Nucleic Acid Measurement Device Using FRET Probe Segmentation
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Solution Overview
Problem
Existing nucleic acid sequence measurement devices face limitations in detection sensitivity due to incomplete quenching between fluorescent and quenching probes, inability to measure offset light, and variations in light intensity between chips and spots, which affect the lower limit of detection and require complex operational procedures.
Innovation Solution
A nucleic acid sequence measurement device utilizing donor and acceptor fluorescent probes with complementary binding parts, where the acceptor fluorescent molecule quenches the donor fluorescent molecule, allowing for measurement of offset light and improved detection sensitivity by calculating fluorescence changes before and after hybridization, and enabling real-time observation without the need for washing or labeling.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If quenching probe and fluorescent probe are bound to maintain quenching, then fluorescence is quenched, but detection sensitivity is limited due to incomplete quenching and offset light
Solution Approach 1:
The probe is divided into two separate probes: a fluorescent probe and a quenching probe. Each probe has a specific binding part that can bind to complementary sequences. When both binding parts bind to the target nucleic acid, the fluorescent probe and quenching probe are brought into close proximity, enabling efficient energy transfer and quenching of fluorescence. This segmentation allows independent optimization of each probe's binding affinity and fluorescent properties.
Solution Approach 2:
The target nucleic acid acts as an intermediary that brings the fluorescent probe and quenching probe into close proximity. When the target is present, it simultaneously binds to both probes, positioning them within the energy transfer distance required for efficient quenching. This intermediary mechanism ensures that quenching only occurs when the target is present, improving detection specificity and sensitivity.
2Measurement precision
If only fluorescent probe detection is used, then device complexity is reduced, but detection sensitivity is insufficient
Solution Approach 1:
The invention utilizes fluorescence resonance energy transfer (FRET) between the fluorescent probe and quenching probe, which involves wavelength-dependent energy transfer. The fluorescent probe emits fluorescence at a specific wavelength, and when in proximity to the quenching probe, energy is transferred to the quenching probe which then emits at a different wavelength or quenches the fluorescence. This wavelength-based detection allows for sensitive target detection while maintaining relatively simple device requirements.
3Measurement precision
If light intensity comparison before and after hybridization is performed, then detection is possible, but operational complexity increases due to multiple imaging steps
Solution Approach 1:
The fluorescent probe and quenching probe are pre-bound to the solid support surface in specific positions before the hybridization reaction. This preliminary arrangement ensures that when the target nucleic acid is introduced, it can simultaneously bind to both probes and bring them into close proximity for FRET. This pre-positioning eliminates the need for complex post-hybridization manipulation and allows for direct fluorescence measurement.
Solution Approach 2:
The FRET-based detection system allows for continuous monitoring of the hybridization reaction. As the target binds to both probes, the fluorescence signal changes continuously, providing real-time detection information. This continuous detection capability eliminates the need for separate pre- and post-hybridization imaging steps, simplifying the overall operation while maintaining detection accuracy.
4Adaptability or versatility
If two-color laser excitation is used, then fluorescence imaging of multiple substances is possible, but time lag occurs between image acquisitions
Solution Approach 1:
The detection system uses periodic excitation of the fluorescent probe and quenching probe at their respective excitation wavelengths. By alternating excitation between the two probes in a rapid periodic manner, the system can capture fluorescence signals from both probes sequentially. This periodic excitation approach allows for multi-substance detection while minimizing time lag between measurements, as the excitation cycles are designed to complete within a short time frame.
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 device achieves high accuracy in detecting fluorescence changes due to hybridization, reduces the lower limit of detection, and improves reproducibility by accounting for variations in light intensity, allowing for real-time monitoring and simplified operation.
Implementation Method 1
the donor fluorescent probe and the quenching probe have a positional relationship in which fluorescence of the donor fluorescent molecule is quenched with the acceptor fluorescent molecule that approaches the donor fluorescent molecule
Data Source
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AI summary
When the target (30) is not supplied, the binding via the binding part is maintained, and when the donor fluorescent molecule (11) is excited, energy is transferred to the acceptor fluorescent molecule (21) that is close to the donor fluorescent molecule (11). Then, the acceptor fluorescent molecule (21) exhibits fluorescence. When the target (30) is supplied, the target (30) is bound to the detection part to release the binding via the binding part, and the acceptor fluorescent molecule (21) is separated from the donor fluorescent molecule (11). Then, the donor fluorescent molecule (11) exhibits fluorescence.