Patent Application
20090280472
The field of the invention relates generally to the detection of antigens, including, but not limited to, quantum dots (Qdots) and metal oxide nanoparticles. More specifically, the invention relates to the detection of antigens on a surface or in a source, which antigens include bacteria, viruses, and small proteins. In some embodiments, the invention can be used to detect biological warfare agents, such as anthrax and ricin. In some embodiments, the invention can be used for early detection of diseases in human and animals. The invention may utilize a swab-test and may further utilize a filtration process, such as with a syringe-disc.
The flurry of anthrax mailings and the contamination of Senator Thomas Daschle's Capitol Hill headquarters complex in late 2001, which later cost an estimated $41.7 million to decontaminate by chlorine dioxide, underscore the need for detection and neutralization technologies to combat civilian bioterrorism and military biowarfare attacks. Development of method that could both aid in detection and decontamination of biological warfare agents (also referred to as biowarfare agents) in building interiors would be a valuable asset in homeland defense and a useful military tool.
Therefore, there is a need to develop a method for the detection of biowarfare agents. Such technology would also be useful in hospitals, surgical suites, industrial clean rooms. There is further a need for such an apparatus and method to afford sensitive detection in a single process, and which utilizes devices that are generally available. Such technologies could promote rapid and much more cost effective detection and decontamination of biological warfare agents in public facilities contaminated by the actions of bioterrorists. Such technologies would also be useful in early detection of diseases in human and animals. And, a rapid, sensitive, and easy to use technology would also be beneficial for point of Care Testing (POCT) in hospitals and doctors offices.
There is further a need that the target antigen may be detected at very low amounts, such as at concentrations as low as about 10 cfu/ml and 4 μg/l.
Quantum dots are particles of matter so small that the addition or removal of an electron changes their properties. Quantum dots (QDs) have high fluorescence efficiency, lack photobleaching, and have long fluorescence (decay) lifetimes [H. Harma, T. Soukka, T. Lovgren, “Europium nanoparticles and time-resolved fluorescence for ultrasensitive detection of prostate-specific antigen,” Clin. Chem. 47 (2001) 561-568; T. Soukka, J. Paukkunen, H. Harma, S. Lonnberg, H. Lindroos, T. Lovgren, “Supersensitive time-resolved immunofluorometric assay of free prostate-specific antigen with nanoparticle label technology,” Clin. Chem. 47 (2001) 1269-1278]. These properties allow QDs to be ultrasensitive and therefore compete with conventional fluorescent dyes for many applications.
A composition and method has been discovered [co-pending and co-assigned U.S. patent application Ser. No. 11/222,093, filed Sep. 8, 2005] for detection and decontamination of antigens by nanoparticle-Raman spectroscopy, which comprises a fluorescent nanoparticle conjugated to a substance capable of binding specifically to an antigen and exposing the location containing the fluorescent nanoparticle and antigen to a wavelength of light capable of exciting the fluorescent nanoparticle. For instance, in an embodiment disclosed therein, a method was described of detecting an antigen comprising: (a) obtaining a fluorescent nanoparticle conjugated to a substance capable of binding specifically to an antigen to form a conjugated fluorescent nanoparticle; (b) placing the conjugated fluorescent nanoparticle in a location where the antigen is suspected to be; (c) exposing the location to a wavelength of light capable of exciting the conjugated fluorescent nanoparticle; (d) measuring fluorescence emission of the conjugated fluorescent nanoparticle; and (e) observing the wavelength of the measured fluorescence emission of step (d) in comparison with the wavelength of the fluorescence emission of the conjugated fluorescent nanoparticles that have not been exposed to the antigen wherein the conjugated fluorescent nanoparticle exhibits a lower emission wavelength upon binding to the antigen.
Furthermore, a method and apparatus was discovered [co-pending and co-assigned U.S. patent application Ser. No. 11/292,604, filed Dec. 2, 2005] for low quantity detection of bioparticles in small sample volumes (i.e., nanoliter/picoliter quantities of a sample). The apparatus involved a very small and low cost apparatus that contains a fluorometer. The detection process used the fluorescence of nanoparticles. Dielectrophoresis can used to concentrate, mix and position the target particles with regard to the light sensor such that maximum detection efficiency could be achieved.
The invention relates to antibody-nanoparticle (NP) or other receptor-NP conjugates for detection of antigens, such as for detection of antigens used as biological warfare agents (like anthrax or ricin) and early detection of diseases in human and animals. An apparatus and method has been discovered utilizing receptor (antibody or aptamer)-conjugated nanoparticles (NPs) or quantum dots (QD) capable of fluorescence scanner-based detection of antigens. Such apparatus and can be in, for example, a swab-base swipe test (i.e., swabbing of antibody-NP conjugates onto building interior surfaces). In embodiments of the invention, agent fluorescent NP-based immunoassay test kits can be used with any general fluorometer used for detection purposes. The invention may be used to detect antigens having concentrations less than 10,000 cfu/ml and may be used to detect the presence of very low concentrations of antigens (even small proteins), such as at concentrations of antigens as low as about 10 cfu/ml and 4 μg/ml.
In an embodiment of the invention, the present invention is a method of detecting an antigen comprising: (a) obtaining a sample from a surface or other source where an antigen is suspected to be (such as by using a swab-test); (b) obtaining a fluorescent nanoparticle conjugated to a substance capable of binding specifically to the antigen; (c) interacting the sample with the fluorescent nanoparticle such that antigen, if present, is bound to the conjugated fluorescent nanoparticles; (d) exposing the conjugated fluorescent nanoparticle of the resulting material to a wavelength of light capable of exciting the conjugated fluorescent nanoparticle; (e) measuring fluorescence emission of the conjugated fluorescent nanoparticle; and (f) observing the wavelength of the measured fluorescence emission of step (e) in comparison with the wavelength of the fluorescence emission of the conjugated fluorescent nanoparticles that have not been exposed to the antigen wherein the conjugated fluorescent nanoparticle exhibits a lower emission wavelength upon binding to the antigen.
Such interacting step may further comprise incubating the sample with the fluorescent nanoparticle.
Embodiments of the invention may further comprise a filtration method (such as a syringe-disc) that may be utilized in embodiments of the present invention. In this embodiment, after interacting the sample with the fluorescent nanoparticle, the resulting material may be passed through a filtering material (such as a disc), that has a pore size selected dependant upon the size of the antigen, to filter out the unbounded conjugated fluorescent nanoparticles from this interacted material. The nanoparticles that do not pass through the disc may them be exposed, measured, and observed as described above. This testing can be done so on, for example, the filter disc itself, by reverse flushing of the disc (to get the bounded conjugated fluorescent nanoparticles out of the filter),or by washing the conjugated fluorescent nanoparticles out in a buffer (such as a Phosphate Buffered Saline (PBS) buffer).
By such embodiments, the emission peaks can be read off and measured quantitatively.
Another embodiment of the present invention is a method for detecting two or more types of antigen comprising a first and second fluorescent nanoparticle conjugated to substances capable of binding specifically to the two or more types of antigen to form a first and second conjugated fluorescent nanoparticles wherein the first and second conjugated nanoparticles emit at different wavelengths and exhibit a lower emission peak wavelength upon binding to the two or more types of antigen.
The foregoing has outlined rather broadly the features and technical advantages of a number of embodiments of the present invention in order that the detailed description of the present invention that follows may be better understood. Additional features and advantages of the invention will be described hereinafter.
The foregoing summary as well as the following detailed description of the preferred s embodiment of the invention will be better understood when read in conjunction with the appended drawings. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown herein. The components in the drawings are not necessarily to scale, emphasis instead being placed upon clearly illustrating the principles of the present invention. Moreover, in the drawings, like reference numerals designate corresponding parts throughout the several views.
The invention may take physical form in certain parts and arrangement of parts. For a more complete understanding of the present invention, and the advantages thereof, reference is now made to the following descriptions taken in conjunction with the accompanying drawings, in which:
An “antibody” is an immunoglobulin molecule that only interacts with the antigen that induced its synthesis in cells of the lymphoid series, or with an antigen closely related to it.
An “antigen” is a substance capable of inducing synthesis of an antibody and being bound by such antibody. This substance is selected from the group including but not limited to bacteria, virus, viral particles and protein.
“Aptamers” are specific RNA or DNA oligonucleotides or proteins which can adopt various three dimensional configurations. Because of this aptamers can be produced to bind tightly to a specific molecular target.
“Bacteria” are one cell organisms.
“CFU” are colony forming units.
“Fluorescence” is the emission of light of one wavelength upon absorption of light of another wavelength.
“Quantum dots” or “QDs” are particles of matter so small that the addition or removal of an electron changes their properties.
“Raman Emission Peak” is the peak at about 460 nm wavelength for water.
“Wavelength” is the distance between two waves of energy.
As disclosed in co-pending and co-assigned U.S. patent application Ser. No. 11/222,093, filed Sep. 8, 2005, NPs (sometimes termed as semiconductor NPs), can be used to sensitively detect antigens, including, but not limited to, bacteria, virus, and proteins. Such NPs, which can be composed of CdSe/ZnS quantum dots (QDs) exhibit change in the Raman Emission Peak when conjugated to antibodies or DNA aptamers that are bound to bacteria or other antigens. Such a Nano-Ab-Tag can be formed, as shown in
Referring to
In step 202, a sample is obtained from that surface or source, such as by swiping the surface or source with a material that will obtain, but not effect or alter, the suspected antigen. One manner in which this can be done is by a swab-test (also referred to as a swab-base swipe test), such as the following. An area that is selected to be tested is swabbed (such as with a wet swab). The swab can then dipped into a release buffer for a period of time, generally at least two minutes, and more typically at least five minutes, to yield a sample (which will include the suspected antigen, if present on the surface or source). The swab can then be disposed of and the sample can be used for testing. In another embodiment, antigens on the surface can be obtained by washing off the surface with a liquid spray and collecting the liquid.
In step 203, a fluorescent nanoparticle is obtained that has conjugated to it a substance capable of binding specifically to the suspected antigen to form a conjugated fluorescent nanoparticle. Such fluorescent nanoparticle may be QDs, such as those made by Quantum Dot Corp. (now Invitrogen Corp., Carlsbad, Calif.), like Qdot 655 nm. As illustrated in
In step 204, the sample obtained in step 202 is interacted with the fluorescent nanoparticle to form a resulting material. If the suspected antigen is present within the sample, the antigen will bind with the conjugated fluorescent nanoparticles as anticipated and the resulting material will comprise bounded conjugated fluorescent nanoparticles. If the suspected antigen is not present within the sample, the conjugated fluorescent nanoparticles will not have any antigen bound to it. In one embodiment of the invention, step 204 includes incubating the sample with the fluorescent nanoparticle. For instance, 1 ml of the required sample can be incubated with about 10 μl (5 μg) of the conjugated fluorescent nanoparticles for at least around 10 minutes, and more particularly at least around 15 minutes.
In step 205, the resulting material is exposed to a wavelength of light capable of exciting the conjugated fluorescent nanoparticle. In step 206, the fluorescence emission of the resulting materials is measured, including, in particular, the emission of the conjugated fluorescent nanoparticle, if any, present in the interacted material. Such exposure and measurement can be performed on any general purpose fluorometer that can read emission from 300 to 700 nm, such as, for example, the Cary Eclipse Fluorometer from Varian, Inc., (Walnut Creek, Calif.) which scans fluorescence emissions from 200-850 nm with picomolar sensitivity or the Picofluor from Turner Biosystems, Inc. (Sunnyvale, Calif.), which is an off-the-shelf handheld or portable fluorometer. Steps 205-206 can be completed in a variety of time frames, including as little as about 15, 10, 5 or 2 minutes
In step 207, the wavelength observed of the measured fluorescence emission of step 206 is compared with the wavelength of the fluorescence emission of the conjugated fluorescent nanoparticles that have not been exposed to the antigen. The conjugated fluorescent nanoparticle exhibits a lower emission wavelength upon binding to the antigen.
Such method may be used to detect the presence of the antigen at concentrations equal to or above about 10 cfu/ml or about 4 μg/ml (i.e., the present invention can detect antigen at a concentration of at least about 10 cfu/ml or about 4 μg/ml).
In step 302, the filter can be prepared by washing it with deionized (DI) water or with a prepared buffer. For instance, as shown in 401 of
In step 303, the resulting material, usually incubated, is then passed through the disc holder and filtrate is collected. For instance, as shown in 402 of
As shown in 404 of
By these filtration process, the unbounded conjugated fluorescent nanoparticles are filtered out, i.e., the filtrate 410 contains the unbounded conjugates, while the bounded conjugated fluorescent nanoparticles do not pass through the filter 412.
In step 304, the bounded conjugated fluorescent nanoparticles are prepared for exposure, measurement, and observation, as reflected in
In another embodiment, a centrifuge method is used rather than a syringe-filtration method, such as the syringe-filtration method described above. After incubating the sample with the fluorescent nanoparticle, the fluid containing the antigen is then spun in a centrifuge, such as at 14,000 g. The supernate can then be taken out; generally, this is done from the middle. A portion is added to PBS in a cuvette (such as 2 ml of the supernate and an equal part of PBS). A standard fluorometer can then used to measure the sample, such as similar to as previously described.
The following examples are provided to more fully illustrate some of the embodiments of the present invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute exemplary modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
These test were performed to simulate testing on building surfaces. In these test, a concrete slab was utilized for testing. The concrete slab was sprayed with 103, 104, 105 and 106 CFU/ml of Male Specific Coliphage (“MS2”) (approved by the U.S. Department of Defense as a viral stimulant). The slab was let to dry. After the concrete slab had dried, a swab was used to rub on the dry area. The swab was then put in 1 ml of PBS buffer and left in the buffer for 15 minutes.
The swab was then taken out and the sample (i.e, the resulting PBS buffer solution) was tested.
QDs, specifically, Qdot 655 nm made by Quantum Dot Corp. (now Invitrogen), were utilized to make the conjugated fluorescent nanoparticle designed to bind to MS2. The standard process for conjugating Quantum Dot's Qdot 655 nm was used and the antibody was obtained from Tetracore, Inc., Rockville, Md. Different amounts of the conjugate material were interacted with the sample and incubated from 15 minutes and the resulting material was exposed, measured, and observed. These trials were repeated to verify the results. From this Example 1, it appeared that 7 μg/ml yielded superior results for MS2, as compared to other concentrations.
Using 7 μg/ml concentration for the amount conjugate for MS2, the slab surface was sprayed with various concentrations of MS2 (103, 104, 105, 106, 107, and 108).
The above Example 1 was repeated for Ovalbumin (“OV”) (approved by the U.S. Department of Defense as a Ricin stimulant), except that the concrete slab in Example 1 was sprayed 31.25 ug/ml, 62.5 ug/ml, and 125 ug/ml of OV and the conjugated fluorescent nanoparticle was designed to bind to OV. The antibody was obtained from Sigma, St. Louis, Mo. From Example 3, it appeared that 5 μg/ml yielded superior results for OV, as compared to other concentrations.
The above Example 2 was repeated from OV, except that the concrete slab in Example 1 was sprayed 31.25 ug/ml, 62.5 ug/ml, 125 ug/ml, 250 ug/ml and 500 ug/ml, the conjugated fluorescent nanoparticle was designed to bind to OV, and the conjugate concentration for OV was 5 μg/ml.
The above Example 1 was repeated for Erwinia herbicola (EH) (approved by the U.S. Department of Defense as a Yersinia pestis stimulant), except that the concrete slab in Example 1 and the conjugated fluorescent nanoparticle was designed to bind to EH. The antibody was obtained from Morphosys USA, Brentwood, N.H. Furthermore, the incubation time required about 20 minutes. From Example 5, it appeared that 6 μg/ml yielded superior results for EH, as compared to other concentrations.
The above Example 2 was repeated from EH, except that the concrete slab in Example 1 was sprayed at 103, 104, 105, 106, and 107 concentrations, the conjugated fluorescent nanoparticle was designed to bind to EH, and the conjugate concentration for EH was 6 μg/ml.
The above Example 1 was repeated for Bacillus Globigii (BG) (approved by the U.S. Department of Defense as an anthrax stimulant), except that the concrete slab in Example 1 the conjugated fluorescent nanoparticle was designed to bind to BG. The antibody was obtained from Tetracore, Inc., Rockville, Md. From Example 7, it appeared that 7 μg/ml yielded superior results for BG, as compared to other concentrations.
The above Example 2 was repeated from BG, except that the concrete slab in Example 1 was sprayed at 103, 104, 105, 106, 107, and 108 concentrations, the conjugated fluorescent nanoparticle was designed to bind to BG, and the conjugate concentration for BG was 7 μg/ml.
Examples 1-8 reflect that each of MS2, OV, EH, and BG were detected utilizing the present invention.
The present invention can also be used to detect a mixture or “cocktail” of various bioterrorism assays, and can be used to discriminate some or all of the components spectrally. In this Example, four different bacterial immuno-QD assays were mixed together. Specifically, these four bacterial immuno-QD assays were (a) dead Listeria monocytogenes, (b) live E. coli O111:B4, (c) dead Campylobacter, and (d) dead E. coli O157:H7. Four different conjugated fluorescent nanoparticle were designed, with each designed to bind to one of these four bacterial immuno-QD assays. Respectively, these were (a) Ab-Lake Placid Blue QDs (498 nm), (b) Ab-Adirondack Green QDs (522 nm), (c) Ab-Birch Yellow QDs (588 nm), and (d) Ab-Fort Orange Qds (605 nm). In this example, Evitag QDs (of Evident Technology, Troy, N.Y.) were utilized.
Food samples (dry soup mix) were prepared with concentrations of 101, 102, 103, and 104 cfu/ml of E. coli O157:H7 bacteria The food sample containing the concentration of 101cfu/ml of E. coli O157:H7 bacteria was prepared as follows. 25 ml of buffer was added to 3 g of the food sample to form a mixture. 101 cfu/ml of E. coli O157:H7 bacteria was spiked into this mixture and then mixed thoroughly. 1 ml was taken of this composition and put into a first tube. This was then centrifuged at 14,000 rpm for 5 minutes. The supernatant was collected and placed in a second tube. The first tube with pellet was discarded. The other food samples with concentrations of 102, 103, and 104 cfu/ml of E. coli O157:H7 bacteria were prepared by a similar process.
These various food samples were tested using an embodiment of the present invention. The collected supernatant in each of the various tubes were incubated in a solution comprising conjugated fluorescent nanoparticles designed to bind to E. coli O157:H7 bacteria. The incubated supernatant were then subjected to the syringe-filtration method. The fluorescence output for the various food samples was then measured.
Example 10 was repeated, except that the food samples were prepared with concentrations of Salmonella typhimurium bacteria and the conjugated fluorescent nanoparticles were designed to bind to Salmonella typhimurium bacteria.
Example 10 was repeated, except that a serum sample was utilized in lieu of food samples, the serum samples were spiked with concentrations of 4 μg, 8 μg, 16 μg, 32 μg, and 64 g of OV, and the conjugated fluorescent nanoparticles were designed to bind to OV. The serum sample containing the 4 μg concentration of OV was prepared as follows. 2 ml of blood was drawn and centrifuged to separate the serum out. Thereafter 500 μl of the serum was spiked with 4 ug/ml of OV. PBS was then added to yield a 1 ml serum sample. The other to serum samples with concentrations of 8 μg, 16 μg, 32 μg, and 64 μg of OV were prepared by a similar process.
Similar to Examples 11 and 12, the serum samples were incubated with the conjugated fluorescent nanoparticles were designed to bind to OV, subjected to the syringe-filtration method, and the fluorescence output for the various serum samples was then measured.
Although the invention has been described with reference to specific embodiments, these descriptions are not meant to be construed in a limiting sense. Various modifications of the disclosed embodiments, as well as alternative embodiments of the invention will become apparent to persons skilled in the art upon reference to the description of the invention. It will be understood that certain of the above-described structures, functions, and operations of the above-described embodiments are not necessary to practice the present invention and are included in the description simply for completeness of an exemplary embodiment or embodiments. In addition, it will be understood that specific structures, functions, and operations set forth in the above and below described referenced patents and publications can be practiced in conjunction with the present invention, but they are not essential to its practice. It is therefore to be understood that the invention may be practiced otherwise than as specifically described without actually departing from the spirit and scope of the present invention as defined by the appended claims.
It is therefore, contemplated that the claims will cover any such modifications or embodiments that fall within the true scope of the invention.
This application claims priority to and benefit of U.S. Provisional Application Ser. No. 60/741,349, filed on Nov. 30, 2005. The following co-pending and co-assigned applications contain related information and are incorporated herein by reference: (1) U.S. patent application Ser. No. 11/292,604, filed Dec. 2, 2005, entitled “Method and Apparatus for Low Quantity Detection of Bioparticles in Small Sample Volumes” having Srinagesh Satyanarayana and Sulatha Dwarakanath as inventors; and (2) U.S. patent application Ser. No. 11/222,093, filed Sep. 8, 2005, entitled “Method for Detection and Decontamination of Antigens by Nanoparticle-Raman Spectroscopy” having Sulatha Dwaraknath and John G. Bruno as inventors.
The present invention was made in connection with research pursuant to Department of Defense Contract No. W9132T-040C-0030.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US06/61382 | 11/30/2006 | WO | 00 | 10/3/2007 |
| Number | Date | Country | |
|---|---|---|---|
| 60741349 | Nov 2005 | US |
