• <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 ?

    AND molecular logic gates based on host-guest complexation operational in live cells

    2022-06-18 03:00:36SiyngJingWeipengMoDkeMoZhnTingLi
    Chinese Chemical Letters 2022年2期

    Siyng Jing, Weipeng Mo, Dke Mo, Zhn-Ting Li,*, D M,b,*

    a Department of Chemistry, Fudan University, Shanghai, Shanghai 200433, China

    b College of Chemistry, and Green Catalysis Center, Zhengzhou University, Zhengzhou 450001, China

    ABSTRACT We report supramolecular AND logic gates based on host-guest complexation between acid-labile acyclic cucurbit[n]uril (CB[n]) molecular container and NaClO-responsive dye.Supramolecular AND logic gate is turned on due to acid-triggered degradation of molecular container and the release of the dye, followed by NaClO-induced fluorescence “switch on” effect of the dye.The reason for AND molecular logic gate is discovered to be the combination of oxidation inhibition and fluorescence “switch off” effect.Supramolecular AND logic gate is confirmed to be operational in live MCF-7 and HeLa cancer cells.

    Keywords:Host-guest chemistry Logic gate Stimuli-responsive systems Cucurbituril Bioimaging

    Inspired by information technology, molecular logic gates are important molecular devices to generate outputs with one or multiple chemical or physical inputs [1,2].Molecular logic gates could be constructed by covalent conjugation or supramolecular complexation [3-6].Molecular logic gates have been used for information processing, drug delivery, and chemical or biological sensing[7–10].

    Supramolecular complexation is a strategy to construct complex systems with simple building blocks [11–20].To this date,it is challenging to develop supramolecular logic gates, which are operational in the complex biological environment.Researchers are interested in special physiological and pathological conditions, which are crucial for diagnostic and therapeutic uses.Mildly acidic condition and reactive oxygen species (ROSs) are two important biomarker signals.ROS is existing in tumor and inflammatory tissues [21,22].Mildly acidic condition is often found in tumor extracellular environment or endosomal compartments [23].Molecular or nanoscale structures responsive to acidic pH, ROS or other biomarker signals are valuable for disease diagnostics or the targeted delivery of pharmaceutical drugs [24,25].Nevertheless,there are only limited examples of biological system-operational supramolecular logic gates [26].

    To construct sophisticated supramolecular logic gates, the major challenge is to introduce two or multiple types of stimuliresponsiveness.We recently develop a type of stimuli-reactive host molecules, which could be used to construct stimuli-responsive supramolecular assemblies [27–30].By introducing one type of stimuli-responsiveness to the host molecule and the other type of stimuli-responsiveness to the guest molecule, the resulting hostguest complexation-based molecular logic gate may be responsive to two input types.This supramolecular molecular logic gate will be composed of simple-to-synthesize building blocks.Hostguest interaction has been proved to be a useful method to build supramolecular assembly for biological applications [31,32].Therefore, this new strategy combines simplicity in structure and complexity in function.

    Here, we report a new strategy to construct host-guest complexation-based AND molecular logic gates.As a proof-ofconcept system, this AND molecular logic gate is composed of an acid-labile acyclic cucurbit[n]uril (CB[n]) host molecule and a ROSresponsive guest dye.By introducing pH-responsiveness to the host molecule and ROS-responsiveness to the guest dye, supramolecular AND logic gate is conveniently assembled by modular design.In the presence of both chemical inputs, fluorescence output is turned on.Mechanistic study is carried out for AND logic gate.This supramolecular complex demonstrates operational AND logic gate in live human cancer cells, which may be used for sophisticated biological sensing.

    The design of supramolecular logic gate involves host-guest complexation between hosts 1, 2 and guest 4.As shown in Fig.1a, hosts 1 and 2 are acid-labile acyclic CB[n] molecular containers, previously developed by us [28].These host molecules undergo acid-induced conversion from anionic hosts 1 and 2 to cationic host 3.The degradation rate is tunable depending on different molecular structures: citraconic amide-based host 1 degrades faster than maleic amide-based host 2 under the same condition.For both host molecules, a higher degradation rate is achieved at a lower pH.Guest 4 is developed by the Yi group,which possesses stimuli-responsiveness towards ROS (NaClO): the presence of NaClO is capable of converting guest 4 to fluorescent dye 5 [33].

    Fig.1.(a) Acid-induced conversion from anionic hosts 1 and 2 to cationic host 3,and NaClO-induced conversion from guest 4 to dye 5.(b) Schematic representation for AND logic gate, and the fluorescence “switch on” in the presence of both acidic condition and NaClO (1, 1).

    We expect supramolecular complexes based on hosts 1-2 and guest 4 demonstrate AND logic gate.As shown in Fig.1b, there are four types of input patterns: (1) in the absence of both low pH and NaClO (0, 0); (2) in the presence of NaClO (0, 1); (3) in the presence of low pH (1, 0); (4) in the presence of both low pH and NaClO (1, 1).When NaClO is absent (0, 0), guest 4 is nonfluorescent under our experimental condition (λex 620 nm,λem 690 nm), and no output is observed.Similarly, when only low pH is present (1, 0), guest 4 is unable to convert to be fluorescent dye 5, and the system is non-fluorescent.When only NaClO is present(0, 1), guest 4 is encapsulated by the host molecule, which inhibits NaClO-induced degradation and results in a slowed formation of fluorescent dye 5.Even if dye 5 is formed under this condition,this fluorescent dye is encapsulated by anionic host, and the fluorescence of the dye is switched off.Therefore, the sole logic operation to generate fluorescence output is (1, 1): acid-induced degradation of hosts 1 and 2 leads to release of guest 4, which allows it to convert to be fluorescent dye 5 in the non-complexed form.

    First, we investigated host-guest interaction between hosts 1-3 and guest/dye 4 and 5.While guest 4 is poorly soluble in water,dye 5 has decent solubility in water for1H NMR spectroscopy.As shown in Figs.S1 and S2 (Supporting information), upfield shifted proton resonances indicated supramolecular encapsulation of dye 5 by the cavity of hosts 1 and 2.In contrast, when mixed with host 3, proton resonances of dye 5 shifted downfield, indicating the supramolecular complexation was out of the cavity (Figs.S3 and S4 in Supporting information).The poor aqueous solubility of guest 4 renders it impossible for1H NMR study.As shown in Figs.S7 andS8 (Supporting information), the addition of hosts 1 and 2 could significantly enhance the weak fluorescence of guest 4, which indicated supramolecular complexation.Direct UV-vis or fluorescence titration and indicator displacement assay were used to determine the value of binding constantKa(Figs.S7-S16 in Supporting information).As summarized in Table 1, anionic hosts 1 and 2 could encapsulate guest 4 and dye 5 with modest affinity.The value ofKafor the complex of anionic host 3 and guest 4/dye 5 was smaller compared to that for hosts 1 and 2.Mildly acidic condition of pH 6.0 was also used to determine the binding affinity for hosts 1-3.Anionic hosts 1 and 2 could bind guest 4 and dye 5 at pH 6.0 with similar binding affinity as that at pH 7.4.The binding affinity between cationic host 3 and guest 4/dye 5 was also significantly weaker compared to that for hosts 1 and 2 at pH 6.0.Therefore,anionic hosts 1 and 2 could encapsulate guest 4 and dye 5 under neutral or acidic condition.After the acid-induced conversion from hosts 1 and 2 to cationic host 3, binding affinity was significantly reduced to release encapsulated guest or dye.

    Table 1 Binding constant value (Ka, L/mol) determined for supramolecular complexation between hosts 1-3 and guest/dye 4-5.

    Next, AND molecular logic gate behavior was studied by fluorescence spectroscopy.Excess quantity of host molecule was used to ensure a high ratio of host-guest complexation.Guest 4 was incubated with host 1 (20 equiv.) at 37 °C under four different conditions: (1) phosphate buffered saline (PBS, pH 7.4) (0, 0); (2)NaClO (20 μmol/L) at pH 7.4 (0, 1); (3) mildly acidic buffer (pH 6.0) (1, 0); (4) NaClO (20 μmol/L) at pH 6.0 (1, 1).All four solutions were colorless in the beginning.After 30 min, the solution in the presence of both chemical inputs (1, 1) turned blue (Fig.2a).UV-vis absorbance was consistent with naked-eye observations,which showed the significantly enhanced absorbance at 670 nm in the presence of both chemical inputs (1, 1) (Fig.2b).Fluorescence spectroscopy showed that the solution in the presence of both chemical inputs (1, 1) had the significantly higher fluorescence intensity compared to the other three input patterns with an 8.2-fold difference.As summarized in Figs.2c and d, the complex of host 1 and guest 4 demonstrates AND logic gate with high fluorescence output in the presence of both chemical inputs (1, 1).

    Fig.2.(a) Image of an aqueous solution for the 1·4 complex under different conditions ([1] = 200 μmol/L, [4] = 10 μmol/L, [NaClO] = 20 μmol/L, pH 6.0 or 7.4,under 37 °C, after 0.5 h).(b) UV-vis absorbance for the 1·4 complex under four different conditions.(c) Fluorescence intensity for the 1·4 complex under four different conditions (λex 620 nm, λem 695 nm).The fluorescence intensity of (1, 0) is 9.2 times that of (0, 1).(d) Logic gate matrix of four input combinations, and their corresponding fluorescence outputs.

    Similarly, host 2 also demonstrated AND logic gate towards low pH and NaClO (Fig.S22 in Supporting information).Since host 2 was more stable under acidic condition compared to host 1, a lower pH of 4.6 was used.As a supramolecular system, host/guest ratio could be varied.As shown in Fig.S22, a lower host/guest ratio resulted in a less obvious AND logic gate effect due to the partial complexation between the host and the guest/dye.With a host/guest ratio of 5 or higher, a decent AND logic gate effect was observed.A similar ratio-dependent logic gate effect was observed for host 1 as well (Fig.S21 in Supporting information).

    We are interested in the mechanism of these supramolecular logic gates.Stimuli-responsive degradation of host and guest was studied.As shown in Fig.3a, NaClO triggered the degradation of guest 4, which led to an enhancement in fluorescence intensity.Acidic pH slightly accelerated degradation rate for guest 4.By contrast, when NaClO was absent, negligible fluorescence intensity enhancement was observed either at pH 7.4 or pH 6.0.The encapsulation of dye 5 by hosts 1 and 2 was confirmed to significantly reduce its fluorescence intensity (Fig.S5 in Supporting information).Therefore, we used dye 5 as an indicator for the host degradation and the release of the encapsulated dye.As shown in Fig.3b, under neutral condition, minimal dye release was observed.By contrast, acidic condition significantly enhanced the dye release.As reported previously, host 1 was less stable at acidic pH compared to host 2.Consequently, supramolecular AND logic gates are tunable by choosing hosts with different acid-responsiveness.

    Fig.3.(a) Degradation curve for guest 4 under four different conditions([4] = 10 μmol/L, [NaClO] = 20 μmol/L).(b) Degradation curve for hosts 1-2 with dye 5 under different pH conditions ([1] = 200 μmol/L, [2] = 200 μmol/L,[5] = 10 μmol/L).(c) Fluorescence intensity change of guest 4 and NaClO with/without host 2 ([2] = 200 μmol/L, [4] = 10 μmol/L, [NaClO] = 20 μmol/L,λex = 620 nm, λem = 695 nm).(d) UV-vis absorbance change of guest 4 and NaClO with/without host 2 at 635 nm ([2] = 200 μmol/L, [4] = 10 μmol/L,[NaClO] = 20 μmol/L).

    The encapsulation of guest 4 by host may inhibit its NaClOinduced degradation.A solution of guest 4 was incubated with the relatively stable host 2 in a neutral solution of NaClO (0,1), and the fluorescence intensity was monitored.As shown in Fig.3c, the fluorescence intensity grew at a much slower pace compared to that for guest 4 alone.Since supramolecular encapsulation resulted in fluorescence quenching, UV-vis absorbance was monitored.While guest 5 showed no absorbance at visible wavelength, dye 4 has strong visible absorbance.An isosbestic point was found at 635 nm based on UV-vis titration of host 2 and dye 5(Fig.S6 in Supporting information).Therefore, we monitor UV-vis absorbance at 635 nm to avoid the impact of supramolecular encapsulation toward absorbance.As shown in Fig.3d, degradation of guest 4 was inhibited in the presence of host 2.Based on the above mechanistic study, we confirmed that there are two reasons for the AND logic gate: (1) supramolecular encapsulation causes the inhibition of NaClO-induced guest degradation; (2) dye fluorescence is“switched off” by supramolecular complexation.

    Lastly, human cancer MCF-7 and HeLa cells were used to validate the operation of AND molecular logic gatein vitro.HeLa cells were incubated with guest 4 and excess host 1 (5 equiv.or 20 equiv.) for 2 h at pH 7.4 or 6.5 to mimic neutral or acidic extracellular environment.The host/guest ratio was varied to investigate its impact towards logic gate effect.Subsequently, cells were washed, and further incubated for another 0.5 h in the presence or absence of NaClO to mimic pathological (ROS) or physiological condition.Cells were then thoroughly washed and imaged by fluorescence microscopy.As shown in Figs.4 and 5, for both cell lines and both host/guest ratios, fluorescence was only turned on when both acidic pH (6.5) and NaClO were present.ImageJ was used to quantify fluorescence intensity, which showed fluorescence intensity in the presence of both biomarker signals (1, 1) was at least 38.1-fold higher compared to that for any other condition when the host/guest ratio is 20:1.For the host/guest ratio of 5:1, fluorescence intensity of (1, 1) was also at least 9.2-fold higher than the other groups.The results showed that logic gate effect was robust to certain extent of host/guest ratio.Therefore, we confirm that AND logic gates are operational in both types of cancer cells.

    Fig.4.Fluorescence microscopic images of MCF-7 cells treated with host 1 and guest 4 under four different logic gate chemical input patterns.(0, 0): pH 7.4; (0,1): NaClO at pH 7.4; (1, 0): pH 6.5; (1, 1): NaClO at pH 6.5 when host:guest molar ratio is 5:1 (a) or 20:1 (b).[4] = 10 μmol/L, [NaClO] = 50 μmol/L.(c) Fluorescence intensity statistics based on ImageJ.

    Fig.5.Fluorescence microscopic images of HeLa cells treated with host 1 and guest 4 under four different logic gate chemical input patterns.(0, 0): pH 7.4; (0, 1): Na-ClO at pH 7.4; (1, 0): pH 6.5; (1, 1): NaClO at pH 6.5 when host:guest molar ratio is 5:1 (a) or 20:1 (b).[4] = 10 μmol/L, [NaClO] = 50 μmol/L.(c) Fluorescence intensity statistics based on ImageJ.

    In summary, we develop supramolecular AND logic gates based on acid-labile host molecules and ROS-responsive guest, which is operational in live cells.Oxidation inhibition and encapsulationinduced fluorescence quenching were discovered to be the reasons for AND logic gate.Convenient-assembled and highly tunable supramolecular design render these AND logic gates to be further developed for diagnostic and therapeutic applications.

    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

    The authors are grateful to National Natural Science Foundation of China (Nos.21921003 and 21672042) for financial support.Guest 4 was kindly provided by Prof.Tao Yi and Dr.Peng Wei.

    Supplementary materials

    Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2021.08.021.

    久久青草综合色| 国产免费又黄又爽又色| 免费观看性生交大片5| 日日撸夜夜添| 久久女婷五月综合色啪小说| 欧美日韩av久久| 亚洲精品av麻豆狂野| 啦啦啦在线观看免费高清www| 久久久久久久久久久久大奶| 亚洲国产毛片av蜜桃av| 亚洲欧美中文字幕日韩二区| 2018国产大陆天天弄谢| 午夜福利在线观看免费完整高清在| 亚洲伊人色综图| 亚洲国产最新在线播放| 亚洲国产欧美在线一区| 香蕉丝袜av| 天天躁狠狠躁夜夜躁狠狠躁| 狂野欧美激情性bbbbbb| 久久综合国产亚洲精品| 26uuu在线亚洲综合色| 午夜福利在线观看免费完整高清在| 久久亚洲国产成人精品v| 妹子高潮喷水视频| 日韩欧美精品免费久久| 午夜久久久在线观看| 一区在线观看完整版| 日本免费在线观看一区| 人妻一区二区av| 18禁动态无遮挡网站| 成年人午夜在线观看视频| 天美传媒精品一区二区| 亚洲精品乱久久久久久| 欧美人与性动交α欧美精品济南到 | 成人国语在线视频| 国产av国产精品国产| 日韩一区二区三区影片| 久久久a久久爽久久v久久| 在线观看人妻少妇| 亚洲国产av新网站| 国产在线视频一区二区| 久久精品aⅴ一区二区三区四区 | 伊人久久大香线蕉亚洲五| 亚洲av综合色区一区| 国产一级毛片在线| 日本av手机在线免费观看| av免费观看日本| 欧美精品人与动牲交sv欧美| 性高湖久久久久久久久免费观看| 久久国产精品男人的天堂亚洲| 麻豆精品久久久久久蜜桃| 人妻人人澡人人爽人人| 97在线视频观看| 久久这里只有精品19| 老司机影院毛片| 亚洲视频免费观看视频| 午夜影院在线不卡| 晚上一个人看的免费电影| 下体分泌物呈黄色| 观看美女的网站| 欧美另类亚洲清纯唯美| 免费在线观看黄色视频的| 极品教师在线免费播放| 亚洲第一av免费看| 欧美日韩视频精品一区| 免费少妇av软件| 天堂俺去俺来也www色官网| 久久国产亚洲av麻豆专区| 老司机福利观看| 黄色片一级片一级黄色片| 亚洲国产精品一区二区三区在线| 99国产精品免费福利视频| 国产av在哪里看| 亚洲人成网站在线播放欧美日韩| 亚洲中文日韩欧美视频| 99久久综合精品五月天人人| 久久九九热精品免费| 99精国产麻豆久久婷婷| 免费看a级黄色片| 九色亚洲精品在线播放| 一级片'在线观看视频| 免费搜索国产男女视频| 欧美老熟妇乱子伦牲交| 欧美+亚洲+日韩+国产| 曰老女人黄片| 亚洲欧美日韩高清在线视频| 精品日产1卡2卡| e午夜精品久久久久久久| av在线天堂中文字幕 | 18禁国产床啪视频网站| 久久欧美精品欧美久久欧美| 国产精品免费视频内射| 成人精品一区二区免费| 欧洲精品卡2卡3卡4卡5卡区| 免费不卡黄色视频| 香蕉国产在线看| 久久亚洲真实| 一夜夜www| 国内毛片毛片毛片毛片毛片| 亚洲精品粉嫩美女一区| 国产高清激情床上av| 天天影视国产精品| 亚洲精品一区av在线观看| 悠悠久久av| 久久精品亚洲熟妇少妇任你| 在线国产一区二区在线| 亚洲精品国产色婷婷电影| 操美女的视频在线观看| 精品久久久久久电影网| 免费日韩欧美在线观看| 国产在线精品亚洲第一网站| 一级片'在线观看视频| av片东京热男人的天堂| 99精品久久久久人妻精品| 一区二区三区精品91| 久久中文字幕一级| 国产三级黄色录像| 日本精品一区二区三区蜜桃| av在线播放免费不卡| 国产精品影院久久| 亚洲国产欧美日韩在线播放| 美女国产高潮福利片在线看| av国产精品久久久久影院| 大码成人一级视频| 欧美日韩一级在线毛片| 又大又爽又粗| 99国产综合亚洲精品| 亚洲,欧美精品.| 一区二区日韩欧美中文字幕| 色在线成人网| 神马国产精品三级电影在线观看 | 亚洲成a人片在线一区二区| 国产伦人伦偷精品视频| 久久久水蜜桃国产精品网| 午夜福利欧美成人| 女人高潮潮喷娇喘18禁视频| 变态另类成人亚洲欧美熟女 | 精品国产一区二区久久| 久久精品成人免费网站| 男女之事视频高清在线观看| 亚洲久久久国产精品| 18禁国产床啪视频网站| 无遮挡黄片免费观看| 午夜免费成人在线视频| 亚洲精品一卡2卡三卡4卡5卡| 精品国产国语对白av| 脱女人内裤的视频| 国产高清国产精品国产三级| 国产高清国产精品国产三级| 一级片'在线观看视频| 亚洲avbb在线观看| 女人精品久久久久毛片| 69精品国产乱码久久久| 视频在线观看一区二区三区| 香蕉丝袜av| 中文字幕人妻丝袜制服| 久久精品国产亚洲av高清一级| 欧美日韩亚洲综合一区二区三区_| 91成年电影在线观看| www.999成人在线观看| 黄色女人牲交| 午夜免费成人在线视频| 亚洲成人久久性| 一边摸一边做爽爽视频免费| 又黄又爽又免费观看的视频| 精品国内亚洲2022精品成人| av天堂久久9| 国产高清videossex| 天堂影院成人在线观看| 人人妻人人添人人爽欧美一区卜| av国产精品久久久久影院| 91在线观看av| 欧美日韩精品网址| 亚洲中文字幕日韩| 老熟妇仑乱视频hdxx| 亚洲自拍偷在线| 男人舔女人下体高潮全视频| 亚洲精品一二三| 精品久久久久久久毛片微露脸| 中文字幕人妻丝袜一区二区| 视频区欧美日本亚洲| а√天堂www在线а√下载| 十八禁人妻一区二区| 国产国语露脸激情在线看| 亚洲一区高清亚洲精品| 搡老乐熟女国产| 国产熟女xx| 一边摸一边做爽爽视频免费| 日韩免费av在线播放| 亚洲国产精品sss在线观看 | 亚洲精品中文字幕一二三四区| 国产精品成人在线| 桃色一区二区三区在线观看| 日本三级黄在线观看| 日日夜夜操网爽| 好看av亚洲va欧美ⅴa在| 亚洲欧美日韩另类电影网站| 纯流量卡能插随身wifi吗| 最好的美女福利视频网| 日本 av在线| 在线天堂中文资源库| av在线天堂中文字幕 | 午夜老司机福利片| 亚洲av美国av| 一级片免费观看大全| 大码成人一级视频| 国产成+人综合+亚洲专区| 亚洲国产欧美日韩在线播放| 老熟妇仑乱视频hdxx| av中文乱码字幕在线| 99re在线观看精品视频| 欧美黑人欧美精品刺激| 亚洲精品国产精品久久久不卡| 免费高清在线观看日韩| 日韩欧美免费精品| 免费看十八禁软件| 免费在线观看黄色视频的| 狂野欧美激情性xxxx| 正在播放国产对白刺激| 免费观看精品视频网站| 国产熟女xx| 欧美日韩精品网址| 老汉色∧v一级毛片| 久久久国产欧美日韩av| 日韩 欧美 亚洲 中文字幕| 久久精品国产清高在天天线| 国产一区二区三区在线臀色熟女 | 欧美日韩瑟瑟在线播放| 97碰自拍视频| 日韩成人在线观看一区二区三区| 男女下面进入的视频免费午夜 | 日本a在线网址| 免费人成视频x8x8入口观看| 成人手机av| 女人精品久久久久毛片| 国产精品 欧美亚洲| 国产亚洲精品一区二区www| 女同久久另类99精品国产91| 黑人巨大精品欧美一区二区蜜桃| 久热这里只有精品99| www.999成人在线观看| 香蕉丝袜av| 桃红色精品国产亚洲av| 别揉我奶头~嗯~啊~动态视频| 欧美成狂野欧美在线观看| 夜夜夜夜夜久久久久| 久久影院123| 99久久99久久久精品蜜桃| 麻豆成人av在线观看| 亚洲精品一区av在线观看| 黄片小视频在线播放| 欧美日韩亚洲综合一区二区三区_| 一个人观看的视频www高清免费观看 | 国产91精品成人一区二区三区| 亚洲精品美女久久久久99蜜臀| 精品久久久久久久毛片微露脸| 久久久久久久久久久久大奶| 日本一区二区免费在线视频| 国产av一区在线观看免费| 一级毛片高清免费大全| 亚洲自偷自拍图片 自拍| 久久婷婷成人综合色麻豆| 啦啦啦免费观看视频1| 国产精品二区激情视频| 国产精品自产拍在线观看55亚洲| 国产精品一区二区三区四区久久 | 亚洲国产精品sss在线观看 | 精品一区二区三区av网在线观看| 成人三级做爰电影| 黄片播放在线免费| 亚洲自偷自拍图片 自拍| 老汉色∧v一级毛片| 久久亚洲精品不卡| 久久香蕉国产精品| 精品国产乱码久久久久久男人| 大型av网站在线播放| 国产精品成人在线| 久久草成人影院| 人人澡人人妻人| 日韩精品中文字幕看吧| 老司机靠b影院| 久久久久亚洲av毛片大全| 日韩欧美一区视频在线观看| 久久久国产精品麻豆| 丝袜人妻中文字幕| 国产成人av教育| 成人18禁高潮啪啪吃奶动态图| 国产精品久久久久成人av| 麻豆久久精品国产亚洲av | 性色av乱码一区二区三区2| 波多野结衣一区麻豆| 夫妻午夜视频| 又黄又粗又硬又大视频| 丰满饥渴人妻一区二区三| 国产在线观看jvid| 国产欧美日韩精品亚洲av| 国产成人精品久久二区二区91| 午夜精品久久久久久毛片777| 多毛熟女@视频| 在线观看舔阴道视频| 妹子高潮喷水视频| 宅男免费午夜| 国产精品 国内视频| av视频免费观看在线观看| 欧美激情极品国产一区二区三区| 久久精品91无色码中文字幕| 亚洲avbb在线观看| 色婷婷久久久亚洲欧美| 亚洲成人免费av在线播放| 午夜福利免费观看在线| 久久香蕉激情| 久久午夜综合久久蜜桃| 久久国产精品影院| 久久精品aⅴ一区二区三区四区| 日本a在线网址| 久久久国产成人精品二区 | 男人舔女人下体高潮全视频| 国产精品av久久久久免费| 中文字幕人妻熟女乱码| 精品国内亚洲2022精品成人| 久久国产精品影院| 黄色女人牲交| 国产精品久久视频播放| svipshipincom国产片| 欧美日韩精品网址| 男人舔女人下体高潮全视频| 黄频高清免费视频| 亚洲成国产人片在线观看| 午夜91福利影院| 亚洲 国产 在线| 久久久久久久午夜电影 | 国产成人欧美| 嫩草影院精品99| 亚洲人成电影免费在线| 亚洲 欧美一区二区三区| 中文亚洲av片在线观看爽| 日本 av在线| 啪啪无遮挡十八禁网站| 欧美中文综合在线视频| 91九色精品人成在线观看| 999精品在线视频| 亚洲男人天堂网一区| 午夜福利在线观看吧| 亚洲国产中文字幕在线视频| 色婷婷久久久亚洲欧美| 久久人人97超碰香蕉20202| 国产精品二区激情视频| 久久久久九九精品影院| 久久久久国产一级毛片高清牌| 日韩免费高清中文字幕av| 999久久久国产精品视频| 在线免费观看的www视频| 午夜免费激情av| 国产高清国产精品国产三级| 午夜两性在线视频| 一区二区日韩欧美中文字幕| 电影成人av| 法律面前人人平等表现在哪些方面| 亚洲专区字幕在线| 女人高潮潮喷娇喘18禁视频| 亚洲人成77777在线视频| 欧美日韩福利视频一区二区| 黄色怎么调成土黄色| 久久香蕉精品热| 久久精品aⅴ一区二区三区四区| 久久人妻熟女aⅴ| 自拍欧美九色日韩亚洲蝌蚪91| 欧美激情久久久久久爽电影 | 国产精品99久久99久久久不卡| 精品一品国产午夜福利视频| 国产精品 国内视频| 亚洲avbb在线观看| 水蜜桃什么品种好| 丰满的人妻完整版| 91国产中文字幕| 国产99久久九九免费精品| 每晚都被弄得嗷嗷叫到高潮| 五月开心婷婷网| 亚洲欧美激情综合另类| 亚洲午夜理论影院| 男女午夜视频在线观看| 中文字幕人妻丝袜制服| 自拍欧美九色日韩亚洲蝌蚪91| 国产精品乱码一区二三区的特点 | 精品国产超薄肉色丝袜足j| www.999成人在线观看| 在线观看免费日韩欧美大片| 怎么达到女性高潮| 久久婷婷成人综合色麻豆| 人人妻人人爽人人添夜夜欢视频| 亚洲色图 男人天堂 中文字幕| 亚洲精品久久午夜乱码| 久久久久久人人人人人| 俄罗斯特黄特色一大片| 99久久国产精品久久久| 久久精品国产综合久久久| 成人三级黄色视频| 免费人成视频x8x8入口观看| 国产av一区在线观看免费| 欧美日韩黄片免| 亚洲精品国产精品久久久不卡| x7x7x7水蜜桃| 色婷婷久久久亚洲欧美| 两性午夜刺激爽爽歪歪视频在线观看 | 一级作爱视频免费观看| 一级毛片高清免费大全| 午夜影院日韩av| 日韩av在线大香蕉| 制服人妻中文乱码| 久久久久久人人人人人| 国产精品爽爽va在线观看网站 | 91老司机精品| 国产成人免费无遮挡视频| 亚洲成人免费av在线播放| av网站免费在线观看视频| 高潮久久久久久久久久久不卡| ponron亚洲| 欧美日韩中文字幕国产精品一区二区三区 | 国产在线精品亚洲第一网站| 成人特级黄色片久久久久久久| 深夜精品福利| 国产成人一区二区三区免费视频网站| 亚洲av成人一区二区三| 波多野结衣av一区二区av| 亚洲黑人精品在线| 人人澡人人妻人| 99国产极品粉嫩在线观看| 精品国产乱码久久久久久男人| 我的亚洲天堂| 一级a爱片免费观看的视频| 18禁黄网站禁片午夜丰满| 在线观看舔阴道视频| 成人三级黄色视频| 18禁美女被吸乳视频| 国内久久婷婷六月综合欲色啪| 国产无遮挡羞羞视频在线观看| 欧美日本中文国产一区发布| 精品无人区乱码1区二区| 亚洲一区中文字幕在线| 日本精品一区二区三区蜜桃| 精品一区二区三区四区五区乱码| 欧美激情久久久久久爽电影 | 巨乳人妻的诱惑在线观看| 18禁裸乳无遮挡免费网站照片 | 一边摸一边抽搐一进一出视频| 亚洲成av片中文字幕在线观看| 性少妇av在线| 97人妻天天添夜夜摸| 亚洲久久久国产精品| 国产黄色免费在线视频| 在线播放国产精品三级| 久久人人97超碰香蕉20202| 交换朋友夫妻互换小说| 日日夜夜操网爽| 精品人妻在线不人妻| 国产精品秋霞免费鲁丝片| a级毛片黄视频| 亚洲av日韩精品久久久久久密| 欧美另类亚洲清纯唯美| 成人特级黄色片久久久久久久| 午夜福利免费观看在线| 日韩免费av在线播放| 欧美乱妇无乱码| 亚洲国产中文字幕在线视频| 精品第一国产精品| 亚洲精品国产区一区二| 亚洲熟妇中文字幕五十中出 | 色尼玛亚洲综合影院| 国产精品98久久久久久宅男小说| 少妇被粗大的猛进出69影院| 一区在线观看完整版| 欧美日韩国产mv在线观看视频| 一区福利在线观看| 69av精品久久久久久| 成年女人毛片免费观看观看9| 9热在线视频观看99| 亚洲激情在线av| 后天国语完整版免费观看| 免费在线观看日本一区| 黄色 视频免费看| 日韩三级视频一区二区三区| 黄片大片在线免费观看| 欧美大码av| 亚洲成人久久性| 91av网站免费观看| 亚洲精品国产一区二区精华液| 亚洲熟女毛片儿| 欧美日本中文国产一区发布| x7x7x7水蜜桃| 老司机亚洲免费影院| 国产高清激情床上av| 欧美人与性动交α欧美精品济南到| 亚洲中文日韩欧美视频| 韩国精品一区二区三区| 亚洲性夜色夜夜综合| 91麻豆av在线| 在线播放国产精品三级| 日本黄色视频三级网站网址| 美女高潮到喷水免费观看| 国产精品美女特级片免费视频播放器 | 亚洲熟女毛片儿| 天堂√8在线中文| 欧美黄色淫秽网站| 日韩精品中文字幕看吧| 十八禁网站免费在线| 桃色一区二区三区在线观看| 午夜激情av网站| 美女高潮喷水抽搐中文字幕| 国产麻豆69| 欧美 亚洲 国产 日韩一| 岛国视频午夜一区免费看| 免费在线观看亚洲国产| 久热爱精品视频在线9| 19禁男女啪啪无遮挡网站| 久久国产精品人妻蜜桃| 国产亚洲精品第一综合不卡| 久久久久国内视频| 啦啦啦在线免费观看视频4| 久久国产精品男人的天堂亚洲| 两性夫妻黄色片| 亚洲自偷自拍图片 自拍| 久久久精品欧美日韩精品| 多毛熟女@视频| 嫩草影视91久久| 国产精品 国内视频| 香蕉久久夜色| 五月开心婷婷网| 日韩大尺度精品在线看网址 | 久久人人爽av亚洲精品天堂| 91成人精品电影| 成人手机av| 欧美精品亚洲一区二区| 久久中文字幕人妻熟女| 久久人人爽av亚洲精品天堂| av在线播放免费不卡| 女人精品久久久久毛片| 老熟妇仑乱视频hdxx| 久久久久国内视频| 久久精品亚洲精品国产色婷小说| 99国产精品免费福利视频| 黑人猛操日本美女一级片| 天天躁狠狠躁夜夜躁狠狠躁| 欧美日韩一级在线毛片| 精品国产亚洲在线| 夜夜爽天天搞| 9191精品国产免费久久| 成年人免费黄色播放视频| 免费av中文字幕在线| 日本欧美视频一区| 别揉我奶头~嗯~啊~动态视频| 国产精品一区二区精品视频观看| 极品教师在线免费播放| 窝窝影院91人妻| 99热只有精品国产| 日本三级黄在线观看| 搡老岳熟女国产| 免费在线观看亚洲国产| 亚洲全国av大片| 成年人黄色毛片网站| 一二三四在线观看免费中文在| 久久人妻熟女aⅴ| 中文欧美无线码| 亚洲av五月六月丁香网| 久久久久久大精品| 黑人猛操日本美女一级片| 午夜免费成人在线视频| 亚洲精华国产精华精| 久久伊人香网站| 亚洲三区欧美一区| 黄色视频不卡| 亚洲一区高清亚洲精品| av免费在线观看网站| 黄色女人牲交| 69av精品久久久久久| 免费观看精品视频网站| 国产男靠女视频免费网站| a级毛片在线看网站| 免费一级毛片在线播放高清视频 | 一本综合久久免费| 欧美激情 高清一区二区三区| 成人国语在线视频| 超碰97精品在线观看| 欧美日韩乱码在线| 国产日韩一区二区三区精品不卡| 好男人电影高清在线观看| 亚洲一码二码三码区别大吗| 人人澡人人妻人| 久久久国产欧美日韩av| 久久久久久久久久久久大奶| 人人妻人人添人人爽欧美一区卜| 男女午夜视频在线观看| 视频在线观看一区二区三区| 9191精品国产免费久久| 91麻豆精品激情在线观看国产 | 美国免费a级毛片| 亚洲精品一二三| 亚洲一区高清亚洲精品| 一区二区三区激情视频| 国产精品av久久久久免费| 亚洲精品国产色婷婷电影| 亚洲伊人色综图| 精品久久久久久电影网| 97碰自拍视频| aaaaa片日本免费| 久久久久亚洲av毛片大全| 日韩一卡2卡3卡4卡2021年| 国产野战对白在线观看| 可以免费在线观看a视频的电影网站| 午夜影院日韩av| 国产成人欧美在线观看| 亚洲第一av免费看| 中文字幕色久视频| 99re在线观看精品视频| 欧美中文综合在线视频| 免费av毛片视频|