Article pubs.acs.org/ac
Monitoring of the Enzymatically Catalyzed Degradation of Biodegradable Polymers by Means of Capacitive Field-Effect Sensors Sebastian Schusser,†,‡ Maximilian Krischer,† Matthias Bac̈ ker,†,‡ Arshak Poghossian,*,†,‡ Patrick Wagner,§,∥ and Michael J. Schöning†,‡ †
Institute of Nano- and Biotechnologies (INB), FH Aachen, Jülich, Germany Peter Grünberg Institute (PGI-8), Forschungszentrum Jülich GmbH, Jülich, Germany § Department of Physics and Astronomy, Catholic University Leuven, Leuven, Belgium ∥ Institute for Materials Research (IMO), Hasselt University, Diepenbeek, Belgium ‡
ABSTRACT: Designing novel or optimizing existing biodegradable polymers for biomedical applications requires numerous tests on the effect of substances on the degradation process. In the present work, polymer-modified electrolyte− insulator−semiconductor (PMEIS) sensors have been applied for monitoring an enzymatically catalyzed degradation of polymers for the first time. The thin films of biodegradable polymer poly(D,L-lactic acid) and enzyme lipase were used as a model system. During degradation, the sensors were read-out by means of impedance spectroscopy. In order to interpret the data obtained from impedance measurements, an electrical equivalent circuit model was developed. In addition, morphological investigations of the polymer surface have been performed by means of in situ atomic force microscopy. The sensor signal change, which reflects the progress of degradation, indicates an accelerated degradation in the presence of the enzyme compared to hydrolysis in neutral pH buffer media. The degradation rate increases with increasing enzyme concentration. The obtained results demonstrate the potential of PMEIS sensors as a very promising tool for in situ and real-time monitoring of degradation of polymers.
B
with desired properties, it is of particular importance to know about the impact of influencing factors in detail. This implies a diversity of quantities for testing due to the complexity of the environment in which the degradation may take place. To perform comprehensive studies, inexpensive analyzing techniques capable of real-time in situ monitoring of the degradation kinetics are highly appreciated. The process of degradation, which actually means the splitting of the polymer chain into shorter fragments, is challenging to measure in situ and in real-time. Common analyzing techniques predominantly utilize destructive test methods and focus on secondary effects confirming degradation, such as mass loss over time,4,8 which can be simply determined by means of analytical balances. Also gel permeation chromatography (GPC) has been used, which allows comparison of the molecular weight distribution prior and after a certain time of degradation. Unfortunately, both techniques require preparation of the sample for analysis that alters the polymer’s constitution. As a consequence, in order to achieve a time-resolved degradation profile, a larger number of samples must be prepared and processed, equal in their
iodegradable polymers have become very important for the field of medical applications. Their particular nature to erode in a biological environment has led to a large number of scenarios that take advantage of this feature. This includes implants that do not need an extra surgery to remove them,1−4 scaffolds for tissue engineering,4−6 and loaded microspheres for drug delivery.7,8 So far, aliphatic polyesters, including poly(glycolic acid) (PGA), poly(lactic acid) (PLA), and poly(caprolactone), are the dominating ones in the biomedical field.1,9 This polymer family is considered as biocompatible and comprises an ester bond in the backbone that is cleaved by hydrolysis. However, even though these polymers have been used for several applications, their applicability is still limited. For instance, in the case of PLA and PGA, several studies have been reported on inflammatory or foreign body reactions.10 The assumed origin of this side effect is the release of acidic products as a result of the degradation process of these types of polymer.6,11,12 To extend the functional capabilities of biodegradable polymers, the development of new materials is inevitable. Associated to this, a large number of studies is necessary in order to characterize new materials, especially, since in addition to the chemical composition of the polymer backbone, different environmental issues have been identified that influence the rate of degradation (e.g., pH, ionic strength, temperature, enzyme activity).11,13−16 Thus, in terms of designing a polymer © XXXX American Chemical Society
Received: February 13, 2015 Accepted: May 27, 2015
A
DOI: 10.1021/acs.analchem.5b00617 Anal. Chem. XXXX, XXX, XXX−XXX
Article
Analytical Chemistry
can be found in refs 33 and 34. In previous experiments, capacitive field-effect electrolyte−insulator−semiconductor (EIS) sensors have been applied for the detection of pH, ionand analyte concentrations, as well as charged molecules (e.g., DNA, proteins, polyelectrolytes). In general, the functioning mechanism of these sensors is based on the effect of charge or potential changes at the electrolyte/gate-insulator interface induced by the particular analyte resulting in a parallel shift of the capacitance−voltage (C−V) curve of the original EIS sensor along the voltage axis. However, in the case of the PMEIS structure, besides interfacial potential changes, other effects, such as the changes in the coverage, thickness, or dielectric constant of the polymer layer, could also modulate the effective gate capacitance and flat-band voltage of an EIS structure and therefore can be utilized as a transducer mechanism. Figure 1 schematically shows the typical shape of the highfrequency C−V characteristic of a bare, p-type EIS sensor with
properties, but degraded for different periods of time. So far, only a few analyzing techniques have been reported applicable to study degradation in situ, among them nuclear magnetic resonance spectroscopy (NMR) and infrared spectroscopy (IR). In comparison to the techniques mentioned before, NMR and IR are technologically limited to trace only a single or at most a very few number of samples at the same time. Sensorbased techniques can help increasing the throughput in terms of the number of samples monitored. One kind of sensor technology that has been frequently used for the determination of mass load changes is the quartz crystal microbalance (QCM).17,18 Primarily employed for studying the mass load change of rigid layers according to the Sauerbrey’s equation,19 the extension toward viscoelastic films involves incorporation of dissipation effects, which can be challenging in accurate interpretation (see, e.g., Domack et al.20). Recently, a sensor system has been reported utilizing capacitive field-effect polymer-modified electrolyte−insulator− semiconductor (PMEIS) sensors for real-time and in situ degradation monitoring.21 The system allows monitoring of multiple sensors in parallel by simple multiplexing of sensors. This option provides the capability to perform studies on numerous samples in parallel and, thus, to get a more detailed insight into degradation processes and parameters by increasing the throughput of studies with little effort and at low costs. In the present work, PMEIS sensors have been used to study the enzymatic degradation of polymers using poly(D,L-lactic acid) (PDLLA) and the enzyme lipase from Rhizomucor miehei (LipaseRM) as the model system. LipaseRM is commonly used to catalyze hydrolysis of lipids in food industry: its active center attacks a number of natural fats, like vegetable oils, beef tallow, and lard oil22,23 and its specificity has also been demonstrated for both types of PLA,24 enantiomerically pure and racemic mixtures of the two enantiomers D- and L-lactic acid.
Figure 1. Typical shape of the high-frequency C−V characteristic of a bare, p-type EIS sensor with common accumulation (Acc), depletion (Dep), and inversion (Inv) regions as well as expected shifts of the C− V curve after the polymer deposition (PMEIS) and during polymer degradation. VG: gate voltage.
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common accumulation, depletion and inversion region, as well as expected shifts of the C−V curve after the polymer deposition and during polymer degradation. In general, the presence of an additional polymer layer onto the gate insulator can lead to the shift of the C−V curve of the original EIS structure along both the capacitance and the voltage axis. The shift of the C−V curve along the capacitance axis in the accumulation region in the direction of smaller capacitances is due to the additional capacitance/impedance of the polymer layer in series with the capacitance of the bare EIS structure. During progressive degradation, shifts of the C−V curve in the direction of larger capacitances can be expected. In the ideal case, if the polymer layer is completely degraded and removed from the gate surface, the C−V curve will become the same shape as for the original bare EIS structure. In the accumulation region, the PMEIS structure works as a capacitively coupled electrode. As can be seen in Figure 1, in contrast to the accumulation region, the shift of the C−V curve in the depletion region is not parallel because of the space-charge capacitance in the Si, which is among others a function of the gate voltage (VG) applied to the PMEIS structure and the possible potential changes at the gate/electrolyte interface after the polymer deposition as well as during the polymer degradation. Therefore, any changes in the polymer resistance/capacitance as well as in the interfacial potential induced by the polymer degradation will modulate the global capacitance/impedance of the PMEIS structure, which can be used as an indicator for polymer degradation. Thus, in contrast to polymer-covered conductive-electrode devices,35−37
MATERIALS AND METHODS Polymer Material. As a model polymer, commercial PDLLA with a syndiotactic order of the two enantiomers Dand L-lactic acid, a molecular mass in the range of Mw = 10 000−18 000 Da and a free carboxylic acid as the end group was used (RESOMER R 202 H, Evonik Röhm GmbH, Germany). The deposition of the polymer layer on the sensor structure was performed in analogy to the procedure described in previous works.21,25−27 The polymer was solved in methyl ethyl ketone with a concentration of 96 mg/mL and was applied to the sensor surface by means of a spin-coating process. The thickness of the prepared polymer layers was measured using a subset of sensor chips, where part of the polymer layer was scraped off and the height difference of the layer was determined by means of profilometry. The average thickness was d = 488 ± 23 nm and was considered as representative for the whole batch of sensor chips. Sensor Structure and Measurement Setup. The PMEIS sensors consist of a Al−Si−SiO2−Ta2O5 layer structure (p-Si with a resistivity of ρ = 5−10 Ω cm, 30 nm thermally grown SiO2, 60 nm Ta2O5, 300 nm Al as rear-side contact) with a size of 10 mm × 10 mm. The Ta2O5 films were prepared by thermal oxidation of Ta in an oxygen atmosphere.28 In contrast to SiO2gate field-effect devices,29,30 the Ta2O5-gate devices exhibit a small signal drift (
Monitoring of the Enzymatically Catalyzed Degradation of Biodegradable Polymers by Means of Capacitive Field-Effect Sensors Sebastian Schusser,†,‡ Maximilian Krischer,† Matthias Bac̈ ker,†,‡ Arshak Poghossian,*,†,‡ Patrick Wagner,§,∥ and Michael J. Schöning†,‡ †
Institute of Nano- and Biotechnologies (INB), FH Aachen, Jülich, Germany Peter Grünberg Institute (PGI-8), Forschungszentrum Jülich GmbH, Jülich, Germany § Department of Physics and Astronomy, Catholic University Leuven, Leuven, Belgium ∥ Institute for Materials Research (IMO), Hasselt University, Diepenbeek, Belgium ‡
ABSTRACT: Designing novel or optimizing existing biodegradable polymers for biomedical applications requires numerous tests on the effect of substances on the degradation process. In the present work, polymer-modified electrolyte− insulator−semiconductor (PMEIS) sensors have been applied for monitoring an enzymatically catalyzed degradation of polymers for the first time. The thin films of biodegradable polymer poly(D,L-lactic acid) and enzyme lipase were used as a model system. During degradation, the sensors were read-out by means of impedance spectroscopy. In order to interpret the data obtained from impedance measurements, an electrical equivalent circuit model was developed. In addition, morphological investigations of the polymer surface have been performed by means of in situ atomic force microscopy. The sensor signal change, which reflects the progress of degradation, indicates an accelerated degradation in the presence of the enzyme compared to hydrolysis in neutral pH buffer media. The degradation rate increases with increasing enzyme concentration. The obtained results demonstrate the potential of PMEIS sensors as a very promising tool for in situ and real-time monitoring of degradation of polymers.
B
with desired properties, it is of particular importance to know about the impact of influencing factors in detail. This implies a diversity of quantities for testing due to the complexity of the environment in which the degradation may take place. To perform comprehensive studies, inexpensive analyzing techniques capable of real-time in situ monitoring of the degradation kinetics are highly appreciated. The process of degradation, which actually means the splitting of the polymer chain into shorter fragments, is challenging to measure in situ and in real-time. Common analyzing techniques predominantly utilize destructive test methods and focus on secondary effects confirming degradation, such as mass loss over time,4,8 which can be simply determined by means of analytical balances. Also gel permeation chromatography (GPC) has been used, which allows comparison of the molecular weight distribution prior and after a certain time of degradation. Unfortunately, both techniques require preparation of the sample for analysis that alters the polymer’s constitution. As a consequence, in order to achieve a time-resolved degradation profile, a larger number of samples must be prepared and processed, equal in their
iodegradable polymers have become very important for the field of medical applications. Their particular nature to erode in a biological environment has led to a large number of scenarios that take advantage of this feature. This includes implants that do not need an extra surgery to remove them,1−4 scaffolds for tissue engineering,4−6 and loaded microspheres for drug delivery.7,8 So far, aliphatic polyesters, including poly(glycolic acid) (PGA), poly(lactic acid) (PLA), and poly(caprolactone), are the dominating ones in the biomedical field.1,9 This polymer family is considered as biocompatible and comprises an ester bond in the backbone that is cleaved by hydrolysis. However, even though these polymers have been used for several applications, their applicability is still limited. For instance, in the case of PLA and PGA, several studies have been reported on inflammatory or foreign body reactions.10 The assumed origin of this side effect is the release of acidic products as a result of the degradation process of these types of polymer.6,11,12 To extend the functional capabilities of biodegradable polymers, the development of new materials is inevitable. Associated to this, a large number of studies is necessary in order to characterize new materials, especially, since in addition to the chemical composition of the polymer backbone, different environmental issues have been identified that influence the rate of degradation (e.g., pH, ionic strength, temperature, enzyme activity).11,13−16 Thus, in terms of designing a polymer © XXXX American Chemical Society
Received: February 13, 2015 Accepted: May 27, 2015
A
DOI: 10.1021/acs.analchem.5b00617 Anal. Chem. XXXX, XXX, XXX−XXX
Article
Analytical Chemistry
can be found in refs 33 and 34. In previous experiments, capacitive field-effect electrolyte−insulator−semiconductor (EIS) sensors have been applied for the detection of pH, ionand analyte concentrations, as well as charged molecules (e.g., DNA, proteins, polyelectrolytes). In general, the functioning mechanism of these sensors is based on the effect of charge or potential changes at the electrolyte/gate-insulator interface induced by the particular analyte resulting in a parallel shift of the capacitance−voltage (C−V) curve of the original EIS sensor along the voltage axis. However, in the case of the PMEIS structure, besides interfacial potential changes, other effects, such as the changes in the coverage, thickness, or dielectric constant of the polymer layer, could also modulate the effective gate capacitance and flat-band voltage of an EIS structure and therefore can be utilized as a transducer mechanism. Figure 1 schematically shows the typical shape of the highfrequency C−V characteristic of a bare, p-type EIS sensor with
properties, but degraded for different periods of time. So far, only a few analyzing techniques have been reported applicable to study degradation in situ, among them nuclear magnetic resonance spectroscopy (NMR) and infrared spectroscopy (IR). In comparison to the techniques mentioned before, NMR and IR are technologically limited to trace only a single or at most a very few number of samples at the same time. Sensorbased techniques can help increasing the throughput in terms of the number of samples monitored. One kind of sensor technology that has been frequently used for the determination of mass load changes is the quartz crystal microbalance (QCM).17,18 Primarily employed for studying the mass load change of rigid layers according to the Sauerbrey’s equation,19 the extension toward viscoelastic films involves incorporation of dissipation effects, which can be challenging in accurate interpretation (see, e.g., Domack et al.20). Recently, a sensor system has been reported utilizing capacitive field-effect polymer-modified electrolyte−insulator− semiconductor (PMEIS) sensors for real-time and in situ degradation monitoring.21 The system allows monitoring of multiple sensors in parallel by simple multiplexing of sensors. This option provides the capability to perform studies on numerous samples in parallel and, thus, to get a more detailed insight into degradation processes and parameters by increasing the throughput of studies with little effort and at low costs. In the present work, PMEIS sensors have been used to study the enzymatic degradation of polymers using poly(D,L-lactic acid) (PDLLA) and the enzyme lipase from Rhizomucor miehei (LipaseRM) as the model system. LipaseRM is commonly used to catalyze hydrolysis of lipids in food industry: its active center attacks a number of natural fats, like vegetable oils, beef tallow, and lard oil22,23 and its specificity has also been demonstrated for both types of PLA,24 enantiomerically pure and racemic mixtures of the two enantiomers D- and L-lactic acid.
Figure 1. Typical shape of the high-frequency C−V characteristic of a bare, p-type EIS sensor with common accumulation (Acc), depletion (Dep), and inversion (Inv) regions as well as expected shifts of the C− V curve after the polymer deposition (PMEIS) and during polymer degradation. VG: gate voltage.
■
common accumulation, depletion and inversion region, as well as expected shifts of the C−V curve after the polymer deposition and during polymer degradation. In general, the presence of an additional polymer layer onto the gate insulator can lead to the shift of the C−V curve of the original EIS structure along both the capacitance and the voltage axis. The shift of the C−V curve along the capacitance axis in the accumulation region in the direction of smaller capacitances is due to the additional capacitance/impedance of the polymer layer in series with the capacitance of the bare EIS structure. During progressive degradation, shifts of the C−V curve in the direction of larger capacitances can be expected. In the ideal case, if the polymer layer is completely degraded and removed from the gate surface, the C−V curve will become the same shape as for the original bare EIS structure. In the accumulation region, the PMEIS structure works as a capacitively coupled electrode. As can be seen in Figure 1, in contrast to the accumulation region, the shift of the C−V curve in the depletion region is not parallel because of the space-charge capacitance in the Si, which is among others a function of the gate voltage (VG) applied to the PMEIS structure and the possible potential changes at the gate/electrolyte interface after the polymer deposition as well as during the polymer degradation. Therefore, any changes in the polymer resistance/capacitance as well as in the interfacial potential induced by the polymer degradation will modulate the global capacitance/impedance of the PMEIS structure, which can be used as an indicator for polymer degradation. Thus, in contrast to polymer-covered conductive-electrode devices,35−37
MATERIALS AND METHODS Polymer Material. As a model polymer, commercial PDLLA with a syndiotactic order of the two enantiomers Dand L-lactic acid, a molecular mass in the range of Mw = 10 000−18 000 Da and a free carboxylic acid as the end group was used (RESOMER R 202 H, Evonik Röhm GmbH, Germany). The deposition of the polymer layer on the sensor structure was performed in analogy to the procedure described in previous works.21,25−27 The polymer was solved in methyl ethyl ketone with a concentration of 96 mg/mL and was applied to the sensor surface by means of a spin-coating process. The thickness of the prepared polymer layers was measured using a subset of sensor chips, where part of the polymer layer was scraped off and the height difference of the layer was determined by means of profilometry. The average thickness was d = 488 ± 23 nm and was considered as representative for the whole batch of sensor chips. Sensor Structure and Measurement Setup. The PMEIS sensors consist of a Al−Si−SiO2−Ta2O5 layer structure (p-Si with a resistivity of ρ = 5−10 Ω cm, 30 nm thermally grown SiO2, 60 nm Ta2O5, 300 nm Al as rear-side contact) with a size of 10 mm × 10 mm. The Ta2O5 films were prepared by thermal oxidation of Ta in an oxygen atmosphere.28 In contrast to SiO2gate field-effect devices,29,30 the Ta2O5-gate devices exhibit a small signal drift (
