Electrochemiluminescence Investigation of Glucose Transporter 4

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Electrochemiluminescence Investigation of Glucose Transporter 4 Expression at Skeletal Muscle Cells Surface Based on a Graphene Hydrogel Electrode Gen Liu, Cheng Ma, Baokang Jin, Zixuan Chen, Faliang Cheng, and Jun-Jie Zhu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05340 • Publication Date (Web): 29 Jan 2019 Downloaded from http://pubs.acs.org on February 3, 2019

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Analytical Chemistry

Electrochemiluminescence Investigation of Glucose Transporter 4 Expression at Skeletal Muscle Cells Surface Based on a Graphene Hydrogel Electrode Gen Liua,c, Cheng Mac, Bao-Kang Jina ,*, Zixuan Chenc ,*, Fa-Liang Chengb and Jun-Jie Zhuc a

College of Chemistry & Chemical Engineering, Anhui University, Hefei, 230601, China

b

Guangdong Engineering and Technology Research Center for Advanced Nanomaterials, School of Environment

and Civil Engineering, Dongguan University of Technology, Dongguan, 523808, China c

State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering,

Nanjing University, Nanjing, 210023, China

*To whom correspondence should be addressed: [email protected] (B.-K. Jin), [email protected] (Z. Chen)

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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

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ABSTRACT In situ detection of the expression level of cell-surface receptors has become a hotspot study in recent years. We propose in this manuscript a novel strategy for sensitive electrochemiluminescence (ECL) detection of glucose transporter 4 (GLUT4) on human skeletal muscle cells (HSMC). Graphene hydrogel (GH) was selected to fabricate a permeable electrode with purpose of overcoming the steric hindrance of cells on electrode, which leads errors in the detection of cell-surface receptors. GLUT4 was labeled with carbon dots (CDs), which generate ECL emission at the interface between GH and cells, so about half amount of GLUT4 expressed at cell-surface could be determined, which provided an accurate GLUT4 expression quantification. The prepared cytosensor exhibited a good analytical performance for HSMC cells, ranging from 500 to 1.0 × 106 cells·mL-1, with a detection limit of 200 cells·mL-1. The average amount of GLUT4 on per HSMC cell was calculated to be 1.88 × 105. Furthermore, GLUT4 on HSMC surface had a 2.3-fold increase under the action of insulin. This strategy is capable of evaluating the receptors on cell-surface, which may push the application of ECL for the disease diagnosis.

Keywords: Electrochemiluminescence, Insulin, Glucose transporters 4, Graphene hydrogel

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Cell-surface receptors play a pivotal physiological roles in regulating a diverse range of cellular processes, and their abnormal expression results in scores of pathological conditions. 1,2 As a result, in-situ assay of receptors is crucial for the accurate diagnosis of diseases. Glucose transporter (GLUT) is significant for metabolism of glucose in cells of diverse organisms from microbes to human, because they take charge of glucose uptake and maintain the balance of the glucose homeostasis. 3,4 As one of 14 human GLUT family members, GLUT4 mainly exists in muscle cells. 4 More importantly, the amount of GLUT4 in plasma membrane is controlled by insulin, 5 whose aberration will bring about type2 diabetes mellitus and obesity. 6,7 Therefore, sensitive analysis of GLUT4 on cell-surface is of great significance for research of disease processes. Current biological techniques for GLUT4 detection mainly include immunofluorescence and western blotting assay.

8-11

However, most of them bear

time-consumption or sophisticated operations. Besides, these methods only offered the relative abundance of GLUT4. ECL integrates the advantages of electrochemistry and chemiluminescence and has been a powerful analytical tool in fields of bioanalysis, imaging and light-emitting devices.

12-16

ECL is a potential way for

studying of cell-surface receptors. Unfortunately, only receptors associated ECL probes at the boundary can generate ECL because of the steric hindrance of cells on electrode surface,

17,18

which increases some

measurement error when we analyze the receptors at cell-surface. 19 In our previous work, 20 we applied permeable chitosan film to increase the space between cells and electrode. ECL reagents in bulk solution could detour around the cell and enter chitosan film. It is concluded that a permeable substrate is crucial to eliminate the steric hindrance effect originated from cells. However, the electrical conductivity of chitosan is poor. Electrode and cells are separated by chitosan film, thus it is difficult to excite the ECL probes labeled on cells to generate ECL. If the permeable substrate also possesses good conductivity, it is possible for the ECL probes between electrode and cells to generate luminescence. Fortunately, conductive hydrogels can meet these requirements. Thus, conductive hydrogels are promising materials to study the ECL events occurred below cells and can be used as an ideal biosensor electrode substrate. Among various kinds of hydrogels, graphene hydrogel (GH) is a strong candidate and has been widely used in the fields of supercapacitors,

21

cell culture,

22

water purification

23

and

electrochemical sensors. 24,25 Here we proposed a GH-based ECL biosensor for quantitative detection of GLUT4 on the surface of HSMC cells. The GH electrode showed a good permeability, avoiding the steric hindrance of cells. As shown in Scheme 1, glucose transporter 1 antibody (GLUT1-Ab)-modified gold nanoparticles were immobilized on the GH electrode to specifically capture GLUT1 on the membrane of HSMC cells. Carbon dots are carbon nanoparticles 3



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less than 10 nm. HSMC cells were labeled with GLUT4-Ab-functionalized carbon dots (CDs-GLUT4-Ab) via the specific recognition of GLUT4-Ab to GLUT4, and then immobilized on the GH-based electrode. When electrochemical reactions occurred, the coreactant K2S2O8 diffused across the permeable structure and reacted with CDs on the bottom surface of cells, generating the ECL emission. Thus, GLUT4 at the bottom surface (about half amount of GLUT4 at cell-surface) of HSMC cells could be determined. The average number of cell-surface GLUT4 using a GH-based electrode was more accurate than that determined with the impenetrable glass carbon electrode (GCE) electrode. The GH-based electrode contributed to improve the accuracy when we analyzed cell-surface receptors and had proved to be a practical tool to investigate the GLUT4 expression.

EXPERIMENTAL SECTION Materials, apparatus, synthesis of CDs, synthesis of GH as well as cell culture are presented in the Supporting Information.

Fabrication of Cytosensor and ECL assay. The schematic diagram of the ECL cytosensor fabrication was displayed in Scheme 1. GH was used as a working electrode (Figure S2A, Supporting Information). AuNPs deposited on GH by amperometric technique (i-t) at -0.8 V in 0.5 mM HAuCl4 solution containing 0.1 M KCl for 1 min. Subsequently, the GH/AuNPs electrode was dipped in GLUT1 antibody solution (GLUT1-Ab, 50 ng·mL−1, to recognize the normally expressed GLUT1 at HSMC surface) and incubated in a moisture-saturated environment overnight at 4 °C to immobilize GLUT1-Ab. Afterwards, the modified electrode (abbreviated as GH/AuNPs/GLUT1-Ab) was soaked in 1% BSA solution at 4 °C for 20 min to block the nonspecific binding sites. This electrode was denoted as GH/AuNPs/GLUT1-Ab/BSA. 15 mM NHS and 75 mM EDC were added to 5 mL of CDs solution (2 mg·mL-1) to activate the carboxylic groups on CDs surface at 37 °C for 60 min. Subsequently, the activated CDs were reacted with GLUT4 antibody (GLUT4-Ab) aqueous solution (50 ng·mL−1) at 37 °C for 12 h. After that, the precipitum was centrifuged at 18000 rpm for 20 min and then washed with ultrapure water. The final product CDs-GLUT4-Ab was redispersed in 0.5 mL of PBS (10 mM, pH 7.4) and stored at 4 °C. The HSMC cells treated by 4% paraformaldehyde (PFA) were incubated with a certain amount of CDs-GLUT4-Ab probes for 90 min at room temperature. The ECL probes were linked on the surface of HSMC through the specific binding of the GLUT4 on HSMC surface with the GLUT4-Ab on CDs-GLUT4-Ab. Here, we denoted such cells as cell@CDs-GLUT4-Ab. Then, the mixed solution was centrifuged twice at 1000 rpm for 5 min, and the final sediment was resuspended with 1.0 mL of phosphate buffer solution (PBS, 10 mM, pH 7.4) for the next stage of experiments. 4


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200 μL of cell@CDs-GLUT4-Ab suspension at a certain concentration was dropped on the as-prepared GH/AuNPs/GLUT1-Ab/BSA electrode and incubated for 60 min at room temperature (Figure S2B, Supporting Information). In this step, HSMC cells were captured by GH/AuNPs/GLUT1-Ab/BSA through the specific interaction between GLUT1-Ab on electrode and GLUT1 at HSMC surface. Finally, the obtained electrode was washed

with

PBS

to

remove

the

free

cells.

This

ECL

cytosensor

was

denoted

as

GH/AuNPs/GLUT1-Ab/BSA/cell@CDs-GLUT4-Ab. Finally, the assembled ECL cytosensor was immersed in PBS (10 mM, pH 7.4) with K2S2O8 (0.1 M) and scanned from 0 to -1.8 V at a scan rate of 0.1 V·s-1. In addition, the ECL signal was recorded under a photmultiplier tube (PMT) voltage of 800 V.

RESULTS AND DISCUSSION Characterization of CDs and GH-based electrodes. The morphology and size of CDs were characterized with high-resolution transmission electron microscopy (HETEM). As shown in Figure 1A, CDs were spherical in shape with an average size of ~ 6 nm. Its lattice spacing was ~ 0.21 nm (inset in Figure 1A), which was close to the (100) facet of graphite carbon.26 Subsequently, CDs were characterized by fluorescence, UV-vis absorption and FT-IR spectrum. When excited at 410 nm, the CDs exhibited a maximum emission peak at 517 nm (Figure 1B). And The UV–Vis absorption peak of CDs occurred at 332 nm and 413 nm (Figure 1C). Besides, CDs had many FT-IR characteristic peaks, as shown in Figure 1D, such as O–H / N–H (3426, 3332, 3199 cm−1), C–H (2813 cm−1), C=O / N-H (1668, 1619 cm−1) and C–O / C–N / C–H (1444, 1326, 1188 cm−1). Additionally, the mean zeta potential of CDs was measured to be -31.4 mV, indicating that there were plenty of carboxyl groups at the surface of the CDs. Figure 2A, B and C were the scanning electron microscopy (SEM) images of freeze-dried GH, GH/AuNPs and GH/AuNPs/GLUT1-Ab, respectively. Figure 2A showed that the freeze-dried GH had a well-defined and interconnected 3D porous network and the pore walls consisted of thin layers of stacked graphene sheets. Figure 2B was the SEM image of AuNPs on GH. AuNPs attached to GH sheets, and the diameter of AuNPs was ∼50 nm, see the inset in Figure 2B. Besides, Figure S3 (Supporting Information) depicted the X-ray diffraction (XRD) patterns of GH and GH/AuNPs. The diffraction peaks at 2θ = 24.7º and 43.2º were assigned to (002) and (100) crystal planes of the graphene, 27 respectively, while the major peaks of AuNPs located at 2θ = 38.0º, 44.2º, 64.3º and 77.5º, corresponding to (111), (200), (220) and (311) lattice plane of the gold, 5


28

respectively. This also


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indicated that GH/AuNPs was the combination of the independent GH and AuNPs. After GLUT1-Ab was modified on GH/AuNPs, as shown in Figure 2C, some sheets stuck together, collapsed and formed an uneven and incompact surface.

Steric hindrance of cells on GH-based electrode. To visually clarify that steric hindrance of cells on GH was eliminated, we performed ECL imaging experiments in L012 solution (8-Amino-5-chloro-7-phenylpyrido [3,4-d]pyridazine-1,4(2H,3H)-dione, a luminol analogue).20 Cells on GCE/AuNPs/GLUT1-Ab/BSA and GH/AuNPs/GLUT1-Ab/BSA were localized with fluorescence 2-NBDG (a nontoxic and fluorescent D-glucose derivative), as shown in Figure 3A and C. GCE/AuNPs/GLUT1-Ab/BSA presented shadows of cells (Figure 3B), because cells on GCE hindered the diffusion of L012 from bulk solution to electrode surface, which made it difficult for L012 to participate in ECL reaction below cells. On the contrary, no shadow was observed on GH/AuNPs/GLUT1-Ab/BSA surface (Figure 3D), confirming that GH-based electrode was beneficial to eliminate the steric hindrance of cells. In addition, this conclusion was verified by the electrochemical impedance spectra (EIS), as shown in Figure S4 (Supporting Information). According to the report, 17,19 only the receptors with ECL probes at cell-edge could emit light due to the blockage of cells on impenetrable electrodes. As a result, the ECL signals obtained using GCE failed to calculate the receptors on the whole cell-surface. Unlike it, in this work, sufficient coreactant K2S2O8 could diffuse from bulk solution to GH and react with the CDs at the cell membrane facing GH. Since the GLUT4 at the bottom surface of HSMC cells was effective, about half amount of GLUT4 expressed at cell-surface could be estimated using the ECL detection system equipped with a photomultiplier tube. Unfortunately, the light of CDs on cells failed to support ECL imaging test.

ECL performance of GH-based electrode. The optimization of ECL detection conditions were described in the Supporting Information. The ECL behavior of the cytosensor was investigated in PBS (10 mM, pH 7.4) containing 0.1 M KCl, 0.1 M K2S2O8 and 10.0 mM Fe(CN)63−/4- (Figure S7, Supporting Information). Compared with GH, GH/AuNPs and GH/AuNPs/GLUT1-Ab/BSA, GH/AuNPs/GLUT1-Ab/BSA/cell@CDs-GLUT4-Ab showed an obvious ECL signal, because the CDs on cell-surface could be excited on GH surface and generate ECL. In contrast, chitosan (CS) film covered electrode, another permeable electrode in our previous work, 20 was also used to detect the CDs-labeled cells. Nevertheless, it was difficult to obtain a distinct ECL response because of the poor conductivity of CS film, though the steric hindrance of cells on CS film was also eliminated. The specific discussion was presented in the Supporting Information (Figure S8 and S9). 6


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In brief, considering the permeability and conductivity, the GCE-based electrode just provided excited ECL probes at cell-edge (Figure 4A), the GH-based electrode supported the excited ECL probes at the bottom surface of cells (Figure 4B), but the chitosan film-based electrode failed to excite the ECL probes on cell-surface (Figure 4C). Therefore, GH was an excellent substrate to study cell-surface receptors.

Quantitative Detection of HSMC Cells. GH-based ECL cytosensor was further employed to quantitatively detect HSMC cells. As shown in Figure S10 (Supporting Information), the ECL intensity increased with the increasing concentration of HSMC cells, and the ECL intensity was proportional to the logarithms of HSMC cell concentrations in range from 500 to 1.0 × 106 cells·mL-1. The linear regression equation was IECL = 1737 log Ccells - 3053 with a correlation coefficient (r) of 0.9904. The detection limit of HSMC cells was ~ 200 cells·mL−1 (S/N = 3). Considering that 200 μL of HSMC cells were used for incubation, the ECL approach achieved a detection limit of 40 HSMC cells.

Evaluation of GLUT4 expression on cell-surface. To evaluate the GLUT4 expression on HSMC cell surface, we adopted a displacement binding method. 29-31 Briefly, a certain concentration of GLUT4 was added in CDs-GLUT4-Ab solution (0.5 mL) to block the GLUT4-Ab on ECL probes. Afterward, the HSMC cells (5.0 × 105 cells·mL−1, 1.0 mL) were incubated with the probes (2 mg·mL-1, 60 μL). The CDs-labeled cells were separated by centrifuging and then diluted to 1.0 mL with PBS. Because the blocked CDs-GLUT4-Ab probes failed to recognize the GLUT4 on HSMC surface, the amount of CDs-GLUT4-Ab on cell-surface decreased, causing the declined ECL in comparison with the cells with unblocked ECL probes. Figure 5A exhibited that the ECL signal was obviously weakened with the increasing amount of recombinant GLUT4 (CGLUT4) on ECL probes. The plot of the decrease in ECL intensity (ΔIECL) and the amount of recombinant GLUT4 grew in range from 0.5 to 3.5 ng·mL-1, and finally reached a plateau at 3.5 ng·mL-1, corresponding to the excess foreign GLUT4 (Figure 5B). The plot of ΔIECL versus CGLUT4 showed a linear calibration in the range of 1.0 ~ 3.5 ng·mL-1. The linear regression equation was: ΔIECL = 1350 CGLUT4 + 459.4 (R = 0.9931). The recombinant GLUT4 of 3.5 ng·mL-1 could be considered as the complete substitute of the GLUT4 on cell-surface, so the average amount of GLUT4 on a single HSMC cell-surface (CGLUT4/CCell × 2NA) was estimated to be 1.88 × 105. Herein, NA was Avogadro’s number, the molecular mass of GLUT4 was ~ 45 kDa, 11 the cell concentration was 5.0 × 105 cells·mL−1 and the sample volume was 200 μL. Importantly, the cell density on electrode was stable, though various concentration of recombinant GLUT4 was introduced to block CDs-GLUT4-Ab probes (Figure S11, Supporting Information), 7



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indicating that the declined ECL signal for the GLUT4 expression resulted form the reduced CDs-GLUT4-Ab on cell-surface. By contrast, GCE-based ECL cytosensor was also used to evaluate GLUT4 on cell-surface, but it exhibited a lower measured value (Figure S12, Supporting Information). This protocol was further applied in the evaluation of alterations of cell-surface GLUT4 under the action of insulin (Figure S13 and S14, Supporting Information ), a model drug to motivate GLUT4 expression. 6,32,33 Based on the proposed method, the average number of GLUT4 on a single HSMC cell-surface was calculated to be 4.28 × 105. This result revealed that insulin could cause a 2.3-fold increase of GLUT4 on HSMC surface, which was close to the literature. 5

Selectivity and stability. The selectivity is essential to evaluate the availability of a cytosensor. Accordingly, the proposed electrode was used to test other kinds of cells, such as human breast adenocarcinoma cells (MCF-7) and murine macrophages (RAW 264.7), which showed no presence or low expression of GLUT1 and GLUT4 at cell-surface. 4 Figure 6A displayed that a strong ECL signal was observed in the presence of HSMC cells (5.0 × 105 cells·mL−1), but the signals of MCF-7 and K562 cells (5.0 × 105 cells·mL−1) were very weak, since few CDs were linked to MCF-7 and K562 cell-surface. And it was difficult to capture these cells using GH/AuNPs/GLUT1-Ab/BSA. The results suggested the good selectivity of GH/AuNPs/GLUT1-Ab/BSA. Figure 6B depicted the ECL intensity upon continuous scanning for 15 cycles. No significant ECL change was observed. Moreover, the cytosensor was stored in the PBS at 4 °C for one week, the ECL signal attenuated almost 5.7%, signifying a satisfactory stability of the cytosensor.

CONCLUSION In summary, a GH-based ECL cytosensor without steric hindrance of cells on electrode was developed, which could rapidly detect cells and evaluate GLUT4 expression at cell-surface. Thanks to the permeability of graphene hydrogel, the steric hindrance of cells on electrode was eliminated, facilitating the diffusion of co-reactant. ECL reaction occurred on the contact interface between graphene hydrogel and HSMC cells rather than at cell-edge, which improved the accuracy of the evaluation of GLUT4 at cell-surface in comparison to the impenetrable GCE-based electrode. This protocol provided a useful tool for investigating receptor expression at cell-surface and had potential application in clinical analysis and disease diagnosis. Meanwhile, this work showed the broad application prospect of conductive hydrogel in cell-based biosensor research, especially the ECL 8


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cytosensors. Additionally, the ECL assays without steric hindrance of cells contributed to pave the way for the development of ECL imaging of receptors at cell-surface for the high-throughput analysis.

ASSOCIATED CONTENT Supporting Information. Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Authors [email protected] (B.-K. Jin), [email protected] (Z. Chen)

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This research is supported by National Natural Science Foundation of China (Grants Nos.21427807, 21335004, 21605081 and 21375001), Guangdong Provincial Key Platform and Major Scientific Research Projects for Colleges and Universities (No. 2015KCXTD029), Natural Science Foundation of Jiangsu Province (Grants No BK20160638), and Fundamental Research Funds for the Central Universities.

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Figure Captions Scheme 1. The fabrication process of the ECL cytosensor.

Figure 1. High-resolution TEM image (A), fluorescence (B), UV-vis absorption (C) and FT-IR (D) spectra of of CDs. The inset in image (A) shows the lattice spacing of CDs.

Figure 2. SEM images of the freeze-dried (A) GH, (B) GH/AuNPs and (C) GH/AuNPs/GLUT1-Ab. The inset in image (B) displays the AuNPs on GH sheets.

Figure 3. Fluorescence images of (A) GCE/AuNPs/GLUT1-Ab/BSA/cell@CDs-GLUT4-Ab and (B) GH/AuNPs/GLUT1-Ab/BSA/cell@CDs-GLUT4-Ab;

ECL

GH/AuNPs/GLUT1-Ab/BSA/cell@CDs-GLUT4-Ab

images

of

and

(C) (D)

GH/AuNPs/GLUT1-Ab/BSA/cell@CDs-GLUT4-Ab in a solution of 10 mM PBS (pH 7.4) and 400 μM L012.

Figure 4. Schematic principle for the excited CDs-GLUT4-Ab on (A) cell-GCE interface, (B) cell-GH interface and (C) cell-chitosan film interface.

Figure 5. (A) ECL curves obtained with recombinant GLUT4 concentrations of (from a to j) 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 ng·mL-1. (B) Plot of the recombinant GLUT4 concentration and the decrease of ECL intensity (ΔIECL). The data obtained on a GH/AuNPs/GLUT1-Ab/BSA/cell@CDs-GLUT4-Ab.

Figure 6. (A) Selectivity of these nanoprobes after incubation with different kinds of cells; (B) stability of ECL intensity of proposed cytosensor as time increases.

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Scheme 1

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Table of Contents (TOC)

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Electrochemiluminescence Investigation of Glucose Transporter 4

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