Ratiometrically Fluorescent Electrospun Nanofibrous Film as a Cu2+-

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Ratiometrically Fluorescent Electrospun Nanofibrous Film as a Cu2+-Mediated Solid Phase Immunoassay Platform for Biomarkers Tong Yang, Chun Mei Li, Jia Hui He, Bin Chen, Yuan Fang Li, and Cheng Zhi Huang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02286 • Publication Date (Web): 24 Jul 2018 Downloaded from http://pubs.acs.org on July 26, 2018

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Analytical Chemistry

Ratiometrically Fluorescent Electrospun Nanofibrous Film as a Cu2+-Mediated Solid Phase Immunoassay Platform for Biomarkers Tong Yang,†, ¶ Chun Mei Li,*, †, § Jia Hui He,† Bin Chen,// Yuan Fang Li,‡ and Cheng Zhi Huang*, †, ‡ †

Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, P. R. China. ‡ Chongqing Key Laboratory of Biomedical Analysis (Southwest University), Chongqing Science & Technology Commission, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, P. R. China. § State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha 410082, P. R. China. // College of Electronic and Information Engineering, Southwest University, Chongqing 400715, P. R. China. ABSTRACT: Increasing demands for the sensitive, selective, visual, conveniently solid phase sensing film for disease-related biomarkers, electrospun nanofibrous films (ENFFs) is a novel sensing paltform because of high surface area ratios, networked structures, and facile functionalization. In this research, carbon dots (CDs) and quantum dots (QDs) were doped inside and electrostatically assembled on electrospun nanofibers (ENFs), affording ratiometrically dual-emitting fluoresecent films. Because of the FRET process between CDs and QDs on NFs, and the strong quenching response of QDs to Cu2+, the as-prepared NFs can visually detect Cu2+ with high sensitivity and selectivity based on ratiometric fluorescent signals. By exploiting the release of numerous Cu2+ from CuO nanoparticles (NPs) under acidic conditions, a sandwich-type immunoassay with CuO NPs-labelled antibody was developed for the detection of biomarker proteins via the response of dual-emitting and FRET-based NFs to different Cu2+ concentrations. Taking advantages of the ratiometrically fluorescent property of these ENFFs as well as Cu2+-mediated signal amplification strategy, alpha fetoprotein can be detected with high sensitivity (detection limit of 8.3 pg/mL) and selectivity. This strategy demonstrated a promise candidate with a point-of-care assay for clinical diagnostics and biomedical research.

INTRODUCTION Solid film-based sensing platforms have attracted considerable research attention for food assays, environmental monitoring, quality control, and biomedical analysis because of their portability, stability, real-time detection, and easy fabrication and integration with flexible devices.1-7 Comparing with numerous methods for film production, including spin-coating or dropcasting, the Langmuir-Blodgett method, layer-by-layer assembly and self-assembled monolayer techniques,8 electrospinning has been considered to be a quite versatile, direct approach for fabricating polymer nanofibrous sensing films, which exhibit characteristics of a large surface area ratio, high porosity and network-like structures.9 Furthermore, polymeric nanofibrous films can be easily modified and functionalized with more target-bonding sites and sensing elements for enhancing their sensitivity and selectivity towards analytes.10-12 Currently, the solid electrospun nanofibrous films (ENFFs)-based sensing platforms can considerably satisfy the requirements of analytical science and portable sensing technology.8,13 With respect to ENFFs-based fluorescent sensing platform, fluorescent materials such as small organic molecules, metal nanoclusters, carbon dots (CDs) and quantum dots (QDs) typically have been doped into polymeric ENFFs for the detection of gas,14 heavy metal ions,15 pH,16 biomolecules,17 antibiotics,18 toxic nerve gas,12 explosive,19 and latent fingerprint discrimination20. These fluorescent ENFFs can provide a method for analysing these metal ions and small molecules based on “turn-off” fluorescence response. Nevertheless, these ENFFs have been reported to be unsatisfactory for visual dis-

crimination and detection using a single emitted wavelength of single fluorescent materials, which are also easily affected by instruments, environmental conditions and sample matrix to cause lower sensitivity. In contrast, ratiometric fluorescence is an analytical strategy by recording the ratios of fluorescence intensities located at two wavelengths, which can provide a built-in-correction, exhibit increasing sensitivity, high selectivity, wide linear ranges, and produce multicolour and visual assay for chemo-/bio-sensor.21,22 Intriguingly, it also provides an opportunity to fabricate ratiometrically fluorescent ENFFs for visual chemo-/bio-sensors. At present, several studies have mainly focused on the implantation or doping of simple or FRET-based organic dyes into electrospun polymeric nanofibers (NFs) for the detection of various metal ions, pH values and small biomolecules.23-26 However, organic-dye-based ratiometric fluorescent probes have been well known to exhibit some notorious features such as the photobleaching, weak luminescence, and complicated synthesized procedures, typically making it difficult to establish steadily and efficiently ratiometric fluorescent sensing films. The use of some fluorescent nanomaterials such as CDs, QDs and metal nanoclusters can avoid the above-mentioned limitations. That being said, it is a challenge to rationally design the ratiometrically FRETbased fluorescent electrospun nanofibrous sensing films based on these luminescent nanomaterials. In addition, it is not appropriate to use the above-mentioned ENFFs to directly detect disease-related macro-targets such as DNA, polypeptides, cells and cancer biomarkers because they cannot permeate into NFs for interaction with fluorescent probes unless the surface of electropsun NFs surface is

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Scheme 1. Schematic Illustration of (A) the Preparation of FRET-Based r-CdTe QDs/g-CDs/CA ENFFs and (B) Application of Cu2+-Mediated Immunoassay for Biomarkers.

subjected to specific functionality to form sensing interfaces.27,28 In a previous study reported by our group, surface engineering is employed to prepare QDs-lit fluorescent aptasensing ENFFs for biomarkers assay, which display high sensitivity, selectivity and reproducibility.10 Despite of these advantages, the design and preparation of these fluorescent electrospun nanofibrous biosensing films are still complicated. Currently, metal-ions-mediated ELISA are popular by coupling traditional sandwich-type immunoreactions with metal oxides nanoparticles (NPs), which can be released amounts of metal ion under acidic environment.29,30 This process can afford signal amplification for enhancing analytical sensitivity. Furthermore, in view of the above ENFF-based metal ions sensor, it is preferable to incorporate fluorescent electrospun NFs with metal-ions-mediated immunoassays for visually detecting biomarkers. With this background, dual-emitting r-CdTe QDs/g-CDs cellulose acetate (CA) ENFFs were prepared by direct doping of CDs inside NFs and electrostatic assembly of CdTe QDs@3-mercaptopropionic acid (MPA) on NF surfaces (Scheme 1A). On the basis of the FRET process between CDs and CdTe QDs@MPA, the specific response to Cu2+ of the CdTe QDs, and superior microstructures of ENFFs, ratiometrically fluorescent CdTe QDs/CDs CA ENFFs can be developed for visually and sensitively detecting Cu2+, which shows superior sensing performance to that of single-emitting CdTe QDs/CA ENFFs. Then, by a sandwich-type immunoreaction, CuO-NP-labelled antibodies were captured in each working zone of 96-well plates (Scheme 1B). Owing to large numbers of Cu2+ releasing from CuO NPs under acidic conditions, the Cu2+-mediated signal amplification strategy indirectly reflects the biomarkers concentration. With alpha fetoprotein (AFP) as an example, this strategy exhibit an excellently sensitive, selective and clearly visual effect. To prove the applicability of this strategy, semi-quatification of AFP by visual detection was carried out in real serum samples.

EXPERIMENTAL SECTION Materials. CA powder (average Mw = 100,000) were obtained from Shanghai Yuanye biotechonology Co., Ltd.. pPhenylenediamine, ethylenediamine, MPA, sodium borohydride (NaBH4), potassium tellurite (K2TeO3), cadmium chloride hemi(pentahydrate) (CdCl2·2.5H2O), ethylene imine polymer (PEI, average Mw = 70, 000), bovine serum albumin (BSA, 96%) were purchased from Aladdin Chemistry Co.,

Ltd. (Shanghai, China). Acetone and N,N-dimethylacetamide (DMAC) were acquired from Chongqing Chuandong Chemical (Group) Co., Ltd.. CuO nanoparticles (~50 nm) were purchased from Sigma-Aldrich (USA). AFP antigen from human were obtained from Shanghai Linc-Bio Science Co. Ltd. (Shanghai, China). A commercial polystyrene 96-well plate substrate with the primary antibody of human AFP, and AFP secondary antibody (Ab2) were acquired from Biocell Biotechnology Co., Ltd. (Zhengzhou, China). All chemicals were used as received without further treatment. Milli-Q purified water (18.2 MΩ·cm−1, deionized water, DIW) was used throughout the experiment. Apparatus. Scanning electron microscopy (SEM, S-48 00, Hitachi, Japan) and transmission electron microscopy (TEM, G2 F20 S-TWIN, Tokyo, Japan) were employed to investigate the morphologies of CA NFs, g-CDs/CA NFs and r-CdTe QDs/g-CDs/CA NFs. Fluorescence and absorption spectrum were performed with a Multi-Mode Microplate Reader (BioTek Instruments, Inc., Synergy H1, USA, the excited wavelength: 365 nm) and a UV-3600 spectrophotometer (Hitachi Ltd., Tokyo, Japan), respectively. Zeta-sizer Nano-ZS instrument (Malvern Inc., UK) was used to determine the hydrodynamic diameter and zeta potential of NPs. Fabrication of CDs and CdTe QDs. CDs (Figure. S1A) were synthesized using p-phenylenediamine and ethylenediamine as reactants and absolute ethyl alcohol as a solvent under hydrothermal condition. The final products were separated and purified by silica gel column chromatography. Solvent was removed by vacuum-rotary evaporation, and the solid powder were dissolved in a certain absolute ethyl alcohol (the final concentration of CDs, 20 mg/mL). The synthetic procedure of CDs were provided by Peng Hou in our group. Red-emission monodispersed CdTe QDs (Figure. S1B) were synthesized based on the reported method with some modification.31 Typically, 0.2 mmol of CdCl2·2.5H2O was dissolved into 50 mL of DIW in conical flask under stirring. Then, 20 µL MPA was dropped in the mixture solution, and pH was adjusted to 10.5‒11.0 by using 1 M NaOH solution. Secondly, 0.04 mmol K2TeO3 was also dissolved in 50 mL of DIW, and then added into the above solution. Thirdly, 90 mg of NaBH4 was directly added into the precursor mixture solutions. After 5 min, conical flask was heated up to 100 oC and refluxed for another 90 min under air with continuous stirring. The revised method can quickly obtain red CdTe QDs at short times. Finally, the CdTe QDs were precipitated and purified


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with absolute ethyl alcohol (the volume ration of CdTe QDs solution and absolute ethyl alcohol, 1:2) by centrifugation. The purified red-emission CdTe QDs were dispersed in DIW and quantified according to the previous research for further use.32 Preparation of CA, g-CDs/CA, r-CdTe QDs/CA and rCdTe QDs/g-CDs/CA NFs. All ENFFs were prepared using

commercial electrospinning equipment (DNF−001, Beijing Kaiweixin Technology Co. Ltd., China) under a high-voltage of 25.0 kV. The needle was located at a distance of 15 cm from the cylinder’s collector wrapped with an aluminium foil, which was pasted with some circular cover glass (diameter of 6 mm). The cylinders collector and syringe pump rates were at 5 rpm and 0.1 mm/min, respectively. The circular cover glass attached with nanofibrous membranes were removed from aluminium foil to further use. First, CA powder (17 wt %) were dissolved in DMAC/acetone (1:2, v/v) mixed solution for 3 h at room temperature. After electrospinning, CA NFs were acquired. Then, the as-synthesized CDs (400 µL, 20 mg/mL) were added into CA electrospun NFs, which could be electrospun to form gCDs/NFs. Third, the CA and g-CDs/CA NFs were immersed into PEI solution (10 mg/mL aqueous solution). Because of the negatively charged property of CA NFs, the surfaces of CA and CDs/CA NFs would be modified by positively charged PEI. Then, the PEI-modified CA and CDs/CA NFs were soaked into negatively charged CdTe QDs solution for a certain time, and red-emission CdTe QDs can be assembled on their surfaces. All modified NFs were washed with DIW and dried with a nitrogen stream for further use. Cu2+ detection. First, 1 mL of different concentrations of Cu2+ in a PBS buffer solution (pH 7.2, 10 mM) were prepared. Then, r-CdTe QDs/g-CDs/CA ENFFs collected on circular cover glass were immersing into the Cu2+ solution. After a certain time period, the r-CdTe QDs/g-CDs/CA ENFFs were dried in the air and placed in 96-well plates (black). The fluorescence spectrum of various NFs were obtained by MultiMode Microplate Reader. In addition, to explore the selectivity of the r-CdTe QDs/g-CDs/CA ENFFs for Cu2+, fluorescent responses of the r-CdTe QDs/g-CDs/CA ENFFs were also performed for other metallic ions (K+, Na+, Ag+, Ca2+, Mg2+, Zn2+, Cd2+, Ni2+, Pb2+, Mn2+, Co2+, Ba2+, Hg2+, Al3+, Cr3+ and Fe3+) following the same procedure as that carried out for Cu2+ mentioned above. The concentrations of Cu2+ and other metal ions were 800 nM and 5 µM, respectively. Preparation of CuO NPs-Labelled Antibody (CuO NPsAb). According to a previously reported method, CuO NPs-Ab conjugates were prepared by physical adsorption.29,33 First, 1 mg of fresh CuO NPs (Figure. S1C) were dispersed in 1 mL of PBS-Tween solution (PBST, pH 7.2, 10 mM, 0.1% Tween 20) under ultrasonication for 20 min. Second, 20 µL of AFP Ab2 (1 mg/mL) were added in 400 µL of CuO NPs solution, and the mixtures were vortex-shaken for 3 h 37 oC. Then, the solution was subjected to centrifugation for 5 min at 10,000 rpm to remove the excess antibodies, and the precipitates were redispersed in 400 µL PBST. 100 µL of 10 % BSA solution was added in the Ab-labeled CuO NPs solution to block the active site and stabilize the solution. The Ab-labeled CuO NPs solution was stored at 4 oC. The hydrodynamic diameter (Figure S2A) and zeta potential (Figure S2B) of CuO NPs or Abattached CuO NPs were determined by Zeta-Sizer Nano-ZS. All measurements were performed in water (PBST, pH 7.2), and the temperature was maintained at 25 oC.

Detection of Disease-Related Biomarkers via Conventional Sandwich-Format Immunoassay. Firstly, 50 µL of different concentrations of AFP standard solution or real serum samples were added to the 96 wells coated with Ab1 and incubated for 1 h at 37 oC under gently shaking. After washing the wells three times using 4.5% of an OP emulsifier, 50 µL of CuO NPs-Ab solution were added into wells, and the plate were gently shaken at 37 oC for 1 h. The plate were washed again, and 20 µL of 10 mM HCl was added into each well and incubated for 40 min at 37 oC to release Cu2+ from CuO NPsAb. The resulting acid solution containing Cu2+ were adjusted to neutral (pH 7.2) using PBS buffer solution and DIW to 400 µL. Similarly, the r-CdTe QDs/g-CDs/CA ENFFs were also soaked into the above mixture solutions and incubated for about 50 min at room temperature. Finally, the fluorescence spectrum of various NFs were collected by Multi-Mode Microplate Reader. Confocal Fluorescence Microscopy Images of ENFs. CA or g-CDs/CA NFs were deposited on cell imaging dishes for 20 s, and r-CdTe QDs were carefully assembled onto their surface on the basis of electrostatic interaction. Fluorescence images were acquired at 60× magnification using an Olympus IX-81 inverted microscope equipped with an Olympus IX2DSU confocal scanning system and a CoolSNAP HQ2 CCD. The green fluorescence images were captured with exciting at 460‒480 nm and detecting with a BA495‒540 nm barrier filter under dichroic mirror of 485 nm (GFP channel), while red fluorescence images were captured with exciting at 360‒370 nm and detecting with a BA605 nm barrier filter (QD 605 channel).

RESULTS AND DISCUSSIONS Preparation of r-CdTe QDs/g-CDs/CA ENFFs. To obtain the excellent electrospun nanofibrous sensing films for the dual-color fluorescence analysis of analytes, the ENFFs performance must meet the following four significant factors: (1) good homogeneity of luminescent probes inside or on the surface of ENFFs, (2) highly specific and sensitive response to analytes, (3) stability under certain test condition, and (4) clear color changes for detection. In this research, the r-CdTe QDs/g-CDs/CA ENFFs were prepared by direct doping and surface assembling strategies. Firstly, the CDs was directly doped inside CA NFs. Secondly, because of the negatively charged property of the CA polymeric NFs, bare CA or gCDs/CA NFs can be modified with the positively charged property of PEI polymer. After modification, the negatively charged r-CdTe QDs were decorated on the surface of CA NFs and g-CDs/CA NFs by electrostatic interaction (Scheme 1A). As seen from Figure. 1A‒D, comparing with the colorless CA NFs, the g-CDs/CA, r-CdTe QDs/CA, r-CdTe QDs/gCDs/CA ENFFs displayed green, red and orange red color under UV light, respectively. From the fluorescence spectrum (Figure. 1E), broad emission bands of g-CDs/CA and r-CdTe QDs/CA ENFFs centered at 500 and 660 nm, respectively. In the fluorescence spectrum of r-CdTe QDs/g-CDs/CA ENFFs, two characteristic emission bands: 500 (g-CDs, weak peak) and 660 nm (r-CdTe QDs, strong peak.) were observed. Notably, the fluorescence intensity of CDs in CA NFs were drastically decreased after assembling r-CdTe QDs, which were related to the FRET process between CDs and r-CdTe QDs. Therefore, coupling the CDs and CdTe QDs with ENFs were employed to provide the green and red luminescence, respectively. The two colors can be regarded as a typical color match



in visual analysis.34 In addition, as compared to the fluorescence emission of bare CDs and r-CdTe QDs solution (Figure. S3A and B), those of g-CDs/CA and r-CdTe QDs/CA NFs showed a certain blue- and red-shift with a decreased bandwidth, respectively, which might be attributed to two reasons: (a) the strong confinement of luminescent nanomaterials in solid NFs matrix;35 (b) the dielectric difference of the local environment of the luminescent nanomaterials.36 According to these relevant photoluminescent characterization, CDs and CdTe QDs can actually exist in CA ENFs.

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larly, the thickness of the ENFFs were optimized by controlling the electrospinning times (Figure. S5A and A’). The fluorescence intensities of CdTe QDs on g-CDs/CA NFs become stronger with the prolongation of electrospinning time. At electrospinning times ranging from 60 to 100 min, the I660/I500 values were quite steady. However, I660/I500 values were decreased after a electropsinning time 2 h, and the color homogeneity of luminescent electrospun films were poor (the inset in Figure. S5A’). Thus, a electrospinning time of 80 min is chosen to ensure a steady fluorescence signal of r-CdTe QDs/g-CDs/CA NFs. In addition, the assembly time of r-CdTe QDs on g-CDs/CA NFs is another important parameter. The fluorescence intensities of CdTe QDs on g-CDs/CA NFs increased with the assembly time ranging from 10 to 140 min (Figure. S5B and B’). After 140 min, because r-CdTe colloidal solution will produce aggregates under longer assembling time, a higher immersion time of g-CDs/CA NFs in CdTe QDs solution were not propitious to the decoration of CdTe QDs on its surfaces. The I660/I500 values were gradually decreased. Therefore, an optimized immersion time of 1 h is used for gCDs/CA NFs in CdTe QDs solution. The above two vital conditions can ensure stable fluorescence signals of r-CdTe QDs/g-CDs/CA NFs and the good sensing performances.

Figure 1. Optical characterizations of relevant ENFFs. Optical photographs of (A) CA ENFFs, (B) g-CDs/CA ENFFs modified with PEI, (C) r-CdTe QDs/CA ENFFs, (D) r-CdTe QDs/gCDs/CA ENFFs under UV-ultraviolet irradiation, and (E) Fluorescent spectrum of CA ENFFs (black curve), g-CDs/CA ENFFs modified with PEI (green curve), r-CdTe QDs/CA ENFFs (blue curve), r-CdTe QDs/g-CDs/CA ENFFs (red curve).

To observe the microstructures of CA, g-CDs/CA, r-CdTe QDs/CA, r-CdTe QDs/g-CDs/CA NFs in detail, SEM, TEM and fluorescence images were recorded. At first, the SEM images of CA, g-CDs/CA, and r-CdTe QDs/g-CDs/CA ENFs (Figure. S4, A‒C) exhibited the good nanofibrous morphologies and networked microstructures, which will provide a good foundation for excellent sensing performance. Comparing with the TEM image of bare CA NFs (Figure. 2A), the magnified TEM images of g-CDs/CA NFs (Figure. 2B, inset) exhibited more black dots inside CA NFs, indicating that the CDs are doped into CA NFs. After the assembly of CdTe QDs on the g-CDs/NFs surface, the g-CDs/CA NFs surface appears coarse (Figure. 2C). In addition, luminescent g-CDs/CA, rCdTe QDs/CA, and r-CdTe QDs/g-CDs/CA NFs can be visualized in virtue of fluorescence microscopy. Bright green, red and orange red were uniformly observed throughout the gCDs/CA, r-CdTe QDs/CA, and r-CdTe QDs/g-CDs/CA NFs, respectively (Figure. 2D‒F). All of the above morphology characterizations confirm that the CDs and CdTe QDs are successfully doped and assembled on ENFs, which exhibit good nanofibrous and networked microstructures. Generally, the thickness of the luminescent ENFFs can affect the luminescent intensity, which can also influence the subsequent sensing performance.17 In a study previously reported by our group,10 the stability of fluorescent signals is related to the thickness of the fluorescent ENFFs, which was a significant precondition for biosensor. In this research, simi-

Figure 2. Morphologies of relevant electropsun NFs. TEM images of (A) CA ENFs, (B) g-CDs/CA ENFs, (C) r-CdTe QDs/gCDs/CA ENFs, FL images of (D) g-CDs/CA ENFs (GFP channels), (E) r-CdTe QDs/CA ENFs (QD 605 channels), FL overlay images of (F) r-CdTe QDs/g-CDs/CA ENFs under GFP and QD 605 channel.

Ratiometric Fluorescent r-CdTe QDs/g-CDs/CA ENFs for Cu2+ Visual Assay. At first, the absorption profile of rCdTe QDs significantly overlapped with the emission spectrum of g-CDs/CA NFs, allowing FRET to occur with the gCDs in CA NFs as a donor and r-CdTe QDs as an acceptor (Figure. 3A). Indeed, these results revealed that the fluorescence intensity of green CDs/CA ENFFs would decrease after assembly of r-CdTe QDs on its surface due to FRET process


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(Figure 1E). With the immersion of the r-CdTe QDs/gCDs/CA NFs into different concentrations of Cu2+ solutions, the red fluorescence of r-CdTe QDs on g-CDs/CA NFs decreased, and the green emission of g-CDs in CA NFs gradually increase (Figure. 3B). According to the previously reported studies,37-39 the high affinity constant of the MPA-Cu2+ led to the chemical displacement of Cd2+ on r-CdTe QDs surface. The nonradiative surface channels induced the electron transfer process between Cu2+ and r-CdTe QDs, leading to the fluorescence quenching of r-CdTe QDs. Furthermore, with the contact of the r-CdTe QDs with different concentrations of Cu2+ solutions, the absorption of r-CdTe QDs-Cu2+ complex decrease (Figure. 3C), thereby leading to a lower spectral overlap between the absorption profile of r-CdTe QDs and the emission spectrum of g-CDs/CA NFs. Thus, the emission peak of g-CDs/CA NFs at 500 nm gradually recovered with the increasing concentration of Cu2+. From above analysis, the FRET process between g-CDs and r-CdTe QDs on ENFFs can form ratiometric fluorescence property for Cu2+ assay. In this process, CDs in CA NFs cannot response to Cu2+ (Figure. S6).

Figure 3. Ratiometric fluorescence response of the r-CdTe QDs/g-CDs/CA ENFFs to Cu2+ through FRET process. (A) Spectra overlap between the emission profile of g-CDs/CA ENFFs (green curve) and the absorption of the r-CdTe QDs solution. (B) Fluorescence spectra of r-CdTe QDs/g-CDs/CA ENFFs after immersing into 0 (black curve), 1 (red curve), 10 µM (blue curve) Cu2+ solution. (C) The absorption of individual r-CdTe QDs solution with 0 (control, black curve), 2 µM (red curve), 20 µM (blue curve) and 40 µM (pink curve) Cu2+. (D) Fluorescence lifetime decay of g-CDs in CA ENFFs (λem = 500 nm): original g-CDs/CA ENFFs (black squares), g-CDs/CA ENFFs modified with PEI (red circles), r-CdTe QDs/g-CDs/CA ENFFs (green triangle) and rCdTe QDs/g-CDs/CA ENFFs after soaking into 2 µM Cu2+ solution (violet star).

Energy transfer between the donor and the acceptor is well known to result in a decreased donor fluorescence lifetime.40,41 Here, to further confirm energy transfer process between the donor (CDs in CA ENFFs) and the acceptor (r-CdTe QDs on the g-CDs/CA nanofibrous surfaces), the fluorescence lifetime of g-CDs in g-CDs/CA ENFFs, g-CDs/CA ENFFs modified with PEI, r-CdTe QDs/g-CDs/CA ENFFs and r-CdTe QDs/gCDs/CA ENFFs with 2 µM Cu2+ were recorded (Figure. 3D) and calculated as 4.20, 4.09, 2.32 and 2.66 ns (Table S1), respectively. Obviously, after assembly of r-CdTe QDs on gCDs/CA ENFFs, the fluorescence lifetime of g-CDs in ENFFs decreased about 50 %. The fluorescence lifetime of g-CDs in r-CdTe QDs/g-CDs/CA ENFFs interacting with 2 µM Cu2+

exhibited a certain recovery. Hence, the r-CdTe QDs and gCDs on CA ENFFs actually exist FRET process. The optimal analytical performance for Cu2+ was examined by investigating the effect of pH in detection solution (Figure. S7A) and reaction time of the r-CdTe QDs/g-CDs/CA NFs in the presence of 800 nM Cu2+ solution under optimal pH (Figure. S7B). The optimum fluorescence intensity ratio (I660/I500) was observed at pH of 7.2 and at a reaction time of 50 min. Under optimal conditions, the standard linear curve between the fluorescence signal ratio changes of I660/I500 and the logarithm of various Cu2+ concentrations were established within the working range from 10 nM to 10 µM (Figure. 4A and B). The limit of detection (LOD) was defined as LOD =Ycontrol + 3σcontrol on the basis of IUPAC standard method, whereYcontrol is the average signal values without target (control sample) and σcontrol is the standard deviation of background.42 In this work, the LOD was considerable less than as low as 2.82 nM (n = 11), which was much lower than the maximum level (1.3 ppm or 20 µM) of Cu2+ in drinking water permitted by the United State Environmental Protection Agency (U.S. EPA), demonstrating that the constructed method exhibits good sensitivity for Cu2+ assay.

Figure 4. r-CdTe QDs/g-CDs/CA ENFFs for detecting Cu2+. (A) Fluorescence spectra of the sensing ENFFs after immersing into Cu2+ solution with different concentrations: (a) 0 (control), (b) 1, (c) 10, (d) 50, (e) 200, (f) 60 nM, (g) 1, (h) 2, (i) 5, (j) 10, (k) 20, (l) 40, (m) 60, (n) 80 µM. (B) Calibration curve obtained by plotting I660/I500 against the logarithm of the Cu2+ concentrations. (C) The selectivity of the sensing ENFFs to various metal ions in pH 7.2 PBS buffer (the concentration of Cu2+: 800 nM, the concentration of the other metal ions: 5 µM, 1‒18: control, K+, Na+, Ag+, Ca2+, Mg2+, Zn2+, Cd2+, Ni2+, Pb2+, Mn2+, Co2+, Ba2+, Hg2+, Cu2+, Al3+, Cr3+, Fe3+, respectively). (D) Photograph of r-CdTe QDs/gCDs/CA NFs for Cu2+ visual analysis. (E) Photograph of r-CdTe QDs/CA NFs for Cu2+ visual analysis. Error bars indicates the standard errors of three independent experiments of three r-CdTe QDs/g-CDs/CA ENFFs.

Besides, to assess the selectively of the dual-emitting fluorescent r-CdTe QDs/g-CDs/CA ENFFs for Cu2+, the fluorescence intensity ratio (I660/I500) were recorded toward K+, Na+, Ag+, Ca2+, Mg2+, Zn2+, Cd2+, Ni2+, Pb2+, Mn2+, Co2+, Ba2+, Hg2+, Cu2+, Al3+, Cr3+ and Fe3+, respectively. The I660/I500 values of rCdTe QDs/g-CDs/CA ENFFs for other metal ions, except for Cu2+, did not exhibit considerable changes (Figure. 4C). Inevitably, Hg2+ also slightly quenched the fluorescence of r-CdTe QDs on g-CDs/CA NFs, but the quenching capability of 5 µM



Hg2+ was far less than that of 800 nM Cu2+. The I660/I500 values of r-CdTe QDs/g-CDs/CA NFs exhibit a considerably higher change toward Cu2+, indicating that r-CdTe QDs/g-CDs/CA ENFFs in this work are also selective to Cu2+. Notably, the slight interference of Hg2+ can be suppressed by a simple sample pretreatment using KI, NaCl and Rhodamine B.38,43

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sandwich-format immunoassay process (Scheme 1B), firstly, the antibody-immobilized 96-well plates captured antigens. Then, the CuO-NP-labelled antibodies was bound with the antigen to form the antibody-antigen-antibody-CuO NPs sandwich complex in 96-well plates. HCl was introduced to react with CuO and release amounts of Cu2+. On account of the different concentrations of target proteins, the different concentrations of Cu2+ released from CuO in a detection system were quantitatively reacted with r-CdTe QDs on gCDs/CA NFs. As a result, the fluorescence variation (I660/I500) of r-CdTe QDs/g-CDs/CA electrospun NFs is also closely associated with the concentration of target proteins.

Figure 5. FL images of r-CdTe QDs/g-CDs/CA ENFs for Cu2+ visual analysis. Various concentrations of Cu2+: 0 nM (control, A1‒C1), 100 nM (A2‒C2), 500 nM (A3‒C3), 1 µM (A4‒C4), 10 µM (A5‒C5). First row shows the FL images collected at GFP channel; second row shows the FL images collected at QD 605 channel; third row shows FL overlay images of first row and second rows.

In addition, the I660/I500 values changes of the r-CdTe QDs/g-CDs/CA ENFFs in different Cu2+ solution with various concentration can bring about the discriminated fluorescence color variations from red to green, which can be further used to visually discriminate Cu2+ by naked eyes under UV light (Figure. 4D). In contrast, the single-color fluorescence quenching of r-CdTe QDs/CA ones are hard to recognize (Figure. 4E), less sensitive and lower LOD for Cu2+ analysis (Figure S8 and Table S2). Comparison experiments clearly revealed that the ratiometric dual-emitting QDs/CDs-based ENFFs can provide higher sensitivity and distinguishable capability as compared to single-color fluorescence sensing films in visual detection. Interestingly, by FL imaging (Figure. 5), the fluorescence color changes of r-CdTe QDs/g-CDs/CA ENFs can be clearly and finely distinguished from red, orange, yellow to green with increasing concentrations of Cu2+. Furthermore, the good and networked microstructures of r-CdTe QDs/g-CDs/CA ENFs revealed that the nanofibrous morphologies cannot be destroyed by aqueous solution. Namely, the rCdTe QDs/g-CDs/CA ENFs exhibit good water-stability. Currently, a large number of ENFFs-based metal ions sensor have been reported, which involved the optical, electro and mass analytical strategies.44 These various functional electrospun nanofibrous sensing films exhibit highly sensitive and specific analytical performance for various metal ions, which were ascribed to the large surface area, high porosity and micro-networked structures of ENFs. Furthermore, fluorescent ENFFs have some advantages over solution-based fluorescent sensing strategies such as easy preparation and functionality, the stability of probes in electrospun carriers, and portability.8 However, metal ions-mediated electrospun nanofibrous bio- or immuno-sensing platform are seldom reported and developed. Therefore, in this work, the excellently analytical performance of r-CdTe QDs/g-CDs/CA ENFFs for Cu2+ with sandwichformat immunoassay method are developed for the Cu2+mediated biomarker assay. r-CdTe QDs/g-CDs/CA ENFFs as Cu2+-Mediated Ratiometric Sensing Films Toward Biomarkers. According to

Figure 6. r-CdTe QDs/g-CDs/CA ENFFs for Cu2+-mediated biosensing. (A) Fluorescence spectra of the r-CdTe QDs/g-CDs/CA ENFFs for AFP analysis at various concentrations: (from top to bottom, a‒k: 0‒200 ng/mL). (B) The linear relationship between the I660/I500 ratio values of r-CdTe QDs/g-CDs/CA ENFFs and the logarithm of the AFP concentrations. (C) Photograph of r-CdTe QDs/g-CDs/CA NFs for various concentrations OF target AFP. (D) Selectivity of the proposed strategy for AFP (10 ng/mL), PSA (100 ng/mL), CEA (100 ng/mL), HSA (100 ng/mL) and control (without any targets), respectively. (E) Stability test of fluorescence intensity ratio (I660/I500) of r-CdTe QDs/g-CDs/CA NFs without target AFP (control) and with AFP (10 ng/mL) at different storage times. The as-fabricated r-CdTe QDs/g-CDs/CA ENNFs were stored in 4 oC refrigerator. Error bars indicates the standard errors of three independent experiments of three r-CdTe QDs/g-CDs/CA NFs.

AFP has been extensively accepted early biomarkers of human hepatocellular carcinomas.45 In this work, AFP was taken as a typical target protein to validate the feasibility of the rCdTe QDs/g-CDs/CA ENFFs for the early detection of biomarkers. The high concentrations of AFP corresponded to the high concentrations of Cu2+, leading to the decreased red fluorescence at 660 nm and the gradual increase of green fluorescence at 500 nm (Figure. 6A). Therefore, I660/I500 also linearly decreases with the logarithm of various AFP concentrations ranging from 0.01 to 200 ng/mL (Figure. 6B), with the detection limit of 8.3 pg/mL (LOD =Ycontrol + 3σcontrol). Visually, the fluorescence color of the r-CdTe QDs/g-CDs/CA ENFFs gradually varied from red to green with increasing AFP con-


Page 7 of 10 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


centrations (Figure. 6C). Comparing with other reported analytical technologies, the dual-emitting r-CdTe QDs/g-CDs/CA ENFFs exhibit a wide linear range, better sensitivity and clearly visual effect for the detection of AFP based on the Cu2+induced biosensing method (Table S3). The good sensitivity of the dual-emitting r-CdTe QDs/g-CDs/CA ENFFs for the detection of AFP is ascribed to the following several crucial factors. First, comparing with the signal elements and target binding sites encapsulated inside films or NFs, the surface assemblies of r-CdTe QDs on electrospun NFs were beneficial for improving the utilization of probes (r-CdTe QDs) and promoting the direct and quick reaction between target (Cu2+) and probes (r-CdTe QDs).46,47 Second, based on the abovementioned excellent sensitivity of the r-CdTe QDs/g-CDs/CA electrospun NFs for Cu2+-mediated biosensing, amounts of Cu2+ released from CuO-NP-labelled antibody will produce signal amplification during biomarkers analysis.48 Third, comparing with conventional ELISA, nanomaterials-based sandwich-format immunoassay can prevent the issues related to facile inactivation of nature enzyme, instability of enzyme substrates, lower sensitivity and unsatisfactory visualization.49 Four, as compared to the complex and inefficient modification of biomacromolecules on ENFs,50,51 it is preferable to incorporate a sandwich-type immunoreaction using CuO-NP-labelled antibody with ENFFs in this work. Five, the inherent advantages of ratiometric fluorescence technique for establishing chemo-/bio-sensors can considerably contribute to the sensitivity improvement for quantitative analysis.52-55 Except for the sensitivity, the specificity and stability of the r-CdTe QDs/g-CDs/CA ENFFs based on the Cu2+-induced biosensing strategy should be further investigated. In this study, carcinoembryonic antigen (CEA), prostate specific antigen (PSA) and human serum albumin (HSA) in biological fluids were chosen as interfering agents (Figure. 6D). Because of the essential specificity of antigen-protein-related antibody in ELISA, I660/I500 values, including those of CEA, PSA and HSA, were almost the same as that of control (without AFP), while I660/I500 apparently decreased in presence of AFP. Similarly, the coexistence of other biomarkers with AFP did not cause the significant change of the fluorescence signal (I660/I500). These results illustrate the satisfactory specificity and selectivity of the Cu2+-meditated fluorescent nanofibrous sensing films. In addition, the stability of the as-fabricated rCdTe QDs/g-CDs/CA ENFFs were explored during 50 days, which were stored in darkness at 4 oC. The I660/I500 ratio values of the sensing films maintained stability without target AFP (Figure. 6E). In the presence of AFP (10 ng/mL), I660/I500 values for the sensing films decreased in comparison to the control and also essentially retained a relative stability within 50 days. Each point in Figure. 6E represents the average values acquired from three parallel and independent samples, and the standard relative deviation (RSD) of control (without AFP) and experimental (with AFP) groups were respectively less than 5.9 % and 13.2 %, indicating a good reproducibility. Preliminary Analysis of Real Human Serum Samples toward AFP. A recovery experiment was confirmed by a standard addition method in real human serum to examine the feasibility of the r-CdTe QDs/g-CDs/CA ENFFs for detecting AFP. Six serum samples were collected from normal patients (The Second Affiliated Hospital of Chongqing Medical University, Chongqing, China). Different AFP concentrations were added into human serum (defined as added), followed by measurement using our method (defined as found). Recovery

was defined as the percentage ratio values between the found concentration and the added concentration, which would directly reveal the accuracy of the analytical methods. According to Table 1, the RSD values of the several detection results range between 2.9 and 8.6 %, and the recoveries are between 96.4 and 104.0%. All these results revealed that the fluorescence visualization and the accuracy of this proposed strategy could be preliminarily applied for the assay of target AFP in human serum. Different concentrations of AFP in serum samples caused the different fluorescence color variations of the rCdTe QDs/g-CDs/CA ENFFs, demonstrating the feasibility of this assay to be applied for semi-quantification in real samples (Figure. 7). Table 1. Detection of AFP in Human Serum Samples by the Proposed Methoda No.

Added (ng/mL)

Found (ng/mL)

RSD (%, n = 3)

Recovery (%)

1

0.10

0.098 ± 0.004

4.1

98.4

2

2.00

1.93 ± 0.11

5.7

96.4

3

30.0

30.8 ± 0.9

2.9

102.6

4

5.00

4.99 ± 0.43

8.6

99.8

5

120.0

120.0 ± 7.0

5.8

100.0

6

70.0

72.8 ± 3.9

5.4

104.0

a In this work, the serum samples were obtained from the Second Affiliated Hospital of Chongqing Medical University (Chongqing, China). The detection of AFP for these real samples were carried out by the same procedures as that employed for the standard samples (mean ± SD, n = 3 ).

Figure 7. Fluorescence photographs of the r-CdTe QDs/gCDs/CA sensing ENFFs for AFP in six spiked human serum corresponding with Table 1.

CONCLUSIONS In conclusion, highly sensitive, selective and ratiometrically fluorescent r-CdTe QDs/g-CDs/CA ENFFs for Cu2+ analysis and Cu2+-mediated biosensing are reported. Comparing with single-emitting r-CdTe QDs/CA ENFFs, the strategy of dualemitting r-CdTe QDs/g-CDs/CA ENFFs not only decrease the detection limit of Cu2+ for two orders of magnitude, but also exhibits multicolour and visual detection. By the incorporation of a sandwich-type immunoreaction using CuO-NP-labelled antibody with r-CdTe QDs/g-CDs/CA ENFFs, the asfabricated electrospun nanofibrous test films can be also indirectly employed for the sensitive, selective and visual quantification of disease-related biomarkers via the quenching response of r-CdTe QDs to Cu2+, which were released from CuO NPs under acidic conditions. Taking advantages of FRET property of ENFFs and Cu2+-mediated signal amplificaiton strategy, these ratiometrically fluorescent ENFFs are capable



of detecting AFP with high sensitivity (detection limit of 8.3 pg/mL) and selectivity. The proposed strategy demonstrates promise for the further development of portable assays in food, environmental, counter terrorism areas.

ASSOCIATED CONTENT Supporting Information Additional data and information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * C.Z.H.: Tel.: (+86) 23 68254659, Fax: (+86) 23 68367257; Email: [email protected] * C.M.L.: E-mail:[email protected]

Present Address ¶ T.Y.: College of Chemistry and Chemical Engineering, Yunnan Normal University, Yunnan, Kunming 650500, P. R. China. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was financially supported by the Natural Science Foundation of China (NSFC, No. 21535006 and No. 21705131), Chongqing Graduate Student Scientific Research Innovation Project (No. CYB17058), the Open Project Foundation of State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University (No. 2016003). Thank Peng Hou in our group for providing the synthesized method of carbon dot to support this research.

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Table of Contents (TOC)

Ratiometric fluorescent r-CdTe QDs/g-CDs/CA electrospun nanofibrous films (ENFFs) were first used for Cu2+-mediated biosensing based on the fluorescence resonance energy transfer (FRET) between QDs and CDs on ENNFs. Taking advantages of ratiometric property of the dual-emitting ENFFs and Cu2+-mediated signal amplifying strategy, a sandwich-type immunoassay with CuO NPs-labelled antibody was developed for visual detection of alpha fetoprotein (AFP) with high sensitivity and selectivity.


Ratiometrically Fluorescent Electrospun Nanofibrous Film as a Cu2+-

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