DiFMUP (6,8-Difluoro-4-Methylumbelliferyl Phosphate) Labeling

DiFMUP (6,8-Difluoro-4-Methylumbelliferyl Phosphate) is a fluorogenic substrate widely used for measuring phosphatase activity. Unlike conventional fluorescent labels that emit light continuously, DiFMUP remains only weakly fluorescent until the phosphate group is enzymatically removed. Following dephosphorylation, the molecule is converted into DiFMU (6,8-Difluoro-4-Methylumbelliferone), which exhibits bright blue fluorescence with excellent sensitivity.

When conjugated to DNA, RNA, PNA, aptamers, or other synthetic oligonucleotides, DiFMUP enables the development of enzyme-activated fluorescent probes for detecting phosphatase activity. These probes are valuable for studying enzyme kinetics, cellular signaling pathways, biomarker detection, and biosensor performance. Because fluorescence is generated only after enzymatic activation, DiFMUP-based probes provide very low background fluorescence and excellent signal-to-noise ratios.

Bio-Synthesis offers custom DiFMUP-modified oligonucleotides for research applications involving DNA, RNA, PNA, aptamers, molecular beacons, and biosensors. Depending on the desired assay format, DiFMUP may be introduced through linker chemistry or post-synthetic conjugation.

Typical applications include:

• Alkaline phosphatase assays
• Protein phosphatase assays
• Fluorogenic biosensors
• Enzyme-responsive probes
• Molecular diagnostics
• High-throughput screening (HTS)
• Cell signaling studies
• Fluorescence microscopy
• Aptamer biosensors
• Enzyme kinetics research

Typical Specifications

Property	Specification
Modification	DiFMUP
Full Chemical Name	6,8-Difluoro-4-Methylumbelliferyl Phosphate
Chemical Class	Coumarin-based fluorogenic phosphatase substrate
Activation Mechanism	Phosphatase-catalyzed dephosphorylation
Fluorescent Product	DiFMU (6,8-Difluoro-4-Methylumbelliferone)
Excitation (DiFMU)	≈358 nm
Emission (DiFMU)	≈455 nm
Fluorescence Color	Blue
Typical Placement	5′ end, 3′ end, or linker conjugation
Compatible Oligonucleotides	DNA, RNA, PNA, aptamers, enzyme-responsive probes
Typical Applications	Phosphatase assays, fluorogenic biosensors, enzyme-responsive probes, molecular diagnostics, high-throughput screening

Technical note

DiFMUP is not a conventional fluorescent dye like FAM, Cy5, or Alexa Fluor®. It is an enzyme-activatable fluorogenic substrate. The phosphate group quenches fluorescence until it is removed by a phosphatase, producing DiFMU, which is highly fluorescent (approximately Ex 358 nm / Em 455 nm). If DiFMUP is attached to an oligonucleotide, it is typically introduced through post-synthetic conjugation using an activated derivative rather than direct phosphoramidite incorporation.