Biosensors and Bioelectronics 62 (2014) 315–319
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Short communication
A novel multi-amplification photoelectrochemical immunoassay based on copper(II) enhanced polythiophene sensitized graphitic carbon nitride nanosheet Rongxia Li a, Yixin Liu b, Xiaojian Li b, Sen Zhang a, Dan Wu b, Yong Zhang b, Qin Wei b, Bin Du a,n a
School of Resources and Environment, University of Jinan, Jinan 250022, China Key Laboratory of Chemical Sensing & Analysis in Universities of Shandong (University of Jinan), School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, China
b
art ic l e i nf o
a b s t r a c t
Article history: Received 14 March 2014 Received in revised form 12 June 2014 Accepted 13 June 2014 Available online 3 July 2014
A new sandwich photoelectrochemical (PEC) sensing strategy was proposed for the first time based on the increasing photocurrent of water-soluble polythiophene sensitized g-C3N4 nanosheet (PT-Cl/g-C3N4) in the presence of copper(II) (Cu2 þ ), which was doped on the surface of titanium dioxide as labels for multi-amplification. Herein, the photoactive films of PT-Cl/g-C3N4 is employed as the photoactive antibody (Ab1) immobilization matrix for the subsequent sandwich-type antibody–antigen affinity interactions. Upon the presence of antigen (Ag), greatly enhanced photocurrent could be triggered in the PEC platform by the labels of second antibody (Ab2) of Cu2 þ doped titanium dioxide (Cu2 þ –TiO2). As a result of the multi-amplification in this Cu2 þ –TiO2 enhanced PT-Cl/g-C3N4-based PEC immunoassay, it possesses excellent analytical performance. The antigen could be detected from 0.01 pg mL 1 to 100.0 ng mL 1 with a detection limit of 5 fg mL 1. This work opens up g-C3N4 nanosheet applied in PEC sensing. More importantly, the strategy of specific positive effect of Cu2 þ on the photocurrent of g-C3N4 opens an alternative horizon for PEC sensing. & 2014 Elsevier B.V. All rights reserved.
Keywords: Polythiophene Sensitized g-C3N4 nanosheet Cu2 þ doped titanium dioxide Photoelectrochemical immunoassay Multi-amplification
1. Introduction Recently, polymer-like semiconductor material graphitic carbon nitride (g-C3N4) has attracted great interest in numerous fields including photocatalytic water splitting, photodegradation of environmental organic pollutants and electrochemical sensors, because the stable and metal-free g-C3N4 possess energy band characteristics of metal oxide semiconductors (Wang et al., 2012a, 2012b, 2009a, 2009b). However, low carrier mobility, insufficient sunlight absorption and high recombination rate of photogenerated electron–hole pairs in the bare g-C3N4 system, limit its potential application in the area of photoelectric conversion (Sun et al., 2012). To solve these problems, some effective approaches have been developed to further optimize the photoelectric performance of g-C3N4, including doping it with metallic or nonmetallic elements, and copolymerizing it with other organic compounds (Zhang et al., 2010; Wang et al., 2009a, 2009b; Cheng et al., 2013). Even though, significant improvement of its photoelectric conversion efficiency has been shown by the methods mentioned above, n
Corresponding author. Tel./fax: þ 86 531 82765730. E-mail address: [email protected] (B. Du).
http://dx.doi.org/10.1016/j.bios.2014.06.061 0956-5663/& 2014 Elsevier B.V. All rights reserved.
the more effective and simple photosensitizers to improve the performance of g-C3N4-based photovoltaic devices are highly deserved. Conjugated polymers (CPs) are attracting more and more interest because of their wide absorption coefficients leading to efficient light harvesting properties, which could reduce the thickness of the semiconductor film (Yanagida et al., 2004). In quest of commercially viable conducting polymers, polythiophene and its derivatives have always been the most promising candidates because they have high absorption coefficiency and chargecarrier mobility, good chemical stability and thermal stability, easy modification and controlling for electrochemical and photoelectrochemical (PEC) behavior (Gazotti et al., 2001; Schilinsky et al., 2002; Valaski et al., 2001; Huynh et al., 2002; Song et al., 2003; Breeze et al., 2001). Although obviously improved performance were obtained through the coupling of CPs and inorganic semiconductors (e.g. TiO2) (Smestad et al., 2003; Arango et al., 1999; Grant and Schwartzberg, 2002; Yanagida et al., 2004) and carbon nanotubes (CNT) (Sgobba et al., 2009), to the best of our knowledge, little work has been done about these CPs as photosensitizers (“dye” molecules) in the g-C3N4 systems. In the present study, a novel water-soluble cationic conjugated polymer denoted
316
R. Li et al. / Biosensors and Bioelectronics 62 (2014) 315–319
as poly 2,5-[3-(1,1-dimethyl-4-piperidine methylene) thiophene] chloride (PT-Cl) was used to be an efficient photo-sensitizer for enhancing PEC sensing of g-C3N4. The novel PT-Cl-sensitized g-C3N4 photoactive films (PT-Cl/g-C3N4) with super-photoelectric performance were fabricated by assembling of positively charged PT-Cl and negative charged carboxylated g-C3N4. Additionally, in order to further improve the crystal morphology of the photoactive films to acquire higher photoelectric conversion efficiency as well as better stability of modified electrode, a method of calcination at 450 °C was employed in this work. In recent reports, g-C3N4 has been applied to the PEC selective sensing of copper (II) (Cu2 þ ) in aqueous solutions, which were based on the photocurrent intensity of the g-C3N4 increased with the increasing concentration of Cu2 þ (Xu et al., 2013, 2014; She et al., 2014). Inspired by this mind, in present work, the Cu2 þ -loaded nanomaterial was explored as labels for the first time to enhance the photocurrent of g-C3N4-based materials. Nanoporous titanium dioxide (TiO2) has been particularly investigated in recent decades since they have high photo-current conversion efficiency, superior optical and electrical properties (Gajjela et al., 2010; Lutz et al., 2012; Wang et al., 2011; Leshuk et al., 2012; Liu et al., 2010). In this study, copper ions surface-doped nanoporous titanium dioxide microspheres (Cu2 þ –TiO2) was employed as labels of secondary antibodies (Ab2) to obtain multi-amplification by using the excellent photoelectric conversion efficiency of TiO2 as well as the specific positive effect of Cu2 þ on the photocurrent of g-C3N4. The multi-amplification in this Cu2 þ –TiO2 enhanced PT-Cl/g-C3N4 -based PEC immunoassay possesses excellent analytical performance and opens an alternative horizon for PEC sensing.
2. Experimental section
which proved the successful loading of Cu2 þ onto the TiO2. The details of preparation of Cu2 þ –TiO2–Ab2 was in Supplementary materials. The utilized water-soluble Poly 2,5-[3-(1,1-dimethyl-4piperidine methylene) thiophene] chloride (PT-Cl) was synthesized according to literature (Li et al., 2012). 2.4. Immunoassay development Scheme 1 shows the developing process of the multi-amplified PEC immunoassay. Firstly, 4 μL negative charged g-C3N4 solution and 4 μL positively charged PT-Cl solution was dropped onto the pretreated ITO surface sequentially and kept for 20 min. After each dropping step, the ITO sample zone was rinsed thoroughly, dried in room temperature and then annealed at 450 °C for 5 min in a muffle furnace to obtain desired film stability and photocurrent intensity. Afterwards, 4 μL of 0.5 mg mL 1 Ab1 was immobilized onto the PT-Cl/g-C3N4 modified ITO electrode via physical process, after incubation for 1 h, the electrode was rinsed with the washing buffer to remove physically adsorbed Ab1. The electrode was then blocked with 4 μL blocking solution for 1 h at 4 °C to block nonspecific binding sites and washed with the washing buffer thoroughly. Next, 4 μL of NMP-22 antigen with different concentrations were dropped onto the Ab1 modified electrodes and incubated for 1 h at 37 °C followed by washing with washing buffer. After the binding reaction between Ab1 and the antigen, the electrodes were allowed for labeling by additional incubation with 4 μL diluted Cu2 þ –TiO2–Ab2 bioconjugates solution for 1 h. Thereafter, the electrodes were washed thoroughly with washing buffer to remove nonspecifically bound conjugations, and immersed in the PBS (pH 5.4) for 5 min before measurements. Then, the photocurrent was measured by the current–time technique at a bias voltage of 0.2 V following a 200 W LED excitation.
2.1. Chemicals and reagents 3. Results and discussion Ethylenediamine (NH2CH2CH2NH2, Z99%), glutaraldehyde (25%), Bovine serum albumin (BSA) (96–99%) were obtained from SigmaAldrich (Beijing, China). All aqueous solutions were prepared using ultrapure water (Milli-Q, Millipore). The details are shown in Supplementary information. 2.2. Apparatus Fourier Transform Infrared Spectrometer (FT-IR Spectrometer) was recorded by VERTEX70 spectrometer (Bruker Co., Germany). The details are shown in Supplementary information. 2.3. Preparation and characterization of materials In this work, carboxylated g-C3N4 was synthesized according to the reference with some slight modifications (Cheng et al., 2013). As shown in Fig. S1, the SEM (Fig. S1A) and TEM images (Fig. S1B) of the carboxylated g-C3N4 illustrated the nanosheet structure, which could provide excellent photoelectric properties and large surface area. Fig. S1C shows the FT-IR spectrum of g-C3N4 (blank line) and carboxylated g-C3N4 (red line) in the wavelength range of 500–4000 cm 1. The peak of 1720 cm 1 is corresponding to the CQO bending band and the absorption band for –COO at 1575 cm 1 and 1380 cm 1, indicating successful functionalization of carboxyl group on g-C3N4 (Dong et al., 2013). The fabrication of TiO2 and Cu2 þ –TiO2 was conducted following a literature procedure with some modifications (Li et al., 2008). As the TEM images of TiO2 and Cu2 þ –TiO2 show (Fig. S1D and E), the as-prepared TiO2 possess uniform size with an average diameter of 250 nm (Fig. S1D). The photo of Fig. S1E showed that there was an obvious change in the color of the powder after the assembling of Cu2 þ ,
Fig. 1A reveals the stepwise photocurrent responses of corresponding assembly. Curve a presented the photocurrent of PT-Cl/g-C3N4 modified ITO electrode. Even though, the photocurrent decreased gradually with Ab1 anchoring, BSA blocking and Ag specific binding (curves b–d), which was due to the steric hindrance of the hydrophobic protein layer (curves c–e, Fig. S3). However, after the final amplification via Cu2 þ –TiO2 by the formation of Cu2 þ –TiO2–Ab2–Ag conjugations, the photocurrent intensity was 3 times of the initial intensity (curve e). Compared with pure g-C3N4 (curve a, Fig. 1B), the coupling of PT-Cl and g-C3N4 resulted in a dramatic enhanced photocurrent (curve b, Fig. 1B). This may be ascribed to the wide absorption coefficients of PT-Cl that leading to efficient light harvesting properties (Yanagida et al., 2004), and the fast separation of charge carriers in the PT-Cl/g-C3N4 system caused by PT-Cl (Sgobba et al., 2009). Moreover, as shown in Fig. 1B, the photocurrent of Cu2 þ –TiO2 (curve d) as labels was clearly higher than that of TiO2 (curve c), indicating the prominent synergy enhancement of TiO2 and Cu2 þ . In comparison with the previous PEC immunoassay protocols, the prominent signal enhancement should be attributed to the Cu2 þ –TiO2 labeling-induced multi-signal amplification. Specifically, the addition of Cu2 þ would significantly increase the photocurrent of g-C3N4. The possible mechanism (see Fig. S2) may be that photo-generated electron of g-C3N4 transmitted to the ITO electrode, and then to the Pt reference electrode and formed loops (Xu et al., 2014). The photoinduced electron could be captured by Cu2 þ that released from Cu2 þ –TiO2–Ab2 in weakly acid solution, thus promoted the separation rate of electron and hole and led to the increasement of the photocurrent response. Besides, TiO2 would expand the light absorption of the underlying
R. Li et al. / Biosensors and Bioelectronics 62 (2014) 315–319
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Scheme 1. Schematic diagram of the construction of PT-Cl/C3N4 nanosheet films (A) and Cu2 þ –TiO2–Ab2 (B) and the fabrication of a NMP-22 biosensor (C).
Fig. 1. (A) Photocurrent response of the modified electrode (a) before and (b) after Ab1 anchoring, (c) after further BSA blocking, (d) after NMP-22 incubation (10 ng mL 1), (e) after further Cu2 þ –TiO2 labeling. (B) Photocurrent response of the modified electrode with (a) g-C3N4, (b) PT-Cl/g-C3N4, (c) PT-Cl/g-C3N4/Ab1/BSA/NMP-22/TiO2–Ab2, (d) PT-Cl/g-C3N4/Ab1/BSA/NMP-22/Cu2 þ –TiO2–Ab2. The PEC tests were performed in 0.1 M PBS (pH ¼5.4) with 0.2 V applied potential and 430 nm excitation light.
PT-Cl/g-C3N4 film enormously, thus caused the further enhancement of photocurrent. Since the novel nanolabel carrying considerable Cu2 þ , using NMP-22 as a model, the presence of trace quantities of NMP-22 would be greatly amplified by the synergy effect of TiO2 and Cu2 þ . The extent of final photocurrent enhancement associates with the NMP-22 concentration intimately. Under the optimal conditions (Figs. S4 and S5), via tracking the increasement of the photocurrent, an exquisite PEC NMP-22 immunoassay could be tailored (Fig. 2A and B). The response had a fairly good logarithmical relationship with the concentration of the NMP-22 ranging from 0.01 pg mL 1 to 100 ng mL 1 with a correlation coefficient of 0.996, and the limit of detection (LOD)
was 5 fg mL 1 at 3 s/slope. The achieved LOD is much lower than the clinical judgment value (6.5 U mL 1 ) in urine set by US Food and Drug Administration (Shariat et al., 2004). The photocurrent enhances as the NMP-22 concentration increases, indicating the NMP-22-controlled enhancement of the synergy effect. Due to the synergy effect as stated above, the NMP-22 could be detected at levels down to 5 fg mL 1 , which is much lower than previous electrochemical method (0.5 ng mL 1 ) (Ning et al., 2007). Control experiments revealed that the developed PEC sensor does not exhibit any obvious changes in photocurrents when incubating the as-fabricated immunosensor in sample solutions containing a 100-fold excess of different interfering agents such as
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Fig. 2. (A) Effect of different NMP-22 concentrations on the differential photocurrent responses, (a)–(f) 0.01, 0.1, 1, 10, 100, 1000, 10,000, 100,000 pg mL 1, respectively. (B) The corresponding calibration curve. The changes of photocurrent was the changes between the photocurrent of the modified electrode prior to NMP-22 immobilization and final photocurrent of the electrode after incubation with Ag of elevated concentrations corresponding to 0.01, 0.1, 1, 10, 100, 1000, 10,000, 100,000 pg mL 1, respectively, and then after the final TiO2–Cu2 þ –Ab2. (C) Selectivity of NMP-22 toward different analytes (ascorbic acid, adenine, guanine and uric acid, 1 mg mL 1) which are 100-fold concentrations of the NMP-22. (D) Time-based photocurrent response of PT-Cl/g-C3N4/Ab1/BSA/NMP-22/Cu2 þ –TiO2–Ab2 modified ITO electrode under several on/off irradiation cycles for 300 s.
ascorbic acid, adenine, guanine and uric acid (Fig. 2C). The results demonstrate that the specific immunobinding should be responsible for the photocurrent enhancement and thus the good selectivity. It is also worth mentioning that the photocurrent response of the PEC sensor was fairly reversible and stable under several on/off irradiation cycles for 300 s. As shown in Fig. 2D, the current could reproducibly increase violently under each irradiation and recover rapidly in the dark, indicating the structural stability of the developed PEC sensors and their potential for biosensing experiments. The reproducibility of this PEC NMP-22 immunoassay is also assessed by an interassay relative standard deviation (RSD). RSD of 6.1% was obtained by assaying the same 10 ng mL 1 NMP-22 samples with five electrodes, indicating satisfactory reproducibility. Compared to other PEC approaches, this immunoassay also had the advantages of low cost (4 mL of each step and no enzyme is used), and being environment-friendly (toxic precursors such as cadmium are not used) and so on. Obviously, the proposed PEC sensor showed promise for application in the monitoring of NMP-22 with high sensitivity and fast response.
4. Conclusions In summary, a novel PEC immunoassay for protein was first proposed. It is based on the in Cu2 þ –TiO2-enhanced PT-Cl/g-C3N4 nanosheet-based photoelectrochemistry via sandwich immunoreaction. This work has shown that NMP-22 can be detected at very low concentrations by a new PEC immunoassay protocol, which provides a feasible alternative technique for NMP-22 screening to facilitate early bladder cancer diagnosis. Upon the introduction of a novel labeling system, synergy effects could be triggered in the PEC platform for multi-signal amplification. With enhanced analytical performance, this strategy opens a different perspective for ultrasensitive protein detection especially for trace cancer markers (or other infectious/biothreatening agents and DNA analysis), in which high sensitivity is strongly required.
Acknowledgments This work was financially supported by the National Natural Science Foundation of China (21375047, 21377046, and 21245007),
R. Li et al. / Biosensors and Bioelectronics 62 (2014) 315–319
the Science and Technology Plan Project of Jinan (No. 201307010), and QW thanks the Special Foundation for Taishan Scholar Professorship of Shandong Province and UJN (No. ts20130937).
Appendix A. Supplementary information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.06.061.
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Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Short communication
A novel multi-amplification photoelectrochemical immunoassay based on copper(II) enhanced polythiophene sensitized graphitic carbon nitride nanosheet Rongxia Li a, Yixin Liu b, Xiaojian Li b, Sen Zhang a, Dan Wu b, Yong Zhang b, Qin Wei b, Bin Du a,n a
School of Resources and Environment, University of Jinan, Jinan 250022, China Key Laboratory of Chemical Sensing & Analysis in Universities of Shandong (University of Jinan), School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, China
b
art ic l e i nf o
a b s t r a c t
Article history: Received 14 March 2014 Received in revised form 12 June 2014 Accepted 13 June 2014 Available online 3 July 2014
A new sandwich photoelectrochemical (PEC) sensing strategy was proposed for the first time based on the increasing photocurrent of water-soluble polythiophene sensitized g-C3N4 nanosheet (PT-Cl/g-C3N4) in the presence of copper(II) (Cu2 þ ), which was doped on the surface of titanium dioxide as labels for multi-amplification. Herein, the photoactive films of PT-Cl/g-C3N4 is employed as the photoactive antibody (Ab1) immobilization matrix for the subsequent sandwich-type antibody–antigen affinity interactions. Upon the presence of antigen (Ag), greatly enhanced photocurrent could be triggered in the PEC platform by the labels of second antibody (Ab2) of Cu2 þ doped titanium dioxide (Cu2 þ –TiO2). As a result of the multi-amplification in this Cu2 þ –TiO2 enhanced PT-Cl/g-C3N4-based PEC immunoassay, it possesses excellent analytical performance. The antigen could be detected from 0.01 pg mL 1 to 100.0 ng mL 1 with a detection limit of 5 fg mL 1. This work opens up g-C3N4 nanosheet applied in PEC sensing. More importantly, the strategy of specific positive effect of Cu2 þ on the photocurrent of g-C3N4 opens an alternative horizon for PEC sensing. & 2014 Elsevier B.V. All rights reserved.
Keywords: Polythiophene Sensitized g-C3N4 nanosheet Cu2 þ doped titanium dioxide Photoelectrochemical immunoassay Multi-amplification
1. Introduction Recently, polymer-like semiconductor material graphitic carbon nitride (g-C3N4) has attracted great interest in numerous fields including photocatalytic water splitting, photodegradation of environmental organic pollutants and electrochemical sensors, because the stable and metal-free g-C3N4 possess energy band characteristics of metal oxide semiconductors (Wang et al., 2012a, 2012b, 2009a, 2009b). However, low carrier mobility, insufficient sunlight absorption and high recombination rate of photogenerated electron–hole pairs in the bare g-C3N4 system, limit its potential application in the area of photoelectric conversion (Sun et al., 2012). To solve these problems, some effective approaches have been developed to further optimize the photoelectric performance of g-C3N4, including doping it with metallic or nonmetallic elements, and copolymerizing it with other organic compounds (Zhang et al., 2010; Wang et al., 2009a, 2009b; Cheng et al., 2013). Even though, significant improvement of its photoelectric conversion efficiency has been shown by the methods mentioned above, n
Corresponding author. Tel./fax: þ 86 531 82765730. E-mail address: [email protected] (B. Du).
http://dx.doi.org/10.1016/j.bios.2014.06.061 0956-5663/& 2014 Elsevier B.V. All rights reserved.
the more effective and simple photosensitizers to improve the performance of g-C3N4-based photovoltaic devices are highly deserved. Conjugated polymers (CPs) are attracting more and more interest because of their wide absorption coefficients leading to efficient light harvesting properties, which could reduce the thickness of the semiconductor film (Yanagida et al., 2004). In quest of commercially viable conducting polymers, polythiophene and its derivatives have always been the most promising candidates because they have high absorption coefficiency and chargecarrier mobility, good chemical stability and thermal stability, easy modification and controlling for electrochemical and photoelectrochemical (PEC) behavior (Gazotti et al., 2001; Schilinsky et al., 2002; Valaski et al., 2001; Huynh et al., 2002; Song et al., 2003; Breeze et al., 2001). Although obviously improved performance were obtained through the coupling of CPs and inorganic semiconductors (e.g. TiO2) (Smestad et al., 2003; Arango et al., 1999; Grant and Schwartzberg, 2002; Yanagida et al., 2004) and carbon nanotubes (CNT) (Sgobba et al., 2009), to the best of our knowledge, little work has been done about these CPs as photosensitizers (“dye” molecules) in the g-C3N4 systems. In the present study, a novel water-soluble cationic conjugated polymer denoted
316
R. Li et al. / Biosensors and Bioelectronics 62 (2014) 315–319
as poly 2,5-[3-(1,1-dimethyl-4-piperidine methylene) thiophene] chloride (PT-Cl) was used to be an efficient photo-sensitizer for enhancing PEC sensing of g-C3N4. The novel PT-Cl-sensitized g-C3N4 photoactive films (PT-Cl/g-C3N4) with super-photoelectric performance were fabricated by assembling of positively charged PT-Cl and negative charged carboxylated g-C3N4. Additionally, in order to further improve the crystal morphology of the photoactive films to acquire higher photoelectric conversion efficiency as well as better stability of modified electrode, a method of calcination at 450 °C was employed in this work. In recent reports, g-C3N4 has been applied to the PEC selective sensing of copper (II) (Cu2 þ ) in aqueous solutions, which were based on the photocurrent intensity of the g-C3N4 increased with the increasing concentration of Cu2 þ (Xu et al., 2013, 2014; She et al., 2014). Inspired by this mind, in present work, the Cu2 þ -loaded nanomaterial was explored as labels for the first time to enhance the photocurrent of g-C3N4-based materials. Nanoporous titanium dioxide (TiO2) has been particularly investigated in recent decades since they have high photo-current conversion efficiency, superior optical and electrical properties (Gajjela et al., 2010; Lutz et al., 2012; Wang et al., 2011; Leshuk et al., 2012; Liu et al., 2010). In this study, copper ions surface-doped nanoporous titanium dioxide microspheres (Cu2 þ –TiO2) was employed as labels of secondary antibodies (Ab2) to obtain multi-amplification by using the excellent photoelectric conversion efficiency of TiO2 as well as the specific positive effect of Cu2 þ on the photocurrent of g-C3N4. The multi-amplification in this Cu2 þ –TiO2 enhanced PT-Cl/g-C3N4 -based PEC immunoassay possesses excellent analytical performance and opens an alternative horizon for PEC sensing.
2. Experimental section
which proved the successful loading of Cu2 þ onto the TiO2. The details of preparation of Cu2 þ –TiO2–Ab2 was in Supplementary materials. The utilized water-soluble Poly 2,5-[3-(1,1-dimethyl-4piperidine methylene) thiophene] chloride (PT-Cl) was synthesized according to literature (Li et al., 2012). 2.4. Immunoassay development Scheme 1 shows the developing process of the multi-amplified PEC immunoassay. Firstly, 4 μL negative charged g-C3N4 solution and 4 μL positively charged PT-Cl solution was dropped onto the pretreated ITO surface sequentially and kept for 20 min. After each dropping step, the ITO sample zone was rinsed thoroughly, dried in room temperature and then annealed at 450 °C for 5 min in a muffle furnace to obtain desired film stability and photocurrent intensity. Afterwards, 4 μL of 0.5 mg mL 1 Ab1 was immobilized onto the PT-Cl/g-C3N4 modified ITO electrode via physical process, after incubation for 1 h, the electrode was rinsed with the washing buffer to remove physically adsorbed Ab1. The electrode was then blocked with 4 μL blocking solution for 1 h at 4 °C to block nonspecific binding sites and washed with the washing buffer thoroughly. Next, 4 μL of NMP-22 antigen with different concentrations were dropped onto the Ab1 modified electrodes and incubated for 1 h at 37 °C followed by washing with washing buffer. After the binding reaction between Ab1 and the antigen, the electrodes were allowed for labeling by additional incubation with 4 μL diluted Cu2 þ –TiO2–Ab2 bioconjugates solution for 1 h. Thereafter, the electrodes were washed thoroughly with washing buffer to remove nonspecifically bound conjugations, and immersed in the PBS (pH 5.4) for 5 min before measurements. Then, the photocurrent was measured by the current–time technique at a bias voltage of 0.2 V following a 200 W LED excitation.
2.1. Chemicals and reagents 3. Results and discussion Ethylenediamine (NH2CH2CH2NH2, Z99%), glutaraldehyde (25%), Bovine serum albumin (BSA) (96–99%) were obtained from SigmaAldrich (Beijing, China). All aqueous solutions were prepared using ultrapure water (Milli-Q, Millipore). The details are shown in Supplementary information. 2.2. Apparatus Fourier Transform Infrared Spectrometer (FT-IR Spectrometer) was recorded by VERTEX70 spectrometer (Bruker Co., Germany). The details are shown in Supplementary information. 2.3. Preparation and characterization of materials In this work, carboxylated g-C3N4 was synthesized according to the reference with some slight modifications (Cheng et al., 2013). As shown in Fig. S1, the SEM (Fig. S1A) and TEM images (Fig. S1B) of the carboxylated g-C3N4 illustrated the nanosheet structure, which could provide excellent photoelectric properties and large surface area. Fig. S1C shows the FT-IR spectrum of g-C3N4 (blank line) and carboxylated g-C3N4 (red line) in the wavelength range of 500–4000 cm 1. The peak of 1720 cm 1 is corresponding to the CQO bending band and the absorption band for –COO at 1575 cm 1 and 1380 cm 1, indicating successful functionalization of carboxyl group on g-C3N4 (Dong et al., 2013). The fabrication of TiO2 and Cu2 þ –TiO2 was conducted following a literature procedure with some modifications (Li et al., 2008). As the TEM images of TiO2 and Cu2 þ –TiO2 show (Fig. S1D and E), the as-prepared TiO2 possess uniform size with an average diameter of 250 nm (Fig. S1D). The photo of Fig. S1E showed that there was an obvious change in the color of the powder after the assembling of Cu2 þ ,
Fig. 1A reveals the stepwise photocurrent responses of corresponding assembly. Curve a presented the photocurrent of PT-Cl/g-C3N4 modified ITO electrode. Even though, the photocurrent decreased gradually with Ab1 anchoring, BSA blocking and Ag specific binding (curves b–d), which was due to the steric hindrance of the hydrophobic protein layer (curves c–e, Fig. S3). However, after the final amplification via Cu2 þ –TiO2 by the formation of Cu2 þ –TiO2–Ab2–Ag conjugations, the photocurrent intensity was 3 times of the initial intensity (curve e). Compared with pure g-C3N4 (curve a, Fig. 1B), the coupling of PT-Cl and g-C3N4 resulted in a dramatic enhanced photocurrent (curve b, Fig. 1B). This may be ascribed to the wide absorption coefficients of PT-Cl that leading to efficient light harvesting properties (Yanagida et al., 2004), and the fast separation of charge carriers in the PT-Cl/g-C3N4 system caused by PT-Cl (Sgobba et al., 2009). Moreover, as shown in Fig. 1B, the photocurrent of Cu2 þ –TiO2 (curve d) as labels was clearly higher than that of TiO2 (curve c), indicating the prominent synergy enhancement of TiO2 and Cu2 þ . In comparison with the previous PEC immunoassay protocols, the prominent signal enhancement should be attributed to the Cu2 þ –TiO2 labeling-induced multi-signal amplification. Specifically, the addition of Cu2 þ would significantly increase the photocurrent of g-C3N4. The possible mechanism (see Fig. S2) may be that photo-generated electron of g-C3N4 transmitted to the ITO electrode, and then to the Pt reference electrode and formed loops (Xu et al., 2014). The photoinduced electron could be captured by Cu2 þ that released from Cu2 þ –TiO2–Ab2 in weakly acid solution, thus promoted the separation rate of electron and hole and led to the increasement of the photocurrent response. Besides, TiO2 would expand the light absorption of the underlying
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Scheme 1. Schematic diagram of the construction of PT-Cl/C3N4 nanosheet films (A) and Cu2 þ –TiO2–Ab2 (B) and the fabrication of a NMP-22 biosensor (C).
Fig. 1. (A) Photocurrent response of the modified electrode (a) before and (b) after Ab1 anchoring, (c) after further BSA blocking, (d) after NMP-22 incubation (10 ng mL 1), (e) after further Cu2 þ –TiO2 labeling. (B) Photocurrent response of the modified electrode with (a) g-C3N4, (b) PT-Cl/g-C3N4, (c) PT-Cl/g-C3N4/Ab1/BSA/NMP-22/TiO2–Ab2, (d) PT-Cl/g-C3N4/Ab1/BSA/NMP-22/Cu2 þ –TiO2–Ab2. The PEC tests were performed in 0.1 M PBS (pH ¼5.4) with 0.2 V applied potential and 430 nm excitation light.
PT-Cl/g-C3N4 film enormously, thus caused the further enhancement of photocurrent. Since the novel nanolabel carrying considerable Cu2 þ , using NMP-22 as a model, the presence of trace quantities of NMP-22 would be greatly amplified by the synergy effect of TiO2 and Cu2 þ . The extent of final photocurrent enhancement associates with the NMP-22 concentration intimately. Under the optimal conditions (Figs. S4 and S5), via tracking the increasement of the photocurrent, an exquisite PEC NMP-22 immunoassay could be tailored (Fig. 2A and B). The response had a fairly good logarithmical relationship with the concentration of the NMP-22 ranging from 0.01 pg mL 1 to 100 ng mL 1 with a correlation coefficient of 0.996, and the limit of detection (LOD)
was 5 fg mL 1 at 3 s/slope. The achieved LOD is much lower than the clinical judgment value (6.5 U mL 1 ) in urine set by US Food and Drug Administration (Shariat et al., 2004). The photocurrent enhances as the NMP-22 concentration increases, indicating the NMP-22-controlled enhancement of the synergy effect. Due to the synergy effect as stated above, the NMP-22 could be detected at levels down to 5 fg mL 1 , which is much lower than previous electrochemical method (0.5 ng mL 1 ) (Ning et al., 2007). Control experiments revealed that the developed PEC sensor does not exhibit any obvious changes in photocurrents when incubating the as-fabricated immunosensor in sample solutions containing a 100-fold excess of different interfering agents such as
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Fig. 2. (A) Effect of different NMP-22 concentrations on the differential photocurrent responses, (a)–(f) 0.01, 0.1, 1, 10, 100, 1000, 10,000, 100,000 pg mL 1, respectively. (B) The corresponding calibration curve. The changes of photocurrent was the changes between the photocurrent of the modified electrode prior to NMP-22 immobilization and final photocurrent of the electrode after incubation with Ag of elevated concentrations corresponding to 0.01, 0.1, 1, 10, 100, 1000, 10,000, 100,000 pg mL 1, respectively, and then after the final TiO2–Cu2 þ –Ab2. (C) Selectivity of NMP-22 toward different analytes (ascorbic acid, adenine, guanine and uric acid, 1 mg mL 1) which are 100-fold concentrations of the NMP-22. (D) Time-based photocurrent response of PT-Cl/g-C3N4/Ab1/BSA/NMP-22/Cu2 þ –TiO2–Ab2 modified ITO electrode under several on/off irradiation cycles for 300 s.
ascorbic acid, adenine, guanine and uric acid (Fig. 2C). The results demonstrate that the specific immunobinding should be responsible for the photocurrent enhancement and thus the good selectivity. It is also worth mentioning that the photocurrent response of the PEC sensor was fairly reversible and stable under several on/off irradiation cycles for 300 s. As shown in Fig. 2D, the current could reproducibly increase violently under each irradiation and recover rapidly in the dark, indicating the structural stability of the developed PEC sensors and their potential for biosensing experiments. The reproducibility of this PEC NMP-22 immunoassay is also assessed by an interassay relative standard deviation (RSD). RSD of 6.1% was obtained by assaying the same 10 ng mL 1 NMP-22 samples with five electrodes, indicating satisfactory reproducibility. Compared to other PEC approaches, this immunoassay also had the advantages of low cost (4 mL of each step and no enzyme is used), and being environment-friendly (toxic precursors such as cadmium are not used) and so on. Obviously, the proposed PEC sensor showed promise for application in the monitoring of NMP-22 with high sensitivity and fast response.
4. Conclusions In summary, a novel PEC immunoassay for protein was first proposed. It is based on the in Cu2 þ –TiO2-enhanced PT-Cl/g-C3N4 nanosheet-based photoelectrochemistry via sandwich immunoreaction. This work has shown that NMP-22 can be detected at very low concentrations by a new PEC immunoassay protocol, which provides a feasible alternative technique for NMP-22 screening to facilitate early bladder cancer diagnosis. Upon the introduction of a novel labeling system, synergy effects could be triggered in the PEC platform for multi-signal amplification. With enhanced analytical performance, this strategy opens a different perspective for ultrasensitive protein detection especially for trace cancer markers (or other infectious/biothreatening agents and DNA analysis), in which high sensitivity is strongly required.
Acknowledgments This work was financially supported by the National Natural Science Foundation of China (21375047, 21377046, and 21245007),
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the Science and Technology Plan Project of Jinan (No. 201307010), and QW thanks the Special Foundation for Taishan Scholar Professorship of Shandong Province and UJN (No. ts20130937).
Appendix A. Supplementary information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.06.061.
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