Functional Gold Nanoparticles as Sensing Probes for Concanavalin A

May 22, 2019 - The Supporting Information is available free of charge on the ACS Publications ... amount of ConA on model cells) and additional figure...
0 downloads 0 Views 4MB Size
Download PDF
Article Cite This: ACS Appl. Nano Mater. 2019, 2, 3348−3357

www.acsanm.org

Functional Gold Nanoparticles as Sensing Probes for Concanavalin A and as Imaging Agents for Cancer Cells Wen-Jie Chen, Karthikeyan Kandasamy, and Yu-Chie Chen* Department of Applied Chemistry, National Chiao Tung University, Hsinchu 300, Taiwan

Downloaded via RUTGERS UNIV on August 8, 2019 at 09:50:21 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

S Supporting Information *

ABSTRACT: Concanavalin A (ConA), which exists in jack bean, can specifically bind to either mannose or glucose units. Given that ConA is a potential anticancer agent for antihepatoma therapy, analytical methods that can be used for ConA detection in plant extracts or biological fluids are important. Labeling agents that can be used to distinguish cancer cells from noncancer cells are also useful for cancer diagnostics. In this study, maltose-directed synthesis of Au nanoparticles (NPs; Au@Maltose NPs) were generated from one-pot reactions and used as sensing probes toward ConA tetrameric forms. Results showed that the binding affinity between Au@Maltose NPs and ConA was desirable with a dissociation constant as low as ∼6.66 × 10−8 M. Moreover, the limit of detection against ConA was estimated to be ∼23 pM, which was the lowest ever reported when Au NPs were used as sensing probes against ConA. Moreover, Au@Maltose NPs were used as imaging agents for ConA-treated cancer cells, such as human hepatoma HepG2 cells and human breast T-47D cancer cells, which can overexpress mannose N-glycan units on the cell membrane. NIH 3T3 fibroblast cells, which are noncancer cells, were used for comparison. Results demonstrated that the imaging agent made from combined ConA and Au@Maltose NPs can be used to distinguish cancer cells from noncancer cells. KEYWORDS: gold nanoparticles, concanavalin A, imaging agents, cancer cells, localized surface plasmon resonance, LSPR

■

INTRODUCTION The surface of human cells is rich in carbohydrate-containing molecules, including glycolipids1 and glycoprotein.2−4 The interactions between carbohydrates on cell surface and specific proteins are involved in many biological processes, such as cellular adhesion,5 cell recognition,6 inflammation,7 cell differentiation,8 virus infections,9 and cancer metastasis.10 Thus, studying carbohydrate−protein interactions is important to realize numerous molecular processes in biological functions. Lectins derived from plants or animals are wellknown carbohydrate-binding proteins.11 For example, concanavalin A (ConA), a lectin derived from Canavalia ensiformis (jack bean) that can specifically bind to mannose and glucose units, is usually chosen as a model for studying carbohydrate− protein interactions.12−14 Given that ConA is considered as a potential anticancer agent for cancer therapy,15 detection of ConA from biological fluids or plant extracts is important. Thus, sensitive sensing methods against ConA must be developed. Conventional methods for studying carbohydrate−protein interactions, such as fluorescence spectroscopy-based approaches 16 and enzyme-linked lectinosorbent assay (ELLA),17 have been performed. Fluorescence labeling is often used for protein−carbohydrate interactions because of its high sensitivity. ELLA is a modified version of enzyme-linked © 2019 American Chemical Society

immunosorbent assay, in which lectins are conjugated with enzyme instead of antibodies. However, both methods are time-consuming because these methods require several steps, including labeling, incubation, and washing.18 Owing to their ease-of-modification, good biocompatibility, and optical properties,19,20 Au nanoparticles (NPs) have been used to probe interactions between carbohydrates and their target proteins.21 Au NPs possess unique localized surface plasmon resonance (LSPR) optical property, instilling Au NPs with size/shape-dependent color.22 Interparticle interactions among Au NPs also affect the resultant color.23,24 When the interparticle distance is less than the diameter of the particles, the absorption band of Au NPs resulting from surface plasmon resonance is broadened accompanied by a dramatic red shift to a prolonged wavelength, which is called surface plasmon coupling effect.25−27 Many carbohydrate-functionalized NPs have been developed as affinity probes to facilitate the detection of carbohydrate− protein interactions.28,29 Glycan ligands are usually immobilized on Au NPs through S−Au binding.30−33 For instance, trivalent α-2,6-thio-linked sialic acid ligand-functionalized Au Received: February 5, 2019 Accepted: May 21, 2019 Published: May 22, 2019 3348

DOI: 10.1021/acsanm.9b00220 ACS Appl. Nano Mater. 2019, 2, 3348−3357

Article

ACS Applied Nano Materials

Selectivity of Au@Maltose NPs. Protein samples including ConA, peanut lectin, Musa paradisiaca lectin (BanLec), ricin B subunit (ricin B), and bovine serum albumin (BSA) prepared in phosphate buffer saline (PBS) were selected as the model samples. PBS was prepared by mixing aqueous sodium phosphate dibasic (10 mM, 40 mL) with aqueous sodium phosphate monobasic (10 mM, 10 mL) followed by adjusting pH to 7.4. Additional Ca2+ (10 nM) and Mn2+ (10 nM) were added to PBS for facilitating the binding between Au@Maltose NPs and target proteins.43 The protein samples (1 nM, 194 μL) were individually shaken (150 rpm) with Au@Maltose NPs (0.16 mg mL−1, 6 μL) at 25 °C for 30 min. The resultant samples were examined by ultraviolet−visible (UV−vis) absorption spectroscopy. Using Au@Maltose NPs as Cellular Labeling Agents. Cellular imaging was conducted by seeding human hepatoma HepG2 cells, human breast T-47D cancer cells, and NIH3T3 cells (2 × 105 cells), which were cultured in minimum essential medium eagle (MEM) medium, Dulbecco’s modified Eagle medium-high glucose (DMEM) medium, and Roswell Park Memorial Institute (RPMI)-1640 medium (containing L-glutamine) medium, respectively, on a cover glass slide in a 6-well plate, placed in an incubator (5% CO2) at 37 °C for 24 h followed by elimination of media. The cells were rinsed twice by PBS (1 mL × 2). Subsequently, the cells were incubated with PBS (1 mL) containing NaCl (100 mM), Ca2+ (10 μM), and Mn2+ (10 μM) for 1 h. After incubation, PBS was removed, and ConA (1 μM, 1 mL) prepared in the same PBS was added followed by incubation at 37 °C for a given time (1, 2, and 3 h). The cells were rinsed by PBS (1 mL × 3). Au@Maltose NPs (1 mL, 16 μg mL−1) prepared in 4-(2hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) buffer (20 mM) containing NaCl (0.045%), Ca2+ (10 nM), and Mn2+ (10 nM)) were added to the rinse cells and incubated at 37 °C for 5 h. The medium containing Au@Maltose NPs was removed, and the remaining cells were rinsed by PBS (1 mL × 3). The cells were added with paraformaldehyde (1 mL, 4%) and incubated for 30 min to fix the cells on the glass slide. The treated cells were rinsed with PBS (1 mL × 3). To stain cellular nuclei, the treated cells were further added with a blocking solution (200 μL) containing 0.25% X-100 Triton and 10% fetal bovine serum prepared in PBS and incubated at 37 °C for 1 h to block nonspecific binding. The block solution was then eliminated from the cell sample. The remaining cells were added with nucleus dye, i.e., Hoechst 33342 (100 μL, 2 mg μL−1) and incubated at 37 °C for 30 min. Subsequently, the cells were rinsed with wash solution (1 mL × 3) containing X-100 Triton (0.25%) prepared in PBS. Glycerol (10 μL, 80%) was deposited on a glass slide, whereas the cover glass slide loaded with cells was covered on the glass slide. The glass slides were sealed by nail polish. The cell images of the resultant samples were examined by fluorescent microscopy.

NPs have been employed for colorimetric detection of human influenza virus.32 Thiolated mannose derivative immobilized NPs are also used as sensing probes for ConA detection with a limit of detection (LOD) of ∼0.04 μM.33 Desirable binding affinity arising between maltose-conjugated Au NPs and ConA with a dissociation constant of ∼12.59 nM has also been reported.34 The above-mentioned approaches using glycan derivatives as probe molecules require time-consuming, multisynthesis steps for the generation of glycan derivatives. Thus, the simplification of these tedious synthesis steps is desirable. The time spent in generation of functionalized NPs can be reduced. One-step generation of functionalized Au NPs using naturally available saccharides as the starting materials has been reported. Given that saccharides contain reducing ends, saccharide immobilized Au NPs can be easily generated by directly reacting saccharides and aqueous tetrachloroaurate through one-pot reactions by properly adjusting reaction conditions, such as temperature, time, and pH.35−37 Maltose is a disaccharide consisting of two units of glucose joined with an α(1→4) bond. Thus, maltose contains a reducing end on one glucose unit. Herein, we report an approach that generates maltose-encapsulated Au NPs (Au@Maltose NPs) through one-pot reactions. The generated functional Au NPs possessing glucose units were used as affinity probes to interact with ConA. ConA possesses the dimensions of approximately 60 Å × 70 Å × 70 Å 38 and contains four binding moieties against glucose/mannose at pH 7.39 If the asprepared Au@Maltose NPs (e.g., 10 nm) are larger than the size of ConA, apparent surface plasmon coupling effects resulting from the binding between ConA and the as-prepared Au NPs may be observed. As a consequence, apparent LSPR shift may be obtained. Therefore, the LOD of ConA using Au@Maltose NPs as sensing probes is expected to be very low. The cell membrane of human cancer cells, such as liver40 and breast cancer cells,41 overexpress much more mannose Nglycan units on membrane proteins than normal cells.42 That is, the cell membrane of liver and breast cancer cells can be immobilized with more ConA than that of normal cells. Thus, ConA-treated cancer cells can be labeled with more Au@ Maltose NPs than normal cells, and cancer cells can be distinguished from normal cells based on the favorable labeling of Au@Maltose NPs on their cell membrane. We also demonstrated the feasibility of using Au@Maltose NPs as labeling agents for cell samples after ConA treatment. An Au NP-based labeling approach that can be used to distinguish cancer cells from normal cells was explored by combining the use of ConA with Au@Maltose NPs as labeling agents.

■

■

RESULTS AND DISCUSSION

Characterization of Au@Maltose NPs. Figure 1A shows the UV−vis absorption spectrum of the generated Au@ Maltose NPs obtained from one-pot reactions by reacting aqueous tetrachloroaurate and maltose under alkaline conditions. The maximum absorption band appeared at a wavelength of ∼525 nm. The results showed that maltose could be used as the reducing agent, and Au@Maltose NPs were successfully produced through the one-pot reactions. The particle size of Au@Maltose NPs was then estimated by TEM. Figure 1B shows the representative TEM image of the generated Au@Maltose NPs, which are spherical. The particle size of Au@Maltose NPs was estimated to be 20.1 ± 4.9 nm by counting ∼200 NPs using ImageJ. The inset shows the size distribution of the generated Au@Maltose NPs. The ζ potentials of Au@Maltose NPs prepared at pH 6.4, 7.4, and 8.4 were estimated to be −17.8 ± 0.8, −19.9 ± 0.05, and −22.2 ± 0.04 mV by using a ζ potential meter, respectively.

EXPERIMENTAL SECTION

The details of reagents, materials, and instrumentation used in this study have been listed in Supporting Information. Synthesis of Au@Maltose NPs. Au@Maltose NPs were prepared by stirring aqueous maltose (61 mg mL−1, 450 μL), deionized water (500 μL), and aqueous tetrachloroaurate(III) (6.25 mM, 50 μL) at room temperature (∼25 °C) for 1 h. Subsequently, aqueous sodium hydroxide (2 M, 2 μL) was added into the mixture. The mixture was stirred continuously in a water bath at 50 °C for another 3 h. The color of mixture turned from purple to red at the end of the reaction. The sample was first centrifuged at 2000 rpm for 5 min to remove large particles, then 5500 rpm for 20 min and 8000 rpm for another 20 min to remove supernatants containing unreacted species. The precipitates were rinsed by deionized water (1 mL) and resuspended in deionized water (0.1 mL) before use. 3349

DOI: 10.1021/acsanm.9b00220 ACS Appl. Nano Mater. 2019, 2, 3348−3357

Article

ACS Applied Nano Materials

surface of Ag NPs. In our case, we also believe that the peak at 1739 cm−1 shown in Figure S1B was due to the binding arising between maltose and the surface of Au NPs. In a previous study, a band above 1727 cm−1 may be attributed to O−C O.45 Other characterization data will be further provided and discussed. Au@Maltose NPs and maltose were further examined by Xray photoelectron spectroscopy (XPS). Figure S2A and Figure S2B show the XPS spectra of C 1s and O 1s derived from maltose, respectively. Figure S3A, Figure S3B, and Figure S3C present the XPS spectra of C 1s, O 1s, and Au 4f derived from Au@Maltose NPs, respectively. The C 1s XPS spectra obtained from matlose and Au@Maltose NPs appeared apparently different. Two peaks at binding energies of ∼85.2 and ∼88.9 eV appeared in the Au 4f XPS spectrum (Figure S3C), indicating the presence of Au(0) and Au(I)46 in the asprepared Au NPs. To analyze the data further, we performed XPS peak fittings to examine the binding details of the Au@ Maltose NPs and maltose. Figure S4A and Figure S4B show the deconvoluted XPS spectra of C 1s derived from maltose and Au@Maltose NPs, respectively. On the basis of the spectrum in Figure S4A, the peaks at 285.3, 286.9, and 288.3 eV corresponded to C−C, C−OH, and CO, respectively.47 The inset table indicates that the majority was C−OH, whereas C−C and CO were minorities (Figure S4A). The deconvoluted C 1s spectrum of Au@Maltose NPs showed that the major peaks at the binding energies of 285.7 and 287.1 eV were derived from C−C and C−OH,47 respectively, whereas the peak appearing at the binding energies of 289.3 eV corresponded to OC−O 47 (Figure S4B). The peaks at

Figure 1. (A) Absorption spectrum of the generated Au@Maltose NPs. (B) Representative TEM image of Au@Maltose NPs. (Inset) The size distribution of the generated Au@Maltose NPs. Scale bar: 50 nm.

The generated Au@Maltose NPs carried negative charges at pH 6−8. Figure S1A and Figure S1B show the infrared (IR) absorption spectra of maltose (black) and Au@Maltose NPs (red), respectively. The two spectra appeared very similar, indicating that the surface of the generated Au@Maltose NPs was immobilized with maltose. The main difference between these two spectra of maltose and Au@Maltose NPs was the absorption band at 1739 cm−1 that only appeared in the spectrum of Au@Maltose NPs, indicating that binding between maltose and the surface of Au NPs occurred. Maltose has been previously used as the reducing agent for Ag NPs (Ag@Maltose NPs).44 In the previous study, the IR absorption peak at 1717 cm−1 also only appeared in the IR spectrum of Ag@Maltose NPs, indicating binding between maltose and the

Scheme 1. Putative Reaction Mechanism of Maltose-Direct Synthesis of Au@Maltose NPs

3350

DOI: 10.1021/acsanm.9b00220 ACS Appl. Nano Mater. 2019, 2, 3348−3357

Article

ACS Applied Nano Materials

Figure 2. (A) Photograph of the resultant samples containing Au@Maltose NPs (0.16 mg mL−1, 10 μL) with ConA at different concentrations (0− 1 μM). (B) Corresponding absorption spectra of the samples shown in panel A. (C) Plot obtained by plotting the ratio of the absorbance at the wavelengths of 620−525 nm (A620nm/A525nm) intensity versus the logarithm of ConA concentration obtained from panel B.

Au7O2−, Au8O2−, Au9O2−, and Au10O2−, respectively. Another group of ions (blue dots) at m/z 1245.7 and 1442.8 with a mass difference of 197.1 appeared in the same mass spectrum, corresponding to Au6O4− and Au7O4−, respectively. The ion peaks marked by yellow, red, and blue dots adjacent to each other and showing a mass unit difference of 32 and 64 that respectively indicated 2 and 4 oxygen atoms were bound onto the surface of the as-prepared Au NPs. The results indicated that a pair of oxygens derived from oxidized maltose bound to the surface of Au NPs. Putative Structure of Maltose-Directed Au NP Synthesis. On the basis of the characterization results above, we proposed a putative reaction mechanism of Au@Maltose NP generation (Scheme 1). The reaction of maltose and tetrachloroauric acid was conducted under the presence of sodium hydroxide. Maltose is a reducing sugar composed of two glucose units, in which the glucose unit on the right-hand side possesses a reducing end. Thus, the presence of aldehydic form when dissolving in water was used to interact with hydroxyl. Au(III) was then reduced in tetrachloroauric acid to Au(I), and Au@Maltose NPs were formed. The other glucose unit remained on the surface of the as-prepared Au@Maltose NPs. Determination of the Dissociation Constant of Au@ Maltose NPs and ConA. We examined the binding affinity between Au@Maltose NPs and ConA based on the determination of dissociation constant (Kd). The details of the experimental steps are described in Supporting Information. Figure S6 shows the plot of the binding amount of Au@ Maltose NPs toward ConA versus the concentration of unbound ConA. To fit the plot, we employed the Hill equation:53 Q* = QmaxCn/Kd + Cn where Kd is the dissociation constant, Qmax is the maximum binding amount, C is the concentration of ConA remaining in the supernatant, n is the Hill coefficient, and Q* is the binding amount of Au@Maltose NPs toward ConA. Kd was estimated to be ∼6.66 × 10−8 M, demonstrating that ConA exhibits good binding affinity toward Au@Maltose NPs. Moreover, n is equal to 4, indicating that the binding is a positive, cooperative process. Using Au@Maltose NPs as Sensing Probes against ConA. To examine whether Au@Maltose NPs can be used as sensing probes against ConA, we examined the samples

289.3 eV were not observed in Figure S4A, indicating that these peaks formed due to binding between maltose and Au@ Maltose NPs. The inset table in Figure S4B shows the percentage of these peaks. All of the binding energies considerably increased presumably due to the oxidation of maltose for binding onto Au NPs. Figure S4C shows the deconvoluted O 1s XPS spectrum of maltose. The peak was dominated by the functional group of C−O (531.5 eV), and only a very small fraction of the band was derived from CO (533.3 eV).49 Figure S4D shows the deconvolution of O 1s XPS spectrum of Au@Maltose NPs. The peak was deconvoluted to two peaks at the bind energies at 531.8 and 533.6 eV, corresponding to CO and C−O,49,50 respectively. Another small peak appeared at 540.2 eV, indicating further oxidation of oxygen-containing functional groups. Presumably, those oxygen-containing functional groups were involved in the reaction during Au@Maltose NP synthesis. Figure S4E shows the deconvoluted Au 4f XPS spectrum of Au@Maltose NPs. The Au 4f7/2 peak was deconvoluted to the peak at 85.2 eV (80.99%) and the peak at 85.6 eV (19.01%), corresponding to Au(0) and Au(I), respectively. The surface of Au@Maltose contained Au(0) and Au(I) at a ratio of 4:1. Thus, binding between maltose and the as-prepared Au NPs occurred. In addition, we also estimated the binding amount of maltose onto the surface of the generated Au@Maltose NPs using colorimetric titration by phenolsulfuric acid.51 Our results showed that ∼0.55 mg of maltose was bound to 1 mg of Au@Maltose NPs. Surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) has been used to clarify the binding elements between the protected groups on the surface of the as-prepared Au NPs and the surface of the Au NPs.52 We also used SALDI-MS to examine the binding elements. Figure S5 shows the SALDI mass spectrum of Au@Maltose NPs obtained in linear negative mode. Ion peaks (yellow dots) in the mass spectrum appeared at m/z 787.8, 984.8, 1181.8, 1378.7, 1575.8, 1772.6, and 1969.6, corresponding to [Au4−], [Au5−], [Au6−], [Au7−], [Au8−], [Au9−], and [Au10−], respectively. A group of ions (red dots) occurred at m/z 819.8, 1016.8, 1213.9, 1410.8, 1607.9, 1804.8, and 2001.5 with a mass difference of 196.9 appearing in the same mass spectrum, which corresponded to Au4O2−, Au5O2−, Au6O2−, 3351

DOI: 10.1021/acsanm.9b00220 ACS Appl. Nano Mater. 2019, 2, 3348−3357

Article

ACS Applied Nano Materials

Maltose NPs, the color of the sample also shifted to blue (cf. Figure 2A). To provide proof of the concept, we used TEM to examine the binding conditions between Au@Maltose NPs and ConA at the concentrations of 1 μM and 1 nM. Figure S7A and Figure S7B show the TEM images of the Au@Maltose NPConA conjugates obtained at ConA concentrations of 1 μM and 1 nM, respectively. Au@Maltose NPs were more distant in the sample containing 1 μM ConA (Figure S7A) than that with 1 nM ConA (Figure S7B). Au@Maltose NPs severely aggregated in the presence of 1 nM ConA. The larger area overview TEM image of Au@Maltose NPs alone indicated that they were not seriously aggregated (Figure S7C). The results corresponded to our hypothesis, as shown in Scheme 2. Examination of the Linear Dynamic Range and LOD. Given that the apparent LSPR shift occurred in the nM range, we further examined the linear dynamic range and LOD of our sensing method. The samples containing ConA at different concentrations were prepared by serially diluting aqueous ConA (1 nM) with a dilution factor of 1.5 with PBS (pH 7.4, 10 mM). Figure 3A shows the UV−vis absorption spectra obtained by mixing Au@Maltose NPs (0.16 mg mL−1, 8 μL) with 0.13−1 nM ConA (192 μL). Figure 3B shows the corresponding plot at A620nm/A525nm versus ConA concentration. Figure 3C shows the calibration curve with a linear dynamic range of 0.13−0.67 nM (y = 1.0998x − 0.0276; R2 = 0.9993) by plotting A620nm/A525nm versus the concentration of ConA. LOD was estimated based on the equation 3Sb/m, where Sb is the standard deviation of the regression line and m is the slope of the calibration curve. According to the results, m is 1.0998 and Sb is 0.00852. Thus, LOD was estimated to be ∼23.2 pM. The LOD of the current approach against ConA was as low as ∼23.2 pM, which was much lower than those obtained in previous reports45−48 that also used glucose- or mannoseimmobilized Au NPs as sensing probes. We suspected that the sizes of Au NPs and the length of the linker from the surface of Au NPs to the probe molecules may have played important roles in affecting the analysis. The size of Au NPs in the current study and in previous studies was ∼22 and 12−17 nm, respectively. Larger Au NPs can result in more apparent LSPR shift. Moreover, according to calculation of the linker based on the composition of atoms,54 the length of the linker in our Au@Maltose NPs was ∼7.72 Å, which was relatively short among those existing Au NPs used as sensing probes against ConA. Presumably, the short linker can cause Au@Maltose NPs to be close to each other to show apparent surface coupling effects. In addition, the concentration of Au@Maltose NPs used in the sensing experiments can affect the sensing sensitivity and the linear dynamic range as well. That is, when the concentration of Au@Maltose NPs was increased in the sensing experiments, the sensing sensitivity became worse and the linear dynamic range shifted to a higher concentration. To achieve low LOD and good sensitivity, Au@Maltose NPs with a relatively low concentration are used as sensing probes against ConA herein. Selectivity of Au@Maltose NPs. We further examined the selectivity of Au@Maltose NPs toward ConA by using several nontarget proteins, including peanut lectin, BanLec, ricin, and BSA as model samples. Peanut lectin, BanLec, and ricin B possess the binding moieties of Gal-β(1−3)-GalNAc, α(1−3) linked glucose/mannose, and β-1,4-linked galactose,

containing ConA at different concentrations (0.01 nM to 1 μM). Figure 2A shows the photographs obtained by mixing Au@Maltose NPs with ConA at different concentrations. The color of the samples changed from pale red (0 nM) to pale purple (0.1 nM) and pale blue (1 nM, 10 nM, and 0.1 μM). The color changes occurred in the concentration range between 0.1 nM and 0.1 μM. Figure 2B shows the corresponding UV−vis absorption spectra. Apparent LSPR shifts also occurred at the concentration range of 0.1 nM to 0.1 μM. The LSPR shifts were quite apparent in the sample containing ConA with the concentrations of 1 nM, 10 nM, and 0.1 μM. Although there were LSPR shifts occurring in the samples containing ConA with the concentrations of 0.01 nM and 1 μM, the shifts were much less apparent than the concentrations at 1 nM, 10 nM, and 0.1 μM. The maximum absorbance band shifted from ∼525 nm (black) to ∼620 nm (pink) (Figure 2B). To show the difference, we plotted the ratio of the absorbance at the wavelengths of 620 to 525 nm (A620nm/A525nm) versus the logarithm of ConA concentration (Figure 2C) according to the results obtained in Figure 2B. The maximum ratio was obtained when the concentration of ConA reached 1 nM. The ratio of A620nm to A525nm increased as the concentration increased from 0.01 nM to 1 nM. However, the ratio declined when the concentration was increased to 10 nM (green) and 0.1 μM (blue). The ratio was further dropped as the concentration was further increased to 1 μM. The results showed that the maximum LSPR shift occurred at a concentration of 1 nM. The results appeared unusual. Presumably, surface coupling effects36−38 were involved during sensing. If the relative ratio of the amounts of Au@Maltose NPs to ConA in a sample was in a proper range, the surface coupling effects would dominate, leading to an apparent LSPR shift. Scheme 2 shows a cartoon illustration of the putative Scheme 2. Putative Binding Mechanisms between Au@ Maltose NPs and ConA at Concentrations of 1 μM (Left) and 1 nM (Right)

mechanism when using Au@Maltose NPs as the sensing probes to interact with ConA at the concentrations of 1 μM (left) and 1 nM (right). At 1 μM ConA, the distance between Au@Maltose NPs was far (cartoon on the left-hand side in Scheme 2), leading to unapparent surface coupling effect and a small LSPR shift (Figure 2B). However, at 1 nM ConA, Au@ Maltose NPs were very close to each other because of the limited amount of ConA (cartoon on the right-hand side in Scheme 2), resulting in apparent surface coupling effects and a large LSPR shift. Owing to the close distance among Au@ 3352

DOI: 10.1021/acsanm.9b00220 ACS Appl. Nano Mater. 2019, 2, 3348−3357

Article

ACS Applied Nano Materials

Figure 3. (A) UV−vis absorption spectra of the resultant samples obtained after mixing Au@Maltose NPs (0.16 mg mL−1, 8 μL) with ConA (192 μL) at different concentrations (0.13−1 nM) for 30 min. (B) Plot obtained by plotting A620nm/A525nm versus the concentration of ConA. (C) Calibration curve showing the linear dynamic range. Three replicates were conducted.

Au@Maltose NPs possessed good selectivity toward ConA and were insensitive to the rest of the model proteins. BanLec also contains glucose-binding sites, but no apparent LSPR shift was observed when our sensing probes were used. Figure S8 shows the cartoon illustration of the structure of banana lectin and the absorption spectra of the samples containing BanLec at different concentrations (1 μM to 0.01 nM, 194 μL) when using Au@Maltose NPs (0.16 mg mL−1, 6 μL) as the sensing probes. LSPR shift was not observed at a BanLec concentration below 10 nM. Presumably, the protein concentration was too low to lead any LSPR shift. Moreover, BanLec is a dimeric protein with each monomer containing two carbohydrate-binding sites.55 Nevertheless, the two binding sites in each monomer (Figure S8) are too close.55 Thus, two Au NPs were unlikely to bind on two binding sites because of steric effects, and no surface coupling effects were involved during sensing. The sensing sensitivity of using Au@ Maltose NPs as sensing probes for BanLec was worse than that for ConA. In addition, we also used dynamic light scattering (DLS) to examine the hydrodynamic size (HS) of Au NP-target species conjugates. Supporting Information Figure S9A shows the summarized bar graphs of the distribution of Au@Maltose NPs alone (Figure S9B), Au@Maltose NPs mixed with ConA at the concentrations of 1 nM (Figure S9C) and 1 μM (Figure S9D), Au@Maltose NPs mixed with peanut lectin at the concentrations of 1 nM (Figure S9E) and 1 μM (Figure S9F). Apparently, the HS shifted to a larger diameter after Au@ Maltose NPs mixed with ConA and the HS even shifted to higher value when the concentration of ConA was increased from 1 nM (Figure S9C) to 1 μM (Figure S9D), indicating the occurrence of binding arising between Au@Maltose NPs and ConA. However, the HS of the mixture between Au@Maltose NPs and peanut lectin at the concentrations of 1 nM (Figure S9E) and 1 μM (Figure S9E) did not change much. The DLS results indicated that Au@Maltose NPs have selectivity toward their target proteins. Using Au@Maltose NPs as Labeling Agents for Cancer Cells. As mentioned in Introduction, the cell membrane of human liver and breast cancer cells overexpress more mannose N-glycan units on the membrane proteins than that of normal cells. Thus, when incubating ConA with cell samples followed by incubation with Au@Maltose NPs, more cancer cells should be labeled with Au@Maltose NPs than normal cells. Therefore, the combination of ConA and Au@ Maltose NPs can be used as labeling agents to distinguish cancer cells from normal cells. We initially examined the binding amount of ConA on noncancer cells and target cancer cells by incubating the cells with ConA for 2 h followed by

respectively. Figure 4A shows the photograph of the resultant samples obtained after shaking Au@Maltose NPs (0.16 mg

Figure 4. Selectivity of Au@Maltose NPs. (A) Photograph of the resultant samples by shaking Au@Maltose NPs (0.16 mg mL−1, 6 μL) with different model proteins (1 nM, 194 μL) and (B) the corresponding absorption spectra. (C) Bar graphs obtained by plotting LSPR shift versus different model proteins according to the results obtained in panel B. (D) Bar graphs obtained by plotting A620nm/A525nm versus different model proteins according to the results obtained in panel B.

mL−1, 6 μL) with different proteins (1 nM, 194 μL) for 30 min. Only the sample containing ConA became pale blue, and the rest of the samples remained pale red. Figure 4B shows the corresponding UV−vis absorption spectra. The maximum absorption band of the samples containing nontarget protein did not considerably change. The maximum absorption band of the sample containing ConA shifted from ∼525 nm to ∼586 nm. Figure 4C shows the corresponding bar graphs obtained by plotting LSPR shift versus different model proteins obtained from panel B. Apparently, only the sample containing ConA caused an apparent LSPR shift on the Au@Maltose NPs. Figure 4D shows the bar graphs obtained by plotting A620nm/ A525nm versus different proteins. The ratio of A620nm/A525nm obtained from the sample containing ConA was the highest. 3353

DOI: 10.1021/acsanm.9b00220 ACS Appl. Nano Mater. 2019, 2, 3348−3357

Article

ACS Applied Nano Materials rinse steps. The concentration of ConA in the supernatant obtained before and after incubated with the cell samples for 2 h was examined. Our results showed that the binding amounts of ConA on HePG2 and T-47D cells were ∼2.02 fmol cell−1 and ∼0.76 fmol cell−1, respectively, whereas the binding amount of ConA on NIH3T3 normal cells was only ∼0.10 fmol cell−1. The results indicated that much more ConA molecules were able to be attached onto the cell membrane of HePG2 and T-47D cancer cells than that of NIH3T3 normal cells. After demonstrating this fact, we further used Au@ Maltose NPs as labeling agents for ConA-treated cell samples, and optical microscopy was used to investigate the labeling effects of Au@Maltose NPs. NIH3T3 normal cells, HePG2, and T-47D cancer cells were fixed on glass slides with paraformaldehyde and underwent nuclear staining with Hoechst 33342 dye. Figure S10A, Figure S10B, and Figure S10C shows the optical microscopic images of the cells including NIH3T3 normal cells, HePG2, and T-47D cancer cells, respectively. Different cells have different shapes. Figure 5A, Figure 5B, and Figure 5C show the obtained optical microscopic images of NIH3T3 normal cells incubated alone in PBS buffer through dark field microscopy, fluorescence microscopy and by overlapping the images derived from Figure 5A and Figure 5B, respectively. Figure 5D, Figure 5E, and Figure 5F present the obtained optical microscopic images of the NIH3T3 normal cells incubated with Au@Maltose NPs for 5 h through dark field microscopy, fluorescence microscopy and by overlapping the images derived from Figure 5D and Figure 5E, respectively. Figure 5G, Figure 5H, and Figure 5I illustrate the obtained optical microscopic images of the NIH3T3 normal cells treated with ConA for 1 h followed with Au@Maltose NPs for 5 h through dark field microscopy, fluorescence microscopy and by overlapping the images derived from Figure 5G and Figure 5H, respectively. Figure 5J, Figure 5K, and Figure 5L show the obtained optical microscopic images NIH3T3 normal cells treated with ConA for 2 h followed with Au@Maltose NPs for 5 h through dark field microscopy, fluorescence microscopy and by overlapping the images derived from Figure 5J and Figure 5K, respectively. Figure 5M, Figure 5N, and Figure 5O present the optical microscopic images of NIH3T3 normal cells incubated with ConA for 3 h followed with Au@Maltose NPs for 5 h through dark field microscopy, fluorescence microscopy and by overlapping images derived from Figure 5M and Figure 5N, respectively. We could observe some yellow dots corresponding to Au NPs from the images. However, these dots were not dense and did not stay close to the cells even when the incubation time with Au@Maltose NPs was extended. The binding of Au@Maltose NPs on NIH3T3 cells was not apparent. We then performed the same treatment to HePG2 cancer cells. Figure 6A, Figure 6B, and Figure 6C show the obtained optical microscopic images of the HePG2 cancer cells incubated with PBS buffer through dark field microscopy, fluorescence microscopy and by overlapping the images derived from Figure 6A and Figure 6B, respectively. Figure 6D, Figure 6E, and Figure 6F show the obtained optical microscopic images of the HePG2 cancer cells incubated with Au@Maltose NPs for 5 h through dark field microscopy, fluorescence microscopy and by overlapping images derived from Figure 6D and Figure 6E, respectively. Some Au NPs were attached around the cells. The result was expected because the cell membrane of HePG2 cells contained glucose-

Figure 5. (A) Dark field microscopic and (B) fluorescence microscopic images obtained after incubating NIH3T3 normal cells (∼2 × 105 cells) with PBS (pH 7.4) alone. (C) Overlapped image by merging the images in panels A and B together. (D) Dark field optical and (E) fluorescence microscopic images obtained after incubating NIH3T3 normal cells (∼2 × 105 cells) with Au@Maltose NPs alone (16 μg mL−1, 1 mL) for 5 h. (F) Overlapped image by merging the images in panels D and E together. (G) Dark field optical and (H) fluorescence microscopic images obtained after incubating ConA (1 μM, 1 mL) with NIH3T3 normal cells (∼2 × 105 cells) for 1 h followed by incubating with Au@Maltose NPs alone (16 μg mL−1, 1 mL) for 5 h. (I) Overlapped image by merging the images in panels G and H together. (J) Dark field optical and (K) fluorescence microscopic images obtained after incubating ConA (1 μM, 1 mL) with NIH3T3 normal cells (∼2 × 105 cells) for 2 h followed by incubating with Au@Maltose NPs alone (16 μg mL−1, 1 mL) for 5 h. (L) Overlapped image by merging the images in panels J and K together. (M) Dark field optical and (N) fluorescence microscopic images obtained after incubating ConA (1 μM, 1 mL) with NIH3T3 normal cells (∼2 × 105 cells) for 3 h followed by incubating with Au@Maltose NPs alone (16 μg mL−1, 1 mL) for 5 h. (O) Overlapped image by merging the images in panels M and N together. The cells were stained with Hoechst 33342 dye prior to observation under microscopy. The exposure time under dark filed was set to 600 ms, whereas the exposure time for obtaining fluorescence images was set to 80 ms. The exposure time for obtaining overlapped images from dark field and fluorescence microscopy was set to 100 ms. The excitation wavelength was set at 330−380 nm. The scale bar is 20 μm.

binding moieties.56 Figure 6G, Figure 6H, and Figure 6I show the obtained optical microscopic images of the HePG2 cancer cells treated with ConA for 1 h followed with Au@Maltose NPs for 5 h through dark field microscopy, fluorescence microscopy and by overlapping the images derived from Figure 6G and Figure 6H, respectively. Apparently, more Au NPs attached on the cells than those not treated by ConA. As the incubation time with ConA was extended to 2 (Figure 6J−L) and 3 h (Figure 6M−O), additional Au@Maltose NPs attached onto the cells. Furthermore, 2 h of ConA treatment caused the cells to bind the maximum amount of Au@Maltose NPs, as shown in Figure 6J. When the cells were incubated with ConA for 3 h, Au NP labeling was less. Presumably, ConA might be internalized by the cells when the incubation time with ConA was extended to longer than 2 h, leading to few Au@Maltose NP binding sites available on the cell membrane. 3354

DOI: 10.1021/acsanm.9b00220 ACS Appl. Nano Mater. 2019, 2, 3348−3357

Article

ACS Applied Nano Materials

cells also overexpressed glucose-binding moieties.57 Thus, Au@Maltose NPs could readily bind to the cell membrane of the cells even without prior ConA treatment. Nevertheless, more Au@Maltose NPs were attached onto the cells after prior ConA treatment (Figure S11G, Figure S11J, and Figure S11M). After 2 h of incubation with ConA, the cells showed maximum Au NP binding. These results indicated that our approach could be used to distinguish cancer cells that overexpress mannose N-glycan units on the cell membrane from normal cells by combining the use of ConA and Au@ Maltose NPs as labeling agents. The efficiency of using our labeling agents was much better than that by merely using Au@Maltose NPs as labeling agents.

■

CONCLUSIONS In this study, maltose was successfully used as the reducing and capping agent for the generation of maltose-immobilized Au NP (Au@Maltose NPs) through one-pot reactions. The synthesis method was simple and straightforward. The binding affinity between ConA and the generated Au@Maltose NPs was desirable. According to the binding experiments, the dissociation constant arising between ConA and Au@Maltose NPs was as low as ∼6.66 × 10−8 M. The sensing results could also be directly observed by the naked eye at a concentration of ConA above 1 μM. The LOD toward ConA using our Au@ Maltose NPs was estimated to be ∼23 pM, which was 2 orders of magnitudes lower than those obtained from existing reports when using UV−vis absorption spectroscopy as the detection tool. To clarify why our results were much better than those reported in previous reports, we compared the particle size and the length of the spacer of our sensing probes with those reported in the previous studies. We found that our NPs (∼20 nm) were larger than those previously used (∼12−17 nm), resulting in increasingly apparent LSPR shifts. The length of the spacer in our NPs was relatively short compared with those previously reported. The short spacer might also help in enhancing surface coupling effects. Given that ConA possesses four binding moieties toward glucose, it can be used as a bridge to bring multi-Au@Maltose NPs very close, leading to apparent LSPR shifts. The large size and short spacer of our Au@Maltose NPs resulted in obvious surface coupling effects and low LOD. We further extended the application of our Au@Maltose NPs for cancer cell imaging. HePG2 and T-47D cancer cells, which can overexpress mannose N-glycan ligands on the cell membrane, and NIH3T3 noncancer cells, which do not overexpress mannose N-glycan ligands, were selected as model cells. The combination of ConA with Au@Maltose NPs as labeling agents for imaging cancer cells was feasible. Relatively high amounts of Au@Maltose NPs were bound to the surface of HePG2 and T-47D cancer cells after treatment with ConA and Au@Maltose NPs. However, negligible Au@ Maltose NPs bound to the cell surface of NIH3T3 cells. The developed protocol can be used to distinguish cancer cells that possess mannose N-glycan ligands on the cell membrane from noncancer cells. Thus, we believe that the labeling method developed herein may show potential for distinguishing breast and liver cancer cells from normal cells.

Figure 6. (A) Dark field microscopic and (B) fluorescence microscopic images obtained after incubating HePG2 cancer cells (∼2 × 105 cells) with PBS (pH 7.4) alone. (C) Overlapped image by merging the images in panels A and B together. (D) Dark field optical and (E) fluorescence microscopic images obtained after incubating HePG2 cancer cells (∼2 × 105 cells) with Au@Maltose NPs alone (16 μg mL−1, 1 mL). (F) Overlapped image by merging the images in panels D and E together. (G) Dark field optical and (H) fluorescence microscopic images obtained after incubating ConA (1 μM, 1 mL) with HePG2 cancer cells (∼2 × 105 cells) for 1 h followed by incubating with Au@Maltose NPs alone (16 μg mL−1, 1 mL) for 5 h. (I) Overlapped image by merging the images in panels D and E together. (J) Dark field optical and (K) fluorescence microscopic images obtained after incubating ConA (1 μM, 1 mL) with HePG2 cancer cell (∼2 × 105 cells) for 2 h followed by incubating with Au@ Maltose NPs alone (16 μg mL−1, 1 mL) for 5 h. (L) Overlapped image by merging the images in panels J and K together. (M) Dark field optical and (N) fluorescence microscopic images obtained after incubating ConA (1 μM, 1 mL) with HePG2 cancer cells (∼2 × 105 cells) for 3 h followed by incubating with Au@Maltose NPs alone (16 μg mL−1, 1 mL) for 5 h. (O) Overlapped image by merging the images in panels M and N together. The cells were stained with Hoechst 33342 dye prior to observation under microscopy. The exposure time under dark field was set to 600 ms, whereas the exposure time for obtaining fluorescence images was set to 80 ms. The exposure time for obtaining overlapped images from dark field and fluorescence microscopy was set to 100 ms. The excitation wavelength was set at 330−380 nm. The scale bar is 20 μm.

HePG2 cells treated with ConA could be labeled with more Au@Maltose than those without ConA treatment. After 2 h of ConA treatment, the cells showed maximum binding of Au@ Maltose NPs. T-47D cells were further examined with similar treatment used for HePG2 cells, as those shown in Figure 6. Figure S11 shows the resultant optical images obtained from the samples containing T-47D cells that were incubated in PBS buffer (Figure S11A−C), incubated with Au@Maltose NPs (Figure S11D−F) for 5 h, and treated with ConA for 1 (Figure S11G− I), 2 (Figure S11J−L), and 3 h (Figure S11M−O), followed by incubation with Au@Maltose NPs for 5 h. The results were very similar to those obtained in Figure 6. Some Au NPs attached to the cells when T-47D cells were incubated with Au@Maltose NPs (Figure S11D). The reason was similar to what we explained earlier. The cell membrane of T-47D cancer

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsanm.9b00220. 3355

DOI: 10.1021/acsanm.9b00220 ACS Appl. Nano Mater. 2019, 2, 3348−3357

Article

ACS Applied Nano Materials

■

Plasmon resonance Sensor for Sensitive Detection of Concanavalin A. Biosens. Bioelectron. 2013, 50, 305−310. (14) Dam, T. K.; Roy, R.; Das, S. K.; Oscarson, S.; Brewer, C. F. Binding of Multivalent Carbohydrates to Concanavalin A and Dioclea Grandiflora Lectin. J. Biol. Chem. 2000, 275, 14223−14230. (15) Lei, H.-Y.; Chang, C.-P. Lectin of Concanavalin A as an Antihepatoma Therapeutic Agent. J. Biomed. Sci. 2009, 16, 10. (16) Lee, Y.-C. Fluorescence Spectrometry in Studies of Carbohydrate-protein Interactions. J. Biochem. 1997, 121, 818−825. (17) McCoy, J. P., Jr; Varani, J.; Goldstein, I. J. Enzyme-linked Lectin Assay (ELLA). II. Detection of Carbohydrate Groups on the Surface of Unfixed Cells. Exp. Cell Res. 1984, 151, 96−103. (18) Zeng, X.; Andrade, C. A.; Oliveira, M. D.; Sun, X. L. Carbohydrate−protein Interactions and Their Biosensing Applications. Anal. Bioanal. Chem. 2012, 402, 3161−3176. (19) Chen, H.; Kou, X.; Yang, Z.; Ni, W.; Wang, J. Shape- and Sizedependent Refractive Index Sensitivity of Gold Nanoparticles. Langmuir 2008, 24, 5233−5237. (20) Sperling, R. A.; Gil, P. R.; Zhang, F.; Zanella, M.; Parak, W. J. Biological Applications of Gold Nanoparticles. Chem. Soc. Rev. 2008, 37, 1896−1908. (21) Yilmaz, G.; Becer, C. R. Glyconanoparticles and Their Interactions with Lectins. Polym. Chem. 2015, 6, 5503−5514. (22) Stewart, M. E.; Anderton, C. R.; Thompson, L. B.; Maria, J.; Gray, S. K.; Rogers, J. A.; Nuzzo, R. G. Nanostructured PlasmoSc sensors. Chem. Rev. 2008, 108, 494−521. (23) Huang, X.; El-Sayed, M. A. Gold Nanoparticles: Optical Properties and Implementations in Cancer Diagnosis and Photothermal Therapy. J. Adv. Res. 2010, 1, 13−28. (24) Guler, U.; Turan, R. Effect of Particle Properties and Light Polarization on the Plasmonic Resonances in Metallic Nanoparticles. Opt. Express 2010, 18, 17322−17338. (25) Ghosh, S. K.; Pal, T. Interparticle Coupling Effect on the Surface Plasmon Resonance of Gold Nanoparticles: from Theory to Applications. Chem. Rev. 2007, 107, 4797−4862. (26) Slistan-Grijalva, A.; Herrera-Urbina, R.; Rivas-Silva, J. F.; Á valos-Borja, M.; Castillón-Barraza, F. F.; Posada-Amarillas, A. Classical Theoretical Characterization of the Surface Plasmon Absorption Band for Silver Spherical Nanoparticles Suspended in Water and Ethylene Glycol. Phys. E (Amsterdam, Neth.) 2005, 27, 104−112. (27) Basu, S.; Panigrahi, S.; Praharaj, S.; Ghosh, S. K.; Pande, S.; Jana, S.; Pal, T. Dipole−Dipole Plasmon Interactions in Self-assembly of Gold Organosol Induced by Glutathione. New J. Chem. 2006, 30, 1333−1339. (28) Marín, M. J.; Schofield, C. L.; Field, R. A.; Russell, D. A. Glyconanoparticles for Colorimetric Bioassays. Analyst 2015, 140, 59−70. (29) Compostella, F.; Pitirollo, O.; Silvestri, A.; Polito, L. Glyco-gold Nanoparticles: Synthesis and Applications. Beilstein J. Org. Chem. 2017, 13, 1008−1021. (30) De la Fuente, J. M.; Barrientos, A. G.; Rojas, T. C.; Rojo, J.; Cañada, J.; Fernández, A.; Penadés, S. Gold Glyconanoparticles as Water-soluble Polyvalent Models to Study Carbohydrate Interactions. Angew. Chem., Int. Ed. 2001, 40, 2257−2261. (31) Schofield, C. L.; Field, R. A.; Russell, D. A. Glyconanoparticles for the Colorimetric Detection of Cholera Toxin. Anal. Chem. 2007, 79, 1356−1361. (32) Marín, M. J.; Rashid, A.; Rejzek, M.; Fairhurst, S. A.; Wharton, S. A.; Martin, S. R.; McCauley, J. W.; Wileman, T.; Field, R. A.; Russell, D. A. Glyconanoparticles for the Plasmonic Detection and Discrimination between Human and Avian Influenza Virus. Org. Biomol. Chem. 2013, 11, 7101−7107. (33) Schofield, C. L.; Haines, A. H.; Field, R. A.; Russell, D. A. Silver and Gold Glyconanoparticles for Colorimetric Bioassays. Langmuir 2006, 22, 6707−6711. (34) Chuang, Y. J.; Zhou, X.; Pan, Z.; Turchi, C. A Convenient Method for Synthesis of Glyconanoparticles for Colorimetric

Additional experimental details (reagents and materials, instrumentation, sample preparation for TEM analysis, sample preparation for ζ potential analysis, sample preparation for FT-IR analysis, characterization of Au@ Maltose NPs by XPS, determination of the amount maltose immobilized on Au@Maltose NPs, characterization of Au@Maltose NPs by SALDI-MS, determination of the dissociation constant between Au@ Maltose NPs and ConA, LOD determination, cell culture, examination of the binding amount of ConA on model cells) and additional figures (Figures S1−S11) (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +886-3-5131527. Fax: +886-3-5723764. ORCID

Yu-Chie Chen: 0000-0003-2253-4049 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank the Ministry of Science and Technology of Taiwan (MOST Grant 105-2113-M-009-022-MY3) for financial support of this research.

■

REFERENCES

(1) Alhadeff, J. A.; Glick, M. C. Malignant Cell Glycoproteins and Glycolipids. Crit. Rev. Oncol. Hematol. 1989, 9, 37−107. (2) Varki, A., Cummings, R. D., Esko, J. D., Freeze, H. H., Stanley, P., Bertozzi, C. R., Hart, G. W., Etzler, M. E., Eds. Essentials of Glycobiology, 2nd ed.; Cold Spring Harbor: New York, 2009. (3) Varki, A. Biological Roles of Oligosaccharides: All of the Theories are Correct. Glycobiology 1993, 3, 97−130. (4) Berg, J. M.; Tymoczko, J. L.; Stryer, L. Biochemistry, 5th ed.; W. H. Freeman: New York, 2002. (5) Kannagi, R.; Izawa, M.; Koike, T.; Miyazaki, K.; Kimura, N. Carbohydrate-mediated Cell Adhesion Involved in Hematogenous Metastasis of Cancer. Cancer Sci. 2004, 95, 377−384. (6) Brandley, B. K.; Schnaar, R. L. Cell-surface Carbohydrates in Cell Recognition and Response. J. Leukocyte Biol. 1986, 40, 97−111. (7) Cockerell, G. L. Changes in Plasma Protein-bound Carbohydrates and Glycoprotein Patterns during Infection, Inflammation andS starvation. Exp. Biol. Med. 1973, 142, 1072−1076. (8) Dabelsteen, E.; Mandel, U.; Clausen, H. Cell Surface Carbohydrates are Markers of Differentiation in Human Oral Epithelium. Crit. Rev. Oral Biol. Med. 1991, 2, 493−507. (9) Stansell, E.; Desrosiers, R. C. Functional Contributions of Carbohydrate on AIDS Virus Glycoprotein. Yale J. Biol. Med. 2010, 83, 201−208. (10) Glavey, S. V.; Huynh, D.; Reagan, M. R.; Manier, S.; Moschetta, M.; Kawano, Y.; Roccaro, A. M.; Ghobrial, I. M.; Joshi, L.; O’Dwyer, M. E. The Cancer Glycome: Carbohydrates as Mediators of Metastasis. Blood Rev. 2015, 29, 269−279. (11) Lis, H.; Sharon, N. Lectins: Carbohydrate-specific Proteins That Mediate Cellular Recognition. Chem. Rev. 1998, 98 (2), 637− 674. (12) Jin, H.-Y.; Li, D.-W.; Zhang, N.; Gu, Z.; Long, Y.-T. Analyzing Carbohydrate−protein Interaction based on Single Plasmonic Nanoparticle by Conventional Dark Field Microscopy. ACS Appl. Mater. Interfaces 2015, 7, 12249−12253. (13) Huang, C.-F.; Yao, G.-H.; Liang, R.-P.; Qiu, J.-D. Graphene Oxide andD dextran Capped Gold Nanoparticles based Surface 3356

DOI: 10.1021/acsanm.9b00220 ACS Appl. Nano Mater. 2019, 2, 3348−3357

Article

ACS Applied Nano Materials Measuring Carbohydrate-protein Interactions. Biochem. Biophys. Res. Commun. 2009, 389, 22−27. (35) Chiu, W.-J.; Chen, W.-Y.; Lai, H.-Z.; Wu, C.-Y.; Chiang, H.-L.; Chen, Y.-C. Dextran-encapsulated Photoluminescent Gold Nanoclusters: Synthesis and Application. J. Nanopart. Res. 2014, 16, 2478. (36) Liu, J.; Qin, G.; Raveendran, P.; Ikushima, Y. Facile “Green” Synthesis, Characterization, and Catalytic Function of β-D-glucosestabilized Au Nanocrystals. Chem. - Eur. J. 2006, 12, 2131−2138. (37) Engelbrekt, C.; Sørensen, K. H.; Zhang, J.; Welinder, A. C.; Jensen, P. S.; Ulstrup, J. Green Synthesis of Gold Nanoparticles with Starch−glucose and Application in Bioelectrochemistry. J. Mater. Chem. 2009, 19, 7839−7847. (38) Waner, M. J.; Gilchrist, M.; Schindler, M.; Dantus, M. Imaging the Molecular Dimensions and Oligomerization of Proteins at Liquid/ solid Interfaces. J. Phys. Chem. B 1998, 102, 1649−1657. (39) McKenzie, G. H.; Sawyer, W. H.; Nichol, L. W. The Molecular Weight and Stability of Concanavalin A. Biochim. Biophys. Acta 1972, 263, 283−293. (40) Dube, D. H.; Bertozzi, C. R. Glycans in Cancer and Inflammation−potential for Therapeutics and Diagnostics. Nat. Rev. Drug Discovery 2005, 4, 477−488. (41) Liu, X.; Nie, H.; Zhang, Y.; Yao, Y.; Maitikabili, A.; Qu, Y.; Shi, S.; Chen, C.; Li, Y. Cell Surface-specific N-glycan Profiling in Breast Cancer. PLoS One 2013, 8, No. e72704. (42) Ho, W. L.; Hsu, W. M.; Huang, M. C.; Kadomatsu, K.; Nakagawara, A. Protein Glycosylation in Cancers and Its Potential Therapeutic Applications in Neuroblastoma. J. Hematol. Oncol. 2016, 9, 100. (43) Brown, R. D.; Brewer, C. F.; Koenig, S. H. Conformation states of concanavalin A: Kinetics of Transitions Induced by Interaction with Mn2+ and Ca2+ Ions. Biochemistry 1977, 16, 3883−3896. (44) Ç ulha, M.; Kalay, Ş .; Sevim, E.; Pinarbaş, M.; Baş, Y.; Akpinar, R.; Karaoğlu, Ş . A. Biocidal Properties of Maltose Reduced Silver Nanoparticles against American Foulbrood Diseases Pathogens. BioMetals 2017, 30, 893−902. (45) Liu, C.; Hu, G. Effect of Nitric Acid Treatment on the Preparation of Ggraphene Sheets by Supercritical N,N-dimethylformamide Exfoliation. Ind. Eng. Chem. Res. 2014, 53, 14310−14314. (46) Schofield, C. L.; Haines, A. H.; Field, R. A.; Russell, D. A. Silver and Gold Glyconanoparticles for Colorimetric Bioassays. Langmuir 2006, 22, 6707−6711. (47) Chuang, Y. J.; Zhou, X.; Pan, Z.; Turchi, C. A Convenient Method for Synthesis of Glyconanoparticles for Colorimetric Measuring Carbohydrate-protein Interactions. Biochem. Biophys. Res. Commun. 2009, 389, 22−27. (48) Karamanska, R.; Mukhopadhyay, B.; Russell, D. A.; Field, R. A. Thioctic Acid Amides: Convenient Tethers for Achieving Low Nonspecific Protein Binding to Carbohydrates Presented on Gold Surfaces. Chem. Commun. 2005, 3334−3336. (49) Pacheco, F. G.; Cotta, A. A. C.; Gorgulho, H. F.; Santos, A. P.; Macedo, W. A.A.; Furtado, C. A. Comparative Temporal Analysis of Multiwalled Carbon Nanotube Oxidation Reactions: Evaluating Chemical Modifications on True Nanotube Surface. Appl. Surf. Sci. 2015, 357, 1015−1023. (50) Lan, M.; Zhang, J.; Chui, Y.-S.; Wang, H.; Yang, Q.; Zhu, X.; Wei, H.; Liu, W.; Ge, J.; Wang, P.; Chen, X.; Lee, C.-S.; Zhang, W. A Recyclable Carbon Nanoparticle- based Fluorescent Probe for Highly Selective and Sensitive detection of Mercapto biomolecules. J. Mater. Chem. B 2015, 3, 127−134. (51) Nielsen, S. S. Phenol-Sulfuric Acid Method for Total Carbohydrates. In Food Analysis Laboratory Manual; Nielsen, S. S., Ed.; Food Science Texts Series; Springer: Boston, MA, U.S., 2010. (52) Li, C.-H.; Bai, Y.-L.; Selvaprakash, K.; Mong, K.-T.; Chen, Y.-C. Selective Detection of Shiga-likeToxin 1 from Complex Samples Using Pigeon Ovalbumin Functionalized Gold Nanoparticles as Affinity Probes. J. Agric. Food Chem. 2017, 65, 4359−4365. (53) Goutelle, S.; Maurin, M.; Rougier, F.; Barbaut, X.; Bourguignon, L.; Ducher, M.; Maire, P. The Hill Equation: A Review

of Its Capabilities in Pharmacological Modelling. Fundam. Clin. Pharmacol. 2008, 22, 633−648. (54) Mack, E. T.; Snyder, P. W.; Perez-Castillejos, R.; Bilgiçer, B.; Moustakas, D. T.; Butte, M. J.; Whitesides, G. M. Dependence of Avidity on Linker Length for a Bivalent Ligand-bivalent Receptor Model System. J. Am. Chem. Soc. 2012, 134, 333−345. (55) Sharma, A.; Vijayan, M. Influence of Glycosidic Linkage on the Nature of Carbohydrate Binding in beta-Prism I Fold Lectins: An Xray and Molecular Dynamics Investigation on Banana Lectincarbohydrate Complexes. Glycobiology 2011, 21, 23−33. (56) Amann, T.; Maegdefrau, U.; Hartmann, A.; Agaimy, A.; Marienhagen, J.; Weiss, T. S.; Stoeltzing, O.; Warnecke, C.; Schölmerich, J.; Oefner, P. J.; Kreutz, M.; Bosserhoff, A. K.; Hellerbrand, C. GLUT1 Eexpression Is Increased in Hepatocellular Carcinoma and Promotes Tumorigenesis. Am. J. Pathol. 2009, 174, 1544−1552. (57) Oh, S.; Kim, H.; Nam, K.; Shin, I. Glut1 Promotes Cell Proliferation, Migration and Invasion by Regulating Epidermal Growth Factor Receptor and Integrin Signaling in Triple-negative Breast Cancer Cells. BMB Rep. 2017, 50, 132−137.

3357

DOI: 10.1021/acsanm.9b00220 ACS Appl. Nano Mater. 2019, 2, 3348−3357