Optimized Fluorescent pH-Sensitive Dextran Conjugates Enable Ratiometric Measurement of Cerebral Blood pH In Vivo and...

Area of Science:

• Biomedical Engineering
• Optical Imaging
• Biochemistry

Background:

• In vivo pH monitoring in blood is challenging due to tissue properties and probe stability requirements.
• Existing pH probes face limitations like aggregation-caused quenching (ACQ) upon conjugation, reducing brightness and loading capacity.

Purpose of the Study:

• To develop a stable, dual-emission ratiometric pH probe for accurate in vivo blood pH monitoring.
• To overcome ACQ limitations of H-Ruby dyes when conjugated to dextran for enhanced performance.

Main Methods:

• Developed a dual-emission ratiometric pH probe using pH-insensitive AF488 and pH-sensitive H-Ruby (HR) conjugated to dextran.
• Engineered a PEGylated H-Ruby (HR-PEG) to improve solubility, two-photon absorption, and reduce ACQ.
• Utilized in vivo two-photon imaging in mice to assess probe performance in blood vessels.

Main Results:

• HR-PEG conjugates showed significantly reduced ACQ, increased brightness, and preserved pKa (7.5).
• A 12-fold fluorescence increase was observed upon acidification from pH 7.4 to 6.4.
• The enhanced probe enabled improved signal detection and accurate quantitative measurement of vascular acidosis during hypercapnia.

Conclusions:

• The developed dextran-conjugated HR-PEG probe offers a robust solution for in vivo blood pH monitoring.
• This approach significantly improves signal detection and quantitative analysis of physiological pH changes in dynamic tissues.
• The probe facilitates accurate measurement of vascular acidosis, advancing diagnostic capabilities for conditions like hypercapnia.