- Email: [email protected]
Cerium oxide-monoclonal antibody bioconjugate for electrochemical immunosensing of HER2 as a breast cancer biomarker
Journal Pre-proof Cerium oxide-monoclonal antibody bioconjugate for electrochemical immunosensing of HER2 as a breast cancer biomarker
Yeni Wahyuni Hartati, Leonard Kristofel Letelay, Shabarni Gaffar, Santhy Wyantuti, Husein H. Bahti PII:
S2214-1804(19)30135-7
DOI:
https://doi.org/10.1016/j.sbsr.2019.100316
Reference:
SBSR 100316
To appear in:
Sensing and Bio-Sensing Research
Received date:
10 August 2019
Revised date:
4 December 2019
Accepted date:
6 December 2019
Please cite this article as: Y.W. Hartati, L.K. Letelay, S. Gaffar, et al., Cerium oxidemonoclonal antibody bioconjugate for electrochemical immunosensing of HER2 as a breast cancer biomarker, Sensing and Bio-Sensing Research(2019), https://doi.org/ 10.1016/j.sbsr.2019.100316
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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.
© 2019 Published by Elsevier.
Journal Pre-proof
Cerium Oxide-Monoclonal Antibody Bioconjugate for Electrochemical Immunosensing of HER2 as a Breast Cancer Biomarker Yeni Wahyuni Hartati1 *, Leonard Kristofel Letelay 1 , Shabarni Gaffar1 , Santhy Wyantuti1 , Husein H. Bahti1 1
Department of Chemistry, Universitas Padjadjaran, Bandung
*Email: [email protected].
ABSTRACT
na
lP
re
-p
ro
of
Metal oxide-based sensors have the advantage of rapid response and of high sensitivity in detecting specific active biological species and are relatively inexpensive. This report of the present study concerns the development of a cerium oxide – monoclonal antibody bioconjugate for its application as a sensitive immunosensor to detect a breast cancer biomarker. A cerium oxide-anti HER2 bioconjugate has been constructed by adding anti HER2 onto cerium oxide that had been previously reacted with APTMS and PEGNHS-Maleimide. The FTIR spectra of the reaction product showed that the cerium oxide-anti HER2 bioconjugate was successfully synthesized. The resulted bioconjugate was then immobilized on a screenprinted carbon-gold nanoparticles electrode surface by using the amine coupling bonding systems. The interaction of the synthesized cerium oxide-anti-HER2 bioconjugate with HER2 was found to inhibit an electron transfer and a decrease in the voltammetric Fe(CN)6 3-/4- peak current, which was proportional to the concentration of HER2. The optimal response of the current signal was generated at an anti-HER2 concentration of 5.0 µg/mL. The two linear ranges of HER2 concentration were found to be from 0.001 to 0.5 ng/mL and from 0.5 to 20.0 ng/mL. By using the first calibration curve, the limit of detection was found to be 34.9 pg/mL. The developed label-free immunosensor has been used to determine HER2 in a human serum sample with satisfactory results, as shown by a consistent result with the addition of standard. Thus, the resulted immunosensor in this study is promising and would have a potential application in clinical bio analysis.
1. Introduction
ur
Keywords: cerium oxide, bioconjugate, electrochemical immunosensor, HER2.
Jo
Cerium belongs to the lanthanide series (with electron configuration [Xe]4f15d16s2) and is one of the most abundant rare earth elements in the Earth’s crust. Several cerium minerals have been mined and processed for industrial applications and pharmaceutical uses [1], [2]. Cerium is a rare-earth element contained at high concentration in monazite minerals, which are widespread in Indonesia, and have been produced as a side -product (or “tailing”) of tin processing [3]. The element is an important material for industrial us e in catalytic converters for removing toxic gases, solid oxide fuel cells, applications of electro -chromic thin-film, glass polishing, catalysts, and sensors. Cerium has also been developed for pharmaceutical uses and shown a role as antioxidants in biolo gical systems, which makes cerium nanoparticles well suited for applications in nano -biology and regenerative medicine [1, 2, 4, 5]. Cerium oxide has been reported to have advantageous properties such as non -toxicity, good electrical conductivity, chemical inertness, large surface area, negligible swelling, oxygen transferability, and good bio compatibility. These characteristics have attracted more attention to the development in fabricating high performance electrochemical biosensors and immunosensors [6-10]. A nano-structured cerium oxide film-based immunosensor has been developed for ochratoxin detection [11,12]. Nano-structured cerium oxide (nano-CeO2 ) film was fabricated onto indium-tin-oxide (ITO) coated glass plate, and then rabbit-immunoglobulin antibodies (r-IgGs) and bovine serum albumin (BSA) were immobilized onto the film. Electrochemical studies revealed that nano-CeO2 particles provide an increased electron communication between rIgGs and electrode electroactive surface area. The resulting immunosensor had a linear response to ochratoxin in the range of 0.5 – 6.0 ng/dl, a low detection limit of 0.25 ng/dl, and a fast response time of 30 s. The high value of the
1
Journal Pre-proof
Jo
ur
na
lP
re
-p
ro
of
association constant, Ka 0.9×1011 l/mol, indicates the high affinity of the BSA/r-IgGs/nano-CeO2 /ITO to ochratoxin [11]. A label-free amperometric immunosensor based on a multi-wall carbon nano-tubes-ionic liquid-cerium oxide film-modified electrode has been proposed for the detection of myeloperoxidase (MPO) in human serum [13, 14]. The cerium oxide was dispersed by chitosan, coated on the glassy carbon electrode, and then antibodies (anti-MPO) were attached to the nano-cerium oxide surface. With a non-competitive-immunoassay format, the antibody-antigen complex was formed between the immobilized anti-MPO and MPO in a sample solution. Under optimal conditions, the immunosensor showed a current change, which was proportional to MPO concentration in the range of 5 to 300 ng/mL, with a limit of detection of 0.2 ng/mL [13]. An improved electrochemical immunosensor for MPO detection was based on nano-gold/cerium oxide-lcysteine composite film has been developed. Cerium oxides were dispersed in 1-butyl-3-methylimidazolium hexafluorophosphate and then were immobilized on the l-cysteine film. The cerium oxides have a positive charge that may be able to bind more anti-MPO. The immunosensor improved some characteristics, such as a linear range to the MPO concentration between 10 ng/mL and 400 ng/mL, and a limit of detection of 0.06 ng/mL [14]. Yang et al. used ceria nanoparticles (CNPs) in the design of an amplification electrochemical immunosensor for influenza biomarker determination. The sensor was constructed based on cerium oxide/graphene oxide composites, as a catalytic signal amplifier for the detection of influenza, and using 1-naphthol that can be hydrolyzed to naphthoquinones by O-acetylesterase enzyme of the influenza virus. The immunosensor showed a linear range of 0.0010 – 0.10 ng/mL with a detection limit of 0.43 pg/mL [15]. An electrochemical biosensor also has been developed based on CeO 2 nano-wires for the determination of Vibrio cholerae O1 (VcO1). The antibodies of VcO1 (anti-VcO1) were immobilized on the surface of a CeO2-nanowire. Interaction of bacterial cells with the anti-VcO1 was analyzed by decreasing the current peak of the biosensor. The biosensor had a linear range of response between 102 CFU/mL and – 107 CFU/mL with a detection limit of 100 CFU/mL for VcO1 [16]. Another development of cerium oxide in immunosensors is cerium nanocomposites biosensors. A nanocomposite with a core-shell structure, cuprous oxide@cericdioxide (Cu 2 O@CeO2 -Au), was proposed for the quantitative detection of prostate-specific antigen (PSA). The amino-functionalized Cu 2 O@CeO2 -NH2 core-shell nanocomposites were prepared to bind gold-nanoparticles (AuNPs) by constructing a stable Au-N bond between AuNP sand-NH2 . The amplified signal sensitivity was achieved by the synergetic effect existing in Cu 2 O@CeO2 core-shell loaded with AuNPs. It showed a good electro-catalytic activity towards the reduction of hydrogen peroxide and was used as transducing materials to achieve efficiently capture antibodies and triple signal amplification of the proposed immunosensor. The developed immunosensor showed a wide linear range between 0.1 pg/mL and 100 ng/mL, with a low detection limit of 0.03pg/mL (S/N3) [17]. The Co 3 O4 @CeO2 -Au@Pt nanocomposite also has been developed as labels to conjugate with secondary antibodies for signal amplification in a sandwich-type electrochemical immunoassay for squamous cell carcinoma antigen (SCCA) detection. The amino functionalized cobaltosicoxide@cericdioxide nanocubes with core-shell morphology were prepared to combine seaurchin like gold @ platinum nanoparticles (Co 3 O4 @CeO2 -Au@Pt). The glassy carbon electrodes (GCE) modified using electro-deposited gold, were used as antibodies carriers and sensing platforms. Because the synergetic effect presents in Co 3 O4 @CeO2 -Au@Pt, the amplification of sensitivity was achieved towards the reduction of hydrogen peroxide. The proposed immunosensor exhibited a wide linear range, from 100 fg/mL to 80 ng/mL, with a low detection limit of 33 fg/mL for detecting SCCA [18]. As discussed, studies of various developments of ceria-based immunosensors have been extensively reported. The functionalized ceria or cerium oxides have attracted much attention in the fabrication of bio-sensing systems due to their unique properties. The use of a linker in the controlled assembly of nanoparticles to a target is the one important of surface modification and functionalization in biomedicine as diagnostic and therapeutic agents in cells or tissues. Polyethylene glycol (PEG), has been reported as a long compatible linker for nanoparticles. The main advantage of using PEG is to provide enough space to bind more recognizing element molecules like monoclonal antibodies to nanoparticles and allow them to stand aside and result in more effective combination with the target molecules [19-22]. Anti HER2 is a monoclonal antibody (mAb) that can bind HER2 protein and has known as one of breast cancer biomarkers because HER2 is over-expressed in some breast cancer patients. HER2 is a receptor tyrosine kinase, which is a member of the epidermal growth factor receptor (EGFR) family involved in cellular signaling pathways, which may lead to proliferation and differentiation. The overexpression of HER2 in some breast cancer patients can be used as a key prognostic marker, and effective therapeutic treatment targets to diagn ose breast cancer that generally occurred in adult females [22, 23]. This research concerns the use of PEGylated nano-structured cerium oxides attached to different concentrations of anti HER2 to form bioconjugates. A screen-printed carbon-gold nanoparticles electrode was used for designing a
2
Journal Pre-proof label-free platform, and the bioconjugates were stabilized covalently on the electrode surface to assay HER2 antigen in serum samples. This highly sensitive and straight forward electrochemical analysis method holds great potential for the detection of this biomarker in clinical diagnoses. 2. Experimental 2.1 Materials Anti HER2 Ab (Herceptin) was purchased from F. Hoffmann -La Roche Ltd. The HER2/CD340 human, 3aminopropyl trimethoxisylane (APTMS), bovine serum albumin (BSA), cysteamine (Cys), 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC), potassium ferricyanide, n -hydroxide succinimide (NHS), 2-iminithiolane, 3mercaptopropionic acid (MPA), and polyethylene glycol-α-maleimide-ω-NHS (NHS-PEG-mal), were purchased from Sigma-Aldrich Ltd. Toluene, hydrochloric acid, ethylene diamine tetraacetic acid (EDTA), ammonium hydroxide, potassium hydrogen phosphate, sodium hydrogen phosphate, cerium sulfate, sodium hydroxide, and methanol were purchased from Merck.
-p
ro
of
2.2 Apparatus Voltammetric measurements were done using Autolab type III Potentiostat/Galvanostat with NOVA 1.10 software (Metrohm). The screen-printed carbon-gold nanoparticles electrodes (SPCE-GNPs) DRP-110GNP (Dropsens) were used for the electrochemical experiments . Other instruments used were Scanning electron microscopy (SEM) images JEOL JSM-6360LA type and an FT-IR spectrophotometer (Perkin Elmer Spectrum 100).
lP
re
2.3 Preparation of Nano Structured Cerium Oxide (NS CeO2 ) NS CeO2 was synthesized based on a simple method [24]. Briefly, 2 g of Ce(SO4 )2 was dissolved in 25 mL double-distilled water and then a drop of sodium hydroxide solution (0.3 M) was added to the 25 mL solution. The mixture was continuously stirred for about 2-3 hours at room temperature until a pale yellowish-white precipitate was obtained. The precipitate was separated and washed several times with double-distilled water, after then it was dried at 150 C for an hour. The obtained yellowish particles were dried at 250 C for 3 hours.
Jo
ur
na
2.4 Preparation of NHS-PEG-Mal-NS CeO2 The Mal-PEG-NHS-NS CeO2 was prepared based on the functionalization of the ferric oxide procedure [22,25] with a modification. Briefly, to an amount of 0.1 g of NS CeO2 powder, 35 mL of toluene and 25 L of APTMS were added. Then the mixture was sonicated for 30 min and heated at 60 C for 5 hours in an oven. The obtained APTMS-coated NS CeO2 was separated by decantation and redispersed in 50 mL of methanol. The result ed of amino-NS CeO2 (0.01 g) was dissolved in 10 mL of redistilled water, and then 31 mg of NHS-PEG-Mal was added to the solution. The mixture was sonicated for 30 min and stirred for 6 hours. At the end of this step, PEGylated nanostructures were separated and redispersed in 5 mL of redistilled water. 2.5 Preparation of Bioconjugate The bioconjugate was synthesized based on reported methods [22,25,26]. The synthesis was started with the thiolation of anti HER2 with 2-iminithiolane. The thiolated HER2 was done by incubating 1000 g/mL of anti HER2 in 0.1 M PBS pH 8.0 in 2-iminithiolane, in a molar ratio of 1:200. Then 5 mM of EDTA was added into the reaction mixture to protect the thiol groups from oxidation. The mixture was stirred at room temperature for 1 h using mini spin. The thiolated anti HER2 thus formed was purified by its dialyzing with 20 mL of PBS pH 8.0. The purified thiolated anti-HER2 (100 μL of 15 g/mL) was subsequently added to 200 L of of 5 mg/mL NHS-PEGMal-NS CeO2 and kept the mixture under constant shaking for 6 hours at room temperature. In this way, the thiol groups of the thiolated anti HER2 would have been covalently attached to the unsaturated bond of maleimides that linked to the NS CeO2 to form the bioconjugates. The resulted bioconjugates were then separated by using magnetic decantation and, finally, redispersed in 1 mL of PBS pH 7.4. Figure 1 shows schematic reaction mechanisms of preparation of cerium oxide-anti HER2 bioconjugate.
3
of
Journal Pre-proof
Figure 1. Schematic reaction mechanisms of preparation of bioconjugate
ro
2.6 Optimization of Experimental Conditions
re
-p
Determination of optimum incubation time for MPA reaction The electrode is immersed in a 0.1 M MPA for 10, 30, 60, and 120 min. Then, the generated electrochemical response was measured by cyclic voltammetry using a redox system of 10 mM of Fe(CN)6 3-/4-in KCl 0.1 M at a potential range of -0.6 V to +0.6 V, with a scanning rate of 50 mV/s.
na
lP
Determination of optimum anti-HER2 antibody concentrations in bioconjugate formation As much as 200 µL of Mal-PEG-NHS-NS CeO2 solution was put into a microtube, then 2.5 µg/mL of anti-HER2 antibody was added into the solution. The mixture was stirred using a mini spin, and then its electrochemical response was measured by cyclic voltammetry using a redox system of 10 mM of Fe(CN) 6 3-/4- in KCl 0.1 M at a potential range of -0.6 V to +0.6 V with a scanning rate of 50 mV/s. The whole experiment was repeated three times, each using a different concentration of anti-H ER2 antibodies, i.e., 5.0, 7.5, and 10.0 µg/mL.
Jo
ur
Optimization of incubation time of the bioconjugate The electrode was immersed in a 0.1 M MPA for 30 min (i.e., the optimum time), then dipped in a 0.1 M cysteamine solution and rinsed with redistilled water. After that, a 40 µL solution containing 0.1 M EDC and 0.1 M NHS with a mole ratio of 1: 1 was dropped onto the electrode, which was subsequently incubated for one hour at room temperature. Then, after dropping 40 µL of bioconjugate onto the electrode, it was incubated for one hour at room temperature, respectively. The electrochemical response was measured by cyclic voltammetry using a redox system of 10 mM of Fe(CN)6 3-/4- in 0.1 M KCl at a potential range of -0.6 V to +0.6 V, with a scanning rate of 50 mV/s. The whole process was repeated but three times, each with different lengths of time (i.e., 2, 3, and 4 hours) of the final incubation process of the bioconjugate. 2.7 Preparation of The Immunosensor and Electrochemical Signals Measurements The (SPC-GNPs) electrode was immersed into 0.5 mL of 0.1 M MPA for 30 minutes at room temperature. This aim would have led to the formation of MPA-GNPs on the electrode. The electrode was then rinsed with doubledistilled water for eliminating any unwanted material absorbed on it physically. Following this, to activate the carboxyl groups of MPA, 40 μL of a solution containing 0.1 M EDC and 0.1 M NHS with a mole ratio of 1:1 was dropped onto the MPA-GNPs electrode surfaces. After that, the electrode was incubated for one hour at room temperature. After washing with double-distilled water, the electrode was immersed into a 0.1 M of cysteamine solution to form Cys-MPA-GNPs on it, via an amide formation, and rinsed with double-distilled water. Thereafter, 40 μL of the bioconjugate was dropped onto the electrode, and incubated for two hours (i.e, the optimum incubation time) at 4 C. Stable carbon-sulfur bonds would have been formed between carbon double bonds in the free maleimides of the bioconjugate and the thiol groups of Cys [22, 26, 27]. Any possible excess of the bioconjugate was rinsed with a 0.1 M PBS pH 7.4 solution and redistilled water, consecutively. Next, 20 µL of 1% BSA solutio n was added to the modified electrode, which was subsequently incubated for 45 minutes at room temperature,
4
Journal Pre-proof followed by rinsing the electrode with 0.1 M PBS pH 7.4 solution, and redistilled water, consecutively. It will be noted that BSA had been used to block any possible non-specific binding sites. To continuing the experimentations, 20 µL of HER2 solution (in a concentration range of 0.01-100 ng/mL) was dropped onto the bioconjugate-modified electrode, which was then left for 30 minutes at room temperatu re. Finally, the electrode was rinsed with 0.1 M PBS pH 7.4 solution and redistilled water, consecutively. To test the performance of the resulted the electrode as an immunosensor, experiments on cyclic voltammetric measurements were done by using the redox system consisted of 10 mM of Fe(CN)6 3-/4- with 0.1 M KCl in 0.05 M PBS pH 7.4 solution, at a potential range of -0.6 V to +0.6 V, with a scanning rate of 50 mV/s. Each measurement was carried out after the addition of MPA, bioconjugates, an d BSA. The whole experiment was finally accomplished by measuring some other HER2 solutions of different concentrations. Figure 2 shows the general schematic diagram of the immunosensor platform for the detection of HER2.
Jo
ur
na
lP
re
-p
ro
of
2.8 Analysis of Blood Serum Samples and Recovery Test Serum samples were obtained from the Bio Fit clinical laboratory in Bandung, Indonesia. Each of the samples was diluted 20 times, with 0.01 M of PBS pH 7.4 solutions. Then 30 µL of a diluted sample was dropped onto the bioconjugate-electrode, which was subsequently and incubated for 30 min utes at 37 °C. After rinsing the electrode with PBS and redistilled water, it was used to measure electrochemical response by cyclic voltammetry using a redox system of 10 mM of Fe(CN)6 3-/4- with 0.1 M KCl in 0.05 M PBS pH 7.4 solution, at a potential range of -0.6 V to +0.6 V, and with a scan rate 50 mV/s. Three replicate measurements were done, and the HER2 level of the sample was calculated using a previously prepared calibration regression equation. Recovery tests were carried out for two samples. Each sample was spiked with two different concentrations of HER2. The standard addition method was used to assay spiked concentration.
5
ur
na
lP
re
-p
ro
of
Journal Pre-proof
3
Jo
Figure 2. Schematic diagram of immunosensor platform for the determination of HER2 using CeO2 -anti HER2 bioconjugate
Results and Discussion
3.1 Characterization of Nanostructured Cerium Oxide, Bioconjugate, and the Modified Electrode Characterization of the NS CeO2 was carried out by using scanning electron microscopy to determine its morphology and size distribution. Figure 3.A displays the results of NS CeO2 analysis at 12.000 × magnifications, which show the average size of the NS CeO2 of about 100 nm. In order to proof the APTMS had been coated on the NS CeO2 , FT-IR spectroscopic analyses were carried out to record the spectra of the APTMS as a reference, and those of the APTMS-NS CeO2 . The resulted IR spectra of the two compounds are overlaid and are presented in Figure 3.B. In the IR spectrum of APTMS (Figure 3.B; a), there are several peaks in the frequency region of 3460-3280 cm−1 , which are originated from stretching vibration of -OH groups of water molecules or from those of surface −OH groups. The peak at 1500 cm−1 is the bending vibration of N-H and thus confirms the presence of a primary N-H bond. The peak at 2930.18 cm−1 is assigned to C-H stretching band, and the peak at 1111.96 cm−1 corresponds to Si-O stretching. Moreover, the peak at 1430 cm−1 is assigned to the bending vibration of C-H, while stretching vibration of the aliphatic C-N bond is characterized by the peak around 1023,33 cm−1 — meanwhile, the spectrum of APTMS-NS CeO2 (Figure 3. B; b) shows several bands. The very large and strong peak around 2557.0 cm−1 originated from the functional group of –CH. The peak at 1505.7
6
Journal Pre-proof
lP
re
-p
ro
of
cm−1 is the bending vibration of the –N-H bond; the moderate peak at 1130.7 cm−1 can be assigned as Si-O bond, and the sharp peak with strong absorption intensity at 579.8 cm−1 is assigned to the Ce−O stretching band. The intense band observed around 852 cm−1 is due to the Ce−O−C stretching vibration [16]. The peak at 3410 cm−1 is related to −OH groups of water molecules or those of surface −OH groups. From the analysis of the two spectra given above, i.e., the spectra of APTMS and those of APTMS-NS CeO2 , it can be concluded that APTMS had successfully been attached to the surface of NS CeO2 . Cyclic voltammetry has been used in this study for the electrochemical characterization of the electro des. The cyclic voltammogram of SPCE-GNPs with MPA, those of SPCE-GNPs-MPA-NSCeO2, and those of SPCE-GNPsMPA bioconjugates are shown in Figure 3.C. As can be seen in Figure 3.C, the presence of NS CeO 2 had decreased the intensity of the redox signal from 116.018 µA to 91.131 µ A, i.e., about 25 %. This will be proof that the nano structure of CeO2 has an electro-active characteristic that facilitates the process of electron transfers to electrodes. Therefore, NS CeO2 has an excellent characteristic and will form bioconjugate with the antibody to be used for a good electrochemical detection of HER2.
B
Jo
ur
na
A
C Figure 3. (A) SEM images of NS CeO2 , (B) FT-IR spectra of APTMS only (a), and those of APTMS-NS CeO2 (b), (C) Cyclic voltammograms of (a) SPCE-GNPs-MPA; (b) SPCE-GNPs-MPA-NS CeO2 ; (c) SPCE-GNPs-MPAbioconjugate. Cyclic voltametric experimental conditions: a redox system of 10 mM K3 [Fe(CN)6 ] with 0.1 M KCl in 0.05 M PBS pH 7.4 at scan rate 50 mV/s, at a potential range -0.6 V to 0.6 V. 3.2 Optimization of Experimental Conditions
7
Journal Pre-proof
Jo
ur
na
lP
re
-p
ro
of
Immunoreagents, including anti-HER2 and HER2 antibodies, are non-electroactive compounds, and thus they reduce the current flow of K3FeCNS redox compounds on the electrode surface. For this reason, optimal experimental conditions resulting in the lowest current were chosen in the experiments. In this present study, the electrode was modified through the immobilizing process of anti-HER2 bioconjugates on the electrode, which was conducted by using the co valent bonding method through the amine coupling system. However, the formation of the covalent bonds in this step is reported to be slow [22]. It has also been noted further that each important step plays an important role and needs to be optimized, inclu ding the incubation time of MPA. Each measurement should be carried out for three replicates. The gold electrode was first immersed in a 0.1 M of MPA solution, forming a robust Au-S bond because the MPA will be impregnated on the gold surface. The carboxylate group of MPA will facilitate the electron transfer process between the bioconjugates and the electrode. Figure 4. A shows optimum incubation time of MPA immersed in 30 minutes, with the average current of about 52 µA. Figure 4.B shows that the optimum concentration of anti-HER2 in bioconjugates was 5 μg/mL because it gave the lowest current response. The bioconjugation process was carried out by conjugating NS CeO 2 with thiolated anti- HER2. The method to bind thiolated anti HER2 at various concentrations, with PEG-NHS-Mal and NS CeO2 , was done as follows: For the first experiment, concentration ratio between PEG-NHS-Mal-NS CeO2 and anti HER2 was 1:0.05 µg/mL (200:400 µL). At this condition, the current obtained was too high, i.e., about 138 µA. Next, anti HER2 of different concentrations (i.e., 2.5; 5; 7.5; and 10 µg/mL) were used, and the results are presented in Figure 4.B. The data in Figure 4.B show that the optimum concentration (i.e., a concentration resulting in t he lowest current) was 5 µg/mL. If the concentration was higher than the optimum, the excess of anti-HER2 on the NS CeO2 had probably resulted in saturation of the nanoparticle surface. It interfered with the binding of the bioconjugate maleimide groups to the electrode surface. However, if the concentration of anti-HER2 were lower than the optimum, the current would be increased because of less anti-HER2 covering the electrode surface. Figure 4.C shows data on the optimization of incubation time of biocon jugates on the electrodes. The data show that the optimum incubation time of the bioconjugate on the electrode surface was two hours, i.e., the time giving the lowest peak current, which was about 115 µA.
A.
B.
C. Figure 4. Optimization of experimental conditions: (A). The effect of incubation time of MPA on the gold electrode surface, on the peak current; (B). The effect of concentrations of anti-HER2 in the bioconjugate, on the peak current; and (C). The result of the incubation time of the bioconjugate on the electrode, on the peak current.
8
Journal Pre-proof
3.3 Immunosensor Response and Calibration Curve If HER2 was dropped onto the developed immunosensor (with a specific concentration) for 30 minutes, a decrease in current was observed. This decreasing in current was caused by the binding of anti-HER2 with HER2 to form an immunocomplex, which acts as a blocking layer to isolate the electron and mass transfer. The immunocomplex also hinders the diffusion of (Fe(CN)6 3-/4-) toward the electrode surface. The detection principle is based on the change in oxidation peak current response (Ipa) before and after the antibody-antigen interaction. This peak current is s evaluated using the following equation [13]: Ipa = I0 −In ,
Jo
ur
na
lP
re
-p
ro
of
where I0 is the response current before the immunoreaction, and I n is the response current after the immunoreaction. In this present study, the responses of the prepared immunosensor towards HER2 of seven different concentrations (in the range of 0.001 ng/mL – 20.0 ng/mL) were studied by cyclic voltammetry using the redox system of 10 mM (Fe(CN)6 3-/4-) in 0.1 M PBS pH 7.4 solution containing 0.1 M KCl, under the optimal experimental conditions. Triplicate experiments for each HER2 solution concentration were done, and the resulting voltammograms are presented in Figure 5.A. A calibration curve was then prepared using peak current data from the seven resulted voltammograms. As can be seen in Figure 5. B, the calibration curve consisted of two segments of the linear curve (line) having different slopes (the two segments of the calibration curve are drawn separately). The first segment of the calibration curve is linear for a HER2 concentration range of 1 – 500 pg/mL. The calibration regression equations for this concentration ranges is Ipa = 0.0179 [HER2] + 0.9515, with R2 = 0.9898. Meanwhile, the second segment of the calibration curve is also linear for a 5.0-20.0 ng/mL range of HER2 concentration. The calibration regression equations for the later concentration range is Ipa = 0.303 [HER2] + 9.8006, with R2 = 0.9974. Limit of detection (LoD) of HER2 using the developed electrochemical immunosensing was evaluated as 3 s/m, where s is the standard deviation of the triplicate measurements of blank peak current, and m is the slope of the calibration curve for HER2 concentration range of 1-500 pg/mL (1; 10; 100; dan 500 pg/mL). By using the developed sensing method and the relevant procedure, it was found that the limit of detection of HER2 is 34.9 pg/mL. This figure of limit detection is much lower than those previously reported (i.e., 6000 pg/mL), using a sandwich immunosensor without bioconjugate [28]. Thus, the near future use of the NS CeO immunosensor developed in the present study for a non -invasive control of HER2 biomarkers in breast cancer patients is very promising and would result in a much better quality of information. Table 1 shows a comparison of the analytical parameter characteristics of the developed immunosensor with the other existing types of HER2 immunosensor.
9
A
B
ro
of
Journal Pre-proof
re
-p
Figure 5. (A) Cyclic voltammograms of SPCE-GNPs toward different concentrations of HER2 (0.001-20.0 ng/mL; a-g); a redox system of 0.1 mM (Fe(CN)6 3-/4-) in 0.1M PBS (pH 7.0) containing 0.1M KCl s olution; scan rate of 50 mV/s; (B) Calibration curve of HER2 for concentration range of 0.001 – 0.5 ng/mL (above), and those for concentration range of 0.5-20.0 ng/mL (below), are prepared using the developed immunosensor.
lP
Table 1. Comparison of analytical parameter characteristics of the developed immunosensor with the other existing types of HER2 immunosensor. Linearity ranges (ng mL-1 ) 5.0×10−4 –50.0
Limit of detection (pg mL-1 ) 2.0 × 10−2
References
Impedimetric immunosensor based on 1,6-hexanedithiol as a cross linker of antiHER2 - AuNPs/MWCILE
10–110
7400
[30]
PbS quantum dots bioconjugates immunosensor
1–100
280
[31]
Fe3 SO4 bioconjugates immunosensor
0.01-10; 10-100
0.995
[22]
AuNPs bioconjugates immunosensor
0.15-100
10.2
[26]
CeO2 NS bioconjugates immunosensor
0.001-20.0
34.9
This present work
na
Methods
Jo
ur
Immunosensor based on antiHER2/APTMS Fe 3 O4 NPs and antiHER2/Hydrazine@AuNPs-APTMS-Fe3 O4 bioconjugates
[29]
From the information given in Table 1, it will be noted that the immunosensor developed in the present study is better than most of the existing immunosensors used to detect HER2.
3.4 Analysis of Serum Samples and Recovery Tests
10
Journal Pre-proof
ro
of
The immunosensor method developed in the present study has been applied for the determination of HER2 concentration in some blood serum samples collected from breast cancer patient suspects. Conclusions on the possibility of patients whether to suffer breast cancer or not are based on the concentration of HER2 in their blood serum. If a suspect patient’s blood serum contains HER2 greater than 15.0 ng/mL, it means that his or her HER2 concentration is excessive, and the suspicious patient presumably has breast cancer [28]. The procedure used to measure HER2 concentration in blood serum samples in this study was similar to those applied to standard HER2 as a reference compound. Two different blood serum samples have been analyzed for their HER2 contents, using the developed immunosensor. It was found that the concentration of HER2 in one blood serum sample was 51.28 ±0.46 ng/mL, and in the other was 4.67 ±0.30 ng/mL. A recovery test has been carried out to evaluate the consistency of the new method, with respect to its precision. This has been done by using the Standard Addition Method. Thus, the two serum samples were spiked with the standard HER2 of known different concentrations (1 and 10 ng mL/mL). As usual, calibration curves were prepared, and the concentration of HER2 in the samples can be found from the calibration curves. Using this standard addition method, with three replicate measurements, it was found that the percentage recoveries of one sample were 101.58 ± 1.03%, and those of the other sample was 102.37± 0.70%. It can be concluded that the resulted recovery figures have verified the good precision of the new method.
re
-p
Conclusion Based on the results it can be concluded that cerium oxide nanostructures were successfully synthesized and conjugated with anti-HER2 to form CeO2 -anti HER2 bioconjugate to detect HER2. Voltammetric immunosensor using CeO2 -anti-HER2 bioconjugate was selective and sensitive to detect HER2. It could be concluded that the proposed immunosensor for the determination of HER2 has good measurement sensitivity and could be applicated for developing alternative clinical bioanalysis.
lP
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article
2. 3.
4. 5. 6.
7.
J.T. Dahle, Y. Arai, Environmental geochemistry of cerium: applications and toxicology of nanoceria , Int J Environ Res Public Health, 12 (2) (2015), pp. 1253-1278 B.C. Nelson, M.E. Johnson, M.L. Walker, K.R. Riley, C.M. Sims , Antioxidant cerium oxide nanoparticles in biology and medicine, Antioxidants, 5 (2016), p. 15 H.H. Bahti, Y. Mulyasih, A. Anggraeni, Extraction and chromatographic studies on rare-earth elements (REEs) from their minerals: the prospect of REEs production in Indonesia. Proceedings of the 2nd International Seminar on Chemistry (2011), pp.421-430, ISBN 978-602-19413-1-7.
Jo
1.
ur
References
na
Acknowledgments This research is financially supported by The Indonesian Ministry of Research, Technology, and Higher Education through the PUPT research scheme 1129/UN6.D/LT/2018, and The Academic Leadership Grant (ALG) research scheme of Universitas Padjadjaran, No.2295/UN.6.D/KS/2018. The authors also would like to thank Drs. Purwanto of the Bio Fit clinical laboratory, for providing some blood serum samples during this study .
Sun, H. Li, L, Chen, Nanostructured ceria-based materials: synthesis, properties, and applications , Energy Environ Sci 5(9) (2012), pp. 8475–8505 S. Das, J.M. Dowding, K.E. Klump, J.F. McGinnis, W. Self, S. Seal, Cerium oxide nanoparticles: applications and prospects in nanomedicine, Nanomedicine (Lond. Engl.), 8 (9) (2013), pp. 1483-1508 K.-J. Feng, Y.-H. Yang, Z.-J. Wang, J.-H. Jiang, G.-L. Shen, and R.-Q. Yu, A Nano-Porous CeO2 /Chitosan Composite Film as the Immobilization Matrix for Colorectal Cancer DNA Sequence-Selective Electrochemical Biosensor. Talanta 70 (2006), pp. 561–565. R. W. Tarnuzzer, J. Colon, S. Patil, and S. Seal, Vacancy Engineered Ceria Nanostructures for Protection from Radiation-Induced Cellular Damage, Nano Lett 5 (2005) pp. 2573–2577.
11
Journal Pre-proof
15.
16.
17.
18.
19. 20. 21. 22.
23. 24. 25. 26.
27.
28.
of
ro
14.
-p
13.
re
12.
lP
11.
na
10.
ur
9.
Y. Y. Tsai, J. O. Cossio, K. Agering, N. E. Simpson, M. A. Atkinson, C. H. Wasserfall, I. Constantinidis, and W. Sigmund, Novel synthesis of cerium oxide nanoparticles for free radical scavenging , Nanomedicine 2 (2007), pp. 325-332 A.A. Ansari, A. Kaushik, P.R. Solanki, B.D. Malhotra, Sol–gel derived nanoporouscerium oxide film for application to cholesterol biosensor, Electrochem Com-mun 10 (2008), pp. 1246–1249 F. Charbgoo, M.B. Ahmad, M. Dorroudi, Cerium oxide nanoparticles: green synthesis and biological application. Int J Nanomed 12 (2017), pp. 1401 - 1413. A. Kaushik, P.R. Solanki, M.K. Pandey, S. Ahmad, B.D. Malhotra, Cerium oxide-chitosan based nanobiocomposite for food borne mycotoxin detection , Appl Phys Lett 95 (2009), pp.173703 A. Kaushik, P.R Solanki, A.A. Ansari, S. Ahmad, B.D.Malhotra, A nanostructured cerium oxide film-based immunosensor for mycotoxin detection. Nanotechnology, 20(5) (2009), 055105. L. Lu, B. Liu, C. Liu, and G. Xie, Amperometric immunosensor for myeloperoxidase in human serum based on a multi-wall carbon nanotubes -ionic liquid-cerium dioxide film-modified electrode, Bull Korean Chem Soc 31 (2010) No. 11. L. Lu, B. Liu, S. Li, W. Zhang, G. Xie, Improved electrochemical immunosensor for myeloperoxidase in human serum based on nanogold/cerium dioxide-BMIMPF6/l-Cysteine composite film. Colloids Surf B Biointerfaces 86(2) (2011), pp. 339-344 Z.H. Yang, Y. Zhuo, R. YuanY.Q. Chai, An amplified electrochemical immunosensor based on in situ produced 1-naphthol as electroactive substance and graphene oxide and Pt nanoparticles functionalized CeO 2 nanocomposites as signal enhancer, Biosens Bioelectron 69 (2015), pp. 321–327. P.D. Tam, N.L. Hoang, H. Lan, P.H. Vuong, T.T.N. Anh, T.Q. Huy, N.T. Thuy, Detection of vibrio cholerae O1 by using cerium oxide nanowires-based immunosensor with different antibody immobilization methods. J Korean Phys Soc. 68(10) (2016), pp. 1235–1245 F. Li, Y. Li, J. Feng, Y. Dong, P. Wang, L. Chen, Z. Chen, H. Liu, Q. Wei, Ultrasensitive amperometric immunosensor for PSA detection based on Cu2O@CeO2-Au nanocomposites as integrated triple signal amplification strategy. Biosens Bioelectron 87 (2017), pp. 630–637. Y. Li, Y. Zhang, F. Li, J. Feng, M. Li, L. Chen, Y. Dong, Ultrasensitive electrochemical immunosensor for quantitative detection of SCCA using Co3O4@CeO2-Au@Pt nanocomposite as enzyme-mimetic labels, Biosens Bioelectron 92 (2017), pp. 33–39. A.S. Karakoti, S. Das, S. Thevuthasan, S. Seal, PEGylated inorganic nanoparticles, Angew Chem 50 (2011), pp. 1980–1994. H. Otsuka, Y. Nagasaki and K. Kataoka, PEGylated nanoparticles for biological and pharmaceutical applications, Adv Drug Delivery Rev 64 (2012), pp. 246–255. J. Suh, K. L. Choy, S. K. Lai, J. S. Suk, B. C. Tang, S. Prabhu and J. Hanes, PEGylation of nanoparticles improves their cytoplasmic transport, Int J Nanomed 4 (2007), pp. 735–741. M. Emami, M. Shamsipur, R. Saber, R. Irajirad, An electrochemical immunosensor for detection of a breast cancer biomarker based on antiHER2-iron oxide nanoparticle bio-conjugates, Analyst 139 (2014), pp. 2858– 2866, http://dx.doi.org/10.1039/c4an 00183d. L. Wang, X. Jia, Y. Zhou, Q. Xie, and S. Yao, Sandwich-type amperometric immunosensor for human immunoglobulin G using antibody-adsorbed Au/SiO2 nanoparticles, Microchim Acta 168 (2010), pp. 245–251. N. Nesakumar, S. Sethuraman, U.M. Krishnan, J.B. Rayappan. Fabrication of lactate biosensor based on lactate dehydrogenase immobilized on cerium oxide nanoparticles. J Colloid Interface Sci 410 (2013), pp. 158-164. M. Niemeyer, Bioconjugation protocols, Humana, New Jersey, 2004. Y.W. Hartati, D. Nurdjanah, S. Wyantuti, A. Anggraeni, S. Gaffar, Gold nanoparticles modified screen-printed immunosensor for cancer biomarker HER2 determination based on anti HER2 bioconjugates , AIP Conference Proceedings 2049 (1) (2018), 020051. W. Scales, A. J. Convertine, C. L. McCormick, Fluorescent labeling of RAFT-generated poly(Nisopropylacrylamide) via a facile maleimide–thiol coupling reaction, Biomacromolecules 7 (2006), pp. 1389–1392 Q.A.M. Al-Khafaji, M. Harris, S. Tombelli, S. Laschi, A.P.F. Turner, M. Mascini, G.Marrazza, An electrochemical immunoassay for HER2 detection, Electroanalysis 24 (2012), pp. 735–742, http://dx.doi.org/10.1002/elan.20110050
Jo
8.
12
Journal Pre-proof
Jo
ur
na
lP
re
-p
ro
of
29. M. Shamsipur, M. Emami, L. Farzin, R. Saber, A sandwich-type electrochemical immunosensor based on in situ silver deposition for determination of serum level of HER2 in breast cancer patients. Biosensors and Bioelectronics, 103 (2018), pp. 54-61. 30. E. Arkan, R. Saber, Z. Karimi, M. Shamsipur, A novel antibody–antigen based impedimetric immunosensor for low level detection of HER2 in serum samples of breast cancer patients via modification of a gold nanoparticles decorated multiwall carbon nanotube-ionic liquid electrode. Analytica chimica acta, 874 (2015), pp. 66-74. 31. S.A.A. Ahmad, M.S. Zaini, M.A. Kamarudin, An Electrochemical Sandwich Immunosensor for the Detection of HER2 using Antibody-Conjugated PbS Quantum Dot as a label. Journal of Pharmaceutical and Biomedical Analysis (2019), pp.
13
Journal Pre-proof CRediT author statement
Title: Cerium Oxide-Monoclonal Antibody Bioconjugate for Electrochemical Immunosensing of HER2 as a Breast Cancer Biomarker
Jo
ur
na
lP
re
-p
ro
of
Yeni Wahyuni Hartati: Conceptualization, Methodology, Supervision, Funding acquisition, Leonard Kristofel Letelay: Visualization, Data curation, Writing- Original draft preparation, Shabarni Gaffar: Supervision , Visualization, Investigation, Santhy Wyantuti: Project administration, Validation, Husein H. Bahti: Writing- Reviewing and Editing, Funding acquisition.
14
Yeni Wahyuni Hartati, Leonard Kristofel Letelay, Shabarni Gaffar, Santhy Wyantuti, Husein H. Bahti PII:
S2214-1804(19)30135-7
DOI:
https://doi.org/10.1016/j.sbsr.2019.100316
Reference:
SBSR 100316
To appear in:
Sensing and Bio-Sensing Research
Received date:
10 August 2019
Revised date:
4 December 2019
Accepted date:
6 December 2019
Please cite this article as: Y.W. Hartati, L.K. Letelay, S. Gaffar, et al., Cerium oxidemonoclonal antibody bioconjugate for electrochemical immunosensing of HER2 as a breast cancer biomarker, Sensing and Bio-Sensing Research(2019), https://doi.org/ 10.1016/j.sbsr.2019.100316
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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.
© 2019 Published by Elsevier.
Journal Pre-proof
Cerium Oxide-Monoclonal Antibody Bioconjugate for Electrochemical Immunosensing of HER2 as a Breast Cancer Biomarker Yeni Wahyuni Hartati1 *, Leonard Kristofel Letelay 1 , Shabarni Gaffar1 , Santhy Wyantuti1 , Husein H. Bahti1 1
Department of Chemistry, Universitas Padjadjaran, Bandung
*Email: [email protected].
ABSTRACT
na
lP
re
-p
ro
of
Metal oxide-based sensors have the advantage of rapid response and of high sensitivity in detecting specific active biological species and are relatively inexpensive. This report of the present study concerns the development of a cerium oxide – monoclonal antibody bioconjugate for its application as a sensitive immunosensor to detect a breast cancer biomarker. A cerium oxide-anti HER2 bioconjugate has been constructed by adding anti HER2 onto cerium oxide that had been previously reacted with APTMS and PEGNHS-Maleimide. The FTIR spectra of the reaction product showed that the cerium oxide-anti HER2 bioconjugate was successfully synthesized. The resulted bioconjugate was then immobilized on a screenprinted carbon-gold nanoparticles electrode surface by using the amine coupling bonding systems. The interaction of the synthesized cerium oxide-anti-HER2 bioconjugate with HER2 was found to inhibit an electron transfer and a decrease in the voltammetric Fe(CN)6 3-/4- peak current, which was proportional to the concentration of HER2. The optimal response of the current signal was generated at an anti-HER2 concentration of 5.0 µg/mL. The two linear ranges of HER2 concentration were found to be from 0.001 to 0.5 ng/mL and from 0.5 to 20.0 ng/mL. By using the first calibration curve, the limit of detection was found to be 34.9 pg/mL. The developed label-free immunosensor has been used to determine HER2 in a human serum sample with satisfactory results, as shown by a consistent result with the addition of standard. Thus, the resulted immunosensor in this study is promising and would have a potential application in clinical bio analysis.
1. Introduction
ur
Keywords: cerium oxide, bioconjugate, electrochemical immunosensor, HER2.
Jo
Cerium belongs to the lanthanide series (with electron configuration [Xe]4f15d16s2) and is one of the most abundant rare earth elements in the Earth’s crust. Several cerium minerals have been mined and processed for industrial applications and pharmaceutical uses [1], [2]. Cerium is a rare-earth element contained at high concentration in monazite minerals, which are widespread in Indonesia, and have been produced as a side -product (or “tailing”) of tin processing [3]. The element is an important material for industrial us e in catalytic converters for removing toxic gases, solid oxide fuel cells, applications of electro -chromic thin-film, glass polishing, catalysts, and sensors. Cerium has also been developed for pharmaceutical uses and shown a role as antioxidants in biolo gical systems, which makes cerium nanoparticles well suited for applications in nano -biology and regenerative medicine [1, 2, 4, 5]. Cerium oxide has been reported to have advantageous properties such as non -toxicity, good electrical conductivity, chemical inertness, large surface area, negligible swelling, oxygen transferability, and good bio compatibility. These characteristics have attracted more attention to the development in fabricating high performance electrochemical biosensors and immunosensors [6-10]. A nano-structured cerium oxide film-based immunosensor has been developed for ochratoxin detection [11,12]. Nano-structured cerium oxide (nano-CeO2 ) film was fabricated onto indium-tin-oxide (ITO) coated glass plate, and then rabbit-immunoglobulin antibodies (r-IgGs) and bovine serum albumin (BSA) were immobilized onto the film. Electrochemical studies revealed that nano-CeO2 particles provide an increased electron communication between rIgGs and electrode electroactive surface area. The resulting immunosensor had a linear response to ochratoxin in the range of 0.5 – 6.0 ng/dl, a low detection limit of 0.25 ng/dl, and a fast response time of 30 s. The high value of the
1
Journal Pre-proof
Jo
ur
na
lP
re
-p
ro
of
association constant, Ka 0.9×1011 l/mol, indicates the high affinity of the BSA/r-IgGs/nano-CeO2 /ITO to ochratoxin [11]. A label-free amperometric immunosensor based on a multi-wall carbon nano-tubes-ionic liquid-cerium oxide film-modified electrode has been proposed for the detection of myeloperoxidase (MPO) in human serum [13, 14]. The cerium oxide was dispersed by chitosan, coated on the glassy carbon electrode, and then antibodies (anti-MPO) were attached to the nano-cerium oxide surface. With a non-competitive-immunoassay format, the antibody-antigen complex was formed between the immobilized anti-MPO and MPO in a sample solution. Under optimal conditions, the immunosensor showed a current change, which was proportional to MPO concentration in the range of 5 to 300 ng/mL, with a limit of detection of 0.2 ng/mL [13]. An improved electrochemical immunosensor for MPO detection was based on nano-gold/cerium oxide-lcysteine composite film has been developed. Cerium oxides were dispersed in 1-butyl-3-methylimidazolium hexafluorophosphate and then were immobilized on the l-cysteine film. The cerium oxides have a positive charge that may be able to bind more anti-MPO. The immunosensor improved some characteristics, such as a linear range to the MPO concentration between 10 ng/mL and 400 ng/mL, and a limit of detection of 0.06 ng/mL [14]. Yang et al. used ceria nanoparticles (CNPs) in the design of an amplification electrochemical immunosensor for influenza biomarker determination. The sensor was constructed based on cerium oxide/graphene oxide composites, as a catalytic signal amplifier for the detection of influenza, and using 1-naphthol that can be hydrolyzed to naphthoquinones by O-acetylesterase enzyme of the influenza virus. The immunosensor showed a linear range of 0.0010 – 0.10 ng/mL with a detection limit of 0.43 pg/mL [15]. An electrochemical biosensor also has been developed based on CeO 2 nano-wires for the determination of Vibrio cholerae O1 (VcO1). The antibodies of VcO1 (anti-VcO1) were immobilized on the surface of a CeO2-nanowire. Interaction of bacterial cells with the anti-VcO1 was analyzed by decreasing the current peak of the biosensor. The biosensor had a linear range of response between 102 CFU/mL and – 107 CFU/mL with a detection limit of 100 CFU/mL for VcO1 [16]. Another development of cerium oxide in immunosensors is cerium nanocomposites biosensors. A nanocomposite with a core-shell structure, cuprous oxide@cericdioxide (Cu 2 O@CeO2 -Au), was proposed for the quantitative detection of prostate-specific antigen (PSA). The amino-functionalized Cu 2 O@CeO2 -NH2 core-shell nanocomposites were prepared to bind gold-nanoparticles (AuNPs) by constructing a stable Au-N bond between AuNP sand-NH2 . The amplified signal sensitivity was achieved by the synergetic effect existing in Cu 2 O@CeO2 core-shell loaded with AuNPs. It showed a good electro-catalytic activity towards the reduction of hydrogen peroxide and was used as transducing materials to achieve efficiently capture antibodies and triple signal amplification of the proposed immunosensor. The developed immunosensor showed a wide linear range between 0.1 pg/mL and 100 ng/mL, with a low detection limit of 0.03pg/mL (S/N3) [17]. The Co 3 O4 @CeO2 -Au@Pt nanocomposite also has been developed as labels to conjugate with secondary antibodies for signal amplification in a sandwich-type electrochemical immunoassay for squamous cell carcinoma antigen (SCCA) detection. The amino functionalized cobaltosicoxide@cericdioxide nanocubes with core-shell morphology were prepared to combine seaurchin like gold @ platinum nanoparticles (Co 3 O4 @CeO2 -Au@Pt). The glassy carbon electrodes (GCE) modified using electro-deposited gold, were used as antibodies carriers and sensing platforms. Because the synergetic effect presents in Co 3 O4 @CeO2 -Au@Pt, the amplification of sensitivity was achieved towards the reduction of hydrogen peroxide. The proposed immunosensor exhibited a wide linear range, from 100 fg/mL to 80 ng/mL, with a low detection limit of 33 fg/mL for detecting SCCA [18]. As discussed, studies of various developments of ceria-based immunosensors have been extensively reported. The functionalized ceria or cerium oxides have attracted much attention in the fabrication of bio-sensing systems due to their unique properties. The use of a linker in the controlled assembly of nanoparticles to a target is the one important of surface modification and functionalization in biomedicine as diagnostic and therapeutic agents in cells or tissues. Polyethylene glycol (PEG), has been reported as a long compatible linker for nanoparticles. The main advantage of using PEG is to provide enough space to bind more recognizing element molecules like monoclonal antibodies to nanoparticles and allow them to stand aside and result in more effective combination with the target molecules [19-22]. Anti HER2 is a monoclonal antibody (mAb) that can bind HER2 protein and has known as one of breast cancer biomarkers because HER2 is over-expressed in some breast cancer patients. HER2 is a receptor tyrosine kinase, which is a member of the epidermal growth factor receptor (EGFR) family involved in cellular signaling pathways, which may lead to proliferation and differentiation. The overexpression of HER2 in some breast cancer patients can be used as a key prognostic marker, and effective therapeutic treatment targets to diagn ose breast cancer that generally occurred in adult females [22, 23]. This research concerns the use of PEGylated nano-structured cerium oxides attached to different concentrations of anti HER2 to form bioconjugates. A screen-printed carbon-gold nanoparticles electrode was used for designing a
2
Journal Pre-proof label-free platform, and the bioconjugates were stabilized covalently on the electrode surface to assay HER2 antigen in serum samples. This highly sensitive and straight forward electrochemical analysis method holds great potential for the detection of this biomarker in clinical diagnoses. 2. Experimental 2.1 Materials Anti HER2 Ab (Herceptin) was purchased from F. Hoffmann -La Roche Ltd. The HER2/CD340 human, 3aminopropyl trimethoxisylane (APTMS), bovine serum albumin (BSA), cysteamine (Cys), 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC), potassium ferricyanide, n -hydroxide succinimide (NHS), 2-iminithiolane, 3mercaptopropionic acid (MPA), and polyethylene glycol-α-maleimide-ω-NHS (NHS-PEG-mal), were purchased from Sigma-Aldrich Ltd. Toluene, hydrochloric acid, ethylene diamine tetraacetic acid (EDTA), ammonium hydroxide, potassium hydrogen phosphate, sodium hydrogen phosphate, cerium sulfate, sodium hydroxide, and methanol were purchased from Merck.
-p
ro
of
2.2 Apparatus Voltammetric measurements were done using Autolab type III Potentiostat/Galvanostat with NOVA 1.10 software (Metrohm). The screen-printed carbon-gold nanoparticles electrodes (SPCE-GNPs) DRP-110GNP (Dropsens) were used for the electrochemical experiments . Other instruments used were Scanning electron microscopy (SEM) images JEOL JSM-6360LA type and an FT-IR spectrophotometer (Perkin Elmer Spectrum 100).
lP
re
2.3 Preparation of Nano Structured Cerium Oxide (NS CeO2 ) NS CeO2 was synthesized based on a simple method [24]. Briefly, 2 g of Ce(SO4 )2 was dissolved in 25 mL double-distilled water and then a drop of sodium hydroxide solution (0.3 M) was added to the 25 mL solution. The mixture was continuously stirred for about 2-3 hours at room temperature until a pale yellowish-white precipitate was obtained. The precipitate was separated and washed several times with double-distilled water, after then it was dried at 150 C for an hour. The obtained yellowish particles were dried at 250 C for 3 hours.
Jo
ur
na
2.4 Preparation of NHS-PEG-Mal-NS CeO2 The Mal-PEG-NHS-NS CeO2 was prepared based on the functionalization of the ferric oxide procedure [22,25] with a modification. Briefly, to an amount of 0.1 g of NS CeO2 powder, 35 mL of toluene and 25 L of APTMS were added. Then the mixture was sonicated for 30 min and heated at 60 C for 5 hours in an oven. The obtained APTMS-coated NS CeO2 was separated by decantation and redispersed in 50 mL of methanol. The result ed of amino-NS CeO2 (0.01 g) was dissolved in 10 mL of redistilled water, and then 31 mg of NHS-PEG-Mal was added to the solution. The mixture was sonicated for 30 min and stirred for 6 hours. At the end of this step, PEGylated nanostructures were separated and redispersed in 5 mL of redistilled water. 2.5 Preparation of Bioconjugate The bioconjugate was synthesized based on reported methods [22,25,26]. The synthesis was started with the thiolation of anti HER2 with 2-iminithiolane. The thiolated HER2 was done by incubating 1000 g/mL of anti HER2 in 0.1 M PBS pH 8.0 in 2-iminithiolane, in a molar ratio of 1:200. Then 5 mM of EDTA was added into the reaction mixture to protect the thiol groups from oxidation. The mixture was stirred at room temperature for 1 h using mini spin. The thiolated anti HER2 thus formed was purified by its dialyzing with 20 mL of PBS pH 8.0. The purified thiolated anti-HER2 (100 μL of 15 g/mL) was subsequently added to 200 L of of 5 mg/mL NHS-PEGMal-NS CeO2 and kept the mixture under constant shaking for 6 hours at room temperature. In this way, the thiol groups of the thiolated anti HER2 would have been covalently attached to the unsaturated bond of maleimides that linked to the NS CeO2 to form the bioconjugates. The resulted bioconjugates were then separated by using magnetic decantation and, finally, redispersed in 1 mL of PBS pH 7.4. Figure 1 shows schematic reaction mechanisms of preparation of cerium oxide-anti HER2 bioconjugate.
3
of
Journal Pre-proof
Figure 1. Schematic reaction mechanisms of preparation of bioconjugate
ro
2.6 Optimization of Experimental Conditions
re
-p
Determination of optimum incubation time for MPA reaction The electrode is immersed in a 0.1 M MPA for 10, 30, 60, and 120 min. Then, the generated electrochemical response was measured by cyclic voltammetry using a redox system of 10 mM of Fe(CN)6 3-/4-in KCl 0.1 M at a potential range of -0.6 V to +0.6 V, with a scanning rate of 50 mV/s.
na
lP
Determination of optimum anti-HER2 antibody concentrations in bioconjugate formation As much as 200 µL of Mal-PEG-NHS-NS CeO2 solution was put into a microtube, then 2.5 µg/mL of anti-HER2 antibody was added into the solution. The mixture was stirred using a mini spin, and then its electrochemical response was measured by cyclic voltammetry using a redox system of 10 mM of Fe(CN) 6 3-/4- in KCl 0.1 M at a potential range of -0.6 V to +0.6 V with a scanning rate of 50 mV/s. The whole experiment was repeated three times, each using a different concentration of anti-H ER2 antibodies, i.e., 5.0, 7.5, and 10.0 µg/mL.
Jo
ur
Optimization of incubation time of the bioconjugate The electrode was immersed in a 0.1 M MPA for 30 min (i.e., the optimum time), then dipped in a 0.1 M cysteamine solution and rinsed with redistilled water. After that, a 40 µL solution containing 0.1 M EDC and 0.1 M NHS with a mole ratio of 1: 1 was dropped onto the electrode, which was subsequently incubated for one hour at room temperature. Then, after dropping 40 µL of bioconjugate onto the electrode, it was incubated for one hour at room temperature, respectively. The electrochemical response was measured by cyclic voltammetry using a redox system of 10 mM of Fe(CN)6 3-/4- in 0.1 M KCl at a potential range of -0.6 V to +0.6 V, with a scanning rate of 50 mV/s. The whole process was repeated but three times, each with different lengths of time (i.e., 2, 3, and 4 hours) of the final incubation process of the bioconjugate. 2.7 Preparation of The Immunosensor and Electrochemical Signals Measurements The (SPC-GNPs) electrode was immersed into 0.5 mL of 0.1 M MPA for 30 minutes at room temperature. This aim would have led to the formation of MPA-GNPs on the electrode. The electrode was then rinsed with doubledistilled water for eliminating any unwanted material absorbed on it physically. Following this, to activate the carboxyl groups of MPA, 40 μL of a solution containing 0.1 M EDC and 0.1 M NHS with a mole ratio of 1:1 was dropped onto the MPA-GNPs electrode surfaces. After that, the electrode was incubated for one hour at room temperature. After washing with double-distilled water, the electrode was immersed into a 0.1 M of cysteamine solution to form Cys-MPA-GNPs on it, via an amide formation, and rinsed with double-distilled water. Thereafter, 40 μL of the bioconjugate was dropped onto the electrode, and incubated for two hours (i.e, the optimum incubation time) at 4 C. Stable carbon-sulfur bonds would have been formed between carbon double bonds in the free maleimides of the bioconjugate and the thiol groups of Cys [22, 26, 27]. Any possible excess of the bioconjugate was rinsed with a 0.1 M PBS pH 7.4 solution and redistilled water, consecutively. Next, 20 µL of 1% BSA solutio n was added to the modified electrode, which was subsequently incubated for 45 minutes at room temperature,
4
Journal Pre-proof followed by rinsing the electrode with 0.1 M PBS pH 7.4 solution, and redistilled water, consecutively. It will be noted that BSA had been used to block any possible non-specific binding sites. To continuing the experimentations, 20 µL of HER2 solution (in a concentration range of 0.01-100 ng/mL) was dropped onto the bioconjugate-modified electrode, which was then left for 30 minutes at room temperatu re. Finally, the electrode was rinsed with 0.1 M PBS pH 7.4 solution and redistilled water, consecutively. To test the performance of the resulted the electrode as an immunosensor, experiments on cyclic voltammetric measurements were done by using the redox system consisted of 10 mM of Fe(CN)6 3-/4- with 0.1 M KCl in 0.05 M PBS pH 7.4 solution, at a potential range of -0.6 V to +0.6 V, with a scanning rate of 50 mV/s. Each measurement was carried out after the addition of MPA, bioconjugates, an d BSA. The whole experiment was finally accomplished by measuring some other HER2 solutions of different concentrations. Figure 2 shows the general schematic diagram of the immunosensor platform for the detection of HER2.
Jo
ur
na
lP
re
-p
ro
of
2.8 Analysis of Blood Serum Samples and Recovery Test Serum samples were obtained from the Bio Fit clinical laboratory in Bandung, Indonesia. Each of the samples was diluted 20 times, with 0.01 M of PBS pH 7.4 solutions. Then 30 µL of a diluted sample was dropped onto the bioconjugate-electrode, which was subsequently and incubated for 30 min utes at 37 °C. After rinsing the electrode with PBS and redistilled water, it was used to measure electrochemical response by cyclic voltammetry using a redox system of 10 mM of Fe(CN)6 3-/4- with 0.1 M KCl in 0.05 M PBS pH 7.4 solution, at a potential range of -0.6 V to +0.6 V, and with a scan rate 50 mV/s. Three replicate measurements were done, and the HER2 level of the sample was calculated using a previously prepared calibration regression equation. Recovery tests were carried out for two samples. Each sample was spiked with two different concentrations of HER2. The standard addition method was used to assay spiked concentration.
5
ur
na
lP
re
-p
ro
of
Journal Pre-proof
3
Jo
Figure 2. Schematic diagram of immunosensor platform for the determination of HER2 using CeO2 -anti HER2 bioconjugate
Results and Discussion
3.1 Characterization of Nanostructured Cerium Oxide, Bioconjugate, and the Modified Electrode Characterization of the NS CeO2 was carried out by using scanning electron microscopy to determine its morphology and size distribution. Figure 3.A displays the results of NS CeO2 analysis at 12.000 × magnifications, which show the average size of the NS CeO2 of about 100 nm. In order to proof the APTMS had been coated on the NS CeO2 , FT-IR spectroscopic analyses were carried out to record the spectra of the APTMS as a reference, and those of the APTMS-NS CeO2 . The resulted IR spectra of the two compounds are overlaid and are presented in Figure 3.B. In the IR spectrum of APTMS (Figure 3.B; a), there are several peaks in the frequency region of 3460-3280 cm−1 , which are originated from stretching vibration of -OH groups of water molecules or from those of surface −OH groups. The peak at 1500 cm−1 is the bending vibration of N-H and thus confirms the presence of a primary N-H bond. The peak at 2930.18 cm−1 is assigned to C-H stretching band, and the peak at 1111.96 cm−1 corresponds to Si-O stretching. Moreover, the peak at 1430 cm−1 is assigned to the bending vibration of C-H, while stretching vibration of the aliphatic C-N bond is characterized by the peak around 1023,33 cm−1 — meanwhile, the spectrum of APTMS-NS CeO2 (Figure 3. B; b) shows several bands. The very large and strong peak around 2557.0 cm−1 originated from the functional group of –CH. The peak at 1505.7
6
Journal Pre-proof
lP
re
-p
ro
of
cm−1 is the bending vibration of the –N-H bond; the moderate peak at 1130.7 cm−1 can be assigned as Si-O bond, and the sharp peak with strong absorption intensity at 579.8 cm−1 is assigned to the Ce−O stretching band. The intense band observed around 852 cm−1 is due to the Ce−O−C stretching vibration [16]. The peak at 3410 cm−1 is related to −OH groups of water molecules or those of surface −OH groups. From the analysis of the two spectra given above, i.e., the spectra of APTMS and those of APTMS-NS CeO2 , it can be concluded that APTMS had successfully been attached to the surface of NS CeO2 . Cyclic voltammetry has been used in this study for the electrochemical characterization of the electro des. The cyclic voltammogram of SPCE-GNPs with MPA, those of SPCE-GNPs-MPA-NSCeO2, and those of SPCE-GNPsMPA bioconjugates are shown in Figure 3.C. As can be seen in Figure 3.C, the presence of NS CeO 2 had decreased the intensity of the redox signal from 116.018 µA to 91.131 µ A, i.e., about 25 %. This will be proof that the nano structure of CeO2 has an electro-active characteristic that facilitates the process of electron transfers to electrodes. Therefore, NS CeO2 has an excellent characteristic and will form bioconjugate with the antibody to be used for a good electrochemical detection of HER2.
B
Jo
ur
na
A
C Figure 3. (A) SEM images of NS CeO2 , (B) FT-IR spectra of APTMS only (a), and those of APTMS-NS CeO2 (b), (C) Cyclic voltammograms of (a) SPCE-GNPs-MPA; (b) SPCE-GNPs-MPA-NS CeO2 ; (c) SPCE-GNPs-MPAbioconjugate. Cyclic voltametric experimental conditions: a redox system of 10 mM K3 [Fe(CN)6 ] with 0.1 M KCl in 0.05 M PBS pH 7.4 at scan rate 50 mV/s, at a potential range -0.6 V to 0.6 V. 3.2 Optimization of Experimental Conditions
7
Journal Pre-proof
Jo
ur
na
lP
re
-p
ro
of
Immunoreagents, including anti-HER2 and HER2 antibodies, are non-electroactive compounds, and thus they reduce the current flow of K3FeCNS redox compounds on the electrode surface. For this reason, optimal experimental conditions resulting in the lowest current were chosen in the experiments. In this present study, the electrode was modified through the immobilizing process of anti-HER2 bioconjugates on the electrode, which was conducted by using the co valent bonding method through the amine coupling system. However, the formation of the covalent bonds in this step is reported to be slow [22]. It has also been noted further that each important step plays an important role and needs to be optimized, inclu ding the incubation time of MPA. Each measurement should be carried out for three replicates. The gold electrode was first immersed in a 0.1 M of MPA solution, forming a robust Au-S bond because the MPA will be impregnated on the gold surface. The carboxylate group of MPA will facilitate the electron transfer process between the bioconjugates and the electrode. Figure 4. A shows optimum incubation time of MPA immersed in 30 minutes, with the average current of about 52 µA. Figure 4.B shows that the optimum concentration of anti-HER2 in bioconjugates was 5 μg/mL because it gave the lowest current response. The bioconjugation process was carried out by conjugating NS CeO 2 with thiolated anti- HER2. The method to bind thiolated anti HER2 at various concentrations, with PEG-NHS-Mal and NS CeO2 , was done as follows: For the first experiment, concentration ratio between PEG-NHS-Mal-NS CeO2 and anti HER2 was 1:0.05 µg/mL (200:400 µL). At this condition, the current obtained was too high, i.e., about 138 µA. Next, anti HER2 of different concentrations (i.e., 2.5; 5; 7.5; and 10 µg/mL) were used, and the results are presented in Figure 4.B. The data in Figure 4.B show that the optimum concentration (i.e., a concentration resulting in t he lowest current) was 5 µg/mL. If the concentration was higher than the optimum, the excess of anti-HER2 on the NS CeO2 had probably resulted in saturation of the nanoparticle surface. It interfered with the binding of the bioconjugate maleimide groups to the electrode surface. However, if the concentration of anti-HER2 were lower than the optimum, the current would be increased because of less anti-HER2 covering the electrode surface. Figure 4.C shows data on the optimization of incubation time of biocon jugates on the electrodes. The data show that the optimum incubation time of the bioconjugate on the electrode surface was two hours, i.e., the time giving the lowest peak current, which was about 115 µA.
A.
B.
C. Figure 4. Optimization of experimental conditions: (A). The effect of incubation time of MPA on the gold electrode surface, on the peak current; (B). The effect of concentrations of anti-HER2 in the bioconjugate, on the peak current; and (C). The result of the incubation time of the bioconjugate on the electrode, on the peak current.
8
Journal Pre-proof
3.3 Immunosensor Response and Calibration Curve If HER2 was dropped onto the developed immunosensor (with a specific concentration) for 30 minutes, a decrease in current was observed. This decreasing in current was caused by the binding of anti-HER2 with HER2 to form an immunocomplex, which acts as a blocking layer to isolate the electron and mass transfer. The immunocomplex also hinders the diffusion of (Fe(CN)6 3-/4-) toward the electrode surface. The detection principle is based on the change in oxidation peak current response (Ipa) before and after the antibody-antigen interaction. This peak current is s evaluated using the following equation [13]: Ipa = I0 −In ,
Jo
ur
na
lP
re
-p
ro
of
where I0 is the response current before the immunoreaction, and I n is the response current after the immunoreaction. In this present study, the responses of the prepared immunosensor towards HER2 of seven different concentrations (in the range of 0.001 ng/mL – 20.0 ng/mL) were studied by cyclic voltammetry using the redox system of 10 mM (Fe(CN)6 3-/4-) in 0.1 M PBS pH 7.4 solution containing 0.1 M KCl, under the optimal experimental conditions. Triplicate experiments for each HER2 solution concentration were done, and the resulting voltammograms are presented in Figure 5.A. A calibration curve was then prepared using peak current data from the seven resulted voltammograms. As can be seen in Figure 5. B, the calibration curve consisted of two segments of the linear curve (line) having different slopes (the two segments of the calibration curve are drawn separately). The first segment of the calibration curve is linear for a HER2 concentration range of 1 – 500 pg/mL. The calibration regression equations for this concentration ranges is Ipa = 0.0179 [HER2] + 0.9515, with R2 = 0.9898. Meanwhile, the second segment of the calibration curve is also linear for a 5.0-20.0 ng/mL range of HER2 concentration. The calibration regression equations for the later concentration range is Ipa = 0.303 [HER2] + 9.8006, with R2 = 0.9974. Limit of detection (LoD) of HER2 using the developed electrochemical immunosensing was evaluated as 3 s/m, where s is the standard deviation of the triplicate measurements of blank peak current, and m is the slope of the calibration curve for HER2 concentration range of 1-500 pg/mL (1; 10; 100; dan 500 pg/mL). By using the developed sensing method and the relevant procedure, it was found that the limit of detection of HER2 is 34.9 pg/mL. This figure of limit detection is much lower than those previously reported (i.e., 6000 pg/mL), using a sandwich immunosensor without bioconjugate [28]. Thus, the near future use of the NS CeO immunosensor developed in the present study for a non -invasive control of HER2 biomarkers in breast cancer patients is very promising and would result in a much better quality of information. Table 1 shows a comparison of the analytical parameter characteristics of the developed immunosensor with the other existing types of HER2 immunosensor.
9
A
B
ro
of
Journal Pre-proof
re
-p
Figure 5. (A) Cyclic voltammograms of SPCE-GNPs toward different concentrations of HER2 (0.001-20.0 ng/mL; a-g); a redox system of 0.1 mM (Fe(CN)6 3-/4-) in 0.1M PBS (pH 7.0) containing 0.1M KCl s olution; scan rate of 50 mV/s; (B) Calibration curve of HER2 for concentration range of 0.001 – 0.5 ng/mL (above), and those for concentration range of 0.5-20.0 ng/mL (below), are prepared using the developed immunosensor.
lP
Table 1. Comparison of analytical parameter characteristics of the developed immunosensor with the other existing types of HER2 immunosensor. Linearity ranges (ng mL-1 ) 5.0×10−4 –50.0
Limit of detection (pg mL-1 ) 2.0 × 10−2
References
Impedimetric immunosensor based on 1,6-hexanedithiol as a cross linker of antiHER2 - AuNPs/MWCILE
10–110
7400
[30]
PbS quantum dots bioconjugates immunosensor
1–100
280
[31]
Fe3 SO4 bioconjugates immunosensor
0.01-10; 10-100
0.995
[22]
AuNPs bioconjugates immunosensor
0.15-100
10.2
[26]
CeO2 NS bioconjugates immunosensor
0.001-20.0
34.9
This present work
na
Methods
Jo
ur
Immunosensor based on antiHER2/APTMS Fe 3 O4 NPs and antiHER2/Hydrazine@AuNPs-APTMS-Fe3 O4 bioconjugates
[29]
From the information given in Table 1, it will be noted that the immunosensor developed in the present study is better than most of the existing immunosensors used to detect HER2.
3.4 Analysis of Serum Samples and Recovery Tests
10
Journal Pre-proof
ro
of
The immunosensor method developed in the present study has been applied for the determination of HER2 concentration in some blood serum samples collected from breast cancer patient suspects. Conclusions on the possibility of patients whether to suffer breast cancer or not are based on the concentration of HER2 in their blood serum. If a suspect patient’s blood serum contains HER2 greater than 15.0 ng/mL, it means that his or her HER2 concentration is excessive, and the suspicious patient presumably has breast cancer [28]. The procedure used to measure HER2 concentration in blood serum samples in this study was similar to those applied to standard HER2 as a reference compound. Two different blood serum samples have been analyzed for their HER2 contents, using the developed immunosensor. It was found that the concentration of HER2 in one blood serum sample was 51.28 ±0.46 ng/mL, and in the other was 4.67 ±0.30 ng/mL. A recovery test has been carried out to evaluate the consistency of the new method, with respect to its precision. This has been done by using the Standard Addition Method. Thus, the two serum samples were spiked with the standard HER2 of known different concentrations (1 and 10 ng mL/mL). As usual, calibration curves were prepared, and the concentration of HER2 in the samples can be found from the calibration curves. Using this standard addition method, with three replicate measurements, it was found that the percentage recoveries of one sample were 101.58 ± 1.03%, and those of the other sample was 102.37± 0.70%. It can be concluded that the resulted recovery figures have verified the good precision of the new method.
re
-p
Conclusion Based on the results it can be concluded that cerium oxide nanostructures were successfully synthesized and conjugated with anti-HER2 to form CeO2 -anti HER2 bioconjugate to detect HER2. Voltammetric immunosensor using CeO2 -anti-HER2 bioconjugate was selective and sensitive to detect HER2. It could be concluded that the proposed immunosensor for the determination of HER2 has good measurement sensitivity and could be applicated for developing alternative clinical bioanalysis.
lP
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article
2. 3.
4. 5. 6.
7.
J.T. Dahle, Y. Arai, Environmental geochemistry of cerium: applications and toxicology of nanoceria , Int J Environ Res Public Health, 12 (2) (2015), pp. 1253-1278 B.C. Nelson, M.E. Johnson, M.L. Walker, K.R. Riley, C.M. Sims , Antioxidant cerium oxide nanoparticles in biology and medicine, Antioxidants, 5 (2016), p. 15 H.H. Bahti, Y. Mulyasih, A. Anggraeni, Extraction and chromatographic studies on rare-earth elements (REEs) from their minerals: the prospect of REEs production in Indonesia. Proceedings of the 2nd International Seminar on Chemistry (2011), pp.421-430, ISBN 978-602-19413-1-7.
Jo
1.
ur
References
na
Acknowledgments This research is financially supported by The Indonesian Ministry of Research, Technology, and Higher Education through the PUPT research scheme 1129/UN6.D/LT/2018, and The Academic Leadership Grant (ALG) research scheme of Universitas Padjadjaran, No.2295/UN.6.D/KS/2018. The authors also would like to thank Drs. Purwanto of the Bio Fit clinical laboratory, for providing some blood serum samples during this study .
Sun, H. Li, L, Chen, Nanostructured ceria-based materials: synthesis, properties, and applications , Energy Environ Sci 5(9) (2012), pp. 8475–8505 S. Das, J.M. Dowding, K.E. Klump, J.F. McGinnis, W. Self, S. Seal, Cerium oxide nanoparticles: applications and prospects in nanomedicine, Nanomedicine (Lond. Engl.), 8 (9) (2013), pp. 1483-1508 K.-J. Feng, Y.-H. Yang, Z.-J. Wang, J.-H. Jiang, G.-L. Shen, and R.-Q. Yu, A Nano-Porous CeO2 /Chitosan Composite Film as the Immobilization Matrix for Colorectal Cancer DNA Sequence-Selective Electrochemical Biosensor. Talanta 70 (2006), pp. 561–565. R. W. Tarnuzzer, J. Colon, S. Patil, and S. Seal, Vacancy Engineered Ceria Nanostructures for Protection from Radiation-Induced Cellular Damage, Nano Lett 5 (2005) pp. 2573–2577.
11
Journal Pre-proof
15.
16.
17.
18.
19. 20. 21. 22.
23. 24. 25. 26.
27.
28.
of
ro
14.
-p
13.
re
12.
lP
11.
na
10.
ur
9.
Y. Y. Tsai, J. O. Cossio, K. Agering, N. E. Simpson, M. A. Atkinson, C. H. Wasserfall, I. Constantinidis, and W. Sigmund, Novel synthesis of cerium oxide nanoparticles for free radical scavenging , Nanomedicine 2 (2007), pp. 325-332 A.A. Ansari, A. Kaushik, P.R. Solanki, B.D. Malhotra, Sol–gel derived nanoporouscerium oxide film for application to cholesterol biosensor, Electrochem Com-mun 10 (2008), pp. 1246–1249 F. Charbgoo, M.B. Ahmad, M. Dorroudi, Cerium oxide nanoparticles: green synthesis and biological application. Int J Nanomed 12 (2017), pp. 1401 - 1413. A. Kaushik, P.R. Solanki, M.K. Pandey, S. Ahmad, B.D. Malhotra, Cerium oxide-chitosan based nanobiocomposite for food borne mycotoxin detection , Appl Phys Lett 95 (2009), pp.173703 A. Kaushik, P.R Solanki, A.A. Ansari, S. Ahmad, B.D.Malhotra, A nanostructured cerium oxide film-based immunosensor for mycotoxin detection. Nanotechnology, 20(5) (2009), 055105. L. Lu, B. Liu, C. Liu, and G. Xie, Amperometric immunosensor for myeloperoxidase in human serum based on a multi-wall carbon nanotubes -ionic liquid-cerium dioxide film-modified electrode, Bull Korean Chem Soc 31 (2010) No. 11. L. Lu, B. Liu, S. Li, W. Zhang, G. Xie, Improved electrochemical immunosensor for myeloperoxidase in human serum based on nanogold/cerium dioxide-BMIMPF6/l-Cysteine composite film. Colloids Surf B Biointerfaces 86(2) (2011), pp. 339-344 Z.H. Yang, Y. Zhuo, R. YuanY.Q. Chai, An amplified electrochemical immunosensor based on in situ produced 1-naphthol as electroactive substance and graphene oxide and Pt nanoparticles functionalized CeO 2 nanocomposites as signal enhancer, Biosens Bioelectron 69 (2015), pp. 321–327. P.D. Tam, N.L. Hoang, H. Lan, P.H. Vuong, T.T.N. Anh, T.Q. Huy, N.T. Thuy, Detection of vibrio cholerae O1 by using cerium oxide nanowires-based immunosensor with different antibody immobilization methods. J Korean Phys Soc. 68(10) (2016), pp. 1235–1245 F. Li, Y. Li, J. Feng, Y. Dong, P. Wang, L. Chen, Z. Chen, H. Liu, Q. Wei, Ultrasensitive amperometric immunosensor for PSA detection based on Cu2O@CeO2-Au nanocomposites as integrated triple signal amplification strategy. Biosens Bioelectron 87 (2017), pp. 630–637. Y. Li, Y. Zhang, F. Li, J. Feng, M. Li, L. Chen, Y. Dong, Ultrasensitive electrochemical immunosensor for quantitative detection of SCCA using Co3O4@CeO2-Au@Pt nanocomposite as enzyme-mimetic labels, Biosens Bioelectron 92 (2017), pp. 33–39. A.S. Karakoti, S. Das, S. Thevuthasan, S. Seal, PEGylated inorganic nanoparticles, Angew Chem 50 (2011), pp. 1980–1994. H. Otsuka, Y. Nagasaki and K. Kataoka, PEGylated nanoparticles for biological and pharmaceutical applications, Adv Drug Delivery Rev 64 (2012), pp. 246–255. J. Suh, K. L. Choy, S. K. Lai, J. S. Suk, B. C. Tang, S. Prabhu and J. Hanes, PEGylation of nanoparticles improves their cytoplasmic transport, Int J Nanomed 4 (2007), pp. 735–741. M. Emami, M. Shamsipur, R. Saber, R. Irajirad, An electrochemical immunosensor for detection of a breast cancer biomarker based on antiHER2-iron oxide nanoparticle bio-conjugates, Analyst 139 (2014), pp. 2858– 2866, http://dx.doi.org/10.1039/c4an 00183d. L. Wang, X. Jia, Y. Zhou, Q. Xie, and S. Yao, Sandwich-type amperometric immunosensor for human immunoglobulin G using antibody-adsorbed Au/SiO2 nanoparticles, Microchim Acta 168 (2010), pp. 245–251. N. Nesakumar, S. Sethuraman, U.M. Krishnan, J.B. Rayappan. Fabrication of lactate biosensor based on lactate dehydrogenase immobilized on cerium oxide nanoparticles. J Colloid Interface Sci 410 (2013), pp. 158-164. M. Niemeyer, Bioconjugation protocols, Humana, New Jersey, 2004. Y.W. Hartati, D. Nurdjanah, S. Wyantuti, A. Anggraeni, S. Gaffar, Gold nanoparticles modified screen-printed immunosensor for cancer biomarker HER2 determination based on anti HER2 bioconjugates , AIP Conference Proceedings 2049 (1) (2018), 020051. W. Scales, A. J. Convertine, C. L. McCormick, Fluorescent labeling of RAFT-generated poly(Nisopropylacrylamide) via a facile maleimide–thiol coupling reaction, Biomacromolecules 7 (2006), pp. 1389–1392 Q.A.M. Al-Khafaji, M. Harris, S. Tombelli, S. Laschi, A.P.F. Turner, M. Mascini, G.Marrazza, An electrochemical immunoassay for HER2 detection, Electroanalysis 24 (2012), pp. 735–742, http://dx.doi.org/10.1002/elan.20110050
Jo
8.
12
Journal Pre-proof
Jo
ur
na
lP
re
-p
ro
of
29. M. Shamsipur, M. Emami, L. Farzin, R. Saber, A sandwich-type electrochemical immunosensor based on in situ silver deposition for determination of serum level of HER2 in breast cancer patients. Biosensors and Bioelectronics, 103 (2018), pp. 54-61. 30. E. Arkan, R. Saber, Z. Karimi, M. Shamsipur, A novel antibody–antigen based impedimetric immunosensor for low level detection of HER2 in serum samples of breast cancer patients via modification of a gold nanoparticles decorated multiwall carbon nanotube-ionic liquid electrode. Analytica chimica acta, 874 (2015), pp. 66-74. 31. S.A.A. Ahmad, M.S. Zaini, M.A. Kamarudin, An Electrochemical Sandwich Immunosensor for the Detection of HER2 using Antibody-Conjugated PbS Quantum Dot as a label. Journal of Pharmaceutical and Biomedical Analysis (2019), pp.
13
Journal Pre-proof CRediT author statement
Title: Cerium Oxide-Monoclonal Antibody Bioconjugate for Electrochemical Immunosensing of HER2 as a Breast Cancer Biomarker
Jo
ur
na
lP
re
-p
ro
of
Yeni Wahyuni Hartati: Conceptualization, Methodology, Supervision, Funding acquisition, Leonard Kristofel Letelay: Visualization, Data curation, Writing- Original draft preparation, Shabarni Gaffar: Supervision , Visualization, Investigation, Santhy Wyantuti: Project administration, Validation, Husein H. Bahti: Writing- Reviewing and Editing, Funding acquisition.
14