Progress in Electromagnetic Research Symposium 2004, Pisa, Italy, March 28 - 31

Semiconductor chemical and biochemical sensors

Abstract In this paper, problems related to the fabrication of semiconductor (bio)chemical sensors and microreactors, including modification of the surface of different materials (semiconductors, dielectric materials, polymers) to create a sensing membrane on the transducers surface and immobilise bioreceptors namely enzymes are discussed. 1. Introduction An accurate and easy monitoring of a concentration of (bio)chemical species at low costs is important for medical diagnosis. Moreover, the modern medical diagnosis requires a minimisation of analytical samples. Semiconductor technology gives a potential possibility for development of reliable and low cost sensors for implantation and home use for in vivo and in vitro monitoring of biochemical parameters. The (bio)chemical sensors e.g. amperometric and potentiometric, show many advantages such as: small size, better durability and reliability, and the possibility of integration of the sensors with the required electronics over conventional sensors. Nowadays thanks to progress in micromechanics, the biosensors have been integrated into micro analytical systems. In many modern analytical systems, instead of conventional ion selective electrodes, ion sensitive field effect transistors (ISFETs) for detection of the following ions: hydrogen (pH), potassium, sodium, calcium, ammonium etc are used. Since in many enzymatic reactions the end products are in the form of hydrogen, hydroxyl and ammonium ions, an important group of biosensors are that based on the detection of changes of pH and ammonium ion concentration. These are for example: glucose, urea, triglycerides and creatynine biosensors. Depending on labelling of immune species (antibodies and antigens), these biosensors can also be applied for immunoassays. In our works, as transducers pH-sensitive ISFETs and chemically modified FETs (ChemFETs) sensitive to ammonium ions were used. To create a biosensor, an enzyme is immobilised on the surface of chemically sensitive layer in the gate area of the transistor. The above-mentioned sensors may be integrated with microreactors, made with the utilisation of semiconductor technology, to create the micro total analytical systems (TAS) or lab-on-a-chip. In our laboratory, microreactors were fabricated as a system of microchannels etched in silicon. The immobilisation of bioreceptors was obtained in alkilation reaction between amino groups on the enzyme and an epoxy ring of the silane. In this paper, problems related to the fabrication of the described sensors and microreactors, including modification of the surface of different materials (semiconductors, dielectric materials, polymers) to create a sensing membrane on the transducers surface and immobilise bioreceptors (enzymes) are discussed. 2. (Bio)chemical sensors construction Biosensor is an analytical device that incorporates a biologically active material with properly selected transducer. Combination of a chemical sensor with biologically active receptor enables widespread use of various substances with high selectivity. The functional principle of an enzymatic sensor consists in the utilization of catalytic properties of enzyme immobilised inside or on the surface of chemically sensitive layer. The substrate transported by diffusion to the enzymatic layer is decomposed to products, penetrating to chemosensitive layer and then the specific product is detected by a basic transducer.

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Progress in Electromagnetic Research Symposium 2004, Pisa, Italy, March 28 - 31

We have developed a number of chemical sensors and biosensors dedicated to different applications among them clinical as well as environmental analysis. As transducers an ion sensitive field effect transistor (ISFET) and chemically modified field effect transistor (ChemFET) [1] were used. A set of enzymatic reactions for determination of different analytes is shown in Table 1.
Table 1. Enzymatic reaction and utilised detection methods

Analyte Urea Triglycerides Acetylcholine (Ach) Butyrlcholine (BCh) Creatynine

Enzymatic reaction CO(NH2)2 + 3 H2O urease> CO2 + 2 triglyceride + 3H2O

NH4+

+ 2 OH

(1)

(2) glycerol + 3 fatty acids CH3COO(CH2)2N(CH3)3 + H2O AChEsterase> (3) HO(CH2)2N(CH3)3 + CH3COO- + H+ CH (CH ) COO(CH ) N(CH ) + H O BChEsterase> (4)
3 2 2

Detection pH, pNH4 pH pH pH pNH4

HO(CH2)2N (CH3)3 + CH3(CH2)2COO + H Creatynine + H2O Creatynine deiminase> NH4+ + N-methylaydantonine

2 2

3 3

(5)

Since products of the enzymatic reaction (1), (3) and (4) cause changes of pH, enzymatically modified pH-ISFETs were used for indirect detection of urea and acetylcholine. Reaction (3) was also used for indirect determination of pesticides and heavy metal ions [2]. A ratio of enzyme inhibition caused by inhibitors (pesticides or heavy metal ions) is expressed in percentage as a relative sensors response to the given concentration of substrate (Ach) in the presence and absence of inhibitors in a sample solution. In many cases inhibition of the enzyme is irreversible, which means that the biosensor cannot be regenerated by any chemical reagent. In these cases biosensors may be regenerated by replacement of the enzymatic membrane with a new one. Since replacement of the membrane may cause some difficulties, another approach has been undertaken. In this approach the problem has been solved by use of a modular system, consisting of a sensor and replaceable microreactor. The microreactor with enzyme immobilized on the internal walls of grooves or filled with the batch with immobilized enzyme, plays a role of enzymatic membrane in multi-layer biosensors. Principle of operation of ChemFETs is analogical to ion-selective electrodes (ISE). However, there are some differences. Since for designs with a direct deposition of an organic ion selective membrane onto inorganic gate of the ISFET the output signal is unstable, an intermediate hydrogel layer, deposited underneath ion-selective membrane, has been introduced (Fig.1) [3]. Thanks to the hydrogel performing 1 : enzymatic membrane 1 2 : ion selective membrane function as similar as function of the internal 2 3 : polyHEMA hydrogel layer 4 : gate layers: 6 6 electrolyte in ISE, the influence of acidic 3 - silicon nitride substances such as CO2 and ill-defined - silicon oxide 4 5 : channel interface between membrane and the gate S D 6 : encapsulation 5 S : source insulator have been eliminated. Based on this B D : drain B : bulk approach ChemFETs for sodium, potassium, calcium and ammonium ions has been developed [1,3,5]. Figure 1. Structure of the urea ENFET 3. Methods of surface modification pH-sensistive ISFETs have been a subject of research work for many years. Inorganic materials have been used as the gate insulator e.g.: SiO2, Si3N4, Al2O3 and Ta2O5. Those and other materials have been tested in our laboratory as the gate chemically sensitive membranes of the pH- ISFETs, but the silicon nitride have been used in practice In the case of utilisation of pH-detection of enzymatic reactions products, a modification of the ISFET gate surface and immobilisation of enzymes on this surface should be performed. In our laboratory several methods of enzyme deposition have been used. Two of the considered methods are

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Progress in Electromagnetic Research Symposium 2004, Pisa, Italy, March 28 - 31

based on chemical immobilization of urease on the silicon nitride surface with the use of glutaraldehyde (GA). The first one is based on Schiffs base formation between amino groups on the silicon nitride surface and enzyme via GA [4]. The scheme of Schiffs base formation (silicon nitrideGA-Enzyme) is as follows:
Si-NH2 + OHC-(CH2)3-CHO + H2N-E Si-N=CH-(CH2)3-CH=N-E + 2H2O (6)

where Si-NH2 amino type groups on the hydrated surface of the silicon nitride and E enzyme. In the second method, the silicon nitride surface has been treated with aminopropyltrietoxysilne (APTS) first and then to the functionalized silicon nitride surface enzyme was coupled through glutaraldehyde, according to the following reactions:
-OH -OH -OH -O

+ (C 2H5O)3Si(CH2)3-NH 2

-O- Si -O

-(CH2) 3-NH2 + 3C2H5OH

-O -O- Si -O

-(CH2)3-NH2 + OHC-(CH2)3-CHO + H 2N-Enzym

-O

-O- Si -O

-(CH2)3 -N=CH-(CH2)3-CH=N-Enzym + 2H 2O

(7) The third method consisted in cross-linking of enzyme molecules with bifunctional reagent glutaraldehyde. Creatynine can be electrochemically detected using indirect method by determination of ammonium ion concentration (5). As it was mentioned, to obtain sensor sensitive to other then hydrogen ions, the gate surface of ISFET was chemically modified. The modification is a multi-stage process, consisting of hydration, silanisation, deposition of liquid HEMA cocktail, and photopolymerisation. The hydrated silicon nitride surface was functionalised with metactrylic groups in the silanisation process. In the next stage, liquid HEMA mixture was deposited onto the wafer and photochemically polymerised by UV-irradiation. In result, a coat of poly(2-hydroxyethyl methacrylate) covalently bound to the substrate is obtained:
H C O O O O Si ( CH ) O
2 3

CH3 C = CH
2

O C OCH CH OH
2 2

H2 C = C

O O O Si ( CH ) O
2 3

O C

CH3 C CH
2

CH3 C CH
2 n 2

fotoinitiator, h

C=O
2

OCH CH OH

(8)

Afterwards, the ion-selective membrane containing nonactine was deposited. The last stage of the procedure consisted in deposition of the enzymatic membrane, obtained by cross-linking of enzyme molecules with glutaraldehyde. In the case of sodium sensitive ChemFETs the ion-selective membrane, deposited on the top of the pHEMA hydrogel layer, contained newly synthesised ionophore selective to sodium ions. The ionophore was of increased lipophilicity, which results in low leakage rate of the ionophore from the membrane [5]. A new approach of biosensing is based on microreactor construction. To types of microreactors has been developed: (1) lamella with a system of parallel microchanels etched in silicon and (2) batch type in a form of column filled with glass microbeads with immobilized enzyme (Fig.2).

Figure 2. View of the silicon microreactors: lamella type(a) and batch type (b).

a).

b).

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Progress in Electromagnetic Research Symposium 2004, Pisa, Italy, March 28 - 31

In general, enzymes immobilized in microreactors were attached using the chemical bond to the surface modified with 3-glycidoxypropyltrimethoxysilane (GOPTMS) [6]. Silanisation process of silicon compounds is based on reaction of Si-OH groups with a silane. Therefore, the first step in the procedure, leading to formation of active hydroxyl groups on the substrates: glass microbeads and native silicon dioxide on walls of channels, was hydration. Next, the silanisation procedure resulted in the substrate surface functionalisation with epoxy rings. The functional groups were used for coupling to amino groups (-NH2) on the enzyme (alkilation reaction), according the scheme 1 - reactions (9).
SCHEME 1
1. silanisation
-OH -OH + -OH (C 2H5O-) 3Si-(CH 2 )3-O-CH2-CH-CH2 O -O

-O- Si-(CH 2 )3-O-CH2-CH-CH2 + 3C2 H5 OH -O O

2. enzyme coupling
-O -O- Si-(CH 2 )3-O-CH2-CH-CH 2 + NH2 -Enzyme -O O -O NH-Enzyme

-O- Si-(CH 2 )3 -O-CH2-CH-CH 2 -O

OH

(9)

For the purpose of microreactors preparation, the method based on APTS has been successfully applied. The methods can be used for preparation not only stand-alone microreactors but also microreactors being part of micro analytical systems. 4. Conclusions
Several biosensors based on different detection for different analytes such as: hydrogen, ammonium and sodium ions as well as urea, creatynine, triglycerides, acetylcholine, butyrylcholine, pesticides and heavy metal ions were presented. Moreover, a general overview concerning specific approaches to construction of biosensors and microreactors as well as their fabrication with special regards to enzyme immobilisation was done.
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