Anal. Chem. 1997, 69, 3115-3118
Technical Notes
Calibration of Micropipets Using the Bioluminescent Protein Aequorin Anne L. Grosvenor,† Czarena L. Crofcheck,† Kimberly W. Anderson,*,† Donna L. Scott,‡ and Sylvia Daunert*,‡
Department of Chemical and Materials Engineering and Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506-0055
A new method for calibrating micropipets and determining accurate injection volumes using a pressure-based injector has been developed. This method employs the bioluminescent protein aequorin and can be used to determine injection volumes as small as 3 pL. The calibration plots are linear over at least 3 orders of magnitude. In contrast to conventional micropipet calibration methods that employ fluorescent molecules, the present method produces small background signals. Although microinjection using micropipets has been used to introduce compounds into cells for many years, only a few reports concerning the reproducibility of micropipet injections and the relationship between injection time and pressure on the injection volume have been published.1-5 The scarcity of quantitative studies may be due to the fact that many researchers are only interested in injecting a large excess of the probe molecule into the cell, and they are not concerned with exactly how much is injected. Often, these researchers rely on purely qualitative observations as the criteria for the evaluation of injected volumes. The increased interest of analytical chemists in microanalyses and single-cell analyses requires reproducible injection or delivery of reagents into cells or sample streams in most cases. In that respect, we are interested in developing methods for controlling and measuring the injection volume for picoliter to nanoliter volume injections of chemical and biochemical species. Previous work involving the microinjection process used a variety of approaches. Van Dongen1 and Schnorf et al.2 used the diameter of drops formed in paraffin to calibrate the volume injected by a micropipet. While this method is widely employed and useful for studying the injection process, it provides no signal that could be used to determine actual volumes injected into cells. McCaman et al. used a radioactive tracer to quantify the injection volume.3 This method could be used for determining volumes of solution injected into cells, but it is undesirable because of the added * E-mail:
[email protected]. Phone: (606) 257-7060. Fax: (606) 3231069. † Department of Chemical and Materials Engineering. ‡ Department of Chemistry. (1) van Dongen, P. A. M. J. Neurosci. Methods 1984, 10, 281-291. (2) Schnorf, M.; Potrykus, I.; Neuhaus, G. Exp. Cell Res. 1994, 210, 260-267. (3) McCaman, R. E.; McKenna, D. G.; Ono, J. K. Brain Res. 1977, 136, 141147. (4) Lee, G. J. Cell Sci. 1989, 94, 443-447. (5) Minaschek, G.; Bereiter-Hahn, J.; Bertholdt, G. Exp. Cell Res. 1989, 183, 434-442. S0003-2700(97)00123-6 CCC: $14.00
© 1997 American Chemical Society
precautions associated with the use of radioactive probes. Lee4 and Minaschek et al.5 used fluorescent probes, which are also quite sensitive and eliminate the necessity of using radioactive material. However, the use of fluorescent probes is limited by background fluorescence from biological samples, which increases the uncertainty at low volumes. This paper describes a new method for calibrating micropipets and determining injection volumes using aequorin, a photoprotein found naturally in the jellyfish Aequorea victoria.6,7 When aequorin binds calcium, it undergoes conformational and chemical changes, which result in the oxidation of the prosthetic group of the protein and the emission of light. The bioluminescence exhibits flashtype kinetics, and the emission peak occurs at 469 nm.6,7 Since its discovery, aequorin has been widely used in the determination of calcium concentrations within cells8 and has recently been used to monitor gene expression.9 Aequorin has also been used as a label in the development of highly sensitive immunoassays and competitive binding assays.10-12 Aequorin offers several advantages when used in determining the volumes injected by micropipets.8 First, since no exciting light is required, the problem of photobleaching is avoided. Likewise, background signals associated with the configuration of the instrument are considerably reduced. In addition, since bioluminescence is much rarer than fluorescence, the number of possible background interferences from biological samples is much smaller. Finally, the photons emitted by aequorin bioluminescence reflect the total number of molecules of the photoprotein and are, therefore, a direct measure of injection amounts. EXPERIMENTAL SECTION Reagents. Aequorin was obtained from Molecular Probes (Eugene, OR). Tris(hydroxymethyl)aminomethane (Tris) was purchased from Research Organics (Cleveland, OH), NaN3 from Janssen (Flanders, NJ), glycerol from Fisher (Pittsburgh, PA), and ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA) from (6) Shimomura, O. Biochem. Biophys. Res. Commun. 1995, 211, 359-363. (7) Ashley, C. C., Campbell, A. K., Eds. Detection and Measurement of Free Ca2+ in Cells; Elsevier: New York, 1979. (8) Miller, A. L; Karplus, E.; Jaffe, L. F. Methods Cell Biol. 1994, 40, 305-338. (9) Inouye, S.; Tsuji, F. F. Anal. Biochem. 1992, 201, 114-118. (10) Witkowski, A.; Ramanathan, S.; Daunert, S. Anal. Chem. 1994, 66, 18371840. (11) Stults, N. L; Stocks, N. F.; Rivera, H.; Gray, J.; McCann, R.; Kane, D.; Cummings, R. D.; Cormier, M. J.; Smith, D. F. Biochemistry 1992, 31, 1433-1441. (12) Galvan, B.; Christopoulos, T. K. Anal. Chem. 1996, 68, 3545-3550.
Analytical Chemistry, Vol. 69, No. 15, August 1, 1997 3115
Aldrich (Milwaukee, WI), bovine serum albumin (BSA) and dithiothreitol were from Sigma (St. Louis, MO). All chemicals were reagent grade or better and were used as received. Solutions were prepared with water purified by distillation followed by deionization using a Milli-Q purification system (Millipore, Bedford, MA). Apparatus. Experiments were performed using an inverted Zeiss Axiovert 35 microscope (Thornwood, NJ) equipped with a 20× objective (NA of 0.75). A Narishige micromanipulator (Sea Cliff, NY) was mounted on the microscope stage. Microinjections were performed using an IM-300 microinjector (Narishige). The micropipets were pulled from borosilicate capillaries of 1.0-mm outer diameter and 0.78-mm inner diameter (Sutter Instrument Co., Novato, CA) on a Flaming/Brown micropipet puller, Model P-97 (Sutter). The inner diameter of the pulled micropipets was determined using the bubble pressure method2 and refers to the hole at the micropipet tip. Bioluminescence was measured using a photomultiplier tube from Photon Technology International (South Brunswick, NJ) and Felix software (Photon Technology International). This software was also used to integrate the response peaks. Equipment used for measuring microdroplet diameters for volume determinations included a Hamamatsu SIT video camera (Bridgewater, NJ), a Panasonic Model AG 6500 VCR (Secaucus, NJ), an image shearing monitor (PMI, San Diego, CA), and a black and white Sony monitor (Tokyo, Japan). Procedures. Aequorin was diluted using the following buffer: 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. Dithiothreitol (1.0 mM) was added to help prevent oxidation of the aequorin. Glycerol was also added to give a final concentration of 20% (v/v). Micropipets were backfilled using a 1-mL syringe with a 26-gauge needle. For the bioluminescence experiments, a relatively large volume (∼100 µL) of 0.100 M CaCl2 in 100 mM Tris-HCl, pH 7.5, was placed on a microscope cover slip (obtained from Sigma). For each point on the calibration plot, three to six injections using the same injection time and pressure settings were made with a 1 × 10-6 M aequorin solution, and the resulting bioluminescence signal was collected. The area that corresponds to the bioluminescence peak was determined through integration using the Felix software. Typically, injections for the same settings were made in succession, while the emitted bioluminescence was continually monitored. Injection time (or pressure) was then changed to give a different injection volume, and the procedure was repeated. This entire procedure was repeated until bioluminescence data for all desired points had been obtained. Next, a drop of Zeiss immersion oil was placed on a new cover slip. Aequorin solution was injected into the oil using one of the settings that had been used in the bioluminescence experiments, and the resulting microdroplet was videotaped. It should be noted that the injected aqueous solution of aequorin is immiscible with the oil drop. Therefore, the spherical aequorin aqueous drop is suspended in the oil. This procedure was repeated two to five times for each time/pressure setting used in the bioluminescence experiments. After completion of injections, the image shearing monitor was employed to visualize the recorded drops as well as to measure the diameter of each imaged drop. Prior to measurement of the diameters, the shearing monitor had been calibrated with a stage micrometer. Then, the volumes of the drops were 3116 Analytical Chemistry, Vol. 69, No. 15, August 1, 1997
Figure 1. Bioluminescence produced by injection of three aliquots (each 8.7 pL) of 1 × 10-6 M aequorin into a solution containing 0.100 M calcium in 100 mM Tris-HCl, pH 7.5.
calculated based on the measured diameters. Outliers in the bioluminescence and volume determination experiments were eliminated using the Q-test at the 95% confidence level. Large-volume experiments were performed on an Optocomp I luminometer (GEM Biomedical, Carrborro, NC) as described in ref 10 using aequorin. RESULTS AND DISCUSSION In order to develop reliable analytical techniques for microanalysis, the issue of reproducibility in the delivery of the reagents is critical. For this purpose, Kennedy and Jorgenson developed a pneumatic microsyringe that was capable of injecting volumes as low as 0.245 nL in a very reproducible manner.13 The development of new microinjectors by several different manufacturers has allowed researchers to inject very small volumes of reagents into a variety of biological samples. Although the manufacturers claim that volumes of less than 1 pL can be injected, there is a general lack of experimental data showing that such low-volume injections can be made reproducibly. This prompted us to develop a method for evaluating the reproducibility of picoliter-volume injections. Our method employs a commercial pressure-based microinjector in conjunction with the photoprotein aequorin. In the system developed, aequorin was injected into a calcium solution on a microscope cover slip as described in the Experimental Section. The injection of the aequorin solution was accomplished by applying constant pressure to the micropipet held by the pressure-based microinjector for a controlled period of time. Then, the bioluminescence emitted by aequorin was measured with a photon-counting photomultiplier. By varying the injection time (or applied pressure), different volumes of aequorin could be injected. Figure 1 shows a representative series of three injections of 8.7 pL of aequorin into the calcium solution using the same micropipet. It should be noted that these data were taken near the detection limit of our system (vide infra). As can be seen in this figure, even when such a low volume of aequorin was injected (8.7 pL is equivalent to an injection of 8.7 amol of aequorin) the typical flash-type kinetics characteristic of aequorin bioluminescence can be observed. Indeed, there is an initial burst of bioluminescence signal followed by an exponential decay to background level. With regard to reproducibility, the relative standard deviation of the total bioluminescence emission from the injections shown in this figure was 17%. Relative standard deviations for injections in the tens of picoliter range are ∼10% and average ∼5% at larger volumes. (13) Kennedy, R. T.; Jorgenson, J. W. Anal. Chem. 1988, 60, 1521-1524.
Figure 2. Calibration plot that relates the bioluminescence of injected aequorin to the volume. Conditions are as in Figure 1. Peak areas were obtained by integrating the area under each bioluminescence peak. Standard deviations are plotted for all points but are, in most cases, smaller than the symbols. The inset shows the plot at the lower injection volumes.
As mentioned previously, bioluminescent techniques have associated background signals that are much lower than those of methods that employ fluorescent probes. A background of ∼5500 counts/s or less is commonly observed for our system in a bioluminescence mode. This is ∼10-fold lower than the background signal typically obtained when using the same microscopy setup in a fluorescence mode (data not shown). The transience (i.e., the flash-type kinetics) of the bioluminescence signal generated by aequorin can also aid in distinguishing the signal from the background. As described in the Experimental Section, the volume of aequorin that corresponds to a given injection time (or pressure) setting was determined by injecting the aequorin solution into Zeiss immersion oil. The spherical microdroplet was videotaped, and the diameter of the droplet was measured using a shear monitor. Then, the volume of the microdroplet was calculated from the diameter obtained by this measurement. Relative standard deviations for the volume determinations using this approach averaged 6.9%. Thus, a large part of the observed standard deviation in our bioluminescence method may be due to the injection reproducibility. A calibration plot that relates the bioluminescence signal of aequorin (obtained from integration of each bioluminescence peak) to the injected volume is given in Figure 2. As shown in this figure, as the volume of aequorin increases, the bioluminescence signal increases in a linear fashion. The inset in Figure 2 shows that the linearity extends to picoliter injection volumes. The limit of detection calculated from this plot and the corresponding standard deviations is 3 pL, which corresponds to 3 × 10-18 mol of aequorin. This is comparable to the detection limit previously observed for biotinylated aequorin10 and aequorin (see below) using a conventional luminometer and a 100 µL sample volume. In a separate experiment, we measured bioluminescence with a luminometer (used in conventional bioluminescence assays) using the same amounts of aequorin as those employed in microscopy experiments. For the microscopy experiments, the different amounts of aequorin were obtained by using different volumes (in the picoliter to nanoliter range) of 1 × 10-6 M aequorin. The luminometer experiments used larger volumes (100 µL) but smaller concentrations of aequorin than the micro-
Figure 3. Calibration plots that relate the bioluminescence signal to the amount of aequorin present as determined by the microscopy setup ([) and the Optocomp I luminometer (O). Inset shows luminometer peak areas (logarithm of the peak area in counts) plotted against microscope peak areas (logarithm of the peak area in counts) for equal amounts of aequorin.
Figure 4. Injection volume as a function of injection time for micropipets with inner diameters of 0.29 (O) and 0.18 (9) µm. Injection pressure was 20 psi. Each point represents a single injection. The method for determining volume is described in the Experimental Section of the text.
scope experiments to obtain identical numbers of moles. The resulting calibration plots (Figure 3) demonstrate that although the luminometer yields a more sensitive (steeper slope) plot, the luminometer detection limit of 2 amol is virtually identical to that seen using the microscope (3 amol). The average standard deviation for the luminometer data was 3.9%. The inset in Figure 3 shows the very good correlation between the signals measured by the luminometer and the microscopy setup for the same amount of aequorin. While previous work with pressure-based microinjection has indicated that injection volumes vary linearly with injection time (or pressure), under some conditions this is not the case.1-5 In our system, it was found that, in the range of volumes of interest, the volumes (and hence, the bioluminescence) vary linearly with both time (Figure 4) and pressure (Figure 5). As one would expect, the micropipet with the larger inner diameter produces a larger injection volume than the micropipet with the smaller inner diameter using the same settings (Figures 4 and 5). Pipet-to-pipet reproducibility is shown in Figures 4 and 5. It is interesting to note that although the inner diameters of the micropipets used to generate the data shown in Figures 4 and 5 Analytical Chemistry, Vol. 69, No. 15, August 1, 1997
3117
Figure 5. Injection volume as a function of injection pressure for micropipets with inner diameters of 0.23 (O) and 0.18 (9) µm. Injection time was 0.1 s. Each point represents a single injection. The method for determining volume is described in the Experimental Section of the text.
were different, the pipet-to-pipet reproducibility of injection volume (see above) was much better than that previously observed for micropipets of the identical nominal diameter.5 It is also noteworthy that our micropipets can be made more reproducibly (inner diameter 0.23 ( 0.05 µm, as determined using the bubble pressure test) than those which are commercially available (outlet diameter 0.5 ( 0.2 µm).14 The variation in the data in Figure 4 represents a worst-case scenario for functional micropipets (clogged or broken micropipets are not considered functional). The diameter of the larger pipet, 0.29 µm, is one standard deviation above the average diameter of 0.23 µm and the diameter of the (14) Eppendorf Brochure ECET Femtotip: The Standard Capillary for Microinjection.
3118 Analytical Chemistry, Vol. 69, No. 15, August 1, 1997
smaller pipet, 0.18 µm, is one standard deviation below the average of 0.23 µm. As previously described, the plots of volume vs time and volume vs pressure intercept the x-axis at positive values, indicating that threshold times and pressures are necessary to produce an injection.1,3,5 The x-intercepts of the time plots converge at ∼0.036 s, which probably indicates that the threshold time is limited by the injection system.3 On the other hand, the pressure plots are parallel, and the micropipet with the larger diameter has a smaller intercept as one would expect. Thus, the micropipet diameter seems to be the major determinant of the threshold pressure. In summary, we have demonstrated that aequorin is a viable alternative to fluorescent probes for determining injection volumes in the range of picoliters to nanoliters. Aequorin should prove even more useful for biological and in vivo studies where background fluorescence limits the use of fluorescence probes. Aequorin may also be used as a label in binding assays, and development of methods for determining concentrations of compounds of biological significance in small volumes using aequorin-labeled ligands is currently underway in our laboratories. ACKNOWLEDGMENT This work was supported by the Department of Energy (Grant DE-FG05-95ER62010) and the National Institutes of Health (Grant GM 47915). Received for review January 30, 1997. Accepted May 21, 1997.X AC970123A X
Abstract published in Advance ACS Abstracts, July 1, 1997.
