text:
An integrated device for nucleic acid analysis having a support (10) and a first tank (8) for introducing a raw biological specimen includes at least one pre-treatment channel (17), a buried amplification chamber (21), and a detection chamber (24) carried by the support (10) and in fluid connection with one another and with the tank (8). The device can be used for all types of biological analyses.
1) an integrated micro-device for analysis of a biological specimen, comprising:
a) a support comprising:
i) a first tank;
ii) a buried channel formed inside said support, and
iii) a detection chamber;
wherein the first tank, the buried channel, and the detection chamber are fluidly coupled and wherein the first tank is accessible from outside of said support.
2) The integrated micro-device of
3) The integrated micro-device of
4) The integrated micro-device of
5) The integrated micro-device of
6) The integrated micro-device of
7) The integrated micro-device of
8) The integrated micro-device of
9) The integrated micro-device of
10) An integrated device for analysis of nucleic acid, said device comprising a support carrying i) a first tank for introducing a biological specimen into said support, ii) at least one pre-treatment channel, iii) a buried channel inside said support, and iv) a detection chamber, each being in fluid connection with each other.
11) The device according to
12) The device according to
13) The device according to
14) The device according to
15) The device according to claims 14, characterized in that said pre-treatment channel is formed above said support and is delimited laterally by a containment structure and on top by a protective plate that covers said containment structure.
16) The device according to
17) The device according to
18) The device according to
19) The device according to
20) The device according to
21) The device according to
22) The device according to
23) The device according to
24) The device according to
25) The device according to
26) The device according to
27) The device according to
28) The device according to
29) The device according to
30) The device according to
31) The device according to
32) A process for manufacturing an integrated device for nucleic acid analysis, comprising the steps of:
a) forming at least one first buried channel inside a body of semiconductor material; and
b) forming at least one second channel on top of said body, said second channel being at least partially arranged on top of said first channel.
33) The process according to
a) depositing a polymeric material layer on top of said body; and
b) defining said polymeric material layer so as to form a containment structure delimiting said second channel.
34) The process according to
a) depositing a heater on top of said body;
b) forming, on top of said body, a first base incorporating said heater, and a second base; and
c) depositing electrodes on top of said first base and detectors on top of said second base.
35) The process according to
36) The process according to
a) functionalizing said detectors; and
b) closing said chamber with a protective plate.
37) The process according to
38) The process according to
39) The process of
40) A method of amplification, comprising amplifying a target nucleic acid in a buried channel inside a substrate having high thermal conductivity, and detecting an amplified nucleic acid on a detector on said substrate, wherein the detector is fluidly connected to said buried channel.
41) The method of
42) The method of
43) The method of
44) The method of
45) A portable device for analysis of a biological material, said portable device comprising:
a) a printed circuit board;
b) a disposable support having a buried channel therein and an inlet port accessible from outside of the disposable support, and a sensor placed thereon;
c) said disposable support and said sensor operably coupled to said printed circuit board.
46) The portable device of
47) The portable device of
48) The portable device of
49) The portable device of
50) The portable device of
51) The portable device of
52) The portable device of
53) The portable device of
[0001] This application claims priority to Italian Patent Application No. TO2002A 000808 filed on Sep. 17, 2002 in the name of STMicroelectronics S.r.l.
[0002] Not applicable.
[0003] Not applicable.
[0004] The present invention relates to an integrated device for biological analyses, such as nucleic acid analyses.
[0005] Typical procedures for analyzing biological materials, such as nucleic acid, protein, lipid, carbohydrate, and other biological molecules, involve a variety of operations starting from raw material. These operations may include various degrees of cell separation or purification, cell lysis, amplification or purification, and analysis of the resulting amplification or purification product.
[0006] As an example, in DNA-based blood analyses samples are often purified by filtration, centrifugation or by electrophoresis so as to eliminate all the non-nucleated cells, which are generally not useful for DNA analysis. Then, the remaining white blood cells are broken up or lysed using chemical, thermal or biochemical means in order to liberate the DNA to be analyzed. Next, the DNA is denatured by thermal, biochemical or chemical processes and amplified by an amplification reaction, such as PCR (polymerase chain reaction), LCR (ligase chain reaction), SDA (strand displacement amplification), TMA (transcription-mediated amplification), RCA (rolling circle amplification), and the like. The amplification step allows the operator to avoid purification of the DNA being studied because the amplified product greatly exceeds the starting DNA in the sample.
[0007] If RNA is to be analyzed the procedures are similar, but more emphasis is placed on purification or other means to protect the labile RNA molecule. RNA is usually copied into DNA (cDNA) and then the analysis proceeds as described for DNA.
[0008] Finally, the amplification product undergoes some type of analysis, usually based on sequence or size or some combination thereof In an analysis by hybridization, for example, the amplified DNA is passed over a plurality of detectors made up of individual oligonucleotide detector fragments that are anchored, for example, on electrodes. If the amplified DNA strands are complementary to the oligonucleotide detectors or probes, stable bonds will be formed between them (hybridization). The hybridized detectors can be read by observation by a wide variety of means, including optical, electromagnetic, electromechanical or thermal means (see e.g., U.S. Pat. No. 5,653,939, U.S. Pat. No. 5,846,708, U.S. Pat. No. 5,965,452, U.S. Pat. No. 6,258,606, U.S. Pat. No. 6,197,503, U.S. Pat. No. 6,448,064, U.S. Pat. No. 6,325,977, U.S. Pat. No. 6,207,3769, U.S. Pat. No. 6,140,045, U.S. Pat. No. 6,066,448, U.S. Pat. No. 5,532,128, U.S. Pat. No. 6,060,023, U.S. Pat. No. 6,203,981, U.S. Pat. No. 6,399,303, U.S. Pat. No. 6,287,776, U.S. Pat. No. 6,338,968, U.S. Pat. No. 6,340,568, U.S. Pat. No. 6,368,795, U.S. Pat. No. 6,376,258, U.S. Pat. No. 6,485,905, U.S. Pat. No. 6,167,748, U.S. Pat. No. 6,123,819, U.S. Pat. No. 6,325,904, U.S. Pat. No. 6,403,317, and all patents and applications related thereto).
[0009] Other biological molecules are analyzed in a similar way, but typically molecule purification is substituted for amplification and detection methods vary according to the molecule being detected. For example, a common diagnostic involves the detection of a specific protein by binding to its antibody. Such analysis requires various degrees of cell separation, lysis, purification and product analysis by antibody binding, which itself can be detected in a number of ways. Lipids, carbohydrates, drugs and small molecules from biological fluids are processed in similar ways. However, we have simplified the discussion herein by focusing on nucleic acid analysis, in particular DNA analysis, as an example of a biological molecule that can be analyzed using the devices of the invention.
[0010] The steps of nucleic acid analysis described above are currently performed using different devices, each of which presides over one part of the process. In other words, known equipment for nucleic acid analysis comprises a number of devices that are separate from one another so that the specimen must be transferred from one device to another once a given process step is concluded.
[0011] The use of separate devices increases cost and decreases the efficiency of sample processing because it is necessary to add dead time for transferring the specimen from one device to another. Further, qualified operators are now required because the handling of the specimens calls for a high degree of specialization due to possible contamination problems. For these reasons an integrated device would be preferred.
[0012] Further, the use of large amounts of specimen fluid is also disadvantageous due to increased reagent costs and increased thermal cycling time. Therefore, in addition to using an integrated device, it would be advantageous to process small quantities of sample.
[0013] Several devices that perform biological analyses have been proposed (see e.g, U.S. Pat. No. 6,303,343, U.S. Pat. No. 6,524,830, U.S. Pat. No. 6,306,590, U.S. 2002, 0,055,149, U.S. Pat U.S. Pat. No. 5,635,358, U.S. Pat. No. 5,726,026, U.S. Pat. No. 5,928,880, U.S. Pat. No. 5,955,029, U.S. Pat. No. 6,184,029, U.S. Pat. No. 6,210,882, U.S. Pat. No. 6,413,766, U.S. 2001 0,029,036, U.S. Pat. No. 6,132,580, U.S. Pat. No. 6,284,525, U.S. Pat. No. 6,261,431, U.S. Pat. U.S. Pat. No. 6,576,549, U.S. Pat. No. 6,180,372, U.S. Pat. No. 6,428,987, U.S. 2001 0,000,752, U.S. Pa U.S. 2001 0,046,703, U.S. Pat. No. 6,379,929, U.S. 2002 0,168,671, U.S. 2002 0,172,969, U.S. 2003 008,286, U.S. 2003 0,129,646, U.S. Pat. No. 5,942,443, U.S. Pat. No. 6,046,056, U.S. P U.S. Pat. No. 6,488,897, U.S. 2002 0,127,149, U.S. Pat. No. 6,167,910, U.S. Pat. No. 6,321,791, U.S. Pat. U.S. Pat. No. 5,856,174, U.S. Pat. No. 5,922,591, U.S. Pat. No. 6,168,948, U.S. Pat. No. 6,197,595, U.S. Pat. No. 6,326,211, U.S. 2001 0,036,672, U.S. 2002 0,022,261, U.S. Pat. No. 6,595,232, U.S. Pat. No. 6,454,945, U.S. 2002 0,100,714, U.S. and applications related thereto) but the devices do not employ truly monolithic structures (e.g., having buried channels or chambers), and thus are more costly to make, more fragile and subject to clogging with glue when the caps are added or when two layers are sandwiched together.
[0014] Further, most devices are not truly integrated. In such cases, it is necessary to provide removable microfluid connections between the different devices, as well as an external micropump for moving the specimen fluid between devices.
[0015] The use of separate devices and removable microfluid connections involves, however, certain drawbacks. Micropump and microfluid connections are difficult to make and frequently leak. In particular, membrane-type micropumps and their valves are commonly used, but are affected by poor tightness. Consequently, it is necessary to process a conspicuous amount of specimen fluid because a non-negligible fraction is lost to leakage. Other types of pumps, such as servo-assisted piston pumps or manually operated pumps, present better qualities of tightness, but currently are not integratable on a micrometric scale.
[0016] The aim of the present invention is to provide an integrated micro-device for bio-analysis that is free from the drawbacks described above. The device can be applied to the analysis of any biological molecule or biological reaction, including nucleic acid such as DNA, RNA or synthetic derivatives such as PNA (peptide nucleic acid), or other synthetic derivatives. Further, by substituting the amplification channels for reaction or purification channels and modifying the detection means, the device can be used with proteins by, for example, antibody detection.
[0017] According to the present invention, an integrated device for nucleic acid analysis is provided, as defined in claim 1.
[0018] As used herein "integrated device" is defined as a single device wherein all sample processing and analysis steps can be performed without physical intervention by an operator, other than electronic control or programming of the analysis.
[0019] As used herein "buried channel" is defined as a channel or chamber that is buried inside of a single monolithic support, as opposed to a channel or chamber that is made by welding or otherwise bonding two supports with a channel or two half channels together.
[0020] "High thermal conductivity" as used herein means a material that provides for very efficient heat transfer, so as to obtain the capacity for thermal cycles with nearly perfect linear profiles, as shown in FIG. 22. As an example of a material with high thermal conductivity, we have used silicon, but other materials such as gallium nitride (GaN), other Group Ill-V and Group II-VI semiconductor substrates, ceramics, and the like may be used.
[0021] In one embodiment, the invention is an integrated micro-device for analysis of a biological specimen, comprising a support having a first tank accessible from outside said support (e.g., an inlet port), a buried channel formed inside a monolithic support, and a detection chamber; each fluidly coupled to the other. The device may also have an integrated micropump on said support for moving a sample fluid through the microreactor. The micropump may be truly monolithic or may be welded to the support. Heaters and sensors may also be provided, and in a preferred embodiment are also integral to the support. However, the micropump, heaters and sensors may also be provided externally (e.g., not on or in said support). Ideally, the support is a material with high thermal conductivity, such as silicon, allowing for excellent thermal response.
[0022] In another embodiment, the invention is an integrated device for analysis of nucleic acid, having a support carrying at least one tank for introducing a biological specimen into said support, at least one pre-treatment channel, at least one buried channel inside said support, and at least one detection chamber, each being in fluid connection with each other. Additional tanks, channels and chambers may be added (or subtracted) as required for the application, and mixing chambers can be formed by the intersection of two channels. Where heaters and/or sensors are integrated into the device, the support is operably mounted on a printed-circuit board, and software and control elements are included. The device may also contain a micropump, preferably an integrated micropump.
[0023] In another embodiment, the inventions are methods of manufacturing or using such devices. A complete portable device, including the various supports described herein (which can be disposable) and having a suitable user interface are also invented.
[0024]FIG. 1 is a three-quarter top perspective view of an integrated device according to a first embodiment of the invention.
[0025]FIG. 2 is a top plan view of the device of FIG. 1.
[0026]FIG. 3 is a cross-section through the device of FIG. 1, taken according to line III-III of FIG. 2.
[0027]FIG. 4 is a top plan view of the device of FIG. 1, sectioned along line IV-IV of FIG. 3.
[0028]FIG. 5 is a enlarged scale view of a detail of FIG. 3.
[0029]FIG. 6 is a bottom view of the detail illustrated in FIG. 5, sectioned along line VI-VI of FIG. 5.
[0030]FIG. 7 is a simplified circuit diagram of the device of FIG. 1.
[0031]FIG. 8 is a top plan view of an integrated device according to a second embodiment of the present invention.
[0032]FIG. 9 is a cross-section of the device of FIG. 8, taken according to line IX-IX of FIG. 8.
[0033] FIGS. 10 to 13 are cross-sections through a semiconductor wafer in successive steps of a process for manufacturing a first part of the device according to the present invention.
[0034] FIGS. 14 to 21 are cross-sections through a semiconductor wafer in successive steps of a process for manufacturing a second part of the device according to the present invention.
[0035]FIG. 22 is a comparison of a thermal profile of a PCR mixture in a typical plastic tube, and the thermal profile under the same cycling conditions of a prototypic silicon channel.
[0036] As illustrated in FIG. 1, an integrated device for DNA analysis (Lab-On-Chip) designated, as a whole, by the reference number 1, comprises a microreactor 2 and a micropump 3. The microreactor 2 is carried on a printed-circuit board (PCB) 5 equipped with an interface 6 for connection to a driving and reading device (of a known type and not illustrated herein). In particular, input/output pins 7 of the microreactor 2 and of the micropump 3 are provided on the interface 6.
[0037] The microreactor 2 has a specimen tank 8 and a plurality of reagent tanks 9 (two, in the example illustrated), which are open on one face 2 a opposite to the PCB base 5 and accessible from outside. The micropump 3 is hermetically seal-welded on the microreactor 2 (see also FIG. 2).
[0038] With reference to FIGS. 3 and 4, the microreactor 2 comprises a first body 10 of semiconductor material, for instance, monocrystalline silicon, and, on top thereof, a first and a second base 11, 12 of silicon dioxide, and a containment structure 13 of polymeric material, for example SU-8. In turn, the containment structure 13 is coated with a protective plate 14, which is open at the specimen tank 8 and the reagent tanks 9. The protective plate 14 is made using a transparent material coated with a conductive film 14′, also transparent, for example, of indium-tin oxide ITO. Alternatively, the protective plate 14 is of conductive glass. A hydraulic circuit 15 is defined inside the containment structure 13 and the first body 10.
[0039] In greater detail, a pre-treatment channel 17, delimited laterally by the containment structure 13, at the top by the protective plate 14, and at the bottom by the first base 11, extends from the specimen tank 8, in the direction opposite to the micropump 3 substantially rectilinearly. Reagent channels 18 of preset length each connect a respective reagent tank 9 to the pre-treatment channel 17. Furthermore, at the outlet of the reagent channels 18, respective mixing chambers 20 are defined.
[0040] One end 17 a of the pre-treatment channel 17, opposite to the specimen tank 8, is connected to an amplification channel 21, which is buried in the first body 10. In particular, the amplification channel 21 extends into the first body 10 underneath the pre-treatment channel 17 and ends into a detection chamber 24 formed in the containment structure 13 above the second base 12. A suction channel 26, which is also buried in the first body 10 and has an inlet into the detection chamber 24, extends underneath the micropump 3, and is connected to the latter via chimneys 23, as explained in greater detail hereinafter. In practice, the pre-treatment channel 17, the amplification channel 21, the detection chamber 24, and the suction channel 26 form a single duct through which a specimen of biological material is made to flow.
[0041] Stations for processing and analysis of the fluid are arranged along the pre-treatment channel 17 and the amplification channel 21; in proximity thereof sensors are provided for detecting the presence of fluid 22 and controlling advance of the specimen to be analyzed. In detail, two dielectrophoresis cells 25 are located in the pre-treatment channel 17 immediately downstream of the specimen tank 8 and, respectively, between the mixing chambers 20. The dielectrophoresis cells 25 comprise respective grids of electrodes 27 arranged above the first base 11 and forming electrostatic cages with respectively facing portions of the protective plate 14. The grid of electrodes 27 are electrically connected to a control device (of a known type and not illustrated) through connection lines (not illustrated either) and enable electric fields to be set up having an intensity and direction that are controllable inside the dielectrophoresis cells 25.
[0042] A heater 28 is arranged on the first body 10 above the amplification channel 21, is embedded in the first base 11 of silicon dioxide and enables heating of the amplification channel 21 for carrying out thermal PCR processes (see also FIG. 4).
[0043] Located downstream of the amplification channel 21 is the detection chamber 24, which, as mentioned previously, is formed in the containment structure 13 and is delimited at the bottom by the second base 12 and at the top by the protective plate 14. An array of detectors 30, here of the cantilever type, is arranged on the second base 12 and can be read electronically. In addition, a CMOS sensor 31, associated to the detectors 30 and illustrated only schematically in FIG. 3, is provided in the first body 10 underneath the detection chamber 24. In practice, then, a CMOS sensor 31 is connected directly to the detectors 30 without interposition of connection lines of any significant length.
[0044] The suction channel 26 extends from the detection chamber 24 underneath the micropump 3, and is connected top the latter by the chimneys 23.
[0045] The micropump 3, which for convenience is illustrated in FIG. 3 in a simplified way, is shown in detail in FIG. 5. The micropump 3 comprises a second body 33 of semiconductor material, for example silicon, accommodating a plurality of fluid-tight chambers 32. In greater detail, the fluid-tight chambers 32 have a prismatic shape, extend parallel to each other and to a face 34 a of the second body 33, and have predetermined dimensions, as will be clarified hereinafter. In addition, the fluid-tight chambers 32 are sealed by a diaphragm 35 of silicon dioxide, which closes respective inlets 36 of the fluid-tight chambers 32 so as to maintain a preset pressure value, considerably lower than atmospheric pressure (for example, 100 mtorr). Preferably, the diaphragm 35 has a thickness of not more than 1 μm.
[0046] As illustrated in FIGS. 3 and 5, the inlets 36 of the fluid-tight chambers 32 are aligned to respective chimneys 23 so as to be set in fluid connection with the suction channel 26 once the diaphragm 35 has been broken. Furthermore, since the micropump 3 is hermetically bonded to the microreactor 2, the fluid-tight chambers 32 can be connected with the outside world only through the duct formed by the suction channel 26, the amplification channel 21, the pre-treatment channel 17, and the reagent channels 18.
[0047] The micropump 3 is then provided with electrodes for opening the fluid-tight chambers 32. In particular, a first activation electrode 37 is embedded in the diaphragm 35 and extends in a transverse direction with respect to the fluid-tight chambers 32 near the inlets 36 (see also FIG. 6). In greater detail, the first activation electrode 37 is perforated at the inlets 36 so as not to obstruct the latter. Second activation electrodes 38 are arranged on a face of the diaphragm 35 opposite to the first activation electrode 37 and extend substantially parallel to the fluid-tight chambers 32. In addition, each second electrode 38 is superimposed to a first electrode 37 at the inlet 36 of a respective fluid-tight chamber 32, thus forming a plurality of capacitors 40 having respective portions of the diaphragm 35 as dielectric.
[0048]FIG. 7 illustrates a simplified electrical diagram of the micropump 3 and of a control circuit 41. In practice, the first activation electrode 37 may be connected, via a switch 42, to a first voltage source 43, supplying a first voltage V1. Through a selector 44, the second activation electrodes 38 can be selectively connected to a second voltage source 45, which supplies a second voltage V2, preferably, of opposite sign to the first voltage V1. In this way, it is possible to select each time one of the capacitors 40 and to apply to its terminals a voltage equal to V1-V2 higher than the breakdown voltage of the diaphragm 35, which functions as a dielectric. Consequently, the corresponding fluid-tight chamber 32 is selectively opened and set in fluid connection with the suction channel 26.
[0049] At the start of the DNA analysis process, a (fluid) specimen of raw biological material is introduced inside the specimen tank 8, while the reagent tanks 9 are filled with respective chemical species necessary for the preparation of the specimen, for instance, for subsequent steps of lysis of the nuclei. In this situation, the inflow of the air from the outside environment towards the inside of the pre-treatment channel 17, the reagent channels 18, and the amplification channel 21 is prevented.
[0050] Next, the micropump 3 is operated by breaking the portion of the diaphragm 35 that seals one of the fluid-tight chambers 32. In practice, by opening the vacuum cell 32, a negative pressure is created and then, after the air present has been suctioned out, the specimen and the reagents previously introduced into the tanks 8, 9 are suctioned along the duct formed by the pre-treatment channel 17, the reagent channels 18, the amplification channel 21, the detection chamber 24, and the suction channel 26. The mass of fluid moved and the distance covered depend upon the pressure value present in the fluid-tight chamber 32 before opening and upon the dimensions of the fluid-tight chamber 32. In practice, the first vacuum cell 32 that is opened is sized so that the specimen will advance up to the dielectrophoresis cell 25 arranged at the inlet of the pre-treatment channel 17, and the reagents will advance by preset distances along the respective reagent channels.
[0051] After a first dielectrophoretic treatment has been carried out, the other fluid-tight chambers 32 of the pump 3 are opened in succession at preset instants so as to cause the specimen to advance first along the pre-treatment channel 17 and then along the amplification channel 21 up to the detection chamber 24. In practice, therefore, the micropump 3 is used as a suction pump that can be operated according to discrete steps. The specimen, whose advance is controlled also by the presence of sensors 22, is prepared in the pre-treatment channel 17 (separation of the reject material in the dielectrophoresis cells 25 and lysis of the cells and nuclei in the mixing chambers 20), and in the amplification channel 21, where a PCR treatment is carried out. Then, in the detection chamber 24, hybridization of the detectors 30 takes place, and the latter are then read by the CMOS sensor 31.
[0052]FIGS. 8 and 9 illustrate an integrated device 100 implemented according to a different embodiment of the invention and comprising a microreactor 102 and a micropump 103, which is similar to the micropump 3 of FIGS. 1 to 5. In this case, a containment structure 104 of plastic or other polymeric material is formed on a PCB 105, which functions as support and is coated with a protective plate 106 having a conductive film 106′ on which the micropump 103 is welded. The microreactor 102 comprises: a specimen tank 107 and a reagent tanks 108; a pre-treatment channel 110, which extends from the specimen tank 107 and ends into an amplification chamber 111; reagent channels 112, which connect a respective reagent tank 108 to the pre-treatment channel 110; a detection chamber 113, arranged downstream of the amplification chamber 111; and a suction channel 115, which extends from the detection chamber 113 and is connected to the micropump 103 through openings 116 formed in the protective plate 106. In addition, a read circuit 117 is carried on the PCB 105 outside the microreactor 102 in the proximity of the detection chamber 1.
[0053] Dielectrophoresis cells 119 are provided along the pre-treatment channel 110 and accommodate electrode grids 120, which form electrostatic cages with the protective plate 106, and mixing chambers 121 are provided at outlet of the reagent channels 108. In addition, a heater 122 is arranged inside the amplification chamber 113. Preferably, a heat sink 123 is connected to the PCB 105 at the heater 55.
[0054] The detection chamber 113 comprises an array of detectors 125 similar to the ones already described, connected to the read circuit 117.
[0055] In the embodiment described, the electrode grids 120 of the dielectrophoresis cells 119, the heater 122, and the detectors 125 are directly printed on the PCB 105. The micropump 103 comprises a semiconductor body 127 accommodating fluid-tight chambers 128 sealed by a diaphragm 130 and having inlets 131 at respective openings 116 of the protective plate 106. The micropump 103 is then provided with a first activation electrode 133, embedded in the diaphragm 130 and extending transversely to the fluid-tight chambers 128, near the inlets 131, and with second activation electrodes 134 arranged on one face of the diaphragm 130 opposite to the first activation electrode 133 and extending substantially parallel to the fluid-tight chambers 128. In addition, each of the second activation electrodes 134 are arranged above the first activation electrodes 133 at the inlet 131 of a respective fluid-tight chamber 128.
[0056] The integrated device according to the invention has numerous advantages. First, all the processing stations necessary for preparation and analysis of the specimen of biological material are made on a single support (i.e., the first body 10 and the PCB 105) and are in permanent fluid connection with one another.
[0057] In particular, also the micropump is directly welded to the microreactor. Thereby, there is no more the need, at the moment of analysis, for connecting devices made on different supports by means of microfluid connections and for handling the specimen of biological material in intermediate steps of the process. Consequently, all the leakage of specimen fluid, which afflict traditional apparatus and which are normally due to imperfect fluid tightness and/or to evaporation, are eliminated. As a result, minimal amounts of raw biological material are sufficient, i.e., of the order of microliters or even nanoliters. Clearly, the use of smaller amounts of specimen fluid affords an advantageous reduction both in costs and in treatment time (shorter thermal cycles).
[0058] In addition, since the device according to the invention carries out preparation, analysis, and moving of the specimen fluid, it is possible to perform DNA analyses even outside of specialized environments or in the absence of qualified personnel. The device according to the invention may also be manufactured at a low cost and is therefore suitable for being used as a disposable product.
[0059] Particularly advantageous is the first embodiment (described with reference to FIGS. 1 to 6) for at least two reasons. On the one hand, in fact, in the amplification channel 21, the high thermal conductivity of silicon is exploited, which enables steep and precise temperature profiles to be imposed during the PCR process.
[0060] On the other hand, the CMOS sensor 31 can be provided in the immediate vicinity of the detectors 30, practically without using connection lines or by providing lines of negligible length. It is known that electronic reading of the hybridized detectors may be based upon different quantities; for example, it is possible to detect variations in capacitance, as in the example described, in impedance, or in other electrical quantities. In addition, reading can be carried out according to different modalities: continuous, dynamic, or by a sweep of variable and controlled frequencies. In all cases, however, very small variations need to be detected. In order to reduce any possible causes of distortion to a minimum, it is therefore extremely important for the read circuit (the CMOS sensor, in the example described) to be as close as possible to the detectors.
[0061] On the other hand, the second embodiment of the invention described enables even simpler and more inexpensive integrated devices to be built.
[0062] Additional advantages derive from the use of the vacuum micropump. First, the micropump is welded in a hermetically sealed way to the microreactor and, consequently, is not subject to leakage. Furthermore, the micropump has no moving parts and does not interact directly with the specimen fluid, so preventing any possible chemical reactions. The micropump is then able to move the specimen fluid in a single direction without the aid of valves and to cause it to advance at each step by a preset distance.
[0063] Both the microreactor 2 and the micropump 3 can then be implemented in a simple way. In particular, a process for manufacturing the microreactor 2 is illustrated hereinafter with reference to FIGS. 10 to 13.
[0064] The amplification channel 21, and the suction channel 26, buried in the substrate 51, and the chimneys 23 are formed. Next (see FIG. 11), after depositing a polysilicon germ layer, not illustrated here, that is removed from the portion of the substrate 51 where electronic components are to be integrated, an epitaxial layer 52 is grown and oxidized on the surface. Then, the CMOS sensor 31 is formed in the monocrystalline portion of the wafer 50; a pad oxide layer 53 is formed, and the heater 28 is deposited thereon. The substrate 51 and the epitaxial layer 53 in practice form the supporting body 10 of the microreactor 2.
[0065] Next (see FIG. 12), a thick layer of silicon dioxide is deposited and defined so as to form the first base 1 1 and the second base 12, on which the electrodes 27 and the detectors 30 are formed. The containment structure 13 is then formed and delimits the pre-treatment channel 17 and the detection chamber 24. In particular, in this step, a polymeric material layer 13′, in this case SU-8, is deposited on the wafer 50 and then defined.
[0066] Then, the body 10 is etched to open up an access to the amplification channel 12 and to the chimneys 23, as illustrated in FIG. 13.
[0067] After bonding of the micropump 3, the detectors 30 are functionalized, i.e., pre-selected segments of DNA or "probes", complementary to the nucleic acid to be analyzed, are anchored. Finally, the protective wafer 14 is bonded over the containment structure 13 and is selectively etched to open up the specimen tank 8 and the reagent tanks 9. Alternatively, the protective plate 14 may be made up of two parts, which are applied for closing the pre-treatment channel 17 and the detection chamber 24, respectively before and after functionalization of the detectors 30.
[0068] Thereby, the structure represented in FIG. 3 is obtained. The method described enables convenient creation of channels on two different levels arranged one above the other (the pre-treatment channel 17, at the more external level, and the amplification channel 21 and the suction channel 26, at the more internal level). The structure thus obtained is compact and of small size.
[0069] The micropump may, instead, be formed following the process illustrated hereinafter with reference to FIGS. 14 to 21.
[0070] According to FIG. 14, a hard mask 62, comprising a silicon dioxide layer 63 and a silicon nitride layer 64, is initially formed on a semiconductor wafer 60 having a substrate 61. The hard mask 62 has groups of slits 65, which are substantially rectilinear and are arranged parallel to one another. The substrate 61 is then etched using tetramethylammoniumhydroxide (TMA) and the fluid-tight chambers 32 are dug through respective groups of slits 65.
[0071] Next (see FIG. 15), a polysilicon layer 68 is deposited and coats the surface of the hard mask 62 and the walls 32 a of the fluid-tight chambers 32. In addition, the polysilicon layer 68 incorporates portions 62 a of the hard mask 62, suspended after formation of the fluid-tight chambers 32. The polysilicon layer 68 is then thermally oxidized (see FIG. 16) so as to form a silicon dioxide layer 70, which grows also outwards and closes the slits 65.
[0072] After depositing a germ layer 71 of polysilicon (see FIG. 17), an epitaxial layer 72 is grown and thermally oxidized on the surface so as to form an insulating layer 74 (see FIG. 18). An aluminum strip is then deposited on the insulating layer 74 and forms the first activation electrode 37.
[0073] Then, an STS etch is performed. As illustrated in FIG. 19, in this step the first activation electrode 37, the insulating layer 74, the epitaxial layer 72 and the hard mask 62 are perforated, and the inlets 36 of the fluid-tight chambers 32 are defined, thus opening again the fluid-tight chambers 32.
[0074] By depositing silicon dioxide at low pressure (for example, 100 mtorr), the diaphragm 35 is then formed, which incorporates the first activation electrode 37 and seals the fluid-tight chambers 32 (see FIG. 20). Consequently, the pressure imposed during deposition of the diaphragm 35 is maintained inside the fluid-tight chambers 32.
[0075] Next, by a new aluminum deposition, the second activation electrodes 38 are formed, and a protective resist layer 75 is then formed and open above the second activation electrodes 38 (see FIG. 21).
[0076] Finally, the semiconductor wafer 60 is cut so as to obtain a plurality of dice, each containing a micropump 3, which is bonded to a respective microreactor 2. Thereby, the structure illustrated in FIGS. 3 and 5 is obtained.
[0077] Finally, it is clear that modifications may be made to the integrated device described herein, without departing from the scope of the present invention.
[0078] For example, the microreactor may comprise a different number or order of dielectrophoresis cells, pre-treatment channels, chambers, reagent tanks, channels, and the like. In particular, the number and succession of electrodes, chambers, channels and their connecting components depends upon the type of treatment to which the specimen fluid is to be subjected. Further, if the sample is premixed with all necessary reagents, the reagents tanks may be eliminated.
[0079] In addition, the microreactor may comprise more than one beater for carrying out different thermal treatment steps (for instance, thermal lysis of the cells, heat denaturation of proteins, and the like) and may also include one or more coolers (for rapid cooling between heating steps, which can shorten the cycle time, and/or protect delicate molecules from degradation).
[0080] Further, although we have described detectors based on hybridization to oligonucleotides, other detection means specific for the biological molecule being analyzed are readily available and each detector technology would require corresponding changes in sensor technology. Also the CMOS sensor could be made in a different way (see e.g., U.S. 2002 0,097,900). For example, it could be manufactured separately, on a dedicated semiconductor chip and then bonded on the body of the microreactor.
[0081] The micropump may comprise a different number of fluid-tight chambers according to the number of steps required by the treatment. The fluid-tight chambers may differ also as regards their shape, dimensions, and arrangement. In particular, the fluid-tight chambers may be arranged according to a matrix array. In this case, the micropump may comprise a plurality of first electrodes 37 (up to the number of rows of the matrix) and a row selector, similar to the selector illustrated in FIG. 7 for selective connection of one of the first electrodes 37 to the first voltage source 43.
[0082] Also, instead of employing dielectrophoresis cells to separate nucleated and non-nucleated blood cells, the device may simply incorporate a chamber to lyse all cells by heat, enzymatic or chemical means. Cell debris can be collected on the chamber walls by charge interactions, may be retained by virtue of exits shaped to retain large debris while allowing small molecules to pass, or can be separated from the nucleic acid via travel through a separation matrix or porous membrane or by electrophoretic transport of the negatively charged nucleic acid.
[0083] Additionally, the microreactor may be coupled to a micropump based upon a different operating principle as compared to the one described herein, such as ferrofluidic magnetic micropumps, electrochemical micropumps, piezoelectric micropump, valve-less planar pumps, and the like.
[0084] Buried channel-based microreactors may be fabricated in a number of ways, in addition to that described herein (see e.g., EP1043770, U.S. Pat. No. 6,376,291, EP1123739, EP1130631, EP1161985, U.S. 2002 0,045,244, and U.S. 2003 0,057,199 and patents and applications related thereto, each incorporated by reference in their entirety).
[0085] A prototype silicon channel was made by bonding 2 etched silicon wafers to produce a 600 μm wide losange shaped channel. A thermocouple was inserted into the channel under oil and the chip placed on a thermo-cycler. Thermal profiles were compared with a regular plastic PCR tube in the same thermo-cycler as shown in FIG. 22. The results confirm that a silicon substrate provides superior thermal performance due to its high thermal conductivity. This will allow the cycling times to be minimized for fastest performance.
[0086] A dummy chip (with no channels) having 18 heating elements and 4 sensors was packaged on an FR4 substrate and isothermy measured by infrared camera. Although the results were somewhat variable, an optimal isothermy of �0.3 was obtained.
[0087] Experiments will be performed in buried channel prototypes complete with thermal resistors in order to determine the minimum cycle time. The preliminary experiments indicate that increased ramp rates, decreased cycle time and decreased denaturation temperatures are possible in the chip (as compared with tube PCR), due to its high thermal conductivity, small size and possibly also due to the surface passivation which may affect the Tm of the DNA molecules in the microenvironment.
[0088] A prototype device was manufactured, as described above, having 20 buried channels and 20 surface detection electrodes. Both PCR and detection were realized on the prototype chip.
[0089] A variety of channel cross sections were tested in prototype devices, and it was discovered that "V" shaped channels (formed by pursuing etching to completion) were superior to trapezoidal channels, in that the channel surfaces were smoother and there were fewer problems with filling, flow, bubbles and sample recovery. In one embodiment, the triangular channels were approximately 200 μm wide by 150 μm deep and contained a total volume of about 3 μl.
[0090] It was also discovered that naked silicon channels, although allowing amplification, produced low yields that could be improved by passivation of the channel surface or washing. Of the various passivation treatments employed, thermal deposition of SiO2 (1 μm) was preferred as least expensive, although other passivation treatments, including silanization and BSA coatings could be effective. Silanization, however, was not compatible with the MICAM process (below).
[0091] For the prototype, MICAM technology (see e.g, U.S. Pat. No. 6,510,237 and related patents and applications) was used to electronically address individual probe detector molecules to specific locations on the chip, although many other technologies are available. The electrodes employed a three layer metallization: Ti for adhesion, Ni as a diffusion barrier and gold for the copolymerization of the pyrrole-pyrrole-DNA probes. The process included Ti/Ni/Au sputtering, photolithography and a final wet etching. Photoresist was found to be compatible with the etching process and was chosen for the prototype wafers. Care should be taken not to overetch the metallic layers.
[0092] The DNA probes were sequentially deposited on the electrodes using pyrrole electropolymerzation at 1V/ECS for 1 second. Experiments with a fluorescent dye confirmed that the polymerization process did not clog the buried channels. Further, the MICAM electrodes were exposed to thermal cycling (30 cycles at 94� C.) and were shown to be compatible with typical cycling conditions. The detectors were able to detect full length biotin-labeled PCR products prepared in a classical tube reaction by hybridization at 42� C. for 1 hour and optical detection using phycoerythrin-streptavidin. The signals were both strong and specific.
[0093] In the prototype model, the detection electrodes were superficial (e.g., on the surface rather than buried). Hence a cap or cover was employed to prevent contamination and evaporation, and it was discovered that a glass cover was not compatible with the MICAM technology. However, the difficulty could also be accommodated by employing a different detection methodology or by using substituting metals or by changing the thickness of the different metallization layers.
[0094] In this prototype, however, a plastic cover (1 mm polycarbonate) was used where applicable. Glues 5008 and 564A from ABLEFIL™ were PCR compatible and were cured at 150� C. or 175� C., respectively, for 2 hours. Care should be taken not to plug the channels during the capping process.
[0095] A test amplification was performed in the channels by filling the channels with PCR mix (target, primers, dNTPs, polymerase, Mg++, buffer and BSA) by capillary action, using a drop of oil to cover the inlet and outlet reservoirs. BSA or another anti-absorbant (such as PVP40, Tween20, gelatin, acrylamide and the like) was found to be required for amplification in the chip environment, and this was believed to prevent adsorption of the enzyme to the surfaces. The chip itself was placed in a thermal-cycler in the preliminary experiments. The cycle profile was typical and products were analyzed by electrophoresis and EtBr stain. Successful amplifications were obtained.
[0096] The use of two thermal zones may be preferred for continuous PCR applications, wherein detection and amplification are to occur simultaneously at two different temperatures. However, in most amplification reactions the two processes occur sequentially and the use of trenches or heat sinks (such as a metal plate) between the thermal zones is not required.
[0097] For continuous PCR an additional prototype with two thermal zones was designed, whereby thermal isolation of the two zones was obtained by back etching and the addition of a heat sink. In the first prototyope, the two heating zones were connected by superficial channels made with SU8 walls and a glass cap. Where back etching of trenches was used to create two thermal zones, simulations showed that each trench contributed to a 10� C. difference in temperature between the two zones.
[0098] Test experiments were performed with fluorescent labeled cells to confirm that red and white blood cells could be separated and lysed in the microchip environment. Superficial channels with a cap and electrodes were configured for dielectrophoresis (DEP) (see e.g., U.S. Pat. No. 6,576,459, U.S. Pat. No. 6,403,367, and all patents and applications related thereto) followed by lysis (see e.g, U.S. Pat. No. 6,287,831, U.S. Pat. No. 6,534,295 and all patents and applications related thereto) further along the channel. Both cell separation and cell lysis were observed by confocal microscopy.
[0099] All patents and applications cited herein are incorporated by reference in their entirety.
Referenced by
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London: At an elegant and colourful ceremony Sunday, the Indian contingent was formally welcomed and ushered into the London Olympics' Games Village here.
Games Village Mayor Charles Allan welcomed the Indian deputy Chef de Mission P.K.M. Raja, athletes and officials at the plush apartment as the tricolour was hoisted while the national anthem was played.
Raja exchanged mementos with Allan and then signed on the peace statue pledging India's support for spreading the gospel of peace and the Olympic movement.
Image: Indian contingent march during the countrys flag hoisting ceremony at the London Olympic Games village.
Text: IANS
Images: PTI/AP

subset: commoncrawl


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You can now invite anyone to meet at your favorite Starbucks.
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45 year old man | Bellevue, WA, USA | Seeking women 35-47 within 50 miles
eat dessert first
hard working chef who likes to have fun enjoys the outdoors and like to meet someone with same interest as me ,some who likes long walks,wine,good food and great conversation, also who can be themseleves around others and laugh and enjoy life as it comes and doesnt mind from time to time just acting like a kid and forgetting about all the troubles and stress of everyday life
Camping , Coffee and conversation , Business networking , Cooking , Dining out , Fishing/Hunting , Gardening/Landscaping , Hobbies and crafts , Movies/Videos , Museums and art , Music and concerts , Exploring new areas , Nightclubs/Dancing , Performing arts , Playing cards , Playing sports , Shopping/Antiques , Travel/Sightseeing , Video games , Watching sports , Wine tasting
Aerobics, Auto racing / Motorcross, Baseball, Basketball, Billiards / Pool, Bowling, Cycling, Dancing, Football, Golf, Hockey, Martial arts, Other types of exercise, Swimming, VolleyBall, Walking / Hiking, Weights / Machines, Yoga
I exercise 5 or more times per week.
I have Dogs
I like Birds, Cats, Exotic Pets, Fish, Gerbils / Guinea Pigs / Etc., Horses, Other, Reptiles
Middle of the Road
new to area but willing to explore with a companion anyone want to get lost while exploring ?.....pike market ,,,,local farmers markets ...Seattle"s different neighborhoods
like to try diffrent cuisines,jump in mud puddles when it rains cuddle on cold nights act like a kid and even alittle insane also like massages and sharing a good wine conversations a great whisky cigar road trips the water
golf, need a partner any takers? ,hockey,baseball,wine tastings,conversation,camping,hiking,cooking,baking,movies going out staying home moonlight walks
mens heatlh guys gourmet
Z{{Cta}} {{#if HasMoreInfo}} ?{{CtaSecondary}} {{/if}}
|What he is looking for
|5'9" (175cms)
|5'5" (165cms) to 8'0" (243cms)
|Body type:
|About average
|Slender, About average, Athletic and toned, A few extra pounds
|No preference
|No preference
|No Way
|No Way
|Social Drinker
|Social Drinker
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enjoy what i do for a living if i could cook for you what would you like ?
|I'll tell you later
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|Have kids:
|Yes, and they live away from home (1)
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|White / Caucasian
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|English, French
|No preference
|Bachelors degree
|No preference


text:
Colombian singer Shakira gave birth to a baby boy at 9.36 p.m in a Barcelona hospital Tuesday was confirmed by her family members. Both the mother and the baby is said to be doing fine. The boy weight 3 kg. At 6:30 pm in the evening, Shakira was admitted to the private Teknon clinic, she was acco
Shakira - Si te vas
Singer Shakira who is pregnant with her first child with boyfriend Gerard Pique posted a picture of baby shower online and asked his fans to honor her child by showering gift and supporting UNICEF underprivileged babies across the globe. Shakira is UNICEF Goodwill Ambassador and she reques
King Khan of Bollywood has roped in sexy Latin singer Shakira to groove with him in the new music video for Kolkata Knight Riders. Shahrukh Khan is a huge fan of Shakira. When Shahrukh approached Shakira, she readily accepted. He will be shooting with the Waka Waka girl after he warps up
Latest Bollywood News
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- Neeraj Pandey's 'Toilet - Ek Prem Katha' to address menace of open defecation
- Aaradhya "off limits" for public, says Abhishek
- 'Calendar Girls' teaser: Bhandarkar captures life behind fame and money
- Shahid marriage: Actor's stepmom Supriya says it's a normal wedding


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Fashion your most stylish self
Culture, tips & hacks for every man.
Modern motherhood. Redefined.
Discover something delicious.
Shape a mindful, balanced life.
Before Leona Lewis took Britian by storm, there was UK singer/songwriter Alesha Dixon. Former member of Mis-Teeq, her tell-it-like-it-is tunes are just as hot as her fashion sense. Inspired by her...


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Houston Gay Pride Parade
The Houston Gay Pride Parade is a Gay Pride festival held annually since 1979 in the Neartown area of Houston. The festival takes place in June to celebrate the lesbian, gay, bisexual, and transgender people and their allies. The route of the parade is usually along Westheimer Road, from Dunlavy Street and ending at Crocker Street. It is currently the most attended and largest gay pride event in the Southwest region of the United States.
This event commemorates the 1969 police raid of the Stonewall Inn on Christopher Street in New York City's Greenwich Village neighborhood, which is generally considered to be the beginning of the modern Gay Rights Movement.
The festivities are held all day on the 4th Saturday of June. The highlight of the event is the parade which is held in the evening after sunset since 1997 (which was arranged by then-Houston City Councilmember Annise Parker where a parade ordinance was revised). Because the event is held at night, the various floats and surroundings are creatively illuminated by the various participants.
Parade organizers (Pride Houston) estimate attendance of 200,000[1]
On September 30, 2014, the Pride Committee of Houston's executive board voted to move the parade to Downtown Houston commencing June 2015 - this was not the first time the board considered the move where it was first discussed in 2008. The 2014 decision resulted in voiced opposition within Houston's GLBT community feeling that the Pride Committee of Houston did not allow transparency concerning community input on having the parade moved where a splinter group will be likely established - much like the former Westheimer Street Festival and the 2001 future of the Houston Art Car Parade (which remained but not affiliated with the Houston International Festival) there has been blog posts about the move - also on social media (Twitter, Facebook) there are those who feel that the GLBT community was cheated. As of February 2015, the proposed Montrose Homecoming Walkabout is in the works to be held in late June.
On June 29, 2015, it was announced that the Houston Pride Festival attracted 700,000 attendees which set a new record.[2]
Entries in the parade are eligible for awards in categories with cultural references significant to LGBT history as well as a judges pick and categories with more localized focus:
- Judges' Choice
- Aria (best creative sound)
- Fabulous (best costumes)
- Rainbow (best lighting)
- Ruby Slipper (best walking)
- Judy Garland (best performance)
- Pink Diamond (best float by a for-profit company)
- Priscilla (best float by a non-profit group)
- Spirit of Montrose (best overall)
Houston Pride Themes & Parade Grand Marshals[edit]
|Grand Marshal(s)
|Honorary Grand Marshal(s)
|Organizational Grand Marshal(s)
|Community Grand Marshal(s)
|Celebrity Grand Marshal(s)
|"United We Stand"
|Thelma Hansel
|"Proud to Be"
|Jim Farmer & Ruth Ravas
|"We the People"
|Ray Hill & Rita Wanstrom
|"A Part Of, Not Apart From"
|Marion Coleman & Andy Mills
|"Unity through Diversity"
|Marion Pantzer & Walter Strickler
|"Unity and More in '84"
|Pokey Anderson & Rick Grossman
|"Alive with Pride"
|Terry Clark & Freda Jerrell
|"Liberty Is In Our Grasp"
|Tony Bicocchi & Dee Lamb
|"Come Out and Celebrate Pride"
|Persons Living With AIDS
|"Rightfully Proud"
|Bruce Cook & Eleanor Munger
|Sharon Kowalski & Harvey Milk
|"Stonewall 20"
|Charles Armstrong & Bettie Naylor
|"Look to the Future"
|Walter Carter & Annise Parker
|Debra Danburg & Craig Washington
|"Take Pride"
|Gene Harrington, Jack Jackson & Linda Morales
|Katy Caldwell & Marvin Davis
|"Pride = Power"
|Sheri Cohen Darbonne & Brian Keever
|Gay & Lesbian Switchboard Houston
|"Out & Proud"
|Brian Bradley & Carolyn Mobley
|Adan Rios
|PFLAG Houston
|"HouStoneWall 25"
|Jay Allen & Cicely Wynne
|Claire Koepsel
|"Silence to Celebration"
|Suzanne Anderson & Don Gill
|Annella Harrison
|"Pride Knows No Borders"
|Bill Havard & Jeanette Vaughn
|Jack Abercia
|The Royal, Sovereign, and Imperial Court of the Single Star
|"Glowing with Pride"
|Deborah Bell & Jimmy Carper
|Krewe of Olympus
|"Unified, Diversified, Electrified"
|Bob Bouton & Jackie Doval
|Barbara Winston
|Texas Gay Rodeo Association
|"Pride, Power & Pizzazz"
|Sean Carter & Nancy Ford
|Colt 45's
|"Take Pride, Take Joy, Take Action"
|Richard Weiderholt & Tori Williams
|Don Sinclair
|People With AIDS Coalition Houston
|"Embrace Diversity"
|Mitchell Katine & Dalia Stokes
|Blake & Gordon Weisser
|PFLAG Houston
|"Pride Worldwide"
|Mela Contreras & Rusty Mueller
|Jane & Irv Smith
|Gay & Lesbian Switchboard Houston
|"Silver Celebration"
|All former Grand Marshals honored
|"Pride As Big As Texas"
|Sonna Alton & Jerry Simoneaux
|Sue & Jim Null
|Lesbian Gay Rights Lobby of Texas
|"Equal Rights! No More! No Less!
|Weldon Hickey & Deb Murphy
|Dennis and Evelyn Schave
|Bayou City Boys Club
|"Say It Out Loud!"
|Phyllis Randolph Frye & Ray Ramirez
|Rev. Marilyn Meeker-Williams
|Bunnies on the Bayou
|"Lone Star Pride"
|Jack Valinski & Maria Gonzalez
|Garnet Coleman
|The Imperial Court of Houston
|"We are Family"
|Dalton DeHart & Kelly McCann
|Julie Eberly
|AIDS Foundation Houston, Inc.
|"Out 4 Justice"
|James Knapp & Fiona Dawson
|Mike & Linda Bratsen
|Legacy Community Healthcare
|"Pride NOT Prejudice"
|Gary Wood & Carol Wyatt
|Ann Robison
|Montrose Counseling Center
|Mayor Annise Parker
|Andy Cohen
|"Live. Love. Be."
|Bryan Hlavinka & Tammi Wallace
|Duane & Judy Roland
|The GLBT Community Center
|Jonathan D. Lovitz
|"Live Out Proud"
|Nicolas Brines, Jenifer Rene Pool and Council Member Ellen Cohen
|Toro, mascot of the Houston Texans
|Madison Hildebrand
See also[edit]
- Gulf Coast Archive and Museum - Houston-based LGBT archive
- Houston Gay and Lesbian Film Festival
- LGBT rights in Texas
- LGBT culture in Houston
External links[edit]
|This Houston-related article is a stub. You can help Wikipedia by expanding it.
|This article about lesbian, gay, bisexual or transgender topics is a stub. You can help Wikipedia by expanding it.
|This festival-related article is a stub. You can help Wikipedia by expanding it.


text:
Staff was extremely friendly when I checked in late at night. They continued to be friendly throughout my stay. The room was very clean and comfortable.
Breakfast area was clean and offered a nice breakfast.
Even though this stay was short, they treated me very well. I will definitely be back.


text:
The UK coroner's report on the deaths of ten RAF servicemen in a Lockheed Martin C-130K Hercules XV179 over Iraq in January 2005 has confirmed that they died as a result of "serious systemic failures" by the Ministry of Defence. Ground fire, including small arms fire, caused a fuel tank explosion that blew off the outboard 7m of […]
Archive | October, 2008
Set up for it
Helicopters need help
That headline may sound patronising, but it's not intended to be. Flying helicopters is difficult. The tasks they are asked to perform are those which no other transport mode could carry out. Helicopters are so expensive to operate they are only called out when nothing else can do the job. The Morecambe Bay report is one of the […]
Taking crew qualifications seriously
Sorry to go on about what Flybe has been doing recently, but actually it's important to the UK airline industry, and other European carriers could learn a trick or two as well. Europe's largest regional carrier has just finished training 21 cabin crew, and the airline has won recognition under the UK's national vocational qualifications system […]
Airport security: why it makes grown men cry
It was at Heathrow Airport that I had a screwdriver confiscated. Okay, you might reasonably say. Unless you knew it was one of those minute devices for tightening the tiny hinge screws in a pair of reading spectacles. It was exactly an inch (24mm) long, plastic-handled, and the metal part measured about a quarter of an inch. […]
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Garee Mel Battad, True Food Kitchen , Fox Restaurant Concepts, THE POINT, El Segundo
The Point (Located at the Northeast Corner of Sepulveda Blvd & Rosecrans Ave)
860 S Sepulveda, Suite 100
El Segundo, CA 90245
True Food Kitchen HIRING FAIR:
WHEN: July 2 – 3 and July 6 – 8
WHERE: The Point is located on the North East Corner of Sepulveda and Rosecrans. Enter at the stop light at the intersection of Village Drive & Rosecrans. Keep to the right, and look for the tent.
Fox Restaurant Concepts is a growing restaurant group encompassing over a dozen unique concepts spanning 8+ states with 4,000-plus employees. Each concept strives to provide guests with a remarkable dining experience that will leave a lasting impression. Varying from contemporary upscale to fast casual and walkup retail, Fox Restaurant Concepts is dedicated to creating flavorful, fresh and innovative cuisine, and offers gracious service in stylish and comfortable settings.
Globetrotting Swedish actress Stina Lundberg
For Swedish actress Stina Lundberg, it all began when her mother put her in dancing school at the tender age of 6 and from there an incredible talent was born.
Stina made it clear that although there wasn't an actual acting school in her hometown, she still managed to snag the lead in all school plays ever produced during the time she was a student. Every chance she had to act, she took describing herself as a wild and unafraid child.
Since then, her career has escalated to amazing heights. She's played the lead role in films like Cheater, Textaholic, Little Screams, The Key and many more. She's been in countless commercials including: Rahja Yoga, Hydroxycut, and Feeding The Actor.
On stage, she has played Cinderella in Happily Ever After, Katharine in Cruel Intentions and Brooke in Other Desert Cities.
Stina says that so far she has two favorite roles that she has played. The first was Sarah, in 'The Uninvited Guest'. She expressed how much she loved this role because the stakes were so high for her and in every scene she faced a surprise. Her other absolute favorite role was playing the lead actress in Bronco and cited she loved it so much because the character was so different from her. She smoked, she didn't have any ambitions, the love of her life left her for someone else and she used another boy to heal that wound. She pointed out both as being two interesting characters to work on.
Proving how in demand she really is, Stina says one of her biggest career highlights thus far was when Innovate International Inc flew her out to Canada to shoot the commercial for Hydroxycut. Her dream is to continue perfecting her craft as a successful actress and keep her fans and audiences glued to their seats. Some of her biggest inspirations include Noomi Rapace for being so gutsy to take on roles very different from her and Meryl Streep for her natural and awe inspiring work. We can't wait to see Stina Lundberg in her next role. This is one beautiful Swedish actress turning heads wherever she goes!
Petra Vasvari – Making a huge mark on the Cinematography industry
Petra Vasyari's visual works are one of a kind. She studied Visual Media Production at University Campus Suffolk. Vasvari said in a recent interview that she has been a creative mind since a very young and that she has always been interested in art. She has worked on multiple commercials, short films, music videos and more. These can be viewed on her website http://www.petravasvari.com/ Vasvari has held a number of different creative positions in filmography. She has been a Production Designer, a Costume Designer, Art Director, and contributed to the success of countless well known productions.
We can find several different genres when searching through her international achievements; short films ("Skyscraper", "Princess", and "The Eucharist") music videos (Angelica Agurbash, Pegasus Warning – Olive Island, Manu Gavassi –Esse Amor Terre, KORR-A – Heart of Glass, Sawyer – Please) feature films; (The Goob, 45 Years, The Spoiler) Velvet stated how she finds it intriguing to switch between genres and that her preference working on a project depends more on the story line and her connection with the idea or concept, rather than the actual genre.
In the same interview Petra Vasvari also highlighted the importance that creativity plays a big part in set designing. Vasvari said "In set design I like how we get a chance to create a space that (hopefully) tells a story on it's own; that allows us to explore, build things and craft a visual story as a team." This quote says a lot about how Vasvari approaches visual concepts in the film industry.
Vasvari's creative mind and fantastic artistic work have earned her respectable titles within the industry. She has worked in multiple areas within film development and according to industry professionals she has been a great asset to the projects. Petra Vasvari is clearly a very talented artist and has had a huge impact on the world of cinematography.


text:
Like many people, I suppose, I had already heard good things about South Jersey's newest upscale daily-fee course, Scotland Run, in Williamstown, N.J.
The word was that it was built on and around an old quarry, making for some beautiful holes and difficult carries over mess you didn't want to get into. It had bent grass from tee to green, which is a must these days. And it was the latest work from Jersey's own Stephen Kay, whose previous courses include the respected, high-end resort tracks Blue Heron Pines and Harbor Pines at the Shore.
But as I stood in the small, temporary pro shop one day in 1998 and plunked down my credit card only to hear, "$85, please," all I could think was: $85? On a Wednesday? This had better be good.
Well, Scotland Run was good. Very good. I wouldn't say it was so good I would pay $85 in the middle of the week too many times, but then maybe owner Chip Ottinger Sr. knows the market for quality daily-fee courses in South Jersey better than I do. And it is true that Scotland Run, open since June 1999, is still a work in progress.


text:
Inition's latest research - an augmented reality / 3D print combination proof-of-concept iPad App. Various augmented layers are presented on to a 3D printed model of the Eli & Edy Broad Art Museum at Michigan State University by architects Zaha Hadid.
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Your WordPress, Streamlined.
I love what you are doing guys, Thanks a lot!
we also got those math-functions after upgrading. When deactivating both Beautiful Math AND Protect plugins in the Jetpack/Settings page the math's no longer were required. We are building a multisite so we had to go and do the same on each site...
- Anne
You will surprise how much time took Jetpack's loading.
On my AVADA theme(3.7.3), It took above 2.5 sec~5sec just for plug in load.
It caused layout crash on old android browser(prior to 4.3).
I have deleted Jet pack. Oh my! All of issues were disappeared.
crashes site, white screen of death. Stay on previous version. Do not upgrade!
Not sure what is going on here, the plugin secretly turned on a maths captcha on one of my sites, the maths captcha code did not work properly, and we were all locked out
so had to delete the plugin from cpanel
not good really
Please solve this math problem to prove that you are not a bot. Once you solve it, you will need to log in again.
Using it for a while now, thank you!
Really good plugin(s). Of course they have the inside track on getting lots of use of their plugin, but unlike many, their stuff is well deserving. I use bunches of their features.
Hi, search with firefox and with chrome Win 7 does not work which is annoying, the plugin removes leading zeros, like letter-spacing: 0.1 px becomes .1px, and even transition 0.2s gets kind of minified to .2s, which is ridiculous. This should have been noticed while testing, It's that much obvious. Which leads me to the thought, that the other plugins might get published without testing, too. I just removed jetpack, it's not safe. I use Simple Custom CSS instead. I have to add the missing 0-value now by hand, I have here more than 1.000 lines to go through. No need to answer this post, thank you, bye.
New update crashed the website. Then Jetpack disappeared from my WordPress admin panel and all the settings along with it.
Seems everyone is having a similar issue.
My team on my website works very hard to drive content and keep track of everything. Jetpack did a good job of this, until now. I am afraid that all that hard work and the settings along with the views tally are now going to be affected and wiped out back to zero.
Tried re-installing Jetpack (because it is not visible and appears to have deleted itself) but I was unable to because the folder exists. I am no longer able to access, delete, or install Jetpack because of this issue.
Over 2 weeks ago you rolled out the update and it updated itself and crashed my site
Finally after spending over half an hour with Bluehost I figured out it was YOUR New update !
After I googled and found all these people complaining
At the moment my jetpack is disabled until I know its going to work
FYI before the update I would have given it a 5 !
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Posted on May 4, 2015 in Body Language Depression
Curses on the May Disorder. Curses on June Gloom. Sixty days before it passes. Sixty not quite miserable, annoying days.
Posted on April 22, 2015 in Anxiety Attitudes Compassion Depression Guilt
The black spiral literally knocked me off my feet. I decided on my own to stop taking Geodon — a horrible drug that left me dizzy for all but the last three to four hours of my waking day — and I crashed and crashed hard. My bedroom was my habitation; my cats my constant […]
Posted on April 17, 2015 in Depression Mania Memory Reflections Stigma
What could they have said to a raging bullshit artist?
Posted on April 11, 2015 in Depression Therapy
You are dealing with an irrational disease not a philosophical system.
Posted on March 31, 2015 in Depression Stigma
Was Lubitz an evil genius? I take exception to the conclusion that his actions were in any way "malicious".
Posted on March 27, 2015 in Depression Stigma Suicide
*TRIGGER WARNING* I must tell the truth here: I do not understand what Andreas Lubitz did. In my suicidal fugues, I thought of many ways that I might kill myself that involved others such as throwing myself in front of a truck or crashing my car into a tree or driving it off a cliff, […]
Posted on March 13, 2015 in Agitation Body Language Depression Weather
It's the damn wind again, a Santa Ana blowing off the mountain and against my door. Combined with the heat, it gives me a headache and a stiff feeling all over my body. Plus I have been sneezing. At first I mistook this for a depression. Friends counseled me to seek out some sunlight. As […]
Posted on February 10, 2015 in Compassion Depression Ettiquette Stigma
When you are depressed, society forces you to lie.
Posted on November 28, 2012 in Depression Disappointment
The brain is a dark country. I travel there alone, lurching over its high roads and through its scourging vales. I believe that what happens to us is only a small part of our moods. The rest is a geology set down when we were in the womb, shaped by the fidgetings of life. At […]
Posted on November 23, 2011 in Bipolar Disorder Depression Grief Mania
Sorry for my absence. I got word a few weeks ago that my mother had a [[glioblastoma]] growing in her head and had only a few weeks to live. Since then, I have been swinging from depression to mania and back again, with a day or two here and there where I feel neither condition. […]
Posted on July 9, 2011 in Depression
I'm surrounded by people who tell me that everyone has a talent. Years of aptitude testing followed by generous explorations into various careers and skills have led me to the conclusion that my special genius is for being depressed about 10% to 90% of the day, depending on whether I have taken my medications, exercised, […]
Posted on June 11, 2011 in Depression Loneliness
Imagine a soft, gray rock domed and striated like a hamburger bun. No life here, you think, then you notice the blue gray beady eyes blink. It opens its maw and takes you in, chewing toothlessly on your chest. Paralysis prevents you from crying. Oh save my soul, oh save my soul, oh save my […]


text: Miss Bellum's face! Flag By: beefsinister (More by author: Most viewed, Newest, Most Thumbs) previous next random - Fav + Fav Comment My life is now complete. Black Maid's Face in Tom & Jerry ---> /funny_pictures/2678575/Black+Maid+s+Face+in+Tom+and+Jerry/ Tags: mis | bellums | power | puff | girls Recommend tagsx +17 Views: 4570 Favorited: 2 Submitted: 10/01/2011 Share On Facebook Remove from favorites Add to favorites Unsubscribe from beefsinister Subscribe to beefsinister


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Nugget makes Fortune's best places to work list again
For the ninth year in a row, Woodland-based Nugget Market Inc. is one of Fortune magazine's best places to work.
The grocer is No. 36 on Fortune's annual list of "100 Best Companies to Work For," released today. Last year, the family owned company placed 37th on the national rankings. It's the only company headquartered in the Sacramento region that appears on the list.
Fortune cites an annual revenue of $280 million for Nugget. The company has 1,151 employees, according to Fortune.
The magazine writes, "This past summer the grocery chain took the entire staff whitewater rafting in the Sierra Foothills and staggered events so that everybody could come."
Founded in 1926, Nugget has nine Northern California locations and three Food 4 Less stores.
Google tops the list, which includes 17 California companies.


text: Jon Stewart on Gun Control by Hilton Hater at January 9, 2013 2:30 pm. Comments Share Tweet Email Jon Stewart tackles the issue of gun control in this segment. Do you agree with his take? Edit Delete Follow Jon Stewart? Star: Jon Stewart Related Videos: Jon Stewart Videos Related Post: Jon Stewart, Plastic Gun Attack NRA, "Paranoid Fear" of Future Uploaded by: Hilton Hater Uploaded: January 09, 2013


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Originally written on Diehardsport | Last updated 2/20/13
It appears that Richard Sherman constantly running his mouth has rubbed another player the wrong way again. Darrelle Revis doesn't seem ready to give up his crown as the best cornerback in the NFL, despite Sherman's belief that he is the best. The Seahawks have reportedly been "highly interested" in acquiring Revis, and I can't imagine that would happen now with Revis and Sherman's feud. Twitter has certainly made sports more interesting with the constant back and forth between athletes now.
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