Sol-gel method for encapsulating molecules

Details Sol-gel method for encapsulating molecules Sol-gel method for encapsulating molecules

2000-04-13

2002-12-17

Ceperley, Mary E. (Department: 1641)

Chemistry: molecular biology and microbiology

Carrier-bound or immobilized enzyme or microbial cell;...

Enzyme or microbial cell is immobilized on or in an...

C435S007930, C435S026000, C435S028000, C436S527000, C436S815000, C436S829000

Reexamination Certificate

active

06495352

ABSTRACT:

BACKGROUND OF THE INVENTION
The invention relates generally to a sol-gel method for encapsulation of organic molecules and more particularly, to a sol-gel encapsulation method using an alkali metal silicate precursor.
Sensors using immobilized organic-based receptors are finding ever-increasing application in a wide variety of fields including clinical diagnostics, environmental monitoring, food and drinking water safety, affinity chromatography, enzyme reactors, and illicit drug monitoring. Important in the development of these sensors is immobilization and integration of biological molecules (biomolecules) and other organic molecules in the sensor platform and retention of the functionality of the molecules. Using biological molecules, numerous techniques such as physical adsorption, covalent attachment, entrapment in polymer and inorganic matrices, have been explored over the years to achieve a high yield, reproducible, robust immobilization technique that preserves the biological activity of the recognition molecule without adversely affecting the performance of the transduction component. No single method has emerged as the universal method of choice for each and every application and ongoing efforts strive to optimize these methods to render them adequate for specific applications. Silica host matrices, made by sol-gel process, have been studied as a platform for encapsulation of organic molecules and for biomolecules, such as proteins (including enzymes and antibodies), peptides, nucleic acids and cells. These sol-gel matrices have been chemically inert, hydrophilic, biocompatible, and inexpensive to produce. The matrices can also exhibit superior mechanical strength, enhanced thermal stability, and negligible swelling in solvents compared to organic polymers. The sol-gel matrix can also be tailored to act as a reservoir for water thereby significantly enhancing the ability to maintain the biological activity of entrapped enzymes, antibodies or cells. Biomolecules can find a more stable environment upon encapsulation in a silica host, because the polymeric framework grows around the biomolecule, creating a cage to protect the biomolecule from aggregation and unfolding. Other advantages of silica supports include biocompatibility and provision of resistance to microbial attack by serving as a nanofiltration material.
Sol-gel matrices offer another advantage in that their optical transparency makes them useful for transduction platforms that rely on transmission of light for detection, such as absorbance or fluorescence measurements. Sol-gel films can be made relatively fast and cast as thin layers on sensor surfaces. For silicon oxide
itride based sensors, such as field-effect transistors or optical fibers, formation of sol-gel films results in minimum alteration in optical, chemical and physical properties of the base material. The hydrophilic nature of silica also allows uninterrupted transport of water and other molecules such as substrates and products of an enzymatic reaction.
In general, sol-gel matrices containing encapsulated organic molecules, and in particular, biomolecules, have been prepared by hydrolysis and condensation of an orthosilicate such as tetramethyl orthosilicate (TMOS) or tetraethyl orthosilicate (TEOS). First, TMOS is partially hydrolyzed in an acidic medium by addition of a controlled amount of water. Next, the biological species is introduced in a suitable buffer to facilitate gelation. The buffer pH is chosen so as to allow the final solution to be close to neutrality in order to avoid denaturation of proteins. However, use of TMOS (or TEOS) as starting material leads to generation of alcohol (e.g., methanol or ethanol), the presence of which in large quantities can be deleterious to biomolecules, such as proteins and cells. In low temperature aging typically used with encapsulation of biological species, the generation of alcohol proceeds for an extended period of time allowing the encapsulated species to denature over time.
Useful would be a method incorporating the advantages of silica sol-gel structures but that does not produce the deleterious alcohols as a by-product.
SUMMARY OF THE INVENTION
According to the present invention, a method is provided to encapsulate molecules by forming a silica sol from a solution of a silicon oxide and alkali metal oxide, such as potassium oxide or sodium oxide, in water, adjusting the pH to a pH value less than approximately 7 to stabilize the silica sol, forming a silica sol matrix solution, adding a solution containing an organic compound to be encapsulated to form a silica sol matrix encapsulating said organic compound, aging said silica sol matrix encapsulating said organic compound, and forming a material selected from the group selected of a thin film and a gel. The ratio of silicon oxide to alkali metal oxide is between approximately 1.5 and approximately 4. Adjustment of the pH to a value less than 7, and generally to a value between approximately 1 and 4, is performed by adding an acid, such as HCl, or by adding a hydrogen-containing ion-exchange compound, such as an acidic cation exchange resin. No alcohols are formed in the method of the present invention.
An important class of organic compounds to be encapsulated include biomolecules, such as peptides, proteins, including enzymes and antibodies, nucleic acids and cells, which require mild processing conditions to preserve the integrity and activity of the biomolecules. Other organic compound classes include, but are not limited to, polysaccharides, carbohydrates, and lipids. Results have shown that certain biomolecules retain greater than 50 percent of their activity compared with the activity of the biomolecules in free solution.
In one embodiment, a silica sol is formed from a solution of a SiO
2
and Na
2
O in water, the pH is adjusted to a value of approximately 2 to stabilize the silica sol, forming a silica sol matrix solution, a solution containing a biomolecule to be encapsulated is added to form a silica sol matrix encapsulating the biomolecules, the silica sol matrix is aged and either a thin film or gel is formed.


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