Sensitive Microscale Thermophoresis Assay Using Aptamer Thermal Switch.

Area of Science:

• Biotechnology
• Analytical Chemistry
• Molecular Diagnostics

Background:

• Rapid and sensitive detection methods are crucial for environmental monitoring, food safety, and molecular diagnostics.
• Existing methods often face limitations in speed, sensitivity, or versatility for diverse targets.

Purpose of the Study:

• To develop an innovative aptamer-based strategy for rapid and sensitive target detection.
• To engineer aptamers that exhibit significant conformational changes upon target binding, modulated by heating.
• To convert these conformational changes into measurable fluorescence signals for quantitative analysis.

Main Methods:

• Rational engineering of aptamer sequences to create a thermal switch with distinct conformational states (bound vs. unbound).
• Incorporation of a conformation-sensitive fluorophore into the aptamer structure.
• Utilizing microscale thermophoresis (MST) to measure heating-induced fluorescence changes.
• Testing the system with diverse targets including cadmium ions (Cd2+), aflatoxin B1, and immunoglobulin E.

Main Results:

• The engineered aptamer thermal switch demonstrated a large difference in heating-induced fluorescence changes between bound and unbound states.
• Successful detection of cadmium ions, aflatoxin B1, and immunoglobulin E within seconds.
• Achieved high sensitivity in target detection using the aptamer thermal switch coupled with MST.

Conclusions:

• The aptamer thermal switch strategy provides a rapid, sensitive, and versatile platform for detecting a wide range of targets.
• This approach offers significant potential for applications in environmental monitoring, food safety, and molecular diagnostics.
• The method is also valuable for studying molecular binding interactions and affinity.