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Female police Lt. demoted for sending racy pics to fellow officer
An investigation was launched after anonymous letters were sent to department chiefs
By PoliceOne Staff
TUCSON, Ariz. — A female police lieutenant who sent sexually explicit photos and videos of herself in uniform to a lower-ranking officer has been demoted, according to reports.
Diana Lopez, who was in a relationship with the man at the time, was the public information officer at the Tucson Police Department.
Police said her actions violated several regulations, its code of ethics and its professional standards, according to the Daily Mail.
"Lopez used extremely poor judgment in sending these images undermining her credibility as a commander," Assistant Chief Kathleen Robinson said.
"Her actions have negatively affected not only her reputation, but the reputation and mission of the Tucson Police Department."
Lopez's boyfriend, who has not been identified, showed the photos and videos to other officers on the department, according to reports.
An investigation was launched after anonymous letters were sent to department chiefs alerting them to the incident. According to KVOA Tucson, the official report was 179-pages long.
Lopez is now working in the operations division. Her attorney, Michael Piccarreta, said she is considering a civil lawsuit against the city or appealing the demotion.
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How did three Danes end up being tortured in Lebanon?
Saving Afghanistan's archaeological sites
13 May 2013 03:12 GMT | Politics, Humanitarian crises, Asia Pacific, Philippines, Taiwan
Taiwan's government has issued a 72-hour ultimatum to the Philippines' government, demanding an apology over the fatal shooting of a Taiwanese fisherman.
The Taiwanese coastguard on Sunday dispatched four vessels to guard the disputed water where the shooting incident took place.
"The government is determined to protect our fishermen," cabinet spokeswoman Cheng Li-wen said in a statement as vessels headed towards the Bashi Strait, claimed by both governments, where the fisherman was killed on Thursday.
If the Filipino government fails to respond in a positive manner within 72 hours, the hiring of Philippine workers will be frozen.
Lee Chia-fei , Taiwan presidential office spokeswoman
Ao Ming-wei, the captain of the coastguard vessel Tainan Jian said his vessel was prepared to face provocation and the crew would fire against possible threats.
Taiwan has demanded Manila apologise and compensate the victim's family or face a freeze on the hiring of its nationals.
It also asked the Philippines to bring to justice the coastguards responsible and start negotiating a fisheries agreement.
"If the Filipino government fails to respond in a positive manner within 72 hours, the hiring of Philippine workers will be frozen," presidential office spokeswoman Lee Chia-fei said on Saturday.
There are about 87,000 Philippine domestic helpers and other workers in Taiwan and they send home hundreds of millions of dollars a year.
Lee said other sanctions could include recalling the Taiwan ambassador to the Philippines and asking the Philippines ambassador to Taiwan to return to Manila to assist the investigation.
'Illegal fishing'
More than 50 bullets hit the ship, killing skipper Hung Shih-cheng.
"This is nothing but a slaughter," prosecutor Liu Chia-kai said after examining the ship.
Several fishermen's groups said they plan to file a protest at the Philippine mission in Taipei Monday.
In Manila Abigail Valte, a spokeswoman for President Benigno Aquino, said on Saturday that authorities had launched a "transparent and impartial investigation" into the incident.
She expressed hope that economic ties with Taipei would not be affected and added that the coastguard crew involved in the incident had been temporarily suspended from duty to ensure a fair inquiry.
Philippine coastguard spokesman Commander Armand Balilo said on Friday that the incident took place in Philippine waters and the crew had been carrying out their duty to stop illegal fishing.
"If somebody died, they deserve our sympathy but not an apology," Balilo told reporters.
The victim's son, who was with his father and two other sailors on the boat at the time, has insisted they did not cross into Philippine waters.
The incident comes at a time of heightened tensions around the region over rival claims to the nearby South China Sea.
China, the Philippines, Taiwan, Vietnam, Malaysia and Brunei all have competing claims to parts of the sea.
China's state media stood by Taiwan over the latest incident, asking Beijing to "teach Manila a lesson".
Source: Agencies
Al Jazeera looks at the row between China and its neighbours over islands in East China Sea and South China Sea.
Interactive, War & Conflict, China, South China Sea, Asia
Navy conducts open-sea defence and offence training around islands that are also claimed by Japan.
Asia, China, Japan, Taiwan
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Changing climate, unpredictable rains dry out the land, threaten the pastoral way of life in Somaliland.
Poverty & Development, Environment, Africa
Tensions rise as Greeks from both side of the bailout debate take to the streets to withdraw money and express angst.
Politics, Europe, Greece
The world's biggest producer and consumer of the metal sees a golden opportunity to expand its pricing power.
Business & Economy, Asia, China
Normalised Cuba-US relations could result in a hybrid economic model blending socialism and entrepreneurial spirit.
Business & Economy, Communism, Politics
Nigerian president under pressure to act as string of bloody attacks hit northern towns, killing more than 160 people.
War & Conflict, Humanitarian crises, Africa, Nigeria, Boko Haram
Government forces pounded Aleppo with air strikes trying to repel most intense attack by rebels in years.
War & Conflict, Middle East, Syria, Syrian crisis
After decades of tension and hostility between the two nations, has change finally come to US-Cuba relations?
As the Baku games close, we examine the challenges facing journalists; plus, Angola, Portugal and 'reverse colonialism'.
Media, Europe, Azerbaijan
How Brazil's football legend turned every Corinthians' match into a political meeting for democracy.
Politics, Football, Latin America
The ICC chief prosecutor discusses Sudan's president escaping justice in South Africa and alleged war crimes in Gaza.
Politics, ICC, South Africa

subset: commoncrawl


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Uniform Costumes
Try one of these fresh and flirty uniform costumes to spice up the party. Take off to fantasy land as a sexy flight attendant, or get ready to put out his fire with one of our frisky firefighter outfits. Ready to step it up a level? Try the ringleader.


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The following address was presented by His Beatitude Metropolitan Tikhon at the 73rd Annual Commencement exercises at St. Tikhon's Orthodox Theological Seminary on May 23, 2015.
2015 Commencement Address
The St. Tikhon's Community is pleased to announce that the 18th Annual Golf Benefit will be held on Saturday, August 22, 2015 at Jack Frost National Golf Course in Blakeslee, PA. Registration for the event begins at 12:00 PM, with the Shotgun Start occurring at 1:00 PM, followed by an Awards Dinner at 6:00 PM.
The cost of the event is $100 per golfer, which includes Golf, cart, lunch, admission to the banquet, drinks, refreshments, and prizes. Checks should be made payable to St. Tikhon's Seminary and mailed to Bernard S. Golubiewski at 19 Main Street, Laflin, PA 18702.
Those wishing to support the event are invited to sponsor a hole at the tournament. Corporate Sponsorship costs $100 and Individual Sponsorship is $25. All sponsor participants will be recognized on the course for all to see and your sponsorship will be indicated in a special remembrance at the awards dinner to follow.
Checks for sponsorships should also be made out to Bernard Golbiewski and mailed to the aforementioned address.
We look forward to seeing you on the course!
Is The Lord Calling You to Serve His Holy Church?
Applications to St. Tikhon's Seminary are being accepted through July, 31, 2015.
Scholarship Assistance Available.
Click here for information on how to begin the Admissions process.
For question you can reach us by phone at 570-561-1818 or you may email the Dean at firstname.lastname@example.com or the Registrar at email@example.com.
Saint Tikhon's Orthodox Theological Seminary here recently initiated a search for a full-time Director for its Office of Student Life, which directly provides for the overall welfare and practical needs of seminarians in formation, with special attention given to their spiritual life.
The Director of Student Life will be responsible for supervising the overall discipline of seminary student body, coordinating with all seminary constituencies in matters related to community events, serving in counseling, advocacy, and advisory roles to students in general academic, spiritual, and personal matters.
A detailed job description may be found on the seminary web site. The seminary is offering a competitive salary, flexible work environment, and benefits package. To apply for the position, please forward a cover letter, resume, three references and three writing samples to the V. Rev. Steven Voyovich, Dean, at P.O. Box 130, South Canaan, PA 18459. Alternatively, applications may be submitted by e-mail to: email@example.com
Each year the St. Tikhon's Monastery and Seminary Community opens its arms in dramatic fashion to the greater Orthodox and non-orthodox community. This is a labor of love, the preparation for which is several weeks long and involves every person associated with the Monastery and the Seminary as well as many friends and families, volunteers from neighboring parishes, and long-time supporters.
This labor of love is, in many ways, an iconic representation of our relationship with the world and an articulation of our place it that world. Each year we open up our lives to everyone who wishes to come and take part in the pilgrimage and each year we are surprised by the response. This year, hundreds of Pilgrims turned out to spend Memorial Day with the St. Tikhon's Community. This was the 111th Memorial Day Pilgrimage.
On Monday the main Liturgy was celebrated by His Beatitude, Metropolitan Tikhon, His Eminence, Archbishop Michael of New York and New York and New Jersey; His Eminence, Archbishop Mark of Philadelphia and Eastern Pennsylvania; His Grace, Bishop Thomas of the Diocese of Charleston, Oakland and the Mid-Atlantic of the Antiochian Orthodox Christian Archdiocese of North America; and His Grace, Bishop John of Naro-Fominsk, Administrator of the Patriarchal Parishes in the USA, Moscow Patriarchate.
Following the Liturgy pilgrims were also able to participate in memorials for the departed and Holy Unction for the healing of soul and body. They enjoyed a wide variety of food from vendors stationed around the Monastery grounds. They beheld and venerated relics, ancient icons, and priceless treasure of the Church at the St. Tikhon's Metropolitan Museum. They were able to venerate the Miraculous Myrrh Streaming Icon from Hawaii. They immersed themselves in the rich history of the Seminary through the latest edition of the Tikhonaire, which chronicles history of the Seminary and Monastery and has been in publication since 1948.
The Memorial Day Pilgrimage is an opportunity to reunite with friends and loved ones each year to pray together, share stories with one another, to teach one another, and to remember together.
Thank you for allowing us to share our lives with you. Thank you for sharing each of your lives with us, and God willing we hope to see you again next year.
On Saturday, May 23, 2015 Seminarian Joel Brady was tonsured a Reader by His Beatitude Metropolitan Tikhon at St. Tikhon's Monastery Chapel as part of the Memorial Day Pilgrimage service on Pilgrimage Saturday. Later that day Reader Joel would also graduate Valedictorian of the graduating Class of 2015.
As a seminarian Reader Joel exemplified what it means to be a model student and servant in Christ. He was active in student government, maintained constant participation at both the Holy Services and Community Events, and was ever vigilant of his community stewardship responsibilities. During his final year the additional responsibility of being one of the two dormitory RA's was placed upon him, a responsibility that was accepted graciously with a profound humility.
On behalf of the entire community we would like to thank the Reader Joel Brady for his service to St. Tikhon's and pray that God continues to bless and keep him as he begins this next phase of his public ministry.
On May 23rd, St. Tikhon's Seminary held its 73rd Commencement. Metropolitan Tikhon offered the Commencement Address. Thirteen graduates completed the requirements for the Master of Divinity Degree, including graduates from India, Austria, and the Middle East. Dn. Theophan received Honors in Church History for his Thesis: "The Mystery of Lawlessness: A Pastoral Examination of the Manifestation of Evil in Contemporary Society."
Archpriest Daniel Donlick, in just having celebrated 50 years of ordination to the priesthood, was awarded an honorary doctorate following his decisive role in moving the seminary toward accreditation with the Association of Theological Schools, and literally decades of service as professor, academic dean, and confessor that continues to the present day! A scholarship was also named in honor of Fr. Daniel in recognition of his fiftieth anniversary.
Participating in commencement exercises were two new board members: Nancy Kohudic, who is a member of the Diocese of Philadelphia and Eastern PA and an ardent supporter of our Married Student Housing Project, and Bishop Thomas (Joseph) of the Antiochian Christian Diocese of Charleston, Oakland, and the Mid-Atlantic, who is a long time supporter and friend of the St. Tikhon's Community.
May God grant our graduates many blessed years of ministry in our Lord's Vineyard!
HistorySchedule of Services These Truths We Hold St. Tikhon of Zadonsk St. Tikhon of Moscow Contact
Books Icons Media Liturgical Items Gifts Contact


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The Boss 302 returns to the road after 42 years.
Shaking Hands With The Devil
Watch as a Cobra Jet lines up with a Super Cobra Jet!
Make Your "Five-Oh" Go Faster and Smoother
If you love Ford performance, this is the place for you!
King of the Road
A Race Car For The Streets
Born on California's highways, the Mustang SSP offered police a high-performance alternative.
How to get up-close and personal to find the finest Ford Mustang you can afford.
A visual guide to telling one vintage Pony Car from its model-year stable mates.
A visual feast for Mustang lovers.
Mustang's third body style emerges.
A Real Jekyll & Hyde Ride
The power plants that gave the Mustang its gallop.
Upgrading a 1966 Mustang's Baer system from two-piston PBR caliper to six-piston Baer caliper.
A fitting end to the S-197 Mustang era.
Daytona Speedway was a fitting corral for these ponies.
Tulsa Raceway Park thundered with Ford power!
Carving corners with Ford power!
Walking around the paddock at Hallett Motor Racing Circuit
A great mix of old and new meet at this year's Atlanta auto show.
The Ultimate Ford Family Reunion
Fords invade downtown Tulsa's Brady Arts District
Walking around Tulsa Raceway Park
The capstone of a great event.
More from the 17th fairway of the Omni Amelia Island Plantation Resort.
Steel may be real, but this plastic is fantastic.
Flat colors are the new "it" color.
From horseless carriages to speedsters, these rolling masterpieces captured everyone's imagination.
Love them or hate them, rat rods were on the prowl in Minnesota.
Race Track Refugees On Display
These builders went "all in" on their designs and it shows!
AutoTrader Classics brings you the best of AutoRama!
A fitting finale for an amazing Ford filled event!
Long flowing running boards, stunning metalwork, and dripping with style.
Another steller year for the Amelia Island show.
These designers dared to be different.
Get your fill of Camaros, Mustangs and more!
These machines only have one purpose: the checked flag.
Tail fins from mild to wild were on display this year.
Classic F-Bodies Storm Las Vegas
Chevrolet's latest evolution of the Corvette was right at home with the classics.
Big block, small block, mod motor, boosted, blown or just plain wild!
These motorcars are a history lesson from the base of the automotive family tree.
If the Vegas strip is packed with cool cars then it must be time for SEMA!
From the earliest days of motorsports to the latest in cutting edge technology and everything in between.
Variety was the name of the game as the Festivals of Speed returned to Amelia Island.
More photos from the Mitty!
Chevrolet's pony car is still going strong!
Back for a second year this early Saturday morning event brought an amazingly diverse group of vehicles.
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|SUMMARY: The rapidly growing popularity of social media has many B2B marketers changing their marketing mix to include it. What are the most commonly used B2B social marketing tactics? Find out in this weeks chart.
Get the latest research on email, lead gen, and social media along with MarketingSherpa updates and promotions.
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Earth Day Network Blog Updates
America Needs Lisa Jackson9/12/11 | | 0 comments
Lisa Jackson is the environmental conscience of the Obama Administration. Throughout her career she has had the backbone to stand up for good science and for strong enforcement of the laws that protect our air and water — regardless of politics. She does not always win, but America can take comfort in knowing that this skillful, tireless champion is working 24/7 to safeguard our neighborhoods and our children.Read More
New: The Green Schools Leadership Center!9/9/11 | | 4 comments
Last night, President Obama delivered his job's speech introducing the American Jobs Act to Congress and the nation. In it, he proclaimed: "We have to look beyond the immediate crisis and start building an economy that lasts into the future!" The future? There is no better place to start than investing in the education of our youth. Spending money on education generates the largest number of jobs (23.1 per $1 million dollars in spending) of any government spending according to Dr. Robert Pollin of the University of Massachusetts-Amherst.Read More
24 Hours of Reality9/9/11 | | 3 comments
Climate change is a global problem — and the solutions will come when we address it together. That's why this month, former Vice President Al Gore will host 24 Hours of Reality, a global event that will reveal the full truth, scope and impact of the climate crisis.Read More
The National Mall is about to be illuminated….with solar energy!9/6/11 | | 1 comment
Did you know that the sun is greatest power source for planet Earth? In fact, it provides more energy to the Earth in one hour than the amount of energy produced by all nations in one year! And with growing prevalence of solar technologies worldwide, solar power is an excellent way to produce energy for your home and also save you tons of money on heating and energy costs. Don't believe us? Then visit the U.S.Read More
How would your town spend 5.7 million dollars?9/6/11 | | 6 comments
That's the question citizens of Wildpoldsried, Germany have to answer every year. How did this little village in southern Germany produce so much revenue? In 1997, the Village Council and newly elected mayor decided to commit to an ambitious goal to create jobs, become more locally focused and generate more revenue. So naturally the citizens of Wildpoldsried wanted to come up with a plan that forced their town to invest in renewable energy, reduce water waste and emissions.Read More
Music moves people to do powerful things9/6/11 | | 3 comments
Music moves people to do powerful things and now artists are using this power to strengthen the environmental movement. The Patagonia Music Collection allows artists to donate the proceeds from certain songs to environmental groups, not only educating their fans, but giving them an easy way to support the causes that are important to the artists. An avid environmentalist himself, Patagonia founder and owner Yvon Chouinard is very excited about this endeavor.Read More
An opportunity to end shark finning in California9/2/11 | | 5 comments
As early as today, members of the California State Senate could be voting on AB 376, the ban on the sale of shark fins in the state of California. The goal is to curb the cruel practice of shark finning, the harvesting technique used by fisherman to obtain the expensive ingredient in shark fin soup, an Asian delicacy. When a shark is caught for their fin, the appendage is cut off and the injured shark is thrown back into the ocean, condemned to cannibalism by other sharks and fish, drowning or bleeding to death.Read More
A groundbreaking new green building for the Bullitt Foundation9/2/11 | | 10 comments
The Bullitt Foundation is breaking new ground, literally. It is not very often that office building construction is something celebrated by the environmental community, but on Monday, August 29th, the Bullitt Foundation started construction of a truly green building. From the construction material to composting sewage, the building will make new strides in combining all of these practices under one roof, so to speak.Read More
The U.S. Department of Education Wants Your Feedback on the new Green Ribbon Schools Program9/2/11 | | 4 comments
Have you shown an interest or been following the events with respect to new Green Ribbon Schools Award program? If so, the U.S. Department of Education would like YOU to give your feedback on their proposed plans for this incredible initiative. This is the first time the federal government has launched a comprehensive green schools initiative, and you can contribute to it!!Read More
Toxic water found in private wells8/31/11 | | 7 comments
A recently released study by the U.S. Geological Society has found that private wells are more likely to contain unsafe levels of trace elements, including arsenic, manganese, radon and uranium. Exposures to these elements in high levels have been known to cause serious health problems. These can range from cancer to kidney disease, affecting child intellectual development and symptoms that mirror Parkinson's disease.Read More


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The present invention relates to a method of preparing a substrate surface supporting a lipid film membrane structure, which method comprises the steps of contacting a substrate surface with an aqueous liquid containing detergent/lipid mixed micelles to adhere detergent/lipid mixed micelles to the substrate surface, and then contacting the substrate surface having detergent/lipid mixed micelles adhered thereto with an aqueous liquid substantially free from detergent to elute the detergent molecules from the adhered mixed micelles and make the remaining lipid molecules assemble into a lipid film membrane structure on the substrate surface. The invention also relates to a substrate supporting a lipid film membrane structure as prepared by the method, the use of a substrate supporting a lipid film membrane structure as prepared by the method for molecular interaction studies, and a substrate surface for use in the method.
1. A method of preparing a substrate surface supporting a lipid film membrane structure, which method comprises the steps of:
a) contacting the substrate surface with an aqueous liquid containing detergent/lipid mixed micelles to adhere detergent/lipid mixed micelles to the substrate surface, and
b) contacting the substrate surface having detergent/lipid mixed micelles adhered thereto with an aqueous liquid substantially free from detergent to elute the detergent molecules from the adhered mixed micelles and make the remaining lipid molecules assemble into a lipid film membrane structure on the substrate surface.
2. The method according to
3. The method according to
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45. A substrate surface supporting a lipid film membrane structure as prepared in
46. Use of a substrate surface supporting a lipid film membrane structure as prepared in
47. The use according to
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50. A method of preparing a substrate surface supporting a lipid film membrane structure, which method comprises the steps of:
a) in a flow cell contacting the substrate surface with an aqueous liquid flow containing detergent/lipid mixed micelles to adhere detergent/lipid mixed micelles to the substrate surface, and
b) contacting the substrate surface having detergent/lipid mixed micelles adhered thereto in the flow cell with an aqueous liquid flow substantially free from detergent to elute the detergent molecules from the adhered mixed micelles and make the remaining lipid molecules assemble into a lipid film membrane structure on the substrate surface.
51. A method of preparing a substrate surface supporting a lipid film membrane structure, which method comprises the steps of:
a) providing a substrate surface having a biomolecule immobilized thereon,
b) contacting the substrate surface with an aqueous liquid flow containing detergent/lipid mixed micelles to adhere detergent/lipid mixed micelles to the substrate surface, and
c) contacting the substrate surface having detergent/lipid mixed micelles adhered thereto with an aqueous liquid flow substantially free from detergent to elute the detergent molecules from the adhered mixed micelles and make the remaining lipid molecules assemble into a lipid film membrane structure on the substrate surface.
52. A substrate surface for use in surface reconstitution of a protein or a polypeptide, comprising a hydrogel modified with lipophilic compounds, and immobilized to the hydrogel one member of a specific binding pair, the other member of the specific binding pair being attached to or part of a protein or peptide to be reconstituted on the surface.
53. The substrate surface according to
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[0001] 1. Field of the Invention
[0002] The present invention relates generally to a preparation method for lipid film membrane type structures on substrate surfaces, particularly sensor surfaces, a supported lipid film membrane prepared by the method, the use of such supported lipid film membranes in lipid membrane interaction studies, and a substrate surface for use in the method.
[0003] 2. Description of the Related Art
[0004] Membranes play a central role in the structure and function of all living cells. Defining the boundaries of the cells as well as compartments within the cells, the membranes not only have a barrier function but also control the transport of substances across the membrane, mediate information between the compartments and are the site of enzymatic reactions.
[0005] The membranes consist of a lipid molecule bilayer with proteins and other components, such as e.g., lipopolysaccharides. The membrane lipids have a polar (hydrophilic) part and a non-polar (hydrophobic) part. In the membrane bilayer, the non-polar parts of the lipids are turned towards each other in the middle of the bilayer with the polar lipid parts forming the external surfaces of the membrane. Proteins in the membranes, which may extend through the membrane (transmembrane proteins) or be anchored to one of the membrane surfaces, are generally bound to the membrane through noncovalent forces, such as the hydrophobic force or electrostatic interactions, but there are also examples of proteins that are covalently bound to lipids.
[0006] Artificial supported bilayer lipid membranes (so-called BLM's) on solids provide a natural environment for the immobilization of proteins, and have therefore gained increasing interest for application on biosensors of various kinds. Such BLM's may be used for studying, for example, ligand-receptor interactions at the membrane-water interface.
[0007] In order to obtain suitable conditions for transmembrane proteins, it is desirable to provide for an aqueous layer between the membrane and the support. For example, EP-A-441120 proposes to join the lower lipid layer to the support by hydrophilic spacer arms.
[0008] The assembly of BLM's on a solid support may be obtained in various ways. Basically, however, three different approaches have generally been used.
[0009] In a first approach, a solid support is immersed into an aqueous solution and a lipid monolayer is then formed at the air-water interface. When the support is retracted from the solution, a lipid monolayer is adhered to the support. The support is then re-immersed into the solution which results in the formation of a lipid bilayer on the support (Suarez-Isla, B. A., et al., Biochemistry (1983) 22:2319-2323).
[0010] In a second approach, a solid support surface is contacted with an aqueous solution of lipid vesicles whereby lipid bilayers under certain conditions may be formed by spontaneous fusion of the lipid vesicles, or liposomes, to the surface (a liposome is a lipid bilayer enclosing a volume). In this way, for example, fluid lipid bilayers floating on a thin water film may be formed on a hydrophilic support, such as SiO2. Similarly, lipid bilayer membranes may be produced which rest on an ultrathin hydrated polymer film, or hydrogel (see e.g., Sackmann, E., Science (1996) 271:43-48). On the other hand, liposomes contacted with a hydrophobic substrate will build up a monolayer on the hydrophobic surface (Cooper, M. A., et al., Biochim. Biophys. Acta (1998) 1373:101-111).
[0011] The membrane may be linked more stably to the support, for instance, through hydrophilic spacers as mentioned above, or as disclosed in, for example, U.S. Pat. No. 5,922,594 by covalently binding the lipid bilayer to a self-assembled monolayer of straight long chain molecules coated on the substrate surface. In the latter case, an aqueous solution of micellar or vesicle liposomes is contacted with the monolayer-supporting surface so that a majority of the liposomes bind covalently to the monolayer to form an anchored bilayer lipid membrane. Due to the covalent anchoring of the membrane to the support, the process may be performed in a flow cell using a controlled laminar flow. In a variant of preparing bound lipid bilayers, a streptavidin-coated surface is contacted with biotinylated liposomes.
[0012] In a third approach, a solid substrate surface is contacted with an aqueous solution of mixed micelles formed by codispersion of detergent with lipid in a receptacle, and the formation of a lipid bilayer on the surface is then effected by selective removal of the detergent by either diluting the micellar solution with buffer, "the micellar dilution technique", or by dialysis.
[0013] Micellar dilution may be exemplified by Lang, H., et al., Langmuir (1994) 10:197-210, where a supported mixed bilayer was formed by contacting a gold electrode supporting a thiolipid monolayer with an aqueous solution of mixed micelles of lipid and detergent, and then stepwise diluting the solutions with electrolyte below the critical micelle concentration (CMC) of the detergent.
[0014] Removal of the detergent by dialysis is disclosed in, for example, U.S. Pat. No. 5,204,239, where mixed phospholipid/detergent micelles in solution were allowed to attach to a gold electrode surface via a bridging arm with a terminal thiol group, and the detergent was then removed by dialysis, which resulted in the formation of a continuous lipid bilayer attached to the electrode.
[0015] Supported lipid bilayer membranes containing proteins or peptides (oligo- or polypeptides) have been prepared by including the protein or peptide in the lipid layer and lipid vesicles used in the first and second approaches, respectively, mentioned above, or in the mixed micelles used in the above third approach.
[0016] Heyse, S., et al., Biochem. (1998) 37:507-522, describe functionalization of a gold surface with a patterned organic monolayer consisting of alternating regions with carboxyl-exposing thiols (CTA) and regions with hydrocarbon-exposing thiolipids. Rhodopsin-containing mixed phospholipid/detergent micelles were applied to the patterned monolayer, and on dilution with buffer, the phospholipids were self-assembled on the structured support and formed membranes that alternated between phospholipid bilayer domains (on CTA) and monolayer domains (on thiolipids), rhodopsin preferentially being in the bilayer domains.
[0017] Bieri, C., et al., Nature Biotechnology (1999) 17:1105-1108, describe covering a sensor chip by a self-assembled monolayer consisting of biotinylated thiols and an excess of ω-hydroxy-undecanethiol to which streptavidin was bound. Biotinylated protein along with lipid/detergent mixed micelles were added to the surface, whereupon a supported lipid bilayer was formed by micellar dilution as above, the biotinylated protein binding to the immobilized streptavidin.
[0018] WO 96/38726 discloses a solid device having a covalently attached coating of a lipid bilayer containing a protein. A proximal phospholipid layer, which may contain e.g., a transmembrane protein, was first covalently attached to a linker layer on the solid device. A distal lipid layer was then deposited by vesicle or mixed micelle fusion to give a lipid bilayer structure. Mixed micelle fusion was effected by depositing a mixed micelle dispersion onto the proximal phospholipid layer, and then diluting with an aqueous buffer.
[0019] U.S. Pat. No. 5,765,355 discloses preparation of a bilayer lipid membrane sensor by contacting a gold surface with a thiolipid/detergent solution to covalently bind a thiolipid layer to the surface. After washing with detergent solution, a transmembrane protein-containing phospholipid/detergent solution was added and stepwise diluted with potassium chloride solution. The resulting lipid bilayer consisted of a mixed monolayer of thiolipid and phospholipid, and a second phospholipid monolayer containing the transmembrane protein.
[0020] Generally, however, the prior art methods for preparing lipid films, and especially bilayers, on solid surfaces are laborious, time-consuming and do not always give the desired result. Also, as regards receptor-containing cell membrane preparations, the receptor density in such preparations has usually not been high enough for successful use in biosensor applications.
[0021] There is therefore a need of improved methods for preparing lipid film membranes on solids, and especially for preparing such membranes containing reconstituted proteins or peptides.
[0022] It is an object of the present invention to provide a method of preparing supported lipid film membrane structures, which method overcomes the disadvantages of the prior art methods and which thus is easy and rapid to carry out and which makes it possible to provide substrate surfaces densely packed with reconstituted membrane proteins.
[0023] According to the present invention, it has now been found that the above object as well as other objects and advantages may be achieved by a method wherein a detergent/lipid mixed micelle preparation is deposited from an aqueous dispersion thereof onto a substrate surface, and the substrate surface is then contacted with an aqueous liquid substantially free from detergent to elute detergent from the micelles, the remaining lipid molecules thereby forming a lipid film on the substrate surface.
[0024] Therefore, in one aspect, the present invention provides a method of preparing a substrate surface supporting a lipid film membrane structure, which method comprises the steps of:
[0025] a) contacting a substrate surface with an aqueous liquid containing detergent/lipid mixed micelles to adhere detergent/lipid mixed micelles to the substrate surface, and then
[0026] b) contacting the substrate surface having detergent/lipid mixed micelles adhered thereto with an aqueous liquid substantially free from detergent to elute the detergent molecules from the adhered mixed micelles and make the remaining lipid molecules assemble into a lipid film membrane structure on the substrate surface.
[0027] In another aspect, the invention provides a substrate supporting a lipid film membrane structure as prepared according to the first aspect above.
[0028] In yet another aspect, the invention provides the use of the method for reconstituting membrane protein function.
[0029] In still another aspect, the invention provides the use of a substrate supporting a lipid film membrane structure as prepared according to the first aspect above for interaction studies with membrane associated proteins or peptides, e.g., in screening of drug candidate molecules.
[0030] In yet another aspect, the invention provides a substrate surface for use in the method.
[0031] These and other aspects of the invention will be evident upon reference to the following detailed description and the attached drawings.
[0032]FIGS. 1A to 1C are a schematic illustrations of the reconstitution of membrane proteins by deposition of detergent/lipid mixed micelles on an amphiphilic biosensor sensing surface with immobilized proteins, and subsequent elution of the detergent to form a lipid bilayer.
[0033]FIG. 2 is a sensorgram showing the response (RU) versus time (s) for the injection of three different mixtures of lipid and detergent into the flow cell of a SPR-biosensor instrument.
[0034]FIG. 3 is a diagram wherein the ratio of the difference between the concentration of detergent (OG) and its CMC to the concentration of lipid (POPC) is plotted against the deposition level of lipid on a sensing surface when passed by aqueous samples of detergent/lipid mixed micelles.
[0035]FIG. 4 is a diagram representing the signaling of reconstituted rhodopsin receptor on a SPR biosensor sensing surface. The relative response (RU) is plotted against time (s) for the illumination of a POPC-reconstituted rhodopsin surface (1) relative to the signal in a POPC-only reference surface (2).
[0036] As mentioned above, the present invention relates to the preparation f lipid film membranes, especially lipid bilayer membranes, supported on a solid substrate surface. Membrane lipids are amphiphilic molecules, or amphiphiles, comprising a hydrophilic (water soluble) part and a hydrophobic (water insoluble) part. The lipids form together a characteristic bilayer where the hydrophobic parts are directed towards the middle and the hydrophilic parts form the two surfaces of the membrane. In this bilayer, there may also be biomolecules, such as proteins or peptides, partly or fully inserted therein.
[0037] The invention is based on the idea of reconstituting lipid membranes, with or without proteins or peptides, by adhering mixed micelles of detergent and lipid to a substrate surface with subsequent depletion of the detergent by aqueous liquid substantially free from detergent. When detergent/lipid mixed micelles in an aqueous solution are contacted with the substrate surface, they attach thereto. With proper detergent/lipid ratio, this attachment is strong enough to allow selective elution of the detergent from the adhered detergent/lipid mixed micelles even when the liquid containing mixed micelles (and detergent) is rapidly replaced by aqueous liquid substantially free from detergent, such as by a liquid flow.
[0038] The replacement of the mixed micelle-containing liquid by detergent-free liquid may be performed in a static system by removal of the micelle-containing liquid from the substrate surface, e.g., by a pipette, with subsequent addition of the detergent-free liquid to the substrate surface. Alternatively, the substrate having detergent/lipid mixed micelles adhered thereto is moved to another compartment or receptacle with detergent-free liquid. Preferably, however, the depletion of the detergent from the substrate surface is performed by a liquid flow over the substrate surface.
[0039] If proteins or peptides are attached to the substrate surface prior to forming the lipid film thereon, the proteins or peptides are reconstituted with lipids when the detergent is selectively eluted from the adhered detergent/lipid mixed micelles.
[0040] The type of lipid film membrane that is formed on the substrate surface, i.e., a monolayer or bilayer, depends mainly on the character of the surface. While the method of the invention performed on a hydrophobic substrate surface usually will produce a lipid monolayer, the formation of a lipid bilayer, which is (at least currently) preferred, requires an amphiphilic substrate surface as will be explained in more detail below.
[0041] The procedure of the invention applied to the preparation of a supported lipid bilayer membrane is schematically illustrated in FIGS. 1A to 1C. Mixed micelles consisting of detergent and lipid are attached to projecting (hydrophobic) alkyl groups and other non-polar groups like hydrophobic parts of immobilized membrane proteins (for example, a pre-immobilized receptor protein) on an otherwise hydrophilic sensor surface (FIG. 1A). When a liquid flow free from amphiphile (detergent) is injected over the sensor surface, a detergent monomer concentration is maintained in the mobile phase leading to a rapid depletion of detergent from the mixed micelles (FIG. 1B). The lipids remain attached to the surface, assembling into a bilayer structure, and are able to reconstitute the function of membrane proteins on the sensor surface (FIG. 1C).
[0042] The term substrate, or support, as used herein refers to any material body or layer onto which it is desired to apply a lipid film membrane. Exemplary substrate materials are gels, beads, polymers, stationary hydrophobic or amphiphilic phases, etc. Reference to the "surface" of the substrate or support includes, for porous substrates, the interior surfaces as well. Currently preferred substrates are sensor surfaces and chromatographic particles.
[0043] As mentioned above, the substrate surface should be amphiphilic for a lipid bilayer to be formed. The term "amphiphilic" is, however, to be construed broadly herein. Basically, the term means that the surface should exhibit hydrophilic and hydrophobic chemical structures (i.e., chemical groups or residues, including whole molecules, e.g., biomolecules) in ratios that may vary within a wide range, including surfaces ranging from partially hydrophobic to partially hydrophilic.
[0044] Preferably, the hydrophobic structures of an amphiphilic surface constitute chemical projections capable of interacting with hydrophobic parts of the lipid bilayer. For example, the surface may support a self-assembled layer of hydrophilic residues mixed with hydrophobic residues (e.g.,alkyl groups), the latter preferably extending out from the hydrophilic residues. As mentioned above, the hydrophobic residues serve to adhere the mixed micelles whereas the hydrophilic residues aid in the formation of the lipid bilayer upon the depletion of the detergent.
[0045] As mentioned above and will be further described below, it is often desired that the supported membrane bilayer includes a biomolecule, such as a membrane protein or peptide (the term peptide including oligopeptides and polypeptides). Such proteins or peptides may be the species that provide the amphiphilic character to an otherwise hydrophilic surface. For example, a hydrophobic protein or peptide may be attached to a self-assembled layer of hydrophilic residues supported on the substrate surface. Alternatively, the amphiphilic character of the surface may be provided by both hydrophobic chemical groups and membrane proteins or peptides.
[0046] It is believed that the meanings of the terms "hydrophilic" and "hydrophobic" are well known to those skilled in the art. Basically, hydrophobic may be defined as water-repelling whereas hydrophilic may be defined as water-attracting. It is also customary to define hydrophilicity and hydrophobicity with regard to the contact angle for a droplet of a liquid on a planar solid surface, the contact angle being measured from the plane of the surface, tangent to the water surface at the three phase boundary line. A hydrophilic liquid will thus have a low contact angle on a hydrophilic surface, whereas a hydrophobic liquid will have a high contact angle. For example, hydrophobic surfaces typically have contact angles in the range of 40 to 110�, while the contact angles with water for hydrophilic surfaces typically are in the range of 1 to 25�.
[0047] The optimum ratio of hydrophilic moieties to hydrophobic moieties on the amphiphilic substrate surface will depend on the particular moieties as well as on the components of the mixed micelles used and may readily be determined by the skilled person for each particular situation.
[0048] A presently preferred substrate surface comprises a biocompatible porous matrix, preferably a hydrogel, modified to contain a certain amount of hydrophobic groups. For the present purposes, a "hydrogel" may be defined as presenting a surface layer of bound molecules which by reason of their chemical nature hold a large fraction of water, in which the molecules are predominantly in an amorphous, water-solvated state, and in which the thickness of the layer is of the order of 30 Å minimum up to any indefinitely higher limit (Merrill, E. W., et al., Hydrogels in Medicine and Pharmacy, Vol. III, N. A. Peppas, Ed., Chapter I, CRC Press, Inc., Boca Raton, Fla. (1986)). Building reconstituted membranes on polymer hydrogels will allow essentially undisturbed lipid bilayer dynamics. An exemplary such modified hydrogel is a carboxymethyl-modified dextran polymer hydrogel on which a substantial fraction of the glucose moieties have been modified by alkyl groups.
[0049] Before describing the additional features of the invention in more detail, the terms micelle, mixed micelle, lipid, and detergent will be discussed.
[0050] "Micelles" are aggregates of amphiphilic molecules (amphiphiles), such as detergents, which aggregates do not enclose an aqueous volume. They are formed when the concentration of the amphiphilic molecule in a liquid exceeds a critical value called the "critical micelle concentration", or CMC. By strict definition, the CMC is the concentration when 50% of the amphiphiles are in the form of micelles, but CMC is often used as the concentration where micelles first appear. The micelles are usually globular but also other shapes, such as e.g., rod-shaped micelles, may form at high amphiphile concentrations.
[0051] "Detergents" are in a broad sense defined as substances capable of lowering the surface tension of liquids, but the term relates in a more narrow sense to surfactants as means for purification purposes. As mentioned above, the detergents are amphiphiles and may form micelles. They may be anionic, cationic, zwitterionic or uncharged. In the context of the present invention, detergents may be said to be micelle-forming amphiphiles.
[0052] "Lipids" are generally esters of long-chain carboxylic esters and include fats, waxes and cell lipids. In the context of the present invention, the lipids are usually membrane lipids (cell and organelle membrane lipids), which exhibit an enormous diversity, are more or less amphiphilic and include inter alia the following classes: phospholipids, lysophospholipids, glycosyl diacylglycerols, plasmalogens, sphingomyelins, gangliosides, and sterols. Depending on the chemical structure of the cell lipids and physical factors like temperature, pH and ionic strength, they may form different types of aggregates in aqueous media. For example, lysophospholipids form micelles, whereas some phospholipids under certain conditions form bilayers.
[0053] If a detergent (or detergent mixture) is co-dispersed with a membrane lipid (or lipid membrane mixture), "mixed micelles" may be formed consisting of alternating bilayer-prone lipid molecules and detergent molecules. As mentioned above, a lipid bilayer will form on an amphiphilic support if the detergent is eluted from the micelles, such as by a continuous flow of substantially detergent-free liquid.
[0054] Lipids suitable for reconstitution purposes according to the present invention may readily be selected by the skilled person and are basically those able to (i) form lamellar aggregation structures, at least somewhere in the temperature interval 15-40� C.; and (ii) be solubilised by any suitable detergent to constitute a nonturbid micellar suspension. These lipids, or lipid mixtures, should preferably have a relatively low CMC and may be selected from natural or synthetic lipids or mixtures thereof.
[0055] Generally, such lipids may be selected from natural or synthetic lipid molecules such as glycerophospholipids, glyceroglycolipids, sphingophospholipids and sphingoglycolipids, and from the classes phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl glycerol, phosphatidyl acid, phosphatidyl inositol, galactopyranoside, glucopyranoside, digalactopyranoside, diglucopyranoside, ceramide-phosphatidyl choline, ceramide-phosphatidyl ethanolamine, ceramide-phosphatidyl serine, ceramide-phosphatidyl glycerol, ceramide-phosphatidyl acid, ceramide-phosphatidyl inositol, sphingomyelin molecules, glucosylceramides, glucocerebrosides, galactoceramides, galactocerebrosides, gangliosides, monoacyl phosphatidyl choline, cardiolipin molecules, that may be linked to saturated or mono-, di or polyunsaturated fatty or fluorocarbon chains ranging from three to thirty carbons in length where fatty chains attached to the head group can be the same or of different structure, cholesterol, lanosterol, ergosterol, stigmasterol, sitosterol and derivatives thereof capable of being incorporated into lipid membranes, N,N-dimethyl-N-octadecyl-1-octadecanammonium chloride or bromide, (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride, N-[2,3-dihexadecyloxy)prop-1-yl]-N,N,N-trimethylammonium chloride, bolaamphiphiles, polyglycerolmonoalkylethers, polyethoxymonoalkylethers, and liposome-forming molecules from the classes amphiphilic polymers, amino acids, crown ether compounds and di(acyloxy)dialkylsilanes, as well as mixtures of the lipids mentioned above.
[0056] Specific exemples of such lipids are: Phosphatidylcholines with acyl chains ranging in length from 14 to 18 carbons, such as di-1,2-myristoyl-SN-phosphatidylcholine, di-1,2-oleoyl-SN-phosphatidylcholine, 1-palmitoyl-2-oleoyl-SN-phosphatidylcholine (POPC) and 1-stearoyl-2-oleoyl-SN-phosphatidylcholine; glyceroglycolipids like di-1,2-myristoyl-3-diglucopyranosyl-SN-glycerol, di-1,2-oleoyl-3-diglucopyranosyl-SN-glycerol, 1-palmitoyl-2-oleoyl-3-diglucopyranosyl-SN-glycerol, 1-stearoyl-2-oleoyl-3-diglucopyranosyl-SN-glycerol, di-1,2-myristoyl-3-digalactopyranosyl-SN-glycerol, di-1,2-oleoyl-3-digalactopyranosyl-SN-glycerol, 1-palmitoyl-2-oleoyl-3-digalactopyranosyl-SN-glycerol and 1-stearoyl-2-oleoyl-3-digalactopyranosyl-SN-glycerol, or sphingomyelins with the corresponding acyl chain lengths and unsaturations as those mentioned above.
[0057] Likewise, detergents, or detergent mixtures, suitable for use in the present invention may readily be selected by the skilled person. Generally, it is preferred that the CMC of the detergent is relatively high. Thus, while the detergent CMC usually should be at least about 1 mM, it is often preferable that the CMC is higher, such as at least about 10 mM.
[0058] Exemplary detergents are 3-[(3-cholamidopropyl)dimethylammoniol]-1-propane sulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propane sulfonate (CHAPSO), N,N-bis-(3-D-gluconeamidopropyl)-deoxycholamide (deoxy-BIGCHAP), sodium taurocholate, cholic acid, deoxycholic acid, n-octylglucoside (OG), n-octylthioglucoside, N-decyl-N,N-dimethyl-3-ammonio-1-propane sulfonate (Zwittergent 3-10), N-dodecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate (Zwittergent 3-12), octanoyl-N-methylglucamide (MEGA-8), decanoyl-N-methylglucamide (MEGA-10), 6-O-(N-heptylcarbamoyl)-methyl-α-D-glucopyranoside (HECAMEG), and sucrose monolaurate.
[0059] The selective elution of detergent from the mixed micelles in accordance with step c) in the method of the invention as defined above is basically driven by the difference in critical micelle concentration, or CMC, of the detergent and lipid, respectively. Thus, generally, the higher this difference is, the better. This is due to the relatively high CMC of the detergent striving to maintain a high monomer concentration in the mobile phase and thereby getting extracted from the stationary phase.
[0060] The ratio of detergent to lipid in the mixed micelles may be a critical factor in the method of the invention. Too detergent rich preparations may result in poor or no attachment at all of mixed micelles to the surface (note that liquids of a high detergent concentration, well above the CMC, are usually used to completely wash away deposited lipids from a sensor surface in a flow cell). On the other hand, too lipid rich preparations tend to be turbid and adhere slowly to the solid support, building aberrant aggregate structures. Therefore, it is crucial that the lipid/detergent ratio in the mixed micelles be balanced to obtain a maximal level of attachment to the solid support while still having a clear, non-turbid solution.
[0061] In the prior art time-consuming micellar dilution methods, a functional detergent/lipid ratio will be reached at some point during the dilution, providing for the formation of a lipid bilayer (although too large dilution steps will give vesicle formation). In contrast, the approach of the present invention is to use mixed micelles of a predetermined detergent/lipid ratio known to give a clear micellar solution which attaches the micelles to the surface sufficiently strongly not to be released when contacted with detergent-free solution.
[0062] The desired ratio may be determined by relating the ratio of the excess concentration of detergent over CMC, i.e., ([detergent]—CMC)/[lipid], to the amount of lipid deposited on the solid support. The optimum ratio varies depending on the particular lipid and detergent selected but can readily be determined by the skilled person, as will be further described below. Usually, the above ratio is in the range of from about 0.1 to about 100, preferably from about 0.5 to about 100, and more preferably from about 0.5 to about 10. In preferred embodiments, the optimum ratio may be in the range from about 0.5 to about 5, an exemplary range being from about 0.5 to about 3, which is applicable to inter alia octylglucoside and POPC.
[0063] The concentration of lipid (or lipids) is usually in the range of from about 0.1 to about 50 mM, preferably from about 0.1 to about 10 mM.
[0064] The concentration of detergent (or detergents) is usually in the range of from about 0.5�CMC to about 10�CMC, preferably from about 0.5�CMC to about 5�CMC for the detergent.
[0065] The method of the invention should generally be performed at conditions, such as e.g., temperature, where the lipid (or lipid mixture) may form a liquid lamellar phase, i.e., above the main transition temperature of the lipid (or lipid mixture). In many cases it may be satisfactory to carry out the method at room temperature.
[0066] The term "elute" is used herein basically in the commonly acknowledged sense, i e., to denote the removal of an adsorbed substance from an adsorbent by means of a solvent, the adsorbent in the present case being the substrate surface.
[0067] The liquid used for eluting the detergent molecules from the mixed micelles should be substantially free from detergent, which means that the eluent liquid should contain no detergent at all or contain only trace amounts thereof. Otherwise the composition of the eluent liquid may vary depending on the particular application of the method of the invention. For example, in the case of chromatographic applications, the eluent liquid may be a conventional type eluent, and when the method is used in flow-cell-based biosensor applications, the eluent liquid may be the conventionally used running buffer.
[0068] The eluent liquid should provide a mobile phase for detergent removal, a salient feature of the invention being that micelle-containing liquid above the substrate surface is completely replaced (e.g., displaced) rather than diluted by the detergent-free liquid. The flow of eluent liquid should be substantially continuous, i.e., it should preferably not be arrested, and if so, at least not for any long time periods, and it should also not be interrupted by air bubbles. Likewise, while the liquid flow rate may vary during the elution, it is preferred that the flow rate is substantially constant. The optimum flow rate, which may vary within wide limits, depends on inter alia the flow system used and may readily be chosen by the skilled person for each particular situation.
[0069] While it is possible to carry out the deposition of the mixed micelles on the substrate surface according to step a) of the method in a stationary liquid, and only perform the elution step b) under flow conditions, it is preferred that the whole procedure is performed using a continuous liquid flow.
[0070] As mentioned above, the method of the invention may be used for chromatographic applications. To this end, the lipid bilayer membranes may be applied to hydrophobic or amphiphilic chromatographic particles, preferably in place in a column or in a channel of a micro- or nanofluidic device. In the latter types of devices, the lipid bilayer membranes may alternatively be applied to a channel wall.
[0071] Currently, however, it is preferred that the method of the invention is applied to a biosensor. As appreciated by those skilled in the art, a biosensor is an analytical device for analyzing minute quantities of sample solution having an analyte of interest, wherein the interaction of the analyte with a sensing surface is detected by a detection device. For the purposes of the invention, the sensing surface or surfaces of the biosensor are preferably located in a flow cell or flow cells, i.e., broadly a channel part(s) or compartment(s) through which a liquid flow may be maintained.
[0072] For many chromatographic as well as biosensor applications, it is desired that the lipid bilayer contains a biomolecule(s), usually a protein or peptide, preferably a so-called membrane protein. As mentioned above, a protein or peptide may be applied to the bilayer lipid membrane by including the protein or peptide in the mixed micelle preparation to be deposited on the substrate surface, or by attaching the protein or peptide to the bilayer lipid membrane after the formation thereof, e.g., by adsorption or covalent binding. For many applications, however, it may be preferable to attach the protein or peptide to the substrate surface before depositing the mixed micelle preparation thereon. The lipid bilayer will then reconstitute the protein or peptide by being formed around the protein or peptide molecules. In this way, a high protein or peptide density may be ensured. As mentioned above, the protein or peptide may then constitute the sole hydrophobic elements of an amphiphilic surface.
[0073] Immobilization of the biomolecule to the substrate surface may be performed by methods well known in the art. For instance, groups on a protein or peptide may be coupled directly to active functional groups on the substrate, for example by amine coupling to surface carboxyl groups as described in the Examples below. It may, however, at least in some cases be advantageous to attach the biomolecule, such as a protein or peptide, to the substrate surface via a coupling member, such as, for instance, a spacer, a linker, or another protein or peptide. Such spacers, linkers etc are well-known to the skilled person and need not be discussed in any detail herein. More particularly, a specific binding pair (sbp) may be used for attaching the biomolecule to the substrate surface. Exemplary specific binding pairs include antibody-antigen, antibody-hapten, biotin-avidin (or streptavidin), carbohydrate-protein, carbohydrate-lectin, nucleic acid duplexes, oligonucleotide pairs, oligonucleotide-polynucleotide pairs, polynucleotide pairs, such as DNA-DNA and DNA-RNA, protein nucleic acid (PNA) pairs, protein-RNA, interacting peptide pairs, and protein-metal chelate.
[0074] For example, G-protein coupled receptors (GPCRs) can be selectively biotinylated in their N-terminal glycosylations (Bieri, C., et al., Nature Biotech. (1999) 17:1105-1108) and subsequently captured on the substrate surface by immobilized streptavidin, which could be followed by reconstitution of the receptors on the surface according to the invention, so-called on surface reconstitution. This would give a uniform orientation of the lipid-reconstituted receptors, facilitating studies of their interactions with intracellular components. Likewise, a histidine-tagged protein or peptide (e.g., a recombinant fusion protein) may be captured on the substrate surface by an immobilized metal chelate, such as, e.g., a nitrilotriacetic acid (NTA) nickel complex.
[0075] "Antibody" as used herein means an immunoglobulin which may be natural or partly or wholly synthetically produced and also includes active fragments, including Fab antigen-binding fragments, univalent fragments and bivalent fragments. The term also covers any protein having a binding domain that is homologous to an immunoglobulin binding domain. Such proteins can be derived from natural sources, or partly or wholly synthetically produced. Exemplary antibodies are the immunoglobulin isotypes and the Fab, Fab′, F(ab′)2, scFv, Fv, dAb, and Fd fragments.
[0076] "Hapten" as used herein means a low molecular species that may give rise to an immune response only when coupled to a larger molecule or cell or by aggregation. After immunisation, however, free haptens may react with antibodies.
[0077] A substrate surface having an array of different (or the same) proteins or peptides in a lipid bilayer membrane may be prepared by attaching these proteins or peptides to the substrate surface, and then depositing a mixed micelle preparation to the surface to reconstitute the proteins or peptides.
[0078] "On surface reconstitution" according to the present invention permits the same handling of membrane proteins as for soluble proteins by standard procedures. No reconstitutions, into more or less stable proteoliposomes, have to be prepared in advance and when the protein is ready for reconstitution on a substrate surface, no elaborate detergent dilution steps are needed.
[0079] Should it, for some reason, be desired that the lipid bilayer membrane contain other biomolecules than proteins or peptides, such molecules may be provided in the same ways as those outlined for proteins and peptides.
[0080] Several analytical applications of lipid bilayer membranes use a lipid bilayer without any protein or peptide. For example, the published U.S. patent application Ser. No. 2002/0019019 A1 (the full disclosure of which is incorporated by reference herein) discloses a method of assaying drug candidates (usually small molecules) with regard to inter alia absorption, i.e., the uptake of a drug compound from the site of administration into the systemic circulation, by estimating the absorption from biosensor data associated with a sensor chip having lipids immobilized thereon. Specifically, the analysis of interactions of a drug candidate with immobilized liposomes can be used to predict whether or not the drug candidate is absorbed by the small intestine. The method of the invention may advantageously be used for such absorption assaying of drug candidates.
[0081] A great advantage of the present method of invention is the promptness by which the lipid reconstitution can be accomplished. Thus, whilst the immobilization of liposomes to a sensor surface is rather time-consuming, the present process of forming a lipid bilayer membrane on a support surface may be performed in a very short time. This means among other things that a fresh lipid bilayer may be formed for each assay, which may be desired for several reasons. For example, an accidentally introduced air bubble may tear away a part of the bilayer, it may not be possible to completely wash away absorbed small molecules (candidate molecules often tend to remain bound to the lipid bilayer in ligand binding assays), etc. Many approaches that before were either very laborious or impossible have now become both possible and easy to carry out. This is important for applications demanding standardized and high throughput processing, like screening and array assays.
[0082] As mentioned above, the method of the present invention is, at least currently, considered to be especially useful for chromatographic and biosensor applications, in particular biosensor applications. Biosensors may be based on a variety of detection methods. Typically such methods include, but are not limited to, mass detection methods, such as piezoelectric, optical, thermo-optical and surface acoustic wave (SAW) device methods, and electrochemical methods, such as potentiometric, conductometric, amperometric and capacitance methods. With regard to optical detection methods, representative methods include those that detect mass surface concentration, such as reflection-optical methods, including both internal and external reflection methods, angle, wavelength or phase resolved, for example ellipsometry and evanescent wave spectroscopy (EWS), the latter including surface plasmon resonance (SPR) spectroscopy, Brewster angle refractometry, critical angle refractometry, frustrated total reflection (FTR), evanescent wave ellipsometry, scattered total internal reflection (STIR), optical wave guide sensors, evanescent wave-based imaging such as critical angle resolved imaging, Brewster angle resolved imaging, SPR angle resolved imaging, and the like. Further, photometric methods based on, for example, evanescent fluorescence (TIRF) and phosphorescence may also be employed, as well as waveguide interferometers.
[0083] In the detailed description and Examples that follow, the present invention is illustrated, by way of example only, in the context of surface plasmon resonance (SPR) spectroscopy. One exemplary type of SPR-based biosensors is sold by Biacore AB (Uppsala, Sweden) under the trade name BIACORE� (hereinafter referred to as "the BIACORE instrument"). These biosensors utilize a SPR based mass-sensing technique to provide a "real-time" binding interaction analysis between a surface bound ligand and an analyte of interest.
[0084] The BIACORE instrument includes a light emitting diode (LED), a sensor chip including a glass plate covered with a thin gold film, an integrated fluid cartridge providing a liquid flow over the sensor chip, and a photo detector. Incoming light from the LED is totally internally reflected at the glass/gold interface and detected by the photo detector. At a certain angle of incidence ("the SPR angle"), a surface plasmon wave is set up in the gold layer, which is detected as an intensity loss "or dip" in the reflected light. More particularly, and as is appreciated by those skilled in the art, the phenomenon of SPR associated with the BIACORE instrument is dependent on the resonant coupling of monochromatic p-polarized light, incident on a thin metal film via a prism and a glass plate, to oscillations of the conducting electrons, called plasmons, at the metal film on the other side of the glass plate. These oscillations give rise to an evanescent field that extends a distance of the order of one wavelength (≈1 μm) from the surface into the liquid flow. When resonance occurs, light energy is lost to the metal film through a collective excitation of electrons therein and the reflected light intensity drops at a sharply defined angle of incidence, the SPR angle, which is dependent on the refractive index within reach of the evanescent field in the proximity of the metal surface.
[0085] As noted above, the SPR angle depends on the refractive index of the medium close to the gold layer. In the BIACORE instrument, dextran is typically coupled to the gold surface, with the analyte-binding ligand being bound to the surface of the dextran layer. The analyte of interest is injected in solution form onto the sensor surface through the fluid cartridge. Because the refractive index in the proximity of the gold films depends on (i) the refractive index of the solution (which is constant), and (ii) the amount of material bound to the surface, the binding interaction between the bound ligand and analyte can be monitored as a function of the change in SPR angle. In the Examples below embodying the present invention, a lipid bilayer membrane is bound to a modified such dextran layer.
[0086] A typical output from the BIACORE instrument is a "sensorgram", which is a plot of response (measured in "resonance units" or "RU") as a function of time. An increase of 1,000 RU corresponds to an increase of mass on the sensor surface of about 1 ng/mm2.
[0087] A detailed discussion of the technical aspects of the BIACORE instrument and the phenomenon of SPR may be found in U.S. Pat. No. 5,313,264. More detailed information on matrix coatings for biosensor sensing surfaces is given in, for example, U.S. Pat. Nos. 5,242,828 and 5,436,161. In addition, a detailed discussion of the technical aspects of the biosensor chips used in connection with the BIACORE instrument may be found in U.S. Pat. No. 5,492,840. The full disclosures of the above-mentioned U.S. patents are incorporated by reference herein.
[0088] In the following Examples, various aspects of the present invention are disclosed more specifically for purposes of illustration and not limitation.
[0089] This example describes the preparation of mixed micelles of detergent and lipid, deposition of the mixed micelles on a sensor chip surface, and elution of detergent to form a lipid bilayer on the surface. A BIACORE 3000 instrument (Biacore AB, Uppsala, Sweden) was used. BIACORE instruments are based on surface plasmon resonance (SPR) detection at gold surfaces, and a micro-fluidic system is used for passing samples and running buffer through four individually detected flow cells (one by one or in series), with very high precision and with small sample volumes needed. As sensor chip was used Pioneer Chip L1 (Biacore AB, Uppsala, Sweden) which has a gold surface with a covalently linked carboxymethyl-modified dextran polymer hydrogel on which a substantial fraction of the glucose moieties is modified with alkyl groups (Cooper, M. A., et al., Anal. Biochem. (2000) 277:196-205). Running buffer was HBS-N (10 mM HEPES pH 7.4 and 150 mM NaCl) (Biacore AB, Uppsala, Sweden).
[0090] A. Preparation of Mixed Micelles
[0091] 1-Palmitoyl-2-oleoyl-phosphatidylcholine (POPC) (lipid; Avanti Polar Lipids Inc., Alabaster, Ala., U.S.A.) at 10 mM in chloroform was pipetted to round bottomed glass tubes, pre-washed in chloroform. The solvent was evaporated under a stream of nitrogen gas and solvent remains were removed under reduced pressure for at least 2 h. Octylglucoside (OG) (detergent; Sigma, St. Louis, U.S.A.) was diluted from a 0.5 M stock (frozen in aliquots) to desired concentrations with HBS-N and water, yielding 9 mM HEPES pH 7.4 and 135 mM NaCl (HBS-OG). HBS-OG was added to the dry lipid film and the mixtures were shaken every 10 min for at least 45 min at room temperature. In this way a number of combinations with 0.12-10 mM POPC and 5-50 mM octylglucoside were prepared. The preparations were checked for turbidity by the eye.
[0092] B. On Surface Reconstitution of Lipid Membranes
[0093] After conditioning of the L1-surface with five 30 s injections of 20 mM 3-[(3-cholamidopropyl)dimethylammonio]propanesulphonic acid (CHAPS) (Sigma, U.S.A.) the mixed micelles (prepared as described in step A above) were injected into BIACORE 3000 for 8 min (during optimization, otherwise 1 min) at 5 μl/min. The washing of the flow system was delayed for 2 min after injection. Lipid deposition quantity data were collected 100 s after the end of injection. The sensor surface was regenerated by two 1 min injections of 20 mM CHAPS or optimally 50 mM octylglucoside. The results are shown in FIGS. 2 and 3.
[0094] As seen in FIG. 2, three groups of samples could be identified: (1) Detergent rich clear preparations giving no attachment of POPC at all; (2) lipid rich, turbid samples that adhered firmly with slow association curves of analogous appearance as liposome capturing curves (Cooper, M. A., et al., supra; and Erb E. -M., et al., Anal. Biochem. (2000) 280:29-35); and (3) balanced, clear preparations leading to fast elution of octylglucoside and high deposition of POPC and having very steep association and dissociation curves, but with a significant and lasting increment of the baseline after the injection. This remaining signal elevation is most likely caused by deposited POPC on the sensor surface. The mechanism behind this can be explained in the following way: Mixed micelles of proper compositions adhere very rapidly to the amphiphilic surface during the injection. When the injection is terminated and detergent free buffer is running through the flow cell, the mixed micelles remain adhered to the amphiphilic hydrogel but the octylglucoside, striving to keep the monomer concentration (CMC), is very rapidly depleted. As the POPC micelles become detergent free, they fuse to build a continuous lipid film. This kind of reconstitution is taking advantage of the efficient fluidics of the instrument. Injections of 0.1 mg/ml of BSA showed that these lipid bilayers equally well protected the Pioneer Chip L1 surface from binding of BSA, as surfaces saturated with LUVs (large unilamellar vesicles). This together with the fact that the optimum deposition level of about 5000 RU is close to the theoretical level for a bilayer, 4600 RU (Cooper, M. A., et al, supra; Cooper, M. A., et al, Biochim. Biophys. Acta (1998) 1373:101-111; and S�vin-Landais, A., et al., Biophys. Chem. (2000) 85:141-152), indicates that the result is a continuous bilayer covering the entire sensor surface.
[0095] The ratio of detergent to lipid in the mixed micelles seems to be a major factor in determining to which of the three groups above the sample belongs. When the ([octylglucoside]-CMC)/[POPC] ratio is related to the amount of POPC deposited after injection, it appears that the optimum is between 0.5 and 3 octylglucoside molecules per POPC in the mixed micelles, as illustrated in FIG. 3 (only clear preparations are included in the diagram). Since this equation is dependent on the CMC of the detergent and the solubility of the current lipid mixture, which can vary considerably (Sch�rholz, T., Biophys. Chem. (1996) 58:87-96), the fine-tuning of composition has to be done in each instance. In this case, the best mixture found was 3.3 mM POPC and 25 mM octylglucoside. It also seems to be of high importance that the detergent is rapidly depleted after the injection. When different concentrations of octylglucoside were included in the running buffer, no deposition at all occurred or the system became very unstable.
[0096] When using lipids other than POPC, by themselves or in mixture with POPC, shifts in the optimal ([octylglucoside]-CMC)/[POPC] ratio were detected. However, most of the variants could mediate as high lipid deposition levels as could pure POPC, as shown in the following Table 1.
[0097] This example describes immobilization of rhodopsin (a G-protein coupled receptor; GPCR) on a sensor surface, and reconstitution of the protein by the formation of a lipid bilayer on the surface. Rhodopsin function was tested by assaying its signaling capacity (transducin activation). BIACORE 3000 and BIACORE X instruments (Biacore AB, Uppsala, Sweden) were used. As sensor chip was used Pioneer Chip L1 (see Example 1 above).
[0098] A. Immobilization of Rhodopsin
[0099] After docking in BIACORE 3000 or BIACORE X (Biacore AB, Uppsala, Sweden), sensor chip Pioneer Chip L1 (Biacore AB, Uppsala, Sweden) was first washed by two 1 min injections of 20 mM CHAPS (Sigma, U.S.A.). The carboxymethyl-modified dextran polymer, which is partially substituted with alkyl groups on Pioneer Chip L1, was activated with an injection of 0.2 M N-ethyl-N-dimethylamino-propylcarbodiimide (EDC) and 50 mM N-hydroxysuccinimide (NHS) for 7 min. The running buffer was as in Example 1 above. Rhodopsin (obtained from Dr. Andreas von Usedom, Institute for Medical Physics and Biophysics, Humboldt University, Berlin) stored at 61 μM in 20 mM BTP, 130 mM NaCl, 1 mM MgCl2, 53 mM octylglucoside and 200 mM α-methyl-mannopyranoside, was diluted to 0.61 μM in 10 mM maleate pH 6.0 and 20 mM octylglucoside. The diluted rhodopsin was injected for 14 min minimum and the surface was then blocked by 0.96 M ethanolamine-HCl pH 8.5 and 20 mM octylglucoside (Sigma, St. Louis, MO, U.S.A.) for 7 min.
[0100] The amine coupling of rhodopsin above resulted in immobilization levels close to 4000 RU on the L1 chip, which corresponds to 4 ng/mm2 (Stenberg, E., et al., J. Colloid Interface Sci. 143, 513-526) or 0.1 pmol/mm2 of rhodopsin. Since the amine coupling is not site specific but can involve any free amine group on the protein (mostly lysine residues), the rhodopsin is not uniformly oriented on the chip surface. However, most of the lysines in rhodopsin are positioned on the C-terminal side (cytosolic side) according to sequence based models of the structure (Hargrave, P. A., et al., Biophys. Struct. Mech. (1983) 9:235-244). Hence, the C-terminal side is probably dominating as the side of attachment, keeping the outside out orientation in favour.
[0101] B. Reconstitution of Rhodopsin by Lipid Bilayer Formation
[0102] The immobilized rhodopsin obtained in step A above was immediately reconstituted by a 2 min injection of mixed micelles, 3.3 mM POPC and 25 mM octylglucoside in HBS-N, prepared as in Example 1 above. Around 4500 RU of POPC were deposited. In a reference flow cell with unmodified Pioneer Chip L1-surface, about 5000 RU of lipid was simultaneously deposited. The always lower amount of lipids that bound in the rhodopsin flow cell is probably due to the space occupied by the immobilized protein, which indicates that lipid and protein coexist in each other's proximity.
[0103] The lipids deposited this way could be completely removed by two consecutive 1 min injections of 50 mM octylglucoside, and then resupplemented with a new 1 min injection of 3.3 mM POPC in 25 mM octylglucoside. This was indicated by the very stable responses achieved during 10 cycles of removal and supplementation.
[0104] C. Assay for Rhodopsin Functionality
[0105] In order to judge if the rhodopsin has a native and functional conformation after immobilization and supplementation with lipids as described in steps A and B above, its signaling capacity was assayed. When rhodopsin is activated by light, it transmits its signal by activating transducin. Activated transducin dissociates from the membrane under consumption of GTP (Heyse, S., et al., Biochemistry (1998) 37:507-522).
[0106] Measurements of transducin dissociation by activated rhodopsin were performed in a BIACORE X instrument into which an optical fiber had been inserted for illumination of the flow cells. The instrument temperature was set to 20� C. All the operations were performed under safe red light. Equal volumes of 11.6 μM transducin (obtained from Dr. Andreas von Usedom, Institute for Medical Physics and Biophysics, Humboldt University, Berlin) and 1 mM GTP, both stored in 20 mM BTP, 130 mM NaCl and 1 mM MgCl2, were mixed with three volumes of HBS-MDE (10 mM HEPES pH 7.4 and 150 mM NaCl, 1 mM MgCl2, 1 mM DTT and 0.2 μM EDTA), giving a five times dilution of both (2.3 μm transducin and 200 mM GTP).
[0107] This solution was injected at 5 μl/min over both the rhodopsin and the reference flow cells in the manual mode. Upon injection, transducin bound readily to both the rhodopsin-POPC surface and the POPC reference surface, but when the injection was finished it dissociated very rapidly from the surface into the mobile phase of running buffer. In order to detect the active dissociation of transducin, the flow was stopped at maximal binding during the injection. FIG. 4 shows the response signal for the POPC-rhodopsin reconstituted surface (curve 1) relative to the signal for the POPC reference surface. The flow was stopped at 459 s (FIG. 4). A few minutes after the flow-stop, at 902 s (FIG. 4), the level of bound transducin was stabilized in both the reference and rhodopsin flow cells. The flow cells were then illuminated with an Ocean Optics 5 W halogen lamp via an optic fibre. The activation of the receptor was recorded as a surface mass decrease caused by dissociation of the activated transducin from the membrane. Since this decrease was not observed in the reference flow cell or in absence of GTP it was concluded that it displays the signalling capacity of the receptor. After the signal decrease had levelled out significantly, the flow was resumed and the injection terminated (FIG. 4). When recharging the rhodopsin on the sensor surface, 9-cis-retinal (Sigma, St. Louis, Mo., U.S.A.) at 10 μM in HBS-MDE and 0.7% DMSO were injected over both flow cells for 13 min. Membrane bound retinal was allowed to dissociate for about 40 min before next round of the assay.
[0108] Following extended illumination of the rhodopsin and over night incubation, the signaling capacity of the receptor was exhausted. However, after injection of 10 μM 9-cis-retinal a recovery of the signalling capacity was detected. This showed that also the ligand binding capacity of the receptor was preserved when reconstituted by the method presented here.
[0109] From these results it was concluded that rhodopsin in a micellar environment can be covalently attached to the Pioneer Chip L1 chip surface by a commonly employed protocol for protein immobilization without irreversible loss of function. Since rhodopsin is sensitive to its lipid environment (Brown, M. F., Chem. Phys. Lipids (1994) 73:159-180) and demands reconstitution with phospholipids (Bubis, J., Biol. Res. (1998) 31:59-71), it was concluded that the lipid deposition method described in Examples 1 and 2 can be generally used for functional reconstitution of membrane proteins on amphiphilic surfaces. The method can also be used generally for building lipid bilayers on amphiphilic surfaces in flow systems, and would also be applicable to chromatography columns.
[0110] From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.
[0111] All of the above U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet, are incorporated herein by reference, in their entirety.
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