Chemiluminescence in Analytical Chemistry

Figure 4 Structures of Lumigen acridan esters: PS-1: R1CR3CH, R2COCH3, PS-2: R1CR3COCH3, R2CH, PS-3: R1CR2CR3CH.

of HRP at the 10 19–10 15 mol range. This constitutes a major improvement over the commercial luminol-based enhanced chemiluminescence reagent.

2.3 Mechanism of Enhanced Chemiluminescence

Little is known about the role of the enhancer in the HRP-catalyzed chemiluminescent oxidation of acridan esters. From the work of Akhavan-Tafti and Schaap’s group the intermediacy of the corresponding acridinium ester can be inferred [14], which in a subsequent reaction with hydrogen peroxide affords N- methylacridone with the concomitant production of light. This last reaction is well known from McCapra [2]. In the corresponding HRP-catalyzed chemiluminescent oxidation of luminol, the enhancer is assumed to rapidly react with the peroxidase reactive intermediates, Compound I (CI) and Compound II (CII), accelerating enzyme turnover and producing enhancer radicals [21]. These enhancer radicals react rapidly with luminol, acting as a redox mediator between HRP intermediates and luminol [22]. Assuming a similar role for the enhancer in acridan ester oxidation, a simplified reaction scheme incorporating some of the hypothesized reactions can be devised (Fig. 5).

As in the luminol case, the main role of the enhancer (EnH) seems to be related to turnover of the enzyme, generating enhancer radicals (En rad) in the process that are capable of oxidizing the acridan ester (AcH). The structure of the enhancer obviously is very important. To accelerate HRP turnover, the enhancer must on the one hand be able to rapidly react with the reactive HRP intermediates CI and especially CII (k2 and k3 large). On the other hand, the oxidized enhancer intermediate (radical or radical cation) must be able to oxidize the acridan ester (light-generating step). This last reaction also depends on the structure of the acridan ester: in a very unfavorable case, adding an ‘‘enhancer’’ for enzyme turnover could actually diminish the light production if k 4 k4 (Fig. 5), i.e., if the enhancer radical would not be able to oxidize the acridan ester.

Figure 5 Proposed reactions involved in the HRP-catalyzed chemiluminescent peroxidation of acridan esters.

Further complicating factors in the choice of an enhancer include degradation of HRP by enhancer radicals [23], pH effects [24] on reduction and oxidation potentials for enhancer and acridan ester, inactivation of enhancer radicals because of dimerization or other reactions, etc. All these, and other, effects of the structures (and because of the kinetics also the concentrations) of enhancer and acridan ester may cause erratic results when optimization studies are conducted. When

Figure 6 Structure of the enhancer as incorporated in the Pierce Supersignal family.

testing new enhancers it is advisable to optimize the enhancer concentration with respect to a complete calibration line for HRP. It is possible that a certain enhancer concentration will give good results at the high end of the HRP calibration curve while performing worse than optimal at the low end of the calibration curve. Studies that show only enhancement factors for one HRP concentration should be treated with suspicion.

Although substituted phenols (e.g., para-iodophenol, para-phenylphenol, firefly luciferin, coumaric acid) are popular enhancers, in both luminol and acridan ester oxidation, enhancers with other functional groups [24], e.g., phenylboronic acids [25–28], phenothiazines [29], are also useful. As an example the structure of the phenothiazine enhancer used in the Supersignal substrate family is shown in Figure 6.

Other additives to the signal reagent for enhanced chemiluminescence signal reagents include Tween20, protein, e.g., bovine serum albumin, and ionene [30] (their main effect seeming to be stabilization of the HRP). In preliminary experiments a stabilizing effect of ionene on the activity of HRP could be shown when phenylphenol was used as the enhancer but not with other enhancers (Zomer, unpublished results). Also, the salts to prepare the buffer solutions have effects on the intensity and duration of the signal [31].

2.4Characteristics of the GZ-11 Acridan Ester Signal Reagent

In the ideal case the kinetics of the HRP-catalyzed oxidation of acridan ester is solely dependent on the enzyme concentration; i.e., the signal is proportional to the HRP concentration irrespective of the point in time. To achieve such a situation the mechanism of the light-generating reaction must be the same for all HRP concentrations. This implies that the concentrations of hydrogen peroxide and acridan ester should be large enough to cover the whole HRP concentration range without affecting the mechanism. For convenient measurement of the signal the plateau of steady-state light emission is chosen. This steady state of light emission

Although the signal reagent can be used over a wide pH range, the optimal pH was found to be 5–7. Most of the results described in this chapter were obtained using a pH of 5.4 [phosphate-buffered saline (PBS), 10 mM].

From the simplified mechanism of enhanced chemiluminescence it might be concluded that the enzyme turnover benefits from a high concentration of enhancer. After all, the rate of enzyme turnover is governed by the rate-limiting step, the reduction of compound II to the resting enzyme, which is proportional to the enhancer concentration. However, when enhancer concentrations are plotted against enhancement factors (at constant HRP concentration) a bell-shaped curve is obtained [21]. Furthermore, the optimal enhancer concentration is not necessarily equal for all HRP concentrations. This implies that secondary reactions of enhancer (or of impurities present in the enhancer [32]) take place. The characteristics of the chemiluminescence signal from the HRP-catalyzed peroxidation of acridan ester GZ-11 are shown in Figures 7 (kinetics of the reaction) and 8 (HRPcalibration curve).

As can be seen from Figure 7, the signal reaches a plateau within 10 min and lasts for a relatively long time (even after 5 h a useful signal can be obtained). Furthermore, the signal reagent allows measurement of HRP over a wide range (Fig. 8).

Figure 8 HRP calibration curve (log-log plot of HRP dose vs. chemiluminescence signal, corrected for background) using signal reagent incorporating GZ-11.

3. APPLICATIONS

To test our new signal reagent based on GZ-11 the detection system was applied to two competitive-format immunoassays. These two assays (for atrazine and clenbuterol) normally use chromogenic detection systems. While these colorimetric assays may be adequate for laboratory use, chemiluminescent detection offers potential advantages in sensitivity and on site screening applications [33].

3.1 Atrazine Immunoassay

3.1.1Introduction

Monitoring water samples for the presence of atrazine is normally done using conventional techniques involving chromatographic separation followed by detection (UV, MS). Recently an enzyme immunoassay for atrazine was reported. This assay uses enhanced luminol chemiluminescence detection of an HRP conjugate and results in an assay with a theoretical sensitivity of 0.05 ppb using 2 h of incubation in an ice-water bath [34]. Using a signal reagent containing GZ11 we decided to develop an enzyme immunoassay for atrazine.

3.1.2Assay Development

The assay conditions were optimized with respect to antibody and tracer concentration to obtain maximal sensitivity at 0.1 ppb, the current maximum admissible concentration (MAC) in drinking water.

The normal protocol using monoclonal antibody (mAb) K4E7 [35] involves the following steps: Coating of plates using goat anti-mouse antibody (0.25 mL, 1:5000 in 50 mM carbonate buffer, pH 9.6) was performed overnight at 4°C. After being washed with washing buffer (PBS, pH 7.6; 4 mM, 0.05% Tween 20), the plates were incubated with 0.2 mL of mAb K4E7 (dilution 1:100,000) in PBS (pH 7.6, 40 mM) for 2 h at room temperature. The plates were washed

with washing buffer. In the competition step 0.15 mL of atrazine standards (0– 50 g/L) and samples were incubated in the wells with 0.05 mL of atrazine-HRP

conjugate (dilution 1:50,000) for 1 h at room temperature. After the plate was washed with PBST, the substrate reaction using the chromogen tetramethylbenzidine (TMB) was performed.

The assay conditions involving the GZ-11 signal reagent were optimized with respect to mAb and tracer dilutions. This resulted in the following protocol: The atrazine assay was performed in coated Lumacuvettes (overnight, room temperature, pH 9.6, goat-anti mouse 1/5000) using antiatrazine monoclonal antibodies (1/160,000 in PBS, pH 7.4, 40 mM), with 2 h of incubation at room temperature. The antibody-coated tubes were incubated with atrazine-HRP conjugate

(1/200,000) and standards and samples. After a 1-h incubation at room temperature, the tubes were washed, and signal reagent was added to all tubes. The chemiluminescence was measured at different points in time. The results are shown in Figure 9.

3.1.3Results

From Figure 9 it can be concluded that the sensitivity of the assay is adequate to screen water samples for the presence of atrazine at a MAC value of 0.1 ppb. In a preliminary experiment 10 river water samples were screened for the presence of atrazine. In eight out of 10 an immunological response corresponding to atrazine levels greater than 0.1 ppb could be confirmed by GC-MS analysis. The other two samples contained atrazine at levels below 0.1 ppb (both with the enzyme immunoassay as with the GC-MS analysis).

Figure 9 Atrazine calibration curve, plotted as percentage binding (B) over binding at zero dose (B0) against dose, as detected after different signal-developing times.

3.2 Clenbuterol Immunoassay

3.2.1Introduction

β-Agonists like clenbuterol are widely used, not only for the treatment of respiratory diseases but also to improve carcass characteristics and growth rates of farm animals. The use of β-agonists as anabolic agents is illegal in the European Community and in North America. Within the framework of Residue Control Directive 86/469/EEC the European Member States try to gain control of the growing black market for β-agonists. To do so a large number of biological samples (urine, hair, eyes, etc.) are screened annually for the presence of clenbuterol and other β-agonists. Enzyme immunoassays are frequently used for these examinations. However, in a recent evaluation of nine commercially available ELISA kits for the detection of clenbuterol in bovine urine it was concluded that none met the requirement of the official residue control plans of the EU Member States to detect β-agonists at the 1-ppb level [36]. The above ELISA methods are suitable for laboratory use only.

Ideally, one prefers to perform a first screening at the place of sampling (farmor slaughterhouses). To do this a tube enzyme immunoassay for β-agonists was developed [37]. This test was capable of detecting clenbuterol at a level of 3–4 ppb when performed in fivefold diluted urine. Here we describe the adaptation of an enzyme immunoassay capable of detecting clenbuterol and other β- agonists at the sub-ppb level in bovine urine using HRP as the label and the chemiluminescence signal reagent based on GZ-11 acridan ester. The immunoassay can be performed in microtiterplates with the signal being detected either in a plate luminometer or on photographic materials (X-ray and instant film).

3.2.2Materials and Methods

Chemiluminescence measurements were performed on X-ray film or Polaroid 20,000 ASA film using a camera luminometer (Tropix, Inc., Bedford, MA). The microplate luminometer we use is the Lucy 1 (Anthos Labtec Instruments, Wals, Austria). Black, white, and transparent microtiterplates and strips were from Corning Costar (Badhoevedorp, The Netherlands).

Immunoassays were performed in microtiter plates or eight-well strips. Briefly, plates were coated with a suitable antibody dilution (1:3000) overnight at 4°C in carbonate buffer (50 mM, pH 9.6). The coated strips were washed four times with PBST (40 mM, pH 7.4 containing 0.1% Tween 20). The strips were incubated with 10-fold diluted urine samples or standards (200 µL) and tracer dilution 1:4000 (10 µL). After standing for 1–2 h the strips were washed with PBST. To each well 200 µL of signal reagent containing GZ-11 was added. The glowing plate was either analyzed in the microplate luminometer or exposed to

tracer. Consequently, the signal reagent must be capable of generating enough light to be detectable on instant film in the low-pg range of the tracer.

3.2.3Results

An example of a clenbuterol assay of 10-fold diluted bovine urine samples as detected on X-ray film is shown in Figure 10. This image results from an incubation of standards (column 1 and 8200 L) or urine samples (columns 2–7 and 9–12) and tracer (1:8000, 10 L) in PBS (100 mM, pH 7.2) in the wells of an antibody-coated (1:3000) microtiter plate for 2 h at room temperature, followed by incubation with signal reagent. After a 10-min incubation, the glowing plate was covered with X-ray film, and exposed for 13 min to the film. After development, the photographic image was scanned into a computer and inverted. From Figure 10 it can be concluded that it should be possible to screen 10-fold-diluted urine samples for the presence of clenbuterol below ppb levels.

4.SYNTHESIS AND CHARACTERIZATION OF ACRIDAN ESTERS

Acridan esters were synthesized according to the general synthetic scheme depicted in Figure 11.

4.1 Acetanilide

Aromatic amine (20 g), acetic acid anhydride (20 mL), acetic acid (glacial, 20 mL), and zinc powder (0.1 g) were mixed and refluxed. After 30 min the solution was poured into 500 mL of ice water and the precipitate was filtered and dried to obtain the acetanilide.

4.2 4-Chlorophenylphenyl Amine

Acetanilide (13.5 g), (substituted) aromatic bromide (25 g), potassium carbonate (13.2 g), and copper iodide (1.9 g) were heated (190°C) and stirred overnight.

After cooling to room temperature toluene was added and the precipitate filtered. The solution was concentrated and the excess of bromide removed by distillation under reduced pressure. The residue was dissolved in ethanol (200 mL), potassium hydroxide (10.3 g) was added, and the mixture refluxed overnight. Ethanol was evaporated, the residue dissolved in dichloromethane, and washed with brine. The organic layer was dried over MgSO4 and concentrated to obtain the crude diphenylamine.