TCEP Biotin Dextran Buffer for Viscous Sample Liquefaction
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Solution Overview
Problem
Current methods for liquefying viscous biological samples, such as mucous secretions, are cumbersome and often hindered by enzyme inhibitors and auto-fluorescent particles, requiring vigorous shaking and toxic reagents like DTT, which can reduce fluorescence and are prone to oxidation.
Innovation Solution
A composition comprising TCEP, biotin, and dextran sulphate, buffered at a pH between 7 and 10, which liquefies samples efficiently without affecting fluorescence and reducing background auto-fluorescence, allowing for rapid analysis of pathogens in bodily secretions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If DTT or other mercaptans are used to reduce disulfide bonds in mucous proteins, then liquefaction is achieved, but oxidation susceptibility and unpleasant/toxic odours occur
Solution Approach 1:
The patent changes the chemical parameter from DTT to TCEP (tris(2-carboxyethyl)phosphine), which has similar reducing capability but superior oxidation stability. TCEP does not form disulfide bonds and is not susceptible to oxidation by atmospheric oxygen, thereby resolving the contradiction between liquefaction capability and oxidation stability.
Solution Approach 2:
The patent employs a combination of TCEP with biotin and dextran sulphate, where TCEP serves as the primary reducing agent that can be used without special precautions against oxidation. The additional components biotin and dextran sulphate provide auxiliary functions (fluorescence suppression and viscosity reduction) that enhance the overall performance while allowing TCEP to maintain its reducing function reliably.
2Reliability
If TCEP is used as a substitute for DTT, then oxidation stability is improved, but fluorescence reduction in proteins occurs
Solution Approach 1:
The patent separates the functions: TCEP is extracted as the dedicated reducing agent for disulfide bond reduction, while biotin and dextran sulphate are added to specifically address the fluorescence suppression function. This functional separation allows TCEP to perform reduction without being blamed for fluorescence issues, which are handled by the biotin-dextran sulphate system.
Solution Approach 2:
The patent introduces biotin and dextran sulphate as intermediary substances that mediate between TCEP and the fluorescent proteins. These intermediaries bind to the proteins and suppress autofluorescence, thereby protecting the fluorescent signal while allowing TCEP to freely perform its reducing function.
3Ease of manufacture
If vigorous shaking with glass beads is used to liquefy samples, then liquefaction is achieved, but handling complexity and time consumption increase
Solution Approach 1:
The patent replaces the mechanical system of vigorous shaking with glass beads with a chemical system using TCEP-biotin-dextran sulphate composition. The chemical agents directly interact with the mucous proteins to reduce viscosity, eliminating the need for complex mechanical agitation and glass beads, thereby simplifying the handling procedure.
Solution Approach 2:
The patent uses a composite material system consisting of TCEP, biotin, and dextran sulphate working together. TCEP provides the reducing function, biotin provides fluorescence suppression, and dextran sulphate contributes to viscosity reduction and background suppression. This composite approach achieves superior liquefaction with simpler handling compared to mechanical methods.
4Quantity of substance
If dextran is added to increase apparent molar concentration, then concentration effect is achieved, but background and auto-fluorescence increase
Solution Approach 1:
The patent merges dextran sulphate with biotin to create a combined system where the harmful auto-fluorescence effect of dextran is counterbalanced by the fluorescence suppression capability of biotin. The two substances work synergistically: dextran sulphate provides the concentration effect and viscosity reduction, while biotin suppresses the background fluorescence, thereby resolving the contradiction between concentration enhancement and fluorescence suppression.
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 composition enables rapid liquefaction of highly viscous samples with mild shaking, suppressing auto-fluorescence and maintaining hybridization conditions, facilitating direct pathogen detection in less than 45 minutes using DNA-beacons in sputum samples.
Implementation Method 1
reducing disulfide bonds readily present in mucous proteins with Sulphur reducing compounds such as Dithioerytrol (DTT) or other mercaptans
Implementation Method 2
A further reduction of background and autofluorescence was achieved by the addition of biotin combined with Dextran (dextran sulphate)
Implementation Method 3
buffered at a pH between 7 and 10, wherein a buffer substance may be provided in concentrations between 10 and 100 mM
Data Source
AI summary
The invention concerns a composition comprising TCEP, biotin and dextran suitable for liquefying a viscous biological sample. The composition according to the invention can be used in diagnostic methods, preferably for use in diagnosis of an infection with a micro-organism, more preferably for use in diagnosis of HCAP.