• <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.

    每晚都被弄得嗷嗷叫到高潮| 欧美不卡视频在线免费观看 | 亚洲aⅴ乱码一区二区在线播放 | 一a级毛片在线观看| 黄色女人牲交| 亚洲av熟女| 午夜老司机福利片| 窝窝影院91人妻| 亚洲中文字幕日韩| 在线永久观看黄色视频| 中亚洲国语对白在线视频| 婷婷丁香在线五月| 手机成人av网站| 精品亚洲成国产av| 成人免费观看视频高清| 一区在线观看完整版| 中文字幕最新亚洲高清| 黄网站色视频无遮挡免费观看| 人妻 亚洲 视频| 欧美激情高清一区二区三区| x7x7x7水蜜桃| 国产免费男女视频| 国产一区在线观看成人免费| 日韩欧美免费精品| 成人三级做爰电影| 下体分泌物呈黄色| 他把我摸到了高潮在线观看| 欧美在线一区亚洲| 一级片'在线观看视频| 无限看片的www在线观看| 国产精品影院久久| 丝袜美腿诱惑在线| 精品午夜福利视频在线观看一区| 女人爽到高潮嗷嗷叫在线视频| 久久精品国产a三级三级三级| 岛国毛片在线播放| 99精品久久久久人妻精品| 黄色怎么调成土黄色| 一区二区三区激情视频| 亚洲av成人av| 热99re8久久精品国产| 亚洲色图综合在线观看| 身体一侧抽搐| 国产免费现黄频在线看| 亚洲成国产人片在线观看| 美国免费a级毛片| 十八禁高潮呻吟视频| 色94色欧美一区二区| bbb黄色大片| 日日夜夜操网爽| 国产成人欧美| 亚洲av电影在线进入| bbb黄色大片| 精品高清国产在线一区| 午夜影院日韩av| 欧美日韩亚洲综合一区二区三区_| 久99久视频精品免费| 国精品久久久久久国模美| 999精品在线视频| 免费在线观看完整版高清| 国产精华一区二区三区| 亚洲成人手机| 乱人伦中国视频| 亚洲中文av在线| 99香蕉大伊视频| 人人澡人人妻人| 亚洲中文日韩欧美视频| 纯流量卡能插随身wifi吗| 人人妻人人爽人人添夜夜欢视频| 男人的好看免费观看在线视频 | 成人黄色视频免费在线看| 国内久久婷婷六月综合欲色啪| 精品福利观看| 中文字幕人妻丝袜一区二区| 91国产中文字幕| 欧美大码av| 国产精品亚洲一级av第二区| 在线永久观看黄色视频| 国产av又大| 怎么达到女性高潮| 人人妻,人人澡人人爽秒播| 亚洲av电影在线进入| 日本vs欧美在线观看视频| 黑人操中国人逼视频| 99国产极品粉嫩在线观看| 免费高清在线观看日韩| 99久久综合精品五月天人人| 欧美日韩av久久| 丰满的人妻完整版| 国产精品美女特级片免费视频播放器 | 美女视频免费永久观看网站| 中文字幕人妻丝袜一区二区| av福利片在线| 久久精品国产综合久久久| 国产男靠女视频免费网站| 精品国产一区二区三区久久久樱花| 两人在一起打扑克的视频| 一本大道久久a久久精品| 亚洲精品在线观看二区| 一级片免费观看大全| www.熟女人妻精品国产| 女同久久另类99精品国产91| 美女高潮喷水抽搐中文字幕| 欧美人与性动交α欧美精品济南到| 一级毛片精品| 搡老岳熟女国产| 天堂俺去俺来也www色官网| 久久中文看片网| 久久午夜亚洲精品久久| 午夜福利影视在线免费观看| 国产xxxxx性猛交| 亚洲黑人精品在线| 亚洲欧洲精品一区二区精品久久久| 亚洲欧美日韩另类电影网站| 深夜精品福利| 99久久精品国产亚洲精品| 老司机深夜福利视频在线观看| 搡老岳熟女国产| 交换朋友夫妻互换小说| 精品国内亚洲2022精品成人 | 欧美乱妇无乱码| av国产精品久久久久影院| 乱人伦中国视频| 久久人妻熟女aⅴ| 国产成人啪精品午夜网站| 午夜两性在线视频| 精品国产一区二区三区久久久樱花| 欧美一级毛片孕妇| 999精品在线视频| 亚洲av日韩精品久久久久久密| 在线av久久热| 老司机在亚洲福利影院| 欧美激情 高清一区二区三区| tocl精华| 国产精品久久久久成人av| 精品视频人人做人人爽| 精品熟女少妇八av免费久了| netflix在线观看网站| 无遮挡黄片免费观看| 精品福利永久在线观看| 亚洲精品av麻豆狂野| 男人舔女人的私密视频| 51午夜福利影视在线观看| 大码成人一级视频| 久久香蕉精品热| 亚洲中文av在线| 欧美丝袜亚洲另类 | 久久国产亚洲av麻豆专区| 久久国产乱子伦精品免费另类| 亚洲aⅴ乱码一区二区在线播放 | 亚洲熟女毛片儿| 久久久水蜜桃国产精品网| 日韩一卡2卡3卡4卡2021年| 免费日韩欧美在线观看| 国产91精品成人一区二区三区| 久9热在线精品视频| 热re99久久国产66热| 国产在线观看jvid| 欧美人与性动交α欧美精品济南到| 91九色精品人成在线观看| 成年动漫av网址| a在线观看视频网站| 啦啦啦视频在线资源免费观看| 国产野战对白在线观看| 高清毛片免费观看视频网站 | 欧美性长视频在线观看| 国产一区二区三区综合在线观看| 99热网站在线观看| 国产精品久久久久久精品古装| 90打野战视频偷拍视频| 久久ye,这里只有精品| 少妇粗大呻吟视频| 啪啪无遮挡十八禁网站| 人人澡人人妻人| 日本五十路高清| 最近最新免费中文字幕在线| 欧美最黄视频在线播放免费 | 久久天堂一区二区三区四区| 在线观看免费午夜福利视频| 亚洲va日本ⅴa欧美va伊人久久| 欧美午夜高清在线| 99久久精品国产亚洲精品| 精品福利观看| 久久中文字幕人妻熟女| 黄色a级毛片大全视频| 999久久久国产精品视频| 51午夜福利影视在线观看| 久久热在线av| 美女午夜性视频免费| 午夜精品在线福利| 国产精品久久久久成人av| 国产精品免费一区二区三区在线 | 欧美成人午夜精品| 欧美日韩中文字幕国产精品一区二区三区 | 波多野结衣av一区二区av| 亚洲中文av在线| 性少妇av在线| 国产成人欧美| 一边摸一边抽搐一进一小说 | 天天操日日干夜夜撸| 淫妇啪啪啪对白视频| 国产日韩欧美亚洲二区| 欧美成狂野欧美在线观看| 美国免费a级毛片| 黄片小视频在线播放| 夜夜夜夜夜久久久久| 亚洲欧美精品综合一区二区三区| av视频免费观看在线观看| 日韩人妻精品一区2区三区| 欧美另类亚洲清纯唯美| 国产精品免费视频内射| 午夜福利乱码中文字幕| 满18在线观看网站| 一级片'在线观看视频| 久久香蕉精品热| 成年人免费黄色播放视频| 国产精品秋霞免费鲁丝片| 天天躁日日躁夜夜躁夜夜| 美女午夜性视频免费| 国产精品 国内视频| 高清av免费在线| 色婷婷久久久亚洲欧美| 一级a爱片免费观看的视频| 夜夜爽天天搞| 久久久久精品国产欧美久久久| 又紧又爽又黄一区二区| 午夜免费观看网址| 男人的好看免费观看在线视频 | 露出奶头的视频| 欧美成狂野欧美在线观看| 黄色a级毛片大全视频| 麻豆乱淫一区二区| 精品一品国产午夜福利视频| 亚洲精品中文字幕一二三四区| 狂野欧美激情性xxxx| 欧美精品亚洲一区二区| 久久精品aⅴ一区二区三区四区| 久久中文字幕一级| 国产成人一区二区三区免费视频网站| 99精品在免费线老司机午夜| 大型黄色视频在线免费观看| www.999成人在线观看| 69精品国产乱码久久久| 不卡av一区二区三区| 老汉色av国产亚洲站长工具| 美女扒开内裤让男人捅视频| 亚洲av成人不卡在线观看播放网| 免费看十八禁软件| 757午夜福利合集在线观看| 国产欧美亚洲国产| 久久人妻av系列| 欧美在线黄色| 国产精品亚洲av一区麻豆| 亚洲国产欧美日韩在线播放| 交换朋友夫妻互换小说| 超碰成人久久| 欧美激情极品国产一区二区三区| 伦理电影免费视频| 亚洲片人在线观看| 精品午夜福利视频在线观看一区| 成人国产一区最新在线观看| 黄片大片在线免费观看| 成年人午夜在线观看视频| 成人免费观看视频高清| 精品国内亚洲2022精品成人 | 久久久久视频综合| 免费看a级黄色片| 欧美人与性动交α欧美精品济南到| 成年版毛片免费区| 亚洲av熟女| 黑人操中国人逼视频| 十八禁网站免费在线| 日韩欧美在线二视频 | 夫妻午夜视频| 免费少妇av软件| 三级毛片av免费| 妹子高潮喷水视频| 久久久久国内视频| 变态另类成人亚洲欧美熟女 | 黑丝袜美女国产一区| 黄色怎么调成土黄色| av视频免费观看在线观看| 狂野欧美激情性xxxx| 日韩制服丝袜自拍偷拍| 女警被强在线播放| 12—13女人毛片做爰片一| 中亚洲国语对白在线视频| 免费在线观看视频国产中文字幕亚洲| 国内毛片毛片毛片毛片毛片| 国产激情欧美一区二区| 久久久水蜜桃国产精品网| 国产高清videossex| 亚洲欧美色中文字幕在线| 精品国产一区二区久久| 自线自在国产av| 午夜精品在线福利| 欧美日韩黄片免| 1024视频免费在线观看| av不卡在线播放| 视频区欧美日本亚洲| 亚洲欧美日韩高清在线视频| 国产精品久久久人人做人人爽| 极品人妻少妇av视频| 国产成人欧美| 久久香蕉激情| 久久婷婷成人综合色麻豆| 1024视频免费在线观看| 又紧又爽又黄一区二区| 一边摸一边抽搐一进一小说 | 如日韩欧美国产精品一区二区三区| 9热在线视频观看99| 国产男女超爽视频在线观看| 国产精品电影一区二区三区 | 在线观看舔阴道视频| 久久久水蜜桃国产精品网| 精品第一国产精品| 午夜福利,免费看| 黑人猛操日本美女一级片| 精品国产一区二区三区四区第35| 国产成人免费观看mmmm| 亚洲美女黄片视频| 少妇的丰满在线观看| 久久精品国产a三级三级三级| 别揉我奶头~嗯~啊~动态视频| 在线观看www视频免费| 久久精品成人免费网站| 又黄又爽又免费观看的视频| 18禁美女被吸乳视频| av电影中文网址| 国产免费现黄频在线看| 亚洲国产欧美网| 岛国毛片在线播放| 人人妻人人添人人爽欧美一区卜| 亚洲视频免费观看视频| 久久ye,这里只有精品| 午夜福利乱码中文字幕| 免费av中文字幕在线| 啦啦啦免费观看视频1| 亚洲精品乱久久久久久| 国产xxxxx性猛交| 少妇的丰满在线观看| 午夜影院日韩av| 日本撒尿小便嘘嘘汇集6| 男人舔女人的私密视频| 国产精品秋霞免费鲁丝片| 精品电影一区二区在线| 午夜老司机福利片| 婷婷精品国产亚洲av在线 | 国产深夜福利视频在线观看| 精品福利永久在线观看| 欧美精品啪啪一区二区三区| 亚洲av日韩在线播放| 搡老熟女国产l中国老女人| 午夜免费观看网址| 91国产中文字幕| 人成视频在线观看免费观看| 色94色欧美一区二区| 亚洲人成伊人成综合网2020| 9热在线视频观看99| 国产男女超爽视频在线观看| 看免费av毛片| 国产真人三级小视频在线观看| 国产精品 国内视频| 99精品在免费线老司机午夜| 精品久久久久久,| 亚洲人成电影观看| 啦啦啦视频在线资源免费观看| 久久国产精品男人的天堂亚洲| 电影成人av| 亚洲成人手机| 欧美激情 高清一区二区三区| 久久草成人影院| 99国产极品粉嫩在线观看| 成人手机av| 国产在视频线精品| 久久精品国产亚洲av香蕉五月 | 老司机深夜福利视频在线观看| 欧美黄色片欧美黄色片| 国产区一区二久久| 丝袜在线中文字幕| 免费人成视频x8x8入口观看| 黄色毛片三级朝国网站| 日韩 欧美 亚洲 中文字幕| 丝袜人妻中文字幕| 黄色毛片三级朝国网站| 精品亚洲成国产av| 在线观看午夜福利视频| 精品卡一卡二卡四卡免费| avwww免费| 女警被强在线播放| 建设人人有责人人尽责人人享有的| 在线看a的网站| 免费在线观看日本一区| 日韩精品免费视频一区二区三区| 18禁裸乳无遮挡免费网站照片 | 高清欧美精品videossex| 午夜福利欧美成人| 亚洲一卡2卡3卡4卡5卡精品中文| 女性被躁到高潮视频| 老汉色av国产亚洲站长工具| 一级片'在线观看视频| 男人舔女人的私密视频| 国产精品偷伦视频观看了| 欧美不卡视频在线免费观看 | 美女 人体艺术 gogo| 色播在线永久视频| 国产精品国产av在线观看| 人人妻人人澡人人看| 国产精品亚洲一级av第二区| 天堂中文最新版在线下载| 夜夜躁狠狠躁天天躁| 在线视频色国产色| 亚洲一卡2卡3卡4卡5卡精品中文| av免费在线观看网站| 极品教师在线免费播放| 精品少妇久久久久久888优播| 午夜福利视频在线观看免费| 久久午夜亚洲精品久久| 亚洲一区中文字幕在线| 久久精品人人爽人人爽视色| 国产在线精品亚洲第一网站| 一级a爱片免费观看的视频| 18禁黄网站禁片午夜丰满| 中文亚洲av片在线观看爽 | 王馨瑶露胸无遮挡在线观看| 一本一本久久a久久精品综合妖精| 成人三级做爰电影| 亚洲伊人色综图| 欧美精品一区二区免费开放| 午夜激情av网站| 91av网站免费观看| 男男h啪啪无遮挡| 精品乱码久久久久久99久播| xxxhd国产人妻xxx| 国产av精品麻豆| 不卡av一区二区三区| 电影成人av| 精品国产亚洲在线| 18禁黄网站禁片午夜丰满| 91成人精品电影| 熟女少妇亚洲综合色aaa.| 国产不卡av网站在线观看| 亚洲精品久久午夜乱码| 俄罗斯特黄特色一大片| 国产精品99久久99久久久不卡| a在线观看视频网站| 欧美日韩亚洲高清精品| 最新在线观看一区二区三区| 国产不卡一卡二| 91大片在线观看| 岛国毛片在线播放| 亚洲自偷自拍图片 自拍| 一级片免费观看大全| 国产99白浆流出| bbb黄色大片| 丁香欧美五月| 久久午夜综合久久蜜桃| 欧美日韩中文字幕国产精品一区二区三区 | 日韩三级视频一区二区三区| 女人爽到高潮嗷嗷叫在线视频| 亚洲aⅴ乱码一区二区在线播放 | 日韩三级视频一区二区三区| 成人永久免费在线观看视频| 久久久久久久久久久久大奶| 老汉色∧v一级毛片| 精品久久久久久电影网| 大香蕉久久网| 高潮久久久久久久久久久不卡| 99在线人妻在线中文字幕 | 国产精品九九99| 中文字幕av电影在线播放| av有码第一页| 女性被躁到高潮视频| 欧美日韩成人在线一区二区| 天天躁夜夜躁狠狠躁躁| 老司机福利观看| 一区福利在线观看| 91老司机精品| 国产一区二区三区在线臀色熟女 | 久久狼人影院| 精品一区二区三区av网在线观看| 大陆偷拍与自拍| 欧美精品亚洲一区二区| 在线观看日韩欧美| 天天影视国产精品| 久久人人爽av亚洲精品天堂| 黄色视频,在线免费观看| 老司机在亚洲福利影院| 精品熟女少妇八av免费久了| 国产真人三级小视频在线观看| 精品第一国产精品| 99精国产麻豆久久婷婷| 精品少妇一区二区三区视频日本电影| 黄色片一级片一级黄色片| 99re在线观看精品视频| 精品国产乱子伦一区二区三区| 欧美日韩亚洲综合一区二区三区_| 欧美精品亚洲一区二区| 日本一区二区免费在线视频| 国产不卡一卡二| 高潮久久久久久久久久久不卡| 精品国产一区二区三区久久久樱花| 老熟妇乱子伦视频在线观看| tocl精华| 天天影视国产精品| 高潮久久久久久久久久久不卡| 国内毛片毛片毛片毛片毛片| 黄网站色视频无遮挡免费观看| 很黄的视频免费| 久久久精品免费免费高清| 国精品久久久久久国模美| 51午夜福利影视在线观看| 天堂俺去俺来也www色官网| 老司机午夜十八禁免费视频| 他把我摸到了高潮在线观看| 王馨瑶露胸无遮挡在线观看| 麻豆av在线久日| 亚洲 国产 在线| 熟女少妇亚洲综合色aaa.| 国产一区二区三区综合在线观看| 免费日韩欧美在线观看| 麻豆国产av国片精品| 丁香欧美五月| 亚洲精品久久午夜乱码| 亚洲一码二码三码区别大吗| 欧美日韩亚洲高清精品| 熟女少妇亚洲综合色aaa.| 成年人免费黄色播放视频| 岛国在线观看网站| 国产精华一区二区三区| tube8黄色片| 人人澡人人妻人| 午夜亚洲福利在线播放| 国产精品久久久久久精品古装| 久久精品国产99精品国产亚洲性色 | 人人妻人人添人人爽欧美一区卜| 久久人妻熟女aⅴ| 亚洲精品国产区一区二| 国产单亲对白刺激| 精品国产美女av久久久久小说| 亚洲欧美激情综合另类| 亚洲美女黄片视频| 精品一区二区三区av网在线观看| 51午夜福利影视在线观看| 久久婷婷成人综合色麻豆| 精品欧美一区二区三区在线| 成年人免费黄色播放视频| 亚洲午夜精品一区,二区,三区| 欧美日韩福利视频一区二区| 亚洲精品久久成人aⅴ小说| 少妇猛男粗大的猛烈进出视频| 老鸭窝网址在线观看| 亚洲av成人不卡在线观看播放网| 欧美精品av麻豆av| 岛国毛片在线播放| 国产精品1区2区在线观看. | 欧美亚洲日本最大视频资源| 大码成人一级视频| 亚洲精品粉嫩美女一区| 97人妻天天添夜夜摸| 精品一区二区三区四区五区乱码| 亚洲视频免费观看视频| 精品久久久久久久久久免费视频 | 免费看十八禁软件| videos熟女内射| av一本久久久久| 叶爱在线成人免费视频播放| 不卡一级毛片| 日韩一卡2卡3卡4卡2021年| 国产一区在线观看成人免费| 中文字幕最新亚洲高清| 国产欧美日韩精品亚洲av| 一级毛片精品| 女人久久www免费人成看片| 欧美人与性动交α欧美软件| 亚洲七黄色美女视频| 欧美人与性动交α欧美软件| 免费人成视频x8x8入口观看| 中文亚洲av片在线观看爽 | 精品国产一区二区久久| 国产亚洲精品第一综合不卡| 亚洲第一欧美日韩一区二区三区| 在线观看免费视频网站a站| 国产人伦9x9x在线观看| 天堂√8在线中文| 咕卡用的链子| 亚洲黑人精品在线| 一区二区三区激情视频| 操出白浆在线播放| 色在线成人网| 色尼玛亚洲综合影院| 日韩三级视频一区二区三区| 成人永久免费在线观看视频| av网站免费在线观看视频| 成人特级黄色片久久久久久久| 亚洲人成电影免费在线| 十八禁高潮呻吟视频| 大片电影免费在线观看免费| 精品乱码久久久久久99久播| 啦啦啦在线免费观看视频4| 久久中文字幕一级| 国产精品99久久99久久久不卡| tocl精华| av线在线观看网站| 欧美黄色淫秽网站| 国产精品香港三级国产av潘金莲| 亚洲国产中文字幕在线视频| 午夜福利,免费看| netflix在线观看网站| 嫁个100分男人电影在线观看| 久久国产精品男人的天堂亚洲| 男男h啪啪无遮挡| 欧美日韩成人在线一区二区| 国产又色又爽无遮挡免费看| 精品久久久久久久久久免费视频 |