pubs.acs.org/Langmuir © 2009 American Chemical Society
Enhanced Protein Immobilization Efficiency on a TiO2 Surface Modified with a Hydroxyl Functional Group Wan-Joong Kim,*,† Sanghee Kim,† Bong Soo Lee,‡ Ansoon Kim,† Chil Seong Ah,† Chul Huh,† Gun Yong Sung,† and Wan Soo Yun*,§ † Biosensor Research Team, Electronics and Telecommunications Research Institute, Daejeon 305-700, South Korea, ‡Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, South Korea, and §Division of Advanced Technology, Korea Research Institute of Standards and Science, Daejeon 305-340, South Korea
Received May 8, 2009. Revised Manuscript Received June 17, 2009 An antibody immobilization was investigated using a self-assembled monolayer (SAM) over the highly refractive coatings with a SiO2, TiO2, or Si3N4 substrate. The immobilization was characterized by analyzing the hydrophilic properties of hydroxyl (OH) groups on surface coatings with contact angle (CA) measurements to enhance protein immobilization. The hydroxyl (OH) group was formed in greater amounts as the oxygen plasma exposure time was increased, which resulted in a large enhancement in antibody immobilization. It indicated that hydroxyl (OH) group formation is critical for developing a label-free optical transducer with a high sensitivity.
1. Introduction Titanium dioxide (TiO2) has been intensively studied for various technological applications in field of heterogeneous catalyst support,1,2 sensory devices,3,4 ductile ceramics,5 H2 storage material,6 and photovoltaic devices.7 In particular, TiO2 has been utilized for photocatalytic application of self-cleaning, deodorizing, self-sterilizing, and antifogging capabilities.8-11 Various physical and chemical surface modification methods which generate specific functional groups on metal oxide substrates were reported.12-15 Generally, the formation of the hydroxyl (OH) functional group on the surface of a metal oxide (TiO2 or SiO2) which allows the binding of biomolecules with a hydroxyl functional group has been performed by either a chemical or a physical method for their future application as a biosensor.16 The chemical method is achieved by hydroxylating the substrate with a strong acid such as 1 N HNO3 and piranha solution [a 7:3 (v/v) 98% H2SO4/30% H2O2 mixture] or by exposing the metal oxide substrate under O2 plasma for the desired period of time. *To whom correspondence should be addressed. (W.-J.K.) E-mail: kokwj@ etri.re.kr. Telephone: þ82-42-860-3979. Fax: þ82-42-860-5404. (W.S.Y.) E-mail:
[email protected]. Telephone: þ82-42-868-5952. Fax: þ82-42-868-5953.
(1) Haruta, M.; Yamada, N.; Kobayashi, T.; Iijima, S. J. Catal. 1989, 115, 301. (2) Valden, M.; Lai, X.; Goodman, D. W. Science 1998, 281, 1647. (3) Wang, R.; Hashimoto, K.; Fijishima, A. Nature 1997, 388, 431. (4) Travera, E.; Gnappi, G.; Monternero, A.; Gusmano, G. Sens. Actuators, B 1996, 31(1-2), 59. (5) Karch, J.; Barringer, R.; Gleiter, H. Nature 1987, 330, 556. (6) Lim, S. H.; Luo, J.; Zhong, Z. Y.; Ji, W.; Lin, J. Inorg. Chem. 2005, 44(12), 4124. (7) Linsebigler, A. L.; Lu, G.; Yates, J. T. Chem. Rev. 1995, 95, 735. (8) Fujishima, A.; Honda, K. Nature 1972, 238, 37. (9) Fujishima, A.; Rao, T. N.; Tryk, D. A. J. Photochem. Photobiol., C 2000, 1, 1. (10) Fujishima, A.; Hashimoto, K.; Watanabe, T. TiO2 Photocatalysis: Fundamentals and Applications; BKC: Tokyo, 1999; p 124. (11) Hoffmann, M. R.; Martin, S. T.; Choi, W.; Bahnemann, D. W. Chem. Rev. 1995, 95, 69. (12) Helmy, R.; Fadeev, Y. Langmuir 2002, 18, 8924. (13) Wang, M.; Liechti, K. M.; Wang, Q.; White, J. M. Langmuir 2005, 21, 1848. (14) Marcinko, S.; Helmy, R.; Fadeev, A. Y. Langmuir 2003, 19, 2752. (15) Hu, M.; Noda, S.; Okubo, T.; Yamaguchi, Y.; Komiyama, H. Appl. Surf. Sci. 2001, 181, 307. (16) Sakataa, T.; Kamahorib, M.; Miyaharaa, Y. Mater. Sci. Eng., C 2004, 24, 827.
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Scheme 1. Possible Mechanisms of (a) the Generation of Hydroxyl Groups by Oxygen Plasma and (b) the Decrease in the Level of Hydroxyl Groups in Contact with Regular Atmospheric Conditions
Hydroxylation of the TiO2 substrate by O2 plasma exposure is similar to that of water photooxidation with UV irradiation proposed by Nosaka et al.,17 as seen in Scheme 1. The hydroxylated TiO2 substrate is constructed in forms of Ti-OH via oxidation by O2 plasma and nucleophilic attack of a H2O molecule present in the atmosphere. In addition, the physical method which allows the adsorption of biomolecules on the surface of the metal oxide uses the physisorption technique.18 In the past several years, however, few studies of the surface modification with TiO2 were reported, although it is still a research area that is being explored since it has potential in biological applications, including biosensors. For the biosensor application, an antibody that is able to bind with a target molecule of antigen would be immobilized on various metal oxide surfaces via the linker of an amine (NH2) functional group. Recently, Nanci et al.19 and Yanagida et al.18 have reported the biological application of the TiO2 substrate as a biosensor following the immobilization of bioactive organic molecules on the surface of TiO2 via chemical and physical methods, respectively. The purpose of surface modification with TiO2 is to introduce (17) Murakami, Y.; Kenji, E.; Nosaka, A. Y.; Nosaka, Y. J. Phys. Chem. B 2006, 110, 16808. (18) Senadeera, G. K. R.; Kitamura, T.; Yanagida, S. J. Photochem. Photobiol., A 2004, 164, 61. (19) Nanci, A.; Wuest, J. D.; Peru, L.; Brunet, P.; Sharma, V.; Zalzal, S.; McKee, M. D. J. Biomed. Mater. Res., Part A 1998, 40, 324.
Published on Web 07/28/2009
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Figure 1. Contact angle measurements as a function of aging time in the laboratory atmosphere after treatment of oxygen plasma for (a) 1 and (b) 5 min. The CA of the SiO2 (O), TiO2 (0), and Si3N4 (4) surfaces was increased with an increase in aging time. Scheme 2. Representation of the Sandwich Immunoassay Using Conjugated Au NPs
terminal functional groups of amine (NH2) by which specific biomolecules such as proteins,20-24 enzymes,25 DNA,26-28 and RNA29 recognizing the target analyte are covalently attached to the TiO2 substrate. To enhance the amine (NH2) functionalization with TiO2, the self-assembly technique30 which develops the molecular assemblies of chemical linkers in symmetric order was applied. To generate a high-efficiency biosensor, the orientation of the immobilized antibody is an important issue since the antigen binding site of the antibody is required to be exposed to antigens in solution.31 As mentioned above, the purpose of surface modification is to form hydroxyl functional groups on the surface of TiO2 which is (20) Rios, F.; Smirnov, S. N. Appl. Mater. Interfaces 2009, 1, 768. (21) Yang, H.; Zhu, X.; Song, W.; Sun, Y.; Duan, G.; Zhao, X.; Zhang, Z. J. Phys. Chem. C 2008, 112, 15022. (22) Hong, S.; Lee, D.; Zhang, H.; Zhang, J. Q.; Resvick, J. N.; Khademhosseini, A.; King, M. R.; Langer, R.; Karp, J. J. Langmuir 2007, 23, 12261. (23) Haginaka, J.; Okazaki, Y.; Matsunaga, H. J. Chromatogr., A 1999, 840, 171. (24) Deng, L.; Mrksich, M.; Whitesides, G. M. J. Am. Chem. Soc. 1996, 118, 5136. (25) Su, J.; Bringer, M. R.; Ismagilov, R. F.; Mrksich, M. J. Am. Chem. Soc. 2005, 127, 7280. (26) O’Brien, J. C.; Stickney, J. T.; Porter, M. D. J. Am. Chem. Soc. 2000, 122, 5004. (27) Sakata, T.; Kamahori, M.; Miyahara, Y. Mater. Sci. Eng., C 2004, 24, 827. (28) Li, Z.; Chen, Y.; Li, X.; Kamins, T. I.; Nauka, K.; Williams, R. S. Nano Lett. 2004, 4, 245. (29) Rozkiewicz, D. I.; Brugman, W.; Kerkhoven, R. M.; Ravoo, B. J.; Reinhoudt, D. N. J. Am. Chem. Soc. 2007, 129, 11593. (30) Wirth, M. J.; Fairbank, R. W. P.; Fatunmbi, H. O. Science 1997, 275, 44. (31) Weiping, W.; Bin, X.; Lei, W.; Chunxiao, W.; Zengding, S.; Danfeng, Y.; Zuhong, L.; Yu, W. J. Inclusion Phenom. Macrocyclic Chem. 1999, 35, 419–429.
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required for further introduction of SAMs and a linker that directly bind with the antibody. In this study, we employed an O2 plasma treatment method with a TiO2 substrate for the formation of hydroxyl group on the surface since a mild surface treatment was required for immobilization of the anti-PSA (prostate-specific antigen) antibody on the hydroxylated TiO2 substrate as a biosensor. The anti-PSA antibody was immobilized to the hydroxylated TiO2 substrate by covalent bonding via a terminal amine (NH2) functional group which linked with the assembled SAM layer formed by treatment with APTES and glutaraldehyde. The immobilized antiPSA antibody on the TiO2 substrate was used to measure the altered number of immunogold particles via a sandwich-type ELISA (enzyme linked immunosorbent assay) as seen in Scheme 2. In addition, changes in the efficiency of anti-PSA antibody immobilization on the hydroxylated TiO2 substrate are determined by the altered hydrophilic property of the TiO2 substrate measured by changes in the wettability of the TiO2 substrate. Our results showed the augmented anti-PSA antibody immobilization efficiency with an increased hydrophilic property of the TiO2 substrate determined by contact angle measurement that showed enhanced wettability. Sandwich-type ELISA results in an increased number of attached immunogold nanoparticles as a result of hydroxylation of the TiO2 substrate. In addition, our results clearly demonstrated that the enhanced hydrophilic property of the TiO2 substrate is dependent on O2 plasma exposure time. DOI: 10.1021/la901615e
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Figure 2. (a) Photographs of a water droplet and (b) comparison of contact angles measured on the SiO2 (O), TiO2 (0), and Si3N4 (4) surfaces as a function of the treatment time of oxygen plasma. The inset shows the contact angles in a 15 s treatment of oxygen plasma. Table 1. Contact Angles Measured on SiO2, TiO2, and Si3N4 Surfaces as a Function of O2 Plasma Exposure Time
contact angle of SiO2 (deg) contact angle of TiO2 (deg) contact angle of Si3N4 (deg)
0s
1.5 s
3s
4.5 s
6s
10 s
15 s
20 s
30 s
45 s
60 s
120 s
135 s
150 s
49 62 72
12 46 27
11 40 26
9 39 22
0 26 17
0 24 11
0 23 0
0 22 0
0 21 0
0 17 0
0 15 0
0 10 0
0 7 0
0 0 0
2. Experimental Section Material and Instrumentation. 3-Aminopropyltriethoxylsilane (APTES), H2SO4 (99.9%), H2O2 [30% (w/v) solution], 25 wt % glutaraldehyde (CHO-CH2CH2CH2-CHO), sodium cyanoborohydride (NaBH4CN), potassium carbonate (K2CO3), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich. The prostate-specific antigen (PSA)-free monoclonal antibody of PSA (host, rabbit, mab-PSA, affinity constant of ∼2 1010 L/M) and the polyclonal antibody of PSA (host, rabbit, pabPSA) were purchased from Fitzgerald. Au nanoparticles (NPs) were synthesized according to the literature procedure.32 Deionized water was distilled with a Milli-Q water purification system. Oxygen plasma was performed using a SPI Plasma-Prep II Plasma Etcher. Contact angle measurement was performed using a Phoenix 300 Goniometer (Surface Electro Optics Co., Ltd.). FE-SEM images were collected on a FEI sirion-400 field emission scanning electron microscope. Surface analysis by XPS was performed using ESCALAB 200R (VG scientific). Measurement of the Hydrophilic Property. The wettability of SiO2, TiO2, and Si3N4 surfaces via contact angle (CA) was characterized after the oxygen plasma (30 Pa, oxygen at 100 mL/ min, and 100 W) was applied to the surface of the samples. The substrate was cleaned by sonication in methanol for 15 min, a water rinse, and a nitrogen blow before it was exposed to oxygen plasma radiation. The CA measurements for the wettability of SiO2, TiO2, and Si3N4 surfaces were performed as a function of the exposure time of oxygen plasma radiation. The typical exposure times were 1.5, 3, 4.5, 6, 10, 15, 20, 30, 45, 60, 90, 120, 135, and 150 s. The water CA was also conducted within ∼1-5 min of oxygen plasma treatment to verify the hydrophilic properties of the substrates, which were exposed to laboratory atmospheric conditions of 22 °C and 32% humidity. Silanization. As shown in Scheme 2, the reactivity steps for the surface of various substrates were performed with the hydrophilic property via an immune reaction, which was mediated by a Au NP-conjugated polyclonal antibody. The hydroxyl groups (OH) on the surface are required, as a binding linker, for immobilization of anti-PSA on SiO2, TiO2, and Si3N4 surfaces. Each substrate was treated with oxygen plasma for 60 or 300 s to add hydroxyl groups to the surface. The hydroxylated surface was silanized in ethanol (0.5% deionized water) containing 2 wt % APTES. The target monolayers of APTES were thus formed on surface of the (32) Grabar, K. C.; Freeman, R. G.; Hommer, M. B.; Natan, M. J. Anal. Chem. 1995, 67, 735.
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hydroxylated wafers. The amino-silanized surface was rinsed with ethanol and was baked at 120 °C for 15 min. Reactive amino groups were then introduced onto the surface. Immobilization of the Antibody. Anti-PSA was immobilized on the modified surfaces using glutaraldehyde as a bifunctional cross-linking agent. The amino-silanized surface wafers were dipped into 10 mL of the 25 wt % glutaraldehyde solution with 0.1% sodium cyanoborohydride for 4 h at room temperature. Then, the wafers were rinsed with deionized water and dried with a nitrogen blow. The amino functional surface wafers were converted into a reactive surface with aldehyde (CHO) groups, which could be coupled with the anti-PSA. For coupling the antibody with aldehyde (CHO) groups, the surface wafers with CHO groups were incubated in a buffer solution [10 mM PBS buffer (pH 7.4)] of anti-PSA (0.1 mg/mL) with 0.1% sodium cyanoborohydride at 4 °C for 30 min. Then, the surface wafers with anti-PSA were soaked in PBS buffer with 3% BSA at 4 °C for 20 min to block any residual CHO groups and were rinsed with a PBS solution and deionized water before the wafers were dried with nitrogen. Conjugation of Au NPs with a Secondary Antibody. To modify gold NPs with a polyclonal antibody, a solution of 1 mL of Au NPs (0.3 nM) was mixed with 5 μL of 0.1 M potassium carbonate (K2CO3), and the mixture was shaken for 1 min. In sequence, the solution was shaken for 5 min with addition of 100 μL of polyclonal PSA (0.1 mg/mL) and was quivered for 5 min with injection of 100 μL of 10% BSA. After the solution had been centrifuged at 12000 rpm, the polyclonal PSA probe was diluted to 1 mL of 50 mM PBS (pH 8.0, 0.1% NaN3). Immunoassay with Conjugated Au NPs. The immobilized surface wafers with anti-PSA were immersed in the antigen solution for 20 min at room temperature. After being washed with a PBS solution, each sample was immersed in the Au NPconjugated polyclonal antibody for 1 h and washed with the PBS solution and deionized water before it was dried with nitrogen. Finally, the SiO2, TiO2, and Si3N4 substrates were characterized for their efficiency of antibody immobilization by a FE-SEM.
3. Results and Discussion In this study, our results elucidate the significant property of the TiO2 substrate as a biosensor following the induction of hydrophilic surface modification which facilitates the immobilization of the target antibody under O2 plasma exposure. We employed an O2 plasma exposure method to generate and enhance the hydrophilic surface property of the TiO2 substrate exhibited as an altered Langmuir 2009, 25(19), 11692–11697
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Figure 3. XPS O 1s core-level spectra for the TiO2 sample, showing the Ti-O and OH components. (a) Atomic percentages of Ti-O and OH peaks are 70.9 and 29.1% after no O2 plasma exposure, (b) 69.4 and 30.6% after O2 plasma exposure treatment for 60 s, and (c) 64.2 and 35.8% after O2 plasma exposure treatment for 300 s, respectively.
Figure 4. FE-SEM images of anti-PSA immobilization efficiency examined by a sandwich-type pab-PSA Au NP conjugate immunoassay: (a-c) treated with oxygen plasma for 1 min and (d-f) treated with oxygen plasma for 5 min. For the immunogold assembly condition, the surfaces immobilized with anti-PSA were immersed in a solution of 0.1 μg/mL PSA antigen (1 PBS and pH7.4) for 20 min and immersed in the Au NP-conjugated polyclonal antibody (0.4 nM) for 60 min.
degree of hydroxylation as seen in Scheme 1a. The ultimate goal of surface modification is to use the hydrophilic TiO2 substrate as a biosensor followed by immobilization of anti-PSA antibody via covalent bonding to a terminal amine (NH2) functional group as seen in Scheme 2. Langmuir 2009, 25(19), 11692–11697
The wettability which was evaluated by water contact angle (CA) measurements clearly showed different hydrophilic surface properties of the metal oxides. Altered hydrophilic properties of each metal oxide surface were determined under the laboratory atmosphere conditions (22 °C and 32% humidity) to determine DOI: 10.1021/la901615e
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Figure 5. Anti-PSA immobilization efficiency examined by a sandwich-type pab-PSA Au NP conjugate immunoassay as the oxygen plasma exposure time. (a) FE-SEM images of the attached Au NPs on TiO2 surfaces as a function of oxygen plasma exposure time. (b) Quantitative relationship between the immobilization efficiency and the oxygen plasma exposure time. Immunogold assembly after treatment of oxygen plasma for 0, 60, 90, 100, 120, 150, 220, and 300 s was accomplished. For the immunogold assembly conditions, the surfaces immobilized with anti-PSA were immersed in a solution of 0.1 μg/mL PSA antigen (1 PBS and pH 7.4) for 20 min and immersed in the Au NP-conjugated polyclonal antibody (0.4 nM) for 60 min.
the presence and level of the OH group by measuring the water CA. Figure 1 shows altered water CAs of SiO2, TiO2, and Si3N4 surfaces following O2 plasma exposure for 60 and 300 s. The measured water CAs of TiO2 have shown the immediate increase from 0° to 16° by 3 h and reached 60° by 480 h, while those of SiO2 and Si3N4 surfaces were maintained at ∼0° for ∼72 h and ∼15° for 480 h, respectively. The water CA value reached to 62° after atmosphere exposure for 480 h under 60 and 300 s exposure conditions. It implies that the original Ti-O-Ti structure experienced transient structural change to [Ti-OOH HO-Ti] by O2 plasma exposure. Figure 1b indicates that the TiO2 surface quickly lost the hydroxyl groups and returned to the hydrophobic condition by showing an increased CA value of ∼62°, while neither SiO2 nor Si3N4 surfaces exhibited recovered original CA values (49° for SiO2 and 72° for Si3N4). The recovered CA values of SiO2 and Si3N4 surfaces were ∼25° and ∼22°, respectively, after exposure to the atmosphere for 480 h. Figure 2 shows the O2 plasma exposure time required for induction of the optimal hydrophilic properties from the surface of metal oxides. Figure 2a shows the image of water droplets for measuring the contact angle with different SiO2, TiO2, and Si3N4 substrates, and Figure 2b shows the measured contact angles as a function of O2 plasma exposure time. Our results demonstrated that the formation of the hydroxyl group on the TiO2 surface requires an O2 plasma exposure time of ∼150 s relatively longer than those of other metal oxide substrates (∼6 and ∼15 s, as seen in Table 1). After O2 plasma exposure, the contact angle values of water droplets on the surfaces of SiO2 and Si3N4 were quickly dropped to 0° by 6 and 15 s, respectively. It is surprising that a CA 11696 DOI: 10.1021/la901615e
Table 2. Number of NPs on SiO2, TiO2, and Si3N4 Surfaces for the SEM Images in Figure 4
2
no. of attached Au Nps/μm for SiO2 no. of attached Au Nps/μm2 for TiO2 no. of attached Au Nps/μm2 for Si3N4
60 s
300 s
116 6 125
125 121 128
of 0° on TiO2 required a plasma surface exposure time of 150 s, which probably resulted from the different structural stabilities of TiO2 and SiO2 surfaces. In addition, the presence and altered level of hydroxyl group formation as a function of O2 plasma exposure time are determined by XPS measurement. Hydroxyl group formation results clearly showed the linear increase from 29% (0 s) to 31 and 36% by 60 and 300 s, respectively, as seen in Figure 3. As a vehicle for a biosensor, the hydroxylated TiO2 substrate was silanized via introduction of APTES upon the completion of O2 plasma exposure. A change in the TiO2 surface by silanization allows the surface to become bioactive for immobilization of the anti-PSA antibody. Figure 4 shows a FE-SEM image of attached immunogold nanoparticles bound to the PSA antigen by antigen-antibody interactions on different types of metal oxide substrates upon O2 plasma exposure for 60 s (a-c) and 300 s (d-f). The densities of attached immunogold particles were ∼116 particles/μm2 (SiO2), ∼6 particles/μm2 (TiO2), and ∼125 particles/μm2 (Si3N4) after exposure for 60 s. After O2 plasma exposure for 300 s, attached immunogold nanoparticles on TiO2 showed almost 20-fold increases (∼121 particles/μm2) as seen in Table 2, Langmuir 2009, 25(19), 11692–11697
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2
no. of attached Au Nps/μm for TiO2
0s
60 s
90 s
100 s
120 s
150 s
220 s
300 s
4
6
15
55
119
121
125
127
while attached immunogold nanoparticles on other metal substrates maintained the same level. In detail, the presence of an amine functional group on the TiO2 substrate was determined by immunogold particle adhesion based on a sandwich-type immunoassay under different time courses. Results showed an increased level of attachment proportional to the O2 plasma exposure time as seen in Figure 5. After O2 plasma exposure for 60 s, the number of attached immunogold particles on the TiO2 substrate was relatively small by showing 6 particles/μm2; however, the number was increased and reached 127 particles/μm2 following exposure for 300 s as seen in Table 3. As seen in Scheme 2, surface modification by using the SAM method is advantageous in measuring the efficiency of antibody immobilization via the amine functional group on the surface that was characterized by counting the number of Au NPs conjugated to the detection antibody appearing during the terminal stage of the immunoassay. This result supports the notion that O2 plasma exposure and following silanization processes successfully induce the active form of Ti-OH structure on the TiO2 substrate during O2 plasma exposure in a time-dependent manner. As seen in Figure 5, increased immunogold nanoparticle density exhibits complete hydroxylization of TiO2 surface with O2 plasma exposure by 300 s. The results imply that APTES was fully deposited on the SiO2 and Si3N4 surfaces via SAMs. However, it was not fully deposited on the TiO2 surfaces, because the TiO2 surface (Figure 4b) was not completely hydroxylated by O2 plasma treatment for 60 s. There is a different state of structural stability for TiO2 and SiO2 surfaces since silanization results exhibited APTES in TiO2 was not fully deposited by O2 plasma exposure for 60 s. Since exposure of 60 s is not enough for the TiO2 surface to induce complete hydroxylation, this could be explained by the fact that the hydroxylation of TiO2 by O2 plasma exposure is a reversible process as seen in Scheme 1, since the final product of [Ti-OH HOO-Ti] is unstable in the atmosphere and it quickly returned to the initial Ti-O-Ti structure from [Ti-OOH HO-Ti] by a selfnucleophilic attack. Si-O-Si on the surface of SiO2 readily changes to Si-OH; however, on the TiO2 surface, the Ti-O-Ti structure indicates a hydrophobic property rather than Ti-OH structure of hydrophilic conditions is the dominant structure as the stabilized form. This could be explained by the electronegativity difference (ΔEN) value that determines the type of bonding between the
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atoms, either ionic or covalent. It has been known that an ionic bond is formed when the value of ΔEN is greater than 1.7 whereas a covalent bond is created with a ΔEN value of
