|Title of Invention||
A DRY METHOD FOR SURFACE MODIFICATION OF SU-8 FOR IMMOBILIZATION OF BIOMOLECULES USING HOTWIRE INDUCED PYROLYTIC PROCESS
|Abstract||Biosensors and bioreactors require immobilization of biological molecules within the device.With advancements of microfabrication technology,such biosensors have been miniaturised using standard microfabrication techniques and substrates such as silicon,its derivatives(e.g.silicon dioxide,silicion nitride,etc.),and noble metals like gold.Off late the polymer called SU-8(Glycidyl ether of bisphenol A)has attracted attention due to the fact that low Young's modulus and/or high aspect ratio structures can be created using SU-8.However there was lack of processes to immobilize biomolecules on SU-8.The method developed and described in this document pertains to a novel and inexpensive method modifying the SU-8 surface in order to make it amenable to immobilization of biomolecules.Further this invention provides SU-8 substrates with biomolecules(e.g.antibody,antigen,DNA,RNA and Enzymes etc.)immobilized on the surface.|
|Full Text||F O R M - 2
THE PATENTS ACT, 1970
(39 OF 1970)
PROVISIONAL /COMPLETE SPECIFICATION
(See section 10; rule 13)
TITLE OF INVENTION
" A Dry Method for Surface Modification of SU-8 for Immobilization of Biomolecules using Hotwire Induced Pyrolytic Process "
(a) INDIAN INSTITUTE OF TECHNOLOGY Bombay (b) having administrative office at Powai, Mumbai 400076, State of Maharashtra, India and (c) an autonomous educational Institute, and established in India under the Institutes of Technology Act 1961.
The following specification particularly describes the nature of the invention and the manner in which it is to be performed.
Field of invention
This invention relates to a rapid and simple process for activation of epoxy surfaces such as SU-8 (Glycidyl ether of bisphenol A) to enable surface immobilization of molecules with carboxyland / or amino functional groups.
Background of the invention
Microsystems for biological applications require immobilization of biological molecules within the device. Conventionally, substrate materials used for these devices have been silicon and noble metals (e.g. gold) which have been found to have shortcomings in terms of their high Young"s modulus, biocompatibility and their suitability for micro fabrication.
Immobilization of substances, especially of bio-molecules onto epoxy surfaces such as SU-8 is of interest because of their application in surface modification, bioMEMS, biomolecule immobilization related assays, biosensors, membrane bioreactors, clinical diagnostics, molecular biology, agriculture, environmental science, chemical or biochemical industry etc.
SU-8 provides a good substrate for fabrication of miniaturized biosensors. However the bare SU-8 pattemed with conventional photolithography techniques does not allow the immobilization of antibodies on its surface. SU-8 surface therefore needs to" be activated to immobilize the biomolecules for the fabrication of bio-MEMS structures.
Several methods are known for the modification of polymer surfaces. However, these are not suitable for SU8 surface modifications.
Publications titled "Study on single-bond interaction between amino-terminated organosilane self-assembled monolayer by atomic force microscopy," (2000) Surface Science 459, 401-412; "Preparation of smooth siloxane surfaces for AFM visualization of immobilized biomolecules" Surface Science, volumes 532-535, 1085-1091; "Covalent immobilization of GL-7-ACA acylase on silica gel through silanization," (2002) Reactive and Functional Polymers 51, 79-92 describe methods of introducing NH2 group on the surface generated by coating aminosilane on the polymer surface in liquid phase. However in these wet methods, strong oxidizing/hydrolyzing agents (acids/bases) are used that damage the sensor surface and the bulk of the substrate. Such multiple steps and time consuming wet methods need fine control of process parameters with repetitive immersion, washing and drying thereby increasing the possibilities of causing structural damage to the microsensor.
US Patents 4415665, 4582875, 4562157, European patent No. 87401000 and PCT Publication WO03083477 describe techniques to prepare hydrophilic polymer surfaces by introducing carboxyl groups, carbonyl groups and hydroxyl groups on the polymer surface using acid/base solutions.
US Patents 5922161, 4833093, 3956179, 5077210 describe methods to create hydroxyl groups on polymer surfaces and further reacting them with multifunctional amino compounds to provide functionality to directly react with proteins.
The strong reagents used in the wet methods may severely damage the sensor surfaces and also affect the bulk properties of the substrate.
Dry methods involving plasma treatment have also been reported. Publication titled "Current trend in biomaterial surface functionalization-nitrogen-containing plasma assisted processes with enhanced selectivity" Vaccum (2003) 71, 391 -406 describes a method in which the polymer surface is activated by introducing hydroxyl or amino groups by a plasma technique, employing oxygen or ammonia. Low-pressure gas discharge plasmas are used for polymer surface modification. Plasma treatment is known to cause plasma damage to surface and bulk of the substrate material. It is known that suspended microstructures when treated with plasma can get damaged.
Similarly Polymer surface modification using UV treatment in NH3 atmosphere has been reported in a publication titled "Cell proliferation on UV-excimer lamp modified and grafted polytetrafluoroethylene," Nuclear instrument and Method of Physical Research (2004) B217, 307-313. In this publication grafting of NH2 on the polymer surface is achieved by exposing the polymer surface to UV radiation in the range 200 to 400 nm in NH3 atmosphere. The time required for polymer surface modification using UV is fairly long (10 to 12 hours). Similarly US Patent 5051312 discloses a process for modifying polymer surfaces using ultraviolet and/or visible radiation while such surfaces are in contact with modifiers. A major shortcoming is that UV radiation is known to affect the bulk of the sensitive polymers.
Polymer surfaces have also been modified using photochemicals. Publication titled "Biomaterial Surface Modification Using Photochemical Coupling Technology," in Encyclopedic Handbook of Biomaterials and Bioengineering, Part A: Materials, New York, Marcel Dekker, pp 895-926, 1995 describes a photochemical method for oovalent attachment of active functional group onto inert solid surface. This method is based on a compound having at least two functional groups of which one is essentially a photoactivable group. There are different methods for activation of inert surfaces through a photo linker. This method uses expensive or difficult to prepare photoactivable compounds. Further substrates derivatized with photochemical species are sensitive to UV light and hence have storage problems.
US Patent 5427779 describes a method for photochemical immobilization of molecules to polystyrene or other plastic materials. The invention is based on the photo reactive poly-cyclic compounds, which are firmly bound to a variety of polymer surfaces when exposed to electromagnetic radiation.
US Patent Application 2003228410 relates to a process for the preparation of activated polymer surface capable of forming a covalent bond with the biomolecules by coating the surface of a polymer with a photo linker (1-fluoro-2-nitro-4-
azidobenzene) and subjecting the dry photo linker-coated polymer surface to UV or sunlight for 1 second to 60 minutes followed by removal of the unattached compound by washing thoroughly with a methanol and drying at ambient temperature. The photo-chemicals used are difficult to synthesize making the process commercially unviable.
PCT Publication WO0119903 describes a method for modifying polymer surfaces by irradiating energized hydrogen ions on the surface of a polymer material at a distance between 15cm and 90cm, while blowing reactive gas or gases around the surface of the polymer material under vacuum. Besides the requirements of high vacuum, it also requires a high irradiation dose of hydrogen ion (1015~1017 ions/cm2) and energy of the order of a few keV which can severely damage the surface and bulk of sensor and adjacent devices.
EP0155252 describes how chemical groups can be grafted on the surface of plastic as a thin film ( Japanese Patent JP60135414 describes a process in which the surface of a polymeric substrate is subjected to glow discharge by applying a high-frequency voltage in vacuum. A desired liquid monomer (or a solution of a monomer) is placed in a sealed container and, after evacuation, is subjected to glow discharge and immediately brought in contact with the glow discharge-treated substrate under exclusion of oxygen to form a graft-polymerized layer. US Patent Application 2003207099 describes the use of a polymeric membrane having a stable low-contact angle and used as a template in forming biological micro arrays. The membrane surface is modified by a first plasma treatment using oxygen, nitrogen, helium or argon and subsequently subjected to second plasma such as Silicon tetrachloride (SiCl4) gas. The resulting membrane allows a solution containing a biological material to wet the surface of the membrane such that the membrane can quickly and easily form a biological micro array on substrate in which the features of the array are distinctly formed on the substrate. Such plasma treatments apart from being complex and expensive can cause damage to the polymer surface and severely degrade the functionality of adjacent devices and electronics. Suspended microstructures when treated with plasma, can suffer damages caused by plasma pressure.
The limitations of the prior art suggests the longstanding need to develop processes for attachment of biomolecules to the polymers amenable to microfabrication techniques without damaging the polymer surface, the adjacent sites and the bulk. Since the biomolecules have to remain functional, these have to be attached to the surface by processes that do not impair their activity. Further, requirements of
miniaturization also dictate that such polymers be amenable to microfabrication techniques (lithography, pattern development etc.).
Summary of the invention
The main object of the invention is to provide a rapid and simple process for activation of epoxy surfaces such as SU-8 without damage to surface of interest, adjacent surfaces and the bulk to enable surface immobilization of molecules with carboxyl and amino functional groups.
Another object of the invention is to provide a process for immobilization of biomolecules on epoxy surface by grafting amino groups on its surface.
Another object of the invention is to develop microfabrication technology compatible process for attachment of amino group on epoxy surfaces such as SU-8 surface.
Yet another object is to develop a dry process for surface modification without damaging the sensor surface and its structure.
Yet another object of the invention is to modify only the epoxy surface without affecting its bulk properties.
Yet another object is to achieve a high throughput of activated substrates by reducing the processing time required for surface modification.
Yet another object is to eliminate the possibility of thermal damage to the surface and bulk while processing the epoxy surface.
Yet another objective is to make the modified epoxy surface amenable for immobilization of biomolecules through linker attachments such as glutaraldehyde etc.
Thus the process of this invention involves grafting of NH2+ groups on the epoxy (e.g. SU-8) surface using hotwire induced pyrolytic decomposition of ammonia under vacuum, followed by antibody immobilization on the treated SU-8 surface.
The process of the invention enables single or multi-step tailored immobilization of biomolecules (antigen, antibodies, proteins, DNA, RNA enzymes etc.) on the modified epoxy surface.
Detailed description of the invention
The process is now described with a preferred embodiment wherein the substrate is SU-8.
SU-8 is patterned on various surfaces like silicon, gold on silicon dioxide, etc. using photolithography techniques. The surface so obtained is tailored by grafting amino groups by dissociation of ammonia using a hotwire induced pyrolytic process.
The process of surface modification of the SU-8 comprises:
• Loading of substrate coated with SU-8 on substrate holder inside a hot wire CVD chamber.
• Evacuating moisture and residual gases from the chamber
• Maintaining the gas pressure of 8 mBar to 1 Bar
• Passing ammonia at a controlled flow rate into the chamber while maintaining preset gas pressure in the chamber.
• Raising the filament temperature (1100 to 1900°C) to disassociate ammonia, and treat the SU-8 surface for preset time 1 - 30 minutes.
• Unload the substrate
The modified surface is investigated using grazing angle method of FTIR. The peak at 1607 cm"1 in the treated sample (Fig 2) shows the presence of NH2 group on the surface of the substrate. It may be observed that the untreated sample does not show the peak at 1607 cm"1 (Fig 1)
Evaluation of the Modified Substrate
The antibody (HIgG) is incubated on the modified SU-8 surface with and without linker molecules. The non specific sites of antibodies on the surface are blocked using BSA solution. To identify the grafted layer of HIgG, FITC tagged Goat anti HIgG is allowed to react with the HIgG immobilized surface. The SU-8 surface is washed with PBS solution and de-ionized water after each step of antibody immobilization. The FITC tagged Goat anti HIgG immobilized SU-8 surface is investigated by fluorescence microscopy. Fluorescence excitation wavelength of 450-490 nm and emission sensitivity above 520 nm is used for the studies. Presence of fluorescence indicates that antibodies have attached to the surface. The intensity of the fluorescence gives an indication of the the number of fluorescent molecules that have attached to the surface, thereby providing a qualitative measure of the immobilization that has taken place. It may be noticed in Figure 3a and 3b that immobilization has not taken place on SU-8 surface that has not undergone the process of surface modification
The invention is illustrated using a few non-limiting examples. The Table 1 presents the effect of process parameter variations on observable immobilization.
SU-8 is coated on the silicon surface using spin coating followed by photolithography without patterning it. The surface is modified by dissociation of ammonia using hotwire induced pyrolytic process for 10 minutes. The process parameters used in the Hotwire chamber are: Gas pressure (Pg) = 200mBar, Filament temperature (Tf) = 1130 deg C,
Substrate temperature = room temperature. The treated surface is dipped in 1% aqueous solution of (glutaraldehyde) for 30 minutes followed by incubation of Human immunoglobulin (HlgG) on it. The non specific sites of antibodies and unsaturated aldehyde sites on the surface are blocked using BSA solution. To identify the grafted layer of HlgG, FITC tagged Goat anti HlgG is allowed to react with the HlgG immobilized surface. The SU-8 surface is washed with PBS solution and deionized water after each step of antibody immobilization. The immobilized SU-8 surface is investigated using a fluorescent microscope.
The antibody immobilized SU-8 surface is observed under optical microscope to understand its surface features (Fig.4a) followed by the observation of the same spot using fluorescent microscope (Fig.4b). Uniform and dense fluorescence is observed on the SU-8 surface which is incubated with FITC tagged goat anti HigG (Fig.4b).
Substrate Filament Temperature
(°C) Gas Pressure
(mBar) Process Time
(minutes) Example and
number Observation and Remarks
SU-8 1130 200 10 Example 1, Figure 4 Good Immobilization
SU-8 and gold pattern 1800 200 10 Figure 5 Good Immobilization
SU-8 1800 8 10 Figure 6 Good Immobilization
SU-8 1800 200 1 Figure 7 Good Immobilization
SU-8 1130 8 1 Figure 8 Moderate Immobilization
SU-8 pattern on gold 1800 200 5 Figure 9 Good Immobilization
SU-8 pattern on Silicon 1800 200 5 Example 2 Figure 10 Immobilization done 45 days after surface activation. Good Immobilization
SU-8 pattern on Silicon 1800 200 5 Example 3 Figure 11 HlgG immobilzed initially. 45 days later FITC tagged GaHIGg was applied. Good Immobilization.
Activity of ammonia treated SU-8 surface towards antibody immobilization is investigated after 45 days. SU-8 is patterned on silicon surface using standard photolithography technique. The surface so obtained is modified by dissociation of ammonia using hotwire induced pyrolytic process for 5 minutes. The process parameters used in the Hotwire chamber are: Gas pressure = 200 mBar, Filament
temperature = 1800 deg C, Substrate temperature = room temperature. This sample is stored for 45 days in refrigerator at 4 deg C. After 45 days, the treated surface is dipped in 1% aqueous solution of homo-bifunctional linker (glutaraldehyde) for 30 minutes followed by incubation of antibodies (HlgG).The non specific sites of antibodies and unsaturated aldehyde sites on the surface were blocked using BSA solution. FITC tagged Goat anti HlgG is allowed to react with the HlgG immobilized surface. The SU-8 surface is washed with PBS solution and deionized water after each step of antibody immobilization. The immobilized SU-8 surface is observation using fluorescent microscope.
The antibody immobilized SU-8 surface is observed under optical microscope to understand its surface features (Fig.10a) followed by observation of the same spot using fluorescent microscope (Fig.tOb). Uniform and dense fluorescence is observed on the SU-8 surface which is incubated with FITC tagged goat anti HlgG.
Activity of ammonia treated SU-8 surface immobilized with Human Immunoglobulin (HlgG) towards FITC tagged goat anti HlgG is investigated after 45 days. SU-8 is patterned on silicon surface using standard photolithography technique. The surface so obtained is modified by dissociation of ammonia using hotwire induced pyrolytic process for 5 minutes. The process parameters used in the Hotwire chamber are: Gas pressure = 200 mBar, Filament temperature = 1800 deg C, Substrate temperature = room temperature. The treated surface is dipped in 1% aqueous solution of homo-bifunctional linker (glutaraldehyde) for 30 minutes followed by incubation of antibodies (HlgG).The non specific sites of antibodies and unsaturated aldehyde sites on the surface were blocked using BSA solution. This sample is stored for 45 days in refrigerator at 4 deg C. After 45 days, FITC tagged Goat anti HlgG is allowed to react with the HlgG immobilized surface. The SU-8 surface is washed with PBS solution and deionized water after each step of antibody immobilization. The immobilized SU-8 surface is characterized using fluorescent microscope.
The antibody immobilized SU-8 surface is observed under optical microscope to understand its surface features (Fig. 11a) followed by observation of the same spot using fluorescent microscope (Fig. 11b). Uniform and dense fluorescence is observed on the SU-8 surface which is incubated with FITC tagged goat anti HlgG.
It is evident that the epoxy surface such as SU-8 when treated using the process of this invention provides a surface amenable to immobilization of biomolecules by grafting amino groups on the epoxy surface using hot wire induced pyrolytic dissociation of ammonia without thermal damage to the surface and bulk of substrate. Further, such a treated surface is compatible with micro fabrication technology and reduces problems of stiction associated with wet processes as described in prior art.
1. A process for modification of epoxy surfaces without substantially damaging the
surface and bulk to enable surface immobilization of molecules with carboxyl
and amino functional groups wherein the said epoxy surface is modified in the
• loading of substrate in a CVD chamber;
• evacuating the chamber;
• passing ammonia at a controlled flow rate into the chamber while maintaining gas pressure in the chamber;
• raising the filament temperature to dissociate ammonia to treat the substrate surface;
• unloading the modified epoxy surface.
2. A process for modification of epoxy surfaces as claimed in claim 1 wherein the epoxy surface is SUB.
3. A process for modification of epoxy surfaces as claimed in claim 1 wherein the filament temperature in the CVD chamber is 1130°C to 1900°C.
4. A process for modification of epoxy surfaces as claimed in claim 1 wherein pressure in the CVD chamber is of 8 mbar to 1 bar.
5. A process for modification of epoxy surfaces as claimed in claim 1 wherein ammonia flow rate is of 10 to 30 sccm.
6. A process for modification of epoxy surfaces as claimed in claim 1 wherein the molecules with carboxyl and amino group are selected from antigen, antibodies, proteins, DNA, RNA and enzymes.
7. A process for modification of epoxy surfaces as claimed in claim 1 wherein SU8 is optionally coated on silicon, gold on silicon dioxide, glass, plastic, or any other substrates on whch SU8 can be coated.
8. A process for surface immobilization of molecules with carboxyl and amino functional groups on the modified SU8 surface comprising of the following steps:
optionally attaching a linker molecule such as glutaraldehyde on the modified SU8 surface
reacting the original modified SU8 surface or the surface derived from step (i) with the biomolecule of interest to obtain biomolecule immobilized SU8 surface.
An amino modified SU8 surface.
Amino modified SU8 surface immobilized with molecules with carboxyl and amino group which are selected from antigen, antibodies, proteins, DNA, RNA or enzymes.
|Indian Patent Application Number||1267/MUM/2004|
|PG Journal Number||13/2008|
|Date of Filing||25-Nov-2004|
|Name of Patentee||INDIAN INSTITUTE OF TECHNOLOGY|
|Applicant Address||INDIAN INSTITUTE OF TECHNOLOGY, POWAI, MUMBAI 400 076|
|PCT International Classification Number||G01N33/543|
|PCT International Application Number||N/A|
|PCT International Filing date|