Universal Multifunctional Nanoplatform Based on Target-Induced in

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A universal multifunctional nanoplatform based on the target-induced in situ promoting Au seeds growth to quench fluorescence of upconversion nanoparticles Qiongqiong Wu, Hongyu Chen, Aijin Fang, Xinyang Wu, Meiling Liu, Haitao Li, Youyu Zhang, and Shouzhuo Yao ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.7b00616 • Publication Date (Web): 29 Nov 2017 Downloaded from http://pubs.acs.org on December 1, 2017

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A universal multifunctional nanoplatform based on the targetinduced in situ promoting Au seeds growth to quench fluorescence of upconversion nanoparticles Qiongqiong Wuǂ, Hongyu Chenǂ, Aijin Fang, Xinyang Wu, Meiling Liu*, Haitao Li, Youyu Zhang*, Shouzhuo Yao Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, China KEYWORDS: Multifunctional nanoplatform, In situ produce reductant; Target-inducing enlarged AuNPs; Upconversion nanoparticles; Fluorescence resonance energy transfer ABSTRACT: Construction of newly multifunctional chem/biosensing platform for small biomolecules and tumor

markers is of great importance in analytical chemistry. Herein, a novel universal multifunctional nanoplatform for biomolecules and enzyme activity detection was proposed based on fluorescence resonance energy transfer (FRET) between upconversion nanoparticles (UCNPs) and the target-inducing enlarged gold nanoparticles (AuNPs). The reductive molecule such as H2O2, can act as the reductant to reduce HAuCl4, which will make the Au seeds grow. The enlarged AuNPs can effectively quench the fluorescence of UCNPs owing to the good spectral overlap between the absorption band of the AuNPs and the emission band of the UCNPs. Utilizing the FRET between the UCNPs and enlarged AuNPs, good linear between the fluorescence of UCNPs and the concentration of H2O2 can be found. Based on this strategy, other H2O2 related molecules such as L-lactate, glucose and uric acid can also be quantified. On the basis of UCNPs and PVP/HAuCl4, a general strategy for other reductants such as ascorbic acid (AA), dopamine (DA) or enzyme activity can be established. Therefore, the universal multifunctional nanoplatform based on UCNPs and the target-inducing in situ enlarged Au NPs will show its potential as a simple method for the detection of some life related reductive molecules, enzyme substrates as well as enzyme activity. Construction of newly chem/biosensing platform for detection of some small biomolecules, tumor markers and enzyme activity is of great importance in the area of analytical chemistry. Much attention has been focused on the development and preparation of novel nanomaterials to fabricate some electrochemical 1, colorimetric and fluorescence sensors 2-4 for sensitive detection of life-related small molecules and biomolecules. However, one signal or single functional nanomaterialbased sensors is not sufficient for multiple targets detection purpose5. And at the same time, developing the different functions of the same nonomaterial is of great importance. Hence, preparation of multifunctional material or construction of simple, multiple-signal nanosensor or multifunctional detection platform is gaining much attention in recent years. For the one hand, combining optical methods such as fluorescence (FL) and colorimetric assays together to construct two signals readout platform are highly favored owing to the high sensitivity, convenience and accessible instrument requirement 6. Liu and her group synthesized N-doped carbon dots and constructed a multiple-signal nanosensor for highly selective detection

of cysteine 7, which is sensitive and effective. However, when considering the multi-signal nanosensors, it is highly required that there are at least two signals which may be confirmed by each other. While for the multifunctional platforms, it is required that the platform can be simply regulated or induced to realize different purpose5, which can realize different functions via modification or through simply changing the external conditions. Therefore, this protocol will show much prospective in application in analytical chemistry areas. To the best of our knowledge, carbon dots8, Au nanomaterials9 and Au@Fe3O410-11, are often utilized as the multifunctional platform components. Among them, AuNPs show some advantages in the fabricating of multifunctional platform owing to their sizedependent or target induced-dependent surface plasmonic absorption (SPR) properties 12, shape-dependent optical properties13 and extremely high extinction coefficients 14. It is reported that in the presence of special targets, the Au seeds can be enlarged or be triggered to aggregate accompanying with color

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and UV-Vis spectra change 15. Therefore, the intensity variation or the shifts of the SPR peak are sensitive to the circumstance, which is notably as the merit for colorimetric detection of many molecules. For example, it is reported that H2O2catalyzed AuNPs growth increases the intensity of the localized SPR and then improves the detection sensitivity 16,17. Baron et al. developed a sensitive platform for neurotransmitters and tyrosinase activity based on the neurotransmitters-induced growth of Au nanoparticles 18. Peng et al. reported a strategy based on plasmonic enzyme-linked immunosorbent assay using alcohol dehydrogenase catalyzed gold nanoparticles seedmediated growth as a colorimetric signal generation method for detecting disease biomarkers2. The mechanism of the target-mediated growth was interesting, which was utilized to develop sensing platforms through the target induced reduction of AuCl4- to Au0 on the surface of Au seeds 16. And in these strategies, the growth or the aggregation of AuNPs are often used to design the nanosensors with colorimetric or UV spectra variations. It is sensitive and selective; however, the SPR peak of AuNPs can be notably influenced by the external factors such as the complicated environment, the proteins or other species in the biological systems. Therefore, if replacing the output signal by other signals with increased sensitivity, that is, combining the SPR peak change of AuNPs with the fluorescence materials and utilization of the FL as the output signal is considered as an alternative strategy to obtain more superior platforms.

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zyme substrates with good performance, which may show great prospective in clinical applications. Experimental Materials Rare-earth oxides used in this work, including yttrium oxide (Y2O3), ytterbium oxide (Yb2O3) and erbium oxide (Er2O3), were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) and dissolved in hot nitric acid and then dissolved in deionized water to achieve final concentrations of 0.4 M, 0.2 M and 0.05 M, respectively. Sodium phosphate dibasic dodecahydrate (Na2HPO4·12H2O), hydrofluoric acid (HF), sodium hydroxide (NaOH), nitric acid (HNO3), ethylenediaminetetraacetic acid disodium salt (EDTA), L-lactate, lactate oxidase (LaX), glucose, uric acid, glucose oxidase (GOX), uric acid oxidase (UOX), alkaline phosphatase (ALP), L-ascorbic acid 2-phosphate (AAP), ascorbic acid (AA), dopamine (DA), glucose tyrosinase and Ltyrosine, cetyltrimethyl ammonium bromide (CTAB), and PVP were purchased from Sigma and Beijing Chemical Corp. All other chemicals (99%, Merck) used were of analytical grade and without further purification, and Milli-Q ultrapure water (Millipore, ≥ 18 MΩ cm) was used throughout the experiments. (A)

Based on these concepts, in this report, rare-earth (RE) doped upconversion nanoparticles (UCNPs) emitting higherenergy visible light when excited by low-energy NIR light was employed as the fluorescence materials. Combining the UCNPs and the target-inducing enlarged AuNPs, a universal multifunctional nanosensing platform for some biomolecules, enzyme activity and enzyme substrates was proposed on the basis of fluorescence resonance energy transfer (FRET). As shown in Scheme 1, poly(vinylpyrrolidone) (PVP) act as a mild reductant and capping agent for the formation of Au seeds from HAuCl4. In the presence of some stronger reductants, PVP-Au seeds will be enlarged which can quench the fluorescence of the UCNPs. Based on this protocol, some reductants, enzyme substrates and enzyme activity can be determined. Therefore, a new universal multifunctional platform for the sensing of H2O2, glucose, L-lactate, dopamine, ascorbic acid and the enzyme activity was developed based on the FRET between UCNPs of NaYF4:Yb, Er and the targetinducing enlarged AuNPs. Unlike previous AuNPs-based assays that depend on the red-to-blue (or purple) color change, the assay we report here is based on a colorless-to-red process. The colorless AuNPs solution turns to red along with an appearance of absorption band at around 525 nm in the visible region. The absorption band of larger size of AuNPs and the emission band of UCNPs overlapped well, which results in the FRET between AuNPs and UCNPs. This strategy will not only improve the sensitivity, but also eliminate the interference from the matrix of the biological system on the UV spectra of AuNPs. Though the selectivity will be affected in the complex samples when coexisting of several reductants, it can become a universal method for detection of some reductants and en-

980 nm

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a: GOX + glucose or UOX +uric acid; b: AA or ALP+AAP or DA.

Scheme 1: Schematic illustration for the construction and principle of the multifunctional nanoplatform.

Apparatus The size and morphology of UCNPs of NaYF4:Yb, Er and AuNPs were characterized by transmission electron microscopy (TEM) images using a JEOL-1230 TEM (JEOL, Japan). The fluorescence spectra were measured using an F4500 fluorescence spectrophotometer (Hitachi Ltd, Japan) with an external 980 nm laser diode as the excitation source (Hi-Tech Optoelectronic Co., Ltd., China) and the UV-Vis absorption spectra were obtained using a UV-2450 UV-Vis spectrophotometer (Shimadzu Co., Japan). The crystalline phases of UCNPs were characterized on a Rigaku 2500 (Japan) X-ray diffractometer (XRD).


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Preparation of UCNPs The UCNPs of NaYF4:Yb3+, Er3+ were synthesized according to the previously reported method of our group with slight modification 19. 1315 µL 0.4 M Y(NO3)3, 420 µL 0.05 M Yb(NO3)3 and 105 µL 0.05 M Er(NO3)3 were added to an aqueous solution (2000 µL) containing different amounts of Na2HPO4·12H2O. Under ultra-sonication, 0.2925 g EDTA was added into the solution and the pH of the mixture was adjusted to 8.5 with HNO3 or NaOH. Then, 10.00 mL glycol and 0.0675 g CTAB were added into the solution. After the solution becoming clear, 500 µL HF aqueous was added dropwisely to the mixture and the pH was adjusted to 2. After vigorous stirring for 30 min at room temperature, the colloidal solution was transferred to a 50 mL Teflon-lined autoclave, sealed and maintained at 180 °C for 5 h. Then, the solution was cooled to room temperature. The nanocrystals were precipitated from the solution by centrifugation. The precipitates were washed with deionized water at first and then washed with ethanol. These washing procedures were repeated for three times. The product was dried under vacuum before use. Determination of H2O2 and L-lactate Briefly, the stock solution of H2O2 was freshly diluted from 30% solution by 0.02 M phosphate buffer solution (PBS, pH 8.0). The Au seeds were prepared by mixing 1.5 mM PVP in PBS (50 µL, pH 8.0) with 50 mM HAuCl4 solution (10 µL) for 3 min. Concurrently, 50.0 µL of H2O2 with various concentrations and 50 µL 1 mg/mL UCNPs were mixed and added to the above PVP-Au seeds solution. Then the total solution was diluted to 500 µL by 0.02 M PBS (pH 8.0) and shaken thoroughly to form homogeneous solution. After incubation at 37 °C for 60 min, the fluorescence emission spectra of the mixture were recorded with excitation at 980 nm. The L-lactate solution was prepared in 0.02 M PBS (pH 8.0). 17.5 µL 1 mg/mL LaX and 50.0 µL L-lactate with various concentrations, and 50 µL 1 mg/mL UCNPs were mixed and added to the PVP-Au seeds solution. Then the detection of L-lactate was similar to that of H2O2. The detection of Llactate in real samples was as follows. Serum samples were obtained from healthy volunteers. Before detection of Llactate, the human serum samples were diluted 20 times with 0.02 M PBS (pH 8.0). A series of serum samples were prepared by spiking with the standard L-lactate stock solution. 50 µL 1 mg/mL UCNPs was added to the PVP-Au seeds (50 µL 1.5 mM PVP mixed with 10 µL 50 mM HAuCl4 for 3 min). Thereafter, 50 µL of the diluted serum samples with different concentrations of L-lactate (including 17.5 µL 1 mg/mL LaX) were added in 1.5 mL vial, respectively. The detection processes were similar to those of H2O2 or L-lactate. Construction of multifunctional nanosensor based on the HAuCl4/PVP and UCNPs To expand the application of the UCNPs/HAuCl4/PVP, several nanosensors were constructed. The multifunctional nanosensor for determination of other reductants such as AA, DA or enzyme substrates such as glucose and uric acid were

investigated. The fluorescence of UCNPs was examined in the presence of GOX and glucose, UOX and uric acid, AA, dopamine, ALP and AAP, tyrosinase and L-tyrosine when coexisted with HAuCl4/PVP. The enzyme activity of ALP was also determined by this multifunctional platform. The detection processes were similar to those of H2O2 and L-lactate. Results and discussion Characterization of UCNPs and the in-situ produced AuNPs UCNPs have aroused considerable attention in the forefront of material science and biomedical fields 20 which have been widely used to construct biosensors. Comparing with other conventional fluorescent labels, the unique luminescence of UCNPs possesses several advantages such as improved detection sensitivity owing to no auto fluorescence, deeper NIR light penetration into biological causing minimum photodamage to living organisms, good chemical and physical stability and bio-compatibility 21. The morphology, structure and optical properties of UCNPs were characterized by TEM, XRD and fluorescence spectra. The TEM image of UCNPs in Fig. 1A shows that UCNPs are spherical and of good dispersion in aqueous with the average diameter of about 40 nm, which was also similar in different batches using the same preparation process. The X-ray powder diffraction (XRD) spectra were scanned at 2θ from 10 to 70o in Fig. S1. The diffraction features appeared at 28.179, 32.563 and 55.439o, which corresponded to the (111), (200) and (311) facets of the cubic crystal structure of Yb, respectively (JCPDS no. 77-2042). All the XRD peaks of UCNPs correspond to the calculated values for hexagonalNaYF4 23, suggesting that the prepared UCNPs are highly crystalline. As shown in Fig. 2A, the fluorescence spectrum of the UCNPs revealed typical emission bands at 435, 550 and 660 nm, which can be assigned to the transitions from the 2H11/2, 4 S3/2 and 4F9/2 excited states to the 4I15/2 ground state of the Er3+. Therefore, the UCNPs with excellent fluorecence property were successfully prepared. At the same time, the stability of the UCNPs is investigated. The fluorescence intensity remained unchanged within 60 min as shown in Fig. S2A, which indicated that they were very stable in aqueous solution. And it is found that the fluorescence intensity of UCNPs was not affected by pH (Fig. S2B) or temperature (Fig. S2C). The typical TEM images of the coexisted enlarged AuNPs and the UCNPs are shown in Fig. 1 B. These AuNPs (the black particles) were spherical in shape with the diameter of about 20 nm, which were close to UCNPs (the gray particles). It indicated that the AuNPs and UCNPs were coexisted in the system. Fig. 2B (b) shows the UV-Vis absorption spectra of AuNPs and the characteristic band located at around 525 nm. At the same time, the fluorescence spectrum of the UCNPs overlapped with the absorption of AuNPs, which showed that FERT occurred between them. Therefore, making use of the reaction between UCNPs and AuNPs, some sensors can be fabricated. In this study, the target-inducing enlarged gold nanoparticles were used which can affect the fluores-


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cence of the UCNPs and hence a universal multifunctional platform can be constructed.

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formation of smaller AuNPs in the presence of H2O2. These results are consisted with those of the TEM.

The principle of the nanosensor Studies have revealed that Au seeds can be enlarged to AuNPs with larger size in the presence of some special reductants. The spectroscopic features of AuNPs with larger size depend on the used reductants and their concentrations, such as nicotinamide adenine dinucleotide (NADH) 22 or neurotransmitters 18. Herein, a simple and important reductant, H2O2 was chosen as a typical reductant. AuNPs could be enlarged in the solution containing HAuCl4 as gold source, PVP as surfactant and H2O2 as the reductant. It is reported that PVP is a frequently used protective agents in preparation of Au seeds and metal nanoparticles 17. As shown in Scheme 1, PVP acted as a mild reductant for the formation of Au seeds from HAuCl4, and it also served as a capping agent for Au seeds. When H2O2 was added, PVP-Au seeds acted as the catalysts for the reduction of AuCl4- by H2O2, the main reaction is shown as follows:

HAuCl4 +

3 3 0 Au seeds H2O2        → Au + 4HCl + O2 2 2

The produced Au0 then deposited on the surface of the seeds and resulted in the enlargement of the AuNPs 23. While UCNPs can be good candidates as donors in FRET assay 21. It is reported that FRET is a highly distance-dependent process, only the emitters (doped rare earth ions) locating near the particles surface can be efficiently quenched 24. It is supposed that the UCNPs and the acceptors such as AuNPs coexisted in the system, the FRET may occur 25-26. The results in Fig. 2B showed that the absorption band of AuNPs and the emission band of UCNPs well overlapped, which would result in the FRET between them and the fluorescence of UCNPs can be quenched by the AuNPs (as shown in Fig. 2A, g, h). Therefore, the nanosensor may be developed for detection of H2O2 based on the FRET of AuNPs and UCNPs. Similarly, the H2O2 related molecules such as L-lactate can also be detected in the presence of its enzyme LaX. In order to confirm this mechanism, the TEM images of AuNPs mediated by H2O2 was conducted as shown in Fig. 1C and the Au seeds prepared by PVP and HAuCl4 are also shown in Fig. 1D for comparison. It can be seen that the PVP-Au seeds (Fig. 1D) are smaller than 6 nm, and they can be enlarged to AuNPs (~20 nm) in the presence of H2O2 (Fig. 1C). It can be found that the diameters of some AuNPs enlarged to about 35 nm with the concentration of H2O2 at 50 µM. The process was also monitored by UV-Vis spectroscopy. As shown in Fig. S3, gold seeds do not show any SPR peak in the absence of H2O2 or the concentration of H2O2 is too low. It indicated that Au seeds are in small size, which agreed with the literature that the SPR of gold AuNPs only existed in the case of the size are larger than 6 nm 27. Upon incubation of PVP-Au seeds with various concentrations of H2O2, the characteristic SPR absorbance of the enlarged AuNPs was well intensified with the increasing concentration of H2O2 and the maximum absorbance were slightly blue shifted, indicating the

Fig. 1. Typical TEM images of UCNPs (A), UCNPs and AuNPs (B), AuNPs mediated by 50 µM H2O2 (C) and the Au seeds prepared by HAuCl4 + PVP (D).

When adding of UCNPs, the newly produced AuNPs would approach to the surface of UCNPs via electrostatic interaction, which can cause the fluorescence quench of UCNPs through FRET. To further confirm the mechanism, the zeta potential was conducted to investigate the surface charge of UCNPs and AuNPs in aqueous system. As shown in Fig. S4, the enlarged AuNPs, the UCNPs and the Au seeds exhibited the zeta potential of -26.5, +38 and -13.8 mV, respectively. Therefore, AuNPs could be adsorbed to the surface of UCNPs closely through electrostatic interaction, which was in accordance with the results in Fig. 2B. Hence, it can be concluded that the FRET between AuNPs and UCNPs could occur. In order to further confirm the feasibility of the detection of H2O2 as well as the H2O2 related molecules such as Llactate, a series of experiments were carried out and the results are shown in Fig. 2A. UCNPs solution exhibits a strong fluorescent signal at approximately 550 nm (a), and its fluorescence intensity and peak location do not show obvious changes when UCNPs incubated with HAuCl4 (b), PVP (c), H2O2 (d), LaX (e) and L-lactate (f), respectively. It showed that the fluorescence of UCNPs cannot be affected by the HAuCl4, PVP or H2O2, which indicated that no obvious interaction occurred between each of them and UCNPs. However, the emission peak visibly be quenched when the mixture of HAuCl4/PVP/ H2O2 (g) or HAuCl4/PVP/LaX/L-lactate (h) were added into the UCNPs solution. According to the above results, the nanosensor based on FRET between UCNPs and the enlarged AuNPs may be used for the detection of H2O2 and L-lactate.


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ensure maximal fluorescence quenching efficiency, 35 µg/mL LaX, 1 mM HAuCl4 and 150 µM PVP (Fig. S5D-F) were chosen as the optimum concentrations.

Fig. 2. (A) The fluorescence spectra of UCNPs solution (a), UCNPs + HAuCl4 (b), UCNPs + PVP (c), UCNPs + H2O2 (d), UCNPs + LaX (e), UCNPs + L-lactate (f), UCNPs + HAuCl4 + PVP + H2O2 (g) and UCNPs + HAuCl4 + PVP + LaX + L-lactate (h). (B) The upconversion spectrum of UCNPs (a) and the SPR absorption spectrum of AuNPs (b).

Detection of H2O2 and L-lactate 0.4

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Fig. 3. Normalized FL intensity of UCNPs nanosensor towards (A) different concentrations of H2O2 and (C) different concentrations of L-lactate. The linearity of the fluorescence quenching efficiency towards different concentrations of H2O2 (B) and Llactate (D). [UCNPs]: 0.1 mg/mL, [PVP]: 150 µM, [HAuCl4]: 0.8 mM. [LaX]: 35 µg/mL.

In order to obtain high detection sensitivity, the parameters including the media pH, temperature, incubation time, concentrations of HAuCl4, PVP and LaX were optimized. In this study, the fluorescence quenching efficiency is used as a standard for optimization of the best condition. Fluorescence quenching efficiency is defined as (I0-I)/I0, where I0 and I represent the fluorescence intensity in the absence and presence of H2O2 or L-lactate, respectively. Although the pH value and temperature have no effect on the fluorescent intensity of the UCNPs, they can affect the enlargement of AuNPs and the activity of LaX. Hence, it is important to choose proper pH value and temperature for the detection of L-lactate with the proposed sensor. As shown in Fig. S5, the most suitable pH was around 8.0 (Fig. S5A) and the appropriate temperature was 37 °C (Fig. S5B). Besides, 60 min was employed as the incubation time for obtaining stable signal (Fig. S5C). On the other hand, concentrations of LaX, HAuCl4 and PVP play important roles in the enlargement of Au seeds to AuNPs. To

Under the optimum conditions, the capability of HAuCl4/ PVP and UCNPs system for quantitative detection of H2O2 and L-lactate were evaluated. As shown in Fig. 3A, the normalized fluorescence intensity of UCNPs were quenched in different concentrations of H2O2 from 1 to 3000 µM. Fig. 3B shows the plot of the fluorescence quenching efficiency vs. concentrations of H2O2, with the linear regression equation of (I0-I)/I0 = 0.0196 + 0.0035[H2O2] (R2= 0.997) in the linear range of 1-100 µM and the detection limit of 0.35 µM, where (I0-I)/I0 refers to the fluorescence quenching efficiency and [H2O2] refers to the concentration of H2O2. This suggests that the platform is suitable for the determination of H2O2. L-lactate is the end product of glycolysis, and it is also one of the most important metabolites in clinical analysis 28-29. It was reported that malignant transformation of normal cells into tumor cells frequently leads to an increased accumulation of L-lactate concentration in extracellular space, and L-lactate can enhance the motility of tumor cells and inhabit migration and cytokine release 28, 30. The features make L-lactate serve as one of biomarkers of cancers in its early stage and play an important role in antitumor treatment. Therefore, estimate of L-lactate is of great importance for biological science. As shown in Fig. 3C, the normalized fluorescence intensity of UCNPs was quenched in different concentrations of L-lactate from 1 to 1000 µM. As shown in Fig. 3D, the plots of the fluorescence quenching efficiency vs. the concentration of Llactate shows good linear, with the linear regression equation of (I0-I)/I0 = 0.0126 + 0.0018 [lactate]. The linear range was 1 to 100 µM with the correlation coefficient (R2) of 0.9900 and the limit of detection (LOD) of 0.39 µM. Τhe comparison of different methods for L-lactate detection is shown in Table S1, which indicated that the proposed nanosensor exhibits good performance in detection limit and linear range for L-lactate detection. To further prove the specificity of the developed fluorescence nanosensor for L-lactate, the potential interfering substances including common inorganic salts, glucose, cysteine, uric acid and some proteins and amino acids were investigated. Fig. S6 shows the changes in the fluorescence intensity of the nanosensor in the presence of other species. In contrast to addition of 50 µM L-lactate, the addition of excess of other potential interferences did not lead to apparent response. These results clearly demonstrated that quantification of L-lactate is feasible when using the proposed sensor owing to the special selectivity of the LaX and the relative stronger interaction between AuNPs and the UCNPs. To test the practicability of the proposed nanosensor, this new developed sensor was applied to detect L-lactate in human serum. Different concentrations (0, 0.005, 0.01, 0.05 mM) of L-lactate were added to the sample and the results are listed in Table 1. The recoveries of L-lactate ranged from 99.5 % to 105.6% and the relative standard deviations of 3.58% to


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5.02%, indicated that the nanosensor is effective and reliable for the detection of L-lactate in complicated serum samples.



the enlarged AuNPs may be expanded to construct of the sensor for other reductants and enzyme substrates, such as AA, DA, glucose, uric acid and so on. As shown in Fig. 4, fluoresTable 1. The application of the method for determination of secence of UCNPs was quenched in the presence of GOX and rum samples with different amounts of L-lactate. glucose (A), UOX and uric acid (B), AA (C), DA (D), ALP and AAP (E), tyrosinase and L-tyrosine (F) when coexisted Sample Added Found Recovery RSD (n=3, with HAuCl4/PVP. These results were similar to those of the (mM) (mM) (%) %) detection of H2O2 and L-lactate. The main mechanism can be explained as follows: the enzyme-catalysis produced H2O2 or 1 0.00 0.00 — 3.15 other reductants such as DA and AA, and these reductants can also induce the Au seeds to enlarge and form AuNPs, which 2 0.005 0.0053 105.6 5.02 also result in the quenching of fluorescence of UCNPs. Similarly, it is also reported that ALP can catalyze the inactive 3 0.050 0.0495 99.5 3.58 substrate AAP to in situ produce AA, which can be used for ALP activity detection. Therefore, in all these cases, the fluo4 0.100 0.1033 103.3 4.08 rescence of the UCNPs can be affected by the enlarged AuNPs, which can be utilized to determine these reductants, enzyme substrates or enzyme activities. Construction of the universal multifunctional nanoplatform It is reported that the detection of some biomolecules and for some biomolecules and enzyme activity detection based activity of enzyme is of great importance11. Therefore, the on the HAuCl4/PVP and UCNPs multifunctional nanosensor based on the HAuCl4/PVP/UCNPs was typically used to detect AA and ALP activity as a model 1.2 1.2 system. As seen in Fig. 5, when different concentration of AA B 1.0 1.0 A was added in the system of UCNPs/HAuCl4/PVP, the fluores0.8 0.8 0.6 0.6 cence of UCNPs was quenched. The linear regression equation 0.4 0.4 was (I0-I)/I0 = 0.0136 [AA] + 0.325, with the correlation coef0.2 0.2 ficient (R2) of 0.9850. The detection linear range was 0.1 to 25 0.0 0.0 µM with the limit of detection (LOD) 0.04 µM. The compari200 300 400 500 600 700 800 900 200 300 400 500 600 700 800 900 son of different methods for AA detection is shown in Table Wavelength (nm) Wavelength (nm) S2, which suggested that our method is better than those of many of the literatures. 1.2 1.2 1.0

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0.7

A 0

1.0

B

0.6

0.8

(I0-I)/I0

C


0.8

Nornalized Intensity

Nornalized Intensity

1.0

0.6 0.4

0.5 0.4

200

0.2

0.3 0.0

0.2

E


1.0 0.8 0.6 0.4 0.2

1.0

300

400

500

600

700

Wavelength (nm)

800

900

500

550

600

Wavelength (nm)

F

650

700

0

5

10

15

20

25

30

CAA (µ µM)

0.8 0.6 0.4 0.2 0.0

0.0 200

450

1.2

1.2 Normalized Intensity

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

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200

300

400

500

600

700

800

900

Fig. 5. (A) Normalized FL intensity of UCNPs/PVP-Au seeds nanosensor towards different concentration of AA. (B) The linearity of the fluorescence quenching efficiency towards different concentration of AA. [UCNPs]: 0.1 mg/mL, [PVP]: 150 µΜ, [HAuCl4]: 0.8 mM.

Wavelength (nm)

Fig. 4. Multifunctional nanosensor based on the UCNPs/PVP-Au seeds: Normalized FL intensity of UCNPs in the absence (black line) and presence (red line) of (A) GOX and glucose; (B) UOX and uric acid; (C) AA; (D) DA; (E) ALP and AAP; (F) tyrosinase and L-tyrosine. [UCNPs]: 0.1 mg/mL, [PVP]: 150 µM, [HAuCl4]: 0.8 mM. Concentrations of enzyme and their substances: 20 µg/mL and 0. 1 mM, respectively.

To make full use of the HAuCl4/PVP and UCNPs and expand its application, the multifunctional nanoplatform was developed. We supposed that the FRET between UCNPs and

Owing to the ALP can catalyze the inactive substrate AAP to in situ produce AA, therefore, this nanoplatform of UCNPs/HAuCl4/PVP can also be used for evaluating the activity of ALP. After the concentration of AAP was optimized (Fig. S7), the fluorescence of the system UCNPs/HAuCl4/PVP was monitored in the presence of different concentrations of ALP and fixed concentration of AAP. As the concentration of the enzyme increased, the fluorescence was quenched intensively and the relationship between the (I0-I)/I0 and concentration of ALP was shown in Fig. 6. Fig. 6B shows the derived calibration curve, the detection range and detection limit of


Page 7 of 9

ALP were 2-100 U·L-1 and 0.06 U·L-1, respectively. The results were also compared with the reported works as shown in Table S3, which showed that our proposed method had better performance. It also indicated that our approach can be used for detection of AA and ALP activity. Therefore, the multifunctional nanosensor based on HAuCl4/PVP and UCNPs may be expanded to the determination of other reductants or those enzyme substrates that can induce to produce reductants. In addition, the enzyme activities can also be evaluated. 1.2 1.0

1.2

A

B

1.0

0.8

(I0-I)/I0


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

ACS Sensors

0.6 0.4

0.8 0.6

0.2

0.4

0.0

0.2 200

300

400

500

600

700

800

900

Wavelength (nm)

0

2

4

6

8

10

* Corresponding author: Tel: +86-731-88872618; fax: +86-73188872618; E-mail address: [email protected], [email protected], zhangyy@ hunnu.edu.cn

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (21645008, 21305042, 21275051 and 21475043), the Scientific Research Fund of Hunan Provincial Education Department (14B116), the Science and Technology Department (14JJ4030), Collaborative Innovation Center of New Chemical Technologies for Environmental Benignity and Efficient Resource Utilization and the Aid Program for Science and Technology Innovative Research Team in Higher Educational Institutions of Hunan Province.

12

CALP (U.L-1)

Fig. 6. Normalized FL intensity of UCNPs nanosensor towards different concentrations of ALP (A) (0, 0.2, 1, 2, 3, 5, 6, 7, 8, 10 U·L-1). (B) Calibration curve of (A). [UCNPs]: 0.1 mg/mL, [PVP]: 150 µM, [HAuCl4]: 0.8 mM. [AAP]: 1 mg/mL.

Conclusion In this study, a simple and sensitive UCNPs-based FRET universal multifunctional nanosensor for some biomolecules and enzyme activity detection was constructed. On the basis of the target-inducing in situ promote Au seeds growth to quench the fluorescence of upconversion nanoparticles, the reductants such as H2O2, ascorbic acid, dopamine can be detected directly with great sensitivity. Some enzyme substrates and their related enzyme activity can also be detected indirectly by the proposed multifunctional platform. It is believed that the multifunctional nanoplaform of UCNPs/PVP-Au will become a universal method for the detection of some life related reductive molecules. This is the first work combining the target induced Au seeds growth strategy with the UCNPs-based fluorescence sensor, which may pave a new way for chemo/biosensing. Though the selectivity of the multifunctional platform may be not good in some complex samples, some masking agents or preprocessing can help its application in practical.

ASSOCIATED CONTENT Supporting Information. Tables for comparison of different typical techniques for the determination of lactate, AA and ALP activity, XRD pattern of NaYF4:Yb3+,Er3+ UCNPs, the stability study of UCNPs, absorbance spectra of Au seeds in the different concentrations of H2O2, Zeta potentials of UCNPs, AuNPs and Au seeds, the effects and interferences for detection of L-lactate, the optimization condition for ALP detection were supplied in the Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION ǂ

Corresponding Author

Qiongqiong Wu and Hongyu Chen contributed equally to this work.

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ACS Sensors

For TOC only

(A)

980 nm

HAuCl4 PVP

L-lactate

UCNPs

550 nm

L-lacate + O2 + H2O

Pyruvate + H2O2

HAuCl4 + 3/2H2O2

Au0 + 4HCl + 3/2O2

980 nm

(B)

HAuCl4

a or b

PVP 550 nm

a: GOX + glucose or UOX +uric acid; b: AA or ALP+AAP or DA.


Universal Multifunctional Nanoplatform Based on Target-Induced in

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