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Development and Application of Dioxetane-based Chemiluminescent Probes

Anchen Fu , Yanjia Mao , Hongbo Wang , Zhijuan Cao

Progress in Chemistry ›› 2023, Vol. 35 ›› Issue (2) : 189 -205.

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Progress in Chemistry ›› 2023, Vol. 35 ›› Issue (2) : 189-205 DOI: 10.7536/PC220731
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Development and Application of Dioxetane-based Chemiluminescent Probes
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Abstract

Optical analysis is non-destructive, real-time with a specific spatial resolution, which has been developed as an essential technology to study the occurrence, development, diagnosis, and treatment of diseases. It contains fluorescent (FL), bioluminescent (BL) and chemiluminescent (CL) methods. Among them, CL probes with an adamantane-dioxetane chemiluminescence (AD-CL) scaffold attracted much attention. Recently, significant improvement on these probes has been achieved with the elimination of external light source, low phototoxicity, high sensitivity, and a facile system without additional reagents, such as oxidants. Until now, the CL probes were further developed with special modifications and new synthesis routes based on the AD-CL scaffold, realizing the detection and optical imaging of various biomolecules in living systems with enhanced properties. Herein, the recent research progress on AD-CL probes has been reviewed. The review is divided into two parts. The first part will mainly introduce the molecular modification strategy of AD-CL probes and the second part will focus on their application in several cases.

Contents

1 Introduction

2 Modification of AD-CL probes

2.1 Intramolecular modification of AD-CL probes

2.2 Supramolecular modification of AD-CL probes

3 Application of AD-CL probes

3.1 Application on small molecules detection

3.2 Application on macromolecules detection

3.3 Application on pathogenic and drug-resistance bacteria detection

3.4 Other application

4 Conclusion and outlook

Graphical abstract

Keywords

dioxetane / chemiluminescence / probes / molecular design / optical imaging

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Anchen Fu, Yanjia Mao, Hongbo Wang, Zhijuan Cao. Development and Application of Dioxetane-based Chemiluminescent Probes. Progress in Chemistry, 2023, 35(2): 189-205 DOI:10.7536/PC220731

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1 Introduction

The ground state electron in the molecule absorbs energy and is excited to the excited state, and when it returns to the ground state, it produces electromagnetic radiation, that is, luminescence[1]. The excitation energy of Chemiluminescence (CL) is stored inside the molecule and released when a specific chemical reaction occurs, resulting in luminescence. Common CL systems include peroxyoxalate, luminol, etc., which usually use hydrogen peroxide to trigger the oxidation reaction to produce CL[2]. Different from Bioluminescence (BL) based on special enzymes (luciferase) and substrates (luciferin, etc.) In organisms and Fluorescence (FL) based on light excitation,The CL method does not require the transfection of enzyme-related genes, the delivery of enzyme substrates, or the excitation of external light sources, so it can avoid the effects of autofluorescence and light scattering, and has the advantages of simple operation, low cost, low background, and high sensitivity, and has broad application prospects in the fields of medicine, environment, clinic, and food detection[3][4,5].
The luminescence of most CL systems goes through the following two steps: first, an unstable high-energy intermediate is formed by oxidation; In the second step, the energetic intermediate decomposes to generate an excited state species, which produces CL when it returns to the ground state[6]. Most CL reactions depend on the participation of oxidants and need to be carried out in alkaline environment. Therefore, most CL systems can not be directly applied to in vivo analysis, but are mostly used to monitor the changes of some oxides in organisms or related substances involved in the reaction in vitro, which can not be applied under physiological conditions and has a limited scope of application.
The dioxetane chemiluminescent probe has a relatively simple detection system. It is important to introduce Adamantane to stabilize the probe structure, and use the phenol-like structure as the detection basis to finally form the probe skeleton of Adamantane-Dioxetane Chemiluminescence (AD-CL), which can realize the detection of other substances including oxides and has a wide range of applications. In recent years, with the in-depth study of AD-CL probe by researchers, its application in probe design, substance detection, bioimaging and other fields, especially in physiological conditions, has been greatly developed. In this paper, the research progress of AD-CL probe is reviewed, including the modification of AD-CL probe molecule, supramolecular modification and its specific application to various detection objects.

2 Modification of chemiluminescent probe based on dioxetane skeleton

In 1963, White et al. Found that the luminescence mechanism of fireflies involves a chemical structure of dioxetane, which is unstable and spontaneously decomposes to form an excited carbonyl structure, which is accompanied by BL phenomenon when it returns to the ground state[7]. Inspired by this mechanism, researchers have synthesized a series of dioxetane-based derivatives, which are very stable at room temperature, but their CL process is difficult to control and their luminous efficiency is low, so their application is limited[8]. In 1982, Schaap et al. First discovered a special luminescence pathway of dioxetane derivatives[9]. The structure of the derivative is characterized in that the dioxetane structure is connected with adamantane to become more stable, and then connected with a phenol-like structure containing a masking group to form an adamantane-dioxetane (AD-CL) luminescent system. At first, AD-CL and the protected phenolic hydroxyl group were located at the para position of the benzene ring, and then derivatives with both in the meta position were developed. It is found that the structure has a high quantum yield. As shown in Figure 1, on the basis of AD-CL skeleton molecules, the recognition structure of the target analyte is designed at the position of the phenolic hydroxyl protection group. When the target analyte reacts with it, the protection group is removed and the phenolic oxygen anion is exposed.It then decomposes through a Chemically Initiated Electron Exchange Luminescence (CIEEL) process to produce an excited benzoate structure that emits blue light when it returns to its ground state[9]. According to this idea, Schaap et al. Synthesized a series of AD-CL probes for the detection of fluoride and esterase[9]. However, the application of these AD-CL probes under physiological conditions is still limited: (1) low luminescence intensity and poor solubility in the physiological environment with aqueous solution as the matrix; (2) The emission wavelength is relatively single and can not reach the near infrared, which is not conducive to the imaging application of biological tissues. In recent years, researchers have proposed a series of improvements to further expand the application of AD-CL probe.

2.1 Modification of the internal structure of AD-CL molecule

The core of the CL phenomenon of AD-CL structure lies in the formation of a benzoate excited state intermediate. There are three main ways for researchers to modify the molecular structure of AD-CL, as shown in Figure 2: (1) introducing electron-withdrawing groups to increase its solubility and luminescent properties in aqueous solution; (2) introducing a long conjugated group to extend the emission wavelength to the near infrared region; And (3) introducing a phenoxy group to shorten the time for generating a luminescent signal and rapidly detect. The above strategies improve the chemiluminescence performance of AD-CL probe from the aspects of luminescence intensity, luminescence efficiency under physiological conditions and response time, which is conducive to enhancing the detection sensitivity and tissue penetration, and expanding its application in biological detection.
First, as shown in fig. 2A, the introduction of an Electron with drawing group (EWG) on the benzene ring, such as acrylic acid, methyl acrylate, acrylonitrile, etc., can effectively increase the CL strength of the structure[6]. At the same time, the introduction of halogen atoms, such as chlorine (Cl), at the ortho position of the phenolic hydroxyl group can effectively increase the photostability of the probe, reduce its pKa value, and increase the dissociation degree of the excited state intermediate under physiological conditions (pH about 7. 4), thus facilitating the application of the probe in vivo[10,11].
Second, the researchers covalently attached long conjugated groups, such as near-infrared small molecules, to the AD-CL structure, which extended the CL wavelength by increasing the degree of conjugation of the AD-CL structure (Figure 2B), facilitating tissue penetration for imaging analysis. Near-infrared fluorescent small molecules (DCMC) with malononitrile structure can be covalently attached to the para or ortho position of the phenolic hydroxyl group, and specific compounds can initiate chemiluminescence at 690 and 700 nm, respectively[12]. Huang et al. Replaced the oxygen atom (O) in the near-infrared small molecule structure with sulfur atom (S) and selenium atom (Se), respectively, and obtained the corresponding probes with CL wavelengths of 760 and 780 nm, respectively. Due to the red shift of CL wavelength, the light signal will penetrate the organism more easily, which will be conducive to biological detection and imaging at the tissue level[13]. In order to adjust the wavelength of CL, as shown in fig. 2B, the researchers covalently attached different fluorescent groups, such as fluorescein and quinone-cyanine (QCy), to the meta position of the phenolic hydroxyl group through a linking group. When the analyte initiates the CIEEL process,The resulting excited benzoate intermediate transfers Energy to the fluorophore through Energy transfer (ET), so that it is excited to the excited state, and when it returns to the ground state, it emits light at the emission wavelength of the fluorophore[14].
Thirdly, the researchers propose that the construction of AD-CL with the characteristics of rapid response to the analyte will be beneficial to improve the sensitivity of the detection and the real-time monitoring ability of in vivo application. Because the photon can be released in a shorter time, its Signal/noise (S/N) will be higher and its sensitivity will be enhanced. The shortening of the response time is beneficial to the real-time response of the concentration change of biomolecules in vivo. The introduction of benzene or phenoxy groups at the ortho-electron withdrawing position of the phenolic hydroxyl group (Fig. 2C) was found to increase the stability of the phenoxy anion radical, resulting in faster chemical excitation, which is conducive to improving the photon release rate[15]. However, it should be pointed out that the requirements for the chemiluminescence rate are different depending on the application purpose. If it is necessary to respond quickly to the real-time changes of biomolecules, it is necessary to emit light immediately after contact with the target molecules, and the duration should not be too long. If biomolecules need to be monitored for a long time, the reaction rate is too fast, and it is very likely to miss the window period of imaging, which is not conducive to the application of in vivo imaging. Therefore, a probe that can emit light for a long time will be more advantageous.
In addition, by increasing the number of AD-CL units in the molecule, multiple luminescence signals are accumulated to achieve the purpose of increasing the CL intensity. For example, when two AD-CL molecules are linked by a linking group, two benzoate excited state intermediates can be obtained when the chemical reaction triggers the departure of the protecting group (Figure 3A), and the luminescence signal is enhanced by superposition, which is applied by the researchers to detect fluoride and hydrogen peroxide[16,17]. Subsequently, a "domino" signal amplification system was further designed (Fig. 3B), and multiple AD-CL structures were connected by ester bonds. When the protecting group leaves, the phenoxide anion is exposed, and then the "self-decomposition" elimination reaction is initiated to generate a plurality of benzoate excited intermediate structures, so that the luminescence signal intensity is greatly improved[18].
The chemiluminescence performance of AD-CL probe is closely related to the aromatic ring combined with the adamantane-dioxetane moiety in the structure, while most of the current probes use m-hydroxybenzaldehyde as the starting material.The subsequent chemical modification is based on this structure, so only benzene ring can be used, which limits the development of AD-CL probes with different chemiluminescent characteristics. In addition, the AD-CL probe mother nucleus obtained by the existing method usually needs to undergo about seven steps of chemical reactions, and the conditions of individual reactions are relatively harsh, such as the Wittig-Horner reaction at -78 ℃, the synthesis steps are relatively complicated, and the yield needs to be improved. Recently, researchers have developed a new synthetic route of AD-CL, in which the halogenation of different aryl groups is first achieved by Stille cross-coupling reaction, and then the halogenated arene is connected with stannane-enol etherified adamantane in a one-step reaction, and finally AD-CL is produced under singlet oxygen conditions[19]. Through this strategy, researchers have realized the connection of various halogenated aryl or conjugated parent nuclei with functionalized adamantane groups, and developed a series of AD-CL probes with different chemiluminescence wavelengths, quantum yields and luminescence intensities, which greatly enriched the types of probes. At the same time, this simplified synthesis procedure also provides an efficient way to accelerate the synthesis of new probes and explore their applications.
To sum up, in recent years, researchers have modified the molecular structure of AD-CL, improved the CL intensity, extended the CL wavelength, and accelerated the CL reaction rate, making the AD-CL probe more suitable for the detection and imaging of substances in biological tissues under physiological conditions. There is still much room for the development of probe research based on the internal structure of molecules, which is in further research.

2.2 Structural modification based on supramolecular formation

Researchers not only modify the structure of a single AD-CL molecule, but also try to introduce other molecules to interact with the AD-CL element, which can further improve the luminescent properties of AD-CL molecules, including enhancing signal intensity, enhancing stability and prolonging signal duration.
Because the light signal of AD-CL molecules is related to the quenching of water molecules, surfactants can be added to the system to create a hydrophobic environment to reduce the quenching effect of water and further enhance the CL signal. For example, Gnaim et al. Added surfactant-fluorescent dye complexes to the system[6]. As shown in Figure 4A, the commercially available Emerald II, composed of a polymer and an Emerald dye, can form micelles by self-assembly, which will facilitate the transfer of energy from the benzoate excited state intermediate to the fluorescent dye, which emits light at the corresponding wavelength of 540 nm after excitation. The whole process does not need an external light source, only involves the transfer of CL energy within the system, and finally achieves the purpose of enhancing the luminescence signal.
The AD-CL probe can also be assembled with β-cyclodextrin (β-CD) to construct a supramolecular compound system, which can effectively enhance the luminescence intensity of the structure in aqueous solution (Fig. 4B). Shabat's group formed a host-guest complex between AD-CL probe and β-CD at a ratio of 1 ∶ 1, and the CL intensity in aqueous solution was about 60 times higher than before[20]. The researchers further modified the supramolecular compound system: fluorescein was covalently modified to β-CD, and the AD-CL probe and the fluorescein-β-CD structure could form a 1 ∶ 1 host-guest complex. When the protecting group reacted with the analyte,After the CIEEL process was initiated, the CL energy of the excited benzoate was transferred to the fluorescein modified by β-CD, and the fluorescein was excited to emit the corresponding wavelength, and the luminescence intensity was about 1500 times higher than that of the original.
In recent years, researchers have combined amphiphilic molecules with both hydrophilic and lipophilic structures with AD-CL systems to construct a series of new supramolecular CL systems based on nano-coprecipitation. Generally speaking, AD-CL small molecules and (or) fluorescent receptor molecules with energy transfer are encapsulated in the lipophilic and hydrophobic core, and the hydrophilic outer part has good water solubility, forming nanoparticles by coprecipitation to construct supramolecular systems.The AD-CL molecule and other fluorescent molecules can be converged and gathered in a nanometer scale, the distance between the AD-CL molecule and the surrounding fluorescent molecules is close, energy transfer can be effectively carried out, and the CL wavelength can be improved through chemiluminescence energy transfer (CRET). While reducing the background, it can also enhance the ability of tissue penetration (Figure 4 C). Ni et al. Prepared nanoparticles by nano-coprecipitation of AD-CL molecular precursor with TPE-Ph-DCM, a fluorescent molecule with Aggregation-Induced Emission (Aggregation- Induced Emission AIE) and amphiphilicity[21]. After irradiation, TPE-Ph-DCM will produce singlet oxygen 1O2, and the precursor molecule will be transformed into AD-CL structure with dioxygen bridge, which can be spontaneously degraded to start the CIEEL process. At this time, CL energy will be transferred to TPE-Ph-DCM, and TPE-Ph DCM will be stimulated to emit light greater than 650 nm. The system can not only prolong the luminescence wavelength, but also the design of the precursor molecule enables the dioxygen bridge to be generated only when the TPE-Ph-DCM is illuminated, and the luminescence molecule can be continuously activated by one time of white light irradiation, and the luminescence signal can last for more than 10 days, so that the system is expected to be used for surgical navigation. The spontaneous degradation process of the above AD-CL structure can also be realized by heating. Andronico et al. Used the nano-coprecipitation method to wrap the AD-CL structure and the fluorescent amphiphilic substance CN-PPV into the nanoparticles. Because the AD-CL structure has good stability, when the temperature rises to 110 ℃,The degradation occurs and the CIEEL process is initiated. At this time, the CL Energy is transferred to the fluorescent CN-PPV through F Förster Resonance Energy Transfer (FRET), and the corresponding emission wavelength is about 640 nm[22]. Similarly, based on the Chemiluminescence Resonance Energy Transfer (CRET) strategy, Cui et al. Designed three kinds of semiconductor amphiphilic polymers SP with different emission wavelengths, namely PFPV, PFBT and PFODBT, which were used to prepare SPNRs nanoparticles with Chemiluminescence Resonance Energy Transfer sensitive AD-CL structure and PEG-PPG-PEG copolymer by nano-coprecipitation method, and the O 2 · - could start the CIEEL process[23]. Through the CRET effect, the excited state intermediate of benzoate transfers energy to the semiconducting polymer, and the three semiconducting polymers are stimulated to emit at the corresponding wavelengths of 540, 540, and 700 nm, respectively.
To sum up, the researchers used surfactants, cyclodextrins and amphiphilic polymers to explore the interaction between AD-CL molecules and other molecules, and constructed a supramolecular AD-CL luminescence system, which increased the CL intensity under physiological conditions, red-shifted the CL wavelength and prolonged the luminescence duration. The construction of diverse supramolecular architectures will also further improve the luminescent properties of AD-CL as well as the in vivo process.

3 Application of dioxetane-based chemiluminescent probe

Researchers have made a series of fruitful modifications to the AD-CL system, which has promoted the application of the probe in the field of substance detection, especially in the detection of bio-related substances under physiological conditions and in the field of biological tissue imaging. The following will introduce the typical application examples of AD-CL skeleton chemiluminescent probes in recent years from the perspective of different analyte types.

3.1 Detection of small molecules

A variety of bioactive small molecules in organisms are very important functional substances, which participate in physiological processes such as cell signal transduction, and can be used as molecular indicators for disease diagnosis and treatment[24,25]. These reactive small molecules are mainly reactive oxygen species, reactive nitrogen species and reactive sulfur species, including hydrogen peroxide (H2O2), singlet oxygen (1O2), hypochlorous acid (HClO), nitroxyl (HNO), peroxynitrite (ONOO-), cysteine (Cys), glutathione (GSH) and so on[12,26,27][28,29][30][31][32][33][11,26]. Designing groups that can specifically respond to active small molecules as protecting groups for AD-CL probes can not only be used for their specific assays, but also play an auxiliary role in evaluating disease States and drug therapeutic effects.
1O2 is a small molecule involved in cell signaling that is associated with mitochondrial membrane permeability and can be used in tumor photodynamic therapy[34][35][36]. In 2017, the Shabat group designed and synthesized the first AD-CL probe SOCL for detecting singlet oxygen 1O2, and the detection principle is shown in Fig. 5A: when 1O2 exists, SOCL can activate the spontaneous degradation of dioxetane probe to produce CL[28]. In order to enhance the cell membrane penetration of SOCL, the researchers modified the structure with cell-penetrating peptide CPP to obtain a probe SOCL-CPP, which can be used to detect the level of 1O2 in photodynamic therapy. Yang et al. Designed and synthesized a probe CL-SO for detecting 1O2, which can produce CL at 700 nm in the near-infrared wavelength, which is conducive to enhancing the detection and imaging ability of the probe for 1O2 in the biological tissue level[29].
Nitroxyl (HNO), also known as nitrosyl, is a one-electron reduction product of nitric oxide, which has significant biological activity and can be used to treat cardiovascular diseases. An et al. Designed and synthesized a probe HNOCL-1 (Fig. 5B) for quantitative detection of HNO, which can rapidly respond to HNO (the signal reaches the maximum within 20 min), so it can monitor the change of HNO level in living cells in real time[31].
Cysteine (Cys) is a small biological thiol molecule, which plays an important role in physiological and pathological processes, including redox, signal transduction, heavy metal detoxification, etc. Abnormal levels of Cys are associated with many diseases, such as rheumatoid arthritis, Parkinson's disease, Alzheimer's disease, etc. Lack of Cys will lead to hematopoietic dysfunction, leukopenia, psoriasis, etc.Zhang Xinrong's research group designed and synthesized an AD-CL probe CL-Cys (Fig. 5B) for specific detection of cysteine. The detection principle is as follows: the acrylate structure of the sensitive group can undergo Michael reaction with Cys, and after cyclization, it will produce phenoxide negative ions, initiate the CIEEL process, and produce CL signals[33]. The probe has high specificity for the detection of Cys and basically has no response to glutathione and homocysteine, and the probe can image endogenous Cys under the skin of mice.
Biological thiols are involved in redox reactions in organisms and are related to cancer, Alzheimer's disease, etc. Our research group designed and synthesized AD-CL probes for detecting biological thiols, SHCL (Fig. 5B) and NCCL, using 2,4-dinitrobenzenesulfonyl chloride (DNBS), a classical thiol reaction sensitive molecule, as a recognition group. The main difference between them is whether they have chlorine substitution structure[37,38][11]. It was found that compared with NCCL, the probe SHCL with chlorine substitution structure had a lower pKa value, which could reflect the level of biological thiols in a wide range of concentrations in vitro, in tumor cells and in tumor tissues, further verifying the importance of chlorine substitution for AD-CL probe.
In addition to the above bioactive molecules, the AD-CL probe can also be used for the determination of other ions and molecules. Excessive fluoride ion can lead to skeletal fluorosis or DNA damage. Turan et al. Designed and synthesized an AD-CL probe (Fig. 5B) for the detection of fluoride ion, which uses tert-butyldimethylsilyl ether as a recognition group to connect two AD-CLs through dimethylolphenol.After the fluoride ion to be detected reacts with it, the phenoxide anion is exposed, and two molecules of AD-CL can be released, which produces higher light intensity than a single AD-CL, and the detection sensitivity of the fluoride ion is higher[39][16]. Formaldehyde is a common harmful compound in food and environment, which can also be produced in human body through a variety of metabolic and epigenetic processes. Shabat's team designed and synthesized AD-CL probes, CFAP540 and CFAP700, which can be used to detect endogenous formaldehyde molecules in nude mice (Fig. 5B)[40]. Among them, CFAP700 modified with DCMC near-infrared group can emit near-infrared light with a wavelength of 700 nm, which is more penetrating to tissues and is conducive to biological imaging, but its cell permeability is not as good as CFAP540 substituted with methyl acrylate, so both of them have their own advantages in cell imaging.
The pre-accumulation of AD-CL chemiluminescent precursor and then concentrated excitation will be beneficial to the improvement of instantaneous luminescence intensity, so as to obtain high-brightness in vivo bioimaging results. For example, Zhang et al. developed a "dual-lock" response probe, which does not contain a dioxygen bridge structure. Under the action of biomolecules in vivo, a stable AD-CL chemiluminescent precursor is generated and accumulated at the focus, and then external light excitation promotes the free radical reaction to generate a large number of unstable dioxygen bridge products in situ, which release high-intensity chemiluminescent signals through the CIEEL process[41]. Compared with the traditional "one-lock", the brightness is improved by 460 times, which is of great significance for bioimaging applications (Fig. 5C). However, in this study, the probe was directly injected into the subcutaneous tumor of nude mice, and the practical application may need to be further combined with the modification of targeting groups to achieve the purpose of real accumulation of lesions.

3.2 Detection of macromolecules

Biomacromolecules, especially various proteases in organisms, such as cathepsin B, nitroreductase, acetylcholinesterase and fibroblast activating protein, play important physiological functions, and their activity levels are closely related to the occurrence and development of diseases, and can also be used as targets for drug action. The development of rapid, accurate and sensitive detection or imaging technology is of great significance for the determination of clinical disease indicators, the study of the relationship between biomarkers and diseases, the evaluation of disease treatment, and the promotion of the integration of diagnosis and treatment. The detection system of AD-CL is relatively simple, which only needs to add the corresponding detection probe to the sample to be detected, providing a new way for rapid clinical diagnosis. Researchers explored its application in the detection of some clinical disease indicators.
In 1987, Schaap et al. First used the AD-CL probe for the detection of biomacromolecule alkaline phosphatase (ALP), which is associated with many diseases such as bone diseases, hepatitis, and prostate cancer (Fig. 6B). The probe was only used for the determination of ALP under alkaline conditions in vitro[42][43]. Because of the weak luminescence of the probe under physiological conditions (pH = 7.4), it is difficult to apply in vivo.
Since 2016, with the introduction of electron-withdrawing groups in AD-CL probe, the luminescence performance under physiological conditions has been greatly improved, and it has been applied to the detection of various biological enzymes. NAD (P) H: quinone oxidoreductase-1 (NQO1, closely related to cell carcinogenesis), the researchers designed a probe to protect the phenoxy group of trimethylp-benzoquinone structure for in vitro, cell assay, and tumor imaging analysis in tumor-bearing nude mice[44][45]. Tyrosinase is associated with melanoma and connects m-hydroxybenzyl alcohol to the AD-CL backbone through a linker, which can be used to measure Tyrosinase in vitro[46]. Alcohol oxidase (AOX, used in the field of synthesis and analytical testing), researchers use AOX to catalyze methanol into H2O2, and design the probe structure, which can indirectly determine AOX[47][48][49]. γ-glutamyl transpeptidase (GGT) is a cell membrane-bound protease that maintains cellular GSH and Cys homeostasis. Measurement of serum GGT activity is a routine clinical blood test and can be used to diagnose liver and obstructive biliary diseases.An et al. designed the recognition group of the AD-CL probe into a γ-glutamic acid structure (Fig. 6B) that can be cleaved by GGT for the determination of GGT. The probe can respond to GGT with high sensitivity in vitro and in living cells, and can accurately reflect the elevated GGT level in the serum of lipopolysaccharide mice, providing a new method for the detection of GGT in clinical serum[50]. Furthermore, the researchers explored the ability of the probe to image the level of GGT activity in tumor-bearing nude mice in real time after tail vein injection, indicating the location of subcutaneous tumors in nude mice. It is worth mentioning that for the first time, the researchers tried to use the AD-CL single-molecule probe solution system to image subcutaneous tumors in the blood circulation system of nude mice. The single-molecule AD-CL first gathered in the liver and kidney, and then transferred to the subcutaneous tumor site. By setting the imaging observation time, the imaging interference of the tumor site could be avoided. Because of the imbalance of oxygen supply and consumption, it usually creates a hypoxic environment in solid tumors. Nitroreductase (NTR) is one of the representative reductases in organisms. It is a common biomarker of hypoxia and is widely used to evaluate the degree of hypoxia in tumors[51]. Zhang Xinrong's team used the 4-nitrobenzyl structure as the protective group of the AD-CL probe (Fig. 6B). In the presence of the cofactor nicotinamide adenine dinucleotide (NADH), NTR reduced the 4-nitrobenzyl structure to the corresponding 4-hydroxylamine benzyl group in a hypoxic environment, and then initiated the CIEEL process through a 1,6-rearrangement elimination reaction to produce CL[52]. The probe can visually measure in vitro and endogenous NTR, and can image and analyze the hypoxia state of tumor-transplanted mice in real time. Acetylcholinesterase (AChE) can degrade thiocholine to produce sulfhydryl compounds. Inspired by this, our group extended the probe SHCL designed for the determination of biological thiols to detect AChE[11]. AChE uses iodoacetylthiocholine as a substrate and hydrolyzes it to produce a nucleophilic thiol substance, which can further react with the probe SHCL to produce a CL signal (Fig. 6B). It was found that the probe SHCL can be used to determine AChE in vitro, in animal tissues, and in human plasma, and is a rapid AC hE analysis method.
The recognition group of AD-CL probe can not only specifically respond to in vivo protease biomarkers, but also explore the relationship between protease and disease. In addition, the water solubility of the recognition group can improve the luminescence performance of the AD-CL probe under physiological conditions and the cell membrane permeability. Therefore, the introduction of many hydrophilic groups, such as amino acids, peptide chains, and polyethylene glycol (PEG), further improves the performance of AD-CL probes and further expands their potential in disease diagnosis and imaging analysis. Aminopeptidase N (APN) has been proved to be associated with many diseases such as liver injury and cancer. Recently, our group developed a probe APN-CL for detecting APN.The protective group of the probe is designed to be a leucine structure that can be specifically recognized and cleaved by APN, so that APN can be selectively detected in vitro with high sensitivity, and can respond to APN activity in human plasma, tumor cells, and tumor sites of tumor-bearing nude mice[53]. Cathepsin B is highly expressed in pancreatic cancer stem-like cells and is associated with cancer. Based on the characteristic that Cathepsin B can specifically recognize and cleave the carboxyl end of citrulline in Val-Cit, Shabat's team covalently linked the group containing Val-Cit peptide to the AD-CL structure through the linkage group, p-aminobenzyl alcohol, and designed an AD-CL probe that can detect Cathep B (Fig. 6B)[54][55]. When the analyte cleaves the peptide fragment, the linker spontaneously undergoes a 1,6-elimination process to obtain the phenoxide anion structure, thereby initiating the CIEEL process and finally producing CL. In order to improve the water solubility and cell permeability of the probe, the researchers also made two structural modifications to the protecting group of the probe: one was to replace the benzyloxycarbonyl (Cbz) at the N-terminal of valine in the probe with a PEG structure with a degree of polymerization of 17; The second is to introduce a maleimide linker to connect the Cys-Gly-Lys-Arg-Lysine (CGKRK) peptide, which contains three positively charged residues to increase water solubility and improve the cell membrane permeability and tumor targeting of the probe. All the probes responded to Cathepsin B, and the probe carrying the polypeptide fragment had better cell membrane penetration, higher luminescence intensity and detection sensitivity. Because Cathepsin B is overexpressed in cancer cells, the probe was used to image cancer cells RAW 264.7 (leukemia-associated), CT26 (colon cancer cells) and 3T3 (mouse fibroblast cells), and it was found that only cancer cells had light signals, which proved that the probe could visually detect Cathepsin B at the cellular level and distinguish cancer cells from normal cells. Fibroblast Activation Protein Alpha (FAPa) is associated with diseases related to tumors, inflammation, and cell fibrosis, and our research group took advantage of the fact that FAPa can specifically recognize and cleave the amino terminus of proline in Gly-Pro (Figure 6B).Three AD-CL probes with different glycine-terminated structures were designed and synthesized: Cbz-protected probe CFCL, Boc-protected probe BFCL, and quinolone-protected QFCL[56]. It was found that CFCL had the best selectivity for FAPa and could reflect FAPa activity in vitro, cells, nude mice, subcutaneous tumors and human plasma. At the same time, we also explored the application of the probe in the screening of FAPa inhibitors. Prostate specific antigen (PSA) is a specific biomarker of prostate cancer. The researchers designed an AD-CL probe with histidine-serine-serine-lysine-leucine-glutamine (HSSKLQ) peptide as a recognition group to determine PSA in human semen[57][58].
In general, the AD-CL probe starts the CIEEL process by exposing the phenoxide anion, producing CL. However, a considerable number of proteases can only cleave in a certain region of the peptide, such as matrix metalloproteinases, which leads to the failure of AD-CL to produce phenoxide negative ions, limiting the application of the probe in protease detection. In order to break this limitation, the researchers proposed a new protease detection mode based on the CRET strategy[59]. A peptide fragment which can be cleaved by the substance to be detected is designed at the ortho position of the phenolic hydroxyl structure, and one end of the peptide fragment is connected with a fluorescence quenching group, so that the CRET process can be started due to the existence of the quenching group, and at this time, the phenoxide anion can only act with the quenching group and cannot carry out electron transfer to cause the cleavage of dioxetane; Only when the analyte cleaves the peptide and the quenching group is separated from the main structure of AD-CL, can the CRET process be blocked, thus initiating the CIEEL process and generating the CL signal (Fig. 6A). Using this strategy, the researchers designed an AD-CL probe to detect matrix metalloproteinases (MMPs), which are associated with cancer, secondary infection, and organ failure and selectively recognize and cleave the G-V bond in the PLGVRG peptide[60][61]. Therefore, the researchers assembled the peptide as a recognition group on the AD-CL probe backbone, which was cleaved by matrix metalloproteinase to block CRET and produce CL.
The AD-CL probe can be used for imaging detection of biomolecules in vivo, and can also be used for evaluation of biological functions of cells in vivo. Natural Killer (NK) cells are immune cells that can kill certain types of cancer cells. Adoptive Transfer of NK cells is a promising tumor immunotherapy[62]. Shabat's team designed an AD-CL probe and used it to evaluate the efficacy of NK-92 cell adoptive transfer immunotherapy at the animal level[63]. The light signal generated by the probe was only observed in the MDA-MB-231 cell tumor-bearing mice after intratumoral injection of NK-92 cells, and the light signal was not observed in the tumor site without injection of NK-92 cells and without injection of the probe. This is the first attempt to evaluate the antitumor activity of NK cells in vivo with the AD-CL probe, demonstrating the possibility of the probe for disease treatment evaluation.
In addition, the AD-CL probe can also be designed in the form of a prodrug, and a drug molecular structure is introduced while a design recognition group is introduced, so that the probe releases the drug after responding to the marker at the focus, and achieves the effects of imaging and treatment at the same time. β-galactosidase (β-galactosidase/β-gal), which is overexpressed in primary ovarian cancer cells and is closely related to cell senescence, Morquio B syndrome, and β-galactose sialidemia, was successfully measured and imaged in Heck293-LacZ cells in vitro by using β-galactose structure as a recognition group design[12,26][64][65]. The Shabat team designed a prodrug probe to monitor the progress of the drug in vivo (Figure 7). The basic principle is as follows: the phenol structure is protected by a recognition group, and the drug molecule is connected to the para position to obtain the prodrug molecule. When the recognition group leaves after the reaction with the analyte, the CIEEL process can be initiated through a spontaneous degradation process to produce CL and release the drug monomer[66]. In practical application, the researchers designed the recognition group as β-galactose, the substrate structure of β-gal highly expressed in tumor cells, and the drug was methylreoxetine E to obtain Prodrug 1. In order to compare with Prodrug 1, the researchers replaced β-galactose with methyl structure to obtain Prodrug 2. The results of cell and animal experiments showed that only when β-gal reacted with the recognition group, the CIEEL process was initiated, CL was produced and the drug was released, while the control molecule could not emit light and release the drug.
The above application examples show that the probe can be widely used for the detection of various types of biomolecules, and provides a new direction for the development of new detection methods for clinical disease indicators, the study of disease mechanisms, the revelation of the relationship between diseases and biomarkers, and especially the study of tumor disease-related substances. On the other hand, AD-CL, as a molecular optical probe, can also be developed into an integrated tool for diagnosis and treatment through structural design, especially combined with the concept of prodrug design, for disease treatment evaluation and drug targeted delivery, which is expected to play an important role in disease diagnosis and treatment.

3.3 Detection of pathogens and drug-resistant bacteria

The detection of bacteria is of great significance in disease prevention, food safety and other fields. In 2019, the Shabat team first used AD-CL probes CLSP and CLLP (Figure 8) for the detection of foodborne pathogens, Salmonella (which can cause intestinal fever and diarrhea) and Listeria monocytogenes (which can cause gastroenteritis, leukemia, cystitis and other diseases)[67][68][69][70]. The recognition group of CLSP is octyl ester structure, and the recognition group of CLLP is inositol-1-phosphate structure. Compared with the fluorescent probe, the detection sensitivity is improved by about 600-fold.
Pulmonary tuberculosis is one of the top ten causes of death in the world, which is usually caused by Mycobacterium tuberculosis infection. Early diagnosis can effectively avoid the occurrence of death. Babin et al. Developed an AD-CL probe for specific detection of Mycobacterium tuberculosis, FLASH, which can be used for drug sensitivity monitoring[71]. The recognition group of the probe is a substrate of Hip1 protease of Mycobacterium tuberculosis, has a femtomole (fmol) response to recombinant Hip1 protease in vitro, can detect thousands of Mycobacterium tuberculosis in human sputum samples within minutes, and has high selectivity. At the same time, the researchers also used the probe to evaluate the killing effect of various antibiotics on Mycobacterium tuberculosis in samples, which can be used for tuberculosis drug screening, drug resistance determination and so on.
Carbapenemase-Producing bacteria (Carbapenemase Producing Organisms, CPOs) can hydrolyze carbapenems to produce resistance, which poses a serious threat to antimicrobial therapy. In order to ensure the clinical efficacy of antimicrobial therapy, carbapenems should be used cautiously. Therefore, early detection and diagnosis of CPOs are very important[72]. Shabat's group designed and synthesized the AD-CL probe CPCL (Fig. 8), which can be used to detect CPOs in living bacteria[73]. Carbapenemase can hydrolyze the β-lactam ring of the recognition group, which is rapidly decomposed by a 1,8-elimination process to initiate the CIEEL process, thereby generating CL.
β-lactamase-positive bacteria can develop resistance to commonly used β-lactam antibiotics, which affects the treatment of diseases and poses a threat to human health. Based on the characteristic that β-lactamase-positive bacteria produce the metabolite hydrogen sulfide (H2S) when degrading β-lactam drugs, the Shabat group designed an AD-CL probe (Fig. 8) that can respond to H2S to detect bacterial antibiotic resistance[74].

3.4 Other

Multi-channel and multi-marker detection probes can expand the detection range of analytes and are suitable for the simultaneous detection of multiple components in samples. Multi-channel fluorescent probes have been a research hotspot[37,75,76]. The researchers combined chemiluminescence with fluorescence to construct a chemiluminescence-fluorescence dual-channel dual-marker detection probe. Cheng et al. connected the AD-CL structure responding to O 2 · - with the near-infrared fluorescent probe responding to Caspase-3 to form a single probe molecule, realizing the simultaneous detection of the two substances. Since the imaging channels are chemiluminescence channel and fluorescence channel respectively, the detection does not interfere with each other (Fig. 9a)[77]; On this basis, Huang et al. Constructed a molecular renal probe (MRP) with high renal clearance efficiency for in vivo optical imaging of drug-induced acute kidney injury (AKI) in mice, which consists of a trifluoromethanesulfonate-protected AD-CL element and an Asp-Glu-Val-Asp (DVED) -protected near-infrared fluorescent element. O 2 · - and Caspase-3, which respond to AKI, respectively, and the cyclodextrin structure (CCD), which can improve the clearance efficiency of mouse kidney, are linked for multi-molecular and multi-channel imaging. In addition, MRP can also be used as an exogenous tracer for urine analysis, and its performance is better than that of existing clinical detection methods, demonstrating the great potential of such probes in disease diagnosis and biological research (Fig. 9B)[78].

4 Conclusion and prospect

CL probes based on AD-CL skeleton have been developed rapidly in recent years, and their applications have become more and more extensive. AD-CL probe has the following characteristics: (1) Low background interference and high sensitivity are the traditional advantages of chemiluminescence technology. A luminescence signal can be obtain without an external excitation light source; And (2) that luminous performance in the body is obviously improve. The introduction of the electron-withdrawing group (— Cl) at the ortho position of the phenoxy group reduces the pKa value of the probe, which is beneficial to the formation of phenoxy negative ions under physiological conditions, thereby improving the chemiluminescence quantum efficiency and luminescence intensity, and significantly improving the luminescence performance under physiological conditions. The modification of some near-infrared groups and conjugated groups further prolongs the emission wavelength, which is beneficial to tissue penetration. (3) The design of the protective group at the phenoxy site further expands the application range of AD-CL related probes, covering various biological small molecules, macromolecules, cells and bacteria. (4) The synthetic scheme based on stannane-enol etherified adamantane and halogenated aryl not only simplifies the synthetic steps and improves the yield, but also helps to introduce aromatic groups with different luminescent properties or other long conjugated groups on the skeleton of AD-CL, which is conducive to the design and synthesis of diversified probes. However, this synthesis method needs halogen group to connect, so it is not easy to introduce electron-withdrawing halogen group at the ortho position of phenoxy group, and the introduction of halogen is conducive to reducing the pKa value, thereby improving the luminescence performance under physiological conditions. Therefore, how to introduce halogen substitution in this synthetic path may become one of the next development directions. On the other hand, there is still much room for development of this kind of probe. First of all, most of these probes are eliminated quickly in vivo, and there is no active targeting group. How to further improve the molecular targeting function is another development direction. Secondly, the probe is easy to degrade in vivo, and properly improving its stability in vivo is helpful for its application in vivo. Therefore, the introduction of long cyclic groups such as polyethylene glycol into the structure, or the combination with other micelles, liposomes, and nano-covalent materials may be a way to further improve the stability of the probe. Thirdly, the connection of the targeting head group will make it possible to improve the active targeting performance of the probe in vivo, improving the stability and targeting. It can be predicted that with the further study of the structure, through modification and optimization, especially the improvement of targeting performance, stability and luminescence performance, its application ability in biological tissue imaging, drug targeted delivery and other fields will be greatly enhanced, which will provide an important tool for disease prevention and diagnosis, biological research and so on.

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Funding

Shanghai Agriculture Science and Technology Support Project(21N31900500)

Shanghai Municipal Health Commission Project(202140016)

project of Basic Medicine funded by Minhang Hospital, Fudan University(2021MHJC10)

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