Redox-Controlled Fluorescent Nanoswitch Based on Reversible

Subscriber access provided by La Trobe University Library

Article

Redox-Controlled Fluorescent Nanoswitch Based on Reversible Disulfide and Its Application in Butyrylcholinesterase Activity Assay Guilin Chen, Hui Feng, Xiaogan Jiang, Jing Xu, Saifei Pan, and Zhaosheng Qian Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02976 • Publication Date (Web): 04 Jan 2018 Downloaded from http://pubs.acs.org on January 7, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Redox-Controlled Fluorescent Nanoswitch Based on Reversible

Disulfide

and

Its

Application

in

Butyrylcholinesterase Activity Assay Guilin Chen,† Hui Feng,† Xiaogan Jiang, Jing Xu, Saifei Pan and Zhaosheng Qian* College of Chemistry and Life Science, Zhejiang Normal University, Jinhua 321004, People’s Republic of China *Corresponding author. E-mail: [email protected]. Tel.: +86-579-82282269. Fax.: +86579-82282269.

1 ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 26

ABSTRACT: Butyrylcholinesterase (BChE) mainly contributing to plasma cholinesterase activity is an important indicator for routinely diagnosing liver function and organophosphorus poisoning in clinical diagnosis, but its current assays are scarce and frequently suffer from some significant interference and instability. Herein, we report a redox-controlled fluorescence nanoswtich based on reversible disulfide bonds, and further develop a fluorometric assay of BChE via thiol-triggered disaggregation-induced emission. Thiol-functionalized carbon quantum dots (thiol-CQDs) with intense fluorescence is found to be responsive to hydrogen peroxide, and their redox reaction transforms thiol-CQDs to nonfluorescent thiol-CQD assembly. The thiols inverse this process by a thiol-exchange reaction to turn on the fluorescence. The fluorescence can be reversibly switched by the formation and breaking of disulfide bonds caused by external redox stimuli. The specific thiol-triggered disaggregation-induced emission enables us to assay BChE activity in a fluorescence turn-on and real-time way using butyrylthiocholine iodide as the substrate. As-established BChE assay achieves sufficient sensitivity for practical determination in human serum, and is capable of avoiding the interference from micromolar glutathione and discriminatively quantifying BChE from its sister enzyme acetylcholinesterase. The first design of reversible redox-controlled nanosiwtch based on disulfide expands the application of disulfide chemistry in sensing and clinical diagnostics, and this novel BChE assay enriches the detection methods for cholinesterase activity.


Page 3 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION Butyrylcholinesterase (BChE; EC 3.1.1.8) is a nonspecific cholinesterase enzyme that hydrolyzes many different choline-based esters like acetylcholine as a neurotransmitter in nerve cells and various drugs acting at the neuromuscular junctions.1 In contrast to its sister enzyme acetylcholinesterase (AChE; EC 3.1.1.7), BChE is abundant in blood plasma and mainly contribute to the cholinesterase activity in human serum, which enables BChE to function as a more useful biochemical marker in clinical diagnosis.2 A variety of diseases including hepatocellular carcinoma, chronic liver diseases and poisoning with organophosphates can be reliably and conveniently diagnosed by assaying BChE activity in blood serum in terms of its activity alternations. As a consequence, its great significance in practical clinical diagnosis urgently demands accurate assay methods for BChE activity. However, very few methods have been developed for BChE activity in sharp contrast to a wide diversity of assays for AChE.3 Except for several electrochemical sensors based on controlled release of substrate for BChE activity,4-7 most assays were established on optical techniques including colorimetric and fluorometric approaches. Among these spectrometric detection protocols, Ellman’s colorimetric method is the most common and universally used assay for BChE activity because of its convenience and accessibility of high-throughput analysis.8 Its inherent drawbacks such as significant interference from biomolecules in serum samples, and instability of the used reagent, push the establishment of the other colorimetric assays based on 2,6-dichloroindophenol acetate9 and self-assembly of gold nanorod.10 In comparison to these improvements, fluorometric approach is more suited for improving sensitivity and lowering interference from samples in quantifying BChE activity. Several fluorogenic substrates like resorufin butyrate and indoxyl acetate,11 a dimethoxyphthallimide derivative,12 and butylryl-modified molecular probes13 have



Page 4 of 26

been employed to assay BChE activity in standard buffer solutions with a good performance. A quantum dot-based nanoprobe was also used for sensing BChE due to its higher stability and lower interference from biomolecules.14 However, none of them are capable of assaying BChE and AChE simultaneously and discriminating BChE from AChE through a small alternation of the assay. Therefore it is still greatly demanded for reliable and discriminative BChE assays based on benign fluorescent materials. The disulfide bond is an extremely important functional groups in chemical synthesis and biological processes because it is stable in ambient environment and is able to be effectively cleaved by small biothiols or some specific medium reductants. Disulfide linker has been broadly employed for drug conjugation in targeted drug delivery and reduction-responsive therapy because of its specific and sensitive thiol-triggered cleavage.15 Disulfide-based functionalization has also shown its powerful and promising application in the development of chemosensors targeting at thiols and metal ions.16 Most of these preceding works focus on the development of fluorogenic probes for the detection and imaging of cellular glutathione in the single17-22 or twophoton modes23,24 apart from a few examples on detecting mercury and cuprous cations.25,26 Recent advance shows that disulfide-based fluorescent probe is also able to image H2Se in living cells;27 however, only one case study utilized the specific thiol-responsive behavior of disulfide bonds to assay enzyme activity up to date.28 In this work, we design a redox-controlled fluorescent nanoswitch based on reversible disulfide bonds, and further develop a reliable and fluorometric BChE assay via disaggregationinduced emission of bonding aggregates of carbon quantum dots. Thiol-functionalized carbon quantum dots (thiol-CQDs) were prepared through covalently incorporating cysteamine to the


Page 5 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Scheme 1. Schematic Illustration of Redox-Controlled Fluorescent Nanoswitch and Detection Strategy for BChE Activity Based on Thiol-Triggered Disulfide Cleavage.

surface of carbon quantum dots via amide linker. As illustrated in Scheme 1, thiol-CQDs possess intense yellowish green fluorescence in aqueous solution, and can be aggregated into disulfidelinked assemblies triggered by hydrogen peroxide. The resulting disulfide-linked assemblies can be inversed to thiol-CQDs in the presence of thiols such as thiocholine because of the effective cleavage of disulfide by thiols. The fluorescence is able to be reversibly switched between ON and OFF state in response to external redox stimuli. This nanoswitch was further employed to develop a fluorometric assay of butyrylcholinesterase (BChE) using butyrylthiocholine iodide (BTCh) as the substrate. In this assay, thiol-CQD assembly functions as the fluorogenic probe, and it remains nonfluorescent without BChE and BTCh. Once the BChE and BTCh are



Page 6 of 26

introduced, thiolcholine hydrolyzed from the substrate rapidly reacts with the probe to produce highly fluorescent thiol-CQDs. BChE activity in the assay can be evaluated from the change of the fluorescence signal. The possible interference from micromolar glutathione in plasma was assessed, and discriminative detection of BChE activity from AChE activity was also achieved by the use of a different substrate. The function of this assay to screen inhibitors of BChE was also assessed using tacrine and parathion-methyl as the examples.

EXPERIMENTAL SECTION Materials and Reagents. Triple-distilled water was utilized throughout the whole experimental process. Activated carbon, cysteamine hydrochloride, glutathione (GSH), penicillamine (PA), sodium dithionite (Na2S2O4), dithiothreitol (DTT), acetylthiocholine chloride (ATCh), S-butyrylthiocholine iodide (BTCh), tacrine, parathion-methyl solution, 1-(3dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were purchased from J&K Chemical Company (Shanghai, China). Butyrylcholinesterase (BChE, EC 3.1.1.8, 0.5 kU) from equine serum and acetylcholinesterase (AChE, EC 3.1.1.7, 0.5 kU) from Electrophorus electricus were bought from Sigma-Aldrich Company (Shanghai, China). HEPES solution (10 mM, pH 7.4) was used as the buffer solution. Three fresh human serum samples were from three healthy male volunteers. All reagents were of analytical grade and without any further purification. Synthesis of Thiol-Functionalized Carbon Quantum Dots (Thiol-CQDs). The detailed procedure of the carbon quantum dots was reported in our previous article.29,30 A brief procedure is described as follows: a fixed amount of activated carbon (2.0 g) was added into a mixture of concentrated sulfuric acid (180.0 mL) and nitric acid (60.0 mL) and then the mixture was heated at 80 °C for 5 h. After that the reaction mixture was neutralized with sodium hydroxide and


Page 7 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


dialyzed in a dialysis bag (1000 Da) for 3 days, and then the resultant solution was further treated with the dialysis bag (50000 Da) to remove large nonfluorescent materials. The acquired solution was concentrated and dried to obtain carbon quantum dots solid. Thiol-functionalized carbon quantum dots were synthesized as follows: carbon quantum dots (0.10 g), cysteamine (0.10 g), EDC (0.10 g) and NHS (0.05 g) were added into 50-mL triple-distilled water. The mixture was stirred and heated at 90 °C for 5 days. The final product solution was dialyzed in a dialysis bag (1000 Da) for 1 day. The inside solution of the dialysis was collected for further use. Redox-Controlled Fluorescent Nanoswitch Based on Thiol-CQDs. Thiol-functionalized carbon quantum dots in aqueous solution are readily turned to thiol-CQDs covalent bonding assemblies in the presence of H2O2 and KI in a typical procedure as follows: a fixed amount of thiol-CQDs solution (2.0 mL, 0.17 mg/mL) was mixed with 60.0 µL of H2O2 (10.0 mM) and KI (0.01 g), and resulting mixture was incubated for 15 min. Because of the formation of thiol-CQD assembly, the yellowish green fluorescence of the solution was gradually reduced during the reaction period under a UV lamp. The luminescence of the resulting mixture solution is easily recovered by a reduction reaction with some specific reductants like Na2S2O4, GSH, DTT and PA. A fixed amount of Na2S2O4 (20.0 µL, 10.0 mM) was add into thiol-CQD assembly solution (2.0 mL, 0.17 mg/mL), and then the mixture was incubated for several seconds. It is observed that the yellowish green luminescence is progressively recovered as the reaction proceeds. The PL spectra were continuously monitored and recorded. Five cycles’ operation were conducted to test the reversibility of this redox-controlled luminescence switch. Fluorescence Turn-on Assay of BChE Activity. Thiol-CQD assembly was first prepared by reacting a certain amount of thiol-CQDs in HEPES solution (2.0 mL, 0.17 mg/mL, pH 7.4) with H2O2 (20.0 µL, 10.0 mM) in the presence of KI (0.01 g). The saturating substrate concentration



Page 8 of 26

was then explored using the assay solution containing thiol-CQD assembly (2.0 mL, 0.17 mg/mL), BChE (100.0 U/L) and different amounts of BTCh in the range of 0.05 – 0.25 mM. The PL intensity of the mixed solutions was recorded as a function of incubation time ranging from 0 – 16 min. Time-course enzymatic reaction kinetics of BChE assay were acquired using a series of assay solutions containing thiol-CQD assembly (2.0 mL, 0.17 mg/mL), BTCh (1.0 mM) and varied levels of BChE (0.0 – 260.0 U/L) as a function of incubation time (0 – 16 min). The PL intensity versus incubation time at each BChE activity was recorded. The influence of GSH on BChE assay was tested as follows: time-course enzymatic reaction kinetics of BChE assay were performed according to the preceding procedure except that a fixed amount of GSH (1.0, 2.0 or 10.0 µM) was added prior to the addition of BTCh (1.0 mM). Discriminative assay of BChE from AChE was conducted as follows: time-course enzymatic reaction kinetics of the assay solution containing a certain level of different enzymes (BChE and AChE) and different substrates (BTCh and ATCh) were recorded and compared. The temperature was maintained at 37 °C throughout the experiments. The measurement of BChE level in fresh human serum samples was carried out as follows: three different volumes (10.0, 20.0, and 30.0 μL) of fresh human serum from a healthy male were separately added into a 2.0mL assay solution containing thiol-CQD assembly (0.17 g/L) and BTCh (0.5 mM), and then the resulting solutions were monitoring by fluorescence spectrophotometer as a function of time within a time course of 16 min at 37 °C, respectively. The BChE activity for each sample was calculated using the reaction rate based on the established calibration curve. Three serum samples from three healthy males were used. Inhibitor Screening Assay. To test the function of inhibitor screening of the BChE assay, tacrine and parathion-methyl were chosen as the examples. The inhibition of tacrine on BChE


Page 9 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


activity was assessed according to the following procedure. A series of different amounts of tacrine (1.0 – 6.0 µM) were mixed first with 20.0 µL of BChE solution (10.0 U/mL), and the resulting mixture was added into the assay solution containing thiol-CQD assembly (2.0 mL, 0.17 mg/mL) and BTCh (1.0 mM). Time-course enzymatic reaction kinetics of each mixed solution was recorded in the range of 0 – 16 min. The inhibition of parathion-methyl on BChE activity was conducted according to the preceding procedure by replacing tacrine with parathionmethyl. The concentration of parathion-methyl used in the assay was 0.025, 0.050, 0.075 and 0.100 µM, respectively. Characterization Methods. Transmission electron microscopy (TEM) was conducted on a JEOL-Model 2100F instrument with an accelerating voltage of 200 kV. A Kratos Axis ULTRA X-ray photoelectron spectroscopy was used for the X-ray photoelectron spectroscopy analyses. The UV-vis spectra were recorded on a Perkin-Elmer Lambda 950 spectrometer. The luminescence

spectra

were

performed

using

SHIMADZU

RF-6000

fluorescence

spectrophotometer.

Figure 1. (A) TEM image of thiol-CQDs. (B) XPS wide spectrum of thiol-CQDs.



Page 10 of 26

RESULTS AND DISCUSSION Synthesis and Characterization of Thiol-Functionalized Carbon Quantum Dots (ThiolCQDs). Carbon quantum dot (CQD) was chosen as the fluorophore in the nanoprobe since it possesses excellent aqueous solution, good biocompatibility and outstanding fluorescence performance.31-33 CQDs used in this work were prepared from chemical oxidation of activated carbon according to our previous papers.29,30 The synthesis protocol for the CQDs shows good reproducibility and can be used for large-scale preparation of CQDs. These CQDs are soluble and stable in aqueous solution, and have an intense yellowish green fluorescence. To introduce thiol functionals into the surface of CQDs, cysteamine was adopted to covalently bind to carboxylic acid groups of CQDs via amide linkers using EDC/NHS coupling reaction. Excess cysteamine was used in the modification of CQDs to guarantee the abundance of thiol groups in the thiol-functionalized CQDs (thiol-CQDs). As-prepared thiol-CQDs were characterized by TEM, XPS and fluorescence spectroscopy, respectively. Figure 1A presents high-resolution TEM image of thiol-CQDs, and it is indicated that these CQDs possess crystalline structures and fall in the size range of 2 – 5 nm. The TEM image also shows that these CQDs exist in welldispersed state for their excellent water solubility. In comparison with the morphology of CQDs reported in the previous paper,29,30 there is no much difference between CQDs and thiol-CQDs in structure and in size distribution. However, apparent difference in XPS spectra between them was observed as shown in Figure S1 and Figure 1B. Only two predominant signals were recorded for CQDs, and they originate from C1s (284.3 eV) and O1s (531.3 eV), respectively. XPS spectrum for thiol-CQDs in Figure 1B displays three additional signals from S2s (227.3 eV), S2p (163.2 eV) and N1s (339.6 eV) compared to that of CQDs, indicating that cysteamine has been successfully incorporated into CQDs.


Page 11 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. (A) Fluorescence excitation and emission spectra of CQDs (0.17 mg/mL) and thiol-CQDs (0.17 mg/mL) in water. Inset: image of thiol-CQDs solution under a UV lamp. (B) Fluorescence response of thiolCQDs to different compositions: (a) thiol-CQDs (0.17 mg/mL), (b) thiol-CQDs (0.17 mg/mL) and H2O2 (300.0 μM), (c) thiol-CQDs (0.17 mg/mL), H2O2 (300.0 μM) and GSH (300.0 μM), (d) thiol-CQDs (0.17 mg/mL), H2O2 (300.0 μM) and PA (300.0 μM), (e) thiol-CQDs (0.17 mg/mL), H2O2 (300.0 μM) and DTT (300.0 μM), (f) thiol-CQDs (0.17 mg/mL), H2O2 (300.0 μM) and Na2S2O4 (100.0 μM). Insets: corresponding fluorescence images under UV light.

Figure 2A presents fluorescence spectra of CQDs and thiol-CQDs, and one can notice that both of them fluoresce in the range of yellowish green light. However, the emission maximum of thiol-CQDs is located at 523 nm, which is blue-shifted by 7 nm relative to that of CQDs, and their optimal excitation peaks are also different by about 20 nm. Thiol-CQD solution shows a brighter fluorescence than CQD aqueous solution under the irradiation of UV light because its quantum yield (7.2%) is much higher than that of CQDs (2.2%)29,30 using quinine sulfate as the standard reference (0.55 in 1M H2SO4). The lifetime of thiol-CQDs was determined to be 1.9 ns in terms of its time-resolved decay curve in Figure S2, and this shorter lifetime than that of CQDs (6.2 ns)29,30 might contribute to higher fluorescence quantum yield of thiol-CQDs. The influence of various pHs ranging from pH 1.0 to pH 11.0 on the fluorescence of thiol-CQDs was



Page 12 of 26

Figure 3. (A) The change of PL spectra of thiol-CQDs solution (0.17 mg/mL) with the continuous addition of H2O2 from 0.0 – 300.0 μM. (B) The change of PL spectra of thiol-CQDs assembly (0.17 mg/mL) in the presence of different amounts of GSH from 0.0 – 125.0 μM. (C) TEM image of thiol-CQDs assemblies from oxidation of thiol-CQDs by H2O2. (D) Reversibility of fluorescent nanoswitch controlled by H2O2 (300.0 μM) and Na2S2O4 (100.0 μM).

also examined, and Figure S3 illustrates that no appreciable change in fluorescence intensity is observed in the pH range of 1.0 – 9.0 and only very slight increase is recorded for pHs over 10.0, indicating that thiol-CQD as the fluorophore has excellent tolerance to various pHs, and is suited for different detection environments relative to CQDs. More importantly, it is discovered that the fluorescence of thiol-CQDs is entirely quenched in the presence of a small amount of H2O2, and


Page 13 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


is almost completely recovered by separately introducing glutathione (GSH), penicillamine (PA), sodium dithionite (Na2S2O4), dithiothreitol (DTT) into the preceding solution as shown in Figure 2B. After quenching the fluorescence by H2O2, these three thiol-containing compounds are able to restore the fluorescence to 90% of its original intensity, and the introduction of the same amount of sodium dithionite leads to almost a full recovery. It is speculated that the reversible response of thiol-CQDs to redox species is attributed to reversible bond making and breaking of disulfide controlled by redox reactions. This hypothesis is partly supported by the abundance of free thiols in thiol-CQDs and the lack of free thiols in oxidized form of thiol-CQDs. Ellman reagent 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) was adopted to prove whether the free thiols are present or not in thiol-CQD and in its oxidized form because DTNB is able to rapidly react with free thiols to generate a color product with a significant absorption at 410 nm. The change in UV-vis spectra in Figure S4A indicates that thiol-CQD is abundant in free thiol whereas its oxidized form possess no free thiol groups, and the thiol content in thiol-CQDs is determined to be 1.36 mM per gram of thiol-CQDs according to the calibration curve between absorbance of the product and GSH concentration in Figure S4B. Redox-Controlled Fluorescent Nanoswitch Based on Thiol-CQDs. This unique fluorescence response to specific redox species enables thiol-CQDs to construct a redoxcontrolled fluorescent nanoswitch. The fluorescence quenching trend as the amount of added H2O2 is shown in Figure 3A, and the fluorescence of thiol-CQDs is almost entirely quenched after adding 300.0 μM H2O2 in the presence of trace amount of KI as the catalyst. We have proven that CQDs from chemical oxidation of activated carbon have similar aggregation-induced quenching (ACQ) property to traditional molecular dyes in our previous work.29,30 Recent advances on the formation of disulfide bonds also verify that disulfide can be effectively formed



Page 14 of 26

by H2O2.33 These observations lend us the reasons to deduce that the efficient quenching of the fluorescence of thiol-CQDs is originated to bonding-induced quenching induced by the formation of a great deal of disulfide bonds among thiol-CQDs under the oxidation of H2O2. This speculation was confirmed by TEM image of oxidized form of thiol-CQDs by H2O2 in Figure 3B. One can note that a multitude of large spheres appeared accompanying the small carbon quantum dots in several nanometers, and the sizes of most large spheres are over ten nanometers in diameter, suggesting that they are composed of several carbon quantum dots as disulfide bonding-induced assemblies. As a result, the sharp fluorescence quenching by H2O2 is ascribed to oxidation-triggered bonding-induced quenching of thiol-CQDs. The fluorescence recovery by reductants was further evaluated using GSH as the example in Figure 3C, which displays the gradual fluorescence enhancement as the rise of GSH in amount and 125.0 μM GSH is required to attain the highest brightness. It is easily deduced that this fluorescence enhancement is due to effective breaking of disulfide bonds among CQDs in the nonfluorescent assemblies by GSH via a disulfide exchange reaction according to the references.15,16 This disaggregation-induced emission phenomenon was further confirmed by TEM technique as shown in Figure S5, which proves that large assemblies linked by disulfide are turned back to small carbon quantum dots after the addition of GSH. The sharp fluorescence change induced by redox reaction was also supported by apparent alternation in UV-visible spectra of thiol-CQDs with continuous addition of H2O2 and GSH in Figure S6. No appreciable absorption in the range of over 250 nm for thiolCQDs solution is observed, but two dominant absorption peaks at 287 nm and 352 nm is recorded after the addition of H2O2, and the bright yellow color is readily observed by naked eyes, which supports the formation of the new species from initial thiol-CQDs via an H2O2triggered oxidation reaction. The introduction of the same amount of GSH to thiol-CQD


Page 15 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. (A) Change of fluorescence spectra of thiol-CQDs solution in the presence of different constituents: (a) thiol-CQDs (0.17 mg/mL), (b) thiol-CQDs (0.17 mg/mL) and H2O2 (100.0 µM), (c) thiol-CQDs (0.17 mg/mL), H2O2 (100.0 mM) and BTCh (1.0 mM), (d) thiol-CQDs (0.17 mg/mL), H2O2 (100.0 mM), BTCh (1.0 mM) and BChE (300.0 U/L). (B) PL intensity of BChE assay solution (thiol-CQD assembly 0.17 mg/mL, pH 7.4 HEPES buffer, 100 U/L BChE) versus substrate (BTCh) concentration in the range of 0.05 – 0.25 mM during the incubation time of 0 – 16 min. (C) PL intensity of BChE assay solution versus BChE activity in the range of 20.0 – 260.0 U/L. The assay solution contains thiol-CQD assembly (0.17 mg/mL) and BTCh (1.0 mM) in HEPES buffer solution (pH 7.4). (D) Enzymatic rates K plotted against BChE activity. Inset: calibration curve between rate constant and BChE activity in the scope of 60.0 – 220.0 U/L.



Page 16 of 26

Figure 5. (A) Time-course enzymatic reaction kinetics of BChE assay in the presence of different concentrations of GSH: (a) 0.0 μM GSH, (b) 1.0 μM GSH, (c) 2.0 μM GSH, (d) 10.0 μM GSH. (B) Timecourse enzymatic reaction kinetics of BChE and AChE in the presence of different substrates BTCh or ATCh: (a) BTCh (1.0 mM) and AChE (100.0 U/L), (b) BTCh (1.0 mM) and BChE (100.0 U/L), (c) ATCh (1.0 mM) and AChE (100.0 U/L), (d) ATCh (1.0 mM) and BChE (100.0 U/L).

assembly solution results in restoration of UV-visible absorption to the original state of thiolCQDs, clearly indicating that disulfide-linked assemblies are converted to thiol-CQDs through an exchange reaction. As a result, the fluorescence on-off-on process corresponds to two different species: highly fluorescent thiol-CQDs and nonfluorescent disulfide-linked assemblies of thiol-CQDs. The conversion of the fluorescence between on and off state is controlled by two distinct redox reactions, and thus the redox-controlled fluorescence nanosiwtch can be constructed in this way. It is found that Na2S2O4 can almost fully recover the quenched fluorescence by H2O2, and consequently the reversibility of the nanoswitch mediated by H2O2 and Na2S2O4 was assessed as shown in Figure 3D. The entirely fluorescence quenching by H2O2 and fully fluorescence recovery by Na2S2O4 with over five cycles demonstrates the excellent reversibility of the fluorescent nanoswitch. In comparison with the other nanoswitches mediated


Page 17 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


by coordination reaction29,30,35 or dynamic covalent reaction,36 redox-controlled nanoswitches show better reversible response to the external stimuli. Fluorescence Turn-on Detection of Butyrylcholinesterase Based on DisaggregationInduced Emission by Cleavage of Disulfide. The specific fluorescence response of thiol-CQD assembly linked by sulfide bonds to thiol-containing compounds provides the possibility of assaying BChE activity as shown in Scheme 1. When BTCh was used as the substrate, it can be rapidly hydrolyzed into butyrate and thiocholine, and then generated thiocholine reacts with thiol-CQD assembly to produce highly fluorescence thiol-CQDs. This design was first tested as displayed in Figure 4A. The transformation of thiol-CQDs to thiol-CQD assembly by the addition of H2O2 leads to severe fluorescence quenching, and the introduction of single BTCh causes almost no change in fluorescence intensity. However, the presence of a small level of BChE results in a sharp fluorescence recovery, and the enhanced fluorescence closely approaches the original intensity. This observation verifies the feasibility of the detection route for BChE activity. According to Michaelis-Menten kinetic theory,37 the initial rate K of formation of the product or depletion of substrate generally follows saturation kinetics with respect to the concentration of substrate [S] during the steady state. At saturating substrate concentration, K tends to a limiting value termed Kmax. As a consequence, saturating substrate concentration for a certain BChE activity was explored to ascertain stable reaction rates at different BChE levels. Figure 4B shows time-course enzymatic reaction kinetics at different substrate concentrations in the range of 0.05 – 0.25 mM. It is readily noted from Figure S7 that the initial rates apparent increase as the concentration of BTCh from 0.05 – 0.20 mM, and no appreciable change is recorded when the amount of substrate is increased to 0.25 mM, indicating that this amount has reached saturation concentration at which the initial rate K approaches the



Page 18 of 26

limiting value Kmax. Thus a larger substrate concentration of 1.0 mM than the saturation concentration was used for the following detection with different BChE levels. Figure 4C illustrates a gradual rise of initial rates during a time course of 14 min at different BChE levels from 20.0 – 260 U/L. It is noted that the enzymatic reaction enters the steady state from 5 – 10 min because initial rates remains constant during this time course for all BChE levels. As a result, the initial rate K was calculated in terms of reaction rates of this time course. The plot of enzymatic reaction rates against BChE levels is shown in Figure 4D, and the reaction rate is found to have a good linear relationship with BChE levels in the range of 60.0 – 220.0 U/L. The detection limit estimated from three standard deviations according to this calibration curve was determined to be 2.7 U/L. The existing optical methods for BChE activity was summarized in Table S1 for comparison, and it is noted that the lower detection limit of our assay is comparable to those of fluorometric assay based on quantum dots (10 U/L)14 and a potentiometric assay (7.5 U/L),4 and is much lower than those of most optical methods which varied from 10 to 1000 U/L.9,12,14 Since it is reported that normal concentration of GSH in healthy human plasma is 2.09 ±1.15 μM,38 micromolar level of GSH in plasma may exert an significant impact on the BChE assay due to non-specific response of thiol-CQDs assembly to thiol-containing species. In order to avoid the interference from GSH in the samples, the assay protocol is slightly changed as follows: the sample containing GSH and BChE is first added into the assay solution without BTCh, and then BTCh is introduced after the reaction between GSH and the nanoprobe is completed. The influence of such amounts of GSH as in the plasma on BChE assay in this way was evaluated as shown in Figure 5A. Three GSH amounts (1.0, 2.0 and 10.0 μM) were chosen to assess this possible interference. It is found that the presence of these amounts of GSH results in the


Page 19 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6. (A) Time-course enzymatic reaction kinetics of BChE assay in the presence of different concentrations of tacrine ranging from 0.0 to 6.0 μM. (B) Time-course enzymatic reaction kinetics of BChE in the presence of different amounts of parathion-methyl in the range of 0.000 – 0.100 μM. Table 1. Measurement of BChE level in three distinct human serum samples. Sample Number 1

Measured BChE level in serum (U/L) using established method 8639.5±15.1

Measured BChE level in serum using Ellman method 8957.3±36.3

2

9053.6±16.5

8964.3±38.0

3

8029.6±13.4

8560.6±32.3

Average

8574.2±8.7

8827.4±20.6

Reported BChE level in normal population (U/L)

9010.0±2041

increase of the fluorescence intensity during all entire time-course relative to that without GSH because existing GSH reacts with thiol-CQDs assembly to produce fluorescent thiol-CQDs. However, the existence of these different amounts of GSH does not change the enzymatic reaction rates with respect to the original one and thus exerts no impact on detection of BChE activity. It consequently is concluded that the presence of a small amounts of GSH in the sample does not interfere with the assay of BChE activity as long as the sample is added prior to the substrate. Another interference for BChE assay in plasma may come from a small amount of



Page 20 of 26

AChE, and thereby possible interference from AChE was also assessed as shown in Figure 5B. This assay has no response to AChE when BTCh is used as the substrate, but the same units of BChE shows excellent catalysis activity to BTCh from its large reaction rate. However, when the substrate BTCh is replaced by ATCh, this assay is also capable of quantifying AChE activity although its reaction rate is much lower than BChE at the same condition. This observation supports the conclusion that AChE has specific catalytic activity while BChE is non-specific to the substrates.3 It is deduced that this assay is able to discriminatively assay BChE activity in real samples as long as BTCh is used as the substrate. To further validate the detection method in this work, this BChE assay was applied in the measurement of BChE activity in fresh human serum samples. Three serum samples from three healthy people were used determine the average BChE level in real sample based on the established calibration curve between reaction rate and BChE activity in Figure 4D. BChE levels of the three serum samples were also measured using traditional Ellman method for comparison. Table 1 lists the measured values of BChE level in three human serum samples based on our method and Ellman method, and the reported normal BChE level in large population. It is found that the average value of measured BChE level (8574.2±8.7 U/L) from our method is very close to that (8827.2±20.6 U/L) obtained based on Ellman method, and both of them fall in the normal range of healthy people (9010.0±2041),39 and this good agreement is convincing that our assay is capable of accurately measuring BChE level in real human serum. Inhibition Assay of BChE. This assay is further extended to evaluate the inhibition of BChE using tacrine and parathion-methyl as the examples to demonstrate the potential application of proposed BChE assay in inhibitor screening. Figure 6A demonstrates the change of PL intensity of the assay as incubation time in the presence of different concentrations of tacrine, and it is


Page 21 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


found that the reaction rate is gradually decreased as increasing the concentration of tacrine, clearly indicating the inhibition effect of tacrine on BChE. Previous study13 has shown that tacrine inhibits BChE in a competitive way, and thus the reciprocal of reaction rate is linearly related with the inhibitor concentration. Our case is consistent with this linear relationship for a competitive inhibition mechanism. Parathion-methyl has also shown an effective inhibition to BChE when its concentration is up to 0.100 μM as displayed in Figure 6B. However, its inhibition mechanism is different from that of tacrine according to the fact that the reaction rate is linearly correlated to parathion-methyl concentration. Its non-competitive inhibition mechanism induced by irreversible binding to BChE is consistent with the finding revealed by the other reports.40

CONCLUSION In summary, we report a unique redox-controlled fluorescent nanoswitch based on reversible disulfide

bonds,

and

develop

a

novel

fluorescence

turn-on

assay

strategy

for

butyrylcholinesterase activity in terms of the specific response of thiol-CQDs assembly to thiols. Thiol-CQDs with intense yellowish green emission can be transformed to nonfluorescent disulfide-linked assembly under the oxidation of H2O2, and the resulting thiol-CQDs assembly can be turned back to thiol-CQDs by thiols. It is the first time to design a reversible redoxcontrolled nanoswitch based on the formation and breaking of disulfide bonds mediated by external stimuli. This specific thiol-triggered disaggregation-induced emission enables us to develop a fluorometric assay of butyrylcholinesterase using butyrylthiocholine iodide as the substrate. The BChE assay is sufficiently sensitive to accurately determine BChE level in human serum, and is able to assess the inhibition of potential inhibitor of BChE. Compared to the previous luminescent detection methods of BChE, as-established assay in this work achieves



Page 22 of 26

discriminative detection of BChE from AChE, and is capable of quantifying AChE activity by changing the substrate. Its desired signal turn-on response to BChE and good tolerance to micromolar glutathione open a new pathway to monitor and visualize BChE in live cells in reliable and real-time manner.

ASSOCIATED CONTENT The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.xxxxxxx. Details of UV-visible, XPS, PL spectra of used materials and data for BChE assay.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Tel: +86-579-82282269. Fax: +86-579-82282269. †

These authors contributed to this work equally.

ACKNOWLEDGMENT We gratefully acknowledge financial support from the National Natural Science Foundation of China (Grant Nos. 21675143, 21775139 and 21775138) and Natural Science Foundation of Zhejiang Province (Grant No. LR18B050001 and LY17B050003).

REFERENCES (1) Lockridge, O. Pharmacol. Therapeut., 2015, 148, 34−46. (2) Pohanka, M. Bratisl. Med. J., 2013, 114, 726−734. (3) Miao, Y. Q.; He, N. Y.; Zhu, J. J. Chem. Rev., 2010, 110, 5216−5234.


Page 23 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(4) Ding, J. W.; Qin, W. Chem. Commun., 2009, 971−973. (5) Ding, J. W.; Qin, W. Electroanal., 2009, 21, 2030−2035. (6) Khaled, E.; Hassan, H. N. A.; Mohamed, G. G.; Ragab, F. A.; Seleim, A. E. Talanta, 2010, 83, 357−363. (7) Pohanka, M. Talanta, 2014, 119, 412−416. (8) Jonca, J.; Zuk, M.; Wasag, B.; Janaszak-Jasiecka, A.; Lewandowski, K.; Wielgomas, B.; Waleron, K.; Jasiecki, J. PLoS ONE, 2015, 10, e0139480. (9) Pohanka, M.; Drtinova, L. Talanta, 2013, 106, 281−285. (10) Lu, L. L.; Xia, Y. S. Anal. Chem., 2015, 87, 8584−8591. (11) Guilbault, G. G.; Kramer, D. N. Anal. Chem., 1965, 37, 120−123. (12) Kang, S.; Lee, S.; Yang, W.; Seo, J.; Han, M. S. Org. Biomol. Chem., 2016, 14, 8814−8820. (13) Yang, S. H.; Sun, Q.; Xiong, H.; Liu, S. Y.; Moosavi, B.; Yang, W. C.; Yang, G. F. Chem. Commun., 2017, 53, 3952−3955. (14) Chen, Z. Z.; Ren, X. L.; Meng, X. W.; Tan, L. F.; Chen, D.; Tang, F. Q. Biosens. Bioelectron., 2013, 44, 204−209. (15) Wong, P. T.; Choi, S. K. Chem. Rev., 2015, 115, 3388−3432. (16) Lee, M. H.; Yang, Z. G.; Lim, C. W.; Lee, Y. H.; Sun, D. B.; Kang, C.; Kim, J. S. Chem. Rev., 2013, 113, 5071−5109.



Page 24 of 26

(17) Pires, M. M.; Chmielewski, J. Org. Lett., 2008, 10, 837−840. (18) Pullela, P. K.; Chiku, T.; Carvan III, M. J.; Sem. D. S. Anal. Biochem., 2006, 352, 265−273. (19) Cao, X. W.; Lin, W. Y.; Yu, Q. X. J. Org. Chem., 2011, 76, 7423−7430. (20) Guo, Y. S.; Wang, H.; Sun, Y. S.; Qu, B. Chem. Commun., 2012, 48, 3221−3223. (21) Zhu, B. C.; Zhang, X. L.; Liu, Y. M.; Wang, P. F.; Zhang, H. Y.; Zhuang, X. Q. Chem. Commun., 2010, 46, 5710−5712. (22) Reddie, K. G.; Humphries, W. H.; Bain, C. P.; Payne, C. K.; Kemp, M. L.; Murthy, N. Org. Lett., 2012, 14, 680−683. (23) Lim, C. S.; Masanta, G.; Kim, H. J.; Han, J. H.; Kim, H. M.; Cho, B. R. J. Am. Chem. Soc., 2011, 133, 11132−11135. (24) Lee, J. H.; Lim, C. S.; Tian, Y. S.; Han, J. H.; Cho, B. R. J. Am. Chem. Soc., 2010, 132, 1216−1217. (25) Yin, B. C.; You, M. X.; Tan, W. H.; Ye, B. C. Chem. Eur. J., 2012, 18, 1286−1289. (26) Pujol, A. M.; Cuillel, M.; Renaudet, O.; Charbonnier, P.; Cassio, D.; Gateau, C.; Dumy, P.; Mintz, E.; Delangle, P. J. Am. Chem. Soc., 2011, 133, 286−296. (27) Kong, F. P.; Zhao, Y. H.; Liang, Z. Y.; Liu, X. J.; Pan, X. H.; Luan, D. R.; Xu, K. H.; Tang, B. Anal. Chem., 2017, 89, 688−693.


Page 25 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(28) Lou, X. D.; Hong, Y. N.; Chen, S. J.; Leung, C. W. T.; Zhao, N.; Situ, B.; Yip, J. W.; Tang, B. Z. Sci. Rep., 2014, 4, 4272. (29) Qian, Z. S.; Chai, L. J.; Tang, C.; Huang, Y. Y.; Chen, J. R.; Feng, H. Anal. Chem., 2015, 87, 2966−2973. (30) Qian, Z. S.; Chai, L. J.; Zhou, Q.; Huang, Y. Y.; Tang, C.; Chen, J. R.; Feng, H. Anal. Chem., 2015, 87, 7332−7339. (31) Lim, S. Y.; Shen, W.; Gao, Z. Q. Chem. Soc. Rev., 2015, 44, 362−381. (32) Hola, K. Zhang, Y.; Wang, Y.; Giannelis, E. P.; Zboril, R.; Rogach, A. L. Nano Today, 2014, 9, 590−603. (33) Zheng, X. T.; Ananthanarayanan, A.; Luo, K. Q.; Chen, P. Small, 2015, 11, 1620−1636. (34) Mandal, B.; Basu, B. RSC Adv., 2014, 4, 13854−13881. (35) Chai, L. J.; Zhou, J.; Feng, H.; Lin, J. J.; Qian, Z. S. Sens. Actuat. B, 2015, 220, 138−145. (36) Ao, H.; Feng, H.; Huang, X. L.; Zhao, M. Z.; Qian, Z. S. J. Mater. Chem. C, 2017, 5, 2826−2832. (37) Schnell, S.; Chappell, M. J.; Evans, N. D.; Roussel, M. R. C. R. Biol., 2006, 329, 51−61. (38) Jones, D. P.; Carlson, J. L.; Samiec, P. S.; Sternberg, P.; Mody, V. C.; Reed, R. L.; Brown, L. A. S. Clin. Chim. Acta, 1998, 275, 175−184. (39) Dimov, D.; Kanev, K.; Dimova, I. Acta Biochim. Pol., 2012, 59, 313−316.



Page 26 of 26

(40) Carter, M. D.; Crow, B. S.; Pantazides, B. G.; Watson, C. M.; Thomas, J. D.; Blake, T. A.; Johnson, R. C. Anal. Chem., 2013, 85, 11106−11111.

For TOC


Redox-Controlled Fluorescent Nanoswitch Based on Reversible

Recommend Documents