Anal. Chem. 1997, 69, 4768-4772
Detecting Biomolecules in Picoliter Vials Using Aequorin Bioluminescence Czarena L. Crofcheck,† Anne L. Grosvenor,† Kimberly W. Anderson,*,† Janet K. Lumpp,‡ Donna L. Scott,§ and Sylvia Daunert*,§
Departments of Chemical and Materials Engineering, Electrical Engineering, and Chemistry, University of Kentucky, Lexington, Kentucky 40506-0055
The quantitative determination of proteins in picolitervolume vials is described. The assay is based on the bioluminescence of the photoprotein aequorin along with photon-counting detection. Using this approach, avidin can be detected at femtomole levels by taking advantage of its inhibitory effect on the bioluminescence signal generated by biotinylated recombinant aequorin. The picoliter vials were fabricated on glass substrates using a laser ablation technique. Parameters that affect the reproducibility of the assay such as the fabrication and calibration of the pipets, the fabrication of the vials, and the composition of the assay solutions were studied. The design of instruments and techniques capable of detecting and quantifying small amounts of biomolecules is essential for gaining further insight into biological processes. Techniques such as microcolumn separations,1 capillary electrophoresis,2-6 and the use of microsensors7-14 have played important roles in the analysis of small-volume samples. Small-volume manipulations have been performed using microtitration techniques with sample volumes in the picoliter and femtoliter ranges.15,16 In addition, recent advances in micromachining techniques have provided invaluable tools for the design and fabrication of microstructures that can be used in microanalysis. For example, small-volume sample vials †
Department of Chemical and Materials Engineering. Department of Electrical Engineering. § Department of Chemistry. (1) Kennedy, R. T.; Oates, M. D.; Cooper, B. R.; Nickerson, B.; Jorgenson, J. W. Science 1989, 246, 57-63. (2) Valaskovic, G. A.; Kelleher, N. L.; McLafferty, F. W. Science 1996, 273, 1199-1202. (3) Orwar, O.; Jardemark, K.; Jacobson, I.; Moscho, A.; Fishman, H. A.; Scheller, R. H.; Zare, R. H. Science 1996, 272, 177-182. (4) Zhang, Y.; Arriaga, E.; Diedrich, P.; Hindsgaul, O.; Dovichi, N. J. J. Chromatogr. 1995, 716, 221-229. (5) Harrison, D. J.; Fluri, K.; Seiler, K.; Fan, Z.; Effenhauser, C. S.; Manz, A. Science 1993, 261, 895-897. (6) Lillard, S. J.; Yeung, E. S. J. Chromatogr. 1997, 689, 321-325. (7) Fan, F.-R. F.; Bard, A. J. Science 1995, 267, 871-874. (8) Gibbon, B. C.; Kropf, D. L. Science 1993, 263, 1419. (9) Tan, W.; Shi, Z.-Y.; Smith, S.; Birnbaum, D.; Kopelman, R. Science 1992, 258, 778-781. (10) Hickman, J. J.; Ofer, D.; Laibinis, P. E.; Whitesides, G. M.; Wrighton, M. S. Science 1991, 252, 688-681. (11) Hughes, R. C.; Ricco, A. J.; Butler, M. A.; Martin, S. J. Science 1991, 254, 74-80. (12) Malinski, T.; Taha, Z. Nature 1992, 358, 676-678. (13) Bratten, C. D. T.; Cobbold, P. H.; Cooper, J. M. Anal. Chem. 1997, 69, 253-258. (14) Clark, R. A.; Hietpas, P. B.; Ewing, A. G. Anal. Chem. 1997, 69, 259-263. (15) Gratzl, M.; Yi, C. Anal. Chem. 1993, 65, 2085-2088. (16) Yi, C.; Huang, D.; Gratzl, M. Anal. Chem. 1996, 68, 1580-1584. ‡
4768 Analytical Chemistry, Vol. 69, No. 23, December 1, 1997
of 118 nL17 and 100 pL18 have been fabricated to be used for sample introduction in capillary electrophoresis. Sample wells with picoliter to attoliter volumes have also been fabricated.19 Aequorin, a calcium-activated photoprotein, has been employed in the highly sensitive determination of intracellular calcium concentrations20 and has recently been used in cell-trafficking studies21 and in the development of competitive binding22-25 and DNA probe assays26 with low detection limits. Key to the low detection limits obtained with aequorin are its intrinsic luminescence properties, which allow aequorin to be detected at subattomole levels. In particular, the bioluminescence of aequorin can be triggered by the addition of Ca2+, which induces a change in the conformation of the protein. This results in the oxidation of its intrinsic chromophore, coelenterazine, and the subsequent emission of photons with a maximum intensity at 469 nm.20 Bioluminescence offers several advantages over other types of detection. For example, in comparison to fluorescence, photodegradation is not a problem because no photoexcitation is required. In addition, background interference from biological and other matrices is much smaller since bioluminescence is much rarer than fluorescence. Because of the high sensitivity associated with bioluminescence detection of aequorin, we have been examining the possibility of using aequorin to quantify biomolecules in very small volumes, approaching the volumes of single cells. Methods for determining analytes within individual cells are necessary in order to study the origin and nature of disease. To this end, a homogeneous binding assay that employs biotinylated aequorin for the quantitative determination of avidin in picoliter-volume vials was designed. EXPERIMENTAL SECTION Reagents. Biotinylated recombinant aequorin (AEQ-biotin), with 2.4 mol of biotin/mol of aequorin, was purchased from Molecular Probes (Eugene, OR). Tris(hydroxymethyl)aminomethane (Tris) was purchased from Research Organics (Cleve(17) Jansson, M.; Emmer, A.; Roeraade, J. J. Chromatogr. 1992, 626, 310-314. (18) Beyer Hietpas, P.; Ewing, A. G. J. Liq. Chromatogr. 1995, 18, 3557-3576. (19) Pantano, P.; Walt, D. R. Chem. Mater. 1996, 8, 2832-2835. (20) Campbell, K. Chemiluminescence; Ellis Horwood: Chichester, England, 1988. (21) Rizzuto, R.; Simpson, A. W.; Brini, M.; Pozzan, T. Nature 1992, 358, 325328. (22) Stults, N. L.; Stocks, N. F.; Rivera, H.; Gray, J.; McCann, R. O.; O’Kanne, D.; Cummings, R. D.; Cormier, M. J.; Smith, D. F. Biochemistry 1992, 31, 1433-1441. (23) Jackson, R. J.; Fujihashi, K.; Kiyono, H.; McGhee, J. R. J. Immunol. Methods 1996, 190, 189-197. (24) Yeh, J. C.; Cummings, R. D. Anal. Biochem. 1996, 236, 126-133. (25) Witkowski, A.; Ramanathan, S.; Daunert, S. Anal. Chem. 1994, 66, 18371840. (26) Galvan, B.; Christopoulos, T. K. Anal. Chem. 1996, 68, 3545-3550. S0003-2700(97)00678-1 CCC: $14.00
© 1997 American Chemical Society
land, OH), EDTA and glycerol were from Fisher, and bovine serum albumin (BSA) and dithiothreitol (DTT) were from Sigma (St. Louis, MO). All chemicals were of reagent grade or better and were used as received. All solutions were prepared using deionized (Milli-Q water purification system, Millipore, Bedford, MA) distilled water and contained 10% glycerol, unless otherwise stated. Solid AEQ-biotin was dissolved with a solution containing 10 mM Tris-HCl, 10 mM EDTA, 1 M KCl, 10 mM MgCl2, and 1 mM DTT with 0.1% (w/v) BSA and 0.1% (w/v) sodium azide, while subsequent dilutions were made using the assay buffer (0.150 M NaCl, 1.0 mM DTT, and 2.00 mM EDTA in 10.0 mM Tris-HCl, pH 8.0, with 0.1% (w/ v) BSA). BSA was added to prevent adsorption of the proteins onto the inner walls of the pipets. The assay buffer was also used to prepare all avidin solutions. Bioluminescence was triggered with a buffered calcium solution (100 mM CaCl2 in 100 mM TrisHCl, pH 7.5). The two buffers, calcium solutions, and avidin solutions were stored at 4 °C, while AEQ-biotin (solid and solutions) was stored at -60 °C. Apparatus. Picoliter-scale experiments were performed using an inverted Zeiss Axiovert 35 microscope (Thornwood, NY) equipped with a Zeiss 20× objective (NA ) 0.75). Microinjections were performed using a Narishige IM-300 microinjector (Sea Cliff, NY). Two micromanipulators (Zeiss and Narishige) were mounted on the microscope stage. Bioluminescence was measured using a modified RM-L microscope photometry system, which includes a single-emission photomultiplier tube (PMT), from Photon Technology International (South Brunswick, NJ). Light emission data were collected and peaks integrated using Felix software, also from Photon Technology International. Equipment used for volume determination included a Hamamatsu SIT video camera (Bridgewater, NJ), a Panasonic Model AG 6500 VCR (Secaucus, NJ), a Sony black-and-white monitor (Tokyo, Japan), and a PIM image-shearing monitor (San Diego, CA). Micropipets were pulled on a Flaming/Brown micropipet puller, Model P-97, made by Sutter Instrument Co. (Novato, CA), using borosilicate glass capillaries (Sutter Catalog No. BF 10078-15) with an outside diameter of 1.0 mm and an inside diameter of 0.78 mm. The picoliter vials were “drilled” into glass slip covers (Sigma, St. Louis, MO) using a laser ablation technique with a KrF laser (248 nm) operating at 10 Hz and under computer control. The vials were washed and stored in assay buffer and used repeatedly. Micropipets. Micropipets were pulled immediately before experimentation and were backloaded using syringes fitted with 26G1/2 needles. Since the fabrication procedure does not produce identical pipets, and because the development of assays requires accurate volume delivery, each pipet had to be individually calibrated. We developed a procedure to roughly calibrate pipets to find time/pressure settings which would produce the desired injection volumes before bioluminescence experiments (see Results and Discussion) and then calibrated them more accurately after the bioluminescence experiments (see below). Exact injection volumes were measured by making aqueous injections into Zeiss immersion oil and recording the image of the suspended aqueous droplets on video tape. Using the imageshearing monitor (calibrated using a stage micrometer), the diameters of the resulting spherical droplets were measured, and the volumes were calculated on the basis of the measured diameters. It was assumed that injection volumes were constant
for the same microinjector settings and the same pipet as long as the pipet remained free from tip clogging and chipping. The latter was verified by inspection under the microscope. Calibration Curves for AEQ-Biotin. Calibration curves relating bioluminescence and the amount of AEQ-biotin were generated by varying the amount of AEQ-biotin injected into picoliter vials containing buffered calcium solution. The PMT was focused on a single picoliter vial loaded with approximately 200250 pL of buffered calcium solution. A micropipet containing AEQbiotin was micromanipulated to the picoliter vial and securely immersed in the calcium solution. The lights were turned off, and the data collection was initiated. Then, the desired amount of AEQ-biotin was injected. The amount of AEQ-biotin injected was varied by varying the volume and/or the concentration of AEQ-biotin injected. Some evaporation of the calcium solution allowed for injection of a total of more than 350 pL into each vial. Once the preceding procedure had been repeated in several picoliter vials, the injection volume was determined using the droplet-in-oil method. This procedure was repeated using various concentrations of AEQ-biotin. For the purpose of quantitation, the total amount of light emitted by the bioluminescence reaction was obtained by integrating the bioluminescence peak. Assay for Avidin. Calibration curves for avidin were generated by using a constant amount of AEQ-biotin and varying the amount of avidin. For each avidin concentration, eight picoliter vials were loaded with approximately 200-250 pL of assay buffer containing 15% (v/v) glycerol, which reduces evaporation and makes subsequent injections easier and more reproducible. Avidin injections of 100 pL were made into four vials, while the remaining four vials received 100 pL of assay buffer. AEQ-biotin (1.0 × 10-6 M) injections of 100 pL were made into each of the eight vials. The vials were allowed to incubate while a calcium pipet was manipulated to the picoliter vials. The rest of the procedure is identical to that used for generating the calibration curve, except that the luminescence was triggered by the injection of 100 pL of the buffered calcium solution. RESULTS AND DISCUSSION Vial Fabrication and Characterization. Two recent papers have reported on the electrochemistry of a model compound (ferrocenecarboxylic acid) in picoliter vials.13,14 Vials used in these studies were fabricated using photolithographic procedures, which typically require many steps and a number of reagents. While the time required to produce vials is reduced by the reuse of a template, changes in the design of the vials require that the whole process be repeated. We have been using a simpler and more versatile procedure for the fabrication of our picoliter vials. The picoliter vials are “drilled” into glass slip covers by laser ablation using an excimer KrF laser with an emission wavelength of 248 nm. This type of laser was chosen due to its ease of operation, high energy output per photon, and flat beam profile. The diameter of the picoliter vials in the current study was 100 µm (rsd < 3%, n ) 8) and represents the upper limit for the laser being used, since it is the approximate size of the laser beam. A photograph of a vial with a diameter of 100 µm is shown in Figure 1. We have fabricated vials with diameters of 63 µm (∼80 pL), and it is theoretically possible to fabricate vials with diameters as small as ∼17 µm (∼2 pL). The depth of the picoliter vials was controlled by the number of pulses used to ablate each vial. The current studies use vials Analytical Chemistry, Vol. 69, No. 23, December 1, 1997
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Figure 1. Photograph of a picoliter volume vial with diameter of 100 µm and a volume of 350 pL.
with depths of ∼45 µm, which can be obtained with 350 laser pulses with a laser power of 26 mW (measured at the source). The relative standard deviation of the depth of vials machined on a given day is
