Anal. Chem. 2006, 78, 5664-5670
Holographic Lactate Sensor Felicity K. Sartain, Xiaoping Yang, and Christopher R. Lowe*
Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QT, UK
Measurement of blood L-lactate is used to assess and monitor exercise performance in sports medicine. This report describes the initial development of a holographic sensor, which employs a synthetic receptor, to enable the selective and continuous real-time measurement of Llactate for eventual in vivo application. Three boronic acidbased receptors have been synthesized, integrated into thin acrylamide hydrogel films, and then subsequently transformed into holographic sensors. Changes in the replay wavelength of the sensors were used to characterize the swelling behavior of the matrix as a function of L-lactate concentration. It was found that the incorporation of 3-acrylamidophenyl boronic acid into an acrylamide hydrogel produced the largest response toward L-lactate. The effects of hydrogel composition, fluctuating L-lactate concentrations, and the response of potential interfering agents to the sensor have been investigated. L-Lactate is a unique metabolite produced by many organisms as a result of anaerobic metabolism. Monitoring the production of such a metabolite provides useful information about the biological system under analysis. Consequently, there is substantial interest in developing simple and inexpensive sensors for reliable measurements of L-lactate within the food industry,1,2 fermentation processes,3 clinical diagnostics,4-14 and sports medicine.15-19
* To whom correspondence should be addressed. Phone: (+44) 1223 334160. Fax: (+44) 1223 334162. E-mail:
[email protected]. (1) Palmisano, F.; Quinto, M.; Rizzi, R.; Zambonin, P. G. Analyst 2001, 126, 866-870. (2) Torreiero, A. A. J.; Salinas, E.; Battaglini, F.; Raba, J. Anal. Chim. Acta 2003, 498, 155-163 (3) Merten, O. W.; Palfi, G. E. Dev. Biol. Stand. 1987, 66, 111-142. (4) D’Auria, S.; Gryczynski, Z.; Gryczynski, I.; Rossi, M.; Lakowicz, J. R. Anal. Biochem. 2000, 283, 83-88. (5) Li, C. I.; Lin, Y. H.; Shih, C. L.; Tsaur, J. P.; Chau, L. K. Biosens. Bioelectron. 2002, 17, 323-330. (6) Liu, X.; Tan, W. Mikrochim. Acta 1999, 131, 129-135. (7) Hirano, K.; Yamato, H.; Kunimoto, K.; Ohwa, M. Biosens. Bioelectron. 2002, 17, 315-322. (8) Nakamura, H.; Murakami, Y.; Yokoyama, K.; Tamiya, E.; Karube, I. Anal. Chem. 2001, 73, 373-378. (9) Moser, I.; Jobst, G.; Urban, G. A. Biosens. Bioelectron. 2002, 17, 297-302. (10) Baker, D. A.; Gough, D. A. Anal. Chem. 1995, 67, 1536-1540. (11) Yang, L.; Kissinger, P. T. Curr. Sep. 1995, 14, 31-35. (12) Jobst, G.; Moser, I.; Varahram, M.; Svasek, P.; Aschauer, E.; Trajanoski, Z.; Wach, P.; Kotanko, P.; Skraba, l F.; Urban, G. Anal. Chem. 1996, 68, 31733179. (13) Lin, C.-N.; Chen, S.-H.; Kou, G.-H.; Kuo, C.-M. Acta Zoo. Tai. 1999, 10, 91-101. (14) Kurita, R.; Hayashi, K.; Fan, X.; Yamamoto, K.; Kato, T.; Niwa, O. Sens. Actuators, B 2002, 87, 296-303.
5664 Analytical Chemistry, Vol. 78, No. 16, August 15, 2006
In sport, blood L-lactate is the primary metabolic variable that indicates the capability of the muscles for athletic performance. Through monitoring L-lactate levels, a coach is able to determine whether an optimal balance of the aerobic and anaerobic systems has been achieved,15 how an athlete has adapted to training, and the training that is required and, hence, prescribe accurate training parameters to bring about optimal progress. The ideal sensor for this application would be one that provides a real-time, reliable, continuous measurement of L-lactate. This would eliminate the need for frequent blood samples to be taken while enabling the athletes to monitor their L-lactate levels in real time as they train. Most current L-lactate sensors tend to be either electrochemical or optical biosensors, using the enzymes L-lactate dehydrogenase4-6 and L-lactate oxidase1,2,7-18 as the means of selective recognition. Hydrogen peroxide-detecting L-lactate biosensors invariably suffer from electroactive interference from ascorbic acid1,7,14 and show sensitivity to O2 concentrations.1,7 Furthermore, such electrochemical biosensors usually employ large overpotentials, which further add to interference problems due to co-oxidation of reducing substances present in biological media.6 The use of optical transduction eliminates these interference problems and also tends to reduce the sample size required.8,15 The use of a simple reflection hologram that combines an analyte-selective “smart polymer” with optical interrogation and a reporting transducer has been introduced.20-31 Holographic (15) Pyne, D. B.; Boston, T.; Martin, D. T.; Logan, A. Eur. J. Appl. Phys. 2000, 82, 112-116. (16) Meyerhoff, C.; Bischof, F.; Mennel, F. J.; Sternberg, F.; Bican, J.; Pfeiffer, E. F. Biosens. Bioelectron. 1993, 8, 409-414. (17) Poscia, A.; Messeri, D.; Moscone, D.; Ricci, F.; Valgimigli, F. Biosens. Bioelectron. 2005, 20, 2244-2250. (18) Gavalas, V. G.; Chaniotakis, N. A. Mikrochim. Acta 2001, 136, 211-215. (19) Smutok, O.; Gayda, G.; Gonchar, M.; Schuhmann , W. Biosens. Bioelectron. 2005, 20, 1285-1290. (20) Blyth, J.; Millington, R. B.; Mayes, A. G.; Frears, E. R.; Lowe, C. R. Anal. Chem. 1996, 68, 1089-1094. (21) Blyth, J.; Millington, R. B.; Mayes, A. G.; Lowe, C. R. Imaging Sci. J. 1999, 47, 87-91. (22) Millington, R. B.; Mayes, A. G.; Blyth, J.; Lowe, C. R. Anal. Chem. 1995, 67, 4229-4233. (23) Millington, R. B.; Mayes, A. G.; Blyth, J.; Lowe, C. R. Sens. Actuators, B 1996, 33, 55-59. (24) Mayes, A. G.; Blyth, J.; Millington, R. B.; Lowe, C. R. J. Mol. Recognit. 1998, 11, 168-174. (25) Mayes, A. G.; Blyth, J.; Kyro¨la¨ianen-Reay, M.; Millington, R. B.; Lowe, C. R. Anal. Chem. 1999, 71, 3390-3396. (26) Mayes, A. G.; Blyth, J.; Millington, R. B.; Lowe, C. R. Anal. Chem. 2002, 74, 3649-3657. (27) Marshall, A. J.; Blyth, J.; Davidson, C. A. B.; Lowe, C. R. Anal. Chem. 2003, 75, 4423-4431. (28) Kabilan, S.; Blyth, J.; Lee, M.-C.; Marshall, A. J.; Hussain, A.; Yang, X.; Lowe, C. R. J. Mol. Recognit. 2004, 17, 162-166. (29) Lee, M.-C.; Kabilan, S.; Hussain, A.; Yang, X.; Blyth, J.; Lowe, C. R. Anal. Chem. 2004, 76, 5748-5755. 10.1021/ac060416g CCC: $33.50
© 2006 American Chemical Society Published on Web 07/15/2006
sensors undergo visible optical transitions in response to fluctuations in analyte concentration. The replay wavelength changes result as a consequence of the swelling/contraction induced in the base hydrogel, which in turn alters the spacing between the embedded diffraction grating and, thus, varies the wavelength of the reflected light. The holographic grating is generated within the polymeric matrix by first pretreating the thin film with a lightsensitive silver halide emulsion, after which upon exposure to a single collimated beam of laser light, which passes through the film and is then reflected back by a mirror, ultrafine fringes of silver nanoparticles are produced within the hydrogel. After development, the silver fringes created in the matrix behave as Bragg gratings, reflecting a narrow band of wavelengths that, when exposed to white light, are perceived as a monochromatic mirror. The wavelength, and hence color, of the reflected light measured from the hologram is determined by the distance between the silver fringes in accordance with Bragg’s law (λpk ) 2nD cos θ). Thus, any change in the spacing between the fringes (D) or the refractive index (n) will result in a proportional change in the reflected wavelength (λpk). It is believed that such transitions are achieved in holographic sensors by altering the electrostatic properties of the “smart polymer”, which in turn induces a shift in the swelling equilibrium of the gel. It has been demonstrated24-31 that through the rational design of a hydrogel that incorporates suitable synthetic receptors for a specific analyte, such volumetric changes can be induced upon the association/dissociation of the analyte under investigation. A responsive gel for L-lactate could be produced through chemical conjugation of a ligand that, upon complex formation with the analyte, results in an overall charge change occurring within the gel phase. Boronic acids are known to bind with bidentate chelating ligands to form five- or six-membered cyclic esters.32,33 The reaction occurs in aqueous solution and tends to be rapid and reversible, which makes boronates ideal compounds to be used as synthetic receptors. Whereas more recent work has focused on the binding and detection of polyols, in particular, saccharides and carbohydrates, previous studies have shown that boronic acids also react reversibly with o-diphenols, o-hydroxy acids, dicarboxylic acids, and R-hydroxy acids, including L-lactate.34-39 In aqueous conditions, an equilibrium between the trigonal (sp2 hybridized) and tetrahedral (sp3 hybridized) boronic acid species is established. This equilibrium is pH-dependent; thus, increasing the pH causes a shift in the equilibrium and generates a greater concentration of the anionic tetrahedral form. The addition of an R-hydroxy acid to such a solution generates a complex equilibrium that includes an anionic tetrahedral boronate cyclic ester (Figure 1). Such a bidentate chelator is believed to bind predominantly (30) Madrigal Gonza´lez, B.; Christie, G.; Davidson, C. A. B.; Blyth, J.; Lowe, C. R. Anal. Chim. Acta 2005, 528, 219-228. (31) Kabilan, S.; Marshall, A. J.; Sartain, F. K.; Lee, M.-C.; Hussain, A.; Yang, X.; Blyth, J.; Karangu, N.; James, K.; Zeng, J.; Smith, D.; Domschke, A.; Lowe, C. R. Biosens. Bioelectron. 2005, 20, 1602-1610. (32) Springsteen, G.; Wang, B. Tetrahedron 2002, 58, 5291-5300. (33) Lorand, J. P.; Edwards, J. O. J. Org. Chem. 1959, 24, 769-774. (34) Babcock, L.; Pizer, R. Inorg. Chem. 1980, 19, 56-61. (35) Pizer, R.; Selzer, R. Inorg. Chem. 1984, 23, 3023-3026. (36) Friedman, S.; Pace, B.; Pizer, R. J. Am. Chem. Soc. 1974, 96, 5381-5384 (37) Pizer, R. D.; Babcock, L. Inorg. Chem. 1977, 16, 1677-1681. (38) Ishihara, K.; Mouri, Y.; Funshashi, S.; Tanaka, M. Inorg. Chem. 1991, 30, 2356-2360. (39) Pizer, R.; Tihal, C. Inorg. Chem. 1992, 31, 3243-3247.
Figure 1. The equilibrium between a boronic acid and its boronate ester on reaction with an R-hydroxy acid.
with the trigonal form of a boronic acid.36 Reaction with the tetrahedral form does occur, but at a significantly slower rate.34 Inclusion of a boronic acid moiety into a hydrogel should not only confer the ability to bind L-lactate, but also generate an anionic tetrahedral boronate complex, producing a more charged gel phase. The Donnan potential produced within the matrix will cause an influx of counterions to maintain electroneutrality, which in turn will cause a swelling of the hydrogel.40 This same principle has been exploited previously to enable the detection of diols, including glucose, but the detection of L-lactate has not been investigated thoroughly.29,31,41 Thus, if this charge change can be realized, then a holographic response for L-lactate is likely to be achieved employing a boronic acid moiety as the functional ligand. This paper reports the initial studies toward the development of a holographic L-lactate sensor utilizing a synthetic receptor for eventual in vivo application. Detailed here is the investigation of the response of three holographic L-lactate sensors that have been fabricated with boronic acid-based “smart” hydrogels and the subsequent optimization of the most responsive sensor. EXPERIMENTAL SECTION Materials. All chemicals were of analytical grade unless otherwise stated. Acryloyl chloride, L-alanine, 1,1′-diethyl-2,2′cyanine iodide (QBS photosensitizing dye), 2,2′-dimethoxy-2phenyl acetophenone (DMPA), dimethyl sulfoxide (DMSO), N,N′methylene-bisacrylamide (electrophoresis grade), 3-(trimethoxysilyl)propyl methacrylate, phosphorus pentoxide, potassium bromide, pyruvate, silver nitrate (1 M, volumetric standard), silver perchlorate, sodium hydroxide, sodium L-lactate, and sodium thiosulfate were purchased from Sigma-Aldrich Chemical Co. 3-Aminophenylboronic acid monohydrate was purchased from Avocado Research Chemicals Ltd., and glucose was purchased from ICN Biomedicals, Inc. 2-Aminophenylboronic acid hydrochloride and 4-aminophenylboronic acid were purchased from Combi-Blocks Inc., and phosphate buffered saline (PBS) tablets were purchased from Oxoid Ltd. Deuterium oxide (D2O), sodium deuterioxide (NaOD), and deuterium chloride (DCl) were purchased from Cambridge Isotope Laboratories Inc. Equipment. Microscope slides (Super Premium, 1-1.2 mm thick, low iron) were purchased from BDH (Merck) Ltd. Alumi(40) Flory, P. J. Principles of Polymer Chemistry; Cornell University Press: Ithaca, NY, 1953. (41) Alexeev, V. L.; Sharma, A. C.; Goponenko, A. V.; Das, S.; Lednev, I. K.; Wilcox, C. S.; Finegold, D. N.; Asher, S. A. Anal. Chem. 2003, 75, 23162323.
Analytical Chemistry, Vol. 78, No. 16, August 15, 2006
5665
Figure 2. (a) The structures of 2-, 3-, and 4-acrylamidophenyl boronates, (2-APB, 3-APB, 4-APB); (b) a comparison of the shifts in replay wavelength recorded from the 12 mol % 2-APB ([), 3-APB (]), and 4-APB (×) holographic sensors toward increasing concentrations of L-lactate. The error bars represent the standard deviation of the average peak shifts of each of the holograms to increasing lactate concentrations, (n ) 3). Inset: Response of a 12 mol % 4-APB hologram to six additions of ∼2 mM L-lactate in PBS pH 7.4 buffer at 30 °C.
nized 100-µm polyester film (grade MET401) was purchased from HiFi Industrial Film Ltd. The UV exposure unit (∼350 nm, model no. 555-279) was purchased from RS components. A standard benchtop pH meter and electrode and calibration buffers were purchased from Hanna Instruments. A micro PerpHect pH electrode and benchtop meter were purchased from Orion. Instrumentation. A frequency-doubled Nd:YAG laser (350 mJ, 532 nm, Brilliant B) was used in silver-hologram recording. Holograms were analyzed using a LOT-ORIEL MS127i model 77480 imaging spectrograph in single-channel mode with a 256 × 1024 pixel InstaSpec IV CCD detector, processing software, and a tungsten halogen light source. Spectrometer calibration was achieved using a spectral calibration lamp (37-4405) purchased from Ealing Electro Optics plc. The setup used is the same as described in Mayes et al.26 Syntheses of 4-, 3-, and 2-Acrylamidophenyl Boronic Acids (4-, 3-, 2-APB). (Figure 2a). The syntheses of 3-APB42 and 2-APB43 have previously been reported. For each compound, the 1H NMR, 13C NMR, 11B NMR, and MS data obtained were (42) Kanekiyo, Y.; Masahito, S.; Ritsuko, I.; Shinkai, S. J. Polym. Sci., Part A: Polym. Chem. 2000, 38, 1302-1310. (43) Yang, X.; Lee, M.-C.; Sartain, F. K.; Pan, X.; Lowe, C. R. Chem.sEur. J. 2006, in press.
5666 Analytical Chemistry, Vol. 78, No. 16, August 15, 2006
consistent with that published. 4-Aminophenylboronic acid-HCl (0.5 g, 2.89 mmol) was dissolved in 5 mL 10% (w/v) NaOH. This solution was cooled in an ice bath to 0 °C, and acryloyl chloride (0.5 mL, 5.77 mmol) added dropwise to it. The mixture was allowed to stir for 3 h while being brought to room temperature. The brown solution was monitored throughout this time, and the pH was adjusted to ∼8. A white-grey precipitate was produced, which was filtered and washed with ∼50 mL of ice cold water. The solid was dried and recrystallized in 30% (v/v) EtOH, and the solution left overnight at 20 °C. The crystals produced were filtered off and dried under high vacuum. The overall yield of this reaction was 32.2%. The structure of 4-APB was confirmed by 1H NMR, 13C NMR, 11B NMR, and MS. 1H NMR (DMSO-d6): 5.80 (d, 1H), 6.38 (d, 1H), 6.55 (dd, 1H), 7.74 (d, 2H), 7.85 (d, 2H), 8.07 (s, 2H), 10.29 (s, 1H). 13C NMR (D2O): 121.44, 129.40, 131.14, 135.38, 139.87, 167.67. 11B NMR (DMSO-d6): 11.14 MS (EI) (m/ z): 191.092 (M+) 214.065 (M+ +23). Synthesis of Polymer Films. All hydrogels were produced via UV-initiated co-polymerization. Appropriate quantities of the monomers were dissolved in 2% (w/v) DMPA in DMSO at a ratio 1:2.21 (w/v) of the monomers to solvent. In the synthesis of the 2-APB polymer, 5 M NaOH was also added to the polymer mixture to encourage the dissolution of 2-APB. A 100-µL aliquot of the polymer mixture was pipetted onto the aluminum side of an aluminum/plastic reflective sheet, and a silanized glass slide was placed on top of the solution. The slides were then left exposed to UV light for 1 h. The polymerized films were subsequently submerged in deionized water, peeled from the aluminum sheet, and washed in some fresh deionized water. Any excess polymer material on the edges of the slide was removed using a scalpel. Hologram Construction. The polymer films initially had to be photosensitized by introducing a silver halide emulsion into the polymer matrix. The technique used was based on the principle of the diffusion method described by Blyth et al.21 For these (2-APB, 3-APB, 4-APB) acrylamide-based copolymers, the following procedure was used: The polymer slide was placed face down onto 400 µL of 0.2 M AgNO3 solution for 2 min. Excess solution was wicked off, and the slide was dried in a stream of warm air, after which the slide was placed polymer side up in a 40-mL solution of 4% (w/v) KBr and 0.05% (w/v) ascorbic acid in 1:1 CH3OH/H2O (v/v) with 1 mL of 0.1% (w/v) QBS dye in CH3OH and agitated for 1 min. The slide was rinsed thoroughly in deionized water before being placed face-down into the hologram exposure bath, containing 100 mL of PBS buffer, for ∼15 min. The whole slide was then exposed to three 10 ns single pulses from the frequency-doubled (532 nm) Nd:YAG laser. After exposure, the slide was then placed in fresh developer solution (40 mL of 20 g/L ascorbic acid, 5 g/L 4-methylaminophenol sulfate, 20 g/L sodium carbonate, 6.5 g/L sodium hydroxide) and agitated for ∼10-20 s. The slide was rinsed under deionized water, placed in “stop” solution (5% (w/v) acetic acid) for ∼30 s to stop development, and rinsed thoroughly in deionized water before being immersed for 2-5 min in 20% (w/v) sodium thiosulfate to remove any undeveloped silver. Finally, the slide was rinsed in methanol and then deionized water. All of this work was carried out under safe red lighting. Monitoring the Holographic Response. The holograms were interrogated using an in-house-built reflection spectropho-
tometer as described in Mayes et al.26 A piece of hologram was cut from the whole slide (∼8 mm wide) and placed in a 4-mL plastic cuvette with the polymer side facing inward. PBS buffer (1 mL, pH 7.4, concentration 10 mM, ionic strength 160 mM) was added, and the cuvette was covered and allowed to stir at a constant rate with a magnetic microflea/stirrer arrangement. This setup was left at 30 °C to allow the system to reach equilibrium. A 0.1 M solution of sodium lactate was made up in PBS buffer, and 20 µL of this solution was added to the cuvette, and the system allowed to equilibrate. An additional 5 aliquots of 20 µL of 0.1 M sodium lactate solution were added, and each time, the system was allowed to stabilize. This method was also used when monitoring the response of the 15 mol % 3-APB hologram toward glucose, chloroacetic acid, L-alanine, pyruvate, and thiolactic acid. To assess reversibility of the sensor response, the 3-APB hologram was left overnight at 30 °C in 1 mL of PBS buffer to equilibrate the system. Solutions of 2, 4, 6, 8, 10, and 12 mM sodium lactate were made up in PBS buffer. The 1 mL of PBS buffer was replaced with 1 mL of 2 mM sodium lactate solution and the system left until a stabilized signal was recorded. Once stable, the hologram was washed with PBS buffer twice and then allowed to stabilize again with 1 mL of PBS buffer. This was repeated with all the lactate solutions up to 12 mM and then stepwise in reversed order. Another similar experiment was performed without the “wash step” between additions. pKa Determination of 2-, 3-, and 4-APB. The pKa values of 2-, 3-, and 4-APB in the hologram were measured as follows: A range of buffers with various pH values at constant concentration and ionic strength were made up. Starting with the most acidic buffer, 1 mL was added to the cuvette, and the holographic system was allowed to equilibrate at 30 °C with stirring. Once a stable peak had been recorded, the first buffer was removed and replaced with a fresh buffer of a higher pH. The range over which the hologram was analyzed was usually 4-11 pH at 0.5 pH unit intervals. In solution: pD was measured using microcombination pH/sodium electrodes. The term “pD” was converted into pH using the following equation: pH ) pD + 0.4. The pKa of the boronates in solution was measured using 11B NMR spectroscopy. The principle for 11B NMR titration relies on the fact that the hydroxyl exchange between the sp2 and sp3 species is fast on the 11B NMR time scale and gives only one mean signal for the uncomplexed boron.44 Boronate (2-3 mg) was dissolved in D2O, and the pD was altered with DCl and NaOD. For each pD, a 11B NMR was run. The data gained from these experiments was inputted into SigmaPlot, where the pKa for each of the boronates was determined in solution and within the hologram. RESULTS AND DISCUSSION Synthesis and Characterization of 2-, 3-, and 4-Acrylamidophenyl Boronates. 2-,3-, and 4-APB were synthesized in basic aqueous conditions and purified through recrystallization in 2030% (v/v) ethanol. Each compound produced was characterized using 1H NMR, 13C NMR, 11B NMR, and MS, and in the case of 2- and 3-APB, the data obtained was consistent with that previously reported.42,43 The purity of the compounds was assessed by 1H NMR integration and revealed each product to be >98% pure. Response of 12 mol % 2-, 3-, and 4-APB Holographic Sensors to L-Lactate. A 12 mol % 4-APB hologram was allowed (44) Chapelle, S.; Verchere, J.-F. Tetrahedron 1988, 44, 4469-4482.
Figure 3. The effect of pH at 30 °C on the replay wavelength (λmax) of the acrylamide-based hologram containing 5 mol % 4-APB (() and in a solution of 4-APB measured by 11B NMR ()). Similar experiments were performed with 2- and 3-APB.41 Table 1. pKa Values for 2-, 3-, and 4-APB Determined in Solution and in the Holographic Sensor boronic acid 2-APB 3-APB 4-APB
pKa determined in solution
apparent pKa determined in the holographic sensor
10.48 8.87 8.93
9.96 8.63 8.88
to equilibrate in 1 mL PBS pH 7.4 buffer at 30 °C prior to the addition of ∼2-11 mM final concentration L-lactate. PBS buffer was chosen to carry out these experiments, since the ultimate aim of this project is to use the sensor in a biological fluid. Therefore, it seemed appropriate to use a buffer that resembles blood in terms of its pH, ionic content, and ionic strength. The increase in replay wavelength observed (Figure 2b inset) on each addition of L-lactate indicates that the polymer swells and suggests that an overall charge was generated within the hydrogel as a result of L-lactate binding to 4-APB. When the same procedure was carried out with 12 mol % 2-APB and 3-APB holographic sensors, it was found that the 3-APB sensor behaved similarly to the 4-APB sensor, but surprisingly, the 2-APB did not respond to L-lactate at all (Figure 2b). The pKa of the boronic acid receptor is believed to influence the binding reaction between itself and a bidentate chelator, such as L-lactate.34-36,39 The stability constant for complex formation has been shown to increase with the increasing acidity of the boronic acid.34 Hence, the pKa values for 2-, and 3-, and 4-APB were determined both in solution and in the hologram in an attempt to understand the observed responses (Figure 3). It is apparent (Table 1) that the microenvironment of the hydrogel causes each of the immobilized boronate receptors to appear more acidic than when in solution. Thus, it is assumed that the tendency for each of these boronate-based receptors to switch from its trigonal to a tetrahedral form is enhanced in the hydrophilic hydrogel. A slight variance in the pKa values for each of the boronates tested was expected due to the difference in the positioning of the acrylamide group around the phenyl ring. 4-APB is thought to be more basic than 3-APB due to the greater separation between the acrylamide group and the boronate; hence, Analytical Chemistry, Vol. 78, No. 16, August 15, 2006
5667
Figure 4. Comparison of the response of holograms containing 5, 8, 10, 12, 15, 20, 25, 30, and 40 mol % 3-APB to increasing L-lactate concentration, (1.97 mM, blank column; 3.85 mM, horizontally striped column; 5.66 mM, diagonally striped column, lower left to upper right; 7.41 mM, solid column; 9.09 mM, vertically striped column; and 10.17 mM, diagonally striped column, upper left to lower right; respectively) in pH 7.4 PBS buffer at 30 °C. The error bars in each instance represent the standard deviation in peak shift (n ) 3).
the -I effect from the acrylamide group is less effective, rendering the boron center fractionally more negative. Surprisingly, the pKa values for 2-APB in both solution and the hologram were ∼1.3 units different, as compared to those recorded for 3- and 4-APB. Analysis of the solution data obtained from the three titrations revealed that the conformation at the boron center in 3- and 4-APB was different from that in 2-APB at low pH.42 Considering the greater upfield 11B NMR resonance for 2-APB (-13.57 ppm), which indicates that the boron center is negatively charged, we suggest that 2-APB adopts a zwitterionic tetrahedral form, in which a pair of electrons from the carbonyl oxygen is donated to the boron, resulting in a strong B-O interaction.43 This conformation explains the higher pKa value of 2-APB and suggests an explanation as to why no response was observed in the hologram upon the addition of L-lactate. Although L-lactate is able to bind to the tetrahedral conformer, the steric hindrance created by the internal B-O bond, along with the negative charge at the boron center, is thought to hinder the complexation reaction between L-lactate and 2-APB. Optimization of the 3-APB Holographic Sensor. The sensitivity of a holographic sensor can be altered by varying the amount of cross-linker utilized or the amount of functional monomer used.26,27,29 The 3-APB hologram with 1.5 mol % methylenebisacrylamide (cross-linker) was found to be more responsive than when a higher mole percent was used. Hydrogels with less cross-linking density were found to be either too soft to be able to record to a hologram, or if the fringes were able to be created, it was not possible to produce a stable signal. Consequently, a cross-linker concentration of 1.5 mol % was used throughout this work. Varying the amount of the recognition ligand within the hydrogel was found to alter the response toward L-lactate, and the largest peak shift was recorded with the 15 mol % 3-APB sensor (Figure 4). This phenomenon of having an optimum response at a particular mole percent of ligand has been observed previously26,31 and is explained by considering the properties of the gel. With a low mole percent of 3-APB (5-10 mol %), there 5668 Analytical Chemistry, Vol. 78, No. 16, August 15, 2006
are fewer binding sites present, and thus, upon complexation with L-lactate, less swelling is induced in the matrix, and smaller color shifts are observed. As the content of 3-APB is increased (>20 mol %), its planar, rigid, and hydrophobic nature is thought to affect the elastic characteristics of the polymer to a greater extent. Consequently, although a greater number of binding sites are available, a reduced swelling is observed upon the addition of L-lactate, since the matrix becomes more rigid. The balance of these factors results in an optimum response toward L-lactate occurring with the 15 mol % 3-APB holographic sensor. Reversibility. Reversible binding needs to be demonstrated to enable continuous measurement of L-lactate with the holographic sensor. From inspection of the literature, it is evident that the reaction between L-lactate and boronic acids is reversible.34,35 Monitoring the response of the 15 mol % 3-APB holographic sensor to fluctuating L-lactate concentrations demonstrated that the replay wavelength for each concentration of L-lactate was within 1-2 nm, whether the concentration was increased or decreased. (Figure 5a,b). Furthermore, the replay wavelength observed in each case returns to the basal level in the absence of L-lactate, suggesting that this sensor is freely reversible. Comparing the peak shift observed for each concentration of L-lactate from the separate experiments (Figure 5c) further demonstrates the reversibility of the system, with the overall peak shift for each concentration of L-lactate falling within 2-4 nm. Evidently, it made no difference whether the sensor was washed between additions or utilized continuously; the response is the same for each individual L-lactate concentration, confirming that this sensor is freely reversible toward L-lactate. Selectivity. The effects of small molecules with a comparable structure to L-lactate have been investigated to assess the selectivity of such a boronic acid based sensor. It was necessary to adjust the pH of the thiolactic acid and chloroacetic acid solutions since it was found that they overwhelmed the buffering capacity of the PBS. Once the pH was adjusted, neither of these solutions induced a significant response in the sensor (Figure 6a). Likewise, addition of L-alanine caused very little response (e2-nm shift) to the replay wavelength of the hologram. This suggests that other carboxylic acids, R-amino acids, and thiols are unlikely to affect the response of the hologram. However, addition of 10.71 mM pyruvate resulted in a ∼13-nm red shift, indicating that pyruvate is able to bind to 3-APB. Funahashi and co-workers38,46 have previously demonstrated that 4-isopropyltropolone, which possesses a functionality similar to pyruvate, can bind to a boronate center. It is assumed that pyruvate is able to bind to 3-APB due to its keto-enol tautomerism, since in its enol form it is able to form two covalent bonds with the boron center. Pyruvate is a potential interfering agent to the 3-APB sensor function; however, only trace levels (∼0.05 mM)46 of pyruvate are present in the blood and, thus, unlikely to interfere significantly with the L-lactate reading. Other selectivity issues do arise, however, when we begin to consider other molecules that may interfere with eventual in vivo L-lactate detection. Boronic acids are known to bind compounds that contain cisdiol moieties;32-36 thus, a sensor that utilizes such a receptor is likely to suffer from interference from such compounds. Lee et (45) Kagawa, S.; Sugimoto, K.-I.; Funahashi, S. Inorg. Chim. Acta 1995, 231, 115-119. (46) Landon, J.; Fawcett, J. K.; Wynn, V. J. Clin. Pathol. 1962, 15, 579-584.
Figure 5. (a) Response of a 15 mol % 3-APB holographic sensor to L-lactate concentrations across the range 2-12 mM with a PBS wash between additions; (b) response of a 15 mol % 3-APB holographic sensor to the stepwise addition of L-lactate from 2 to 12 mM and reversed, repeated twice; (c) comparison of the peak shifts observed for each L-lactate concentration obtained from the previous experiments, a ([) and b (]).
Figure 6. (a) Response of a 15 mol % 3-APB holographic sensor to increasing concentrations of L-lactate ([), thiolactic acid (]), chloroacetic acid (×) (both pH-adjusted), pyruvate (b), L-alanine (-) and glucose (4)29 at pH 7.4. The error bars represent the standard deviation in peak shift (n ) 3). (b) Response of 5 mol % 3-APB holographic sensor to increasing concentrations of L-lactate ([), thiolactic acid (]), chloroacetic acid (×) (both pH-adjusted), pyruvate (b), L-alanine (-), and glucose (4)29 at pH 7.4. The error bars represent the standard deviation in peak shift (n ) 3).
al.29 have previously reported the response of a 12 mol % 3-APB holographic sensor to R1-acid glycoprotein as well as a number of mono- and disaccharides. Although the glycosylated protein did not induce a response in the sensor, the addition of the smaller saccharides resulted in significant red shifts. In terms of this study, only fructose, galactose, and glucose are potential sources of interference, since they are the main monosaccharides present in blood.47 In a healthy adult, typical resting L-lactate concentrations of 0.36-0.75 mM4,48 can rise to 12 mM during very strenuous exercise.48 Comparing this concentration to that of fructose and
galactose, for which the levels are said to be
