(Adapted with permission from [23], copyright by the American Chemical Society.). Binding of conjugate to transferrin and hence a significant signal was observed with 12 g mL1 of conjugate at 2 hours incubation whereas, at this conjugate concentration and incubation time, a relatively small signal was obtained for IgG on the NPG surface. Figure 1. Even though array based methods do not directly provide detailed information about carbohydrate structure, often information on only the terminal or functional carbohydrate units is enough for screening of glycoproteins and other glycoconjugates. An increase in Km for the ALP enzyme is expected both as a consequence of conjugation to Con A and immobilization within NPG for a number of reasons. The rate of a reaction on the electrode surface can be obtained by dividing the peak difference current by the product nF, where, n is the number of electron involved in the redox process and F is Faraday's constant[97]. The enzyme reaction rate is assumed to be proportional to the peak difference current of the square wave voltammogram. Soybean agglutinin binds preferentially to terminal N-acetylgalactosamine units, and less strongly to galactose residues. Continue reading for detailed information about the fields on each unique tab. Serum transferrin consists of two biantennary glycan units and glycosylation changes in transferrin have been reported for disease conditions including hepatocellular carcinoma and sepsis [76, 77]. Square Wave Voltammetry (SWV) Pulse Sequence Detail, Figure 9. Bertok T, Sediva A, Katrlik J, Gemeiner P, Mikula M, Nosko M, Tkac J. Marradi M, Chiodo F, Garca I, Penade's S. Shulga OV, Zhou D, Demchenko AV, Stine KJ. It is hypothesized based on these initial results that an NPG array format with use of a larger number of lectin ALP conjugates each responding differently to an immobilized glycoprotein could be used as a method of electrochemical profiling of glycosylation. To perform a square wave voltammetry experiment in AfterMath, choose. Methyl -D-mannopyranoside is a ligand for Con A, and binding of Con A to other glycoproteins and glycoconjugates has been reported to be inhibited by Me--D-Man. Miller et al. 2741 Campus Walk Avenue Cell surface carbohydrates are also important for cell-cell communication, cell-pathogen interactions, and metastasis [14]. The ability of square wave voltammetry to rapidly scan wide range to rapidly scan a wide potential range has been used to advantage by researchers. Thus, we chose a 200 M substrate concentration for the kinetic assay, a concentration which falls in the pseudo-linear range. Time,, for One Square-Wave Cycle and Step. Cells are covered with glycocalyxes which vary with cell type, and in different stages of cell growth and differentiation [12]. Lead leaching from lead service lines, the main contamination source, and other plumbing materials is controlled by the plumbosolvency of water. After incubation with conjugate, the NPG coated wires with bound conjugate were incubated with 0.2 mM p-APP in glycine buffer (pH 9.0, 100 mM) for 2 minutes and then a square-wave voltammetry scan was conducted. Alkaline phosphatase labeling kits were purchased from Dojindo Molecular Technologies Inc. (Rockville, Maryland). Thus, detection of variant glycoforms of transferrin of lower concentration seems at least worth considering using electrochemical ELLA provided the transferrin fraction can be isolated and comparison with other methods such as mass spectrometry made. The SWV peak difference current was measured after incubation for 17 hours with glycoprotein in these solutions. Although glycans would not be directly identified, the response patterns could potentially indicate general glycan types or the likely presence of certain terminal sugars. Concanavalin A is one of the most commonly used lectins in glycan assays [85]. Changes in the response of a lectin-enzyme conjugate upon cell binding have also been used[42], as has a method based on competition between mannose units in an SAM and on the cell surfaces[43]. Hydroquinone undergoes oxidation at 21 mV on a flat gold surface[103]. During this period, data are not collected. The https:// ensures that you are connecting to the Hence, any estimate of fractional surface coverage is approximate in the absence of orientational information. The current response for SCP is shown in Fig3. The traditional approach of ELLA involves immobilization of glycoproteins or glycoconjugates on 96 well plates by physisorption followed by washing and then incubation with biotinylated lectins and then exposure to avidin or extravidin labeled alkaline phosphatase or horseradish peroxidase as an enzyme label, for the purpose of detection [32]. Solutions of 50 mM K[Ag(CN)2] and K[Au(CN)2] in 0.25 M Na2CO3 were prepared. In contrast, attempts to measure current peaks due to oxidation of p-aminophenol using linear sweep voltammetry were much less successful due to the high background currents arising from charging of the electric double layer. The square wave frequency in Hz is 1/. Gyurcsanyi RE, Bereczki A, Nagy G, Neuman MR, Lindner E. Pemberton RM, Hart JP, Stoddard P, Foulkes JA. Application notes discuss practical aspects of conducting specific experiments. Optimal experimental and operational parameters have been selected for the drug preconcentration and determination in aqueous medium. Figure 11.18 shows the shapes of SWV difference current curves under experimental conditions where the reduction of Zn2+ is reversible (10 Hz) and nearly irreversible (500 Hz). Differential Pulse Voltammetry (DPV) is a potentiostatic method that offers some advantages to common techniques like Cyclic Voltammetry (CV), in that the waveform is a series of pulses increasing along a linear baseline. Adding another ion in solution (e.g., Pb+2) would add a second peak at a different Vstep. Lectins are used as recognition elements in glycoanalytical techniques including ELLA, immunohistochemistry, and affinity chromatography [30]. Added to the staircase waveform is a square wave, so that as the voltage suddenly jumps with each step, the square wave is coincident with the jump. You can read how we use them in our privacy policy. When the pulse amplitude is sufficiently large that the pulse potential is close to the redox potential, there is a faradaic reaction in response to the potential pulse (assuming moderately fast electron transfer kinetics), and the magnitude of this current may depend on both the rate of diffusion and the rate of electron transfer. The following is a brief introduction to the theory of SWV. Square-wave voltammetry at = 0.1 s for 6 ppm Cd+2 in an aqueous acetate buffer. Dammer U, Hegner M, Anselmetti D, Wagner P, Dreier M, Huber W, Gntherodt HJ. The WaveDriver 100. Immobilization of the Bauhinia monandra (BmoLL) lectin onto gold nanoparticles dispersed in polyaniline on a gold electrode surface was also able to detect dengue fever glycoproteins via EIS. Gold wire (0.2 mm diameter, 99.99%) was obtained from Electron Microscopy Sciences (Fort Washington, PA). Carcinoembryonic antigen (CEA) and prostate specific antigen (PSA) were purchased from Fitzgerald (North Acton, MA). Prostate specific antigen is a biomarker for prostate cancer; it is also heavily glycosylated and its different glycosylation patterns have been reported as related to malignant versus benign conditions [84]. Samanman S, Thammakheta C, Kanatharanaa P, Buranachai C, Thavarungkula P. Landberg E, Pahlsson P, Lundblad A, Arnetorp A, Jeppsson JO. Building 100 Tech. The use of other enzymes in the Con A enzyme conjugate, such as horseradish peroxidase, could also be considered. The potential was scanned from 0.1 V to 0.2 V at a rate of 5.0 mV sec1. Sampling Mode (circled in red) has three choices, including Surface mode. The height of the peak is directly related to the solution concentration of that species. The properties of NPG electrodes present opportunities for the development of new electrochemical assay formats. (a) Square-wave voltammograms (SWV) of Con A-ALP conjugate immobilized on gold wire and on NPG. Square-wave voltammograms of the flavonoids (10 M) and their structures - quercitrin (b), rutin (c), quercetin (d), chrysin (e) and 100 M of diosmin (f). Some of the advantages of p-APP over other substrates that make p-APP an attractive substrate include the low oxidation potential, less electrode fouling and electrochemical reversibility, and it is commercially available [110]. CV scans were performed in 3 mL of solution in a three electrode cell arrangement as mentioned before for other CV measurements. Voltamperograms for 10-ng ml solutions of lead and of cadmium were recorded under the various experimental conditions. This Application Note describes what square-wave voltammetry is, and the parameters involved. Square Wave Voltammetry. Consider a reduction. After the induction period, the potential of the working electrode is stepped through a series of increasing forward and reverse pulses from the Initial potential to the Final potential. Based on the adsorption and accumulation of Tarabine PFS using Osteryoung square-wave anodic stripping voltammetry (OSWASV) at MFE, the drug is easily detected as 0.134 ng/ml (5.51 x 10(-10) M). SWV was conducted using a pulse height of 50 mV, a pulse width of 0.2 sec, a step height of 2 mV and scan rate of 5.0 mV sec1 (vs. Ag|AgCl reference electrode) in pH 9.0 glycine buffer (100 mM). The square wave frequency in Hz is 1/. Conjugation to Con A will introduce steric hindrance to access of the substrate to the active site of ALP. A decrease in amount of the bound conjugate with an increase in amount of free glycoprotein in solution is observed. 1.0 5/28/2019 Copyright 2019 Gamry Instruments, Inc. Interface, Reference, and Framework are trademarks of Gamry Instruments, Inc. KCl) reference electrode. WaveVortex 10 Electrode Rotator Samples were cooled and absorbance measurement was recorded at 562 nm using a Cary-50 UV-visible spectrophotometer. Reference 3000 With Auxiliary Electrometer. More on Induction Period is found within the knowledgebase. A competitive type enzyme multiplied immunoassay (EMIT) for digoxin was reported by Chang et. SEM shows the expected network of pores and ligaments. Cell voltage is measured between the working and reference electrodes. If a biotinylated lectin is allowed to bind, then it is subsequently reacted with a streptavidin or avidin labeled enzyme. IC50 values using methyl -D-mannopyranoside were calculated using Graphpad Prism 5.04 software. First, it increases the discrimination against the charging current, since any residual charging current is subtracted out. The concentration of p-APP of 200 M is close to the Km value, and p-APP concentrations less than 100 M did not produce current peaks of sufficient magnitude in the SWV scans. Abstract. Waiting till the end of the pulse to sample the current avoids involving the charging current. Difference current peak heights from SWV scans (pulse height of 50 mV, pulse width of 0.2 sec, step height of 2 mV and scan rate of 5.0 mV sec1, Ag|AgCl reference electrode) vs. p-APP concentrations from 0.01 mM to 1.0 mM in pH 9.0 glycine buffer (100 mM) for the Con A ALP conjugate immobilized on lipoic acid SAMs fit to the Michaelis-Menten equation. An official website of the United States government. Binding affinity has been shown to be higher for multivalent mannose units compared to similar monovalent units in the glycoconjugates [122]. Measurement Requirements The basis of all pulse techniques is the difference in the rate of the decay of the charging and the faradaic currents following a potential step (or "pulse"). Instruments. The terminal carbohydrate units of CEA can vary with the source of the CEA sample [134]. During this period, data are not collected. In this report, theory for cyclic square wave voltammetry for single and consecutive reversible electron transfer reactions is presented and experimentally verified. In the kinetic enzyme-linked lectin assay, the peak difference current for p-AP oxidation on each wire was measured before incubating with glycoprotein and again after incubating with glycoprotein. Since alkanethiol SAMs are reported to be less stable at basic pH, and most of the protein binding studies are performed in phosphate buffer close to neutral pH, phosphate buffer at pH 7.4 was chosen for the incubation of electrodes with the conjugates and for longer storage [107, 108]. Carbohydrate-protein interaction studies have predominantly been performed on substrate supported gold films [43, 58], glass [59] or polystyrene [60] with a growing number of studies on Au nanoparticles [6166]. Fig. Lectin-carbohydrate interactions were subsequently studied in a competitive format assay, in a format using immobilized glycoproteins for screening lectin-enzyme conjugate binding, and in a kinetic assay approach based on the rate of enzymatic reaction before and after lectin-glycoprotein binding. Peracaula R, Tabars G, Royle L, Harvey DJ, Dwek RA, Rudd PM, de Llorens R. Edelman GM, Cunningham BA, Reeke GN, Becker JW, Waxdal MJ, Wang JL. Below is an example of the experimental setup window* from Gamry Instruments Framework software: Fig. At the end of the relaxation period, the post-experiment idle conditions are applied to the cell and the instrument returns to the idle state. The use of self-assembled monolayers of lipoic acid allows easy immobilization of the conjugate onto the NPG surface. The surface morphology and general structure of NPG was studied by scanning electron microscopy (JEOL JSM-6320F field emission SEM). Below are the typical results for SWV (current difference) for the oxidation of a 1 mM solution of ferrocene n 0.1 M Bu. who immobilized lectin onto the mixed SAM modified gold surface and then performed a competitive assay using CdS nanoparticle labeled and unlabeled sugars. Eraso LH, Ginwala N, Qasim AN, Mehta NN, Dlugash R, Kapoor S, Schwartz S, Schutta M, Iqbal N, Mohler ER, Reilly MP. Differential protein glycosylation has been found to be associated with different disease conditions and malignancies [9]. This is a consequence of the subtraction of the forward and reverse currents, as in the case of reduction of Zn2+. A period of 2 minutes was found to be sufficient to obtain the maximal difference current and longer times of exposure to substrate up to 30 minutes did not increase the difference current. government site. Sampled current polarograph (SCP) is a modification of the classical DC polarography experiment, and was designed to reduce the effect of the changing surface area of the mercury drop electrode. The absolute value of the difference in the maximum peak difference currents |(I (before incubation) I (after incubation))| was plotted vs. the glycoprotein concentration to obtain the response plots. SWV peak difference current (I) was determined using different concentrations of glycoprotein in solution. The Data on protein-carbohydrate interactions is important for improved understanding of carbohydrate associated biological processes. Different peak difference currents were observed depending on the amount of conjugate bound onto the glycoprotein attached to the SAM on the NPG surface, which is anticipated to vary with the structure of the glycans present on the immobilized glycoproteins, the number and orientation of the glycans presented by the surface, and any contribution from non-specific adsorption of conjugate. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Reuel NF, Mu B, Zhang J, Hinckley A, Strano MS. Kuno A, Uchiyama N, Koseki-Kuno S, Ebe Y, Takashima S, Yamada M, Hirabayashi J. Pei Z, Anderson H, Aastrup T, Ramstrm O. Sanchez-Pomales G, Morris TA, Falabella JB, Tarlov MJ, Zangmeister RA. onds using single-sweep square-wave voltammetry, to a couple of minutes for a cyclic voltammogram, to possibly 30 min (or more) for a very-low-concentration ASV determination. Sodium carbonate (enzyme grade, >99%), N-hydroxysuccinimide (NHS, 97%), sulfuric acid (certified ACS plus), nitric acid (trace metal grade), hydrogen peroxide (50%), and sodium bicarbonate (certified ACS) were all from Fisher Scientific (Pittsburg, PA). Fig. The potential wave form consists of a series of pulses of increasing amplitude, with the potential returning to the initial value after each pulse. [1] It has found numerous applications in various fields, including within medicinal and various sensing communities. Multivalency and increased affinity have been achieved on solid surfaces by appropriately controlling carbohydrate density on the surface [56]. Various concentrations of acidified copper ions in solution, recorded with square-wave voltammetry. We also demonstrate that glycoproteins immobilized onto NPG exhibit a differential response to the binding of a lectin-enzyme conjugate (Con A or soybean agglutinin bound to alkaline phosphatase) as detected by SWV detection of the product formed from the action of alkaline phosphatase on the substrate p-aminophenyl phosphate (p-APP). Development of array based methods for the screening of glycoforms is gaining increasing interest among researchers in glycomics [19]. Gonzalez-Quintela A, Alende R, Gude F, Campos J, Rey J, Meijide LM, Fernandez-Merino C, Vidal C. Green ED, Adelt G, Baenziger J, Wilson S, Van Halbeek H. Safina G, Duran IB, Alasel M, Danielsson B. Yamashita K, Totani K, Kuroki M, Matsuoka Y, Ueda I, Kobata A. Chandrasekaran EV, Davila M, Nixon DW, Goldfarb M, Mendicino J. Tabares G, Radcliffe CM, Barrabes S, Ramirez M, Aleixandre RN, Hoesel W, Dwek RA, Rudd PM, Peracaula R, De Llorens R. Meany DL, Zhang Z, Sokoll LJ, Zhang H, Chan DW. ALP can act against a number of other reported substrates that can be oxidized at an electrode surface. Measurements of current are timed to occur at the end of each forward and reverse pulse. The rigorous theory applies to wide mechanistic diversity and SWV parameters. SWV was conducted in both cases using a pulse height of 50 mV, a pulse width of 0.2 sec, a step height of 2 mV and scan rate of 5.0 mV sec1 (vs. Ag|AgCl reference electrode) in pH 9.0 glycine buffer (100 mM).