Membrane-based scintillation proximity assays I. Detection and quantification of 14CO2

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MEMBRANE SCIENCE ELSEVIER

Journal of Membrane Science 98 (1995) 275-280

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Membrane-based scintillation proximity assays I. Detection and quantification of 1 4 C O 2 Colin D. Mattingly, Robert K. Mansfield, Dibakar Bhattacharyya, Michael Jay * Division of Medicinal Chemistryand Pharmaceutics, Departmentof Chemical Engineering, Universityof Kentucky, Lexington, KY 40536-0082, USA Received 9 June 1994; accepted in revised form 18 August 1994

Abstract 14CO2 is a radioactive byproduct of numerous clinical exams and enzymatic assays. Traditional methods for capturing and quantifying ~4CO2produce a large quantity of radioactive liquid organic waste, which is both difficult and expensive to handle. A polymeric membrane which can simultaneously capture and quantify ~4CO2has been developed to circumvent both problems. The membrane contains a quaternary amine trapping agent (HyamineTM) and an organic lluor for/3 - detection. A 12% (w/v) polysulfone/sulfonated polysulfone blend membrane containing 0.5 mmol of HyamineTM captured 4.0 × 10-5 mmol/cm2 of ~4CO2 with a counting efficiency of 70%. The membrane retained all of the 14CO2for at least a three-day period. Keywords: Affinity membranes; Microporous and porous membranes

1. Introduction Carbon dioxide is commonly stripped from gas streams by absorption into amine solutions through formation of a salt. The same principle can be used in detecting ~4CO2, a radioactive byproduct of a number of oxidative and enzymatic processes as well as an analyte of interest in clinical breath tests. Typically, product gasses containing 14CO2 are passed through a solution of HyamineTM, a methanolic solution of the quaternary amine methylbenzethonium hydroxide. Hyamine is the trapping agent of choice because it is relatively nontoxic and nonflammable, and it binds CO2 in a 1:1 molar ratio rather than in a 1:0.5 ratio typical of more traditional amines such as DEA [ 1 ]. * Corresponding author. Tel.: (606)-257-5288; Fax: (606)-2577585; E-mail: [email protected]. 0376-7388/95/$9.50 © 1995 Elsevier Science B.V. All fights reserved

SSD10376-7 38 8 ( 94 ) 00194-4

14C is one of a family of radionuclides that decay by the emission of energetic electrons (negatrons, internal conversion electrons, etc.) and are commonly quantified by liquid scintillation (LS) counting in which a small amount of a solution containing the electronemitting radionuclide is mixed with a solution containing fluors (LS cocktail). This allows the radionuclide to be in close contact with the fluors so that the emitted electrons, which have short ranges (typically 1-100 /zm in solution), can interact with the fluors. The interaction of the electron with the fluors results in the emission of UV light (scintillations) which can be quantified in an LS counter. LS counting is an extremely sensitive technique which can accurately detect minute amounts of radionuclide in solution. LS cocktails are primarily composed of organic solvents, e.g., xylene or pseudocumene, containing specific concentrations of primary fluors (molecules that

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- emlsslon

Photon

Fig. 1. Detectionof a/3 - emmisionby a scintillationproximity(SP) bead. emit light following interaction with radiation) and secondary fluors (wavelength shifters) for optimal detection by the LS counter. Wavelength shifters are present to increase the detection efficiency by shifting the wavelength of the primary fluor's emitted photons to a higher wavelength that is more efficiently detected by the LS counter. Once the sample is counted, it must be disposed of using a complex and expensive series of treatment procedures. The disposal of this "mixed" (organic and radioactive) liquid waste is a serious environmental concern [ 2]. One of the first attempts to circumvent the generation of such an environmental hazard was the development of scintillation proximity (SP) beads [ 3 ]. Glass beads containing fluors were functionalized with recognition groups to selectively bind specific ligands, such as drug molecules, proteins, or other molecules of interest. Only the ligands bound to the bead were in close enough proximity to excite the fluors, thereby accomplishing selective removal of the desired ligand from solution and the production of scintillations without the need for scintillation cocktail or a separation step. Fig. 1 shows the emissions of a typical SP bead. SP beads allow a large proportion of the emitted negatrons ( / 3 - ) to escape into the solution without being detected. Therefore, we embarked on the development of a solution to the problem of generation of mixed waste and the inefficiencies inherent in SP beads by the creation of reactive SP membranes. This is

Fig. 2. Cross sectionof a scintillationproximity(SP) membrane. achieved by preparing porous, functionalized membranes in which fluors have been incorporated in the membrane matrix. A typical cross section of an SP membrane is shown in Fig. 2. The radioligand bound to the pore interior has a much higher geometric counting efficiency than on an SP bead if the pore diameter is less than or equal to the path length of the negatron. In this paper, we describe the preparation of fluorcontaining membranes designed to trap carbon dioxide for the purpose of quantifying ~4CO2. In current research, we are investigating the use of membranes functionalized with antibodies for carrying out one-step radioimmunoassays.

2. Experimental 2.1. Materials

Polysulfone (PS) resin (MW 752,000), polyvinylpyrrolidone (PVP, MW 40,000), and diphenyloxazole (POP) were purchased from Aldrich Chemicals. Cellulose acetate (CA, 40% acetyl content) and methylbenzethonium hydroxide (HyamineTM, 1 M solution in methanol) were purchased from Sigma Chemicals. p-Bis-o-methylstyrylbenzene (bis-MSB) was purchased from Research Products International. Radiolabeled sodium bicarbonate (Nanl4co3) was purchased from New England Nuclear/DuPont. Sulfonated polysulfone (PSS) was prepared by the method of Johnson et al. [4]. Hydroxylated polysul-

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277

Table 1 Compositionof membranes,solvents,and nonsolventsused in 14CO2trappingexperiments Polymer (w/v)

POW (w/v)

bis-MSBb (w/v)

PVP¢ (w/v)

Solvent

Nonsolvent

12% PS 15% CA 12.5% CA 12% PSS 6% PS/6% PSS 12% PS-OH

20% 25% 25% 20% 20% 20%

1% < 1% < 1% 1% 1% 1%

4% 2% 2% 4%

NMPa Acetone 50:50 Acetone/Water NMP NMP NMP

Water Water Water 95:5 Water/NMP 95:5 Water/NMP 95:5 Water/NMP

a Diphenyloxazole,primaryfluor. bp-Bis-o-methylstyrlbenzene,secondaryfluor. Polyvinylpyrolidone,MW 40,000. N-Methylpyrrolidinone. fone (PS-OH) was prepared by reacting ethanolamine with chloromethylated polysulfone. 2.2. Membrane preparation

All membranes were prepared by phase inversion to produce a high surface area, microporous, asymmetric membrane. Each membrane was made by applying a uniform film of polymer solution to a glass plate and then immediately submerging the plate into a nonsolvent bath. The conditions for the preparation of the various membranes, including the composition of the casting solution and the nonsolvent, are summarized in Table 1. The concentrations of the primary and secondary fluors (POP and bis-MSB, respectively) were chosen on the basis of previous results that indicated that these concentrations yielded maximum counting efficiency [ 4]. The volume of the nonsolvent gel bath was 1 I in each case, and the thickness of all prepared membranes was ~ 220 mm. Because of the tendency of the organic fluors to leach into gel baths containing acetone or methanol, water or 95:5 water/N-methylpyrrolidinone (NMP) mixtures were used exclusively as the nonsolvent gelation baths. The addition of 5% NMP to the gelation baths to prevent skin formation did not result in appreciable fluor loss. 2.3. Hyamine loading

Hyamine was incorporated into the membranes by loading under a transmembrane pressure gradient. This method was found to be superior to equilibrium loading and the direct incorporation of Hyamine in the membrane casting solution. Equilibrium loading

resulted in low loading, and direct incorporation had deleterious effects on the mechanical integrity. Pressure loading was accomplished by placing dried membranes inverted in an ultrafiltration apparatus. After washing the membranes with methanol, 10 ml of 1 M Hyamine solution were passed through the membranes under carbon dioxide-free nitrogen pressure. The membranes were then stored in purged vials and sealed. The amount of Hyamine loaded onto the membranes was determined by titration of the effluent Hyamine solution with a standardized acid. 2.4. Exposure to 14C02

All membranes used in 14CO2 trapping experiments were made by phase inversion in an aqueous bath and loaded with Hyamine under pressure as previously described. The membranes were exposed to ~4CO2 using the apparatus shown in Fig. 3. The apparatus consisted of a series of chambers for generating and trapping ~4CO2. To the first chamber, which served to generate ~4CO2, 5 /zCi of Nanl4co3 was added, and the system was purged with CO2-free nitrogen. A fresh membrane was added to the second chamber and the system was again purged. 10 ml of 12 N HCI was then rapidly added to the first chamber using a glass syringe. The 14CO2 that was formed was swept through the system in a single pass (to simulate exhaled air in a clinical breath test) using nitrogen as a flow gas. Hyamine and NaOH traps were added on-line to capture any ~4C02 that was not trapped by the membrane. After exposure to the 14C02, the membranes were placed in vials, sealed, and counted in an LS counter without addition of LS cocktail. The ~4C-bicarbonate

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Figure 3

q HCI

Nitrogen

12 N NaOH trap

\ 14C.NallCO3

HyamineTM trap

Fig. 3. Diagramof the apparatususedto exposecarbondioxidetrappingmembranesto 14CO2. salt formed by the interaction of 14CO2 with the membrane-bound Hyamine was in close enough proximity to the fluors in the membrane matrix to produce scintillations.

3. Results 3.1. Membrane criteria

tion of CA in acetone was very brittle and difficult to handle. The membranes cast from the 12.5% ( w / v ) solution of CA in 50:50 acetone/water and the 12% ( w / v ) solution of PS/PSS in NMP possessed good physical strength and flexibility. The membrane cast from the 12% ( w / v ) solution of PSS in NMP did not possess a coherent structure. The choice of nonsolvents was limited by the solubility of POP in the casting bath. In a 40:60 methanol/ water bath, over 80% of the fluor partitioned out of the membrane. A 40:60 acetone/water bath removed almost 100% of the fluor. A bath of 95:5 water/NMP was used in all other experiments to minimize fluor loss while still yielding a skinless, microporous membrane. The membranes in which the greatest amount of

The ideal scintillation proximity membrane must fulfil several key criteria. It must have enough mechanical integrity to withstand the physical manipulation needed for laboratory and clinical applications. The membrane matrix must be able to incorporate a fluor and wavelength shifter for energetic electron detection in sufficient quantities to ensure a practical detection efficiency. The membrane polymer should also have good energy transfer characteristics to amplify the range of interaction between the fluor and radionuclide,

Hyamine could be pressure loaded were those composed of PS-OH and the PS/PSS composite membranes. Approximately 0.5 mmol of Hyamine could be loaded onto 5 cm 2 samples for each of these membranes.

3.2. Formation o f membranes

3.3. ~4C02 trapping

The fluor-containing membranes formed from the various polymers had greatly varying properties, some of which were unsuitable for use in the present application. The membrane cast from the 15% ( w / v ) solu-

After exposing the membranes to 14CO2,the membranes were counted in an LS counter. The number of disintegrations per minute obtained were used to calculate the number of millimoles of 14CO2 trapped by

C.D. Mattingly et aL / Journal of Membrane Science 98 (1995) 275-280

Figure 4

279

Two sources of error could have possibly affected the results. The first of these involves quench correction by the LS counter. The instruments are designed for the counting of homogeneous solutions, not membranes. Therefore, the quench correction parameters used by the counter may not accurately convert the observed number of counts detected to the actual number of radioactive disintegrations. Secondly, the calculations assume that no endogenous CO 2 was present in the apparatus. It is likely that small amounts of CO2 were present in the exposure chamber and sweep gas; this would lead to an underestimation of the amount of ~4CO2 trapped by the membrane. However, it is expected that these errors would be relatively constant for all membranes tested.

70 60 50 40 30 20 10

A

B

C

D

Membrane Type Fig. 4. Binding results of ~4CO2 trapping membranes containing Hyaminel. Membrane types: (A) 12% (w/v) PS-0H; (B) 12% (w/v) PS; (C) 15% (w/v) CA; (D) 12% (w/v) PS/PSS blend.

each membrane. The results of the exposure experiments are shown in Fig. 4. It can be seen from this figure that the 12% ( w / v ) PS/PSS composite membrane demonstrated the greatest ability to trap 14CO2 and have it detected by the LS counter. This may be a function of the membrane porosity and the availability of the Hyamine within the membrane to be accessible to the 14CO2. It can also be seen from this figure that the number of radioactive events did not diminish over a three-day period for any of the membranes tested. This indicated that the 14CO2 was tenaciously bound to the membrane and did not desorb over time. This is an important feature because it allows one to trap ~4CO2 with these membranes and count the trapped radioactivity at a later time without loss of activity. To ensure that the amount of ~4CO2 trapping was due to reaction with Hyamine and not simply due to adsorption onto the membrane surface, samples of each membrane that had not been loaded with Hyamine were also exposed to 14CO2. The amount of 14CO2 trapped by these membranes was less than 0.1% of the amount trapped by Hyamine-loaded membranes.

4. Discussion There are several important applications for trapping and quantifying radioactive carbon dioxide. A number of biochemical assays involve measuring the activity of decarboxylase enzymes. This is usually accomplished by feeding the appropriate substrate labeled with ~4C in the carbonyl position to the enzyme and measuring the amount of 14C02 generated. The 14C02 can be adsorbed onto a piece of NaOH-saturated filter paper suspended above the reaction media; the filter paper is then added to an LS cocktail, and the radioactivity quantified in an LS counter. This approach has been used to quantify the amount of ornithine decarboxylase present in specific cells. Some clinical tests of human health involve the administration of a 14C-labeled substrate followed by the measurement of exhaled ~4C02. One such test of growing clinical significance is for the detection of Helicobacterpylori, a microorganism whose presence in the stomach is associated with gastritis and ulcer [ 5 8]. This test involves the oral administration of 14Curea; the urease-rich H. pylori metabolize the urea resulting in the appearance of 14CO2 in the breath. Patients are generally directed to blow through a straw or some other breath collection system at various time intervals into a methanolic Hyamine solution. An LS cocktail is subsequently added to this solution and the sample is quantified in an LS counter. One of the problems with these kinds of tests is that the use of meth-

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anolic solutions and LS cocktails is not always compatible and convenient in a clinical setting. One problem common to all LS counting experiments is the disposal of the mixed waste. In the two examples above, large amounts of mixed waste containing only small amounts of radioactivity are generated for each experiment/study. This is where the use of membranes can aid in reducing the volume of waste generated. Fluor-containing membranes functionalized with CO2-trapping agents can be employed in the place of cocktails. After quantifying the amount of 14CO2 in an LS counter (without the need to add cocktail), the waste product is simply a thin, solid membrane. This is much easier and less costly to dispose of than an organic solution containing long-lived isotopes (14C has a half-life of 5730 years). In the current study, several membranes were evaluated for their ability to incorporate fluor and to trap ~4CO2. The membrane that yielded the greatest number of detectable photons after exposure to a standard amount of ~4CO2 was found by LS counting It appears that the membranes composed of the mixture of PS and PSS was the most efficient. When coagulated in water at 25°C, it forms a flexible, coherent membrane that requires a minimum of care to handle. This membrane showed a high capacity for entrapping fluors and Hyamine. It was capable of trapping up to 6 mmol of 14CO2/cm2 per mmol of Hyamine trapped. The high binding of the PS/PSS membrane could be due to its highly negatively charged character. Assuming equal porosities between the PS, PS-OH, and the PS/PSS composite, the amount of t4CO2 trapped increases as the negative charge on the membrane increases. Hyamine is a quaternary amine, so it is reasonable to conjecture that the more negative the membrane, the tighter the binding of the trapping agent. In addition, the ability of this membrane to retain the trapped radioactivity over a three-day period is important since it means that samples do not have to be counted immediately after trapping the 14CO2The long-term retention capacity will be an important factor when considering radiation waste disposal.

5. Conclusions Polymeric membranes were loaded with fluors during phase inversion using appropriate gelation baths that would not leach the fluors. The membranes were also pressure loaded with Hyamine in amounts sufficient to trap radioactive carbon dioxide. Membranes composed of a mixture of polysulfone and sulfonated polysulfone yielded the maximum photon output after exposure to 14CO2. The radioactivity on these membranes was retained for at least a three-day period.

Acknowledgements This work was supported in part by NSF grant EHR9108764, NSF/REU grant DMR-9200164, and the Kentucky EPSCoR Program.

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