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Ag NPs composites for electrochemical immunoassay of prostate-specific antigen
Author’s Accepted Manuscript Enhanced conductivity of rGO/Ag NPs composites for electrochemical immunoassay of prostatespecific antigen Lu Han, Cheng-Mei Liu, Shi-Lei Dong, Cai-Xia Du, Xiao-Yong Zhang, Lu-Hai Li, Yen Wei www.elsevier.com/locate/bios
PII: DOI: Reference:
S0956-5663(16)30763-1 http://dx.doi.org/10.1016/j.bios.2016.08.004 BIOS9001
To appear in: Biosensors and Bioelectronic Received date: 8 May 2016 Revised date: 25 July 2016 Accepted date: 2 August 2016 Cite this article as: Lu Han, Cheng-Mei Liu, Shi-Lei Dong, Cai-Xia Du, XiaoYong Zhang, Lu-Hai Li and Yen Wei, Enhanced conductivity of rGO/Ag NPs composites for electrochemical immunoassay of prostate-specific antigen, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2016.08.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Enhanced conductivity of rGO/Ag NPs composites for electrochemical immunoassay of prostate-specific antigen
Lu Han a, Cheng-Mei Liu a, Shi-Lei Dong a, Cai-Xia Du a, Xiao-Yong Zhang b, Lu-Hai Li a, **, Yen Wei a,b,* a Beijing Engineering Research Center of Printed Electronics, Beijing Institute of Graphic Communication, Beijing 102600, PR China
b Department of Chemistry and Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education), Tsinghua University, Beijing 100084, PR China
* Corresponding author. Tel.: +86 10 62772674; Fax: +86 10 62771149.
** Corresponding author. Tel.: +86 10 60261064; fax: +86 10 60261108.
E-mail addresses: [email protected] (L.H. Li), [email protected] (Y. Wei).
Abstract Electrode materials play a vital role in the development of electrochemical immunosensors (EIs), particularly of label-free EIs. In this study, composites containing reduced graphene oxide with silver nanoparticles (rGO/Ag NPs) were synthesized using binary reductants, i.e. hydrazine hydrate and sodium citrate. Due to the fact that graphene oxide (GO) was fully restored to rGO, and rGO stacking was effectively inhibited by insertion of small Ag NPs between the graphene sheets, the electrical conductivity of rGO/Ag NPs composites was significantly improved compared to rGO alone, with an enhancement factor of 346% at 40 wt% of rGO. Moreover, the conducting path between rGO and Ag NPs formed because the structural defects in rGO were effectively repaired by decoration with Ag NPs. Subsequently, based on a screen-printed three-electrode system, a label-free EI for detecting prostate-specific antigen (PSA) was constructed using rGO/Ag NPs composites as a support material. The fabricated EIs demonstrated a wide linear response range (1.0 to 1000 ng/ml), low detection limit (0.01 ng/ml) and excellent specificity, reproducibility and stability. Thus, the proposed EIs based on rGO/Ag NPs composites can be easily extended for the ultrasensitive detection of different protein biomarkers.
Keywords: rGO/Ag NPs composites; enhanced conductivity; label-free immunosensor; prostate-specific antigen (PSA)
1. Introduction Electrochemical immunosensors (EIs) have aroused much interest due to their high specificity and sensitivity, as well as the possibility that they can be miniaturized for in vivo analyses (Burcu Bahadır and Kemal Sezgintürk 2015). Thus, EIs offer tremendous potential for application in point-of-care (POC) diagnostics for early detection of diseases. The realization of POC testing requires that EIs possess features including simple operation, fast detection, low cost and low detection limit (LOD) for disease biomarkers (Wan et al. 2013). In contrast to sandwich-type EIs, label-free EIs have more advantages in POC testing, owing to the fact that they directly detect antibody-antigen binding, instead of time-consuming, costly labeling processes (Jang et al. 2015; Li et al. 2011). In order to fabricate label-free EIs with lower LOD values, it is important to focus on the development of electrode materials, since they can provide a large specific surface area to immobilize more antibodies and superior electrical conductivity to accelerate electron transportation on the electrode surface. Graphene (Chen et al. 2016), carbon nanotubes (Prieto-Simón et al. 2015), conducting polymers (Barton 2008), metal nanoparticles (Wang et al. 2014) and their derived materials (Jang et al. 2015) have been widely applied as electrode materials to amplify the electrochemical signal from immunocomplexes. Graphene, a hexagonal monolayer network of sp2-hybridized carbon atoms, is a preferred support material for label-free EIs because of its large surface area, outstanding electronic and mechanical properties (Ball 2014). Furthermore, to reduce the manufacturing cost of EIs, liquid-phase exfoliation of pristine graphite is usually employed to produce graphene in high-yield after chemical reduction of graphene oxide (GO) (Nie et al. 2015). Nevertheless, during the synthetic process, oxides, i.e. epoxy bridges, hydroxyl groups and carboxyl groups, are unavoidably introduced on the surface of GO (Ciesielski and Samorì 2014). In addition to heteroatomic contamination, topographical defects remaining on graphene dramatically reduce the performance of graphene, especially its fast electron transport (Bourlinos et al. 2009; Lee et al. 2014). Therefore, graphene-based composites, such as graphene-gold nanoparticles composites (Jang et al. 2015), rGO-TEPA-PTC-NH2 and
AuPt-modified C60 bimetallic nanoclusters (Chen et al. 2016) and ternary hollow Pt/PdCu nanocubes anchored on graphene (Liu et al. 2016), were widely exploited to modify the working electrodes of EIs. However, despite these sophisticated signal amplification strategies with impressive LOD, these EIs may not be suitable for use in clinical applications owing to their use of noble metal nanoparticles and their complicated manufacturing procedures. Compared to the aforementioned metal nanoparticles, silver nanoparticles (Ag NPs), which have a very low price and superior conductivity, are undoubtedly the best option for reducing the cost of synthesizing graphene-based composites. However, the polymers that are usually used as stabilizers for Ag NPs, such as polyvinyl pyrrolidone (PVP) (Chen et al. 2013), glucose (Xu and Wang 2009) and vitamin C (Dinh et al. 2014), impeded electron transport between Ag NPs and graphene sheets, resulting in lower electroconductivity of rGO/Ag NPs composites. Furthermore, small Ag NPs are better able to improve the electrical properties of rGO than large Ag NPs (Dinh et al. 2014). Based on the above considerations, in our present study, we used a small molecule, i.e. sodium citrate, and a strong reducing agent, i.e. hydrazine, to obtain rGO/Ag NPs composites with enhanced electrical conductivity. In order to construct a simple, low-cost and label-free EIs, rGO/Ag NPs composites were successfully synthesized by one-pot reaction under mild conditions. The composites were then used to modify screen-printed carbon electrodes (SPCEs) to immobilize antibodies and enhance the electrochemical signals. Compared with rGO, the rGO/Ag NPs exhibited higher conductance due to the insertion of small Ag NPs into rGO sheets. Using PSA as a model biomarker, the electrochemical performance of the prepared EIs was thoroughly evaluated. The proposed EIs showed high sensitivity for PSA analysis with an LOD value of 0.01 ng/ml, and can also be applied to the measurement of other protein biomarkers.
2. Experimental 2.1 Materials Graphite flakes (～325 mesh), silver nitrate (AgNO3), sodium citrate, sodium nitrate
(NaNO3) and hydrazine hydrate were purchased from Alfa Aesar. PSA antibody was procured from Cell signaling technology (CST, MA, USA). Screen-printed three-electrode systems were obtained from DropSens (Llanera, Asturias, Spain). K3[Fe(CN)6], K4[Fe(CN)6] and bovine serum albumin (BSA) were received from Sigma-Aldrich (St.Louis., MO, USA). Vitamin C, glucose, hydrogen peroxide (H2O2) and all other chemicals were of analytical reagents grade and were obtained from Sinopharm Chemical Reagent Co., Ltd (Beijing, China). The ultrapure water used throughout all experiments was purified to 18.2 MΩ·cm with the ELGA system.
2.2 Preparation of rGO/Ag NPs composites The formation process of rGO/Ag NPs is illustrated in Scheme 1. GO was prepared by oxidizing graphite according to the Hummers method (Hummers Jr and Offeman 1958) with some modification. In brief, concentrated H2SO4 (46 ml) was added into a 500 ml flask filled with graphite flakes (1 g) and NaNO3 (1 g) at 0 ℃ with stirring for 1 h. Then KMnO4 (6 g) was slowly added while the temperature of the solution was kept below 20 ℃ by carefully controlling the rate of addition. The temperature was then increased to 35 ℃ for 1 h. Ultrapure water (280 ml) and 30% H2O2 (10 ml) were added dropwise with vigorous stirring at 95 ℃ for 0.5 h. The obtained yellowish-brown solution was centrifuged at 1000 rpm for 2 min, then at 8000 rpm for 15 min to collect the GO precipitate, which was then sequentially washed with 5% HCl, anhydrous ethanol and ultrapure water until the pH was near 7. The resulting GO precipitate was lyophilized at -50 ℃ for 12 h. GO (25 mg) in ultrapure water (50 ml) was treated with ultrasound sonication for 1.5 h. AgNO3 and sodium citrate were added in sequence at 95 ℃ with stirring for 1 h. Then hydrazine hydrate (28 mg) was added to the mixture solution at 95 ℃ for 4 h. The resulting rGO/Ag NPs composites were washed with ethanol and ultrapure water by centrifugation. The final precipitate was lyophilized at -50 ℃ for 12 h. The sizes of the Ag NPs on the surface of rGO were varied by changing the amount of AgNO3 (10, 25 and 250 mg), and the resulting composites were labeled as rGO/Ag NPs(a)-(c), respectively. For the synthesis of rGO, the method was identical to that for rGO/Ag NPs, only without addition of AgNO3.
2.3 Fabrication of a label-free immunosensor based on rGO/Ag NPs
Scheme.1 Preparing process of rGO and rGO/Ag NPs and schematic illustration of fabricated electrochemical immunosensor for PSA detection
Scheme.1 shows the fabrication procedure of the label-free immunosensor based on rGO/Ag NPs composites using a three-electrode system (DropSens, Spain) containing an SPCEs (Φ=4 mm) as the working electrode, and carbon and silver electrodes as the counter and reference electrodes, respectively. First, 8 μl of rGO/Ag NPs solution at different concentrations was deposited onto the SPCEs. After the rGO/Ag NPs-coated working electrode was infrared-dried for 2 h and washed with PBS buffer, it was incubated in anti-PSA antibody solution at a dilution of 1:1000 (v/v) for 1 h at room temperature and then overnight at 4℃. Anti-PSA was then immobilized onto the surface of the rGO/Ag NPs through the Ag-N coordinate bonds between the Ag atoms in the NPs and the available NH2 groups of the antibody. Subsequently, after washing, the electrodes were incubated in 1wt.% BSA solution for 1 h to eliminate nonspecific binding between PSA and the electrode surface. PSA solution at various concentrations was then dropped onto the electrode surface and incubated for 1 h at room temperature. After that, the working electrode was washed extensively to remove unbounded PSA molecules. Finally, the screen-printed three-electrode
system was ready for electrochemical measurement.
2.4 Electrochemical measurements The electrical properties of rGO and rGO/Ag NPs composites were thoroughly investigated. Specifically, pastes were firstly prepared by dispersing lyophilized powders of rGO and rGO/Ag NPs in DMF, to obtain pre-determined concentrations of solids by weight. For measurement of their electrical conductivity, the pastes were evenly coated on the surface of glass slides using a 0.9 mm diameter capillary. The surface resistance and the thickness of the conductive patterns fabricated with rGO or rGO/Ag NPs pastes were detected with a four-point probe resistivity measurement system (RTS-9, Guangzhou, China) and a Surface Profiler (Dektak 150, Bruker, USA). The conductivity κ is calculated according to the following equation:
1 ＲＳ·Ｗ / 1 0
Where Rs and W represent the surface resistance (Ω/□) and the thickness of the coated samples (mm), respectively. The electrochemical properties of all the samples were studied using electrochemical workstation (PGZ 402, VoltaLab, France) with a three-electrode configuration. 0.8 μl of 1 mg/ml rGO or rGO/Ag NPs was spread on the working area of SPECs using a micropipette and the film was dried at room temperature. All the cyclic voltammetry (CV) measurements were recorded in 0.1 M phosphate buffer (PBS, pH 7.4) containing 5 mM K3[Fe(CN)6] and K4[Fe(CN)6] in the potential range of -200 to +400 mV. A reproducible voltammogram was obtained under steady-state conditions after about three cycles. All the electrochemical impedance spectroscopy (EIS) measurements were carried out in 0.01 M PBS containing 5 mM K3[Fe(CN)6] and K4[Fe(CN)6] in a frequency range from 0.1 to 104 Hz at 220 mV.
3. Results and discussion
3.1 Characteristics of rGO and rGO/Ag NPs composites
Fig.1 TEM images of GO (A), rGO (B), rGO/Ag NPs (C, D and E)
The morphology and structure of rGO/Ag NPs were carefully characterized by a number of analytical methods. TEM was used to observe the appearance of the as-synthesized GO, rGO and rGO/Ag NPs composites. Fig.1A shows a typical TEM image of GO, which was fully exfoliated into individual sheets with corrugated surfaces. After sufficient reduction, the obtained rGO formed aggregats due to van der Waals forces, and this aggregation was also observed in rGO dispersion (Fig.S1B). As seen in the enlarged image of Fig. 1B, the agglomerated rGO sheets were composed of around 5 or 6 layers. However, it has been reported that high-quality rGO monolayers can be obtained after decoration with Ag NPs, and this is thought to be because Ag NPs in the composites function as spacers between the adjacent rGO sheets, preventing their aggregation (Salgado et al. 2012). As shown in Figure.1 C, D and E, the rGO sheets were almost transparent and could hardly be distinguished from the carbon-supported films on the copper grid. This illustrated that, rGO formed monolayers by combing with Ag NPs. In Figure.1C-E, we can see that the anchored Ag NPs are distributed uniformly on surface of the rGO sheets and few Ag NPs are scattered out of the
carbon sheet. This indicates that there is a strong interaction between Ag NPs and the rGO supports, and this can be explained by the introduction of the residual epoxy, hydroxyl and carboxylic moieties on the surface of rGO during the one-step synthesis process, which offer a sufficient number of chemically active sites for deposition of Ag NPs (Lim et al. 2013). In addition, the size of the Ag NPs can be effectively controlled between 16.6 and 38.5 nm by increasing the input amount of AgNO3. The functional groups of the as-obtained GO, rGO and rGO/Ag NPs(a) were confirmed by FT-IR spectroscopy (Fig. S2A). The IR spectrum of rGO/Ag NPs demonstrated that Ag NPs may interact with rGO sheets through physical absorption including electrostatic interaction and charge-transfer interaction. In addition, the crystalline nature and phase purity of raw graphite, GO, rGO and rGO/Ag NPs(a) were further characterized by XRD (Fig.S2B). The interlayer d-spacing of rGO sheets was 0.37 nm, much smaller than that of GO (d=0.82 nm), illustrating that rGO sheets had aggregated due to the strong Van der Waals forces. However, the characteristic peak of rGO/Ag NPs shifted to 23.06° with d-spacing of 0.39 nm, which is higher than the 0.37 nm d-spacing of rGO. This suggests that the insertion and uniform distribution of Ag NPs on rGO prevents restacking between rGO sheets (Lin-jun et al. 2012; Zhai et al. 2012).
3.2 Electrical properties of rGO/Ag NPs composites
Fig.2 (A) Comparison of the conductivity of rGO and rGO/Ag NPs(a) pastes with the same contents of rGO; (B) Electrical conductivity of rGO/Ag NPs pastes as a function of the Ag NPs content.
To clarify whether decoration of rGO with Ag NPs can indeed improve the electrical
conductivity of rGO, the four-probe technique with silver electrodes was used to measure the conductance of the as-synthesized rGO and rGO/Ag NPs. The results are plotted in Fig.2. The conductivity of rGO pastes with a content of 40% reached 7.9 S/cm, which was significantly superior to the conductance of rGO reported previously (Chen et al. 2013). Such high electrical conductivity of rGO pastes is mainly attributed to the sufficient reduction of GO to rGO by the binary reductants. In Fig.2A, the rGO/Ag NPs(a) paste showed more outstanding electrical properties than rGO, with an enhancement factor of 346% at 40 wt% of rGO, almost two times higher than that of 50 wt % Ag nanowires (NWs)/CVD graphene papers (191%) (Chen et al. 2013) and comparable to that of Ag NWs-doped graphene fibers (330%) (Xu et al. 2013). Furthermore, the conductivities of rGO/Ag NPs pastes increased dramatically as the Ag NPs fraction incrementally increased, and approached 35.5 S/cm at 59.6 wt% of Ag NPs (Fig.2B). These data demonstrated that the Ag NPs had an enhanced effect on the electrical properties of rGO. In order to further confirm the enhanced conductivity of rGO/Ag NPs due to insertion of Ag NPs, CV curves of the bare, GO-modified and rGO/Ag NPs-modified SPCEs were measured in 0.1 M PBS (pH 7.4) containing 5 mM K3[Fe(CN)6] and K4[Fe(CN)6] as electrolyte. A pair of symmetric and well-separated redox peaks was observed (Fig.3A), indicating that the measured electroconductivity was based on the redox mechanism. It was also noted that rGO/Ag NPs had another cathodic peak at -50 mV due to the redox of Ag NPs. In Fig.3A, rGO/SPCEs exhibited higher current density than bare SPCEs due to the accelerated electron transfer by rGO. The peak currents of the redox couple of rGO/Ag NPs/SPCEs continued to increase significantly, implying that immobilized Ag NPs enhanced the electron transfer on rGO sheets. Fig.3B shows the CV curves of rGO/Ag NPs electrodes at different scan rates ranging from 80 to 800 mV s-1. The results demonstrate that the anodic and cathodic peak currents were directly proportional to the square root of the scan rate (v1/2), suggesting that the rGO/Ag NPs electrode reactions were involved in the surface-confined charge transfer process.
Fig.3 (A) CV curves of SPCEs, rGO/ SPCEs and rGO/Ag NPs/SPCEs at the scan rate of 100 mV s-1 in 0.1 M PBS (pH 7.4) at RT. (B) CV of the rGO/Ag NPs electrode at different scan rates (from inner to outer): 80, 100, 200, 300, 400, 500, 600, 700 and 800 mV s-1. The inset shows the dependence of redox peak currents on the potential sweep rates. (C) Nyquist plots of SPCEs, rGO/SPCEs and rGO/Ag NPs/SPCEs in 0.01 M PBS containing 5×10-3 M K3[Fe(CN)6]/K4[Fe(CN)6].
EIS is an effective tool for monitoring changes in the surface features of the modified electrodes. The impedance spectra include a semicirclular portion and a linear portion, which correspond to the electron transfer process at higher frequencies and the electron diffusion process at lower frequencies, respectively. The semicircle diameter is equal to the electron-transfer resistance, Ret. Fig.3C shows the EIS of the electrodes at different stages. For the bare SPCEs, the redox process of Fe(CN)63-/ Fe(CN)64- probe showed a Ret value of 42.03 Ω·cm2. The rGO/SPCEs showed a lower resistance, which implied that the presence of rGO improved the electrical conductivity. After rGO was decorated with Ag NPs, the Ret value further decreased to 10.1 Ω·cm2, probably because the rGO/Ag NPs layers could generate a conductivity-increased surface that enhanced the Fe(CN)63-/ Fe(CN)64- probe to
access the modified rGO/Ag NPs layer. These EIS results were consistent with the CV curves, and confirm that electron transport on the rGO sheets was indeed strengthened by the immobilized Ag NPs. The in situ-generation of Ag NPs on rGO sheets significantly improved the conductivity of rGO, most likely because Ag NPs, as effective nanoscale spacers, increase the interlayer spacing (Fig.S2B), thus preventing face-to-face aggregation of rGO sheets. In addition, the thickness of the sodium citrate layer around the Ag NPs was theoretically calculated as approximately 6 Å (Giersig and Mulvaney 1993). Therefore, electrons can tunnel through this insulating barrier (Ruschau et al. 1992) and shuttle back and forth between rGO and Ag NPs. Importantly, Ag NPs can remove functional oxygen groups (Fig.S2A) and further repair the defects which are introduced during the oxidation process (Fig.S3A), hence providing conductive paths (Sundaram et al. 2008), as shown in the enlarged image in Scheme.1. Moreover, the embedded Ag NPs that are distributed at the edges of holes or vacancies in the rGO plane further decrease the contact resistance of graphene flakes (Liu et al. 2013). Therefore, rGO/Ag NPs could be potentially applied as an electrode material in the development of label-free EIs.
3.3 Electrochemical behaviors of the fabricated immunosensor based on rGO/Ag NPs composite The electrochemical behavior of rGO/Ag NPs-modified SPCEs was also determined using [Fe(CN)6]3-/4- as a model redox-active compound. As indicated in Fig.S4, a typical reversible electrochemical reaction occurred, in which the reaction rate is governed by the diffusion of electroactive species on the surface of a planar electrode (Holze 2002). This reversible behavior implied that no side reactions took place, and that the kinetics of electron transfer is rapid enough to maintain the surface concentration of redox-active species required by the Nernst equation. Subsequently, a label-free immunosensor for PSA detection was fabricated using rGO/Ag NPs composites as efficient substrate materials. The CV curves of ferricyanide act as an indicator to investigate the changes in SPCE behavior before and after each assembly step,
as shown in Fig.4A. We observed that the peak current of SPCEs was dramatically enhanced after modification with rGO/Ag NPs. However, there was a decrease in the peak current after the sequential addition of anti-PSA, BSA and PSA antigen (1 ng/ml), illustrating that the electron-transfer kinetics of the [Fe(CN)6]4-/[Fe(CN)6]3- probe was obviously reduced at the SPCE interface. This confirmed the successful immobilization of these substances. The addition of rGO/Ag NPs enhanced the response of the fabricated immunosensor and increased the peak current because rGO/Ag NPs serve as a conductor, which amplified the signals.
Fig.4 CVs (A) and corresponding impedance (B) of different components modified working electrodes in pH7.4 PBS containing 5 mM [Fe(CN)6]3-/4-. (C) Relationship between linear sweep stripping currents and PSA concentration, eleven measurements for each point. (D) Logarithmic calibration curve for PSA.
Fig.4B displays the corresponding electrochemical impedance spectroscopy (EIS) of the developed immunosensor. As observed, the bare SPCEs showed the maximum resistance. After the SPCEs were modified with rGO/Ag NPs composites, there was a distinct decrease in the Ret value, which can be attributed to the fact that rGO/Ag NPs greatly facilitate the interfacial electron transfer of the [Fe(CN)6]3-/4- probe. Nevertheless, the Ret values grow
steadily after sequential modification with anti-PSA, BSA and PSA, which can be ascribed to the successful immobilization of these components. The anodic peak current change (△i) was proportional to the PSA concentration because the electrochemical signal was inhibited when the antibody-antigen immunocomplex formed on the electrode surface. The calibration plot and the logarithmic calibration curve for PSA detection under optimum experimental conditions are illustrated in Fig.4C and Fig.4D. As expected, the current response decreased with increasing PSA concentration. There was a good linear relationship between the current change (△i) and the logarithm values of PSA concentrations in the range from 1.0 to 1000 ng/ml. The linear equation is 4.2552+1.5194x with a correlation coefficients of 0.9523. The limit of PSA detection was 0.01 ng/ml at a signal-to-noise ratio of 3. The serum PSA concentration of a normal person (＜4 ng/ml) and of a prostate cancer patient (＞10 ng/ml) fall into the linear range of this immunosensor. Thus, our fabricated immunosensor provides a novel method to quantify PSA concentration, indicating its potential for clinical application. The analytical performance of the proposed immunosensor in PSA detection was compared with previously reported electrochemical immunosensors based on graphene-metal composites, which are listed in Table.S1. It can be seen that our immunosensor displayed a superior detection limit and linear detection range for PSA, which may be attributed to the following factors. First, anti-PSA was immobilized on rGO/Ag NPs composites via Ag-N coordinate bonds instead of covalent linkage, thus greatly preserving the active sites of the anti-PSA. Secondly, as discussed earlier, the synthesized rGO/Ag NPs composites are highly conductive
electrode
materials.
Consequently,
even
though
the
anti-PSA/PSA
immunocomplexes blocked the electron transport, the electrochemical signal of Fe(CN)63-/ Fe(CN)64- was amplified, leading to the enhanced sensitivity of the proposed immunosensor.
3.4 Specificity, reproducibility and stability of the immunosensor In order to investigate the selectivity of the immunosensor based on rGO/Ag NPs, the immunosensor was incubated with PSA in the presence of interfering agents. 100 ng/ml of interfering substances including vitamin C, glucose and BSA were mixed with 1 ng/ml of PSA, respectively. As shown in Fig.5A, the current responses of the immunosensors exposed
to the interfering agents were all within 5 % of the immunosensor without interference, indicating that the specificity of the immunosensor was outstanding.
Fig.5 (A) The specificity study of the PSA immunosensor: (a) 1.0 ng/ml PSA; (b) 1.0 ng/ml PSA+100 ng/ml vitamin C; (c) 1.0 ng/ml PSA+100 ng/ml glucose; (d) 1.0 ng/ml PSA+100 ng/ml BSA. (B) The stability assay of the PSA immunosensor.
Reproducibility is also an important factor to be considered for biosensors. Therefore, we need to determine it to confirm the reliability of our developed immunosensor. By analysis of 1 ng/ml of PSA using five identically prepared BSA/anti-PSA/rGO-Ag NPs/SPCEs, the current variations of the immunosensor were thoroughly investigated (results not shown). The relative standard deviation (RSD) was 3.52%, less than 5%, illustrating that the immunosensor exhibited acceptable precision and reproducibility. Stability
is
another
essential
feature
of
immunosensors.
In
this
study,
BSA/anti-PSA/rGO-Ag NPs/SPCEs immunosensors were stored in pH 7.4 PBS at 4℃ and their current responses were examined every 7 days. After 14 days, the current response of the immunosensor decreased by only 8.73%. After 3 weeks, the current response attained 87.54% of its initial value, suggesting that the immunosensor shows good long-term stability. We presume that this stability can mainly be ascribed to the large surface area of rGO/Ag NPs and their supreme affinity for biomolecules as well as the carbon electrode surface.
4. Conclusions A label-free electrochemical immunosensor for PSA detection was fabricated using
rGO/Ag NPs composites as electrode materials on a screen-printed three-electrode system. The rGO/Ag NPs composites were synthesized using binary reductants, i.e. hydrazine hydrate and sodium citrate, at high yields by one-pot reaction under mild conditions. Moreover, the rGO/Ag NPs possessed superior electrical conductivity compared to rGO because the small Ag NPs, stabilized by sodium citrate were anchored onto the rGO sheets. The EIs demonstrated a wide linear response range (1.0 to 1000 ng/ml) and low detection limit (0.01 ng/ml). Furthermore, the as-prepared EIs also exhibited excellent specificity, reproducibility and stability. The proposed EIs provided a simple, low-cost method for PSA detection and have potential applications in clinical diagnosis of prostate cancer, which can be further extended to the ultrasensitive determination of other disease-related protein biomarkers.
5. Acknowledgements This work was financially supported by Chinese Natural Science Foundation project (No. 31300820), Beijing Municipal Commission of Education project (No.18190114/006), a project founded by Beijing Institute of Graphic Communication (No.27170115004/025), a foundation for raising the scientific research level of materials and technologies for printed electronics (No.Eb201530) and a grant from Beijing collaborative innovation for green printing and publication.
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Highlights: ■
rGO/Ag NPs composites with enhanced electrical conductivity were
successfully prepared using the binary reductants, namely hydrazine hydrate and sodium citrate. ■Using rGO/Ag NP composites as electrode materials, a low-cost label-free electrochemical immunosensor was easily constructed. ■ The proposed immunosensor exhibited the widest linear detection range (1.0-1000 ng/ml) and a low detection limit ( 0.01 ng/ml) for PSA.
PII: DOI: Reference:
S0956-5663(16)30763-1 http://dx.doi.org/10.1016/j.bios.2016.08.004 BIOS9001
To appear in: Biosensors and Bioelectronic Received date: 8 May 2016 Revised date: 25 July 2016 Accepted date: 2 August 2016 Cite this article as: Lu Han, Cheng-Mei Liu, Shi-Lei Dong, Cai-Xia Du, XiaoYong Zhang, Lu-Hai Li and Yen Wei, Enhanced conductivity of rGO/Ag NPs composites for electrochemical immunoassay of prostate-specific antigen, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2016.08.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Enhanced conductivity of rGO/Ag NPs composites for electrochemical immunoassay of prostate-specific antigen
Lu Han a, Cheng-Mei Liu a, Shi-Lei Dong a, Cai-Xia Du a, Xiao-Yong Zhang b, Lu-Hai Li a, **, Yen Wei a,b,* a Beijing Engineering Research Center of Printed Electronics, Beijing Institute of Graphic Communication, Beijing 102600, PR China
b Department of Chemistry and Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education), Tsinghua University, Beijing 100084, PR China
* Corresponding author. Tel.: +86 10 62772674; Fax: +86 10 62771149.
** Corresponding author. Tel.: +86 10 60261064; fax: +86 10 60261108.
E-mail addresses: [email protected] (L.H. Li), [email protected] (Y. Wei).
Abstract Electrode materials play a vital role in the development of electrochemical immunosensors (EIs), particularly of label-free EIs. In this study, composites containing reduced graphene oxide with silver nanoparticles (rGO/Ag NPs) were synthesized using binary reductants, i.e. hydrazine hydrate and sodium citrate. Due to the fact that graphene oxide (GO) was fully restored to rGO, and rGO stacking was effectively inhibited by insertion of small Ag NPs between the graphene sheets, the electrical conductivity of rGO/Ag NPs composites was significantly improved compared to rGO alone, with an enhancement factor of 346% at 40 wt% of rGO. Moreover, the conducting path between rGO and Ag NPs formed because the structural defects in rGO were effectively repaired by decoration with Ag NPs. Subsequently, based on a screen-printed three-electrode system, a label-free EI for detecting prostate-specific antigen (PSA) was constructed using rGO/Ag NPs composites as a support material. The fabricated EIs demonstrated a wide linear response range (1.0 to 1000 ng/ml), low detection limit (0.01 ng/ml) and excellent specificity, reproducibility and stability. Thus, the proposed EIs based on rGO/Ag NPs composites can be easily extended for the ultrasensitive detection of different protein biomarkers.
Keywords: rGO/Ag NPs composites; enhanced conductivity; label-free immunosensor; prostate-specific antigen (PSA)
1. Introduction Electrochemical immunosensors (EIs) have aroused much interest due to their high specificity and sensitivity, as well as the possibility that they can be miniaturized for in vivo analyses (Burcu Bahadır and Kemal Sezgintürk 2015). Thus, EIs offer tremendous potential for application in point-of-care (POC) diagnostics for early detection of diseases. The realization of POC testing requires that EIs possess features including simple operation, fast detection, low cost and low detection limit (LOD) for disease biomarkers (Wan et al. 2013). In contrast to sandwich-type EIs, label-free EIs have more advantages in POC testing, owing to the fact that they directly detect antibody-antigen binding, instead of time-consuming, costly labeling processes (Jang et al. 2015; Li et al. 2011). In order to fabricate label-free EIs with lower LOD values, it is important to focus on the development of electrode materials, since they can provide a large specific surface area to immobilize more antibodies and superior electrical conductivity to accelerate electron transportation on the electrode surface. Graphene (Chen et al. 2016), carbon nanotubes (Prieto-Simón et al. 2015), conducting polymers (Barton 2008), metal nanoparticles (Wang et al. 2014) and their derived materials (Jang et al. 2015) have been widely applied as electrode materials to amplify the electrochemical signal from immunocomplexes. Graphene, a hexagonal monolayer network of sp2-hybridized carbon atoms, is a preferred support material for label-free EIs because of its large surface area, outstanding electronic and mechanical properties (Ball 2014). Furthermore, to reduce the manufacturing cost of EIs, liquid-phase exfoliation of pristine graphite is usually employed to produce graphene in high-yield after chemical reduction of graphene oxide (GO) (Nie et al. 2015). Nevertheless, during the synthetic process, oxides, i.e. epoxy bridges, hydroxyl groups and carboxyl groups, are unavoidably introduced on the surface of GO (Ciesielski and Samorì 2014). In addition to heteroatomic contamination, topographical defects remaining on graphene dramatically reduce the performance of graphene, especially its fast electron transport (Bourlinos et al. 2009; Lee et al. 2014). Therefore, graphene-based composites, such as graphene-gold nanoparticles composites (Jang et al. 2015), rGO-TEPA-PTC-NH2 and
AuPt-modified C60 bimetallic nanoclusters (Chen et al. 2016) and ternary hollow Pt/PdCu nanocubes anchored on graphene (Liu et al. 2016), were widely exploited to modify the working electrodes of EIs. However, despite these sophisticated signal amplification strategies with impressive LOD, these EIs may not be suitable for use in clinical applications owing to their use of noble metal nanoparticles and their complicated manufacturing procedures. Compared to the aforementioned metal nanoparticles, silver nanoparticles (Ag NPs), which have a very low price and superior conductivity, are undoubtedly the best option for reducing the cost of synthesizing graphene-based composites. However, the polymers that are usually used as stabilizers for Ag NPs, such as polyvinyl pyrrolidone (PVP) (Chen et al. 2013), glucose (Xu and Wang 2009) and vitamin C (Dinh et al. 2014), impeded electron transport between Ag NPs and graphene sheets, resulting in lower electroconductivity of rGO/Ag NPs composites. Furthermore, small Ag NPs are better able to improve the electrical properties of rGO than large Ag NPs (Dinh et al. 2014). Based on the above considerations, in our present study, we used a small molecule, i.e. sodium citrate, and a strong reducing agent, i.e. hydrazine, to obtain rGO/Ag NPs composites with enhanced electrical conductivity. In order to construct a simple, low-cost and label-free EIs, rGO/Ag NPs composites were successfully synthesized by one-pot reaction under mild conditions. The composites were then used to modify screen-printed carbon electrodes (SPCEs) to immobilize antibodies and enhance the electrochemical signals. Compared with rGO, the rGO/Ag NPs exhibited higher conductance due to the insertion of small Ag NPs into rGO sheets. Using PSA as a model biomarker, the electrochemical performance of the prepared EIs was thoroughly evaluated. The proposed EIs showed high sensitivity for PSA analysis with an LOD value of 0.01 ng/ml, and can also be applied to the measurement of other protein biomarkers.
2. Experimental 2.1 Materials Graphite flakes (～325 mesh), silver nitrate (AgNO3), sodium citrate, sodium nitrate
(NaNO3) and hydrazine hydrate were purchased from Alfa Aesar. PSA antibody was procured from Cell signaling technology (CST, MA, USA). Screen-printed three-electrode systems were obtained from DropSens (Llanera, Asturias, Spain). K3[Fe(CN)6], K4[Fe(CN)6] and bovine serum albumin (BSA) were received from Sigma-Aldrich (St.Louis., MO, USA). Vitamin C, glucose, hydrogen peroxide (H2O2) and all other chemicals were of analytical reagents grade and were obtained from Sinopharm Chemical Reagent Co., Ltd (Beijing, China). The ultrapure water used throughout all experiments was purified to 18.2 MΩ·cm with the ELGA system.
2.2 Preparation of rGO/Ag NPs composites The formation process of rGO/Ag NPs is illustrated in Scheme 1. GO was prepared by oxidizing graphite according to the Hummers method (Hummers Jr and Offeman 1958) with some modification. In brief, concentrated H2SO4 (46 ml) was added into a 500 ml flask filled with graphite flakes (1 g) and NaNO3 (1 g) at 0 ℃ with stirring for 1 h. Then KMnO4 (6 g) was slowly added while the temperature of the solution was kept below 20 ℃ by carefully controlling the rate of addition. The temperature was then increased to 35 ℃ for 1 h. Ultrapure water (280 ml) and 30% H2O2 (10 ml) were added dropwise with vigorous stirring at 95 ℃ for 0.5 h. The obtained yellowish-brown solution was centrifuged at 1000 rpm for 2 min, then at 8000 rpm for 15 min to collect the GO precipitate, which was then sequentially washed with 5% HCl, anhydrous ethanol and ultrapure water until the pH was near 7. The resulting GO precipitate was lyophilized at -50 ℃ for 12 h. GO (25 mg) in ultrapure water (50 ml) was treated with ultrasound sonication for 1.5 h. AgNO3 and sodium citrate were added in sequence at 95 ℃ with stirring for 1 h. Then hydrazine hydrate (28 mg) was added to the mixture solution at 95 ℃ for 4 h. The resulting rGO/Ag NPs composites were washed with ethanol and ultrapure water by centrifugation. The final precipitate was lyophilized at -50 ℃ for 12 h. The sizes of the Ag NPs on the surface of rGO were varied by changing the amount of AgNO3 (10, 25 and 250 mg), and the resulting composites were labeled as rGO/Ag NPs(a)-(c), respectively. For the synthesis of rGO, the method was identical to that for rGO/Ag NPs, only without addition of AgNO3.
2.3 Fabrication of a label-free immunosensor based on rGO/Ag NPs
Scheme.1 Preparing process of rGO and rGO/Ag NPs and schematic illustration of fabricated electrochemical immunosensor for PSA detection
Scheme.1 shows the fabrication procedure of the label-free immunosensor based on rGO/Ag NPs composites using a three-electrode system (DropSens, Spain) containing an SPCEs (Φ=4 mm) as the working electrode, and carbon and silver electrodes as the counter and reference electrodes, respectively. First, 8 μl of rGO/Ag NPs solution at different concentrations was deposited onto the SPCEs. After the rGO/Ag NPs-coated working electrode was infrared-dried for 2 h and washed with PBS buffer, it was incubated in anti-PSA antibody solution at a dilution of 1:1000 (v/v) for 1 h at room temperature and then overnight at 4℃. Anti-PSA was then immobilized onto the surface of the rGO/Ag NPs through the Ag-N coordinate bonds between the Ag atoms in the NPs and the available NH2 groups of the antibody. Subsequently, after washing, the electrodes were incubated in 1wt.% BSA solution for 1 h to eliminate nonspecific binding between PSA and the electrode surface. PSA solution at various concentrations was then dropped onto the electrode surface and incubated for 1 h at room temperature. After that, the working electrode was washed extensively to remove unbounded PSA molecules. Finally, the screen-printed three-electrode
system was ready for electrochemical measurement.
2.4 Electrochemical measurements The electrical properties of rGO and rGO/Ag NPs composites were thoroughly investigated. Specifically, pastes were firstly prepared by dispersing lyophilized powders of rGO and rGO/Ag NPs in DMF, to obtain pre-determined concentrations of solids by weight. For measurement of their electrical conductivity, the pastes were evenly coated on the surface of glass slides using a 0.9 mm diameter capillary. The surface resistance and the thickness of the conductive patterns fabricated with rGO or rGO/Ag NPs pastes were detected with a four-point probe resistivity measurement system (RTS-9, Guangzhou, China) and a Surface Profiler (Dektak 150, Bruker, USA). The conductivity κ is calculated according to the following equation:
1 ＲＳ·Ｗ / 1 0
Where Rs and W represent the surface resistance (Ω/□) and the thickness of the coated samples (mm), respectively. The electrochemical properties of all the samples were studied using electrochemical workstation (PGZ 402, VoltaLab, France) with a three-electrode configuration. 0.8 μl of 1 mg/ml rGO or rGO/Ag NPs was spread on the working area of SPECs using a micropipette and the film was dried at room temperature. All the cyclic voltammetry (CV) measurements were recorded in 0.1 M phosphate buffer (PBS, pH 7.4) containing 5 mM K3[Fe(CN)6] and K4[Fe(CN)6] in the potential range of -200 to +400 mV. A reproducible voltammogram was obtained under steady-state conditions after about three cycles. All the electrochemical impedance spectroscopy (EIS) measurements were carried out in 0.01 M PBS containing 5 mM K3[Fe(CN)6] and K4[Fe(CN)6] in a frequency range from 0.1 to 104 Hz at 220 mV.
3. Results and discussion
3.1 Characteristics of rGO and rGO/Ag NPs composites
Fig.1 TEM images of GO (A), rGO (B), rGO/Ag NPs (C, D and E)
The morphology and structure of rGO/Ag NPs were carefully characterized by a number of analytical methods. TEM was used to observe the appearance of the as-synthesized GO, rGO and rGO/Ag NPs composites. Fig.1A shows a typical TEM image of GO, which was fully exfoliated into individual sheets with corrugated surfaces. After sufficient reduction, the obtained rGO formed aggregats due to van der Waals forces, and this aggregation was also observed in rGO dispersion (Fig.S1B). As seen in the enlarged image of Fig. 1B, the agglomerated rGO sheets were composed of around 5 or 6 layers. However, it has been reported that high-quality rGO monolayers can be obtained after decoration with Ag NPs, and this is thought to be because Ag NPs in the composites function as spacers between the adjacent rGO sheets, preventing their aggregation (Salgado et al. 2012). As shown in Figure.1 C, D and E, the rGO sheets were almost transparent and could hardly be distinguished from the carbon-supported films on the copper grid. This illustrated that, rGO formed monolayers by combing with Ag NPs. In Figure.1C-E, we can see that the anchored Ag NPs are distributed uniformly on surface of the rGO sheets and few Ag NPs are scattered out of the
carbon sheet. This indicates that there is a strong interaction between Ag NPs and the rGO supports, and this can be explained by the introduction of the residual epoxy, hydroxyl and carboxylic moieties on the surface of rGO during the one-step synthesis process, which offer a sufficient number of chemically active sites for deposition of Ag NPs (Lim et al. 2013). In addition, the size of the Ag NPs can be effectively controlled between 16.6 and 38.5 nm by increasing the input amount of AgNO3. The functional groups of the as-obtained GO, rGO and rGO/Ag NPs(a) were confirmed by FT-IR spectroscopy (Fig. S2A). The IR spectrum of rGO/Ag NPs demonstrated that Ag NPs may interact with rGO sheets through physical absorption including electrostatic interaction and charge-transfer interaction. In addition, the crystalline nature and phase purity of raw graphite, GO, rGO and rGO/Ag NPs(a) were further characterized by XRD (Fig.S2B). The interlayer d-spacing of rGO sheets was 0.37 nm, much smaller than that of GO (d=0.82 nm), illustrating that rGO sheets had aggregated due to the strong Van der Waals forces. However, the characteristic peak of rGO/Ag NPs shifted to 23.06° with d-spacing of 0.39 nm, which is higher than the 0.37 nm d-spacing of rGO. This suggests that the insertion and uniform distribution of Ag NPs on rGO prevents restacking between rGO sheets (Lin-jun et al. 2012; Zhai et al. 2012).
3.2 Electrical properties of rGO/Ag NPs composites
Fig.2 (A) Comparison of the conductivity of rGO and rGO/Ag NPs(a) pastes with the same contents of rGO; (B) Electrical conductivity of rGO/Ag NPs pastes as a function of the Ag NPs content.
To clarify whether decoration of rGO with Ag NPs can indeed improve the electrical
conductivity of rGO, the four-probe technique with silver electrodes was used to measure the conductance of the as-synthesized rGO and rGO/Ag NPs. The results are plotted in Fig.2. The conductivity of rGO pastes with a content of 40% reached 7.9 S/cm, which was significantly superior to the conductance of rGO reported previously (Chen et al. 2013). Such high electrical conductivity of rGO pastes is mainly attributed to the sufficient reduction of GO to rGO by the binary reductants. In Fig.2A, the rGO/Ag NPs(a) paste showed more outstanding electrical properties than rGO, with an enhancement factor of 346% at 40 wt% of rGO, almost two times higher than that of 50 wt % Ag nanowires (NWs)/CVD graphene papers (191%) (Chen et al. 2013) and comparable to that of Ag NWs-doped graphene fibers (330%) (Xu et al. 2013). Furthermore, the conductivities of rGO/Ag NPs pastes increased dramatically as the Ag NPs fraction incrementally increased, and approached 35.5 S/cm at 59.6 wt% of Ag NPs (Fig.2B). These data demonstrated that the Ag NPs had an enhanced effect on the electrical properties of rGO. In order to further confirm the enhanced conductivity of rGO/Ag NPs due to insertion of Ag NPs, CV curves of the bare, GO-modified and rGO/Ag NPs-modified SPCEs were measured in 0.1 M PBS (pH 7.4) containing 5 mM K3[Fe(CN)6] and K4[Fe(CN)6] as electrolyte. A pair of symmetric and well-separated redox peaks was observed (Fig.3A), indicating that the measured electroconductivity was based on the redox mechanism. It was also noted that rGO/Ag NPs had another cathodic peak at -50 mV due to the redox of Ag NPs. In Fig.3A, rGO/SPCEs exhibited higher current density than bare SPCEs due to the accelerated electron transfer by rGO. The peak currents of the redox couple of rGO/Ag NPs/SPCEs continued to increase significantly, implying that immobilized Ag NPs enhanced the electron transfer on rGO sheets. Fig.3B shows the CV curves of rGO/Ag NPs electrodes at different scan rates ranging from 80 to 800 mV s-1. The results demonstrate that the anodic and cathodic peak currents were directly proportional to the square root of the scan rate (v1/2), suggesting that the rGO/Ag NPs electrode reactions were involved in the surface-confined charge transfer process.
Fig.3 (A) CV curves of SPCEs, rGO/ SPCEs and rGO/Ag NPs/SPCEs at the scan rate of 100 mV s-1 in 0.1 M PBS (pH 7.4) at RT. (B) CV of the rGO/Ag NPs electrode at different scan rates (from inner to outer): 80, 100, 200, 300, 400, 500, 600, 700 and 800 mV s-1. The inset shows the dependence of redox peak currents on the potential sweep rates. (C) Nyquist plots of SPCEs, rGO/SPCEs and rGO/Ag NPs/SPCEs in 0.01 M PBS containing 5×10-3 M K3[Fe(CN)6]/K4[Fe(CN)6].
EIS is an effective tool for monitoring changes in the surface features of the modified electrodes. The impedance spectra include a semicirclular portion and a linear portion, which correspond to the electron transfer process at higher frequencies and the electron diffusion process at lower frequencies, respectively. The semicircle diameter is equal to the electron-transfer resistance, Ret. Fig.3C shows the EIS of the electrodes at different stages. For the bare SPCEs, the redox process of Fe(CN)63-/ Fe(CN)64- probe showed a Ret value of 42.03 Ω·cm2. The rGO/SPCEs showed a lower resistance, which implied that the presence of rGO improved the electrical conductivity. After rGO was decorated with Ag NPs, the Ret value further decreased to 10.1 Ω·cm2, probably because the rGO/Ag NPs layers could generate a conductivity-increased surface that enhanced the Fe(CN)63-/ Fe(CN)64- probe to
access the modified rGO/Ag NPs layer. These EIS results were consistent with the CV curves, and confirm that electron transport on the rGO sheets was indeed strengthened by the immobilized Ag NPs. The in situ-generation of Ag NPs on rGO sheets significantly improved the conductivity of rGO, most likely because Ag NPs, as effective nanoscale spacers, increase the interlayer spacing (Fig.S2B), thus preventing face-to-face aggregation of rGO sheets. In addition, the thickness of the sodium citrate layer around the Ag NPs was theoretically calculated as approximately 6 Å (Giersig and Mulvaney 1993). Therefore, electrons can tunnel through this insulating barrier (Ruschau et al. 1992) and shuttle back and forth between rGO and Ag NPs. Importantly, Ag NPs can remove functional oxygen groups (Fig.S2A) and further repair the defects which are introduced during the oxidation process (Fig.S3A), hence providing conductive paths (Sundaram et al. 2008), as shown in the enlarged image in Scheme.1. Moreover, the embedded Ag NPs that are distributed at the edges of holes or vacancies in the rGO plane further decrease the contact resistance of graphene flakes (Liu et al. 2013). Therefore, rGO/Ag NPs could be potentially applied as an electrode material in the development of label-free EIs.
3.3 Electrochemical behaviors of the fabricated immunosensor based on rGO/Ag NPs composite The electrochemical behavior of rGO/Ag NPs-modified SPCEs was also determined using [Fe(CN)6]3-/4- as a model redox-active compound. As indicated in Fig.S4, a typical reversible electrochemical reaction occurred, in which the reaction rate is governed by the diffusion of electroactive species on the surface of a planar electrode (Holze 2002). This reversible behavior implied that no side reactions took place, and that the kinetics of electron transfer is rapid enough to maintain the surface concentration of redox-active species required by the Nernst equation. Subsequently, a label-free immunosensor for PSA detection was fabricated using rGO/Ag NPs composites as efficient substrate materials. The CV curves of ferricyanide act as an indicator to investigate the changes in SPCE behavior before and after each assembly step,
as shown in Fig.4A. We observed that the peak current of SPCEs was dramatically enhanced after modification with rGO/Ag NPs. However, there was a decrease in the peak current after the sequential addition of anti-PSA, BSA and PSA antigen (1 ng/ml), illustrating that the electron-transfer kinetics of the [Fe(CN)6]4-/[Fe(CN)6]3- probe was obviously reduced at the SPCE interface. This confirmed the successful immobilization of these substances. The addition of rGO/Ag NPs enhanced the response of the fabricated immunosensor and increased the peak current because rGO/Ag NPs serve as a conductor, which amplified the signals.
Fig.4 CVs (A) and corresponding impedance (B) of different components modified working electrodes in pH7.4 PBS containing 5 mM [Fe(CN)6]3-/4-. (C) Relationship between linear sweep stripping currents and PSA concentration, eleven measurements for each point. (D) Logarithmic calibration curve for PSA.
Fig.4B displays the corresponding electrochemical impedance spectroscopy (EIS) of the developed immunosensor. As observed, the bare SPCEs showed the maximum resistance. After the SPCEs were modified with rGO/Ag NPs composites, there was a distinct decrease in the Ret value, which can be attributed to the fact that rGO/Ag NPs greatly facilitate the interfacial electron transfer of the [Fe(CN)6]3-/4- probe. Nevertheless, the Ret values grow
steadily after sequential modification with anti-PSA, BSA and PSA, which can be ascribed to the successful immobilization of these components. The anodic peak current change (△i) was proportional to the PSA concentration because the electrochemical signal was inhibited when the antibody-antigen immunocomplex formed on the electrode surface. The calibration plot and the logarithmic calibration curve for PSA detection under optimum experimental conditions are illustrated in Fig.4C and Fig.4D. As expected, the current response decreased with increasing PSA concentration. There was a good linear relationship between the current change (△i) and the logarithm values of PSA concentrations in the range from 1.0 to 1000 ng/ml. The linear equation is 4.2552+1.5194x with a correlation coefficients of 0.9523. The limit of PSA detection was 0.01 ng/ml at a signal-to-noise ratio of 3. The serum PSA concentration of a normal person (＜4 ng/ml) and of a prostate cancer patient (＞10 ng/ml) fall into the linear range of this immunosensor. Thus, our fabricated immunosensor provides a novel method to quantify PSA concentration, indicating its potential for clinical application. The analytical performance of the proposed immunosensor in PSA detection was compared with previously reported electrochemical immunosensors based on graphene-metal composites, which are listed in Table.S1. It can be seen that our immunosensor displayed a superior detection limit and linear detection range for PSA, which may be attributed to the following factors. First, anti-PSA was immobilized on rGO/Ag NPs composites via Ag-N coordinate bonds instead of covalent linkage, thus greatly preserving the active sites of the anti-PSA. Secondly, as discussed earlier, the synthesized rGO/Ag NPs composites are highly conductive
electrode
materials.
Consequently,
even
though
the
anti-PSA/PSA
immunocomplexes blocked the electron transport, the electrochemical signal of Fe(CN)63-/ Fe(CN)64- was amplified, leading to the enhanced sensitivity of the proposed immunosensor.
3.4 Specificity, reproducibility and stability of the immunosensor In order to investigate the selectivity of the immunosensor based on rGO/Ag NPs, the immunosensor was incubated with PSA in the presence of interfering agents. 100 ng/ml of interfering substances including vitamin C, glucose and BSA were mixed with 1 ng/ml of PSA, respectively. As shown in Fig.5A, the current responses of the immunosensors exposed
to the interfering agents were all within 5 % of the immunosensor without interference, indicating that the specificity of the immunosensor was outstanding.
Fig.5 (A) The specificity study of the PSA immunosensor: (a) 1.0 ng/ml PSA; (b) 1.0 ng/ml PSA+100 ng/ml vitamin C; (c) 1.0 ng/ml PSA+100 ng/ml glucose; (d) 1.0 ng/ml PSA+100 ng/ml BSA. (B) The stability assay of the PSA immunosensor.
Reproducibility is also an important factor to be considered for biosensors. Therefore, we need to determine it to confirm the reliability of our developed immunosensor. By analysis of 1 ng/ml of PSA using five identically prepared BSA/anti-PSA/rGO-Ag NPs/SPCEs, the current variations of the immunosensor were thoroughly investigated (results not shown). The relative standard deviation (RSD) was 3.52%, less than 5%, illustrating that the immunosensor exhibited acceptable precision and reproducibility. Stability
is
another
essential
feature
of
immunosensors.
In
this
study,
BSA/anti-PSA/rGO-Ag NPs/SPCEs immunosensors were stored in pH 7.4 PBS at 4℃ and their current responses were examined every 7 days. After 14 days, the current response of the immunosensor decreased by only 8.73%. After 3 weeks, the current response attained 87.54% of its initial value, suggesting that the immunosensor shows good long-term stability. We presume that this stability can mainly be ascribed to the large surface area of rGO/Ag NPs and their supreme affinity for biomolecules as well as the carbon electrode surface.
4. Conclusions A label-free electrochemical immunosensor for PSA detection was fabricated using
rGO/Ag NPs composites as electrode materials on a screen-printed three-electrode system. The rGO/Ag NPs composites were synthesized using binary reductants, i.e. hydrazine hydrate and sodium citrate, at high yields by one-pot reaction under mild conditions. Moreover, the rGO/Ag NPs possessed superior electrical conductivity compared to rGO because the small Ag NPs, stabilized by sodium citrate were anchored onto the rGO sheets. The EIs demonstrated a wide linear response range (1.0 to 1000 ng/ml) and low detection limit (0.01 ng/ml). Furthermore, the as-prepared EIs also exhibited excellent specificity, reproducibility and stability. The proposed EIs provided a simple, low-cost method for PSA detection and have potential applications in clinical diagnosis of prostate cancer, which can be further extended to the ultrasensitive determination of other disease-related protein biomarkers.
5. Acknowledgements This work was financially supported by Chinese Natural Science Foundation project (No. 31300820), Beijing Municipal Commission of Education project (No.18190114/006), a project founded by Beijing Institute of Graphic Communication (No.27170115004/025), a foundation for raising the scientific research level of materials and technologies for printed electronics (No.Eb201530) and a grant from Beijing collaborative innovation for green printing and publication.
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Highlights: ■
rGO/Ag NPs composites with enhanced electrical conductivity were
successfully prepared using the binary reductants, namely hydrazine hydrate and sodium citrate. ■Using rGO/Ag NP composites as electrode materials, a low-cost label-free electrochemical immunosensor was easily constructed. ■ The proposed immunosensor exhibited the widest linear detection range (1.0-1000 ng/ml) and a low detection limit ( 0.01 ng/ml) for PSA.