Gold modified screen printed carbon electrode (SPCE)

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biotin were immobilized respectively, then other immunoreagents were added (1% BSA, 0.001. mL BNP, and 10. mL Anti-BNP-HRP) in the volume 20 uL each.

Gold modified screen printed carbon electrode (SPCE) with steptavidin-biotin system for detection of heart failure by using immunosensor Shabarni Gaffar, Dede Udamas, Yeni Wahyuni Hartati, and Toto Subroto

Citation: AIP Conference Proceedings 2049, 030017 (2018); doi: 10.1063/1.5082518 View online: https://doi.org/10.1063/1.5082518 View Table of Contents: http://aip.scitation.org/toc/apc/2049/1 Published by the American Institute of Physics

Gold Modified Screen Printed Carbon Electrode (SPCE) with Steptavidin-Biotin System for Detection of Heart Failure by Using Immunosensor Shabarni Gaffar1,a), Dede Udamas1,b), Yeni Wahyuni Hartati2,c), Toto Subroto1,d) 1

2

Laboratory of Biomolecul, Food and Health Laboratory of Analytical Chemistry and Saparation, Chemistry Departmen Faculty of Mathematic and Natural Sciences, Universitas Padjadjaran Jl. Raya Bandung-Sumedang Km. 21, Jatinangor, Indonesia a

Corresponding author: [email protected] b [email protected] c [email protected] d [email protected] Abstract

Immunosensor for heart failure detection using the BNP (B type Natriuretic Peptide) biomarker was developed in this study. BNP can be identified with anti-BNP antibodies using immunosensor method electrochemically. Anti-BNP antibodies are immobilized on the SPCE surface with the biotin-streptavidin system. Modification of SPCE with gold can increase the detection limit in determining BNP concentration. This study aims to develop a BNP detecting immunosensor for the diagnosis of heart failure using gold-modified SPCE. The SPCE surface is characterized before and after modification with gold. Anti-BNP antibodies were immobilized on the surface of gold-modified SPCE with the biotin-streptavidin system and optimized antibody concentrations and incubation times were measured. BNPs with varied concentration variations were tested on immunosensors to determine their sensitivity and detection limits. SPCE surface characteristics before and after modification with gold can increase surface area and electroactive properties on the surface of the electrode. Better current signal response is generated at both primary and secondary antibody mL as well as the incubation time of secondary antibody and BNP antigen for 30 min. c The developed immunoensensor showed a linear response at a BNP concentration of 10 to 100 fg/mL, with a detection limit of 34 fg/mL. Keywords: Gold-SPCE, heart failure, immunosensor.

INTRODUCTION Heart failure is a physiological condition when the heart is unable to pump enough blood to meet the metabolic needs of the body. B-type Natriuretic Peptide (BNP) is one of the neurohormons derived from the granular membrane of the heart's ventricle that contributes to the high volume and high pressure in the ventricles (1,2,3). BNP levels can be measured using the point of care Triage Meter Plus assay (Biosite Diagnostic) with a detection limit of 5-5000 pg/mL (1, 4). Another BNP assay is sandwich immunofluorescent using monoclonal antibodies that increase the detection limit to 0.4 pg/mL (5, 6). In addition, another group was reported the BNP immunosensors using a microfluidic device equipped with Surface Plasmon Resonance spectroscopy (SPR) to detect BNP with a concentration range of 5-100 ng/mL. (7). Liu et al. (2010) used anti-BNP antibodies with streptavidin systems using micro magnetic particles to detect BNP and show lower detection limits that is 10 pg/mL (8). Surface plasmon resonance-based with nanobeads for detection of brain natriureticpeptide also gave sensitive immunosensing result (9). The method that combining

The 3rd International Seminar on Chemistry AIP Conf. Proc. 2049, 030017-1–030017-8; https://doi.org/10.1063/1.5082518 Published by AIP Publishing. 978-0-7354-1775-5/$30.00

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streptavidin with nano bead has been patented (10). The method allows the SPR sensor to detect BNP at the picogram level (pg/mL) is a concept with potential applications for biosensor methods (11) Several immunosensor devices have been developed by using Screen Printed Carbon Electrode (SPCE). The main advantages of Screen Printed Electrode are simplicity, flexibility, simple cost, portability, ease of operation, reliability, small size, and have the ability to mass production. Regardless of the working electrode, the orientation of immobilized antibodies on the surface of the immunosensor is another important factor to consider in order to increase the sensitivity and specificity of the immunosensor (12). Streptavidin-biotin systems with high affinity and good reaction specificity have attracted widespread attention in recent years. Streptavidin is a tetrameric protein; each of which is equivalent to its subunit can conjugate with one biotin molecule, which means a streptavidin molecule can bind four biotin molecules. The hydrogen bonding and van der Waals forces also contribute to the stability of the proposed system (13, 14). Nano-material modified screen printed electrodes to enhance the sensitivity of detection devices have been widely used. The use of gold nano-particles on the SPCE can increase the electroactive surface area, increasing the electron transfer between the electrode and the analyte. The modified electrode provides fast, accurate, reproducible measurements for the determination of antigens with low detection limits and high sensitivity and selectivity (15). This study is expected to provide actual information on the condition of BNP detecting immunosensors made with variations of anti-BNP-biotin and anti-BNP-HRP concentrations as well as the incubation time. Hartati (2018) showed that anti-BNP antibodies can be immobilized on the surface of the SPCE electrode with the streptavidin-biotin system (16). Immunosensors developed showed a linear response at BNP concentrations between 0.01 to 100 ng/mL, with a detection limit of 3.3 ng/mL. Results from Hu et al. (2014) show that electron transfer is promoted by electrodeposition of the gold nanoparticles, so that electron communication can be increased and the conductivity of the SPCE electrode can also be amplified. In addition, the amount of conjugated biomolecules is very high with the electrodeposition of gold nanoparticles and the biotin-streptavidin system which exhibits excellent sensor performance with increased detection limits for detecting S.pullorum & S. gallinarum microorganisms (17). EXPERIMENTAL Material The equipment used in this study includes sterilizer (Hirayama Autoclave HVE-50), screen printed carbon electrode (Dropsens), mini spin (Eppendorf), weighing scales (Mettler Toledo AL204), pH meters (Mettler Toledo InLab pH combination polymer elecrodes) micro pipettes (Eppendo connected to a computer using NOVA 1.10 software, micro tube, micro pipette tip, vortex (Boeco V-1 plus). Scanning electron microscope (SEM) (Hitachi SU03500, Japan), rotatory evaporator (Rotavapor R-114, Buchi) and desiccator. The substances used in the study were streptavidin and biotin (Abcam), Anti-BNP-biotin antibody, AntiBNP-HRP antibody (Abcam), Synthetic B-type Natriuretic Peptide (BNP) (Abcam), Phosphate Buffer Saline (PBS) pH 7.4, 10 mM K3[Fe (CN) 6], 0.1 M KCl, and 3.5.3, 5'-tetramethylbenzidine (TMB) (Abcam) solution. The solution of the gold salt (HAuCl4.3H2O) with a concentration of 1021.8 ppm. Modification of SPCE electrode SPCE was dipped in HAuCl4 solution with concentration of 1021,8 ppm and was electro-deposition by Differential Pulse Voltammetry (DPV) with the potential range from -1,5V to 0,6V in 10 cycle at scan rate equal to 0,1V/s. After the electro-deposition, the electrode is carefully rinsed with Q water and dried in air. After the gold modified SPCE ( SPCE-Gold) was characterized by Cyclic Voltametry (CV) and Scaning Electron Microscope (SEM) (17). Ammobilization of Streptavidin, Antibody Anti-BNP-biotin Antibody and BNP on SPCE-Gold surfaces Briefly, of streptavidin solutio mL was dropped on the SPCE-Gold surface and incubated overnight at 4ºC. Then the electrode was washed 3 times using PBS solution pH 7.4. There -BNP-biotin solution with a concent -Gold surface and incubated for 60 min at room temperature. After incubation, the electrode was washed again 3 times using PBS solution pH 7.4. Determination of the attachment of streptavidin, Anti-BNP-biotin and BNP on the SPCE-Gold surfaces was performed by an indirect method. Cyclic voltammetry was performed by using SPCE and SPCE-Gold at each stage of immobilized immune-reagent by using a redox system of ferricyanide 10 mM K3[Fe(CN)6] solution in 0.1 M KCl at a potential range of -0.6 V to 0, 6 V with a scan rate of 50 mV/s.

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Optimization of Anti-BNP-biotin concentration To the surface of SPCE-Gold, L mL streptavidin was added, then 5 ug/mL and 10 ug/mL anti-BNPbiotin were immobilized respectively, then other immunoreagents were added (1% BSA, 0.001 mL BNP, and 10 mL Anti-BNP-HRP) in the volume 20 uL each. After incubation, the electrochemical response was measured amperometrically at a potential of -0.2 V for 10 min. Optimization of Anti-BNP-HRP concentration SPCE-Gold was dipped mL streptavidin, followed by mL anti-BNP-biotin of 1% BSA, of 10-3 mL BNP. Furthermore, SPCE-Gold of AntiBNP-HRP with mL). After incubation, the electrochemical response was measured amperometrically at potential -0 , 2 V for 10 minutes. Optimization of BNP and Anti-BNP-HRP incubation time The procedure for determination of optimum incubation time for BNP and Anti-BNP-HRP was done as above. The incubation time of 30 and 60 minutes was tested for both immune-reagens. While the concentration of antibodies used was the optimum result of previous study. Determination of calibration curve and detection limit BNP solutions with varying concentrations (0, 10, 25, 50, 75, and 100 fg /mL) were tested on immunosensors. The electrochemical response resulted from the catalytic reaction of conjugated HRP enzyme on Anti BNP antibody with the TMB and H2O2 as a substrate. The electrochemical response was measured using differential pulse voltammetry methods in the potential range -0.6 V to +1.0 V with a 50 mV / s scan rate. The detection limit was calculated by measuring the blank response three times then calculating the standard deviation, using the equation: y = yb + 3Sb With y being the response to the blank signal, Sb is the standard deviation of the blank, and b is the slope of the regression equation y = a + bx (18). RESULT AND DISCUSSION In this study we developed the immunosensor for detection of B-type Natriuretic Peptide (BNP) as a diagnosis of heart failure by using SPCE modified with gold. The BNP hormone is considered as antigen to be recognized by the anti-BNP antibody-recognizing compound, the interaction occurring in the formation of an antibody-antigen complex. Anti-BNP antibodies are immobilized on the electrode with the streptavidin-biotin system. Electrochemical immunosensor for BNP detection can be performed by two type of measurement, that is as current (amperometry) and as potential (potentiometry). The immunosensor has 3 electrodes that is working, comparator and auxiliary electrodes. In this study the working electrode was modified with gold particles, to increase the sensitivity of BNP immunosensor. The scheme of fabrication process for immunosensor BNP is shown in Figure 1.

FIGURE 1. Ilustration of fabrication process electrochemical immunosensor on SPCE-Gold for BNP detection

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Caracterization of gold on electrode surface before and after modification SPCE before and after modified with gold was characterized by Scaning Electron Microscope (SEM) and Cyclic Voltametry (CV) to determine the nature of the electrode. In Figure 2 the SPCE surface alone exhibits fragments of graphite particles that make up the cavity typically of a large size and the surface is less evenly distributed. While a gold-modified electrode produces an electrode surface with an Au particle attached evenly so that the distribution of the Au particles on the electrode surfaces are homogenously as shown in Figure 2.

.

FIGURE 2. Surface of SPCE characterized by SEM (A) SPCE before modified (B, C) SPCE after modified with gold. The electroactive properties of the electrode was measured by cyclic voltammetry (CV) using a redox system of 10 mM potassium ferricianide solution in 0.1 M KCl at the potential range -0.6 to 1.0 Volt with a scan rate of 0.05 Volt/second. The result shown that characterization by dipped the electrode into the gold solution was produces a higher current than by dropped the gold solution to the electrode. These indicated that by dripping the electrode into the HAuCl4 solution, the more gold will attached to the electrode (Figure 3). Figure 3 also show that medified electrode was more electroactive compared to unmodified.

FIGURE 3. Cyclic voltammograms of unmodified SPCE (1) modified SPCE with gold by dipped (2) and by dropped (3). Scan rate 50.0 mV/s in 10.0 mM K3Fe(CN)6 containing 100.0 mM KCl.

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Figure 4 showed the cyclic voltammogram of a redox system of 10 mM potassium ferricianide solution in 0.1 M KCl after addition of immunoreagent; streptavidin, anti BNP-biotin, BSA, BNP, and Anti BNP-HRP, respectively. It can be seen that after addition of streptavidin the peak current of SPCE-Gold was reduce because streptavidin will covered some of electrode surface, therefore the electron transfer will be block. The peak current will become lower after addition of Anti BNP-biotin. Streptavidin will interacted with biotin that already attaced to Anti BNP. BSA will covered all the surface of electrode that does not attached by streptavidin, therefore the electron transfer will be blocked. Furthermore, addition of BNP, the BNP will interacted with Anti-BNP-biotin and finally Anti BNP-HRP will interact with BNP at another site. Addition of all immunoreagent will reduce the peak current that is produce by redox activity of 10 mM potassium ferricianide solution in 0.1 M KCl on SPCE-Gold (Figure 4).

FIGURE 4 . Cyclic voltammogram of 10 mM (K3[Fe(CN)6]) in 0,1 M KCl on SPCE-Gold. Scanning potential -0.6 to +6.0 V. Scan rate 50.0 mV/s. The immunosensor developed in this study was based on sandwich ELISA that used horseradish peroxidise (HRP) enzyme attached to Anti BNP. The enzyme will oxidize the substrate TMB/H2O2, produce the electron that can be detect on SPCE-Gold. Here we also study some of parameter that influences the analysis, that is concentration of Anti BNPbiotin, Anti BNP-HRP, incubation time of BNP and Anti BNP-HRP. This parameter was considered will effected the sensitivity of developed immunosensor. Fabricated of immunosensor was tested with Anti BNP-biotin concentration was 5 and 10 μg/mL. Mesurement was done by amperometric. The result show that 5 μg/mL of Anti BNP-biotin give high peak current compare to 10 μg/mL. We compare the ratio of current produce to the blank. Higher concentration of Anti BNPbiotin might be head off the electron transfer of enzymatic reaction of HRP, therefore the current ratio become lower (Figure 5).

3 2.5 2 1.5 1 0.5 0 5 μg/mL

10 μg/mL

Anti-BNP-biotin concentration FIGURE 5. Anti-BNP-biotin optimization. Current ratio was measure at 1,0 ng/mL of BNP, compare to current at no BNP at all.

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The optimization of anti BNP-HRP concentration was determined at 0,5; 1 and 5 μg/mL. The result was shown in Figure 6. It can be seen that 1 μg/mL gave highest peak current. Higher concentration will block the electron transfer but if the Anti BNP-HRP was to low, the number of Anti BNP-HRP molecule will not enough to interact with BNP molecule, therefore the peak current was low. The Anti BNP-biotin and Anti BNP-HRP has different site to interact with BNP.

5 4 3 2 1 0 0,5 μg/mL

1 μg/mL

5 μg/mL

Anti-BNP-HRP antibodies concentration FIGURE 6. Anti-BNP-HRP optimization. Current ratio was measure at 1,0 ng/mL of BNP, compare to current at no BNP at all. The incubation time of BNP and Anti BNP-HRP also influence the measurement. We compare 30 minutes and 60 minutes incubation time. Longer incubation time of BNP reduce peak current significantly compare to Anti BNP-HRP.

FIGURE 7. The incubation time optimization of BNP and Anti-BNP-HRP. Calibration curve and detection limit The linear relationship between the peak current and the concentration of BNP in various concentration is shown in Figure 7. The linear respond was measured at 10 to 100 fg/mL of BNP, with the regretion equation is y= 0,0653x + 1,5478 and R2 value is = 0,9136 and detection limit is 34 fg/mL. If we compare to the result by using unmodified SPCE (Hartati 2018), the used of SPCE-Gold improve the sensitivity of measurement. These migh be due to modification with gold can absorbed more streptavidin on electrode surface and also because of gold atoms had strong interaction with streptavidin, therefore the stability of

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Anti BNP-biotin as primary antibody on the surface electrode will also strong. As a result the identification site of anti BNP-biotin to BNP become expose and available to interact with Anti BNP-HRP.

FIGURE 8. The calibration curve for BNP detection by immunosensor with SPCE-Gold.

. CONCLUSION The modification of SPCE with gold improve surface area and electroactive properties of SPCE. The optimum concentration: 5.0 μg/mL of Anti-BNP-biotin dan 1,0 μg/mL of Anti-BNP-HRP, and 30 minutes of BNP and AntiBNP-HRP incubation time was gave optimum response of BNP immunosensor. The BNP immunosensor with SPCE-Gold show linear relationship at 10 to 100 fg/mL of BNP, with the detection limit was 34 fg/mL.

ACNOWLEDGEMENT Author are grateful to Directorate General for Higher Education, Ministry of Ristek Dikti through the PUPT research fund number 718/UN6.3.1/PL/2016, and to Universitas Padjadjaran for the Academic Leaderships Grant (ALG) 2016.

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