Analytical Biochemistry 465 (2014) 172–178
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Sensitive detection of acetylcholine based on a novel boronate intramolecular charge transfer fluorescence probe Chang Liu, Youming Shen, Peng Yin, Lidong Li, Meiling Liu, Youyu Zhang, Haitao Li ⇑, Shouzhuo Yao Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, People’s Republic of China
a r t i c l e
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Article history: Received 25 April 2014 Received in revised form 25 July 2014 Accepted 1 August 2014 Available online 14 August 2014 Keywords: Boronate probe 1,8-Naphthalimide Acetylcholinesterase Choline oxidase Acetylcholine Fluorescence detection
a b s t r a c t A highly sensitive and selective fluorescence method for the detection of acetylcholine (ACh) based on enzyme-generated hydrogen peroxide (H2O2) and a new boronate intramolecular charge transfer (ICT) fluorescence probe, 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-N-butyl-1,8-naphthalimide (BN), was developed. This strategy involves the reaction of ACh with acetylcholinesterase (AChE) to produce choline, which is further oxidized by choline oxidase (ChOx) to obtain betaine and H2O2. The enzymegenerated H2O2 reacts with BN and results in hydrolytic deprotection of BN to generate fluorescent product (4-hydroxyl-N-butyl-1,8-naphthalimide, ON). Two consecutive linear response ranges allow determining ACh in a wide concentration range with a low detection limit of 2.7 nM (signal/noise = 3). Compared with other fluorescent probes based on the mechanism of nonspecific oxidation, this reported boronate probe has the advantage of no interference from other biologically relevant reactive oxygen species (ROS) on the detection of ACh. This study provides a new method for the detection of ACh with high selectivity and sensitivity. Ó 2014 Elsevier Inc. All rights reserved.
Acetylcholine (ACh)1 is one of the most important neurotransmitters contained in the central and peripheral nervous systems and is widely involved in controlling nerve conduction in the body [1]. Acetylcholine comes from choline in the presence of choline acetyltransferase and acetyl-coenzyme A and has a significant influence on human memory and sleep [1]. The metabolic abnormalities of ACh in the brain may cause neuropsychiatric disorders such as Huntington’s disease, Alzheimer’s disease, schizophrenia, and Parkinson’s disease [1,2]. Therefore, highly sensitive and selective detection of ACh is very necessary and of great importance. A number of analytical techniques have been reported for the detection of ACh. Direct analytical techniques include mass spectrometry (MS), gas chromatography (GC), high-performance liquid chromatography (HPLC), and liquid chromatography coupled with mass spectrometry (LC/MS) [3,4]. Although these techniques are sensitive and reliable, most of them require tedious sample pre-
⇑ Corresponding author. Fax: +86 731 8865515. E-mail address: [email protected] (H. Li). Abbreviations used: ACh, acetylcholine; AChE, acetylcholinesterase; ChOx, choline oxidase; H2O2, hydrogen peroxide; HRP, horseradish peroxidase; UV–vis, ultraviolet– visible; ROS, reactive oxygen species; Amplex Red, 10-acetyl-3,7-dihydroxyphenoxazine; ICT, intramolecular charge transfer; BN, 4-(4,4,5,5-tetramethyl-1,3, 2-dioxaborolan-2-yl)-N-butyl-1,8-naphthalimide; ON, 4-hydroxyl-N-butyl-1,8-naphthalimide; PBS, phosphate buffer solution; FTIR, Fourier transform infrared. 1
http://dx.doi.org/10.1016/j.ab.2014.08.003 0003-2697/Ó 2014 Elsevier Inc. All rights reserved.
treatment, complex separation processes, and skilled operators [1,3]. The enzyme-based method is an indirect detection technique of ACh with high sensitivity. Acetylcholinesterase (AChE) and choline oxidase (ChOx) could catalyze their substrates to produce electrochemically active product (hydrogen peroxide, H2O2) or spectral (absorbance and fluorescence) sensing products. Therefore, the enzyme-based detection method usually depends on electrochemical and optical techniques. However, electrochemical detection of ACh by measuring its electrochemical response (enzymegenerated H2O2) is not sensitive enough [5], and the complex pretreatment of electrode is needed. Alternatively, optical detection methods, such as colorimetric Ellman assays and fluorescence assays, are widely used due to the simple operations and easy pretreatment processes. Colorimetric assays always depend on the use of peroxidase- or oxidase-like enzymes, such as horseradish peroxidase (HRP) and various of nanoparticles (NPs), and also need various substrates, including 3,3,5,5-tetrame-thylbenzidine (TMB), o-phenylenediame (OPD), pyrogallol, and 2,20 -azino-bis(3ethylbenzo-thiazoline-6-sulfonic acid) diammonium salt (ABTS) [1]. The detection can be performed through the color change by the naked eye or ultraviolet–visible (UV–vis) absorption. Colorimetric assays are simple but not sensitive enough [6]. Nowadays, more and more attention is paid to fluorescence analysis for high sensitivity [7].
Detection of ACh based on boronate ITC fluorescence probe / C. Liu et al. / Anal. Biochem. 465 (2014) 172–178
Up to now, there have been few reports about the detection of ACh directly using fluorescence probe. Based on the principle of indirect detection, it should be useful for the detection of ACh through detecting enzyme-generated H2O2 by using H2O2-sensitive fluorescent probes. There are several fluorescent probes that have been developed for measuring H2O2, superoxide, and peroxynitrite in biological systems or detecting enzyme-generated H2O2 [8,9]. Unfortunately, the assays based on dichlorodihydrofluorescein diacetate (DCFH–DA), hydroethidine (HE), and Mito-SOX always have high fluorescence background produced by their reaction with reactive oxygen species (ROS) [7,9–11]. To the best of our knowledge, it is reported that only the 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red) ACh/AChE Assay Kit provides ultrasensitive measurement of ACh based on enzyme-generated H2O2 [10]. However, HRP is an indispensable factor in the catalyzing oxidation of Amplex Red in the presence of H2O2. In addition, the maximum absorption band and fluorescence emission peak of the oxidation product of Amplex Red are extraordinarily close (absorbance at 571 and emission at 585 nm) [9,10], which may cause spectral interferences. There are some fluorescence approaches for detection of ACh based on nanoparticles such as C-dots [2] and Au/Ag nanoparticles [1]. These methods are highly sensitive and selective; however, they always suffer from potential toxicity and chemical instability. Because of the limitations of currently available H2O2-responsive probes, many researchers are focusing their studies on boronate probes for their reaction with H2O2 [7,9,12]. So far, boronate probes, fluorescein peroxyfluor 1 (PF1) probes, and peroxyxanthone 1 (PX1) probes have been synthesized and used to detect H2O2. The interaction of H2O2 and boronate probes is ambiphilic and nucleophilic reactivity which is beneficial to hydrolytic deprotection of the boronates. In comparison with other probes taking the mechanism of nonspecific oxidation, specific interaction of H2O2 with boronates will achieve higher selectivity than other biologically relevant ROS probes [7,13]. Based on the advantages of boronate probes, a novel intramolecular charge transfer (ICT) boronate probe, 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-N-butyl-1,8-naphthalimide (BN) is designed and synthesized for the selective detection of ACh with significant spectral shift (455/555 nm). BN contained 1,8-naphthalimide as the fluorophore and boronic ester as the recognition unit. Because of the structure of BN, it has a high quantum yield at 76.24% (stated in Section S1 of the online supplementary material). The novel boronate probe is highly sensitive and selective toward enzyme-generated H2O2 that is produced from the enzyme catalytic reaction of ACh. When H2O2 coexists with BN under appropriate conditions, BN is transferred to a highly fluorescent product, 4-hydroxyl-N-butyl-1,8-naphthalimide (ON) by the hydrolytic deprotection of the boronates. The novel detection strategy is based on the fluorescence change depending on
O
AC h
E
N
choline
O B O
O
ACh H2O 2
Ox Ch
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enzyme-generated H2O2, as shown in Scheme 1. This method is sensitive and does not require a complex pretreatment process or enzyme immobilization. Materials and methods Materials and apparatus Acetylcholine, amino acetic acid, alanine, dopamine, glutamic acid, proline, tryptophane, arginine, and aspartate were purchased from Wako Pure Chemical Industries (Osaka, Japan). Acetonitrile was obtained from Sinopharm Chemical Reagent. Acetylcholinesterase and choline oxidase were obtained from Sigma–Aldrich. All other chemicals used here were of analytical grade. The detection buffer was phosphate buffer solution (PBS, pH 7.5). Milli-Q ultrapure water (P18 MX cm, Millipore) was used throughout. Unless otherwise noted, solvents were purified by distillation. Human serum samples were provided by the Hospital of Hunan Normal University (Changsha, China). A Bruker AVB-500 spectrometer and an API 4000 QTRAP LC/MS/ MS System with ESI Ion Source (AB Sciex, USA) were used to characterize BN. Fourier transform infrared (FTIR) spectra were collected on a Nicolet Nexus 670 FTIR instrument (Nicolet Instrument, USA). UV–vis and fluorescence spectra were recorded on a UV-2450 spectrophotometer (Shimazu, Japan) and an F4500 fluorescence spectrophotometer (Hitachi, Japan), respectively. Synthesis of 4-bromo-N-butyl-1,8-naphthalimide The synthesis of 4-bromo-N-butyl-1,8-naphthalimide was similar to that reported by Zhengneng and coworkers [14]. 4-Bromo1,8-naphthalic anhydride (486.4 mg, 2 mmol) and butylamine (204.8 mg, 2.8 mmol) were dissolved in 50 ml of ethanol, and the resulting solution was stirred at 80 °C for 12 h. After being cooled to room temperature, the precipitated solid was filtered and recrystallized in ethanol to give a light yellow solid (386 mg, 65%) [15]. Synthesis of 4-boronate-N-butyl-1,8-naphthalimide A nitrogen-flushed three-neck round-bottom flask was charged with 4-bromo-N-butyl-1,8-naphthalimide (99.7 mg, 0.3 mM), bis(pinacolato)diboron (91.4 mg, 0.36 mM), sodium acetate (73.8 mg, 0.9 mM), bis(triphenylphosphine)palladium(II) chloride (11.0 mg, 0.015 mM), CuI (6.0 mg, 0.03 mM), and triphenylphosphine (19.0 mg, 0.075 mM). 1,2-Dimethoxyethane (210 ml) was then added, and the mixture was bubbled with nitrogen for 15 min. After stirring at 60 °C for 24 h, the reaction mixture was cooled to room temperature and then poured into icewater (200 ml). It was then extracted with methylene chloride (20 ml), and the combined organic layer was dried over anhydrous magnesium sulfate. Then, the organic solvent was removed by rotary evaporation, and the residue was passed through a flash silica gel column with mineral ether as the eluent to give white crystals (73.51 mg, yield 65%) [16].
O N O
Fluorescent detection of ACh OH
Scheme 1. Schematic representation of the sensing strategy using 4-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)-N-butyl-1,8-naphthalimide to detect ACh.
The experiments were repeated three times to ensure the accuracy of the measurements. The fluorescent measurements for the detection of ACh were carried out as follows [12]. First, 150 ll of PBS containing 3 U/ml AChE, 1 U/ml ChOx, 0.01 to 200 lM ACh, and 36.5 lM BN was incubated under gentle shaking at 37 °C for 30 min [1]. Subsequently, fluorescence spectra were recorded at
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the excitation wavelength of 455 nm. The relative fluorescence intensity at 555 nm was used for the quantitative analysis of ACh. Determination of ACh in serum samples Serum samples (15 ll) were diluted with 135 ll of PBS. Prior to the test, 135 ll of PBS containing 3 U/ml AChE, 1 U/ml ChOx, 0 to 30 lM ACh, and 36.5 lM BN was equilibrated under gentle shaking at 37 °C for 1 h. Then, fluorescence spectra were recorded. Results and discussion Spectral properties of the probe 1
The structure of BN was confirmed by H nuclear magnetic resonance (NMR), 13C NMR, and FTIR spectroscopy (see Figs. S1–S3 in supplementary material). 1H NMR (500 MHz, CdCl3): d 9.12 (d, 1H), 8.61–8.56 (m, 2H), 8.30 (d, 1H), 7.79 (t, 1H), 4.20 (t, 2H), 1.74–1.71 (m, 2H), 1.58 (m, 2H), 1.45 (s, 12H), 0.99 (t, 3H); 13C NMR (125 MHz, CdCl3): 136.4, 135.9, 135.5, 131.4, 130.3, 127.7, 85.2, 77.9, 77.6, 77.4, 40.9, 30.8, 25.6, 21.0, 14.4 ppm. FTIR spectrum was used to study the functional groups of BN (Fig. S3). The broad peak centered at 2956 cm1 is assigned to the stretching vibration of the C@C stretching mode of aromatic hydrocarbons, whereas the peak at 1355 cm1 is attributed to the bending vibration of the CAH bond [17]. The stretching vibration of OAH (3451 cm1) may be attributed to the residual water. The peak at 1660 cm1 correspond to the CAO stretching vibration. The band around 1157 cm1 is the CAN stretching mode [18]. The absorption, excitation, and emission spectra of the probe in PBS are presented in Fig. 1. The probe exhibited a well-defined absorption peak at 344 nm, which was considered as a p–p transition of the naphthalimide backbone [19,20]. In addition, the peak appearing at 345 nm in the excitation spectrum of BN was consistent with the UV–vis absorption spectrum. As shown in Fig. 1, a narrow emission spectrum ranging from 400 to 500 nm was observed under excitation at 345 nm, and its maximum emission is centered at 440 nm, suggesting that BN has strong fluorescent emission intensity. As shown in Fig. S4 of the supplementary material, the maximum emission wavelength (455 nm) was not changed with different excitation wavelengths, which indicates the spectral purity of the probe.
Fig.2. Normalized fluorescence spectra of probe on interaction with 3.0 U/ml AChE and 1.0 U/ml ChOx in the presence (1) and absence (2) of 1.0 mM ACh in PBS.
ing the change in the relative fluorescence intensity of the system, the concentration of ACh can be measured. To confirm the interaction of the probe with enzyme-generated H2O2, we incubated the probe with PBS containing AChE and ChOx in the absence and presence of ACh. As shown in Fig. 2, there was no obvious change of the normalized fluorescence intensity in the absence of ACh. However, the fluorescence intensity was obviously increased at 555 nm in the presence of ACh. Therefore, AChE converts ACh to choline, which is further oxidized by ChOx to produce betaine, and H2O2 should be responsible for the change in the fluorescence intensity. The enzyme-generated H2O2 causes the hydrolytic deprotection of the boronates and elicits a significant fluorescence increase [21]. To further confirm the detection mechanism, the reaction of the probe with H2O2 was also investigated; the results are shown in Fig. 3. An absorption peak at around 345 nm was found in the spectrum of BN. However, in the presence of H2O2, an absorption peak at around 455 nm occurred, suggesting that a new compound (ON) was produced. As shown in Fig. 3B, the probe displayed a large turn-on fluorescence response. On excitation at 455 nm, the probe of BN had low fluorescence (Fig. 3B). However, the product (ON) had strong emission at 555 nm, and the normalized fluorescence intensity was increased with the increase of H2O2 concentrations [21]. In addition, experimental facts revealed that the fluorescence intensity of BN in alkaline medium was preponderant to acidic medium (Fig. S4), indicating that alkaline medium was beneficial to the hydrolytic deprotection of the boronates [2].
Mechanism for detection of ACh Effect of probe on enzyme activity The general principle for detection of ACh based on BN is shown in Scheme 1. First, AChE hydrolyzes ACh to choline. Then, choline is oxidized by ChOx to produce betaine and H2O2. The resulting H2O2 reacts with BN to produce the fluorescent product of ON. By detect-
As a probe used in the enzyme reaction system, it is important to evaluate the influence of the probe on the enzyme activity. The method supported by Hestrin [22] has been selected to monitor the
Fig.1. (A) UV–vis absorption, excitation spectrum, and emission spectrum of the probe 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-N-butyl-1,8-naphthalimide (BN). (B) Excitation spectrum and emission spectrum of the resultant 4-hydroxyl-N-butyl-1,8-naphthalimide (ON).
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Fig.3. UV–vis absorption spectra (A) and normalized fluorescence spectra (B) of 20.0 lM probe in 1 ml of acetonitrile–PBS (1:1, v/v) solution with different concentrations of H2O2 (0.2, 0.4, and 1.0 mM). kex = 455 nm.
Fig.4. Plots of the absorbance at 520 nm of the resulting solution of ACh interacting with alkaline hydroxylamine and ferric chloride versus the concentration of ACh in the presence (A) and absence (B) of 36.5 lM probe in PBS. The inset shows the color change with increasing concentrations of ACh in PBS containing 3.0 U/ml AChE and 1.0 U/ml ChOx.
enzyme activity change in the presence or absence of BN. Acetylcholine reacting with hydroxylamine hydrochloride would produce acetyl hydroxamic acid, which could further interact with ferric chloride to produce ferric–cethydroxamic acid complex. The yield of acetyl hydroxamic acid is a direct function of ACh concentration, which indicates that the absorbance is proportional to the concentration of ACh. The absorbance spectra and the color change of different concentrations of ACh on its interaction with AChE, ChOx, and alkaline hydroxylamine are shown in Fig. 4. In the wavelength ranging from 520 to 540 nm, little or no absorption is shown by ferric chloride, whereas absorption-related to ferric– cethydroxamic acid complex is nearly the maximum. Therefore, the absorbance at 520 nm that has lower background was selected to monitor the enzyme activity change. Obviously, there are no significant differences between the cases of presence (Fig. 4A) and absence (Fig. 4B) of BN, and it indicates that the BN does not affect the enzyme activity. Optimization conditions for detection To obtain a highly sensitive response, it is necessary to optimize the conditions. To generate more H2O2, it is of paramount importance to optimize the concentrations of ChOx and AChE [23]. Fig. 5A shows the relative fluorescence intensity of BN on interaction with 1.00 mM ACh and different ratios of ChOx/AChE. It shows that the maximum response can be obtained when the concentrations of ChOx and AChE are 1.0 and 3.0 U/ml, respectively. Therefore, 1.0 U/ml ChOx and 3.0 U/ml AChE were used in further experiments [23]. After determining the best ratio of the enzyme, the effect of BN concentration was investigated. There is a direct correlation
between the relative fluorescence intensity and the concentrations of the probe. The lower concentration will result in lower fluorescence intensity. However, the higher concentration will lead to high background. Fig. 5B indicates that the optimal concentration of the probe was 36.5 lM. Fig. 5C shows the effect of pH on the relative fluorescence intensity in the presence of ACh. The relative fluorescence intensity was the highest at pH 7.5, which indicated that the fluorescence of BN was much higher in alkaline medium than in acidic or neutral medium. In addition, it is advantageous in alkaline medium for the hydrolytic deprotection of the boronates and subsequent generation of an open, colored, and fluorescent product. It is reported that different pH values would result in different enzyme activities. The optimal pH values for ChOx and AChE are 7.0 to 8.0 and 8.0 to 9.0, respectively, due to higher pH leading to the denaturation of AChE and ChOx. Therefore, pH 7.5 PBS was used in the further experiments. As shown in Fig. 5D, the relative fluorescence intensity of BN was the highest at 37 °C. This observation should be highly correlated with the enzyme kinetics and decomposition rate of H2O2. Therefore, 37 °C was chosen as the optimal reaction temperature in this work. As shown in Fig. 5E, the experiments demonstrated that the relative fluorescence intensities reached stable values after 15 min; therefore, 15 min was selected as the reaction time for recording the fluorescence intensity of the system [23]. Detection of ACh Under the optimal conditions, the detection of ACh was conducted. As shown in Fig. 6, relative fluorescence intensity increased
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Fig.5. (A) Relationship between relative fluorescence intensity and ratio of AChE/ChOx in PBS containing 1.00 mM ACh and 36.5 lM probe. (B) Dependence of relative fluorescence intensity on concentration of probe in PBS containing 1.0 mM ACh, 3.0 U/ml AChE, and 1.0 U/ml ChOx. (C,D) Effect of pH (C) and temperature (D) on relative fluorescence intensity of 36.5 lM probe in PBS containing 1.0 mM ACh, 3.0 U/ml AChE, and 1.0 U/ml ChOx. (E) Time-dependent relative fluorescence changes of 36.5 lM probe on interaction with different concentrations of ACh in PBS containing 3.0 U/ml AChE and 1.0 U/ml ChOx. kex = 455 nm.
Fig.6. (A) Normalized fluorescence spectra of 36.5 lM probe with different concentrations of ACh in PBS (pH 7.5) thermostated at 37 °C after 15 min. (B) Linearity regression analysis of relative fluorescence intensity of 36.5 lM probe against concentrations of ACh in PBS containing 3.0 U/ml AChE and 1.0 U/ml ChOx. kex = 455 nm.
on increasing concentrations of ACh with a linear response (R2 = 0.9961) in the range of 0.01 to 80 lM. When the concentration of ACh was larger than 80 lM, the fluorescence was also proportional to the ACh concentration (R2 = 0.991). The evaluation of
fluorescence intensity is suitable for the determination of ACh within a wide range of 0.01 to 80 lM and 80 to 200 lM over two consecutive linear ranges with a low detection limit of 2.7 nM. It revealed that the assay was comparable to, or even better than,
Detection of ACh based on boronate ITC fluorescence probe / C. Liu et al. / Anal. Biochem. 465 (2014) 172–178
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Table 1 Comparison of various approaches to ACh detection. Method
LOD (nM)
Detection mode
Advantage(s)
Disadvantage(s)
Reference
LC/MS/MS Biosensor Biosensor Biosensor FIA–CLD Biosensor
0.01 49.3 10000 50 2 2.7
Mass spectrometry Amperometry Potentiometry Chemiluminescence Chemiluminescence Fluorescence
Highly sensitive Simple Simple Quick Highly sensitive Simple and selective
Tedious sample pretreatment and complex separation processes Not sensitive Complex pretreatment Lowly sensitive Complex process
[3] [24] [25] [22] [26] This study
Note. LOD, limit of detection; LC/MS/MS, liquid chromatography tandem mass spectrometry; FIA, flow injection analysis; CLD, chemiluminometric detection.
Detection of ACh in serum samples The excellent specificity and high sensitivity of the assay suggested that the developed method might be applied to detect ACh in real samples. The fluorescence responses were linearly related to the concentrations of ACh added into the serum in the concentration range of 0 to 30 lM (Table 2). The ACh concentration in the serum was determined to be 77.2 lM, and it was consistent with the result obtained by using a hospital instrument (80.0 lM). Consequently, it indicated that fluorescent probe of BN holds great potential for practical applications. Conclusion
Fig.7. Relative fluorescence responses of 36.5 lM probe in PBS containing 3.0 U/ml AChE, 1.0 U/ml ChOx, and 80 lM ACh or 8 mM amino acetic acid, alanine, dopamine, glutamic acid, proline, tryptophane, arginine, and aspartate, respectively. kex = 455 nm.
most reported optical sensors. In comparison with other methods (Table 1), our assay for ACh detection is relatively simple and selective. Thus, the fluorescent probe of BN holds great potential for practical applications.
A novel fluorescent boronate probe was synthesized and used to quantitatively detect ACh. The mechanism of ACh detecting is based on enzyme-generated H2O2, which reacts with BN to produce a fluorescence product of ON. On observation of the fluorescence change, the concentration of ACh can be determined. With favorable biocompatibility and high quantum yield, the fluorescent probe of BN may become a promising compound for ACh detection in biological samples. Furthermore, the method based on the fluorescent boronate probe has many advantages such as simple operation, high sensitivity, and good selectivity. In addition, this research will enrich the design of novel sensors based on the assembly of boronate probes with other enzymes to monitor valuable molecule in the future. Acknowledgments
Selectivity of probe To evaluate the selectivity of the assay toward ACh (80 lM) detection over some possible interfering substances (8 mM), amino acetic acid, alanine, dopamine, glutamic acid, proline, tryptophane, arginine, and aspartate were tested as the interfering substances. Remarkably, only ACh resulted in a significant increase in the fluorescence of the system, revealing that the assay was highly selective toward ACh (Fig. 7). Besides, on reaction with ACh in the presence of AChE and ChOx, hydrolytic deprotection of the boronates was a specific reaction that subsequently generated an open, colored, and fluorescent product (ON). Thus, interference from other organic substances can be ignored.
Table 2 Determination of ACh levels using the BN in serum sample. ACh concentration (lM)
Normalized intensity
Recovery (%)
Relative error (%)
0 10 15 20 25 30
0.20 ± 0.009 0.45 ± 0.006 0.56 ± 0.014 0.74 ± 0.011 0.85 ± 0.003 0.96 ± 0.007
– 98.0 93.8 105.4 101.4 98.9
4.54 1.27 2.49 1.53 0.30 0.77
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Sensitive detection of acetylcholine based on a novel boronate intramolecular charge transfer fluorescence probe Chang Liu, Youming Shen, Peng Yin, Lidong Li, Meiling Liu, Youyu Zhang, Haitao Li ⇑, Shouzhuo Yao Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 25 April 2014 Received in revised form 25 July 2014 Accepted 1 August 2014 Available online 14 August 2014 Keywords: Boronate probe 1,8-Naphthalimide Acetylcholinesterase Choline oxidase Acetylcholine Fluorescence detection
a b s t r a c t A highly sensitive and selective fluorescence method for the detection of acetylcholine (ACh) based on enzyme-generated hydrogen peroxide (H2O2) and a new boronate intramolecular charge transfer (ICT) fluorescence probe, 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-N-butyl-1,8-naphthalimide (BN), was developed. This strategy involves the reaction of ACh with acetylcholinesterase (AChE) to produce choline, which is further oxidized by choline oxidase (ChOx) to obtain betaine and H2O2. The enzymegenerated H2O2 reacts with BN and results in hydrolytic deprotection of BN to generate fluorescent product (4-hydroxyl-N-butyl-1,8-naphthalimide, ON). Two consecutive linear response ranges allow determining ACh in a wide concentration range with a low detection limit of 2.7 nM (signal/noise = 3). Compared with other fluorescent probes based on the mechanism of nonspecific oxidation, this reported boronate probe has the advantage of no interference from other biologically relevant reactive oxygen species (ROS) on the detection of ACh. This study provides a new method for the detection of ACh with high selectivity and sensitivity. Ó 2014 Elsevier Inc. All rights reserved.
Acetylcholine (ACh)1 is one of the most important neurotransmitters contained in the central and peripheral nervous systems and is widely involved in controlling nerve conduction in the body [1]. Acetylcholine comes from choline in the presence of choline acetyltransferase and acetyl-coenzyme A and has a significant influence on human memory and sleep [1]. The metabolic abnormalities of ACh in the brain may cause neuropsychiatric disorders such as Huntington’s disease, Alzheimer’s disease, schizophrenia, and Parkinson’s disease [1,2]. Therefore, highly sensitive and selective detection of ACh is very necessary and of great importance. A number of analytical techniques have been reported for the detection of ACh. Direct analytical techniques include mass spectrometry (MS), gas chromatography (GC), high-performance liquid chromatography (HPLC), and liquid chromatography coupled with mass spectrometry (LC/MS) [3,4]. Although these techniques are sensitive and reliable, most of them require tedious sample pre-
⇑ Corresponding author. Fax: +86 731 8865515. E-mail address: [email protected] (H. Li). Abbreviations used: ACh, acetylcholine; AChE, acetylcholinesterase; ChOx, choline oxidase; H2O2, hydrogen peroxide; HRP, horseradish peroxidase; UV–vis, ultraviolet– visible; ROS, reactive oxygen species; Amplex Red, 10-acetyl-3,7-dihydroxyphenoxazine; ICT, intramolecular charge transfer; BN, 4-(4,4,5,5-tetramethyl-1,3, 2-dioxaborolan-2-yl)-N-butyl-1,8-naphthalimide; ON, 4-hydroxyl-N-butyl-1,8-naphthalimide; PBS, phosphate buffer solution; FTIR, Fourier transform infrared. 1
http://dx.doi.org/10.1016/j.ab.2014.08.003 0003-2697/Ó 2014 Elsevier Inc. All rights reserved.
treatment, complex separation processes, and skilled operators [1,3]. The enzyme-based method is an indirect detection technique of ACh with high sensitivity. Acetylcholinesterase (AChE) and choline oxidase (ChOx) could catalyze their substrates to produce electrochemically active product (hydrogen peroxide, H2O2) or spectral (absorbance and fluorescence) sensing products. Therefore, the enzyme-based detection method usually depends on electrochemical and optical techniques. However, electrochemical detection of ACh by measuring its electrochemical response (enzymegenerated H2O2) is not sensitive enough [5], and the complex pretreatment of electrode is needed. Alternatively, optical detection methods, such as colorimetric Ellman assays and fluorescence assays, are widely used due to the simple operations and easy pretreatment processes. Colorimetric assays always depend on the use of peroxidase- or oxidase-like enzymes, such as horseradish peroxidase (HRP) and various of nanoparticles (NPs), and also need various substrates, including 3,3,5,5-tetrame-thylbenzidine (TMB), o-phenylenediame (OPD), pyrogallol, and 2,20 -azino-bis(3ethylbenzo-thiazoline-6-sulfonic acid) diammonium salt (ABTS) [1]. The detection can be performed through the color change by the naked eye or ultraviolet–visible (UV–vis) absorption. Colorimetric assays are simple but not sensitive enough [6]. Nowadays, more and more attention is paid to fluorescence analysis for high sensitivity [7].
Detection of ACh based on boronate ITC fluorescence probe / C. Liu et al. / Anal. Biochem. 465 (2014) 172–178
Up to now, there have been few reports about the detection of ACh directly using fluorescence probe. Based on the principle of indirect detection, it should be useful for the detection of ACh through detecting enzyme-generated H2O2 by using H2O2-sensitive fluorescent probes. There are several fluorescent probes that have been developed for measuring H2O2, superoxide, and peroxynitrite in biological systems or detecting enzyme-generated H2O2 [8,9]. Unfortunately, the assays based on dichlorodihydrofluorescein diacetate (DCFH–DA), hydroethidine (HE), and Mito-SOX always have high fluorescence background produced by their reaction with reactive oxygen species (ROS) [7,9–11]. To the best of our knowledge, it is reported that only the 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red) ACh/AChE Assay Kit provides ultrasensitive measurement of ACh based on enzyme-generated H2O2 [10]. However, HRP is an indispensable factor in the catalyzing oxidation of Amplex Red in the presence of H2O2. In addition, the maximum absorption band and fluorescence emission peak of the oxidation product of Amplex Red are extraordinarily close (absorbance at 571 and emission at 585 nm) [9,10], which may cause spectral interferences. There are some fluorescence approaches for detection of ACh based on nanoparticles such as C-dots [2] and Au/Ag nanoparticles [1]. These methods are highly sensitive and selective; however, they always suffer from potential toxicity and chemical instability. Because of the limitations of currently available H2O2-responsive probes, many researchers are focusing their studies on boronate probes for their reaction with H2O2 [7,9,12]. So far, boronate probes, fluorescein peroxyfluor 1 (PF1) probes, and peroxyxanthone 1 (PX1) probes have been synthesized and used to detect H2O2. The interaction of H2O2 and boronate probes is ambiphilic and nucleophilic reactivity which is beneficial to hydrolytic deprotection of the boronates. In comparison with other probes taking the mechanism of nonspecific oxidation, specific interaction of H2O2 with boronates will achieve higher selectivity than other biologically relevant ROS probes [7,13]. Based on the advantages of boronate probes, a novel intramolecular charge transfer (ICT) boronate probe, 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-N-butyl-1,8-naphthalimide (BN) is designed and synthesized for the selective detection of ACh with significant spectral shift (455/555 nm). BN contained 1,8-naphthalimide as the fluorophore and boronic ester as the recognition unit. Because of the structure of BN, it has a high quantum yield at 76.24% (stated in Section S1 of the online supplementary material). The novel boronate probe is highly sensitive and selective toward enzyme-generated H2O2 that is produced from the enzyme catalytic reaction of ACh. When H2O2 coexists with BN under appropriate conditions, BN is transferred to a highly fluorescent product, 4-hydroxyl-N-butyl-1,8-naphthalimide (ON) by the hydrolytic deprotection of the boronates. The novel detection strategy is based on the fluorescence change depending on
O
AC h
E
N
choline
O B O
O
ACh H2O 2
Ox Ch
173
enzyme-generated H2O2, as shown in Scheme 1. This method is sensitive and does not require a complex pretreatment process or enzyme immobilization. Materials and methods Materials and apparatus Acetylcholine, amino acetic acid, alanine, dopamine, glutamic acid, proline, tryptophane, arginine, and aspartate were purchased from Wako Pure Chemical Industries (Osaka, Japan). Acetonitrile was obtained from Sinopharm Chemical Reagent. Acetylcholinesterase and choline oxidase were obtained from Sigma–Aldrich. All other chemicals used here were of analytical grade. The detection buffer was phosphate buffer solution (PBS, pH 7.5). Milli-Q ultrapure water (P18 MX cm, Millipore) was used throughout. Unless otherwise noted, solvents were purified by distillation. Human serum samples were provided by the Hospital of Hunan Normal University (Changsha, China). A Bruker AVB-500 spectrometer and an API 4000 QTRAP LC/MS/ MS System with ESI Ion Source (AB Sciex, USA) were used to characterize BN. Fourier transform infrared (FTIR) spectra were collected on a Nicolet Nexus 670 FTIR instrument (Nicolet Instrument, USA). UV–vis and fluorescence spectra were recorded on a UV-2450 spectrophotometer (Shimazu, Japan) and an F4500 fluorescence spectrophotometer (Hitachi, Japan), respectively. Synthesis of 4-bromo-N-butyl-1,8-naphthalimide The synthesis of 4-bromo-N-butyl-1,8-naphthalimide was similar to that reported by Zhengneng and coworkers [14]. 4-Bromo1,8-naphthalic anhydride (486.4 mg, 2 mmol) and butylamine (204.8 mg, 2.8 mmol) were dissolved in 50 ml of ethanol, and the resulting solution was stirred at 80 °C for 12 h. After being cooled to room temperature, the precipitated solid was filtered and recrystallized in ethanol to give a light yellow solid (386 mg, 65%) [15]. Synthesis of 4-boronate-N-butyl-1,8-naphthalimide A nitrogen-flushed three-neck round-bottom flask was charged with 4-bromo-N-butyl-1,8-naphthalimide (99.7 mg, 0.3 mM), bis(pinacolato)diboron (91.4 mg, 0.36 mM), sodium acetate (73.8 mg, 0.9 mM), bis(triphenylphosphine)palladium(II) chloride (11.0 mg, 0.015 mM), CuI (6.0 mg, 0.03 mM), and triphenylphosphine (19.0 mg, 0.075 mM). 1,2-Dimethoxyethane (210 ml) was then added, and the mixture was bubbled with nitrogen for 15 min. After stirring at 60 °C for 24 h, the reaction mixture was cooled to room temperature and then poured into icewater (200 ml). It was then extracted with methylene chloride (20 ml), and the combined organic layer was dried over anhydrous magnesium sulfate. Then, the organic solvent was removed by rotary evaporation, and the residue was passed through a flash silica gel column with mineral ether as the eluent to give white crystals (73.51 mg, yield 65%) [16].
O N O
Fluorescent detection of ACh OH
Scheme 1. Schematic representation of the sensing strategy using 4-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)-N-butyl-1,8-naphthalimide to detect ACh.
The experiments were repeated three times to ensure the accuracy of the measurements. The fluorescent measurements for the detection of ACh were carried out as follows [12]. First, 150 ll of PBS containing 3 U/ml AChE, 1 U/ml ChOx, 0.01 to 200 lM ACh, and 36.5 lM BN was incubated under gentle shaking at 37 °C for 30 min [1]. Subsequently, fluorescence spectra were recorded at
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the excitation wavelength of 455 nm. The relative fluorescence intensity at 555 nm was used for the quantitative analysis of ACh. Determination of ACh in serum samples Serum samples (15 ll) were diluted with 135 ll of PBS. Prior to the test, 135 ll of PBS containing 3 U/ml AChE, 1 U/ml ChOx, 0 to 30 lM ACh, and 36.5 lM BN was equilibrated under gentle shaking at 37 °C for 1 h. Then, fluorescence spectra were recorded. Results and discussion Spectral properties of the probe 1
The structure of BN was confirmed by H nuclear magnetic resonance (NMR), 13C NMR, and FTIR spectroscopy (see Figs. S1–S3 in supplementary material). 1H NMR (500 MHz, CdCl3): d 9.12 (d, 1H), 8.61–8.56 (m, 2H), 8.30 (d, 1H), 7.79 (t, 1H), 4.20 (t, 2H), 1.74–1.71 (m, 2H), 1.58 (m, 2H), 1.45 (s, 12H), 0.99 (t, 3H); 13C NMR (125 MHz, CdCl3): 136.4, 135.9, 135.5, 131.4, 130.3, 127.7, 85.2, 77.9, 77.6, 77.4, 40.9, 30.8, 25.6, 21.0, 14.4 ppm. FTIR spectrum was used to study the functional groups of BN (Fig. S3). The broad peak centered at 2956 cm1 is assigned to the stretching vibration of the C@C stretching mode of aromatic hydrocarbons, whereas the peak at 1355 cm1 is attributed to the bending vibration of the CAH bond [17]. The stretching vibration of OAH (3451 cm1) may be attributed to the residual water. The peak at 1660 cm1 correspond to the CAO stretching vibration. The band around 1157 cm1 is the CAN stretching mode [18]. The absorption, excitation, and emission spectra of the probe in PBS are presented in Fig. 1. The probe exhibited a well-defined absorption peak at 344 nm, which was considered as a p–p transition of the naphthalimide backbone [19,20]. In addition, the peak appearing at 345 nm in the excitation spectrum of BN was consistent with the UV–vis absorption spectrum. As shown in Fig. 1, a narrow emission spectrum ranging from 400 to 500 nm was observed under excitation at 345 nm, and its maximum emission is centered at 440 nm, suggesting that BN has strong fluorescent emission intensity. As shown in Fig. S4 of the supplementary material, the maximum emission wavelength (455 nm) was not changed with different excitation wavelengths, which indicates the spectral purity of the probe.
Fig.2. Normalized fluorescence spectra of probe on interaction with 3.0 U/ml AChE and 1.0 U/ml ChOx in the presence (1) and absence (2) of 1.0 mM ACh in PBS.
ing the change in the relative fluorescence intensity of the system, the concentration of ACh can be measured. To confirm the interaction of the probe with enzyme-generated H2O2, we incubated the probe with PBS containing AChE and ChOx in the absence and presence of ACh. As shown in Fig. 2, there was no obvious change of the normalized fluorescence intensity in the absence of ACh. However, the fluorescence intensity was obviously increased at 555 nm in the presence of ACh. Therefore, AChE converts ACh to choline, which is further oxidized by ChOx to produce betaine, and H2O2 should be responsible for the change in the fluorescence intensity. The enzyme-generated H2O2 causes the hydrolytic deprotection of the boronates and elicits a significant fluorescence increase [21]. To further confirm the detection mechanism, the reaction of the probe with H2O2 was also investigated; the results are shown in Fig. 3. An absorption peak at around 345 nm was found in the spectrum of BN. However, in the presence of H2O2, an absorption peak at around 455 nm occurred, suggesting that a new compound (ON) was produced. As shown in Fig. 3B, the probe displayed a large turn-on fluorescence response. On excitation at 455 nm, the probe of BN had low fluorescence (Fig. 3B). However, the product (ON) had strong emission at 555 nm, and the normalized fluorescence intensity was increased with the increase of H2O2 concentrations [21]. In addition, experimental facts revealed that the fluorescence intensity of BN in alkaline medium was preponderant to acidic medium (Fig. S4), indicating that alkaline medium was beneficial to the hydrolytic deprotection of the boronates [2].
Mechanism for detection of ACh Effect of probe on enzyme activity The general principle for detection of ACh based on BN is shown in Scheme 1. First, AChE hydrolyzes ACh to choline. Then, choline is oxidized by ChOx to produce betaine and H2O2. The resulting H2O2 reacts with BN to produce the fluorescent product of ON. By detect-
As a probe used in the enzyme reaction system, it is important to evaluate the influence of the probe on the enzyme activity. The method supported by Hestrin [22] has been selected to monitor the
Fig.1. (A) UV–vis absorption, excitation spectrum, and emission spectrum of the probe 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-N-butyl-1,8-naphthalimide (BN). (B) Excitation spectrum and emission spectrum of the resultant 4-hydroxyl-N-butyl-1,8-naphthalimide (ON).
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175
Fig.3. UV–vis absorption spectra (A) and normalized fluorescence spectra (B) of 20.0 lM probe in 1 ml of acetonitrile–PBS (1:1, v/v) solution with different concentrations of H2O2 (0.2, 0.4, and 1.0 mM). kex = 455 nm.
Fig.4. Plots of the absorbance at 520 nm of the resulting solution of ACh interacting with alkaline hydroxylamine and ferric chloride versus the concentration of ACh in the presence (A) and absence (B) of 36.5 lM probe in PBS. The inset shows the color change with increasing concentrations of ACh in PBS containing 3.0 U/ml AChE and 1.0 U/ml ChOx.
enzyme activity change in the presence or absence of BN. Acetylcholine reacting with hydroxylamine hydrochloride would produce acetyl hydroxamic acid, which could further interact with ferric chloride to produce ferric–cethydroxamic acid complex. The yield of acetyl hydroxamic acid is a direct function of ACh concentration, which indicates that the absorbance is proportional to the concentration of ACh. The absorbance spectra and the color change of different concentrations of ACh on its interaction with AChE, ChOx, and alkaline hydroxylamine are shown in Fig. 4. In the wavelength ranging from 520 to 540 nm, little or no absorption is shown by ferric chloride, whereas absorption-related to ferric– cethydroxamic acid complex is nearly the maximum. Therefore, the absorbance at 520 nm that has lower background was selected to monitor the enzyme activity change. Obviously, there are no significant differences between the cases of presence (Fig. 4A) and absence (Fig. 4B) of BN, and it indicates that the BN does not affect the enzyme activity. Optimization conditions for detection To obtain a highly sensitive response, it is necessary to optimize the conditions. To generate more H2O2, it is of paramount importance to optimize the concentrations of ChOx and AChE [23]. Fig. 5A shows the relative fluorescence intensity of BN on interaction with 1.00 mM ACh and different ratios of ChOx/AChE. It shows that the maximum response can be obtained when the concentrations of ChOx and AChE are 1.0 and 3.0 U/ml, respectively. Therefore, 1.0 U/ml ChOx and 3.0 U/ml AChE were used in further experiments [23]. After determining the best ratio of the enzyme, the effect of BN concentration was investigated. There is a direct correlation
between the relative fluorescence intensity and the concentrations of the probe. The lower concentration will result in lower fluorescence intensity. However, the higher concentration will lead to high background. Fig. 5B indicates that the optimal concentration of the probe was 36.5 lM. Fig. 5C shows the effect of pH on the relative fluorescence intensity in the presence of ACh. The relative fluorescence intensity was the highest at pH 7.5, which indicated that the fluorescence of BN was much higher in alkaline medium than in acidic or neutral medium. In addition, it is advantageous in alkaline medium for the hydrolytic deprotection of the boronates and subsequent generation of an open, colored, and fluorescent product. It is reported that different pH values would result in different enzyme activities. The optimal pH values for ChOx and AChE are 7.0 to 8.0 and 8.0 to 9.0, respectively, due to higher pH leading to the denaturation of AChE and ChOx. Therefore, pH 7.5 PBS was used in the further experiments. As shown in Fig. 5D, the relative fluorescence intensity of BN was the highest at 37 °C. This observation should be highly correlated with the enzyme kinetics and decomposition rate of H2O2. Therefore, 37 °C was chosen as the optimal reaction temperature in this work. As shown in Fig. 5E, the experiments demonstrated that the relative fluorescence intensities reached stable values after 15 min; therefore, 15 min was selected as the reaction time for recording the fluorescence intensity of the system [23]. Detection of ACh Under the optimal conditions, the detection of ACh was conducted. As shown in Fig. 6, relative fluorescence intensity increased
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Fig.5. (A) Relationship between relative fluorescence intensity and ratio of AChE/ChOx in PBS containing 1.00 mM ACh and 36.5 lM probe. (B) Dependence of relative fluorescence intensity on concentration of probe in PBS containing 1.0 mM ACh, 3.0 U/ml AChE, and 1.0 U/ml ChOx. (C,D) Effect of pH (C) and temperature (D) on relative fluorescence intensity of 36.5 lM probe in PBS containing 1.0 mM ACh, 3.0 U/ml AChE, and 1.0 U/ml ChOx. (E) Time-dependent relative fluorescence changes of 36.5 lM probe on interaction with different concentrations of ACh in PBS containing 3.0 U/ml AChE and 1.0 U/ml ChOx. kex = 455 nm.
Fig.6. (A) Normalized fluorescence spectra of 36.5 lM probe with different concentrations of ACh in PBS (pH 7.5) thermostated at 37 °C after 15 min. (B) Linearity regression analysis of relative fluorescence intensity of 36.5 lM probe against concentrations of ACh in PBS containing 3.0 U/ml AChE and 1.0 U/ml ChOx. kex = 455 nm.
on increasing concentrations of ACh with a linear response (R2 = 0.9961) in the range of 0.01 to 80 lM. When the concentration of ACh was larger than 80 lM, the fluorescence was also proportional to the ACh concentration (R2 = 0.991). The evaluation of
fluorescence intensity is suitable for the determination of ACh within a wide range of 0.01 to 80 lM and 80 to 200 lM over two consecutive linear ranges with a low detection limit of 2.7 nM. It revealed that the assay was comparable to, or even better than,
Detection of ACh based on boronate ITC fluorescence probe / C. Liu et al. / Anal. Biochem. 465 (2014) 172–178
177
Table 1 Comparison of various approaches to ACh detection. Method
LOD (nM)
Detection mode
Advantage(s)
Disadvantage(s)
Reference
LC/MS/MS Biosensor Biosensor Biosensor FIA–CLD Biosensor
0.01 49.3 10000 50 2 2.7
Mass spectrometry Amperometry Potentiometry Chemiluminescence Chemiluminescence Fluorescence
Highly sensitive Simple Simple Quick Highly sensitive Simple and selective
Tedious sample pretreatment and complex separation processes Not sensitive Complex pretreatment Lowly sensitive Complex process
[3] [24] [25] [22] [26] This study
Note. LOD, limit of detection; LC/MS/MS, liquid chromatography tandem mass spectrometry; FIA, flow injection analysis; CLD, chemiluminometric detection.
Detection of ACh in serum samples The excellent specificity and high sensitivity of the assay suggested that the developed method might be applied to detect ACh in real samples. The fluorescence responses were linearly related to the concentrations of ACh added into the serum in the concentration range of 0 to 30 lM (Table 2). The ACh concentration in the serum was determined to be 77.2 lM, and it was consistent with the result obtained by using a hospital instrument (80.0 lM). Consequently, it indicated that fluorescent probe of BN holds great potential for practical applications. Conclusion
Fig.7. Relative fluorescence responses of 36.5 lM probe in PBS containing 3.0 U/ml AChE, 1.0 U/ml ChOx, and 80 lM ACh or 8 mM amino acetic acid, alanine, dopamine, glutamic acid, proline, tryptophane, arginine, and aspartate, respectively. kex = 455 nm.
most reported optical sensors. In comparison with other methods (Table 1), our assay for ACh detection is relatively simple and selective. Thus, the fluorescent probe of BN holds great potential for practical applications.
A novel fluorescent boronate probe was synthesized and used to quantitatively detect ACh. The mechanism of ACh detecting is based on enzyme-generated H2O2, which reacts with BN to produce a fluorescence product of ON. On observation of the fluorescence change, the concentration of ACh can be determined. With favorable biocompatibility and high quantum yield, the fluorescent probe of BN may become a promising compound for ACh detection in biological samples. Furthermore, the method based on the fluorescent boronate probe has many advantages such as simple operation, high sensitivity, and good selectivity. In addition, this research will enrich the design of novel sensors based on the assembly of boronate probes with other enzymes to monitor valuable molecule in the future. Acknowledgments
Selectivity of probe To evaluate the selectivity of the assay toward ACh (80 lM) detection over some possible interfering substances (8 mM), amino acetic acid, alanine, dopamine, glutamic acid, proline, tryptophane, arginine, and aspartate were tested as the interfering substances. Remarkably, only ACh resulted in a significant increase in the fluorescence of the system, revealing that the assay was highly selective toward ACh (Fig. 7). Besides, on reaction with ACh in the presence of AChE and ChOx, hydrolytic deprotection of the boronates was a specific reaction that subsequently generated an open, colored, and fluorescent product (ON). Thus, interference from other organic substances can be ignored.
Table 2 Determination of ACh levels using the BN in serum sample. ACh concentration (lM)
Normalized intensity
Recovery (%)
Relative error (%)
0 10 15 20 25 30
0.20 ± 0.009 0.45 ± 0.006 0.56 ± 0.014 0.74 ± 0.011 0.85 ± 0.003 0.96 ± 0.007
– 98.0 93.8 105.4 101.4 98.9
4.54 1.27 2.49 1.53 0.30 0.77
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