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Mariner Selected to Provide Advanced Maritime Situation Management System at Port Everglades, Florida .
The Mariner Group, LLC, (Mariner) announced today that it has been selected to provide its industry-leading situation management and security system, CommandBridge™, to Port Everglades, Florida. Located in the heart of Greater Fort Lauderdale and the City of Hollywood, FL, Port Everglades is one of the busiest cruise ports in the world. As a leading container port in Florida and among the most active cargo ports in the United States, Port Everglades is South Florida's primary seaport for receiving petroleum products including gasoline and jet fuel.
Mariner provides integrated physical and logical security systems and services for mission-critical clients world-wide, including an industry-leading advanced security integration platform, CommandBridge.
"Mariner is very proud to have been chosen by Port Everglades for this important project. Our CommandBridge security integration platform provides overall security situation awareness to significantly enhance incident management. CommandBridge is designed to adapt to virtually any environment requiring advanced situation awareness, automated anomaly detection, real-time event collaboration, and response & recover work-flow processing all in an easy to use application," said Steve Dryden, President & CEO Mariner.
"Mariner's CommandBridge platform uses advanced software technology to cut through security-information clutter, surface vital data, place it in a relevant context, give users various options for action, and allow them to easily share the information inside their organization and with outside agencies" said Erin Phelps, Sr. Vice President Mariner. "With more than ten years of proven field experience, CommandBridge has evolved to meet maritime & coastal domain awareness needs throughout the U.S. and we are very pleased to include Port Everglades as a client"
About The Mariner Group:
Founded in 2000, Mariner provides security system integration and advanced situation management software, CommandBridge, delivering results which are essential to the needs of clients across multiple industries. Client-focused and results-driven, Mariner partners with clients to deliver technology solutions and services that deliver tangible results and business value. For more information, visit www.themarinergroup.net.
Maritime Today
The Maritime Industry's original and most viewed E-News Service
LNG? Methanol as fuel? Hybrid systems? Tier 4? Reducing noise? Cutting emissions without crushing fuel economy? Marine News readers have questions and Bob Kunkel has answers.
Longtime waterjet propulsion equipment provider NAMJet introduces key advancements for the workboat sector.
Back in 2011, when boat builder Birdon went looking
The emerging trend of marrying the design of an articulated tug and barge (ATB) to a particular propulsion system to maximize efficiencies of that system has, of late,
Further progress towards identifying and designating Particularly Sensitive Sea Areas (PSSAs) in south-east Asia has been made during a regional meeting in Lombok, Indonesia (27-28 July).
Libyan oil exports from closed ports should resume in no more than one to two weeks after a deal was signed between the government and an armed brigade controlling the terminals,
Standing NATO Mine Countermeasures Group 2 (SNMCMG2) has completed its third deployment into the Black Sea under the command of Captain Ramazan Kesgin, Turkish Navy.
In June 2016 Horizon Shipbuilding, Inc. of Bayou La Batre, Ala. delivered the M/V Marty Cullinan, a 120' towboat with a retractable pilot house, to its home port of New Orleans, La.
Integrated ship control systems supplier Servowatch said it will use SMM to launch its new automation and control software, WINMON 9.
WINMON 9 is a singular,
The maritime software development company SDSD are launching a new web based VSnP - Vessel Sale and Purchase module, enabling Sale and Purchase brokers to buy and


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Binge drinking problem for young bisexuals
Relevant offers
Many young people attracted to more than one gender tend to binge drink because they feel stigmatised and socially excluded, a study by the University of Otago in Wellington has found.
Passing legislation for marriage equality - also known as same sex marriage - is suggested as a way to help alleviate the problem.
Binge drinking was higher among young people attracted to more than one gender than for other sexual minorities or for heterosexual young people, lead author, Frank Pega, from the university's public health department and the Harvard School of Public Health, said.
He noted it was still a minority of those attracted to more than one gender who were binge drinking.
The study involved in-depth interviews this year with 32 people aged 18-25 in 11 focus groups in Auckland, Wellington, and Dunedin.
A significant factor leading to binge drinking was found to be wide-ranging social exclusion experienced by young people attracted to more than one gender, from heterosexual, lesbian and gay communities.
"Most study participants reported that they commonly experienced biphobia (an aversion toward bisexuality and bisexual people) and discrimination, and some had been verbally harassed and physically abused for their sexual attraction.
''For many, these experiences resulted in a sense of being stigmatised, which caused daily stress and anxiety," Pega said.
"While many participants were very resilient and responded positively, some participants binge drank to manage this stress."
Sexual minority communities, health practitioners, and policy makers had long wanted to tackle the issue, but too little information had been available.
The report suggested more attention needed to be paid to reducing social stigma towards young people attracted to more than one gender.
Proposed measures included increasing opportunities for more-than-one-gender attracted young people to meet, socialise and organise, along with broad anti-stigma campaigns.
The study also called for social policies that ensured equal rights for sexual minorities.
"One example is the marriage equality legislation, currently before parliament," Pega said.
"Going from US evidence, we can expect marriage equality and similar legislation to improve the health of sexual minority populations in general, including to reduce binge drinking."
The report said the demographic of people sexually attracted to more than one gender included people attracted to both men and women.
It also included those attracted to people regardless of whether they were cisgender, transgender or gender queer.
Cisgender meant people whose gender identity and biological sex were congruent, while transgender referred to those whose gender identity differed from their biological sex. Gender queer encompassed all alternative gender identities, other than cisgender.
The 32 participants in the study included 24 who identified their gender as female, seven who identified as male, and two who identified as female-to-male transgender - one of whom also identified as female.
The study said it had been estimated that 2.9 to 3.8 per cent of young people at high school in this country were more-than-one-gender attracted.
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The WIlliam J. Perry PRoject Was created by the
former secretary of defense to work towards a world in which
nuclear weapons are never used again
William J. Perry's memoir about coming of age in the nuclear era
and his role in trying to shape and contain its urgent threat.
From the Blog
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America at the Atomic Crossroads
July 25, 2016
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July 20, 2016
Foreign Policy in Focus
In an age of 'smart' weapons, we can live without nukes
July 19, 2016
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July 17, 2016
The New Yorker
Former SecDef: Long-Range Standoff Missile a Step to New Cold War
July 14, 2016
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RT @Kalianeke: Ich habe ein @YouTube-Video von @secdef19 positiv bewertet: https://t.co/fa1U6n1cRX Nuclear Nightmare DC
RT @wellerstein: 70 years ago today: The Crossroads Baker test. I have written an article for @NewYorker's @elements blog: https://t.co/LrHEhssuR0
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A beautiful pendant with a good weight and a surprisingly good quality snake chain. I actually feel like I'm wearing it. Slightly larger than expected, so it's not a subtle as I thought it would be. However the stone is a wonderful size if you aren't going for subtle which moonstone is hard to be anyway. Immediately after I put it on I received compliments that it was a beautiful stone that was "always like a rainbow."
I rated this a 3 because of the steel chain. It seems to get kinks in it Otherwise I love the stone!
Beautiful! I bought the earrings to match.
Beautiful comfortable, very soft
Beautiful Moonstone Teardrop Necklace with Stainless Steel chain. Goes beautifully with the Moonstone Teardro earrings & for a great price!!
The setting is simple and doesn't detract from the stone. What I like about moonstone in the first place is the warmth and depth of the stone. The faceting enhances both of those traits in the stone, and I am very pleased with the purchase. The picture does not do the stone credit - it doesn't show the depth of the blue/green 'inner glow' of the stone.
I can't say enough good things in here, but here are a few. The order was speedily raced to my door, and in such a cute little drawstring bag that I can use foe when I don't have the pendant on. The pendant itself is so beautiful and catches the light no matter what angle. I adore my new Opaline. Thank you.
It feels like plastic to the touch. I have a hard time believing this is a natural gemstone. It still is pretty though. I am not sure if I should keep it or return it.
Love wearing this...very exotic looking and elegant at the same time. Truly stands out when I wear a dark colored top which makes this stand out.
Good quality at low price and quick delivery. Easy to shop at home
Terrible quality. This just looked like cloudy glass with none of the sparkle that real moonstone has. Its also huge, which I knew from the description, but you dont quite realize how big til you put it on. The chain is very short for such a large piece. Returning it for a different option.
I bought this for my girlfriend. It's not designer but it is beautiful. She wears it work & when we go out. Couldn't ask for anything more at this $. Very happy!
This was a gift for my daughter, and she wears it for special occasions.
Very pretty. Only problem with this necklace is the last piece of wire wrapped around the necklace is not secure. I decided to keep the necklace anyway because it is pretty. At times when I wear the necklace the wire scratches me.


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By Dan Moisand, CFP |
June 7, 2011
Reflections on the FPA in China trip include Dan Moisand's musings on what he learned about China and its financial planners, and newfound appreciation for his own country.
By Marie Swift |
October 1, 2008
In my last article, I relayed the startling statistic that by the end of this year, the number of non-U.S. CFP certificants will almost surely...
By Marie Swift |
September 1, 2008
Here's a startling statistic: By the end of this year, the number of non-U.S. CFP certificants will likely surpass the number of U.S. certificants. It's...
By Staff Writer |
August 1, 2008
Laura Brook, director of international relations for the Financial Planning Association, points out that advisors should be sure to determine their clients' citizenship status. "Do...
By David J. Drucker |
March 1, 2007
American culture and behaviors from movies to fast food, get exported every day -- and wealth management is no exception. Of course, private banking, the precursor...
Learn how to build a better business and create loyal clients and advocates from the start.
Don't allow high-value prospects to leave you for more connected, more digitally-capable, advisors. Download this white paper and learn six distinctive strategies you can...
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NSW boy hit by car while riding scooter
A boy has been taken to hospital with a leg injury after being trapped under a 4WD while he was riding a scooter in Sydney's west.
A boy has been taken to hospital with a leg injury after being trapped under a 4WD while he was riding a scooter in Sydney's west.
Police believe a man shot outside his mother's house near Fairfield overnight is the victim of a gangland attack. Peter Fegan reports.
The NSW state government has gone to the Supreme Court in a bid to keep Ahmed Elomar, one of the notorious Hyde Park rioters, behind bars. Samantha Brett reports.
The NSW Labor Party says National MPs should vote against the government's statewide greyhound racing ban.
A summary of three letters former prime minister Kevin Rudd sent to Malcolm Turnbull in his quest for nomination for the role of UN secretary-general.
It may be all downhill from here for a teenage skateboarder who posted a video of himself speeding at 80 kilometres an hour down a steep road on the central coast.
Kevin Rudd says he had assurances of support from Prime Minister Malcolm Turnbull in his nomination bid for the role of UN secretary general.
A gunman remains on the run after a man was shot in the back during a targeted shooting in Sydney's west.
Executive officers should leave tactical decisions to those on the frontline during sieges, the Lindt Cafe inquest has heard.
It took commanders at least two minutes to find out Man Monis could be heard barking orders at hostage Tori Johnson in the final minutes of the Sydney siege.
The Australian dollar fell on Friday as the yen gained in the wake of BoJ stimulus announcements.
A tiny yellow-bellied glider joey has found some motherly love at Taronga Zoo after surviving a crash into a barbed-wire fence.
Minister for Transport and Infrastructure Andrew Constance has asked people to help name the city's six new ferries, the first of which will hit the water this summer.
The decision to grant parole to an allegedly radicalised Sydney rioter is being challenged in the NSW Supreme Court.
A man is set to face a NSW court after he allegedly groomed a teenage boy for sexual activity.
Sydney man Zane Alchin, 25, will have a conviction recorded after he posted dozens of abusive comments about women online.
Family of a 17-year-old Sydney youth who has an intellectual disability and Asperger's syndrome wept as he was refused bail on a terrorism-related charge.
A selection of posts allegedly made by a teen accused of doing an act in preparation for a terrorist act.
This week economists focused on weak CPI figures but were divided on what they meant for interest rates. They also saw some positive signs for the economy.
- °


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I have no doubt that within the Halls of Power in Washington, the gig was already out on the IRS long before, since it was doing the bidding of the Obama Administration anyway. Us Bible and Gun totters deserve another look if we are organizing, right? In the words of Rahm Emanuel, "Never let a good Crisis go to waste.
When the temperature was reaching boiling point, The IRS Temporary Commissioner fell on his sword,like a good solider, for the good of those in power.
The bright lights of exposure have now been directed to the IRS scandal as the script reads. Does anyone actually believe that it just appeared out of the blue ?
Not me and I doubt any of you who see throught this nonsense as well. The IRS is just another example of the "Chicago Way". They made Americans' life a nightmare because they were organizing against the Regime.
The saying "Remember the Alamo" has been replaced with "Remember Benghazi".
Well, I am sure I am probably on some list somewhere because of my opinions too. Well, why not. I don't make enough money to file taxes, ha.
Mitch Thurmer
July 29, 2016
I just read the article on Mark Siedleki's allegedly illegal corporate donations in his campaign. Well, it is clear that Democrats think laws and rules apply to everyone but them. ... (click for more)
Once upon a time, there was a kingdom. This kingdom was a prosperous kingdom and it was filled with peoples from many lands. The people were happy in this kingdom and took great pride in ... (click for more)
These are wonderful times for the families whose children attend the Hamilton County schools in District 2. One can sense renewed energy and commitment by all stakeholders who are connected to ... (click for more)
I just read the article on Mark Siedleki's allegedly illegal corporate donations in his campaign. Well, it is clear that Democrats think laws and rules apply to everyone but them. Hillary and Obama will be proud of you, Mark. Derek Kukura * * * No wonder the Democrat running for assessor of property doesn't understand the law preventing any assessor from freezing ... (click for more)
Once upon a time, there was a kingdom. This kingdom was a prosperous kingdom and it was filled with peoples from many lands. The people were happy in this kingdom and took great pride in being a part of it. One day, a Swen Merchant came to the kingdom. He looked in amazement at the prosperity of the kingdom and at all the peoples that lived and work in it. The ... (click for more)
A 28-year-old man was shot and killed in East Chattanooga early Friday morning. The victim was identified as Jeremy Clark, a brother of House candidate Dennis Clark. Dennis Clark said, "Jeremy, a beloved son, my younger brother and dear friend to many, was taken from us too soon. The pain from our sudden and tragic loss is Indescribable. He was a compassionate and fun-loving ... (click for more)
Democratic assessor of property candidate Mark Siedlecki filed an amended financial disclosure form on Friday afternoon after county Republicans said he accepted far above the legal limit from a corporation he heads. The new disclosure said he made personal loans totaling $38,288 to the campaign. It does not include any in-kind contributions. The earlier forms listed ... (click for more)
With the score tied at three with one out in the bottom of the ninth inning, Lookouts outfielder Zack Granite singled home Edgar Corcino to give the team with 4-3 walk-off win over the Smokies. Tied going into the bottom of the ninth inning, Corcino led off with his second hit of the game. He advanced to second base on a bunt. A Levi Michael single pushed Corcino to third ... (click for more)
All eyes are on Saturday as Chattanooga FC prepares for the NPSL National Championship Semifinal match against Sonoma County Sol at Finley Stadium. In efforts to FILL "FORT" Finley, CFC has extended their discount on $7 tickets until Thursday at 2:00. After 2:00, tickets will be $10 until 6:00 on Saturday, when the price will rise to $12. Tickets are available online at ChattanoogaFC.com. ... (click for more)


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Takato has been missing for 20 years,and so have 4 others in the digidestined realm. A Tamers&Digidestined crossover that is like none other. Where did Takato go? And who is behind it? What are the...0Unrated
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Jay-Z said Auto-Tune is dead. But based on the sheer amount of music utilizing the famous vocal-editing software, it's already an undead interloper set to haunt music for years to come. A good example is Discovery, the collaboration between Vampire Weekend keyboardist/producer Rostam Batmanglij and Ra Ra Riot singer Wes Miles that's filled with processed vocals and synthetic blue-eyed soul. If Chromeo is sweaty and sweet and Junior Boys is icy yet romantic, Discovery is sappy and as bright as its Pantonelike album cover.
Filled with innocent, lovelorn lyrics and the last 30 years' worth of guilty-pleasure keyboard cliches, the music can be sunny and indulgent, like with the swooning vocals during the half-time break in "So Insane" (some of the best and most unprocessed of the bunch) or Angel Deradoorian's chopped up one-woman chorus on "I Wanna Be Your Boyfriend." But many parts—like the ineffective, bedroom-begging lyrics of "Can You Discover?" and the wobbly, gimmick-filled Jackson 5 cover "I Want You Back"—have too much of a childlike rhythm and feel. It's a fitting undercurrent for a group named Discovery, but it doesn't always translate into great pop. [3 out of 5 stars]
The Woodstock Invitational Luthiers Showcase Announced for October 2016
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Hatebreed Premiere New Music Video, "Something's Off"
82-Year-Old Performs Rob Zombie's "Dragula" on 'America's Got Talent'
Applying Modes to Improvised Guitar Solo Ideas, Part 1
How to Use the Dark Sound of the Phrygian Mode's "Flatted†Intervals
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The Next 20 Star Wars Spin-Offs
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Sep 25
Fitty Smallz faces off with other local talent in the Friday night music challenge hosted by Jasmine Rhey and Madiline Skinner.
Jul 2
Beautiful Bodies Competition during the Art Walk in downtown Jacksonville, Florida. Hosted by @JasmineRhey. Live performance on the main stage during intermission. It's gonna be HOT!
Sounds Like: LMFAO
Bio: Fitty Smallz, AKA Notorious F.I.T. is a fitness expert, entrepreneur and author whose experience has spanned over three decades. As a lyricist, producer, rap star and performer, Fitty has pushed the envelope of creative thinking to deliver his message through music and music videos. Breaking into...See Full Bio
"When we first heard about Fitty Smallz, America's first fitness rapper, at first we were like "what?" And then we were like "oh yes." His deal is that he's quite literally a rapper who's all about staying fit. We chatted with him to find out more, and landed the honor of premiering his brand new video for the vampy "Thunda Thighs," which you can see after the Q&A."


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How Much Will Switching Motherboards Affect Performance?
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Call our experts: 888.808.FINE
Fall Wedding Favorites $10 off order $75+ | Code: SUNNY10 Back to School Stationery
Select a quantity and printing method. Changes are easily made later.
Send a letter to friends and family via this ecru lettersheet, featuring an embossed border and ample space for your personalization. Shown with optional monogram, M-797.PLEASE NOTE: Thermography printing is not recommended for use in laser printers.


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Lyrics Depot is your source of lyrics to Take Me Back To Tulsa by George Jones. Please check back for more George Jones lyrics.
Take Me Back To Tulsa Lyrics
Artist: George Jones
Album: Sings Bob Wills
Where's that gal with the red dress on some folks called her Dinah
Stole my heart away from me down in Louisiana
Take me back to Tulsa I'm too young to marry
Take me back to Tulsa I'm too young to marry
[ steel ]
Little bee sucks the blossom big bee gives the honey
Dark man raise the cotton white man gets the money
Take me back to Tulsa...
[ fiddle ]
Walk and talk to Suzy walk and talk to Suzy
Walk and talk to Suzy walk and talk to Suzy
We always wear a great big smile we never do look sour
Travel all over the country playing by the hour
Take me back to Tulsa...
George Jones Lyrics
George Jones Sings Bob Wills Lyrics
More George Jones Music Lyrics:
George Jones - Baby You've Got What It Takes Lyrics
George Jones - I Could Never Be Ashamed Of You Lyrics
George Jones - Life Turned Her That Way Lyrics
George Jones - Never Bit A Bullet Like This Lyrics
George Jones - She's Mine Lyrics
George Jones - Shine On (shine All Your Sweet Love On Me) Lyrics
George Jones - Talk To Me Lonesome Heart Lyrics
George Jones - Yesterday's Wine Lyrics


text:
The Way of Wisdom - eBook
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Publisher's Description
Wisdom is supreme; therefore get wisdom. Though it cost all you have, get understanding. Proverbs 4:7 (NIV) When a mans life embodies the pursuit of wisdom, it bears among its fruits the deep love and respect of his family, friends, and colleagues. Bruce K. Waltke is such a man. Wisdom has defined Dr. Waltke, both as one of his personal qualities and as the core of his many years of biblical study, invoking the highest efforts of his formidable intellect and etching itself indelibly on his character. In tribute to Dr. Waltke, we present this collection of writings exploring the wisdom perspective of the Bible. The Way of Wisdom displays a level of scholarship and insight in keeping with Bruce Waltkes high academic standards, and a breadth of outlook reflective of his own broad grasp of Gods Word and its application to all of life. May you, the reader, benefit.


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Fender Electric Guitar Christmas Ornament
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|Publication number
|Publication type
|Application number
|Publication date
|Feb 6, 2007
|Filing date
|Jan 24, 2003
|Priority date
|Jan 24, 2003
|Fee status
|Also published as
|Publication number
|Jonathan S. Dordick, Moo-Yeal Lee, Aravind Srinivasan, Bosung Ku
|Original Assignee
|Rensselaer Polytechnic Institute
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (12), Non-Patent Citations (7), Referenced by (6), Classifications (7), Legal Events (6)
|External Links: USPTO, USPTO Assignment, Espacenet
The invention was supported, in whole or in part, by grants from the Defense Advanced Research and Projects Agency, the Biotechnology Research and Development Corporation and the National Science Foundation under grant number BES-0118820. The Government has certain rights in the invention.
Microfluidics is the field of microscale fluid flow and control, typically through microscale fluid control features constructed on a substrate such as a glass chip. These devices can be used to manipulate liquid chemical and biological samples in order to conduct analysis, perform synthetic reactions, and the like.
A motive force often used in microfluidics is a phenomenon known as electroosmotic flow (EOF). EOF depends strongly on various aspects of charge mobility in these systems, and in particular, depends on having a high density of ionic groups of the same polarity on the walls or flow surfaces of a microfluidics component. For example, a typical microfluidics feature is a microchannel etched in a glass substrate. The interior surface of the channel is treated to expose free silanol groups, which can then be deprotonated to provide siloxide anions. A voltage is applied across the flow direction of the channel causing solvated counterions of the charged groups on the channel walls to move. Because the dimensions of a microfluidic channel are small, the layer of counterions at the flow surface contact a significant portion of the fluid in the channel. Thus, when the counterion layer moves, the entire volume of the channel moves nearly simultaneously, a mechanism known as "plug flow". For many applications, plug flow is advantageous because it means that the components of a particular volume portion of the flow travel together, and are not spread out as they would be in a conventional pressure driven flow. Thus, on a microfluidics chip, precise volumes can be delivered from one location to another with a high degree of control.
Many features of a macroscale laboratory can thus be miniaturized using microfluidics, allowing significant reductions in the amount of costly, rare, or hazardous materials that are used. For example, microfluidics has the potential to make efficient use of biomolecules such as enzymes or catalytic antibodies, which are typically expensive or difficult to prepare in large quantities. It is particularly desirable that these molecules be reusable or recoverable, for example, by immobilizing them to a solid support, in order to further limit the quantities that are required.
A significant problem that must be solved, however, is the difficulty of immobilizing biomolecules with high biological activity while simultaneously maintaining acceptable EOF capability.
For example, enzymes have been attached to glass chips by covalent attachment to free silanol groups on the glass surface. However, the high pH necessary to provide the charged siloxide anions significantly decreases the stability and catalytic activity of enzymes. Another attempt functionalized the silanol groups with a linker group ending in an amine, which can then be covalently attached to the enzyme. However, this leads to a reduction in the number of available siloxide groups at the surface, and further, shields the groups that are present from the flow, resulting in poor EOF characteristics.
Other attempts have been made to provide polymers that specifically enhance EOF, for example, by using a charged polymer such as dextran sulfate. These polymers, however, are dynamically unstable coatings, i.e., are not substantially adhered, and so are eventually washed away by the flow. Furthermore, they are not easily functionalized with biocatalysts such as enzymes, and they do not maintain the catalytic activity of enzymes.
High enzyme activity can be maintained by encapsulating and/or covalently attaching enzymes to matrices, such as solgels, but such matrices typically fill the entire microfluidic channel, providing a severe impediment to fluid flow. Furthermore, many other examples of polymers exist to immobilize enzymes with high activity, but they are not designed to support EOF.
Therefore, there is a need to immobilize biomolecules on a microfluidics apparatus, while simultaneously maintaining high biological activity and high EOF capability.
It has now been found that certain polymers containing both ionic groups and hydrophobic groups can be substantially adhered to microfluidic channels and can be used to simultaneously immobilize biocatalysts with good catalytic activity while supporting electroosmotic flow.
One embodiment of the invention is method of immobilizing a biomolecule in the presence of electroosmotic flow. One step is providing a microfluidic biochip. The biochip includes a microfluidic component comprising a flow surface; at least two electrodes whereby an electroosmotic flow can be generated at the flow surface; and an immobilizing polymer that is substantially adhered to the flow surface. The polymer includes a first substituent selected from ionic groups of the same polarity and covalent precursors of the ionic groups, wherein the first substituent is optionally a biomolecule immobilizing group. The polymer includes a second substituent that is a hydrophobic group. The polymer optionally includes a third substituent that is a biomolecule-immobilizing group. Between the first substituent and the optional third substituent, the polymer includes at least one substituent that is a biomolecule-immobilizing group. Another step of the method is applying a motive force selected from pressure, electroosmotic force, capillary action, and centrifugal force, thereby generating flow. Yet another step is directing a biomolecule from a source to the polymer by employing the flow. An additional step is reacting the biomolecule with the biomolecule-immobilizing group under suitable reaction conditions, whereby the biomolecule is immobilized.
Another embodiment of the invention is the biochip, wherein the substituents of the immobilizing polymer include a first substituent selected from ionic groups of the same polarity and covalent precursors of the ionic groups; a second substituent that is a hydrophobic group; and a third substituent comprising an immobilized biomolecule.
Another embodiment of the invention is a method for conducting one or more reactions by using the biochip. The biochip additionally includes at least one reservoir, wherein the reservoir contains a starting reactant. All the microfluidic components are in microfluidic communication. The substituents of the immobilizing polymer include ionic groups of the same polarity; a hydrophobic group; a first immobilized biomolecule; and optionally a second, chemically distinct immobilized biomolecule; A step of the method is applying a voltage to the electrodes, thereby generating electroosmotic flow. Another step is directing the reactant from the reservoir to the first biomolecule by employing the electroosmotic flow, then reacting the first reactant with the first biomolecule under suitable reaction conditions, thereby producing a first reaction product. Another step is optionally contacting the first product with the second biomolecule, if present, and reacting the first product with the second biomolecule under suitable reaction conditions, thereby producing a second reaction product.
The invention can be used to immobilize biomolecules on a microfluidics biochip to conduct reactions. The invention retains the activity of the biomolecules while simultaneously supporting EOF. Also, the invention allows multiple reactions using catalytically distinct biomolecules. Furthermore, the invention allows sequential reactions using catalytically and spatially distinct immobilized biomolecules. Thus, using the invention, a wide array of sequential, stepwise reactions can be conducted with high activity while minimizing the use of costly or dangerous reactants.
The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention.
The invention is generally directed to methods of immobilizing biomolecules on a microscale device using an immobilizing polymer. In particular, the invention is directed to immobilizing biomolecules in microfluidic channels, using an immobilizing polymer that simultaneously maintains good biological activity of the biomolecule and good EOF characteristics.
A microfluidic device is a system of microscale fluid control components, such as channels, reservoirs, junctions such as T-junctions, and the like. Typically, these components are incorporated into a single solid substrate, for example, by chemically etching channels and reservoirs into the surface of a chip, e.g., a glass microscope slide, a polymer slab, a silicon wafer, and the like. These features can be made in such solid substrates by other techniques known to the art, for example, precision mechanical machining, laser machining, polymer molding from a machined or etched master, and the like.
The cross-sectional area of a microfluidic channel is less than 0.1 mm2, generally less than 0.05 mm2, more preferably less than 0.025 mm2, and most preferably about 0.003 mm2. The length of a microfluidic channel can be at a much larger scale, for example, a single channel could run up to the length of the substrate, e.g. almost 75 mm on a standard microscope slide, or a channel could loop back and forth on a chip, becoming many times longer than the chip itself. One or more reservoirs are typically connected to microchannel, e.g., to supply buffer solution, reactant solution, to collect products or waste, and the like. Fluid flow in a microfluidic chip can be generated by applying a motive force, for example, pressure, electroosmotic force, capillary action, centrifugal force, and the like. Preferably, the force used is electroosmotic. The biochip typically includes, or is used in combination with, a plurality of electrodes, i.e., at least two, which can be used to apply a voltage for causing EOF.
The ease of generating EOF in a microchannel depends on the number of ionic groups attached to the interior flow surface of the microchannel. When a voltage is applied to electrodes at opposite ends of a channel, negatively charged ions tend to flow to the anode and positively charged ions tend to flow to the cathode. In a channel designed for EOF, the ionic groups attached to the flow surface cannot move. However, the dissolved counterions of the ionic groups can move, and cause the surrounding solvent to move, resulting in EOF.
The rate of fluid flow is (VEOF) is affected by several factors (Eq. 1), where E is the electric field (equal to the voltage divided by the distance between the electrodes for microchannels of uniform resistance per unit length), ε is the relative permittivity of liquid, ε0 is the permittivity of free space, ζ is the zeta potential of the microchannel/liquid interface and η is the liquid viscosity (Fletcher, PDI; Haswell, S J; Paunov, V N. Analyst, 1999, 124, 1273–1282).
The sign of Eq 1 indicates the charge of the ionic groups attached to the channel flow surface, i.e. when the channel wall is negatively charged, the ζ-potential is negative, the diffuse charge in the liquid is positive (i.e., due to the positive counterions of the channel wall negative ions) and the liquid flows towards the cathode.
The microfluidic biochip can be made of any material, for example, glass, ceramics, polymers, silicon wafers, metals, and the like, in which microfluidic features can be fabricated. The microfluidic biochip can be a solid slab of a relatively hydrophobic polymer (distinct from the immobilizing polymer) that is readily coated by hydrophobic groups in the immobilizing polymer. For example the biochip can be made of polydialkyl siloxanes such as polydimethyl siloxane (PDMS), polyalkylenes such as polyethylene and polypropylene, polystyrenes, polyvinyl alcohol alkyl ethers, polyacrylate alkyl esters, and the like.
Alternatively, the microfluidic biochip is made of a material that has groups on its surface that are reactive, or can be made reactive, whereby the immobilizing polymer can be covalently attached to the flow surface. For example, a biochip made out of glass possesses —Si—O— groups, which can be treated to expose reactive siloxide anion or silanol surface groups that can react with the immobilizing polymer or with a linker that reacts with the immobilizing polymer. For example, siloxide anion groups on a glass surface can be reacted with a linker, e.g., aminopropyl triethoxysilane (APTES), thus covering the surface with amino groups:
which can be reacted with the immobilizing polymer. For example, in
The "immobilizing polymer" is a multifunctional, substituted polymer that is coated on, or exposed at, a flow surface of the biochip. Through its substituents, the immobilizing polymer can simultaneously provide four functions: biomolecule immobilization, maintenance of biomolecule activity, support of EOF characteristics, and substantial adherence of the polymer to the flow surface of the biochip. The immobilizing polymer contains at least two substituents that can perform these four functions. Preferred polymers possess simple polymer structural chemistry, such as a copolymer with the minimum set of substituents necessary to achieve multiple functions, e.g., a minimal set can be an ionic group (or precursor thereof) and a hydrophobic group. For example, an immobilizing polymer can be substituted with carboxylate and a C24 alkyl chain. The carboxylate can immobilize an enzyme by forming an amide bond and can support EOF through its negative charge, while the alkyl chain can help to maintain the enzyme's activity and can help to adhere the polymer to a hydrophobic microchannel wall through hydrophobic interactions. Alternatively, adherence to the microchannel wall can be provided through the carboxylate, either through ionic association with ionic groups on the microchannel wall or through creation of a covalent bond to the wall. An immobilizing polymer can also contain more than two substituents to perform the four functions, for example, a polymer could have a sulfonate for EOF, a carboxylate or thiol for enzyme immobilization, and a hydrophobic group for enzyme stability and adherence to the surface.
Substituents that support EOF are ionic substituents (or precursors thereof) that are the same polarity. For example, ionic groups can include negatively charged groups such as carboxylate, carbamate, sulfate, thiosulfate, sulfonate, phosphate, phosphonate, and hydroxyl. Alternatively, ionic groups include carboxylate, carbamate, sulfate, and hydroxyl. Preferably, negatively charged ionic groups are carboxylates.
In an alternative embodiment, ionic groups that are positively charged are used. For example, positive ionic groups can be ammonium, phosphonium, and sulfonium groups, optionally substituted with one or more groups selected from C1–C8 alkyl and aryl. Alternatively, positive ionic groups are selected from ammonium groups optionally substituted with C1–C4 alkyl groups. Preferably, positively charged ionic groups are ammonium groups.
Precursors of ionic groups are uncharged groups that can be reacted under appropriate reaction conditions to produce an ionic group, for example, an ester can be hydrolyzed to produce a carboxylate anion. Precursors of ionic groups can include optionally substituted alkyl esters, acids, amides, alkyl anhydrides, cyclic anhydrides, and aryl esters of ionic groups. Alternatively, precursors of ionic groups include optionally substituted alkyl esters, alkyl anhydrides, amides, and cyclic anhydrides. More preferably, precursors are alkyl esters, alkyl anhydrides, or cyclic anhydrides. Most preferably, a precursor is derived from a maleic anhydride group.
Substituents that are biomolecule-immobilizing groups are those that react to form a covalent bond with the biomolecule while maintaining some biological activity. Biomolecule-immobilizing groups can be ionic groups or precursors thereof, such as carboxyl or an anhydride, or can be other groups separate from the ionic groups used to support EOF. Biomolecule immobilizing groups include aldehydes, amines, alcohols, acid halides, alkyl halides, photoprecursors to radicals such as diazo and substituted diazenes, carboxyls, esters, alkyl anhydrides, cyclic anhydrides, thiols, disulfides, and the like. Alternatively, a biomolecule immobilizing group is selected from aldehyde, amine, carboxyls, esters, alkyl anhydrides, cyclic anhydrides, thiols, and disulfides. More preferably, a biomolecule immobilizing group is selected from amines, carboxyls, esters, alkyl anhydrides, cyclic anhydrides, and thiols. Most preferably, a biomolecule immobilizing group is a carboxyl or a cyclic anhydride, for example, maleic anhydride that is part of the polymer backbone.
Substituents that maintain the biological activity of an immobilized biomolecule include hydrophobic groups. A hydrophobic group, as the term is used herein, means a group which, as a separate entity, is more soluble in octanol than water. For example, the octyl group (C8H17) is "hydrophobic" because its parent alkane, octane, has greater solubility in octanol than in water. Specific examples of suitable hydrophobic groups include n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, n-tetradecyl, n-octadecyl, 2-ethylhexyl, 3-propyl-6-methyl decyl, phenyl, (Para n-dodecyl)phenyl, naphthyl, anthracyl, 3-cholesteryl, and the like. The hydrophobic substituent can be an alkyl, cycloalkyl, aliphatic, cycloaliphatic, non-aromatic heterocyclic, aryl, or heteroaryl group, or combinations thereof. The hydrophobic substituents can be the same, for example, all n-C18, or different, such as a range, for example, C24 through C28, or a mixture of structural isomers, for example, branched and linear C16, or mixtures of different groups, such as alkyl and aromatic, and the like. For example, a hydrophobic group can be C8–C30 aliphatic, polycyclic aliphatic, polycyclic aryl, or polycyclic heteroaryl, or a C8–C30 alkyl optionally interrupted or substituted by, for example, a heteroatom, a cycloaliphatic, aryl, or heteroaryl group, and the like. Alternatively, a hydrophobic substituent is an optionally substituted C8–C30 alkyl, polycyclic alkyl, polycyclic aryl, or polycyclic heteroaryl. Preferably, a hydrophobic substituent is a C8–C30 alkyl, and most preferably, a C12–C28 alkyl.
Substituents that substantially adhere the immobilizing polymer to the biochip are those that can create strong noncovalent or covalent interactions between the polymer and the biochip. "Substantially adhere" means that the polymer is not washed away during normal operation of the biochip. For example, hydrophobic substituents can cause substantial adherence to a hydrophobic microchannel wall through hydrophobic interactions, e.g., between octadecyl groups and a polydimethyl siloxane wall. In another example, ionic substituents can cause substantial adherence to a ionic microchannel wall through electrostatic interactions, e.g., between tetraalkyl ammonium cations and siloxide wall anions. In yet another example, a group can form a covalent bond with a microchannel wall, e.g, an ionic group such as a carboxyl can form a covalent bond with a channel surface functionalized with an amine as in structure A. Preferably, the immobilizing polymer is adhered to the wall through hydrophobic interactions or through covalent interactions. More preferably, the immobilizing polymer is substantially adhered through covalent interactions. Most preferably, the immobilizing polymer is covalently attached to the microchannel wall through a linker containing an amide bond, e.g., an amide bond formed between a carboxylate on the polymer and an amine on the linker. In a particularly preferred embodiment, the immobilizing polymer is covalently attached to a glass microchannel wall through a linker represented by structure B:
wherein Rs represents a surface —Si—O— group on the glass microchannel, Ra is a C1–C8 alkylene chain; each Rb is independently a C1–C4 alkyl group; and D represents an amide bond to the immobilizing polymer. For example, amide D can be formed by amidation of a group on the polymer, for example, an acyl halide, an ester, or an anhydride. For example, if the polymer is derived from a maleic anhydride copolymer, amide D can be formed by amidation of one of the maleic anhydride groups on the polymer.
In another alternative, the microfluidic biochip is made of the immobilizing polymer itself, and is thus effectively "substantially adhered" to itself by virtue of being a solid slab.
The immobilizing polymer itself, i.e, prior to reaction with a biocatalyst or adherence to a biochip, can be represented as a copolymer of monomers, wherein the collective substituents of the monomers perform the four functions discussed above. As used herein, a copolymer involves a combination of monomers wherein at least one of each monomer are present in the resulting polymerized polymer in any order. "Can be represented" means that the monomers are the hypothetical monomers that describe the repeat units of the polymer, not necessarily the actual monomer used to make the polymer (i.e. acetylene is the hypothetical monomer of polyacetylene, which can also be made from cyclooctatetraene). The polymer could be made from a single monomer that has appropriate substituents, for example, 2-octadecyl acrylate.
More preferably, the immobilizing polymer can be represented as copolymer of a monomer with an ionic group, e.g., carboxylate, and a monomer with a hydrophobic group, e.g., C16 alkyl. Even more preferably, the immobilizing polymer can be represented as a copolymer of ethylenically unsaturated monomers, e.g., acrylic acid and 1-octadecene. For example, an immobilizing polymer can be represented as a copolymer including a first monomer such as acrylic acid, itaconic anhydride, methyl methacrylate, allyl amine, vinyl alcohol, maleic anhydride, maleimide, carboxylate, vinyl carbamate, allyl sulfate, vinyl thiosulfate, vinyl sulfonate, allyl phosphate, vinyl phosphonate, vinyl alcohol, vinyl trimethylammonium chloride, other ethylenically unsaturated monomers substituted with ionic groups or precursors thereof, and the like. A second monomer includes a hydrophobic substituent, for example, styrene, C8–C30 α-olefin, acrylic acid-n-dodecyl ester, vinyl naphthalene, vinyl anthracene, (para n-dodecyl)styrene, other ethylenically unsaturated monomers substituted with hydrophobic groups, and the like.
A particularly preferred polymer is a maleic anhydride-α-olefin copolymer, for example, poly(maleic acid-alt-α-olefin, C24–C28) (PMA-OL), poly(maleic anhydride-alt-1-tetradecene) (PMA-TD), poly(maleic anhydride-alt-1-octadecene) (PMA-OD), and the like.
Other alternatives for the the immobilizing polymer can be any polymer that includes the functional substituents described above, i.e., at least an ionic group, or precursor thereof, and a hydrophobic group. Such polymers include appropriately substituted polyvinylalcohol, polyethers, polyacrylic acids, polyalkylacrylates, polymethacrylic acids, polyalkylmethacrylates, polystyrene, polystyrene sulfonates, polyvinylnaphthalene, polyethylvinylbenzene, polyaminostyrene, polyvinylbiphenyl, polyvinylanisole, polyvinylimidazolyl, polyvinylpyridinyl, polydimethylaminomethylstyrene, polyvinylamine, poly-N-alkylvinylamine, polyallylamine, poly-N-alkylallylamine, polydiallylamine, poly-N-alkyldiallylamine, polyalkylenimine, other polyamines, polyamides, polyacrylamides, polymethacrylamides, poly-N-alkylacrylamides, poly-N-alkylmethacrylamides, polydiallylmethylammonium chloride, polytrimethylammonium ethyl methacrylate, polytrimethylammonium ethyl acrylate, and copolymers thereof.
The molecular weight of the immobilizing polymer itself, i.e., prior to reaction with a biocatalyst or attachment to a biochip, should be large enough that there are significant interpolymer hydrophobic interactions between hydrophobic groups to allow the immobilizing polymer to stably coat the flow surfaces, yet small enough so that the polymer can be dissolved in a suitable solvent for coating. This is highly polymer dependent, e.g., polyacrylate can be much higher molecular weight than polystyrene sulfonate. The number average molecular weight of the polymer can be, for example, between about 2,000 and about 50,000, alternatively between about 2,500 and about 25,000, more preferably between about 5,000 and about 20,000, and most preferably between about 7,500 and about 15,000. A suitable solvent includes any solvent that can dissolve the immobilizing polymer, for example, protic solvents such as water, and alcohols; aprotic polar solvents such as N,N-dimethyl formamide, dimethyl sulfoxide, nitromethane; halogenated solvents such as chloroform, carbon tetrachloride, and trichloroethylene; ethereal solvents such as tetrahydrofuran; aryl solvents such as benzene, toluene, and xylenes; and the like. Suitable solvents can be made acidic or basic, for example, a polymer with carboxyl substituents can be more soluble in a basic alcohol.
The immobilizing polymer itself can be further, or alternatively, characterized by the numerical ratio of ionic groups, or precursors thereof, to hydrophobic groups. In this characterization, ionic groups, or precursors thereof, are counted by the number of charges that can be generated, e.g., a carboxylate and a carboxylic acid are each counted as one, and a maleic anhydride and a phosphate are each counted as two. Each hydrophobic group counts as one. In this fashion, the ratio of ionic groups to hydrophobic groups in the immobilizing polymer itself can be between about 1:20 to about 20:1, alternatively from about 1:8 to about 8:1, more preferably between about 1:5 to about 5:1, even more preferably between about 1:2 to about 2:1, or alternatively about 1:1. In a particularly preferred embodiment, the ratio of ionic groups to hydrophobic groups in the immobilizing polymer itself is about 2:1.
Alternatively, the polymer can be characterized by the molar ratio of monomers, separated into ionic monomers, i.e., those contributing an ionic group (or precursor thereof) and hydrophobic monomers, i.e., those contributing a hydrophobic group. The ratio between ionic and hydrophobic monomers can be between about 1:20 to about 20:1, alternatively from about 1:8 to about 8:1, more preferably between about 1:5 to about 5:1, even more preferably between about 1:2 to about 2:1, or most preferably about 1:1. In a particularly preferred embodiment, the ratio of ionic monomers to hydrophobic monomers is about 1:1.
The polymer can be produced in situ, or can be produced in whole or in part and then coated and adhered to the solid substrate. For example, maleic anhydride and an α-olefin can be polymerized using a radical initiator in the presence of the amine-functionalized biochip of structure A. The biomolecules can then be reacted with the immobilized polymer under suitable reaction conditions. Alternatively, the polymer can be produced separately (e.g., polymerizing maleic anhydride and an α-olefin) added to the amine-functionalized biochip, thereby immobilizing the polymer and coating the flow surface. Subsequently, the biomolecule can be added under suitable reaction conditions, thereby immobilizing the biomolecule. Alternatively, the polymer can be produced, the biomolecule can be immobilized thereon, and the biomolecule-polymer product reacted with the amine-functionalized biochip.
As used herein, a biomolecule is any biologically derived molecule that has biological activity, for example, binding activity, catalytic activity, and the like. A biomolecule is derived from any appropriate source, such as an enzyme domain extracted from a natural organism, a protein genetically engineered to be expressed in a different organism, a naturally derived peptide synthesized to contain an unnatural amino acid, and the like. A biomolecule can be a protein, a peptide, an antibody, an enzyme, an enzyme cofactor, a catalytic antibody, an oligonucleotide such as ribonucleic acid or deoxyribonucleic acid, a lipid, an oligosaccharide, a polysaccharide, a purified cell component, and the like. A biocatalyst is a biomolecule that can catalyze a chemical or biochemical reaction, and includes any associated cofactors necessary to carry out a particular catalytic transformation. A biocatalyst can include, for example, a catalytically functional portion of a catalytic antibody, an enzyme, an enzyme domain, a catalytic peptide, an oligonucleotide enzyme such as a ribonucleic acid enzyme or a deoxyribonucleic acid enzyme, and the like. Alternatively, the biocatalyst can be a catalytic antibody, an enzyme, or an enzyme domain. More preferably, the biocatalyst is an enzyme, most preferably, a catalytically functional portion of an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase, or a ligase, i.e., IUBMB (International Union of Biochemistry and Molecular Biology, www.chem.qmul.ac.uk/iubmb/ systematic enzyme classification (EC) numbers 1, 2, 3, 4, 5, and 6, respectively).
In one embodiment, a biomolecule is directed from a source on the biochip to a biomolecule-immobilizing group under suitable reaction conditions, thereby immobilizing the biomolecule to the immobilizing polymer at a flow surface. For example,
In another embodiment, at least two chemically distinct biomolecules are immobilized on a polymer at a flow surface. The chemically distinct biomolecules can be immobilized in the same location or in distinct locations. Alternatively, at least two chemically distinct biomolecules are separately directed and immobilized on polymers at spatially distinct flow surfaces, whereby the biomolecules are catalytically and spatially distinct. For example,
As used herein, "chemically distinct" means biomolecules that differ in chemical structure, molecular formula, molecular weight, isotopic composition, cofactors, sequence (of monomers, amino acids, DNA base pairs, RNA base pairs, and the like), function, i.e., catalysis or binding, oxidation state, secondary structure, tertiary structure, and the like. "Catalytically distinct" or "distinct biocatalysts" means that two catalysts are capable of catalyzing different chemical or biochemical reactions.
The biomolecule immobilized on the polymer at the flow surface can be characterized relative to the amount of biomolecule theoretically needed to form a monolayer on the flow surface. It is believed that interpolymer hydrophobic interactions form a highly porous, three dimensional matrix at the flow surface, allowing immobilization of more biomolecules than needed for a monolayer. Thus, in one embodiment, the amount of biomolecule immobilized on the polymer at each flow surface is between about 100% and about 800% of a theoretical monolayer, alternatively between about 110% and about 500% of a theoretical monolayer, more preferably between about 125% and about 500% of a theoretical monolayer, and most preferably between about 150% and about 300% of a theoretical monolayer. Of course, in many embodiments, much less biomolecule will be required.
The biochip with an immobilized biomolecule can be used to conduct chemical or biochemical reactions, for example, catalysis, binding, polymerization, molecular recognition, and the like. For example, in one embodiment, a reactant is directed from a source to an immobilized biocatalyst under suitable reaction conditions, thereby producing a first reaction product. In another embodiment, the first reaction product is reacted with a second catalytically distinct immobilized enzyme, thereby producing a second reaction product. Alternatively, the first reaction product is directed to a second catalytically distinct biocatalyst that is immobilized at a spatially distinct flow surface, thereby producing a second reaction product in a sequential manner.
One particularly preferred embodiment of the invention is a microfluidics biochip, comprising at least one microfluidic reservoir and a plurality of microfluidic channels. The channels are each less than about 0.1 mm2 in cross-sectional area, and the channels each comprise a flow surface. Furthermore, the channels are in microfluidic communication with each other and with the reservoir, and each channel is in microfluidic communication with at least two electrodes, whereby an electroosmotic flow can be generated at the flow surfaces. A polymer is coated on at least one flow surface, wherein the polymer is substantially adhered to the surface. The polymer comprises one or more ionic substituents of the same polarity selected from optionally substituted carboxylate, carbamate, sulfate, thiosulfate, sulfonate, phosphate, phosphonate, or hydroxyl. The polymer also includes one or more hydrophobic substituents selected from optionally substituted C8–C30 alkyl, a polycyclic alkyl, or a polycyclic aryl. The polymer also includes at least two catalytically distinct biocatalysts that are each immobilized to the polymer through an amide bond. The electrodes can be used to generate an electroosmotic flow to direct a reactant solution from the reservoir to a first catalytically distinct biocatalyst, whereby a first reaction product can be produced; and the first product from the first biocatalyst can be contacted with a to a second catalytically distinct biocatalyst, whereby a second reaction product can be produced.
As used herein, "suitable reaction conditions" include appropriate values for temperature, pressure, reaction time, pH, solvent, presence of biocatalyst cofactors, consumable reagents required such as adenosine triphosphate (ATP) or nicotinamide adenine dinucleotide phosphate (NADPH), and the like, that permit biological or biocatalytic activity.
Several microfluidic biochips were constructed by etching microfluidic channels into glass microscope slides or by molding channels into polydimethyl siloxane slabs. Slides were prepared as EOF controls (i.e., plain glass microchannels treated to activate siloxide anions on the channel surfaces), as immobilization controls (i.e., siloxide-activated channels functionalized with aminopropyl triethoxysilane (APTES) and then with glutaraldehyde), and as a test system to demonstrate the invention (i.e., siloxide-activated channels functionalized with APTES and PMA-OL, and PDMS (polydimethyl siloxane) channels coated with PMA-OD). The enzymes that were immobilized included soybean peroxidase (SBP), lipase, invertase, and glucose oxidase.
In these experiments, the ionic groups necessary to support the electroosmotic flow in the PMA-OL and PMA-OD coated biochips are provided by carboxyl groups. These groups are freed from the maleic anhydride groups in the PMA polymer when the maleic anhydride group reacts with either the enzyme, or, in the case fo the APTES-functionalized glass biochips, when the anhydride reacts tith the surface amine groups. The resulting negative charges ensured significant flow in the enzyme-bound microchannels.
A single step reaction consisting of SBP catalyzed oxidation of p-cresol was examined:
The reactions were conducted by SBP catalyzed reaction of cresol and H2O2 using a 2% PMA-OL-coated chip with 0.4 μg immobilized SBP, 20 mM p-cresol and 0.125 mM H2O2 in phosphate buffer containing 20% (v/v) DMF. Initially, the microchannel, with immobilized SBP, and product reservoir were filled with the DMF-buffer solution, and substrates were added to both substrate reservoirs. Substrates were supplied to the microchannel by EOF allowing product accumulation to take place.
As shown in
In this equation, CAf is the residual H2O2 concentration in the microchannel, T is residence time of substrate in the microchannel, and CA0 is initial H2O2 concentration (0.125 mM). The term CAf/Vmax was assumed to be small relative to the other term on the left-hand side of Eq. 4 and was, therefore, ignored to simplify the expression. This expression can be used when the limiting substrate (e.g., H2O2) conversion is high at long residence times, and the plot in
Calculation of SBP's kinetics of p-cresol oxidation reveals that the kinetics are, surprisingly, largely unaffected by immobilization to PMA in the 90 nL microchannels. Specifically, the Km of SBP in the biochip was 0.98�0.12 mM, compared with 0.95�0.14 mM for p-cresol oxidation in soluble forms in both 5-mL volumes (in 20 mL scintillation vials) and 20 μL volumes in 384-well plates. Similarly, the Vmax was 0.21�0.03 μmol H2O2 converted/mg SBP-min, which was essentially identical (0.20�0.03 μmol H2O2 converted/mg SBP-min) for the soluble enzyme at larger scales12. These results indicate that SBP displays intrinsically native activity even in the immobilized form at the microscale, and further attests to the mild immobilization conditions afforded by a polymer containing ionic and hydrophobic groups such as PMA-OL. Moreover, the PMA-OL matrix appeared to be highly porous, as the enzyme is immobilized at a higher concentration than would be expected in a single monolayer, yet no diffusional limitations are apparent, as the immobilized SBP in the microchannel has a Vmax identical to that in solution.
A coupled lipase/peroxidase biochip was prepared where the enzymes were co-immobilized using 5% (w/v) PMA solution. Lipase B from C. antarctica was used for the first reaction, the hydrolysis of p-tolyl acetate, which yields acetic acid and p-cresol, followed by coupling of the cresol to poly(p-cresol) using SBP and H2O2:
The lipase was used at a 10-fold higher concentration than SBP because of the expected low reactivity of the lipase on the cresol ester, which was not expected to be a highly reactive substrate of the enzyme. Reactions were initiated upon flowing solutions of 15 mM p-tolyl acetate and 0.25 mM H2O2 in phosphate buffer (pH 7.0) containing 20% (v/v) DMF. The solubility of the tolyl acetate was greatly facilitated by the presence of DMF. Reactions were performed in the microchannel with 0.6 μg total enzyme, 15 mM p-tolyl acetate, 0.25 mM H2O2 and in the 384-well plate with 100 μg/mL lipase, 10 μg/mL SBP, 15 mM p-tolyl acetate, and 0.25 mM H2O2 in phosphate buffer containing 20% (v/v) DMF.
The two-step enzymatic transformation was sufficiently reactive to give complete conversion of the H2O2 supplied with a residence time of 3 min
The higher reactivity of the bienzymic system in the microfluidic biochip is likely due to the higher concentration of the rate limiting enzyme, in this case the lipase, in the biochip than in free solution. Assuming 0.6 μg total protein loading (the lipase and SBP are of similar size), then ca. 0.55 μg of lipase was immobilized to the microchannel, giving ca. 5.5 mg/mL lipase concentration. This is far higher than the 100 μg/mL lipase, even if the aforementioned calculation was an overestimate (we did not determine the loading density of lipase in the microchannel) used in free solution in 384-well plates.
In Example 4, a three-enzyme biochip was constructed to demonstrate a 3 step reaction sequence that is purely synthetic, i.e., is a non-natural pathway. Invertase cleaves sucrose into glucose and fructose:
glucose oxidase oxidizes glucose using O2 to produce H2O2;
and SBP uses the H2O2 to produce poly(p-cresol):
The immobilized and solution phase systems used the three enzymes, invertase, glucose oxidase, and SBP in a ratio of 10:10:2. Reactions were performed in the microchannel with 0.6 μg total enzyme, 1 mM sucrose, and 20 mM p-cresol and in solution with 100 μg/mL invertase, 100 μg/mL glucose oxidase, 20 μg/mL SBP, 1 mM sucrose, and 20 mM p-cresol in phosphate buffer containing 20% (v/v) DMF.
In Example 5, the three-enzyme system of Example 4 was established on a PDMS biochip, wherein the three enzymes were immobilized at distinct locations.
As in Example 4, the immobilized and solution phase systems used the three enzymes, invertase, glucose oxidase, and SBP in a ratio of 10:10:2. Reactions were performed in the microchannel with 0.6 μg total enzyme, 1 mM sucrose, and 20 mM p-cresol and in solution with 100 μg/mL invertase, 100 μg/mL glucose oxidase, 20 μg/mL SBP, 1 mM sucrose, and 20 mM p-cresol in phosphate buffer containing 20% (v/v) DMF.
SBP (54 purpurogallin units/mg solid; RZ=1.3), fluorescein isothiocyanate (FITC)-labeled horseradish peroxidase (HRP), invertase from Saccharomyces cerevisiae and glucose oxidase from Aspergillus niger were purchased from Sigma (St. Louis, Mo.). Lipase B from Candida antarctica (Chirazyme L-2) was purchased from BioCatalytics (Pasadena, Calif.). 3-Aminopropyltriethoxysilane (APTES), poly(maleic anhydride-alt-α-olefin) (PMA-OL), glutaraldehyde, p-cresol, tolyl acetate, toluene, methanol (MeOH) and dimethylformamide (DMF) were purchased from Aldrich (Milwaukee, Wis.). Hydrogen peroxide (H2O2), NH4OH, NaOH, and borosilicate glass microscope slides were obtained from Fisher Scientific (Pittsburgh, Pa.). Buffered oxide etch was purchased from Doe and Ingalls, Inc (Boston, Mass.). All other solvents and reagents were obtained commercially at the highest purity available and used without further purification.
Unless otherwise specified, substrates (20 mM p-cresol and 0.125 mM H2O2) were dissolved in 0.1 M sodium phosphate buffer (pH 7.0) containing 20% (v/v) DMF (to aid in p-cresol solubility). The substrates were loaded into opposite reservoirs on the upstream side of the T-channel; each with 80 μL of 40 mM p-cresol and 0.25 mM H2O2 in phosphate buffer containing 20% DMF. The product reservoir was initially filled with 40 μL of phosphate buffer containing 20% DMF. After initiating different flow rates by applying different voltages between the substrate (anode) and product (cathode) reservoirs for 1 h, 20 μL of sample was withdrawn from the product reservoir. Minimal electrolysis gases were observed to form at voltages less than 2500 V.
Glass Biochip Fabrication
Borosilicate microscope slides were cleaned with isopropyl alcohol and acetone in a Class 100 clean room facility. The biochip consisted of a simple T-shaped microchannel with two substrate reservoirs and a product reservoir, and was fabricated using standard photolithographic techniques, shown schematically in
The silanol groups on the surfaces of the etched glass slide and the cover plate were activated by treating with a 1:1 solution of NH4OH and H2O2 at 70� C. for 25 min. Channels activated in this manner could be used as the EOF control without further surface modification. After the glass slide and cover plate were rinsed with distilled water and dried using a nitrogen gun, bonding was performed using UV-cured glue, lens bond type SK-9 (Summers Optical, Fort Washington, Pa.). The glue was spread between the cover plate and the etched glass slide by capillary action and cured at room temperature by UV irradiation at 365 nm for 1 h. Subsequently, additional glass tubes of 100 μL volume as substrate and product reservoirs and platinum electrodes were attached to the drilled holes using 2-ton epoxy glue (ITW Devcon, Danvers, Mass.).
Surface Modification of Glass Biochip with PMA-OL and Glutaraldehyde
A thin PMA-OL coating was prepared on the microchannel using 5% (w/v) of PMA-OL in toluene, and supplying 4 μL of this solution by vacuum. After removing excess PMA-OL in the microchannel, the biochip was dried at 90� C. for 1 h. To wet the microchannel, a series of washings were performed with acetonitrile (ACN), ACN:H2O (1:1), and deionized distilled H2O; each kept in the microchannel for 15 min and then removed by vacuum. Excess PMA-OL in the substrate and product reservoir was then removed by washing with toluene.
For glutaraldehyde modification, the microchannel was rinsed with toluene, ACN:H2O (1:1), and deionized distilled H2O three times following APTES treatment8. Glutaraldehyde (5%, v/v) in 0.1 M sodium phosphate buffer (pH 7.0) was loaded into the substrate reservoirs at room temperature and supplied to the biochip for 10 min by vacuum, and then for 2 h by EOF at 500 V. Excess glutaraldehyde was rinsed out of the microchannel by vacuum with 0.1 M sodium phosphate (pH 7.5) containing 1 mM MgCl2 for 30 min. A high voltage power supply (Model 215, Bertan Associates, Inc., Syosset, N.Y.) was used to maintain a constant voltage difference for EOF between substrate reservoirs and product reservoir.
Enzyme Immobilization
Soybean peroxidase (SBP) (2 mg/mL) in 0.1 M sodium phosphate buffer (pH 7) was supplied to the PMA-coated or glutaraldehyde-derivatized microchannel for 8 h by EOF at 500 V to immobilize enzyme. All enzyme and substrate solutions were filtered prior to use to prevent particulates from clogging the microchannel. A hydrophilic syringe-driven filter unit with 0.45 μm pore size was used for the filtration (Millex-LCR, Millipore Co., Bedford, Mass.). The enzyme solution was replaced every 2 h due to the pH change in the reservoirs as a result of the electrolysis of water generating H+ and OH− ions. Small bubbles that often formed in the microchannel were removed by vacuum. To remove excess enzyme after immobilization and before reaction, the microchannel was rinsed with phosphate buffer solution for 4 h by EOF at 1000 V. Immobilized FITC-labeled HRP was visualized in the microchannel using a Spot RT camera attached to a Nikon Eclipse TE 200 inverted microscope with TE-FM Epifluorescence attachment (Micro Video Instruments, Avon, Mass.).
PDMS Biochip Fabrication
A 1:1 mask was created by printing a high-resolution drawing of the desired microfluidic features as a negative (i.e., with the desired microfluidic features transparent) on a standard transparency film. A silicon master was created in a class-100 clean room facility from a silicon wafer (Silicon Quest International, Inc., Santa Clara, Calif.). The silicon wafers were cleaned with isopropyl alcohol and acetone, a layer of SU-8 negative photoresist (Microlithography Chemicals Co., MA) was spin-coated at 2000 rpm and 40 sec, and the coated wafer was pre-baked at 90� C. for 20 min. The coated wafer was aligned and exposed to ultraviolet light at 8.4 mW/cm2 (365 mm) for 50 sec using a mask aligner through direct contact with the transparency film mask. After post baking the wafer for 5 min at 90� C., it was developed by immersion into SU-8 thinner solution (Microlithography Chemicals Co., MA) for 1 min followed by immersion into SU-8 developing solution (Microlithography Chemicals Co., MA) until unmodified photoresist was removed, about 15 min. The dimensions of the pattern on the silicon master were determined using a profilometer (Alpha-Step 2000, Tencor Insturments, Mountain View, Calif.).
A 10:1 weight ratio of monomer and curing agent (Sylgard 184, Dow Corning Co., MI) was mixed and degassed under vacuum. This mixture was poured over the silicon master and cured at 70� C. for 2 hours. The patterened PDMS substrate thus formed was peeled off, cut to working dimensions, and sealed using a cover slab of PDMS. This was achieved by oxidizing the common surfaces of the substrate and the cover slab using oxygen plasma for 10 sec and then bringing them into conformal contact.
PDMS Biochip Surface Polymer Coating and Enzyme Immobilization
A thin PMA-OD coating of the microchannels was obtained by flowing a 0.5% (w/v) of PMA-OD in acetone (4 μL) through the whole microfluidic network (
Measurement of Enzyme Loading Density
To estimate the immobilized enzyme content in the microchannel, the amount of immobilized SBP on a glass surface at different PMA concentration was measured using the micro-BCA method (Pierce Biotechnology, Inc., Rockford, Ill.). Glass (1 cm�1 cm) was incubated for 16 h in 10 mg/mL SBP solution after coating with different concentrations of PMA and washed with phosphate buffer and distilled water for 4 h before BCA analysis. The glass squares with SBP covalently attached were incubated at 60� C. for 1 h in the micro-BCA working reagent (see www.piercenet.com). The chromogenic product of the micro-BCA assay is soluble in the assay reagent and was removed from the immobilized enzyme preparation for spectrophotometric measurements. The protein content was then determined based on a standard curve with SBP in solution.
Fluorescence and Flow Velocity Measurement
Spectrofluorophotometry was used to determine the activity of SBP. The intrinsic fluorescence of oligo- and polyphenols can be used to monitor the formation of product as the monomeric phenols have relatively minimal fluorescence. Sample analysis was performed in a 384-well plate (Simport Plastics, Quebec, Canada) and the reactions were monitored by measuring the relative fluorescence intensity (RFU) using a BioAssay Reader HTS 7000 Plus (Perkin Elmer, Norwalk, Conn.) at an excitation wavelength of 325 nm and emission wavelength of 405 nm.
To determine the flow rate of substrate in the microchannel at a particular voltage, a known concentration of the fluorescent p-cresol oligomeric product (original RFU of 8,500) was transported through the microchannel by EOF. The product reservoir was filled with 40 μL of phosphate buffer with 20% DMF. After 1 h of EOF operation, 20 μL samples were withdrawn from the product reservoir and the RFU measured. The flow rate was calculated by correlating the measured RFU as a function of the dilution ratio to known oligomeric concentration.
Calculation of Reaction Yield
SBP catalysis gives a diverse array of oligomers that appear to have widely different fluorescence properties. Moreover, fluorescence intensity does not scale linearly with concentration, as shown in Eq. 2, where where φ is the quantum efficiency, I0 is the incident radiation power, ε is the molar absorptivity, b is the path length of the cell, and c is the molar concentration16.
F=φI 0(1−e −εbc) (2)
Thus, the biochip operating at different flow rates, thereby resulting in different degrees of product dilution in the product reservoir, will give different fluorescence intensities even if the conversion is identical. To overcome this problem, we generated poly(p-cresol) in a 5 ml reaction containing 20 mM p-cresol, 0.125 mM H2O2, and 20 μg/ml SBP in phosphate buffer (pH 7.0) with 20% (v/v) DMF to obtain a product with a net relative fluorescence (RFU) of 8,500. This product was then diluted up to 100 fold and the fluorescence intensity measured as a function of dilution. As expected, the RFU was logarithmically dependent on dilution (curve not shown), and could be represented by the expression in Eq. 3, where X is the dilution ratio in the product reservoir and A is the original RFU of the sample after correcting for dilution.
Log Y=−0.85 Log X+Log A (3)
Based on this expression the dilution ratio (X) in the product reservoir at different voltages was calculated from the original RFU (8,500) in the substrate reservoir (A) and diluted RFU measured in the product reservoir (Y).
While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.
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|U.S. Classification
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|International Classification
|Cooperative Classification
|European Classification
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Owner name: RENSSELAER POLYTECHNIC INSTITUTE, NEW YORK
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