An Immunosensor Based on the Glucose Oxidase Label and Optical

A novel optical immunosensor setup is described which uses glucose oxidase enzyme as a label in conjunction with a luminescence lifetime-based oxygen ...
0 downloads 0 Views 66KB Size
Download PDF
Anal. Chem. 1999, 71, 1568-1573

An Immunosensor Based on the Glucose Oxidase Label and Optical Oxygen Detection Dmitri B. Papkovsky,*,† Toma´s C. O’Riordan,† and George G. Guilbault‡

Biochemistry Department and Chemistry Department, National University of Ireland, Cork, Ireland

A novel optical immunosensor setup is described which uses glucose oxidase enzyme as a label in conjunction with a luminescence lifetime-based oxygen sensor and phase measurements. The oxygen sensor membranes prepared on microporous filters were used as a solid phase on which the immunoassay was carried out. These sensing materials in combination with a new measurement setup provided high sensitivity for the detection of oxidase enzymes, being at nanogram per milliliter level, i.e., 10-11-10-12 M, with respect to glucose oxidase and its conjugates. Experimental data on the sensitivity were validated using theoretical equations and calculations. Using the new measurement setup and IgG-anti-IgG as a model, a number of different sensing materials were studied aimed to optimize the immunosensor and evaluate its performance. This approach was then applied to a practical system for the detection of human lactate dehydrogenase isoenzymes. It provided similar sensitivity of ∼1 ng/mL, which is comparable to that of standard ELISA. The attributes of the new immunosensor approach are discussed with respect to performance and versitility. In the past decade there was marked progress in luminescencebased oxygen sensors which included sensor material chemistry, instrumentation, and practical applications.1 Optical oxygen sensors usually comprise a long-lived photoluminescent dye embedded in a suitable polymer matrix having good permeability for oxygen which is applied as a thin-film coating onto a suitable support such as a glass slide, plastic foil, or optical fiber.1-3 Sample oxygen penetrates the polymer coating and efficiently quenches the luminescence of the dye by a dynamic mechanism; oxygen is not consumed by the sensor. At present, fluorescent complexes of ruthenium4-6 and phosphorescent porphyrin dyes7-9 are com* Corresponding author: (fax) 353-21-274034; (tel) 353-21-904257; (e-mail) [email protected]. † Biochemistry Department. ‡ Chemistry Department. (1) Wolfbeis, O. S., Ed. Fiber Optic Chemical Sensors and Biosensors; CRC Press: Boca Raton, 1991. (2) Papkovsky, D. P.; Olah. J.; Troyanovsky, I. V.; Sadovsky, N. A.; Rumyantseva, V. D.; Mironov, A. F.; Yaropolov, A. I.; Savitsky, A. P. Biosens. Bioelectron. 1991, 7, 199-206. (3) Hartmann, P.; Trettnak, W. Anal. Chem. 1996, 68, 2615-2620. (4) Lippitsch, M. E.; Pusterhofer, J.; Leiner, M. P. J.; Wolfbeis, O. S. Anal. Chim. Acta 1988, 205, 1-6.

1568 Analytical Chemistry, Vol. 71, No. 8, April 15, 1999

monly used as oxygen probes. Some other luminescent dyes such as pyrene, pyrenebutyric acid, fluoranthene, deacyclene, perylene dibutyrate, and trypaflavin have also been proposed;1 their performance with respect to oxygen sensing, however, is not always as good. The principle of an optical oxygen sensor is to exploit changes in luminescence intensity or lifetime10 as a function of oxygen concentration. It has become clear that although intensity-based oxygen sensors are advantageous in their optical simplicity, using inexpensive and noncomplex light-emitting diodes and photodiodes,11 their mode of measurement is unsatisfactory for certain applications. Thus, they are susceptible to many interfering factors such as geometry, sensor positioning, fluorophore degradation, light source and detector fluctuations, and aging. The use of luminescence lifetime as information carrier provides the whole system with higher stability and versatility, essential independence of the dye concentration, geometrical factors, and instability of optical components. Lifetime-based oxygen sensors allow for use of optical fibers and also “contactless sensing” in closed systems, e.g., through the wall of a (semi)transparent vessel.12 Luminescence lifetime can be measured either directly using pulsed excitation or by means of the phase measurements using intensitymodulated excitation.10 Phase measurements are more convenient for practical sensing, as they are more simple in terms of optics and electronics and allow real-time monitoring. A number of simple phase detectorssprototype oxygen sensorsshave been described in recent years.12,13 Various techniques have been used for the application of sensitive coatings onto solid supports. These include simple spreading of the dissolved sensor material on planar surfaces (5) Hartmann, P.; Leiner, M. P. J.; Lippitsch, M. E. Anal. Chem. 1995, 67, 88-93. (6) Wolfbeis, O. S.; Weis, L. J.; Leiner, M. P. J.; Ziegler, W. E. Anal. Chem. 1988, 60, 2028-2032. (7) Papkovsky, D. P. Sens. Actuators B 1993, 11, 293-300. (8) Papkovsky, D. P.; Ponomarev, G. V.; Trettnak, W.; O’Leary, P. Anal. Chem. 1995, 67, 4112-4117. (9) Vinogradov, S. A.; Wilson, D. F. J. Chem. Soc. Perkin Trans. 2 1995, 103111. (10) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Plenum Press: New York, 1991. (11) Trettnak, W.; Gruber, W.; Reininger, F.; Klimant, I. Sens. Actuators B 1995, 29, 219-225. (12) Trettnak, W.; Kolle, C.; Reininger, F.; Dolezal, C.; O’Leary, P. Sens. Actuators B 1996, 35-36, 506-512. (13) Trettnak, W.; Gruber, W.; Reininger, W.; O’Leary, P.; Klimant, I. Adv. Space Res. 1996, 18, 139-148. 10.1021/ac9810617 CCC: $18.00

© 1999 American Chemical Society Published on Web 03/09/1999

(glass, polyester foils),6 dip-coating on fibers,14 spin-coating,15 solgel process,16 Langmuir-Blodgett technique,17 photopolymerization,18 and capillary coatings,19 while immobilization of dye on solid carriers by physical20 or chemical21 adsorption has also been employed. Sensors fabricated using these techniques have been adapted to the detection of O2,5,8,9 CO2,14 pH changes,15 glucose,22 and a number of other important analytes, with the ability to detect two or more of these by the same device having been reported.23,24 The effect of the carrier polymer on sensor performance has been examined in great detail,2,3 while reduction in sensor size, i.e., miniaturization, has also been achieved.12,25 Recently we proposed the use of microporous light-scattering support materials such as membrane or fiber filters for sensor fabrication. It was demonstrated that such materials enable improvement of the oxygen sensor performance and open the possibilities for new assay formats, e.g., enzymatic analysis of metabolites.26 In the present paper, we describe application of the oxygen sensors prepared on microporous support for the development of sensitive bioassays, such as measurement of trace activity of oxidase enzymes and an immunosensor in which glucose oxidase enzyme is used as a label.

EXPERIMENTAL SECTION Materials. Glucose oxidase (GOx) from Aspergilus niger, 229.2 units/mg, mouse serum, human lactate dehydrogenase (LDH) isoenzymes LDH-1, LDH-2, and LDH-5, bovine serum albumin, polystyrene (MW 280 000), glutaraldehyde, β-D-glucose, and Durapore (PVDF), glass fiber, and nitrocellulose membranes (pore size 0.22, 2.3, and 5.0 µm, respectively) were from SigmaAldrich. The anti-mouse-IgG-GOx conjugate was from Calbiochem (Nottingham, England). The polyclonal sheep anti-humanLDH-1 antibodies were from The Binding Site (Birmingham, UK). The 96-well microtiter plates were from Nunc; 12-well plates were from Falcon. Apparatus. The fiber-optic phosphorescence phase detector was a simple homemade instrument described in detail earlier.8 It had a yellow light-emitting diode (LED, emission maximum, 586 nm, light output, 2500 mcd; Toshiba Corp., Tokyo, Japan) as a light source whose intensity was modulated at a frequency of 3683 Hz. The photodetector was an S2386-44K silicone photodiode (Hamamatsu). A 591-nm interference filter, 15-mm diameter, 3-mm thickness (DiaM, Moscow, Russia) and an RG9 glass filter, 10 × (14) Munkholm, C.; Walt, D. R. Talanta 1988, 35, 109-112. (15) Jones, T. P.; Porter, M. D. Anal. Chem. 1988, 60, 404-406. (16) MacCraith, B. C.; McDonagh, C. M.; O’Keeffe, G.; McEvoy, A. K.; Butler, T.; Sheridan, F. R. Sens. Actuators B 1995, 29, 51-57. (17) Beswick, R. B.; Pitt, C. W. Chem. Phys. Lett. 1988, 143, 589-594. (18) Tan, W.; Shi, Z. Y.; Smith, S.; Birnbaum, D.; Kopelman, R. Science 1992, 258, 778-782. (19) Weigl, B. H.; Wolfbeis, O. S. Anal. Chem. 1994, 66, 3323-3327. (20) He, H.; Fraatz, R. J.; Leiner, M. P. J.; Rehn, M. M.; Tusa, J. K. Sens. Actuators B 1995, 29, 246-250. (21) Mohr, G. J.; Wolfbeis, O. S. Anal. Chim. Acta 1994, 292, 41-48. (22) Schaffar, B. P. H.; Wolfbeis, O. S. Biosens. Bioelectron. 1990, 5, 137148. (23) Ferguson, J. A.; Healey, B. G.; Bronk, K. S.; Barnard, S. M.; Walt, D. R. Anal. Chim. Acta 1997, 340 (1-3), 123-131. (24) Li, L.; Walt, D. Anal. Chem. 1995, 67, 3746-3752. (25) Rosenzweig, Z.; Kopelman, R. Anal. Chem. 1996, 68, 1408-1413. (26) Papkovsky, D. B.; Ovchinnikov, A. N.; Ogurtsov, V. I.; Ponomarev, G. V.; Korpela, T. Sens. Actuators B 1998, 58, 137-145.

10 mm2, 2 mm thick (Schott, Germany) were used for effective separation of the excited light and phosphorescence. The analog electronic scheme of the instrument provided amplification, lowfrequency filtering, and acquisition of the phosphorescence signal from the (PtOEPK-PS) oxygen membrane via its fiber-optic probesa 60-cm bifurcated glass fiber bundle, 4 mm in diameter. It allowed real-time continuous measurement of the phosphorescence intensity and phase shift signals. The instrument had an analog 10-mV output to the chart recorder (Recorder Co.) to monitor both the intensity signal (arbitrary units) and phase shift (degrees). The response of the instrument (integration constant) was variable from 3 to 30 s. All measurements were carried out under ambient light without special precautions.

METHODS (a) Preparation of the Phosphorescent Oxygen-Sensitive Membrane. A coating “cocktail” was prepared that contained 1 mg/mL platinum(II) complex of PtOEPK (Joanneum Research, Graz, Austria) in a 5% (w/w) solution of polystyrene in toluene. Aliquots of 5-10 µL of this solution were applied with a micropipet onto pieces of microporous filter membranes (see above) and allowed to spread and dry for ∼15 min in air. Thus, uniformly colored pink spots having a diameter of ∼10 mm were obtained which were cut into disks of 12-mm diametersphosphorescent oxygen sensor membranes. The membranes were stored in the dark at +4 °C for later use. (b) Immobilization of Proteins on the Oxygen Membranes. Two procedures were used to immobilize the protein antigens (mouse serum and anti-LDH-1-antibodies) on the oxygen membranes: cross-linking with glutaraldehyde and direct passive adsorption on the polystyrene-based oxygen-sensitive coating. In the glutaraldehyde method, a solution containing 880 µL of H2O, 100 µL of PBS buffer, pH 7.4, and 10 µL of the stock protein solution (mouse serum, 51.4 mg/mL or anti-LDH-1-Ab, 10 mg/ mL) was prepared. To this solution was added 10 µL of a 5% solution of glutaraldehyde and 20-µL aliquots were applied immediately to each of the oxygen sensor membranes, which were then left to incubate for 1 h at 37 °C. Alternatively, the oxygen membranes were simply soaked in 20 µL of an antigen solution, the latter contained 890 µL of H2O, 100 µL of carbonate buffer (0.1 M, pH 9.5), and 10 µL of the stock antigen solution, and incubated for 1 h at 37 °C to passively adsorb the protein to the polymer surface. Following immobilization, the membranes were incubated for 1 h in PBS buffer containing 5 mg/mL BSA (PBSBSA) and then washed with the same buffer to block sites of nonspecific binding and remove any unbound antigen from the sensor membranes. The immunoaffinity membranes thus obtained were rinsed with water and stored at +4 °C for later use. As blank controls, the oxygen membranes with immobilized nonspecific antibodies or other proteins such as BSA were used; immobilization was performed in the same way as above. (c) Preparation of GOx Conjugate with Anti-LDH-1 Antibodies. A 2-mg sample of GOx (Sigma) was dissolved in 1 mL of water, mixed with a 100 mM solution of sodium metaperiodate, and incubated for 20 min at room temperature. This solution was then passed through a PD10 desalting column (Pharmacia) equilibrated with a 1 mM acetate buffer (pH 4.4), collecting the Analytical Chemistry, Vol. 71, No. 8, April 15, 1999

1569

fractions of the protein peak (using A280 nm). The 1.3 mL of the resulting solution of oxidized protein was mixed with 1 mg of polyclonal anti-LDH-1 antibodies which had been dissolved in 125 µL of 0.1 M carbonate buffer (pH 9.5). This solution was stirred for 2 h at room temperature, and then 125 µL of a 4 mg/mL solution of sodium borohydride was added to the reaction and shaken for a further 2 h. The resulting anti-LDH1-Ab-GOx conjugate was purified on a PD10 desalting column equilibrated with 0.1 M sodium phosphate buffer (pH 7). The protein fraction collected had a concentration of 0.358 mg/mL, determined spectrophotometrically. The conjugate was stored at +4 °C. (d) Measurement of Activity of Glucose Oxidase in Solution. A series of standard solutions of GOx were prepared in PBS-BSA buffer. A 0.2-mL aliquot of GOx standard solution was put in a well of a 12-well microtiter plate, and 0.1 mL of substrate solution (1.0 M glucose in PBS buffer, pH 7.0) was added. The oxygen sensor membrane was then soaked in this solution, covered with a piece of cover glass, 10 × 10 mm, 2 mm thick, to limit oxygen access to the membrane (bubbles must be avoided), and the kinetics of the phase shift signal was recorded, with the fiber-optic probe of the phase detector positioned at the bottom of the well. Slopes of the phase shift (deg/min), which reflect enzymatic consumption of the dissolved oxygen inside the sensor membrane, were determined and plotted as a function of GOx concentration, to produce a calibration curve. (e) Immunoreaction and Quantitation. The immunoaffinity membranes containing mIgG were extensively washed prior to use with PBS-BSA buffer using Swinnex cartridges (Millipore) and a 10-mL syringe. The membranes were placed in wells of 12well microtiter plates (Falcon), incubated with 1 mL of different dilutions of labeled antibodies in PBS-BSA for 1 h at 37 °C, and then washed extensively with PBS-BSA. In the case of antiLDH1-Ab membranes, they were treated in a similar way, but with two incubation steps (sandwich assay). The first incubation was with different concentrations of antigen (LDH1, LDH2, or LDH5) and then with anti-LDH1-GOx conjugate, 7 µg/mL for each membrane. At the final stage of quantitation, 0.2 mL of substrate buffer (100 mM glucose in 0.1 M potassium phosphate, pH 7.0) was added to each membrane and the activity of specifically bound GOx determined, similar to the above. Again, a cover glass was used to limit oxygen access and improve the sensitivity. Slopes of the phase shift (deg/min) were determined for each membrane and plotted as a function of anti-mIgG-GOx or LDH concentration, to produce calibration curves. RESULTS Principles of the Immunosensor Operation and Experimental Setup. The immunosensor format resembles the wellknown ELISA: the immunoreagent immobilized on a solid-phase participates in binding interactions with the analyte (antigen) and labeled antibodies; the bound fraction of the label is then quantified. GOx, which has excellent catalytic properties and stability characteristics, was employed as a label in immunoassays and electrochemical immunosensors.27 Its activity is quantified via the reaction of oxidation of β-D-glucose: GOx

Glu + O2 98 gluconic acid + H2O2

(1)

Monitoring of trace activity of GOx via dissolved oxygen con1570 Analytical Chemistry, Vol. 71, No. 8, April 15, 1999

Figure 1. Diagram showing the experimental setup for measurement of GOx activity both in the immunosensor format and in solution: 1, oxygen sensor membrane; 2, fiber-optic probe; 3, cover glass; 4, transparent polystyrene well; 5, substrate buffer.

sumption is usually complicated by the vast excess of oxygen in the environment. Special precautions are required with respect to oxygen diffusion from air and the capacity of the measurement cell for oxygen.28 However, the use of microporous sensor membranes and contactless optical oxygen detection provides a simple solution and measurement setup which is shown in Figure 1. As the sensor membrane is sandwiched between the two oxygen-impermeable surfaces, bottom of the well and cover glass, the enzymatic reaction is monitored only in a small part of the liquid sample that is trapped inside the microporous membrane. Since such membranes usually have uniform and well-defined structure, thickness, and capacity, oxygen sensors based on them comprise standard cells where the enzymatic reaction is monitored on a microscale. Thus, according to the specifications of the manufacturer, the capacity of a Durapore membrane (and corresponding oxygen sensor) is ∼10 µL/1 cm2.29 Moreover, it was found that such a setup provides a highly sensitive detection of GOx activity, due to the fact that the oxygen diffusion from the external space (both solution and air) into the membrane is restricted. Theoretical Sensitivity of Optical Detection of GOx. A simple optical oxygen sensor can be considered as a homogeneous population of the dye molecules in a polymer matrix (however, this is not the case for most practical systems1). In this case, the parameter that is monitored in phase measurements, i.e., the phase shift φ, relates to the luminescence lifetime of the oxygen probe (τ) as follows:

τ ) tan φ/2πf

(2)

where f is the working frequency of modulation of excitation and π ) 3.14. The luminescence lifetime, in turn, relates to the oxygen concentration according to the relationship derived from the Stern-Volmer equation10 as

[O2] ) (τo - τ)/kqτoτ

(3)

where kq is the bimolecular quenching rate constant and τo and τ are the luminescence lifetimes without and with oxygen, respectively. This gives the relationship between the oxygen concentra(27) Scheller, F.; Schubert, F. Biosensors; Elsevier Press: Amsterdam, 1992. (28) Gyss, C.; Bourdillon, C. Anal. Chem. 1987, 59, 2350-2355. (29) Millipore Laboratory Catalogue, 1996.

tion and the phase shift:

[O2] ) (2πf/kq)(1/tan φ - 1/tan φo)

(4)

where φ and φo are the luminescence phase shifts in the presence and in the absense of oxygen, respectively. Then the rate of oxygen consumption will be the derivative of eq 4:

d[O2]/dt ) - (2πf/kqsin2 φ)(dφ/dt)

(5)

The kinetics of GOx is described by the Michaelis-Menten equation which also involves the initial rate V:

V ) - (d[O2]/dt) ) kcat[E][O2]o/(Km + [O2]o)

(6)

where [E] is the enzyme activity and kcat and Km are the catalytic and Michaelis constants, 800 s-1 and 2 × 10-4 M, respectively.30 Operating with eqs 5 and 6, the following expression can be obtained which defines the minimal activity of enzyme that can be detected by quenched-luminescence oxygen sensing and phase measurements:

Figure 2. Calibration graph for (a) GOx quantitation in solution using the PtOEPK-PS oxygen sensors membranes prepared on 0.22-µm Durapore (2) and 5.0-µm nitrocellulose (×) microporous filters and (b) for the detection of specific binding of anti-mIgG-GOx conjugate to the immunosensor membranes prepared on 0.22-µm Durapore ([) and 5.0-µm nitrocellulose (9) filters. Antigen (mouse serum) was immobilized by cross-linking with glutaraldehyde. Buffer: air-saturated 0.1 M potassium phosphate, pH 7.0, 23 °C.

Calculation of [E]min requires the knowledge of the main parameters for the enzyme, oxygen sensor, and measurement system. For the system used in the present work, the parameters were as follows: kq ) 1.12 × 109 M-1 s-1; f ) 3683 Hz; φo ) 53°, φ ) 18°, at 23 °C and [O2]o ) 200 µM (air-saturated solution). The minimal resolution of the instrument, i.e., (∆φ/∆t)min, was ∼0.01°/min in kinetic mode. Putting all these figures into eq 7, gives the theoretical sensitivity for GOx of ∼1.5 × 10-12 M. The absolute amount of GOx measurable in a 1-cm2 oxygen sensor membrane (i.e., in 10-µL volume) is equal to 1.5 × 10-17 M (i.e., 15 amol). Evaluation of Practical Sensitivity for GOx Detection in Solution. Measurement of the activity of pure GOx in solution using the new setup is shown on Figure 2. One can see that the system is capable of detecting ∼10 ng/mL of GOx (∼5 × 10-11 M, 2.3 × 10-3 units/mL) using sensors prepared on 0.22-µm Durapore membranes and 1 ng/mL (5 × 10-12 M, 2.3 × 10-4 units/mL) for the sensors on 5.0-µm nitrocellulose membranes. The analytical range was at least 1.5 orders of concentration for both types of sensors, with background signals of 0.05° and 0.111°/min, respectively. It was shown that such high sensitivity of detection of GOx activity can only be achieved in conditions of limited oxygen access, i.e., with a microporous membrane soaked in substrate buffer and covered on both sides with oxygenimpermeable material. However, optimization of the measurement setup, namely, the sensor size, amount of liquid added, and shape of the cover slide, is required to achieve the highest sensitivity.

Evaluation of the Immunosensor with IgG-Anti-IgG Model. With a simple model system, mouse IgG (mouse serum) as an antigen and anti-mouse-IgG antibodies labeled with GOx, the sensitivity of Ab-GOx detection was ∼10 ng/mL for Durapore and 5 ng/mL (i.e., 2.6 × 10-11 M) for nitrocellulose membranes, which is quite similar to that of pure GOx in solution (see above). For nitrocellulose membranes, blank signals (i.e., drift of the phase shift) were observed, which does not seem to be associated with the enzymatic reaction. These were rather small and stable and enabled reliable quantitation of the antigen. Results are shown in Figure 2. Since the PtOEPK-PS oxygen sensor membranes provide in fact a polystyrene-based surface, i.e., the material commonly used in immunoassays (microtiter plates and test tubes), immobilization of antigens by passive adsorption was tested for preparation of the immunoaffinity membranes. The procedure was simply transferred from the one commonly used in ELISA.31 This method, which is more convenient than the glutaraldehyde method, was shown to give good and reliable results in immunoassay and reasonably low background signals. Results are shown in Figure 3. The reproducibility of the immunosensor was tested by repeating four sets of assays in a reproducible manner using the Durapore membrane. The results obtained are shown in Figure 4; relative standard deviations were as follows: 200 ng/mL, 10.1%; 100 ng/mL, 13.2%; 50 ng/mL, 9.1%; 10 ng/mL, 26% (n ) 4). Such precision and reproducibility are reasonably good for disposable handmade sensor elements and nonautomated multistep assay procedures. Glass fiber membranes (2.3-µm pore size, 270 µm thick), which are expected to have low intrinsic capacity for oxygen, were also tested as a sensor support. The results are shown in Figure 4. Compared to Durapore and nitrocellulose membranes, a ∼10-fold increase in sensitivity was obtained with this type of membrane, the limit of detection being 0.5 ng/mL (or 2.6 × 10-12 M) GOx conjugate. This is comparable to the sensitivity of standard ELISA, using horseradish peroxidase as a label and colorimetric substrates.31 Therefore, the glass fiber membranes were considered the most promising for use in the immunosensor.

(30) Gibson, Q. H.; Bennett, E.; Swoboda, P.; Massey, V. J. Biol. Chem. 1964, 239, 3927-3934.

(31) Crowther, J. R. ELISA: Theory and Practice; Methods in Molecular Biology 42; Humana Press: Totowa, NJ, 1995.

[E]min ) -

2πf(Km + [O2]o)

(∆φ/∆t)min kq sin2 φ kcat[O2]o

(7)

Analytical Chemistry, Vol. 71, No. 8, April 15, 1999

1571

Figure 3. Reproducibility of the immunosensor (four identical sets of experiments) for the 0.22-µm Durapore-based immunoaffinity membranes, with respect to the detection of anti-mIgG-GOx conjugate. Conditions are the same as in Figure 2.

Figure 5. Calibration graph of the LDH-1 immunosensor based on the oxygen sensor membranes prepared on 2.6-µm glass fiber filters, with anti-LDH-1 antibodies immobilized by passive adsorption. Sandwich assay; for conditions, see text.

with LDH-5 is more likely due to the quality of the antibodies and/or LDH isoenzyme standards. Conventional ELISA in similar sandwich format showed similar cross-reactivity values for different LDH isoenzymes.

Figure 4. Calibration graphs for the detection of anti-mIgG-GOx conjugate using (a) oxygen sensor membranes prepared on 0.22µm Durapore filters, with antigen (mouse serum) passively adsorbed from PBS, pH 7.4 (9) and from 0.1 M carbonate, pH 9.5, ([) and (b) using oxygen sensor membranes prepared on 2.6-µm glass fiber filters, with antigen (mouse serum) passively adsorbed from 0.1 M carbonate buffer, pH 9.5 (2). Conditions are the same as in Figure 2.

Application of the Immunosensor To Detection of LDH Isoenzymes. The immunosensor for the detection of human LDH-1 isoenzyme was constructed on the basis of oxygen sensor membranes prepared on the glass fiber support. Polyclonal antihuman LDH-1 antibodies were immobilized by passive adsorption, and a sandwich assay was performed in which the immunoaffinity membrane was first incubated with the antigen and then with antiLDH-1 antibodies labeled with GOx. The concentrations of adsorbed antibody and GOx-antibody conjugate were optimized to 15 and 9 µg/mL, respectively. In this format, the limit of detection was ∼2.5 ng/mL, which corresponds to ∼1 × 10-11 M or 0.65 units of LDH-1. Three blank values were obtained with a mean slope of 0.162°/min and a standard deviation of 0.0114. Results are shown in Figure 5. Cross-reactivity of LDH-1 immunosensor with respect to LDH-2 and LDH-5 isoenzymes was tested. Since LDH has a tetrameric structuresLDH-1 consists of four H subunits, LDH-2 contains three H and one M subunit, and LDH-5 is made up of four M subunits, none of which are present in LDH-132ssignificant crossreactivity of the LDH-1 immunosensor was found toward LDH-2 (43.8%) and only minor toward LDH-5 (12.8%), for 10 ng/mL concentrations of the isoenzymes. Relatively high cross-reactivity (32) Moss, D. W.; Henderson, R.; Kachmar, J. F. In Textbook of Clinical Chemistry; Tietz, N. W., Ed.; W. B. Saunders Co.: Philadelphia, 1986; Chapter 5.

1572 Analytical Chemistry, Vol. 71, No. 8, April 15, 1999

DISCUSSION The use of the oxygen sensor on a microporous support described here allows certain advantages with respect to measuring the trace activity of oxidase enzymes, and for immunosensor applications. These are not associated with other methods of oxygen detection such as the Clark electrode27 and conventional optical oxygen sensors. The electrochemical systems consume oxygen and are heavily influenced by hydrodynamic conditions in the measurement cell such as flow rate, stirring, and mass transfer. Optical systems overcome these drawbacks as oxygen is not consumed. However, for conventional oxygen sensors, such as planar nonporous oxygen membranes, it is hard to standardize sample volume on a microscale. Experiments performed with conventional oxygen sensor membranes on the measurement of the trace activity of GOx showed poor reproducibility and low sensitivity. Monitoring of the dissolved oxygen consumption by phase measurements in the kinetic mode is a sensitive and reliable technique. Changes of the phase shift of as low as 0.01°/min can be reliably detected over a period of few minutes. Principally, it is possible to process several membranes at once in a way similar to that in a microplate reader, using repetitive measurement of the phase shift for each sensor membrane for the determination of the slope (e.g., two-point). In addition to a small sample volume, which usually falls within the range of 10-20 µL/cm2 for different microporous membranes, their three-dimensional structure and large surface area allows the efficient mass exchange at the interface between the solid phase (both the oxygen-sensitive layer and the bioactive layer) and solution. This enables flow-through sampling with the sensor membranes rather than simple soaking, development of simple express assays, and automation. In contrast to other luminescence-based immunosensors, which usually deal with very low intensity signals and have strict requirements for background light and optical and electronic components of the detection system, the new immunosensor always operates with high levels of signals coming from the

oxygen sensor. It realizes contactless measurements under ambient light; the latter has no interference as it is filtered by electronics of the phase detector (nonmodulated light). All this is of particular importance for application to biological systems, where detachment of the measurement system from the sample may be a serious advantage and/or requirement. Disposable active elements can be produced very reproducibly, at low cost, and on a small and large scale using simple technology. Other advantages of microporous oxygen sensor membranes in terms of optical performance are discussed elsewhere.26

ACKNOWLEDGMENT Financial support of this work by the Irish Research Foundation Enterprise Ireland (Forbairt), Grants ST/95/809A and SC/98/486, is gratefully acknowledged. Received for review September 23, 1998. Accepted February 1, 1999.

AC9810617

Analytical Chemistry, Vol. 71, No. 8, April 15, 1999

1573