KR20060131311A - A self-assembled monolayer for urease immobilization of biosensor - Google Patents

Abstract

A self-assembled monolayer for urease immobilization of a biosensor is provided to enhance sensitivity at low urea concentration, reduce reaction time of the biosensor by direct electron transfer of the self-assembled monolayer, and especially enhance sensitivity of the biosensor when porous silicone is used as a substrate. The self-assembled monolayer for urease immobilization of the biosensor is used as an immobilization membrane of the biosensor and is manufactured with 3-mercaptopropionic acid, wherein the biosensor is prepared by depositing the gold thin layer on the surface of a planar electrode and porous silicone to produce an electrode, and forming the self-assembled monolayer on the gold thin layer. The urea sensor comprises the self-assembled monolayer made of 3-mercaptopropionic acid as the immobilization membrane.

Description

A self-assembled monolayer for urease immobilization of biosensor}

1 shows an SEM photograph of a gold thin film electrode formed on porous silicon.

2 is a simplified illustration of immobilization via covalent bonding of a self-assembled monolayer and urease.

FIG. 3 shows an overall process diagram for semiconductor processes such as porous silicon layer formation, Au and Ag thin film deposition, self-assembled monolayer formation, urease immobilization, and the like.

Figure 4 shows the results of measuring the amount of current according to the shape of each electrode and the shape of the electrode by the time zone current method.

FIG. 5 shows a cyclic voltammogram of the gold plane electrode and the porous silicon substrate in phosphate buffer.

FIG. 6 shows a cyclic voltammogram after forming a self-assembled monolayer on a gold plane electrode and a porous silicon substrate in phosphate buffer.

7 shows the XPS spectrum of the Urease / SAM / Au / PSi layer.

8 is a graph showing the sensitivity of the sensor electrode according to the urea concentration by the time zone current method.

9 shows the sensitivity by the time zone current method as a correction curve.

The present invention relates to a self-assembled monolayer for an immobilization film of a biosensor. More specifically, the present invention relates to a self-assembled monolayer for use as an immobilization film of a biosensor considering the electrochemical properties of a self-assembled monolayer of 3-mercaptopropionic acid. .

Biosensors use biomaterials such as enzymes, biomolecules, antibodies, and cells as cognitive substances to cause reactions with analytes to be measured with high selectivity and detect the results with existing physical and chemical sensors. Therefore, it is a trend to replace the conventional medical chemical sensor.

The difference between the biosensor and the conventional sensor is that it utilizes selective reaction and binding of biomaterials. Therefore, the most important thing in the practical use of the biosensor is the immobilization technology of the bioreactive substance and the selection of the immobilization membrane.

Many studies have been conducted in terms of biosensor solid surface and cognitive substance immobilization technology in terms of suppressing structural deformation of biomaterials and securing a space to play a unique role on the surface. In particular, biosensors using enzymes have problems due to structural deformation and steric hindrance due to functional groups capable of many binding to solid surfaces. In addition, the immobilization technology using physical and chemical methods such as adsorption and entrapment has a disadvantage that it is difficult to maintain the orientation of the active site in the enzyme in a certain direction.

Self-assembled monolayers (SAMs) can easily obtain dense and stable monolayers due to van der Waals interactions, so that in addition to biomaterial immobilization films, protection or semiconductor processes can be patterned to prevent corrosion. It is also used as a mask for. Recently, researches on electron transfer mechanisms using the electrically active properties of self-assembled monolayers and researches for using them as gate materials of transistors have been actively conducted.

As a representative biosensor, urea sensors using a constant voltage method have been studied a lot, but they are not commercialized due to disadvantages due to a decrease in sensitivity at low concentrations.

Accordingly, an object of the present invention is to prepare a self-assembled monolayer of 3-mercaptopropionic acid as an immobilization film for use in urea sensor and to investigate its electrochemical properties.

The object of the present invention is to fabricate an electrode in which a thin film of gold is deposited on the surface of a planar electrode and porous silicon using a semiconductor process, to form a self-assembled monolayer on the gold electrode, and to self-assemble a monolayer as a film for immobilizing urease. Observe the electrochemical characteristics of the working electrode, consider the change in current magnitude with respect to the voltage applied to the electrode by cyclic voltammetry, and generate the amount of urea decomposed by urease by electrolysis of the generated water. This was achieved by measuring sensitivity according to the concentration of urea using the time-domain constant voltage method, which is an indirect method of measuring current.

The present invention comprises the steps of fabricating an electrode deposited with a thin film of gold on the surface of the planar electrode and porous silicon; Forming a self-assembled monolayer on the gold thin film to observe electrochemical properties; Considering a current magnitude change with respect to the voltage applied to the electrode through the cyclic voltammetry; It is composed of sensitivity measurement step according to concentration of urea by using time constant voltage method.

Hereinafter, the contents of the present invention will be described in more detail with reference to Examples. However, these examples are only for explaining the contents of the present invention and do not limit the scope of the present invention.

EXAMPLE

Example 1 Preparation and Fixation of Self-Assembly Monolayers

A self-assembled monolayer for use as the urease immobilization film of the urea sensor was manufactured according to the following semiconductor process.

First step: formation of porous silicon

The boron-doped resistivity 14-17 Pa.cm, 100, p-type silicon wafer as an impurity was washed by RCA standard process, and then a porous silicon layer was formed by an electrochemical method using a working electrode (anode electrode). To explain this concretely,

RCA cleaning process consists of RCA1 wet cleaning process and RCA2 wet cleaning process. RCA1 washing process is to remove organic dirt. NH ₃ (25% aqueous solution) and DI H ₂ O are mixed at a ratio of 1: 5 and heated to add H ₂ O ₂ (NH ₃ : H ₂ O : H ₂ O ₂ = 1: 5: 1), the wafer is submerged for 10 minutes. RCA2 washing process is to remove metal ions, HCl and DI H ₂ O is mixed in a 1: 6 ratio and heated, then H ₂ O ₂ is added (HCl: DI H ₂ O: H ₂ O ₂ = 1: 6: 1), the wafer is submerged for 10 minutes. RCA2 cleaning processes are especially needed for oxidation and diffusion furnaces free of metal contamination.

Both cleaning processes leave a thin oxide on the wafer. The wafer is briefly immersed in a 1% HF aqueous solution to remove the oxide prior to Si etching. Water is sprayed onto the oxide (hydrophilic) surface and drops onto the bare Si (hydrophobic) surface. In this way, it is possible to confirm the presence of residual oxides.

Most IC laboratories forbid the exposure of wafers to KOH in advance. Potassium may ruin the IC fabrication process. In relatively less stringent environments, wafer cleaning with RCA1 and RCA2 is carefully allowed (After Madou, M.J., Fundamentals of microfabrication, CRC press, Florida, USA (2002)).

After the RCA standard cleaning process, to form a porous silicon layer, a p-type silicon wafer with a resistivity of 14-17 Ωcm and a (100) orientation boron-doped and polished on one side was used as a starting material. The PSi layer was deposited on the silicon wafer via electrochemical etching. The oxidation treatment system consists of a specially manufactured Teflon cell, Pt (99.99%) wire cathode, and the wafer itself as an anode and an electrolyte solution. The composition of the electrolyte solution was HF (49%, CMOS grade, JTBaker): C ₂ H ₅ OH (95%, Ep, Daejung): Secondary distilled water = 1: 2: 1 by volume and high purity (99.99%) of platinum. The electrode was used as a counter electrode (reduction electrode). The current density was maintained at -7 mA / cm ² . The oxidation treatment time is preferably within 5 minutes, it was carried out for 300 seconds. The PSi substrate was oxidized with dry oxygen at 400 ° C. for 1 hour and then annealed with nitrogen at the same temperature for 15 minutes. Next, the AZ1512 photoresister was coated with a spin coater at 400 rpm.

As described above, as a result of forming the porous silicon layer under a condition of flowing a current density constant current, a very uniform porous silicon layer having a diameter of 2 μm and a depth of about 10 μm was formed (FIG. 1).

Second Step: Depositing Au and Ag Thin Films

Ti and Pt thin films were deposited by a RF-sputtering method as a counter electrode. In order to improve the adhesion between the oxide and Au formed on the porous silicon layer, the Au electrode was sputtered about 200Å of Ti and evaporated 2,500Å of Au. At this time, the base pressure of the vacuum chamber was 2 × 10 ⁵ mbar, the temperature of the substrate was maintained at 100 ℃ or less in consideration of the patterning process using a photoresist.

The working electrode was patterned using AZ-1512 positive photoresist, and the etching was performed using potassium iodide solution.

Pt target (99.98% purity, Heesung Metal Ltd.) is 9 cm x 1.5 mm suitable for loading into a vacuum coater system (Lecuold, LS560, Korea basic science institute, Seoul branch).

The deposition of Ag thin film for the fabrication of the reference electrode was sputtered with Ti at about 200 로서 and evaporated to 2,000Å. The metal deposition was followed by a heat treatment process at about 400 ° C. for 2 hours.

An electrochemical method was used to convert the Ag electrode into an Ag / AgCl reference electrode. Also known as "chloridation", the chloridation of Ag electrode was performed electrochemically in 1 mmol / L NaCl solution with a potential range of 0.6 to 1.2V.

The sputtering conditions are summarized in Table 1 below.

Sputtering Conditions for Ti, Pt Metal Deposition thickness Ti: 200 Hz, Pt: 2,000 Hz Substrate temperature 100 ℃ Bass pressure 2 × 10 ^-5 mbar Temperament-Target Distance 40 mm Sputtering pressure 5 × 10 ^-3 mbar Sputtering power 150 W Sputtering rate 5 ms / sec

Step 3: Form Self-Assembly Monolayer and Immobilize Urease

The gold thin film electrode deposited on the silicon wafer was washed for about 5 minutes at 80 ° C using piranha solution (30% H ₂ O: H ₂ SO ₄ = 1: 3). Soak for about 12 hours in 3-mercaptopropionic acid (2 mmol / L, ACROS) solution to form a self-assembled monolayer. After washing sufficiently with ethanol, NHS (3 mmol / L, N-hydroxysulfosuccinimide, Sigma Chemical Co.) and EDC (100 mmol / L, 1-ethyl-3- [3- (dimethylamino) -propyl] carbodiimide, Sigma The reaction was performed in 0.1 mol / L sodium phosphate buffer (pH 7.4) containing Chemical Co.) for about 3 hours. 24 hours in 5 mg / cm ³ urease (EC 3.5.1.5, type III, from Jack Bean, Sigma Chemical Co.) solution, washed with phosphate buffer and diluted in 0.1 mol / L sodium phosphate buffer (pH 7.0) solution The degree of soaking induced covalent bonding of the self-assembled monolayer and the enzyme at room temperature. The electrodes were then washed thoroughly in the following solution order: phosphate buffer (pH6.3), 1 mol / L NaCl, Milli Q Plus water and sodium phosphate buffer (pH 7.0).

2 briefly illustrates the immobilization through covalent bonding of a self-assembled monolayer and urease. 3 shows an overall process diagram for the above semiconductor process.

Experimental Example 1: Comparison of Conductivity in Body Fluids According to Electrode Types

The electrode configuration for comparing the conductivity in the body fluid according to the shape of each electrode was produced as shown in Figure 4 (a) to compare the characteristics.

Figure 4 (b) is a result of measuring the amount of current according to the shape of each electrode by the time zone current method, it was found to be proportional to the area of the electrode irrespective of the length of the interface of the electrode, the working electrode on both sides Among the four electrode patterns shown in FIG. 4 (a) where the reference electrode and the counter electrode are located, type 1 showed the best sensitivity characteristic.

Experimental Example 2: Investigation of Electrochemical Characteristics of Working Electrodes

The amount of current of the Au flat electrode and the electrode made of porous silicon as a substrate was considered by cyclic voltammetry.

As shown in Figure 5, it can be seen that the increase in surface area and the amount of current due to the structural difference of the substrate is proportional. An increase of about 10 times the amount of current was observed when the flat electrode.

FIG. 6 is a cyclic voltammogram obtained by forming a self-assembled monolayer on a gold thin film electrode, and since the amount of current is decreased after forming a single layer, the characteristics as an insulator can be observed.

Experimental Example 3: Surface Analysis of Enzyme Immobilized Membrane

The surface of the enzyme-immobilized working electrode was surface analyzed by X-ray photoelectron spectroscopy. Fig. 7A is a spectrum of the C 1s region, where peaks representing carbon-oxygen double bonds, carbon-oxygen single bonds, and carbon-carbon single bonds can be observed. (b) is a peak of a carbon-oxygen double bond and a carbon-oxygen single bond in the spectrum of the O 1s region. (c) shows the binding strength of nitrogen to the amide bond of the polypeptide chain in the spectrum of the N 1s region. XPS spectra indicate that urease and self-assembled monolayers are covalent.

Experimental Example 4: Sensitivity Measurement

The sensitivity of the sensor electrode according to urea concentration by time-phase ammeter was investigated by investigating the change of current with the concentration of serotonin over time under constant voltage (600mV).

As shown in Figure 8, it can be seen that the diffusion limit current also increases with increasing urea concentration. In particular, the time of 90% of the sensitivity was about 20 seconds, showing a fast response time. At this time, the composition of the electrolyte solution was KCl 0.1mol / L, PBS 1mmol / L, hydrogen ion concentration 7.4, the applied voltage was 600mV.

9 shows the sensitivity by the time zone current method as a correction curve. It can be seen that the diffusion limit current increases linearly with the concentration of urea. Urea was measured by concentration in the range of 0μmol / L ~ 100mmol / L, and the sensitivity in Urease / SAM / Au / Porous Si (device 1) was 11.21 μA / mMcm ² , and Urease / SAM / Au / Planar The sensitivity at Si (device 2) was measured to 4.39 μA / mMcm ² . In case of device 1, the sensitivity is increased by about 3 times than device 2.

In summary, as a result of fabricating a gold thin film electrode using a semiconductor process and forming a self-assembled monolayer on the gold electrode, the self-assembled monolayer was used as a film for immobilizing urease. Fast response time was shown within seconds. When the substrates were made of porous silicon and planar electrodes, the sensitivity was increased by about three times due to the effect of increasing the surface area. Self-assembled monolayers used in biosensors have the advantage of achieving uniform molecular thin films with ease of handling and stable bonding between proteins and solids. However, in the case of a protein having many functional groups that can be bound, a study should be conducted to compensate for the disadvantages such as steric hindrance effect due to loss of function and dense accumulation of proteins due to structural modification.

As described through the above examples and experimental examples, the present invention relates to a self-assembled monolayer for an immobilized membrane of a biosensor, and a self-assembled monolayer using 3-mercaptopropionic acid is excellent at low urea concentration due to direct electron transfer. Sensitivity and fast reaction time are exhibited. In particular, when porous silicon is used as a substrate, the sensitivity is increased by about three times than that of the planar electrode. Therefore, the present invention is a very useful invention in the pharmaceutical industry.

Claims (2)

Self-assembled monolayer, which is used as an immobilization film of a biosensor and is made of 3-mercaptopropionic acid. An element sensor comprising a self-assembled monolayer according to claim 1 as an immobilization film.

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