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Surface Plasmon Resonance Enhanced Real-time, Photoelectrochemical Protein Sensing by Au Nanoparticle-Decorated TiO2 Nanowires Peimei Da, Wenjie Li, Xuan Lin, Yongcheng Wang, Jing Tang, and Gengfeng Zheng Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac501406x • Publication Date (Web): 29 May 2014 Downloaded from http://pubs.acs.org on June 1, 2014
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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Analytical Chemistry
Surface Plasmon Resonance Enhanced Real-time, Photoelectrochemical Protein Sensing by Au Nanoparticle-Decorated TiO2 Nanowires Peimei Da, Wenjie Li, Xuan Lin, Yongcheng Wang, Jing Tang and Gengfeng Zheng*
Laboratory of Advanced Materials, Department of Chemistry, Fudan University, Shanghai, 200433, People’s Republic of China * Address correspondence to: [email protected]
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ABSTRACT The recently developed photoelectrochemical sensing represents a unique and potential detection method for real-time analysis of chemical/biological molecules, while the low absorption of TiO2 nanomaterials in the visible wavelength region and the slow surface charge transfer efficiency limit the ultimate sensitivity. Here we develop a gold nanoparticle-decorated TiO2 nanowire sensor for PEC detection of protein binding. The direct attachment of Au nanoparticles to TiO2 nanowires offers strong surface plasmon resonance for electrochemical field effect amplification, yielding a ~ 100% increase of photocurrent density. In addition, the surface functionalization of gold nanoparticles allows for direct capturing of target proteins near the Au/TiO2 interface, and thus substantially enhance the capability of attenuation of energy coupling between Au and TiO2, leading to much improved sensor performance. As a proof-of-concept, cholera toxin subunit B has been robustly detected by the TiO2-Au NW sensor functionalized with ganglioside GM1, with a high sensitivity of 0.167 nM and excellent selectivity. Furthermore, the real-time feature of the photoelectrochemical sensing enables direct measurement of binding kinetics between cholera toxin subunit B and GM1, yielding the association, disassociation rate constants, and an equilibrium constant Kd as 4.17 nM. This surface plasmon resonance-enhanced real-time, photoelectrochemical sensing design may lead to exciting biodetection capabilities with high sensitivity and real-time kinetic studies.
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Analytical Chemistry
Sensitive detection of chemical and biological targets is important to many areas of life and medical sciences, from early disease diagnosis, fast drug screening and bio-safety.1-4 Key to the detection is the selective recognition of a molecular target, along with rationally designed signal generation and transduction pathways. A large variety of optical,5-7 electrical8,9 and mechanical10,11 methods have been developed for detection of biomolecular species, with the ultimate goal of fast, sensitive, large-scale and low-cost analysis in complex samples. Among those detection methods, the recently developed photoelectrochemical (PEC) sensing approach represents a unique signal transducing modality, in which light and electricity as two different forms of signals are used for the excitation and detection of sensing signal, thus enabling reducing of background noise and increasing of sensitivity.12 TiO2 nanostructures are one of the most promising candidates for the PEC sensing, due to their excellent chemical stability, favorable band edge positions and low cost, and have been demonstrated for detection of H2O2,13 rabbit immunoglobulin G (IgG),14 adenosine,15 glucose16 and prostate specific antigen.17 More recently, it has been reported that the co-functionalization of hemin and IrO2 nanoparticles on TiO2 nanowires (NWs) can enhance both light absorption and charge transfer kinetics upon photo-excitation, resulting in sensitive detection of glutathione in both buffer and cell extracts.18 Nonetheless, a well-known challenge for the TiO2-based light-to-electricity conversion is the wide bandgap of TiO2 and thus predominant UV photoabsorption,19,20 which substantially limits its solar energy conversion efficiency and corresponding PEC sensitivity. More importantly, all 3 ACS Paragon Plus Environment
Analytical Chemistry
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the previous reports of the PEC sensing are based on the binding of molecular targets on TiO2 sensor surfaces and subsequent charge transfer (Fig. 1a). This sensor design, however, is only capable of affecting charge carrier transfer and chemical reactions near the surface, thus limiting the ultimate sensitivity. The incorporation of noble metal nanoparticles (NPs) with surface plasmonic resonance (SPR) properties has been recently discovered for enhancing the photoconversion efficiency of TiO2 and other wide bandgap semiconductors.21 In particular, gold NPs, due to their photo-, thermo-, and chemical stability, easiness to prepare, and excellent electronic and optical properties,22 have been the focus of the SPR-enhanced photoactivity.23,24 For instance, Au nanostructure-decorated TiO2 NWs were reported to exhibit photoactivity across entire UV-visible region for the PEC water splitting.25 Zhang et al. showed the coupling of SPR of Au NPs with slow-photo-effect of TiO2 photonic crystal for synergistically enhanced PEC water splitting.26 These works suggest that SPR-enhanced energy conversion scheme is mainly attributed to the electrical field amplification effect as well as the injection of SPR-generated hot electrons into the conduction band of TiO2. Thus, the field effect and charge transfer efficiency should strongly depend on the local chemical environment fluctuation between Au and TiO2. Therefore, it is rational to design a sensing scheme that couples Au SPR nanostructures with TiO2 PEC sensors (Fig. 1b), in which not only the photoconversion efficiency is increased by the SPR enhancement, but also the binding of molecular targets can effectively tune the energy coupling and charge transfer across the interface between these two materials, leading to a much 4 ACS Paragon Plus Environment
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Analytical Chemistry
improved sensitivity. In this paper, we have developed an SPR-enhanced PEC sensor, in which one-dimensional (1D) TiO2 NWs are first hydrothermally grown on a fluorine-doped tin oxide (FTO) substrate, followed by the in-situ reduction of HAuCl4 to deposit Au NPs on TiO2 surface (Fig. 1c). The PEC measurement shows that the Au NP-decorated TiO2 NWs present a photocurrent density of 1.60 mA cm-2 at 0 V vs. Ag/AgCl at 1-sun illumination, approximately 100% increase compared to the pristine TiO2 NWs. The incident photo-to-current conversion efficiency (IPCE) measurement shows that the photocurrent increase is attributed to the absorption enhancement in both the UV and visible regions, suggesting the electrical field enhancement effect and the electron injection upon the SPR excitation. In addition, the direct receptor functionalization and subsequent capturing of protein targets on Au NP surface enable a sensitive method for tuning of the coupling of field effect and charge injection between Au and TiO2, leading to a much enhanced PEC sensitivity. As a proof-of-concept, Au NPs are functionalized with ganglioside GM1 (Fig. S1), a cell membrane receptor for binding of cholera toxin subunit B (CTB).27 A high sensitivity of 0.167 nM has been achieved for selective detection of CTB, substantially better than that obtained from the direct PEC sensing of TiO2-surface binding. Moreover, by analyzing the time-dependent photocurrent signal, this direct, real-time detection method enables measuring the association, disassociation rate constants, and equilibrium constants between protein targets with their receptors, thus suggesting a unique approach for both high sensitivity and binding kinetic studies. 5 ACS Paragon Plus Environment
Analytical Chemistry
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EXPERIMENTAL SECTION
Synthesis of TiO2 NWs and Au NP-decorated TiO2 NWs. TiO2 NWs were directly grown on fluorine-doped tin oxide (FTO) coated glass substrates (14 Ω per square, Wuhan Ge-Ao Ltd., China) by a hydrothermal method, in which tetrabutyl titanate (TBOT) was used for the Ti precursor. After the synthesis, TiO2 NWs were subsequently decorated with Au NPs by a modified solution reduction method. In brief, a FTO substrate with TiO2 NWs was immersed into an aqueous solution containing 0.01 M HAuCl4 (with pH tuned to 4.5 by adding 0.2 M NaOH solution) for 2 h. Afterwards, the sample was thoroughly washed by de-ionized (DI) water, dried in air and annealed in air at 300 oC for 2 h to generate Au NPs on TiO2 surface. Surface functionalization with ganglioside GM1. For functionalization of GM1 on Au NPs, the TiO2-Au NW samples were first immersed in a solution of 2% (v:v) octadecanethiol in anhydrous toluene for overnight. The samples were then rinsed with anhydrous toluene and ethanol, and dried with compressed N2. Afterwards, a 0.1 mg mL-1 monosialoganglioside GM1 solution in 0.1 M phosphate buffer saline (PBS) was deposited onto the NW surface for 4 h. For functionalization of GM1 on TiO2 NWs, the TiO2 NW samples were first incubated overnight with 2% (v:v) dimethyloctadecylchlorosilane of anhydrous toluene solution, and then rinsed with anhydrous toluene and ethanol, followed by baking at 120 oC for 15 min. Afterwards, the insertion of ganglioside GM1 was performed followed the same method described above. 6 ACS Paragon Plus Environment
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Analytical Chemistry
Photoelectrochemical (PEC) measurement. The PEC measurement was carried out using a 3-electrode electrochemical quartz cell, with an Ag/AgCl reference electrode and a Pt wire as counter electrode, under simulated sunlight coupled with an AM 1.5G filter (Newport 94022A, USA). A PBS (pH = 7.4) was used as the electrolytes for the PEC measurements, with a volume of ~ 5 mL in the electrochemical cell. The sensor area was defined around 0.02−0.06 cm2 by epoxy sealing. Linear sweeps and amperometry (I−t) scans were measured by an electrochemical workstation (CHI660D, CH Instrument, USA). The IPCE spectra were collected by a Newport electrochemical station with a solar simulator (Newport 66902), coupled with a filter (Newport 74010) and an aligned monochrometor (Newport 74125). Kinetics measurement. The binding/unbinding signals can be rigorously analyzed to extract the kinetics parameters using the Langmuir binding algorithm, which is frequently used to study the molecule adsorption to surface. The reaction between proteins and surface receptors can be regarded as a simple reaction: P + S ↔ P·S, where P, S, and P·S stand for free protein (CTB), immobilized surface receptor (GM1), and protein-receptor complex, respectively. Here it is assumed that all the surface-immobilized receptors are equivalent and independent. Since only a very small fraction of protein inside the electrochemical cell is bound to receptors on the sensor surface, the free proteins can be regarded as constant and equal to the total concentrations of the proteins. Then, the reaction between the immobilized surface receptors and free proteins can then be assumed to follow the pseudo-first order reaction kinetics. If we assume the NW PEC sensor signal (∆Conductance) is directly proportional to the concentration of protein-receptor complex, 7 ACS Paragon Plus Environment
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[P·S], therefore, the real-time binding curve can be fitted by a time (t)-dependent single-exponential plot, i.e. A × exp (−kobs × t), while A stands for the signal magnitude and kobs stands for the observed kinetics rate constant of this reaction (see the Appendix). In addition, based on the theoretical equation, kobs is linear to the CTB concentration for the binding phase, that is kobs = kon × C + koff, where C stands for the CTB concentration. The association rate constant (kon) and the disassociation rate constant (koff) can be extracted from the fitting. The dissociation equilibrium constant (Kd) can be calculated as koff / kon.
RESULTS AND DISCUSSION
Materials Characterization. The synthesis of Au NP-decorated TiO2 (designated as TiO2-Au) NWs and the surface receptor functionalization of Au NPs with GM1 receptors for the CTB detection are schematically illustrated in Figure. 1c (Experimental Section). After the TiO2 NW growth, the substrate surface is covered by a white thin film, which turns to pink after the deposition of Au NPs. Scanning electron microscopy (SEM) images show that TiO2 NWs form a packed arrays that are perpendicularly orientated from the growth substrate, and small Au NPs are uniformly dispersed on the surface of TiO2 NWs (Fig. 2a, b). Transmission electron microscopy (TEM) images show that these as-synthesized Au NPs have a spherical morphology and an average diameter of 8 ± 2 nm (Fig. 2c). High-resolution TEM (HRTEM) images clearly exhibit the single crystalline nature of both the TiO2 NWs and Au NPs (Fig. 2d). Two lattice 8 ACS Paragon Plus Environment
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Analytical Chemistry
spacings of the TiO2 NW of 0.324 and 0.248 nm are assigned to the (110) and (101) planes of rutile TiO2,28 and one lattice spacing of 0.236 nm corresponds to the (111) planes of Au crystal with a face-center-cubic (fcc) structure,29 in good accordance with previous reports.25 X-ray diffraction pattern (XRD) display characteristic peaks at 36.1o, 54.3o and 62.7o (Fig. S2), corresponding to the 101, 211 and 002 reflections of a rutile TiO2 crystalline structure (JCPDS No. 21-1276). The energy dispersive X-ray (EDX) spectroscopy shows that the Au/Ti molar percentage in this structure is ~ 0.2% (Fig. S3). The elemental mapping data confirm the uniform distribution of Au over the entire TiO2 NWs (Fig. S4). Photoelectrochemical Measurement. The photoelectrochemical properties of Au NPs-decorated TiO2 NWs are investigated in a standard 3-electrode electrochemical cell in 1 M KOH electrolyte, with Ag/AgCl and a Pt wire as the reference and counter electrodes, respectively (Experimental Section). For both the pristine TiO2 NW and TiO2-Au NW photoanodes, the linear sweep voltammagrams between −0.6 and 0.6 V (vs. Ag/AgCl) present clear photo-responses well correlated to on/off cycles under simulated 1-sun illumination (AM 1.5G), and the photocurrent densities increase with the applied potential (Fig. 3a). Compared to the pristine TiO2 NWs, the TiO2-Au NW photoanode exhibits a substantially higher photoactivity. For instance, a photocurrent density of ~ 1.6 mA cm-2 at 0 V vs. Ag/AgCl is obtained for the TiO2-Au NW photoanode, which is ~ 100% higher than that of the pristine TiO2 NWs. The IPCE spectrum of the pristine TiO2 NWs shows a maximum value of ~ 28% at 390 nm, which quickly drops to negligible above 400 nm (Fig. 3b and inset). In contrast, the 9 ACS Paragon Plus Environment
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TiO2-Au NWs exhibit a significant increase of IPCE to ~ 48% at 390 nm, substantially higher than that of the pristine TiO2 NWs in the UV range. Although the IPCE spectrum of the TiO2-Au NWs decreases to a low value (< 1%) in the visible light range, it is still well above that of the pristine TiO2 NWs for the wavelength range up to 620 nm, with a local maximum IPCE centered ~ 530−550 nm. The much increased IPCE spectrum in both the UV and visible regions can be attributed to the enhanced photoabsorption and hot electron injection from the Au NP-induced SPR enhancement, leading to higher charge carrier density and larger photocurrent density inside TiO2 NWs.30 Surface Functionalization. The conversion of TiO2-Au NW photoanodes into a PEC sensor for CTB detection is achieved via a two-step surface functionalization process of Au NPs by octadecanethiol and ganglioside GM1, respectively.31 First, octadecanethiol is self-assembled into a monolayer on the surface of Au NPs, due to the high affinity between thiol groups and Au (Fig. S1). Afterwards, the long alkyl chains of monosialoganglioside GM1 are inserted into the close packing of octadecanethiol molecules, forming an interdigitate molecular layer. The contact angle measurement performed before and after each functionalization step shows that the surface of TiO2-Au NW arrays changes from hydrophilic (contact angle ~ 21o) to highly hydrophobic (contact angle ~ 133o) after the first step of octadecanethiol modification, and then back to highly hydrophilic (contact angle
Article
Surface Plasmon Resonance Enhanced Real-time, Photoelectrochemical Protein Sensing by Au Nanoparticle-Decorated TiO2 Nanowires Peimei Da, Wenjie Li, Xuan Lin, Yongcheng Wang, Jing Tang, and Gengfeng Zheng Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac501406x • Publication Date (Web): 29 May 2014 Downloaded from http://pubs.acs.org on June 1, 2014
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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Analytical Chemistry
Surface Plasmon Resonance Enhanced Real-time, Photoelectrochemical Protein Sensing by Au Nanoparticle-Decorated TiO2 Nanowires Peimei Da, Wenjie Li, Xuan Lin, Yongcheng Wang, Jing Tang and Gengfeng Zheng*
Laboratory of Advanced Materials, Department of Chemistry, Fudan University, Shanghai, 200433, People’s Republic of China * Address correspondence to: [email protected]
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Analytical Chemistry
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ABSTRACT The recently developed photoelectrochemical sensing represents a unique and potential detection method for real-time analysis of chemical/biological molecules, while the low absorption of TiO2 nanomaterials in the visible wavelength region and the slow surface charge transfer efficiency limit the ultimate sensitivity. Here we develop a gold nanoparticle-decorated TiO2 nanowire sensor for PEC detection of protein binding. The direct attachment of Au nanoparticles to TiO2 nanowires offers strong surface plasmon resonance for electrochemical field effect amplification, yielding a ~ 100% increase of photocurrent density. In addition, the surface functionalization of gold nanoparticles allows for direct capturing of target proteins near the Au/TiO2 interface, and thus substantially enhance the capability of attenuation of energy coupling between Au and TiO2, leading to much improved sensor performance. As a proof-of-concept, cholera toxin subunit B has been robustly detected by the TiO2-Au NW sensor functionalized with ganglioside GM1, with a high sensitivity of 0.167 nM and excellent selectivity. Furthermore, the real-time feature of the photoelectrochemical sensing enables direct measurement of binding kinetics between cholera toxin subunit B and GM1, yielding the association, disassociation rate constants, and an equilibrium constant Kd as 4.17 nM. This surface plasmon resonance-enhanced real-time, photoelectrochemical sensing design may lead to exciting biodetection capabilities with high sensitivity and real-time kinetic studies.
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Analytical Chemistry
Sensitive detection of chemical and biological targets is important to many areas of life and medical sciences, from early disease diagnosis, fast drug screening and bio-safety.1-4 Key to the detection is the selective recognition of a molecular target, along with rationally designed signal generation and transduction pathways. A large variety of optical,5-7 electrical8,9 and mechanical10,11 methods have been developed for detection of biomolecular species, with the ultimate goal of fast, sensitive, large-scale and low-cost analysis in complex samples. Among those detection methods, the recently developed photoelectrochemical (PEC) sensing approach represents a unique signal transducing modality, in which light and electricity as two different forms of signals are used for the excitation and detection of sensing signal, thus enabling reducing of background noise and increasing of sensitivity.12 TiO2 nanostructures are one of the most promising candidates for the PEC sensing, due to their excellent chemical stability, favorable band edge positions and low cost, and have been demonstrated for detection of H2O2,13 rabbit immunoglobulin G (IgG),14 adenosine,15 glucose16 and prostate specific antigen.17 More recently, it has been reported that the co-functionalization of hemin and IrO2 nanoparticles on TiO2 nanowires (NWs) can enhance both light absorption and charge transfer kinetics upon photo-excitation, resulting in sensitive detection of glutathione in both buffer and cell extracts.18 Nonetheless, a well-known challenge for the TiO2-based light-to-electricity conversion is the wide bandgap of TiO2 and thus predominant UV photoabsorption,19,20 which substantially limits its solar energy conversion efficiency and corresponding PEC sensitivity. More importantly, all 3 ACS Paragon Plus Environment
Analytical Chemistry
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the previous reports of the PEC sensing are based on the binding of molecular targets on TiO2 sensor surfaces and subsequent charge transfer (Fig. 1a). This sensor design, however, is only capable of affecting charge carrier transfer and chemical reactions near the surface, thus limiting the ultimate sensitivity. The incorporation of noble metal nanoparticles (NPs) with surface plasmonic resonance (SPR) properties has been recently discovered for enhancing the photoconversion efficiency of TiO2 and other wide bandgap semiconductors.21 In particular, gold NPs, due to their photo-, thermo-, and chemical stability, easiness to prepare, and excellent electronic and optical properties,22 have been the focus of the SPR-enhanced photoactivity.23,24 For instance, Au nanostructure-decorated TiO2 NWs were reported to exhibit photoactivity across entire UV-visible region for the PEC water splitting.25 Zhang et al. showed the coupling of SPR of Au NPs with slow-photo-effect of TiO2 photonic crystal for synergistically enhanced PEC water splitting.26 These works suggest that SPR-enhanced energy conversion scheme is mainly attributed to the electrical field amplification effect as well as the injection of SPR-generated hot electrons into the conduction band of TiO2. Thus, the field effect and charge transfer efficiency should strongly depend on the local chemical environment fluctuation between Au and TiO2. Therefore, it is rational to design a sensing scheme that couples Au SPR nanostructures with TiO2 PEC sensors (Fig. 1b), in which not only the photoconversion efficiency is increased by the SPR enhancement, but also the binding of molecular targets can effectively tune the energy coupling and charge transfer across the interface between these two materials, leading to a much 4 ACS Paragon Plus Environment
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Analytical Chemistry
improved sensitivity. In this paper, we have developed an SPR-enhanced PEC sensor, in which one-dimensional (1D) TiO2 NWs are first hydrothermally grown on a fluorine-doped tin oxide (FTO) substrate, followed by the in-situ reduction of HAuCl4 to deposit Au NPs on TiO2 surface (Fig. 1c). The PEC measurement shows that the Au NP-decorated TiO2 NWs present a photocurrent density of 1.60 mA cm-2 at 0 V vs. Ag/AgCl at 1-sun illumination, approximately 100% increase compared to the pristine TiO2 NWs. The incident photo-to-current conversion efficiency (IPCE) measurement shows that the photocurrent increase is attributed to the absorption enhancement in both the UV and visible regions, suggesting the electrical field enhancement effect and the electron injection upon the SPR excitation. In addition, the direct receptor functionalization and subsequent capturing of protein targets on Au NP surface enable a sensitive method for tuning of the coupling of field effect and charge injection between Au and TiO2, leading to a much enhanced PEC sensitivity. As a proof-of-concept, Au NPs are functionalized with ganglioside GM1 (Fig. S1), a cell membrane receptor for binding of cholera toxin subunit B (CTB).27 A high sensitivity of 0.167 nM has been achieved for selective detection of CTB, substantially better than that obtained from the direct PEC sensing of TiO2-surface binding. Moreover, by analyzing the time-dependent photocurrent signal, this direct, real-time detection method enables measuring the association, disassociation rate constants, and equilibrium constants between protein targets with their receptors, thus suggesting a unique approach for both high sensitivity and binding kinetic studies. 5 ACS Paragon Plus Environment
Analytical Chemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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EXPERIMENTAL SECTION
Synthesis of TiO2 NWs and Au NP-decorated TiO2 NWs. TiO2 NWs were directly grown on fluorine-doped tin oxide (FTO) coated glass substrates (14 Ω per square, Wuhan Ge-Ao Ltd., China) by a hydrothermal method, in which tetrabutyl titanate (TBOT) was used for the Ti precursor. After the synthesis, TiO2 NWs were subsequently decorated with Au NPs by a modified solution reduction method. In brief, a FTO substrate with TiO2 NWs was immersed into an aqueous solution containing 0.01 M HAuCl4 (with pH tuned to 4.5 by adding 0.2 M NaOH solution) for 2 h. Afterwards, the sample was thoroughly washed by de-ionized (DI) water, dried in air and annealed in air at 300 oC for 2 h to generate Au NPs on TiO2 surface. Surface functionalization with ganglioside GM1. For functionalization of GM1 on Au NPs, the TiO2-Au NW samples were first immersed in a solution of 2% (v:v) octadecanethiol in anhydrous toluene for overnight. The samples were then rinsed with anhydrous toluene and ethanol, and dried with compressed N2. Afterwards, a 0.1 mg mL-1 monosialoganglioside GM1 solution in 0.1 M phosphate buffer saline (PBS) was deposited onto the NW surface for 4 h. For functionalization of GM1 on TiO2 NWs, the TiO2 NW samples were first incubated overnight with 2% (v:v) dimethyloctadecylchlorosilane of anhydrous toluene solution, and then rinsed with anhydrous toluene and ethanol, followed by baking at 120 oC for 15 min. Afterwards, the insertion of ganglioside GM1 was performed followed the same method described above. 6 ACS Paragon Plus Environment
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Analytical Chemistry
Photoelectrochemical (PEC) measurement. The PEC measurement was carried out using a 3-electrode electrochemical quartz cell, with an Ag/AgCl reference electrode and a Pt wire as counter electrode, under simulated sunlight coupled with an AM 1.5G filter (Newport 94022A, USA). A PBS (pH = 7.4) was used as the electrolytes for the PEC measurements, with a volume of ~ 5 mL in the electrochemical cell. The sensor area was defined around 0.02−0.06 cm2 by epoxy sealing. Linear sweeps and amperometry (I−t) scans were measured by an electrochemical workstation (CHI660D, CH Instrument, USA). The IPCE spectra were collected by a Newport electrochemical station with a solar simulator (Newport 66902), coupled with a filter (Newport 74010) and an aligned monochrometor (Newport 74125). Kinetics measurement. The binding/unbinding signals can be rigorously analyzed to extract the kinetics parameters using the Langmuir binding algorithm, which is frequently used to study the molecule adsorption to surface. The reaction between proteins and surface receptors can be regarded as a simple reaction: P + S ↔ P·S, where P, S, and P·S stand for free protein (CTB), immobilized surface receptor (GM1), and protein-receptor complex, respectively. Here it is assumed that all the surface-immobilized receptors are equivalent and independent. Since only a very small fraction of protein inside the electrochemical cell is bound to receptors on the sensor surface, the free proteins can be regarded as constant and equal to the total concentrations of the proteins. Then, the reaction between the immobilized surface receptors and free proteins can then be assumed to follow the pseudo-first order reaction kinetics. If we assume the NW PEC sensor signal (∆Conductance) is directly proportional to the concentration of protein-receptor complex, 7 ACS Paragon Plus Environment
Analytical Chemistry
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[P·S], therefore, the real-time binding curve can be fitted by a time (t)-dependent single-exponential plot, i.e. A × exp (−kobs × t), while A stands for the signal magnitude and kobs stands for the observed kinetics rate constant of this reaction (see the Appendix). In addition, based on the theoretical equation, kobs is linear to the CTB concentration for the binding phase, that is kobs = kon × C + koff, where C stands for the CTB concentration. The association rate constant (kon) and the disassociation rate constant (koff) can be extracted from the fitting. The dissociation equilibrium constant (Kd) can be calculated as koff / kon.
RESULTS AND DISCUSSION
Materials Characterization. The synthesis of Au NP-decorated TiO2 (designated as TiO2-Au) NWs and the surface receptor functionalization of Au NPs with GM1 receptors for the CTB detection are schematically illustrated in Figure. 1c (Experimental Section). After the TiO2 NW growth, the substrate surface is covered by a white thin film, which turns to pink after the deposition of Au NPs. Scanning electron microscopy (SEM) images show that TiO2 NWs form a packed arrays that are perpendicularly orientated from the growth substrate, and small Au NPs are uniformly dispersed on the surface of TiO2 NWs (Fig. 2a, b). Transmission electron microscopy (TEM) images show that these as-synthesized Au NPs have a spherical morphology and an average diameter of 8 ± 2 nm (Fig. 2c). High-resolution TEM (HRTEM) images clearly exhibit the single crystalline nature of both the TiO2 NWs and Au NPs (Fig. 2d). Two lattice 8 ACS Paragon Plus Environment
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Analytical Chemistry
spacings of the TiO2 NW of 0.324 and 0.248 nm are assigned to the (110) and (101) planes of rutile TiO2,28 and one lattice spacing of 0.236 nm corresponds to the (111) planes of Au crystal with a face-center-cubic (fcc) structure,29 in good accordance with previous reports.25 X-ray diffraction pattern (XRD) display characteristic peaks at 36.1o, 54.3o and 62.7o (Fig. S2), corresponding to the 101, 211 and 002 reflections of a rutile TiO2 crystalline structure (JCPDS No. 21-1276). The energy dispersive X-ray (EDX) spectroscopy shows that the Au/Ti molar percentage in this structure is ~ 0.2% (Fig. S3). The elemental mapping data confirm the uniform distribution of Au over the entire TiO2 NWs (Fig. S4). Photoelectrochemical Measurement. The photoelectrochemical properties of Au NPs-decorated TiO2 NWs are investigated in a standard 3-electrode electrochemical cell in 1 M KOH electrolyte, with Ag/AgCl and a Pt wire as the reference and counter electrodes, respectively (Experimental Section). For both the pristine TiO2 NW and TiO2-Au NW photoanodes, the linear sweep voltammagrams between −0.6 and 0.6 V (vs. Ag/AgCl) present clear photo-responses well correlated to on/off cycles under simulated 1-sun illumination (AM 1.5G), and the photocurrent densities increase with the applied potential (Fig. 3a). Compared to the pristine TiO2 NWs, the TiO2-Au NW photoanode exhibits a substantially higher photoactivity. For instance, a photocurrent density of ~ 1.6 mA cm-2 at 0 V vs. Ag/AgCl is obtained for the TiO2-Au NW photoanode, which is ~ 100% higher than that of the pristine TiO2 NWs. The IPCE spectrum of the pristine TiO2 NWs shows a maximum value of ~ 28% at 390 nm, which quickly drops to negligible above 400 nm (Fig. 3b and inset). In contrast, the 9 ACS Paragon Plus Environment
Analytical Chemistry
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TiO2-Au NWs exhibit a significant increase of IPCE to ~ 48% at 390 nm, substantially higher than that of the pristine TiO2 NWs in the UV range. Although the IPCE spectrum of the TiO2-Au NWs decreases to a low value (< 1%) in the visible light range, it is still well above that of the pristine TiO2 NWs for the wavelength range up to 620 nm, with a local maximum IPCE centered ~ 530−550 nm. The much increased IPCE spectrum in both the UV and visible regions can be attributed to the enhanced photoabsorption and hot electron injection from the Au NP-induced SPR enhancement, leading to higher charge carrier density and larger photocurrent density inside TiO2 NWs.30 Surface Functionalization. The conversion of TiO2-Au NW photoanodes into a PEC sensor for CTB detection is achieved via a two-step surface functionalization process of Au NPs by octadecanethiol and ganglioside GM1, respectively.31 First, octadecanethiol is self-assembled into a monolayer on the surface of Au NPs, due to the high affinity between thiol groups and Au (Fig. S1). Afterwards, the long alkyl chains of monosialoganglioside GM1 are inserted into the close packing of octadecanethiol molecules, forming an interdigitate molecular layer. The contact angle measurement performed before and after each functionalization step shows that the surface of TiO2-Au NW arrays changes from hydrophilic (contact angle ~ 21o) to highly hydrophobic (contact angle ~ 133o) after the first step of octadecanethiol modification, and then back to highly hydrophilic (contact angle
