Electrochemical pH Modulation for Multiplexed Fluorescent Detection
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Solution Overview
Problem
Current multiplexing techniques in PCR and qPCR are limited by the number of fluorescent probes with non-overlapping spectral properties and optical channels, leading to restricted analyte detection capabilities, with existing methods for expanding detection often causing sample loss, dilution, or being unsuitable for real-time applications.
Innovation Solution
Utilizing pH-dependent fluorescent properties of organic dyes and electrochemical pH modulation to differentiate dyes with overlapping spectral properties, allowing for reversible and repeatable pH changes within a qPCR process, enabling the detection of multiple analytes using the same optical channel without altering existing detection instruments.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If buffer exchange, desalting, or dialysis is used to change pH, then pH modulation is achieved, but sample loss or dilution occurs and the process is time-consuming
Solution Approach 1:
The patent replaces mechanical separation methods (buffer exchange, desalting, dialysis) with an electrochemical system. An electrode directly modulates pH in the reaction solution through electrochemical reactions, eliminating the need for physical sample manipulation and associated losses.
Solution Approach 2:
The patent introduces an electrochemically active agent as an intermediary substance. This agent undergoes oxidation-reduction reactions at the electrode surface, indirectly controlling pH without requiring direct addition of strong acids or bases that would dilute or contaminate the sample.
2Adaptability or versatility
If photobleaching-based multiplexing is used, then fluorescent stability differentiation is achieved, but only end-point detection is possible
Solution Approach 1:
The patent changes the control parameter from irreversible photobleaching (cumulative damage) to reversible pH modulation. By dynamically adjusting pH levels, the same fluorescent probes can be selectively activated or deactivated multiple times, enabling real-time monitoring throughout the qPCR process rather than only end-point detection.
3Adaptability or versatility
If more optical channels are added to detect more analytes, then multiplexing capability increases, but device complexity and cost increase
Solution Approach 1:
The patent introduces dynamic pH control to a previously static detection system. By modulating pH over time, the same optical channel can selectively detect different fluorescent probes at different time points, effectively multiplying the detection capacity without adding physical channels.
Solution Approach 2:
The patent employs periodic pH modulation cycles to sequentially activate different fluorescent probes. During each qPCR cycle, pH is adjusted to favor specific dye fluorescence, allowing multiple analytes to be detected in sequence through a single optical channel, similar to time-division multiplexing.
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
This approach increases the number of detectable analytes per channel by modulating fluorescence based on pH changes, preserving sample integrity and enabling real-time quantitative analysis without the need for sample exchange or irreversible processes.
Implementation Method 1
the concentration of hydronium ions is altered in situ as the result of a controlled electrochemical reaction (oxidation or reduction) occurring at the surface of an electrode
Implementation Method 2
If two dyes fluoresce at the same wavelength (i.e., have overlapping spectral properties), but have distinctly different pH profiles
Data Source
AI summary
An apparatus to detect more than one analyte in a solution comprising at least one electrode in contact with the solution, at least two dyes including a first dye and a second dye, and an electrochemically active agent, where the solution has a pH, the electrode is configured to modulate the pH of the solution by oxidizing or reducing the electrochemically active agent, the first dye and the second dye fluoresce at different pH levels, fluorescence of the first dye is used to indicate the presence of a first analyte, and fluorescence of the second dye is used to indicate the presence of a second analyte. Methods of detecting multiple analytes in a solution are also provided.