Chapter 1 Introduction
1.2 Thesis organization
In this thesis, the development processes of FET based biosensor were described. In chapter 2, the operation principles of the pH-ISFET, reference systems, readout circuits and biosensors will be introduced. Those are the fundamentals of developing a miniaturized biosensor. In chapter 3, the optimization for the developed ZrO2 gated pH-ISFET was performed. The optimal post-annealing process conditions were determined. Based on the optimized pH-ISFET, the ion-interference was also characterized. A novel method was proposed to improve the drift behavior in chapter 4. Through the measurement method, the pH-dependent drift can be improved significantly.
Chapter 5 will describe the development of a polymer based REFET. The ISFET/REFET pair with differential arrangement demonstrated good sensitivity of hydrogen ions and achieved the mini feature of sensors. In chapter 6, the urease biosensor
with the integration of the solid-state reference system was developed. The considerations of electrical match for the ENFET/ISFET differential pair were also addressed. The last chapter concludes the whole work.
References
[1.1] Bergveld, P. The impact of MOSFET-based sensors. Sens. Actuat. B 1985, 109-127.
[1.2] Madou, M. J.; Morrison, S. R. Chemical sensing with solid state devices, New York:
Academic Press 1989.
[1.3] Sze, S.M. Semiconductor sensors, New York: John Wiley & Sons 1994.
[1.4] Kim, Dong-Sun; Park, Jee-Eun; Shin, Jang-Kyoo; Kim, Pan Kyeom; Lim Geunbae;
Shoji, Shuichi. An extended gate FET-based biosensor integrated with a Si microfluidic channel for detection of protein complexes. Sens. Actuat. B 2006, 117, 2, 488-494.
[1.5] Newman, J. D.; Turner, A.P.F. Home blood glucose biosensors: a commercial perspective. Biosens. Bioelectron. 2005, 20, 12, 2435-2453.
[1.6] Wilson, G. S.; Hu, Yibai. Enzyme-based biosensors for in vivo measurements.
Chem. Rev. 2000, 100, 7, 2693-2704.
[1.7] Wilson, G. S.; Gifford, R. Biosensors for real-time in vivo measurements. Biosens.
Bioelectron. 2005, 20, 12, 2388-2403.
Chapter 2
Principles of ISFET-based biosensors: pH-ISFETs, reference systems and readout circuits
In this chapter, the composition and working principles of pH-ISFET based biosensors are introduced. The biosensors composed of pH-ISFETs, reference systems and readout circuits incorporate the immobilization technology transforming the detecting capability of chemical signals to biological ones. The pH-ISFETs perform the detecting function. The reference systems provide stable potential as measurement basis. The readout circuits deal with the signals and noises come from the detecting stage and send them to post treatment tools. The immobilization techniques and detecting principles are different depend on the characteristics of tested biological materials.
2.1 pH-ISFETs
In its most common interpretation, pH is used to specify the degree of acidity or basicity of an aqueous solution. In formal, the definition of pH is expressed as
log H log [ ]
pH = − a + = − γ H+ (2-1) where aH+ is the hydrogen ion activity, γ is the activity coefficient which equals to 1 for diluted solution, and is the molar concentration of solvated protons in units of moles per liter. In practice, the measurement of pH is not accomplished by the direct determination of the hydrogen ion activity but relative to standard solution of known pH.
[H+]
The ISFET, which is a MOSFET with the gate connection separated in the form of a reference gate immersed in aqueous solution which is contact with the sensing layer above gate oxide. It is working based on the approach of electrochemical measurement of pH.
The general expression for the drain current of the MOSFET and thus also of the ISFET in the non-saturated mode is
( )
12 2 channel, respectively, and μ is the electron mobility in the channel.Another important MOSFET equation describes the physical properties in nature is that of the threshold voltage
f Where the first two terms describe the work function difference between the gate metal (Φ ) and the silicon (M ), the second term is describes the effect of accumulated charge in the oxide ( ), at the oxide-silicon interface (
ΦSi
Qox Q ) and the depletion charge in the ss
silicon bulk ( ), the last term determines the onset of inversion depending on the doping level of the silicon.
QB
The measurement of pH with the ISFET was that it immersed in a liquid and the electrical circuits are connected to the reference electrode and source/drain contacts. When the device operating in linear mode, the constant drain-source voltage Vds was applied. The hydrogen activity influences the gate voltage was described in terms of Vt, hence the expression for the ISFET threshold voltage becomes
0 sol Si ox ss B 2
where Eref is the constant potential of the reference electrode, Ψ is the chemical input 0 parameter and χsolis the surface dipole potential of the solvent and thus having a constant
value [2.1]. As a result, the termΨ dominates the pH sensitivity of the ISFET since all 0 the other terms are constant.
Prior to determine the termΨ , the properties of the electrode/electrolyte interface, as 0 shown in Figure 2-1, should be studied. There are four possible properties of the interface:
first, the local concentration of both cation and anion changes at the interface; second, the ion-electron exchange, i.e., redox reaction; third, the ion-electron interaction, no redox reaction; and last, the ion-molecular interaction. Yates et al. [2.2] firstly introduced the site-binding model, as illustrated in Figure 2-2, for the electrode/electrolyte interface. This model describes the equilibrium between the amphoteric SiOH surface sites and the
-ions in the solution. The reactions are H+ where denotes the protons in the bulk of the solution. An originally neutral surface hydroxyl site can bind a proton from the bulk solution, becoming a positive site and leaving a negative site on the oxide surface. It is called amphoteric site.
HB+
It is known that the background electrolyte has a large influence on the surface charge [2.3]. This dependence is ascribed to variations in the double layer capacitance. The Gouy-Chapman-Stern model is most widely used to describe the double layer structure in ISFET literature [2.4]. Gouy and Chapman proposed independently the idea of a diffuse layer to interpret the capacitive behavior of an electrode/electrolyte solution interface. The excess charge in the solution side of the interface is equal in value to that on the solid state surface, but is of opposite sign. The ions in the solution are therefore electrostatically attracted to the solid-state surface but the attraction is counteracted by the random thermal motion which acts to equalize the concentration throughout the solution. Stern modified the
model with proposing a diffuse layer of charge in the solution starting at a distance X from the surface.
A new model was introduced by van Hal and Eijkel [2.5, 2.6] and is in fact nothing else than the well-known equation for capacitors Q CV= , where is surface charge in the form of protonized or deprotonized
Q
(
OH2+) ( )
O− OH groups of the oxide surface, is the double-layer capacitance at the interface and is the resulting surface potential The potential between the gate insulator surface and the electrolyte solution causes a proton concentration difference between bulk and surface. According to Boltzmann equation:C V constant and is the absolute temperature. The subscripts
K
T B and refer to the bulk
and the surface, respectively.
S
Here define two parameters: βS andCdiff . The intrinsic buffer capacity βS represents the ability of the oxide surface to deliver or take up protons, it is the capability to buffer small changes in the surface pH (pH ) but not in the bulk pH (s pH ); the B is the differential double-layer capacitance, of which the value is mainly determined by the ion concentration of the bulk solution via the corresponding Debije length. We have
Cdiff and positive surface sites per unit area, and
ν− ν+
( )
pHS
−ν βS ≡ ΔνΔ− + .
An equal but opposite charge is built up in the electrolyte solution side of the double layerσDL because of the charge neutrality. The σDLcan be described as a function of the integral double layer capacitance, Ci, and the electrostatic potential
0
0 σ
σDL =−CiΨ =− (2-10) The integral capacitance will be used later to calculate the total respons
e of the ISFET on the change in pH. The ability of the electrolyte solution to adjust the amount the of stored charge as result of a small change in the electrostatic potential is the differential capacitance, Cdiff
From the Equations 2-7 ~ 2-11, we obtain that
0
Rearrange Equation 2-12 gives a general express for the sensitivit potential to changes in the bulk pH
ion y of the electrostatic
0 2.3 kT B
Note thatαis a dimensionless sens
buffer capacity and the differential capacitance, and the value ofαvaries between 0 and 1.
2-17)
itivity parameter which determined by the intrinsic
Only in the case α approach 1, the maximum Nernstian response of 58.2 mV /pH at 298K can be achieved. In other word, the high pH sensitivity requires a large value of the surface buffer capacity βS and a low value of the double layer capacityCdiff . Meanwhile, the highβ reduces the importance of theC ; as a result, a Nernstian sensitivity can be
achieved over wide pH range due to the independency of the electrolyte concentration. In
2.2 Reference systems for pH-ISFETs
conclusion, the selectivity and chemical sensitivity concerning the surface potential of the ISFET are dominated by the conditions of the electrode/electrolyte interface.
According to the operational principle of the ISFET, the reference system with . The stability of the reference electrode is portant factor in any electroanalytical procedure since any variation can affect the
2.2.1 Reference electrode
electrode potential. The high stability of the electrode potential is usually reached by employing a each participants of the redox reaction. The
reference electrode. A typical aqueous reference electrode is providing stable reference potential is essential
an im
response of the working electrode. Ideal reference electrodes are required to meet the following characteristics: (1) Stable and reproducible potential (2) Low temperature dependence of potential (3) Low electrical resistance (4) Application in variety of media (5) Reproducible and small liquid junction potentials.
To fabricate the miniaturized ISFET, there are two types of on-chip reference systems were proposed: the reference electrode and the reference FET (REFET).
A Reference electrode is an electrode which has a stable and well-known
redox system with constant concentrations of
reference electrodes can be divided into 3 categories: aqueous, solid-state and pseudo reference electrodes.
2.2.1.1 Aqueous reference electrode The first type is aqueous
the glass electrode. It is a type of ion-selective electrode made of a doped glass membrane that
(2-18)
This ng that a sufficiently high
d through the electrode with
is sensitive to a specific ion. The electric potential of the electrode system in solution is sensitive to changes in the content of a certain type of ions, which is reflected in the dependence of the electromotive force (EMF) of galvanic element concentrations of these ions. Another popular aqueous reference electrode is the silver chloride electrode. The electrode functions as a redox electrode and the reaction is between the silver metal (Ag) and its salt — silver chloride (AgCl).
The corresponding equations can be presented as follows:
Ag0(s)+Cl− ↔AgCl )(s + e−
reaction characterized by fast electrode kinetics, meani
current can be passe the 100% efficiency of the redox reaction (dissolution of the metal or cathodic deposition of the silver-ions). The Nernst equation below shows the dependence of the potential of the Ag/AgCl electrode on the activity or effective concentration of chloride-ions:
− −
= RT aCl
E
E 0 ln (2-19) The standard electrode potential E0 agains
F
t standard hydrogen electrode is 0.230V ± 0.01V.
Based on the principles, some min
Traditional reference electrodes used for electrochemical measurements, such as the odes, have a limited range of applicability. The iaturized Ag/AgCl reference electrodes with encapsulated liquid reference solutions were developed [2.7–2.9]. However, the activity of potential determining ions can vary due to an outflow of inner electrolyte via the liquid–liquid connection. Meanwhile, the leakage of the reference solutions limits the device lifetime and affects the measurement accuracy.
2.2.1.2 Solid-state reference electrode
calomel and silver/silver chloride electr
liqui
ped reference electrode (2) To reali
macro electrode in which a Ag/AgCl wire is emb
pseudo-reference electrode is a class of electrodes, they do not maintain a constant d junction is problematic with these electrodes, and they cannot be used either with wholly solid-state electrochemical cells or for very high-temperature reactions such as those in molten electrolytes. As an alternative to standard commercial reference electrodes, a solid state reference electrode is fabricated for in situ voltammetric analysis in solutions containing little or no added supporting electrolyte. It can be used under specific conditions in which traditional reference electrodes cannot be used.
The activities in the improvement of solid-state reference electrodes are divided into four main groups: (1) To improve the classical rod-sha
ze the planar conventional reference electrode (3) To implement the reference systems for ISFETs (4) To establish an all-solid-state reference. Different approached for ISFET reference systems are shown in Figure 2-3.
Many approaches to fabricate solid state reference electrode were investigated. Daniel Rehm et. al. fabricated an all solid-state
edded directly in the salt-doped resin [2.10]. I-Yu Huang et. al. introduced a novel agarose-stabilized KCl-gel membrane to serve both as a polymer-supported solid reference electrolyte and an ionic bridge for Ti/Pd/Ag/AgCl electrode [2.11, 2.12]. A nanoporous platinum oxide electrodes work as a noble solid-state reference electrode was also reported [2.13]. Two different strategies for the use of sensors have been pursued. On the one hand, low-cost disposables with simple designs and cheap component parts have been produced—especially in the field of medical sensors. On the other hand, sensor systems have been developed with sophisticated technologies to increase the lifetime of the devices without reduction of the sensor performances.
2.2.1.3 Pseudo reference electrode A
potential but vary predictably with conditions. The potential of such electrodes is not estab
r ISFETs
Like all other potentiometric electrochemical sensors, the ISFET pH indicator erence electrode, the potential of which should be mp
all closed epoxy compartment EC
the ion-sensitive membrane of an ISFET is lished in accordance with the Nernst Equation but rather by some other interaction with its environment. As long as its environment remains constant, its reference potential will likely remain constant.
2.2.2 Reference electrodes fo
electrode must be complemented by a ref
co letely independent of the pH value and the presence of other ions in the measuring solution. Three main approaches for ISFET reference systems development: (1) ISFET with pH buffer compartment (2) miniaturised Ag/AgCl, Cl− reference electrode (3) ISFET with modified pH-insensitive membrane.
The earliest ISFET on-chip reference system, Figure 2-3 (a), was described by Comte and Janata [2.14]. At this Janata-type configuration, a sm
containing a pH-buffered gel is placed on one of a matched pair of pH ISFETs. The electrolytic connection to the solution is made via a glass capillary. However, since it has only restricted lifetime and cannot be produced completely by using IC technology, it did not obtain noticeable acceptance. Figure 2-3 (b) shows the second type of reference electrodes. It is more like a miniaturized planar version of a conventional Ag/AgCl, Cl− reference electrode. It is called Prohaska-type microelectrode, which was prepared in thin film technology using IC-compatible techniques. The general aim is to eliminate the need of a separate reference electrode and to facilitate the use of ISFET. Many researches based on this type were reported [2.11, 2.12, 2.15].
The most popular version of reference electrodes is reference FET (REFET). As shown schematically in Figure 2-3 (c), where
covered with a pH-insensitive membrane or is itself modified to become pH insensitive.
This version was initiated by Matsuo et al. [2.16, 2.17], who deposited an ion-blocking parylene film on the Si3N4 layer on the gate of an ISFET. Errachid et al. [2.18] described a simple REFET for pH detection in differential mode measurements. The device is based on a pH-insensitive polymeric PVC membrane cast on the gate insulator of an ISFET device that has been previously silylated by chemical grafting of a silane compound. The REFET shows low pH sensitivity (1.8 mV/pH) and is only slightly affected by the concentration of Na+ and K+.
Two types of REFET structures can be distinguished with respect to the penetration of ions into the polymeric layer, resulting in two different mechanisms of the REFET oper
ind of the reference electrode, different measuring circuits for FETs are recommended. If any really potential-stable reference electrode is available, a simp
ation. In an ion-unblocking REFET structure, ion exchange occurs between the solution and the polymer, whereas in an ion-blocking REFET structure ion exchange is negligible. In the first case, the electrical potential is a membrane potential; in the second, however, it is a surface potential resulting from reversible ion-complexation reactions at the surface of the polymer.
2.3 Readout circuits
Depending on the k IS
le one-ended circuit is suitable, as shown in Figure 2-4 (a). This structure can be considered as an optimal among the interfaces without feedback. In order to achieve stable signal detection under noisy and unstable environmental conditions and to maintain the response linearity, sophisticated sensor interfaces are used. However, since the development of miniaturized reference electrode with thermodynamically defined interface
potential is still challenging, the ISFET/REFET differential measuring circuit as shown in Figure 2-4 (b) is widely accepted. In contrast to a real reference electrode, this electrode must not exhibit a stable potential because its potential fluctuations are eliminated by the common mode rejection of the differential amplifier. An electrode from any chemically resistant material, such as a platinum wire or a Pt layer deposited on the ISFET sensor chip, can be applied for this purpose. This arrangement seems to be particularly favorable with regard to the compensation of influences of temperature, light and other disturbance variables. However, the compensating effect must not be overestimated because it is only noticeable if the ISFET and the REFET have identical operating parameters. Chodavarapu et al. designed a differential circuit without any post-fabrication processing or material
deposition [2.19]. This system has two identical ISFETs as the inputs to a pair of ISFET operational transconductance amplifiers (IOTAs) arranged in a novel differential architecture. The IOTAs have different sized p-MOSFET load transistors with different amplification factors. The CMOS ISFET chip is fabricated in an unmodified 1.5 mm commercial process. However, the sensitivity is only 20 mV/pH, which is insufficient for biological applications. Another Fully CMOS-integrated pH-ISFET interface circuit design was reported [2.20], the sensor chip is fabricated in a standard 0.35,um 4-metal and 2-poly layer CMOS process to which extra post processing steps are added for depositing membranes. The ISFET/REFET pair was fabricated with Ta2O5 ion sensitive layer and the other with PVC insensitive layer. The differential measurement result achieved 40.76 mV/pH, demonstrated the necessary and importance of REFET development.
2.4 Biosensors
2.4.1 Overview
Since the biosensor was first described by Clark and Lyons in 1962 [2.21], the biosensors were vigorous. Many biological materials provide a broad platf
itive material to electrical signals.
The
d sensitivity. This is the capability to select and to measure the specific bioch
nsor can be used with limited
osensor is often not reaction-limited, but is rather
inical diagnosis (2) Agriculture development and monitoring (3) Food development of
orm of functional units for their integration with electronic elements. For example, the biomolecules of optimal recognition and binding capabilities lead to high selectivity and specific biopolymer compexes (antigen-antibody, hormone-receptor, or duplex DNA complexes). Biosensors are small analytical bioelectronic devices that combine a transducer with a sensing biological component.
The transducer transforms the weight, electrical charge, potential, current, temperature or optical activity measured with the biologically sens
schematic diagram was shown in Figure 2-5. The biological sensing element was in
schematic diagram was shown in Figure 2-5. The biological sensing element was in