Competitive Binding Assay for Glucose Based on Glycodendrimer

This assay has been shown to have a large response to glucose within the biological ... Data showing that the sensor is fully reversible and specific ...
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Anal. Chem. 2005, 77, 7039-7046

Competitive Binding Assay for Glucose Based on Glycodendrimer-Fluorophore Conjugates Bennett L. Ibey,* Hope T. Beier, Rebecca M. Rounds, and Gerard L. Cote´

Department of Biomedical Engineering, Texas A&M University, College Station, Texas 77843 Vamsi K. Yadavalli and Michael V. Pishko

Department of Chemical Engineering and the Materials Research Institute, The Pennsylvania State University, University Park, Pennsylvania 16802

A new fluorescent glucose assay has been created using Alexa Fluor 647-labeled concanavalin A (Con A) and a fourth-generation PAMAM Alexa Fluor 594-labeled glycodendrimer. This assay has been shown to have a large response to glucose within the biological range and to be capable of functioning within a polymer hydrogel. In this paper, the glucose response is shown to be a single fluorophore-based quenching reaction. Data showing that the sensor is fully reversible and specific through competitive binding between the dendrimer and glucose with Con A are presented. Overall, the assay is shown to have potential over the traditional dextran-based assay because it has a larger dynamic response to physiological glucose concentrations, incorporates longer wavelength dyes that improve signal penetration through dermal tissue, and provides an internal reference in the form of a nonreactive fluorescent label. Diabetes mellitus is a debilitating disease that results in enormous medical costs each year in the United States.1 Diabetes is the fifth leading cause of death by disease and affects 6.2% of the U.S. population.1 It is estimated that over 132 billion dollars a year (2002) are spent to help regulate type 1 and type 2 diabetes.1 However, what is needed most is an alternative to the standard “finger prick” method, an approach that limits the ability of a patient to monitor his/her blood sugar multiple times a day because of painful drawbacks, potential infection, and cost.2 Multiple or near-continuous (5 or more times a day) monitoring of blood glucose concentrations has been shown to dramatically reduce the secondary complications and further progression of diabetes.3 To effectively improve the overall health of those suffering from diabetes and reduce overall costs associated with this disease, routine monitoring and proper control of blood glucose is mandatory. * To whom correspondence should be addressed. E-mail: bli6339@ tamu.edu. Phone: (979) 862-1076. Fax: (979) 845-4450. (1) U.S. Department of Health and Human Services, Centers for Disease Control and Prevention, Atlanta, GA, 2002. (2) de Graaff, J. C.; Hemmes, G. J.; Bruin, T.; Ubbink, D. T.; Robert, P. J.; Jacobs, M.; Sanders, G. T. B. Clin. Chem. 1999, 45, 2200-2206. (3) Diabetes Control and Complication Trial Research Group. N. Engl. J. Med. 1993, 329, 977-986. 10.1021/ac0507901 CCC: $30.25 Published on Web 09/28/2005

© 2005 American Chemical Society

One potential technology that has been investigated for nearcontinuous blood glucose detection is an implantable chemical assay based on a competitive binding reaction between the protein concanavalin A (Con A), dextran, and glucose.4-7 One embodiment of this sensing approach is that it will be implanted superficially into the skin tissue and an external probe will be used to monitor the interstitial fluid glucose concentration, which is comparable to that of the blood.8-13 Traditional assay sensors have been based on a fluorescence resonance energy-transfer (FRET) reaction between fluorescein isothiocynanate (FITC)-labeled dextran and tetramethylrhodamine isothiocyanate (TRITC)-labeled Con A.14-15 This heterogeneous FRET reaction occurs when one fluorophore is placed in proximity (20-100 Å) to a second fluorophore whose absorption spectrum heavily overlaps the emission spectrum of the first fluorophore.16 In the absence of glucose, the Con A tetramer binds to the dextran backbone bringing the TRITC dye (acceptor) within the Fo¨rster radius of the FITC dye (donor), reported to be 54 Å6,16 (Scheme 1). Within the Fo¨rster radius, the FRET efficiency is above 50%, which appears optically as an increase in the TRITC emission and a decrease in the FITC emission. Due to a higher affinity of Con A for glucose over dextran, glucose will bind to the Con A protein preferentially and displace the dextran chain, resulting in a change in fluorescent (4) Chowdhury, T. K.; Weiss, A. K. Concanavalin A: Advances in experimental medicine and biology; Plenum Press: New York, 1975; p 55. (5) Lakowicz, J. R.; Maliwal, B. Anal. Chim. Acta 1993, 271, 155-164. (6) Schultz, J. S.; Mansouri, S.; Goldstein, I. J. Diabetes Care 1982, 5, 245253. (7) Al-Ati, T. Cornell University Poisonous Plants Database, 2001; http:// www.ansci.cornell.edu/plants/toxicagents/lectins/lectins.html. (8) Russell, R. J.; Pishko, M. V.; Gefrides, C. C.; McShane, M. J.; Cote´, G. L. Anal. Chem. 1999, 71, 3126-3132. (9) McShane, M. J.; Russell, R. J.; Pishko, M. V.; Cote´, G. L. IEEE Eng. Med. Biol. Mag. 2000, 19, 36-45. (10) McShane, M. J.; O’Neal, D. P.; Russell, R. J.; Pishko, M. V.; Cote´, G. L. SPIE Proc. 2000, 3923, 78-87. (11) O’Neal, D. P.; McShane, M. J.; Pishko, M. V.; Cote´, G. L. SPIE Proc. 2001, 4263, 20-24. (12) Ibey, B. L.; Meledeo, M. A.; Gant, V. A.; Yadavalli, V.; Pishko, M. V. SPIE Proc. 2003, 4965-01. (13) Aussedat, B.; Dupire-Angel, M.; Gifford, R.; Klein, J. C.; Wilson, G. S.; Reach, G. Am. J. Physiol. Endocrinol. Metab. 2000, 278, 716-728. (14) Meadows, D. L.; Schultz, J. S. Anal. Chim. Acta 1993, 280, 21-30. (15) Ballerstadt, R.; Schultz, J. S. Anal. Chem. 2000, 72, 4185-4192. (16) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 2nd ed.; Kluwer Academic/Plenum Publishers: New York, 1999.

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Scheme 1. Pictorial Representation of the Traditional FITC Dextran/TRITC Con A Assaya

a The Forster distance is maintained while the components are in the bound state and, upon exposure to glucose, the molecules dissociate to a distance greater than the Forster distance.

emission. Using the ratio between the two emission peaks, a quantitative measure of glucose concentration can be made.6,12 Schultz et al. proved the feasibility of this assay for glucose detection by embedding unlabeled Con A protein into the wall of a hollow dialysis membrane. This technology proved the ability of the assay to measure glucose but was difficult to calibrate due to the use of a single fluorescent emission.6 This research was expanded by labeling the Con A with TRITC to create a FRET pair and exciting each of the fluorophores separately using two fibers to improve the response of the sensor. In this embodiment of the sensor, the Con A protein was allowed to move freely within the membrane. Results showed an increased fluorescent change due to glucose beyond that seen in the single fluorophore sensor (45% quenching and 60% return with glucose). This sensor used the emission of the TRITC Con A as an internal reference and monitored the quenching of the FITC dextran, which indirectly correlated to glucose concentration.14 Ballerstadt et al.15 furthered the hollow fiber membrane technology by using a macroporous Sepharose bead to immobilize the QSY21-labeled Con A protein (improving long-term stability) and allowing it to bind to Alexa Fluor 647 (AF647)-labeled dextran (70 kDa) to generate a FRET reaction. The dextran was then displaced from the Con A with the addition of glucose. Also included in this sensing technology is a separate hollow fiber sensor of polysulfone fibers labeled with LD800 to serve as a reference. This sensing technology showed about a 20% recovery due to glucose binding across the biological range.17 The change in fluorescent emission across the biological range of glucose concentrations has been shown to be widely variable in past sensing modalities (roughly 20-30%).6,14,17 It is important to maximize the fluorescent change seen in vitro because dermal attenuation of fluorescent emission will reduce the perceived change through increased scattering and absorption of the light. It has been reported that the dextran molecule, specifically, its molecular weight and degree of branching, can cause a significant (17) Ballerstadt, R.; Gowda, A.; McNichols, R. Diabetes Technol. Ther. 2004, 6, 191-200.

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reduction in the dynamic response of the assay due to incomplete dissociation from the Con A protein. 6,18 This incomplete dissociation results in a smaller fluorescent emission change due to limited increase in the separation distance between the fluorescent labels.19 Lakowicz et al., in attempt to create a Con A assay for use in luminescence decay detection based on a FRET reaction between Malachite Green and Cy5, replaced the dextran molecule with a maltose-insulin-Malachite Green conjugate.20 An improved dynamic response (84% quenching and 50% return with glucose) through a FRET reaction between the two dyes was seen. The purpose of the work was to monitor fluorescent lifetime using longer lifetime dyes, but due to the need for high labeling ratios, the system showed increased aggregation of protein, limiting its potential for reversibility.20 In attempt to increase the dynamic response of an assay-based glucose monitoring system, our group describes a system that uses a globular dendrimer with Con A in place of the dextran molecule. The dendrimer has had its surface amine groups modified to contain both glucose-like binding sites and Alex Fluor 594 (AF594) dye. The spherical shape of the dendrimer molecule ensures that only a single bond can be formed with each Con A tetramer, thus eliminating the potential for incomplete dissociation (Scheme 2). This paper will focus on the this new assay based on an affinity binding reaction between AF647 Con A, AF594 glycodendrimer, and glucose for development of an implantable blood glucose sensor for diabetic patients. In this paper, data will be provided that describe the assay response to glucose, the single fluorophore quenching reaction, the reversibility of the assay, the specificity of the assay, and the assay time response. PROCEDURE Preparation of AF594 Glycodendrimer and AF647 Con A. Polyamidoamine (PAMAM) dendrimer-generation 4 (G4) (18) Aslan, K.; Lakowicz, J. R.; Geddes, C. Anal. Biochem. 2004, 330, 145-155. (19) Yadavalli, V. K.; et al., submitted to Anal. Chem. (20) Tolosa, L.; Malak, H. K.; Raob, G.; Lakowicz, J. R. Sens. Actuators, B 1997, 93-99.

Scheme 2. Theoretical Representation of the New Assay Systema

a This shows single binding between the assay components and complete dissociation upon introduction of glucose, eliciting a large optical response.

(Sigma, St. Louis, MO) has a measured diameter of 45 Å, a molecular weight of 14 215, and 64 surface amine groups. To synthesize the glycosylated dendrimer, glucosyloxyethyl methacrylate (MW 292.3) (50% w/w in water, 1.36 mL) (Polysciences, Warrington, PA) was added dropwise to a solution containing the PAMAM G4 dendrimer (10% w/w in methanol, 21.1 µmol) and LiCl (4 mg) in methanol (10 mL) (Sigma). The mixture was stirred for 48 h at room temperature, and the solvent was evaporated in a centrifuge with low heat into a yellowish gel. The gel was allowed to redissolve into distilled water for 24 h, and the solution was filtered using a 0.2-µm syringe filter. Using centrifuge filters, the solution was heavily rinsed with distilled water until the filtrate no longer possessed absorbance at 250 nm (unbound glucosyloxyethyl methacrylate) using a UV spectrometer (Ocean Optics). AF594 succinimidyl ester (Molecular Probes, Eugene, OR) was attached to the PAMAM G4 dendrimer by the protocol for linking amine reactive probes (2.5:1 dye mol/dend).21 The mass of the glycodendrimer was determined prior to fluorescent labeling (17.67 kDa) to ensure proper glycolsylation using MALDI-TOF mass spectroscopy and after to determine the degree of labeling (19.6 kDa). The final concentration of the AF594 labeled glycodendrimer was estimated to be 52.10 µM (∼1 mg/mL). A 5-mg sample of AF647 Con A (Molecular Probes) was dissolved into 5 mL of buffer consisting of 10 mM HEPES, 150 mM NaCl, 100 mM CaCl2, 10 mM MnCl2, and 0.08% NaN3 (Sigma). The pH of the buffer was adjusted to 7.4 using NaOH prior to the addition of protein. The protein solution was centrifuged to remove any aggregates, and the resulting supernatant was separated into 500-µL aliquots. The dissolved concentration of the stock protein solution was determined using the absorption ratio between 650 and 280 nm obtained from a UV/visible spectrometer (Beckman, Fullerton, CA)21 as per the Molecular Probes labeling protocol (2.73 µM, 3 dyes/molecule). Fluorescence Detection Systems. In the following experiments, two fluorescence detection systems are used. The first is (21) Handbook of Fluorescent Probes and Research Products, 8th ed.; Molecular Probes: Eugene, OR, 2002.

Figure 1. Fluorescent detection system based on a CCD spectrometer, a HeNe laser, and a long-pass 610-nm interference filter. A cuvette holder is used in the first configuration (A) and light collected at 90° to the detector, whereas in the second system (B) backscattered fluorescence is collected through a dual fiber-optic probe.

a fluorescence detection system from Photon Technologies Inc. (PTI, Lawrenceville, NJ) set to collect fluorescence at 90°. This system consists of a mercury arc lamp, an excitation monochromator, a sample chamber with a liquid cooled cuvette holder, an emission monochromator, and a PMT. The second system (Figure 1A) is composed of a custom cuvette holder, a CCD-based spectrometer (Roper Scientific, Tucson, AZ), a HeNe laser source (594 nm) (Hughes, Carlsbad, CA), a lens, and a 610-nm highpass filter (Chroma, Rockingham, VT) also set at 90° collection. The second system was altered by replacing the custom cuvette holder with a dual fiber probe (Figure 1B), which allowed for the collection of fluorescent backscattered light. The custom system was required to reduce the acquisition time per spectrum, to improve stability, and to provide for a more reliable measurement of single fluorescent emissions. Glucose Titration into the Assay. To prove the effectiveness of the devised sensor chemistry in detecting glucose within the physiological range, a series of experiments were performed. The first experiment was a glucose titration using AF594 glycodendrimer and AF647 Con A. Con A was diluted in 2 mL of 0.01 M phosphate-buffered saline (PBS) solution to 273 nM and placed into a fluorometer containing a 610-nm high-pass filter. Spectra Analytical Chemistry, Vol. 77, No. 21, November 1, 2005

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were taken with a wavelength range of 610-835 nm with an integration time of 1 s/2 nm step. AF594 glycodendrimer (26.05 nM) was titrated into the cuvette, and spectra were taken until the fluorescence of the AF647 dye was minimized. The optimum ratio of AF647 Con A to AF594 glycodendrimer to maximize quenching was determined to be a 1:1 molar ratio. A glucose solution was generated by dissolving dextrose (2 g) (Sigma) into a PBS buffer solution (10 mL) to a final concentration of 1.1 M. Small, highly concentrated, aliquots of glucose solution (10 µL into 2 mL or 5.5 µM) were added to the assay and stirred using a pipet. The solution was given 5 min to equilibrate, and triplicate spectra were taken using a fluorometer. Single Quenching Experiments. The previous experiment showed a large response to glucose within the 0-33 mM physiological range (0-600 mg/dL), and the mechanism of this fluorescence change was hypothesized to be a single fluorophore quenching reaction. To test this hypothesis, an unlabeled glycodendrimer was created by omitting the amino reactive probe labeling procedure from the protocol. An assay solution was created by adding diluted AF647 Con A and unlabeled glycodendrimer at a molar ratio of 1:1, and the assay was then diluted to 2 mL (273 nM:260.5 nM). Spectra were taken in triplicate using the custom fluorescence detection system (Figure 1A) within an integration time of 0.1 s from 600 to 700 nm. A glucose titration was performed similarly to the one in the previous experiment. Time Response of the Assay. To test the response time of the assay, a solution containing Con A protein (68.75 nM), glycosylated dendrimer (63 nM), and PBS was created. The solution was allowed 1 h at room temperature to stabilize. Small aliquots of glucose solution (10 µL of 1.1 M solution) were added to the cuvette and stirred for 5 s. Using a fluorometer, the fluorescent response of the assay was recorded every second for 10 min/aliquot from 0 to 33 mM. Due to small noise artifacts, the data was averaged every 10 s. Specificity of the Assay. To prove the specificity of the AF647 Con A/AF594 glycodendrimer sensor, three titrations were performed in a buffer solution containing the assay. The assay was prepared at a molar ratio of 1:1 Con A to glycodendrimer at a volume of 200 µL diluted to 2 mL. Stock solutions of mannose (Sigma), glucose (Sigma), and lactic acid (Sigma) were prepared at 1 M concentrations and dissolved at 50 °C. The assay solution was placed into a methacrylate cuvette, and spectra were taken using the custom fluorescence system (Figure 1A). Spectra were recorded from 600 to 750 nm. Three titrations were performed on each of the three separate assay samples. The peak emission of each spectrum was recorded to generate the response curve of the assay. Reversibility of the Assay. To demonstrate the reversibility of our devised sensor, a 10 000 MWCO dialysis membrane cassette (Pierce, Rockford, IL) was used. The assay was created, as in previous experiments, and injected into the dialysis cassette. The cassette was then placed in a 500-mL beaker containing alternating solutions of 55 mM glucose and pure buffer for a period of 7 days. The cassette was allowed to remain in each solution for a period of 24 h before spectra were taken; then it was placed into fresh solution. Using a dual fiber-optic cable, spectra were recorded by placing the probe against the membrane and recording the backscattered fluorescence (Figure 1B). Multiple 7042 Analytical Chemistry, Vol. 77, No. 21, November 1, 2005

Figure 2. Optimal concentration of each assay component determined by titrating the glycodendrimer into a known concentration of protein (273 nM). By monitoring the fluorescent quenching of AF647 dye, this plot shows the optimal concentration of dendrimer is roughly 260 nM, after which further titration shows no change in fluorescence.

Figure 3. Response of the assay to glucose determined by adding concentrated aliquots of glucose solution to a large assay volume. This figure shows the resultant fluorescent emission of the AF647 dye as a function of glucose concentration. It can be seen that the fluorescent emission recovers to nearly the intensity seen before the addition of dendrimer to the Con A solution.

areas of the solution were probed, and the average spectrum was generated to remove any artifacts. RESULTS AND DISCUSSION Glucose Titration into the Assay. In preparing an assay it is important to optimize both components to ensure the greatest signal change possible for a given analyte concentration. Optimization of this assay was done through a simple titration of one component into the other while monitoring the fluorescent signal to achieve maximum quenching. Figure 2 shows the response of the AF647 Con A to increasing concentrations of glycodendrimer. It can be seen that this curve levels off at ∼260.5 nM, and further additions of glycodendrimer have little quenching effect on the AF647 dye (total 65% reduction). Following that experiment, glucose solution was added at an interval of 2.75 mM and a 75% reversal of the quenching reaction was seen (Figure 3). These experiments were repeated in triplicate to demonstrate repeatability. A sample spectrum is shown in Figure 4. The fluorescent response of the assay to the analyte of interest is very important because it will determine both the complexity

Figure 4. Trace of the peak emission of each of the two fluorophores in the assay versus glucose concentration. It can be seen that the AF594 emission changes very little with addition of glucose while the AF647 emission changes dramatically. This plot suggests that the AF594 label does not contribute to the response of the assay. The spectra are inset within the figure.

of the final sensor and the ability of the sensor to monitor small changes accurately. Previous work has shown that the dynamic response of the traditional FITC dextran/TRITC Con A assay was 25% in solution and the Sephadex bead approach was 20%.14,17 It can be seen in Figure 4 that the newly devised assay has a much larger dynamic response of greater than 70%. The glucose response curve in a traditional FRET-based assay is generated by taking the ratio between the two peak emissions of the FITC and TRITC dyes. In this system, the ratio is also taken, but it is evident that AF594 peak emission does not change due to glucose while the AF647 peak emission increases greatly as depicted in Figure 4. This figure shows a tracing of the peak emission of the two independent fluorophores as derived from the acquired fluorescent spectra (Figure 4 inset). Single Quenching Experiments. It was hypothesized that the assay is based on a quenching event rather than a FRET reaction as were previous Con A-dextran-based sensors. The rationale behind this is that the “acceptor” peak gains a large amount of intensity with the addition of glucose, which would go against the notion of a heterogeneous FRET reaction.16 In addition, no quantifiable change is seen in the “donor” peak fluorescence.16 Figure 5 demonstrates the basic fluorescent phenomenon that is observed. When Con A is placed in a cuvette alone there is substantial acceptor excitation from the 594-nm laser source due to a broad absorption spectrum inherent to the AF647 dye. With the addition of the AF594 glycodendrimer, a large loss of AF647 Con A fluorescent signal is observed. If there is not a reaction between the two dyes, a simple superposition of the two dye emission spectra would be expected; however, loss of fluorescent emission from the AF647 dye is seen. With addition of glucose, a return of the acceptor peak fluorescence to a near superposition of the two dyes is seen. Therefore, it appears that the glucose molecules displace the dendrimer from the Con A tetramer, allowing the AF647 dye to be free to fluoresce. It is noted that, compared to previous FRET based approaches, the background fluorescence of our assay at zero glucose concentration is high. Our theoretical explanation for this high background fluorescence

Figure 5. Excitation of the AF647 molecule by the 594-nm excitation source. With addition of the AF594 glycodendrimer, a large reduction in the emission of the AF647 label is seen. This reduction is reversed with the addition of glucose. This figure strongly suggests that the assay is based on a single fluorophore quenching reaction.

is that some dyes on the Con A molecule are not being quenched through binding to dendrimer. However, due to the ratiometric nature of the assay, the background signal has little effect on the overall response of the sensor. Further optimization of the assay will focus on reduction of background fluorescence. To confirm the independence of the reaction from the AF594 dye, unlabeled glycodendrimer was added to the assay in place of the labeled dendrimer. Figure 6 shows the AF647 spectral response to a glucose titration. This response, compared to the previous response curve using two dyes (Figure 4), appears similar in amplitude and shape proving that the response seen is based upon a single fluorescent event. This titration confirms that the observed phenomenon is essentially independent of other fluorescent dyes, namely, the AF594 label on the glycodendrimer. However, the exact mechanism for the reduction in fluorescent Analytical Chemistry, Vol. 77, No. 21, November 1, 2005

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Figure 6. Normalized peak intensity versus glucose concentration. By removing the AF594 label from the glycodendrimer within the assay and titrating glucose into the solution, this figure proves that the devised assay does not require multiple fluorophores and that the AF594 label can serve as an internal reference. The spectra are inset within the figure.

intensity is still unknown. Potentially, this reduction may be due to a steric quenching introduced by the binding of multiple components and reversed by the unbinding of components with the addition of glucose. Also, dendrimer molecules are very alkaline (pH ∼11)22 due to the large number of free amine groups on the dendrimer surface. By our calculations, glycoside coverage on the dendrimer surface averages 10 of the 64 possible binding locations, resulting in many unbound amine groups. This creates a higher local pH around the dendrimer molecules, which could adversely affect a fluorescent molecule placed within proximity of the dendrimer. Additional vigorous testing using methods such as dynamic light scattering and fluorescent lifetime detection are currently being pursued to further explain the fluorescent phenomena. Time Response of the Assay. The speed of a reaction is paramount to its eventual clinical usage. A simple time titration was performed across the physiological range, and a monochromator was used to monitor the emission wavelength (680 nm). The results (Figure 7) show the response of the assay. It can be seen from the data set that 90% of the assay’s response occurs under 5 min for a large transition in glucose concentration (5.5 mM). This change is larger than what is expected in a physiological system where glucose changes typically occur within 30 min to 1 h following consumption.23-24 The speed of this reaction is however, slower than reported in popular literature for the traditional Con A/dextran system (∼1 s).6,15 This small lag in reaction time would suggest that the there is a potential for multiple binding between different assay components, which exaggerates the reaction time due to molecular hindrances and multiple unbinding processes. Specificity of the Assay. Specificity is one of the most important parameters when designing a glucose sensor, as it is important that the sensor not be confounded by other molecules. (22) Dendritech. (23) Kulcu, E.; Tamada, J. A.; Reach, G.; Potts, R. O.; Lesho, M. J. Diabetes Care 2003, 26, 2405-2409. (24) Heinemann, L.; Kramer, U.; Klotzer, H.; Hein, M.; Volz, D.; Hermann, M.; Heise, T.; Rave, K. Diabetes Technol. Ther. 2000, 2, 211-220.

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Figure 7. Time response of the assay shown with increasing glucose concentration by recording the emission collected from the AF647 dye at 680 nm. The time response is at 90% within 5 min for a 5.5 mM glucose change, which corresponds to 100 mg/dL.

In Figure 8, the results from the specificity experiments are shown as normalized peak intensity versus analyte concentration. Dilution affects were removed by using a simple ratio of total volume plus the volume of the aliquot divided by the total volume. Con A is a protein that has different binding affinities for various sugar molecules. Mannose, a six-carbon sugar not present in interstitial fluid, elicits a stronger binding affinity for the Con A protein than glucose. Titration of mannose to the assay solution results in a quick loss of the fluorescent quenching with less concentration of analyte as compared to the glucose response curve. Lactate is a byproduct of glucose metabolism and is usually at 20% the concentration of glucose.25 It can be seen that no quantifiable change is present with addition of lactate to the assay solution. The small changes seen in the peak amplitude are attributed to system noise. Following this experiment, glucose was added to the sample of the titrated lactate and the expected fluorescence (25) Fournier, R. L. Basic Transport Phenomena in Biomedical Engineering; Taylor and Francios: Lillington, NC, 1998.

Figure 8. Specificity of the sensor shown by exposing the assay to mannose, glucose, and lactate. Mannose, known to bind strongly to Con A, fully binds at lower concentrations than glucose. Lactate, known to not bind to Con A, did not elicit a quantifiable change in fluorescent emission.

recovery was seen. These results provide data that show the specificity of the newly created assay to glucose and suggest that this assay could be used as an interstitial fluid monitoring implant. This test provided insight into the assay response and most importantly ruled out nonspecific interactions between the Con A and glycodendrimer. This result also re-emphasizes the power of the Con A-dendrimer system over other approaches (scattering, boronic acid) by showing that the fluorescent response is not confounded by other analytes in the body.24,26 Reversibility of the Assay. To truly create a sensor for tracking dynamic concentrations of biological analytes, reversibility of the sensing chemistry is necessary. In previous research, the reversibility of the Con A-dextran system has been suspect and shown to have debatable response to glucose. With the introduction of the dendrimer, a spheroid molecule in place of the dextran, the potential for glucose binding without full displacement of the secondary molecule is eliminated and the dynamic range and reversibility should improve. We have seen that displacement of the secondary molecule (dendrimer) has a

profound effect on the fluorescence emission of the Con A dye. Reversiblity was tested by placing this assay into a dialysis cassette where glucose can not only be added to the system but also be removed. In Figure 9, the ratio between the two fluorescent emission dye peaks across days is shown. Each solution was given 24 h to diffuse into the membrane. Kinetic testing was not done because the speed of the reaction was attributed to the distance between the membranes within the cassette and their respective porosity and not the response time of the assay. What is shown in the figure is the average of triplicate spectra, taken from three locations on the cassette, with error bars indicating the range of values for each concentration for each day. From this graph it can be seen that the assay is reversible over long periods of time when left at room temperature. However, there is also a slight drop in dynamic range of the sensor as time progresses, which is due to deactivation of the protein over the course of the 10-day experiment as reported in the literature.14 By pairing up the consecutive days (0 and 55 mM), the loss of response can be tracked independent of system fluctuations and intensity loss. Figure 10 shows the overall response of the assay to glucose over the 10-day period to be dropping. This is expected due to the fragile nature of free Con A. This graph is also independent of intensity losses, which were evident due to protein inactivation and aggregation within the dialysis membrane, but due to the ratiometric design of the sensor it did not heavily influence the change in fluorescence seen from glucose exposure. Overall, the assay is reversible over the physiological range and maintains its large dynamic response to glucose, which has not been previously shown in the literature for the traditional free Con A/dextran system. CONCLUSION In this paper, an assay for eventual use in dermal glucose sensing using fluorescently labeled Con A with a fluorescently labeled glycodendrimer was described. It was established that the assay was capable of measuring glucose across the biological range with a dynamic range even broader than that seen in previous assays. By removing the label from the glycodendrimer, it was proven that the assay is not based upon a fluorescent

Figure 9. Reversibility of the sensor tested by placing the assay into a dialysis membrane cassette. For repeating 24-h periods, the cassette was placed into a solution of glucose or buffer and spectra were taken. The ratio between the two fluorescent emission peaks was taken in triplicate and recorded. This figure shows that the assay is reversible despite some protein denaturation due to the extended time of the experiment.

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Figure 10. Glucose response decay over the course of the reversibility experiment plotted as overall change over the course of 48-h periods corresponding to 0 and 55 mM glucose concentrations. It can be seen that over time the glucose response is dropping due to the inactivation of Con A in solution within the dialysis cassette. Temperature of study, 26 °C.

transfer or a heterogeneous reaction between two fluorophores, but rather on a single fluorophore quenching reaction. It was also shown that the sensor assay is reversible over the course of many days when placed within a dialysis membrane permeable to glucose. Finally, by using buffered lactate, a byproduct of glucose metabolism for which Con A does not have an affinity, and glucose, the molecule of interest in this study, it was shown that the sensor assay follows the affinity binding curve of Con A by responding well to titration of glucose and not responding to lactate. This new assay has the benefit of an expanded dynamic range since the assay is based upon a single fluorophore phenomenon (26) Badugu, R.; Lakowicz, J. R.; Geddes, C. D. Anal. Chem. 2004, 76, 610618. (27) Ballerstadt, R.; Polak, A.; Beuhler, A.; Frye, J. Biosens. Bioelectron. 2004, 19, 905-914. (28) Huet, C.; Lonchampt, M.; Huet, M.; Bernadac, A. Biochim. Biophys. Acta 1974, 365, 28-39.

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rather than a FRET-based phenomenon. It also contains fluorophores that emit well into the red and near-infrared wavelength range, which will result in improved transmission of fluorescent emission through dermal tissues. Also, since the assay is based upon a single fluorophore phenomenon, the glycodendrimer label is able to act as an internal reference capable of eliminating motion artifact and spurious changes in fluorescent emission that are not attributed to glucose. This paper thus provides advantages for this assay as a potential over previous FRET-based systems for implantation into dermal tissue. It should be noted that we saw the stability of the Con A protein at body (37 °C) temperature decrease over a 72-h period (data not shown) as has been described in many publications.14,27,28 However, in this study, the use of Con A is not the focus, but rather the advantages for the use of a glycosylated dendrimer as a replacement for competing ligands in FRET- and non-FRET-based affinity sensors. In addition, previous publications have shown dramatically increased stability of Con A in the presence of glucose (as would be present in the body).27 Current work is focused on improving the stability of the Con A protein as well as seeking more robust alternatives to Con A (e.g., APO-GoX). Techniques for encapsulation of this assay into a PEG hydrogel for use as an implantable sensor are also being developed. ACKNOWLEDGMENT The authors acknowledge the support of the National Aeronautics and Space Administration (Grant NNJ04HB04G). B.L.I. acknowledges the support of a Department of Education Graduate Assistance in Areas of National Need (GAANN) Fellowship. H.T.B. acknowledges the support of a National Science Foundation Graduate Research Fellowship.

Received for review May 6, 2005. Accepted August 20, 2005. AC0507901