Pyruvate Optical Sensor for Real-Time In Situ Detection
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Solution Overview
Problem
Current methods for detecting pyruvate levels in cells are time-consuming, invasive, and unsuitable for real-time, high-throughput analysis, lacking the capability for in situ detection within living cells and subcellular organelles.
Innovation Solution
Development of a pyruvate optical sensor comprising a pyruvate-sensitive polypeptide fused with an optically active polypeptide, such as fluorescent proteins, allowing for real-time localization and quantitative detection of pyruvate within and outside cells through gene manipulation, eliminating the need for invasive sample processing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional detection methods (UV spectrophotometry, HPLC, dehydrogenase colorimetry) are used, then pyruvate content can be detected, but sample processing is time-consuming and invasive
Solution Approach 1:
The patent replaces mechanical/chemical sample processing methods with an optical detection system. The optical sensor uses fluorescence excitation and emission to detect pyruvate directly in cells without requiring cell disruption, separation, extraction, or purification steps, thereby eliminating time-consuming mechanical processing while maintaining detection precision
Solution Approach 2:
The patent introduces an optical sensor as an intermediary between the pyruvate target and the detection system. The sensor comprises a pyruvate-sensitive polypeptide fused with an optically active polypeptide, which acts as a mediator that binds to pyruvate and produces a fluorescent signal, enabling direct detection without invasive sample processing
2Measurement precision
If conventional detection methods are used, then pyruvate can be measured, but in situ real-time detection in living cells is not possible
Solution Approach 1:
The patent replaces invasive mechanical detection methods with non-invasive optical detection. The optical sensor allows real-time monitoring of pyruvate in living cells and subcellular organelles through fluorescence microscopy or plate reading, eliminating the need for cell disruption and enabling straightforward in situ detection operations
Solution Approach 2:
The optical sensor is designed to function autonomously within living cells. The pyruvate-sensitive polypeptide component automatically binds to pyruvate molecules in their natural cellular environment, and the optically active polypeptide automatically generates the fluorescent signal, enabling the system to serve itself without external intervention or invasive processing
3Measurement precision
If conventional detection methods are used, then pyruvate levels can be determined, but high-throughput and high spatiotemporal resolution detection is not achieved
Solution Approach 1:
The optical sensor system is designed with universal applicability across multiple detection formats. The same sensor can be used for single-cell fluorescence microscopy imaging for high spatiotemporal resolution, or for multi-well plate reading for high-throughput screening, allowing one detection system to serve multiple productivity requirements simultaneously
Solution Approach 2:
The patent replaces low-throughput mechanical processing methods with parallelizable optical detection. Multiple samples can be detected simultaneously using multi-well plates or high-content imaging systems, and the optical detection process itself is inherently parallelizable, enabling high-throughput measurement without the bottleneck of sequential sample processing
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 pyruvate optical sensor enables rapid, high-throughput, and quantitative detection of pyruvate in cells, demonstrating enhanced sensitivity and specificity, with the ability to localize pyruvate in various cellular structures and perform compound screening, as evidenced by significant fluorescence responses and accurate quantitation in blood samples.
Implementation Method 1
an optically active polypeptide or a functional variant thereof... demonstrating enhanced sensitivity and specificity, with the ability to localize pyruvate in various cellular structures and perform compound screening, as evidenced by significant fluorescence responses
Data Source
AI summary
Disclosed in the present invention are a pyruvic acid optical probe, a preparation method therefor, and an application thereof. One aspect of the present invention is the disclosure of an optical probe, which includes a pyruvic acid-sensitive polypeptide and an optically active polypeptide, wherein the optically active polypeptide is located within the sequence of the pyruvic acid-sensitive polypeptide. The present invention also discloses a preparation method for the probe and an application of said probe in pyruvic acid measurement.