Fluorescence monitoring of a benzo[a]pyrene metabolite using a regenerable immunochemical-based fiber-optic sensor

Analytrca Chrmrca Acta, 236 (1990) 237-244 Elsevter Sctence Pubhshers B V., Amsterdam

237

Fluorescence monitoring of a benzo[ alpyrene metabolite using a regenerable immunochemical-based fiber-optic sensor JEAN PIERRE

ALARIE,

JAMES

R. BOWYER

and MICHAEL

J SEPANIAK

*

Department

of Chemrstry, Umverslty of Tennessee, Knoxvrlle, TN 37996-1600 (US A )

Department

of Chemutry,

ARTHUR

M. HOYT

Unrversrty of Central Arkansas, TUAN

VO-DINH

*

Healih and Safety Research Dwlslon, Oak Ridge National Laboratory, (Received

25th January

Conway, AR 72032 (US A )

Oak Ridge, TN 378314101

(U.S A )

1990)

ABSTRACT

Microscale regenerable biosensors are described and utthzed to measure the natural fluorophor benzo[a]pyrene tetraol (BPT). The sensors combme laser-excited/fiber-optic remote sensmg pnnctples wtth a umque captllary tube dehvery system to make repetitive, heterogeneous fluorounmunoassay measurements Two sensor conftgurations and modes of operation are described.. Concentrattons of BPT m the nanomolar range are easily measured with a reproductbthty of 10% or better, depending on the sensor design, selective measurements can be made m ca 20 mm, then the sensor can be regenerated by dehvenng new reagents to the sensmg chamber, wtthout removmg the sensor from the sample Keywords

Btosensors,

Ftbre-optic

sensor,

Benzo[a]pyrene

tetraol

Remote spectroscopic-based measurements of physical parameters and chemical concentrations have been made possible with the advent of small-diameter optical fibers that are capable of efficrently transmitting radiation over relatively long distances. By providing an optical link between the sample, located in its native environment, and the spectroscopic instrumentation located m the laboratory, many analytical advantages can potentially be realized. Recently, fiber-optic chemical sensors (FOCS) have been the focus of considerable research activity [l-3]. Spectroscopic signals with FOCSs result from interactions between the analyte and a reagent phase that is immobilized at the sensing terminus of the 0003-2670/90/$03

50

0 1990 - Elsevler

Science Publishers

fiber-optic by various methods, including direct covalent attachment to the fiber [4], entrapment withm a gel [5] or entrapment within a chamber having an analyte-permeable membrane [6]. The analyte-reagent interaction can involve the formation of fluorescent adducts or the quenching of fluorescent reagent phases by analytes such as 0,. FOCSs have been used most often to measure small analytes such as H+, CO,, 0, and metal ions [7-lo]. However, the FOCS measurement of large molecules has also been accomplished through the use of “bioaffmity” reagent phases [1,6,11-131. Our recent work has involved the use of immunochemical reagent phases for performing FOCS-implemented fluoroimmunoassays. Direct BV

238

measurements of natural fluorophors [4] and competitive binding assays [6] have been performed in this fashion. The specificity of antibody-antigen recognition and the excellent sensitivity of fluoroimmunoassays indicate potential analytical utility for “ fluoroimmunosensors”. For a FOCS to be considered a “true” sensor, at least one of the following criteria must be satisfied: the interaction between the analyte and reagent must be reversible and rapid; there must be a sufficient amount of reagent such that it is not depleted over long periods of time; or there must be some means of regenerating the reagent, preferably in situ. Many of the bioaffinity FOCS that have been reported might be better referred to as probes, as they only marginally, if at all, satisfy any of the above criteria. This is partly due to the large equilibrium constanti associated with most affinity reactions, which render the interactions between the affinity reagent and analyte highly selective but, unfortunately, not very reversible (i.e., they respond very slowly to changes in analyte concentration). An example of a bioaffinity FOCS is the direct sensor developed for the measurement of benzo[a]pyrene tetraol (BPT), a fluorescent hydrolysis by-product of the interaction of the carcinogenic polynuclear aromatic compound benzo[a]pyrene with DNA [6]. This FOCS employed a reagent phase consisting of a small sensmg chamber that contained either a solution of anti-BPT or a slurry of anti-BPT immobilized on silica beads. Operation was based on diffusion of BPT into the sensing chamber where it was bound by the “entrapped” antibody. This process concentrated the BPT within the sensing region and enhanced signals, permitting the detection of subnanomolar concentrations of BPT (attomole amounts in the low-p1 samples that could be analyzed with this diminutive sensor). Because of the moderately large amount of antibody in the chamber, the second criterion stated above was marginally satisfied and “true sensing” was possible over a short time interval (about 2 h). Nevertheless, this sensor was basically a “one-measurement” device. Significant analytical selectivity was also exhibited, although this reqmred removing

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ET AL

the sensor from the sample and dialyzing to remove interferents, again diminishing the true sensing character of the device. It has recently been demonstrated how a single-fiber FOCS could be combined with a capillary delivery system to permit a variety of affimty assay procedures (e.g., adding reagents, rinsing to remove impurities and recycling) to be done at the sensing terminus of the FOCS [ll]. In principle, this sensor can be used to perform m situ fluoroimmunoassays on a repetitive basis, thereby satisfying the third criterion above. The results of a preliminary evaluation of this “microscale regenerable biosensor” (MRB) for the direct assay of BPT is the subject of this paper. Two types of MRBs were employed in the assays. The first used an analyte-permeable membrane to entrap a liquid antibody solution in the sensing chamber and relied on diffusion through the membrane to deliver the analyte to the sensing region. The sensing chamber of the second MRB consisted of a “hollowed” stainless-steel frit which served to entrap antibodies bound to silica beads, “immunobeads”. Aspiration was employed to deliver analyte rapidly to the sensing chamber. The usual analytical figures of merit, sensitivity, precision, linear dynamic range and selectivity are reported for both types of MRBs.

EXPERIMENTAL

Reagents Phosphate-buffered saline (PBS), pH 7.4, was obtained from Sigma Chemical (Saint Louis, MO). Monoclonal anti-BPT was provided by Dr. Regina Santella, Columbia University (New York). The results of an immunochemical characterization of this antibody have been reported [14]. BPT and chrysene were obtained from Midwest Research Institute (Kansas City, MO) and Aldrich Chemicals (Milwaukee, WI), respectively. The BPT, chrysene and anti-BPT solutions used were prepared by diluting to volume with PBS solution. Fluorescence emission spectra were obtained using a Perkm-Elmer Model MPF-43A spectrofluorimeter.

FLUORESCENCE

MONITORING

239

OF BPT

Optical configuratton A block diagram of the optical and aspirationbased sensor configurations employed is shown in Fig. 1. Radiation from an Omnichrome (Chino, CA), Model 356 XM He-Cd laser (15 mW, 325 nm) is passed through a beam sphtter constructed by bormg a 2-mm hole through the center of a 25-mm diameter mirror, and focused onto the incident end of a 200- or 400~pm core diameter plastic-clad fused-silica or silica-silica optical fiber (General Fiber Optics, Cedar Grove, NJ). A laser line filter is used to isolate the 325-nm output line of the laser. The focusing lens is chosen to have an f number that is approximately equal to that of the fiber. Laser radiation exiting the fiber at its termmus can excite fluorophors m the sensing chamber. A fraction of the resulting fluorescence emission is collected by the fiber and transmitted back to the detection instrumentation. Thts radiation exits the incident end of the fiber as a cone that 1s deterrnmed by the numerical aperture of the fiber, is collimated by the lens, reflected by the beam splitter and directed to a detector consisting of a secondary filter (band-pass filter centered at 395 nm), a Hamamatsu Model R760 photomultiplier tube and a Keathley Model 485 autoranging picoammeter. Photothermal complications are minimized by employmg a Uniblitz Model SD-10 shutter (Vincent Associates, Rochester, NY) to provide a l-s laser exposure every 40-90 s, depending on the experiment. Sensor construction The sensing tips employed are shown m Fig. 2. They are constructed by surrounding a 200- or 400~pm core diameter fiber (protective coating removed) with six fused-silica capillary columns (200 pm i.d., 300 pm o.d. and about 50 cm m length) supplied by Scientific Glass Engineering (Austm, TX). The fiber/capillary assembly is coated with epoxy; a piece of heat-shrink tubing is placed around the assembly as the epoxy cures. The heat shrmk tubing is removed after epoxy polymerization, the tip is polished and finally the assembly is slipped into a cylindrical chamber. The diffusion-based MRB chamber (see Fig. 2a) is formed using heat-shrink tubing and is capped with a 10000 M.W. cut-off cellulose membrane

(ref. 10.17, Diachema, Zurich, Switzerland). In this work the chamber diameter and depth were ca. 1.1 and 1.0 mm, respectively, resulting in a chamber volume of about one ~1. Three pairs of capillary columns, oriented on opposite sides of the assembly, function to deliver anti-BPT or rmse solutions, or are used as outlets. Each capillary pair is secured m a Luer adapter that includes an on-off valve to facilitate connection to a syringe-syringe pump for reagent delivery. The entire MRB is purged with liquid in all applications so as to be as bubble-free as possible. The aspiration-based MRB (see Fig. 2b) is constructed by affixing a hollowed stainless-steel frit (Newmet Krebsoge, Terryville, CT) onto the end of the capillary assembly. The 0.5~pm porous frit is 4.8 mm long, 2.5 mm o.d., and was formed wtth

Fig 1 Block diagram of optlcal and aspuatlon-based confIguratIons employed wth the MRB

sensor

J P ALARIE

a

b

Fig. 2. (a) Diffusion-based MRB employmg heat-k&able tubing and membrane to define the sensmg chamber and (b) asplrauon-based MRB employmg a hollowed fnt as the sensmg chamber

a 4.0 mm deep, 1.5 mm diameter hole (see Fig. 2b). Two capillary columns are attached to a three-way Luer lock stopcock and used to aspirate sample solution (sampling volume ca. 100 ~1) and, subsequent to introducing immunobeads, push the sample back through the sensing chamber to permit binding to the entrapped immunobeads. The latter step is performed with the outlets closed so that the immunobeads remain within the sensing chamber and, hopefully (see later discussion), in the field of view of the optical fiber. Two capillaries are used as outlets, one for immunobead injection via an LC six-port-injection valve and the other for rinsing (the inset in Fig. 1 shows the arrangement of capillary columns for this MRB). Sensor operation Normal operation for the diffusion-based MRB direct assay of BPT involves the following steps: the capillary columns are filled with the appropriate reagent solutions, the sensor is placed in the sample, anti-BPT solution is delivered to the sensing chamber and a baseline signal level is measured, analyte is allowed to diffuse into the sensing chamber, signal (i.e., the increase in signal relative to the established baseline signal level) is measured and the sensor is recycled by flushing out the used reagents and delivering fresh antibody solution to the sensing chamber. Ideally, the

ET AL

effects of non-immunospecific interferents can be minimized by rinsing the antibody-BFT complex, with the outlets sealed, to expel interferents through the membrane. Thts procedure proved unsuccessful with this design and led to the utilization of the aspiration-based immunobead MRB. The aspiration-based MRB is operated by filling the capillaries with the appropriate solutions, placing the sensor m the sample, aspirating sample through the frit and into the aspiration capillaries, injecting and delivering immunobeads mto the sensing region, passmg the aspirated sample through the immunobeads with the outlets sealed, washing the chamber and immunobeads to remove interferents, measuring the resulting signal and, finally, recycling. These steps were performed in about 20 min. The three-way stopcock permits the delivery of reproducible volumes (about 100 ~1 in this instance) of aspirated sample [15]. The reproducibility of the delivery of immunobeads is improved by the use of the fixed-loop LC injection valve. In this work a 20-~1 volume of 22 mg ml-’ immunobead slurry was introduced into the chamber. Anti-BPT was covalently attached to silica beads using carbonyldiimidazole as previously described [16]. The bead coverage was 11 mg anti-BF’T gg’ beads. A detailed evaluation of the MRB is described elsewhere [15].

RESULTS

AND

DISCUSSION

The sensors (MRBs) described are designed to perform remote, in situ fluoroimmunoassays in a repetitive fashion. Although many assay protocols are possible, this work focused on the measurement of BPT, a natural fluorophor, which can be measured using a direct assay protocol (i.e., no fluorescent label is required). The usual analytical figures of merit were evaluated. The unique virtue of the MRB is that it is capable of repetitive, relatively rapid and selective (with appropriate rinsing) equilibrium measurements without being removed from the sample. Dlffwon-based MRB The response characteristics of this sensor were evaluated by establishing a dose-response curve

FLUORESCENCE

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OF BF”l-

over the range 5.3 x 10e9-5.3 x lop7 M BPT using the procedure described under Experimental. The response (signal level over background after a 20-min incubation with the investigated sample) is plotted in Fig. 3A. The last data point on the curve exhibits considerable saturation (i.e., the 1.9 x 10e6 M solution of anti-BPT in the sensing chamber is significantly depleted via the formation of an immune complex for the highest concentration sample). Ignoring the last data point, the correlation coefficient of the curve is 0.9996. The dynamic range can be extended, at the expense of sensitivity, by simply measuring signals after shorter incubation times. The relative standard deviation (r.s.d.) of the peak signals resulting from l-s exposures to laser excitation (baseline noise) was ca. 1 nA. Extrapolation of the dose-response curve to a signal level of 2 nA yields a limit of detection for these measurement conditions of 3.8 X lop9 M BPT or 1.2 X lo-” g in a loo-p1 sample. Laser fluorimetry is inherently sensitive; however, the detectability observed for this MRB

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i 5 ii5

200 -

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100 -

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.,.,.I.,.,’

10

0 BPT

CONCENTRATION,

20 MOLAR

is substantially enhanced by the “concentrating effect” of the anti-BPT [6]. For example, the signal level after a 20-min mcubation with a 1.1 X lop7 M BPT solution was 17.6 and 84.0 n4 without and with anti-BPT m the sensing chamber, respectively. Measurement precision was evaluated by performing eight replicate measurements on a 2.7 x lo-* M BPT solution. The mean signal of 21.7 nA exhibited an r.s.d. for these measurements of 10%. A membrane “memory effect” probably degrades the precision. The BPT is very soluble m the thin (7-pm) membrane and requires thorough rinsing to remove it completely. The measured background depended on the membrane history. Nevertheless, by rinsing between measurements with anti-BPT solution (a costly procedure) and plotting increases m signal over background, reliable dose-response curves could be generated, even if standard solutions of decreasing BPT concentration were sequentially measured. Although the above experiments demonstrate the true sensing capability of this MRB, efforts to exploit the specificity of antibody-antigen binding to enhance the analytical selectivity were unsuccessful. Large numerical aperture optical fibers have viewing depths of about two fiber diameters [l]. In order to restrict signals to the sensing chamber, the distance between the fiber terminus and the transparent membrane used in this MRB was made relatively large. The process of rinsing the immune complex with the outlet capillaries sealed after mcubation (to eliminate mterferents) forces the complex to the membrane. Thus, signal levels were nearly eliminated and required long periods to recover following the rinsing step. Moreover, when the system was not completely purged of air bubbles, the signal often did not completely recover. These problems were largely eliminated with the implementation of the aspiration-based MRB that employs solid-phase antibody and a non-transparent frit to isolate the antibody.

30 10-B

Fig. 3. Dose-response (cahbratlon) graphs for BIT obtamed wth (A) the dlffuslon-based and (B) the asptratlon-based MRBs

Aspwatlon-based MRB Most heterogeneous fluoroimmunoassays are done with solid-phase reagents. Work in progress includes the development of MRBs that utilize

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immunochemicals chermcally bonded to small diameter (< 10 pm) silica or latex beads to perform competitive-binding and sandwich assays. As a result of the lirmtations of the diffusion-based MRB that were discussed above, the analytical performance of the aspiration-immunobead MRB, described under Experimental, was evaluated. Previous work demonstrated that relatively large amounts of anti-BPT could be bonded to the silica beads without substantial denaturing [16]. Other experiments demonstrated that beads could be reproducibly delivered and removed from the sensing chamber [ll], albeit not as easily as liquid phase anti-BPT. Moreover, the beads could be kept within in the field of view of the fiber optic while rmsing. In order to retain the beads without rupturing, the chamber was constructed from a specially fabricated frit. Among the advantages of the frit are that it is rigid and provides a stable

380

400

420

380

EMISSION

Rg

4

sensing chamber even after numerous rinsing and filling steps, it is not transparent and elimmated the “see-through” problem mentioned above and it did not exhibit the memory effects experienced with the cellulose membrane. As a consequence of the slow rate of diffusion of BPT through the frit, sampling was performed via mild aspiration through two of the capillary columns (as shown in Fig. 1). Although this added a step to the assay protocol, it actually decreased the analysis time relative to the diffusion-based measurement and was demonstrated to have an r.s.d. of less than 8’%, even in a biological matrix [15]. The response characteristics of the aspirationbased sensor were evaluated by establishing a dose-response curve over the range 2.1 X 10e82.7 x lo-’ M BPT (see Fig. 3b). In this case the correlation coefficient was 0.9989. The r.s.d. for replicate measurements of a 2.7 X lo-’ M solu-

b

a

Fluorescence emsslon spectra for (a) 6 9

x

ET AL

C

400

WAVELENGTH

10e6 M chrysene, (b) 2 7 X lo-’

420

380

400

(nm)

M BF’T and (c) a mture

of the two

420

FLUORESCENCE

MONITORING

OF BPT

tion was 5%. The dynamic range of this MRB was limited, relative to the diffusion-based MRB, at the low concentration end by poorer detectability. It is not believed that this is an inherent limitation of the aspiration-based MRB but rather a consequence of the fact that operational and design parameters were not extensively investigated and optimized in this initial investigation. Among the important parameters are the shape, symmetry and depth of the sensing chamber, the amount of immunobeads delivered to the chamber and the flow-rates for delivery of sample and rinse solutions to the chamber-entrapped beads. In addition, the hollowed frits used in this study had greater end thickness than wall thickness, resulting in greater permeability through the walls when solutions were delivered to the chamber during operation. This tended to force the beads toward the walls of the chamber and out of the field of view of the fiber optic. Future work will focus on optimizing MRB operational and design parameters. The selective removal from the sensing chamber, via rinsing, of a non-immunospecific interferent is demonstrated usmg the highly fluorescent polynuclear aromatic compound chrysene as the interferent. Figure 4 shows the emission spectra (A,, = 325 nm) for 6.9 X lop6 M chrysene, 2.7 X lop7 M BPT and a rmxture of the two. It can be seen that the compounds interfere and their spectral signals are roughly additive. The bar graph in Fig. 5 depicts the relative signals for these three solutions during aspiration and rmsing (rinsing flow-rate ca. 20 ~1 nun-‘). The mixture signal is roughly equal to the sum of the individual BPT and chrysene signals during aspiration and in the early stages of rinsing. However, after a 20-min rinse the interferent is essentially completely removed whereas a substantial signal from the immunobead-bound BPT remams. At that point the mixture and BPT signals are approximately equal. Opening the outlet capillaries and rinsmg allows the complete remova? of the immunobeads and the re-establishment of a baseline signal. Although the entire measurement process required about 20 min m this experiment, by optimizing the parameters mentioned above and increasing the flow-rates it should be possible to perform measurements

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Fig. 5 Bar graph deplctlon of the signals obtamed solutions gwen m Fig. 4 dunng an MRB expenment

for the

more rapidly and with larger post-rinsing signals. It should be stated that the total BPT binding capacity of the beads introduced mto the sensmg chamber is greater than the amount of BPT sampled in this experiment. Hence, with appropriate modifications of the chamber geometry (hollowed frits used m the future will employ a tapered interior and have a greater permeability at the tip) it is possible that the post-rmsmg signal could be significantly larger than the signal observed during the aspiration step shown in Fig. 5. In conclusion, the ability to perform repetitive direct fluoroimmunoassays remotely using microscale regenerable biosensors has been demonstrated. A diffusion-based sensor that employed a membrane-capped chamber to isolate antibody provided excellent sensitivity but was not durable and did not perrmt exploitation of the selectivity of immunochermcal reactions. An aspiration-based sensor that employed unmunobeads isolated withm a special hollowed frit proved to be durable and capable of selective measurements. Although the sensing chamber design clearly requires optimization to enhance sensitivity and speed of response, it is considered that the aspiration-based sensor shows substantial promise for the performance of a variety of affinity chemical measurements. This research was sponsored by the National Science Foundation under contract CHE-8708581

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and the Division of Chemical Sciences, Office of Basic Energy Research, U.S. Department of Energy under contract DE-FGO586ER13613 with the University of Tennessee, Knoxville, and the Office of Health and Environmental Research, U.S. Department of Energy, under contract DEAC05840R21400 with Martin Marietta Energy Systems.

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