Short communication
A new membrane electrode detection of antibody C. Ran-Minh, Laboratoire de Biotechnologie,
for the
P. C. Pandey*+ & 0. Chavanne Ecole des Mines, 42023 Saint-Etienne Cedex 2, France
(Received 12 September 1990; revised version received 19 December 1990; accepted 10 May 1991)
Ahstnet: A new membrane electrode sensitive to specific antibody is described which incorporates dinitrophenyl antigen in polyvinyl chloride matrix membrane on to the surface of a solid-state graphite-loaded epoxy-resin electrode. The sensitivity of the electrode is based on the ionophoric property of the dini~phenyl antigen. Response curves for the potassium ion and its specific antibody are reported. Keywords: membrane electrode, antibody detection, potentiometry.
There are a large number of reports on the measu~ment of antibodies (Solsky & Rechnitz, 1979, 1981; Connell etai., 1983; Keating & Rechnitz, 1983, 1984; Bush & Rechnitz, 1987) based on the incorporation of antigen-ionophore conjugate/ionopho~c antigens in polyvinyl using chloride (PVC) matrix membrane conventional ion-selective electrode bodies and reference solution. The sensitivity of the electrode to the specific antibody is based on the ionophoric character of the membrane material and on the selective binding of the antibody to the antigens. Based on this argument, at least two
*Permanent address: Department of Chemistry, Banaras Hindu University, Vamnasi-221005, India. +To whom correspondence should be addressed.
categories of antibody-sensitive membrane electrode can be designed by incorporating (a) antigen-ionophore conjugate and (b) ioncarrier antigens (Bush & Rechnitz, 1987),in PVC matrix membrane. The antigens which are not ion-carriers require conjugation to an ionophore to obtain an antibody response. In this paper we report a new antibodysensitive electrode without reference solution. The membrane electrode is designed by incorporating dinitrophenyl (DNP) antigen in PVC matrix membrane on to the surface of a graphite-loaded epoxy resin electrode, since DNP has affinity for potassium ion (IUPAC, 1979). The use of solid-state graphite-loaded epoxy resin electrode eliminates the need for a reference solution with the excellent recovery of baseline potential. Response curves for the potassium ion and antiserum to DNP are reported.
0956-5663/92/$05.00 0 1992 Elsevier Science Publishers Ltd.
147
C. Tran-Minh et al.
Biosensors & Bioelectronics
EXPERIMENTAL
electrode was placed in a reaction cell containing 2 ml of working buffer (Tris-HCI 0.05 M, pH 7.4) and the baseline potential was recorded, Following establishment of the steady baseline potential, varying concentrations of potassium ion were added and the steady-state potential was recorded. For measurements of the DNP antibody, first the appropriate concentration of the potassium ion was selected for obtaining maximum response since the response of the membrane electrode to its antibody is dependent on the potassium ion concentration. Buffer (O-05 M TrisHCl, pH 7.4) containing the appropriate concentration of potassium ion (2.0 X 10m3M) was used as background solution. The membrane electrode was placed in a reaction cell containing 2 ml of background solution and the baseline potential was recorded. Following the establishment of baseline potential, varying concentrations of antibody solution in 0.05 M Tris-HCl buffer pH 7.4 were injected and the steady-state potential was recorded. After an antibody measurement, the baseline potential was re-established when the membrane electrode was placed in 25 ml of background solution. The steady value was reached within lo-15 min.
Materials DNP antigen and antisera to the DNP group were obtained from Sigma Chemical Co.; PVC powder and anhydrous tetrahydrofuran were obtained from Aldrich Chemical Co.; trioctyl phosphate and graphite fine powder were obtained from Merck. Preparation of graphite-loaded epoxy-resin electrode Epoxy resin (Ciba-Geigy, AY 103 and HY 956) was thoroughly mixed with graphite powder (Merck graphite fine powder, < 50 pm) and filled inside a PVC tubing (i.d. 4 mm) containing a Pt disc of the same diameter. The length of the graphite-loaded epoxy column was about 56 mm above the Pt disc. The loaded column was dried at 95°C for 12 h. The reverse of the Pt disc was connected to Pt wire using silver-loaded epoxy resin. A hole of O-2 mm depth was drilled inside the dried resin. Preparation of solid-state PVC matrix DNP antigen-based antibody electrode For the construction of antibody-sensitive electrode, a 4% solution of PVC (w/v) was made in anhydrous tetrahydrofuran containing 10 &ml DNP antigen and 15% (w/v) trioctyl phosphate. Care was taken to ensure the complete dissolution of PVC and DNP in the solvent. Ten microlitres of the solution was poured into the graphite-loaded epoxy hole. The electrode was then allowed to dry at room temperature overnight under controlled drying conditions.
RESULTS AND DISCUSSION Figure 1 shows the potassium ion response of the solid-state PVC matrix DNP-based antibody
Measurement of the potential Potential measurements using the membrane electrode were made in a cell with a working volume of 5 ml, equipped with magnetic stirrer and thermostated at 25°C using an ultrathermostat K5 Colora Messtechnik, GMBH LORCHI WURTT. The potential was recorded with a Radiometer PHM 64 pH meter connected to a Sefram SRD no. 429 recorder. The reference electrode was a Radiometer calomel electrode (K401) containing Tris-HCl buffer (O-05 M, pH 7.4) as internal filling solution. The membrane 148
1O-2
1o-3 Potassium
Ion
[MMI L]
Fig. 1. Potassium ion response of solid-state PVC matnk antibody electrode in 0.05 M Tris-HCI buffer pH 7.4 at 25°C containing (a) 01 pg DNP and (b) 0.3 pg DNP
Biosensors & Bioelectronics
Membrane electrode for detection of antibody
electrode. The response of the DNP antigen to potassium ion is due to its afftnity to K+ (IUPAC, 1979). This result supports the observation of Bush & Rechnitz (1987). The slope of the response curve became greater when the DNP concentration in the PVC matrix membrane was increased (Fig. 1). The membrane electrode is also sensitive to other ions (e.g. Na+); however, K+ yields the greatest response. The response of the membrane electrode is also sensitive to pH. However, in order to simulate physiological conditions, all the experiments were performed at pH 7.4. The response of the membrane electrode to DNP antibody is due to the ionophoric character of DNP. It is supposed that the potentiometric response is due to the selective binding of the antibody to the ionophoric site of the antigen, and this can be confirmed by performing competitive binding experiments (Solsky & Rechnitz, 1981; Keating & Rechnitz, 1984, Bush & Rechnitz, 1987). Figure 2 shows the response of the membrane electrode to DNP antibody in 0.05 M
60 Antibody
concentration
[pg I ml]
Fig. 2. Antibody response of solid-state PVC matrix DNPbased membrane electrode containing 01 pg DNPantigen. The measurements were made in a05 M Tris-HCI buffer containing 2 X 10-j M KC1 at 25°C.
Tris-HCl buffer pH 7.4 containing 2.0 X 10m3M potassium ion. This response was obtained when the amount of DNP antigen in PVC matrix membrane was O-1pg. The response was less when the DNP concentration in the PVC matrix membrane was increased. This is in accordance with the earlier observation of Keating & Rechnitz (1984). Potentiometric responses within the 550 pg ml-’ antibody concentration range were found to be fully reversible, with excellent recovery of baseline potential. The time required for attaining steady-state potential after the addition of antibody was found to be 4-10 min. The response time was decreased by l-2 min after several sets of antibody measurement. The baseline potential was re-established after antibody measurement by placing the membrane electrode in 25 ml of background solution. Approximately lo-15 min were required for steady baseline potential to be reached after placing the membrane electrode in the background solution. REFERENCES Bush, D. L. & Rechnitz. G. A. (1987). Antibody of polymer membrane electrodes response incorporating antigenic ionophore. J Membrane Sci., 30, 313-22. Connell, M. Y.. Sanders, K. M. &Williams, R. L. (1983). Electroimmunoassay: a new competitive proteinbinding assay using antibody-sensitive electrode. Biophys. 1. 44, 123. Keating, M. Y. & Rechnitz, G. A. (1983). Cortisol antibody electrode. Analyst (London), 108, 764. Keating. M. Y. & Rechnitz, G. A. (1984). Potentiometric digoxin antibody measurements with antigenionophore based membrane electrode. Anal. Chem., 56, 801-6. IUPAC (1979). Stability Constants of Metal-Zon Complexes, Part B. Pergamon Press, New York. Solsky, R. L. & Rechnitz, G. A. (1979). Antibodyselective membrane electrodes. Science, 204,1308. Solsky, R. L. & Rechnitz. G. A. (1981). Preparation and properties of an antibody-selective membrane electrode. Anal. Chim. Acta, 123, 135-41.
149
A new membrane electrode detection of antibody C. Ran-Minh, Laboratoire de Biotechnologie,
for the
P. C. Pandey*+ & 0. Chavanne Ecole des Mines, 42023 Saint-Etienne Cedex 2, France
(Received 12 September 1990; revised version received 19 December 1990; accepted 10 May 1991)
Ahstnet: A new membrane electrode sensitive to specific antibody is described which incorporates dinitrophenyl antigen in polyvinyl chloride matrix membrane on to the surface of a solid-state graphite-loaded epoxy-resin electrode. The sensitivity of the electrode is based on the ionophoric property of the dini~phenyl antigen. Response curves for the potassium ion and its specific antibody are reported. Keywords: membrane electrode, antibody detection, potentiometry.
There are a large number of reports on the measu~ment of antibodies (Solsky & Rechnitz, 1979, 1981; Connell etai., 1983; Keating & Rechnitz, 1983, 1984; Bush & Rechnitz, 1987) based on the incorporation of antigen-ionophore conjugate/ionopho~c antigens in polyvinyl using chloride (PVC) matrix membrane conventional ion-selective electrode bodies and reference solution. The sensitivity of the electrode to the specific antibody is based on the ionophoric character of the membrane material and on the selective binding of the antibody to the antigens. Based on this argument, at least two
*Permanent address: Department of Chemistry, Banaras Hindu University, Vamnasi-221005, India. +To whom correspondence should be addressed.
categories of antibody-sensitive membrane electrode can be designed by incorporating (a) antigen-ionophore conjugate and (b) ioncarrier antigens (Bush & Rechnitz, 1987),in PVC matrix membrane. The antigens which are not ion-carriers require conjugation to an ionophore to obtain an antibody response. In this paper we report a new antibodysensitive electrode without reference solution. The membrane electrode is designed by incorporating dinitrophenyl (DNP) antigen in PVC matrix membrane on to the surface of a graphite-loaded epoxy resin electrode, since DNP has affinity for potassium ion (IUPAC, 1979). The use of solid-state graphite-loaded epoxy resin electrode eliminates the need for a reference solution with the excellent recovery of baseline potential. Response curves for the potassium ion and antiserum to DNP are reported.
0956-5663/92/$05.00 0 1992 Elsevier Science Publishers Ltd.
147
C. Tran-Minh et al.
Biosensors & Bioelectronics
EXPERIMENTAL
electrode was placed in a reaction cell containing 2 ml of working buffer (Tris-HCI 0.05 M, pH 7.4) and the baseline potential was recorded, Following establishment of the steady baseline potential, varying concentrations of potassium ion were added and the steady-state potential was recorded. For measurements of the DNP antibody, first the appropriate concentration of the potassium ion was selected for obtaining maximum response since the response of the membrane electrode to its antibody is dependent on the potassium ion concentration. Buffer (O-05 M TrisHCl, pH 7.4) containing the appropriate concentration of potassium ion (2.0 X 10m3M) was used as background solution. The membrane electrode was placed in a reaction cell containing 2 ml of background solution and the baseline potential was recorded. Following the establishment of baseline potential, varying concentrations of antibody solution in 0.05 M Tris-HCl buffer pH 7.4 were injected and the steady-state potential was recorded. After an antibody measurement, the baseline potential was re-established when the membrane electrode was placed in 25 ml of background solution. The steady value was reached within lo-15 min.
Materials DNP antigen and antisera to the DNP group were obtained from Sigma Chemical Co.; PVC powder and anhydrous tetrahydrofuran were obtained from Aldrich Chemical Co.; trioctyl phosphate and graphite fine powder were obtained from Merck. Preparation of graphite-loaded epoxy-resin electrode Epoxy resin (Ciba-Geigy, AY 103 and HY 956) was thoroughly mixed with graphite powder (Merck graphite fine powder, < 50 pm) and filled inside a PVC tubing (i.d. 4 mm) containing a Pt disc of the same diameter. The length of the graphite-loaded epoxy column was about 56 mm above the Pt disc. The loaded column was dried at 95°C for 12 h. The reverse of the Pt disc was connected to Pt wire using silver-loaded epoxy resin. A hole of O-2 mm depth was drilled inside the dried resin. Preparation of solid-state PVC matrix DNP antigen-based antibody electrode For the construction of antibody-sensitive electrode, a 4% solution of PVC (w/v) was made in anhydrous tetrahydrofuran containing 10 &ml DNP antigen and 15% (w/v) trioctyl phosphate. Care was taken to ensure the complete dissolution of PVC and DNP in the solvent. Ten microlitres of the solution was poured into the graphite-loaded epoxy hole. The electrode was then allowed to dry at room temperature overnight under controlled drying conditions.
RESULTS AND DISCUSSION Figure 1 shows the potassium ion response of the solid-state PVC matrix DNP-based antibody
Measurement of the potential Potential measurements using the membrane electrode were made in a cell with a working volume of 5 ml, equipped with magnetic stirrer and thermostated at 25°C using an ultrathermostat K5 Colora Messtechnik, GMBH LORCHI WURTT. The potential was recorded with a Radiometer PHM 64 pH meter connected to a Sefram SRD no. 429 recorder. The reference electrode was a Radiometer calomel electrode (K401) containing Tris-HCl buffer (O-05 M, pH 7.4) as internal filling solution. The membrane 148
1O-2
1o-3 Potassium
Ion
[MMI L]
Fig. 1. Potassium ion response of solid-state PVC matnk antibody electrode in 0.05 M Tris-HCI buffer pH 7.4 at 25°C containing (a) 01 pg DNP and (b) 0.3 pg DNP
Biosensors & Bioelectronics
Membrane electrode for detection of antibody
electrode. The response of the DNP antigen to potassium ion is due to its afftnity to K+ (IUPAC, 1979). This result supports the observation of Bush & Rechnitz (1987). The slope of the response curve became greater when the DNP concentration in the PVC matrix membrane was increased (Fig. 1). The membrane electrode is also sensitive to other ions (e.g. Na+); however, K+ yields the greatest response. The response of the membrane electrode is also sensitive to pH. However, in order to simulate physiological conditions, all the experiments were performed at pH 7.4. The response of the membrane electrode to DNP antibody is due to the ionophoric character of DNP. It is supposed that the potentiometric response is due to the selective binding of the antibody to the ionophoric site of the antigen, and this can be confirmed by performing competitive binding experiments (Solsky & Rechnitz, 1981; Keating & Rechnitz, 1984, Bush & Rechnitz, 1987). Figure 2 shows the response of the membrane electrode to DNP antibody in 0.05 M
60 Antibody
concentration
[pg I ml]
Fig. 2. Antibody response of solid-state PVC matrix DNPbased membrane electrode containing 01 pg DNPantigen. The measurements were made in a05 M Tris-HCI buffer containing 2 X 10-j M KC1 at 25°C.
Tris-HCl buffer pH 7.4 containing 2.0 X 10m3M potassium ion. This response was obtained when the amount of DNP antigen in PVC matrix membrane was O-1pg. The response was less when the DNP concentration in the PVC matrix membrane was increased. This is in accordance with the earlier observation of Keating & Rechnitz (1984). Potentiometric responses within the 550 pg ml-’ antibody concentration range were found to be fully reversible, with excellent recovery of baseline potential. The time required for attaining steady-state potential after the addition of antibody was found to be 4-10 min. The response time was decreased by l-2 min after several sets of antibody measurement. The baseline potential was re-established after antibody measurement by placing the membrane electrode in 25 ml of background solution. Approximately lo-15 min were required for steady baseline potential to be reached after placing the membrane electrode in the background solution. REFERENCES Bush, D. L. & Rechnitz. G. A. (1987). Antibody of polymer membrane electrodes response incorporating antigenic ionophore. J Membrane Sci., 30, 313-22. Connell, M. Y.. Sanders, K. M. &Williams, R. L. (1983). Electroimmunoassay: a new competitive proteinbinding assay using antibody-sensitive electrode. Biophys. 1. 44, 123. Keating, M. Y. & Rechnitz, G. A. (1983). Cortisol antibody electrode. Analyst (London), 108, 764. Keating. M. Y. & Rechnitz, G. A. (1984). Potentiometric digoxin antibody measurements with antigenionophore based membrane electrode. Anal. Chem., 56, 801-6. IUPAC (1979). Stability Constants of Metal-Zon Complexes, Part B. Pergamon Press, New York. Solsky, R. L. & Rechnitz, G. A. (1979). Antibodyselective membrane electrodes. Science, 204,1308. Solsky, R. L. & Rechnitz. G. A. (1981). Preparation and properties of an antibody-selective membrane electrode. Anal. Chim. Acta, 123, 135-41.
149
