Associating biosensing properties with the morphological structure of multilayers containing carbon...

Phys. Status Solidi A 207, No. 4, 781–786 (2010) / DOI 10.1002/pssa.200983301 p s sapplications and materials science

a

statu

s

soli

di

www.pss-a.comph

ysi

ca

Associating biosensing properties
with the morphological structureof multilayers containing carbon nanotubeson field-effect devices
Jose R. Siqueira Jr.1,2,3, Matthias Backer2,3, Arshak Poghossian2,3, Valtencir Zucolotto1,Osvaldo N. Oliveira Jr.*,1, and Michael J. Schoning**,2,3

Editor’s Choice

1 Instituto de Fısica de Sao Carlos, Universidade de Sao Paulo, 13560-970 Sao Carlos, Brazil2 Institute of Nano- and Biotechnologies, Aachen University of Applied Sciences, 52428 Julich, Germany3 Institute of Bio- and Nanosystems (IBN-2), Research Centre Julich, 52425 Julich, Germany

Received 25 August 2009, accepted 24 November 2009

Published online 18 March 2010

Keywords inorganic/organic multilayers, carbon nanotubes, morphology, biosensors

*Corresponding author: e-mail [email protected], Phone: þ55 16 3373-9825, Fax: þ55 16 3371-5365** e-mail [email protected]

The control of molecular architecture provided by the layer-by-

layer (LbL) technique has led to enhanced biosensors, in which

advantageous features of distinct materials can be combined.

Full optimization of biosensing performance, however, is only

reached if the film morphology is suitable for the principle of

detection of a specific biosensor. In this paper, we report a

detailed morphology analysis of LbL films made with

alternating layers of single-walled carbon nanotubes (SWNTs)

and polyamidoamine (PAMAM) dendrimers, which were then

covered with a layer of penicillinase (PEN). An optimized

performance to detect penicillin G was obtained with 6-bilayer

SWNT/PAMAM LbL films deposited on p-Si-SiO2-Ta2O5

chips, used in biosensors based on a capacitive electrolyte-

insulator-semiconductor (EIS) and a light-addressable poten-

tiometric sensor (LAPS) structure, respectively. Field-emission

scanning electron microscopy (FESEM) and atomic force

microscopy (AFM) images indicated that the LbL films were

porous, with a large surface area due to interconnection of

SWNT into PAMAM layers. This morphology was instrumen-

tal for the adsorption of a larger quantity of PEN, with the

resulting LbL film being highly stable. The experiments to

detect penicillin were performed with constant-capacitance

(ConCap) and constant-current (CC)measurements for EIS and

LAPS sensors, respectively, which revealed an enhanced

detection signal and sensitivity of ca. 100mV/decade for the

field-effect sensors modified with the PAMAM/SWNT LbL

film. It is concluded that controlling film morphology is

essential for an enhanced performance of biosensors, not only in

terms of sensitivity but also stability and response time.

� 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

1 Introduction The search for hybrid inorganic/organic supramolecular structures is motivated by the newphysical and chemical properties that may be achieved withspecific molecular-level interactions [1–3]. In biosensing,in particular, enhanced features have been obtained withnanostructured films, including high sensitivity, fastresponse time, stability, and reproducibility [4–6]. Many ofsuch films are produced with the layer-by-layer (LbL)technique [1–5, 7, 8], based on the deposition of oppositelycharged layers, which permits fine control of film architec-ture with maximized interactions between distinct materials.The LbL method has been effective in immobilizing

biomolecules owing to the capability of molecular engin-eering and the mild conditions for film fabrication, thusimparting high sensitivity and selectivity to the sensing units.A diversity ofmaterials exist whichmay be used to build LbLfilms, either to serve as templates or scaffolds forimmobilization of biomolecules or to enhance the signal tobe measured [1–5, 7, 8]. Among the latter type of materialare the carbon nanotubes (CNTs) whose electric andmorphological properties may be useful [9–13]. CNTs canbe single-walled (SWNTs) or multiwalled (MWNTs), withtheir large length-to-diameter aspect ratio providing a highsurface-to-volume ratio. Biomolecules such as proteins can


782 J. R. Siqueira Jr. et al.: Associating biosensing properties with the morphological structure of multilayersp

hys

ica ssp st

atu

s

solid

i a

Figure 1 (online colour at: www.pss-a.com)Schematic representation of a capacitive EISstructure and a LAPS device functionalizedwith a PAMAM/SWNT LbL film and theenzyme PEN, as well as their operationprinciple.

be immobilized on their surfaces without losing biologicalactivity [9–11]. The latter is important, as the integration oftheCNT/biomoleculemay lead to the fabrication of uniquelytailored devices with enhanced performance [9–11, 14].

We have instituted a project aimed at exploiting theadvantageous features of CNTs in two types of devices,namely a capacitive electrolyte-insulator-semiconductor(EIS) structure [15] and a light-addressable potentiometricsensor (LAPS) [16], where a suggestion was made that theenhanced performance could be attributed to the changes inmorphology induced by the CNTs (see Fig. 1). In this paper,we present a detailed morphological investigation of LbLfilms made with layers of SWNT alternated with thepositively charged polyamidoamine (PAMAM) dendrimer,on which a layer of the enzyme penicillinase (PEN) wasadsorbed. These films were incorporated in the EIS andLAPS structures [17] and used as sensing units to detectpenicillin G. Information from the morphology study wasemployed in optimizing the film architecture to achieveenhanced performance for the biosensors.

2 Experimental2.1 Materials and solutions Carboxylic acid func-

tionalized SWNTs and fourth generation G4 PAMAMdendrimer were purchased from Sigma–Aldrich. TheSWNT-COOH were produced with the arc dischargetechnique, having mainly semiconductor features and anominal purity of 95%, length of 0.5–1.5mm, and diameterof 1.5 and 3–5 nm for individual and bundled samples,respectively. PAMAM and SWNT solutions were preparedat concentrations of 1.0 g/L, using Titrisol buffered solutionsat pH 4 and 8, respectively. The SWNT dispersion wasobtained in a buffer solution by adding 10mg of thematerial into 10mL of water under ultra-sonication for 2 h,


and then filtrated. The enzyme PEN (Bacillus cereus,1670 units/mg protein) and Penicillin G (benzyl penicillin,C16H17N2O4SNa) were acquired from Sigma.

The enzyme solution was prepared in a 0.2M triethanol-amine (TEA) buffer at pH 8, adsorbed by dropping 80mL ofroom temperature for 4 h. The penicillin solution wasprepared at different concentrations ranging from 25mM to10mM in a 0.5mM polymix buffer, pH 8, containing 0.1MKCl solution as ionic strength adjuster.

2.2 Fabrication of EIS sensors and LAPSdevices The capacitive EIS and LAPS structures of p-Si-SiO2-Ta2O5 were prepared on a p-type Si(100) wafer (356–406mm in thickness, 1–10Vcm) via thermal oxidation of Siin an O2 atmosphere at 1050 8C for 30min to form a 30 nmSiO2 insulating layer. The Ta2O5 was prepared via electron-beam evaporation of 30 nm Ta, followed by thermaloxidation at 515 8C during 30min to form a 55 nm-thickinsulating, pH-sensitive layer. A contact layer ofAl filmwith300 nm was deposited on the rear side of the p-Si wafer,which was then cut into single chips of 10� 10 and20� 20mm2 sizes for EIS and LAPS, respectively. BothEIS and LAPS chips were mounted into their respectivehome-made measuring cells and sealed by an O-ring toprotect the side walls and rear side contact of the chip fromthe electrolyte solution. The contact area of the EIS sensorwith the solution was 0.5 cm2, while for the LAPS chips theAl rear contact was partly removed for illumination. An areaof 2.25 cm2 on the sensor surface was exposed to the testsolution and illuminated by 16 infrared light-emitting diodes(IR-LEDs) from the rear side. For details of preparation ofthe sensor transducer structures and their functional principlesee, e.g., Ref. [17–27]. Figure 1 illustrates a representation ofthe EIS and LAPS setup and their operation principle.

www.pss-a.com

Phys. Status Solidi A 207, No. 4 (2010) 783

Editor’s

Choice

Figure 2 Top view FESEM image (a) for a 6-bilayer PAMAM/SWNT LbL film on a field-effect structure and its zoomed area (b).

2.3 Fabrication and characterization of LbLfilms The LbL films of PAMAM/SWNT were assembledin a home-made measuring cell, sealed with silicone, viaalternated dropping of PAMAM for 5min and during 10minfor SWNT solutions into the cell, and then rinsed withdeionized water and dried under a N2 flow. The idealizedLbL film structure and the materials employed are alsorepresented in Fig. 1. The morphology of the PAMAM/SWNT LbL films was analyzed with a field-emissionscanning electron microscope (FESEM) Gemini1 ColumnLEO 1550 VP (Germany) and an atomic force microscope(AFM) BioMatWorkstation (JPK Instruments, Germany) inthe tapping mode, using Si cantilevers with silicon nitridetips. Images of the functionalized EIS and LAPS with LbLfilms (EIS-NT and LAPS-NT, respectively) were taken in aheight measuring mode at scanned areas of 5.0� 5.0 and8.0� 8.0mm2. The same was applied to the EIS and LAPSstructures containing the PEN immobilized atop the sensorsurface.

The penicillin biosensor based on a bare EIS and an EIS-NT structure was characterized by capacitance–voltage(C/V) and constant-capacitance (ConCap) methods using aZahner Elektrik (Germany) impedance analyzer at afrequency of 50Hz. All measurements were carried outapplying an ac voltage with an amplitude of 20mV. Inaddition, in the ConCap mode the capacitance of the EISsensor was kept constant by using a feedback-control circuitthat allowed the direct monitoring of potential changes at thesensor surface. The bare LAPS and LAPS-NT penicillinbiosensor were characterized using an electronic unit withsurface mounted devices (SMD) with 16 IR-LEDs [25–27]by means of current–voltage (I/V) measurements with a biasvoltage range from�0.8 to 1.2V, and a step width of 20mVat a frequency of 1 kHz. Moreover, measurements wereperformed in the constant-current (CC)mode, where the biasvoltagewas controlled by a feedback loop tomaintain a fixedphotocurrent. In both sensor structures a conventional liquid-junction Ag/AgCl electrode (Metrohm) was used as thereference electrode and themeasurementswere carried out ina dark Faraday cage at room temperature.

3 Results and discussion3.1 Morphological features of LbL films on

field-effect structures Analyzing the morphology ofnanostructured films may be essential to optimize theperformance of biosensors, for it is important in theintegration of the sensor electrode or chip with a biologicalcompound. A fine, careful control of film formation isrequired to obtain a homogeneous film surface; otherwise,morphological differences may affect the sensor response.Figure 2 shows FESEM images for a 6-bilayer PAMAM/SWNT LbL film on a p-Si-SiO2-Ta2O5 structure. The topview image in Fig. 2a exhibits a randomly oriented SWNTsnetwork into dendrimers layers over the entire sensorsurface. The strong interconnection of SWNTs intoPAMAM layers created a film with high surface area and

www.pss-a.com

porosity as visualized in the zoomed area in Fig. 2b. Suchfilm structure, in particular, is appropriate for biosensorsbased on the field-effect devices platform, as the nano-porosity creates channels for the penetration of ions, whilethe large film surface area acts as a nanomembrane for moreefficient immobilization and distribution of biomolecules.Furthermore, the field-effect devices using multilayerPAMAM/SWNT LbL films were highly stable, and werenot affected by several rinsing processes with aqueoussolutions at distinct pHs and by drying steps. In contrast, insubsidiary experiments we observed that cast films ofSWNTs dissolved upon several rinsing processes.

To obtain further details of the LbL film structure, themorphology of the films was investigated with AFM imagesfor a scanned area of 8.0� 8.0mm2, as shown in Fig. 3 for a2-bilayer PAMAM/SWNT LbL film. The height image inFig. 3a and its inverted image in Fig. 3b show the samerandomly oriented SWNTs network into dendrimers layersdepicted in the FESEM images of Fig. 2. However, thevisualization of the nanotubes distribution on a largerscanned area is more straightforward in the AFM image.The PAMAM/SWNTfilm covers the sensor surface entirely,with the nanotubes distributed as individual and small



hys

ica ssp st

atu

s

solid

i a

Figure 3 (online colour at: www.pss-a.com) AFM height image(a) and inverted image (b) in the tapping mode for a 2-bilayerPAMAM/SWNT LbL film and their cross-section plot.

bundles with an average diameter of 3–5 nm,while PAMAMappears in agglomerates as displayed in the white and blackspots in Figs. 3a and b, respectively. The interconnectionbetween the SWNTs network and PAMAM led to a highlyrough film with a root-mean-square (RMS) roughness of ca.5–7 nm, while the valley-to-peak height varied from 5 to28 nm, as shown in the cross-section plot. The filmmorphology tends to be different after 9-bilayers, with adense packing of PAMAMaggregates arising for an increasein number of bilayers [16].


3.2 Morphological characterization of thepenicillinase enzyme atop the LbL film The enzymePEN was adsorbed on the surface of modified EIS-NT andLAPS-NT structures, which depended on the film andsubstrate. Figures 4a and b showAFM images with the whiteand small pink spots representing the enzyme immobilizedon Ta2O5 and a 6-bilayer LbL film, respectively. The topview images and the corresponding 3D images revealed amuch more efficient adsorption of PEN on the LbL film thanon the bare substrate. This may be associated with the larger,rougher surface area created by the LbL film, with theuncompensated positive charges from PAMAM for theadsorption of the negatively charged PEN molecules.

3.3 Modified field-effect devices for penicillindetection The EIS-NT-PEN and LAPS-NT-PEN weretested to detect penicillin G within the concentration rangefrom 25mM to 10mM, with the results being compared withthe corresponding bare substrates. The motivation to detectthis analyte comes from its importance in veterinarytreatment and food control [14, 28]. The detection principleof this biosensor is based on changes in the concentration ofHþ-ions that are released from the hydrolysis of penicillininto penicilloic acid by the enzyme PEN [14, 28]. Figure 5presents ConCap measurements for a capacitive EIS sensor(a) and CC curves using a LAPS platform (b). For bothmodified field-effect devices, a clear improvement in thedetection signal is noted, especially at the lowest (25, 50, and100mM) and highest (2.5, 5, and 10mM) penicillinconcentrations. Biosensors built with PAMAM/SWNTLbL films had a highly stable signal, fast response time,and small drift for all the penicillin concentrations used.Indeed, the sensitivity improved from ca. 83 and 79mV/decade for bare EIS and LAPS, respectively, to ca. 100mV/decade for the sensors with the LbL films, with a linearresponse in the range between 0.25 and 10mM.

These values are higher than in similar works in theliterature [28, 29]. The enhanced performance of EIS-NT-PEN and LAPS-NT-PEN biosensors can be also associatedwith the film morphology. The nanoporous film architectureinduced by the interconnection of SWNTs networks intoPAMAM layers leads to the formation of channels throughwhich Hþ-ions from the enzymatic reaction penetrate.

It is stressed that a systematic investigation led to anoptimized architecture for both EIS and LAPS structureswith 6-bilayer PAMAM/SWNT LbL films. This number ofbilayers was chosen from an analysis of morphology inFESEM and AFM images for LbL films containing differentnumbers of bilayers (not shown). Films with more than6-bilayers had increasingly close packing of the dendrimers,which may hamper the sensor signal. On the order hand,films with less than 6-bilayers had less homogeneousdistribution of adsorbed enzyme. Moreover, the resultspresented here demonstrated that the enhanced properties offield-effect biosensors can be correlated with themorphology of the nanostructure comprising dendrimersand SWNTs.

www.pss-a.com

Phys. Status Solidi A 207, No. 4 (2010) 785

Editor’s

Choice

Figure 4 (online colour at: www.pss-a.com) AFM height imagesof theenzymePEN(whiteandpinkspots)adsorbedonTa2O5surface(a) and on a 6-bilayer PAMAM/SWNT LbL film (b), with theircorresponding 3D images.

Figure 5 (online colour at: www.pss-a.com) ConCap and CCresponse at different penicillin concentrations for a bare EIS andEIS-NT sensor (a), and a bare LAPS and a LAPS-NT sensor (b),respectively. Penicillin concentration ranging from 25mM to10mM.

www.pss-a.com

4 Conclusions We have found that the morphology ofLbL films containing CNTs has an important effect on thesensing performance of field-effect devices with the EIS andLAPS structures. FESEM and AFM images indicated thatthe film had randomly oriented SWNTs highly intercon-nected into the PAMAM layers, giving rise to a porous, largesurface area nanostructure. These features were responsiblefor amore efficient adsorption and distribution of the enzymePEN atop the film surface. As a consequence, enhancedbiosensing properties were observed in the detection ofpenicillin G with improved sensitivity and stability. It isconcluded that an investigation of film morphology andarchitecture is essential to understand and optimize sensingproperties of sensors containing nanohybrid films.

Acknowledgements We gratefully acknowledge thefinancial support from CAPES, FAPESP, and CNPq (Brazil).



hys

ica ssp st

atu

s

solid

i a

References

[1] P. T. Hammond, Adv. Mater. 16, 1271 (2004).[2] Z. Y. Tang, Y. Wang, P. Podsiadlo, and N. A. Kotov, Adv.

Mater. 18, 3203 (2006).[3] J. F. Quinn, A. P. R. Johnston, G. K. Such, A. N. Zelikin, and

F. Caruso, Chem. Soc. Rev. 36, 707 (2007).[4] J. L. Lutkenhaus and P. T. Hammond, Soft Matter 3, 804

(2007).[5] W. Zhao, J. J. Xu, and H. Y. Chen, Electroanalysis 18, 1737

(2006).[6] R. Nohria, R. K. Khillan, Y. Su, R. Dikshit, Y. Lvov, and K.

Varahramyan, Sens. Actuators, B 114, 218 (2006).[7] K. Ariga, J. P. Hill, and Q. M. Ji, Phys. Chem. Chem. Phys. 9,

2319 (2007).[8] L. Grossin, D. Cortial, B. Saulnier, O. Felix, A. Chassepot,

G. Decher, P. Netter, P. Schaaf, P. Gillet, D. Mainard,J. C. Voegel, and N. Benkirane-Jessel, Adv. Mater. 21,650 (2009).

[9] K. Balasubramanian and M. Burghard, Anal. Bioanal. Chem.385, 452 (2006).

[10] B. L. Allen, P. D. Kichambare, and A. Star, Adv. Mater. 19,1439 (2007).

[11] S. N. Kim, J. F. Rusling, and F. Papadimitrakopoulos, Adv.Mater. 19, 3214 (2007).

[12] J. R. Siqueira, Jr., L. H. S. Gasparotto, O. N. Oliveira, Jr., andV. Zucolotto, J. Phys. Chem. C 112, 9050 (2008).

[13] S. W. Lee, B. S. Kim, S. Chen, Y. Shao-Horn, and P. T.Hammond, J. Am. Chem. Soc. 131, 671 (2009).

[14] J. R. Siqueira, Jr., M. H. Abouzar, M. Backer, V. Zucolotto,A. Poghossian, O. N. Oliveira, Jr., and M. J. Schoning, Phys.Status Solidi A 206, 462 (2009).

[15] J. R. Siqueira, Jr., M. H. Abouzar, A. Poghossian, V. Zuco-lotto, O. N. Oliveira, Jr, and M. J. Schoning, Biosens.Bioelectron. 25, 497 (2009).


[16] J. R. Siqueira, Jr., C. F.Werner, M. Backer, A. Poghossian, V.Zucolotto, O. N. Oliveira, Jr., and M. J. Schoning, J. Phys.Chem. C 113, 14765 (2009).

[17] M. J. Schoning and A. Poghossian, Electroanalysis 18, 1893(2006).

[18] M. J. Schoning, Sensors 5, 126 (2005).[19] A. Poghossian, M. Thust, P. Schroth, A. Steffen, H. Luth, and

M. J. Schoning, Sens Mater. 13, 207 (2001).[20] A. Poghossian, H. Luth, J. W. Schultze, and M. J. Schoning,

Electrochim. Acta 47, 243 (2001).[21] Yu. G. Mourzina, T. Yoshinobu, J. Schubert, H. Luth, H.

Iwasaki, and M. J. Schoning, Sens. Actuators, B 80, 136(2001).

[22] A. Poghossian, M. Thust, M. J. Schoning, M. Muller-Veggian, P. Kordos, and H. Luth, Sens. Actuators, B 68,260 (2000).

[23] M. J. Schoning and H. Luth, Phys. Status Solidi A 185, 65(2001).

[24] M. J. Schoning, D. Brinkmann, D. Rolka, C. Demuth, and A.Poghossian, Sens. Actuators, B 111/112, 423 (2005).

[25] J. P. Kloock, L. Moreno, A. Bratov, S. Huachupoma, J. Xu, T.Wagner, T. Yoshinobu, Y. Ermolenko, Y. G. Vlasov, and M.J. Schoning, Sens. Actuators, B 118, 149 (2006).

[26] T. Wagner, T. Yoshinobu, C. Rao, R. Otto, and M. J.Schoning, Sens. Actuators, B 117, 472 (2006).

[27] T. Wagner, C. Rao, J. P. Kloock, T. Yoshinobu, R. Otto, M.Keusgen, and M. J. Schoning, Sens. Actuators, B 118, 33(2006).

[28] M. H. Abouzar, A. Poghossian, A. Razavi, A. Besmehn, N.Bijnens, O. A. Williams, K. Haenen, P. Wagner, and M. J.Schoning, Phys. Status Solidi A 205, 2141 (2008).

[29] M. H. Abouzar, A. Poghossian, A. Razavi, O. A.Williams, N.Bijnens, P. Wagner, and M. J. Schoning, Biosens. Bioelec-tron. 24, 1298 (2009).

www.pss-a.com

Associating biosensing properties with the morphological structure of multilayers containing carbon...

Documents

Transcript of Associating biosensing properties with the morphological structure of multilayers containing carbon...