text:
|November 4, 1999, 04:29 PM
Join Date: February 26, 1999
Posts: 28
has anyone come up with a simple mod to improve the trigger on a bushmaster m17s bullpup? it's a great handy little poodle shooter but the trigger pull could definitely use some improvement.
|November 4, 1999, 10:48 PM
Staff Alumnus
Join Date: October 12, 1998
Location: Earlington KY
Posts: 2,299
Kampilan, you might try one of the drop-in triggers. They aren't cheap but they are pretty easy to install. George
|November 11, 1999, 11:56 PM
Senior Member
Join Date: April 10, 1999
Location: Idaho
Posts: 112
I don't know of anyone publicizing their work on the Bullpup, but I understand your desires! Watch out that that hammer spring legs don't drop below the sear. It will render the fire control system inoperable, and it can happen when just cleaning the rifle. I'm not impressed with this, but it's a fun little rifle.
I have two suggestions. Buy a tube of Moly-Slide (from NECO Enterprises, and available from Brownell's, Natchez, etc.). It's a high-grade, concentrated Molybdenum Disulfide grease.
First, apply a little of this to the bearing surfaces. Then, coat your thumb and index finger and apply a little moly to the entire trigger bar (that run from front to rear). You won't think this is a match trigger, but it will substantially improve the quality of the trigger.
If you learn better ideas, let me know!
|November 14, 1999, 10:05 AM
Senior Member
Join Date: July 1, 1999
Location: Southern Maine, Bushmaster Country
Posts: 275
I used to work for Bushmaster, and now that I don't represent the company anymore, I can throw out some hints.
First, yank the spring out of the middle of the trigger. It really doesn't need to be there at all and only increases the trigger pull. This is the spring between the trigger and the cam in the middle of the trigger. Removing that will help a lot.
Lubing all contact surfaces will also help, especially where the trigger cam rides on the inside of the frame, lots of friction right there.
If you're really handy, you might want to make another trigger cam out of aluminum or something, change the location of the holes slightly, and try that. You can change the leverage that way and change the trigger pull a lot.
Because of the linkage, smoothing the contact surfaces in the firing pocket usually doesn't really accomplish much, and I always recommend leaving them alone. You can do a lot more just by smoothing the link rod itself and lubricating it well.
That's pretty much it without getting too crazy and making custom springs and stuff like that. These fairly simple mods will improve the stock trigger a lot, but it will still be heavier than a regular trigger. Bullpup triggers are tough to tinker up, it's the design that complicates matters. Anyway, hope this helps.
With my shield or on it...
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The owner of a Northern California bar has been charged with misdemeanor disorderly conduct for installing a hidden camera in the bathroom.
Panama Bar owner Robert Stephen Mowry II tells the Chico Enterprise-Record he installed the cameras in a fake smoke detector to catch vandals and protect the safety of customers. The camera was installed in 2005.
Butte County prosecutors say a customer notified police after seeing an employee watching a live video feed from inside the bathroom.
California law prohibits videotaping in public bathrooms or dressing rooms where there's a reasonable expectation of privacy.
Information from: Chico Enterprise-Record, http://www.chicoer.com


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Next Oracle User Group meeting - Log Miner - Feb 21st
Next Oracle User Group meeting - Log Miner - Feb 21st
Thank you to Tim Robinson and El Paso for providing the facilities and
to Rick Goudeau of Clearwinds Technologies for doing the presentation.
Please RSVP if you plan on attending. Just reply to this message
to RSVP.
NOTE: This is a bring your own lunch meeting. We'll have several door
prizes, including proceedings CD's from the IOUG-A Live 2001.
Oracle LogMiner
-Presented by Rick Goudeau of Clearwinds Technologies-
DATE: Thursday, February 21st
TIME: 11:30 am
PLACE: El Paso Corporation
LUNCH: Bring you own, including drink
(food court in the Harbert Center)
RSVP REQUIRED: Yes
Oracle LogMiner
LogMiner is an Oracle 8.1 new feature. It allows you to format and
display data from the archive redo logs. This is useful in tracking
data changes, or planning a point in time recovery, and perhaps
doing some data trend analysis. Logminer consists a series of Pl/Sql
procedures, and v$sql views to extract and display log data.
Rick Goudeau is an Oracle database consultant with Clearwinds
Technologies. Rick has been a dba for more years than he would like
to remember, and has been using Oracle in a variety of environments
since 1990 starting with release 6.0. Clearwinds provides local database,
systems engineering, networking, and security services.
El Paso Corporation
1900 5th Avenue North
Birmingham, AL 35202-2563
The procedure when you get to the building is to go to the 20th floor and
sign in. You will be directed to the 29th floor and we'll have a sign
There will be some limited parking available in the deck attached to the
building (enter on 19th street). Around lunch time it may be enough. There's
plenty of parking in the BPA deck #7 at the corner of 18th Street and 5th
Ave (enter on 17th street or 18th street).
We hope to see you there.
Jim Hill


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Labyrinths: Walking Earth's Transformational Paths
Sunday Apr 21, 2013
Bachechi Open Space
9521 Rio Grande Blvd NW
Albuquerque, NM 87114
Website: Click to Visit
Led by BJ Harris, this hands-on workshop will teach participants about the history and styles of labyrinths and their connection to the earth.
Using natural ephemeral materials, workshop attendees will create several temporary labyrinths together at the Bachechi Open Space, providing inspiration to create your own labyrinths at home. Part of the Bernalillo County Open Space Program's 'Naturalist Series.' Awaken your senses to the natural world around you by observing the wondrous plants and animals in the Bosque and learn from local experts about this extraordinary ecosystem. Bernalillo County Open Space presents the Naturalist Series featured at Bachechi Open Space located at 9521 Rio Grande Blvd. Parking is available at the Alameda parking area. The workshops are FREE; however, space is limited and participations must register. Register online at www.bernco.gov/openspace. Contact Bernco Open Space Program Coordinator Colleen Langan-McRoberts with any questions at 505-314-0398 or firstname.lastname@example.com.
Contact:Colleen Langan-McRoberts
Phone: 505-314-0398
Website: Click to Visit
Save this Event:iCalendar
Google Calendar
Yahoo! Calendar
Windows Live Calendar


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Founders Day Weekend April 20-22
Founders Day weekend at Southwestern College will be Friday through Sunday, April 20-22. Four Southwestern College halls of fame will open their doors to new inductees, and the newly-refurbished Reuter Organ will be dedicated. The celebration will include recognition of inductees entering the Leaders in Service Hall of Fame for the Social Sciences, Fine Arts, Business, and Educators Halls of Fame.
Hall of Fame festivities will begin at 5:30 p.m. on Friday, April 20, with the unveiling of plaques of those entering the Leaders in Service Hall of Fame. At 6 p.m. there will be a dinner in Deets Library followed by the induction ceremony at 6:45. Limited seating is available and RSVP is necessary.
Saturday's schedule begins with the unveiling of plaques honoring the inductees into the Fine Arts Hall of Fame at 10 a.m. in the Darbeth Fine Arts Center lobby. The inauguration ceremony will be at 10:30 a.m. in Messenger Auditorium.
At 11:30 a.m., there will be a walking tour of the Richardson Performing Arts Center.
At 1:30 p.m., a reception for the Business Hall of Fame inductees will be held in the Deets Library lobby followed by the induction ceremony at 2 p.m.
At 3:30 p.m. in Deets Library, there will be a reception for the Educators Hall of Fame inductees followed by the induction ceremony beginning at 4 p.m.
At 8 p.m., the college will dedicate the Reuter Organ followed by an organ concert featuring James Leland and James Strand.
Founders Day weekend will conclude Sunday at 3 p.m. with the Spring Choral Concert in Richardson Performing Arts Center. There is no admission charge to the concert.
"Founders Day weekend is always an exciting time at Southwestern and this year's celebration certainly promises to fulfill that tradition," says Susan Lowe, director of alumni programs at SC. "The college is honored to recognize several outstanding Moundbuilders at the four halls of fame that will take place, and we hope alumni, friends, and members of the community will join us."
Individuals entering the various halls of fame include:
• Leaders in Service: Larry Eason '55; Eugene Lowry '55; Myrne (Richards) Roe '58.
• Fine Arts: Orcenith Smith, James Strand, Jerry Thomas '81.
• Business: Terri (McCorkle) Dalenta '87; Ron Richardson '83. Business Builder Award: Bob Redford.
• Educators: E. Katherine (White) Davidson '38/'56; Anne Farmer '97; James Wilson '67. Marilyn McNeish Special Education award: Andrew Brenn '01/'06.
For more information about any of the events for Founders Day, contact Lowe at (620) 229-6334.


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Neurocardiogenic Syncope
View Single Post
Senior Member
Join Date: Jun 2003
Posts: 153
Hi everyone
I was recently diagnosed with NCS after being misdiagnosed with epilepsy for the last 20 years! Grrrr.
Zazzaz: I live in Portland too! I use the Yahoo group for NCS that someone else mentioned - it's a pretty good group, very supportive, but maybe not as informative as I'd like it to be.
Analyzethis: I started taking those same supplements too and also found them to be very helpful. However, the calcium and magnesium tend to cause "the trots" a little, so I've stopped taking them and am trying to figure out how to take them without the bad effects... does anyone have any ideas?
to Mamaslittlehelper - I think you should take your daughter in to see a cardiologist. They can more accurately diagnose the NCS as there are many different paths it can take - some people start with a too fast heart beat, some too slow, or too hard, but ultimately we all end up with a drop in blood pressure that causes the fainting. Some people who have this have a type of heart murmer called a mitral valve prolapse (this is fairly common and I believe not dangerous).
One more question - does anyone else get really sick before of after fainting? Before I faint, I get hot and clammy, nauseaus, short of breath, and feel sort of anxious... then afterwards I get really sick to my stomach and often will throw up. I'm curious if anyone else has this happen. ALso, although most of the time I've been standing or sitting, I've had a few while lying down - anyone else?
View Public Profile
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By Anthony Faiola
Tuesday, September 28, 2010; 8:28 PM
LONDON - Fears that Europe could see another Greek-like debt crisis unfold in Ireland or Portugal escalated Tuesday, with investors selling off their bonds and analysts warning that both nations might be heading into critical periods that could trigger bids for bailouts.
The mounting problems in two of Europe's smallest economies are emerging as the biggest threat to an otherwise robust recovery underway in the major economies of the region, including Germany and Britain. Deteriorating conditions in Ireland and Portugal have already pushed the euro off a five-month high against the dollar, causing fresh jitters in stock markets across the region and beyond.
A sharp turn for the worst, analysts say, could spark a new round of financial turbulence in global markets from New York to Hong Kong, last seen when Greece's finances spiraled out of control over the summer. That crisis led to an emergency rescue by the Washington-based International Monetary Fund and the European Union.
Concern has been growing for weeks. But on Tuesday, investors punished Ireland in particular after a warning by Standard & Poor's that it might further downgrade Irish bonds as the cost of that nation's government bank rescue potentially reaches backbreaking levels. In Portugal, attempts to rein in runaway spending are falling far short of promises, prompting fear that it might need to follow Greece, which late last spring secured a $145 billion sovereign debt bailout from the IMF and E.U.
Ireland remained the primary focus of attention. Initially, the island nation of 4 million escaped the worst of the fallout from the Greek crisis by vowing to make painful spending cuts, reducing payments to everyone from widows to the blind. Since then, however, the cost of rescuing its troubled financial institutions, particularly the massive Anglo Irish Bank stung by the collapse of the Irish real estate boom, have soared. S&P warned the cost could reach a staggering $46 billion, making the budget deficit markedly worse that Greece's was before it was forced to seek a bailout. In comments to the press Tuesday, a senior analyst from the Fitch rating agency also raised fresh doubts about Ireland's rating.
At the same time, the Irish economic recovery fell apart in the second quarter, diving 5 percent at an annualized rate. The drop is fueling worries that the nation might be heading into another deep recession after its economy shrank by a whopping 10 percent in 2009.
Irish officials have rejected speculation the nation might need an IMF/E.U. bailout. On Thursday officials there are set to outline the cost of the Irish bank rescue. If those costs exceed current estimates, however, analysts say it could spark a full-blown crisis in Dublin. Adding to the government's woes, the opposition Fine Gael party announced this week that it would try to force snap elections and oust the administration of unpopular Prime Minister Brian Cowen.
"Many thought Ireland was on the right path, but if they don't stop this market panic soon, it is going to be a real problem," said Sonia Pangusion, senior economist for IHS Global Insight in London. "The bigger effect will come from the euro, since all this could put new downward pressure on it."
But the problems with Ireland and Portugal might not generate the same kind of upset seen when Greece reached a tipping point. During the first half of the year, global financial markets convulsed as the Greek debt crisis raised doubts about the ability of overextended nations in Europe to meet their obligations to investors, who lent them hundreds of billions of dollars by buying their bonds. The measure triggered a wave of government cutbacks from Greece to Spain, sparking a number of protests, with the most violent demonstrations on the streets of Athens.
The European Union has since established a $1 trillion bailout fund for countries in fiscal crisis, though just how willing other European nations will be to offer up those funds remains in question.
In addition, during the height of Greece's problems last summer, panicked investors drove up borrowing costs for a number of financially overextended nations across the region. They demanded higher interest rates to buy the bonds of nations including those with large economies, such as Spain and Italy. But in recent months, investors have been differentiating more between nations, with pressure easing on Spain and Italy, even as it escalated in Ireland and Portugal.
Still, a worsening situation in Ireland or Portugal could yet reignite a stronger panic across Europe, particularly in Spain. A Moody's rating review that is concluding this week could downgrade the nation's coveted AAA rating on its debt. Such a move could trigger a fresh sell-off in Spanish bonds, and worsen the plight of Ireland and Portugal.
On Tuesday, the cost of insuring against a default leapt in Ireland, Portugal and Greece. But it also increased, albeit more moderately, in Italy and Spain.
"There is still a lot of uncertainty, and the market is responding to that risk," said Ioannis Sokos, bond analyst at BNP Paribas in London.


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February Lady in Mushishi
Happy February! This week I wanted to feature a knit that was named after the shortest month of the year.
February Lady is designed by Pamela Wynne, who was inspired by Elizabeth Zimmerman's Baby Sweater on Two Needles.
**Jessica's version is cropped, so you'll most likely need more than one skein if you plan on making the full length version.
Category Design/Patterns, Yarn | Tags: mushishi february lady sweater pamela wynne plymouth yarn company
by Carmel
On February 3, 2011 at 10:12 am
That is the best February Lady sweater I've seen……I normally harbor a viscious dislike for it!! Plus I love mushishi it's one of my favorite Plymouth Yarns!!


text:
WASHINGTON, June 2 (UPI) -- Two women protesting Peruvian President Alan Garcia's visit to Washington created a stir outside of the White House, the Secret Service said.
A Secret Service spokesman said one woman chained herself to the fence ringing the White House Tuesday and her mother poured a black fluid on her, The Washington Post reported Wednesday. The liquid, determined to be neither flammable nor toxic, apparently was meant to symbolize petroleum, the spokesman said.
President Barack Obama was meeting with Garcia at the time of the incident.
The Secret Service said the women were arrested. The younger woman was charged with disorderly contact and her mother was charged with defacing government property, because the liquid apparently stained the pavement at the White House, the Post said.


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Measurement instrument delivers jitter analysis up to 80 MHz
Post a comment
Menlo Park, Calif. SyntheSys Research, Inc. has introduced the BERTScope 11000A digital communications receiver with jitter analysis (DCRj). The company claims it's the first 10 Gbits/s telecommunications jitter solution to test device compliance and show the full 80-MHz spectrum to precisely see and troubleshoot device behavior. The DCRj is aimed at telecom applications, particularly for developers of XFP and SFP+ transceivers.
The DCRj is a standalone jitter measurement solution with built-in compliant clock recovery that also provides OC-192/STM-64 and 10GbE reference receiver capability to the BERTScope series of analyzers including the BERTScope S 12500B signal integrity analyzer and third-party instruments used for transmitter compliance. Offering broad wavelength coverage, the DCRj has an amplified receiver for testing single-mode or multimode transmitters.
The DCRj also provides an electrical input to enable jitter spectral analysis on electrical signals. While the majority of the optical or electrical signal is passed through to provide an electrical output for other test instrumentation, a small portion is used to drive a standards-compliant, flexible clock recovery, said the company. As well as providing the correct loop bandwidths for transmitter measurements, such as the 4-MHz loop bandwidth called for by SONET, the DCRj provides the engine for one of the most sensitive jitter generation measurements available, said the company.
Availability: 12 weeks ARO.
Product information: DCRj
SyntheSys Research, Inc., www.bertscope.com


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Tag: the entrance
Pancakes by the Seawritten by erinwoodgatesphotography on 2012-08-14 #places
Pancakes by the sea, is as the name suggests, right by the water at The Entrance on the New South Wales Central Coast. I headed there with some friends one sunny morning and almost went into chocolate overload. It is everything you expect from a pancake restaurant and more!5 Share Tweet


text:
Written on: 21/03/2013 by Tahhgreen (1 review written)
I ordered a print for my girlfriend for mother's day. It arrived today (21st March) so needless to say she didn't get it on the day in question. Regarding quality though I can't complain and I would buy from these again. I would just order well in advance. I also want to mention that when ordering I accidentally ordered 2; anyway I sent an email explaining everything and within a week or so I'd received an email back and a refund for my accidental purchase. Other than the slow postage I was greatly impressed and for the price it's we. well worth it. Thanks Matt.


text:
Credits & Info
- Uploaded
- Oct 22, 2010 | 7:10 PM EDT
- File Info
- Song
- 3 min 1 sec
- Score
- 4.47 / 5.00
Related Content
Heard in 1 Entry!
View all submissions featuring this audio file.
Licensing Terms
Please contact me if you would like to use this in a project. We can discuss the details.
- Score:
- Rated 4.47 / 5 stars
- Plays & Downloads:
- 7,970 Plays | 870 Downloads
- Share Links:
- Genres:
- Electronic - Drum N Bass
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Author Comments
Rated 5 / 5 stars
Roses are red...
Roses are red
violets are blue
all my ears
belong to you
Rated 5 / 5 stars
Just wrote this to give ya the 5stars, frankly beyond words [cliche but true] if i used words to describ this it wouldnt be english yo haha. itd have to be like some martian shit.? fuck now i sound like lil wayne.... idk but yeah this is epic. 'nuff said.
Rated 5 / 5 stars
A lot of your songs...
...seem to be Hybrid inspired, but I'm loving single one of them, this ain't no exception. Your stuff is tight, hope to hear you keep pushing songs out!
hi old boy
go to my soundcloud
Rated 4.5 / 5 stars
Very well done.
Agreed with the other use, I thought it was Hybrid as well.
The crisp drums are well done, as well as the bass drops are very nice. A good song to play when in a spaced out mood to just flow with your thoughts. The bass carries you away. Well done.
ahaha. I seriously love how you people seem to catch the Hybrid likeness so well. Thanks for the appreciation
Rated 4.5 / 5 stars
I thought this sounded like hybrid while playing ether: space defense! :D
Loved this track, it sounds spacey, and I love the beats and tune. It fit the game very well too, and made it more enjoyable to play. :)
Great track, keep em coming! ^^


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Do you love to learn new things but never seem to have enough time? The Guelph Public Library is hosting a HOW-TO Expert Festival on Saturday, March 29, where everyone can learn something new in just 10 minutes!
Fourteen Experts. 10 Minutes. 100% Free. Think of all the wonderful skills you can learn in such a short time…HOW-TO: become a juggling wizard, a "checkmate" master, learn to wrap a sari in style, choose the right camera for the best shot, trim your cat's nails, repair a flat bicycle tire with ease, tie a knot like the pros, dance Zumba, prepare for a good hike, look and feel great every day, become an origami whiz, draw cartoons, create a coin collection with personality, and more!Guelph Public Library's HOW-TO Festival poster
The HOW-TO Expert Festival will be held on Saturday, March 29 from 11:00 a.m. to 2:00 p.m. at the Main Library (100 Norfolk Street). Come join us and learn something new!


text:
Oh! Oh!
We can have a clubhouse at Comdex! Only members allowed to enter--and no girls! We can read comic books and everything! =)
I do like the idea of a Comdex booth, though.
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|Publication number
|Publication type
|Application number
|Publication date
|Jun 29, 2010
|Filing date
|Sep 13, 2006
|Priority date
|Sep 13, 2005
|Fee status
|Also published as
|Publication number
|Randall True
|Original Assignee
|Affymetrix, Inc.
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (63), Non-Patent Citations (24), Referenced by (8), Classifications (23), Legal Events (5)
|External Links: USPTO, USPTO Assignment, Espacenet
This U.S. patent application claims priority from co-pending U.S. provisional application Ser. No. 60/762,238 filed Jan. 25, 2006 and U.S. provisional application Ser. No. 60/716,694 filed Sep. 13, 2005, the subject matter of each being incorporated herein by reference in their entirety.
The present invention relates to the art of microstructures, and more particularly to encoded microparticles.
Microparticles or nanoparticles are often referred to as structures whose characteristic dimensions are on the order of micrometers or less, such as those with volumes of 1 mm3 or less. Due to their unique properties arising from their small characteristic dimensions, microparticles have found distinguishable applications in laboratory research and many industrial fields. Encoded microparticles possess a means of identification and are an important subclass of the general field of microparticles. Because encoded particles carry information and can be physically tracked in space and time, they greatly extend the capabilities of non-encoded particles. A particularly important application for encoded microparticles is multiplexed bioassays, including those involving DNA and proteins. Other important fields for encoded microparticles include combinatorial chemistry, tagging, etc. Many biochemical and non-biochemical applications as will be discussed herein below.
For many applications, one more desirable attributes include: a large number of identifiable codes (i.e. a high codespace), accurate and reliable identification of the encoded particles, material compatibility for a particular application, low cost manufacturing of the microparticles (on a per batch, per particle, and per code set basis), and flexibility in the detection systems.
Several approaches to produce encoded microparticles have been developed in the past, such as fragmented colored laminates, colored polystyrene beads, quantum dot loaded polymer beads, rare-earth doped glass microbarcodes, electroplated metal nano rods, diffraction grating based fiber particles, and pattern bars and disks, and other types of microparticles. These technologies however suffer from any of a number of limitations, such as, insufficient codespace, high cost, inadequate precision, poor performance in applications, problematic clumping incapability of large scale manufacture, and complicated preprocessing or assay procedures.
Therefore, what is desired is an encoded microparticle or a set of encoded microparticles carrying coded information, methods of making the same, methods for providing the codes for microparticles, methods for fabricating the microparticles, methods and systems for detecting microparticle, and methods and systems for using.
As an example of the invention, a method of forming a plurality of encoded microparticles with a lithography process to define codes for identifying the particles is disclosed herein. The method comprises: providing a planar substrate on which the microparticles are to be formed; providing a 1st pattern having a 1st portion of an encoding pattern; exposing the substrate to the 1st pattern, thereby printing the 1st portion of the encoding pattern; providing a 2nd pattern having a 2nd portion of an encoding pattern; exposing the substrate to the 2nd pattern, thereby printing the 2nd portion of the encoding pattern; and wherein a complete encoding pattern is provided.
As another example of the invention, a method of forming a plurality of encoded microparticles with a lithography process to define codes for identifying the particles comprising the steps of: providing a planar substrate on which the microparticles are to be formed; performing a first lithography step having a 1st portion of an encoding pattern; performing a second lithography step having a 2nd portion of an encoding pattern; and wherein a complete encoding pattern is provided.
Such objects of the invention are achieved in the features of the independent claims attached hereto. Preferred embodiments are characterized in the dependent claims. In the claims, only elements denoted by the words "means for" are intended to be interpreted as means plus function claims under 35 U.S.C. §112, the sixth paragraph.
While the appended claims set forth the features of the present invention with particularity, the invention, together with its objects and advantages, may be best understood from the following detailed description taken in conjunction with the accompanying drawings of which:
An encoded microparticle is provided carrying a code, and a set of encoded microparticles are provided with distinguishable codes, wherein the codes comply with a pre-determined coding scheme. Preferably, the microparticles in the examples below have a volume of 1 mm3 or less. The microparticle of the invention enables fast, precise and less complicated detection of the code. Methods for providing the codes on microparticles, methods for fabricating the microparticles, methods and systems for detecting the microparticle, and methods and systems for using the microparticles are also disclosed.
In the following, the invention will be discussed with reference to specific examples. It will be appreciated by those skilled in art that the following discussion is for demonstration purposes, and should not be interpreted as a limitation. Instead, other variations without departing from the spirit of the invention are also applicable.
Overall Structure of the Microparticle
As an example,
The microparticle in this particular example has a set of segments (e.g. segment 102) and gaps (e.g. gap 104) intervening the segments. Specifically, segments with different lengths (the dimension along the length of the microparticle, e.g. along the Y direction) represent different coding elements; whereas gaps preferably have the same length for differentiating the segments during detection of the microparticles. The segments of the microparticle in this example are fully enclosed within the microparticle, for example within body 106. As an alternative feature, the segments can be arranged such that the geometric centers of the segments are aligned to the geometric central axis of the elongated microparticle. A particular sequence of segments and gaps represents a code. The codes are derived from a pre-determined coding scheme.
Segments of the microparticle can be any suitable form. In an example of the invention, each segment of the microparticle has a substantially square cross-section (i.e. the cross-section in the X-Z plane of a Cartesian coordinate as shown in
The microparticle example of
The code elements, i.e. the segments and gaps, may take any desired dimensions. As an example of the invention, each coding structure has a characteristic dimension that is 5 um (microns) or less, such as 3 microns or less, and more preferably 1 micron or less, such as 0.8 or 0.5 microns or less. In particular, when gaps are kept substantially the same dimension while the segments vary in dimension, each gap preferably has a characteristic dimension that is 1.5 microns or less, such as 0.8 or 0.5 microns or less.
As one example, if forming the microparticles on a 12-inch silicon wafer with 0.13 line widths, the gap areas can be made to have 0.13 um minimum widths, with the less transparent segments having widths of from 0.13 um to much larger (depending upon the desired length of the particle and the encoding scheme and code space desired). Minimum gap widths, as well as minimum segment widths, of from 0.13 to 1.85 um (e.g. from 0.25 to 0.85 um) are possible depending upon the wafer fabrication used. Of course larger minimum gap and segment lengths (e.g. 1.85 to 5.0 um, or more) are also possible. Other sized wafers (4 inch, 6 inch, 8 inch etc.) can of course be used, as well as wafers other than silicon (e.g. glass), as well as other substrates other than silicon (larger glass panels, for example).
Though the microparticle may have the same length in the X, Y and/or Z directions, preferably the encoded microparticle has a ratio of the length to width of from 2:1 to 50:1, e.g. from 4:1 to 20:1. In an example of the invention, the microparticle has a length (e.g. the dimension along the Y direction) of 70 microns or less, 50 microns or less, 30 microns or less, such as 20 microns or less, 16 microns or less, or even 10 microns or less. The width (e.g. the dimension along the X direction), as well as the height (the dimension along the Z direction), of the microparticle can be 15 microns or less, 10 microns or less, 8 microns or less, 4 microns or less, or even 1 microns or less, such as 0.13 micron. Widths as small as from 0.5 to 2 microns are also possible. Other than the shape as shown in
The coding structures and gaps of the microparticles can take any suitable form as long as the coding structures and gaps together represent detectable codes. As mentioned above, the cross-section of the microparticles, as taken perpendicular to the length of the particle, can be square, rectangular, circular, elliptical, or any desired shape such as jagged or curved shapes or other profiles. When the cross-section is rectangular, the rectangle preferably has an aspect ratio (the ratio of the length to the width or height) of 2:1 or higher, such as 4:1 or higher, 10:1 or higher, 20:1 or higher, or even 100:1 or higher, but preferably less than 500:1. The ratio of the width to height can be around 1:1 (square cross section), or have a ratio of from 1:4 to 1:1—preferably a ratio that allows the particle to rest on either the sides defining the width or height of the particle such that the code of the microparticle can be detected regardless of which of the elongated sides the particle rests.
To facilitate fast, cost-effective, reliable, and easy detection of the code represented by the coding structures and gaps, it is preferred that each coding structure is as omni-directional as possible to the detection means. That is—each coding structure exhibits substantially the same geometric appearance or detectable properties when observed from at least two directions, more preferably from four (or all, if not four-sided in cross section) directions perpendicular to the length of the microparticle. Accordingly, the coding structures preferably possess rotational symmetry along the length of the microparticle, such as 2-folded or 4-folded rotational symmetry.
A microparticle of the invention can have any suitable number of coding structures depending upon the shape or length of the particle, and the code space desired. Specifically, the total number of coding structures of a microparticle can be from 1 to 20, or more typically from 3 to 15, and more typically from 3 to 8.
The desired code can be incorporated in and represented by the microparticle in many ways. As an example, the coding elements of the pre-determined coding scheme can be represented by the segment(s)—e.g. segments of different lengths represent different coding elements of the coding scheme. Different spatial arrangements of the segments with the different (or the same) lengths and intervened by gaps represent different codes. In this code-incorporation method, the intervening gaps preferably have substantially the same dimension, especially the length in the direction to which the segments are aligned. As another example, the codes are incorporated in the microparticle by arranging gaps that vary in lengths; while the segments have substantially the same dimension and are disposed between adjacent gaps. In another example, the both segments and gaps vary in their dimensions so as to represent a code. In fact, the code can also be represented in many other alternative ways using the segments, gaps, and the combination thereof.
For representing a code derived from the predetermined coding scheme, the segments and gaps are arranged along the length (the Y direction) of the elongated microparticle (2D, or even 3D, arrangements however are also possible). Specifically, the segments and gaps are alternately aligned along the length with the each segment being separated (possibly fully separated and isolated) by adjacent gaps; and each gap is separated (possibly fully separated and isolated) by adjacent segments, which is better illustrated in a cross-sectional view in
In an example of the invention, any suitable number of segments can be used—e.g. from 2 to 20, or more typically from 3 to 15 segments (more typically from 3 to 8 segments) of less transparent material (as compared to the intervening gaps between the segments) are provided within the encoded microparticle. To form the code, it is possible that the segments of less transparent material are varying lengths. Alternatively, the segments of less transparent material could each have substantially the same length whereas the intermediate segments of more transparent material could have varying lengths. Of course, the segments of more transparent material and the intermediate segments of less transparent material could both have varying lengths in order to represent the code.
Referring to
In order to enable detection of codes incorporated in microparticles, the segments and gaps in each microparticle can be composed of materials of different optical, electrical, magnetic, fluid dynamic, or other desired properties that are compatible with the desired detection methods. In one example the segments and gaps are directly spatially distinguishable under transmitted and/or reflected light in the visible spectrum. For example, when the code detection relies upon optical imaging, the distinguishable property (segments vs. gaps) can be a difference in transmissivity to the particular light used for imaging (which can be any desired electromagnetic radiation—e.g. visible and near-visible light, IR, and ultra-violet light. The segments can be made to be more light absorbing (or light reflecting) than the intervening spacing material (or vice versa). When the code detection relies upon the electrical property measurements, the property can be resistance and conductance. When the code detection involves magnetic methods, the properties can be inductance and electro-inductance. When the code detection involves fluid dynamic methods, the property can be viscosity to the specific fluid used in the code detection. Regardless of which specific property is relied upon, the segments and gaps are preferred to exhibit sufficient difference in the specific property such that the difference is detectable using the corresponding code detection method. In particular, when the code is to be detected by means of optical imaging, the segments and gaps are composed of materials exhibiting different transmissivity (in an optical transmittance mode) or reflectivity (in optical reflectance mode) to the specific light used in imaging the microparticles. For example, the segments of the microparticle of the less transparent material can block and/or reflect 30% or more, preferably 50% or more, or e.g. 80% or more, of the visible light or near visible light incident thereon.
Given the fact that transmissivity of electromagnetic radiation through an object varies with the thickness of the object, it is preferred that the segments that are capable of blocking and/or reflecting 30% or more, preferably 50% or more, or e.g. 80% or more (or even 90% or more), of the detection light; while the gaps between the coding structures are provided from materials and at dimensions that are capable of transmitting 50% or more, 70% or more, 80% or more, or even 90% or more of the detecting light. Alternatively, the segments and gaps are composed of different materials such that the ratio of the transmissivity difference is sufficient to detect the codeγ, e.g. is 5% or more, 10% or more, 20% or more, 50% or more, and 70% or more. The transmissivity is defined as the ratio of the light intensities of the passed light to the incident light.
The microstructure can be made of organic and/or inorganic materials or a hybrid of organic and inorganic material. Specifically, the gaps (which are preferably more transmissive to visible or near-visible light) and segments (which are preferably less transmissive to visible or near-visible light as compared to gaps) each can be composed organic or inorganic materials, or a hybrid organic-inorganic material. The segments can be composed of a metal (e.g. aluminum), an early transition metal (e.g. tungsten, chromium, titanium, tantalum or molybdenum), or a metalloid (e.g. silicon or germanium), or combinations (or nitrides, oxides and/or carbides) thereof. In particular, the segments can be composed of a ceramic compound, such as a compound that comprises an oxide of a metalloid or early transition metal, a nitride of a metalloid or early transition metal, or a carbide of a metalloid or early transition metal. Early transition metals are those from columns 3b (Sc, Y, Lu, Lr), 4b (Ti, Zr, Hf, Rf), 5b (V, Nb, Ta, Db), 6b (Cr, Mo, W, Sg) and 7b (Mn, Tc, Re, Bh) of the periodic table. However, preferred are early transition metals in columns 4b to 6b, in particular tungsten, titanium, zirconium, hafnium, niobium, tantalum, vanadium and chromium.
The gaps which are in this example more transparent, can comprise any suitable material that is more transparent than the segments. The spacing material can be a siloxane, siloxene or silsesquioxane material, among others, if a hybrid material is selected. The spacing material, if inorganic, can be a glass material. Thin film deposited silicon dioxide is a suitable material, with or without boron or phosphorous doping/alloying agents. Other inorganic glass materials are also suitable such as silicon nitride, silicon oxynitride, germanium oxide, germanium oxynitride, germanium-silicon-oxynitride, or various transition metal oxides for example. A spin on glass (SOG) could also be used. If an organic material is used for the gap material, a plastic (e.g. polystyrene or latex for example) could be used.
Both the segments and the gaps can be deposited by any suitable methods such as CVD (chemical vapor deposition), PVD (physical vapor deposition), spin-on, sol gel, etc. If a CVD deposition method is used, the CVD could be LPCVD (low pressure chemical vapor deposition), PECVD (plasma enhanced chemical vapor deposition), APCVD (atmospheric pressure chemical vapor deposition), SACVD (sub atmospheric chemical vapor deposition), etc. If a PVD method is used, sputtering or reactive sputtering are possible depending upon the desired final material. Spin on material (SOG or hybrid organic-inorganic siloxane materials
As a more specific example, the segments can be comprised of a any suitable silicon material such as CVD (chemical vapor deposition) deposited amorphous silicon. Polysilicon or single crystal silicon area also suitable as are a wide range of other materials as mentioned above. It is preferred, but not necessary, that the material selected for the segments has a high degree of deposition thickness control, low surface roughness, control of etching—both patterning and release (e.g. using a dry plasma etch for patterning and a wet or dry chemical etch for release), and CMOS process compatibility. The gap material can be CVD deposited silicon dioxide. The silicon dioxide may include doping/alloying materials such as phosphorous or boron. Temperature considerations may be taken into account in choosing a combination of more and less transparent materials for the segments and gaps.
In the examples as discussed above, the microparticle is composed of materials of selected distinguishable properties, such as distinguishable optical properties. In the example above, one material has a greater transparency or optical transmissivity than the other material, which difference is detectable under magnification. A specific example of the above is where one material is a light absorbing material, and the other material is a translucent or transparent material with greater light transmittance in the visible spectrum (or in another spectrum should a different detection system be used—e.g. UV, IR etc). In another example, one material is a light reflecting material whereas the other material is either light absorbing or light transmitting. A detectable difference where one material is more opaque and the other material is less opaque, or where one material is more reflective and the other material is less reflective, are within the scope of this example. As mentioned above, the alternating portions of opaque and transparent materials can be made of silicon and glass among other materials. Given the fact that transmissivity (and reflectivity) of almost all materials exhibit dependencies from the thickness of the material, the microparticle may be formed such that the coding structures (i.e. the structures representing coding elements of a code) are derived from a single material.
Referring to
For facilitating the application of the microparticles, especially biological/biochemical/biomedical/biotechnology applications wherein the sample bio-molecules are to be attached to the surfaces of the microparticles, an immobilization layer may be desired to be coated on the surfaces of the microstructures.
The microparticle of
A coding structure layer is deposited and patterned so as to form the coding structures, such as structures 218, 222, 220, 224. After forming the coding structures, surrounding layer 224 is deposited on the formed coding structures. Because the surrounding layer will be exposed to the target sample in the assay, it is desired that layer 224 is composed of a material that is resistant to chemical components in the assay solution wherein the microparticles are to be dispensed. Moreover, for holding the probe molecules, such as nucleic acids (e.g. DNA or RNA), proteins, antibodies, enzymes, drugs, receptors, or ligands, molecules on the surface of the layer, layer 224 is desired to be capable of immobilizing the probe molecules.
Fabrication Process
The following exemplary fabrication processes will be discussed in reference to microparticles with segments and gaps, however it should be noted that the following methods are applicable to many other types of code elements.
The microstructure of the invention can be fabricated with a method that fall into the broad field of micro-machining, such as MEMS fabrication methods. MEMS use the techniques of the semiconductor industry to form microscale structures for a wide variety of applications. MEMS techniques typically, but not in all circumstances, include the deposition of thin films, etching using dry and/or wet methods, and lithography for pattern formation. Because MEMS is an offshoot of the semiconductor industry, a vast worldwide manufacturing infrastructure is in place for cost-effective, high volume, precision production. Generally speaking, the more similar the full MEMS process is to existing integrated circuit processes, e.g. CMOS compatible, the more accessible this infrastructure is.
The microstructure of the invention can be fabricated in many ways, such as fabrication methods used for integrated circuits (e.g. interconnects) or MEMS. In the following, an exemplary fabrication method compatible with the MEMS fabrication for making a microparticle will be discussed with reference to
Referring to
Referring to
The patterning of the layers can be done in many methods, one of which is photolithography that is widely used in standard fabrication for semiconductor integrated circuits and MEMS devices. The most common form of photolithography used in the MEMS industry is contact photolithography. A reticle (aka mask) is typically composed of a binary chrome pattern on a glass plate. The reticle is placed very near or in contact with a photoresist covered wafer (or other substrate). UV light is shone through the mask, exposing the photoresist. The wafer is then developed, removing the photoresist in the exposed regions (for positive-tone photoresist). The pattern on the reticle is thus transferred to the photoresist where it serves as a mask for a subsequent etching step.
Projection photolithography is another type of photolithography that is used exclusively in modern integrated circuit manufacturing. Instead of bringing the mask into physical contact, projection photolithography uses a system of lenses to focus the mask pattern onto the wafer. The primary advantage of this system is the ability to shrink the mask pattern through the projection optics. A typical system has a five times reduction factor. In general, much smaller feature sizes can be printed with projection as compared to contact lithography. A projection photolithography system is also known as a step-and-repeat system (or stepper for short). The maximum pattern or field size on the mask is significantly smaller than the wafer diameter. The mask pattern is repeatedly exposed ("stepped") on the wafer forming an array of "dies". The stepping distance is the distance the wafer stage travels in X and Y between exposures and is usually equal to the die size. This typical scheme produces a non-overlapping array of identical dies, allowing for subsequent parallel processing of the dies on the wafer.
The hard mask layer (150) is further patterned so as to form discrete areas, as shown in
In the example above, the patterning of the hard mask layer is performed in two separate lithography steps. In an alternative example, the reticle may comprise a pattern such that the patterning of the hard mask can be accomplished with a single lithography step. As a further alternative, the hard mask can be omitted and either a two step or single step lithography process used.
After patterning the top hard mask layer, silicon layer 148 is etched so as to form corresponding discrete silicon areas on the substrate, such as silicon segments 168 and 172, with areas there between for material of greater transparency (e.g. gap areas 170 and 172, as shown in
After patterning silicon layer 148, transmissive layer 168 is then deposited as shown in
The microparticles are then separated from each other, while still attached to the underlying substrate, as shown in
The indentations are as a result of the particular fabrication method; and can remain in the final product, or can be removed by, for example, planarization—e.g. chemical-mechanical-polishing (CMP) techniques. In fact, the indentations in some situations can be beneficial for code detection and/or fluorescence quantitation using fluorescent methods because the binding of a fluorescently tagged material to the surface of the microbarcode is greater in the indentation areas (per unit length of the microbarcode), the so called indentation signal enhancement, fluorescence can be greater in the indentation areas and can be used to determine the code (with or without other transmissive or reflective techniques discussed herein below). The same indentation signal enhancement would be applicable with reporter systems other than fluorescence, e.g. radioactive reporters, etc.
Though a silicon wafer was mentioned as the substrate in the example given above, a glass substrate, such as a glass wafer or larger glass sheet or panel (e.g. like those used in the flat panel display industry) could be used. Glass (or silicon) wafers can be of any suitable size—e.g. 4 in., 6 in., 8 in. or 12 in. When a glass wafer is used, typically an additional sacrificial layer will first be deposited (for later removal during the release step). The sacrificial layer can be semiconductor material, such as silicon, an early transition metal, such as titanium, chromium, tungsten, molybdenum, etc. or a polymer, such as photoresist, as mentioned earlier herein.
A scanning-electron-microscopy (SEM) image of a segment (e.g. segment 102) in
An SEM image of a multiplicity of microparticles fabricated with the exemplary fabrication method as discussed above is presented in
The microparticles of the invention can be fabricated at the wafer-level, and released either at the wafer level or die level. Specifically, a plurality of dies each comprising a set of microparticles can be formed on a wafer. The microparticles on each die may or may not be the same—that is the microparticles on each die may or may not have the same code. After forming the microparticles, the dies can be separated from the wafer; and the wafer(s) on the singulated dies can be then removed. An exemplary wafer-level fabrication method is demonstrated in
Referring to
After the formation of the microparticles, the wafer is then broken into dies as shown in
The releasing step can be performed in many ways, such as dry etch, wet etch, and downstream plasma etch. In an exemplary bulk wet etch, shown schematically in
After pelleting the particles through centrifugation or lapse of time, the liquid (so called supernatant) is removed and the particles are washed several times in water or a solvent. "Washing" refers to the successive replacement of the supernatant with a new liquid, usually one involved in the next chemical processing step. After detaching the microparticles from the substrate (or wafer), the substrate can be removed from etchant—leaving the microparticles in tubes. The released microparticles can then be transferred to containers for use.
The microparticles can be fabricated on the wafer level, as shown in
The tendency of the microparticles to form a monolayer is not trivial. Monolayer formation involves many factors, such as the surface charge state (or zeta potential) of the microparticles, the density of microparticles in a specific solution, the fluid in which microparticles are contained, and the surface onto which the microparticles are disposed. Accordingly, the microparticles of the invention are comprised of materials and are constructed in a form that favors the maintenance of a charged state sufficient to substantially overcome stiction forces; and thus microparticles are capable of undergoing Brownian motion which facilitates the formation of a reasonably dense monolayer of particles.
In biological applications, the microparticles are often used to carry biochemical probe molecules. For immobilizing such probe molecules, the microstructure preferably comprises a surface layer, such as a silicon dioxide layer, which can be chemically modified to attach to the probe molecules. In accordance with an example of the invention, the microparticles are constructed such that the microparticles are capable of forming a monolayer, for example, at the bottom of a well containing a liquid; and the monolayer comprises 500 or more particles per square millimeter, more preferably 1,000 or more, 2,000 or more, or 3,000 or more microparticles per square millimeter. In an alternative example, the microparticles can form a monolayer that such that the detectable particles occupy 30% or more, 50% or more, or 70% or more of the total image area (i.e. the image field of view). In connection with the example mechanism of self-assembled monolayer formation, it is preferred that the 2D diffusion coefficient of the microparticles of the invention is greater than 1×10−12 cm2/s . For accommodating the monolayer of the microparticles, the container for holding the microparticles in detection preferably has a substantially flat bottom portion.
The system depicted in
In this image, the particles are in a solution that contains suspended fluorescent molecules. The fluorescent molecules, when excited by the microscope light source, provide illumination from above (i.e. behind with respect to the collection optics, see
For successfully identifying the microparticles, e.g. reading the codes incorporated therein, the images of the microparticles may be processed. Such image processing can be performed with the aid of software programs. According to exemplary examples of software programs and algorithms, pairs of raw and processed image are presented in
Referring to
Table 3 shows identification data for image sets that include those images shown in
Full ID %
Limited ID %
30,069 Codes
1,035 Codes
40x objective
~500 particles/image
9866 particles measured
60x objective
~250 particles/image
2733 particles measured
The microparticles included in Table 3 have a codespace of 30,069, wherein the codespace is defined as the total number of possible codes with the particular particle design, i.e. with the chosen coding scheme and coding scheme parameters. A pre-determined identification method assigns one of the 30,069 possible codes based on the analysis of the particle segment information. 1035 codes were randomly selected, manufactured, and mixed to form the collection. When analyzing the identification of the collection, if the software assigned code is one of the 1035, it is assumed to be correct. The number of "correctly" identified particles divided by the total is called the "ID %". This assumption underestimates the error rate (1-ID %) by the probability that a random error falls within the 1035 present codes, or 1035 divided by 30,069=about 3%. The assumption therefore ignores this 3% deviation and provides a close approximation to the true identification accuracy.
In an alternate example, a flow-cell enabling the microparticles flowing in a fluid can be provided for detection by continuous imaging, as shown in
Other Structures
Another alternative microparticle of the invention is schematically illustrated in
In another example, the fluorescent layer may be comprised of fluorophores, or other luminescent materials. The fluorescent layer may interact with molecular species in an assay, for example with fluorescently labeled nucleic acids or protein samples via Fluorescence Resonant Energy Transfer processes. In yet another example, the microparticles may have a non-fluorescent layer, wherein incorporated in or on the layer are molecules, for example quenchers that interact with luminescent emitter molecules.
In exemplary examples of encoded microparticles comprising indentations, the surface of the particles have fluorescent, or otherwise emitting, molecules attached to or in the surface, as shown in
Method for Producing Codes
The invented general method of generating the codes on microparticles consists of the use of multiple lithographic printing steps of a single code element per particle region. The multiple printing steps create multiple code elements per particle region. The code elements taken together form the code for the microparticle. In a preferred example, the printing steps are performed on many particles in parallel using a master pattern. A master pattern comprises an array of single code elements per particle region. A code element may represent more than one physical feature, such as holes, stripes, or gaps. The master pattern is printed multiple times such that a multiplicity of microparticles with complete codes is formed, wherein the multiplicity of microparticles comprises identical particles (e.g. all particles have the same code). Variations upon this theme, for example wherein the multiplicity of microparticles are not identical, are anticipated and will be described in detail below. Between multiple print steps, a component of the overall printing system changes to translate the code element within the particle region. In a most preferred example, this change is a movement of the substrate on which the particles are formed. In another preferred example, this change is the movement of the master pattern. In yet other examples this change is the movement of an optical element such as a mirror.
An exemplary example of the general method of generating code using multiple print steps involves photolithography as the printing mechanism, e.g. contact photolithography and projection photolithography. An exemplary example of projection photolithographic utilizes a step and repeat system (aka stepper). A reticle contains a code pattern that has a single code element per particle. Through multiple exposures of this code pattern at different lateral offsets, a multiplicity of code elements (per particle) is created. Combined, these code elements form a complete code. The lateral offsets define the code and are programmed into the stepper software. The offsets, and therefore the code, can be changed on a per die or per wafer basis. The codes printed on different dies on a wafer and/or different wafers in a lot are thus controlled by software and can be arbitrarily changed. This enables a powerful flexibility in the manufacture of large sets of codes. A single mask set, having one to a few masks, can be used to generate an arbitrary number of codes, numbering into the 105 range and beyond.
The code pattern, shown in
An exemplary example of the invented method for producing codes uses photolithography and positive-tone photoresit. Positive-tone means that the areas exposed to light are developed away. For a negative-tone resist, exposed regions are what remain after development. The photocurable epoxy SU-8 is an example of a negative-tone resist. In an alternate example using a negative-tone resist such as SU-8, the regions that are to be segments are exposed to light instead of the regions that are to be gaps.
An alternate example of the general method of generating code using multiple print steps utilizes stamping (aka imprint lithography) as the printing mechanism, and is schematically depicted in
In addition to the microparticle as illustrated in
Referring to
Another example of a system for organizing the multi print method using a stepper is to exposure all of the code elements within a single die before moving on to the next die. Of course, a number of offsets other than four could be used. Though this and other examples of the general method of producing codes on microparticles has been described with respect to using a projection photolithography and a stepper, contact lithography and other patterning methods may also be used.
The invented method of producing codes, for example the use of a photolithographic step and repeat system to form a complete code through multiple exposure steps of a single reticle field, may be used to apply unique codes to many types of components, e.g. MEMS and IC devices.
Coding Scheme
The microparticles as discussed above have incorporated therein codes derived from any desired coding scheme, such as binary or non-binary coding.
By way of example,
Number of
Number of
In a exemplary example, the coding scheme utilizes code elements placed at locations spanned by interval lengths smaller than the code element size itself. This deviates from the standard binary coding where the code consists of the absence or presence of a feature at discrete, evenly spaced locations. In the preferred embodiment of this coding scheme, naturally applicable to the above structure manufactured using the multiple print technique, the code element is the gap in the segmented inner opaque material. The gap size is chosen to be one that is reliably defined by the stepper and photolithography process and also resolvable by the microscope (working at the desired magnification). The gap size, interval length, and particle length determine the codespace (number of codes possible). The determination of a codespace involves tradeoffs between particle density on the wafer, identification accuracy, optical detection system complexity, and particle number per microscope image. Codespaces of over a million can be produced and accurately identified using practical parameter combinations.
In the example of a standard binary coding scheme, the particle would be divided into units of equal length. Each unit could then be black or white, 0 or 1. Because the particle is symmetric, there are two codes that are the same when one is reversed (so called "degenerate" codes). When counting the codes, one from each of the pair of degenerate codes is preferably discarded. Without the degeneracy there would be 2N possible codes, where N is the number of bits (units). With the degeneracy, there are about half that number. Exactly, the number of possible codes with the standard binary format is [2N+2floor[(N+1)/2]]/2. In the example of the high contrast encoded microparticle structures of the present invention, previously shown in
The non binary coding scheme mentioned above has many advantages in the fabrication and detection of microparticles, including providing for high codespaces and robust code identification. In the example of the coding scheme, the reliability of the microparticle fabrication process is improved by permitting optimization of patterning and etch conditions for features, of a single size, e.g. gaps in the segments having a single width.
In the exemplary examples of encoded microparticles and methods of determining codes therein, e.g. as shown in
In general, a high codespace is desirable. In the field of genomics, having a codespace in the tens of thousands is especially important because it enables full genomes of complex organisms, such as the human genome, to be placed on a single particle set. The top portion of Table 1 shows the effect of varying the delta parameter, d, on the codespace. Shrinking d gives many more codes but places increased demand on the optical system. The need to resolve a smaller d means that a more expensive objective would typically be used. Practically, the lower limit of the gap interval distance is set by the resolution by the optical system (manifested as the pixel size of the digital image captured using a CCD camera). Using a 60× objective and 6.2 mm 1024×1024 CCD chip, an interval distance of d=0.4 um equals approximately 4 pixels. If the interval distance is reduced to 0.3 um (3 pixels), there are 105,154 codes. The codespace can be extended into the millions for longer particle lengths, L, and/or smaller gap widths, w.
The lower portion of Table 1 shows the effect of varying the length of the particle at fixed w and d. The length L is inversely proportional to the density of particles on the die (number of particles per unit area). The length also affects the number of particles in an image and thus throughput (particles detected per second). Tradeoffs exist between codespace, density, identification, and throughput. Optimization of the coding scheme parameters will determine the selected coding scheme for a particular application.
Large Particle Sets
In the upper right photograph of
The encoded microparticles, systems, and methods of the invention have a wide range of applications in the fields of biology, chemistry, and medicine, as well as in security and commercial fields involving the tagging of monetary bills, identification cards and passports, commercial products, and the like. In one example, the microparticles can be used in for molecular detection, such for as analyzing DNA, RNA, and proteins. In other examples, combinatorial chemistry or drug screening assays are performed as known in the art.
Referring to the flowchart shown in
The mating of the encoded particles and biomolecules produces a "pooled probe set" through step 414. The pooled probe set is a mixture of encoded particles where each code has a particular probe attached to the particle surface. The pooled probe set can then be used to determine the amount of individual targets present in a mixture of targets. The mixture of targets is referred to as the sample and is typically derived from a biological specimen. The sample is then labeled, typically with a fluorophore at step 416. When the sample is mixed with the pooled probe set, the probes and targets find each other in solution and bind together. With nucleic acids, this reaction, step 418, is called hybridization and is very selective. After the reaction, the particles are imaged to read the codes and quantify the fluorescence at step 420. Referring to the code-probe lookup table, the amounts of the different target species in the mixed sample can now be measured and as a the assay result determined at step 422.
The samples reacted with the microparticles may be a purified biological extract or a non-purified sample, including but not limited to whole blood, serum, cell lysates, swabs, or tissue extracts. The samples reacted with the microparticles may be produced by culturing, cloning, dissection, or microdissection. Cells may serve as either the sample or probe in a bioassay utilizing the microparticles and other aforementioned inventions.
It is noted that multiple different samples may be identified in a single bioassay as discussed above. Before the detection and after the hybridization, the microparticles can be placed into wells of a well plate or other container for detection. In one detection example, the microparticles settle by gravity onto the bottom surface of the well plate. The microparticles in the well can be subjected to centrifugation, sonication, or other physical or chemical processes (multiple washing steps, etc.) to assist in preparing the particles for detection. In another example, the microparticles can be placed onto a glass slide or other specially prepared substrate for detection. In yet other examples, the particles are present in a flow stream during detection, or present in a suspended solution.
Term conjugation is used to refer to the process by which substantially each microparticle has one or more probe molecules attached to it's surface. Methods of conjugation are well known in the art, for example in Bioconjugate Techniques, First Edition, Greg T. Hermanson, Academic Press, 1996: Part I (Review of the major chemical groups that can be used in modification or crosslinking reactions), Part II (A detailed overview of the major modification and conjugation chemicals in common use today), and Part III (Discussion on how to prepare unique conjugates and labeled molecules for use in applications).
The molecular probes attached to the surface of the particles typically have known attributes or properties. In an example, the molecular probes can be derived from biological specimens or samples and used in the screening, including but not limited to genetic sequencing, of large populations where typically, the derivatives from one member of the population is applied to a single code, typically a multiplicity of particles of a single code. Preferably, microparticles having the same code have attached substantially the same probe molecules; whereas microparticles having different codes likewise have different probe molecules.
One of the most powerful features of a multiplexed assay using solution arrays of encoded particles as the platform instead of planar microarrays is the flexibility to add functionality to the assay by simply adding new particles. With standard microarrays, once the arrays are printed or synthesized, the array typically cannot be changed. If the researcher wants to change the probes for genes on the array or add probes for new genes, typically entirely new arrays would then be produced. With pooled probe sets of particles, new probe and particle conjugates (probes for short) can easily be added to the existing pooled probe set. In practice the new probes could be different probes for an already represented gene, probes for alternative splicing variants of genes, or tiling probes for genes.
A Bioassay Process Using the Microparticle
The microparticles of the invention can be used as major functional members of biochemical (or chemical) analysis systems, including but not limited to solution based arrays, biochips, DNA microarrays, protein microarrays, lab-on-a-chip systems, lateral flow devices (immunochromatographic test strips). Applications include but are not limited to gDNA and protein sequencing, gene expression profiling, genotyping, polymorphism analysis, comparative genomic hybridization (CGH), chromatin immunoprecipitation (CHiP), methylation detection, as well as discovering disease mechanisms, studying gene function, investigating biological pathways, and a variety of other biochemical and biomolecular related applications such as inspection and analyses of proteins, peptides, polypeptide, and related biochemical applications. Assay architectures may include those well known in the art, including but not limited to direct DNA hybridization, hybridization of DNA to RNA or RNA to RNA, enzymatic assays such as polyemerase extension, ligation. The microparticles can also be used in microfluidic or lab-on-a-chip systems or any flow based systems, including but not limited to those systems wherein sample preparation, biochemical reaction, and bio-analyses are integrated.
For example, fluorescent tags can be employed when an optical imaging method based on the presence of fluorescence can be used. Radioactive labels can be used when the microparticles are utilized to expose or develop relevant photographic films. Alternatively, enzymatic tags can be used when the detection involves detection of the product of the enzyme tag that is released when the sample molecules bind to or react with the probe molecules on the microparticles. Other tagging methods are also possible, as set forth in "Quantitative monitoring of gene expression patterns with a complementary DNA microarray" by Schena et al. Science, 1995, 270-467, the subject matter of which is incorporated herein by reference in its entirety.
Samples without labels can also be reacted with the microparticles. For example, molecular beacon probes can be applied to the microparticle. Molecular beacon probes typically contains a hairpin structure that, upon binding the labelless, or in some examples labeled, sample molecules unfold, thus producing a signal indicative of the binding events. Such molecular beacon probes, as well as other probes, may be used in assays involving FRET (Fluorescence Resonant Energy Transfer), where for example fluorophores or quenchers are placed on or in the surface of the microparticles.
For facilitating fast, reliable, and efficient bioassay for large number of sample molecules, it is preferred that the microparticles are capable of arranging themselves substantially in a monolayer on a surface, such as the bottom surface of the well in which the microparticles are contained. The microparticles are preferred to be able to undergo Brownian motion in the specific liquid in which the optical detection is performed. Given the specific liquid in which the microparticles are hybridized and detected, it is preferred that the 2D diffusion coefficient of the microparticles is equal to or greater than 1×1012 cm2/s and/or 10% or more, such as 15% or more, or even 20% or more, and 50% or more of the microparticles are measured to undergo a lateral displacement of 20 nm or greater, such as 30 nm or greater, or even 50 nm or greater—in a time interval of 1 second or less, or preferably 3 seconds or less, or five seconds or less.
The detectable microparticles, which are referred to as those that are able to be accurately detected by the desired detection means, such as optical imaging using visible light, are capable of occupying 30% or more, 40% or more, and typically 50% or more of the surface area on which the microparticles are collected together, such as a portion of the bottom surface of the container in which the microparticles are contained. Defining an area in which at least 90% of all the microparticles are disposed (typically at least 95% or more typically at least 99%, and often 100%), the microparticles can be seen to have a density of 1000 particles/mm2 or more, such as 1500 particles/mm2 or more, 2000 particles/mm2 or more, and typically 3000 particles/mm2 or more (e.g. 5000 particles/mm2 or more). The detection rate within the above-mentioned area, which rate is defined as the ratio of the total number of detected microparticles (microparticles with spatial codes detected) of a collection of microparticles under detection to the total number of the collection of microparticles, is preferably 80% or more, typically 90% or more, or more typically 99% or more.
Another preferred example of the invention is a kit comprising biochemically active encoded microparticles that contains 200 or more, more preferably 500 or more, 1000 or more, or even 10,000 or more different codes within the kit (due to the large codespace enabled by the invention, even larger numbers of codes.) Due to statistical sample requirements of convenient liquid pipetting and a desired redundancy of particular codes within the kit, more than 10 particles of the same code are typically provided (20 or more, or even 30 or more microparticles of the same code) within the kit, as in some example applications the redundancy improves the overall assay performance. The term "biochemically active encoded microparticles" is refers to microparticles that have biological or chemical moieties on surfaces and thus can be used in assays; and the term "moieties" are referred to as molecular species; including but are not limited to nucleic acids, synthetic nucleic acids, oligonucleotides, single stranded nucleic acids, double stranded nucleic acids, proteins, polypeptides, antibodies, antigens, enzymes, receptors, ligands, and drug molecules, cells, and complex biologically derived samples.
Universal adapter schemes may be used to provide a set of non-interacting synthetic sequences that are complementary to sequences provided on the probes. Genotyping can be performed using common probes and allele specific reporters or allele specific probes and common reporters. Amplification assays such as those involving PCR, padlock probes, or Molecular Inversion Probes can be performed using the particles of the current invention. Examples of two of these assays are shown in
Protein based assays are also applicable. These include but are not limited to sandwich immunoassays, antibody-protein binding assays, receptor-ligand binding assays, or protein-protein interaction assays. Examples of these assays are shown in
A single type of protein can be applied to microparticles of a single code. Upon mixing of the particle-protein conjugates and reaction in a particular biochemical environment, proteins that interact and bind to one another are determined by the presence of adjacent particles during detection. The square cross section of the microparticle structures of the present invention provide an improvement over the prior art by providing an increased area of contact in the shape of a flat, rectangular surface. Prior art particles that are spherical or cylindrical in shape limit the contact areas to single points or lines respectively. This invention is not limited to proteins: any interacting molecules may be used with this assay architecture. Also, the omni-directional encoded microparticles of the present invention may be used in conjunction with any other encoded particles including but not limited to fluorophores, quantum dots, latex or glass beads, colloidal metal particles, spectroscopically active particles, SERS particles, or semiconductor nanorods.
The encoded microparticles may be used in conjunction with a 2D planar array of molecules. Interaction between molecules on the surface of the particles and those contained in spots on the 2D planar array are determined by the binding of the particles to the spots. The presence of the particles in the predetermined spot locations, preferably after washing steps, indicates a binding interaction between the molecules on the particles and the molecules on the 2D planar array. The assay result can be determined by identifying 1) the particle code, and 2) the spot location. This is shown in
The microparticles of the invention may have other applications. For example, by placing protein-detection molecules (e.g., ligands, dyes which change color, fluoresce, or cause electronic signal upon contact with specific protein molecules) onto the microparticles, bioassay analyses can be performed (i.e., evaluation of the protein and/or gene expression levels in a biological sample). As another example, by placing (cellular) receptors, nucleic acids/probes, oligonucleotides, adhesion molecules, messenger RNA (specific to which gene is "turned on" in a given disease state), cDNA (complementary to mRNA coded-for by each gene that is "turned on"), oligosaccharides & other relevant carbohydrate molecules, or cells (indicating which cellular pathway is "turned on", etc.) onto the microparticles, the microparticles can be used to screen for proteins or other chemical compounds that act against a disease (i.e., therapeutic target); as indicated by (the relevant component from biological sample) adhesion or hybridization to specific spot (location) on the microarray where a specific (target molecule) was earlier placed/attached. In fact, the microparticles of the invention can be applied to many other biochemical or biomolecular fields, such as those set forth in the appendix attached herewith, the subject matter of each is incorporated herein by reference.
It will be appreciated by those of skill in the art that a new and useful microparticle and a method of making the same have been described herein. The large sets of encoded microparticles produced by this invention can be a fundamental technology that will have far reaching applications, especially in the field of biotechnology and more specifically genomics. It has the potential to dramatically reduce the cost of highly multiplexed bioassays. Moreover, enables researchers to easily design custom content solution arrays. The researcher can also easily add new particle types to the pooled set, for instance including new found genes of interest with the microparticles of the invention.
In view of the many possible embodiments to which the principles of this invention may be applied, however, it should be recognized that the embodiments described herein with respect to the drawing figures are meant to be illustrative only and should not be taken as limiting the scope of invention. Those of skill in the art will recognize that the illustrated embodiments can be modified in arrangement and detail without departing from the spirit of the invention.
For example, the microparticle may have a six sided shape with four elongated sides and two end sides. The encoded microparticle can be configured such that the code of the encoded microparticle can be detectable regardless of which of the four elongated sides the barcode is disposed on. The microparticle may have a ratio of the length to width is from 2:1 to 50:1, from 4:1 to 20:1. The length of the microparticle is preferably from 5 to 100 um and more preferably less than 50 um. The width of the microparticle can be from 0.5 to 10 um. In other examples, the length of the microparticle can be less than 10 um, less than 25 um, less than 25 um; less than 5 um, less than 27 um; and the width of the microparticle can be less than 3 um. The ratio of width to height of the microparticle can be from 0.5 to 2.0. The ratio of the length to width of the microparticle can be from 2:1 to 50:1. The cross section taken along the length of the microparticle is substantially rectangular with a length at least twice the width.
The microparticle may have a glass body with segments embedded therein. The difference of the transmissivity of the glass body and segments can be 10% or more. The glass body may have a length of less than 50 um and a width of less than 10 um with the glass body having a volume of from 5 to 500 um3. The encoded microparticle may have 2 to 15, 3 to 10, or 4 to 8 portions of less transparent material within the encoded microparticle. The code incorporated in the microparticle can be binary or non-binary or any other desired codes. The microparticle may have biochemical molecules attached to one or more surfaces of the microparticle, such as DNA and RNA probes with a density of from 102 to 106/um2. When fabricated on the wafer-level, the wafer may have a surface area of from 12.5 in2 to 120 in2, and wherein there are at least 3 million microparticles per in2 of the wafer. The wafer may have at least one million codes are formed on the substrate, or at least two hundred different codes are present within the one million codes, or at least 3000 different codes are present within the one million codes. When placed in a liquid buffer, for example in a bioassay, the microparticles can form a single monolayer with a 2 dimensional diffusion coefficient of the microparticles greater than 1×10−12 cm2/s and more preferably greater than 1×11−11 cm2/s.
Therefore, the invention as described herein contemplates all such embodiments as may come within the scope of the following claims and equivalents thereof.
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|U.S. Classification
|430/270.1, 430/330, 430/311, 430/5, 430/394, 430/396
|International Classification
|Cooperative Classification
|European Classification
|May 8, 2007
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