Subscriber access provided by University of Florida | Smathers Libraries
Article
Gold Nanoparticle-Based Photoluminescent Nanoswitch Controlled by HostGuest Recognition and Enzymatic Hydrolysis for Arginase Activity Assay Hao-Hua Deng, Xiao-Qiong Shi, Hua-Ping Peng, Quanquan Zhuang, Yu Yang, Ai-Lin Liu, Xing-Hua Xia, and Wei Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b19513 • Publication Date (Web): 26 Jan 2018 Downloaded from http://pubs.acs.org on January 26, 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.
ACS Applied Materials & Interfaces 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 7 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
ACS Applied Materials & Interfaces
Gold Nanoparticle-Based Photoluminescent Nanoswitch Controlled by Host-Guest Recognition and Enzymatic Hydrolysis for Arginase Activity Assay Hao-Hua Deng,a‡ Xiao-Qiong Shi,a‡ Hua-Ping Peng,a Quan-Quan Zhuang,a Yu Yang,a Ai-Lin Liu,a Xing-Hua Xia,b Wei Chen*a a
Higher Educational Key Laboratory for Nano Biomedical Technology of Fujian Province, Department of Pharmaceutical Analysis, Fujian Medical University, Fuzhou 350004, China b
State Key Laboratory of Analytical Chemistry for Life Science and Collaborative Innovation Center of Chemistry for Life Sciences, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China
ABSTRACT: The development of simple yet powerful methods for monitoring enzyme activity is of great significance. Herein, a facile, convenient, cost-effective, and continuous fluorescent method for the detection of arginase and its inhibitor has been reported based on a host-guest interaction- and enzymatic hydrolysis-controlled luminescent nanoswitch. The fluorescence intensity of 6-aza-2-thiothymine-stabilized gold nanoparticle (ATT-AuNP) is enhanced by L-arginine, owing to the formation of supramolecular host-guest assembly between the guanidine group of L-arginine and ATT molecules capped on AuNP surface. However, hydrolysis of L-arginine, catalyzed by arginase, leads to a decrease of fluorescence intensity of L-arginine/ATT-AuNPs hybrids. Upon incorporation of the arginase inhibitor L-norvaline, the fluorescence of the ATT-AuNP-based detecting system is restored. The linear range of arginase activity determination is from 0.0625 to 1.15 U/mL and the limit of detection is 0.056 U/mL. The half-maximal inhibition value IC50 of L-norvaline is determined to be 5.6 mM. The practicability of this luminescent nanoswitch is validated by assaying arginase activity in rat liver and monitoring the response of rat liver arginase to pharmacological agent. Compared to the existing fluorescent method of arginase activity assay, the approach demonstrated here does not involve any complicated technical manipulation, thereby greatly simplifying the detection steps. We propose that this AuNP-based luminescent nanoswitch would find wide applications in the field of life sciences and medicine. KEYWORDS: gold nanoparticle, host-guest recognition, photoluminescence, arginase, L-arginine
■INTRODUCTION Arginase (L-arginine amidinohydrolase, EC 3.5.3.1) is a ureohydrolase enzyme, containing an unusual binuclear manganese(II) center as the active site.1 It is the terminal enzyme of the Krebs-Henseleit urea cycle that metabolizes L-arginine to yield L-ornithine and urea. This enzyme is very widespread in nature, ranging from bacteria, yeasts, plants, invertebrates, and vertebrates.2 It serves to regulate L-arginine homeostasis in three key metabolic pathways: (1) regulation of L-arginine level for the biosynthesis of nitric oxide; (2) regulation of Lornithine level for the biosynthesis of L-proline to facilitate collagen formation; (3) regulation of L-ornithine level for the biosynthesis of polyamine to promote cell growth and repair.3 Experimental data provide overwhelming evidence supporting the close association of abnormal arginase activity with diseases such as hypertension, atherosclerosis, multiple sclerosis, asthma, wound healing, and hyperargininemia.4 Therefore, design and development of simple and convenient methods for estimating activities of arginase and its inhibitor is of utmost significance for point-of-care diagnostics, drug discovery, and biomedical research. So far, a number of analytical techniques, including colorimetry,5-7 fluorescence,8 high performance liquid chromatography,9 radiochemistry,10 and electrochemistry11 have been
proposed for assaying arginase activity based on the determination of residual arginine or one of the products after arginine hydrolysis. Among those, fluorescence detection is the most attractive because of its high sensitivity, specificity, simplicity, rapidity, and high-throughput capability. However, the existing fluorescence-based method relies on arginase-catalyzed hydrolysis of L-arginine to generate urea, urease-catalyzed conversion of urea to ammonia, and reduced nicotinamide adenine dinucleotide-coupled reaction,8 which is expensive, inconvenient, and involves rather complex procedures. As a result, a facile, cost-effective, and continuous approach for the detection of arginase activity is considered preferable. In recent years, fluorescent few-atom metal nanoparticles (MNPs) have received considerable attention in the detection of chemical and biological analytes and in cell imaging, mainly owing to their distinct advantages in terms of easy preparation and modification, high biocompatibility and stability, low toxicity, ultrasmall size, and large Stokes shifts.12-22 Till now, MNP-based sensing probes have been used for detecting enzymes (e.g. deoxyribonuclease,23 alkaline phosphatase,24,25 inorganic pyrophosphatase,26-28 phospholipase C,29 acetylcholinesterase,30,31 trypsin,32 protein kinase,33,34 urease,35 and posttranslational modification enzymes36) by various mechanisms such as enzyme-triggered hydrolysis of metal-substrate complex, enzyme-triggered reduction, enzyme-triggered decompo-
ACS Paragon Plus Environment
ACS Applied Materials & Interfaces 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
sition or modification of the protector, enzymatic reactioninduced aggregation, enzymatic reaction-induced fluorescence resonance energy transfer, etc. Although burgeoning progress has been made in this aspect, there is no report yet on the construction of a detection system for the arginase-catalyzed hydrolysis reaction using fluorescent MNPs as the signaling subunits. The underlying reason could be that arginase has strict specificity for its natural substrate arginine but most of the existing MNPs cannot selectively and sensitively recognize arginine or its hydrolysates.37 In our previous study, we demonstrated a novel method to enhance the luminescence of gold nanoparticles (AuNPs) by rigidifying their ligand shell via host-guest recognition.38 It was found that 6-aza-2-thiothymine-stabilized AuNPs (ATTAuNPs) exhibit weak photoluminescent emission; however, their photoluminescence intensity could be strikingly improved by introducing L-arginine into the capping layer. The restriction of intramolecular vibration and rotation of ligands capped on the gold surface is responsible for the enhanced emission efficiency, which has also been considered as an important mechanism for aggregation-induced emission of thiolate-protected MNPs.39-41 The L-arginine-induced enhancement of ATT-AuNP luminescence has a strong correlation with its chemical structure, which is why amino acids lacking a guanidine group cannot trigger the enhanced luminescence. Moreover, the resulting L-arginine/ATT-AuNPs hybrids exhibit good water-solubility and long-term stability. These outstanding advantages motivated us to further investigate the potential applications of such AuNPs. In this study, we introduced a simple procedure for the detection of arginase activity and its inhibitor based on the fluorometric determination of residual arginine after its hydrolysis by arginase, using ATT-AuNP as a fluorescent nanoswitch. The feasibility of the newly developed method for monitoring arginase activity in rat liver has also been demonstrated. Moreover, the response of rat liver arginase to the administered pharmacological agent triptolide was studied by applying this AuNP-based nanoswitch. The sensing protocol described here is sure to open up a new path for arginase activity assay and arginaserelated drug screening.
■EXPERIMENTAL SECTION
50 mM glycine-NaOH buffer, pH 9.8). The fluorescence spectra were recorded, after a 2-h incubation at 37 °C, employing a Cary Eclipse Fluorescence Spectrophotometer with an excitation wavelength of 472 nm. For assay of arginase inhibitor, 150 µL of 2.5 mM Larginine (pH 9.8) was mixed with 150 µL of 2 mg/mL ATTAuNPs (pH 9.8), 100 µL of 4 U/mL arginase (in 50 mM glycine-NaOH buffer, pH 9.8), and different concentrations of Lnorvaline. The fluorescence spectra were recorded, after a 2-h incubation at 37 °C, using a Cary Eclipse Fluorescence Spectrophotometer with an excitation wavelength of 472 nm. Arginase Activity Assay in Rat Liver. To evaluate the practicability of the proposed method, arginase activity in rat liver was analyzed. Liver sample was obtained from a Sprague-Dawley (SD) rat. The following procedures were performed at 4 °C throughout. The liver was homogenized, sonicated in an ice-cold sucrose solution, and then centrifuged. Proteins, precipitated in saturated (NH4)2SO4 solution, were collected by centrifugation at 6,000 rpm for 10 min. The pellet was re-suspended with glycine-NaOH buffer (50 mM, pH 9.8) and purified by ultrafiltration (Millipore, 10 kDa) at 8,100 rpm for 35 min. The pretreated sample was used for arginase activity determination according to the procedure described above. Standard addition experiments were carried out by adding three different concentrations of arginase in the pretreated sample. Monitoring the Response of Rat Liver Arginase to the Administration of Triptolide. Male SD rats weighing between 180-200 g were housed in air-conditioned laboratories and fed a commercial rat diet. Eleven rats were randomly divided into two groups: blank control group (n = 5) and triptolide-treated group (n = 6). In the triptolide-treated group, the rats were administered triptolide (1.34 mg/kg) by intravenous injection in the tail. The control group rats underwent the same operation with only physiological saline injected. After 12 h of administration, all rats were sacrificed. Sample pretreatment and detection procedure were same as described above. Statistical comparison of the groups was made by one-way ANOVA, with P < 0.05 regarded as significant. Data were expressed as mean ± standard error of mean (SEM).
■RESULTS AND DISCUSSION
Materials and Apparatus. ATT was bought from Alfa Aesar Chemicals Co. Ltd. HAuCl4·3H2O, L-arginine, Lnorvaline, L-ornithine, urea, NaOH, glycine, sucrose, and (NH4)2SO4 were obtained from Aladdin Reagent Co. (Shanghai, China). Arginase was acquired from Sigma-Aldrich. Triptolide was purchased from Nanjing Zelang Medical Technology Co. Ltd. (Nanjing, China) and the purity was above 99.0%. Other reagents were of at least analytical grade and used without further purification. Deionized water was used in all experiments. All glassware was cleaned with aqua regia and rinsed with water prior to use. The PL spectra were performed on a Cary Eclipse Fluorescence Spectrophotometer (Agilent). Synthesis of Water-Soluble ATT-AuNPs. ATT-AuNPs were synthesized according to our previous report.38 The resulting ATT-AuNP solution could be stored in the dark at 4 °C for at least six months with no obvious changes in its fluorescent property. Measurement of Arginase and Its Inhibitor. For measurement of arginase activity, 150 µL of 2.5 mM L-arginine (pH 9.8) was mixed with 150 µL of 2 mg/mL ATT-AuNPs (pH 9.8) and 100 µL of different concentrations of arginase (in
Sensing Strategy. A facile method of arginase activity assay which avoids the multiple technical manipulations involved in existent traditional measurements, has been successfully realized by employing ATT-AuNP as a fluorescent nanoswitch. A schematic representation of the sensing procedure is displayed in Scheme 1. The ATT-AuNPs show fluorescence emission at 526 nm when excited at 472 nm (curve a in Figure 1). In presence of L-arginine, the fluorescence of ATT-AuNPs is greatly enhanced (curve b in Figure 1). As described in our previous study, the enhancement of fluorescence intensity is ascribed to the hydrogen bonding-enabled supramolecular host-guest assembly formation between the ATT capped on the Au core and the guanidine group of L-arginine, which rigidifies the capping layer of ATT thereby suppressing the energy-loss process on AuNP surface.38 L-Arginine can competitively bind to the active site of arginase through its guanidine group by a hydrogen bond interaction and subsequently gets decomposed by arginase through a metal-activated hydroxide mechanism.42 After hydrolysis, the host-guest interaction is de-
ACS Paragon Plus Environment
Page 2 of 7
Page 3 of 7 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
ACS Applied Materials & Interfaces Scheme 1. Schematic Representation of the ATT-AuNP-Based Fluorescent Assay of Arginase Activity
Figure 1. Fluorescence emission spectra of (a) ATT-AuNPs, (b) ATT-AuNPs + 2.5 mM L-arginine, and (c) ATT-AuNPs + 2.5 mM L-arginine + 1.5 U/mL arginase. stroyed and the fluorescence is significantly decreased (curve c in Figure 1). To verify the feasibility of this AuNP-based nanoswitch in arginase activity assay, several control experiments were carried out. As shown in Figure S1, addition of only arginase had negligible influence on the fluorescence intensity of ATT-AuNPs. In addition, no obvious fluorescence change was observed with the addition of L-ornithine and urea (the hydrolytic products of L-arginine) into the ATT-AuNP or L-arginine/ATT-AuNP system (Figure S2). The above results together suggest that the hydrolysis of L-arginine could lead to fluorescence change of ATT-AuNPs, thereby providing great promise for arginase activity assay. Optimization of Detection Conditions. We optimized the ATT-AuNP-based sensing system for the determination of arginase activity; the effects of operational parameters for arginase-catalyzed hydrolysis reaction, including substrate Larginine concentration, solution pH, and incubation temperature, were investigated. The change in fluorescence intensity ∆F (∆F = F0−F, where F0 and F are the fluorescence intensities of L-arginine/ATT-AuNPs at 526 nm before and after the arginase-catalyzed hydrolysis reaction, respectively) was used for monitoring arginase activity. To begin with, we estimated the effect of substrate L-arginine concentration on the ∆F values. Figure 2A shows that ∆F increases with increasing concentration of L-arginine (from 0.5 to 2 mM) and varies only
slightly beyond that concentration. Thus, the concentration of L-arginine chosen for subsequent experiments was 2.5 mM. Next, the effect of solution pH was examined between pH 8 and 11.5. As depicted in Figure 2B, ∆F increases gradually with increasing pH, reaches a maximum at pH 9.8, and decreases with further increase of pH. Hence, the solution pH was set at 9.8 in the subsequent experiments. As displayed in Figure 2C, ∆F increases with increasing incubation temperature from 15 to 37 °C, but decreases at temperature beyond that range. Therefore, 37 °C was selected as the incubation temperature for further assay. Sensitivity and Selectivity. Under optimal conditions (2.5 mM L-arginine, pH 9.8, 37 °C), we tested the sensitivity of this nanoswitch for the detection of arginase activity. With increasing concentrations of arginase, the host-guest interaction between ATT and L-arginine on the surface of AuNPs was interrupted and the rigidifying ability of L-arginine for ATT-AuNPs became weaker. As shown in Figure 3A, the fluorescence intensity of the ATT-AuNP-based sensing system decreased with increasing enzyme activity of arginase. A linear correlation (∆F = 373.25[Arginase] + 22.69, [Arginase]: U/mL, r = 0.998) was found between the value of ∆F for ATTAuNPs/L-arginine hybrids and the arginase activity over the range of 0.0625−1.15 U/mL (Figure 3B). The limit of detection (LOD) was calculated to be 0.056 U/mL using the equation LOD = 3S/K, where S represents the standard deviation of the blank signal and K is the slope of the working curve. The relative standard deviation was 0.9% for the detection of 0.5 U/mL arginase (n = 10), validating the high reliability of this approach. To evaluate the specificity of this nanoswitch towards arginase, the sensing system was tested with other enzymes including ascorbic oxidase, urease, pepsin, choline oxidase, acetylcholinesterase, alkaline phosphatase, horseradish peroxidase, sarcosine oxidase, superoxide dismutase, glucose oxidase, catalase, and lysozyme. As shown in Figure 4, in comparison to that in case of arginase, negligible change in fluorescence was observed for these enzymes, suggesting that this assay possesses excellent selectivity/specificity towards arginase. Detection of Arginase in Rat Liver. Arginase is most abundantly expressed in mammalian liver, besides being found in non-hepatic tissues, such as red blood cells, kidney, and lactating mammary glands.43 In order to assess the perfor-
ACS Paragon Plus Environment
ACS Applied Materials & Interfaces 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 4 of 7
Figure 2. Effect of (A) L-arginine concentration, (B) solution pH, and (C) incubation temperature on the assay system for the determination of arginase activity. The concentrations of ATT-AuNPs and arginase used in all experiments were 2 mg/mL and 1.5 U/mL, respectively. Error bars represent the standard deviations across three repetitive experiments.
Figure 3. (A) PL emission spectra of the L-arginine/ATT-AuNPs hybrids in presence of different concentrations of arginase. a−i: 0, 0.0625, 0.06875, 0.125, 0.25, 0.4, 0.5, 0.7, 0.8, 0.9, 1.0, 1.15 U/mL. (B) Linear plot of ∆F against the arginase activity. Experimental conditions: L-arginine concentration: 2.5 mM, pH: 9.8, temperature: 37 °C, and reaction time: 2 h. Error bars represent the standard deviations across three repetitive experiments. recoveries, known amounts of arginase were spiked into the liver samples. As can be seen from Table 1, this sensing system shows satisfactory recoveries in the range of 102.8−118% and good reproducibility with RSD values less than 1.8%, indicating that the ATT-AuNP-based sensing platform is feasible for determination of arginase activity in biological matrices. Table 1. Analysis of arginase activity in rat liver sample
Figure 4. Selectivity test of the newly developed method for the detection of arginase activity. Samples marked as 0−13 correspond to blank, arginase (1 U/mL), ascorbic oxidase (1 U/mL), urease (2 U/mL), pepsin (2 U/mL), choline oxidase (2 U/mL), acetylcholinesterase (2 U/mL), alkaline phosphatase (4 U/mL), horseradish peroxidase (4 U/mL), sarcosine oxidase (5 U/mL), superoxide dismutase (8 U/mL), glucose oxidase (10 U/mL), catalase (10 U/mL), and lysozyme (10 U/mL), respectively. Error bars represented the standard deviations across three repetitive experiments. mance of this newly constructed method, we applied it to detect arginase activity in real biological samples. The standard addition method was employed to monitor the recoveries (related to the proposed method) in rat liver sample. To measure
a
Added
Found
Recoverya
RSDb
(U/mL)
(U/mL)
(%)
(%, n=3)
0
493.80
-
-
80
576.06
102.8
1.4
240
776.87
118.0
0.5
400
916.94
105.8
1.8
Recovery (%) = [(Found value - 493.80)/Added value] × 100
b
RSD represents relative standard deviation, which was calculated from each of three measurements.
Monitoring the Response of Rat Liver Arginase to Pharmacological Agent. As is already known, the enzymatic activity in living organisms might change with environmental variables such as altitude, climate, X-irradiation, and pharmacological agent, also called enzymatic adaptation. In medical terms, monitoring the changes in enzymatic activity can con-
ACS Paragon Plus Environment
Page 5 of 7 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
ACS Applied Materials & Interfaces tribute to elucidating the mechanisms involved in pharmacology and toxicology of drug therapy. To further validate its applicability, this sensing platform was applied to monitor the response of rat liver arginase to pharmacological agent. In our assay, triptolide was selected as a model drug to explore the possibility of such use. As shown in Figure 5, a significant increase of arginase level occurred in rat liver after administration of triptolide, compared to the control group (66.75±1.60 U/mg vs. 91.06±9.30 U/mg, P
