Anal. Chem. 2004, 76, 292-297
Glucose Biosensor Based on the Microcantilever Jianhong Pei,† Fang Tian, and Thomas Thundat*
Life Science Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-6123
Diagnosis and management of diabetes require quantitative and selective detection of blood glucose levels. We report a technique for micromechanical detection of biologically relevant glucose concentrations by immobilization of glucose oxidase (GOx) onto a microcantilever surface. Microfabricated cantilevers have traditionally found utility in atomic force microscope imaging. During the past decade, however, microcantilevers have been increasingly used as transducers in chemical-sensing systems. This paper describes the combination of this technology with enzyme specificity to construct a highly selective glucose biosensor. The enzyme-functionalized microcantilever undergoes bending due to a change in surface stress induced by the reaction between glucose in solution and the GOx immobilized on the cantilever surface. Experiments were carried out under flow conditions. The common interferences for glucose detection in other detection schemes have been tested and have shown to have no effect on the measurement of blood glucose level by this technique. Diabetes is among the most prevalent and costly diseases in the world. Diabetes and its associated complications are leading causes of death and disability in the world. Approximately 17 million people in the United States, or 6.2% of the population, have diabetes. The diagnosis and management of diabetes require daily monitoring of blood glucose levels. Tight control of glucose levels in the blood is a very important way to delay the onset and dramatically slow the progression of complications from diabetes. The challenge of providing such tight and reliable glycemic control remains the subject of an enormous amount of research.1,2 An easy-to-use, inexpensive, and reliable method is therefore required for the measurement of glucose in a selective, sensitive, and quantitative manner for diabetes care. The development of an implantable glucose sensor for use in battling diabetes has been the focus of intense research.3-6 Most advanced glucose sensors are based on a device in which glucose * To whom correspondence should be addressed. Fax: +1(865) 574-6210. E-mail:
[email protected]. † Current address: Nova Biomedical Corp., 200 Prospect St., Waltham, MA 02454. (1) Wilson, G. S.; Hu, Y. Chem. Rev. 2000, 100, 2693-2704. (2) Wang, J. Electroanalysis 2001, 13, 983-988. (3) Henry, C. Anal. Chem. 1998, 70, 594A-598A. (4) Schmidtke, D. W.; Freeland, A. C.; Heller, A.; Bonnecaze, R. T. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 294-299. (5) Gough, D. A.; Armour, J. C. Diabetes 1995, 44, 1005-1009. (6) Bindra, D.; Zhang, Y.; Wilson, G.; Sternberg, R.; Trevenot, D.; Reach, G.; Moatti, D. Anal. Chem. 1991, 63, 1692-1696.
292 Analytical Chemistry, Vol. 76, No. 2, January 15, 2004
oxidase (GOx) is coupled to an electrochemical system.1-10 Quantitative analysis is based on the measurement of the consumption of oxygen or the production of hydrogen peroxide. These detection methods suffer a disadvantage because a number of potentially interfering species (such as ascorbic acid, catechol, uric acid, and acetaminophen) are electroactive at the applied potential required for peroxide oxidation. Therefore, improvement in selectivity during blood glucose measurement has attracted much attention.1,2 Recent advances in designing and fabricating microcantilever beams capable of detecting extremely small forces, mechanical stresses, and mass additions offer the promising prospects of chemical, physical, and biological sensing with unprecedented sensitivity and dynamic range.11,12 A microcantilever is the simplest microelectromechanical systems device that can easily be micromachined and mass-produced. This sensor transduction mechanism is particularly attractive because it can be integrated with on-chip electronic control circuitry.13 Microcantilever-based sensors can operate in two fundamentally different modes: (1) static mode, in which measurement of cantilever deflection is based on the stress changes induced by molecular interaction on the cantilever surface,14-16 and (2) dynamic mode, in which the cantilever undergoes excitation in its fundamental vibration mode and the change in resonance frequency upon mass loading is measured.2,13,17 The two methods impose completely different constraints on cantilever design for optimum sensitivity. Method 1 requires a long, soft cantilever to achieve large deflections and can be used in both gaseous18,19 and liquid environments.14-16,20-24 (7) Karyakin, A. A.; Kotel’nikova, E. A.; Lukachova, L. V.; Karyakina, E. E.; Wang, J. Anal. Chem. 2002, 74, 1597-1603. (8) Pei, J. H.; Li, X. Y. Electroanalysis 1999, 11, 1266-1272. (9) Ramanathan, K.; Jo ¨nsson, B. R.; Danielsson, B. Anal. Chim. Acta 2001, 427, 1-10. (10) Wang, J.; Zhang, X. J. Anal. Chem. 2001, 73, 844-847. (11) Thundat, T.; Warmack, R. J.; Chen; G. Y.; Allison, D. P. Appl. Phys. Lett. 1994, 64, 2894-2896. (12) Thundat, T.; Wachter, E. A.; Sharp, S. L.; Warmack, R. J. Appl. Phys. Lett. 1995, 66, 1695-1697. (13) Lange, D.; Hagleitner, C.; Hierlemann, A.; Brand, O.; Baltes, H. Anal. Chem. 2002, 74, 3084-3095. (14) Xu, X. H.; Thundat, T. G.; Brown, G. M.; Ji, H. Anal. Chem. 2002, 74, 36113615. (15) Stevenson, K. A.; Mehta, A.; Sachenko, P.; Hansen, K. M.; Thundat, T. Langmuir 2002, 18, 8732-8736. (16) Fritz, J.; Baller, M. K.; Lang, H. P.; Rothuizen, H.; Vettiger, P.; Meyer, E.; Gu ¨ ntherodt, H. J.; Gerber, Ch.; Gimzewski, J. K. Science 2000, 288, 316318. (17) Battiston, F. M.; Ramseyer, J.-P.; Lang, H. P.; Baller, M. K.; Gerber, Ch.; Gimzewski, J. K.; Meyer, E.; Gu ¨ ntherodt, H.-J. Sens. Actuators, B 2001, 77, 122-131. (18) Hu, Z.; Thundat, T.; Warmack, R. J. J. Appl. Phys. 2001, 90, 427-431. (19) Jensenius, H.; Thaysen, J.; Rasmussen, A. A.; Veje, L. H.; Hansen, O.; Boisen, A. Appl. Phys. Lett. 2001, 76, 2615-2617. 10.1021/ac035048k CCC: $27.50
© 2004 American Chemical Society Published on Web 12/03/2003
Method 2 requires a short, stiff cantilever to achieve high operational frequencies; unlike method 1, method 2 is rather difficult to apply in liquid media.13,17 In recent years, the unique ability of biomolecules to recognize other molecules has been investigated in the development of microcantilever-based biosensors. Microcantilever-based biosensors can offer many advantages over other biosensor designs; for example, the microcantilevers can easily be fabricated into multiple-element arrays, and the sensors do not require the use of external probes or labeling. General applications of this label-free detection method have been shown for DNA hybridization, the detection of single-base mismatches,16,20-22 nanomechanical motion induced by antibodyantigen recognition,16,23 and the detection of the prostate-specificantigen cancer marker.24 Here we report the study of a glucose biosensor based on the microcantilever platform operated in static mode. The microcantilever deflection response mechanism is discussed. Glucose detection was achieved by immobilizing a layer of GOx on the surface of a microcantilever and then detecting the mechanical bending induced by the enzyme reaction that took place on the microcantilever surface in the presence of glucose. The bending was sensed by reflecting a laser beam from the cantilever surface to a position-sensitive detector. Movement of the microcantilever beam is a function of physiologically relevant concentrations of glucose. A major advantage of such a direct transduction is the high selectivity due to the high specificity of GOx. EXPERIMENTAL SECTION Reagents and Procedures. Glucose oxidase (EC 1.1.3.4, Type VII-S, purified from Aspergillus niger, 245 900 units/g), bovine serum albumin (BSA), and glutaraldehyde (GA) (50% aqueous solution) were purchased from Sigma Chemical Co. β-D-Glucose, ascorbic acid, 3-hydroxytyramine hydrochloride, uric acid, 4-acetamidophenol, catechol, D-fructose, and D-mannose were used as received from Aldrich. A phosphate buffer (PB, 100 mM, pH 7) solution was prepared by dissolving 8.5 g of NaH2PO4 and 11.0 g of Na3PO4 in 1000 mL of water; dilute HCl and NaOH were used to adjust the pH to 7. Other chemicals employed were all of analytical grade. High-purity deionized water was obtained with a NanoPure water system (g18 MΩ) (Barnstead). For all of the experiments, standard rectangular silicon tipless cantilevers from MikroMasch were used. The dimensions of the cantilever are 350µm length, 35-µm width, and 1-µm thickness with a force constant of 0.03 N/m. On one side of the microcantilever, a 2.5-nm adhesion layer of chromium and then a 25-nm layer of Au was deposited by using an e-beam evaporator (Thermionics, Port Townsend, WA). Before film deposition, the microcantilevers were carefully cleaned for 10 min in acetone and for 10 min in absolute ethanol and were then immersed in piranha solution (three parts 30% (20) Hansen, K. M.; Ji, H.-F.; Wu, G.; Datar, R.; Cote, R.; Majumdar, A.; Thundat, T. Anal. Chem. 2001, 73, 1567-1571. (21) Wu, G.; Ji, H.; Hansen, K.; Thundat, T.; Datar, R.; Cote, R.; Hagan, M. F.; Chakraborty, A. K.; Majumdar, A. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 1560-1564. (22) McKendry, R.; Zhang, J.; Arntz, Y.; Strunz, T.; Hegner, M.; Lang, H. P.; Baller, M. K.; Certa, U.; Meyer, E.; Gu ¨ ntherodt, H.-J.; Gerber, C. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 9783-9788. (23) Grogan, C.; Raiteri, R.; O’Connor, G. M.; Glynn, T. J.; Cunningham, V.; Kane, M.; Charlton, M.; Leech, D. Biosens. Bioelectron. 2002, 17, 201-207. (24) Wu, G.; Datar, R. H.; Hansen, K. M.; Thundat, T.; Cote, R. J.; Majumdar, A. Nat. Biotechnol. 2001, 19, 856-860.
hydrogen peroxide, seven parts concentrated sulfuric acid) for 30 s. (Caution: Piranha solution reacts violently with many organic materials and is highly corrosive. It should be handled with care.) They were rinsed with water and ethanol and then dried in an oven at 80 °C for more than 1 h. The cleaned cantilevers were stored in vacuum desiccators and ready for the deposition. Immobilization of GOx on the Surface of the Cantilever. Before the GOx enzyme layer was immobilized onto the Au layer on the cantilever surface, the cantilevers were rinsed for ∼5 min each with acetone and ethanol. Then they were equilibrated with the PB solution (pH 7) and dried by purging with nitrogen gas. The GOx was immobilized onto the silicon cantilever surface by being cross-linked with GA in the presence of BSA. The enzyme stock solution was prepared by dissolving a defined amount of GOx (typically 20 mg/mL; other solution concentrations were also tested), 5 mg of BSA, and 40 µL of 50% GA in sequence in 1 mL of the PB solution (pH 7). A microsyringe was used to withdraw 5 µL of the enzyme solution and to dispense it onto the cantilever surface. The cantilever was stored in a refrigerator and dried at 4 °C overnight. The cantilever was rinsed with PB solution thoroughly before it was used for the measurements. Deflection Measurements. Optical deflection measurements were carried out with an atomic force microscope head with an integrated laser and a position-sensitive detector (Digital Instruments, Santa Barbara, CA). The experiments were performed in a flow-through glass cell in which the microcantilevers were immersed in the solution. Details of the experimental setup are similar to those described elsewhere.14,15 The liquid flow rate was controlled by a syringe pump (IITC, Inc, Woodland Hill, CA) equipped with a low-pressure liquid chromatography injector valve and a 1-mL injection loop (Upchurch Scientific, Oak Harbor, WA). The flow cell, which has a volume of ∼200 µL, was connected to the syringe valve via plastic tubing. The small volume ensured a fast replacement of solution. Once the cantilever was mounted and the laser spot was positioned onto the cantilever, continuous washing or injection was performed through the flow system. Cantilever deflection was monitored throughout each experiment by an HP Data Logger (model 34970A). Before glucose was injected into the flow cell, the cantilever was placed in the flow cell in the same PB solution (pH 7), and the syringe was set up to deliver the buffer at a flow rate of 2 mL/h. The slow drift in the cantilever’s output reached a steady state ∼2 h after the start of the experiments. After a stable baseline was obtained, 1 mL of various concentrations of glucose or an interfering agent in the PB solution was injected at the same flow rate. After 30 min, the syringe pump resumed delivering the buffer solution as a washing solution. A schematic of the above static mode microcantilever device setup is shown in Figure 1. RESULTS AND DISCUSSION Deflection Response of Glucose on the Microcantilever. A GOx-immobilized microcantilever was prepared by dropping 5 µL of the enzyme solution (20 mg/mL GOx, 5 mg/mL BSA, and 2% GA) onto a gold-coated microcantilever. After being stored overnight in a refrigerator at 4 °C, the enzyme-functionalized microcantilever was initially used to study its deflection response to the injection of a 5 mM concentration of glucose. The cantilever was equilibrated with the PB solution in the flow cell system until a stable baseline was obtained. Then, 5 mM β-D-glucose in the Analytical Chemistry, Vol. 76, No. 2, January 15, 2004
293
Figure 1. Schematic diagram of static mode microcantilever device setup. Fluid flow was controlled by a syringe pump connected to an in-line injection valve and 1-mL sample loop. Figure 3. Effect of GOx concentrations in the enzyme solution on the cantilever deflection response of 5 mM glucose. Flow rate, 2 mL/ h. The enzyme solution contains 5 mg/mL BSA, 2% GA, and various concentrations of GOx.
Figure 2. Comparison of the cantilever bending response of 5 mM glucose in a PB solution as function of time for (a) a GOxfunctionalized microcantilever and (b) a blank microcantilever. Carrier solution, PB solution (pH 7); flow rate, 2 mL/h.
same PB solution was injected into the flow cell by syringe pump at a flow rate of 2 mL/h and the deflection versus time profile was recorded (Figure 2a). A clear change in the deflection of the cantilever was observed as the glucose sample solution passed over it. The cantilever continuously bent toward the Au side while glucose solution flowed through the cell. When the glucose sample solution was replaced by the PB washing solution, the deflection of cantilever arrived at a plateau. A delay in the response time during injection and washing corresponds to the time needed for solution in the sample loop to pass through the tubing and fill the flow cell. To check whether the deflection response was caused by the specific enzyme reaction, the same measurement procedure was carried out on a blank Au/Si cantilever (Figure 2b). When the 5 mM glucose in PB solution passed over the cantilever, the cantilever bent slightly toward the silicon side. The cantilever maintained its deflected position when the glucose solution fully filled and stayed in the flow cell. When the washing process began, the cantilever returned to its original position. It displayed response properties totally different from those obtained in the glucose solution (Figure 2a). The slight downward deflection at 30 min was thought to be due to a change in the refractive index because the buffer solution contains a 5 mM concentration of glucose. To confirm this assumption, more control experiments 294 Analytical Chemistry, Vol. 76, No. 2, January 15, 2004
were carried out, in which the laser was focused on the chip body instead of on the cantilever and the same glucose concentration was injected into the flow cell. Deflection response curves similar to those shown in Figure 2b were obtained whether the cantilever was functionalized by GOx or not. A slight apparent deflection (toward silicon side) due to a change in the refractive index was observed. Another control experiment was carried out on a cantilever prepared by using a 5-µL solution containing only 5 mg/mL BSA and 2% GA without GOx. The laser was directed at the cantilever, and the same concentration of glucose was injected into the flow cell. A response profile similar to that in Figure 2b was obtained (data not shown). All of these results confirm that the deflection response in Figure 2a is due to the reaction of solution glucose with GOx immobilized on the cantilever surface. The enzyme reaction that takes place on the cantilever surface causes the cantilever to bend. These results were further confirmed by the observation that the apparent change in deflection caused by the change in refractive index happened on the cantilever whether it was functionalized by the enzyme or not and also occurred when the laser was focused on either the cantilever or the chip body. The refractive index change only related to the composition of the solution in the flow cell. Effect of GOx Concentration on Cantilever Response. The relationship between the cantilever deflection responses to 5 mM glucose and the concentrations of GOx in the enzyme solution used for the preparation of the cantilevers was tested for concentrations ranging from 0 to 25 mg/mL. The deflection response is greatly affected by the concentration of GOx in the enzyme solution; deflection response increases with the GOx concentration (Figure 3). It is easy to understand that a greater number of enzyme molecules on the cantilever surface will result in higher activity of the sensor to glucose. In all of our subsequent measurements, a 20 mg/mL solution of GOx was used in the enzyme solution; it showed very high response sensitivity. This result also confirms that the cantilever deflection is really closely related to the surface
Figure 4. Curve a: cantilever deflection as a function of time for the injection of various concentrations of glucose on a GOxfunctionalized cantilever. Glucose concentrations: (1) 0, (2) 0.2, (3) 0.5, (4) 1, (5) 2, (6) 5, (7) 10, and (8) 20 mM. Curve b: the calibration plot. Flow rate, 2 mL/h. The carrier solution is a phosphate buffer solution (pH 7).
concentration of the GOx enzyme and to the enzyme reaction taking place on the cantilever. Cantilever Deflection Response Properties. Normally, human blood glucose levels stay within narrow limits throughout day (4-8 mM). They are usually highest after meals and usually lowest in the morning. The blood glucose level of people with diabetes will move outside these limits. To monitor blood glucose level effectively, a glucose sensor must reliably measure the glucose concentration at a relevant range for diabetics (1-20 mM). The GOx-functionalized cantilever deflections were tested by recording profiles of the deflection as function of time with the injection of different concentrations of glucose in a PB solution at a flow rate of 2 mL/h. A typical set of deflection-time response profiles for individual cantilevers exposed to single concentrations of glucose in a PB solution is shown in Figure 4a. Curve 1 in Figure 4a, which shows the deflection profile after the injection of PB alone, represents the control experiments. The injection of PB alone produces no change in deflection. From curve 2 to curve 8 in Figure 4a, the cantilever deflection increases with concentrations of glucose within the range studied. The calibration plot is shown in Figure 4b (inset). The plotted values of deflection signals shown are the maximal deflections observed after passage of the sample. The GOx-functionalized cantilever specifically deflected when exposed to glucose, and the magnitude of the deflection was dependent on concentration over a large concentration range. To characterize the reproducibility of the GOx-functionalized cantilever, continuous measurements of repetitive injections of 5 mM glucose were carried out with a single cantilever (see Figure 5). Between each injection, the PB solution was used to fully replace the solution in the flow cell. The deflection responses of a single cantilever decreased with the repeated injections. Hydrogen peroxide is produced by the enzyme-substrate reaction, and the decrease in cantilever response may be due to the corrosive effect of H2O2 on the enzyme layer. Another possibility is that enzyme activity decreases over time with continuous exposure to H2O2. The effect of long-term storage on the stability of the functionalized cantilever was also tested (data not shown). The
Figure 5. Reproducibility of a single GOx-functionalized cantilever response to sequential injections of 5 mM glucose. (Other experimental conditions were the same those for the results in Figure 2.)
Figure 6. Specificity experiments of the GOx-functionalized cantilever. The cantilever deflection-time profiles recorded from the injection of (1) 5 mM D-glucose, (2) 5 mM D-fructose, and (3) 5 mM D-mannose. (Other experimental conditions were the same as those for results in Figure 2.)
experimental results show that the cantilever could retain ∼70% of its original response after three weeks stored in refrigerator at 4 °C. Longer times have not been tested. Specificity and Interferences. The specificity and the selectivity of the cantilever sensor depend on the enzyme chosen. GOx possesses a very high specificity toward glucose (5 × 104 greater activity) compared with its specificity for any other sugars (e.g., D-fructose or D-mannose).25 The specificity of the enzyme-functionalized cantilever sensor was measured, and the results are shown in Figure 6. The cantilever displays a sensitive response to the injection of glucose (curve 1 in Figure 6). The deflection of cantilever continuously bends up while the glucose passes through the cantilever until the washing step begins, and then the deflection remains constant. Conversely, injections of D-fructose and D-mannose elicit completely different responses (curves 2 and 3 in Figure 6, respectively). When D-fructose and D-mannose pass through the flow cell, the cantilever appears to bend slightly (25) Cass, A. E. G. Biosensors: A Practical Approach; Oxford University Press: Oxford, U.K., 1990; p.15.
Analytical Chemistry, Vol. 76, No. 2, January 15, 2004
295
Figure 7. Selectivity experiments of the GOx-functionalized cantilever. The deflection-time profiles recorded for the injection of (1) 5 mM glucose, (2) 5 mM 4-acetaminophenol, (3) 5 mM catechol, (4) 5 mM 3-hydroxyramine, and (5) 5 mM ascorbic acid. (Other experimental conditions were the same as those for results in Figure 2.)
downward because the refractive index changes. The sugar solutions produce a flat response curve when they fill the flow cell. After the washing process begins, the effect of the sugars on the refractive index of the solution is reversed. This behavior indicates a highly specific reaction of GOx with glucose at the surface of cantilever. It also confirms that the deflection responses on the cantilever originate from the enzyme reaction on the cantilever surface between glucose oxidase and glucose. Due to the specificity of the enzyme, the enzyme-functionalized cantilever displays a high selectivity. We can call this device an “interference-free” glucose biosensor. Given the current state of the art, one can expect that it will have great potential for use in blood monitoring for diabetics. For example, amperometric enzyme electrodes, based on GOx bound to electrode transducers, currently play a leading role in monitoring blood glucose. The amperometric measurement of hydrogen peroxide requires application of a potential at which coexisting species, such as ascorbic acid, 4-acetaminophen, catechol, and 3-hydroxyramine, are also electroactive.1,2 The contributions of these and other oxidizable species present in biological fluids can compromise the selectivity and hence the overall accuracy of amperometric monitoring devices. Therefore, extensive efforts were needed to minimize the error of electroactive interferences using amperometric measurements.26,27 In general, the bending of a cantilever can be attributed to the surface stress change induced by the molecular interaction between the cantilever surface and analytes in the solution.1,2,28 No external physical parameters, such as potential or current, were applied during the measurements, so the selectivity only depends on the enzyme reaction. It provides a potential way to detect glucose in a highly selective manner. Other coexisting species in the blood sample were also checked using the enzyme-functionalized cantilever sensor; the results are shown in Figure 7. The effects of species such as ascorbic acid, 4-acetaminophen, catechol, and 3-hydroxy(26) Zhang, Y.; Hu, Y.; Wilson, G. S.; Moatti-Sirat, D.; Poitout, V.; Reach, G. Anal. Chem. 1994, 66, 1183-1188. (27) Emr, S.; Yacynych, A. M. Electroanalysis 1995, 7, 913-923. (28) Datskos, P. G.; Thundat, T. J. Nanosci. Nanotechnol. 2002, 2, 369-373.
296
Analytical Chemistry, Vol. 76, No. 2, January 15, 2004
ramine on the deflection of a cantilever at a concentration of 5 mM were studied, and deflection-time profiles were recorded separately (curves 2-5 in Figure 7). The deflection response of 5 mM glucose is shown in curve 1 in Figure 7 for comparison. At this concentration, all of these species appear to cause the cantilever to slightly bend toward the untreated side and to show a flat response. When the washing step begins, the refractive index changes and the cantilever appears to bend back to its initial level. All of these results suggest that this enzyme cantilever sensor could be a highly selective glucose sensor. This single-cantilever approach has inherent problems, namely, the lack of a reference cantilever. Multiple-cantilever devices are under development in our laboratory and incorporate at least one reference cantilever, which could avoid some problems of temperature and flow vibration. Preliminary experimental results obtained with a two-cantilever device (data not shown) were identical to the single-cantilever experimental results presented in this paper. Response Mechanism. Because the microcantilever is an extremely sensitive temperature sensor, it was initially assumed that cantilever deflection was the result of an increase in temperature caused by the heat released from the enzyme reaction.29 If this assumption were true, the cantilever would bend toward the untreated silicon side because the expansion coefficient of Au is much larger than that of silicon. A diagnostic test in which a cantilever was heated showed that the cantilever bends down toward the silicon side when heated. Because experimental results show that the cantilever bends toward the Au side when glucose passed through the enzyme-functionalized cantilever, it can be reasonably concluded that the deflection response is not due to the heat released by the chemical reaction. An alternative possible explanation is that the measured deflection response of the cantilever is due to surface forces other than heat. Changes in the local chemical environment that result from the glucose conversion to gluconic acid and hydrogen peroxide may induce the surface stress change. The H2O2 produced from the enzyme reaction is highly corrosive; it can break some chemical bonds in the enzyme layer and can remove some of the material deposited on the cantilever surface, thus changing the surface stresses. That is why the reproducibility of results for a cantilever sensor is not good; its response decreases with the repeated injections of same concentration of glucose solution. Details of the response mechanism are still under study. Regardless of the signal transduction mechanism, coating the cantilever with an enzyme can lead to highly specific chemical detection that can be exploited for biological applications. CONCLUSIONS Our study has shown that a glucose biosensor has been developed based on microcantilever technology. This sensor was found to be highly selective and specifically responsive to glucose over a wide range of concentrations. These results clearly show the usefulness of this platform for biosensing without the added complications of sample labeling. The mechanism of deflection response appears to be the result of a change in surface stress instead of a thermal effect. This work demonstrated the combination of microcantilever sensor technology with an enzyme-specific (29) Subramanian, A.; Oden, P. I.; Kennel, S. J.; Jacobson, K. B.; Warmack, R. J.; Thundat, T.; Doktycz, M. J. Appl. Phys. Lett. 2002, 81, 385-387.
reaction to construct a glucose biosensor. This can be regarded as a model for this combination of two techniques. The same procedure can be applied with other enzymes. Experiments are presently under way to develop enzyme-functionalized microcantilever sensors for other analytes. Moreover, arrays of microcantilevers may be developed to perform multiple assays by immobilizing a different enzyme on each cantilever. The potential advantages of a label-free assay that can measure multiple analytes in a single step without addition of other reagents are enormous and could ultimately translate to much lower cost per test.
ACKNOWLEDGMENT This research work was supported by the Office of Biological and Environmental Research (OBER), U.S. Department of Energy under contract DE-AC05-00OR22725 with Oak Ridge National Laboratory managed by UTsBattelle. Helpful discussions were provided by Drs. K. Stevenson, K.M. Hansen, and S. Cherian. Received for review September 8, 2003. Accepted October 28, 2003. AC035048K
Analytical Chemistry, Vol. 76, No. 2, January 15, 2004
297
