Sanford A. Asher* and John H. Holtz
Department of Chemistry
University of Pittsburgh
Pittsburgh PA 15260
412-624-8570
Fax 412-624-0588
asher+@pitt.edu
* corresponding author
We have developed novel glucose and galactose sensing materials that report on analyte concentrations via diffraction of visible light from polymerized crystalline colloidal arrays (PCCAs)1-4. These PCCAs are mesoscopically periodic crystalline colloidal arrays (CCA) of spherical polystyrene colloids polymerized within thin intelligent polymer hydrogel films (Fig 1). CCAs are brightly colored; they efficiently diffract visible light meeting the Bragg condition. The intelligent hydrogel contains molecular recognition agents that cause the gel to swell (Fig. 2) in response to the presence of analyte5-10.
CCAs
self assemble from suspensions of highly charged, monodisperse colloidal particles (Fig.
1).4,11-14
At low ionic strengths, the colloidal particles repel each other, and the system assumes a
minimum energy configuration, which is usually a body- or face-centered cubic lattice. The
colloidal particles may be composed of inorganic materials such as silica, or organic
polymers such as poly(methylmethacrylate), polystyrene, or poly(N-isopropyl acrylamide).2, 15,16
The periodicity of the CCA is on the order of ~200 nm, so the CCA diffracts visible light. The diffraction is in the “dynamical diffraction regime”, and almost obeys Bragg’s law: 11,17
ml=2 n d sin q
where m is the order of diffraction, l is the diffracted wavelength in vacuum, n is the refractive index of the system (solvent, hydrogel and colloids), d is the spacing between the diffracting planes (for the CCAs here, the 110 planes of a BCC lattice), and q is the glancing angle between the incident light propagation direction and the diffracting planes.
We polymerize the CCA into an acrylamide hydrogel film to form a PCCA1-3 by dissolving non-ionic polymerizable monomers, crosslinkers and photoinitiators into the liquid CCA, and then photopolymerizing the mixture to make a thin, diffracting PCCA film. These PCCA films have applications as tunable filters, optical switches and non-linear optical devices.2-4,18,19
To make an intelligent polymerized crystalline colloidal array (IPCCA) sensor, we incorporate glucose oxidase or b-D-galactosidase as the chemical recognition elements. We attach glucose oxidase or galactose oxidase by coupling some of the acrylamide amide groups in the PCCA to biotin functional groups. This biotinylated hydrogel then binds avidinated glucose oxidase, and b-D-galactosidase.
Figure
3 and 4 show the response of the glucose and galactose sensors, to varying concentrations
of glucose or galactose. For example, 0.1 mM glucose concentrations causes the diffraction
to shift from yellow at 550 nm to red at 600 nm. The IPCCA continues to shift to diffract
in the deep red for glucose concentrations of 0.2 mM; the shifts saturate for
concentrations above 0.2 mM for this sensor. In the case of the galactose sensor the
shifts occur for concentrations even beyond 0.3 mM galactose. Neither sensor responds to
sucrose, or to the substrate of the other enzyme (glucose or galactose).
As discussed below these shifts result from a steady
state concentration of reduced enzyme where a competition occurs between the reduction of
the enzyme by reaction with glucose and reoxidation of the enzyme by oxygen. At low levels
of oxygen, where the enzyme oxidization rate is very small, the glucose sensor swells in
sub-nanomolar concentrations of glucose. We observed a 30 nm diffraction wavelength shift
in 10-10 M
glucose, and an 8 nm wavelength shift in 10-12 M glucose. 
These IPCCA hydrogels are crosslinked polymeric networks that are swollen in water. These hydrogel networks change volume as their environment changes; hydrogels swell into good solvents, and this tendency to swell is resisted by the elastic restoring force of the hydrogel network, which arises from the crosslinks. Hydrogels with covelently attached ionic groups swell in water, due to Donnan-type equilibria established by the mobile counterions of the fixed charges inside gel and by electrolytes in the gel or its bathing solution.20-23 Tanaka has shown that the degree of swelling of a polyelectrolyte gel is proportional to the number of ionic side groups per polymer chain in the gel (a polymer chain is defined as the length of polymer between crosslinks).21,22 In addition, repulsions between charged groups will further swell the gel at high charge densities.
The equilibrium hydrogel volume is determined by the sum of the free energy of mixing of the polymer chains with the solvent medium, the free energy of elasticity of the crosslinked network, and the ionic electrostatic energy due to the Donnan equilibrium and electrostatic repulsions between charged side groups on the polymer backbone:
DFtot=DFmix+DFelas+DFion
Glucose
oxidase converts glucose to gluconic acid in a two-step process. In the first step,
glucose is converted to gluconic acid, and the enzyme is reduced. In the second step, the
enzyme is reconverted to its oxidized form by oxygen in the solution, producing H2O2 as a
by-product:23,24
EnzymeH(ox) + Glucose ® Enzyme1-
(red) + Gluconic Acid + H+
H+ + Enzyme1- (red) +
O2 ®H2O2 + Enzyme(ox).
When the glucose oxidase reacts with glucose the enzyme flavin prosthetic group becomes reduced and negatively charged24,25. The hydrogel then expands, primarily due to an increased osmotic pressure from a Donnan-type potential arising from mobile counterions.
In the absence of oxidizer, the sensor should eventually reach its maximum volume even if the bath solution contains only one stoichiometric amount of glucose relative to the glucose oxidase in the gel. In principle, we could detect diffraction from a 1 (mm)3 IPCCA sensor. Since the glucose oxidase concentration in our IPCCA is ~ 10-4 M, and since we can detect a ~ 10 nm shift in diffraction, this suggests a detection limit of less than ~ 106 molecules of glucose which would be required to fully reduce the glucose oxidase in this 1mm3 IPCCA
These IPCCAs are a new motif for fabricating sensors. The utility of this motif is limited only by the availability of suitable molecular recognition agents. We previously demonstrated, that the IPCCA can be coated onto optical fibers such that they can be used to fabricate optrodes to remotely measure analytes.
These sensors have the great advantage of having a response which is easily detectable by the human eye. The sensitivity and detection range of these sensors can be tailored by controlling the gel composition, as well as the molecular recognition agents used. We are now modifying this glucose sensor for use at the high physiological ionic strengths of biological fluids such as blood.
Acknowledgments.
This work was supported by the Office of Naval Research grant N00014-94-1-0592, AFOSR AASERT grant F 49620-94-1-0268 and National Science Foundation (grant CHE-9633561).
(1) a) Holtz, J. H and Asher, S. A. Nature 1997, 389, 829.
(2) Asher, S.A.; Holtz, J.; Liu, L.; Wu Z.; J. Am. Chem. Soc. 1994, 116, 4997.
(3) Weissman, J.M.; Sunkara, H.B.; Tse, A.S.; Asher, S.A. Science 1996, 274, 959.
(4) a) Asher, S.A.; Weissman, J.M.; Sunkara, H.B.; Pan, G.; Holtz, J.; Liu, L.; Kesavamoorthy, R.; In Polymers for Advanced Optical Applications, S. A. Jenekhe and K. J. Wynne (eds.), Washington, DC, (1997).; b) Asher, S. A. U.S. Patents 4,627,689 and 4,632,517; c) Haacke, G., Panzer H.P.; Magliocco, L.G.; d) Asher, S.A. U.S. Patents 5,266,238 and 5,368,781; e) Asher, S.A.; Jagannathan, S. U.S. Patent 5,281,370; Asher, S.A., f) Pan, G. in Nanoparticles in Solids and Solutions J.H.Fendler and I. Dekany eds, NATO ASI Series Vol. 18 pp.65-69, Kluwer Academic Publishers, Dordrect (1996).
(5) Irie, M. Adv. Polym. Sci. 1993, 110, 49.
(6) Gerkhe, S. H. Adv. Polym. Sci. 1993, 110, 81.
(7) Kishi, R.; Hara, M.; Sawahata, K.; Osada, Y. In Polymer Gels, DeRossi et al. eds. Plenum, New York, 1991.
(8) Irie, M.; Misumi, Y.; Tanaka, T. Polymer 1993, 34, 4531.
(9) Kikuchi, A.; Suzuki, K.; Okabayashi, O.; Hoshino, H.; Kataoka, K.; Sakurai, Y.; Okano, T. Anal. Chem. 1996, 68, 823.
(10) Sheppard, N.F.; Lesho, M. J.; McNally, P.; Francomacaro A. S. Sensors and Actuators B 1995, 28, 95.
(11) Carlson, R.J.; Asher, S.A. Appl. Spectrosc. 1984, 38, 297.
(12) Krieger, I.M.; O’Neil, F.M. J. Am. Chem. Soc. 1968, 90, 3114.
(13) Clark, N.A.; Hurd, A.J.; Ackerson, B.J. Nature 1979, 281, 57.
(14) Aastuen, D.J.W.; Clark, N.A.; Cotter L.K.; Ackerson, B.J. Phys. Rev. Lett. 1986, 57, 1733.
(15) Chang, S-Y.; Liu, L.; and Asher, S.A. J. Am. Chem. Soc. 1994, 116, 6739.
(16) Chang, S-Y.; Liu, L.; and Asher, S.A. J. Am. Chem. Soc. 1994, 116, 6745.
(17) a) Rundquist, P.A.; Kesavamoorthy, R.; Jagannathan, S.; Asher, S.A.; J. Chem. Phys. 1991, 95, 1249.b) Rundquist, P.A.; Photinos, P.; Jagannathan, S.; Asher, S.A. J. Chem. Phys. 1989, 91, 4932.
(18) Pan, G.; Kesavamoorthy, R.; Asher, S.A. Phys. Rev. Lett. 1997, 78, 3860.
(19) Asher, S.A.; Flaugh, P.L.; Washinger, G. Spectroscopy 1986, 1, 26.
(20) Li, Y.; Tanaka, T. Annu. Rev. Mater. Sci. 1992, 22, 243.
(21) Tanaka, T.; Fillmore, D.; Sun, S. T.; Nishio, I.; Swislow, G.; Shah, A. Phys. Rev. Let. 1980, 45, 1636.
(22) Hirotsu, S.; Hirokawa, Y.; Tanaka, T.; J. Chem. Phys. 1987, 87, 1392.
(23) Flory, P.J. Principles of Polymer Science Cornell University Press, Ithaca, 1953.
(24) Raba, J.; Mottola, H. Crit. Rev. in Anal. Chem. 1995, 25, 1.
(25) Bright, H.J.; Porter, D.J.T. in The Enzymes Vol. XII 2nd ed. (Academic Press, New York, 1975). Chapter 7, p. 421.