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Tissue Engineering and Regenerative Medicine, Vol. 11, No. 1, pp 10-15 (2014)

DOI 10.1007/s13770-013-1119-4

｜Original Article｜

Immobilization of Fibrinogen Antibody on Self-Assembled Gold Monolayers

for Immunosensor Applications

Hongsik Cho1,2,3, Justin Zook4, Todd Banner5, Sang-Hyug Park6, Byoung-Hyun Min7,8,9,

Karen A Hasty1,2,3

, Eugene Pinkhassik5*, and Erno Lindner

4,5*

1Department of Orthpaedic Surgery & Biomedical Engineering, University of Tennessee Health Science Center, Memphis,

TN, 38104 USA2Department of Orthopaedic Surgery, Campbell Clinnic, Memphis, TN,38104 USA

3Research 151, Veterans Affairs Medical Center, Memphis, TN, 38104 USA

4Department of Biomedical Engineering, University of Memphis, Memphis, TN, 38152 USA5Department of Chemistry, University of Memphis, Memphis, TN, 38152 USA

6Department of Biomedical Engineering, Jungwon University, Geosan, 367-700 Korea

7Department of Orthopeadic Surgery, Medical School, Ajou Univeristy, Suwon, 443-749 Korea8Department of Molecular Science and Technology, Ajou Univeristy, Suwon, 443-749 Korea

9Cell Therapy Center, Ajou University Medical Center, Suwon, 443-749 Korea

(Received: July 8th, 2013; Revision: August 8th, 2013; Accepted: August 20th, 2013)

Abstract : Self-assembled gold monolayers offer several advantages for the realization of novel modified electrodes

for biosensor applications. This is due to their ability to decrease non-specific adsorption and provide for covalent

attachment of biomolecules. Surfaces for these applications require the precise control of ligand density, the ability

to immobilize ligands, and in situ-modulation of ligand activity. In this study, we focused our studies on the immo-

bilization of antibody on a gold monolayer surface. We self-assembled thioctic acid onto the gold surface as an

anchor point for the immobilization of anti-fibrinogen onto the surface. The modifications to the gold surface were

characterized by ELISA, ellipsometry, and AFM.

Key words: self-assembled monolayers (SAMs), anti-fibrinogen, immunosensors, enzyme-linked immuno sorbent

assay (ELISA), ellipsometry, atomic force microscopy (AFM), gold monolayer, antibody immobilization

1. Introduction

Chemical reactions on solid surfaces are important to the

essential understanding of interfacial phenomena and for

technological applications to immunosensors.1-3 Gold substrates

have played an important role in the advancement of surface-

based nanotechnology research. Gold is useful because it can

chemically bind to thiol and disulfide functionalized compounds

which provide the foundation for self-assembled monolayers

(SAMs).4-6 SAMs have been studied extensively and many new

research areas and applications have subsequently evolved.6,7

SAMs offer several advantages for the realization of novel

modified electrodes for biosensor applications because of their

role in decreasing non-specific adsorption and allowing for

covalent attachment of biomolecules, such as antibodies and

other proteins.8-10 Antibodies can be covalently immobilized

onto a gold surface using thiols.11-13 The immobilization of

antibodies onto surfaces provides enhanced or maximized

interactions between the immobilized biomolecules and

ligands.In this respect, various chemistry and biology-based

methods have been developed utilizing antigen-antibody

interactions and protein-ligand interactions.14-16

To extend the benefit of these surfaces to generate new types

of immunosensors and to study complex biological processes,

it is necessary to develop substrates with more sophisticated

and flexible surface properties. Surfaces for these applications

require the precise control of ligand density, the ability to

immobilize the ligand, and the in-situ modulation of ligand

activity.12

Fibrinogen is the most important protein involved in coagula-

*Corresponding author

Tel: 901-678-5641; Fax: 901-678-3447

e-mail: elindner@memphis.edu (Erno Lindner)

Tel: 901-678-4430; Fax: 901-678-3447

e-mail: epnkhssk@memphis.edu (Eugene Pinkhassik)

Fibrinogen Antibody Immobilization on SAM

11

tion and the concentration of fibrinogen in plasma is 1.5-4.5 mg/

mL in normal condition.17,18 An abnormal concentration of

plasma fibrinogen is a strong risk factor for thrombotic disorders

and must be accurately determined with short-term diagnosis.18

In this study, we set up a common intermediate method to

functionalize gold surfaces with terminal carboxylic acid

groups (via thioctic acid) in order to immobilize anti-fibrinogen

onto the surface. We then used N-(3-dimethylaminopropyl)-N'-

ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS)

to activate the carboxylic acid functionalized surface so that it

would couple with anti-rabbit immuno-globulin G (IgG) and

horseradish peroxidase (HRP). Figure 1 is showing the concept

of biosensor using polyclonal fibronogen antibody immobiliza-

tion on gold monolayer surface. We focused our studies on the

immobilization of fibrinogen antibody onto the funcionalized

gold monolayer surface. The coupling reaction on the gold

surface was characterized by enzyme-linked immuno sorbent

assay (ELISA), ellipsometry, atomic force microscopy (AFM)

and the coupling efficiency was estimated by comparing the

experimentally determined absorbance of 3,3´,5,5´-

tetramethylbenzidine (TMB) reaction of ELISA.

2. Materials and Methods

2.1 Chemicals

11-Mercaptoundecanoic acid (MUA), thioctic acid, N-

hydroxysuccinimide (NHS), 1-(3-dimethylaminopropyl)-N’-

ethylcarbodiimidehydro-chloride (EDC) were purchased from

Aldrich (St. Louis. Mo, USA). Rabbit IgG, goat anti-rabbit IgG

with HRP, goat anti-mouse IgG with HRP, and bovine serum

albumin (BSA) were purchased from Pierce (Rockford, IL,

U.S.A). All solvents were reagent-grade. Reagents were used

without any further purification. Experiments were carried out

at room temperature.13,19

2.2 Preparation of Gold Substrate

Gold template substrates (catalog number AU.1000.SL2NOTI)

were purchased from Platypus Technologies (Madison, WI). The

substrates were 100 mm diameter silicon disks covered with

electron-beam deposited gold to a thickness of 1000Å. A titanium

adhesion layer between the silicon and gold layers was not used.

2.2.1 Preparation of Supporting Substrate

Silicon supporting substrate was purchased from AlSil-

Supply Division (Palm Beach, Florida). The substrates were 3

inch diameter silicon (N-type, phosphorous doped) disks with

thicknesses of 1 mm ± 20 µm. It was polished on a single side

with mean roughness < 6 Å. The substrates were cut with a

scoring stylus and then cleaved into pieces of approximately 1

×2 cm. The pieces were rinsed with ethanol, then water, and

finally blown dry using a strong stream of argon gas.

2.2.2 Adhesion of Supporting Substrate

About 1 mg of freshly prepared Epotek 377 epoxy glue

(Bellerica, MA; parts A and B were mixed 1:1 by weight) was

placed onto the polished side of the cleaved supporting silicon

pieces using a disposable syringe with needle. The silicon pieces

were then placed, epoxy side down, onto the top of the gold

template substrate. The gold template substrate wafer was then

placed into an oven at 150oC for overnight to cure the epoxy. The

gold template/supporting silicon substrate sandwich disk was

cooled at ambient temperature for at least 10 minutes before

stripping the gold from the template substrate.6

2.2.3 Stripping of the Template Substrate

A razor blade was used to gently scrape near the edges of the

adhered supporting substrate. The supporting substrate would

typically release from the surface yielding gold surface on top

of the supporting silicon substrate.

Figure 1. Schematic illustration of the concept of biosensor using

polyclonal fibronogen antibody immobilization on gold monolayer

surface; A self-assembled (SAM) gold monolayers with immobi-

lized antibodies on short thiol molecules design for immunosensor

that measuring fibrinogen concentration in small amount of blood

sample base on enzyme kinetic method in the future.

Hongsik Cho et al.

12

2.3 Activation of Gold Monolayer and Immobilization

of Antibody

We first prepared and characterized gold substrate on silica.

Then, the bare gold substrate films were modified with thioctic

acid by submersing them in 10 mM ethanolic solutions for 6

hours. Longer immersion times did not noticeably improve the

efficiency. The substrates were then submersed in a 2 mL

solution of NHS (20 mM) and EDC (10 mM) in ultra pure water

for 20 min, followed by the antibody (poly anti-fibrinogen, 1 µg/

mL) solution in PBS for 1 hour. The residual NHS esters were

blocked by submersing the modified substrates in 1 M

ethanolamine (pH 9.0) for 20 min. After washing with ultra

pure water, the substrates were finally immersed in a 1% (w/v)

solution of BSA in 10 mM PBS pH 7.4 for 1 h.19

2.4 Ellipsometry

Ellipsometric measurements were performed using a

Gaertner Scientific ellipsometer (model: LSE) equipped with a

He-Ne laser (λ = 6328Å), set at an angle of incidence of 70o.

The gold substrate constants were derived from ellipsometric

measurements conducted at 10 or more locations on a bare gold

substrate. For the substrate coated with SAMs, the thickness of

the SAM was determined from ellipsometric measurements at

five different locations (separated by at least 0.5 cm) using the

recorded substrate constants. We assumed that the refractive

index of the film was 1.46 and the film was completely

transparent to laser beam.

2.5 Analysis Enzymatic Activities (ELISA) by TMB

Substrate Assay

After the antibody immobilized surface was washed with

PBS and Tween 20, the unreacted sites on the surface were then

blocked by reaction with a 1% solution of bovine serume

albumin (BSA). The 2nd antibody (anti-rabbit IgG) with HRP

reacted to 1st antibody during 1 hr at room temperature. The

peroxidase enzyme, in the presence of H2O2, catalysed the

oxidation of colorless 200 ul TMB substrate, (Pierce, IL, USA)

yielding to a blue colored product. After a fixed reaction time

(30 min), the reaction was stopped with 50 µL of 2M H2SO4 and

the solutions were placed into a 96-well plate and the absorbance

of the solutions was measured at 450 nm by spectrophotometer.

For this ELISA, we prepared an acrylic cage apparatus which

can securely hold the gold monolayer and has a hole that is

same size of well in 96-well plate (Fig 2).

2.6 Characterization of Gold Surface by AFM Imaging

AFM images were gathered using an MFP-3D-BIO instrument

purchased from Asylum Research (Santa Barbara, CA). All

images were collected in repulsive AC mode. The cantilevers

used were AC240TS manufactured by Olympus and purchased

from Asylum Research. The imaging of samples was done in

AC mode in air. AFM provides topographical information

about the gold surface and has the ability to detect individual

protein molecules on the surface.

2.7 Set up of Experimental Group

We first prepared and characterized ultra-smooth gold

surfaces on silica. Then, the bare gold substrate films were

modified with thioctic acid from 10 mM ethanolic solutions for

a period of 6 hr. The antibody was coupled with the carboxylic

acid group on gold surfaces via EDC and NHS activation of the

carboxylic acid group. After the primary antibody was

immobilized the surface, we detected its presence with a secondary

HRP-anti-rabbit IgG antibody and TMB. In order to determine

if the antibody was immobilized on the surface, we performed

the experiments outlined in Table 1.

3. Results and Discussion

The immunoosensors are used to detect or quantify disease-

related substances known as biomarkers in clinical diagnostics.

In immunosensor applications, antibodies are immobilized

onto the biosensor surface to capture specific biomarkers.20 The

most important consideration is the development of a technique to

immobilize antibodies onto the sensor substrate surface without

decreasing their binding affinities and binding capacities.21,22 In

this project, the resulting structures were characterized by

ellipsometry, ELISA, and AFM imaging.

3.1 Ellipsometry Analysis

In this experiment, we used two different types of SAM that

Figure 2. Diagram of the apparatus for ELISA on gold mono-

layer. This apparatus was design to support the ELISA reaction

directly onto the gold SAMs.

Fibrinogen Antibody Immobilization on SAM

13

vary based on their chain length. Long-chain SAM (11- MUA)

has been widely used as linker molecule due to its strong van

der Waals forces among the chain molecules. This allows for it

to have a relatively high packing density in addition to well-

ordered structure. Conversely, having high densities in the

surface terminal groups leads to steric hindrance. To overcome

these limitations, short-chain SAM such as thiotic acid (alpha-

lipoic acid) has recently been suggested as an alternative linker

molecule. Short-chain SAMs offer higher sensitivity and require

shorter incubation time than long-chain SAMs, therefore, for

these experiments we used the thiolate molecules based upon

prior reports that shorter thiol molecules are better for gold

substrate immunosensors than longer thiols.16,19 The thickness

of MUA and thiotic acid monolayers were measured by

Ellipsometry. The thickness of the thioctic acid and MUA

monolayer measured by ellipsometry were 9.8 ± 1.03 Å and

19.16 ± 1.75Å, respectively (Fig 3).

The additional thickness for only BSA (without antibody)

was 12.94 ± 3.23 Å. The thickness of the anti-fibrinogen

(without BSA) was 15.91 ± 1.14 Å. When blocking with BSA

after anti-fibrinogen, a significant difference in thickness could

not be detected. Also, the binding of the HRP-labeled antibody

did not result in a significant difference in thickness. When no

primary antibody was incubated with the surface (only

activation with EDC and NHS), the change was less than Å

(Fig 4). These ellipsometry results shown that the thiotic acid

molecule is 50% shorter than MUA and that there is no affect

on fibronogen antibodies binding efficiency.

Table 1. Experimental Procedure and Group. The Group 1 is our established method for immobilized antibody on gold substrate,

Group 2 is negative control for non specific binding to gold monolayer, Group 3 is control for 2nd Ab nonspecific binding to unre-

acted site on gold substrate, Group 4 is prepare for measurement of BSA thickness and density.

Procedure Chemical & LigandExperimental groups

Measurement1 2 3 4

7 Immunochemical reaction 2nd Ab-HRP Y Y Y Y Ellipsometry, ELISA, AFM image

6 Blocking 1% BSA Y N N Y Ellipsometry

5 Remove ramaining EDC/NHS groups Ethanolamine Y Y Y Y N/A

4 Immobilization 1st Ab (antifibrinogen) Y N Y N Ellipsometry

3 Activation of gold monolayer EDC+NHS Y Y Y Y N/A

2 Modified gold surface Thioctic acid Y Y Y Y Ellipsometry

1 Preparation of bare gold on Si Y Y Y Y Ellipsometry, AFM image

Note] To measurements the gold substrate take off and washed by DW and then dried by streamed argon gas at the point of each step. The

group 1 is our established method for immobilized antibody on gold subatrate, Group 2 is negative control for non specific binding to gold

monolayer, Group 3 is control for 2nd

Ab nonspecific binding to unreacted site on gold substrate, Group 4 is prepare for measurement of BSA

thickness and density. (2nd

Ab : anti-rabbit IgG with HRP, BSA : bovine serum albumin)

Figure 3. Change of thickness of bare gold surface after modified MUA or Thioctic Acid (A) Comparison to thickness after use shorter

thio (thioctic acid) instead of MUA (11-mercaptonudecanoic acid) using Ellipsometry, (B) Chemistry information (from The United

States Pharmacopeial Convention; USP.org)

Hongsik Cho et al.

14

3.2 ELISA

For the ELISA measurements of the same surfaces (Fig 5), the

absorbances for the surfaces with immobilized anti-fibrinogen

were higher than the absorbances for surfaces without immobilized

antibody. Therefore, this confirms the ellipsometry results that

the antibody is immobilized on the surface. It also demonstrates

that the HRP-labeled antibody attaches to the immobilized

antibody. Interestingly, even though the HRP-labeled antibody

could not be detected by ellipsometry, its presence was verified

by ELISA. Therefore, it is present on the surface, but it is most

likely present in much smaller concentrations than the primary

antibody.

Enzymatic Assay (ELISA): ELISA measurements showed

that the antibody immobilized groups (#1, 3) have 5 times

higher absorbance than non-immobilized groups (# 2, 4). They

did not demonstrate a significant difference of absorbance in

immobilized groups (#1 and #3) and non-immobilized groups

(#2 and #4), respectively. It is good evidence that after antibody

immobilization they do not have as much non-specific binding.

The independent control experiments were carried out to

demonstrate that the immobilization results, specifically from

interaction between the 1st antibody with blocking agent (#1)

and without blocking (#3) and that nonspecific protein

absorption does not occur.

The enzymatic assay of group 1 and 3 are providing clear

evidence that the antibody is immobilized and they have high

density. In addition, results for groups 2 and 4 show the 2nd is

specifically binding to primary antibody (Fig 5).

3.3 Morphology by AFM

The immobilized antibodies on short thiol-modified surfaces

were submitted to AFM. A scan size of 4×4 µm were used for

Figure 4. Ellipsometry measurements. To determine the average

thickness of the organic layer after each step (n = 9). Note that the

thickness after the thioctic acid monolayer is subtracted from all

of the above measurements. (Unit: Å)

*Note : Group 1 : Normal group (our established method), Group

2 : without 1st antibody and without blocking (BSA), Group 3 :

without blocking (BSA), Group 4 : without 1st antibody but with

blocking step.

Figure 5. Enzymatic Activities. After the antibody immobilized

the surface washed and blocked by BSA and reacted by 2nd anti-

body. After another buffer wash, immunochemical reactions may

be performed by TMB. (UV-vis absorbance for reaction with

immobilization of antibody and 2nd Anti rabbit IgG-HRP mea-

sured at 450 nm by TMB assay. *: p < 0.05)

*Note : Group 1 : Normal group (our established method), Group

2 : without 1st antibody and without blocking (BSA), Group 3 :

without blocking (BSA), Group 4 : without 1st antibody but with

blocking step

Figure 6. Image of the gold substrate surface by AFM. Tapping

mode AFM images of evaporated gold surface following colloi-

dal Au-amplified immunoassays of solution containing. (A) bare

gold surface, (B) bare gold phase, (C) anti-fibrinogen antibodies

immobilized surface, (D) Phase image of immobilization of anti-

fibrinogen antibodies.

Fibrinogen Antibody Immobilization on SAM

15

these images. An AFM image of clean gold surface is shown in

Figures 6 (a, b). The binding of antibody to gold-coated silicon

was highly uniform as shown in Figure 6 (c, d). On the SAM

containing andibodies, the white spots were about 2.5 nm high,

with some higher defaults (using MFP-3D software; Asylum

Res, CA). The antibody immobilized gold surface is significantly

rougher than pure bare gold substrate. AFM was utilized to

assess the uniformity of biomolecules immobilized on gold-

coated silicon at high spatial resolution (Fig 6). More detailed

studies on effects of linker flexibility and functionality on the

immobilization of antibody and the subsequent antibody-

antigen interactions will be investigated in the near future.

In conclusion, we have confirmed this immobilization

platform for immunosensor applications using fibronogen

antibody. Our results showed that these antibodies were directly

immobilized onto the gold substrate with high binding ability

and homogeneity of the binding affinity. We believe that this

gold SAM system using polyclonal anti-fibronogen antibody is

suitable for the next step of immunosensor applications such as

antibody microarrays, in which it is important to immobilize the

active antibody or their fragment at a high density in a very

small area. This method of immunosensor technology, is easy

to monitor, its speedy and accurate and only requires a small

sample blood. This immunosensor technology will have

potential for use in point-of-care diagnostics in the near future.

Acknowledgement: This work was supported by the National

Science Foundation CAREER award (CHE-0349315), the

FedEx Institute of Technology Innovation Award (EP) and NIH

R01(079147-01) grant (EL).

Disclosure Statements: Hongsik Cho, Justin Zook, Todd

Banner, Sang-Hyug Park, Byoung-Hyun Min, Karen A Hasty,

Eugene Pinkhassik, and Erno Lindner declare that they have no

conflict of interest. This article does not contain any studies

with human or animal subjects performed by the any of the

authors.

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