Anal. Chem. 1980, 61, 2362-2365
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Electrochemical Method for Measuring C-Reactive Protein Using Crown Ether-Phosphate Ester Ionophores Carey M. Merritt* a n d J a m e s W. Winkelman' The Department of Pathology, Health Science Center a t Syracuse, Syracuse, New York 13210
A new dectrochemlcal method for measurement of C-reactive proteln (CRP) Is presented. We have syyntheslred new crown ether-phosphate ester Ionophores havlng hJgh afflnlty for CRP. A study uslng proton nuclear magnetlc resonance shows CRP binding to the Ionophores In stolchiometrlc amounts. The Incorporation of these crown ether-phosphate ester Ionophores Into poly(vlnyl chlorlde) membrane electrodes yleMs a CRP-senslthre electrode with senSnlVny In the mlcrogram per mllllliter range. The Ionophore synthesis and prellmlnary electrode characteristics are described.
INTRODUCTION C-Reactive protein (CRP) is a so-called "acute phase protein" found in human serum shortly after the onset of acute inflammation. Concentrations of CRP are reported to be elevated up to 1000-fold (to 200 pg/mL) during many disease states including post myocardial infarction (I),rheumatoid diseases (2),systemic lupus erythematosis (3),and a host of chronic infections (4). The level of CRP in serum has provided a useful clinical index for these disease states for many years. Since the discovery of CRP, much work has been devoted to studying its binding properties. The binding of CRP to many phosphate esters including phosphocholine, phosphoethanolamine, and a number of their mono- and diester derivatives is well characterized ( 5 , 6 ) . Oliveira et al. presented evidence that one binding site of CRP consists of two loci, a primary locus for a phosphoryl ester moiety (-OP03H) and a secondary locus for the binding of a cationic amine group (-NR3+). The binding of CRP to phosphate esters is known to be Ca2+dependent (7). Crown ethers are a class of compounds containing an oxygen-dense ether ring structure (8). They have ion complexing properties and may have been found useful in biosensor applications (9-11). Those applications employ antigens covalently bonded to a crown ether and use the derivatized ionophore as the basis for an electrochemical sensor for specific antibody. Electrochemical sensors for dinitrophenol, cortisol, and digoxin antibodies have been introduced that use a crown ether-antigen ionophore. Since improved methods for the detection of CRP may be important in the study of disease, we have developed electrochemical sensors for CRP. It is based upon new compounds consisting of the crown ether methyl benzo-15-crown-5 bound to phosphoethanolamine and the p-aminophenyl derivative of phosphocholine. The two ionophores synthesized are 4(phosphoethanolamine) methyl benzo-15-crown-5 (CrownPEA) and 4-( (p-aminopheny1)phosphocholine)methyl benzo-15-crown4 (Crown-PC) (Scheme I). These ionophores have ion complexing ability and high affinity for CRP. The degree of CRP binding to the ionophore was determined by observing changes in proton nuclear magnetic resonance (H NMR) spectra of Crown-PC before and after addition of CRP. A biosensor for CRP was created by incorporating the ionophore in a polymeric membrane and affixing the membrane Current address: Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115. 0003-2700/89/0361-2362$01.50/0
to the sensing module of an ion selective electrode body. The response to various K+ and CRP concentrations of such electrodes demonstrates that analyses in a useful range can be made.
EXPERIMENTAL SECTION Apparatus. Potentiometric measurements were recorded with a Corning Model 125 pH/Mv meter in junction with a Yew type 3066 strip chart recorder. Proton NMR data were acquired on a Varian XL 400 H NMR. For each spectrum we scanned 1000 times at a rate of 1scan/s. Chemical shifts were reported as (ppm) relative to an internal HzO signal. Mass spectra (60 ev or lower) were acquired on a Finnigan 4000 quadrapole MS/DS system using electron impact and chemical ionization (isobutene). Reagents. All chemicals were analytical grade or better. (pAminophenyl)phosphocholine, phosphoethanolamine, bovine serum albumin, human y-globulins (Sigma Biochemical), poly(vinyl chloride) MW 60OO0,and dipentyl phthalate (Polysciences) were used without further purification. 4Methyl catechol (Aldrich Chemical Co.) and tetraethylene glycol dichloride (supplied by Dr. Johannes Smid) were distilled under reduced pressure immediately prior to use. NMR grade CDC13,DzO,and CH4Si(EM Science) were used for the NMR studies. Preparation of 4-(Bromomethyl)benzo-15-crown-5. Methylbenzo-15-crown-5was prepared from 4-methylcatechol and tetraethylene glycol dichloride according to the procedure used by Petranek and Ryba (12). A typical bromination of the crown ether proceeded w follows: 0.7 mmol of 4methylbenml5crown-5 and 0.7 mol of n-bromosuccinimide were dissolved in 40 mL of dried CC14and stirred for 5 min under N2 The reaction mixture was placed in a 60 "C oil bath and irradiated with a 500-W tungsten lamp for 20 min. After cooling in an ice bath, the mixture was filtered and the solvent removed under reduced pressure at room temperature. The yellow oil was extracted 3 times with 10 mL of boiling hexane. The aliquots were combined and cooled to 4 "C for 4 h. The hexane was decanted and the 4-(bromomethyl)benzo-15-crown-5 (white solid) was used immediately without further purification. 'H NMR (in CDC13): 6.9 (3 H, aromatic); 4.5 (2 H, -CHz-); 3.6-4.2 (16 H, -CHz-0-). Synthesis of 4 4 (pAminopheny1)phosphochohne)Methyl Benzo-15-crown-5 (Crown-PC). fpAminopheny1)phosphocholine, 0.27 mmol, and 50 mg of sodium bicarbonate were dissolved in 15 mL of 4:l methanol/butanol. To this solution, 0.28 in mL of 4:l butammol of 4-(bromomethyl)benzo-15-crown-5 nol/methanol solvent was added dropwise over 10 min with stirring. The pinkish yellow solution was stirred at 45 "C for 2 days. The methanol was removed under reduced pressure and the mixture was cooled to 10 "C and filtered and the butanol evaporated at 50 "C under reduced pressure. The remaining tarlike substance was washed twice with 5-mL aliquots of CHC1,. The light brown oily solid weighing 43 mg was the desired product. This compound was stored in a desiccator under PzOsat 4 "C. Mass spectrometry: (m/z+)509 (M+ - 3CH3);397 (M+ - CH(OCHzCHz)zOCHz-); 297 (M+- NHCHZ - C,H,(CHZCHzO)J; 281 (M+- CH~C~H~(CHZCHZ-O)); 192 (M+- (CH,CHz-O)@); 179 (M+ - C6H,NC,H,O(CHZCH3O),). 'H NMR (in DzO): 6.8-7.0 (7 H, aromatic); 3.1 (9 H, CH3-N); 3.6-3.9(16 H, ether ring); 4.1-4.3 (4H, -CHz-CHz);3.4 (2 H, NCH,-O). FT-IR (em-'): 3400-3300 (-NH- stretching); 2500 (P-OH); 1600 (aromatic); 1510 (-NHbending); 1250 (P=O stretching); 110 (C-0). Synthesis of 4-(Phosphoethanolamine) Methyl Benzo15-crown-5(Crown-PEA). 4-(Bromomethyl)benzo-15-crown-5, 0.28 mmol, was dissolved in 10 mL of 1-butanol. Phosphoethanolamine, 1.4mmol, in a 1:l methanol/water solvent was 0 1989 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 61, NO. 21, NOVEMBER 1, 1989 2363
Scheme I. Preparation of Crown-PC and Crown-PEA
t
n
0
H
CH3-YH-CHZ-CH2-O-P-OH
ii
I
OH
(phosphoeihandamine)methyl benzel5crown-5
4-((aminq~henyl)phosphotylcholine) methyl benzel5crown-5
(Crown-PC)
added slowly and the temperature raised to 50 "C for 1h. The solution was stirred at room temperature for 24 h. The product was isolated by removing the solvent under reduced pressure and extracting with methanol. A yellowish brown tar was the desired product. The compound was stored in similar conditions as the Crown-PC compound. Mass spectrometry: (m/z+)354 (M+ 40,5H+);326 (M+ - PO4H2) 312 (M+ - PO4Hz - CH,); 298 (M+ - P04H2- CH, - CH,); 282 (M+ - P04H2- CH2 - CH2- N'H,). FT-IR (cm-l): 1600 (N-H bending), 2920-2870 (C-H stretch aliphatic); 1210 (C-N stretching); 1255 (0-o-Cstretching); 1506 (C-C ring); 3300-3400 (N-H stretching); 940 (P-OH). NMR Binding Study. Human C-reactive protein purchased from Calbiochem-Behring (San Diego, CA) in a saline Tris buffer was exhaustively dialyzed overnight against deionized distilled water and lyophilized. The powdered CRP was reconstituted to 1mg/mL concentration in a 100 mM NaCl, 5 MM KC1,lO mM CaC12,and 20 mM Tris buffer at pH 7.4 D20 solution. A stock 5 mg/mL Crown-PC DzO solution was prepared and spiked with absolute methanol to a concentration of 1pL/mg Crown-PC. The methanol yielded an NMR peak that wa9 inert and could be used as a reference peak. Five solutions were prepared containing 1 X lo4 mol of CRP and Crown-PC concentrations ranging from 0.9 X lo4 to 9.0 x lo-* mol. The resultant mole ratios of Crown-PC to CRP were 0.11,0.22, 0.37, and 1.11. An 'H NMR spectrum of each solution was obtained. Peak heights were obtained by direct measurement of the spectrum peaks. Electrochemical Measurements. Sensor membranes were prepared by mixing 0.25 mL dipental phthalate with 50 pg of Crown-PC, Crown-PEA, or methylbenzo-15-crown-5 and slowly adding the mixture to 200 mg of poly(viny1 chloride) dissolved in 5 mL of tetrahydrofuran. The resulting solutionswere poured into 48-mm glass petri dishes and allowed to slowly evaporate overnight to produce pliable transparent membranes. A 7 mm diameter round piece of each membrane was separately affixed to a Model 93 Orion ion selective electrode body using a modified electrode tip manufactured from Delrin and containing a threaded flange for affixing the membrane to the end of the electrode. The internal r i n g solution was 0.01 m KCl. A Lazar Model DJM-146 micro double junction reference electrode with a KC1 filled inner junction and KN03gel fiied outer junction served as the reference electrode. A reference solution containing 0.01 M KC1,O.Ol M CaCl? and 0.01 M Tris at pH 7.4 was prepared. Three commerciallyavtulable proteins, C-reactive protein, bovine serum albumin, or human y-globulins,were exhaustively dialyzed and further diluted with this solution. Careful preparation ensured equal K+ concentrations and activities between all CRP, BSA, y-globulin dilutions and the reference solution, as shown by K+ measurements using a K+ ion selective electrode. One set of solutions contained CRP concentrations of 0,10,50,100 pg/mL. The BSA and y-globulin solutions resulted in concentrations of 0, 10,20, and 50 mg/mL. Conditioning of the electrode required overnight soaking in the reference solution. Electrochemical measurements were obtained by measuring the potential of the CRP electrode, first in the reference solution
(Crown-PEA)
t
1
ieot \ loo
60
i
4
0
t
,
-,
,
~
, o ,
a9 20 0
0.2 0.4 0.6 0.8 [CRP]/[CROWN-PC]Molar
1.0
1.2
Flgure 1. Plot of binding data from Crown-PC In the presence of CRP. The graph shows the result of tiirating a CRP solution (1 X 10" mol)
with varying amounts of Crown-PC (0.9 X 10" mol). and then, in one of the protein solutions immediately afterward. All solutions were gently stirred. The electrodes were blotted dry after potential measurements were obtained from the reference solution and were rinsed with 50 mM EDTA and then with distilled H20 after potential measuremenb were obtained from the protein solutions. Electrode equilibration occurred in 5-10 min. The differences in potential between the reference and protein solutions were attributed to the presence of protein.
RESULTS AND DISCUSSION We have synthesized and characterized two novel crown ether-phosphate ester ionophores. The phosphate esters vary in structure and point of attachment to the crown ether. The Crown-PEA molecule has the phosphoethanolamine bound to the crown ether via an amino group as a secondary amine. Below pH 9 this amino group assumes a positive charge (6). The Crown-PC molecule is a phosphodiester bound to the crown ether via the phosphate group, with a charged trimethylammonium group a t the terminus. Both ionophore structures satisfy the primary and secondary binding site requirements for binding CRP. The binding of CRP to Crown-PC was demonstrated by the reduction of the -N(CH3)3+ peak of the Crown-PC NMR spectrum in the presence of CRP. The four solutions studied had CRP to Crown-PC mole ratios ranging from 0.11 to 1.11 and peak reduction was observed for all solutions. Integrated peak areas remained constant in all spectra, suggesting that the change in NMR spectra is not associated with a loss in -CH,- hydrogens. A methanol peak adjacent to the -N(CH3)3+peak provided a useful reference peak for measuring peak heights. Figure 1 is a plot of this peak reduction effect at various CRP/Crown-PC mole ratios. The ordinate represents the percent -N(CH3)3+/methanol NMR peak height ratios. The
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ANALYTICAL CHEMISTRY, VOL. 61, NO. 21, NOVEMBER 1, 1989
1 20
1
>
& Crown-PC I Crown-PEA -cMB15cr5 4
$ 1
-
-20
-
0 crown PC Crown PEA MB15C15
20
40
80
60
100
120
[CRP] ugh1
Figure 3. Potentiometric differences between a reference buffer solution and the same solution containing various CRP concentrations measured with a PVC membrane electrode incorporated with (0) 1 pg/disk Crown-PC, (W) 1 pg/disk Crown-PEA, and (0) 1 pg/disk MB15cr5.
100
Log [K’I
Figure 2. Potentiometric response of ionophore incorporated PVC membrane electrode to various K+ concentrations in 0.01 M Tris buffer pH 7.4: (0) 1 pg/disk Crown-PC, (W) 1 Fg/disk Crown-PEA, (0)40 pg/disk MB15Cr5.
Crown-PC mole ratios reach 0.22 suggesting that one CRP molecule can interact or bind up to five Crown-PC molecules. This agrees well with reports that show CRP to be a pentamer consisting of five subunits with each subunit containing a binding site (13). An electrochemical sensor was constructed by incorporation of the ionophores in plasticized poly(viny1 chloride) (PVC) membrane electrodes. The basis for this type of biosensor system requires that the ionophore complex a cation in the membrane phase and also bind a substrate in the aqueous phase. When the incorporated biosensor is in contact with a solution containing a cation capable of complexing the crown ether moiety, a base-line potential is established across the membrane. It is speculated that the binding of the substrate to the ionophore occurs at the membrane/solution interface and reduces the mobility of the ionophore-cation complex in the membrane phase (9). This mobility reduction of the ionophore causes a change in membrane potential that is proportional to the amount of substate in the solution. In our system, the ionophore can complex a K+ ion and the base-line potential is determined by K+ concentration in solution. Figure 2 shows the electrode’s response to various K+ concentrations. Although the electrode displays sub-Nerstian behavior toward changes in K+ concentration, the response is comparable to similar electrodes reported elsewhere (9-11). The integrity of the biosensor as a potassium sensor is quite good and it is imperative that K+ concentrations remain constant in all solutions being studied for a substrate binding effect. Furthermore, the presence of a phosphate ester on the crown ether molecule is shown not to effect response to changing K+ concentrations. A C-reactive protein molecule can act as the substrate for the biosensor we incorporated in the PVC membrane. The change in base-line potential associated with CRP binding in this system is shown in Figure 3. A nearly linear relationship between increased potential and sample CRP concentration for both biosensors presented is shown. The Crown-PC was a slightly better CRP biosensor than Crown-PEA. This finding is not surprising since binding studies have shown phosphocholine derivatives to be more efficient binders of
Table I. Potentiometric Differences between a Reference Buffer Solution and the Same Solution Containing Various Concentrations of BSA and y-Globulins Measured with PVC Membrane Electrode Incorporated with 1 pg/disk Crown-PC
amt, mg/mL
BSA
0 5
0.0
10 20
0.6 0.4 1.2
AE, mV y -globulins 0.0 0.4 0.0 0.5
CRP than phosphoethanolamine derivatives (6). Figure 3 also shows that the response to CRP cannot be attributed to the crown ether. Incorporated methylbenzo-15-crown-5 had a negligible response to changing CRP concentrations. Potential measurements were generally obtained in a matter of minutes, Figure 4. After potential measurements in CRP solutions were obtained, the electrode was easily restored to base-line potential by rinsing in a EDTA solution. EDTA, a well-known Ca2+chelator, has been shown to be very effective in regulating CRP binding to phosphate esters (14). This allowed for an easy way to cleanse the membrane surface of bound CRP. The electrode’s response was remarkably stable (draft rate of less than 0.4 mV/h) however, with continuous use the sensitivity for CRP decreased gradually after 2 weeks. This is attributable to ionophore leaching from the PVC membrane. We observed the Crown-PC incorporated electrode’s response to BSA and human yglobulins, Table I. The response could be considered an initial indication of any cross reactivity the new ionphore might have with serum proteins. Our fiidings show almost no response to either BSA or y-globulins at concentrations up to 20 mg/mL. In summary, we have described a unique but simple electrochemical method for determining CRP. The detection limits for this method are well within the range needed for clinically useful CRP measurements. This method has the advantages of being both rapid and inexpensive compared to
Anal. Chem. 1989, 61, 2365-2372
other methods currently in use. The simplicity of ionophore synthesis and electrode fabrication should make this method attractive for future application in determining clinical CRP levels.
ACKNOWLEDGMENT The authors thank Ruth Silberman and Johanne Smid for their aasktance in ionophore synthesis, David Rice for assisting in the NMR study, and Paul Turner for his technical assistance.
LITERATURE CITED Kushner, Irving; Broder, Martin L.; Karp, David J. Clin. Invest. 1978, 61, 235-242. Gitlin. Jonathan D.: Gitlin. Joan I.: Gitlin. David, Arthrits Rheum. 1977, 20, 1491-1499. Pereira, De Sika J. A.: Elkon, Keith 6.;Hughes, G. R. v.; Dyck, Roland F.; Pepys, Mark B. Arthrnis Rheum. 1980, 23, 770-771.
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(4) Oewurz, Henry, Hosp. Practice 1982, (June), 67-81. (5) Robey, Frank A.; Liu, Teh-Yung J. Bkl. Chem. 1982, 258,3895-300. (6) Oliveira, Eduado B.; Gotschlich, Emil C.; Liu, Teh-Yung, J. Immunol. 1980, 124, 1396-1402. (7) Tanaka. Taeko; Robey, Frank A. J. Immunol. Methods 1983, 65, 333-341. (8) Pedersen, C. J. J. Am. Chem. SOC.1967, 89, 7017-7036. M. Y.; Rechnitz, G. A. Anal. Chem. 1984. 56. 801-806. Keating, (9) (10) Solsky, Robert L.; Rechnitz, G. A. Anal. Chlm. Acta 1881, 123, 135-141. (11) Keating, M. Y.; Rechnitz, G. A. Analyst 1983, (June), 766-766. (72) Petranek, J.; Ryba, 0. Anal. Chim. Acta 1974. 72, 375-380. (13) Osmond, Alexander P.; Freidenson, Bernard Gerwurz, Henry; Painter, Robert H.; Hofmann, Theo; Shelton, Emma Roc. Natl. Aced. Sci. U.S.A. 1977, 74, 739-743. (14) Volanakis, John E.; Clement, W. L.; Schrohenloher, Ralph E. J. Immunol. Methods 1978, 23, 285-295.
RECEIVED for review August 15, 1988. Revised manuscript received April 26, 1989. Accepted August 7, 1989.
Potentiometric Combination Ion/Carbon Dioxide Sensors for in Vitro and in Vivo Blood Measurements M. E. Collison,' G. V. Aebli, Jennifer Petty, and M. E. Meyerhoff* Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109
The development and analytical performance of a novel potentiometric combhation lon/pCO, sensor design for in vitro and In VIVO measurements are reported. The design is based on Incorporating an appropriate Ionophore wlthln the outer slilcone gas permeable membranes of both conventlonai macro and new catheter-type pC0, sensors. Simultaneous measurement of the potentials across the ion-selectlve/gas permeable membrane and the inner glass or polymer pH sensltlve membrane provldes the basis for continuous monltorlng of both Ionic and pC0, levels wlth the same devlce. A macro-sized K+/pC02 embodlment of the sensor Is constructed from a commercial Severinghaus COPsensor and is used to demonstrate the prlnciples and capablilties of the proposed design. A flexible, minlaturized (outer diameter = 1.2 mm) combination K+/pCO, catheter sensor is also described. The catheter-type sensor is fabricated by Inserting a tubular polymer membrane pH electrode Into an outer siilcone rubber tube doped with valinomycin. Continuous measurements of K+ and pC0, during 6-h blood pump studles uslng both the macro and catheter-type combination sensors correlate well wtth those of conventlonaibenchtop analyzers. I n addltkn, continuous (4 h) Intravascular measurementswlth the combhation catheter sensor in dogs show good agreement wlth those of commercial blood analyzers ( R = 0.984 and 0.962 for pC0, and K', respectively).
In the modern health care setting, clinicians are increasingly dependent upon the reliable and rapid measurement of chemical variables for accurate diagnostic and therapeutic decisions (1). Consequently, there has been growing interest in the monitoring of blood gases and electrolytes continuously
Present address: Eli L i l l y Corp., Indianapolis,
IN.
0003-2700/89/0361-2365$01.50/0
using invasive chemical sensors (2-4). From the standpoint of patient safety (i.e. danger of infection and blood vessel damage), the invasiveness of implantable sensors will be a prime consideration in the clinical acceptance of these devices. Hence, a primary goal in the development of in vivo chemical sensors is the incorporation of multiple sensing elements per implant probe (3). In this report we describe the design and analytical performance of both macro in vitro and miniature catheter-type in vivo ion (K+)/pC02sensors. The proposed catheter device is a disposable, multiple-analyte sensor that is capable of continuous, simultaneous in vivo monitoring of a selected ion (e.g. K+) and carbon dioxide gas. Several approaches to the fabrication of multiple-analyte in vitro and in vivo sensors have been reported previously. Ion-selective field effect transistor (ISFET) devices have very small sensing areas, allowing a single miniaturized solid-state chip to be configured for simultaneous monitoring of several analytes (2, 5). While the ISFET concept has generated considerable interest, fundamental problems regarding sensor encapsulation, drift, and interference from electrically neutral acidic sample species (e.g. COJ must be resolved before these devices become practical for in vivo monitoring applications (3, 6,7). More recently, multiple-analyte fiber-optic sensors, particularly for pH, pCO,, and PO,, have been described (8). However, optical electrolyte sensors (for K+, Ca2+,Na+) that respond in a rapid and reversible fashion have not yet been developed, owing to the absence of appropriate indicators for these clinically important ions (9). Moreover, fundamental concerns regarding the effect of sample ionic strength on the accuracy of optical ion activity and pH measurements remain the subject of some debate in the literature (10). Miniature potentiometric combination ion/gas sensing devices have also been described previously. In 1976 General Electric Corporation developed a novel combination pH/pCO2 catheter sensor (11). Carbon dioxide sensing was achieved by using a metal oxide (Pd/PdO) pH electrode as the internal transducer in a classical Severinghaus-style gas sensing ar0 1989 American Chemical Society
