- Email: [email protected]
A facile route to glycated albumin detection
Author’s Accepted Manuscript A facile route to glycated albumin detection Nadra Bohli, Olivier Meilhac, Philippe Rondeau, Syrine Gueffrache, Laurence Mora, Adnane Abdelghani www.elsevier.com/locate/talanta
PII: DOI: Reference:
S0039-9140(18)30272-8 https://doi.org/10.1016/j.talanta.2018.03.027 TAL18461
To appear in: Talanta Received date: 23 November 2017 Revised date: 7 March 2018 Accepted date: 11 March 2018 Cite this article as: Nadra Bohli, Olivier Meilhac, Philippe Rondeau, Syrine Gueffrache, Laurence Mora and Adnane Abdelghani, A facile route to glycated albumin detection, Talanta, https://doi.org/10.1016/j.talanta.2018.03.027 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. 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.
A facile route to glycated albumin detection Nadra Bohlia,*, Olivier Meilhacb,c, Philippe Rondeaub, Syrine Gueffrachea, Laurence Morad, Adnane Abdelghania a
Carthage University, UR17ES22 Research Unit of Nanobiotechnology and valorisation of medicinal plants, National Institute of Applied Science and Technology, Bp 676, Centre Urbain Nord, 1080 Charguia Cedex, Tunisia. b Université de La Réunion, INSERM, UMR 1188 Diabète athérothrombose réunion Océan Indien (DéTROI), Saint-Denis de La Réunion, France. c CHU de La Réunion, Saint-Denis de La Réunion, France. d Université Paris13, Inserm, U1148, Laboratory for Vascular Transitional Science, Institut Galilée, Sorbonne Paris Cité, F-93430, Villetaneuse, France.
Abstract: In this paper we propose an easy way to detect the glycated form of human serum albumin which is biomarker for several diseases such as diabetes and Alzheimer. The detection plateform is a label free impedimetric immunosensor, in which we used a monoclonal human serum albumin antibody as a bioreceptor and electrochemical impedance as a transducing method. The antibody was deposited onto a gold surface by simple physisorption technique. Bovine serum albumin was used as a blocking agent for non-specific binding interactions. Cyclic voltammetry and electrochemical impedance spectroscopy were used for the characterization of each layer. Human serum albumin was glycated at different levels with several concentrations of glucose ranging from 0 mM to 500 mM representing physiological, pathological (diabetic albumin) and suprapathological concentration of glucose. Through the calibration curves, we could clearly distinguish between two different areas related to physiological and pathological albumin glycation levels. The immunosensor displayed a linear range from 7.49 % to 15.79 % of glycated albumin to total albumin with a good sensitivity. Surface plasmon resonance imaging was also used to characterize the developed immunosensor.
*
Corresponding author: Email: [email protected], [email protected]
1
graphical abstract
Keywords: Electrochemical immunosensor.
1
Impedance
Spectroscopy,
Glycated
albumin,
SPRi,
Introduction
Advanced glycation end products (AGEs) are the result of a non-enzymatic non reversible reaction between reducing sugars such as glucose and proteins like hemoglobin or albumin. Compared to their native form, the resulting glycated proteins undergo structural changes and several of their functions are often impaired [1]. AGEs have attracted a growing of interest during the past decade as they seem to be related to several health complications associates
2
with the aging process, diabetes mellitus, Alzheimer, as well as cardiovascular disease and could be used as biomarkers for these pathologies [2, 3]. Thus, a lot of effort is now aimed at developing methods to detect and quantify glycated proteins in order to predict potential complications [4, 5, 6, 7]. Quantification of glycated hemoglobin is routinely used to monitor long-term glycemic control in diabetic patients. The turnover of albumin in plasma is much shorter than that of hemoglobin, and assessment of albumin glycation may be more appropriate in diabetic patients with chronic kidney disease [8, 9]. Sandwich assay, like ELISA (Enzyme-Linked ImmunoSorbent Assay) or RIA (radio-immuno-assay) techniques are the most known systems under development in AGE detection systems. They require a label or enzyme conjugated to a direct or indirect ligand of the target analyte. The ligand used is often an antibody that recognizes specifically an epitope and allows the capture of the target. In sandwich ELISA, a different antibody directed to the target is required; it can be directly conjugated to a label, or a secondary antibody carrying the label may be used to amplify the signal. The amount of label detected is assumed to be proportional to the number of bound targets. Labels can be fluorophores, radioisotopes, active enzymes, etc. In addition to the cost and time required for ELISA testing, some concerns are often underlined. In fact, the yield of the target-label coupling reaction can be highly variable for proteins and their labeling can drastically change the protein binding properties [4]. Impedance biosensors offer an alternative to ELISA assay as they allow the development of cost effective label-free detection systems [10, 11, 12]. They are also easily miniaturized and can provide good start for the development of point of care testing devices (POCT) [13]. In this paper we propose a simple method for the detection of glycated albumin based on label-free impedimetric immunosensor using only one type of anti-human serum albumin as a capture antibody followed by the detection of electrochemical changes associated with albumin glycation. The glycating agent used is glucose, as it is the major circulating glycating agent for humans.
2 2.1
Material and methods Reagents
Anti-Human Serum Albumin monoclonal antibody (mAb) was purchased from ABBIOTEC (USA). The Goat anti-rabbit IgG and Rabbit IgG were purchased from Pierce Biotechnology. Bovine serum albumin (BSA) was obtained from Sigma-Aldrich (France). Lyophilized powder of HSA with purity above 97 % was bought from Sigma Aldrich. Phosphate buffered
3
saline (PBS) containing 140 mM NaCl, 2.7 mM KCl, 0.1 mM Na2HPO4, 1.8mM KH2PO4, pH = 7.4 was used for all experiments with K4Fe(CN)63-/K4Fe(CN)64− redox couple at 5 mM concentration. All reagents were of analytical grade. The used ultrapure water, with a resistance of 18.2 MΩ.cm−1, was produced by a Millipore Milli-Q system. 2.2
GA and HSA preparation
The samples used are referenced as HSA and GA respectively corresponding to native nonglycated and glycated human serum albumin. For the preparation of glycated serum albumin, lyophilized powder of commercial HSA with purity above 97 % was used with glucose powder (99.5 % of purity). GA samples were produced by incubating 0.6 mM HSA protein with different concentrations of glucose and in PBS under sterile conditions and nitrogen gas in capped vials at 37 °C for three weeks. The used glucose concentrations are 25 mM, 50 mM, 100 mM and 500 mM. More details on HSA and GA preparation are described in detail in previous reference [14]. HSA and GA preparations were characterized in terms of glycation level (%), advanced glycation end-products (AGE) level (%) and average molecular mass. The glycation level was determined by boronate affinity chromatography (100 % corresponding to complete glycation of HSA). The percent increase in fluorescent AGE level was obtained by the maximal fluorescence emission at an excitation wavelength of 340 nm. The average molecular mass was obtained by ESI/MS. Table 1 indicates the level of glycation for each HSA sample. These three methods were well described in a previous study [14].
2.3
Gold electrode cleaning
The gold electrodes were purchased from the LAAS of Toulouse (France). Evaporated gold (300 nm thickness) was coated on silicon, using titanium under layer (30 nm thickness) as a substrate. Prior to their modification, the gold electrodes were cleaned in an ultra-sonic bath for 10 min in acetone. Then, they were dried under a nitrogen flow and then dipped for 15 min into ‘piranha solution’ (3:1 (v/v) 95 % H2SO4 / H2O2). Finally, the gold substrates were rinsed two to three times with ultra-pure water. 2.4
Cyclic voltammetry
Cyclic voltammetry was performed at room temperature in a conventional voltammetric cell with a three separated electrodes configuration using Chi 604e impedance analyser (CH Instruments, USA). The gold electrode (0.16 cm²) was used as working electrode, and platinum (1 cm2) and Ag/Ag-Cl electrodes were used as counter and reference electrodes,
4
respectively. All the electrochemical measurements were carried out in PBS buffer solution with a scan rate of 100 mV/s and a potential scan from -600 mV to 600 mV. 2.5
Electrochemical Impedance Spectroscopy
The impedance measurement was performed with a three separate electrodes configuration cell using Chi 604e impedance analyser (CH Instruments, USA) in the frequency range from 100 mHz to 100 kHz, using a modulation voltage of 10 mV. All electrochemical measurements were carried out in PBS. More details on electrochemical impedance spectroscopy can be found in reference [15]. EIS results were analysed by fitting the experimental impedance data to electrical equivalent circuit models. Parameters of the electrical-equivalent circuits were obtained by fitting the impedance function to the measured Nyquist plots with a program built into the Chi 604e impedance analyser. 2.6
Surface Plasmon Resonance Imaging
SPRi measurements were performed with an SPR imager® II (from GWC Technologies, USA), with an excitation wavelength of 850 nm. The experimental setup is based on a Kretschmann configuration, in which the light from a light-emitting diode integrated with a prism (prism refractive index = 1.7) is firstly p-polarized and then internally reflected from a metal surface. The used metal surface is a 16 gold spots array format glass substrate. Each gold spot has a 0.004 cm2 surface on which the different layers of the immunosensor were deposited. The resonance angle was determined according to the minimum reflectivity signal and was set for all the measurements. A couple charge device camera was used to capture all data for all the gold spots simultaneously and converts the reflectivity changes to pixels data, allowing a real time refractive index reading. All measurements were performed with PBS buffer at 29 °C. The substrate was exposed to the appropriate analyte using the 1mL flow cell.
3 Results and discussion 3.1
HSA and GA structural and biochemical characterizations
The biochemical characterization of HSA and GA preparations, summarized in Table 1, revealed a glucose dose-dependent increase in glycation level (up to 66.7% [GA/Total Albumin] for G500) and in fluorescent AGE formation (up to 35.2% for G500). In parallel, an increase in average molecular mass in a dose dependent manner was also noticed. For
5
instance, the molecular mass in G100 was approximately increased by 625 Da corresponding to a condensation of ~4 glucose units per molecule of albumin (one glucose unit is equivalent to a ass increase of 160 Da). For G500, this increase in molecular mass is about 1621 Da corresponding to the attachement of ~10 glucose units per albumin molecule.
3.2
Immuosensor design
The immunosensor construction is realized following three main steps. The first one is the physical adsorption of the HSA antibody solution onto the cleaned gold electrode surface. Unlike self-assembled monolayers (SAMS) treatment, physical adsorption is a simple procedure that is not time consuming. It consisted in depositing 50 microliters of anti-HSA monoclonal antibody at a concentration of 50 µg/mL on the gold electrode followed by an overnight incubation at 4 ºC. The second step consisted in blocking the reactive sites on the gold electrode through the deposition of 100 µL of BSA solution (0.1 %, w/v) on the surface for 45 min. The aim of this step is to prevent non-specific adsorption of antigens onto the uncovered gold surface, thus making sure that the antigens will be only bound to the antibodies. Finally, human serum albumin at different glycation levels were deposited on the modified gold electrode surface and incubated for 15 minutes at room temperature at a fixed concentration, thus allowing the antigen/antibody specific reaction to take place. All incubations were carried out in a humid chamber. After each immobilisation step of the antibody, BSA or the several analyte samples used, the electrode is washed three times with PBS to remove the excess unbound molecules, and appropriate measurements were performed. 3.3
Characterization of the immunosensor layers
Cyclic voltammetry and electrochemical
impedance
spectroscopy were used
as
characterization techniques after each step in the immunosensor building. Cyclic voltammetry is a potentiodynamic electrochemical technique performed by cycling the potential of a working electrode, and measuring the resulting current. Figure 1 shows the cyclic voltammogram of the composing layers of the immuosensor measured after each deposition step which are the bare gold electrode, the gold/mAB
6
(monoclonal antibody to human serum albumin) and the gold/mAB/BSA layers. All voltammogramms were cycled at least three times in order to verify the signal stability. The cyclic voltammogramm obtained for the bare gold electrode displays a reversible nature, with two current peaks corresponding to Fe(CN)64− oxidation and Fe(CN)63− reduction, typical of the redox couple used. These peak intensities decreased after the deposition of the antibody layer. They are severely diminished upon the addition of the BSA layer, showing that almost all the faradic current was blocked. The successful immobilization of each functionalized layer was confirmed through electrochemical impedance spectroscopy (EIS) measurements. EIS is a powerful technique for the investigation of interfacial reaction mechanisms. It measures the response of an electrochemical system to an oscillating potential as a function of frequency. This technique allows the explicit observation of different processes occurring in the monolayer and taking place at different rates such as diffusion currents, double layer charging and solution resistance. Figure 2 shows the Nyquist plots of the composing layers of the immunosensor measured after each deposition step which are the bare gold electrode, the gold/mAB and the gold/mAB/BSA layers. The impedance spectrum was fitted using Randles equivalent electrical circuit presented in figure 3. The elements composing this circuit are briefly described as follows. Rs results from the resistance of the electrolyte, the contacts and connections. It is generally not affected by binding. Zw is the Warburg impedance. It represents the delay arising from the diffusion of the electroactive species to the electrode, and is only detectable at low frequencies. ZCPE is the constant phase element modelling the non-pure capacitor formed between the electrode and ions in solution. The capacitor combines the surface modification capacitance and the double layer capacitance and is generally dependent on the thickness and dielectric constant of the probe layer. CPE behaviour can be explained by dispersion in local capacitance values due to microscopic roughness, and inhomogeneous current distribution resulting from microscopic chemical heterogeneities and ion adsorption. The complex impedance of a CPE is given by equation 1: =
( )!×"
(1)
Where Q is analogous to a capacitance, ω is the frequency (rad/s), and 0.5 < n < 1. Finally, Rct is the charge transfer resistance. It is associated to the energy potential of the oxido-reduction event occurring at the electrode alongside with the energy barrier of the
7
redox species reaching the electrode owing the electrostatic repulsion and/or steric hindrance. Rct is the commonly used element to indicate affinity binding for faradaic systems. The measured spectra of the impedance and phase were analysed in terms of electrical equivalent circuits. The electric parameters of the system were calculated with the computer program and the fit error was kept under a maximum of 10 %. All of the values of the electrical parameters are presented in Table 2. In the Nyquist plots, the diameter of the semi-circles corresponds to the charge transfer resistance of the electrode/electrolyte interface. The bare gold electrode displayed a small circle at high frequencies, suggesting a very low electron transfer resistance to the redox probe dissolved in the electrolyte solution. When the HSA antibody was immobilized on the electrode, the charge transfer resistance increased from 274.4 Ω to 768.1 Ω. Then, when BSA was added, Rct increased to 7683 Ω. We clearly observe that the BSA layer drastically increases the Rct. This increase is due to the conductivity decrease at the working electrode– electrolyte interface where the grafted layer becomes more insulating. These results confirm those obtained with cyclic voltammetry and indicate the successful immobilization of the antibody and BSA layers onto the gold surface. 3.4
Glycated albumin detection
Electrochemical impedance spectroscopy measurements for the detection of different ratios of glycated albumin to total albumin were made for a concentration of 50 ng/mL as presented in Figure 4. Nyquist plots show a significant variation of the impedance spectral curves with different HSA glycation ratios. We observe then that the charge transfer resistance decreases with the increase in the ratio of GA to total albumin. This observation can be explained by the global charge displayed by the protein. In fact, previous studies measured the global charge of these proteins at physiological pH, known as the isoelectric point. And it was found to be quite weak (global negative charge) and to decrease upon glycation [16, 17]. The morphology changes operating on HSA upon glycation can also lead to a favorable configuration to the transport of negative charges (through more heterocycles with mobile electrons or double links). We clearly observe that for the same dilution, the response of the immunosensor to G0 and G5 glycation levels are very different from those of higher glycation levels. R ct is very low for high glycation levels. The same measurement was done for 10 ng / mL concentration. In order to better understand the behaviour shown above, impedance data was fitted using the
8
Randles equivalent circuit. According to the Rct values obtained from data fitting, we calculated the change of the charge transfer resistance upon HSA glycation level following equation 2: ∆%&'
#
%*
#=
%&' (+,)- %*
(2)
%*
Where Rct(Ag) is the value of charge transfer resistance after the immunoreaction and R 0 is the value of the charge transfer resistance obtained for the fully functionalized immunosensor (Gold/mAB/BSA). The calibration curves consisting of the variation of the percentage values of relative %* versus the ratio of glycated electrode charge transfer resistance variation #∆%% &'# = %&'(+,)% *
*
albumin to total albumin at two different concentrations with each concentration is presented in the figure 5. According to these calibration curves, the linear range of the immunosensor depends on the concentration used for the analyte. For a concentration of 50 ng/mL, the linearity was observed for an albumin glycation ratio from 7.49 % to 15.79 % and up to 22.56 % for a concentration of 10 ng/mL. The sensitivity of the immunosensor, quantified by the slope of the linear part of the calibration curve is evaluated by linear fitting to 5 [%. [% (GA/Total albumin])]−1] for the concentration of 50 ng/mL and to 0.6 [%. [% (GA/Total albumin)] −1] for the concentration of 10 ng/mL. The saturation phase is reached faster for high analyte concentration as it seems that the glycated HSA proteins induce a more conductive layer on the immunosensors’ surface. These results confirm previous studies in which it has been demonstrated that the glycation human serum albumin has an impact on its dielectric properties as well as on its structure and morphology [18, 19, 20]. It is important to note that for a healthy person, albumin glycation levels generally range from 11 % to 16 %. So, the proposed immunosensor can definitely provide an easy detection of the protein and an easy assessment of its glycation levels, associated with unbalanced diabetic conditions. To confirm that the above observed impedance changes generated from the result of specific antibody-antigen interaction, we replaced the anti-HSA antibody with anti-IgG. The immunosensor was exposed to the target analyte at a concentration of 50 ng/mL. The glycation ratio of the analyte was increased in order to observe if there was a significant effect on the immunosensor by EIS. The associated calibration curve is given in figure 6. As
9
we can see, the sensor was not subjected to the non-specific binding and is therefore applicable to the selective determination of glycated HSA. In order to better characterise the developed immunosensor at a concentration of 50 ng/mL, we then used surface plasmon resonance imaging as an alternative transducing technique. 3.5
Detection of Glycated albumin with SPR Imaging
SPR imaging is an optical method used for label free detection of analytes and for evaluating molecular interactions [21, 22, 23]. 100 microliters of HSA antibody were deposited on the cleaned SPRi substrate and incubated overnight at 4 °C. After washing out the excess unbound antibody with PBS, the substrate was treated with a solution of BSA (0.1 %, w/v) for 45 minutes to prevent unspecific binding. The substrate was then mounted in the microfluidic cell and exposed to the injection of the different 50 ng/mL analyte concentration samples. After each antigen injection, an injection of PBS was performed with the pumping device to wash out the unbound antigen to the surface of the immunosensor. All the data were collected on real time using differential imaging. The obtained signals are presented in figure 7. We observe that the SPRi signal changes after each antigen injection. This change is due to the specific recognition by the anti-HSA. This recognition induces a thin layer deposition, which leads to a resonance angle variation at the interface. For the 50 ng/mL concentration, an SPRi calibration curve is plotted and presented in figure 8. The dynamic range was observed for 7.49 % up to 46.5 % of GA to total albumin.
4
Conclusion
In this paper, we developed a label free impedimetric immunosensor for the detection of glycated human serum albumin based on a simple physisorbed layer of monoclonal antibody to HSA. The immunosensor was tested for the detection of different glycation levels of albumin prepared through incubation in glucose concentrations ranging from 0 mM to 500 mM, at two distinct concentrations of 10 ng/mL and 50 ng/mL. High and low glycation levels are easily distinguished from the calibration curves. A severe drop in the charge transfer resistance is observed for HSA with high glycation levels. Dynamic range for 50 ng/mL was observed for G5 and G25 corresponding to a level of glycation obtained respectively with 5 (G5) and 25 mM of glucose (G25). SPRi optical transducing technique was also used for the characterisation of the immunosensor.
10
Acknowledgements This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
References [1]
R. Singh, A. Barden, T. Mori, L. Beilin, Advanced glycation end-products: a review, Diabetol. 44 (2001) 129-146.
[2]
A. Prasad, P. Bekker, S. Tsimikas, Advanced glycation end products and diabetic cardiovascular disease, Cardiol. Rev. 20 (2012) 177-83.
[3]
G. Münch, J. Thome, P. Foley,
R. Schinzel,
P. Riederer, Advanced glycation
endproducts in ageing and Alzheimer's disease, Brain Res Brain Res Rev. 23 (1997) 134-43. [4]
A. Jalaluddin Mohd, A. Saheem, C. Inho, A. Nashrah, F. Mohd, T. Godovikova, S. Uzma, Critical Review, Recent Advances in Detection of AGEs: Immunochemical, Bioanalytical and Biochemical Approaches, Int. Union Biochem. Mol. Biol. 67 (2015) 897-913.
[5]
X. Wang, N. Xia, L. Liu, Review: Boronic Acid-Based Approach for Separation and Immobilization of Glycoproteins and Its Application in Sensing, Int. J. Mol. Sci. 14 (2013) 20890-20912.
[6]
O. S. Zhernovaya, V. V. Tuchin, I. V. Meglinski, Monitoring of blood proteins glycation by refractive index and spectral measurements, Laser Phys. Lett. 5 (2008) 460-464.
[7]
N. Vigneshwarana, G. Bijukumarb, N. Karmakara, S. Ananda, A. Misra, Autofluorescence characterisation of advanced glycation end products of hemoglobin, Spectrochim. Acta Part A. 61 (2004) 163-170.
[8]
F. E. Vos, J. B. Schollum, R. J. Walker, Glycated albumin is the preferred marker for assessing glycaemic control in advanced chronic kidney disease, Nephrol. Dial. Transplant. Plus. 4, (2011) 368-375.
[9]
K. Neelofar, A. Jamal, Review Article: Amadori albumin in diabetic nephropathy, Indian. J. Endocrinol. Metab. 19 (2015) 39-46.
11
[10]
H. Chammem, I. Hafaid, N. Bohli, A. Garcia, O. Meilhac, A. Abdelghani, L. Mora, A Disposable Electrochemical Sensor based on Protein G for High-Density Lipoprotein (HDL) Detection, Talanta. 144 (2015) 466-473.
[11]
Q. Xu, J. J. Davis, The Diagnostic Utility of Electrochemical Impedance, Electroanal. 26 (2014) 1249-1258.
[12]
F. Lisdat, D. Schäfer, The use of electrochemical impedance spectroscopy for biosensing, Anal. Bioanal. Chem. 391 (2008) 1555-1567.
[13]
G. Abel, Current status and future prospects of point-of-care testing around the globe, Expert. Rev. Mol. Diagn. 15 (2015) 853-5.
[14]
J. Baraka-Vidot, C. Planesse, O. Meilhac, V. Militello, J. Van den Elsen, E. Bourdon, P. Rondeau, Glycation alters ligand binding, enzymatic, and pharmacological properties of human albumin, Biochem. 54 (2015) 3051-62.
[15]
J. R. MacDonald, Impedance Spectroscopy, Wiley, New York, 1987.
[16]
I. Bundschuh, I. Jäckle-Meyer, E. Lüneberg, C. Bentzel, R. Petzoldt, H. Stolte, Glycation of serum albumin and its role in renal protein excretion and the development of diabetic nephropathy, Eur. J. Clin. Chem. Clin. Biochem. 30 (1992) 651-6.
[17]
J. Figge, T. Rossing, V. Fencl. The role of serum proteins in acid–base equilibria. J. Lab. Clin. Med. 117 (1991) 453-67.
[18]
N. Bohli, H. Chammem, O. Meilhac, L. Mora, A. Abdelghani, Electrochemical Impedance Spectroscopy on Interdigitated Gold Microelectrodes for Glycosylated Human Serum Albumin Characterization, IEEE Trans. NanoBiosci.16 (2017) 676681.
[19]
P. Rondeau, N. R. Singh, H. Caillens, F. Tallet, E. Bourdon, Oxidative stresses induced by glycoxidized human or bovine serum albumins on human monocytes, Free. Radic. Biol. Med. 45 (2008) 799-812.
[20]
P. Rondeau, E. Bourdon, The glycation of albumin: Structural and functional impacts, Biochim.93 (2011) 645-658.
12
[21]
M. Puiu, C. Bala, SPR and SPR Imaging: Recent Trends in Developing Nanodevices for Detection and Real-Time Monitoring of Biomolecular Events, Sens.16 (2016) 116.
[22]
H. Chammem, I. Hafaid, O. Meilhac, F. Menaa, L. Mora, A. Abdelghani, Surface Plasmon Resonance for C-Reactive Protein Detection in Human Plasma, J. Biomater. Nanobiotechnol. 5 (2014) 153-158.
[23]
V. Kodoyianni, Label-free analysis of biomolecular interactions using SPR imaging, BioTech. 50 (2011) 32-40.
Figure Captions Figure 1: Cyclic voltammograms of each layer composing the immunosensor: (a) bare gold electrode, (b) Gold/mAb (monoclonal antibody to human serum albumin) and (c) Gold/mAb/BSA. The measurements were made in a PBS buffer solution of pH 7.4 in the presence of 5 mM Fe(CN6)3–/4– redox probe with a scan rate of 100 mV/s. Figure 2: Nyquist plots of impedance spectra for each layer composing the immunosensor: (a) bare gold electrode, (b) Gold/mAb and (c) Gold/mAb/BSA. The measurements were made in a PBS buffer solution of pH 7.4 in the presence of 5 mM Fe(CN6)3–/4–
13
redox probe at a frequency range between 100 mHz to 100 kHz, using a modulation voltage of 10 mV. Figure 3: Randles equivalent electrical circuit. Figure 4: Nyquist plots of impedance spectra for different ratios of glycated albumin to total albumin at a concentration of 50 ng/mL. The measurements were made in a PBS buffer solution of pH 7.4 in the presence of 5 mM Fe(CN6)3–/4– redox probe at a frequency range between 100 mHz to 100 kHz, using a modulation voltage of 10 mV. Figure 5 : Calibration curves of the immunosensor for 10 ng/mL and 50 ng/mL concentrations of different ratios of glycated albumin to total albumin. Figure 6: Calibration curves of the immunosensor for 50 ng/mL concentrations of different ratios of glycated albumin to total albumin with anti-HSA and Anti-IgG antibodies. Figure 7 : SPRi signal versus time for 50 ng/mL concentration of different HSA glycation levels at fixed temperature of 29 °C. Figure 8 : SPRi signal shift versus different ratios of glycated albumin to total albumin at 50 ng/mL antigen concentration.
14
Table 1: Structural and biochemical parameters in different HSA samples.
HSA
Glycation level
AGE level
Average molecular mass
samples
% [GA/Total Albumin]
(% G0)
(kDa)
G0
0
0
66.237 ± 0.036
G5
7.49
3.38 ± 0.95
66.360 ± 0.096
G25
15.79
8.65 ± 1.72
66.562 ± 0.068
G50
22.56
12.79 ± 5.89
66.631 ± 0.045
G100
46.50
17.92 ± 4.36
66.862 ± 0.069
G500
66.73
35.19 ± 5.11
68.060 ± 0.013
Table 2: Electric parameters obtained from the fitting of experimental results for the immunosensor design to the Randles electrical circuit. Rs
Rct
W
Q
(W.)
(W.)
(W-1.s1/2)
(W-1.sn)
Bare Gold
229.0
274.4
1.832 10-3
13.41
0.8198
mAb
225.7
768.1
1.698 10-3
11.33
0.8273
BSA
216.8
7683
629.2 10-3
10.24
0.8242
Layers
n
15
Highlights
·
Design of an immunosensor for glycated human serum albumin with anti-HSA antibody.
·
It can differentiate between physiological and pathological HSA glycation levels.
·
EIS and SPRi techniques were used.
·
The linear range goes from 7.49 % to 15.79 % of glycated albumin to total albumin.
16
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
PII: DOI: Reference:
S0039-9140(18)30272-8 https://doi.org/10.1016/j.talanta.2018.03.027 TAL18461
To appear in: Talanta Received date: 23 November 2017 Revised date: 7 March 2018 Accepted date: 11 March 2018 Cite this article as: Nadra Bohli, Olivier Meilhac, Philippe Rondeau, Syrine Gueffrache, Laurence Mora and Adnane Abdelghani, A facile route to glycated albumin detection, Talanta, https://doi.org/10.1016/j.talanta.2018.03.027 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. 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.
A facile route to glycated albumin detection Nadra Bohlia,*, Olivier Meilhacb,c, Philippe Rondeaub, Syrine Gueffrachea, Laurence Morad, Adnane Abdelghania a
Carthage University, UR17ES22 Research Unit of Nanobiotechnology and valorisation of medicinal plants, National Institute of Applied Science and Technology, Bp 676, Centre Urbain Nord, 1080 Charguia Cedex, Tunisia. b Université de La Réunion, INSERM, UMR 1188 Diabète athérothrombose réunion Océan Indien (DéTROI), Saint-Denis de La Réunion, France. c CHU de La Réunion, Saint-Denis de La Réunion, France. d Université Paris13, Inserm, U1148, Laboratory for Vascular Transitional Science, Institut Galilée, Sorbonne Paris Cité, F-93430, Villetaneuse, France.
Abstract: In this paper we propose an easy way to detect the glycated form of human serum albumin which is biomarker for several diseases such as diabetes and Alzheimer. The detection plateform is a label free impedimetric immunosensor, in which we used a monoclonal human serum albumin antibody as a bioreceptor and electrochemical impedance as a transducing method. The antibody was deposited onto a gold surface by simple physisorption technique. Bovine serum albumin was used as a blocking agent for non-specific binding interactions. Cyclic voltammetry and electrochemical impedance spectroscopy were used for the characterization of each layer. Human serum albumin was glycated at different levels with several concentrations of glucose ranging from 0 mM to 500 mM representing physiological, pathological (diabetic albumin) and suprapathological concentration of glucose. Through the calibration curves, we could clearly distinguish between two different areas related to physiological and pathological albumin glycation levels. The immunosensor displayed a linear range from 7.49 % to 15.79 % of glycated albumin to total albumin with a good sensitivity. Surface plasmon resonance imaging was also used to characterize the developed immunosensor.
*
Corresponding author: Email: [email protected], [email protected]
1
graphical abstract
Keywords: Electrochemical immunosensor.
1
Impedance
Spectroscopy,
Glycated
albumin,
SPRi,
Introduction
Advanced glycation end products (AGEs) are the result of a non-enzymatic non reversible reaction between reducing sugars such as glucose and proteins like hemoglobin or albumin. Compared to their native form, the resulting glycated proteins undergo structural changes and several of their functions are often impaired [1]. AGEs have attracted a growing of interest during the past decade as they seem to be related to several health complications associates
2
with the aging process, diabetes mellitus, Alzheimer, as well as cardiovascular disease and could be used as biomarkers for these pathologies [2, 3]. Thus, a lot of effort is now aimed at developing methods to detect and quantify glycated proteins in order to predict potential complications [4, 5, 6, 7]. Quantification of glycated hemoglobin is routinely used to monitor long-term glycemic control in diabetic patients. The turnover of albumin in plasma is much shorter than that of hemoglobin, and assessment of albumin glycation may be more appropriate in diabetic patients with chronic kidney disease [8, 9]. Sandwich assay, like ELISA (Enzyme-Linked ImmunoSorbent Assay) or RIA (radio-immuno-assay) techniques are the most known systems under development in AGE detection systems. They require a label or enzyme conjugated to a direct or indirect ligand of the target analyte. The ligand used is often an antibody that recognizes specifically an epitope and allows the capture of the target. In sandwich ELISA, a different antibody directed to the target is required; it can be directly conjugated to a label, or a secondary antibody carrying the label may be used to amplify the signal. The amount of label detected is assumed to be proportional to the number of bound targets. Labels can be fluorophores, radioisotopes, active enzymes, etc. In addition to the cost and time required for ELISA testing, some concerns are often underlined. In fact, the yield of the target-label coupling reaction can be highly variable for proteins and their labeling can drastically change the protein binding properties [4]. Impedance biosensors offer an alternative to ELISA assay as they allow the development of cost effective label-free detection systems [10, 11, 12]. They are also easily miniaturized and can provide good start for the development of point of care testing devices (POCT) [13]. In this paper we propose a simple method for the detection of glycated albumin based on label-free impedimetric immunosensor using only one type of anti-human serum albumin as a capture antibody followed by the detection of electrochemical changes associated with albumin glycation. The glycating agent used is glucose, as it is the major circulating glycating agent for humans.
2 2.1
Material and methods Reagents
Anti-Human Serum Albumin monoclonal antibody (mAb) was purchased from ABBIOTEC (USA). The Goat anti-rabbit IgG and Rabbit IgG were purchased from Pierce Biotechnology. Bovine serum albumin (BSA) was obtained from Sigma-Aldrich (France). Lyophilized powder of HSA with purity above 97 % was bought from Sigma Aldrich. Phosphate buffered
3
saline (PBS) containing 140 mM NaCl, 2.7 mM KCl, 0.1 mM Na2HPO4, 1.8mM KH2PO4, pH = 7.4 was used for all experiments with K4Fe(CN)63-/K4Fe(CN)64− redox couple at 5 mM concentration. All reagents were of analytical grade. The used ultrapure water, with a resistance of 18.2 MΩ.cm−1, was produced by a Millipore Milli-Q system. 2.2
GA and HSA preparation
The samples used are referenced as HSA and GA respectively corresponding to native nonglycated and glycated human serum albumin. For the preparation of glycated serum albumin, lyophilized powder of commercial HSA with purity above 97 % was used with glucose powder (99.5 % of purity). GA samples were produced by incubating 0.6 mM HSA protein with different concentrations of glucose and in PBS under sterile conditions and nitrogen gas in capped vials at 37 °C for three weeks. The used glucose concentrations are 25 mM, 50 mM, 100 mM and 500 mM. More details on HSA and GA preparation are described in detail in previous reference [14]. HSA and GA preparations were characterized in terms of glycation level (%), advanced glycation end-products (AGE) level (%) and average molecular mass. The glycation level was determined by boronate affinity chromatography (100 % corresponding to complete glycation of HSA). The percent increase in fluorescent AGE level was obtained by the maximal fluorescence emission at an excitation wavelength of 340 nm. The average molecular mass was obtained by ESI/MS. Table 1 indicates the level of glycation for each HSA sample. These three methods were well described in a previous study [14].
2.3
Gold electrode cleaning
The gold electrodes were purchased from the LAAS of Toulouse (France). Evaporated gold (300 nm thickness) was coated on silicon, using titanium under layer (30 nm thickness) as a substrate. Prior to their modification, the gold electrodes were cleaned in an ultra-sonic bath for 10 min in acetone. Then, they were dried under a nitrogen flow and then dipped for 15 min into ‘piranha solution’ (3:1 (v/v) 95 % H2SO4 / H2O2). Finally, the gold substrates were rinsed two to three times with ultra-pure water. 2.4
Cyclic voltammetry
Cyclic voltammetry was performed at room temperature in a conventional voltammetric cell with a three separated electrodes configuration using Chi 604e impedance analyser (CH Instruments, USA). The gold electrode (0.16 cm²) was used as working electrode, and platinum (1 cm2) and Ag/Ag-Cl electrodes were used as counter and reference electrodes,
4
respectively. All the electrochemical measurements were carried out in PBS buffer solution with a scan rate of 100 mV/s and a potential scan from -600 mV to 600 mV. 2.5
Electrochemical Impedance Spectroscopy
The impedance measurement was performed with a three separate electrodes configuration cell using Chi 604e impedance analyser (CH Instruments, USA) in the frequency range from 100 mHz to 100 kHz, using a modulation voltage of 10 mV. All electrochemical measurements were carried out in PBS. More details on electrochemical impedance spectroscopy can be found in reference [15]. EIS results were analysed by fitting the experimental impedance data to electrical equivalent circuit models. Parameters of the electrical-equivalent circuits were obtained by fitting the impedance function to the measured Nyquist plots with a program built into the Chi 604e impedance analyser. 2.6
Surface Plasmon Resonance Imaging
SPRi measurements were performed with an SPR imager® II (from GWC Technologies, USA), with an excitation wavelength of 850 nm. The experimental setup is based on a Kretschmann configuration, in which the light from a light-emitting diode integrated with a prism (prism refractive index = 1.7) is firstly p-polarized and then internally reflected from a metal surface. The used metal surface is a 16 gold spots array format glass substrate. Each gold spot has a 0.004 cm2 surface on which the different layers of the immunosensor were deposited. The resonance angle was determined according to the minimum reflectivity signal and was set for all the measurements. A couple charge device camera was used to capture all data for all the gold spots simultaneously and converts the reflectivity changes to pixels data, allowing a real time refractive index reading. All measurements were performed with PBS buffer at 29 °C. The substrate was exposed to the appropriate analyte using the 1mL flow cell.
3 Results and discussion 3.1
HSA and GA structural and biochemical characterizations
The biochemical characterization of HSA and GA preparations, summarized in Table 1, revealed a glucose dose-dependent increase in glycation level (up to 66.7% [GA/Total Albumin] for G500) and in fluorescent AGE formation (up to 35.2% for G500). In parallel, an increase in average molecular mass in a dose dependent manner was also noticed. For
5
instance, the molecular mass in G100 was approximately increased by 625 Da corresponding to a condensation of ~4 glucose units per molecule of albumin (one glucose unit is equivalent to a ass increase of 160 Da). For G500, this increase in molecular mass is about 1621 Da corresponding to the attachement of ~10 glucose units per albumin molecule.
3.2
Immuosensor design
The immunosensor construction is realized following three main steps. The first one is the physical adsorption of the HSA antibody solution onto the cleaned gold electrode surface. Unlike self-assembled monolayers (SAMS) treatment, physical adsorption is a simple procedure that is not time consuming. It consisted in depositing 50 microliters of anti-HSA monoclonal antibody at a concentration of 50 µg/mL on the gold electrode followed by an overnight incubation at 4 ºC. The second step consisted in blocking the reactive sites on the gold electrode through the deposition of 100 µL of BSA solution (0.1 %, w/v) on the surface for 45 min. The aim of this step is to prevent non-specific adsorption of antigens onto the uncovered gold surface, thus making sure that the antigens will be only bound to the antibodies. Finally, human serum albumin at different glycation levels were deposited on the modified gold electrode surface and incubated for 15 minutes at room temperature at a fixed concentration, thus allowing the antigen/antibody specific reaction to take place. All incubations were carried out in a humid chamber. After each immobilisation step of the antibody, BSA or the several analyte samples used, the electrode is washed three times with PBS to remove the excess unbound molecules, and appropriate measurements were performed. 3.3
Characterization of the immunosensor layers
Cyclic voltammetry and electrochemical
impedance
spectroscopy were used
as
characterization techniques after each step in the immunosensor building. Cyclic voltammetry is a potentiodynamic electrochemical technique performed by cycling the potential of a working electrode, and measuring the resulting current. Figure 1 shows the cyclic voltammogram of the composing layers of the immuosensor measured after each deposition step which are the bare gold electrode, the gold/mAB
6
(monoclonal antibody to human serum albumin) and the gold/mAB/BSA layers. All voltammogramms were cycled at least three times in order to verify the signal stability. The cyclic voltammogramm obtained for the bare gold electrode displays a reversible nature, with two current peaks corresponding to Fe(CN)64− oxidation and Fe(CN)63− reduction, typical of the redox couple used. These peak intensities decreased after the deposition of the antibody layer. They are severely diminished upon the addition of the BSA layer, showing that almost all the faradic current was blocked. The successful immobilization of each functionalized layer was confirmed through electrochemical impedance spectroscopy (EIS) measurements. EIS is a powerful technique for the investigation of interfacial reaction mechanisms. It measures the response of an electrochemical system to an oscillating potential as a function of frequency. This technique allows the explicit observation of different processes occurring in the monolayer and taking place at different rates such as diffusion currents, double layer charging and solution resistance. Figure 2 shows the Nyquist plots of the composing layers of the immunosensor measured after each deposition step which are the bare gold electrode, the gold/mAB and the gold/mAB/BSA layers. The impedance spectrum was fitted using Randles equivalent electrical circuit presented in figure 3. The elements composing this circuit are briefly described as follows. Rs results from the resistance of the electrolyte, the contacts and connections. It is generally not affected by binding. Zw is the Warburg impedance. It represents the delay arising from the diffusion of the electroactive species to the electrode, and is only detectable at low frequencies. ZCPE is the constant phase element modelling the non-pure capacitor formed between the electrode and ions in solution. The capacitor combines the surface modification capacitance and the double layer capacitance and is generally dependent on the thickness and dielectric constant of the probe layer. CPE behaviour can be explained by dispersion in local capacitance values due to microscopic roughness, and inhomogeneous current distribution resulting from microscopic chemical heterogeneities and ion adsorption. The complex impedance of a CPE is given by equation 1: =
( )!×"
(1)
Where Q is analogous to a capacitance, ω is the frequency (rad/s), and 0.5 < n < 1. Finally, Rct is the charge transfer resistance. It is associated to the energy potential of the oxido-reduction event occurring at the electrode alongside with the energy barrier of the
7
redox species reaching the electrode owing the electrostatic repulsion and/or steric hindrance. Rct is the commonly used element to indicate affinity binding for faradaic systems. The measured spectra of the impedance and phase were analysed in terms of electrical equivalent circuits. The electric parameters of the system were calculated with the computer program and the fit error was kept under a maximum of 10 %. All of the values of the electrical parameters are presented in Table 2. In the Nyquist plots, the diameter of the semi-circles corresponds to the charge transfer resistance of the electrode/electrolyte interface. The bare gold electrode displayed a small circle at high frequencies, suggesting a very low electron transfer resistance to the redox probe dissolved in the electrolyte solution. When the HSA antibody was immobilized on the electrode, the charge transfer resistance increased from 274.4 Ω to 768.1 Ω. Then, when BSA was added, Rct increased to 7683 Ω. We clearly observe that the BSA layer drastically increases the Rct. This increase is due to the conductivity decrease at the working electrode– electrolyte interface where the grafted layer becomes more insulating. These results confirm those obtained with cyclic voltammetry and indicate the successful immobilization of the antibody and BSA layers onto the gold surface. 3.4
Glycated albumin detection
Electrochemical impedance spectroscopy measurements for the detection of different ratios of glycated albumin to total albumin were made for a concentration of 50 ng/mL as presented in Figure 4. Nyquist plots show a significant variation of the impedance spectral curves with different HSA glycation ratios. We observe then that the charge transfer resistance decreases with the increase in the ratio of GA to total albumin. This observation can be explained by the global charge displayed by the protein. In fact, previous studies measured the global charge of these proteins at physiological pH, known as the isoelectric point. And it was found to be quite weak (global negative charge) and to decrease upon glycation [16, 17]. The morphology changes operating on HSA upon glycation can also lead to a favorable configuration to the transport of negative charges (through more heterocycles with mobile electrons or double links). We clearly observe that for the same dilution, the response of the immunosensor to G0 and G5 glycation levels are very different from those of higher glycation levels. R ct is very low for high glycation levels. The same measurement was done for 10 ng / mL concentration. In order to better understand the behaviour shown above, impedance data was fitted using the
8
Randles equivalent circuit. According to the Rct values obtained from data fitting, we calculated the change of the charge transfer resistance upon HSA glycation level following equation 2: ∆%&'
#
%*
#=
%&' (+,)- %*
(2)
%*
Where Rct(Ag) is the value of charge transfer resistance after the immunoreaction and R 0 is the value of the charge transfer resistance obtained for the fully functionalized immunosensor (Gold/mAB/BSA). The calibration curves consisting of the variation of the percentage values of relative %* versus the ratio of glycated electrode charge transfer resistance variation #∆%% &'# = %&'(+,)% *
*
albumin to total albumin at two different concentrations with each concentration is presented in the figure 5. According to these calibration curves, the linear range of the immunosensor depends on the concentration used for the analyte. For a concentration of 50 ng/mL, the linearity was observed for an albumin glycation ratio from 7.49 % to 15.79 % and up to 22.56 % for a concentration of 10 ng/mL. The sensitivity of the immunosensor, quantified by the slope of the linear part of the calibration curve is evaluated by linear fitting to 5 [%. [% (GA/Total albumin])]−1] for the concentration of 50 ng/mL and to 0.6 [%. [% (GA/Total albumin)] −1] for the concentration of 10 ng/mL. The saturation phase is reached faster for high analyte concentration as it seems that the glycated HSA proteins induce a more conductive layer on the immunosensors’ surface. These results confirm previous studies in which it has been demonstrated that the glycation human serum albumin has an impact on its dielectric properties as well as on its structure and morphology [18, 19, 20]. It is important to note that for a healthy person, albumin glycation levels generally range from 11 % to 16 %. So, the proposed immunosensor can definitely provide an easy detection of the protein and an easy assessment of its glycation levels, associated with unbalanced diabetic conditions. To confirm that the above observed impedance changes generated from the result of specific antibody-antigen interaction, we replaced the anti-HSA antibody with anti-IgG. The immunosensor was exposed to the target analyte at a concentration of 50 ng/mL. The glycation ratio of the analyte was increased in order to observe if there was a significant effect on the immunosensor by EIS. The associated calibration curve is given in figure 6. As
9
we can see, the sensor was not subjected to the non-specific binding and is therefore applicable to the selective determination of glycated HSA. In order to better characterise the developed immunosensor at a concentration of 50 ng/mL, we then used surface plasmon resonance imaging as an alternative transducing technique. 3.5
Detection of Glycated albumin with SPR Imaging
SPR imaging is an optical method used for label free detection of analytes and for evaluating molecular interactions [21, 22, 23]. 100 microliters of HSA antibody were deposited on the cleaned SPRi substrate and incubated overnight at 4 °C. After washing out the excess unbound antibody with PBS, the substrate was treated with a solution of BSA (0.1 %, w/v) for 45 minutes to prevent unspecific binding. The substrate was then mounted in the microfluidic cell and exposed to the injection of the different 50 ng/mL analyte concentration samples. After each antigen injection, an injection of PBS was performed with the pumping device to wash out the unbound antigen to the surface of the immunosensor. All the data were collected on real time using differential imaging. The obtained signals are presented in figure 7. We observe that the SPRi signal changes after each antigen injection. This change is due to the specific recognition by the anti-HSA. This recognition induces a thin layer deposition, which leads to a resonance angle variation at the interface. For the 50 ng/mL concentration, an SPRi calibration curve is plotted and presented in figure 8. The dynamic range was observed for 7.49 % up to 46.5 % of GA to total albumin.
4
Conclusion
In this paper, we developed a label free impedimetric immunosensor for the detection of glycated human serum albumin based on a simple physisorbed layer of monoclonal antibody to HSA. The immunosensor was tested for the detection of different glycation levels of albumin prepared through incubation in glucose concentrations ranging from 0 mM to 500 mM, at two distinct concentrations of 10 ng/mL and 50 ng/mL. High and low glycation levels are easily distinguished from the calibration curves. A severe drop in the charge transfer resistance is observed for HSA with high glycation levels. Dynamic range for 50 ng/mL was observed for G5 and G25 corresponding to a level of glycation obtained respectively with 5 (G5) and 25 mM of glucose (G25). SPRi optical transducing technique was also used for the characterisation of the immunosensor.
10
Acknowledgements This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
References [1]
R. Singh, A. Barden, T. Mori, L. Beilin, Advanced glycation end-products: a review, Diabetol. 44 (2001) 129-146.
[2]
A. Prasad, P. Bekker, S. Tsimikas, Advanced glycation end products and diabetic cardiovascular disease, Cardiol. Rev. 20 (2012) 177-83.
[3]
G. Münch, J. Thome, P. Foley,
R. Schinzel,
P. Riederer, Advanced glycation
endproducts in ageing and Alzheimer's disease, Brain Res Brain Res Rev. 23 (1997) 134-43. [4]
A. Jalaluddin Mohd, A. Saheem, C. Inho, A. Nashrah, F. Mohd, T. Godovikova, S. Uzma, Critical Review, Recent Advances in Detection of AGEs: Immunochemical, Bioanalytical and Biochemical Approaches, Int. Union Biochem. Mol. Biol. 67 (2015) 897-913.
[5]
X. Wang, N. Xia, L. Liu, Review: Boronic Acid-Based Approach for Separation and Immobilization of Glycoproteins and Its Application in Sensing, Int. J. Mol. Sci. 14 (2013) 20890-20912.
[6]
O. S. Zhernovaya, V. V. Tuchin, I. V. Meglinski, Monitoring of blood proteins glycation by refractive index and spectral measurements, Laser Phys. Lett. 5 (2008) 460-464.
[7]
N. Vigneshwarana, G. Bijukumarb, N. Karmakara, S. Ananda, A. Misra, Autofluorescence characterisation of advanced glycation end products of hemoglobin, Spectrochim. Acta Part A. 61 (2004) 163-170.
[8]
F. E. Vos, J. B. Schollum, R. J. Walker, Glycated albumin is the preferred marker for assessing glycaemic control in advanced chronic kidney disease, Nephrol. Dial. Transplant. Plus. 4, (2011) 368-375.
[9]
K. Neelofar, A. Jamal, Review Article: Amadori albumin in diabetic nephropathy, Indian. J. Endocrinol. Metab. 19 (2015) 39-46.
11
[10]
H. Chammem, I. Hafaid, N. Bohli, A. Garcia, O. Meilhac, A. Abdelghani, L. Mora, A Disposable Electrochemical Sensor based on Protein G for High-Density Lipoprotein (HDL) Detection, Talanta. 144 (2015) 466-473.
[11]
Q. Xu, J. J. Davis, The Diagnostic Utility of Electrochemical Impedance, Electroanal. 26 (2014) 1249-1258.
[12]
F. Lisdat, D. Schäfer, The use of electrochemical impedance spectroscopy for biosensing, Anal. Bioanal. Chem. 391 (2008) 1555-1567.
[13]
G. Abel, Current status and future prospects of point-of-care testing around the globe, Expert. Rev. Mol. Diagn. 15 (2015) 853-5.
[14]
J. Baraka-Vidot, C. Planesse, O. Meilhac, V. Militello, J. Van den Elsen, E. Bourdon, P. Rondeau, Glycation alters ligand binding, enzymatic, and pharmacological properties of human albumin, Biochem. 54 (2015) 3051-62.
[15]
J. R. MacDonald, Impedance Spectroscopy, Wiley, New York, 1987.
[16]
I. Bundschuh, I. Jäckle-Meyer, E. Lüneberg, C. Bentzel, R. Petzoldt, H. Stolte, Glycation of serum albumin and its role in renal protein excretion and the development of diabetic nephropathy, Eur. J. Clin. Chem. Clin. Biochem. 30 (1992) 651-6.
[17]
J. Figge, T. Rossing, V. Fencl. The role of serum proteins in acid–base equilibria. J. Lab. Clin. Med. 117 (1991) 453-67.
[18]
N. Bohli, H. Chammem, O. Meilhac, L. Mora, A. Abdelghani, Electrochemical Impedance Spectroscopy on Interdigitated Gold Microelectrodes for Glycosylated Human Serum Albumin Characterization, IEEE Trans. NanoBiosci.16 (2017) 676681.
[19]
P. Rondeau, N. R. Singh, H. Caillens, F. Tallet, E. Bourdon, Oxidative stresses induced by glycoxidized human or bovine serum albumins on human monocytes, Free. Radic. Biol. Med. 45 (2008) 799-812.
[20]
P. Rondeau, E. Bourdon, The glycation of albumin: Structural and functional impacts, Biochim.93 (2011) 645-658.
12
[21]
M. Puiu, C. Bala, SPR and SPR Imaging: Recent Trends in Developing Nanodevices for Detection and Real-Time Monitoring of Biomolecular Events, Sens.16 (2016) 116.
[22]
H. Chammem, I. Hafaid, O. Meilhac, F. Menaa, L. Mora, A. Abdelghani, Surface Plasmon Resonance for C-Reactive Protein Detection in Human Plasma, J. Biomater. Nanobiotechnol. 5 (2014) 153-158.
[23]
V. Kodoyianni, Label-free analysis of biomolecular interactions using SPR imaging, BioTech. 50 (2011) 32-40.
Figure Captions Figure 1: Cyclic voltammograms of each layer composing the immunosensor: (a) bare gold electrode, (b) Gold/mAb (monoclonal antibody to human serum albumin) and (c) Gold/mAb/BSA. The measurements were made in a PBS buffer solution of pH 7.4 in the presence of 5 mM Fe(CN6)3–/4– redox probe with a scan rate of 100 mV/s. Figure 2: Nyquist plots of impedance spectra for each layer composing the immunosensor: (a) bare gold electrode, (b) Gold/mAb and (c) Gold/mAb/BSA. The measurements were made in a PBS buffer solution of pH 7.4 in the presence of 5 mM Fe(CN6)3–/4–
13
redox probe at a frequency range between 100 mHz to 100 kHz, using a modulation voltage of 10 mV. Figure 3: Randles equivalent electrical circuit. Figure 4: Nyquist plots of impedance spectra for different ratios of glycated albumin to total albumin at a concentration of 50 ng/mL. The measurements were made in a PBS buffer solution of pH 7.4 in the presence of 5 mM Fe(CN6)3–/4– redox probe at a frequency range between 100 mHz to 100 kHz, using a modulation voltage of 10 mV. Figure 5 : Calibration curves of the immunosensor for 10 ng/mL and 50 ng/mL concentrations of different ratios of glycated albumin to total albumin. Figure 6: Calibration curves of the immunosensor for 50 ng/mL concentrations of different ratios of glycated albumin to total albumin with anti-HSA and Anti-IgG antibodies. Figure 7 : SPRi signal versus time for 50 ng/mL concentration of different HSA glycation levels at fixed temperature of 29 °C. Figure 8 : SPRi signal shift versus different ratios of glycated albumin to total albumin at 50 ng/mL antigen concentration.
14
Table 1: Structural and biochemical parameters in different HSA samples.
HSA
Glycation level
AGE level
Average molecular mass
samples
% [GA/Total Albumin]
(% G0)
(kDa)
G0
0
0
66.237 ± 0.036
G5
7.49
3.38 ± 0.95
66.360 ± 0.096
G25
15.79
8.65 ± 1.72
66.562 ± 0.068
G50
22.56
12.79 ± 5.89
66.631 ± 0.045
G100
46.50
17.92 ± 4.36
66.862 ± 0.069
G500
66.73
35.19 ± 5.11
68.060 ± 0.013
Table 2: Electric parameters obtained from the fitting of experimental results for the immunosensor design to the Randles electrical circuit. Rs
Rct
W
Q
(W.)
(W.)
(W-1.s1/2)
(W-1.sn)
Bare Gold
229.0
274.4
1.832 10-3
13.41
0.8198
mAb
225.7
768.1
1.698 10-3
11.33
0.8273
BSA
216.8
7683
629.2 10-3
10.24
0.8242
Layers
n
15
Highlights
·
Design of an immunosensor for glycated human serum albumin with anti-HSA antibody.
·
It can differentiate between physiological and pathological HSA glycation levels.
·
EIS and SPRi techniques were used.
·
The linear range goes from 7.49 % to 15.79 % of glycated albumin to total albumin.
16
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8