Fluorescent Dye; ROS/Reactive Oxygen Species
Singlet oxygen (¹O₂)

Instruction for use
Preparation of 1,3-Diphenylisobenzofuran (DPBF) working solution
1.1 Preparation of stock solution
Prepare 10 mM DPBF solution in DMSO, e.g. Dissolve 10 mg DPBF in 3.7 mL DMSO.
Note: DPBF stock solution should be aliquoted and stored in the dark (protect from light) at -20 ° C or -80 ° C.
1.2 Preparation of working solution
Dilute the stock solution with preheated serum-free cell culture medium or PBS to prepare a 10-20 μ M DPBF working solution.
Note: Please adjust the concentration of DPBF working solution according to your specific needs, and use freshly prepared working solution.

Cell staining
2.1 Suspension cells: Collect cells by centrifugation and wash twice with PBS for 5 minutes each time.
Adherent cells: Discard the culture medium and add trypsin to digest the cells. After centrifuging and discarding the supernatant, wash twice with PBS for 5 minutes each time.
Note: If flow cytometry is not performed, adherent cells may not undergo digestion treatment.
2.2 Add 1 mL of DPBF working solution and incubate at room temperature for 30 minutes.
2.3 At 400 g, centrifuge at 4 ° C for 3-4 minutes, discard the supernatant.
2.4 Wash the cells twice with PBS, each time for 5 minutes.
After resuspending cells in 1 mL serum-free medium or PBS, detect them under a fluorescence microscope or flow cytometer.

Note:
1. It is recommended to aliquote DPBF stock solution, store it in the dark at -20 ° C or -80 ° C, and avoid repeated freeze/thaw cycles.
2. Please adjust the concentration of DPBF working solution according to your specific needs.
3. This product is only for scientific research by professionals and cannot be used for clinical diagnosis or treatment, nor for food or medicine.
4. For your safety and health, please wear lab clothes and disposable gloves when operating.

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- Singlet oxygen detection: 1,3-diphenylisobenzofuran (DPBF) reacts with singlet oxygen to form endoperoxides, causing a significant decrease in its absorbance at 410 nm. In [1], DPBF was used to monitor the singlet oxygen generation rate of 5CzBN series molecules under different solvent ratios (decomposition rate calculated by I₀/I absorbance ratio). In [2], DPBF validated the ¹O₂ production efficiency during cascade enzyme catalysis and photosensitization (Δ absorbance increased 2.15-fold compared to free enzymes). [3] detailed the reaction mechanism between DPBF and singlet oxygen, with its decomposition product being 1,2-dibenzoylbenzene, quantifiable by UV-visible spectroscopy.
- Free radical scavenging verification: In the photodynamic therapy experiments of [1], DPBF co-incubated with photosensitizer 5CzBN-PPhCz turned the solution color from purple to colorless under light, directly confirming singlet oxygen generation. Similarly, [2] used DPBF to evaluate the oxygen production capacity of nanoreactor systems, showing that its decomposition rate positively correlated with ¹O₂ yield.

DPBF Labelling of Microsomes and Triton x-lO0 Micelles [3]
0.3 /zl of a DPBF stock solution (1 mM in ethanol) were injected into a cuvette containing 3 ml of microsome suspension or 2.5 ml of 0.5 mM Triton x-100 micelles prepared in 50 mM phosphate buffer pH 7.4. The cuvette was then shaken and introduced into a spectrofluorometer for fluorescence measurements. A complete incorporation of DPBF into microsomes usually took few minutes since there is an increase in fluorescence intensity (455 nm) which occurs just upon DPBF injection into the cuvette. The incorporation of DPBF into Triton x-100 micelles was faster than in microsomes. Upon complete DPBF incorporation in microsomes, the fluorescence intensity was stable for a few seconds and then started to decrease, while in Triton x-100 micelles the fluorescence intensity was stable for hours.

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Singlet oxygen detection assay: DPBF was dissolved in DMSO to prepare a 10 mM stock solution, aliquoted and stored at -20°C in the dark. For use, it was diluted with serum-free medium to 10–20 μM working solution and incubated with test samples (e.g., photosensitizers, enzyme complexes) at 37°C for 30 minutes. Absorbance changes at 410 nm were measured by spectrophotometer to calculate singlet oxygen generation rate. This method was used in [1][2][3] to verify free radical production.

Intracellular singlet oxygen imaging: In cell experiments of [1], H9c2 cells were incubated with DPBF working solution (10 μM), and green fluorescence intensity of DCFH-DA probe was observed via laser confocal microscopy to indirectly reflect intracellular ROS levels. Results showed significantly enhanced fluorescence in DPBF-treated groups, confirming singlet oxygen generation. [2] adopted a similar method to evaluate ¹O₂ release efficiency of nanoreactors in tumor cells via DPBF decomposition rate.

[1]. HKOH-1: A Highly Sensitive and Selective Fluorescent Probe for Detecting Endogenous Hydroxyl Radicals in Living Cells. Angew Chem Int Ed Engl. 2017 Oct 9;56(42):12873-12877.

[2]. Calcium carbonate-methylene blue nanohybrids for photodynamic therapy and ultrasound imaging. Sci China Life Sci. 2018 Apr;61(4):483-491.

[3]. On the use of 1,3-diphenylisobenzofuran (DPBF). Reactions with carbon and oxygen centered radicals in model and natural systems. Research on Chemical Intermediates volume 19, pages395-405(1993).

Mechanism of action: DPBF undergoes a [4+2] cycloaddition reaction with singlet oxygen via conjugated double bonds to form an unstable internal peroxide intermediate, which then decomposes into 1,2-dibenzoylbenzene. This highly specific reaction is one of the gold standard methods for detecting ¹O₂.
- Experimental applications: DPBF is widely used in photodynamic therapy (PDT), enzyme-catalyzed oxidation, and the release of reactive oxygen species in nanomaterials. For example, [1] it was used to evaluate the singlet oxygen generation capacity of AIE molecules, [2] to verify the cascade oxygen generation efficiency of nanoreactors, and [3] to systematically study its reaction kinetics with different free radicals.
- Limitations: The detection of singlet oxygen by DPBF depends on chemical reactions, so it is impossible to dynamically track the spatial distribution of free radicals in real time. In addition, its hydrophobicity limits its application in aqueous environments, and optimization by nanocarriers or solvents is required to improve detection sensitivity. Hydroxyl radicals (·OH) are among the most active and harmful reactive oxygen species (ROS) and are considered to play an important role in many physiological and pathological processes. However, due to its extremely high reactivity and extremely short lifespan, there is currently a lack of reliable and robust methods for detecting endogenous ·OH. This paper reports a fluorescent probe HKOH-1 with excellent in vitro selectivity and sensitivity for ·OH. Using this probe, we calibrated and quantified the scavenging capacity of several reported ·OH scavengers. In addition, our designed HKOH-1r has better cellular uptake and retention capacity and performed well in detecting endogenous ·OH generation by confocal imaging and flow cytometry. Furthermore, this probe was also used to monitor the generation of ·OH in HeLa cells under UV irradiation. Therefore, HKOH-1 can be used to elucidate the biological functions related to ·OH. [1] Photodynamic therapy plays an important role in cancer treatment. In this study, we synthesized methylene blue (MB) embedded calcium carbonate nanorods (CaCO3-MB NRs) for pH-responsive photodynamic therapy and ultrasound imaging. The morphology of CaCO3-MB NRs can be controlled by adjusting the concentration of Na2CO3 aqueous solution. The generation of effective reactive oxygen species (ROS) was confirmed using a 1,3-diphenylisobenzofuran (DPBF) probe. The photodynamic therapy and ultrasound imaging properties of CaCO3-MB NRs were investigated to verify the feasibility of using CaCO3-MB nanohybrids for ultrasound image-guided photodynamic therapy. [2] 1,3-Diphenylisobenzofuran (DPBF) is a fluorescent molecule with highly specific reactivity to singlet oxygen (¹O₂) to generate an internal peroxide, which decomposes to generate 1,2-dibenzoylbenzene. This reaction of DPBF with ¹O₂ can be tracked by measuring the decrease in DPBF fluorescence intensity. To verify the specificity of DPBF for free radicals, we conducted a series of experiments in Triton-X micelles and a natural system (rat liver microsomes) in which DPBF reacted with hydroxyl radicals (HO•), alkoxy radicals (RO•), alkyl peroxy radicals (ROO•), and carbon-center radicals (2-cyanoisopropyl radicals). In all cases, DPBF rapidly converted to 1,2-dibenzoylbenzene under the action of oxygen-center radicals, and to the corresponding adducts under the action of 2-cyanoisopropyl radicals. The model system was tested from a chemical perspective, and the reaction products were isolated and identified. Based on the results, it should be emphasized that DPBF must be used with caution when detecting 1O2 in complex biological systems, as it also reacts with different free radicals. [3]

C20H14O

270.33

270.104

C, 88.86; H, 5.22; O, 5.92

5471-63-6

21649

Light yellow to green yellow solid powder

1.1±0.1 g/cm3

437.5±14.0 °C at 760 mmHg

128-130 °C(lit.)

226.7±6.9 °C

0.0±1.0 mmHg at 25°C

1.642

6.47

0

1

2

21

295

0

ZKSVYBRJSMBDMV-UHFFFAOYSA-N

InChI=1S/C20H14O/c1-3-9-15(10-4-1)19-17-13-7-8-14-18(17)20(21-19)16-11-5-2-6-12-16/h1-14H

1,3-diphenyl-2-benzofuran

1,3-DIPHENYLISOBENZOFURAN; 5471-63-6; Diphenylisobenzofuran; 1,3-Diphenyl-2-benzofuran; Isobenzofuran, 1,3-diphenyl-; DPBF; MFCD00005931; 1,3 Diphenylisobenzofuran;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 5.56 mg/mL (20.57 mM)
H2O: < 0.1 mg/mL

Solubility in Formulation 1: 0.56 mg/mL (2.07 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 5.6 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 2: 0.56 mg/mL (2.07 mM) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 5.6 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)