Real-time intracellular hydroxyl radical 

detection for ROS monitoring in live cells:

CytoCHECK SPAchip® OHrad ROS Single-Detection Kit

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Reactive oxygen species (ROS) term is commonly used to define the reactive molecules and free radicals originating from molecular oxygen. A large number of studies have proved the messenger role of ROS in cell survival, proliferation, differentiation, cell death, apoptosis, and ferroptosis (1,2). CytoCHECK SPAchip® technology facilitates surface modification with a fluorescent dye, enabling real- time monitoring of intracellular ROS through population to single-cell analysis. CytoCHECK SPAchip® OHrad ROS Single-Detection Kit specifically enables the detection of hydroxyl radicals (•OH) via changes in green fluorescence intensity within living cells. In addition, this product has been designed to only be active when in contact with intracellular esterases. Now, CytoCHECK SPAchip® OHrad ROS Single- Detection Kit offers an innovative solution: it can monitor intracellular hydroxyl radical levels without being affected by pH changes of the media; thereby enabling the dynamic detection inside living cells without any perturbation of their physiological or metabolic state over time.



How oxidative stress contributes to neurodegenerative diseases, including neuron damage and mitochondrial dysfunction. For example, Amyotrophic Lateral Sclerosis (ALS) studies on the role of hydroxyl radicals in the degeneration of motor neurons.


How antitumoral drugs may induce hydroxyl radicals leading to apoptotic cell death and ferroptosis (1,2). Therapy assays using antioxidants to reduce oxidative stress in cancer cells to improve the effectiveness of traditional treatments such as chemotherapy and radiotherapy.


How oxidative stress and hydroxyl radicals contribute to atherosclerotic plaque formation and endothelial dysfunction. Studies on oxidative damage induced by reperfusion of ischemic tissues.


How excess glucose and metabolic disorders induce the generation of hydroxyl radicals, contributing to diabetes complications. 


How oxidative stress contributes to cellular aging and the deterioration of biological functions with age. Research on the relationship between oxidative stress and diseases such as osteoporosis and macular degeneration. 


Studies on how ROS, including hydroxyl radicals, act as mediators of inflammation and contribue to pathogenesis of chronic inflammatory and autoimmune diseases.

Monitoring intracellular radical hydroxyl (•OH) for reactive oxygen species (ROS) detection in 1095SK fibroblast cell line

SPAchip® technology is optimal to work in the intracellular environment and has the ability to remain in the cytoplasm for long periods of time (3). 

CytoCHECK SPAchip® OHrad ROS Kit has been designed to be active ONLY when in contact with intracellular esterases, thus obtaining a fluorescent signal with internalized SPAchips. 

CytoCHECK SPAchip® OHrad ROS Single-Detection Kit is selective for hydroxyl radicals (•OH). In cells, •OH radical can be generated spontaneously from H₂O₂ through the Fenton and Haber-Weiss reactions catalyzed by metal ions (4). H₂O₂ is formed by superoxide dismutase (SOD) from superoxide radical anion (O2•-). Hence, this kit enables to measure •OH radicals generated either as primary ROS or as secondary species downstream of other ROS.

Additionally, OHrad ROS SPAchips are not sensitive to pH changes (Fig. 1), avoiding measurement artifacts due to pH variations in experimental setup.

The use of Doxorubicin (DOXO), an anthracycline that has been broadly used in the treatment of malignant tumors due to its broad spectrum, efficiency, and induction to oxidative stress, results in excessive reactive oxygen species (ROS) generation (5,6). DOXO produces ROS by redox cycling at its semiquinone structure (6,7). As reported in the Fenton‘s reaction, iron catalyses the generation of •OH radicals from H₂O₂ (7,8). 

Here, we analyzed reactive oxygen (ROS) changes in 1095SK fibroblast cells using CytoCHECK SPAchip® OHrad ROS Single-Detection Kit. Firstly, we observed internalized OHrad ROS SPAchips in green fluorescence channel in fibroblast cells (Fig. 2). Secondly, ROS concentration analysis in 1095SK cell line was performed, observing that SPAchip signal is dependent on the ROS amount produced by the cells due to drug concentration (3 and 6 hours after DOXO treatment) (Fig. 3). SPAchip fluorescence signals showed an increment in the intensity (compared with basal condition) as a function of the drug concentration (Fig. 3).

Thus, CytoCHECK SPAchip® OHrad ROS Single-Detection Kit allows to easily detect intracellular hydroxyl radical dynamics inside live cells (e.g. 1095SK fibroblast cells; Fig. 2) without causing cytotoxicity over long periods of time. In addition, our SPAchip biosensors facilitate accurately monitoring and measurement of reactive oxygen species without being affected by the pH of media (Fig. 1)


  • ROS sensor: intracellular activation by ctosolic esterases.
  • ROS specificty: hydroxyl radical (OH).
  • ph-insenstive.

Figure 1: CytoCHECK SPAchip® OHrad ROS Single-Detection Kit is not pH-sensitive. 

No significant effect of pH on the fluorescence of CytoCHECK OHrad ROS SPAchips®.

Figure 2:

CytoCHECK SPAchip® OHrad ROS Single- Detection Kit in fibroblast

1095SK cell line. 

Images showing intracellular ROS SPAchip® in green, 40x magnification objective. In blue, nuclear staining by Hoechst 33342. In red, cytoplasmatic staining by Cell Mask™ Plasma Membrane. Scale bar: 20 µm. acquired with  green, 40x magnification objective. In blue, nuclear staining by Hoechst 33342. In red, cytoplasmatic staining by Cell Mask™ Plasma Membrane. Scale bar: 20 µm.


a)  CytoCHECK SPAchip® ROS Green 1095sk 3 hours

b)  CytoCHECK SPAchip® ROS Green 1095sk 3 hours

Figure 3: Doxorubicin dose-response with SPAchip® OHrad ROS Single-Detection Kit in fibroblast 1095SK

Fluorescence corrected intensity of ROS detection probe coupled to SPAchips within fibroblast 1095sk cell line after 3 hours a) and

6 hours b) of treatment with different Doxorubicin (DOXO) drug concentrations. ROS SPAchips exhibit a correlation with the treatment concentration. Bars represent mean values for each condition and error bars correspond to SEM values. ROUT method was used for identifying outliers. For statistical analysis, two-way ANOVA with alpha value set to 0.05 (95% confidence interval) was performed.


1.Chen Z., Wang W., Abdul Razak SR., Han T., Ahmad NH., & Li X. Ferroptosis as a potential target for cancer therapy. Cell Death Dis. 2023 Jul 24;14(7):460.

2.Yu Y., Yan Y., Niu F., Wang Y., Chen X., Su G., Liu Y., Zhao X., Qian L., Liu P., & Xiong Y.. Ferroptosis: a cell death connecting oxidative stress, inflammation and cardiovascular diseases. Cell Death Discov. 2021 Jul


3.Torras, N.; Agusil, J. P.; Vázquez, P.; Duch, M.; Hernández-Pinto, A. M.; Samitier, J.; De La Rosa, E. J.; Esteve, J.; Suárez, T.; Pérez-García, L.; Plaza, J. A. Suspended Planar-Array Chips for Molecular Multiplexing at

the Microscale. Advanced Materials 2016, 28 (7), 1449–1454.

4.Henninger, C.; Fritz, G. Statins in anthracycline-induced cardiotoxicity: Rac and Rho, and the heartbreakers. Cell death & disease, 2018, vol. 8, no 1, p. e2564-e2564. 5.Espinoza, E. M.; Røise, J. J.; Li, I. C.; Das, R.; Murthy, N. Advances in Imaging Reactive Oxygen Species. Journal of Nuclear Medicine 2021, 62 (4), 457–461. 6.He, H., Wang, L., Qiao, Y., Zhou, Q., Li, H., Chen, S., ... & He, M. Doxorubicin induces endotheliotoxicity and mitochondrial dysfunction via ROS/eNOS/NO pathway. Frontiers in Pharmacology, 2020, vol. 10, p. 1531.

7.Štěrba, M., Popelová, O., Vávrová, A., Jirkovský, E., Kovaříková, P., Geršl, V., & Šimůnek, T. Oxidative stress, redox signaling, and metal chelation in anthracycline cardiotoxicity and pharmacological

cardioprotection. Antioxidants & redox signaling, 2013, vol. 18, no 8, p. 899-929.

8.Van Acker, H.; Gielis, J.; Acke, M.; Cools, F.; Cos, P.; Coenye, T. The Role of Reactive Oxygen Species in Antibiotic-Induced Cell Death in Burkholderia Cepacia Complex Bacteria. PLoS One 2016, 11 (7).

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