Patent Application
20050019901
Portions of the disclosure of this patent document contain material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the Patent and Trademark Office file or records, but otherwise reserves all copyright rights whatsoever.
1. Field of the Invention
This invention relates in general to chemical and biological assays and, in particular, to synthesis of nanoparticles and nanocapsules for use in optical bio-disc assays. More specifically, but without restriction to the particular embodiments hereinafter described in accordance with the best mode of practice, this invention relates to the synthesis of bio-active nanoparticles and nanocapsules for use in disc assays and optical analysis discs adapted for use therewith.
2. Discussion of the Background Art
It is known in the prior art that microparticles can be synthesized by polymerization from monomers to give linear or cross-linked polymer particles. Polymerization is initiated by an initiator and occurs fast as a chain reaction. As a result, particles of very big sizes and even continuous gel-like structures can form if the reaction is not stopped after some time. Even when the polymerization reaction is stopped, the resulting particles significantly differ in size and shape. It is an object of the present invention to provide methods for synthesis of polymer nanoparticles or nanocapsules of uniform size and shape and to provide suitable means for disc assays and disc analysis for analyte detection.
The present invention is directed to the synthesis of nanoparticles or nanocapsules for use in disc assays and optical analysis discs adapted for use therewith.
More specifically, the present invention is directed to a method for preparing synthetic nanoparticles comprising the steps of forming reverse micelles (RM) having an outer non-polar shell and an inner polar cavity by contacting one or more surfactants or emulsifiers with a non-polar organic solvent. Once the micelles are formed a polymerizing mixture comprising one or more monomers or co-monomers and an initiator of polymerization is solubilized into the micelle. The mixture is then polymerized. In another embodiment of the present invention, the surfactants or emulsifiers are in part or totally polymeric surfactants that are cross-linked during polymerization.
The present invention is also directed to a method for preparing labeled synthetic nanoparticles including the steps of forming reverse micelles having an outer non-polar shell and an inner polar cavity by contacting one or more surfactants or emulsifiers with non-polar organic solvent. Once the micelles are formed a polymerizing mixture including one or more monomers or co-monomers, an initiator of polymerization and a label including, for example absorbing, luminescent, or fluorescent dyes or particles is incorporated into the micelle. The mixture is then polymerized.
The present invention is further directed to a method for preparing synthetic semiconductor nanoparticles, including the steps of forming reverse micelles having an outer non-polar shell and an inner polar cavity by contacting one or more surfactants or emulsifiers with non-polar organic solvent. The next steps include solubilizing into the reverse micelles a polymerizing mixture having one or more monomers or co-monomers, an initiator of polymerization and metal semi-conductor material. The mixture is then polymerized. Optionally, the surfactants or emulsifiers may be, in part or totally, polymeric surfactants that are cross-linked during polymerization.
The present invention is still further directed to method for preparing synthetic magnetic nanoparticles, including the concomitant or sequential steps of forming reverse micelles having an outer non-polar shell and an inner polar cavity by contacting one or more surfactants or emulsifiers with non-polar organic solvent. In another embodiment of the present invention, the surfactants or emulsifiers may be in part or totally polymeric surfactants that are cross-linked during polymerization. The next step in this method of the present invention, includes solubilizing a polymerizing mixture in the reverse micelles. The polymerizing mixture may include one or more monomers or co-monomers and an initiator of polymerization. The solubilizing step is then followed by adding magnetic particles into the micelles. These magnetic particles are incorporated into the nanoparticles during polymerization. The mixture, including the nanoparticles that have been incorporated in to the micelles, is then polymerized. The method for preparing synthetic magnetic nanoparticles may further include the step of coating the synthetic magnetic nanoparticles with a bio-compatible polymer.
In all the methods according to the present invention, the polymerizing mixture may be comprised of acrylic or methacrylic compounds forming a linear or cross-linked polymer and optionally additional cross-linking agents. The surfactants or emulsifiers are selected from anionic, cationic, and non-ionic surfactants. The non-polar organic solvent is a hydrocarbon.
Further embodiments of the present invention include synthetic nanoparticles adapted for use in disc assays having size of about 1 to 1000 nanometers obtained according to the above-recited methods. The synthetic nanoparticles may be synthesized from acrylic or methacrylic linear or cross-linked polymers, optionally further including cross-linking agents.
In yet another embodiment of the present invention, the synthetic nanoparticles comprise a label absorbing, luminescing or fluorescing molecules having absorbance and/or emission properties at suitable wavelength detectable by an optical disc reader.
In a still different embodiment of the present invention, the nanoparticles are synthetic semiconductor nanoparticles or synthetic magnetic nanoparticles adapted for use in disc assays having size of about 1 to 1000 nanometers and obtained according to the above-recited methods. These magnetic nanoparticles, are optionally coated with bio-compatible polymer.
Still further objects of the present invention are methods for immobilizing a bio-active substance into synthetic nanoparticles including the steps of (1) forming reverse micelles having an outer non-polar shell and an inner polar cavity by mixing surfactants or emulsifiers with a non-polar organic solvent, (2) solubilizing a polymerizing mixture including one or more monomers or co-monomers, an initiator of polymerization and the biologically active substance into the reverse micelles, and (3) polymerizing the mixture. The bio-active substance may also be immobilized into labeled synthetic nanoparticles according to another method including the steps of forming reverse micelles having an outer non-polar shell and an inner polar cavity by contacting one or more surfactants or emulsifiers with a non-polar organic solvent; solubilizing a polymerizing mixture including one or more monomers or co-monomers, an initiator of polymerization, the biologically active substance and one or more labels in the reverse micelles; and polymerizing the mixture. In some embodiments, the labels may be dyes that preferably absorb or fluoresce at a wavelength detectable using an optical disc drive.
The present invention is also directed to methods for immobilizing a bio-active substance into synthetic semiconductor nanoparticles including the steps of forming reverse micelles having an outer non-polar shell and an inner polar cavity by contacting one or more surfactants or emulsifiers with non-polar organic solvent; and solubilizing a polymerizing mixture including one or more monomers or co-monomers, an initiator of polymerization, and a biologically active substance into the reverse micelles. The next step in this method is the addition of a metal semi-conductor material into the reverse micelles and polymerizing the resulting mixture.
Further embodiments of the present invention are methods for immobilizing a bio-active substance into synthetic magnetic nanoparticles. These include the steps of forming reverse micelles having an outer non-polar shell and an inner polar cavity by contacting one or more surfactants or emulsifiers with a non-polar organic solvent; incorporating a polymerizing mixture comprising one or more monomers or co-monomers, an initiator of polymerization, and a biologically active substance into the reverse micelles; adding to the reverse micelles magnetic nanoparticles; and polymerizing the resulting mixture.
An alternative method for immobilizing a bio-active substance onto synthetic magnetic nanoparticles includes the steps of coating the synthetic magnetic nanoparticles, free of bio-active substance, with a bio-compatible polymer and conjugating the biologically active substance to the bio-compatible polymer.
In all the above mentioned methods the biologically active substance may include proteins, antigens, antibodies, enzymes, drugs, and functionally active subunits, parts and mixtures thereof. The nanoparticles, nanospheres, or complexes obtained with the above-recited methods, including nanoparticles with a bio-active substances, labeled nanoparticles, semiconductor nanoparticles, magnetic nanoparticles, or magnetic coated nanoparticles, are additional objects of the invention.
Another embodiment of the present invention is a method for making a nanocapsule for use in optical bio-disc assays. This method includes the steps of forming reverse micelles having an outer non-polar shell and an inner polar cavity by mixing micelle-forming surfactants with a non-polar organic solvent; adding weakly polar monomers that solubilized near the shell of the reverse micelles; solubilizing in the reverse micelles an initiator of polymerization; and polymerizing the weakly polar monomers to thereby form the nanocapsule.
The present invention is further directed to optical bio-disc assays for analyte detection including the step of using the above-described nanoparticles, nanospheres, nanocapsules, or complexes in chemical, biological, biochemical, immunochemical, and biomedical assays in optical analysis discs.
The present invention also includes a method of using the above-mentioned nanocapsules or nanoparticles to test for the presence of a target nucleic acid in a test sample. This method include the steps of providing a bio-disc that includes a substantially circular substrate having a center and an outer edge, a target zone disposed between the center and the outer edge, at least one strand of capture DNA attached to the substrate in target zone, the capture DNA and the target nucleic acid having at least some complementary sequence. The method continues with the steps of depositing the test sample on the target zone; allowing any target nucleic acid present in the test sample to hybridize with the capture-DNA; attaching a signal DNA onto the nanocapsule or nanoparticle; depositing the nanocapsule or nanoparticle on the target zone; and hybridizing the signal DNA to the target nucleic acid such that the nanocapsule or nanoparticle is immobilized within the target zone. This method further includes washing the target zone to remove any unbound nanocapsule; depositing onto the target zone at least one enzyme substrate that reacts with the enzyme inside the bio-active nanoparticle to produce at least one detectable signal; and detecting any signal in the target zone to thereby determine whether target-nucleic acid is present in the test sample.
The present is further directed to an optical assay disc implemented to perform any of the above methods, use of such discs in performing any of these methods, and the manufacturing or assemblying of these specific optical disc assemblies as made to perform any of the above methods or assays.
This invention or different aspects thereof may be readily implemented in, adapted to, or employed in combination with the discs, assays, and systems disclosed in the following commonly assigned and co-pending patent applications: U.S. patent application Ser. No. 09/378,878 entitled “Methods and Apparatus for Analyzing Operational and Non-operational Data Acquired from Optical Discs” filed Aug. 23, 1999; U.S. Provisional Patent Application Ser. No. 60/150,288 entitled “Methods and Apparatus for Optical Disc Data Acquisition Using Physical Synchronization Markers” filed Aug. 23, 1999; U.S. patent application Ser. No. 09/421,870 entitled “Trackable Optical Discs with Concurrently Readable Analyte Material” filed Oct. 26, 1999; U.S. patent application Ser. No. 09/643,106 entitled “Methods and Apparatus for Optical Disc Data Acquisition Using Physical Synchronization Markers” filed Aug. 21, 2000; U.S. patent application Ser. No. 09/999,274 entitled “Optical Biodiscs with Reflective Layers” filed Nov. 15, 2001; U.S. patent application Ser. No. 09/988,728 entitled “Methods and Apparatus for Detecting and Quantifying Lymphocytes with Optical Biodiscs” filed Nov. 20, 2001; U.S. patent application Ser. No. 09/988,850 entitled “Methods and Apparatus for Blood Typing with Optical Bio-discs” filed November, 19, 2001; U.S. patent application Ser. No. 09/989,684 entitled “Apparatus and Methods for Separating Agglutinants and Disperse Particles” filed Nov. 20, 2001; U.S. patent application Ser. No. 09/997,741 entitled “Dual Bead Assays Including Optical Biodiscs and Methods Relating Thereto” filed Nov. 27, 2001; U.S. patent application Ser. No. 09/997,895 entitled “Apparatus and Methods for Separating Components of Particulate Suspension” filed Nov. 30, 2001; U.S. patent application Ser. No. 10/005,313 entitled “Optical Discs for Measuring Analytes” filed Dec. 7, 2001; U.S. patent application Ser. No. 10/006,371 entitled “Methods for Detecting Analytes Using Optical Discs and Optical Disc Readers” filed Dec. 10, 2001; U.S. patent application Ser. No. 10/006,620 entitled “Multiple Data Layer Optical Discs for Detecting Analytes” filed Dec. 10, 2001; U.S. patent application Ser. No. 10/006,619 entitled “Optical Disc Assemblies for Performing Assays” filed Dec. 10, 2001; U.S. patent application Ser. No. 10/020,140 entitled “Detection System For Disk-Based Laboratory and Improved Optical Bio-Disc Including Same” filed Dec. 14, 2001; U.S. patent application Ser. No. 10/035,836 entitled “Surface Assembly for Immobilizing DNA Capture Probes and Bead-Based Assay Including Optical Bio-Discs and Methods Relating Thereto” filed Dec. 21, 2001; U.S. patent application Ser. No. 10/038,297 entitled “Dual Bead Assays Including Covalent Linkages for Improved Specificity and Related Optical Analysis Discs” filed Jan. 4, 2002; U.S. patent application Ser. No. 10/043,688 entitled “Optical Disc Analysis System Including Related Methods for Biological and Medical Imaging” filed Jan. 10, 2002; U.S. Provisional Application Ser. No. 60/348,767 entitled “Optical Disc Analysis System Including Related Signal Processing Methods and Software” filed Jan. 14, 2002 U.S. patent application Ser. No. 10/086,941 entitled “Methods for DNA Conjugation Onto Solid Phase Including Related Optical Biodiscs and Disc Drive Systems” filed Feb. 26, 2002; U.S. patent application Ser. No. 10/087,549 entitled “Methods for Decreasing Non-Specific Binding of Beads in Dual Bead Assays Including Related Optical Biodiscs and Disc Drive Systems” filed Feb. 28, 2002; U.S. patent application Ser. No. 10/099,256 entitled “Dual Bead Assays Using Cleavable Spacers and/or Ligation to Improve Specificity and Sensitivity Including Related Methods and Apparatus” filed Mar. 14, 2002; U.S. patent application Ser. No. 10/099,266 entitled “Use of Restriction Enzymes and Other Chemical Methods to Decrease Non-Specific Binding in Dual Bead Assays and Related Bio-Discs, Methods, and System Apparatus for Detecting Medical Targets” also filed Mar. 14, 2002; U.S. patent application Ser. No. 10/121,281 entitled “Multi-Parameter Assays Including Analysis Discs and Methods Relating Thereto” filed Apr. 11, 2002; U.S. patent application Ser. No. 10/150,575 entitled “Variable Sampling Control for Rendering Pixelization of Analysis Results in a Bio-Disc Assembly and Apparatus Relating Thereto” filed May 16, 2002; U.S. patent application Ser. No. 10/150,702 entitled “Surface Assembly For Immobilizing DNA Capture Probes in Genetic Assays Using Enzymatic Reactions to Generate Signals in Optical Bio-Discs and Methods Relating Thereto” filed May 16, 2002; U.S. patent application Ser. No. 10/194,418 entitled “Optical Disc System and Related Detecting and Decoding Methods for Analysis of Microscopic Structures” filed Jul. 12, 2002; U.S. patent application Ser. No. 10/194,396 entitled “Multi-Purpose Optical Analysis Disc for Conducting Assays and Various Reporting Agents for Use Therewith” also filed Jul. 12, 2002; U.S. patent application Ser. No. 10/199,973 entitled “Transmissive Optical Disc Assemblies for Performing Physical Measurements and Methods Relating Thereto” filed Jul. 19, 2002; U.S. patent application Ser. No. 10/201,591 entitled “Optical Analysis Disc and Related Drive Assembly for Performing Interactive Centrifugation” filed Jul. 22, 2002; U.S. patent application Ser. No. 10/205,011 entitled “Method and Apparatus for Bonded Fluidic Circuit for Optical Bio-Disc” filed Jul. 24, 2002; U.S. patent application Ser. No. 10/205,005 entitled “Magnetic Assisted Detection of Magnetic Beads Using Optical Disc Drives” also filed Jul. 24, 2002; U.S. patent application Ser. No. 10/230,959 entitled “Methods for Qualitative and Quantitative Analysis of Cells and Related Optical Bio-Disc Systems” filed Aug. 29, 2002; U.S. patent application Ser. No. 10/233,322 entitled “Capture Layer Assemblies for Cellular Assays Including Related Optical Analysis Discs and Methods” filed Aug. 30, 2002; U.S. patent application Ser. No. 10/236,857 entitled “Nuclear Morphology Based Identification and Quantification of White Blood Cell Types Using Optical Bio-Disc Systems” filed Sep. 6, 2002; U.S. patent application Ser. No. 10/241,512 entitled “Methods for Differential Cell Counts Including Related Apparatus and Software for Performing Same” filed Sep. 11, 2002; U.S. patent application Ser. No. 10/279,677 entitled “Segmented Area Detector for Biodrive and Methods Relating Thereto” filed Oct. 24, 2002; U.S. patent application Ser. No. 10/293,214 entitled “Optical Bio-Discs and Fluidic Circuits for Analysis of Cells and Methods Relating Thereto” filed on Nov. 13, 2002; U.S. patent application Ser. No. 10/298,263 entitled “Methods and Apparatus for Blood Typing with Optical Bio-Discs” filed on Nov. 15, 2002; U.S. patent application Ser. No. 10/307,263 entitled “Magneto-Optical Bio-Discs and Systems Including Related Methods” filed Nov. 27, 2002; U.S. patent application Ser. No. 10/341,326 entitled “Method and Apparatus for Visualizing Data” filed Jan. 13, 2003; U.S. patent application Ser. No. 10/345,122 entitled “Methods and Apparatus for Extracting Data From an Optical Analysis Disc” filed on Jan. 14, 2003; U.S. patent application Ser. No. 10/347,155 entitled “Optical Discs Including Equi-Radial and/or Spiral Analysis Zones and Related Disc Drive Systems and Methods” filed on Jan. 15, 2003; U.S. patent application Ser. No. 10/347,119 entitled “Bio-Safe Dispenser and Optical Analysis Disc Assembly” filed Jan. 17, 2003; U.S. patent application Ser. No. ______ entitled “Multi-Purpose Optical Analysis Disc for Conducting Assays and Related Methods for Attaching Capture Agents” filed on Jan. 21, 2003; U.S. patent application Ser. No. ______ entitled “Processes for Manufacturing Optical Analysis Discs with Molded Microfluidic Structures and Discs Made According Thereto” filed on Jan. 21, 2003; U.S. patent application Ser. No. ______ entitled “Methods for Triggering Through Disc Grooves and Related Optical Analysis Discs and System” filed on Jan. 23, 2003; U.S. patent application Ser. No. ______ entitled “Bio-Safety Features for Optical Analysis Discs and Disc System Including Same” filed on Jan. 23, 2003; U.S. patent application Ser. No. ______ entitled “Manufacturing Processes for Making Optical Analysis Discs Including Successive Patterning Operations and Optical Discs Thereby Manufactured: filed on Jan. 24, 2003; U.S. patent application Ser. No. ______ entitled “Processes for Manufacturing Optical Analysis Discs with Molded Microfluidic Structures and Discs Made According Thereto” filed on Jan. 27, 2003; and U.S. patent application Ser. No. ______ entitled “Method and Apparatus for Logical Triggering” filed on Jan. 28, 2003. All of these applications are herein incorporated by reference in their entireties. They thus provide background and related disclosure as support hereof as if fully repeated herein.
The above described methods and apparatus according to the present invention as disclosed herein can have one or more advantages which include, but are not limited to, simple and quick on-disc processing without the necessity of an experienced technician to run the test, small sample volumes, use of inexpensive materials, and use of known optical disc formats and drive manufacturing. These and other features and advantages will be better understood by reference to the following detailed description when taken in conjunction with the accompanying drawing figures, technical examples, and claims.
Further objects of the present invention together with additional features contributing thereto and advantages accruing therefore will be apparent from the following description of the preferred embodiments of the invention which are shown in the accompanying drawing figures with like reference numerals indicating like components throughout, wherein:
The present invention is directed in general to optical bio-disc assays, to synthesis of active micro-particles suitable for bio-disc assays, to disc drive systems, optical bio-discs, image processing techniques, counting methods, and related software. In particular, the invention relates to the synthesis of micro-particles for use in bio-disc assays and optical analysis discs adapted for use therewith. Each of the aspects of the present invention is discussed below in further detail.
Drive System and Related Discs
The second element shown in
The third element illustrated in
Referring now to
The second element shown in
The third element illustrated in
In addition to Table 1,
With reference next to
With continuing reference to
The final principal structural layer in this transmissive embodiment of the present bio-disc 110 is the clear, non-reflective cap portion 116 that includes inlet ports 122 and vent ports 124.
Referring now to
As shown in
Counting Methods and Related Software
By way of illustrative background, a number of methods and related algorithms for white blood cell counting using optical disc data are herein discussed in further detail. These methods and related algorithms are not limited to counting white blood cells, but may be readily applied to conducting counts of any type of particulate matter including, but not limited to, red blood cells, white blood cells, beads, microparticles and any other objects, both biological and non-biological, that produce similar optical signatures that can be detected by an optical reader.
For the purposes of illustration, the following description of the methods and algorithms related to the present invention as described with reference to
With continuing reference to
Referring next to
During the analog-to-digital transformation, each consecutive sample point 224 along the laser path is stored consecutively on disc or in memory as a one-dimensional array 226. Each consecutive track contributes an independent one-dimensional array, which yields a two-dimensional array 228 (
With particular reference now to
Referring next to
Referring now to
The computational and processing algorithms of the present invention are stored in analyzer 168 (
With reference now to
The next principle step 246 is selecting an area of the disc for counting. Once this area is defined, an objective then becomes making an actual count of all white blood cells contained in the defined area. The implementation of step 246 depends on the configuration of the disc and user's options. By way of example and not limitation, embodiments of the invention using discs with windows such as the target zones 140 shown in
In embodiments of the invention using a transmissive disc without windows, as shown in
As for the user options mentioned above in regard to step 246, the user may specify a desired sample area shape for cell counting, such as a rectangular area, by direct interaction with mouse selection or otherwise. In the present embodiment of the software, this involves using the mouse to click and drag a rectangle over the desired portion of the optical bio-disc-derived image that is displayed on a monitor 114. Regardless of the evaluation area selection method, a respective rectangular area is evaluated for counting in the next step 248.
The third principal step in
The next step in the flow chart of
As shown in
An optional step 254 directed to removing bad components may be performed as indicated in
The next principal processing step shown in
In some hardware configurations, some cells may appear without bright centers. In these instances, only a dark rim is visible and the following two optional steps 258 and 260 are useful.
Step 258 is directed to removing found cells from the picture. In step 258, the circular region around the center of each found cell is filled with the value 2000 so that the cells with both bright centers and dark rims would not be found twice.
Step 260 is directed to counting additional cells by dark rims. Two transforms are made with the image after step 258. In the first substep of this routine, sub step (a), the value v at each point is replaced with (2000-v) and if the result is negative it is replaced with zero. In sub step (b), the resulting picture is then convolved with a ring of inner radius R1 and outer radius R2. R1 and R2 are, respectively, the minimal and the maximal expected radius of a cell, the ring being shifted, subsequently, in sub step (d) to the left, right, up and down. In sub step (c), the results of four shifts are summed. After this transform, the image of a dark rim cell looks like a four petal flower. Finally in sub step (d), maxima of the function obtained in sub step (c) are found in a manner to that employed in counting step 256. They are declared to mark cells omitted in step 256.
After counting step 256, or after counting step 260 when optionally employed, the last principal step illustrated in
Additional computer science methodologies and apparatus directed to extracting and visualizing data from bio-discs and/or optical analysis discs are discussed in commonly assigned U.S. patent application Ser. No. ______ entitled “Method and Apparatus for Visualizing Data” filed Jan. 13, 2003 and U.S. patent application Ser. No. ______ entitled “Methods and Apparatus for Extracting Data From an Optical Analysis Disc” filed on Jan. 14, 2003 both of which have been herein incorporated by reference.
Synthetic Nanoparticles and Nanocapsules
A particle of a few micrometers in size is detectable with a CD and DVD disc drive. If a particle is used as a reporter in a bio-disc assay there are density and size requirements specific for an assay such that the particles in buffer of pre-determined density move with proper speed at a given centrifugal force through the channel of the disc in the drive. If the particles are too light, they move too slow and full separation of non-bound and bound particles does not occur. If the particles are too heavy, they move too fast and may have too much mass such that their movement can result in breaking of specifically bound microparticles or nanoparticles.
There are two major technical challenges when synthesizing particles as labels for a disc assay. The first is how to get particles with narrow size and shape distribution. The second is how to incorporate bio-active substances such as enzymes and antibodies within the particles to thereby form a bio-particle or bio-active particle. It is an object of the present invention to synthesize bio-particles of essentially uniform size and shape.
A bio-active nanoparticle or bioparticle may be synthesized by polymerization from monomers to form a linear or cross-linked polymer particle. Polymerization is catalized by an initiator including, for example, a radical formed as a result of a decay of the initiator molecule under UV light or temperature action. Polymerization may occur fast as a chain reaction. As a result, particles of very big sizes and even continuous gel-like structures can form if the reaction is not stopped after some time. Even when the polymerization reaction is discontinued after a specific time, resulting particles may be of different shapes and sizes.
Synthesis of nanoparticles of essentially uniform size and shape may be achieved if polymerization is performed in the presence of a surfactant, emulsifier, or micelle-forming surfactant that forms small oil bubbles or micelles. Many surfactants form ‘micelles’ or ‘normal micelles’ in an aqueous solution. As shown in
With reference next to
Surfactants that may be used to form reverse micelles include anionic, cationic, and non-ionic surfactants. The most common reverse micelle forming surfactant is an anionic surfactant bis-(2-ethylhexyl) sulfosuccinate sodium salt, (AOT). The structure of AOT is shown in
Surfactants may either form a microemulsion in non-polar organic solvents. Terminology of ‘micelles’ and ‘microemulsions’ is used according to the size of surfactant aggregates. Micelles normally range in size from approximately 1 to about 1000 nanometers, are stable, and result in a transparent or clear solution. Microemulsions, however, have aggregates that are about 1 micrometer or more in size and normally result in a cloudy solution and the aggregates are not so stable.
The diameter of the inner cavity 322 of reverse micelles 307 normally ranges from 1 nm to 1000 nm and can be varied by changing the degree of hydration (Wo) of the aqueous inner cavity 322. One of the important characteristics of the reverse micellar system is the degree of hydration (Wo) which is the ratio of water concentration and surfactant concentration, as expressed by the following formula:
Wo=[H2O]/[surfactant]
This parameter defines the size of the reverse micelles. As illustrated in
The size of the synthesized polymeric nanoparticles 310 can be varied by changing the concentrations of the monomers 308, co-monomers, and initiator in the reverse micellar solution. Alternatively, the size of the inner cavity 322 of the reverse micelle 307 may be varied by changing its degree of hydration thus incorporating more polymerizable components including monomers 308, co-monomers, and initiator thereby increasing the size of the nanoparticle 310 formed after polymerization.
The size of polymeric nanoparticles 310 may increase after polymerization relative to the size of micelles before polymerization. This can be explained by the fact that besides intramicellar polymerization, intermicellar polymerization takes place to a certain extent. Despite the increase in size during polymerization, nanoparticles comprising polyacrylamide are still realtively small. An example of such particles are those obtained by polymerization of the mixture of acrylamide (AA) and N,N′-methylene-bis-acrylamide (MBAA) induced by UV-irradiation in the system of AOT micelles in toluene. The size of resulting particles, in this example, can be varied according to the polymerization conditions, from a few nanometers up to about 0.6 micrometers.
The micellar solution may became either more opaque or a polymer precipitate may appear after polymerization. The size of polymeric nanoparticles 310 is strongly influenced by the composition of the RM system as discussed above, but the formation of uniform species in each case makes it possible to easily select systems with particles of an appropriate size.
The polymeric nanoparticles 310 of the present invention may be modified for use in an optical-bio disc system. For instance, bigger particles that are detectable using an optical disc reader 112,
Another way to improve the signal is to change the particle material, or to add an absorbing or luminescing label to the micelle before polymerization, so that the label becomes embedded in the particle after polymerization. The label may also be covalently bound to the polymers of the nanoparticle 310. Dyes or labels can be easily incorporated into polymeric nanoparticles 310 when added to reverse micelles 307 before polymerization. Many dyes absorbing or fluorescing at a pre-determined wavelength are suitable for such purpose. In one preferred embodiment of the present invention, infrared absorbing dyes are embedded into nanoparticles 310. These infrared dyes absorb electromagnetic radiation at or near the wavelength of the laser of a CD player. In this case, the signal generated by the nanoparticles will be good despite small particle size.
Semiconductor nanoparticies may also be synthesized using the same methods described above. In this embodiment of the present invention, semiconductor components are mixed with the polymerizing solution in the RMs prior to polymerization thereby generating nanoparticles having semiconductor properties.
Another embodiment of the present invention is the synthesis of magnetic nanoparticles using the same RM methods described above. In this embodiment metallic substances such as iron oxide are mixed with the polymerizing solution in the RMs prior to polymerization thereby generating magnetic nanoparticles. Further details relating the use of magnetic particles in bio-disc assays are described in, for example, commonly assigned and co-pending U.S. patent application Ser. No. 10/307,263 entitled “Magneto-Optical Bio-Discs and Systems Including Related Methods” filed on Nov. 27, 2002 which is herein incorporated by reference in its entirety.
Yet another embodiment of the present invention is the synthesis of nanocapsules 326, illustrated in
Any nanoparticle 310 or nanocapsule 326 synthesized using the methods of the present invention can be coated with a chemically active substance for conjugation to bio-active molecules or agent and used in disc assays. Suitable bio-compatible molecules include, for instance saccharide material such as dextran, cellulose, or protein material. Further details relating methods for conjugating bio-active molecules onto various surfaces are described in, for example, commonly assigned and co-pending U.S. patent application Ser. No. ______ entitled “Multi-Purpose Optical Analysis Disc for Conducting Assays and Related Methods for Attaching Capture Agents” filed on Jan. 21, 2003 which is herein incorporated by reference in its entirety. In addition, synthesis of a nanoparticle 310 or nanocapsule 326 having free active groups can be achieved by using monomers with such groups for polymerization.
Alternatively, the polymeric nanoparticle may be modified by grafting active groups onto the nanoparticle surface. See for instance Braybrook et al., Prog. Polym. Sci. 15:715-734, 1990. Most of the modification procedures known in the art involve sequential treatment of surfaces with chemical reagents. Examples include sulfonation of polystyrene, Gibson, et al., Macromolecules 13:34, 1980; base hydrolysis of polyimide, Lee, et al., Macromolecules 23:2097, 1990; and base treatment of polyvinylidene fluoride, Dias et al., Macromolecules 17:2529, 1984. Another conventional method for modifying polymer surfaces includes exposing the surface of the hydrocarbon such as polyethylene with nitrene or carbene intermediates generated in a gas phase (Breslow in “Azides and Nitrenes”, Chapter 10, Academic Press, New York, 1984). Perfluorophenyl azides (PFPAs) have been shown to be efficient in the insertion in CH bonds over their non-fluorinated analogues (Keana, et al., Fluorine Chem. 43:151,1989). Recently, bis-(PFPA)s have been shown to be efficient cross-linking agents for Polystyrene (Cai, et al., Chem. Mater. 2:631,1990).
Chemical modification of the inert polymer substrate surface, such as the surface of a nanoparticle 310, is efficiently done through grafting procedures that allow the deposition of a thin interphase layer, active layer, or interlayer on the surface of the nanoparticle 310. Ideally, the interphase layer should make a stable linkage of the grafted material to the substrate surface and contain a spacer molecule ending in a functional group or variety of chemically different functional groups. This allows the selection of specific surface chemistries for efficient covalent immobilization of a variety of bio-active agents with different demand for spatial orientation, side directed attachment within the structure of the binding protein. The introduction of spacer molecules, especially hydrophilic spacers as part of the graft, contributes significantly to the flexibility and accessibility of the immobilized bio-active substance or agents. By placing a spacer layer between the solid phase of the nanoparticle surface modified or grafted with different functional groups and the bio-active substance or agent, a potentially denaturing effect of the direct contact of the bio-active agent with the functional groups is eliminated.
Integration of Bio-Active Substances into Nanoparticles and Nanocapsules
With reference now generally to
Immobilized biologically active substances, antibodies and enzymes, in particular, are widely used in biotechnology. A great number of carriers with different properties have been developed for immobilization of bio-active substances. However, most of the carriers used suffer from the fact that their properties, particularly large size, cannot be varied to a great extent. A problem of diminishing the size of carrier particle stems from the necessity of increasing the surface of a catalyst and overcoming diffusion limitations. The use of small colloid particles would allow combining the advantages of heterogeneous and homogeneous catalysis. In a preferred embodiment of the present invention, a single bio-active substance is immobilized inside its own nanoparticle 310 or contained in a single nanocapsule 326.
Bio-active substances can be immobilized into the nanoparticles 310 if they are solubilized inside the aqueous inner cavity 322 of the reverse micelle 307 and polymerization is performed in their presence. Biological properties of bio-active substances such as proteins and drugs entrapped in nanoparticles 310 may change, but is it known that bioactivity (for example, enzyme catalytic activity) can be retained after polymerization up to 90% of initial activity. One advantage in using the nanoparticles of the present invention for bio-disc assays is the substantial improvement in the stability of a bio-active substance due to protection by the polymeric layer of the nanoparticle 310 from denaturing effects of temperature and aggressive solvents. For example, nanoparticle embedded enzymes possess high thermostability (exceeding by a factor of 1000 the thermostability of the native enzyme) and are soluble and stable both in aqueous solutions and non-polar organic solvents as described below in connection with
Referring now specifically to
Referring next to
With reference to
In another embodiment of the present invention, a water-soluble substance 320 may be encapsulated in a hydrophobized nanocapsule 328.
Referring now to
With reference next to
Immunochemical Assays Using Bio-Active Nanoparticles on the Optical Bio-Disc
There are three general classes of binding assays as related to the present invention. These include binding protein capture assays, analyte capture assays, and sandwich type assays. The latter assay type can have a binding protein-analyte-binding protein or analyte-binding protein-analyte format.
A specific implementation of a binding assay is an immunoassay. In such an immunoassay, the binding protein may be represented by a capture antibody or a capture antigen and the analyte may be an antigen/hapten or a target antibody, respectively. The product of the reaction is an antigen-antibody immune complex.
The following discussion will concentrate on the immunoassay implementation of binding assays but will in most cases apply also to the broader definition of binding assays. More detailed information on immunoassays can be found in “Radioimmunoassay Methods”, K. E. Kirkham and W. M. Hunter (Eds.), Churchill Livingston Edinburgh and London (1973) and “Principles of Competitive Protein Binding Assays”, W. D., Odel, W. H. Daughaday, JB Lippincot Co., Philadephia, Pa. (1971) which is herein incorporated by reference in its entirety. Both, a target or analyte antigen and a target antibody can be quantified by an immunoassay designed in analogy to one of the formats as described below in conjunction with
Referring now specifically to
Referring next to
With reference now to
Conversely, an antigen-antibody-antigen sandwich assay (
Quantification of antigen molecules is preferably most efficiently done by the two-antibody sandwich assay represented by
Detection or quantification of an antibody or any immunoglobulin is alternatively done by a solid phase immobilized antigen test device, as shown in
More recently, antibodies are determined by antigen sandwich, dubbed “inverse sandwich” immunoassays as illustrated in
Turning now to
Referring now specifically to
After binding, the flow channel 130 may be washed to clear the target zone 140 of any unattached target agents in the sample. After removing the unattached target agents in the sample, signal agents, probes, or antibodies 346 conjugated to the bio-active nanoparticle 316 are introduced in the flow channel 130,
After the signal agent binding step, the flow channel 130 may be washed to clear the target zone 140 of any unattached nanoparticles 316. Upon removal of unattached nanoparticles 316, enzyme-reactive substrates 348 are then introduced in the channel as shown in
As would be apparent to those of skill in the art, in view of the present disclosure, the method for performing an immunochemical assay illustrated above in conjunction with
Conjugation of the signal agent onto nanoparticles or nanocapsules may be achieved by passive absorption of the signal agent onto the nanoparticle or covalent binding of signal agents onto nanoparticles having free functional groups on its surface. The functional groups may include hydroxyl, carboxyl, aldehyde, sulfhydryl, maleimide, succinyl, anhydride, and amino functional groups. For example, monomers having carboxyl groups may be used to synthesize the nanoparticle resulting in a carboxy-modified nanoparticle having free carboxyl groups on its surface. The free carboxyl groups on the surface of the nanoparticle may be activated using N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylamino)propyl carbodiimide (CDI or EDAC) which generates an NHS ester active group. Once the NHS ester has been generated, free reactive amino groups on lysine residues of antibodies or the amino group on the PEG derivatized antibodies are then allowed to covalently bind to the carbon of the NHS ester in a substitution reaction. In this reaction, the nitrogen on the amino group on the antibodies acts as a nucleophile binding onto the carbon of the carboxyl group in the NHS ester removing the NHS leaving group thereby tethering the antibody on the surface of the nanoparticle. Further details relating to binding of biological material to functionalized surfaces are disclosed in commonly assigned and co-pending U.S. patent application Ser. No. 09/997,741 entitled “Dual Bead Assays Including Optical Biodiscs and Methods Relating Thereto” filed Nov. 27, 2001; U.S. patent application Ser. No. 10/038,297 entitled “Dual Bead Assays Including Covalent Linkages For Improved Specificity And Related Optical Analysis Discs” filed Jan. 4, 2002; U.S. patent application Ser. No. 10/086,941 entitled “Methods For DNA Conjugation Onto Solid Phase Including Related Optical Biodiscs and Disc Drive Systems” filed Feb. 26, 2002; and the above incorporated by reference U.S. patent application Ser. No. ______ entitled “Multi-Purpose Optical Analysis Disc for Conducting Assays and Related Methods for Attaching Capture Agents”. All of which are herein incorporated by reference in their entireties.
Use of the Bio-Active Nanoparticle in Optical Bio-Disc Genetic Assays
With reference now to
Referring to
Referring now to
Referring next to
Next,
The method for performing the genetic assay described above may be carried out on an open disc or inside a flow channel of the optical bio-disc. Further details regarding various methods for enzyme based detection of genetic materials on optical bio-discs are disclosed in the above referenced U.S. patent application Ser. Nos. 10/035,836 and 10/150,702. Furthermore, the genetic assays described above may be implemented using any of the bio-active hydrophobized nanoparticles, nanocapsules, and hydrophobized nanocapsules described above in conjunction with
Having generally described the invention, the same will be more readily understood through reference to the following examples, which are provided by way of illustration, and are not intended to be limiting of the present invention.
Synthesis of Bio-Active Nanoparticles
Cross-linked polyacrylamide bio-active nanoparticles were prepared by polymerization of an aqueous solution of acryloylated α-chymotrypsin, acrylamide (Koch-Light, U.K.), and N—N′-methylenebisacrylamide (Reanal, Hungary), solubilized in a solution of AOT (Fluka, Germany) in purified toluene. Acryloylated α-chymotrypsin was produced by modification of α-chymotrypsin with acryloyl chloride (Serva, Germany) at 0° C. and pH 8.0. Toluene was purified by shaking with sulfuric acid and redistillation. Polymerization was initiated by UV-irradiation of degassed solution of reverse micelles containing solubilized acrylamide, methylene bisacrylamide, acryloylated α-chymotrypsin, and azobisisobutyronitrile (initiator for radical polymerization). UV-irradiation was perfomed for 10 minutes at a distance of 33 cm from the light source (XBO-200 xenon lamp, Germany). The nanoparticles were isolated from the organic solution by salting out with a 10-fold excess of cold acetone. The resulting bio-active nanoparticle may be dissolved in water or in an organic solvent with the addition of a micelle-forming surfactant and a small quantity of water as described above in conjunction with
As would be apparent to one of skill in the art, in view of the procedure described in this Example 1, any enzyme, antibody, DNA, receptors, ligands, or other labels including fluorescent dyes may be incorporated into the nanoparticle using the method described above.
Genetic Assays
Fresh polystyrene solution was prepared by adding 3 g polystyrene pellets (Sigma cat. no. 182427; molecular weight=280,000) to 310 ml toluene and stirring for 1 hour using a teflon stir bar and a stir plate. After the polystyrene was completely dissolved in the toluene, 68 ml reagent grade isopropanol was slowly added while stirring.
A stock solution of nitrocellulose was prepared by diluting a nitrocellulose collodium solution (4-8% in ethanol/diethylether, Fluka cat. no. 09986, lot no. 389973/1 30299) 1:5 in reagent grade ethanol. Prior to spin coating, the stock solution was diluted 1:10 with reagent grade ethanol and filtered using a 0.2 μm syringe filter.
A polycarbonate disc having a 200 Angstrom gold semi-reflective layer (BTI Optical Bio-disc Set FDL21:E001308) was placed on a “spin coater,” or modified centrifuge, with the reflective surface up. While rotating the disc on the spin coater, the reflective surface was cleaned with reagent grade alcohol.
The spin coater was set to start spinning at 2500 rpm, followed by acceleration to 4000 rpm within 10 seconds. During this 10-sec acceleration, a steady stream of polystyrene solution was applied to the disc using a pasteur pipette, with the polystyrene solution applied from the outer edge to the inner side in one smooth stroke.
The spin coater speed was then adjusted to 1500 rpm, and the diluted and filtered nitrocellulose solution was applied onto the inner portion of the disc in a steady stream using a pasteur pipette.
The disc from Example 2 was placed on a CD assembler/spindle with the nitrocellulose layer up. Between about 0.5 to 2.0 μl of 1 μM oligo probes (capture DNA) in 1 M NH4OAc were applied to the disc at defined target zones. The droplets of capture DNA were dried onto the nitrocellulose at 37° C.
A cover disc containing U-shaped fluidic circuits (50 μM adhesive; Fraylock, DBL243a) was applied using a disc assembler spindle, and the disc was run through a wringer to seal the two discs.
DNA blocking solution (1% bovine serum albumin [BSA], 5× Denhardt's solution, 0.1 mg/ml salmon sperm DNA, 200 mM KCl, 10 mM MgCl2, 50 mM Tris, pH 7.4) was degassed in a vacuum desiccator and injected into the fluidic circuits of a bio-disc prepared as in Example 3, taking care that no air bubbles remained in the circuits. The bio-disc was then incubated at room temperature for 30 to 60 minutes.
The DNA blocking solution was removed, and the fluidic circuits washed with hybridization buffer (200 mM NaCl, 10 mM MgCl2, 50 mM Tris, pH 7.4) injected into the fluidic circuits using a syringe. PCR amplicons (target DNA amplified using biotinylated primer sets, purified using the Qiagen QIAquick PCR Purification Kit, cat. no. 28104, lot no. 10927932, and eluted using hybridization buffer) were denatured at 95° C. for 5 minutes and immediately placed on ice for 5 minutes.
The denatured amplicons were added to the appropriate fluidic circuits (10 μl per fluidic circuit) and allowed to hybridize for 1.5 to 2 hours at room temperature. Following hybridization, the fluidic circuits were washed with hybridization buffer using a syringe.
Neutravidin-Horseradish Peroxidase Conjugated enzyme (N-HRP; Pierce product no. 31001, lot no. BK46404) was diluted 1:5000 in hybridization buffer, and 12 μl was applied to each fluidic circuit. The disc was then incubated at room temperature for 15 minutes.
The fluidic circuits were then washed with hybridization buffer using a syringe, and 12 μl of TMB Substrate in Stable hydrogen peroxide buffer (Calbiochem cat. no. 613548, lot B34202) was added to each fluidic circuit. The enzyme reaction was allowed to proceed for 5 minutes, after which the reaction was stopped by flushing the fluidic circuits with distilled water using a syringe.
Each fluidic circuit was sealed with tape, and the bio-disc was then placed in a disc-reader, similar to that shown in
Alternatively, the Horse Radish Peroxidase enzyme (HRP) may be immobilized or embedded into the nanoparticles of the present invention as descibed above in Example 1. After the bio-active nanoparticle containing the HRP is synthesized, Neutravidin or Streptavidin is then bound or conjugated to the surface of the bio-active nanoparticle through passive adsorption or covalently if monomers having functional groups are used as described above. Once the Neutravidin is bound to the bio-active nanopaticles, the bio-active nanoparticles may then be used in the following examples of genetic assays.
A bio-disc with 6 target zones was prepared as in Example 3, with 1.6 μl of 10 μM DNA oligonucleotides specific to one of the Brucella strains applied to three of the target zones, as indicated in Table 2, below. One target zone contained a mix of all three Brucella species, one target zone contained biotinylated DNA (positive control), and one target zone contained no capture DNA (background).
Brucella sp. genomic DNA was subjected to PCR amplification using forward/reverse primer sets directed to B. melitensis to generate target B. melitensis amplicons. Each reaction contained 1 ng/μl Brucella DNA, 0.2 μM biotinylated forward and reverse primers, 0.2 mM dNTPs, 0.05 U/μl Taq polymerase, 3.0 mM MgCl2 and 1×PCR buffer (Qiagen; 15 mM MgCl2). The thermocycle conditions were:
The enzyme DNA assay was performed as described in Example 4, both without the addition of the target B. melitensis amplicons (results indicated in Table 2) and with the addition of purified target B. melitensis amplicons (results indicated in Table 3). Values from Tables 2 and 3 are event counts collected using an optical disc reader using the reflective disc format. The counting area was a rectangle, 940 μm×2300 μm; event count amplitude was between 225-500.
B. abortus
B. melitensis
B. suis
B. mix
As shown above, the B. melitensis target zone produces a much higher signal than the other Brucella species when the B. melitensis amplicon is used in the present invention.
Bio-discs were prepared as in Example 3, with NosT 52-mer oligonucleotide capture probes applied to the target zones. NosT is a marker gene for genetically modified plant material.
GMO reference materials of soya bean powder containing different mass fractions of powder from genetically modified soya beans, were obtained from Fluka BioChemika, certified reference material IRMM-410R (0, 0.1, 0.5, 1, 2, 5% Roundup Ready Soya). DNA was extracted from each of the six reference materials, using the WIZARD® Magnetic DNA Purification System for Food (Promega, Madison, Wis.).
The extracted DNA was subjected to PCR amplification using biotinylated forward/reverse primer sets directed to NosT to generate biotinylated 280-mer target NosT amplicons. Each reaction contained 50 ng extracted DNA, 0.2 μM biotinylated forward and reverse primers, 0.2 mM dNTPs, 0.05 U/μl Taq polymerase, 3.0 mM MgCl2 and 1×PCR buffer.
The enzyme assay was performed as in Example 4, using the NosT amplicons as target probes. The results are shown in Table 4.
As shown in Table 4, the detectable signal (event counts) increased as the percentage of GMO material increased. Between 0% and 1% GMO, there was a 90% correlation between the amount of GMO material and the resulting signal.
Immunochemical Assays
The following immunochemical assays were perfomed using fluorescent microspheres as reporters. As discussed above, fluorescent nanoparticles may be synthesized by including fluorescent dyes in the polymerization mixture during synthesis of the nanoparticles. Hence, the examples described below in Examples 7, 8, 9, 10, and 11 may be perfomed using the fluorescent nanoparticles of the present invention.
A 2 mg amount of affinity purified anti-HCG-alpha capture antibody (Biocheck, Burlingame, Calif.) was dissolved in 2% glycerol in PBS, pH 7.4 to obtain a 100 ug/ml stock solution. A pin stamper was used to directly apply multiple spots of 0.2-0.3 ul of the capture antibody stock solution on the gold metal layer (150 Angstroms thick) of the transmissive disc. The disc was then incubated in a humid environment using a humidity chamber at room temperature overnight. After incubation, the disc was washed with a gentle stream of deionized water to remove excess unbound capture antibodies and spun dried at 1000-1500 rpm. The cap portion having attached thereto the adhesive layer, having fluidic circuits formed therein, is then applied onto the substrate. After the disc is fully assembled, the fluidic channels are then filled with blocking buffer (10 ul/channel) containing 1% BSA, 1% Sucrose, 0.1% Tween-20 in PBS, pH 7.4. The disc is incubated for 2 hours to allow the blocking agents sufficient time to bind to unoccupied sites on the capture zone, cover disc, and substrate to prevent or minimize non-specific binding of reporter agents onto unwanted sites. Also, loosely bound capture antibodies will be displaced through dissociation of the antibody-antibody bonds by the detergent in the blocking buffer. After blocking, the excess blocking buffer is aspirated and the channels are filled with deionized water (10 ul/channel) to remove excess salt from the blocking buffer. The water is then aspirated and the disc is kept at 4 degrees Celsius prior to use.
Microspheres may be purified using dialysis or centrifugation. With centrifugation, bead suspensions are centrifuged at a speed required to precipitate the particles. The speed is determined empirically and depends on the mass of the microspheres or nanoparticles and the density of the buffer containing the microspheres or nanoparticles [e.g., 0.2 um Fluospheres (Molecular Probes) in PBS or conjugation buffer may be centrifuged at 6000 rpm for 30 mins and 0.5 um Fluospheres (Molecular Probes) in PBS may be centrifuged at 14,000 rpm for 20 mins.). After the initial centrifugation of the bead suspension, the supernantant is discarded and the beads are resuspended in a conjugation buffer. The conjugation buffer is preferably a low ionic strength sodium phosphate buffer (PBS) having a pH slightly above the isoelectric point of the signal agent to be conjugated to the microspheres. The centrifugation, aspiration, and resuspension steps are repeated three times and the final pellet of beads is resuspended in conjugation buffer to obtain a suspension containing 10 mg/ml microspheres. The purified bead suspension is then stored at 4 degrees Celsius and sonicated for 30 seconds prior to use. Microspheres ranging in size from 0.01 um to 10 um in diameter and colloidal particles between 4 to 50 nm in diameter may be used in the present invention.
A 5.0 mg amount of purified and sonicated 0.2 um polystyrene carboxylate Fluospheres (Molecular Probes, Eugene, Oreg.), prepared as described in Example 8, were dispensed into 250 ul of 20 mM Sodium Phosphate buffer, pH 7.2 in a 1.7 ml Costar centrifuge tube. The beads were mixed in a vortex mixer and an additional 250 ul of Sodium Phosphate buffer was then added to the bead suspension. Then 250 ug of anti-HCG-beta was added to the bead suspension and immediately mixed using a vortex mixer. The tube containing the bead suspension was then placed on a Dynal mixer and rotated to 40 hours at 4 degrees Celsius shielded from light. After incubation, the beads were spun at 6000 rpm for 15 mins, the supernatant was aspirated and the pellet was resuspended with 500 ul of 20 mM Sodium Phosphate buffer, pH 7.2, sonicated for 30 seconds. After the initial washing step, the beads were further washed 3 times with 500 ul of 20 mM Sodium Phosphate buffer, pH 7.2 by repeated aspiration and spin cycles of 6000 rpm for 30 mins. The final pellet was then reconstituted with 1.0 ml 20 mM Sodium Phosphate buffer, pH 7.2 to obtain a final microsphere concentration of 5.0 mg/ml. The anti-HCG-beta conjugated microspheres were then stored at 4 degree Celsius.
A 400 ul bead suspension containing 4 mg of 0.5 um carboxylate polystyrene Fluospheres (Molecular Probes, Eugene, Oreg.) in PBS, prepared as described in Example 2, was dispensed into a 1.7 ml Costar centrifuge tube. Then 200 ug of anti-HCG-beta antibody in 15 mM potassium phosphate, 145 mM sodium chloride, pH 7.4 buffer was added to the bead suspension. The resulting antibody-bead suspension was then mixed using a Dynal rotator at room temperature for 4 hours. The suspension was then further incubated at 4 degrees Celsius without mixing for an additional 36 hours. After incubation, the beads were spun at 14000 rpm for 20 mins, the supernatant was aspirated and the pellet was resuspended with 500 ul of 20 mM Sodium Phosphate buffer, pH 7.2. After the initial washing step, the beads were further washed 3 times with 500 ul of 20 mM Sodium Phosphate buffer, pH 7.2. The final pellet was then reconstituted with 800 ul 20 mM Sodium Phosphate buffer, pH 7.2 containing 0.05% sodium azide. The anti-HCG-beta conjugated microspheres were then stored at 4 degree Celsius.
The following materials were utilized in this Example: (1) Fully assembled optical bio-disc made according to Example 7; (2) HCG standard or unknown in 1% BSA PBS 7.4, 0.05% sodium azide; (3) Bead Conjugate Dilution Buffer (BCDB): 1% BSA, 0.1% Tween-20, and 0.05% sodium azide in PBS 7.4 (Note: The BSA concentration may be 0.1-10%; sucrose may be replaced with other sugars including glucose, fructose, trehalose, or lactose at a concentration of 0.1-10%; Tween-20 may be replaced with other non-ionic detergents including Triton X-100 and Tween-80 at a concentration of 0.1-5%; and sodium azide concentration may range from 0.01 to 1%); and (4) 0.2 um or 0.5 um Fluospheres conjugated with Anti-HCG-beta, respectively made according to either Example 9 or 10, washed and resuspended in BCDB.
The Assay: Various concentrations (0, 12.5, 25, 50, 250, and 500 mlU/ml) of HCG standard were mixed with an equal volume (10 ul) of 0.5 um Fluospheres conjugated with anti-HCG-beta in BCDB. Prior to use, the Fluospheres were washed and reconstituted in BCDB to obtain a bead concentration of 25 ug Fluospheres/ml of BCDB. The assay solutions were mixed and a 10 ul aliquot of each suspension was applied, using a pipette, through the inlet port into various channels in the bio-disc such as those shown and described above in conjunction with
Synthesis of Nanoparticles
Enzyme immobilization on the nanoparticle can be achieved by immobilization during polymerization process (inside the particle) as described above in Example 1, or immobilization as a second step after polymerization is complete and nanoparticles are separated from the organic solvent (on the surface of the particle). Enzyme immobilization on the nanoparticle can be performed non-covalently or covalently. For covalent immobilization, active polymerizable groups of the enzyme molecule (such as double bonds and triple bonds), and active groups of monomers (double bonds and triple bonds) can be used.
An enzyme molecule can be chemically modified by attaching needed functional groups before solubilizing the enzyme into the RM solution as described below in Example 12.
A 5 mL volume of Horse Radish Peroxidase (HRP) enzyme solution was prepared in 0.2 M phosphate buffer (pH=8.0). Then 10-20 uL of acryloylchloride was added to the enzyme solution at intensive stirring at 0° C., under a fumehood. The mixture was stirred for 20-30 min.
The degree of enzyme modification (amount of active monomers per enzyme molecule) was determined by titration of free amino groups of the enzyme, as described in Yakunitskaya, L. M. et al. Biokhimiya (“Biochemistry”), 1983, Vol. 48, No. 10, p.1596-1603 (in Russian).
A 0.5 M solution of AOT was prepared in toluene. Pre-calculated quantities of acrylamide, methacrylamide, and N,N′-methylene-bis-acrylamide, and 2-3 vol. % of water were added to the micellar solution. The resulting mixture was stirred until the monomers were fully dissolved. Then a polymerization initiator (azo-bis-isobutyronitrile) at 0.1 mg/mL concentration and a pre-determined amount of aqueous enzyme solution (non-modified enzyme or modified enzyme as in Example 12) was added into the mixture. The resulting solution was mixed until the solution became clear.
The solution prepared in Example 13 was transfered into a quartz vial and degassed using a vacuum. The degassed solution was then irradiated with UV light for 5-40 min, at 33 cm distance between UV source and solution and 1 cm solution thickness. The degree of polymerization was monitored by NMR spectroscopy following the disappearance of “vinyl” protons peaks.
After the polymerization step in Example 14, 10-20 fold excess of cold (+4 C or below) acetone was added to the solution containing the newly synthesized nanoparticles or bio-active nanoparticles. The resulting mixture was centrifuged at 3000 rpm for 3-5 min. The supernatant was decanted. The precipitate was washed with cold acetone 2 times by repeated addition of cold acetone, centrifugation, and removal of the supernatant. The final precipitate was dried on air at room temperature for 10-30 min. The precipitate or nanoparticles was stored +4° C.
Concluding Summary
All patents, provisional applications, patent applications, technical specifications, and other publications mentioned in this specification are incorporated herein in their entireties by reference.
While this invention has been described in detail with reference to certain preferred embodiments, it should be appreciated that the present invention is not limited to those precise embodiments. Rather, in view of the present optical bio-system disclosure that describes the current best mode for practicing the invention, many modifications and variations would present themselves to those of skill in the art without departing from the scope and spirit of this invention. The scope of the invention is, therefore, indicated by the following claims rather than by the foregoing description. All changes, modifications, and variations coming within the meaning and range of equivalency of the claims are to be considered within their scope.
Furthermore, those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are also intended to be encompassed by the following claims.
The present application is a continuation in part of U.S. patent application Ser. No. 10/150,702 filed May 16, 2002. This application also claims the benefit of priority from U.S. Provisional Application Ser. No. 60/353,949 filed on Jan. 31, 2002 which is herein incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 60353949 | Jan 2002 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10150702 | May 2002 | US |
| Child | 10356666 | Jan 2003 | US |
