Graphene Oxide Sensor for Fluorescent Gene Detection
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Solution Overview
Problem
Current methods for screening cells with completed gene introduction, such as those using selectable markers, introduce additional elements that can influence target gene expression or protein structure, and are not suitable for mass screening due to labor and cost requirements.
Innovation Solution
A method involving the use of a graphene oxide sensor with a water-soluble polymer and a fluorescent conjugated probe to detect fluorescent emission in cells, allowing for the identification and selection of foreign gene-introduced cells without the need for reporter genes or selectable markers, utilizing techniques like CRISPR-Cas9 for gene introduction.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If selectable markers such as reporter gene or antibiotic-resistant gene are used for screening, then positive cells can be identified, but additional elements are introduced that may influence target gene expression or protein structure
Solution Approach 1:
The invention extracts and removes the selectable marker elements from the gene introduction construct. Instead of using reporter genes or antibiotic-resistant genes, the method relies on direct detection of the target gene product or integration event, thereby eliminating the harmful influence of additional genetic elements on target gene expression and protein structure.
Solution Approach 2:
The invention introduces an intermediary detection system that uses antibodies or nucleic acid probes to specifically detect the target gene product or integration event. This intermediary approach allows for accurate identification of positive cells without requiring selectable markers, thus resolving the contradiction between screening accuracy and target gene integrity.
2Measurement precision
If quantitative PCR and DNA sequencing are used for screening, then gene introduction can be detected, but human labor and costs increase making it unsuitable for mass screening
Solution Approach 1:
The invention replaces the mechanical and labor-intensive processes of quantitative PCR and DNA sequencing with a simplified detection system based on antibody binding or nucleic acid hybridization. This substitution maintains detection accuracy while dramatically reducing human labor requirements and enabling mass screening of cells.
Solution Approach 2:
The invention employs colorimetric or fluorescent detection methods where antibodies or probes produce detectable color or fluorescence signals upon binding to the target. This allows for rapid, high-throughput screening of large numbers of cells without the complex procedures and labor associated with PCR and sequencing, thereby increasing productivity while maintaining detection accuracy.
3Measurement precision
If classical screening methods with selectable markers are used, then positive cells can be identified, but the process is labor-intensive and costly
Solution Approach 1:
The invention extracts the essential detection function from the complex selectable marker system. By removing the need for reporter genes, antibiotic resistance markers, and associated selection procedures, the method simplifies the screening process while maintaining the ability to accurately identify positive cells through direct detection of target gene presence or expression.
Solution Approach 2:
The invention uses antibodies or nucleic acid probes as intermediaries to simplify the detection process. These intermediaries provide specific, easy-to-read signals (such as color changes or fluorescence) that enable straightforward identification of positive cells without the complex multi-step procedures required by classical selectable marker methods, thereby reducing overall process complexity.
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
Enables efficient and reliable detection of foreign gene introduction in cells, overcoming limitations of existing methods by facilitating selective enrichment of gene-edited cells with high specificity and reduced labor and cost, suitable for mass screening.
Implementation Method 1
treating the foreign gene-introduced cells with a graphene oxide sensor in which a water-soluble polymer and a fluorescent conjugated probe are bound to a surface thereof; and detecting fluorescent emission in the cells
Data Source
AI summary
A method for introducing a foreign gene into a cell according to an embodiment of the present disclosure can easily identify foreign gene introduction by detecting whether there is fluorescent emission or not, and can reduce influence of additional elements other than a target gene since any reporter gene or selectable marker is not required. Further, the inventive method does not need an additional sampling process and therefore may implement a relatively accurate and simple screening process.