Morphology Effects on the Biofunctionalization of Nanostructured ZnO

Letter pubs.acs.org/Langmuir

Morphology Effects on the Biofunctionalization of Nanostructured ZnO Yan Cao,† Elena Galoppini,*,† Pavel Ivanoff Reyes,‡ Ziqing Duan,‡ and Yicheng Lu*,‡ †

Department of Chemistry and ‡Department of Electrical and Computer Engineering, Rutgers, The State University of New Jersey, Newark, New Jersey 07102, United States S Supporting Information *

ABSTRACT: A stepwise surface functionalization methodology was applied to nanostructured ZnO films grown by metal organic chemical vapor deposition (MOCVD) having three different surface morphologies (i.e., nanorod layers (ZnO films-N), rough surface films (ZnO films-R), and planar surface films (ZnO filmsP). The films were grown on glass substrates and on the sensing area of a quartz crystal microbalance (nano-QCM). 16-(2-Pyridyldithiol)-hexadecanoic acid (PDHA) was bound to ZnO films-N, -R, and -P through the carboxylic acid unit, followed by a nucleophilic displacement of the 2-pyridyldithiol moiety by single-stranded DNA capped with a thiol group (SH-ssDNA). The resulting ssDNA-functionalized films were hybridized with complementary ssDNA tagged with fluorescein (ssDNA-Fl). In a selectivity control experiment, no hybridization occurred upon treatment with non complementary DNA. The ZnO films' surface functionalization, characterized by FT-IR-ATR and fluorescence spectroscopy and detected on the nano-QCM, was successful on films-N and -R but was barely detectable on the planar surface of films-P.

1. INTRODUCTION Zinc oxide (ZnO) is a wide-band-gap semiconductor that has attracted tremendous interest in the past decade with respect to advanced electronics, optical devices, and renewable energy applications.1−3 In particular, nanostructured ZnO is increasingly used in sensors,4−6 biosensors,7−11 and solar cells,12−14 as demonstrated by the exponential growth of publications in this area.15 ZnO is a robust, biocompatible, multifunctional material that can be integrated with microelectronic components16 and is particularly attractive for developing transducers for miniaturized biosensors. A unique advantage of ZnO, when compared to other wide-band-gap semiconductors, is that it forms highly anisotropic nanocrystals, such as nanorods, because of the propensity of ZnO crystals to grow along the c-axis direction of the hexagonal wurtzite structure. Numerous low-temperature growth methods with excellent morphology control are available, and ZnO films can be grown or patterned in microarrays on a variety of substrates, including sapphire, ITO, silicon, gold, and flexible polymeric materials,17 or directly onto sensors. A key step toward the realization of biosensors having nanostructured ZnO as the sensing area is to tune the surface chemistry in order to achieve high sensitivity and selectivity.18 To this end, we recently reported the functionalization of ZnO nanorod films with a reactive monolayer of molecules that binds a biomolecule. As a proof-of-concept experiment, we developed a layer designed to hybridize DNA19 and applied this methodology to nanostructured ZnO quartz crystal microbalances (nano-QCM)20 and thin film bulk acoustic resonators (nano-TFBAR).21 © 2012 American Chemical Society

In this work, we address two open questions about this methodology: selectivity and sensitivity. First, we wanted to determine whether the functionalization method is applicable to other ZnO morphologies and how the sensor properties change depending on the morphology of the ZnO layer. We anticipate that the sensors prepared from nanorod films (i.e., with morphology over a larger surface area) will exhibit higher sensitivity. Second, we wanted to probe whether the functionalization scheme, in this case, the hybridization of immobilized DNA, is selective.

2. EXPERIMENTAL SECTION ZnO Film Growth. Metal organic chemical vapor deposition (MOCVD) growth methods were used for the growth of ZnO films from diethylzinc and oxygen, which were used as the metalorganic Zn source and oxidizer, respectively, on glass with three different morphologies using procedures described previously.22 The morphology was controlled by varying the growth temperature: ZnO films-P were grown at ∼250 °C, films-R were grown at ∼330 °C, and the nanorods required a relatively high growth temperature (>400 °C). In the nano-QCM, the ZnO-covered sensing area was exposed to UV light to make it superhydrophilic.23 Field emission scanning electron microscope (FESEM) images for the three types of nanostructured ZnO films are shown in Figure 1. Films-N consisted of columnar, perpendicularly aligned ZnO nanorods about 0.5 μm long and 40 nm in diameter (Figure 1a). Films-R exhibited a rough surface (Figure 1b), and films-P had a mostly planar surface (Figure 1c). In all cases, the films were about 0.5 μm thick. Received: February 10, 2012 Revised: April 20, 2012 Published: April 27, 2012 7947

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Figure 1. FESEM images of 0.5-μm-thick MOCVD-grown ZnO films on glass with three different morphologies: (a) films-N (nanorods), scale bar 0.6 μm; (b) films-R (rough), scale bar 0.75 μm; and (c) films-P (planar), scale bar 0.5 μm. Functionalization of the ZnO Nanorod Surface. Each step in the functionalization process (experimental details in the Supporting Information) was performed on ZnO films-N, -R, and -P in a sealed Petri dish to avoid evaporation at room temperature. The commonly used method of binding molecules to metal oxide films involves the immersion of the films in solutions. Here we used a method in which the solution with the reagent is deposited as droplets, henceforth known as the droplet method24 (Supporting Information, Figures S1− S3). This method allowed the deposition of the binding solutions onto the ZnO sensor area without full immersion of the entire sensor. The films were stored for days in the dark without any apparent change. ZnO-Modified QCM Sensor. We utilized a ZnO-modified quartz crystal microbalance (QCM) sensor to verify the morphologydependent chemical binding on ZnO. The sensor consists of a standard QCM with a piezoelectric AT-cut quartz layer that is sandwiched between two 100 nm gold electrodes. The quartz substrates have a diameter of 1.37 cm, and the sensing area is 0.2047 cm2. The sensing area (top electrode) of the QCM device consists of ZnO nanostructured arrays grown directly on the top electrode using MOCVD (Figure 2). We prepared three sensors corresponding to the similar ZnO morphologies that are shown in Figure 1.

The functionalization sequence was monitored by FT-IRATR on ZnO films-N, -R, and -P. In step A, the binding of the COOH group resulted in spectral changes in the carbonyl region (Figure S4). The characteristic carbonyl asymmetric stretching band (νas(CO)) of the free acid at 1706 cm−1, which is present in the spectrum of neat PDHA, was replaced by bands assigned to the carboxylate asymmetric stretch, νas(O···C···O), at 1540 and 1400 cm−1. The C−O stretching band at 1250 cm−1 disappeared upon binding, and the C−H stretching bands of the long saturated alkyl chain were visible in the region below 3000 cm−1 in the bound films. Overall, the observed spectral changes were indicative of PDHA binding on ZnO nanorod films through the COOH group and were consistent with our previous observations.19 Steps B and C are more difficult to monitor by FT-IR-ATR because bands that are characteristic of the phosphate groups of DNA (∼1100 cm−1)25 are in part obscured by the bands of the ZnO substrate (Figure S4). The broad bands in the 1100 cm−1 region are intensified after the hybridization step with ssDNAF1 (step C), as shown in the FT-IR-ATR spectra in Figure 3, using the 1540 cm−1 band as an internal reference, and a shoulder appears in the 1600 cm−1 region. Other bands that can be assigned to DNA26 were observed in the 1600 cm−1 region (arrow). Similar spectral changes were observed for all three morphologies, but very weak spectra were observed for the planar morphology films (ZnO films-P), as illustrated in Figures S5 and S6. In summary, the quality of the FT-IR-ATR spectra was satisfactory only for films with higher surface areas and only to monitor the first step, step A. Although the reason for this observation is not clear, the low surface area of the films and possibly the inability to obtain good surface contact may play a role. All three types of ZnO films, after the hybridization step with ssDNA-Fl, were studied by monitoring the fluorescence of the 56-FAM fluorescein (F1) tag (λex = 495 nm), which exhibits an intense band centered at 520 nm (Figure 4a). A comparison between the free ssDNA-Fl in PBS buffer solution and the fluorescence spectrum of the immobilized and hybridized DNA-Fl on ZnO films-N and -R shows that the spectrum broadens upon binding, probably as a result of the distribution of different arrangements of the adsorbed fluorophore and surface heterogeneity. The weak fluorescence in films-P may be indicative of low surface coverage, consistent with the previous results,19 and/or the formation of a disordered layer where the Fl unit is in close proximity to ZnO and the fluorescence is quenched. Selectivity of the Hybridization Process. To determine the selectivity of the method, we probed whether only complementary ssDNA would hybridize with the DNA-

Figure 2. Schematic of a ZnO-nanoQCM device used to verify the morphology-dependent chemical binding; top view and cross section of the multilayer structure.

3. RESULTS AND DISCUSSION Functionalization. The three-step functionalization of ZnO, illustrated in Scheme 1, followed the previously reported methodology.19 The functionalization scheme can be summarized as follows: step A, linker binding; step B, ssDNA immobilization; step C, hybridization with complementary ssDNA (fluorescein-tagged); and step D, treatment with noncomplementary ssDNA′ (fluorescein-tagged). 7948

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Scheme 1. Functionalization Sequence of ZnO Films-N, -R, and -P

Figure 3. FT-IR-ATR spectra of ZnO film-N bound to PDHA (top, black line) following the immobilization of SH-DNA (middle, green line) and following the hybridization with complementary F1-ssDNA (bottom, red line).

functionalized ZnO layer. Non complementary 5′-fluoresceinmodified DNA (5′-/56-FAM/ATGCTTCGATGCAACG-3′, ssDNA-Fl′) was used in step D instead of complementary ssDNA-Fl. Step D with ssDNA-Fl′ was monitored by fluorescence spectroscopy and fluorescence microscopy (Figure 4b). The ZnO N films did not fluoresce after treatment with noncDNA (Figure 4b, red dashed line and inset), indicating that the hybridization step on the functionalized films is selective and a necessary requirement for biosensor applications. Morphology Effects on ZnO-Modified QCM Sensors. The same functionalization procedures were made on three ZnO-modified QCM sensors shown schematically in Figure 2. Three ZnO-modified QCM sensors were prepared with ZnO films-N, -R, and -P on the sensing area. The impedance spectra of the sensors were recorded using an HP8753D network analyzer after every chemical step outlined in Scheme 1. The frequency shifts of the impedance spectra due to mass loading on the ZnO-coated sensing area were recorded after steps A, B, and C for films N, R, and P, and the impedance spectra are reported in Figure 5. Table 1 summarizes the frequency shifts for each chemical step for the ZnO-modified QCM sensors with different sensing

Figure 4. (a) Fluorescence spectra of ssDNA-Fl in 10 mM PBS buffer (black dashed line): ZnO films-N after hybridization with complementary ssDNA-Fl (short red dashed line); ZnO films-R after hybridization with F1-ssDNA (blue dashed−dotted line); ZnO filmsP after hybridization with Fl-ssDNA (purple dotted line) in step C; ZnO films-N after the immobilization of SH-ssDNA (solid green line) (λex= 495 nm). (b) Fluorescence spectra of noncomplementary ssDNA-Fl′ in 10 mM PBS buffer (top, black dotted line): ZnO films-N modified with SH-ssDNA (bottom, green solid line); ZnO films-N after step D, the treatment with ssDNA′-Fl (middle, red dashed line). (Inset) Fluorescence microscopy image of ZnO films-N after the reaction with ssDNA′-Fl, step D.

area morphologies. The data indicate that the sensor with ZnO film-N yields the highest sensitivity as evidenced by the largest frequency shifts due to mass loading (>10 times compared to 7949

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the QCM device because its sensitivity is directly proportional to the frequency shift and is given by Sf = kΔf/fo, where Sf is the sensitivity of the device, k is a constant that is proportional to the effective surface area available for sensing, Δf is the frequency shift, and fo is the operating frequency of the device. The enhanced sensitivity can be attributed to the larger effective surface sensing area provided by the ZnO film-N morphology, compared to that of the other surfaces.

4. CONCLUSIONS Advanced applications of nanostructured ZnO sensors with enhanced sensitivity and selectivity require careful surface engineering. Here we probed the morphology effect of nanostructured ZnO films on a stepwise surface functionalization methodology that allows us to hybridize DNA selectively on the films. Three morphologies of ZnO nanorod film with comparable thickness (∼500 nm) were studied: nanorods (films-N), rough surfaces (films-R), and planar films (films-P). FT-IR-ATR spectra and fluorescence emission studies indicated that the ZnO nanorod films with larger surface areas (i.e., R or N) are needed for the immobilization and detection of biomolecules. Additionally, a control experiment with noncDNA tagged with fluorescein proved that the methodology is highly selective for the detection of biomolecules, in this case, ssDNA. The methodology was applied to ZnO-modified QCM sensors with ZnO nanorods (films-N), rough surfaces (filmsR), and planar films (films-P) on the sensing area. The measurements indicate that the sensor with the ZnO nanorod film provides the largest frequency shifts (>10-fold greater than the planar morphology) because of mass loading. In summary, we have demonstrated that the selectivity of nanostructured ZnO sensors is achieved through surface engineering via chemical modification of the films, and improved sensitivity is achieved through the morphology control of nanostructured ZnO.

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ASSOCIATED CONTENT

S Supporting Information *

Experimental and binding method. Schematic illustration of the droplet method. FT-IR-ATR of ZnO films-N and ZnO films-R after step A functionalized by the droplet method and immersion method. FT-IR-ATR of ZnO films-N after step A bound to a bifunctional linker. FT-IR-ATR of ZnO films-N, ZnO films-R, and ZnO films-P after steps A and C. This material is available free of charge via the Internet at http:// pubs.acs.org.

Figure 5. Impedance spectrum (Z(ω)) after step A (black squares), step B (red circles), step C (blue triangles) on the ZnO-modified QCM with sensing areas of (a) ZnO film-N (top), (b) ZnO film-R, and (c) ZnO film-P. The inset of each plot is the fluorescence microscope image of the sensing area of the QCM device after step C, confirming binding on the ZnO nanostructures (bar = 100 μm).

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Table 1. Frequency Shifts and Mass Detection

ZnO film-P ZnO film-R ZnO film-N

AUTHOR INFORMATION

Corresponding Author

Δf (kHz) step B

Δf (kHz) step C

Δm (ng) step B

Δm (ng) step C

1.523

1.675

20.17

22.19

Notes

7.037

7.431

93.23

98.45

The authors declare no competing financial interest.

13.49

11.06

178.8

146.5

*E-mail: [email protected].

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ACKNOWLEDGMENTS We thank Professor Mendelsohn and Dr. Flach for generous access to FT-IR-ATR instrumentation in the initial stage of this work and the Rutgers Research Council for the partial support of this work.

that on the ZnO film-P surface). This 10-fold increase in the frequency shift directly implies the increase in the sensitivity of 7950

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Nanostructure-Based Quartz Crystal Microbalance Device for Biochemical Sensing. IEEE Sens. J. 2009, 9, 1302−1307. (21) Chen, Y.; Reyes, P. I.; Duan, Z.; Saraf, G.; Wittstruck, R.; Lu, Y.; Taratula, O.; Galoppini, E. Multifunctional ZnO-Based Thin Film Bulk Acoustic Resonator for Biosensors. J. Electron. Mater. 2009, 38, 1605− 1611. (22) Muthukumar, S.; Gorla, R. C.; Emanetoglu, W. N.; Liang, S.; Lu, Y. Control of Morphology and Orientation of ZnO Thin Films Grown on SiO2/Si Substrates. J. Cryst. Growth 2001, 225, 197−201. (23) Zhang, Z.; Chen, H.; Zhong, J.; Saraf, G.; Lu, Y. Fast and Reversible Wettability Transitions on ZnO Nanostructures. TMS IEEE J. Electron. Mater. 2007, 36, 895−899. (24) Oshige, M.; Yamaguchi, K.; Matsuura, S.; Kurita, H.; Mizuno, A.; Katsura, S. A New DNA Combing Method for Biochemical Analysis. Anal. Biochem. 2010, 400, 145−147. (25) Mao, Y.; Daniel, L.; Whittaker, N.; Saffiotti, U. DNA Binding to Crystalline Silica Characterized by Fourier-Transform Infrared Spectroscopy. Environ. Health Perspect. 1994, 102, 165−171. (26) Stepanyugin, A.; Samijlenko, S.; Martynenko, O.; Hovorun, D. ATR-IR Spectroscopy as Applied to Nucleic Acid Films. Spectrochim. Acta, Part A 2005, 61, 2267−2269.

REFERENCES

(1) Morkoç, H.; Ö zgür, Ü . Bandgap Engineering. Zinc Oxide: Fundamentals, Materials and Device Technology; Wiley-VCH: Weinheim, Germany, 2009. (2) Lu, Y.; Zhong, J. Zinc Oxide-Based Nanostructures. In Semiconductor Nanostructures for Optoelectronic Application; Steiner, T., Ed.; Artech House: Boston, 2004; Chapter 6, pp 187−228. (3) Ö zgür, Ü .; Alivov, Ya, I.; Liu, C.; Teke, A.; Reshchikov, M. A.; Doğan, S.; Avrutin, V.; Cho, S.-J.; Morkoç, H. A Comprehensive Review of ZnO Materials and Devices. J. Appl. Phys. 2005, 98, 041301. (4) Mai, L.; Kim, D.-H.; Yim, M.; Yoon, G. A Feasibility Study of ZnO-Based FBAR Devices for an Ultra-Mass-Sensitive Sensor Application. Micro. Opt. Tech. Lett. 2004, 42, 505−507. (5) Zhang, Z.; Chen, H.; Zhong, J.; Chen, Y.; Lu, Y. ZnO NanotipBased QCM Biosensors. Proc. IEEE Int. Freq. Control Symp. 2006, 545−549. (6) Gabl, R.; Green, E.; Schreiter, M.; Feucht, H. D.; Zeininger, H.; Primig, R.; Pitzer, D.; Eckstein, G.; Wersing, W.; Reichl, W.; Runck, J. Novel Integrated FBAR Sensors: A Universal Technology Platform for Bio- and Gas-Detection. Proc. 2003 IEEE Sens. 2003, 2, 1184−1188. (7) Dorfman, A.; Kumar, N.; Hahm, J.-I. Highly Sensitive Biomolecular Fluorescence Detection Using Nanoscale ZnO Platforms. Langmuir 2006, 22, 4890−4895. (8) Kumar, N.; Dorfman, A.; Hahm, J.-I. Ultrasensitive DNA Sequence Detection Using Nanoscale ZnO Sensor Arrays. Nanotechnology 2006, 17, 2875−2881. (9) Zhao, J.; Wu, L.; Zhi, J. Fabrication of Micropatterned ZnO/SiO2 Core/Shell Nanorod Arrays on A Nanocrystalline Diamond Film and Their Application to DNA Hybridization Detection. J. Mater. Chem. 2008, 18, 2459−2465. (10) Israr, M. Q.; Sadaf, J. R.; Nur, O.; Willander, M.; Salman, S.; Danielsson, S. Electric Field Induced Bacterial Flocculation of Enteroaggregative Escherichia coli 042. Appl. Phys. Lett. 2001, 98, 253701. (11) Ali, S. U.; Alvi, H. N.; Ibupoto, Z.; Nur, O.; Willander, M.; Danielsson, B. Selective Potentiometric Determination of Uric Acid with Uricase Immobilized on ZnO Nanowires. Sens. Actuators, B 2011, 152, 241−247. (12) Lira-Cantu, M.; Gonzalez-Valls, I. Vertically-Aligned Nanostructures of ZnO for Excitonic Solar Cells: A Review. Energy Environ. Sci. 2009, 2, 19−34. (13) Ellmer, K., Klein, A., Rech, B.; Eds. Transparent Conductive Zinc Oxide: Basics and Applications in Thin Film Solar Cells; Springer Series in Materials Science; Springer: New York, 2008; Vol. 104. (14) Galoppini, E.; Rochford, J.; Chen, H.; Saraf, G.; Lu, Y.; Hagfeldt, A.; Boschloo, G. Fast Electron Transport in Metal Organic Vapor Deposition Grown Dye-Sensitized ZnO Nanorod Solar Cells. J. Phys. Chem. B 2006, 110, 16159−16161. (15) For instance, in 2007 there were about 400 publications, compared to just a handful in 2002, devoted exclusively to 1D ZnO nanocrystals: Wang, Z. L. A New Nanomaterial Family for Nanotechnology. ACS Nano 2008, 2, 1987−1992 . Splendid onedimensional nanostructures of zinc oxide. (16) Calzolari, A.; Ruini, A.; Catellani, A. Anchor Group versus Conjugation: Toward the Gap-State Engineering of Functionalized Zn(1010) Surface for Optoelectronic Applications. J. Am. Chem. Soc. 2011, 133, 5893−5899. (17) Morin, S. A.; Amos, F. F.; Jin, S. Biomimetic Assembly of Zinc Oxide Nanorods onto Flexible Polymers. J. Am. Chem. Soc. 2007, 129, 13776−13777. (18) Taratula, O.; Galoppini, E.; Wang, D.; Chu, D.; Zhang, Z.; Chen, H.; Saraf, G.; Lu, Y. Binding Studies of Molecular Linkers to ZnO and MgZnO Nanotip Films. J. Phys. Chem. B 2006, 110, 6506− 6515. (19) Taratula, O.; Galoppini, E.; Mendelsohn, R.; Reyes, P. I.; Zhang, Z.; Duan, Z.; Zhong, J.; Lu, Y. Stepwise Functionalization of ZnO Nanotips with DNA. Langmuir 2009, 25, 2107−2113. (20) Reyes, P. I.; Zhang, Z.; Chen, H.; Duan, Z.; Zhong, G.; Saraf, G.; Lu, Y.; Taratula, E.; Galoppini, E.; Boustany, N. N. A ZnO 7951

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