June 2011, Volume 2, No.

3 International Journal of Chemical and Environmental Engineering

New Impedance Measurements Based Microsystems Setup for Characterizing Biomaterial Hydrogel
* a

R. S. Bajwa-1a,b, M. Al-Halhouli -2a , Gregory Dame -3a, G.A. Urban -4a University Freiburg, Institute for Microsystem Technology, Freiburg 79110 Germany b Preston Institute of Nanoscience and Technology, Islamabad, Pakistan
*

Corresponding Author E-mail: rizwan.bajwa@hotmail.com

Abstract New design of an Impedance measurement based sensor is integrated into the standard 96 wells microtiter plate which will be used for charactering different stimulus hydrogels. The online monitoring of swelling and deswelling behavior of pH sensitive hydrogel is possible with this new system. Hydrogels of Poly-Hydroxyethyle Methacrylate Poly (HEMA) with different compositions of mixing monomers were used for measurements. These hydrogels which absorbs/desorbs solution in response to changes in surrounding environmental condition polymerized over the sensor with roughly 200m height. The pH-dependent swelling behavior of hydrogel observed in Phosphate buffer solution for pH ranging from 5 to 9. The sensor output voltage response to pH value changes with swelling and contracting of hydrogel. The reversible pH-dependent swelling results indicates that the Poly (HEMA) hydrogel under use can remain stable even after undergoing many swelling and deswelling cycles without any loss of mass or changing its mechanical properties. Keywords: pH-Sensitive, Online Monitoring, Poly (HEMA), Thin Film, swelling/deswelling.

1. Introduction
Hydrogels are three-dimensional networks of hydrophilic polymers that can absorb large account of water but remain insoluble. Three-dimensional network are usually formed by chemical cross-linking of hydrophilic polymer chains.Poly Hydroxyethyle Methacrylate is one of the well studied hydrogel due to its good swelling capabilities, non toxic and biocompatibilities [1, 2]. Hydrogels are extensively desired material in fabrication technology of sensors and actuators due to its unique swelling/deswelling behavior in response to environmental conditions [3-5]. Mainly four different types of Hydrogels are working as sensing elements of hydrogel based sensors. These four types are pHSensitive, Ions-Sensitive, Antigen Sensitive and Glucose Sensitive Hydrogels [6-8]. PH-sensitive hydrogel is synthesized in sensor under discussion. In literature wide range of pH-sensitive hydrogel based sensors available in different applications [9-14].

1.1 Hydrogel Network structure and Basic Properties

Two significant phases of hydrogel structures are solid phase and liquid phase. Cross-linker monomers make the solid portion of the network with acidic or basic groups bound to polymer chains of the network. When solution start diffusing to the hydrogel cross-linkers provide the restoring force to prevent the complete dissolving of polymer chains and the solvent. Over all gel is consist of Polymer network and absorbed solution as shown in Figure: 1

Figure 1. Structure of Hydrogel[15]

New Impedance Measurements Based Microsystems Setup for Characterizing Biomaterial Hydrogel The chains network is surrounded by both polymer network and the diffused solution as indicated from Figure: 2. the ionisable groups (basic or acidic) not completely dissociated [15] .The polymer solvent interaction cause to develop an osmotic pressure mix. This expansion due to polymer-solvent interaction is counteract elastic force elastic due to polymer-polymer interaction [16]. At hydrogel swelling equilibrium state these force are balanced which can be describe by force given below, and mobile counter ions within the hydrogel which leading to hydrogel shrinking/deswelling[17].

Hydrogels with basic groups bounded to their polymer chain shows opposite pH-sensitivity they swell in acidic solutions due to ionization of their basic groups by protonation. The swelling of hydrogels get stops with complete ionization of ionisable contents any further increase in pH only increases the ionic strength which decrease the osmotic pressure which lead to the shrinking of hydrogel see Figure 3. which shows 3different types of hydrogel behavior at different pH value.

Figure: 2. The swelling phenomena of hydrogel in Buffered pH solution[15]

1.2 Swelling/Deswelling Behavior of Hydrogels

As mentioned above the acidic or basic groups are bound with polymer chains, these groups can be ionized in the presence of aqueous solutions. Hydrogel bounded by acidic groups with their polymer chains ionized by deprotonation of in basic solution, whereas hydrogel bounded by basic groups with their polymer chains ionized by protonation in acidic solutions. This ionization of acidic and basic groups of hydrogel generates three kinds of forces which cause for the swelling and deswelling of hydrogel in aqueous solution. For example, a hydrogel bounded by acidic groups to its polymer chains will ionized in basic by deprotonation and will release H+ ions in basic solution which combine with OH- to form H2O as given in Equation (2).

Figure 3.Phase transition behavior of polyelectrolyte hydrogel. Acidic hydrogels () are ionized by deprotonation in basic solutions, which have an excess of hydroxyl groups. Basic hydrogels () swell in acidic solutions due to ionization of their basic groups by protonation. Amphiphilic hydrogels () contains both acidic and basic groups; therefore they show two phase transition[18]

1.3 Equilibrium Swelling Theory

Flory and Renher give theory [19] about the equilibrium swelling of hydrogels in term of Gibbs free energy. This thermodynamic theory state that when crosslinked hydrogels are immersed in solution and allowed to reach its equilibrium, the equilibrium degree of swelling is governed by the change in total free energy F see Equation (4) .

Eq. 2

Where F mix is the thermodynamic force of polymersolvent mixing, F el is the elastic force in polymerpolymer interaction due to expansion of polymer chains network structure, F ion is the ionic force. In the equilibrium state of swelling all these forces get balanced as describe in Equation (5).

Consequently, the density of likewise charges increases within the hydrogel, which induces electrostatic repulsion. Charge is compensated by diffusion of cations that enter the gel with another OH- to maintain charge neutrality. The increased flux of these new cations gives rise to Osmotic pressure which drives the actual swelling of hydrogel. While in acidic solution this acidic hydrogel protonate which cause in decreasing of the charge density

In this study Basic Hydrogel is synthesize with HEMA/TEGDMA/Water/Irgacure651 Which swell in acidic solution due to ionization of their basic groups by protonation. The output voltage of the sensor response to swelling deswelling behavior of

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New Impedance Measurements Based Microsystems Setup for Characterizing Biomaterial Hydrogel pH-Sensitive hydrogel in a series of buffer solution of pH value 5 to 9.

2. Simulation and Sensor Assembly

The hydrogel layer height for new 96 wells microtiter plate chip has been verified by using

(a)

(b)

Figure 4. Simulation model (a) electrodes top view (b) current density distribution at the cross section, y = 0m, CI-width = 250m, gap = 30m, CE-width = 400m and CS-width 540m.

Multiphysics Modelling and Simulation software COMSOL. The purpose of introducing hydrogel with specific height is to have two separated current regions, one for CE current limited inside the hydrogel layer and the second for CS current which passes through the hydrogel layer into the suspension region (we will not consider suspension region in this study). Three parameters CE width, CI width and insulation gap between these two electrodes play important role in controlling the current density and electric field distribution within the well. In simulation these parameters addressed such that the current density and electric field between the CI and CE electrodes decays as much as possible at the height of 200m, to prevent the CE current penetration from hydrogel layer to suspension layer. The design for 96well microtiter plate format consists of configuration of three ring shaped electrodes. The simulations performed using 2D electrostatic model of AC/DC module. Figure: 4 (a) shows the top view of 3D model well and electrode inside the well. Figure 4.1(b) shows the current density distribution at the cross section in the middle of the well. The CS-electrode is set as electrical insulation boundary in electrodes dimensions simulation because the current region limited inside the hydrogel is controlled by current between CI-electrode and CE-electrode. To see simulation influence different CE-widths remodeled, keeping two variables the gap between CI-CE electrode and CI-width fixed 30m and 250m respectively.

Figure 5. the current density decay curve between CI and CE electrodes with different CE-electrode widths.

Figure 5. shows current density decay curve with respect to height for different widths of CE-electrode (100m, 170m, 300m, 460m). The highest current density point is at 0m height which gradually decays with increasing height. The decay rate is sharper for thinner electrodes dimensions. The current density of CE-electrode for width 100m at height 200m decays 1.3% of its maximum value. From

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New Impedance Measurements Based Microsystems Setup for Characterizing Biomaterial Hydrogel simulation the best simulated dimensions, CI-electrode width is 250m, CE-electrode width is 100m, the gap between CI-electrode and CE-electrode is 30m and CSelectrode 540m.
Table 1: 1mL Poly (HEMA) composition formulations

Sample Code

HEMA vol% 84.9 83 99.9 98

TEGDMA vol% 0.1 2 0.1 2

Water vol% 15 15 0 0

Initiator wt% 0.5 0.5 1.5 1.5

2.1. Sensor Assembly 2.2.1 Chip Assembly

Thin film technology used in the fabrication of chip on Pyrex substrate. First 500nm layer of silicon nitride (SiNx) deposited on the substrate as insulation layer using Plasma Enhanced Chemical Vapor Deposition (PECVD). Physical Vapor Deposition is used for metal layers of Titanium (Ti) and Platinum (Pt). Titanium used as adhesion layer for Platinum with silicon nitride (SiNx). First Titanium layer of 20nm then Platinum layer of 100nm and again Titanium layer of 50nm. To cover the metal layers another silicon nitride layer of 500nm deposited using PECVD. Finally, Reactive Ion Etching (RIE) process is used for exposing the Platinum electrodes by etching the silicon nitride and Titanium layers.

A1 A2 B1 B2

After completion of polymerization process all wells filled with deionised water for overnight to ensure the removal of residual monomers, cross-linker and water soluble Irgacure Photoinitiator. All hydrogel samples synthesized were clear and transparent which mean there was no phase separation. This membrane thickness of about 200m was acceptable to keep the electric field between the current injection and electrolyte electrodes within the hydrogel layer.

2.2.3

Readout Circuit

The voltage divider circuit is used for measuring the current pass the microtiter plates chamber. Figure 7(b) shows the readout voltage divider circuit for one chamber. The output voltage through output resister Ro changes due to the current value which correlates to the conductivity change of the electrolyte inside the hydrogel layer. Figure 7(a) shows Solartron 1260 connection to microtiter plate chip through voltage divider readout circuit PCB.

Figure 6. Chip is integrated to Standard 96well PMMA substrate holder.

The chip is integrated to 16wells, 2 x 8-array of PMMA substrate made by the Laboratory for sensors IMTEK see Figure 6. The chip has terminals connecting pads for 8wells on each side. The hydrogel chamber of 200m height is made inside the wells. 2.2.2 Synthesis of Cross-Linked Hydrogel Four hydrogels were made of different compositions, see Table 1. Aqueous solutions were prepared by mixing different ratios of cross-linker, HEMA monomers, water and Photoinitiator. The 5L of prepared solution of hydrogel pipette over the chip integrated in standard 96well microtiter plate. Immediately chip placed in a box of transparent cover sheet and irradiated with UV light (366 nm, 14W/cm2) in Bungard Hellas Exposure Unit under continuous flushing of Nitrogen (to maintain oxygen free environment) for 35minuts.

Figure 7, 2x8-Standard microtiter plate Chip connection to Voltage divider Readout circuit PCB.

3. Experimental Methods 3.1. Impedance Measurements Setup

Impedance measurement setup consists of three interconnected parts, Solartron 1260 an impedance gain-

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New Impedance Measurements Based Microsystems Setup for Characterizing Biomaterial Hydrogel phase analyzer, a computer and standard microtiter plate chip. The computer is the controlling unit of the system through Zplot and Zview software. The computer communication with Solartron 1260 performed through GPIB-card. The standard microtiter plate is interconnected with Solartron 1260 through coaxial cables. Figure 8. system setup connection to computer. In order to study the reversibility of the swelling process, the last experiment was continued from pH-9 in reverse order by exchanging buffer solution in 96well from pH-9 to pH-5. The reversibility swelling showed that each sample get the almost same equilibrium point which was observed in forward swelling at the same pH value.

4. Results and Discussion 4.1. NaCl Measurements Results

Figure 9. shows the swelling time measured result of different formulations of Poly (HEMA) hydrogels from their try state to wet state. Figure 9(a) shows for hydrogel samples B2 and B3 with TEGDMA cross-linker ratio of 0.1% and 2% respectively, for 0.1 % TEGDMA the swelling time of hydrogel from its dry state to wet state is about 80min with 0.1M concentration of NaCl, from stable wet of 0.1M NaCl to stable wet state of 0.2M NaCl it take 20min and the same time it takes to back from 0.2M NaCl to 0.1M NaCl. The equilibrium state output signal for 0.1M NaCl before and after pipetting 0.2M NaCl is the same value which indicates that the ions for new concentration of 0.2M is properly exchanged suspension phase and hydrogel phase and hydrogel has not stored them. The swelling time and ions exchanged time were measured for 2% TEGDMA but with less swelling ratio which is due to dens crosslinking microstructure of hydrogel which created at barrier for diffusion of solute, consequently the swelling ratio is decreased.

Figure 8. System setup

3.2. NaCl Measurements

NaCl experiments were performed for studying initial characterization of hydrogels for its swelling and deswelling behavior. Two different NaCl ionic concentrations of 0.1 M and 0.2 M have been used for measuring different hydrogels swelling stability time and ions exchange time from its dry state to wet state. Further the effect of different composing compartments (crosslinker, initiator etc) on its swelling behavior is also experimentally studied. Voltage divider setup is used to monitor output voltage across the out resister of CE-electrode. The applied voltage is 300mv at 1MHz frequency. First 200l of 0.1 M solution of NaCl pipette to the well and let the hydrogel to swell till its equilibrium state and noted its swelling time from dry state to wet state, when its get stable in equilibrium state then experiment is pause and 0.1 M NaCl solution is pipette out and exchanged with 0.2 M solution. With exchange of new concentration of 0.2 M it stated again swelling and takes some time to again get equilibrium state for new concentration. This new equilibrium state used to measure the ions exchange time.

3.3.

pHMeasurements

Phosphate buffer solutions with pH values between 5 and 9 of 0.1 M ionic strength were prepared from solutions Hna2PO4 and NaH2PO4 and NaCl. The pH varied (5, 6, 7, 8 and 9) while the salt concentration remained constant (0.1 M NaCl). To study the swelling behavior of the Poly (HEMA) hydrogel in medium of different pH, First 200l buffer solution of pH-5 was pipette to standard 96well microtiter plate and allowed the hydrogel to equilibrate, when it get equilibrium pH-5 solution pipette out and pH-6 buffer solution pipette to the 96well and again hydrogel allowed to get equilibrate, similar process was performed for pH7, pH-8 and pH-9 for online monitoring of Poly (HEMA) swelling behavior in different pH buffer solution. The sensor output voltage dependent to hydrogel behavior. After each consecutive pH change hydrogel change its volume until a new equilibrium is gained.

Figure 9. The swelling time of Poly (HEMA) in 0.1M and 0.2M NaCl Solution.

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New Impedance Measurements Based Microsystems Setup for Characterizing Biomaterial Hydrogel To reduce the hydrogel dens structure new formulation is prepared with adding 15% waster see sample (A1, A2) in Table 1. With new formation of hydrogel the swelling time from dry state to wet state and ion exchange is reduced from 80min -> 25min and 20min - > 8min for 0.1% TEGDMA (A1) and about 30min and 10min for 2% TEGDMA (A2) see Figure 9(b). This significant reduction in swelling time is due to enhanced porous microstructure of hydrogel with addition of water which increased average molecular weight between the crosslinks which decrease the hydrogel resistance to solute diffusion, consequently hydrogel swell very fast. indicates that the poly (HEMA) hydrogel with this composition can remain stable even after undergoing as many swelling and deswelling cycles without any loss of mass or changing its mechanical properties.
Figure: 11 Comparison output voltage of sensor covered with

4.2. pHMeasurementsResults
Figure 4.2-1 shows output voltage of sensor in response to long time measurement in different pH value buffer solution ranging from 5 to 9 and vice versa to show output voltage for reversible swelling behavior of Poly (HEMA). As expected basic hydrogel swell in acidic groups solution due to higher degree ionization of their basic groups by protonation see Figure 3. As Measured results showing in Figure 10. At low pH, the output voltage is increasing due to higher degree of ionization inside hydrogel which lead increasing osmotic pressure making hydrogel swell, as pH increasing the degree of ionization inside hydrogel decreases, consequently output voltage deceasing. With less degree of ionization there is less osmotic pressure making the gel deswells. Current results are consistent with previous shown results [10, 20, 21].

hydrogel (+HG) and without hydrogel (-HG)

Figure 11 shows the comparative output voltages results of sensor with hydrogel (+HG) and without hydrogel (HG). These results indicate that output voltage is changing with response to pH value and not with conductivity of buffer solution at different pH value. The gap between curves shows the resistivity of the hydrogel.

5. Conclusion
We have developed new conductometric sensor with three electrodes. In this study only two electrodes measurements are discussed for characterizing the hydrogels. Three electrodes measurements deal with the monitoring of bacteria growth. Initially Poly(HEMA) hydrogels synthesized and successfully polymerized over the sensor electrodes with roughly thickness of 200m. Following conclusions can be drawn from two electrode measurements All hydrogel samples synthesized were clear and transparent which mean there was no phase separation. NaCl experimental results show that Poly (HEMA) hydrogel is good electrically passive material with fast swelling time and ions exchange time without any loss of mass and changing its mechanical properties. The sensor output voltage response to pH sensitive hydrogel swelling and deswelling behavior. At low pH, the output voltage is increasing due to higher degree of ionization inside hydrogel which lead increasing osmotic pressure making hydrogel swell, as pH increasing the degree of ionization inside hydrogel decreases, consequently output voltage deceasing. With less degree of ionization there is less osmotic pressure making the gel deswell.

Figure10. Long time measurements, effect of pH value on sensor output voltage.

less osmotic pressure making the gel deswells. Current results are consistent with previous shown results [10, 20, 21]. To study the reversibility behavior of the hydrogel, the solution in the well exchanged consecutively from pH-9 to pH-5 after getting equilibrate for each sample. As expected, the output voltage increasing from pH-9 to pH5 (Basic groups to Acidic groups). The equilibrium point output voltage for each pH value in reverse swelling is the same which was in forward swelling. This observation

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New Impedance Measurements Based Microsystems Setup for Characterizing Biomaterial Hydrogel The reversibility measurements indicates that the poly (HEMA) hydrogel with this composition under use can remain stable even after undergoing many swelling and deswelling cycles without any loss of mass or changing its mechanical properties.
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ACKNOWLEDGMENT
Im very grateful to Prof. Dr. Gerald Urban and Dr. Gregory Dame at IMTEK to give me opportunity to work on this project. Im also very thankful to M. Al-Halhouli who always helps me many times during my work.

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