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Highly sensitive electrogenerated chemiluminescence biosensor for galactosyltransferase activity and inhibition detection using gold nanorod and enzymatic dual signal amplification
JEAC-02666; No of Pages 7 Journal of Electroanalytical Chemistry xxx (2016) xxx–xxx
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Highly sensitive electrogenerated chemiluminescence biosensor for galactosyltransferase activity and inhibition detection using gold nanorod and enzymatic dual signal amplification Zhuhai Chen, Ling Zhang, Yang Liu, Jinghong Li ⁎ Department of Chemistry, Beijing Key Laboratory for Analytical Methods and Instrumentation, Tsinghua University, Beijing 100084, China
a r t i c l e
i n f o
Article history: Received 29 April 2016 Received in revised form 17 May 2016 Accepted 20 May 2016 Available online xxxx Keywords: Glycobiology Galactosyltransferase Electrogenerated chemiluminescence Biosensor Gold nanorod Dual signal amplification
a b s t r a c t As one of the glycosyltransferase involved in protein glycosylation, β-1,4-galactosyltransferase (Gal T) plays an important role in the cellular process and progression of cancer. Here, using the bovine serum albumin conjugated N-acetylglucosamine (GlcNAc-BSA) as a receptor to fabricate bioelectrode interface, a sensitive electrochemiluminescence (ECL) biosensor was constructed for Gal T activity analysis based on the recognition between artocarpus integrifolia lectin (AIA) and galactose, integrating with a dual signal amplification strategy from the xanthine oxidase (XOD) and AIA multi-labeled gold nanorod nanoprobes. The gold nanorods promoted the electron transfer on the electrode interface and were also employed as carriers of AIA and XOD due to their large surface area. Furthermore, both the gold nanorods and XOD catalyzed the ECL reaction, which dramatically amplified the ECL signal of luminol in the presence of hypoxanthine (HA) and oxygen. The as-proposed ECL biosensor exhibited high sensitivity on the detection of Gal T activity and a detection limit of 9 × 10−4 U mL−1 was obtained. This assay was successfully applied for the analysis of Gal T activity expression in different cell lines and inhibition detection, showing great potential for glycosyltransferase activity analysis and inhibitors screening in clinic diagnostics. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Carbohydrates on the surface of eukaryotic cells play significant roles in a broad range of crucial biological processes, including cell growth and differentiation, cell adhesion and signaling, immune response, and progression of cancer [1–4]. Galactosyltransferase is a type of glycosyltransferase which catalyzes the transfer process of galactose to form the cellular glycoconjugates [5]. Previous studies found that the enzyme β-1,4-galactosyltransferase (Gal-T), which catalyzes the transfer of a galactose residue from UDP-galactose (UDP-Gal) to N-acetylglucosamine, is closely associated with some vital cellar processes such as cell adhesion [6], and diseases such as lung cancer [7]. Developing rapid and sensitive biosensor to evaluate the Gal-T activity in biological samples is important in clinic diagnostics and biomedical research. Various approaches for the determination of Gal T activity have been developed, such as chromatography [8], radiochemical assay [9], fluorescence [10], and colorimetry [11]. However, these methods often require costly labeling, sophisticated sample pretreatment and
⁎ Corresponding author. E-mail address: [email protected] (J. Li).
complicated instruments. Electrogenerated chemiluminescence (ECL), which involves electron-transfer reactions and light-emitting process of the luminophores on the electrode, combines the electrochemical and luminescent techniques [12,13]. In comparison to the conventional methods, ECL is a powerful analytical tool with its advantages of low cost, low background noise, wide dynamic concentration response range and high sensitivity [14]. By integrating some biomolecular recognition strategy, ECL biosensors have been widely applied in DNA analysis [15], immunoassay [16,17], protein analysis [18,19], cell analysis [20, 21], clinical diagnosis [22] and environment monitoring [23]. Recently, the developments of nanostructures and nanomaterials have greatly enhanced the performance of electrochemical biosensors owing to their remarkable electrocatalytic activity, large surface area, and good biocompatibility [24,25]. These excellent optical, electrical, and electrochemical properties allow the nanomaterials to promote the surface area and improve the electron transfer at the electrode interface [26–28]. Also, they can be used as carriers to load more active biomolecule ligands for target recognition and ECL labels for signal amplification. Owing to their unique properties, some nanomaterials such as gold nanoparticles [29,30], and gold nanorods [31] have been extensively used in ECL biosensors in previous works. Herein, a sensitive sandwich type ECL biosensor for Gal T activity and inhibition detection is designed. The glycosylation receptor N-
http://dx.doi.org/10.1016/j.jelechem.2016.05.034 1572-6657/© 2016 Elsevier B.V. All rights reserved.
Please cite this article as: Z. Chen, et al., Highly sensitive electrogenerated chemiluminescence biosensor for galactosyltransferase activity and inhibition detection using go..., Journal of Electroanalytical Chemistry (2016), http://dx.doi.org/10.1016/j.jelechem.2016.05.034
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Acetylglucosamine-BSA (GlcNAc-BSA) was immobilized onto the gold electrode to form the bioelectrode interface. After the glycosylation catalyzed by Gal T, galactose was conjugated to the GlcNAc-BSA. And the galactose was then specifically recognized by the artocarpus integrifolia lectin (AIA) on gold nanorods (GNRs), resulting the absorption of the AIA and xanthine oxidase (XOD) conjugated GNR (AIA-XOD@GNR) nanoprobes. The ECL signal was markedly enhanced due to the gold nanorod and enzymatic dual amplification of the luminol ECL signals. The ECL biosensor showed high sensitivity toward Gal T activity detection with a low detection limit, and further was applied for cell lysate detection and inhibition evaluation.
2. Experimental section 2.1. Reagents Artocarpus integrifolia lectin (AIA) was obtained from BioSun Sci&Tech Co. (Shanghai, China). N-Acetylglucosamine-BSA (GlcNAcBSA) was received from Professor Lokesh Joshi, National University of Ireland, Galway, Ireland. Luminol, HAuCl4·3H2O, hypoxanthine (HA), β-1,4-galactosyltransferase, and uridine 5′-diphosphogalactose disodium (UDP-Gal) were purchased from Sigma-Aldrich. Cetyltrimethyl Ammonium Bromide (CTAB), poly(sodium 4styrenesulfonate) (PSS, molecular weight = 7000), 1-(3Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and N-hydroxysulfosuccinimide sodium salt (NHSS) were obtained from Alfa Aesar Co. (Ward Hill, MA, USA). 11-mercaptoundecanoic acid (MUA) and 6-mercapto-1-hexanol (MEH) were purchased from J&K Scientific Ltd. (Beijing, China). Xanthine oxidase (XOD) was purchased from Yuanye Biotech. Ltd. (Shanghai, China). Bovine serum albumin (BSA), 2,2′-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) and bicinchoninic acid (BCA) protein assay kit were from Dingguo Biological Products Co. (Beijing, China). Other regents of analytical grade were obtained from Beijing Chemical Co. (Beijing, China).
2.2. Preparation of AIA and XOD conjugated gold nanorods The gold nanorods (GNRs) are synthesized according to previous works [32–34]. In brief, 0.25 mL HAuCl4·3H2O (10 mM) was mixed with 0.6 mL of 10 mM NaBH4 solution and 5 mL cetyltrimethylammonium bromide (CTAB) (100 mM) to get a pale brown gold seed solution. Then, 0.25 mL of 10 mM AgNO3 and 0.27 mL of 100 mM ascorbic acid solution were added to a mixed solution of 40 mL CTAB (100 mM) and 1.7 mL HAuCl4·3H2O (10 mM) successively to get the growth solution. Finally, 0.42 mL of gold seed solution was added to the growth solution. The mixture was incubated for 15 h at 28 °C before centrifugation. To remove the extra free CTAB, the fresh GNRs solution was centrifuged at 14,000 rpm for 10 min, and the products at the bottom were then re-dispersed in PBS solution. The prepared GNRs were characterized by transmission electron microscopy (TEM) and UV–Vis spectra (Fig. S1 in Supporting information). Statistical analysis of them shows that the GNRs had an average diameter of ~10 nm and an average length of ~36 nm with an aspect ratio of ~ 3.5. And a strong adsorption at 750 nm was observed for the gold nanorods. Then, 40 μL of 5 mg mL−1 PSS was incubated with 1 mL of the re-dispersed GNRs solution for 40 min to get a PSS functionalized GNRs. The mixture was centrifuged at 10,000 rpm to get rid of excessive PSS and the pH value was adjusted to 7.5 with 10 mM PBS buffer. To obtain the AIA and XOD conjugated GNRs, 1 mL colloidal solution of PSS caped GNRs was mixed with the solution of 100 μL of 5 mg mL−1 AIA and 6 μL of 1 mg mL− 1 XOD. The mixture was incubated for 60 min under shaking, and was centrifuged to remove extra AIA and XOD and re-dispersed in PBS solution containing 1 mM Ca2+ and Mn2+.
2.3. Fabrication of ECL biosensor Prior to fabrication of ECL sensors, a gold electrode (diameter of 3 mm) was successively polished with 0.3, and 0.05 μm α-Al2O3 powder and ultrasonically cleaned with ethanol and water. After being dried with a nitrogen flow, the electrode surface was immersed into 100 μL solution of 0.01 mM MUA and 0.09 mM MEH in 4:1 ethanol/H2O overnight at room temperature. To activate carboxyl groups, the electrode was then immersed in 30 μL of a mixture aqueous solution containing 50 mM EDC and NHSS at 37 °C for 1 h. For GlcNAc-BSA immobilization, the electrode was incubated with 5 mL of 0.2 mg mL−1 GlcNAc-BSA at 37 °C for 80 min. Next, the electrode was washed with PBS and followed by incubation in solution of 1 mg mL−1 BSA for 1 h at 37 °C to block the additional active groups. The solution for glycosylation reaction was prepared by mixing 75 mL of 50 mM HEPES, 5 mL of 20 mM UDP-Gal, 10 mL of 20 mM Mn2+ and 5 mL Gal T at a certain concentration. The GlcNAc-BSA modified electrode was soaked in 25 mL of reaction solution with Gal T and UDP-Gal at 37 °C for 3 h to conjugate galactose to the GlcNAc-BSA. After being thoroughly rinsed with PBS, the electrode was incubated with 5 μL solution containing AIA-XOD@GNR at 37 °C for 1 h to capture the nanoprobes via the specific binding between galactose and AIA. The resultant electrode was washed with PBS to remove the extra nanoprobes before being used for ECL assays. ECL measurements were performed in a 0.1 M PBS (pH 8.5) containing 5 mM HA and 100 μM luminol using Ag/AgCl electrode with saturated KCl solution and platinum wire as the reference electrode and counter electrode, respectively. The ECL measurements were performed from 0 to 0.6 V with scan rate of 100 mV s−1. The experiments for Gal T activity measurements of cell lysate protein were the same as described above except for substituting 50 μg of cell lysate protein for 5 mL Gal T. 2.4. Cell culture and lysis The HeLa cells, CCRF-CEM cells, SMMC-7721 cells and HL-7702 cells were kindly provided by the Medicine School of Tsinghua University, Beijing, China. HeLa cells were cultured with Dulbecco's Modified Eagle Medium (DMEM) (Hyclone, Logan, UT, USA). CCRF-CEM cells and SMMC-7721 cells were cultured in RPMI 1640 medium (Dingguo Biological Products Co., Beijing, China). Each medium was supplemented with 10% fetal calf serum (Zhejiang Tianhang Biological Technology Co., Ltd., Zhejiang, China), 100 U mL−1 penicillin, and 100 μg mL− 1 streptomycin. The cells were cultured at 37 °C in a humidified atmosphere of 5% CO2. And the HL-7702 cells were cultured with Dulbecco's Modified Eagle Medium (DMEM) (Hyclone) supplemented with 20% fetal calf serum (Zhejiang Tianhang Biological Technology Co., Ltd., Zhejiang, China). The cells were grown to midlog phase and then collected and separated from the medium by centrifugation at 1000 rpm for 5 min. Then the cells were washed with sterile phosphate buffer saline (PBS, pH 7.4) twice. The cells were added with cell lysis buffer containing 1 mM PMSF (Beyotime biotechnology Co., Shanghai, China) at 4 °C for 30 min. The suspension was centrifugated by refrigerated centrifugation at 14,000 rpm for 5 min, and the supernatant was retained and stored at 80 °C for further Gal T analysis. Before Gal T analysis, the proteins in each cell lysate sample were quantified with the bicinchoninic acid (BCA) protein assay kit (Dingguo Biological Products Co., Beijing, China). Cell lysate containing 50 μg proteins was added in the glycosylation reaction solution for each ECL test. 2.5. Apparatus and characterization Scanning electron microscope (SEM) images were obtained with an SU8010 Scanning transmission electron microscope (Hitachi, Japan). UV–vis experiments were carried out with a UV-3900 spectrophotometer (Hitachi, Japan). The cyclic voltammetry was conducted on a CHI 660b instrument (CH Instrument Co., USA). Electrochemical impedance
Please cite this article as: Z. Chen, et al., Highly sensitive electrogenerated chemiluminescence biosensor for galactosyltransferase activity and inhibition detection using go..., Journal of Electroanalytical Chemistry (2016), http://dx.doi.org/10.1016/j.jelechem.2016.05.034
Z. Chen et al. / Journal of Electroanalytical Chemistry xxx (2016) xxx–xxx
spectroscopy (EIS) was carried out on a PARSTAT 2273 potentiostat/ galvanostat (Advanced Measurement Technology Inc. USA). The cyclic voltammetry and Impedance measurements were performed in a 5 mM K3[Fe(CN)6]/K4[Fe(CN)6] redox probe solution with 0.5 M KCl. Impedance measurements were completed by applying an AC voltage of 10 mV amplitude with frequency from 0.1 Hz to 105 Hz. The ECL measurements were carried out on an MPI-B multifunctional electrochemical analytical system (Xi'an Remex Analytical Instrument Ltd. Co., China). The voltage of the photomultiplier tube (PMT) was maintained at 600 V. 3. Results and discussion Scheme 1 shows the principle and construction of the ECL biosensor for Gal T activity analysis. Firstly, a self-assembly monolayer (SAM) of MUA and MEH was formed on the gold electrode to provide the carboxyl groups for glycoprotein modification. The glycoprotein GlcNAc-BSA was immobilized onto the electrode surface as a receptor for the glycosylation reaction. Then, the galactose was transferred to GlcNAc-BSA in the presence of UDP-Gal donor and Gal T, followed by the capture of AIA-XOD@GNR nanoprobes onto the gold electrode surface via the specific affinity between galactose and AIA. The ECL reaction was generated and greatly amplified by the gold nanorods and the enzyme-catalyzed reaction, which can be applied for Gal T activity detection. 3.1. Characterization of AIA-XOD@GNR nanoprobes To evaluate the modification and catalytic activity of as-prepared AIA-XOD@GNR nanoprobes, the HRP-catalyzed colorimetric reaction was used. As shown in Fig. S2 (Supporting information), in the presence of H2O2, HRP could catalyze the oxidation of ABTS, generating a bluegreen colorimetric signal, which can be easily monitored by the naked eye and the UV–Vis absorption spectra. However, when the H2O2 was replaced by HA, no significant signal was observed. Then, by adding XOD into the solution containing HRP, ABTS and HA, the oxidation of HA could be catalyzed and H2O2 was generated, resulting the colorimetric signal. Sequentially, the as-prepared AIA-XOD@GNR nanoprobes were added into the solution containing HRP, ABTS and HA, similar colorimetric signal was obtained, demonstrating the successful modification of XOD on the GNRs and the good catalytic property of the AIAXOD@GNRs. While, as a control test, the GNRs were mixed with HRP,
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ABTS and HA, and no colorimetric signal was observed, furtherly confirming the catalytic activity of the nanoprobes. 3.2. Electrochemical characterizations of the biosensor The assembly processes of the modified electrodes were characterized using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) step by step. Fig. 1A displays the CV curves of the modified gold electrode using Fe(CN)4−/3− as the electroactive probes. 6 Firstly, on the bare electrode, a couple of reversible redox peaks were observed (curve a). After the modification of the SAM, the peak currents decreased and the potential gap increased (curve b) owing to the poor conductivity of the SAM. After the modification of GlcNAc-BSA, the peak current further dropped dramatically and the potential gap increased as well (curve c) due to the electronic inert property of the glycoprotein. After being blocked the excess active sites by BSA, the conjugation of galactose to GlcNAc-BSA was made under the glycosylation reaction catalyzed by Gal T (curve d), inducing a slight decrease of the peak currents. Finally, the electrode was incubated the AIA-XOD@ GNR nanoprobes. As shown in curve (e), the binding of nanoprobes resulted in an increase in peak currents and a decrease in the potential gap between the anodic and cathodic peaks, indicating the nanoprobes possessed good feature of electron transfer and mass transfer of Fe(CN)4−/3− on the electrode interface. These results confirm the 6 successful construction of the ECL biosensor. Meanwhile, the impedance spectra shown in Fig. 1B also verify the fabrication processes of the biosensor. The nyquist plot comprises a semicircular part at higher frequency range and a straight linear part at lower frequency range. The diameter of the semicircle equals to the electron transfer resistance (Ret) at the electrode interface. Due to the good electronic transfer ability, the bare gold (curve a) exhibited the lowest Ret. Then the diameter of the semicircles increased successively with sequential formation of SAM (curve b), GlcNAc-BSA immobilization (curve c) and glycosylation reaction (curve d). Finally, with AIAXOD@GNR modification (curve e), the Ret again decreased. These changes were similar to those CV results, further confirming the successful assembly of the biosensor. 3.3. ECL behaviors of the biosensor The ECL behavior of the biosensor was characterized with the different modifications on the electrode in 0.1 M PBS (pH 8.5) containing
Scheme 1. The procedures for the fabrication of the ECL biosensor and β-1,4-galactosyltransferase activity analysis.
Please cite this article as: Z. Chen, et al., Highly sensitive electrogenerated chemiluminescence biosensor for galactosyltransferase activity and inhibition detection using go..., Journal of Electroanalytical Chemistry (2016), http://dx.doi.org/10.1016/j.jelechem.2016.05.034
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cannot be transferred from UDP-Gal to the N-acetylglucosamine groups on GlcNAc-BSA, and then nanoprobes cannot be adsorbed onto the electrode for signal amplification without the specific recognition between AIA and galactose. As shown in curve c and d, after the Gal T catalyzed glycosylation reaction on the electrode and the following AIA-XOD@ GNRs incubation, the GlcNAc-BSA modified electrode exhibited a sensitive ECL emission starting at about 0.4 V with a peak at around 0.58 V. While the AIA-XOD@GNRs were not immobilized onto the electrode, the ECL intensity was quite slight, close to the baseline. These results demonstrate the AIA-XOD@GNR nanoprobes can increase ECL intensity effectively and this ECL biosensor can be applied for sensitive and selective analysis of Gal T activity. 3.4. Optimization of assay conditions
Fig. 1. Cyclic voltammograms and electrochemical impedance spectra of bare gold (curve a), SAM modified gold electrode (curve b), GlcNAc-BSA modified electrode (curve c), β1,4-Glycosylated modified electrode (curve d) and after the incubation with probe (curve e) in 0.5 M KCl solution with 5 mM [Fe(CN)6]3−/4− electroactive probes, respectively. CV Scan rate: 100 mV s−1. EIS Frequency range: 0.1 ~ 105 Hz.
5 mM HA and 100 μM luminol during the CV scanning. As shown in Fig. 2, when the modified electrode was incubated in the enzymic precursor solution without Gal T or UDP-Gal followed by incubating AIA-XOD@ GNR nanoprobes, no detectable ECL signal was observed (curve a and b). This result indicates that, without Gal T and UDP-Gal, galactose
Fig. 2. ECL-potential curves of GlcNAc-BSA modified electrodes before (a) and after (b) the incubation with probe (AIA-XOD@Au NRs), and GlcNAc-BSA modified electrodes before (c) and after (d) the incubation with probe after the β-1,4-galactosylation reaction. The ECL measurements were performed in 0.1 M PBS (pH 8.5) containing 100 μM luminol and 5 mM hypoxanthine. Scan rate: 100 mV s−1. PMT: 600 V.
As for the enzymatic glycosylation reaction, it is generally influenced by some external factors, such as pH and temperature. In order to improve the Gal T activity detection performance, the effects of pH, temperature and incubation time of glycosylation reaction were investigated. As shown in Fig. 3A, the ECL signal gradually increased from pH 5.5 to pH 7.4, and then decreased. This result indicates Gal T possesses high activity at pH 7.4. In the study of temperature for glycosylation reaction (Fig. 3B), it is observed that relative ECL intensity increased with the temperature up to 37 °C. After that, the ECL changed decreasingly by the continue increase of temperature, suggesting Gal T had high activity at 37 °C. The resultant optimal pH and temperature of 7.4 and 37 °C were in agreement with the physiological conditions. In addition, as shown in Fig. 3C, it was found that with the increase of the reaction time, the ECL signal increased and finally reached a plateau in 3 h, suggesting a tendency to complete the glycosylation process. Accordingly, the optimal reaction time was chosen to be 3 h. Besides, to get a sensitive ECL signal, the optimization of the pH value of ECL electrolyte solution was performed (Fig. S3 in Supporting information). The pH 8.5 was selected as the optimal condition for ECL electrolyte solution preparation. Then, the molar ratio of XOD to AIA immobilized on gold nanorods was also optimized (Fig. S4 in Supporting information). The optimal molar ratio 4.5/1 of XOD to AIA was used for nanoprobe preparation. 3.5. ECL biosensor for Gal T activity detection On the basis of the optimal experimental conditions, the ECL biosensor was applied for detecting Gal T activity. The relationship of the ECL responses with Gal T concentrations is displayed in Fig. 4. Measurements were performed three times for each test. It can be seen that the ECL signals gradually increased with the increasing of Gal T activity and reached a plateau when the concentrations of Gal T rose up to 0.5 U mL−1. The ECL intensity changes are proportional to the logarithmic value of Gal T activity in the range from 0.001 U mL− 1 to 0.1 U mL−1. The linear relationship can be described as ΔIECL (a.u.) = 1167 × Log UGal T (U mL− 1) + 4398 with the correlation coefficient R2 = 0.990, where ΔIECL is ECL intensity of glycosylated GlcNAc-BSA modified electrode at different Gal T concentration after the AIAXOD@GNR incubation, and UGal T is the concentration of Gal T. The detection limit was obtained to be 9 × 10−4 U mL−1 at signal-to-noise ratio of 3, which was much lower than the earlier reported method [35]. The high sensitivity of the ECL biosensor could be ascribed to the dual amplification of the ECL signal by the GNRs and the enzyme-catalyzed reaction. Here, the GNRs are not only used to electrocatalyze the ECL process of luminol but also acted as a carrier to load more AIA and XOD. Owing to its excellent electron-transfer ability, the gold nanorods also improved the electron transfer at the electrode interface, thus further improved the performance of the as-designed ECL sensor. Moreover, the stability of the ECL biosensor was characterized by successively scanning in 0.1 M PBS containing 5 mM HA and 100 μM luminol for ten cycles (Fig. S5 in Supporting information). The relative
Please cite this article as: Z. Chen, et al., Highly sensitive electrogenerated chemiluminescence biosensor for galactosyltransferase activity and inhibition detection using go..., Journal of Electroanalytical Chemistry (2016), http://dx.doi.org/10.1016/j.jelechem.2016.05.034
Z. Chen et al. / Journal of Electroanalytical Chemistry xxx (2016) xxx–xxx
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Fig. 4. (A) ECL-potential curves with various activity units of β-1,4-galactosyltransferase (top to bottom, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0 U mL−1) in 0.1 M PBS (pH 8.5) containing 100 μM luminol and 5 mM hypoxanthine. Scan rate: 100 mV s−1. PMT: 600 V. (B) The dependence of ECL intensity on the activity concentration of Gal T. inset: the linear relationship between the ECL intensity with the logarithm of Gal T concentration.
Fig. 3. Optimization of the pH of enzymatic β-1,4-galactosylation (A), enzymatic β-1,4galactosylation temperature (B), and enzymatic β-1,4-galactosylation time (C). The ECL measurements were performed in 0.1 M PBS (pH 8.5) containing 100 μM luminol and 5 mM hypoxanthine. CV Scan rate: 100 mV s−1. PMT: 600 V. β-1,4-galactosyltransferase concentration: 0.05 U mL−1.
(human hepatocyte cell line) were measured. Fig. S6 (Supporting information) displays the ECL response of cellular Gal T in HeLa cell lysate from 500 cells. The detection limit of cell number for Gal T activity analysis in cell lysates was obtained as 150 cells. As shown in Fig. 5, various Gal T activity levels were obtained in different cell lines. Clearly, as a normal cell line, HL-7702 cell line expressed lower level of Gal T than the other three disease related immortal cell lines. This result reveals that the intracellular galactosyltransferases and glycans have significant roles in cell carcinogenesis. These experimental results furtherly confirm that the as-prepared ECL biosensor can be used to determine Gal
standard deviation was 4.2%, signifying that the ECL sensor possessed excellent potential cycling stability. 3.6. Detection of Gal T activities in cell lysates Gal T plays important roles in a variety of cellular processes such as cell apoptosis, proliferation, differentiation, and cell–cell communications. It is also served as a biomarker for diagnostic and prognostic. Here, The Gal T activities of four cells, HeLa cell (human cervical cancer cell line), CCRF-CEM cell (human leukemic lymphoblasts), SMMC-7721 cell (human hepatocellular carcinoma cell line) and HL-7702 cell
Fig. 5. The Gal T activity analyzed from HeLa, CCRF-CEM, SMMC-7721 and HL-7702 cell lysates. Cell lysate containing 50 μg proteins was used for each ECL test.
Please cite this article as: Z. Chen, et al., Highly sensitive electrogenerated chemiluminescence biosensor for galactosyltransferase activity and inhibition detection using go..., Journal of Electroanalytical Chemistry (2016), http://dx.doi.org/10.1016/j.jelechem.2016.05.034
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T activity in complex biological samples with high sensitivity and accuracy.
Conflict of interest There is no conflict of interest.
3.7. Screening inhibitors of Gal T Inhibitors of glycosyltransferases are invaluable as tools for understanding the function of these enzymes and relative glycans. The proposed biosensor was applied to rapidly evaluate the effectiveness of various Gal T inhibitors. Ten candidates, uridine-5′-diphosphogalactose (UDP-Gal), uridine-5′-diphosphoglucose (UDP-Glu), uridine-5′diphospho-N-acetylglucosamine (UDP-N-GlcNAc), uridine-5′monophosphate (UDP), uridine-5′-triphosphate (UMP), uridine-5′-triphosphate (UTP), adenosine-5′-diphosphate (ADP), Benzyl 2acetamido-2-deoxy-α-D-galactopyranoside (BG), galactose (Gal) and glucose (Glu) were chosen and detected. The screening result, in Fig. 6, shows that UDP, UTP, ADP and Gal had potent inhibition, especially UDP. It is consistent with the previous reported work that Gal T enzymic reaction was observably perturbed by UDP and UTP [36]. However, BG, a well known inhibitor of α-2,3-sialyltransferase which extends a GalNAc [37,38], didn't show effective inhibition to galactosylation in the detection. In addition, the IC50 (50% inhibitory concentration) of UDP was further determined by the biosensor. Fig. S7 (Supporting information) shows the value of IC50 for UDP to be 211 μM, which was in agreement with that based on mass spectrometry assay [36]. From the results above, it is indicated that the constructed biosensor provides a sensitive method for rapidly screening glycosyltransferases inhibitors.
4. Conclusion In summary, a sensitive ECL biosensor for β-1,4galactosyltransferases (Gal T) activity analysis has been developed based on the specific recognition between lectin and galactose, integrating with the gold nanorods and enzymatic dual amplification of ECL signal. Via the specific interaction between AIA and galactose, the AIA and XOD conjugated gold nanorods were absorbed onto the bioelectrode interface and greatly enhanced the ECL signals due to the excellent electrocatalytic activity, large surface area, and good conductivity. The proposed ECL biosensor showed high sensitivity and specificity for Gal T activity analysis with a low detection limit of 9 × 10−4 U mL−1. The platform has been applied for the Gal T activity analysis in different cell lines and screening of Gal T inhibitors, which provides a valuable tool for glycosyltransferase activity evaluation and inhibition in clinic diagnostic.
Fig. 6. Evaluation of Gal T activity inhibition. The ECL intensity as a function of different inhibitors with concentration of 1 mM in 0.1 M PBS (pH 8.5) containing 100 μM luminol and 5 mM hypoxanthine. Scan rate: 100 mV s−1. PMT: 600 V. The enzymatic reaction was carried out with 0.05 U mL−1 Gal T.
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Journal of Electroanalytical Chemistry journal homepage: www.elsevier.com/locate/jelechem
Highly sensitive electrogenerated chemiluminescence biosensor for galactosyltransferase activity and inhibition detection using gold nanorod and enzymatic dual signal amplification Zhuhai Chen, Ling Zhang, Yang Liu, Jinghong Li ⁎ Department of Chemistry, Beijing Key Laboratory for Analytical Methods and Instrumentation, Tsinghua University, Beijing 100084, China
a r t i c l e
i n f o
Article history: Received 29 April 2016 Received in revised form 17 May 2016 Accepted 20 May 2016 Available online xxxx Keywords: Glycobiology Galactosyltransferase Electrogenerated chemiluminescence Biosensor Gold nanorod Dual signal amplification
a b s t r a c t As one of the glycosyltransferase involved in protein glycosylation, β-1,4-galactosyltransferase (Gal T) plays an important role in the cellular process and progression of cancer. Here, using the bovine serum albumin conjugated N-acetylglucosamine (GlcNAc-BSA) as a receptor to fabricate bioelectrode interface, a sensitive electrochemiluminescence (ECL) biosensor was constructed for Gal T activity analysis based on the recognition between artocarpus integrifolia lectin (AIA) and galactose, integrating with a dual signal amplification strategy from the xanthine oxidase (XOD) and AIA multi-labeled gold nanorod nanoprobes. The gold nanorods promoted the electron transfer on the electrode interface and were also employed as carriers of AIA and XOD due to their large surface area. Furthermore, both the gold nanorods and XOD catalyzed the ECL reaction, which dramatically amplified the ECL signal of luminol in the presence of hypoxanthine (HA) and oxygen. The as-proposed ECL biosensor exhibited high sensitivity on the detection of Gal T activity and a detection limit of 9 × 10−4 U mL−1 was obtained. This assay was successfully applied for the analysis of Gal T activity expression in different cell lines and inhibition detection, showing great potential for glycosyltransferase activity analysis and inhibitors screening in clinic diagnostics. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Carbohydrates on the surface of eukaryotic cells play significant roles in a broad range of crucial biological processes, including cell growth and differentiation, cell adhesion and signaling, immune response, and progression of cancer [1–4]. Galactosyltransferase is a type of glycosyltransferase which catalyzes the transfer process of galactose to form the cellular glycoconjugates [5]. Previous studies found that the enzyme β-1,4-galactosyltransferase (Gal-T), which catalyzes the transfer of a galactose residue from UDP-galactose (UDP-Gal) to N-acetylglucosamine, is closely associated with some vital cellar processes such as cell adhesion [6], and diseases such as lung cancer [7]. Developing rapid and sensitive biosensor to evaluate the Gal-T activity in biological samples is important in clinic diagnostics and biomedical research. Various approaches for the determination of Gal T activity have been developed, such as chromatography [8], radiochemical assay [9], fluorescence [10], and colorimetry [11]. However, these methods often require costly labeling, sophisticated sample pretreatment and
⁎ Corresponding author. E-mail address: [email protected] (J. Li).
complicated instruments. Electrogenerated chemiluminescence (ECL), which involves electron-transfer reactions and light-emitting process of the luminophores on the electrode, combines the electrochemical and luminescent techniques [12,13]. In comparison to the conventional methods, ECL is a powerful analytical tool with its advantages of low cost, low background noise, wide dynamic concentration response range and high sensitivity [14]. By integrating some biomolecular recognition strategy, ECL biosensors have been widely applied in DNA analysis [15], immunoassay [16,17], protein analysis [18,19], cell analysis [20, 21], clinical diagnosis [22] and environment monitoring [23]. Recently, the developments of nanostructures and nanomaterials have greatly enhanced the performance of electrochemical biosensors owing to their remarkable electrocatalytic activity, large surface area, and good biocompatibility [24,25]. These excellent optical, electrical, and electrochemical properties allow the nanomaterials to promote the surface area and improve the electron transfer at the electrode interface [26–28]. Also, they can be used as carriers to load more active biomolecule ligands for target recognition and ECL labels for signal amplification. Owing to their unique properties, some nanomaterials such as gold nanoparticles [29,30], and gold nanorods [31] have been extensively used in ECL biosensors in previous works. Herein, a sensitive sandwich type ECL biosensor for Gal T activity and inhibition detection is designed. The glycosylation receptor N-
http://dx.doi.org/10.1016/j.jelechem.2016.05.034 1572-6657/© 2016 Elsevier B.V. All rights reserved.
Please cite this article as: Z. Chen, et al., Highly sensitive electrogenerated chemiluminescence biosensor for galactosyltransferase activity and inhibition detection using go..., Journal of Electroanalytical Chemistry (2016), http://dx.doi.org/10.1016/j.jelechem.2016.05.034
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Acetylglucosamine-BSA (GlcNAc-BSA) was immobilized onto the gold electrode to form the bioelectrode interface. After the glycosylation catalyzed by Gal T, galactose was conjugated to the GlcNAc-BSA. And the galactose was then specifically recognized by the artocarpus integrifolia lectin (AIA) on gold nanorods (GNRs), resulting the absorption of the AIA and xanthine oxidase (XOD) conjugated GNR (AIA-XOD@GNR) nanoprobes. The ECL signal was markedly enhanced due to the gold nanorod and enzymatic dual amplification of the luminol ECL signals. The ECL biosensor showed high sensitivity toward Gal T activity detection with a low detection limit, and further was applied for cell lysate detection and inhibition evaluation.
2. Experimental section 2.1. Reagents Artocarpus integrifolia lectin (AIA) was obtained from BioSun Sci&Tech Co. (Shanghai, China). N-Acetylglucosamine-BSA (GlcNAcBSA) was received from Professor Lokesh Joshi, National University of Ireland, Galway, Ireland. Luminol, HAuCl4·3H2O, hypoxanthine (HA), β-1,4-galactosyltransferase, and uridine 5′-diphosphogalactose disodium (UDP-Gal) were purchased from Sigma-Aldrich. Cetyltrimethyl Ammonium Bromide (CTAB), poly(sodium 4styrenesulfonate) (PSS, molecular weight = 7000), 1-(3Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and N-hydroxysulfosuccinimide sodium salt (NHSS) were obtained from Alfa Aesar Co. (Ward Hill, MA, USA). 11-mercaptoundecanoic acid (MUA) and 6-mercapto-1-hexanol (MEH) were purchased from J&K Scientific Ltd. (Beijing, China). Xanthine oxidase (XOD) was purchased from Yuanye Biotech. Ltd. (Shanghai, China). Bovine serum albumin (BSA), 2,2′-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) and bicinchoninic acid (BCA) protein assay kit were from Dingguo Biological Products Co. (Beijing, China). Other regents of analytical grade were obtained from Beijing Chemical Co. (Beijing, China).
2.2. Preparation of AIA and XOD conjugated gold nanorods The gold nanorods (GNRs) are synthesized according to previous works [32–34]. In brief, 0.25 mL HAuCl4·3H2O (10 mM) was mixed with 0.6 mL of 10 mM NaBH4 solution and 5 mL cetyltrimethylammonium bromide (CTAB) (100 mM) to get a pale brown gold seed solution. Then, 0.25 mL of 10 mM AgNO3 and 0.27 mL of 100 mM ascorbic acid solution were added to a mixed solution of 40 mL CTAB (100 mM) and 1.7 mL HAuCl4·3H2O (10 mM) successively to get the growth solution. Finally, 0.42 mL of gold seed solution was added to the growth solution. The mixture was incubated for 15 h at 28 °C before centrifugation. To remove the extra free CTAB, the fresh GNRs solution was centrifuged at 14,000 rpm for 10 min, and the products at the bottom were then re-dispersed in PBS solution. The prepared GNRs were characterized by transmission electron microscopy (TEM) and UV–Vis spectra (Fig. S1 in Supporting information). Statistical analysis of them shows that the GNRs had an average diameter of ~10 nm and an average length of ~36 nm with an aspect ratio of ~ 3.5. And a strong adsorption at 750 nm was observed for the gold nanorods. Then, 40 μL of 5 mg mL−1 PSS was incubated with 1 mL of the re-dispersed GNRs solution for 40 min to get a PSS functionalized GNRs. The mixture was centrifuged at 10,000 rpm to get rid of excessive PSS and the pH value was adjusted to 7.5 with 10 mM PBS buffer. To obtain the AIA and XOD conjugated GNRs, 1 mL colloidal solution of PSS caped GNRs was mixed with the solution of 100 μL of 5 mg mL−1 AIA and 6 μL of 1 mg mL− 1 XOD. The mixture was incubated for 60 min under shaking, and was centrifuged to remove extra AIA and XOD and re-dispersed in PBS solution containing 1 mM Ca2+ and Mn2+.
2.3. Fabrication of ECL biosensor Prior to fabrication of ECL sensors, a gold electrode (diameter of 3 mm) was successively polished with 0.3, and 0.05 μm α-Al2O3 powder and ultrasonically cleaned with ethanol and water. After being dried with a nitrogen flow, the electrode surface was immersed into 100 μL solution of 0.01 mM MUA and 0.09 mM MEH in 4:1 ethanol/H2O overnight at room temperature. To activate carboxyl groups, the electrode was then immersed in 30 μL of a mixture aqueous solution containing 50 mM EDC and NHSS at 37 °C for 1 h. For GlcNAc-BSA immobilization, the electrode was incubated with 5 mL of 0.2 mg mL−1 GlcNAc-BSA at 37 °C for 80 min. Next, the electrode was washed with PBS and followed by incubation in solution of 1 mg mL−1 BSA for 1 h at 37 °C to block the additional active groups. The solution for glycosylation reaction was prepared by mixing 75 mL of 50 mM HEPES, 5 mL of 20 mM UDP-Gal, 10 mL of 20 mM Mn2+ and 5 mL Gal T at a certain concentration. The GlcNAc-BSA modified electrode was soaked in 25 mL of reaction solution with Gal T and UDP-Gal at 37 °C for 3 h to conjugate galactose to the GlcNAc-BSA. After being thoroughly rinsed with PBS, the electrode was incubated with 5 μL solution containing AIA-XOD@GNR at 37 °C for 1 h to capture the nanoprobes via the specific binding between galactose and AIA. The resultant electrode was washed with PBS to remove the extra nanoprobes before being used for ECL assays. ECL measurements were performed in a 0.1 M PBS (pH 8.5) containing 5 mM HA and 100 μM luminol using Ag/AgCl electrode with saturated KCl solution and platinum wire as the reference electrode and counter electrode, respectively. The ECL measurements were performed from 0 to 0.6 V with scan rate of 100 mV s−1. The experiments for Gal T activity measurements of cell lysate protein were the same as described above except for substituting 50 μg of cell lysate protein for 5 mL Gal T. 2.4. Cell culture and lysis The HeLa cells, CCRF-CEM cells, SMMC-7721 cells and HL-7702 cells were kindly provided by the Medicine School of Tsinghua University, Beijing, China. HeLa cells were cultured with Dulbecco's Modified Eagle Medium (DMEM) (Hyclone, Logan, UT, USA). CCRF-CEM cells and SMMC-7721 cells were cultured in RPMI 1640 medium (Dingguo Biological Products Co., Beijing, China). Each medium was supplemented with 10% fetal calf serum (Zhejiang Tianhang Biological Technology Co., Ltd., Zhejiang, China), 100 U mL−1 penicillin, and 100 μg mL− 1 streptomycin. The cells were cultured at 37 °C in a humidified atmosphere of 5% CO2. And the HL-7702 cells were cultured with Dulbecco's Modified Eagle Medium (DMEM) (Hyclone) supplemented with 20% fetal calf serum (Zhejiang Tianhang Biological Technology Co., Ltd., Zhejiang, China). The cells were grown to midlog phase and then collected and separated from the medium by centrifugation at 1000 rpm for 5 min. Then the cells were washed with sterile phosphate buffer saline (PBS, pH 7.4) twice. The cells were added with cell lysis buffer containing 1 mM PMSF (Beyotime biotechnology Co., Shanghai, China) at 4 °C for 30 min. The suspension was centrifugated by refrigerated centrifugation at 14,000 rpm for 5 min, and the supernatant was retained and stored at 80 °C for further Gal T analysis. Before Gal T analysis, the proteins in each cell lysate sample were quantified with the bicinchoninic acid (BCA) protein assay kit (Dingguo Biological Products Co., Beijing, China). Cell lysate containing 50 μg proteins was added in the glycosylation reaction solution for each ECL test. 2.5. Apparatus and characterization Scanning electron microscope (SEM) images were obtained with an SU8010 Scanning transmission electron microscope (Hitachi, Japan). UV–vis experiments were carried out with a UV-3900 spectrophotometer (Hitachi, Japan). The cyclic voltammetry was conducted on a CHI 660b instrument (CH Instrument Co., USA). Electrochemical impedance
Please cite this article as: Z. Chen, et al., Highly sensitive electrogenerated chemiluminescence biosensor for galactosyltransferase activity and inhibition detection using go..., Journal of Electroanalytical Chemistry (2016), http://dx.doi.org/10.1016/j.jelechem.2016.05.034
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spectroscopy (EIS) was carried out on a PARSTAT 2273 potentiostat/ galvanostat (Advanced Measurement Technology Inc. USA). The cyclic voltammetry and Impedance measurements were performed in a 5 mM K3[Fe(CN)6]/K4[Fe(CN)6] redox probe solution with 0.5 M KCl. Impedance measurements were completed by applying an AC voltage of 10 mV amplitude with frequency from 0.1 Hz to 105 Hz. The ECL measurements were carried out on an MPI-B multifunctional electrochemical analytical system (Xi'an Remex Analytical Instrument Ltd. Co., China). The voltage of the photomultiplier tube (PMT) was maintained at 600 V. 3. Results and discussion Scheme 1 shows the principle and construction of the ECL biosensor for Gal T activity analysis. Firstly, a self-assembly monolayer (SAM) of MUA and MEH was formed on the gold electrode to provide the carboxyl groups for glycoprotein modification. The glycoprotein GlcNAc-BSA was immobilized onto the electrode surface as a receptor for the glycosylation reaction. Then, the galactose was transferred to GlcNAc-BSA in the presence of UDP-Gal donor and Gal T, followed by the capture of AIA-XOD@GNR nanoprobes onto the gold electrode surface via the specific affinity between galactose and AIA. The ECL reaction was generated and greatly amplified by the gold nanorods and the enzyme-catalyzed reaction, which can be applied for Gal T activity detection. 3.1. Characterization of AIA-XOD@GNR nanoprobes To evaluate the modification and catalytic activity of as-prepared AIA-XOD@GNR nanoprobes, the HRP-catalyzed colorimetric reaction was used. As shown in Fig. S2 (Supporting information), in the presence of H2O2, HRP could catalyze the oxidation of ABTS, generating a bluegreen colorimetric signal, which can be easily monitored by the naked eye and the UV–Vis absorption spectra. However, when the H2O2 was replaced by HA, no significant signal was observed. Then, by adding XOD into the solution containing HRP, ABTS and HA, the oxidation of HA could be catalyzed and H2O2 was generated, resulting the colorimetric signal. Sequentially, the as-prepared AIA-XOD@GNR nanoprobes were added into the solution containing HRP, ABTS and HA, similar colorimetric signal was obtained, demonstrating the successful modification of XOD on the GNRs and the good catalytic property of the AIAXOD@GNRs. While, as a control test, the GNRs were mixed with HRP,
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ABTS and HA, and no colorimetric signal was observed, furtherly confirming the catalytic activity of the nanoprobes. 3.2. Electrochemical characterizations of the biosensor The assembly processes of the modified electrodes were characterized using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) step by step. Fig. 1A displays the CV curves of the modified gold electrode using Fe(CN)4−/3− as the electroactive probes. 6 Firstly, on the bare electrode, a couple of reversible redox peaks were observed (curve a). After the modification of the SAM, the peak currents decreased and the potential gap increased (curve b) owing to the poor conductivity of the SAM. After the modification of GlcNAc-BSA, the peak current further dropped dramatically and the potential gap increased as well (curve c) due to the electronic inert property of the glycoprotein. After being blocked the excess active sites by BSA, the conjugation of galactose to GlcNAc-BSA was made under the glycosylation reaction catalyzed by Gal T (curve d), inducing a slight decrease of the peak currents. Finally, the electrode was incubated the AIA-XOD@ GNR nanoprobes. As shown in curve (e), the binding of nanoprobes resulted in an increase in peak currents and a decrease in the potential gap between the anodic and cathodic peaks, indicating the nanoprobes possessed good feature of electron transfer and mass transfer of Fe(CN)4−/3− on the electrode interface. These results confirm the 6 successful construction of the ECL biosensor. Meanwhile, the impedance spectra shown in Fig. 1B also verify the fabrication processes of the biosensor. The nyquist plot comprises a semicircular part at higher frequency range and a straight linear part at lower frequency range. The diameter of the semicircle equals to the electron transfer resistance (Ret) at the electrode interface. Due to the good electronic transfer ability, the bare gold (curve a) exhibited the lowest Ret. Then the diameter of the semicircles increased successively with sequential formation of SAM (curve b), GlcNAc-BSA immobilization (curve c) and glycosylation reaction (curve d). Finally, with AIAXOD@GNR modification (curve e), the Ret again decreased. These changes were similar to those CV results, further confirming the successful assembly of the biosensor. 3.3. ECL behaviors of the biosensor The ECL behavior of the biosensor was characterized with the different modifications on the electrode in 0.1 M PBS (pH 8.5) containing
Scheme 1. The procedures for the fabrication of the ECL biosensor and β-1,4-galactosyltransferase activity analysis.
Please cite this article as: Z. Chen, et al., Highly sensitive electrogenerated chemiluminescence biosensor for galactosyltransferase activity and inhibition detection using go..., Journal of Electroanalytical Chemistry (2016), http://dx.doi.org/10.1016/j.jelechem.2016.05.034
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cannot be transferred from UDP-Gal to the N-acetylglucosamine groups on GlcNAc-BSA, and then nanoprobes cannot be adsorbed onto the electrode for signal amplification without the specific recognition between AIA and galactose. As shown in curve c and d, after the Gal T catalyzed glycosylation reaction on the electrode and the following AIA-XOD@ GNRs incubation, the GlcNAc-BSA modified electrode exhibited a sensitive ECL emission starting at about 0.4 V with a peak at around 0.58 V. While the AIA-XOD@GNRs were not immobilized onto the electrode, the ECL intensity was quite slight, close to the baseline. These results demonstrate the AIA-XOD@GNR nanoprobes can increase ECL intensity effectively and this ECL biosensor can be applied for sensitive and selective analysis of Gal T activity. 3.4. Optimization of assay conditions
Fig. 1. Cyclic voltammograms and electrochemical impedance spectra of bare gold (curve a), SAM modified gold electrode (curve b), GlcNAc-BSA modified electrode (curve c), β1,4-Glycosylated modified electrode (curve d) and after the incubation with probe (curve e) in 0.5 M KCl solution with 5 mM [Fe(CN)6]3−/4− electroactive probes, respectively. CV Scan rate: 100 mV s−1. EIS Frequency range: 0.1 ~ 105 Hz.
5 mM HA and 100 μM luminol during the CV scanning. As shown in Fig. 2, when the modified electrode was incubated in the enzymic precursor solution without Gal T or UDP-Gal followed by incubating AIA-XOD@ GNR nanoprobes, no detectable ECL signal was observed (curve a and b). This result indicates that, without Gal T and UDP-Gal, galactose
Fig. 2. ECL-potential curves of GlcNAc-BSA modified electrodes before (a) and after (b) the incubation with probe (AIA-XOD@Au NRs), and GlcNAc-BSA modified electrodes before (c) and after (d) the incubation with probe after the β-1,4-galactosylation reaction. The ECL measurements were performed in 0.1 M PBS (pH 8.5) containing 100 μM luminol and 5 mM hypoxanthine. Scan rate: 100 mV s−1. PMT: 600 V.
As for the enzymatic glycosylation reaction, it is generally influenced by some external factors, such as pH and temperature. In order to improve the Gal T activity detection performance, the effects of pH, temperature and incubation time of glycosylation reaction were investigated. As shown in Fig. 3A, the ECL signal gradually increased from pH 5.5 to pH 7.4, and then decreased. This result indicates Gal T possesses high activity at pH 7.4. In the study of temperature for glycosylation reaction (Fig. 3B), it is observed that relative ECL intensity increased with the temperature up to 37 °C. After that, the ECL changed decreasingly by the continue increase of temperature, suggesting Gal T had high activity at 37 °C. The resultant optimal pH and temperature of 7.4 and 37 °C were in agreement with the physiological conditions. In addition, as shown in Fig. 3C, it was found that with the increase of the reaction time, the ECL signal increased and finally reached a plateau in 3 h, suggesting a tendency to complete the glycosylation process. Accordingly, the optimal reaction time was chosen to be 3 h. Besides, to get a sensitive ECL signal, the optimization of the pH value of ECL electrolyte solution was performed (Fig. S3 in Supporting information). The pH 8.5 was selected as the optimal condition for ECL electrolyte solution preparation. Then, the molar ratio of XOD to AIA immobilized on gold nanorods was also optimized (Fig. S4 in Supporting information). The optimal molar ratio 4.5/1 of XOD to AIA was used for nanoprobe preparation. 3.5. ECL biosensor for Gal T activity detection On the basis of the optimal experimental conditions, the ECL biosensor was applied for detecting Gal T activity. The relationship of the ECL responses with Gal T concentrations is displayed in Fig. 4. Measurements were performed three times for each test. It can be seen that the ECL signals gradually increased with the increasing of Gal T activity and reached a plateau when the concentrations of Gal T rose up to 0.5 U mL−1. The ECL intensity changes are proportional to the logarithmic value of Gal T activity in the range from 0.001 U mL− 1 to 0.1 U mL−1. The linear relationship can be described as ΔIECL (a.u.) = 1167 × Log UGal T (U mL− 1) + 4398 with the correlation coefficient R2 = 0.990, where ΔIECL is ECL intensity of glycosylated GlcNAc-BSA modified electrode at different Gal T concentration after the AIAXOD@GNR incubation, and UGal T is the concentration of Gal T. The detection limit was obtained to be 9 × 10−4 U mL−1 at signal-to-noise ratio of 3, which was much lower than the earlier reported method [35]. The high sensitivity of the ECL biosensor could be ascribed to the dual amplification of the ECL signal by the GNRs and the enzyme-catalyzed reaction. Here, the GNRs are not only used to electrocatalyze the ECL process of luminol but also acted as a carrier to load more AIA and XOD. Owing to its excellent electron-transfer ability, the gold nanorods also improved the electron transfer at the electrode interface, thus further improved the performance of the as-designed ECL sensor. Moreover, the stability of the ECL biosensor was characterized by successively scanning in 0.1 M PBS containing 5 mM HA and 100 μM luminol for ten cycles (Fig. S5 in Supporting information). The relative
Please cite this article as: Z. Chen, et al., Highly sensitive electrogenerated chemiluminescence biosensor for galactosyltransferase activity and inhibition detection using go..., Journal of Electroanalytical Chemistry (2016), http://dx.doi.org/10.1016/j.jelechem.2016.05.034
Z. Chen et al. / Journal of Electroanalytical Chemistry xxx (2016) xxx–xxx
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Fig. 4. (A) ECL-potential curves with various activity units of β-1,4-galactosyltransferase (top to bottom, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0 U mL−1) in 0.1 M PBS (pH 8.5) containing 100 μM luminol and 5 mM hypoxanthine. Scan rate: 100 mV s−1. PMT: 600 V. (B) The dependence of ECL intensity on the activity concentration of Gal T. inset: the linear relationship between the ECL intensity with the logarithm of Gal T concentration.
Fig. 3. Optimization of the pH of enzymatic β-1,4-galactosylation (A), enzymatic β-1,4galactosylation temperature (B), and enzymatic β-1,4-galactosylation time (C). The ECL measurements were performed in 0.1 M PBS (pH 8.5) containing 100 μM luminol and 5 mM hypoxanthine. CV Scan rate: 100 mV s−1. PMT: 600 V. β-1,4-galactosyltransferase concentration: 0.05 U mL−1.
(human hepatocyte cell line) were measured. Fig. S6 (Supporting information) displays the ECL response of cellular Gal T in HeLa cell lysate from 500 cells. The detection limit of cell number for Gal T activity analysis in cell lysates was obtained as 150 cells. As shown in Fig. 5, various Gal T activity levels were obtained in different cell lines. Clearly, as a normal cell line, HL-7702 cell line expressed lower level of Gal T than the other three disease related immortal cell lines. This result reveals that the intracellular galactosyltransferases and glycans have significant roles in cell carcinogenesis. These experimental results furtherly confirm that the as-prepared ECL biosensor can be used to determine Gal
standard deviation was 4.2%, signifying that the ECL sensor possessed excellent potential cycling stability. 3.6. Detection of Gal T activities in cell lysates Gal T plays important roles in a variety of cellular processes such as cell apoptosis, proliferation, differentiation, and cell–cell communications. It is also served as a biomarker for diagnostic and prognostic. Here, The Gal T activities of four cells, HeLa cell (human cervical cancer cell line), CCRF-CEM cell (human leukemic lymphoblasts), SMMC-7721 cell (human hepatocellular carcinoma cell line) and HL-7702 cell
Fig. 5. The Gal T activity analyzed from HeLa, CCRF-CEM, SMMC-7721 and HL-7702 cell lysates. Cell lysate containing 50 μg proteins was used for each ECL test.
Please cite this article as: Z. Chen, et al., Highly sensitive electrogenerated chemiluminescence biosensor for galactosyltransferase activity and inhibition detection using go..., Journal of Electroanalytical Chemistry (2016), http://dx.doi.org/10.1016/j.jelechem.2016.05.034
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Z. Chen et al. / Journal of Electroanalytical Chemistry xxx (2016) xxx–xxx
T activity in complex biological samples with high sensitivity and accuracy.
Conflict of interest There is no conflict of interest.
3.7. Screening inhibitors of Gal T Inhibitors of glycosyltransferases are invaluable as tools for understanding the function of these enzymes and relative glycans. The proposed biosensor was applied to rapidly evaluate the effectiveness of various Gal T inhibitors. Ten candidates, uridine-5′-diphosphogalactose (UDP-Gal), uridine-5′-diphosphoglucose (UDP-Glu), uridine-5′diphospho-N-acetylglucosamine (UDP-N-GlcNAc), uridine-5′monophosphate (UDP), uridine-5′-triphosphate (UMP), uridine-5′-triphosphate (UTP), adenosine-5′-diphosphate (ADP), Benzyl 2acetamido-2-deoxy-α-D-galactopyranoside (BG), galactose (Gal) and glucose (Glu) were chosen and detected. The screening result, in Fig. 6, shows that UDP, UTP, ADP and Gal had potent inhibition, especially UDP. It is consistent with the previous reported work that Gal T enzymic reaction was observably perturbed by UDP and UTP [36]. However, BG, a well known inhibitor of α-2,3-sialyltransferase which extends a GalNAc [37,38], didn't show effective inhibition to galactosylation in the detection. In addition, the IC50 (50% inhibitory concentration) of UDP was further determined by the biosensor. Fig. S7 (Supporting information) shows the value of IC50 for UDP to be 211 μM, which was in agreement with that based on mass spectrometry assay [36]. From the results above, it is indicated that the constructed biosensor provides a sensitive method for rapidly screening glycosyltransferases inhibitors.
4. Conclusion In summary, a sensitive ECL biosensor for β-1,4galactosyltransferases (Gal T) activity analysis has been developed based on the specific recognition between lectin and galactose, integrating with the gold nanorods and enzymatic dual amplification of ECL signal. Via the specific interaction between AIA and galactose, the AIA and XOD conjugated gold nanorods were absorbed onto the bioelectrode interface and greatly enhanced the ECL signals due to the excellent electrocatalytic activity, large surface area, and good conductivity. The proposed ECL biosensor showed high sensitivity and specificity for Gal T activity analysis with a low detection limit of 9 × 10−4 U mL−1. The platform has been applied for the Gal T activity analysis in different cell lines and screening of Gal T inhibitors, which provides a valuable tool for glycosyltransferase activity evaluation and inhibition in clinic diagnostic.
Fig. 6. Evaluation of Gal T activity inhibition. The ECL intensity as a function of different inhibitors with concentration of 1 mM in 0.1 M PBS (pH 8.5) containing 100 μM luminol and 5 mM hypoxanthine. Scan rate: 100 mV s−1. PMT: 600 V. The enzymatic reaction was carried out with 0.05 U mL−1 Gal T.
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