- Research article
- Open Access
Anthraquinonyl glycoside facilitates the standardization of graphene electrodes for the impedance detection of lectins
© Zhu et al.; licensee Springer. 2014
Received: 9 July 2014
Accepted: 30 October 2014
Published: 25 November 2014
Construction of electrochemical impedance sensors by the self-assembly technique has become a promising strategy for the `label-free' detection of protein-ligand interactions. However, previous impedance sensors are devoid of an inherent electrochemical signal, which limits the standardization of the sensors for protein recognition in a reproducible manner.
We designed and synthesized an anthraquinonyl glycoside (AG) where the anthraquinone (AQ) moiety can bind to the surface of a graphene-based working electrode while the glycoside serving as a ligand for lectin. By measuring the inherent voltammetric signal of AQ, the glycosides decorated on the working electrode could be simply quantified to obtain electrodes with a unified signal window. Subsequently, impedance analysis showed that the `standardized' electrodes gave a reproducible electrochemical response to a selective lectin with no signal variation in the presence of unselective proteins.
Anthraquinone-modified ligands could be used to facilitate the standardization of electrochemical impedance sensors for the reproducible, selective analysis of ligand-protein interactions.
Sugars distributed on the surface of mammalian cells are key informational molecules for cell-cell recognition and adhesion through the interaction with lectins (sugar recognition proteins). Unquestionably the ability to probe sugar-lectin recognitions may boost the advancement of the glycomics. However, conventional approaches for analysis of these interactions mainly rely on immunofluorescence techniques, which are time-consuming and expensive. As a result, a number of `label-free' methods for the quick and economic detection of lectins have been developed -.
Among the various methods introduced, electrochemistry, because of its ease in manipulation and good sensitivity, has been widely employed for lectin analyses ,,. In addition, electrochemical techniques generally do not require heavy facilities for signal output. Electrochemical impedance spectroscopy (EIS) can sensitively interpret the resistive ability of an interfacial species, which has been broadly applied in the study of corrosion science as well as development of label-free sensors. EIS sensors for lectins, based on the gold-alkenethiol self-assembly technique, have provided promising means for the concise, label-free detection of lectins and live cells that express a glyco-receptor -.
Nevertheless, while the use of gold as working electrode may increase the detection cost, the standardization of electrodes remains difficult due to the lack of an inherent signal `reporter'. To address these issues, we report here the design and synthesis of an anthraquinonyl glycoside (AG) in which the anthraquinone moiety can simultaneously serve as a `binder' for a graphene-based electrode and a reporter that produces an electrochemical signal to standardize the sensor fabrication. By using voltammetry, the AGs decorated on the graphene-based electrodes can be easily quantified, thereby facilitating the standardization of the electrodes to produce a unified signal window for lectin detection. Subsequently, EIS analyses showed that the standardized electrodes gave a highly reproducible electrochemical response to a selective lectin, suggesting the promise of using anthraquinone-modified glyco-ligands for the impedance detection of lectins.
Results and discussion
where R ct and R ct(0) are the charge transfer resistance in the presence and absence of lectin, respectively.
We then tested the selectivity of the biosenor with a series of unselective lectins and proteins including the N-acetyl glucosamine-selective wheat germ agglutinin (WGA), the galactose-selective peanut agglutinin (PNA), bovine serum albumin (BSA) and pepsin (Pep). As shown in Figure 3d, the electrode only showed an impedance change in the presence of the selective Con A with no insignificant response to the unselective proteins. This suggests the usefulness of the EIS sensor developed for the selective detection of lectins.
All purchased chemicals and reagents are of analytical grade. Nano-graphene (lateral diameters ranging from 1-10 nm) was purchased from Nanjing XFNano Materials Tech. Co., Ltd. Solvents were purified by standard procedures. Reactions were monitored by TLC (thin-layer chromatography) using E-Merck aluminum precoated plates of Silica Gel. 1H NMR spectrum was recorded on a Bruker AM-400 spectrometer using tetramethylsilane (TMS) as the internal standard (chemical shifts in parts per million). High resolution mass spectrum was recorded on a Waters LCT Premier XE spectrometer using standard conditions (ESI, 70 eV). High performance liquid chromatogram (HPLC) was taken on an Agilent 1100 equipment.
Synthesis of ZBW1
To a solution of b (250 mg, 0.50 mmol) and a (68.6 mg, 0.25 mmol) in a solvent mixture of CH2Cl2 (5 mL) and H2O (5 mL) were added CuSO4 · 5H2O (2.0 equiv.) and Na ascorbate (4.0 equiv.). This mixture was stirred over night and then diluted with CH2Cl2 and washed with brine. The combined organic layer was dried over MgSO4, filtered and concentrated in vacuum to provide a crude product. The product was purified by column chromatography (EtOAc/MeOH = 20:1, v/v) to give an intermediate as a yellow solid (249.4 mg, 0.19 mmol). To a solution of the intermediate in a solvent mixture of MeOH (5 mL) and H2O (5 mL) was added excessive Et3N. This mixture was stirred at room temperature for 36 h. Then, solvent was removed in vacuum to directly afford ZBW1 as a yellow solid (148.5 mg, 2-step yield 66.4%). R f = 0.48 (EtOAc/MeOH = 1:1, v/v). 1H NMR (400 MHz, D2O): Г 8.02 (s, 2H), 7.25-7.14 (m, 6H), 5.06 (s, 4H), 4.63 (s, 2H), 4.51 (t, J = 4.0 hz, 4H), 3.83 (t, J = 4.0 hz, 4H), 3.75-3.74 (m, 2H), 3.69 (d, J = 2.0 hz, 1H), 3.66 (d, J = 2.0 hz, 1H), 3.62 (d, J = 3.6 hz, 1H), 3.61 (s, 1H), 3.60-3.55 (m, 4H), 3.53 (s, 1H), 3.51 (s, 1H), 3.48 (s, 1H), 3.42 (t, J = 3.2 hz, 6H), 3.36 (s, 8H), 3.32-3.28 (m, 1H). HR-ESI-MS: calcd. for [C44H58N6O20 + Na]+ 1013.3604, found 1013.3606. HPLC: t R = 3.9 min over 17 min of eluent (acetonitrile/H2O = 9:1, v/v), purity 96.4%.
Cyclic voltammetry (CV)
CVs were recorded with a computer controlled CHI 1211B electrochemical station (Chenhua Co. Ltd., Shanghai, China) between -0.8 V and -0.2 V (vs. Ag/AgCl) at a scan rate of 100 mV/s. The electrolyte (Tris hCl, 0.01 M, pH 7.3) used was degassed with N2 for 20 min before measurements. Screen-printed electrodes (SPEs) were pretreated in a PBS (0.05 M, pH 7.0) containing 0.1 M KCl by applying an anodic potential of 2 V (vs. Ag/AgCl) for 200 s, and were then washed with water three times. The circular area (2 Mm in diameter) was used as the working electrode; the reference electrode was printed with 40% AgCl in silver paste, and the auxiliary electrode printed with carbon ink. For functionalization of the SPEs, a drop (4 μL) of aqueous nG solution was dripped onto the working electrode and dried under vacuum. Then a drop (4 μL) of the aqueous ZBW1 solution was dripped onto the working electrode and dried under vacuum. The functionalized SPEs were finally immersed into Tris hCl solutions for recording the CVs.
Differential pulse voltammetry (DPV)
DPVs were recorded with an amplitude of 0.05 V, a pulse width of 0.2 s, a standing time of 2 s, and a scanning range from -0.8 V to -0.2 V in Tris hCl (0.01 M, pH 7.3). For detection of analytes, a drop (4 μL) of the Tris hCl solution of ZBW1 (5 × 10−3 M) was dripped onto the GO functionalized working electrode area of SPE, and then incubated for 30 min. Then the electrodes were rinsed with the buffer solution three times, dried at room temperature, and then immersed in degassed buffer for measurement.
Electrochemical impedance spectroscopy (EIS)
Fourier Transform Infrared Spectroscopy (FTIR)
FTIR spectra were recorded on a Nicolet 380 FTIR spectrometer (Thermo Electron Corporation, USA). The samples were mixed with KBr and then compressed into pellets for analysis in the spectral range of ν = 4000 to 500 cm-1. All baselines of the spectra were corrected.
Raman spectra were performed on a Renishaw InVia Reflex Raman system (Renishaw plc, Wotton-under-Edge, UK) that employs a grating spectrometer with a Peltier-cooled charge-coupled device (CCD) detector coupled to a confocal microscope, which were then processed with Renishaw WiRE 3.2 software. The Raman scattering was excited by an argon ion laser (I = 514.5 nm).
In summary, we have reported the synthesis of an anthraquinonyl glycoside to fabricate EIS-based electrochemical sensors, where the AQ moiety served as a binder that promotes self-assembly of the glyco-ligands to the working electrode. Importantly, AQ also acted as a signal reporter that facilitates the sensor standardization. By using voltammetry and EIS, we determined that the electrodes with unified current signals showed reproducible impedance response to a selective lectin adhered to the electrode with high selectivity over other unselective proteins. This study provides insights into the simple construction of readily standardizable EIS sensors for the general, economic electrochemical analysis of ligand-protein interactions.
We thank the 973 project (2013CB733700), the National Natural Science Foundation of China (21176076, 21202045), the Key Project of Shanghai Science and Technology Commission (13NM1400900) and the Fundamental Research Funds for the Central Universities.
- Chen Y, Star A, Vidal S: Sweet carbon nanostructures: carbohydrate conjugates with carbon nanotubes and graphene and their applications. Chem Soc Rev. 2013, 42: 4532-4552. 10.1039/c2cs35396b.View ArticleGoogle Scholar
- Vedala H, Chen Y, Cecioni S, Imberty A, Vidal S, Star A: Nanoelectronic detection of lectin-carbohydrate interactions using carbon nanotubes. Nano Lett. 2011, 11: 170-175. 10.1021/nl103286k.View ArticleGoogle Scholar
- He XP, Wang XW, Jin XP, Zhou H, Shi XX, Chen GR, Long YT: Epimeric monosaccharide-quinone hybrids on gold electrodes toward the electrochemical probing of specific carbohydrate-protein recognitions. J Am Chem Soc. 2011, 133: 3649-3657. 10.1021/ja110478j.View ArticleGoogle Scholar
- Zhang HL, Wei XL, Zang Y, Cao JY, Liu S, He XP, Chen Q, Long YT, Li J, Chen GR, Chen K: Fluorogenic probing of specific recognitions between sugar ligands and glycoprotein receptors on cancer cells by an economic graphene nanocomposite. Adv Mater. 2013, 25: 4097-4101. 10.1002/adma.201300187.View ArticleGoogle Scholar
- Li Z, Deng SS, Zang Y, Gu Z, He XP, Chen GR, Chen K, James TD, Li J, Long YT: Capturing intercellular sugar-mediated ligand-receptor recognitions via a simple yet highly biospecific interfacial system. Sci Rep 2013, 3:2293.,Google Scholar
- Bertók T, Katrlík J, Gemeiner P, Tkac J: Electrochemical lectin based biosensors as a label-free tool in glycomics. Microchim Acta. 2013, 180: 1-13. 10.1007/s00604-012-0876-4.View ArticleGoogle Scholar
- Min IM, Choi L, Ahn KS, Kim BK, Lee BY, Kim KS, Choi HN, Lee WY: Electrochemical determination of carbohydrate-binding proteins using carbohydrate-stabilized gold nanoparticles and silver enhancement. Biosens Bioelectron. 2010, 26: 1326-1331. 10.1016/j.bios.2010.07.038.View ArticleGoogle Scholar
- Bertok T, Sediva A, Katrlik J, Gemeiner P, Mikula M, Nosko M, Tkac J: Label-free detection of glycoproteins by the lectin biosensor down to attomolar level using gold nanoparticles. Talanta. 2013, 108: 11-18. 10.1016/j.talanta.2013.02.052.View ArticleGoogle Scholar
- Hu Y, Zuo P, Ye BC: Label-free electrochemical impedance spectroscopy biosensor for direct detection of cancer cells based on the interaction between carbohydrate and lectin. Biosens Bioelectron. 2013, 43: 79-83. 10.1016/j.bios.2012.11.028.View ArticleGoogle Scholar
- Oliveira MDL, Andrade CAS, Correia MTS, Coelho LCBB, Singh PR, Zeng X: Impedimetric biosensor based on self-assembled hybrid cystein-gold nanoparticles and CramoLL lectin for bacterial lipopolysaccharide recognition. J Colloid Interface Sci. 2011, 362: 194-201. 10.1016/j.jcis.2011.06.042.View ArticleGoogle Scholar
- Loaiza OA, Lamas-Ardisana PJ, Jubete E, Ochoteco E, Loinaz I, Cabañero G, García I, Penad s S: Nanostructured disposable impedimetric sensors as tools for specific bimolecular interactions: sensitive recognition of concanavalin A. Anal Chem. 2011, 83: 2987-2995. 10.1021/ac103108m.View ArticleGoogle Scholar
- Bertok T, Gemeiner P, Mikula M, Gemeiner P, Tkac J: Ultrasensitive impedimetric based biosensor for glycoproteins containing sialic acid. Microchim Acta. 2013, 180: 151-159. 10.1007/s00604-012-0902-6.View ArticleGoogle Scholar
- Zhang X, Teng Y, Fu Y, Zhang S, Wang T, Wang C, Jin L, Zhang W: Lectin-based electrochemical biosensor constructed by functionalized carbon nanotubes for the competitive assay of glycan expression on living cancer cells. Chem Sci. 2011, 2: 2353-2360. 10.1039/c1sc00562f.View ArticleGoogle Scholar
- Chen H, Xi F, Gao X, Chen Z, Lin X: Bienzyme bionanomultilayer electrode for glucose biosensing on functional carbon nanotubes and sugar-lectin biospecific interaction. Anal Biochem. 2010, 403: 36-42. 10.1016/j.ab.2010.04.011.View ArticleGoogle Scholar
- Zhang Y, Luo S, Tang Y, Yu L, Hou KY, Cheng JP, Zeng X, Wang PG: Carbohydrate-protein interactions by "clicked" carbohydrate self-assembled monolayers. Anal Chem. 2006, 78: 2001-2008. 10.1021/ac051919+.View ArticleGoogle Scholar
- Otaman O, Boullanger P, Lafont D, Hamaide T: New amphiphilic glycopolymers based on a polycaprolactone-maleic anhydride copolymer backbone: characterization by 15N NMR and application to colloidal stabilization of nanoparticles. Macromol Chem Phys. 2008, 209: 2410-2422. 10.1002/macp.200800300.View ArticleGoogle Scholar
- Song W, Li DW, Li YT, Li Y, Long YT: Disposable biosensor based on graphene oxide conjugated with tyrosinase assembled gold nanoparticles. Biosens Bioelectron. 2011, 26: 3181-3186. 10.1016/j.bios.2010.12.022.View ArticleGoogle Scholar
- Huang X, Zeng Z, Fan Z, Liu J, Zhang H: Graphene-based electrodes. Adv Mater. 2012, 24: 5979-6004. 10.1002/adma.201201587.View ArticleGoogle Scholar
- Cai L, Fu Q, Shi R, Tang Y, Long TY, He XP, Jin Y, Liu G, Chen GR, Chen K: `Pungent' copper surface resists acid corrosion in strong HCl solutions. Ind Eng Chem Res. 2014, 53: 64-69. 10.1021/ie402609g.View ArticleGoogle Scholar
- He XP, Deng Q, Cai L, Wang CZ, Zang Y, Li J, Chen GR, Tian H: Fluorogenic resveratrol-confined graphene oxide for economic and rapid detection of Alzheimer s disease. ACS Appl Mater Interfaces. 2014, 6: 5379-5382. 10.1021/am5010909.View ArticleGoogle Scholar
- Sun X, Zhu B, Ji DK, Chen Q, He XP, Chen GR, James TD: Selective fluorescence detection of monosaccharides using a material composite formed between graphene oxide and boronate-based receptors. ACS Appl Mater Interfaces. 2014, 6: 10078-10082. 10.1021/am500801g.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.