Anal. Chem. 1988, 6 0 , 853-855
853
Determination of Avidin and Biotin by Fluorescence Polarization Keith J. Schray* and Pamela G. Artz Department of Chemistry, Lehigh University, Bethlehem, Pennsylvania 18015
Richard C. Hevey CPH Biomedical, Inc., 881 King Road, Malvern, Pennsylvania 19355
Assays for avldln and blotln are presented whlch comblne high sendtlvlty with short assay tlmes. Mlnlmum detectable concentrations are 5 ng/mL avldln and 100 pg/mL Moth and assay thne Is under 10 mln. The fluorescence polarlzatlon of a blotln-fluorescein conjugate Is monitored. Polarlzatlon varies as a functh of avidin concentration and, at ftxed avldln levels, as a functlon of competing blotln concentrations. This slmple, r a w assay has a detecth llmn about IOOO-fokl lower than the commonly used 4-hydroxyazobenzene-2tarboxyllc acid dye blndlng method.
The use of biotin and avidin as a tool in biochemistry, microbiology, and immunochemistry (1-4) has experienced a very rapid growth. This interest together with biotin's role as an essential component in human biochemistry (5) has spurred the creation of a significant number of assay methods (6-22) including microbiological, colorimetric, enzymatic, radiometric, electrochemical, and fluorescent approaches. The most widely used method is that of Green (7,8 ) which is a rapid, facile spectrophotometric procedure. In general, a lower detection limit has been obtained at a cost of considerably more complex and time-consuming techniques. The assay reported here is a fluorescence polarization assay relying on the use of a biotin-fluorescein conjugate. A combination of lower detection limit and a simple, rapid protocol makes this assay a good candidate for wider use.
EXPERIMENTAL SECTION Materials. The following materials were obtained from Sigma Chemical Co.: avidin, biotin, N-hydroxysuccinimide biotin ester and fluorescein isothiocyanate. All solvents and buffers were of reagent grade quality. Apparatus. Polarization values were determined by using an SLM fluorometer equipped with a xenon arc lamp, a monochromator in the excitation beam set to 491 nm, and a 10-nm bandwidth 520-nm filter in the emission beam. Polarizing filters were placed in the excitation and emission beams so the emission filter could be aligned parallel or perpendicular to the excitation filter. An SLM SPC-822 data processing module was used in the photon counting mode to determine polarization values. Synthesisof Biotin-Fluorescein. Fluorescein isothiocyanate, 10 mg (25 pmol), was dissolved in 2 mL of dimethylformamide and 0.008 mL of ethylenediamine (120 pmol) was added at room temperature. An orange product formed immediately as indicated by a new component on thin-layer chromatography (silica gel G, methanol solvent). Product crystallizes over a period of several hours at room temperature. The precipitate was recrystallized in dimethylformamide,filtered, and dried (mp 300-304 "C). Yield of purified material was 56%. This synthesis is considerablymore facile than that reported previously (23). The fluoresceinethylenediamine adduct, 6.5 mg (14 pmol), was dissolved in 2 mL of dimethyl sulfoxide and 9 mg (26 pmol) of N-hydroxysuccinimide biotin ester was added. The mixture was followed by monitoring the effect of avidin on the polarization of the reaction mixture. This reached a maximum after 60 h at room temperature. The conjugate was purified by chromatographing 100 NLof the above reaction mixture on a silica gel column (20 X 1.5cm,230-400 mesh, 0003-2700/88/0360-0853$0 1.50/0
60A pore size), preequilibrated and eluted with methanol. Three fluorescent peaks separated with the rapidly eluted lead fraction exhibiting a large (p = 0.29) fluorescence polarization change upon complexation with avidin. This fraction constituted 85% of the pooled, eluted fluorescence. The other fractions were 9% and 6% of the fluorescence, respectively, and showed no significant polarization change upon addition of avidin. Avidin Assay. To glass or plastic tubes or disposable cuvettes was added 1.3 mL of biotin-fluorescein in 0.1 M phosphatebuffered saline, pH 7.4. The concentration of biotin-fluorescein conjugate essentially determines the detection limit of the assay because the high binding constant to avidin allows virtually complete complexation at equimolar concentrations. The concentration was chosen to yield a 201 sample/buffer fluorescence ratio with no special precautions to reduce buffer fluorescence. The concentration is estimated to be ca. 4 x lo4 M based on the avidin titration curve equivalence point. To this solution was routinely added samples of sufficient avidin to yield the concentrations shown in Figure 1. As the biotin-fluorescein may be supplied to the assay in as little as 0.001 mL volumes, the remainder of the assay volume (1.35 mL total in this instrument configuration) may be the avidin sample which thus experiences virtually no dilution. These were allowed to incubate a minimum of 3 min at room temperature and fluorescence polarization was determined. The standard curve may then be constructed for use in determinations of unknowns. Detection limit is defined as the lowest concentration in which the response values do not overlap those of the zero value. Biotin Assay. To samples of various concentrations of biotin as shown in Figure 2 was added avidin, 40 ng/mL (5 X M) in the above buffer. These mixtures were incubated at room temperature for 3 min and then biotin-fluorescein was added to yield a final concentration of 4 X M. The assay mixtures were then incubated at room temperature for an additional 3 min, polarizations were determined, and a standard curve was constructed.
RESULTS AND DISCUSSION Because of rapid molecular tumbling, fluorescence radiation from small molecules is depolarized when excited by polarized light. Complexation by a high molecular weight molecule slows tumbling of the resultant complex and allows the emitted radiation to remain partially polarized. Thus, complexed and uncomplexed fluorescent molecules may be distinguished by the polarization of the emitted light. This assay makes use of this fact by forming a small fluorescent biotin species by covalently linking fluorescein and biotin. Fluorescein isothiocyanate was reacted with ethylenediamine to form a crystallizable amino derivative of the fluorophore. This derivative was then reacted with N hydroxysuccinimide biotin ester to obtain the conjugate in approximately 50% yield based on fluorescein. Chromatographic purification on a silica gel column yielded a fraction whose polarization was p = 0.02 and when complexed with avidin increased to p = 0.31. The assay for avidin relies on this change of polarization when complexation of the conjugate occurs with avidin. A standard curve is shown in Figure 1. The minimum detectable avidin concentration in the assay is approximately 5 ng/mL with an assay range of ca. 20-fold. 0 1988 Amerlcan Chemlcal Soclety
854
ANALYTICAL CHEMISTRY, VOL. 60, NO. 9,MAY 1, 1988
Table I. Detection Limits of Available Avidin-Biotin Assays
method fluorometric colorimetric colorimetric radioisotopic radioisotopic potentiometric fluorometric spectrophotome-
detection limits [avidin], [biotin], ng/mL ng/mL 170000
250
50000 3 5000
100 100
50
100
Assay
time
ref
minutes minutes
6 7,8 9
minutes minutes
10
hours 20
minutes
11 12
20
minutes
20
5
minutes
13
0.6 0.5
hours
14
40
100
0.2
5000
3000
tric 20
100
60
140
160
[AVIDIN]
np/ml
220
260
300
Flgure 1. Standard curve for assay of avldln measwing the change in polerizatkn of Motln-Ruoresoehas a furctkn of avidin concmtmtton. Biotln-fiuoresceln concentration is 4 X io-' M in 0.1 M phosphate buffered saline, pH 7.4. Error bars represent the high, low, and average values of triplicates.
0.lE
0.14
z
-I-
0
a
0.12
i
N
6.
0.1 I
-1
0
a
0.IC
0.06
I
I
I
0.00
0.16
0.24
I
0.32
I
0.40
Flgure 2. Standard curve for assay of biotin measuring the change in polarirdon of a soiutlon of 4 X lo4 M biotln-fluorescein and 40 ng/ml avidin In 0.1 M phosphete buffered sake, pH 7.4, as a functlon of biotln c o n w t b n . Error bars represent the hlgh, bw, and average values of triplicates.
The effect of avidin is prevented by biotin due to competition for the limited avidin binding sites. A biotin standard curve is shown in Figure 2. This is generated by selection of an avidin concentration (40 ng/mL), which gives approximately 50% of the maximum polarization change seen in Figure 1. This avidin concentration is chosen to minimize the detection limit of the biotin assay. The resultant smaller polarization change gives rise to the larger percent errors in Figure 2. The assay range is approximately 40-400 pg/mL in the assay. The biotin assay was performed at room temperature with the sequential addition of biotin, avidin, and biotin-fluorescein with incubation times of ca. 3 min after addition of both avidin and biotin-fluorescein. Polarization values reach a maximum
radioisotopic fluorometric radioisotopic chemiluminescent fluorometric bacterial radioisotopic immunoassay bacterial
10000
0.2
5 100
0.1 0.026
minutes hours minutes minutes
0.01
days day
0.2-0.002 0.001
1-20 h day
15 21 22 this work 17
16 19
18
in less than 1min at all avidin concentrations studied and are stable for at least several hours. The 3-min incubations are chosen as a convenient interval for multiple sample assays. Though theoretically a competitive binding assay, the sequential addition and rapid association kinetics allow biotin-fluorescein to occupy only avidin sites left vacant by less than saturating biotin amounts. This yields lower detection limits than those obtained for a true competitive process. Dissociation kinetics are too slow on this time scale to allow competitive equilibration based on binding constants. Human serum up to the level of 1% does not interfere with assay results. The assay described is a very rapid, simple assay in which incubation and assay times and conditions need not be stringently controlled. These conditions compare favorably with the most facile of the currently available assays. A comparison of this and other assays with regards to detection limit is shown in Table I. It can be seen that the detection limit of this assay is 3 orders of magnitude lower than that acid dye of the widely used 4hydroxyazobenzene-2-carboxylic binding method (7,8). Other assays with lower detection limita are more complex and time-consuming. This combination of low detection limit and ease of method should make this a very useful technique. Biotin levels of biotinylated compounds may also be determined by this assay using very small amounts of these compounds. Two requirements of this basically facile method are beyond those of the dye binding assay. The first is the biotinfluorescein conjugate. This is now commercially available from CPH Biomedical, Paoli, PA. The assays described here utilize M conjugate so very little is needed. The second is, of course, the requirement for fluoreacence polarization capability achieved by placing polarizing filters in the excitation and emission beams of the fluorometer. One additional aspect of working at these avidin concentrations is the tendency to lose significant avidin activity due to adsorption at glass or air interfaces. For this reason standards should be freshly prepared or prepared in the presence of 30 p g / m L bovine serium albumen or 0.1% Triton X-100 if storage is intended. Avidin solutions may be stored at least 1 week at 4 "C. Registry No. Biotin, 58-85-5.
LITERATURE CITED ( 1 ) Wilchek, M.; Bayer. E. A. Immunol. Today 1984, 5 . 39-43. (2) Fucillo. D. A. Blofechnlques 1085, 3 , 494-501.
Anal. Chem. 1988, 6 0 , 855-858 (3) Bayer, E. A.; Wllchek, M. Methods Biochem. Anal. 1980, 2 6 , 1-45. (4) Korpela, J. Med. 8/01.1984, 6 2 , 5-26. (5) Romberg, P. E. I n The Metabollc Basis of Inherited Dlsease, 5th ed.; Stanbury, J. B., Wyngarden, J. E., Frederickson, D. S., Goldstein, J. L., Eds.; McGraw-Hill: New York, 1982. (6) Mock, D. M.; Langford, G.; Dubois, D.; Criscimagna, N.; Horowitz, P. Anal. Blochem. 1905, 151, 178-181. (7) Green, N. M. Blochem. J. 1965, 9 4 , 23c-24c. (8) Green, N. M. I n Methods In Enzymology; McCormlck, D. B.,Wright, L. D., Eds.; Academic: New York, 1970; Vol. 18A, pp 418-424. (9) Nledbala, R. S.; Gergitz, F., 111; Schray, K. J. J. Biochem. Biophys. Methods 1986, 13, 205-210. ( I O ) Green, N. M. Biochem. J. 1983, 8 9 , 585-591. (11) O’MaHey, E. W.; Korenman, S . G. Life Sci. 1967, 6 , 1953-1959. (12) Gebause, C. R.; Rechnitz, G. A. Anal. Biochem. 1980, 103, 280-284. (13) Carrico, R. J.; Chrlstner, J. E.; Boguslaskl, R. C.; Yeung, K. K. Anal. Bbchem. 1978, 7 2 , 271-282. (14) Rettenmeier, R. Anal. Chim. Acta 1980, 113, 107-112.
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(15) AI-Haklem, M. H. H.; Landon, J.; Smith, D. S.; Nargessl, R. D. Anal. Blochem. 1981, 116, 264-267. (16) Chan, P. W.; Bartlett, K. Clin. Chlm. Acta 1988, 159, 185-196. (17) Mock, D. M.; DuBols, D. B. Anal. Biochem. 1986, 153, 272-278. (18) DeMoll, E.; Shlve, W. Anal. Biochem. 1986, 158, 55-58. (19) Bayer, E. A.; Ben-Hur, H.; Wllchek, M. Anal. Biochem. 1986, 754. 367-370. (20) Lin, H. J.; Kirsch, J. F. Anal. Blochem. 1977, 8 1 , 442-446. (21) Goldstein, L.; Yankofsky, S. A.; Cohen, G. I n Methods h Enzymology, Chytll, F., McCormick, D. E., Eds.; Academic: New York, 1988; Vol. 122, pp 78-82. (22) Williams, E. J.; Campbell, A. K. Anal. Biochem. 1986, 155, 249-255. (23) Rypacek, F.; Drobnik, J.; Kalal, J. Anal. Biochem. 1980, 104, 141- 149.
RECEIVED for review June 18, 1987. Accepted December 29, 1987.
Measurement of Femtogram Quantities of Trace Elements Using an X-ray Microprobe Robert D. Giauque,* Albert C. Thompson, James H. Underwood, and Yan Wu
Lawrence Berkeley Laboratory, University of California, Berkeley, California 94720 Keith W. Jones
Brookhaven National Laboratory, Upton, New York 11973 Mark L. Rivers
University of Chicago, Chicago, Illinois 60637
Femtogram quantltles of trace elements in blologkai materials have been measured with an X-ray microprobe. The microprobe uses a pair of concave spherical mirrors which were coated with tungsten-carbon multllayers. Arranged in the Kirkpatrick-Baez geometry, the mirrors produce a focused and quaslmonochromated beam from the synchrotron white radiation beam. The beam spot sire is less than 10 X 10 pm and the monochromated beam has a band-pass of 1 keV at 10 keV. Minimum detectable limits achleved varied from 3 to 70 fg for the elements Zn ( 2 = 30) to K (2 = 19).
monochromatize synchrotron radiation. Using an irradiation area of 3500 pm2, a minimum detectable limit (MDL) of 0.03 pg was ascertained for Zn adsorbed on chelate resin beads. In this paper, we describe the application of an X-ray microprobe for the measurement of femtogram quantities of trace elements. The instrument has been used to determine the spatial distribution of trace elements using count intervals of 60 s or less per 10 X 10 hm pixel. Calibration of the microprobe was accomplished by using National Bureau of Standards thin glass film Standard Reference Materials. A variety of specimens have been scanned to demonstrate the applications that are possible with this probe.
The development of intense synchrotron radiation beams has led to significant interest in their application for the measurement of trace element concentrations of very small specimens by X-ray fluorescence. Sparks (I) reported the use of an X-ray fluorescence microprobe using synchrotron radiation for the analysis of monazite giant halo inclusions in biotite in the search for superheavy primordial elements. A curved mosaic graphite crystal was employed to both focus and monochromatize the synchrotron radiation beam. Photons of energy 37 keV with a full width at half maximum (fwhm) of 0.4 keV were used. A flux of 15 X 1O’O photons/(s mm2) was realized. The actual beam spot size was 0.64 mm2. Since 1981 a number of papers have been published regarding the use of X-ray microprobes (2-5). In most cases, the synchroton white radiation beam has been collimated by using vertical and horizontal slits or doubly curved crystals have been used to focus the radiation. Typical usable beam spot sizes attained are on the order of 700-2500 pm2. More recently Iida and Gohshi ( 6 ) have demonstrated the use of a reflection/transmission mirror combination to focus and
EXPERIMENTAL SECTION The experiments were carried out at the Brookhaven National Synchrotron Light Source (NSLS). White radiation from the X-26C X-ray microprobe beam line was used as the primary source for the measurements undertaken. The useful area of the white radiation incident beam was set to a 0.5 x 0.5 mm cross section using horizontal and vertical slits. The slits were located at approximately 20 m from the electron beam. The beam flux utilized was monitored by an ionization chamber that was positioned after the slits and in front of the X-ray microprobe. The microprobe that was employed to serve as a wide band-pass monochromator and focus the X-ray beam is illustrated in Figure 1. The principal components of the instrument are a pair of concave spherical mirrors coated with tungsten-carbon multilayers. The mirrors are arranged in the Kirkpatrick-Baez geometry. The focusing elements were “superpolished” quartz mirrors with a 6-m concave spherical radius of curvature. By use of a dual source sputtering system, each mirror was coated with tungsten-carbon multilayer pairs. The mirror nearest the synchrotron radiation source was coated with 200 multilayer pairs. The second mirror was coated with 100 multilayer pairs. The 2d spacings of the tungsten-carbon multilayer-coatedmirrors were
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0 1988 American Chemical Society
