Multianalyte Pin-Printed Biosensor Arrays Based

ments are on the order of 100 µm in diameter. Arrays are formed (1) ... the first time the combination sol-gel processing and pin ... particular anti...
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Anal. Chem. 2002, 74, 6177-6184

Multianalyte Pin-Printed Biosensor Arrays Based on Protein-Doped Xerogels Eun Jeong Cho, Zunyu Tao, Elizabeth C. Tehan, and Frank V. Bright*

Department of Chemistry, Natural Sciences Complex, University at Buffalo, The State University of New York, Buffalo, New York 14260-3000

We report the first biosensor arrays based on pin printing protein-doped xerogels. The individual biosensor elements are on the order of 100 µm in diameter. Arrays are formed (1) onto a planar substrate that is excited by an external source (laser) or (2) directly on the face of a light-emitting diode. We illustrate the potential of our approach by fabricating, testing, and characterizing four types of pin-printed biosensor arrays (PPBSA) for the simultaneous detection of glucose and O2. The analytically reliable operating ranges for the PPBSAs are 0.1-10 mM for glucose and 0.1-100% for O2. The PPBSAs exhibit short- and long-term reproducibilities of no worse than 4 and 8%, respectively. The overall array-to-array response reproducibilities are e12%. These results demonstrate for the first time the combination sol-gel processing and pin printing methods as a way to rapidly form ensembles of integrated, reusable, and stable biosensor arrays for simultaneous multianalyte detection.

INTRODUCTION Biosensors exploit immobilized biomolecules to selectively recognize a target analyte, and the binding/catalytic event leads to an electrochemical, mass, optical, or thermal signal that can be related to the analyte concentration in the sample.1 Biosensors have traditionally been designed to detect one analyte at a time in a sample; however, there are significant advantages associated with the parallel detection of several analytes in a sample. More recently, several research teams have developed biosensor arrays2-6 that can be used for the simultaneous detection of multiple analytes in a single sample. Representative examples of * Corresponding author: (phone) 716-645-6800 ext 2162; 716-645-6963; (e-mail) [email protected]. (1) (a) Biosensors with Fiberoptics; Wise, D. L., Wingard, L. B. Jr, Eds.; Humana Press: Clifton, NJ, 1991. (b) Commercial Biosensors: Applications to Clinical, Bioprocess, and Environmental Samples; Ramsay, G., Ed.; John Wiley & Sons: New York, 1998. (c) Biosensors and Chemical Sensor Technology: Process Monitoring and Control, Rogers, K. R., Mulchandani, A., Zhou, W., Eds.; American Chemical Society: Washington, DC, 1995; Vol. 613. (d) Wolfbeis, O. S. Anal. Chem. 2002, 74, 2663-2678. (2) Liu, X.; Farmerier, W.; Schuster, S.; Tan, W. Anal. Biochem. 2000, 283, 56-63. (3) Revzin, A. F.; Sirkar, K.; Simonian, A.; Pishko, M. V. Sens. Actuators, B: Chem. 2002, B81, 359-368. (4) Moser, I.; Jobst, G.; Urban, G. A. Biosens. Bioelectron. 2002, 17, 297-302. (5) Biran, I.; Walt, D. R. Anal. Chem. 2002, 74, 3046-3054. (6) Christodoulides, N.; Tran, M.; Floriano, P. N.; Rodriguez, M.; Goodey, A.; Ali, M.; Neikirk, D.; McDevitt, J. T. Anal. Chem. 2002, 74, 3030-3036. 10.1021/ac020454+ CCC: $22.00 Published on Web 11/12/2002

© 2002 American Chemical Society

biosensor array strategies include the Tan group’s molecular beacon-based DNA biosensors,2 the electrostatically complexed monolayers deposited on photolithographically patterned gold microelectrodes described by Revzin et al.,3 the photopatternable enzyme membranes reported by Moser et al.,4 the Walt group’s high-density fiber-optic arrays that are based on intact bacteria and yeast cells,5 and the McDevitt laboratories’ efforts to develop “electronic taste chips” based on immunochemistry.6 An ideal biosensor array should be entirely self-contained, rapid and simple to fabricate, and stable to avoid calibration and drift difficulties. Over the past several years, we7 and others8 have been exploring the potential of sol-gel-processed xerogels as platforms for chemical sensors and biosensors. The literature is ripe with examples7,8 of protein-doped xerogels in which (1) the biomolecule’s kcat and Km or Kbinding within the xerogels does not change substantially from the values in solution and (2) the xerogel-doped biomolecules remain stable for relatively long periods of time. For example, work from our laboratory7m showed that polyclonal antidansyl antibodies were stable for several months and they exhibited a high binding affinity (∼108 M-1) when they were sequestered within a xerogel even when stored under ambient conditions. In an aqueous buffer at room temperature, this particular antibody lost all its affinity in ∼10 h. (7) (a) Wang, R.; Narang, U.; Prasad, P. N.; Bright, F. V. Anal. Chem. 1993, 65, 2671-2675. (b) Narang, U.; Jordan, J. D.; Prasad, P. N.; Bright, F. V. J. Phys. Chem. 1994, 98, 8101-8107. (c) Narang, U.; Prasad, P. N.; Bright, F. V.; Ramanathan, K.; Kumar, N. D.; Malhotra, B. D.; Kamalasanan, M. N.; Chandra, S. Anal. Chem. 1994, 66, 3139-3144. (d) Narang, U.; Prasad, P. N.; Bright, F. V.; Ramanathan, K.; Kumar, N. D.; Malhotra, B. D.; Kamalasanan, M. N.; Chandra, S. Chem. Mater. 1994, 6, 1596-1598. (e) Narang, U.; Rahman, M. H.; Wang, J. H.; Prasad, P. N.; Bright, F. V. Anal. Chem. 1995, 67, 1935-1939. (f) Jordan, J. D.; Dunbar, R. A.; Bright, F. V. Anal. Chem. 1995, 67, 2436-2443. (g) Dunbar, R. A.; Jordan, J. D.; Bright, F. V. Anal. Chem. 1996, 68, 604-610. (h) Jordan, J. D.; Dunbar, R. A.; Bright, F. V. Anal. Chim. Acta 1996, 332, 83-91. (i) Baker, G. A.; Jordan, J. D.; Bright, F. V. J. Sol-Gel Sci. Technol. 1998, 11, 43-54. (j) Jordan, J. D.; Dunbar, R. A.; Hook, D. J.; Zhuang, H.; Gardella, J. A., Jr.; Colo´n, L. A.; Bright, F. V. Chem. Mater. 1998, 10, 1041-1051. (k) Hartnett, A. M.; Ingersoll, C. M.; Baker, G. A.; Bright, F. V. Anal. Chem. 1999, 71, 12151224. (l) Pandey, S.; Baker, G. A.; Kane, M. A.; Bonzagni, N. J.; Bright, F. V. Chem. Mater. 2000, 12, 3547-3551. (m) Doody, M. A.; Baker, G. A.; Pandey, S.; Bright, F. V. Chem. Mater. 2000 12, 1142-1147. (8) (a) Dave, B. C.; Dunn, B.; Valentine, J. S.; Zink, J. I. Anal. Chem. 1994, 66, 1120A-1127A. (b) Avnir, D.; Braun, S.; Lev, O.; Ottolenghi, M. Chem. Mater. 1994, 6, 1605-1614. (c) Avnir, D. Acc. Chem. Res. 1995, 28, 328-341. (d) Ingersoll, C. M.; Bright, F. V. CHEMTECH 1997, 27, 26-35. (e) Chen, Q.; Kenausis, G. L.; Heller, A. J. Am. Chem. Soc. 1998, 120, 4582-4585. (f) Brennan, J. D. Appl. Spectrosc. 1999, 53, 106A-121A. (g) Bradshaw, J. T.; Mendes, S. B.; Saavedra, S. S. Anal. Chem. 2002, 74, 1751-1759. (h) Jin, W.; Brennan, J. D. Anal. Chim. Acta 2002, 461, 1-36.

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Over the past two years, we have been developing chemical sensor array platforms based on sol-gel-processed xerogels. To date we have developed the following: (1) an optical sensor array and integrated light source (OSAILS) where the chemical sensor elements are formed directly within wells that were machined into the face of a light-emitting diode (LED),9 (2) a pin-printed chemical sensor array (PPCSA)10 where we contact print the sensor array onto a planar substrate and excite the array with and external light source, and (3) a pin-printed optical sensor array and integrated light source (PPOSAILS)11 where the sensor array is formed by pin printing onto the face of an LED. These pin printing strategies combine the attraction of xerogels, and they offer a simple method to rapidly fabricate reusable, integrated chemical biosensor arrays ( D-gluconolactone

D-gluconolactone

+ H2O2 (1)

+ H2O ))))> D-gluconic acid (2)

Luminophore quenching by O2 is governed by many factors.14 In the simplest scenario, the Stern-Volmer expression suffices to describe the quenching:

I0/I ) 1 + KSV[O2]

(3)

where I0 and I represent the steady-state luminescence intensities in the absence and presence of O2, respectively, [O2] is the O2 concentration in the sample, and KSV is the dynamic Stern-Volmer quenching constant. (KSV depends on the luminophore excitedstate luminescence lifetime in the absence of quencher, τ0, and the bimolecular quenching rate, kq, that describes the diffusional encounters between the luminophore and quencher; KSV ) τ0kq.) (9) Cho, E. J.; Bright, F. V. Anal. Chem. 2001, 73, 3289-3293. (10) Cho, E. J.; Bright, F. V. Anal. Chem. 2002, 74, 1462-1466. (11) Cho, E. J.; Bright, F. V. Anal. Chim. Acta 2002, 470, 101-110.

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In our sensors, luminophore quenching by O2 is described by the Demas model,15 a common occurrence for many luminophoredoped xerogels:16,17

Io/I ) [(f1/(1 + KSV,1[O2])) + (f2/(1 + KSV,2[O2]))]-1

(4)

Here, fi represents the fractional contribution of the total emission from luminophores located at site type i within the xerogel that exhibits Stern-Volmer quenching constant KSV,i. EXPERIMENTAL SECTION Reagents and Materials. The following reagents were used: tris(4,7′-diphenyl-1,10′-phenanathroline)ruthenium(II) chloride pentahydrate ([Ru(dpp)3]Cl2‚5H2O) (GFS Chemicals); tetraethylorthosilane (TEOS), and tetramethoxysilane (TMOS) (United Chemical Technologies); n-propyltrimethoxysilane (Pro-TriMOS) (Hu¨ls America); HCl (Fisher Scientific); EtOH (Quantum Chemical); glucose oxidase type VII-S (EC 1.1.3.4 from Aspergillus niger, 100-200 units mg-1), β-D-glucose, poly(ethylene glycol) (PEG 400), D-sorbitol, phosphate-buffered saline (PBS, pH 7.4), Trizma hydrochloride, and Trizma base (Sigma). Pluronic P104 (polyoxypropylene-polyoxyethylene block copolymer, MW 5900) was a generous gift from BASF. [Ru(dpp)3]2+ was purified as described in the literature.18 All other reagents were used as received. All aqueous solutions were prepared with deionized water that had been treated with a Barnstead NANOpure II system to a specific resistivity of 18.3 MΩ‚cm. Standard glass microscope slides were purchased from Fisher Scientific Co.. LEDs were from Nichia America (Part No. NSPB520S). Navy blue spray paint (Gloss, No. 1922) was a RustOleum product. All glucose working solutions were prepared by dilution from a 0.1 M glucose stock solution (PBS) that had been stored for at least 24 h at 4 °C. Solutions of varying O2 tension were prepared (12) (a) Ziauddin, J.; Sabatini, D. M. Nature 2001, 411, 107-110. (b) Rose, D. In Microarray Biochip Technology; Schena, M., Ed.; Eaton Publishing: Natick, MA, 2000; pp 19-38. (c) Cheung, V. G.; Morley, M.; Aguilar, F.; Massimi, A.; Kucherlapati, R.; Childs, G. Nat. Genet. Suppl. 1999, 21, 15-19. (d) Lemieux, B.; Aharoni, A.; Schena, M. Mol. Breeding 1998, 4, 277-289. (e) Marshall, A.; Hodgson, J. Nat. Biotechnol. 1998, 16, 27-31. (f) Shalon, D.; Smith, S.; Brown, P. O. Genome Res. 1996, 6, 639-645. (g) Schena, M.; Shalon, D.; Heller, R.; Chai, A.; Brown, P. O.; Davis, R. W. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 10614-10619. (h) Schena, M.; Shalon, D.; Davis, R. W.; Brown, P. O. Science 1995, 270, 467-470. (13) (a) Michael, A. C.; Justice, J., Jr. Anal. Chem. 1987, 59, 405-410. (b) Dermal, B. A. A.; Il, S.-Y.; Schmid, R. D. Biosens. Bioelectron. 1992, 7, 133139. (c) Bindra, D. S.; Zhang, Y. N.; Wilson, G. S.; Sternberg, R.; Thevenot, D. R.; Moatti, D.; Reach, G. Anal. Chem. 1991, 63, 1692-1696. (d) Amine, A.; Patriarche, G. J.; Marrazza, G.; Masvini, M. Anal. Chim. Acta 1991, 242, 91-98. (e) Rishpon, J.; Shabtai, Y.; Rosen, I.; Zibenberg, Y.; Tor, R.; Freeman, A. Biotechnol. Bioeng. 1990, 35, 103-107. (14) Eftink, M. R. In Topics in Fluorescence Spectroscopy; Lakowicz, J. R., Ed.; Plenum: New York, 1991; Vol. 2, Chapter 2. (15) Demas, J. N.; DeGraff, B. A.; Xu, W. Anal. Chem. 1995, 67, 1377-1380. (16) McDonagh, C. M.; MacCraith, B. D.; McEvoy, A. K. Anal. Chem. 1998, 70, 45-50. (17) Murtagh, M. T.; Shahriari, M. R.; Krihak, M. Chem. Mater. 1998, 10, 38623867. (18) Lin, C.-T.; Bo ¨ttcher, W.; Chou, M.; Creutz, C.; Sutin, N. J. Am. Chem. Soc. 1976, 98, 6536-6544.

Figure 1. Outline of the four PPBSA fabrication schemes. (A) Fabrication of PPBSAs onto glass substrates. (B) Fabrication of PPBSAs onto LEDs. Abbreviations: PP, pin printing O2-sensing elements; SC, spin casting glucose-sensing elements; OP, overprinting glucose-sensing elements; PPCSA, pin-printed chemical sensor array; and PPOSAILS, pin-printed optical sensor array and integrated light source.

by bubbling the appropriate gas mixture into PBS for at least 10 min. Instrumentation. The pin printer and sensor array characterization system have be described in detail elsewhere.9,10 Stock Sol-Gel Processing Solutions. Stock solution A was prepared by physically mixing 0.5 mL of Pro-TriMOS (2.84 mmol), 0.5 mL of TMOS (3.40 mmol), 1.2 mL of EtOH (20.6 mmol), and 0.4 mL of 0.1 N HCl (40 µmol).19 This mixture was hydrolyzed for 1 h with stirring under ambient conditions. Solution B was prepared by physically mixing 2.25 mL of TEOS (10.1 mmol), 0.7 mL of water (38.9 mmol), and 50 µL of 0.1 N HCl (5 µmol). This mixture was then sonicated (VWR Scientific Products, model 75HT) under ambient conditions until the solution became clear (∼1 h). Solution C was prepared by adding 0.50 g of a P104 solution (13.6% (w/v) dissolved in deionized water) to 1.00 g of solution B followed by stirring under ambient conditions for 30 min. PEG, sorbital, and P104 are used to help produce crack-free, GOx-doped xerogels with active enzyme. We also had to contend with the issue of buffering8 the enzyme within the sol-gel processing solution and simultaneously avoiding gelling within the pin printer’s quill pins. In our hands, traditional sol-gel processing solutions8 were universally inadequate for pin printing. A wide variety of xerogel formulations and compositions were tested and screened20 to yield a combination of adequate working (19) Wolfbeis, O. S.; Oehme, I.; Papkovskaya, N.; Klimant, I. Biosens. Bioelectron. 2000, 15, 69-76. (20) E. J. Cho; Tao Z.; Tang, Y.; Tehan, E. C.; Bright, F. V.; Hicks, W. L. Jr.; Gardella, J. A. Jr.; Hard, R. Appl. Spectrosc., in press.

times prior to gelation, high GOx activity, sensor element uniformity, and sensor element stability. PPBSA Fabrication. Figure 1 presents a simplified schematic describing the four types of biosensor arrays we have fabricated. Parts A and B of Figure 1 outline the methods of producing PPBSAs onto glass microscope slides and LEDs, respectively. The basic fabrication steps include pin printing the O2-sensing layer (PP) and forming a glucose-sensing layer or element by spin casting (SC) or overprinting (OP), respectively. (A) Fabrication of PPBSAs onto Planar Glass Substrates. As shown in Figure 1A, we initially prepare an O2-responsive PPCSA.10 The O2-sensing elements are formed from a sol-gel processing solution that is composed of 4 µL of 25.0 mM [Ru(dpp)3]2+ (dissolved in EtOH) and 50 µL of solution A. All O2responsive PPCSAs were aged in the dark under ambient conditions for at least 4 h before further use. In the second step, a GOx-doped sol-gel processing solution is either spin cast (SC, PPBSA 1) or overprinted (OP, PPBSA 2) on top of the O2responsive PPCSAs. To prepare the glucose-responsive layer on PPBSA 1, we prepared a GOx-doped sol-gel processing solution by mixing 10 µL of a GOx stock solution (6 mg of GOx dissolved in 500 µL of PBS) with 30 µL of solution C. An O2-responsive PPCSA is mounted in the rotor of a spin coater with the O2-responsive sensing elements facing up, the rotor is engaged, and the rotational velocity is adjusted to 2000 rpm. A 10-µL aliquot of the GOx-doped sol-gel processing solution is delivered to the center Analytical Chemistry, Vol. 74, No. 24, December 15, 2002

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Figure 2. Summary of PPBSA 1 performance. (A) Array challenged with air-saturated buffer that contains 10 mM glucose. (B) Array challenged with buffer alone, no glucose. (C) Typical composite glucose calibration curve for 100 elements. (D) Array challenged with N2-saturated buffer. (E) Array challenged with O2-saturated buffer. (F) Typical composite O2 calibration curve for 100 sensor elements. The line passing through the data is the best fit to eq 4.

of the PPCSA by using a micropipet, and spinning is continued for 10 s. Profilometry showed that the GOx-doped xerogel film was 0.5 ( 0.1 µm thick. To prepare PPBSA 2, we mixed 100 µL of a GOx stock solution (6 mg of GOx, 25 mg of sorbitol, and 15 mg of PEG 400 in 500 µL of Tris buffer (5 mM, pH 7.4)) with 100 µL of solution B. The GOx-doped sol-gel processing solution was printed directly on top of the PPCSAs O2-responsive sensor elements. Scanning electron microscopy showed that the printed glucose-responsive sensing element were 1.0 ( 0.1 µm thick. (B) Fabrication of PPBSAs onto LEDs. Figure 1B illustrates the procedure we used to form PPBSAs on LEDs. An O2responsive PPOSAILS is formed first.11 The glucose sensor elements were formed by spin casting (SC, PPBSA 3) or overprinting (OP, PPBSA 4) by using the same strategies and formulations described for PPBSA 1 and PPBSA 2, respectively. General Guidelines on Pin Printing. Sensor elements were typically printed with the sensor element column-to-column center spacing adjusted to between 200 and 400 µm and the sensor element row-to-row center spacing set at 200 µm. The time required to print each sensor element is on the order of 1 s. The print chamber relative humidity was maintained between 30 and 40%. The sensor element appearance, diameter, and performance are functions of the sol-gel processing solution composition, hydrolysis time, relative humidity in the print chamber, pin contact time with and velocity toward the substrate, and substrate’s surface 6180 Analytical Chemistry, Vol. 74, No. 24, December 15, 2002

chemistry.12,20 In our hands, the individual xerogel-based sensor elements are between 100 and 180 µm in diameter and they are reproducible within a given PPBSA to (10 µm. PPBSA Storage and Testing. All PPBSAs were aged in the dark for at least 24 h prior to being tested. All measurements were performed at room temperature. All experiments were performed on at least three separate occasions using separate reagent batches. Average results from all experiments are reported along with the corresponding standard deviations. RESULTS AND DISCUSSION Figure 2 summarizes the PPBSA 1 response characteristics. Parts A and B of Figure 2 present false color fluorescence images when the array is subjected to air-saturated buffer that contains 10 mM glucose or air-saturated buffer without glucose, respectively. Figure 2C presents the composite calibration curve for the PPBSA 1 responding to glucose. The data points represent the average response from 100 individual sensor elements (10 × 10 array), and the error bars reflect the standard deviation for all 100 sensor elements. Parts D and E of Figure 2 present false color fluorescence images from PPBSA 1 when the array is subjected to N2- or O2-saturated buffer, respectively. Figure 2F presents the composite calibration curve for the PPBSA 1 responding to O2. The line that passes through the data points in Figure 2F is the best fit to eq 4. Figure 3 presents results from a dual-analyte PPBSA 2 in which one column, labeled (G), contains glucose-responsive sensor

Figure 3. Summary of PPBSA 2 sensor performance. The columns labeled (O) and (G) denote O2- and glucose-responsive sensor elements, respectively. (A) Array challenged with air-saturated buffer that contains 10 mM glucose. (B) Array challenged with buffer alone, no glucose. (C) Calibration curves for the O2- (2) and glucose-responsive (b) sensor elements responding to glucose. (D) Array challenged with N2-saturated buffer. (E) Array challenged with O2-saturated buffer. (F) Calibration curves for the O2- (2) and glucose-responsive (b) sensor elements responding to O2. The line passing through the data is the best fit to eq 4.

elements. The O2-responsive sensor elements are located in the columns labeled (O). To properly pin print the GOx-doped solgel-derived solutions, we had to completely reformulate the GOxdoped sol-gel processing solution. Specifically, we needed to extend the solution working time to avoid the sol-gel processing solutions from gelling within the pin, avoid xerogel cracking, and produce uniform and stable biosensor elements. The gelation times for sol-gel-processed solutions depend on the composition of sol-gel-processed solution, volume ratio of sol-gel-processed solution and buffer solution, buffer pH, and buffer ionic strength.21 In our hands, a switch from PBS to Tris buffer (vide supra) yielded an extended solution working time.20 Further, by modifying the sol-gel processing solution composition as described (vide supra), we could readily and reproducibly overprint the GOx-doped solgel solutions directly on top of the O2-responsive sensor elements. Parts A and B of Figure 3 present false color fluorescence images when PPBSA 2 is subjected to air-saturated buffer solutions that contain 10 or 0 mM glucose, respectively. Parts D and E of Figure 3 present false color fluorescence images when PPBSA 2 is subjected to N2- or O2-saturated buffer, respectively. Parts C and E of Figure 3 respectively show the corresponding glucose and O2 calibration curves from 5 glucose sensor elements (b) and 10 O2 sensor elements (2) within a typical PPBSA 2. The glucose-responsive biosensor elements selectively respond to (21) Doong, R.-A.; Tsai, H.-C. Anal. Chim. Acta 2001, 434, 239-246.

changes in the glucose level whereas the O2-responsive sensor elements do not respond to glucose. Figure 3F presents the composite calibration curves for the PPBSA 2 responding to O2. The line that passes through the data points in Figure 3F is the best fit to eq 4. Figure 4 summarizes the PPBSA 3 response characteristics. Parts A, B, D, and E of Figure 4 present false color fluorescence images when the biosensor array is subjected to air-saturated buffer that contains 10 mM glucose, air-saturated buffer without glucose, N2-saturated buffer, and O2-saturated buffer, respectively. Compared to the PPBSA 1, which is fabricated onto a glass substrate and excited with an external laser light source, the absolute intensity from each sensor element in PPBSA 3 varies across the LED face. Because the luminophores in PPBSA 3 are excited directly by the LED optical output below the sensor elements, the absolute intensity is greatest near the LED’s p-n junction (located in the bottom right region of each image) and decreases as one moves radially away from the p-n junction. As we reported in our previous work,11 this intensity variability is compensated for completely by our ratiometric measurements [I0/ I or ((I - I0)/I0) × 100%] and the thin blue paint filter on the LED face. Parts C and F of Figure 4 present the PPBSA 3 composite calibration curves for glucose and O2, respectively. In Figure 5, we present results from PPBSA 4. In this biosensor array, one column, labeled (G), has glucose-responsive biosensor Analytical Chemistry, Vol. 74, No. 24, December 15, 2002

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Figure 4. Summary of PPBSA 3 performance. The LED p-n junction is located in the bottom right segment of the images. (A) Array challenged with air-saturated buffer that contains 10 mM glucose. (B) Array challenged with buffer alone, no glucose. (C) Typical composite glucose calibration curve for 100 elements. (D) Array challenged with N2-saturated buffer. (E) Array challenged with O2-saturated buffer. (F) Typical composite O2 calibration curve for 100 sensor elements. The line passing through the data is the best fit to eq 4.

elements, and the O2-responsive sensor elements are located in the columns labeled (O). Parts A, B, D, and E of Figure 5 present false color fluorescence images when PPBSA 4 is subjected to 10 mM glucose, buffer alone without glucose, N2-saturated buffer, and O2-saturated buffer, respectively. Parts C and F of Figure 5 present the composite calibration curves for the individual types of sensor elements responding to glucose and O2, respectively. Table 1 summarizes the PPBSA’s analytical performance. Overall, the four PPBSAs (Figure 1) exhibit similar analytical figures of merit. The response time and detection limits for the O2 sensor elements were 10-12 s and 0.1% O2, respectively. These results argue that the GOx-doped xerogel-based overlayer, regardless of its composition, does not affect the performance of the underlying O2-responsive sensor elements. The response time for the glucose sensor elements is generally a factor of 3-4 greater in comparison to the O2 sensors, and the best-case response times are seen with the entirely pin-printed glucose sensors (i.e., PPBSA 2 and PPBSA 4). The 3-4-fold slower response is likely due to differences in the O2 versus glucose diffusivity in water. The 25% difference in response time between PPBSA 1/3 and PPBSA 2/4 is consistent with differences in the actual xerogel composition (PPBSA 1/3: P104 and PBS; PPBSA 2/4: sorbital, PEG and Tris). (Note: The thickness of the glucose-responsive elements proper in PPBSA 1/3 and PPBSA 2/4 are 0.5 and 1.0 µm, respectively. Thus, if the diffusivity were equivalent within each biosensor element, one would expect PPBSA 1/3 to exhibit a more prompt response in comparison to PPBSA 2/4. We see the opposite 6182

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trend.) However, these data alone cannot rule out intrinsic differences in the actual sensor elements derived from pin printing versus spin coating, an issue under current investigation in our laboratories. Regardless of the origin, glucose appears to exhibit a greater diffusivity within the pin-printed sorbitol/PEG/Tris xerogels in comparison to the spin-coated P104/PBS xerogels. The detection limits for glucose were between 0.1 and 0.2 mM. The LED-based sensor platforms (PPBSA 3/4) yield biosensor arrays with poorer detection limits; however, the detection limits for all four PPBSAs exceed clinical needs.13 The response of the glucose sensor elements (scaled to the actual amount of GOx within each sensor element) was a function of the xerogel composition. In general, the spin cast glucose biosensors exhibited a response that is 30-50% poorer in comparison to the pin-printed biosensors. If we operated sets of PPBSAs and rapidly cycled the sensor between 0 and 10 mM glucose and N2- and O2-saturated buffer, we observed responses that were reproducible to within 5-7%. If we operated calibrated PPBSAs over a 12-h period with regular cycling between 0, 2, and 10 mM glucose solutions (20% O2) and 0, 10, and 100% O2-saturated buffer solutions, the array response deviated by 3-5%. When individual PPBSAs are removed from the testing system, stored, remounted in the system, and recalibrated on a weekly basis for 6 weeks using one randomly selected biosensor element in the array, the biosensor element response deviated by no more than 4-8%. When we prepared five PPBSAs

Figure 5. Summary of PPBSA 4 sensor performance. The columns labeled (O) and (G) denote O2- and glucose-responsive sensor elements, respectively. (A) Array challenged with air-saturated buffer that contains 10 mM glucose. (B) Array challenged with buffer alone, no glucose. (C) Calibration curves for the O2- (2) and glucose-responsive (b) sensor elements responding to glucose. (D) Array challenged with N2-saturated buffer. (E) Array challenged with O2-saturated buffer. (F) Calibration curves for the O2- (2) and glucose-responsive (b) sensor elements responding to O2. The line passing through the data is the best fit to eq 4. Table 1. Pooled Analytical Figures of Merit for PPBSAs Responding to Glucose and O2a response reproducibility (%) array format PPBSA 1 glucose O2 PPBSA 2 glucose O2 PPBSA 3 glucose O2 PPBSA 4 glucose O2

response time (s)b

detection limitsc

responsed

47 ( 17 12 ( 1

0.1 mM 0.1%

34 ( 8 12 ( 2

reversibilitye (%)

short-termf

long-termg

PPBSA-toPPBSAh

29 ( 2% 3.2 ( 0.1

6 5

3 4

7 4

12 10

0.1 mM 0.1%

35 ( 3% 3.5 ( 0.1

5 5

3 3

8 6

12 11

48 ( 14 10 ( 2

0.2 mM 0.1%

17 ( 2% 3.1 ( 0.1

7 5

5 4

7 5%

10 9

35 ( 7 12 ( 3

0.2 mM 0.1%

25 ( 3% 3.4 ( 0.2

5 5

3 4

6 5

8 10

a For 100 sensor elements on a single PPBSA. b Based on the time required to reach 90% of the full response following a switch between air-saturated buffer that contained 10 mM glucose and air-saturated buffer alone without glucose or O2- and N2-saturated buffer solution. Laser stability ∼2% RSD. c Minimum quantity of glucose or O2 that can be detected. d Defined as (I - I0)/I0 × 100% at 4 mM glucose or I0/I at 100% O2. The glucose results are scaled to the actual concentration on GOx in the sensor element. e Results of five cycles between air-saturated buffer that contained 10 mM glucose and air-saturated buffer alone without glucose or O2- and N2-saturated buffer solution. f Based on the analysis of a single calibrated PPBSA after being repeatedly challenged for 12 h with 0, 2, and 10 mM glucose solution (20% O2) and 0, 10, and 100% O2 saturated buffer solution (pH 7.0). g Based on the analysis of a single calibrated PPBSA after being repeatedly challenged with 0, 2, and 10 mM glucose solution (20% O2) and 0, 10, and 100% O2 saturated buffer solution (pH 7.0) following full shutdown, weekly PPBSA recalibration, and reuse for 6 weeks. h Based on the response profiles of five separate PPBSAs fabricated at 2-3-week intervals over the course of 2.5 months using separate reagent batches and preparations following complete PPBSA calibration.

at 2-3-week intervals using different reagent batches, the sensor element responses are reproducible to within 8-12%. Overall, these results show the potential of the PPBSAs.

CONCLUSIONS We report the first pin-printed biosensor arrays based on protein-doped xerogels and their analytical performance. We Analytical Chemistry, Vol. 74, No. 24, December 15, 2002

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demonstrate that the combination of pin printing and sol-gel processing techniques provides a simple method to rapidly fabricate (