WO2006122083A2 - Water relaxation-based sensors - Google Patents
Water relaxation-based sensors Download PDFInfo
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- WO2006122083A2 WO2006122083A2 PCT/US2006/017842 US2006017842W WO2006122083A2 WO 2006122083 A2 WO2006122083 A2 WO 2006122083A2 US 2006017842 W US2006017842 W US 2006017842W WO 2006122083 A2 WO2006122083 A2 WO 2006122083A2
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/72—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating magnetic variables
- G01N27/74—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating magnetic variables of fluids
- G01N27/745—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating magnetic variables of fluids for detecting magnetic beads used in biochemical assays
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/145—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue
- A61B5/14503—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue invasive, e.g. introduced into the body by a catheter or needle or using implanted sensors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01R—MEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES
- G01R33/00—Arrangements or instruments for measuring magnetic variables
- G01R33/20—Arrangements or instruments for measuring magnetic variables involving magnetic resonance
- G01R33/44—Arrangements or instruments for measuring magnetic variables involving magnetic resonance using nuclear magnetic resonance [NMR]
- G01R33/46—NMR spectroscopy
- G01R33/465—NMR spectroscopy applied to biological material, e.g. in vitro testing
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01R—MEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES
- G01R33/00—Arrangements or instruments for measuring magnetic variables
- G01R33/20—Arrangements or instruments for measuring magnetic variables involving magnetic resonance
- G01R33/44—Arrangements or instruments for measuring magnetic variables involving magnetic resonance using nuclear magnetic resonance [NMR]
- G01R33/48—NMR imaging systems
- G01R33/50—NMR imaging systems based on the determination of relaxation times, e.g. T1 measurement by IR sequences; T2 measurement by multiple-echo sequences
Definitions
- This invention relates to magnetic resonance-based sensors and related methods.
- Magnetic resonance (MR)-based reporting methods such as magnetic resonance imaging (MRI)
- MRI magnetic resonance imaging
- MRI magnetic resonance imaging
- MRI can be used at tissue depths where optical reporting methods can sometimes be complicated by light scattering and absorption by the tissue, e.g., tissue depths greater than about 250 ⁇ m.
- nanotechnology in medicine is the development of biocompatible nanomaterials as environmentally sensitive sensors and molecular imaging agents.
- Preparations of magnetic particles designed for separation and extraction use particles that are amenable to easy manipulation by weak applied magnetic fields. These materials are typically micron sized and have a high magnetic moment per particle.
- nanoparticles do not respond to the weak, magnetic fields of hand held magnets.
- This invention relates generally to magnetic resonance-based sensors (e.g., water relaxation and equilibrium-based sensors) and related methods, and is based, in part, on the discovery that sensors having magnetic nanoparticles encapsulated within a semipermeable enclosure can be used as remote sensors for detecting various analytes in an aqueous, e.g., a water-containing, sample and can be used for the continuous monitoring of changing levels of the analytes.
- the invention provides a water relaxation-based sensor for detecting the presence of an analyte in a sample.
- the sensor includes an enclosure defining an opening for entry of the analyte, e.g., a semipermeable membrane, and confined within said enclosure, a plurality of nanoparticles.
- the nanoparticles are suspended or suspendable in an aqueous liquid phase, have a magnetic moment, e.g., comprise crystalline iron oxide or other magnetic material, and are covalently or non covalently linked to, or otherwise have immobilized thereon, one or more moieties selected to alter the state of aggregation of the nanoparticles as a function of the presence or concentration of the analyte in the enclosure.
- this invention features water relaxation-based sensors for detecting the presence of an analyte (e.g., an exogenous analyte) in a sample.
- the sensors include: (i) a walled enclosure enveloping a chamber, wherein the wall includes one or more openings (e.g., a single opening or a plurality of openings) for passage of the analyte into and out of the chamber; (ii) a plurality of magnetic nanoparticles located within the chamber, each nanoparticle having at least one moiety that is covalently or noncovalently linked to (immobilized on) the nanoparticle; and optionally, (iii) at least one binding agent located within the chamber, in which the opening can be smaller in size than the nanoparticles and the binding agent and larger in size than the analyte; and the moiety and the analyte can each bind reversibly to the binding agent, when present; or the analyte can bind reversibly to
- Embodiments can include one or more of the following features.
- the nanoparticles can be suspended or be suspendable in an aqueous liquid phase.
- the nanoparticles can have a magnetic moment generally, or under certain conditions.
- the wall can include more than one opening. " When the wall contains more than one opening (e.g., a plurality of openings), then at least one of the openings is smaller in size than the nanoparticles and optionally the binding agent, and larger in size than the analyte.
- the wall includes a plurality of openings, in which each of the openings is smaller in size than the nanoparticles and the binding agent, and each of the openings is larger in size than the analyte.
- the moiety can be selected to alter the state of aggregation of the nanoparticles as a function of the presence or concentration of the analyte in the enclosure.
- the senor may exploit different detection formats.
- the moiety may be selected to bind to the analyte to produce an aggregate of plural linked nanoparticles as a function of the presence or concentration of the analyte in the enclosure.
- the sensor may include an aggregate of plural linked nanoparticles, which is disaggregated as a function of the presence or concentration of the analyte in the enclosure.
- the moiety may be a fragment of an authentic sample of the analyte or a structural mimic thereof, in which case the sensor further includes a multivalent binding agent, which binds to the analyte and the mimic (if used) to produce an aggregate of plural linked nanoparticles.
- the sensor can further include a multivalent binding agent which binds to the moiety to produce an aggregate.
- the sensor also can include a binding agent, which binds to the moiety in the presence of the analyte to disassociate an aggregate, hi yet another form, the sensor can include a binding agent that binds to the moiety in the presence of the analyte to produce an aggregate.
- the sensor further includes a plurality of aggregates confined within the enclosure.
- the sensor includes a sample flow path in communication with the interior of the enclosure. Thus, the sample can flow into, or into and out of, the enclosure to permit periodic sampling of the analyte.
- the sensors include features (i), (ii), and (iii) above; the moiety can be, or can include as part of its chemical structure, a molecular fragment of the analyte being detected or a molecular fragment of a derivative, isostere, or mimic of the analyte being detected; and the binding agent can be a protein.
- the chamber can include one or more nanoparticle aggregates.
- Each of the nanoparticle aggregates can include one or more nanoparticles and the binding agent. Formation of the nanoparticle aggregate can occur through binding of a moiety on the nanoparticle to the binding agent (e.g., the binding agent can include one or more binding sites that are recognized for binding by the moiety).
- the chamber can include substantially disaggregated nanoparticles.
- the analyte when present, can displace the nanoparticles from the nanoparticle conjugates, thereby providing substantially disaggregated nanoparticles (e.g., the analyte and the moiety can be selected such that the analyte and the nanoparticles can compete for binding with the binding agent, and the analyte, when present, can displace the nanoparticles from the binding agent in the aggregate to provide disaggregated nanoparticles).
- the chamber when the analyte is absent, can include a nanoparticle aggregate, wherein the nanoparticle aggregate can include nanoparticles bound to the binding agent through the moiety; and (b) when the analyte is present, the nanoparticles are displaced from the binding agent by the analyte, and the chamber comprises substantially disaggregated nanoparticles.
- the change in nanoparticle aggregation alters the proton relaxation of water inside of the chamber, but does not substantially alter the proton relaxation of water outside of the chamber.
- the sensors include features (i) and (ii) above, feature (iii) is absent; and the moiety can be, or can include as part of its chemical structure, a protein.
- the chamber can include substantially disaggregated nanoparticles.
- the chamber can include one or more nanoparticle aggregates.
- Each of the nanoparticle aggregates can include one or more nanoparticles and the analyte. Formation of the nanoparticle aggregate can occur through binding of the analyte to the moiety on the nanoparticle (e.g., the moiety can include one or more binding sites that are recognized for binding by the analyte).
- the chamber when the analyte is absent, the chamber comprises substantially disaggregated nanoparticles; and (b) when the analyte is present, the chamber comprises a nanoparticle aggregate, wherein the nanoparticle aggregate comprises nanoparticles bound to the analyte through the moiety.
- the change in nanoparticle aggregation alters the proton relaxation of water inside of the chamber, but does not substantially alter the proton relaxation of water outside of the chamber.
- this invention features methods of detecting an analyte in an aqueous sample (e.g., monitoring the presence or concentration of an analyte in a sample stream), the methods include: (i) providing a sensor as described herein; (ii) measuring relaxation times (e.g., T2 or Tl relaxation times) of the water inside of the chamber of the sensor in the absence of the analyte or under conditions that mimic the absence of the analyte; (iii) contacting the sensor with the sample (e.g., the nanoparticles can be suspended, or suspendable, in an aqueous liquid phase and can also have a magnetic moment); (iv) measuring relaxation times (e.g., T2 or Tl relaxation times) of the water inside of the chamber of the sensor; and (v) comparing the T2 relaxation times measured in step (ii) and step (iv). A change (e.g., an increase or decrease) in T2 relaxation times measured in step (iv) relative to the T2 relaxation
- nanoparticles e.g., suspended nanoparticles
- Embodiments can include one or more of the following features.
- the nanoparticles can be suspended or be suspendable in an aqueous liquid phase.
- the nanoparticles can have a magnetic moment.
- the wall can include more than one opening.
- the wall contains more than one openings (e.g., a pluralit/of openings), then at least one of the opening is smaller in size than the nanoparticles and the binding agent, and larger in size than the analyte.
- the wall includes a plurality of openings, in which each of the openings is smaller in size than the nanoparticles and the binding agent, and each of the openings is larger in size than the analyte.
- the moiety can be selected to alter the state of aggregation of the nanoparticles as a function of the presence or concentration of the analyte in the enclosure.
- the moiety and the analyte can each bind reversibly to the binding agent, when present; or the analyte can bind reversibly to the moiety.
- the analyte can be a monovalent or multivalent analyte.
- the moiety can be, or can include as part of its structure, a carbohydrate, an antibody, an amino acid, a nucleic acid, an oligonucleotide, a therapeutic agent or a metabolite thereof, a peptide, or a protein.
- the moiety can be a covalently or noncovalently linked analyte (e.g., the analyte that is being detected, sometimes referred to as a bound analyte or a bound binding protein), a covalently or noncovalently linked analyte derivative, or a covalently or noncovalently linked analyte isostere or mimic (e.g., a derivative, isostere, or mimic of the analyte that is being detected).
- the moiety can be, or can include as part of its structure, a molecular fragment of the analyte being detected or a molecular fragment of a derivative, isostere, or mimic of the analyte being detected.
- a moiety that includes "a molecular fragment of the analyte being detected" is one in which a portion (e.g., a substantial portion) of the chemical structure of the analyte being detected (or a derivative, isostere, or mimic thereof) is incorporated into the chemical structure of the moiety.
- the nanoparticle can have the general formula (A): (A) Z -NP C ; in which "A” is a molecular fragment of an analyte, A-X, in which X is a hydrogen atom or a functional group that is present in the analyte, but not incorporated into the nanoparticle of formula (A); "NP C " is the nanoparticle core, "-” is a covalent linkage (e.g., a chemical bond or linking functional group) that connects any atom of the fragment to the nanoparticle; and z is 1-50 (e.g., 1-40, 1-30, 1-25, 1-20, 2-20).
- "A” in the above formula can also be the molecular fragment of a derivative, isostere, or mimic of the analyte being detected.
- the moiety can be a protein or a nucleic acid.
- the binding agent can be absent.
- the moiety can be, or include as part of its structure, a protein.
- the chamber includes substantially disaggregated nanoparticles; and (b) when the analyte is present, the chamber can include one or more nanoparticle aggregates.
- Each of the nanoparticle aggregates can include one or more nanoparticles and the analyte. Formation of the nanoparticle aggregate can occur through binding of the analyte to the moiety on the nanoparticle (e.g., the moiety can include one or more binding sites that are recognized for binding by the analyte).
- the chamber comprises substantially disaggregated nanoparticles; and (b) when the analyte is present, the chamber comprises a nanoparticle aggregate, wherein the nanoparticle aggregate comprises nanoparticles bound to the analyte through the moiety.
- the binding agent can be present, it can be, for example, a protein or a monoclonal antibody.
- the moiety can be, or can include as part of its structure, a molecular fragment of the analyte being detected or a molecular fragment of a derivative, isostere, or mimic of the analyte being detected.
- the chamber when the analyte is absent, can include one or more nanoparticle aggregates; and (b) when the analyte is present, the chamber can include substantially disaggregated nanoparticles.
- Each of the nanoparticle aggregates can include one or more nanoparticles and the binding agent. Formation of the nanoparticle aggregate can occur through binding of a moiety on the nanoparticle to the binding agent (e.g., the binding agent can include one or more binding sites that are recognized for binding by a chemical group that is present as all or part of the chemical structure of the moiety).
- the analyte when present, can displace the nanoparticles from the nanoparticle conjugates, thereby providing substantially disaggregated nanoparticles (e.g., the analyte and the nanoparticle substituent can be selected such that the analyte and the nanoparticle moiety can compete for binding with the binding agent, and the analyte, when present, can displace the nanoparticles from the binding agent in the aggregate to provide disaggregated nanoparticles).
- the analyte and the nanoparticle substituent can be selected such that the analyte and the nanoparticle moiety can compete for binding with the binding agent, and the analyte, when present, can displace the nanoparticles from the binding agent in the aggregate to provide disaggregated nanoparticles).
- the chamber when the analyte is absent, can include a nanoparticle aggregate, wherein the nanoparticle aggregate can include nanoparticles bound to the binding agent through the moiety; and (b) when the analyte is present, the nanoparticles are displaced from the binding agent by the analyte, and the chamber comprises substantially disaggregated nanoparticles.
- the change in nanoparticle aggregation between (a) and (b) can alter the proton relaxation of water inside of the chamber, but does not substantially alter the proton relaxation of water outside of the chamber.
- the change in nanoparticle aggregation between (a) and (b) can produce a measurable change in the T2 relaxation times of water inside the chamber, and the change in the T2 relaxation times can be measurable using a magnetic resonance imaging or nonimaging method.
- the moiety can be linked to the nanoparticle by a functional group such as -NH-, -NHC(O)-, -(O)CNH-, -NHC(O)(CH 2 ) n C(O), -(O)C(CH 2 ) n C(O)NH-, - NHC(0)(CH 2 ) n C(0)NH-, -C(O)O-, -OC(O)-, or -SS-, in which n can be 0-20, e.g., 2, 5, 10, or 15.Each of the openings can have a size (pore size) of from about 1 kDa to about 1 ⁇ m (e.g., about 1 kDa to about 300,000 kDa; about 1 kDa to about 100,000 kDa; about 1 kDa to about 5 kDa; 1 kDa to about 3 kDa; 1 kDAto about 1 ⁇ m).
- a functional group such as -NH
- Each of the nanoparticles can have a particle size or overall size of from about 10 nm to about 500 nm (e.g., about 10 nm to about 60 nm, about 30 nm to about 60 nm).
- the overall size is the largest dimension of a particle.
- the nanoparticle aggregate can have an overall size (particle size) of at least about 100 nm.
- the nanoparticles can be substantially aggregated (e.g., include on or more nanoparticle aggregates) or substantially disaggregated.
- the analyte can be a carbohydrate (e.g., glucose).
- the analyte can be chiral.
- the chiral analyte can be present together with one or more optically active moieties in the sample.
- the chiral analyte can be present together with a stereoisomer of the chiral analyte in the sample.
- the chiral analyte can be present together with the enantiomer of the chiral analyte in the sample.
- the chiral exogenous analyte can be an amino acid.
- the analyte can be a nucleic acid or an oligonucleotide.
- the analyte " can be a therapeutic agent, which as used herein refers to a bioactive moiety, which when administered to a subject (e.g., a human or animal subject) confers a therapeutic, biological, or pharmacological effect (e.g., treats, controls, ameliorates, prevents, delays the onset of, or reduces the risk of developing one or more diseases, disorders, or conditions or symptoms thereof) on the subject, or a metabolite thereof.
- the analyte can be, e.g., folic acid.
- the analyte can be a peptide or a protein (e.g., influenza hemagglutinin peptide).
- W 2 a protein (e.g., influenza hemagglutinin peptide).
- the moiety can be, or can include as part of its structure, a chiral moiety.
- the moiety can be, or can include as part of its structure, an amino acid, a nucleic acid, an oligonucleotide, a therapeutic agent, a metabolite of a therapeutic agent, a peptide, or a protein.
- the moiety can be, or can include as part of its structure, a carbohydrate (e.g., having the structure:
- the binding agent can be a protein that includes at least two binding sites or at least four binding sites.
- the binding agent can be a recombinant protein or be a complex of proteins each with binding sites.
- the complex of proteins may be assembled by crosslinking.
- the binding agent can be a protein that binds to a carbohydrate (e.g., glucose).
- the protein can be conconavalin A.
- the binding agent can be a monoclonal antibody, a polyclonal antibody, or a oligonucleotide.
- the binding agent can be, e.g., an antibody to folic acid or an antibody to influenza hemagglutinin peptide.
- the analyte and the nanoparticle can bind reversibly to the binding agent.
- the magnetic nanoparticles each can include a magnetic metal oxide (e.g., a superparamagnetic metal oxide).
- the metal oxide can be iron oxide.
- Each of the magnetic nanoparticles can be an arnino-derivatized cross-linked iron oxide nanoparticle.
- the sensor can be configured to be an implantable sensor.
- the sensor can be implanted subcutaneously.
- the sensor can be implanted in an extremity of a subject (e.g., a human or animal).
- Steps (ii) and (iv) can include measuring T2 relaxation times or Tl relaxation times.
- An increase in T2 relaxation times measured in step (iv) relative to the T2 relaxation times measured in step (ii) can indicate the presence of the analyte.
- a decrease in T2 relaxation times measured in step (iv) relative to the T2 relaxation times measured in step (ii) can indicate the presence of the analyte.
- analyte or “exogenous analyte” refers to a substance or chemical constituent (e.g., glucose, folic acid, or influenza hemagglutinin peptide) in a sample (e.g., a biological or industrial fluid) that can be analyzed (e.g., detected and quantified) and monitored using the sensors described herein.
- a sample e.g., a biological or industrial fluid
- subject includes mice, rats, cows, sheep, pigs, rabbits, goats, horses, primates, dogs, cats, and humans.
- a nanoparticle having at least one moiety described herein that is covalently or noncovalently linked to the nanoparticle and that can switch from being in an aggregated and disaggregated state is sometimes referred to herein as a “magnetic nanoswitch” or “nanoswitch.”
- Embodiments can have one or more of the following advantages. While not wishing to be bound by theory, it is believed that nanoparticle aggregation (formation of nanoparticle aggregates, e.g., microaggregates) and disaggregation (formation of disaggregated or dispersed nanoparticles from microaggregates or nanoparticle agregates) is an equilibrium controlled process, and that the position of this equilibrium is dependent upon (and therefore maintained by) analyte concentration. When analyte concentration changes, the position of the equilibrium changes, at least in a range of sensitivity depending on several factors. This change in the position of this equilibrium is manifested by changes in proton relaxation of the water inside of the sensor chamber, which is measurable.
- the sensors have the advantage of being useful for the continuous monitoring of changing levels of analytes because there is generally no need to re-condition or replace the sensors during the course of most ongoing (e.g., long term) measurements because essentially nothing is created or produced during detection; the equilibrium between aggregated and disaggregated nanoparticles is shifted by the analyte.
- the equilibrium between aggregated and disaggregated nanoparticles is shifted by the analyte.
- one can continuously monitor changes in the aforementioned equilibrium that occur inside the sensor chamber e.g., by periodically or continuously monitoring T2 relaxation times of the water inside of the chamber, then one can continuously monitor changing levels of analytes as those changes occur.
- the sensors can be used to detect a chemically diverse array of analytes, which include without limitation, carbohydrates (e.g., glucose), peptides (e.g., influenza hemagglutinin peptide), and therapeutic agents (e.g., folic acid).
- carbohydrates e.g., glucose
- peptides e.g., influenza hemagglutinin peptide
- therapeutic agents e.g., folic acid
- the sensors are relatively simple devices lacking moving parts, electronics and any connection to an outside recording device such as a sampling tube or wire. Instead, the sensor operates by absorbing and emitting radiation at the Larmour precession frequency of water protons, which is interpretable as T2 and exogenous analyte (e.g., glucose) concentration.
- the radiation employed e.g., 60 MHz for the 1.5 T MRI
- the sensor can therefore be essentially a remote sensor, reporting on its local environment through water relaxation measurements while unconnected to an outside recording device or power source.
- Analyte detection can take place in solution rather than on a surface, so as to avoid the need for developing and optimizing sensor surface chemistry. This enables the features of an assay (sensitivity, specificity, kinetics) to be determined in a tube format, independently from the semi-permeable device or instrumentation needed to distinguish sensor water from bulk water. Binding agents, e.g., proteins and antibodies, and nanoparticles can readily be tested as reagents for new water relaxation assays, leading a panel of relaxation-based sensors for different analytes.
- binding agents e.g., proteins and antibodies, and nanoparticles
- the production or consumption of molecules is avoided as compared with an assay that includes irreversible reactions (e.g., single use assays).
- an assay that includes irreversible reactions (e.g., single use assays).
- the sensors can be prepared for reuse, for example, by equilibrating in the absence of the analyte or under conditions chosen to mimic the absence of the analyte (e.g., relatively low concentrations of the analyte).
- the radiofrequency radiation used with the water relaxation sensor interacts with water protons, rather than nanoparticles or biological molecules, thereby minimizing the likelihood of radiation induced damage.
- the sensors are amenable for use in the detection of two or more analytes (e.g., a panel of different sensors each having, e.g., a different binding agent and moiety, can be used in the same screening or testing environment to detect multiple analytes).
- FIG. IA is a cross-sectional view of an embodiment of a water relaxation-based sensor for detecting a monovalent analyte. Also shown are the equilibrium controlled processes that occur in the sensor chamber in the absence and presence of the analyte and a summary of the water relaxation properties of the aggregated and dispersed nanoparticles.
- FIG. IB is a cross-sectional view of an embodiment of a water relaxation-based sensor for-detecting a monovalent analyte. Also shown are the equilibrium controlled processes that occur in the sensor chamber in the absence and presence of the analyte and a summary of the water relaxation properties of the aggregated and dispersed nanoparticles.
- FIG. 1C is a schematic representation of a sensor configuration embodiment in which the nanoparticles can bind directly to each other, and the analyte can mediate aggregation/disaggregation.
- the surface of this nanoparticle (left side of equation) is capable of binding to an analyte and binding of the analyte can alter the physical properties of the surface, e.g., charge or hydrophobicity, resulting in aggregation/disaggregation (right side of equation).
- aggregation/disaggregation is mediated by changes in pH (H+).
- ID is a schematic representation of a sensor configuration embodiment in which the detection of a sequence of bases on a nucleic acid fragment can mediate self- assembly of the nanoparticles.
- the two types of nanoparticles can self-assemble via the hybridization that occurs between bases of the oligonucleotides (left side of equation).
- An analyte that can bind to one of the types of particles can induce the dissociation of aggregates (right side of equation) .
- FIG.2 is a reaction scheme showing the synthesis of glucose linked cross-linked iron oxide nanoparticle (GIu-CLIO).
- FIG. 3 A is a graphical representation of changes in T2 relaxation times obtained in a tube based water relaxation assay for glucose using the Glu-CLIO/ConA configuration.
- Amino-CLIO does not react with ConA binding protein, as indicated by a stable T2 relaxation time after ConA addition.
- Attachment of 2-amino-glucose (G) results in a functionalized nanoparticle, GIu-CLIO, which shows a T2 drop upon addition of a glucose-binding protein (ConA).
- the T2 drop is reversed by the addition of glucose.
- the data is indicative of nanoparticle aggregation and disaggregation.
- 0.5 mL of GIu-CLIO (10 ug Fe/mL), 800 ug/mL ConA in PBS with 1 mM CaCl 2 and 1 mM MgCl 2 were used.
- FIG. 3B is a photograph of the assay apparatus. Iron concentration in the photograph was increased to 100 ug Fe/mL for better contrast.
- FIG. 4A is a graphical representation of changes in T2 relaxation times obtained in a tube based assay for glucose using the GIu-CLIO nanoswitch/ConA configuration. Addition of ConA to GIu-CLIO caused a initial T2 drop, which was reversed by the addition of increasing concentrations of glucose.
- FIG. 4B is a graphical representation of changes in T2 values (at the plateau) obtained with different glucose concentrations.
- FIGS. 4C, 4D, and 4E are graphical representations of particle size distribution as obtained by light scattering experiments.
- FIG. 4C shows the particle size distribution for dispersed GIu-CLIO nanoparticles.
- FIG. 4D shows the switch from dispersed nanoparticles (dark bars) to the microaggregate state (light bars) upon ConA addition.
- FIG. 4E shows the switch of the GIu-CLIO nanoparticles back to the dispersed state upon glucose addition.
- FIG. 5A is a graphical representation of changes in T2 relaxation times obtained by contacting a water relaxation sensor with solutions of varying external glucose concentrations. Sensor was first placed in PBS with 0.1 mg/rnL glucose, then in buffer with 1.0 mg/rnL glucose and returned to a solution of 0.1 mg/mL glucose. Conditions were essentially the same as described with respect to FIGS. 3A and 3B.
- FIG. 5B is a photograph of the sensor and the apparatus for containing the glucose-containing sample media.
- FIGS. 6A, 6B, and 6C are images corresponding to the reaction of a water relaxation sensor to glucose visualized by MRI.
- FIG. 5A shows sensors with ConA and GIu-CLIO placed in 50 mL tubes.
- FIG. 5B shows an MR image of a 50 mL tube with external glucose of 0.5 mg/mL
- FIG. 5C shows an MR image of with an external glucose concentration of 1.4 mg/mL. Conditions were essentially the same as described with respect to FIGS. 3A and 3B.
- FIG. 7 is a graphical representation of time dependent changes in T2 with increasing and decreasing glucose concentrations.
- the GIu-CLIO nanoswitch/ConA system used in the experiments described in FIGS. 4A-4E was placed in a semipermeable device so that glucose could be cycled between low and high concentrations, causing nanoparticles in the sensor to shift back and forth between a low T2 state and high T2 state.
- FIG. 8 A is a graphical representation of changes in T2 relaxation times obtained in a tube based assay for influenza hemagglutinin peptide (HA) using the HA-CLIO nanoswitch/antibody to HA (anti-HA) configuration. Addition of anti-HA to HA-CLIO caused an initial T2 drop, which was reversed by the addition of increasing concentrations of HA.
- FIG. 8B is a graphical representation of changes in T2 values obtained with different HA concentrations.
- FIGS. 8C, 8D, and 8E are graphical representations of particle size distribution as obtained by light scattering experiments.
- FIG. 8C shows the particle size distribution for dispersed HA-CLIO nanoparticles.
- FIG. 8A is a graphical representation of changes in T2 relaxation times obtained in a tube based assay for influenza hemagglutinin peptide (HA) using the HA-CLIO nanoswitch/antibody to HA (anti-HA) configuration. Addition of anti-HA
- FIG. 8D shows the switch from dispersed nanoparticles (dark bars) to the microaggregate state (light bars) upon anti-HA addition.
- FIG. 8E shown the switch of the HA-CLIO nanoparticles back to the dispersed state upon HA addition.
- FIG. 9A is a graphical representation of changes in T2 relaxation times obtained in a tube based assay for folic acid (FA) using the FA-CLIO nanoswitch/antibody to FA (anti-FA) configuration. Addition of anti-FA to FA-CLIO caused a initial T2 drop, which was reversed by the addition of increasing concentrations of FA.
- FIG. 9B is a graphical representation of changes in T2 values obtained with different FA concentrations.
- FIGS. 9C, 9D, and 9E are graphical representations of particle size distribution as obtained by light scattering experiments.
- FIG. 9C shows the particle size distribution for dispersed FA-CLIO nanoparticles.
- FIG. 9D shows the switch from dispersed nanoparticles (dark bars) to the microaggregate state (light bars) upon anti-FA addition.
- FIG. 9E shows the return of the FA-CLIO nanoparticles back to the dispersed state upon FA addition.
- This invention relates generally to magnetic resonance-based sensors (e.g., water relaxation-based sensors) and methods for detecting various analytes (e.g., exogenous analytes) in water-containing media (e.g., in vitro or in vivo media).
- various analytes e.g., exogenous analytes
- water-containing media e.g., in vitro or in vivo media
- the sensors described herein include magnetic nanoparticles, or nanoparticles with a magnetic moment under certain conditions, encapsulated within a semipermeable walled enclosure, e.g., an enclosure that retains the nanoparticles, but allows for passage of the analyte into and out of the confines of the sensor chamber.
- the walled enclosure can have one or more openings sized to enable the passage of the analyte, but not the nanoparticles (and binding agent, when present).
- Each of the nanoparticles has at least one moiety (e.g., a molecular fragment of the analyte being detected or a molecular fragment of a derivative, isostere, or mimic of the analyte being detected; or a protein) that is covalently or noncovalently linked to the nanoparticle.
- the sensor can further include a binding agent (e.g., a protein or a monoclonal antibody) also encapsulated within a semipermeable enclosure.
- the binding agent when present, is W 2
- the moiety is capable of binding to the analyte and the moiety; and the moiety is capable of binding to the analyte or the binding agent, hi general, the moiety and the analyte can each bind reversibly to the binding agent, when present; or the analyte can bind reversibly to the moiety.
- preferred chemistries for use in the practice of the invention are described herein.
- the chemistry of the analyte, binding moiety, and binding agent, per se, unless indicated otherwise herein, may be and typically is conventional, and may be adapted from other arts for use in the novel sensors and methods disclosed herein.
- a water relaxation-based sensor 10 for detecting a monovalent analyte includes a walled enclosure 12, a plurality of nanoparticles 20, and at least one binding agent (e.g., a protein) 18.
- the walled enclosure encapsulates a chamber 16 and is perforated with a plurality of openings 14. Both the nanoparticles 20 and the binding agent 18 are located within the confines of the chamber 16.
- Each of the nanoparticles 20 has at least one moiety (A b in FIG. IA) that is covalently or noncovalently linked to the nanoparticle and that includes a molecular fragment of the analyte being detected.
- the binding agent 18 is capable of binding (e.g., reversibly binding) to the analyte and the moiety A b .
- the analyte is smaller in size than either the nanoparticles 20 or the binding agent 18.
- the openings 14 are (i) larger in size than the analyte so as to allow the analyte to pass freely into and out of the chamber 16 (arrows 17) and (ii) smaller in size than either the nanoparticles 20 or the binding agent 18 so as to retain the nanoparticles 20 and the binding agent 18 within the chamber 16.
- the nanoparticles 20 bind to the binding agent 18 to form a nanoparticle aggregate 22 within the sensor chamber 16. It is believed that binding of the nanoparticles 20 to the binding agent 18 occurs through the moiety A b (see FIG. IA)- In general, formation of the nanoparticle aggregate 22 is an equilibrium controlled process (arrows 23).
- the binding agent-bound (e.g., binding protein-bound) nanoparticles of aggregate 22 are displaced from the binding agent by the analyte (A ex in FIG. IA), thereby altering the nanoparticle-binding agent (binding protein) equilibrium (arrows 23).
- a second equilibrium is established (arrows 25) in the chamber 16 among the analyte, s analyte-binding agent (binding protein complex 24, and (regenerated) nanoparticles 20.
- the regenerated nanoparticles produced in the second equilibrium controlled process (arrows 25) are substantially disaggregated relative to the bound nanoparticles of aggregate 22 formed in the first equilibrium controlled process (arrows 23).
- a water relaxation-based sensor 10 for detecting a multivalent analyte includes a walled enclosure 12 as described elsewhere and a plurality of nanoparticles 26.
- the nanoparticles are located within the confines of the chamber, and each of the nanoparticles has at least one moiety (e.g., at least one protein; at least 2, at least 3, at least 4) that is linked to the nanoparticle (hollow wedges in FIG. IB).
- the exogenous analyte is smaller in size than the nanoparticles 26.
- the openings 14 are (i) larger in size than the exogenous analyte so as to allow the analyte to pass freely into and out of the chamber 16 (arrows 17) and (ii) smaller in size than the nanoparticles 26 so as to retain the nanoparticles 26 within the chamber 16.
- the nanoparticles 26 are substantially disaggregated within the sensor chamber 16.
- the nanoparticles 26 bind to the multivalent analyte ( ⁇ A ex in FIG. IB) to form a nanoparticle aggregate 28 within the chamber (arrows 27).
- the nanoparticles that form part of aggregate 28 are substantially aggregated relative to nanoparticles 26. It is believed that binding of the nanoparticles 26 to the analyte occurs through the moiety (e.g., a protein). In general, formation of the nanoparticle aggregate 28 is an equilibrium controlled process (arrows 27). Referring to FIG.
- the sensors can be configured such that an analyte (e.g., a proton, H + ) can directly mediate self-assembly (aggregation and disaggregation of the nanoparticles).
- Sensors having the configuration shown in FIG. 1C can have one or more of the following properties: (i) the nanoparticles can bind directly to each other (i.e., there are no molecules serving a "bridges" between nanoparticles, as shown in Figures IA and IB); (ii) a single type of nanoparticle can be employed, and (iii) an analyte can control the self-assembly by binding to the surface of the nanoparticle.
- an analyte e.g., a proton, H +
- Sensors having the configuration shown in FIG. 1C can have one or more of the following properties: (i) the nanoparticles can bind directly to each other (i.e., there are no molecules serving a "bridges" between
- the surface of the nanoparticle is capable of binding to an analyte. Binding of the analyte can alter the physical properties of the surface, e.g. charge or hydrophobicity. While not wishing to be bound by theory, it is believed that the change in surface properties can alter the attraction between the nanoparticles, and self-assembly (or disassembly) of nanoparticles can occur.
- the surface of the nanoparticle can be designed to have a surface that can be charged or uncharged, as the pH is varied over some range of interest.
- a bifunctional crosslinking agents such as SPDP or SIA, (see, e.g., Koch et al. "Uptake and metabolism of a dual fluorochrome Tat-nanoparticle in HeLa cells.” Bioconjug Chem. 2003;74(6):l 115).
- SPDP bifunctional crosslinking agents
- SIA see, e.g., Koch et al. "Uptake and metabolism of a dual fluorochrome Tat-nanoparticle in HeLa cells.” Bioconjug Chem. 2003;74(6):l 115).
- the histidine is substantially unprotonated, and aggregation can occur through self-association among the hydrophobic leucine side chains.
- the sensors can be configured such that detection of a sequence of bases on a nucleic acid fragment can mediate self- assembly of the nanoparticles.
- Sensors having the configuration shown in FIG. ID can have one or more of the following properties: (i) two types nanoparticles can be prepared which have an affinity for each other, and (ii) one of the two types of nanoparticles is capable of binding the analyte.
- two types of nanoparticles can be synthesized, each having a specific sequence of synthetic oligonucleotide attached.
- nanoparticles When the two types of nanoparticles are mixed, they can self-assemble via the hybridization that occurs between bases of the oligonucleotides.
- An example of such double-stranded, oligonucleotide-mediated, nanoparticle aggregate is given hrPerez et; al., "DNA-based magnetic nanoparticle assembly acts as a magnetic relaxation nanoswitch allowing screening of DNA-cleaving agents.” JAm Chem Soc. 2002;124:2856.
- An analyte e.g. a sequence of bases present on a nucleic acid fragment
- can then enter the sensor and by binding to one of the types of particles can induce the dissociation of aggregates.
- the concentration dependent reaction of the analyte with the nanoparticle aggregate alters the nanoparticle aggregation state
- the presence and quantity of the exogenous analyte can be sensed, for example, as a change in the T2 relaxation times of water inside of the sensor chamber. It is known, for example, that water T2 relaxation times shorten upon aggregation or clustering of previously dispersed (e.g., monodispersed, polydispersed) magnetic nanoparticles.
- the superparamagnetic iron oxide core of individual nanoparticles becomes more efficient at dephasing the spins of the surrounding water protons (i.e., enhancing spin-spin relaxation times, e.g., T2 relaxation times).
- the analyte can be detected and quantified in the sampling media by monitoring the relaxation properties of the water that is present within the sensor chamber 16 (e.g., measuring changes, e.g., increases and decreases, in T2 relaxation times of water that is present within the sensor chamber).
- the T2 relaxation times of the water inside of the sensor chamber 16 are expected to decrease in the absence of analyte (due to formation of the nanoparticle aggregate 22) and then increase relative to these depressed values in the presence of analytes (due to displacement and subsequent disaggregation of the bound nanoparticles of aggregate 22).
- FIG. IA the T2 relaxation times of the water inside of the sensor chamber 16 are expected to decrease in the absence of analyte (due to formation of the nanoparticle aggregate 22) and then increase relative to these depressed values in the presence of analytes (due to displacement and subsequent disaggregation of the bound nanoparticles of aggregate 22).
- FIG. IA the T2 relaxation times of the water inside of the sensor chamber 16 are
- the T2 relaxation times of the water inside of the sensor chamber 16 are expected to increase in the absence of multivalent analyte and then decrease relative to these values in the presence of the multivalent analytes. Since the binding agent and/or the nanoparticles are confined within the chamber 16, the changes in nanoparticle aggregation occurring within the sensor chamber 16 in general do not substantially alter the proton relaxation of water outside of the chamber (i.e., bulk water).
- any water relaxation phenomena associated with nanoparticles or with their change in aggregation state can be used.
- T2 can generally be determined in a relatively fast and facile manner.
- measurements of nanoparticle aggregation can use T2 in conjunction with other relaxation processes such as Tl.
- Measurements of Tl and T2 can be used to correct for small changes in nanoparticle aggregation state within the sensor, due to a small expansion of contraction of the chamber. Accordingly, as used herein, references to measurement of relaxation phenomenon or magnetic relaxivity is intended to embrace all such relaxation related processes, including measurement of Tl.
- the size and shape of the sensor 10 can be selected as desired, hi some embodiments, the sensors can be, for example, tubular, spherical, cylindrical, or oval shaped. The sensors described herein can have other shapes as well. hi some embodiments, the size and shape of the sensor can be selected to accommodate a desired or convenient sample holder size and/or sample volume (e.g., in in vitro sensing applications). In general, the volume of sensor can be selected to enable the sensor to distinguish between the relaxation properties of water inside of the chamber and the water outside of the chamber.
- the senor size can be selected so as to accommodate a sample volume of from about 0.1 microliters ( ⁇ L) to about 1000 milliliters (mL) (e.g., about 1 ⁇ L (e.g., with animal imagers), 10 ⁇ L (e.g., with clinical MRI instruments) or 0.5 mL.
- the sensor can have a tubular shape in which the open end of the tube has a diameter of from about 1 millimeter (mm) to about 10 mm (e.g., 5 mm 7.5 mm).
- the sensor size and shape can be selected on the basis of the spatial resolution capabilities of conventional magnetic resonance technology (e.g., in in vitro sensing applications), hi certain embodiments, the longest dimension of the sensor can be from about 0.01 mm to about 2 mm (e.g., 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1).
- the applied magnetic field can be, for example, about 0.47 Tesla (T), 1.5 T, 3 T, or 9.4 T (animal assays generally).
- the walled enclosure 12 separates the chamber 16 from the bulk sample media and provides one or more conduits (e.g., openings 14) for entry of the exogenous analyte (if present) from the bulk sample media.
- the walled enclosure 12 can be any semipermeable material (e.g., a biocompatible semipermeable material) that is permeable to the exogenous analyte and water and substantially impermeable to the nanoparticles and the binding agent.
- the semipermeable material can be an ultrafiltration or dialysis membrane, hi some embodiments, the semipermeable material can be a polymeric substance (e.g., polymeric substances used for encapsulating transplanted cells, see, e.g., M.S. Lesney, Modern Drug Discovery 2001, 4, 45). In some embodiments, the semipermeable material can be a material used in small implantable, sustained release devices (e.g., those used in implantable, sustained release birth control devices, e.g., Depo-Provera, Norplant, Progestasert; or those described in C. I. Thompson et al, Can J Physiol Pharmacol 80, 180-92 (Mar, 2002) or D. C. Stoller, S. R. Thornton, F. L. Smith, Pharmacology 66, 11-8 (Sep, 2002)).
- sustained release devices e.g., those used in implantable, sustained release birth control devices, e.g., Depo-Provera, Norplant,
- the walled enclosure is relatively resistant to fouling or coating under the sampling conditions, thereby increasingly the likelihood that the walled enclosure can maintain the specified pore size of the openings 14 (e.g., increasing the likelihood that openings 14 will remain substantially unblocked during sensing).
- Fouling is the closure of pores (e.g., openings 14) due to the adsorption of protein that blocks pore. Fouling can be ascertained by placing materials in biological fluids (e.g., blood) and evaluating their performance using biocompatibility testing methods known in the art.
- the walled enclosure 12 can be essentially nonimmunogenic, thereby minimizing the likelihood of causing unwanted immune or toxic side effects in a subject (e.g., a human).
- biocompatible, semipermeable materials include without limitation polysaccharide based materials (cellulose), modified carbohydrate (cellulose ester), or polyvinyl pyrolidine.
- the walled enclosure 12 can be made of a relatively inflexible semipermeable material, meaning that the encapsulated chamber 16 is a true space or void that does not substantially change in volume when contacted with the fluid sample media.
- the walled enclosure can be a relatively flexible semipermeable material, meaning, for example, that the encapsulated chamber can expand in volume when contacted with the fluid sample media (e.g., by intake of the fluid sample media).
- the walls of the enclosure 12 are sufficiently thin to allow rapid sensor equilibration to changes in exogenous analyte levels.
- the membrane that forms the wall can have a thickness of from about 1 and about 500 hundred microns.
- the pore size of the openings 14 can be selected so as to meet the molecular exclusion criteria described herein (i.e., permeable to the exogenous analyte and water and substantially impermeable to the nanoparticles and the binding agent).
- molecular exclusion can be exclusion by molecular weight.
- each of the openings can have a pore size of from about 1 KDa to about 500,000 kDa (e.g., a pore size that allows passage of molecules that have a certain molecular weight).
- Each of the openings can have a size (pore size) of from about 1 kDa to about 1 um (e.g., about 1 kDa to about 300,000 kDa; about 1 kDa to about 100,000 kDa; about 1 kDa to about 5 kDa; 1 kDa to about 3 kDa; 1 kDA to about 1 ⁇ m).
- the openings can have a pore size of about 1 kDa or about 3 kDa.
- the semipermeable material can be Spectra/Por® tubing, Slide-A-Lyzer® microcassetes or dialysis fibers. Such materials are generally preferred for applications not involving implantation.
- the semipermeable material has a pore size that is larger in size than the analyte to permit passage of the analyte into and out of the chamber, but sufficiently small to retain magnetic nanoparticles and other reagents such as binding agents (e.g., a binding protein) within the confines of the chamber.
- the semipermeable material can be selected for the stability (long term function) in the fluid, which contains the analyte to be measured (e.g., blood plasma, interstitial fluid, cerebral spinal fluid of a human or animal subject).
- the semipermeable material can be further selected on the basis of whether the sensor is implanted or whether the fluid to be assayed is contained within a vessel that is outside of the subject (e.g., a bioreactor, tube or pipe).
- the magnetic particles can be nanoparticles (e.g., having a particle size of from about 10 nanometers (nm) to about 200 nm) or particles (e.g., having a particle size of from about 200 nm to about 5000 nm) provided that the particles remain essentially suspended (i.e., the particles do not settle).
- the term "magnetic nanoparticles” refers to any particle that is always magnetic and any particle that has a magnetic moment under certain conditions (e.g., in an applied electromagnetic field). Particle settling can generally be avoided by using relatively small particles (e.g., nanoparticles) or relatively large particles whose density is comparable to that of water.
- the density of particles can be altered by using polymers of different densities in their synthesis.
- the nanoparticles or particles have a surface that permits the attachment of biological molecules.
- the magnetic particles can be nanoparticles having a particle size of from about 10 ran to about 500 nm (e.g., about 15 to about 200 nm, about 20 to about 100 nm, about 10 nm to about 60 nm, about 20 nm to about 40 nm, about 30 nm to about 60 nm, about 40 to 60 nm; or about 50 nm).
- the unfunctionalized metal oxides are generally crystals of about 1-25 nm, e.g., about 3-10 nm, or about 5 nm in diameter.
- Magnetic materials larger than nanoparticles can be used.
- such particles can have one or more of the following properties: (i) it is desirable that the particles have a relatively high R2, i.e., alter water relaxation, (ii) it is desirable that the particles not to have a high susceptibility to settle significantly by gravity during the time course of the assay, (iii) it is desirable that the particles have a surface for the attachment of biomolecules, preferably amino or carboxyl groups. Examples include microspheres of from about 1-5 micron in diameter.
- Such particles can be obtained, e.g., from commercial suppliers, which include Dynbead magnetic microspheres from Invitrogen (Carlsbad, CA), microspheres from Bangs Laboratories (Fishers, IN), and Estapor® Microspheres from Merck or EMD Life Sciences (Naperville, IL).
- the particles can be unfunctionalized magnetic metal oxides, such as superparamagnetic iron oxide.
- the magnetic metal oxide can also include cobalt, magnesium, zinc, or mixtures of these metals with iron.
- the term "magnetic” as used herein means materials of high positive magnetic susceptibility such as paramagnetic or superparamagnetic compounds and magnetite, gamma ferric oxide, or metallic iron.
- the nanoparticles 20 include those having a relatively high relaxivity, i.e., strong effect on water relaxation.
- the particles can have a relatively high relaxivity owing to the superparamagnetism of their iron or metal oxide.
- the nanoparticles e.g., 20 or 26
- the nanoparticles have an Rl relaxivity between about 5 and 30 mM "1 sec "1 , e.g., 10, 15, 20, or 25 mM "1 sec “1 .
- the nanoparticles e.g., 20 or 26
- nanoparticles e.g., 20 or 26
- the nanoparticles e.g., 20 or 26
- concentrations of iron can be from about 2 micrograms ( ⁇ g)/mL to about 50 ⁇ g/mL Fe.
- the iron concentration is selected so as to be sufficiently high to alter the relaxation properties of water.
- lower iron concentrations can be used.
- higher iron concentrations can be used.
- Each of the nanoparticles e.g., 20 or 26
- includes at least one moiety e.g., at least 2, at least 3, at least 4) that is covalently or noncovalently linked to the nanoparticle.
- the moiety can be linked to the nanoparticle via a functional group.
- the functional group can be chosen or designed primarily on factors such as convenience of synthesis, lack of steric hindrance, and biodegradation properties.
- Suitable functional groups can include -O-, -S-, -SS-, -NH-, -NHC(O)-, -(O)CNH-, - NHC(O)(CH 2 ) ⁇ C(O)-, -(O)C(CH 2 ) n C(O)NH-, -NHC(O)(CH 2 ) n C(O)NH-, -C(O)O-, - OC(O)-, -NHNH-, -C(O)S-, -SC(O)-, -OC(O)(CH 2 ) n (O)-, -O(CH 2 ) n C(O)O-, - OC(O)(CH 2 ) n C(O)
- Functional groups having cyclic, unsaturated, or cyclic unsaturated groups in place of the linear and fully saturated alkylene linker portion, (CH 2 ) n can also be used to attach the moiety to the nanoparticle.
- the functional group can be selected so as to render the nanoparticle larger in size than the opening(s) in the wall of the chamber so as to retain the nanoparticle within the confines of the chamber (e.g., where a relatively large analyte is being detected such as a lipoprotein).
- the functional group can be -NHC(O)(CH 2 ) n C(O)NH-, in which n can be 0-20. In certain embodiments, n can be 2, 3, 4, 5, or 6 (preferably, 2).
- the functional group can be present on a starting material or synthetic intermediate that is associated with either the nanoparticle portion or the moiety portion of the nanoparticles (e.g., 20 or 26).
- a nanoparticle-based starting material can contain one or more functional groups for attachment of one or more moieties, (e.g., 2, 4, 6, 8, 10, 15, 20, 25, 30, 35, 40, 45, or 50 functional groups).
- the nanoparticle can be an amino-derivatized cross-linked iron oxide nanoparticle (e.g., NH 2 -CLIO).
- the number of moieties e.g., a molecular fragment of the analyte being detected or a molecular fragment of a derivative, isostere, or mimic of the analyte being detected; or a protein
- the number of moieties can be equal to or less than the number of functional groups that are available for attachment of the moiety(ies) to the nanoparticle.
- the number of moieties per nanoparticle can be selected as desired (e.g., depending on the number and location of binding sites on the binding agent (e.g., a protein) and/or if it is desired to have the nanoparticle cross link binding agents (e.g., proteins) when more than one binding agent is present),
- the magnetic particles can be multivalent particles in which multiple copies of a monovalent material are attached to the same particle, hi general, the valency can be from about 2.5 to about 20 copies of bound moiety per nanoparticle (i.e., average numbers of copies per nanoparticle, thus some particles can be monovalent within a given population of generally multivalent particles). Higher levels are not necessarily believed to be needed for function.
- Multivalent nanoparticles can be prepared by attaching two or more functional groups per nanoparticle. Multivalent nanoparticles can also be prepared by attaching either multivalent or monovalent binding agents (e.g., proteins).
- moieties can include, without limitation, carbohydrates (e.g., glucose, polysaccharides), antibodies (e.g., monoclonal antibodies, biotinylated anti-GFP polyclonal antibody), amino acids as well as derivatives and stereoisomers thereof (e.g. D-phenylalanine), chiral moieties, lipids, sterols, lipopolysaccharides, lipoproteins, nucleic acids, oligonucleotides, therapeutic agents (e.g., folic acid), metabolites of therapeutic agents, peptides (e.g., influenza hemagglutinin peptide), or proteins.
- carbohydrates e.g., glucose, polysaccharides
- antibodies e.g., monoclonal antibodies, biotinylated anti-GFP polyclonal antibody
- amino acids as well as derivatives and stereoisomers thereof (e.g. D-phenylalanine)
- chiral moieties e.g
- the moiety can be, or include as part of its chemical structure, a molecular fragment of the analyte being detected or a molecular fragment of a derivative, isostere, or mimic of the analyte being detected.
- such a moiety is one that is (i) recognized by the binding agent (e.g., protein, e.g., a binding protein) and (ii) displaceable from the binding agent by the analyte (i.e., the analyte can compete with the moiety for binding to the binding agent (e.g., a protein)).
- the binding agent e.g., protein, e.g., a binding protein
- the analyte can compete with the moiety for binding to the binding agent (e.g., a protein)
- the moiety and the analyte being detected can be substantially similar in structure to one another and have substantially similar binding affinities towards the binding agent.
- the moiety and the analyte being detected may not necessarily be substantially similar in structure, but may have substantially similar binding affinities towards the binding agent.
- Nanoparticles having at least one moiety that is a molecular fragment of the analyte being detected or a molecular fragment of a derivative, isostere, or mimic of the analyte being detected can have the general formula (A):
- NP 0 is the nanoparticle core
- - is a covalent linkage (e.g., a chemical bond or any linking functional group described herein) that connects any atom of the fragment to the nanoparticle
- z is 1-50 (e.g., 1-40, 1-30, 1-25, 1-20, 2-20, 2, 4, 6, 8, 10, and 15).
- X can be a hydrogen atom that forms part of an amino or hydroxy group that is present in the analyte; or X can be functional group, such as an amino group or a hydroxy group.
- the corresponding nanoparticle can have, for example and without limitation, the structure (A-O) Z -NP C or (A-NH) 2 -NP 0 .
- the moiety can include a carbohydrate as part of its chemical structure (e.g., glucosyl).
- the moiety can include a molecular fragment of a carbohydrate analyte being detected or a molecular fragment of a derivative, isostere, or mimic of a carbohydrate analyte being detected.
- the moiety can have formula (I):
- the moiety of formula (I) can be used in conjunction with a sensor for detecting and quantifying glucose.
- the moiety can be a monovalent or multivalent protein (e.g., having at least two (e.g., three, four, five, or six)) binding sites.
- the nanoparticle can further include a substituent that serves to render the nanoparticle larger in size than the opening(s) in the wall of the chamber so as to retain the nanoparticle within the confines of the chamber (e.g., where a relatively large analyte is being detected such as a lipoprotein).
- a substituent that serves to render the nanoparticle larger in size than the opening(s) in the wall of the chamber so as to retain the nanoparticle within the confines of the chamber (e.g., where a relatively large analyte is being detected such as a lipoprotein).
- the binding agent can be absent (e.g., when detecting multivalent analytes with nanoparticles in which the covalently or noncovalently linked moiety is, e.g., a protein; see, e.g., assay configuration delineated in FIG. IB).
- the assay can measure multivalent analyte proteins, using a sensor with a pore size that is large enough to allow analyte to enter and leave the chamber, while retaining nanoparticles, i.e. the pore size in FIG. IA and FIG IB. can be adjusted.
- Various assay configurations are described herein.
- the binding agent can be present (e.g., when detecting monovalent analytes with nanoparticles, which include a molecular fragment of the analyte being detected or a molecular fragment of a derivative, isostere, or mimic of the analyte being detected; see, e.g., assay configuration delineated in FIG. IA).
- the binding agent can be, for example, a protein, an antibody, a lectin, a receptor binding protein, a binding domain of a protein, a synthetic material, or a non-protein material, hi some embodiments, the binding agent can be a protein.
- the binding protein can be a multi-valent binding protein having at least two binding sites (e.g., three, four, five, or six).
- binding between the binding agent e.g., a protein or antibody
- the nanoparticle via the moiety
- binding between a moiety and an analyte is reversible.
- the analyte can be detected and quantified in a non- consumptive manner (i.e., the binding agent or the moiety reversibly binds, but does not consume, the analyte). This reversibility provides a steady state condition for bound and unbound analyte that can be quantitated.
- Analyte concentration can then be mathematically calculated using conventional methods.
- the protein binding agent can bind a therapeutic agent or metabolite thereof; a carbohydrate (e.g., glucose; e.g., the protein can be conconavalin A); or an amino acid (e.g., the binding protein can selectively bind one enantiomer over another, e.g., D-alanine versus L-alanine).
- the protein can be green fluorescent protein (GFP).
- the protein binding agent can be a monomer, hi other embodiments, the protein binding agent can be multimeric binding agent (e.g., prepared by making a fusion protein that includes several copies of one protein or cross-linking monomers to create a multivalent binding moiety).
- the binding agent can be an enzyme, which is modified so that it can bind to a substrate, but does not catalyze a reaction.
- the binding agent can bind a lipid, a sterol, a lipopolysaccharide, or a lipoprotein.
- the binding agent can be a protein that binds a sterol (e.g., apoSAAp for binding cholesterol, see, e.g., Liang and Sipe, 1995, "Recombinant human serum amyloid A (apoSAAp) binds cholesterol and modulates cholesterol flux", J. Lipid Res, 36(1): 37).
- the binding agent can be a receptor that binds a lipoprotein (e.g., a soluble low density lipoprotein and/or mutants thereof, see, e.g.,Bajari et al, 2005, "LDL receptor family: isolation, production, and ligand binding analysis", Methods, 36: 109-116; or Yamamoto et al., 2005,
- the binding agent can be a protein that binds a fatty acid (e.g.,human serum albumin, see, e.g., Fang et al, 2006, "Structural changes accompanying human serum albumin's binding of fatty acids are concerted", 1764(2):285-91. Epub 2005 Dec 27).
- the binding agent can be a monoclonal antibody, a polyclonal antibody, or a oligonucleotide.
- the binding agent can be, e.g., an antibody to folic acid or an antibody to influenza hemagglutinin peptide.
- the analyte can be chiral.
- the chiral analyte can be present together with one or more optically active moieties in the sample (e.g., a stereoisomer of the chiral analyte in the sample, e.g., the enantiomer of the chiral analyte in the sample).
- the chiral analyte can be an amino acid.
- the sensors described herein can be used to detect, monitor, and quantify a of analytes that can include, without limitation, ions, small molecules, proteins, viruses and lipoproteins " (see table 1).
- the analyte can be a carbohydrate (e.g., glucose); a lipid, a sterol, a lipopolysaccharide, a lipoprotein, a nucleic acid or an oligonucleotide; therapeutic agents (e.g., folic acid), metabolites of therapeutic agents, peptides (e.g., influenza hemagglutinin peptide), or a protein.
- a carbohydrate e.g., glucose
- a lipid, a sterol, a lipopolysaccharide, a lipoprotein, a nucleic acid or an oligonucleotide e.g., folic acid
- peptides e.g., influenza hemagglutinin peptide
- nanoparticles having reactive functional groups e.g., electrophilic functional groups such as carboxy groups or nucleophilic groups such as amino groups
- electrophilic functional groups such as carboxy groups or nucleophilic groups such as amino groups
- Carboxy functionalized nanoparticles can be made, for example, according to the method of Gorman (see WO 00/61191). In this method, reduced carboxymethyl (CM) dextran is synthesized from commercial dextran. The CM-dextran and iron salts are mixed together and are then neutralized with ammonium hydroxide. The resulting carboxy functionalized nanoparticles can be used for coupling amino functionalized groups, (e.g., a further segment of the functional group or the substrate moiety).
- CM carboxymethyl
- Carboxy-functionalized nanoparticles can also be made from polysaccharide coated nanoparticles by reaction with bromo or chloroacetic acid in strong base to attach carboxyl groups.
- carboxy-functionalized particles can be made from arnino- functionalized nanoparticles by converting amino to carboxy groups by the use of reagents such as succinic anhydride or maleic anhydride.
- Nanoparticle size can be controlled by adjusting reaction conditions, for example, by using low temperature during the neutralization of iron salts with a base as described in U.S. Patent No. 5,262,176. Uniform particle size materials can also be made by fractionating the particles using centrifugation, ultrafiltration, or gel filtration, as described, for example in U.S. Patent No. 5,492,814.
- Nanoparticles can also be synthesized according to the method of Molday (Molday, R. S. and D. MacKenzie, "Immunospecific ferromagnetic iron-dextran reagents or the labeling and magnetic separation of cells, " J. Immunol. Methods, 1982, 52(3):353-67, and treated with periodate to form aldehyde groups.
- the aldehyde- containing nanoparticles can then be reacted with a diamine (e.g., ethylene diamine or W
- Dextran-coated nanoparticles can be made and cross-linked with epichlorohydrin.
- the addition of ammonia will react with epoxy groups to generate amine groups, see, e.g., Josephson et al., Angewandte Chemie, International Edition 40, 3204-3206 (2001); Hogemann et al., Bioconjug. Chem., 2000, ll(6):941-6; and Josephson et al., "High- efficiency intracellular magnetic labeling with novel superparamagnetic-Tat peptide conjugates," Bioconjug. Chem. , 1999, 10(2): 186-91.
- This material is known as cross- linked iron oxide or "CLIO" and when functionalized with amine is referred to as amine- CLIO orNH 2 -CLIO.
- Carboxy-functionalized nanoparticles can be converted to amino-functionalized magnetic particles by the use of water-soluble carbodiimides and diamines such as ethylene diamine or hexane diamine.
- Nanoparticles 20 having a moiety corresponding to formula (I) can be prepared by contacting amino-CLIO (NH 2 -CLIO) with succinic anhydride (pH 8.5) followed by 2- aminoglucose in the presence of a carbodiimide (e.g. a water soluble carbod ⁇ mide, e.g., l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) /N- hydroxysuccinimide (NHS), pH 6.0 (see FIG.2).
- a carbodiimide e.g. a water soluble carbod ⁇ mide, e.g., l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) /N- hydroxysuccinimide (NHS), pH 6.0 (see FIG.2).
- a carbodiimide e.g. a water
- Such nanoparticles are referred to herein as “G-CLIO 5 " "GIu-CLIO,” or “GIu-CLIO nanoswitcb.es.”
- Folic acid (FA) can be conjugated to NH 2 -CLIO using a water soluble carbodiimide (e.g. EDC/NHS, pH 6.0) to provide nanoparticles 20 having a folic acid- containing moiety linked to the nanoparticle.
- a water soluble carbodiimide e.g. EDC/NHS, pH 6.0
- Such nanoparticles are referred to herein as “FA-CLIO” or “FA-CLIO nanoswitches.”
- Influenza hemagglutinin peptide can be conjugated to NH 2 -CLIO with, e.g., N-succinirnidyl 3-(2-pyridyldithio) propionate (SPDP) (PBS buffer, pH 7.4 to provide nanoparticles 20 having an HA-containing moiety linked to the nanoparticle.
- SPDP N-succinirnidyl 3-(2-pyridyldithio) propionate
- PBS buffer pH 7.4
- Such nanoparticles are referred to herein as "HA-CLIO” or "HA-CLIO nanoswitches.”
- the nanoparticles used in the sensors described herein can also be prepared using the conjugation chemistry described in, e.g., Sun, E.Y., Josephson, L., Kelly, K., Weissleder, R. Bioconjugate Chemistry 2006, 17, 109-113, which is incorporated by reference herein.
- the nanoparticles used in the sensors described herein can also be prepared using "click chemistry" methodology described in, e.g., KoIb et al, Angew Chem IntEd Engl., 2001, 40:2004-2021.
- the combination of nanoparticles 20, analyte, and protein binding agent 18 used in the sensors described herein can be selected as desired.
- the combination of nanoparticles, analyte, and binding agent used in the sensors can be based on the assay configurations described in, e.g., Josephson, et al., Angewandte Chetnie, International Edition 40, 3204-3206 (2001); Perez et al., Nat Biotechnol 20, 816-20 (2002); J. M. Perez et al. JAm Chem Soc 124, 2856-7 (2002); and Tsourkas et al.
- Table 2 illustrates representative assay configurations.
- the sensors described herein can be used to monitor physiological concentrations of glucose (see Examples section).
- any MR-based method that is capable of discriminating water T2 relaxation times in the sensor chamber from those outside of the sensor chamber can be used to monitor the sensors.
- Such methods can be MR imaging or MR non-imaging methods.
- the sensor can be implanted in a tube of flowing tube of fluid, minimizing the volume of a homogeneous magnetic field needed but still using the spatial encoding methods of MRI instrumentation.
- the applied magnetic field need not be homogeneous, a requirement of magnets used to generate MR images.
- Selective excitation magnets were considered in early MR imager designs (see, e.g., Z. Abe, K. Tanaka, Y. Yamada, Radiat Med 2, 1-23).
- a variable field strength hand-held magnet and excitation/receiver coil are used for analyzing the relaxation properties of samples within several millimeters of the magnet in commercial devices, see, e.g., http://www.minispec.com/products/ProFiler.htm. These devices can be used with the new sensors.
- T2 relaxation times can be determined by relaxation measurements using a nuclear magnetic resonance benchtop relaxometer.
- T2 relaxation time measurements can be carried out at 0.47 T and 40 0 C (Bruker NMR Minispec, Billerica, MA) using solutions with a total iron content of 10 ⁇ g Fe/mL.
- T2 relaxation times can be determined by magnetic resonance imaging of 384-well plates (50 ⁇ L sample volume), allowing parallel measurements at higher throughput.
- This technique is described in, for example, Perez, J. M., et al. Nat Biotechnol 2002, 20, 816- 820; and Hogemann, D., et al. Bioconjug Chem 2002, 13, 116-121.
- the measurement of Tl can be used to determine nanoparticle concentration, while measurements of T2 can be used to determine nanoparticle aggregation.
- [A] the concentration of analyte, can be a simple or complex function of R2, which reflects the aggregation state of the particles.
- Nanoparticle aggregation can also be determined without measurement of T2 as the examples below indicate.
- relaxation based sensors can be used to monitor exogenous analytes in any enclosed, aqueous system including bioreactors or fluids in a variety of industrial applications.
- the senor can be an implantable sensor implanted subcutaneously (e.g., the sensor can be implanted in an extremity of a subject so as to avoid having the entire body of the subject surrounded by the magnetic field).
- the invention is further illustrated by the following Examples.
- the Examples are provided for illustrative purposes only, and are not to be construed as limiting the scope or content of the invention in any way.
- EDC l-ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride
- sulfo-NHS sulfosuccinimidyl ester of N-hydroxysuccinimide
- SPDP N-succinimidyl 3-(2- pyridyldithio) propionate
- All other chemicals were purchased from Sigma Aldrich and used as received.
- Amino-CLIO nanoparticles were synthesized by crosslinking the dextran coating with epichlorohydrin and reacting it with ammonia, to provide primary amine groups (see, e.g., Josephson, L.; Tung, C. H.; Moore, A.; Weissleder, R. Bioconjug Chem. 1999, 10, (2), 186-91.; and Josephson, L.; Perez, J. M.; Weissleder, R. Angewandte Chemie, International Edition 2001, 40, (17), 3204-3206).
- the number of amines was determined by reaction with SPDP and treatment with dithiothreitol that releases pyridine-2-thione (P2T) (see, e.g., Zhao, M.; Kircher, M. F.; Josephson, L.; Weissleder, R. Bioconjug Chem 2002, 13, (4), 840-4).
- P2T pyridine-2-thione
- Protein binding agents ConA, anti-folate acid antibody (anti-FA) and anti-HA antibody (anti-HA) were purchased from Sigma.
- T2 measurements a 0.47T relaxometer (Bruker) was used. Measurements were made in 0.5 mL ofPBS at 40 °C. The concentration of surface functionalized nanoparticles was between 8 and 15 ⁇ g/ml Fe, adjusted to give a starting T2 of about 150 msec. The concentrations of binding proteins were 1 mg/mL (Con A), 0.1 mg/mL (anti- HA) and 0.1 mg/mL (anti-FA). After addition of each amount of analyte, T2 was recorded several times until it reached a stable value. The size of nanoparticles was measured by a laser light scattering (Zetasizer,
- Example 1 GIu-CLIO Nanoswitches
- Conconavalin A is a tetravalent lectin that is known to react with glucose.
- Some of the sensors were prepared having a walled enclosure with a pore size of 3 kDa. In general, the walled enclosure of the sensors retained the GIu-CLIO nanoparticle and ConA, while permitting glucose to freely enter or leave the sensor.
- MION-47 and amino-CLIO were prepared as described elsewhere.
- D-Glucose, D-(+)-Glucosamine hydrochloride, succinic anhydride, Concanavalin A (ConA) and Sephadex G-25 were from Sigma Aldrich Co.
- l-Ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride (EDC) and sulfo-N- hydroxysuccinimide (sulfo-NHS) were from Pierce (Rockford, IL).
- NH 2 -CLIO was first converted to a carboxylic group functionalized nanoparticle, followed by coupling of 2-amino-glucose using a water-soluble carbodiimide.
- a carboxylic functionalized CLIO 2.0 mg succinic anhydride was added into 200 uL NH 2 -CLIO (10 mg Fe/mL, 42 NH 2 per 2064 Fe) with 300 uL (0.1 M) NaHCO 3 buffer, pH 8.5.
- the mixture was incubated at room temperature for two hours and succinic acid removed using a Sephadex G-25 column eluted with MES buffer (0.5 M NaCl, 0.05 M MES), pH 6.0.
- MES buffer 0.5 M NaCl, 0.05 M MES
- Glu-CLIO-ConA-Glucose Tube Assay Relaxation times were obtained at 0.47T, 40 0 C using a Minispec relaxometer (Bruker). To demonstrate the interaction between GIu-CLIO, ConA and glucose, experiments were performed directly in NMR tube (no semi-permeable walled enclosure), 10 ug Fe/mL, 800 ug/mL ConA. AU experiments were performed in PBS with 1 mM CaCl 2 and 1 mM MgCl 2 . Transverse relaxation times (T2's) were measured in the relaxometer, Bruker Minispec® NMS 120 at 0.47 T and 40 0 C.
- Example 1C Glu-CLIO-ConA-Glucose Tube Assay GIu-CLIO nanoswitches were diluted into tubes to obtain a T2 of 153 msec. Upon addition of ConA, T2 decreased and reached a plateau value of 65 msec (see FIG. 4A). Addition of increasing concentrations of glucose reversed this effect, with a constant T2 value observed at each glucose concentration (see FIG. 4B). The change in plateau T2 values occurred in a linear fashion over the physiological range of glucose concentrations (see FIG. 4B).
- Example IE Glu-CLIO-ConA-Glucose Sensor Assay with MRI Imaging
- GIu-CLIO 10 ug Fe/mL
- Con A 800 ug/mL
- nanoswitch/binding protein equilibrium would be maintained if the nanoswitches and binding proteins were enclosed in a semipermeable device, with pores that would allow analyte to enter but which would retain nanoparticles and binding protein. This would allow analyte concentrations to be raised or lowered, depending on the sensor environment (dialysate).
- a number of different units were investigated as possible including Spectra/Por tubing, Slide-A-Lyzer microcassettes and dialysis fibers.
- Spectra/Por tubing permitted the rapid and repeated transfer of the sensor from a 100 mL beaker, where glucose concentrations were cycled between 20 mg/dl and 400 mg/dl to an MR relaxometer, where T2 measurements were made in less than a minute.
- the T2 of sensor water changed in cycled between about 68 and 100 msec as it responded to changing concentrations of glucose (see FIG. 7). Both increases and decreases in glucose concentration resulted in alteration of the nanoswitch microaggregation state, evident by changes in T2, further demonstrating the equilibrium nature of nanoswitches.
- hemagglutinin peptide-CLIO a thiolated influenza hemagglutinin (HA) peptide with a C-terminal cysteine (YPYDVPDVAGGC) was synthesized by using Fmoc chemistry on Rink amide resin (Calbiochem, NovaBiochem) and was purified by reverse phase HPLC. The molecular weight of HA was confirmed by MALDI-TOF.
- amino-CLIO was first reacted with SPDP. After purification, 200 ⁇ L SPDP modified CLIO (5.0 mg/mL Fe) in PBS buffer, pH 7.4 was mixed with 100 ⁇ L of HA (50 mM) in DMSO.
- Example 2B HA-CLIO-anti-HA-HA Tube Assay Relaxation times were obtained at 0.47T, 40 0 C using a Minispec relaxometer
- Nanoswitches had similar properties when HA-CLIO replaced GIu-CLIO and antibody to HA (anti-HA) replaced ConA as shown in FIGS. 8A-8E. As shown in FIG.
- T2 dropped from 162 to 141 msec with the addition of anti-HA.
- Plateau values of T2 changed over a range of HA concentrations between 50 and 400 nM (FIG. 8B), which was about 80 fold lower than the concentrations of glucose needed to change T2 (2.5 ⁇ M-20 ⁇ M, FIG. 4B).
- FIGS. 8C, 8D, and 8E show that the analyte (HA) was capable of essentially completely reversing microaggregate formation (see FIGS. 8C, 8D, and 8E).
- HA analyte
- Example 3B FA-CLIO-anti-FA-FA Tube Assay Relaxation times were obtained at 0.47T, 40 0 C using a Minispec relaxometer
- the properties of the nanoswitch system were examined with FA-CLIO nanoparticles and anti-FA as the binding protein.
- T2 dropped from 155 msec to 113 msec with anti-FA addition.
- the range or concentrations of FA associated with changing T2 values are shown in FIG. 9B (5-20 nM) and was about 1000 fold lower than the concentrations of glucose measured (2.5 ⁇ M-20 ⁇ M, FIG. 4B).
- light scattering data indicated that the analyte (HA) was capable of essentially completely reversing microaggregate formation (see FIGS. 9C, 9D, and 9E).
- nanopartic ⁇ es and binding proteins maintained an equilibrium, continuously switching between a dispersed (disaggregated), low T2 state (20-40 nm) and microaggregated high T2 state (200-250 nm), depending the concentration of exogenous analyte. Based on light scattering data, high concentrations of exogenous analytes completely reversed microaggregate formation, returning the system to its original dispersed state expected for an equilibrium process. As indicated by the use of nanoswitches and binding proteins for glucose, FA, and HA, nanoswitches were able to detect chemically diverse analytes over a relatively wide range of concentration.
Abstract
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Also Published As
Publication number | Publication date |
---|---|
CA2606924C (en) | 2013-04-16 |
US8535949B2 (en) | 2013-09-17 |
CA2606924A1 (en) | 2006-11-16 |
WO2006122083A3 (en) | 2007-05-03 |
US20140017816A1 (en) | 2014-01-16 |
CN101253416A (en) | 2008-08-27 |
JP5021629B2 (en) | 2012-09-12 |
US20060269965A1 (en) | 2006-11-30 |
EP1880228B1 (en) | 2011-07-06 |
CN101253416B (en) | 2011-08-24 |
JP2008541089A (en) | 2008-11-20 |
US20100120174A1 (en) | 2010-05-13 |
EP2385386A1 (en) | 2011-11-09 |
EP1880228A2 (en) | 2008-01-23 |
ATE515711T1 (en) | 2011-07-15 |
EP2385386B1 (en) | 2012-11-14 |
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