Not applicable.
Not applicable.
This invention relates to novel signal detector molecules that are longer lived than most background fluorescence and thus can be assayed by time resolved fluorescence. These can be employed in a variety of different assays types based on any ligand/target binding, and can be used in solid phase, bead based, or solution based assays.
Enzyme-linked immunosorbent assays (ELISAs) are a standard workhorse technique in widespread use for the sensitive detection of biomolecules. A major advantage of ELISA is that it uses antibodies for detection. There are well-known techniques for raising high-affinity antibodies against almost any biomolecule of interest, and plenty of existing antibodies that enable ELISA-type techniques with little to no antibody development work.
However, ELISAs suffer from a series of disadvantages, including: (1) batch-to-batch variations in enzyme activity requiring the generation of a standard curve in every ELISA plate run; (2) signal instability necessitating extra quench steps after the proper development time, or the ability to read plates at defined time points, complicating its application in high throughput settings; and (3) both fluorescence and absorbance based ELISAs often require high-cost, high-sensitivity equipment for detection. These disadvantages are especially acute for deployment of ELISAs to field use in resource-poor regions.
Some of these disadvantages have been addressed by using single lanthanide ions or lanthanide nanoparticles incorporating lanthanide chelates to label detection antibodies1. Herein, we describe an alternative to ELISAs that uses lanthanides to yield higher consistency and greater ease of detection, which promises to give even higher sensitivity than existing lanthanide-based techniques. It is called Lanthanide Nanoparticle-based Immunosorbent Assay (LANISA) and is based on our previously-published work using iron oxide nanoparticles2.
At the core of LANISA is a lanthanide-containing metal oxide nanoparticle (
These passivated, coated nanoparticles can then be used to perform immunoassays (
The invention includes any one or more of the following embodiment(s), in any combination(s) thereof:
A fluorogenic reagent comprising:
a) a lanthanide-containing metal oxide nanoparticle;
b) means for water dispersion of said lanthanide-containing metal oxide nanoparticle;
c) functionalization of said lanthanide-containing metal oxide nanoparticle with a linker or a ligand; and
d) said fluorogenic reagent being dissolvable at acid pH to release said lanthanide.
A fluorogenic reagent, said fluorogenic reagent comprising:
a) a passivated lanthanide-containing oxide nanoparticle that is water soluble;
b) said nanoparticle being covalently bound to a ligand;
c) said fluorogenic reagent being dissolvable at acidic pH to release said lanthanide.
A fluorogenic reagent, said fluorogenic reagent comprising:
a) a lanthanide-containing oxide nanoparticle coated with a polar lipid on an exterior surface thereof, said lanthanide being europium;
b) said polar lipid being covalently bound to a tag selected from the group consisting of an antibody, an antibody derivative, a biotin, a lectin, a streptavidin, a polyHIS tag, maleimide, a nucleic acid, a peptide, a protein, and a polysaccharide.
c) wherein said fluorogenic reagent is capable of dissolving at acid pH to release said lanthanide.
A fluorogenic reagent, said fluorogenic reagent comprising:
a) a lanthanide-containing oxide nanoparticle coated with DSPE-PEG on an exterior surface thereof, wherein said lanthanide is terbium or dysprosium or europium;
b) said exterior surface being functionalized with maleimide;
c) wherein said fluorogenic reagent is capable of dissolving at acid pH to release said lanthanide.
A fluorogenic reagent, said fluorogenic reagent comprising
a) a lanthanide-containing metal oxide nanoparticle;
b) a coating on said lanthanide-containing metal oxide nanoparticle to allow water solubility;
c) an optional functionalization of said coating to allow binding to a linker or a ligand; and
d) said fluorogenic reagent being dissolvable at acid pH (pH<7) to release said lanthanide.
Any fluorogenic reagent herein described, wherein said lanthanide is europium, terbium or dysprosium.
Any fluorogenic reagent herein described, wherein said metal oxide is terbium oxide or dysprosium oxide or europium oxide or lanthanide-doped iron oxide.
Any fluorogenic reagent herein described, wherein said coating is a polar lipid.
Any fluorogenic reagent herein described, wherein said coating is a functionalized polar lipid.
Any fluorogenic reagent herein described, wherein said coating is a polar lipid covalently bound to a tag selected from an antibody, an antibody derivative, a biotin, a lectin, a streptavidin, a polyHIS tag, a nucleic acid, a peptide, a protein, and a polysaccharide.
Any fluorogenic reagent herein described, wherein said coating is selected from DSPE-PEG, DSPE-PEG-NH2, DSPE-PEG-FA, DSPE-PEG-CHO, DSPE-PEG-NPC, DSPE-PEG-NHS, DSPE-PEG-MAL, DSPE-PEG-PDP, Bis-DSPE-PEG, DSPE-PEG-Cyanur, DSPE-PEG-Azide, DSPE-PEG Succinyl, DSPE-PEG-TMS, DSPE-PEG-Carboxylic Acid, DSPE-RGD, phosphatidyl choline, phosphatidyl ethanolamine, phophatidyl serine, phospholipids, and glucolipids.
Any fluorogenic reagent herein described, wherein the metal oxide is europium-doped iron oxide and the coating is DSPE-PEG or a functionalized derivative of DSPE-PEG.
A method of assaying a target; said method comprising:
a) contacting a sample suspected of containing a target with a surface capable of capturing said target;
b) contacting a solution containing any fluorogenic reagent herein described for a time sufficient for said reagent to directly or indirectly bind said target;
c) washing away unreacted fluorogenic reagent and assay components;
d) acidifying said sample to release said lanthanide;
e) chelating said lanthanide;
flash or continuously activating said sample for less than a second with a light of a wavelength absorbable by said lanthanide; and
measuring fluorescence of said lanthanide at least 1 millisecond after said flash or continuous activation.
A method of assaying a target; said method comprising:
a) contacting a solution containing a target with any fluorogenic reagent herein described for a time sufficient for said reagent to directly or indirectly bind said target;
b) washing away unreacted fluorogenic reagent and assay components;
c) acidifying said sample to release said lanthanide;
d) flash activating said sample for less than a second with a light of a wavelength absorbable by said lanthanide; and
e) measuring fluorescence of said lanthanide at least 1 millisecond after said flash activation.
Any method herein, wherein said fluorogenic reagent is on a solid substrate, or on an antibody which is on a solid substrate.
Any method herein, wherein said target is captured is on a solid substrate or an antibody or an antibody which is captured on a solid substrate. Antibodies can also be captured via yet another antibody. The solid substrate can be a multiwell microtiter plate, any container, a flat substrate such as glass or plastic slide with an array of spots thereon, a bead, a bead with an antibody thereon, a bead with magnetic element thereon, etc., etc.
Any method herein, wherein said method is a competitive assay; a non-competitive assay; a homogeneous immunoassay; a two-site, noncompetitive immunoassay; a competitive, heterogeneous immunoassay; a one-site, non-competitive immunoassay; a two-site, non-competitive immunoassay; or an array LANISA.
As used herein a “nanoparticle” is less than 1 micron in any one dimension, usually <500 nm, most preferred in the 5-25 or about 10 nm range. The nanoparticles described herein are generally spherical, but this is not essential.
As used herein, “flash” activation refers to a brief burst of the light at which the lanthanide will absorb energy, so that the decay fluorescence can be measured. The flash is <1 second, preferably less than a microsecond, and the signal is then measured over time or at least 1 millisecond later, preferably 0.2 seconds later. The times will of course vary depending on the context, what the background radiation sources are and which lanthanide was chosen for use.
As used herein, “passivation” prevents or minimizes non-specific binding to surfaces, such as the glass or plastic used in assay containers.
As used herein, the “coating” is a layer that surround the nanoparticle, and is preferably non-covalently bound thereto, but ionically or covalently bound coatings could also be used.
The coating is preferably a polar lipid. As used herein, a “polar lipid” will spontaneously form a bilayer in water, typically having a polar head and an apolar tail. Exemplary polar lipids include phosphatidyl choline (PC), phosphatidyl ethanolamine (PE), phophatidyl serine (PS), phospholipids, glucolipids.
Polar lipids alone may have difficulty forming the coating layer of nanoparticles, and thus may benefit from conjugating the nanoparticles with a molecule that assists in the coating, such as a hydrophobic residue, such as the oleyl used herein. The nanoparticle coating or “capping” molecules can be a mixture of oleic acid and oleylamine, oleic acid alone, or oleylamine alone, depending on the synthesis methods. Therefore, polar lipids can form a micellar coating via hydrophobic interactions with the capping molecules. Potentially, however, the polar lipids could form liposomes to encapsulate nanoparticles that are conjugated with hydrophilic capping molecules, such as citric acid, poly(acrylic acid) or polyamines.
A particularly preferred coating is DSPE-PEG or 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) or any other lipid-PEG. Exemplary coatings include PLGA-PEG, PLA-PEG, PCL-PEG, lipid-PEGs, poly(L-lysine)-PEG, and poly(L-glutamic acid)-PEG, to name a few.
There are also a large number of functionalized derivatives of DSPE-PEG that could be used and conveniently are commercially available. These include, but are not limited to, DSPE-PEG-NH2 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)), DSPE-PEG-CHO (1,2-distearoyl-sn-glycero-3-phosphoethanolamine), DSPE-PEG-NPC, DSPE-PEG-NHS (alpha-(1,2-distearoyl-sn-glycero-3-phosphoethanolamine)-omega-succinimidyl carbonate poly(ethylene glycol)), DSPE-PEG-MAL (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)), DSPE-PEG-PDP (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[PDP(polyethylene glycol)), Bis-DSPE-PEG Bis(1,2-distearoyl-sn-glycero-3-phosphoethanolamine)-N-[(polyethylene glycol), DSPE-PEG-Cyanur (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[cyanur(polyethylene glycol)), DSPE-PEG-Azide (“1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[azido(polyethylene glycol)), DSPE-PEG Succinyl (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[succinyl(polyethylene glycol)), DSPE-PEG-TMS (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[10-(trimethoxysilyl)undecanamide(polyethylene glycol)), DSPE-PEG Carboxylic Acid (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)), and DSPE-RGD (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N44-(p-(cysarginylglycylaspartate-maleimidomethyl)cyclohexane-carboxamide]).
As used herein, the term “ligand” refers to a molecule that binds to a particular “target” molecule and forms a complex, usually a binary complex, but trinary complexes are also possible. The binding is preferably highly specific binding, however, in certain embodiments, the binding of an individual ligand to the target molecule can be with relatively low affinity and/or specificity. The “target” molecule is the molecule of interest (e.g., the target of the assay), and obviously, a molecule can be a ligand in one context, but a target in another.
Examples of ligand/target binding pairs include, but are not limited to, streptavidin/biotin; peptide/peptide such as spytag/spycatcher; nucleic acid/complementary nucleic acid; ligand/receptor; steroid/steroid receptor; antibody/antigen; enzyme/substrate; polyhistidine tag/metal ion (e.g., nickel, cobalt and copper); aptamer/target; zinc finger/DNA, and the like, and more are being discovered every day.
As used herein, “tag” refers to a member of a binding pair or binding triplet. It functions to provide a tag or handle, that the other member(s) of the binding pair/triplet can grab onto to form a complex.
A “lanthanide” is one of the 15 metallic chemical elements with atomic numbers 57 through 71 (see below). A preferred lanthanide is europium. The lanthanides are:
Lanthanum—atomic number 57 with symbol Ln
Cerium—atomic number 58 with symbol Ce
Praseodymium—atomic number 59 with symbol Pr
Neodymium—atomic number 60 with symbol Nd
Promethium—atomic number 61 with symbol Pm
Samarium—atomic number 62 with symbol Sm
Europium—atomic number 63 with symbol Eu
Gadolinium—atomic number 64 with symbol Gd
Terbium—atomic number 65 with symbol Tb
Dysprosium—atomic number 66 with symbol Dy
Holmium—atomic number 67 with symbol Ho
Erbium—atomic number 68 with symbol Er
Thulium—atomic number 69 with symbol Tm
Ytterbium—atomic number 70 with symbol Yb
Lutetium—atomic number 71 with symbol Lu
The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims or the specification means one or more than one, unless the context dictates otherwise.
The term “about” means the stated value plus or minus the margin of error of measurement or plus or minus 10% if no method of measurement is indicated.
The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or if the alternatives are mutually exclusive.
The terms “comprise”, “have”, “include” and “contain” (and their variants) are open-ended linking verbs and allow the addition of other elements when used in a claim.
The phrase “consisting of” is closed, and excludes all additional elements.
The phrase “consisting essentially of” excludes additional material elements, but allows the inclusions of non-material elements that do not substantially change the nature of the invention.
The following abbreviations are used herein:
As an example, a full protocol for detection of Vascular Cell Adhesion Molecule 1 (VCAM1) using the scheme shown in
This protocol was modified from the published literature3. Europium oxide (Eu2O3) powder was dissolved to 75 mM in deionized water with HNO3. It is then complexed to 1-benzoylacetone (HBA) by mixing in two volumes of 0.3 M HBA, and precipitated by dropwise addition of ammonia. The precipitate was collected by filtration, washed with water, and dried under vacuum. 0.2 mmol of the precipitate was then added to 15 ml of a 3:5 mixture of oleic acid and oleylamine. The solution was bubbled with argon, incubated under vacuum at 100° C. for 1 hr to remove solvents, and heated to 310° C. under argon at 20 K/min. It was then held at this temperature under argon for 1 hr. After the reaction, an excess of ethanol was added and the nanoparticles isolated by centrifugation. Nanoparticles were then washed with ethanol and dispersed in toluene. We note that similar synthesis protocols can be used for samarium, gadolinium, and terbium oxides3, the lanthanide ions of which all exhibit strong fluorescence in aqueous solution4.
Alternatively, nanoparticles may be mostly iron oxide and doped with lanthanides. This protocol was developed according to a published method5. Lanthanide-dope iron oxide nanoparticles with uniform size distribution were synthesized in two steps: seed synthesis and seed-mediated growth. The first step was to synthesize nanocrystals of 5-6 nm in diameter as the seeds. 5 mmol Fe(acac)3, 1 mmol Eu(acac)3, 24 mmol oleic acid, 24 mL benzyl ether and 12 mL of squalane were mixed in a 100 mL flask. The mixture was heated to 200° C. at a ramping rate of 4° C. per minute and incubated at this temperature for 2 hours under an argon flow. Then the mixture was heated to reflux at a ramping rate of 4° C. per minute and incubated for 1 hour under an argon flow. After reaction, the mixture was cooled down to room temperature and the nanocrystals collected by precipitation with acetone.
In a typical seed mediated growth step, 86 mg Fe of seeds dispersed in 10 mL of toluene, 2.5 mmol Fe(acac)3, 0.5 mmol Eu(acac)3, 24 mmol oleic acid, 24 mL benzyl ether and 12 mL of squalane are mixed in a 100 mL flask. The mixture was heated to 200° C. for 2 hours and to 300° C. for 30 minutes. The resulting nanocrystals were collected by precipitation with acetone. A TEM image of EuFeO nanoparticles is shown in
The protocol for this and subsequent steps was adapted from previously published work. 0.4 ml of a Eu2O3 nanocrystal suspension (15 mg Eu/ml in toluene) were mixed with 2 ml chloroform containing 12 mg DSPE-mPEG (the PEG chain ends with a methoxy group) and 0.24 mg DSPE-PEG-maleimide in a 250 ml flask 10 ml DMSO added to the flask dropwise with gentle shaking over 20 min.
Chloroform and toluene were removed by putting the suspension under vacuum until 8.8 g suspension remains. 16 ml of deionized water was then slowly added to the mixture, and the DMSO removed by filtration through a Vivaspin centrifugal filter tube (m.w. cutoff=100 kD). Coated nanoparticles were collected by two centrifugations at 50000×g, 4° C. for 1 hr, and resuspended in deionized water.
100 μg goat anti-human VCAM1 antibodies (IgG) were reduced by mixing with 200 μl PBS-EDTA containing 100 mM 2-mercaptoethylamine-HCl and incubating at 37° C. for 2 hr. The antibody fragments were washed 6× with sodium acetate (100 mM, pH 5.5) in Amicon centrifugal filters (m.w. cutoff=10 kD). Reduced antibody fragments are then mixed with coated Eu2O3 nanoparticles (3 antibody fragments per coated nanoparticle), and phosphate buffered saline (PBS) added to adjust the pH of the solution to 7.2. This mixture was incubated at room temperature overnight. After incubation, nanoparticles were collected by two centrifugations at 50000×g, 4° C. for 1 hr, and the supernatant removed. Conjugated nanoparticles were then resuspended at 300 μg Eu/ml in deionized water.
To generate capture surfaces for VCAM1 immunoassays, wells in high-binding microplates (e.g. Nunc™ Maxisorp™ 96 plates) were filled with 100 μl of capture antibody solution (mouse anti-VCAM1, 10 μg/ml) and incubated for 24 hrs at 4° C. They were then washed 3× with PBS+0.05% tween-20 and incubated for another 24 hrs at 4° C. with 300 μl 1% BSA in PBS, after which they were ready to use. To capture VCAM1 molecules, 100 μl VCAM1-containing cell lysate (0.8-3 μg/ml protein) was incubated in wells at 37° C. with shaking for 1 hr. The lysate was then removed, and the well washed 3× with PBS+0.05% Tween-20. Conjugated nanoparticle suspension from step III was then added (100 μl), incubated at 37° C. with shaking for 1 hr, and unbound suspension removed. The well was washed 3× with PBS+0.05% tween-20.
Bound nanoparticles were dissolved by adding 50 μl 6 M HCl to the well and incubated at room temperature for 15 min. Then 35 μl 8 M NaOH and 50 μl of 4 M ammonium acetate solution were added sequentially to neutralize the solution. Luminescence was then developed by adding 50 μl DELFIA enhancement solution (Perkin Elmer).
The DELFIA® Enhancement Solution is an acidic chelating detergent solution intended for use in the quantitative determination of Eu3+/Sm3+ when using a time-resolved fluoroimmunoassay. The solution dissociates Eu3+/Sm3+ from solid phase bound Eu-labeled antibodies or proteins during a time period of a few minutes to form a homogeneous and highly fluorescent micellar chelate solution. The solution allows highly sensitive Eu3+/Sm3+ measurements to be made when using the time-resolved fluorometer. The DELFIA® Enhancement Solution is also used as part of Tb3+/Dy3+ measurement together with DELFIA® Enhancer.
The well can then be read in a standard plate reader in time-resolved fluorescence mode, with excitation at 320 nm, 400 μs delay, and 400 μs integration time.
The use of lanthanide nanoparticles can potentially achieve large signal gains: a 40 nm diameter pure Eu2O3 nanoparticle is estimated to contain approximately 1.0 million europium atoms, thereby yielding 1 million fluorophores from a single binding event. As far as we are aware, this level of amplification gain would be one of the highest reported for an immunosorbent assay.
The fluorophores so generated have the additional advantage that their fluorescence is long-lived. Therefore, they can be quantitated using time-resolved fluorescence. In this mode, fluorophores in the sample are excited by a burst of light but quantitated a short time (>100 μs) after the end of that burst. Since most contaminating fluorescence has much shorter lifetimes (ns), these will all have decayed by the time signal integration starts, so that background autofluorescence will not affect measurements. This reduces the noise floor of quantitation and increases detection sensitivity. Furthermore, because excitation light is not present during quantitation, no optical filters are needed in the quantitation device6 as long as the detection apparatus can be made sufficiently light-tight. This significantly reduces instrument cost and complexity.
In a preliminary experiment (
There is a good linearity when the concentration of europium oxide increases from 20 pg/mL to 2500 pg/mL (
To functionalize EuFeO nanoparticles, we modified streptavidin as shown in
In a subsequent experiment, we synthesized europium-doped iron oxide (EuFeO) nanoparticles and coated them with DSPE-PEG. These nanoparticles were then functionalized with streptavidin (
After that, the plate was washed with PBS with 0.05% Tween-20. Each well was then incubated with a biotinylated rabbit anti-human IgA antibody (50 μL, 2 μg/mL) for 1 hr. After washes, the plate was incubated with the streptavidin-functionalized EuFeO nanoparticles (50 μL, 50 μg/mL) for 1 hr. After unbound EuFeO nanoparticles were removed by additional washes, the fluorescence signal was developed and detected as mentioned above. As shown in
We also demonstrated the high sensitivity and wide dynamic range of LANISA in quantifying human PSA (
We further quantify the pancreatic cancer marker CA19-9 in the sera of pancreatic cancer patients using a sandwich LANISA assay. We first generated a standard curve by measuring fluorescence signals of serum samples with known concentrations of purified human CA19-9 (
Lanthanide nanoparticles. Many variants can be made to the above protocol. Several different synthetic routes to Eu2O3 nanoparticles have been reported8-10, and other studies have used commercially purchased nanoparticles11. In addition to europium oxide nanoparticles, other lanthanide nanoparticles may be used, including terbium oxide and dysprosium oxide nanoparticles. The use of different lanthanide nanoparticles in LANISA may enable multiplexing.
Coating moieties. Further, there are several commercially available DSPE-PEG conjugates of interest here, including those with amine, carboxy, biotin, azide, and DBCO groups (Avanti Polar Lipids, Inc.). These groups allow crosslinking via NHS esters, carbodiimide, streptavidin, copper-catalyzed click chemistry, and copper-free click chemistry, respectively.
Detection molecules. In addition to biomolecule detection with antibodies, other capture systems include lectins that bind to carbohydrate groups with specificities that match those of antibody-antigen interactions12 and use of nucleic acids that can be designed to specifically hybridize to sequences of interest with tunable specificity and sensitivity13. Nanoparticle functionalization with lectins can be accomplished using NHS or carbodiimide14, and functionalization with nucleic acids can be accomplished with any of the above-mentioned techniques by using oligonucleotides synthesized with the relevant functionalization groups.
Solution phase assays. One major drawback of surface-based immune assays is that analyte binding requires extended incubation periods. While this is less of a problem in laboratory experiments, use of these assays in clinical settings can benefit from having shorter assay times. We propose to overcome this drawback by using solution phase analyte capture with magnetic beads. As shown in
A magnetic bead-based immunoassay protocol would be as follows:
Conjugate 100 μg mouse anti-VCAM1 antibodies to 100 μl ProMag® Bind-IT™ pre-activated magnetic microspheres (Bangs Labs) according to manufacturer's recommendation (protocol attached). Wash 10 μl antibody-conjugated microspheres with PBS+1% BSA. Resuspend to 10 μl in PBS+1% BSA and mix with 100 μl VCAM-1-containing cell lysate in a plain polystyrene 96-well plate. Incubate with gentle shaking at 37° C., 15 min. Add 50 μl suspension of antibody-conjugated lanthanide oxide nanoparticles and incubate with gentle shaking at 37° C. for another 15 min. Chill the plate to 4° C. and bind magnetic beads to the well bottom using a rare earth magnet. Remove supernatant and resuspend with 200 μl ice-cold PBS+1% BSA. Repeat binding, supernatant removal, and resuspension three times. Bind beads, remove supernatant, and resuspend using 50 μl 6 M HCl. Incubate at room temperature with gentle shaking, 15 min. Mix in 50 μl DELFIA enhancement solution and read on plate reader, as described previously.
Array LANISA. LANISA can be generalized to a microarray format to have multiplexed detection of analytes (
In another embodiment, a microarray of spots printed on paper will be used instead of a microplate. The capture molecules corresponding to a specific analyte will be immobilized at the spot through the thickness of the paper, together with lanthanide chelators, such as Tris chelates, tetrakis chelates, phthalates, picrate, diethylenetriaminepentaacetic acid (DTPA), triethylenetetraminehexaacetic acid (TTHA), 2,2′,2″,2′″-[[4′-(aminobiphenyl-4-yl)-2,2′:6′,2″-terpyridine-6,6″-diyl]bis(methylenenitrilo)]tetrakis(acetato) (ATBTA) and its derivatives, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(acetamidoacetic acid) (DOTA), and salicylate complexes. Acid (such as hydrochloric acid, nitric acid, acetic acid, and sulfuric acid) will be applied to the entire microarray for signal development and the fluorescence signal at each spot will be quantified. In this embodiment, chelators would be immobilized on paper fibers and be concentrated enough to suppress diffusion of lanthanide ions released by the dissolution of the oxide nanoparticles. The array LANISA can be used for multiplexed detection of proteins, carbohydrate groups, nucleic acids and their combinations.
Competitive, homogeneous immunoassays. In a competitive, homogeneous immunoassay, unlabeled analyte in a sample competes with labeled analyte to bind an antibody. The amount of labeled, unbound analyte is then measured. In theory, the more analyte in the sample, the more labeled analyte gets displaced and then measured; hence, the amount of labeled, unbound analyte is proportional to the amount of analyte in the sample.
Two-site, noncompetitive immunoassays. These usually consist of an analyte “sandwiched” between two antibodies. ELISAs are often run in this format.
Competitive, heterogeneous immunoassays. As in a competitive, heterogeneous immunoassay, unlabeled analyte in a sample competes with labeled analyte to bind an antibody. In the heterogeneous assays, the labeled, unbound analyte is separated or washed away, and the remaining labeled, bound analyte is measured.
One-site, noncompetitive immunoassays. The unknown analyte in the sample binds with labeled antibodies. The unbound, labeled antibodies are washed away, and the bound, labeled antibodies are measured. The intensity of the signal is directly proportional to the amount of unknown analyte.
Two-site, noncompetitive immunoassays. The analyte in the unknown sample is bound to the antibody site, then the labeled antibody is bound to the analyte. The amount of labeled antibody on the site is then measured. It will be directly proportional to the concentration of the analyte because the labeled antibody will not bind if the analyte is not present in the unknown sample. This type of immunoassay is also known as a sandwich assay as the analyte is “sandwiched” between two antibodies.
The following reference are incorporated by reference in its entirety for all purposes.
This application claims priority to U.S. Ser. No. 62/701,734, filed Jul. 21, 2018, and incorporated by reference in its entirety for all purposes.
Filing Document | Filing Date | Country | Kind |
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PCT/US19/42589 | 7/19/2019 | WO | 00 |
Number | Date | Country | |
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62701734 | Jul 2018 | US |