The application of evanescent wave sensing to a high-sensitivity fluoroimmunoassay

Biosensors L Bioelectronics 7 (1992) 39-48

The application of evanescent wave sensing to a high-sensitivity fluoroimmunoassay Irene M. Walczak”, “Ciba Coming Diagnostic bComing

Walter

F. Loveb, Thomas A. Cookb & Rudolf E. Slovaceka,*

Corporation, Incorporated,

(Received 9 November

Research and Development, Sullivan

63 North Street, Medtield, MA 02052, USA

Research Park, Coming,

NY 14831, USA

1990; revised version received 7 March 1991; accepted 11 March 1991)

Abstract: The principle of evanescent wave sensing was applied to immunological measurements of the clinically important human enzyme creatine kinase (CK; EC 2.7.3.2) isoenzyme MB form (CK-MB) with a molecular weight of about 84 000. The measurements were obtained using a green helium-neon laser based fluorimeter and the antibody conjugated fluorophor B-phycoerthrin (BPE). With conservative optical launch and collection angles of 25”, a detection limit of 0.1 ng ml-’ CK-MB was observed in less than 15 min for a fiber optic based assay in which there were no subsequent washing, handling or processing steps after exposure to the sample. Keywords: fiber-optic,

immunosensor,

fluorescence,

wave.

amplitude E decays exponentially distance from the interface, i.e.

INTRODUCTION When a dielectric

material of refractive index nt is surrounded by a medium of lower refractive index n2 an optical waveguide is formed whereby light rays exceeding the critical angle a, measured normal to the interface are totally internally reflected and hence propagated along the waveguide. If the cladding or lower refractive

where depth l/e of depth d,

(1)

x is the distance and d, is the penetration into the cladding for the amplitude to fall to its value at the surface, E,. The penetration is given in eqn. (2) (Hat-rick, 1967) as =

A

2 TIn2 [(nl/n2)2 sin2 Q, - l] ‘I2

(2)

The penetration depth becomes larger when the ray angle @ (defined relative to the normal to the approaches @c = sin-’ (n2/n1), interface) indicating a transition from an evanescent to a refracted wave in the lower-index or surrounding medium. Typically, dp is less than A, the wavelength of the incident light in a vacuum. The

should be addressed.

0965-5663/92/$05.00 @ 1992 Elsevier Science Publishers

to zero with

E = E, exp(-x/d,)

index medium contains fluorescent material, then the waveguide may be used as a sensor which both excites and collects the fluorescent radiation. In the rarer medium @z2) there is a nonpropagating or evanescent wave which is nonzero for distances close to the core/cladding interface. In particular, the electric field *To whom correspondence

evanescent

Ltd.

39

I. M. Walczak et al.

evanescent electromagnetic field can interact strongly with molecules in a surrounding medium if they are near the interface. Thus, absorption of incident light of appropriate wavelength causes a subsequent fluorescence emission, which can be collected by the waveguide. Although this phenomenon was first partially described for dilute solutions of fluorescein using a planar geometry (Hirschfeld, 1965), it was subsequently applied (Kronick & Little, 1975) to detection of an analyte attached to the surface of a glass slide by an immunological binding reaction. If one employs the same principles of evanescent wave interaction at the coresurrounding medium interface of a fiber optic waveguide (Andrade et al., 1985), a simple nonseparation immunoassay can be constructed in which a serum- or water-based sample takes the place of a conventional cladding whereas the fiber core itself serves as a solid phase on which analyte may be concentrated by a combination of diffusional movement to the surface and trapping via antigen-antibody binding reactions, adsorption processes, or by non-specific binding mechanisms. Detection limits with fibers of lo-* and 10e7 M were first described (Sutherland er al., 1984) for a two-site fluoroimmunometric assay of immunoglobulin (IgG, mol. wt. 150000) and a competitive binding assay for methotrexate (mol. wt. 454) respectively. This work was in part limited by the use of low numerical launch designs of 0.3 or less as predetermined by the fiber core and cladding indices of refraction which were initially developed for telecommunications work but adapted for use in such immunoassay studies. The determination of a strong signal dependence (Hirschfeld, 1987) upon the launch and collection numerical aperture (i.e. NAtaunch = sin emax, where 6,,, denotes the maximum external angle for the launch and collection cone of light) and the use of high-NA sensors in experimental work (Love & Slovacek, 1986) have made possible improvements in detection limits to cover clinically relevant ranges for most analytes. In particular, detection sensitivities of 1.1 X 10-t’ M and 6 X 10-i’ M for the iron storage protein ferritin (mol. wt. 450 000) and the cardiac glycoside digoxin (mol. wt. 780) respectively, have been reported (Sutherland ef al., 1988). It has also been demonstrated from both a theoretical and experimental approach (Love & 40

Biosensors & Bioelectronics

Button, 1988) that the total fluorescent signal, Sr , obtained from one end of such a fiber sensor is given by ST Q: c ZOL a (r,&a)2

sin’ 8,,

(NAmat1)-4 (3)

where E is the absorbance coefficient, IO is the optical source radiance, L the fiber length, a the fiber radius, r,,, the radius of the launch spot, 8,, the maximum external angle of the launch and acc;p;;nce cone and N&,,t is given by h2-n2)

*

From eqn. (3) it should be evident that modulation of several dependent variables could lead to enhanced signal and hence a greater detection sensitivity. For the purpose of this investigation we have chosen to increase the molar absorption coefficient by changing from fluorescein, with a molar absorption coefficient of .s495nm = 72 000 mol-’ L cm-‘, to B-phycoerythrin, with e545nm = 2 410 000 mol-’ L cm-’ as a fluorescent label (Oi et al., 1982). The emission maximum for B-phycoerythrin is at 578 nm. Also, the source radiance was increased by the use of a green helium-neon laser (543.5 nm) chosen to match closely the peak absorbance wavelength of B-phycoerythrin at 545 nm. Finally, we have extended the application of this method to the cardiac isoenzyme creatine kinase MB form (CK-MB, mol. wt. 84 000) as there is considerable interest in the use of a sensitive immunodiagnostic assay for early detection of myocardial infarctions. Although comparative studies of the efficacy of activity versus mass measurements are currently a topic of clinical interest (Delanghe et al., 1990) we have chosen to restrict our attention to the measurement of the immunologically detectable mass of CK-MB. Current sensitivities lie in the 2 ng ml-’ or 2.4 X lo-” M range (see, for example, Delanghe et al. (1990) and Pearson & Carrea (1990).

EXPERIMENTAL Chemicals Antibodies to human CK-MB were raised in mice by methods similar to those previously described (Slovacek & Harvey, 1984). Ascites fluid fractions containing the antibodies were further purified on Aftigel@ blue columns before dilution in phosphate-buffered saline solutions for either fiber-coating processes or preparation of a

~vunes~~t wuve sensing for~uoroimmun~~

Biosensors dt Bi~l~~nics

INLET

fluorescent-labeled antibody conjugate. Custom B-phycoerythrin anti-C&MB antibody conjugates were prepared by the services of Molecular Probes Inc. (Eugene, OR). The conjugate fractions, containing approximately 1.2-l -3 B-phycoerythrin molecules per antibody were puritied by passage over a size exclusion column of Sepharose G-25.

M

Immunoassays Two-site fluoroimmunometric assays for the cardiac isoenzyme CK-MB were performed using fibers with selective antibodies attached to the lateral surface. The samples themselves were

END CAP EMISSION

Fiber optic sensors Fused silica fiber rods of l-00 mm diameter were fabricated by Corning Inc. (Corning, NY). Lengths of fiber were then bundled in a hard fixative wax for batch handling and precut to approximately 6.5 cm cores for placement in a polishing tool to produce an optical finish on the fiber end-faces. After dewaxing and acid cleaning, the fiber surfaces were silanized to promote protein adsorption (Jonsson et al., 1982) with a dichloromethyl silane obtained from Petrarch Systems Inc. Bristol, PA, Antibody was subsequently adsorbed on the fiber, by imme~ion in phosphate-buffered saline solutions containing 100 lug ml-’ of protein, a concentration previously shown (Jonsson et al., 1985) to be on the plateau of binding isotherms. The sensors were rinsed with phosphate-buffered saline and stored in a fresh aliquot of the same buffer at 4°C until use. Under these conditions, coatings remained stable for up to several months, when radioimmunometrically. Protein measured coverage values, for this process, were virtually identical to those reported earlier (Jonsson et al., 1985) and produced active binding sites in the range of 1 X 1O-‘5 mol mm2 as determined from equilibrium radiolabeling studies (Zettner, 1973). A flow cell, similar to that described previously (Sutherland et al., 1988) and holding 059 ml in the reaction space, was constructed using a 5 cm length of 4 mm id. glass tubing with sample inlet and outlet ports located near the ends. Thin silicone end-caps, formed by a casting method and with an aperture of slightly less than 1 mm diameter, were placed on each end of the reusable cell to provide a liquid seal when the fibers were inserted as in Fig. l(A).

M

FLUOROPHOR

(A)

Fig. 1. (A) Optical sensor cell. Solid line represents a single ray jkom the laser excitation souse impinging on the proximalface at an angle of 25” andpropagated within the jiber by total int~ai reunion. The dashed fine represents one ray ofemitted light captured as evanescentfluorescence propagated by total internal refllectionand refracted out of thefiber in the direction of the collection optics. (B) Optical research apparatus Block diagram of the laser SOUFW, optical components, sensor, and photomultipIier detector for evanescent wave sensing studies.

constructed from serum standards of CK-MB (Ciba Corning Diagnostic Corp., Walpole, MA), which were further diluted into human serum which had been previously stripped of any endogenous CK-MB with an anti-OK-MB affinity immunoadsorbent. The stripped serum contained no measurable CK-MB when analyzed with a Ciba Coming Diagnostics Corp. CK-MB Magic-Lite @ kit. Before measurement 250 ng ml-’ anti-CK-MB antibody-BPE conjugate (also prepared in serum) was mixed with an equal volume of sample and injected into the flow cell containing a fiber. One minute after the fiber sensing region was covered by the solution mixture, a 10 s integration of the photometric signal was performed and continued once a minute thereafter. It should be noted that the 41

Biosensors & Bioelmmnics

I. M. Walczak et al.

usual protocol of a wash step before measurement has been eliminated. Optical instrumentation

Figure l(B) displays a block diagram of the optical apparatus used for fluorescence measurements. The beam from a single-mode, TEMoo, randomly polarized helium-neon laser (Particle Measurement Systems Model LHGR-0050) operating at 543.5 nm was first passed through a 543.5 nm/lO nm FWHM Melles Griot (03~FIL-048) interference filter to remove plasma tube fluorescence and a 1.0 mm pinhole to reduce stray speckle. A 20 cm focal length (fl) fused quartz lens was then used to focus the beam to a 180 Frn spot diameter on the tiber end-face. The launch or insertion power was measured with a Photodyne Model 88 XLC Radiometer to be 200 p W. The laser, filter, pinhole and lens were mounted on a movable optical rail whose pivot point was located in the fiber end-face plane. This allowed for changes in the launch angle between 0 and 45” to be rapidly implemented. A 50 mm diameter 200 mm fl achromatic collection lens (Melles Griot 01 LAU 045) was located at a fixed angle of 25” to the fiber axis and the collimated beam passed through two Omega Optical DF 580/20 nm interference filters. Stray light was reduced by spatial filtering of the collected light. This was accomplished by refocusing with an identical achromatic lens onto a pinhole aperture (2.0 mm), then passing the light with a Melles Griot 0.16 NA microscope objective onto a thermoelectrically cooled Hamamatsu R928P tube (PMT). Photoelectron photomultiplier pulses were amplified with an EG&G model 1120 amplifier discriminator and counted with an EG&G Model 1112 photon counter. The dark count rate was determined by integration over a 100 s interval to be approximately 2 counts s-i so that all values in excess of this number may be attributed to light-dependent phenomena. The Fresnel reflection from the fiber end-face was simulated by replacing the fiber with the flat face of a cleaned fused quartz plano-convex lens and the signal measured upon varying the launch angle between 0 and 40”. Only a slight monotonic increase between 15 and 24 counts s-t was observed and could not be eliminated by blocking the laser beam. This is consistent with the signal, at this level, being due to stray light at 580 nm which enters the collection optics. It also 42

indicates that the launch beam power, which reached the detector, was attenuated by 12 orders of magnitude or lo- ‘* . In contrast, fluorescent emission light, also collected from the proximal end of a fiber sensor (see Fig. l(A)), was transmitted through the combined emission filters with a 25% efficiency at the 580 nm band pass. This high contrast allowed detection of weak fluorescence at 580 nm with a good signalto-noise ratio. THEORY One of our first considerations in making an idealized fluorescent measurement, with a fiber, was to understand the nature and source of background fluorescent emission. When analyzing background sources from the sensor, several possibilities come to mind. The first is that color centers or impurities in the bulk fiber material itself may contribute to the background. Similarly, scattering sites within the fiber may direct light to and from fluorescent materials located in the general vicinity of the fiber. In this case, the background fluorescence should be proportional to the optical pathlength through the sensor with an Llcos 0, dependence, where L denotes the fiber length and 0, the internal angle of the propagating laser beam relative to the fiber axis. Alternatively, with optically pure materials, the background may be caused by either surface contaminants from handling and preparation, or fluorescent materials embedded near the surface. Consequently, the fluorescence should show a dependence on the regions of evanescent wave surface interaction or an (L/h) tan 0, dependence, where a is the fiber radius and 0, is the angle with respect to the fiber axis for meridional rays. A consideration of skew rays would enhance this dependence somewhat. If the signal of interest is assumed to be a fluorescent molecule brought to the surface by an immunological binding reaction (Kronick & Little, 1975) or an adsorptive mechanism, or present near the surface through a statistical distribution of molecules in solution, one might expect a signal dependence derived in the following manner. A differential element of the fluorescence signal can be defined by the quantities d(signa1) ot N Pabs g

dA

Evarrescentwavesensingfor~uoroimmunoa~say

Biosensots d5Bioel~t~ni~

where N is the number of reflections, Pabs is the optical power absorbed per reflection, daldA is the transformation of the lateral surface onto the fiber end surface area and dA is a differential element of the fiber end-surface. For propagation angles 0, with respect to the fiber axis and in the more general case for skew rays in which the azimuthal angle component is given by 19,, the number of surface interactions may be defined as N=:_--

L sin@,

1

2a cos 0, [ sin 5,

SILICA FIBER TRIS BUFFER

: i

25” DETECT

,

1

The optical power absorbed

will be given by

Pabs cc E [t]* = .s sin2 8, sin* 0,

2(6)

where [t] is the absolute value of the Fresnel transmission coefficient and E is the absorbance coefficient of a dye solution assuming unpolarized light and random molecular orientation. The transformation of the lateral surface to the fiber end-face is given by da --=: dA

in^I 8 B-

cos e, sin 0,

(8)

The differential element of the signal can now be integrated for the laser source over the end-face illuminated area A, and angular launch conditions. As the laser light is launched primarily at a single angle, we expect the total signal to vary as sin2 0,. Using SnelPs law of refraction to relate &, to &, we further expect the signal to be propo~onal to sin* BeXt , where 0,, is the external launch angle relative to the fiber axis. The generalized relationship for the signal will thus be given by signal a c$ sin2 e,,, sin 0, A

(9)

RESULTS AND DISCUSSION Actual measurements of background are shown in Fig. 2 for a fiber in the a 10 IIIM Trizma hydrochloride solution at pH 7.6 serving as the

W

5

0

10

20

30

8,40

EXTERNAL LAUNCH ANGLE (“) Fig. 2. Angular dependencefor the excitationoffluorescent ba~k~o~nd s&als@om aJirfed silica~b~ surrounded by Tris buffer in a frow cell.

(7)

As the probability of emission into the fried-angle detection optics is constant, substitution of eqns. (5), (6) and (7) into (4) gives a generalized dependence of d(signa1) cc a$ sin2 0, sir@ dA

!

fluorescence flow cell with (Tiis) buffer surrounding

medium. The refractive index, measured at 1.33, is about the same as that of water. The angle 6, in the figure is the external critical angle. Light rays launched at angles less than 0, (external to the fiber) are bound or waveguided in the fused silica fiber. For increasing launch angles there is a fairly close relation to the number of reflections which are proportional to tan 0,, indicating that the background is of a surface origin. It is also apparent that the fluorescent signal is greatly diminished at 40” when the launched light rays are no longer propagated as bound modes. Even with a 0” launch angle there is still significant background. From calculations it can be easily shown that the Raman contribution, from either fused silica or water, to the background signal is less than 0.01%. Acausal nature has thus not been completely identified at 0”; however, we expect that some beam spreading occurs within the fiber, leading to absorption and fluorescence near the surface. From a practical viewpoint, it is the statistical variation in the photon arrival rate from this background component which will determine the minimum signal detection limit. For a 25” launch angle this corresponds to a background of 6000 counts s-l or a statistical noise variation of 77 counts s-l. Thus, approximately 80 counts s-’ above background would be required to begin elevation of the signal43

Z. M Walczak et al.

Bioserrsors & Bioelectronicr

to-noise ratio above one. In the more complex case described below, where serum samples containing the B-phycoerythrin antibody conjugate surrounded the fiber, a background level of about 16 800 counts s-l was observed. In this situation, more than 130 counts s-l above the background signal would be required for a signalto-noise of greater than one. To establish a measure of sensitivity, dilute solutions of B-phycoerythrin, made up in a Tris buffer, were placed in the flow cell and the signal levels were recorded with either full or attenuated optical launch power. As illustrated in Fig. 3, variation in launch power has the effect of diminishing the signal amplitude in a manner proportional to the input power. One concludes that the input power, at least for this particular measurement, is below the threshold for observations of irreversible photobleaching. Based on a linear extrapolation from the O-98 ND value at a lo-l2 M concentration, a S&I’d intensity of 50-100 counts s-’ may be estimated to occur with full power at lo-l4 M and with a signalto-noise of one for the simple case without serum present. To determine whether the fluorescence emanating from a fiber sensor is consistent with

1.0 1

10-‘OM

BPE n

/

I I

8

5

53 # 0.1 0 2

B b # g 0

I I

sin e,,

0.01 .08

.15

.25 .4 S’” %xt

.6

Fig. 4. Angular dependence for the excitation of a lo-to M solution of jluorescent B-phycoetythnm. Arbitrary values are background corrected and normalized to throughput power

FUSED SILICA (lmm)

10-12

10-11

10-10 10-g MOLAR CONCENTRATION

Fig. 3. Fluorescence sensitivity measurements with free solutions of B-phycoerythrin at three power levels. Neutral densiq filters (ND) as indicated were used to attenuate the input power Fluorescentvalues were computed by subtmcting measured background signals from the signals observed with thefluorescent dye present. Full power refers to the 200 u W of unattenuated beam power. 44

an evanescent wave interaction, the launch angle dependence for the signal was measured using a lo-” M solution of B-phycoerythrin and is shown in Fig. 4. The net signal, after correction for a background term measured in the absence of the dye, (i.e. signal - background), was normalized to the throughput power of the fiber and is plotted as a sine function of the external launch angle, 8,, , on logarithmic coordinates. At least-squares tit (linear regression) of the data gives a slope of 2.1, indicating experimental agreement with the sin28,,, dependence predicted by eqn. (9). As it is possible that some Bphycoerythrin in solution may have become adsorbed on the fiber surface, one cannot accurately assess whether this relation holds specifically for material in solution near the fiber, material absorbed by the fiber, or a combination of both. A second important test of the angular dependence was thus performed using a fiber

l&sensors & Bioelectronics

specifically coated with fluorescent material instead of measurements with the dye molecules added free in solution. The fiber, originally coated with anti-CK-MB antibodies, was incubated in a solution ~on~ining both 50 ng ml-’ CK-MB and B-phycoerythrin labeled anti-CK-MB antibody at 4°C for 20 h overnight to promote a high degree of antigen-antibody binding or adsorption of the fluorescent material. The fiber was rinsed with phosphate-buffered saline to eliminate carry-over of any fluorescent conjugate solution, then mounted in the flow cell and covered with fresh buffer before measurement, It should be noted here that, in contrast to the free solution results of Fig. 4, continuous illumination was observed to cauSe some photoblea~hing when the fluorescent dye molecules were more rigidly attached to the fiber and could not readily diffuse either into or out of the evanescently pumped surface region. The problem was, however, minimized by making the angular adjustments and then ~luminating the fiber for only the required integration interval (10 s) used in the photon counting process. This procedure was subsequently adopted for all the following examples where antibody binding reactions bound the fluorescently labeled material to the fiber surface. measurements of the resulting fluorescent signal vs. launch angle are presented in Fig. 5 on logarithmic axes. A linear regression analysis gives a slope of 1.7 for the dependence on sin f&, . This is also reasonably close to the value predicted by the model considering the presence of some photobleaching effects and the possibility that some fluorescent material was inadvertently fixed to the end surfaces during the coating and handling stages. For the lowest solution concentration of Bphycoerythrin actually measured here (lo-‘* M), one can estimate the approximate number of molecules sensed in the evanescent region. From the total projected beam area along the sensor lateral surface (1.37 mm2) and the evanescent wave penetration depth, calculated to be about 100 nm (angle dependent), a total solution volume of l-37 X lo-” 1 is interrogated. This would correspond to approximately 1.37 X lo-** mol or 82 molecules. It is interesting to note that in using a similar analysis, a calculated comparison between fluorescent immunological samples and free fluorescein solutions (Sutherland ec al., 1988) displayed reasonably good agreement with radiolabeling methods in determining the amount of

Evanescent wave~~ingfor~uoroimm~noassay 1X10

77

sin

e

&

1x1o.i; sin. . .

3

0 ext

Fig. 5. Angular dependewceforthe excitationoffIuore.wence Jiom B-phycoerythrincoated on a@ber by immunological rn~h~s.

molecules sensed near a fiber surface. The clinical analyte CK-MB was subsequently determined by constructing a two-site fluoroimmunomet~c assay and using a conservative 25” launch and 25” collection angle in the optical measurements. Although more signal intensity could be obtained at higher angles of up to 0, = 36.8” (see Fig. 5), the ease of optical alignment and insensitivity to perturbations of the light into solution yielded the more conservative approach. Samples, made up with serum standards of the isoenzyme, were mixed with the B-phycoerythrin conjugated antibody and then each mixture was injected into a flow cell loaded with a fresh antiCK-MB antibody coated fiber. A binding reaction between CK-MB and the B-phycoe~h~n antibody conjugate followed by a subsequent binding of the isoenzyme complex to a second, but different, fiber antibody completes the two-site reaction, although it is also possible for the binding sequence to be reversed. Through a combination of diffusion and antibody-antigen binding reactions, increasing amounts of the fluorescent B-phycoerythrin labeled material 45

I. M. Walczak et al.

Biosensors & Bioelectronics

were expected to concentrate on the fiber surface in a manner directly dependent on the analyte concentration. From measurements of lo-‘* M B-phycoerythrin solutions, shown in Fig. 3, one intuitively expects that immunochemical leading to an enhancement of reactions, fluorescent material on the fiber surface, should allow measurements to be made of analytes at equivalent or lower concentrations. Fluorescent signals, representative of the binding kinetics for subnanogram per milliliter levels of CK-MB, are presented in Fig. 6. As there is very little signal accumulation (i.e. 900 counts s-’ or less) in the absence of added CK-MB, but in the presence of the B-phycoerythrin antibody conjugate, it is unlikely that non-specific adsorption of labeled conjugate or an exchange with surface-adsorbed antibody accounts for much of the signals observed in Figs 5-7 where CK-MB was present during the incubation process. A sample of 0.1 ng ml-’ or 1.2 X lo-‘* M CK-MB gives a signal increase of greater than 3000 counts s-l over that observed as non-specific background (NSB) associated with a zero control standard. Non-specific adsorption mechanisms can thus account for, at most, 25% of the time-dependent signal accumulated with the lowest CK-MB sample and a proportionally smaller fraction of the signals with higher levels of CK-MB. The results strongly favor antibody-antigen binding

.

. 2.0

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l

4O~!~fAA*+q+qtTi . l** 2

4

. . . ::t

. .

.

.

.

.

l.::: 8.0.

6 8 10 12 TIME (Minutes)

.

.

l

1.0

: .0.5 . .O.l fp& 14

16

Fig. 6. Time-dependentjluorescence kinetics for subnanogram samples of CK-MB giving antigen and jluorescent conjugate accumulation on optical Jibres. Sample concentrations aregiven next to curves. Curves represent values obtained after a point-by-point subtraction of the initial measured value on each individual fiber. NSB refers to a jiber exposed to the labeled BPE-antibody conjugate in serum without added CK-MB. 46

280 .

.

f2400 “* 200 5 w 160Y

. . . .

.

.

L&J 120

2 ;

-

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g 40B! 0

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. l

. .

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.

.

020

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l

l

5

l

6 8 10 12 TIME (Minutes)

8 rl

t2

14

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16

Fig. 7. Kinetics of the fluorescence increases for clinically equivalent samples of CK-MB. Curves represent values obtained after a point-by-point subtraction of the initial measured value on each individualjber.

reactions specific for CK-MB as the predominant cause for signal increases. Optical signals for higher-level samples which span the clinical range between 2 and 50 ng ml-’ are plotted in Fig 7 as a function of the incubation time. In view of the expected dependences upon conjugate concentration, number of fiber binding sites, and the analyte concentration, it is noteworthy that the kinetic curves display nearly linear rates over the time period and range of sample values examined. The premixing of sample with at least a five-fold excess of labeled conjugate was chosen to minimize the reaction dependence upon the secondary labeled antibody. Although the number of antibody binding sites on a fiber (see Experimental section) may approximate the total CK-MB antigen available in the sample chamber after dilution and mixing of the highest 50 ng ml-’ standard, it is unlikely that diffusion rates permit sampling of many molecules greater than 5OOpm from the fiber or from a surrounding volume greater than one-fifth of the chamber volume during the 15-min incubation in serum. For comparison, a large protein complex such as unease (EC 3.5.1.5),with a molecularweight of 480 000, which begins to approach the size of a BPE-antibody conjugate, and topomyosin, whose molecular weight (93 000) approximates that of CK-MB, have diffusion constants of 3.46 X 10F7 cm* s-l and 2.24 X 10m7cm* s-l respectively in water (Tanford, 1961) and may be shown to travel a distance of only about 500600 pm. The deviation from linear rates would be

Biosensom (B Bioelectmnics

Evanescent wave sensing for~uoroimmunoa~ay

expected to occur over short time intervals only if moderate to high analyte levels were available in the diffusion space to saturate the fiber binding sites quickly and bring about an equilibrium value. Such a case was previously noted (Bluestein et al., 1990) for lidocaine, an antiarrhythmia drug whose therapeutic range (micromolar) is more than four log orders greater than that covered in the work described here. Alternatively, when the analyte is equivalent to or less than the number of binding sites, as in work described here, one might still expect a rate diminution at longer time intervals, such as 30 min or more, when the diffusion distance for the mobile binding partner begins to approach that of the vessel wall. Examples of longer-term kinetic behavior, described above, have been reported (Sutherland et al., 1988). Similar rate decreases (data not shown) can be observed for more prolonged incubations under conditions like those employed in Figs. 6 and 7 or when inadequate labeled conjugate, or fiber binding sites, are used in the assay. The apparent first-order dependence on the CK-MB concentration is clearer in the graph of Fig. 8. Readings, taken at 15 min, were corrected for non-specific binding (NSB) and the averages computed for duplicate samples. These corrected fluorescence values are plotted vs. CK-MB concentration on logarithmic axes. It is also evident that detection of subnanogram quantities of C&MB occurs with a photon event frequency well above the Shot noise limitation of the photon counting method used. If the external launch NA

is raised to 0.6 or &, = 36.8” to match the sensor NA more closely, the signal amplitude might be expected to double, as indicated by Fig. 5. Additional improvements in the collection optics should also further enhance the signal, leading to even lower detection limits for CK-MB. ACKNOWLEDGMENTS We would like to thank Ralph Westwig and Leslie Button for fruitful discussion and encouragement. This work was partially supported by Ciba Coming Diagnostics Corp. and Coming Inc. EVINCES Andrade, J. D., Van Wagenen, R A, Gregonis, D. E., Newby, K &ILin, J. N. (1985). Remote tiber-optic biosensors based on evanescent-excited fluorimmunoassay: concept and progress. IEEE Trans. Electron Devices, ED 32(7), 1175-g. Bluestein, B., Walczak, I. & Shin-Yen, C. (1990). Fiber optic evanescent wave immunosensors for medical diagnostics. Trends Biotechnol,, 8(6), 161-8. Delanghe, J. R, De Mol, A M., De Buyzere, M. L., De Scheerder, I. E. & Wieme, R J. (1990). Mass concentration and activity concentration of creatine kinase isoenzyme MB compared in serum after acute myocardial infarction. Clin. Chem., 36(l), 149-53. Harrick, N. J. (1967). Principles of internal reflection spectroscopy. In Internal Reflection Spectroscopy. Wiley, New York, pp. 13-63. Hirschfeld, T. (1%5). Total n&&ion fluorescence. .X Can. Spectrosc., 10, 128. Hirschfeld, T. (1987). Apparatus for improving the numerical aperture at the input of a fiber optics device. US Patent 4654,532. Jonsson, U., Ivarsson, B., Lundstrom, I. & Berghem, L. (1982).Adsorption behavior of fibronectin on well characterized silica surfaces. J; Colloid Inter&e Sci., !JO(l), 148-63.

Jonsson, U., Malmqvist, M. & Ronnberg, I. (1985) Immobilization of immunoglobulins on silica surfaces. B&hem. 1, 227,373-g. Eronick, M. N. & Little, W. A. (1975).A new immunoassay based on fluorescence excitation by internal reflection spectroscopy. J. Immunol. Methods, 8, 0.5 1 2 CONCENTRATION

5 10 20 50 CKMB (ng/ml)

Fig. 8. Fluorescence-based standarrl curve for the analyte CK-MB in serum. Corrected value on ordinate axis were obtained by subtraction of non-spec@e binding values at 15 min (see NSB trace, Fig. 6).

235-40. Love, W. & Button, L. (1988). Optical characteristics of fiber optic evanescent wave sensors. In Chemical, Biochemical and En~~nrn~t~ Appi~o~ of Fibers,

ed. R Lieberman & M. Wlodarczyk. SF&ZFroc.. 999,175-80. Love, W. F. & Slovacek, R E. (1986). Fiber optic 47

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