WO2012115501A1

WO2012115501A1 - Protective coating for biosensor membrane and method of forming said coating

Info

Publication number: WO2012115501A1
Application number: PCT/MY2012/000025
Authority: WO
Inventors: Mohd Rais Ahmad; Sagir Alva; Aiman Sajidah ABD AZIZ; Norazah ABDUL RASHID
Original assignee: Mimos Berhad
Priority date: 2011-02-22
Filing date: 2012-02-21
Publication date: 2012-08-30
Also published as: MY159008A

Abstract

A method of forming a protective coating over a chemical-sensing membrane of a biochemical sensor comprising polymerizing an alkyl methacrylate monomer with a polar monofunctional acrylate monomer, the acrylates preferably are methyl methacrylate and tetrahydrofurfurylacrylate (THFA). The copolymerization reaction shown below The methyl methacrylate is preferably mixed with tetrahydrofurfuryl acrylate in a ratio to derive a desired polarity of the resultant copolymer, and the polymerization occurs under UV photo-chemical reaction which a photo-initiator and a cross- linker. Lipophilic salt and ionophore may be added to the polymerization.

Description

Protective coating for biosensor membrane and method of forming said coating

TECHNICAL FIELD

This invention discloses a method for forming a protective coating over chemically-sensitive surfaces, such as chemically-sensing surfaces of a biosensor. In particular, the method includes a polymerisation process in which a copolymer coating is adhesively formed over the chemically-sensing surface.

BACKGROUND ART

Biochemical sensors such as those deployed wirelessly outdoors to monitor in situ moisture, temperature and various soil macronutrients (by way of their representative ions such as hydrogen for soil pH, potassium, nitrate and phosphate) for agriculture purposes typically comprised of miniaturized ion- selective electrodes (ISE) with chemical sensor array and integrated with reference electrode, moisture and temperature sensors, readout circuits, microcontroller, transmitter module and power supply unit. These components are thus exposed to the harsh outdoor elements, especially the sensitive ion-selective electrodes with polymeric chemically-sensing membranes placed in situ for the detection of the aforesaid soil macronutrients and parameters.

The electrode's sensing surface is typically a membrane made from polymeric matrix doped with plasticizer, lipophihc ionic agent and ionophores for recognizing and carrying a specific ion through the membrane. Common polymeric matrices are polyvinyl chloride (PVC), polyurethane (PU), acrylic, silicone rubber and polystyrene. PVC is preferred due to its long-term established reliability, plasticizer additive approach and solvent casting method in its fabrication method. PVC is suitable as a polymer base for the chemical-sensing membrane to detect ionic as well as neutral chemical species. PVC has, however, poor adhesion on electrode surface and would easily peel off, resulting in functional failure of the biosensor. U.S. Patent No. 4,743,352 is an example of an anion-selective membrane made of PVC on a sodium ion-selective electrode system used on a patient. In this prior art electrode, only a porous ceramic plug at the tip end is provided with an ion- selective membrane to minimise exposing the biosensing surface. Another approach is to monitor for deviation from reference values to indicate impairment or damage of the electrode such as that proposed in U.S. Patent Publication No. 2002/0027085. PCV-based sensing membranes is known to provide poor adhesion to ion-selective electrode (ISE) surfaces. Thus, sensing membrane peeling off electrode surface is a serious problem with most polymer matrices resulting in shortened lifetime of the biochemical sensor. This problem is agravated in the harsh outdoor, field environment particularly when exposed to high temperature and humidity. Thus, it would be desirable to provide a protective layer over the ion-selective membrane. In WO 92/04438, the glucose sensor's sensing and reference electrodes and enzyme layer are provided with a substrate-limiting layer made of polyurethane and a protection layer made of cellophane. Polyhydroxyethyl methacrylate is mentioned as a preferred hydrophilic material suitable as the protection layer; it is clear that such protection is not meant for harsh outdoor use. US 2005/0173245 discloses a protective encapsulating polymeric membrane which complements or even enhances the multiple chemical sensing capability of an electrode's sensing layer. This protective membrane is comprised of crosslinked polymers containing heterocyclic nitrogen groups. No acrylate co-monomers or copolymer is disclosed.

SUMMARY OF DISCLOSURE

It is thus an endeavour of our invention to provide a protective coating for the chemically sensing membrane of a biosensor so that the biosensor may be deployed outdoors, particularly in remote and harsh environment such as that for agriculture purposes. It would be desirable that such protective coating does not impede or affect the properties of the chemical-sensing membrane. In particular, the protective coating should have an electrical impedance matching that of the sensing membrane and not affect its ion-selectivity. The proposed protective coating should also have good adhesion to the sensing membrane and, consequently, assist the sensing membrane layer to adhere better onto the electrode so that peeling off the electrode surface may be prevented. Additionally, the coating should strive to protect and ensure the biosensing functions of the electrode to continue unaffected by the high field temperatures which could reach 40° - 60°C.

In the general aspect, our invention comprises a method of forming a protective coating over a chemical-sensing membrane of a biochemical sensor comprising polymerizing an alkyl methacrylate monomer with a polar monofunctional acrylate monomer to form said coating over said chemical-sensing membrane. Preferably, the alkyl methacrylate is methyl methacrylate while the polar monofunctional acrylate is tetrahydrofurfurylacrylate (THFA). The copolymerization may be represented in the following reaction.

In one aspect, methyl methacrylate is mixed with tetrahydrofurfuryl acrylate in a ratio to derive a desired polarity of the resultant copolymer, preferably, ranging from 2:8 to 3:7. Preferably, our method provides that the polymerization occurs under photo-chemical reaction. A photo-initiator and a cross-linker, such as hexanediol diacrylate, may preferably be used with ultraviolet radiation under constant nitrogen gas flow in the polymerization process. A lipophilic salt, which may be borate and/or ammonium salt, may preferably be added to the co-monomer mixture to be polymerized. Ionophore may also be added to the polymerization process.

In another aspect, the copolymer coating protectively formed on a chemical-sensing membrane of a biochemical sensor comprises of an alkyl methacyrlate monomer (preferably methacrylate is methyl) polymerized with a polar monofunctional acrylate monomer (preferably tetrahydrofurfuryl acrylate (THFA), said copolymer having the following molecular structure:

formed on the chemical-sensing membrane of a biosensor such as an ion-selective electrode (ISE) or ion-sensitive field effect transistor (ISFET).

LIST OF ACCOMPANYING DRAWINGS

The drawings accompanying this specification as listed below may provide a better understanding of our invention and its advantages upon reference with the detailed description that follows. These drawings are illustrative and exemplary only and may illustrate one or more non-limiting embodiments or examples of our invention in whole or in part. FIGURE 1 shows a graph plotting electrical response (mV/pH) against hydrogen ion activity for a pH sensor with a layer of protective coating according to our invention. FIGURE 2 illustrates a graph plotting electrical response (mV) versus potassium ion activity for a potassium sensor on PCB substrate with protective coat layer.

FIGURE 3 embodies a graph plotting electrical response (mV/dec) against potassium ion activity for a potassium sensor on FR4 substrate coated with a protective layer according to our invention.

FIGURE 4 exemplifies a graph plotting electrical response (mV) versus nitrate ion activity for a nitrate sensor with protective coating layer according to our invention.

FIGURE 5 shows a graph plotting electrical response (mV) against phosphate ion activity for a phosphate sensor with protective coating layer according to our invention.

DETAILED DESCRIPTION OF EMBODIMENTS

We have found a method for protecting the polymeric sensing membrane of a biochemical sensor which typically includes ion-sensitive electrode and the like. As will be presented in detail in the following description, the preferred embodiment of our method includes forming a protective layer over the sensing membrane using a photo-curable, plasticizer-free, polar copolymer adhesive coating layer. The proposed protective layer has shown good adhesion onto the sensing membrane as well as to the surrounding areas and walls of the electrode. More importantly, our layer of protective coating does not interfere with the functionality and performance of the biochemical sensor apart from enabling the electrode to tolerate operating above room temperature, i.e. at 40° - 60°C. Our general method of forming a protective coating over a chemical- sensing membrane of a biochemical sensor comprising polymerizing an alkyl methacrylate monomer with a polar monofunctional acrylate monomer to form said coating over said chemical-sensing membrane. The alkyl methacrylate may preferably be methyl methacrylate, which molecular structure is shown below.

Methyl methacrylate For the polar monofunctional acrylate co-monomer, we have found that tetrahydrofurfurylacrylate (THFA) is most advantageous for providing properties such as low impedance, ease of handling, non-stickiness while promoting adhesion to surfaces being coated. A molecular structure of THFA is shown below.

Tetrahydrofurfurylacrylate

We have attempted co-polymerizing the methyl methacrylate with THFA in different volume ratios under ultra-violet photocuring conditions to arrive at a most desirable self-plasticizing coating over the sensing membrane. The preferred ratios may range from 2:8, i.e. 2 parts of methyl methacrylate being mixed with 8 parts of tetrahydrofurfurylacrylate, to 3:7. The ratios may also be varied to arrive at a resultant copolymer with the desired polarity and impedance properties. The polymerization may preferably occur under photo-chemical reaction conditions which may be defined as including photo-initiation, photo- polymerization, photo-curing and like photo-reactions. Preferably, at least a photo- initiator or a cross-linker may be advantageously used with the UV radiation in the polymerization process. The cross-linker may preferably be a diacrylate; most preferably hexanediol diacrylate, specifically 1,6-hexanediol diacrylate (as shown in the molecular formula below), which is commonly used as a cross-linking agent in UV curing, inks and coatings.

hexanediol diacrylate

Other additives advantageous to the reaction may be added as preferred embodiments. For example, the photo-chemical reaction may be allowed to run under constant nitrogen gas flow for a predetermined time to enable the coating layer to be cured. Additionally, a lipophilic salt may be added to the co-monomers mixture to be polymerized together so that the resulting copolymer coating may have a lower impedance. The lipophilic salt may be any one or combination of borate and ammonium salt, which may be added to mimic the ionic selectivity of the chemical sensing membrane of the biosensor electrode. Another advantageous additive that may be included is a suitable ionophore for the specific ion to be detected by the biosensor electrode. In combination, the lipophilic salt and the ionophore help to prevent blockage of ions and signal flow at the protective coating.

The overall copolymerization process may be represented by the following reaction wherein monomer 1 is methyl methacrylate is reacted with monomer 2 tetrahyrofurfuryl acrylate under UV photo-reaction to produce the resultant copolymer 3.

The resultant co-polymer have been successfully coated over ion-selective electrodes (ISE) and ioon-sensitive field effect transistors (ISFET) as would be shown in the following examples as some of our actual tests and trials conducted.

Example 1

Preparation of co-monomers cocktail

A co-monomers cocktail comprising 2 parts of methyl methacrylate and 8 parts of tetrahydrofurfurylacrylate by volume is prepared. In addition to 200 μΐ. of methyl methacrylate 800 μΐ tetrahydrofurfuryacrylate (THFA), cross-linking additives comprising 1.05 μ] of 2-hexanedioldiacrylate (HDDA) was also added using micro-pipette into a 5 ml vial. The vial was capped tightly and wrapped with paraffin film to avoid evaporation of volatile components. The mixture was then sonicated at room temperature for 2 minutes to get a homogenous mixture. The vial is stored in the refrigerator if not used.

Example 2

Preparation of protective coating for pH sensor The freshly prepared co-monomers cocktail (about 100 μΙ) from Example 1 in a 5 ml vial is now added with a photoinitiator and two ionophores, specifically 1 mg 2,2-dimethoxy-2-phenylacetophenone (DMPA), 3 mg sodium tetrakis[bis-3,5 trifluoromethyl)phenyl]borate (NaTFPB) and 10.6 mg hydrogen ionophore I (tridodecylamine or [CH3(CH2)n]3N) were added into the cocktail. The vial was capped tightly and wrapped with paraffin film to avoid evaporation of volatile components. The mixture was sonicated at room temperature for 2 minutes to get homogenous mixing of the components.

The protective coating cocktail in about 1 μΐ was dispensed along the edge of freshly fabricated and characterized pH-sensing membrane of an ion-selective electrode and photocured under UV radiation in nitrogen ambient for 180 seconds. The pH ISE sensor was prepared using solvent cast technique using high molecular weight PVC matrix with hydrogen ionophore I in tetrahydrofuran (THF) solvent. The protected pH sensor was characterized for response to hydrogen ion at pH 4, 7 and 10 in Table 1 below.

Table 1

The plot of potentiometric response versus hydrogen ion activity gives a near Nernstian-slope and a high correlation coefficient may be seen in FIGURE 1. The result shows that the protective coating layer does not block the flow of signal.

Example 3

Preparation of protective coating layer for potassium sensor on PCB substrate

The freshly prepared co-monomers cocktail (100 μΐ) from Example 1 was pipetted into a 5 ml vial and added thereto with a photoinitiator, i.e. 1 mg 2,2- dimethoxy-2-phenylacetophenone (DMPA), and two ionophores, i.e. 1.4 mg potassium tetrakis(4-chlorophenyl)borate and 6.3 mg valinomycin respectively were carefully weighed and mixed with the co-monomers cocktail mixture of the protective coating. The vial was capped tightly and wrapped with paraffin film to avoid evaporation of volatile components. The mixture was sonicated at room temperature for 2 minutes to get homogenous mixing of the components.

1 μΐ of the coating layer cocktail was dispensed on top, and the surrounding edges, of the potassium sensing membrane which has been freshly fabricated on the PCB board. The electrode's coated potassium sensing membrane is then irradiated under UV lamp in nitrogen ambient for 180 seconds. The potassium ISE sensor was prepared using solvent cast technique using high molecular weight PVC matrix and valinomycin ionophore in THF solvent. The potassium ISE sensor on PCB board with protective coating layer was then characterized for potassium response in 10 ¹ M to 10⁴ M potassium chloride solutions versus conventional double-junction reference electrode as per Table 2 below.

Table 2:

Response of Potassium Sensor with

Protective Coating Layer on PCB Substrate

The plot of emf response versus activity of potassium ion for the potassium sensor on PCB substrate is shown in FIGURE 2 wherein the graph is close to ideal slope and good linearity. The result shows that the coating layer is compatible with the potassium membrane.

Example 4

Preparation of Protective Coating Layer for Potassium Sensor on FR4 Substrate The protective coating layer has also been applied on potassium ISE sensor fabricated on FR4 substrate with poly(pyrrole) conductive electrode. The freshly prepared co-monomers cocktail (100 μΐ) prepared in Example 1 above was pipetted into a 5 ml vial and a photoinitiator, i.e. 1 mg of 2,2-dimethoxyl-2- phenylacetophenone (DMPA) with two ionophores, i.e. 1.4 mg potassium tetrakis(4- chlorophenyl) borate and 6.3 mg valinomycin were carefully weighed and mixed with the co-monomers cocktail. The vial was capped tightly and wrapped with paraffin film to prevent evaporation of volatile components. The mixture was sonicated at room temperature for 2 minutes to get a homogenous mixing of the components.

1 μΐ of the co-monmers cocktail was dispensed over and on surrounding the edges of a freshly fabricated potassium sensing membrane on FR4 substrate and irradiated under UV lamp in nitrogen ambient for 180 seconds. The potassium ISE sensor was prepared using solvent cast technique using high molecular weight PVC matrix and valinomycin ionophore in THF solvent. The potassium ISE sensor on FR4 substrate protective coating layer was characterized for potassium response in 10 ¹ M to 10⁴ M potassium chloride solutions versus conventional double-junction reference electrode according to the figures in Table 3 below.

Table 3

Response of Potassium Sensor with

Protective Coating Layer on FR4 Substrate

The plot of emf response versus activity of potassium ion is shown in FIGURE 3 wherein it may be seen that the graph affords Nernstian slope and ideal correlation coefficient. The result shows that the coating layer does not block flows of ions or signals and is suitable for the potassium sensing membrane.

Example 5 Preparation of Protective Coating Layer for Nitrate Sensor

The protective coating layer has also been applied on nitrate ISE sensor fabricated on PCB board with poly(pyrrole) conductive electrode. The freshly prepared co-mononer cocktail (100 μΐ) prepared in Example 1 was pipetted into a 5 ml vial and a photoinitiator, i.e. 2.0 mg 2,2-dimethoxyl-2-phenylacetophenone (DMPA) and an ionophore, i.e. 3.0 mg tetraoctylammonium nitrate were carefully weighed and mixed with the co-monomer cocktail. The vial was capped tightly and wrapped with paraffin film to avoid evaporation of volatile components. The mixture was sonicated at room temperature for 2 minutes to get homogenous mixing of the components.

The coating layer cocktail (1 μΐ) was dispensed on top and surrounding the edges of freshly fabricated nitrate sensing membrane on PCB board and irridiated under UV lamp in nitrogen ambient for 180 seconds. The nitrate ISE sensor was prepared using solvent cast technique using high molecular weight PVC matrix in THF solvent. The nitrate ISE sensor on FR4 substrate protective coating layer was characterized for nitrate response in 10 ¹ M to 10⁴ M potassium nitrate solutions versus conventional double-junction reference electrode, which response is tabulated in Table 4 below.

Table 4:

Response of Nitrate Sensor with Protective Glue Layer

The plot of emf response versus activity of nitrate ion is shown in FIGURE 4 wherein is shown that the graph affords near Nernstian slope and acceptable linearity. The result shows that the co-polymer coating layer is also compatible th anion sensing membrane and applicable for nitrate sensor.

Example 6

Preparation of Protective Coating Layer for Phosphate Sensor

The protective coating layer has also been applied on dibasic phosphate ISE sensor fabricated on PCB board with poly(pyrrole) conductive electrode. The freshly prepared co-monomer cocktail (100 μΐ) prepared in the above was pipetted into a 5 ml vial. A photointiator, i.e. 2.0 mg 2,2-dimethoxyl-2-phenylacetophenone (DMPA), and 3.0 mg tetradodecyl ammonium chloride were carefully weighed and mixed with the glue cocktail. The vial was capped tightly and wrapped with paraffin film to avoid evaporation of volatile components. The mixture was sonicated at room temperature for 2 minutes to get homogenous mixing of the components.

The coating layer cocktail (1 μΐ) was dispensed on top and surrounding the edges of freshly fabricated dibasic phosphate sensing membrane on PCB board and irradiated under UV lamp in nitrogen ambient for 180 seconds. The dibasic phosphate ISE sensor was prepared using solvent cast technique using high molecular weight PVC matrix in THF solvent. The dibasic phosphate ISE sensor on FR4 substrate protective glue layer was characterized for dibasic phosphate response in 10 ¹ M to 10⁴ M potassium phosphate dibasic solutions, buffered at pH 8 with acetic acid-sodium hydroxide versus conventional double-junction reference electrode (see Table 5 below).

Table 5

Response of Dibasic Phosphate Sensor

with Protective Coating Layer

HP04

Activity K063 K064 K069

-4 424.9 401.0 413.3

-3 358.4 345.0 357.8

-2 292.6 281.4 293.4

-1 254.0 242.6 254.4

Slope -57.87 -53.86 -54.09

C 187.82 182.83 194.52

R2 0.9865 0.9913 0.9914 The plot of emf response versus activity of nitrate ion is shown in FIGURE 5 and which affords near Nernstian slope and acceptable linearity. The result shows that the glue layer is also compatible with anion sensing membrane and applicable for nitrate sensor.

While it might be possible to engineer a copolymerization process to form a copolymer coating over the chemically-sensing membrane of the biosensor electrode, the choice of methyl methacrylate and tetrahydrofurfyl acrylate under UV photo-reaction offers the advantage of spatial and temporal control of the copolymerization process over the target area, in addition to a solventless process. Photoinitiated free radical polymerization of multifunctional monomers also produces highly crosslinked networks with high thermal stability, mechanical strength and resistance to solvent absorption. These copolymers offer good characteristics that include colorless film, good flexibility, not sticky, and good adhesion on electrode surface. Additionally, the polarity of these copolymer can be varied depending on the ratios of methyl methacrylate and tetrahydrofurfural acrylate.

It should be noted that apart from the aforedescribed embodiments of our process, solution and the resultant copolymer coating, there are many aspects or advantages of our invention that may be presented or achieved in other variations, substitution or modifications to the many compounds and reagents suggested above without departing from the essence and working principles of the invention. Such suitable variations, alternates, analogs or equivalents are to be considered as falling within the letter and scope of the following claims.

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Claims

METHOD CLAIMS: 1. A method of forming a protective coating over a chemical-sensing membrane of a biochemical sensor comprising polymerizing an alkyl methacrylate monomer with a polar monofunctional acrylate monomer to form said coating over said chemical-sensing membrane.

2. A method of forming a protective coating according to Claim 1 wherein the alkyl methacrylate is methyl methacrylate.

3. A method for forming a protective coating according to Claim 2 wherein the polar monofunctional acrylate is tetrahydrofurfurylacrylate (THFA).

4. A method of forming a protective coating according to Claim 3 wherein methyl methacrylate is mixed with tetrahydrofurfurylacrylate in a ratio of 2 to 8.

5. A method of forming a protective coating according to Claim 3 wherein methyl methacrylate is mixed with tetrahydrofurfurylacrylate in a ratio of 3 to 7.

6. A method of forming a protective coating according to Claim 3 wherein the methyl methacrylate is mixed with tetrahydrofurfurylacrylate in an

appropriate ratio to derive a desired polarity of the resultant copolymer.

7. A method of forming a protective coating according to Claim 3 wherein the polymerization occurs under photo-chemical reaction.

8. A method of forming a protective coating according to Claim 7 wherein at least one of a photo-initiator and a cross-linker is used with ultra-violet radiation in the polymerization process.

9. A method of forming a protective coating according to Claim 8 wherein ultra-violet radiation is extended with a constant flow of nitrogen gas to cure the coating.

10. A method of forming a protective coating according to Claim 8 wherein the cross-linker is a diacrylate, including hexanediol diacrylate.

11. A method of forming a protective coating according to Claim 3 wherein a lipophilic salt is added to the co-monomer mixture to be polymerized.

12. A method of forming a protective coating according to Claim 11 wherein the lipophilic salt is any one or combination of borate and ammonium salt.

13. A method of forming a protective coating according to Claim 3 wherein ionophore is added to the polymerization process. PRODUCT CLAIMS:

14. A copolymer coating protectively formed on a chemical-sensing membrane of a biochemical sensor, wherein said copolymer comprising an alkyl methacyrlate monomer polymerized with a polar monofunctional acrylate monomer.

15. A copolymer coating according to Claim 14 wherein the alkyl

methacrylate is methyl methacrylate and the polar monofunctional acrylate is tetrahydrofurfuryl acrylate (THFA) having the following molecular structure.

16. A copolymer coating according to Claim 15 wherein methyl methacrylate is mixed with tetrahydrofurfurylacrylate in any one ratio ranging from 2:8 to 3:7.

17. A copolymer coating according to Claim 15 further including a lipophilic group derived from a lipophilic salt added in the copolymerization process.

18. A copolymer coating according to Claim 17 wherein the lipophilic salt is any one or combination of borate and ammonium salt.

19. A copolymer coating according to Claim 15 further including at least an ionophore which is added in the copolymerization process.

20. A biochemical sensor incorporating a copolymer coating according to any one of Claims 14 to 19 wherein the chemical-sensing membrane is comprised in any one of an ion-selective electrode (ISE) or ion-sensitive field effect transistor

(ISFET).

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