Tacrine

A fluorescent probe for butyrylcholinesterase activity in human serum based on a fluorophore

Soyeon Yoo 1, Min Su Han 1

Summary

Non-specific binding of a fluorescent probe to human serum albumin is problematic because it induces signal interference when the probe detects the target biomarker in human serum. To eliminate this problem, we used intrinsically problematic non-specific fluorescence in designing a fluorescent probe for butyrylcholinesterase activity in serum. The probe containing a fluorophore with specific binding affinity for albumin could sensitively detect butyrylcholinesterase activity in serum with high selectivity to acetylcholinesterase and screen the efficiency of butyrylcholinesterase inhibitors.
The detection of a biomarker in human serum is crucial for diagnosing a disease associated with an abnormal concentration of the biomarker.1 Fluorescence-based sensors have been widely studied for the identification of various biomarkers in buffer systems owing to numerous merits, such as simplicity, non-destructiveness, high sensitivity, and the possibility of realtime analysis.2 Nevertheless, it is difficult to apply most fluorescent probes to a real human serum sample because of the interference between such a probe and the excess serum components, such as human serum albumin (HSA), which is the most abundant protein in human serum.3
The non-specific binding of HSA to a probe causes numerous issues, including non-specific changes in the fluorescence of the probe.3 These affect the relationship between a specific fluorescence signal and the concentration of the target biomarker, which prevents quantitative detection of the latter. Hence, the concentration of a biomarker may be overestimated or underestimated, resulting in inaccurate diagnosis of the associated disease. Thus, for quantitative detection of a biomarker in serum, the interference caused by HSA should be eliminated. Recently, to address the non-specific fluorescence caused by HSA, the Tan group reported an excellent sensing system using a biotinylated fluorophore and avidin. However, it still required an additive process, such as biotinylation of the fluorophore and addition of avidin to the assay solution.3a
In this study, we design a new sensing strategy with a turnon-type fluorescent probe for the sensitive detection of a biomarker in human serum based on a fluorophore with high binding affinity for HSA. This fluorophore, whose fluorescence is dramatically enhanced after strongly binding to HSA, allows HSA to be directly used as a part of the sensing system for detecting the target biomarker in the serum, instead of acting as an interfering agent affecting the fluorescence signal. Caging of the fluorophore can reduce the binding affinity of the fluorophore for HSA, reducing the fluorescence. Subsequently, the caged-fluorophore is irreversibly de-caged by a selective reaction with the target biomarker, leaving it bare to bind strongly to HSA, thereby increasing the fluorescence intensity. Using the non-specific fluorescence induced by HSA, which was previously problematic, our sensing strategy allows a quantitative detection of the biomarker in serum containing excess HSA without any pre-treatment and modification processes.
With our sensing strategy using HSA and a caged-fluorophore, an assay of butyrylcholinesterase (BChE, EC 3.1.1.8) is tested. BChE catalyzes the hydrolysis of various choline-based esters, such as neurotransmitters acetylcholine and butyrylcholine, and it is mainly present in blood serum.4 BChE is emerging as a significant biomarker for various diseases, such as Alzheimer’s disease, diabetes, and organophosphate poisoning.5 In particular, the activity of BChE in the serum of a patient suffering from Alzheimer’s disease is observed to be higher than the normal level, and the development of a selective BChE inhibitor has remarkably advanced for the treatment of Alzheimer’s disease.5b,6 Therefore, it is necessary to develop an assay method that enables the identification of novel inhibitor candidates as well as a quantitative detection of the BChE activity in serum.
Dansyl-L-sarcosine-choline (DSC) was designed as the cagedfluorophore for detecting the BChE activity in the assay. Dansyl-Lsarcosine (DS) is a well-known fluorophore whose fluorescence intensity dramatically increases when bound to HSA.7 The carboxylic acid group of DS binds strongly to binding site II of HSA via hydrogen bonding with two residues: Lys414A and Ser489A in HSA.7b Thus, caging the carboxylic acid group of DS with choline transforms bare DS into a caged-fluorophore form, which results in a low binding affinity for HSA and low fluorescence intensity. In the presence of BChE, DSC is de-caged by a cleavage reaction and returns to the bare DS form, which then exhibits an increase in fluorescence by binding to HSA (Scheme 1).
The mechanism was investigated via fluorescence spectroscopy. DSC does not significantly change its fluorescence in the presence of HSA or BChE alone, whereas the fluorescence of DSC incubated with BChE tremendously increases when HSA is present (Fig. 1). This suggests that the DSC is de-caged by the activity of BChE to become bare, allowing it to bind to HSA. The above was also demonstrated via high-resolution mass spectroscopy (HR-MS) and liquid chromatography MS (LC-MS). The retention time and mass value of the product that was formed by the DSC incubated with BChE completely corresponded to those of DS (Fig. S1 and S2, ESI†). In addition, this assay system has been shown to work with various serum albumins (bovine, rat, and rabbit) as well as HSA, indicating that DSC can be used to detect BChE activity in serum from other animal models (Fig. S3, ESI†).
The relation between the fluorescence intensity of DSC in the presence of BChE and the concentration of HSA was evaluated to prove that the HSA was directly used as a part of the sensing system for detecting the target biomarker in the serum, instead of being an interfering agent affecting the fluorescence signal. The fluorescence intensity of DSC in the presence of BChE increases with increasing HSA concentration and is saturated at 20 mM (Fig. 2). By contrast, in the absence of BChE, the HSA concentration has little effect on the fluorescence change of DSC. In addition, the assay system also worked well in the entire pH range in which BChE works (pH 6.0–8.0) (Fig. S4, ESI†). These results suggest that the DSC-based assay system could appropriately detect BChE activity in serum because it was effective when the HSA concentration in the assay solution exceeds 20 mM and the assay solution pH is 6.0–8.0. Hence, experiments were conducted under the following conditions: pH 7.0 buffer solution (phosphate, 20 mM) containing 20 mM HSA and 10 mM DSC.
Using the assay system under the above conditions, the BChE activity was monitored by measuring the change in the fluorescence intensity in real time. The rate of change of the fluorescence intensity gradually increases as the activity of BChE increases from 0 to 2 U mL1 (Fig. 3a). In particular, when the concentration of BChE is 2 U mL1, at 485 nm, the fluorescence intensity is saturated immediately within 2 min. The plot of kobs, which is the rate constant of the enzymatic reaction, shows a high linearity in the range of 0–2 U mL1 for BChE activity with R2 = 0.99746. Moreover, the detection limit of the BChE activity, 0.0012 U mL1, is low (Fig. 3b). Numerous reported fluorescent probes for BChE activity are prepared by indirect methods, combining a thiocholine derivative as the substrate for BChE and a fluorescent probe for the thiol group, which results in a low detection rate because of the two-step reaction (BChE enzymatic reaction and probethiocholine reaction). In particular, the assay methods may be affected by several factors including oxidants or thiol-containing molecules in the serum.8 A few fluorescent probes have been developed to detect the activity of BChE directly, which change the fluorescence signal when they are cleaved by BChE. However, these probes have some drawbacks: a slow response or turn-off signal.9 In this aspect, because our probe was rapidly de-caged by the BChE activity and the de-caged probe immediately exhibited fluorescence with HSA and the system was not significantly affected by biothiols and oxidant, our probe addressed the problems faced with previous methods (Fig. S6, ESI†).
To verify the selectivity of the assay system for BChE, excess AChE (20 U mL1), whose enzymatic activity is similar to that of BChE, was added to the assay solution containing DSC and HSA. Despite excess AChE, there is no significant increase in the fluorescence intensity of the assay solution, indicating that AChE does not hydrolyze DSC to DS (Fig. 4a and Fig. S7a, ESI†). In addition, the fluorescence spectra of the assay solutions in the presence of BChE were the same regardless of the presence or absence of AChE. Our method shows that AChE does not interfere with the assay system for the detection of the BChE activity. In addition, the presence of other various enzymes in the serum does not induce a fluorescence increase at 485 nm: glucose oxidase (GOx), lysosome, phospholipase C (PLC), thrombin, trypsin, lipase, and alkaline phosphatase (ALP) (Fig. 4b and Fig. S7b, ESI†). Thus, DSC is proven to be a highly selective probe for BChE, suggesting that this system can be applied to a real serum sample assay for detecting BChE activity.
An actual human serum sample, purchased from Sigma Aldrich, was used to evaluate the practical applicability of this assay system for detecting BChE activity. Considering that the actual serum contains 526–827 mM (35–55 g L1) of HSA, it is not necessary to add extra HSA to the assay solution because our assay system works well with HSA of concentration 20 mM or higher. Hence, the serum (10 mL) was directly added to the buffer solution (190 mL) containing only DSC without any further treatment, and the change in the fluorescence intensity at 485 nm was measured with time. The positions of the measured values in the linear calibration plot of kobs for the BChE activity yield a BChE activity of 5.723 0.0014 U mL1 in the serum (Table 1). This is extremely similar to the result (5.720 0.0085 U mL1) obtained by the well-known Ellman method (Table 1 and Fig. S8, ESI†). This outcome verifies that the BChE activity in human serum can be detected quantitatively and simply using only DSC in the buffer solution without any treatment, suggesting that this assay system could be a simple tool for diagnosing Alzheimer’s disease.
It is also necessary to develop an inhibitor of BChE for the treatment of Alzheimer’s disease. Accordingly, high-throughput screening methods are needed to rapidly and easily screen large amounts of inhibitor candidates. To verify the applicability of our assay system for screening inhibitors, IC50 values were obtained for two well-known BChE inhibitors, tacrine and galantamine (18 nM and 8.5 mM, respectively), using our assay system containing DSC, HSA, and BChE (Fig. 5a and b), similar to those obtained in other reported studies.9a,10 Inhibition efficiencies of selective BChE inhibitor (chlorpromazine), selective AChE inhibitor (huperzine A), and carbamate-based AChE/ BChE inhibitor (rivastigmine) were also confirmed, and the IC50 values of these inhibitors were determined to be 5.0 mM, 194.4 mM, and 1.4 mM, respectively (Fig. S9, ESI†).11 In addition, the fluorescence of the assay solution containing the inhibitors on the microwell plate was photographed with a UV hand lamp (365 nm) for 5 min. Indeed, the assay solution containing the inhibitors, exhibiting a high inhibition efficiency, was observed to emit low intensity fluorescence with the naked eye (Fig. 5c). Thus, our assay system is suitable for high-throughput screening of new inhibitors of BChE.
In conclusion, we devised a fluorescent probe for the detection of a biomarker in human serum using the specific binding affinity of a fluorophore for HSA. The sensing system utilized HSA, which acts as an interfering agent for other reported sensors, as a part. Accordingly, it eliminated the fluorescence changes Tacrine induced by the non-specific binding of a fluorophore to various proteins in serum and allowed quantitative detection of BChE activity in the same. The assay system for BChE activity showed excellent linearity over a wide range, low detection limit, and high selectivity over other enzymes containing AChE as well as the possibility of high-throughput screening for BChE inhibitors. In particular, the detection of BChE activity in human serum could be used as a diagnosis method for Alzheimer’s disease with only one drop of the serum and fluorescent probe. The strategy utilizing the specific binding affinity of the fluorescent probe for HSA is extremely simple and can be applicable to effectively develop detectors for various biomarkers such as enzymes and small molecules in human serum.

Notes and references

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