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5 Tips for an Organised Home
January 18, 2018
If the clutter and chaos is starting to get you down follow our 5 easy steps to a calm and organised home.
#1 Clear your Clutter
Let's face it, de-cluttering is good for the soul. It can be hard to know where to start though, especially if you are drowning in a sea of paperwork, lego bricks, random plastic toys, unloved ornaments and shoes! If your house has become a clutter hot spot then keep reading…
Tackle your home one room at a time, sorting your stuff into 3 piles;
-Throw Away
We all have nostalgic items we wouldn't dream of getting rid of. From boxes of old photos to your daughter's first shoes and not forgetting those much-loved books.
Invest in good quality storage boxes (that you can stack) and store in 'out of the way 'areas such as your attic, garage or shed. If you opt for clear boxes you give yourself a good chance of quickly finding any stored items fast in the future.
When it comes to paperwork create two piles; one for the shredder and the other to be filed. Bin all old leaflets catalogues and junk mail that may have found its way into your paper piles.
Ensure that you shred all paperwork that you don't need to avoid the risk of identity fraud.
If you haven't already got a hot filing system then grab that Really Useful Box (again)! The 19 Litre Extra Large is the perfect size to use with suspension files and store your A4 bills, letters and important documents. Alternatively a home or expanding file is a great place to store and easily access your more frequently referenced paperwork such as school letters and bills to be paid.
Upcycling your junk helps to prevent excess waste going into landfills, in turn reducing emissions. The upside of upcycling (like what we did there?) is that you could even make a little bit of extra cash…
Give away your unwanted clothes, shoes, toys, books and games to charity shops who will be most grateful for it.
If it's old gadgets and tech that you are hoarding, check websites such as musicMagpie , MuzumaMobile or Money4mytech to easily recoup some lost cash. This goes for unwanted DVDs and CDS as well.
If you are clearing out some larger items such as old furniture why not list for free on websites like Shpock and Gumtree If you are feeling especially brave you could even set up a stall at your local car boot…but be ready to stand firm with the hagglers!
Throw Away
It's pretty surprising to discover that a massive 70% of our household waste can be recycled in some way. So it's a good idea to resist the urge to throw all of your unwanted stuff into a black bin bag and shoe-horn it into your wheelie bin.
Instead take your rubbish to a local tip (now known as Household Waste Recycling Sites) where you can dispose of broken electrical items, batteries, plastics, wood and plenty more.
#2 Label EVERYTHING!
If you want to organise your home…and actually keep it organised then labelling everything is a must! There is no end to the things that you can label; jars and Tupperware in the kitchen, cardboard boxes in the garage, the filing system previously discussed and of course; your grand collection of storage boxes!
You can go analogue with a sharpie marker and pack of sticky labels or opt for a slightly more high tech approach with a label printer Labels could save you hours hunting for mislaid items but more importantly than that they could actually save the day! If you have children labelling lunch boxes, school bags, PE kits and even your child if the urge takes you (just kidding) could prevent lost items over the course of the year.
#3 Get the Whole Family Involved
Teaching your children to be organised from a young age is going to be a life skill that will benefit them forever.
It's much easier to get the kids involved than you think, turn the de-clutter mission into a game. You could race each other, and the winner gets a prize (think a walk to the park or choccie biscuit!).
Put on some 'banging' age appropriate tunes to help pick up the pace, classics from their favourite Disney movie will be a hit with younger kids although you may want to avoid whistle while you work...
Educating the youngsters in your home to the benefits of a clean and organised living space will not only help you keep your house tidy right now, but more importantly give them the skills to keep their own home tidy in the future.
#4 "A place for everything and everything in its place"
Are visitors to your home greeted with piles of shoes and coats slung over the bannister? Are you fed up with looking at stacks of papers, junk mail and magazines in your kitchen? Tired of the debilitating pain of standing on yet ANOTHER piece of abandoned lego?
There is a miracle cure to all of the above problems, simply create a place for EVERYTHING in your home.
Invest in a key cupboard, boxes for shoe storage and somewhere to hang coats to instantly transform halls and keep daily life organised. A magazine file is the perfect holding place for mail, paperwork and catalogues before you store or bin them.
Organise children's bedrooms and playrooms with plenty of storage boxes, labelled with the toys, books and games stored within for quick access.
Keep dressing tables or bathroom shelves clutter free with an acrylic storage tidy Ideal for organising makeup and jewellery.
If storage space is at a premium in your home then get creative and start to think (pardon the pun) outside of the box. Look at every spare nook and cranny as a potential storage space; the top of your fridge or kitchen cabinets, under the stairs, bottom of your wardrobe or the top of an airing cupboard to name a few.
#5 List Friendly
Ever wonder why it's hard to get organised and then stay organised? Well one reason could be that the stresses and fast pace of family life distract you from chores that need to be done.
Create to-do lists to help you stay on top of cleaning, tidying and general day to day organising and prioritise your tasks.
Bullet Journals and creative journaling have become popular recently, so harness these tools to manage your own home. With so many things to do and so little time in most of our schedules to do them, simply write down a daily to –do list in your journal and tick them off each night when they have been done.


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The food and beverage manufacturing are a very competitive industry. Producers want to be the first to introduce new products, provide quality, and build a loyal brand following.
In such a competitive industry, efficiency and productivity require powerful automated inspection systems to monitor production lines. Manufacturers need inspection systems that inspect flaws at a high-speed with precision and accuracy.
While there is a wide range of inspection system choices, manufacturers should consider the value of an integrated system. A few years ago, an all-in-one packaging system was considered an emerging trend. Today, integrated systems are dominating the market and as technology continues to evolve, will become the system of choice for manufacturers. Integrated systems provide manufacturers the ability to inspect container flaws, fill levels, labels, and cases with the ability to reject those that do not meet quality standards.
While going through the inspection system selection process, manufacturers must consider the following factors:
- Is the system future-proof? In other words, as your production grows and expands, is your inspection system upgradeable, adaptable, and expandable?
- How much will it cost to train your staff? Will it be an on-going investment or is it one and done?
- Will the system add value to your production line? Will it increase productivity and efficiency? Is it measurable?
- Will the machine require increased staffing or reduced staffing? What are the internal implications?
- Will you have to carry additional parts inventory or will the integrated system reduce the number of parts required? What are the financial implications?
- As we all know, return on investment is paramount. In the case of an integrated system, how does the integrated system increase productivity, is it worth the investment, and can you calculate the savings?
- The all-important intangible that we know is important, is there a monetary value of eliminating consumer complaints and recalls?
FILTEC offers a variety of inspection systems for all applications. Contact us to learn about our inspection system solutions.

subset: commoncrawl


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This ultra-pigmented, lustrous gloss glides on the lips smoothly and evenly leaving behind a luminous and voluminous effect. Ceramide is added to the lip glaze to help thicken the stratum corneum, improve the tolerance of the entire skin, avoid the occurrence of sensitivity, and moisturize. Great gloss with lasting color. A high-coverage liquid lip color with ultimate shine, comfort, and all-day wear.Vinyl Cream dresses lips in full coverage color and high-impact vinyl shine. The diamond shaped applicator allows for perfect lip contouring and color glides on evenly in just one stroke. The creamy formula provides 10 hour wear and all day comfort so lips look and feel beautiful. Make it personal and extra special with complimentary engraving.
Focallure Devil Cream Long Lasting Lip Satin -6 Auburn
$5.42 Regular Price
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Product will be delivered to you within 3-7 business days after you placed an order.


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Hybrid Exchange
The hybrid exchanges will unite the advantages of both centralized and decentralized exchanges, which include the cooperation with large investors as well as the trust of many users. The concept of a hybrid exchange is to add components of centralized and decentralized cryptocurrency exchange.
In some instances, users' trade directly from their wallets with tokens deposited into a smart contract before the trading begins. This ensures that no third-party entity maintains custody for user's funds which reduces potential security risks.
Hybrid crypto exchange development is gaining popularity, and hybrid cryptocurrency exchanges are known to be the next-generation crypto trading marketplace.
- Complete integration of present traditional solutions used in the blockchain infrastructure.
- Interfaces and tools used by financial experts such as reliability, modularity, and scalability.
- Rapid operation even with the rate of transaction processing being inferior to that of other payment systems e.g. Visa.
- Access to real-time operation
Location, 25 Business Avenue, Houston, USA
Call us : +1 213 269 4908
101, Kumaran Colony, Vadapalani, Chennai
Call us : +91 7397 2244 61
MALAYSIA108, Level 9, Brunsfield Oasis Tower, Petaling Jaya, Malaysia
Call us : +60 39 2121 566
Standard Chartered Tower, Level 5, Emaar Square, Downtown Burj Khalifa Dubai – UAE
Skype : Digikolma


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Ratiometric fluorescent pH sensor for non-invasive monitoring
The present invention provides ratiometric fluorescent pH sensors for non-invasive, continuous monitoring of pH in such applications as fermentation processes. The ratiometric fluorescent pH sensors comprise a fluorescent dye that exhibits a shift in excitation wavelength with a corresponding shift in pH in the local environment of said fluorescent dye. Ratiometric measurements of the emission intensities at dual excitation maxima correlate to pH. Also provided is a fluorescent dye 6-methacryloyl-8-hydroxy-1,3-pyrene disulfonic acid (MA-HPDS). Further provided are systems and methods to non-invasively and continuously monitor pH.
This non-provisional application claims benefit of U.S. provisional application Ser. No. 60/478,051, filed Jun. 12, 2002, now abandoned and U.S. provisional application Ser. No. 60/434,034, filed Dec. 17, 2002, now abandoned.FEDERAL FUNDING LEGEND
This invention was made with government support under contract No. 0091705 awarded by the National Science Foundation. The government has certain rights in the invention.BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates in general to fluorescent pH sensors and ratiometric fluorescence measuring techniques. More particularly, the present invention relates to a fluorescent dual excitation-ratiometric pH sensor for non-invasive monitoring of pH.
2. Description of the Related Art
In recent years, there has been considerable research effort toward the development of techniques for continuous on-line monitoring of pH for environmental, biomedical and bioprocess applications. In particular, optical techniques based on fluorescence measurements offer many advantages over conventional electrochemical approaches including high sensitivity and ease of miniaturization. In addition, since fluorescence emission from an indwelling patch can be monitored without direct contact, in situ pH measurements can be made non-invasively with external instrumentation. The latter is highly desirable for bioprocesses using large, instrumented fermenters, by circumventing the cumbersome task of probe sterilization. For benchtop shake flasks and small scale, high throughput operations that do not readily accommodate the larger probes, in situ fluorescence-based patches delivering continuous real time pH values could be used to monitor and even control the acidity of the environment.
With judicious choice of the fluorescent indicator and immobilization conditions, measurements can be made over the desired region with high sensitivity. Sensors have been developed that measure the pH-dependent change in emission intensity, including those based on fluorescein, cyanine (1) and transition metal complexes (2-4). However, the inherent drawbacks of intensity-based measurements include signal variations due to probe photobleaching and fluctuations in source intensity, and sensitivity to position and orientation of the sensor patch that precludes non-invasive detection.
Furthermore, measurements in highly scattering or fluorescent media are difficult at best, even with an opaque membrane backing (5). Sensors based on the pH-dependent change in fluorescent lifetime of an immobilized ruthenium metal ligand complex (6) do not suffer the same drawbacks, but require more complicated instrumentation. In addition, sensitivity to collisional quenching by oxygen results in an additional calibration parameter or operation under anaerobic conditions.
An alternate approach that circumvents the problems associated with intensity-based measurements is ratiometric detection. Given a fluorescent indicator that exhibits a shift in excitation or emission wavelength with pH, the ratio of the emission intensity at the two wavelengths can be used as a robust measure of the pH that is insensitive to orientation, probe concentration and background fluorescence. Dual emission fiber-optic sensors based on seminapthofluorescein (7) and carboxynaphthofluorescein (8) have been described that rapidly and reliably correlate intensity ratios to pH. The extensive photobleaching that is observed for these dyes is accounted for by the ratiometric approach, but would still limit the useful lifetime of the sensor.
The fluorescent dye 8-hydroxy-1,3,6-pyrene trisulphonic acid trisodium salt (HPTS) consists of a pyrene core with three sulfonic acid groups and a hydroxyl group that imparts pH sensitivity around a pka of approximately 7.3 (9). HPTS exhibits two excitation wavelengths, one UV at 405 nm and one blue at 457 nm, that correspond to the acid and its conjugate base (9-10). The subsequent pH-dependent shift in excitation maximum about the pKa of 7.3 enables dual-excitation ratiometric detection in the physiological range. This, together with a low toxicity (11) and insensitivity to oxygen concentration (12), makes HPTS a suitable probe for physiological and bioprocess pH measurements.
To date, covalent attachment of HPTS has been via sulfonamide coupling (13). The presence of the three strongly anionic sulphonic acid groups allows for HPTS to be immobilized by ionic binding to cationic supports. While effective in immobilizing the dye and preserving pH sensitivity, polymer substrates are limited to those that contain primary amines. In addition, amine groups which remain on the substrate after coupling will affect the local pH inside the polymer matrix. The dye has been covalently attached to controlled pore glass (15) and aminoethyl cellulose (16) in the development of fluorescence-based pH sensors that operate in neutral and acidic environments, as well as an intravascular blood gas monitoring system where it was used for both pH and pCO2 detection (17). Fiber-optic pH sensors have been described with HPTS bound to an anion exchange membrane (12) or resin (18) and fixed to the tip of the optical fiber.
For example U.S. Pat. No. 5,114,676 provides a pH sensor with a fluorescent indicator which may be covalently attached to a particle or to a microcrystalline cellulose fiber. The sensor comprises an optically transparent substrate, a thermoplastic layer and a hydrogel. Part of the particle with the indicator attached thereto is imbedded in a thermoplastic layer that is coated on the substrate and mechanically adhered using heat and pressure. The majority of the particle/indicator is imbedded within a hydrogel layer that is applied over the thermoplastic layer. The pH sensor is applied to the tip of an optical waveguide.
Furthermore, with the recent availability of low cost UV LEDS, the dye can be measured with relatively inexpensive instrumentation that combines UV and blue LEDs and a photodiode module. Such a setup has been described (14) to detect the pH of a high throughput microbioreactor system via HPTS directly dissolved in the fermentation media.
The inventors have recognized a need for improvement in HPTS sensors and uses thereof in inexpensively collecting ratiometric emission data externally from a biosystem. Specifically, the prior art is deficient in sensors comprising HPTS or derivatives thereof which is easily immobilized in a polymer matrix having suitable optical and diffusion properties. Additionally, the prior art is deficient in the use of substrates other than those having substituent primary amines. Furthermore, the prior art is deficient in sensors comprising HPTS or derivatives thereof which are easily assembled and which can be used as indwelling sensors in a reaction vessel to collect ratiometric emission data. The present invention fulfills this long-standing need and desire in the art.SUMMARY OF THE INVENTION
The present invention is directed to a fluorescent dye having the structure:
The present invention also is directed to a ratiometric fluorescent pH sensor comprising a fluorescent dye that exhibits a shift in excitation wavelength with a corresponding shift in pH in the local environment of the dye, a polymer matrix where the fluorescent dye can be immobilized therein, a means of optically isolating the polymer matrix comprising the fluorescent dye, and an adhesive means.
The present invention also is directed to a system for non-invasive monitoring of pH comprising a reaction vessel in which to monitor the pH, the ratiometric fluorescent pH sensor described herein whereby the adhesive means adheres the front surface of the pH sensor to the reaction vessel, a means for delivering a first pH-sensitive excitation maximum wavelength to the front surface of the fluorescent pH sensor; a means for delivering a second pH-sensitive excitation maximum wavelength to the front surface of the fluorescent pH sensor, a means for detecting maximum emission intensity of a wavelength emitted from the fluorescent pH sensor at both of the first and second pH-sensitive excitation maxima wavelengths, and a means for evaluating a ratio of the first and second maximum emission intensities as pH.
The present invention is further directed to a method of continuous monitoring of pH of a growth media during a fermentation process comprising placing the growth media or a sample therefrom into the reaction vessel of the system described herein. The fluorescent pH sensor is excited with the first of the pH-sensitive excitation maxima wavelengths and the intensity of fluorescent light emitted from the sensor at the emission maximum wavelength upon the first excitation is measured. The fluorescent pH sensor is further excited with the second of the pH-sensitive excitation maxima wavelengths and the intensity of fluorescent light emitted from the sensor at the emission maximum wavelength upon the second excitation is measured. A ratio of the emission intensity at the first excitation maxima wavelength to the emission intensity at the second excitation maxima wavelength is calculated and the ratio is correlated the pH of the growth media thereby monitoring pH during fermentation.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.BRIEF DESCRIPTION OF THE DRAWINGS
So that the matter in which the above-recited features, advantages and objects of the invention, as well as others which will become clear, are attained and can be understood in detail, more particular descriptions of the invention briefly summarized above may be had by reference to certain embodiments thereof which are illustrated in the appended drawings. These drawings form a part of the specification. It is to be noted, however, that the appended drawings illustrate preferred embodiments of the invention and therefore are not to be considered limiting in their scope.
In one embodiment of the present invention there is provided a fluorescent dye having the structure:
In this embodiment the fluorescent dye is 6-methacryloyl-8-hydroxy-1,3-pyrene disulfonic acid (MA-HPDS).
In another embodiment of the present invention there is provided a ratiometric fluorescent pH sensor comprising a fluorescent dye which fluorescent dye exhibits a shift in excitation wavelength with a corresponding shift in pH in the local environment of the fluorescent dye, a polymer matrix which immobilizes the fluorescent dye therein and where the polymer matrix comprises a front surface and a back surface, a means of optically isolating the polymer hydrogel comprising the fluorescent dye, and an adhesive means.
In one aspect of this embodiment the ratiometric fluorescent pH sensor comprises the fluorescent dye 6-methacryloyl-8-hydroxy-1,3-pyrene disulfonic acid (MA-HPDS). In this aspect the polymer matrix may comprise a p(ethylene glycol) hydrogel. The MA-HPDS covalently binds p(ethylene glycol) diacrylate in a polymerization reaction thus functioning as a comonomer and becoming immobilized within the p(ethylene glycol) hydrogel. Further to this aspect the fluorescent pH sensor additionally may comprise a comonomer and/or a cross-linking agent. The comonomer may be a siloxane-based monomer. The cross-linking agent may be trimethylolpropane triacrylate or ethylene glycol dimethacrylate.
In another aspect of this embodiment the ratiometric fluorescent pH sensor may comprise the fluorescent dye 8-hydroxy-1,3,6-pyrene trisulphonic acid trisodium salt (HPTS). The polymer matrix may comprise a p(ethylene glycol). The monomer p(ethylene glycol) diacrylate is polymerized around the anionic exchange resin to form the hydrogel. The anionic exchange resin immobilizes the HPTS within the hydrogel via ionic binding.
In yet another aspect of this embodiment the ratiometric fluorescent pH sensor again comprises the fluorescent dye HPTS. In this aspect the polymer matrix comprises a copolymer poly(2-hydroxyethyl methacrylate)-co-(methacryloyl ethyltrimethyl ammonium chloride) (pHEMA-METMA) hydrogel. The HPTS ionically binds to the copolymer thus becoming immobilized within the pHEMA-METMA copolymer hydrogel.
In all aspects of this embodiment the optically isolating means may be a microfiltration membrane. The adhesive means may be an adhesive tape, a hydrophobic adhesive substrate or a transfer adhesive. A representative example of adhesive tape is a polyethylene tape. An example of a hydrophobic adhesive substrate is a silicon rubber substrate or an acrylate-functional adhesive. Again in all aspects the fluorescent dye may exhibit dual excitation maxima and an emission maximum wavelength. Additionally, the fluorescent sensor responds to a shift in pH within a range of about pH 6.0 to about pH 9.0. Furthermore the fluorescent pH sensor may be sterilized.
Further to all aspects the sensor may comprise a patch. The sensor patch may be assembled by placing the adhesive means on a flat surface and adhering the front surface of the polymer matrix to the adhesive means. The back surface of the polymer matrix is covered with the optically isolating means. The adhesive means and the optically isolating means are as described supra.
In yet another embodiment of the present invention there is provided a system for non-invasive monitoring of pH comprising a reaction vessel in which to monitor the pH, the ratiometric fluorescent pH sensor described supra where the front surface of the pH sensor adheres to the reaction vessel via the adhesive means, a means for delivering a first pH-sensitive excitation maximum wavelength to the front surface of the fluorescent pH sensor, a means for delivering a second pH-sensitive excitation maximum wavelength to the front surface of the fluorescent pH sensor, a means for detecting maximum emission intensity of a wavelength emitted from the fluorescent pH sensor at both of the first and second pH-sensitive excitation maxima wavelengths, and a means for evaluating a ratio of the first and second maximum emission intensities as pH. This embodiment may further comprise means for closed-loop feedback control of the system.
In all aspects of this embodiment the ratiometric fluorescent pH sensors have the dual excitation maxima wavelengths and the emission maximum wavelength as described supra. An example of a delivery means is a light-emitting diode. The light-emitting diode may emit light in the UV range or in the blue wavelength range. A representative detecting means is a photodiode.
In still another embodiment of the present invention there is provide a method of continuous monitoring of pH of a growth media during a fermentation process comprising the steps of placing the growth media or a sample therefrom into the reaction vessel of the system described supra, exciting the fluorescent pH sensor with the first of the pH-sensitive excitation maxima wavelengths, measuring the intensity of fluorescent light emitted from the sensor at the emission maximum wavelength upon the first excitation, exciting the fluorescent pH sensor with the second of the pH-sensitive excitation maxima wavelengths, measuring the intensity of fluorescent light emitted from the sensor at the emission maximum wavelength upon the second excitation calculating a ratio of the emission intensity at the first excitation maxima wavelength to the emission intensity at the second excitation maxima wavelength, and correlating the ratio to the pH of the growth media thereby monitoring pH during fermentation. In all aspects of this embodiment the excitation and emission wavelengths and the ratiometric fluorescent pH sensors are as described supra.
The following terms shall be interpreted according to the definitions set forth below. Terms not defined infra shall be interpreted according to the ordinary and standard usage in the art.
As used herein, "polymer hydrogel" shall refer to a polymer matrix that absorbs water in an aqueous environment.
The present invention provides fluorescent excitation-ratiometric, non-invasive pH sensors and applications thereof, e.g., continuous on-line fermentation monitoring. The ratiometric approach is robust and insensitive to factors such as source intensity, photobleaching or orientation of the sensor patch. The pH sensors of the present invention are capable of timely and reversible pH measurement and demonstrate linearity and sensitivity over a physiologically and bioprocess-relevant range. Measurements may be made with external instrumentation and without direct contact with the patch and thus detection is completely non-invasive.
The external instrumentation comprises an inexpensive excitation light source and emission photodetector thereby significantly lowering the cost of the hardware used to monitor pH. An excitation light source may be UV or blue LEDs and light emitted by the dyes incorporated into the sensors are detected by a photodiode module. The components are inexpensive, and represent a simple and potentially portable way to obtain reliable, on-line pH information from a bioprocess without the use of an invasive probe or optical fiber. The sensor is easily adaptable to closed-loop feedback control systems.
Provided herein is a simple and raid method for the covalent incorporation of HPTS into the backbone of a polymer matrix used to prepare a pH sensor. Covalently linking the dye overcomes problems associated with ionic immobilization of HPTS within a polymer matrix. As such, a derivative of HPTS, 6-methacryloyl-8-hydroxy-1,3-pyrenedisulfonic acid (MA-HPDS), in which one of the sulfonate groups is replaced by a methacrylate moiety provides a dye capable of effecting covalent binding with a polymer hydrogel. The single-step synthetic method uses a related dye, 6,8-dihydroxy-1,3-pyrenedisulfonic acid disodium salt (DHDS), which is similar in structure to HPTS except for an additional hydroxyl group which replaces one of the sulfonic acid groups. As a result, the dye has a second pKa at approximately 8.5 (9).
Nucleophilic substitution of one hydroxyl group with methacrylic anhydride results in a methacryloyl functional dye (MA-HPDS). The pendant methacrylate group can participate as a monomer in free radical polymerization reactions and can thereby be incorporated into a number of substrates. Furthermore, the crude product yield from the synthesis need not be purified to remove unreacted DHDS or methacrylate. The choice of comonomer(s) determines the properties of the matrix and can be engineered for specific properties, e.g., mass transport, mechanical strength and biocompatibility. The co-monomer may be, although not limited to, poly(ethylene glycol) diacrylate (PEG-DA). Additionally, a novel method for assembling the sensor patch is described herein. Generally, the swollen hydrogel layer is sandwiched between a microfiltration membrane such as white polyethersulfone microfiltration membrane backing and a layer of transfer adhesive.
Comonomers containing anionic groups might be incorporated to improve proton transport through the hydrogel or to shift the local pH inside the hydrogel. Mechanical strength might be improved through the addition of siloxane-based monomers or crosslinking agents such as trimethylolpropane triacrylate or ethylene glycol dimethacrylate. The free radical polymerization reaction described herein is not limited to photochemical initiation. Other radical initiators might be considered including thermal (e.g. azobis(4-cyanovaleric acid)) and redox initiators (e.g. sodium metabisulfite/ammonium persulfate). Additionally, it is contemplated that this modification technique may be used with other dyes. Fluorescein indicators contain two hydroxyl groups that may participate in nucleophilic substitution reactions with methacrylic anhydride. For example, dichlorofluorescein with a pka of ˜4 may be used to extend the working range of the sensor to a lower pH.
Other embodiments of the invention presented herein are contemplated. For example other means of attachment may be used including cyanoacrylate adhesives and heterofunctional crosslinkers. Additionally, the sensors may be sterilized by means such as ethylene oxide treatment provided the hydrogel is kept hydrated. Also, it is contemplated that a temperature-independent sensor may be constructed since emission intensities normally decrease with increasing temperature.
While the pH range overlapped by the sensor patches described herein is somewhat narrow, i.e., the sensors function well over the pH range of 6 to 9, it is suitable for many industrially-relevant microbial fermentations. Since many relevant bioprocesses operate at a lower pH, e.g., yeast or fungi, dyes with lower pKa's may be used to expand the range of operation for monitoring in the non-invasive manner presented herein. These include, but are not limited to, 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) and 5-(and-6)-carboxy-2′-7′-dichlorodifluorescein which can both be detected ratiometrically or other probes such as Rhodol Green™ and the LysoSensor™ dyes (23).
Applicability for non-invasive bioprocess monitoring and effectiveness of the sensors was demonstrated using an HPTS patch assembled by polymerization of HPTS-Dowex bound resin beads into a highly swollen poly(ethylene glycol) hydrogel layer. The hydrogel layer serves to fix the Dowex resin in place but still promotes proton diffusion in the vicinity of the dye. Additionally, poly(ethylene glycol) hydrogels have excellent biocompatibility (19, 20). Lastly, a white microfiltration membrane backing provides an optical barrier between the sensing layer and the background media.
The HPTS-Dowex sensor was successfully used in a shake flask setup to provide continuous, on line measurements of the pH of an E. coli fermentation over a range of about pH 6 to about pH 9. The dual-excitation ratiometric measuring technique, combined with the presence of an opaque membrane backing for optical isolation, eliminates any interference from the nutrient broth and cells. The pH recorded by the sensor on line was in good agreement with samples measured off line with a conventional pH electrode. The HPTS-Dowex-PEG sensor is suitable for bioprocess monitoring and can be adapted for pH control with a decrease in thickness and corresponding response time. However, the manufacturing process may be cumbersome and the presence of the micron scale Dowex resin beads may lead to difficulties in measurement. These include background fluorescence and position-dependent signal intensity.
Alternatively, HPTS may be immobilized within a homogeneous co-polymer layer that is hydrophilic and contains cationic functional groups similar to those on the Dowex resin to ionically bind HPTS. The sensor layer is a copolymer of poly(2-hydroxyethyl methacrylate)-co-(methacryloylethyltrimethyl ammonium chloride) (pHEMA-METMA) that is synthesized by the bulk free radical polymerization of the monomers in the presence of a crosslinker, e.g., ethylene glycol dimethacrylate (EGDMA). The poly(alkylacrylate) family of polymers is well-suited to bioprocess applications. These polymers have a typically high thermostability, which enables steam sterilization and a resistance to hydrolysis, which eliminates potentially toxic degradation by-products (21). In addition, the high water content of pHEMA-METMA promotes proton diffusion and rapid response times.
Again the use of a high water-content layer to immobilize HPTS is advantageous for proton diffusion but may present difficulties associated with adhesion of this layer to a reaction vessel as was observed with the HPTS-Dowex sensor. Since the pHEMA-METMA layer is highly swollen, conventional adhesives are ineffective. Additionally, the hydrogel must be kept fully hydrated to prevent cracking precluding the use of adhesives with long cure times.
As an alternative, it is contemplated that a two-layer patch comprising a pHEMA-METMA sensor layer chemically attached to a second, hydrophobic adhesive layer may be used. Acrylate-functional adhesives and silicone rubbers are available (Gelest Inc.; Tullytown, Pa.) with pendant methacryloyl functional groups that participate in free radical reactions. If the HEMA-METMA reaction mixture is polymerized on such a substrate, the result is a two-layer composite with excellent adhesion between the two polymers. The pHEMA-METMA layer is fully hydrated while the adhesive layer is hydrophobic and remains "sticky".
The METMA comonomer contains a quaternary ammonium group that imparts cationic functionality on the polymer for uptake of the anionic HPTS. While the methacrylate polymers in general are known to be hydrolytically stable, the presence of the quaternary ammonium group has been shown to increase the susceptibility of the ester linkage to hydrolysis at elevated temperatures (22). Indeed, following an autoclave cycle, a loss of fluorescence was observed (˜50%) indicating the possibility that the cationic group is cleaved and lost. As the remaining HPTS is fully responsive and sufficient for detection in the spectrophotometer it is further contemplated that a higher loading of HPTS in the pHEMA-METMA layer can be used for detection with less expensive instrumentation. It is also contemplated that alternate comonomers that impart cationic functionality to the polymer but eliminate the unstable ester linkage be used, such as ar-(vinyl benzyl) trimethyl ammonium chloride.
The HPTS-pHEMA-METMA sensor described herein immobilizes the dye within a cationic matrix via ionic binding. However, while this immobilization strategy is sufficient for use in most buffer solutions, long term leaching of the dye can occur. Furthermore, ionic immobilization precludes the use of media containing strong anions, including the common cell culture media component Phenol Red that is used as a visual indicator of pH. Since Phenol Red contains sulfonic acid groups, it competes with HPTS for the cationic sites on the resin. This results in rapid loss of HPTS to the media causing signal loss over time until the sensor is no longer useable.
As described herein, the invention provides a number of therapeutic advantages and uses. The embodiments and variations described in detail herein are to be interpreted by the appended claims and equivalents thereof. The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion.EXAMPLE 1
The fluorescent dye 8-hydroxy-1,3,6-pyrene trisulphonic acid trisodium salt (HPTS) was obtained from Molecular Probes Inc. (Eugene, Oreg.). 6,8-dihydroxy-1,3-pyrenedisulfonic acid disodium salt (DHDS) was urchased from Fluka (Milwaukee, Wis.). Methacrylic anhydride (94% purity), anhydrous dimethyl formamide (DMFP, otassium carbonate and phenolsulfonephthalein sodium salt (Phenol Red) were purchased from Sigma-Aldrich Inc. (St. Louis, Mo.). Poly(ethylene glycol) diacrylate with an average molecular weight of 4000 was obtained from Polysciences Inc. (Warrington, Pa.). The photoinitiator Darocur® 1173, was supplied by Ciba Specialty Chemicals (Tarrytown, N.Y.).
Dowex 1×8-400 strongly basic anion exchange resin was obtained from Sigma Chemical Co. (St. Louis, Mo.). White microfiltration membranes in mixed esters of cellulose with a nominal pore diameter of 0.2 μm (Kimble) were used to optically isolate the sensor and as a support backing. The monomer 2-hydroxyethyl methacrylate (HEMA), initiator 2,2-dimethoxy-2-phenyl acetophenone (DMPA) and crosslinker ethylene glycol dimethacrylate (EGDMA) were obtained from Sigma-Aldrich (St. Louis, Mo.). The co-monomer methacryloyl ethyl trimethylammonium chloride (METMA) was obtained from Polysciences Inc. (Warrington, Pa.) as a 70% v/v aqueous solution.
Analytical grade sodium chloride, sodium dihydrogen phosphate and tris(hydroxymethyl)aminomethane (Tris) were obtained from Sigma Inc. (St. Louis, Mo.). Tryptone and yeast extract (Difco) used in the preparation of nutrient broth were from Beckton-Dickinson (Sparks, Md.).EXAMPLE 2
The overnight seed culture consisted of a 1% inoculum of Escherichia coli JM105 strain frozen stock in 20 mL LB nutrient broth, incubated at 37° C. with shaking at 260 rpm (Orbit Environ Shaker, Lab-line Instruments, Melrose Park, Ill.). The fermentation was carried out at room temperature in a 500 mL shake flask containing 100 mL of LB broth that was inoculated with 2.5% seed culture. The shake flask was placed on a rotating shaker (LaPine Scientific Co., Berkeley, Calif.) set at 100 rpm. Dissolved oxygen and pH were not controlled.EXAMPLE 3 Synthesis of 6-methacryloyl-8-hydroxy-1,3-pyrenedisulfonic acid (MA-HPDS)
The indicator DHDS (100 mg) was dissolved in DMF (10 mL) in a 25 mL reaction vessel. Potassium carbonate (1 g) and methacrylic anhydride (1:1 eq., 36 μl L) were added. The vessel was stoppered and placed in a 70° C. water bath to react for 12 hours. The cooled reaction mixture was filtered. The solvent was removed from the filtrate in a rotary evaporator to yield the crude solid product (MA-HPDS).
Presumably, this product contains a mixture of the modified dye MA-HPDS with an unknown amount of reaction by-products that include unmodified DHDS and methacrylic acid salts. Further purification is unnecessary prior to copolymerization of the modified dye MA-HPDS and poly(ethylene glycol) diacrylate (PEG-DA). The reaction scheme is depicted in
Copolymerization of MA-HPDS with Hydrogel Monomer
A stock solution of MA-HPDS in deionized water (10 mg/mL) was prepared. Polymer precursor solution was prepared by combining 45 mg of PEG-DA, 200 μl of deionized water, 100 μl of the HPDS-MA stock solution and 6 μl of the photoinitiator Darocur and vortexing for 30 minutes. The precursor solution was polymerized between glass plates to prevent oxygen inhibition and volume contraction of the gel. Thickness was controlled with 2-inch wide, 0.004-inch thick aluminum spacer tape (McMaster-Carr Inc.; Atlanta, Ga.). Free radical polymerization of the acrylate end groups was initiated by exposure to a 100-W long wave UV spot lamp (UVP Inc.; Upland, Calif.) for 4 minutes. After polymerization, the PEG-HPDS layer was peeled from the glass plate and hydrated in deionized water for at least 48 hours. This both hydrates the matrix and removes any unbound dye. The resulting hydrogel layers were approximately 120 um thick.
Any residual DHDS is free to diffuse out of the polymer matrix. This separation is readily accomplished in aqueous buffer because of the high water solubility of DHDS. Any methacrylic acid in the crude product will also be incorporated into the backbone of the polyme matrix and does not interfer with the spectral or pH behavior of the indicator. In order to verify modification of the dye and covalent attachment to the hydrogel matrix, the fraction of MA-HPDS in the crude product was estimated through measurement of the amount of dye that remained in the polymer matrix after extensive washing in buffer solution. These sensors were placed in phosphate-buffered saline at pH 7.2 and monitored for fluorescence over several days, with frequent changes of the soaking solution. After 10 days of soaking in aqueous buffer, the copolymer of PEG DA and MA-HPDS lost 26.7±3.3% of its original fluorescence intensity with the greatest loss of 20.8±6.5% occurring in the first 24 hours. Beyond this time fluorescence of the copolymer remained relatively stable. The fraction of dye retained in the matrix is a crude indicator of the conversion of the substituion reaction as it includes dye that is both covalently and nonspecifically bound. It is contemplated to increase the yield by optimizing the ratio of methacrylic anhydride to DHDS beyond the 1:1 equivalent used herein. Copolymerization is schematically depicted in
HPDS-PEG Sensor Assembly
The polymer hydrogel layer with the indicator dye covalently attached was fashioned into a sensor patch to facilitate testing. The polymer hydrogel layer was sandwiched between a bottom layer of transfer adhesive (Adhesives Research Inc.; Glen Rock, Pa.) and a top layer of white polyethersulfone microfiltration membrane (0.45 um, Pall Life Sciences; Port Washington, N.Y.). A 0.8×0.8 cm square was cut from the transfer adhesive and placed, liner down, on a flat surface. A smaller square of HPDS-hydrogel was blotted dry and placed in the center of the adhesive.
Immediately, the hydrogel was covered with a piece of white microfiltration membrane large enough to overlap the hydrogel and form a seal with the exposed edges of the adhesive. The opaque backing serves as an optical barrier between the sensing layer and the background solution, but still allows diffusion of protons. This is a critical component for the sensor's use in bioprocesses where culture media that is often colored and fluorescent will interfere with the optical signal from the dye. The liner was removed from the adhesive and the patch was secured to the inside wall of a cuvette for testing. A schematic diagram of the sensor patch is depicted in
Instrumentation and HPDS-PEG Sensor Evaluation
Fluorescence measurements were recorded with a Varian Cary Eclipse fluorescence spectrophotometer. A solid sample/cuvette holder designed for front-face measurements was used to measure sensor patches that were fixed to the inside front wall of a PMMA disposable cuvette. Excitation light was fixed at an angle of incidence of 30°. Solutions were measured in regular 90° configuration. Cuvettes containing sensor patches were equilibrated in buffered saline solutions ranging in pH from 5 to 10. Uncorrected excitation and emission spectra were recorded for each sample. For comparison, solutions of DHDS and HPTS were also prepared and tested at the same pH values.EXAMPLE 7
Excitation and Emission Spectra for DHDS, HPTS and the Immobilized HPDS
The excitation and emission spectra for DHDS, HPTS and the immobilized HPDS are shown in
As with HPTS, the acid-base equilibrium of the modified dye results in a pH-dependent change in the excitation spectrum toward increased blue excitation at higher pH. At the fixed pH of 7.2 DHDS exhibits relatively greater absorbance in the blue region, 460 nm, because of the second hydroxyl group on the pyrene core. The emission spectrum of the modified dye closely resembles that of HPTS and DHDS, with a single emission maximum at 502 nm. The relative locations of the emission maxima for these dyes may be subject to environmental effects in the polymer hydrogel matrix.EXAMPLE 8
Calibration Curves of Excitation Intensity Ratio Versus pH for HPDS Solution, HPTS Solution and the Immobilized HPDS
Calibration curves for HPTS and MA-HPDS were fit to the following expression (25) describing intensity ratio (R) of monoprotic indicator dyes as a function of pH:
R=([H+]Rmax+ka,appRmin)/ka,app+[H+] (1)
where ka,app is the apparent dissociation constant of the immobilized dye. A least-squares linear regression was used to determine the parameters Rmin, Rmax and ka,app. The pka,app of the immobilized dye was calculated to be 7.61±0.05 for these conditions. This is in good agreement with the pka,app of HPTS in solution, which was calculated to be 7.72±0.02 under the same conditions.
The modified dye is sensitive over the same range as HPTS. The calibration curve for DHDS was fit to an expression similar to that for monoprotic acids, but for diprotic acids. The second hydroxyl group causes DHD to be sensitive over an extended range with two dissociation constants calculated to be pka1,app=7.03±0.02 and pka1,app=9.05±0.02 under these conditions. The calibration curve for MA-HPDS exhibits only a single inflection point and does not demonstrate sensitivity in the upper pH region >9. This further indicates that the substitution reaction has successfully modified the additional hydroxyl group.
The calibration curve for MA-HPDS depicted in
Sterilizing the HPDS-PEG Sensor
An additional advantage of HPDS-PEG sensors over those utilizing electrostatic immobilization to a resin is that the components are fully autoclavable. The poly(alkyl acrylate) family of polymers have a typically high thermostability which enables steam sterilization and a resistance to hydrolysis which elimates potentially toxic degradation by-products (21). Sensor assemblies were fixed to the inside of a glass vessel, covered with pH 7.2 buffer and autoclaved. Minimal loss of dye was observed and sensors remained intact and fixed to the wall.
However, a volume transition at the elevated temperature resulted in a reversible shrinking of the gel matrix between the membrane backing and the adhesive base. Upon rehydration, the gel layer did not return to its original configuration but remained somewhat "crinkled". The pH sensitivity and function of the sensor was preserved. This effect may be prevented by covalently coupling the hydrogel matrix to the adhesive base in order to minimize lateral contraction.EXAMPLE 10
Ionic Strength Dependence of MA-HPDS Sensors
Because of its highly charged nature, HPTS is sensitive to changes in local ionic strength (15), as is methacrylate-modified dye, MA-HPDS.
pKIa=(pKtha+0.512(zA2−zHA2))×(I0.5/I+1.6I0.5) (2)
where pKath is the thermodynamic pka of the dye, zA and zHA are the charges on the acid and the conjugate base respectively and I is the ionic strength of the buffer. This relationship arises due to changes in the activity of the electrolyte species. The ionic strength dependence of the immobilized dye over the observed range of 30-300 mM is well described by equation 2, as demonstrated by the linearity of the plot in
HPDS-PEG Emission Intensity in Phenol Red Solution
Sensor patches containing MA-HPDS copolymerized with PEG-DA were equilibrated in pH 7.2 buffer with 150 mM ionic strength containing 0.01 g/L Phenol Red. A slight loss of emission intensity, i.e., 16.6±2.0%, was observed, owing to the absorbance of the Phenol Red dye in the wavelength region under investigation, however the intensity ratio remained constant. The signal loss was reversible and returned to the original value within 3.6% when the sensor was returned to clear buffer (data not shown).EXAMPLE 12
Instrumentation and Sensor Evaluation HPTS-Dowex-PEG Sensor
Fluorescence measurements were performed using a Varian Cary Eclipse fluorescence spectrophotometer with a solid sample/cuvette holder designed for front-face measurements with excitation light at an angle of incidence between 20 and 35 degrees. Excitation and emission spectra of both solutions and sensor assemblies were recorded in polystyrene disposable cuvettes fixed at 20 degrees from the incident beam. For measurement of the HPTS-Dowex-PEG sensor, the assembly was mounted to the inside front surface of the cuvette using double-sided adhesive tape. Unless otherwise stated, all measurements were performed at room temperature (25+/−2° C.).
In order to measure the response time of the sensor, buffer solutions were continuously pumped from larger reservoirs into a sealed cuvette containing a sensor assembly, using a peristaltic pump operated at 40 mL/min. At this flow rate, the well-stirred solution, ˜4 ml, inside the cuvette would be replaced in approximately 40 seconds (6τ).
Since the solid sample/cuvette holder is not equipped for temperature control, a separate single-cell Peltier accessory was used to collect excitation spectra at temperatures other than ambient. Temperature control in the jacket surrounding the cuvette was varied between 10° C. and 40° C. Since the single-cell Peltier unit is not adaptd for front-face measurements, the sensor assembly was suspended in the cuvette from a rubber stopper at 20 degrees from the incident beam.EXAMPLE 13
HPTS-Dowex-PEG Sensor Fabrication
The Dowex resin beads used to immobilize HPTS consist of a polystyrene support with quaternary ammonium groups capable of ionic binding with the sulphonic acid groups of HPTS. One gram of Dowex resin was suspended in 10 mL of a 50 μM solution of HPTS in deionized water. The resin suspension was allowed to equilibrate at room temperature for 24 hours with occasional stirring. Polymer precursor solution was prepared by combining 50 mg PEG-diacrylate, 0.2 mL of the HPTS-Dowex suspension and 2 μL Darocur® and vortexed for 10 minutes. The precursor solution was polymerized directly onto the membrane backing to promote adhesion and between glass slides to prevent oxygen inhibition. The beads are then physically entrapped in a hydrogel layer formed by the free radical polymerization of poly(ethylene glycol) diacrylate. The resulting matrix is highly swollen, at approximately 85% water contend (26) and promotes proton diffusion.
Specifically, the microfiltration membrane was cut to 2 cm×4 cm and placed on a glass microscope slide. The precursor solution was pipetted directly onto the membrane backing and covered with a second glass slide, using aluminum spacer tape at the edges to control the gap thickness. Free radical polymerization of the acrylate end groups was initiated by exposure to a 100 W long wave UV spot lamp (UVP, Inc., Upland, Calif.) for 30 seconds. After polymerization, the peg-Dowex layer was peeled from the glass slides with the microfiltration membrane attached to the bottom surface and allowed to hydrate in deionized water for 24 hours. Sensors constructed in this manner were approximately 250 μm thick. For comparison, control PEG-Dowex layers without HPTS were also prepared.EXAMPLE 14
Recording of Fluorescence Spectra to Reduce Scatter and Reflection
To facilitate insertion of the sensor into cuvettes for reproducible analysis, the HPTS-Dowex-PEG layer was affixed to rigid transparency film. A 0.8 cm square of hydrogel was blotted dry and placed backing-up on a transparency film. The layer was fixed to the surface with black polyethylene waterproof tape (3M, St. Paul, Minn.) from which a 0.5 cm square had been removed to provide exposure to the sample solution. With this setup, the sensor assembly could be easily attached to the inside surface of polystyrene disposable cuvettes using adhesive or double-sided tape, enabling front face measurement of the HPTS-Dowex-PEG layer.
As depicted schematically in
HPTS-Dowex-PFG Sensor Calibration
For calibration of the sensor assemblies, buffered saline solutions (0.15 M or 0.3 M overall ionic strength) were prepared by reconstitution of sodium chloride and the appropriate base (10 mM) in deionized water and titration to the desired pH using 1 M HCl or 1M NaOH. Calibration buffers with pH between 6.2 and 8.2 were buffered with phosphate, while those solutions of higher pH (>8.2) were buffered with Tris. Luria-Bertani (LB) nutrient broth was prepared that contained sodium chloride (5 g/L), tryptone (10 g/L) and yeast extract (5 g/L) with pH adjusted to 7.2 with strong acid or base. The pH of each solution was recorded using a Model IQ240 Benchtop/Portable ISFET sensor pH probe (IQ Scientific Instruments; San Diego, Calif.).EXAMPLE 16
Comparison of HPTS and HPTS-PEG-Dowex Sensor Assembly Excitation and Emission Spectra
The excitation spectra of the control layer prepared without HPTS is also depicted in
pH-Dependent Excitation Spectra of HPTS-PEG-Dowex Sensor Assemblies
In addition, the precision of these measurements indicates that the scatter described earlier is consistent and does not interfere with the operation of the sensor. The apparent pKa of the immobilized dye is approximately 7.7, slightly higher than that of HPTS in solution. This shift toward a more basic value is not surprising. Given that both the highly anionic sulfonic acid groups on the dye and the polyether segments of the PEG hydrogel are attractive to protons, the apparent pH inside the sensor matrix is reduced. The range of linearity of the HPTS-Dowex-PEG sensor extends from approximately 6.7 to 8.7. The increase in pKa is therefore favorable in terms of bioprocess measurement, as it corresponds to a working range that is consistent with that observed during many microbial and mammalian fermentations including Escherichia coli and Klebsiella pneumoniae (5, 19).EXAMPLE 18
Performance of HPTS-Dowex-PEG Sensor Assembly Under Conditions of Varying Ionic Strength and Temperature
The effect of various operating parameters on the performance of the HPTS-PEG-Dowex was evaluated.
The performance of the HPTS-PEG-Dowex sensor assembly is also sensitive to temperature.
Changes in temperature and ionic strength can influence the degree of hydration of both the Dowex resin and the PEG hydrogel and, therefore, the local dye concentration. However, such a change in swelling alone would be expected to affect both the UV and blue excitation peaks equally. Unexpectedly, this contradicts what is expected based on the increase in nonradiative decay at higher temperatures, indicating possible interactions between the dye and the immobilization resin. Since the sensor is targeted for use in bioprocesses that are largely temperature-controlled, the impact is minimal.
The effect of aging on sensor performance was also evaluated. Since PEG hydrogels must be stored hydrated to prevent cracking of the matrix, it is simportant to monitor the potential for leaching of HPTS into the soaking solution. Sensor assemblies were shielded from light and stored in deionized water for three weeks at ambient temperature. While loss of signal intensity was minor, less than 10%, and expected, owing to equilibrium leaching of HPTS from the matrix into the storage solution, there was no observable change in the sensor calibration curve. The sensor assembly was also exposed to 70% ethanol/water to determine the feasibility of this method of sterilization. Apart from temporary and reversible dehydration of the hydrogel matrix, no change in the performance of the sensor was detected.
The dynamic behavior of the sensor was investigated by pumping different pH buffers into the cuvette. The sensor was allowed to equilibrate in each buffer with constant, open-loop circulation from a larger reservoir while the excitation intensity ratio was monitored. The response time for a 95% change was approximately 9 minutes. While this is adequate for the purpose of bioprocess monitoring, the faster response times that are desirable for control schemes should be easily attained with a reduction in sensor thickness. The change in intensity ratio was completely reversible and could be cycled back and forth with no observable drift.EXAMPLE 19
Use of HPTS-PEG-Dowex Sensor in a Hatch Fermentation
In preparation for use in a batch fermentation, the performance of the HPTS-PEG-Dowex sensor was evaluated in spent nutrient broth from an Escherichia coli fermentation. LB broth containing E. coli at an optical density of 600 nm of 0.25 was titrated to produce samples of varying pH, between 6 and 9.
A polystyrene disposable cuvette containing a sensor assembly was sterilized thoroughly with 70 v/v % ethanol solution, rinsed extensively in sterile water and mounted in the spectrophotometer. To prevent optical interference from the media beyond the edges the sensor, the outside of the cuvette was covered with black tape except for a small square that exposed the sensor. The fermentation broth was pumped into the cuvette and recirculated to the shake flask by a peristaltic pump operating at 40 mL/minute. The spectrophotometer recorded the emission intensity at 515 nm with excitation at 408 nm and 468 nm, at 1 minute intervals. At 20 minute intervals, samples of the media were removed and analyzed for pH using the ISFET probe and optical density at 600 nm using a Milton Roy Spectronic 401.
The PEG-Dowex sensor assembly was used to perform continuous on-line measurement of the pH during an E. coli fermentation.
Furthermore, the raw intensities recorded by the sensor, as well as the calculated ratio, were remarkably stable with very little noise, in spite of the high flow rate of 40 mL/min and degree of mixing inside the cuvette. Toward the end of the fermentation, greater than 8.5 hours, a significant and consistent overprediction of approximately 0.15 pH units was observed. Examination of the used patch revealed evidence of microbial growth on the nside surface of the hydrogel, indicating that the patch had been poorly sealed to the transparency film. It is difficult to attach a highly swollen layer to a hydrophobic surface. The polyethylene tape used here to fix the patch in place may allow media to accumulate in stagnant areas between the hydrogel and the transparency film. Other methods of attachment may be more effective and could extend the lifetime of the sensor.EXAMPLE 20
HPTS-pHEMA-METMA Sensor Assembly
The reaction mixture consisted of HEMA, METMA and EGDMA in a (10:1:0.8) volume ratio with 10 mg DMPA initiator per ml of HEMA. The mixture was sandwiched between two glass plates using aluminum spacer tape to maintain a gap width of approximately 100 μm. Free radical polymerization was initiated by exposure to a 100 W longwave UV spot lamp for 2 minutes. The polymer was hydrated overnight in deionized water and peeled from the glass plate.
A 1 cm square of pHEMA-METMA was placed in a solution of HPTS in 200 μM deionized water. The membrane was allowed to equilibrate for 24 hours with occasional stirring. Excess HPTS was removed by soaking in deionized water for a further 24 hours with several changes of the soaking solution. HPTS-membranes were mounted into polystyrene cuvettes for front face measurement as before, using 0.2 μm microfiltration membrane as a backing.EXAMPLE 21
Excitation Spectra of HPTS-pHEMA-METMA Sensor in PBS at Varying pH
Hydrogel membranes of pHEMA-METMA are optically clear with good strength and flexibility. The cationic functionality incorporated via the comonomer results in visibly high HPTS uptake. Response times are on the order of a few minutes for a 200 μm thick sensor layer.
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Any patents or publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. Further, these patents and publications are incorporated by reference herein to the same extent as if each individual publication was specifically and individually incorporated by reference.
One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The present examples along with the methods, procedures, treatments, molecules, and specific compounds described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention as defined by the scope of the claims.
1. A fluorescent dye having the structure of
2. The fluorescent dye of claim 1, wherein said fluorescent dye is 6-methacryloyl-8-hydroxy-1,3-pyrene disulfonic acid (MA-HPDS).
3. A ratiometric fluorescent pH sensor comprising:
- a fluorescent dye, said fluorescent dye exhibiting a shift in excitation wavelength with a corresponding shift in pH in the local environment of said fluorescent dye;
- a polymer matrix, said fluorescent dye immobilized therein, said polymer matrix comprising a front surface and a back surface;
- a means of optically isolating said polymer matrix comprising said fluorescent dye; and
- an adhesive means.
4. The ratiometric fluorescent pH sensor of claim 3,
- wherein said fluorescent dye is 6-methacryloyl-8-hydroxy-1,3-pyrene disulfonic acid (MA-HPDS); and
- wherein said polymer matrix is a p(ethylene glycol) hydrogel.
5. The ratiometric fluorescent pH sensor of claim 4, wherein said p(ethylene glycol) immobilizes said MA-HPDS via covalent binding therewith.
6. The ratiometric fluorescent pH sensor of claim 4, further comprising a comonomer, a cross-linking agent or a combination thereof.
7. The ratiometric fluorescent pH sensor of claim 6, wherein the comonomer is a siloxane-based monomer.
8. The ratiometric fluorescent pH sensor of claim 6, wherein the cross-linking agent is trimethylolpropane triacrylate or ethylene glycol dimethacrylate.
9. The ratiometric fluorescent pH sensor of claim 3,
- wherein said fluorescent dye is 8-hydroxy-1,3,6-pyrene trisulphonic acid trisodium salt (HPTS); and
- wherein said polymer matrix is p(ethylene glycol) diacrylate monomer polymerized around an anionic exchange resin.
10. The ratiometric fluorescent pH sensor of claim 9, wherein said anionic exchange resin immobilizes HPTS via ionic binding therewith.
11. The ratiometric fluorescent pH sensor of claim 3,
- wherein said fluorescent dye is 8-hydroxy-1,3,6-pyrene trisulphonic acid trisodium salt (HPTS); and
- wherein said polymer matrix is a poly(2-hydroxyethyl methacrylate)-co-(methacryloyl ethyltrimethyl ammonium chloride) (pHEMA-METMA) copolymer hydrogel.
12. The ratiometric fluorescent pH sensor of claim 11, wherein said pHEMA-METMA immobilizes HPTS via ionic binding therewith.
13. The ratiometric fluorescent pH sensor of claim 3, wherein the optically isolating means is a microfiltration membrane.
14. The ratiometric fluorescent pH sensor of claim 3, wherein the adhesive means is an adhesive tape, a hydrophobic adhesive substrate or a transfer adhesive.
15. The ratiometric fluorescent pH sensor of claim 14, wherein said adhesive tape is a polyethylene tape.
16. The ratiometric fluorescent pH sensor of claim 14, wherein said hydrophobic adhesive substrate is a silicon rubber substrate or an acrylate-functional adhesive.
17. The ratiometric fluorescent pH sensor of claim 3, wherein the fluorescent dye comprising the sensor exhibits dual excitation maxima wavelengths and an emission wavelength maxima.
18. The ratiometric fluorescent pH sensor of claim 3, wherein the fluorescent dye comprising the sensor responds to a shift in pH within a range of about pH 6.0 to about pH 9.0.
19. The ratiometric fluorescent pH sensor of claim 3, wherein the sensor is sterilized.
20. The ratiometric fluorescent pH sensor of claim 3, wherein the sensor comprises a patch.
21. The ratiometric fluorescent pH sensor of claim 20, wherein assembly of said sensor patch comprises the steps of:
- placing the adhesive means on a flat surface;
- adhering the front surface of said polymer matrix comprising said fluorescent dye to the adhesive means; and
- covering the back surface of said polymer matrix with the optically isolating means.
22. A system for non-invasive monitoring of pH comprising:
- a reaction vessel in which to monitor the pH;
- the ratiometric fluorescent pH sensor of claim 3 wherein the front surface of said pH sensor adheres to the reaction vessel via said adhesive means;
- a means for delivering a first pH-sensitive excitation maximum wavelength to the front surface of said fluorescent pH sensor;
- a means for delivering a second pH-sensitive excitation maximum wavelength to the front surface of said fluorescent pH sensor;
- a means for detecting maximum emission intensity of a wavelength emitted from said fluorescent pH sensor at both of the first and second pH-sensitive excitation maxima wavelengths; and
- a means for evaluating a ratio of the first and second maximum emission intensities as pH.
23. The system of claim 22, further comprising means for closed-loop feedback control of the system.
24. The system of claim 22, wherein said delivery means is a light emitting diode.
25. The system of claim 24, wherein said light emitting diode emits light in the ultraviolet wavelength range or in the blue wavelength range.
26. The system of claim 22, wherein said detecting means is a photodiode.
27. A method of continuous monitoring of pH of a growth media during a fermentation process comprising the steps of:
- placing the growth media or a sample therefrom into the reaction vessel of the system of claim 22;
- exciting the fluorescent pH sensor with the first of said pH-sensitive excitation maxima wavelengths;
- measuring the intensity of fluorescent light emitted from the sensor at said emission maximum wavelength upon said first excitation;
- exciting the fluorescent pH sensor with the second of said pH-sensitive excitation maxima wavelengths;
- measuring the intensity of fluorescent light emitted from the sensor at said emission maximum wavelength upon said second excitation;
- calculating a ratio of said emission intensity at the first excitation maxima wavelength to said emission intensity at the second excitation maxima wavelength; and
- correlating said ratio to the pH of the growth media thereby monitoring pH during fermentation.
Type: Application
Filed: Jun 30, 2003
Publication Date: Apr 28, 2005
Patent Grant number: 7390462
Inventors: Govind Rao (Columbia, MD), Iordan Kostov (Baltimore, MD), Haley Kermis (Baltimore, MD), Peter Harms (Ellicott City, MD)
Application Number: 10/609,720


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For the first time the exhibition Picturex will present to the public a vast selection of art works from the Proximus Art Collection. This project fits perfectly within the series of collection presentations that the museum organizes since 2007. By examining and exhibiting important private collections one can study the acceptance of contemporary art and the impact of the collector on the Belgian art scene. It is also a unique opportunity to discover significant private art patrimony!
The Proximus collection is one of the most important corporate collections in Belgium and is a fascinating display of the art production of the last four decades. A committee of art experts, consisting of art critics and curators, compiled the collection. Since 1996 they have brought together works of some hundred artists. Most of the works were given a place in the corridors and offices of the Brussels Proximus premises.
The exhibition Picturex focuses on contemporary photography and displays several photograph series from the collection. More than half of the works acquired by Proximus Art between 1996 and 2014 is photography and many artists who, in the last two decades, have made a difference with this medium are represented in the collection with a series of photographs. In addition to photography other art media are also represented in the collection. To show the diversity we decided to show some absolute masterpieces of our recent art history.
With works by John Baldessari, Lothar Baumgarten, Pierre Bismuth, Balthasar Burkhard, Jean-Marc Bustamante, Jan De Cock, Rodney Graham, Andreas Gursky, Seydou Keïta, Saul Leiter, Robert Mapplethorpe, Gabriel Orozco, Sigmar Polke, Thomas Ruff, Ana Torfs,en Marijke Van Warmerdam.


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Mulan Chinese Restaurant
Mulan, established in 2008, reflects the concept of new China. Banished were the outmoded western ideas of what traditional chinese restaurants should look like. The new creation would be cutting edge and contemporary. Beautiful design lines and shapes draw clients into a colourfoul world of stule and panache. The interior invites the customer to participate in an ambience of reflective fine dining. The meal becomes an opportunity to indulge tastes for unique cuisine, shared with friends and family.
▶ Mulan Chinese Restaurant? Like it? Hate it? Review it! ▼
Opening Hours:
Reservation needed: ---
Tel :24629508
Fax: 24626433
Address: 1 Areos, 6016, Larnaka
Email: [email protected]
Listed under the following categories:
- Smoking Area
- Open for Lunch
- Open for Dinner
- Vegetarian Items on Menu
- Parking Available
- Credit Cards Accepted
- Outdoor Seating
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Providing information and support on the keeping, breeding and care of parrots in captivity and encouraging 'best practice' for all aspects of parrot aviculture.
Can we help?
Tel: 07 3208 5610
Member login below.
Saturday 16th May 2020
Welcome to our hotspot for the largest Bird Sale on Brisbane's northside, and a great event to be hosted in the Moreton Bay region! The next NBBS will be on Saturday 16th May 2020. To book tickets online, and receive fast-tracked entry to our event, click below:
Every Autumn we return to the Caboolture Showgrounds, with a wide array of bird and trade vendors collecting for the general public to come and explore. This is a family friendly event, and wheelchair accessible. All birds are examined prior to entry by an Avian Veterinarian, and we adhere to all relevant Codes of Practice.
To book table space, please contact Chris Jaffrey on 0404 494 485 or E-Mail Chris by clicking the below link:
Here's a quick video from long-term Parrot Society of Australia Member Mike Hannant (Brisbane Birds). A great insight to what we offer in Moreton Bay!


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open view news web desk
6th edition of Miss Himalaya conducted by Mr Lobsang Wangyal founder director Miss himalya pageant
Jammu: 999 Productions welcomed Miss Himalaya 2nd runner-up Miss Ashima Sharma in Press Club Jammu and interacted with Miss Ashima Sharma. Speaking on her experience, she told that this was 6th edition of Miss Himalaya conducted by Mr Lobsang Wangyal founder director Miss himalya pageant. Among 40 participants from all over region of Himalaya only 7 girls were selected as finalist and she is the first ever girl from J&K to qualify and selected for such prestigious pageant. After selection she got grooming session in Machlodganj Himachal for 3 day during which she visited many places and got chance to interact with each other cultures of Himalaya region. Finale was held on 6th of October after 4 rounds-Talk round in which she had to talk on a topic for 5-7 minutes, Talent round, traditional costume and interview round judges gave their decision and she marked her 2nd runner-up place and was awarded by the jury members. She also thanked to those persons who helped her to bag this crown. Mrs Parul Bhateja (trained classical dancer and choreographer from Delhi), Mrs Neelam Kumari (Designer), Miss Mehak Sharma (Property Designer). During this press conference Parvinder Pal Singh owner 999 productions congratulated Ashima for her success and wished her luck for future events. She is first female fashion choreographer of J&K and recently awarded by Indian Institute of Science and Technology for her contribution in her field. Ashima is now national director J&k of Miss Himalaya peagent. After now she will send the contestant to this pegeant every year. Social activist Aditi Sharma present on the occasion congratulated Ashima for her success and also appreciated her hard work to achieve that mile stone in early age of her career.
posted on 12/10/2018
updated on 13/10/2018
Categories: Jammu & Kashmir


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If nothing else, this faux-pandemic has unmasked just how many people there are in this country who truly hate the United States of America (and by default, Donald Trump) and who will do whatever is necessary to ensure the downfall of this still great country. Or better yet in their eyes, its transformation into a Confederation of Socialist states tightly controlled by an elite group of politburo members. (Eh...sound familiar?) I hate them all, these anarchists. But they are not the only ones upon whom I vent my base emotion; there are others...
I hate the Chinese...not the people themselves, but their overlords - the Chinese Communists. It is they who have succeeded in bringing to its knees the world's most vibrant economy and who have directly caused:
- the permanent closing of numerous small businesses;
- the curtailment of civil liberties;
- the neutering of Constitutional guarantees, and;
- the unprecedented birth of mini-dictators at the State level.
I hate the Federal Government's medical professionals (Fauci & Co.) who essentially have shut down the national economy to control a virus that can't be controlled, based upon computer models that have repeatedly been proven false and whose past and still current affiliations with Bill Gates mean a further erosion of both the economy and, especially, personal liberties. To paraphrase an oft-quoted Winston Churchill: "Never before have so few
un-electeds taken away more civil liberties from so many citizens." A true omniscient, was Churchill.
I hate the Democratic Party and the Socialist/Marxist Democrats within its members: the Pelosi's; the Schumer's; the Cuomo's. And let's not forget the young, up-andcoming Marxist Democrats: Alexandra Oscasio-Cortez, Ilhan Ohmar and Rashida Tlaib. (And...the now ubiquitous Obama's, of course.) These people have NO allegiance whatsoever to this country, but only to their Party and their Personal ideals. Collectively, they seek to tear-down, destroy, amend, transform and, ultimately, to re-birth this country into The United Socialist States of America. How do these people get elected and how do they stay elected? (See below for part of that answer.)
I hate the United States Conference of Catholic Bishops, and their minion diocesan Bishops. The USCCB is just another branch of the Democratic Party and has been for the last 5 decades. For each Democratic Party cause celebre, you will find open support from the USCCB or, at the least, a large group of their progressive Bishops. Maureen Mallarkey rightly describes them as "...an open borders lobby, a fifth column in cope and chasuble." She is not far from wrong and her 2018 article demands a full read.
I hate Progressive Catholics who use their Faith when its convenient (Pelosi's "I am a Catholic" comes to mind) but who otherwise keep it under tight control and well hidden. It is an open secret that enough Catholics profess allegiance to the Democratic Party (and therefore, allegiance to its main platforms - unrestricted abortion and homosexuality) to keep Democrats in power (or at least power enough to affect the political ebb & flow of this country). Around 45% to well over 50% of Catholics vote Democratic and have for a goodly number of years, knowingly putting their Spiritual lives in grave danger through formal cooperation in the grave sins of others. In the post-Vatican II age, even nominal Catholics cannot claim invincible ignorance about abortion, homosexuality or adultery.
I could go on with more "I hate (??????)", but I think you get my point by now, and, that I've proven hate can be a virtue when done for a just reason, as noted in the above examples.
I have nothing, and no one, to fear...
|"Thou shalt not hate thy brother in thy heart, but reprove him openly, lest thou incur sin through him."
Copyright 2020 M3 Squared - All Rights Reserved


text: I have a old CD/DVD player and a old VCR a new Blu-Ray and a new TV LG smart UHD4K. I don't have cable tv,I have a TV converter box so we can watch TV. How do I connected them together to work?Is it possible?If so how do you do it and what cables do I need?I'm tech challenged!


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Miral Welfare Foundation believes that every child has an equal right to access to inclusive, quality education.
We provide education opportunities to children to help them grow healthy and become leaders in the world.
Our holistic approach for children includes not only education programs but nutrition supports, life skills,
water, sanitation and hygiene(WASH) program.
Hope School Construction10Schools
2019 Miral Yearly Report
300 children die from starvation every hour.
15% of children in the world have never access to education.
70% of children of developing countries study without textbooks.
53,000 children die from violence each year.
1 in 6 children in developing countries is engaged in child labor.
230,000 children die of HIV / AIDS a year.
An average of 20,000 children per day die before they reach their fifth birthday.
One in four children around the world develop poorly.
To advocate survival, protection, growth and right to participate of children under the guideline of UNCRC.
To provide education for all children based on "Education for All'.
To help children in poverty to become leaders through holistic education; intellectual, physical, socio-emotional, and spiritual areas.
To achieve "quality education" with quantity growth based on "Education for All".
Educational environment
school supplies support,
financial support
Nutrition, WASH program,
Medical Care,
Myanmar, Ethiopia, Uganda, Côte d'Ivoire, Thailand
You can check the country name when mouse hover.
Miral Welfare Foundation have implemented "Hope School Project" in Africa region in conjunction with "SBS hope TV" to protect rights to safe and quality education for all children. As a part of this project, Miral built 12 community-based schools including special schools for children with disabilities. Now children have equal access to safe and nurturing school environment where they can unlock their potential and fulfill their dreams with trained teachers, teaching and learning materials, etc. We work with children , communities, families, and government to support children's education.
|School Building
|Hope School
|Grace Evangelical Elementary School, Grace Evangelical Special School, Grace Evangelical Junior High School
|Association Aina Fitiavana, EPP Fanantenana Velona Anjabetrongo
|Hanbyul Christian Academy, Hanbyul Christian Academy High School, Guangua Green Light School
|Kim's Christian School, Bethel Secondary School
|Côte d'Ivoire
|College Prive MIC-CI Espoir de Brofodoume


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Architectural Photography | Exteriors
Architectural photography Portland Maine region by Mike Shaw. Serving Maine, New Hampshire, Boston area, and beyond. If your looking for professional photography to document your beautiful architecture, building project, construction work, engineering, or commercial real estate investment, please reach out to me so we can discuss your needs, and I'll provide you with a quote. Thank you!
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- /goht"suk'euhr/, n.nightjar (def. 2).[1605-15; so called because formerly believed to suck the milk of goats; trans. of L caprimulgus, itself trans. of Gk aigothélas]
* * *alternative name of birds of the family Caprimulgidae, otherwise known as nightjars (nightjar) (q.v.). The name goatsucker is sometimes applied to the order, Caprimulgiform.
* * *
Universalium. 2010.
Look at other dictionaries:
Goatsucker — Goat suck er, n. (Zo[ o]l.) One of several species of insectivorous birds, belonging to {Caprimulgus} and allied genera, esp. the European species ({Caprimulgus Europ[ae]us}); so called from the mistaken notion that it sucks goats. The European… … The Collaborative International Dictionary of English
goatsucker — [gōt′suk΄ər] n. [transl. of L caprimulgus < capri, goat + mulgere, to milk: it was thought to suck milk from goats] any of an order (Caprimulgiformes) of large mouthed, usually nocturnal birds that feed on insects or fruit; esp., any of the… … English World dictionary
goatsucker — noun Date: 1611 nightjar … New Collegiate Dictionary
goatsucker — noun Any bird in the nightjar family Caprimulgidae … Wiktionary
goatsucker — n. nightjar, species of insectivorous nocturnal bird from the genus Caprimulgus … English contemporary dictionary
goatsucker — noun another term for nightjar … English new terms dictionary
goatsucker — goat•suck•er [[t]ˈgoʊtˌsʌk ər[/t]] n. orn any of numerous insect eating, mostly nocturnal birds of the family Caprimulgidae, of nearly worldwide distribution, including nightjars and nighthawks • Etymology: 1605–15; trans. of L caprimulgus,… … From formal English to slang
goatsucker — /ˈgoʊtsʌkə/ (say gohtsukuh) noun 1. the Eurasian nightjar, Caprimulgus europaeus, formerly supposed to suck the milk of goats. See nightjar (def. 2). 2. (formerly) any bird of the order Caprimulgiformes, as nightjars, frogmouths, owlet nightjars … Australian English dictionary
goatsucker — n. = NIGHTJAR … Useful english dictionary
European goatsucker — noun Old World goatsucker • Syn: ↑European nightjar, ↑Caprimulgus europaeus • Hypernyms: ↑goatsucker, ↑nightjar, ↑caprimulgid • Member Holonyms: ↑Caprimulgus, ↑ … Useful english dictionary