Two-Photon Fluorescence Probe in Bio-Sensor

Hui Zhao; Wenbo Hu; Quli Fan

doi:10.7536/PC210408

Progress in Chemistry ›› 2022, Vol. 34 ›› Issue (4) : 815-823 DOI: 10.7536/PC210408

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Two-Photon Fluorescence Probe in Bio-Sensor

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Abstract

Fluorescent probes have become a powerful tool for the study of complex biological systems due to their high sensitivity and selectivity. Compared with single-photon fluorescence probe, two-photon fluorescence probe (TPFP) plays an irreplaceable role in biosensor due to its larger penetration depth, lower interference of tissue autofluorescence and better spatial selectivity. In this review, starting from the design strategies of TPFP, we systematically and comprehensively introduce the application of TPFP in the determination of metal ions, celluar microenvironment, reactive species (including reactive oxygen species ROS, reactive nitrogen RNS, reactive sulfur RSS), enzymes, organelles (mitochondria, lysosomes) and so on. After that, we look forward to the key opportunities and challenges in the development and application of organic TPFP.

Contents

1 Introduction

2 Design principle of two-photon fluorescence probes

3 Bio-application of two-photon fluorescence probes

3.1 Two-photon probes for metal ions

3.2 Two-photon probes for cellular microenvironment

3.3 Two-photon probes for reactive species

3.4 Two-photon probes for enzymes

3.5 Two-photon probes for organelles

4 Conclusion and outlook

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Keywords

fluorescent probe / two-photon absorption / bio-sensor

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Hui Zhao, Wenbo Hu, Quli Fan. Two-Photon Fluorescence Probe in Bio-Sensor. Progress in Chemistry, 2022, 34(4): 815-823 DOI:10.7536/PC210408

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1 Introduction

Fluorescent probe refers to a substance that can interact with the analyte and determine the analyte with a change in its spectral properties. Among them, small molecule fluorescent probes are widely used in the study of biological systems because they can be easily loaded by cells and can quickly detect target molecules in the biological environment^[1,2]. Most of the common small molecule fluorescent probes are designed and developed based on common fluorophores (such as fluorescein, coumarin, BODIPY, rhodamine, etc.)^[3]. However, these fluorophores are usually excited by single photon, and the excitation wavelength is in the ultraviolet-visible region (350 ~ 550 nm).These fluorescent probes will be interfered by light scattering, autofluorescence and photodamage in the detection process, which restricts the development and application of small molecule fluorescent probes in biological detection^[4].

In order to solve the above problems, the concept of two-photon excited fluorescent probe was proposed. Two-photon excitation refers to the process in which a material absorbs two long-wavelength photons at the same time under the excitation of an intense laser source, transitions to a high-energy state through a virtual intermediate state, and emits a photon with a shorter wavelength (Fig. 1)^[5]. Compared with the single-photon excitation fluorescent probe, on the one hand, the excitation wavelength of the two-photon absorption material is in the bio-optical transparent window (650 ~ 950 nm), which can effectively improve the tissue penetration depth (> 500 μm); Two-photon excitation, on the other hand, occurs only at the focal plane of the excitation source, increasing the spatial selectivity^[6]. At present, fluorescent probes based on two-photon excitation have been widely used to detect the localization, movement and concentration of some substances in cells, and their development is of great significance for the development of clinical diagnosis and drug screening^[7].

In This review, based on the design principle of two-photon fluorescence probes, we systematically and comprehensively introduce the application of organic two-photon fluorescence probes in the detection of metal ions, cellular microenvironment, reactive substances (including reactive oxygen species ROS, reactive nitrogen species RNS, reactive sulfur species RSS), enzymes and cellular organelles (mitochondria, lysosomes).Finally, the application prospects of organic two-photon fluorescent probes are summarized, and the opportunities and challenges in the development and application of organic two-photon fluorescent probes are prospected, which provides a reference for the design of more excellent organic two- photon fluorescent probes in the future.

2 Design principle of two-photon fluorescence probe

The design principle of two-photon fluorescence probe is basically the same as that of single-photon fluorescence probe. It usually consists of three parts. The first part is a two-photon fluorophore. When the two-photon fluorescence probe binds or reacts with the target molecule, the fluorescence emission intensity and wavelength of the two-photon fluorophore will change^[8]. The second part is the recognition receptor, which is used to bind or react with the target molecule. The third part is used to connect the two-photon fluorophore and the acceptor^[9].

The mechanism of action of two-photon fluorescent probes reported in the literature can be divided into the following three types. The first type is photoinduced electron transfer (PET) (Figure 2a). PET process refers to the process of photoexcitation.The electron donor group can transfer the electron in the highest energy level to the empty electron orbit of the fluorophore in the excited state, so that the excited electron can not directly jump to the original ground state orbit to emit fluorescence, resulting in fluorescence quenching. When combined with the target molecule, the electron-donating ability of the electron-donating group is reduced, the PET process is inhibited, and the photoexcited electrons in the fluorophore can directly jump back to the original ground state orbital, thereby enhancing the fluorescence emission of the two-photon fluorophore.

The second is intramolecular charge transfer (ICT). The ICT-based two-photon fluorescent probe is mainly composed of electron-donating groups and electron-withdrawing groups, which shows a polarized structure in the ground state, while under photoexcitation conditions, the dipole moment increases, which is prone to ICT process (Figure 2B). For example, when the target molecule is a cation, if it is combined with an electron-withdrawing group, it will increase the degree of intramolecular charge transfer, resulting in a red shift in the fluorescence spectrum, while if it is combined with an electron-donating group, it will reduce the push-pull electron characteristics of the conjugated system, resulting in a blue shift in the fluorescence spectrum.

The third is fluorescence resonance energy transfer (FRET), which generally consists of two fluorophores connected by a flexible acceptor, one of which is an energy donor and the other is an energy acceptor. The occurrence of FRET process is related to many factors, such as the degree of spectral overlap, the relative direction of the transition dipole, the distance between the donor and the acceptor, and so on. Before binding to the target molecule, the two fluorophores are closely linked, resulting in resonance energy transfer and fluorescence quenching. Once the receptor binds to the target molecule, the two closely linked fluorophores are separated from each other, the resonance energy transfer is blocked, and the probe fluorescence is restored (Fig. 2C). In the following, we will analyze the implementation process of the action mechanism of these two-photon fluorescent probes in biological detection through specific examples.

3 Biological application of two-photon fluorescence probe

3.1 Metal ion detection

Intracellular metal ions play an important role in regulating cell function and participating in many cellular processes as cofactors required for structure or catalysis^[10]. Disruption of metal ion homeostasis leads to aging and diseases, including cancer, neurological diseases, and diabetes. In order to ensure normal metabolic processes, the homeostasis of metal ions must be strictly controlled. Therefore, it is important to develop two-photon fluorescent probes that can detect metal ions at the living cell, tissue, and organism levels.

In general, metal ion-responsive two-photon fluorescent probes are composed of a two-photon fluorophore and a receptor capable of binding metal ions. These probes can be divided into two categories according to the change of fluorescence intensity or the change of emission peak position. The common turn-on/off probe mainly depends on the change of fluorescence intensity, because the binding of metal ions will change the two-photon absorption cross section of the fluorescent probe, and finally show the change of fluorescence brightness. As a trace element in human body, zinc plays an important role in gene transcription, cell proliferation, protein metabolism and antioxidation. Detection of changes in zinc ion (Zn²⁺) concentration is important for the study of Zn²⁺ related diseases such as Alzheimer's disease, Parkinson's disease, and prostate cancer. Ahn et al. Developed a novel two-photon fluorescence probe for the detection of Zn²⁺ in lysosomes by conjugating a morpholine group and an N, N-di- (2-pyridyl) ethylenediamine (DPEN) ligand to a naphthalimide dye (Fig. 3). After the probe was added to the Zn²⁺, the PET process was inhibited, resulting in a significant fluorescence enhancement at pH = 5.0^[11]. However, at pH = 7.4, the probe showed only weak fluorescence, indicating that it could specifically recognize Zn²⁺ in lysosomes. When excited by 900 nm light, the probe could detect the Zn²⁺ in the lysosome of NIH 3T3 cells. In addition, it can also image endogenous Zn²⁺ in deep brain tissue of mice by two-photon microscopic imaging technology. Similar to the above detection principle, a series of other types of two-photon fluorescent probes have also been reported.They react with monovalent copper ion (Cu⁺), calcium ion (Ca²⁺), magnesium ion (Mg²⁺),Metal ions such as sodium ion (Na⁺), iron ion (Fe³⁺) and mercury ion (Hg²⁺) all showed obvious fluorescence enhancement when combined^[12]^[13]^[14]^[15]^[16,17]^[18].

However, the above on/off type probe only changes the brightness of the probe luminescence by quenching the luminescence of the fluorophore, but does not change the inherent properties of the chromophore. Due to intracellular photobleaching, uneven distribution, and the presence of noise signals, it is difficult to accurately distinguish the intensity changes of the actual fluorescence signal. Therefore, it is to some extent unreliable to rely solely on a single fluorescence intensity change to obtain quantitative information on intracellular metal ions. Comparatively speaking, the change of emission peak position when combined with metal ions can offset the difference of fluorescence intensity caused by the above factors. In the design of typical two-photon fluorescence probes, the binding of metal ions can change both the fluorescence intensity and the emission peak position. Tian et al. Reported an ICT-based Zn²⁺ ratio two-photon fluorescence probe (Fig. 4). It is composed of 2,2 '-bipyridyl group as electron acceptor, phenyl group as electron donor and alkene as linker, showing D-π-A-π-D structure^[19]. Due to the long conjugation length of the alkene and the strong electron donating ability of the phenyl group, the probe has a large two-photon absorption cross section (516 ± 77 GM) at the wavelength of 700 nm, and the two-photon absorption cross section increases to 958 ± 144 GM when the probe is coordinated with Zn²⁺. In addition, the fluorescence quantum efficiency of the probe was enhanced from 0.59 to 0.71 after the addition of Zn²⁺. The two-photon fluorescence emission spectrum of the probe showed a red shift from 465 nm to 550 nm after the addition of Zn²⁺. Furthermore, the fluorescence emission ratio (I_red/I_green) of the red channel (495 ~ 585 nm) to the green channel (460 ~ 495 nm) of the two-photon fluorescence probe in SHSY-5Y cells was dose-dependent on the concentration of Zn²⁺, which was observed by two-photon confocal fluorescence imaging. In addition, the probe can also be used to monitor the increase of Zn²⁺ levels in mouse brain and zebrafish with Alzheimer's disease, demonstrating that it can be used as a powerful two-photon fluorescence probe to detect endogenous levels of Zn²⁺ in vivo. Similar ratiometric two-photon fluorescence probes for the detection of copper ions (Cu²⁺), magnesium ions (Mg²⁺), palladium ions (Pd²⁺), etc. Have also been successfully developed^[20]^[21]^[22].

3.2 Cell microenvironment detection

pH, polarity and viscosity are important parameters of cell microenvironment, which play an important role in controlling intracellular signal transduction, transport, and interaction with biomacromolecules. Imbalance of these parameters can lead to cell dysfunction.

Among them, pH, as an important cellular microenvironmental factor, is closely related to many cell behaviors, such as cell proliferation, apoptosis, endocytosis and enzyme activity regulation. In different cell compartments, the pH varies from 4.0 to 8.0, where the cytoplasmic pH is close to 7.2, while the mitochondrial pH is about 8.0, and the lysosomal pH is acidic (pH 4.0 to 5.5). Abnormal changes in pH can be considered a hallmark of many diseases, including inflammation, Alzheimer's disease, and cancer, among others. At present, the common two-photon fluorescent probes used to detect pH usually rely on the shift of their absorption or emission spectra during protonation. For example, Kim et al. Reported a 2-naphthol based ratiometric two-photon fluorescence probe with benzimidazol-2-one as fluorophore and triphenylphosphonium salt as mitochondrial targeting unit (Fig. 5)^[23]. After protonation of benzimidazole, the fluorescence of benzimidazole increases at 604 nm and decreases at 540 nm due to the enhancement of its electron-withdrawing ability and ICT effect. Under the excitation of two-photon light source at 820 nm, the heterogeneity of mitochondrial pH in probe-labeled HeLa cells could be determined by fluorescence ratio (I_red/I_green).

Similarly, Park et al. Developed a ratiometric two-photon fluorescence probe, which connected pyridine to the fluorophore of 2-methoxy-6- (5-oxazolyl) naphthalene as the protonation site. Because the pK_a of pyridine is about 5.0, it is suitable for detecting weak acidic environment. After the protonation of pyridine nitrogen atom, the electron-withdrawing ability is enhanced, and the ICT effect is enhanced, which makes the fluorescence red shift, so that the pH value can be detected by the ratio method^[24].

Most of the probes designed for the polarity of the cell microenvironment are also based on the ICT principle. When the polarity of the surrounding solvent changes, the fluorescence of the probe will have a significant blue/red shift or change in fluorescence intensity. Lin et al. Reported a two-photon fluorescent probe with a "D-π-A" structure that facilitates the detection of lysosomal polarity in living systems (Fig. 6)^[25]. It uses 1,8-naphthalimide as a two-photon fluorophore and electron acceptor, and diphenylamine as an electron donor, which are linked by a phenyl group. Among them, morpholino is a lysosomal targeting group, which can promote the accumulation of probes in lysosomes. When the solvent was changed from toluene to DMSO, the fluorescence spectrum of the probe showed a red shift of up to 106 nm, and the fluorescence intensity decreased from 0. 308 to 0. 009, a 43-fold decrease. The fluorescence of the probe was strong in low polarity lysosomes and weak in high polarity lysosomes, which was mainly caused by positive solvation. Finally, the probe can be used to image the polarity changes of embryonic and adult zebrafish models, and it is found that the fluorescence of embryonic zebrafish increases by 1. 8 times at 500 ~ 550 nm, indicating that the polarity environment of embryonic zebrafish is lower than that of adult zebrafish.

As a major property of living cells, subcellular viscosity plays an important role in controlling diffusion and bimolecular reaction rates in diffusion-mediated cellular processes. Most viscosity-responsive probes contain a rotatable conjugated moiety, and the change of environmental viscosity can change the fluorescence intensity by controlling the degree of molecular rotation. Based on this, Lin et al. Designed an open-type two-photon fluorescence probe with deep red fluorescence emission to monitor mitochondrial viscosity (Fig. 7)^[26]. The design of the probe is mainly based on limiting the free rotation of the hemicyanine to improve the fluorescence intensity of the probe, and using carbazole and hemicyanine as a mitochondrial targeting group and a rotatable group respectively. When detected in a low viscosity (1. 4 cP) methanol-glycerol system, the probe showed weak fluorescence at 520 nm excitation, indicating that the fluorescence of the probe was quenched by the free rotation of the hemicyanine. However, in the high-viscosity system (956 cp), the emission intensity was enhanced by 32-fold due to the suppression of the free rotation of the single bond between the carbazole moiety and the indole salt. HeLa cells and zebrafish loaded with monensin or nystatin showed enhanced fluorescence in both one-photon and two-photon modes, and the ability of the probe to track viscosity was verified in inflammatory, fatty liver, and tumor models, respectively.

3.3 Active Substance Detection

Reactive oxygen species (ROS) are natural by-products of the normal metabolism of oxygen, including hydrogen peroxide (H₂O₂), singlet oxygen (¹O₂), superoxide anion radical (

O 2 · -

), hydrogen peroxide radical (HO₂·), hydroxyl radical (HO ·), peroxyl radical (RO₂·) and hypochlorous acid (HOCl).

Among them, the most important primary reactive oxygen species produced in living systems is

O 2 · -

, which plays an important role in maintaining cellular redox homeostasis. The concentration of

O 2 · -

is an important reflection of the level of other ROS, because

O 2 · -

, which is first generated within the mitochondria, will be rapidly converted to H₂O₂, leading to the formation of other ROS. However, excessive

O 2 · -

levels contribute to ischemia-reperfusion, degenerative diseases, and many other diseases caused by oxidative stress. Therefore, the development of a novel fluorescent probe for tracking

O 2 · -

is of increasing importance. Therefore, Zhang et al. Designed a novel turn-on two-photon fluorescence probe based on the interaction between caffeic acid phenethyl ester and

O 2 · -

. In the presence of

O 2 · -

, the catechol structure of caffeic acid was oxidized from the original electron donor to the electron acceptor benzoquinone, which enhanced the fluorescence of the probe. It can be used to dynamically and reversibly study

O 2 · -

fluctuations in hepatocytes, zebrafish, and mice during simulated ischemia-reperfusion injury with high resolution and large tissue penetration depth^[27]. Inspired by previous work on highly nucleophilic

O 2 · -

and ONOO^- sensitive phosphate groups, Yoon et al. Developed a new open-form thiophosphate-based two-photon fluorescence probe for the detection of

O 2 · -

(Fig. 8)^[28]. It was the first to use the thiophosphate moiety as a specific reactive site for

O 2 · -

. The nucleophilic

O 2 · -

first reacts with the electron-deficient phosphorus group, followed by the destruction of the relatively weak π-bond in the phosphorus group to form negatively charged oxygen, followed by the formation of a phosphorus-oxygen double bond with the production of diphenyl phosphate (DPPA) as a by-product, and the production of highly fluorescent 5- (dimethylamino) -naphthalene-1-thiol. It is worth mentioning that the probe can be easily synthesized by one-step reaction, has good photostability and biocompatibility, and has high specificity and sensitivity to

O 2 · -

. The results show that the emission intensity of the probe at 470 nm is linear with the concentration of

O 2 · -

H₂O₂ is a key regulator of cell signaling and is derived from

O 2 · -

by activation of the NADPH oxidase complex. H₂O₂ is involved in a variety of biological responses, stimulating cell proliferation, differentiation, migration, and death. There is growing evidence that excessive H₂O₂ is associated with many diseases, such as cardiovascular disease, neurodegenerative diseases, and cancer. Therefore, the development of efficient fluorescent probes for H₂O₂ response is of great significance for elucidating the biological function of H₂O₂. Masanta et al. Developed a novel ratiometric fluorescent probe for the detection of mitochondrial H₂O₂, which uses arylboronate carbamate as the H₂O₂ reaction site and triphenylphosphonium as the mitochondrial targeting moiety^[29]. The probe has a large two-photon absorption cross-section, has obvious luminescence color change to the H₂O₂, and has the characteristics of good selectivity to the H₂O₂, low cytotoxicity, insensitivity to biomembrane pH and the like, so that the change of mitochondrial H₂O₂ level in living cells and living tissues at the depth of 100-180 mm can be visualized, and is not interfered by other biologically related species. Similarly, Tang et al. Also synthesized a two-photon fluorescence sensor based on a flavonol-borate conjugate, which can achieve specific H₂O₂ response and precise CO release in an optically controllable manner (Fig. 9). In the presence of H₂O₂,The maximum absorption wavelength of the probe is red-shifted, and two fluorescence emission peaks appear at 480 nm and 585 nm, in which the emission at 585 nm is mainly due to the release of the protected hydroxyl group, which restores the excited state intramolecular proton transfer^[30].

Hypochlorous acid (HOCl) is a potent neutrophil antioxidant antibacterial agent, which is mainly produced by myeloperoxidase catalyzed H₂O₂ and chloride ion (Cl^-). HOCl has a special antibacterial effect in the innate immune system, which can oxidize and modify various protein functional groups (sulfhydryl and amino groups) to produce chloramines and aldehydes, and improve the immunity of the body. However, excess HOCl is associated with many diseases, such as rheumatoid arthritis, Parkinson's disease, Alzheimer's disease, and cancer. Therefore, the development of HOCl-responsive probes is necessary to understand its biological effects. Mao et al. Developed a quinolone-based ratiometric two-photon fluorescence probe for the detection of HOCl (Fig. 10)^[31]. Oxidation and deprotection of oxathiolyl groups by HOCl resulted in an enhanced ICT effect and a red-shifted fluorescence emission. In addition, the reaction product with a typical "push-pull" structure can promote its two-photon absorption, enabling it to monitor the fluctuation of HOCl by two-photon fluorescence microscopy. The results showed that the addition of HOCl decreased the emission intensity at 492 nm and increased the emission intensity at 562 nm. In addition, endogenous HOCl was able to be detected in RAW264.7 cells under two-photon excitation at 820 nm. More importantly, excess HOCl generated during wound healing was also able to be monitored by ratiometric two-photon fluorescence imaging.

Reactive nitrogen species (RNS) are chemically active molecules derived from nitric oxide (NO), which act on cells together with reactive oxygen species (ROS). It has been found that nitric oxide is an important signaling molecule involved in the regulation of many physiological and pathological processes. In particular, NO can act as a neurotransmitter by diffusing in the central nervous system to neighboring neurons. In addition, the cardiovascular protective effect of NO has been demonstrated. Dysregulation of NO homeostasis has been implicated in many diseases, such as Alzheimer's disease, pulmonary fibrosis, ischemia-reperfusion injury, and cancer. Therefore, monitoring of NO in intact tissues is essential to understand its biological role. Xiao et al. Designed the first lysosome-targeted two-photon fluorescent probe for NO detection based on the structure of naphthalimide, in which naphthalimide derivatives are a class of fluorophores with excellent two-photon properties.It has been widely used in the design of two-photon fluorescent probes. By introducing o-phenylenediamine into this structure, the specific recognition of NO can be achieved by PET effect^[32]^[33]. Seo et al. Developed a rapid and specific two-photon fluorescence probe for NO reaction, in which acetamido was used as a reporter and diaminobenzene was proline-linked as the reaction site of NO^[34]. After reaction with NO, the two-photon excited fluorescence intensity increased by 68 times within 5 min, which may be due to the blockage of PET after oxidation. In addition, Mao et al. First developed a two-photon fluorescence probe for detecting the far-infrared emission of NO (Fig. 11)^[35]. It is constructed with o-phenylenediamine as the NO recognition site and Nile red as the two-photon fluorophore. Initially, the fluorescence of Nile red is blocked by the o-phenylenediamine unit through the PET process. When the probe is in contact with NO, o-phenylenediamine is converted to triazole, thereby disrupting the PET process, resulting in far-infrared emission (maximum of 650 nm). The probe can perform two-photon fluorescence imaging of exogenous and endogenous nitric oxide in HeLa and RAW264.7 cells, respectively, at 820 nm excitation. In addition, it is capable of imaging NO in mouse liver tissue at a depth of 168 mm with high resolution.

Hydrogen sulfide (H₂S) is a reactive sulfur that can interact strongly with ROS and RNS. Various physiological processes, including regulation of neuronal transmission, regulation of insulin release, relaxation of smooth muscle, and reduction of metabolic rate, have been implicated in H₂S. Therefore, highly selective recognition of H₂S is important for understanding its biological and pathological functions. Azido has a strong electron-withdrawing ability, which is a commonly used method for H₂S recognition. The introduction of azide can destroy the typical D-π-A structure, resulting in no two-photon excitation fluorescence. When this azido group is specifically reduced to the electron donor amino group by H₂S, it will exhibit intense fluorescence emission. Based on this mechanism, Bae et al. Reported a mitochondria-localized two-photon fluorescent probe with naphthalene derivative as the two-photon chromophore, 4-azidobenzylcarbamate as the H₂S reaction site, and triphenylphosphonium salt as the mitochondria-targeting moiety^[36]. Quantitative detection of H₂S in living astrocytes and brain slices was achieved by changing the emission peak position and increasing the two-photon absorption cross section after the reaction of thiocyanate with azido. In addition, Tang et al. Also reported a two-photon fluorescence probe that can simultaneously detect H₂S and HOCl (Fig. 12), which was constructed by linking 7-aminocoumarin and rhodamine B with piperazine, in which, thiolactone and azido were used as HOCl and H₂S reactive groups, respectively^[37]. When HOCl alone was added, the fluorescence at 580 nm gradually increased. The addition of H₂S alone leads to a gradual enhancement of the emission at 445 nm, which is mainly due to the formation of D-π-A structure by coumarin derivatives. The probe can separately image endogenously produced H₂S and HOCl in RAW264.7 cells under two-photon excitation at 750 nm. Compared with the single-response probe, this dual-response probe can obtain different information by measuring two analytes in the same biological system, which is of great significance for exploring the biological effects of multiple analytes related to a variety of diseases and disorders.

3.4 Enzyme assay

Enzymes are highly selective catalysts that play a vital role in accelerating the speed and specificity of metabolic reactions. Abnormalities in enzyme activity are associated with the development of a variety of diseases, including Parkinson's disease, Alzheimer's disease, and cancer. Determining the location and expression level of these enzymes in living cells is essential for early diagnosis and monitoring the effectiveness of disease treatment.

Hu et al. First reported a two-photon fluorescent probe for enzyme detection by incorporating a two-photon fluorophore into a modular probe design^[38]. This modular design requires the inclusion of an enzyme substrate, a fluorophore, and a quencher. The probe is normally in the off state due to FRET action because the fluorophore is efficiently quenched by the quencher. Cleavage of the enzyme substrate, when introduced into the cell, results in the release of the quenching group, along with a highly fluorescent intermediate that has a half-life long enough to diffuse out of the enzyme active site.Available proteins in the same suborganelle as the intended target are labeled, resulting in fluorescence amplification due to the accumulation of the fluorophore at the active site in situ. This new fluorescent probe with good spatiotemporal resolution is expected to expand the breadth of active probes in vivo and open up many new ways for cell biology research. Recently, Tian et al. Developed a two-photon ratiometric fluorescent probe capable of detecting β-secretase (BACE1), also based on FRET^[39]. BACE1 is an important enzyme in Alzheimer's disease. Cyanine derivative (mCyd) was used as the energy donor and Alexa Fluor 633 (AF633) was used as the energy acceptor. They are linked by a peptide chain that is highly selectively cleaved by BACE1 (fig. 13). When the probe was excited by 820 nm two-photon light source, the emission of AF633 was dominant, so the maximum emission was at 651 nm. Subsequently, with the increase of BACE1, the emission intensity at 651 nm gradually decreased and the emission at 578 nm increased due to the disappearance of FRET between mCyd and AF633, which mainly showed the emission of mCyd. Finally, BACE1 in the hippocampal region of Alzheimer mice was detected by two-photon fluorescence imaging to a depth of 300 mm.

In addition, steroid sulfatase (STS) is also an important enzyme, which is mainly derived from the endoplasmic reticulum and is responsible for the hydrolysis of aryl and alkyl steroid sulfates. The activity and response of STS are associated with physiological processes and pathological conditions including estrogen-related breast cancer. STS expression was increased in breast tumor tissues compared with normal tissues. Recently, Li et al. Designed and synthesized a two-photon fluorescent probe based on the two-photon fluorescent dye 4-hydroxy-1,8-naphthalimide for ratiometric detection of STS^[40]. Among them, the sulfate group and the methylsulfonamide group serve as the STS recognition site and the endoplasmic reticulum targeting group, respectively. It is mainly based on the ICT mechanism, which regulates the fluorescence intensity by changing the electron-donating ability of the hydroxyl group. When the probe was added with STS, the red-shifted emission of the probe could be observed due to enzymolysis and phenol release. Finally, the enhancement of STS activity in breast tumor tissue can be monitored by two-photon three-dimensional imaging.

In addition to being able to detect enzymes, two-photon fluorescent probes can also be used for protein tagging. Xiao et al. Used naphthalimide to develop a highly efficient two-photon fluorophore for specific labeling and imaging of subcellular proteins^[41]. Protein-labeled naphthalimide has a large two-photon absorption cross section, which has potential application in protein-specific two-photon fluorescence imaging.

3.5 Subcellular organelle detection

The development of two-photon fluorescent probes that specifically localize subcellular organelles is of great significance for elucidating cellular activities. Lysosome is a typical acidic organelle with a pH value of 4.5 to 5.5. Its main function is to digest food or decompose dead cells. In addition, the localization of lysosomes often changes from the perinuclear region to the surrounding region when the tumor invades. Therefore, the development of lysosomal probes is of great significance for the localization and movement of lysosomes in living cells and the study of cell activity during tumor invasion. Normally, a tertiary amine as a protonation site is often introduced into lysosomal probes, protonating in acidic vesicles with a concomitant enhancement of two-photon fluorescence. To determine the location of the probe in the cell, it is often necessary to colocalize the cell with a fluorescent probe to detect the location of the target and lysosomes. Wang et al. Designed and synthesized a fluorene-based lysosomal probe LT1, which has a pair of 10-unit polyethylene glycol (PEG) groups at the 9-position of fluorene, so that the probe has good water solubility and biocompatibility^[42]. Single-photon fluorescence images of HCT116 cells co-stained with LT1 and a commercially available single-photon fluorescence probe (LT-Red) showed that the localization of LT1 in cells was almost the same as that of LT-Red, indicating that LT1 has a high lysosomal specificity. Preliminary two-photon fluorescence imaging of HCT116 cells was then performed, demonstrating the advantage of the LT1 probe as a two-photon fluorescence probe for the detection of lysosomes. However, it is controversial to show the distribution of lysosomes in cells by using single-photon fluorescent probes coupled with lysosomal two-photon fluorescent probes, because two-photon fluorescence imaging and single-photon fluorescence imaging must be obtained at intervals where lysosomes can move. To overcome this controversy, simultaneous imaging with two lysosomal two-photon fluorescent probes is a method of choice. Son et al. Developed two two-photon fluorescent probes (CTL-blue and CTL-yellow), whose emission peaks are far away from each other, which can be used for colocalization experiments^[43]. Two-photon fluorescence imaging maps of HeLa cells co-labeled with CTL-blue and CTL-yellow exhibited almost perfect overlap.

The most prominent role of mitochondria is to produce energy in the form of ATP. In addition, mitochondria are involved in the control of various cellular biological processes, such as biosynthesis of amino acids and lipids, programmed cell death, calcium ions, and ROS levels. Given that mitochondria play a key role in the regulation of apoptotic pathways, many anti-cancer drugs targeting mitochondria have been developed. Tang et al. Designed a dual-mode mitochondria-targeting probe with aggregation-induced emission (AIE) and Raman output for live mitochondrial imaging (Fig. 14)^[44]. The probe uses cyanostyrene as an AIE backbone, pyridinium as a mitochondrial targeting group, diphenylacetylene as a Raman signal reporter factor, and the Raman signal peak is at the 2225 cm^-1. The probe absorbed at 380 nm and emitted fluorescence at 500 nm in DMSO. It has a two-photon absorption cross section of 14.2 GM under two-photon excitation at 800 nm. When the water content increases from 0% to 70%, the fluorescence intensity decreases due to the enhancement of the intramolecular twisted charge transfer process caused by the increased polarity. AIE action induced fluorescence enhancement with increasing water content from 70% to 99%. In addition, the probe exhibited maximum two-photon excitation at 780 nm, and the two-photon fluorescence intensity increased with increasing probe concentration.

4 Conclusion and prospect

Based on the principle of two-photon fluorescent probes, this paper reviews the research of two-photon fluorescent probes in biosensing applications by many research groups. However, most of the current two-photon fluorescent probes can be excited by near-infrared light, but their emission wavelength is relatively short. The development of two-photon fluorescent probes with near-infrared emission can reduce the scattering of emission light during imaging and achieve deeper penetration depth^[45]. In addition, the absorption cross-section of the current common two-photon fluorophore is small, and its two-photon absorption cross-section will be further reduced due to the quenching effect of the surrounding water molecules. In the biological application of fluorescent probes, the brightness of probe imaging is not enough to obtain high-quality images. In this case, increasing the laser intensity is a way to make up for the above shortcomings. However, increasing the laser intensity can easily exceed the threshold of photothermal damage. Therefore, it is of great significance to explore two-photon fluorophores with large two-photon absorption cross section for their application in biosensing.

In addition, the development of ratiometric two-photon fluorescent probes is also very important, which can facilitate quantitative measurements due to internal calibration in biomedical applications, and FRET-based systems can provide a potential strategy for the design of ratiometric two-photon fluorescent probes. Finally, factors currently well known in the design of single-photon fluorescent probes, including water solubility, fluorescence quantum efficiency, photostability, cytotoxicity, and cell permeability, should also be considered in the development of efficient two-photon fluorescent probes.

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Funding

Natural Science Foundation of Jiangsu Province of China(19KJB140014)

Natural Science Foundation of Jiangsu Province of China(NY219125)

Natural Science Foundation of Jiangsu Province of China(NY220024)

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1 Introduction

2 Design principle of two-photon fluorescence probe

3 Biological application of two-photon fluorescence probe

3.1 Metal ion detection

3.2 Cell microenvironment detection

3.3 Active Substance Detection

3.4 Enzyme assay

3.5 Subcellular organelle detection

4 Conclusion and prospect

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