ISFET pH Sensing for Probe-Free Nucleic Acid Amplification
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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 use of pH-sensitive ion-sensitive field effect transistors (ISFETs) can monitor proton release during PCR cycling, enabling probe-free detection in small volumes.
Innovation Solution
A sensing apparatus employing pH-sensitive ISFETs is integrated into a microfluidic device to detect proton release during nucleic acid amplification, overcoming the need for labeled probes by generating an electrical output signal in response to pH changes, allowing for real-time monitoring of PCR in low reaction volumes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If labeled probes are used for detection in qPCR, then detection accuracy is improved, but device complexity and cost increase
Solution Approach 1:
The invention extracts the detection function from complex labeled probes and implements it through a simplified ISFET-based pH sensing system. The ISFET directly detects proton release during nucleic acid amplification, eliminating the need for fluorescent labels, optical systems, and complex probe structures while maintaining detection capability
Solution Approach 2:
The invention replaces the optical detection system (fluorescent probes, light sources, detectors) with an electrical sensing system using ISFET. This substitution transitions from optical-mechanical complexity to electrical signal detection, simplifying the overall device architecture while preserving real-time monitoring capability
2Measurement precision
If small reaction volumes are used with ISFET, then sensitivity is improved, but buffering capacity is reduced
Solution Approach 1:
The invention changes the buffering parameters by using low concentrations of Tris buffer (e.g., 10 μM) and controlling pH conditions to be near the pKa of the buffer system. This optimization allows the small reaction volume to maintain sufficient buffering capacity during early amplification cycles while still enabling detectable pH changes when amplification reaches threshold levels
Solution Approach 2:
The invention accepts that buffering capacity is partially compromised in small volumes but compensates by optimizing other parameters (buffer concentration, pH, reaction conditions) to ensure that pH changes become detectable once amplification reaches the threshold. The system is designed to detect the point where proton release exceeds the limited buffering capacity, which occurs at the desired sensitivity threshold
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 real-time monitoring of nucleic acid amplification, facilitating applications in low-volume reactions and providing a cost-effective and efficient method for DNA sequencing and genetic testing.
Implementation Method 1
pH sensitive ion-sensitive field effect transistor (ISFET) to detect proton release
Implementation Method 2
ion-sensitive field effect transistor (ISFET) arranged to generate an electrical output signal in response to change of pH at said transistor surface
Implementation Method 3
one or more heating elements arranged to heat a sample
Implementation Method 4
one or more temperature sensors and, in a preferred option, the temperature sensors may be arranged to control the one or more heating elements
Data Source
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Figure 5
AI summary
The present invention provides a sensing apparatus for monitoring nucleic acid amplification in a sample comprising a silicon substrate integrating one or more heating elements arranged to heat a sample and one or more ions-sensitive field effect transistors (ISFETs) to measure the pH of said sample.