Advances in design strategies and imaging applications of specific butyrylcholinesterase probes
Tianyu Sun , Zhoujun Dong , Paul Michael Malugulu , Tengfei Zhen , Lei Wang , Yao Chen , Haopeng Sun
Butyrylcholinesterase (EC 3.1.1.8) is an enzyme synthesized by the endoplasmic reticulum of the liver, which along with its homologous enzyme AChE (EC 3.1.1.7), catalyzes the degradation of the neurotransmitter acetylcholine (ACh) in organisms [1]. As the vital neurotransmitter in the cholinergic system, ACh plays a crucial role in neural signal transmission and participates in various essential physiological functions such as learning, memory, cognition, and attention [2-4]. Cholinergic signal transmission relies on the dual regulation of two cholinesterases (ChEs) for ACh hydrolysis and the choline acetyltransferase for ACh synthesis. Both AChE and BChE in the synaptic cleft can break down ACh into choline and acetic acid, thus terminating the neural signal transmission (Fig. 1) [5,6].
The amino acid sequence of BChE shares 65% homology with AChE, and both enzymes possess similar active site compositions, including a peripheral anionic site (PAS), a hydrophobic gorge with a depth of 20 Å and a catalytic anionic site (CAS) located deep within the gorge [7,8]. The primary structural difference between AChE and BChE lies in the size of the gorge, where the narrower gorge of AChE confers high specificity towards the substrate ACh, making AChE a critical enzyme involved in the termination of cholinergic signaling in the central nervous system. In contrast, BChE has three residues in the PAS region and another three residues in the gorge replaced by smaller aliphatic amino acids, corresponding to Asn68, Gln119, Ala277, Leu286, Val288, and Ala328 (Fig. 1) [9,10]. These substitutions in BChE create an additional volume of approximately 300 Å3, leading to decreased specificity towards substrates and enabling BChE to degrade larger molecules such as butyrylcholine, succinylcholine, and benzoylcholine [7,11].
Furthermore, BChE possesses the catalytic ability to break the ester bond in the neurostimulant cocaine, converting it into inactive benzoylecgonine and ecgonine methyl ester. Consequently, BChE demonstrates significant pharmacological application potential in treating cocaine addiction (Fig. 2) [12]. BChE can also shield organisms from the harmful impacts of nerve agents like organophosphorus (OP) pesticides. The administration of exogenous BChE, either before or after exposure to OPs, can promote OP consumption and effectively reduce the risk of toxic exposure to AChE [13,14].
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by memory dysfunction, cognitive decline and motor dysfunction [15]. Research has uncovered that in healthy brains, AChE activity comprises approximately 80% of the total ChEs activity, whereas BChE contributes to the remaining 20% [7]. However, in late-stage AD, AChE activity gradually diminishes to a range of 62% to 67% of its physiological levels, whereas BChE activity undergoes a significant surge, increasing up to 140% to 165% [16]. Notably, BChE inhibitors have been demonstrated to exhibit significant neuroprotective effects and can effectively alleviate cognitive impairment in amyloid β-protein (Aβ)-induced or scopolamine-induced AD-like mice [17-20]. Some BChE inhibitors have also exerted anti-inflammatory and antioxidant effects by inhibiting the formation of proinflammatory cytokines, resisting the damage of toxic substances to nerve cells, and clearing oxidative stress products [21,22]. BChE is also believed to participate in the process of converting non-toxic primary Aβ into toxic β-pleated conformations, and the lack of BChE leads to a diminution of fibrillar Aβ in 5xFAD transgenic mouse models [23,24]. Besides, BChE is linked to the formation of neurofibrillary tangles in the central nervous system of AD patients and can stimulate the phosphorylation of microtubule-associated Tau protein [25]. These findings offer novel insights into the pathogenesis of AD and provide theoretical foundations for the development of AD treatment strategies targeting BChE.
BChE also demonstrates a correlation with multiple sclerosis (MS), an autoimmune disease characterized by demyelination of white matter in the central nervous system [26,27]. Compared to control subjects, the level of ACh in the serum and cerebrospinal fluid of MS patients exhibits a substantial decrease. In contrast, the mRNA level and activity of BChE in serum are significantly increased [28,29]. Additionally, BChE is likely to be involved in the deacylation of proteolipid protein (PLP), which is the most abundant protein in the myelin. This process can undermine the stability of the myelin structure, leading to myelin decompaction and exacerbating the formation of lesioned white matter, thereby making BChE a crucial player in the demyelination process of white matter in MS [30].
Moreover, BChE has progressively been unveiled as a critical participant in diverse metabolic-related physiological and pathological processes, offering a novel perspective for the intervention and management of disorders stemming from metabolic dysregulation [31,32]. Intriguingly, studies have revealed that under identical high-fat diet conditions, BChE-knockout mice exhibited significantly higher body weights compared to their wild-type littermates, and this obesity phenotype could be reversed by knocking in BChE gene into BChE-deficient mice [33,34]. BChE also participates in the inactivation of acyl ghrelin, which can reduce food intake and energy consumption, and effectively lower aggressiveness in mice [35]. Beyond hydrolyzing the octanoyl group of ghrelin, BChE has been found to hydrolyze 4-methylumbelliferyl palmitate and arachidonoylcholine under physiological conditions, suggesting its lipolysis activity [36-38]. Further research has shown that BChE inhibits low-density lipoprotein (LDL) uptake by influencing the stability of protein arginine methyltransferase 5 (PRMT5) and its downstream extracellular regulated protein kinases (ERK) signaling pathway [39]. Additionally, significant upregulation of serum BChE activity has been observed in diabetic mice, rats, dogs, and humans [40,41]. BChE has been shown to regulate glucose and insulin homeostasis in BChE-knockout mice by lowering plasma ghrelin levels [42]. It also effectively mitigates the formation of amylin oligomers, thus alleviating the toxicity of protein oligomers to pancreatic β-cells and maintaining their normal function [43]. These findings establish a strong link between BChE and diabetes.
Serum BChE also serves as an essential indicator for assessing the protein synthesis function of hepatocytes and can be used for clinical diagnosis and disease monitoring of hepatocyte dysfunction [44,45]. The average activity range of BChE in plasma is 5000–12,000 U/L, but it is significantly reduced in the case of liver damage [45,46]. In addition, BChE content has also been found to show fluctuating changes during the development of various malignant tumors [47,48]. Specifically, BChE expression was high in some tumor tissues like neuroblastoma [49], oral cancer [50], and breast cancer [51], while it was significantly down-regulated in most other cancers such as endometrial cancer [47], cervical cancer [52] and pancreatic cancer [53]. These findings provide new potential biomarkers for cancer diagnosis and prognosis, enabling better intervention and treatment of cancers. To sum up, BChE plays a crucial role in a wide range of physiological and pathological processes (Fig. 2), so quantitative detection and real-time monitoring of BChE activity are particularly critical in clinical diagnosis, disease surveillance and drug development.
At present, the methods used to detect BChE activity include ultraviolet spectrophotometry, fluorescence spectroscopy, electrochemical methods, radiometric assay, isothermal titration calorimetry and inorganic nanomaterials (Fig. 3) [54-57]. Among them, the Ellman method is the most widely used spectrophotometric method [58]. It involves two reaction processes that is, BChE first catalyzes the substrate butyrylthiocholine (BTC) to thiocholine, and then thiocholine reacts with 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) to produce yellow 2-nitro-5-thiobenzoic acid (TNB). The absorbance of the yellow solution at 412 nm is positively correlated with enzyme activity so that it can be used for quantitative monitoring of BChE activity [58]. Nevertheless, the poor stability of DTNB and the similar absorption wavelength of hemoglobin make this method unsuitable for use in vivo [59].
Additionally, since the detection of the potentiometric method and titration method completely depend on the change of system pH caused by acetic acid generated from the degradation of ACh, slight pH fluctuations in the measurement process will seriously affect the accuracy of the two methods [55]. Voltammetry also fails to avoid the interference of AChE and other proteins in the detection system, resulting in its limited practical application [56,59]. In contrast, radiometric and calorimetric assays are more accurate methods. The radiometric assay typically employs ACh labeled with radioactive isotopes (such as 3H or 14C) as a substrate, and it indirectly determines enzyme activity by measuring the radioactive intensity of the unhydrolyzed substrate or labeled product (acetic acid). Calorimetric assay is also an indirect method for assessing enzyme activity, which utilizes the heat changes captured during the enzymatic reaction as a quantitative indicator. However, due to the complex operation and long stabilization time, neither of these methods is suitable for high-throughput screening [54,55,60]. Also, many inorganic nanomaterials for the detection of BChE have constantly emerged, such as quantum dots, carbon dots and gold nanoclusters, but the poor biocompatibility limits their further development and application [57,61-63]. It is worth noting that fluorescence spectroscopy has attracted much attention in recent years due to its advantages like low limit of detection (LOD), high accuracy, simple operation and non-destructive [54]. Moreover, the chemiluminescent probe can successfully avoid the interference of photobleaching and self-fluorescence because it does not need to rely on an excitation light source. It effectively eliminates the inherent background fluorescence and significantly improves the signal-to-noise ratio and detection sensitivity, showing its great potential in analysis and detection [64]. Therefore, fluorescent and chemiluminescent probes targeting BChE have garnered considerable attention, and research has received extensive attention in recent times. Herein, the highly selective optical probes for BChE that emerged in recent years are briefly reviewed. This review aims to dissect the intricate design strategies of these probes and delve into their practical applications, hoping to provide some valuable ideas for relevant researchers and promote further development. The reported BChE probes are categorized into two principal classes based on their fluorescence response mechanism: single-responsive probes and dual-responsive probes.
Single-responsive probe is the sensor that responds to one analyte. Probes react specifically with the target physically or chemically, causing a significant change in its molecular structure, which triggers a significant change in the detection signal. By utilizing detection systems to capture and identify these signal changes, accurate identification and real-time monitoring of the target analyte are achieved [65,66]. According to the role of BChE in fluorescence production, single-responsive probes are further differentiated into two subclasses: probes that are directly activated by BChE and those that are indirectly activated by BChE.
Probes directly activated by BChE are a class of molecules that can be activated effectively under the enzymatic hydrolysis of BChE. When BChE reacts with the probes, the fluorescence or other signals of the probes will change significantly, thus completing the quantification of BChE activity and monitoring of expression [67]. On the basis of the different response modes, the probes directly activated by BChE are also subdivided into on-off probes (also called fluorescence quenching), off-on probes (also called fluorescence enhancement) and ratiometric probes. Each of these three types of BChE probes possesses distinct characteristics, providing a diverse range of experimental tools and methods for biochemical research and drug screening.
On-off probes refer to a type of probe in which the fluorescence signal changes "all or nothing" before and after identification with the target substance. Specifically, the probe itself possesses strong fluorescence, but following a specific reaction with the target, the fluorescence intensity is significantly reduced or even undergoes quenching. The fluorescence quenching phenomenon can arise from various mechanisms, including excited-state reactions, energy transfer, complex formation, and intermolecular collisions [68].
In 2016, Han et al. ingeniously employed 4,5-dimethoxyphthalimide to label choline and successfully designed a BChE-responsive probe 1 with the unique "turn-off" effect [69]. When exposed to excitation light with a wavelength of 355 nm, probe 1 emitted a robust green fluorescence at 520 nm. Following enzymatic hydrolysis of ester bonds by BChE, this molecule converted into N-carboxylated 4,5-dimethoxyphthalimide derivatives, triggering the intramolecular photoinduced electron transfer (PET) quenching under physiological pH conditions (Fig. 4). This phenomenon led to a rapid quenching of the original fluorescence and the whole system showed a noticeable fluorescence shutdown effect. Probe 1 had also been successfully used to evaluate the inhibitory effect of the ChEs inhibitors tacrine and galantamine. The detection system exhibited visible color changes during testing, making this probe a promising candidate as a tool molecule for the visual high-throughput screening of BChE inhibitors.
Off-on probes are those that emit negligible fluorescence prior to reacting with the analyte, but yield a remarkable fluorescence after the reaction. Due to the absence of high background signals, off-on probes are able to circumvent the inherent background interference in on-off probes, leading to higher accuracy in trace detection. This significant change in fluorescent signals renders the interaction between probes and analytes more intuitive and precise, making off-on probes an effective tool for detecting specific analytes [68].
The majority of the specific BChE probes reported are obtained by substrate-based design strategy, and these probes are mainly composed of fluorescence fragments and recognition groups [70]. In recent years, the unique targeting properties of cyclopropyl fragment have promoted the development of structurally diverse BChE fluorescent sensors. According to the hydrolysis mechanism of ACh and OP pesticides by BChE, the breaking process of the cyclopropyl ester bond is inferred as shown in Fig. 5. First, the imidazole nitrogen atom of His438 in the catalytic triad of BChE seizes hydrogen from the residue Ser198, and the exposed oxygen anion of Ser198 attacks the carbonyl oxygen of the ester bond, releasing the fluorophore with fluorescence through electron transfer. Subsequently, under the mediation of water, the acyl part is separated from the BChE, thus restoring the enzyme activity [71,72].
In 2017, Yang et al. engineered the first specific BChE fluorescent probe 3 by simulating endogenous substrate and implementing stepwise optimization strategies (Fig. 6) [60]. The researchers discovered that the volume of recognition groups onto methoxyfluorescein influenced the selectivity of probes towards two ChEs, and that probe 3 with cyclopropyl moiety as the masking unit exhibited the highest BChE selectivity and relative reaction rate. It could generate a 275-fold increase in the fluorescence intensity when the presence of BChE in the buffer under physiological conditions compared to the absence of BChE. However, limitations such as short excitation and emission wavelengths and potential tissue damage hindered further in vivo investigations. Therefore, Yang and his colleagues continued to develop near-infrared (NIR) fluorescent probes 4, 5 and 6 for BChE in the following year [73]. Among them, probe 6 possessed the longest emission wavelength of 705 nm, enabling it to conduct living imaging in zebrafish and APP/PS1 transgenic mice. Additionally, the researchers experimentally confirmed that the presence of Aβ fibrils and glucocorticoid-induced insulin resistance both upregulated BChE levels, indicating that these two conditions are likely significant factors contributing to the abnormal increase in BChE during AD progression.
Subsequently, Guo et al. found probe 7 with a significantly increased ability to resist AChE interference by adding a chlorine atom to the ortho position of the hydroxyl group in hemicyanine (Fig. 6) [74]. Molecular docking results indicated that four critical amino acid residues in AChE and BChE, respectively, contribute to maintaining the stability of the probe-protein complex. In AChE, the average spatial distance between the four amino acid residues and probe 7 is 3.60 Å, which is significantly greater than that of probe 6 without chlorine atom substitution (2.95 Å), indicating that the presence of chlorine atoms affected the hydrolysis of AChE. However, the mean spatial distance between the two probes and the amino acids in BChE was not much different (3.38 Å for probe 6 and 3.58 Å for probe 7), suggesting that both were suitable substrates for BChE. Probe 7 has been proven to be a tool molecule for screening BChE inhibitors, and it could also image endogenous BChE in HepG2 cells and L02 cells, demonstrating its good membrane permeability and application potential.
Liu et al. constructed probe 8 using a similar chromene-indole fluorophore structure, which could quickly and accurately respond to endogenous BChE and produce a 130-fold fluorescence enhancement before and after hydrolysis [75]. After probe 8 was sprayed in the liver of tumor-bearing mice with abdominal skin excised, the tumor area showed extremely strong fluorescence intensity. Within the same fluorescence threshold range, normal mouse livers showed slight fluorescence. Ex vivo imaging results showed that the fluorescence signal of the tumor liver was 48 times stronger than that of the normal liver. In addition, within 60 min after injection of probe 8, the fluorescence signals in the brains of normal mice and APP/PS1 transgenic mice showed a time-dependent trend, and the fluorescence intensity of each detection time was enhanced in the order of normal mice, 2-month-old mice and 6-month-old mice.
On this basis, Sun et al. successfully synthesized probe 9 by replacing the ethyl group on the quaternary ammonium nitrogen in the hemicyanine with a pentynyl group (Fig. 6). It exhibited excellent selectivity for BChE, and its fluorescence signal maintained excellent stability in a complex environment of multiple ions and amino acids. Subsequently, researchers utilized click reaction to fuse the highly active and highly selective BChE inhibitor with probe 9, and innovatively designed the first probe for theranostics of AD [20,76]. Probe 10 had good blood-brain barrier permeability and photostability. Since the inhibitor part of probe 10 had a high affinity with BChE, it could inhibit the degradation of ACh by BChE and effectively alleviate the spatial cognitive impairment and memory impairment of APP/PS1 transgenic mice. Researchers used this probe to explore the changes in fluorescence signals in the brains of AD model mice of different months. They found that the fluorescence intensity increased with age, which was consistent with the previously reported phenomenon that BChE gradually increased with the progression of AD. These not only proved that probe 10 could accurately reflect the change of BChE content but also revealed its potential application value in the diagnosis and treatment of AD.
In addition, Liu et al. skillfully utilized fluorescent probe 11 composed of nitrobenzoxadiazole (NBD) dye and cyclopropyl moiety to investigate the distribution and expression of BChE in biological systems for the first time in 2021 (Fig. 7) [77]. Cell imaging revealed that selective BChE inhibitors tacrine and tetraisopropyl pyrophosphoramide (iso-OMPA) could significantly prevent the generation of red fluorescence, while selective AChE inhibitor donepezil had no impact on fluorescence, highlighting the high selectivity of probe 11 for BChE. Notably, the red fluorescence of probe 11 in the cell highly overlapped with the green fluorescence used to track mitochondria, indicating that the probe could accurately recognize and locate BChE in mitochondria within organelles. Furthermore, in vitro imaging results showed that the fluorescence intensity of healthy mouse livers was significantly higher than that of other organs, providing strong evidence for the widespread distribution of BChE in liver tissue and new ideas for developing of liver-targeting drugs.
In 2022, Guo et al. adopted a similar design strategy to synthesize probe 12 and evaluated the stress changes of BChE in apoptotic cells using this probe [78]. Probe 12 possessed a large Stokes shift of 130 nm, which helped reduce the overlap between its absorption and emission spectra, thereby significantly minimizing background interference and improving the accuracy. Further research revealed that probe 12 was activated in the hydrogen peroxide (H2O2)-induced apoptosis model of human normal liver cells. The dose of H2O2 was positively correlated with the fluorescence intensity, indicating that probe 12 could effectively reflect the changes of BChE in apoptotic cells. These results also suggested that cell apoptosis was accompanied by pathological up-regulation of BChE activity.
In the same year, Liu's group designed the fluorescent molecule 13 using 2-dicyanomethyldiene-3-cyano-2,5-dihydrofuran as the fluorophore, and cyclopropyl as the BChE recognition moiety. It had good reaction sensitivity and could rapidly recognize the target for 7 min [79]. Good membrane permeability and biocompatibility enabled probe 13 to track BChE in HepG2 cells and HEK293T cells in real time, and identify BChE in the liver and brain of healthy mice. Additionally, researchers developed another probe 14 bearing the butyryl group and found that its performance was similar to probe 13 in vitro, but did not explore the in vivo imaging capabilities in depth.
Similarly, Ding et al. designed probe 15 with no background interference and a high signal-to-noise ratio by coupling the BChE-specific recognition element with a spontaneous cyclization fragment (Fig. 8) [80]. BChE released the oxygen anion by catalyzing ester bond hydrolysis in probe 15, and subsequently, oxyanion attacked the cyano group to trigger an in situ cyclization reaction concomitantly, ultimately leading to fluorescence generation. Probe 15 could not only effectively recognize BChE in HEK293, HeLa, and HepG2 cells, but also had the potential to localize cytoplasm precisely. When incubated with cells for 30 min using a concentration of 5 µmol/L probe 15, fresh mouse brain slices exhibited bright green fluorescence. Interestingly, it could also respond to OP and carbamate pesticides, providing broad prospects for its application in food safety, especially in the detection of trace pesticides.
Based on its intramolecular charge transfer (ICT) process of the donor-π-acceptor (D-π-A) structure, long fluorescence emission wavelength, large Stokes shift and good photostability, the dicyanoisophorone (DCI) fragment is considered to be the classic fluorophore for constructing NIR fluorescent probes [81,82]. The introduction of an electron-withdrawing group (EWG) to the hydroxyl group of DCI-OH (18) will increase the energy gap between its highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO), thereby preventing electron transitions and subsequently causing fluorescence quenching [83,84]. When the EWG is triggered to depart, the previously blocked ICT process is restored. At this point, electrons transfer from the donor group (hydroxyl group) to the acceptor group (cyano group), and this process promotes the regeneration of the fluorescent signal (Fig. 9A) [85,86].
By incorporating cyclopropyl into the DCI-OH derivative, Zhou et al. in 2022 devised a far-red/NIR probe 19 (Fig. 9B) [82]. Probe 19 was used to detect the target activity of BChE inhibitor, and the median inhibitory concentration (IC50) of tacrine on BChE measured by this probe was close to 23.34 nmol/L reported in the literature, indicating that it could be used as a tool molecule for screening BChE inhibitors. Additionally, this probe tracked BChE in HepG2 cells and reflected BChE activity in tumor tissue of HepG2 tumor-bearing mice, demonstrating its potential application in diagnosing BChE-related diseases.
In 2024, Kang et al. also obtained probe 20 with a similar structure [87]. The activity of human serum BChE detected by probe 20 was 10,329.30 U/L, which was similar to the results obtained using a commercially available kit via the enzyme-linked immunosorbent assay (ELISA) method, and both of them were within the normal range of BChE activity. Notably, researchers took advantage of probe 20 to discover for the first time that the content of BChE in human thyroid cancer TPC-1 cells is lower than in normal thyroid Nthy-ori-3–1 cells, suggesting that the occurrence of thyroid cancer is accompanied by a decrease in BChE expression or BChE activity.
Building on these, Jiang et al. further modified the DCI-OH skeleton by introducing furan between the two six-membered rings to construct a fluorescent group FDCI-OH (21) with an extensive conjugation range and longer emission wavelength. Utilizing this fragment, they developed the fast-responding and highly sensitive fluorescent probe 22 [88]. Probe 22 could accurately quantify BChE in human serum, and its fluorescence intensity was highly positively correlated with BChE content in the in vitro test system. It was able to track BChE in both in vivo and in vitro biological systems, bearing weak fluorescence signals in liver injury cell models and significant fluorescence intensity in diabetic mouse models. Yang's group also observed that the diabetic mouse models exhibited more robust abdominal fluorescent responses than normal mice, by the means of conducting intraperitoneal injections of probe 23 into streptozotocin-induced diabetic mouse models and healthy mice [89]. This finding further corroborated the upregulation of BChE in diabetes and revealed the promising widespread application of this probe in assisting diabetes diagnosis.
Inspired by the fact that Asp70 in the PAS region of BChE induce butyrylcholine to rapidly enter the CAS region through the electrostatic interaction, wang et al. sought to introduce positive charges into the probe structure, aiming to increase the targeting effect between the probe and Asp70, and improving the affinity. In the study, probe 24 demonstrated the fastest response to BChE within 5 min, could image BChE in AD model cells and effectively distinguish normal cells from AD cells. Noticeably, an extra negative charge caused the probe to be repelled by PAS, thereby extending the time required for the probe to enter the CAS region of BChE. Conversely, introducing more positive charges induced the molecule to form a cation-π interaction with Trp82. This interaction delayed the binding of subsequent probe molecules to BChE, ultimately resulting in a reduced detection rate of the fluorescence signal [90].
In 2024, Zhang et al. developed probe 25 featuring an ICT effect and a large Stokes shift. This probe was capable of effectively recognizing and localizing BChE in HepG2 cells and healthy mice, and exhibited high sensitivity in detecting oxon pesticides both in vitro and in cells. Furthermore, the recoveries of paraoxon in spiked vegetable samples ranged from 104% to 108%, emphasizing the great potential of probe 25 in detecting OPs residues in plant samples [91].
The same year, Yang's team also designed a novel NIR fluorescent probe utilizing the donor-dual-acceptor π-electron cyanine skeleton as the fluorophore [92]. To effectively shorten the distance between the recognition group and the catalytic triad deep within the BChE pocket, researchers ingeniously employed 4-hydroxybenzyl alcohol with self-immolative characteristics to precisely link the cyclopropyl with fluorescent moiety, thereby constructing probe 26 (Fig. 10A). Further, probe 28 was synthesized by extending the conjugate system of probe 26. Probes 26 and 28 exhibited 25-fold and 17-fold selectivity for BChE over AChE, respectively. However, after incubation with BChE for 60 min at 37 ℃, the fluorescence signals of probe 28 and fragment 29 decreased by 38% and 54%, respectively. In contrast, probe 26 maintained relative stability, indicating its value for further investigation. In the 100-fold diluted human plasma, the results of BChE activity determined by probe 26 were close to those by the traditional Ellman method, with recovery rates of standard BChE ranging from 97.51% to 104.01%. This probe could precisely identify BChE in HT22 and U87MG cells and was successfully applied to discover the significant inhibitory effect of flavonoid compounds on BChE.
Chen et al. designed and synthesized two innovative fluorescent probes, 30 and 33, for BChE on the basis of the benzopyrylium structure (Fig. 10B) [93]. Under the catalysis of BChE, the cyclopropyl moiety in probe leaves, accompanied by the release of hydroxyl groups. Subsequently, the electron transfer occurred on the hydroxyl group, triggering the ring-opening reaction of the spirocyclic group in the fluorescent moiety. This led to a significant expansion of the conjugated structure, thus activating fluorescence. Probes 30 and 33 exhibited outstanding selectivity and sensitivity to BChE, with the LOD of 0.0072 U/mL and 0.0019 U/mL, respectively. Probe 30 could indicate endogenous BChE in HeLa cells, and the fluorescence intensity obviously increased after adding 1 U/mL of exogenous BChE. However, compound 33 only showed weak fluorescence at the cellular level, possibly due to the lack of N,N-diethylamino group that helps the molecule enter cells. Additionally, probe 30 exhibited stronger red fluorescence in the liver tissue of non-alcoholic fatty liver disease (NAFLD) zebrafish compared to the control group, and this fluorescence could be blocked by tacrine, indicating its potential as a visual tool molecule for NAFLD diagnosis.
Furthermore, by responding to two distinct and independent mechanisms, dual-mode probes enable self-calibration of testing backgrounds or inherent system errors, significantly reducing the occurrence of false positives or false negatives [94-97]. Based on this, Dong et al. ingeniously designed a dual-mode probe 36 in 2023, combining electrochemical and fluorescent signal technologies (Fig. 11A) [98]. In the electrochemical detection system, as the concentration of BChE increases, the oxidation peak current density of probe 36 decreased at −0.28 V, while that of 37 increased at −0.18 V. Calculating the ratio of electrical signals between compounds 36 and 37 allowed quantification of BChE content. Regarding fluorescence properties, the fluorescence intensity of probe 36 at 600 nm surged within the range of 0 to 0.1 mg/mL of BChE levels. The LODs of probe 36 for BChE measured by the fluorescent and electrochemical methods are 0.05 µg/mL and 0.08 µg/mL, respectively. Moreover, the concentrations of BChE in human and mouse serum measured using both the electrochemical and fluorescent detection modes of probe 36 were comparable to those obtained using commercially available ELISA kits. In clinical sample testing, the recovery rates of standard BChE ranged from 91.0% to 106.0%, with a relatively low relative standard deviation (RSD) maintained between 1.8% and 4.5%, further demonstrating the excellent performance and high reliability of the dual-mode probe in BChE determination.
Song et al. also designed a dual-mode probe 38 in 2023, verifying that the level of BChE was down-regulated in liver cancer and liver injury while up-regulated in diabetic patients (Fig. 11B) [99]. The emission peak and absorption peak of 38 increase significantly at 642 and 575 nm in the presence of BChE, respectively, aligning with the emission and absorption wavelengths of compound 39. Probe 38 enabled the identification and detection of BChE through fluorescent strategy (0.000056 U/mL) and colorimetric method (0.00492 U/mL). It was resilient to potential interference from various analytes in complex environments and could effectively recognize BChE even at AChE concentrations up to 1000 U/L. Additionally, L02 cells treated with probe 38 exhibited distinct red fluorescence signals while HepG2 cells displayed weaker signals, indicating that the probe could distinguish between normal liver cells and liver cancer cells. This further proved that the occurrence of liver cancer may lead to a decline in liver protein synthesis function, triggering the downregulation of BChE expression.
Distinct from the approach above, Acari et al. designed the first chemiluminescent probe 40 for specifically targeting BChE, taking into account the advantages of chemiluminescent probes with high sensitivity, high signal-to-noise ratio, and no need for external light excitation, and they [100]. Chemiluminescence arises from the chemical reaction process in which a substance in an excited state returns to its ground state. It does not require photon absorption, thus avoiding the drawbacks of photobleaching, light scattering, and autofluorescence associated with fluorescent probes [101,102]. Probe 40 underwent BChE-mediated hydrolysis to yield an unstable phenolic-dioxirane (41), which subsequently decomposed into the excited intermediate benzoate ester (42) and adamantanone through a chemiexcitation process to produce. The excited-state benzoate derivative transitioned to the ground state by emitting photons, resulting in the generation of bright chemiluminescence (Fig. 12) [100]. With good biocompatibility and chemical stability, probe 40 could not only detect BChE in different cellular environments like HEK293T, HepG2, and SH-SY5Y cells, but also localized to the liver in healthy mice and tumor tissue in SY5Y tumor-bearing mice.
The design strategy and activation principle of probe 43 are identical to those of probe 40, with the primary difference being the absence of chlorine substitution on the benzene ring [103]. Docking results indicated that probe 43 entered the active pocket of BChE and formed interactions with multiple amino acids related to BChE hydrolysis. However, it failed to successfully enter the active cavity of AChE, demonstrating the specificity of the probe from a computational perspective. The probe could distinguish between different types of pesticides and effectively detect pesticide residues in vegetables such as vulgaris, celery, cucumber, and carrot. The luminescence intensity of probe 43 increased with the increase of HepG2 cell count, enabling it to accurately locate the tumor tissue of HepG2 tumor-bearing mice.
Ratiometric fluorescent probes, as opposed to single-emission fluorescent probes, offer significant advantages in the quantitative analysis of biochemical targets. Traditional single-emission probes are susceptible to interference from instrument parameters, system errors, and some inherent properties of fluorophores, seriously affecting the accuracy and sensitivity. This limitation has spurred the development of ratiometric probes, which utilize the ratio of fluorescence intensities at two different wavelengths as the response signal [104,105]. With the unique dual-wavelength emission characteristics, these probes demonstrate remarkable self-calibration capabilities, greatly enhancing their detection sensitivity, accuracy, and resistance to interference [106,107]. Upon recognition of target analytes, ratiometric probes exhibit a red shift or blue shift in their fluorescence spectra, and most probes experience a noticeable color change in solution, allowing for visual observation with the naked eye [108-110].
In 2022, Ding et al. disclosed a ratiometric probe 46 towards BChE utilizing hemicyanine as the fluorophore (Fig. 13) [111]. After activation, the yellow fluorescence of probe decreased at 556 nm, and the blue fluorescence increased at 446 nm. In HEK293 cells, the blue fluorescence signal became apparent 10 min after administration of probe 46, with the ratio of the two fluorescent signals approaching the maximum value at 30 min and remaining stable for up to 55 min, demonstrating the rapid response of probe and the stability of its fluorescent signal. Probe 46 was also used for analyzing BChE levels in serum samples from healthy individuals, osteoarthritis, breast cancer, and colorectal cancer patients. The results showed that serum BChE expression in patients was significantly lower than in healthy individuals. In living cell and mouse brain slice imaging, the BChE inhibitor iso-OMPA dimmed or even disappeared the blue fluorescence, while the yellow fluorescence was enhanced, again providing a strong basis for the fluorescence recovery mechanism of compound 46.
Aggregation-induced emission (AIE) effect is the phenomenon where the luminescence of a compound is significantly enhanced after aggregation [112]. Generally speaking, organic molecules with AIE effects have highly twisted conformations in the aggregated state that limit the free rotation of carbon single bonds. It weakens molecular stacking and intermolecular interactions, significantly reducing non-radiative energy consumption and thus exhibiting higher fluorescence emission efficiency [113]. Most traditional fluorescent dyes have the phenomenon of aggregation-caused quenching (ACQ), which leads to poor sensitivity of sensing systems and limits their application range. The appearance of AIE materials just makes up for this defect, and the advantages of high quantum yield and light stability make it an ideal choice for the development of efficient fluorescent probes [114-116].
In 2022, Gong et al. constructed the ratiometric probe 47 with the AIE effect by introducing the cyclopropyl group onto the hydroxyl group of tricyanofuranyl iminosalicylaldehyde, which was an AIE molecule [117]. Probe 47 also exhibited a weak ICT effect, while the molecule produced had a strong ICT effect after removing the masking group. In a water/tetrahydrofuran system, the ratiometric signal of probe 47 increased with the increase in water proportion until it reached 70%. Once the water content exceeded 70%, the ratiometric fluorescence signal decreased sharply, which may be attributed to the intramolecular twisted charge transfer effect induced by the highly polar environment within the molecule. Probe 47 was capable of responding to the increase in BChE concentration in HeLa cells, with its fluorescence signal gradually increasing at 626 nm and decaying at 760 nm. Furthermore, NAFLD model mice receiving probe 47 produced signals three times stronger than those in the control group, strongly supporting the idea that the onset of NAFLD leads to BChE overexpression.
Similar to the AIE mechanism, the aggregation-induced emission enhancement (AIEE) effect also originates from the restriction of intramolecular motion, but this emphasizes the significant increase in fluorescence intensity or efficiency in the aggregated state [118]. Noteworthily, some molecules exhibiting AIEE phenomena are also equipped with the characteristic of excited state intramolecular proton transfer (ESIPT), such as 2-(2′-hydroxyphenyl)benzothiazole (HBT)-based compounds [119-123]. The ESIPT refers to a four-level proton transfer process between the proton donor and adjacent proton acceptor after the organic molecule is excited by light [124]. This unique process gives molecules a large Stokes shift, effectively preventing fluorescence self-absorption, making fluorescent dyes with ESIPT properties exhibit high potential and application value in fluorescence imaging and sensing [124-126].
In 2023, Chen et al. reported a BChE fluorescent probe 48 with both AIEE and ESIPT effects on the basis of the HBT framework, which enabled precise imaging of HeLa cells and livers in zebrafish NAFLD model (Fig. 14) [127]. Compound 48 underwent BChE-mediated ester bond cleavage and spontaneous formaldehyde release to enol form (50) with the weakly yellow fluorescent at 527 nm, which then isomerized through the ESIPT process to keto form (51) with the intensely orange fluorescent at 614 nm. The increase in system hydrophilicity was accompanied by a decrease in enol emission and an enhancement of the keto signal. Probe 48 emitted a ratio fluorescence intensity in a mixed system of tetrahydrofuran and water (5:95 ratio) that exceeded 200 times that in pure tetrahydrofuran, which was conducive to its application in aqueous solutions.
What is more, the signal generation of the NIR ratiometric fluorescent probe 52 designed by Ding et al. in 2023 relies not only on the enzyme-catalyzed reaction mediated by BChE but also on the subsequent 1,6-elimination reaction (Fig. 15) [128]. After the catalytic reaction was triggered by BChE, the fluorescence signal emitted by probe 52 intensified at 637 nm and weakened at 816 nm. The color of the solution also changed significantly from green to red, which could be observed by the naked eye. In the RAW264.7 cell line overexpressing BChE, the red fluorescence signal of the probe was significantly enhanced, while the NIR fluorescence signal (green fluorescence) was relatively weaker. However, the opposite trend was observed in HeLa cells and B16 cells with relatively low BChE expression levels, revealing the crucial role of BChE in the probe activation process. It was worth mentioning that this innovative sensing system not only could identify BChE in mouse brain tissue slices accurately, but also showed potential applications in detecting pesticide residues in agricultural products.
Furthermore, inspired by the fact that BChE can catalyze the hydrolysis of substrates containing benzoyl groups, for example, benzoylcholine, tetracaine, and cocaine, Yan et al. designed a specific fluorescent probe 57 with benzoyl as the recognition group for BChE using the bioinspired design strategy in 2024 (Fig. 16) [129]. Compared with the other four probes with masking units of acetyl, cyclopropanoyl, cyclobutanoyl, and cyclopentanoyl, benzoyl derivative 57 displayed superior BChE selectivity. Notably, as the volume of the recognition moiety increased, the ratio of fluorescence signal triggered by AChE gradually decreased, indicating that large-volume fluorescent quenching groups effectively resisted AChE interference. Researchers further utilized probe 58 to explore the relationship between Aβ and BChE in AD progression. Cellular fluorescence imaging experiments revealed that Aβ could induce an increase in fluorescent signals in MCF-7 cells, while BChE inhibitors reversed this phenomenon. Additionally, low oxygen conditions, exogenous administration of H2O2, or phorbol 12-myristate 13-acetate (PMA) all induced cell apoptosis. The enhanced green fluorescent signals were observed in these treated cells, indicating cell apoptosis accompanied an upregulation of BChE expression. Combining the phenomenon that Aβ protein induces neuronal apoptosis by causing mitochondrial energy disorders, researchers believe that in AD models, Aβ protein promotes the upregulation of BChE expression by inducing cell apoptosis. This discovery provided a new perspective for better understanding the pathogenesis of AD.
Probes indirectly activated by BChE are those activated by thiocholine, which is produced by the hydrolysis of BTC by BChE. The principle of this detection mechanism is similar to the Ellman method, both utilizing the reaction between the sulfhydryl group of the thiol and the specific part of the probe, thereby resulting in a change in the fluorescent signal and indirectly reflecting the activity of BChE [67].
In 2018, Qian et al. first designed a redox-regulated fluorescent nanoswitch 59 based on reversible disulfide bonds, serving as one of the new means for BChE fluorescence detection (Fig. 17) [130]. The disulfide bonds in polymer 59 were sensitive to thiols, and in the presence of thiols, they underwent decomposition triggered by thiols to generate thiol-functionalized carbon quantum dots (thiol-CQDs) with strong fluorescence. Oxidant H2O2 induced the exposed sulfhydryl group on the surface of thiol-CQDs to connect into disulfide bond components, resulting in fluorescence quenching. The nanoswitch 59 was used to measure the BChE activity in serum samples from three healthy individuals, and the obtained data was close to the results measured by the Ellman method, suggesting the credibility of this detection method. This approach has a certain universality, as when the substrate is replaced with acetylthiocholine, it could also quantitatively measure the activity of AChE.
Utilizing the principle of thiol-click reaction, the same team further designed a fluorescent probe 61 that was sensitive to thiocholine in 2019 [131]. Probe 61 itself had a faint blue fluorescence, while the introduction of thiocholine reduced a rapid thiol-click reaction to produce highly fluorescent thioether, resulting in a sharp increase in fluorescence signal at 460 nm, thus indirectly reflecting BChE activity (Fig. 18). Within the BChE concentration range of 0.2–9.0 U/mL, the fluorescence signal of the probe 61 presented a highly positive linear relationship with the protein concentration (R2 = 0.997). Unexpectedly, the nonspecific response of the probe to glutathione (GSH) caused micromolar levels of GSH in normal human plasma to affect the detection accuracy. Therefore, during detection, it was advisable first to add probe 61 to the sample and then introduce thiocholine after the GSH depletion to minimize the impact of GSH.
Förster or fluorescence resonance energy transfer (FRET) is an experimental technique that detects the relationship between two chromophores based on the spatial distances. When the emission and absorption spectra of two fluorescent groups (donor and acceptor) overlap, the excited donor can transfer energy non-radiatively to the acceptor, altering the overall fluorescence intensity of the system [132]. The efficiency of this energy transfer is related to the distance between the donor and acceptor, typically within the range of 1–10 nm [133]. Among them, 5-(2-aminoethylamino)−1-naphthalenesulfonic acid (EDANS) and 4-[4-(dimethylamino)phenylazo]benzoic acid (DABCYL) are a commonly used fluorescent donor-acceptor pair. When EDANS is excited, its excited-state energy can be transferred non-radiatively to DABCYL. Since DABCYL is a non-fluorescent quencher molecule, no photon emission is detected from the entire system [134-138]. However, when the distance between the two groups extends, the efficiency of FRET between them decreases, allowing the fluorescence of EDANS to be released and detected. This change in signal can be used as a means for quantitative analysis of enzyme activity or detection of specific molecular interactions [139,140].
In 2020, Gong et al. employed the principles of bioorthogonal copper-free strain-promoted azide-alkyne cycloaddition (SPAAC) reaction to react a disulfide-containing bicyclononyne with an EDANS derivative, followed by a reaction with DABCYL, successfully synthesizing a FRET probe 63 with "turn-on" fluorescence (Fig. 19). This probe was designed to specifically and quantitatively detect the activity of BChE in serum in the presence of GSH [141]. Probe 63 could make a response to thiols rapidly, and EDANS fluorescence was induced by molecules containing sulfhydryl groups except GSH. Similarly, thiocholine could react with probe 63, which was resistant to the interference of GSH at concentrations up to 50 µmol/L or even 50 mmol/L. It was also applied to determine BChE activity in healthy human plasma samples with physiological GSH concentrations, yielding an average activity of 7460 ± 597 U/L, which fell within the normal range of BChE.
The progression of diseases is generally accompanied by abnormal fluctuations in biomolecular levels, and these biomolecules are likely to serve as essential biomarkers for distinguishing pathological states from normal conditions [142-145]. Conventional probes are active by only one biomarker or an analyte, but the complex biological pathways and pathological factors shared by multiple diseases make it difficult to accurately determine the specific type of disease [66,146,147]. For that reason, dual-responsive probes (also known as AND-logic probes) capable of recognizing and detecting two or more substances have attracted significant attention [148-152]. Dual-responsive probes are only activated in systems where both biomarkers coexist, and this feature gives them high specificity and sensitivity, greatly reducing the possibility of false results [153-155].
To eliminate the interference from the non-specific binding of various components in human serum samples, particularly the high content of human serum albumin (HSA), Han's team in 2019 innovatively integrated HSA into a fluorescent sensing system, and successfully developed the fluorescent probe 66 with a high affinity for HSA by utilizing choline to block the carboxyl group of the fluorescent moiety dansyl-l-sarcosine (DS) (Fig. 20) [156]. The carboxyl group of DS could form stable hydrogen bonds with two residues in HSA, Lys414A and Ser489A, leading to a dramatic increase in fluorescence intensity. Caging of the fluorophore weakened the binding affinity and fluorescence intensity which was reversed under the enzymatic catalysis of BChE. The probe 66 remained inactive when only HSA or BChE was present, but released fluorescence when HSA and BChE coexisted. Probe 66 could also effectively quantify BChE activity in human serum, and the detection result (5.723 ± 0.0014 U/mL) was basically consistent with the traditional Ellman method (5.720 ± 0.0085 U/mL).
It is reported that oxidative stress in the brain is a major contributor to the aging of the central nervous system and the progression of multiple diseases, including AD. This pathological manifestation significantly drives the progression of AD by exacerbating Aβ deposition, excessive phosphorylation of Tau protein, and the loss of synapses and neurons [157]. During this process, a large amount of reactive oxygen species (ROS) was generated, which can damage cell structures and impair cell functions, ultimately leading to the injury and death of nerve cells, causing persistent damage to the brains of AD patients [158,159]. In order to develop more accurate and efficient diagnostic tools for AD, Ding et al. innovatively designed the AND-logic fluorescent probe 68 based on the dual upregulated biomarkers, BChE and ROS (Fig. 21) [160]. The dual-responsiveness of probe 68 originated from its structure, which contained both a BChE-specific cyclopropyl moiety and a ROS-sensitive phenolic hydroxyl group. Under the synergistic effect of BChE and ROS, probe 68 experienced ester hydrolysis, redox reaction, and spontaneous decarboxylation, releasing the NIR fluorescent dye methylene blue. Compared to HEK293 cells treated only with probe 68, cells pretreated with lipopolysaccharide and PMA to stimulate ROS generation exhibited stronger fluorescent signals, while cells incubated with the antioxidant N-acetylcysteine or tacrine quenched fluorescence. The probe possessed good tissue penetration ability and biocompatibility, and had excellent fluorescence imaging performance on the brains of APP/PS1 mice. Since the possibility of these two biomarkers rising simultaneously in other diseases is minimal, this fluorescence restoration strategy provides high accuracy and reliability for diagnosing AD.
Moreover, hypochlorous acid (HClO) is also an essential member of the ROS family, which is produced by the oxidation reaction mediated by myeloperoxidase (MPO) [161,162]. HClO plays a crucial role in the human immune defense system, including eliminating external pathogens, regulating cell apoptosis, and participating in cell signaling transmission [163,164]. However, excessively high concentrations of HClO causes oxidative damage to biological macromolecules like nucleic acids, proteins, and lipids, leading to cellular oxidative stress and tissue damage, which may further induce neurodegenerative diseases, cardiovascular diseases, ischemic stroke, and other conditions [164-166]. Studies have shown that MPO, which is closely related to the production of HClO, is highly expressed in Aβ plaques in the brains of AD patients but not in normal aging brain tissue [167]. In 2019, Govindaraju et al. used a fluorescence probe concerning HClO to confirm the pathological increase of HClO in the brains of AD mice and found that the aggregation of Aβ could induce the production of HClO. It elucidated the correlation between HClO levels and amyloid plaques in the brains of AD patients, further indicating that HClO can also be considered a reliable biomarker for AD diagnosis [168].
Therefore, taking the high levels of HClO and BChE in the AD brain as the starting point, Li et al. developed a dual-responsive probe 72 based on BChE and HClO in 2023, utilizing the two-photon fluorescence technology with deeper penetration depth (Fig. 22) [169]. By using the coumarin lactone derivative as the fluorophore, the HClO recognition site was obtained by replacing the oxygen atom of the carbonyl group with a sulfur atom, and the cyclopropyl fragment was introduced on the hydroxyl group to construct the BChE recognition group. Only when both factors were present did probe 72 release 4-methylumbelliferone (74) with strong fluorescence properties. The probe was capable of detecting endogenous and exogenous HClO in PC12 cells, and its fluorescence signal was blocked by the MPO inhibitor 4-aminobenzoic acid hydrazide. Probe 72 could distinguish between normal cells and AD model cells induced by Aβ1–42 or glutamate, and successfully crossed the blood-brain barrier to release two-photon fluorescence in the brains of AD mice. Notably, the green fluorescence generated by this probe partially overlapped with the red fluorescence yield by the Aβ protein probe CRANAD-2 in AD mouse brain slices, further confirming previous reports about the co-localization of BChE and Aβ deposition in the pathological process of AD [170,171]. However, due to limitations in optical properties and instrumentation, it failed to achieve non-invasive imaging in tracking brain biomarkers [169].
According to the order of probes appearing in the article, Table S1 (Supporting information) summarizes and gives important information on BChE-specific probes disclosed in recent years. On the basis of the information presented in Table S1, BChE probes exhibit an extensive range of applications, encompassing scientific research, disease diagnosis, and environmental monitoring. Notably, due to their reliance on exogenous substances for fluorescence recovery, probes indirectly activated by BChE are confined to quantitative analysis of BChE activity in plasma samples in an in vitro setting, precluding direct application to cells or living animals.
Of particular significance is probe 38, demonstrating the lowest LOD through fluorescence methods among probes using enzyme activity as the unit. This feature underscores its exceptional sensitivity and analytical performance, which is conducive to enhancing diagnostic sensitivity in liver injury and diabetes. Comparing the LODs of probes 9 and 10, it is found that the introduction of BChE inhibitors reduces the detection sensitivity for BChE. This phenomenon suggests that large molecules may disturb the access of cyclopropyl to the BChE active site to some extent, thereby affecting the detection efficacy. Moreover, the LODs of two structurally similar probes, 19 and 20, reveal a nearly 50-fold difference. Based on the docking mode of probe 20 with BChE provided in the primary literature, it is speculated that the chlorine atom in probe 19 may form specific interactions with amino acids within the active site of BChE. These interactions may be conducive to strengthening the affinity between the probe and BChE, giving probe 19 a more sensitive detection capability.
Specific BChE probes play a pivotal role in biochemical research, providing powerful tools for monitoring and evaluating the activity of BChE. Among them, fluorescent and chemiluminescent probes play an essential role in the detection of BChE. As visual tool molecules, they greatly assist researchers in gaining deeper insights into the physiological and pathological functions of BChE. Features in the visualization of these probes greatly facilitate the high-throughput screening of BChE inhibitors and aid in the early assessment and diagnosis of BChE-related diseases such as AD, NAFLD, diabetes, and thyroid cancer. This article provides a review of the probes with high specificity for BChE, aiming to offer detailed and comprehensive information for a deeper understanding and exploration of research progress in this field.
However, in the retrieved literature, most BChE probes utilize cyclopropyl as the recognition moiety, indicating the singularity of activation principles and limitations in structural design. Employing the bioinspired design strategy to develop novel probes may be an innovative and promising approach. This method can draw inspiration from the unique recognition and catalytic mechanisms between BChE and its substrates within biological systems, thus providing ideas for designing recognition units with diverse backbones and structures. Therefore, exploring recognition units with different backbones through biomimetic techniques to enrich the structural diversity of probes and enhance their recognition efficiency and specificity towards target molecules could be one of the significant research directions in this field. Furthermore, the excitation wavelength and emission wavelength of reported BChE probes are mainly in the visible region (400–760 nm) and the NIR-Ⅰ region (760–1000 nm), which have generally poor skull penetration. The development of NIR-Ⅱ (1000–1700 nm) fluorescence probes with centimeter-level penetration, lower background fluorescence, and higher sensitivity is expected to achieve deep imaging of central nervous system diseases.
What is more, the recent research by Sun et al. has brought significant breakthroughs in the diagnosis and treatment of AD. Researchers reported the first probe integrated diagnostic and therapeutic effects for AD, which not only effectively imaged BChE in cells and animals but also significantly improved cognitive dysfunction in APP/PS1 transgenic mice. This innovative investigation highlights the importance and feasibility of developing multifunctional BChE probes with simultaneous detection, imaging, and therapeutic capabilities, offering new perspectives and directions for the practical application and clinical translation of probes. At the same time, considering the widespread distribution of BChE in serum, probes activated solely depending on the catalytic capacity of BChE may also generate fluorescence signals in non-disease sites, causing signal weakening in the target region or false positive diagnosis. To address this issue, it is important to develop dual or multiple controlled release strategies based on two or more triggering factors of pathological conditions. This strategy can improve the specificity and accuracy of probes by detecting multiple biomarkers simultaneously, leading to more reliable diagnostic results. In addition, by covalently linking two or more fluorophores with distinct functions, multi-targeted probes can also be designed. These approaches enable the simultaneous detection of multiple biomarkers and reveal the interactions and complex relationships, offering novel perspectives and insights into the pathogenesis of diseases.
It is also noteworthy that current exploration into the in vivo safety and metabolic stability of specific-BChE probes remains limited, underscoring the urgency and significance in this respect. This comprehensive assessment of probe performance is crucial for ensuring the safe and effective application in clinical or practical settings. Also, enhancing probe stability in complex biological environments and durability during long-term storage and transportation through chemical modifications, packaging improvements and other methods, will further broaden their application scope. In the field of drug development, utilizing BChE probes to compare the activity or distribution differences of BChE between normal and disease states can further validate the rationality and efficacy of targeting BChE. Additionally, the unique targeting capability of BChE probes renders them promising candidates as part of targeted drug delivery systems, enabling direct transport of drugs to the lesion, thereby enhancing therapeutic efficacy and reducing systemic side effects. The introduction of probes also visualizes the drug delivery process in vivo, with this real-time tracking capability providing vital information for optimizing drug delivery strategies. Given the abundant distribution of BChE in plasma, developing disease diagnostic kits targeting plasma holds broad application prospects. As a routinely tested clinical sample, plasma is easy to obtain and process, which may facilitate the widespread clinical adoption and promotion of such diagnostic kits. These kits may have potential applications in diagnosing and monitoring various diseases, including neurodegenerative disorders, malignant tumor, and liver diseases, providing crucial diagnostic and therapeutic references for physicians.
In terms of food safety, future attempts can be made to develop BChE probes for specific classes of pesticides. By optimizing probe structures and enhancing the combination ability of probes and pesticides, the accuracy and sensitivity of detection can be improved, which is conducive to the rapid screening of pesticide residues in vegetables and soil, ensuring food safety. In conclusion, specific BChE probes play a vital role in biomedical research, environmental monitoring and food safety detection, deserving further research and attention to meet the demands of various fields.
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Tianyu Sun: Writing – original draft, Conceptualization. Zhoujun Dong: Writing – review & editing. Paul Michael Malugulu: Writing – review & editing. Tengfei Zhen: Writing – review & editing. Lei Wang: Writing – review & editing. Yao Chen: Supervision. Haopeng Sun: Writing – review & editing, Supervision, Funding acquisition.
We gratefully acknowledge the financial support from the National Natural Science Foundation of China (Nos. 82173652 and 81872728) and the Natural Science Foundation of Jiangsu Province (No. BK20221522). Support from Jiangsu "333 High Level Talents Cultivation" Leading Talents (No. 2022–3–16–203) and the Qing Lan Project is also appreciated.
Supplementary material associated with this article can be found, in the online version, at doi:
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Figure 1 Schematic diagram of ACh generation and degradation at the synaptic junction, and comparison of active pockets between hAChE (PDB ID: 4EY4) and hBChE (PDB ID: 6QAA). Proteins appear light brown and six residues related to the size of the active gorge are shown in wheat. Created with BioRender.com.
Figure 3 Conventional and emerging BChE detection methods along with their advantages and disadvantages.
Figure 5 The mechanism of fluorescence signal generation in a probe induced by BChE-mediated cleavage of an ester bond. His438 and Ser198 are key residues in the catalytic triad of BChE.
Figure 9 (A) The ICT effect and fluorescence signal recovery mechanism of DCI-OH derivatives. (B) Structures of probes 19-25.
Figure 10 (A) Structures of probes 26 and 28, and the corresponding fluorescence generation mechanism. (B) Structures of probes 30 and 33, and the corresponding fluorescence generation mechanism.
Figure 11 (A) Structure of the dual-mode probe 36 and its fluorescence generation mechanism. The red characters are the LOD for BChE measured by fluorescent and electrochemical methods. (B) Structure and fluorescence generation mechanism of dual-mode probe 38. The red characters are the LOD for BChE measured by the fluorescent and colorimetric methods.
Figure 12 Structures of probes 40 and 43 with the corresponding fluorescence generation mechanism.
Figure 17 Schematic diagram of fluorescence recovery mechanism of redox-regulated fluorescence nanoswitch 59.
Figure 21 Structure of dual-responsive probe 68 and its fluorescence generation mechanism.
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