• <tr id="yyy80"></tr>
  • <sup id="yyy80"></sup>
  • <tfoot id="yyy80"><noscript id="yyy80"></noscript></tfoot>
  • 99热精品在线国产_美女午夜性视频免费_国产精品国产高清国产av_av欧美777_自拍偷自拍亚洲精品老妇_亚洲熟女精品中文字幕_www日本黄色视频网_国产精品野战在线观看 ?

    Enhanced oxidase-like activity of g-C3N4 nanosheets supported Pd nanosheets for ratiometric fluorescence detection of acetylcholinesterase activity and its inhibitor

    2022-06-18 03:00:18ChenghuiZhngPengjunNiBoWngWendongLiuYunyunJingChunxiChenJinSunYizhongLu
    Chinese Chemical Letters 2022年2期

    Chenghui Zhng, Pengjun Ni, Bo Wng, Wendong Liu, Yunyun Jing,Chunxi Chen,*, Jin Sun, Yizhong Lu,*

    a School of Materials Science and Engineering, University of Jinan, Ji’nan 250022, China

    b State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China

    ABSTRACT The undesirable enzymatic activity of nanozymes under near neutral pH condition and the traditional single signal output always restrict the analytical application of nanozyme-based biosensors.Herein,graphitic carbon nitride nanosheets supported palladium nanosheets composite (Pd/g-C3N4) with both oxidase-like activity and fluorescent property is synthesized.Notably, Pd/g-C3N4 exhibits enhanced oxidase-like activity compared to Pd NSs under pH 7.4.By combining Pd/g-C3N4 with o-phenylenediamine(OPD), a ratiometric fluorescence assay for acetylcholinesterase (AChE) activity detection is developed.Pd/g-C3N4 can catalyze oxidation of nonfluorescent OPD to fluorescent oxidized OPD (oxOPD, Em = 565 nm), which can quench fluorescence of g-C3N4 supporter (Em = 441 nm) through fluorescence resonance energy transfer (FRET).However, in presence of AChE, acetylthiocholine can be hydrolyzed into thiocholine, which will block the oxidase-like activity of Pd/g-C3N4 and then hamper the FRET process.This ratiometric fluorescence assay is also viable to screen AChE inhibitor.This work will guide design of ratiometric fluorescence assay based on nanozymes with improved enzymatic activity.

    Keywords:Oxidase-like activity Ratiometric fluorescence Acetylcholinesterase g-C3N4 nanosheets Pd nanosheets

    Acetylcholinesterase (AChE), an acetylcholine (ACh) hydrolase,plays a vital role in determining the contents of ACh that participates in learning and memory processes [1].Low ACh level will cause Alzheimer’s disease (AD) [2].As for AD, some inhibitors,such as tacrine, are used to clinically treat AD by restraining activity of AChE [3].So, it is significantly important to develop reliable and simple methods for AChE activity detection and inhibitors screening.To date, many efforts have been dedicated to colorimetry [4], fluorometry [5], electrochemistry [6], chemiluminescence[7].Among them, fluorometry holds distinct advantages for its simplicity and sensitivity [8,9].However, conventional fluorometric assays based on the single signal output are inevitably disturbed by various objective conditions.By comparison, ratiometric fluorescent method based on self-calibration of double signals has been proved to give more reliable results [10-15].

    Nanozymes are believed to have lower cost with convenient preparation process and higher stability than natural enzymes[16].To date, several kinds of nanomaterials, including noble metals [17], metal oxides [18] and so on [19-21] have been proved to exhibit enzymatic activities.Among them, noble metals-based nanozymes have attracted great interesting due to their good stability [17].To this end, ultrathin two-dimensional (2D) noble metal nanosheets (NSs) with high ratio of surface to volume have become a hot research topic in fields of sensing and catalysis, benefiting from their smaller diffusion barrier and more accessible active sites for substrates, compared to their 3D counterparts [22-24].Unfortunately, like other nanozymes, though exhibit good activity under acidic condition, the activity of noble metal-based nanozymes is significantly decreased under near neutral pH condition, which is inconvenient in practical applications.So, it is meaningful to develop efficient method to enhance enzymatic activity of noble metal, especially under physiological pH condition.

    It is well known that the catalytic performance of metal catalysts can be facilely improved by loading them onto appropriate supporters [25].Among various supporters, graphitic carbon nitride nanosheets (g-C3N4NSs) have attracted enormous attention in bio-sensing filed due to their easy preparation and good biocompatibility [10].Besides, g-C3N4NSs with intrinsicπelectronic structure and abundant nitrogen sites can enrich some reactants throughπ-πstacking and/or hydrogen bonding interactions, which will shorten the distance between reactants and catalytic sites supported on g-C3N4NSs [23].Moreover, the fluorescence property of g-C3N4NSs displays great potential to construct ratiometric fluorescence assay [26-28].Hence, developing composites by supporting the 2D noble metals NSs on g-C3N4NSs will be expectable to construct ratiometric fluorescence assay.

    Scheme 1.Illustration of the Pd/g-C3N4 based ratiometric fluorescence strategy for determination of AChE activity.

    Herein, a neoteric Pd/g-C3N4composite possessing both effi-cient oxidase-like activity and fluorescent property is synthesized.Notably, the oxidase-like activity of Pd NSs is successfully enhanced under neutral pH condition after loaded onto g-C3N4NSs.By combining Pd/g-C3N4witho-phenylenediamine (OPD), a ratiometric fluorescence assay is developed for AChE activity detection with acetylthiocholine (ATCh) as substrate (Scheme 1).Pd/g-C3N4as oxidase mimic can catalyze the transformation of nonfluorescent OPD to fluorescent oxidized OPD (oxOPD, 2,3-diaminophenazine).Then the fluorescence of g-C3N4NSs supporter is quenched by ox-OPD through fluorescence resonance energy transfer (FRET).However, in presence of AChE, ATCh is hydrolyzed into thiocholine(TCh), which poisons the active sites by forming Pd-S bond and blocks the Pd/g-C3N4catalyzed OPD oxidation.Thus, the fluorescence of oxOPD is weakened and fluorescence of Pd/g-C3N4is recovered, providing a ratio signal for AChE detection.Furthermore,tacrine is detected as a proof-of-concept to investigate the AChE inhibitor screening ability of this ratiometric fluorescence assay.

    Firstly, Pd NSs is synthesized by reducing palladium(II) acetylacetonate in presence of tungsten hexacarbonyl (W(CO)6), which can produce CO, serving as capping and reducing agent [29].Secondly, melamine is pyrolyzed to give bulk g-C3N4, which is refluxed by HNO3, followed by ultrasonic exfoliated to produce g-C3N4NSs [26].Lastly, Pd/g-C3N4is obtained by mixing aqueous solution of Pd NSs and g-C3N4NSs under stirring.Transmission electron microscopy (TEM) shows that hexagonal Pd NSs are obtained with a lateral diameter ofca.8.44 nm and an average thickness ofca.1.0 nm (Fig.1a and Fig.S1 in Supporting information).Fig.S2 (Supporting information) indicates that g-C3N4NSs are multiple layers with thickness of below 7.0 nm.And the obtained g-C3N4NSs have better dispersibility in water than bulk g-C3N4(Fig.S3 in Supporting information), which is beneficial for bioanalysis.As shown in Fig.1b, Pd NSs are successfully deposited on the surface of g-C3N4NSs without changing both of their morphologies.

    As for Pd NSs, X-ray diffraction (XRD) pattern shows two weak diffraction peaks centered at 40.42° and 68.34°, which are ascribed to (111) and (220) planes of Pd (JCPDS-46-1043), demonstrating the cubic structure of Pd NSs (Fig.1c).Similar to other Pd NSs synthesized in presence of W(CO)6[22], the obtained Pd NSs show lower crystallinity compared to those obtained by direct filling CO[30].For bulk g-C3N4, there are one strong peak centered at 27.70°and one weak peak centered at 13.34°, which are assigned to the typical (002) plane of inter-planar stacking in graphitic-like materials and (100) plane of in-plane structural packing motif among the sheets, respectively (Fig.1c) [31,32].Comparatively, the intensity of peak ascribed to (002) plane decreases accompanied with shifting from 27.70° to higher degree of 27.82°, whereas the weak peak corresponding to (100) plane disappears in g-C3N4NSs, suggesting that bulk g-C3N4is successfully exploited into nanoscale layers after acidic treatment and ultrasonic exfoliation (Fig.1c and Fig.S4 in Supporting information) [27].However, no obvious peak of Pd is found in Pd/g-C3N4due to the low loading content(4.03 wt%) and undesirable crystallinity of Pd NSs (Fig.1c)[22].In addition, Pd/g-C3N4exhibits similar diffraction patterns to g-C3N4NSs, suggesting that the loading of Pd NSs does not disturb the structure of g-C3N4NSs, which is also confirmed by the similar Fourier transform infrared characteristic peaks of g-C3N4NSs and Pd/g-C3N4(Fig.S5 in Supporting information).

    X-ray photoelectron spectroscopy (XPS) measurements are conducted to further disclose chemical composition of those materials.Compared to g-C3N4NSs that contain C, N, and O peaks, a new peak arising from Pd is found in Pd/g-C3N4(Fig.S6 in Supporting information).Taken TEM images of Pd/g-C3N4in mind, the existence of Pd peak further demonstrate that Pd NSs are successful supported onto g-C3N4NSs.The high-resolution XPS spectra of Pd 3d, C 1s, and N 1s are analyzed.For Pd in Pd/g-C3N4, the resolved peaks located at 335.7 and 340.9 eV are attributable to Pd03d5/2and Pd03d3/2, while the other pair of peaks centered at 337.6 and 343 eV originate from Pd2+3d5/2and Pd2+3d3/2, respectively (Fig.1d) [23].The C 1s peak can be divided into four peaks centered at 284.7, 286.2, 287.9, and 288.5 eV, ascribing to graphitic carbon (C-C), sp3-hybridized carbon with oxygen (C-O),sp2-hybridized carbon with nitrogen (N-C=N), and oxygen (C=O),respectively (Fig.1e) [33,34].The N 1s spectrum is deconvolved into four peaks, in which the binding energies at 398.5, 399.7, and 400.9 eV are assigned to the sp2-hybridized nitrogen (C-N=C) in triazine ring, the tertiary nitrogen HN–(C)2, and N–(C)3groups, respectively.While the relative weaker and broad peak centered at 404.1 eV belongs to the quaternary nitrogen, arising from the positive charge localization and/or delocalization ofπelectron in the heterocycles (Fig.1f) [33,34].No obvious shifting of binding energy of N 1s can be found in Pd/g-C3N4in comparison with g-C3N4NSs,excluding the possibility that Pd NSs attach on g-C3N4NSsviaPd-N chemical bond [23].Furthermore, zeta potentials of Pd NSs and g-C3N4NSs dispersed in ultrapure water are measured to be –13.9 and 15.2 mV (Fig.S7 in Supporting information), suggesting that there exists electrostatic interaction between them.These results demonstrate that Pd/g-C3N4composites are successfully obtained based on electrostatic interaction between Pd NSs and g-C3N4NSs.

    Fig.1.TEM images of (a) Pd NSs and (b) Pd/g-C3N4.(c) XRD patterns and high-resolution XPS spectra of (d) Pd 3d, (e) C 1s, (f) N 1s.

    The oxidase-like activities of Pd NSs, g-C3N4NSs and Pd/g-C3N4are examined by the catalytic oxidation of OPD under pH 7.4 that is similar to physiological environment.As depicted in Fig.2a,both Pd NSs and Pd/g-C3N4can trigger the oxidation of colorless OPD to orange with corresponding absorption peaks at 419 nm.In contrast, the control solution with g-C3N4NSs shows no color change, indicating that it is Pd NSs rather than g-C3N4NSs that exhibit oxidase-like activity toward catalytic oxidation of OPD to oxOPD.Despite its inactive when exist alone, g-C3N4NS effectually strengthen the catalytic activity of Pd NSs.As proof, the absorbance of Pd/g-C3N4+ OPD is higher (about 1.43 times) than that of Pd NSs + OPD.To probe into the enhancement mechanism, steady-state kinetic parameters including Michaelis constant(Km) and maximal reaction rate (Vmax) are calculated from typical Michaelis-Menten curves of Pd/g-C3N4and Pd NSs.As shown in Fig.2b, theKmof Pd/g-C3N4(0.086 mmol/L) is lower than that of Pd NSs (0.121 mmol/L), indicating Pd/g-C3N4show higher affinity for OPD.Meanwhile, theVmaxof Pd/g-C3N4is higher than that of Pd NSs, further revealing Pd/g-C3N4has enhanced catalytic efficiency compared to Pd NSs.This result can be explained by the fact that g-C3N4NSs can adsorb OPDvia π-πstacking and hydrogen bonding interaction, endowing higher concentration of OPD near to Pd NSs supported on g-C3N4NSs, resulting Pd/g-C3N4with enhanced catalytic activity toward OPD oxidation [23].

    Fig.2.UV-vis absorption spectra and the corresponding color change of OPD oxidation catalyzed by Pd NSs, Pd/g-C3N4 or g-C3N4.(b) Michaelis-Menten kinetics for the oxidation of OPD.

    To reveal the origin of catalytic activity of Pd NSs as oxidase mimics, the absorbance of Pd NSs + OPD system in O2/air/N2-saturated solutions are explored.Those results show that the catalytic activity of Pd NSs in different atmosphere is in the order of O2>air>N2(Fig.S8a in Supporting information), indicating that O2plays vital role for Pd NSs as oxidase mimics.During this reaction process, O2might be activated to generate several reactive oxygen species including superoxide anion (O2·–), singlet oxygen (1O2), hydroxyl radicals (·OH) [35].Initially, the reactive oxygen species are determined based on scavenger mechanism with superoxide dismutase (SOD), tryptophan, and isopropanol as scavengers for O2·–,1O2, and·OH, respectively [36,37].From Fig.S8b(Supporting information), isopropanol has little effect on Pd NSscatalyzed OPD oxidation, suggesting·OH is not involved in this reaction.Whereas, as the concentration of SOD and tryptophan increased, the absorbance is gradually decreased, indicating that O2·–and1O2are all produced.In addition, electron paramagnetic resonance (EPR) spectra also prove that no signal of·OH can be found(Fig.S8c in Supporting information).However, the four peaks with intensity of 1:1:1:1 corresponding to O2·–and the triplet peaks ascribing to1O2all present in EPR spectra (Figs.S8d and e in Supporting information).The above phenomena display that Pd NSs can activate the dissolved O2into O2·–and1O2, which is responsible for OPD oxidation.

    Pd/g-C3N4exhibits abroad excitation band ranging from 306 to 410 nm (Fig.S9 in Supporting information) and a fluorescence emission peak centered at 441 nm upon exciting at 390 nm (Fig.S10a in Supporting information).And a new peak centered at 565 nm derived from oxOPD is recorded when OPD is introduced,whereas the intensity of peak centered at 441 nm is significantly decreased.This phenomenon indicates that oxOPD not only emits fluorescence but also quenches fluorescence of g-C3N4supporter,which provides basis for developing ratiometric fluorescence assay.Then the quenching mechanism of oxOPD toward g-C3N4supporter is investigated.From Fig.S10b (Supporting information), an obvious overlap between the emission spectrum of Pd/g-C3N4and the absorption spectrum of oxOPD is found, suggesting the existence of FRET and/or inner-filter effect between Pd/g-C3N4and oxOPD [15].Furthermore, the fluorescence lifetime of Pd/g-C3N4decays from 4.0 ns to 1.8 ns (Fig.S10c in Supporting information), and the zeta potential of Pd/g-C3N4changes from –6.9 mV to –4.9 mV after incubation with OPD (Fig.S10d in Supporting information).All of those above results disclose an electrostatic interaction-based FRET process between positively charged oxOPD and negatively charged Pd/g-C3N4.

    It is known that thiols tend to coordinate with metal atoms[38], thus can tailor the enzymatic activity of Pd NSs.L-Cysteine(Cys) is selected as mercapto molecule to verify the interaction between Pd NSs and thiols.Compared to Cys, XPS curve of S 2p in Pd NSs + Cys shows a new peak with higher binding energy, proving that there exists strong coordination interaction between Pd NSs and Cys (Fig.S11 in Supporting information).Besides, the electrocatalytic oxygen reduction reaction of Pd NSs is immediately inhibited upon addition of Cys, further indicating the interaction between Pd NSs and Cys (Fig.S12 in Supporting information) [21].So, it is reasonably believed that the active sites of Pd NSs can be blocked by forming Pd-S bond upon addition of thiols, thus inhibiting the oxidase-like activity of Pd NSs.As further proved, the absorbance of Pd NSs + OPD system gradually decreases accompanying the increased contents of Cys.And the calculated variation of absorbance shows a good linearly correlated with Cys concentration ranging of 1-15 μmol/L (Fig.S13 in Supporting information).

    Inspired by such phenomenon, a ratiometric fluorescence strategy for AChE is proposed by taking ATCh as substrate (Scheme 1).From Fig.S14 (Supporting information), Pd/g-C3N4+ OPD system shows no significant change with individual ATCh or AChE.However, in presence of AChE, ATCh can be transformed into thiolcontaining TCh, which inhibits the oxidase-like activity of Pd/g-C3N4by forming Pd-S bond.As a result, the oxidation of OPD and the subsequent FRET process are impeded, accompanied with the weakened and recovered fluorescence of oxOPD (Em = 565 nm)and Pd/g-C3N4(Em = 441 nm), respectively.Therefore, the quantitative determination of AChE activity through change ofF441/F565can be realized.

    In order to realize satisfactory detection performance, the loading amount of Pd NSs; the needed time for hydrolysis of AChE into ATCh and the oxidation of OPD into oxOPD; the concentration of ATCh, OPD, and Pd/g-C3N4; the pH and temperature of Pd/g-C3N4catalyzed oxidation of OPD, are all explored in detail to give optimal sensing parameters (Figs.S15-S17 in Supporting information).Additionally, to ensure the accuracy and reproducibility of this dissolved oxygen-dependent sensing system, all experiments are performed under the same temperature, atmospheric pressure and salt concentration.Then, under the optimal sensing condition,the AChE activity detection is performed.As expected, the emission intensity at 441 nm of Pd/g-C3N4is gradually increased, while the emission intensity at 565 nm of oxOPD is gradually decreased upon the increased AChE activity ranging from 0.2 mU/mL to 6 mU/mL (Fig.3a).Meanwhile, the value ofF441/F565increases accompanied with increased AChE activity (Fig.3b).The linear dependence relationship (R2= 0.996) with AChE activity ranging from 0.2 mU/mL to 4.0 mU/mL is realized (Fig.3b).The detection limit of 0.06 mU/mL (3σ/S) is comparable to or even lower than previously reported AChE activity assays (Table S1 in Supporting information).

    Fig.3.(a) Fluorescence emission spectra of Pd/g-C3N4 + OPD system toward various AChE activities.(b) The plot of F441/F565 as a function of AChE activity (the inserted graph is the liner response to AChE activity).(c) Fluorescence emission spectra of Pd/g-C3N4 + OPD system with ATCh (30 μmol/L), AChE (6 mU/mL) and different concentrations of tacrine.(d) The IE plot of tacrine toward AChE as a function of tacrine concentrations.

    To assess anti-interference performance and selectivity of this assay, some biomolecules and common ions, includingαglucosidase, glucose oxidase, pepsin, lysozyme, pancreatin, bull serum albumin, trypsin, D-glucose, L-aspartic, glycine, L-alanine,L-leucine, Ca2+, K+, Mg2+and NO3–are selected as interferents.The results in Fig.S18 (Supporting information) indicate that those potential interferents show no significant influence on this ratiometric fluorescence assay either with or without AChE, suggesting good anti-interference performance and high selectivity toward AChE of this assay, which is due to the specificity of AChE toward its substrate.Furthermore, the feasibility of this ratiometric fluorescence assay is investigated by detecting AChE activity in human serums.The human serum samples were provided by the Hospital of University of Jinan (Ji’nan, China) and obtained from healthy donors.Besides, all experiments were approved by the Life-Science Ethics Review Committee of University of Jinan and obtained the informed consent from the blood donors of this project.The interferences from biothiols in serums are negligible (Fig.S19 in Supporting information), or can be eliminated byN-ethylmaleimide[15].Acceptable recoveries and relative standard deviation within the ranges of 99.0%–108.3% and 0.80%–3.40% are achieved, respectively (Table S2 in Supporting information), indicating this ratiometric fluorescence assay is suitable for detecting AChE activity in real samples.

    To further develop potential application to inhibitor screening of this ratiometric fluorescence assay, tacrine, an unequivocable AChE inhibitor used in AD therapy, is selected as a model to assess the inhibitory activity assay.Feasibility analysis demonstrates that tacrine puts no effect on the oxidase-like activity of Pd/g-C3N4(Fig.S20 in Supporting information).With the concentration increase of tacrine, the fluorescence emissions centered at 441 nm and 565 nm are gradually decreased and increased, respectively, indicating that the activity of AChE is successfully inhibited by tacrine(Fig.3c).By using the plot of inhibition efficiency (IE) as function of tacrine concentration (Fig.3d), IC50value, the needed concentration of tacrine for the inhibition of 50% enzyme activity, is obtained to be 3.8 nmol/L, which is similar to previous literatures[39,40].

    In summary, Pd/g-C3N4nanocomposite with enhanced oxidaselike activity compared to Pd NSs under pH 7.4 is successfully synthesized.By combining the obtained Pd/g-C3N4composite with OPD, a sensitive and selective ratiometric fluorescence platform is developed for AChE activity determination.This strategy is suitable for serology test benefitting from the reliable signal output.Moreover, this method is feasible to screen AChE inhibitor.Look forward to the future, such ratiometric fluorescent assay can be extended to monitoring other thiol-containing species and thiolgenerating or consuming biological process.This work demonstrates a novel guidance for designing nanozymes with improved activity in biosensing system, especially under physiological pH condition.

    Declaration of competing interest

    The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

    Acknowledgments

    This work was supported by the Natural Science Foundation of Shandong Province (Nos.ZR2020QB033 and ZR2019YQ10), the National Natural Science Foundation of China (Nos.21904048,21974132, 21902061 and 21902062) and the Young Taishan Scholars Program (No.tsqn201812080).

    Supplementary materials

    Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2021.08.017.

    麻豆精品久久久久久蜜桃| 欧美激情极品国产一区二区三区| 永久免费av网站大全| av福利片在线| 日本爱情动作片www.在线观看| 亚洲精品第二区| 日韩精品免费视频一区二区三区| 亚洲国产日韩一区二区| 国产日韩欧美在线精品| 97在线人人人人妻| 啦啦啦啦在线视频资源| 国产黄色视频一区二区在线观看| 建设人人有责人人尽责人人享有的| 热99国产精品久久久久久7| 我要看黄色一级片免费的| 久久久久久免费高清国产稀缺| 亚洲人成网站在线观看播放| 午夜日本视频在线| 欧美另类一区| 日韩中文字幕欧美一区二区 | 我的亚洲天堂| av国产久精品久网站免费入址| 一边摸一边做爽爽视频免费| 国产亚洲精品第一综合不卡| 美女中出高潮动态图| 日本欧美视频一区| 五月开心婷婷网| 午夜av观看不卡| 两个人看的免费小视频| 亚洲av综合色区一区| 久久毛片免费看一区二区三区| 纵有疾风起免费观看全集完整版| av网站在线播放免费| 亚洲,一卡二卡三卡| 在线 av 中文字幕| 亚洲av综合色区一区| 熟女av电影| freevideosex欧美| 亚洲综合色惰| 一边亲一边摸免费视频| 观看美女的网站| 日韩一区二区三区影片| 国产伦理片在线播放av一区| 如日韩欧美国产精品一区二区三区| 久久影院123| 久久精品夜色国产| 午夜福利,免费看| 久久免费观看电影| 久久久久精品久久久久真实原创| 啦啦啦在线观看免费高清www| 亚洲综合精品二区| 国产xxxxx性猛交| 免费观看av网站的网址| a级毛片在线看网站| 亚洲美女黄色视频免费看| 桃花免费在线播放| 女性被躁到高潮视频| 亚洲欧美色中文字幕在线| 中文字幕人妻丝袜制服| 中文精品一卡2卡3卡4更新| 午夜老司机福利剧场| 丁香六月天网| 精品国产一区二区三区久久久樱花| 这个男人来自地球电影免费观看 | 午夜久久久在线观看| 中文字幕精品免费在线观看视频| 香蕉精品网在线| 久热久热在线精品观看| 国产日韩欧美亚洲二区| 国产免费福利视频在线观看| 欧美国产精品一级二级三级| 一区二区三区激情视频| 精品少妇黑人巨大在线播放| 精品国产一区二区久久| 免费日韩欧美在线观看| 午夜免费观看性视频| 国产av精品麻豆| 91aial.com中文字幕在线观看| 久久久国产一区二区| 女性被躁到高潮视频| 久久久久久人妻| 国产精品香港三级国产av潘金莲 | 婷婷成人精品国产| 久久国产精品大桥未久av| 极品人妻少妇av视频| 日韩欧美精品免费久久| 秋霞伦理黄片| 日产精品乱码卡一卡2卡三| 在线观看美女被高潮喷水网站| 一边摸一边做爽爽视频免费| 青春草视频在线免费观看| 高清视频免费观看一区二区| av免费观看日本| 国产av国产精品国产| 高清欧美精品videossex| 亚洲精品成人av观看孕妇| 国产日韩欧美亚洲二区| 亚洲,欧美,日韩| 成人黄色视频免费在线看| 啦啦啦视频在线资源免费观看| 99久久人妻综合| 精品国产乱码久久久久久男人| 亚洲精品乱久久久久久| 一级毛片我不卡| 午夜精品国产一区二区电影| 久久久欧美国产精品| 国产黄频视频在线观看| 日日摸夜夜添夜夜爱| 免费日韩欧美在线观看| 女的被弄到高潮叫床怎么办| av有码第一页| 桃花免费在线播放| 青春草国产在线视频| 国产毛片在线视频| 久久久久精品久久久久真实原创| 欧美日韩av久久| 午夜福利乱码中文字幕| 免费观看av网站的网址| 国产极品粉嫩免费观看在线| 啦啦啦中文免费视频观看日本| 麻豆乱淫一区二区| 97在线人人人人妻| 另类亚洲欧美激情| 亚洲激情五月婷婷啪啪| 久久午夜综合久久蜜桃| 免费黄网站久久成人精品| 日韩中文字幕欧美一区二区 | 91久久精品国产一区二区三区| 国产精品熟女久久久久浪| 成年动漫av网址| 免费观看av网站的网址| av免费观看日本| 欧美另类一区| 在线观看www视频免费| 久久久久国产网址| 高清av免费在线| 日韩中文字幕欧美一区二区 | 熟女av电影| 91aial.com中文字幕在线观看| 欧美少妇被猛烈插入视频| 亚洲精品日本国产第一区| 涩涩av久久男人的天堂| 视频在线观看一区二区三区| 黄色毛片三级朝国网站| 女的被弄到高潮叫床怎么办| 丝袜喷水一区| 久久精品国产亚洲av涩爱| 免费人妻精品一区二区三区视频| 午夜91福利影院| 国产免费又黄又爽又色| 制服诱惑二区| 在线 av 中文字幕| 大香蕉久久网| 一级片'在线观看视频| 又黄又粗又硬又大视频| 新久久久久国产一级毛片| 看十八女毛片水多多多| 成年女人毛片免费观看观看9 | 久久久久久久久免费视频了| 涩涩av久久男人的天堂| 街头女战士在线观看网站| 国产免费视频播放在线视频| 国产淫语在线视频| 国产麻豆69| 在线 av 中文字幕| 国产成人午夜福利电影在线观看| 男女下面插进去视频免费观看| 七月丁香在线播放| 天美传媒精品一区二区| 日韩电影二区| 美女主播在线视频| 少妇人妻精品综合一区二区| 亚洲经典国产精华液单| 成人18禁高潮啪啪吃奶动态图| 中文字幕制服av| 免费女性裸体啪啪无遮挡网站| 国产精品亚洲av一区麻豆 | 久久久久网色| 亚洲在久久综合| 色吧在线观看| 中文字幕精品免费在线观看视频| 麻豆精品久久久久久蜜桃| 男女无遮挡免费网站观看| 久久久久久久久久久久大奶| 国产精品久久久久久av不卡| kizo精华| 少妇人妻 视频| 99久久精品国产国产毛片| 国产不卡av网站在线观看| 国产极品粉嫩免费观看在线| 午夜91福利影院| 熟妇人妻不卡中文字幕| 欧美激情极品国产一区二区三区| 777米奇影视久久| 人妻系列 视频| 久久久久精品人妻al黑| 中文欧美无线码| xxx大片免费视频| 久久午夜福利片| 考比视频在线观看| 美女国产视频在线观看| 久久久精品区二区三区| 国产成人精品无人区| 天美传媒精品一区二区| 日韩欧美一区视频在线观看| 亚洲国产av新网站| 国产一级毛片在线| 亚洲中文av在线| 最黄视频免费看| 亚洲精品久久午夜乱码| 一区二区日韩欧美中文字幕| 日韩一卡2卡3卡4卡2021年| 国产精品嫩草影院av在线观看| 亚洲少妇的诱惑av| 九草在线视频观看| 国产欧美日韩综合在线一区二区| 侵犯人妻中文字幕一二三四区| 午夜福利在线免费观看网站| 校园人妻丝袜中文字幕| 精品人妻在线不人妻| 欧美日韩一级在线毛片| 三级国产精品片| 老司机影院毛片| 欧美中文综合在线视频| 免费黄色在线免费观看| 久久亚洲国产成人精品v| 亚洲美女黄色视频免费看| 欧美 亚洲 国产 日韩一| 美女中出高潮动态图| 久久人妻熟女aⅴ| 国产成人精品婷婷| 午夜激情av网站| 岛国毛片在线播放| 国产日韩一区二区三区精品不卡| 久久这里有精品视频免费| 久久这里只有精品19| 最近的中文字幕免费完整| 90打野战视频偷拍视频| 91在线精品国自产拍蜜月| 日韩一区二区视频免费看| 只有这里有精品99| 美女福利国产在线| av在线app专区| 亚洲国产精品一区二区三区在线| 久久 成人 亚洲| 久久狼人影院| 母亲3免费完整高清在线观看 | 丝袜脚勾引网站| 久久毛片免费看一区二区三区| 国产成人91sexporn| 亚洲欧美精品综合一区二区三区 | 色哟哟·www| 建设人人有责人人尽责人人享有的| 日本-黄色视频高清免费观看| 青青草视频在线视频观看| 中文字幕亚洲精品专区| 日本免费在线观看一区| 亚洲成国产人片在线观看| 最近的中文字幕免费完整| www.av在线官网国产| 日韩一卡2卡3卡4卡2021年| 天天躁日日躁夜夜躁夜夜| 天天影视国产精品| 18在线观看网站| 各种免费的搞黄视频| 老汉色∧v一级毛片| 美女大奶头黄色视频| 好男人视频免费观看在线| 日韩一区二区视频免费看| 亚洲精品自拍成人| 国产一区二区 视频在线| 欧美成人精品欧美一级黄| 纵有疾风起免费观看全集完整版| 免费观看无遮挡的男女| 亚洲欧美色中文字幕在线| 午夜福利在线免费观看网站| 18禁裸乳无遮挡动漫免费视频| 国产精品久久久久久精品电影小说| 亚洲av电影在线进入| 人人澡人人妻人| 考比视频在线观看| 多毛熟女@视频| 欧美av亚洲av综合av国产av | 久久精品久久精品一区二区三区| 在线精品无人区一区二区三| 欧美另类一区| 亚洲图色成人| 久久久亚洲精品成人影院| 国产精品女同一区二区软件| 国产在线一区二区三区精| 国产精品成人在线| 天堂俺去俺来也www色官网| 男的添女的下面高潮视频| 久久精品国产综合久久久| 捣出白浆h1v1| xxxhd国产人妻xxx| 精品少妇黑人巨大在线播放| 搡老乐熟女国产| 日韩精品免费视频一区二区三区| av网站在线播放免费| 亚洲国产欧美日韩在线播放| 国产白丝娇喘喷水9色精品| 各种免费的搞黄视频| 亚洲欧美精品综合一区二区三区 | 午夜免费鲁丝| 91精品三级在线观看| 欧美精品av麻豆av| 久久久欧美国产精品| 熟女少妇亚洲综合色aaa.| 新久久久久国产一级毛片| 国产极品粉嫩免费观看在线| 天天躁夜夜躁狠狠躁躁| 狠狠婷婷综合久久久久久88av| 男女高潮啪啪啪动态图| 18禁动态无遮挡网站| a级毛片在线看网站| 老司机影院成人| 99精国产麻豆久久婷婷| 国语对白做爰xxxⅹ性视频网站| 看十八女毛片水多多多| 嫩草影院入口| 久久久国产精品麻豆| 在线观看三级黄色| 欧美日韩亚洲高清精品| 性高湖久久久久久久久免费观看| 亚洲精品国产一区二区精华液| 蜜桃国产av成人99| 日本色播在线视频| 久久久久久人人人人人| 哪个播放器可以免费观看大片| 亚洲精品成人av观看孕妇| 老鸭窝网址在线观看| 久久久久网色| 美女大奶头黄色视频| 五月天丁香电影| 成人亚洲精品一区在线观看| av一本久久久久| 国产在线一区二区三区精| 这个男人来自地球电影免费观看 | 久久国产精品男人的天堂亚洲| 只有这里有精品99| 黄色毛片三级朝国网站| 日韩一区二区视频免费看| 少妇人妻 视频| 久久久久久人人人人人| 人人妻人人澡人人看| 久久久久久久大尺度免费视频| 在线观看免费日韩欧美大片| 天堂俺去俺来也www色官网| 亚洲精品久久久久久婷婷小说| 9热在线视频观看99| 久久99一区二区三区| 国产精品熟女久久久久浪| 亚洲成人av在线免费| 色视频在线一区二区三区| 欧美av亚洲av综合av国产av | 久久久精品国产亚洲av高清涩受| 99热全是精品| 国产高清不卡午夜福利| 欧美国产精品一级二级三级| 亚洲国产成人一精品久久久| 久久久亚洲精品成人影院| av女优亚洲男人天堂| 中文字幕另类日韩欧美亚洲嫩草| 又黄又粗又硬又大视频| 午夜福利网站1000一区二区三区| 人妻少妇偷人精品九色| 2022亚洲国产成人精品| 一区二区三区激情视频| 欧美人与性动交α欧美精品济南到 | 欧美日韩国产mv在线观看视频| 热99久久久久精品小说推荐| 最近最新中文字幕免费大全7| 国产日韩欧美亚洲二区| 国产一区亚洲一区在线观看| www.自偷自拍.com| 18在线观看网站| av卡一久久| 黄片小视频在线播放| 亚洲国产欧美在线一区| 久久这里有精品视频免费| √禁漫天堂资源中文www| h视频一区二区三区| 免费av中文字幕在线| 欧美变态另类bdsm刘玥| 在线观看国产h片| 人妻一区二区av| 人人妻人人添人人爽欧美一区卜| 亚洲精品美女久久久久99蜜臀 | 国产av精品麻豆| 久久 成人 亚洲| 90打野战视频偷拍视频| 9191精品国产免费久久| 高清视频免费观看一区二区| 大片电影免费在线观看免费| 午夜精品国产一区二区电影| 亚洲一区二区三区欧美精品| 麻豆精品久久久久久蜜桃| 成人手机av| 亚洲av综合色区一区| 美女主播在线视频| av在线app专区| 中文字幕色久视频| 日韩一区二区视频免费看| www.熟女人妻精品国产| 精品少妇黑人巨大在线播放| 国产视频首页在线观看| 青春草视频在线免费观看| 少妇被粗大猛烈的视频| 成年人午夜在线观看视频| 久久久久久久久久久久大奶| 性色av一级| 国产精品无大码| 在线亚洲精品国产二区图片欧美| 美女国产视频在线观看| 美女中出高潮动态图| 丰满乱子伦码专区| 午夜影院在线不卡| 日本vs欧美在线观看视频| 国产成人aa在线观看| 久久人妻熟女aⅴ| 亚洲欧美一区二区三区久久| 亚洲第一区二区三区不卡| 国产 精品1| 日产精品乱码卡一卡2卡三| 老汉色∧v一级毛片| 亚洲欧洲国产日韩| 久久久久久久亚洲中文字幕| 国产片内射在线| 精品少妇黑人巨大在线播放| 午夜91福利影院| 香蕉丝袜av| 一区二区三区乱码不卡18| 亚洲经典国产精华液单| 亚洲情色 制服丝袜| 免费看不卡的av| 啦啦啦视频在线资源免费观看| 久久国产精品大桥未久av| 不卡av一区二区三区| 一级,二级,三级黄色视频| 亚洲三区欧美一区| 搡女人真爽免费视频火全软件| tube8黄色片| 亚洲欧洲日产国产| 久久精品国产亚洲av涩爱| 色播在线永久视频| 国产熟女欧美一区二区| 97精品久久久久久久久久精品| 香蕉国产在线看| 狠狠精品人妻久久久久久综合| 亚洲av.av天堂| 日本欧美视频一区| 亚洲国产精品一区三区| 亚洲av电影在线进入| 在线 av 中文字幕| 男女无遮挡免费网站观看| 欧美国产精品一级二级三级| 久久精品国产亚洲av涩爱| 亚洲婷婷狠狠爱综合网| 成人毛片a级毛片在线播放| 韩国av在线不卡| 一区在线观看完整版| 亚洲少妇的诱惑av| 欧美另类一区| www.av在线官网国产| 一本大道久久a久久精品| 人成视频在线观看免费观看| 一区二区三区激情视频| 精品一区二区免费观看| 精品国产一区二区三区四区第35| 在线观看美女被高潮喷水网站| 波野结衣二区三区在线| 黄色视频在线播放观看不卡| 欧美精品一区二区免费开放| 涩涩av久久男人的天堂| 亚洲精品国产色婷婷电影| 日韩,欧美,国产一区二区三区| 亚洲国产最新在线播放| 久久免费观看电影| 三上悠亚av全集在线观看| 中文字幕色久视频| 午夜福利,免费看| 好男人视频免费观看在线| 人成视频在线观看免费观看| 丝袜在线中文字幕| 一本色道久久久久久精品综合| 午夜免费男女啪啪视频观看| 久久韩国三级中文字幕| 欧美精品一区二区大全| 女人被躁到高潮嗷嗷叫费观| av又黄又爽大尺度在线免费看| 久久久欧美国产精品| 卡戴珊不雅视频在线播放| 日本-黄色视频高清免费观看| 秋霞在线观看毛片| 日韩不卡一区二区三区视频在线| 成年动漫av网址| 免费观看性生交大片5| 免费女性裸体啪啪无遮挡网站| 韩国av在线不卡| 国产麻豆69| 人妻 亚洲 视频| 黄色毛片三级朝国网站| 欧美精品一区二区大全| 中文字幕制服av| 欧美精品国产亚洲| 不卡视频在线观看欧美| 在现免费观看毛片| av免费观看日本| 午夜影院在线不卡| xxxhd国产人妻xxx| 国产成人精品久久二区二区91 | 亚洲精品一区蜜桃| 国产视频首页在线观看| 免费高清在线观看日韩| 亚洲五月色婷婷综合| 久久久精品国产亚洲av高清涩受| 80岁老熟妇乱子伦牲交| 嫩草影院入口| 国产黄色免费在线视频| 国产在线一区二区三区精| 欧美国产精品一级二级三级| 亚洲成色77777| 国产成人aa在线观看| 又大又黄又爽视频免费| 两个人免费观看高清视频| 黄色怎么调成土黄色| 国产成人精品久久久久久| 婷婷成人精品国产| 欧美精品国产亚洲| www.av在线官网国产| 亚洲国产精品成人久久小说| 免费在线观看黄色视频的| 久久婷婷青草| 男男h啪啪无遮挡| 日韩一本色道免费dvd| 亚洲美女搞黄在线观看| 激情五月婷婷亚洲| 久久精品人人爽人人爽视色| 在线免费观看不下载黄p国产| 视频区图区小说| 日韩欧美一区视频在线观看| 嫩草影院入口| 国产精品国产三级专区第一集| 亚洲精品一区蜜桃| 久久影院123| 欧美激情高清一区二区三区 | 久久鲁丝午夜福利片| 国产片特级美女逼逼视频| 久久精品国产自在天天线| h视频一区二区三区| 国产成人a∨麻豆精品| 老鸭窝网址在线观看| 丝瓜视频免费看黄片| 亚洲欧洲精品一区二区精品久久久 | 国产午夜精品一二区理论片| 午夜91福利影院| 国产精品成人在线| 美女午夜性视频免费| 亚洲,一卡二卡三卡| 精品人妻偷拍中文字幕| 伊人久久国产一区二区| 黑人欧美特级aaaaaa片| 在线看a的网站| 国产欧美日韩一区二区三区在线| 免费av中文字幕在线| 99精国产麻豆久久婷婷| 免费看av在线观看网站| 高清av免费在线| 人人妻人人澡人人爽人人夜夜| 亚洲五月色婷婷综合| 亚洲一级一片aⅴ在线观看| 久久精品国产亚洲av涩爱| 久久久久精品性色| 人人妻人人澡人人看| 1024香蕉在线观看| 亚洲四区av| 99热国产这里只有精品6| 国产午夜精品一二区理论片| 国产在视频线精品| 69精品国产乱码久久久| 日韩一区二区视频免费看| 久久精品国产综合久久久| 欧美精品一区二区免费开放| 日韩精品免费视频一区二区三区| 精品卡一卡二卡四卡免费| 国产黄频视频在线观看| 男人添女人高潮全过程视频| 亚洲成人手机| 91国产中文字幕| 欧美精品高潮呻吟av久久| 国产1区2区3区精品| 欧美亚洲 丝袜 人妻 在线| 免费少妇av软件| av卡一久久| 中文欧美无线码| 久久婷婷青草| 美国免费a级毛片| 欧美日韩精品成人综合77777| 日韩一区二区视频免费看| 精品久久久精品久久久| 五月伊人婷婷丁香| 国产又色又爽无遮挡免| 日本欧美国产在线视频| 亚洲欧洲国产日韩| 国产伦理片在线播放av一区| 国产片特级美女逼逼视频| 性少妇av在线| 国产一区二区三区综合在线观看| 侵犯人妻中文字幕一二三四区| 中文字幕色久视频| 亚洲经典国产精华液单| 亚洲av综合色区一区| 男女高潮啪啪啪动态图| 午夜福利在线观看免费完整高清在|