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Copyright 2011 American Scientific PublishersAll rights reservedPrinted in the United States of America

SENSOR LETTERSVol. 9, 15, 2011

Development of Locaslized Surface Plasmon ResonanceBased Biosensor Using Au Deposited Nano-Porous

Aluminum Anodic Oxide Chip

Se-Hyuk Yeom1, Heng Yuan1, Woo-Youp Choi1, Nyeon-Sik Eum2, and Shin-Won Kang1

1School of Electrical Engineering and Computer Science, Kyungpook National University, Daegu, Republic of Korea2Kyungnam 2Center for IT-Medical Device Convergence, Keimyung University, Daegu, Korea

(Received: 13 November 2009. Accepted: 5 August 2010)

In this study, we fabricated a highly sensitive and real-time monitoring biosensor using a nano-porous aluminum anodic oxide (AAO) chip. We improved the surface uniformity by two-step anodiz-

ing and fabricated an AAO chip having a pore size of 50 nm. The device uses interference and

localized surface plasmon resonance (LSPR) phenomena, and it has high sensitivity and selectivity

relative to a sensing device that use a single sensing mechanism. We deposited gold on an Al2O3layer to induce the LSPR phenomenon, and fabricated a sensing membrane using the SAM method

to immobilize troponin T antibody on gold. A real time biosensor system is constructed using optical

spectroscopy and an optical fiber reflectance probe. We measured troponin T Antigen quantitatively

using fabricated sensor, and confirmed its applicability as a diagnostic tool for myocardial infarction.

The troponin T is known as an unusual myocardial injury marker, and as a protein that forms the

complexes with troponin I, and troponin C, which control muscle contraction.

Keywords: AAO, LSPR, Biosensor, Interferometry, Troponin T.

1. INTRODUCTION

With developments in medical technology, there has beenincreasing interest in diagnostic techniques for pathogensand for the detection and analysis of bio-materialswithin or outside the body. Many studies have aimed toimprove the sensitivity and speed of existing measurementmechanisms. Methods for detecting bio-materials includefluorescence-, luminescence-, evanescent field, and sur-face plasmon resonance (SPR) based approaches usingoptical and piezoelectric devices, electrochemical basedpotentiometric approaches, amperometric approaches, andacoustic based conductimetric approaches. Among these,optical biosensors have advantages such as high accu-racy, and sensitivity and short measurement time. Opticalbiosensors include sensors with an optical fiber, SPR typebiosensors, waveguide biosensors that use the change inrefractive index, and interferometric biosensors.12 In this

Corresponding author; E-mail: swkang@knu.ac.kr

study, we developed biosensor with a nano-porous struc-ture that uses the localized SPR (LSPR) and the inter-ference phenomena. LSPR is sensitive to changes in therefractive index of a sensing membrane that are inducedby reactions with bio-materials, and has great advan-tages that label-free technique.36 In addition, the usesof these phenomena enable a lightweight sensor to bemanufactured, and therefore, such sensors can be usedportably.

Fabrication methods for nano-porous structures includean electrolytic polishing method developed by Uhlir andTurner7 in 1950 and a method developed by Canham8 forforming a porous structure on silicon. When a porous alu-minum substrate is irradiated with white light, a Fabry-Perot fringe pattern is produced because of the opticalpath difference, reflection between the pores and the bulk-head, and changed in optical path length. In addition,the wavelength change of fringe pattern is very sensi-tive in small materials, this can be highly advantageousfor detecting small molecules such as bio-materials. Fur-thermore, measurements can be carried out in real-time.9

Sensor Lett. 2011, Vol. 9, No. 1 1546-198X/2011/9/001/005 doi:10.1166/sl.2011.1426 1

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We fabricated various depths of porous, thin films havingthe aforementioned advantages using a two-step anodizingmethod. A white light tungsten halogen lamp (480 nm1100 nm) was used as a light source, and the change inthe wavelength and intensity of light were measured usinga spectrometer. We measured the changes in wavelengthinterference from the refractive index and mass change atthe sensing membrane. By measuring the intensity changein reflected light and wavelength shift from an antigenantibody reaction of The troponin T, we confirmed theapplicability of the developed device as a biosensor.

2. EXPERIMENTAL DETAILS

2.1. Sensing Mechanism

We designed an LSPR-based interferometry biosensor con-sisting of a white-light source (Ocean Optics), reflectance

optical probe bundle, antibodyantigen reaction cham-ber, spectroscope (Ocean Optics), and a nano-porousaluminum anodic oxide (AAO) chip. The troponin T anti-body was immobilized in the nano-porous AAO chipby irradiating it with visible light perpendicularly usingan optical fiber probe bundle. The reflected light wasdetected using the same optical probe and measuredas a reference signal using a UV-visible spectrometer.Next, we analyzed the reflection spectrum by chang-ing the concentration of the troponin T antigen. Theexperimental setup and sensing mechanism are shown inFigure 1.

The fringe pattern of the porous AAO chip can be ana-lyzed by measuring the effective optical thickness. Whenwe irradiate white light on the device, the optical pathdifference depends on the thickness (d) of the aluminumoxide. If white light is irradiated on the porous thinfilm, an interference pattern that depends on the effec-tive optical thickness can be generated. Rossi10 definedthe effective optical thickness (d) as the product of

Fig. 1. Experimental setup and sensing mechanism.

the thickness and the refractive index (n), as given byEq. (1).

m= 2nd (1)

Here, m is the order of the fringe and , the wavelengthof light.

The thickness can be controlled by changing the concen-

tration of the electrolyte solution, voltage, and anodizingtime. The number of fringes and the wavelength changewith an increase in the thickness.

2.2. Fabrication of Nano-Porous AAO Chip

Generally, a passive metal like aluminum has an approx-imately 1 nm thick native oxide layer at the surface. Toincrease the thickness of this layer, the anodizing methodis most widely used.

In the anodizing method, if aluminum serves as theanode and carbon or platinum, the cathode, in an elec-

trolyte solution, aluminum cation dissociate into the elec-trolyte solution and oxygen and hydroxide ions move tothe aluminum anode and combine with aluminum ions,forming an Al2O3 layer. If the electrolyte solution is acidic,a partial solution is formed in the oxide, and therefore,the oxide surface is rough and partially eroded. When anelectric field is applied on the eroded oxide, it grows upto form pores.11

Matsuda et al. suggested the use of two-step anodiz-ing in order to fabricate AAO that has three times betteruniformity than that fabricated using one-step anodizing.After anodizing aluminum under certain conditions, thegenerated aluminum is removed and anodizing is carriedout again under the same conditions. We can control thesize of the pore by phosphoric acid etching and also con-trol the concentration of oxalic, sulfuric, and phosphoricacids in the electrolyte solution.12

In this study, we applied the aforementioned two-stepanodizing method to improve the uniformity of pores. Alu-minum foil (99.999% Al, Aldrich) was used as a substrateand 0.3 M oxalic acid was used as the electrolyte solution.To remove the native oxide and impurities, and smooththe surface of aluminum, we placed the aluminum foil ina solution of 30% ethanol and 70% perchloric acid andperformed electropolishing with a bias voltage of 20 V.

The electropolished aluminum foil was dipped into theelectrolyte solution as the anode and a carbon electrodewas used as the counter electrode; anodizing was then per-formed with a bias voltage of 40 V.

In two-step anodizing, the anode oxide generated in thefirst step was placed in a solution consisting 1.8 wt%chromic acid and 6 wt% phosphoric acid for 90 min at60 C to remove the first anodized aluminum oxide. Byperforming the second step under the same conditions, theAl2O3 layer was grown up. We can establish a conditionfor growing the oxide by controlling the time of the sec-ond anodizing step. The temperature was maintained at

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9 C using a circulator, the concentration of the electrolytesolution was 0.3 M, the bias voltage was 40 V, and the cur-rent in the electrolyte solution was 0.010 A. Under theseconditions, we fabricated five devices by controlling theanodizing time every 16 min, and we then measured thethicknesses of the devices using FE-SEM. As a result, afteranodizing times of 16, 33, 50, 67, and 83 min, the thick-nesses were found to be 1.009, 2.024, 3.089, 3.96, and4.965 m, respectively. Additionally, we confirmed thatuniform pore diameters of 50 nm could be obtained. Basedon the above results, we analyzed the growth rate of theanode oxide in terms of time and current, and confirmed agrowth rate of 1 nm/s. The growth rate is given by Eq. (2).

T= A time 107m (2)

where, T is the thickness of the anode oxide and A, thecurrent flowing in the electrolyte solution; the time isexpressed in units of seconds.

Figure 2 shows SEM and AFM images of the fivedevices fabricated for various anodizing times. The ana-lyzed growth rate of the anode oxide was rather linear ascompared to the actually fabricated devices, and the errorrate was less than 3%.

2.3. Fabrication of Sensing Membrane

After fabricating the AAO plate, to improve the LSPRoptical properties, we deposited a 50--thick Ni layerand a 150--thick Au layer as buffer layers by e-beamevaporation. The troponin T antibody was immobilizedon an Au-deposited nano-porous AAO chip by the self-assembled monolayer (SAM) method.

To form a SAM, we dissolved 11-mercaptoundecanoicacid (MUA) in ethanol at a concentration of 1 mMand stirred it for 2 hr, and then cleaned it usingethanol and distilled water. Next, we dissolved 1-ethyl-3-3-dimethylaminopropyl carbodiimide hydrochloride (EDC)and n-hydroxysuccinimide (NHS) in distilled water at a

Fig. 2. SEM and AFM images of AAO chip.

Fig. 3. Growth rate of AAO according to anodizing time.

concentration of 100 mM; these were functionalized after

stirring at a 1:1 ratio for 1 hr. Finally, we conjugated theantibody by stirring 10 g/ml of troponin T antibody atroom temperature for 1 hr. After cleaning the surface ofthe fabricated device, we measured the shift of the reflec-tion spectrum from the conjugation of the antibody usingthe constructed system.

3. RESULTS AND DISCUSSION

3.1. Optical Characteristics of Sensing Membrane

To measure the shift of the reflection spectrum caused

by the change in the refractive index of the sensingmembrane, we used glycerin solutions of various concen-trations. Solutions of glycerin in distilled water were pre-pared at various concentrations (6, 9, 12, and 15 wt%) inorder to obtain solutions with different refractive indices.We measured the refractive indices of these solutions usingan Abbe refractometer. The measured values are listed inTable 1.

Glycerin solutions with various refractive indices werespin-coated on the fabricated sensing membranes, and theshift of the reflection spectrum was measured by using thereflection measurement system. Figure 4 shows the shiftof the reflection wavelength, as measured the change inthe refractive index of the sensing membrane. We con-firmed a linear shift of the wavelength from the changein the refractive index. The next set of results (Fig. 5)shows the shift of the reflection spectrum for the case

Table I. Refractive index of glycerin solutions.

Glycerin % by weight Refractive index

15 1.349112 1.34589 1.34306 1.3392

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Fig. 4. Wavelength shift by various refractive index of Glycerinsolutions.

in which a solution with a different refractive index wascoated on the surface of a device. The results indicatethat the wavelength shifts with changes in the refractiveindex.

3.2. Characteristics of LSPR Based Biosensor

We measured the change in the refractive index afterincubating the troponin T antigen on a sensing membraneconjugated with troponin T antibody for 30 min, the con-centration of the target molecule varied from 1 pg/ml to1 g/ml. We also measured the shift and the increase inthe intensity of the reflection spectrum with an increase inthe concentration of the target material. The sensor can-

not detect changes in the wavelength for a concentrationof 1 pg/ml; therefore, a measurement limit of 1 ng/mlwas selected. Under this limit, the sensor showed a linearsensing characteristic with respect to increasing concen-tration of the troponin T antigen. Figure 6(a) shows theresult of reflection spectrum for various concentration ofthe troponin T antigen, Figure 6(b) shows the result of

Fig. 5. Optical property of Au deposited AAO chip.

(a)

(b)

(c)

Fig. 6. Reflectance spectrum shift by antibodyantigen reaction ofTroponin T. (a) The results as the wavelength from 480 nm to 1000 nm,(b) the results as the wavelength from 700 nm to 760 nm, (c) the peak

wavelength shift by detecting Troponin T antigen.

spectrum change from 700 nm to 760 nm. Figure 6(c)shows the change in the wavelength peak dependingon the increase in the concentration of the troponinT antigen.

We examined whether our sensor system showed non-specific binding of antigens. We confirmed the change ofthe reflection spectrum after the reaction of the Salmonellaantigen 1 g/ml with the troponin T antibody for 30 min.We could confirm that the sensing membrane only reactswith the target material, namely, the troponin T antigen,

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Fig. 7. Selectivity test of biosensor system.

and not with the Salmonella antigen or other kinds of

proteins (Fig. 7). We verified that the developed sensorsystem did not show nonspecific binding and was selective.

4. CONCLUSIONS

In this study, we manufactured a biosensor system by usinga nano-porous AAO chip as a sensing membrane that candetect the antibodyantigen reaction in biomolecules; thedetection is based on LSPR and optical interference phe-nomena. We fabricated devices by the anodizing methodand improved the surface uniformity by the two-stepanodizing method. The thickness of the AAO can be con-

trolled by varying the anodizing time. We used an Al2O3sensing membrane (thickness: 3 m) and determined theoptimum membrane length by performing tests for mem-branes of various thicknesses.

In order to enhance the LSPR signal and promote pro-tein bonding, we deposited Au on the sensing mem-brane for binding the Troponin T antibody by the SAMmethod. The various concentrations of troponin T antigenwere measured by the fabricated sensor. The current

measurement limit of the system is 10 g/ml but weexpect to enhance the sensitivity by modifying the methodfor immobilizing biomolecules. By performing a testinvolving reactions of an antibody with the target mate-rial and other viruses, we confirmed the selectivity of thefabricated sensor.

The biosensor system fabricated in this study is highlysensitive, lightweight, and inexpensive. In particular, itenables real-time measurements, unlike other similar sys-tems developed using AAO chips. In the future, we intendto study the impact of the size and shape of biomoleculeson sensor performance in further detail; we will also tryto fabricate an array-type sensing membrane. This mayenable us to use the developed biosensor as a tool fordetecting and analyzing various biomolecules.

Acknowledgment: This work was supported byKorea Science and Engineering Foundation (KOSEF)grant funded by the Korea government (MEST)

(No. 2009-0063405)

References and Notes

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