Enzyme-coated single ZnO nanowire FET biosensor for detection of uric acid

Accepted Manuscript Title: Enzyme-coated single ZnO nanowire FET biosensor for detection of uric acid Author: Xi Liu Pei Lin Xiaoqin Yan Zhuo Kang Yanguang Zhao Yang Lei Chuanbao Li Hongwu Du Yue Zhang PII: DOI: Reference:

S0925-4005(12)00857-X doi:10.1016/j.snb.2012.08.043 SNB 14480

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

7-5-2012 12-8-2012 21-8-2012

Please cite this article as: X.L. , Pei Lin, X. Yan, Z. Kang, Y. Zhao, Y. Lei, C. Li, H. Du, Y. Zhang, Enzyme-coated single ZnO nanowire FET biosensor for detection of uric acid, Sensors and Actuators B: Chemical (2010), doi:10.1016/j.snb.2012.08.043 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Enzyme-coated single ZnO nanowire FET biosensor for detection of uric acid Xi Liu1, Pei Lin1, Xiaoqin Yan1*, Zhuo Kang1, Yanguang Zhao1, Yang Lei1, Chuanbao Li2, Hongwu Du2, Yue Zhang1, 3* State Key Laboratory for Advanced Metals and Materials, School of Materials Science and

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Engineering, University of Science and Technology Beijing, Beijing 100083, China

Department of Biotechnology, School of Chemistry and Biology Engineering, University of Science

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and Technology Beijing, Beijing 100083, China

Key Laboratory of New Energy Materials and Technologies, University of Science and Technology

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Beijing, Beijing 100083, China

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* Author to whom correspondence should be addressed; E-Mail: [email protected], [email protected]

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Tel.: +86 10 62334725. Fax: +86 10 62333113

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2 Biographies Xi Liu received the B.S. degree from Harbin Institute Of Technology in 2008 and the M.S. degree in material physics from University Of Science and Technology Beijing in 2011. His work focuses on the

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biosensors based on one-dimensional nanomaterial.

Pei Li received the B.S. degree in material physics from University Of Science and Technology

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Beijing, China, in 2009. Now, he is a Ph.D. candidate in nanomaterial and devices.His current research

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interests include synthesis of sensing materials and fabrication of sensors.

Zhuo Kang received the B.S. degree in material physics from University Of Science and Technology

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Beijing ,China, in 2011. Now ,he is a Ph.D. candidate in nanomaterial and devices. His current research is the fabrication of biosensor and its application in biological field.

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Yanguang Zhao is a Ph.D. candidate in material physics, University Of Science and Technology Beijing. Her current research is the fabrication of biosensor and its application in biological field.

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Yang Lei received his Ph.D. degree from University Of Science and Technology Beijing in 2011.His

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main research interests include the fabrication of electrochemical biosensor based on one-dimensional

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nanomaterial and the characterization.

Chuanbao Li is now a Ph.D. candidate in Department of Biotechnology, School of Chemistry and Biology Engineering, University of Science and Technology Beijing. His current research focuses on biological engineering and sensing technology. Yue Zhang received his Ph.D. in Materials Physics from University of Science & Technology Beijing in 1993. He is now a professor in School Of material science and engineering. As an excellent pioneer in nanoscience, his research focuses on in-situ techniques for nano-scale measurements, self-assembly nanostructures, chemical and biochemical sensors and nanoscale behavior and failure of devices. Xiaoqin Yan received her Ph.D. in material science from Chinese Academy of Sciences. She is now a professor in School Of material science and engineering in University Of Science and Technology Beijing. Her current research focuses on zinc oxide nanowires and nanobelts, in-situ techniques for nano-scale measurements, self-assembly nanostructures, nanodevices and nanodamages. Page 2 of 19

Hongwu Du received his Ph.D. from School Of Medicine, Tsinghua University in 2005. He is now a

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professor in Department of Biotechnology, School of Chemistry and Biotechnology Engineering, University of science and technology Beijing. His current research focuses on biological engineering

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and sensing technology.

Abstract: Qualitative and quantitative detection of biological and chemical species is crucial in many

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areas, ranging from clinical diagnosis to homeland security. Due to the advantages of ultralow detection limit, label-free, fast readout and easy fabrication over the traditional detection systems,

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semiconductor nanowire based electronic devices have emerged as a potential platform. In this paper,

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we fabricated a single ZnO nanowire-based biologically sensitive field-effect transistors (BioFETs) for detection of different concentration uric acid solution at the same time. The addition of uric acid

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with the concentrations from 1pM to 0.5mM resulted in the electrical conductance changes of up to 227 nS, and the response time turns out to be in the order of millisecond. The ZnO NW biosensor could easily detect as low as 1 pM of the uric acid with 14.7 nS of conductance increase, which implied that the detection limit of the biosensor can be as low as 1pM. Keywords: Field-effect transistor, Uricase, Chemical functionalization, Sensors, ZnO nanowire

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1. Introduction

Currently, fabrication of nanoscale biosensors based on one-dimensional nanomaterials such as Si,

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In2O3, ZnO nanotetrapod, Carbon Nanotubes have attracted much attention [1-5]. Nanowire-based

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sensing devices can be configured as high-performance FETs by linking the specific receptor to the surface, and the fundamental principle of the FET biosensor relies on its sensitive response to the

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variation of electric field or potential at the surface resulting from the binding of charged molecules [6]. In comparison to planar and bulk materials, the one-dimensional morphology and nanometer-scale cross-section of nanowires lead to depletion or accumulation of carriers in the ‘bulk’ of the device when a charged species binds to the surface. Based on the specific feature of nanowire, the detection limit of the biosensor may even go down to the ultimate level of a single molecule [7-8]. What’s more, the detection is monitored in terms of significant change in electrical properties which eliminates the need of labor-intensive labeling and complex measurement equipment. Most of the current works are focused on silicon nanowire and carbon nanotube, only a limited number of studies focusing on the detection of bio species by using oxide semiconductor. However, the unstable silicon nanowire surfaces can be easily oxidized and form an insulating layer which may degrade the device reliability

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and sensitivity [9], while the chirality of carbon nanotube remains an unsolved problem. Thus, it’s

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worthwhile to investigate metal oxide material for biosensing application. Among the oxide semiconductor, ZnO nanowires are believed to be one of the best candidates as the future integrated biosensors because of its excellent electrical properties and biocompatibility [10-11].

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Through proper thermal annealing and surface passivation, the ZnO nanorod based FET could exhibit high electron mobility above 1000 cm2/Vs [12]. Meanwhile, the ZnO nanowires have active surfaces

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that can be easily modified for the immobilization of numerous biomolecules [13]. Above all, the ZnO

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nanowire based FET biosensor can be integrated with Si-based signal processing and communication circuits. These significant advantages over other non-oxide semiconductors make ZnO a promising

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material for nano-scaled biosensors [14].

Uric acid is created when the body breaks down purine nucleotides. High blood concentrations of

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uric acid can lead to a type of arthritis known as gout. Also, the monitoring of urea concentration in blood is a way to evaluate kidney disease. When uric acid reaches the enzyme-functionalized surface,

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the uricase catalyzes the following reaction

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Uric Acid +O2 H2O2

Uricase Allantoin+H O +CO 2 2 2 O2+2H++2e-

(1)

(2)

Here, we report the preparation of single ZnO nanowire based FET biosensor to detect uric acid concentration in vitro. Firstly, uniform and single crystal nanowires were obtained through Chemical Vapor Deposition (CVD) method. Secondly, using the covalent modification method, uricase was linked to the surface closely and retained its full pristine activity. Thirdly, current-voltage (Ids-Vds) was used to monitor the change in the conductance during the successive addition of different concentrations of uric acid. The results demonstrated applications of ZnO nanowire devices for labelfree, ultrasensitive and real-time detection of a wide dynamics range of biological and chemical species.

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6 2. Materials and methods 2.1 Synthesis and characteristics of ZnO nanowires Ultralong ZnO nanowires were synthesized in CVD furnace without any catalysts as reported

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previously [15-16].The mixture of ZnO and carbon power with the molar ratio of 1:1 were initially milled to a fine grade, and suitable amount of powder was placed in an Al2O3 boat inside a quartz tube

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as the evaporation source. A silicon substrate without any catalysts was then fixed on the top of the source boat to collect the as-synthesized nanowires. Argon was used here as the carrier gas, and O2

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was the reaction gas. Then the whole system was maintained at the temperature of 980! C with the flow

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rate of Ar/O2 at 297/3 SCCM (standard-state cubic centimeter per minute) for 20 minutes. After the reaction, the furnace was cooled in Argon for about 1hr and then the substrate with a white flocky

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product was taken out for further use. 2.2 Fabrication of bioFET by Single ZnO NW

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ZnO NW-based FET biosensors were fabricated for the real time detection of biomolecular

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interactions. The widely used fabrication methods reported by other groups for NW FET fabrication was used here [17]. The as-grown high quality ZnO NWs were dispersed from ethanol suspension onto

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the silicon substrate with a thin oxide layer [18]. Ti/Au metal layers were defined using electron-beam lithography, and the single ultralong ZnO NW was immobilized by silver paste carefully. After that, the electrodes were passivated with polymethyl methacrylate (PMMA) to reduce the leak current and eliminate the effect of metal-nanowire contact region, which make sure the entire conductance changes originate from the nanowire [19].

2.3 Surface modification of ZnO nanowire In order to enhance the immobilization efficiency, the widely used cross-linking method was used here rather than the physical absorption via the following steps [20].The ZnO NWs were treated with an oxygen plasma (0.3Torr, 25W power for 60 s) to remove contaminants and add hydroxyl groups to the surfaces [21], and were then immersed in a 2% ethanol solution of 3-aminopropyltriethoxysilane (APTES). After the reaction, the amino-silanized surface was rinsed with ethanol thrice and then was Page 6 of 19

7 baked at 120 ! C for 10 min under N2 gas condition. After wire bonding, 5µL uricase (5 units/mL) was deposited on the surface of ZnO nanowire, and the bioFET device was placed in saturated glutaraldehyde vapor for 40 minutes. The device was then rinsed with 0.01×PBS and deionized water before air-dried for 15 minutes.

After modification, the device was covered with the solution

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2.4 Measurement of real-time conductance change of ZnO NW bioFET

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exchanging chamber, and kept in 0.01 × PBS at 4! C before further calibration and measurements.

The conductance change of the ZnO NW bioFET functionalized with uricase was measured with

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semiconductor parameter analyzer (Keithley 4200) for exposure to uric acid with the concentration tenfold increased every time from 1pM to 0.5mM in a 0.01×PBS, the Vg (gate voltage) and Vsd

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(source-drain voltage) were fixed at 0V and -1V, respectively. All the electrical measurements were

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performed at room temperature in an ambient air environment.

3. Results and Discussion

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The NWs were grown in a disorderly manner, but their morphologies were uniform. Fig.1 (a) shows

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the typical Field Emission Scanning Electron Microscope (FE-SEM, LEO1530, Japan) image of assynthesized ZnO nanowires grown on silicon substrate. The average diameter is about 500nm, and the

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length could be longer than 1mm. The crystal structure and phase purity of the bulk nanowire samples was assessed using X-ray diffraction (XRD, D/MAX-RB) and shown in Fig.1 (b). All the relatively sharp diffraction peaks are in good agreement with the standard ZnO wurtzite structure (a = 0.3250nm and c = 0.5206nm). The strongest peak (002) means that the growth of zinc oxide has a very clear Caxis orientation. Fig. 1(c) shows the room temperature photoluminescence spectrum of the NWs measured by using a continuous He-Cd laser (325 nm) as an excitation source. The extremely strong UV emissions at about 380 nm and negligible deep-level emissions at about 520 nm imply that the assynthesized NWs have defect-suppressed crystal structures [22-23]. Using the as-synthesized high quality NWs, the ZnO NW FETs were fabricated using standard procedures with back gate geometry [24].The Figure 2(a) illustrates the schematic of ZnO nanowire biosensor system and (b) shows the optical image of it. We have then measured the device Page 7 of 19

characteristics as shown in Figure 2(c), the drain current (Ids) versus drain voltage (Vds) characteristics

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of ZnO NWs-based transistors obtained as a function of different gate voltages (Vg) indicate that the pronounced gate effect is indicative of an n-type semiconductor. The on/off ratio and transconductance were ~4.6×106 and ~8.2nS, respectively.

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Prior to the electrical measurement of the ZnO NW bioFET, the modification of the ZnO NW surface with uricase was investigated. Under the high magnification optical microscope (×2000), the

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Fig.3 (b) shows the surface of the ZnO NW becomes lumpy and has a transparent layer on it. Since the

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fluorescent images can provide visual evidence of efficient surface modification, we applied the microscopic fluorescence imaging technique to further demonstrate the successful functionalization

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[25]. The left part of Fig.3 (c) shows the red fluorescence after the modification of Cy5 labeled uricase (Cy5-uricase) on the ZnO surface via the procedures shown in Fig.3 (a). The right part shows the

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association of fluorescein isothiocyanate-labeled antibody (FITC-antiuricase) against the uricase, the green fluorescence indicate that the modified uricase keeps its enzymatic activity well without

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of ZnO NW surface.

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denaturing. The result indicates that the functionalization methods were available for the modification

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The fabricated NW FET was loaded into the home-made liquid exchanging chamber with the sensor electrically wired to the semiconductor parameter analyzer. After the baseline signal was established in pure 0.01×PBS buffer, aliquots of a 10 mg/mL solution of BSA were added for the two main reasons: First, BSA can efficiently block the unmodified site to reduce the nonspecific binding interactions which may lead to false positive results and hence decrease the signal to noise ratio; Second, the increase of the protein concentration in the buffer could help to keep the enzymatic activity of uricase. After BSA addition, the baseline re-equilibrated to a lower value. When the conductance baseline was stable in the protein-rich medium, the uric acid was added to the solution from 1pM to 0.5 mM with the concentration tenfold increased every time in 0.01×PBS, as Fig. 4(a) below. From the Enzyme catalysis equation (1) and (2), one uric acid molecule yields two hydrogen ions, and it changes the local hydrogen ion activity of the solution. Changes in the local hydrogen ion concentration alter the surface potential and, hence, the electric field which modulates the conductance of the BioFET. [26] Page 8 of 19

The conductance of the device rapidly increased upon exposing the nanowire sensor to uric acid.

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Under such measurement conditions, the response time turned out to be in the order of millisecond which can be considered relatively short compared to other diagnostic technologies [27]. The comparison of some uric acid sensors based on different ZnO structures was shown in Table 1.

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The calibrated relationship between the stable current and the concentration of uric acid was plotted in Fig.4 (b). From the plot, we can come to the conclusion that the sensor as constructed was more

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sensitive at low uric acid concentrations than at high concentrations. Furthermore, lactate and glucose

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were selected to affirm the selectivity of BioFET in our work. Upon adding 300μM lactate and 300mM glucose, the signal changed only slightly as is shown by the output response in Fig.4 (c). That is, the

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uricase functionalized ZnO BioFET only led to significant increase in current in the presence of uric acid, because the enzyme catalysis reaction is very specific due to the nature of the uricase All electrical measurements were performed at room temperature in an ambient air

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functionality.

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4. Conclusions

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ZnO NWs were directly synthesized through the CVD method. The characterization results of XRD

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and PL spectrum implied that the NWs had a well-crystallized structural quality. Using the assynthesized high quality nanowire, electrical biosensors based on FET were fabricated for the highly sensitive detection of uric acid at the low and high concentrations simultaneously. It is shown that the ZnO NW bioFET sensors could easily detect uric acid down to a concentration of 1 pM with 14.7 nS of conductance increase, and the response time turns out to be in the order of millisecond. In conclusion, a ZnO NW with an actual n-type property was easily fabricated as a nanobiosensor without any doping, and showed feasibility and a lower detection limit compared to other NW-based biosensors. This cost effective process can be further exploited by expanding into arrays so that the technology can lead to portable, reliable and real-time detecting with applications in many areas.

Acknowledgments

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This work was supported by the Major Project of International Cooperation and Exchanges

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(2012DFA50990), NSFC (51172022, 50972011), the Research Fund of Co-construction Program from Beijing Municipal Commission of Education, the Fundamental Research Funds for the Central Universities, the Program for Changjiang Scholars and Innovative Research Team in University, the

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Beijing novel program (2008B19) and the Program for New Century Excellent Talents (NCET-09-

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0219).

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Figure Captions

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Fig.1 (a) Top-view SEM image of ZnO nanowires grown on silicon substrate; (b) XRD pattern of ZnO

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nanowires and (c) Photoluminescence spectrum of ZnO nanowires.

Fig.2 (a) Schematic of single ultralong ZnO nanowire biosensor system and (b) Optical image of

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biosensor (c) Ids-Vds measurements under varying Vg,Vds=-1V.

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Fig.3 (a) Immobilization of uricase onto the ZnO NW surface via crosslinking surface modification method (1)2% 3-APTES in ethanol; (2) 25wt% GAD; (3) 5µl uricase (5units/ml) (b) High optical

image;

(c)

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magnification

Left:

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microscopic

fluorescence

image

of

Cy5-

uricase/GAD/APTES/ZnO; Right: After association of fluorescein isothiocyanate-labeled antibody against the uricase (FITC-antiuricase).

Fig.4 (a) Conductance of the device versus time following the addition of uric acid in the buffer solution. The upper inset picture is the home-made reaction cell for sensing, and the optical image of silver paste immobilized ultralong ZnO nanowire; the lower inset is the configuration of our device during active sensing measurements. (b) Plot of stable current vs. different concentrations of uric acid. (c) Effect of potentially interfering substances on sensor response upon adding 300μM lactate and 300mM glucose solution

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15 Table 1. Comparison of some uric acid sensors based on different ZnO structures. Range 1 1 µM-1 mM

Reference

Transducer Potentiometric

Matrix Response time ZnO NWs 6-9 s

Potentiometric

ZnO NTs

Potentiometric

ZnO nanoflakes ~8 s

500 nM-1.5 mM

Amperometric

ZnO NW

1 pM-0.5 mM

8s

[28]

0.5 µM-1.5 mM

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[30]

[present]

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~millisecond

[29]

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