A new type of glucose sensor based on luminescence quenching (LUQUEN) is proposed. In this LUQUEN principle the concentration induced quenching is enlarged by radiationless (excited-state) energy transfer within the luminescent material. We describe the integrated optical detection part and the chemo-optical interface, in which the concentration induced optical changes are based on the binding of glucose. Some experiments on the internal energy transfer are reported, and the results are discussed

Bovenkamp, H.J. van de

Engbersen, J.F.J.

Hesselink, G.L.J.

Kreuwel, H.J.M.

Lambeck, P.V.

Popma, Th.J.A.

Reinhoudt, D.N.

English

G.L.J. Hesselink

H.J.M. Kreuwel

P.V. Lambeck

H.J.V.D. Bovenkamp

J.F.J. Engbersen

D.N. Reinhoudt

Th. J.A. Popma

Crossref

Sensors and Actuators B Chemical

Glucose sensor based on the LUQUEN principle

Hesselink, Gerlo

Kreuwel, Harrie

Lambeck, Paul

van de Bovenkamp, H.J.

Engbersen, Johannes F.J.

Reinhoudt, David

Popma, T.J.A.

NARCIS 

Glucose-sensor based on the LUQUEN-principle

Glucose sensor based on the LUQUEN-principle

University of Twente Research Information

Sensors and Actuators A, 7 (1992) 363-366 363 Glucose sensor based on the LUQUEN principle G. L. J. Hesselink, H. J. M. Kreuwel, P. V. Lambeck, H. J. v. d. Bovenkamp, J. F. J. Engbersen, D. N. Reinhoudt and Th. J. A. Popma MESA, Institute for Micro Electronics, Materials Engineering, Sensor & Actuators, University of Twente, P.O. Box 217, 7500 AE Enschede (Netherlands) Abstract A new type of glucose sensor based on luminescence quenching (LLJQUEN) is proposed. In this LUQUEN principle the concentration induced quenching is enlarged by radiationless (excited-state) energy transfer within the lumines- cent material. We describe the integrated optical detection part and the chemo-optical interface, in which the concentration induced optical changes are based on the binding of glucose. Some experiments on the internal energy transfer are reported, and the results are discussed. Introduction There is a need for the development of a glucose sensor that can be used easily and on line for analytical purposes in chemical laboratories, food and fermentation industry, and medical applica- tions. Instead of the well known enzymatic conver- sion [I], we have chosen another approach, based on the measurement of optical effects caused by reversible binding of glucose to a selective opti- cally labeled receptor. This receptor system will function as an interface between the chemical and the optical domain. In principle, the optical re- sponse upon the glucose binding can be a change in either the absorptive, the refractive or the lu- minescent properties of the receptor. If we pay first attention to the absorption a most promising method is based on quenching of luminescence emission (LUQUEN) by these recep- tor molecules, applied as a interface layer on top of a luminescent material. Using luminescent ma- terials, showing a large radiationless transfer be- tween the centres, an optical gain is achieved. Strong points of the LUQUEN principle are the great sensitivity achieved by this gain and the small sensitivity for changes in optical properties of the chemical environment. By using wave- guides, which can be used for the transport of the light to and from the luminescent material, a small versatile sensor can be obtained. It has been shown by Kreuwel [2], using a non-optimized pro- totype based on this principle, that detection of a 0925-4005/92/$5.00 fractional complexation as small as 10e3 to 10e5 of the reported molecules (in this case bromo cresol purple) is feasible. This detection limit will be strongly dependent on the extent of the energy transfer in the luminescent material and some experimental results about this phenomena will be presented and discussed in this paper. But before doing so, we will describe the sensor principle in more detail, in particular the optical detection part and the opto-chemical interface aimed for glucose detection. Optical detection Energy transfer An excited luminescent molecule can return to the ground state by emission of light, but also by transferring its excitation energy (often called exci- ton) radiationless to another molecule. The physi- cal origin of this exciton transfer is often an electric dipole-dipole interaction between the exci- ton donor and the exciton acceptor. It can be calculated [3] that the probability of this transfer is strongly dependent on the distance R between both centres, involved in the transfer: (1) where r. is the luminescent decay time of the luminescent centre, if no transfer occurred. & is the critical distance, i.e. the distance for which the @ 1992 - Elsevier Sequoia. AI1 rights reserved 364 probability for transfer is equal to the probability for decay. For dipole-dipole interaction the criti- cal distance is given by the classical Fiirster-Dex- ter expression: -‘F dE (2) receptor molecule L-l ip-$p-r lumjmxexe centre Herein Q is the integrated absorption cross sec- tion, f&E) the normalized emission spectrum of the donor, F*(E) the normalized absorption spec- trum of the acceptor, n is the refractive index of the surrounding medium. The above formula holds as well for two identical molecules as for Fig. 1. Cross section of the LUQUEN sensor. two molecules of different type. Sensing based on luminescence quenching For sensing we apply principally a simple setup consisting of a layer of luminescent material with on top of it a thin interface layer consisting of receptors. It is required that complexed receptors show a quenching potential completely different from that of uncomplexed receptors. This can be achieved in such a way that the emission spectrum of the luminescent centre shows a large overlap with the absorption spectrum of the complexed receptor molecules but a small overlap (preferen- tially zero) with the absorption spectrum of the uncomplexed ones. Besides the receptors have to return to the ground state radiationless. By choos- ing a luminescent material with large internal exci- tation transfer, we increase the probability for an exciton entering the effective region of a com- plexed receptor and thus the quenching effect of that receptor. In that way the quenching centre is able to consume a lot of photons, originally cap- tured by the luminescent centres, and presented to it as excitons. By this mechanism, comparable to antenna action, a photon consumption by a com- plexed receptor molecule is obtained that is much larger than can be reached by pure absorption in the absence of the luminescent material. Hence we have introduced an optical gain. The following factors will affect this gain: (i) Exciton transfer. Increasing the internal ex- citon transfer in the luminescent material, e.g. by increasing the concentration, and by choosing cen- tres with large Ro. (ii) The quenching action of the complexed re- ceptor. For increasing the gain we must make an effective quencher. This means matching its ab- sorption spectrum to the emission of the lumines- cent centre and radiationless return to the ground state with a decay time small compared to the decay time of the luminescent centre. The latter causes the receptor to be almost continuously available for the excitons. Because of the short range of energy transfer, there is only a small sensitivity for molecules out- side the interface. Layout The system part described above can be incorpo- rated in a waveguiding system (fiber or integrated optics) that takes care of the transport of the excitation light the emission light, taking advantage of the fact that a large part ( z 60%) of the emission light can be collected as a waveguide mode [2]. The prototype system is depicted in Fig. 1. For the waveguide we used a SiON deposited on top of a SiOZ layer, and partly screened from the environ- ment by a SiOZ cladding layer. In the part without cladding a 1 urn thick luminescent layer is spin- coated which consists of polyurethane (Neorenz) with luminescent Rhodamine-6G incorporated to a typical concentration of 6 x lop4 Mol. The Rho- damine is excited with green light (typically 475 nm) and gives a red emission (typically 600 nm). The interface layer on top of it is de- scribed in the next section. Interface For the receptor we use a glucose-binding protein (GPB). In the first stage we will use the commercially available molecule concanavaline A (Con A) showing an association constant of about 4 x lo2 mol-‘. Later on proteins with larger spe- cificity and larger association constants will be used. Because the glucose binding of Con A does not introduce absorption changes in the visible, a reporting function has to be added. Several report- ing schemes are possible. A first option is a compe- tition between glucose and the reporting group (the latter being covalently bound to the protein) at the binding site of the receptor molecule; bind- ing of glucose will result in a variation of the microenvironment of the reporter molecule. This can be signalled by choosing strongly solva- tochromic dyes, for example the merocyanines, spirans and the pyridinium-N-phenolate betaines, the solvatochromic properties of which have been reported elsewhere [4]. A shift in the absorption band, corresponding with our demands, is feasible. The protein can be immobilized on the poly- urethane by covalent binding [5]. Measurements The most essential point in this sensor is the energy transfer. Energy transfer in the luminescent material can be measured in different ways. By using the method of concentration quenching of fluorescence polarization [6], information on the critical distance R,, between the luminescent cen- tres can be obtained. By measuring the lumines- cence quenching obtained in a system as presented in Fig. 1, the excitation diffusion length can be calculated from the dependency of the quenching on the thickness of the layer. Polarization measuremen Is If an excited molecule itself emits radiation, the polarization states of excited and emitted light are correlated. A common assumption in most theo- ries about fluorescence depolarization is that this correlation will already be lost on an immediate exciton transfer step between two identical molecules [6]. This means that measuring the po- larization of the emitted light as a function of the mean mutual distances gives information about the critical distance R,. This is called concentra- tion quenching of fluorescence polarization (cqfp). The quantity to be measured is the anisotropy, defined as: A - Iit - 1l III + 211 365 (3) where 11, and 1, are the intensities of the light with polarization parallel and perpendicular, respec- tively, with respect to the incoming light. From the dependency of the normalized an- isotropy A/& on the normalized concentration c/co, with co = ($nRi)-‘, of the luminescent cen- tres, the critical distance R. can be obtained by using the method given by Craver and Know [6c] (Fig. 2). For 6 x 10m4 M Rhodamine in pol urethane we have calculated in this way R,, = 65 8: . Measurements of luminescence quenching Exciton concentration (c*) in a luminescent layer can be described by a continuity equation: c*(r) de*(u) Dvc*(r) + crPEX&) - - = - 70 dt (4) wherein the first term describes a diffusion, the second the excitation, and the third term the decay of the luminescent molecules in the system given in Fig. 1, in which a monomolecular acceptor layer is applied, the problem can be approximated as one- dimensional. By assuming, the PExC is position independent (a rather rough approximation) the steady state condition can be found from the equation D&‘(X) +a. PExc-- c*(x) = o TO (5) applying the proper boundary conditions. Assum- ing the probability of the energy transfer to the monolayer receptors to be much larger than the probability of decay, from this equation a simple 0.8 - 0.6 - 0.4 - 0.2 - 0 0.01 Fig. 2. Fluorescence polarization measurement with fit from Craver and Knox [6c]. 366 01 I I I 8 I I I I I I 8 I I I I I I 8 I 0 12 3 4 5 6 7 8 9 10 - h[Cunl Fig. 3. Exiton diffusion length measurement and fit with eqn. (6). expression for the quenching can be deduced [2] sion length (d = 1 pm) by considering the mean mutual distance between the centres at the experi- mental concentration: R,, = 87 A. It means that an exciton should have to be transferred more than 100 times in order to diffuse over 1 pm. This seems to be in contradiction to the fact that for this value of the mutual distance the hopping probability would be smaller than the decay prob- ability. By applying relations between R, and d from Gouchanour et al. [7] this discrepancy can also be seen. This contradiction could be explained either by assuming chains of Rhodamine molecules with smaller distances which form gateways for the exciton, or by assuming that the polyurethane matrix has a certain influence on the transfer, e.g. by affecting the mutual orientations. Further re- search is in progress. wherein h is the thickness of the luminescent layer and d represents the exciton diffusion length d = ,/& which is the mean distance an exciton can travel before it decays. The quenching Q is defined as Q = 1 - Z/lo, where Z is the lumines- cent intensity with and I,, without quenching. Ap- plying bromo cresol purple (BCP) in the basic state as acceptor molecules, from Q(d) measure- ments (Fig. 3) a diffusion length of 1 urn can be calculated. Discussion By substitution of experimentally obtained ab- sorption and emission data of the Rhodamine in relation (2) we calculate R. x 76 A, a value about 10% larger than the experimental one. This differ- ence can be caused by the assumption of dipole- dipole interaction or by experimental uncertainties. The microscopic experimental data (& = 65 A) could be related to the macroscopic exciton diffu- References Sl. Brocks, R. E. Ashby, A. P. F. Turner, M. R. Clader and D. J. Clarke, Biosensors, 3 (1987) 45. H. J. M. Kreuwel, Planar waveguide sensors for the chemical domain, Ph.D. Thesis, Enschede, The Netherlands, 1988, Ch. 6. Th. Forster, Zwischenmolekulare Energiewanderung und Fluo- reszenz, Ann. Phys., 2 (1948) 55. H. J. van de Bovenkamp, G. L. J. Hesselink, P. V. Lambeck, Th. J. A. Popma, J. F. J. Engbersen and D. N. Reinhoudt, Synthesis and screening of dyes for the development of an optical glucose sensor based on a new concept, Lectines: Bio., Biochem., Clin. Biochem. (1991) to be published. G. H. M. Engbers, Development of heparinized materials with an improved blood compatibility, Ph.D. Thesis, Enschede, The Netherlands, 1990. (a) R. S. Knox, Theory of polarization quenching by excitation transfer, Physica, 39 (1968) 361, (b) F. W. Craver, Theory of concentration quenching of fluorescence polarization in two-dimen- sional solutions, Mol. Phys., 22 (1971) 403-420, (c) F. W. Craver and R. W. Knox, Theory of polarization quenching by excitation transfer, II. Anisotropy and second-neighbour considerations, Mol. Phys., 22 (1971) 3855402. C. R. Gouchanour, H. C. Anderson and M. D. Fayer, Electronic excited state transport in solution, J. Chem. Phys., 70 (1979) 4254-4270. 

Development of heparinized materials with an improved blood compatibility,

Electronic excited state transport in solution,

Planar waveguide sensors for the chemical domain,

Synthesis and screening of dyes for the development of an optical glucose sensor based on a new concept,

Theory of polarization quenching by excitation transfer,

Zwischenmolekulare Energiewanderung und Fluoreszenz,

Glucose sensor based on the LUQUEN-principle

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University of Twente Research Information

University of Twente Research Information