首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 15 毫秒
1.
In the present study, an electrochemical aptasensor for highly sensitive detection of thrombin was developed based on bio-barcode amplification assay. For this proposed aptasensor, capture DNA aptamerI was immobilized on the Au electrode. The functional Au nanoparticles (DNA–AuNPs) are loaded with barcode binding DNA and aptamerII. Through the specific recognition for thrombin, a sandwich format of Au/aptamerI/thrombin/DNA–AuNPs was fabricated. After hybridization with the PbSNPs-labeled barcode DNA, the assembled sensor was obtained. The concentration of thrombin was monitored based on the concentration of lead ions dissolved through differential pulse anodic stripping voltammetric (DPASV). Under optimum conditions, a detection limit of 6.2 × 10−15 mol L−1 (M) thrombin was achieved. In addition, the sensor exhibited excellent selectivity against other proteins.  相似文献   

2.
We report here for the first time on the fabrication of highly dispersed PtM (M = Ru, Pd and Au) nanoparticles on composite film of multi-walled carbon nanotubes (MWNTs)–ionic liquid (IL, i.e., trihexyltetradecylphosphonium bis(trifluoromethylsulfonyl)imide) by using ultrasonic-electrodeposition method. The PtM nanoparticles are characterized by scanning electron microscopy, X-ray photoelectron spectroscopy and X-ray diffraction, and we find that they are well-dispersed and exhibit alloy properties. Electrochemical experiments show that the PtRu(1:1, i.e., ratio of c(H2PtCl6)/c(RuCl3))–MWNT–IL nanocomposite modified glassy carbon electrode (PtRu(1:1)–MWNT–IL/GCE) has smaller electron transfer resistance and larger active surface area than PtRu(1:1)/GCE, PtRu(1:1)–MWNT/GCE, PtPd(1:1)–MWNT–IL/GCE and PtAu(1:1)–MWNT–IL/GCE. The PtRu(1:1)–MWNT–IL/GCE also presents stronger electrocatalytic activity toward the glucose oxidation than other electrodes. At −0.1 V, the electrode responds linearly to glucose up to 15 mM in neutral media, with a detection limit of 0.05 mM (S/N = 3) and detection sensitivity of 10.7 μA cm−2 mM−1. Meanwhile, the interference of ascorbic acid, uric acid, acetamidophenol and fructose is effectively avoided. The as-made sensor was applied to the determination of glucose in serum and urine samples. The results agreed closely with the results obtained by a hospital. This novel nonenzyme sensor thus has potential application in glucose detection.  相似文献   

3.
Aluminum (Al) chemistry was studied in soils and waters of two catchments covered by spruce (Picea abies) monocultures in the Czech Republic that represent geochemical end-members of terrestrial and aquatic sensitivity to acidic deposition. The acid-sensitive Lysina catchment, underlain by granite, was compared to the acid-resistant Pluhův Bor catchment on serpentine. Organically-bound Al was the largest pool of reactive soil Al at both sites. Very high median total Al (Alt) concentrations (40 μmol L−1) and inorganic monomeric Al (Ali) concentrations (27 μmol L−1) were observed in acidic (pH 4.0) stream water at Lysina in the 1990s and these concentrations decreased to 32 μmol L−1 (Alt) and 13 μmol L−1 (Ali) in the 2000s. The potentially toxic Ali fraction decreased in response to long-term decreases in acidic deposition, but Ali remained the largest fraction. However, the organic monomeric (Alo) and particulate (Alp) fractions increased in the 2000s at Lysina. In contrast to Lysina, marked increases of Alt concentrations in circum-neutral waters at Pluhův Bor were observed in the 2000s in comparison with the 1990s. These increases were entirely due to the Alp fraction, which increased more than 3-fold in stream water and up to 8-fold in soil water in the A horizon. Increase of Alp coincided with dissolved organic carbon (DOC) increases. Acidification recovery may have increased the content of colloidal Al though the coagulation of monomeric Al.  相似文献   

4.
Red tide blooms of Cochlodinium polykrikoides in a coastal cove   总被引:1,自引:0,他引:1  
Successive blooms of the dinoflagellate Cochlodinium polykrikoides occurred in Pettaquamscutt Cove, RI, persisting from September through December 1980 and again from April through October 1981. Cell densities varied from <100 cells L−1 at the onset of the bloom and reached a maximum density exceeding 3.4 × 106 cells L−1 during the summer of 1981. The bloom was mainly restricted to the mid to inner region of this shallow cove with greatest concentrations localized in surface waters of the southwestern region during summer/fall periods of both years. Highly motile cells consisting of single, double and multiple cell zooids were found as chains of 4 and 8 cells restricted to the late August/September periods. The highest cell densities occurred during periods when annual temperatures were between 19 and 28 °C and salinities between 25 and 30. A major nutrient source for the cove was Crying Brook, located at the innermost region at the head of the cove. Inorganic nitrogen (NH3 and NO2 + NO3) from the brook was continually detectable throughout the study with maximum values of 57.5 and 82.5 μmol L−1, respectively. Phosphate (PO4-P) was always present in the source waters and rarely <0.5 μmol L−1; silicate always exceeded 30 μmol L−1 with maximum concentrations reaching 226 μmol L−1. Chlorophyll a and ATP concentrations during the blooms varied directly with cell densities. Maximum Chl a levels were 218 mg m−3 and ATP-carbon was >20 g C m−3. Primary production by the dinoflagellate-dominated community during the bloom varied between 4.3 and 0.07 g C m−3 d−1. Percent carbon turnover calculated from primary production values and ATP-carbon varied from 6 to 129% d−1. The dinoflagellates dominated the entire summer period; other flagellates and diatoms were present in lesser amounts. A combination of low washout rate due to the cove dynamics, active growth, and life cycles involving cysts allowed C. polykrikoides to maintain recurrent bloom populations in this area.  相似文献   

5.
Edge plane pyrolytic graphite electrode (EPPGE) modified with single-wall carbon nanotubes (SWNTs) has been used as a sensor to determine triamcinolone, abused by athletes for doping. A comparison of the voltammetric behavior between SWNTs modified EPPGE and fullerene – C60-modified EPPGE indicated that SWNTs modified EPPGE is more sensitive. The electrode exhibited an effective catalytic response with good reproducibility and stability. The effect of several parameters such as pH, square wave frequency and steroid concentration were studied. The square wave voltammetric response of the electrode to triamcinolone is linear in the range 0.1–25 nM with a detection limit and sensitivity of 8.9 × 10−10 M and 2.06 μA nM−1, respectively. The method was applied for the determination of triamcinolone in several commercially available pharmaceuticals and real urine samples obtained from patients undergoing pharmacological treatment with triamcinolone. A comparison of the observed results with HPLC analysis indicated a good agreement. The product obtained after reduction of triamcinolone was also characterized using 1H NMR and GC–MS and the site of reduction is found to be carbonyl group at position 20. The method described is rapid, simple and accurate and can be easily applied for detecting cases of doping.  相似文献   

6.
The red tide dinoflagellate Karenia brevis (Davis) G. Hansen and Moestrup is noted for causing mass mortalities of marine organisms in the Gulf of Mexico. Most research has focused on culture isolates from the eastern Gulf of Mexico. In this investigation, we examine the effects of light, temperature and salinity on the growth rate of K. brevis from the western Gulf of Mexico. Growth rates of K. brevis were determined under various combinations of irradiance (19, 31, 52, 67, and 123 μmol m−2 s−1), salinity (25, 30, 35, 40 and 45), and temperature (15, 20, 25, and 30 °C). Maximum growth rates varied from 0.17 to 0.36 div day−1 with exponential growth rates increasing with increasing irradiance. Little or no growth was supported at 19 μmol photons m−2 s−1 for any experiment. Maximum growth rates at 15 °C were much lower than at other temperatures. Maximum growth rates of the Texas clone (SP3) fell within the range of Florida clones reported in the literature (0.17–0.36 div day−1 versus 0.2–1.0 div day−1). The Texas clone SP3 had a very similar light saturation point compared to that of a Florida isolate (Wilson's clone) (67 μmol m−2 s−1 versus 65 μmol m−2 s−1), and light compensation (20–30 μmol m−2 s−11). The upper and lower salinity tolerance of the Texas clone was similar than that of some Florida clones (45 versus 46 and 25 versus 22.5, respectively). In our study, the Texas clone had the same temperature tolerance reported for Florida clones (15–30 °C). While individual clones can vary considerably in maximum growth rates, our results indicate only minor differences exist between the Texas and Florida strains of K. brevis in their temperature and salinity tolerance for growth. While the literature notes lower salinity occurrences of K. brevis in nearby Louisiana, our isolate from the southern Texas coast has the higher salinity requirements typical of K. brevis in the eastern Gulf of Mexico.  相似文献   

7.
A sequential on-line preconcentration and separation system for Cr(VI) and Cr(III) species determination was developed in this work. For this purpose, a microcolumn filled with nanostructured α-alumina was used for on-line retention of Cr species in a flow-injection system. The method involves the selective elution of Cr(VI) with concentrated ammonia and Cr(III) with 1 mol L−1 nitric acid for sequential injection into an electrothermal atomic absorption spectrometer (ETAAS).Analytical parameters including pH, eluent type, flow rates of sample and eluent, interfering effects, etc., were optimized. The preconcentration factors for Cr(VI) and Cr(III) were 41 and 18, respectively. The limit of detection (LOD) was 1.9 ng L−1 for Cr(VI) and 6.1 ng L−1 for Cr(III). The calibration graph was linear with a correlation coefficient of 0.999. The relative standard deviation (RSD) was 8.6% for Cr(VI) and 6.1% for Cr(III) (c=10 μg L−1, n=10, sample volume=25 mL). Verification of the accuracy was carried out by analysis of a standard reference material (NIST SRM 1643e “Trace elements in natural water”) with a reported Cr content of 20.40±0.24 μg L−1. Using the proposed methodology the total Cr content, computed as sum of Cr(III) and Cr(VI), in this SRM was 20.26±0.96 μg L−1. The method was successfully applied to the determination of Cr(VI) and Cr(III) species in parenteral solutions. Concentration of Cr(III) species was found to be in the range of 0.29–3.62 μg L−1, while Cr(VI) species was not detected in the samples under study.  相似文献   

8.
The effect of elicitation with linoleic (C18:2) and α-linolenic (C18:3) fatty acids (LLA and α-LNA) was investigated in Panax ginseng C.A. Meyer adventitious roots cultured in 5 l balloon-type bioreactors. Fatty acids were added in culture medium at 0.0, 1.0, 2.5, 5.0, 10.0, and 20.0 μmol l−1 at day 40, at the end of exponential growth phase. Roots were harvested and assayed at day 47. Elicitation with both LLA and α-LNA enhanced accumulation of total polyphenolics and flavonoids in roots compared with control without elicitation. The highest accumulation of flavonoids was observed at 5.0 μmol l−1 for both elicitors. Total phenolics production reached its highest value of about 4.0 mg g−1 DW under the elicitation with 5.0 μmol l−1 LLA and 5.0–20.0 μmol l−1 α-LNA. Meanwhile, α-LNA was more effective than LLA for increasing biomass and ginsenoside production. The biomass of 11.1 g DW l−1 and maximal total ginsenoside content of 7.9 mg g−1 DW were achieved at 5 μmol l−1 α-linolenic acid. The essential polyunsaturated linoleic (C18:2) and α-linolenic (C18:3) fatty acids were accumulated in roots in response to elicitation while content of palmitic (C16:0) and oleic (C18:1) acids declined. The activities of SOD, G-POD and CAT were enhanced by two elicitors to similar extent while APX activity was preferably stimulated by α-LNA. Our results demonstrate that elicitation with α-linolenic acid stimulates production of biomass and secondary metabolites in bioreactor-cultured P. ginseng adventitious roots.  相似文献   

9.
A novel glucose biosensor was developed, based on the immobilization of glucose oxidase (GOD) with cross-linking in the matrix of bovine serum albumin (BSA) on a Pt electrode, which was modified with gold nanoparticles decorated Pb nanowires (GNPs-Pb NWs). Pb nanowires (Pb NWs) were synthesized by an l-cysteine-assisted self-assembly route, and then gold nanoparticles (GNPs) were attached onto the nanowire surface through –SH–Au specific interaction. The morphological characterization of GNPs-Pb NWs was examined by transmission electron microscopy (TEM). Cyclic voltammetry and chronoamperometry were used to study and to optimize the electrochemical performance of the resulting biosensor. The synergistic effect of Pb NWs and GNPs made the biosensor exhibit excellent electrocatalytic activity and good response performance to glucose. The effects of pH and applied potential on the amperometric response of the biosensor have been systemically studied. In pH 7.0, the biosensor showed the sensitivity of 135.5 μA mM−1 cm−2, the detection limit of 2 μM (S/N = 3), and the response time <5 s with a linear range of 5–2200 μM. Furthermore, the biosensor exhibits good reproducibility, long-term stability and relative good anti-interference.  相似文献   

10.
Three microalgal species (Dictyosphaerium chlorelloides (D.c.), Scenedesmus intermedius (S.i.) and Scenedesmus sp. (S.s.)) were encapsulated in silicate sol–gel matrices and the increase in the amount of chlorophyll fluorescence signal was used to quantify simazine. Influence of several parameters on the preparation of the sensing layers has been evaluated: effect of pH on sol–gel gelation time; effect of algae density on sensor response; influence of glycerol (%) on the membrane stability. Long term stability was also tested and the fluorescence signal from biosensors remained stable for at least 3 weeks. D.c. biosensor presented the lowest detection limits for simazine (3.6 μg L−1) and the broadest dynamic calibration range (19–860 μg L−1) with IC50 125 ± 14 μg L−1. Biosensor was validated by HPLC with UV/DAD detection. The biosensor showed response to those herbicides that inhibit the photosynthesis at photosystem II (triazines: simazine, atrazine, propazine, terbuthylazine; urea based herbicides: linuron). However, no significant increases of fluorescence response was obtained for similar concentrations of 2,4-D (hormonal herbicide) or Cu(II). The combined use of two biosensors that use two different genotypes, sensitive and resistant to simazine, jointly allowed improving microalgae biosensor specificity.  相似文献   

11.
Canna indica L. is an upright perennial rhizomatous herb, and Schoenoplectus validus (Vahl) A. Löve and D. Löve is a tall, perennial, herbaceous sedge. The nutrient uptake kinetics of C. indica and S. validus were investigated using the modified depletion method after plants were grown for 4 weeks in simulated secondary-treated wastewater. The maximum uptake rate (Imax) and Michaelis–Menten constant (Km) were estimated by iterative curve fitting. The Imax for NH4N (623 μmol g−1 dry root weight h−1) was significantly higher than that for NO3N (338 μmol g−1 dry root weight h−1) in S. validus. In contrast, no difference was observed in C. indica. The Imax values for NO3N and NH4N were higher in S. validus than in C. indica. A significantly lower Km was detected for NO3N uptake in C. indica (385 μmol L−1) compared to that in S. validus (1908 μmol L−1). The Imax for PO4P did not differ between the plant species. The Km for PO4P was significantly higher in C. indica (157 μmol L−1) than in S. validus (60 μmol L−1). In conclusion, we found that S. validus preferred NH4N over NO3N, had greater capacity for N uptake and higher affinity for PO4P, but C. indica had greater affinity for NO3N. Nutrient uptake capacity is likely related to habitat preference, and is influenced by the structure of roots and rhizomes.  相似文献   

12.
Seasonal changes of field populations and growth rates of two dinoflagellates, Ceratium furca and Ceratium fusus, were examined in the temperate coastal water of Sagami Bay, Japan. Weekly field sampling was conducted from August 2002 to August 2003, and laboratory experiments were also carried out to investigate effects of temperature, irradiance and photoperiod on the growth rates of these two Ceratium species. In the field, the abundances of both species increased significantly from April to August 2003, were gradually decreased from November 2002 and were not observed in January 2003. C. fusus was able to increase at lower temperatures in February 2003 compared to C. furca. In the laboratory, the two species did not grow at <10 °C or >32 °C. The highest specific growth rate of C. furca was 0.72 d−1 at 24 °C and 600 μmol m−2 s−1. Optimum growth rates (>0.4 d−1) of C. furca were observed at temperatures from 18 to 28 °C and at irradiances from 216 to 796 μmol m−2 s−1. The highest growth rate of C. fusus was 0.56 d−1 at 26 °C and 216 μmol m−2 s−1. Optimum growth rates of C. fusus were observed at the same irradiance rage of C. furca, whereas optimum temperature range was narrower (26–28 °C). The growth curves of both species indicated saturation of the growth rates when light intensity was above 216 μmol m−2 s−1, and did not show photoinhibition at irradiances up to 796 μmol m−2 s−1. The specific growth rates of both Ceratium species were clearly decreased at L:D = 10:14 relative to those at L:D = 14:10 and L:D = 12:12. The present study indicates the two Ceratium species can adapt to a wide range of temperature and irradiance.  相似文献   

13.
Highly ordered Ni nanowire arrays (NiNWAs) were synthesized for the first time using a template-directed electropolymerization strategy with a nanopore polycarbonate (PC) membrane template, and their morphological characterization were examined by scanning electron microscopy (SEM) and transmission electron microscope (TEM). A NiNWAs based electrode shows very high electrochemical activity for electrocatalytic oxidation of glucose in alkaline medium, which has been utilized as the basis of the fabrication of a nonenzymatic biosensor for electrochemical detection of glucose. The biosensor can be applied to the quantification of glucose with a linear range covering from 5.0 × 10−7 to 7.0 × 10−3 M, a high sensitivity of 1043 μA mM−1 cm−2, and a low detection limit of 1 × 10−7 M. The experiment results also showed that the sensor exhibits good reproducibility and long-term stability, as well as high selectivity with no interference from other oxidable species.  相似文献   

14.
The “unprotected” Pt nanoclusters (average size 2 nm) mixed with the nanoscale SiO2 particles (average size 13 nm) were used as a glucose oxidase immobilization carrier to fabricate the amperometric glucose biosensor. The bioactivity of glucose oxidase (GOx) immobilized on the composite was maintained and the as-prepared biosensor demonstrated high sensitivity (3.85 μA mM−1) and good stability in glucose solution. The Pt–SiO2 biosensor showed a detection limit of 1.5 μM with a linear range from 0.27 to 4.08 mM. In addition, the biosensor can be operated under wide pH range (pH 4.9–7.5) without great changes in its sensitivity. Cyclic voltammetry measurements showed a mixed controlled electrode reaction.  相似文献   

15.
The present paper reports the graft copolymerization of N-vinylformamide onto sodium carboxymethylcellulose by free radical polymerization using potassium peroxymonosulphate/thiourea redox system in an inert atmosphere. The reaction conditions for maximum grafting have been optimized by varying the reaction variables, including the concentration of N-vinylformamide (12.0 × 10−2–28.0 × 10−2 mol dm−3), potassium peroxymonosulphate (4.0 × 10−3–12.0 × 10−3 mol dm−3), thiourea (1.2 × 10−3–4.4 × 10−3 mol dm−3), sulphuric acid (2.0 × 10−3–10.0 × 10−3 mol dm−3), sodium carboxymethylcellulose (0.2–1.8 g dm−3) along with time duration (60–180 min) and temperature (25–45° C). Water swelling capacity, metal ion sorption and flocculation studies of synthesized graft copolymer have been performed with respect to the parent polymer. The graft copolymer has been characterized by FTIR spectroscopy and thermogravimetric analysis.  相似文献   

16.
In the Crassulacean acid metabolism (CAM) plants Clusia alata Triana and Planch., decarboxylation of citrate during phase III of CAM took place later than malate decarboxylation. The interdependence of these two CO2 and NADPH sources is discussed. High light accelerated malate decarboxylation during the day and lowered citrate levels. Strong light stress also activated mechanisms that can protect the plant against oxidative stress. Upon transfer from low light (200 μmol m−2 s−1) to high light (650–740 μmol m−2 s−1), after 2 days, there was a transient increase of non-photochemical quenching (NPQ) of fluorescence of chlorophyll a of photosystem II. This indicated acute photoinhibition, which declined again after 7 days of exposure. Conversely, after 1 week exposure to high light, the mechanisms of interconversion of violaxanthin (V), antheraxanthin (A), zeaxanthin (Z) (epoxydation/de-epoxydation) were activated. This was accompanied by an increase in pigment levels at dawn and dusk.  相似文献   

17.
One and a half year-old Ginkgo saplings were grown for 2 years in 7 litre pots with medium fertile soil at ambient air CO2 concentration and at 700 μmol mol−1 CO2 in temperature and humidity-controlled cabinets standing in the field. In the middle of the 2nd season of CO2 enrichment, CO2 exchange and transpiration in response to CO2 concentration was measured with a mini-cuvette system. In addition, the same measurements were conducted in the crown of one 60-year-old tree in the field. Number of leaves/tree was enhanced by elevated CO2 and specific leaf area decreased significantly.CO2 compensation points were reached at 75–84 μmol mol−1 CO2. Gas exchange of Ginkgo saplings reacted more intensively upon CO2 than those of the adult Ginkgo. On an average, stomatal conductance decreased by 30% as CO2 concentration increased from 30 to 1000 μmol mol−1 CO2. Water use efficiency of net photosynthesis was positively correlated with CO2 concentration levels. Saturation of net photosynthesis and lowest level of stomatal conductance was reached by the leaves of Ginkgo saplings at >1000 μmol mol−1 CO2. Acclimation of leaf net CO2 assimilation to the elevated CO2 concentration at growth occurred after 2 years of exposure. Maximum of net CO2 assimilation was 56% higher at ambient air CO2 concentration than at 700 μmol mol−1 CO2.  相似文献   

18.
A novel direct readout colorimetric optical glucose sensor strip was constructed based on a three-layer film, including a green-emitted CdTe/CdS quantum dots (QDs) layer as a stable color background, a red-fluorescent platinum-porphyrin oxygen-sensing layer and a glucose oxidase layer. The sensor achieved high resolution (up to 0.2 mmol L−1) glucose determination with a detection range from 0 to 3.0 mmol L−1. A “glucose ruler” which acts as a glucose standard colorimetric card was obtained. Glucose concentration could easily be directly readout using the “glucose ruler”, which made the glucose determination rapid, convenient and easy. The effects of pH, salinity and temperature were systematically investigated. The prepared sensor was finally applied for glucose sample analysis, compared with the “glucose ruler”, accurate results could be directly readout.  相似文献   

19.
Glassy carbon (GC) electrode was modified using multi-wall carbon nanotubes (MWCNTs), quercetin (Q) and Nafion® in this sequence. The thus modified electrode was used for the detection of dopamine (DA) in the presence of equimolar ascorbic acid (AA). It is demonstrated in this study that MWCNTs can increase the current response of DA by five-fold and Q can reduce the oxidation overpotential of DA by about 60 mV, compared to these parameters obtained with a bare GC electrode. It is also shown that a layer of Nafion® can virtually eliminate the interference of AA for the detection of DA. The GC/MWCNTs/Q/Nafion® electrode (hereafter also called composite electrode) shows a current density of about 900 μA cm−2 for DA, compared to the value of 80 μA cm−2 of the GC electrode and to the value of 390 μA cm−2 of the GC/MWCNTs electrode. The 11-fold enhancement in the sensitivity of the GC electrode for DA determination is attributed to the composite modification of the electrode, and is substantiated through various cyclic voltammetric experiments. Cyclic voltammetry (CV) and linear sweep voltammetry were used to characterize the electrodes. Calibration curves of batch and flow systems were obtained by amperometry for the detection of DA. Additionally, the composite modified electrode was tested with a human serum sample for the determination of DA and was found to be promising at our preliminary experiments.  相似文献   

20.
An immunosensor based on the electrochemiluminescence (ECL) of peroxydisulfate was firstly proposed by coupling the cooperation of two enzymes to in situ generate coreactant with palladium nanoparticles (PdNPs) as catalyst for the ECL reaction. PdNPs were previously synthesized, which successfully attached to functional carbon nanotubes (FCNTs), to bind the secondary antibody and bienzyme (horseradish peroxidase and glucose oxidase). Then the prepared bioconjugates were introduced to the electrode via sandwich immunoreactions. Accordingly, a dramatically amplified ECL signal was obtained for that GOD catalyzed glucose to produce H(2)O(2) which was subsequently reduced by HRP to in situ generate O(2), then PdNPs as catalyst for the ECL reaction of peroxydisulfate/O(2). The present immunosensor was used to detect α-1-fetoprotein (AFP) and showed a wide linear range of 1×10(-5)-100ngmL(-1), with a low detection limit of 3.3fgmL(-1)(S/N=3). This new signal amplification strategy for preparation of the ECL immunosensor could be easily realized and has a potential application in ultrasensitive bioassays.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号