenza viral anti-serum. The antibody was

directly attached to the CDI surface, and the antibody molecules

were clearly observed on the surface (Fig. 7e). This clear

discrimination between the attachment on the Glu and CDI

surface based on roughness might be attributed to crowdedness

and orientation of the molecular arrangements. In the case of

Glu, there are two aldehyde groups at the ends, and there is,

therefore, a higher probability for formation of a randomly

arranged chemical surface which will lead to a surface with more

roughness. Due to the rough nature of the Glu-immobilized

surfaces, there will be more surface area to receive lower

molecular sized molecules. Diagrammatic illustrations repre-

senting the molecular ordered-ness with different-sized molecules

on different surface chemistries are shown in the ESI, Fig. 2†.

Conclusion

Based on these results, it is clear that even though there is

a higher probability for more protein attachments on the Glu-

modified surface, it can be inferred that the CDI surface might be

the best choice for larger molecules (particles and viruses) as well

as for various molecular interactive analyses or biomolecular

assembly; and this surface can make direct linkage with the

molecules. However, for direct observation of proteins or protein

interactions with lower-sized molecules such as drugs, Glu might

be helpful and ideal for molecules with restricted sizes. The

3526 | Analyst, 2012, 137, 3520–3527

protein biochips with these chemical modifications will be useful

as diagnostic tools for proteomic analyses to obtain information

about the protein functions and interactions. Further, the

nanoparticle-conjugated proteins on these chemically modified

surfaces will enable the development of surfaces for nano-

biosensors, and nano-alignment using the particles can be carried

out on these sensor chip surfaces for nano-patterning purposes.

Acknowledgements

This study was supported by a grant from the Industrial Tech-

nology Research Program, 2009, of the New Energy and

Industrial Technology Development Organization (NEDO),

Japan. Part of this work was conducted at the AIST Nano-

Processing Facility, which is supported by the ‘‘Nanotechnology

Support Project’’ of the Ministry of Education, Culture, Sports,

Science and Technology (MEXT), Japan. The authors would

also like to thank the Advanced Functional Materials Research

Center of Shin-Etsu Chemical Co., Ltd. for supplying the

samples.

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