Ion-Sensitive Field Effect Transistors for ph and Potassium ion concentration sensing, towards detection of Myocardial Ischemia.
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1 Ion-Sensitive Field Effect Transistors for ph and Potassium ion concentration sensing, towards detection of Myocardial Ischemia. Pratyush Rai a, Soyoun Jung b, Taeksoo Ji b, Vijay K Varadan b a Biomedical Engineering, 203 Engineering Hall, University of Arkansas, Fayetteville, Arkansas b Organic Electronics and Devices Laboratory (OEDL), Department of Electrical Engineering, University of Arkansas, Fayetteville, Arkansas Abstract: Ion Sensitive Field Effect Transistors (ISFETs) for sensing change in ionic concentration in biological systems can be used for detecting critical conditions like Myocardial Ischemia. Having the ability to yield steady signal characteristics can be used to observe the ionic concentration gradients which mark the onset of ischemia. Two ionic concentrations, ph and [K + ], have been considered as the indicator for Myocardial Ischemia in this study. The ISFETs in this study have an organic semi-conductor film as the electronically active component. Poly-3 hexylthiophene was chosen for its compatibility to the solution processing, which is a simple and economical method of thin film fabrication. The gate electrode, which regulates the current in the active layer, has been employed as the sensor element. The devices under study here were fabricated on a flexible substrate PEN. The ph sensor was designed with the Tantalum Oxide gate dielectric as the ion selective component. The charge accumulated on the surface of the metal oxide acts as the source of the effecter electric field. The device was tested for ph values between 6.5 and 7.5, which comprises the variation observed during ischemic attack. The potassium ion sensor has got a floating gate electrode which is functionalized to be selective to potassium ion. The device was tested for potassium ion concentration between 5 and 25 mm, which constitutes the variation in extra cellular potassium ion concentration during ischemic attack. The device incorporated a monolayer of Valinomycin, a potassium specific ionophore, on top of the gate electrode. 1. Introduction 1.1 Deviation from normal electrophysiology of Myocardial cells during Ischemic Stroke: In the resting (polarized) state the cationic concentrations across the cell membrane is stated as 150mM K + intracellular, 5mM K + extracellular and 10mM Na + intracellular, 145mM Na + extracellular. A cardiovascular muscle flex involves an influx of extracellular sodium ions (depolarization), which is later balanced by efflux of intracellular potassium ions (repolarization) through the cell membrane. The Myocardial cells normally derive most of the energy from aerobic metabolism of glucose while drawing a small fraction of it from the anaerobic metabolism of glycogen. [1] Local blood circulation prevents accumulation of extracellular potassium ion. The local circulation arrest in the coronary artery preceding the stroke cuts off the oxygen supply to the cells triggering the anaerobic metabolism. The potassium ions accumulate in the extracellular region up to four fold concentration. The lactic acid produced from the anaerobic metabolism produces protons which changes the extracellular protons. Both the processes happen simultaneously and can be used as markers for detection of the on set of the Ischemic stroke. [1] Nanosensors and Microsensors for Bio-Systems 2008, edited by Vijay K. Varadan, Proc. of SPIE Vol. 6931, 69310I, (2008) X/08/$18 doi: / SPIE Digital Library -- Subscriber Archive Copy Proc. of SPIE Vol I-1
2 The electrochemical sensors used for ion concentration detection in body fluids are based on different types of electrode assembly, which provide voltametric or amperometric readouts. These microelectrodes are very sensitive, but they are vulnerable to background noise, which affect their ability to provide with consistent status of ionic concentration. Field Effect Transistors are robust transducers of electrode potentials and can provide the options of potentiometric and amperometric sensing simultaneously. The also outscore the electrodes in power consumption and bulk production (IC fabrication techniques which also increases the possibility of miniaturization). [2] 1.2 Ion-Sensitive Field Effect Transistor (ISFET) Design: The Field Effect Transistors (FETs) have found applications in field of in-vitro and in-vivo (online) biosensing. In sensor devices like Enzyme-FETs, they form the transducer element. Unlike electrode sensors, they act as sturdy transducers which are not influenced by small perturbations in their surroundings. ISFETs, based on MOSFET design, have been used by many research groups for detection of metabolites (Urea) [3] and chemicals (Penicillin anti-biotic) [4]. The FET designed in this study has a flexible substrate with source-drain, gate dielectric, floating type gate electrode and semiconductor assembled in a bottom contact orientation. Modifications like zigzag pattern source-drain, for high widthlength ratio; help enhance the transduction capability of the FET. [5] Conventional ISFETs do not have a gate electrode. They rather have a reference electrode suspended in the analyte. [6, 7] The ion accumulation in the vicinity of the gate insulation layer provide for the effecter potential to drive current through the transistor. Some of the ISFETs have floating gate electrode to provide for transduction of the charge accumulated on the sensing surface into gate potential. It also helps the designers to revert back to unchanged CMOS architecture. [6] A floating gate electrode is employed in the potassium ion sensor for the purpose of tapping the charge in a better way and also surfaces like gold facilitate functionalization in a better way. 1.3 Materials and Fabrication techniques: The materials used for fabrication contribute to the sensitivity and easy fabrication of the device. Regioregular P3HT has been used as semiconductor for ISFETs in many cases. P3HT spin coated on a preformed monolayer of hexamethyldisilazene (HMDS) followed by heat treatment (annealing) results in comprehensively aligned crystalline semiconductor film with field-effect mobility of cm2v-1s-1.[8] Solution processing is an economic fabrication option which can be used for bulk production in form of offset printing. Use of gold as the material for source and drain electrodes is suggested because of lower work function barrier in case of P3HT. [9] Tantalum Oxide with dielectric constant between 21 and 24 [10], belongs to the category of high κ dielectrics. As the gate dielectric material it will provide for higher drain current corresponding to small stimuli (gate potentials). [11] Tantalum oxide film deposition can be done in different ways. Chemical vapor deposition can be reactive (with tantalum) as wells as direct (with tantalum oxide). [12] Oxygen is needed in both cases to provide as a reactant or to prevent diffusion of oxygen resulting in leaky film. In both cases oxygen gas has to be provided in to the chamber to maintain uniform quality of the film. Reactive ion sputtering uses tantalum target with oxygen gas inlet for reaction to form the oxide and be deposited. [13] Anodization is also employed which involves deposition of tantalum and the electrochemically oxidizing it to form the oxide film, leaving a very thin layer of tantalum underneath. The architecture of the device does not allow for Anodization and the quality of the film obtained by chemical vapor deposition is not as uniform as that by sputtering. [14] The film deposited by Magnetron Sputtering is amorphous in nature. Normally annealing is used to crystallize the film at 700ºC. Since the PEN substrate cannot withstand heat treatment beyond 175ºC, the film was etched out in its amorphous state. The etching process used is Reactive Ion Etching (Dry Etching). The wet etchants (1-10 wt% HF in water) used for Tantalum Oxide are highly corrosive and can damage Proc. of SPIE Vol I-2
3 the underlying polymer semiconductor film. The amorphous Tantalum Oxide film shall be hard to etch out because unlike the crystalline Tantalum Oxide it does not have well defined grain boundaries for the reagent (in this case plasma) to diffuse through and etch underneath. [12] Research groups have used a mixture of argon and SF 6 or CCl 4 for RIE. The process never exceeds 200Watts of power to keep the photo resist from getting damaged. The chamber pressure is kept around mTorr. Some research groups have analyzed the variation in etching rate with varying O 2 and SF 6 partial pressure. Picard et. al (1985) [15] have found that a ratio of approximately 1:10 for O 2 :SF 6 is optimum for etching process. Gate electrode Gate dielectric Polymer and drain Device Functioning for potassium ion detection Sensor is 0.75mm x 0.75mm ionophores Gate electrode 2000A nm Charge accumulation 1000A Electric field 1000A (a) charge Drain Gate dielectric Polymer semiconductor ph sensor for monitoring Myocardial Acidosis Sensor is 0.75mm x 0.75mm and drain ))I -p 'FJ 2000A Charge accumulation nm Electric field Drain charge 1000A (b) Fig 1: Layout and Working Principle of (a) Potassium ion sensor and (b) ph sensor The surfaces of the gate dielectric or the pseudo gate electrode can be functionalized deposition of selectively permeable films of ionophores or selectophores (word coined by Sigma Aldrich). In the development of Ischemia sensors, Valinomycin (ionophore for potassium ion) and Tantalum Oxide (gate dielectric surface) are used. Isolated from prokaryotic Streptomyces fulvissiumus, Valinomycin membrane molecule has a hydrophilic core to bind to the ionic molecule (NH 4 + or K + ) and a hydrophobic exterior to Proc. of SPIE Vol I-3
4 act as a lipophile. It is a 18-crown-6 ionophore which has a cavity of inner-diameter of Aº. This is compatible with the diameter of hydrated K + ion (diameter 2.66Aº). [16] Valinomycin is soluble in solvents of low or moderate polarity like chloroform. It is known to form a monolayer at oil-water interface. Being lipophilic it can be incorporated in a lipid bilayer. The molecules donut shaped geometry is highlighted by a hydrophobic exterior which can be used to position the molecules on the gate surface to form monolayer of limited friability. [16] 2. Methodology The PEN substrate was mounted on the silicon wafer by high pressure lamination. The PEN films are heath treated at 175ºC for 3 hours. Metallization was done using E-Beam evaporation process to deposit 50Aº of chromium and 500Aº of gold. The source and drain electrodes were patterned by photolithography and wet etching of chromium-gold film. The substrate was then coated with HMDS layer to render it hydrophobic to help form a seed layer of poly 3-Hexylthiophene (P3HT). P3HT was deposited by spin coating the substrate using P3HT-chlorofrom solution at 1000 rpm. The film (~1000Aº) was annealed at 70ºC for 2hrs in N 2 environment to enhance crystallization in the film. Tantalum Oxide (1000Aº thick) was deposited over P3HT by Magnetron Sputtering at a pressure of 2.0 x 10-4 mtorr using a Tantalum Oxide target. The gas pumped inside is a mixture of argon and oxygen. For the fabrication of potassium ion sensor an additional layer of 50Aº chromium and 500Aº gold was deposited by e-beam evaporation. The devices were patterned out by successive etching processes for the constituent layers. The chromiumgold film was etched out using wet etching. Reactive Ion Etching was used to remove the Tantalum Oxide and P3HT film. The recipe used is 30sccm argon, 100sccm SF 6 and 10sccm O 2. The process was carried out at 200mTorr pressure and 100W of power. The RIE process was done in steps of 10 minutes duration. After each step the chamber was conditioned by running a conditioning process using titanium wafer. The photo resist was also stripped off and reapplied for the next step. This was done to keep the photo resist from getting affected by the fluoride plasma. The photo resist used for this process was AZ-4620 coated at 1000 rpm to give a resist film 18µm thick for providing sturdier protection to the circuitry underneath. The device was encapsulated by photo-cross linked PVA, leaving a window open to expose the gate electrode (K + sensor)/ tantalum oxide surface (ph sensor). The gate electrode of potassium sensor was functionalized with Valinomycin. The Valinomycin monolayer was formed by pre treating the gate electrode was HMDS and the apply Valinomycin by solution casting (chloroform as the solvent). During solution casting the Valinomycin + chloroform solution forms a receding droplet (chloroform is evaporating. In this case the Valinomycin will also recede to form a blob in the center of the electrode. HMDS was used to help the Valinomycin molecules to adhere to the electrode surface and not recede along with the chloroform droplet. The formation of a Valinomycin monolayer was confirmed by an AFM scan. A standard ISFET setup consists of a reference electrode incorporated in a circuit which has gate voltage (V g ) applied between the reference electrode and the drain while a voltage is applied between drain and source electrode (V ds ). The source is grounded (potential at the source is zero). During device characterization the V g is changed from 0V to -5.0V and V ds was kept constant at 5.0V. Agilent 4156B Precision Semiconductor Parameter Analyzer was used as the power source and analysis tool. The ph buffer solutions were made from mixing the stock buffer solutions CH 3 COOH + CH 3 COOMn.H 2 O 0.1M (ph 4.75) and NH 3 in H 2 O + (NH 4 ) 2 SO 4 0.1M (ph 9.25). The buffers prepared were of the ph 6.09, 6.68, 7.09, 7.46, 7.88, 8.47, 8.81, 9.33, 9.5. The potassium ion solution was made by mixing potassium chloride (0.1M stock solution) in a 0.33M Na+ solution. The sample solutions were prepared with K+ concentration of 0.33M, 0.43M, 0.5M, 0.55M. The sample solutions were applied (droplet) on the sensing component using pipette (1000µl) and the reference electrode (2mm diameter Ag/AgCl) was inserted into the droplet Proc. of SPIE Vol I-4
5 and readings were taken at 1minute intervals till concurrent reading were obtained. The Id values at Vg = 1.5V were noted for different concentrations and calibration graphs were plotted. The thin film dielectric was characterized by using both optical as well as electrical methods. The film thickness was measured using a nano-spectrometer, where the refractive index of the film was expected to be 2.1 approximately. The tantalum oxide thin film was sand witched between a layer of 750Aº of chromium (1.7mm diameter contact pads made by using shadow mask) and heavily doped n-type silicon substrate to form a floating plate type capacitor. The capacitance was measured using AT&T flying prober. The capacitance values help provide with the dielectric constant (expected to be 21-28) of the film. 3. Results: The Tantalum Oxide film, 1000Aº in accordance with its blue color, has a dielectric constant of The value was calculated using equation (1) where the averaged capacitance value i.e. 2.5 nf, film thickness and contact pad diameter. The diameter of the chromium pad and the distance between the pads were measured using a wire gauge (dia = 1.7mm). These coordinated were required to be uploaded on to the computer aided control for the robotic arm to measure the capacitance of the floating plate capacitors. C A κε d 3 ( ) 9 12 π = κ κ = 10 = 24.9( approximately) 8.85 π = 0 2 (1) n I 0.0 flm Fig 2:Atomic Force Micrograph of the Valinomycin Monolayer deposited on gold JiM Valinomycin solution was prepared in chloroform (5mg in 10 ml) and solution caste on the gold surface coated with HMDS. Atomic force micrograph (Fig. 2) of the substrate revealed that the monolayer has been formed and hence the process was repeated on the gate electrodes of the potassium sensors. The ph sensor and potassium ion sensors were tested within a range of ph and potassium ion concentration 0.3M-0.5M. The IV characteristics were compiled to show the response of the devices at low operating voltages. The device response at V gs = -1.5V was monitored for calibration purposes. In addition to that, the potassium ion sample prepared in Na + helped check the functioning of the Valinomycin monolayer. The baseline was obtained in both the cases by applying DI water to the sensing component. When 0.33M Na + solution was applied to the sensing component the response matched that of the base line to great extent. This indicates that the Valinomycin monolayer is functioning as intended. Proc. of SPIE Vol I-5
6 Potassium ion sensor showed sensitivity towards potassium in sodium ion environment. The ph sensor showed sensitivity even around the ph values 7.88, 7.46 and 7.09 also which are near neutral conditions. Also, these correspond to the physiological ph range. The calibration curves obtained in both the cases were also linear (R 2 values in excess of 0.9). The sensors had sensitivity of 0.07µA/pH and 0.29µA/pK (Fig 3 (a) and (b)).s However, the signal strength was in question (of the order of micro amperes) and needed to be looked into calibration graph for ph sensor ph Id (ua) (a) calibration graph for potassium ion sensor pk Id (ua) (b) Fig 3: Calibration graphs for devices at Vg= -1.5V : (a) ph sensor and (b) potassium ion sensor 4. Discussion The ph sensor showed discernable sensitivity at near neutral (ph 7.0) concentrations. The sensor showed the ability to detect ph changes in the region of (the physiological range during ischemic strokes). The consistency of monolayer formation is confirmed by the AFM scan. The functioning of the monolayer meets the expectation as it is able to maintain the ion-selectivity of the sensor in the presence of Sodium ion, the other abundant cation in the extra cellular region. The sensor shows sensitivity at ionic concentration of the order of mm. The voltages applied are between 0 and -5V, which can be provided by batteries. This helps towards development of an implantable sensor with in-built power source. Proc. of SPIE Vol I-6
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