Anal. Chem. 1987, 5 9 , 2115-2118
LITERATURE CITED Cheng, F. S.; Christian, G. D. Anal. Chem. 1977.49, 1765. Yastrebova, E. A.; Osipov, I. V.; Varfolomeev, S. D.; Agasyan, P. K. Zh. Anal. Khlm. 1982,37, 1278. (3) Laval, J. M.; Bourdillon, C.; Molroux. J. J . Am. Chem. SOC. 1984, 106, 4701. (4) Wlngard, L. 6.; Shaw, C. H.; Castner, J. F. Enzyme Mlcrob. Techno/. 1982,4 , 137. Molroux, J.; Ehrlng. P. J. Anal. Chem. 1978,50, 1056. Jaegfeldt, H. J . Elecffoanal. Chem. 1980. 110, 295. Eking, P. J.; Bresnahan, W. T.; Moiroux, J.; Samec, 2. Bloelectrochem Bhnerg 1982,9 , 365. Ottoway, J. H. Blochem. J . 1968.99, 253. Smith, M. D.; Olsson, C. L. Anal. Chem. 1975,47, 1074. Winartasaputra. H.; Kuan, S. S.; Gullbault, 0. 0. Anal. Chem. 1982, 54. 1967. Smith, M. D.; Olsson, C. L. Anal. Chem. 1974,46, 1544. Sulalman, S. T.; Najeeb. M. M. Microchem. J . 1985,31, 37. Matsumoto, K.; Ukeda. H.; Osajlma, Y. Agrlc. Blol. Chem. 1984s48, 1679. Jaegfeidt, H.; Kuwana, T.; Johansson, G. J . Am. Chem. SOC. 1983, 105, 1805. Tse. D. C. S.; Kuwana, T. Anal. Chem. 1978,50, 1315. Jaegfektt, H.; Tortensson, A.; Gorton, L.; Johansson. G. Anal. Chem. 1981,53, 1979. Carlson, B. W.; Mlller, L. L. J . Am. Chem. SOC. 1985, 107, 479.
.
.
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(18)Gorton, L.; Johansson. 0.; Tortensson, A. J . Elecffoanal. Chem.
iaas. ----. 198. ---.s- i- .-
(19) Gorton, L. J . Chem. Soc.,Farahy Trans. 1 1988,82, 1245. (20) Albery, W. J.; Hillman, A. R. Annu. Rep. Rog. Chem., Sect. C 1981, 377. (21) Schlapfer, P.; Mindt, W.; Raclne, P. Clin. Chlm. Acta 1974,57, 283. (22) Ianniello, R. M.; Lindsay, T. J.; Yacynych, A. M. Anal. Chem. 1982, 54, 1980. (23) Powell, M. F.; Wu, J. C.; Bruice, T. C. J . Am. Chem. SOC.1984, 106, 3850. (24) Bocarsly. A. 6.; Sinha, S. J . Electroanal. Chem. 1982, 140, 167. (25) Schneemeyer, L. F.: Spengler, S. E.; Murphy, D. W. Inorg. Chem. 1985,24. 3044. (26) Thomas, D.; Broun, G. J . Biochim. 1973,55, 975. (27) Lane, R. F.; Hubbard. A. T. J . Phys. Chem. 1973, 77, 1401. (28) Laviron, E.; Rouiiler, R. J . Electroanal. Chem. 1980, 115, 65. (29) Sharp, M.; Petersson, M.; Edstrom, K. J . Electroanel. Chem. 1979, 95, 123. (30) Laviron, E. J . Elecffoanal. Chem. 1979, 101, 19. (31) Wenger, J. I.; Bernofsky, C. Blochlm. Blophys. Acta 1971.227, 479.
RECEIVEDfor review February 12,1987. Accepted May 18, 1987. The financial support of the Science and Engineering Research Council is gratefully acknowledged.
Potentiometric Biosensor for Riboflavin Based on the Use of Aporiboflavin-Binding Protein Toshio Yao' and G. A. Rechnitz*
Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716
A new potentlometric blosensor uslng blndlng proteln Is proposed for the selective detennhatkn of rlbdlavin (vttamh B& The sensor Is based on the prlnclple of rlboflavln Mndlng dlsplacement of aporlbotlavln-blndlng protein (apoRBP) from the rlboflavln analogue bound membrane to form a stable holoRBP. Flavin adenlne dlnucleotkle (FAD) or acriflavine Is bound on the membrane surface as rlboflavln analogue to provlde charged groups. The dlsplacement of apoRBP brlngs about a change In the membrane charge and results In a change of the membrane potential. the resulting apoRBPFAD bound membrane electrode showed good senslng characterlstlcs for the selectlve determlnatlonof rlboflavln In the 0.1 to 2 pM concentration range wlth rapid and reproduclble response.
A variety of methods for determining riboflavin (vitamin B,) are available. Some (1-3) depend on the use of highperformance liquid chromatography with fluorescence detection while others use adsorptive stripping voltammetry as a highly sensitive method for the determination of riboflavin (4-6). However, these methods involve time-consuming steps and are subject to interference from other compounds. It is known that riboflavin-binding protein (RBP) binds riboflavin, through noncovalent interaction, more tightly than it binds riboflavin analogues (7,8). The binding of RBP to riboflavin is reversible and RBP dissociates into apoRBP and free riboflavin at low pH. The present paper describes the development of a new potentiometric sensor for riboflavin 'On leave from Department of Applied Chemistry, College of Engineering, University of Osaka Prefecture, Japan. 0003-2700/87/0359-2115$01.50/0
utilizing this effect. The response principle of the sensor depends on the change in membrane charge induced by displacement of apoRBP from the riboflavin analogue bound membrane to form stable holoRBP with riboflavin to produce a shift in membrane potential. In this study, both negatively charged flavin adenine dinucleotide (FAD) and positively charged acriflavine are used as riboflavin analogues for immobilization at the membrane surface.
EXPERIMENTAL SECTION Apparatus. All potentiometric measurements were made with a Corning Model 12 pH/mV meter and recorded on a HeathSchlumberger SR 204 strip chart recorder. Measurements were made in twin cells thermostated at 30 OC with a Haake Model FS temperature controller. Prepared membranes were assembled in Orion &search 92 series electrode bodies. A Corning saturated calomel reference electrode was also employed. Reagents and Materials. All chemicals were of analytical reagent grade. Deionized water was used throughout. Riboflavin, acriflavine, flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), glutaraldehyde (25%aqueous solution), riboflavin-binding protein (apo form from chicken egg whites; 1 mg of protein binds 10.3 fig of riboflavin), retinol (vitamin A), thiamine (vitamin B,) and L-ascorbic acid (vitamin C) were obtained from Sigma (St. Louis, MO), acetylcellulose and cyanogen bromide were obtained from Eastman Kodak (Rochester, NY), and octadecylamine was obtained from Aldrich (Milwaukee,WI). Riboflavin Stock Solution. Stock solution of 0.1 mM riboflavin in 0.01 M sodium acetate, pH 5.0,was prepared from recrystallized riboflavin. this solution was stored at 4 "C and protected from light. Periodic checks showed that this solution was stable for 1 month at 4 "C. Preparation of Acriflavine- and FAD-Bound Membranes. The membrane film was cast with acetylcellulose (0.1 g dissolved in 6 mL of acetone) and octadecylamine (10 mg dissolved in 1 mL of methylene chloride) as a support in a 90-mm culture dish. 0 1987 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 17, SEPTEMBER 1, 1987 CH?
4
*a
b+;,;FD
AF
FAD
AF
I
1s t step
(acriflavine)
FAD
apoRBP
cHWbJ-" H2
2nd step
HO OH (FAD)
Figure 1. Structural characteristics of acriflavine and FAD bound on
the electrode membrane. Table I. Bioaffinity Constants for the Binding of Some Flavin Analogues by Aporiboflavin-Binding Protein (ref 8) flavin
Kd, M
riboflavin acriflavine flavin mononucleotide (FMN) flavin adenine dinucleotide (FAD)
1.3 x 10-9 1.8 X 1.4 X lo* 1.4 x 10-5
The acriflavine-bound membrane was prepared by immersing an acetylcellulose-octadecylamine membrane into a 2.5% solution of glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.5) for at least 1.5 h and then coupling for 2 h in a 0.05 M solution of acriflavin in 0.1 M sodium phosphate buffer (pH 7.5). FAD-bound membrane was prepared as follows. The ribityl moiety of FAD was activated with cyanogen bromide (9),using equimolar amounts (30 mM) of FAD and cyanogen bromide, at pH 11for 12 min. The acetylcellulose-octadecylamine membrane was immersed into the activated FAD solution and the pH was adjusted to 8.5 using 0.01 M hydrochloric acid. The coupling proceeded overnight at room temperature. The membrane was exhaustively washed with 0.1 M NaC03 to remove unreacted cyanogen bromide. Construction of the apoRBP-Complexed Membrane Electrode. The apoRBP-complexed membrane was prepared by immersing the acriflavine or FAD bound membrane into an apoRBP solution (2 mg of apoRBP in 0.02 M Tris-HC1 buffer at pH 7.7) for 1h. Membrane disks of 3-mm diameter were cut for placement into the Orion 92 series electrode bodies with 0.01 M sodium phosphate buffer adjusted with 0.1 M HCl to pH 7.7 as an internal filling solution. Prior to measurements, each electrode was immersed for approximately 30 min in the background electrolytes until the electrode achieved a constant potential. RESULTS AND DISCUSSION From the structural formulas in Figure 1 and the binding data in Table I it is clear that acriflavine and FAD possess the necessary charge characteristics for attachment to membrane surfaces and for selective binding with aporiboflavin binding properties. These properties are utilized for the construction and operation of potentiometric sensors according to the scheme represented in Figure 2. The first step is the preparation of apoRBP-complexed membranes. The interaction between the riboflavin analogue (acriflavine or FAD) and apoRBP depends on noncovalent binding of these components. This interaction blocks the positively or negatively charged group of the riboflavin analogue bound membrane, because apoRBP is a macromolecule (molecular weight 36 000 (10))compared to riboflavin ana-
+
+
0
0
Figure 2. Schematic diagram of apoRBP-riboflavin analogue bound membrane electrodes: a, acriflavine (AF') bound membrane; b, flavin adenine dinucleotide (FAD-) bound membrane; c, internal solution; d, aporiboflavln-binding protein (apoRBP); RF riboflavin. The first step is the preparation of apoRBP-complexed membranes and the second is
the sensing step for riboflavin. A
apoRBP
k
-1
.E
L.
T
-
I
OQORBP
Flgure 3. Responses of riboflavin analogue bound membrane electrodes to apoRBP (90 pg) and riboflavin (1.3 p M ) additions: A, acriflavine-bound membrane electrode; B, FAD-bound membrane eiectrode.
logues. It should be noted that apo-RBP is negatively charged at pH values near neutrality with an isoelectric point of 4.6 (10). However, apoRBP binds riboflavin with a bioaffinity constant (Kd) of about 1.3 nM, e.g., several orders of magnitude more tightly than it binds FAD, acriflavine, or related flavins (Table I). The addition of riboflavin, therefore, causes the displacement of apoRBP from the membrane surface to form stable holoRBP (second step). The displacement of apoRBP-riboflavin complex causes a shift of the Donnan equilibrium in the direction of increasing charge on the membrane surface accompanied by a potential change. The overall potential difference across the membrane is the sum of the two Donnan potentials a t the membrane-solution interfaces and the diffusion potential across the membrane. However, displacement of apoRBP occurs only at the external side of membrane, since the internal side of the membrane remains unchanged provided the apoRBP-riboflavin analogue complex is stable over a reasonable time interval when exposed
ANALYTICAL CHEMISTRY, VOL. 59, NO. 17, SEPTEMBER 1, 1987
b
d
1
/
2117
1OmV
-
PH
10 min
Flgure 4. Responses of apoRBP-acriflavlne bound membrane electrode to riboflavin (1.3 pM) in a variety of electrolyte solutions (pH 7.7): a, 0.1 M sodlum phosphate buffer which Is free of chloride b n ; b, 0.02 M Trls-HCI buffer; c, 0.05 M Tris-HCI buffer; d, 0.1 M Tris-HCI buffer.
Flgure 5. Effect of pH on the response to riboflavin: 0, apoRBPacriflavine bound membrane electrode (0.1 M Tris-HCI buffer); 0 apoRBP-FAD bound membrane electrode (0.02 M sodium phosphate buffer); concentration of riboflavin, 1.6 pM.
to the internal electrolyte solution. Figure 3 illustrates the potentiometric behavior resulting from the two-step processes just described. In the case of the acriflavine-bound membrane electrode (Figure 3A), the potential changed in the positive direction when riboflavin was added. Also, the complexation of apoRBP to the acriflavine-bound membrane went essentially to completion in ca. 12 min at 30 "C. In addition to the potential change induced by the apoRBP displacement, the apoRBP-acriflavine bound membrane electrode exhibited a response to concentration changes of chloride ion from 0.013 to 0.16 M with a slope of 8.7 mV per decade. As shown in Figure 4, when C1- free phosphate buffer was used as an electrolyte (curve a), no marked potential change was generated and potentials shifted gradually in a positive direction after the addition of riboflavin. However, by use of Tris-HC1 buffers (curve b, c, and d), potential changes were generated and sustained a t buffer concentrations of more than 0.05 M. These results suggest that potential changes take place slowly with the removal of apoRBP from the membrane surface and that chloride ion facilitates the displacement of apoRBP. This is reasonable in view of the small difference in the bioaffiiity constants for riboflavin and acriflavin (see Table I). Additional experiments were carried out on blank membranes not containing acriflavine. Such membranes showed negligible riboflavin response, suggesting that nonspecific interactions do not contribute substantially to the potential responses observed with the complete sensing membrane. Experiments were carried with constant levels of Na+ and C1- to eliminate any effect of these ions on the overall response. As shown in Figure 3B, the apoRBP-FAD bound membrane electrode gave a well-defined response for the addition of riboflavin. Since the FAD has a negatively charged site, the direction of the potential change for this electrode was opposite to that of the apoRBP-acriflavine bound membrane electrode. In addition, the response time of this electrode was shorter than that of the apoRBP-acriflavine bound membrane electrode because the response time is dependent, in part, upon the difference in the bioaffinity constants of riboflavin and its analogues. The bioaffinity constant, Kd, for the displacement of apoRBP-FAD complex by riboflavin can be derived from the Kd values of riboflavin and FAD by apoRBP according to
5:
apoRBP-FAD
+ riboflavin F= apoRBP-riboflavin
Kd = (1.3 x 10-9)/(1.4 X
= 9.3 X
+ FAD M (1)
This value shows that the equilibrium lies far to the right so that the displacement reaction is completed in a relatively short period of time; the experimentally obtained response
E 12 u :lo C
2V 8
-
6
0
';4 C
2 2
d
01
I
I
-2
-1 log C
0
(MI
Figure 6. Effect of electrolyte concentration on the apoRBP-FAD bound membrane electrode. Sodium phosphate buffers (pH 7.7) were used as electrolyte solution. Concentration of riboflavin was 1.3 pM.
time was ca. 6-8 min. Figure 5 shows the potential changes caused by the addition of riboflavin at various pH values. The response of the sensors decreased at pH values below ca. 7 , suggesting that apoRBP complexed to the membranes dissociates at pH values below neutrality, since that complex is metastable. Similarly, the decrease of the response observed at pH values higher than 8.2 is probably due to instability of apoRBP at higher pH values. The effect of electrolyte concentration on the response of FAD bound membrane electrodes was also examined (Figure 6). Sodium phosphate buffers were used as an electrolyte and internal solution; the concentration ratio of electrolyte and internal solutions was held constant at 2:l throughout this experiment. Several membrane theories have shown that the induced Donnan potential makes a large contribution to the overall potential at relatively low concentrations of electrolyte since the fixed charge on the membrane is influenced by the surrounding electrolyte concentration (11). Figure 6 supports such a concept and the results favor 0.02 M sodium phosphate buffer (pH 7.7) as an optimal electrolyte solution. Figure 7 shows the analytical relationship between the potential change and riboflavin concentration for both sensors. These curves were constructed by making successive additions of riboflavin. Since such experiments involve only shifts in the binding equilibrium, no reconditioning of the electrode is required. The direction of the potential change was positive and negative for the apoRBP-acriflavine and apoRBP-FAD bound membrane electrodes, respectively. The sensitivity was almost the same for both sensors, but the apoRBP-FAD bound membrane electrode surpasses the apoRBP-acriflavine bound membrane electrode with regard to its rapid-response characteristic and good reproducibility of the measurements. The relative standard deviations (n = 6) were 6.8% for the apoRBP-FAD electrode and 11.8% for the apoRBP-acriflavine electrode at a concentration of 1.0 WMriboflavin for
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 17, SEPTEMBER 1, 1987
is given in eq 2.
Therefore, the present method is nearly
apoRBP-FAD
Figure 7. Plots of potential change vs. concentration of riboflavin: 0,
apoRBP-acriflavine bound membrane electrode (the direction of the potential change was positive); 0 , apoRBP-FAD bound membrane electrode (the dlrection of the potential change was negative).
+
Riboflavin (1.3pM)
FAD(5pM)
FMN(5PM)
7 - 73
+ FMN
6
apoRBP-FMN
+ FAD
specific for the determination of riboflavin in mixed samples of riboflavin, FAD, and FMN. Other vitamins (vitamin A, B,, and C) did not interfere with the measurement of riboflavin even in 50-fold excess. Although the present sensor functions over a limited riboflavin concentration range, it represents a novel concept for biosensors with possible applicability to more general sensor design for other analytes. Finally, the time stability of the membrane electrode was examined in terms of storage and operational stabilities. After the FAD-bound membrane was freshly prepared, it was stored in a 0.02 M phosphate buffer (pH 7.7) in the refrigerator. The FAD-bound membrane retained binding activity for apoRBP for 20-25 days. Operational stability for repeated use was inferior to the storage stability with a useful lifetime of approximately 1week. It was observed that the yellow color of the membrane faded when the membrane lost binding activity for apoRBP. This probably means that internal reduction of FAD takes place, resulting in fragmentation. The active part of the molecule, the isoalloxazine ring, may leak out into the solution during repeated use and only the ribityl moiety, which binds covalently, remains bound to the membrane. Registry No. FAD, 146-14-5; riboflavin, 83-88-5;acriflavine, 65589-70-0.
LITERATURE CITED F l g m 8. Response comparison to riboflavin and FAD or FMN for the apoRBP-FAD bound membrane electrode.
experiments carried out with freshly conditioned electrodes. The apoRBP has a strong affinity for riboflavin and lower affinity for the analogue compounds of riboflavin. It is important to investigate the effect of the FAD and FMN on the electrode response, because they are biologically important analogue compounds of riboflavin. Figure 8 compares the response of riboflavin with those of FAD and FMN for the case of the apoRBP-FAD bound membrane electrode proved to be superior in the present study. The electrode did not respond to FAD at all, but gave a slight shift in potential in the negative direction when FMN was added to the cell. However, FMN is not a serious interference for riboflavin determinations even in 80-fold excess. The calculated Kd value for the displacement of apoRBP-FAD complex by FMN
(1) Ichinose, N.; Adachi, K.; Schwedt, G. Ana&& (London) 1985, 770, 1505-1508. (2) Lambert, Willy E.; Cammaert, Piet M.; DeLeenheer, Andre P. Clin. Chem. (Winston-Salem, N.C.)1985, 37, 1371-1373. (3) Watada, A. E.; Tran, T. T. J . Liq. Chromatogr. 1985, 8 , 1651-1682. (4) Sawamoto, Hiromichi J . fiectroanal. Chem. 1885, 786, 257-265. (5) Grigor’ev, V. I.; Mllyaev, Y. F.; Balyatinskaya, L. M. Z h . Anal. Khim. 1985 40, 736-739. (6) Wang, Joseph; Luo, Den B.; Farias, Percio A. M.; Mahmoud, Jaward S. Anal. Chem. 1985, 57,156-162. (7) Lotter, Sharon E.; Miller, Mark S.; Bruch, Richard C.; White, Harold B., 111 Anal. Biochem. 1982, 725,110-117. (8) Becvar, James; Palmer, Graham J. Blol. Chem. 1982, 257, 5607-56 17. (9) Mgnsson, M. 0.; Mattiasson, B.; Gestrelius, S.; Mosbach, K. Biotechno/. Bioeng. 1978, 78, 1145-1159. (10) Rhodes, Marvin B.; Bennett, Nelle; Feeney, Robert E. J . Bioi. Chem. 1959, 234, 2054-2060. (11) Ohki, Shinpei J . Colloid Interface Sci. 1971, 37,318-324.
RECEIVED for review February 20,1987. Accepted May 8,1987. We gratefully acknowledge the support of NIH Grant GM25308.
