Plasmonic photocatalyst-like fluorescent proteins for generating reactive oxygen species
The recent advances in photocatalysis have opened a variety of new possibilities for energy and biomedical applications. In particular, plasmonic photocatalysis using hybridization of semiconductor materials and metal nanoparticles has recently facilitated the rapid progress in enhancing photocatalytic efficiency under visible or solar light. One critical underlying aspect of photocatalysis is that it generates and releases reactive oxygen species (ROS) as intermediate or final products upon light excitation or activation. Although plasmonic photocatalysis overcomes the limitation of UV irradiation, synthesized metal/semiconductor nanomaterial photocatalysts often bring up biohazardous and environmental issues. In this respect, this review article is centered in identifying natural photosensitizing organic materials that can generate similar types of ROS as those of plasmonic photocatalysis. In particular, we propose the idea of plasmonic photocatalyst-like fluorescent proteins for ROS generation under visible light irradiation. We recapitulate fluorescent proteins that have Type I and Type II photosensitization properties in a comparable manner to plasmonic photocatalysis. Plasmonic photocatalysis and protein photosensitization have not yet been compared systemically in terms of ROS photogeneration under visible light, although the phototoxicity and cytotoxicity of some fluorescent proteins are well recognized. A comprehensive understanding of plasmonic photocatalyst-like fluorescent proteins and their potential advantages will lead us to explore new environmental, biomedical, and defense applications.
KeywordsPlasmonic photocatalysis Fluorescent proteins Photosensitization Reactive oxygen species Visible light
Photocatalysis has extensively been used in a variety of applications, including energy generation, environment remediation, and biomedicine, as mentioned in numerous review articles on photocatalysis [1, 2, 3, 4, 5, 6, 7, 8]. Conventional photocatalysis requires three essential components of a semiconductor photocatalyst, a light source with appropriate wavelengths, and an oxidizing agent (e.g. water or oxygen molecules). In semiconductor photocatalysis, the wide bandgap energy (e.g. 3.0–3.2 eV) of semiconductor photocatalysts intrinsically limits light absorption to only the ultraviolet (UV) region (wavelength of light λ < 420 nm), which accounts for only about 4% of the total solar energy. Furthermore, the requirement of UV irradiation is commonly considered as a serious biohazard, potentially leading to premature aging of the skin, suppression of the immune system, damage to the eyes, and skin cancer [9, 10, 11, 12]. Thus, to avoid the use of UV as an activation light source, plasmonic effects of metal nanoparticles (mNPs), such as Au, Ag, and Pt, have been successfully hybridized, resulting in broad and strong light absorption in the visible region [13, 14, 15, 16], as summarized in several recent review articles [17, 18, 19, 20, 21].
One of the important aspects of photocatalysis is photoinduced production of reactive oxygen species (ROS), which often have direct applications for environment remediation and biomedicine, such as disinfection, water purification, and air purification. Typical semiconductor photocatalysts, such as titanium dioxide (TiO2) and zinc oxide (ZnO), were extensively studied for efficient and stable photogeneration of ROS [1, 2, 3, 4, 5, 6, 22]. As intermediate or final products, semiconductor photocatalysis generates several different types of ROS, including superoxide anion (O 2 •‒ ), singlet oxygen (1O2), hydrogen peroxide (H2O2), and hydroxyl radical (–OH•). Regarding ROS produced by plasmonic photocatalysis, O 2 •‒ and 1O2 are typically generated via electron transfer under visible light excitation [13, 14]. Overall, O 2 •‒ and 1O2 play a key role in electrochemistry and photochemistry related to photocatalysis.
There is always an imperative need for cost-effective, eco-friendly, and nontoxic photocatalytic nanomaterials and their photoexcitation using visible (or solar) light. Although plasmonic photocatalysis overcomes the requirement of UV irradiation, it still has concerns with respect to environmental and biomedical utilizations. For example, nano-sized plasmonic photocatalysts (e.g. 1 − 100 nm) could potentially have hazardous and adverse (e.g. carcinogenic and cytotoxic) biological effects, which often result in the limited utilizations for environmental remediation and biomedicine [23, 24]. Noble metals (e.g. Ag, Au, and Pt) also have some drawbacks, including rarity, high cost, and easy dissolution (especially for Ag) upon exposure to air or humidity. In this respect, nontoxic organic photosensitizers (e.g. natural dyes or proteins) could potentially be an excellent alternative to noble mNP-based plasmonic photocatalysts, as photosensitization has a great similarity with visible light-driven plasmonic photocatalysis.
In this review article, we introduce plasmonic photocatalyst-like fluorescent proteins for ROS generation upon visible (or solar) light activation. Several recent review articles have extensively covered photosensitizing molecules found in nature (e.g. porphyrin and chlorophyll) [25, 26, 27] and genetically-encoded ROS-generating proteins for cellular functions and redox signaling pathways [28, 29, 30]. To the best of our knowledge, a systematic review on ROS photoproduction from fluorescent proteins has not yet been available, compared to plasmonic photocatalysis. First, we briefly describe the basic mechanisms of plasmonic photocatalysis and photosensitization in terms of ROS photogeneration. Second, we review selected photosensitizing proteins that can be compared with plasmonic photocatalytic nanomaterials in a parallel manner. Third, we discuss outlook based on the current state of understanding on ROS utilizations. An enhanced understanding of plasmonic photocatalysis and fluorescent protein photosensitization will allow us to take advantage of ROS generated from light-induced fluorescent proteins for unexplored environmental, biomedical, and defense applications.
2 Basic mechanisms of plasmonic photocatalysis and photosensitization
2.1 Visible light-driven plasmonic photocatalysis
2.2 Type I and Type II reactions of photosensitization
2.3 ROS lifetime and migration distance in plasmonic photocatalysis and photosensitization
As explained above, both plasmonic photocatalysis and photosensitization under visible light activation can produce short-lived ROS, given that O 2 •‒ and 1O2 are highly unstable and reactive [39, 40]. ROS photogenerated from plasmonic photocatalysis and photosensitization is only effective in the vicinity to semiconductor photocatalyst nanomaterials or photosensitizing molecules. Typically, O 2 •‒ exhibits a lifetime of ~ 50 μs, depending on the local environments . On the other hand, the typical lifetime of 1O2 is ~ 3.1–3.9 μs in H2O. The lifetime of 1O2 can be as long as 68 μs in deuterium oxide (D2O), because it is mainly determined by energy transfer to the vibrational energy levels of the surrounding molecules [38, 42]. Short-lived ROS from plasmonic photocatalysis and photosensitization allows the migration distance to be as long as ~ 320 and ~ 200 nm for O 2 •‒ and 1O2, respectively [41, 43]. Overall, the short lifetime and the relatively short migration (or damage) distance can be considered as a disadvantage requiring a high concentration for a prolonged effect or an advantage for a safeguard, given O 2 •‒ and 1O2 are extremely reactive and toxic.
3 Identification of phototoxic fluorescent proteins from biological studies
The phototoxicity and cytotoxicity of some fluorescent proteins are well known in different scientific communities. In cellular imaging, several nontoxic variants of phototoxic fluorescent proteins were successfully developed for cellular labeling and imaging in vivo [44, 45, 46]. In a contrary manner, phototoxic fluorescent proteins have extensively been employed as a means of selectively damaging target molecules in a localized region and at a particular time-point upon light activation [47, 48, 49]. Chromophore photoreduction in red fluorescent proteins (RFPs) is considered to be mainly responsible for photobleaching and phototoxicity, forming dianionic open-shell states of the chromophore in RFPs . This method is known as chromophore-assisted light inactivation (CALI) that can be used to inactivate target cells and ablate tissue of interest. In particular, CALI using fluorescent proteins can allow for spatiotemporal knockdown or loss-of-function of targeted proteins, which can be microscopically controlled with light activation in situ [30, 47, 49, 51]. In addition, some fluorescent proteins can be used for photodynamic therapy (PDT) to destruct diseased tissue without affecting the surrounding healthy tissue [52, 53, 54].
CALI and PDT using fluorescent proteins can offer an initial overview to identify major ROS-generating fluorescent proteins. There are several studies on CALI using photosensitizing proteins, such as enhanced green fluorescent protein (EGFP) [55, 56], mini Singlet Oxygen Generator (miniSOG) , KillerRed , and SuperNova . CALI with EGFP was used to inactivate α-actinin in fibroblasts, which resulted in stress fiber detachment . EGFP variants, including enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP), were used for CALI. In general, the efficiency followed an order of EGFP > EYFP > ECFP . The use of miniSOG for CALI was demonstrated , in which miniSOG was fused with the succinate dehydrogenase complex subunit of the mitochondrial respiratory complex II to disrupt complex II activity. Mitochondrion-targeted miniSOG caused rapid and effective death of neurons in a cell-autonomous manner without detectable damages to the surrounding cells . Immunophotosensitizer 4D5 single chain variable fragment (4D5scFv)-miniSOG was used to selectively recognize the extracellular domain of human epidermal growth factor receptor 2 (HER2/neu) . KillerRed was used for CALI of Escherichia coli and eukaryotic cells [58, 60, 61]. KillerRed was also tested for PDT by fusing to an antibody to target tumor cells, resulting in tumor-specific cell death . SuperNova, which is a monomeric variant of KillerRed, was used to suppress actin filament motility by illuminating orange light .
4 ROS photogeneration of phototoxic fluorescent proteins
Optical excitation and emission of phototoxic fluorescent proteins and their detected ROS types
Fluorescent protein variant
Excitation maximum (nm)
Emission maximum (nm)
Detected ROS type
Pp2FbFP L30 M
O 2 •‒ and 1O2
O 2 •‒ and 1O2
O 2 •‒ and 1O2
4.1 GFP and EGFP
4.3 Pp2FbFP L30 M
As far as ROS photogeneration is concerned, KillerRed is known to undergo Type I photosensitization reaction to yield O 2 •‒ . Irradiated KillerRed exhibited a tenfold increase in fluorescence signals (λem = 440 nm) of 4-((9-acridinecarbonyl)amino)-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO-9-ac), compared to unchanged levels of controls (Fig. 6c) . As a turn-on fluorescent free radical probe for sensing ROS related Type I photosensitization, the original status of TEMPO-9-ac is not fluorescent as the acridine moiety is initially quenched by the stable paramagnetic nitroxide moiety. ROS (mostly long-lived carbon- or sulfur-centered) can convert nitroxide to the corresponding piperidine, resulting in fluorescence turn-on (λex = 358 nm and λem = 440 nm) [68, 87, 88]. Irradiated KillerRed with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) in PBS showed a broad singlet with a peak-to-trough width of 15 Gauss in the electron paramagnetic resonance (EPR) spectrum, supporting O 2 •‒ generation. (Fig. 6d). As controls, non-irradiated KillerRed and irradiated PBS did not produce EPR signals . ROS associated with Type I photosensitization reaction was also detected with spin trapping of DMPO using steady-state EPR [89, 90]. In addition to O 2 •‒ , 1O2 was detected in irradiated KillerRed using a radical scavenger (sodium azide, NaN3) and a fluorescent probe (ADPA) [59, 69]. Thus, ROS photoinduced by KillerRed is primarily associated to O 2 •‒ with a possibility of 1O2 photogeneration.
4.8 Fluorescent proteins with a cleft-like structure in β-barrels
5 Outlook and conclusion
We have discussed the similarities between plasmonic photocatalysis and phototoxic fluorescent proteins in terms of ROS generation under visible light activation. Like plasmonic photocatalysis, protein photosensitization requires three essential components of a fluorescent protein, a light source with appropriate wavelengths, and an oxidizing agent. A proper interaction of these elements leads to the photogeneration of ROS in the close vicinity. Among the current active applications in environment remediation and biomedicine, O 2 •‒ and/or 1O2 photogeneration from fluorescent proteins could highly be useful for inactivating harmful microorganisms and pathogens, such as bacteria, viruses, and fungi [111, 112], as well as contaminants and endocrine disrupting compounds . In particular, ROS (i.e. 1O2) can be effective in inactivating viruses, impairing genome replication [114, 115, 116, 117]. ROS could be useful for insect eradication [118, 119] and water disinfection for control of water-borne pathogens [120, 121].
Protein photosensitization can offer several pivotal advantages over conventional photocatalysis: (i) Fluorescent proteins can rule out biohazardous concerns on the byproducts and residuals of foreign synthesized metal/semiconductor nanomaterial photocatalysts. Thus, fluorescent proteins can overcome the limitation of hazardous and adverse (e.g. carcinogenic and cytotoxic) effects associated with photocatalytic nanoparticles [23, 24]. Fluorescent proteins are degradable and digestible, eliminating the potential risk of exposure and consumption. (ii) Without a need of additional nanoconjugations (e.g. mNPs, photosensitizers, and quantum dots), fluorescent proteins can generate selective ROS by being activated under solar (visible) light without UV irradiation. (iii) As ROS-generating nanomaterials, fluorescent proteins could potentially be mass-produced in an eco-friendly manner using biological reactors (e.g. microorganisms and insects) [71, 103, 122, 123, 124].
JWL and YLK mainly wrote the manuscript. All of the authors participated in discussion. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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This work was supported by Cooperative Research Program for Agriculture Science & Technology Development (PJ0120892018) from Rural Development Administration, Republic of Korea and Air Force Office of Scientific Research (FA2386-17-1-4072), USA.
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