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Split Hybridization Probe Utilizing a DNA Fluorescent Light-up Aptamer as a Signal Reporter for Sequence-Specific Nucleic Acid Analysis

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Protein quantification using controlled DNA melting transitions in bivalent probe assemblies.

Joonyul Kim¹, Juan Hu¹, Andresa B Bezerra¹

¹Department of Chemistry and Biochemistry, 179 Chemistry Building, Auburn University , Auburn, Alabama 36849, United States.

Analytical Chemistry

|September 16, 2015

Summary

This summary is machine-generated.

A new homogeneous protein assay, thermofluorimetric analysis of bivalent probe (TFAB) assemblies, offers multiplexable detection in biological samples. This method provides robust and generalizable protein quantification with superior performance.

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Area of Science:

Biochemistry
Molecular Biology
Assay Development

Background:

Homogeneous protein assays face challenges with biological matrix interferences and limited multiplexing capabilities.
Existing methods often require physical separation, complicating mix-and-read workflows.

Purpose of the Study:

To develop a robust and generalizable homogeneous protein assay using standard qPCR instrumentation.
To enable quantitative protein detection through multiplexable DNA melting transitions.

Main Methods:

Utilized thermofluorimetric analysis of bivalent probe (TFAB) assemblies.
Employed differential thermal analysis to overcome background interferences without physical separation.
Validated TFAB using antibody-oligonucleotides or aptamers for protein quantification in various matrices.

Main Results:

Translated protein levels into DNA melting transitions within 30 minutes.
Demonstrated analytical removal of background interferences in buffer, human serum, and human plasma.
Achieved sensitive detection of 1 amol of protein in 100 pL microfluidic channels.

Conclusions:

TFAB is a robust and generalizable homogeneous protein assay.
The method offers superior performance and scalability for protein quantification in complex biological samples.
Direct optical detection via TFAB overcomes limitations of previous homogeneous assays.