enzyme immunoassay with amperometric flow-injection analysis using horseradish peroxidase as a...
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ANALYTICA CHIMICA ACTA
Analytica Chimica Acta 336 (1996) 167-174
Enzyme immunoassay with amperometric flow-injection analysis using horseradish peroxidase as a label. Application to the
determination of polychlorinated biphenyls
Michele Del Carlo, Marco Mascini”
Dipartimento Sanitci Pubblica, Epidemiologia e Chimica Analitica Ambientale, Sezione di Chimica Analitiw.
Universirir degZi Studi di Firenze, Via G. Capponi 9, 50121 Firenze, Italy
Received 21 March 1996; revised 1 August 1996; accepted 10 August 1996
Abstract
An amperometric detection system for horseradish peroxidase (HRP) activity was optimized using flow injection analysis (PIA) with glassy carbon as a working electrode. Ferroceneacetic acid was investigated as a co-substrate for the electrochemical detection of HRP. The calculated detection limit for HRP was 2.6x lo-” M with incubation of 30 min. The substrate was used in an electrochemical enzyme immunoassay for polychlorinated biphenyls (PCB). We used a competitive assay, where PCB-protein conjugate (gelatin) was immobilized to the solid phase (microtitre assay plate) and the competition
was carried out with PCB standards using a limiting amount of anti-PCB IgG. The extent of the competition was evaluated using a secondary, HRP labelled, IgG; the amount of the enzyme label was detected after 30min of incubation with the substrate. The PCB range was 0.1-50 pgml-‘. The overall assay time was 2 h and 30min. The within assay precision, over 6 measurements, was lower than 10% for the entire range.
Ke~or&: Immunoassay; Amperometry; Flow injection; Horseradish peroxidase; Polychlotinated biphenyls
1. Introduction
Polychlorinated biphenyls (PCB) have been recog- nized for several years as ubiquitous environmental
pollutants [ 11. These complex mixtures of variable chlorinated biphenyls, have occurred in nearly all environmental mediums (e.g. surface water, soil, rain,
and snow) [2,3]. The PCB mixtures are usually referred as Aroclors followed by a number (i.e.,
* Corresponding author. Fax: (+39 55) 2476972; e-mail:
Masciniacesitl .unifi.it.
Aroclor 1260: 12 atoms of carbons and 60% of chlorine) which indicates the number of carbons and the chlorination percentage present in the molecule and in the total mixture, respectively. Even though the production of PCB has stopped they are still employed in capacitors and transformers, thus they may still be introduced in the environment and do
represent a health risk f4]. In fact, the toxicity for the
209 congeners ranges from moderately, to highly toxic [5]. Therefore the cleaning-up of contaminated sites is a major problem for the environmental agencies [6]. PCB analysis is usually carried out during both the monitoring and cleaning procedures
0003.2670/96/$15.00 NJ‘! 1996 Elsevier Science B.V. All rights reserved
PII SOOO3-2670(96)00377-7
168 M. Del Carlo, M. MascidAnalytica Chimica Acta 336 (1996) 167-174
using gas chromatography equipped with an electron capture detector, although other detecting strategies (e.g. mass spectrometry) are used [7]. These techni-
ques require expensive instruments which are not suitable for on-site analysis, and usually the analysis
procedure is time consuming. Immunochemical techniques are envisaged for
application as rapid and simple analytical tools that
can be used on-site for screening of samples prior to further laboratory analysis; furthermore, in many cases simple procedures can be developed that can be performed by non-specialists [8]. Within this class of techniques, the enzyme linked immunosorbent assay (ELISA) is a well established analytical method which combines the extreme specificity of antigen/ antibody recognition with the high amplification
capability of enzymatic reactions [9]. The great interest in ELISA as a quantitation method for environmental analysis is shown by the increasing
number of publications on immunoassays for pesti- cide and industrial pollutants detection [ 10,111. Immunochemistry based methods for PCB analysis have been described in the literature ranging from the classical use of radioimmunoassay (RIA) [ 121 to the use of liposome based immunoassay [ 131. There are also commercially available PCB test kits [Millipore, Ensys, and Ohmicron] based on spectrophotometric
determination of the enzymatic product. Their major limitation, as a screening test, is the high cost for
analysis, which is still estimated at $25-50 per sample (as obtained from the manufacturers litera- ture). The present approach attempts to introduce electrochemical measurements in PCB detection as a
first step in the development of an electrochemical disposable immunosensor, based on screen printing technology.
Horseradish peroxidase (HRP) is one of the most commonly used enzyme label for immunoassay. The
enzyme activity can be measured either by absor- bance in the visible spectrum [14,15], fluorescence [ 161, or electrochemistry [ 171.
The capability of HRP to directly exchange
electrons with a carbon electrode when the enzyme is immobilized onto that material was first described by Yaropolov [ 181. Some authors [ 19,201 have used this ‘mediatorless’ electron exchange to construct HRP enzyme electrodes for detection of low levels of peroxide at 50mV vs. Ag/AgCl. The amperometric
detection of HRP used as an enzyme label in immunoassays needs the use of an electron mediator which is able to shuttle electrons between the
oxidized enzyme and the cathodically polarized working electrode, as the enzyme is physically far
from the electrode. Ferrocene and ferrocene deriva- tives have been employed for this purpose with various electrode materials [2 1,221. The mediator
used in this work, ferroceneacetic acid, was stable at pH 7.4 in accordance with the stability conditions of
the enzyme [23]. This paper reports the optimization of ferrocenea-
cetic acid as a substrate for HRP, and the develop- ment of a microtitre plate based on competitive ELISA for PCB (Aroclor 1260), coupled with amperometric detection. The format of the assay is the competitive one. To immobilize the antigen onto
the solid phase, a conjugate of PCB with gelatin was used. In a competition between the bound and the sample antigens for the anti-PCB, IgG was carried
out and finally the extent of the competition was evaluated using a HRP labelled secondary antibody.
The reaction product was determined by flow injection analysis (FIA).
The PCB mixture chosen as analyte was Aroclor 1260. A PCB mixture with a similar congener profile
has been used in the Italian electricity plants and represents the contamination present in those polluted
sites.
2. Experimental
2. I. Chemicals
The Aroclor 1260 was supplied by ENEL (Brindi- si). The PCB-gelatin conjugate was provided by the Department of Experimental Medicine, University Tor Vergata, Rome, Italy; Aroclor 1260 was used for the protein conjugation. The preparation of the PCB-
gelatin conjugate consisted in the carboxylation of the PCB mixture by treatment with acetic anhydride in the presence of SnC14 as catalyst, followed by purification by flash-chromatography. The purified aldehydes were finally oxidized using bromine to obtain a carboxylic group. The latter was crosslinked with gelatin (molar ratio 10: 1) using carbodiimide under acid conditions. The polyclonal antibodies
M. Del Carlo, M. MascirdAnalytica Chimica Acta 336 (1996) 167-I 74 169
(IgG), against the polychlorinated biphenyls, were
from Bioreclamation (New York); they were raised in chicken against the whole spectrum of PCB con- geners and information about their congeners speci- ficity was not supplied by the producer. The secondary HRP labelled antibody against chicken
IgG, HRP and ferroceneacetic acid were from Sigma (Milan), microtitre plates were purchased from
Corning (New York). Skimmed milk was acquired from a local shop.
All the other reagents were analytical grade and were from Merck (Milan).
2.2. Cyclic voltammetrq
Cyclic voltammetry (CV) of ferroceneacetic acid (500pM) was carried out with an AMEL 433-
electrochemical analyzer (Amel, Milan), using 1OOmM phosphate buffer at pH 7.4, containing
10 mM NaCl as an electrolyte phosphate buffer saline (PBS). The voltammetric cell consisted of the
working and reference electrode blocks, the BAS Unijet cell (Bioanalytical System), which are a glassy carbon (id. 3 mm) and an Ag/AgCl electrode, respectively, and the circuit was completed with a Pt counter electrode. The scan rate was 5 mV s-‘.
2.3. Amperometric FIA hydrodynamic voltammetv
The aforementioned BAS Unijet Cell, was used in combination with an AMEL 599 amperometric
detector and connected with an AMEL chart recorder. The FIA peak currents were measured. Solutions
were injected using a glass syringe and a Rheodyne
7125 Valve equipped with a 5 ~1 sample loop. The flow rate (75 ul mini’) was driven by a peristaltic pump (Gilson). In the hydrodynamic voltammetry experiment, the peak currents were measured at successive potentials in the +lOO mV to -5OOmV range vs. Ag/AgCl. In all the other FIA experiments,
the potential was -3OOmV vs. the same reference electrode.
2.4. Substrate stability
Using the foregoing electrochemical arrangement, the stability of the 1 mM hydrogen peroxide solution containing 500 uM ferrocene acetic acid was tested at
three different pH in PBS (7.0, 7.5, 8.0). The
hydrogen peroxide solution was freshly prepared for each measurement and after the addition of the mediator the stability was evaluated. Measurements were made in triplicates.
2.5. Optimizution of HRP substrate
A 3mm i.d. glassy carbon working electrode was in combination with an AglAgCl and a Pt counter electrode, and at the applied potential of -3OOmV
vs. Ag/AgCl. The electrochemical measurements were performed with an AMEL 433-electrochemical analyzer (Amel, Milan). Three different concentra- tions of ferroceneacetic acid (25, 100, 500uM) were evaluated for hydrogen-peroxide solutions in the 1 O-
2000uM range. The procedure was as follows: the circuit was switched on and after 3min the current reached a stable value (noise <5nA) in a stirring
solution, the HRP was added (0.1 U ml-‘) and the reaction monitored over a period of 2 min; the initial
rate (dlldt) was extrapolated by a linear regression, the current data collected at fixed times within 20s after enzyme addition.
2.6. Electrochemical determination of HRP
A calibration curve for HRP was obtained using an enzyme/substrate (1 mM H202, 500 uM ferrocenea- cetic acid) and incubation time of 30min. All the
other conditions were the same as described in Section 2.3.
2.7. Amperometric immunoassay for PCB
The 96 well polysterene microtitre plate was coated with 1OOul of PCB-protein conjugate diluted to 20 ug ml-’ in carbonate buffer (0.1 M pH 9.6 KC1 1 M) for 16 h at 4°C. The unspecific binding was
blocked by 2 h treatment at 25°C with 1% milk in a coating buffer, containing 0.05% Tween 20. The plate was then washed three times with PBS. The PCB
standards, prepared with 5% methanol for their solubilization, were allowed to incubate with 4ugml-’ of anti-PCB IgG in PBS for 1Omin and
then 100 ~1 of each calibration standard was added to each well. The competition reaction was allowed to proceed for 50min, after which period of time the
170 M. Del Carlo, M. h4ascidAnalytica Chimica Acta 336 (1996) 167-174
plate was washed thrice with PBS containing 0.1% Tween 20, the secondary HRP labelled antibody was added and incubated for 1 h. After the final washing
procedure (PBS, Tween-20), the electrochemical
substrate was added and 30min later, the enzymatic product was detected by amperometric flow injection
analysis.
3. Results and discussion
1000 ,
:‘P 600 _ . . . . .___ <...- ,_._ _ ____ __.._ -- . . . . -.-..--..
$ 0 _ .__.__.._. _ ._.-.- ------
_ .__._.. ~..__.,.._...
5
-7 c ~
-500 -,
3.1. Amperometric determination of peroxidase
activity
Peroxidase is often used as enzyme label both in calorimetric and fluorimetric immunoassays, its activity is determined by measuring the signal which
is produced (either a colour change or a fluorescent emission) by a co-substrate molecule in the perox- idase reaction:
H202 + Cos H2 + 2H20 + Cos
This reaction can be used for electrochemical detection of HRP activity, where the co-substrate
molecule serves as electron mediator transferring electrons from one electrode to the oxidized catalytic site of the enzyme according to the following
scheme:
The suitability of the investigated electron mediator (ferroceneacetic acid) was evaluated by cyclic voltammetry in phosphate buffer (pH 7.4 1OmM NaCI). As expected for a ferrocene derivative, the voltammogram in Fig. 1 shows that the reac-
tion is almost perfectly reversible (Zpa/Zpc=l. l), this means a rapid exchange of electrons at the working electrode; and that an electron is involved in the process (the actual separation between the peak potentials was 62mV) thus ensuring the capability of the proposed mediator to measure HRP activity. The cathodic peak potential was OmV.
-woo 7 400 0 200 400 600
ElmV vs Ag/AgCi
Fig. 1. DC cyclic voltammograms of ferroceneacetic acid 500 PM
in PBS lOOmM, NaCl lOmM, pH 7.4. The scan rate was 5 mV s-’
in the range -600 to +600mV vs. Ag/AgCl. Ipa: anodic peak
current, Ipc: cathodic peak current.
In order to establish the working potential, a FIA hydrodynamic voltammogram for the mediator solu- tion was measured at potentials in the +lOO to -500 mV range, in steps of 50mV. Mediator oxidation was observed for potentials ranging between +lOO mV and -200 mV (Fig. 2). For potential values below -300 mV, the cathodic current became independent of the applied potential, there-
fore this value was chosen as working potential. The spontaneous oxidation of ferroceneacetic acid
in presence of I mM Hz02 was evaluated with FIA procedure. Fig. 3 shows the results of stability experiments performed at three different pH: 7.0, 7.5, 8.0. At more acid pH the H202 readily oxidizes the ferroceneacetic acid in a few minutes. Therefore, as a compromise between the stability of ferrocenea- cetic acid in the presence of H202 at room temperature and the HRP activity, pH 7.5 was chosen as working pH. In these conditions, the mediator and hydrogen peroxide react in the first 10 min after H202 was added, then the reagents were stable for at least
the following 50 min. The slopes of the linear portion of the current,
recorded during the initial 20s was considered in order to establish the optimum concentration ratio of mediator and hydrogen peroxide. Fig. 4 shows the recording for 500uM mediator and 500 uM hydrogen peroxide, enzyme concentration being 0.1 U ml-‘.
M. Del Carlo, M. MascidAnalytica Chimica Acta 336 (1996) 167-174 171
Fig. 2. Hydrodynamic voltammogram for (0) PBS, the support-
ing electrolyte and (0) ferroceneacetic acid 500 uM in PBS using
FIA. The injection loop was 5 ~1, the flow rate was 75 ~1 min-‘.
The scan was in the 1OOmV to --500mV range, each step was
50 mV; all the potentials were vs. Ag/AgCl.
Wmin
Fig. 3. Stability evaluation for peroxidase substrate (ferroceneace-
tic acid .500uM, 1 mM HaOa) over 60min using FIA at three
different pH, (0) pH 8,0, (0) pH 7.4, (A) pH 7.0. The injection loop was 5 ~1, the flow rate was 75 ul mine’, and the injections were
performed every 2 min in triplicates.
Fig. 5 shows the slope as a function of concentra- tions of mediator and hydrogen peroxide.
The enzyme activity increased up to 0.5 mM with the hydrogen peroxide concentration. For hydrogen peroxide concentrations higher than 1 mM, the current initially decreased, possibly due to enzyme inhibition. Therefore, the optimized substrate/media- tor concentrations were 1 mM hydrogen peroxide, and 500 pM ferroceneacetic acid in PBS.
Fig. 4. Time dependent current change after the addition of HRP
(final enzyme concentration 0.1 Urn1 ‘). The current data were
collected by computer during the initial 20s of the enzymatic
reaction. The conditions of the present experiment were: 5OOuM
ferroceneacetic acid, 500 uM hydrogen peroxide, stirring speed
300 rpm, enzyme concentration 0.1 U ml ‘. The slope of the linear
regression was used for the evaluation of the optimal hydrogen
peroxide/ferroceneacetic acid concentrations as shown in Fig. 5.
20 -
Fig. 5. The dependence of the current generation (time change of
current after the addition of HRP, final concentration 0.1 U ml-‘)
on hydrogen peroxide concentrations measured with glassy carbon
working electrode polarized at -300mV vs. Ag/AgCl, the
ferroceneacetic acid was used at (A) 25, (0) 100, and (m)
500 PM.
Using these parameters the activity of HRP was evaluated. Fig. 6 shows a typical calibration curve for HRP in the 10~‘2-10-7M range. With an incubation of 30min, the calculated detection limit was
172 M. Del Carlo, M. MasciniLAnalytica Chimica Acta 336 (1996) 167-I 74
1800 -
1600 -
1400 -
1200 -
-
P
1000
zz 800 -
600 -
400 -
200 -
H RP (moUL)
ot--ft- 1 I I I I I
0 le-12 le-11 le-10 le-9 le-8 le-7
HRP (mol/L)
Fig. 6. Calibratrion curve for HRP determination using the proposed substrate, using FIA. The enzyme/substrate incubation time of 30min
showed to be suitable for detection of low levels of HRP (lo-“M).
2.6 x lO_” M, and was evaluated by the linear regression equation in the 10-‘2-10~10M concentra- tion range [y=8.3x lO”x+17.3, ?=0.978], using the signal of the blank solution (O.OM HRP) plus three times its standard deviation [15+(3x 1.5) nA]. The highest sensitivity was in the 10~‘“-10-8M range.
The optimized substrate was finally used in the electrochemical enzyme immunoassay for Aroclor
1260. The calibration curve in the 0.1-50 yg ml-’
range (Fig. 7) was obtained by evaluating the peak current of single injection of the enzymatically produced ferricinium ion. Six measurements were carried out for each standard. The blank (0 ug ml-’ standard) response was 325f29 nA, and the ICse was estimated to 1 ugml-‘. The lowest detectable cali- brator was estimated to be 0.1 ug ml-’ which produced a signal -10% below the blank signal. The within assay reproducibility was lower than 10% for the all range.
4. Conclusion
The use of ferroceneacetic acid as a mediator for the electrochemical detection of HRP was investi- gated and optimized with respect to substrate concentration, stability and suitability for the detec- tion of low levels of HRP. The suitability of the HRP detection system and its use in electrochemical
immunoassay for PCB was here demonstrated. The
assay performance was comparable in terms of detection limits with the commercially available kits for PCB detection. The overall assay time (2 h and 30min) was longer than the time reported for these test kits (40min), though a shorter assay time can be obtained by reducing the incubation times; this may possibly lead to a reduced assay sensitivity. The optimized detection system can now be used with other immunochemical systems using peroxidase as the enzyme label. Future work will be carried out to
M. Del Carlo, M. Mascini/Analytica Chimica Acta 336 (3996) 167-174 173
350
300
250
200
P :
150
100
50
0 I I I I I I I I
0 0.1 0.5 1.0 5.0 10.0 50.0
Aroclor 1260&g ml -’
Fig. 7. The calibration curve of the electrochemical immunoassay for Aroclor 1260. The enzyme reaction time was 30 min.
couple the presented optimized system with dispo-
sable electrochemical sensors already developed in our laboratory [24,25].
I41
161
Acknowledgements [71
This work was supported by the Environment and Climate Program from the Commission of the European Community (Contract no EV5V-CT94- 0407).
ISI
[91 [lo1 1111
[I21
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