Target-Induced Nano-Enzyme Reactor Mediated Hole-Trapping for

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Target-Induced Nano-Enzyme Reactor Mediated HoleTrapping for High-Throughput Immunoassay Based on a Split-Type Photoelectrochemical Detection Strategy Junyang Zhuang, Dianyong Tang, Wenqiang Lai, Mingdi Xu, and Dianping Tang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b02676 • Publication Date (Web): 20 Aug 2015 Downloaded from http://pubs.acs.org on August 24, 2015

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Analytical Chemistry

Target-Induced Nano-Enzyme Reactor Mediated Hole-Trapping for High-Throughput Immunoassay Based on a Split-Type Photoelectrochemical Detection Strategy †

†

†

†,

Junyang Zhuang, Dianyong Tang,‡,* Wenqiang Lai, Mingdi Xu, and Dianping Tang *

†

Institute of Nanomedicine and Nanobiosensing, Key Laboratory of Analysis and Detection for Food

Safety (MOE & Fujian Province), Department of Chemistry, Fuzhou University, Fuzhou 350108, People's Republic of China ‡

Chongqing Key Laboratory of Environmental Materials & Remediation Technologies, Chongqing University of Arts and Sciences, Chongqing 402160, People's Republic of China

CORRESPONDING AUTHOR INFORMATION Phone: +86-23-6116 2836; fax: +86-23-6116 2836; e-mail: [email protected] (D.Y.T.) Phone: +86-591-2286 6125; fax: +86-591-2286 6135; e-mail: [email protected] (D.P.T.)

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ABSTRACT: Photoelectrochemical (PEC) detection is an emerging and promising analytical tool. However, its actual application still faces some challenges like potential damage of biomolecules (caused by itself system) and intrinsic low-throughput detection. To solve the problems, herein we design a novel split-type photoelectrochemical immunoassay (STPIA) for ultrasensitive detection of prostate specific antigen (PSA). Initially, the immunoreaction was performed on microplate using secondary antibody/primer-circular DNA-labeled gold nanoparticle as the detection tag. Then, numerously repeated oligonucleotide sequences with many biotin moieties were in situ synthetized on the nanogold tag via RCA reaction. The formed biotin concatamers acted as a powerful scaffold to bind with avidin-alkaline phosphatase (ALP) conjugates and construct a nano-enzyme reactor. By this means, enzymatic hydrolysate (ascorbic acid) was generated to capture of the photogenerated holes in the CdS quantum dot-sensitized TiO2 nanotube arrays, resulting in amplification of photocurrent signal. To elaborate the microplate-based immunoassay and the high-throughput detection system, a semiautomatic detection cell (installed with a three-electrode system) was employed. Under optimal conditions, the photocurrent increased with the increasing PSA concentration in a dynamic working range from 0.001 to 3 ng mL-1 with a low detection limit (LOD) of 0.32 pg mL-1. Meanwhile, the developed split-type photoelectrochemical immunoassay exhibited high specificity and acceptable accuracy for analysis of human serum specimens in comparison with referenced electrochemiluminescence immunoassay method. Importantly, the system was not only suitable for the sandwich-type immunoassay mode, but also utilized for the detection of small molecules (e.g., aflatoxin B1) with a competitive-type assay format.

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■ INTRODUCTION The increasing interest to life science, food safety and environmental protection has led to a boom in the development of various analytical tools for the rapid and selective monitoring of trace-amount analyte. Photoelectrochemical (PEC) detection method has received the great attention because of its natural merits.1 First of all, the excitation source (light) and the readout signal (electricity) in the PEC method belong to different energy forms, endowing it with a low background signal and high sensitivity.2,3 Secondly, the use of electrochemical signal readout with the simple instrumentation and low cost gives the PEC method a huge advantage over conventional optical methods in simplicity.4 Moreover, ongoing progress on the semiconductor nanomaterials and molecular recognition technique has brought the extensive application of the PEC method in many fields including immunoassay,5 aptamer-based analysis,6,7 cell-related detection8

and enzymatic

sensing.9,10 Nowadays, however, the actual application of PEC method in routine laboratory detection still faces some obstacles. One major problem is that PEC detection system may inevitably cause the damage of biomolecules (both the immobilized bioprobes and captured target molecules on the electrode) because of the requirement for light illumination (especially using the UV light) as well as the strong oxidation characteristics of photo-generated holes in semiconductors.11 For example, TiO2 (one of the most commonly used semiconductor material) is of wide band gap (3.0-3.2 eV) and requires UV (λ < 400 nm) excitation. The corresponding photo holes can harm or even destroy biomolecules.12,13 Although the PEC-based biosensors could utilize the narrow-gap semiconductors with visible-light absorption ability (e.g., CdS and CdTe quantum dot),14,15 this unfavorable factor cannot be completely eliminated yet. Meanwhile, the absorption or scatter of biomolecules to visible light may also interfere with photocurrent signal.16 Similar to conventional electrochemical method, the sample incubation and signal monitoring in the PEC method are carried out on the same modified electrode that only one sample can be analyzed in a given period of time. This disadvantage determines its inherent low-throughput. To meet the need of high-throughput analysis, the detection of PEC method should be improved. Unfortunately, rather limited effort has been exerted in this 3



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area. Rolling circle amplification (RCA) is a simple and efficient isothermal enzymatic process, in which short nucleic acid fragment can be extended to a concatamer containing tens to hundreds of tandem repeats that are complementary to the circular template.17,18 By customizing the sequence of circular template, RCA products can be tailor-designed to include multifarious functional sequences including DNAzyme,19 aptamer20 and restriction enzyme sites.21 More importantly, the RCA-synthesized long DNA concatamer can massively carry fluorescent reagents, electroactive species or nano-bio-label materials through intercalative binding, electrostatic adsorption or DNA hybridization to produce strong electronic and optical signals.22-25 These excellent characteristics have made the RCA as a powerful tool to amplify detection signals of various biosensing methods on the basis of different signal-generation principles. To the best of our knowledge, there is still no report focusing on the RCA to amplify photocurrent signal in the PEC detection method. Herein we present a novel split-type photoelectrochemical immunoassay (STPIA) method for ultrasensitive and high-throughput detection of prostate-specific antigen (PSA, as a model analyte). To achieve this design, RCA-triggered formation of enzymatic concatamers on the nanogold particle was coupled with enzymatic hydrolysate-enhanced photocurrent response of CdS quantum dot (QD)-modified TiO2 nanotube array (Scheme 1). The enzymatic concatamer is formed via the biotin-avidin reaction

between

RCA-synthesized biotinylated concatamer and avidin-labeled alkaline phosphatase (ALP) after the immunoreaction and rolling circle amplification. To eliminate the potential damage of the biomolecules (e.g., antigen or antibody), the immunoreaction is initially carried out using secondary antibody/primer-circle DNA-labeled gold nanoparticle as the detection antibody in a 96-well microplate. Accompanying the RCA and the biotin-avidin reaction, the concatenated ALP molecules hydrolyze the ascorbic acid 2-phosphate (AAP) to ascorbic acid (AA). The as-produced ascorbic acid can be used as an efficient hole-trapping reagent to amplify the photocurrent of CdS quantum dot (QD)-modified TiO2 nanotube array with high sensitivity. The aim of this work is to design a new semiautomatic photoelectrochemical detection system for quantitative monitoring of low-abundant proteins or biocompounds. 4


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■ EXPERIMENTAL SECTION Materials and Chemicals. Oligonucleotides, T4 DNA ligase, Phi29 DNA polymerase, exonuclease I & III, dNTP and prostate specific antigen (PSA) standards were purchased from Dingguo Biotechnol. Co., Ltd. (Beijing, China). Mouse monoclonal anti-human PSA capture antibody (designated as mAb1) and goat polyclonal anti-human PSA detection antibody (designated as pAb2) were obtained from Abcam Inc. (Cambridge, MA) and ImmunoReagents Inc. (Raleigh, NC), respectively. All high-binding polystyrene 96-well microplates were achieved from Greiner Bio-One (Frickenhausen, 705071, Germany). Gold colloids with 16 nm in diameter were prepared and characterized as described in our previous work.26 Biotin-14-dATP conjugate and avidin-alkaline phosphatase (ALP) conjugate were acquired from Invitrogen (Carlsbad, CA) and Mabtech (Nacka Strand, Sweden), respectively. All other chemicals used in this work were of analytical grade and used without further purification. Ultrapure water used in all runs was obtained from a Millipore water purification system (18.2 MΩ cm-1, Milli-Q). All buffers including Tris-HCl buffer and phosphate-saline buffer were the products of Sigma-Aldrich. The sequences of the used oligonucleotides in this work are listed as follows: Primer DNA: 5'-SH-TGAGGTAGTAGGTTGTATAGTT-3' Linear padlock DNA: 5'-phosphate-CTACTACCTCATACACCTATATCGTCTAGTTAGTGTTGCTAGT TACTCTTCGCAATTTTAACTATACAAC-3'

The underlined sequence of the linear padlock DNA was the binding region of primer DNA. To ligate the linear padlock DNA into a circular template, a primer DNA without the thiol at 5' end was also customized. Preparation of CdS Quantum Dot-Modified TiO2 Nanotube Array. TiO2 nanotube array (designated as TiO2 NTA) was prepared by using a two-step anodization method according to the literature with minor modification.27 Prior to anodization, a titanium sheet (50 mm × 20 mm × 0.1 mm, 99.9%) was subjected to successive sonication in acetone, ethanol and distilled water for 15 min. Afterwards, titanium sheet was chemically polished in a mixture containing HF-HNO3-H2O with the volume ratio of 1:4:5 for 30 s, washed with distilled water, and dried under nitrogen. Electrochemical anodization was carried out in an

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ethylene glycol solution containing 0.3% (w/v) NH4F and 2% (v/v) distilled water at 50 V for 2 h, with a two-electrode system by using the titanium sheet as the working electrode and a graphite sheet as the counter electrode. After that, the resulting titanium sheet was washed again with distilled water and sonicated for 5 min to remove the surface layer. Following that, the second anodization was implemented by using the same method. Finally, the as-prepared TiO2 NTA was cleaned via sonication in distilled water and annealed at 450 °C for 2 h to crystallize the amorphous TiO2. Next, the TiO2 NTA was used as the support for the modification of CdS quantum dot (CdS QD) by using a sequential chemical bath deposition method.28 Briefly, the above-prepared TiO2 NTA was initially immersed into 0.05 M Cd(NO3)2, H2O, 0.05 M Na2S and H2O in turn, and then incubated for 30 s (each solution) at room temperature (Note: The whole process was repeatedly executed for 30 times, which was described in Figure S1). Finally, CdS QD-functionalized TiO2 NTA (designated as CdS/TiO2 NTA) was obtained by washing and drying dried under nitrogen. Preparation of Circular DNA Template. The circular DNA template was prepared by the ligation and circularization of the linear padlock. Before ligation reaction, the liner padlock DNA and primer DNA were mixed and denatured at 65 °C for 5 min, and slowly cooled to room temperature. The ligation reaction was carried out in a 10-µL reaction buffer containing 1× ligation buffer [40 mM Tris-HCl, 10 mM MgCl2, 10 mM dithiothreitol (DTT), 0.5 mM ATP (pH 7.8)], 12 U of T4 DNA ligase, 1.0 µM liner padlock DNA and 1.0 µM primer DNA (without the thiol at 5' end) at 37 °C for 4 h. After that, the reaction was terminated by heating at 90 °C for 10 min. Finally, 5 U of exonuclease I and 10 U of exonuclease III were added to the resulting solution to remove primer and unreacted linear padlock DNA. After being incubated for 2 h, the exonucleases were inactivated by heating at 95 °C for 10 min. The obtained circular DNA template was stored at 4 ºC for further use. Bioconjugation of Gold Nanoparticle with pAb2 and Primer-Circular DNA Template (pAb2-AuNP-pcDNA). Prior to bioconjugation with pAb2 and primer-circular DNA template, colloidal gold nanoparticles with 16 nm in diameter were adjusted to pH 9.2 by using Na2CO3 aqueous solution (0.1 M). Afterwards, 100 µL of pAb2 (1.0 mg mL-1) was injected to 3.0 mL of gold colloids (gold conc.: 24 nM) and incubated for 2 h at 4 ºC under 6


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gentle shaking (Note: The pAb2 antibody was covalently bound to gold nanoparticle by the dative binding between colloidal gold and free –SH group of the antibody during this process).29 Following that, the thiolated primer DNA (0.5 OD) was added to the resultant gold colloids. After being incubated for 2 h under the above-same conditions, 100 mM of Tris-HCl buffer containing 0.5 M NaCl (pH 7.5) was added to the mixture until a final concentration of 0.1 M NaCl and 20 mM Tris-HCl buffer (pH ≈ 7.4). The resulting mixture was further incubated for 6 h at 4 ºC, followed by centrifugation for 15 min at 14,000 rpm in order to remove excess pAb2 and primer DNA. The obtained pellets were dispersed into 1.0 mL of 10 mM PBS containing 1.0 wt % BSA. Subsequently, excess circular DNA template prepared above was added into the suspension and reacted for 4 h at 37 ºC to make the circular DNA hybridize with the primer DNA. Finally, gold nanoparticle labeled with pAb2 and primer-circular DNA (designated as pAb2-AuNP-pcDNA) was acquired by centrifugation and dispersed into 1.0 mL PBS, which was stored at 4 °C for further usage. Split-Type Photoelectrochemical Immunoassay (STPIA) for PSA. Scheme 1 gives the schematic illustration of split-type photoelectrochemical immunoassay for target PSA by coupling with target-induced nano-enzyme reactor mediated hole-trapping and pAb2-AuNP –pcDNA-based rolling cycle amplification. A high-binding polystyrene 96-well microplate was coated overnight at 4 °C with 50 µL per well of mAb1 antibody (10 µg mL-1) in 50 mM sodium carbonate buffer (pH 9.6). The microplates were covered with adhesive plastics plate sealing film to prevent evaporation. On the following day, the plates were washed three time with washing buffer (10 mM PBS containing 0.05% Tween 20, pH 7.4), and then incubated with 300 µL per well of blocking buffer (10 mM PBS containing 1.0 wt % BSA, pH 7.4) for 60 min at 37 °C with gentle shaking. The plates were then washed as before. Following that, 50 µL of PSA standard or sample was added into the microplate, and incubated for 40 min at 37 °C under gentle shaking. After being washed for three times with washing buffer, 50 µL of the above-prepared pAb-AuNP-pcDNA suspension was injected into the well and incubated for 40 min under the same conditions to form the sandwiched immunocomplex. Following that, the RCA reaction (induced by the primer-circular DNA on the AuNP) in the microplate was carried out by incubation with 50 µL of RCA reaction solution [Tris-HAc buffer (33 mM, pH 7.9) containing 10 mM MgAc2, 7



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66 mM KAc, 0.1% (v/v) Tween 20, 1.0 mM DTT, 200 nM biotin-14-dATP, 200 nM dTTP, 200 nM dGTP, 200 nM dCTP and 5 U Phi29 DNA polymerase] for 50 min at 37 °C. After that, 50 µL of avidin-alkaline phosphatase (ALP) conjugate (2 U) was injected into the well and incubated for 45 min to form the nano-enzyme reactor. During this process, the avidin-ALP conjugate was bound to the RCA product through the biotin-avidin chemistry. Subsequently, 200 µL Tris-HCl buffer (pH 9.0, 10 mM) containing 10 mM ascorbic acid 2-phosphate (AAP) and 1.0 mM MgCl2 was added into the well (Note: The microplate was washed with the washing buffer after each reaction). Finally, the enzymatic hydrolysate (ascorbic acid, AA) by the ALP was transferred to a homemade semiautomatic detection cell for photocurrent measurement.

Scheme 1 Schematic Illustration of (A) Immunoreaction-Induced Alkaline Phosphatase (ALP)-Based Nano-Enzyme Reactor Formation through Rolling Circle Amplification (RCA), and (B) Enzymatic Hydrolysate (Ascorbic Acid, AA) Mediated Hole-Trapping in CdS Quantum Dot (QD)-Sensitized TiO2 Nanotube Array (NTA) for the Amplification of Photocurrent Signal (Ascorbic Acid 2-Phosphate, AAP). 8


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Photocurrent Measurement. Photoelectrochemical (PEC) measurement was carried out on a homemade detection cell equipping with a peristaltic pump, a 500 W Xe lamp and a 420 nm cutoff filter (NBET, Beijing) at a constant potential of 0 V (versus Ag/AgCl) (Figure 1D). All photocurrent measurements were registered and recorded on an AutoLab electrochemical workstation (µAUTIII.FRA2.v, Eco Chemie, Netherlands) with a conventional three-electrode system including a CdS/TiO2 NTA-based working electrode, a Pt-wire counter electrode and an Ag/AgCl reference electrode. All determinations were made at least in duplicate. ■ RESULTS AND DISCUSSION Construction and Design of STPIA-Based Immunoassay. Scheme 1 represents the assay process of split-type photoelectrochemical immunoassay (STPIA) toward target PSA on the basis of target-induced rolling circle amplification and formation of ALP concatamer. Design of the split-type detection device for immunoreaction and photocurrent measurement is expected to eliminate the potential damage toward the biomolecules (e.g., antigen or antibody, caused by the photoelectrochemical system itself, the light radiation) and enhance the detectable throughput. The photocurrent is amplified by the concatenated ALP molecules on the basis of the RCA products toward the hydrolysis of AAP. Upon addition of target PSA in the microplate, the sandwiched immunocomplex is formed between the immobilized mAb1 capture antibody and the labeled pAb2 detection antibody on the pAb2-AuNP-pcDNA. Accompanying the AuNP, the carried primer-circular DNA can be used as the template for the RCA reaction in the presence of biotin-dATP, dTTP, dGTP and dCTP. In this case, numerous biotin moieties are present on the DNA backbone followed by dATP base. The biotinylated concatamer can be utilized for the reaction with the avidin-ALP conjugate through the biotin-avidin chemistry, thereby resulting in the formation of ALP concatamer on the AuNP (like a nano-enzyme reactor). In this way, each ALP molecule in the concatamer can hydrolyze the ascorbic acid 2-phosphate to ascorbic acid (an ideal hole-trapping reagent). Followed by the homemade flow-through detection device, the injected ascorbic acid captures the photo-generated hole (h+) of CdS/TiO2 NTA

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and donates the electron to inhibit the electron-hole recombination, thus increasing the photocurrent signal for the detection of target PSA. By monitoring the change in the photocurrent, we can evaluate the amount of target PSA in the sample. In addition, the microplate-based immunoreaction enables simultaneous detection of multiple samples, so enhancing the detection throughput of the STPIA. Characterization of CdS/TiO2 NTA. As described above, a photoelectrochemical material with high photoelectric transformation efficiency would be preferable for the successful development of STPIA-based immunoassay (which can sensitively probe the generating AA in this case). Compared with pure TiO2 nanoparticles (which is only photoactive in ultraviolet region), the light-utilization range of one-dimensional TiO2 nanotube array (NTA) can be extended to visible light region due to its high surface area and uniformly ordered structure. In addition, the morphology of TiO2 nanotube array was also useful for separating and directing electrons to the collecting electrode surface, making it an ideal candidate for photocurrent response.30 However, the large band gap of TiO2 nanotube array (3.2 eV) determined its inherent low photoelectric conversion efficiency in visible region, limiting its further applications.31 Hence, CdS QD-modified TiO2 NTA could be employed as the photoelectric transducer in this work, because CdS QD with narrow band gap was proven to greatly sensitize to the visible light photoresponse.32 Figure 1A shows the typical scanning electron microscopy (SEM) image of the as-synthesized TiO2 NTA, which exhibited a large scale of vertically aligned and highly ordered tubular structure, indicating the successful fabrication of TiO2 NTA. The magnified SEM image further demonstrated that the pore diameter of TiO2 NTA was about 100 nm (Figure 1A, insert). After chemical bath deposition of CdS, a layer of CdS QD was observed to cover on the surface of TiO2 nanotube (Figure 1B), and the size of CdS QD was about 5 nm according to the magnified SEM image (Figure 1B, insert). The results clearly demonstrated the successful synthesis of CdS QD-modified TiO2 NTA.

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Figure 1. SEM images of (A) TiO2 nanotube array and (B) CdS quantum dot-modified TiO2 nanotube array (Insets: the corresponding magnification images); (C) Photocurrent responses of (a) TiO2 NTA and (b) CdS QD-modified TiO2 NTA [Insets: (left) the photocurrent-transducer mechanism (left) and (right) photocurrents of CdS QD-modified TiO2 NTA toward different-concentration ascorbic acid]; and (D) Photograph of the semiautomatic STPIA electrochemical detection cell equipped with a three-electrode system and a peristaltic pump.

Next, the PEC property of CdS QD sensitized-TiO2 NTA was also investigated by transient photocurrent measurement under visible light illumination (≥ 420 nm). As indicated in Figure 1C, the photocurrent response of CdS QD modified-TiO2 NTA (curve 'b') was 3 - 4 times higher than that of TiO2 NTA alone (curve 'a'), demonstrating that sensitization of CdS QD on TiO2 NTA could efficiently enhance its visible-light photocurrent response. The sensitization mechanism was exhibited in the insert of Figure 1C. When illuminated by visible light, CdS QD with low band gap can effectively excite electrons and holes. Since the CB of TiO2 NTA was more positive than that of CdS QD, the excited electrons in CdS QD could rapidly inject to the CB of TiO2 NTA followed by 11



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directly transfer to the collector electrode (Ti substrate). Meanwhile, the excited holes of TiO2 NTA could transfer to CdS, causing the accumulation of holes in VB of CdS QD to form the hole center. In this case, the excited e-/h+ pairs could be separated effectively, thereby contributing to the enhancement of photocurrent. Especially, the capture of the hole center by AA would further inhibit the recombination of e-/h+ pairs, which also had a positive effect on photocurrent improvement. Experimentally, the photocurrent response of CdS QD sensitized-TiO2 NTA was observed to increase with the increasing AA concentration (Figure 1C, insert). More importantly, the response to AA was sensitive enough that even 1 nM of AA solution could result in a significant increase in photocurrent, providing a key precondition for the development of the STPIA-based immunoassay method. To further facilitate the PEC measurement and improve analysis speed of this method, the CdS QD-sensitized TiO2 NTA fabricated on Ti foil was equipped in an integrated PEC detection cell to directly serve as the photoelectrode. As shown in Figure 1D, the customized detection cell could not only integrate a three-electrode system, but also rapidly remove the test solution after measurement using a peristaltic pump (detailed structure of the PEC cell is shown in Figure S2 of the Supporting Information). More importantly, the sample volume required for PEC measurement in the integrated cell could be as low as 150 µL, thus the generating AA solution could be directly tested without dilution and sacrifice of sensitivity.

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Figure 2. (A) SPR measurements of the surface binding reactions starting from the exposure of gold substrate to (a) thiol-primer DNA, followed by (b) 3-mercapto hexanol, (c) circle template DNA, (d) Phi29 DNA polymerase + (biotin-dATP + dTTP + dGTP + dCTP), and finally avidin-ALP; (B) UV-vis absorption spectra of (a) pAb2/primer DNA-labeled AuNP, (b) sample 'a' + circle template DNA, and (c) sample 'b' after rolling circle amplification; (C) SPR responses of (a) mAb1-modified gold substrate, (b) substrate 'a' + 0.1 ng mL-1 PSA, (c) substrate 'b' + pAb2-AuNP-pcDNA, (d) substrate 'c' + RCA, and (e) substrate 'e' + avidin-ALP; and (D) Photocurrent responses of STPIA-based immunoassay toward 0.1 ng mL-1

PSA

using

different

signal-amplification

strategies:

(b)

pAb2-AuNP-pcDNA,

(d)

pAb2-AuNP-pcDNA without the labeled biotin on the dATP, (e) ALP-AuNP-pAb2 and (f) ALP-labeled pAb2 [Notes: photocurrent responses of (a) AAP on CdS/TiO2 NTA and (c) the STPIA toward 0 ng mL-1 PSA with the pAb2-AuNP-pcDNA].

Characteristics of Split-Type Photoelectrochemical Immunoassay. In addition to the fabrication of CdS QD sensitized-TiO2 NTA based photoelectrode, several issues in this protocol should be further investigated and evaluated to ensure its successful implementation: (i) whether the immunoreaction could trigger the progression of RCA reaction, (ii) whether the RCA-based product could be carried out on the

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pAb2-AuNP-pcDNA to scaffold the formation of ALP concatamer, and (iii) whether the photocurrent signal could

be specifically achieved by immunoreaction-induced

nano-enzyme reactor. To clarify the issues, we initially used surface plasmon resonance (SPR) with angle scanning mode to real-time investigate the RCA reaction and assembly of avidin-ALP. As shown in Figure 2A, the SPR response (∆θ versus time) initially increased with injection of thiol-primer (curve 'a'), indicating the self-assembly of thiol-primer on the gold chip. When 3-mercapto hexanol was introduced to block the electrode surface, the SPR response increased slightly (curve 'b'). The subsequent injection of circle template continued to increase the SPR response due to the hybridization of circle template on the primer (curve 'c'). It is worth noting that a remarkable angle shift occurred with the introduction of RCA buffer (containing all materials required for RCA reaction) (curve 'd'). This significant SPR response clearly demonstrated that RCA reaction could be successfully carried out with the aid of primer and circle template to synthesize long DNA concatemers with high molecular weight. Furthermore, another remarkable angle shift was observed when incubated avidin-ALP with RCA product (curve 'e'), suggesting that the long concatemers were in-situ decorated with biotin moieties that they could act as polymeric scaffolds to load a lot of ALP through biotin-avidin linkage. Importantly, the RCA could also be readily carried out on the pAb2-AuNP-pcDNA according to UV-visible spectra characterizations. As shown in Figure 2B, the UV-visible spectra of pAb2-AuNP-pcDNA exhibited two absorption peaks at 260 and 520 nm (curve 'a'), which were characteristic peaks of DNA molecules and colloidal gold, respectively. The hybridization of circle template on the AuNP further increased the absorption peak at 260 nm (curve 'b') with the absorption peak at 520 nm remained unchanged. Moreover, the absorption peak of 260 nm increased sharply when the RCA was implemented on the pAb2-AuNP-pcDNA to synthesize long DNA concatemers (curve 'c'). Based on these results, we could conclude that pAb2-AuNP-pcDNA were useful for mediating the RCA and constructing nano-enzyme reactor. SPR measurement was also employed to investigate the feasibility of the sandwiched immunoreactions-triggered formation of nano-enzyme reactor. As shown in Figure 2C, the SPR

response

continuously

increased

with

the

immunoreactions,

14


capture

of

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pAb2-AuNP-pcDNA, implementation of biotinylated RCA and formation of nano-enzyme reactor. This observation clearly demonstrated that sandwiched immunoreactions could trigger the formation of nano-enzyme reactor. Furthermore, the catalytic capability of the nano-enzyme reactor for AA production and signal amplification was studied through photocurrent measurement achieved by controlled experiments. AAP solutions as substrates for ALP were added into the microwell plate with different treatments, and then injected into the integrated PEC cell to perform photocurrent measurement. As shown in Figure 2D, the AAP solution incubated in bare microwell plate (only immobilized with mAb1) could only cause a low background photocurrent due to the lack of hole-traping dopant (curve 'a'). In contrast, when the sandwiched immunoassay with signal amplification by the nano-enzyme reactor was performed in microplate, the AAP solution could cause a significant increase in photocurrent response after incubating in the resulting microplate (curve 'b'). The reason was that the immunoreactions induced ALP-based nano-enzyme reactor could catalyze the hydrolysis of AAP to product AA as hole-traping dopant. Importantly, no obvious change in photocurrent was observed when the immunoreactions and biotinylated RCA were performed in the absence of target PSA (curve 'c'), indicating that the photocurrent response was specifically triggered by target PSA. Also, there was almost no photocurrent increase could be observed when RCA was carried out without the participation of biotin-dATP (curve 'd'). It was clearly that the RCA-synthesized concatemer without biotin moiety could not capture avidin-ALP to form the nano-enzyme reactor. To further demonstrate the signal amplification ability of ALP-based nano-enzyme reactor, two other signal tags containing the ALP-labeled pAb2 (curve 'e') and pAb2-ALP-AuNPs conjugates (curve 'f') were used in the STPIA to catalyze the production of AA and photocurrent. The comparison of curve 'b' with curve 'e' and curve 'f' clearly demonstrated the excellent signal amplification of the biotinylated RCA-triggered nano-enzyme reactor. Based on these results, we might make a conclusion that the STPIA could be preliminary applied for PSA detection by coupling with the biotinylated RCA-triggered nano-enzyme reactor.

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Figure 3. Effects of (A) incubation time for the antigen-antibody reaction, (B) RCA reaction time, (C) AAP concentration, and (D) hydrolytic time of ALP-based nano-enzyme reactor toward AAP on the (A,B,D) photocurrent and (C) the signal-to-background (S/B) ratio of STPIA-based immunoassay (0.1 ng mL-1 PSA used in this case).

Optimization of Experimental Conditions. To achieve an optimal analytical performance of the STPIA-based immunoassay, several experiment conditions including the reaction time for the immunoreaction, the reaction time for RCA, AAP concentration and hydrolytic time for AAP should be investigated (0.1 ng mL-1 PSA used as an example). The incubation time for the antigen-antibody reaction was evaluated by using the transient photocurrent as judging criteria. As shown in Figure 3A, the photocurrent increased with the increment of incubation time and leveled off after 30 min. Longer incubation did not cause the significant increase in photocurrent. Hence, 30 min was used as the incubation time for the antigen-antibody reaction. At this condition, we also investigated the effect of the reaction time for RCA on the photocurrent of STPIA-based immunoassay since the amount of ALP concatamers would directly affect the assay sensitivity. Figure 3B displays 16


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the dependence of the photocurrents on the time of RCA reaction. By the same token, the photocurrents increased with the increasing reaction time and an optimal signal could be achieved at 50 min. To save the assay time, 50 min was used for the RCA reaction after the antigen-antibody reaction. In this work, the photocurrent mainly derives from the enzymatic hydrolysate toward the substrate (AAP) to capture the photo-generated hole in the CdS QD sensitized-TiO2 nanotube array. Thus, the concentration of AAP in the detection system was expected to affect

the

response

signal.

Figure

3C

depicts

a

correlation

between

the

signal-to-background (S/B) ratio (i.e., relative to the background signal) as a function of the concentration of AAP. With the increasing AAP concentration, the S/B ratio was observed to increase to a maximum (at 10 mM) and then decrease substantially. We attributed the initial increase in the S/B ratio to the fact that high-concentration AAP as enzymatic substrate could result in a high reaction rate. However, excess AAP might cause a high background signal, which was the reason for the later decrease of S/B ratio. So, 10 mM AAP was employed as enzyme substrate. Finally, the enzymatic hydrolytic time toward AAP was also investigated by using 10 mM AAP as the enzymatic substrate, and an optimal signal was achieved at 25 min (Figure 3D). Analytical Performance of STPIA-Based Immunoassay. Under the optimal conditions, the sensitivity and dynamic range of the STPIA-based immunoassay were studied by assaying routine samples with different PSA standards based on the developed strategy. The transient photocurrents increased with increasing PSA concentration in the sample (Figure 4A). A linear dependence between the change of photocurrent (relative to background signal) and the logarithm of PSA concentration was obtained in the range from 0.001 ng mL-1 to 3 ng mL-1 (Figure 4B). The linear regression equation was i (µA) = 181 × lg(C[PSA]/ng mL-1) + 559 (R2 = 0.995, n = 8) with a detection limit (LOD) of 0.32 pg mL-1 estimated at a signal-to-noise ratio of 3σ (where σ is the standard deviation of a blank sample, n = 11, i.e. IUPAC recommended method, 1978). Obviously, the LOD of the STPIA-based immunoassay was lower than those of commercialized human PSA ELISA kits from different companies (e.g., 8 pg mL−1 for Sigma-Aldrich; 0.5 ng mL-1 for Biocell. Biotechnol. Co., Ltd., Zhengzhou, China). Such a high sensitivity was ascribed to the 17



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strong signal-amplification ability of RCA-synthesized nano-enzymatic reactor and the free diffusion of the generated ascorbic acid to the CdS QD-sensitized TiO2 nanotube array.

Figure 4. (A) Photocurrent responses of the STPIA-based immunoassay toward different-concentration PSA standards; (B) the corresponding calibration curve of the photoelectrochemical immunoassay; (C) the specificity of the STPIA-based immunoassay against target PSA (0.1 ng mL-1), CA 19-9 (1.0 U mL-1), CEA (1.0 ng mL-1), CA 15-3 (1.0 U mL-1), CA 125 (1.0 U mL-1), AFP (1.0 ng mL-1), and HIgG (1.0 ng mL-1); and (D) comparison of the results for human serum samples obtained between the STPIA-based immunoassay and the referenced ECLIA-based immunoassay method.

To validate the specificity and selectivity of the STPIA-based immunoassay, we challenged the system against other biomarkers in human serum, e.g., carcinoembryonic antigen (CEA), cancer antigen 15-3 (CA 15-3), cancer antigen 125 (CA 125), alpha-fetoprotein (AFP), human immunoglobulin G (HIgG) and cancer antigen 19-9 (CA 19-9). As shown in Figure 4C, the photocurrents of the STPIA-based immunoassay toward CEA, CA 15-3, CA 125, AFP, HIgG and CA 19-9 were almost the same as the background

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signal (i.e., blank sample), while the strong photocurrent was obtained toward target PSA. More significantly, the co-existence of interfering materials with target PSA did not cause the significant change in the photocurrent toward target analyte, indicating the high specificity of the developed STPIA. To further monitor the application potential of the STPIA-based immunoassay for the real samples, 8 human serum specimens containing different-concentration PSA (collected from Department of the Medical Diagnostics of local Provincial Hospital, Fujian, PR China) were measured by the developed STPIA and a referenced electrochemiluminescence immunoassay (ECLIA) method, respectively. Inspiringly, the regression equation for the data obtained from two methods displayed a well positive correlation with a slope of 0.987, an intercept of 0.004 and a correlation coefficient of 0.9862 (Figure 4D). These results revealed that the developed STPIA could hold great potential as a reliable technique for the detection of PSA in complex system. The universality of the developed STPIA method was further investigated by applying it for the detection of small biomolecules (aﬂatoxin B1, AFB1, a highly toxic mycotoxin in food) with a competitive-type immunoassay formation. Results revealed that our strategy could be also utilized for monitoring the small-molecular AFB1 with high sensitivity and selectivity (please see Figure S3 and the corresponding description in the Supporting Information). ■ CONCLUSIONS In summary, this work reports on a novel split-type photoelectrochemical immunoassay for ultrasensitive detection of disease-related proteins (PSA used as the model analyte) coupling with the nano label and RCA-synthesized nano-enzyme reactor for the signal amplification. The photocurrent originated from the as-synthesized CdS QD-sensitized TiO2 nanotube array. Compared with traditional photoelectrochemical immunoassays, our developed STPIA-based immunoassay could effectively avoid the biomolecular damage caused by photoelectrochemical system itself (Please see Figure S4 in the Supporting Information) since target recognition and photocurrent measurement were separated into

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two parts. Moreover, immunoreaction-induced RCA reaction could promote the formation of numerous alkaline phosphatase concatamers on gold nanoparticle (similar to a nano-enzymatic reactor), which could largely amplify the photocurrent signal via the enzymatic hydrolysate (ascorbic acid, AA) to capture the photogenerated holes in the CdS QD-sensitized TiO2 nanotube array. Furthermore, introduction of microplates and semi-automatic detection device could provide an accessible route to enhance the detection throughput of the SPTIA-based immunoassay. More importantly, our strategy is not only suitable for the sandwich-type immunoassay mode, but also can be applied to the competitive-type assay protocol for the detection of small biomolecules, thereby representing a universal and useful scheme with great potential in the field of clinical diagnostics and food safety testing. ■ ACKNOWLEDGEMENT Support by the National Natural Science Foundation of China (grant nos. 41176079 & 21475025), the National Science Foundation of Fujian Province (grant no. 2014J07001), and the Program for Changjiang Scholars and Innovative Research Team in University (grant no. IRT1116) is gratefully acknowledged.

■ ASSOCIATED CONTENT Supporting Information Additional information as noted in the text. The Supporting Information contains Optimization of CdS QD deposition time, immunoassay for AFB1, effect of light illumination on the activity of ALP, and Figure S1 to Figure S4.

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■ For TOC only

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Target-Induced Nano-Enzyme Reactor Mediated Hole-Trapping for

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