- Email: [email protected]
A prototype biosensor based on transport proteins: electrical transducers applied to lactose permease
Sensorsand Actuators B, 13-14 (1993) 173-175
173
A prototype biosensor based on transport proteins: electrical transducers applied to lactose permease D. Ottenbachera, F. JShnigb and W. G8pel” ‘InstiMe of Physicaland TheoreticalChemisby, Univem@~of Tdbinpt, Auf der Motgenstelk 8, D-7400 TUbingen(Gemany) ?UavPlan&-Insrinttefor Biology Comtmsse
38, D-7400 Tiibingen (Gemany)
Abstract We report on tbe development of a lactose sensor based on the transport protein lactose pennease (LP) which cotransports lactose and H+ ions in a strictly stoichiometric ratio of 1:l. The sensor is realized by reconstituting LP in E. co& lipid vesicles which spread on hydrophilic oxide surfaces thereby forming supported phospholipid bilayers @PBS). Essential for the function of this biosensor is first the buffer capacity of the solution and second the small volume of water entrapped above the gate of an ion-sensitive field-effect transistor (ISPET) to monitor pH changes upon lactose changes. In the present study the influence of these two parameters on the sensor performance was investigated. Ellipsometty was used to estimate thicknesses and refractive indices of the ISFET overlayer sandwich. Thicknesses were independently determined by a biochemical tracer technique.
1. Introduction For the detection of lactose we make use of the transport protein lactose permease (LP) of the cyto-
plasmic membrane of E. cofi,which catalyses the transport of lactose into the cell [l]. Thereby the sugar transport is coupled to the proton transport in a stoichiometric ratio of 1:l [2]. To build a lactose sensor we utilize the ‘symport’ of substrate (lactose) and cosubstrate (H+), i.e. the cotransported protons are detected via a pasensitive fluorophore [3,4] or a pHIFSET [5]_ The lactose permease serves as a model system for many other transport proteins [6] to build a new type of biosensor based on molecular or ionic transport through biological membranes. We used a pH-ISFET coated with the SPB membrane which contains the lactose/H+ cotransporter LP (Fig. 1). In order to optimize this new type of lactose sensor we characterized the SPBs by a biochemical tracer technique as well as by spectral ellipsometry to obtain information about the mean thicknesses of the SPB and of the entrapped water layer and to correlate these data with the sensor performance. In the sensor test experiments, Al,03- and S&N.,ISFETs were mounted in a flow-through cell of a flow injection analysis system and covered with an SPB. First, pH step measurements were performed to determine preparation conditions under which the bilayer itself shows a minimum permeability towards protons. In the second step, vesicles including the reconstituted LP were used to prepare thin film ISFET coatings. In
0!25-4OCW3136.00
the third step, the lactose sensor was tested. In these tests, the H+/lactose cotransport was triggered by adding lactose to the medium and, subsequently, the pH change was monitored in the entrapped volume. The present study concerns the optimization of the buffer capacity by varying the buffer concentration and the small volume of water entrapped above the gate s of the ISFET by using different types of ISFETs (Al,O, and Si,N.,) with different roughnesses.
2. Experimental
The experimental set-up for spectral ellipsometry is described in ref. 7. We used a flow-through cell with flow injection analysis (FIA) for ISFET measurements as introduced in ref. 5. Lactose permease and E. coli lipid vesicles were prepared as described in ref. 5. A biochemical tracer technique was used to estimate the thickness of the lipid layer and the entrapped water layer between the lipid layer and the supports. Several supports (silicon wafers with SiO, and Al,O, and quartz slides) were coated by spreading E. coli lipid vesicles (0.2 mg/ml, 12 h exposure) including 0.5% (wt./wt.) of fluorescein phosphatidylethanolamine (FPE). After rinsing five times in pure buffer solution (20 mM Na,HPO,jNaH,PO,, 100 mM Na$O,, pH 7.3), the lipid layer was washed off with 1% (wt./vol.) sodium d6decylsulfate (SDS) solution. With a spectrofluorimeter (Perkin-Elmer MPF-3), the fluorescence intensity was measured and from this the total amount of lipid
0 1993 - Elsevier Sequoia. AU rights resewed
174
H,O. H+ , H,POa-. HPOa*-. K+. SO,+*.
waste
outer compartment drpid *
5 nm
$tiw~0.5-8nm @
thickness
Fig. 1. Schematic set-up of the lactose sensor based upon the IWET pH-transducer thickness of the entrapped water layer (2) have to be optimized.
was calculated. We repeated the coating and washing procedure using pure E. coli lipid vesicles (0.2 mg/ml) in the presence of the fluorescent dye pyranine (10 mM). From the amount of entrapped pyranine, the thickness of the water layer between the lipid layer and support was estimated.
3. Determination of the thickness of the entrapped water layer In the first tracer experiment the total amount of lipid was determined from the fluorescence intensity of the fluorescent lipids. Assuming 0.6 nm’ per lipid head-group we estimate the area of the bilayer to be a factor of 1.1 rt 0.1 larger than the area of the support. This indicates the support to be covered with one single bilayer (dlipid= 5 run). In the second set of tracer experiments pure lipid vesicles were spread on different supports in the presence of the fluorescent dye pyranine. Since we observed fluorescence after the washing procedure we conclude that the pyranine was entrapped between bilayer and support, i.e., an entrapped layer of water had formed underneath the bilayer. From the fluorescence intensity we calculated the entrapped thickness d,,,, (Table 1). These results are in line with those from independent scanning electron microscopy (SEM) showing the S&N, and SiO, surface to be smoother than the A&O3surface and therefore the entrapped volume on A&O, supports is larger than on SiO, substrates. Using refractive indices of water (1.33) and of lipids (1.44) from the literature, the ellipsometric data yield layer thicknesses which are two to three times higher if compared with the tracer results (Table 1). Using the data on water and lipid layer thicknesses from the tracer experiments to interpret the ellipsometric data, one obtains ‘strange’ refractive indices. These differences between tracer and ellipsometry results may be caused by the model assumptions in the evaluation of
principle. Tbe buffer capacity (1) and the
TABLE 1. Results from tracer experiments and ellipsometry (A =589.3 nm) on AlsOs, SiOl and quartz substrates coated with SPBs. The digits in italics indicate the ellipsometric results obtained by using the other parameters as given values (for one bilayer C&,-S nm is known from literature) Tracer
Ellipsometxy
&id (nm)
dw,,., (nm)
4, (nm)
dwcr (nm)
A1203
5
8
5 10.2
SiOt
5
0.5
8 21.6 0.5 21.6
Quartz
5
2
12
--) + + +
1.64 1.44 1.59 1.44
1.27 1.33 1.23 1.33
ellipsometric data: the model assumes sharp transitions between the water and lipid phase and therefore neglects undulations of the lipid bilayer [8] as well as the roughness of the support.
4. Sensor signals of ISFETs with different gate materials 4.1. ISFETs with Al,O, gate ISFET measurements were performed with an FIA system to test first the itiuence of the SPB on stepwise changes of the pH value (Fig. 2). The A&O,-based ISFET with SPB coating (without LP) shows a twostep kinetics where the slow component represents the proton diffusion across the SPB [5]. The quality of the coating was checked by embedding gramicidin in the SPB to short-circuit the membrane for H’ ions. The results of as-treated lipid bilayers are identical with those observed for ISFETs without SPB. Figure 3 shows sensor results from ISFETs with LP incorporated in the SPB. At low buffer concentrations the SPB/LP-coated ISFET shows a rapid response to lactose which results from the active cotransport of
175
buffer: 10mM flow rate: 0.8 mllmln
, pH-step ,
diffusion of H*
pti 7.86
A
.”
1 without SPS I.“‘,
‘..I
2
1
3
4.2. ISFETs with Sifl, gate These measurements were also performed with S&N,based ISFETs. After a stepwise change of pH no twostep kinetics occurs as observed for SPB coatings of AI,O,-ISFETs (cf. Fig. 2). Also, the response of bare and SPB/LP-coated Si$J4-ISFETs upon lactose exposure was negligible within experimental error. This may be caused by the smaller roughness of the Si,N., gate causing a smaller entrapped water volume (see Table 1) and therefore reducing the H+ enrichment in the inner compartment. We plan further measurements with ISFETs of different gate material and different roughness to check for eventual specific water interactions in the monolayer range.
time flmin) Fig. 2. Influence of the supported lipid biiayers (SPB) on stepwise changes Gf the pH value with AV,, as ISFBT response: (1) uncoated Ai,O,-ISFEZ (2) SPB-coated AI&ISFET.
Lactose:
We thank Ch. Striebel for the ellopsometric and DC H. Kiefer for the biochemical tracer experiments. The work was supported by the Bundesministerium ftir Forschung und Technologie (BMFT, project 0319325A).
OmM
20mM
Acknowledgements
References
Sensor Effect
1 W. Gopel, G. Gauglita, G. Jung and F. J&dg,
-5
1,2: 0.5 mM sodium borate buffer $4: 10 mM potassium phosphate buffer 0
2
4
6
a
10
12
time tlmin) Fii. 3. Typical sensor response upon addition of lactose (see arrows) to the ISFET gate coated with SPB/LP (1, 3) and to the bare gate (2, 4). Results (1, 2) refer to low and (3, 4) to high buffer concentrations.
lactose and protons through the LP. The bare ISFET also shows a response which is due to a changed pH value in the solution after dissolving the lactose. The difference signal in Fig 3 depends on the lactose concentration as it is monitored by an additional active H+ transport with a strong influence from the buffer concentration.
Biosensor systems based upon receptor functions, in R. D. S&mid and F. ScheUer (edr), GBFMonogmphs, Vol. 13, VCH, Weinbeim, 1989, pp. 165-177. 2 J. K Wright, R. Seclder and P. Overath, Moiecular aspects of sugar:ion cotransport, Ann. Rev. Biochem., 55 (1986) 225-248. 3 H. Kiefer. B. Klee, E. John, Y. D. Stierhof and F. Jtinig, Biosensors based on membrane transport proteins, Eioscnsors L&n&bon., 6 (1991) 233-237. 4 B. Kiee, E. John and F. Jiihnig, A biosensor based on the membrane protein lactose permease, Sensors and Actuators a 6 (1992) 376-379. D. Okenbacher. R. Kindervater, P. Gimmel, B. Kiee. F. Jahnig and W. Gi5pel, Developing biosensors with pH-ISFET transducers utilizing lipid bilayer membranes with transport proteins, Sensors and Acruutors B, 6 (1992) 192-l%. W. D. Stein, Channels, Catriers, and &ups An Introai~~fion to Membnme Tmnpmt, Academic Press, London, 1990, p. 2&t. Ch. Striebei, A. Brecht and G. Gaughtz. Characterization of biomembranes by spectral eilipsometty, surface piasmon resonance and interferometry with regard to biosensor application, Biosentors Bioek~, submitted for publication. J. N. Israelachvili and H. Wennerstrgm, Hydration or steric forces between amphiphihc surfaces?, Ln~ir, 6 (1990) 873-876.
173
A prototype biosensor based on transport proteins: electrical transducers applied to lactose permease D. Ottenbachera, F. JShnigb and W. G8pel” ‘InstiMe of Physicaland TheoreticalChemisby, Univem@~of Tdbinpt, Auf der Motgenstelk 8, D-7400 TUbingen(Gemany) ?UavPlan&-Insrinttefor Biology Comtmsse
38, D-7400 Tiibingen (Gemany)
Abstract We report on tbe development of a lactose sensor based on the transport protein lactose pennease (LP) which cotransports lactose and H+ ions in a strictly stoichiometric ratio of 1:l. The sensor is realized by reconstituting LP in E. co& lipid vesicles which spread on hydrophilic oxide surfaces thereby forming supported phospholipid bilayers @PBS). Essential for the function of this biosensor is first the buffer capacity of the solution and second the small volume of water entrapped above the gate of an ion-sensitive field-effect transistor (ISPET) to monitor pH changes upon lactose changes. In the present study the influence of these two parameters on the sensor performance was investigated. Ellipsometty was used to estimate thicknesses and refractive indices of the ISFET overlayer sandwich. Thicknesses were independently determined by a biochemical tracer technique.
1. Introduction For the detection of lactose we make use of the transport protein lactose permease (LP) of the cyto-
plasmic membrane of E. cofi,which catalyses the transport of lactose into the cell [l]. Thereby the sugar transport is coupled to the proton transport in a stoichiometric ratio of 1:l [2]. To build a lactose sensor we utilize the ‘symport’ of substrate (lactose) and cosubstrate (H+), i.e. the cotransported protons are detected via a pasensitive fluorophore [3,4] or a pHIFSET [5]_ The lactose permease serves as a model system for many other transport proteins [6] to build a new type of biosensor based on molecular or ionic transport through biological membranes. We used a pH-ISFET coated with the SPB membrane which contains the lactose/H+ cotransporter LP (Fig. 1). In order to optimize this new type of lactose sensor we characterized the SPBs by a biochemical tracer technique as well as by spectral ellipsometry to obtain information about the mean thicknesses of the SPB and of the entrapped water layer and to correlate these data with the sensor performance. In the sensor test experiments, Al,03- and S&N.,ISFETs were mounted in a flow-through cell of a flow injection analysis system and covered with an SPB. First, pH step measurements were performed to determine preparation conditions under which the bilayer itself shows a minimum permeability towards protons. In the second step, vesicles including the reconstituted LP were used to prepare thin film ISFET coatings. In
0!25-4OCW3136.00
the third step, the lactose sensor was tested. In these tests, the H+/lactose cotransport was triggered by adding lactose to the medium and, subsequently, the pH change was monitored in the entrapped volume. The present study concerns the optimization of the buffer capacity by varying the buffer concentration and the small volume of water entrapped above the gate s of the ISFET by using different types of ISFETs (Al,O, and Si,N.,) with different roughnesses.
2. Experimental
The experimental set-up for spectral ellipsometry is described in ref. 7. We used a flow-through cell with flow injection analysis (FIA) for ISFET measurements as introduced in ref. 5. Lactose permease and E. coli lipid vesicles were prepared as described in ref. 5. A biochemical tracer technique was used to estimate the thickness of the lipid layer and the entrapped water layer between the lipid layer and the supports. Several supports (silicon wafers with SiO, and Al,O, and quartz slides) were coated by spreading E. coli lipid vesicles (0.2 mg/ml, 12 h exposure) including 0.5% (wt./wt.) of fluorescein phosphatidylethanolamine (FPE). After rinsing five times in pure buffer solution (20 mM Na,HPO,jNaH,PO,, 100 mM Na$O,, pH 7.3), the lipid layer was washed off with 1% (wt./vol.) sodium d6decylsulfate (SDS) solution. With a spectrofluorimeter (Perkin-Elmer MPF-3), the fluorescence intensity was measured and from this the total amount of lipid
0 1993 - Elsevier Sequoia. AU rights resewed
174
H,O. H+ , H,POa-. HPOa*-. K+. SO,+*.
waste
outer compartment drpid *
5 nm
$tiw~0.5-8nm @
thickness
Fig. 1. Schematic set-up of the lactose sensor based upon the IWET pH-transducer thickness of the entrapped water layer (2) have to be optimized.
was calculated. We repeated the coating and washing procedure using pure E. coli lipid vesicles (0.2 mg/ml) in the presence of the fluorescent dye pyranine (10 mM). From the amount of entrapped pyranine, the thickness of the water layer between the lipid layer and support was estimated.
3. Determination of the thickness of the entrapped water layer In the first tracer experiment the total amount of lipid was determined from the fluorescence intensity of the fluorescent lipids. Assuming 0.6 nm’ per lipid head-group we estimate the area of the bilayer to be a factor of 1.1 rt 0.1 larger than the area of the support. This indicates the support to be covered with one single bilayer (dlipid= 5 run). In the second set of tracer experiments pure lipid vesicles were spread on different supports in the presence of the fluorescent dye pyranine. Since we observed fluorescence after the washing procedure we conclude that the pyranine was entrapped between bilayer and support, i.e., an entrapped layer of water had formed underneath the bilayer. From the fluorescence intensity we calculated the entrapped thickness d,,,, (Table 1). These results are in line with those from independent scanning electron microscopy (SEM) showing the S&N, and SiO, surface to be smoother than the A&O3surface and therefore the entrapped volume on A&O, supports is larger than on SiO, substrates. Using refractive indices of water (1.33) and of lipids (1.44) from the literature, the ellipsometric data yield layer thicknesses which are two to three times higher if compared with the tracer results (Table 1). Using the data on water and lipid layer thicknesses from the tracer experiments to interpret the ellipsometric data, one obtains ‘strange’ refractive indices. These differences between tracer and ellipsometry results may be caused by the model assumptions in the evaluation of
principle. Tbe buffer capacity (1) and the
TABLE 1. Results from tracer experiments and ellipsometry (A =589.3 nm) on AlsOs, SiOl and quartz substrates coated with SPBs. The digits in italics indicate the ellipsometric results obtained by using the other parameters as given values (for one bilayer C&,-S nm is known from literature) Tracer
Ellipsometxy
&id (nm)
dw,,., (nm)
4, (nm)
dwcr (nm)
A1203
5
8
5 10.2
SiOt
5
0.5
8 21.6 0.5 21.6
Quartz
5
2
12
--) + + +
1.64 1.44 1.59 1.44
1.27 1.33 1.23 1.33
ellipsometric data: the model assumes sharp transitions between the water and lipid phase and therefore neglects undulations of the lipid bilayer [8] as well as the roughness of the support.
4. Sensor signals of ISFETs with different gate materials 4.1. ISFETs with Al,O, gate ISFET measurements were performed with an FIA system to test first the itiuence of the SPB on stepwise changes of the pH value (Fig. 2). The A&O,-based ISFET with SPB coating (without LP) shows a twostep kinetics where the slow component represents the proton diffusion across the SPB [5]. The quality of the coating was checked by embedding gramicidin in the SPB to short-circuit the membrane for H’ ions. The results of as-treated lipid bilayers are identical with those observed for ISFETs without SPB. Figure 3 shows sensor results from ISFETs with LP incorporated in the SPB. At low buffer concentrations the SPB/LP-coated ISFET shows a rapid response to lactose which results from the active cotransport of
175
buffer: 10mM flow rate: 0.8 mllmln
, pH-step ,
diffusion of H*
pti 7.86
A
.”
1 without SPS I.“‘,
‘..I
2
1
3
4.2. ISFETs with Sifl, gate These measurements were also performed with S&N,based ISFETs. After a stepwise change of pH no twostep kinetics occurs as observed for SPB coatings of AI,O,-ISFETs (cf. Fig. 2). Also, the response of bare and SPB/LP-coated Si$J4-ISFETs upon lactose exposure was negligible within experimental error. This may be caused by the smaller roughness of the Si,N., gate causing a smaller entrapped water volume (see Table 1) and therefore reducing the H+ enrichment in the inner compartment. We plan further measurements with ISFETs of different gate material and different roughness to check for eventual specific water interactions in the monolayer range.
time flmin) Fig. 2. Influence of the supported lipid biiayers (SPB) on stepwise changes Gf the pH value with AV,, as ISFBT response: (1) uncoated Ai,O,-ISFEZ (2) SPB-coated AI&ISFET.
Lactose:
We thank Ch. Striebel for the ellopsometric and DC H. Kiefer for the biochemical tracer experiments. The work was supported by the Bundesministerium ftir Forschung und Technologie (BMFT, project 0319325A).
OmM
20mM
Acknowledgements
References
Sensor Effect
1 W. Gopel, G. Gauglita, G. Jung and F. J&dg,
-5
1,2: 0.5 mM sodium borate buffer $4: 10 mM potassium phosphate buffer 0
2
4
6
a
10
12
time tlmin) Fii. 3. Typical sensor response upon addition of lactose (see arrows) to the ISFET gate coated with SPB/LP (1, 3) and to the bare gate (2, 4). Results (1, 2) refer to low and (3, 4) to high buffer concentrations.
lactose and protons through the LP. The bare ISFET also shows a response which is due to a changed pH value in the solution after dissolving the lactose. The difference signal in Fig 3 depends on the lactose concentration as it is monitored by an additional active H+ transport with a strong influence from the buffer concentration.
Biosensor systems based upon receptor functions, in R. D. S&mid and F. ScheUer (edr), GBFMonogmphs, Vol. 13, VCH, Weinbeim, 1989, pp. 165-177. 2 J. K Wright, R. Seclder and P. Overath, Moiecular aspects of sugar:ion cotransport, Ann. Rev. Biochem., 55 (1986) 225-248. 3 H. Kiefer. B. Klee, E. John, Y. D. Stierhof and F. Jtinig, Biosensors based on membrane transport proteins, Eioscnsors L&n&bon., 6 (1991) 233-237. 4 B. Kiee, E. John and F. Jiihnig, A biosensor based on the membrane protein lactose permease, Sensors and Actuators a 6 (1992) 376-379. D. Okenbacher. R. Kindervater, P. Gimmel, B. Kiee. F. Jahnig and W. Gi5pel, Developing biosensors with pH-ISFET transducers utilizing lipid bilayer membranes with transport proteins, Sensors and Acruutors B, 6 (1992) 192-l%. W. D. Stein, Channels, Catriers, and &ups An Introai~~fion to Membnme Tmnpmt, Academic Press, London, 1990, p. 2&t. Ch. Striebei, A. Brecht and G. Gaughtz. Characterization of biomembranes by spectral eilipsometty, surface piasmon resonance and interferometry with regard to biosensor application, Biosentors Bioek~, submitted for publication. J. N. Israelachvili and H. Wennerstrgm, Hydration or steric forces between amphiphihc surfaces?, Ln~ir, 6 (1990) 873-876.