Preparation of α-mannoside hydrogel and electrical detection of saccharide-protein interactions using the smart gel-modified gate field effect transistor
© Maeda et al; licensee Springer. 2012
Received: 30 November 2011
Accepted: 7 February 2012
Published: 7 February 2012
The purpose of this study was to detect saccharide-protein interaction capitalizing on the gel-modified field effect transistor [FET]. A lectin-sensitive polymer gel that undergoes volume changes in response to the formation of molecular complex between 'pendant' carbohydrate and a 'target' lectin concanavalin A [Con A] was synthesized. It was revealed that direction and magnitude of the gel response (swelling or deswelling) could be readily designed depending on composition and network density of the gel. The Con A-sensitive polymer gel has shown the ability to transduce the detection of saccharide-protein interactions into electrical signals for FET.
PACS: 87.85.jf, bio-based materials
KeywordsSaccharide-protein interaction field effect transistor potentiometry soft interface concanavalin A stimuli-responsive gel molecular recognition.
Field effect transistor [FET]-based bio-sensing, so-called bio-FET, is an emerging class of label-free sensing format applicable to a range of biological targets, which can be readily miniaturized and integrated by virtue of advanced semi-conductor processing technology. In principle of bio-FET, any 'programmed' charge density changes on the gate surface can be detected as a mode of the modified channel characteristics of the FET in synchronization with electro-static interactions between these charges and the thin insulator-segregated silicon electrons. The bio-FET, however, while offering a number of promising applications, is susceptible to the charge-screening effect caused by counter ions [1, 2]. As a result, the technique yields a short detectable length limit (from the gate surface), which corresponds to the thickness of the electrical double layer or the Debye length of up to a few nano-meters at most with minimized ionic strength of the environment . This leads to an upper limit of the molecular weight for which quantitative charge detection can be feasibly performed [1, 3, 4].
To address this, we have recently proposed exploitation of a stimuli-responsive polymer gel as a signal-transducing material bridging between the target and the gate insulator [5, 6]. Stimuli-responsive polymer gels or 'smart gels' are a unique class of material capable of undergoing marked changes in their physicochemical properties in response to a series of specific stimuli. In particular, those sensitive to chemical stimuli, i.e., concentration fluctuations of specific molecules, are of significant interest due to their potential impact on clinical applications including bio-materials, drug delivery systems and actuators. In the gate-introduced configuration, smart gels, upon applied stimuli, can evoke an abrupt volume change termed as 'volume phase transition' causing other physical parameter changes such as thickness, charge density and permittivity. As a key feature, these physicochemical changes commencing at the gel/outer aqueous media interface can geometrically propagate across a macroscopic thickness of the gel layer and are, thus, able to transport the signal beyond the 'barrier' of the Debye length.
A monomer bearing a pendant mannose (p-acrylamidophenyl-α-D-mannoside [α-man]) was prepared via procedures reported previously . N, N-dimethylacrylamide [DMAAm] (Wako Pure Chemical Industries, Ltd., 3-1-2, Doshomachi, Chuo-Ku, Osaka, 540-8605, Japan) was purified under reduced pressure (13 mmHg) in a nitrogen atmosphere before use. N, N'-methylenebisacrylamide [MBAAm], 2,2'-Azobis(2-methylpropionamidine) dihydrochloride (V50), acryloyl chloride, dimethyl sulfoxide [DMSO], anhydrous ethanol, manganese (II) chloride tetrahydrate, calcium chloride (Wako Pure Chemical Industries, Ltd.), 1-[4-(2'-hydroxyethoxy)-phenyl]-2'-hidroxy-2"-methyl-1-propane-1-one (Irgacure2959) (Ciba Specialty Chemicals, Inc., Klybeckstrasse, 141 CH-4002 Basel, Switzerland), Con A (lectin from Canavalia ensiformis) (J-Oil Mills, 1-11-1, Marunouchi, Chiyoda-Ku, Tokyo, 100-6226, Japan), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES] and 11-amino-1-undecanethiol, hydrochloride [AUT] (Dojindo Molecular Technologies, Inc., Kumamoto Techno Research Park, 2025-5 Tabaru, Mashikimachi, Kamimashiki gun, Kumamoto 861-2202, Japan) were all used without further purification. Deionized ultra-pure Milli-Q water (Millipore, 290 Concord Road, Billerica, MA, 01821, USA) (18.2 MΩ cm-1) was used throughout the experiments. Hydrofluoric acid [HF] (23%) was prepared by diluting 46% HF (Morita Chemical Industries, Co., Ltd., 4-1-3, Kyutaromachi, Chuo-Ku, Osaka, 541-0056, Japan) with water.
Preparation of buffers
HEPES buffers (20 mM) were prepared using recipes from buffer calculator [10, 11] developed by R. Beynon at the University of Liverpool, UK. The buffers were titrated to a specific pH with KOH solution, and the overall ionic strengths were fixed with KCl.
Preparation of α-man hydrogel
Composition of functional monomer in pregel solution
Content of α-man (mol%)
Modification of gold electrode with α-man gel
A gold electrode (5 × 5 mm2) was fabricated by the sputter deposition of an adhesion layer of titanium (10 nm) and then of a gold layer (90 nm, 99.99% purity) on a silicon substrate (Ferrotec silicon; Ferrotec Corporation, 1-4-14 Kyobashi, Chuo-ku, Tokyo, 104-0031, Japan). The gold electrode was cleaned before use with acetone, ethanol, water and, finally, piranha solution (H2O2/H2SO4 = 25/75 v/v) (extreme caution must be exercised when using piranha etch; an explosion-proof hood should be used). The surface was rinsed thoroughly with water and ethanol, and was dried with nitrogen gas. An AUT self-assembled mono-layer [AUT SAM] was formed on a clean gold electrode by immersing the electrode in a 2-mM AUT ethanol solution under nitrogen atmosphere at room temperature for 24 h. After repeated rinses and sonication in pure ethanol for 5 min, the electrode was dried with nitrogen gas. The AUT SAM, thus, obtained was then treated with acryloyl chloride under nitrogen atmosphere at room temperature for 6 h to modify the SAM terminus amino groups with acryloyl groups. After the reaction, the electrode was washed by sonication in ethanol for 5 min and rinsed with water. The electrode was covered with a teflon tape with a round hole (thickness = 50 μm, φ = 6 mm), in a way, exposing the central gold surface to the air.
Composition of functional monomer in pregel solution for photo-polymerization
Content of α-man (mol%)
Fabrication of extended gate-FET bio-sensor
A reaction chamber (200 μL volume) was made on the electrode. A glass tube (ID = 5 mm) was immobilized on the gold surface with a thermosetting-insulating epoxy resin (XA-1295/HQ-1) (Pelnox, Ltd., 8-7 Bodai, Kanagawa, 259-1302, Japan) by heating at 60°C for 12 h. The rest of the substrate, including bonding wires, had to be completely protected against water penetration from the reaction solution so the periphery of the chamber was completely covered with the epoxy resin. HEPES buffer (pH = 6.9, 4°C, I = 0.3 M) containing CaCl2 (1 mM) and MnCl2 (1 mM) was pipetted into the reaction chamber. The source and drain of a commercially available FET (N-Channel Depletion-Mode MOSFET, LND150) (Supertex Inc., 1235 Bordeaux Drive, Sunnyvale, CA, 94089, USA) and Ag/AgCl reference electrode were connected to a real-time FET analyzer (Optogenesis, 2-1-8 Kenpuku, Honjo-Shi, Saitama, 367-0044, Japan), and the gate of the FET was connected to the wire, which was connected to separated α-man gel-modified gold electrode.
Detection of Con A using α-man gel-modified FET
The α-man gel-modified gold electrode was equilibrated in HEPES buffer (pH = 6.9, 4°C, I = 0.3 M) containing CaCl2 (1 mM) and MnCl2 (1 mM) prior to use. Real-time change in the gate potential was monitored and recorded at source current (IS) of 1,800 μA and gate voltage (VG) of 0 V (versus Ag/AgCl) when fixing source-drain voltage (VD) at 1 V. Various concentrations of Con A solution were prepared in the HEPES buffer. Con A detection was carried out by adding 5 μM Con A into the reaction chamber and incubated at 25°C.
Results and discussion
Synthesis of α-man gels
Con A-responsive behavior of α-man gels
Lectin binding to a single saccharide ligand is typically a low-affinity interaction. However, the multi-valent nature of lectin-saccharide interactions allows many low-affinity binding events to occur, resulting in high overall avidity . Multimeric lectins can cross-link multi-valent carbohydrate ligands, and they selectively cross-link with a single species of glycoprotein to form uniform lectin-carbohydrate lattices . Since tetrameric Con A can bind with four independent carbohydrates at once, one can anticipate that Con A-α-man complexation would result in inter- or intra-chain cross-linkages and, thus, deswelling of the gel (Figure 1a).
Con A detection using α-man gel-modified FET
where Eref is the constant potential of the reference electrode, ψ0 is the potential drop in the electrolyte at the insulator-electrolyte interface, χsol is the surface dipole potential of the solution, is the silicon electron work function, the fifth term is due to accumulated charge in the oxide(QOX) at the oxide-silicon interface (QSS) and the depletion charge in the silicon (QB), Φ f is the potential difference between the Fermi levels of doped and intrinsic silicon, Qgel is the charge in the gel layer and CCom is the combined capacitance of the gate oxide (COX) and the α-man gel layer (Cgel). All other variables than Qgel and Cgel are regarded constant throughout the chemical stimulation to the gel. On the basis of the operation function of FET (Equation 2), an increased anionic density on the gate surface gives rise to a positive directional shift of VT, whereas a decreased anionic density gives a negative directional shift of VT. Likewise, an increase in the gel permittivity on the gate surface leads to a negative direction shift of VT and vice versa.
In this study, lectin-sensitive polymer gels showing volume changes in response to the formation of molecular complex between pendant carbohydrate and target lectin Con A were prepared. The direction and magnitude of the gel response (swelling or deswelling) could be readily designed depending on composition and network density of the gel. The α-man gel-modified FETs not only showed the ability to electrically detect weakly charged proteins of large molecular weights but also revealed kinetics of the signal transduction.
The authors acknowledge the financial support grant-in-aid for Scientific Research on Innovative Areas "Molecular Soft-Interface Science" from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
- Bergveld P: The future of biosensors. Sens Actuators A Phys 1996, 56: 65–73. 10.1016/0924-4247(96)01275-7View ArticleGoogle Scholar
- Schasfoort RBM, Bergveld P, Kooyman RPH, Greve J: Possibilities and limitations of direct detection of protein charges by means of an immunological field-effect transistor. Anal Chim Acta 1990, 238: 323–329.View ArticleGoogle Scholar
- Schoning MJ, Poghossian A: Recent advances in biologically sensitive field-effect transistors (BioFETs). Analyst 2002, 127: 1137–1151. 10.1039/b204444gView ArticleGoogle Scholar
- Bergveld P: A critical evaluation of direct electrical protein detection methods. Biosens Bioelectron 1991, 6: 55–72. 10.1016/0956-5663(91)85009-LView ArticleGoogle Scholar
- Matsumoto A, Endo T, Yoshida R, Miyahara Y: Electrical visualization of chemo-mechanical signal transduction using a smart gel-modified gate field effect transistor. Chem Commun (Camb) 2009, 5609–5611.Google Scholar
- Matsumoto A, Sato N, Sakata T, Yoshida R, Kataoka K, Miyahara Y: Chemical-to-electrical-signal transduction synchronized with smart gel volume phase transition. Adv Mater 2009, 21: 4372–4378. 10.1002/adma.200900693View ArticleGoogle Scholar
- Taylor ME, Drickamer K: Introduction to Glycobiology. New York: Oxford University Press; 2006.Google Scholar
- Jelinek R, Kolusheva S: Carbohydrate biosensors. Chem Rev 2004, 104: 5987–6016. 10.1021/cr0300284View ArticleGoogle Scholar
- Toyoshima M, Miura Y: Preparation of glycopolymer-substituted gold nanoparticles and their molecular recognition. J Polym Sci A Polym Chem 2009, 47: 1412–1421. 10.1002/pola.23250View ArticleGoogle Scholar
- Beynon RJ: Buffer calculator.[http://www.liv.ac.uk/buffers]
- Beynon RJ, Easterby JS: Buffer Solutions. Oxford: IRL Press; 1996.Google Scholar
- Lee W-F, Yen S-H: Thermoreversible hydrogels. XII. Effect of the polymerization conditions on the swelling behavior of the N-isopropylacrylamide gel. J Appl Polym Sci 2000, 78: 1604–1611. 10.1002/1097-4628(20001128)78:9<1604::AID-APP50>3.0.CO;2-VView ArticleGoogle Scholar
- Zhang X, Zhuo R, Yang Y: Using mixed solvent to synthesize temperature sensitive poly(-isopropylacrylamide) gel with rapid dynamics properties. Biomaterials 2002, 23: 1313–1318. 10.1016/S0142-9612(01)00249-6View ArticleGoogle Scholar
- Tokuyama H, Ishihara N, Sakohara S: Effects of synthesis-solvent on swelling and elastic properties of poly(N-isopropylacrylamide) hydrogels. Eur Polym J 2007, 43: 4975–4982. 10.1016/j.eurpolymj.2007.09.016View ArticleGoogle Scholar
- Tokuyama H, Ishihara N, Sakohara S: Porous poly(N-isopropylacrylamide) gels polymerized in mixed solvents of water and N, N-dimethylformamide. Polym Bull 2008, 61: 399–405. 10.1007/s00289-008-0961-3View ArticleGoogle Scholar
- Dam TK, Roy R, Das SK, Oscarson S, Brewer CF: Binding of multivalent carbohydrates to concanavalin A and Dioclea grandiflora lectin. J Biol Chem 2000, 275: 14223–14230. 10.1074/jbc.275.19.14223View ArticleGoogle Scholar
- Sacchettini JC, Baum LG, Brewer CF: Multivalent protein-carbohydrate interactions. A new paradigm for supermolecular assembly and signal transduction. Biochemistry 2001, 40: 3009–3015. 10.1021/bi002544jView ArticleGoogle Scholar
- Matsumoto A, Kurata T, Shiino D, Kataoka K: Swelling and shrinking kinetics of totally synthetic, glucose-responsive polymer gel bearing phenylborate derivative as a glucose-sensing moiety. Macromolecules 2004, 37: 1502–1510. 10.1021/ma035382iView ArticleGoogle Scholar
- Agrawal BBL, Goldstein IJ: Physical and chemical characterization of concanavalin A, the hemagglutinin from jack bean ( Canavalia ensiformis ). Biochim Biophys Acta 1967, 133: 376–379.View ArticleGoogle Scholar
- Agrawal BBL, Goldstein IJ: Protein-carbohydrate interaction: VII. Physical and chemical studies on concanavalin A, the hemagglutinin of the jack bean. Arch Biochem Biophys 1968, 124: 218–229.View ArticleGoogle Scholar
- Akedo H, Mori Y, Kobayashi M, Okada M: Changes of isoelectric points of concanavalin A induced by the binding of carbohydrates. Biochem Biophys Res Commun 1972, 49: 107–113. 10.1016/0006-291X(72)90015-0View ArticleGoogle Scholar
- Bhattacharyya L, Brewer CF: Isoelectric focusing studies of concanavalin A and the lentil lectin. J Chromatogr A 1990, 502: 131–142.View ArticleGoogle Scholar
- Entlicher G, Kostír JV, Kocourek J: Studies on phytohemagglutinins. VIII. Isoelectric point and multiplicity of purified concanavalin A. Biochim Biophys Acta 1971, 236: 795–797.View ArticleGoogle Scholar
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