Anal. Chem. 1989, 6 1 , 1069-1072
and acceptor because at certain temperatures the size of the micelle is fixed. From this we can deduce that for larger Sm(lTA),L and En(TTA),L (L is a neutral ligand) molecules, the nonionic micelles will weaken the intermolecular transfer of energy, while for smaller molecules the micelles will reinforce the intermolecular transfer of energy. Our last experimental results agree with this deduction. Registry No. TTA, 326-91-0; Phen, 66-71-7; Triton X-100, 9002-93-1; La, 7439-91-0; Gd, 7440-54-2; Tb, 7440-27-9; Lu, 7439-94-3; Y, 7440-65-5; Sm, 7440-19-9; Eu, 7440-53-1; Sm(TTA),(Phen), 18078-88-1;YzO3, 1314-36-9;Gdz03,12064-62-9. LITERATURE CITED (1) Kononenko, L. I.; Poluektov, N. C.; Nikonova, M. P. Zavod. Lab. 1964, 3 0 . 779. (2) Kononenko, L. I.; Tishchenko, M. A. Zh.Anal. Khim. 1969, 2 4 , 1823. (3) Fisher, R . P.; Winefordner, J. D. Anal. Chem. 1971, 43, 454. (4) Ci, Y.-X.; Hu, K.-R.; Liu, J.-R.; Ma, H.J. Fengxi Huaxue 1982, 70(4). 232. (5) Shi, H.-M.; Cui, W.-C. Fengxi Huaxue 1982, 10(9), 561.
1069
(6) Aihara, M.; Arai, M.; Taketaksu, T. Analyst (London) 1986, 177(6), 641. (7) Huang, H.; Geng, X. Bunseki Kagaku 1986, 35(7), 584. (8) Sun, S.-S.; He, J.-K. Huaxue Tongbao 1982, (I), 16. (9) Ci, Y.-X.; Yang, R.-M. Fenxi Ceshi Tongbao 1984, 3(4), 9. (IO) Ramirez, A. A.; Linares, C. J.; Barrero, f . A.; Ceba, M. R. Talanta 1986, 33, 1021. (11) Ci, Y.-X.; Lan, Z.-H.; Liu, W. Analyst(London) 1968, 713, 933. (12) Ci, Y.-X.; Lan, 2.-H. Anal. Left. 1988, 21(8),1499. (13) Ci, Y.-X.; Lan. 2.-H.; Liu, W. Anal. Chim. Acta 1986, 212, 285. (14) Yang, J.-H.; Zhu, G.-Y.; Wu, B. Anal. Chim. Acta 1987, 798, 287. (15) Ci, Y.-X.; Gao, X.-S.; Hu, K.-R. Xitu Vingyong 1978, 3 , 10. (16) Melby, L. R.; Rose. N. J.: Abramson, E.: Caris, J. C. J . Am. Chem. SOC. 1964, 86, 5117. (17) Bauer. H.: Blanc, J.; Ross, D. L. J. Am. Chem. SOC.1964, 8 6 , 5125. (18) Liang, Y.-Q.; Zhao,W.-Y.; Zhang, Y.-F.; Zhao,Y.-N. Acta fhys.-Chim. Sinica 1985, 1 , 493. (19) Subbarao, E. C.: Wallace, W. E. Science and Technology of Rare Earth Materials; Academic Press: 1989; p 308.
RECEIVEDfor review July 19,1988. Accepted January 23,1989. This research was supported by the National Natural Science Foundation of China.
Avidin-Biotin Coupling as a General Method for Preparing Enzyme-Based Fiber-optic Sensors S h u f a n g Luo a n d David R. Walt*
Max Tishler Laboratory for Organic Chemistry, Department of Chemistry, T u f t s University, Medford, Massachusetts 02155
A general method for preparing enzyme-based fiber-optlc sensors is described. Three different sensors, capable of detecting peniclilln, organic esters, and urea, were developed based on this method. Each flber was constructed first with a layer of polymer containing pendant amino groups that react further with succinimidyl-derivatized biotin. Later biotin and fluorescein-labeled enzymes were Introduced to the fiber through a sandwich biotin-avidin-biotin Interaction. The substrate concentration is detected indirectly through the pH-sensitive dye, fluorescein, which responds to the microenvironmental pH changes produced by the enzyme catalyzed reactlons. The response times for the penicillin, organic ester, and urea optrodes are less than 90, 30, and 70 s, respectlveiy. Each sensor has a detectlon ilmit of 0.1 mM and responds to substrate concentrations over 2 orders of magnitude.
INTRODUCTION Recently, special emphasis has been placed on developing new sensors for monitoring biological substances. This development is due in part to the proliferation of production scale bioreactions and to the desire to develop continuous clinical monitoring devices. Fiber-optic chemical sensors offer significant advantages over conventional sensors for both in vitro and in vivo clinical applications such as their small size and lack of a direct electrical connection to the solution being measured. These sensors operate by detecting changes in the absorptive or emissive properties of an indicator fixed to the fiber's distal tip. Many indicators do not respond directly to concentration changes of biological substances, which has led to the development of numerous enzyme-based fiber-optic 0003-2700/89/0361-1069$0 1.50/0
biosensors (1-9). These sensors are comprised of a thin layer of polymer containing both an immobilized enzyme and an indicator that responds to the analyte concentration as a consequence of the enzyme-catalyzed reaction. Fiber-optic chemical sensors for detecting glucose and penicillin have been developed previously (1-6). These sensors are made in the conventional manner by entrapping enzymes in a polymer layer covalently attached to the fiber tip or by immobilizing enzymes on a solid support placed manually on the fiber tip. Both immobilization techniques are used widely for preparing both optical and electrochemical enzyme sensors. In this paper we describe a general approach for attaching enzymes to fibers based on the biotin-avidin interaction. Three enzyme sensors have been made by using this technique and operate on the same principle-the enzyme catalyzes the transformation of substrate into a product that changes the microenvironmental pH. This pH change is detected through the fluorescence intensity change of a pH-sensitive dye (FITC) attached to the enzyme. Any enzyme that converts substrates to either basic or acidic products can be employed with this approach. The method has been demonstrated for the substrates penicillin, ethyl butyrate, and urea according to the following reactions: penicillin G ethyl butyrate urea
penicillinase
+ H+ ethanol + butyrate + H+
esteraae
urease
penicilloate
NH4++ HC03-
EXPERIMENTAL SECTION Instrumentation. The instruments employed for excitation of the distal end of the fiber and for measuring the returning fluorescence signal have been described previously (IO). In brief, 0 1989 American Chemical Society
1070
ANALYTICAL CHEMISTRY, VOL. 61, NO. 10, MAY 15, 1989
an argon ion laser providing 4 S n m excitation or a xenon arc lamp source with excitation selected a t 488 nm with a double monochromator are focused into an optical fiber. The fluorescence signal returns over the same fiber and is discriminated from the incident light with a dichroic mirror and band-pass filter and then focused into the entrance slit of the emission monochromator. Materials. Penicillinase (EC 3.5.2.6, type I, from Bacillus cereus),penicillin G (potassium salt), esterase (EC 3.1.1.1, type I, from porcine liver), avidin (from egg white), and fluorescein isothiocyanate, isomer I (FITC), were purchased from Sigma Chemical Co. Acrylamide, NJ-methylenebis(acry1amide) (BIS), ethyl butyrate, y-(methacry1oxy)propyltrimethoxysilane were obtained from Aldrich Chemical Co. Sulfosuccinimidyl6-(biotinamido)hexanoate (NHS-LC-Biotin) was from Pierce Chemical Co., Rockford, IL. N-(3-Aminopropyl)methacrylamidehydrochloride (APMA) was from Eastman Kodak Co., Rochester, NY. All reagents were used without further purification. Glass-on-glass optical fibers (200/250 wm) used in this work have NA = 0.28 and length approximately 1 m. They were terminated with amp connectors on one end and polished on both ends. Enzyme Labeling ( 1 1 ) . Five to ten milligrams of enzyme was dissolved in 0.5-1.0 mL of 0.05 M bicarbonate buffer, pH 8.5-9.0. The solution was stirred gently on ice while 50-100 equiv of NHS-LC-Biotin was added. The reaction was allowed to proceed for 2 h. One milligram of FITC was then added and the reaction continued for another 2 h. Next the insoluble impurities were filtered and the labeled enzyme was separated from free biotin and fluorescein by dialyzing exhaustively against 0.015 M phosphate buffer, pH 7.4. Optrode Construction. Surface Silanization. The polished ends of the fibers were cleaned in concentrated sulfuric acid for several hours and then rinsed with distilled water. The fiber tips were silanized by soaking for 24 h in a dry toluene solution of 10% y-(methacry1oxy)propyltrimethoxysilaneand then rinsed with water and acetone. Polymerization Reagents. In solution A, 1.54 g of BIS was dissolved in 50 mL of 0.1 M phosphate buffer, pH 7.0. In solution B, 2.13 g of acrylamide and 5.37 g of APMA were dissolved in 10 mL of 0.1 M phosphate buffer, pH 7.0. Polymerization. One milliliter of solution A was mixed with 2 mL of solution B. The mixture was deoxygenated with nitrogen for 15 min. To this mixture, 100 pL of ammonium persulfate (10 mg dissolved in 1 mL of 0.05 M phosphate buffer, pH 7.0) was added. The silanized fibers were then inserted into the mixture and heated with an oil bath to 45-55 "C. After the polymer gelled (within half an hour), the fibers were withdrawn. Biotin-Auidin Complexation. The polymerized fibers were placed in 1mL of buffer (0.05 M NaHC03, pH 8.5-9.0) containing 5 mg of NHS-LC-Biotin for more than 3 h. After reaction the fibers were rinsed with distilled water. The fibers were then transferred to an avidin solution (3-5 mg of avidin dissolvd in 1 mL of phosphate buffer, pH 7.0) and kept in the refrigerator for 24 h and then rinsed with distilled water. Finally, the biotin-avidin fibers were placed in a reaction vessel containing labeled enzyme and were allowed to react for more than a day in the refrigerator. After reaction the enzyme sensors were soaked in 0.005 M, pH 7.0, phosphate buffer (penicillin and organic ester sensors) or in 0.002 M, pH 6.5, phosphate buffer (urea sensor) for 1 day to remove adsorbed enzymes. Sensor Measurements. The enzyme optrodes were excited at 488 nm and the intensity of emitted light was measured at 530 nm. Stock solutions of 0.1 M penicillin G, 0.01 M ethyl butyrate, and 0.1 M urea were prepared fresh daily. The penicillin G, ethyl butyrate stock solutions, and diluted solutions were prepared with pH 7.0,0.005 M phosphate buffer containing 0.1 M KC1. For urea solutions, a pH 6.5, 0.002 M phosphate buffer with 0.1 M MgSO, was used. Before each measurement, the optrode was soaked in substrate-free buffer until it reached a stable signal. Then the optrode was transferred to the substrate solution. Upon reaching a steady signal, the optrode was placed back in the original buffer. The process was repeated a t different substrate concentrations. All optrodes were stored in buffer at 4 OC when not in use. RESULTS A N D DISCUSSION T h e highly specific and extremely strong noncovalent binding of the vitamin biotin t o the glycoprotein avidin has
Scheme I. General Reaction Scheme for Preparing an Enzyme Optical Sensor
i l
NHS-LC-Biotin
*
$1 Biotin
Fluorescein
Enzyme - Biotin
..\;,idin
Ahdin Fluorescein
I
Biotin -Enzyme
made it widely accepted as a useful method to bind biological species in both analytical and preparative applications (12). T h e avidin-biotin interaction has been used in affinity chromatography for isolating proteins, peptides, and hormone receptors (13-19). The strong binding constant of avidin and biotin ( K , = l O I 5 M-I) enables the interaction t o tolerate extremes of pH, organic solvents, temperature, and other denaturing agents. On the basis of these properties and avidin's tetravalency for biotin, we designed a new general method for immobilizing enzymes on optical fibers. Enzyms-based optrodes for penicillin, organic esters, and urea have been prepared by immobilizing biotin-fluorescein labeled enzymes t o a fiber tip containing covalently bound avidin. The general reaction scheme for preparing an enzyme optrode is shown in Scheme I. The fiber is silanized initially with y-(methacry1oxy)propyltrimethoxysilaneand then coimmobilized with acrylamide, BIS, and APMA. The resulting fiber has free amino groups in the polymer that react further with NHS-LC-Biotin to form covalently attached biotin. Next the fiber is incubated with avidin to furnish a fiber containing residual biotin binding sites. T h e fiber can be used, in principle, to immobilize any biotinylated species. The enzyme is treated sequentially with NHS-LC-Biotin and FITC and then dialyzed exhaustively t o remove the free derivatizing agents. NHS-LC-Biotin primarily couples to the lysine residues of enzymes. T h e extended spacer arm of this biotin derivative reduces steric hindrance around the enzyme. No detailed experiments were performed to optimize the ratio of biotin to protein or fluorescein t o protein. T h e enzymes were always found to be reactive after labeling with biotin and fluorescein except that the residual activity varied from enzyme to enzyme. The resulting biotin and fluorescein-labeled enzyme is incubated with the avidin fiber and attached through a sandwich biotin-avidin-biotin interaction. This method of immobilizing enzymes has several advantages over conventional physical entrapment and covalent cross-linking methods. Physical entrapment suffers from
NO. 10, MAY
ANALYTICAL CHEMISTRY, VOL. 61, I " " I " " 1 " " I " " I "
-
200 -
l'O
1071
15, 1989
k
0.8c
I 0.610
0.45x10% 0.2 -
1x10-BM 5xlO-*M
1
4
OO
100
200
400
300
500
Time( s e c ) Figure 1. Response of penicillin optrode to different penicillin G concentrations in pH 7.0, 0.005 M phosphate, 0.1 M KCI. l
1.0-
t
"
'
I
'
~
"
'
'
'
"
'
1
'
'
'
1
'
"
1
I
0.a -
-
0.6 L
--1
1
1
1
-
0.41
o ~ " " l " 50" l Time (sec)
100
1
1
1
1
~
/
/
1
1
/
1
1
1
1
~
1
1
1
1
1
1
" " 150
Flgure 2. Response of organic ester optrode to different ethyl butyrate concentrations in pH 7.0, 0.005
M
phosphate, 0.1 M KCI.
/
1x1o-BM
d .
1
I
lxlo-Q/
d
420
'G 410 400
riI \
r-i 0.95
10
5~10% 1x10-L
4
3900
100
50
150
Time(sec)
Figure 3. Response of urea optrode to different urea concentrations in pH 6.5, 0.002 M phosphate, 0.1 M MgSO,.
continuous loss of enzyme. Also, it suffers from large diffusional barriers to the transport of substrates and products leading to reaction retardation particularly with high molecular weight substrates. Covalent cross-linking of enzyme to the solid support usually requires sophisticated chemical reactions, is difficult to carry out, is time-consuming, and requires immobilization procedures tailored to the individual enzyme. In this method avidin is coupled to the fiber by readily available reagents using mild reaction conditions. The resulting fiber is a useful precursor for all immobilized enzyme optical sensors. The derivatized enzyme is strongly bound to the fiber due to the stability of the avidinlbiotin interaction. In addition, the diffusion barrier is kept minimal because the en-
0'a5 0.00
0.01
0.02
0.03
0.04
0.05
0.06
Urea Concentration (M)
Figure 6. Calibration curve of urea optrode.
zymes bind only to sites in the polymer where the pore size is large. The results of exposing the three optrodes to different substrate concentrations are depicted in Figures 1-3. The time required to reach a 95% steady-state signal was less than 90 s for the penicillin optrode (Figure l),less than 30 s for the ester optrode (Figure 2), and less than 70 s for the urea optrode (Figure 3). These response times are faster than typical optrodes prepared by immobilizing enzymes on membranes (usually several minutes or longer) ( 3 , 4 ) . The short response times of these optrodes may be attributed to the tiny size of the fiber, the low diffusional barrier around the enzyme, and the intimate contact between the enzyme and the fluorescent indicator. Calibration curves for the sensors are shown in Figures 4-6. I, represents the fluorescence intensity of the fiber in the
~
1
1072
ANALYTICAL CHEMISTRY, VOL. 61, NO. 10, MAY 15, 1989
Table I. K , Values of Free Enzymes and Immobilized Enzymes enzyme
Km"
penicillinase esterase urease
5 x 10-5 4.4 x 10-4 1 x 10-2
K,' 2 x 10-2 1 x 10-3
6X
"Values are from ref 20. bValries are determined by Lineweaver-Burk plots.
buffer containing no substrate and I corresponds to the fluorescence intensity measured in the different substrate solutions. From the results it can be seen that the penicillin optrode has the greatest sensitivity and responds to concentrations over more than 2 orders of magnitude. Both penicillin and ester optrodes operate in the same way and measure the decreased fluorescence signal with increased substrate concentration. When there is no substrate in the buffer, the local pH of the fiber is the same as the pH 7 phosphate buffer. As the substrate concentration increases, the local pH of the fiber decreases and causes the fluorescence signal to decrease. The pH sensitivity was determined by calibrating the various sensors in different pH buffers containing no substrate. In this way, a local or microenvironmental p H can be assigned to the steady-state signals obtained with substrate present. The local pH generated around a penicillin optrode is about 4.1 when it is placed in 5 X M penicillin G. For an ester optrode, M ethyl butyrate generates a local pH of 4.8 around the fiber. The urea optrode operates in the opposite way. The enzyme catalyzes the conversion of urea to ammonium and bicarbonate and results in an increased microenvironmental pH of the fiber. K', Na+, and NH4+ are inhibitors of urease (ZO), so 0.1 M MgS04 was used instead of 0.1 M KCl to control the ionic strength of the buffer. Since fluorescein has a pK, value of 6.5, the fluorescence intensity does not increase significantly in the presence of urea when the starting buffer p H is 7.0. Consequently, for all measurements of the urea optrode, a pH 6.5 buffer was used. However, even with this change, the sensitivity of the urea optrode is still not as good as that of the penicillin and ester optrodes. This reduced sensitivity is due either to product inhibition of urease with ammonia or to the low activity of urease after labeling. An alternative way of increasing the sensitivity of a urea optrode may lie in the use of a pH-sensitive fluorescent dye with a higher pK,, such as hydroxypyrene trisulfonic acid. However this dye must first be derivatized before it can be used as an enzyme label. The buffer strength is an important factor in determining the detection limit and working range of the optrode. Kulp and co-workers ( I , 2) demonstrated that 5 mM phosphate/O.l M KCl buffer gave the most linear response of penicillin in the working range of 0-10 mM. Fuh e t al. (3)found that in 1 mM citric acid/Na2HP04buffer, penicillin could be detected as low as 0.1 mM with a working range of 0-10 mM. On the basis of the above results, 5 mM phosphate buffer was used and the same detection limit and working range were achieved as in the previous study. All three enzyme fiber-optic sensors have a detection limit of 0.1 mM and operate over more than 2 orders of magnitude in substrate concentration. T h e enzyme stability was monitored during storage in buffer about twice per month. The penicillin optrode has demonstrated storage stability over a 3-month period with 20% activity loss. The ester optrode survives for more than 11/2months. On the other hand, urea
optrodes show a lifetime of less than a week. In solution, labeled urease activity is lost completely after a week. Apparently, labeling causes a significant loss of urease activity and immobilization with avidin/biotin does not enhance its stability. The Michaelis-Menten constant K , is an inverse measure of the affinity of the enzyme for the substrate. When an enzyme is placed in a polymeric environment the physical factors present may act to either impede or facilitate the approach of the substrate to the enzyme. Thus the apparent affinity of the immobilized enzyme for the substrate may be either decreased or increased. Table I summarizes the K , values for free enzymes and K,' values for the immobilized enzymes. K,' values were determined by Lineweaver-Burk plots based on initial enzyme reaction kinetics. The increases in apparent K,' for penicillinase and esterase may be attributed to mass transfer limitations on the substrate's access to the immobilized enzyme. In summary we have successfully utilized the avidin/ biotin interaction as a general method to immobilize enzymes onto optical fibers. The procedure is both simple and mild and should be applicable to the immobilization of other biomolecules such as antibodies. The response time of these optrodes is about 1 min, which is as good as the best enzyme optrodes developed to date. The method is applicable to any kind of enzyme except that new indicators (transducers) must be developed for those enzyme reactions not involving changes of microenvironmental pH. The major disadvantage is one inherent to all biosensors-the enzyme gradually loses its activity, requiring frequent calibrations. The penicillin, ester, and urea optrodes demonstrated here are selective, sensitive, and completely reversible and demonstrate the generality of this technique for biosensor design. LITERATURE CITED Kulp, T. J.; Camins. 1.; Angel, S. M.; Munkholm, C.; Walt, D. R. Anal. Chem. 1987, 59, 2849-2853. Kulp, T. J.; Camins, I.; Angel, S. M. SPIE Opt. Fibers Med. I I I 1988, 906, 134-138. Fuh, M . S.; Burgess, L. W.; Christian, G. D. Anal. Chem. 1988, 6 0 , 433-435. Abdel-Latif, M. S.; Suleiman. A. A.; Guilbault, G. G. Anal. Lett. 1988, 2 1 , 943-951. Narayanaswamy, R ; Sevilla, F., 111 Anal. Lett. 1988, 2 1 , 1165-1175. Schultz, J . S. US-Pat.. 4.344, 1982, 438. Wangsa. J.; Arnold, M. A. Anal. Chem. 1988. 6 0 , 1080-1082. Arnold, M . A. SPIE Opt. Fibers Med. I I I 1988- 906, 128-133. Klainer, S. M.; Harris, J. M. SPIE Opt. Fibers Med. I I I 1988, 906, 139-147. Luo, S.; Walt, D.R . Anal. Chem. 1989, 6 1 , 174-177. Hnatowich, D . J.; Virzi, F.; Rusckowski, M. J . Nucl. Med. 1987, 2 8 , 1294- 1302. Bayer, E. A.; Wilchek, M. Methods 8iochem. Anal. 1980, 2 6 , 1-45. Hofmann, K.; Finn, F. M.: Kiso, Y . J . Am. Chem. SOC.1978. 100, 3585-3590. Orr, G. A.; Heney. G. C.; Zeheb, R. Methods Enzymol. 1988, 122, 83-81
Oir,G. A. J . 8iol. Chem. 1981, 256, 761-766. Hofmann, K.; Wood, S. W.; Brinton, C . C.; Montibeller, J. A,; Finn, F. M. Proc. Natl. Acad. Sci. U . S . A . 1980, 7 7 , 4666-4668. Hofmann, K.; Titus, G.; Montibeller, J. A,; Finn, F. M. 8iochemisfry 1982. 2 1 , 976-984. Romovacek, H.; Finn, F. M.; Hofmann, K. Biochemistry 1983, 22. 904-909 Redeiih, G.; Secco, C ; Baulieu, E. E J . B i d . Chem. 1985, 2 6 0 , 3996-4002. Barman. T. E. Enzyme Handbook, Vo/. I I : Springer-Verlag: BerlinHeidelberg, 1969; p 648.
RECEIVED for review November 28,1988. Accepted February 15,1989. Financial support was generously provided by the Environmental Protection Agency through Tufts Center for Environmental Management.
