Near-Infrared Light-Excited Core-Core-Shell UCNP@Au@CdS

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Near-Infrared Light-Excited Core-Core-Shell UCNP@Au@CdS Upconversion Nanospheres for Ultrasensitive Photoelectrochemical Enzyme Immunoassay Zhongbin Luo, Lijia Zhang, Ruijin Zeng, Lingshan Su, and Dianping Tang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02421 • Publication Date (Web): 25 Jun 2018 Downloaded from http://pubs.acs.org on June 26, 2018

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Analytical Chemistry

Near-Infrared Light-Excited Core-Core-Shell UCNP@Au@CdS Upconversion Nanospheres for Ultrasensitive Photoelectrochemical Enzyme Immunoassay Zhongbin Luo, Lijia Zhang, Ruijin Zeng, Lingshan Su, and Dianping Tang*

Key Laboratory for Analytical Science of Food Safety and Biology (MOE & Fujian Province), State Key Laboratory of Photocatalysis on Energy and Environment, Department of Chemistry, Fuzhou University, Fuzhou 3501116, People's Republic of China

CORRESPONDING AUTHOR INFORMATION Phone: +86-591-2286 6125; fax: +86-591-2286 6135; e-mail: [email protected] (D. Tang)

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ABSTRACT: A novel photoelectrochemical (PEC) enzyme immunoassay was designed for the ultrasensitive detection of alpha-fetoprotein (AFP) based on near-infrared (NIR) light-excited core-core-shell UCNP@Au@CdS upconversion nanospheres. Plasmonic gold (Au) between the sandwiched layers was not only utilized as an energy harvester for the collection of the incident light, but acted as an energy conveyor to transfer the energy from upconversion NaYF4:Yb3+, Er3+ (UCNP) to semiconductor CdS, thus exciting the efficient separation of electron-hole pairs by the generated H2O2 of enzyme immunoreaction under the irradiation of a 980-nm laser. By virtue of high catalytic activity of natural enzymes, gold nanoparticles heavily functionalized with glucose oxidase (GOx) and polyclonal anti-AFP antibody were utilized to generate H2O2. A sandwiched immunoreaction was firstly carried out in monoclonal anti-AFP antibody-coated microplate by using antibody-labeled gold nanoparticle as secondary antibody. Accompanying gold nanoparticle, the carried GOx oxidized glucose in H2O2, thereby resulting in the enhanced photocurrent via capturing holes on the valence band of CdS to promote the electron-hole pairs separation. Under optimum conditions, NIR light-based PEC immunosensing system exhibited good photocurrent responses toward target AFP within the dynamic working range of 0.01 – 40 ng mL-1 at a detection limit of 5.3 pg mL-1. Moreover, NIR light-based sensing platform had good reproducibility and high selectivity. Importantly, good well-matched results obtained from NIR light-based PEC immunoassay were acquired for the analysis of human serum specimens by using AFP ELISA kit as the reference.

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Photoelectrochemical (PEC) immunoassay sensing technique, as an innovative and promising analytical tool by integrating photochemistry with electrochemistry, has been developed rapidly in recent years.1-3 During typical PEC process, the separation of electron-hole pairs and charge transfer occurred on photoactive materials under light irradiation cause the photon-to-electricity signal conversion, whereas the corresponding photocurrent can be significantly affected by the electron donor/acceptor.4,5 Typically, the electron donor/acceptor can be produced by biological reactions after the target-recognition process. Thanks to the merits (e.g., low-background signal, high sensitivity, accessible operation and device portability), ongoing efforts have been made worldwide to develop and improve photoelectrochemical sensing systems with high efficiency in a very predictable manner to meet the needs of specific applications.6-8 Routine approaches are usually designed on the basis of the visible/ultraviolet light as the light source.9,10 Recent research has looked to explore new light sources for the development of PEC immunosensing platforms. One major advantage of using near-infrared (NIR) light is that its region accounts for a large proportion (about 50%) of the solar spectrum, and can efficiently avoid destruction of the biological system because of low phototoxicity.9,10 Upconversion materials have attracted increasing attention due to their brilliant performance such as excellent penetrability, low light damage and high light stability.11,12 Generally speaking, the upconversion materials convert a long-wavelength incident light into a shorter one with the higher energy through an anti-Stokes process,13 and are usually applied to convert near-infrared light into the visible light and ultraviolet light.13,14 These materials have been widely utilized in biomedical imaging,15 solar energy conversion,16 photodynamic therapy,17 photocatalysis18 and other research fields. For instance, Chen's group utilized lanthanide-doped LiLuF4 upconversion nanoprobes for the detection of disease biomarkers.19 Hao et al. reported a method for designing the upconversion material, Zn3Ga2GeO8:Yb/Er/Cr, as a rechargeable fluorescent probe for the bioimaging in vivo.20 Using near-infrared light as the indirect excitation light, the photosensitive material in the mode of the semiconductor-coated upconversion nanoparticle (UCNP@S) has been rapidly developed.21-23 As a result of the small absorption cross section of UCNP and the strong interfacial reflection between UCNP and semiconductor, however, utilization of light always turns out to be unsatisfactory.24,25 To utilize light energy efficiently, we combine the 3



plasmonic gold (Au) to synthesize core-core-shell UCNP@Au@CdS upconversion nanospheres by using NaYF4:Yb3+,Er3+ as the upconversion nanoparticle (UCNP) and CdS as the semiconductor material. To this end, the emission spectrum of UCNP and the absorption spectrum of CdS are well-matched with surface plasmon resonance (SPR) effect of plasmonic Au. In this regard, not only can plasmonic Au serve as the energy harvester to efficiently collect the incident light (980 nm), but also can act as an energy conveyor to gather energy from UCNP through fluorescence resonance energy transfer (FRET) and transfer the accumulated energy to CdS via the plasma resonance energy transfer (PRET), thereby minimizing the energy dissipation at the interface of UCNP and CdS.

Scheme 1. Schematic representation of near-infrared (NIR) light-triggered photoelectrochemical (PEC) immunoassay for target alpha-fetoprotein (AFP) based on core-core-shell UCNP@Au@CdS upconversion nanospheres: (A) Immunoreaction process on monoclonal anti-AFP capture antibody (Ab1)-coated microplate using glucose oxidase and polyclonal anti-AFP antibody-labeled gold nanoparticle (GOx-AuNP-Ab2) as the detection antibody with a sandwich-type assay format, and (B) diagram of energy transfer (ET) among lanthanide ion, Au and CdS with H2O2 under NIR irradiation (980 nm) (UCNP: NaYF4:Yb3+,Er3+-based upconversion nanoparticle; FRET: fluorescence resonance energy transfer; PRET: plasma resonance energy transfer; ETU: energy-transfer upconversion; BSA: bovine serum albumin).

Alpha-fetoprotein (AFP; an oncofetal glycoprotein produced by the liver and the yolk sac) is 4


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widely used as the cancer biomarker of yolk sac cancer, nasopharyngeal cancer, hepatocellular cancer and other diseases.26-28 Thus, sensitive detection of AFP is absolutely crucial in clinic diagnostics. Herein we report a sensitive and feasible PEC sensing strategy for the detection of AFP coupling core-core-shell UCNP@Au@CdS upconversion nanospheres (as the photoactive material) with target-controlled enzyme immunoreaction system (Scheme 1). The assay consists of enzyme immunoreaction (panel A) and NIR light-activated photocurrent measurement (panel B). In the presence of target AFP, glucose oxidase (GOx) and detection antibody-labeled gold nanoparticles accompanying specific antigen-antibody reaction is introduced in the microplate. The carried GOx oxidizes glucose to generate H2O2, which serves as the hole-trapping reagent to increase the photocurrent of UCNP@Au@CdS via fluorescence resonance energy transfer (FRET) and plasma resonance energy transfer (PRET). This study aims to explore a novel NIR light-based photoelectrochemical immunoassay for ultrasensitive detection of low-abundance protein by utilization of highly efficient upconversion nanomaterials.

EXPERIMENTAL SECTION Synthesis of Core-Core-Shell UCNP@Au@CdS Upconversion Nanospheres. For typical synthesis, core-core-shell UCNP@Au@CdS upconversion nanospheres were prepared by using the wet-chemistry method referring to the literature.24 Prior to synthesis, a mixture of RECl3 with the molar ratio of Y : Yb : Er = 44 : 20 : 1 was firstly prepared by mixing YbCl3·6H2O, YCl3·6H2O and ErCl3·6H2O. Then, 20 mL of the above-prepared RECl3 mixture was injected into 20-mL ultrapure water containing 1.6-mmol EDTA under vigorous stirring. Following that, NaF aqueous solution (20 mL, 0.72 M) was dropped into the resulting mixture. After stirring for 60 min at rt, the suspension was transferred into a 100-mL Teflon-lined autoclave and heated for 3 h at 180 °C. Finally, the obtained NaYF4:Yb3+, Er3+ upconversion nanoparticles (UCNP) were washed with ultrapure water and ethanol alternately for three times by centrifugation (10 min, 13,000g), and dried at 60 °C. Next, the as-prepared UCNP was utilized to synthesize core-core-shell UCNP@Au@CdS as follows. Initially, HAuCl4 aqueous solution (20 mL, 1.0 µM) was adjusted to pH ~9.0 by using 1.0 M Na2CO3 for displacing the Cl- ions with OH-, and then 400-mg UCNP was added into the 5



mixture under vigorous stirring and dried in a 60 °C water bath, thus Au(III) was completely precipitate on the surface of bare UCNP. The obtained precipitate was added to NaBH4 aqueous solution (50 mL, 1.0 M), and vigorously stirred for 2 h at rt for the reduction of Au(III) to Au0. Following that, the core-shell UCNP@Au nanospheres were collected by centrifugation (10 min, 13,000g). Subsequently, the obtained UCNP@Au nanospheres were added into 10-mL L-cysteine

aqueous solution (0.04 M) containing 0.2-mmol Cd(NO3)2 under vigorous stirring (2

h, rt), followed by introduction of ethanol (70 mL). Finally, the resultant mixture was transferred into a Teflon-lined autoclave and heated at 160 °C for 12 h. After cooling to rt, core-core-shell UCNP@Au@CdS upconversion nanospheres were washed and centrifuged as before, and dried at 60 °C for further use.

Immunoreaction and NIR Light-Based Photoelectrochemical (PEC) Measurement. Prior to measurement, gold nanoparticle (AuNP) with 16 nm in diameter, glucose oxidase (GOx) and polyclonal anti-human AFP antibody (Ab2)-labeled gold nanoparticle (Ab2-AuNP-GOx), and monoclonal mouse anti-human AFP antibody (Ab1)-coated in the microplate were prepared on the basis of our reports (Please see the detailed process in the Supporting Information).29-31 Scheme 1 gives the schematic illustration of NIR light-mediated PEC immunosensing system toward target AFP based on core-core-shell UCNP@Au@CdS upconversion nanospheres after typical sandwich-type immunoreaction on anti-AFP capture antibody-coated microplate using Ab2-AuNP-GOx as the secondary antibody. Initially, 50 µL of AFP standards or samples with various concentrations was added into the microplate, and incubated for 50 min at 37 °C under shaking. After washing, Ab2-AuNP-GOx suspension (50 µL, C[Au] ≈ 250 nM) was added into the well and incubated for 50 min at 37 °C with shaking. The plates were washed three times with washing buffer and one time with water, and then 50 µL of 5.0 mM glucose in PBS (10 mM, pH 6.0) was added to each well and incubated at 37 °C for 20 min. Subsequently, the resulting solution was transferred into the detection cell for photoelectrochemical measurement on the UCNP@Au@CdS-modified Fluorine-doped tin oxide (FTO) electrode (Please see the Supporting Information for measurement procedure).

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RESULTS AND DISCUSSION Characterization of Core-Core-Shell UCNP@Au@CdS Upconversion Nanospheres. For the successful development of NIR light-based photoelectrochemical sensing system, design of upconversion materials is very important since it directly affects the sensitivity of this strategy. Herein, core-core-shell UCNP@Au@CdS nanospheres are used as the photosensitive materials for the generation of photocurrent under the irradiation of 980 nm. The sandwiched plasmonic Au between UCNP upconversion material and CdS semiconductor can be efficiently employed as an energy harvester to collect the incident light, meanwhile, undertake an energy conveyor to transfer the energy from NaYF4:Yb3+, Er3+ UCNP to CdS, thereby exciting the high-efficiency separation of electron-hole pairs. In this case, the photocurrent is enhanced with the assistance of H2O2. The assay mainly consists of the sandwiched immunoreaction and NIR light-induced PEC measurement. The immunoreaction is carried out on monoclonal anti-AFP Ab1 capture antibody-coated microplate by using Ab2-AuNP-GOx as the secondary antibody. AuNP heavily functionalized with GOx and Ab2 detection antibody is expected to increase the immobilized amount of natural enzyme. Accompanying the immunoreaction, the carried GOx can catalyze glucose to generate H2O2, thus resulting in the amplification of photocurrent signal by capturing the holes on the valence band of CdS to promote the electron-hole pairs separation. To realize our design, the structure and morphology of the core-core-shell UCNP@Au@CdS nanospheres were firstly characterized using high-resolution transmission electron microscopy (HRTEM; Tecnai G2 F20 S-TWIN FEI, USA) (Figure 1A-C). Figure 1A shows typical TEM image of the as-prepared NaYF4:Yb3+, Er3+ upconversion nanoparticles, and the average size was 350 nm in diameter. Moreover, the surface was also relatively smooth (Figure 1A, inset). Upon introduction of HAuCl4 with the help of NaBH4, the surface of nanoparticles became rougher than that of UCNP alone (Figure 1B), and numerous nanoparticles were also observed on the surface of the UCNP (Figure 1B, inset). After reaction of the formed nanospheres with Cd(NO3)2 and L-cysteine under heating at 160 °C for 12 h, significantly, a fluff-like structure was acquired with a large number of nanostructures on the surface (Figure 1C). Obviously, the topology of nanoparticle on the surface (Figure 1C, inset) was different from that in the inset of Figure 1B. The sizes of the nanospheres after each step were further verified by using dynamic 7



light scattering (DLS; Zetasizer Nano S90, Malvern, U.K.). The average sizes were 352 ± 3.2, 357 ± 1.3 and 361 ± 3.2 nm for UCNP, UCNP@Au and UCNP@Au@CdS, respectively. Such a unique construction was favorable for the light absorption and mass transfer.23

Figure 1. HRTEM images of (A) NaYF4:Yb3+,Er3+ UCNP, (B) core-shell UCNP@Au and (C) core-core-shell UCNP@Au@CdS (insets: magnification images); XPS spectra of (D) core-core-shell UCNP@Au@CdS nanospheres (insets: XPS spectra of Yb4d and Au4f orbits, respectively) and (E) Er4d orbits; (F) XRD patterns of core-core-shell UCNP@Au@CdS nanospheres (insets: magnification portions of regions I and II).

Logically, one question arises to whether the synthesized nanospheres were really composed of NaYF4:Yb3+, Er3+, Au and CdS. To clarify this concern, X-ray photoelectron spectroscopy (XPS; VG Scientific ESCALAB 250 spectrometer, USA) was employed for verification of the obtained nanospheres. As shown from XPS spectrum in Figure 1D-E, all the elements including Na, Y, F, Yb, Er, Au, S and Cd were present in the single architecture, indicating the successful synthesis of UCNP@Au@CdS nanospheres. Two peaks at 84.0 eV (Au 4f5/2) and 87.7 eV (Au 4f7/2) were assigned to noble metallic Au (Figure 1D, right inset), whereas the peak at 186.6 eV derived from the spectra of Yb 4d (Figure 1D, left inset).32 Moreover, the binding energy peak of element S could be covered at 168.5 eV due to its low content except for binding energies at 172.7 and 174.8 eV from Er3+ ions relative to Er 4d (Figure 1E). Further, the crystal form of the UCNP@Au@CdS was monitored by X-ray diffraction (XRD; PANalytical X'Pert spectrometer, Netherlands). As seen from Figure 1F, XRD patterns of the as-prepared nanospheres matched 8


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well with the standard cubic phase of NaYF4 (JCPDS no. 77-2042).24 In addition, the enlarged XRD patterns of regions I and II were revealed in the illustrations which the diffraction peaks at 24.8°, 26.5° and 28.2° (left inset) were respectively corresponding to the (100), (002) and (101) crystal planes of wurtzite CdS (JCPDS no. 70-2553), and the peak at 38.2° (right inset) could be assigned to metallic Au (111) (JCPDS no. 89-3697).24 On the basis of these results, we might make an affirmative conclusion that core-core-shell UCNP@Au@CdS nanospheres could be successfully synthesized through a chemical impregnation reduction process and a solvothermal process without any phase changes, which provided a necessary prerequisite for development of NIR light-activated PEC sensing system.

Feasibility Evaluation of NIR Light-Activated PEC Sensing System. Figure 2A gives the real samples of UCNP, UCNP@Au and UCNP@Au@CdS under the irradiation of natural light and 980 nm laser, respectively. With the attachment of plasma Au and the coating of CdS on the surface of UCNP, the nanospheres changed from white (a) to purple (b) then yellow-brown (c) under natural light, whereas the upconversion emission intensities decreased in sequence (d-f) under a 980-nm laser (Figure 2A). These results could be further demonstrated in Figure 2B. Yb3+ was served as a sensitizer to adsorb the incident light from the 980 laser, whilst Er3+ was acted as an activator to give the multi emissions: the single emission peak at 660 nm and double peaks at 530 and 550 nm could be assigned to 4F9/2 → 4I15/2, 4S3/2 → 4I15/2, and 2H11/2 → 4I15/2 transitions of Er3+, respectively.33 To further demonstrate that the decrement of light intensities originated from the plasmonic Au and semiconductor CdS, UV-vis diffuse reflectance spectra (DRS) was used to characterize the as-synthesized nanospheres (Figure 2C). Bare UCNP (curve 'a') was inactive in the visible and ultraviolet range while a strong broad absorption curve located in 500 ~ 600 nm arose after the modification with plasmonic Au (curve 'b'), which was attributed to the absorption of SPR effect of gold nanoparticles. In addition, the general increment over the entire spectral range of UCNP@Au (curve 'b') illustrated that plasmonic Au indeed enhanced the absorption to incident light.34 In addition to the absorption of SPR, the UV-vis absorption curve of UCNP@Au@CdS (curve 'c') also clearly described the bandgap absorption of CdS which energy band gap of 2.24 eV could be calculated from the absorption edge at 554.8 nm (using a tangent line) by following the relationship of Eg = 1240/λ.35 By combining the Upconversion luminescence (UCL) 9



spectral of UCNP (region 'd'), we could observe that both the SPR characteristic absorption of Au and the band gap absorption of CdS matched with the emission well, suggesting stable and effective energy transfer.

Figure 2. (A) Pictures of UCNP, UCNP@Au and UCNP@Au@CdS powder under (a,b,c) natural light and (d,e,f) 980 nm laser; (B) Upconversion photoluminescence spectra of upon NIR excitation at 980 nm from UCNP, UCNP@Au and UCNP@Au@CdS (inset: the corresponding magnification spectra for UCNP@Au and UCNP@Au@CdS); (C) UV-vis diffuse reflectance spectra of (a) UCNP, (b) UCNP@Au and (c) UCNP@Au@CdS [note: (d) upconversion photoluminescence spectra of UCNP]; (D) Photocurrent responses of (a) UCNP, (b) UCNP@Au and (c,d) UCNP@Au@CdS on the FTO electrode under NIR light in Na2SO4 solution (0.1 M) in the (a,b,c) absence and (d) presence of 1.0 ng mL-1 target AFP (inset: magnification curves 'a,b,c').

Before the successful establishment of NIR light-activated PEC sensing systems for detection of cancer biomarker based on core-core-shell UCNP@Au@CdS upconversion nanospheres, a puzzling concern to be produced was whether the photocurrents of the synthesized nanospheres could be effectively enhanced by the products of enzyme-linked immunoreaction. As shown in 10


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Figure 2D, the increment in the photocurrent from curve 'a' to curve 'c' further corroborated the successful coating of plasmonic Au and CdS. Unfavorably, the photocurrent obtained from the UCNP@Au@CdS upconversion nanospheres alone was relatively small in the absence of any promoters, e.g., H2O2. Another question was whether UCNP@Au@CdS-modified electrode could be utilized for the determination of target AFP with high sensitivity under the irradiation of 980-nm laser. To verify this issue, NIR light-mediated PEC immunosensing platform was investigated in the absence and presence of target AFP (1.0 ng mL-1 AFP used as an example) by coupling UCNP@Au@CdS upconversion nanospheres with Ab2-AuNP-GOx-based immune assay format. Experimental results indicated that the photocurrent in the absence of target AFP was almost the same as curve 'c' of UCNP@Au@CdS alone (data not shown). In the presence of 1.0 ng mL-1 AFP, however, the photocurrent (curve 'd') largely increased in comparison with curve 'c'. The increased photocurrent stemmed from the generated H2O2 by catalytic reaction of GOx toward substrate (glucose) to efficiently capture the holes on the valence band of CdS to promote the electron-hole pairs separation (Scheme 1B), thereby resulting in the amplification of the detectable signal. These results further revealed that core-core-shell UCNP@Au@CdS upconversion nanospheres could be utilized as the signal-generation tags for the development of GOx-based sandwich-type enzyme immunoassay.

Optimization of PEC Immunosensing Systems. As mentioned above, the photocurrent of the UCNP@Au@CdS is enhanced by the by-product (H2O2) of the enzyme immunoassay. The produced amount of H2O2 is directly dependent on catalytic efficiency of GOx toward glucose. In this regard, the pH of glucose solution affects the catalytic activity of GOx. As shown in Figure 3A, the photocurrent first increased with the increasing pH values, and then decreased. A maximum photocurrent was obtained at pH 6.0 by using 1.0 ng mL-1 AFP as an example. So, pH 6.0 glucose solution was used as the substrate for GOx catalytic reaction. At this condition, we investigated the effect of the immunoreaction time on the photocurrent of UCNP@Au@CdS upconversion nanospheres from 5 min to 80 min (Figure 3B). To avoid confusion, the immunoreaction times of Ab1 antibody with target AFP were paralleled with those of Ab1-AFP with Ab2-AuNP-GOx. The photocurrent increased with the increasing incubation time, and tended to level off after 50 min, indicating that the antigen-antibody reaction reached a dynamic equilibrium. By the same token, the catalytic time of GOx toward 11



glucose was also optimized even if GOx used in this study has high catalytic activity. Typically, GOx can oxidize glucose into gluconic acid and H2O2. The reaction can be accomplished within a certain time. As seen from Figure 3C, the photocurrent reached a plateau after 20 min. A longer reaction time did not significantly increase the photocurrent. To save the assay time, 50 and 20 min were selected for the antigen-antibody reaction and glucose oxidization, respectively.

Figure 3. Effects of (A) pH of glucose solution, (B) the antigen-antibody reaction time and (C) GOx catalytic time on the NIR light-activated PEC sensing system (1.0 ng mL-1 AFP used in this case).

Dose Responses of UCNP@Au@CdS-Based PEC Immunoassay toward Target AFP. By combination the advantages of the as-synthesized UCNP@Au@CdS upconversion nanospheres with high-efficient enzyme immunoassay mode, PEC immunosensing platform was utilized for quantitative detection of target AFP standards with different concentrations under the irradiation of 980-nm laser. The assay was carried out in a separate setup: immunoassay in the microplate and photocurrent measurement in the homemade detection cell. As indicated from Figure 4A, the photocurrents increased with the increment of target AFP concentrations from 0 to 40 ng mL-1 in 0.1 M Na2SO4 containing the enzymatic product (H2O2) after enzyme immunoreaction. A good linear relationship between photocurrent density and the logarithm of AFP level could be acquired within a dynamic working range of 0.01 – 40 ng mL-1 (Figure 4B). The regression equation could be fitted as follows: y = 133.89 + 58.81 × logC[AFP] (ng mL-1, R2 = 0.9964, n = 8). The limit of detection (LOD) was estimated to 5.3 pg mL-1 on the basis of the 3σ/m criterion (where σ is the standard deviation of the blank and m is the slope of calibration plot). Since the normal threshold (cutoff value) of AFP in human serum is 20 ng mL-1, the NIR light-based PEC immunosensing strategy can completely meet the requirement of clinic diagnostics. 12


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Figure 4. (A) Photocurrent responses of photoelectrochemical immunosensing platform in 0.1 M Na2SO4 for AFP standards from 0 to 40 ng mL-1 based on NIR light-excited UCNP@Au@CdS upconversion materials under the irradiation of 980-nm laser; (B) calibration curve within the dynamic linear range of 0.01 – 40 ng mL-1; (C) the specificity of NIR light-mediated PEC immunoassay against 1.0 ng mL-1 AFP, 100 ng mL-1 CEA, 100 ng mL-1 PSA and 100 ng mL-1 human IgG; and (D) the stability and reproducibility of UCNP@Au@CdS-modifed FTO electrode.

Specificity and Reproducibility of NIR Light-Based PEC Immunoassay. To consider the practical application for a newly developed detection scheme, the specificity and reproducibility of this method are very crucial for detection of target analyte.36,37 To this end, PEC immunoassay based on NIR light-mediated UCNP@Au@CdS upconversion nanospheres was utilized for the screening of other cancer biomarkers, e.g., carcinoembryonic antigen (CEA), prostate-specific antigen (PSA) and immunoglobulin G (IgG), by using the same assay mode. In this case, the evaluation was carried out by comparison with the change in the photocurrent in the presence and absence of target AFP. As shown in Figure 4C, the high-concentration non-targets could not almost increase the photocurrents relative to background/blank signal, whereas only target AFP could cause the increasing photocurrent even if using a 100-fold low 13



level. More inspiringly, coexistence of non-targets with AFP did not cause the significant increment in the photocurrent. Thus, NIR light-mediated PEC immunoassay can be used for specific detection of target AFP. Next, the reproducibility of NIR light-mediated PEC immunoassay was studied on the basis of two aspects.38 Firstly, we investigated the batch-to-batch reproducibility of the as-prepared Ab2-AuNP-GOx and UCNP@Au@CdS by measuring 1.0 ng mL-1 AFP. Experimental results suggested that the coefficient of variation (CV) with the same batch was 6.7% (n = 6), whilst that with different batches was 10.7% (n = 6). Further, the reproducibility of UCNP@Au@CdS modified onto the FTO electrode was monitored by intermittently controlling the 980-nm laser under the 'on-off' switch. Meanwhile, the photocurrents were registered at the different states. As seen from Figure 4D, all the baselines and photocurrent responses (n = 10) were relatively stable at the 'on' and 'off' state, respectively. Therefore, the as-synthesized UCNP@Au@CdS upconversion nanospheres were reproducible and could be used for the batch-preparation. Monitoring of Human Serum Samples and Interlaboratory Validation. To evaluate the accuracy of NIR light-mediated PEC immunoassay method, we collected several human serum specimens containing different-concentration AFP from Mengchao Hepatobiliary Hospital of Fujian Medical University (Fuzhou, China), which was detected by NIR light-mediated PEC immunosensing platform. AFP levels in these specimens were calculated on the basis of calibration curve in Figure 4A referring to the obtained photocurrents. For comparison, these samples were determined by commercialized available human AFP ELISA kit as the reference. The method accuracy and interlaboratory validation were evaluated by using a Student's t-test method (Please see the Supporting Information).39 As analyzed from these results in Table 1, no significant differences were encountered between two methods for assaying six serum samples since all texp values were below 2.77 (tcrit[0.05,4] = 2.77), indicating that NIR light-mediated PEC immunoassay can be used as an optional detection protocol for the quantitative determination of target AFP in biological fluids. CONCLUSIONS In conclusion, this contribution reports on the proof-of-concept of novel photoelectrochemical 14


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immunosensing approach for ultrasensitive screening of disease-related biomarker, AFP used in this case, based on near-infrared light-excited core-core-shell UCNP@Au@CdS upconversion nanospheres in a separation setup. Introduction of plasmonic Au in the nanospheres efficiently concentrates UCNP-generated upconversion luminescence to excite separation of electron-hole pairs of semiconductor CdS. H2O2, as a hole-trapping reagent, readily aggregates with enzyme immunoreaction and photocurrent measurement as a whole. The signal is amplified via gold nanoparticle heavily functionalized with GOx and efficient UCNP@Au@CdS upconversion. Compared with conventional photoelectrochemical immunoassays, highlights of this work can simply summarize as follows: (i) plasmonic Au, as the light concentrator and energy conveyor, can efficiently enhance the absorption cross section and interfacial energy in the upconversion system; (ii) use of NIR light has low phototoxicity and corrosivity, and minimal photobleaching with a large proportion in solar spectrum in comparison with the visible light. Nevertheless, one limitation of this method is that the generated H2O2 by enzyme immunoassay is artificially injected into the photocurrent detection cell. Future work should focus on design of integrated detection system to simplify the operation steps and reduce experimental errors. Impressively, NIR light-excited core-core-shell UCNP@Au@CdS accompanying the photocurrent generation provides a new perspective for development of PEC immunosensing systems. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.0000. Materials and reagents, conjugation of gold nanoparticle with Ab2 antibody and GOx (Ab2-AuNP-GOx), preparation of Ab1-coated microplate, immunoreaction protocol, NIR light-excited photoelectrochemical measurement, enzyme-linked immunosorbent assay (ELISA) for AFP, monitoring of human real sample, statistical analysis, calculation method for t-test statistics (PDF).

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]. Tel: +86-591-2286 6125. Fax: +86-591-2286 6135. ORCID(iD) Dianping Tang : 0000-0002-0134-3983 15



Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENT The authors thank the National Natural Science Foundation of China (21675029, 21475025), the National Science Foundation of Fujian Province (2014J07001), and the Program for Changjiang Scholars and Innovative Research Team in University (IRT15R11) for financial assistance.

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Table 1. Comparison of Analytical Results for Human AFP Serum Samples by NIR Light-Activated PEC Sensing System and AFP ELISA Kit Method; Conc. (mean ± SD, ng mL-1, n = 3)a

a

Sample no.

PEC sensing system

AFP ELISA kit

texp

1

13.65 ± 0.95

14.25 ± 0.89

0.8

2

6.87 ± 0.47

6.76 ± 0.51

0.27

3

0.093 ± 0.006

0.101 ± 0.008

1.39

4

2.55 ± 0.18

2.64 ± 0.22

0.55

5

28.18 ± 1.83

27.65 ± 1.79

0.36

6

0.53 ± 0.06

0.62 ± 0.08

1.56

Each sample was determined in triplicate, and the high-concentration AFP samples were determined with an appropriate dilution.

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FOR TOC ONLY

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Near-Infrared Light-Excited Core-Core-Shell UCNP@Au@CdS

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