• <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 ?

    Jigsaw-like mini-pillar platform for multi-mode biosensing

    2022-09-16 05:24:46YongchaoSongDongdongWangZehuaLiLirongWangChuanFanXuechengHeTailinXuXuejiZhang
    Chinese Chemical Letters 2022年8期

    Yongchao Song, Dongdong Wang, Zehua Li, Lirong Wang, Chuan Fan,Xuecheng He, Tailin Xu,?, Xueji Zhang

    a College of Chemistry and Materials Engineering, Beijing Technology and Business University, Beijing 100048, China

    b Research Center for Bioengineering and Sensing Technology, University of Science and Technology Beijing, Beijing 100083, China

    c School of Biomedical Engineering, Health Science Center, Shenzhen University, Shenzhen 518060, China

    ABSTRACT The multiple sensing provides booming options to eliminate interference and ensure the accuracy of detection by mutually coupling and validating multiple data sets.Here, we integrate the jigsaw-like multifunctional mini-pillar platform to perform multi-mode (electrochemical, fluorescence, surface-enhanced Raman scattering (SERS) and colorimetric) sensing in individual microdroplets.Each mini-pillar connector can parallelize together by specific concave-convex interface to form integrated jigsaw-like platform for multi-mode sensing, and each specific mini-pillar can be modified into the individual sensing unit to read the prescribed signals.We successfully implemented electrochemical, fluorescence, SERS and colorimetric detection by multiple signals coupling to reduce the false positive analysis.Such platform brings a promising clue of in-situ analysis and point-of-care testing for disease diagnosis and health monitoring.

    Keywords:Multiple sensing Jigsaw-like Mini-pillar platform Signal coupling Microdroplets array

    The sequential operation droplet array has emerged as a powerful tool for medicine, biology, chemistry, physics, and materials science [1–7].For example, microfluidic-based droplet operations that integrate droplet generation, splitting, fusion, addressing, capture and sorting are famous for cell-related applications [8–11].Recently, emerging open-channel droplet platform such as superwettable surface, paper-based and mini-pillar microchips with the capabilities of easy solution manipulation also have been advanced in diverse areas such as high-throughput screening, single cell analysis and miniaturized cell experiments [12–18].By integrating with signal-output approaches such as colorimetric, electrochemical, SERS and fluorescence, the open-channel droplet platform has achieved widespread high-throughput biosensing applications [19–25], providing appreciated blueprint of thein-situanalysis and point-of-care testing for disease diagnosis and health monitoring.

    Chemical and biological sensors are currently one of the most active areas of analytical research [26–30].Most advances in sensing technologies and devices are catalyzing key breakthroughs in the detection limit, which have reached the single-molecule level[31–33], while the single-signal output strategy is difficult to provide complete information and unconvincing due to the signal interference from complex sample component and the intrinsic drawbacks of each sensing technology in terms of selectivity and reproducibility.Recently, much efforts have been devoted to coupling the multiple sensing technologies in one chip for parallel detection, and the results are superior to traditional single-signal platforms owing to multiple sets of data coupling and mutual validation [34–39].For example, the combination of fluorescence and colorimetry on paper-based platform has achieved aptamerinitiated isothermal amplification analysis of glutamate dehydrogenase in clinical samples [36], the coupling of plasmonic circular dichroism and luminescent signals realizes ultrasensitive miRNAin-situquantitation in live cells [40].These above encouraging results provide unlimited possibilities for accurate, sensitive, rapid and parallel multi-mode sensing in one chip to fulfill the high demands of clinical sensing.

    Here, we demonstrate the jigsaw-like mini-pillar platform that confines the analyses in array of open-channel microreactors for electrochemical, fluorescence, SERS and colorimetric multi-mode sensing.Such mini-pillar jigsaw-like platform is established by the programmable 3D printing, modification, splicing and integration.We successfully accomplish electrochemical, fluorescence,SERS and colorimetric multimode sensing on splitable mini-pillar platform, and couple the multi-signal to accurate result output in complex samples.This is the first trial of such jigsaw-like platform to improve the accuracy of detection by multiple signals coupling,which provides an opportunity to develop multi-mode sensing device toward precise physiological and pathological diagnosis.

    Fig.1.Schematic of jigsaw-like mini-pillar platform for electrochemical, SERS, colorimetrical, and fluorescence biosensing.

    The programmable procedures of jigsaw-like mini-pillar platform including construction and modification, splicing and integration, and multi-mode sensing were illustrated in Fig.1.In brief,the customized individual mini-pillars with the specific concaveconvex interface were first manufactured by 3D printing.Then, in response to different testing requirements, diversified functionalization was applied to mini-pillars.The circuit board stabilized the detachable platform, which is used as a medium for reading batch electrochemical data, and the mini-pillar unit modified by electrodeposition of nanodendritic gold is used to read the SERS signal.Furthermore, with its advantage of droplets anchoring and enrichment, the mini-pillar without other modification could complete the fluorescence and colorimetric detection.The individual and functionalized mini-pillars could realize the array splicing through specific concave-convex interface to form a patterned multi-mode platform for multi-mode sensing.The coupling of multiple results can significantly reduce false positive analysis caused by interference and ambiguous signals, showing huge potentialsin-situanalysis and point-of-care testing for disease diagnosis and health monitoring.

    The jigsaw-like mini-pillar platform exhibits excellent characteristics, which can anchor and enrich trace solutions to achieve multi-mode sensing.The single mini-pillar was manufactured by 3D printing, and the printed polylactic acid (PLA) mini-pillars were successfully assembled into a matrix platform for immobilizing droplets, as shown in Fig.2a.We further investigated the droplets anchoring ability of the mini-pillar platform in Figs.2b and c.The droplet (20 μL) was stable on the surface of the inclined and inverted, while Rhodamine 6 G labelled microdroplets on as-prepared platforms presented two diametrically opposed appearances.On the ordinary glass platform, microdroplet was distributed in a relatively large area (Fig.S1a in Supporting information), and there is a clear "coffee ring" after evaporation (Fig.2d).In contrast, the droplets are uniformly distributed on the surface of the mini-pillar in the designated smaller area (Fig.2e and Fig.S1b in Supporting information), revealing its excellent enrichment performance.Subsequently, we use 4×4 array as a concept-of-proof to show the splicing and integration process of jigsaw-like minipillar platform.The mini-pillars with specific concave-convex interface (Fig.2f) can be spliced to a complete high-throughput platform for carrying microdroplets.

    Fig.2.Characterization of jigsaw-like mini-pillar platform.(a) Individual mini-pillar fabrication by 3D printing.Investigation of the microdroplet adhesion of the mini-pillar platform by (b) sloping and (c) inversing.The enrichment ability of (d) common glass and (e) mini-pillar platform.(f) The splicing and integration process from individual mini-pillar array to jigsaw-like mini-pillar platform.(g) Cyclic voltammetry curves of electrochemical mini-pillar platform at increasing scan rates from 10 mV to 100 mV.(h)The SERS spectra comparison of bare gold mini-pillar platform (red) and nanodendritic gold platform (black).(i) The comparison of colorimetric analysis between common glass and mini-pillar platform.(j) The fluorescence images and corresponding fluorescence intensities of droplets evaporation on glass and mini-pillar.Scale bar: 2 mm.

    Fig.3.Jigsaw-like mini-pillar platform toward multi-mode sensing.(a) Schematic illustration of mini-pillar platform for multi-mode glucose detection.(b) Amperometry responses of droplets microarray with glucose concentrations from 2 mmol/L to 10 mmol/L.(c) Raman intensity at 1368 cm?1 (2,3-diaminophenazine) of glucose-containing microdroplets from 2 mmol/L to 10 mmol/L.(d) Colorimetrical calibration curve of glucose (2, 4, 6, 8 and 10 mmol/L).(e) The fluorescence images and corresponding intensities of glucose (2, 4, 6, 8 and 10 mmol/L) in microdroplets.

    The functional modification of mini-pillar is the prerequisite for multi-modal sensing.The electrochemical mini-pillar retains the hole array during the 3D printing process, and the embedded electrode array was connected to the circuit board to realize complete electrical signal transmission.The cyclic voltammograms (?0.1~0.4 V; 10 μL 5 mmol/L hydroxymethyl-ferrocene in PBS buffer) at scan rates from 10 mV/s to 100 mV/s on electrochemical mini-pillar were recorded as shown in Fig.2g.With the increase of the scan rate, the anode and cathode peak currents increased, and the peak potential remained almost unchanged, indicating that the redox process on the mini-pillars was a surfacelimited process.For SERS sensing, first sputter a layer of gold on the micro-pillars to ensure conductivity, as shown in Fig.S2a (Supporting information).Then, the electrolyte composed of HAuCl4(1 mg/mL) and sulfuric acid (0.5 mol/L) was employed to electrodeposit the nanodendritic gold on the surface of mini-pillar at ?1.0 V for 1000 s.In Fig.2h, we tested the Raman signal enhancement of nanodendritic gold mini-pillar compared with bare gold minipillar for possible SERS detection of Rhodamine 6 G (R6G).For the bare gold mini-pillar, all the R6G characteristic peaks were detected with fairly weak signals intensity.In comparison, the Raman intensity on the nanodendrites gold mini-pillar was significantly enhanced as shown in Figs.S2b and c (Supporting information).Benefit from the performance of droplet anchoring and sample enrichment, the mini-pillar can sensitively carry out the fluorescence and colorimetric sensing without additional modification.As displayed in Fig.2i, the microdroplet (R6G, 10 μL) on mini-pillar presented the more regular and smaller sphere than on common glass and had the obvious enhancement of the gray value.After evaporation, the microdroplets on common glass tended to spread to a larger area and form obvious “coffee ring” spot with stronger fluorescence intensity than the interior of spot, while the distribution of R6G was very homogeneous on mini-pillar with the uniform fluorescence intensity as shown in Fig.2j.These results indicate that the jigsaw-like mini-pillar platform has excellent performance for electrochemical, SERS, fluorescence and colorimetric multi-mode sensing.

    The glucose concentration as the important physiological indicator is tested by electrochemical, SERS and fluorescence and colorimetric multi-mode sensing on such jigsaw-like mini-pillar platform to evaluate its versatility and feasibility (Fig.3a).In brief,for electrochemical sensing zone, glucose oxidase (1 kU/mL) is modified on the working electrode and then 10 μL glucose (2,4, 6, 8 and 10 mmol/L) with different concentrations are dropped on electrochemical mini-pillar platform for sensing.The electrochemical detection is performed using chronoamperometry.The glucose is oxidized to gluconic acid and generates hydrogen peroxide, which is then catalyzed at the working electrode under current fluctuations.For SERS sensing zone, 10 μL microdroplet containing glucose oxidase, horseradish peroxidase (HRP) andophenylenediamine dihydrochloride (OPD) is dropped on mini-pillar,and glucose solution with different concentrations are added for SERS sensing that glucose is catalyzed by glucose oxidase to generate hydrogen peroxide and HRP catalytically oxidized OPDH2O2with the production of 2,3-diaminophenazine.Then, the Raman spectra of each mini-pillar sites are measured by using a 532 nm laser source with exposure time of 10 s.The colorimetric sensing is performed with the assistance of signal screening by smartphone.The microdroplet containing KI and GOx is dispended on mini-pillar and the colorimetric intensities were increased with the increasing glucose concentration, which are recorded by smartphone and analyzed by using ImageJ analysis software to read the grayscale value.For fluorescence imaging sensing, 3-nitrophenylboronic acid in the droplet can effectively quench the fluorescence of esculetin, and the fluorescence will be restored when glucose is combined with 3-nitrophenylboronic acid.

    The results of multi-mode sensing of glucose have simultaneously been recorded by jigsaw-like mini-pillar platform as shown in Figs.3b–e.In the electrochemical measurements, the electrochemical signal (ΔI) increased gradually with the increase of the glucose concentration, and showed a good linear relationship with a correlation coefficient of 0.9988 (Fig.3b).For the SERS sensing,specific Raman signals of generated 2,3-diaminophenazine presented at 1368 and 1572 cm?1and increased upon an increase of glucose from 2 mmol/L to 10 mmol/L.It was found that the corresponding calibration chart of the 1368 cm?1peak intensity and the glucose concentration was linear, with a correlation coefficient of 0.9987 (Fig.3c).In terms of colorimetric verification, color intensities increased with the concentration of glucose with a correlation coefficient of 0.9937 as shown in Fig.3d.Meanwhile, the fluorescence intensities increased gradually upon an increase of glucose concentration, and showed a good linear relationship, with a correlation coefficient of 0.9743 (Fig.3e).Thus, we successfully realized electrochemical, SERS, fluorescence and colorimetric multimode detection of the glucose on such jigsaw-like mini-pillar platform.

    Fig.4.Jigsaw-like mini-pillar platform toward prescribed sample multi-signal coupling and analysis.(a) Multi-signal integration of glucose and fructose samples in PBS.(b) The electrochemical, SERS, colorimetric and fluorescent results of glucose in diluted serum.?P > 0.05, ??P < 0.001, Student’s T-test.

    Multi-signal coupling and analysis can effectively avoid the results fluctuation.We further verified ability of such jigsaw-like mini-pillar platform for multi-mode analysis of the specified samples as shown in Fig.4.Firstly, such platform is employed to detect the glucose concentration in PBS with glucose (6 mmol/L)and fructose (6 mmol/L), the results indicate that the concentration of glucose by electrochemical, SERS and colorimetric are similar(5.90±0.14, 5.96±0.09 and 5.91±0.08 mmol/L;P >0.05) while the fluorescent signal has large fluctuations due to the weak selectivity of the fluorescence method for sugars (11.59±0.62 mmol/L;P <0.001) (Fig.4a).Thus, by multi-signal coupling, error signals of fluorescence can be removed, the final glucose concentration was finally calculated as 5.92±0.11 mmol/L.Then, we further employ such platform to detect the glucose (6 mmol/L) in diluted human serum (containing various proteins, 1:10 diluted with 0.1 mol/L PBS, pH 7.0).The results of four signals have no obvious difference (6.05±0.08, 6.08±0.17, 6.01±0.06 and 5.98±0.17 mmol/L;P >0.05) indicate that each method can realize the accurate detection of glucose in serum sample without obvious interference(Fig.4b).Such multi-mode results of the same analyses greatly reduce the false positive analysis due to the intrinsic drawbacks of the signal sensing technology by multiple data coupling, difference analyzing and results confirmation, which brings a promising clue ofin-situanalysis and point-of-care testing for disease diagnosis and health monitoring.

    In conclusion, we have demonstrated a jigsaw-like mini-pillar platform for electrochemical, fluorescence, SERS and colorimetric multi-mode sensing.Each mini-pillar as individual sensing unit can read out the relevant signal from microdroplets and parallelize mini-pillars splice by specific concave-convex interface to form an integrated multi-signal transmission platform for multimode sensing.Multifunctional modifications allow such jigsawlike mini-pillar platform to achieve the multiple and quantitative detection.The excellent abilities of mini-pillar for anchoring and enriching the trace solution provide an ideal platform for fluorescence and colorimetric sensing, and the embedded electrode array can achieve electrochemical analysis, and electrodeposited nanodendritic gold substrate can realize SERS sensing.We employed such jigsaw-like platform to detection of trace glucose in PBS and diluted serum by multiple signals coupling, the results indicate that the integrated platform can discharge the interference and ensure the accuracy of detection by multiple signals coupling.Such jigsaw-like mini-pillar platform can easily perform high-throughput detection with the advantages of high-yield, low sample volume, multiple signal options, no cross contamination and simple to disassemble and splice, demonstrating the potential application ofin-situanalysis and point-of-care testing for disease diagnosis and health monitoring.

    Declaration of competing interest

    The authors declare no competing interests.

    Acknowledgments

    We acknowledge funding from National Natural Science Foundation of China (Nos.21804007 and 21890742), Youth Scholars of Beijing Technology and Business University (No.QNJJ2020–04), and SZU Top Ranking Project (No.86000000210), Shenzhen Stability Support Plan (No.20200806163622001).

    Supplementary materials

    Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2021.12.059.

    在线国产一区二区在线| 男女做爰动态图高潮gif福利片| www.色视频.com| 精品午夜福利在线看| 久久99热这里只有精品18| 国产男人的电影天堂91| 日韩av在线大香蕉| 精品欧美国产一区二区三| 亚洲第一电影网av| 中文资源天堂在线| 亚洲av二区三区四区| 永久网站在线| 成人特级黄色片久久久久久久| 尾随美女入室| 黄色一级大片看看| 欧美区成人在线视频| 在线观看66精品国产| av福利片在线观看| 国产女主播在线喷水免费视频网站 | 哪里可以看免费的av片| 久久久色成人| or卡值多少钱| 女生性感内裤真人,穿戴方法视频| 色哟哟·www| 深爱激情五月婷婷| 国产精品精品国产色婷婷| 亚洲最大成人手机在线| 在线免费十八禁| 非洲黑人性xxxx精品又粗又长| 蜜桃亚洲精品一区二区三区| 亚洲av不卡在线观看| 精品久久久久久久久久免费视频| av专区在线播放| 欧美高清成人免费视频www| 波多野结衣巨乳人妻| 日日摸夜夜添夜夜添小说| 99热全是精品| 亚洲综合色惰| 日韩在线高清观看一区二区三区| 18禁在线无遮挡免费观看视频 | 国产一区二区在线av高清观看| av免费在线看不卡| 国产私拍福利视频在线观看| 午夜a级毛片| 男人舔女人下体高潮全视频| 久久精品人妻少妇| 无遮挡黄片免费观看| 国产三级中文精品| ponron亚洲| 村上凉子中文字幕在线| 在线观看一区二区三区| 丰满的人妻完整版| 精华霜和精华液先用哪个| 国产精品久久久久久精品电影| 午夜影院日韩av| 99国产极品粉嫩在线观看| 午夜影院日韩av| 久久99热这里只有精品18| 国产精品人妻久久久影院| 激情 狠狠 欧美| 男女视频在线观看网站免费| 成人无遮挡网站| 日韩欧美精品免费久久| 久99久视频精品免费| 99久国产av精品| 乱码一卡2卡4卡精品| 九九爱精品视频在线观看| 男女那种视频在线观看| 波多野结衣巨乳人妻| a级一级毛片免费在线观看| 国产91av在线免费观看| 色av中文字幕| 级片在线观看| 99热6这里只有精品| 人妻久久中文字幕网| 男人狂女人下面高潮的视频| 亚洲成a人片在线一区二区| 欧美色欧美亚洲另类二区| 桃色一区二区三区在线观看| 韩国av在线不卡| 桃色一区二区三区在线观看| 中出人妻视频一区二区| 国产综合懂色| 青春草视频在线免费观看| 男女那种视频在线观看| 午夜精品一区二区三区免费看| 免费一级毛片在线播放高清视频| 淫妇啪啪啪对白视频| 午夜免费男女啪啪视频观看 | 校园春色视频在线观看| 欧美极品一区二区三区四区| 又爽又黄a免费视频| 国内精品久久久久精免费| 一级毛片aaaaaa免费看小| 99视频精品全部免费 在线| 久久久久久久久大av| 国产亚洲91精品色在线| 给我免费播放毛片高清在线观看| 亚洲在线观看片| 在线观看66精品国产| 亚洲乱码一区二区免费版| 亚洲av一区综合| 午夜激情福利司机影院| 国产精品电影一区二区三区| 成人亚洲欧美一区二区av| 日本五十路高清| 欧美精品国产亚洲| 国产片特级美女逼逼视频| 十八禁国产超污无遮挡网站| 久久精品国产亚洲av涩爱 | www日本黄色视频网| 成年女人永久免费观看视频| 国内精品宾馆在线| 尾随美女入室| 国产精品久久久久久精品电影| 插阴视频在线观看视频| 亚洲在线自拍视频| 神马国产精品三级电影在线观看| 国产一区二区三区在线臀色熟女| 非洲黑人性xxxx精品又粗又长| 啦啦啦韩国在线观看视频| 男女啪啪激烈高潮av片| or卡值多少钱| 久久精品夜色国产| 网址你懂的国产日韩在线| 女人被狂操c到高潮| 91在线观看av| 免费av毛片视频| 欧美成人a在线观看| 真实男女啪啪啪动态图| 99久久久亚洲精品蜜臀av| 国产高清视频在线观看网站| 国产黄色视频一区二区在线观看 | 99久久精品国产国产毛片| 亚洲图色成人| 你懂的网址亚洲精品在线观看 | 亚洲欧美精品自产自拍| 哪里可以看免费的av片| 99久久精品国产国产毛片| 中文字幕久久专区| 欧美又色又爽又黄视频| 亚洲av熟女| 成人二区视频| 国产伦一二天堂av在线观看| 久久亚洲精品不卡| 毛片女人毛片| 男女那种视频在线观看| 国产精品综合久久久久久久免费| 长腿黑丝高跟| 亚洲精品456在线播放app| 成人永久免费在线观看视频| 国产三级在线视频| 欧美成人a在线观看| 亚洲不卡免费看| h日本视频在线播放| 国产成年人精品一区二区| 禁无遮挡网站| 天堂影院成人在线观看| 久久欧美精品欧美久久欧美| 91久久精品国产一区二区三区| 国产v大片淫在线免费观看| .国产精品久久| 国产一区亚洲一区在线观看| 最近的中文字幕免费完整| 亚洲中文日韩欧美视频| 久久精品夜夜夜夜夜久久蜜豆| 久久6这里有精品| 一级av片app| 国产精品亚洲一级av第二区| 国产精品免费一区二区三区在线| 观看美女的网站| 国产精品永久免费网站| 悠悠久久av| 超碰av人人做人人爽久久| 国产亚洲欧美98| 欧美+日韩+精品| 九九久久精品国产亚洲av麻豆| 久久天躁狠狠躁夜夜2o2o| 国产乱人偷精品视频| ponron亚洲| 综合色av麻豆| 麻豆精品久久久久久蜜桃| 午夜日韩欧美国产| 国产精品无大码| 女人十人毛片免费观看3o分钟| 亚洲av电影不卡..在线观看| 赤兔流量卡办理| 国产高清视频在线播放一区| 国产精品三级大全| 级片在线观看| 欧美一区二区精品小视频在线| 一夜夜www| 人妻久久中文字幕网| 人妻制服诱惑在线中文字幕| 免费高清视频大片| 成年女人毛片免费观看观看9| 成人特级黄色片久久久久久久| 欧美精品国产亚洲| 免费看av在线观看网站| av.在线天堂| 国产成人福利小说| 免费看日本二区| 白带黄色成豆腐渣| 国产精品,欧美在线| 久久久久性生活片| 欧美日韩国产亚洲二区| 亚洲美女视频黄频| 久久精品夜色国产| 精品福利观看| 18禁在线播放成人免费| 一本精品99久久精品77| 91久久精品电影网| 亚洲高清免费不卡视频| 可以在线观看毛片的网站| 亚洲精品成人久久久久久| 久久韩国三级中文字幕| 国内精品久久久久精免费| 亚洲精品日韩av片在线观看| 丝袜美腿在线中文| 国产精品不卡视频一区二区| 精品久久久久久久久亚洲| 美女内射精品一级片tv| 成人精品一区二区免费| 草草在线视频免费看| 天天躁日日操中文字幕| 搡老熟女国产l中国老女人| 亚洲精品日韩av片在线观看| 欧美色视频一区免费| 成人精品一区二区免费| 日本 av在线| 午夜爱爱视频在线播放| 成人永久免费在线观看视频| eeuss影院久久| 精品一区二区三区视频在线| 在线播放无遮挡| 日本与韩国留学比较| 超碰av人人做人人爽久久| 九九爱精品视频在线观看| 欧美+亚洲+日韩+国产| 综合色丁香网| 尤物成人国产欧美一区二区三区| 最好的美女福利视频网| 国模一区二区三区四区视频| 欧美+日韩+精品| 九九久久精品国产亚洲av麻豆| 一级黄片播放器| 少妇熟女aⅴ在线视频| 日韩制服骚丝袜av| 一级黄片播放器| 欧美激情久久久久久爽电影| 国产私拍福利视频在线观看| 成人性生交大片免费视频hd| 91狼人影院| 人人妻,人人澡人人爽秒播| 国产精品精品国产色婷婷| av在线老鸭窝| 亚洲美女黄片视频| а√天堂www在线а√下载| 国产欧美日韩精品一区二区| 久久人人精品亚洲av| 亚洲欧美日韩卡通动漫| 真实男女啪啪啪动态图| 国产男靠女视频免费网站| 好男人在线观看高清免费视频| 久久久久久久久中文| 美女免费视频网站| 国产麻豆成人av免费视频| 亚洲欧美日韩东京热| 精品久久国产蜜桃| 国产欧美日韩精品一区二区| 精品久久久久久久久亚洲| 亚洲精品久久国产高清桃花| 国产精品野战在线观看| 在线观看一区二区三区| 国产麻豆成人av免费视频| 国产成人freesex在线 | 日韩欧美国产在线观看| 美女大奶头视频| 国产精品久久久久久av不卡| 尾随美女入室| 亚洲18禁久久av| 晚上一个人看的免费电影| 国产极品精品免费视频能看的| 18禁在线播放成人免费| 美女被艹到高潮喷水动态| 日韩中字成人| 国产亚洲欧美98| 国产亚洲精品久久久久久毛片| 久久这里只有精品中国| 女人被狂操c到高潮| 高清午夜精品一区二区三区 | 午夜精品在线福利| 欧美日本视频| 精品午夜福利在线看| 久久精品国产亚洲av天美| 91久久精品电影网| 午夜免费激情av| 色5月婷婷丁香| 久久精品国产清高在天天线| 午夜福利成人在线免费观看| 国产精品综合久久久久久久免费| 精品免费久久久久久久清纯| 精品久久久久久久人妻蜜臀av| 色噜噜av男人的天堂激情| 国产日本99.免费观看| 一个人看的www免费观看视频| 99热只有精品国产| 中文字幕人妻熟人妻熟丝袜美| 国产乱人视频| 好男人在线观看高清免费视频| 两性午夜刺激爽爽歪歪视频在线观看| 我要搜黄色片| 久久久久久久亚洲中文字幕| 亚洲av中文av极速乱| 在线免费十八禁| 黄色一级大片看看| 搡老妇女老女人老熟妇| 日韩一区二区视频免费看| av中文乱码字幕在线| 简卡轻食公司| 插逼视频在线观看| 亚洲aⅴ乱码一区二区在线播放| 久久久精品大字幕| 欧美日韩精品成人综合77777| 欧美色欧美亚洲另类二区| 全区人妻精品视频| 亚洲高清免费不卡视频| 内射极品少妇av片p| 国产免费一级a男人的天堂| 一区二区三区免费毛片| 亚洲,欧美,日韩| 99久久中文字幕三级久久日本| 女人十人毛片免费观看3o分钟| 国产av麻豆久久久久久久| 精品久久久久久久久亚洲| 性欧美人与动物交配| 国产午夜福利久久久久久| 亚洲va在线va天堂va国产| 欧美人与善性xxx| 成人漫画全彩无遮挡| 亚洲一区高清亚洲精品| 亚洲第一区二区三区不卡| 亚洲av一区综合| 男女那种视频在线观看| 亚洲综合色惰| 校园春色视频在线观看| 五月伊人婷婷丁香| 美女 人体艺术 gogo| 悠悠久久av| 少妇猛男粗大的猛烈进出视频 | 老师上课跳d突然被开到最大视频| 日本 av在线| 免费在线观看成人毛片| 22中文网久久字幕| 久久国内精品自在自线图片| 国产高清不卡午夜福利| 日韩成人av中文字幕在线观看 | 亚州av有码| 老司机午夜福利在线观看视频| 久久人人精品亚洲av| 亚洲欧美日韩高清专用| 日韩精品中文字幕看吧| 日日撸夜夜添| 免费看日本二区| 看免费成人av毛片| 女人十人毛片免费观看3o分钟| 真人做人爱边吃奶动态| 久久久精品欧美日韩精品| 99热这里只有是精品50| 日本精品一区二区三区蜜桃| 欧美高清性xxxxhd video| 少妇被粗大猛烈的视频| 国产黄色视频一区二区在线观看 | 夜夜夜夜夜久久久久| .国产精品久久| 亚洲高清免费不卡视频| 亚洲国产精品合色在线| 日日摸夜夜添夜夜爱| 国产精品不卡视频一区二区| 噜噜噜噜噜久久久久久91| 欧美一级a爱片免费观看看| 少妇丰满av| 午夜福利成人在线免费观看| 午夜影院日韩av| 桃色一区二区三区在线观看| 国产乱人视频| 最近手机中文字幕大全| 1000部很黄的大片| 国产亚洲精品久久久com| 亚洲欧美精品自产自拍| 俺也久久电影网| 中文亚洲av片在线观看爽| 精品免费久久久久久久清纯| 中文字幕人妻熟人妻熟丝袜美| 91麻豆精品激情在线观看国产| 中出人妻视频一区二区| 久久精品久久久久久噜噜老黄 | 我要搜黄色片| 欧美最黄视频在线播放免费| 成人特级av手机在线观看| 国产成人a区在线观看| 国产一区亚洲一区在线观看| 99久久成人亚洲精品观看| 亚洲综合色惰| 国产精品永久免费网站| www日本黄色视频网| 午夜激情福利司机影院| 一进一出抽搐gif免费好疼| 午夜免费激情av| 黄色日韩在线| 91在线精品国自产拍蜜月| 在线播放国产精品三级| 国产一区二区在线观看日韩| 免费在线观看成人毛片| 亚洲精品成人久久久久久| 桃色一区二区三区在线观看| 91久久精品电影网| 午夜精品在线福利| 精品一区二区三区视频在线观看免费| 最近在线观看免费完整版| 成人亚洲欧美一区二区av| 最近手机中文字幕大全| 亚洲人成网站在线播放欧美日韩| 99久国产av精品国产电影| 在线观看66精品国产| 99国产精品一区二区蜜桃av| 国产午夜精品论理片| 国产一区二区三区在线臀色熟女| 精品少妇黑人巨大在线播放 | 亚洲欧美日韩无卡精品| 国产精品永久免费网站| 波多野结衣高清作品| 国产伦精品一区二区三区四那| 最近视频中文字幕2019在线8| 波多野结衣巨乳人妻| 18禁在线播放成人免费| 午夜日韩欧美国产| av在线播放精品| 亚洲欧美成人精品一区二区| av在线播放精品| 熟妇人妻久久中文字幕3abv| 亚洲精品影视一区二区三区av| 久久久久国产精品人妻aⅴ院| 夜夜爽天天搞| 婷婷精品国产亚洲av| 亚洲欧美清纯卡通| 亚洲av二区三区四区| 给我免费播放毛片高清在线观看| 在线天堂最新版资源| 亚洲国产色片| 午夜福利高清视频| 色视频www国产| 国产男靠女视频免费网站| 免费看日本二区| 国产真实伦视频高清在线观看| 能在线免费观看的黄片| 欧美一区二区精品小视频在线| 亚洲欧美清纯卡通| 亚洲av中文av极速乱| 成人性生交大片免费视频hd| 悠悠久久av| 国产精品久久久久久av不卡| 亚洲欧美日韩高清在线视频| av国产免费在线观看| 国产人妻一区二区三区在| 91在线精品国自产拍蜜月| 国产亚洲精品综合一区在线观看| av视频在线观看入口| 欧美激情国产日韩精品一区| 中国国产av一级| 欧美激情久久久久久爽电影| 丝袜美腿在线中文| 国产午夜福利久久久久久| 国内精品宾馆在线| 亚洲真实伦在线观看| 免费在线观看成人毛片| 久久精品国产自在天天线| 国产精品久久久久久久久免| 国产精品久久久久久精品电影| 蜜桃久久精品国产亚洲av| 亚洲精品色激情综合| 成人美女网站在线观看视频| 久久99热这里只有精品18| 欧美高清性xxxxhd video| 国产高清激情床上av| 好男人在线观看高清免费视频| 国产视频一区二区在线看| 熟女人妻精品中文字幕| 神马国产精品三级电影在线观看| 精品久久久久久成人av| 99久久精品热视频| 国产激情偷乱视频一区二区| 国产精品日韩av在线免费观看| 内射极品少妇av片p| 嫩草影院新地址| 国产精品久久久久久久电影| 久久久色成人| 亚洲精品日韩av片在线观看| 欧美极品一区二区三区四区| 国产av不卡久久| 99久久精品一区二区三区| 午夜亚洲福利在线播放| 又黄又爽又免费观看的视频| 99在线视频只有这里精品首页| 91久久精品电影网| 老司机福利观看| 国产蜜桃级精品一区二区三区| 99久久精品一区二区三区| 精品久久国产蜜桃| 97超碰精品成人国产| 国产精品免费一区二区三区在线| 日韩欧美国产在线观看| 十八禁网站免费在线| 欧美日本视频| 狠狠狠狠99中文字幕| 永久网站在线| 可以在线观看的亚洲视频| av国产免费在线观看| 国产精品久久电影中文字幕| 长腿黑丝高跟| 午夜日韩欧美国产| 国产成人a∨麻豆精品| 小说图片视频综合网站| 国产成人aa在线观看| 神马国产精品三级电影在线观看| 一级黄片播放器| av中文乱码字幕在线| 在线播放国产精品三级| 亚洲av免费在线观看| 亚洲国产日韩欧美精品在线观看| 少妇裸体淫交视频免费看高清| 在线观看66精品国产| 精品人妻偷拍中文字幕| 啦啦啦啦在线视频资源| 精品国产三级普通话版| 成人一区二区视频在线观看| a级毛片a级免费在线| av天堂中文字幕网| 一本一本综合久久| 3wmmmm亚洲av在线观看| 内地一区二区视频在线| 丰满乱子伦码专区| 国产精品野战在线观看| 床上黄色一级片| 成人精品一区二区免费| 免费看美女性在线毛片视频| 日本熟妇午夜| 搡老妇女老女人老熟妇| 日本成人三级电影网站| 久久精品夜色国产| 久久国产乱子免费精品| 国产色婷婷99| 国产一区二区三区在线臀色熟女| 国产老妇女一区| 精品一区二区三区视频在线| 男人舔女人下体高潮全视频| 一本精品99久久精品77| 观看美女的网站| 国产黄片美女视频| 久久久色成人| 国产熟女欧美一区二区| 男人和女人高潮做爰伦理| 大香蕉久久网| 最近手机中文字幕大全| 国产爱豆传媒在线观看| 亚洲国产精品久久男人天堂| 五月玫瑰六月丁香| 久久久久国产精品人妻aⅴ院| 午夜老司机福利剧场| 人人妻,人人澡人人爽秒播| 老司机福利观看| 成年女人永久免费观看视频| 搡老熟女国产l中国老女人| 国产亚洲av嫩草精品影院| 老熟妇仑乱视频hdxx| 亚洲图色成人| 精品一区二区免费观看| 欧美最黄视频在线播放免费| 精品午夜福利在线看| 小蜜桃在线观看免费完整版高清| 国产欧美日韩精品亚洲av| 国产精品人妻久久久久久| 国产精品日韩av在线免费观看| 国产探花在线观看一区二区| 夜夜爽天天搞| 成年女人看的毛片在线观看| 国产精品久久久久久久久免| 丰满的人妻完整版| 欧美绝顶高潮抽搐喷水| 人妻少妇偷人精品九色| 搡老岳熟女国产| 看片在线看免费视频| 国产高清有码在线观看视频| 国内少妇人妻偷人精品xxx网站| 久久99热6这里只有精品| 日韩三级伦理在线观看| 久久久久久久久中文| 男插女下体视频免费在线播放| 久久久久久九九精品二区国产| 午夜精品国产一区二区电影 | 一级黄色大片毛片| 亚洲欧美日韩卡通动漫| 婷婷精品国产亚洲av| 国产美女午夜福利| 婷婷色综合大香蕉| 精品一区二区三区av网在线观看| 变态另类丝袜制服| 91狼人影院| 久99久视频精品免费| 国产av在哪里看| 欧洲精品卡2卡3卡4卡5卡区| 舔av片在线| 波野结衣二区三区在线| 国产精品一二三区在线看| 精品一区二区免费观看|