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Development of Binding Assays in Microfabricated Picoliter Vials: An Assay for Biotin Anne L. Grosvenor,† Agatha Feltus,‡ Richard C. Conover,§ Sylvia Daunert,*,‡,§ and Kimberly W. Anderson*,†
Departments of Chemical and Materials Engineering, Chemistry, and Pharmaceutical Sciences, University of Kentucky, Lexington, Kentucky 40506
A homogeneous binding assay for the detection of biotin in picoliter vials was developed using the photoprotein aequorin as the label. The binding assay was based on the competition of free biotin with biotinylated aequorin (AEQ-biotin) for avidin. A sequential protocol was used, and modification of the assay to reduce the number of steps was examined. Results showed that detection limits on the order of 10-14 mol of biotin were possible. Reducing the number of steps provided similar detection limits but only if the amount of avidin used was decreased. These binding assays based on picoliter volumes have potential applications in a variety of fields, including microanalysis and single-cell analysis, where the amount of sample is limited. In addition, these assays are suitable for the high-throughput screening of biopharmaceuticals. The need to analyze samples in increasingly smaller volumes, including those of single cells, has driven research toward the miniaturization of analytical techniques.1 Scientists are looking to miniaturization for preparing microsensors,2,3 for fast-throughput diagnostics,4,5 for genomics and proteomics, and for the analysis of individual cells. Small-volume analysis requires detection limits on the femtomole to attomole and even subattomole levels. Methods with such detection limits often involve either the use of hazardous radioactive compounds, which present disposal problems, or the use of chemiluminescence detection, which must be carried out at extreme pH, making them unsuitable for highthroughput analyses or for use with biological samples. Therefore, a need exists for small-volume analysis techniques that have sufficient detection limits and that are reliable, fast, and compatible with biological systems. Scientists currently use tools such as microscopic diffusional titrations,6,7 mass spectrometry,8 microelectrodes,9 and microcolumn techniques5,9-15 to analyze small-volume samples. Addition†
Department of Chemical and Materials Engineering. Department of Pharmaceutical Sciences. § Department of Chemistry. (1) Xue, Q.; Yeung, E. S. J. Chromatogr., A 1994, 661, 287-295. (2) Li, L.; Walt, D. R. Anal. Chem. 1995, 67, 3746-3752. (3) Achtnich, U. R.; Tiefenauer, L. X.; Andres, R. Y. Biosens. Bioelectron. 1992, 7, 279-290. (4) Li, P. C. H.; Harrison, D. J. Anal. Chem. 1997, 69, 1564-1568. (5) Bratten, C. D. T.; Cobbold, P. H.; Cooper, J. M. Anal. Chem. 1997, 69, 253-258. (6) Gratzl, M.; Yi, C. Anal. Chem. 1993, 65, 2085-2088. (7) Yi, C.; Huang, D.; Gratzl, M. Anal. Chem. 1996, 68, 1580-1584. ‡
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ally, advances in micromachining, such as photolithography,5,10,16,17 photopolymerization,18 and chemical etching,19 have made it possible to prepare sample vials with nanoliter,19 picoliter,16 and even attoliter volumes.18 These microstructures can be used in conjunction with current analytical methods to produce smaller and simpler diagnostic testing devices. For example, microvials can hold samples for introduction in capillary electrophoresis10 or individual cells can be manipulated and reacted with various chemicals using electroosmotic pumping on micromachined glass chips.4 Another method that could be used for single-cell analysis involves the production of miniature electrochemical cells capable of analyzing picomole, and possibly femtomole, levels of analytes in subnanoliter samples.5 Other researchers have performed electrochemical detection in vials as small as 1 pL.16 Such methods are, however, limited to analysis of electrochemically active species. One way to detect other types of molecules in small volumes is to develop highly sensitive binding assays. Our work in this area has focused on using the photoprotein aequorin as a label in developing such assays. Aequorin emits a flash of light (λmax ) 469 nm) when bound to calcium ions.20 The total time required to release the majority of photons is less than 3 s, leading to extremely low backgrounds, which permit detection of attomole amounts of the protein.21 For small-volume analyses, aequorin has been used extensively as an indicator of intracellular calcium22-25 and has also been used to calibrate micropipets26 and to detect (8) Odom, R. W.; Lux, F. R. H.; Chu, P. K.; Niemeyer, I. C.; Blattmer, R. J. Anal. Chem. 1988, 60, 2070-2075. (9) Paras, C. D.; Kennedy, R. T. Electroanalysis 1996, 9, 203-208. (10) Jansson, M.; Emmer, A.; Roeraade, J.; Lindberg, U.; Hok, B. J. Chromatogr. 1992, 626, 310-324. (11) Chen, G.; Ewing, A. G. Crit. Rev. Neurobiol. 1997, 11, 59-90. (12) Ewing, A. G.; Mesaros, J. M.; Gavin, P. F. Anal. Chem. 1994, 66, 527A536A. (13) Fishman, H. A.; Scheller, R. H.; Zare, R. N. J. Chromatogr., A 1994, 680, 99-107. (14) Hietpas, P. B.; Ewing, A. G. J. Liq. Chromatogr. 1995, 18, 3557-3576. (15) Hogan, B. L.; Yeung, E. S. Anal. Chem. 1992, 64, 2841-2845. (16) Clark, R. A.; Hietpas, P. B.; Ewing, A. G. Anal. Chem. 1997, 69, 259-263. (17) Mooney, J. F.; Hunt, A. J.; McIntosch, J. R.; Liberko, C. A.; Walba, D. M.; Rogers, C. T. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 12287-12291. (18) Healey, B. G.; Foran, S. E.; Walt, D. R. Science 1995, 269, 1078-1080. (19) Pantano, P.; Walt, D. R. Chem. Mater. 1996, 8, 2832-2835. (20) Shimomura, O. Biol. Bull. 1995, 189, 1-5. (21) Casadei, J.; Powell, M. J.; Kenten, J. H. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 2047-2051. (22) Blinks, J. R. Methods Enzymol. 1989, 172, 165-203. (23) Blinks, J. R.; Mattingly, P. H.; Jewell, B. R.; VanLeeuwen, M.; Harrer, G. C.; Allen, D. G. Methods Enzymol. 1979, 57, 292-328. 10.1021/ac991289+ CCC: $19.00
© 2000 American Chemical Society Published on Web 04/29/2000
avidin.27 In this last assay, biotinylated aequorin (AEQ-biotin) was used to detect avidin on the basis of the inhibitory effect of avidin binding on the bioluminescence of the aequorin, a phenomenon which was also used to detect 4 amol of biotin in microliter volumes in a conventional luminometer.28 The assay depends on AEQ-biotin competing with biotin for avidin binding sites. The more biotin present, the less avidin is bound to AEQ-biotin and a larger bioluminescence signal is observed. The objective of this study was to demonstrate that binding assays for biomolecules can be developed in picoliter volumes and that these assays yield detection limits that make them suitable for small-sample volumes (e.g., single cells). In this respect, we chose to develop an assay using aequorin as the label and biotin as our model analyte. Biotin was chosen because of our previous experience in the development of the assay in microliter volumes. A sequential protocol was used in which the free biotin and avidin were allowed to interact prior to the addition of AEQ-biotin. Additionally, modification of the assay by premixing the avidin and calcium was examined in order to reduce the number of injections into each vial. EXPERIMENTAL SECTION Reagents. Biotinylated recombinant aequorin (AEQ-biotin), with 2.4 mol of biotin/mol of aequorin, and avidin were purchased from Molecular Probes (Eugene, OR). Tris(hydroxymethyl)aminoethane (Tris) was purchased from Research Organics (Cleveland, OH). The disodium salt of ethylenediaminetetraacetic acid (Na2EDTA) and glycerol were from Fisher. Bovine serum albumin (BSA), dithiotreitol (DTT), biotin, and all other chemicals were from Sigma (St. Louis, MO). All solutions were prepared using deionized (Milli-Q water purification system, Millipore, Bedford, MA) distilled water and, unless otherwise stated, contained 10% glycerol. AEQ-biotin was dissolved in 10 mM Tris-HCl, pH 7.5, containing 10 mM EGTA, 1.0 M KCl, 10 mM MgCl2, 0.1% (w/v) NaN3, and 0.1% (w/v) BSA to a concentration of 1.2 × 10-6 M. Dithiotreitol (1.0 mM) was added to prevent oxidation of aequorin. The stock solution was then divided into aliquots and frozen at -80 °C. Individual aliquots of the stock solution were removed when needed and diluted to the necessary concentration using 50 mM Tris-HCl, pH 7.8, containing 0.15 M NaCl, 0.1% (w/v) NaN3, 20 mM Na2EDTA, 10% (v/v) glycerol, and 0.1% BSA. This buffer was also used to prepare and dilute avidin and biotin stock solutions. CaCl2 (250 mM) was used to trigger the bioluminescence of AEQ-biotin and was dissolved in either the avidin solution or the assay buffer, depending on the protocol of the assay. Equipment. The experiments described in this paper were performed using an inverted Axiovert 35 microscope (Zeiss, Thornwood, NY) equipped with a Zeiss 20× objective (NA ) 0.75). An IM-300 air pressure driven microinjector (Narashige, Sea Cliff, NY), was used for injecting reagents. Micropipets were pulled on (24) Rizzuto, R.; Simpsoin, A. W.; Brini, M.; Pozzani, T. Nature 1992, 358, 325327. (25) Miller, A. L.; Karplus, E.; Jaffe, L. F. Methods Cell Biol. 1994, 40, 305-335. (26) Grosvenor, A. L.; Crofchek, C. L.; Anderson, K. W.; Scott, D. L.; Daunert, S. Anal. Chem. 1997, 69, 3115-3118. (27) Crofcheck, C. L.; Grosvenor, A. L.; Anderson, K. W.; Lumpp, J. K.; Scott, D. L.; Daunert, S. Anal. Chem. 1997, 69, 4766-4722. (28) Witkowski, A.; Ramanthan, S.; Daunert, S. Anal. Chem. 1994, 66, 18371840.
a Flaming/Brown micropipet puller, made by Sutter Instrument Co. (Novato, CA), using borosilicate glass capillaries (Sutter Catalog No. BF 100-78-15) with outside diameters of 1.0 mm and inside diameters of 0.78 mm. Micropipets were pulled immediately before experimentation and were backloaded using Eppendorf Microloader tips. Bioluminescence was quantified using a modified Rm-L microscope photometry system, which included a singleemission photomultiplier tube, 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 an SIT video camera (Hamamatsu, Bridgewater, NJ), an AG 6500 VCR (Panasonic, Seaucus, NJ), a black and white monitor (Sony, Tokyo), and an image shearing monitor (IPM, San Diego, CA). Micropipet Calibration. Because the fabrication procedure did not produce identical micropipets and there was always the possibility that a micropipet might be chipped during handling, each micropipet was individually calibrated in terms of volume using either of two methods. In the first, the micropipet tip was placed in a droplet of oil and an aqueous solution (i.e., AEQ-biotin, avidin, etc.) was injected. The diameter of the resulting spherical droplet was then measured using a calibrated image shearing monitor, and the volume was calculated. Injection parameters (time and pressure) could then be adjusted to obtain the desired volume. In the other method, used by Crofcheck et al.,27 the aqueous solution contained in the micropipet was injected onto a coverslip. The diameter of the resulting droplet was measured, and the injection volume was determined by comparison to a calibration curve relating droplet size to injection volume in oil. The advantage of the second method is that it is faster than the first and can be performed at any time during the experiment to check for changes in injection volume. Both methods gave consistent results. In all experiments, the injection volume for each micropipet was rechecked by injection into oil after experimentation. Fabrication of the Microvials. Vials with approximate volumes of 350 pL were drilled onto glass coverslips (Sigma, St. Louis, MO) by laser ablation using a KrF laser (248 nm) operating at 10 Hz and under computer control. The microvials were prepared in 4 × 5 arrays with 4 arrays on a single coverslip (Figure 1). They had diameters of approximately 100 µm and depths of 45 µm. The microvials could be used up to 50 times before becoming chipped and irregular. After experiments, the coverslip was washed with deionized water and stored in assay buffer. Sequential Assay. A sequential assay for biotin was performed by incubating varying amounts of biotin with a constant amount of avidin in the microvials for 5 min prior to injecting a constant amount of AEQ-biotin (Figure 2a). Biotin was injected into 10 microvials, and an equal volume of assay buffer was injected into another 10 microvials. The concentration of biotin ranged from 2 × 10-3 to 2 × 10-9 M; the injection volume was typically 200250 pL. Avidin was injected from a separate micropipet into all microvials. AEQ-biotin (1 × 10-16 mol) was injected into each microvial via a third micropipet and allowed to incubate for an additional 5 min. Next, the PMT was focused on an individual microvial and light collection started. An excess of CaCl2 (250 mM) was injected into the microvial and light collection continued for Analytical Chemistry, Vol. 72, No. 11, June 1, 2000
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Figure 1. SEM image of an array of picoliter vials used for smallscale assay. The volume of each vial is approximately 350 pL.
Figure 2. Schematic diagram of the difference between the sequential assay (a) with a 5 min incubation of AEQ-biotin in the vial and (b) without the incubation. The identities of the injections are shown below the pictures.
15 s to ensure complete discharge of AEQ-biotin. This step was repeated for all microvials. Typically, 150-200 pL of AEQ-biotin and avidin was injected and was kept constant for all concentrations of biotin in a single dose-response curve. Two different amounts of avidin were used (2.2 × 10-14 and 1.2 × 10-14 mol) to generate dose-response curves. The relative signal for each concentration of biotin was calculated as described in the Analysis section below. Dose-response curves were constructed by plotting the relative signal vs the log of the amount of biotin added. Sequential Assay with Simultaneous Injection of Avidin and Calcium. Because the assays described above required four injections per microvial, assays were also performed in which the avidin and calcium were premixed prior to injection (Figure 2b). Before this assay was perfomred an association study was performed to determine the amount of avidin that provided the maximum inhibition of AEQ-biotin. A constant amount of AEQbiotin (200 pL of a 5.8 × 10-7 M solution) was injected into microvials containing 250 pL of calcium and varying amounts of avidin. Bioluminescence was measured as described above, and the percent inhibition was determined by dividing the bioluminescence signal in the presence of avidin by that in the absence 2592
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of avidin and subtracting from 1. The binder dilution curve was then constructed by plotting the percent inhibition vs the amount of avidin added to the microvial. For the sequential assay itself, biotin was first injected into 10 microvials and an equal volume of assay buffer was injected into another 10 microvials. Avidin (1.7 × 10-14 mol) with 250 mM CaCl2 was injected from a separate micropipet into all microvials and allowed to incubate with the free biotin for 5 min. AEQ-biotin (2.9 × 10-16 mol) was injected last into the microvials, and light was collected as described above for 15 s. This step was repeated for all microvials. The relative signal for each concentration of biotin was calculated as described in the Analysis section below. Dose-response curves were constructed by plotting the relative signal vs the logarithm of the amount of biotin added. Analysis. The resulting bioluminescence peak for each microvial was integrated for 15 s, and the background was subtracted. The average light intensity from five to tem microvials for each concentration of biotin was compared to the average light intensity obtained from injecting the same volume of AEQ-biotin into microvials containing avidin and assay buffer with no biotin. The effect of biotin at each concentration was evaluated by dividing the average light intensity for each biotin standard by the average light intensity without biotin. Because each value was normalized by the theoretical maximum inhibition, the error due to slight differences in injection volume over the course of an experiment was reduced. The ratio of the average light intensity was then computed for each biotin concentration. The same procedure for analysis was repeated for all experiments. The detection limit for biotin was defined as the minimum amount of biotin examined that showed a significant increase (S/N ) 2) in the AEQ-biotin intensity compared to the control sample containing avidin and AEQ-biotin only. RESULTS AND DISCUSSION In a typical assay, reagents are incubated for a certain amount of time after mixing of all components. In many cases, the samples are also mixed during the incubation itself. One advantage of small-volume assays is that the diffusion of the components across the microvial makes mixing unnecessary. For example, a molecule with a diffusion coefficient of 1 × 10-5 cm2/s would take just over 1 s to cross a 45 µm diameter microvial but 50 000 s to cross a 1 cm test tube. Added to this is the force of subsequent injections on the liquid in the microvial; it is assumed that complete mixing of the reagents takes place. In our assays, this is verified by examination of the bioluminescence peak of the AEQ-biotin itself. AEQ-biotin exhibits flash kinetics, releasing nearly all photons within 5 s. In all cases, the width of the bioluminescence peak from the microvials was 5 s. The first experiments focused on assays in which the avidin and biotin were incubated for 5 min before the addition of AEQbiotin (Figure 2a). A dose-response curve from the sequential assay using 2.2 × 10-14 mol of avidin is shown in Figure 3. This amount of avidin was selected on the basis of the amount necessary to provide maximum inhibition of AEQ-biotin as seen in the binder dilution curve described by Crofcheck et al.,27 which uses the same amount of AEQ-biotin. Using this assay, the detection limit was observed to be 4.0 × 10-14 mol of biotin.
Figure 3. Dose response curve for biotin using a sequential assay with a 5 min incubation using either 2.2 × 10-14 (white diamonds) or 1.2 × 10-14 mol (black squares) of avidin. The amount of AEQ-biotin used was 1 × 10-16 mol. Error bars represent standard errors (n ) 5-8).
On the basis of microliter-volume assays, Witksowki et al.28 reported that the best detection limits are usually reached when the concentration of avidin used is 85% of the amount required to reach maximum inhibition of the bioluminescence signal generated by the AEQ-biotin conjugate. Using a model binder dilution curve (unpublished results), this would correspond to 25.5% inhibition and 1.2 × 10-14 mol of avidin. Hence, a second doseresponse curve was generated using 1.2 × 10-14 mol of avidin in the sample. As seen in Figure 3, the assay with less avidin has response characteristics similar to those of the first curve. However, less biotin (1 × 10-14 mol) is required to observe a significant decrease in inhibition. This is to be expected, since there is a smaller amount of avidin to bind the unlabeled biotin in solution. The less avidin in the sample, the less biotin required to block the avidin binding sites. The linear range of the assay is also greater, increasing to 1.5 orders of magnitude. Therefore, decreasing the amount of avidin by half in the picroliter-volumescale assay not only improves the detection limit of the assay by 75% but also gives a greater sensitivity. These sequential assays require four injections into the microvial (namely, injections of biotin, avidin, AEQ-biotin, and calcium) and need an incubation step of AEQ-biotin with avidin for 5 min prior to light collection (Figure 2a). The purpose of the incubation time was to be certain that the AEQ-biotin had reached steady state with avidin. Avidin binds biotin very quickly with a rate constant of 108 s-1,29 while calcium also binds aequorin quickly, but with a slower rate constant of 100 s-1.30 If the mixing is sufficient in the microvials, then the avidin should bind the AEQbiotin before the light reaction occurs, thereby making it possible to omit the incubation step. This should provide a reduction in micromanipulations, which is not only the greatest time-consuming factor but also the single greatest source of error in the assay (data not shown). Reducing the number of injections would also make the assay more amenable to high-throughput screening and (29) Green, M. N. In Avidin-Biotin Technology; Wilchek, M., Bayer, E. A., Eds.; Academic Press: San Diego, CA, 1990; Vol. 184, pp 51-68. (30) Hirano, T.; Mizoguchi, I.; Yamaguchi, M.; Chen, F.; Ohashi, M.; Ohmiya, Y.; Tsuji, F. I. J. Chem. Soc., Chem. Commun. 1994, 165-167.
Figure 4. Binder dilution curve with simultaneous addition of calcium and avidin. The amount of AEQ-biotin used was 200 pL of a 5.8 × 10-7 M solution. Error bars represent standard errors (n ) 5-8).
single-cell analysis, where the cell can only accept a certain number of injections. The sequential assay was thus performed without incubation of AEQ-biotin and avidin. This was accomplished by simultaneously exposing AEQ-biotin to avidin and calcium (Figure 2b). Prior to undertaking these studies, we constructed a new binder dilution curve without incubation to determine the optimum amount of avidin to be used in the construction of the doseresponse curve. Figure 4 shows the binder dilution curve obtained upon using 200 pL of AEQ-biotin (5.8 × 10-7 M), varying the amounts of avidin, and omitting the incubation step. It was observed that this binder dilution curve is slightly different from the one obtained using an incubation time of 5 min (see Figure 6 of Crofcheck et al.27). In this case, the maximum inhibition was observed to be 22%, while in the binder dilution curve with incubation, the maximum inhibition obtained was 30%. In addition, more avidin was required to reach maximum inhibition of the bioluminescence signal emitted by the AEQ-biotin conjugate. When the incubation step was omitted, the curve began to reach a plateau at 20 fmol of avidin, compared to leveling off at about 10 fmol with incubation. Other studies (data not shown) have indicated that a 5 min incubation is necessary to reach a plateau in the inhibition of the AEQ-biotin. These phenomena can be attributed to two factors: diffusional limitations of avidin and AEQbiotin in the picoliter-volume vials and the time necessary for the avidin-biotin binding event. In this study, where the AEQ-biotin was reacted with calcium and avidin at the same time, the calcium concentration was much higher than the avidin concentration. Thus, a higher concentration of avidin may be necessary to bind AEQ-biotin before the calcium can initiate the triggering of the emission of light. The concentration of calcium in the vials was 3 orders of magnitude higher than the concentration of avidin; therefore, the diffusional limitations of calcium reaching AEQbiotin are smaller than those for avidin reaching AEQ-biotin. The dose-response curve generated from the sequential assay without incubation is shown in Figure 5. The assay was performed using 3 × 10-16 mol of AEQ-biotin and 1.7 × 10-14 mol of avidin. The shape of the curve is slightly different than that observed in the assay with incubation (Figure 3) in terms of sensitivity (the curve without incubation is somewhat less sensitive); however, Analytical Chemistry, Vol. 72, No. 11, June 1, 2000
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Figure 5. Dose response curve for biotin using a sequential assay without incubation with 1.7 × 10-14 mol of avidin. The amount of AEQbiotin used was 2.9 × 10-16 mol. Error bars represent standard errors (n ) 5-8).
the detection limits are approximately the same (1.1 × 10-14 mol of biotin). Decreasing the number of injections, as expected, also significantly decreases the overall error, compared to the assay using four injections (see Figures 3). The most important factor is the simplification of assay performance, which reduces the overall time it takes to perform the assay, while sacrificing some of the sensitivity of the assay. In summary, we have developed a homogeneous assay for biotin in picoliter-volume microvials. A sequential protocol was developed that yields femtomole detection limits. The assay requires two incubations involving four separate injections: First,
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the free biotin must be incubated with avidin before the second in which AEQ-biotin is introduced and a further 5 min incubation is performed before triggering with Ca2+. It was shown that the assay could be performed by premixing the avidin and calcium, eliminating the incubation of AEQ-biotin and avidin and reducing the number of injections. We could obtain detection limits similar to those observed with incubation. Further, the last reagent to be added is AEQ-biotin, reducing the chance for adsorption of this reagent onto the walls of the vial which could, in some situations, lead to a decrease in signal. To the best of our knowledge, this is the first demonstration of a binding assay for a small biomolecule such as biotin performed in picoliter-volume vials. Binding assays based on picoliter volumes have potential applications in a variety of fields such as microanalysis and singlecell analysis, where the amount of sample is limited. In addition, these assays may be suitable for high-throughput screening of biopharmaceuticals. ACKNOWLEDGMENT This work was supported by the Department of Energy (Grant DE-FG05-95ER62010), the National Institutes of Health (Grant GM47915, to S.D.), and the National Science Foundation IGERT Program (Grant DGE-9870691). A.L.G. acknowledges support from a National Science Foundation Graduate Engineering Education Fellowship. A.F. is a National Science Foundation Graduate Fellow. Received for review November 10, 1999. Accepted March 8, 2000. AC991289+
