Branched Polyethylenimine-Modified Upconversion Nanohybrid

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Branched Polyethylenimine-Modified Upconversion Nanohybrid-Mediated Photoelectrochemical Immunoassay with Synergistic Effect of Dual-Purpose Copper Ions Zhongbin Luo, Qingan Qi, Lijia Zhang, Ruijin Zeng, Lingshan Su, and Dianping Tang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05959 • Publication Date (Web): 22 Feb 2019 Downloaded from http://pubs.acs.org on February 22, 2019

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Analytical Chemistry

Branched Polyethylenimine-Modified Upconversion Nanohybrid-Mediated Photoelectrochemical Immunoassay with Synergistic Effect of Dual-Purpose Copper Ions Zhongbin Luo, Qingan Qi, Lijia Zhang, Ruijin Zeng, Lingshan Su, and Dianping Tang* †Key

Laboratory of Analytical Science for Food Safety and Biology (MOE & Fujian Province), State Key Laboratory of Photocatalysis on Energy and Environment, Department of Chemistry, Fuzhou University, Fuzhou 350116, People's Republic of China *Corresponding Author: Phone: +86-591-2286 6125. Fax: +86-591-2286 6135. E-mail: [email protected]. ABSTRACT: This work developed a near-infrared (NIR) light-activated nonenzymatic signal-off photoelectrochemical (PEC) immunoassay for the ultrasensitive detection of alpha-fetoprotein (AFP) on the basis of branched polyethylenimine (BPEI)-modified upconversion nanoparticle (UCNP)@CdTe quantum dot (QD) nanostructures by coupling with the synergistic effect of dual-purpose copper ions. Emission light originated from NaYF4:Yb,Er UCNP was directly utilized through the electrostatic bonding of CdTe QDs to excite the separation of electron-hole pairs, resulting in the generation of obvious photocurrent under a 980-nm laser. By using polyclonal antibody-labelled cupric oxide nanoparticle as the secondary antibody, the nano label was introduced into the monoclonal anti-AFP antibody-modified microplates in the presence of target AFP. After treatment with acid, the as-released copper ion decreased the photocurrent through the synergistic effect with two issues: one was initially to form coordination with BPEI on the surface of UCNP, and then the NIR excitation light and upconversion luminescence were attenuated due to the internal filter effect; another was to snatch the electrons flowing from the valence band of CdTe QD as the exciton trapping sites. Under optimal conditions, the dualpurpose Cu2+-activated signal-off PEC immunosensing platform exhibited a dynamic linear range from 10 pg mL-1 to 50 ng mL-1 accompanying the decreasing photocurrent with the increment of AFP concentration at an experimental detection limit of 1.2 pg mL-1. Importantly, good accuracy was achieved by this method in comparison of the results with human AFP ELISA kit for analysis of human serum samples. This dual-purpose Cu2+-activated PEC immunoassay brings a promising, enzyme-free and innovative thinking for the detection of low-abundance biomarkers.

nanomaterials in photoelectrochemical (PEC) bioassays, but these methods paid the emphasis on the effective combination of semiconductors with the upconversion materials and the efficient energy utilization.10,11 Despite some advances in this field on upconversion nanoparticle (UCNP) integrating with the semiconductors (e.g., CdTe QDs), there is still the requirement to expand continuously other methods, e.g., coupling with adding the efficient signal amplification strategies. At the same time, we noticed that the surface functionalization has a significant influence on the application of UCNP and the construction of detection patterns. Typically, synthesis of most UCNPs is oleic acid (OA) ligand-capped, which is contrary to the aqueous environment required by biosensing applications. Therefore, many methods on ligand modification were reported to improve the applicability of UCNPs, e.g., coating with silica shell,12 incorporation with amphiphilic molecules13 and decorating with cellular membranes,14 etc. In this work, a new type of branched polyethylenimine (BPEI)-functionalized water-soluble UCNPs is directly prepared via a solvothermal method. Besides the enhanced water solubility and the positive electricity, a large number of amine groups are stationed on the surface of UCNPs. These all equipped the UCNP with the reaction capacity, such

Upconversion materials, uniting with their unique excitation light (near infrared, NIR), have drawn more and more attention in recent years, and systems involving these components usually have excellent properties such as high photoelectrochemical stability, large Stokes shift, low biotoxicity and low background interference.1-3 Generally, upconversion materials convert the NIR light into different wavelengths ranging in visible and ultraviolet light because of the lanthanide doping.4 As we know, utilization of clean renewable energy like solar power has become an important issue in today's world and nearly half of the solar spectrum are distributed in the infrared region. So upconversion materials, of which the absorption is located in this region, urgently needs to be extended to apply in more aspects. Many works involving upconversion materials have been reported in photodynamic drug delivery,5 fluorescence analysis6 and magnetic resonance imaging,7 etc. For instance, Xian et al. prepared label-free NaGdF4:Yb,Tm upconversion nanoparticles-based fluorescent probes for the sequential sensing of Cu2+, pyrophosphate and alkaline phosphatase activity.8 Li's group devised a new upconversion luminescence-functionalized DNA nanodevice for the ATP sensing in living cells.9 In addition, our group has reported several works on the application of upconversion

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Scheme 1. Illustration of the Signal-Off Photoelectrochemical (PEC) Immunoassay for Alpha-Fetoprotein (AFP): (A) Synthesis of Electrode Materials and Immunoreaction Process; Synergistic Effect between (B) the Internal Filter Effect (IFE) of BPEI-Cu(II) Complex on the Surface of NaYF4:Yb,Er Upconversion Nanoparticle (UCNP) and (C) the Electron snatching Effect of Exciton Trapping Sites on the BPEI/UCNP@CdTe QD Nanocomposites-Modified FTO Electrodea

aBPEI:

branched polyethylenimine; MPA: 3-mercaptopropionic acid; Ab1: capture antibody; Ab2: detection antibody; NIR: near infrared; UCL: upconversion luminescence; QD: quantum dot; FTO: fluorine-doped tin oxide. as adsorption and complexation with the metal cations (e.g., Cu2+), which means the better integration with immunoreactionbased signal amplification strategies. Copper ion, combining with other nanomaterials or as a kind of target, has been widely used for a long time in fluorescence analysis,15,16 colorimetric analysis,17,18 etc. Conversely speaking, it is also regarded as a kind of special probe such as the fluorescence quencher. Take the advantage of quenching effect of Cu2+ on the fluorescence of CdTe QDs, Li et al. constructed a ratiometrically fluorescence sensing immunoassay platform for detecting of biomarkers based on electrospun nanofibrous film.19 Our group utilized copper ions, acquired from acidtreated copper nanostructures on the DNA, to decrease the photocurrent of carbon dots for the analysis of tumor biomarkers.20 Liu et al. reported a colorimetric and fluorometric assay based on the complexation between Cu2+ and BPEI.21 In this regard, this paper employs the detection antibody labelled cupric oxide nanoparticle (CuO NP) as the source of copper ion by treating the CuO NP with acid. The introduced Cu2+ can bring internal filter effect and the exciton trapping sites on the BPEI/UCNP@CdTe QD nanocomposite-modified electrode, thereby achieving the purpose of ultrasensitive detection of biomarkers by regarding Cu2+ as the photocurrent signal quencher. Alpha-fetoprotein (AFP, a kind of embryo-specific glycoprotein originated from the liver and the yolk sac during the fetal periods) decreases in concentration after birth, while rises obviously when suffering hepatocellular cancer (HCC), yolk sac tumor and other diseases. 22,23 As an important tumor marker using for the early diagnosis of cancer, only about 25 ng mL-1 of AFP exits in the serum of healthy adults,24 therefore it is absolutely crucial to develop precise and ultrasensitive detection strategies for biomarkers. As shown in Scheme 1 (panel A), in the presence of target AFP, the detection antibodylabelled CuO NP (CuO-Ab2) is introduced into the microplates coated with capture antibody (Ab1) via the sandwiched immunoreaction. After being treated by acid, the released Cu2+ ions are transferred for the PEC measurement for two purposes: internal filter effect (IFE) and electrons snatching. On the one hand, the BPEI-Cu(II) complex forming on the surface of

UCNP appears to be a barrier to deaden the NIR excitation light and upconversion luminescence (UCL) because of the IFE after the complexation reaction between amino groups of BPEI and copper ions,21 and thus the photocurrent declined with the weakened exciting light (Scheme 1, panel B). Except to coordinate with BPEI, part of the introduced Cu2+ also tends to form exciton trapping sites on the surface of CdTe QDs, leading to further decline of PEC signal by inhibiting the escape of photogenerated electrons to conductive band (CB) of QDs (Scheme 1, panel C).25 The amount of Cu2+ released into the PEC detection cell is proportional to the target AFP, and the photocurrent of the BPEI/UCNP@CdTe QD nanocompositebased electrode is weakened due to the synergistic effect of the IFE and the electrons snatching effect mentioned above, which achieves the purpose of ultrasensitive detecting of target AFP concentration.

EXPERIMENTAL SECTION Synthesis of BPEI/UCNP@CdTe QDs Composites. BPEIfunctionalized upconversion nanoparticles were obtained by a typical solvothermal method in accordance with the literature.26 Initially, the mixture including NaCl (7.5 mmol), YCl3·6H2O (2.34 mmol), YbCl3·6H2O (0.6 mmol), ErCl3·6H2O (0.09 mmol) and BPEI (1.2 g) were dissolved in 45-mL ethylene glycol under 50 °C. NH4F solution (4.0 mmol in 30-mL ethylene glycol) was dropwise added into the foregoing solution under vigorous stirring. After stirring for another 20 min at 50 °C, the suspension was transferred into a 100-mL Teflon-lined autoclave and heated for 120 min at 200 °C. Finally, the BPEIfunctionalized NaYF4:Yb,Er upconversion nanoparticles (BPEI/UCNP) were washed with ethanol for three times by centrifugation (8 min, 13 000g) and redispersed into 10-mL ultrapure water for further use. Next, MPA-modified CdTe QDs with different absorption wavelengths were prepared in a one-pot method.27 Briefly, 11.1 mg of Na2TeO3 and 18.9 mg of NaBH4 were added in sequence the mixture containing 59 mg of Cd(NO3)2, 100 mg of trisodium citrate, 25 µL of MPA and 25 mL of ultrapure water after adjusting the pH to 10.5 by using 1.0 M NaOH under stirring. Following by a reflux for 60 min under 100 °C oil bath, CdTe

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Analytical Chemistry QDs were precipitated and washed by isopropanol [note: distinctly-sized CdTe QDs were obtained under different reflux durations by following the same procedure]. At last, 2.0 mL of CdTe QD aqueous solution (20 mg mL-1) was dropped into 10mL UCNP solution and stirring for 2 h. The BPEI/UCNP@CdTe QDs composites were obtained by centrifugation (10 min, 13 000g) and dried at 60 °C. Immunoreaction Protocol and Upconversion MaterialsBased Photoelectrochemical (PEC) Measurement. Prior to measurement, polyclonal anti-human AFP antibody (Ab2)labelled CuO nanoparticle (CuO-Ab2) 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).28,29 Initially, 50 μL of AFP standards or real serum samples with various concentrations were added into the well and incubated for 50 min at 37 °C under shaking. After washing, CuO-Ab2 suspension (50 µL) was added into the microplate and incubated for another 50 min at 37 °C with shaking. These plates were washed three times with washing buffer and one time with water, and then 50 µL of 1.0 mM HCl was added to per well and shaking at 37 °C for 30 min to decompose CuO NPs into Cu2+. Subsequently, the resulting solution was transferred into the detection cell (5 mL of PBS, pH 7.0) for PEC detection on the BPEI/UCNP@CdTe QDs-modified Fluorine-doped tin oxide (FTO) electrode (See the Supporting Information for measurement details).

RESULTS AND DISCUSSION Construction of BPEI/UCNP@CdTe QD CompositesBased PEC Immunoassay Platform. As shown in Scheme 1, the BPEI/UCNP@CdTe QD composites-based signal-off PEC immunoassay platform for the determination of target AFP is constructed coupling with dual-purpose copper ions, obtained from the sandwiched immunoreaction. Accompanying the typical sandwiched immunoreaction process in the presence of AFP, Ab2-labelled CuO NPs are converted into Cu2+ by HCl

and transferred to the buffer solution of PEC detection cell. The introduced partial copper ions are coordinated with the amino groups of BPEI coating on the surface of UCNPs. The NIR exciting light and upconversion light (UCL) are weakened due to the internal filter effect (IFE) of these BPEI-Cu(II) complex, which means less separation of photogenerated electrons and holes in CdTe QDs.21 In addition, exciton trapping sites are generated through the interaction between another partial copper ions and MPA-capped CdTe QDs accompanying the partial decomposition of QDs.25 Electrons flowing from FTO to valence band (VB) of CdTe QDs were then snatched by these trapping sites instead of the conductive band (CB). Thanks to the copper ions-mediated synergism effects, the photocurrent obviously decreased with the increase of AFP concentration and ultrasensitive detection is thus achieved by this method. Characterization of BPEI/UCNP Upconversion Composites in Different Steps. Obviously, the changes of upconversion composites went throughout the whole process, and it was the key point for the successful implementation of this strategy, so we first characterized it. As shown in Figure 1A-B, the transmission electron microscope (TEM; H-7650, Hitachi, Japan) image of BPEI/UCNP nanospheres and BPEI/UCNP@CdTe QDs were in sharp contrast in surface topography. The average size of UCNP nanospheres is about 40 nm in diameter and the light-colored substance capping around might be attributed to the modification of organic polymer BPEI (Figure 1A, inset). After the electrostatic bonding with MPAmodified CdTe QDs, the surface of these nanospheres obviously turned rough (Figure 1B). It can be seen clearly from the X-ray diffraction patterns (XRD; DY5261/Xpert3 CEM, USA) in Figure 1C, characteristic diffraction peaks of UCNP and CdTe QDs all matched well with the standard hexagonal phase card of NaYF4 (JCPDS no.77-2042) and CdTe standard card (JCPDS no.15-0770), respectively.30,31 In addition, peaks of the NaYF4@CdTe QDs composites were in accordance with the peaks of these two substances alone, suggesting the successful integration without any destruction. Thus, it is

Figure 1. HRTEM images of (A) BPEI/UCNP (inset: magnification images) and (B) BPEI/UCNP@CdTe QD composites; (C) XRD patterns of UCNP nanospheres, CdTe QDs and composites; XPS spectra of (D) BPEI/UCNP@CdTe QD composites before (a) and after (b) the reaction with Cu2+, (E) Cd3d and Te3d orbits before (a) and after (b) reaction with Cu2+, and (F) Cu2p orbits.



believed that the UCNP nanospheres and CdTe QDs could be successfully synthesized by the solvothermal method and onepot method based on these characterizations, and could be successfully electrostatically bonded without any phase change or destruction due to the opposite ζ potential of +35.9 mV (BPEI/UCNP) and -25.1 mV (MPA/CdTe QDs) [note: Parallel measurement for three times by using Malvern Zetasizer Nano ZS90, U.K.]. To observe the changes of the designed photosensitive materials before and after the measurement and the effect of adding Cu(II) in this strategy, the BPEI/UCNP@CdTe composites were analyzed by X-ray photoelectron spectroscopy (XPS; Scientific ESCALAB 250 spectrometer, USA). As shown in Figure 1D, the appearance of all the peaks matched well with the corresponding elements including Na, Y, F, Yb, Er, Cd and Te, further demonstrating the successful synthesis of the complex (curve 'a') and after the reaction with copper ions obtained from acid-treated immune complex (curve 'b'), and the XPS signature of Cd3d and Te3d decreased in different degree. Figure 1E illustrates the molar ratio of Cd3d and Te3d approximately, felling from 8:1 to 3:1 (panels 'a-b'). Expectedly, XPS peak of Cu2P3/2 emerged with three parts (Figure 1F). These indicated the majority of Cd ions were stabilized by MPA on the surface of QDs instead of Te ions, and the depressed ratio revealed that Cd2+ ion was exchanged by Cu2+, which was restored to the exciton trapping site CuI (931.4 eV).25 Notably, the spilled peak at 933.4 eV might be attributed to BPEI-Cu(II) complex and the rest 932.2 eV could be ascribed to transitional valence.32 These indicated that the CdTe QDs to adhere on the BPEI/UCNP nanospheres were destroyed partially and the redox energy level of product CuI was just in the range from the VB to the CB of the QDs, resulting in the formation of the exciton trapping sites. Feasibility Evaluation of BPEI/UCNP@CdTe QD-Based PEC Immunoassay. First of all, as the most basic requirements of this method, the upconversion luminescence (UCL) must be absorbed by semiconductor CdTe QDs to excite the separation of electrons and holes, which was expressed as the photocurrent. Figure 2A shows the UCL spectra of UCNP (green region) and the absorption spectra of CdTe QDs (orange curve). The absorption peaks of CdTe QDs in 500 ~ 600 nm were perfectly matched with the UCL peak distributing in 530 and 550 nm, suggesting that UCNP could be successfully utilized as an efficacious light source and UCNP@CdTe QDs composites indeed exhibited strong photocurrent (Figure 2D, curve 'a'). In addition, BPEI was designed to bring a large amount of positively charged amine and made the surface of UCNP cationic and adsorbent.33,34 The FT-IR spectra comparison chart of UCNP and BPEI-UCNP are displayed in Figure 2B. The unique IR absorption peaks could be classified into N-H stretching vibration (3300-3100 cm-1), C-H stretching vibration (2935-2832 cm-1), N-H bending vibrations (1650,1581 cm-1), C-H in-pale bending vibration (1455 cm-1), C-N stretching vibration (1362-1073 cm-1) and tertiary amine peak.35,36,37 These characteristic peaks fully demonstrated the successful modification of BPEI, which laid the foundation for electrostatic bonding of negatively charged quantum dots and the amine-based complexation with copper ions. As the synergistic effect leading to the attenuation of photocurrent, the internal filter effect (IFE) is also demonstrated in Figure 2C. The upconversion emission peaks at 500, 530, 550 and 660 nm were originating from 2H9/2 → 4I15/2, 2H11/2,4S3/2 → 4I 4F 4I 3+ 15/2, and 9/2 → 15/2 transitions of activator Er , 38 2+ respectively. After adding 100 μL of Cu solution (10 μM),

the UCL intensity obviously declined from curve 'a' to curve 'b'. Taking the absorption spectrum of Cu2+ solution (Figure 2A, purple curve) into consideration, the attenuation could be attributed to the absorption of incident light (980 nm) and UCL by the BPEI-Cu(II) complex layer. As a result, these led to the weakened NIR light and the weakened UCL irradiating to semiconductor CdTe QDs and finally weakened the photocurrent.21,25

Figure 2. (A) Upconversion luminescence (UCL) spectra of UCNP, absorption spectra of CdTe QDs and Cu(II) solution. (inset: UCNP powder under the 980 nm laser and CdTe QDs under the UV-vis light); (B) FT-IR spectra of UCNP and BPEImodified UCNP; (C) Upconversion luminescence spectra of (a) BPEI/UCNP and (b) BPEI/UCNP + Cu2+; (D) Photocurrent responses of (a,b,c) UCNP@CdTe QDs and (d) UCNP on the FTO electrode under 980-nm laser in PBS (pH 7.0) [note: in the (b) absence and (c) presence of 0.1 ng mL-1 AFP]. According to the design of this scheme, a puzzling concern arises whether this weakening optical signal and the electron snatching effect mentioned above can be smoothly converted into an attenuation of the electrical signal. To this end, the signal-off method was further explored in PEC measurement in Figure 2D. The as-prepared UCNP (curve 'd ') nanospheres practically behaved no photocurrent while the UCNP@CdTe QDs (curve 'a') composites showed strong photocurrent (about 230 nA) at the external bias voltage of 0 V, which provided enough background photocurrent for the subsequent detection. With the intervention of the sandwiched immunoreaction, UCNP@CdTe QD composites exhibited distinct-different current signal in the absence (curve 'b') and presence (curve 'c') of target AFP (0.1 ng mL-1). The intensity of UCNP@CdTe QDs composites under the condition of no target was similar to the background signal, while it declined nearly a half in the presence of 0.1 ng mL-1 target. Under this circumstance, we could infer that it was the product Cu2+ obtaining from acidtreating immunocomplex diminished the photocurrent rather than other substance transferred into the PEC detection cell. On the basis of the above finding and exploration, the BPEI/UCNP@CdTe QD-based NIR light-activated signal-off ultrasensitive PEC immunoassay could be successfully constructed by combining the synergistic effect of the dualpurpose copper ions as the signal quencher. Optimization of PEC Immunosensing Platform. Besides electrostatic bonding with QDs and complexation with copper ions, the BPEI-modified UCNPs were mainly utilized to convert the NIR incident light into proper wavelength which


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Analytical Chemistry

Figure 3. (A) Different absorption spectrum of distinctly-sized CdTe QDs in different reflux times (a ~ j: 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 min (insets: the corresponding fluorescence emission spectrum and the photos under ultraviolet light); effects of (B) pH of PBS and (C) the antigen-antibody incubation time on the NIR light-activated PEC sensing platform (0.1 ng mL-1 AFP used in this case). could match with the absorption range of CdTe QDs. Thus, distinctly-sized CdTe QDs need to be selected because of distinguishing absorbable wavelength (Figure 3A). These distinctly-sized CdTe QDs (curve 'a' ~ 'j') were obtained in different reflux times (note: increased by 10 min in turn until 100 min) and the size of the as-synthesized CdTe QDs could be calculated by the equation proposed by Yu et al.39: D = (9.8127  10-7) λ3 – (1.7147  10-3) λ2 + 1.0064 λ – 194.84 where λ refers to the maximum absorption wavelength. We could find the maximum absorption wavelength increases with the growth of QD size. And those with particle size greater than 3.24 nm (where λ > 550 nm) was the better choice for the asprepared UCNPs mentioned above. Compared with small-sized QDs (curve 'a' ~ 'e'), absorption of QDs in this range (curve 'f ' ~ 'j') perfectly overlapped the emission of UCNP, which mainly distributes in 530 and 550nm. To save synthesis time, 60 min (curve 'f ') was chosen and that also guaranteed the UCL spectrum (mainly 530 and 550 nm) would be overlapped well. Generally speaking, the larger the initial background signal of BPEI/UCNP@CdTe QD composites is, the better a signal weakening strategy is.40,41 The pH of PBS buffer in the PEC detection cell had a significant impact on it, as illustrated in Figure 3B. The suitable pH was optimized at 7.0 in the range of 6.0 ~ 8.0. The underlying cause was that the BPEI/UCNP@CdTe QD-modified electrode tended to be decomposed in acidic solution, while the hydroxide radical was easily captured by Cd2+ at the surface of QDs in an alkaline environment, thus blocking the transmission of electrons to O2 to cause the photocurrent attenuation.25 Under these conditions, we further explored the effect of incubation time on the photocurrent by using 1.0 ng mL-1 AFP as an example [note: the immunoreaction time between antibody Ab1 and target AFP was paralleled with that of Ab1-AFP with CuO-Ab2]. As revealed in Figure 3C, the photocurrent intensity of BPEI/UCNP@CdTe QDs under the 980 nm NIR laser dropped rapidly from 5 to 50 min and tended to be stabilized after 60 min, indicating that the dynamic equilibrium of the antigenantibody was achieved in the near horizontal segment. To save assay time, 50 min was selected for the whole incubation process. Dose Responses of BPEI/UCNP@CdTe QD compositesBased PEC Immunoassay toward Target AFP. To investigate the analytical performance of the proposed signaloff strategy, the NIR light-driven PEC platform by BPEI/UCNP@CdTe QD nanocomposites was applied to quantitatively determine AFP standards at different concentrations under the optimized conditions. As seen from

Figure 4A-B, the obtained photocurrent decreased gradually with the increment of the corresponding AFP concentration in the dynamic range of 0.01 to 50 ng mL-1. A good linear relationship was achieved between the photocurrent intensity y (nA) and the decimal logarithm of target AFP concentration log x (ng mL-1), which the regression equation could be expressed as y = 98.05 – 31.64 log x (R2 = 0.9919, n = 8). The limit of detection (LOD) was estimated to be 1.2 pg mL-1 at a signal-tonoise ratio of 3. Taking the actual clinic diagnostics request for AFP in human serum into account of which the normal threshold (cutoff) value is 20 ng mL-1, the NIR light-activated signal-off PEC immunosensing method was absolutely competent based on the powerful synergistic effect of IFE and exciton trapping sites.

Figure 4. (A) Photocurrents of PEC detection platform in PBS (pH 7.0) toward different AFP standards (0.01 ~ 50 ng mL-1) based on NIR light-activated BPEI/UCNP@CdTe QD composites using a 980-nm laser as light source; (B) calibration curve; (C) the stability of BPEI/UCNP@CdTe QD-modified FTO electrode; and (D) the specificity of the PEC immunoassay against AFP (0.1 ng mL-1), PSA (100 ng mL-1), CEA (100 ng mL-1) and human IgG (100 ng mL-1). Stability and Specificity. As the important basis of the generation of photocurrent, the stability of QD materials-coated FTO electrode was crucial for this signal-off method. However, the stability of individual water-soluble MPA/CdTe QDs was not usually satisfactory.42 In contrast, the BPEI/UCNP@CdTe QD composites-coated FTO presented a surprising performance in this aspect (Figure 4C). Photocurrents in different periods



were similar in data in the 12 cycles for 340 seconds by switching the NIR light source intermittently, suggesting the acceptable stability with the relative standard deviation (RSD) of 8.52%. As one of the significant requirements for clinical application, the specificity of NIR light-activated PEC immunoassay was also explored as indicated in Figure 4D. Target AFP and some other cancer biomarkers that were present in the serum which might cause interference (e.g., prostate-specific antigen, PSA; carcinoembryonic antigen, CEA; human immunoglobulin G, IgG) were used as the analyte. In contrast, AFP at a lowconcentration (0.1 ng mL-1) could cause a substantial decline in photocurrent for about 125 Na, and thus did other interfering analytes which mixed with AFP at the same level, while the decline was negligible in high-concentration individual interfering (non-target) because the signals were approximate to the background signal. Therefore, these results further revealed that the developed NIR light-activated PEC immunoassay platform had a satisfactory specific detection for target AFP. Analysis of Human Serum Specimens. To assess the accuracy of BPEI/UCNP@CdTe QD-based PEC immunoassay protocol, 8 human serum samples including target AFP with different concentrations, which were gifted from Mengchao Hepatobiliary Hospital of Fujian Medical University (Fuzhou, China), were monitored using our designed PEC immunoassay on the basis of calibration curve in Figure 4B. The results were compared with those obtained by using commercialized human AFP ELISA kit as the reference. The data calculated by these two methods are exhibited in Table 1, and the accuracy of the PEC immunoassay was examined by an independent twosample t-test method (please see the evaluation method in the Supporting Information). Meanwhile, we could find that the values of experimental texp were all lower than 4.30, which is the critical value of tcrit[0.05,4]. Therefore, no significant differences at the 0.05 significance level were acquired between these two methods through the detection of human serum samples. We believe that the proposed strategy is highly accurate and can be used for further practical clinical application for quantitative detection of AFP.

CONCLUSIONS In summary, this contribution introduces a novel NIR lightmediated signal-off photoelectrochemical immunoassay by coupling with the synergistic effect of dual-purpose copper ions for the ultrasensitive determination of biomarker (AFP used in this case) on the basis of BPEI/UCNP@CdTe QD nanohybrids. The copper ions were obtained via the acidic treatment toward the sandwiched immunocomplexes and introduced into the three-electrode detection system. Compared with previously reported CuO nanoparticles43 or Cu2+ ion44,45-based photoelectrochemical detection systems, the released copper ions could initially absorb near-infrared incident light and then weaken the upconversion luminescence through the IFE effect of the BPEI-Cu(II) complex, thus reducing the generation of photogenerated electron-hole pairs. In addition, the asgenerated exciton trapping sites on the surface of CdTe QDs could snatch the photogenerated electrons that transferred to the conduction band of the QDs. Thanks to such a synergistic effect of IFE and electron trapping, the photocurrent signal significantly reduced to improve the sensitivity of NIR-based strategy. Impressively, this novel approach, which attenuates photoelectric signals by weakening original light sources and snatching the photogenerated electrons synchronously,

provides a new and interesting perspective for photoelectrochemical biosensings. More importantly, high sensitivity, good stability and specificity of BPEI/UCNP@CdTe QDs-based 'signal-off' PEC method fully meets the needs of the clinical application and opens opportunities for protein diagnostics and biosecurity.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.0000. Reagent and material, conjugation of CuO nanoparticle with Ab2 antibody (CuO-Ab2), preparation of Ab1-coated microplate, preparation of BPEI/UCNP@CdTe QDsmodified electrode, NIR light-activated photoelectrochemical measurement, enzyme-linked immunosorbent assay (ELISA) for AFP, statistical analysis, calculation method for t-test statistics (PDF)

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

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was financially supported by the National Natural Science Foundation of China (21675029, 201874022) and the Health-Education Joint Research Project of Fujian Province (WKJ2016-2-15).

REFERENCES (1) (2)

(3) (4) (5)

(6)

(7)

(8)

Zhu, Z.; Su, Q.; Feng, W.; Li, F. Anti-stokes shift luminescent materials for bio-applications. Chem. Soc. Rev. 2017, 46, 10251039. Liang, T.; Li, Z.; Wang, P.; Zhao, F.; Liu, J.; Liu, Z. Breaking through the signal-to-background limit of upconversion nanoprobes using a target-modulated sensitizing switch. J. Am. Chem. Soc. 2018, 140, 14696-14703. Fan, W.; Bu, W.; Shi, J. On the latest three-stage development of nanomedicines based on upconversion nanoparticles. Adv. Mater. 2016, 28, 3987-4011. Naik, G.; Welch, A.; Briggs, J.; Solomon, M.; Dionne, J. Hotcarrier-mediated photon upconversion in metal-decorated quantum wells. Nano. Lett. 2017, 17, 4583-4587. Xiang, J.; Ge, F.; Yu, B.; Yan, Q.; Shi, F.; Zhao, Y. Nanocomplexes of photolabile polyelectrolyte and upconversion nanoparticles for near-infrared light-triggered payload release. ACS Appl. Mater. Interfaces 2018, 10, 20790-20800. Gulzar, A.; Xu, J.; Yang, D.; Xu, L.; He, F.; Gai, S.; Yang, P. Nano-graphene oxide-UCNP-Ce6 covalently constructed nanocomposites for NIR-mediated bioimaging and PTT/PDT combinatorial therapy. Dalton Trans. 2018, 47, 3931-3939 Liu, M.; Shi, Z.; Wang, X.; Zhang, Y.; Mo, X.; Jiang, R.; Liu, Z.; Fan, L.; Ma, C.; Shi, F. Simultaneous enhancement of red upconversion luminescence and CT contrast of NaGdF4:Yb,Er nanoparticles via Lu3+ doping. Nanoscale 2018, 10, 2027920288. Wang, F.; Zhang, C.; Xue, Q.; Li, H.; Xian, Y. Label-free upconversion nanoparticles-based fluorescent probes for sequential sensing of Cu2+, pyrophosphate and alkaline phosphatase activity. Biosens. Bioelectron. 2017, 95, 21-26.


Page 6 of 8

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Analytical Chemistry (9) (10)

(11)

(12)

(13) (14)

(15) (16)

(17)

(18) (19)

(20)

(21)

(22)

(23)

(24) (25)

(26)

Zhao, J.; Gao, J.; Xue, W.; Di, Z.; Xing, H.; Lu, Y.; Li, L. Upconversion luminescence-activated DNA nanodevice for ATP sensing in living cells. J. Am. Chem. Soc. 2018, 140, 578-581. Qiu, Z.; Shu, J.; Tang, D. Near-infrared-to-ultraviolet lightmediated photoelectrochemical aptasensing platform for cancer biomarker based on core-shell NaYF4:Yb,Tm@TiO2 upconversion microrods. Anal. Chem. 2018, 90, 1021-1028. Luo, Z.; Zhang, L.; Zeng, R.; Su, L.; Tang, D. Near-infrared light-excited core-core-shell UCNP@Au@CdS upconversion nanospheres for ultrasensitive photoelectrochemical enzyme immunoassay. Anal. Chem. 2018, 90, 9568-9575. Kang, F.; He, J.; Sun, T.; Bao, Z.; Wang, F.; Lei, D. Plasmonic dual-enhancement and precise color tuning of gold nanorod@SiO2 coupled core-shell-shell upconversion nanocrystals. Adv. Mater. 2017, 27, 1842-1850. Jo, E.; Byun. J.; Mun, H.; Bang, D.; Son, J.; Lee, J.; Lee, L.; Kim, M.; Single-step LRET aptasensor for rapid mycotoxin detedtion. Anal. Chem. 2018, 90, 716-722. Rao, L.; Bu, L.; Cai, B.; Xu, J.; Li, A.; Zhang, W.; Sun, Z.; Guo, S.; Liu, W.; Wang, T.; Zhao, X.; Cancer cell membrane-coated upconversion nanoprobes for highly specific tumor imaging. Adv. Mater. 2016, 28, 3460-3466. He, F.; Wang, J.; Yin, B.; Ye, B. Quantification of exosome based on a copper-mediated signal amplification strategy. Anal. Chem. 2018, 90, 8072-8079. Ju, E.; Dong, K.; Chen, Z.; Liu, Z.; Liu, C.; Huang, Y.; Wang, Z.; Pu, F.; Ren, J.; Qu, X. Copper(II)-graphitic carbon nitride triggered synergy: improved ROS generation and reduced glutathione levels for enhanced photodynamic therapy. Angew. Chem., Int. Ed. 2016, 55, 11467-11471. He, X.; Liu, H.; Li, Y.; Wang, S.; Li, Y.; Wang, N.; Xiao, J.; Xu, X.; Zhu, D. Gold nanoparticle-based fluorometric and colorimetric sensing of copper(II) ions. Adv. Mater. 2005, 17, 2811-2815. Yin, B.; Ye, B.; Tan, W.; Wang, H.; Xie, C. An allosteric dualDNAzyme unimolecular probe for colorimetric detection of copper(II). J. Am. Chem. Soc. 2009, 131, 14624-14625. Yang, T.; Li, C.; He, J.; Chen, B.; Li, Y.; Huang, C. Ratiometrically fluorescent electrospun nanofibrous film as a Cu2+-mediated solid-phase immunoassay platform for biomarkers. Anal. Chem. 2018, 90, 9966-9974. Lv, S.; Li, Y.; Zhang, K.; Lin, Z.; Tang, D. Carbon dots/g-C3N4 nanoheterostructures-based signal-generation tags for photoelectrochemical immunoassay of cancer biomarkers coupling with copper nanoclusters. ACS Appl. Mater. Interfaces 2017, 9, 38336-38343. Shao, H.; Xu, D.; Ding, Y.; Hong, X.; Liu, Y. An "off-on" colorimetric and fluorometric assay for Cu(II) based on the use of NaYF4:Yb(III),Er(III) upconversion nanoparticles functionalized with branched polyethylenimine. Microchim. Acta 2018, 185, 211. Wu, Y.; Guo, W.; Peng, W.; Zhao, Q.; Piao, J.; Zhang, B.; Wu, X.; Wang, H.; Gong, X.; Chang, J. Enhanced fluorescence ELISA based on HAT triggering fluorescence "turn-on" with enzyme-antibody dual labeled AuNP probes for ultrasensitive detection of AFP and HBsAg. ACS Appl. Mater. Interfaces 2017, 9, 9369-9377. Xu, D.; Liu, C.; Li, C.; Song, C.; Kang, Y.; Qi, C.; Lin, Y.; Pang, D.; Tang, H. Dual amplification fluorescence assay for alpha fetal protein utilizing immunohybridization chain reaction and metal-enhanced fluorescence of carbon nanodots. ACS Appl. Mater. Interfaces 2017, 9, 37606-37614. Wang, F.; Liu, X. Upconversion multicolor fine-tuning: visible to near-infrared emission from lanthanide-doped NaYF4 nanoparticles. J. Am. Chem. Soc. 2008, 130, 5642-5643. Wang, P.; Ma, X.; Su, M.; Hao, Q.; Lei, J.; Ju, H. Cathode photoelectrochemical sensing of copper(II) based on analyteinduced formation of exciton trapping. Chem. Commun. 2012, 48, 10216-10218. Yang, S.; Zhang, F.; Wang, Z.; Liang, Q. A graphene oxidebased label-free electrochemical aptasensor for the detection of alpha-fetoprotein. Biosens. Bioelectron. 2018, 112, 186-192.

(27) Zou, L.; Gu, Z.; Zhang, N.; Zhang, Y.; Fang, Z.; Zhu, W.; Zhong, X. Ultrafast synthesis of highly luminescent green-to near infrared-emitting CdTe nanocrystals in aqueous phase. J. Mater. Chem. 2008, 18, 2807-2815. (28) Qu, W.; Liu, Y.; Liu, D.; Wang, Z.; Jiang, X. Copper-mediated amplification allows readout of immunoassays by the naked eye. Angew Chem., Int. Ed. 2011, 50, 3442-3445. (29) Shu, J.; Qiu, Z.; Zhou, Q.; Lin, Y.; Lu, M.; Tang, D. Enzymatic oxydate-triggered self-illuminated photoelectrochemical sensing platform for portable immunoassay using digital multimeter. Anal. Chem. 2016, 88, 2958-2966. (30) Feng, W.; Zhang, L.; Zhang, Y.; Yang, Y.; Fang, Z.; Wang, B.; Zhang, S.; Liu, P. Near-infrared -activated NaYF4:Yb3+,Er3+/ Au/CdS for H2 production via photoreforming of bioethanol: plasmonic Au as light nanoantenna, energy relay, electron sink and co-catalyst. J. Mater. Chem. A 2017, 5, 10311-10320. (31) Taniguchi, S.; Green, M. The synthesis of CdTe/ZnS core/shell quantum dots using molecular single-source precursors. J. Mater. Chem. C 2015, 3, 8425-8433. (32) Wen, G.; Ju, H. Ultrasensitive photoelectrochemical immunoassay through tag induced exciton trapping. Talanta 2015, 134, 496-500. (33) Fan, Y.; Liu, H. J.; Zhang, Y.; Chen, Y. Adsorption of anionic MO or cationic MB from MO/MB mixture using polyacrylonitrile fiber hydrothermally treated with hyperbranched polyethylenimine. J. Hazard. Mater. 2015, 283, 321-328. (34) Zhao, R.; Li, X.; Sun, B.; Li, Y.; Li, Y.; Yang, R.; Wang, C. Branched polyethylenimine grafted electrospun polyacrylonitrile fiber membrane: a novel and effective adsorbent for Cr(VI) remediation in wastewater. J. Mater. Chem. A 2017, 5, 11331144. (35) Wang, Y.; Shen, P.; Li, C.; Wang, Y.; Liu, Z. Upconversion fluorescence resonance energy transfer based biosensor forultrasensitive detection of martrix metalloproteinase-2 in blood. Anal. Chem. 2012, 84, 1466-1473. (36) Pang, Y.; Zeng, G.; Tang, L.; Zhang, Y.; Liu, Y.; Lei, X.; Li, Z.; Zhang, J.; Xie, G. PEI-grafted magnetic porous powder for highly effective adsorption of heavy metal ions. Desalination 2011, 281, 278-284. (37) Jin, J.; Gu, Y.; Man, C.; Cheng, J.; Xu, Z.; Zhang, Y.; Wang, H.; Lee, V.; Cheng, S.; Wong, W. Polymer-coated NaYF4:Yb3+,Er3+ upconversion nanoparticles for charge-dependent cellular imaging. ACS Nano. 2011, 5, 7838-7847. (38) Suo, H.; Zhao, X.; Zhang, Z.; Wu, Y.; Guo, C. Upconverting LuVO4:Nd3+/Yb3+/Er3+@SiO2@Cu2S hollow nanoplatforms for self-monitored photothermal ablation. ACS Appl. Mater. Interfaces 2018, 10, 39912-39920. (39) Yu, W.; Qu, L.; Guo, W.; Peng, X. Experimental determination of the extinction coefficient of CdTe, CdSe, and CdS nanocrystals. Chem. Mater. 2003, 15, 2854-2860. (40) Zhao, W.; Xu, J.; Chen, H. Photoelectrochemical immunoassays. Anal. Chem. 2018, 90, 615-627. (41) Kong, Q.; Cui, K.; Zhang, L.; Wang, Y.; Sun, J.; Ge, S.; Zhang, Y.; Yu, J. “On-Off-On” photoelectrochemical/visual lab-onpaper sensing via signal amplification of CdS quantum dots@leaf-shape ZnO and quenching of Au-modified prismanchored octahedral CeO2 nanoparticles. Anal. Chem. 2018, 90, 11297-11304. (42) Lin, Y.; Zhou, Q.; Tang, D.; Niessner, R.; Yang, H.; Knopp, D. Silver nanolabels-assisted ion-exchange reaction with CdTe quantum dots mediated exciton trapping for signal-on photoelectrochemical immunoassay of mycotoxins. Anal. Chem. 2016, 88, 7858-7866. (43) Sun, G.; Zhang, Y.; Kong, Q.; Zheng, X.; Yu, J.; Song, X. CuOinduced signal amplification strategy for multiplexed photoelectrochemical immunosensing using CdS sensitized ZnO nanotubes arrays as photoactive material and AuPd alloy nanoparticles as electron sink. Biosens. Bioelectron. 2015, 66, 565-571. (44) Liu, Y.; Li, R.; Gao, P.; Zhang, Y.; Ma, H.; Yang, J.; Du, B.; Wei, Q. A signal-off sandwich photoelectrochemical



immunosensor using TiO2 coupled with CdS as the photoactive matrix and copper(II) ions as inhibitor. Biosens. Bioelectron. 2015, 65, 97-102.

Page 8 of 8

(45) Wang, P.; Lei, J.; Su, M.; Liu, Y.; Hao, Q.; Ju, H. Highly efficient visual detection of trace copper(II) and protein by the quantum photoelectric effect. Anal. Chem. 2013, 85, 8735-8740.

Table 1. Comparison of Analytical Results for Human AFP Serum Samples by BPEI/UCNP@CdTe QD-Based PEC Sensing System with AFP ELISA Kit as the Referencea Method; Conc. (mean ± SD, ng mL-1, n = 3)a Sample no.

PEC detection

AFP ELISA kit

texp

1

5.4 ± 0.3

5.3 ± 0.2

0.65

2

0.6 ± 0.04

0.6 ± 0.05

0.81

3

11.7 ± 0.9

12.2 ± 0.9

0.77

4

19.2 ± 1.5

17.9 ± 1.5

1.09

5

0.9 ± 0.05

0.8 ± 0.04

1.89

6

2.3 ± 0.2

2.3 ± 0.2

0.28

7

23.3 ± 1.9

22.1 ± 1.8

0.82

8

0.09 ± 0.006

0.09 ± 0.007

0.94

a Samples

with high concentrations were properly diluted and each sample was determined in parallel three measurements.

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Branched Polyethylenimine-Modified Upconversion Nanohybrid

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