An Upconversion Luminescence Nanoprobe for the Ultrasensitive

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An Upconversion Luminescence Nanoprobe for the Ultrasensitive Detection of Hyaluronidase Zhe Wang, Xiaohua Li, Yanchao Song, Lihong Li, Wen Shi, and Huimin Ma Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b01131 • Publication Date (Web): 07 May 2015 Downloaded from http://pubs.acs.org on May 11, 2015

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Analytical Chemistry

An Upconversion Luminescence Nanoprobe for the Ultrasensitive Detection of Hyaluronidase Zhe Wang, Xiaohua Li,* Yanchao Song, Lihong Li, Wen Shi and Huimin Ma* Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China.

Abstract A new upconversion luminescence nanoprobe for the detection of hyaluronidase has been developed by coupling the hyaluronic acid-bearing upconversion fluorescence nanoparticles (HA-UCNPs) with poly(m-phenylenediamine) (PMPD) nanospheres via covalent linkage. The nanoprobe alone exhibits an extremely low background signal owing to the effective fluorescence quenching by electron-rich PMPD and the near-infrared excitation characteristic (λex = 980 nm) of HA-UCNPs; upon reaction with hyaluronidase, however, a more than 31-fold fluorescence enhancement is produced. Compared with the corresponding nanosystem assembled via physical adsorption, the prepared nanoprobe shows a largely increased stability and a much higher signal-to-background ratio, which offers an ultrasensitive assay for hyaluronidase, with a detection limit of 0.6 ng/mL. The nanoprobe has been successfully used to determine hyaluronidase in human serum samples from both colorectal cancer patients and healthy people, disclosing that the serum hyaluronidase level in colorectal cancer patients is roughly 3 times higher than that in healthy people. Furthermore, the nanoprobe has also been employed to study the activity change of hyaluronidase affected by different concentrations of arsenate (a potential carcinogen), and the results show that even a low dosage of arsenate (50 μg/L) can raise the activity of hyaluronidase by about one third, revealing the relationship between arsenate and the enzyme. The proposed method is not only simple, but also highly sensitive, making it useful to assay hyaluronidase in relevant clinical samples. 1 Environment ACS Paragon Plus


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■ INTRODUCTION Hyaluronic acid (HA), a class of high molecular weight polysaccharide, is an important component of extracellular matrix, and its synthesis or degradation is closely related to various biological processes, such as cell proliferation, differentiation and migration.1 Hyaluronidase is a family of enzymes capable of degrading HA,2 whose overexpression has been reported to be associated with many malignant tumors, like prostate,3 bladder,4,5 brain6 and colorectal cancers (CRC).7 Therefore, sensitive detection of hyaluronidase is of great importance for clinical diagnosis and therapy of cancer at its early stage. Traditional methods for hyaluronidase detection mainly include turbidimetry,8 viscosimetry9 and colorimetry;10 however, none of these assays shows high sensitivity and selectivity. Immunoassay11 and zymography12 have also been reported for monitoring hyaluronidase. The former is sensitive and specific but requires expensive antibodies and complex washing steps; the latter is only suitable for qualitative but not for sensitive quantitative determination. Alternatively, fluorescence methods have been widely utilized for detecting a variety of enzymes because of their high sensitivity, 13-18 and some novel fluorescence probes have been designed for hyaluronidase assay.19-22 For instance, Wu et al. have developed an approach via electrostatic adsorption of the negatively charged HA with positively charged tetraphenylethylene and anthracene derivatives,19 which showed a ratiometric fluorescence response but suffered from poor selectivity due to the unavoidable non-specific electrostatic adsorption of other charged components in the reaction system. Additionally, some nanoprobes have been proposed by using the fluorophore-labeled HA and gold nanoparticles,20-22 in which the fluorescence of the fluorophore is quenched by gold nanoparticles, and reaction with hyaluronidase results in the cleavage of the HA chain, accompanying the release of the fluorophore-labeled segment and thereby the recovery of fluorescence. Nevertheless, such nanoprobes themselves are not stable because gold nanoparticles are apt to precipitate in biological high-salt environments.23-26 Thus, new fluorescent hyaluronidase probes with high stability, selectivity and in particular sensitivity to monitor trace hyaluronidase in biosystems are still needed for practical applications.

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In this work, we have developed such a nanoprobe for ultrasensitive detection of hyaluronidase by combining

upconversion

fluorescence

nanoparticles

(UCNPs)

with

electron-rich

poly(m-

phenylenediamine) (PMPD). We chose these two components on the basis of the following considerations. On one hand, lanthanide-doped UCNPs emit strong visible fluorescence under the excitation of near infrared (NIR) light (typically 980 nm). This excitation is achieved by absorbing two or multiple photons of NIR light, which can effectively eliminate the auto-fluorescence of biological samples and scattering background,27 thereby improving the signal-to-background ratio. Additionally, compared with traditional organic fluorophores, UCNPs also have other attractive features, such as large Stokes shifts, high resistance to the effect of pH or temperature, and good photostability. 28,29 On the other hand, the electron-rich PMPD can efficiently quench the fluorescence of most fluorophores including UCNPs,30-32 and plenty of amine groups on the surface of PMPD facilitate the conjugation with fluorophores bearing carboxyls through covalent linkage, which would largely decrease the background fluorescence and increase the stability of a nanoprobe. Specifically, our nanoprobe can be prepared by linking the HA-bearing UCNPs (HA-UCNPs) to PMPD through covalent bond formation, as shown in Scheme 1. In this nanoprobe, the hexagonal phase Yb/Er co-doped NaYF4 (-NaYF4:Yb,Er) serves as a fluorophore, because -NaYF4:Yb,Er(Tm) has been reported to be the most efficient NIR-tovisible upconversion luminescence material among current UCNPs.33-35 Monodispersed, water-soluble and carboxyl-functionalized HA-UCNPs were solvothermally synthesized by using HA as a versatile capping reagent (HA can coordinate with the lanthanides, and parts of the uncoordinated HA chains can expose on the surface of UCNPs; the exposed HA provides not only carboxyls for further convenient modification with PMPD, but also the cleavable chain by hyaluronidase). As a result, the nanoprobe shows an extremely low background signal due to the effective fluorescence quenching by electron-rich PMPD and the near-infrared excitation characteristic (λex = 980 nm); upon reaction with hyaluronidase, however, the quenched fluorescence is recovered by the selective cleavage of the HA chain and thus the release of the UCNPs-containing segment, which leads to the establishment of a highly selective and sensitive method for hyaluronidase assay, with a rather low detection limit of 0.6 ng/mL. Using this 3 Environment ACS Paragon Plus


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nanoprobe the concentrations of hyaluronidase in serum samples from both CRC patients and healthy people have been determined and compared, revealing that the serum hyaluronidase level in CRC patients is about 3 times higher than that in healthy people. Moreover, the activity change of hyaluronidase affected by different concentrations of arsenate (a potential carcinogen) was monitored by our nanoprobe, which shows that even a low dosage of arsenate (50 μg/L) can raise the activity of hyaluronidase by about one third, disclosing the relationship between arsenate and the enzyme. The superior performance of the nanoprobe makes it useful for hyaluronidase assay in relevant clinical samples. Scheme 1. Fluorescence response of the designed nanoprobe to hyaluronidase.

■ EXPERIMENTAL SECTION Reagents. HA (Mw = 3500) was purchased from Yangzhou Zhongfu New Materials Co., Ltd. Hyaluronidase, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), human serum albumin (HSA), bovine serum albumin (BSA), L-cysteine, glutathione, 6-O-palmitoyl-L-ascorbic acid (PAA) and matrix metalloproteinase 2 (MMP2) were obtained from Sigma-Aldrich. Matrix metalloproteinase 1 (MMP1) was obtained from Sino Biological Inc. N-Hydroxysuccinimide (NHS), morpholinoethanesulfonic acid (MES), tris(hydroxymethyl)amino-methane (Tris) and 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) were purchased from J＆K Chemical. Na2HAsO4, C17H35COONa, hydrochloric acid, 4 Environment ACS Paragon Plus

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glucose and vitamin B1 were obtained from Beijing Chemicals, Ltd. The spectrally pure rare-earth oxides were purchased from Grinm Advanced Materials Co., Ltd. All other solvents and reagents used were local products of anaylytica1 grade. HEPES buffer (10 mM, pH 7.4) was employed in the experiments. Enzyme-linked immunosorbent assay (ELISA) kit for hyaluronidase was purchased from Shanghai Yanxin Biological Technology Co., Ltd. Human sera from four healthy individuals and four CRC patients were provided by Xijing Hospital, and an informed consent was obtained from each donor. The mixed healthy serum sample was purchased from ZhongKeChenYu (Beijing) Trading Co., Ltd. The mixed serum sample from four cancer patients with liver cancer, cervical cancer, stomach cancer and CRC cancer, respectively, was kindly provided by Prof. Wang’s group of our laboratory. PMPD nanospheres were synthesized using a reaction time of 8 h by room-temperature chemical oxidation polymerization of m-phenylenediamine monomer with (NH4)2S2O8 according to our previous procedure.30 Ultrapure water (over 18 M·cm) from a Milli-Q Reference system (Millipore) was used throughout. Apparatus. Upconversion fluorescence measurements were performed in 1010 mm quartz cells on a Hitachi F-4600 spectrophotometer (Tokyo, Japan), in which the xenon lamp was replaced by using an external 500-mW 980-nm laser diode equipped with 1-m fiber (Beijing Viasho Technology Co.) as the excitation source. UV-vis absorption spectra were recorded in 1-cm quartz cells with a TU-1900 spectrophotometer (Beijing, China). Fourier transform infrared (FT-IR) spectra were taken in KBr disks on a Tensor 27 spectrometer (Bruker, Germany). Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analyses were made on Hitachi S-4800 field emission scanning electron microscope and JEM-1011 instrument, respectively. Powder X-ray diffraction (XRD) patterns of the dried UCNPs were recorded on a X’pert diffractometer (Philips, Holland) using Cu Kα radiation at room temperature. Zeta-potential measurements were performed on a Nano-ZS Zetasizer ZEN3600 (Malvern Instruments Ltd., U.K.). The absorbance for ELISA analysis was recorded on a microplate reader (BIO-TEK Synergy HT, U.S.A.) at 450 nm. A model HI-98128 pH-meter (Hama



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Instruments Inc., U.S.A.) was employed for pH measurements. The incubation was carried out in Shaker incubator (SKY-100C, Shanghai Sukun Industry & Commerce Co., Ltd.). Synthesis of HA-UCNPs. First, rare-earth stearates [(C17H35COO)3RE] were prepared by the substitution reaction of C17H35COONa with rare-earth chlorides obtained by dissolving the corresponding rare-earth oxides in hydrochloric acid, and HA stock solution (1%, w/v) was prepared by dissolving HA in ultrapure water. Then, HA-UCNPs were synthesized following a previously reported procedure.36 Typically, 1.2 mmol of (C17H35COO)3RE (Y: Yb: Er = 80: 18: 2) used as the precursor was dissolved in 8 mL ethanol and 20 mL diethylene glycol to form a homogeneous solution. Then, 5 mL of aqueous solution containing 2.4 mmol of NaCl and 5 mmol of NH4F, and 2 mL of HA stock solution were added to the above homogeneous solution under stirring. The pH of the mixed solution was adjusted to 3.0 with 2 mM of hydrochloric acid, and then the reaction solution was stirred under argon for 30 min at room temperature. The reaction mixture was transferred into a 40 mL Teflon-linked autoclave, which was then sealed and heated at 240 C for 24 h. After cooling down to room temperature, the reaction mixture was poured into an ether/ethanol (1:4, v/v) solution, and the resulting white precipitate product was collected by centrifugation at 1,1000 rpm. The product was purified by washing several times with ethanol and ultrapure water successively, and then dried in an oven at 60 C. Preparation of the Nanoprobe. EDC (1.2 mg) and NHS (2.1 mg) were added to 2 mL of MES buffer (10 mM, pH 5.5) containing 2 mg of HA-UCNPs, and the mixture was incubated by shaking at 35 C for 1 h to activate the surface carboxyl groups. After centrifugation and washing twice with water, the precipitate was collected and then added to 2 mL of HEPES (10 mM, pH 7.4) containing 5 mg of PMPD, followed by reaction at 35 C for 1 h under shaking. Then, 50 mg of Tris was added to block the unreacted NHS. After 20 min, the product of PMPD-HA-UCNPs (referred to the nanoprobe) was centrifuged, washed three times with water, and then dispersed in 0.5 mL of HEPES (10 mM, pH 7.4) for use. General Procedure for Hyaluronidase Detection. In a test tube, different concentrations of hyaluronidase were incubated with the nanoprobe (final concentration of 0.2 mg/mL) at 37 C for 50 6 Environment ACS Paragon Plus

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min in HEPES buffer (10 mM, pH 7.4). Then the upconversion fluorescence of the reaction solution was measured at room temperature with λex/em = 980/545 nm. Determination of Hyaluronidase in Human Serum Samples by the Nanoprobe. Human serum samples were diluted 3 times before analysis. Then appropriate aliquots of the serum samples were transferred to the HEPES buffer (10 mM, pH 7.4) containing 0.2 mg/mL nanoprobe. After reaction at 37 C for 50 min, hyaluronidase in the serum samples was quantified according to the general procedure as described above. The detection of the activity change of hyaluronidase in human serum samples affected by arsenate was carried out following the above general procedure, except that different concentrations of Na2HAsO4 solution were additionally introduced with the final concentrations of 0, 5, 50, 500 and 5000 μg/L, respectively. Determination of Hyaluronidase in Human Serum Samples by ELISA. The concentrations of hyaluronidase in human serum samples were also determined by measuring the absorbance values at 450 nm using a commercial ELISA kit for human hyaluronidase. First, the standard curve (OD = 0.02  [hyaluronidase] (ng/mL) + 0.11, R = 0.999) was obtained following the direction of the kit in the concentration range of hyaluronidase from 0 to 50 ng/mL. Then, human serum sample (50 μL) and horseradish peroxidase-conjugate reagent (100 μL) were added to the ELISA kit wells. After incubation at 37 C for 60 min, all the samples were washed five times with 400 μL wash solution, followed by the addition of 50 μL chromogen solution A and 50 μL chromogen solution B to each well. The reaction mixture was incubated at 37 C for 15 min in the dark. Finally, 50 μL of the stop solution was added to each well to stop the reaction, and the optical density was read immediately on the microplate reader (BIO-TEK Synergy HT, U.S.A.) at 450 nm.

■ RESULTS AND DISCUSSION Preparation and Characterization of PMPD Nanospheres and HA-UCNPs. As shown in Figure 1A, the PMPD nanospheres, prepared following our previous procedure30 with a reaction time of 8 h, 7 Environment ACS Paragon Plus


have a rather uniform spherical shape and an average diameter of ca. 100 nm. Figure 1B is the FT-IR spectrum, which confirms the existence of Ar–NH2 moiety on the surface of PMPD, as evidenced by NH stretching (3151 cm-1), N-H bending (1623 cm-1), and C-N stretching (1402 and 1118 cm-1). In addition, Figure 1C shows that the UV-vis absorption spectrum of PMPD spans a wide range of wavelengths from 200 to 800 nm,30,31 which can overlap the fluorescence spectra of most fluorochromes, suggesting that the electron-rich PMPD is a good fluorescence quencher. Furthermore, the PMPD nanospheres possess excellent dispersibility in water (see the inset of Figure 1C), as usually required for biological and biomedical applications. C

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0.6

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1000

Wavenumber (cm-1)

200

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Figure 1. (A) SEM image, (B) FT-IR spectrum and (C) UV-vis absorption spectrum (0.5 mg/mL; against water) of the PMPD nanospheres. The inset shows the dispersibility of PMPD nanospheres (0.1 mg/mL) in water. As is known, pure -NaYF4:Yb,Er UCNPs have high upconversion luminescence efficiency but are difficult to obtain,36 because there is a rather high dynamical energy barrier for  →  phase transition.37,38 To get pure -NaYF4:Yb,Er in the presence of HA (i.e., HA-UCNPs), we made a systematic study on the reaction temperature. As shown in Figure S1 (Supporting Information), with the increase of reaction temperature, the undesired cubic phase -NaYF4:Yb,Er is gradually decreased, and the purest hexagonal phase -NaYF4:Yb,Er can be prepared at about 240 C (curve d); higher temperature could cause the organic components to become charred. Figure 2A shows the TEM image of the as-prepared HA-UCNPs at 240 C, which has a rather uniform spherical shape with an average diameter of ca. 20 nm. FT-IR spectra (compare curves a and b in Figure 2B) show that HA-UCNPs have the characteristic absorption bands of HA, such as 3417 cm-1 (O-H stretching), 1637 cm-1 (C=O 8 Environment ACS Paragon Plus

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stretching), 1384 cm-1 (O-H bending), and 1087 cm-1 (C-O stretching), suggesting the presence of HA. The fluorescence spectrum of HA-UCNPs (Figure 2C) shows three emission peaks at 529, 545 and 662 nm, which can be assigned to 4H11/2－4I15/2, 4S3/2－4I15/2 and 4F9/2－4I15/2 transitions for Er3+, respectively. All of these emissions are within the absorption range of PMPD (Figure 1C), further indicating that the prepared HA-UCNPs can be used as a fluorophore for our nanoprobe construction. Furthermore, HAUCNPs also exhibit excellent dispersibility in water (see the inset of Figure 2C). B

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Figure 2. (A) TEM image of HA-UCNPs, (B) FT-IR spectra of (a) HA-UCNPs and (b) HA, (C) fluorescence spectrum (1.0 mg/mL) of HA-UCNPs. The HA-UCNPs were prepared at 240 C, and the inset of Figure 2C shows the dispersibility of HA-UCNPs in water (0.2 mg/mL) under irradiation of 980 nm light. In addition, zeta-potential values of the prepared PMPD nanospheres and HA-UCNPs in HEPES buffer (10 mM, pH 7.4) were determined to be +15.8 mV and -21 mV, respectively. This suggests that the PMPD nanospheres are positively charged while HA-UCNPs are negatively charged in aqueous solutions, which can obviously be ascribed to the existences of the -NH2 and -COOH functional groups on the surface of the corresponding nanomaterials. Importantly, these functional groups are desirable for preparing the nanoprobe through covalent linkage. Preparation of the Nanoprobe and Its Fluorescence Response to Hyaluronidase. As mentioned above, the nanoprobe was synthesized by linking HA-UCNPs to PMPD nanospheres through covalent bond formation. First, the amount of PMPD as a quencher was optimized for preparing the nanoprobe. As shown in Figure 3, the fluorescence of HA-UCNPs can be quenched at least by 98% when the concentration of PMPD is higher than 0.5 mg/mL (curve a), which is thus used for the nanoprobe 9 Environment ACS Paragon Plus


preparation in this work. Interestingly, only 86% of the fluorescence is quenched in the simple mixture of PMPD and HA-UCNPs, even if the concentration of PMPD is as high as 0.8 mg/mL (curve b), further indicating that a much lower background signal can be obtained when the fluorophore and the quencher are combined through covalent linkage rather than through physical adsorption. Then, reaction time for the nanoprobe preparation was examined. As shown in Figure S2 (Supporting Information), addition of PMPD to the solution of activated HA-UCNPs leads to a rapid decrease in fluorescence intensity, and this fluorescence decrease reaches a plateau after 50 min. Hence, a reaction time of 50 min was employed for preparing the nanoprobe. 500

Fluorescence Intensity

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Figure 3. Effect of the amount of PMPD as a quencher on the fluorescence of different systems. (a) The nanoprobe prepared through covalent linkage in the presence of UCNPs (0.2 mg/mL) and PMPD with different concentrations (0 - 0.9 mg/mL); (b) simple mixture of UCNPs (0.2 mg/mL) and PMPD at different concentrations (0 - 0.9 mg/mL). To confirm the successful preparation of the nanoprobe, a comparative study was conducted by FT-IR spectroscopy with the following four samples: the first one was PMPD; the second one was HA-UCNPs; the third one was the simple mixture of PMPD and HA-UCNPs; and the last one was the nanoprobe. As shown in Figure S3 in Supporting Information, the FT-IR spectrum from the simple mixture (curve c) displays the characteristic peaks of both PMPD (curve a) and HA-UCNPs (curve b), whereas that from the nanoprobe (curve d) shows the typical amide bond signals including the amide I band (1655 cm-1) and amide II band (1532 cm-1) as well as stronger N-H stretching vibration (3314 cm-1). These results


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clearly indicate no new covalent bond formation in the simple mixture but the successful preparation of a nanoprobe with the covalent linkage. Next, the analytical performance of the nanoprobe was studied in detail. Figure 4 shows the fluorescence spectra of the nanoprobe before and after reaction with hyaluronidase. As can be seen, HA-UCNPs as a control exhibit intense upconversion fluorescence emission at 545 nm under 980 nm excitation (curve a). In contrast, the nanoprobe obtained from the reaction of HA-UCNPs with PMPD gives an extremely low fluorescence (curve b), resulting from the efficient fluorescence quenching of HA-UCNPs bound to PMPD as expected. Nevertheless, addition of hyaluronidase to the nanoprobe solution causes large fluorescence enhancement (curve c), which is attributed to the specific cleavage of the substrate (HA) by hyaluronidase and the release of the UCNPs moiety. It is also noted that the upconversion fluorescence of HA-UCNPs is scarcely influenced by the addition of hyaluronidase in the absence of PMPD (curve d), and PMPD itself exhibits nearly no fluorescence (curve e). All the above observations reveal that the nanoprobe may be applicable for hyaluronidase detection. a, d

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Figure 4. Fluorescence emission spectra (λex = 980 nm) of different reaction systems. (a) UCNPs (0.2 mg/mL); (b) the nanoprobe prepared from the covalent reaction of UCNPs (0.2 mg/mL) with PMPD (0.5 mg/mL); (c) the nanoprobe solution (0.2 mg/mL) + hyaluronidase (300 ng/mL); (d) UCNPs (0.2 mg/mL) + hyaluronidase (300 ng/mL); (e) PMPD (0.5 mg/mL). Furthermore, the effect of pH on the reaction system was studied. As shown in Figure S4 (Supporting Information), the fluorescence intensities of both HA-UCNPs and the nanoprobe are hardly influenced by the pH value changing from 4 to 9, whereas the largest fluorescence enhancement of the nanoprobe 11 Environment ACS Paragon Plus


in the presence of 150 ng/mL of hyaluronidase is observed at around pH 7, which may arise from the highest activity of hyaluronidase under the physiological pH of about 7. The above data show that the nanoprobe is rather stable against pH change and functions well under physiological conditions. Fluorescence kinetic curves of the nanoprobe reacting with hyaluronidase at varied concentrations are depicted in Figure S5, which reveals that higher concentrations of hyaluronidase result in faster cleavage reaction and larger fluorescence enhancement. For hyaluronidase of no more than 250 ng/mL, the fluorescence intensity increases to a plateau in about 50 min. By contrast, the fluorescence of the nanoprobe without hyaluronidase (control) hardly changes during the same period of time, also indicating that the nanoprobe is stable under the experimental conditions. Thus, an incubation time of 50 min was selected for the nanoprobe reacting with hyaluronidase in further analytical applications. To test the selectivity of the nanoprobe for hyaluronidase, various potentially interfering substances, such as inorganic salts (NaCl, KCl, MgCl2, CaCl2, CuCl2, Na2HAsO4), L-cysteine, glutathione, glucose, vitamin B1, HSA, BSA, cytochrome c and some proteases (carboxylesterase, MMP2, MMP1, thrombin), were examined in parallel under the same conditions. As shown in Figure 5, the fluorescence intensity enhanced by hyaluronidase is 15 to 140 times higher than those by the other species tested, showing the pronounced selectivity of nanoprobe for hyaluronidase over the other species, which may be ascribed to the specific cleavage of the substrate by hyaluronidase. 160 120

F

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N aC K l M Cl gC C 2l uC N Ca 2l a 2 H Cl L- As 2 C O G ys 4 lu te ta in th e G ion l Vi uco e ta m se in B 1 H C S y C to ar c B A bo h S xy r o m A le e st C er a M se M P M 2 H T h MP ya r 1 o lu m ro b ni in da se

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Figure 5. Fluorescence responses of the nanoprobe (0.2 mg/mL) to various species: NaCl (100 mM), KCl (100 mM), MgCl2 (2.5 mM), CuCl2 (100 μM), CaCl2 (1 mM), Na2HAsO4 (30 μM), L-cysteine (1 mM), glutathione (5 mM), glucose (10 mM), vitamin B1 (1 mM), HSA (100 nM), BSA (100 nM), 12 Environment ACS Paragon Plus

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cytochrome C (10 nM), carboxylesterase (10 nM), MMP2 (5 nM), MMP1 (5 nM), thrombin (5 nM) and hyaluronidase (80 ng/mL). F is the difference of the fluorescence intensity of the nanoprobe in the presence and absence of a species. The results are the mean  standard deviation of three separate measurements. λex/em = 980/545 nm. The fluorescence response of the nanoprobe to hyaluronidase at varied concentrations was investigated under the optimized conditions (incubation at 37 C for 50 min in HEPES buffer of pH 7.4). As shown in Figure 6, the fluorescence intensity of the reaction system increases with increasing the concentration of hyaluronidase from 0 to 300 ng/mL, with a linear equation of F = 1.62 × [hyaluronidase] (ng/mL) – 1.42 (R = 0.997) in the range of 0.9 to 150 ng/mL hyaluronidase (Figure S6 in Supporting Information), where ΔF is the difference of fluorescence intensity of the nanoprobe in the presence and absence of hyaluronidase. The detection limit (3S/m, in which S is the standard deviation of blank measurements, n = 11, and m is the slope of the linear equation)30 is determined to be 0.6 ng/mL, which is much lower than those of the other reported detection systems for hyaluronidase.19-22 400 Fluorescence Intensity

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700

Wavelength (nm)

Figure 6. Fluorescence intensities of the nanoprobe in the presence of varied concentrations of hyaluronidase (from bottom to top: 0, 5, 10, 20, 40, 80, 120, 150, 200, 250 and 300 ng/mL) in HEPES buffer of pH 7.4 at 37 C. λex = 980 nm.

To verify that the nanoprobe can be cleaved by hyaluronidase, the reaction filtrates of the nanoprobe in the presence and absence (control) of hyaluronidase were collected by centrifugating an ultrafiltration 13 Environment ACS Paragon Plus


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tube (with a membrane of molecular weight cutoff of 10 kD), and then the reaction filtrates were subjected to MALDI-TOF-MS analysis. As shown in Figure S7, the reaction filtrate of the nanoprobe in the presence of hyaluronidase produces a peak at m/z = 396, which is characterized to be the monomeric segment (m/z = 396 [M]–) from the polymeric HA chain; in contrast, the reaction filtrate of the nanoprobe in the absence of hyaluronidase does not show the peak at m/z = 396. This comparative analysis supports that the enzymetic cleavage occurs, thus causing fluorescence enhancement. Moreover, the effect of PAA (a common inhibitor of hyaluronidase) was also investigated on the activity of the enzyme. As shown in Figure S8 (Supporting Information), introduction of PAA into the nanoprobe solution does not change the fluorescence intensity (compare curves a and b), demonstrating that the inhibitor has no effect on the nanoprobe. In contrast, the fluorescence intensity in the presence of both hyaluronidase and the inhibitor (curve d or curve e) is much weaker than that in the presence of the enzyme only (curve c), indicating the effective inhibition of the hyaluronidase activity by PAA. Further, more inhibitor can lead to a larger decrease of the fluorescence intensity (compare curve d with curve e). On the other hand, PAA also did not affect the fluorescence of the reaction product of the nanoprobe with hyaluronidase (curve f). These results indicate that the fluorescence off-on response indeed arises from the enzyme-specific cleavage action. Determination of Hyaluronidase in Human Serum Samples. Then, the concentrations of hyaluronidase in human serum samples were determined using the nanoprobe. In this experiment, eight serum samples from four healthy people and four CRC patients were analyzed, and the results are shown in Table 1. As is seen, the average concentration of serum hyaluronidase in CRC patients is 138.2 ng/mL, which is roughly 3 times higher than that (44.6 ng/mL) in the healthy people.7 This result demonstrates that the level of hyaluronidase is associated with the progress of tumor cells, which in fact provides another proof that hyaluronidase as a clinical marker is overexpressed in tumors. Moreover, we also used a commercial ELISA kit to detect hyaluronidase in the above serum samples (Figure S9, Supporting Information), and the obtained results (Table 1) were compared with those obtained by our method using a Student’s t-test,30 which showed that no significant difference between the two methods 14 Environment ACS Paragon Plus

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was statistically found at the 95% confidence level, suggesting a remarkably good agreement. The above results also demonstrate the practicability of the proposed method for the quantitative determination of hyaluronidase in complex biological samples. Table 1. Determination of the Hyaluronidase Level in Human Serum Samples Serum samples

Current method a [ng/mL]

Average value b [ng/mL]

ELISA a [ng/mL]

Healthy 1

34.8 ±2.4

Healthy 2

29.6 ±1.5

Healthy 3

65.5 ±2.8

Healthy 4

48.5 ±1.4

47.8 ±1.9

CRC 1

141.0 ±5.0

137.0 ±3.0

CRC 2

138.8 ±1.8

CRC 3

148.2 ±2.4

CRC 4

124.8 ±3.7

Average value b [ng/mL]

31.0 ±2.4 44.6

138.2

27.5 ±1.6 60.0 ±5.2

133.8 ±2.8 144.2 ±4.1

41.6

133.6

119.3 ±5.0

a

Mean of three determinations ±standard deviation.

b

The average value from four individuals for different groups.

Detection of Activity Change of Hyaluronidase in Human Serum Treated with Different Concentrations of Arsenate. Further application of the nanoprobe has been demonstrated by detecting the activity change of hyaluronidase in human serum affected by different concentrations of arsenate. As is known, arsenic (a potential carcinogen) often exists in the pentavalent arsenate form in drinking water.39,40 Chronic exposure of humans to arsenic-contaminated water may cause cancer of the skin, liver, lung and bladder,40 but the exact mechanism behind the cancer is still unclear. Here, by using our sensitive nanoprobe, we explored a potential relationship between hyaluronidase activity and pentavalent arsenate. In this experiment, two mixed human serum samples (one from healthy group; the other from 4 cancer patients with liver cancer, cervical cancer, stomach cancer and CRC cancer, respectively) were exposed to different concentrations of Na2HAsO4. Then, the activity variation of hyaluronidase in human serum, reflected by its apparent concentration, was monitored with the 15 Environment ACS Paragon Plus


nanoprobe. As shown in Figure 7, the activity of hyaluronidase in either healthy group or cancer patients increases gradually with increasing the concentration of Na2HAsO4 up to 50 μg/L, and then again decreases. All the activity changes between two adjacent values show a significant difference at the 95% confidence level, as evaluated by using the Student’s t-test.30 Compared with the control, 50 μg/L of Na2HAsO4 causes the maximum enhancement of the enzyme activity, whereas higher concentrations of Na2HAsO4 show less enhancing effect and even depression under the present conditions. These results reveal for the first time the relationship between arsenate and hyaluronidase. Interestingly, it is noted that even a low dosage of arsenate (50 μg/L) can raise the activity of hyaluronidase by about one third, supporting the view that arsenate is a potential carcinogen. Furthermore, this activity enhancement from the low dosage of arsenate (50 μg/L) was confirmed by the inhibitor experiment with PAA, that is, the fluorescence enhancement decreases with the increase of the inhibitor of PAA (Figure S10, Supporting Information). On the other hand, the reason for the depression of the hyaluronidase activity by more arsenate is not completely clear, but a possible explanation may be due to the denaturation of the enzyme in the presence of higher concentration of arsenate. The above observations also suggest that the pentavalent arsenate might influence the progress of hyaluronidaserelated diseases by affecting the enzyme activity in body fluids, tissues and cells (the exact mechanism is yet not clear). 500 Apparent Conc. (g/L)

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400 300

0 g/L Na2HAsO4 (control) 5 g/L Na2HAsO4 50 g/L Na2HAsO4 500 g/L Na2HAsO4 5000 g/L Na2HAsO4

200 100 0

Healthy Group

Cancer Patients

Figure 7. The effect of sodium arsenate on the activity of hyaluronidase in human serum samples (expressed as the apparent concentration of the enzyme). Two mixed human serum samples from healthy group and cancer patients, respectively, were exposed to different concentrations of Na2HAsO4 16 Environment ACS Paragon Plus

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(0 - 5000 μg/L) for 50 min at 37 C. The results are the mean ± standard deviation of three separate measurements.

■ CONCLUSIONS In summary, a new nanoprobe for hyaluronidase detection has been developed by combining the nearinfrared excitation characteristic of upconversion luminescence nanoparticles with the strong fluorescence quenching ability of electron-rich PMPD. These actions make the resulting nanoprobe exhibit an extremely low background fluorescence signal, thereby achieving an ultrasensitive assay of hyaluronidase with a detection limit of 0.6 ng/mL. Moreover, the nanoprobe shows high selectivity, and has been used not only to determine directly hyaluronidase in human serum but also to monitor the activity change of the enzyme affected by toxic pollutants such as arsenate. It is found that the serum hyaluronidase level in colorectal cancer patients is about 3 times higher than that in healthy people, and a low dosage of arsenate such as 50 μg/L can raise the activity of hyaluronidase by about one third. The excellent performance of the nanoprobe makes it useful to detect hyaluronidase in relevant clinical samples.

■ ASSOCIATED CONTENT Supporting Information Additional information, as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org. ■ AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]; [email protected]. Phone: +86-10-62554673. ■ ACKNOWLEDGMENT



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We are grateful to the financial support from the NSF of China (Nos. 91432101, 21435007, 21275147 and 21321003), the Ministry of Science and Technology (Nos. 2015CB932001 and 2015CB856301), and the Chinese Academy of Sciences (XDB14030102).

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For TOC only

serum


An Upconversion Luminescence Nanoprobe for the Ultrasensitive

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