Anal. Chem. 2008, 80, 6594–6601
Ultrasensitive Detection of Cytokines Enabled by Nanoscale ZnO Arrays Viktor Adalsteinsson,† Omkar Parajuli,† Stephen Kepics,† Abhishek Gupta,† W. Brian Reeves,‡ and Jong-in Hahm*,† Department of Chemical Engineering, The Pennsylvania State University, 160 Fenske Laboratory, University Park, Pennsylvania 16802, and Division of Nephrology, The Pennsylvania State University College of Medicine, Milton S. Hershey Medical Center, Hershey, Pennsylvania 17033 Early detection of disease markers can provide higher diagnostic power and improve disease prognosis. We demonstrate the use of zinc oxide nanorod (ZnO NR) arrays in a straightforward, reliable, and ultrasensitive detection of the cytokines interleukin-18 and tumor necrosis factor-r. Specifically, we exploit the fluorescenceenhancing properties of ZnO NR platforms in cytokine assays involving both a pure buffer and urine. The detection sensitivity achieved using this ZnO NR method is in the subfemtogram per milliliter level, which is 3-4 orders of magnitude more sensitive than conventional assay detection limits. This unparalleled detection sensitivity is achieved without the need for indirect enzyme reactions or specialized instrumentation. We highlight various advantages of using ZnO NR arrays in the ultrasensitive profiling of cytokine levels. Key advantages include robustness of NR arrays, simple and direct assay schemes, high-throughput and multiplexing capabilities, and the ability to correlate directly measured signals to cytokine levels. In conjunction with the extremely high sensitivity demonstrated in this work, our ZnO NR arraybased approach may be highly beneficial in early detection of many cytokine-implicated diseases. Measurement of the concentrations of specific proteins in biological fluids is crucial in many clinical and laboratory settings. Protein biomarkers, for example, are increasingly used to assess risk, to diagnose or monitor the activity of diseases, and to guide therapy or assess therapeutic response.1 Antibody-based assays are convenient and provide good levels of sensitivity and specificity for many proteins. However, conventional antibody-based assays may lack sufficient sensitivity to accurately quantify low-abundance proteins. Chemokines and cytokines, for example, are small proteins that are often produced in the setting of inflammation.2,3 In normal individuals, the levels of certain cytokines in plasma or urine may be below the detection limits of conventional immuno* To whom correspondence should be addressed. E-mail:
[email protected]. † The Pennsylvania State University. ‡ The Pennsylvania State University College of Medicine. (1) Altar, C. A. Clin/ Pharmacol/ Ther/ 2008, 83, 361–364. (2) Choy, E. H. S.; Panayi, G. S. N. Engl. J. Med. 2001, 344, 907–916. (3) Allen, S. J.; Crown, S. E.; Handel, T. M. Annu. Rev. Immunol. 2007, 25, 787–820.
6594
Analytical Chemistry, Vol. 80, No. 17, September 1, 2008
assays.4 Therefore, improved sensitivity of protein assays is an area of active investigation.5 In this report, we demonstrate for the first time the ultrasensitive fluorescence detection of cytokines at extremely low concentrations using a novel zinc oxide nanorod (ZnO NR) platform. ZnO thin films and micro/nanostructures exhibit certain desirable optical properties, including a wide band gap of 3.37 eV and a large exciton binding energy of 60 meV at room temperature, which have led to their use in a broad range of optical and optoelectric applications.6-8 However, applications of ZnO in biological or clinical testing schemes have remained largely unexplored, even though many biological assay systems rely heavily on optical detection techniques. Fluorescence is a widely used bioanalytical technique in genomics, proteomics, drug discovery, disease diagnostics, cell studies, and environmental analysis. Major challenges associated with such fluorescence techniques include enhancing detection sensitivity and increasing signal-to-noise ratio. Novel methods that overcome current drawbacks and enable rapid, facile, high-throughput, ultrasensitive, and specific optical detection are in great demand. We have previously carried out proof-of-concept studies in order to demonstrate that nanometer-scale ZnO can be used successfully in the fluorescence detection of duplex DNA formation and protein interaction.9-11 Although these previous studies employed simplified bioassays that did not involve biological fluids, they demonstrated the potential for ZnO NRs to facilitate the highly sensitive detection of DNA and proteins. In this paper, we assess the lowest detection limits of ZnO NR arrays in more clinically meaningful assays. ZnO NR arrays can be easily assembled as assay platforms upon the material’s synthesis, ready to be employed in bioanalysis without any postsynthetic assembly or purification processes.10,12 The physical and chemical structures of ZnO NRs remain stable and inert in commonly used bioassay (4) Nowlan, M. L.; Drewe, E.; Bulsara, H.; Esposito, N.; Robins, R. A.; Tighe, P. J.; Powell, R. J.; Todd, I. Rheumatology 2006, 45, 31–37. (5) Eisenstein, M. Nature 2006, 444, 959–964. (6) Saito, N.; Haneda, H.; Sekiguchi, T.; Ohashi, N.; Sakaguchi, I.; Koumoto, K. Adv. Mater. 2002, 14, 418–421. (7) Liu, C. H.; Zapien, J. A.; Yao, Y.; Meng, X. M.; Lee, C. S.; Fan, S. S.; Lifshitz, Y.; Lee, S. T. Adv. Mater. 2003, 15, 838–841. (8) Cui, J. B.; Daghlian, C. P.; Gibson, U. J.; Pu ¨ sche, R.; Geithner, P.; Ley, L. J. Appl. Phys. 2005, 97, 044315. (9) Kumar, N.; Dorfman, A.; Hahm, J. Nanotechnology 2006, 17, 2875–2881. (10) Dorfman, A.; Kumar, N.; Hahm, J. Langmuir 2006, 22, 4890–4895. (11) Dorfman, A.; Kumar, N.; Hahm, J. Adv. Mater. 2006, 18, 2685–2690. (12) Kumar, N.; Dorfman, A.; Hahm, J. J. Nanosci. Nanotechnol. 2005, 5, 1915– 1918. 10.1021/ac800747q CCC: $40.75 2008 American Chemical Society Published on Web 08/06/2008
conditions. Detection of a wide range of fluorophores typically utilized in many bioassays can be benefited from ZnO NRs regardless of their spectroscopic characteristics.11 The absorption and emission spectra of our high crystalline and atomic defectfree ZnO NRs do not overlap with those of fluorophores, and this property of ZnO NR arrays makes them suitable as nonautofluorecent platforms. Combined with these advantages, the reduced size inherent to ZnO NRs can allow miniaturized, high-throughput, and multiplexed detection. Acute renal failure (ARF) occurs in 5-7% of hospitalized patients13,14 and results in a mortality rate of ∼50%.15 It is unlikely that this high mortality and associated cost will be reduced until we have better tools for the early diagnosis of renal injury. Early diagnosis of acute kidney injury (AKI) in hospitalized patients has proved problematic due to the inadequacy of currently available laboratory tests. Cytokines are small proteins produced by a variety of cells and play a central role in coordinating the host response to infectious diseases. Dysregulation of cytokine production is implicated in the pathogenesis of such diseases as cancer, atherosclerosis, diabetes, arthritis, and neurodegenerative diseases. In some instances, elevated levels of cytokines in body fluids may serve as markers of either disease severity or diagnosis. There is experimental evidence that a number of cytokines and chemokines, including tumor necrosis factor-R (TNFR), keratinocyte chemoattractant, monocyte chemoattractant protein-1, interferon-γ inducible protein-10, and interleukin-18 (IL-18), participate in the pathogenesis of AKI.16-24 Elevated levels of certain cytokines and chemokines have been reported in blood, kidney tissue, or urine in experimental models of AKI18,22,25-28 and in (13) Hou, S. H.; Bushinsky, D. A.; Wish, J. B.; Cohen, J. J.; Harrington, J. T. American Journal of Medicine 1983, 74, 243–248. (14) Nash, K.; Hafeez, A.; Hou, S. Am. J. Kidney Dis. 2002, 39, 930–936. (15) Thadhani, R.; Pascual, M.; Bonventre, J. V. N. Engl. J. Med. 1996, 334, 1448–1460. (16) Fiorina, P.; Ansari, M. J.; Jurewicz, M.; Barry, M.; Ricchiuti, V.; Smith, R. N.; Shea, S.; Means, T. K.; Auchincloss, H.; Luster, A. D.; Sayegh, M. H.; Abdi, R. J. Am. Soc. Nephrol. 2006, 17, 716–723. (17) Miura, M.; Fu, X.; Zhang, Q. W.; Remick, D. G.; Fairchild, R. L. Am. J. Pathol. 2001, 159, 2137–2145. (18) Furuichi, K.; Wada, T.; Iwata, Y.; Kitagawa, K.; Kobayashi, K. I.; Hashimoto, H.; Ishiwata, Y.; Asano, M.; Wang, H.; Matsushima, K.; Takeya, M.; Kuziel, W. A.; Mukaida, N.; Yokoyama, H. J. Am. Soc. Nephrol. 2003, 14, 2503– 2515. (19) Furuichi, K.; Wada, T.; Iwata, Y.; Kitagawa, K.; Kobayashi, K.; Hashimoto, H.; Ishiwata, Y.; Tomosugi, N.; Mukaida, N.; Matsushima, K.; Egashira, K.; Yokoyama, H. J. Am. Soc. Nephrol. 2003, 14, 1066–1071. (20) Kielar, M. L.; John, R.; Bennett, M.; Richardson, J. A.; Shelton, J. M.; Chen, L. Y.; Jeyarajah, D. R.; Zhou, X. J.; Zhou, H.; Chiquett, B.; Nagami, G. T.; Lu, C. Y. J. Am. Soc. Nephrol. 2005, 16, 3315–3325. (21) Patel, N. S. A.; Chatterjee, P. K.; Di Paola, R.; Mazzon, E.; Britti, D.; De Sarro, A.; Cuzzocrea, S.; Thiemermann, C. J. Pharmacol. Exp. Ther. 2005, 312, 1170–1178. (22) Ramesh, G.; Reeves, W. B. J. Clin. Invest. 2002, 110, 835–842. (23) Donnahoo, K.; Meng, X.; Ayala, A.; Cain, M.; Harken, A.; Meldrum, D Am. J. Physiol. 1999, 277, R922–R929. (24) Melnikov, V. Y.; Ecder, T.; Fantuzzi, G.; Siegmund, B.; Lucia, M. S.; Dinarello, C. A.; Schrier, R. W.; Edelstein, C. L. J. Clin. Invest. 2001, 107, 1145–1152. (25) Molls, R. R.; Savransky, V.; Liu, M. C.; Bevans, S.; Mehta, T.; Tuder, R. M.; King, L. S.; Rabb, H. Am. J. Physiol.-Renal Physiol. 2006, 290, F1187–F1193. (26) Lemay, S.; Rabb, H.; Postler, G.; Singh, A. K. Transplantation 2000, 69, 959–963. (27) Takada, M.; Nadeau, K. C.; Shaw, G. D.; Marquette, K. A.; Tilney, N. L. J. Clin. Invest. 1997, 99, 2682–2690. (28) Hoke, T. S.; Douglas, I. S.; Klein, C. L.; He, Z. B.; Fang, W. F.; Thurman, J. M.; Tao, Y. X.; Dursun, B.; Voelkel, N. F.; Edelstein, C. L.; Faubel, S. J. Am. Soc. Nephrol. 2007, 18, 155–164.
patients with AKI.25,29-32 Focusing on the clinically relevant applications of ZnO nanomaterials, we herein report the ultrasensitive detection of cytokines in urine, using IL-18 and TNFR as model systems. EXPERIMENTAL METHODS Preparation of Stripe- and Square-Array ZnO NR Platforms and Polymeric Substrates. Stripe and square arrays of ZnO NR platforms were fabricated using a gas-phase growth method as described in an earlier report.10 Si wafers (resistivity
