1,3-Diphenylisobenzofuran (DPBF)   中文名称: 1,3-联苯基异香豆酮

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

使用方法
1,3-二苯基异苯并呋喃（DPBF）工作液的制备
1.1 储备液的制备
在DMSO中制备10 mM DPBF溶液，例如将10 mg DPBF溶解在3.7 mL DMSO中。
注意：DPBF储备液应分装，并在-20°C或-80°C避光储存。
1.2 工作液的制备
用预热的无血清细胞培养基或PBS稀释储备液，制备10-20μM DPBF工作液。
注意：请根据您的具体实验需求调整DPBF工作液的浓度。为保证最佳效果，工作液请现配现用。

细胞染色
2.1 悬浮细胞：通过离心收集细胞，用PBS洗涤两次，每次5分钟。
贴壁细胞：吸弃培养基，加入胰蛋白酶消化细胞。离心并吸弃上清液后，用PBS洗涤两次，每次5分钟。
注意：如果不进行流式细胞术，贴壁细胞可不进行消化处理。
2.2 加入1 mL DPBF工作液，在室温下孵育30分钟。
2.3 在400 g下，在4°C下离心3-4分钟，吸弃上清液。
2.4 用PBS洗涤细胞两次，每次5分钟。
将细胞重新悬浮在1 mL无血清培养基或PBS中后，在荧光显微镜或流式细胞仪下检测。

注意事项
1.建议对DPBF储备液进行分装，并在-20°C或-80°C避光储存，避免反复冻融。
请根据您的具体需求调整DPBF工作液的浓度。
3.本产品仅供专业人员科研使用，不得用于临床诊断或治疗，也不得用于食品或药品。
4. 为了您的安全和健康，请在操作时穿着实验服和一次性手套。

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- 单线态氧检测：DPBF与单线态氧反应生成内过氧化物，导致其在410 nm处的吸光度显著下降。在文献[1]中，DPBF用于监测5CzBN系列分子在不同溶剂比例下的单线态氧生成速率（分解速率通过I₀/I吸光度比值计算）。文献[2]中，DPBF被用于验证级联酶催化和光敏化过程中¹O₂的产生效率（Δ吸光度变化与游离酶相比提升2.15倍）。文献[3]详细描述其与单线态氧的反应机制，分解产物为1,2-二苯甲酰苯，可通过紫外-可见光谱定量。
- 自由基清除能力验证：在文献[1]的光动力治疗实验中，DPBF与光敏剂5CzBN-PPhCz共孵育后，光照下溶液颜色从紫色变为无色，直接证明单线态氧的产生。类似地，文献[2]中DPBF用于评估纳米反应器系统的产氧能力，结果显示其分解速率与¹O₂生成量呈正相关。

将0.3/zl的DPBF储备溶液（1 mM乙醇溶液）注射到含有3 ml微粒体悬浮液或2.5 ml 0.5 mM Triton x-100胶束的试管中，这些胶束是在pH 7.4的50 mM磷酸盐缓冲液中制备的。然后摇动试管并将其引入荧光分光光度计进行荧光测量。DPBF完全掺入微粒体通常需要几分钟，因为DPBF注射到试管中时荧光强度（455nm）会增加。DPBF在Triton x-100胶束中的掺入速度比在微粒体中快。在微粒体中完全掺入DPBF后，荧光强度在几秒钟内稳定，然后开始下降，而在Triton x-100胶束中，荧光强度稳定数小时。

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单线态氧检测实验：将DPBF溶解于DMSO配制10 mM储存液，分装后-20°C避光保存。使用时用无血清培养基稀释至10–20 μM工作液，与待测样品（如光敏剂、酶复合物）在37°C孵育30分钟。通过分光光度计检测410 nm处吸光度变化，计算单线态氧生成速率。该方法在文献[1][2][3]中均用于验证自由基产生。

细胞内单线态氧成像：在文献[1]的细胞实验中，H9c2细胞与DPBF工作液（10 μM）孵育后，通过激光共聚焦显微镜观察DCFH-DA探针的绿色荧光强度，间接反映细胞内ROS水平。结果显示，DPBF处理组荧光信号显著增强，证实单线态氧的生成。文献[2]采用类似方法，通过DPBF分解率评估纳米反应器在肿瘤细胞内的¹O₂释放效率。

[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 through conjugated double bonds, forming unstable endoperoxide intermediates that subsequently decompose 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 reactive oxygen species release from nanomaterials. For example, [1] used it to assess singlet oxygen generation capacity of AIE molecules, [2] validated cascade oxygen production efficiency of nanoreactors, and [3] systematically studied its reaction kinetics with different free radicals.
- Limitations: DPBF’s detection of singlet oxygen relies on chemical reactions, preventing real-time dynamic tracking of radical spatial distribution. Additionally, its hydrophobicity limits application in aqueous environments, requiring optimization via nanocarriers or solvents to improve detection sensitivity.

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The hydroxyl radical (. OH), one of the most reactive and deleterious reactive oxygen species (ROS), has been suggested to play an essential role in many physiological and pathological scenarios. However, a reliable and robust method to detect endogenous . OH is currently lacking owing to its extremely high reactivity and short lifetime. Herein we report a fluorescent probe HKOH-1 with superior in vitro selectivity and sensitivity towards . OH. With this probe, we have calibrated and quantified the scavenging capacities of a wide range of reported . OH scavengers. Furthermore, HKOH-1r, which was designed for better cellular uptake and retention, has performed robustly in detection of endogenous . OH generation by both confocal imaging and flow cytometry. Furthermore, this probe has been applied to monitor . OH generation in HeLa cells in response to UV light irradiation. Therefore, HKOH-1 could be used for elucidating . OH related biological functions.[1]

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Photodynamic therapy plays an important role in cancer treatment. In this work, methylene blue (MB)-embedded calcium carbonate nanorods (CaCO3-MB NRs) have been synthesized for pH-responsive photodynamic therapy and ultrasound imaging. The morphology of CaCO3-MB NRs can be controlled by modulating the concentration of Na2CO3 aqueous solution. The generation of effective reactive oxygen species (ROS) were confirmed by 1,3-diphenylisobenzofuran (DPBF) probe. Both photodynamic therapy performance and echogenic performance of CaCO3-MB NRs were investigated to confirm the feasibility of CaCO3-MB nanohybrids for ultrasound image-guided photodynamic therapy.[2]

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1,3-diphenylisobenzofuran (DPBF) is a fluorescent molecule which possesses a highly specific reactivity towards singlet oxygen (1O2) forming an endoperoxide which decomposes to give 1,2-dibenzoylbenzene. This reaction between DPBF and 1O2 can be followed by measuring the decrease in fluorescence intensity of DPBF. In order to check the specificity of DPBF toward free radicals a series of experiments was carried out in Triton-X micelles and in natural systems (rat liver microsomes), in which DPBF was reacted with hydroxy (HO•), alkyloxy (RO•), alkylperoxy (ROO•), and C-centered radicals (2-cyanoisopropyl radical). In all cases, the DPBF is rapidly transformed to 1,2-dibenzoylbenzene in the case of O-centered radicals and to the corresponding adduct in the case of 2-cyanoisopropyl radical. The experiments in the model systems were also carried out from the chemical point of view and the reaction products were isolated and identified. From the results obtained, it should be stressed that DPBF must be used with caution in complex biological systems for the detection of 1O2, as it also reacts with different radical species.[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

13.14

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

注意： (1). 本产品在运输和储存过程中需避光。  (2). 请将本产品存放在密封且受保护的环境中（例如氮气保护），避免吸湿/受潮。

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

配方 1 中的溶解度: 0.56 mg/mL (2.07 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 悬浮液；超声助溶。
例如，若需制备1 mL的工作液，将 100 μL 5.6 mg/mL 澄清 DMSO 储备液加入 900 μL 20% SBE-β-CD 生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 2 中的溶解度: 0.56 mg/mL (2.07 mM) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
例如，若需制备1 mL的工作液，可将 100 μL 5.6 mg/mL 澄清 DMSO 储备液添加到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。