201 (2006) 2540–2546www.elsevier.com/locate/surfcoat

Surface & Coatings Technology

A new method for immunosensor preparation: Atmospheric plasma torch

Ebru Akdoğan a, Dilek Çökeliler a, Liutauras Marcinauskas b, Pranas Valatkevicius b,Vitas Valincius b, Mehmet Mutlu a,⁎

a Hacettepe University, Engineering Faculty, Plasma Aided Bioengineering and Biotechnology Research Group, 06532 Ankara, Turkiyeb Lithuanian Energy Institute, Plasma Processing Laboratory, Breslaujos 3, LT-44403 Kaunas, Lithuania

Received 1 November 2005; accepted in revised form 21 April 2006Available online 30 June 2006

Abstract

This paper describes a new method for the modification of piezoelectric quartz crystals for immunosensor preparation. The surfaces of thepiezoelectric quartz crystals were modified employing Atmospheric Pressure Plasma System using different monomers such as Ethylene diamine(EDA), 1,4-Diaminobutane (DAB) and n-Butylamine (BA). The atmospheric pressure plasma system was adjusted to use the temperaturesensitive and volatile monomers. A tunnel from stainless steel was constructed and combined with the plasma torch to introduce the monomersinto the plasma jet and preventing the mixing of atmospheric air with the plasma jet. Plasma parameters were; power: 1 kW, pressure: 1 atm, speedof plasma particles: 219 m/s, and exposure time 10 min for EDA and BA monomers, 15 min for DAB monomer. Crystals were characterized byfrequency shift measurements, Atomic Force Microscopy (AFM) imaging, contact angle measurements and Infrared (IR) spectra. Frequency shiftsfor EDA, DAB and BA modified crystals after plasma modification were 56±10, 143±3 and 904±3 Hz respectively. AFM images showed thepresence of a film on the surfaces for all monomers. N–H, C–N, C–H and C_O group bands were determined in the IR spectra of the crystals.Decrease in the contact angle values of the modified crystals indicated the increase in hydrophilicity. Those results showed that amine containingfilms on the crystal surfaces were successfully deposited using atmospheric pressure plasma torch. The chemical groups on quartz surface werefurther activated with gluteraldehyde (GA) and protein immobilisation properties were checked with test antibody (anti-aflatoxin B1) in liquidphase quartz crystal microbalance system. The dip coating method was chosen for immobilisation procedure. After the immobilisation step,chemical structure of surfaces was analyzed by X-ray Photoelectron Spectroscopy.© 2006 Elsevier B.V. All rights reserved.

Keywords: Atmospheric plasma; Plasma polymerisation; Piezoelectric quartz crystal; Biosensor; Antibody immobilisation; Plasma polymerised films

1. Introduction

In recent decades, immunosensors with various kinds of de-tection formats have received rapid development and wide ap-plications [1–5]. The general preparation procedure of such aminiaturized device involves the immobilisation of antibodies ontopiezoelectric, optical, electrochemical, and thermoelectric trans-ducer surfaces by appropriate chemical or physical treatments. It isclear that the choice of immobilisation strategies for immunoactiveentities is one of the key fundamental issues to pursue. Immo-bilisation of antibodies simply by physical adsorption might haveproblems associated with loss of sensitivity and low reproducibil-

⁎ Corresponding author.E-mail address: gmehmet@hacettepe.edu.tr (M. Mutlu).

0257-8972/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.surfcoat.2006.04.032

ity of sensors because of the gradual elution of adsorbed proteinsduring the analytical performances. Alternatively, considerableinterest has gone into the development of firm immobilisationmethods for antibodies by chemically covalent interaction usinglinkage reagents, such as gluteraldehyde, carbodiimide, and suc-cinimide, etc. [6–9]. In this study plasma polymerisation withatmospheric discharge was used to create functional groups ontoquartz crystal surfaces to immobilize test antibody (anti-aflatoxinB1). Compared with conventional polymerisation methods, plas-ma polymerisation has many advantages; it changes only thesurface properties of the introduced material without affecting itsbulk properties, it is a very efficient method and considered asenvironment friendly. Plasma-deposited polymers possess anextremely different structure, capable of forming deposited ma-terial containing active groups with high cross-linkages, with goodstability, homogeneity and adhesiveness.

Fig. 1. The schematic representation of the plasma system for deposition of themonomers on crystal surfaces. 1-cathode, 2-anode.

2541E. Akdoğan et al. / Surface & Coatings Technology 201 (2006) 2540–2546

Over the last years, atmospheric pressure plasma spray systemshave been widely adopted for industrial application. This is due toversatility of the available process, the possibility of achievingcoatings from almost all kind of materials (ceramics, metals, cer-mets and composites), fast growth rates (up to 1 mm h−1) andpossibility of deposition on large area of substrates. Thermal plas-ma jets are characterized by the high temperature (2000–14,000K)and high velocities (100–2500m s−1). Argon, nitrogen, hydrogen,helium or their mixtures are used as plasma forming gas. Differentpowders, liquids and gases are used as precursors. During theplasma spray operation; coating properties are influenced by thefollowing parameters: precursor injection places, pressure, sub-strate temperature, torch operating regime, plasma flow temper-ature and velocity [10–12].

Fig. 2. The schematic representation of the plasma gun and the tunnel. G0 –argon gas injection point, G1 and G2 – monomer injection points.

Since low pressure systems are used in most of the plasmasurface modification for biosensor applications [9], the aim of thestudy described in this paper was to investigate the possibility touse the atmospheric pressure plasma torch system for the coating ofpiezoelectric quartz crystals with temperature sensitive and volatilemonomers for an immunosensor development. Atmospheric pres-sure plasmas combine several advantages compared to the wellestablished low-pressure plasmas. For the operation of atmosphericpressure plasmas, no vacuum equipment is required, thus they canbe operated at very low costs and may easily be integrated into in-line processes and because of the small electron mean free path ofapproximately 0.1 mm, atmospheric pressure discharges can beignited in very tiny geometries on the micrometer scale. Thereforethey can be used to generate in new areas like biosensors.

2. Experimental

2.1. Materials

5 MHz, AT-cut Ti/Au polished piezoelectric quartz crystalswere purchased from Maxtek Inc., USA. Monomers Ethylene-diamine (EDA), 1, 4-Diaminobutane (DAB, 99%) and n-Butyl-amine (BA, 99.5%) were obtained from Acros Organics, USA.Gluteraldehyde (GA, %50 and antibody (anti-aflatoxin B1)were purchased from Sigma, USA.

2.2. Experimental set up and plasma torch characteristics

The atmospheric pressure plasma system used in the experi-ments described here has been developed by the Lithuanian En-ergy Institute researchers. The system is originally designed toobtain hard coatings and diamond like surfaces and was adjustedfor piezoelectric quartz crystal surfaces modification. Experimen-tal set up consisting of power generator, gas feeding and coolingsystems, plasma torch (PT), feeders, cooling systemof the crystals,the plasma jet tunnel, control devices, auxiliaries and airingdevices is shown in Fig. 1.

Fig. 3. Generalized current-voltage characteristics of plasma torch. Circles showexperimental value, straight line – equation.

Fig. 4. Reaction sequence for treatment steps of the QCM gold electrodes, followed by plasma modification, activation and antibody immobilisation.

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Linear direct current PT was used for modification of surfacesof piezoelectric quartz crystals (Fig. 2). The PT consists of a hothafnium (Hf) cathode (1), the gas injecting ring (3) containing twoblowholes (G0) of tangential gas supply for the arc stabilization,water-cooled copper anode (2) with blowhole (G1). The workinggas-argon passes through the arc column affecting by dissociationand ionisation until it forms plasma. The geometry of the copperanode forces acceleration of rapidly expanding gas and results theformation of a high velocity, high-temperature jet. The coatingmaterial is introduced as evaporated liquid into the torch eitherinternally or externally as shown in Fig. 2. Plasma torch parametersare: arc current 12–80 A, voltage 25–35 V, power 0.35–2.4 kW,gas (argon) flow rate 0.05–0.45×10−3 kg s−1, temperature andvelocity of the gas leaving the plasma torch 2100–7200 K and140–310 m s−1, respectively.

The characteristics of the PTand the parameters of plasma flowwere determined using theory of similarity, presented elsewhere[11]. Generalized current-voltage characteristics (Fig. 3) of PT aredescribed by the following relationship:

UdI

¼ 44dI2

Gd

� �−0;52

dGd

� �−0;42

ð1Þ

The generalized current-voltage characteristics show that PTworks stable and is suitable for deposition process.

The efficiency of PT was obtained as

1−gg

¼ 1; 16d10−4I2

Gd

� �−0;32

dGd

� �−0;32

ð2Þ

With the knowledge of the geometry of PT, its arc currentand voltage drop, it is possible to determine approximately theaverage temperature of the gas leaving the anode:

Tf ¼ 1; 62dI2

Gd

� �0;35

ð3Þ

where U is voltage (V), I – current (A), G – argon gas flow rate(kg s−1), d – anode diameter (m), η is PT efficiency,. Tf is theplasma mean temperature leaving the anode (K).

2.3. Surface modification of piezoelectric quartz crystals

The monomers used for the deposition of coatings on thepiezoelectric quartz crystals are sensitive to high temperature; andthe high temperature (TN4000 K) at the injection place G1 in theoriginal system may cause the decomposition of the monomers.The wind tunnel made from stainless steel with separate hole (G2)was designed and constructed for the prevention of decomposition.The tunnel also prevents substrate and coating from the injection ofambient air to plasma jet and the formation of oxidized groupson crystal surfaces. Monomers were heated in a water bath at aconstant temperature of 373K, vaporised in a stainless steel vesseland were introduced at two points (G1 and G2) into plasma jet.The distance between quartz crystal and tunnel was 6.10−3 m. It isvery well known that the piezoelectric quartz crystals lose theirpiezoelectric properties above 600 K, so during modificationprocesses quartz crystals substrates were cooled by water to keeptemperature below 600 K. The temperature distribution in theplasma jet above the substrate has been determined using chromel/alumel thermocouple.

The surfaces of the piezoelectric quartz crystals were modifiedusing three different monomers, Ethylenediamine, 1,4-Diamino-butane and n-Butylamine, as precursors. These three monomerswere chosen for they are the most common monomers used in theliterature for the preparation of amine containing films by lowpressure plasma polymerisation system [9,13,14]. Moreover, thesethree monomers have different boiling points (117 °C for EDA,118 °C for DAB and 78 °C for BA), since this type of applicationwill be performed in the plasma torch system for the first time, theeffect of different monomers with different properties had to beinvestigated. The argon gas flow rate was kept constant at 0.2810−3 kg s−1. Temperature of gas leaving the anode (calculated by

Table 1Frequency shifts and the calculated changes in mass on the surfaces of theplasma modified crystals*

Modification Frequency shift Δf (Hz) Change in mass (g(mm2)

EDA 56±10 0,68±0,12DAB 143±3 1,73±0,03BA 904±3 10,94±0,03

*The mass changes are the averages of 5 repeat experiments. The errorsrepresent the standard deviation of repeat experiments.

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the Eq. (3)) was 4227 K, the temperature at the central part of thelocation of crystals was less then 353 K. After modification pro-cess the piezoelectric quartz crystals were kept in argon atmo-sphere for 1 day. Themain process parameters were, power; 1 kW,pressure; 1 atm, speed of plasma particles; 219 m/s, and exposuretime; 10 min for EDA and BA monomers, 15 min for DABmonomer.

2.4. Analysis of modified piezoelectric quartz crystals

2.4.1. Frequency shiftBase frequencies of quartz crystals before and after plasma

modification were measured in deionised water using Agilent53181A 225 MHz Frequency Counter. The plain crystals wereplaced to the probe of the quartz crystal microbalance (QCM)system and were immersed to a magnetically stirred vessel filledwith deionised water. The frequencies were measured for 5 minat 25 °C and collected using Agilent Frequency Counter soft-ware. QCM measurements in aqueous solution were possibleonly when one side of the crystal was sealed in a rubber casing.The same procedure has been followed to determine the fre-quencies of the plasmamodified samples. The change in mass asdeposited polymer film is related to the frequency shift by theSauerbery equation,

Dm ¼ Df ðqv=f 20 Þ ð4Þ

where Δm is the mass loading per unit area of the piezoelectricsurface, Δf is the change in the base frequency of the modified

Fig. 5. AFM images of the piezoelectric quartz crystals: a) unmodified surface, b

crystal (MHz), ρ is the density of quartz (2650 kg/m3), v is thespeed of sound propagation in quartz (3340 m/s) and f0 is thefundamental oscillation frequency of the piezoelectric quartzcrystal (5 MHz). [12,15–18].

2.4.2. Chemical activation of modified piezoelectric quartzcrystals

The modified quartz crystals were immersed in GA solution(2.5% w/w in phosphate buffer, pH: 7) for 20 h at 20 °C. Thisactivation procedure was explained and optimised in our previousstudies [9]. The frequency changes of the crystals were monitoredduring the chemical activation step. GA is a dialdehyde and onealdehyde group covalently interacts with an amine group on EDA,DAB and BA modified crystal while the other aldehyde group isfree for biomolecule immobilisation. Reaction sequence for plas-ma treatment and chemical activation is shown in Fig. 4.

2.4.3. Test antibody (anti-aflatoxins) immobilisationAntibody immobilisation was performed by dip coating meth-

od. 0.25 mg/ml antibody solution was prepared from a 1 ml/mgantibody stock and immobilisation was performed at roomtemperature for 2 h. Subsequently, quartz crystal was washedwith distilled to remove the possibly unbound remains on thecrystal surfaces. Frequency changes of the crystals were de-termined with QCM system. In this step, amine group of theantibody covalently interacts with the free aldehyde group ofGA obtained in the activation step, thus being immobilised oncrystal surface.

2.5. Surface analysis of piezoelectric quartz crystals

Surface characteristics of the crystals before and after plasmamodification were determined using Atomic Force Microscopy(AFM), Infrared Spectroscopy (IR), and contact angle measure-ments. Atomic ForceMicroscopy (AFM) imagingwas performedwith using Topometrix TMX 200. 80° reflectance spectroscopywas used in combination with a Bruker Equinox 55 to deter-mine the chemical changes on the surface of the crystals afterthe plasma modification. Contact angle measurements were

) EDA modified surface, c) DAB modified surface, d) BA modified surface.

Fig. 6. IR spectra of crystals.

Table 2Plasma conditions and frequency shifts after the activation and antibodyimmobilisation steps

Precusors Δf1 (Hz) Glutaraldeyhde activation Δf2 (Hz) Antibodyimmobilisation

DAB 18590 112BA 60 26EDA 58 514

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performed by sessile bubble technique using an Intel®Play Q*3Computer Microscope with deionised water. Contact angleswere calculated using WettabilityPro software. XPS was per-formed on a VG CLAM2 spectrometer with a (non-mono-chro-mated)MgKR source for quartz crystals after the immobilisation.Survey scans were taken at a pass energy of 100 eVand core levelspectra at 20 eV. Elemental compositions for these films werecalculated by using peak areas from the survey scans and exper-imentally derived sensitivity factors; core level spectra were fittedwith Gaussian–Lorentzian curves according to a protocol [6].

3. Results and discussion

The base frequencies of each crystal were determined beforeand after the plasma modification with the frequency counter.After the exposure of the crystals to the atmospheric plasmatorch, a decrease in the base frequencies was observed due to themonomer deposition on the crystal surfaces. The mass of thedeposited film was calculated from Eq. (4). The frequency shiftsof the crystals occurring after plasma modification along withthe change in mass are given in Table 1. Frequency shifts forEDA, DAB and BA modified crystals were 56±10, 143±3 and904±3 Hz respectively. The relatively higher frequency shift forBA modified crystal is probably due to its lower boiling point(117 °C for EDA, 118 °C for DAB and 78 °C for BA).

The AFM images of plain and plasma modified crystals areshown in Fig. 5. From the AFM images, it is possible to say thata thick film deposition is obtained for the EDA and BA mod-ified surfaces. AFM images showed a very rough surface and anon-homogeneous deposition. Mass accumulation in some partsof the surface was observed for DAB modified surface.

The changes of the chemical composition of the atmosphericpressure plasma modified piezoelectric quartz crystal surfaceswere studied by IR spectroscopy. The spectra of the samples withvariation of the monomers are shown in Fig. 6. The assign-ments have been suggested for some characteristic bands; forEDA modified surface, 3242 cm−1 and 1553 cm−1 (vs, N–H),2924 cm−1 (vs, C–H), 1739 cm−1 (vs, C_O); for DABmodifiedsurface 3258 cm−1 (vs, N–H); and for BA modified surface3254 cm−1 (vs, N–H), 2943 cm−1 (vs, C–H), 1732 cm−1 (vs,C_O), 1293 cm−1 (vs, C–N). The presence of C_O groups onEDA and BA modified surface shows that an oxidation reactionpossibly occurred. This may be due to the mixing of air into theplasma jet from the 6 mm opening between the end of the tunneland sample surfaces during the modification.

Contact anglesweremeasuredwith sessile bubble technique. Inthis technique, the change on the contact angle gives an idea aboutthe hydrophilicity of the surface compared to the plain surface. Adecrease in the contact angle means increased hydrophilicity. It isknown that amine groups make the surface more hydrophiliccompared to the plain surfaces. Thus a decrease in the contactangle values were expected due to the presence of the aminegroups to be formed on the surface of the crystals after plasma

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modification. Contact angle values for the plain, EDA, DAB andBAmodified surfaceswere obtained as 63±2, 56±3, 54±3, 58±1respectively. A decrease on the contact angle values is observed asexpected. Contact angle values along with the IR results confirmthe formation of amine groups on the modified surfaces.

Frequency shifts after gluteraldehyde activation were 58,18,590 and 60 Hz for EDA, DAB, and BA modified crystalsrespectively (Table 2). FTIR results showed some oxidation onEDA and BA modified surfaces while no oxidation occurred on

Fig. 7. XPS widespectrum a) plain crystal b) EDA

DAB modified surfaces, contact angle results showed that theDAB modified surfaces the most hydrophilic surface. Since theamino groups on the surface are the criteria for the hydro-philicity, it can be assumed that DAB modified surface containsmore amino groups than the EDA and BAmodified surfaces. Sothe greater frequency shift observed for the DAB modifiedcrystal may be due to the increased amino groups present on thissurface leading to greater interaction between GA and aminogroups.

modified and antibody immobilised crystal.

Table 3Elemental composition of EDA modified crystal surface after antibodyimmobilisation and plain crystal surface

Sample ID % C % O % Au % N

EDA 67 21 1.2 9.8Plain 35.8 13.5 50.6 –

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Frequency shifts of the crystals after antibody immobilisationare 514, 112 and 26 Hz for EDA, DAB and BA modified crystalsrespectively (Table 2). The frequency shifts showed that antibodyimmobilisation on crystal surfaces are successfully achieved.Frequency shifts for DAB and BA modified crystals are lowercompared to previous studies performedwith low pressure plasmapolymerisation systems while the frequency shift for EDAmodified crystal is comparable with the literature [9,13,14]. It isimportant to point to the sensitivity of crystal. After the modi-fication of crystals with atmospheric plasma torch, the frequencyshifts (mass deposition) for EDA, DAB and BA treated crystalswere 56.00, 143.00 and 904.00 Hz respectively. Also after thechemical activation step, the film on the EDA modified crystal'ssurface remained the thinnest film compared with BA and DABtreated crystals surfaces. The antibody immobilisation on the EDAmodified surface the most effective compared to the other crystals(514, 112 and 26 Hz for EDA, DAB and BA modified crystalsrespectively). So there is a linear relationship between the thick-ness of the film deposited and the sensitivity of the crystals. Thethinner film means a more sensitive surface for the immobilizedantibody. Since the most successful antibody immobilisation isachieved with EDA modified crystals, the chemical compositionof this crystal and the plain crystal was determined by X-rayPhotoelectron Spectroscopy (Fig. 7). The atomic composition ofthe EDA modified crystal surface consisted of 67% C, 21% O,9.8% N and 1.2% Au which shifts from plain crystal's surfaceconsisted of 35.8% C, 13.5% O, 50.6% Au (Table 3). Althoughthere were no N on plain crystal, on the EDAmodified and furtherantibody immobilised crystal surface consisted of 9.8% N. The50.6% of the plain crystal consisted of Au while this value was1.2% for the biomolecule immobilised crystal as expected. C andO were in higher proportion on the EDA modified surface.

These results show that the atmospheric plasma torch can beused for amino containing film deposition for biosensor develop-ment. By means of atmospheric plasma torch, chemical functionalgroups could be generated by film deposition or surface modifi-cation on materials like quartz crystal. Especially amino and alde-hyde groups are of great interest since they have high chemicalreactivity, therefore they can be used for the immobilisation ofbiomolecules such as antibodies.

4. Conclusions

Piezoelectric quartz crystal surfaces were modified with anatmospheric pressure plasma torch system using EDA, BA andDAB asmonomers to obtain amine group containing film coatings

on the surfaces. After activationwithGA, antibody immobilisationon quartz crystals was successfully achieved. The atmosphericplasma torch system used for the coating process was successivelyadapted to the volatile and temperature sensitive monomers for animmunosensor preparation.

Using atmospheric pressure discharges for chemical func-tionalization of surfaces does not only have economical advan-tages over low pressure plasma processes; the ability to generatethese discharges in volumes of submillimeter dimensions opencompletely new perspectives for the patterned surface modifica-tion e.g. of polymers. Due to the low investment and running costs,these new processes are expected to gain importance for the treat-ment of quartz crystals for immunosensor applications.Our studieson preparing thinner films on the quartz crystal surfaces forreaching higher sensitivity and the antigen tests to complete theimmunosensor are still being performed.

Acknowledgement

This research has been partly supported by ESF-COST 527“Plasma Polymers and Related Materials” Action. The authorswish to thank Dr. Alex G. Shard, The Sheffield University, Mat-erials Engineering Department for the XPS analysis.

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