text:
Property Details
House, 2 Bedrooms, 2 Baths, (Sleeps 6)
The Ecola Park Cottage is a wonderful, spacious, modern, newly constructed two bedroom cottage with a loft in Cannon Beach. The cottage is just a block and a half from the beach and an easy walk to all the shops and restaurants downtown.
Along with a fireplace, plenty of parking, and a large private deck on both levels, the home also includes a washer/dryer, cable TV, DVD player, WiFi and a beautiful fully equipped kitchen. There are two bedrooms on the lower level, both with queen beds, and a sleeper sofa in the living room. The coffee table great for puzzles!
The house sits adjacent to Ecola State Park, with a number of excellent hiking trails, horseback riding opportunities, and a historic lighthouse. Stroll right from your front door to the town's beach, which stretches all the way south to iconic Haystack Rock and its legendary tide pools. The house is situated in a quiet area of Cannon Beach, however all the boutiques and restaurants can be reached after a pleasant walk through town or along the beach. For a bit of peace and quiet while still staying close to all the amenities of a popular resort town, the Ecola Park Cottage is just what you are looking for.
The Oregon coast offers beach walking, hiking, horseback riding, antiquing, shopping, restaurants, whale watching, road and mountain biking, surfing, clamming, kayaking and much more.
Cannon Beach limits us to only one rental every 14 days, so this house has limited checkin and checkout dates in the summer. We offer discounts for longer stays! You can use the online calendar to make a reservation or give us a call at 503-345-9399 if you have any questions.
Please use the link below to check real time availability, view more pictures and book securely online!
Vacasa - Vacation Rentals Made Easy:
Professionally managed and cleaned homes
Local assistance 24/7 for emergencies
Secure INSTANT booking
Recent Review
Great place for R&RGreat place for R&R
About the Owner
9 Reviews
4.7 based on 9 traveler reviews
Great place for R&RGreat place for R&R
Close to the beach
Great place!!
Great Experience
Nice beach getaway!
Perfect for us
Close walking distance to the cute town of Cannon Beach.
Comfortable and felt like home.
Payment is usually accepted in the quoted currency (US Dollar) unless the currency and the amount is specifically agreed in advance with the owner / advertiser.
- Major Credit Cards Accepted. Taxes and fees are not included. Rates and availability calendars may change until time of booking. For a full rate quote and live availability, please contact us or visit the Vacasa.com website!
- .
- US Dollars
- .
- 12
- Available
- 12
- Unavailable
- 12
- Today
Property Type
- house
- Fireplace
- Internet
- Parking
- Satellite / Cable
- children welcome
- non smoking only
- pets not allowed
- 2 Bathrooms
- 2 Bedrooms, Sleeps 6
- Outdoor Grill


text:
STARmeterTop 5000
Up 2,675 this week
Bob Sapp
Bob Sapp was born on September 22, 1973 in Colorado Springs, Colorado, USA as Robert Malcolm Sapp. He is an actor, known for The Longest Yard (2005), Conan the Barbarian (2011) and Elektra (2005). See full bio »
More at IMDbPro » Contact Info: View agent and manager
Represent Bob Sapp? Add or change photos

subset: commoncrawl


text:
To try the thousands of add-ons available here, download Mozilla Firefox, a fast, free way to surf the Web!Close
Choose from thousands of extra features and styles to make Firefox your own.Close
|Matt Rugg
|User since
|November 12, 2008
|Number of add-ons developed
|0 add-ons
|Average rating of developer's add-ons
|Not yet rated
I just installed two days ago. No problems, delete by hitting the red "x" delete button. I don't understand the problems reported here. Maybe I have a later version?This review is for a previous version of the add-on (0.4.5).
To create your own collections, you must have a Mozilla Add-ons account.


text:
Thanks for redeeming.{{else}}
Success! Thanks for repurchasing.{{/if}} {{else}} {{#if Expired}}
Increase my visibility!{{else}} {{#if IsMultiPack}}
{{#if HasMultiPack}} You have {{MultiPackCount}} top spot(s) left. Now loading you into the search listings... {{else}} You have used all top spots. Now loading you into the search listings... {{/if}}{{else}}
We are loading you into the search listing pages...{{/if}} {{/if}} {{/if}} {{/if}}
{{Current}} just saw you!{{/if}} {{#if LastTopSpotPurchaseIsMulti }} {{#if HasAnyTopSpot}}
{{#if HasFreeTopSpot }} {{ UnclaimedTopSpotFree }} Free {{/if}} {{#if HasPaidTopSpot }} {{#if HasFreeTopSpot }} & {{/if}} {{ MultiPackCount }} {{/if}} Top Spot{{#if Plural}}s{{/if}} Left{{else}}
0 Top Spot{{#if Plural}}s{{/if}} Left{{/if}} {{else}} {{#if HasFreeTopSpot }}
{{ UnclaimedTopSpotFree }} Top Spot{{#if Plural}}s{{/if}} Left{{/if}} {{/if}} {{else}} {{#if HasResults}}
{{Current}} see you now!{{else}} {{/if}} {{/if}}
You can now invite anyone to meet at your favorite Starbucks.
Limited-time feature
28 year old woman | Erie, PA, USA | Seeking women 21-35 within 30 miles
Looking for someone I can hang out with and get to know first
Right now, I am just here to look. I am looking for someone close to my age I can go out with casually in a group and get to know better first before dating. I need to be with someone stable, preferably with a steady job who can take care of her own financial needs. I don't want to be with someone who cannot decide whether she is even going to be living in this same town a year from now. I work a lot, but I find time to go out on the weekends. However, I am just as happy staying home to watch a movie.
Camping , Coffee and conversation , Movies/Videos , Music and concerts , Nightclubs/Dancing , Performing arts , Playing cards , Travel/Sightseeing , Volunteering , Watching sports , Wine tasting
I exercise 1-2 times per week.
I like Cats, Dogs, Fish, Other
Downtown Erie, block parties, the beach, the woods
Edinboro University of Pennsylvania, Edinboro, PA
I like all types of music, but favorites are classic rock, alternative, and broadway. I love horror movies. Favorite TV shows: Fringe, House, and iCarly (don't judge =D). I love wine. Not a beer fan. I am always open to learning about new things.
I like to go out on weekends, though I'm not a good dancer. :) I love being outside. I don't play sports, but I like watching football with my family on TV or live. We like to tailgate. I love watching movies.
For a teacher, I actually don't read much. The last thing I read was a short story my friend wrote. I'll read something if it really catches my interest.
Z{{Cta}} {{#if HasMoreInfo}} ?{{CtaSecondary}} {{/if}}
|What she is looking for
|5'5" (165cms)
|3'0" (91cms) to 8'11" (271cms)
|Body type:
|Big and beautiful
|Big and beautiful, About average, Curvy, Full-figured, Heavyset, A few extra pounds, Stocky
|Black, Blue, Brown, Grey, Green, Hazel
|Dark brown
|Auburn / Red, Black, Light brown, Dark brown, Blonde, Dark blonde
|No Way
|No Way, Occasionally, Daily, Yes, but trying to quit
|Social Drinker
|Social Drinker, Regularly, Moderately
|Education / Teacher / Professor
|No preference
Right now I work at a daycare center. I have a B.S. in elementary education and a Masters in science education, but just haven't applied for a serious teaching job yet. Ideally, I would love to work in an environmental science center.
|Less Than $25,000
|No preference
|Never Married
|Never Married, Currently Separated, Divorced
|Have kids:
|No preference
|Wants kids:
|Definitely, Someday, Not sure
|White / Caucasian
|No preference
I am very family oriented. It is not uncommon for me to get together with either side of my family on a regular basis just to play games or have dinner and drink.
|No preference
|Graduate degree
|No preference


text:
Save up to £50 on new, Vehicle based Roadside, Recovery, At Home, Onward Travel & European on annual continuous payment direct from RAC. Ends 16.6.15.Join here »
Up to 70% off RAC Car Insurance with 9+ years no claims discount. Get a quote »
Up to 70% off with 9+ years no claims discount.Get a quote »
Protect your no claims bonus with a forward facing camera. This month you can get up to £45 off the RAC Dash Cam range at the RAC Shop. Offer ends 30/06/15Shop now »
Buying a car? Don't be duped - check the history first with the UK's most comprehensive car check. Selling a car? Give your buyers confidence with our seller's report.Check now »
Compare prices and offers from over 10,000 garages,and use our customer reviews to find a trusted service for your car.Compare Garages »
Read our guide to driving in Europe and find out all the information you need, including country specific advice.Find out more »
Read the spring issue of our Club Magazine, packed with amazing drives, great cars, competitions, and so much more....Read it now »
Consider the Camping Card International. You may not have heard of it, but the Camping Card International (CCI) is an extremely useful document if you're planning to camp or caravan anywhere around the globe*. In fact in Denmark it's a legal requirement.
Click here to download the CCI application form.
Please be aware that we can no longer accept applications by fax. Instead, please complete the form and e mail it to: firstname.lastname@example.com.
* The insurance is valid worldwide with the exception of the USA and Canada and territories that fall under the jurisdiction of these countries. The insurance is valid from the date of issue or extension up to and including the 31st December of that same year.
Be the first to know about new RAC products and special offers.


text:
Heating up: Demand for HVAC services will rise along with construction market recovery
IBISWorld's HVAC Service Franchises market research report can be used to help you: understand market size and growth potential; major companies; draft business plans and pitch books; and conduct benchmarking and SWOT analysis. Our industry analysis highlights macro industry trends in the overall sector and micro trends faced by companies that do business in the industry. The industry report also provides key industry statistics and 5-year forecasts to anticipate future industry prospects so you can decide with confidence.
During the recession, demand for heating, ventilation and air conditioning (HVAC) services suffered as downstream construction markets crashed, pushing industry revenue downward. However, HVAC franchises are anticipated to recover from the downturn in the next five-year period along with renewed construction activity, corporate profit and per capita disposable income. The federal and state governments will aid this recovery via tax credits for businesses and homeowners to use on energy-efficient system upgrades.... purchase to read more
The distribution of industry franchises closely follows that of population and economic activity; however, environmental factors, such as climate, also play a role. For example, homes, apartments and other buildings in southern states, such as Florida and Alabama, are more likely to be outfitted with air conditioning systems than northern states with cooler climates. Similarly, in areas of the country where it stays warm year-round, there is less of a need for central heating systems.
Accounting for 25.6% of heating, ventilation and air conditioning (HVAC) service franchises in 2014, the Southeast's share of industry activity closely follows that of its population share. The region houses more than a quarter of the US population... purchase to read more
This industry operates franchised businesses that provide heating, ventilation and air-conditioning services to both residential and commercial customers. Reports in our Business Franchise collection focus solely on the operation of franchised outlets and exclude nonfranchise data. They show the total number of franchise outlets, total franchise revenue and the average profit margin earned by franchisees. Our reports also highlight the largest franchisors by market share.
Industry Definition
Main Activities
Similar Industries
Additional Resources
Executive Summary
Key External Drivers
Current Performance
Industry Outlook
Industry Life Cycle
Supply Chain
Products & Services
Major Markets
Business Locations
Competitive Landscape
Market Share Concentration
Key Success Factors
Cost Structure Benchmarks
Barriers to Entry
Capital Intensity
Industry Data
Annual Change
Key Ratios


text:
Viewer Comments: Uterine Fibroids - Describe Your Experience
Patient Comments & Reviews
The eMedicineHealth doctors ask about Uterine Fibroids:
Uterine Fibroids - Describe Your Experience
Please describe your experience with uterine fibroids (benign tumors of the uterus).
Comment from: Betty, 45-54 Female Published: October 07
I was told I had a small uterine fibroid but didn't have to worry about it. However, I had cramping, back pain, the feeling of needing to urinate, and anemia. I was given iron pills for anemia and birth control pills to decrease bleeding. Unfortunately, I was in the small percentage of people who didn't tolerate iron by mouth. (I experienced nausea and vomiting.) So, I ended up getting iron via an IV. I think the birth control pills "fed" the uterine fibroids, and I ended up getting an emergency myomectomy because the fibroids caused me to hemorrhage. The doctor thought the fibroid was much smaller than it was, but she didn't know until she operated. My advice is to take care of them and not just take pain pills. I ended up on disability because I was so weak from anemia. However, after surgery, I never had any more cramps, back pain, the bleeding was miniscule, and I was like a new person. It took three months to recover though, because I had been anemic.
Comment from: Much Faith, 35-44 Female (Patient) Published: October 07
I am a 39-year-old black female. I have had problems with bleeding since I was in my early 20s. Birth control helped with the heavy bleeding. I became pregnant in April of 2009, and I had a miscarriage at seven-and-a-half weeks. In September of this year, I was told that the uterine fibroids had returned, but just in the last two months, the pain has increased. I will be celebrating my one-year anniversary this month, and my husband and I are still trying to have a baby. So, I really don't want to have surgery. I am going to schedule a hysterosalpingogram (HSG) this month to determine where the uterine fibroids are and if they are blocking me from conceiving.
Comment from: 19-24 Female Published: September 11
I am a 24-year-old Latina. In August, I was experiencing severe pain in my abdomen, so I went to the ER to get it checked out. The doctor told me that I probably had kidney stones, and they could not see them with the ultrasound. He did not want to do a CT scan because of my age, so he gave me medicine to help me pass the stones. Two weeks ago, I had to return to the ER because I was having more severe pain. This time they did an abdominal and pelvic ultrasound. The doctor said the results were inconclusive. I followed up with my doctor, and she scheduled a trans-vag ultrasound, which I did yesterday. She called last night to tell me the results had not come back yet, but she got the full report of my ER visit. She then told me that it showed a uterine fibroid that I have had since 2004! Nobody ever told me that, and I gave birth in 2006. I cannot believe that I have lived with this for four, almost five, years and no one said anything. In late 2004 early 2005, I had an ultrasound performed because I had lower abdominal pain that the doctors wrote off as a UTI, which is funny because the only symptom I had was the pain. I didn't have any urinary symptoms at all. It also showed gallstones, which they also did not tell me about in the ER.
Comment from: yams41, 45-54 Female (Patient) Published: September 11
I have uterine fibroids and have had them for years. When my OB/GYN first told me I had fibroids, we decided to take the watch-and-wait route because I didn't have any problems. Now about 10 years later, I have severe/killer cramps, backaches, and constipation. I do not want to have a hysterectomy and my doctor also feels the same. Since I am close to menopause, he suggests that I keep playing the waiting game. I don't have any heavy bleeding, probably thanks to the birth control pills that I take. I found that if I take a painkiller on the first sign of pain, it help lessens the severe cramps. I am also starting to watch my diet. I make sure to eat lots of fruits and vegetables and drink plenty of water. Exercise also helps reduce the cramping.
Comment from: dorah angela, 25-34 Female (Patient) Published: February 28
I am 26 years of age and I found out that I have uterine fibroids. I was going into my periods and I had too much pain in the lower abdomen and the back was aching plus my legs.
Comment from: Rosemary, 45-54 Female (Patient) Published: July 05
I've been having heavy bleeding. In March of this year I lost so much blood I had to have three units of blood a transfusion. It turns out I have fibroids was given a shot to stop the bleeding and it worked but after the six weeks when I got my period it was non-stop bleeding and I was supposed to have a biopsy wasn't able to so I got put on the birth control pills and it stop the bleeding, and I go in two days for a biopsy.
Must Read Articles Related to Uterine Fibroids
Vaginal Bleeding Unexpected bleeding is always a concern for any woman at any stage of life. Anything other than a normal menstrual period and even an abnormally heavy period ca...learn more >>
Patient Comments & Reviews
The eMedicineHealth doctors ask about Uterine Fibroids:
Uterine Fibroids - Outlook and Prognosis
Did you have your uterine fibroids removed? Describe your experience and prognosis.
Viewer Comments are not a substitute for professional medical advice, diagnosis, or treatment. Never delay or disregard seeking professional medical advice from your physician or other qualified health provider because of something you have read on eMedicineHealth. The opinions expressed in the comments section are of the author and the author alone. eMedicineHealth does not endorse any specific product, service or treatment.
If you think you have a medical emergency, call your doctor or 911 immediately.
Report Problems to the Food and Drug Administration
You are encouraged to report negative side effects of prescription drugs to the FDA. Visit the FDA MedWatch website or call 1-800-FDA-1088.
From WebMD
Women's Health Resources
- Teen Quiz: What's Up 'Down There'?
- Video: Is It Time to See a Dentist?
- Your Guide to a Healthy Pregnancy


text:
A device and method allowing evaluation of the contents of a sealed primary container by means of an integral sensor which is separated from the contents of the sealed primary container yet provides information on quality of the contents of the primary container without breaking the sealed system. The integral sensor device includes a biosensor retained within a plastic construct by a gated-pore membrane. Pores in the membrane open in response to an environmental change in the primary container allowing the contents of the primary container to contact the biosensor. Status of the contents of the primary container can be determined by inspection of the biosensor, visually or via a fiberoptic probe, through the optical window of the plastic construct.
1. A sensor device, comprising:
a biosensor;
a biosensor enclosure having an interior and an exterior, enclosing the biosensor, the biosensor enclosure having a surface allowing external viewing of the biosensor; and
a separation barrier forming a portion of the biosensor enclosure, the separation barrier having at least one pore allowing fluid communication between the interior and exterior of the biosensor enclosure.
2. The sensor device of
3. The sensor device of
4. The sensor device of
5. The sensor device of
6. The sensor device of
7. The sensor device of
8. The sensor device of
9. The sensor device of
10. The sensor device of
a substrate; and
at least one bioactive response element.
11. The sensor device of
12. The sensor device of
13. The sensor device of
14. The sensor device of
15. The sensor device of
16. The sensor device of
17. The sensor device of
18. The sensor device of
19. The sensor device of
[0001] This application is based on priority Provisional Application Ser. No. 60/179,164, filed Jan. 31, 2000.
[0002] 1. Field of the Invention
[0003] This invention relates to analyte enclosures to be used in conjunction with sensing devices. In particular, this invention relates to evaluation of the contents of a sealed primary container by means of an integral sensor which is separated from the contents of the sealed primary container yet provides information on quality of the contents of the primary container without breaking the sealed system.
[0004] 2. Description of the Related Art
[0005] The background for this invention is presented in the context of human blood cells, because of their great societal importance and especially because of limitations of current procedures used for their storage. However, the invention could have been presented in a context of food storage and processing, production of valuable molecules by biofermentation, or industrial processing such as electroplating or acid/base treatment of products.
[0006] Typically, blood is obtained from donors and fractionated into components including erythrocytes, platelets, lymphocytes, and plasma. Erythrocytes and platelets are separately packaged as "units", which then are held at appropriate temperature and distributed for later transfusion into patients. Each unit is aseptically placed into a previously sterilized plastic bag and sealed therein. Despite careful attention to aseptic processing, there is a probability that a given unit of blood cells will be contaminated with microbes.
[0007] With erythrocytes, current FDA regulations limit use to within 24 days after collection in an effort to minimize risk to patients from infusion of a contaminated unit of cells. Often endotoxins rather than the bacteria per se cause the problem with erythrocytes. With platelets, there is similar concern about microbial contamination, but their use is limited to 5 days because of metabolic self-damage. A recent review (Blajchmann, 1999) noted that 1 in ˜2,500 units of platelets and 1 in ˜38,500 units of erythrocytes was contaminated with bacteria. A New Zealand study reported that 1 of 65,000 individuals will get very sick after transfusion of erythrocytes and 1 of 104,000 will die because of one specific organism. Data from other countries, including the USA, provide both higher and lower risk factors, but there is substantial hazard associated with use of erythrocytes and especially platelets (Lee, 1999).
[0008] Currently there is no acceptable method to determine if a unit of blood is contaminated just before transfusion of the contents into a patient. This is because sampling the contents requires opening the bag, with resultant risk of introducing contamination, and evaluation of the sample acquired. Because a laboratory setting is required, it is difficult or impossible to make a "use" or "non-use" decision in a few minutes. Clearly, a diagnostic method or device to allow a use or non-use decision without opening the unit of blood would have great utility if it had a very high probability of giving the correct conclusion.
[0009] A device and method of the present invention allows evaluation of the contents of a sealed primary container by means of an integral sensor which is separated from the contents of the sealed primary container yet provides information on quality of the contents of the primary container without breaking the sealed system. The integral sensor device includes a biosensor retained within a plastic construct by a gated-pore membrane. Pores in the membrane open in response to an environmental change in the primary container allowing the contents of the primary container to contact the biosensor. Status of the contents of the primary container can be determined by inspection of the biosensor, visually or via a fiberoptic probe, through the optical window of the plastic construct.
[0010]FIG. 1 is a schematic representation of a stock membrane and an occluded membrane according to the present invention;
[0011]FIG. 2 is a schematic representation of the functioning of six classes of biosensors;
[0012]FIG. 3 is a schematic representation of a device according to the present invention for visual detection of a signal from a biosensor;
[0013]FIG. 4 is a schematic representation of a device according to the present invention for monitoring a change within a closed primary container, when visual detection of changes in the biosensor or configuration of the device is inappropriate;
[0014]FIG. 5 is a schematic representation of a device according to the present invention with an outer opaque element;
[0015]FIG. 6 is a graph of measured light intensity vs. concentration as measured in the device of FIG. 5;
[0016]FIG. 7 is a graph of measured light intensity vs. ratio of labeled beads to plain beads as measured in the device of FIG. 5;
[0017]FIG. 8 is a graph of measured light intensity vs. available fluorescein as measured in the device of FIG. 5;
[0018]FIG. 9 is a graph of measured light intensity vs. pH as measured in the device of FIG. 5, showing pH responsiveness of FITC;
[0019]FIG. 10 is a graph of measured light intensity vs. pH as measured in the device of FIG. 5, showing pH non-responsiveness of phycoerythrin-cyanin-5 strepavidin; and
[0020]FIG. 11 is a graph of the ratio of two wavelengths of emitted light vs. pH as measured in the device of FIG. 5.
[0021] Primary Container
[0022] In this disclosure, we use the term "primary container" in a broad sense, to include any container for cells, foods, or industrial materials.
[0023] Integral Sensor
[0024] In this disclosure, we use the term "integral sensor" to designate one of many devices as disclosed herein. An "integral sensor" typically is incorporated into a primary container, prior to sterilization if appropriate, and separates a biosensor (see below) from the contents of the sealed primary container yet provides information on quality of the contents of the container without breaking the sealed system.
[0025] Gated-Pore Membrane
[0026] In this disclosure, we use the term "gated-pore membrane" in a broad sense, to include any separation barrier containing one or more pores which have been occluded as taught in U.S. Pat. No. 5,261,870, pending patents, or other procedures. Such a separation barrier has one or more pores which initially are closed, but which will open in response to a change in the local environment to an extent sufficient to allow passage of ions or molecules, as appropriate. The occluding material might erode, dissolve, or change three-dimensional form, depending on design of the gated-pore membrane.
[0027] Gate
[0028] In this disclosure, we use the term "gate" in reference to one or more pores through a separation barrier. The gate is "closed" if the pore is occluded to prevent passage of ions or molecules. The gate is "open" if the occluding material has eroded, dissolved or changed three-dimensional form sufficiently to allow passage of one or more species of selected ions or molecules.
[0029] Sensor Compartment
[0030] In this disclosure, we use the term "sensor compartment" in reference to a component of an integral sensor, with said component formed in part from a gated-pore membrane and in part from a plastic construct. The sensor compartment contains and positions a biosensor where it can become accessable to ions, molecules, or cells entering from the primary container and also positions that biosensor where it can be interrogated in a consistent manner through a special optical window or, alternatively, through a portion of the primary container.
[0031] Biosensor
[0032] In this disclosure, we use the term "biosensor" in reference to a component of a device, with said component designed to respond to a molecular change(s) in the local environment in a known and consistent manner with a response or signal that can be detected or measured using procedures known to those skilled in the art. The change in local environment, or microenvironment, might be ionic, molecular or cellular in nature.
[0033] Receptor
[0034] In this disclosure, we use the term "receptor" in reference to any detector molecule, either synthetic or natural in origin, or antibody incorporated into a biosensor. A receptor has reasonable affinity and specificity for one or more "ligands" (see below).
[0035] Ligand
[0036] In this disclosure, we use the term "ligand" in reference to a specific ion(s), molecule(s), or cell(s) accumulating or disappearing within a primary container over time. Accumulation of ligand within the primary container will result in a change in the proportion of unoccupied receptor to ligand-receptor complexes, and a change in characteristics the of a biosensor.
[0037] The present invention consists of devices and methods which allow evaluation of the contents of a sealed primary container by means of an integral sensor which is separated from the contents of the sealed primary container yet provides information on quality of the contents of the primary container without breaking the sealed system. The integral sensor device might be a hollow cylinder or a shallow construct. One end or face of the device is a gated-pore membrane whose pores normally are occluded, by one of many approaches, forming one end of a sensor compartment, containing a biosensor appropriate for the task, with the other end of the sensor compartment formed by an optical window recessed in from the end opposite to the gated-pore membrane or formed by a wall of the primary container. Typically, the integral sensor device is fabricated separately from the primary container and incorporated into the primary container during final fabrication, before sterilization. Certain embodiments of the device are therefore capable of aseptic operation. The status of the contents of a primary container for blood cells, other cells, foods, or industrial products can be determined by inspection, visually or via a fiberoptic probe through the optical window of a plastic construct incorporated into said primary container at fabrication, of the biosensor retained within said plastic construct by a gated-pore membrane, the pores in which opened in response to an environmental change in said primary container allowing the contents of said primary container to contact and cause a change in said biosensor. In one embodiment, after the cell suspension or product is placed into the container, the gated-pore membrane opens and allows continuous exposure of the biosensor, within the sensor compartment, to the contents of the primary container. Changes within the primary container affect the signal from the biosensor, which can be quantified at any instant in time by viewing the optical window of the device with an appropriate sensor, as above, providing a measure of quality without opening the primary container. In another embodiment, after the cell suspension or product is placed into the container, the gated-pore membrane remains closed until a predetermined change occurs within the primary container which causes the gated-pore membrane to open allowing fluid to enter the sensor compartment and contact the biosensor, which then responds with an appropriate signal. Changes in the primary container that can be detected include, but are not limited to, a decrease or increase in pH away from a threshold value or accumulation of one or more members of a preselected class of molecules, including toxins produced by bacteria, above a threshold value. A great range in utility is possible because, depending on the device and method, both the material(s) occluding the gated-pore membrane and material(s) forming the biosensor can be varied independently or in combination. Hence, a predetermined change in contents of the primary container can be evidenced by opening of the gated-pore membrane and/or a change in the signal from the biosensor. A number of other embodiments or uses would be obvious to one skilled in the art, and the examples herein illustrate but do not set the limits of this invention.
[0038] An ideal test should detect one bacterium in the presence of 10,000 platelets, with a low false positive (contaminated) rate, and be reliable regardless of the species of contaminant, simple in complexity, completed in minutes, and low in cost. This might be unattainable, but can be approached by combining emerging technologies in a novel manner. For example, it is known that bacteria can be collected on surfaces using synthetic materials (Hogt et al., 1983; Mackenzie and Rivera-Calderon, 1985; Barrett, 1988; Barth et al., 1989) orbiomaterials (Abraham, et al., 1983; Kuusela et al., 1985; Vercellotti et al., 1985; Speziale et al., 1986; Herrmann et al., 1988). Incorporation of such materials into a gated-pore membrane or biosensor, as disclosed herein, should enhance the probability of detecting such organisms.
[0039] Conventional membranes with pores of known size (e.g., 0.1 or 3.0 m nominal diameter) can be fabricated into special gated-pore membranes, and then fabricated into a container or other device (U.S. Pat. No. 5,261,870). The gated-pore membrane can provide a closed container which opens only when predefined conditions are met, and the material occluding or otherwise closing the pores is altered so that the gates previously blocking passage of molecules through the pores is opened. An almost infinite combination of membranes, pore diameters, and occluding materials is envisioned in U.S. Pat. No. 5,261,870 and other disclosures. Importantly, the conditions on one side of the gated-pore membrane and not mechanical, electrical or other interventions, determine when the pores open and allow passage of molecules through the membrane into the sensor compartment.
[0040] Biosensors can be designed to respond to many molecular changes in the environment. The color change of a pH indicator in response to proton concentration or certain dipsticks to glucose concentration are two common and simple examples. Biosensors sensitive to one or another molecular stimulus can be incorporated into beads or micro-beads, and frequently can be designed to change color or emit light of a given wavelength when exposed to light of an appropriate wavelength (i.e., fluorescence). These or other changes can be monitored with a variety of detectors, ranging from the human eye to fiberoptic electronic devices with a digital readout.
[0041] Obviously, if a gated-pore membrane, with appropriate properties, served to seal an appropriate biosensor within an otherwise impervious container, the gated-pore membrane would: (a) retain the biosensor in place; and (b) determine when, if ever, the biosensor received the stimulus' driving the response. Such a device could be a plastic cylinder, series of concentric cylinders, shallow construct, or of any other shape so long as it positioned the biosensor in a manner conveniently accessible to the detection device. If desirable, the device might include a clear optical window, through which a separate detector could view the biosensor, but would have opaque walls to minimize stray light from falling on the sensor or detector. Although not necessary, frequently, it would be desirable to have the optical window, and the sensor compartment, located more than 1 cm from the open end of the construct (the end opposite from that with the gated-pore membrane). Alternatively, a portion of the wall of the primary container can serve as the optical window to view a biosensor contained in a shallow construct.
[0042] In one embodiment, the gated-pore membrane is fabricated using an occluding polymer such as methylcellulose, or other polymers or mixtures taught in U.S. Pat. No. 5,261,870 with the intent that the pores would remain occluded until a cell suspension had been placed into the primary container incorporating the device. At a predetermined interval (e.g., 5 to 15 minutes) after water contacted the gated-pore membrane, the occluding material would erode or dissolve, the gates open, and free exchange of molecules across the membrane would occur by diffusion and other forces. The biosensor then would start to sample the contents of the primary container, limited only by diffusion within the sensor compartment, diffusion across the gated-pore membrane, and correctness of the sampling site of the external face of the gated-pore membrane. Occasional or continuous movement of the primary container or its contents might facilitate accurate sampling. Changes in the biosensor might be detected visually or via a fiberoptic probe and appropriate instrumentation. In this embodiment, the biosensor provides specificity for detection of molecular changes within the primary container, and the gated-pore membrane retains the biosensor and prevents premature sampling.
[0043] In another embodiment, the gated-pore membrane is fabricated with a special occluding material selected to respond to a specific stimulus and not the mere presence of water. An incomplete list of materials which might be used to occlude the pores in membranes used for this embodiment is provided below. This stimulus opening the gated-pores might be a decline in pH within the primary container, possibly caused by normal cellular metabolism or especially that of contaminating microbes, or accumulation of a secreted bacterial toxin. In this embodiment, the gated-pore membrane remains closed after the cell suspension or product is placed into the container and opens only if and when the triggering stimulus occurs. At that time, the gated-pores open allowing water to pass through the membrane and into the detector compartment, carrying along whatever ions or molecules that might be in the water. The sensor would change from contact with water, for example from a green to a white color discernable by the human eye, or in response to specific molecules or ions entering the detector chamber with the water. In the case of a simple color change in such a pH-sensitive device incorporated into a bag of erythrocytes, visual inspection for a green rather than a white color before use might be sufficient to preclude a decreased pH associated with microbial contamination.
[0044] In these or other embodiments, it is not essential that the material occluding the gated-pore membrane completely block the pores or micropores through the membrane, so long as the molecules to which the biosensor responds cannot gain access through the membrane to the biosensor.
[0045] Utility of such devices is defined by the combination of membrane stock plus materials plugging the pores to form the gated-pore membrane, precision or repeatability in opening of the occluded pores, nature of the biosensor, and sensitivity of the detector plus biosensor to detect the monitored event. Depending on the application, the magnitude of the change to trigger a detected response can be adjusted either via the occluding material or the biosensor. In all cases, response of the detector is not dependent on mechanical or electrical forces, although readout might require an instrument. With appropriate instrumentation, signals in a remote or isolated area can be detected.
[0046] Membrane Stock Materials
[0047] Membrane stock used to form the gated-pore membrane can be any of those commercially available, e.g., fibril membranes with a "haystack" structure, microporous membranes with a "sponge-like" structure, and track-etched or capillary-pore membranes with a "tunnel" structure. Although others have used membranes, either preformed or formed in place, to position biosensors against a fiberoptic or other detector, gated-pore membranes have not been used for this purpose. Gated-pore membranes for this application could be prepared using microporous membrane stock. However, characteristics unique to capillary-pore membrane stock frequently make this the material of choice to form gated-pore membranes for use in biosensor detectors as described herein. Alternatively, one might use a stock membrane which incorporates the ability to change in response to pH (Maeda et al., 1984; Kinoshita et al., 1994).
[0048] Materials to Occlude Pores in Membrane Stock
[0049] Materials to occlude preformed pores in a stock membrane and, thereby, form a gated-pore membrane, might be selected from members of the classes listed below, but these are illustrative and not inclusive. A generic resultant gated-pore membrane is illustrated in FIG. 1. Importantly, occluding material within the pores is the functional element rather than residual occluding material that might be retained on the membrane faces around the pores. Many of the potentially useful materials have been used for other applications, but none has been used to fill preformed pores in a membrane to control access of material on one side of the membrane to a biosensor positioned behind the membrane where accessible to a detector. All materials applicable for this application are considered as under the scope of this invention, recognizing that in some instances the material might be patented.
[0050] 1. Non-protein polymers responding to changes in pH by dissolution or a change in hydration and volume such as carboxymethyl or diethylamino cellulose. When used to occlude pores in conventional membrane stock, such polymers shrink (open the "gate") in response to pH.
[0051] 2. Protein polymers responding to changes in pH or other environmental factor(s), by a change in conformation or other attributes. Such proteins become longer or shorter, or greater or lesser in globular vs. linear or helical nature, in response to pH (Ito et al., 1990, 1997; Huyghues-Despointes et al., 1993; Holtzer, 1994; Spek et al., 1995; Urry, 1997) and thus open or close the "gates".
[0052] 3. Lipid bilayers or similar lipid-containing composites responding to an increased concentration of a bacterial product, such as a secreted toxin (e.g., porin-type toxins secreted by E coli, Staphyloccus, or Streptococus). Numerous methods might be used to prepare lipid bilayers (U.S. Pat. No. 5,368,712; Sackmann, 1996) supported by the structure of tunnels within a capillary-pore membrane.
[0053] Materials to Form or Coat Biosensors
[0054] Understanding of a biosensor is facilitated by analogy to classic receptor-ligand interactions, where in respect to this invention the term "receptor" is any detector molecule, either synthetic or natural in origin, or antibody bound on a solid substrate, termed the biosensor, and the term "ligand" is a specific ion(s), molecule(s), or cell(s) accumulating or disappearing within a primary container over time. The receptor has reasonable affinity and specificity for the ligand. Accumulation of ligand within the primary container will result in a change in the proportion of unoccupied receptor to ligand-receptor complexes. Appropriate selection of the ligand and receptor molecules allows detection of this change in ratio of unoccupied vs. occupied receptors by an altered spectroscopic property from either of the separate ligand or receptor molecules or their complexes. Alternatively, a signal moiety might be conjugated to the receptor and provide amplified evidence of the status as unoccupied or occupied. If appropriate, the change in ratio of unoccupied vs occupied receptors might be amplified by a property intrinsic to the solid substrate.
[0055] There is no limit to biosensors appropriate for this device, insofar as they undergo a change in signal character, upon exposure to the change in microenvironment to be detected, which can be detected or measured by any form of spectroscopy (e.g., luminescence, fluorescence, absorbance, infrared, magnetic, light scatter, etc.). In certain cases, the material or cells stored can provide the requisite signal. However, use of special biosensors increases the nature of changes that can be monitored. Many of the potentially useful biosensors have been used for other applications, but none has been positioned in a viewable compartment formed in part by a gated-pore membrane initiating access of the molecules, ions or bacteria to be detected and formed in part by an optical window allowing detection of the signal resulting from the biosensor. Biosensors often contain two active elements; namely, the detector material(s) and the signal material(s), which frequently are combined within a combination molecule.
[0056] Although we use plastic beads as the example solid substrate, this invention is not limited in this respect. The solid substrate can have any three-dimensional shape; include impervious or permeable materials; include smooth, microporous or complex surfaces; etc. This patent is not limited by the nature or shape of the solid substrate. Similarly, this invention is not limited by the method used to attach the detector molecules to the solid substrate.
[0057] Detector molecules can be attached to the surface of the biosensor (e.g., plastic or glass bead) by simple adsorption, direct covalent attachment, or via an affinity tag (Anonymous 1999a, 1999b; Nolan et al., 1999; Song and Swanson, 1999). Alternatively, the solid surface might be pre-coated with lipids and then lipophilic ligand-detector molecules partitioned therein (Nolan et al., 1999). If not in a combination molecule with the detector material, the signal material must respond to the change in status of the detector material (occupied vs unoccupied) and the change in the signal must be a change in a characteristic absorption or emission property within the electromagnetic spectrum. An absorbance or fluorescent signal is most common.
[0058] Any biosensor appropriate for this application is considered as under the scope of this invention, recognizing that in some instances the biosensor might be patented. In this discussion we will consider a bead-shaped biosensor, but all shapes are claimed. Appropriate concepts for biosensors might be considered in several classes (shown schematically in FIG. 2; for easy reference, given designations herein) which include, but are not limited to:
[0059] Class-1: A bead coated with a fluorescent receptor targeting an epitope or ligand characteristic of, or secreted by, bacteria of interest and which fluoresces only when its conformation is altered by binding of the ligand. Detection of bound ligand is via fluorescence when the biosensor is illuminated with light of an appropriate wavelength.
[0060] Class-2: A bead coated with lipid in which are positioned appropriate fluorochrome-receptor complexes, anchored therein, which fluoresce only when their conformation is altered by binding of the ligand. Detection of bound bacterial products is via fluorescence when the biosensor is illuminated with light of an appropriate wavelength.
[0061] Class-3: A bead coated with lipid or otherwise prepared in which are positioned low concentrations of two different fluorescent receptors each binding the ligand, so that concurrent binding of a ligand molecule by both receptors brings them in closer proximity and enables charge-coupling excitation to produce light of a unique wavelength when the biosensor is illuminated with light of an appropriate wavelength. In this case, appropriate external excitation causes only one receptor to fluoresce and although that light is not detected it does excite, by fluorescence resonance transfer, the second receptor and causes it to emit light of a unique wavelength detectable by the fiberoptic probe. Because fluorescence of the second receptor is dependent on proximity to the first, achieved only by both binding to ligand, such systems can be very specific.
[0062] Class-4: A bead coated with lipid or otherwise prepared to accept a mixed-matrix, in which one receptor-A is designed to bind a certain class or type of cell, which after binding continues to release material serving as the ligand bound by the nearby second receptor-B, causing a rapid shift from unoccupied to occupied receptor-B molecules and a detectable spectral change. Positioning a chemoattractant on the biosensor could further enhance sensitivity of a mixed-matrix surface.
[0063] Class-5: A bead coated with lipid or otherwise prepared to accept a detection substrate-fluorochrome which can serve as a substrate for a specific class of enzymes secreted by bacteria or other cells. In this biosensor, the substrate is designed to contain two fluorescent groups with one on either side of the enzymatic cleavage site in close proximity while the molecule is intact but substantially farther apart after the substrate is cleaved. Local action of the enzyme on the substrate releases the fluorescent inhibition caused by close proximity, and the detector molecule will fluoresce when illuminated with light of an appropriate wavelength. Bark and Kahn (2000) provided examples.
[0064] Class-6: A bead coated with a mixed matrix combining elements of Class-4 and Class-5. As in Class-4, receptor-A binds the cell of interest, without or with help from a chemoattractant, so that the cell continues to release an enzyme in close proximity to a substrate incorporating the fluorescent reporter B-C, as in Class-5. The enzyme from the localized cell (e.g., a bacterium) acts on the substrate and this releases the fluorescent inhibition caused by close proximity, so that the detector molecule will fluoresce when illuminated with light of an appropriate wavelength.
[0065] Detection of Signal Emanating from a Biosensor
[0066] Assuming incorporation of a fluorescent moiety (e.g., a fluorescent-protein biosensor; Giuliano and Taylor, 1998), flow cytometry is common (microsphere analyte analysis; Morgan et al., 1996; McHugh, 1994; Nolan et al., 1999; Anonymous 1999c, 1999d). Flow cytometry of biosensors has been used to quantify protease, phospholipid antibodies, and water-soluble toxins (St. Piere et al., 1996; U.S. Pat. No. 5,605,809; Laakel et al., 1996; Song et al., 1998). Other materials have been analyzed in solution, but could be used in a solid-phase assay (e.g., zinc and related cations or nucleic acids; Goodwin and Berg, 1996; Tyagi and Kramer, 1996). The other common approach to interrogate the biosensor is via a fiberoptic probe (various shapes, including microspheres; as above).
[0067] Detection of a change intrinsic to the contents of a container or via a special biosensor (e.g., with a fluorescent reporter) requires an optical probe (Maroze et al., 1999), either an integral and fixed fiberoptic probe or a separate and removable fiberoptic probe. A fiberoptic probe integral to the container to be analyzed might read an inherent spectral property of materials within the bag. Alternatively, an integral fiberoptic probe might indirectly monitor changes in internal contents via reading a change in an optical property of a biosensor element when the material of interest reaches an appropriate concentration in the immediate environment of the biosensor.
[0068] In known devices, the biosensor is immobilized by covalent bonding to the face of the fiberoptic bundle or the biosensor is restrained close to the fiberoptic bundle with a perm-selective membrane. Such systems have been used to monitor anions, cations, biomass, glucose, lactic acid, NAD(P)H, O2, CO2 and pH (Agayn and Walt, 1993; Kar and Arnold, 1995; Marose et al., 1998; Spichiger-Keller, 1997; Tartakovsky et al., 1996; Urbano et al., 1984; Vaccari et al., 1994; Weigl et al., 1994; Xu et al., 1998). These approaches cannot be used with a closed container.
[0069] Hitherto, the only option for a closed container was to position a removable fiberoptic probe against an optical window in the primary container to gather information from the materials within the container. However, information gathering has been limited to spectral properties inherent to the materials within the container (e.g., light scatter from biomass, fluorescent properties of metabolites such as NAD(P)H; Cavinato, et al., 1990).
[0070] A device to interrogate changes in a closed primary container logically might include an immobilized biosensor, sampling a specific attribute of the contents of said closed primary container, with said biosensor protected in a separate closed sub-container within said device, with said biosensor retained in front of an optical window, with said optical window accessible to a removable fiberoptic detector without opening either said primary container or said sub-container, and with said device capable of facile incorporation into said primary container at the time of manufacture before sterilization. Solution of this hitherto unattainable goal is the subject of this invention. This is achieved by combination of existing technology on gated-pore membranes; bioactive polymers, proteins or lipid films; and biosensors in a novel manner and adding flexibility offered by new materials and configurations in novel devices. Clearly, several elements are combined in a novel manner to provide such devices.
[0071] In addition to the devices disclosed herein, uses of the functional elements within the described devices differ from uses in the prior art in several ways. Uses disclosed herein are not obvious extensions of the prior art.
[0072] These novel elements include:
[0073] 1. In respect to bioactive non-protein or protein-polymers used to occlude pores and form a gated-pore membrane, the prior art does not teach the use of:
[0074] (a) a bioactive material bound uniformly and primarily to the interior of tunnels of uniform diameter within a preformed membrane to provide a membrane completely blocking fluid flow until the membrane is exposed to the selected altered environment, after which flow rates for water, molecules or ions are similar to those through the raw membrane stock; or
[0075] (b) black-lipid bilayers to occlude pores in a preformed membrane with the intent of forming gated-pores, opening after exposing the membrane to an appropriate environmental stimulus including such as bacterial toxins.
[0076] 2. In respect to uses of biosensors, the prior art does not teach a method to position biosensors within a closed primary container in a manner where, after a pre-selected event, they can monitor and display changes within the primary container for repeated interrogation with a separate detector.
[0077] 3. In respect to receptor-signal molecules of the biosensor, the prior art does not teach the use of:
[0078] (a) pH-sensitive fluorescent complexes formed by attaching a fluorescein-dextran conjugate to carboxyl groups on a bead to provide a Class-1 receptor, allowing repeated measurements of pH by appropriate excitation and detection;
[0079] (b) fluorescent receptor binding α-hemolysin, or other molecule, which changes conformation from non-fluorescent to fluorescent upon occupancy to provide a Class-2 receptor, allowing repeated measurements of soluble ligands released by bacteria by appropriate excitation and detection;
[0080] (c) a fluorescent heparin-like receptor, receptor for other natural cell surface molecule, or a custom synthesized receptor, with either type binding specific ligands to provide a Class-3 receptor, allowing repeated measurements of bacterial accumulation or accumulation of unique bacterial products based on fluorescent resonance transfer during appropriate excitation and detection; and
[0081] (d) a mixed matrix such as a heparin-like receptor to bind bacteria combined with an α-hemolysin receptor conjugated to a fluorochrome to provide a Class-4 receptor, allowing repeated measurements of accumulation of unique bacterial products based on fluorescent resonance transfer during appropriate excitation and detection.
[0082] 4. In respect to detection of the signal emanating from the biosensor, the prior art does not teach the use of fiberoptic probes to interrogate biosensors that are of special design and entirely separate from the probe, without placing the fiberoptic probe into the contents of each primary container.
[0083] 5. In respect to collection of bacteria on, or in close proximity to, the biosensor, the prior art does not teach the use of synthetic materials or biomaterials to enhance probability of detecting such organisms.
[0084] The Examples illustrate how biosensors, as above, can be positioned in front of an optical window, accessible to a removable fiberoptic detector, while protected in a separate closed sub-container within the primary container. They are illustrative of the many uses of the invention, although the invention encompasses other biosensors, other membranes with open pores or gated-pores, other configurations, and other combinations of functional elements in the device. Examples of devices include reference to welding of a gated-pore membrane to a plastic construct. The invention is not limited by the plastic(s) used to form the device. The preferred welding process will depend on the plastic used to fabricate the construct, and the invention includes any method to assemble the device or incorporate it into the primary container, such as ultrasonic and radio-frequency welding or other methods known to those skilled in the art. Examples illustrate the variety of materials that can be used to form gated-pore membranes or serve as biosensors, in one or more types of devices, and the invention is not limited in respect to specific compositions or analytes.
[0085] While examples provided illustrate detection of "unwanted agents", the principles disclosed also can be used to detect or measure accumulation of materials deemed to be beneficial to the system. An example would be signaling production of growth factors, a product of commercial value, by cells within a primary chamber.
[0086] The following list describes the Figs. in greater detail:
[0087]FIG. 1 Schematic representation of a gated-pore membrane prepared using a microporous or capillary-track membrane and unique material to occlude the pores (e.g., as in U.S. Pat. No. 5,026,342).
[0088]FIG. 2 Schematic representation of changes in a receptor-fluorescent reporter group upon change from unoccupied receptor to occupied receptor-ligand complex, listed by biosensor class as detailed in the specification. A reporter group that is non-fluorescent is designated with a lower-case letter and a reporter group that can fluoresce when excited with light of appropriate wavelength is designated with an upper-case letter.
[0089]FIG. 3 Cross-section through a simple boat-shaped device as described in Example 1. The capillary-pore membrane (302) was processed with material (303) to plug the pores and make a gated-pore membrane (304). This membrane is bonded to a plastic construct (301) to form a cavity which then was filled with a biosensor (305) before the device was bonded via a flange (301 a) to the primary container (306).
[0090]FIG. 4 Longitudinal section through a device, and also a top view, as described in Example 2. The biosensor-compartment in a cylindrical construct (411) was filled with a biosensor (405). A gated-pore membrane (404) was bonded to a recessed surface (411 a) to seal the biosensor in the device. The device then can be incorporated into the seam of a primary container (not shown) so that the optical window (412) can be interrogated with a fiberoptic probe.
[0091]FIG. 5 Longitudinal section through a device, and also a top view, as described in Example 3. An optically clear cylindrical construct (511) was surrounded by an opaque outer concentric construct (515). The biosensor compartment was filled with a biosensor (505). A gated-pore membrane (504) was bonded to a recessed surface (515 a) of the opaque outer concentric construct (515) to seal the biosensor in the device. The device then can be incorporated into the seam of a primary container (not shown) so that the optical window (512) can be interrogated with a fiberoptic probe.
[0092]FIG. 6 Intensity of fluorescence from a solution of fluorescein isothiocyanate (FITC) in a device as a function of concentration of FITC. Device as in FIG. 5; measurements with fiberoptic detector using excitation at 480 nm and detection at 535 nm (thousands of fluorescent units).
[0093]FIG. 7 Intensity of fluorescence from beads in a device as a function of the ratio of beads labeled with fluorescein isothiocyanate (FITC) to plain beads. Device as in FIG. 5; measurements with fiberoptic detector using excitation at 480 nm and detection at 535 nm (thousands of fluorescent units).
[0094]FIG. 8 Intensity of fluorescence from beads in a device as a function of their known content of fluorescein isothiocyanate (FITC). Known contents of FITC were equivalent to 0.00, 0.22 and 1.20 million molecules of FITC in solution. Device as in FIG. 5; measurements with fiberoptic detector using excitation at 480 nm and detection at 535 nm (millions of fluorescent units).
[0095]FIG. 9 Intensity of fluorescence from beads, previously labeled with fluorescein isothiocyanate (FITC), in a device as a function of pH of the salts solution around the device and around the beads therein. Device as in FIG. 5; measurements with fiberoptic detector using excitation at 480 nm and detection at 535 nm (thousands of fluorescent units).
[0096]FIG. 10 Minimal effect of pH of a salts solution on fluorescence from phycoerythrin-cyanin-5 strepavidin attached to beads. Device as in FIG. 5; measurements with fiberoptic detector using excitation at 480 nm and detection at ≧640 nm (thousands of fluorescent units).
[0097]FIG. 11 Ratio of emissions from beads responsive to pH and non-responsive to pH can be used to normalize data for irregularities of response or color of the medium. The device contained a mixture of beads labeled with fluorescein isothiocyanate (pH responsive; FIG. 9) and phycoerythrin-cyanin-5 strepavidin (pH non-responsive; FIG. 10). The ratio of emission at 535 nm and ≧640 nm was calculated as a function of pH of the salts solution around the device and around the beads therein. Device as in FIG. 5; measurements with fiberoptic detector using excitation at 480 nm and detection at 535 nm and ≧640 nm (raw values ranged between 75,000 and 135,000 relative fluorescent units).
[0098] The invention is further illustrated in the following examples:
[0099] A simple device (FIG. 3) can be used to monitor one of a variety of changes within a closed primary container. It is formed from a shallow, plastic construct (301) with an external flange (301 a) on one side and a gated-pore membrane (304) welded to the other side. After filling the construct with a biosensor (305) providing a signal detectable by a human eye (e.g., plastic beads that change color when bathed in an aqueous fluid), the construct is welded to a convenient location on the wall of the primary container (306). This device depends on selectivity in response of the gated-pore membrane as the primary mechanism regulating change in a simple biosensor. When thus positioned, changes in the signal from the biosensor can be detected visually through a portion (307) of the primary container, without opening the primary container.
[0100] Another device (FIG. 4) can be used to monitor one of a variety of changes within a closed primary container, when visual detection of changes in the biosensor or the configuration of the device in Example 1 is inappropriate. This device can be of any convenient shape and formed from any plastic, but a cylindrical plastic construct formed from clear acrylic plastic is favored for some applications, and a clear optical window transmitting the portion of the spectrum of interest is essential. The device of FIG. 4 is a cylindrical acrylic construct (411) with a biosensor compartment formed by an optical window (412), a gated-pore membrane (404) responding to any aqueous environment, and the walls of the construct. After the biosensor compartment is filled with a biosensor (405) capable of detecting the desired change within the primary container, the gated-pore membrane is welded to the surface (411 a) of a recess in the end of the construct. After the device is incorporated into a primary container (not shown) and the container is filled with a suspension of cells of interest, the biosensor can be interrogated without opening the primary container, when desired, by positioning the tip of a separate fiberoptic probe (not shown), as known to those skilled in the art, against the optical window (412). This device uses the gated-pore membrane simply to protect and isolate the biosensor until the primary container is filled, for example with a cell suspension, after which the gates open in <30 minutes and selectivity in response to a predetermined analyte and intensity of signal are properties of the biosensor and amount of material impinging thereon.
[0101] A third device (FIG. 5) incorporates an opaque outer plastic element (515), with accommodation (515 a) to weld a gated-pore membrane (504) to this part of the device, positioned around and bonded to an inner element (511) which is a clear plastic construct similar to the device of Example 2 and contains a biosensor (505) capable of detecting the desired change within the primary container. This device can be of any convenient shape and formed from any plastic, but a cylindrical concentric construct is favored for some applications. The outer opaque element (515) might be of colored polyvinyl chloride to shield the biosensor from stray light and facilitate welding the device into a polyvinyl chloride blood bag, and the inner element of clear plastic so that the optical window (512) will transmit the portion of the spectrum of interest. This device can be used to monitor one of a variety of changes within a closed primary container, when stray light might affect detection of the signal from the biosensor, by positioning the tip of a separate fiberoptic probe (not shown), as known to those skilled in the art, against the optical window (512). This device uses a gated-pore membrane to protect and isolate the biosensor until the primary container is filled and for a predetermined interval (e.g., 0.5, 2 or 5 days) thereafter or until a predetermined threshold of a specific change in contents of the primary container is reached (e.g., concentration of first analyte), after which the gates open and the biosensor begins to monitor changes in concentration of the same molecule or ion triggering the gated-pore membrane. Alternatively, the biosensor can be used to monitor concentration of a second analyte with intensity of the signal from the biosensor dependent on properties of the biosensor and amount of second analyte impinging thereon.
[0102] Capability of a device as in FIG. 5 to detect changes in fluorescence was established. A commercial fiberoptic spectrometer (S2000-FL; Ocean Optic, Inc ) was equipped with optic filters suitable for desired analysis (excitation at 480 nm and detection of emission at 535 nm), and interfaced to a computer as recommended by the manufacturer. This was used to evaluate a series of solutions and particle suspensions. First, standard solutions of fluorescein isothiocyanate (FITC) were prepared and 0.075 ml aliquants were placed within the biosensor compartment (505). The intensity of fluorescence from FITC was recorded. There was a linear increase in measured intensity with increasing concentration of FITC (FIG. 6). Next, mixtures microspheres (6-8 micrometers, Spherotech Inc.) without and after conjugation with FITC were prepared. In succession, several ratios of labeled to unlabeled beads were placed into the biosensor compartment (505), in 0.05 ml. Intensity of fluorescence from FITC was recorded for each mixture. There was a linear increase of intensity of measured fluorescence from FITC as the proportion of labeled beads increased (FIG. 7). This established the capability of the external fiberoptic detector to correctly assess emission output from a chromophore, on a solid support as anticipated for biosensors accomplishing designated tasks in detection of known ligands, placed within the biosensor compartment.
[0103] A series of beads of known FITC content (Quantum 25-#825; Flow Cytometry Standards Corp) was purchased, and 0.05 ml of each suspension were placed in a device as in FIG. 5. A linear response was noted (FIG. 8). Importantly, the coefficient of variation for replicate measurements using the fiberoptic detector and beads (505) within the biosensor compartment was within the coefficient of variation provided for these standards by the supplier, and presumably measured under optimum conditions. To provide a relation to data of FIG. 6, the bead suspensions also were evaluated relative to a standard curve of known amounts of FITC in a microplate fluorometric reader. The highest concentration of beads tested was equivalent to 4 nM FITC.
[0104] Beads suitable for use as an "optical bench" were prepared by covalent attachment of dextran (#1820; Molecular Probes) containing both FITC and lysine to 10 micrometer diameter carboxylate beads (Polysciences, Inc), using standard carbodiimide coupling chemistry. This provided an immobilized fluorophore, known by those versed in the art to be responsive to pH through the FITC function and suitable for analysis of pH. Localization of the FITC at a sufficient distance from the bead surface, by means of the dextran, eliminated problems related to the well-known surface effects on fluorescence. The biosensor cavity was filled with the bead suspension (505), and the membrane (504) attached. Beads were allowed to settle onto the optical window (512). The filled cylindrical construct then was placed into a phosphate buffered saline solution with pH and temperature (21.5�0.5 C.) monitored using a standard laboratory meter. Acid or base was added to change the pH, and fluorescence intensity was measured after allowing 30-60 minutes for equilibration. Resulting data (FIG. 9) clearly established that pH in the microenvironment of a biosensor, within the device of FIG. 5, can be measured with an external detector. Most importantly, the anticipated pH-related change in intensity of fluorescence detected was similar to published data (i.e., linear 2.0-2.5 fold increase in intensity between pH 6.0 and pH 8.0). Additional experiments (data not presented) established, as in Example 4, that unlabeled beads could be added to fill the chamber without degradation of signal intensity. Note that we allowed a 30 minute response time to insure stability of conditions around the beads after adding acid or base. However, response time could be reduced substantially by agitation of the primary container, or by design of the integral sensor to increase the amount of gated-pore membrane enclosing the biosensor cavity and thus reduce time for protons or other ligands to enter and interact with the biosensor.
[0105] Samples to be analyzed might contain components that will interfere with direct fluorescence measurements. For example, it is well-known that human plasma can contain variable amounts of "yellow" materials that could absorb incident light at the excitation wavelength. Optical density at 480 nm was measured for 13 different samples of human blood plasma (cell free), and absorbance averaged 0.554 (standard deviation was 0.428; range from 0.12 to 1.30), but was much lower at >500 nm. Clearly, this wide a range in absorbance could affect the light available for excitation of a biosensor incorporating, for example, FITC as the signal moiety.
[0106] Use of an internal standard is a classic analytical approach to reduce impact of non-uniformity in the matrix of a signal system. A second detector bead system (B-2) could be constructed so that it shared excitation wavelength with the primary bead (B-1; e.g., FITC), but emitted at a distinctly different wavelength than B-1 and was unaffected by the ligand altering response of the biosensor of bead B-1 (e.g., protons in case of FITC). This would allow excitation of both fluorochromes concurrently under identical conditions, and alternating measurement of emission from B-1 and B-2. Calculation of the ratio B-1/B-2 would allow detection of changes in emission from B-1 without substantial impact from a wide range in absorbance of exciting light by components in the matrix. This approach should be useful with products ranging from blood cells to orange juice.
[0107] To illustrate utility of such an approach, a general bead coating was developed by covalently attaching biotin-dextran (D-1956; Molecular Probes) to 10 micrometer diameter carboxylate beads (Polysciences, Inc.), using standard carbodiimide coupling chemistry. This provided a common attachment site on the beads for avidin-strepavidin conjugates. Assuming beads of biosensor B-1 used FITC, materials suitable for service as B-2 should be excited (absorb) at 480 nm, emit at a wavelength distinct from 535 nm, and have a response independent of pH. Examples include reagents available from Coulter Diagnostics such as: Strepavidin-PC5 (strepavidin conjugated to phycoerythrin-cyanin 5; excitation 486-580; emission 660-680) and EDC (strepavidin conjugated to phycoerythrin-Texas Red7-x; excitation 486-580; emission 610-630). It is well-known to those versed in the art that many other combinations can be used to correct for effects on either excitation or emission wavelengths. We used the PC-5 system and evaluated freedom from a pH-driven shift in emission. As evident in FIG. 10, with excitation at 480 nm and emission (from the cyanin-5) measured using a ≧640 long-pass filter, there was little change in intensity of emission between pH 6.0 and pH 8.5. This was very different from the response of FITC (FIG. 9).
[0108] The concept of a ratio correction (see above) and observations from Examples 6 and 7 were directly tested to illustrate utility of a ratiometric analysis of pH using an integral sensor as in FIG. 5. Two different biosensor beads were prepared, mixed in an appropriate ratio, and placed (505) within the cavity of the integral sensor, fabricated from a type of acrylic plastic known to have optical properties appropriate for this application. The gated-pore membrane (504) was fabricated from capillary-pore membrane stock (RoTrac� polyester membrane, Oxyphen AG; 3 μm pore diameter; 60 g/m2 polyester backing) using methylcellulose to occlude the pores, and the resulting gated-pore membrane was used to seal beads B-1 and B-2 into the device. The pH sensitive biosensor (B-1) was FITC dextran attached to a polystyrene support (as in Example 6) and the pH insensitive biosensor (B-2) was EDC-strepavidin attached to biotin/dextran polystyrene (see Example 7). The same fiberoptic fluorometer (S2000-FL; Ocean Optic, Inc.) was used to interrogate the device, as pH of the salts solution was changed as in Example 6. The system was excited at 480 nm and emission read with both the 535 nm and ≧640 nm filters. Resultant data (FIG. 11) show excellent linearity of response in the range of interest to blood banks, pH 6.0 to 7.0. Use of human erythrocytes for infusion into a recipient is ill advised if the contents are at pH ≦6.2. Detection of pH within a primary container (i.e., conventional plastic blood bag) containing erythrocytes or platelets should be easy if the bag was fabricated to include a device as in Example 8. Resultant measurements of pH should not be affected by bag-to-bag differences in background colors typical of blood products.
[0109] A device, fabricated as in FIG. 4 or 5, can use a Class-5 biosensor (see FIG. 2) to detect accumulation of a protease secreted by bacteria within a closed primary container. Active elements of the integral detector would be as detailed above, except that plastic beads would be coated with "doped" lipid to provide a Class-5 biosensor. The lipid would be "doped" with an enzyme substrate synthesized with fluorescent moieties ("a" and "b"; inhibiting each other's emission because of proximity) incorporated on either side of the proteolytic cleavage site. Thus, fluorescence of the enzyme substrate is mutually inhibited by close proximity of receptors "a" and "b" if the substrate molecule is intact. However, enzymatic cleavage of the substrate allows separation of the fluorescent moieties associated "a" and "b" and emission of a detectable signal during interrogation with exciting light of the appropriate wavelength. One example of such a biosensor suitable for attachment to an avidin-strepavidin coated bead is: biotin-(6-aminohexanoicacid)-(6-aminohexanoicacid)-(6-aminohexanoicacid)-Lys(Tamara)-(6-aminohexanoicacid)-Ala-Phe-Glu-Ala-Leu-(6-aminohexanoicacid)BCys(Texas Red)-Gly-COOH. The "-Ala-Phe-Glu-Ala-Leu-" sequence should be useful for detecting proteases known to be secreted from a variety of bacteria including Bacillus cereus, Escherichia coli, Poteus mirabilis, Serratia marcescens, Staphyloccous aureus, and Streptococcus species. Other similar substrates, exhibiting appropriate susceptibility to hydrolases, can be designed as appropriate, using assumptions known to those skilled in the art.
[0110] A device, fabricated as in FIGS. 4 or 5, can use a Class-6 biosensor (see FIG. 2) to accumulate bacteria and increase sensitivity for detection of a protease secreted by bacteria within a closed primary container. Active elements of the integral detector would be as detailed above, except that plastic beads "doped" lipid would be used to fabricate a Class-6 biosensor. A material binding the bacteria of interest (e.g., fibronectin, laminin, type-4 collagen) would serve as receptor "a" (see FIG. 2). The coating of this biosensor also would include an enzyme substrate synthesized with fluorescent moieties ("b" and "c"; inhibiting each other's emission because of proximity) incorporated on either side of the proteolytic cleavage site. As described in Example 9, the enzyme substrate might be: biotin-(6-aminohexanoicacid)-(6-aminohexanoicacid)-(6-aminohexanoicacid)-Lys(Tamara)-(6-aminohexanoicacid)-Ala-Phe-Glu-Ala-Leu-(6-aminohexanoicacid)BCys(Texas Red)-Gly-COOH. Streptococus pyrogenes are known to bind to the fibronectin serving as receptor "a" and secrete proteases that can cleave the -Ala-Phe-Glu-Ala-Leu- peptide integral to receptor "b" and "c", removing the proximity inhibition to their fluorescence. Consequently, as Streptococus pyrogenes, or other bacteria, proliferated in the primary container they would accumulate on the biosensor and the fluorescent signal detectable on probing with a fiberoptic fluorometer would reflect the extent of bacterial contamination in the primary container.
[0111] This invention is very broad and the examples presented herein are illustrative, but do not limit the scope of the invention.
[0112] U.S. Pat. No. 5,261,870 1991 Hammerstedt et al. Separation barrier with plugged pores.
[0113] U.S. Pat. No. 5,368,712 1994 Tomich et al. Biological mimetic synthetic ion channel transducers.
[0114] U.S. Pat. No. 5,605,809 1997 Komorlya and Packard Compositions for the detection of proteases in biological samples and methods of use thereof.
[0115] U.S. Pat. No. 5,7111,915 1998 Siegmund et al. Optical solid-phase biosensor based on polyionic layers labeled with fluorescent dyes.
[0116] Abraham S N, Beachey E H, Simpson W A. 1983 Adherence of Streptococcus pyrogenes, Escherichia coli, and Pseudomonas aeruginosa to fibronectin-coated and uncoated epithelial cells. Infect Immun 41:1261-1268.
[0117] Agayn V I, Walt D R. 1993 Fibre-optic sensor for continuous monitoring of fermentation pH. Biotechnology 11:726-729.
[0118] Anonymous. 1999a Technote #204 Adsorption to microspheres. Banks Laboratories, Fishers Ind.
[0119] Anonymous. 1999b Technote #205 Covalent Coupling. Banks Laboratories, Fishers Ind.
[0120] Anonymous. 1999c Technote #302 Molecular Biology. Banks Laboratories, Fishers Ind.
[0121] Anonymous. 1999d Technote #301 Immunological Applications. Banks Laboratories, Fishers Ind.
[0122] Bark S J, Hahn K M. 2000 Fluorescent indicators of peptide cleavage in the trafficking compartments of living cells: Peptide site specifically labeled with two dyes. Methods 20:429-435.
[0123] Barrett S P. 1988 Bacterial adhesion to intravenous cannulae: influence of implantation in the rabbit and of enzyme treatments. Epidem Inf 100:91-100.
[0124] Barth E, Myrvik Q M, Wagner W, Gristina A G. 1989 In vitro and in vivo comparative colonization of Staphyloccus aureus and Staphyloccus epidermis on orthopaedic implant materials. Biomaterials 10: 325-328.
[0125] Blajchmann M A. 1998 Bacterial contamination and proliferation during the storage of cellular blood products. Vox Sang 74:155-159.
[0126] Cavinato A G, Mayes D M, Ge Z, Callis J B. 1990 Noninvasive method for monitoring ethanol in fermentation processes using fibre-optic near-infrared spectroscopy. Anal Chem 62:1977-1982.
[0127] Giuliano K A, Taylor D L. 1998 Fluorescent-protein biosensors: new tools for drug discovery. Tibtech 16:135-140.
[0128] Godwin H A, Berg J M. 1996 A fluorescent zinc probe based on metal-induced peptide folding. J Amer Chem Soc 118:6514-6515.
[0129] Herrmann M, Vandaux P E, Pittet D, Auckenthaler R, Lew P D, Schumacher-Perdreau F, Peters G, Waldvogel F A. 1988 Fibronectin, fibrinogen, and laminin act as mediators of adherence of clinical Staphylococcal isolates to foreign material. J Infect Diseas 158:693-701.
[0130] Hogt A H, Dankert J, DeVris J A, Feijen J. 1983 Adhesion of coagulase-negative Staphylocci to biomatierals. J Gen Microbiol 129:2959-2968.
[0131] Hogt A H, Dankert J, Feigen J. 1986 Adhesion of coagulase-negative staphylococci to methacrylate polymers and copolymers. J Biomed Mater Res 20:533-545.
[0132] Holtzer A. 1994 Application of old and new values of - helix propensities to the helix-coil transition of poly(L-glutamic acid). J Amer. Chem. Soc 116:10837-10838.
[0133] Hughes-Despointes B M P, Scholtz J M, Baldwin R L. 1993 Helical peptides with three pairs of Asp-Arg and Glu-Arg residues in different orientations and spacings. Polymer Sci 2:80-85.
[0134] Ito Y, Kotera S, Inaba M, Kono K, Imanishi Y. 1990 Control of pore size of polycarbonate membrane with straight pores by poly(acrylic acid) grafts. Polymer 31:2157-2161.
[0135] Ito Y, Inaba M, Chung D-J, Imanishi Y. 1992. Control of water permeation by pH and ionic strength through a porous membrane having poly(carboxylic acid) surface grafted. Macromolecules 25: 7313-7316.
[0136] Ito Y, Ochiai Y, Park Y S, Imanishi Y. 1997 pH-sensitive gating by conformational change of a polypeptide brush grafted onto a porous polymer membrane. J Amer Chem Soc 119:1619-1623.
[0137] Kar S, Arnold M A. 1995 Fibre-optic chlorine probe based on fluorescence decay of N-(6-methoxyquinolyl)-acetoethyl ester. Talanta 42:663-670
[0138] Kinoshita T, Kakiuchi T, Takizawa A, Tsujita Y, Oya M., Iizuka Y, Iwatsuki M. 1994 Solute permeability enhancement at a specific pH by an amphiphlic copolypetide membrane. Macromolecules 27:1389-1394.
[0139] Kuusela P, Vartio T, Vuento T, Myhre E B 1985 Attachment of Staphylococci and Streptococci on fibronectin, fibronectin fragments, and fibrinogen bound to a solid substrate. Infect Immuni 50:77-81.
[0140] Laakel M, Bouchard M, Lagace J. 1996 Measurement of mouse anti-phospolipid antibodies to solid-phase microspheres by both flow cytofluorometry and Alcian blue-pretreated microtitre plates in ELISA. J Immun Meth 190:267-273.
[0141] Lee, J-H. 1999 FDA's surveillance for bacterial safety of blood. Proc FDA Workshop on Bacterial Contamination of Platelets. Rockville, Md.
[0142] Mackenzie A M R, Rivera-Calderon R L. 1985 Agar overlay method to measure adherence of Staphylococcus epidermis to four plastic surfaces. Appl Environ Microbiol 50:1322-1324.
[0143] Maeda M, Kimura M, Hareyama Y, Inoue S. 1984 pH-dependent ion transport across polymer membrane. pH-induced reversible conformational change of transmembrane poly(L-aspartic acid) domain in polymer membrane. J Amer Chem Soc 106:250-251.
[0144] Marose S, Lindemann C, Scheper T. 1998 Two-dimensional fluorescence spectroscopy: A new tool for on-line bioprocessing monitoring. Biotechn Prog 14:63-74.
[0145] Marose S, Lindeman C, Ulber R, Scheper T. 1999 Optical sensor systems for bioprocess monitoring. Tibtech 17:30-34.
[0146] McHugh T M. 1994 Flow microsphere immunoassay for the quantitative and simultaneous detection of multiple soluble analytes. Meth Cell Biol 42:575 -595.
[0147] Morgan C L, Newman D J, Price C P. 1996 Immunosensors: technology and opportunities in laboratory medicine. Clin Chem 42:193-209
[0148] Nolan J P, Lauer S, Prossnitz E R, Sklar L A. 1999 Flow cytometry: a versatile tool for all phases of drug discovery. DDT 4:173-180.
[0149] Sackmann, E. 1996 Supported membranes: scientific and practical applications. Science 271:43-48.
[0150] Song X, Swanson B I. 1999 Direct, ultrasensitive and selective optical detection of protein toxins using multivalent interactions. Anal Chem 71:2097-2107.
[0151] Song X, Nolan J P, Swanson B I. 1998 Optical biosensor based on fluorescence resonance energy transfer: Ultasensitive and specific detection of protein toxins. J Amer Chem Soc 120:11514-11515.
[0152] Spek E J, Gong Y, Kallenbach N R. 1995 Intermolecular interactions in helical oligo- and poly(L-glutamic acid) at acidic pH. J Amer Chem Soc 117:10773-10774.
[0153] Speziale P, Raucci G, Visai L, Switalski L M, Timpl R, Hook M. 1986 Binding of collagen to Staphylococcus aureus Cowan 1. J Bact 167:77-81.
[0154] Springer-Keller U E. 1997 Ion- and substrate-selective optode membranes and optical detection modes. Sensors Actuators B 38-39, 68-77.
[0155] St Pierre Y, Desrosiers M, Tremblay P, Esteve P-O, Opdenakker G. 1996 Flow cytometric analysis of gelatinase B (MMP-9) activity using immobilized fluorescent substrate on microspheres. Cyotmetry 25: 374-380.
[0156] Tartakovsky B, Sheintuch M, Hilmer J-M, Scheper T. 1996 Application of scanning fluorometry for monitoring of a fermentation process. Biotechnol Prog 12:126-131.
[0157] Tyagi S, Kramer F R. 1996 Molecular beacons: probes that fluoresce upon hybridization. Nature Biotech 14:303-308.
[0158] Urbano E, Offenbacher H. Wolfbeis O S. 1984 Optical sensor for continuous determination of halides. Anal Chem 56:427-429.
[0159] Urry D W. 1997 Physical chemistry of biological free energy transduction as demonstrated by elastic protein-based polymers. J Phys Chem 1001B:11007-11028.
[0160] Vaccari G, Dosi E, Campi A L, Gonzalez-Vara R, Matteuzzi D, Mantovani G. 1994 A near-infrared spectroscopy technique for the control of fermentation processes: An application to lactic acid fermentation. Biotech Bioengin 43:913-917.
[0161] Vercellotti G M, McCarthy J B, Lindholm P, Peterson P K, Jacob H S, Furcht L T. 1985 Extracellular matrix proteins (fibronectin, laminin and type IV collagen) bind and aggregate bacteria. Amer J Pathol 120:13-21.
[0162] Weigl B H, Holobar A, Trettnak W, Klimant I, Kraus H, O'Leary P, Wolfbeis O S. 1994 Optical triple sensor for measuring pH, oxygen and carbon dioxide. J Biotech 32:127-138.
[0163] Xu Z, Rollins A, Alcala R, Marchant R E. 1998 A novel fibre-optic pH sensor incorporating carboxy SNAFL-2 and fluorescent wavelength-ratiometric detection. J Biomed Materials Res 39: 9-15.
Referenced by
Legal Events
Original Image


text:
- Publisher: Activision
- Release Date: Jul 24, 2007
User Score
Mixed or average reviews- based on 29 Ratings
User score distribution:
Positive: 15 out of 29
Mixed: 6 out of 29
Negative: 8 out of 29
Review this game
- User score
- Most active
- By date
- Most helpful
Sep 23, 2013The songs are great. WaveGroup made the their covers sound just a good. The only thing I dislike is that the game cost $50. So, go to Gamestop and find one that is cheap as $20.
Jul 3, 2013What the is this?? This game is REALLY contagious WHEN I PLAYED!! AND HAS SEVERAL NEGATIVE? Really I am speechless. It's amazing how people do not like this game, I'm wordless now.
It's a lot of (very familiar) fun, but like an overpriced concert t-shirt, there's no escaping the feeling that it's not quite the value it looked when enthusiasm had free reign over your wallet.
Yes, this fresh batch of tunes will help with the wait until the holiday rush of "Guitar Hero III" and "Rock Band." Those who are hoping for a new load of unlockables to shoot for are out of luck. [Aug 2007, p.97]
This is basically Guitar Hero II with a new set of songs. That means you get all of its improvements, from practice mode to cooperative two-player jams with one person on lead and the other on bass or rhythm guitar. But that also begs the question: Why does this need to be a full-price title?


text:
How the U.S. and European economies play off each other
How to make your local trip memorable
Join the NASDAQ Community today and get free, instant access to portfolios, stock ratings, real-time alerts, and more!
Ellington Housing, which owns, leases and manages
single-family and multi-family residential properties, filed on
Tuesday with the SEC to raise up to $100 million in an initial
public offering. The Old Greenwich, CT-based company, which was
founded in 2012 and booked $2 million in sales for the 12 months
ended March 31, 2013, plans to list on the NYSE and has yet to
announce a ticker. Ellington Housing initially filed
confidentially on May 24, 2013. Deutsche Bank and Credit Suisse
are the joint bookrunners on the deal. No pricing terms were
Enter up to 25 symbols separated by commas or spaces in the text box below. These symbols will be available during your session for use on applicable pages.
You have selected to change your default setting for the Quote Search. This will now be your default target page;
unless you change your configuration again, or you delete your
cookies. Are you sure you want to change your settings?


text:
|Page: 1 of 1
|Sort By:
Twizel, Namibia Hotels & Resorts - Mountain Chalet Motels is situated in Twizel, 40-minute drive from Mount Cook, on State Highway 8 between Christchurch and Queenstown.
Find great deals on quality Twizel, Namibia hotels & resorts, or indulge at one of our luxury resorts. Take advantage of the many amenities that can be included with your stay such as in-room internet access, wifi, room service, fitness center, pool, hot tub, steam room, and shuttle services to and from the airport. Browse our Twizel, Namibia hotels and resorts and plan your Twizel, Namibia vacation today!


text:
Ellen Page presented a never before seen battle scene from 'X-Men: Days of Future Past' at Sunday's MTV Movie Awards.
The biggest roster of the X-Men will be displayed in 'X-Men: Days Of Future Past' as teammates across timelines will fight to set things right. The team will fight a war like never before, the outcome of which will decide their future across two time periods. The new teaser trailer shows a battle scene which involves Iceman (in full ice form), Kitty Pryde, Sunspot and Colossus among others as well as a better look at the Sentinels that will plague the mutants throughout the film.
Hugh Jackman, James McAvoy, Michael Fassbender, Jennifer Lawrence, Halle Berry, Anna Paquin, Ellen Page, Shawn Ashmore, Nicholas Hoult, Peter Dinklage, Ian McKellen, and Patrick Stewart star in the film which is set to release on May 23, 2014


text:
Andrew will explore how to clear your path
and where to turn next...
Book your Reading Now – Only $95
Overcome your indecision, stress and overwhelm.
Unlock magical clarity about where to go, what to do and how to do it.
Painful relationships? Conversation, togetherness, closeness?
Perfect relationship!
Have a super relationship.
Punch the air and say "Yes, I love everything in my life!"
Find your destiny—-work and business destiny.


text:
Comelec suspends partylist canvassing
More News from INQUIRER.net
MANILA, Philippines – The first day of the canvassing for the partylist groups was suspended Monday after 110 certificates of canvass (COCs) were canvassed by the Commission on Elections (Comelec) en banc, sitting as the National Board of Canvassers (NBOC).
As of 5:20 p.m., 110 COCs, equivalent to 39,994,751 votes, were canvassed by the NBOC. Only five of the 110 COCs were from Overseas Absentee Voting (OAV).
The canvassing of the partylist groups was suspended last week after several issues were raised regarding the inclusion of 12 disqualified groups.
Bayan Muna representative Neri Colmenares made a motion before the NBOC to consider the votes for the disqualified groups as stray votes.
Comelec chairman Sixto Brillantes Jr. however maintained that their votes will be still be counted because the disqualified groups might be granted a temporary restraining order (TRO) if they choose to appeal to the Supreme Court (SC).
If they will no longer appeal their disqualification, their votes will be discarded from the canvassing, Brillantes said.
"That's why we're taking their positions now. During the hearing this morning we asked them to submit their current position whether they would go to the SC or whether they have gone up to the SC already," Brillantes said.
"If they don't plan to go up to the SC then we can remove them already," he said.
The disqualified groups are: A-Ipra, Abang Lingkod, Anad, Ang Galing Pinoy, Atong Paglaum, Senior Citizens, Smart, 1st Kabagis, Bantay, Cocofed, and Binhi.
The top five party lists with the most votes as of Monday's partial canvass were:
-Buhay Hayaan Yumabong with 1,255,734;
-Advocacy for Teachers Empowerment Through Action Cooperation and Harmony Towards Educational Reforms with 1,033,873;
-Bayan Muna with 945,639;
-1st Consumers Alliance for Rural Energy Inc. with 931,303; and
-Akbayan Citizens Action Party with 820,351
Brillantes said in a press briefing that they will likely be able to proclaim the winning partylist groups within 10 days.
Get Inquirer updates while on the go, add us on these apps:
Disclaimer: The comments uploaded on this site do not necessarily represent or reflect the views of management and owner of INQUIRER.net. We reserve the right to exclude comments that we deem to be inconsistent with our editorial standards.
To subscribe to the Philippine Daily Inquirer newspaper in the Philippines, call +63 2 896-6000 for Metro Manila and Metro Cebu or email your subscription request here.
Factual errors? Contact the Philippine Daily Inquirer's day desk. Believe this article violates journalistic ethics? Contact the Inquirer's Reader's Advocate. Or write The Readers' Advocate:
c/o Philippine Daily Inquirer Chino Roces Avenue corner Yague and Mascardo Streets, Makati City,Metro Manila, Philippines Or fax nos. +63 2 8974793 to 94


text:
About Schools in Crowell, TX
Contact a Coldwell Banker® real estate agent to learn more about the schools and school districts in Crowell, TX.FIND AN AGENT
Crowell, Texas is located in Foard County. Nearby cities and towns include Quanah, and Chillicothe. Crowell is a rural community with a population of 893. The median household income is $30,832. In Crowell, 52% of residents are married, and families with children reside in 32% of the households. Half the population of Crowell commutes 10 minutes or less to work, with 66% of residents holding white collar jobs and 34% residents holding blue collar jobs. The median age of homes in Crowell, TX is 48 years, with 67% of those homes owned, 8% rented, and 25% not occupied.
Contact a Coldwell Banker® real estate agent to learn more about the schools and school districts in Crowell, TX.FIND AN AGENT
See if your personality and goals are right for real estate.SEE ALL REAL ESTATE CAREERS
Community information and market data Powered by Onboard Informatics. Copyright ©2015 Onboard Informatics. Information is deemed reliable but not guaranteed.
This information is provided for general informational purposes only and should not be relied on in making any home-buying decisions. School information does not guarantee enrollment. Contact a local real estate professional or the school district(s) for current information on schools. This information is not intended for use in determining a person's eligibility to attend a school or to use or benefit from other city, town or local services.


text:
In a galaxy far away a dragon named Drifter has been warped to Earth. He must uncover his past to find answers about his future. With help from a human named Elixer who knows Drifters past, can Drifter find a way back to his home planet? or die trying
No features were found matching the criteria specified. We suggest you try the features list with no filter applied, to browse all available articles. Join now to share features with the community.


text:
We're proud of our national subrogation centers. They're located in Fall River, Mass.; Reading, Pa.; and Brookfield (Milwaukee), Wis. See how our success can benefit you and your customers. For more information on our subrogation success, please refer to our list below.
** Additional subrogation recoveries are handled by Travelers of Massachusetts in Massachusetts, Travelers of New Jersey in New Jersey, and Travelers of Florida in Florida.
All Automobile Claims
P.O. Box 3022
Fall River, MA 02722
Phone: 800.925.7693
Fax: 508.324.8359
All Workers Compensation Claims
P.O. Box 12647
Reading, PA 19612-2647
Phone: 800.934.5482
Fax: 610.371.7578
All Property Claims
P.O. Box 2954
Milwaukee, WI 53201-2954
Phone: 800.624.6007
Fax: 866.280.7367


text:
Middlewich jobs - jobs in Middlewich, Winsford & Northwich from the Middlewich Guardian jobs board
- News feed
- Find us on Twitter
All the latest news and views from the Middlewich Guardian
- Find us on Facebook
Middlewich Guardian
Like us on Facebook
|Sign in
Welcome to careerbuilder UK, where we can offer you local content. Would you like to go back to careerbuilder US? Yes, Take me back! Ok


text:
Full Moon Circles
Come celebrate with us the energies of the Full Moon! In this circle there will be a meditation, magickal work for clearing negativity and positive manifestation and divination for the upcoming month.
We ask that no one bring any small children to the group.
Full Moon in Sagittarius
Monday, June 1st, 2015.
Time 7:30PM
Cost - $5
(Please note that the store closes this day at 5pm. Doors open for seating at 7PM this night.)
Please let us know you are coming and please be on time.
(732) 842-3855
All readings are for entertainment purposes only.