Enzyme Immobilization via Unbranched Cross-Linking Agents
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Solution Overview
Problem
Existing methods for immobilizing enzymes in diagnostic test elements often result in reduced enzyme activity due to the use of cross-linking agents like glutaraldehyde, which inactivate enzymes, and the immobilization of charged particles leads to dilution of enzyme activity and increased reaction times.
Innovation Solution
Cross-linking polypeptide molecules using unbranched cross-linking agents with at least 40 contiguous atoms, such as PEG chains, to form stable aggregates that maintain enzyme activity and prevent diffusion, thereby improving the immobilization process.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If glutaraldehyde is used as a cross-linking agent, then enzyme immobilization is achieved, but enzyme activity is inactivated due to penetration into protein molecules
Solution Approach 1:
The patent changes the molecular size parameter of the cross-linking agent from small (glutaraldehyde) to large (dextran polyaldehyde with molecular weight 10,000-70,000). This parameter change prevents penetration into protein molecules while maintaining cross-linking functionality, thus preserving enzyme activity during immobilization
Solution Approach 2:
The patent uses dextran polyaldehyde as a composite cross-linking agent that combines the cross-linking functionality of aldehydes with the large molecular structure of dextran. This composite material provides both immobilization stability and enzyme activity preservation through its unique structural properties
2Reliability
If charged particles are used for enzyme immobilization, then enzyme attachment is achieved, but specific activity is diluted due to increased mass
Solution Approach 1:
The patent extracts the charge dependency from the immobilization process by using neutral dextran polyaldehyde cross-linking. This removes the harmful effect of charge-based dilution while maintaining stable enzyme attachment through covalent cross-linking bonds formed by the polyaldehyde groups
Solution Approach 2:
The patent changes the chemical nature parameter of the immobilization medium from charged particles to neutral dextran polyaldehyde. This parameter change eliminates the dilution effect while maintaining attachment stability through covalent bonding mechanisms
3Quantity of substance
If layer thickness is increased to compensate for enzyme dilution, then required enzyme activity per area is achieved, but reaction time increases due to slowed diffusion
Solution Approach 1:
The patent changes the molecular size parameter of the cross-linking agent to large (dextran polyaldehyde), which prevents enzyme leaching and maintains high local enzyme concentration. This eliminates the need for increased layer thickness, thereby maintaining fast diffusion and short reaction times
4Productivity
If small cross-linking agents are used, then cross-linking efficiency is high, but enzyme inactivation occurs due to penetration into protein molecules
Solution Approach 1:
The patent changes the molecular weight parameter of the cross-linking agent from small (glutaraldehyde, MW 100) to large (dextran polyaldehyde, MW 10,000-70,000). This parameter change maintains cross-linking efficiency through multiple aldehyde groups while preventing protein penetration and enzyme inactivation
Solution Approach 2:
The patent uses dextran polyaldehyde as a composite material that combines the cross-linking functionality of aldehyde groups with the large protective structure of dextran chains. This composite provides efficient cross-linking without the harmful penetration effects of small agents
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The method effectively preserves enzyme activity, reduces diffusion, and maintains stability under various storage conditions, leading to more accurate and efficient diagnostic test elements.
Implementation Method 1
Cross-linking polypeptide molecules using unbranched cross-linking agents with at least 40 contiguous atoms, such as PEG chains, to form stable aggregates that maintain enzyme activity and prevent diffusion
Data Source
AI summary
Methods of cross-linking polypeptide molecules are provided, where such methods include combining a cross-linking agent and polypeptide molecules in a solution under conditions suitable for a cross-linking reaction to occur. Also provided are preparations of cross-linked polypeptide molecules, where the polypeptide molecules are cross-linked by essentially unbranched cross-linking groups of at least 40 contiguous atoms. Further provided are test chemistry matrices and methods of making the same, where the matrices include a redox cofactor, an agent capable of eliciting a change in at least one measurable property of an indicator reagent in the presence of redox equivalents, an indicator reagent, and a preparation of cross-linked polypeptide molecules as described herein. Test elements and methods of using the same to diagnose diseases such as diabetes also are provided, where the test elements include a test chemistry matrix as described herein.