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Wedding Planner: The Burlap Elephant - Amazing! Couldn't have done it without Katie and her assistant! They really turned my vision into my dream wedding, and didn't miss a detail! She is organized, has helpful tips, keeps you budget, and took care of all the details so I could relax and enjoy my day. The wedding was perfect and I received so many compliments about the décor and set-up. Katie was quick to respond to all emails and coordinated all vendors and contracts for me. She was definitely one of my favorite vendors and I would recommend her to anyone.
Venue: Governor Thomas Bennett House – We fell in love with this venue from the moment we set eyes on it. It's beautiful inside and out. Linda was absolutely wonderful to work with. We were able to bring in our own caterer and rentals, which made the wedding more affordable. We got married outside in front of the gorgeous house, followed by cocktail hour on inside and on the balcony. The reception was outside in the gardens. The gardens were lovely and made for beautiful pictures.
Photographer: Jennings King Photography – Fantastic! Loved working with her and the pictures turned out to be stunning! I felt at ease with Jennings instantly when I spoke with her on the phone, and knew she was the one for us. She captured all the shots we wanted and more. She had our first look up on Facebook the next day and a blog posting after 1 week. I will be using her in the future for family portraits, and would recommend her to anyone. Photographer was the most important vendor to us and we chose well!
Caterer: Newton Farms – The food was amazing. Sarah was really flexible, and helped us find a menu that fit into our budget. She didn't say anything when we changed our minds on stations and sides several times. The food turned out perfect and we received numerous compliments from our guests. The bar service was great a well, every time my drink was almost empty I found them replacing without me even having to go to the bar! Sarah was quick to respond to email and changes in menu (even when she was on vacation!). They even made plates for my husband and I so we could socialize! They were very attentive and I couldn't have asked for better food!
Cake: Wildflour – I love, love, loved my cakes! We did 3 small cakes instead of one big cake and they turned out just as I imagined. I emailed Lauren pictures of cakes I liked and she recreated them perfectly. There were so many good cakes at the tasting that we had a hard time just choosing 3. Lauren let us do different flavors in each tier and we ended up with 5 different types of cake! The Carrot cake and red velvet were by far my favorite. All our guests raved about the hummingbird flavor. She is a little slow to respond to emails, but her quality is well worth the wait! I will be getting anniversary and birthday cakes from her for the rest of my life!
Flowers: Sara Grimshaw Designs – Amazing! I went into my meeting with SGD and gave them my vision and showed them my Pinterest board. I had no idea what my flowers would like until the day of, but I was blown away! They couldn't have designed more perfect floral arrangements for me. My guest loved the teacup arrangements on each table and my bouquet was absolutely stunning! Loved the team and the final product.
Ceremony music: Clelia Reardon – She was great to work with. Very reasonably priced, played all the music I asked for and showed up early. I didn't get to hear much since I was getting pictures during cocktail hour and walking down the aisle was a blurr, but what I did hear was absolutely perfect.
Makeup: Elina Mille (Bellelina Makeup and Spa) – Loved it! I did my trial over a year prior to the wedding, but Elina was still able to recreate the perfect look. Loved the airbrush, my makeup stayed all night and no mascara down my face from tears. I loved my makeup for once and felt like I looked natural and completely like myself. I also had an amazing facial the week before the wedding at her spa and my skin looked and felt amazing!
Hair: Alayna Sullivan at Serendipity Salon – She did all my bridesmaids, and I thought they all looked beautiful. No complaints. She showed up early and was able to get 9 girls done in time for pictures!
Wedding Officiant: Doug Farmer (Charleston Wedding Officiant) – He did a great job.I met with him and felt immediately at ease. He listened to what we wanted and wrote up a ceremony that was exactly what we wanted. It was quick but included everything important to us. I wouldn't have changed a thing.
Transporation: Durham County School Bus – No complaints. They were on time, clean and stuck to the schedule. We were able to bring food and drinks on the transportation (no alcohol).
Wedding Stationary: A. Fitterer Designs – Amanda did an amazing job! I emailed her pictures of invitations, menus and save-the-dates and she was able to recreate similar designs with her special artistic touch! We loved all the stationary and received lots of compliments on the invitations!
Berlins: This was my only vendor I was disappointed in. Allt he groomsmen called in the measurements and picked up their suits from the downtown location where they were supposed to take care of any alterations. They gave everyone the wrong size shirts. We had to run back on the wedding day twice to get correct size shirts. My Husband tried his suit on in the store and told her his suit was too big and she said it looked fine to her. He didn't want to argue so he took it. All I notice in our pictures is how baggy his suit is. Plus they charged all the groomsmen the rate of renting the suit with shoes when we specifically said that we did not need to rent shoes, nor did anyone receive shoes. Would never go back or recommend.
DreamPop Media – I haven't received the final product yet but loved Amanda
& Jeff. They were quick to respond to emails and showed up early to shoot.
They were the cutest couple/team. I felt comfortable with them and didn't feel
like they were intrusive and barely knew they were there. They are affordable
and I can't wait to see the final product. Loved all the videos they have on
their site so I'm sure mine will be just as amazing.


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Zoe Saldana & Shailene Woodley: Armani Show in Paris!
Zoe Saldana and Shailene Woodley arrive at the Giorgio Armani Prive Haute-Couture show as part of Paris Fashion Week at Palais de Chaillot on Tuesday (July 3) in France.
The ladies took in the show from the front row along with Leslie Mann.
New stills of Zoe and her co-star Bradley Cooper from their upcoming flick The Words were recently released. Check them out inside!
The film follows young writer Rory Jansen (Cooper) who finally achieves long sought after literary success after publishing the next great American novel. There's only one catch – he didn't write it.
FYI: Zoe is wearing Jennifer Zeuner jewelery.
15+ pictures inside Zoe Saldana, Shailene Woodley, and Leslie Mann at the Armani show…

subset: commoncrawl


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A word from some of our clients
We're passionate about helping our clients to reach their customers. Below are some of the clients we have had the privilege to work with.
Tyre Fitters. Graphic & Logo Design. Facebook Campaign.
Blaenavon Music Festival. Logo Design and Graphic Design.
Wedding Stationery. Branding, Logo and Graphic Design.
Dog Grooming Service. Graphic & Logo Design.
Indie Band. Logo Design and Website coming soon.


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Hi Everybody!
I'd like to welcome you to the next Wobble Wednesday Challenge! The last winner has been announced, so be sure to check the Action Wobbles Challenge Blog to see if it's you.
This week's theme is:
Awesome Animals
Create any project using this an animal. And of course an Action Wobble :)
Here's the project for today's challenge:
*Silhouette - Wildcat Head
*Jaded Blossom - School Rules
*Prima - patterned paper
*My daughter's school mascot is a wildcat,
so I was excited to find this perfect one in the Silhouette store!
*Action Wobbles add such great dimension!
*Wobbling wildcat!
I hope you liked my project! Now it's your turn! Head on over to the Action Wobbles Challenge Blog to enter this week's challenge.
(Now if you don't have any Wobbles yet, don't worry! There is a special offer for all of you to be able to purchase just a few Wobbles and get in on this challenge. You can purchase a special pack HERE.)


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President Bush announced an agreement with the Russian Federation that will benefit pork producers' bottom lines by increasing the sale of pork products from the U.S. to Russia.
"The Russian Federation has taken numerous actions during the past few years that have negatively impacted U.S. pork producers and others in U.S. agriculture," observes Jon Caspers, president of the National Pork Producers Council. "President Bush committed to resolve these problems and he delivered.
"Our producers were really harmed by Russia's restrictions on U.S. poultry exports. Russia is the U.S. poultry industry's number one export market, and the Russian restrictions backed up the poultry supply in the U.S. and brought down the prices of U.S. pork and live hogs," he remarks. Russian restrictions on U.S. pork imports compounded the problem.
The administration's action sets a country-specific quota that will help shield U.S pork from dumped and subsidized pork exports from Brazil and the European Union to Russia.


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The sea is a passion that carries some risks. One of the most common is boat accidents. Of course, an accident is a situation that has not been foreseen, but it can clearly be avoided. Acting to avoid it becomes a necessity when one knows that some of them are fatal. This site has been designed especially to avoid this situation which is in no way inevitable.
Accidents are not worldly
People think that accidents are situations that are out of their control. Thus, these are special situations that are rare and then become worldly. In the case of boat accidents, do not dress this way. Indeed, accidents are harmful situations and can even become fatal for people who are subject to the accident. In addition, accidents occur because of a lack of control over a given situation. At sea or simply on the water, it is important to take into account all the variables in order to avoid accidents. This will be, after all, a good way to avoid death at sea or just to avoid the inconvenience coming with any injuries. Note that boat accidents affect all water-related activities such as fishing, scuba diving or just hiking at sea.
Avoiding them is an obligation
There are simple gestures that can avoid all types of boat accidents. Of course, many provisions are presently present to really prevent fatal accidents. Only, apart from these provisions, taking these few small steps will save lives significantly. Above all, during the aquatic activities, it is necessary to avoid diving head first. Before embarking on such activities as diving, it is imperative to listen well to the briefings. Of course, it is necessary to be always attentive to what happens around, because the sea is not always an ally. Still diving, after diving into the sea, it will be necessary to move away from the place of fall to avoid being the point of landing of the following person. In case of a glitch, even less, it will be necessary to interrupt the activity in itself.


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Holland Tunnel, twin-tube tunnel under the Hudson River connecting Canal Street in Manhattan, New York City, with 12th and 14th streets in Jersey City, N.J. The tunnel was completed in 1927 and opened for traffic on November 13 of that year. It was named for Clifford M. Holland, the engineer who designed it. The north tube is 8,558 feet (2,608 m) long and the south tube 8,371 feet (2,551 m) long. The roadway is 20 feet (6.1 metres) wide and reaches a maximum depth below mean high water of 93 feet 5 inches (28.5 metres).
Considered a notable engineering achievement because it solved the problem of ventilating a long vehicular tunnel, it has 84 powerful fans capable of replacing air in the tunnel every 90 seconds. The polluted air is drawn off through a duct in the roof of the tube with the aid of suction fans. The system was severely tested on May 13, 1949, when a truck loaded with poisonous and flammable carbon disulfide caught fire in the tunnel and exploded. Despite the destruction of 23 trucks and the ruin of 500 feet of tunnel ceiling, no fatalities resulted.


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With MCM London Comic Con once again setting records, we here at MCM Buzz decided to take a look back at just a few of the many memorable moments, some you may not have even known about, from this past weekend's event.
Bats Amazing!
Following a long trip from LA and over four hours to put together, the life size Batmobile based on the design from Batman Arkham Knight was unveiled to attendees by Catwoman aka Poison Cosplays. Throughout the day this stunning replica drew huge gasps from the crowd and was the deserved background of many selfies and pictures. For more information about this stunning replica check out our post here and watch our video of the reveal below.
Batman: Arkham Knight is released on next gen consoles on June 23rd
Mad about Max
As part of the Mad Max panel on Sunday afternoon, fans in attendance were given a chance to appear in a new trailer for the game thanks to director John Fuller. All they had to do was stand on their feet and chant in unison the name of the game's villian, "Scrotus". The chant was then recorded and would potentially be used in a forthcoming trailer for the new title. It's no surprise that those in attendance rose to their feet and delivered a thunderous chant. Following this Fuller, joined by his Avalanche Studios colleague, Marcus Andrews, then went on to do commentary on 14 minutes of unseen gameplay footage before taking questions from the fans.
Click play below to watch a story trailer for the game, which is released on September 4th on PlayStation 4, Xbox One and PC.
The Truth about our Overlords is out there….
In front of a packed Platinum Suite, Gillian Anderson made her long awaited MCM London Comic Con debut to talk about the British sci-fi movie Robot Overlords. Joined by the movie's director Jon Wright, they discussed everything from the onscreen robots, to working with Sir Ben Kingsley, and of course, inevitably, the forthcoming X-Files series. During one such answer Anderson confirmed that Scully will be having red hair once again. The panel ended with smiles all round when an attendee gifted Anderson with a pair of Funko figures based on Agent Fox Mulder and her character, Agent Dana Scully, which was then followed by a cheeky confession about the effect the actress had on the attendee's life.
DiNozzo is set to vist LA
Sunday saw the stars of NCIS and its spin-off series, NCIS: Los Angeles, take to the stage to talk about their experiences on the hit franchise. The panel featured Emily Wickersham, Bishop from NCIS, alongside two returning NCIS: Los Angeles stars, Barrett Foa (Eric Beale) and Renée Felice Smith (Nell Jones). Amongst the topics discussed in this lively panel were Foa being part of an a cappella group, which then prompted the panelists to do an impromptu performance. There were also discussions about the need for a special NCIS version of the series Lip Sync Battles, which is hosted by their co-star, LL Cool J. But one thing that left the audience salivating was when Emily Wickersham revealed that her NCIS co-star, Michael Weatherly (Very Special Agent Anthony DiNozzo), had recently just finished filming a crossover episode with the NCIS: Los Angles team and that it would air as part of NCIS: Los Angles season seven.
All in fair warned!
The second panel of the Friday in MCM Theatre B proved to be a treat for fans of the series Storage Hunters, when auctioneer Sean Kelly made his MCM London Comic Con debut. In a fascinating discussion he talked about how he developed the concept, how he comes up with the quirky names for the characters on the show and how the UK version came into being. There was also a discussion about the weirdest things Sean had found in lockers, such as an actual murder scene complete with body, and a unit that for seven years had been paid just to house a pot of human urine. One of the most exciting parts of the panel came when Sean revealed that the new season of the UK show will include appearances by stars from the US version of the show, T-Money and Jessie.
In true Storage Hunters fashion the panel ended with a treat for those in attendance as Sean conducted a live auction for fans, in which bidders got the chance to win a signed and personalised t-shirt, a personalised video message/answerphone message/call from Sean to a friend and a package that featured both.
Storage Hunters UK returns to Dave on June 2nd and Sean will also be performing a series of UK stand up comedy dates throughout the rest of 2015.
Will you be my Princess?
The MCM London weekend saw plenty of sweet moments, ranging from babies in cosplay to people being wished happy birthday on the various stages. Our next memorable moment revolves around another of life's big events: marriage.
The Saturday of the event saw a large Disney cosplay photoshoot and our photographer J-Po was on hand to capture the moment when a Cinderella cosplayer, Chelsea-Ann Bicker, was proposed to by her Prince Charming, literally in fact, as her partner, Mark Athos Green, was cosplaying as Prince Charming. See below for the image and on behalf of MCM Buzz we wish this happy couple best wishes and a long happy future together.
Agents of S.H.I.E.L.D. help make an attendees DAY!
Picture this. You attend MCM Comic Con with your girlfriend, you get the last ticket for the Felicia Day photoshoot and decide to keep it for yourself. Understandably your girlfriend is going to be upset. Enter Nick Blood and Iain de Caestecker, who after finding out about this decided to help her, inviting the attendee up for a picture with them there and then. But they also spoke to Felicia Day and helped secure a personalised photo for her, giving this lucky young lady an MCM moment to treasure. It just goes to show that these agents are heroes off camera too. Don't forget, if you missed the Agents of S.H.I.E.L.D. panel on Sunday, or just want to re-live this amusing 45 minutes, then you can watch it by clicking play below.
Well there you have it, just a few of the many memorable moments of MCM London Comic Con. If you have a memorable moment then why not tweet us @MCMBuzz using the hashtag #MCMMoment. You can also post yours on our Facebook page too.
For more coverage check out our pieces on the Tomorrowland panel, Gideon Emery panel, Splatoon hands On, Cyanide and Happiness panel. Also you can watch the sequel to our horror trailer from last October as the ever talented MCM Buzz video team from Southampton Solent University return with the MCM London Comic Con infomercial.
Images provided by: Kay Ibrahim (Storage Hunters panel image), J-Po cosplay (Cinderella Image and NCIS panel image) and Sarah Tsang (Gillian Anderson Panel image).
For more video coverage from the event you can visit our YouTube Channel.


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Hilton is one of the world's largest hotel groups, and has properties in almost every major city and resort destination in the world. The UK based resort management and development arm of the business is responsible for most territories outside the US.
Currently, the company manages a total of 3,800 resorts and hotels worldwide, and many of these include timeshare and residences alongside the main hotel properties that have been specifically designed with the Timeshare audience in mind. Global reach and a household name mean that some 194000 member families have chosen Hilton International Grand Vacations Company for their holiday timeshare.
Hilton International Grand Vacations Company Contact Details
For more information about Hilton International Grand Vacations Company, or to contact them directly for details of their resorts and properties, their contact information is below:
00 44 1339 755858
For the latest news and information about Hilton International Grand Vacations Company from Go Timeshare, please click on the following link:


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Develops the sales plan for designated region of responsibility and upon approval ensures the accurate, effective, and efficient implementation of all sales business operations in order to support the company's business growth and profitability. To optimize sales from both travel trade and consumer direct via relevant channels within the assigned geographical sales area. By utilizing sales data to pro-actively identify any areas for improvement or development and growing the sales business to agreed targets. Ensuring the highest level of customer and client satisfaction and to contribute to departmental compliance with company policies and industry guidelines (USTOA & CLIA).
The BDM is a client/customer facing role and have the ability to be new business hunter, able to self-generate leads, cold call and attend meetings to present to clients/customers and close business through commercial contract negotiations.
• Degree in Business Management or related field from an accredited college or university
• At least 5 years of relevant work experience at management level within the Hospitality and Tourism / Cruising industry
• Must be able to travel a high percentage of the time, hold a valid U.S. passport, and hold a valid driving license
• Ability to speak French a plus
• Account management skills
• Strong negotiation and analytical skills
• Excellent presentation skills
• Communication and interpersonal skills
• Planning, scheduling and forecasting skills
• Problem solving skills
• Knowledge of selling techniques
• High degree of attention to detail


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Another winter blast has arrived in southern Saskatchewan bringing with it a fair amount of snow.
Environment Canada said this system is a continuation from southern B.C. which has resulted in snow falling in Vancouver. The moisture associated with that system has made its way through Alberta and will affect areas in southwest Saskatchewan.
As of Sunday morning, a snowfall warning was in place for areas like Leader, Swift Current, Maple Creek, Shaunavon, and the surrounding communities.
Environment Canada stated the snow started falling Saturday, will continue Sunday and Monday. Up to 30 centimetres – or 12 inches – of snow could fall in some spots by the end of Monday. The sky will clear out Tuesday as a ridge of high pressure builds in the area.
Environment Canada confirmed about 10 cm of snow fell in the Swift Current area Saturday.
The Regina area is not expected to see as much precipitation as the southwest, however flurries are expected throughout Sunday and Monday. In total, as much as 20 cm could be on the ground once the system clears out.
Environment Canada said snowfall could be heavy at times, but it should lighten up on Monday.


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Gents (and ladies ) came across this rather funny photo, although I doubt that the owner of the Bimmer thinks so
Having owned several later model BMW's, I can attest to the lack of quality they appear to exhibit these days
I kinda laughed when my brake MC finally went out 2 years ago, after "only" 35 years of service
The missus current ride, a 2011 F10 538i, amazes me at what has already crapped out or was otherwise needing repair and/or replacement
Case in point, the weather "stripping" under the hood was crumbling, thinking that these strips could be replaced without much ado
But no, the parts counter guy comes out with these 2 HUGE boxes
I figured that can't be right, but sure enough, these weather strips are attached to giant plastic covers and can only be replaced as such
I bit the bullet and picked them up, and no, don't bother asking me how much they set me back
It also occurred to me that since they were in stock, I wasn't the only person replacing them
So keep your old Bimmers up and running fellas
Epic fail - newer BMW "quality"...
Talk women (or men), politics, gripe about your 6er (or other ride), and speak your mind.
Moderators: ron, bfons, GRNSHRK
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3 posts • Page 1 of 1
- E12 Based Guru
- Posts: 2630
- Joined: Thu Apr 08, 2004 3:24 pm
- Location: Sunnyvale (SF Bay Area) CA
Epic fail - newer BMW "quality"...
- Attachments
- Lock-N-Key.jpg (60.98 KiB) Viewed 1080 times
1980 633 CSi
Cypress Green/Pearl Beige
2011 F10 528i
2005 E46 330 Ci ZHP Cabrio
You can never have too many BMW's
- Bigcoupe supporter
- Posts: 3903
- Joined: Tue Mar 16, 2010 8:32 pm
- Location: usually in the barn, Kent, England.
Re: Epic fail - newer BMW "quality"...
Doesn't surprise me. I bought a new front bumper blade for my M635CSi about ten years ago. It went rusty within a few weeks. I returned it to my local Main Dealer (now gone bust) for replacement. That one went rusty as well and I was told that BMW quality control isn't what it once was and that any further replacement was a waste of time
Re: Epic fail - newer BMW "quality"...
It's like they're trying to experiment on their already enamored clientele. Plastic water pump impellers, complex VANOS mechanisms, 15,000 miles oil changes. http://f30.bimmerpost.com/forums/showth ... p?t=868291
I'll be quiet now.
I'll be quiet now.
Karmann, German for extra rusty.
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This article is a collaboration between MedPage Today® and:
In general, the diet of U.S. children improved between 1999 and 2012, but it still remains poor, researchers reported.
The mean Healthy Eating Index-2010 (HEI-2010) scores, which measured dietary quality, significantly improved in children and adolescents from 42.5 in 1999 (95% CI 41.2-43.8) to 50.9 in 2012 (50.0-51.8, (P<0.001 for linear trend), according to Xiao Gu, a graduate student at Brown University School of Public Health in Providence, R.I., and Katherine L. Tucker, PhD, of the University of Massachusetts in Lowell.
While there were gains in consumption of whole grains and fruit, as well as a significant drop in empty calorie intake, the HEI-2010 component score for sodium decreased significantly, which reflected an increase in sodium consumption, they wrote in the American Journal of Clinical Nutrition.
"The average score for whole grains is only 2, which is far below its maximum of 10, even though we observed a significant increasing trend," Gu explained in a press release. "For whole fruit the optimal is 5 but the average we observed is 2.1. I think the increasing trend is encouraging but the current dietary quality level is disappointing."
Also, there was notable socioeconomic and ethnic disparity, with African-American children having the lowest reported mean HEI-2010 value 48.4 in 2012 (95% CI 46.9-49.9, P<0.001 for linear trend).
Dietary behaviors in youth can carry the "life-long risk of chronic disease," such as type 2 diabetes and obesity, the authors pointed out.
"Dietary quality is a major problem in the U.S. among children, and much effort is being put into improving food choices. We wanted to see if progress has been made over time," Tucker explained in an interview with MedPage Today.
In an email to MedPage Today, Gu added that he and Tucker wanted to "see if there are any disparities in dietary quality among different socioeconomic and racial/ethnicity groups," particularly in evaluating the outcomes of the nutritional assistance programs.
Using data from the National Health and Nutrition Examination Survey, they examined a cohort of 38,487 participants, ages 2 to 18 years. Data from a 24-hour diet recall was collected through interviews. Subgroups analyzed in the study included individuals on four various federal nutrition assistance programs: Supplemental Nutrition Assistance Program (SNAP), Special Supplemental Nutrition Program for Women, Infants, and Children (WIC), the National School Lunch Program (NSLP), and the School Breakfast Program (SBP).
The HEI-2010 model used for measuring diet quality adhered to the 2010 Dietary Guidelines for Americans, measuring for 12 various food groups, and three moderation areas, which analyzed empty calorie intake, as well as refined grains and sodium.
In addition to a reduction of empty calorie intake, the researchers reported a significant increase in consumption from 1999 to 2012 in foods such as whole grains, dairy, whole and total fruit, seafood and plant protein, greens and beans, and fatty acids.
Sodium intake was the only HEI-2010 score that did not improve during the study period, dropping by about 0.2 mean points, the authors noted.
"The major improvements in dietary quality have been in reducing empty calories, like soft drinks, which is great," Tucker told MedPage Today. "This needs to continue as well as improving intakes of vegetables, whole grains, and dairy products. Reducing sodium intake is another important goal."
When comparing the participants on a federal nutrition assistance program, the lowest diet quality was reported among the NSLP and SBP participants, while the highest diet quality was seen in the WIC cohort.
However, the authors cautioned that they could not determine a causal effect of federal nutritional assistance programs on dietary quality outcomes due to the study design.
Despite the gains in overall diet quality since 1999, there is still much room for improvement. "To achieve this will require continued efforts in nutrition education and improvements in food programs," Tucker said. "School meals are a key place to continue this effort. Other considerations include expanding taxes on soft drinks, and limiting the food purchases allowed with SNAP."
Gu suggested education is key to healthy food choices in children, adding that "parents ought to be careful about the healthfulness of the foods, snacks, and beverages they choose," because they "play an essential role in forming children's dietary behavior."
Study limitations included the potential lack of accuracy of the 24-hour dietary recall method.
Tucker said follow-up studies are planned "to examine the specific food group intake changes in relation to subsets of the population."
Gu and Tucker disclosed no relevant relationships with industry.
- Reviewed by Henry A. Solomon, MD, FACP, FACC Clinical Associate Professor, Weill Cornell Medical College and Dorothy Caputo, MA, BSN, RN, Nurse Planner
Primary Source
American Journal of Clinical NutritionSource Reference: Gu X and Tucker KL "Dietary quality of the US child and adolescent population: trends from 1999 to 2012 and associations with the use of federal nutrition assistance programs" Am J Clin Nutrition 2016; DOI: 10.3945/ajcn.116.135095.


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A > ACT > Apricot Portable
mini forum is intended to provide a simple means of discussion
about the ACT Apricot Portable computer. If you want to share your own experience or memories,
or add relevant information about this system: post a message!
For other purposes like sales messages, hardware & software
questions or information requests, please use our main
I have an old manual for the apricot portable if anyone wants it . firstname.lastname@example.com
My dad worked at apricot from the early 80�s until there demise, and I�ve inherited many portable apricots also many prototypes of most models endless amounts.I�m looking to move them on to an enthusiast that will appreciate them Thanks Matt
Hello,I''''m looking for the original Apricot mic for my act portable, if someone has one to sell please contact me.
Got the portable, in special case, mouse, manuals, discs etc. complete. Want an offer or what have you?Roy
The Portable/FP was only ever available with an 8086 and strangely will not support the NEC V20 as a $-in speed upgrade. The systems originally selling with only 256kb of ram had the black finish and the better 512kb systems came in white. You also can not repopulate the system board (512kb units came with half their banks filled) with an additional 512kb of ram, thus maxing the machine out, and get the memory to immediately be detected by the BIOS. The service manual also does not state how exactly you get around this, so that nibble of info might be lost to time.The floppy drive (Sony OA-D32W) is a twin head drive unlike the OA-D32V and thus works with double-sided diskettes.The only truly awful design issues with the system was the glossy plastic over the LCD which makes for a near mirror finish (and incredible glare) and the absolutely dreadful IBM text mode emulation which all Apricots had up until the XEN 386.
Didn''t the apricot portable use a 80186 cpu?
I still have mine and its in working order. It was a wonderfull machine and I learnt word processing and spread sheets on it. It was well ahead of its time and has to be one of the best of its time.
The specs on this page say it''s a 8086 processor. Mine has a Z80 processor. Also the Sony diskette drive was blocked. The grease used in those days turns into solid glue after so many years. Had to use solvent to make the thing move again. In the text at the top it says single sided 720k and in the specs at the bottom it says 720k double sided. It is 720k and 720k is always double sided as far as I know. To (dis)assemble the unit is a bit of a where to start and where to finish puzzle. Unfortunately have not been able to get it to work so far. No display and the diskette drive just starts the motors (spinning and head movement) and never stops. Any suggestions?
This machine, released over 25 years ago, was very early on several new tecnologies$ LCD display, GUI, infrared connection and speech recognition. SuperWriter and Supercalc both worked very well. And, of course, I''ve still got mine, in perfect condition!
I also bought an Apricot portable from Dixons, although the Voice Recognition was almost useless the fact it came with SuperCalc 5 rescued it from being a complete waste. Although Apricot hardware was not 100% PC compatible it gave me a cheap (�500) PC when I wanted to move from ZX81's and Atari 400's and see what the real world was like.I remember I quite liked the infra-red keyboard and it came with a fibre optic "light pipe" in case the sun was on the sensor or objects got in the way. Later I got hold of some PC emulatin software which allowed me to run Procomm on it and it became quite a usefull tool.
This computer is bizarre, fantastic, odd & brilliant all at the same time. Way ahead of its time, in more sense than one - and check that case design! I would love to own one of these one day, however the chances of that are highly remote :-(
I bought a second hand Apricot portable around 1985 or 1986 and wrote lots of articles on it. The screen was not very good, but the voice recognition and wireless keyboard were way ahead of its time. Sadly, I traded the unit in for an IBM compatible pc with more memory and a CRT screen, so I don't have it anymore. Haven't seen one since, except for on this excellent site.
Apricot Portable is the most beautifull portable PC I've ever seen. Way ahead of it's time in the mid 80's


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About Us
Mud Foods, or "Mud Pies" as we have been nicknamed, is a small family company based in West Sussex.
We produce delicious, wholesome and unashamedly tasty pies, pasties, sausage rolls and squiches (that's square quiches to the uninitiated!) made from the finest quality fresh, free-range and local ingredients.
Christian Barrington (Chief Pie Maker and Inventor) and his lovely wife, Toni, are the husband and wife team behind Mud Pies. Their aim is to bring tasty, gourmet pies to a farmers' market near to you for the ultimate homely, wholesome and delicious pie experience.
Inspired by a fond childhood memory of his mother and grandmother making homemade pies and disillusioned by a fairly dull work life in sales, Christian set about creating his very own pie company. Armed with a passion for tasty pies, a dream and mere fifty pie tins, Christian started producing his first batch of pies in a small unit in Chichester. One man and his pies!
More than 5 years later, Christian and Toni, have moved to larger premises in Midhurst. They now produce over 5,000 pies per week as well as pasties, sausage rolls and squiches based on family recipes and using delicious locally grown, reared or produced ingredients.
Their secret? A 'butter rich' pastry that is 50% thinner than the standard pie but ssshh, don't tell anybody!
Home is where the pie is!
Mud Foods is proud to have an array of Great Taste Awards to their name. In 2017, we have been awarded 8 Great Taste Awards, which is a phenomenal achievement. The awards had 12,366 products entered of which only 35% receive an award but we achieved an 80% success rate with judges comments such as:
"Judges loved this pie and could easily have finished off the lot. Good pastry, plenty of filling, distinct pieces of tender meat and kidney - we would buy it. Please make more"
"Clearly good quality ingredients used"
"Home-made with a touch of finesse"
"generous filling, with a good level of chunky meat and a decent gravy"
At the British Pie Awards this year we were crowned 'Champions' for our Game Pie and in addition we won Gold for our Steak & Ale Pie, and Silver for our Lamb & Rosemary Pie and our Chip Shop Chicken Curry Pie.
In addition to these fantastic awards, the much acclaimed, and their most important one to date, as voted for by customers, Hampshire Farmers' Market Producer of the Year 2010 and Product of the Year 2013.
These annual awards are like the 'Oscars' for the county's food and drink producers and Mud Foods beat off over 100 local producers from bakers and butchers to artisan cake and cheese makers to land the prize. It just goes to show - a dream, a childhood memory and talent for gourmet food and... the pie is the limit!
Ahh, the life of pie!
Where to Buy
Regular Markets
Alton Farmers' Market 2nd Saturday of each month
Chichester Farmers' Market 1st and 3rd Friday of each month
Elvetham Heath Farmers' Market 2nd Saturday of each month
Emsworth Farmers' Market 3rd Saturday of each month
Farnham Farmers' Market 4th Sunday of each month
Guildford Farmers' Market 1st Tuesday of each month
Ham Parade Farmers' Market 1st Saturday of each month
Kew Village Market 1st Sunday of each month
Petersfield Farmers' Market 1st Sunday of each month
Petworth Farmers' Market 4th Saturday of each month
Ringwood Farmers' Market 4th Saturday of each month
Romsey Farmers' Market 1st Sunday of each month
Shoreham Farmers' Market 2nd Saturday of each month
Southsea Farmers' Market 3rd Sunday of each month
Winchester Farmers' Market 2nd and last Sunday of each month
Forthcoming Foodie Events
Alresford Watercress Festival
20th May 2018
Surrey County Show
28th May 2018
Fleet Food Festival
23rd and 24th June 2018
New Forest Show
24th/25th/26th July 2018
Trout 'n' About
5th August 2018
27th August 2018
Alresford Agricultural Show
1st September 2018
Hamble Food Festival
1st September 2018
Farnham Food Festival
30th September 2018
Farnham Christmas Market
9th December 2018
Upcoming Markets
Saturday 26th May 2018
Petworth Farmers' Market 9am-1pm
Ringwood Farmers' Market 10am-2pm
Friday 1st June 2018
Chichester Farmers' Market 9am-2pm
Saturday 2nd June 2018
Ham Farmers' Market 9am-2pm
Sunday 3rd June 2018
Petersfield Farmers' Market 10am-2pm
Romsey Farmers' Market 10am-2pm
Kew Village Market 10am-2pm
Tuesday 5th June 2018
Guildford Farmers' Market 9am-3pm
Saturday 9th June 2018
Elvetham Heath Farmers' Market 10am-2pm
Shoreham Farmers' Market 10am-2pm
Sunday 10th June 2018
Winchester Farmers' Market 9am-2pm
Friday 15th June 2018
Chichester Farmers' Market 9am-2pm
Saturday 16th June 2018
Alton Farmers' Market 9am-2pm
Emsworth Farmers' Market 9am-2pm
Sunday 17th June 2018
Southsea Farmers' Market 9am-2pm
Saturday 23rd June 2018
Petworth Farmers' Market 9am-1pm
Sunday 24th June 2018
Farnham Farmers' Market 9am-2pm
Winchester Farmers' Market 9am-2pm
(opposite Jigsaw in the High Street)
Saturday 30th June 2018
Ringwood Farmers' Market 10am-2pm
Find us elsewhere...
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Pigs can't fly... but our pies do!
Wholesale & Trade Enquiries
If you are retailer, hotelier, pub landlord or local farm shop, you may be interested in selling our tasty pies! The Chief Pie Maker welcomes all manner of wholesale and trade enquiries so please get in touch to discuss your requirements...
Those pies are moo-vin', moo-vin'!
We are always on the look out for budding pie makers and sellers! If you have a passion for tasty pies and thrive working in a busy foodie environment then our Chief Pie Maker would love to hear from you...
Send us a message!
Mud Pie HQ
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Process to create biomolecule and/or cellular arrays on metal surfaces and product produced thereby
Process to create biomolecule and/or cellular arrays on metal surfaces and product produced thereby
Patent Drawings:
(7 images)
Corn, et al.
Date Issued:
October 3, 2000
August 5, 1999
Brockman; Jennifer M.
(Madison, WI)
Corn; Robert M.
(Madison, WI)
Frutos; Anthony G.
(Big Flats, NY)
Wisconsin Alumni Research Foundation
(Madison, WI)
Primary Examiner:
Campbell; Eggerton A.
Assistant Examiner:
Lundgen; Jeffrey S
Attorney Or Agent:
DeWitt Ross & Stevens S.C.
U.S. Class:
Field Of Search:
International Class:
U.S Patent Documents:
Foreign Patent Documents:
0 305 108
Other References:
Pale-Grosdemage et al., "Formation of Self-Assembled Monolayers by Chemisorption of Derivatives of Oligo(ethylene glycol) of StructureHS(CH2)11(oCH2CH2)mOH on Gold" J. Am. Chem. Soc. 113, pp. 12-20, 1991..
Brockman et al., "A Multistep Chemical Modification Procedure to Create DNA Arrays on Gold Surfaces for the Study of Protein-DNA Interactions with Surface Plasmon Resonance Imaging" J. Am. Chem. Soc. 121, pp. 8044-8051, 1999..
Frutos, A. G. et al, "Demonstration of a Word Design Strategy for DNA Computing on Surfaces," Nucleic Acids Research 25:4748-4757 (1997)..
Frutos, A. G. et al, "Enzymatic Ligation Reactions of DNA "Words" on Surfaces for DNA Computing," J. Am. Chem. Soc. 120:102777-10282 (1998)..
Gross, C. A. et al, "Application of (3-Mercaptopropyl)trimethoxysilane as a Molecular Adhesive in the Fabrication of Vapor-Deposited Gold Electrodes on Glass Substrates," Anal. Chem. 63:85-88 (1991)..
Jordon, C. E. et al, "Surface Plasmon Resonance Imaging Measurements of Electrostatic Biopolymer Adsorption onto Chemically Modified Gold Surfaces," Anal. Chem. 69:1449-1456 (1997)..
Jordan, C. E. et al, "Surface Plasmon Resonance Imaging Measurements of DNA Hybridization Adsorption and Streptavidin/DNA Multilayer Formation at Chemically Modified Gold Surfaces," Anal. Chem. 69:4939-4947 (1997)..
Knobloch, H. et al, "Dispersive Surface Plasmon Microscopy for the Characterization of Ultrathin Organic Films," Appl. Phys. Lett. 69:2336-2337 (1996)..
Lideberg, B. et al, Surface Plasmon Resonance for Gas Detection and Biosensing, Sensors and Actuators, 4:299-304 (1983)..
Pease, A. C. et al, "Light-Generated Oligonucleotide Arrays for Rapid DNA Sequence Analysis," Proc. Natl. Acad. Sci. 91:5022-5026 (1994)..
Thiel, A. J., et al, "In Situ Surface Plasmon Resonance Imaging Detection of DNA Hybridization to Oligonucleotide Arrays on Gold Surfaces," Anal. Chem. 69:4948-4956 (1997)..
Thomas, R. C. et al, "Probing Adhesion Forces at the Molecular Scale," J. Am. Chem. Soc. 117:3830-3834 (1995)..
Winzeler, E. A. et al, "Direct Allelic Variation Scanning of the Yeast Genome," Science 281:1194-1197 (1998)..
Zizlsperger, M. et al, "Multispot Parallel On-Line Monitoring of Interfacial Binding Reactions by Surface Plasmon Microscopy," Progr. Colloid. Polym. Sci. 109:244-253 (1998)..
Disclosed is a process to construct multi-component biomolecule or cellular arrays suitable for use in SPR imaging studies of large molecule, cellular/molecular, and cell/cell interactions. The success of the procedure hinges on the use of a reversible protecting group to modify reversibly .omega.-functionalized alkanethiols self-assembled on metal substrates.
What is claimed is:
1. A method of making a biomolecule or cellular array on a metal substrate comprising:
(a) depositing an .omega.-modified alkanethiol monolayer on a metal substrate;
(b) reacting hydrophobic protecting groups with the monolayer;
(c) patterning the monolayer to create an array of exposed metal substrate areas;
(d) depositing .omega.-modified alkanethiol in the areas of exposed metal substrate, thereby yielding an array of discrete, unprotected .omega.-modified alkanethiol spots;
(e) attaching biomolecules or cells to the discrete, unprotected .omega.-modified alkanethiol spots, thereby yielding an array of discrete spots having biomolecules or cells immobilized thereon;
(f) removing the protecting groups of step (b); and then
(g) making the monolayer resistant to non-specific protein binding wherein the method results in a biomolecule or cellular array.
2. The method of claim 1, wherein in step (a), an amino-C.sub.8 -C.sub.24 -alkanethiol is deposited on the metal substrate.
3. The method of claim 1, wherein in step (a), 11-mercaptoundecylamine is deposited on the metal substrate.
4. The method of claim 1, wherein in step (b), 9-fluorenylmethoxycarbonyl-N-hydroxysuccinimide is reacted with the monolayer.
5. The method of claim 1, wherein in step (c), the monolayer is patterned by selective exposure to UV radiation.
6. The method of claim 1, wherein in step (d), 11-mercaptoundecylamine is deposited in the areas of exposed metal substrate.
7. The method of claim 1, wherein in step (e), nucleic acid molecules are attached to the discrete, unprotected .omega.-modified alkanethiol spots using a bifunctional linker.
8. The method of claim 1, wherein in step (e), DNA molecules are attached to the discrete, unprotected .omega.-modified alkanethiol spots.
9. The method of claim 1, wherein in step (e), RNA molecules are attached to the discrete, unprotected .omega.-modified alkanethiol spots.
10. The method of claim 1, wherein in step (e), a heterobifunctional linker is used.
11. The method of claim 10, wherein in step (e), sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate is used as a linker.
12. The method of claim 1, wherein in step (f), the protecting groups are removed by treating the monolayer with a secondary amine.
13. The method of claim 12, wherein in step (f), the protecting groups are removed by treating with a solution selected from the group consisting of tris(2-aminoethyl)amine, ethanolamine, and piperidine.
14. The method of claim 1, wherein in step (g), the monolayer is made resistant to non-specific protein binding by attaching poly(ethylene glycol) moieties thereto.
15. The method of claim 1, wherein in step (a) the .omega.-modified alkanethiol monolayer is deposited on a gold substrate.
16. The method of claim 15, wherein in step (a), an amino-C.sub.8 -C.sub.24 -alkanethiol is deposited on the gold substrate.
17. The method of claim 15, wherein in step (a), 11-mercaptoundecylamine is deposited on the gold substrate.
18. The method of claim 15, wherein in step (b), 9-fluorenylmethoxycarbonyl-N-hydroxysuccinimide is reacted with the monolayer.
19. The method of claim 15, wherein in step (c), the monolayer is patterned by selective exposure to UV radiation.
20. The method of claim 15, wherein in step (d), 11-mercaptoundecylamine is deposited in the areas of exposed gold substrate.
21. The method of claim 15, wherein in step (e), nucleic acid molecules are attached to the discrete, unprotected .omega.-modified alkanethiol spots using a bifunctional linker.
22. The method of claim 15, wherein in step (e), a heterobifunctional linker is used.
23. The method of claim 22, wherein in step (e), sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate is used as a linker.
24. The method of claim 15, wherein in step (f), the protecting groups are removed by treating the monolayer with a secondary amine.
25. The method of claim 15, wherein in step (g), the monolayer is made resistant to non-specific protein binding by attaching poly(ethylene glycol) moieties thereto.
The invention is directed to the fabrication of biomolecular or cellular arrays on metal surfaces for use in the study of interactions between large molecules, between cells and large molecules, and between cells, such as nucleic acid-proteininteractions or cellular interactions with antigens.
The binding of proteins to DNA plays a pivotal role in the regulation and control of gene expression, replication and recombination. In addition, enzymes that recognize and modify specific oligonucleotide sequences are critical components ofbiological nucleic acid manipulation and repair systems. An enhanced understanding of how these proteins recognize certain oligonucleotide sequences would aid in the design of biomedical systems which could, for example, be used to regulate theexpression of therapeutic proteins. For this reason, the study of protein-nucleic acid interactions (i.e., protein-DNA and protein-RNA interactions) is a rapidly growing area of molecular biology, aided in part by recent advances in NMR and X-raystructural determination methods. At the same time, the explosive increase in the amount of available genomic and extra-genomic (i.e., ribosomal) sequence information obtained from large-scale nucleic acid sequencing efforts creates a need to surveythis vast amount of new sequence data for protein binding sites. The present invention addresses this need by using surface plasmon resonance (SPR) imaging techniques as a rapid and efficient method for screening the sequence or structure-specificbinding of proteins to large arrays of nucleic acid molecules immobilized at chemically-modified metal surfaces.
Arrays of DNA molecules attached to planar surfaces are currently employed in hybridization adsorption experiments to sequence DNA, Pease et al. (1994) Proc. Natl. Acd. Sci. USA 91:5022-5026; to screen for genetic mutations, Winzeler et al.(1998) Science 281:1194-1197: and in DNA computing applications, Frutos et al. (1997) Nucleic Acids Res. 25:4748-4757; and Frutos et al (1998) J Am. Chem. Soc. 120:10277-10282. These arrays are exposed to solutions containing fluorescently labeledcomplementary DNA sequences, rinsed, and then "read-out" using fluorescence imaging methods.
The technique of surface plasmon resonance (SPR) is a surface-sensitive, optical detection method well suited to the monitoring of reversible, protein-nucleic acid interactions. The commercially successful "BIAcore" SPR instrument (Biacore AB,Uppsala, Sweden) has been used previously, for example, to study the interaction of DNA molecules with various enzymes. Although powerful, the "BIAcore" instrument has no imaging capabilities. This severely limits the number of DNA sequences that canbe screened in a single experiment.
Surface plasmon resonance (SPR) is a surface optical technique which is sensitive to the thickness and index of refraction of material at the interface between a free electron metal (e.g. gold, silver, copper, cadmium, aluminum) and a bulkmedium, such as air or water. Surface plasmon resonance may be achieved by using the evanescent wave which is generated when a laser beam linearly polarized parallel to the plane of incidence impinges onto a prism coated with a thin metal film. Themetal may also be coated onto a thin transparent substrate such as glass, and this glass brought into optical contact with the prism. SPR is most easily observed as a reduction of the total internally reflected light just past the critical angle of theprism. This angle of minimum reflectivity (denoted as the SPR angle) shifts to higher angles as material is adsorbed onto the metal layer. The shift in the angle can be converted to a measure of the thickness of the adsorbed or added material by usingcomplex Fresnel calculations and can be used to detect the presence or absence of materials on top of the metal layer.
In using SPR to test for biological, biochemical, or chemical substances, a beam of light from a laser source is directed through a prism onto a biosensor consisting of a transparent substrate, usually glass, which has one external surfacecovered with a thin film of a noble metal, which in turn is covered with an organic film that interacts strongly with an analyte, such as a biological, biochemical, or chemical substance. The organic film can contain substances, such as antibodies orantigens, which can bind with an analyte in a sample to cause an increased thickness which will shift the SPR angle. By monitoring either the position of the SPR angle or the reflectivity at a fixed angle near the SPR angle, the presence or absence ofan analyte in the sample can be detected. Various types of equipment for using SPR with a biosensor for biological or biochemical or chemical substances are described by the Liedberg et al. article found in "Sensors and Actuators," Vol. 4, 1983, page299. See also European Patent Application 0 305 108 and U.S. Pat. No. 5, 374, 563.
The use of conventional SPR as a testing tool offers several advantages and disadvantages. For example, it is relatively fast, it requires no labeling, and it can be performed on site. However, as noted above, commercially-available devices,such as the "BIAcore" instrument, offer no imaging capabilities. Additionally, to achieve the high through-put demanded by large-scale users, there is a need for a simple, practical biosensor which can be readily modified or adapted to test a widevariety of compounds simultaneously.
In SPR imaging, a light source (typically a HeNe laser) is used to illuminate a prism/thin gold film sample assembly at an incident angle that is near the SPR angle, and the reflected light is detected at a fixed angle with a CCD camera toproduce an SPR image. The SPR image arises from variations in the reflected light intensity from different parts of the sample; these variations are created by any changes in organic film thickness or index of refraction that occur upon adsorption ontothe modified gold surface. Since SPR imaging is sensitive only to molecules in close proximity to the surface (within.about.200 nm), unbound molecules remaining in solution do not interfere with in situ measurements.
The formation of robust, reproducible arrays of oligonucleotides tethered to metal-coated surfaces (most often gold) is an essential requirement for SPR imaging of protein-nucleic acid binding interactions. To use SPR imaging techniques, it isessential that the nucleic acid array be constructed on a noble metal surface, and for this reason DNA arrays on glass supports from commercially available sources such as Affymetrix (Santa Clara, Calif.) are not a viable option. Using self-assembledmonolayers of substituted alkanethiols as a starting point, others have previously developed schemes to attach single-stranded DNA molecules to chemically modified gold surfaces. See, for instance, U.S. Pat. No. 5, 629,213). In the subject invention,however, UV photopatterning and microcontact printing techniques are brought to bear to allow alkanethiols to be assembled in a site-directed manner on the metal surface, thereby enabling the creation of multi-component arrays. A combination of theseprocessing techniques along with novel surface chemical reactions enables the manufacture of nucleic acid arrays as described herein.
Disclosed is a multi-step chemical modification procedure to create biomolecule and/or cellular arrays on metal substrates, the arrays being specifically tailored for the study of biomolecular and cellular interactions using surface plasmonresonance imaging. Arrays fabricated by this procedure meet three specific requirements, namely (i) the biomolecules are covalently attached to the surface and remain active and accessible to hybridization and protein binding; (ii) the array backgroundis, at first, sufficiently hydrophobic so as to allow for the "pinning" of aqueous solutions of biomolecules or cells at specific array locations; and (iii) the final array background acts to inhibit the non-specific binding of protein molecules to thesurface. The key components of this fabrication scheme are the utilization of a reversible hydrophobic protecting group, preferably Fmoc, to control the surface hydrophobicity of a tethered .omega.-modified alkanethiol monolayer and the attachment of apoly(ethylene glycol) (PEG) group to render the surface protein resistant. Polarization-modulation Fourier Transform infrared (PM-FTIR) spectroscopy, contact angle, and SPR measurements are used to characterize each step in the surface modificationprocedure and confirm that the array background inhibits the non-specific binding of proteins. As a final test, an SPR imaging experiment which measures the adsorption of single-stranded DNA binding protein (SSB) to a dual component, oligonucleotidearray demonstrates the utility of these surfaces for the monitoring of protein-nucleic acid interactions.
The multi-step procedure disclosed herein is used to create an array of spots that are surrounded first by a hydrophobic background which allows for the pinning of aqueous biomolecule or cell solutions onto individual array elements and then toreplace the hydrophobic background with one that resists the non-specific adsorption of proteins during in situ SPR imaging measurements, thereby yielding an array of biomolecule or cell "islands" in a "sea" which resists non-specific adsorption ofproteins.
In the preferred embodiment, amine-terminated alkanethiol monolayers are employed as the base layer, and Fmoc and PEG modifiers are used to create the sequentially hydrophobic and protein adsorption-resistant surfaces, respectively. In thepreferred embodiment, the chemical modification steps are: (i) the adsorption and self-assembly of an 11-mercaptoundecylamine (MUAM) monolayer on an evaporated gold thin film; (ii) the reaction of the MUAM monolayer with an Fmoc protecting group tocreate a hydrophobic surface; (iii) the photopatterned removal of the alkanethiol followed by (iv) the re-adsorption of MUAM to create an array of MUAM squares (approximately 750 .mu.m.times.750 .mu.m, although smaller or larger squares are attainable)surrounded by a hydrophobic MUAM-Fmoc background that can pin drops of aqueous solution; (v) the attachment of oligonucleotide sequences onto the MUAM squares by the reaction of the amine-terminated surface with the heterobifunctional cross-linker(preferably SSMCC), followed by a coupling reaction to a small volume (0.1 .mu.L) of thiol-modified DNA; (vi) the removal of the Fmoc protecting group followed by (vii) a pegylation reaction of the MUAM with PEG-NHS to create a proteinadsorption-resistant background.
A combination of polarization-modulation FTIR spectroscopy, contact angle and scanning angle SPR measurements are used to characterize the surface modification procedure. An SPR imaging measurement of the adsorption of single-stranded DNAbinding protein (SSB) onto an oligonucleotide array created by this procedure is used to demonstrate the utility of these surfaces to probe nucleic acid interactions with protein and other analytes.
A primary advantage of the subject invention is that it allows an array of immobilized biomolecules or cells to be constructed in which each "island" of bound molecules or cells may differ from the other islands in the array. This allows formassive and simultaneous analysis of a tremendous number of different molecules or cells for their individual affinities and/or binding characteristics to a selected analyte. The fabrication method described herein is well-suited to automation and SPRexperiemnts can be analyzed using standard-format microtiter plates and lab automation equipment (i.e., 96-well, 384-well, and larger formats).
The arrays described herein are useful for any number of analyses wherein a biomolecule or cell interacts with a protein, antigen, or some other molecule, such as in determining binding affinities, epitope mapping, restriction site mapping,measuring the binding effects of short-range secondary structure in nucleic acids, etc. For example, by building an array wherein islands of nucleic acids differ systematically, as by length or primary sequence, the interactions of any given nucleic acidsequence
for any given analyte can be quickly and exhaustively investigated. Likewise, the effects of short-range secondary structure in nucleic acids can be investigated by building an array wherein the islands of nucleic acids differ in sequence suchthat the islands contain nucleic acid sequences which progressively contain more stable secondary structures and then scanning the array after exposure to a given analyte.
FIG. 1 is a schematic representation of a fabrication scheme for constructing multi-element DNA arrays according to the present invention. A clean gold surface is reacted with an amine-terminated alkanethiol and subsequently reacted with aprotecting group to create a hydrophobic surface. This surface is then exposed to UV radiation through a quartz mask and rinsed with solvent to remove the alkanethiol+protecting group from specific areas of the surface, leaving bare gold pads. Thesebare gold areas on the sample surface are filled in with the alkanethiol, resulting in an array of alkanethiol pads surrounded by a hydrophobic protecting-group background. Solutions of nucelic acid are then delivered by pipette onto the specific arraylocations and are covalently bound to the surface via a bifunctional linker. In the final two steps, the protecting groups on the array background are removed and replaced by functional groups which prohibit the non-specific binding of analyte proteinsto the background.
FIG. 2 depicts a surface reaction scheme showing the steps involved in the reversible modification of the array background. The starting amine-terminated alkanethiol surface is reacted with the Fmoc-NHS protecting group to form a carbamatelinkage thus creating a hydrophobic Fmoc-terminated surface. After nucleic acid immobilization, the surface is deprotected, resulting in the return of the original alkanethiol surface. In the final array fabrication step, the deprotected alkanethiolsurface is reacted with PEG-NHS to form an amide bond which covalently attaches PEG to the array surface (to prevent non-specific binding of analyte proteins to the background).
FIG. 3 depicts PM-FTIRRAS spectra in the mid-IR region for the surfaces involved in the array background modification. (A) The starting MUAM surface. (B) After reaction with Fmoc-NHS, bands indicative of the carbamate linkage and the Fmoc ringstretch appear in the spectrum. (C) The surface is deprotected and reverts back to the MUAM surface as evidenced by the similarities between spectra A and C. (D) After reaction with PEG-NHS, bands indicative of the amide linkage as well as thoseassociated with the ethylene glycol groups are present.
FIG. 4 depicts a surface reaction scheme showing the steps involved in immobilizing biomolecules, in this case DNA, to the array locations. A bifunctional linker, such as SSMCC, is used to link thiol-modified DNA to the MUAM pads.
FIG. 5 depicts a series of line profiles showing in situ hybridization and the adsorption of single stranded DNA binding protein (SSB) onto a dual component DNA array containing oligonucleotide sequences D1 and D2. The solid line is the percentreflectivity measured for the starting surface composed of alternating DNA probe spots D1 and D2. The dashed line is the % R measured after exposing the surface to a solution containing the complement to D2. Apparent is an increase in % R at positionD2 upon binding of the complementary DNA sequence. The dot-dashed line is the % R measured after exposing the surface to a 200 nM solution of SSB. While measurable binding did occur at array location D2 (which contained double stranded DNA), theprotein clearly bound more abundantly to the single-stranded sequence D1.
FIG. 6 depicts an in situ SPR difference image showing the binding of single stranded DNA binding protein (SSB) to a checkerboard array of single- and double-stranded oligonucleotide sequences. Images collected immediately before and afterexposure of the surface to SSB were subtracted to produce the image shown. Significant binding of the protein to array locations with covalently bound single-stranded DNA sequences occurred, whereas very little binding occurred at the array locationswhich contained double-stranded DNA sequences.
FIG. 7 depicts a series of line profiles showing the in situ adsorption of bovine serum albumin (BSA) onto a patterned C.sub.18 /MUAM+PEG surface. The solid line is the % R measured for a surface array of 350 mm spots of C.sub.18 surrounded by apegylated MUAM background. The dotted line is the % R measured after exposing the surface to a 1 mg/mL solution of BSA. The much lower change in % R for the MUAM-PEG regions indicates that the pegylated background is much more efficient than C.sub.18in resisting the non-specific binding of the BSA.
Abbreviations and Commercial Suppliers
The following abbreviations and terms are used throughout the specification and claims. All other terms have their standard, accepted meaning in the relevant art. "biomolecule"=any molecule found in biological material, expressly including, butnot limited to nucleic acids, proteins, peptides, antibodies, enzymes, cell-wall components such as phospholipids, etc., and modified and synthetic forms thereof, such as labeled biomolecules and recombinant biomolecules.
"BSA"=bovine serum albumin (Sigma Chemical, St. Louis, Mo.).
"Fmoc-NHS"=9-fluorenylmethoxycarbonyl-N-hydroxysuccinimide (Novabiochem, La Jolla, Calif.).
"metal substrate" or "metal film"=a noble-metal thin film (gold, silver, copper, platinum, etc.). Gold is preferred.
"MUAM"=11-mercaptoundecylamine (a generous gift from the laboratory of Professor George M. Whitesides, Harvard University, Boston, Mass.).
"NHSS"=N-hydroxysulfosuccinimide ester.
"nucleic acids"=deoxyribonucleic acids (DNA), ribonucleic acids (RNA), and peptide nucleic acids from any source, and modified forms thereof, including, without limitation, labeled (radioactive, fluorescent, etc.) nucleic acids, and nucleic acidsmodified to include a binding moiety such as a thiol group or a biotin tag.
"PEG"=poly(ethylene glycol).
"PEG-NHS"=N-hydroxysuccinimidyl ester of methoxypoly(ethylene glycol) propionic acid MW 2000 (Shearwater Polymers, Inc., Huntsville, Ala.).
"poly(ethylene glycol)-modified alkanethiol"=HS(CH.sub.2 ).sub.11 (OCH.sub.2 CH .sub.2).sub.3 OH (from Dr. Whitesides' laboratory).
"SSB"=single-stranded DNA binding protein (Pharmacia Biotech, Piscataway, N.J.).
"SSMCC"=sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Pierce Chemical, Rockford, Ill.).
"TAEA"=tris(2-aminoethyl)amine (Aldrich Chemical, Milwaukee, Wis.).
"TEA"=triethanolamine hydrochloride (Sigma)
".omega.-modified alkanethiol"=an alkanethiol whose terminal carbon atom has been modified by the addition of a chemically-reactive moiety such as an amino, hydroxy, carboxy, or thiol moiety.
The above chemicals and were all used as received. Solvents were of standard laboratory grade and Millipore (Marlborough, Mass.) filtered water was used for all aqueous solutions and rinsing.
The chemical modification of a metal substrate to create a nucleic acid array thereon proceeds in seven general steps These steps are illustrated schematically in FIG. 1 and are as follows:
(1). Self-assembly of an .omega.-modified alkanethiol monolayer on a metal substrate. The .omega.-modification to the alkanethiol may be the addition of any moiety with enables further covalent linkages to be made the .omega.-terminus of thealkanethiol. Such modifications include, without limitation, the addition of an amine group, a hydroxyl group, a carboxyl group, or a thiol group to the .omega. carbon of the alkanethiol chain. The alkanethiol monolayer is preferably an amino-C.sub.8-C.sub.24 -alkanethiol, a straight-chain alkane being much preferred to branched alkane; the most preferred .omega.-modified alkanethiol is MUAM.
(2). Reaction of the .omega.-modified alkanethiol surface with a hydrophobic protecting group, most preferably Fmoc.
(3). Photopatterning of the surface to create an array of bare metal areas.
(4). Re-assembly using additional (.omega.-modified alkanethiol to fill in the bare metal array elements, thereby yielding islands of .omega.-modified alkanethiol.
(5). Covalently attaching biomolecules or cells to the islands of .omega.-modified alkanethiol.
(6). Removal of the protecting group from the array background.
(7). Reaction of the background with a material, preferably PEG, to make the background resistant to non-specific protein binding. (The numbers in parentheses directly above are reference numerals in FIG. 1.)
To ensure the quality of the finished product, each of the above steps may be monitored using PM-FTIRRAS, contact angle measurements, and scanning-angle SPR.
The above steps are now described in greater detail, with specific reference being made to FIG. 1.
Step (1). In step (1), a monolayer of .omega.-modified alkanethiol, preferably an amine-terminated alkanethiol, most preferably MUAM, is self-assembled from an ethanolic solution onto a silanized substrate (glass or other substrate transparentto the wavelengths of radiation to be used in subsequent analysis) coated with a thin noble-metal film. In the preferred embodiment, a film of gold about 450 .ANG. thick is used. The thickness of the metal film is not overly critical insofar as thefilm is uniformly applied and will function in SPR imaging analysis. Self-assembled monolayers of .omega.-modified alkanethiols on gold have been described previously, see, for example, Thomas et al. (1995) J Am. Chem. Soc. 117:3830-3834, and aregenerally accepted by most to form well-ordered, monomolecular films. However, if left exposed for extended periods of time, the terminal amine groups of amino-modified alkanthiols will react with CO.sub.2 to form carbamate salts on the surface. Consequently, amino-terminated alkanethiol-coated substrates should be handled with care and their exposure to CO.sub.2 minimized.
The PM-FTIRRAS spectrum of MUAM in the mid-IR region is shown in FIG. 3, plot (A). The small peak centered at 1545 cm.sup.-1 has been assigned as the NH.sub.3.sup.+ deformation. The presence of this peak suggests that after a rinsing withethanol and Millipore water (pH.about.6), a significant portion of the terminal amine groups exist in the protonated form. Variation in the intensity of the 1545 cm.sup.-1 peak can be effected by rinsing the surface in solutions of differing pH. Bandsat 1465 and 1258 cm.sup.-1 in this same plot have been assigned to the CH.sub.2 scissoring and twist deformations of the alkane chains respectively. The frequencies of the peaks due to the CH.sub.2 asymmetric stretching mode at 2923 cm.sup.-1 and theCH.sub.2 symmetric stretching mode at 2853 cm.sup.-1 (spectrum not shown) indicate that the monolayer exists in a relatively ordered state. Absent from the spectrum in the CH stretching region is a band due to the N-H stretch (.about.3200-3500cm.sup.-1) of the amine groups; it is assumed that this band is too weak to be detected. Due to its terminal amine groups, a MUAM monolayer surface is quite hydrophilic, which is verified by a contact angle measurement of 36.2.+-.2.5 and is consistentwith monolayer formation. Ex situ scanning SPR was used to measure a thickness of 17.5 .ANG..+-.0.4 .ANG. for a gold surface modified with MUAM; this thickness is consistent with that expected for a fully extended MUAM monolayer oriented nearly normalto the surface.
Step (2). In step (2) of the array fabrication, the MUAM covered surface is reacted with a reversible protecting group to create a hydrophobic surface. In the case of MUAM, an amine-modified alkanethiol, the protecting group is, appropriately,an amino protecting group, preferably Fmoc. Fmoc is a bulky, hydrophobic, base labile, amine protecting group routinely used in the solid phase synthesis of peptides. The choice of protecting group used is dependent in large measure upon the nature ofthe .omega.-modification made to the alkanethiol. If the .omega.-modification is the addition of a carboxyl group, a hydrophobic carboxy protecting group would be used. Likewise, if the .omega.-modification is the addition of a hydroxyl or thiol group,a hydrophobic hydroxy or thiol protecting group, respectively, would be used. Any type of hydrophobic protecting suitable for protecting the .omega.-modification used on the alkanethiol can be utilized in the present invention. Numerous such protectinggroups, for any number of reactive moieties, such as amine, hydroxy, and carboxy functionalities, are known to the art. For example, chloride derivatives of both Fmoc and trityl to can be used to reversibly modify hydroxyl-terminated alkanethiols.
The specific chemical reaction for Fmoc is shown in FIG. 2, reference number (2). The N-hydroxysuccinimide ester of Fmoc (Fmoc-NHS) reacts with the terminal amine moiety of the MUAM molecule to form a stable carbamate (urethane) linkage,covalently attaching the Fmoc group to the surface. The IR spectrum of Fmoc linked to a MUAM-coated gold substrate is shown in FIG. 3, plot (B). This spectrum provides evidence that the surface reaction proceeds as expected. Prominent peaks at 1720,1544, and 1267 cm.sup.-1 are due to the carbamate (urethane) linkage which tethers the Fmoc group to the MUAM surface. (The band at 1720 cm.sup.-1 has been assigned to the carbonyl stretching vibration (amide I), that at 1544 cm.sup.-1 to the CHN groupvibration, and that at 1267 cm.sup.-1 to the coupled C--N and C--O stretches (amide IV).) The peak at 1450 cm.sup.-1 is ascribed to the C.dbd.C ring stretch of the fluorenyl group and the band centered at 1147 cm.sup.-1 is attributed to the Fmoc C--O--C(ether) stretch. After reaction with Fmoc-NHS, the surface properties of the array are changed significantly; the surface is extremely hydrophobic as confirmed by the measured contact angle of 74.4.+-.2.5. In addition, an increase in the film thicknessto 22.8 .ANG..+-.0.5 .ANG. is measured with scanning angle SPR.
Step (3). In step (3) the bond anchoring the .omega.-modified alkanethiol to the metal substrate is selectively cleaved to yield a patterned surface of exposed metal. UV photopatterning is preferred to create the patterned surface, although themeans to create the patterned surface is not critical so long as the method reliable yields the desired pattern. For example, microcontact printing methods can also be used to yield a patterned surface. Using UV patterning, the surface is exposedthrough a quartz mask to UV radiation which photo-oxidizes the gold-sulfur bond that anchors the alkanethiol monolayers to the surface. The surface is then rinsed, removing the photo-oxidized alkanethiol and leaving an array of bare metal padssurrounded by a hydrophobic MUAM+Fmoc background. Using photopatterning, features with dimensions as small as 50 mm have been achieved; using microcontact printing methods, arrays with features as small as.about.100 nm are achievable.
Step (4). In step (4), the surface is again exposed to an .omega.-modified alkanethiol solution (in the preferred embodiment an ethanolic solution of MUAM) whereby the alkanethiol assembles into the bare gold regions producing a surface composedof hydrophilic MUAM pads surrounded by the hydrophobic Fmoc background. This difference in hydrophobicity between the reactive MUAM regions and the background is essential for the pinning of small volumes of aqueous biomolecule or cell solutions ontoindividual array locations.
Step (5). At step (5) in the process, biomolecules or cells (preferably nucleic acids) are then covalently attached to the surface. As illustrated, the MUAM reactive pads are first exposed to a solution of a bifunctional linker. To be used inthe invention, the linker must be capable of binding at one end to the .omega.-modified alkanethiol surface and at the other end to the biomolecule or cell to be immobilized to form the desired array. Any bifunctional linker having these characteristicscan be used in the present invention. The preferred bifunctional linker is SSMCC, a heterobifunctional linker which contains both an N-hydroxysulfosuccinimide (NHSS) ester and a maleimide functionality. The NHSS ester end of the molecule reacts withthe free amine groups on an
amino-modified surface, such as the MUAM spots, creating pads terminated in maleimide groups which are reactive towards thiols. Small volumes (0.08 to 0.1 L) of 1 mM solutions of 5'-thiol-modified DNA sequences are then spotted at discretearray locations and react to form a covalent attachment to the surface. See FIG. 2, reference numeral (5). Using this technique, a whole host of biomolecules and/or whole cells can be spotted at different array locations.
A variation on this attachment scheme whereby thiol-DNA is linked via SSMCC to a MUA/PL (11-mercaptoundecanoic acid/poly-L-lysine) bilayer has been used quite extensively in this laboratory, see U.S. Pat. No. 5,629,213. Other researchers haveused the direct self-assembly of thiol-terminated DNA molecules on gold to prepare functionalized surfaces, but this method has the disadvantage that only weak forces exist for the self-assembly of oligonucleotide molecules and hence, the DNA can alsonon-specifically adsorb to the bare gold surface.
Here, a bifunctional linker is used to attach 5'-thiol-modified oligonucleotide sequences to reactive pads of aminoalkanethiol. The bifunctional linker preferably contains a functionality reactive towards amines and a functionality reactivetowards aminoalkanethiols. The surface is first exposed to a solution of the linker, whereby one end of the molecule reacts with the aminoalkanethiol surface. Excess linker is rinsed away and the array surface is then spotted with 5'-thiol-modifiednucleic acid which reacts with the other end of the bifunctional linker, forming a covalent bond between the nucleic acid and the surface monolayer.
Step (6). In step 6 the protecting group, depicted here as Fmoc is removed from the array surface. Preferably, this is accomplished by exposure to a 1M solution of the secondary amine, TAEA, in DMF. Many basic secondary amines can be used toremove Fmoc from the surface; for example, 1M solutions of ethanolamine and piperidine can be used with equal success. TAEA was chosen specifically as the deprotection agent since it effectively scavenges the dibenzofulvene byproduct and is efficientlyrinsed from the array surface. After this deprotection step, the array background has been converted back to the original .omega.-modified alkanethiol surface. The spectrum of a deprotected MUAM surface, the preferred embodiment, is shown in FIG. 3,plot (C); note the strong similarity between it and the original MUAM spectrum. The prominent bands due to the carbamate linkage no longer appear, indicating that the Fmoc protecting group has been completely removed from the surface. The deprotectedsurface was also measured with scanning SPR; the thickness measured was within .+-.1 .ANG. of that measured for the starting MUAM surface and this gives additional proof that the Fmoc protecting group is removed completely from the surface.
Step (7). In the final step of the array fabrication, the .omega.-modified alkanethiol background is reacted with a compound to create a background that is resistant to the non-specific binding of proteins. The preferred compound for thispurpose is PEG-NHS, although any compound which will selectively bind to the .omega.-modified alkanethiol surface and inhibit non-selective protein binding can be used. In order to effectively monitor the binding of proteins to arrays of surface-boundbiomolecules or cells, it is critical that the array background prohibit the non-specific adsorption of protein molecules. Significant amounts of such non-specific binding obscures the measurement of small amounts of protein binding at specific arraylocations.
To create a background that is resistant to the non-specific binding of proteins, the MUAM surface was reacted with PEG-NHS as is shown in FIG. 2 reference number (7). As was the case in the Fmoc-NHS+MUAM reaction, PEG-NHS reacts with theterminal amine groups of the MUAM to form an amide linkage, covalently attaching the PEG polymer chain to the surface. The preferred PEG-NHS polymer has an average molecular weight of 2000 and contains one NHS ester moiety per molecule, allowing for asingle point of attachment. The spectrum collected for a MUAM surface reacted with PEG-NHS is shown in FIG. 3, plot (D). The peaks which appear at 1660 cm.sup.-1 and 1576 cm.sup.-1 have been assigned as amide I and II bands, respectively. The bands at1457 cm.sup.-1 and 1250-1260 cm.sup.-1 are ascribed to the scissoring and twist deformations of the CH.sub.2 groups contained in both the MUAM alkyl chains and the ethylene glycol (EG) groups. The band at 1352 cm.sup.-1 is due to an EG CH.sub.2 waggingmode, and the band centered at 1148 cm.sup.-1 is due to the C--O--C (ether) stretch of the ethylene glycol units. After the reaction of the deprotected surface with PEG-NHS, the surface remains hydrophilic and has a measured contact angle of37.3.+-.2.6. A total thickness of 23.8 .ANG..+-.0.8 .ANG. was measured for a MUAM monolayer film after reaction with PEG-NHS. This increase of only 6 .ANG. of PEG suggests that only a small fraction of the amine groups of the MUAM are modified andthat the oligo(ethylene glycol) chains are lying flat across the surface.
An SPR imaging experiment (see Example 2 and FIG. 7) was used to measure the non-specific adsorption of BSA to a dual component surface (C.sub.18 -thiol/MUAM+PEG) and shows quite clearly that MUAM+PEG effectively resists the non-specificadsorption of proteins.
The following Examples are included solely to provide a more complete understanding of the present invention. The Examples do not limit the scope of the invention disclosed and claimed herein in any fashion.
Standard Procedures for All Examples
Gold substrates used in PM-FTIR and contact angle measurements were purchased commercially (Evaporated Metal Films) and those used in scanning or imaging SPR measurements were prepared by vapor deposition onto microscope slide covers that hadbeen silanized with (3-mercaptopropyl)trimethoxysilane (Aldrich) in a manner similar to that reported by Goss et al. (1991) Anal. Chem. 63:85-88.
All oligonucleotides were synthesized on an ABI (Foster, Calif.) DNA synthesizer at the University of Wisconsin Biotechnology Center. Glen Research's (Sterling, Va.) "5'-Thiol-Modifier C6"and ABI's "6-FAM" were used for 5'-thiol-modified and5'-fluorescein-modified oligonucleotides respectively, and "Spacer Phosphoramidite 18" (Glen Research) was used for the addition of an ethylene glycol spacer region. Thiol-modified oligonucleotides were deprotected as outlined by Glen Research's productliterature.(Glen Research Corp. (1990) "User Guide to DNA Modification and Labeling"). Before use, each oligonucleotide was purified by reverse-phase binary gradient elution HPLC (Shimadzu (Columbia, Md.) "SCL-10AVP") and DNA concentrations wereverified with an HP8452A UV-VIS spectrophotometer (Hewlett-Packard, Palo Alto, Calif.).
The sequences of the DNA molecules used in the SSB experiment of Example 1 were as follows:
The buffer used in the BSA and SSB SPR imaging experiments contained 20 mM phosphate, 100 mM NaCl, 1 mM EDTA, 1 mM DTT, and 5 mM MgCl.sub.2 and was buffered at pH 7.4.
Multi-step array fabrication: A clean gold substrate was immersed in a 1 mM ethanolic solution of MUAM for at least one hour to allow for the adsorption and self-assembly of the aminoalkanethiol monolayer. The substrate was rinsed with ethanoland water, dried under a stream of N.sub.2, and was then reacted with a solution of Fmoc-NHS (3 mM in 1:1 DMSO:100 mM TEA buffer, pH 7). The sample was soaked briefly in DMSO to remove unreacted Fmoc-NHS from the surface and then photopatterned byirradiating the sample with UV light from a mercury-xenon arc lamp through a quartz mask. Subsequent rinsing of the sample with ethanol and water removed alkanethiol from the exposed areas. The sample was re-exposed to the ethanolic MUAM solutionresulting in an array of MUAM elements surrounded by a hydrophobic MUAM+Fmoc background. Single-stranded, 5'-thiol modified DNA was then immobilized onto the array locations using an attachment scheme modified slightly from that used previously. Briefly, the amine-terminated MUAM array elements were spotted with 0.1 .mu.L of a 1 mM solution (in 100 mM TEA, pH 7) of the heterobifunctional linker SSMCC, creating a thiol-reactive, maleimide-terminated surface. 5'-Thiol-modified DNA sequences werethen covalently attached to these maleimide-terminated array elements by spotting the sample with 0.1 .mu.L drops of solutions containing 1 mM DNA onto the specific array locations and reacting for at least 2 hours in a humid environment to preventsolvent evaporation. After exposure to the DNA solution, the surface was rinsed with water and soaked in buffer to remove unbound DNA sequences. The Fmoc was then removed from the background by immersing the array in a 1M solution of TAEA in DMF for 10minutes. The deprotected surface was rinsed with water and subsequently reacted with 4 mM PEG-NHS (in 100 mM TEA, pH 8) to pegylate the array background, rendering it resistant to protein non-specific binding.
PM-FT-IRRAS Measurements: PM-FT-IRRAS spectra were collected on a Mattson RS-1 spectrometer equipped with either a narrow band HgCdTe detector (for spectra in the mid-IR region, 2000-1000 cm.sup.-1) or an InSb detector (for spectra in the CHstretching region, 3400-2600 cm.sup.-1). The optical layout and previously developed real-time interferogram sampling methods have been described elsewhere and need not be elaborated upon here. The PM-FT-IRRAS differential reflectance values (% R/R)were converted to absorbance units for comparison with conventional IRRAS data. Spectra are an average of 1000 scans collected at 2 cm.sup.-1 resolution.
Contact Angle Measurements: Water contact angles were determined at ambient laboratory temperatures using standard and well known procedures. Ten microliter droplets were dispensed from a Gilson pipette onto the surface and the angle measurementwas recorded immediately. Reported contact angle values for both the Fmoc and PEG functionalized surfaces are the average of 12 different measurements taken on 4 individually prepared samples and the value for MUAM is the average of 30 measurementstaken on 10 different samples.
Scanning Angle SPR Measurements: The optical technique of ex situ scanning SPR was used to determine the thickness (reported hereinabove) of MUAM, MUAM+Fmoc, and MUAM+PEG assembled on BK7 coverslips (Fisher Scientific, Pittsburgh, Pa.) onto which475 .ANG. of Au was vapor deposited. Details of the SPR experiment and thickness calculations have been reported elsewhere. Briefly, the reflectivity (R) of a p-polarized HeNe laser beam (632.8 nm) from a sample assembly (BK7 prism/Au/thin film/air)is monitored as a function of incident angle, to generate a SPR curve (% R vs. angle). A steep drop in the reflectivity occurs at angles just past the critical angle (.about.44). The exact position of the minimum is determined by the thickness andindex of refraction of material adsorbed at the gold surface. A 4-phase complex Fresnel calculation was used to determine the film thickness and a refractive index of 1.45 was assumed for all the thin films measured here.
SPR Imaging Apparatus: The in situ SPR imaging instrument is a modified version of that described previously, see Jordan & Corn (1997) Anal. Chem. 69(7):1449-1456; Thiel et al. (1997) Anal. Chem. 69:4948-4956; Jordan et al. (1997) Anal. Chem.69(24):4939-4947; and Frutos et al. (1998), supra, in which the HeNe laser and beam expander have been replaced by a collimated white light source/bandpass filter combination. A more thorough discussion of this modification in the context of near IR(NIR) SPR imaging is reported elsewhere, see Nelson et al. (1999), in preparation. In short, a collimated, polychromatic beam of light was used to illuminate an SF10 prism/Au/thin film/buffer assembly at a fixed incident angle near the SPR angle. Thereflected light was passed through a 10 nm bandpass filter (830 nm) and was collected with an inexpensive CCD camera. Differences in the reflected light intensity measured at various locations on the sample create the image and are a direct result ofdifferences in the thickness or refractive index of the material bound at the gold surface. The image shown in FIG. 6 was collected in situ for a sample constructed on SF10 substrates onto which 450 .ANG. of Au had been deposited. Data work-up wasdone using NIH Image v.1.61 software.
SPR imaging measurements of the binding of single stranded DNA binding protein to arrays of single and double-stranded DNA sequences
To demonstrate that nucleic arrays can be used in conjunction with imaging SPR to monitor protein-nucleic acid binding, a checkerboard surface was constructed containing both single-stranded DNA (D1, SEQ. ID. NO: 1) and double-stranded DNA (D2and its complement, SEQ. ID. NOS. 2 and 3, respectively), by the methods described immediately above. The binding of single-stranded DNA binding protein, SSB, to the array surface was then monitored by SPR. As its name implies, SSB (a tetramer offour identical subunits with a total molecular weight of 75,000 D) binds tightly, selectively, and cooperatively to single-stranded DNA and plays a central role in DNA replication, repair, and recombination. FIG. 6 shows the difference between twoimages collected immediately before and after the exposure of the surface to SSB. The raised areas on the image are a measure of the change in % R upon adsorption of the protein to the surface. The array locations at which the protein bound correspondto those regions which were modified with single-stranded DNA sequences.
FIG. 5 shows various line profiles taken from images collected during the course of the experiment. These "line profiles," which provide quantitative information, are constructed by averaging the % R values measured for each column of pixels ina selected rectangular region drawn across the image and then plotting this average value against that column's lateral position. The solid line shows the starting surface in which two 5'-thiol-modified, single-stranded DNA sequences, D1 and D2, wereimmobilized in a checkerboard pattern onto the array surface. The sequences of these two DNA probe strands are noted above. Each sequence contains a 5'-thiol modifier, a spacer region, and a 16 base-long variable sequence. The variable regions werespecifically chosen from a library developed for the purposes of DNA computing, Frutos et al. (1997) supra, they and their complements exhibit no cross hybridization. To position the DNA sufficiently far from the surface so that stearic hindrance doesnot interfere with the hybridization adsorption process, a spacer region is incorporated. A 15T spacer region was used for D1, but sequence D2 contained a similar length EG spacer instead. This was necessary given the fact that SSB is known to bindquite strongly to polyT sequences. The dashed line shows the effects of exposing the surface, in situ, to a solution containing the 16-mer complement to D2 (SEQ. ID. NO: 3). A measurable change in % R occurred at location D2, indicating thathybridization adsorption of the complementary sequence took place; no increase in signal was seen at the D1 locations. The dot-dashed line shows the surface after exposure to a 200 nM solution of SSB. As expected, the protein bound strongly tolocations on the array which were single stranded but also bound slightly to those locations that contained double-stranded sequences. Since SSB does not bind to double-stranded DNA, we attribute the increased signal at location D2 to the binding of SSBto single-stranded DNA present at these locations as a result of incomplete hybridization. It is important to note that the array background successfully resisted the non-specific binding of both complementary DNA molecules and single-stranded bindingprotein; this allows the measurement of small changes in % R without interference from a high background signal.
This Example shows that the arrays fabricated according to the present invention can be used to probe nucleic acid-protein interactions.
Demonstration that PEG block non-specific protein binding
Here, an array of C.sub.18 alkanethiol spots were assembled on a MUAM+PEG background using the techniques described above. The array was then exposed to BSA and the percent reflectivity measured as described in Example 1. The results are shownin FIG. 7. In FIG. 7, the solid line shows the percent reflectivity of the array prior to exposure to BSA. The dashed line shows the presence reflectivity after exposure to BSA.
While some BSA did adsorb to the MUAM+PEG background, a vastly large amount
of BSA adsorbed to the untreated C.sub.18 spots. This Example thus shows that PEG can be used to inhibit the non-specific binding of proteins to the background surface.
__________________________________________________________________________ # SEQUENCE LISTING - - - - <160> NUMBER OF SEQ ID NOS: 3 - - <210> SEQ ID NO 1 <211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial - #Sequence: synthetic DNA molecule - - <400> SEQUENCE: 1 - - tttttttttt ttttttaacg atgcaggagc aa - # - # 32 - - - - <210> SEQ ID NO 2 <211> LENGTH: 16 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial - #Sequence: Synthetic DNA sequence - - <400> SEQUENCE: 2 - - gcttatcgagctttcg - # - # - # 16 - - - - <210> SEQ ID NO 3 <211> LENGTH: 16 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial - #Sequence: Synthetic DNAsequence - - <400> SEQUENCE: 3 - - cgaaagctcg ataagc - # - # - # 16 __________________________________________________________________________
* * * * *
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