ISFET pH Sensor for Probe-Free qPCR Detection
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Solution Overview
Problem
Current quantitative real-time polymerase chain reaction (qPCR) methods require labeled probes for detection, which can be cumbersome and limit their application, whereas the proposed method employs pH-sensitive ion-sensitive field effect transistors (ISFETs) to detect proton release during PCR, enabling probe-free monitoring of nucleic acid amplification.
Innovation Solution
The method involves using pH-sensitive ISFETs to monitor proton release in a buffered nucleic acid amplification mixture, preferably in a low volume chamber within a microfluidic device, where the ISFET generates an electrical output signal in response to pH changes, allowing for real-time monitoring of nucleic acid amplification without the need for labeled probes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If labeled probes are used for qPCR detection, then detection accuracy is improved, but device complexity and cost increase
Solution Approach 1:
The invention extracts and detects the proton release signal directly from the PCR amplification process itself, removing the need for external labeled probes. The ISFET sensor directly monitors pH changes caused by proton release during nucleotide incorporation, simplifying the detection system while maintaining accuracy.
Solution Approach 2:
The PCR reaction mixture itself serves as the detection medium through its inherent proton release during amplification. The buffer system and pH changes generated by the chemical reactions provide the detection signal, eliminating the need for separate labeling reagents and reducing device complexity.
2Stability of the object's composition
If buffer capacity is increased to maintain stable pH, then pH stability is improved, but detection sensitivity deteriorates
Solution Approach 1:
The invention changes the buffer capacity parameter to an optimal low level that allows sufficient pH stability for the reaction while enabling detectable pH changes during amplification. This parameter optimization allows the system to maintain stability when needed while remaining sensitive to amplification-induced pH changes.
Solution Approach 2:
The buffer capacity is dynamically adjusted through formulation to provide stability during early cycles while allowing pH to change detectably as amplification progresses. The system transitions from a stable state to a detectable state as the reaction proceeds, enabling both stability and sensitivity.
3Measurement precision
If reaction volume is reduced to increase proton concentration, then detection sensitivity is improved, but manufacturing precision requirements increase
Solution Approach 1:
The invention replaces mechanical handling of small volumes with a microfabricated ISFET sensor system that is inherently designed for nanoliter-scale reactions. The integrated sensor and reaction chamber eliminate the need for precise manual manipulation, substituting mechanical precision requirements with integrated sensor design.
Solution Approach 2:
The ISFET sensor is merged with the reaction chamber in an integrated device, combining the sensing function with the reaction volume containment. This integration allows the system to operate at reduced volumes without requiring separate precise alignment and positioning of sensor and reaction components.
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 enables sensitive and probe-free quantification of nucleic acid amplification, facilitating efficient monitoring of PCR processes in small volumes, suitable for applications like DNA sequencing and genetic testing, by leveraging rapid pH changes detectable by the ISFETs.
Implementation Method 1
employing a pH sensitive ion-sensitive field effect transistor (ISFET) to detect proton release arising from primer extension as amplification proceeds
Implementation Method 2
pH sensitive ISFET sensing of proton release consequent to PCR cycling
Data Source
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AI summary
Use of a pH sensor comprising an ion-sensitive field effect transistor (ISFET) to perform real time detection/ quantification of nucleic acid amplification, e.g. polymerase chain reaction (PCR) nucleic acid amplification, based on detection of protons released during the primer extension phase.