text:
|Publication number||US6783992 B2|
|Application number||US 09/754,687|
|Publication date||Aug 31, 2004|
|Filing date||Jan 3, 2001|
|Priority date||Jan 3, 2001|
|Also published as||US20020121487|
|Publication number||09754687, 754687, US 6783992 B2, US 6783992B2, US-B2-6783992, US6783992 B2, US6783992B2|
|Inventors||Karla M. Robotti, Hongfeng Yin|
|Original Assignee||Agilent Technologies, Inc.|
|Export Citation||BiBTeX, EndNote, RefMan|
|Patent Citations (38), Non-Patent Citations (4), Referenced by (39), Classifications (20), Legal Events (4)|
|External Links: USPTO, USPTO Assignment, Espacenet|
The field of this invention is micro-fluidic devices.
Carrying out chemical or biochemical analyses, syntheses or preparations, even at the simplest levels, requires one to perform a large number of separate manipulations on the material components of that analysis, synthesis or preparation, where such manipulations include, but are not limited to: measuring, aliquoting, transferring, diluting, concentrating, separating, detecting, etc. One step that is performed in many of the above described processes is the separation of one or more components from a multi-component fluid sample. Such separation steps are common in applications where a given sample is concentrated with respect to one or more of its constituents and/or “desalted.”
In many situations, it is desirable to work with small volumes of fluid, e.g., from femtoliter to μl quantities of fluid. Such situations include sample analysis in which small volumes of initial sample are analyzed; chemical synthesis, in which small quantities of chemical are desired and/or expensive reagents are employed; and the like. As such, there has been much interest in the development of micro-fluidic devices in which fluid is manipulated through one or more micro-channels present in the device.
Many of the above described manipulations easily lend themselves to such miniaturization and integration. For example, the use of these microfluidic technologies has been described in a number of applications, including, e.g., amplification (U.S. Pat. Nos. 5,587,128 and 5,498,392) and separation of nucleic acids (Woolley et al., Proc. Nat'l. Acad. Sci. 91:11348-352 (1994) and hybridization analyses (WO 97/02357 to Anderson).
However, not all of the above described manipulations have been successfully adapted to be carried out in a microfluidic device. One type of application that has not yet had great success in the microfluidic setting is the separation of components from a multi-component sample. While protocols have been developed or suggested to both concentrate and desalt a sample in a microfluidic device, these technologies are not entirely satisfactory for a number of reasons, e.g., unsuitability for use with a wide range of fluid samples, difficulty in fabrication of the microfluidic device, etc.
As such, there is great interest in the development of a technology that can easily and reliably perform sample separations in a microfluidic format. The present invention satisfies this need.
References of interest include: U.S. Pat. No. 5,869,004; as well as Matson et al., SPIE (1998) 3519:200; F. Xiang et al, Anal. Chem. (1999) 71:1485Feil et al., J. Membrane Sci. (1991) 64:283.
Micro-fluidic devices are described in U.S. Pat. Nos. 5,770,029; 5,755,942; 5,746,901; 5,681,751; 5,658,413; 5,653,939; 5,653,859; 5,645,702; 5,605,662; 5,571,410; 5,543,838; 5,480,614, the disclosures of which are herein incorporated by reference.
Reversible gel compositions are described in U.S. Pat. Nos.: 5,720,717; 5,672,656; 5,631,337; 5,569,364; 5,670,480; 5,658,981; 5,470,445; 5,432,245; 5,298,260; 5,162,582; 4,439,966, the disclosures of which are herein incorporated by reference.
Methods for selectively separating at least one component from a multi-component fluidic sample are provided. In the subject methods, the fluidic sample is introduced into a micro-fluidic device that includes at least one micro-valve made up of a phase reversible material. The multi-component fluidic sample is then contacted with the microvalve in the microfluidic device under conditions sufficient for the at least one component to enter, and often pass through, the microvalve, while the remaining constituents of the fluidic sample remain outside of the microvalve. Also provided are kits for use in practicing the subject methods, where the kits include at least a microfluidic device having a microvalve and instructional material, or at least means for obtaining the same, on how to use the device in the subject methods. The subject devices find use in a variety of applications, including sample desalting and concentration applications.
FIG. 1 provides a schematic view of a microfluidic device that can be used to desalt a sample according to the subject invention.
FIG. 2 provides a schematic view of a microfluidic device that can be used to concentrate a sample according to the subject invention.
Methods for selectively separating at least one component from a multi-component fluidic sample are provided. In the subject methods, the fluidic sample is introduced into a micro-fluidic device that includes at least one micro-valve made up of a phase reversible material. The multi-component fluidic sample is then contacted with the microvalve in the microfluidic device under conditions sufficient for the at least one component to enter, and often pass through, the microvalve, while the remaining constituents of the fluidic sample remain outside of the microvalve. Also provided are kits for use in practicing the subject methods, where the kits include at least a microfluidic device having a microvalve and means for obtaining instructional material, or a means for obtaining the same, on how to use the device in the subject methods. The subject devices find use in a variety of applications, including sample desalting and concentration applications. In further describing the subject invention, the subject methods are discussed first in greater detail, followed by a review of various representative applications in which the subject methods find use and a description of the kits of the subject invention.
Before the subject invention is described further, it is to be understood that the invention is not limited to the particular embodiments of the invention described below, as variations of the particular embodiments may be made and still fall within the scope of the appended claims. It is also to be understood that the terminology employed is for the purpose of describing particular embodiments, and is not intended to be limiting. Instead, the scope of the present invention will be established by the appended claims.
In this specification and the appended claims, the singular forms a, an and the include plural reference unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs.
As reviewed above, the subject invention provides a method for separating at least one component from a multi-component fluidic sample. By multi-component fluidic sample is meant a fluidic sample that comprises a plurality of different components or constituents, i.e., a complex fluidic sample. By plurality of components is meant at least about 2, usually at least about 5 and more usually at least about 10, where the sample comprises even more distinct components in many embodiments, e.g., 50, 100, 200, etc. The multi-component fluidic sample is a sample with a low viscosity, where the viscosity of the sample generally does not exceed about 10 cP and usually does not exceed about 5 cP. In many embodiments, the sample is an aqueous sample, by which is meant that it includes water. Representative samples of interest that may be subjected to manipulation by the subject methods include, but are not limited to: physiological derived fluid samples, e.g., blood or a fraction/derivative thereof, saliva, urine, etc., biological samples, e.g., tissue and cell homogenates and derivatives thereof; and the like. The sample that is manipulated via the subject methods may be obtained and processed, where necessary, using any convenient protocol, where the particular protocol employed in any given sample procurement and processing step will necessarily depend on the nature of the sample to be manipulated.
Once the sample is obtained and any requisite/desired processing steps are finalized, the sample is introduced into a microfluidic device having at least one microvalve made up of a phase reversible material. A feature of the subject invention is that the microvalve of the microfluidic device plays a prominent role in the separation of the at least one component from the multi-component fluid sample. As the microfluidic devices, and particularly the microvalve(s) present therein, play such an important and central role in the subject methods, microfluidic devices that may be employed in the subject methods are now reviewed in greater detail.
As used herein, the term “micro-fluidic” device refers to any device in which micro-volumes of fluid are manipulated along a fluid flow path during any given use or operation, e.g., sample preparation, sample separation, chemical synthesis, etc., where “micro-volume” means from about 10 femtoliters to 500 μl, usually from about 100 femtoliters to about 200 μl. The micro-fluidic devices contain at least one fluid flow path through which fluid flows through the device, where a plurality of flow paths that may or may not be intersecting and may be positioned in any convenient configuration may be present in the device. Generally, the micro-fluidic device with which the subject methods are practiced have at least one micro-compartment positioned at some point in the fluid flow path, where the term “micro-compartment” means any type of structure in which micro-volumes of fluid may be contained, and includes micro-chambers, micro-channels, micro-conduits and the like. Depending on the nature of the micro-compartment, the micro-compartment may be the entire fluid flow path through the device, e.g., where the fluid flow path is a micro-channel, or occupy only a portion of the fluid flow path of the device. The term micro-chamber, as used herein, means any structure or compartment having a volume ranging from about 1 μl to 500 μl, having cross-sectional areas ranging from about 0.05 cm2 with a chamber depth of 200 μm to 5 cm2 with a chamber depth of 1 mm; usually from about 10 μl to 500 μl, having a cross-sectional area ranging from about 0.5 cm2 with a chamber depth of 200 μm to about 5 cm2 with a chamber depth of 1 mm; and more usually from about 20 μl to 200 μl, having a cross-sectional area ranging from about 1 cm2 with a chamber depth of 200 μm to about 4 cm2 with a chamber depth of 500 μm. The micro-compartment structure may have any convenient configuration, including square, circular, rectangular, octagonal, irregular etc. Micro-channels or micro-conduits are micro-compartments that are dimensioned or configured such that fluid is capable of flowing through the micro-channel by capillary flow, i.e., the micro-channel is of capillary dimensions. By capillary dimensions is meant a structure or container in which any cross-sectional dimension from one side to another, e.g., diameter, widest point between two walls of a channel, etc., does not exceed about 250 μm. Generally, any cross-sectional dimension of the micro-channel will range from about 10 to 250 μm, usually from about 50 to 200 μm. The flow through the micro-channels may also be pressurized.
The micro-channel(s) of the device may have a linear configuration, a curved configuration, or any other configuration, e.g., spiral, angular, etc., depending on the intended use of the device. In addition, there may be more than one micro-channel in the device, where the micro-channels may: (a) intersect at various points to form complicated flow paths or patterns through the device, e.g., Y-shaped intersections, T-shaped intersections, crosses; and (b) be separated by one or more micro-chambers, etc, depending on the intended use of the device.
In many embodiments, the micro-channel(s) of the microfluidic devices employed in the subject methods, as well as any other components, e.g., entry ports, etc., will be present in an essentially planar-shaped substrate, e.g., a card-shaped substrate, disk-shaped substrate, etc. The substrate may be fabricated from a variety of different materials, including polymeric substrates, such as polyimides, polycarbonates, polyesters, polyamides, polyethers, polyolefins, and mixtures thereof, as well as silicon or silicon dioxide based materials, such as quartz, fused silica, glass (borosilicates) etc, ceramics and composites thereof.
A variety of different micro-fluidic devices have been developed that may be modified to include at least one microvalve for use in the subject methods, where such devices include, but are not limited to, those described in: U.S. Pat. Nos. 5,770,029; 5,755,942; 5,746,901; 5,681,751; 5,662,787; 5,661,028; 5,658,413; 5,653,939; 5,653,859; 5,645,702; 5,632,876; 5,605,662; 5,599,432; 5,585,069; 5,571,410; 5,543,838; 5,540,826; 5,480,614; and 5,458,761; the disclosures of which are herein incorporated by reference. Of particular interest in many embodiments are the μ-TAS devices described in U.S. Pat. Nos. 5,658,413; 5,571,410 and 5,500,071; the disclosures of which are herein incorporated by reference.
As indicated above, at least one micro-valve that modulates the flow of fluid along at least one fluid flow path in the device is present in the devices employed in the subject methods. The micro-valve of the subject devices is characterized by comprising a phase reversible material which modulates fluid flow along a flow path in the device. By phase reversible material is meant a material that changes its physical state, e.g., going from a permeable to an impermeable state or going from an impermeable to a permeable state, in response to an applied stimulus.
As such, the phase reversible material is a material that is capable of going from a first stage that is substantially permeable to fluid, i.e., allows free flow of fluid, to a second stage that is substantially impermeable to fluid i.e., substantially inhibits fluid flow. Specifically, the material is one that is capable of going from a first, porous state to a second, substantially non-porous state. Any phase reversible material may be employed, so long as the material changes in phase in response to an applied stimulus in a manner sufficient to modulate its fluid permeability, i.e., the ability of fluid to flow through the material. Put another way, the porosity of the phase reversible material varies in response to an applied stimulus, such that by applying various stimuli to the phase reversible material of the microvalve, the porosity of the microvalve and therefore its permeability to molecules of different molecular weight, may be varied or modulated, generally in a manner proportional to the applied stimulus and magnitude thereof.
The phase reversible material of the microvalve is a material that responds to an applied stimulus with at least a porosity or permeability change, if not a phase change depending on the magnitude of the applied stimulus. The material may be responsive to a number of distinct stimuli, where stimuli of interest include, but are not limited to: temperature, pH, electrical current, light, magnetic field, etc. Specific materials of interest as phase reversible materials are polymers.
In many embodiments, the phase reversible material is a reversible gel, where by reversible gel is meant a gel composition that is capable of changing its physical state, e.g., from soluble to semi-solid gel state, in response to a particular stimulus, e.g., temperature, pH, chemical agent, electrical current, light, etc. Such gel compositions are known in the art as “smart” gels, “intelligent” gels, hydrogels, etc. The subject micro-valves of this embodiment in which the phase reversible material is a gel composition may comprise any suitable phase reversible gel, as long as the gel is capable of changing its physical state, or at least porosity/permeability, in response to an applied stimulus.
Of particular interest in many embodiments of the subject invention are reversible gels that change their physical state, e.g., change their fluid permeability, by going from a first state of large pores to second state of small pores in response to a change in temperature, i.e., thermoreversible or temperature sensitive gels. The thermoreversible or temperature sensitive gels of the microvalves of the microfluidic devices that find use in the subject invention are those gels that are capable of changing their physical state, e.g., gels that go from a permeable to an impermeable state or an impermeable to a permeable state, over a narrow temperature range, e.g., the lower critical solution temperature (LCST). In the thermoreversible gels finding use as a phase reversible material in the micro-valves of the subject invention, both gels which go from a permeable to an impermeable form as well as gels that go from an impermeable to a permeable form as the temperature increases find use, where in many embodiments, those thermoreversible gels which go from a permeable to an impermeable form as the temperature increases of are particular interest.
A variety of thermoreversible or temperature sensitive gels have been identified and are suitable for use in the micro-valves of the microfluidic devices that find use in the subject invention. Thermoreversible polymeric gels of interest include those comprising polymers such as: partially saponified polyvinyl acetates, polyvinyl methyl ether, methyl cellulose, polyalkylene oxides, polyvinyl methyloxazolidinone, and polymacrylamides, and the like, where polyacrylamides and polyalkylene oxide based polymers are of particular interest.
Specific polyacrylamides of interest include: poly-N-ethylacrylamide; poly-N-n-propyl(meth)acrylamides; poly-N-isopropyl(meth)acrylamides; poly-N-cyclopropyl(meth)acrylamides; poly-N,N-diethylacrylamide; poly-N-methyl-N-ethylacrylamide; poly-N-methyl-N-n-propylacrylamide; poly-N-methyl-N-isopropylacrylamide; poly-N-acryloylpiperidine; poly-N-acryloylpyrrolidine; poly-N-tetrahydrofurfuryl(meth)acrylamide; poly-N-methoxypropyl(meth)acrylamide; poly-N-ethoxypropyl(meth)acrylamide; poly-N-isopropoxypropyl(metho)acrylamide; poly-N-ethoxyethyl(meth)acrylamide; poly-N-(2,2-dimethoxyethyl)-N-methylacrylamide; poly-N-1 -methyl-2methoxyethyl(meth)acrylamide; poly-N-1-methoxymethylpropyl(meth)acrylamide; poly-N-(1,3-dioxolan-2-yl)-N-methylacrylamide; and poly-N-8-acrylyl-1,4-dioxa-8-azaspiro [4,5]decane, N-(2-methoxyethyl)-N-isopropylacrylamide; and the like. Of particular interest in this class of the thermoreversible polymeric compositions are those made up of N-isopropylacrylamide graft copolymers, where polymers of interest include graft copolymers of hydrophobic polymers, e.g. butyl methacrylate; and hydrophilic polymers, e.g. N,N-dimethylacrylamide. See also Takei et al., Bioconjugate Chem. (1993) 4:341-346, which discloses polymers of interest.
Also of particular interest are gels comprising polyalkylene oxides, particularly block copolymers of two or more different polyalkylene oxides, more particularly block copolymers of both hydrophobic and hydrophilic polyalkylene oxides. In many embodiments, block copolymers of polyethylene oxide and polypropylene oxide are preferred, particularly triblock copolymers thereof. Such copolymers are known in the art and sold under the tradenames PLURONIC™ and POLOXAMER™. Specific polyalkylene triblock copolymers of interest include: F-68, F-88; F-98; F-108, F-127 and the like, all available from BASF corporation.
In one type of microfluid device finding use in the subject invention, the micro-valve is made solely of the phase reversible material. In this embodiment, the phase reversible material may be positioned at one or more distinct locations along the fluid flow path, or along substantially the entire fluid flow path. Where the phase-reversible material occupies substantially the entire fluid flow path, during use its phase is generally switched from a fluid permeable to a fluid impermeable state at one or more distinct locations along the fluid flow path, but not along the entire fluid flow path.
Where the micro-valve consists essentially of the phase reversible material, the phase reversible material will be stably associated with the region of the device in which fluid flow modulation is desired. Stable association may be achieved in a number of ways, including bonding, and the like. In many embodiments, the phase reversible material may be bonded directly to the region of interest of the micro-fluidic device, where the nature of the bond may be covalent or non-covalent. For example, where the phase reversible material is a polymeric gel, the polymeric constituents of the phase reversible material may be bonded directly to the micro-compartment wall of the device in the region in which valve fluid control is desired, where the nature of the bond may be covalent or non-covalent, but will usually be covalent. The length of the micro-compartment occupied by the micro-valve in this second embodiment, i.e., the length of the micro-compartment to which the phase reversible material is stably associated, e.g., to which the polymeric components of the gel have been bound and in which the physical state of the gel is controllable, will vary depending upon the desired characteristics of the micro-valve, i.e., strength, rate of fluid flow modulation, etc., but will generally be at least about 50 μm, usually at least about 100 μm and more usually at least about 500 μm long, and may be as long as 1 cm or longer, but will generally not exceed about 10 cm, and usually will not exceed about 5 cm. In the region of the micro-compartment occupied by the micro-valve, the phase reversible material will generally be stably associated, e.g., bonded, to all surfaces of the compartment in a manner that provides substantially no void space through which fluid may freely flow, e.g., the polymeric constituents of a phase reversible gel will be bonded to all of the surfaces of the micro-compartment, e.g., the top, bottom, left side and right side of a micro-channel having a cross-sectional square shape.
In other microvalve configurations, the phase reversible material is present in combination with one or more additional mechanical elements, such as a high surface area mechanical means, i.e., the micro-valve is a composite of a phase reversible material and a mechanical element, e.g., a reversible gel in combination with one or more high surface area components, e.g., rods, pins, etc, such as the structures described in U.S. Pat. No. 5,427,663, the disclosure of which is herein incorporated by reference. This embodiment is further characterized by having the high surface area stably associated with one or more walls of the flow path, as described in greater detail infra. The substantial surface area structures may be fabricated from a variety of materials, including quartz, fused silica, sapphire, polymeric materials, e.g., polyimides, etc. The substantial surface area structures of this embodiment (and therefore the phase reversible material associated therewith) are stably associated with the surface of the micro-compartment in which they are located. Stable association of the structures in the compartment is achieved in a number of ways, such as bonding of the structures to the micro-compartment surface. For example, the micro-valve may comprise a phase reversible material, (e.g., reversible gel) in combination with a plurality of polymeric rods covalently attached to one or more sides of the fluid flow path, e.g., micro-channel, where such a rod configuration is described in Austin et al., Electrophoresis (1996) 17:1075-1079. In such embodiments, the phase reversible material, e.g., the polymeric constituents of the reversible gel, will be attached to the high surface area component, e.g., rod or pin, either non-covalently or covalently, but usually covalently.
In yet another embodiment, the micro-valve comprises a phase reversible material in combination with one more high surface area components, where the phase reversible material/high surface area composite structure is not attached to one or more of the walls of the fluid flow path. Instead, the otherwise mobile or detached composite structure is retained at one or more locations along the fluid flow path with a retaining means, e.g., a mechanical restriction means. Examples of such means include: physical constrictions provided by appropriate configuration of the walls of the flow path, e.g., micro-compartment, in the region in which the phase reversible material is located; stably positioned frits, filters or other solid permeable structures positioned on either side of the phase reversible material in the fluid flow path of the device; and the like. The frits or analogous structural retention means keep the phase reversible material from shifting location in the flow path of the device. The mobile or detached high surface area component of the composite structure in this embodiment may vary widely. Suitable high surface area components of this embodiment include: beads or particles, e.g., porous silica beads, membranes, mesh structures, and the like.
The micro-valves present in the microfluidic devices employed in the subject methods are actuated by an actuation means, e.g., a switch, that is external to the device, where the actuation means actuates a phase reversing means that may be entirely external to the device or at least partially internal to the device. As such, the subject device may or may not further comprise one or more internal components of a means for reversing the phase of the phase reversible material in the micro-valve.
The phase changing means which influences the state of a micro-valve in any given device will necessarily depend on the nature of the phase reversible material in the micro-valve, and will be a means capable of applying the requisite stimulus to the material to achieve the desired phase change. Thus, the phase changing means may be a means capable of applying thermal energy, light, electrical current, chemical agents, hydrogen ions, etc., to the phase reversible material. For example, where the micro-valve comprises a thermosensitive gel, the phase changing means will be a means for changing the temperature of the gel in a manner sufficient to change to the phase of the gel from one state to another, e.g., soluble state to semi-solid or solid state. In other words, the phase changing means will be a means capable of taking the gel above and/or below the phase critical temperature or lower critical solution temperature of the gel. An example of such a temperature changing means is a resistance heater. Another example of a suitable temperature changing means is a Peltier device.
As mentioned above, the phase changing means may be completely external to the device, i.e., the phase changing means may be entirely peripheral to the device, or one or more components of, but generally not all of, the phase changing means may be internal to the device. For example, where the phase changing means is an external heating element on which the subject device is placed during operation, the entire phase changing means is external or peripheral to the device. Alternatively, where the phase changing means includes a resistor element integrated into the device which interacts with external circuitry to provide the requisite electrical current to the internal resistor, a portion or component of the phase changing means is internal to the device.
Microfluidic devices as described above, as well as methods for their fabrication, are further detailed in U.S. patent application Ser. No. 09/294,867, the disclosure of which is herein incorporated by reference.
As indicated above, the first step in the subject methods is to introduce the fluid sample, which or may not have been preprocessed, into the microfluidic device. The manner by which the fluid sample is introduced into the microfluidic device necessarily varies depending on the nature of the device, and may include injection, pipette, valve and the like. The volume of fluid that is introduced into the device may vary, but generally ranges from about 1 pl to 10 ml, usually from about 1 nl to 1000 μl and more usually from about 100 nl to 10 μl.
Following introduction of the sample fluid into the microfluidic device, the fluid is contacted with at least one microvalve made up of a phase reversible material (as described above) under conditions sufficient for at least one component of the multi-component fluid sample to separate from the remaining components of the multi-component fluid sample, e.g., by at least entering the valve, and often passing through it. Contact of the fluid sample with the valve may be achieved using any convenient protocol for moving fluid through the microfluidic devices. In certain embodiments, a differential pressure gradient is created inside the fluid flow channel(s) of the microfluidic device such that the fluid sample moves through the device. One convenient means of moving the fluid sample through the device is to introduce additional fluid, e.g., via injection into the device, etc., behind the fluid sample that moves or flushes the fluid sample along the flow path of the device, e.g., by creating an area of higher pressure behind the fluid sample and a concomitant pressure differential in the flow path of the device.
At some time prior to contact with the microvalve, the microvalve will have been modulated through application of the appropriate stimulus to have a pore size that selectively allows sample constituents that have a molecular weight below a threshold value to pass into, and often through, the microvalve while excluding sample constituents having a molecular weight above a threshold value. The manner in which the microvalve is modulated, e.g., the nature of and the magnitude of the applied stimulus, necessarily depends on the nature of the phase reversible material that makes up the microvalve.
For purposes of further illustration of the invention, the invention will now be further described in terms of microvalves that are made up of phase reversible polymeric materials that are substantially impermeable above a lower critical solution temperature (LCST) and which are permeable to varying degrees below the LCST. It should be understood, however, that the below described methods using such valves are readily adaptable to other types of microvalves, including microvalves that are substantially impermeable below their LCST but permeable above their LCST. Representative phase reversible polymeric materials from which microvalves may be prepared to produce microvalves that are substantially impermeable above an LCST but proportionally permeable as their temperature moves further below the LCST include, but are not limited to: N-isopropylacrylamide-copolymers and the like.
In those embodiments where the microvalve is made up of a polymer whose permeability proportionally increases as the temperature moves below the LCST but is substantially impermeable when at a temperature above the LCST, the temperature of the micro-valve and polymer from which it is made will have been modulated to provide for the desired porosity at some point prior to contact with the sample. For example, where the threshold molecular weight is about 1000 daltons, the temperature of the microvalve will have been changed prior to contact with the fluid sample to a temperature that provides for a pore size in the phase reversible polymeric material of the microvalve that selectively allows passage of molecules that have a molecular weight that does not exceed about 1000 daltons but excludes molecules of higher molecular weight. In many embodiments, this temperature will be a high temperature that is close to, but does not exceed, the LCST of the material. While this high temperature may vary depending on the nature of the phase reversible polymeric material of the microvalve, in many embodiments this high temperature ranges from about 20° to 100°, usually from about 25° to 80° and more usually from about 30° to 60° C.
In other embodiments where the threshold molecular weight is about 10,000 daltons, the temperature of the microvalve will have been changed prior to contact with the fluid sample to a temperature that provides for a pore size in the phase reversible polymeric material of the microvalve that selectively allows passage of molecules that have a molecular weight that does not exceed about 10,000 daltons but excludes molecules of higher molecular weight. In many embodiments, this temperature will be a medium temperature that is substantially below the LCST of the material but above room temperature. While this medium temperature may vary depending on the nature of the phase reversible polymeric material of the microvalve, in many embodiments this medium temperature ranges from about 25° to 70°, usually from about 30° to 60° and more usually from about 35° to 50°.
By selectively modulating the temperature of microvalve, as exemplified above, the porosity of the microvalve can be adjusted to allow selective passage of molecules through the microvalve that have a molecular weight below a threshold amount, and exclude molecules having a molecular weight above a threshold amount. As such, the temperature of the microvalve can be set to provide for a threshold molecular weight value that ranges from about 1000 to 100,000 daltons, where specific threshold values of interest in many instances include: 1000 daltons, 10,000 daltons, 100,000 daltons and the like.
Contact of the fluid sample with the temperature set microvalve as described above results in selective passage of at least one component of the fluid sample through microvalve, assuming a sufficient driving force, e.g., pressure differential, is present in the flow channel of the microfluidic device to provide for the movement of the at least one component at least into, and often through, the valve. Generally, a portion of the components of the sample will move into and through the valve, while the remainder of the sample components, i.e. constituents, will remain excluded from the valve. In this manner, the subject methods provide for selective separation of at least one component from a multi-component fluidic sample, i.e., separation of the at least one component from the remaining components of the multi-component fluidic sample.
The subject methods find use in a variety of different applications in which the separation of at least one component from a multi-component fluidic sample inside a microfluidic device is desired. One representative application in which the subject methods find use is in the desalting of a fluidic sample. In such applications, the fluid sample is contacted inside the microfluidic device with a microvalve whose porosity has been modulated to a have a threshold value of 1,000 daltons. Upon contact and in the presence of a sufficient driving force, e.g., pressure differential, sample constituents having a molecular weight that do not exceed about 1,000 daltons pass into, and through, the microvalve, where such constituents include salts and other low molecular weight analytes. In this manner, the sample is desalted. Another representative application in which the subject methods find use is the concentration of a fluid sample, where by concentration is meant the selective enrichment of one or more of the sample constituents in the sample, i.e., an increase in the amount of one or more of the sample constituents for a given volume of sample. For example, where one wishes to concentrate the sample with respect to all constituents having a molecular weight above 10,000 daltons, the sample is contacted with a microvalve whose temperature is set to provide a threshold value of 10,000 daltons. Contact results in passage of all of the sample constituents having a molecular weight below 10,000 and “stacking” of the remaining sample constituents at the polymer interface. The above describe desalting and concentration applications are merely representative of the different applications in which the subject methods find use.
Also provided are kits for use in practicing the subject methods. The kits typically at least include a microfluidic device that includes at least one microvalve made up of a phase reversible material, as described above. The kits may further include a phase reversing means or components thereof, e.g., a heating means. The kits of the subject invention will also typically include: (a) instructions for practicing the subject methods with the microfluidic device; and/or (b) means for obtaining such instructions from a remote location, e.g., an internet website address, where the website contains the instructions for practicing the methods. The instructions or means for obtaining the same from a remote source will be recorded onto a substrate, where the particular substrate necessarily depends on the form of the instructions/means for obtaining the same. For example, where the instructions/or means for obtaining the same are in a print format, they will typically be recorded onto a printable substrate, e.g. paper, where the substrate may be the packaging, labeling or a package insert of the kit, etc. Where the instructions/means for obtaining the same are present in electronic format, they will be present on an electronically recordable, e.g., computer readable, medium, e.g., floppy disk, CD, digital tape, etc. In addition, the kits may comprise one or more additional elements that find use in the particular application for which the device has been fabricated, such as: elements used in electrophoretic or chromatographic applications, such as a separation medium, labels for use in separation, buffer mediums, and other reagents for practicing electrochromatographic and liquid chromatographic protocols; etc.
The following examples are offered by way of illustration and not by way of limitation.
I. Synthesis of Microvalve Testing Device
A. Synthesis of Micro-Valve on Nylon Substrate
Nylon mesh filters (Spectrum #148130) with 5 μm opening were treated with a solution of 3.72 g of calcium chloride and 3.72 g of water in 20 ml methanol for 20 min at 50° C. The filter was then placed into 20 ml of 3.6 M HCl for 40 min at 45° C. Finally, the substrate was left in water for 20 hr.
This nylon substrate was covered with a solution of 50 mg of sulfo-HSAB dissolved in 5 ml of phosphate buffer (pH 8.5). The substrate was left in the dark at room temperature overnight, then rinsed with water and air-dried.
Approximately 1 g of the temperature-sensitive polymer was dissolved in 10 ml of water. This solution was placed over the azido-activated nylon substrate and exposed to UV light at 265 nm for ca. 30 min from a distance of 3-6 cm. The nylon substrate was then rinsed with water and air-dried.
B. Valve Set Up
A polymer-modified nylon substrates was prepared as described above. The polymer-modified nylon substrate or valve was placed into a high pressure semi-prep filter assembly (Upchurch Scientific #A330) fitted with 2 short capillary lines (in and out). The assembly was fitted into a drilled hole within an aluminum block. The block was controlled by a Peltier device and heat sink. The device allowed warming and cooling of the block and the filter assembly to within 1° C. accuracy. Fluids flowed through the nylon substrate (valve) and filter assembly via applied pressure on the fluid introduction syringe via the entry line.
A. Stock Solution
A stock solution was made as follows:
A 100 ml stock solution containing 5.8 g NaCl; 2 mg Danzyl Phenylalanine; 2 mg Danzylserine; 1 mg Angiotensin (1046 MW) and 1 mg Bradykinin (1060 MW) and water was prepared.
The entrance line to the device described in I.B above was attached to a 5 ml syringe. The device and valve assembly therein was set to 48° C. to render the valve impermeable by substantially closing the pores. The device was maintained at this temperature for 5 minutes. The syringe was then loaded with 5 ml of stock solution and aliquots were run through the valve as follows:
Valve Temperature in ° C.
Collect ˜0.5 ml (vial #)
Each of the resultant vials was then analyzed by HPLC.
C. HPLC Analysis
The LC column was a VYDAC column C-18 peptide/protein column. A sequence of 5 runs was performed on the stock solution described above as follows: injection=5 ml; A gradient solvent system was used that begins with 100% H2O (0.1% TFA) & runs to 50% H2O:CH3CN at the end of the 30 min; this is followed with 5 min 90% CH3CN (0.1% TFA). Column temperature=37°.
Following the above protocol, four peaks were observed: 15 min/21 min/22 min and 23.5 min.
The middle two peaks were confirmed to be angiotensin and bradykinin.
2. HPLC Analysis of Vials Prepared in II.B
All 9 vials prepared in II.B above were analyzed by HPLC. The peptide hormones appears in vials 2 to 7.
Vials 1 to 3 were analyzed by HPLC as described above. Vial 1 shows both amino acids and potentially some peptide. In Vial 2, the peptides Angiotensin II and Bradykinin begin to appear, with the hydrophilic peptide being the more prominent of the two. In Vial 3, all four stock solution components are present.
The above results demonstrate that the porosity of the valve proportionally changes as the temperature decreases and that microfluidic separation according to the methods of the subject invention works.
III. Representative Applications
Sample is introduced into the microfluidic device shown in FIG. 1. The microfluidic device of FIG. 1 has two temperature sensitive valves, V1 and V2. The temperature of V1 is first set just below the LCST such that only molecules having a molecular weight of less than about 1000 daltons are capable of passing through the valve. As sample passes through the device in the direction of the indicated arrows, low molecular weight solutes, e.g., salts, pass through valve V1 and are sent to waste, while the remaining constituents stack up on the upstream side of the valve. The temperature of the valve is then brought down to room temperature and fluid is flushed in the opposite direction through V1 towards V2, carrying the sample constituents towards V2. V2 is set at a temperature that provides for a porosity in V2 that excludes passage of any constituents having a molecular weight in excess of 10,000 daltons. As such, the fluid that passes through V2 and onward for further processing/manipulation in the microfluidic device is one that contains species that range in size from 1000 to 10,000 daltons.
The device shown in FIG. 2 having three temperature sensitive valves: V1, V2 and V3; is used according to the following parameters. In a first state, the temperature of VI is close to the LCST, the temperature of V2 is room temperature and the temperature of V3 is greater than the LCST. In the second state, the temperature of V1 is at a state where molecules in excess of 10,000 daltons do not pass through it; the temperature of V2 is greater than LCST and the temperature of V3 is room temperature. Fluid is passed through the microfluid device under the first state and then under the second state. As a consequence, effluent from V3 is desalted and concentrated, and contains sample components having a molecular weight of from 1000 to 10,000 daltons.
It is evident from the above results and discussion that the subject invention provides for simple and effective methods for selectively separating sample constituents of a multi-component fluidic sample in a microfluidic device format. The subject methods overcome disadvantages associated with placement of traditional chromatographic media into a microfluidic device, e.g., excessive pressure buildup, or placement of dialysis membranes into a microfluidic device, e.g., incomplete sealing of membrane and flow channel, and provide for proportional control of the porosity of the microvalve separation means, which is not achievable with other technologies. As such, the subject invention represents a significant contribution to the art.
All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the scope of the appended claims.
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|U.S. Classification||436/177, 436/178, 204/605, 204/455, 422/504, 422/537|
|International Classification||B01L3/00, G01N30/20, G01N30/14, G01N30/08|
|Cooperative Classification||Y10T436/25375, Y10T436/255, B01L2300/0681, B01L2400/0677, G01N30/08, G01N30/20, B01L2400/0661, B01L3/502738, G01N30/14|
|Jan 3, 2001||AS||Assignment|
Owner name: AGILENT TECHNOLOGIES, CALIFORNIA
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:ROBOTTI, KARLA M.;YIN, HONGFENG;REEL/FRAME:011459/0973
Effective date: 20001227
|Feb 1, 2008||FPAY||Fee payment|
Year of fee payment: 4
|Feb 1, 2012||FPAY||Fee payment|
Year of fee payment: 8
|Feb 17, 2016||FPAY||Fee payment|
Year of fee payment: 12


text:
Building Set Rainbow Lion by Grimms New in 2020
The Grimm’s Building Set Rainbow Lion, comes in a beautiful wooden frame. It combines organic shapes in oiled natural wood and colourful geometric shapes.
This is both a stunning building set and a puzzle and a tribute to an endangered animal.
Frame Size: 44.5cm x44.5cm
27 wooden pieces: H:30mm and 40mm
- Brand: Grimm's
***Limit to 1 per customer - If you purchase more than one, you will be refunded and item re-listed**
This item includes a $30 bulky item shipping surcharge not applicable if purchased in store.
* Please note if this item is out of stock, subscribe to our newsletter to receive updates on our next Grimms release.

domain: design


The St. Patrick's Day Parade in D.C. will close multiple streets on Sunday and will likely create delays.
The Rock 'n' Roll Marathon will close streets on Saturday in D.C., creating delays and snarling Metrobus service
Biltmore Street is closed between 19th Street and Cliffbourne Place NW.
Red line trains are facing 25- to 30-minute delays in both directions due to a disabled train outside Bethesda.
Residential street sweeping is returning to D.C., and with it comes the related parking restrictions.
Slick road conditions due to overnight snow and ice are causing accidents and delays across the Washington area Thursday morning.
Last night's heavy rains may have subsided, but residual flooding, high water and downed trees are causing problems on local roadways this morning.
The District Department of Transportation closed a stretch of the eastbound Southeast Freeway on Thursday evening, and it won't reopen until mid-2014 or later.
We're keeping an eye on the roads and rails this morning.
Several streets near the White House are closed as D.C. police investigate a suspicious car or package in the 1700 block of H St., NW, according to authorities.


Just when we thought life couldn’t get better than bacon floss, along comes cupcake floss.
Seriously silly, like all good things in life, and oddly appealing, we can’t wait to get our pearly whites on this sweet treat that will have us brushing and flossing more times a day than Matthew McConaughey-hey-hey.
While we’re at it, we’ll be scooping up a package of cupcake bandages too. Just in case we get an owie. Which is quite likely as we do strollerskate in our Skechers Britney Spears rollerskates — and don’t know how to stop.
Perhaps these will help us cut down on our real cupcake consumption. Or have the opposite effect. It’s an experiment we’re willing and able to try.
Want in on the experiment?
Wondering where to find caftans? I've rounded up the 100 of the most fabulous caftans in all price ranges, so you can dress like you live in a Slim Aarons photo!read more
How to wear a yellow hostess gown like Veruschka's yellow caftan in Vogue!read more
How to wear a cape dress like a fashion superhero!read more
Best Street Style at New York Fashion Week!read more
Discover a fun styling idea -- how to wear a party skirt in an unexpected way!read more


text:
Allen County Commissioners narrow down jail sites to four
The Allen County Commissioners are narrowing down potential locations for a new jail.
Eight properties around the county have been screened, with officials looking at proximity to medical, judicial and law enforcement facilities. They are also considering impact on existing or potential development as well as the capacity to handle increased traffic.
According to a press release sent out earlier this week, the commissioners are down to four potential sites.
The county decided not to expand the current jail, currently nestled downtown alongside Headwaters Park, and two blocks from the Allen County Courthouse and other county and city offices.
A federal judge ruled earlier this year that Allen County must address overcrowding at the jail in response to a class-action lawsuit alleging poor conditions.
County officials decided that a new jail is the easiest way to meet the requirements of the federal court order. Adding onto the current jail would cost more than $25 million and only add about 235 beds.

domain: news


Water Saving Aerator 2L
|Mixer Thread||Inner Threaded (Female) F24x1|
|Aerator Housing||Outer Threaded (Male) M24x1|
|Stream Pattern||Spray Flow (Rain Spray Stream)|
|Flow Rate at 1-8 Bar Pressure||Max. Flow Rate 2 Litres/Minute.|
|Saving Rate||Water Saving up to 90%|
|Guarantee||5 Year Guarantee|
– Constant flow rate at 2 litres/minute (flow rate per minute)
– Provides water saving up to 90%
– Provides a non-splashing, non-aerated spray (rain spray stream)
– Ensures a virtually constant flow rate irrespective of sudden pressure fluctuations.
– Pressure compensating for constant flow from 1 to 8 bar, mitigating the effects of pressure fluctuations
– Provides a splash free crystal clear (non-aerated) stream
– Considered for drinking water only (certification of materials and products used in drinking water)
– Anti-liming features
– Standard housing (chrome plated brass material)
– 5 Year Guarantee – helps to achieve minimum 2 credit points for “green” building certification
– Water efficiency rate ★★★★★★ (6/6)
– RST® by MANNESMANN Türkiye
Field of Usage:
Recommended for application in series lavoratory faucets (public bathrooms exposed to walk-in traffic and factory facilities).
Mixer and Housing Dimension:
Mixer Thread: Female F24x1 – Housing: Male M24x1.


text: December 18, 2017 "It's a Major Award" Finals Party at Kapones There are 94 stockings there, I promise The CSX Elf was ready for the games to begin I hope you've seen A Christmas Story! CSX Elves reminding you to drink your Ovaltine. They all got their limited edition 30th Anniversary posters Kapones Sports Tavern in St. Clair Shores was packed on Friday night Everyone had a great time Erin made sure our 94 qualifiers knew what to do Having some fun picking up their prizes So many cool prizes Santa helped Erin, Ryan and Dahmer with the Major Award Having some fun with Santa Dahmer had the whole bar's attention Dahmer with a loyal listener Taking a close-up of Santa Who doesn't want an ornament from the best classic rock station in Detroit! Santa and his Elves We Triple Dog Dared him to put on his Pink Bunny winnings He was a good sport and even got a couple free drinks for being so courageous Santa getting ready for the big reveal! It's A Major Award and she won it - a trip to Mexico! Another Major Award winner! Triple Dog Dare winner scored himself an Electric Sex Leg Lamp! This Triple Dog Dare winner better not shoot her eye out!

domain: entertainment


text:
SF Masonic Auditorium | San Francisco, California
Enanitos Verdes comes to Nob Hill Masonic Center on Thursday, 7th July 2022 as part of their U.S Summer tour 2022. The Argentinian rock band will begin the tour on 6th July and will be at several theatres across the U.S before wrapping up on 13th August. They are one of the biggest rock bands that emerged from Latin America and have a massive following in the U.S. The tour features performances in Dallas, Chicago, San Antonio, Boston, L.A and many other cities. Fans are pretty excited about the upcoming tour as Enanitos Verdes is set to display another set of enthralling performances. If you’re a rock fan, you cannot miss out on this! However, you will need to hurry up and buy your tickets ASAP before they are sold out!
Enanitos Verdes is known as the band that transformed and revolutionized the Spanish Genre in the 80s and 90s. The band was one of the most famous Latin pop-rock music outfits at that time, and they have carried on with their performances to this day. They hail from Mendoza, Argentina and have been extremely successful during their career. The band currently consists of Marciano Cantero, Jota Morelli and Felipe Staiti.
Enanitos Verdes has released 15 studio albums and has won several awards. Their 1999 E.P., Nectar, was nominated at the 1999 Grammies. They were the first Argentine band to sign with a U.S. company back in 1988. They signed with Polygram and released their fifth album, Traccion Acustica, with them. It was also nominated at the Grammies for Best Latin Rock Album. They have performed in U.S. several times and often to sold-out shows. A similar response is expected as they are set to celebrate some of their greatest hits and their fans in the U.S. cannot wait for it. If you’re a Latin or Rock music fan, this could be one of the best experiences of 2022. Catch Enanitos Verdes live on 7th July 2022 at the Nob Hill Masonic Center by getting your tickets right now!

domain: geography


text:
Corporate actions have become a focus point in Latin America. The volume of notifications in the region is on the rise, and organizations are devoting more resources to deal with the issue to meet expectations from local and foreign investors. But the problem is international standardization. Should countries adopt the messaging type standard ISO 15022 or wait for the upcoming ISO 20022?
So far the region has adopted a proactive approach. Americas' Central Securities Depositories Association, ACS
Bill Murphy, CTO of Blackstone, once again joins the podcast to discuss the private equity firm's new offices, designed to house its innovations team.Subscribe to Weekly Wrap emails


text:
Sexual interest is a piece this is certainly key of love relationship between a girl and an individual. The bond without the drive that is sexual either company or relationship. The provided attraction that emerges when their along with her eyes satisfy might be the inspiration of intimate interest. It might be unanticipated, or it may accumulate step by step.
In relation to a relationship this is certainly long-lasting the specific situation of low lib > within our article, we’ll shed light with this particular matter.
Sexual drive: how it works and simply simple tips to boost It
Intimate interest or libido is simply a need that is person’s have intimate closeness while using the function of getting pleasure, lessening the worries, and procreation. The need to have sex could possibly be the way of self-assertion or compensation.
Intimate drive is simply thing this is certainly strictly specific. Many individuals have actually bad libido, even though many are prepared to have intercourse as much as possible. Many individuals concentrate on the closeness that is intimate the contrary sex, even though many are intimately thinking about the people linked to the intercourse that is exact exact same. Intercourse love and drive are closely linked, in order that they shouldn’t individually be looked at.

domain: sociology


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This object is in collection
Many previous approaches to detecting urban change from LIDAR point clouds interpolate the points into rasters, perform pixel-based image processing to detect changes, and produce 2D images as output. We present a method of LIDAR change detection that maintains accuracy by only using the raw, irregularly spaced LIDAR points, and extracts relevant changes as individual 3D models. We then utilize ... read morethese models, alongside existing GIS data, within an interactive application that allows the chronological exploration of the changes to an urban environment. A three-tiered level-of-detail system maintains a scale-appropriate, legible visual representation across the entire range of view scales, from individual changes such as buildings and trees, to groups of changes such as new residential developments, deforestation, and construction sites, and finally to larger regions such as neighborhoods and districts of a city that are emerging or undergoing revitalization. Tools are provided to assist the visual analysis by urban planners and historians through semantic categorization and filtering of the changes presented.read less
- Butkiewicz, Thomas, Remco Chang, Zachary Wartell, and William Ribarsky. "Visual Analysis and Semantic Exploration of Urban LIDAR Change Detection." Presentation at EuroVis2008, Eindhoven, NL, May 26-28, 2008.


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Art & Art History
Sistah Sinema Chicago: Thick Relations
400 South Peoria
Gallery 400 is pleased to partner with the UIC Gender and Sexuality Center, Quare Square Collective, and the national organization Sistah Sinema to launch Sistah Sinema Chicago, a monthly moderated film event focused on queer women of color.
Quare Square Collective, established in 2013 on Chicago’s South Side, is a community-based organization that supports LGBTQ artists of color in the Midwest through collaborative projects, marketing, publications and performances. Sistah Sinema Chicago screenings are held at Gallery 400 and will be followed by a moderated discussion to foster dialogue examining the struggles and opportunities unique to queer women of color.
All screenings are at 2pm. Admission is $10 for the general public and free for the UIC community (with I-card). Snacks and non-alcoholic beverages will be provided.
This month’s film, Thick Relations, is an organic blend of documentary and narrative film elements that create a snapshot of an urban, queer community. Featuring a motley crew of compelling queer anti-heroes, the film constructs and calls forth queer accounts of time, space, and relationality that are unscripted by conventional or institutional forms of social belonging. Thick Relations is a Chosen Family affair.
Director Jules Rosskam uses recurring water images as a metaphor for the fluid promises and potentialities of queer desires and kinships, making his film a celebration of the intimacies that exist outside the normative trappings of proper or mature romances. In this way, Thick Relations exists as a kind of Anti-RomCom; it defies narrative resolution and allows for relational ambiguity.
Thick Relations subverts romantic tropes and opts to trace the ways that chosen family arrangements embrace a different logic of love, alliance, and identification.
Rosskam s film is not a place to define things, to define people, or relationships. It is an ambiguous cinematic space where every day tasks, dinner conversations, and sexual themes all run parallel in an open and unresolved fashion. Inside of this current, the film s central characters are permitted to express love in incalculable ways.
The film celebrates a queer counterpublic that embraces desire/pleasure as its primary, orienting ethos. In one scene, someone casually remarks, Once I date someone, they become family. This is the principle tenet that underscores each character s respective journey through the film. How is chosen family built, maintained, and mourned?
A post-film discussion will be moderated by Jackie Boyd (co-star, Thick Relations).

domain: artistic


The video content is inappropriate. Update Unrecognized Zip Code. Rob Pegoraro, The Washington Post. The video content is misleading. Will Greenwald has been covering consumer technology for a decade, and has served on the editorial staffs of CNET.
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Get it as soon as Nov. To record a show I have to leave my box set to the right channel and leave the EyeTV set to record channel 3, which is what the cable comes in as. Page 1 of 1 Start Over Page 1 of 1.
Reviews 0 Write a review. Mac OS X So, after a bit of trouble, it was up and running.
The small external device connects to your computer via a standard USB 2. OK finding the device and installing OK, but not usable Design and Features Without any attachments, the EyeTV Hybrid looks like an upscale USB flash drive, smooth and metallic silver with a distinguishing black line running around the edge.
See any errors on this page? A weird problem seemed to also be what looks like the “old” horizontal hold stutter like the good old days of analog tube TV’s.
The video does not play. What other items do customers buy after viewing this item? And I can watch on my 24 inch screen.
I know it is not advertised as Windows compliant on ElGato’s site, but Microsoft does claim compliancy on it’s site. Share Facebook Twitter Pinterest.
Elgato EyeTV Hybrid
The availability and amount of program information varies depending on the channel. Amazon Music Stream millions of songs. Ships from and sold by transbiz.
The fact that I can’t provide the serial number of my actual device for a download is pretty silly. The antenna was super easy to set up, just pop on the blade and connect to Elgato device and plug antenna into a power source. Use them to connect a set top box to watch cable and satellite TV on your Mac, or to capture video from your old VHS tapes.
Even set up automatic schedules record your favorite series, or create Smart Schedules to record just the shows you like. AmazonGlobal Ship Orders Internationally.
Elgato EyeTV Hybrid Analog/digital TV HDTV Tuner for Mac | eBay
It has performed flawlessly with a few minor setbacks that are understandable, so I can’t really complain too much. Alexa Actionable Analytics for the Web. For warranty information about this product, please click here. Best features are Mac-only.
Elgato EyeTV Hybrid Review & Rating |
The ad does not inform my purchase. EyeTV can even handle the exporting automatically when recordings finish, so it doesn’t take any clicks at all. This allows you to connect a game console to the device so that you can play on your computer’s display.
It’s a great way for gamers who don’t have a TV to play console games on a computer. TV Software for Mac. Most users will likely prefer using the EyeTV with a mouse and keyboard rather than the remote, since it’s a chunky and poorly designed device. Eyev page of related Sponsored Products.


Smart homes for healthier ageing
The UK faces a major demographic shift, with the number of people aged 65 and older growing and the number of young people in decline. This change in the country’s age profile has serious implications for numerous areas of society; the economy, healthcare, pensions and taxation.
As people become older, their ability to live independent lives declines with the onset of a range of health issues. Some will find their mobility increasingly limited as they age. Others will experience a decline in cognitive function with conditions such as Alzheimer’s Disease. A growing number of people will require help to live healthy, independent lives. This demographic shift will mean fewer people in the workforce able to care for the larger number of elderly people who require it.
This paper illustrates how digital technology could augment what humans are able to do to care for senior citizens. The report points out the opportunities that exist for embedding digital technology into more smartly built properties, while we intensify efforts to integrate technology with the existing building stock, such as peoples’ private homes, sheltered accommodation or residential care homes.


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Explore The Mines of Eternal Darkness. It's a lot of fun for the whole party! Expect Mindflayers, Drow Elves and a Good Kickin'.
This is a very cool print on a soft, 100% cotton t-shirt.
For D&D fans everywhere. Impress your DM with this super-fine tee from Stanko's Mead Hall
Available for Lady Warriors and Gentleman Wizards.
Join Joe Blackmagic's Necromantic Academy today for 10% off Speak With Dead and Animation spells. Why not stop by at Stanko's Alehouse for a quick jar or two after a night in the graveyard?
A great Dungeons and Dragons inspired print on a soft 100% Cotton t-shirt. Noice.
Joe Blackmagic is wearing a large tee.
Stanko Steelbeard's Mead Hall invites you to pop in for a drink, some questionable food and maybe a bed for the night. Our prices are competitive and you might just catch Flipwhistle the bard singing songs and telling a few of the old stories.

domain: hobby


Bedfordale sees biggest house price jump in past five years in Perth
The outer suburbs of Perth have enjoyed far bigger price gains than some of the city's more sought-after property hotspots, while bargains can still be found close to the CBD.
Figures from CoreLogic show parts of the Armadale, Swan and Serpentine-Jarrahdale council areas have had house values jump by close to 50 per cent over the past five years as the rest of the city has stagnated.
The biggest increase in house values since 2012 has been in Bedfordale, where they have climbed 49.7 per cent to a median value of $797,621.
To the north-east, house values in Dayton have improved 43 per cent to $393,374 while the neighbouring suburb of Brabham has seen values lift by 34.1 per cent to $395,674.
North Coogee (up 41.5 per cent), Jarrahdale (31.9 per cent) and Oakford (30.5 per cent) all enjoyed strong gains during a period when median house values across Perth actually edged down.
Closer to the city, CoreLogic found a string of suburbs where prices remain low.
Westminster, in the City of Stirling, had the lowest median house value of any suburb within 10 kilometres of the CBD this year at $383,712.
Nearby Nollamara was the second cheapest part of Perth closer to the CBD, with a median value of $404,537 across 202 sales.
Glendalough had the cheapest units of any area close to the city at $289,078.
The figures also confirmed Peppermint Grove as the most expensive part of the State with a median house value of $3.8 million this year. Dalkeith was second at $2.6 million.
Someone selling up in Peppermint Grove would have plenty of cash to invest in Mt Magnet, which had the lowest median house value of any part of the nation. At a median value of $68,605, Mt Magnet was ahead of two other WA centres, Norseman ($80,314) and Kambalda East ($90,978), as Australia’s cheapest.
The most expensive suburb in Australia is Sydney’s Darling Point where the median house price is $5.7 million.
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Songcatcher (2001, 105 minutes)
After her university refuses to grant her a promotion, musicologist Dr. Lily Penleric heads to Appalachia to record 16th century Scottish and Irish folk songs that the earthy mountain people have kept alive for generations. While in Appalachia, Lily joins her sister and the locals in their struggle against the coal companies and falls in love with gritty musician Tom Bledsoe.
- Thursdays at Noon film series - The Triplets of Belleville
- Thursday, May 28, 2015Main Library, Koret Auditorium
- Film: Godzilla
- Saturday, May 30, 2015Western Addition, Western Addition Meeting Room
- Films for Adults: Lady From Shanghai
- Monday, June 1, 2015Golden Gate Valley, Golden Gate Valley Meeting Room

domain: library


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This week I received a call from one of the California Police Activities League phone drones. His tactics were so over the top, and completely inappropriate, that I later did some research after first telling him that the “children” would indeed be going without my donation.
By consulting the authoritative Web site called charitynavigator.org, I learned that the California Police Activities League manages to spend just under 69 cents of each dollar raised not “helping the kids” but rather helping themselves.
Charitynavigator.org uses a four-star rating system to compare various nonprofits. Example: Big Brothers and Big Sisters and also The Boys and Girls Clubs rate four stars (Charity Navigator’s highest ranking). A four-star rating means the nonprofit has been classified as follows: “Exceptional. Exceeds industry standards and outperforms most charities in its cause.”
The California Police Activities League, however, received the following rating: “Exceptionally Poor. Performs far below industry standards and below nearly all charities in its cause.”
I just hope there an adequate number of prison cells reserved at Pelican Bay State Prison for the con artists at the California Police Activities League. Last time I checked, fraud was still illegal.
Message to the California Police Activities League: It might be time to relocate and change your name to the Florida Police Activities League.
— Tim Elliott, Chico

domain: law


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Need 2x 4x4in designs for diy wedding invitations. Rustic themed but not OTT. Simple and classy
28 pekerja bebas membida secara purata $28 untuk pekerjaan ini
Hi how are you ? i am interested in this project and andt to work with you i am expert in this field please give me a chacne Very experienced logo and graphic designer send me a message so we can discuss further
If you're not happy, I'm not finished! I have done this before but I have a few questions, can we chat please? As I'm expert on it with more than 5 years experience. I am looking forward to hear from you.


An English black Labrador named Marlie is lucky to be alive after being bit on the nose by a large rattlesnake at the Mihaich family home just south of Steamboat Springs.
Hunter Mihaich, the son of Patti and Dave Mihaich, was walking down the driveway to close a gate Thursday afternoon when he heard the rattle.
“The dogs looked around and started chasing it,” Hunter said.
Hunter said he yelled to his mother to get a gun, and the dogs started playing with the snake. Before being shot and killed, the snake decided to bite 125-pound Marlie.
The family rushed Marlie to Pet Kare Clinic, where he was treated by Dr. Erica Dissmore. Patti said it was clear the snake had delivered a venomous bite because Marlie’s face swelled up. Marlie also was in shock so he was given fluids through a tube as well as pain medications. Unable to find antivenom at any local veterinary clinics, Pet Kare called Yampa Valley Medical Center.
“The hospital said, ‘Come and get it at the pharmacy,'” Patti said.
Once given the antivenom, Dr. Dissmore monitored Marlie overnight, and he was able to go home Friday, though it was obvious he still was not feeling well.
“He’s not himself, that’s for sure,” Dave said.
While it was thought Marlie was out of danger, the Mihaichs needed to make sure Marlie was kept calm and on a leash.
During the weekend, Dave said he planned to knock down the tall grass around their home just in case there are more snakes hiding.
Patti described the encounter with the rattlesnake as crazy.
“The dogs were standing right next to the kids,” Patti said.
Dr. Dissmore said it is not very common that dogs get bitten by rattlesnakes in Routt County. This was the first one she had seen at clinic in her two years working at the clinic.
Dr. Dissmore said this particular case is a little more concerning because rattlesnakes are not often seen near the Mihaich residence at Deerwood Ranches off Colorado Highway 131 between Steamboat and Oak Creek. Mihaich said this was the second rattlesnake they had seen in three years at the property.
Dr. Dissmore said rattlesnakes are more often found in the area of Sleeping Giant and to the north along the Red Dirt and Mad Creek trails off of Routt County Road 129.
She recommended that if someone has an animal that is bit by a rattlesnake, they should seek medical attention for the animal immediately.
To reach Matt Stensland, call 970-871-4247 or email mstensland@SteamboatToday.com


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Huntsville Police release body-worn camera footage from 2019 officer-involved shooting
Published on November 5, 2021
The Huntsville Police Department (HPD) has released a video presentation that includes body-worn camera footage from the 2019 officer-involved fatal shooting of Crystal Ragland.
Until now, the footage was subject to a federal court order limiting its release. The media, specifically AL.com and Ms. Ragland’s family, requested that the federal court order the City of Huntsville to publicly release footage shortly after the dismissal of a federal civil rights claim for excessive force.
Upon considering the positions of both sides in the case, the court directed its release and the City has complied. Without a court order, the City nor HPD would have released the footage, due to its sensitive and graphic nature. The footage is unedited, but the faces of bystanders have been redacted for their privacy.
Following the incident, an Incident Review Board found that two officers acted within HPD policy, and the Madison County District Attorney’s Office determined the officers’ actions were justified.
In May 2021, Ms. Ragland’s family filed a federal lawsuit against the City of Huntsville and the two HPD officers involved in the shooting. The lawsuit included the federal civil rights claim for excessive force.
The City and HPD officers subsequently moved to dismiss all claims. They contended the use of force was lawful because Ms. Ragland presented an imminent threat of serious harm to the officers when she reached for the visible gun in her pocket during the encounter.
In dismissing the federal civil rights claim, the court determined the use of deadly force by the HPD officers was reasonable and therefore justified under the Fourth Amendment to the United States Constitution based on settled legal precedents, including Graham v. Connor.
Specifically, the court said:
“[A]s pleaded and as the bodycam shows, the officers could see the handle of a pistol protruding from Ragland’s right pocket, and just before the officers opened fire, Ragland reached towards that pocket and appeared to grasp the handle of the weapon. Robinson is correct that the officers did not wait for Ragland to draw her weapon, but the court must view the facts from the perspective of a reasonable officer at the scene. In light of the reports that Ragland had been waving a handgun at others, when she reached for and grasped the handle of her firearm, a reasonable officer, given the circumstances, could have believed that Ragland posed a threat of serious harm. Moreover, under the relevant caselaw, the officers were not required to wait until Ragland had ‘drawn a bead on the officer or others before using deadly force.’ As the [Eleventh] Circuit has found, ‘[t]he law does not require officers in a tense and dangerous situation to wait until the moment a suspect uses a deadly weapon to act to stop the suspect.’ Therefore, because the video evidence shows Ragland reaching for her weapon prior to the officers opening fire, the . . . officers acted reasonably.”
In addition to fulfilling its legal obligation, HPD believes the presentation will provide background and context into events leading up to the tragic incident as well as the immediate aftermath.
“This was a tragic incident, for the victim, for our community and for the police department,” said HPD Public Information Officer Sgt. Rosalind White. “Our sincerest condolences remain with Ms. Ragland’s family.”


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If there’s one place that Scott Disick absolutely loves to party in, it’s Las Vegas. I don’t think there’s a weekend that goes by that he’s not there! The reality television star looked like his dapper self while on the red carpet for the “Hamptons In The Desert” party at the Sapphire Pool and Dayclub in Vegas on Saturday afternoon.
And yes, I totally love the fact that Scott is wearing leopard-print loafers in these pics here. He’s probably the only person on the planet who could pull off the look, too! Check out our photos below and let us know your thoughts!
Photos via FameFlynet

domain: celebrity


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At least one field is required!
we found 2 Jobs
Technical Assistant Grade 2 7 days ago Armidale
NSW Health Pathology
Duties include the performance of venepuncture on Hospital patients as well as our collection centres in Armidale and Guyra....
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domain: medical


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Have you began a new business, or are you considering finally launching that venture that has been in your thoughts for a long time? Many small business owners cover their begin-up prices solely by means of loans, with the expectation that they will start paying back the loans with the profits from their new enterprise. New businesses can take months or years to generate a profit, nonetheless, and loan funds can really turn into a millstone around the neck of a fledgling operation.
Where your company does enterprise could affect your clients’ take on economics, however total, the engrossing pattern I’m seeing is in the direction of more belief in smaller, independently owned firms. In actual fact, communities throughout the US are beginning to map out futures for themselves which might be as self-sustaining as potential. Earlier, I referenced small enterprise house owners undergoing a psychological shift from lone service provider to group advocate, and here, I’ve mapped out a basic mannequin for towns and cities to shift toward independence.
On-line visibility is extremely important for companies at the present time. If your business does not have a website, your presence within your market is basically nonexistent. But the thing is, if you happen to’re wasting our time marketing on podcasts and nobodyÂ from the podcast-listening world ever converts in your website, it’s most likely time to cut off podcasting out of your promotion technique.
Defending your corporation, your self, employees, and customers during this time. Assist local search engines like google and yahoo – and new prospects – find your business on-line. She also shares three elements that small business homeowners should take into accout when creating go to market methods.
Our management group huddles on how we may help less lucky companies. Our social media efforts will spotlight businesses that we can assist. We are engaging in more ZOOM free teaching sessions on leadership to share ideas to calm the thoughts and spirit during this uncertain time.

domain: economics


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A 7 story quiet hotel with all twin type rooms.?We are an affiliate hotel with Daiichi Takimotokan， which enable guests to enjoy the large shared bath as well as other facilities of Daiichi Takimotokan.
Noboribetsu is located in the southwestern part of Hokkaido and is home to many hot springs such as the Noboribetsu Onsen, Karulurus Onsen, and Shin-Dinbim Onsen, as well as beautiful natural scenery of seas and lakes. Parts of the city has been designated as part of the Shikotsu Toya National Park. Located 200 meters above sea level, the Noboribetsu Hot Spring, which is known as a representative hot spring of Hokkaido, is a resort surrounded by old growth forests. The nine kinds of hot springs here all have their own unique characteristics, some are made up of hydrogen sulfide water, some contain salt water, and others contain iron. The excellent water quality here has made the hot springs well known around the world.
The most notable part of the hot spring area in Noboribetsu is Hell Valley. Here, volcanic gas is emitted from the surface of the grayish yellow rocks and the surrounding air contains a strong sulfur odor. The valley is a burst crater with a diameter of 450 meters and the hot springs there produce up to 3,000 liters water per minute.
The northeastern part of the hot springs is the Noboribetsu old growth forest. Mixed with various hardwood trees, it has been designated as a natural monument. The Noboribetsu Bear Park is also located near the forest and is a place where visitors can check out the brown bears native to the area. Looking from the top of the mountains, Lake Kuttara can be seen in the distance.


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Use spicy honey in hot tea, marinades, to coat meats, in salad dressing, with roasted veggies like sweet potatoes, with cheese, etc. Make a compound butter by mixing some with soft butter.
1 cup honey
2-3 teaspoons red pepper flakes, depending on how hot you want it
Heat the honey and red pepper flakes in a small pot over medium-low heat until it just barely begins to simmer. Remove pan from heat and let sit for about 10 minutes to let it steep. Strain into a container through a fine mesh strainer.

domain: food


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As the government proceeds with preparation for negotiating the Wafi-Golpu, Woodlark and Kainantu gold mines MOA reviews and other projects in the pipeline, it will be interesting to see how these negotiations take place and how agreements are entered into.
Head of the National Secretariat of the Extractive Industries Transparency Initiative (EITI), Lucas Alkan, made the remarks following the government’s recent announcement in the media about the upcoming project MOA reviews.
“In this project development process, transparency mechanisms must be considered and built into these project agreements and the MOA reviews,” he said.
Mr Alkan said the government’s commitment to implementing EITI will not increase transparency overnight, however, it will assist the government to manage the extractive sector in a more inclusive and transparent manner.
He added that highlighting transparency will also assist in minimising public speculations and distrust towards the government.
“The EITI framework works to assist in bringing various sectors of the economy together and builds trust amongst them and creates a platform for these issues to be discussed so that solutions could be found together for better management of the sector.”
Now that PNG is an EITI implementing country, the government should not lose traction on EITI implementation.
It stands to gain from implementing EITI because of the potential governance gains associated with it.
Mr Alkan said that PNG still has a lot of work to do when it comes to citizen engagement.
“According to the World Bank’s World Governance Index 2018 edition, PNG scored very low on the “voice and accountability” indicator which indicates weak performance,” he said.
“As an EITI implementing country, mining and petroleum impacted communities and civil societies have a platform to access information and constructively engage with companies and the government on resource governance issues,” Mr Alkan said.

domain: politics


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All the CHEN family want is a quiet life but as daughter Lucy’s behavior changes from strange to violent, they are forced to confront dark secrets they have hidden for years. One by one, their desperate poverty, the mother’s rape and the father’s revenge by murder return to haunt them…until a searing TRUTH no one knew is revealed. Will this truth give them a second chance to right the wrongs in their lives or destroy them all?
Like millions of kids around the world, Santiago harbors the dream of being a professional footballer…However, living in the Barrios section of Los Angeles, he thinks it is only that–a dream. Until one day an extraordinary turn of events has him trying out for Premiership club Newcastle United.
When trucker, Jack Burton agreed to take his friend, Wang Chi to pick up his fiancee at the airport, he never expected to get involved in a supernatural battle between good and evil. Wang’s fiancee has emerald green eyes, which make her a perfect target for immortal sorcerer, Lo Pan and his three invincible cronies. Lo Pan must marry a girl with green eyes so he can regain his physical form.


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Positive effects of interracial dating
These are not fact lists to apply indiscriminately.
What does bother me, however, is how she presents the information. It was also a blind date — which, as anyone will tell you, can be a thorny situation no matter where in the world that date happens.
She titled the post “On Dating Chinese Men.” A title like that suggests that the author is bringing a great deal of experience to her topic — that perhaps she has dated a number of Chinese men, or at least dated one long enough to get a better sense of the dating culture, and she wants to tell others about that overall experience. Now, she does preface this date as her “first Chinese date,” suggesting she probably did have more than one experience.
Additionally, I also posted about memoirs that feature Asian men and Western women in relationships, since many of their experiences have resonated with me and others in the community.
One of my favorites, which I frequently recommend for anyone dating an Asian guy in a Western country, is Diane Farr’s . Another great way to explore dating Chinese men is through the many other women who have been there and written about it.
The Muricay Beach is located further away, at 4 kilometers, but the white sands and clear seas more than make up for the distance.