text:
US20040043511A1 - Membrane-based assay devices - Google Patents
US20040043511A1
US20040043511A1 US10228836 US22883602A US20040043511A1 US 20040043511 A1 US20040043511 A1 US 20040043511A1 US 10228836 US10228836 US 10228836 US 22883602 A US22883602 A US 22883602A US 20040043511 A1 US20040043511 A1 US 20040043511A1
US10228836
US7285424B2 (en )
Xuedong Song
a) a porous membrane in fluid communication with detection probes capable of generating a detection signal and magnetic calibration probes capable of generating a calibration signal, the porous membrane defining a detection zone; and
b) a magnetic device positioned adjacent to the detection zone;
ii) contacting the detection probes and calibration probes with the test sample to form a solution;
[0023]FIG. 1 is a perspective view of one embodiment of a membrane-based device of the present invention;
[0024]FIG. 2 is a graphical illustration of the mechanism used for one embodiment of a sandwich assay format of the present invention;
[0025]FIG. 3 is a graphical illustration of the mechanism used for another embodiment of a sandwich assay format of the present invention;
[0026]FIG. 4 is a graphical illustration of the mechanism used for one embodiment of a competitive assay format of the present invention;
[0027]FIG. 5 is a graphical illustration of the mechanism used for another embodiment of a competitive assay format of the present invention;
[0028]FIG. 6 is a graphical illustration of one embodiment for covalently conjugating an antibody to carboxylate nanoparticles;
[0029]FIG. 7 shows the excitation (EX) and emission (EM) spectra of a calibration probe (C) and a detection probe (FP) in accordance with one embodiment of the present invention;
[0030]FIG. 8 shows the normalized fluorescent intensity versus the amount of leutinizing harmone (LH) as discussed in Example 1;
[0031]FIG. 9 shows the normalized fluorescent intensity versus the amount of leutinizing harmone (LH) as discussed in Example 2; and
[0032]FIG. 10 shows the normalized fluorescent intensity versus the amount of C-reactive protein (CRP) as discussed in Example 4.
In some embodiments, the detection probes 41 and/or the calibration probes 43 are fluorescent and magnetic. Fluorescent magnetic probes are generally well known in the art and often include a magnetically responsive component and a fluorescent component. In some embodiments, for example, one or more fluorescent dyes can be applied to magnetic particles to form the probes, while in other embodiments, fluorescent dye(s) can be applied to non-magnetic particles that are coupled with magnetic particles. Some examples of suitable fluorescent dyes include, but are not limited to, monomethine dyes, trimethine dyes, pentamethine dyes, quinoline dyes, squaric acid-based dyes, and the like. The monomethine dyes that are pyridines typically have a blue or blue-green fluorescence emission, while quinolines typically have a green or yellow-green fluorescence emission. The trimethine dyes are substantially shifted toward red wavelengths, while the pentamethine dyes are shifted even further, often exhibiting infrared fluorescence emission. Specific examples of such fluorescent dyes include, but are not limited to, phthalocyanines, 2,3-naphthalocyanines, squaraines and croconic acid derivatives. Other examples of suitable fluorescent magnetic particles are believed to be described in U.S. Pat. Nos. 4,731,337 to Luotola, et al. and 6,268,222 to Chandler, et al., which are incorporated herein in their entirety by reference thereto for all purposes.
At the detection zone 31, the complexes 49 and any unbound conjugated, fluorescent magnetic particles 43 are then captured by a magnetic device 60 and separated from the rest of the sample using conventional techniques. A magnetic field generator, for instance, can be used to generate a magnetic field that elicits a response from the magnetically responsive probes. Suitable magnetic field generators include, but are not limited to, permanent magnets and electromagnets. The magnetic separation process typically involves mixing the sample with the magnetic particles in a liquid medium to bind the analyte by affinity reaction, and then separating the unbound magnetic particles and analyte complexes from the sample medium by applying a magnetic field. Most, if not all of the magnetic particles, except those particles that are colloidal, settle in time. The liquid medium, therefore, can be agitated to keep the particles suspended for a sufficient period of time to allow the bioaffinity binding reaction to occur. Examples of known agitation methods include shaking, swirling, rocking, rotation, or similar manipulations of a partially filled container. Some commercially available examples of suitable magnetic separation devices include the Dynal MPC series of separators manufactured by Dynal, Inc., Lake Success, N.Y., which employ a permanent magnet located externally to a container holding a test medium and provide only for separation. Mixing of the magnetic particles in the test medium for affinity binding reaction is done separately. In addition, other methods for capturing magnetic particles may be described in U.S. Pat. Nos. 5,200,084 to Liberti, et al.; 5,647,994 to Tuunanen, et al.; 5,795,470 to Wang, et al.; and 6,033,574 to Siddigi, which are incorporated herein in their entirety by reference thereto for all purposes.
Further, if desired, a technique known as “time-resolved fluorescence detection” may also be utilized in the present invention. Time-resolved fluorescence detection is designed to reduce background signals from the emission source or from scattering processes (resulting from scattering of the excitation radiation) by taking advantage of the fluorescence characteristics of certain fluorescent materials, such as lanthamide chelates of europium (Eu (III)) and terbium (Tb (III)). Such chelates can exhibit strongly red-shifted, narrow-band, long-lived emission after excitation of the chelate at substantially shorter wavelengths. Typically, the chelate possesses a strong ultraviolet absorption band due to a chromophore located close to the lanthamide in the molecule. Subsequent to light absorption by the chromophore, the excitation energy can be transferred from the excited chromophore to the lanthamide. This is followed by a fluorescence emission characteristic of the lanthamide. The use of pulsed excitation and time-gated detection, combined with narrow-band emission filters, allows for specific detection of the fluorescence from the lanthamide chelate only, rejecting emission from other species present in the sample that are typically shorter-lived or have shorter wavelength emission. Other time-resolved techniques for measuring fluorescence are described in U.S. Pat. No. 5,585,279 to Davidson and 5,637,509 to Hemmila, et al., which are incorporated herein in their entirety by reference thereto for all purposes.
(1) 25 microliters of covalently conjugated, non-fluorescent magnetic particles (3 milligrams per milliliter in PBS buffer);
(2) 15 microliters of covalently conjugated, fluorescent non-magnetic particles (2 milligrams per milliliter in PBS buffer);
(3) 10 microliters of fluorescent magnetic particles blocked by β-casein (3 milligrams per milliliter in PBS buffer); and
(4) Leutinizing hormone (LH) analyte ranging from 0,10 microliters (1 microgram per milliliter), 20 microliters (1 microgram per milliliter), 40 microliters (1 microgram per milliliter), 40 microliters (2 microgram per milliliter) and 80 microliters (2 micrograms per milliliter).
To each of the Eppendorf vials, an appropriate amount of PBS buffer was added to a final volume of 150 microliters. The samples were incubated at room temperature for 10 minutes with gentle shaking. The magnetic particles were then separated by a magnetic separator obtained from Dynal, Inc. The supernatant from each vial was discarded and the magnetic particles were re-suspended in 1.5 milliliters of PBS. 300 microliters of the fluorescent magnetic particle suspension was used for each fluorescence measurement. A “Flourolog III Spectrofluorometer”, which was obtained from SPEX Industries, Inc. of Edison, N.J., was used to measure the fluorescence of the sample using a right angle mode. An excitation wavelength of 470 nanometers and an emission wavelength of 560 nanometers were used for the fluorescent magnetic particles, while an excitation wavelength of 570 nanometers and an emission wavelength of 605 nanometers were used for the fluorescent, non-magnetic particles. The integration time was 0.2 seconds.
125 microliters of 10% carboxylate-modified paramagnetic particles (0.35 microns, Estapor® Superparamagnetic microspheres, obtained from Bang's Laboratories, Inc.) were washed once with 1.5 milliliters of carbonate buffer and twice with PBS using a magnetic separator. The washed particles were re-suspended in 0.6 milliliters PBS and 15 milligrams carbodiimide (from Polysciences, Inc.). The mixture was allowed to react at room temperature (RT) for 30 minutes on a shaker. The activated particles were then washed twice with a borate buffer. The activated particles were again re-suspended in 1.2 milliliters of a borate buffer. Thereafter, 30 microliters of LH β-monoclonal antibody (9.8 mg/ml, obtained from Fitzgerald Industries International, Inc.) was added to the activated particles. The reaction mixture was allowed to react at room temperature on a shaker overnight. The activated particles were then collected and incubated in 1 milliliter of 0.1 molar ethanolamine under gentle shaking for 15 minutes. The particles were then washed twice with PBS and stored at 4° C. in a buffer that contained 0.1 molar PBS, 0.15 molar NaCl, 1% β-casein, 5% glycerol and 0.1% NaN3.
The “fluorescent non-magnetic” particles were covalently conjugated according to the procedure described above, except that the binding member was LH α-monoclonal antibody (9.8 milligrams per milliliter, obtained from Fitzgerald Industries International, Inc.) instead of LH β-monoclonal antibody. The particles utilized were FluoSpheres® carboxylate-modified microspheres, which were obtained from Molecular Probes, Inc. The particles had a particle size of 0.5 microns, and were red fluorescent with an excitation wavelength of 580 nanometers and an emission wavelength of 605 nanometers.
100 microliters of a 2.76% solids suspension of fluorescent superparamagnetic particles (obtained from Polysciences, Inc. of Warrington, Pa.) were combined with 1 milliliter of a borate buffer (0.1 molar, pH=8.5) in an Eppendorf tube. Such particles have a mean diameter of between 1 to 2 microns and are believed to be iron-containing microspheres that have a polystyrene surface that allows for passive adsorption and functional group reactions with proteins. The particles were separated by a magnetic separator obtained from Dynal, Inc. and re-suspended in 200 microliters of a 10 milligram per milliliter solution of β-casein in a 0.1 molar borate buffer. The suspension was incubated for 30 minutes with gentle mixing. The above step was repeated twice. The separated particles were re-suspended in 200 microliters of PBS and stored at 4° C.
(1) 5 microliters of covalently conjugated, fluorescent non-magnetic particles (2 milligrams per milliliter in PBS buffer);
(2) 15 microliters of physical absorption conjugated, fluorescent magnetic particles (3 milligrams per milliliter in PBS buffer); and
(3) Leutinizing hormone (LH) analyte ranging from 0, 5, 10 microliters, 20, 40, and 100 microliters (2 micrograms per milliliter).
To each of the Eppendorf vials, an appropriate amount of PBS buffer was added to a final volume of 150 microliters. The samples were incubated at room temperature for 25 minutes with gentle shaking. The magnetic particles were then separated by a magnetic separator obtained from Dynal, Inc. The supernatant from each vial was discarded and the magnetic particles were re-suspended in 1.5 milliliters of PBS. 300 microliters of the fluorescent magnetic particle suspension was used for each fluorescence measurement. A “Flourolog III Spectrofluorometer”, which was obtained from SPEX Industries, Inc. of Edison, N.J., was used to measure the fluorescence of the sample using a right angle mode. An excitation wavelength of 470 nanometers and an emission wavelength of 560 nanometers were used for the fluorescent magnetic particles, while an excitation wavelength of 570 nanometers and an emission wavelength of 605 nanometers were used for the fluorescent, non-magnetic particles. The integration time ranged from 0.2 to 1 second.
(1) 15 microliters of covalently conjugated, non-fluorescent magnetic particles (3 milligrams per milliliter in 0.1 molar PBS buffer);
(3) 20 microliters leutinizing hormone (LH) analyte (1 microgram per milliliter); and
(4) 20 microliters of PBS.
A control Eppendorf vial was also formed with only 20 microliters of PBS (Vial No. 1 in Table I).
Vial No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 Dev %
Day 1 13 254 215 263 285 291 11
Day 2 12 235 207 300 263 299 15
Day 3 12 183 176 213 270 266 20
Day 4 18 265 226 275 282 293 10
Day 5 9 207 193 246 236 244 10
Day 6 14 227 202 252 262 274 12
Std. 23 13 8 11 6 7
(3) 20 microliters of fluorescent magnetic particles blocked by β-casein (3 milligrams per milliliter in PBS buffer); and
(4) 20 microliters leutinizing hormone (LH) analyte (1 microgram per milliliter); and
(5) 20 microliters of PBS.
A control Eppendorf vial was also formed with only 20 microliters of PBS (Vial No. 8 in Table II).
Vial No. 8 No. 9 No. 10 No. 11 No. 12 No. 13 Dev %
Day 1 31/32 352/47 344/43 300/41 318/44 369/39 12
Day 2 31/42 324/42 329/41 323/46 338/47 418/43 14
Day 3 28/39 307/40 333/42 282/42 288/40 425/46 12
Day 4 30/41 267/36 292/36 271/41 281/38 356/43 8.8
Day 5 21/29 252/33 292/34 258/38 275/36 328/37 10
Day 6 21/25 237/33 307/38 265/40 288/35 358/39 12
Std. 13 3 3 4 5 6
As can be seen from the comparisons of each set of samples for the two systems, the standard deviations (Std. Dev%) for the self-calibrated system were significantly smaller than the standard deviations without self-calibration, even under carefully controlled conditions. Because the self-calibrated system is less dependent on the measurement conditions, it is anticipated that the standard deviations for the self-calibrated system would be even smaller than the standard deviations without self-calibration when the conditions are not carefully controlled.
(1) 30 microliters of covalently conjugated, non-fluorescent magnetic particles (2 milligrams per milliliter in PBS buffer);
(2) 20 microliters of covalently conjugated, fluorescent non-magnetic particles (2 milligrams per milliliter in PBS buffer);
(3) 15 microliters of fluorescent magnetic particles blocked by β-casein (1 milligram per milliliter in PBS buffer); and
(4) C-reactive protein (CRP) analyte ranging from 0, 5, 10, 20, 50, and 100 microliters (0.2 micrograms per milliliter in PBS).
The samples were incubated at room temperature for 20 minutes with gentle shaking. The magnetic particles were then separated by a magnetic separator obtained from Dynal, Inc. The supernatant from each vial was discarded and the magnetic particles were re-suspended in 1.5 milliliter of PBS. 300 microliters of the fluorescent magnetic particle suspension was used for each fluorescence measurement. A “Flourolog III Spectrofluorometer”, which was obtained from SPEX Industries, Inc. of Edison, N.J., was used to measure the fluorescence of the sample using a right angle mode. An excitation wavelength of 470 nanometers and an emission wavelength of 560 nanometers were used for the fluorescent magnetic particles, while an excitation wavelength of 570 nanometers and an emission wavelength of 605 nanometers were used for the fluorescent, non-magnetic particles. The integration time ranged from 0.2 to 1 second. The normalized fluorescence intensity as a function of the dose of CRP in each sample is shown in FIG. 10.
125 microliters of 10% carboxylate-modified paramagnetic particles (0.35 microns, Estapor® Superparamagnetic microspheres, available from Bang's Laboratories, Inc.) were washed once by 1.5 ml carbonate buffer and twice by phosphate buffer saline (PBS) using a magnetic separator. The washed particles were re-suspended in 0.6 milliliters PBS and 15 milligrams carbodiimide (from Polysciences, Inc.). The mixture was allowed to react at room temperature (RT) for 30 minutes on a shaker. The activated particles were then washed twice with a borate buffer. The activated particles were again re-suspended in 1.2 ml borate buffer. Thereafter, 30 microliters of anti-C-reactive protein (anti-CRP1) monoclonal antibody (Mab A5804, 2 mg/ml, obtained from BiosPacific, Inc.) were added to the activated particles. The reaction mixture was allowed to react at room temperature on a shaker overnight. The activated particles were then collected and incubated in 1 milliliter of 0.1 molar ethanolamine under gentle shaking for 15 minutes. The particles were then washed twice with PBS and stored at 4° C. in a buffer that contained 0.1 molar PBS, 0.15 molar NaCl, 1%-casein, 5% glycerol and 0.1% NaN3.
The “fluorescent non-magnetic” particles were covalently conjugated according to the procedure described above, except that the binding member was anti-C-reactive protein (anti-CRP2) monoclonal antibody (2 mg/ml, obtained from BiosPacific, Inc.) instead of anti-CRP1. The particles utilized were FluoSpheres® carboxylate-modified microspheres, which were obtained from Molecular Probes, Inc. The particles had a particle size of 0.5 μm, and were red fluorescent with an excitation wavelength of 580 nanometers and an emission wavelength of 605 nanometers.
100 microliters of a 2.76% solids suspension of fluorescent superparamagnetic particles (obtained from Polysciences, Inc. of Warrington, Pa.). Such particles have a mean diameter between 1 to 2 microns, and are believed to be iron-containing microspheres that have a polystyrene surface that allows for passive adsorption and functional group reactions with proteins. 1 milliliter of a borate buffer (0.1 molar, pH=8.5) was then added to the particles in an Eppendorf tube. The particles were separated by a magnetic separator obtained from Dynal, Inc. and the particles were re-suspended in 200 microliters of a 10 mg/ml solution of β-casein in a 0.1 M borate buffer. The suspension was incubated for 30 minutes with gentle mixing. The above step was repeated twice. The separated particles were re-suspended in 200 microliters of PBS and stored at 4° C.
The sample pad of each sample was then treated with 2% polyoxyethylene sorbitan monolaurate (a nonionic surfactant available from Sigma-Aldrich under the name “Tween 20”) and dried at 37° C. for 1 hour. The conjugate pad was soaked with non-fluorescent magnetic particles, fluorescent non-magnetic particles, fluorescent magnetic particles, Tween 20 and sucrose, and then dried at 37° C. for 1 hour. The “non-fluorescent magnetic particles”, “fluorescent non-magnetic particles”, and “fluorescent magnetic particles” were formed as described above in Example 1.
A magnet strip was laid below the middle portion of each sample to form a detection zone. Thereafter, 40 microliters of PBS buffer was applied to the sampling pad of a first sample, 40 microliters of leutinizing hormone (LH) (0.5 micrograms/milliliter) was applied to the sampling pad of a second sample, and 40 microliters of LH (5 micrograms/milliliter) were applied to the sampling pad of a third sample. After 30 minutes, the fluorescent magnetic particles and fluorescent non-magnetic particles captured at the detection zone were measured with a “Flourolog III Spectrofluorometer” using a front face mode and an angle of 30° relative to the sample. An excitation wavelength of 470 nanometers and an emission wavelength of 560 nanometers were used for the fluorescent magnetic particles, while an excitation wavelength of 560 nanometers and an emission wavelength of 605 nanometers were used for the fluorescent, non-magnetic particles.
1. A membrane-based assay device for detecting the presence or quantity of an analyte residing in a test sample, said device comprising:
a porous membrane in fluid communication with detection probes capable of generating a detection signal and magnetic calibration probes capable of generating a calibration signal, said porous membrane defining a detection zone; and
a magnetic device positioned adjacent to said detection zone, wherein said magnetic device is capable of separating said detection probes and said calibration probes from a test sample applied to said porous membrane, wherein the amount of the analyte within the test sample is proportional to the intensity of the detection signal generated by said separated detection probes at the detection zone calibrated by the intensity of the calibration signal generated by said separated calibration probes at the detection zone.
2. A device as defined in claim 1, further comprising one or more conjugate pads in fluid communication with said porous membrane, said detection probes and said calibration probes being applied to said one or more conjugate pads.
3. A device as defined in claim 1, wherein said porous membrane is in fluid communication with a sampling pad to which the test sample is applied.
4. A device as defined in claim 1, further comprising a wicking pad in fluid communication with said porous membrane to facilitate the flow of the test sample therethrough.
5. A device as defined in claim 1, wherein said detection probes and said calibration probes are fluorescent compounds, chemiluminescent compounds, phosphorescent compounds, or combinations thereof.
6. A device as defined in claim 1, wherein said detection probes are fluorescent non-magnetic compounds.
7. A device as defined in claim 1, wherein said calibration probes are fluorescent magnetic particles.
8. A device as defined in claim 1, wherein said detection probes are capable of binding to the analyte.
9. A device as defined in claim 1, further comprising non-fluorescent magnetic particles.
10. A device as defined in claim 9, wherein said non-fluorescent magnetic particles are capable of binding to the analyte.
11. A device as defined in claim 1, wherein said separated detection probes include complexes formed by said detection probes.
12. A device as defined in claim 1, wherein said separated calibration probes include complexes formed by said calibration probes.
13. A device as defined in claim 1, wherein the amount of the analyte within the test sample is proportional to the intensity of the detection signal generated by said separated detection probes at said detection zone divided by the intensity of the calibration signal generated by said separated calibration probes at said detection zone.
14. A lateral flow membrane-based assay device for detecting the presence or quantity of an analyte residing in a test sample, said device comprising:
a porous membrane in fluid communication with detection probes capable of generating a detection signal, fluorescent magnetic calibration probes capable of generating a calibration signal, and optionally non-fluorescent magnetic particles, said porous membrane defining a detection zone; and
15. A device as defined in claim 14, further comprising one or more conjugate pads in fluid communication with said porous membrane, said detection probes and said calibration probes being applied to said one or more conjugate pads.
16. A device as defined in claim 14, wherein said porous membrane is in fluid communication with a sampling pad to which the test sample is applied.
17. A device as defined in claim 14, further comprising a wicking pad in fluid communication with said porous membrane to facilitate the flow of the test sample therethrough.
18. A device as defined in claim 14, wherein said detection probes are fluorescent non-magnetic compounds.
19. A device as defined in claim 14, wherein said separated detection probes include complexes formed by said detection probes.
20. A device as defined in claim 14, wherein said separated calibration probes include complexes formed by said calibration probes.
21. A device as defined in claim 14, wherein the amount of the analyte within the test sample is proportional to the intensity of the detection signal generated by said separated detection probes at said detection zone divided by the intensity of the calibration signal generated by said separated calibration probes at said detection zone.
22. A method for detecting the presence or quantity of an analyte residing in a test sample, said method comprising:
i) providing a membrane-based device, said device comprising:
a) a porous membrane in fluid communication with detection probes capable of generating a detection signal and magnetic calibration probes capable of generating a calibration signal, said porous membrane defining a detection zone; and
b) a magnetic device positioned adjacent to said detection zone;
ii) contacting said detection probes and said calibration probes with the test sample to form a solution;
iii) separating said detection probes and said calibration probes from said solution at said detection zone using said magnetic device;
iv) exciting said separated detection probes and said separated calibration probes, wherein said excitation causes said separated detection probes to emit said detection signal and said separated calibration probes to emit said calibration signal;
23. A method as defined in claim 22, wherein said membrane-based device further comprises one or more conjugate pads in fluid communication with said porous membrane, said detection probes and said calibration probes being applied to said one or more conjugate pads.
24. A method as defined in claim 22, wherein said porous membrane is in fluid communication with a sampling pad to which the test sample is applied.
25. A method as defined in claim 22, further comprising a wicking pad in fluid communication with said porous membrane to facilitate the flow of the test sample therethrough.
26. A method as defined in claim 22, wherein said detection probes are non-magnetic.
27. A method as defined in claim 22, wherein said separated detection probes include complexes formed by said detection probes.
28. A method as defined in claim 27, wherein said complexes are formed with the analyte.
28. A method as defined in claim 22, wherein said separated calibration probes include complexes formed by said calibration probes.
29. A method as defined in claim 28, wherein said complexes are formed with the analyte.
30. A method as defined in claim 22, wherein said first emission wavelength is different than said second emission wavelength.
31. A method as defined in claim 22, further comprising generating a calibration curve by plotting the intensity of the detection signal calibrated by the intensity of the calibration signal for a plurality of predetermined analyte concentrations.
32. A method as defined in claim 22, wherein said separated detection probes and said separated calibration probes are excited simultaneously.
33. A method as defined in claim 22, wherein said separated detection probes and said separated calibration probes are excited separately.
34. A method as defined in claim 22, wherein the intensity of the detection signal and the calibration signal are measured simultaneously.
35. A method as defined in claim 22, wherein the intensity of the detection signal and the calibration signal are measured separately.
36. A method as defined in claim 22, wherein said detection and calibration probes are chemiluminescent.
37. A method as defined in claim 22, wherein said detection and calibration probes are phosphorescent.
38. A method as defined in claim 22, wherein said detection and calibration probes are fluorescent.
39. A method as defined in claim 38, wherein a fluorescence reader is used to measure the intensity of said detection signal and said calibration signal.
40. A method as defined in claim 39, wherein said fluorescence reader uses time-resolved fluorescence spectroscopy to measure said detection and said calibration signals.
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US10228836 US7285424B2 (en) 2002-08-27 2002-08-27 Membrane-based assay devices
US10286342 US7632653B2 (en) 2002-08-27 2002-11-01 Membrane-based assay devices that utilize time-resolved fluorescence
KR20057002264A KR101032172B1 (en) 2002-08-27 2003-07-10 Flow-through assay with an internal calibration system using magnetic particles
KR20117014422A KR20110089194A (en) 2002-08-27 2003-07-10 Membrane-based assays using time-resolved fluorescence
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DE2003632539 DE60332539D1 (en) 2002-08-27 2003-07-10 Membrane-based method using time-resolved fluorescence measurements
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US12551048 US20090314946A1 (en) 2002-08-27 2009-08-31 Membrane-Based Assay Devices that Utilize Time-Resolved Fluorescence
US10286342 Continuation-In-Part US7632653B2 (en) 2002-08-27 2002-11-01 Membrane-based assay devices that utilize time-resolved fluorescence
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text:
Grit in the Gears: 16-Aug-2012
In some of my life, I'm a gas heating engineer, and a part of the job is carrying out service and safety inspections. This is a childcare facility.
Last year they were issued with a warning notice about storing flammable items (cans of paint, cleaning fluids, aerosol cans, cardboard, plastic toys) in the compartment holding a very big 60 Kilowatt ducted space-heater. Oh. And they were also warned about blocking the ventilation grilles.
anything wrong with this scenario?
Would you trust these 'trained professional childminders' with your child's life?

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text:
CM : Princess Purity by *kachima on deviantART
199 4 74 CM : Princess Purityby *kachima
Chibi version : COMMISSION BY PROGRAM : Photoshop/SAICharacter : Princess Purity ........................................................................... SPECIAL COMMISSION CLICK HERE [link] TO SEE MORE DETAIL Hope you enjoy it .
©2013 *kachima Category: Digital Art / Drawings & Paintings / People / Fantasy Details
Submitted onJanuary 16Image Size471 KBResolution600×849
Views1,714Favourites199 (who?)
Comments4Downloads74 URL
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CM : Princess Purity by *kachima on deviantART
I love the coloring Reply *Miss-TaDa
Hobbyist Digital Artist Love it!!!!!!!!!!!
Digital Artist This is pretty nice. Looks good Reply PreviousNext

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text:
Arsenal defender Bellerin leaves Barcelona transfer door ajar as he admits interest is ‘special’ – These are the champions!!!
Arsenal defender Bellerin leaves Barcelona transfer door ajar as he admits interest is ‘special’
The Spain international has always maintained he does not want to go back to Camp Nou, but the Gunners full-back appears to have softened that stance.
Arsenal full-back Hector Bellerin admits it is”special” to hear about reported interest from former club Barcelona.The 22-year-old has regularly been linked with a potential move back to Camp Nou, despite having extended his Gunners contract – reportedly until 2023 – last November.
Bellerin stated towards the end of last year that he had no interest in rejoining Barca but, despite repeating that he felt underappreciated at the club, he has conceded that the latest rumours are
flattering.”It’s nice there are a lot of big clubs that are interested in me, but if one of those is Barcelona, that’s special,” he told Sport. “I don’t forget I grew up in that home and I will never forget my stay at Barcelona.”
Arsenal manager Arsene Wenger said last week that it was difficult to imagine Bellerin signing for Barca so soon after renewing his contract at Emirates Stadium.”Barcelona’s alleged interest in Bellerin is very hard to take seriously,” he told a news conference. “He has just signed a new contract and still has many years left.”
Tottenham’s Kyle Walker, Southampton’s Cedric Soares and Joao Cancelo of Valencia have also been linked with the La Liga champions.
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