CAP is rich in reactive species, including reactive oxygen species (ROS), reactive nitrogen species (RNS), charged particles, excited state atoms and molecules, electric fields, and UV radiation. The active species in CAP can initiate a cascade of chain reactions upon contact with a substrate, generating diverse reactive species, including long-lived species such as O₃, H₂O₂, NO₂ ⁻, and NO₃ ⁻, as well as short-lived species such as O, OH, and NO. Different types of reactive species exhibit distinct permeation abilities. Generally, long-lived species penetrate to greater depths than short-lived ones. Moreover, various physicochemical properties, such as solubility, molecular size, and reactivity, also influence the penetration depth of reactive species (Verlackt et al., 2018; Gelker et al., 2020). Table 1 contains some common short-lived species and long-lived species as well as their main biomedical effect. Some factors, including the parameters of the CAP device, the type of working gas, and whether the application is direct or indirect, can all influence the types of reactive species.

The commonly used low-temperature plasma devices mainly include atmospheric pressure plasma jet (APPJ) and dielectric barrier discharge (DBD). Figure 2 illustrates the main differences between DBD and APPJ. There is also a modified form of DBD, namely the floating-electrode DBD (FE-DBD), in which biological tissue can serve as part of the grounded electrode. Increasing the applied power can enhance the penetration depth of CAP within a defined range (Gelker et al., 2018; Gelker et al., 2019). In addition, the power supply frequency can also significantly affect the penetration behavior. Some studies have reported that microsecond-pulsed DBD exhibits stronger penetration than nanosecond-pulsed DBD (Gelker et al., 2018; Gelker et al., 2019).

For DBD, the working gas is mainly air, whereas APPJ commonly uses inert gases, reactive gases, or gas mixtures. When pure inert gases (typically Ar or He) are used as the working gas of APPJ, the resulting effects are primarily physical, with limited radical generation and shallow penetration depth. Moreover, studies have demonstrated that under identical discharge parameters, Ar-based APPJ can generate more reactive species than He-based APPJ and is more effective in disrupting intercellular E-cadherin, thereby enhancing the permeability of the epidermis (Lee et al., 2018). However, when reactive gases or inert–reactive gas mixtures are used, the specific gas type can significantly influence the types of reactive species generated, which in turn results in markedly different penetration depths. In terms of the gas flow rate, Szili et al. reported that when in the absence of gas flow, the penetration of reactive species in deionized water treated with He plasma decreased. Therefore, it can be considered that gas flow can influence the penetration of reactive species (Szili et al., 2015).

Direct treatment refers to CAP being applied in direct contact with tissues, where all reactive components generated by the plasma (including charged particles, short-lived and long-lived species, UV radiation, electric fields, and heat) act simultaneously on the target cells or tissues (Malyavko et al., 2020).

Indirect treatment refers to CAP first being used to activate a liquid medium, producing a plasma-activated medium (PAM) enriched with long-lived reactive species. PAM is subsequently applied to the target tissues (Dai et al., 2023). Since the primary components in PAM are long-lived species, PAM may exhibit stronger permeability and exert its effects for a longer duration than direct treatment, a finding also confirmed in the study by Liu et al. (Liu X. et al., 2018). Another unique advantage of PAM is that it can be delivered (usually injected) into deep tissues, thereby exerting its effects rapidly in vivo. Therefore, PAM is considered a promising new therapeutic approach for treating various diseases like tumors within the body. Numerous studies have already applied PAM in mechanism research and animal experiments, confirming the efficacy of this treatment method (Nakamura et al., 2017; Yao et al., 2025; Jo et al., 2022; Takeda et al., 2017; Cheng et al., 2020; Saadati et al., 2018). Figure 3 illustrates the main differences between direct treatment and indirect treatment of CAP.

In a study in which a gelatin model was used as the target of CAP treatment, it was found that within a certain range, the penetration depth increased monotonically with the treatment time and decreased with the distance from the plasma source to the tissue surface (Wang et al., 2024a; von Woedtke et al., 2020). Notably, excessively long treatment durations or excessively short treatment distances may result in tissue dehydration and damage.

Several studies have demonstrated that dense tissues like the stratum corneum have a significant barrier effect on the penetration of reactive components. Although previous studies have confirmed that CAP can temporarily weaken the barrier properties of the stratum corneum through mechanisms such as lipid peroxidation and electroporation, CAP permeability in intact skin remains substantially lower than that in skin from which the stratum corneum has been removed (Liu X. et al., 2018).

Tissue components can affect the efficiency of active substance uptake as well as their subsequent penetration into deeper layers of the tissue. In the cell membrane, aquaporins (AQPs) facilitate the entry of hydrophilic RONS into cells (Yusupov et al., 2019; Bogaerts et al., 2019). In contrast, cholesterol inhibits oxidation and pore formation, thereby maintaining membrane stability and limiting RONS entry (Chen et al., 2014). For instance, cancer cells typically exhibit elevated AQP expression and reduced cholesterol in their membranes, which facilitates the entry of RONS into these cells. The extent of CAP effects in tissues is not necessarily directly correlated with the capacity of the entry of RONS into cells (Svarnas et al., 2017), but is instead influenced by a complex interplay of multiple factors. In some instances, a substantial uptake of RONS by superficial cells may reduce their availability for diffusion into deeper layers. Conversely, under other conditions, the entry of RONS into superficial cells may initiate intercellular signaling processes that propagate biological effects into deeper tissue regions. In addition, factors such as the content and fluidity of tissue fluid, the number and functional status of mitochondria within cells can also influence the depth of CAP effects in tissues (von Woedtke et al., 2020; Chen et al., 2014; Zorov et al., 2014; Dan Dunn et al., 2015).

When tissue metabolism is highly active, the extracellular matrix exhibits increased hydration and fluidity, which facilitates the diffusion of RONS generated by plasma. Moreover, CAP can activate redox signaling pathways and modulate metabolism-related immune responses, thereby more readily eliciting systemic effects in metabolically active tissues. Some researchers have argued that the direct impact of plasma on tissues is confined to superficial layers and generally persists for only a few minutes, suggesting that more durable and deeper effects depend on host metabolism and immune mechanisms (Graves, 2014). Mizuno et al. demonstrated that in mice bearing multiple tumors, CAP treatment of a single tumor significantly suppressed the growth of distant, untreated tumors, which supports the above concept (Mizuno et al., 2017). This finding highlights the potential of CAP in inducing systemic immune effects, underscoring the pivotal role of metabolic and immunological interplay in mediating its therapeutic efficacy.

In the presence of H₂O₂, HRP catalyzes the oxidation of OPD, producing the yellow compound 2,3-diaminophenazine (DAP) (Hempen et al., 2005; Szili et al., 2014; Szili et al., 2017a).

Ozone (O₃) oxidizes indigo dyes (e.g., indigo trisulfonate, indigo disulfonate sodium), resulting in the decolorization and the formation of colorless isatin derivatives.

The DPD colorimetric method is primarily used to measure chlorine levels; however, it measures any oxidants present. Some previous studies used DPD to detect O₃ (Nie et al., 2018b).

Nitrite reacts with the Griess reagent to form an Azo dye with a maximum absorption wavelength at 540 nm (Liu et al., 2015; Nie et al., 2018a; He et al., 2016; He et al., 2017; Zhang et al., 2019).

The KI–starch reagent serves as a universal ROS detector that can detect several ROSs with oxidation potentials >0.54 V (Kawasaki et al., 2020; Liu D. et al., 2018; Kawasaki et al., 2016; Kawasaki et al., 2019; Ghimire et al., 2019).

An increasing number of Colorimetric Assay are currently being developed, though they may not yet have been applied to CAP’s detection of biological tissue activity. For instance, a research reported a novel colorimetric and near-infrared fluorescent probe (pyridin-4-ylmethyl (Z)-2-cyano-2-(3-((E)-4-hydroxystyryl)-5,5-dimethylcyclohex-2-en-1-ylidene)acetate diphenyl phosphinate group (AN-DP)) based on isophorone and phosphinate groups for ONOO⁻detection (Gu et al., 2020).

DCFH-DA and its analogues are commonly used for detecting RONS within cells. However, their signals may be affected by interference from other cellular components.

Amplex® Red is a sensitive probe for H₂O₂ and peroxidases, producing red fluorescent resorufin upon reaction (Liu et al., 2015; He et al., 2016; Kim et al., 2011; Zhang et al., 2019; Dobrynin et al., 2012).

When assessing the penetration depth of CAP into hydrogels, CF can be encapsulated at high concentration in vesicles where it is self-quenched. Because CAP causes vesicle rupture, CF is diluted, and quenching is relieved, resulting in enhanced fluorescence (Szili et al., 2017a; Marshall et al., 2013).

DHE probe provides high sensitivity and enables visualization of intracellular superoxide generation (Bernhardt et al., 2019).

Other fluorescent probes can also detect RONS; however, they have not yet been applied to assess the effects of CAP on tissue models or tissues. These include dihydrorhodamine 123, indigo green, 1,3-diphenylisobenzofuran, Azulene-Derived Fluorescent Probe (Murfin et al., 2019) and so on.

Lucigenin is a chemiluminescent probe commonly used to detect superoxide. Lucigenin is membrane-impermeable and therefore detects extracellular ROS only (Caldefie-Chézet et al., 2002).

Luminol can be oxidized by various ROS in the presence of catalysts to produce chemiluminescence. Typical catalysts include multivalent metal ions and peroxidase enzymes such as horseradish peroxidase (Szili et al., 2017b).

There are still other Chemiluminescence Assays used for detecting RONS in biological samples. Cypridina luciferin, a kind of Chemiluminescence Assay originally extracted from sea fireflies, could emit blue light in the presence of luciferase and oxygen. People subsequently developed analogs of cypridina luciferin to detect ROS (Yang et al., 2020).

These cells express compartment-specific ROS probes (e.g., the Hycer reporter and firefly luciferase gene) (Vandamme et al., 2010; Markvicheva et al., 2011; Gu et al., 2009; Bilan et al., 2013; Gast et al., 2022; Belousov et al., 2006).

Raman microspectroscopy can detect chemical bonds in living cells (e.g., lipids, proteins, nucleic acids) without exogenous fluorescent dyes or probes and is non-destructive (Smith et al., 2016; Wenzel et al., 2019; Ember et al., 2017). However, its shallow tissue penetration (typically <500 μm) limits deep-structure imaging and hence, its usefulness for assessing CAP effects in tissue depths (Ember et al., 2017; Imanbekova et al., 2022).

Some studies developed computer simulation methods that analyze the physical and chemical interaction mechanisms between plasma and liquids, primarily to model CAP-induced reactions in liquids (Chen et al., 2014; Zhang et al., 2019; Liu et al., 2016; Tian and Kushner, 2014).