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Autoinjector for the determination of picomolar quantities of ATP with a liquid scintillation counter
ANALYTICAL
BIOCHEMISTRY
63, 418-422
Autoinjector Picomolar
(1975)
for the Determination of Quantities of ATP with
a Liquid
Scintillation
RICHARD
Counter
L. BRUNKER
Department of Limnology, Stroud Water Research Center, The Academy Natural Sciences of Philadelphia, and The Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19103
of
Received July 12, 1974; accepted August IO, 1974 The design, construction, and use of an autoinjection device that when used in conjunction with a liquid scintillation counter allows the detection and quantification of ATP in quantities as low as 2 pmoies is described. It provides a method for the detection and recording of the initial seconds of luminescence that occur when ATP and the luciferin-luciferase enzyme system are combined. This device can be quickly and easily fabricated at a negligable cost from readily available materials and has been routinely used for moderate numbers of determinations.
The use of extracts of firefly lanterns (Photinus pyrafis) in quantitative determinations of submicromolar amounts of ATP has proven to be a valuable assay procedure in recent years. Tal et al. (1) demonstrated that a liquid scintillation counter could be employed to measure quantities as low as 50 ng and Addanki et al. (2) reported the detection of 5 ng using a similar technique. The rapid kinetics of the light reaction immediately on combining ATP and the luciferin-luciferase system produces the maximum number of flashes with 3 set of mixing (3). Neither of these investigators were able to detect these flashes because of the time required to lower the vial into the counting chamber after mixing. Lyman et al. (3) assembled a system consisting of a photomultiplier tube mounted in combination with a specially designed mirror assembly fitted with an electronically timed injection system that allowed for uniform mixing of reactants and the detection and recording of the initial luminescence. They demonstrated the importance of detecting the initial flashes as well as providing for uniform mixing and reported a linear relationship between ATP concentration and luminescence at quantities as low as 10 pg. In this paper we report the design of a very inexpensive autoinjection device from materials already available in most laboratories for use with a liquid scintillation counter. It provides for both the uniform mixing of constituents and the detection of the initial seconds of luminescence such that picomole amounts of ATP may be measured. 418 Copyright @ 1975 by Academic Press, Inc. Printed All rights of reproduction in any form reserved.
in the United
States
AUTOINJECTOR
MATERIALS
FOR ATP DETERMINATION
AND
419
METHOD
The device can be fabricated in about an hour. A 1 ml disposable syringe (Plastipak, Becton-Dickenson, Rutherford NJ) is disassembled. A fine-toothed saw was used to shorten the barrel at the 0.4 ml mark. The plunger was shortened to 25 mm by sawing through the shaft 24 mm from the top end and reshaping the cut end with a razor blade to accommodate the rubber plunger tip (the original tip retaining knob was used as a model). The 26 gauge needle was shortened to 5 mm with a fine triangular file (it may be necessary to reopen the bore tip with a sewing needle after this operation). Three turns of plastic tape 1 cm wide were wrapped around the top of the shortened syringe barrel (Fig. 1). A + in. hole was drilled through the center of the cap of a polyethylene vial (Poly Q II, Beckman Instr., Fullerton, Calif.) and the modified syringe barrel was inserted with the edge of the tape support resting against the surface of the vial cap (Fig. 1). A presser tab was fashioned by cutting an octagonal 25 x 20 mm from a flexible ruler that was approximately 1 mm thick. A hinge was provided for this presser tab by using a loop of 1 cm plastic tape (Time
FIG. 1. Longitudinal section of autoinjector: (a) presser tab, (b) tape retainer on shortened syringe barrel, (c) tape hinge, (d) test tube retainer fashioned from a serum stopper, (e) test tube containing enzyme mix.
420
RICHARD
L.
BRUNKER
Tape, Professional Tape Co., Hinsdale, 111. (Fig. 1). This hinged tab was fastened to the vial cap with a single turn of tape secured around the edge of the cap. The presser tab was lubricated with a thin film of silicon grease. A 4 ml test tube 75 X 10 mm was shortened to an overall length of 37 mm. The outer flange and center membrane were cut from a 1 in. serum stopper. This was used to position the test tube in a suspended position in the scintillation vial so that the specimen contained by the test tube was in approximately the geometric center of the vial (fig. 1). The enzyme was prepared from dessicated firefly lanterns (Sigma Chemical Co.) by the method of Strehler and Totter (4). To eliminate the high background caused by residual ATP 2 units (1 mg) of apyrase (Sigma Chemical Co.) was added for each 50 mg of lanterns used. This mixture was incubated at 32°C for 60 min. Standards were prepared daily from dilutions of disodium ATP stock solutions and kept in an ice bath. The autoinjector was prepared for operation by placing 0.2 ml of enzyme extract and 0.4 ml of double distilled water into the test tube which was then positioned in the vial (Fig. 1). The syringe tip was wiped clean and the syringe was flushed five times with the ATP solution to be analyzed and then filled to the 0.05 ml mark. Care was taken to exclude all bubbles from the syringe during this procedure. The cap containing the syringe was then screwed into place on the vial containing the test tube. The entire assembly was then ready for use in the scintillation counter. The liquid scintillation counter (Beckman, model LS 133) was prepared for operation by setting the gain control on 520, the variable discriminator module assembly on 050-1000, the action controls on manual, the preset error on 0.2%, and the preset minute selector on 0.1. The prepared autoinjector was placed into the conveyor position directly over the elevator with the tape hinge positioned toward the back of the counter. The action controls knob was turned to the sample repeat position lowering the autoinjector into the detector well and causing the closing rotor to depress the presser tab of the autoinjector. This pressure brings about the injection of the ATP substrate into the enzyme solution at almost the same moment that the counting commences. The reaction was allowed to count for three successive 6 set intervals each separated by a 7 set interval for printout. The initial 6 set interval was used for quantitative determinations. The autoinjector was then removed and an internal standard could be then carried out if desired. RESULTS
The results indicated a linear relationship between counts per minute and the quantity of ATP injected in amounts between 2.0 x IO-I2 moles (4.0 X lo-” moles ml-‘) and 4.0 X IO-” moles (8.0 X lo-lo moles ml-‘) (Fig. 2). Reactions involving 8.0 X IO-” (1.6 X 10Ws moles ml-‘) of ATP
AUTOINJECTOR
FOR
FIG.
ATP
2. Standard
DETERMINATION
421
curve.
appeared to be limited by the enzyme concentration. Those involving less than 2.0 x lo-l2 moles (4.0 X IO-” moles ml-‘) yielded too few counts in 6 set to produce significant data. DISCUSSION
The counting of three successive 6 set cycles was carried out to assure that the amount of enzyme present was not the limiting factor in the reaction. The use of apyrase reduced the background to such a low level t< 50 cpm) that it could be ignored in tabulating the results. The counts of the subsequent 6 set intervals fell sharply until background levels were approached. If the data from the second and third counts were not a small fraction of the initial count the investigator should dilute the ATP solution 1 : 10 and repeat the analysis. When the unit is completely assembled the needle tip should be at least 2 mm above the enzyme level in the test tube. This not only assures uniform delivery but allows for the entrainment of sufficient ox-
422
RICHARD
L.
BRUNKER
ygen during delivery to preclude the possibility of poor results caused by a lack of oxygen during the reaction. The small bore of this needle (26 gauge) also provides a sufficient constriction to cause a slight delay before the ATP substrate is completely delivered into the enzyme. The selection of plastic of the proper rigidity for the presser tab is important as it must yield to the closing of the rotor but assure complete delivery after the rotor has closed. After approximately a hundred determinations the presser tab hinge and syringe barrel should be replaced. It was important to check the completeness of delivery by inspecting the syringe barrel after each measurement. Incomplete delivery usually resulted from either the syringe barrel being positioned too far down in the cap ( 1 cm should protrude) (Fig. l), or a plunger that was too short. A presser tab hinge that is not securely fastened to the cap may also cause inconsistant volumes to be delivered. While instruments specifically designed for the detection of ultramicroquantities of ATP have recently become commercially available any laboratory possessing a liquid scintillation counter can use this device in the processing of moderate numbers of samples in which a lower limit of 2 pmoles is satisfactory. ACKNOWLEDGMENTS This work was supported by a Grant from the Rockefeller Foundation and a fellowship from the Environmental Protection Agency. Thanks are due to T. Bott and R. Larson for helpful discussions.
REFERENCES 1. TAL, R., DICKSTEIN, B., AND SULMAN, F. G. (1964) Experientia 20, 652. 2. ADDANKI, S., SOTOS, J. F., AND REARICK, P. D. (1966) Anal. Biochem. 14, 261. 3. LYMAN, G. E., AND DEVINCENZO, J. P. (1967) Anal. Biochem. 21,435. 4. STREHLER, B. L., AND TOTTER, J. R. (1952) Arch. Biochem. Biophysics. 40, 28.
BIOCHEMISTRY
63, 418-422
Autoinjector Picomolar
(1975)
for the Determination of Quantities of ATP with
a Liquid
Scintillation
RICHARD
Counter
L. BRUNKER
Department of Limnology, Stroud Water Research Center, The Academy Natural Sciences of Philadelphia, and The Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19103
of
Received July 12, 1974; accepted August IO, 1974 The design, construction, and use of an autoinjection device that when used in conjunction with a liquid scintillation counter allows the detection and quantification of ATP in quantities as low as 2 pmoies is described. It provides a method for the detection and recording of the initial seconds of luminescence that occur when ATP and the luciferin-luciferase enzyme system are combined. This device can be quickly and easily fabricated at a negligable cost from readily available materials and has been routinely used for moderate numbers of determinations.
The use of extracts of firefly lanterns (Photinus pyrafis) in quantitative determinations of submicromolar amounts of ATP has proven to be a valuable assay procedure in recent years. Tal et al. (1) demonstrated that a liquid scintillation counter could be employed to measure quantities as low as 50 ng and Addanki et al. (2) reported the detection of 5 ng using a similar technique. The rapid kinetics of the light reaction immediately on combining ATP and the luciferin-luciferase system produces the maximum number of flashes with 3 set of mixing (3). Neither of these investigators were able to detect these flashes because of the time required to lower the vial into the counting chamber after mixing. Lyman et al. (3) assembled a system consisting of a photomultiplier tube mounted in combination with a specially designed mirror assembly fitted with an electronically timed injection system that allowed for uniform mixing of reactants and the detection and recording of the initial luminescence. They demonstrated the importance of detecting the initial flashes as well as providing for uniform mixing and reported a linear relationship between ATP concentration and luminescence at quantities as low as 10 pg. In this paper we report the design of a very inexpensive autoinjection device from materials already available in most laboratories for use with a liquid scintillation counter. It provides for both the uniform mixing of constituents and the detection of the initial seconds of luminescence such that picomole amounts of ATP may be measured. 418 Copyright @ 1975 by Academic Press, Inc. Printed All rights of reproduction in any form reserved.
in the United
States
AUTOINJECTOR
MATERIALS
FOR ATP DETERMINATION
AND
419
METHOD
The device can be fabricated in about an hour. A 1 ml disposable syringe (Plastipak, Becton-Dickenson, Rutherford NJ) is disassembled. A fine-toothed saw was used to shorten the barrel at the 0.4 ml mark. The plunger was shortened to 25 mm by sawing through the shaft 24 mm from the top end and reshaping the cut end with a razor blade to accommodate the rubber plunger tip (the original tip retaining knob was used as a model). The 26 gauge needle was shortened to 5 mm with a fine triangular file (it may be necessary to reopen the bore tip with a sewing needle after this operation). Three turns of plastic tape 1 cm wide were wrapped around the top of the shortened syringe barrel (Fig. 1). A + in. hole was drilled through the center of the cap of a polyethylene vial (Poly Q II, Beckman Instr., Fullerton, Calif.) and the modified syringe barrel was inserted with the edge of the tape support resting against the surface of the vial cap (Fig. 1). A presser tab was fashioned by cutting an octagonal 25 x 20 mm from a flexible ruler that was approximately 1 mm thick. A hinge was provided for this presser tab by using a loop of 1 cm plastic tape (Time
FIG. 1. Longitudinal section of autoinjector: (a) presser tab, (b) tape retainer on shortened syringe barrel, (c) tape hinge, (d) test tube retainer fashioned from a serum stopper, (e) test tube containing enzyme mix.
420
RICHARD
L.
BRUNKER
Tape, Professional Tape Co., Hinsdale, 111. (Fig. 1). This hinged tab was fastened to the vial cap with a single turn of tape secured around the edge of the cap. The presser tab was lubricated with a thin film of silicon grease. A 4 ml test tube 75 X 10 mm was shortened to an overall length of 37 mm. The outer flange and center membrane were cut from a 1 in. serum stopper. This was used to position the test tube in a suspended position in the scintillation vial so that the specimen contained by the test tube was in approximately the geometric center of the vial (fig. 1). The enzyme was prepared from dessicated firefly lanterns (Sigma Chemical Co.) by the method of Strehler and Totter (4). To eliminate the high background caused by residual ATP 2 units (1 mg) of apyrase (Sigma Chemical Co.) was added for each 50 mg of lanterns used. This mixture was incubated at 32°C for 60 min. Standards were prepared daily from dilutions of disodium ATP stock solutions and kept in an ice bath. The autoinjector was prepared for operation by placing 0.2 ml of enzyme extract and 0.4 ml of double distilled water into the test tube which was then positioned in the vial (Fig. 1). The syringe tip was wiped clean and the syringe was flushed five times with the ATP solution to be analyzed and then filled to the 0.05 ml mark. Care was taken to exclude all bubbles from the syringe during this procedure. The cap containing the syringe was then screwed into place on the vial containing the test tube. The entire assembly was then ready for use in the scintillation counter. The liquid scintillation counter (Beckman, model LS 133) was prepared for operation by setting the gain control on 520, the variable discriminator module assembly on 050-1000, the action controls on manual, the preset error on 0.2%, and the preset minute selector on 0.1. The prepared autoinjector was placed into the conveyor position directly over the elevator with the tape hinge positioned toward the back of the counter. The action controls knob was turned to the sample repeat position lowering the autoinjector into the detector well and causing the closing rotor to depress the presser tab of the autoinjector. This pressure brings about the injection of the ATP substrate into the enzyme solution at almost the same moment that the counting commences. The reaction was allowed to count for three successive 6 set intervals each separated by a 7 set interval for printout. The initial 6 set interval was used for quantitative determinations. The autoinjector was then removed and an internal standard could be then carried out if desired. RESULTS
The results indicated a linear relationship between counts per minute and the quantity of ATP injected in amounts between 2.0 x IO-I2 moles (4.0 X lo-” moles ml-‘) and 4.0 X IO-” moles (8.0 X lo-lo moles ml-‘) (Fig. 2). Reactions involving 8.0 X IO-” (1.6 X 10Ws moles ml-‘) of ATP
AUTOINJECTOR
FOR
FIG.
ATP
2. Standard
DETERMINATION
421
curve.
appeared to be limited by the enzyme concentration. Those involving less than 2.0 x lo-l2 moles (4.0 X IO-” moles ml-‘) yielded too few counts in 6 set to produce significant data. DISCUSSION
The counting of three successive 6 set cycles was carried out to assure that the amount of enzyme present was not the limiting factor in the reaction. The use of apyrase reduced the background to such a low level t< 50 cpm) that it could be ignored in tabulating the results. The counts of the subsequent 6 set intervals fell sharply until background levels were approached. If the data from the second and third counts were not a small fraction of the initial count the investigator should dilute the ATP solution 1 : 10 and repeat the analysis. When the unit is completely assembled the needle tip should be at least 2 mm above the enzyme level in the test tube. This not only assures uniform delivery but allows for the entrainment of sufficient ox-
422
RICHARD
L.
BRUNKER
ygen during delivery to preclude the possibility of poor results caused by a lack of oxygen during the reaction. The small bore of this needle (26 gauge) also provides a sufficient constriction to cause a slight delay before the ATP substrate is completely delivered into the enzyme. The selection of plastic of the proper rigidity for the presser tab is important as it must yield to the closing of the rotor but assure complete delivery after the rotor has closed. After approximately a hundred determinations the presser tab hinge and syringe barrel should be replaced. It was important to check the completeness of delivery by inspecting the syringe barrel after each measurement. Incomplete delivery usually resulted from either the syringe barrel being positioned too far down in the cap ( 1 cm should protrude) (Fig. l), or a plunger that was too short. A presser tab hinge that is not securely fastened to the cap may also cause inconsistant volumes to be delivered. While instruments specifically designed for the detection of ultramicroquantities of ATP have recently become commercially available any laboratory possessing a liquid scintillation counter can use this device in the processing of moderate numbers of samples in which a lower limit of 2 pmoles is satisfactory. ACKNOWLEDGMENTS This work was supported by a Grant from the Rockefeller Foundation and a fellowship from the Environmental Protection Agency. Thanks are due to T. Bott and R. Larson for helpful discussions.
REFERENCES 1. TAL, R., DICKSTEIN, B., AND SULMAN, F. G. (1964) Experientia 20, 652. 2. ADDANKI, S., SOTOS, J. F., AND REARICK, P. D. (1966) Anal. Biochem. 14, 261. 3. LYMAN, G. E., AND DEVINCENZO, J. P. (1967) Anal. Biochem. 21,435. 4. STREHLER, B. L., AND TOTTER, J. R. (1952) Arch. Biochem. Biophysics. 40, 28.