Multiple-Analyte Fluoroimmunoassay Using an Integrated Optical

IOW/IRE, 9, etched wells, r-, m-, gIgGs, fluorescence, 100s of ng/mL. 14b, na, proposed, interferometry, na. 5b, IC technology, proposed, fluorescence...
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Anal. Chem. 1999, 71, 4344-4352

Multiple-Analyte Fluoroimmunoassay Using an Integrated Optical Waveguide Sensor T. E. Plowman, J. D. Durstchi,† H. K. Wang,† D. A. Christensen,† J. N. Herron,† and W. M. Reichert*

Center for Emerging Cardiovascular Technologies, Department of Biomedical Engineering, Duke University, Durham, North Carolina 27710

A silicon oxynitride integrated optical waveguide was used to evanescently excite fluorescence from a multianalyte sensor surface in a rapid, sandwich immunoassay format. Multiple analyte immunoassay (MAIA) results for two sets of three different analytes, one employing polyclonal and the other monoclonal capture antibodies, were compared with results for identical analytes performed in a singleanalyte immunoassay (SAIA) format. The MAIA protocol was applied in both phosphate-buffered saline and simulated serum solutions. Point-to-point correlation values between the MAIA and SAIA results varied widely for the polyclonal antibodies (R2 ) 0.42-0.98) and were acceptable for the monoclonal antibodies (R2 ) 0.93-0.99). Differences in calculated receptor affinities were also evident with polyclonal antibodies, but not so with monoclonal antibodies. Polyclonal antibody capture layers tended to demonstrate departure from ideal receptorligand binding while monoclonal antibodies generally displayed monovalent binding. A third set of three antibodies, specific for three cardiac proteins routinely used to categorize myocardial infarction, were also evaluated with the two assay protocols. MAIA responses, over clinically significant ranges for creatin kinase MB, cardiac troponin I, and myoglobin agreed well with responses generated with SAIA protocols (R2 ) 0.97-0.99). Contemporary trends in biosensing focus on the detection of multiple analytes through immunoassay. In a typical solid-phase multiple-analyte immunoassay (MAIA), an array of capture antibodies is first immobilized on a solid support by physical adsorption, covalent linkages, indirect anchors, or laser printing and stamping methods.1 A mixture of analytes, derived from a single sample, is then reacted with the multicomponent capture layer and detected with labeled, tracer antibodies. Detection is routinely achieved using radiometric, enzymometric, gravimetric, amperometric, or fluorometric labels.2 Optical methods for detecting biochemical events at surfaces are well characterized and generally possess equivalent, if not better, sensitivities than competing measurement techniques.2 * Corresponding author: [email protected]; (phone) (919) 660-5151; (fax) (919) 684-8886. † Current address: Center for Biopolymers at Interfaces, Department of Pharmaceutics and Bioengineering, University of Utah, Salt Lake City, UT 88504. (1) Go ¨pel, W.; Heiduschka, P. Biosens. Bioelectron. 1995, 10, 853-883. (2) Brecht, A.; Gauglitz, G. Biosens. Bioelectron. 1995, 10, 923-936.

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The literature relevant to MAIA employing optical detection is categorized by transducer type in Table 1. Kakabakos et al. and Ekins et al. were first to propose methods for MAIA based on time-resolved fluorescence and confocal microscopy, respectively.3,4 More recent techniques make use of multiple internal reflection elements such as planar waveguides, 5-12 planar waveguide interferometers,13-15 and bundled fiber optics,16,17 which tend to achieve higher sensitivities due to their large optical path lengths. Also employed in optical MAIA are the reflectometric method of surface plasmon resonance (SPR)18 and capillary flow systems with fluorescent detection.19 Fluorescent chemometric methods have also been proposed that, as an advantage, do not require spatial separation to detect multiple analytes.20 The optical transducers used most often in MAIA are the integrated optical waveguide (IOW) or the internal reflection element (IRE). Although many have proposed different MAIA schemes, no one to our knowledge has presented complete binding curves (i.e., response of the sensor from background to saturating levels) for IOW MAIA when all analytes are assayed (3) Kakabakos, S.; Christpoulus, T.; Diamandis, E. Clin. Chem. 1992, 38, 338342. (4) Ekins, R.; Chu, F.; Biggart, E. Clin. Chim. Acta 1990, 194, 91-114. (5) Klainer, S.; Coulter, S.; Pollina, R.; Saini, D. Sens. Actuators B 1997, 3839, 176-182. (6) Ligler, F.; Conrad, D.; Golden, J.; Feldstein, M.; MacCraith, B.; Bladerson, S.; Czarnaski, J.; Rowe, C. Proc. SPIE 1998, 3258, 50-55 (Proceedings of Micro- and Nanofabricated Structures and Devices for Biomedical Environmental Applications). (7) Wadkins, R.; Golden, J.; Ligler, F. J. Biomed. Opt. 1997, 2, 74-79. (8) Wadkins, R.; Golden, J.; Pritsiolas, L.; Ligler, F. Biosens. Bioelectron. 1998, 13, 407-415. (9) Zhou, Y.; Magill, J. V.; De La Rue, R. M.; Laybourn, P. J. R.; Cushley, W. Sens. Actuators, B 1993, B11, 245-250. (10) Brecht, A.; Klotz, A.; Barzen, C.; Gauglitz, G.; Harris, R.; Quigley, G.; Wilkinson, J.; Sztajnbok, P.; Abuknesha, R.; Gascon, J.; Oubina, A.; Barcelo, D. Anal. Chim. Acta 1998, 362, 69-79. (11) Misiakos, K.; Kakabakos, S. Biosens. Bioelectron. 1998, 13, 825-830. (12) Silzel, J.; Cercek, B.; Dodson, C.; Tsay, T.; Obremski, R. Clin. Chem. 1998, 44, 2036-2043. (13) Schneider, B.; Edwards, J.; Hartman, N. Clin. Chem. 1997, 43, 1757-1763. (14) Lukosz, W.; Stamm, C.; Moser, H.; Ryf, R.; Dubendorfer, J. Sens. Actuators, B 1997, B39, 316. (15) Klotz, A.; Brecht, A.; Gauglitz, G. Sens. Actuators, B 1997, B39, 310-315. (16) Healey, B.; Li, L.; Walt, D. Biosens. Bioelectron. 1997, 12, 521-529. (17) Michael, K.; Taylor, L.; Schultz, S.; Walt, D. Anal. Chem. 1998, 70, 12421248. (18) Berger, C.; Beumer, T.; Kooyman, R.; Greve, J. Anal. Chem. 1998, 70, 703706. (19) Narang; Gauger, P.; Kusterbeck, A.; Ligler, F. Anal. Biochem. 1998, 255, 13-19. (20) Piehler, J.; Brecht, A.; Giersch, T.; Kramer, K.; Hock, B.; Gauglitz, G. Sens. Actuators, B 1997, 38-39, 432-437. 10.1021/ac990183b CCC: $18.00

© 1999 American Chemical Society Published on Web 08/27/1999

Table 1. Groups That Have Demonstrated or Are Developing MAIA Technologya transducer IOW/IRE

fiber SPR microscope slide/other

ref

MAIA configuration

reagents assayed

detection method

MDC

9 14b 5b 15 13b 7, 8 6 10 11 12 17 18b 4 3 20

etched wells na IC technology na spatial arrangement spatial arrangement spatial arrangement spatial arrangement spatial arrangement DeskJet printing encoded microspheres spatial arrangement spatial arrangement spatial arrangement chemometrics multiple capillary tubes

fluorescence interferometry fluorescence interferometry interferometry fluorescence fluorescence fluorescence fluorescence fluorescence fluorescence angle of reflection confocal microscopy time-resolved fluorescence relflectometric interference spectroscopy fluorescence

100s of ng/mL na na na pM ng/mL ng/mL NA NA ng/mL na na pg/mm2 100s of ng/mL pg/mm2

19

r-, m-, gIgGs proposed proposed proposed proposed three toxic agents ovalbumin, SEB proposed Av, biotin-bSA IgG sublasses AP, Av, biotin hCG only TNF, TSH LH, FSH, hCG, PRL three s-triazine derivatives TNT, RDX

ng/mL

a Abbreviations: AP, alkaline phosphatase; Av, avidin; bSA, bovine serum albumin; FSH, follicle stimulating hormone; g, goat; hCG, human chorionic gonadotropin; IC, integrated circuit; IOW, integrated optical waveguide; IRE, internal reflection element; LH, leutinizing hormone; MDC, minimum detectable concentration; m, mouse; na, not available; PRL, prolactin; r, rabbit; SEB, staphyloccocal enterotoxin B; SPR, surface plasmon resonance. b Known industrial affiliation.

simultaneously. This paper examines how accurately MAIA data correlate to those data obtained when each element of the MAIA is assayed separately in a single-analyte immunoassay (SAIA). Such comparisons can provide information on the assay format’s tendencies to misidentify proper analyte levels due to crossreactivity, receptor heterogeneity, and nonspecific binding, as false positives are a typical problem in MAIA sensing.21 Our approach to performing IOW MAIA, Figure 1, is an improvement on the MAIA method of Kakabakos et al.3 because it is performed in one simple step. The surface is an array of three millimeter-sized protein “channels” adsorbed onto a previously described, grating coupled silicon oxynitride (SiON) IOW transducer operating in an evanescently excited fluorescence detection format.22 The channels contained either model monoclonal or polyclonal antibodies or cardiac-specific antibodies to cardiac troponin I (cTnI), myoglobin (Mb), and creatine kinase of the muscle-brain form (CK-MB). A series of assays, in both SAIA and MAIA formats, was conducted to assess binding specificity and interchannel cross reactivity in terms of the correlation between assay data and therefore the tendency to generate false positives. Responses between buffer and serum matrix solutions were subsequently compared by correlation. Biophysical parameters, obtained via Langmuir curve fitting, provided further comparison between formats for both the model monoclonal and polyclonal antibody systems. Assay responses for the model monoclonal antibodies correlated well between MAIA and SAIA formats. The goodness of fit (R2) values for a straight-line correlation between formats were in the range 0.93-0.99. None of the predicted binding constants were statistically different between assay formats and none of the reactions displayed a departure from ideal binding. Two of the three polyclonal capture antibodies, however, showed a significant difference between the predicted binding constants and a departure from ideal binding in the MAIA format. Comparison of the (21) Kricka, L. Clin. Chem. 1992, 38, 327-328. (22) Plowman, T. E.; Saavedra, S. S.; Reichert, W. M. Biomaterials 1998, 19, 341-355.

Figure 1. Schematic representation of a MAIA as performed on a grating coupled SiON IOW. An idealized view of the surface (not to scale) shows three capture antibodies adsorbed to the surface in channels separated by areas of blocking protein. Premixed analyte and tracer antibodies, specific for one channel each, are introduced by the sample inlet port and react with accessible binding sites. Fluorescent labels attached to the tracer antibodies absorb energy from the evanescent wave and re-emit it as fluorescence.

polyclonal antibody results between formats by a point-to-point correlation did not suggest a linear relationship for these same two capture areas. When assays were performed in 10% simulated serum versus assays performed in a phosphate-buffered saline (PBS) solution, only two of the six capture antibodies studied were sensitive to the presence of the 10% serum component. Finally, MAIA conducted for the cardiac proteins correlated well with SAIA Analytical Chemistry, Vol. 71, No. 19, October 1, 1999

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Table 2. Analyte Cocktails Used in the Various Assay Formatsa assay proteins analyte

capture

analyte concentrations tracer

channel tests

single- and multiple-analyte tests

buffer PBS

10% pSA

polyclonal CA SAIA MAIA CA SAIA MAIA CA SAIA MAIA

rIgG-FITC

p RrIgG

m RFITC-Cy5

10 nM

300 pM-100 nM

gIgG-FITC

p RgIgG

m RFITC-Cy5

1 nM

30 pM-30 nM

hIgG-FITC

p RhIgG

m RFITC-Cy5

10 nM

300 pM-30 nM

Av-FITC

m RAv

m RFITC-Cy5

IgG-FITC from goat

m RgIgG

m RFITC-Cy5

1 nM

30 pM-30 nM

bSA-FITC

m RbSA

m RFITC-Cy5

10 nM

30 pM-30 nM

m RhCG-R

m RhCG-β-Cy5

50 nM

cTnI

m RcTnI

m RcTnI-Cy5

3-900 ng/mL

MAIA

CK-MB

m RCK-MB

m RCK-MB-Cy5

1-100 ng/mL

MAIA

Mb

m RMb

m RMb-Cy5

5-500 ng/mL

MAIA

monoclonal

hCG cardiac

30 pM-100 nM

SAIA MAIA CA SAIA MAIA CA SAIA MAIA CA

MAIA MAIA MAIA

SAIA MAIA SAIA MAIA SAIA MAIA

a Abbreviations: R, anti; Av, avidin; bSA, bovine serum albumin; CK-MB, creatine kinase of the muscle brain form; CA, channel addressability experiment; FITC, fluorescein isothiocyanate (isomer 5); g, goat; h, human; IgG, immunoglobin G; Mb, myoglobin; PBS, phosphate-buffered saline; pSA, porcine serum albumin; r, rabbit; cTnI, cardiac troponin I; p, polyclonal; m, monoclonal; hCG, human chorionic gonadotropin.

measurements (R2 ) 0.97-0.99), but only one of the three calculated detection limits in the MAIA format suggested the ability to reliably detect trace amounts of analyte. MATERIALS AND METHODS Reagents. Polyclonal Immunoassay System. Capture antibodies were anti (R) goat-, human-, and rabbit-IgG (RgIgG, RhIgG, and RrIgG) from Pierce (piercenet.com). Antibodies were of variable subclass and their affinities were not provided. Analytes consisted of gIgG, hIgG, and rIgG-fluorescein isothiocyanate (FITC) in 1% bovine serum albumin (bSA) from Sigma (sial.com/sigma). The tracer antibody was the monoclonal RFITC, isomer 5 from Genzyme Diagnostics, Inc. (genzyme.com). Monoclonal Immunoassay System. Capture antibodies were RbSA, RgIgG, and Ravidin (RAv) (Sigma Product B9655, antibody to avidin, had attached biotin groups.) from Sigma or RhCGR from Calbiochem (calbiochem.com). All antibodies were of the IgG1 subclass, and their affinities were not provided. Analytes were any three of the following: Av-FITC, bSA-FITC, gIgG-FITC (Sigma), or hCG (Calbiochem). The tracer was, again, the RFITC from Genzyme Diagnostics, Inc., or RhCGβ for the hCG analyte (Calbiochem). Cardiac Immunoassay System (Lumenal Technologies, Salt Lake City, UT). Both capture and tracer antibodies were one of two monoclonal clones to the analytes: CK-MB, Mb, and cTnI (typically IgG1, affinities not provided). Miscellaneous. Cyanine 5 dye (Cy5) was obtained from Research Organics (resorg.com) while porcine serum albumin (pSA) and trehalose were from Sigma. 4346 Analytical Chemistry, Vol. 71, No. 19, October 1, 1999

Protein Preparations. Antibodies. Polyclonal antibodies from Pierce, the monoclonal tracer antibody from Genzyme, and the cardiac analytes from Lumenal Technologies were used as received without further purification, having already been subjected to fractionation and ion-exchange chromatographic purification procedures. Antibodies from Sigma were shipped in ascites fluid or 1% bSA. In such cases, IgGs were purified with protein G columns (Pharmacia Biotech, biotech.pharmacia.se). The final concentration of capture antibody was generally in the micromolar range, as measured by absorbance (A280). Aliquots (100 µL) of all purified proteins were frozen and stored until needed. Labeling Tracer Antibody. The RFITC tracer antibody was labeled with a Cy5 dye23 following the manufacturer’s procedure (Amersham Life Science, www.apbiotech.com). Generally four dyes were attached to each antibody tracer. Aliquots of 100 µL (µM) were stored frozen. Analyte Cocktails. The assays presented here required several analyte components and multiple-analyte cocktail mixtures, as outlined in Table 2. Channel addressability and generic SAIA tests involved analyzing the binding of one analyte only. Therefore, nanomolar starting solutions containing the analyte of interest were prepared in either PBS or 10% pSA. Generic SAIA assays required diluting the starting solution through the ranges listed in Table 2. All solutions contained nanomolar amounts of RFITCCy5 tracer for detection. MAIA test solutions, in both the polyclonal and monoclonal cases, contained a mixture of all three (23) Mujumdar, R. B.; Ernst, L. A.; Mujumdar, S. R.; Lewis, C. J.; Waggoner, A. S. Bioconjugate Chem. 1993, 4, 105-111.

analytes in nanomolar tracer backgrounds as indicated in Table 2. To simulate the presence of a serum background, 10% pSA was added to some of the monoclonal MAIA test solutions, to some of the cardiac MAIA, and to all of the cardiac SAIA test solutions (see Table 2). Surface Preparation. IOW Silanization. Grating etched SiON surfaces were immersed in a 5% (v/v) solution of hydrogen peroxide in sulfuric acid for 10 min, rinsed in ultrapure water and ethanol, and allowed to dry under room-temperature vacuum. All SiON surfaces were then modified using dimethyldichlorosilane (DDS, Sigma) in toluene to render the surface hydrophobic. After 30 min, the IOWs were removed and cleaned in ethanol, ultrapure water, and ethanol once again. SiON surfaces were then vacuumdried at room temperature for at least 30 min. A 1% DDS/toluene solution was found to produce a water contact angle of about 85°. Other DDS/toluene solutions (10, 0.5, and 0%) showed decreasing contact angles with decreasing DDS concentration (105, 80, and 0°). Test immunoassays at each DDS level indicated that a 1% solution produced the best capture layer (data not shown). Capture Layer Depositions. To pattern antibody capture layers, a three-hole rubber gasket was clamped to an IOW to produce three adjacent rectangular wells (0.5 × 2 cm each) in which capture protein was adsorbed; the clamped wells also prevented cross-contamination of the different antibody solutions. Each capture antibody solution (0.5 mL of 0.1 µM solution), pipetted into its designated well, was allowed to adsorb to the surface for 2 h. At that point, the rubber gasket was removed and the surface rinsed with PBS to yield three distinct regions of hydrated antibody separated by two sections of antibody-free SiON (0.33 × 2 cm). Physical adsorption was chosen over other deposition techniques, e.g., covalent attachment, primarily for its simplicity. While covalent attachment is desirable for sensor stability and regeneration aspects, it was not necessary for our one-shot transducers. Postcoating Technique. After adsorbing capture antibody, the entire sensor surface (except the grating region) was coated with a passivating and preserving solution of pSA and trehalose sugar (5 and 1%, respectively, in water) for 30 min. The pSA was used to block nonspecific sites while the sugar was used to stabilize and preserve the adsorbed antibodies. The surface was removed from solution, rinsed in PBS, and vacuum-dried. Prepared surfaces were stored under refrigeration (4 °C). All treated IOWs were used within a day of preparation. Assay Method. Rehydrated sensor surfaces, capture layer side up, were sealed in a custom sample cell (Lumenal Technologies, Salt Lake City, UT) and placed in the path of a 10-mW, 635-nm diode laser. The m ) 0 guided mode of the IOW was coupled via grating diffraction, establishing a continuous streak of light at the waveguide interface (although not explicitly shown in Figure 1).24 The evanescent field of the guided mode excites fluorescence very near the waveguide/solution interface. After the sample cell was situated and the guided mode coupled, the control fluorescence signal from a plasma/PBS background solution (injected through a 1-cm3 syringe fitted with a plastic pipet tip), was measured over a 5-s exposure, every 30 s, for 5 min. Fluorescence measurements were made using a spatially binned CCD camera (ST-6, Santa (24) Lee, D. L. Electromagnetic Principles of Integrated Optics; John Wiley and Sons: New York, 1986.

Barbara Instrument Group) fitted with a band-pass filter to transmit only the fluorescence from the Cy5 dye. Time course fluorescence readings were stored via LabView (National Instruments, natinst.com) in a KestrelSpec data file (KestrelSpec, rheacorp.com). This process was repeated until all samples in the experiment had been assayed. Acquired data contained arrays of fluorescence intensity as a function of both time and binned IOW position. Since the IOW sensor is ideally designed to be a one-shot device, regeneration of the surface was not considered, and each entire assay represents a fresh surface on a different waveguide. Calibration of the assay did not involve correction for drift (which, unlike mass sensors, is not a major problem for fluorescence transducers); however, a background was collected, as mentioned above, and subtracted from all future intensity profiles acquired in an assay. This background corrected for two effects: (1) fluorescence measured from bulk excitation and nonspecifically bound tracers and (2) attenuation of the guided mode as a function of propagation distance. Thus, there will not be a noticeable depreciation in signal intensity with propagation distance. Two nonspecific channels between the specific binding areas were also present and acted as on-board reference channels that gauged the level of tracer antibody nonspecific binding. Data Analysis. Fluorescence intensities were collected as a function of position over 5-s exposure times every 30 s during a 5-min reaction. Since the exposure time was intermittent, effects due to photobleaching were insignificant. Integrated fluorescence values for each binding channel were plotted on a curve as a function of assay time. A series of sample concentrations produced a step isotherm. Fraction bound, Fb, with respect to bulk analyte concentration, c, was calculated by normalizing the data to the maximum intensity obtained in each experiment. Individually measured intensities were compared between SAIA and MAIA methods by a direct point-to-point correlation. As in our previous work,25 the reactions were also treated as if in a quasi-equilibrium state and modeled analytically with a Langmuir equation that included a term for receptor nonideality, R (this symbol should not be confused with the same symbol that denotes an antibody)

Fb ) (Kc)R/(1 + (Kc)R)

(1)

where K is the binding constant for any particular antibodyantigen pair and R represents the standard deviation of an antibody affinity probability density function, centered about K, for any given population of receptors. This equation is an immunological analogue to one originally intended to model a catalyst surface.26 It predicts well the sigmoid response typical of antibody-antigen reactions and has been applied to immunoassays previously.27 This model is also especially useful when the response is not completely homogeneous due to diffusion limitations, surface activity, average affinity, and avidity with polyclonal antibodies. RESULTS Channel Addressability. Experiments were devised to examine the response of individually addressed sensing areas (i.e., (25) Plowman, T. E.; Reichert, W. M.; Wang, H. K.; Christensen, D. A.; Herron, J. N. Biosens. Bioelectron. 1996, 11, 149-160. (26) Sips, R. J. Chem. Phys. 1948, 16, 490-496. (27) Kaufman, E.; Jain, R. Cancer Res. 1992, 52, 4157-4167.

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Figure 2. (a) Plots of fluorescence intensity versus relative waveguide position for polyclonal capture antibody areas specific to (left to right) hIgG, rIgG, and gIgG. (b) Three plots of integrated intensity versus time for the separate polyclonal capture channels.

channels) arranged on the surface of an IOW. Two separate tests were run, one for the polyclonal capture antibodies and one for the monoclonal capture antibodies. In each test, three analyte solutions, each specific to one of the three channels, were sequentially exposed to the sensor surface. Analyte concentrations were selected to ensure that each channel bound sufficient analyte to elicit a signal. Results for the polyclonal and monoclonal antibody surfaces are displayed in Figures 2 and 3, respectively. Figure 2a consists of three graphs, one for each analyte injection, that display the channel-specific changes in the measured fluorescence. Figure 2b plots the changes in integrated intensity of Figure 2a for each channel as a function of assay time. Similar representations are made in Figure 3a and b for monoclonal antibodies. The final integrated intensities reached in each sample channel, and in one of the two nonspecific areas between channels, are summarized in Table 3. The jagged nature of the traces in Figure 3a is most likely a result of inhomogeneities in the surface chemistry and possible aggregation of tracer antibodies. It is not believed to be due to gaps in the evanescent field (the field is essentially continuous for an IOW) or attenuation of the guided mode (since the traces have already been background subtracted). A portion of the rightmost channel in both assays was obscured from the camera’s field of view; however, this did not affect the results. Polyclonal and Monoclonal Assays. Dose-response curves were constructed for the polyclonal and model monoclonal 4348 Analytical Chemistry, Vol. 71, No. 19, October 1, 1999

Table 3. Comparison of Integrated Intensities for Polyclonal and Monoclonal Antibody Capture Areasa integrated intensities, polyclonal capture antibodies injected analytes injection 1 2 3

rIgG-FITC (10 nM) gIgG-FITC (1 nM) hIgG-FITC (10 nM)

injected analytes injection 1 2 3

hCG (50 nM) gIgG-FITC (1 nM) bSA-b (10 nM)

RhIgG

RrIgG

RgIgG

nsb

71925 31654 372863

564950 159259 6624

9248 194038 9544

5196 6370 3271

integrated intensities, monoclonal capture antibodies RbSA

RhCG

RgIgG

nsb

40492 270763 1194710

815290 155830 294640

149637 1848772 362391

22740 47288 249178

a Abbreviations: R, anti; bSA-b, bovine serum albumin, biotin labeled; g, goat; h, human; IgG, immunoglobin G; nsb, nonspecific binding; r, rabbit.

antibody systems, in both SAIA and MAIA formats, by monitoring the integrated fluorescence of spatially arranged antibody channels with increasing analyte concentration. Figure 4 shows the polyclonal binding curves for the SAIA (top panel) and MAIA (bottom panel) measurements. Figure 5 shows the monoclonal binding curves for the SAIA (top panel) and MAIA (bottom panel)

Figure 3. (a) Plots of fluorescence intensity versus relative waveguide position for monoclonal capture antibody areas specific to (left to right) bSA, hCG, and gIgG. (b) Three plots of integrated intensity versus time for the separate monoclonal capture channels. Table 4. Comparison of SAIA and MAIA Binding Constants and Nonideality Indexes for the Polyclonal and Monoclonal Antibody Systemsa K (nM-1) analyte polyclonal gIgG-FITC rIgG-FITC hIgG-FITC monoclonal bSA-FITC gIgG-FITC Av-FITC

SAIA

MAIA

R SAIA

MAIA

1.40 ( 0.10 1.60 ( 0.10 1.10 ( 0.07 1.11 ( 0.10 0.12 ( 0.01 0.05 ( 0.00b 1.02 ( 0.08 0.51 ( 0.02c 0.11 ( 0.01 1.59 ( 0.21b 1.06 ( 0.07 0.72 ( 0.06c 0.76 ( 0.10 0.54 ( 0.07 0.28 ( 0.04 0.24 ( 0.03 0.13 ( 0.01 0.16 ( 0.02

0.87 ( 0.06 0.98 ( 0.10 1.00 ( 0.06 1.01 ( 0.04 0.79 ( 0.09 0.97 ( 0.05

a Abbreviations: R, nonideality index; K, binding constant. b Statistically different from corresponding SAIA value. c Statistically different from 1.

measurements. Three replicates of each entire titration were performed. The solid curves in Figures 4 and 5 are best fits of the data to eq 1. The binding and nonideality constants derived from these fits are listed in Table 4. According to the R values, polyclonal antibodies acted in an ideal manner in SAIAs; i.e., the antibodies bound antigen in a largely monovalent fashion. However, when the polyclonal antibodies were tested together in a MAIA, changes in the index were apparent; most noticeably, the values for the RrIgG and RhIgGs

dropped from an ideal value of 1 to 0.51 ( 0.02 and 0.72 ( 0.06, respectively. Similar differences were noticed in the binding constants, i.e., 0.12 ( 0.01 to 0.05 ( 0.00 nM-1 (RrIgG) and 0.11 ( 0.01 to 1.59 ( 0.21 nM-1 (RhIgG). The only antibody unaffected by the MAIA cocktail was the RgIgG antibody (Table 4). Monoclonal antibodies displayed similar values between assay formats for each analyte tested. Nonideality constants were not significantly different from unity (p < 0.05). The binding constants for all monoclonal antibodies showed only slight variations between formats and were indistinguishable at or above the 90% confidence level. Cardiac Assays. SAIA and MAIA were performed for three cardiac proteins: CK-MB, Mb, and cTnI.28-30 Table 2 gives the clinically relevant concentration ranges over which the measurements were conducted (units of ng/mL are used in agreement with clinical convention). The immobilized capture proteins used in all cases were monoclonal antibodies. All of the SAIAs employed simulated serum solutions containing 10% pSA, while only one of the MAIAs included the 10% pSA matrix. Parts a and b of Figure 6 show the average (n ) 3) dose-response binding relationships (28) Antman, E.; Tanasijevic, M.; Thompson, B.; Schactman, M.; McCabe, C.; Canno, C.; Fischer, G.; Fung, A.; Thompson, C.; Wybenga, D.; Braunwald, E. N. Engl. J. Med. 1996, 335, 1342-1349. (29) Ruzich, R. Postgrad. Med. 1992, 92, 85-89. (30) Adams, J.; Abendschein, D.; Jaffe, A. Circulation 1993, 88, 750-763.

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Figure 4. (a) Plots of fraction bound (Fb) versus bulk analyte concentration in SAIA format using polyclonal capture antibodies to gIgG, rIgG, and hIgG. (b) Fb versus bulk analyte concentration in MAIA format using polyclonal capture to gIgG, rIgG, and hIgG. Table 5. Analytical Sensitivities for the Cardiac Assays in Both Single-Analyte and Multianalyte Immunoassay Formats analyte

SAIA (ng/mL)

MAIA (ng/mL)

CK-MB cTnI Mb

2.8 15.9 7.0

4.5 26.9 33.8

for the cardiac-specific SAIA and MAIAs, respectively. The responses between assay formats follow similar trends, except for the SAIA measurement of cTnI, which was assayed over a much wider range and thus displayed inflection in the transition from lower to higher analyte concentrations. Analytical sensitivities were calculated (Table 5) according to the clinical chemistry definition, i.e., average background value plus two standard deviations divided by assay sensitivity. Analytical sensitivities were between 3 and 15 ng/mL for the SAIA format, while operating in the MAIA format caused the analytical sensitivity to rise significantly in all cases. Since these data do not represent complete isotherms but 4350 Analytical Chemistry, Vol. 71, No. 19, October 1, 1999

Figure 5. (a) Plots of fraction bound (Fb) versus bulk analyte concentration in SAIA format using monoclonal capture antibodies to bSA, gIgG, and avidin (Av). (b) Fb versus bulk analyte concentration in MAIA format using monoclonal capture antibodies to bSA, gIgG, and Av.

only clinically significant ranges, binding and nonideality constants were not calculated. DISCUSSION In general, the individual channels for MAIA were addressable when either polyclonal and monoclonal antibodies were used. However, the polyclonal rIgG capture antibody was cross reactive with the single-analyte gIgG solution and the monoclonal bSA antibody was cross reactive with the single-analyte gIgG solution. The results further suggested that MAIAs with monoclonal antibodies were more selective than polyclonal antibodies as judged by comparison with SAIA results. Monoclonal antibodies were also more well behaved, according to the nonideality constant. Two of the three polyclonal antibodies displayed significant nonideality, indicative of cross-reactivity, nonspecific binding, and/or, polyvalent binding. The results from the cardiac analyte MAIAs also agreed well with SAIA results, although the analytical sensitivity for two of the three analytes rose significantly.

Figure 7. Comparing the SBRs achieved with polyclonal antibodies and monoclonal antibodies. Each bar represents one analyte and the percentage of total intrachannel SBR it achieves during the experiment. The number above each set of bars indicates the order of analyte injection while the spatial arrangement of the capture areas is identical to their position on the IOW.

Figure 6. Dose-response curves for the cardiac analytes CK-MB, cTnI, and Mb in both (a) SAIA and (b) MAIA formats.

Using the information contained in Table 3, it is possible to express the channel addressability data (Figures 2 and 3) into a signal-to-background ratio (SBR) format by dividing the channelspecific integrated intensities in each row by the tabulated nonspecific integrated intensity. The SBR measures how much greater an analyte signal is compared to the nonspecific background level. Summing the corresponding SBR values down each column provides a total SBR level for each capture area. The percentage of an analyte’s SBR to the total capture area SBR is a measure of the influence a particular analyte has on a given capture area. Figure 7 graphs the SBR calculations for both polyclonal and monoclonal arrays. Considering both experiments, there does not seem to be a dependency on capture area position (advancing from the incoupling site toward the injection site is from left to right) or analyte injection order (number above each column) with percent SBR. It is apparent that analyte binds primarily to the target channel; i.e., the tallest column of each set corresponds to the specific analyte of the capture area (except for the RbSA channel, which is discussed below). The SBR values for the addition of gIgG to the polyclonal capture array suggest that the cross-reactive component of gIgG with the rIgG antibodies was not as pronounced as originally portrayed by the raw data in Figure 2.

Figure 7 also highlights two interesting features that appeared in the RbSA channel with monoclonal capture antibodies. First, the SBR produced by the bSA analyte with its capture layer was about 3 times less than that of hCG and IgG analytes with their specific capture areas. Although the maximum fluorescence attained by the RbSA capture area (Table 3) was similar to the previous two analyte responses, nonspecific binding across the entire sensor surface decreased the final SBR. This result was not completely unexpected, as bSA is known to be a surface-active analyte, especially at high concentrations.31 Improvements in surface chemistry to reduce nonspecific binding could potentially minimize or prevent this effect. (See for instance, http://www. biotul.com.) Second, the RbSA antibodies appeared to actively bind analyte from the IgG sample (striped column). A trivial, but possible, explanation for the false positive displayed by the RbSA antibodies to IgG can be attributed to residual bSA in the IgG stock solutions. Even though the purification process was assessed by SDS-PAGE (data not shown), not every stock solution’s purity was verified. From the comparison of MAIA results with SAIA results, it is clear that two of the three polyclonal capture areas were not suitable when used together in MAIA. Specifically, both the affinity and nonideality constants (Table 4) for rIgG and hIgG capture antibodies point to some form of cross-reactivity between analytes and capture antibodies. One possible explanation for this behavior could be that antibody complexes, which form in the mixing and binding stages of the assay, may attach to the capture areas with enhanced affinity. Since the assays were performed over identical concentrations between MAIA and SAIA, point-to-point correlations were calculated, as a function of MAIA fraction bound versus SAIA fraction bound, to further compare the reponses for polyclonal and monoclonal antibody capture layers (Table 6). Correlations for the hIgG and rIgG antibodies are not quite significant (R2 < 0.90), suggesting that the relationship between SAIA and MAIA is not necessarily linear. All other correlations for the polyclonal and monoclonal antibody test assays performed in buffer were significantly linear and demonstrated one-to-one agreement (i.e., slope, 1) between SAIA and MAIA formats. (31) Ahluwalia, A.; Giusto, G.; De Rossi, D. Mater. Sci. Eng. C 1995, 3, 267271.

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Table 6. Correlation Values between Various Assay Formats fraction bound MAIA vs fraction bound (R2)

analyte

pAb SAIA (n ) 3)

hIgG 1.39 ( 0.19 (0.89)a rIgG 0.67 ( 0.10 (0.42)a gIgG 1.00 ( 0.09 (0.98) bSA av CK-MB cTnI Mb a

mAb SAIA (n ) 3)

10% pSA, mAb MAIA (n ) 3)

1.00 ( 0.11 (0.99) 0.95 ( 0.08 (0.92) 1.03 ( 0.09 (0.93) 0.97 ( 0.01 (0.99) 0.86 ( 0.14 (0.95) 1.17 ( 0.04 (0.98)b 0.72 ( 0.21 (0.97) 1.16 ( 0.03 (0.99)b 0.96 ( 0.36 (0.99)

Linear fit not necessarily valid. b Statistically different from 1.

Serum albumin is the most prevalent blood plasma protein and has the greatest potential to interfere with assay results.32 In an attempt to simulate the interfering effect of dilute serum, MAIAs using monoclonal antibodies were conducted with 10% pSA added to the injected analyte/tracer solution (Table 2). The correlation values for a MAIA run with monoclonal antibodies in buffer versus an identical MAIA run in 10% pSA are also listed in Table 6. Only the avidin channel displayed departure from one-to-one correlation. In this case, the added serum component impeded response; i.e., the signal derived from buffer was 1.17 times that from the serum matrix over the range of concentrations tested (30 pM to 0.1 mM). Herron et al. performed a SAIA for CK-MB over the 0-100 ng/ mL range in whole serum and observed a 60% decrease in assay sensitivity.33 A likely cause for this change in sensitivity is a decrease in analyte transport due to the viscosity of the serum solution, possibly leading to the 15% decrease in the response for avidin. A similar decrease was observed for the cTnI cardiac analyte. The assayed cardiac samples were within the clinically significant ranges for assessing myocardial infarction, i.e., 0.5-100 ng/mL for cTnI, 1.5-100 ng/mL for CK-MB, and 5-500 ng/mL for Mb. However, detection limits were near to, or less than, 10 ng/mL for all analytes in SAIA format (Table 5). When performed in MAIA format, the detection limit for CK-MB and cTnI rose by about 60% while that for Mb increased by a factor of 4. As for comparisons, to our knowledge, there are no published results (32) Werner, M.; Dietz, Ed. Handbook of Clinical Chemistry; CRC Press: Boca Raton, 1989. (33) Herron, J.; Wang, H.-K.; Terry, A.; Durtschi, J.; Tan, L.; Astill, M.; Smith, R.; Christensen, D. Proc. SPIE 1998, 3259, 54-64 (Systems and Technologies for Clinical Diagnostics and Drug Delivery).

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for a CK-MB, cTnI, or Mb assay on an integrated optical waveguide. The CK-MB and cTnI detection limits can be compared to those obtained by Herron et al.33 using a thick-film internal reflection element. Our device performs comparably for CK-MB (2.8 ng/mL SAIA and 4.5 ng/mL MAIA vs 2.3 ng/mL) and less well for cTnI (15.9 ng/mL SAIA and 26.9 ng/mL MAIA vs 0.2 ng/mL). The 2 orders of magnitude difference for the cTnI detection limit is difficult to explain, even in light of the fact that no accepted convention exists for the conversion of cTnI international units to true mass units. CONCLUSIONS The goal of this work was to demonstrate that MAIA was possible on a planar SiON IOW using the methods of spatial arrangement, physical adsorption, and fluorescent tracer antibodies. Channel addressability experiments showed that it was, indeed, possible to separately activate each capture area intended for use in a MAIA. Comparison of SAIA and MAIA affinity constants revealed that two of the six model capture antibodies, both of which were polyclonal, bound analyte differently between formats. Further, the nonideality constants of these two antibodies were significantly different from the theoretical value and a pointto-point correlation between formats for each antibody did not infer a linear relationship. Two of the analytes showed a decrease in assay sensitivity with the addition of a serum component. Of the three cardiac analytes tested, only assays for CK-MB showed a comparable detection limit between formats while those for cTnI and Mb were adversely affected by the MAIA format. These results suggest that polyclonal antibodies are prone to crossreactivity in MAIA and that nonspecific binding can lead to degradation in the quality of MAIA, particularly in the presence of serum. The solution to the latter lies in carefully controlled surface chemistry. ACKNOWLEDGMENT We gratefully acknowledge support from NIH Grant HL 32132, NSF grant BES-9402355, the NSF-sponsored Duke/North Carolina Center for Emerging Cardiovascular Technologies, the Center for Biopolymers at Interfaces at the University of Utah, and Lumenal Technologies of Salt Lake City, UT. The authors also thank anonymous reviewers for identifying critical omissions in the literature review.

Received for review February 17, 1999. Accepted June 23, 1999. AC990183B