Patent Application
20090227044
1. Field of the Invention
This invention relates to the fields of chemistry and biology.
2. Description of the Related Art
Fluorescence is a widely used tool in chemistry and biological science. Fluorescent labeling of molecules is a standard technique in biology. The labels are often organic dyes that give rise to the usual problems of broad spectral features, short lifetime, photobleaching, and potential toxicity to cells. A further drawback of fluorescent dye technology is that the conjugation of dye molecules to biological molecules requires a chemistry that generally is unique to each pair of molecules. Alternative labels may be based on lanthanide-derived phosphors. The recent emerging technology of quantum dots has spawned a new era for the development of fluorescent labels using inorganic complexes or particles. These materials offer substantial advantages over organic dyes including larger Stokes shift, longer emission half-life, narrow emission peak and minimal photo-bleaching. However, quantum dot technology still is in its infancy, and is plagued by many problems including difficulties associated with reproducible manufacture, coating, and derivatization of quantum dot materials.
In addition, although the quantum yield of an individual quantum dot is high, the actual fluorescence intensity of each tiny dot is low. Grouping multiple quantum dots into larger particles is one approach for increasing the fluorescence intensity, but this nascent technology still suffers from drawbacks including difficulties in generating and maintaining uniform particle size distributions. Wider application of quantum dot technology therefore has been limited by the difficulties referred to above.
Alternative labels may be based on lanthanide-derived phosphors. Rare-earth metal elements such as europium are known for their unique optical (fluorescent/phosphorescent) properties. When their salts are dissolved in water, their fluorescence is quenched. Thus, many investigators have used europium and other rare-earth chelates to label biological molecules for the sensitive detection of proteins and nucleic acids, to carry out time-resolved fluorometric assays, and as labels in immunoassays. However, this chelation chemistry often is expensive and complex, and so application of rare-earth chelation technology also has been limited to date.
Recently, nanoparticles have received much attention in biology. These particles can have strong fluorescence that exhibits a spectrally sharp emission peak, large Stokes shift, and less quenching influence by other chemicals. Nanoparticles such as Eu2O3 particles also have been recognized as offering tremendous potential in obtaining large enhancement of emission intensity. However, Eu2O3 and other nanoparticles are easily dissolved by acid during activation and conjugation, thereby losing their desirable properties. In addition, nanoparticles lack reactive groups that allow them to be easily derivatized and linked to analytes and other reagents, thus increasing the difficulty associated with using nanoparticles as labeling reagents for the study of biological and other molecules.
Silica and alumina surfaces have wide-ranging surface reactivities; in particular, silica can be used as a cap to keep europium oxide from dissolving in acid in the conjugation process. However, coating with silica and alumina may increase the particle size, thereby compromising the advantageous properties of nanoparticles that render them suitable as labeling reagents.
Magnetic beads are another type of particle traditionally used in biochemical and clinical analysis for magnetic separation. Usually, they consist of a magnetic core covered by a polymer shell having a functionally modified surface. Particles having magnetic properties and light emitting properties provide additional benefits such as, e.g., permitting optimized biochemical protocols to be developed useful for both analyte detection and analyte separation or purification. U.S. Pat. No. 6,773,812 describes particles having magnetic and light emitting properties, but the light-emitting properties of those particles are derived from conventional dyes such as fluorescent dyes and so suffer from the associated disadvantages of photobleaching, small Stokes shifts, and short lifetimes.
A large variety of nanoparticles with different properties have been subject to intense research focused on their synthesis, characterization and application in biochemistry [1]. Fluorescent nanoparticles have been demonstrated as promising alternatives to widely used organic fluorescent dyes [2]. Quantum dots [3,4], dye-doped silica [5], chelate-doped polystyrene [6] and lanthanide oxides [7,8] each offer unique advantages and find different applications as fluorescent labels in biotechnology such as cell staining, molecular recognition, immunoassays [3, 9-13], visualization of DNA and protein microarrays [14, 15].
Core/shell structured magnetic nanoparticles are currently of interest in a wide variety of applications. For example, Fe/Au core/shell structured nanoparticles [16, 17], due to the possibility of remote magnetic manipulation [18], may be used in biological applications as magnetic resonance imaging (MRI) agents [19, 20], cell tagging and sorting [21] and targeted drug delivery [22] (for reviews see [23, 24]).
The combination of magnetic and fluorescent properties is a new powerful tool allowing manipulation by magnetic fields and visualization/detection by fluorescence. There are commercially available tools and automatic instruments for manipulating magnetic particles and for detecting a variety of fluorescent labels over the entire visible spectrum.
Recently, several groups reported the successful synthesis of particles that possess both fluorescent and magnetic properties. In most of these works, colloidal solution techniques were employed. Lu et al [25] and Levy et al [26] coated iron oxide particles with silica shells containing organic dyes which provided the fluorescent properties of the nanoparticles. Sahoo et al [27] employed covalent binding of organic dyes on the surface of magnetite particles. Using covalent binding, Wang et al [28] formed a fluorescent shell of quantum dots on polymer-coated iron oxide beads, while Mulvaney et al [29] incorporated organic dyes and quantum dots into the polystyrene shell of magnetic beads. Lu et al [30] formed a shell of up-converting phosphor (ytterbium and erbium co-doped sodium yttrium fluoride) on an iron oxide core that made use of the luminescent properties of the lanthanide ions. In most of the cases, the synthesis of particles with magnetic and fluorescent properties is complicated and expensive. For example, the beads of Wang et al [28] are expensive, toxic (due to the use of quantum dots), are less mechanically stable, have shorter lifetimes, and poorer multiplexing than the particle described in conjunction with the present invention. The need for up-converting as described in Lu et at [30] requires more than one excitation source. An additional drawback is that organic dyes have broad emission spectra and poor photostability. An efficient and low-cost method for synthesis of magnetic/fluorescent particles would be highly beneficial for applications that demand significant amounts of reagents and are economical—environmental monitoring for bioterror agents is a good example. A low-cost synthesis route would allow improvements in biotechnologies and facilitate the creation of new widely applicable biochemical protocols.
A great deal of effort has gone in the development of immunodiagnostics for detecting plant disease and pathogens. For example, nanotechnology has been used to provide rapid detection of single bacterial cells. Fluorophore-doped silica nanoparticles were chemically functionalized for the attachment of antibodies against E. coli. A solution of nanoparticles was mixed with a solution of bacteria, then centrifuged to separate bound and unbound antibodies; the fluorescence of the bound antibody fraction was measured. Zhao [31]. Although sensitive, this method is somewhat slow due to the centrifugation step. A number of other schemes, e.g., as demonstrated by Muhammad-Tahir [32], Lagally [33], Vo-Dinh [34], Dill [35], Sapsford [36], Taitt [37], and Weeks [38], have been advanced over the years to provide portable or disposable, miniaturized analytical systems for the detection of pathogens.
Despite these efforts, novel biosensors are still needed to identify multiple toxins and pathogens in individual samples within a complex background matrix. Increased sensitivity is needed to eliminate the need for enrichment of the pathogens using culture methods and to detect the low levels of toxin that can cause disease. Faster real-time assays will enable more samples to be analyzed and timely results obtained before products enter commerce, thereby ensuring statistically more reliable monitoring of the integrity of the food supply.
Foodborne illness, commonly referred to as food poisoning, results from consumption of food contaminated with various materials including pathogenic bacteria, toxins, viruses, prions or parasites. Most food-borne pathogens have animal reservoirs from which they can infect humans. However, recent outbreaks of a variety of pathogens have been associated with fresh produce or water. Beuchat [39]. Outbreaks on fresh produce may pose a more serious food safety problem since these items are not routinely cooked. Illness also can be caused by exotoxins that are excreted by the bacterium as they grow. Thus, exotoxins are capable of causing illness even when the microbes have been killed. Examples include toxins produced by Clostridium botuliiium, Clostridium perfringens, and Staphylococcus aureus. Recently, prions have been shown as a new form of food-borne pathogen with the discovery that Bovine Spongiform Encephalopathy can be transmitted to humans via contaminated meat, resulting in a variant form of Creutzfeldt-Jakob Disease, a 100% fatal neurological disease. Food borne infections cause millions of illnesses and thousands of deaths every year in the United States. Recent estimates suggest that there are 76 million illnesses caused by food-borne diseases resulting in 325,000 hospitalizations and 5,000 deaths in the United States. Mead [40].
Superimposed on these natural causes of food poisoning, we now face the daunting challenge of intentional adulteration of foods. Moon [41]. Along with the bacterial toxins mentioned above, potent plant-derived toxins are candidates for biological warfare and terrorism Two examples are ricin and abrin. Ricin is widely available, easily produced, and derived from the beans of the castor plant (e.g., Ricinus communis Mirarchi [42]), More toxic than ricin, abrin is a glycoprotein found in the precatory bean (Abrus precatorius). Budavari [43].
Identification of these agents is difficult and time-consuming. For example pathogen detection often involves cell culture and sophisticated chemical identification, along with the need for dedicated laboratories and large reference collections. Toxin detection may require a bioassay or polymerase chain reaction (PCR) method that can take 1-2 days. Arnon [44]. Alternatively, toxins can undergo complex extraction steps followed by some form of chromatography and mass spectrometry for identification. Wannemacher [45]. Identification of prions is even more difficult, necessitating an immunohistochemical and biochemical analysis of the sample. All of these approaches are time-consuming, requiring from days to weeks for a result. By the time such lengthy analyses have been completed, the product has moved into commerce. Thus, the needs of quality control technician in the field and the laboratory scientist for testing the integrity of our food supply are several-fold: (1) fast turn around; (2) multi analyte capability; (3) specificity to particular compounds without false positives; (4) high sensitivity.
In addition to semiconductor quantum dots, simple inorganic phosphors such as Eu2O3 have been recognized as offering potential in obtaining high emission intensity. Nogami [46]; Patra [47].
Europium containing nanoparticles have been used previously as labels for time-resolved bioassays. Harma et al. [48] entrapped chelated europium in polystyrene particles that had diameters ranging from 100 to about 400 nm. Carboxyl groups on the surface of the polymer particles were used in the conjugation to biomolecules. Although this scheme was employed successfully, we have devised several simpler methods for using europium oxide nanoparticles directly, without the need for chelation or entrapment in another medium. We found that natural or untreated Eu2O3 particles are insoluble in water but are easily dissolved by acid during activation and conjugation, losing their desirable optical properties. Coating the particles by silanization protected the particle from being dissolved by acid, and provided useful functional groups for biological conjugation. Feng [49]. Passive absorption of proteins is an alternative for surface functionalization that we have recently explored. Dosev [50].
The present invention addresses these and other limitations of the prior art by providing improved nanoparticle compositions and methods of use. Derivatized nanoparticle compositions of the present invention retain the optical properties of the native particles and enable the efficient and low-cost use of these nanoparticles to label and optionally separate or purify biological and other materials.
The present invention is defined by the following claims, and nothing in this section should be taken as a limitation on those claims. Disclosed herein is an assay comprising magnetic/luminescent nanoparticles. The luminescence of the particles serves as an internal calibration for the assay and helps to avoid experimental error from particle loss. Also disclosed is a microchannel in which the assay may be performed.
In one aspect, the present invention consists of using a single microchannel combined with external electromagnets for performing a fast immunoassay within a very small volume. Magnetic/luminescent nanoparticles provide an internal luminescent standard. According to another aspect of the present invention, a binding reaction is accelerated by applying an alternating magnetic field by alternatingly energizing a plurality of electromagnets external to a microchannel, thus inducing oscillation and/or agitation of the particles and achieving better diffusion during incubation. Using the electromagnets, the particles are held in the channel for washing and luminescence detection steps. The luminescence of the particles serves as an internal calibration for the assay and helps to avoid experimental error from particle loss.
Also described herein is the synthesis and the properties of magnetic/luminescent core/shell particles useful in conjunction with the above-described methods, including magnetic cores of iron oxide doped with cobalt and neodymium (Nd:Co:Fe2O3) that are encapsulated in luminescent shells of europium-doped gadolinium oxide (Eu:Gd2O3). Cobalt [51] and neodymium [52] were shown to improve the magnetic properties of iron oxides. In addition, doping of Eu ions into the Gd2O3 matrix gives unique luminescent properties [53, 54]. The methods described herein employ flame spray pyrolysis as a cost-effective, high throughput and versatile synthesis method, allowing a variety of doped materials to be obtained, such as described in U.S. patent application Ser. Nos. 10/393,702 and 10/576,776, each of which is hereby incorporated by reference hereto for all purposes.
Various particle types that may be used in conjunction with the inventive methods described herein are detailed. In one aspect of the invention a silica glass nanoparticle is co-doped with a rare earth element and another metal element. In another aspect, the invention uses nanoparticles having a magnetic oxide core and a shell comprising a rare earth element and optionally another metal element. In one aspect, the particles useful in conjunction with the methods of the present invention are prepared using gas-phase combustion and/or pyrolysis synthesis. These types of particles provide the additional advantage of absorbing and emitting light at multiple wavelengths further expanding the use of these particles as labels in, e.g., multiplexed applications.
These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, and accompanying drawings, where:
a-2i are schematic illustrations depicting one embodiment of an assay as described herein performed in a microchannel.
a shows an Eu2O3 excitation spectrum monitored at 612 nm.
b shows an Eu2O3 emission spectrum excited at 466 nm (full line bare particles without functionalization; dashed line with silence functionalization).
a-7c illustrate magnetic separation of magnetic particles.
a is a transmission electron micrograph (TEM) of pure Eu2O3 nanoparticles.
b shows fluorescence emission spectra for pure Eu2O3 nanoparticles (monoclinic phase) excited at 466 nm showing short fluorescence lifetime.
a is a TEM of Eu:Y2O3 nanoparticles.
b shows fluorescence emission spectra for pure Eu:Y203 nanoparticles excited at 260 nm showing fluorescence lifetime on order of 2 msec.
a illustrates a comparison of magnetic characteristics of Co:Nd:Fe2O3 powder with Co:Nd:Fe2O3/Eu:Gd2O3 core/shell particles and Eu:Gd2O3 particles.
b illustrates an emission spectrum of Co:Nd:Fe2O3/Eu:Gd2O3 core/shell particles under excitation at 260 nm.
a shows an X-ray diffraction (XRD) spectrum of the primary Nd:Co:Fe2O3 particles compared to the typical XRD peaks of Fe3O4.
b shows an XRD of the core/shell Nd:Co:Fe2O3/Eu:Gd2O3 particles
c shows typical XRD spectral peaks of Fe2O3.
d shows the typical XRD spectral peaks of monoclinic Gd2O3.
a illustrates an excitation spectrum of IgG-488 (primary) excited at 480 nm.
b illustrates an excitation spectrum of IgG-635 (secondary) excited at 620 nm.
c illustrates an excitation spectrum of the labels of
The figures depict various embodiments of the present invention for purposes of illustration only. One skilled in the art will readily recognize from the following description that alternative embodiments of the structures and methods illustrated herein may be employed without departing from the principles of the invention described herein.
Briefly, and as described in more detail below, described herein are methods, and apparatus for performing an assay with internal luminescent calibration.
Referring to
Referring now to
An important advantage of the proposed assay on the particle surface is that it permits any type of fluorophore to be used as an analyte label. The intensity of the secondary label can be tuned by varying the amount of used core/shell particles, and therefore the amount of surface binding sites. This way, more particles (larger surface) can be used in order to obtain higher intensity of the secondary label which will not change the quantitative (ratiometric) measurement but will increase the sensitivity. In addition, by using a large number of particles in the assay error from particle size non-uniformity is minimized because the size distribution of the particles does not change and hence the total particle surface area per unit mass of particles is constant. The sharp emission spectrum and long lifetime of Eu3+ ions (about 1 ms) allows us to use any other fluorophores as secondary labels such as quantum dots or polystyrene beads. Even if their spectra overlap with that of the Eu3+ ion, the signal from the long-lifetime Eu3+ ions can be resolved with time-gated detection.
The advantages provided by the synthesis method described herein useful in conjunction with the methods of the present invention include: a simple and low-cost single-step process is used to produce nanoparticles that are more uniform and less prone to aggregation than those produced using prior art methods such as ball milling or solution phase syntheses; the functionalization methods disclosed also are simple and low-cost and result in high quality nanoparticles and largely avoid agglomeration problems such as encountered with similar procedures that take place in the liquid phase, and greatly reduces or eliminates the need for post-functionalization washing of the nanoparticles; and because the spectral properties of the nanoparticles of the present invention do not depend on the particle diameter, the size distribution of a population of the particles need not be monodisperse. Nanoparticles, as used herein in conjunction with the present invention, are particles that are less than 1 micron in diameter, preferably less than 500 nm, and more preferably 100-200 nm.
According to some embodiments, Europium oxide (Eu2O3) particles are used. These particles have a useful excitation region from about 250-410 nm with a maximum at 260 nm as seen in
Nanoparticles of simple lanthanide oxides can offer all the advantages of lanthanide chelates without the complex synthesis and the somewhat uncertain composition, the latter issue leading to uncertainties in the conjugation chemistry. Due to their chemical inertness, and the fact that the lanthanide ion is sequestered in a crystal lattice, they are not susceptible to photo-bleaching or oxygen quenching. The synthesis method disclosed herein allows production of a range of lanthanide oxides that span the useful optical spectrum. The lanthanide family offers multi-wavelength labels for multiplexed assays with high throughput, using a number of the lanthanides (Eu-red, Tb-green and Dy-blue, Sm-red) doped into suitable host materials such as Y2O3.
The advantage offered by simple, single material nanoparticles can be extended by multi-functional materials that are engineered on the nanoscale. The multiple properties may include optical, magnetic, and electrical characteristics that can be usefully employed in an assay. A magnetic moment can allow a particle to be separated from its matrix prior to measurement.
The use of magnetic beads is not new, nor is the use of lanthanide labels for immunoassays in food monitoring. The implementation of magnetic bead separation with fluoroimmunoassays for pathogen detection is being pursued, for example, at the USDA Eastern Region Research Center. Tu et al. [55] recently reported a clever scheme to use Eu and Sm (samarium, another lanthanide) for the detection of E. coli 0157 and Salmonella on alfalfa seeds with an immunoassay. Magnetic beads were used in a sandwich assay in which the beads were separated with a magnetic concentrator. Antibodies that were labeled with Eu and Sm were incubated to bind with the E. coli and Salmonella. A chelation step was then necessary to remove the lanthanide ions from the antibodies prior to detection. The authors made use of the long phosphorescence lifetime of the lanthanide chelate to carry out gated detection of the emission. Sensitivity and specificity were good but the procedure was complicated and impeded by the requirement for multiple steps, especially the chelation step.
The present invention combines magnetic and fluorescence properties in a nanoparticle label. One approach is to use polymer beads with a magnetic core and doped with a fluorophore. Mulvaney [56]; Wang [57]. Drawbacks to this approach are that the beads are large (0.8 μm), subject to photobleaching with organic dye-doped beads, and are expensive. Our proposed method of production combines magnetic and fluorescent properties into one nanoparticle in a controlled manner. They can provide low cost reagents with the ability to perform multiplexed assays.
Few others have succeeded in applying fluorescent nanoparticle formats in a quantitative manner. Feng [49]. Generally, quantum dots and similar materials have been used primarily for qualitative labeling and staining in microscopy. Qhobosheane [58]. However, the use of simple Eu2O3 nanoparticles in a fluoroimmunoassay for atrazine is described herein. Feng [49]. The results are shown in
Small equipment size permits parallel processing of samples with a range of bioassays that are designed to detect a variety of food toxins and pathogens. Miniaturized systems permit faster analyses than are otherwise possible, with less time-consuming human involvement in the various steps. For example, the rate of diffusion of analytes to surfaces scales with the dimensions of the system to the second power. Hence, diffusion times, that may limit rates of binding of Ag (antigen) to Ab (antibody) in some cases, are reduced by a factor of 100 if the length scale for diffusion is reduced by a factor of 10.
While miniaturization offers considerable benefits in terms of size, portability, cost, automation and throughput, further advances in detection technology are afforded by recent developments in nanotechnology. The engineering of materials on the nanoscale provides new properties and functionalities that were not possible in the past. The combination of nanotechnology with microfabricated miniaturized analysis systems opens up new vistas for securing our nation's food supply.
Thus, the inventive materials and methods described herein system provide the following advantages: (1) the working volumes and the amounts of consumed reagents are reduced by several orders of magnitude; (2) the time for magnetic separation is significantly reduced because the solution will be situated very close (a few micrometers) to the magnet surface, where the magnetic field is strongest and the magnetic particles will only need to move a very short distance in order to be separated from the solution; (3) multiple parallel microchannels may be fabricated on a single substrate and controlled simultaneously by one electromagnet. This allows many parallel assays to be performed at the same time; (4) poly(dimethylsiloxane) (PDMS)-based channels can be made cheaply enough to be disposable; (5) the use of an electromagnet to switch electrically between retention (separation) and elution regimes of the nanoparticles in a continuous flow eliminates the need for stop-flow and waiting periods and decreases the reaction time since diffusion no longer is a rate limiting step for binding; (6) the excitation/detection of the fluorescent signal can be performed through the channel wall as the particles are attracted by the magnet. Excitation by an evanescent wave on the top of a waveguide structure can be used to restrict excitation to only the nanoparticles that are closest to the wall. In another embodiment “off-chip” detection in a plate reader after the elution of particles can be used to provide fully automated devices for precise and high-throughput detection.
Nanoparticles have found tremendous applications in the field of biotechnology. Their unique size-dependent properties make these materials superior and indispensable in many areas of human activity. Out of many size-dependent properties available optical and magnetic effects are the most used for biological applications. [59, 60] Due to their controllable size ranging from a few nanometers up to tens of nanometers, comparable to most of the cell parts and even close to the size of proteins, nanoparticles can be used to probe the cell machinery without introducing too much interference. Applications of nanomaterials to biology or medicine includes fluorescent biological labels [61-63], Drug and gene delivery [64], Bio detection of pathogens [65], Detection of proteins [66], Probing of DNA structure [67], Tissue Engineering [68], Tissue destruction via heating [69], Separation and purification of biological molecules and cells [70], MRI contrast enhancement [71] and phagokinetic studies.
A nanoparticle in itself is rarely sufficient for the above-outlined uses. Usually, a nanoparticle must be coated with a layer of material that functions as a bioinorganic interface. The coating may include antibodies, biopolymers like collagen or monolayers of small molecules which make the nanoparticles biocompatible. In addition the particles should be either fluoresce or magnetic for detection purposes. Nanoparticles can also be fluorescent-dye coated for optical detection. A nanoparticle usually forms the core of nano-biomaterial. It can be used as a convenient surface for molecular assembly and may be composed of inorganic or polymeric materials. The core itself might have several layers and be multifunctional. For example, combining magnetic and luminescent layers one can both detect and manipulate the particles.
The core particle is often protected by several monolayers of inert material. Organic materials that are adsorbed or chemisorbed on the surface of the particle can serve the same purpose and also act as a biocompatible material. However an additional layer of linker molecules is required for further functionalization. This linker molecule has reactive groups at both ends. One group is aimed at attaching the linker to the particle surface and other is used to bind various molecules like biocompatibles, antibodies, fluorophores etc. depending on the function required by the application.
Magnetic nanoparticles offer some attractive possibilities in the field of biosciences as they can be manipulated by an external magnetic field gradient. [72] External manipulation and ability of magnetic fields to penetrate most of the bio-surfaces leads to many applications involving the transport and/or immobilization of magnetic nanoparticles or magnetically tagged nanoparticles. They can be used to deliver drugs to a targeted region [73]. The magnetic particles can be made to resonantly respond to a time varying magnetic field consequently transferring energy from the exciting field to the particle itself, so they can be used as hypothermia agents for targeted bodies such as tumors [69]. Also because of there easy maneuverability, small size and direct detection, magnetic nanoparticles have found use as labels in new generation of immunoassays [74]. Magnetically labeled targets are detected directly with a magnetometer [75, 76], with a microscope based on a high transition temperature dc superconducting quantum interference device (SQUID) for rapid detection of superparamagnetic nanoparticles [77, 78]. In this technique a suspension of magnetic particles carrying antibodies is added to targets and the mixture is placed on the microscope. Magnetic field pulses are applied parallel to the SQUID which causes the nanoparticles to develop a net magnetization. This magnetization relaxes when the field is turned off. Unbound nanoparticles relax rapidly by Brownian rotation and contribute no measurable signal. Nanoparticles bound to the target are captured and undergo Nëel relaxation, producing a slowly decaying magnetic flux, which is detected by SQUID.
Recent technology research in the field immunoassays using magnetic-nanoparticles is toward accelerating the process [79], miniaturization of the system and alternative detection capability using magnetic microbeads loaded with fluorophores and quantum dots [80]. The present invention provides a novel assay inside a micro channel using magnetic nanoparticles coated with fluorophores as an assay surface and analyte detection using fluorescent-labeled antibodies. In most conventional immunoassays, observation and final quantification is based on absolute value of a signal that is proportional to the amount of captured analyte. Consequently, the number of incubation and washing steps used for the immunoassay can affect the final values, as a percentage of both analyte and detecting body will be lost during these procedures. Luminescent magnetic particles of the present invention (including fluorescent nanoparticles and particles conjugated to fluorescent dye) and coated with, e.g., an antibody provide an assay surface for, e.g., a sandwich format assay. The signal associated with the particle introduces an internal standard that can be used to improve assay result accuracy by normalizing the signals derived from the particle and the analyte for magnetic nanoparticles of known size range and with controlled coating procedures the number of antibodies attached on the surface can be characterized and used as a base for comparison.
In general, the nanoparticle compositions used in conjunction with the present invention comprise a metal oxide particle having a desirable optical property that has been coated with a functionalizing reagent. The functionalizing reagent used may comprise a silane as disclosed in co-owned pending U.S. Patent Publication 2003/0180780, incorporated herein by reference for all purposes, or comprise a protein or peptide such as, e.g., BSA or an immunoglobulin, or may be a polyionic polymer, such as, e.g., (poly-L-lysine hydrobromide, PL).
Preferred particle diameters are in the range of between about 10 and 1000 nm, more preferably between about 10 and 200 nm and even more preferably between about 10 and 100 nm, or between about 20 and 50 nm. In preferred embodiments, the metal oxide particles have the generic formula MexOy, wherein 1≦x≦2, and 1≦y≦3, and wherein preferably, Me is a rare earth element selected from the lanthanide series and includes, but is not limited to, europium (Eu), cerium (Ce), neodymium (Nd), samarium (Sm), terbium (Th), dysprosium (Dy), gadolinium (Gd), holmium (Ho), thulium (Tm), or Me may be chromium (Cr), yttrium (Y), iron (Fe). Other suitable metal oxide particles include silicon oxide (SiO2), and aluminum oxide (Al2O3) mixed with Eu2O3. or Eu3+.
In other preferred embodiments, the metal oxide particle comprises a doped metal oxide particle by which is meant a metal oxide, and a dopant comprised of one or more rare earth elements. Suitable metal oxides include, but are not limited to, yttrium oxide (Y2O3), zirconium oxide (ZrO2), zinc oxide (ZnO), copper oxide (CuO or Cu2O), gadolinium oxide (Gd2O3), praseodymium oxide (Pr2O3), lanthanum oxide (La2O3), and alloys thereof. The rare earth element comprises an element selected from the lanthanide series and includes, but is not limited to, europium (Eu), cerium (Ce), neodymium (Nd), samarium (Sm), terbium (Tb), gadolinium (Gd), holmium (Ho), thulium (Tm), an oxide thereof, and a combination thereof. Nanoparticles of such oxides may be manufactured according to the methods of the present invention, purchased from commercial suppliers, or fabricated using methods known to those of ordinary skill in the art.
The desirable optical properties of the compositions of the present invention include optical properties that allow the compositions to be useful as labeling agents, such as, e.g., fluorescence, fluorescence resonance energy transfer (“FRET”), and phosphorescence. Thus, the compositions of the present invention may be used by one of skill in the art in the same manner as fluorescent dyes, FRET pairs and other labeling reagents, but with the advantages that nanoparticles bring to labeling technology in terms of larger Stokes shift, longer emission half-life (for lanthanide-containing nanoparticles), diminished emission bandwidth, and less photobleaching as compared with, e.g., traditional fluorescent dyes.
Surface modification and other methods may be used in the practice of the invention for surface modification (i.e., functionalization) and conjugation of the nanoparticles of the invention. In one embodiment, surface modification and conjugation comprises direct coating of the nanoparticles with a protein such as, e.g., BSA, ovalbumin or immunoglobulin. In another embodiment, surface modification is accomplished by physical adsorption and functionalizing with a polyionic polymer such as, e.g., poly-L-lysine hydrobromide, PL.
Using appropriate buffer conditions (pH and concentration), a variety of proteins can be adsorbed spontaneously on the surface of the nanoparticles without affecting their fluorescence properties. The protein coated particles are purified by 3 rounds of centrifugation and are stable for more than 1 month in buffer solution. Adsorption of bovine serum albumin (BSA) provides multiple functional groups (amine, carboxylic) for covalent conjugation to other biomolecules using standard cross-linking procedures. If BSA-biotin is used as a coating protein, biotinylated particles are produced for a variety of applications in bioassays. If the particles are coated with BSA-hapten (small molecule), such as the coating antigens commonly used in ELISA, the modified particles may be used as fluorescent competitors in immunoassays. The nanoparticles are efficiently coated with immunoglobulin molecules, preserving the functionality of the nanoparticles and the functionality and activity of the immunoglobulins. The number of binding sites (biotin, hapten, antibody) may be controlled during the coating procedure by mixing a specific protein (i.e., the protein providing the binding site) and a non-specific blocking protein (i.e., one that does not provide a binding site) in different ratios. Blocking proteins are well-known to those in the biochemical arts and include, e.g., BSA, casein, milk proteins, and other agents useful for blocking non-specific binding in biochemical reactions such as, e.g., ligand binding assays, Western blots, ELISAs, etc. Examples of pairs of specific proteins and non-specific blocking proteins include, e.g. BSA-biotin:BSA, specific anti-rabbit IgG:non-specific sheep IgG. The blocking protein prevents possible non-specific binding of the nanoparticles to other proteins and/or surfaces during the performance of bioassays improving in this way the signal/noise ratio.
PL is a polycationic polymer that adsorbs spontaneously from aqueous solutions onto the negatively charged metal oxide surfaces via electrostatic interactions. The excess of PL is washed off by centrifugation. The formed layer of PL is stable under the most commonly used buffers. The introduced amino groups on the surface of the particles permit their conjugation to a variety of small molecules (haptens) and biomolecules with appropriate functionalizations.
The immunoassay technique is the fastest growing analytical technology for the detection and quantification of biomolecules. It takes advantage of affinity binding between antibodies and the corresponding antigens that allows the detection of one of these, even if it is present at very low concentrations and in complex biological matrixes such as whole blood, serum and other biological fluids. The measurement of binding reaction can be performed by monitoring changes in the different physical phenomenon associated with the biomolecules and the labels used, as well as the configuration of the assay.
The antibody-antigen specific reaction, which is the basis of all immunological techniques, can be characterized by its structure, its strength, also known as affinity, and its stability, also known as avidity. The detection and quantification of the antibody-antigen interaction can be achieved using a variety of different labels, present either in the antigen or antibody. Different techniques can be employed in the design and development of immunoassays. Radio labeled immunoassays use reagents incorporating radioisotopes as tracers to monitor the distribution of free and bound antigen in radioimmunoassay or free and bound antibody in immunoradiometric assays. Fluoroimmunoassays involve in situ detection with antibodies linked to the fluorescent label by using external laser excitation and then measuring the fluorescence signal or by taking optical micrographs. Enzyme immunoassays involve the use of enzyme activity as a means of detecting the binding of an antibody/enzyme conjugate. Enzymes are specific in both reaction they catalyze and the substrate they recognize and are subject to the regulation of their activity by other molecules. Enzyme immunoassays are similar to immunofluoroscence assays. Only difference is in type of detection, which is spectrophotometric when label produces colored product or electrochemical when enzyme catalyzes a redox reaction.
Several other techniques and configurations are used in immunoassay design such as chemiluminescence, which describes the emission of light that occurs as a result of certain chemical reactions producing high amount of energy which is lost in the form of photons when electronically excited product molecules relax to their stable ground state, light-scattering, based on the reaction between an antigen and an antibody to produce an aggregate large enough to scatter light to a greater degree than do the constituents of the reaction, electrochemistry, based on the measurement of the redox potential, by measuring either a current or potential of a reaction, disposable tests that are often membrane based assays which provide visual results and can be designed as rapid tests.
There are three basic configurations of an immuno-reaction, with either antibodies or antigens present in the sample to be analyzed. In the sandwich configurations of an immuno-reaction, the capture antibody is immobilized onto the surface and binds to the antigen present in the sample. A second antibody labeled with a fluorophore binds to a different epitope of the antigen, leading to a fluorescent signal that is proportional to the amount of antigen present in the sample. In some embodiments of the present invention a sandwich configuration immunoassay is used. The use of different labels for the capture bead and secondary antibody provides the ability to normalize analyte signal to capture bead signal, thereby improving the accuracy of the assay result.
The movement of micron sized magnetic particles in a non-flowing aqueous solution can be classified as quasi-static motion (no inertial effects), because of high viscous forces acting on them. See
F
m=(VΔχ/2μo)grad(B2) (1)
where Δχ is the volumetric susceptibility difference between particle and solution, V is the particle volume and μo is the magnetic permeability of free space. Also from vector calculus for static irrotational field grad (B2)=2B grad (B). The drag force on the particle in a fluid is given by Stokes equation
Fd=3πηDpυy (2)
where η is the fluid velocity, Dp is the particle diameter and υy is the component of particle velocity in the direction of magnetic field application. The force balance on a particle yields
(mass)*(acceleration)=Fm−Fd
Using equations (1), (2) & (3) we get the following differential equation in terms of particle position
d2y/dt2+[(π2dp4η)/(2ρ)]dy/dt−[(Δχπ2Dp6)/(36μoρ)]Bgrad(B) (4)
Referring to
Two NPN transistors (NTE263), a NAND logic gate (74HCT04N, Philips) were used in circuitry to alternatively turn on the two magnets. Signal for switching was generated using 15 MHz arbitrary waveform generator (Agilent 33120A) which was also used to specify the frequency at which the electromagnets switch. Laboratory DC power supply (GPS-3030D, GW) was used to power the NAND logic gate and the electromagnets.
Ohmicron magnetic rack (Strategic Diagnostics, Newark, Del.), such as shown in
A high aspect ratio (ratio of channel length to channel depth) microchannel was used to get the closest possible approximation of a perfect laminar flow. The channel was fabricated using two glass slides (75×50×0.9 mm, Corning Glass Works) with epoxy as spacer, which also defined the walls of the channel. Inlet and outlets ports were drilled in the glass for external fluid connections. The channel was rectangular in shape with dimensions 60 mm/10 mm/110 μm (length/width/depth) with approx. volume of 40 μl.
The magnetic particles used in one example comprise 2.52% solids-latex (by wt) of polystyrene Superparamagnetic microspheres, (1-2 μm, Polysciences). The antibodies used were Alexa Fluor-488 anti-mouse IgG, whole molecule developed in goat (Molecular Probes) and Alexa Fluor-635 anti-mouse IgG, whole molecule developed in goat (Molecular Probes), 1×PBS (0.1M PBS diluted 1:10 with deionized water (18 mΩcm), PBS: 90 g/L NaCl, 10.9 g/L Na2HPO4, 3.2 g/L NaH2PO4 in deionized water), Borate buffer (12.4 g/L Boric acid, 19.1 g/l Sodium Tetraborate in distilled water, pH adjusted to 8.5 using NaOH). The antigen used was Mouse IgG (SigmaAldrich, St Louis, Mo.).
A channel is formed by a glass substrate and a poly(dimethylsiloxane) (PDMS) layer bonded to the top of the glass The PDMS—based microfluidic devices is fabricated by placing a PDMS template in contact with the glass surface and applying pressure to create a fluid-tight and air-tight seal in the manner of Chabinyc et al. [82]. The reagents for the assay are introduced into the microchannel with a flow rate controlled by automatic syringe pump.
Functionalized magnetic nanoparticles interacted with the sample within a reservoir built on the same substrate as a part of the microfluidic system. Following the binding reactions in the reservoir, the magnetic nanoparticle substrates were separated from the rest of the sample by magnetic retention at the channel wall. Flow rates were optimized in such a way that long enough time will be given for binding reactions to occur in the reservoir and magnetic particles to be separated in the channel. This can be achieved by varying the ratios between the volumes of the reservoir and the channel. For given flow rate, increasing the reservoir volume will give longer times for the binding to occur.
Magnetic separation assays use magnetic beads to facilitate the separation of bound labeled molecules from the free molecules in the solution. Taking advantage of the unique combination of magnetic and optical properties of our nanoparticles, a sandwich immunoassay for toxins (proteins) was performed on the surface of magnetic Eu nanoparticles coated with a capture antibody, as shown in
In one aspect of the present invention, the fluoroimmunoassay based on magnetic Fe2O3/Eu:Y2O3 nanoparticles was tested in a sandwich format in buffer solution for the detection of model proteins (IgG, BSA or botulinum toxoid) using secondary antibodies labeled initially with organic dyes (e.g. Alexa Fluor or cyanine dyes). To achieve a highly sensitive assay, the parameters of the immunoassay performance were optimized for the magnetic separation and the fluorescence detection of the internal standard (the Eu magnetic nanoparticle) and the second fluorescent label. The conditions for efficient magnetic extraction, such as the amount of magnetic nanoparticles used and time of separation, were determined. In addition, the amount of antibody immobilized on the surface of the magnetic nanoparticles, the optimal concentration of the secondary antibody, and the kinetics of the immunoassay were crucial in determining the dynamic range and the detectability of the assay. The detection parameters were optimized to achieve high signal/noise ratio using the least concentration of immunoreagents. The analytical performance of the magnetic based immunoassay, such as precision, accuracy and reproducibility, was be evaluated with these conventional fluorophores labels.
Several detection schemes were evaluated. The organic dyes have short lifetimes compared to the magnetic Eu nanoparticle substrate. Using time-gated detection we make use of the difference in lifetime of emission from the highly efficient organic dye labels compared to the long lifetime lanthanide to detect the potentially weaker emission from the Eu substrate. In some embodiments, lanthanides or quantum dots are used as secondary labels. The long lifetimes of lanthanides allow time-gating of detection and thereby the ability to discriminate against strong background fluorescence that may be encountered in measurements in foods. Among the lanthanides terbium (Th-green), dysprosium (Dy-blue) and samarium (Sm-red) nanoparticles were explored as secondary antibody labels. In some aspects, quantum dots are used to probe up to six or eight different analytes on a single Eu substrate particle.
Depending on the fluorophores used, a large difference may be present between the intensities of the detected fluorophores. The intensity of the secondary label should preferably be comparable to the intensity of the Fe2O3/Eu:Y2O3 nanoparticles. Possible differences in intensities can be overcome by controlling the binding sites per Fe2O3/Eu:Y2O3 particle and by optimizing the total number of particles used. If the intensity of the secondary label is too low compared to Fe2O3/Eu:Y2O3 nanoparticles, the number of binding sites per particle are increased and the amount of particles used are reduced, or vice versa. Finally, the difference in lifetimes between fluorophores or quantum dot and the lanthanide substrate nanoparticle can be used to optimize the sensitivity of the assay.
Reducing non-specific binding and minimizing nanoparticle agglutination are also crucial steps in using nanomaterials as labels in sensitive and reproducible bioanalysis. There is no universal solution to this difficult problem. The critical parameters are the charge, size and surface functionality of the nanoparticles and the buffer (sample). The surfaces of the nanoparticles were efficiently blocked with proteins to avoid non-specific binding in immunoassays, as has been demonstrated with earlier microprinting experiments. Dosev [50].
The demonstration of nanotechnology with conventional laboratory instrumentation such as micro plate readers was the first step towards the development of a useful, portable, and automated measurement system. Miniaturization was the next logical step. Once the proposed assays based on magnetic fluorescent nanoparticles were optimized, they were downscaled to nanoliter sample volumes. Magnetic bead immunoassays have been performed in volumes of several μL using a capillary tube [31]. Therein, Zhao used commercially available magnetic beads that were not fluorescent and measured the fluorescent signal from the supernatant. This indirect detection approach suffers from lack of precision and poor reproducibility.
Table 1 provides a non-limiting listing of the reagents, abbreviations for the reagents, formulae, suppliers, form of usage of the reagent in the described syntheses and examples of alternative reagents useful for producing the particles and for practicing the methods of the invention. The listing is intended to be exemplary and to provide guidance to an ordinarily skilled artisan as to other materials useful for practice of the invention. Those materials are readily ascertained by the ordinarily skilled artisan provided with the teachings of this specification.
Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.
The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., T. E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A. L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3rd Ed. (Plenum Press) Vols A and B (1992).
Methods
The syntheses of the particles useful in conjunction with the methods of the invention, as described herein, have been conducted in a manner that involves a flame as the reaction zone, utilizing an apparatus illustrated in
Based on the different forms of usage of the starting materials, the syntheses can be divided into two classes, gas-phase synthesis in which all the starting materials are fed into the flame in the vapor phase, and, spray-pyrolysis synthesis in which one or more of the starting materials is fed into the flame in the form of droplets containing the starting material, or solid particles derived from the droplets. The functionalization methods of the present invention may be practiced with nanoparticles synthesized using the disclosed gas-phase combustion and/or pyrolysis synthesis method disclosed herein, or with nanoparticles produced using other manufacturing techniques.
50 mg Eu(TMHD)3 was placed in furnace A shown in
An ethanol solution containing 1 mM Eu(NO3)3 and 30 mM Y(NO3)3 was pumped with a syringe pump (Cole-Parmer, Vernon Hills, Ill.) at 7 mL/h into the inner nozzle of the nebulizer illustrated in
In an alternate method, the spray generated by the nebulizer can be introduced into furnace A, along with 2 standard Liter/min H2. The spray then is preheated in furnace A to remove the solvent from the droplets, to form an aerosol containing dry particles. This aerosol can be ignited at the outlet of furnace A to form a diffusion flame, in which the synthesis reactions take place. Post-synthesis treatment of the nanoparticles produced by the spray-pyrolysis synthesis is optional with furnace B. Post-synthesis treatment helps to remove impurities and improve the crystallographic properties of the nanoparticles formed in the flame. In addition to ethanol, other solvents useful for spray pyrolysis include aqueous ethanol, water, acetone or other lower alcohols, ketones, or any other solvent in which the reagents are stable for the time necessary to carry out the synthesis, and that have a density and molecular weight appropriate to allow atomization of the reagents.
Magnetic particles of Nd:Co:Fe2O3 mixed oxide were obtained by a spray pyrolysis method, previously reported for synthesis of Eu:Y2O3 nanoparticles [83]. Briefly, an ethanol solution containing Fe(NO3)3, Co(NO3)2 and Nd(NO3)3 was sprayed into a hydrogen diffusion flame through a nebulizer. The flame was formed by an H2 flow at 2 l min-1 and an air co-flow at 10 l min-1, surrounding the outlet of the nebulizer. A flame temperature of about 2000° C. was measured. Pyrolysis of the precursor solution within the flame yielded Nd:Co:Fe2O3 nanoparticles. A cold finger was used for collecting the particles thermophoretically. The production rate of this synthesis procedure was about 400-500 mg h-1. The ratio between Fe/Co/Nd salts was optimized experimentally to achieve the best magnetic characteristics.
The Co:Nd:Fe2O3 powder with a Fe:Co:Nd ratio of 80:20:5 was used for the synthesis of core/shell particles, building the luminescent Eu:Gd2O3 shell via a second spray pyrolysis process. 10 mg of Co:Nd:Fe2O3 nanoparticles were dispersed in 50 ml ethanol containing 201mM Eu(NO3)3 and 80 mM Gd(NO3)3. The solution was subjected to an ultrasonic bath for 30 min in order to break any weak agglomerates. Afterwards it was sprayed through the hydrogen flame, as shown in
Analysis with a transmission electron microscope (TEM) revealed that most of the synthesized particles were in the size range between 200 nm and 1 μm depending on the number and the size of the embedded primary Co:Nd:Fe2O3 particles. A representative TEM image of a core/shell particle can be seen in
b shows the luminescent emission spectrum of the synthesized core/shell particles which is typical for the shell material Eu:Gd2O3. Under UV excitation at 260 nm, the particles with Eu:Gd2O3 shell emitted red luminescence with a narrow peak centered at 615 nm that was identical to the spectrum recently reported by the inventors for Eu:Gd2O3 nanoparticles [15]. The compatibility of Gd2O3 with the proposed synthesis process introduces the possibility for a variety of luminescent spectra to be achieved by using different lanthanides such as Th, Sm or Dy for doping [54]. X-ray diffraction studies have been performed on the primary obtained Nd:Co:Fe2O3 powder and the final core/shell particles in order to clarify the origin of the observed changes in the magnetic properties. The XRD spectrum of the primary magnetic particles reveal the presence of FeO4 iron oxide along with other phases, as shown in
The final core/shell particles were successfully separated from an aqueous solution by a commercially available permanent magnet for biochemical purposes, as shown in
Functionalization is carried out using the apparatus illustrated in
The reaction chamber contains two inlets and one outlet. Nanoparticles are collected with a probe located 2-5 cm from the burner illustrated in
The magnetic luminescent nanoparticles were coated with antirabbit IgG via spontaneous physical adsorption according to a previously reported procedure for the biofunctionalization of Eu:Gd2O3 nanoparticles [7]. Briefly, a suspension of the nanoparticles in 25 mM phosphate buffer, pH=7, was incubated overnight in the antibody solution in a rotating mill at room temperature. The particles were extracted from the solution on a magnetic rack and washed three times. After that their surface was blocked with BSA to ensure that no bare particle surface remained.
The concentrations of the coating antibody and labeled antigen were optimized in a titration experiment where 1 mg of particles, coated with anti-rabbit IgG in the concentration range of 10-500 μg mg-1 particles, were incubated for 1 h with different concentrations of rabbit IgG-Alexa Fluor 350. Negative controls were performed with magnetic nanoparticles coated with sheep IgG. After magnetic extraction, the nanoparticles were resuspended in 100 μl of PBS and the fluorescence of the resulting complex was measured on a Spectramax M2 microplate reader (Molecular Devices, Sunnyvale, Calif.). Both Eu:Gd2O3 and Alexa Fluor 350 were excited at 350 nm and their emission spectra were detected in the interval 430-670 nm.
A typical titration curve representing the saturation of the immobilized antibody by the labeled antigen is presented in
Tech data sheet #238E, Polysciences, Inc and Technote #204, Bangs Laboratories, Inc were used as protocols for direct physical adsorption of the antibodies on the magnetic particle surface. For IgG monolayer on the 1-2 μm diameter magnetic particles approximately 9.5 mg IgG/g beads are required [85]. It is recommended to use 3-10 times the amount of protein required for monolayer. Eight times the monolayer requirement (76 mg IgG/g beads) were used in this example. 50 μl of 2.52% polystyrene Superparamagnetic microspheres (1.26 mg particles) were separated from the solution using a magnetic rack and washed once with 1×PBS. Particles were mixed with required amount of antibodies (76 mg IgG/g beads≈48 μl of 2 mg/ml anti-mouse IgG-488 solution) and 3 ml of Borate buffer as the buffer solution. Solution was left for overnight (12 hrs) incubation at 4° C. After incubation magnetic particles were extracted from the solution using magnetic rack and supernatant was discarded. Particles were then washed once with Borate buffer (8.5pH) and stored in 2 ml of 0.1% BSA/PBS solution24 (0.63 mg/ml solution).10 ml stock solution of 0.63 mg/ml of antibody coated magnetic particles was prepared for using above mentioned procedure.
A competitive magnetic immunoassay for detection of rabbit IgG (target analyte) was performed on the functionalized particle surface. Half a mg of anti-rabbit IgG-coated magnetic nanoparticles was pre-incubated with the target analyte in 1 ml of 0.2% BSA/PBS for 1 h in a rotating mill at room temperature. After magnetic separation, the particles were incubated with a solution of 20 μg ml-1 of rabbit IgG-Alexa Fluor 350 for 1 h at room temperature. During this incubation the labeled IgG bound to the available binding sites on the particle surface. The amount of labeled antigen bound on the nanoparticle surface is inversely proportional to the amount of analyte in the sample during the first incubation. Finally the particles were extracted magnetically from the solution. In the detection step, the amount of bound, labeled antigen was quantified by the ratio between the intensities of Alexa 350 and Eu:Gd203. The measured rabbit IgG competitive curve is presented in
Different concentrations (˜0.2 to 25.6 μg/ml, 2× variation) of unlabeled mouse IgG were used as target antigen. The target antigen was diluted in 0.1% BSA/PBS solution and 100 μl of each concentration was incubated with 0.063 mg of anti-mouse IgG-488 coated magnetic particles in 12×75-mm borosilicate glass test tubes for 1 hr at 25° C. After incubation particles were extracted from the solution using magnetic rack and washed twice with 1×PBS solution. 100 μl of 15 μg/ml anti-mouse IgG-647 was then added to the particles and solution was incubated at 25° C. for 1 hr. Particles were again separated using magnetic rack, washed twice with 1×PBS solution and re-suspended in 100 μl 0.1% BSA/PBS. Solution was then poured into 96-well opaque microplate and fluorescence intensities were measured using Spectramax M2 cuvette/microplate spectrofluorometer with excitation at 485 nm, 620 nm and 260 nm.
For the kinetic studies only 0.2 μg/ml target antigen concentration was used. The time period of first incubation of primary antibody coated particles with target antigen was varied (˜0 to 180 mins, 10 min time step) and rest of the assay was performed as explained above.
The organic dyes used for labeling the antibodies used in this experiment have a problem of spectral overlap due to small Stokes shift. To avoid spectral overlap in the spectrum author uses an excitation wavelength which is 10-20 nm lesser than the peak excitation wavelength of corresponding label. The emission spectrums of IgG-488 and IgG-635 after completion of the conventional immunoassay are presented in
The peak emission wavelength values for IgG-635 and IgG-488 were used to create a standard base curve, as shown in
Use of fluorescence intensity ratio to create the base curve eliminates the possible experimental error due the particle loss during washing steps. Fluorescence intensity ratio acts as internal standard unlike if the absolute value of fluorescence emission intensity from secondary label was to be used to create the curve.
For immuno-reaction kinetic studies 10 mins time-step incubations were done for constant analyte concentration (0.2 μg/ml) and emission spectrum from each incubation were used to create another curve, as shown in
The linear variation of fluorescence intensity ratio with respect to time ends somewhere around 45-55 mins, which agrees with the standard protocol value.
The same incubations as in Example 10 were performed inside the microchannel using known concentrations of magnetic particles and analytes. 31.5 μg (50 μl of stock solution) of IgG-635 coated magnetic particles were introduced in the channel. Particles were held against the channel wall by turning on one of the electromagnet and fluid was extracted from the channel using micro-pipet. 50 μl of analyte solution with known concentration (˜0.1 to 1.6 μg/ml, 2× variation) was then introduced in the channel. The electromagnets were turned on and off alternatively for 5 mins at 5 Hz switching frequency for mixing. After mixing the particles were again held against the channel wall and the channel was washed using 0.1% BSA solution. 50 μl solution of 15 μg/ml anti-mouse IgG-488 was then introduced in the channel and particles were again made to oscillate in the solution using the electromagnets. After mixing for 5 mins at 5 Hz switching frequency the second washing was done by holding the particles against the wall. Three samples were collected for each target antigen concentration and particles were re-suspended in 100 μl of 0.1% BSA/PBS solution. Solution was then poured into 96-well opaque microplate and fluorescence intensities were measured using Spectramax M2 cuvette/microplate spectrofluorometer with excitation at 260 nm.
To investigate the effect of electromagnet augmented mixing on antigen-antibody reaction, inside the micro-channel, same incubations were done inside the channel without using electromagnets as control system.
The foregoing description of the embodiments of the invention has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Persons skilled in the relevant art can appreciate that many modifications and variations are possible in light of the above teaching. Concentrations, sizes and other parameters stated in the specification and the claims are for example only and are intended to include variations consistent with the practice of the present invention. Such permissible variations are readily determined by persons of skill in the art in light of the instant disclosure and typically encompass between about +10% to about +20% of the stated parameter. It is therefore intended that the scope of the invention be limited not by this detailed description, but rather by the claims appended hereto. References to publications, patent applications and issued patents contained in this specification are herein incorporated by reference in their entirety for all purposes.
This application claims priority from U.S. Provisional Patent Application Ser. No. 60/762,620, which is hereby incorporated by reference in its entirety for all purposes.
The U.S. Government has certain rights in this invention pursuant to Grant No. DBI-0102662 awarded by the National Science Foundation, Grant No. 5P42ES04699 awarded by the National Institutes of Health (National Institute of Environmental Health Sciences), and Grant No. 05-35603-16280 awarded by the U.S. Department of Agriculture.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US07/02138 | 1/26/2007 | WO | 00 | 4/6/2009 |
| Number | Date | Country | |
|---|---|---|---|
| 60762620 | Jan 2006 | US |
