Development of highly sensitive electrochemical immunosensor based on single-walled carbon nanotube modified screen-printed carbon electrode

Materials Chemistry and Physics 227 (2019) 123–129

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Materials Chemistry and Physics journal homepage: www.elsevier.com/locate/matchemphys

Development of highly sensitive electrochemical immunosensor based on single-walled carbon nanotube modified screen-printed carbon electrode

T

Nguyen Xuan Vieta,b,∗, Nguyen Xuan Hoana, Yuzuru Takamurab a b

Faculty of Chemistry, VNU University of Science, Hanoi, 19 Le Thanh Tong, Hoan Kiem, Hanoi, Viet Nam School of Materials Science, Japan Advanced Institute of Science and Technology (JAIST), 1-1 Asahidai, Nomi City, Ishikawa, 923-1292, Japan

HIGHLIGHTS

GRAPHICAL ABSTRACT

coated material suppresses non• The specific binding on the surface of SWCNT.

fabrication, cost-effective, • Easy reliability to prepare the electro-

and

chemical immunosensor.

the sensitivity to hCG detec• Improve tion on the Oxi-SWCNT modified SPCE.

limit of detection was found to be • The 5.0 pg/mL for linear range 10–1000 pg/mL with sample volume usage as low as 2 μL.

ARTICLE INFO

ABSTRACT

Keywords: Oxi-SWCNT Screen-printed carbon electrode Electrochemical immunosensor hCG Non-specific binding

An electrochemical immunosensors have great potential for use in medical applications because of their high sensitivity and ease of measurement. However, the main challenge associated practical use of electrochemical immunosensors is an achievement of high sensitivity, reliable and reproducible fabrication on a low-cost platform. In this report, a sensitive electrochemical immunosensor has been developed for the detection of hCG, a pregnancy marker, that is based on a single-walled carbon nanotube (SWCNT) modified screen-printed carbon electrode. This electrochemical immunosensor is a sandwich-type immunoassay, where the gold-linked with the second antibody (Au-Mab-hCG) used as a label. The differential pulse voltammetry was employed to measure the signal current response obtained from dissolved Au-Mab-hCG. The signal amplification strategy-using gold nanoparticles as bio-tracker and SWCNT enhanced electron transfer nearly twice comparing with bare SPCE. The performance of the electrochemical immunosensor was also improved by the use of the coated materials in preventing non-specific binding. The developed electrochemical immunosensor shows the linear range of the hCG concentration from 10 pg/mL to 1000 pg/mL, the limit of detection of 5 pg/mL with the sample volume usage as low as 2 μL.

1. Introduction An immunosensor for the detection of antigen-antibody reactions

∗

has a broad scope of applications in numerous fields such as the clinical diagnosis [1,2], food industry [3,4], and environmental monitoring [5]. As the development of biosensors continues, the demand for higher

Corresponding author. Faculty of Chemistry, VNU University of Science, Hanoi, 19 Le Thanh Tong, Hoan Kiem, Hanoi, Viet Nam. E-mail address: [email protected] (N.X. Viet).

https://doi.org/10.1016/j.matchemphys.2019.01.068 Received 1 August 2018; Received in revised form 27 December 2018; Accepted 26 January 2019 Available online 01 February 2019 0254-0584/ © 2019 Elsevier B.V. All rights reserved.

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specificity and sensitivity, low-cost, rapid response, and easy handling keep growing and pushing the researchers to achieve single molecule detection level [6]. In comparison with optic-based methods, which require complex arrangement and expensive instruments, the electrochemical-based techniques have attractive advances such as high sensitivity, simplicity in experimental procedure, fast, low-cost and ease of miniaturization that enable to develop smart portable electrochemical sensors [7,8]. Carbon nanotubes (CNTs) possess a high aspect ratio, high electrical conductivity, excellent chemical stability, and extremely high mechanical strength and modulus [9,10]. These unique properties of CNTs and its large surface area have attracted much attention in electrochemistry and chemical sensors/biosensors [11]. In addition, due to the natural versatility of carbon surface, many works using CNTs for performance-enhanced biosensors have been conducted. For instance, the sidewall of SWCNT was functionalized with polyethylene oxide (PEO) chain to enable the selective recognition and binding of target proteins by conjugation of their specific receptors to PEO-functionalized nanotubes [12]. A new glucose sensor was developed via direct electron transfer between glucose oxidase and boron-doped CNTs [13]. Carboxylated multi-walled carbon nanotube (MWCNT) was used as a platform to obtain highly sensitive detection of aflatoxin-B1 via electrochemical technique [14]. The surface of MWCNT was used as a platform for monitoring the oxidation signal of guanine to detect miRNA in a sensitive electrochemical biosensor [15]. In most works, CNTs sidewall functionalization is essential to soluble nanotubes [16], or self-assembly on the platform's surfaces [17]. However, the biosensors based on CNTs, the non-specific adsorption (NSB) of protein on the surface of CNTs is not desirable, especially when tested in real bio-fluid samples that contain many co-existing proteins [12]. The NSB directly affects the selectivity and sensitivity of the devices. More complicated sensors, therefore, need to overcome issues like the signal on target enhancement, reduce of undesired interferences, and stable operation v.v. Fine-tuning of protein adsorption at the solid/liquid interface is essential in developing high-performance biosensor [18]. There are a few types of research from the viewpoint of selectivity enhancement or minimizing the NSB on SWCNT [12,19–22]. Besides, many promises SWCNT-based biosensor devices required chemical vapor deposition method and clean-room facilities for microfabrication process, which would lead the limitation of practical use due to its complicated procedure and high cost [9,21,23]. In order to realize wide application possibilities for a biosensor, it is necessary to develop cost-effective, disposable, highly sensitive, and reproducible devices. Screen printing technology has a strong advantage to create a large number of near-identical electrodes that can be used in a single shot context at a low cost [2]. The establishment of a simple and quick method for fabricating immunosensor onto a screenprinted electrode surface would contribute as a new approach to biosensor applications [24]. In this report, SWCNT was integrated onto screen-printed carbon electrode (SPCE), a compact three-electrode system that is disposable, low cost and easy to modify [25], to develop a sandwich-type electrochemical immunosensor for highly sensitive detection of the biomarker molecule hCG, used as a model of detection. Moreover, the coated materials were explored to suppress NSB of protein on the surface of SWCNT, while remaining selective and specific binding between antibody-antigen in the immunosensor. This coated layer contributed to improving the performance of the electrochemical immunosensor.

Fig. 1. a) Electrochemical measurement setup; b) a photo of a screen-printed carbon electrode.

constant of 4.9 × 10−9 M−1 was purchased from Medix Biochemica (Finland). The molecular weight of the recombinant human chorionic gonadotropin (hCG) was determined as 57.1 kDa using SDS-PAGE (Rohto Pharmaceutical Co., Ltd., Japan). Au nanoparticles with 40 nm were purchased from British Biocell International Ltd., (Cardiff, UK). HCl, NaH2PO4.2H2O, polyethylene glycol (PEG), KH2PO4 and dimethyl sulfoxide (DMSO) were purchased from Wako Pure Chemical Industries (Japan). 1-pyrenebutanoic acid succinimidyl ester purchased from Life Technologies Corporation, Japan. 1-pyrenebutanol, ethanolamine, bovine serum albumin (BSA) purchased from Sigma-Aldrich, Japan. Other reagents were of analytical grade, and all solutions were prepared and diluted using de-ionized water (18.2 MΩ cm) from the Milli-Q system (Millipore, USA). 2.2. Instruments Scanning electron microscopy (SEM) images were obtained using Hitachi S-4100 with accelerating voltage 20 kV. Electrochemical measurements were performed on ALS/CH Instruments electrochemical analyzer, model 730C (USA), in which SPCE was used as a three-electrode system (Fig. 1). 2.3. Adsorption of protein on SWCNT 2.3.1. Oxidized single-walled carbon nanotube SWCNT was oxidized and shorted by sonication in the solution of concentrated HNO3eH2SO4 3:1 (v/v) for 24 h at room temperature (RT). Then, the dispersion was diluted with de-ionized water and filtered through the 0.2 μm cellulose membrane. This product was washed by de-ionized water until pH reached neutral. The Oxi-SWCNT was dried in a vacuum oven at 60 °C for overnight and re-dispersed in deionized water or other solvents. 2.3.2. SWCNT treated with coated materials Bare-SWCNT (un-oxidized SWCNT) were immersed in a solution of 6 mM 1-pyrenebutanol/DMSO (dimethyl sulfoxide) or solution of 6 mM 1-pyrenebutanoic acid succinimidyl ester and 40 mM ethanolamine in DMSO and sonicated 1 h in an ice bath. Then, they were left standing for 24 h at RT. These mixtures were centrifugated and washed several times with de-ionized water to remove excess amount of coated materials. Finally, SWCNT was re-suspended in de-ionized water at a concentration of 300 μg/mL. The treatment of Oxi-SWCNT with coated materials was the same manner as Bare-SWCNT.

2. Experimental 2.1. Reagents Monoclonal anti-human α-subunit of follicle-stimulating hormone (Mab-FSH) with an affinity constant of 2.8 × 10−9 M−1 and monoclonal anti-human chorionic gonadotropin (Mab-hCG) with an affinity

2.3.3. Adsorption of bovine serum albumin onto SWCNT Bovine serum albumin (BSA) was selected as a model protein to 124

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quantify physical adsorption onto SWCNT. A sample of targeted SWCNT was suspended in de-ionized water to a concentration of 300 μg/mL then sonicated for 30 min to obtain a uniform dispersion. The SWCNT sample was then dispensed at an equal volume of 100 μL into the different concentrations of BSA prepared in phosphate buffered saline (PBS), pH 7.4. The mixture was allowed to mix in a rotary shaker (180 rpm) at 4 °C for 24 h. Then the samples were centrifugated at rate 8000 g for 30 min. The supernatant was collected and analyzed for protein content using the DC protein assays (Bio-rad). The amount of BSA loaded onto SWCNT was deduced from a simple mass balance based on the difference in protein concentration before and after the SWCNT addition.

complex biological mixtures such as blood or serum [28]. The sensitivity of the immunosensors was improved by minimizing non-specific binding (NSB), which is usually caused by the labeled secondary antibody [29]. There are various ways to minimize NSB in immunosensors such as the use of coating and self-assembled monolayers of polymers [30]. Polyethylene glycol (PEG) was recognized as one of most effective and widely used as NSB suppression reagents. However, Shim et al. found that PEG (average MW 10,000) coverage on the SWCNT sidewall was not uniform, thus reduced the effect in preventing NSB. To overcome that the coadsorption of a surfactant, Triton X-100 or Triton X405, and PEG was used and exhibited highly effective in preventing NSB of streptavidin (∼60 kDa) on the surface of SWCNT, while streptavidin found nonspecifically adsorbed to as-grown SWCNT [21]. The mixture of 0.4% of saturated casein +0.05% Tween-20 in another approach was used as NSB reagents, which was evolved as a compromise for the 4 individual analytes [29]. In this studying, we used different coated materials such as 1-pyrenbutanol and product of 1-pyrenbutanoic acid succinimidyl ester, named compound 1, and ethanolamine to suppress protein adsorption on the surface of SWCNT. The pyrene group in the bifunctional compound, being highly aromatic, can irreversibly adsorb on the surface of SWCNT via π-π stacking and hydrophilic moiety prevent the adsorption of protein on SWCNT. Scheme 1 demonstrates the way coated materials preventing the adsorption of protein on the surface on SWCNT. Fig. 2 exhibits the effects of coated materials: 1-pyrenebutanol and mixture of 1-pyrenebutanoic acid succinimidyl ester and ethanolamine in reducing the adsorption of BSA on Bare-SWCNT and Oxi-SWCNT. The ‘loading amount of BSA/SWCNT’ and ‘adsorbed amount of BSA’ shown on the x-axis and y-axis of Fig. 2 describes initial ratio amount of BSA upon SWCNT, and the amount of BSA adsorbed on SWCNT in weight, respectively. The adsorption on SWCNT is attributed to the hydrophobic interaction between the hydrophobic domains of protein and SWCNT surface [12,31]. This adsorption was irreversible and efficient in a wide range of condition such as various temperatures, pH values, and ionic strengths [12,19]. Fig. 2 also shows the adsorbed amount of BSA increases when the loading amount of BSA/SWCNT increases. SWCNT treated with acid solution (Oxi-SWCNT) produced higher protein adsorption compared with non-treated SWCNT (BareSWCNT). This result is assumed due to the increase in the surface area of SWCNTs after acid treatment process [32]. The adsorbed amount of BSA on both SWCNTs seem become saturation when the loading amount was reaching from 1000 μg/mg.

2.4. Fabrication of electrochemical immunosensor with SWCNT modified SPCE 2.4.1. Modification of SPCE with SWCNT and sandwich-type electrochemical immunosensor Oxi-SWCNT was dispersed in de-ionized water at a concentration of 1.0 mg/mL. SPCE consisted of a carbon working electrode, a carbon counter electrode, and Ag/AgCl reference electrode (Fig. 1b). The total length of the SPCE was 11 mm, and the geometric working area was 2.64 mm2 [24]. SPCEs was purchased from Bio Device Technology Ltd (Ishikawa, Japan). The working electrode of SPCE was dropped with 3.0 μL of Oxi-SWCNT solution, then left to dry at RT. Then, the 2.5 μL freshly prepared of 0.1 mM 1-pyrenebutanoic acid succinimidyl ester in water was placed onto Oxi-SWCNT modified SPCE electrodes at RT and washed off after 30 min. The working electrodes were immediately followed by 3 h incubation with 2.5 μL of 200 μg mL−1 Mab-FSH (priab) in PBS solution at 37 °C. The electrodes were then washed in two steps, with 0.05% Tween 20 in PBS and only PBS 10 mM respectively. To deactivate reactive groups of bifunctional molecules and suppress nonspecific binding, 40 mM ethanolamine in PBS 10 mM was added onto the resulting electrodes and incubation for 1 h. Then, the electrodes were rinsed with PBS. These electrodes were stored at 4 °C until use. Antigen, hCG, was diluted in PBS solution containing 1.0% BSA to form different concentrations for detecting process. For the detection of antibody-antigen reaction, 2.0 μL of antigen solution was placed on the working electrode in 1 h at RT. After rinsing with PBS, 2.0 μL of AuMab-hCG (second antibody linked with gold nanoparticles) solution were applied onto the surface and incubated for 30 min at RT. Finally, the Oxi-SWCNT modified SPCE electrodes were rinsed by PBS. The procedure prepared Au-Mab-hCG was fully described in elsewhere [26]. 2.4.2. Electrochemical measurement The electrochemical measurement setup and a photo of SPCE are shown in Fig. 1. The direct redox reaction of Au nanoparticles label was performed in HCl 0.1 M solution (30 μL) covering the full three-electrode zone at RT (Fig. 1a). The pre-oxidation of Au nanoparticles was performed at a constant potential 1.2 V for the 40 s, immediately followed by DPV, while scanning the potential range from 1.0 V to 0.0 V with a step potential of 4.0 mV, a pulse amplitude of 50 mV, and a pulse period of 0.2 s. The potentials were recorded against the built-in Ag/ AgCl as the reference electrode [24,26,27].

(1)

The presence of 1-pyrenebutanoic acid succinimidyl ester and ethanolamine has more effective than 1-pyrenebutanol substance in

3. Results and discussion 3.1. Effect of coated materials on reducing of protein adsorption on the surface of SWCNT Protein immunosensors are promising tools that can potentially enable high throughput proteomic screening in areas such as diagnosis and drug discovery. A critical aspect in the development of protein immunosensors is the optimization of the platform's surface chemistry to achieve the high sensitivity required for detection of proteins in

Scheme 1. Demonstration of the way of coated materials prevents the adsorption of protein on the surface of SWCNT. 125

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pyrenebutanoic acid succinimidyl ester and ethanolamine after immersing in hCG Antibody linked Au nanoparticles, respectively. Fig. 3a clearly shows the adsorption of hCG Antibody labeled Au nanoparticles (bright points, indicated by black arrows) on the Bare-SWCNT. This observation is consistent with the adsorption of BSA on SWCNT previously mentioned and also similar with other proposed results on the adsorption of protein A or streptavidin on the surface of as-grown SWCNT [17,21]. On the other hand, Fig. 3b illustrates almost SWCNT free from the adsorption of hCG Antibody labeled Au nanoparticles. The coated layer used here has similar ability in preventing NSB of streptavidin or BSA on the surface of SWCNT as the coated layer of coadsorption of Triton X-100 and PEG [21] or Tween-20 treated SWCNT [35]. However, this coated layer used here is a nonsurfactant-based material, which facilitates the handling in a drop-casting step of preparing the electrochemical immunosensor. The result means the coated layer with hydrophilic and neutral charge has the substantial effect in depressing the NSB of hCG Antibody linked Au nanoparticles on the surface of SWCNT. Fig. 2. Effect of coated materials in depressing the adsorption of BSA on BareSWCNT and Oxi-SWCNT with 1-pyrenebutanol and mixture of 1-pyrenebutanoic acid succinimidyl ester and ethanolamine.

3.2. Highly sensitive electrochemical immunosensor As illustrated in SEM images (Fig. 4), the surface of SPCE is the high degree of roughness with particles and the small flake of graphite while a thin and uniform layer of an SWCNT is obtained on the surface of SPCE after modifying with Oxi-SWCNT. Most the SPCE's surface area is covered with SWCNT, although SWCNT exists in the small bundle rather than in the individual tube. The typical cyclic voltammograms of Bare-SPCE and Oxi-SWCNT modified SPCE in 2.5 mM K4[Fe(CN)6]/K3[Fe(CN)6] (1:1 M ratio) are shown in Fig. 5. The presence of Oxi-SWCNT on the surface of SPCE enhanced the electrochemical performance of SPCE by lower the peak potential separation of forward and backward curves in a redox couple, ΔEP = Epa - Epc. The ΔEP is 140 mV, and 116 mV on Bare-SPCE and SWCNT modified SPCE, respectively. The ΔEP = 116 mV on OxiSWCNT modified SPCE is quite far from the ideal value ΔEP = 59 mV on one electron exchange redox system [36], but it is the significant improvement in comparison with that on Bare-SPCE. The smaller in the value of ΔEP, the higher in electron driven on the surface of the electrode. Besides, Oxi-SWCNT modified SPCE also improved anode the current intensity at peak position to 13.8 μA from 9.25 μA while cathode current intensity to 14.05 μA from 12.0 μA on Bare-SPCE. The ratio of peak current intensity Ipa/Ipc≈1 suggests the perfect reversible redox reaction on the surface of Oxi-SWCNT modified SPCE [4]. The high electroactivity of Oxi-SWCNT modified SPCE might be explained by the following reasons: First, the presence of rich-edge plane on OxiSWCNT, which is the highest electrochemical activity among carbon materials [37,38]. Second, the treatment with a mixture of the strong acid of H2SO4/HNO3 introduced many active groups on the surface of SWCNT, i.e., eOH, eC=O, or eCOOH which were observed to increase electron transfer rate to inner sphere probe such as [Fe(CN)6]3-/4- [39]. Third, due to the unique electronic structures of the SWCNT, they can act as a promoter to enhance the electrochemical reaction [40,41] and also came from the increase of surface area after SWCNT oxidation in concentrate acid solution.

preventing the adsorption of BSA on both Bare-SWCNT and OxiSWCNT. 1-pyrenebutanol adsorbed on the surface of SWCNT through the π-π stacking between pyrene moiety and carbon nanotubes surface [12,17]. It will convert the hydrophobic surface of SWCNT into more hydrophilic through the eOH moiety of 1-pyrenebutanol. The hydrophilic and neutral charge (at PBS buffer, pH 7.4) of eOH moiety on 1pyrenebutanol will exhibit “hydrophilic repulsion” effect, which was reported on poly(ethylene oxide), PEO [18], thereby eliminating hydrophobic interactions with proteins [33]. Similar to the case of 1pyrenebutanol, the mixture of 1-pyrenebutanoic acid succinimidyl ester and ethanolamine also creates the hydrophilic layer on the surface of SWCNT, shown in Scheme 1. However, this layer having carbon chain is longer than that of 1-pyrenebutanol due to the reaction between ethanolamine and 1-pyrenebutanoic acid succinimidyl ester, illustrated in equation 1. Thus, the steric repulsion was also capable of further minimizing the adsorption of protein on the surface of SWCNT beside the effect of hydrophilic repulsion. To confirm the effect of coated materials in reducing the adsorption of protein on the surface of SWCNT, we have synthesized low-density SWCNT on the Si wafer by chemical vapor deposition (CVD) method as our previous procedure [9,34]. The obtained SWCNT were also treated with coated materials as the manner mentioned above and then immersed in the solution of second hCG antibody linked with 5 nm Au nanoparticles as the indicator in AFM observation. Fig. 3 shows AFM images of Bare-SWCNT and SWCNT coated with the mixture of 1-

Fig. 3. AFM images of a) Bare-SWCNT and b) SWCNT coated with a mixture of 1-pyrenebutanoic acid succinimidyl ester and ethanolamine were immersed in hCG antibody labeled Au in 2 h at RT.

Figure 4. a) SEM image of Bare-SPCE and b) Oxi-SWCNT modified SPCE. 126

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Scheme 2. Steps show the procedure to fabricate electrochemical immunosensor based on Oxi-SWCNT modified SPCE.

Fig. 5. Cyclic voltammogram of Bare-SCPE and Oxi-SWCNT modified SPCE in 2.5 mM K4[Fe(CN)6]/K3[Fe(CN)6] (1:1 M ratio) with KCl 0.1 M as supporting electrolyte, scan rate 50 mV/s.

immobilized immunosensor with various concentration of hCG (from 10 pg/mL to 1000 pg/mL) in HCl 0.1 M. The reduction signal of complex ion [AuCl4]- in DPV was observed approximately at +0.35 V .vs Ag/AgCl (Fig. 6a). This peak position is accorded with the results proposed in Refs. [26,27] and the peak current intensity increased in proportion with the increasing hCG concentrations (Fig. 6b). The calibration curves (n = 3) show the relationship of hCG concentration and current intensity on Oxi-SWCNT modified SPCE and Bare-SPCE are y (10−7A) = 9.4 × 10−3x + 3.31 and y (10−7A) = 5.1 × 10−3x + 2.19, respectively. The current intensity obtained with Oxi-SWCNT modified SPCE is nearly twice comparing with Bare-SPCE. The enhancement in current intensity might be assigned to a synergistic effect of the high electrochemical activity of Oxi-SWCNT and the efficiency of coated materials in preventing NSB on the surface of SWCNT. The limit of detection (LOD) determined from calibration on Oxi-SWCNT modified SPCE is 5.0 pg/mL. Table 1 summarizes the comparison of the feature of this developed electrochemical immunosensor with various proposed sensors for the determination of hCG. As can be observed, the LOD of this developed immunosensor is fifty times lower than that immunoassay with gold nanoparticles as label using open circuit potential measurement (280 pg/mL) [45], almost seven times lower than that on bare SPCE (36 pg/mL) [27], five times lower on immunochromatographic test strips with Au nanoparticles as signal-enhancement (25 pg/mL) [46], immunoassay based on MWCNT-chitosan matrix (30 pg/mL) [47], two order of LOD lower than that of amperometric technique based on PteAu alloy nanotube array (1200 pg/mL) [48]. This LOD is comparable with LOD of the electrochemical immunosensor based on gold-silicon carbide nanocomposites (4.2 pg/mL) [49]. However, this LOD is higher than that of electrochemical immunoassay based on micro-SWCNT electrodes (2.4 pg/mL) [26], and electrochemical immunosensor based on Au nanoparticles dotted carbon nanotube-graphene composite and functionalized mesoporous materials (0.26 pg/mL) [50]. Table 2 provides a comparison of the sample volumes, reagent usage, dynamic ranges, and LOD of a commercial ELISA Kit and this developed electrochemical immunosensor using gold nanoparticles as the label for the detection of hCG. The combination of SWCNT with disposable SPCE electrodes making this electrochemical immunosensor becomes compact design, high sensitivity, low-cost and user friendly. The LOD (5.0 pg/mL) of our electrochemical immunosensor for detection of hCG is much better than that of the commercially available kit. The sample volume and the reagent consumption are also considerably lower than that of the ELISA Kit and other methods (see Table 1). This considerable low volume usage of sample and reagents is important because most of the reagents employed in immunosensor, such as antibodies, aptamers, enzymes, and fluorescence labels are very expensive, and additionally, analytes such as blood from a neonate or spinal fluid are very precious commodities [42]. However, the dynamic

Another challenge accompanying the minimization of NSB in the fabrication of immunosensors is the effective immobilization of antibodies on the surface of SWCNT. This can be achieved by using either covalent or noncovalent method [42]. A simple adsorption approach can be used to form noncovalent protein-nanotube conjugates via hydrophobic interactions between the nanotubes and hydrophobic domains of the proteins [19]. O'Connor et al. reported a method of antibody adsorption onto SWCNT on the basal plane of graphite disk electrode by incubating anti-biotin antibody on SWCNT surface for 3 h at pH 7.2 [43]. However, there are inherent difficulties in attaching the antibodies to the SWCNT by noncovalent methods. The relative size of the several proteins (7–8 nm, human IgG, 150 kDa) compared to the diameter of SWCNTs (1–2 nm) may affect the binding and stability of the antibody adsorbed on CNT [44]. For example, proteins such as ferritin or fibrinogen (∼340 kDa) were found not to be able to directly adsorb on the as-grown SWCNT surface [17,21]. To address this issue, we firstly functionalized the sidewall of SWCNT with a bifunctional molecule, 1-pyrenbutanoic acid succinimidyl ester. The aromatic moiety of this bifunctional molecule irreversibly adsorbed on the surface of SWCNT as discussed above. This adsorption makes a fixation point for the bifunctional molecule on the surface of SWCNT while succinimidyl ester groups that are highly reactive to nucleophilic substitution by primary and secondary amines that exist in abundance on the surface of most proteins similar to the way that reacted with ethanolamine in equation (1) [17]. The use of bifunctional molecule not only enables the immobilization of pri-Ab (Mab-FSH) on the sidewall of SWCNT with specificity and efficiency but also is an effective way to preserve the structure of SWCNT and forms the proteins resistant layer to minimized the NSB as shown in earlier part. A scheme illustrating the principle of electrochemical immunosensor on Oxi-SWCNT modified SPCE is shown in Scheme 2. In the signal detection step of the electrochemical immunosensor, the concentration of hCG will be quantified via the signal of Au nanoparticles in the differential pulse voltammograms (DPV) technique (equation 3). The reaction of Au nanoparticles on DPV measurement was followed by two steps. First, Au nanoparticles were oxidized into complex ion [AuCl4]- in a pre-oxidation step, and then the complex ion [AuCl4]- is reduced into Au nanoparticles again on the DPV scan. Au + 4Cl- — > [AuCl4]- + 3e− (2) oxidation step at potential of +1.2 V [AuCl4]- + 3e− — > Au + 4Cl- (3) reduction occurs at potential of +0.35 V Fig. 6a shows the DPV curves obtained from Au-Mab-hCG 127

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Fig. 6. a) Differential pulse voltammograms (DPV) of the second antibody linked Au nanoparticles on Oxi-SWCNT modified SPCE in HCl 0.1 M. The concentration of hCG ranged from 10 pg/mL to 1000 pg/mL; b) calibration curves of electrochemical immunosensor using Oxi-SWCNT modified SPCE and bare SPCE.

range of this developed method is shorter than that of commercial hCG ELISA kit, but the low LOD and less complicated in preparation and measurement would enable this immunosensor to become a valuable choice for detection of biomarkers in an early stage of people with high potential cancers. In addition, this sandwich-type electrochemical immunosensor using Au nanoparticles as the label has several advantages over the use of enzyme as the label. In the case of enzyme-based detection systems, the electrode surface is covered with the immune-complexes and blocking agents; these biomolecules remain on the surface during electrochemical measurement and may disturb the performance of the electrode. In our method, the pre-oxidation of Au nanoparticles at a high potential and the denaturation of the biomolecules in highly acidic conditions were carried out simultaneously. Thus, the detachment of possible blocking molecules from the surface provided a sizeable electroactive area for oxidized Au ions to be reduced again efficiently during the DPV scan. Besides, the loss of oxidized Au ions by diffusion was avoided because of the negative charge of the chelated compounds with the high concentration of chloride ions in the acidic electrolyte. The constant application of highly positive voltage rapidly attracted negatively charged Au chelates and promoted their electrodeposition on the carbon surface [27].

Table 2 Comparison of Sample volume, Reagent usage, Dynamic range, and LOD of this developed immunosensor with commercial ELISA Kit for the detection of hCG.

Sample volume Usage of reagents Dynamic range Limit of detection

Electrochemical immunosensor in this study

Commercial hCG alpha Human ELISA Kit (Invitrogen)

2.0 μL 7.0–10.0 μL 10–1000 pg/mL 5.0 pg/mL

50–100 μL 50–100 μL 54.87–40,000 pg/mL 50.0 pg/mL

nanoparticles liked anti-body as the label. The coated materials with hydrophilic properties and neutral charge at pH 7.4 showed the high efficiency in reducing the adsorption of NSB on the surface of SWCNT. The longer in carbon chain of coated material resulted in better effect in the resistance to BSA adsorbed on the surface of SWCNT. The synergistic effect of coated materials and SWCNT modified SPCE have contributed to the enhancement of the current intensity of electrochemical immunosensor nearly twice compared to Bare-SPCE, and the LOD was 5.0 pg/mL with sample volume usage as low as 2 μL. This is a reliable technique to fabricate the high sensitivity, compact design, and low-cost electrochemical immunosensor, which becomes a potential candidate in the lack resource place to detect early stage of cancers via their biomarker or prognosis the effect of therapy process on the cancer patients.

4. Conclusion We developed a method to enhance the sensitivity of electrochemical immunosensor based on SWCNT modified SPCE with Au

Table 1 Comparison of the feature of this developed immunosensor and proposed sensors for the determination of hCG. Method

Electrode material

Dynamic range (pg/mL)

LOD (pg/mL)

Sample volume (μL)

Ref

DPV OCP DPV Amperometry DPV DPV CAb Amperometry Color

SWCNT/SPCE SPCE SPCE Pt − Au alloy nanotube array Gold–silicon carbide nanocomposites Au/MWCNTs/GSa/GCE Gold nanotubes array/GCE MWCNT/Chitosan/GCE High-flow Nitrocellulose membrane

10–1000 50–10,000 0–2000 2500–40,000 10–55,000 0.5–50,000 10–10,000 80–50,000 –

5 280 36 1200 4.2 0.26 8 30 25

2.0 2.0 2.0 500 > 500 – > 500 – 10

This work [45] [27] [48] [49] [50] [51] [47] [46]

CA: Chronoamperometry. DPV: Differential pulse voltammetry. OCP: Open circuit potential. SPCE: Screen-printed carbon electrode. GCE: Glassy carbon electrode. a - Graphene nanosheets. b - Resonance scattering assay. 128

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Acknowledgments [26]

This research is funded by Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number 103.99-2016.38.

[27]

References

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