Fluorescent Method for the Detection of Biothiols Using an Ag⁺-Mediated Conformational Switch

Area of Science:

• Biochemistry
• Analytical Chemistry
• Molecular Biology

Background:

• Biothiols, including glutathione and cysteine, are crucial biological molecules.
• Accurate detection of biothiols is vital for understanding cellular processes and diagnosing diseases.
• Existing detection methods can be complex or time-consuming.

Purpose of the Study:

• To develop a novel, simple, and rapid fluorescence-based assay for detecting biothiols.
• To utilize a 2-aminopurine-labeled DNA probe for sensitive biothiol quantification.
• To explore the potential of this assay as a diagnostic tool in clinical settings.

Main Methods:

• A DNA probe labeled with 2-aminopurine (a fluorescent adenine analogue) was synthesized.
• The fluorescence of 2-aminopurine was modulated by interactions with Ag⁺ and subsequent displacement by biothiols.
• The assay involved observing fluorescence quenching upon biothiol addition.

Main Results:

• The assay demonstrated significant fluorescence enhancement in the presence of Ag⁺ due to cytosine-Ag⁺-cytosine pair formation.
• Glutathione and cysteine disrupted the cytosine-Ag⁺-cytosine structure, leading to fluorescence quenching.
• The method achieved low detection limits: 3 nM for glutathione and 5 nM for cysteine.
• Successful determination of biothiols in human blood serum was demonstrated.

Conclusions:

• A novel, simple, and time-saving fluorescence assay for biothiol detection was successfully developed.
• The assay leverages the specific interactions between biothiols, Ag⁺, and a DNA probe.
• The method's sensitivity and applicability in human serum suggest its potential as a valuable diagnostic tool.