text:
Disc Fanatics of Kansas
76 following
Alan Fox April 14, 2013 at 5:45am
I posted pictures of all the holes at Westmoreland's course today was a good time and if anyone was interested now you can see before you go
Josh Parker April 13, 2013 at 1:39pm
Grats to Alan on his skip Ace!
woot woot!
In the bucket. Well, tub:) Good job Alan.
thanks man lol short hole but still counts!
Bang! What disc?
roc from Break the Chains!
Travis Bailey April 12, 2013 at 9:01pm
Left a Purple Lucid Escape on hole #14 at Tuttle on Thursday night by accident. If you happen to stumble across it please let me know!
Will do Travis. hopefully someone gets it back to you.
found it was actually on hole #12 tried calling your phone must of been off
Awesome. Must have been. Thanks! Try texting again today and I can stop by and grab it or something?
sorry i worked 3-11 but will be playing tuttle between ten and 1
and will give u a buzz
On Sunday? I'd love to meet you there but I am going to watch the GBO in Emporia.
Rick Petrie April 10, 2013 at 11:09pm
Good luck to all DFOK members who are playing in this weekends Glass Blown Open.
That's a big time tournament that takes a big time performance to compete.
For those of you who are not playing, it's totally worth it to go watch for a while.
Go see the biggest pro's in the sport... I highly recommend watching them play the Country Club course.
Mark Kewley April 10, 2013 at 9:20pm
Looks like the GBO player's pack is nice: http://www.dgcoursereview.com/forums/showthread.php?t=83557&page=5
Mark Kewley April 9, 2013 at 10:27pm
I ordered a DFOK shirt like this today: http://www.discgolfscene.com/profile/10252/pictures/album_7907/145683#145683
at GTM. They have the logo in the system if you want one. The shirt has to be Green, Black, or White only (DFOK colors).
what was the cost?
The shirt I got was a little less than $30 with tax. It is dri fit type with collar. They have other styles that may be + or -.
Well, if you guys want to order a bunch (as in talk about it the next couple of nights at league) I work at GTM and get a bit of a discount on team orders on top of them already being cheaper when bought in bulk. Just a thought.
AND, if DFOK is okay with it you can get sport grip in just white or black or both for cheaper than what I'm assuming is embroidery Mark?
im down for a shirt. i vote black or green shirts
Wow! I got a call before noon that my shirt was ready! At one time it was decided that only Black, Green, and White were ok. Someone is wanting to include another color. Probably need some sort of group decision.
Josh Carroll April 9, 2013 at 8:43pm
I'll be bringing a box full of plastic tomorrow for Warner Wednesday ::: Innova, Discraft, and Millennium ::: Also have some leftover Gateway Karmas from the Spring Invitational ::: Price Range $10-17 (see my photo albums for a list of inventory)
Kevin RevKev Clark April 9, 2013 at 3:29pm
Just wanted to let you all know that Registration for the 3rd Annual Break the Chains tournament is now open. Go to KSULCM.ORG for more info.
Awesome, registered and paid, thanks Revkev!
league is still on right
Is the $15 price just for KSU students?
Does Open play for cash? Or no prize at all?
Travis Bailey April 7, 2013 at 10:02pm
Put this together earlier.
Rick Petrie April 7, 2013 at 7:20pm
With our Fairmont Fundraiser 3 Disc Challenge coming up, I thought I should start figuring out what driver(s) I want to order.
To do so I'm planning on practicing with what I already have, plus trading or borrowing a few discs with fellow DFOK members...
the Astra I got yesterday was straight flying and hauls ass! I loved it
Ya. I've aquired 3 Astras over the last couple years. I'll be dusting off a couple of those to see how they do too. What weight Astra was you throwin'?
If you are looking for an overstable mid range I've got a sentinel you can borrow.
172 Rick
Thanks Alan. Both of mine are 175. Travis, I appreciate the offer but I think the Sentinel's are going to be too overstable for me.
Rick Petrie April 7, 2013 at 1:18pm
For those of you who care to post their scores (or even just reviews of the layout) from yesterday's DU Open tourney, I added a 20 hole layout option to the course in Herrington.
I think you need to take out the spaces between the hole numbers on your custom layout. Might be causing the funky look when it shows up in the News feed.
Good call Josh. I made that change & it worked... except it does not apply to scores already entered (so I removed & reposted my scores)
Rick Petrie April 6, 2013 at 11:30pm
It was awesome to see so many DFOK members at the DU Open today!
How cool was it to hear the Tournament Director, Dennis Vahsholtz, say "Disc Golf is alive & well in Manhattan."
congrats to rick and kewely for getting 1st and 2nd in the am masters division
Thanks Dakota. Hope to see you at the new & improved Fairmont Park on May 4th for our 3 Disc Challenge (if not sooner).
Thanks Dakota, That tournament is always a blast. I'm glad I managed to keep it together.
Travis Bailey April 6, 2013 at 2:04am
When is check-in for DU Open? I'm finding no information online...
We are planning to get there around 9:00
i heard check in was 9. remember an hour drive from manhattan
Rick Petrie April 4, 2013 at 11:04pm
Shout out to the following people for coming out to work at Fairmont Park tonight.
Josh Carroll
Kevin Clouse
John Helle
Mark Kewley
Tyler Lett
William Mitchell
Thad Pultz
Josh Parker
Daisuke Takahashi
Bryce Williams
A little bit of sweat equity by each of us went a long way tonight. Good job!
great work today, when there is turnout like that then project will just fly by!
Yep. Strength in numbers! (please forgive me if I forgot to name anybody)
Jeremy was there with Tyler. Working? :)
Oops, I missed Jeremy.
I missed Bill Pryor too.
Josh Parker April 1, 2013 at 9:11am
Happy Birthday Today Alan Fox
Alan Fox March 31, 2013 at 11:46pm
Just so everyone knows this weekend at tuttle we set our lights out for night golf Friday and left them out to play again Saturday and I had 5 of my flashlights stolen off the baskets and a few of the milk jugs were destroyed. Just a heads up in case other people had this idea pick your lights up each night
Travis Bailey March 31, 2013 at 7:58pm
Anyone going to the DU open this weekend? If so, any idea when it will end? I'm super interested in going, but need to be home on Sunday by 6:00. Thanks!
I've played in it all of the past three years & they keep it rolling. I don't know exactly what time it usually ends... maybe 4:30ish? Jon Tveite probably knows though. He had to get back to MHK at a specific time last year.
...and by Sunday, do you mean Saturday? :P I'm planning on going!
Haha yeah, Saturday. Thanks, I'll definitely consider signing up now.
I made it back by 6, but I had to miss the awards. Luckily some DFOK guys collected my prizes for me. I think I left Herrington around 5:15.
Cool. Thanks!
Dimond Cutter March 30, 2013 at 11:41am
Who, when, where???? I originally thought Moon Lake this afternoon but I am open fot anything!
I am in for whatever and whenever
some people are meeting at Fairmont to start taking closer looks at fairways late this morning and into the afternoon. however, this won't really be "playing" per say...
I rented a cabin for my bday weekend we are playong day and night at tuttle all day!
im going to tuttle , 1245 ish.
Have a good time up there guys. Be sure to play for tags... (ha)
Happy Birthday Alan!
Damen W March 25, 2013 at 9:22pm
after some heated discussion......the lions club decided to fund seven more baskets tonight and 3 additional sleeves which will give us five alternates now. Hopefully everything will be finished by the end of april with a three weeks to practice before the tournament.
great work!
Mark Kewley March 23, 2013 at 2:17pm
It looks like Monkey Island is Cancelled: http://www.pdga.com/tournament_results/101623


text:
The official Bulgarian website of car manufacturer Renault (Renault.bg) has been breached and several of its subdomains have been defaced by a hacker of the Turkish Ajan Hacker Group that goes by the name of Maxn3y.
The hacker leaked the email addresses of over 6,000 users who had subscribed to the site's newsletter. He also published the usernames and encrypted passwords of the website's administrators, CWN reports.
Besides the Renault subdomains, the hacker also defaced the Bulgarian site of Romanian car manufacturer Dacia (dacia.bg). It's worth noting that Dacia is also owned by Renault.
At the time of writing, the defacement pages have been removed from the sites of Renault and Dacia.
Judging by the hacker's Zone-H account, over the past period he has focused his efforts on car-related websites. Over the Christmas holidays, Maxn3y has also defaced the site of a German dealership and the one of a Russian automobile club dedicated to Subaru.

subset: commoncrawl


text:
By Ann-Sofie Ruf
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text:
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We are all home!
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Replies (1-10):
Quoting Clubpenguin:
Oh my goodness. How adorable. I love the purple sweater.
Thank you. I made it just for her to come home in and it just barely fit her she was so big, lol
Quoting Godspitgrl:
Opps didn't mean to reply twice. You can tell someone made it it's lovely and congratulations.
by Jill - Admin on Apr. 28, 2012 at 9:18 PM
Awww yay! She's sooo cute! I love her chubby chubby cheeks! Glad you're home. I'm sure you're happy to be!
by Gold Member on Apr. 28, 2012 at 9:46 PM
Thank God! Glad to see your all doing well!
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text:
|Publication number
|Publication type
|Application number
|Publication date
|May 10, 1994
|Filing date
|Aug 27, 1991
|Priority date
|Jun 15, 1990
|Fee status
|Also published as
|Publication number
|Rick T. Smethers, Lev J. Leytes, Brian D. Warner, Robert R. Shadel, Michael S. Urdea
|Original Assignee
|Chiron Corporation
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (15), Referenced by (121), Classifications (20), Legal Events (8)
|External Links: USPTO, USPTO Assignment, Espacenet
This application is a continuation-in-part of U.S. application Ser. No. 539,002, filed Jun. 15, 1990, now abandoned.
The present invention relates to an assembly for assaying an analyte in a liquid sample, and in particular, to a self-contained assembly which provides prepacked reagents which can be added to the sample in a defined sequence.
Many analyte assays involve a sequence of reaction steps in which an analyte is reacted sequentially with two or more, reagents. Typically in this type of assay, a reagent is added and the mixture is allowed to react for a preselected time before addition of the next reagent. Where the assay involves analyte binding to a solid support, the reaction chamber containing analyte bound to the support may be washed between reagent-addition steps.
Ideally, it is desired to carry out multiple-addition assays of this type in a simple self-contained device which can be adapted for automated or semi-automated operation in a clinical setting, or alternatively, may be reliably practiced in a home or doctor's office setting by simple assay procedures.
Heretofore, a variety of self-contained diagnostic devices which provide multiple, prepackaged reagents have been proposed. U.S. Pat. No. 4,806,316 describes a disposable diagnostic device in which a liquid sample is distributed to multiple reaction wells which may contain prepackaged reagents for reaction with the analyte. The device may be adapted for sequential-reaction assays by transferring each sample successively through two or more wells, where the different wells contain the desired prepackaged reagents.
U.S. Pat. No. 4,519,981 shows a rotary diagnostic device containing a series of side-by-side, radially overlapping reaction chambers, each of which may contain a prepackaged reagent. The cells are interconnected to allow sample fluid flow from through each chamber to a successive chamber by centrifugal motion alternately in opposite directions. That is, fluid sample is moved from a first chamber to a second, side-adjacent chamber by rotating the device in one direction, and after a selected reaction time, the sample fluid is moved from the second-chamber to a side-adjacent third chamber by rotary motion in the opposite direction.
Another multi-reagent rotary device is disclosed in U.S. Pat. No. 4,390,499. The device includes a series of compartments separated by rupturable seals. In operation, pneumatic and centrifugal forces are used to break the seals between compartments, for sequential mixing of the reagent material with the sample.
U.S. Pat. No. 4,469,793 discloses a rotary diagnostic device in which a sample fluid is deposited in a radially inner well, and distributed in a predetermined volume to each of a plurality of outer receptor cells under centrifugal forces applied by spinning the device in one direction to force the sample first into overflow wells, then in the opposite direction to dispense the predetermined volumes of sample into the outer cells. The compartments of the cells may be supplied with different liquids for use in simultaneous analyses. Also disclosed in this patent is a solid reactor bead which is designed to pick up successively, a quantity of the analyte being measured, then a quantity of a reagent containing a biological indicator.
The above-described assay devices all involve relatively complicated pressure and/or centrifugal force mechanism for releasing prepackaged reagents into a sample or for transferring the sample from one reagent chamber to another. For this reason, the devices may be relatively expensive in manufacture, and also may be unreliable in operation, due to variations in centripetal or pneumatic force applied to the device during operation, or variations in the force needed to rupture seals or advance the sample between chambers, and/or variable volume losses which occur as a liquid is transferred from chamber to chamber.
The invention includes, in one aspect, a self-contained assembly for detecting an analyte ligand by detectable, analyte-specific binding to a solid support. The assembly includes a reaction plate having a well containing the solid support, and a transfer plate containing first and second reagents contained in separate first and second reservoirs. The transfer plate is mounted on the reaction plate for movement thereon to a sample-addition position, at which sample can be added to the well, one reagent-transfer position at which one reservoir is aligned with the well, a wash position at which wash solution can be introduced into the well, and another reagent-transfer position at which the second reservoir is aligned with the well. The transfer and reaction plates are designed to prevent release of reagents from their corresponding reservoirs until the associated reservoir is aligned with the well.
In one embodiment, the reagent is in the form of a liquid solution, the reservoir containing the solution includes a channel formed in the plate, and the channel is sealed at its opening in the transfer plate. Preferably the channel and seal are formed by an elastomeric sleeve held in a channel within the transfer plate, and the confronting planes of the transfer and reaction plates are spaced, adjacent the region of the sleeve, to form a capillary lock, to prevent liquid from leaking from the reservoir by capillarity. Also in a preferred embodiment, the transfer plate includes a chamfered opening communicating a reservoir in the transfer plate with the well in the reaction plate.
In another general embodiment, at least one of the reagents includes a pelletized form of the reagent, and the pelletized reagent is delivered from the associated reagent reservoir to the well by gravity, when the reagent reservoir is aligned with the reaction well.
The reaction well in the reaction plate may be formed by an elongate channel containing a solid-phase assay support. In this embodiment, the transfer plate may include ports which are alignable with spaced areas in the channel, when the transfer plate is moved to a wash position, forming an enclosed passageway for passing a solution through the channel.
In one method, the assembly is used for detecting a nucleic acid with a known target sequence. Here the solid-phase support in the reaction is coated with immobilized nucleic acid fragments, and the assembly includes reagent reservoirs for sequential addition to the reaction channel of (1) a probe effective to hybridize with both the target sequence and the immobilized fragments, and (2) a reporter molecule effective to bind, directly or indirectly, to the probe.
In another method, the assembly is used in an immunoassay for detection of a ligand effective to bind immunospecifically to the solid support. Here the assembly includes reagent reservoirs for sequential addition to the reaction channel of (1) an antiligand reagent capable to binding immunospecifically with the ligand, when the ligand is bound to the support, and (2) a reagent capable of reacting with the antiligand reagent to produce a detectable signal on the solid support.
In another aspect, the invention includes apparatus for detecting an analyte ligand by detectable, ligand-specific binding to a solid support. The apparatus includes a self-contained assembly of the type described above, and a device for holding the reaction plate, and rotating said transfer plate to its various positions.
In one embodiment, the device is effective to rotate the assembly as a unit, the reaction well includes a radially extending channel, the solid support surface includes at least two solid-phase particles carried in the channel for movement therein from inner toward outer radial positions, and the transfer plate includes a receiving slot into which the particles can be received, when the particles in the channel are subjected to an outward force, and the channel is aligned with said slot. The reaction plate includes particle-receiving wells into which particles in the slot can be deposited, when the reagent plate containing deposited particles is rotated with respect to the reaction plate.
In an embodiment of the device designed for heating the liquid contents of the well, the assembly further includes structure for sealing the well when heating is applied. The assembly includes a cover plate attached to the reaction plate, and the sealing structure includes (a) a sealing pad carried in the transfer plate floating in a direction normal to the plane of the plate, and (b) a cam surface in the cover plate for biasing the pad against the well platform, when the transfer plate is moved to a heating position.
Also in an embodiment of the device, the solid support includes a bead in the well, and the device includes a detector having a light sensor, and a tube extending from the light sensor and positionable to encompass a spherical surface portion of the bead.
These and objects and features of the invention will become more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying drawings.
FIG. 1 is an exploded perspective view of the components in an assay apparatus constructed according to the present invention;
FIG. 2 is an enlarged, partially cutaway perspective view of an assay assembly constructed according to the invention;
FIG. 3 is a plan view of a cover plate in the FIG. 2 assembly;
FIG. 4 is a plan view of a transfer plate in the FIG. 2 assembly;
FIG. 5 is a plan view of a reaction plate in the FIG. 2 assembly;
FIGS. 6A and 6B are fragmentary sectional views taken at relative plate positions in the FIG. 2 assembly at which a reagent reservoir in the transfer disc in the assembly is isolated from a reaction channel in the reaction plate (6A), and the reservoir is in a transfer position with respect to the channel (6B);
FIGS. 7A and 7B are enlarged, fragmentary sectional views of reservoir regions seen along lines A--A in FIG. 6A and along line B--B in FIG. 6B, respectively;
FIG. 8 is an enlarged, fragmentary sectional view of sealed well in the assembly, during a heating step in the operation of the assembly;
FIGS. 9A and 9B are sectional views similar to 6A and 6B, respectively, but illustrating an embodiment of an assembly in which the reagent in the reservoir is in dried particle form;
FIG. 10 is an enlarged, fragmentary sectional view of the assembly showing the assembly in a position for irrigating the assembly well;
FIG. 11 is an enlarged sectional view of a well region of the assembly, showing detector structure for detecting an optical signal from a bead in the well;
FIGS. 12A-12C illustrate the steps in transferring solid-phase particles from the reaction channel in the reaction plate into a slot formed in the transfer plate (12A, 12B), and from this slot into separate wells in the reaction plate (12C);
FIG. 13 is a flow diagram of the steps carried out by the apparatus in executing an exemplary analyte assay; and
FIGS. 14A-14D are schematic illustrations of the sequential nucleic acid binding reactions in a DNA analyte assay which can be carried out in an automated fashion by the apparatus of the invention.
FIG. 1 illustrates, in exploded view, an assay apparatus 20 constructed according to the present invention. The apparatus includes an assembly 22 which also forms part of the invention and which will be detailed below with reference to FIGS. 2-12. Briefly, the assembly is formed of reaction and cover plates 24, 26, respectively which are joined together at their outer edges, and an intermediate transfer plate 28 which can be rotated relative to the two outer plates about a central axis 30. To this end, the transfer plate includes a hub 32 having an elongate opening 34 (FIG. 2) which is engageable for rotating the transfer plate, with the reaction and cover plates supported in a stationary position.
A control device 36 in the apparatus includes a base 38 which defines an annular seat 40 in which the assembly is supported during an assay operation. The base provides five pins, such as pins 42, arranged asymmetrically about the seat, as shown. These pins engage corresponding holes in the bottom of the assembly, described below, to immobilize the cover and reaction plates against rotation in the seat.
The base is supported on and attached to a drive motor which is indicated by a drive shaft 44 in the figure, the motor itself being contained with a motor housing. A three-arm support 46 attached to the end of the drive shaft has a projection 48 designed to engage the opening in hub 32, with the reaction surface of the assembly resting on the support arms, as can be appreciated in the figure. Thus, when the assembly is received and immobilized in seat 40, with projection 48 received in opening 34 in the hub, the intermediate transfer plate in the assembly can be rotated to selected positions with respect to the cover and reaction plates by the drive motor.
The motor in the control device can be moved from a lowered transfer position in which the assembly is immobilized in seat 40, as just described, to a raised free-rotation position in which the assembly is disengaged from the seat and is free to rotate on drive shaft 44. Because of a relatively high coefficient of internal (transfer plate) movement, the assembly can be rotated as a unit by the drive motor, such that the assembly and motor in a free-rotation position can function as a centrifuge to force material radially outwardly in the assembly, for a purpose to be described. The assembly can also be agitated, for mixing reaction components, by oscillating the assembly on the motor in a free-rotation position.
A wash unit 50 in the device is designed to circulate wash solution(s) through the assembly, as will be described below with respect to FIG. 10. Briefly, when the assembly is placed in a wash condition, openings 52a, 52b formed in cover plate of the assembly form the ends of a closed passageway which includes a portion of the reaction well in the assembly. Unit 50, which is mounted on base 38 as indicated, includes a vertically positionable arm 54 which holds a pair of tubes 56a, 56b, for insertion into openings 52a, 52b, respectively, when the arm is moved to a lowered, wash position. In a wash operation, wash solution is supplied through tube 56a under pressure, and is removed through tube 56b.
Also shown in FIG. 1 is a heating unit 62 in the apparatus used for heating a reaction well in the region in the assembly to selected reaction temperatures. The unit is attached to the lower side of base 38, as indicated, to position a heating element 64 in the unit directly below the reaction well in the assembly, with the assembly seated on the base.
Although not shown in FIG. 1, device 36 may also include an optical sensing unit for detecting analyte in the assembly. One preferred unit will be described below with respect to FIG. 11.
The assay assembly of the invention is illustrated particularly with respect to FIGS. 2-12. Reaction plate 24 in the assembly (FIGS. 2 and 5) provides an elongate, radially extending channel or well 66 which serves as the reaction chamber in the assembly, i.e., the chamber where the analyte and various reagents required for analyte binding and detection are brought into liquid contact with a solid-phase support contained in the well. In the embodiment shown, the well is formed in a metal, e.g., aluminum, insert 166 which is attached to the lower side of a disk-like plate member 168. The insert is carried below a platform 170 defined by the upper surface of plate member 168, above the insert. Formed in the platform are radially spaced openings 172a and 172b adjacent opposite end regions of the channel, and a central opening 172c. Openings 172a and 172b are aligned with openings 52a and 52b in plate 26, for a purpose to be described.
Details of the well construction in the plate are seen in FIG. 7B which shows an enlarged cross-section of the well region in plate 24 and overlying regions of plates 26, 28. The channel or well formed in the insert has a substantially U-shaped cross-section extending between opposite ends, corresponding to the regions below openings 172a and 172b in FIG. 5, with the outer radial end region of the channel forming a tapered ramp 70, as seen in FIG. 10. The insert is received in a cavity 174 formed in the lower region of plate member 168, and attached to the plate member by flanges, such as seen at 176. Each opening in platform 170, such as opening 172a shown in FIG. 7B, communicates with the well, and includes a short cylindrical-bore section 178 and a chamfered section 180. This construction serves to draw liquid into and through the opening from a reservoir in plate 28 into the channel in the reaction plate, as will be described below.
Carried in the channel are one or more solid-phase beads or particles, such as particles 68a, 68b, 68c, which form solid phase supports in the assay reaction. In the embodiment described in Section C below for solid-phase DNA determination, the three particles are for (1) positive control, (2) negative control, and (3) analyte binding. The channel and particles are shown in cross section in FIGS. 10 and 12A. The invention also contemplates a reaction format containing only a single solid-phase support.
Located just beyond the outer end of the channel in plate 24 is an arcuate groove 72 which serves as a guide for the particles, also for a purpose to be described. Adjacent this groove are three wells 74a, 74b, 74c into which the three particles may be transferred from channel 66 after the chemical reaction, also as will be described below. The three wells may contain a detection solution for detecting the presence of reporter molecules bound to the solid particles.
The reaction plate is secured to the cover plate by fasteners, such as rivets 75 (FIG. 2), connecting the outer annular rim regions of the two plates. The fasteners are received through holes, such as holes 76, formed in the plate. Also formed in the reaction plate is a central opening 82 through which projection 48 is received when the assembly is seated on the control device.
Cover plate 26 in the assembly is illustrated in plan view in FIG. 3. The plate is a circular disc having the same diameter as the reaction plate. Formed in an outer annular rim region 83 of the plate are holes, such as holes 84, for fastening an upper and reaction plates (dotted line 85 in FIG. 3 indicates the outer edge of transfer plate 28 in the assembly). Also formed in the cover plate are: (i) a central opening 86 through which the cover portion of hub 32 is received, as seen in FIG. 2; (ii) a series of windows 88a, 88b, 88c which are aligned with wells 74a, 74b, 74c, respectively, in the reaction plate; (iii) above described openings 52a, 52b, which are aligned with openings 172a, 172b, respectively, for circulating wash solution through the channel; and (iv) an opening 52c between ports 52a and 52b, in alignment with port 172c.
With reference now to FIGS. 2 and 4, transfer plate 28 includes a plate member 190 having a central opening 90 in which hub 32 is fixed for rotation with the plate. The plate includes four reagent reservoirs 92, 94, 96, and 98 which take the form of cylindrical channels extending through the plate. With reference particularly to FIG. 7A, reservoir 92, which is representative, is formed of a sleeve 100 which extends through plate member 184, extending beyond the plane of the plate member on both upper and lower sides of the plate member. In the completed assembly, the sleeves forming the several reservoirs are axially compressed, forming liquid-tight seals against confronting faces of the cover plate and reaction plate, as illustrated in FIG. 7A. The sleeves are preferably formed of Teflon or polyethylene. The sealing ends of the sleeves are also referred to herein as means for preventing release of liquid reagent in a reservoir until the reservoir is aligned with one of the openings in the transfer plate communicating with channel 66.
The spacing between the plate member forming plate 28 and the confronting surfaces of the cover and reaction plates in the assembly function to prevent liquid contained in each reservoir from being drawn by capillarity into the gap between the two plates. That is, the gap between confronting plate surfaces, such as gap 186, is too great to produce capillary flow between the two surfaces. The gap in the embodiment shown is provided by the relatively greater lengths of the sleeves forming the reservoirs compared with the thickness of plate member 184. Alternatively, the region of the transfer plate member immediately adjacent each sleeve may be recessed to form a capillary-block gap around each sleeve.
As just indicated, each of the reservoirs in plate 28 is alignable with one of the openings 172a-172c in the reaction plate communicating with channel 66, for delivering fluid reagents to the reaction chamber, to be described below with reference to FIGS. 6A, 6B and 7B. In particular, it is noted that some of the reservoirs, such as reservoirs 94, 96, and 98 are radially spaced, preventing cross-contamination of liquid reagents in the two reservoirs, as the reservoirs are moved relative to the cover and reaction plates.
Also formed in transfer plate is an openings 102a-102c alignable with opening 172a-172c, respectively, in the reaction plate and at the same time with openings 52a-52c in the cover plate. Any of the three sets of alignable openings are referred to herein collectively as means for introducing sample into the assembly's reaction region.
Other openings formed in the transfer plate include three windows 104a, 104b, 104c which are alignable with windows 88a, 88b, 88c, respectively, for viewing wells 74a, 74b, 74c, respectively, when the two sets of windows are aligned. In addition, the transfer plate provides three sets of radially spaced openings, such as openings 106a, 106b, which are alignable, at one of three different plate positions, with openings 172a, 172b, respectively, in the reaction plate, at the same time, with ports 52a, 52b in the cover plate, for forming a continuous passageway 108 (FIG. 10) for circulating wash solution through the reaction region.
The transfer plate also includes a plurality of floating sealing pads, such as pads 192, 194, which are designed for sealing the channel openings in the reaction plate, when the liquid contents of the reaction plate are heated. Details of the operation of the pads in a heating operation are seen in FIG. 8. Shown here are an enlarged sectional view taken through the assembly in the region of the reaction channel, and a portion of heater element 64 used in heating the liquid contents in the channel. As seen, the heating element makes contact with the lower side of insert 166 during heating.
Pad 192, which is representative, is an elongate elastomeric pad, such as formed from polyethylene or other compressible, low-friction polymer material, which is dimensioned to cover openings 172a-172c, when the pad is positioned on platform 170, as indicated in FIG. 8. The pad is carried in a radially extending slot 194 formed in the transfer plate member, for floating in a direction normal to the plane of the plate. The pad's height dimension allows the pad to be moved easily and without pad compression, between the cover and reaction plates, as the transfer plate is rotated relative to the other two plates. However, as the pad is moved toward a sealing position above the reaction well, it makes contact with a radially extending cam member 195 formed on the lower side of the cover plate, immediately above openings 172a-172c. As can be appreciated from FIG. 8, this contact acts to bias, i.e., compress, the pad against the platform openings, sealing the openings to the reaction well.
The transfer plate also provides an L-shape slot 112 formed in its lower side, i.e., the side confronting the reaction plate. The slot includes radially and circumferentially extending portions 112a, 112b which are alignable with the outer end region of channel 66 and groove 72 in the reaction plate, respectively. The slot is positioned and dimensioned to receive the particles from channel 66 (FIG. 12A) as will be described in Section B.
The plates in the assembly are preferably formed by injection molding of a suitable plastic. For forming the transfer plate, plate member 184 can be injected molded about the four sleeves forming the four reservoirs in the transfer plate, and hub 32, and subsequently fitted with the four sealing pads. For forming the reaction plate, plate member 168 can be injected molded about insert 166. With the transfer plate placed between the cover and reaction plates, the two outer plates are secured together, such as by rivets, glue or heat welding. As noted above, construction and plate attachment is such as to bias the cover and reaction plates firmly against confronting sides of the transfer plate, to compress the elastomeric seals in the assembly. Liquid reagent can be added to the reservoirs and wells by suitable positioning of the transfer plate after assembly.
In another general embodiment of the assembly, the reagent reservoirs are designed for delivery of a dehydrated reagent particle composed of the dried reagent, preferably formulated in pelletized form. This embodiment is illustrated in FIGS. 9A and 9B which shows a fragmentary portion of an assay assembly 120, with cover plate 122, transfer plate 124, and reaction plate 126. The cover and reaction plates in the assembly are substantially identical to those in assembly 22. The transfer plate includes a plurality of reservoirs, such as reservoir 128, which communicate with the reaction plate, but not the cover plate.
Each reservoir contains a dried reagent particle, such as particle 130 in reservoir 128, which is held within the reservoir, for deposit into reaction channel 132 in the reaction plate, when the reservoir is aligned with the transfer plate, as seen in FIG. 9B. The lower end of the reservoir is sealed by the sleeves forming the reagent reservoirs, to prevent exposure of the reagent to moisture or contamination by other reagents.
The pelletized reagent may be formulated, if desired, with a variety of water-soluble bulking agents, such as water-soluble polymers, according to well-known methods. Where the assembly includes one or more solid-phase particles, these are added to the reaction channel in the reaction plate before the transfer and reaction plates are joined.
The assemblies described and illustrated above are designed particularly for use in a solid-phase DNA analyte assay involving (i) at least one solid-phase particle, (ii) four separate liquid and/or solid reagents which are added sequentially, (iii) a heating step following each reagent addition, and (iv) three individual wash steps which follow the addition of the second, third, and fourth reagents, as will be described below.
A variety of alternative assembly configurations are contemplated in the present invention. For example, the assay may involve sequential addition of two reagents, requiring only two reservoirs, or two or more of the reagents may be added to the reaction region simultaneously, in a configuration in which the reservoirs are carried along a common radial line.
Further, the solid-phase particles in the reaction may be replaced by one or more separate solid-phase supports formed on the channel wall, and/or the solid phase particles may be carried within separated wells in the reaction chamber, obviating the need for particle transfer after the assay reaction. Alternatively, the assay reaction may occur in a liquid phase, either in a free solution or in a absorbent filter, eliminating wash steps. In the case of an absorbent-filter type reaction region, a liquid reagent may be drawn onto the filter by wicking or capillarity, eliminating the need for venting liquid-carrying reservoirs.
In operation, the assembly is placed in the seat of the control device, with the pins of the device being received in the corresponding holes in the assembly reaction plate, and with projection 48 being received in hub 32, as described above. Hub 32 is initially oriented with respect to the pins such that the transfer plate is in a home position at which all of the reservoirs and the reaction region are sealed.
The following exemplary operation will be described with reference to FIG. 13, which is a flow diagram of the assay steps in the DNA-analyte assay described in Section C below. The steps shown in solid-line boxes are preferably executed in an automated fashion by a suitable microprocessor control unit (not shown) in the control device. The sample and/or wash-solution addition steps shown in the dashed-line boxes may be performed either manually or under the control of the apparatus.
The control device is first actuated to move the transfer plate in a clockwise direction in FIG. 2, to align opening 102a in the transfer plate with opening 52a in the cover plate and opening 172a in the reaction plate, and a liquid sample is introduced through the aligned opening into the reaction channel. The analyte sample may be any fluid sample, such as a blood, serum, or plasma sample, in a suitable amount, typically between about 10-200 μl.
The transfer plate is rotated further in the same direction to align reservoir 92 with opening 172a in the reaction plate, as shown in FIGS. 6B and 7B, to transfer the liquid reagent in the reservoir into the channel. As the reservoir seal first overlaps the edge of opening 172a, the fluid seal in the reservoir is broken, allowing fluid to flow readily out of the reservoir. As discussed above, the capillary lock feature of the transfer plate prevents the fluid from flowing by capillarity out of the reservoir into the region between transfer and reaction plates. The chamfered opening 172a serves to direct fluid from the reservoir into the well by a combination of capillarity, provided by narrower upper section of the opening, and rapid fluid flow provided by the lower chamfered section of the opening.
As mentioned above, in an alternative embodiment of the invention, the reservoir may contain a dehydrated, pelletized reagent which is deposited by gravity into the reaction channel, as indicated in FIG. 9B. To mix the reagent with the liquid sample, the transfer plate may be rotated back to a channel sealing position, the assembly lifted from the seat in the control device, by raising the drive motor to a free-rotation position, and the assembly is oscillated slowly as a unit by the motor.
After introducing the liquid (or solid) reagent into the reaction well, the transfer plate is rotated slightly to position pad 192 over the reaction well. As described with respect to FIG. 8, the pad at this position is compressed between the cover and reaction plates, forming a tight seal about the reaction well. The heating element in the device is then activated to heat the well for a selected reaction period. During the heating cycle, heated fluid from the reaction mixture is prevented from escaping from the well by the seal over openings 172a-172c.
The transfer plate is now rotated to transfer the contents of reservoir 94 into the channel, and the reaction mixture is again mixed and reacted for a given reaction period at a selected temperature. As can be appreciated with reference to FIG. 4, continued movement of the transfer plate in a clockwise direction aligns the first set of wash-solution ports, indicated at 106a, 106b with openings 52a, 52b, respectively, in the cover plate, and openings 172a, 172b, respectively in the reaction plate. When this alignment is achieved, the wash unit is lowered to insert the wash-solution tubes into the aligned ports, and wash solution is circulated through the reaction channel as illustrated in FIG. 10, to wash the initial sample and first two reagents from the reaction chamber.
The above steps are repeated to (i) transfer, mix and incubate a third reagent with the washed solid-phase particles in the reaction chamber, (ii) remove the third reagent by washing, (iii) transfer, mix and incubate a fourth reagent with the washed particles, and (iv) remove the fourth reagent by washing. These steps are shown in FIG. 13, where the three separate wash steps are indicated at (1), (2), and (3).
After completing the reaction, an analyte-dependent signal on the solid-phase surface, i.e., the solid-phase beads, may be read with the beads in the reaction well. In order to minimize optical-reading artifacts from the other beads in the reaction well, the signal detector in the device (or in a separate optical reading device) preferably has a construction like that shown for detector 196 in FIG. 11, allowing direct reading of each bead. The detector includes an optical sensor 198 and a refractory tube 200 dimensioned to cover a segment of the sphere as indicated, when the tube is lowered through an opening in the assembly (formed by a opening in the cover, transfer, and reaction plates) into the reaction well. After each bead is read, the tube is raised and then lowered into another assembly opening to read the next bead.
Alternatively, the beads may be distributed into separate reading chambers, according to the operation following described with respect to FIGS. 12A-12C. Briefly, the transfer plate is rotated to the position shown in FIG. 12B to align the radial portion of slot 112 with the reaction channel. With the drive motor moved to its raised position, the assembly is now rotated at a speed sufficient to force the particles in the reaction channel radially outwardly into the slot, as shown in FIG. 12A. The particles are forced ultimately into the circumferential portion of the slot, as shown in FIG. 12B. The particles are captured in groove 72 (FIG. 5) at this position, to prevent their return to the reaction channel at the end of centrifugation.
To distribute the particles into the respective wells in the reaction plate, the motor is moved to its lowered position, the assembly reseated on the control device, and the transferred plate rotated in a clockwise direction, i.e., in a direction which tends to force the particles toward the back of the slot (the particle positions shown in FIG. 12B). As the slot is moved over each well in the reaction plate, the forwardmost particle in the slot drops into that well, until particle transfer into all three wells is completed.
The amount of analyte associated with each of the solid phase particles may be determined by a variety of known methods. Typically, the analyte, which is specifically bound to the particle, itself binds a label probe (reporter molecule) which contains fluorescent, or enzyme reporter moieties which can be detected and/or quantitated by standard photodetector or spectrophotometric means. As indicated above, the wells may contain a detection solution which is reactive with the reporter moieties to produce the desired detection signal.
One advantage of the particle transfer feature of the invention is that several solid-phase particles can be reacted under identical conditions in a single chamber, then isolated for detection. The separation of the particles for detection allows accurate detection by chemiluminescence, fluorescence, or enzyme activity which is not possible when the particles are closely spaced and/or in the same reaction chamber. However, it will be recognized that the invention is also advantageous for carrying out a solid-phase reaction employing one or more beads, or other solid-support surfaces in the reaction well, and reading the solid surfaces in the well, as described with respect to FIG. 11.
Section C below describes an exemplary assay method for detection of analyte DNA by a solid-phase reaction method. The method is illustrates various advantages of the assembly and apparatus, including (i) the ability to carry out complex, multiple reagent assays in a simple, self-contained assembly, (ii) quantitative reagent transfer from the transfer plate to the reaction plate, (iii) the ability to intersperse reaction steps with wash cycles, (iv) the ability to carry out heated incubation steps without liquid loss, and (v) the ability to conduct multiple solid phase reactions in a single chamber, then separate the solid-phase supports for analyte detection. Although the DNA analyte assay is illustrative of one type of assay which may be carried out in the assembly and apparatus of the invention, it will be understood that the invention is readily adapted to a wide variety of assay procedures in which solid or liquid reagents are to be added to a reaction region, preferably sequentially, for detection or quantitation of an analyte in a reaction zone.
In particular, the invention contemplates detecting a ligand analyte in which the solid-support surface in the reaction well includes molecules which bind specifically with the ligand, or which can bind specifically to the ligand through a bivalent intermediate molecule provided by way of one of the added reagents. In a preferred format, the ligand is an antigen, and the ligand-binding molecule carried on the solid support is a ligand-specific antibody. After addition of a ligand-containing sample to the solid support, and a wash step to remove unbound sample material, a first reporter reagent is added to the well. This reagent contains a reporter-labeled molecule capable of binding specifically to the ligand, with such bound to the solid support. Following a second wash step to remove unbound material, a second reagent containing a substrate for detection of the bound reporter is added to the reaction well. Preferably the reporter is an enzyme, such as alkaline phosphatase or peroxidase, and the second reagent contains substrate capable of reacting with the enzyme reporter to produce a detectable color reaction in the reaction well.
In both the DNA probe format, and the ligand format just described the multiple reagents carried in the assembly for producing analyte-specific binding to the solid support, and detectable signal of the bound analyte are referred to herein, collectively, as reaction reagents required for binding of analyte to the solid support in detectable form.
It will be appreciated that the assembly of the invention may be manually operated, particularly where the assembly has a simplified format in which reagents are added to a liquid reaction chamber, for production of an analyte-dependent signal in a liquid-phase. Here the user can readily manipulate the assembly to its reagent transfer positions, and perform any necessary mixing by manual shaking.
FIGS. 14A-14D illustrate schematically the sequence of reaction steps in a solid-phase DNA analyte assay carried out using the assembly and apparatus of the invention.
Considering first the solid-phase particles and reagents which are included in the assembly, the three solid-phase particles serve as positive and negative controls (particles a and b) and for specific analyte assay (particle c). The negative-control particle is uncoated and the positive-control and analyte particles are coated with single-stranded DNA fragments which are complementary to positive-control and analyte-specific capturing probes, respectively. The nature of these probes is described below. The positive-control and analyte particles are prepared by derivatizing polymer or glass beads with the selected DNA fragments, according to standard coupling methods. The analyte solid-phase particle is indicated at 130 in FIGS. 14A-14D, and the DNA fragments coating this particle which are complementary to the analyte probes are indicated by dashed lines, such as at 132.
A first liquid reagent, contained in reservoir 92 in the assembly, includes a denaturation agent, such as NaOH, positive-control and analyte-specific capturing probes, a positive-control DNA, and positive-control and specific amplifier probes. The positive-control DNA is a duplex DNA fragment which has a number of known sequence regions which are unique to that fragment, i.e., not present in the analyte DNA. The amplifier probes include a series of probes having a first region which is complementary to one of several different sequences in the positive control DNA and in the analyte nucleic acid, and a second, common region which is complementary to a sequence in a branched DNA contained in the third reagent. In the amplifier probe illustrated at 134 in FIG. 14A, a first region which is complementary to a sequence in an analyte nucleic acid is indicated by a square-wave pattern at 136, and a second common region, indicated by coiled line 138, which is complementary to the sequence in the DNA on a branched DNA.
The positive-control capturing probes include a series of probes which have a common first-sequence which is complementary to a sequence in the DNA fragment carried on the positive control particle, and different second sequences which are complementary to different known sequence regions in the positive-control DNA. The analyte capturing probes similarly include a series of probes which have a common first-sequence which is complementary to a sequence in the DNA fragment carried on the analyte particle, and different second sequences which are common to the different known sequence regions in the analyte nucleic acid. One such analyte capturing probe is illustrated at 140 in FIG. 14A, which shows a first region, indicated by a sawtooth pattern at 142, which is complementary to a sequence in an analyte nucleic acid, and a second common region, indicated by dashed line 144, which is complementary to the sequence in the DNA carried on the analyte particle.
A second liquid reagent, contained in reservoir 94, includes an annealing agent or buffer which allows DNA annealing, such as by neutralizing a base denaturation reagent.
A third liquid reagent, carried in reservoir 96, includes the above-mentioned branched DNA molecule, which is indicated at 146 in FIGS. 14B-14D. The molecule includes a sequence indicated by coiled region 148, which is complementary to region 138 of the binding probes, and a group of branched-chain sequences, such as that indicated by dotted line 150, which are complementary to a label probe sequence. In this embodiment, the branched DNA is adapted for indirect binding to analyte and positive control nucleic acid, i.e., through the amplifier probes. Alternatively, the branched DNA may be designed for direct hybridization to sequences in the analyte and positive-control nucleic acid.
A fourth liquid reagent, contained in reservoir 98, includes a labeling probe, indicated at 152 in FIGS. 14C-14D, which includes a DNA fragment moiety 154 with a nucleotide sequence complementary to sequence 150 in the branched DNA molecule, and a reporter moiety 156 which is used for signal detection.
The reporter moiety in the present assay assembly is preferably an enzyme, such as alkaline phosphatase, which can generate a chemiluminescent signal in a suitable detection solution.
Completing the description of the chemical components of the assembly, the wells in the assembly are partially filled with a detection solution which are reactive with the reporter moiety to generate a detectable signal. For example, a solution containing doxetane is reactive with an alkaline phosphatase reporter moiety to generate a chemiluminescent signal which can be detected and quantitated with a photomultiplier conventionally.
Initially the assembly is positioned for sample addition and a sample containing a DNA analyte 160 is added to the assembly's reaction chamber. As indicated above, the analyte includes multiple sequences, such as sequence 162, which are complementary to regions of the analyte capturing probes in the assay, and one or more sequences, such as sequence 164, which are complementary to the binding probes in the assay.
After sample addition, the assembly is manipulated to add and mix the first reagent, then incubated for 10 minutes at 65� C. to denature the analyte. With addition of the annealing buffer, mixing, and further incubation for 10 minutes, the following probe-mediated binding reactions occur: (1) positive-control capturing probes hybridize with the positive-control particle and with the positive-control DNA to link this DNA to the positive-control particle; (2) amplifier probes hybridize with the positive control DNA; (3) analyte capturing probes hybridize with the analyte particle and with the analyte to link analyte DNA to the particle; and (4) amplifier probes hybridize with the analyte DNA. Steps (3) and (4) are illustrated in FIG. 14B.
After the annealing reaction, the assembly reaction chamber is manipulated for one or more wash steps in which wash solution is circulated through the reaction channel to remove unbound reagents. The third reagent is now added, mixed, and allowed to anneal at 55� C., to hybridize the branched DNA with particle-bound amplifier probes, as is illustrated in FIG. 14C. Following annealing, unbound branched DNA is removed by a second wash step.
The final labeling probe is added to the particles, mixed and allowed to anneal with the bound branched DNA, to bind reporter molecules to the positive-control and analyte particles. Binding of the labeling probe molecules to the analyte particle is illustrated in FIG. 14D. Unbound labeling probe is then removed by a third wash step, as indicated.
The assembly is now manipulated by the steps shown at the right in FIG. 10, and described above, to transfer the three particles into the respective wells for signal detection. Reaction of the bound reporter with the detection fluid in the wells produces a detectable chemiluminescent signal which is measured and converted to a quantitative assay value which is displayed. In the three particle assay, the positive-control value is used to calculate a standard curve of analyte concentration as a function of concentration, with background (negative control) subtraction. Analyte concentration is determined from the standard curve, after background subtraction.
The assembly and apparatus provide several advantages in a solid-phase DNA assay, as can be appreciated. The assay can be carried out in a substantially automated fashion, and without addition of reagents from external sources. Accordingly, a minimum of laboratory training and user manipulations are required.
The ability to carry out multiple solid-phase reactions in a single chamber minimizes variations in quantitative analyte measurements due to variations in reaction conditions, allowing self-corrected analyte determinations based on a standard curve with background subtraction.
Although the invention has been described with respect to certain embodiments, configurations, and applications, it will be apparent to those skilled in the art that various changes and modifications may be made without departing from the invention.
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|Fluidic circuits for sample preparation including bio-discs and methods relating thereto
|Jul 20, 2007
|Sep 23, 2008
|Burstein Technologies, Inc.
|Method for triggering through disc grooves and related optical analysis discs and system
|Aug 27, 2004
|Nov 4, 2008
|3M Innovative Properties Company
|Sample processing devices and carriers
|Mar 16, 2005
|Aug 4, 2009
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|Systems for using sample processing devices
|May 3, 2005
|Aug 4, 2009
|Method for separating analyte from a sample
|Aug 2, 2006
|Sep 29, 2009
|3M Innovative Properties Company
|Sample processing devices and carriers
|Mar 14, 2006
|Jan 26, 2010
|Research International, Inc.
|Optical assay apparatus and methods
|Apr 6, 2006
|Mar 16, 2010
|3M Innovative Properties Company
|Sample processing devices
|May 26, 2000
|Apr 6, 2010
|Affymetrix, Inc.
|Combinatorial strategies for polymer synthesis
|Oct 9, 2003
|May 18, 2010
|3M Innovative Properties Company
|Multilayer processing devices and methods
|Jun 17, 2002
|Jun 15, 2010
|Affymetrix, Inc.
|Combinatorial strategies for polymer synthesis
|Jul 5, 2005
|Jul 13, 2010
|3M Innovative Properties Company
|Sample processing device compression systems and methods
|Jul 5, 2005
|Jul 27, 2010
|3M Innovative Properties Company
|Compliant microfluidic sample processing disks
|Oct 31, 2007
|Aug 3, 2010
|3M Innovative Properties Company
|Modular sample processing kits and modules
|Apr 6, 2006
|Dec 21, 2010
|3M Innovative Properties Company
|Sample processing devices
|Feb 12, 2009
|Mar 29, 2011
|Method for separating an analyte from a sample
|Aug 5, 2004
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|3M Innovative Properties Company
|Sample processing device positioning apparatus and methods
|Jun 25, 2009
|Aug 23, 2011
|3M Innovative Properties Company
|Thermal structure for sample processing systems
|Jun 4, 2010
|Dec 20, 2011
|3M Innovative Properties Company
|Sample processing device compression systems and methods
|Jun 23, 2010
|Jan 10, 2012
|3M Innovative Properties Company
|Compliant microfluidic sample processing device
|Nov 10, 2010
|Jan 17, 2012
|3M Innovative Properties Company
|Sample processing devices
|Mar 7, 2011
|Aug 21, 2012
|Method for separating an analyte from a sample
|Feb 26, 2013
|Rheonix, Inc.
|Self-contained biological assay apparatus, methods, and applications
|Dec 30, 2005
|May 7, 2013
|3M Innovative Properties Company
|Sample processing devices
|Jul 19, 2012
|Nov 26, 2013
|Method for separating an analyte from a sample
|Nov 13, 2009
|Sep 16, 2014
|3M Innovative Properties Company
|Systems for processing sample processing devices
|Mar 30, 2010
|Oct 21, 2014
|3M Innovative Properties Company
|Multilayer processing devices and methods
|May 18, 2012
|Jan 13, 2015
|3M Innovative Properties Company
|Systems and methods for volumetric metering on a sample processing device
|May 18, 2012
|Jun 30, 2015
|3M Innovative Properties Company
|Systems and methods for valving on a sample processing device
|Jul 29, 1999
|Dec 6, 2001
|Jorma Virtanen
|Laboratory in a disk
|Dec 10, 2001
|Jun 13, 2002
|Worthington Mark Oscar
|Methods for detecting analytes using optical discs and optical disc readers
|Nov 30, 2001
|Jun 20, 2002
|Cohen David Samuel
|Apparatus and methods for separating components of particulate suspension
|Dec 14, 2001
|Jun 20, 2002
|Jorma Virtanen
|Detection system for disk-based laboratory and improved optical bio-disc including same
|Nov 19, 2001
|Jul 25, 2002
|Gordon John F.
|Methods and apparatus for blood typing with optical bio-disc
|Nov 16, 2001
|Sep 26, 2002
|The University Court Of The University Of Glasgow
|Apparatus and method for carrying out analysis of samples using radiation detector split beam radiation inspection
|Feb 28, 2002
|Nov 21, 2002
|Phan Brigitte Chau
|Methods for decreasing non-specific binding of beads in dual bead assays including related optical biodiscs and disc drive systems
|Nov 27, 2001
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|Phan Brigitte C.
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|Jun 17, 2002
|May 13, 2004
|Affymetrix, Inc.
|Combinatorial strategies for polymer synthesis
|Nov 18, 2003
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|Per Andersson
|Microfluidic device
|Jul 24, 2002
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|Phillip Bruce
|Magnetic assisted detection of magnetic beads using optical disc drives
|Jan 21, 2003
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|Chen Yihfar
|Microfluidic structures with circumferential grooves for bonding adhesives and related optical analysis discs
|Nov 27, 2002
|Dec 9, 2004
|Coombs James Howard
|Magneto-optical bio-discs and systems including related methods
|Sep 4, 2002
|Dec 9, 2004
|Percival David Alan
|Device for use in fluid assay
|Jan 28, 2003
|Dec 30, 2004
|Worthington Mark Oscar
|Methods and apparatus for logical triggering
|Sep 20, 2004
|Jan 20, 2005
|Norbert Staimer
|Chromatographic analysis on optical bio-discs and methods relating thereto
|Jul 6, 2004
|Jan 27, 2005
|Worthington Mark O.
|Multiple data layer optical discs for detecting analytes
|Jan 30, 2003
|Jan 27, 2005
|Evgenia Matveeva
|Methods for synthesis of bio-active nanoparticles and nanocapsules for use in optical bio-disc assays and disc assembly including same
|Jan 23, 2003
|Feb 3, 2005
|Coombs James Howard
|Bio-safety features for optical analysis disc and disc system including same
|Aug 27, 2004
|Feb 10, 2005
|3M Innovative Properties Company
|Sample processing devices and carriers
|Jan 14, 2003
|Feb 10, 2005
|Pal Andrew Attila
|Methods and apparatus for extracting data from and optical analysis disc
|Apr 21, 2004
|Feb 17, 2005
|Norbert Staimer
|Optical bio-discs including spiral fluidic circuits for performing assays
|Sep 29, 2004
|Feb 24, 2005
|Integrated Chemical Synthesizers, Inc.
|Nanoscale chemical synthesis
|Jun 18, 2004
|Mar 3, 2005
|Horacio Kido
|Fluidic circuits for sample preparation including bio-discs and methods relating thereto
|Jul 12, 2004
|Mar 31, 2005
|Mullis Kary Banks
|Dual bead assays using cleavable spacers and/or ligation to improve specificity and sensitivity including related methods and apparatus
|Oct 9, 2003
|Apr 14, 2005
|Dufresne Joel R.
|Multilayer processing devices and methods
|Jul 26, 2004
|Apr 21, 2005
|Horacio Kido
|Fluidic circuits for sample preparation including bio-discs and methods relating thereto
|Nov 24, 2004
|Jun 9, 2005
|Affymetrix, Inc.
|Combinatorial strategies for polymer synthesis
|Jan 17, 2003
|Aug 11, 2005
|Pal Andrew A.
|Bio-safe dispenser and optical analysis disc assembly
|Mar 16, 2005
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|3M Innovative Properties Company
|Systems for using sample processing devices
|Jan 23, 2003
|Aug 25, 2005
|Coombs James H.
|Method for triggering through disc grooves and related optical analysis discs and system
|May 3, 2005
|Sep 8, 2005
|Method for separating analyte from a sample
|Jan 24, 2003
|Oct 6, 2005
|James Rodney Norton
|Manufacturing processes for making optical analysis discs including successive patterning operations and optical discs thereby manufactured
|Jun 15, 2004
|Dec 22, 2005
|Per Andersson
|Microfluidic system
|Nov 16, 2004
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|Applera Corporation
|Bead dispensing system
|Aug 5, 2004
|Feb 9, 2006
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|Nov 18, 2005
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|Werner Martin E
|Surface assembly for immobilizing DNA capture probes in genetic assays using enzymatic reactions to generate signal in optical bio-discs and methods relating thereto
|Aug 24, 2005
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|Hideki Kambara
|Luminescence detection apparatus
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|Apr 6, 2006
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|Sample processing devices
|Dec 30, 2005
|Oct 12, 2006
|3M Innovative Properties Company
|Sample processing devices
|Aug 2, 2006
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|3M Innovative Properties Company
|Sample processing devices and carriers
|Jul 5, 2005
|Jan 11, 2007
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|Compliant microfluidic sample processing disks
|Jul 5, 2005
|Jan 11, 2007
|3M Innovative Properties Company
|Sample processing device compression systems and methods
|Nov 13, 2009
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|3M Innovative Properties Company
|Sample processing disk cover
|Nov 13, 2009
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|3M Innovative Properties Company
|Sample processing disk cover
|Feb 4, 2011
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|3M Innovative Properties Company
|Sample processing disk cover
|May 18, 2011
|Dec 11, 2012
|3M Innovative Properties Company
|Rotatable sample processing disk
|Sep 7, 2012
|Mar 5, 2013
|3M Innovative Properties Company
|Rotatable sample processing disk
|Feb 22, 2011
|Feb 18, 2015
|Self-contained biological assay apparatus, methods, and applications
|Nov 12, 1998
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|David B. Goodman
|Self-contained assay device and method
|Mar 6, 2000
|Jan 2, 2002
|Thermo Biostar, Inc.
|Optical assay device and method
|Feb 22, 2011
|Sep 1, 2011
|Rheonix, Inc.
|Self-contained biological assay apparatus, methods, and applications
|Jan 11, 2013
|Aug 15, 2013
|Bio-Rad Laboratories, Inc.
|Self-contained multi-reagent assay device
|U.S. Classification
|422/404, 422/72, 436/518, 435/287.3, 435/7.1, 435/287.2, 422/430, 422/425, 435/6.11
|International Classification
|Cooperative Classification
|European Classification
|Aug 27, 1991
Owner name: CHIRON CORPORATION, A CORPORATION OF DE, CALIFORNI
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text:
Filter by:
Content type:
Stage 1&2
Stage 2&3
Stage 3&4
Stage 4&5
Challenge level:
Make and prove a conjecture about the value of the product of the Fibonacci numbers $F_{n+1}F_{n-1}$.
Find the smallest positive integer N such that N/2 is a perfect
cube, N/3 is a perfect fifth power and N/5 is a perfect seventh
Find all the solutions to the this equation.
Find the largest integer which divides every member of the
following sequence: 1^5-1, 2^5-2, 3^5-3, ... n^5-n.
An account of methods for finding whether or not a number can be written as the sum of two or more squares or as the sum of two or more cubes.
The nth term of a sequence is given by the formula n^3 + 11n . Find
the first four terms of the sequence given by this formula and the
first term of the sequence which is bigger than one million. . . .
Tom writes about expressing numbers as the sums of three squares.
Investigate the number of points with integer coordinates on
circles with centres at the origin for which the square of the
radius is a power of 5.
Prove that if a is a natural number and the square root of a is
rational, then it is a square number (an integer n^2 for some
integer n.)
Let a(n) be the number of ways of expressing the integer n as an
ordered sum of 1's and 2's. Let b(n) be the number of ways of
expressing n as an ordered sum of integers greater than 1. (i)
Calculate. . . .
Take a complicated fraction with the product of five quartics top
and bottom and reduce this to a whole number. This is a numerical
example involving some clever algebra.
The sums of the squares of three related numbers is also a perfect
square - can you explain why?
Factorial one hundred (written 100!) has 24 noughts when written in full and that 1000! has 249 noughts? Convince yourself that the above is true. Perhaps your methodology will help you find the. . . .
Find a connection between the shape of a special ellipse and an
infinite string of nested square roots.
Take any pair of two digit numbers x=ab and y=cd where, without
loss of generality, ab > cd . Form two 4 digit numbers r=abcd
and s=cdab and calculate: {r^2 - s^2} /{x^2 - y^2}.
Powers of numbers behave in surprising ways. Take a look at some of these and try to explain why they are true.
When if ever do you get the right answer if you add two fractions
by adding the numerators and adding the denominators?
Which is the biggest and which the smallest of $2000^{2002}, 2001^{2001} \text{and } 2002^{2000}$?
Prove that if the integer n is divisible by 4 then it can be written as the difference of two squares.
A introduction to how patterns can be deceiving, and what is and is not a proof.
Find all real solutions of the equation (x^2-7x+11)^(x^2-11x+30) = 1.
Take any prime number greater than 3 , square it and subtract one.
Working on the building blocks will help you to explain what is
special about your results.
Given that a, b and c are natural numbers show that if sqrt a+sqrt
b is rational then it is a natural number. Extend this to 3
Peter Zimmerman from Mill Hill County High School in Barnet, London
gives a neat proof that: 5^(2n+1) + 11^(2n+1) + 17^(2n+1) is
divisible by 33 for every non negative integer n.
Can you convince me of each of the following: If a square number is
multiplied by a square number the product is ALWAYS a square
A polite number can be written as the sum of two or more
consecutive positive integers. Find the consecutive sums giving the
polite numbers 544 and 424. What characterizes impolite numbers?
Take a number, add its digits then multiply the digits together,
then multiply these two results. If you get the same number it is
an SP number.
An article which gives an account of some properties of magic squares.
Peter Zimmerman, a Year 13 student at Mill Hill County High School
in Barnet, London wrote this account of modulus arithmetic.
We continue the discussion given in Euclid's Algorithm I, and here we shall discover when an equation of the form ax+by=c has no solutions, and when it has infinitely many solutions.
In this article we show that every whole number can be written as a continued fraction of the form k/(1+k/(1+k/...)).
Suppose A always beats B and B always beats C, then would you
expect A to beat C? Not always! What seems obvious is not always
true. Results always need to be proved in mathematics.
In this 7-sandwich: 7 1 3 1 6 4 3 5 7 2 4 6 2 5 there are 7 numbers between the 7s, 6 between the 6s etc. The article shows which values of n can make n-sandwiches and which cannot.
Start with any whole number N, write N as a multiple of 10 plus a remainder R and produce a new whole number N'. Repeat. What happens?
Follow the hints and prove Pick's Theorem.
This is the second article on right-angled triangles whose edge lengths are whole numbers.
The first of two articles on Pythagorean Triples which asks how many right angled triangles can you find with the lengths of each side exactly a whole number measurement. Try it!
Caroline and James pick sets of five numbers. Charlie chooses three of them that add together to make a multiple of three. Can they stop him?
We only need 7 numbers for modulus (or clock) arithmetic mod 7
including working with fractions. Explore how to divide numbers and
write fractions in modulus arithemtic.
It is impossible to trisect an angle using only ruler and compasses
but it can be done using a carpenter's square.
The first of five articles concentrating on whole number dynamics, ideas of general dynamical systems are introduced and seen in concrete cases.
This article extends the discussions in "Whole number dynamics I". Continuing the proof that, for all starting points, the Happy Number sequence goes into a loop or homes in on a fixed point.
Solve this famous unsolved problem and win a prize. Take a positive
integer N. If even, divide by 2; if odd, multiply by 3 and add 1.
Iterate. Prove that the sequence always goes to 4,2,1,4,2,1...
Some puzzles requiring no knowledge of knot theory, just a careful
inspection of the patterns. A glimpse of the classification of
knots and a little about prime knots, crossing numbers and. . . .
Professor Korner has generously supported school mathematics for more than 30 years and has been a good friend to NRICH since it started.
The final of five articles which containe the proof of why the sequence introduced in article IV either reaches the fixed point 0 or the sequence enters a repeating cycle of four values.
In this third of five articles we prove that whatever whole number we start with for the Happy Number sequence we will always end up with some set of numbers being repeated over and over again.
This article looks at knight's moves on a chess board and introduces you to the idea of vectors and vector addition.
This article discusses how every Pythagorean triple (a, b, c) can be illustrated by a square and an L shape within another square. You are invited to find some triples for yourself.
If you take two tests and get a marks out of a maximum b in the first and c marks out of d in the second, does the mediant (a+c)/(b+d)lie between the results for the two tests separately.


text:
Star Trek: The Original Series
The original Star Trek series focuses on the 23rd century adventures of Captain James T. Kirk and the U.S.S. Enterprise (NCC-1701), a powerful interstellar spacecraft dispatched by Earth-based Starfleet Command to explore the galaxy. Kirk commands a crew of 430 men and women aboard his starship, which can travel at speeds surpassing the speed of light. Kirk's five-year mission—and his mandate from Starfleet—is to seek out new life and new civilizations, and to boldly go where no man has gone before.
Main Characters
Featured Character
Leslie, Mr.
Low-key humanoid male who served aboard the U.S.S. Enterprise NCC-1701 between 2266 and 2268.
Featured Alien
Episode: TOS 056 - Spectre of the Gun
Each person on Megas-Tu is a specialist in a specific kind of magic, and while the satyr-like Lucien says there are no rivals, "generalists" such as himself are branded as troublemakers. The Megans...
Featured Creative Staff
Justman, Robert
Episode: TOS - Galileo Seven, The
Bob Justman, who passed away in 2008 at the age of 81, went from gofer to Executive Producer during a career that spanned more than 35 motion pictures and 500 television episodes, pilots and telemo...


text: CF760 FEDERAL AND STATE GRANT STATUS REPORT The Federal and State Grants Status Report provides information on current year projects and any open prior years' projects which have been received and logged by the New York State Education Department. Please use this report to reconcile your grant records. Report fields are defined as follows: PROJECT # - The # assigned by SED at the time of approval. CONTRACT # - For projects running through the grant contract process, the New York State contract number will be listed. FUNDING SOURCE - The name of the grant program. SCHEDULED/ - The total amount of payments processed including scheduled PAID TO DATE but not paid. An asterisk (*) indicates that the Final Expenditure Report (FS-10-F) has been received. If the FS-10-F has been reviewed and closed, the Paid-to-Date will equal the budget. Projects listed as UNDER REVIEW have been received and logged by the program office but have not been forwarded to Grants Finance for processing. The project will continue to show as UNDER REVIEW until (1) the program office approves the application and forwards the budget to Grants Finance or (2) the application is disapproved by the program office. Disapproved projects do not appear on this report. START/END - The funding dates of the project, including any extensions. Funding dates are the dates in which project encumbrances can be made. SED CODE: 304000010040 AGENCY NAME: NYC BOARD OF EDUCATION RUN DATE: 10/02/15 2016 PROJECTS SCHEDULED/ PROJECT # CONTRACT # FUNDING SOURCE BUDGET PAID TO DATE START END 0294160243 TITLE IIB-MATH/SCI 2,300,000 460,000 07/01/15 06/30/16 0294160244 TITLE IIB-MATH/SCI 780,842 156,168 07/01/15 06/30/16 OPEN PROJECTS 2015 AND PRIOR 0021151744 TITLE I A&D IMPROV 2,595,806 2,299,081 09/01/14 08/31/15 0294150243 TITLE IIB-MATH/SCI 2,300,000 460,000 07/01/14 06/30/15 0294150244 TITLE IIB-MATH/SCI 800,000 160,000 07/01/14 06/30/15 0636151004 EXTENDED LEARNING 5,891,938 1,666,651 07/01/14 06/30/15 TOTAL 14,668,586 5,201,900 USE YOUR BROWSER BACK BUTTON TO RETURN TO PREVIOUS PAGE FOR QUESTIONS, PLEASE CONTACT VIA EMAIL firstname.lastname@example.com OR PHONE 518/474-4815


text:
Clark County
Crews extinguish multiple fires in Clark County
VANCOUVER � Multiple small brush fires flared up in Clark County on Tuesday afternoon, but fire officials say they were all out.
Date: 08/04/2015 | Northwest
3 dead in homicide-suicide at home in Hazel Dell
HAZEL DELL � A Clark County sheriff�s officer says two women and one man have been found fatally shot in a home in Hazel Dell, apparently in a double homicide-suicide.
Date: 07/14/2015 | Northwest
Clark County health officials say whooping cough on rise
VANCOUVER, Wash. � Clark County health officials say whooping cough has reached �outbreak levels� in the region.
Date: 07/13/2015 | Northwest
Whooping cough advisory issued for Clark County
VANCOUVER, Wash. � Health officials are worried about the 300 cases of whooping cough that have been reported in Washington state so far this year.
Date: 04/21/2015 | Northwest
Clark County jail inmate dead in apparent suicide
VANCOUVER, Wash. � The Clark County sheriff�s office says an inmate found unresponsive late Monday afternoon in his Vancouver jail cell apparently killed himself.Corrections deputies, medical staff and Vancouver Fire Department responders tried unsuccessfully to revive the man.
Date: 03/31/2015 | Northwest
Uncertain if gun range bullet struck Brush Prairie woman
VANCOUVER, Wash. � The Clark County sheriff�s office says it has been unable to confirm whether or not a bullet that hit a woman in the head outside her Brush Prairie home came from the nearby Clark Rifles gun range.
Date: 02/10/2015 | Northwest
Clark County prosecutor: Shooting of bystander justified
VANCOUVER, Wash. � The Clark County prosecutor says three members of a SWAT team were justified in shooting a bystander during a manhunt last Halloween.
Date: 02/06/2015 | Northwest


text:
Dave Grohl Reveals Never-Before-Heard Solo Track 'Hooker on the Street' From Nirvana Years
Dave Grohl's 'Hooker on the Street' finally debuted over 20 years after its creation.
Dave Grohl's 'Hooker on the Street' finally debuted over 20 years after its creation.
In a new interview with Rolling Stone, Grohl remembers how 'Dimebag' Darrell shaped Foo Fighters' philosophy of backstage hospitality.
Grohl swore off the stuff after an incident with TV's most superfluous family.
Courtney Love discussed her reconciliation with Nirvana's Dave Grohl and Krist Novoselic during an appearance on ABC's 'Jimmy Kimmel Live.'
In a new interview, Grohl says, "Wait until you see what we're doing for the next record."
In a new interview, frontman Dave Grohl reveals the new song 'Subterranean' was inspired by the end of Nirvana.
Nobody ever set out to be a grunge icon — at least nobody who actually became one.
Foo Fighters are making the most of their New York stay, inviting Heart's Ann and Nancy Wilson to join them on 'Letterman' and playing a post-show book bash.
Dave Grohl joined a who's who of music to record a cover of the Beach Boys classic 'God Only Knows' for BBC Music.
Metallica and Dave Grohl will salute U.S. Veterans and their families at 'The Concert for Valor,' which will take place in Washington, D.C., on Veterans Day.
The '80s Washington D.C. hardcore punk scene is easily one of the most important underground movements in music.
This month has brought both wonderful and tragic events to Dave Grohl and his family.


text:
On this past Sunday on Seattle's Lake Union almost 500 rowing shells racing in nearly 50 events were greeted by a glorious fall day that could have been described by the 19th century...
Ready to get your pain on? Vashon Island Rowing Club's biggest fundraiser of the year comes at the expense of your tired legs. The Passport2Pain is an amazingly fun but grueling ride...
Tail of the Lake Regatta 20151
Passport2Pain 2015 is Almost H...2
Vashon Island Rowing Club Cele...3
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Master Rowers Beat the Heat at Regionals
Rule one: Apply sunscreen liberally. Rule two: Stay continually hydrated. Rule three: Stay under the shade of the cottonwood trees whenever possible. Racing in sweltering heat well north of 90 degrees most of the weekend, Vashon Island's Masters rowers medalled in half the events they entered at the Northwest Masters Regional Championships June 26-28 on Vancouver Lake near the...Read More
Posted | 0 Comments
U.S. Rowing Junior Nationals – 2015
Most rowing venues on the annual crew calendar are rivers, lakes, estuaries or bays that have been either permanently or temporarily set up as a racing course. Nathan Benderson Park near Sarasota, Florida, is a noteworthy exception, having been designed and constructed solely as a world-class rowing venue. Nine lanes wide and evenly dredged, the racing course is flanked by a...Read More
Posted | 0 Comments
Vashon Junior Alumni Perform Well on...
The transition from Junior to Collegiate rowing is best understood in the...
Posted | 0 Comments
2015 Youth Regional Championships
Two and a half seconds. In this weekend's NASCAR race that distance equated...
Posted | 0 Comments
Windermere Cup 2015
Prior to all of this pageantry of the annual Opening Day boat parade through...


text:
The Ice Cream Trunk is to multifunctional toy combined with a storage toy chest inside! Added feature of free wheeling will provide lots of fun for play time, transporting and storing toys. Fun to clean up when play time is over.
Manufacturer's Suggested Age: 3-6 Years
Dimensions: 14.010H 8.000W 8.500D
Product Weight: 2.300
other Info.
- Online Item #: 14762904
- Store Item Number (DPCI): 204-12-0055
- Imported


text:
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Included with purchase ($20 value).
Weights & Dimensions
|Pen Type: Rollerball (Gel)
|Retractable: No
|Pack Size: Individual
|Point: Bold
|Item: Pens
|Country of Manufacture: Japan
About the ManufacturerOffering more than 3,000 products and brands that include Sharpie, Paper Mate, uni-ball, Parker, Grumbacher and Waterman, Sanford is an undisputed leader in the writing instrument and art industry.
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Uniball Gel Impact Roller Ball Capped Gel Pen, Metallic Silver Ink, Medium Point
Sanford Ink Corporation


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