- Email: [email protected]
Au hybrid structure amplified by quenching effect of Ab2@V2+ conjugates
Biosensors and Bioelectronics 77 (2016) 339–346
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Ultrasensitive photoelectrochemical immunoassay for CA19-9 detection based on CdSe@ZnS quantum dots sensitized TiO2NWs/Au hybrid structure amplified by quenching effect of Ab2@V2 þ conjugates Hua Zhu a,1, Gao-Chao Fan a,1, E.S. Abdel-Halim c, Jian-Rong Zhang a,b,n, Jun-Jie Zhu a,nn a State Key Laboratory of Analytical Chemistry for Life Science, Collaborative Innovation Center of Chemistry for Life Sciences, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, PR China b School of Chemistry and Life Science, Nanjing University Jinling College, Nanjing 210089, PR China c Chemistry Department, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia
art ic l e i nf o
a b s t r a c t
Article history: Received 2 August 2015 Received in revised form 2 September 2015 Accepted 23 September 2015 Available online 26 September 2015
A novel, enhanced photoelectrochemical immunoassay was established for sensitive and specific detection of carbohydrate antigen 19-9 (CA19-9, Ag). In this protocol, TiO2 nanowires (TiO2NWs) were first decorated with Au nanoparticles to form TiO2NWs/Au hybrid structure, and then coated with CdSe@ZnS quantum dots (QDs) via the layer-by-layer method, producing TiO2NWs/Au/CdSe@ZnS sensitized structure, which was employed as the photoelectrochemical matrix to immobilize capture CA19-9 antibodies (Ab1); whereas, bipyridinium (V2 þ ) molecules were labeled on signal CA19-9 antibodies (Ab2) to form Ab2@V2 þ conjugates, which were used as signal amplification elements. The TiO2NWs/Au/ CdSe@ZnS sensitized structure could adequately absorb light energy and dramatically depress electron– hole recombination, resulting in evidently enhanced photocurrent intensity of the immunosensing electrode. While target Ag were detected, the Ab2@V2 þ conjugates could significantly decrease the photocurrent detection signal because of strong electron-withdrawing property of V2 þ coupled with evident steric hindrance of Ab2. Thanks to synergy effect of TiO2NWs/Au/CdSe@ZnS sensitized structure and quenching effect of Ab2@V2 þ conjugates, the well-established photoelectrochemical immunoassay exhibited a low detection limit of 0.0039 U/mL with a wide linear range from 0.01 U/mL to 200 U/mL for target Ag detection. This proposed photoelectrochemical protocol also showed good reproducibility, specificity and stability, and might be applied to detect other important biomarkers. & 2015 Elsevier B.V. All rights reserved.
Keywords: Photoelectrochemistry Immunoassay Quenching effect Synergy effect CA 19-9
1. Introduction Sensitive and accurate detection of disease-related targets is critical to many areas of life and medical sciences, from food safety, environmental monitoring to clinical diagnosis. Especially, highly sensitive detection of cancer biomarkers shows great promise for early diagnosis and disease monitoring (Kitano, 2002; Srinivas et al., 2001). Carbohydrate antigen 19-9 (CA19-9), a Lewis antigen of the cell surface associated mucin 1 (MUC1) protein with an average molecular weight of 1000 kDa, is a gold standard for pancreatic cancer diagnosis (Gui et al., 2013; Gold et al., 2006). n Corresponding author at: State Key Laboratory of Analytical Chemistry for Life Science, Collaborative Innovation Center of Chemistry for Life Sciences, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, PR China. Fax: þ 86 25 83317761. nn Corresponding author. Fax: þ 86 25 83317761. E-mail addresses: [email protected] (J.-R. Zhang), [email protected] (J.-J. Zhu). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bios.2015.09.051 0956-5663/& 2015 Elsevier B.V. All rights reserved.
Elevated levels of CA19-9 also are associated with gastric, urothelial, and colorectal carcinomas (Xiao et al., 2014; Jha et al., 2013; Narita et al., 2014). Thus, sensitive detection of CA19-9 is of great importance in early prediction for related cancers and diseases. To date, a variety of methods have been developed for CA199 detection, such as enzyme-linked immunoassay (Heidari et al., 2014), photoluminescence (Gu et al., 2011), chemiluminescence immunoassay (Shi et al., 2014; Lin and Ju, 2005), and electrochemical immunoassay (Tang et al., 2013; Yang et al., 2015). Despite many advances of these assays, some of them have drawbacks such as evident sample volume, complicated equipment, limited sensitivity, difficult automation and high cost. Thus, development of highly sensitive, simple and inexpensive techniques for CA19-9 detection is very desirable. Photoelectrochemical analysis is a newly emerged yet dynamically developing technique for the detection of various biological molecules. Recently, it has aroused a great research interest because of the features of simple devices, low cost and easy miniaturization than optical methods such as chemiluminescence
340
H. Zhu et al. / Biosensors and Bioelectronics 77 (2016) 339–346
(Kang et al., 2009; Zhao et al., 2015), fluorescence (Sheng et al., 2009; Zhu et al., 2011), and Raman scattering (Ko et al., 2013; Wang et al., 2015). Moreover, photoelectrochemical assays own potentially higher selectivity than traditional electrochemical methods, due to reduced background signals originating from different energy forms of excitation source and detection signal (Haddour et al., 2006; Wang et al., 2009). To date, semiconductor nanomaterials have proved to be the most popular photoactive materials to construct photoelectrochemical biosensors. Thereinto, TiO2 is an excellent substrate photoactive material owing to its photoelectric activity, good biocompatibility, inexpensiveness, environmental safety, and chemical and physical stability (Qiu et al., 2011; Sano et al., 2012). Recently, one-dimensional TiO2 crystalline films including nanowires, nanotubes and nanorods have been caught particular attention since they have enlarged surface area and can provide direct pathway for photogenerated electron transfer, which accordingly leads to evident enhancement of charge separation, and effective prohibition of charge recombination (Shankar et al., 2009; Mor et al., 2006; Zhu et al., 2001; Liu and Aydil, 2009; Wu and Yu, 2004; Chen et al., 2010). However, as a wide energy band gap semiconductor (3.2 eV), TiO2 can only absorb the ultraviolet light ( o387 nm), leading to great limitation to utilization of light energy (Qiu et al., 2011). As a result, many efforts have been poured on the exploitation of TiO2-based hybrid structures to develop visible-light-motivated photoelectrochemical biosensors, which could adequately increase the light absorption efficiency, significantly enhance the photocurrent conversion efficiency and evidently promote the sensitivity of the related biosensors (Fan et al., 2014a, 2014b; Li et al., 2012). According to signal changes for detection, photoelectrochemical immunoassays can be divided into two types: signalon and signal-off. Currently, most of the developed photoelectrochemical immunoassays belong to the latter type, because steric hindrance generated by immunized recognition between antibody and antigen would apparently obstacle electron transfer, leading to photocurrent decrease. In order to enhance the sensitivity of photoelectrochemical immunoassays, enzymes are often employed for signal amplification (An et al., 2010; Zhao et al., 2012; Li et al., 2012). However, the introduction of enzyme not only increased the cost of sensor preparation but also made the testing process more complicated. Hence, establishing other simple, low cost and effective photoelectrochemical protocols for signal amplification would be highly expected. Inspired by several researches conducted by Willner and his co-workers, N-(2-
carboxymethyl)-N′-methyl-4,4′-bipyridinium (V2 þ ) possesses strong electron-withdrawing capability due to evident electron deficiency of its structure (Tel-Vered et al., 2008; SheeneyHaj-Ichia et al., 2002a, 2002b). Specifically, when semiconductor nanomaterials such as CdS or CdSe connected with V2 þ molecules, the photocurrent intensity was obviously lower than that of CdS or CdSe alone, because V2 þ molecules acted as traps for the conduction-band electrons (Tel-Vered et al., 2008; Zhang et al., 2012). Accordingly, V2 þ can be well used as signal-off labels linking with signal antibodies (Ab2) to form Ab2@V2 þ conjugates for signal amplification. As both V2 þ and Ab2 jointly facilitate decrease of photocurrent signal, using Ab2@V2 þ conjugates as signal amplification elements can contribute to an excellent sensitivity for signal-off photoelectrochemical immunoassays. However, to be of our knowledge, this kind of signal amplification protocol has not appeared in photoelectrochemical immunoassays. Herein, we presented an enhanced, promising platform to construct an ultrasensitive photoelectrochemical immunoassay for CA19-9 (antigen, Ag) detection based on TiO2NWs/Au/CdSe@ZnS sensitized structure and signal amplification of Ab2@V2 þ conjugates, as illustrated in Scheme 1. Firstly, TiO2NWs were synthesized by a hydrothermal growth method, and then were modified onto a bare ITO (indium tin oxide) electrode. Next, the ITO/TiO2NWs electrode was coated with Au nanoparticles, and then was modified with CdSe@ZnS film via layer-by-layer assembling oppositely charged polyelectrolyte and CdSe@ZnS QDs, forming TiO2NWs/Au/CdSe@ZnS sensitized structure to significantly enhance the photocurrent intensity. Afterwards, Ab1 was immobilized on the electrode by EDC coupling reaction between carbonyl and amino groups. After BSA blocked unbound sites on the electrode surface, the immunosensing electrode was ready. For target CA19-9 determination, different concentrations of Ag were first bound on the sensing electrode by specific immunoreaction between Ag and Ab1, and then the fixed concentration of Ab2@V2 þ conjugates as signal amplification elements were further immobilized through specific immunoreaction between Ag and Ab2, which led to significantly reduced photocurrent. The proposed photoelectrochemical protocol exhibited ultrahigh sensitivity, reproducibility, specificity, and stability.
Scheme 1. Construction process of the photoelectrochemical immunoassay for CA19-9 detection.
H. Zhu et al. / Biosensors and Bioelectronics 77 (2016) 339–346
341
2. Experimental
2.3. Synthesis of TiO2NWs
2.1. Materials and reagents
The TiO2 nanowires (TiO2NWs) were synthesized by a modified hydrothermal growth method according to the literature (Nikhil et al., 2014). Briefly, 2 g of TiO2 nanoparticles mixed with 35 mL of 15 M NaOH aqueous solution was transferred into a Teflon-lined digestion vessel and heated to 170 °C for 48 h, and then cooled down to room temperature. The supernatant was gently removed from the product formed at the bottom of the Teflon vessel. After the white products were soaked in 0.1 M HCl solution for 4 h, the solution was washed several times with DI water until it became neutral. Finally the wet white products were dried under vacuum at 100 °C for 4 h to obtain the dry solid of TiO2NWs.
Indium tin oxide (ITO) electrodes (type JH52, ITO coating 30 75 nm, sheet resistancer10 Ω/sq) were purchased from ZhongJingkeyi Technology Co., Ltd. (Nanjing, China). TiO2 powder (P25) was purchased from the Degussa Co. (Germany). Cadmium chloride (CdCl2 2.5H2O), zinc chloride (ZnCl2), sodium hydroxide (NaOH) and chloroauric acid (HAuCl4 4H2O) were purchased from Shanghai Chemical Reagent Co. (China). 4,4′-bipyridine was purchased from J&K Scientific Ltd. (Beijing, China). Methyl iodide (CH3I) was purchased from Xiya reagent (China). Chloroform (CHCl3) and acetonitrile (CH3CN) were purchased from Nanjing Chemical Reagent Co., Ltd. (China). Selenium powder (Se), sodium borohydride (NaBH4), thiolglycolic acid (TGA), poly(diallyldimethylammonium chloride) (PDDA, 20 wt% in water), N-hydroxysuccinimide (NHS), 3-mercaptopropionic acid (MPA), 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), acrylic acid (C3H4O2), and bovine serum albumin (BSA) were all obtained from Sigma-Aldrich (USA). Ascorbic acid (AA) was purchased from Sinopharm Chemical Reagent Co., Ltd. (China). Carbohydrate antigen 19-9 (CA19-9, Ag), capture CA19-9 antibody (Ab1), signal CA19-9 antibody (Ab2), carbohydrate antigen 15-3 (CA15-3), and prostate specific antigen (PSA) were obtained from Shanghai Linc-Bio Science Co. Ltd. (China). Human IL-6 and matrix metalloproteinase-2 (MMP-2) were obtained from Wuhan Uscn Life Science Inc. (China). All reagents were of analytical grade. All aqueous solutions were prepared with deionized water (DI water, 18.2 MΩ/cm) obtained from a Milli-Q water purification system. Phosphate buffer solution (PBS, pH 7.4, 10 mM) was used for the preparation of the antibody and antigen solution, washing buffer solution, and blocking buffer solution which contained 1% (w/v) BSA. 2.2. Apparatus The UV–visible (UV–vis) absorption spectra were tested on a UV-3600 UV–visible spectrophotometer (Shimadzu, Japan). Electrochemical impedance spectroscopy (EIS) was recorded on an Autolab potentiostat/galvanostat (PGSTAT 30, Eco Chemie B.V., Utrecht, Netherlands) with a three-electrode system in 0.1 M KCl solution containing 5.0 mM K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) mixture as a redox probe and recorded in the frequency range of 0.01 Hz to 100 kHz with an amplitude of 50 mV. Photoelectrochemical measurements were performed with a homemade photoelectrochemical system. A 500 W Xe lamp was used as the irradiation source with the light intensity of 400 μW/cm2 estimated by a radiometer (Photoelectric Instrument Factory of Beijing Normal University). Photocurrent was measured on a CHI 660D electrochemical workstation (Shanghai Chenhua Apparatus Corporation, China) with a three-electrode system: a 0.25 cm2 modified ITO as working electrode, a Pt wire as counter electrode and a saturated Ag/AgCl electrode as reference electrode. Transmission electron microscopy (TEM) was performed with a JEOL-2100 transmission electron microscope (JEOL, Japan). Field-emission scanning electron microscopy (FE-SEM) was performed on a Hitachi S-4800 scanning electron microscope (Hitachi Co., Japan) equipped with EX-250 Energy-dispersive X-ray spectroscopy (EDX, HORIBA Co., Japan). The 1HNMR spectra were measured on a Bruker AVANCE III-300 spectrometer using D2O as solvent and the data was reported as: chemical shift (δ ppm), multiplicity, and coupling constant (Hz).
2.4. Synthesis of V2 þ and Ab2@V2 þ conjugates V2 þ (N-(2-carboxymethyl)-N′-methyl-4,4′bipyridinium) was synthesized based on the literature method with appropriate modifications (see Supporting information). The typical preparation process for Ab2@V2 þ conjugates was as below. 1.0 mL saturated V2 þ solution was mixed gently with 600 μL of 10.0 mg/mL freshly prepared EDC for 30 min at room temperature. Subsequently, 50 μL of 0.2 mg/mL Ab2 was mixed with the above solution and incubated 2 h under shaking at 30 °C, and then stored overnight at 4 °C, which makes the unreacted EDC to lose its activity. Finally, the Ab2@V2 þ conjugates were acquired by centrifugation at 5500 rpm for 15 min using Millipore ultrafiltration centrifuge tube (MWCO¼ 100,000), and diluted to 1 mL by PBS (10 mM, pH 7.4). 2.5. Synthesis of CdSe@ZnS QDs The synthesis process of the CdSe@ZnS core–shell QDs included two procedures as below. The CdSe cores were synthesized based on the literature (Fan et al., 2014c). Typically, NaHSe solution was obtained by the reaction of Se powder with NaHB4 under nitrogen atmosphere at room temperature. After 50 mL of 2 mM CdCl2 was mixed with 20 μL of TGA, the pH of the system was adjusted to 10.0 by 1 M NaOH and bubbled with highly pure N2 for 30 min. And then 0.7 mL of 70 mM NaHSe solution was injected into this mixture to acquire a light yellow solution of CdSe precursors. The molar ratio of Se2 /Cd2 þ /TGA was 0.5/1/2.5. The resulting solution was then refluxed at 100 °C for 3 h to form CdSe cores. For the synthesis of CdSe@ZnS core–shell QDs, 70 μL of MPA and 8 mL of 0.5 M ZnCl2 were added into the CdSe cores solution under stirring. The mixture solution was then refluxed at 100 °C for 1 h to form CdSe@ZnS QDs. The resulting CdSe@ZnS QDs were purified by centrifugation at 8000 rpm for 15 min twice using Millipore ultrafiltration centrifuge tube (MWCO¼3000). 2.6. Preparation of ITO/TiO2NWs/Au/CdSe@ZnS electrode Before preparation, ITO slices were cleaned by ultrasonic treatment for 10 min in acetone, 1 M NaOH of water/ethanol mixture (1:1, v/v), and water, respectively, and then dried at 100 °C for 6 h. After certain amount of TiO2NWs was ultrasonically dispersed in DI water, 20 μL of the homogeneous suspension was coated onto a piece of ITO slice with fixed area of 0.25 cm2 and dried in air. The deposition of Au nanoparticles on ITO/TiO2NWs electrode was according to literature method (Li et al., 2014). The TiO2NWs modified electrode was firstly immersed into 0.01 M HAuCl4 aqueous solution (pH ¼ 4.5) for 4 h, and followed by washed with DI water, and then was annealed at 450 °C for 2 h in air atmosphere to generate Au nanoparticles on the TiO2NWs surface. And the desired ITO/TiO2NWs/Au electrode was obtained. The CdSe@ZnS multilayer film was fabricated by alternately
342
H. Zhu et al. / Biosensors and Bioelectronics 77 (2016) 339–346
dipping the ITO/TiO2NWs/Au electrode into a 0.5% positively charged PDDA solution and the negatively charged CdSe@ZnS QDs solution for 10 min, respectively. The film was carefully washed with DI water after each dipping step. The two-step dipping procedure was termed as “one coating”. The coating process was repeated three times, and the ITO/TiO2NWs/Au/CdSe@ZnS electrode was prepared. 2.7. Fabrication of the immunosensor Ab1 was immobilized onto the ITO/TiO2NWs/Au/CdSe@ZnS electrode via the EDC coupling reaction between carbonyl groups on the surface of the CdSe@ZnS QDs and NH2 groups of the Ab1. Initially, the electrode was immersed in a solution containing 20 mM EDC and 10 mM NHS for 30 min at room temperature. After rinsing with washing buffer solution to remove the unreacted EDC and NHS, 20 μL of 100 μg/mL Ab1 was spread onto the electrode surface and incubated at 4 °C in a moisture atmosphere overnight. After that, the electrode was rinsed with the washing buffer solution to remove unbound and physically absorbed Ab1. The electrode was then blocked by covering with 20 μL of BSA blocking buffer solution for 1 h at room temperature to block nonspecific binding sites and washed with the washing buffer solution thoroughly. Next, 20 μL of different concentrations of target Ag was covered on the BSA blocked electrode for an incubation of 1 h at 37 °C, followed by washing with washing buffer solution. After the specific immunoreaction between Ab1 and Ag, the electrode was labeled by incubation with 20 μL of Ab2@V2 þ conjugates solution for 1 h at 37 °C. Finally, the resulting electrode was washed thoroughly with washing buffer solution and was ready for photocurrent measurement. 2.8. Photoelectrochemical detection Photoelectrochemical detection was carried out in PBS (pH 7.4, 0.1 M) containing 0.1 M AA, which served as a sacrificial electron donor during the photocurrent measurement. White light produced by the Xe lamp, with a spectral range of 200–2500 nm, was utilized as excitation light and was switched on and off every 10 s. The applied potential was 0.0 V. The AA electrolyte was deaerated by pumping pure nitrogen for 10 min before photocurrent measurement.
3. Results and discussion 3.1. Characterization of TiO2NWs and TiO2NWs/Au hybrid structure Fig. 1A and B presents the FE-SEM images of the TiO2NWs and TiO2NWs/Au hybrid structure, respectively. It can be seen that the
TiO2NWs were about 3–6 μm in length and 60–80 nm in diameter (Fig. 1A). After Au nanoparticles subsequently grew on the TiO2NWs, many relatively small particles with the average size of about 10–15 nm were appeared on the TiO2NWs (Fig. 1B). Fig. S2 (in Supporting material) shows the elemental mapping analysis of TiO2NWs/Au, which suggested the presence of Ti, O and Au components in the hybrid structure and the deposited Au element was well scattered on the TiO2NWs. Besides, according to color changes from white to violet of the electrode surfaces (which was not shown here), it also confirmed the successful formation of the TiO2NWs/Au hybrid structure. 3.2. Characterization of CdSe cores and CdSe@ZnS QDs Fig. 2A and B shows high-resolution TEM (HRTEM) images of the prepared CdSe cores and CdSe@ZnS QDs, respectively. The lattice fringes of the two kinds of materials are clearly observed. The average size of the CdSe cores was about 3 nm (Fig. 2A), whereas the average size of CdSe@ZnS QDs was about 4.5 nm (Fig. 2B) which was larger than that of the CdSe cores, indicating successful synthesis of the CdSe@ZnS core-shell QDs. Fig. 2C presents the UV–vis absorption spectrum of the synthesized core– shell CdSe@ZnS QDs. It exhibited two sharp absorption peaks at around 450 nm and 500 nm, and a broad continuous absorption spectrum up to the wavelength of 570 nm which potentially indicated effective absorption under visible wavelengths. 3.3. Photoelectrochemical property of ITO/TiO2NWs/Au/CdSe@ZnS electrode As mentioned above, TiO2 can only absorb the ultraviolet light. Yet, CdSe with a lower band gap of 1.7 eV can extend the absorption range to longer-wavelength light. Moreover, CdSe possesses a higher conduction band edge than that of TiO2, which is beneficial for the injection of excited electrons from CdSe to TiO2 (Robel et al., 2006). Thus, combination of CdSe QDs with TiO2 could evidently increase the utilization of light energy and promote the photocurrent response. To further improve the photoelectrochemical properties of CdSe QDs, a shell of ZnS was introduced outside of the CdSe core to form the CdSe@ZnS core– shell structure, because ZnS shell with high conduction band and wide band gap could serve as a passivation layer to confine excitation electrons in CdSe core, reduce the formation of surface defects and the light corrosion, and inhibit recombination between electrons and redox couples (Arora and Manoharan 2007; Sambur and Parkinson, 2010). The introduction of Au nanoparticles could further improve the photocurrent intensity, because Au nanoparticles acted as light scattering centers for more light absorption of CdSe@ZnS QDs (Liu et al., 2011). In order to illustrate the positive effect of Au nanoparticles to photocurrent
Fig. 1. FE-SEM images of the (A) TiO2NWs (B) TiO2NWs/Au hybrid structure.
H. Zhu et al. / Biosensors and Bioelectronics 77 (2016) 339–346
343
Fig. 2. HRTEM images of the (A) CdSe cores and (B) CdSe@ZnS QDs; (C) UV–vis spectrum of the CdSe@ZnS QDs.
intensity, the control experiment was conducted, and it surely demonstrated this point (see Fig. S3 in Supporting material). Thus, herein, we first utilized the TiO2NWs/Au/CdSe@ZnS sensitized structure as photoelectrochemical matrix for the immunosensing electrode. As the concentration of TiO2NWs suspension, deposition of Au nanoparticles and coating number of CdSe@ZnS QDs could influence the photocurrent intensity of the ITO/TiO2NWs/Au/ CdSe@ZnS electrode, to achieve maximum photocurrent intensity, the optimal preparation conditions were exploited (see Fig. S4 in Supporting material). Thus, 0.8 mg/mL TiO2NWs, 4 h of soaking time in HAuCl4 solution, and three coating numbers of CdSe@ZnS QDs were selected to fabricate the ITO/TiO2NWs/Au/CdSe@ZnS electrode. 3.4. EIS characterization of the immunosensor EIS could provide effective information about the electrode surfaces. Generally, the electrochemical impedance spectrum includes a semicircle portion and a linear portion. The semicircle diameter equals the electron-transfer resistance (Ret), which reflects the electron-transfer kinetics of the redox probe at the electrode interface. Fig. 3 shows the Nyquist plots of impedance spectra corresponding to the different construction steps. For ITO/TiO2NWs electrode, the impedance spectrum exhibited a relatively small Ret (curve a), indicating a small electron transfer resistance. After Au nanoparticles deposition (curve b), the Ret reduced. It could be attributed to the good conductivity of Au nanoparticles (Zhou et al., 2014). After CdSe@ZnS QDs, Ab1, BSA, Ag and Ab2@V2 þ were modified onto the electrode surface step by step, the Ret gradually increased owing to low conductivity of semiconductor QDs or insulating effect of organic molecules (curve c–g), indicating that the layer-by-layer assembled immunosensor was successfully constructed.
Fig. 3. Nyquist plots of the (a) ITO/TiO2NWs electrode, (b) after Au nanoparticles deposition, (c) after CdSe@ZnS QDs immobilization, (d) after Ab1 immobilization, (e) after BSA blocking, (f) after incubation with 20 μL of 500 U/mL Ag, and then (g) further incubation with Ab2@V2 þ conjugates.
Fig. 4. Photocurrent responses of the (a) ITO/TiO2NWs/Au electrode, (b) after CdSe@ZnS QDs immobilization, (c) after Ab1 immobilization, (d) after BSA blocking, (e) after incubation with 20 μL of 500 U/mL Ag, and then (f) further incubation with Ab2@V2 þ conjugates.
3.5. Photoelectrochemical characterization of the immunosensor The fabrication process of the photoelectrochemical immunosensor was monitored by photocurrent responses, as shown in Fig. 4. The ITO/TiO2NWs/Au electrode exhibited a relatively small photocurrent response (curve a, I¼ 10.58 μA). After CdSe@ZnS QDs modification, the photocurrent intensity (curve b, I¼ 130.43 μA) increased to 12 times higher than that of the ITO/TiO2NWs electrode, which was because that the TiO2NWs electrode possessed a large surface area for much more CdSe@ZnS QDs QDs loading, and the modified CdSe@ZnS QDs extended the absorption range to longer-wavelength light ( 570 nm), and meanwhile the deposited Au nanoparticles could act as light scattering centers to further increase the light absorption of CdSe@ZnS QDs (Liu et al., 2011; Zarazua et al., 2011). After Ab1 and BSA were successively immobilized on the electrode, the photocurrent intensity decreased (curve d, I ¼83.23 μA; curve e, I¼ 70.70 μA), which could be attributed to the steric hindrance of those modified protein molecules. After the sensing electrode was incubated with target Ag and then Ab2@V2 þ conjugates, the photocurrent intensity decreased to 78% and 25% of the initial sensing electrode (curve f, I¼ 55.24 μA; curve g, I ¼17.47 μA). Obviously, the decrement of photocurrent intensity for target Ag modification was evidently less than that of the subsequently modified Ab2@V2 þ conjugates. This could be attributed to strong electron-withdrawing property of V2 þ coupled with obvious steric hindrance of Ab2 (Tel-Vered et al., 2008). Besides, in order to verify the synergy effect of Ab2 and V2 þ in Ab2@V2 þ conjugates, Ab2 was also used as signal amplification elements for photocurrent test, and the results exhibited that the decrement of photocurrent response to Ab2 was only about 38% of that to Ab2@V2 þ conjugates, indicating the superiority of the Ab2@V2 þ conjugates as signal amplification elements. Thus, the photocurrent responses
344
H. Zhu et al. / Biosensors and Bioelectronics 77 (2016) 339–346
Table 1 Analytical performances of various bioassays for CA19-9 detection. Immunoassay
Linear range [U/mL]
Detection limit [U/mL]
Reference
Electrochemical
0.015–150
0.006
Yang et al. (2015) Sha et al. (2015) Shi et al. (2014) Gu et al. (2011) Zhao et al. (2009) This work
Electrochemiluminescence 0.001–5
0.0005
Chemiluminescence
0.016
Photoluminescence
0.1–180
0.04
Fluorometric
3.0–500
2.30
Photoelectrochemical
Scheme 2. Photogenerated electron–hole transfer mechanism of the immunosensor for target Ag detection.
demonstrated the successful construction of the proposed immunoassay. In a word, two major reasons contributed to ultrahigh sensitivity of the proposed photoelectrochemical immunoassay, as illustrated in Scheme 2. Before target Ag detection, TiO2NWs/Au/ CdSe@ZnS sensitized structure could evidently promote the photocurrent response of the sensing electrode, because it could adequately absorb the light energy and effectively inhibit the electron–hole recombination. However, in the presence of target Ag, the Ab2@V2 þ conjugates could significantly decrease the photocurrent detection signal because of strong electron-withdrawing capability of V2 þ coupled with obvious steric hindrance of Ab2. Accordingly, based on the two main aspects mentioned above, ultrahigh sensitivity was realized. 3.6. Photoelectrochemical detection for CA19-9 CA19-9 detection was based upon the sandwich immunoreactions between Ab1 and target Ag as well as target Ag and Ab2@V2 þ conjugates. The photocurrent response of this immunoassay was directly related to the concentration of target Ag. Thus, the photoelectrochemical immunosensor toward CA19-9 detection can be achieved by monitoring the photocurrent change. Fig. 5A presents the photocurrent responses of the immunosensor after being incubated with different concentrations of target Ag and then with fixed concentration of Ab2@V2 þ conjugates. Along with the concentration of target Ag increased, more and more Ab2@V2 þ conjugates were specifically bound on the electrode, leading to gradually decreased photocurrent intensity. As shown in Fig. 5B, the
0.025–1
0.01–200
0.0039
photocurrent response linearly decreased with an increasing logarithm of the concentration of target Ag in the range from 0.01 U/mL to 200 U/mL. The regression equation was I¼ 44.56 8.27log CCA19-9 (U/mL), with the correlation coefficient of 0.9986. The limit of detection (LOD, S/N ¼ 3) for target Ag concentration was estimated to be 0.0039 U/mL. In order to evaluate the sensitivity of the proposed photoelectrochemical immunoassay, other highly sensitive bioassays for CA19-9 detection reported previously were listed out, as shown in Table 1, which demonstrated the well-designed photoelectrochemical immunoassay also exhibited wider linear range as well as a lower detection limit. 3.7. Reproducibility, specificity and stability of the immunoassay The reproducibility of the immunoassay was assessed by both intra-assay (within-batch) and inter-assay (between-batch) relative standard deviation (RSD). Analyzed from the testing results of five replicate determinations, the intra-assay RSDs were 3.2%, 2.7%, and 2.4% towards 0.05, 0.1, and 0.2 U/mL of CA19-9, respectively. The inter-assay RSDs of 4.4%, 3.6%, and 3.7% were acquired via measuring the same samples with five electrodes fabricated independently under identical experimental conditions. All of these results suggested an acceptable reproducibility of the proposed immunoassay. Specificity is a crucial criterion for immunoassay, since the nonspecific adsorption can influence the sensitivity. To verify that the photocurrent response originated from specific binding, some representative interfering proteins including carcinoma antigen 15-3 (CA15-3), human interleukin-6 (IL-6), matrix metalloproteinases-2 (MMP-2), and prostate-specific antigen (PSA) were selected for the interference test. As shown in Fig. 6, no significant
Fig. 5. (A) Photocurrent responses and (B) calibration curve of the immunoassay for the detection of different concentrations of CA19-9 from 0.01 U/mL to 200 U/mL. The error bars showed the standard deviation of five replicate determinations.
H. Zhu et al. / Biosensors and Bioelectronics 77 (2016) 339–346
345
Saud University for funding the work through the research group project No. RGP-VPP-029.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2015.09.051.
References
Fig. 6. Photocurrent responses of the immunoassay for 1 U/mL CA19-9 detection (a) in the absence of interference or in the existence of 1 U/mL of (b) CA15-3, (c) IL6, (d) MMP-2, (e) PSA, and (f) their mixture. The error bars showed the standard deviation of five replicate measurements.
difference of photocurrent response could be observed as compared to the result obtained in the presence of only CA19-9 at the concentration of 1 U/mL. It indicates that the photocurrent response of the immunosensor was not affected by CA15-3, IL-6, MMP-2, PSA and their mixture. The relative deviations of the photocurrents tested in the presence of single interfering protein or their mixture were well within 4.2% for the measurement of 1 U/mL CA19-9 compared with the photocurrent detected in the absence of interfering protein. And the relative standard deviation (RSD) of five replicate determinations for each interference test was within 3.8%. All these results proved that the designed immunoassay possessed satisfactory specificity. The stability of the constructed immunoassay was also evaluated. After the sensing electrode was stored in PBS (pH 7.4, 10 mM) at 4 °C in a refrigerator for over two weeks, the photocurrent intensity kept 95.6% of its initial response, indicating it possessed good storage stability.
4. Conclusion In summary, we have presented an enhanced, promising protocol of photoelectrochemical immunoassay for ultrasensitive detection of CA19-9 based on cooperation effect of TiO2NWs/Au/ CdSe@ZnS sensitized structure and signal amplification of Ab2@V2 þ conjugates. Compared to the results reported previously, the well-established immunoassay exhibited a lower detection limit of 0.0039 U/mL as well as a wider linear range from 0.01 U/mL to 200 U/mL for CA19-9 detection. The greatly enhanced sensitivity was attributed to following reasons. Firstly, the sensitized structure of TiO2NWs/Au/CdSe@ZnS could evidently promote the photocurrent intensity due to its prominent properties of adequate absorption of light energy and effective depression of charge recombination. Secondly, the well-designed signal amplification elements of Ab2@V2 þ conjugates could significantly reduce the photocurrent detection signal resulting from its strong electron-withdrawing property coupled with obvious steric hindrance effect. The proposed photoelectrochemical protocol is highly expected to the detection of trace levels of disease-related biomarkers.
Acknowledgments We gratefully appreciate the National Natural Science Foundation (21375059, 21175065 and 21335004) and the National Basic Research Program (2011CB933502) of China. The authors extend their appreciation to the Deanship of Scientific Research at King
An, Y.R., Tang, L.L., Jiang, X.L., Chen, H., Yang, M.C., Jin, L.T., Zhang, S.P., Wang, C.G., Zhang, W., 2010. Chem. Eur. J. 16, 14439–14446. Arora, S., Manoharan, S., 2007. Solid State Commun. 144, 319–323. Chen, H., Fu, W.Y., Yang, H.B., Sun, P., Zhang, Y.Y., Wang, L.R., Zhao, W.Y., Zhou, X.M., Zhao, H., Jing, Q., Qi, X.F., Li, Y.X., 2010. Electrochim. Acta 56, 919–924. Fan, G.C., Han, L., Zhu, H., Zhang, J.R., Zhu, J.J., 2014a. Anal. Chem. 86, 12398–12405. Fan, G.C., Han, L., Zhang, J.R., Zhu, J.J., 2014b. Anal. Chem. 86, 10877–10884. Fan, G.C., Ren, X.L., Zhu, C., Zhang, J.R., Zhu, J.J., 2014c. Biosens. Bioelectron. 59, 45–53. Gold, D.V., Modrak, D.E., Ying, Z.L., Cardillo, T.M., Sharkey, R.M., Goldenberg, D.M., 2006. J. Clin. Oncol. 24, 252–258. Gu, B.X., Xu, C.X., Yang, C., Liu, S.Q., Wang, M.L., 2011. Biosens. Bioelectron. 26, 2720–2723. Gui, J.C., Yan, W.L., Liu, X.D., 2013. Clin. Exp. Med. 14, 225–233. Haddour, N., Chauvin, J., Gondran, C., Cosnier, S., 2006. J. Am. Chem. Soc. 128, 9693–9698. Heidari, M.H., Porghasem, M., Mirzaei, N., Mohseni, J.H., Heidari, M., Azargashb, E., Movafagh, A., Heidari, R., Molouki, A., Larijani, L., 2014. J. Environ. Radioact. 128, 64–67. Jha, D.K., Mittal, A., Gupta, S.P., Sathian, B., 2013. Asian Pac. J. Cancer Prev. 14, 4067–4069. Kang, H.Y., Miao, J.R., Cao, Z.J., Lu, J.Z., 2009. Analyst 134, 2246–2252. Kitano, H., 2002. Science 295, 1662–1664. Ko, J.H., Lee, S., Lee, E.K., Chang, S.I., Chen, L.X., Yoon, S.Y., Choo, J., 2013. Phys. Chem. Chem. Phys. 15, 5379–5385. Li, J.T., Cushing, S.K., Zheng, P., Senty, T., Meng, F.K., Bristow, A.D., Manivannan, A., Wu, N.Q., 2014. J. Am. Chem. Soc. 136, 8438–8449. Li, Y.J., Ma, M.J., Zhu, J.J., 2012. Anal. Chem. 84, 10492–10499. Lin, J.H., Ju, H.X., 2005. Biosens. Bioelectron. 20, 1461–1470. Liu, B., Aydil, E.S., 2009. J. Am. Chem. Soc. 131, 3985–3990. Liu, L.P., Wang, G.M., Li, Y., Li, Y.D., Zhang, J.Z., 2011. Nano Res. 4, 249–258. Mor, G.K., Shankar, K., Paulose, M., Varghese, O.K., Grimes, C.A., 2006. Nano Lett. 6, 215–218. Narita, Y., Taniguchi, H., Komori, A., Nitta, S., Yamaguchi, K., Kondo, C., Nomura, M., Kadowaki, S., Takahari, D., Ura, T., Andoh, M., Muro, K., 2014. Cancer Chemother. Pharmacol. 73, 409–416. Nikhil, A., Thomas, D.A., Amulya, S., Raj, S.M., Kumaresan, D., 2014. Sol. Energy 106, 109–117. Qiu, J.X., Zhang, S.Q., Zhao, H.J., 2011. Sens. Actuators B 160, 875–890. Robel, I., Subramanian, V., Kuno, M., Kamat, P.V., 2006. J. Am. Chem. Soc. 128, 2385–2393. Sambur, J.B., Parkinson, B.A., 2010. J. Am. Chem. Soc. 132, 2130–2131. Sano, M., Saotome, M., Urushida, T., Katoh, H., Satoh, H., Ohlishi, K., Hayashi, H., 2012. Intern. Med. 51, 2337–2340. Sha, Y.H., Guo, Z.Y., Chen, B.B., Wang, S., Ge, G.P., Qiu, B., Jiang, X.H., 2015. Biosens. Bioelectron. 66, 468–473. Shankar, K., Basham, J.I., Allam, N.K., Varghese, O.K., Mor, G.K., Feng, X., Paulose, M., Seabold, J.A., Choi, K.S., Grimes, C.A., 2009. J. Phys. Chem. C 113, 6327–6359. Sheeney-Haj-Ichia, L., Willner, I., 2002a. J. Phy. Chem. B 106, 13094–13097. Sheeney-Haj-Ichia, L., Wasserman, J., Willner, I., 2002b. Adv. Mater., 1323–1326. Sheng, Z.H., Han, H.Y., Hu, D.H., Liang, J.G., He, Q.G., Jin, M.L., Zhou, R., Chen, H.C., 2009. Chem. Commun., 2559–2561. Shi, M., Zhao, S.L., Huang, Y., Zhao, L.M., Liu, Y.M., 2014. Talanta 124, 14–20. Srinivas, P.R., Kramer, B.S., Srivastava, S., 2001. Lancet Oncol. 2, 698–704. Tang, D.P., Hou, L., Niessner, R., Xu, M.D., Gao, Z.Q., Knopp, D., 2013. Biosens. Bioelectron. 46, 37–43. Tel-Vered, R., Yehezkeli, O., Yildiz, H.B., Wilner, O.I., Willner, I., 2008. Angew. Chem. 120, 8396–8400. Wang, G.L., Yu, P.P., Xu, J.J., Chen, H.Y., 2009. J. Phys. Chem. C 113, 11142–11148. Wang, Y., Vaidyanathan, R., Shiddiky, M.J.A., Trau, M., 2015. ACS Nano 9, 6354–6362. Wu, J.J., Yu, C.C., 2004. J. Phys. Chem. B 108, 3377–3379. Xiao, J.C., He, X.Y., Wang, Z.Y., Hu, J.Y., Sun, F., Qi, F., Yang, S.G., Xiao, Z.Y., 2014. Tumor Biol. 35, 1331–1334. Yang, F., Yang, Z.H., Zhuo, Y., Chai, Y.Q., Yuan, R., 2015. Biosens. Bioelectron. 66, 356–362. Zarazua, I., Rosa, E.D., Lopez-Luke, T., Reyes-Gomez, J., Ruiz, S., Chavez, C.A., Zhang, J. Z., 2011. J. Phys. Chem. C 115, 23209–23220. Zhang, X.R., Xu, Y.P., Yang, Y.Q., Jin, X., Ye, S.J., Zhang, S.S., Jiang, L.L., 2012. Chem. Eur. J. 18, 16411–16418. Zhao, J.J., Jin, X., Vdovenko, M., Zhang, L.L., Sakharov, I.Y., Zhao, S.L., 2015. Chem.
346
H. Zhu et al. / Biosensors and Bioelectronics 77 (2016) 339–346
Commun. 51, 11092–11095. Zhao, W.W., Ma, Z.Y., Yu, P.P., Dong, X.Y., Xu, J.J., Chen, H.Y., 2012. Anal. Chem. 84, 917–923.Zhao, Y.J., Zhao, X.W., Pei, X.P., Hua, J., Zhao, W.J., Chen, B., Gu, Z.Z., 2009. Anal. Chim. Acta 633, 103–108. Zhou, Y.L., Xu, Z.N., Wang, M., Sun, B., Yin, H.S., Ai, S.Y., 2014. Biosens. Bioelectron.
53, 263–267. Zhu, P.X., Shelton, D.R., Li, S.H., Adams, D.L., Karns, J.S., Amstutz, P., Tang, C.M., 2011. Biosens. Bioelectron. 30, 337–341. Zhu, Y., Li, H., Koltypin, Y., Hacohen, Y.R., Gedanken, A., 2001. Chem. Commun., 2616–2617.
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Ultrasensitive photoelectrochemical immunoassay for CA19-9 detection based on CdSe@ZnS quantum dots sensitized TiO2NWs/Au hybrid structure amplified by quenching effect of Ab2@V2 þ conjugates Hua Zhu a,1, Gao-Chao Fan a,1, E.S. Abdel-Halim c, Jian-Rong Zhang a,b,n, Jun-Jie Zhu a,nn a State Key Laboratory of Analytical Chemistry for Life Science, Collaborative Innovation Center of Chemistry for Life Sciences, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, PR China b School of Chemistry and Life Science, Nanjing University Jinling College, Nanjing 210089, PR China c Chemistry Department, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia
art ic l e i nf o
a b s t r a c t
Article history: Received 2 August 2015 Received in revised form 2 September 2015 Accepted 23 September 2015 Available online 26 September 2015
A novel, enhanced photoelectrochemical immunoassay was established for sensitive and specific detection of carbohydrate antigen 19-9 (CA19-9, Ag). In this protocol, TiO2 nanowires (TiO2NWs) were first decorated with Au nanoparticles to form TiO2NWs/Au hybrid structure, and then coated with CdSe@ZnS quantum dots (QDs) via the layer-by-layer method, producing TiO2NWs/Au/CdSe@ZnS sensitized structure, which was employed as the photoelectrochemical matrix to immobilize capture CA19-9 antibodies (Ab1); whereas, bipyridinium (V2 þ ) molecules were labeled on signal CA19-9 antibodies (Ab2) to form Ab2@V2 þ conjugates, which were used as signal amplification elements. The TiO2NWs/Au/ CdSe@ZnS sensitized structure could adequately absorb light energy and dramatically depress electron– hole recombination, resulting in evidently enhanced photocurrent intensity of the immunosensing electrode. While target Ag were detected, the Ab2@V2 þ conjugates could significantly decrease the photocurrent detection signal because of strong electron-withdrawing property of V2 þ coupled with evident steric hindrance of Ab2. Thanks to synergy effect of TiO2NWs/Au/CdSe@ZnS sensitized structure and quenching effect of Ab2@V2 þ conjugates, the well-established photoelectrochemical immunoassay exhibited a low detection limit of 0.0039 U/mL with a wide linear range from 0.01 U/mL to 200 U/mL for target Ag detection. This proposed photoelectrochemical protocol also showed good reproducibility, specificity and stability, and might be applied to detect other important biomarkers. & 2015 Elsevier B.V. All rights reserved.
Keywords: Photoelectrochemistry Immunoassay Quenching effect Synergy effect CA 19-9
1. Introduction Sensitive and accurate detection of disease-related targets is critical to many areas of life and medical sciences, from food safety, environmental monitoring to clinical diagnosis. Especially, highly sensitive detection of cancer biomarkers shows great promise for early diagnosis and disease monitoring (Kitano, 2002; Srinivas et al., 2001). Carbohydrate antigen 19-9 (CA19-9), a Lewis antigen of the cell surface associated mucin 1 (MUC1) protein with an average molecular weight of 1000 kDa, is a gold standard for pancreatic cancer diagnosis (Gui et al., 2013; Gold et al., 2006). n Corresponding author at: State Key Laboratory of Analytical Chemistry for Life Science, Collaborative Innovation Center of Chemistry for Life Sciences, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, PR China. Fax: þ 86 25 83317761. nn Corresponding author. Fax: þ 86 25 83317761. E-mail addresses: [email protected] (J.-R. Zhang), [email protected] (J.-J. Zhu). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bios.2015.09.051 0956-5663/& 2015 Elsevier B.V. All rights reserved.
Elevated levels of CA19-9 also are associated with gastric, urothelial, and colorectal carcinomas (Xiao et al., 2014; Jha et al., 2013; Narita et al., 2014). Thus, sensitive detection of CA19-9 is of great importance in early prediction for related cancers and diseases. To date, a variety of methods have been developed for CA199 detection, such as enzyme-linked immunoassay (Heidari et al., 2014), photoluminescence (Gu et al., 2011), chemiluminescence immunoassay (Shi et al., 2014; Lin and Ju, 2005), and electrochemical immunoassay (Tang et al., 2013; Yang et al., 2015). Despite many advances of these assays, some of them have drawbacks such as evident sample volume, complicated equipment, limited sensitivity, difficult automation and high cost. Thus, development of highly sensitive, simple and inexpensive techniques for CA19-9 detection is very desirable. Photoelectrochemical analysis is a newly emerged yet dynamically developing technique for the detection of various biological molecules. Recently, it has aroused a great research interest because of the features of simple devices, low cost and easy miniaturization than optical methods such as chemiluminescence
340
H. Zhu et al. / Biosensors and Bioelectronics 77 (2016) 339–346
(Kang et al., 2009; Zhao et al., 2015), fluorescence (Sheng et al., 2009; Zhu et al., 2011), and Raman scattering (Ko et al., 2013; Wang et al., 2015). Moreover, photoelectrochemical assays own potentially higher selectivity than traditional electrochemical methods, due to reduced background signals originating from different energy forms of excitation source and detection signal (Haddour et al., 2006; Wang et al., 2009). To date, semiconductor nanomaterials have proved to be the most popular photoactive materials to construct photoelectrochemical biosensors. Thereinto, TiO2 is an excellent substrate photoactive material owing to its photoelectric activity, good biocompatibility, inexpensiveness, environmental safety, and chemical and physical stability (Qiu et al., 2011; Sano et al., 2012). Recently, one-dimensional TiO2 crystalline films including nanowires, nanotubes and nanorods have been caught particular attention since they have enlarged surface area and can provide direct pathway for photogenerated electron transfer, which accordingly leads to evident enhancement of charge separation, and effective prohibition of charge recombination (Shankar et al., 2009; Mor et al., 2006; Zhu et al., 2001; Liu and Aydil, 2009; Wu and Yu, 2004; Chen et al., 2010). However, as a wide energy band gap semiconductor (3.2 eV), TiO2 can only absorb the ultraviolet light ( o387 nm), leading to great limitation to utilization of light energy (Qiu et al., 2011). As a result, many efforts have been poured on the exploitation of TiO2-based hybrid structures to develop visible-light-motivated photoelectrochemical biosensors, which could adequately increase the light absorption efficiency, significantly enhance the photocurrent conversion efficiency and evidently promote the sensitivity of the related biosensors (Fan et al., 2014a, 2014b; Li et al., 2012). According to signal changes for detection, photoelectrochemical immunoassays can be divided into two types: signalon and signal-off. Currently, most of the developed photoelectrochemical immunoassays belong to the latter type, because steric hindrance generated by immunized recognition between antibody and antigen would apparently obstacle electron transfer, leading to photocurrent decrease. In order to enhance the sensitivity of photoelectrochemical immunoassays, enzymes are often employed for signal amplification (An et al., 2010; Zhao et al., 2012; Li et al., 2012). However, the introduction of enzyme not only increased the cost of sensor preparation but also made the testing process more complicated. Hence, establishing other simple, low cost and effective photoelectrochemical protocols for signal amplification would be highly expected. Inspired by several researches conducted by Willner and his co-workers, N-(2-
carboxymethyl)-N′-methyl-4,4′-bipyridinium (V2 þ ) possesses strong electron-withdrawing capability due to evident electron deficiency of its structure (Tel-Vered et al., 2008; SheeneyHaj-Ichia et al., 2002a, 2002b). Specifically, when semiconductor nanomaterials such as CdS or CdSe connected with V2 þ molecules, the photocurrent intensity was obviously lower than that of CdS or CdSe alone, because V2 þ molecules acted as traps for the conduction-band electrons (Tel-Vered et al., 2008; Zhang et al., 2012). Accordingly, V2 þ can be well used as signal-off labels linking with signal antibodies (Ab2) to form Ab2@V2 þ conjugates for signal amplification. As both V2 þ and Ab2 jointly facilitate decrease of photocurrent signal, using Ab2@V2 þ conjugates as signal amplification elements can contribute to an excellent sensitivity for signal-off photoelectrochemical immunoassays. However, to be of our knowledge, this kind of signal amplification protocol has not appeared in photoelectrochemical immunoassays. Herein, we presented an enhanced, promising platform to construct an ultrasensitive photoelectrochemical immunoassay for CA19-9 (antigen, Ag) detection based on TiO2NWs/Au/CdSe@ZnS sensitized structure and signal amplification of Ab2@V2 þ conjugates, as illustrated in Scheme 1. Firstly, TiO2NWs were synthesized by a hydrothermal growth method, and then were modified onto a bare ITO (indium tin oxide) electrode. Next, the ITO/TiO2NWs electrode was coated with Au nanoparticles, and then was modified with CdSe@ZnS film via layer-by-layer assembling oppositely charged polyelectrolyte and CdSe@ZnS QDs, forming TiO2NWs/Au/CdSe@ZnS sensitized structure to significantly enhance the photocurrent intensity. Afterwards, Ab1 was immobilized on the electrode by EDC coupling reaction between carbonyl and amino groups. After BSA blocked unbound sites on the electrode surface, the immunosensing electrode was ready. For target CA19-9 determination, different concentrations of Ag were first bound on the sensing electrode by specific immunoreaction between Ag and Ab1, and then the fixed concentration of Ab2@V2 þ conjugates as signal amplification elements were further immobilized through specific immunoreaction between Ag and Ab2, which led to significantly reduced photocurrent. The proposed photoelectrochemical protocol exhibited ultrahigh sensitivity, reproducibility, specificity, and stability.
Scheme 1. Construction process of the photoelectrochemical immunoassay for CA19-9 detection.
H. Zhu et al. / Biosensors and Bioelectronics 77 (2016) 339–346
341
2. Experimental
2.3. Synthesis of TiO2NWs
2.1. Materials and reagents
The TiO2 nanowires (TiO2NWs) were synthesized by a modified hydrothermal growth method according to the literature (Nikhil et al., 2014). Briefly, 2 g of TiO2 nanoparticles mixed with 35 mL of 15 M NaOH aqueous solution was transferred into a Teflon-lined digestion vessel and heated to 170 °C for 48 h, and then cooled down to room temperature. The supernatant was gently removed from the product formed at the bottom of the Teflon vessel. After the white products were soaked in 0.1 M HCl solution for 4 h, the solution was washed several times with DI water until it became neutral. Finally the wet white products were dried under vacuum at 100 °C for 4 h to obtain the dry solid of TiO2NWs.
Indium tin oxide (ITO) electrodes (type JH52, ITO coating 30 75 nm, sheet resistancer10 Ω/sq) were purchased from ZhongJingkeyi Technology Co., Ltd. (Nanjing, China). TiO2 powder (P25) was purchased from the Degussa Co. (Germany). Cadmium chloride (CdCl2 2.5H2O), zinc chloride (ZnCl2), sodium hydroxide (NaOH) and chloroauric acid (HAuCl4 4H2O) were purchased from Shanghai Chemical Reagent Co. (China). 4,4′-bipyridine was purchased from J&K Scientific Ltd. (Beijing, China). Methyl iodide (CH3I) was purchased from Xiya reagent (China). Chloroform (CHCl3) and acetonitrile (CH3CN) were purchased from Nanjing Chemical Reagent Co., Ltd. (China). Selenium powder (Se), sodium borohydride (NaBH4), thiolglycolic acid (TGA), poly(diallyldimethylammonium chloride) (PDDA, 20 wt% in water), N-hydroxysuccinimide (NHS), 3-mercaptopropionic acid (MPA), 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), acrylic acid (C3H4O2), and bovine serum albumin (BSA) were all obtained from Sigma-Aldrich (USA). Ascorbic acid (AA) was purchased from Sinopharm Chemical Reagent Co., Ltd. (China). Carbohydrate antigen 19-9 (CA19-9, Ag), capture CA19-9 antibody (Ab1), signal CA19-9 antibody (Ab2), carbohydrate antigen 15-3 (CA15-3), and prostate specific antigen (PSA) were obtained from Shanghai Linc-Bio Science Co. Ltd. (China). Human IL-6 and matrix metalloproteinase-2 (MMP-2) were obtained from Wuhan Uscn Life Science Inc. (China). All reagents were of analytical grade. All aqueous solutions were prepared with deionized water (DI water, 18.2 MΩ/cm) obtained from a Milli-Q water purification system. Phosphate buffer solution (PBS, pH 7.4, 10 mM) was used for the preparation of the antibody and antigen solution, washing buffer solution, and blocking buffer solution which contained 1% (w/v) BSA. 2.2. Apparatus The UV–visible (UV–vis) absorption spectra were tested on a UV-3600 UV–visible spectrophotometer (Shimadzu, Japan). Electrochemical impedance spectroscopy (EIS) was recorded on an Autolab potentiostat/galvanostat (PGSTAT 30, Eco Chemie B.V., Utrecht, Netherlands) with a three-electrode system in 0.1 M KCl solution containing 5.0 mM K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) mixture as a redox probe and recorded in the frequency range of 0.01 Hz to 100 kHz with an amplitude of 50 mV. Photoelectrochemical measurements were performed with a homemade photoelectrochemical system. A 500 W Xe lamp was used as the irradiation source with the light intensity of 400 μW/cm2 estimated by a radiometer (Photoelectric Instrument Factory of Beijing Normal University). Photocurrent was measured on a CHI 660D electrochemical workstation (Shanghai Chenhua Apparatus Corporation, China) with a three-electrode system: a 0.25 cm2 modified ITO as working electrode, a Pt wire as counter electrode and a saturated Ag/AgCl electrode as reference electrode. Transmission electron microscopy (TEM) was performed with a JEOL-2100 transmission electron microscope (JEOL, Japan). Field-emission scanning electron microscopy (FE-SEM) was performed on a Hitachi S-4800 scanning electron microscope (Hitachi Co., Japan) equipped with EX-250 Energy-dispersive X-ray spectroscopy (EDX, HORIBA Co., Japan). The 1HNMR spectra were measured on a Bruker AVANCE III-300 spectrometer using D2O as solvent and the data was reported as: chemical shift (δ ppm), multiplicity, and coupling constant (Hz).
2.4. Synthesis of V2 þ and Ab2@V2 þ conjugates V2 þ (N-(2-carboxymethyl)-N′-methyl-4,4′bipyridinium) was synthesized based on the literature method with appropriate modifications (see Supporting information). The typical preparation process for Ab2@V2 þ conjugates was as below. 1.0 mL saturated V2 þ solution was mixed gently with 600 μL of 10.0 mg/mL freshly prepared EDC for 30 min at room temperature. Subsequently, 50 μL of 0.2 mg/mL Ab2 was mixed with the above solution and incubated 2 h under shaking at 30 °C, and then stored overnight at 4 °C, which makes the unreacted EDC to lose its activity. Finally, the Ab2@V2 þ conjugates were acquired by centrifugation at 5500 rpm for 15 min using Millipore ultrafiltration centrifuge tube (MWCO¼ 100,000), and diluted to 1 mL by PBS (10 mM, pH 7.4). 2.5. Synthesis of CdSe@ZnS QDs The synthesis process of the CdSe@ZnS core–shell QDs included two procedures as below. The CdSe cores were synthesized based on the literature (Fan et al., 2014c). Typically, NaHSe solution was obtained by the reaction of Se powder with NaHB4 under nitrogen atmosphere at room temperature. After 50 mL of 2 mM CdCl2 was mixed with 20 μL of TGA, the pH of the system was adjusted to 10.0 by 1 M NaOH and bubbled with highly pure N2 for 30 min. And then 0.7 mL of 70 mM NaHSe solution was injected into this mixture to acquire a light yellow solution of CdSe precursors. The molar ratio of Se2 /Cd2 þ /TGA was 0.5/1/2.5. The resulting solution was then refluxed at 100 °C for 3 h to form CdSe cores. For the synthesis of CdSe@ZnS core–shell QDs, 70 μL of MPA and 8 mL of 0.5 M ZnCl2 were added into the CdSe cores solution under stirring. The mixture solution was then refluxed at 100 °C for 1 h to form CdSe@ZnS QDs. The resulting CdSe@ZnS QDs were purified by centrifugation at 8000 rpm for 15 min twice using Millipore ultrafiltration centrifuge tube (MWCO¼3000). 2.6. Preparation of ITO/TiO2NWs/Au/CdSe@ZnS electrode Before preparation, ITO slices were cleaned by ultrasonic treatment for 10 min in acetone, 1 M NaOH of water/ethanol mixture (1:1, v/v), and water, respectively, and then dried at 100 °C for 6 h. After certain amount of TiO2NWs was ultrasonically dispersed in DI water, 20 μL of the homogeneous suspension was coated onto a piece of ITO slice with fixed area of 0.25 cm2 and dried in air. The deposition of Au nanoparticles on ITO/TiO2NWs electrode was according to literature method (Li et al., 2014). The TiO2NWs modified electrode was firstly immersed into 0.01 M HAuCl4 aqueous solution (pH ¼ 4.5) for 4 h, and followed by washed with DI water, and then was annealed at 450 °C for 2 h in air atmosphere to generate Au nanoparticles on the TiO2NWs surface. And the desired ITO/TiO2NWs/Au electrode was obtained. The CdSe@ZnS multilayer film was fabricated by alternately
342
H. Zhu et al. / Biosensors and Bioelectronics 77 (2016) 339–346
dipping the ITO/TiO2NWs/Au electrode into a 0.5% positively charged PDDA solution and the negatively charged CdSe@ZnS QDs solution for 10 min, respectively. The film was carefully washed with DI water after each dipping step. The two-step dipping procedure was termed as “one coating”. The coating process was repeated three times, and the ITO/TiO2NWs/Au/CdSe@ZnS electrode was prepared. 2.7. Fabrication of the immunosensor Ab1 was immobilized onto the ITO/TiO2NWs/Au/CdSe@ZnS electrode via the EDC coupling reaction between carbonyl groups on the surface of the CdSe@ZnS QDs and NH2 groups of the Ab1. Initially, the electrode was immersed in a solution containing 20 mM EDC and 10 mM NHS for 30 min at room temperature. After rinsing with washing buffer solution to remove the unreacted EDC and NHS, 20 μL of 100 μg/mL Ab1 was spread onto the electrode surface and incubated at 4 °C in a moisture atmosphere overnight. After that, the electrode was rinsed with the washing buffer solution to remove unbound and physically absorbed Ab1. The electrode was then blocked by covering with 20 μL of BSA blocking buffer solution for 1 h at room temperature to block nonspecific binding sites and washed with the washing buffer solution thoroughly. Next, 20 μL of different concentrations of target Ag was covered on the BSA blocked electrode for an incubation of 1 h at 37 °C, followed by washing with washing buffer solution. After the specific immunoreaction between Ab1 and Ag, the electrode was labeled by incubation with 20 μL of Ab2@V2 þ conjugates solution for 1 h at 37 °C. Finally, the resulting electrode was washed thoroughly with washing buffer solution and was ready for photocurrent measurement. 2.8. Photoelectrochemical detection Photoelectrochemical detection was carried out in PBS (pH 7.4, 0.1 M) containing 0.1 M AA, which served as a sacrificial electron donor during the photocurrent measurement. White light produced by the Xe lamp, with a spectral range of 200–2500 nm, was utilized as excitation light and was switched on and off every 10 s. The applied potential was 0.0 V. The AA electrolyte was deaerated by pumping pure nitrogen for 10 min before photocurrent measurement.
3. Results and discussion 3.1. Characterization of TiO2NWs and TiO2NWs/Au hybrid structure Fig. 1A and B presents the FE-SEM images of the TiO2NWs and TiO2NWs/Au hybrid structure, respectively. It can be seen that the
TiO2NWs were about 3–6 μm in length and 60–80 nm in diameter (Fig. 1A). After Au nanoparticles subsequently grew on the TiO2NWs, many relatively small particles with the average size of about 10–15 nm were appeared on the TiO2NWs (Fig. 1B). Fig. S2 (in Supporting material) shows the elemental mapping analysis of TiO2NWs/Au, which suggested the presence of Ti, O and Au components in the hybrid structure and the deposited Au element was well scattered on the TiO2NWs. Besides, according to color changes from white to violet of the electrode surfaces (which was not shown here), it also confirmed the successful formation of the TiO2NWs/Au hybrid structure. 3.2. Characterization of CdSe cores and CdSe@ZnS QDs Fig. 2A and B shows high-resolution TEM (HRTEM) images of the prepared CdSe cores and CdSe@ZnS QDs, respectively. The lattice fringes of the two kinds of materials are clearly observed. The average size of the CdSe cores was about 3 nm (Fig. 2A), whereas the average size of CdSe@ZnS QDs was about 4.5 nm (Fig. 2B) which was larger than that of the CdSe cores, indicating successful synthesis of the CdSe@ZnS core-shell QDs. Fig. 2C presents the UV–vis absorption spectrum of the synthesized core– shell CdSe@ZnS QDs. It exhibited two sharp absorption peaks at around 450 nm and 500 nm, and a broad continuous absorption spectrum up to the wavelength of 570 nm which potentially indicated effective absorption under visible wavelengths. 3.3. Photoelectrochemical property of ITO/TiO2NWs/Au/CdSe@ZnS electrode As mentioned above, TiO2 can only absorb the ultraviolet light. Yet, CdSe with a lower band gap of 1.7 eV can extend the absorption range to longer-wavelength light. Moreover, CdSe possesses a higher conduction band edge than that of TiO2, which is beneficial for the injection of excited electrons from CdSe to TiO2 (Robel et al., 2006). Thus, combination of CdSe QDs with TiO2 could evidently increase the utilization of light energy and promote the photocurrent response. To further improve the photoelectrochemical properties of CdSe QDs, a shell of ZnS was introduced outside of the CdSe core to form the CdSe@ZnS core– shell structure, because ZnS shell with high conduction band and wide band gap could serve as a passivation layer to confine excitation electrons in CdSe core, reduce the formation of surface defects and the light corrosion, and inhibit recombination between electrons and redox couples (Arora and Manoharan 2007; Sambur and Parkinson, 2010). The introduction of Au nanoparticles could further improve the photocurrent intensity, because Au nanoparticles acted as light scattering centers for more light absorption of CdSe@ZnS QDs (Liu et al., 2011). In order to illustrate the positive effect of Au nanoparticles to photocurrent
Fig. 1. FE-SEM images of the (A) TiO2NWs (B) TiO2NWs/Au hybrid structure.
H. Zhu et al. / Biosensors and Bioelectronics 77 (2016) 339–346
343
Fig. 2. HRTEM images of the (A) CdSe cores and (B) CdSe@ZnS QDs; (C) UV–vis spectrum of the CdSe@ZnS QDs.
intensity, the control experiment was conducted, and it surely demonstrated this point (see Fig. S3 in Supporting material). Thus, herein, we first utilized the TiO2NWs/Au/CdSe@ZnS sensitized structure as photoelectrochemical matrix for the immunosensing electrode. As the concentration of TiO2NWs suspension, deposition of Au nanoparticles and coating number of CdSe@ZnS QDs could influence the photocurrent intensity of the ITO/TiO2NWs/Au/ CdSe@ZnS electrode, to achieve maximum photocurrent intensity, the optimal preparation conditions were exploited (see Fig. S4 in Supporting material). Thus, 0.8 mg/mL TiO2NWs, 4 h of soaking time in HAuCl4 solution, and three coating numbers of CdSe@ZnS QDs were selected to fabricate the ITO/TiO2NWs/Au/CdSe@ZnS electrode. 3.4. EIS characterization of the immunosensor EIS could provide effective information about the electrode surfaces. Generally, the electrochemical impedance spectrum includes a semicircle portion and a linear portion. The semicircle diameter equals the electron-transfer resistance (Ret), which reflects the electron-transfer kinetics of the redox probe at the electrode interface. Fig. 3 shows the Nyquist plots of impedance spectra corresponding to the different construction steps. For ITO/TiO2NWs electrode, the impedance spectrum exhibited a relatively small Ret (curve a), indicating a small electron transfer resistance. After Au nanoparticles deposition (curve b), the Ret reduced. It could be attributed to the good conductivity of Au nanoparticles (Zhou et al., 2014). After CdSe@ZnS QDs, Ab1, BSA, Ag and Ab2@V2 þ were modified onto the electrode surface step by step, the Ret gradually increased owing to low conductivity of semiconductor QDs or insulating effect of organic molecules (curve c–g), indicating that the layer-by-layer assembled immunosensor was successfully constructed.
Fig. 3. Nyquist plots of the (a) ITO/TiO2NWs electrode, (b) after Au nanoparticles deposition, (c) after CdSe@ZnS QDs immobilization, (d) after Ab1 immobilization, (e) after BSA blocking, (f) after incubation with 20 μL of 500 U/mL Ag, and then (g) further incubation with Ab2@V2 þ conjugates.
Fig. 4. Photocurrent responses of the (a) ITO/TiO2NWs/Au electrode, (b) after CdSe@ZnS QDs immobilization, (c) after Ab1 immobilization, (d) after BSA blocking, (e) after incubation with 20 μL of 500 U/mL Ag, and then (f) further incubation with Ab2@V2 þ conjugates.
3.5. Photoelectrochemical characterization of the immunosensor The fabrication process of the photoelectrochemical immunosensor was monitored by photocurrent responses, as shown in Fig. 4. The ITO/TiO2NWs/Au electrode exhibited a relatively small photocurrent response (curve a, I¼ 10.58 μA). After CdSe@ZnS QDs modification, the photocurrent intensity (curve b, I¼ 130.43 μA) increased to 12 times higher than that of the ITO/TiO2NWs electrode, which was because that the TiO2NWs electrode possessed a large surface area for much more CdSe@ZnS QDs QDs loading, and the modified CdSe@ZnS QDs extended the absorption range to longer-wavelength light ( 570 nm), and meanwhile the deposited Au nanoparticles could act as light scattering centers to further increase the light absorption of CdSe@ZnS QDs (Liu et al., 2011; Zarazua et al., 2011). After Ab1 and BSA were successively immobilized on the electrode, the photocurrent intensity decreased (curve d, I ¼83.23 μA; curve e, I¼ 70.70 μA), which could be attributed to the steric hindrance of those modified protein molecules. After the sensing electrode was incubated with target Ag and then Ab2@V2 þ conjugates, the photocurrent intensity decreased to 78% and 25% of the initial sensing electrode (curve f, I¼ 55.24 μA; curve g, I ¼17.47 μA). Obviously, the decrement of photocurrent intensity for target Ag modification was evidently less than that of the subsequently modified Ab2@V2 þ conjugates. This could be attributed to strong electron-withdrawing property of V2 þ coupled with obvious steric hindrance of Ab2 (Tel-Vered et al., 2008). Besides, in order to verify the synergy effect of Ab2 and V2 þ in Ab2@V2 þ conjugates, Ab2 was also used as signal amplification elements for photocurrent test, and the results exhibited that the decrement of photocurrent response to Ab2 was only about 38% of that to Ab2@V2 þ conjugates, indicating the superiority of the Ab2@V2 þ conjugates as signal amplification elements. Thus, the photocurrent responses
344
H. Zhu et al. / Biosensors and Bioelectronics 77 (2016) 339–346
Table 1 Analytical performances of various bioassays for CA19-9 detection. Immunoassay
Linear range [U/mL]
Detection limit [U/mL]
Reference
Electrochemical
0.015–150
0.006
Yang et al. (2015) Sha et al. (2015) Shi et al. (2014) Gu et al. (2011) Zhao et al. (2009) This work
Electrochemiluminescence 0.001–5
0.0005
Chemiluminescence
0.016
Photoluminescence
0.1–180
0.04
Fluorometric
3.0–500
2.30
Photoelectrochemical
Scheme 2. Photogenerated electron–hole transfer mechanism of the immunosensor for target Ag detection.
demonstrated the successful construction of the proposed immunoassay. In a word, two major reasons contributed to ultrahigh sensitivity of the proposed photoelectrochemical immunoassay, as illustrated in Scheme 2. Before target Ag detection, TiO2NWs/Au/ CdSe@ZnS sensitized structure could evidently promote the photocurrent response of the sensing electrode, because it could adequately absorb the light energy and effectively inhibit the electron–hole recombination. However, in the presence of target Ag, the Ab2@V2 þ conjugates could significantly decrease the photocurrent detection signal because of strong electron-withdrawing capability of V2 þ coupled with obvious steric hindrance of Ab2. Accordingly, based on the two main aspects mentioned above, ultrahigh sensitivity was realized. 3.6. Photoelectrochemical detection for CA19-9 CA19-9 detection was based upon the sandwich immunoreactions between Ab1 and target Ag as well as target Ag and Ab2@V2 þ conjugates. The photocurrent response of this immunoassay was directly related to the concentration of target Ag. Thus, the photoelectrochemical immunosensor toward CA19-9 detection can be achieved by monitoring the photocurrent change. Fig. 5A presents the photocurrent responses of the immunosensor after being incubated with different concentrations of target Ag and then with fixed concentration of Ab2@V2 þ conjugates. Along with the concentration of target Ag increased, more and more Ab2@V2 þ conjugates were specifically bound on the electrode, leading to gradually decreased photocurrent intensity. As shown in Fig. 5B, the
0.025–1
0.01–200
0.0039
photocurrent response linearly decreased with an increasing logarithm of the concentration of target Ag in the range from 0.01 U/mL to 200 U/mL. The regression equation was I¼ 44.56 8.27log CCA19-9 (U/mL), with the correlation coefficient of 0.9986. The limit of detection (LOD, S/N ¼ 3) for target Ag concentration was estimated to be 0.0039 U/mL. In order to evaluate the sensitivity of the proposed photoelectrochemical immunoassay, other highly sensitive bioassays for CA19-9 detection reported previously were listed out, as shown in Table 1, which demonstrated the well-designed photoelectrochemical immunoassay also exhibited wider linear range as well as a lower detection limit. 3.7. Reproducibility, specificity and stability of the immunoassay The reproducibility of the immunoassay was assessed by both intra-assay (within-batch) and inter-assay (between-batch) relative standard deviation (RSD). Analyzed from the testing results of five replicate determinations, the intra-assay RSDs were 3.2%, 2.7%, and 2.4% towards 0.05, 0.1, and 0.2 U/mL of CA19-9, respectively. The inter-assay RSDs of 4.4%, 3.6%, and 3.7% were acquired via measuring the same samples with five electrodes fabricated independently under identical experimental conditions. All of these results suggested an acceptable reproducibility of the proposed immunoassay. Specificity is a crucial criterion for immunoassay, since the nonspecific adsorption can influence the sensitivity. To verify that the photocurrent response originated from specific binding, some representative interfering proteins including carcinoma antigen 15-3 (CA15-3), human interleukin-6 (IL-6), matrix metalloproteinases-2 (MMP-2), and prostate-specific antigen (PSA) were selected for the interference test. As shown in Fig. 6, no significant
Fig. 5. (A) Photocurrent responses and (B) calibration curve of the immunoassay for the detection of different concentrations of CA19-9 from 0.01 U/mL to 200 U/mL. The error bars showed the standard deviation of five replicate determinations.
H. Zhu et al. / Biosensors and Bioelectronics 77 (2016) 339–346
345
Saud University for funding the work through the research group project No. RGP-VPP-029.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2015.09.051.
References
Fig. 6. Photocurrent responses of the immunoassay for 1 U/mL CA19-9 detection (a) in the absence of interference or in the existence of 1 U/mL of (b) CA15-3, (c) IL6, (d) MMP-2, (e) PSA, and (f) their mixture. The error bars showed the standard deviation of five replicate measurements.
difference of photocurrent response could be observed as compared to the result obtained in the presence of only CA19-9 at the concentration of 1 U/mL. It indicates that the photocurrent response of the immunosensor was not affected by CA15-3, IL-6, MMP-2, PSA and their mixture. The relative deviations of the photocurrents tested in the presence of single interfering protein or their mixture were well within 4.2% for the measurement of 1 U/mL CA19-9 compared with the photocurrent detected in the absence of interfering protein. And the relative standard deviation (RSD) of five replicate determinations for each interference test was within 3.8%. All these results proved that the designed immunoassay possessed satisfactory specificity. The stability of the constructed immunoassay was also evaluated. After the sensing electrode was stored in PBS (pH 7.4, 10 mM) at 4 °C in a refrigerator for over two weeks, the photocurrent intensity kept 95.6% of its initial response, indicating it possessed good storage stability.
4. Conclusion In summary, we have presented an enhanced, promising protocol of photoelectrochemical immunoassay for ultrasensitive detection of CA19-9 based on cooperation effect of TiO2NWs/Au/ CdSe@ZnS sensitized structure and signal amplification of Ab2@V2 þ conjugates. Compared to the results reported previously, the well-established immunoassay exhibited a lower detection limit of 0.0039 U/mL as well as a wider linear range from 0.01 U/mL to 200 U/mL for CA19-9 detection. The greatly enhanced sensitivity was attributed to following reasons. Firstly, the sensitized structure of TiO2NWs/Au/CdSe@ZnS could evidently promote the photocurrent intensity due to its prominent properties of adequate absorption of light energy and effective depression of charge recombination. Secondly, the well-designed signal amplification elements of Ab2@V2 þ conjugates could significantly reduce the photocurrent detection signal resulting from its strong electron-withdrawing property coupled with obvious steric hindrance effect. The proposed photoelectrochemical protocol is highly expected to the detection of trace levels of disease-related biomarkers.
Acknowledgments We gratefully appreciate the National Natural Science Foundation (21375059, 21175065 and 21335004) and the National Basic Research Program (2011CB933502) of China. The authors extend their appreciation to the Deanship of Scientific Research at King
An, Y.R., Tang, L.L., Jiang, X.L., Chen, H., Yang, M.C., Jin, L.T., Zhang, S.P., Wang, C.G., Zhang, W., 2010. Chem. Eur. J. 16, 14439–14446. Arora, S., Manoharan, S., 2007. Solid State Commun. 144, 319–323. Chen, H., Fu, W.Y., Yang, H.B., Sun, P., Zhang, Y.Y., Wang, L.R., Zhao, W.Y., Zhou, X.M., Zhao, H., Jing, Q., Qi, X.F., Li, Y.X., 2010. Electrochim. Acta 56, 919–924. Fan, G.C., Han, L., Zhu, H., Zhang, J.R., Zhu, J.J., 2014a. Anal. Chem. 86, 12398–12405. Fan, G.C., Han, L., Zhang, J.R., Zhu, J.J., 2014b. Anal. Chem. 86, 10877–10884. Fan, G.C., Ren, X.L., Zhu, C., Zhang, J.R., Zhu, J.J., 2014c. Biosens. Bioelectron. 59, 45–53. Gold, D.V., Modrak, D.E., Ying, Z.L., Cardillo, T.M., Sharkey, R.M., Goldenberg, D.M., 2006. J. Clin. Oncol. 24, 252–258. Gu, B.X., Xu, C.X., Yang, C., Liu, S.Q., Wang, M.L., 2011. Biosens. Bioelectron. 26, 2720–2723. Gui, J.C., Yan, W.L., Liu, X.D., 2013. Clin. Exp. Med. 14, 225–233. Haddour, N., Chauvin, J., Gondran, C., Cosnier, S., 2006. J. Am. Chem. Soc. 128, 9693–9698. Heidari, M.H., Porghasem, M., Mirzaei, N., Mohseni, J.H., Heidari, M., Azargashb, E., Movafagh, A., Heidari, R., Molouki, A., Larijani, L., 2014. J. Environ. Radioact. 128, 64–67. Jha, D.K., Mittal, A., Gupta, S.P., Sathian, B., 2013. Asian Pac. J. Cancer Prev. 14, 4067–4069. Kang, H.Y., Miao, J.R., Cao, Z.J., Lu, J.Z., 2009. Analyst 134, 2246–2252. Kitano, H., 2002. Science 295, 1662–1664. Ko, J.H., Lee, S., Lee, E.K., Chang, S.I., Chen, L.X., Yoon, S.Y., Choo, J., 2013. Phys. Chem. Chem. Phys. 15, 5379–5385. Li, J.T., Cushing, S.K., Zheng, P., Senty, T., Meng, F.K., Bristow, A.D., Manivannan, A., Wu, N.Q., 2014. J. Am. Chem. Soc. 136, 8438–8449. Li, Y.J., Ma, M.J., Zhu, J.J., 2012. Anal. Chem. 84, 10492–10499. Lin, J.H., Ju, H.X., 2005. Biosens. Bioelectron. 20, 1461–1470. Liu, B., Aydil, E.S., 2009. J. Am. Chem. Soc. 131, 3985–3990. Liu, L.P., Wang, G.M., Li, Y., Li, Y.D., Zhang, J.Z., 2011. Nano Res. 4, 249–258. Mor, G.K., Shankar, K., Paulose, M., Varghese, O.K., Grimes, C.A., 2006. Nano Lett. 6, 215–218. Narita, Y., Taniguchi, H., Komori, A., Nitta, S., Yamaguchi, K., Kondo, C., Nomura, M., Kadowaki, S., Takahari, D., Ura, T., Andoh, M., Muro, K., 2014. Cancer Chemother. Pharmacol. 73, 409–416. Nikhil, A., Thomas, D.A., Amulya, S., Raj, S.M., Kumaresan, D., 2014. Sol. Energy 106, 109–117. Qiu, J.X., Zhang, S.Q., Zhao, H.J., 2011. Sens. Actuators B 160, 875–890. Robel, I., Subramanian, V., Kuno, M., Kamat, P.V., 2006. J. Am. Chem. Soc. 128, 2385–2393. Sambur, J.B., Parkinson, B.A., 2010. J. Am. Chem. Soc. 132, 2130–2131. Sano, M., Saotome, M., Urushida, T., Katoh, H., Satoh, H., Ohlishi, K., Hayashi, H., 2012. Intern. Med. 51, 2337–2340. Sha, Y.H., Guo, Z.Y., Chen, B.B., Wang, S., Ge, G.P., Qiu, B., Jiang, X.H., 2015. Biosens. Bioelectron. 66, 468–473. Shankar, K., Basham, J.I., Allam, N.K., Varghese, O.K., Mor, G.K., Feng, X., Paulose, M., Seabold, J.A., Choi, K.S., Grimes, C.A., 2009. J. Phys. Chem. C 113, 6327–6359. Sheeney-Haj-Ichia, L., Willner, I., 2002a. J. Phy. Chem. B 106, 13094–13097. Sheeney-Haj-Ichia, L., Wasserman, J., Willner, I., 2002b. Adv. Mater., 1323–1326. Sheng, Z.H., Han, H.Y., Hu, D.H., Liang, J.G., He, Q.G., Jin, M.L., Zhou, R., Chen, H.C., 2009. Chem. Commun., 2559–2561. Shi, M., Zhao, S.L., Huang, Y., Zhao, L.M., Liu, Y.M., 2014. Talanta 124, 14–20. Srinivas, P.R., Kramer, B.S., Srivastava, S., 2001. Lancet Oncol. 2, 698–704. Tang, D.P., Hou, L., Niessner, R., Xu, M.D., Gao, Z.Q., Knopp, D., 2013. Biosens. Bioelectron. 46, 37–43. Tel-Vered, R., Yehezkeli, O., Yildiz, H.B., Wilner, O.I., Willner, I., 2008. Angew. Chem. 120, 8396–8400. Wang, G.L., Yu, P.P., Xu, J.J., Chen, H.Y., 2009. J. Phys. Chem. C 113, 11142–11148. Wang, Y., Vaidyanathan, R., Shiddiky, M.J.A., Trau, M., 2015. ACS Nano 9, 6354–6362. Wu, J.J., Yu, C.C., 2004. J. Phys. Chem. B 108, 3377–3379. Xiao, J.C., He, X.Y., Wang, Z.Y., Hu, J.Y., Sun, F., Qi, F., Yang, S.G., Xiao, Z.Y., 2014. Tumor Biol. 35, 1331–1334. Yang, F., Yang, Z.H., Zhuo, Y., Chai, Y.Q., Yuan, R., 2015. Biosens. Bioelectron. 66, 356–362. Zarazua, I., Rosa, E.D., Lopez-Luke, T., Reyes-Gomez, J., Ruiz, S., Chavez, C.A., Zhang, J. Z., 2011. J. Phys. Chem. C 115, 23209–23220. Zhang, X.R., Xu, Y.P., Yang, Y.Q., Jin, X., Ye, S.J., Zhang, S.S., Jiang, L.L., 2012. Chem. Eur. J. 18, 16411–16418. Zhao, J.J., Jin, X., Vdovenko, M., Zhang, L.L., Sakharov, I.Y., Zhao, S.L., 2015. Chem.
346
H. Zhu et al. / Biosensors and Bioelectronics 77 (2016) 339–346
Commun. 51, 11092–11095. Zhao, W.W., Ma, Z.Y., Yu, P.P., Dong, X.Y., Xu, J.J., Chen, H.Y., 2012. Anal. Chem. 84, 917–923.Zhao, Y.J., Zhao, X.W., Pei, X.P., Hua, J., Zhao, W.J., Chen, B., Gu, Z.Z., 2009. Anal. Chim. Acta 633, 103–108. Zhou, Y.L., Xu, Z.N., Wang, M., Sun, B., Yin, H.S., Ai, S.Y., 2014. Biosens. Bioelectron.
53, 263–267. Zhu, P.X., Shelton, D.R., Li, S.H., Adams, D.L., Karns, J.S., Amstutz, P., Tang, C.M., 2011. Biosens. Bioelectron. 30, 337–341. Zhu, Y., Li, H., Koltypin, Y., Hacohen, Y.R., Gedanken, A., 2001. Chem. Commun., 2616–2617.