Patent Publication Number: US-2022221447-A1

Title: SYNTHESIS OF nanoSCINT PARTICLES

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims the priority benefit of U.S. Provisional Application No. 62/855,240, filed May 31, 2019, which is incorporated herein by reference in its entirety. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to a process for producing a nanocomposite particle (“nanoSCINT particle”) adapted for use in scintillating proximity assay in a relatively high quantity. The nanoSCINT particle comprises a nanoparticle organic polymer doped with at least one scintillating compound. The nanoparticle organic polymer is then encapsulated with a silica shell. In particular, the process of the invention includes adding a plurality of batch-wise portions of a polymerization initiator to an emulsion comprising a relatively high amount of polymerizable organic compound to produce a nanoparticle organic polymer, which is then doped with at least one scintillating compound, and then encapsulated with a silica shell to produce nanoSCINT particles in gram quantities in a single process. 
     BACKGROUND OF THE INVENTION 
     One of the primary challenges in developing nanoSCINT is synthesizing this material in a quantity large enough to divide and distribute to multiple labs, as well as to use throughout the course of an entire series of testing and characterization. Previously disclosed processes allowed preparation of only a few mg of nanoSCINT in a single batch and had to combine several batches to complete even a single scintillation experiment. See, for example, commonly assigned U.S. Patent Application Publication No. 2018/0118916, which is incorporated herein by reference in its entirety. 
     Unfortunately, even with optimization of previously disclosed process, at best only about 100 mg of nanoSCINT material could be obtained per batch, or enough for at most 1-5 experiments, depending on the design of the experiment. Though an improvement, this was still a major limitation to moving nanoSCINT forward. Such a small amount of nanoSCINT production per batch of reaction resulted in a significantly high overall cost and time in preparing a useful amount of nanoSCINT. 
     Therefore, there is a need for a process that can produce a significantly higher amount of nanoSCINT per reaction in order to reduce the overall material and/or labor cost as well as provide a uniform nanoSCINT that can be used in assays. 
     SUMMARY OF THE INVENTION 
     Surprisingly and unexpectedly, the present inventors have discovered that synthesizing nanoSCINT in a quantity large enough to divide and distribute to multiple labs, as well as to use throughout the course of an entire series of testing and characterization was more difficult than simple scale up process. In fact, simply scaling up the reaction conditions only led the present inventors to a few mg of nanoSCINT in a single batch. In order to conduct an entire series of testing and characterization, the reaction had to be run multiple batches and combined to complete even a single scintillation experiment. A large scale up reaction only resulted in approximately 100 mg of nanoSCINT per batch. Though an improvement, this was still a major limitation to moving nanoSCINT forward. 
     It was discovered by the present inventors that use of the process disclosed herein resulted in heretofore unobtainable amount of nanoSCINT in a single reaction batch. As used herein, the term “single reaction batch” refers to producing the desired nanoSCINT from start to finish without repeatedly conducting the same reaction to produce nanoSCINT and combining the nanoSCINT produced. 
     One particular aspect of the invention provides a process for producing a nanocomposite particle (“nanoSCINT particle”) adapted for use in scintillating proximity assay, wherein said nanoSCINT particle comprises a nanoparticle organic polymer doped with at least one scintillating compound, and wherein said nanoparticle organic polymer is encapsulated with a thin layer of silica shell. The process includes:
         adding a plurality of batch-wise portions of a polymerization initiator to an emulsion comprising at least 0.1 mole/L of polymerizable organic compound and water under conditions sufficient to produce said nanoparticle organic polymer;   adding at least one scintillating compound to said nanoparticle organic polymer to produce a nanocomposite material, wherein said nanocomposite material comprises said nanoparticle organic polymer that is doped with said scintillating compound; and   encapsulating said nanocomposite material with a silica shell to produce said nanocomposite particle.       

     Another aspect of the invention provides a nanoSCINT produced by the process disclosed herein. 
     Still another aspect of the invention provides a nanocomposite particle (“nanoSCINT particle”) adapted for use in scintillating proximity assay, wherein said nanoSCINT particle comprises a nanoparticle organic polymer doped with at least one scintillating compound, and wherein said nanoparticle organic polymer is encapsulated with a silica shell, wherein a thickness of said silica shell is about 100 nm or less. 
     Yet in further embodiments, processes of the invention provide improved uniformity and coating thickness of nanoSCINT. In some embodiments, it has been discovered by the present inventors that by sequential addition of initiator to the reaction mixture emulsion, at least about 5 g, typically at least about 10 g, and often at least about 15 g of nanoSCINT can be prepared in a single reaction batch, thereby narrowing the overall nanoparticle size distribution and decreasing the shell thickness by at least about 30%, typically by at least about 40%, and often by at least about 50% compared to conventional methods such as those disclosed in commonly assigned U.S. Patent Application Publication No. 2018/0118916. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a schematic illustration of nanoSCINT  100  of the present invention. 
         FIG. 2  is transmission electron microscopy (“TEM”) image of nanoSCINT  100  produced using one embodiment of the invention. The contrast between the silica (darker) shells on the outside of the polystyrene (lighter) cores can be seen. The smaller image is an expanded view from the image enclosed within the box, in which silica shell can be more easily seen. 
         FIG. 3  is TEM images of nanoSCINT particles (A) synthesized in a batch of less than 100 mg polystyrene core nanoparticles using conventional methods, e.g., as disclosed in U.S. Provisional Patent Application No. 62/803,448, and (B) synthesized using processed disclosed herein. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Some aspects of the invention provide a process for producing a nanocomposite particle (“nanoSCINT particle”) adapted for use in scintillating proximity assay. The terms “nanoSCINT” and “nanoSPA” are used interchangeably herein and refer to nanocomposite particles that are adapted for use in scintillating proximity assay. In particular, nanoSCINT comprises a nanoparticle sized organic polymer core particle comprising at least one scintillator compound or material. As used herein, the term “nanoparticle” means organic polymer core particle having a mean particle size of about 1,000 nm or less, typically about 500 nm or less, often about 300 nm less, more often about 250 nm or less, and most often 150 nm or less. Still in other embodiments, D90 particle size of the organic polymer core particle is about 900 nm or less, typically about 800 nm or less, often about 600 nm or less, more often 400 nm or less, still more often about 200 nm or less, and most often about 100 nm or less. Alternatively, the average particle size of the organic polymer core particle ranges from about 10 nm to about 1,000 nm, typically from about 5 nm to about 500 nm, often from about 10 nm to about 300 nm, more often from about 20 nm to about 250 nm, and most often from about 20 nm to about 150 nm. 
     The nanoSCINT also includes at least one scintillator compound. Exemplary scintillator compounds that can be used in the invention include, but are not limited to, p-terphenyl (pTP); 1,4-bis(4-methyl-5-phenyl-2-oxazolyl)benzene (dimethyl POPOP); 1-phenyl-3-(2,3,6-trimethylphenyl)-2-pyrazoline (PMP); tris(1,3-diphenyl-1,3-propanedionato)-(1,10-phenanthroline) europium (III); diphenylanthracene; 1,4-bis(5-phenyl-2-oxazolyl)benzene; 1,4-bis(2-methylstyryl)benzene; anthracene; and a combination thereof. As used herein, the term “combination thereof” means two or more, e.g., two, three, four, five, etc., of the compounds or the materials listed can be present. It should be appreciated that the scope of the invention is not limited to these particular scintillator compounds. In general, any and all known scintillator compounds can be used in the invention. The scintillator compound(s) are typically admixed or doped with, adsorbed onto, attached to, or linked to the organic polymer core particle using any of the methods known to one skilled in the art. 
     In one particular embodiment, the scintillating compound comprises a mixture of pTP and POPOP. In some instances, the ratio of pTP and POPOP is at least about 50:1, typically about 25:1, and often 10:1. In other instances the ratio of pTP and POPOP may be about 1:50, typically 1:25, and often 1:10. 
       FIG. 1  schematically illustrates a typically nanoSCINT of the invention. The nanoSCINT  100  of the invention includes a nanoparticle sized organic polymer core particle  104  comprising at least one scintillator compound or material  108 . The nanoSCINT also includes a silica shell  112  of thickness d. As can be seen in  FIG. 1 , the silica shell  112  encapsulates nanoparticle sized organic polymer core particle  104 . 
     As discussed in more detail below, surprisingly and unexpectedly, processes of the invention can result in a significantly thinner silica shell compared to other methods known to one skilled in the art. See, for example, U.S. Patent Application Publication No. 2018/0118916, which has been previously incorporated by reference in its entirety. In some embodiments, the thickness of silica shell is about 90% or less, typically about 80% or less, and often 75% or less of the silica shell thickness produced using conventional methods such as those disclosed in U.S. Patent Application Publication No. 2018/0118916. 
     Surprisingly and unexpectedly, compared to the process for producing nanoSCINT disclosed in previous methods such as those disclosed in U.S. Patent Application Publication No. 2018/0118916, processes of the invention result in a significantly higher yield of nanoSCINT and requires a significantly less amount of solvents and other materials, thereby resulting in a substantial reduction in cost and time. In general, processes of the invention result in about 100% or more, typically about 200% or more, often about 300% or more, and more often about 500% or more yield of the nanoSCINT compared to conventional methods. Furthermore, the amount of solvent required in processes of the invention is about 25% or less, typically about 50% or less, often about 75% or less, and more often about 90% or less compared to the amount of solvent required in conventional methods. 
     Processes of the invention include adding a plurality of batch-wise portions of a polymerization initiator to an emulsion comprising at least about 0.2, typically at least about 0.25, often at least about 0.3, and more often at least about 0.4 mole/L of polymerizable organic compound. Exemplary polymerizable organic compounds that can be used in the present invention include, but are not limited to, styrene, divinylbenzene, polyvinyltoluene, polyvinylpyrrolidone, or a combination thereof. As used herein, the term “about” when referring to a numeric value means being within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system or the degree of precision required for a particular purpose. For example, the term “about” typically means within 1 standard deviation, per the practice in the art. Alternatively, the term “about” when referring to a numerical value can mean±20%, typically ±10%, often ±5% and more often ±1% of the numerical value. In general, however, where particular values are described in the application and claims, unless otherwise stated, the term “about” means within an acceptable error range for the particular value. 
     In some embodiments, the initial concentration of polymerizable organic compound in said emulsion ranges from about 0.1 mole/L to about 0.5 mole/L. In other embodiments, the ratio of said polymerizable organic compound to water is at least about 5 g per 200 mL of water. 
     Typical solvent used in producing the organic polymer core particles  104  comprises water. In some embodiments, the solvent used consists of water. Since the organic compound that is used to produce the organic polymer core particles  104  is typically not readily miscible with water and forms emulsion, one may add a small amount of other organic solvents to aid in solubilizing the organic compound in water. Exemplary organic solvents that can be used in solubilizing the organic compound include alcohol-based solvents, such as methanol, ethanol, isopropanol; ethers such as diethyl ether and tetrahydrofuran (THF); as well as other organic solvents such as dimethylformamide (DMF), dimethylsulfoxide (DMSO), etc. When organic solvent is used, generally the amount of organic solvent used is about 25 v/v % or less, typically about 20 v/v % or less, often about 10 v/v % or less, and most often about 5 v/v % or less relative to the amount water. However, typically no organic solvent is used, i.e., only water is used as the solvent. 
     In some embodiments, water is purged, e.g., with inert gas such as helium, nitrogen, or argon, to remove any dissolved oxygen. Thus, in some embodiments, the amount of dissolved oxygen in water is about 50% or less, typically about 75% or less, and often about 90% or less relative to water that has been exposed to atmosphere for at least 24 hours. Alternatively, water used in processes of the invention has about 250 ppm, typically about 100 ppm, often 50 ppm, more often 10 ppm, and most often 5 ppm or less of oxygen. 
     As can be seen, the concentration of polymerizable organic compound is substantially higher compared to other methods known to one skilled in the art. Typically, the polymerization initiator is added to the reaction at least two, typically at least three, often at least four, and more often at least five times during the reaction. Typically, the next portion or batch of the polymerization initiator is added about 30 min after, typically about 60 min after, and often at least 90 min after addition of the previous portion. Exemplary polymerization initiators that can be used in processes of the invention include, but are not limited to, 2,2″-azobis(2-methylpropionamidine) dihydrochloride (AIBA), 2,2″-azobis(2-methylpropionitrile) (AIBN), or a combination thereof. It should be appreciated, however, that any polymerization initiator known to one skilled in the art can be used. 
     The amount of each portion of the polymerization initiator added is typically about 20 mole % or less, often about 15 mole % or less, and more often about 10 mole % or less relatively to the total amount of polymerizable organic compound. Alternatively, the total amount of polymerization initiator added is typically about 50 mole % or less, often about 25 mole % or less, more often about 10 mole % or less, still more often about 5 mole % or less, and most often about 1 mole % or less relatively to the total amount of polymerizable organic compound. Still in other embodiments, the total amount of said polymerization initiator added is no more than about 1 mmol per 1,000 mmol, typically about 1 mmol per 500 mmol, and often 1 mmol per 100 mmol of said polymerizable organic compound. 
     Typically, the reaction temperature in the polymerization step (i.e., polymerization of polymerizable organic compound) to produce nanoparticle organic polymer is at least about 50° C., often at least about 80° C., and more often at least about 100° C. It should be appreciated that the reaction temperature is not limited to those provided herein. The reaction temperature can vary widely depending on a variety of factors such as, solvent, polymerizable organic compound, amount of polymerization initiator, polymerization initiator, etc. 
     The nanoparticle organic polymer produced can be concentrated prior to adding one or more scintillating compound. For example, water and any unreacted polymerizable organic compound can be removed under reduced pressure (e.g., using a rotary evaporator), by distillation, extraction, or other methods known to one skilled in the art. 
     The nanoparticle organic polymer is then combined or admixed with at least one scintillating compound to produce a nanocomposite material. The nanocomposite material comprises a nanoparticle organic polymer that is doped with scintillating compound(s). When two or more different scintillating compounds are used, the ratio of each scintillating compounds can vary. For example, when pTP and POPOP are used as scintillating compounds, the ratio between pTP and POPOP ranges from about 50:1, typically from about 25:1, often about 20:1, more often about 10:1, still more often about 5:1 and most often about 1:1. It should be appreciated that the scope of the invention is not limited to this particular ratio. In general any ratio of two more different scintillating compounds can be used depending on a particular assay and scintillators used. For example, in an alternative embodiment, the ratio between pTP and POPOP can range from about 1:50, typically from about 1:25, often about 1:20, more often about 1:10, and still more often about 1:5. 
     Doping of nanoparticle organic polymer with scintillant fluorophores typically includes dissolving scintillation compound(s) in an organic solvent such as chloroform, dichloromethane, tetrahydrofuran (THF), diethyl ether, benzene, toluene, methanol, isopropanol, ethanol, etc. This solution is then added to an aqueous mixture comprising the nanoparticle organic polymer. The resulting mixture is stirred or sonicated to produce the nanoparticle sized organic polymer core particle  104 . This is typically stored as a mixture. It should be appreciated that at least a portion of the organic solvent can be removed from the reaction mixture prior to storage or the next reaction. 
     The process of the invention also includes encapsulating the nanoparticle sized organic polymer core particle  104  with a silica shell to produce nanoSCINT nanocomposite particle  100 . For silica shell encapsulation, a concentration of at least about 10 mg/mL, typically at least about 12 mg/mL, often at least about 15 mg/mL, and more often at least about 20 mg/mL of nanoparticle sized organic polymer core particle  104  in aqueous solution is dispersed in an alcohol solvent, such as ethanol, methanol, isopropanol, or a mixture thereof. Typically, at least about 250 mL, often at least about 300 mL, more often at least about 400 mL, and most often at least about 500 mL of nanoparticle sized organic polymer core particle  104  solution is dispersed in about 2 L of an alcohol solvent. The encapsulation process also includes adding a base, e.g., ammonium hydroxide or other amine base (e.g., trialkyl amines, such as triethyl amine). Typically, excess amount of base is added relative to the amount of nanoparticle sized organic polymer core particle  104 . For example, when 400 mL of 12.5 mg/mL of polystyrene nanoparticle sized organic polymer core particle  104  is encapsulate with a silica shell, about 50 mL, typically about 60 mL, often about 70 mL, and more often at least 75 mL of ammonium hydroxide is used. As can be seen, the amount of solvent used compared to those of previous methods (e.g., see, for example, U.S. Patent Application Publication No. 2018/0118916) is significantly lower, thereby greatly reducing the time and cost for synthesis of nanoSCINT of the present invention. 
     The silica shell encapsulation process also includes stirring the mixture for a few minutes while a silica shell precursor compound is added, typically in a dropwise manner. Exemplary silica shell precursor compounds of the invention include, but are not limited to, tetraethylorthosilicate (“TEOS”), 3-aminopropyltriethoxysilane, 3-mercaptopropyltrimethyoxy-silane, or a combination thereof. 
     The resulting mixture is stirred for at least about 30 min, typically at least about 45 min, often at least about 60 min, and more often at least about 90 min. The nanoSCINT particles  100  that are formed are then collected, e.g., by filtration, separation, centrifugation, etc. The crude nanoSCINT particles  100  are then rinsed several times by dispersing the nanoSCINT particles  100  in water and again collecting the nanoSCINT particles  100 , e.g., by filtration, separation, centrifugation, etc. 
     Another aspect of the invention provides a nanocomposite particle (“nanoSCINT particle”)  100  that is adapted for use in scintillating proximity assay, wherein said nanoSCINT particle comprises a nanoparticle organic polymer doped with at least one scintillating compound, and wherein said nanoparticle organic polymer is encapsulated with a silica shell, wherein a thickness of said silica shell is about 200 nm or less, typically 150 nm or less, often about 100 nm or less, and more often about 50 nm or less. 
     In some embodiments, the concentration of nanoSCINT  100  in the solution is about 200 mg/mL or less, typically about 150 mg/mL or less, often about 100 mg/mL or less, and most often about 75 mg/mL or less. 
     As stated above, in some embodiments, the resulting nanoSCINT  100  of the invention have a significantly thinner silica shell compared to conventional methods, such as those described in U.S. Patent Application Publication No. 2018/0118916. In some embodiments, the silica shell thickness (“d” in  FIG. 1 ) of nanoSCINT  100  produced using the process of the invention is about 100 nm or less, typically about 75 nm or less, often about 50 nm or less, more often about 25 nm or less, and most often about 20 nm or less. In one particular embodiment, the thickness of said silica shell is about 20 nm or less, typically about 15 nm or less, and often about 10 nm or less. 
     Some of the advantages of having a thinner silica shell include nanoSCINT  100  that are less dense than those produced by other methods. In some embodiments, the density of nanoSCINT  100  of the invention is about 2 g/mL or less, typically about 1.75 g/mL or less, and often about 1.5 g/mL or less. 
     Unlike conventional methods, a single process of the invention provides at least about 5 g, typically at least about 7.5 g, and often at least about 10 g of nanocomposite particles. 
     Additional objects, advantages, and novel features of this invention will become apparent to those skilled in the art upon examination of the following examples thereof, which are not intended to be limiting. In the Examples, procedures that are constructively reduced to practice are described in the present tense, and procedures that have been carried out in the laboratory are set forth in the past tense. 
     EXAMPLES 
     Materials: Styrene, alumina, p-terphenyl (pTP), and 1,4-bis(4-methyl-5-phenyl-2-oxazolyl)benzene (dimethyl POPOP) were purchased from Acros Organics (NJ). Tetraethylorthosilicate (TEOS), and 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AIBA), 2,2′-azobis(2-methylpropionitrile) (AIBN), Triton X-100, cyclohexane, sodium citrate (tribasic) hydrate, sodium tetraborate hydrate, and 2-(N-morpholino)ethanesulfonic acid hydrate (MES) were obtained from Sigma Aldrich (St. Louis, Mo.). Sodium chloride, sodium phosphate hydrate (monobasic), isopropyl alcohol (IPA), chloroform, tetrahydrofuran (THF), and aqueous ammonium hydroxide (28%) were obtained from EMD Millipore (Billerica, Mass.). Hexyl alcohol and divinylbenzene (DVB) were purchased from Alfa Aesar (Haverhill, Mass.) and ethyl alcohol was purchased from Decon Laboratories (King of Prussia, Pa.). BioCount liquid scintillation cocktail was acquired from Research Products International (Mt. Prospect, Ill.).  3 H-labeled sodium acetate was purchased from Perkin Elmer (Waltham, Mass.). All chemicals except styrene were used as received. Inhibitor was removed from styrene by passing the monomer through 0.5 cm diameter by 3 cm long alumina columns immediately prior to use. 
     Preparation of nanoSCINT Particles: Styrene (6 g, inhibitor-free) was added to 100 mL degassed H 2 O in an Ar-flushed 500 mL round-bottomed flask heated to 70° C. in an oil bath. Polymerization was initiated by adding 100 mg of MBA dissolved in approximately 200 of H 2 O to the reaction flask every 3 h for a total of 3 additions. The H 2 O/styrene mixture was stirred rapidly for a total of 18 h. Excess styrene and some H 2 O were removed from the nanoparticle solution under reduced pressure using a rotary evaporator. The total volume of the nanoparticle solution was reduced to approximately 100 mL, and a small aliquot (1-2 mL) of the solution was removed and lyophilized to determine the weight per volume of nanoparticles. 
     Polystyrene nanoparticles were doped with scintillant fluorophores by dissolving dimethyl POPOP and of pTP in 100 mL of 1:9 isopropanol chloroform (v:v) to make 10 mM dimethyl POPOP and 100 mM pTP. Scintillant fluorophores dissolved in solvent were added directly to 500 mL of 10 mg/mL aqueous polystyrene nanoparticle solution in a 1 L round-bottomed flask. The nanoparticle solution was sonicated using a bath sonicator for several minutes to disperse organic solvent droplets throughout the H 2 O and the solution was stirred rapidly for at least 1 h. Organic solvents were then removed under reduced pressure using a rotary evaporator. Once solvent was removed, the scintillant fluorophore doped polystyrene nanoparticle solution was stored at room temperature until use. 
     Silica shells were added to scintillant fluorophore doped polystyrene nanoparticles by dispersing 400 mL of 12.5 mg/mL polystyrene nanoparticle stock solution in 2000 mL isopropanol with 75 mL NH 4 OH. The dispersion was stirred briskly for several minutes while 20 mL TEOS was added dropwise. Stirring was continued for 1 h before nanoSCINT particles were collected by centrifugation and rinsed several times by dispersing particles in H 2 O and centrifuging the solution to collect rinsed particles. 
     Results: It was found that under some conditions, the silica shell was thinner (10-20 nm) under syntheses performed at maximum-scale (10× concentration and 10× volume) batches than in previous methods disclosed in commonly assigned U.S. Patent Application Publication No. 2018/0118916 (typically 20-50 nm, sometimes 50-100 nm), which is incorporated herein by reference in its entirety. 
     Transmission electron microscopy ( FIGS. 2 and 3 ) of the nanoSCINT particles  100  produced by the process of the present invention showed that the silica shell coverage of the polystyrene cores was complete and uniform. The decrease in shell thickness (see  FIG. 3 , where panel A is produced from conventional method and panel B is produced using the process disclosed herein) did not affect the function of nanoSCINT or nanoSPA. In fact, surprisingly and unexpectedly, decreased shell thickness proved to be advantageous in some situations. For example, decreasing shell thickness led to decrease in overall cost of materials. It also provided a means for controlling average nanoparticle density. 
     NanoSPA or nanoSCINT particles with thicker shells have a greater average density and can be used in assays where settling or collection of the nanoparticles by centrifugation of nanoSPA or nanoSCINT is beneficial. Shells can also be used as a support or attachment point for other sensor components, or to entrap components within the silica matrix. If the shell is porous, some components may reside within the pores while others, possibly different from those inside the pores, are attached to the exterior of the particle. 
     NanoSPA or nanoSCINT with thinner shells can stay suspended in solution for longer times without agitation and may be better suited in time-resolved assays, or for use with delicate samples. Thinner shells can also be advantageous in cases where nanoSCINT or nanoSPA are packed into columns, capillary tubing, or other enclosed spaces, where minimizing the distance between polymer cores is important. 
     The foregoing discussion of the invention has been presented for purposes of illustration and description. The foregoing is not intended to limit the invention to the form or forms disclosed herein. Although the description of the invention has included description of one or more embodiments and certain variations and modifications, other variations and modifications are within the scope of the invention, e.g., as may be within the skill and knowledge of those in the art, after understanding the present disclosure. It is intended to obtain rights which include alternative embodiments to the extent permitted, including alternate, interchangeable and/or equivalent structures, functions, ranges or steps to those claimed, whether or not such alternate, interchangeable and/or equivalent structures, functions, ranges or steps are disclosed herein, and without intending to publicly dedicate any patentable subject matter. All references cited herein are incorporated by reference in their entirety.