WO2011053435A1 - Détection de cellules au moyen de nanoparticules ciblées et de leurs propriétés magnétiques - Google Patents

Détection de cellules au moyen de nanoparticules ciblées et de leurs propriétés magnétiques Download PDF

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WO2011053435A1
WO2011053435A1 PCT/US2010/051417 US2010051417W WO2011053435A1 WO 2011053435 A1 WO2011053435 A1 WO 2011053435A1 US 2010051417 W US2010051417 W US 2010051417W WO 2011053435 A1 WO2011053435 A1 WO 2011053435A1
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cells
nanoparticles
sample
magnetic
particles
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Edward R. Flynn
Richard S. Larson
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Scientific Nanomedicine, Inc.
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Priority to US13/249,994 priority Critical patent/US8447379B2/en

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B10/00—Other methods or instruments for diagnosis, e.g. instruments for taking a cell sample, for biopsy, for vaccination diagnosis; Sex determination; Ovulation-period determination; Throat striking implements
    • A61B10/02—Instruments for taking cell samples or for biopsy

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  • This invention relates to the detection and measurement of cells using targeted nanoparticles and a magnetic needle, a magnetic sensor, or a combination thereof, and is particularly useful in determining minimum residual disease in leukemia patients and in finding rare cells such as circulating tumor cells.
  • the bone marrow biopsy After initial treatment and while in remission, the bone marrow biopsy is expected to reveal zero leukemia cells.
  • Current techniques cannot reliably measure very low concentrations of leukemia cells. Consequently, harmful chemotherapy can be continued longer than necessary, and recurrence or incomplete treatment can be missed until the leukemia has progressed farther than desired (and produced enough cells to be measured by conventional means).
  • the targeted nanoparticles comprise magnetic nanoparticles conjugated an antibodies specific to cells of the first type.
  • the magnetic device comprises an elongated member having disposed thereon a plurality of magnetic regions.
  • the magnetic device further comprises a nonmagnetic sheath removably mounted over the elongated member.
  • the elongated member comprises a stainless steel rod, and the plurality of magnetic regions comprises a plurality of permanent magnets.
  • the permanent magnets comprise NdFeB magnets.
  • the sheath comprises a polyimide sheath.
  • the present invention provides an apparatus for the extraction of cells of a first type from a sample, comprising a plurality of targeted nanoparticles, each comprising a magnetic nanoparticle conjugated with a targeting agent that preferentially binds to cells of the first type; a sample holder configured to contain a sample which can include cells of the first type; and a magnetic device configured to be disposed in relation to the sample such that the magnetic device acts on targeted nanoparticles that have been bound to cells of the first type in the sample holder and extracts said cells from the sample.
  • the targeted nanoparticles comprise magnetic nanoparticles conjugated an antibodies specific to cells of the first type.
  • the magnetic device comprises an elongated member having disposed thereon a plurality of magnetic regions. In some embodiments, the magnetic device further comprises a nonmagnetic sheath removably mounted over the elongated member. In some embodiments, the elongated member comprises a stainless steel rod, and the plurality of magnetic regions comprises a plurality of permanent magnets. In some embodiments, the permanent magnets comprise NdFeB magnets. In some embodiments, the sheath comprises a polyimide sheath.
  • the present invention provides a method of detecting the presence of cancer in a patient, comprising: (a) obtaining a first sample of fluid, serum, tissue, or other substance collected from the patient, wherein the presence of a predetermined substance in the sample indicates the presence of cancer in the patient; (b) providing targeted nanoparticles, comprising magnetic nanoparticles conjugated with targeting agents, wherein a targeting agent comprises an agent that preferentially binds with the predetermined substance; (c) providing a second sample by subjecting the first sample to the targeted nanoparticles under conditions that allow binding of the targeting agents to the predetermined substance; (d) imposing a known magnetization on the nanoparticles in the second sample; (e) measuring the relaxation of the magnetization of the nanoparticles after imposition of the known magnetization; and (f) determining the presence of the predetermined substance in the second sample from the relaxation of the magnetization.
  • the predetermined substance is cells of a specific type of cancer, and the targeting agent comprises one or more antibodies that bind to those cells.
  • the predetermined substance is PSA, and the targeting agent comprises a PSA-specific antibody.
  • the predetermined substance and targeting agent comprise one or more or the pairs set forth in the specification.
  • the present invention provides a method of localizing a recurrence or metastasis of cancer in a patient, comprising: (a) detecting the presence of cancer according to the methods described herein; (b) injecting the patient with targeted nanoparticles, wherein the targeted nanoparticles preferentially bind with cells indicative of the recurrence or metastasis of cancer; (c) scanning the patient with a magnetic relaxation instrument, and identifying when the measured magnetic relaxation indicates bound nanoparticles; (d) Indicating the presence of recurrence or metastasis of cancer responsive to the identification of bound nanoparticles.
  • the cells indicative of recurrence or metastasis comprise cancer cells.
  • the targeted nanoparticles comprise magnetic nanoparticles conjugated with antibodies that preferentially bind with cancer cells.
  • Fig. 2 Normalized probability distribution function,w(D) based on TEM measurements on 2602 particles. A cubic spline interpolates between the measured points for both the susceptibility and relaxometry. A lognormal fit is also shown.
  • Fig. 3 Measured susceptibilities on curves of constant frequency, from top down on left: 0.1, 0.3, 1.0, 3, 10, 30, 100, 300, 1000 Hz. The measurements are 5°K apart and joined by straight lines. The ordinates are volume susceptibility in rationalized MKS. (a) Real part, (b) Imaginary part.
  • Fig.4 Representative samples of fitted constant temperature curves for susceptibility.
  • the 3 parameters in the model, Ms, K and T 0 are adjusted for each temperature to give best fit to the 9 frequency points, (a) Real part for temperatures starting right bottom up: 260 to 350 K in 10 K increments, (b) Imaginary part for 290 K, diamonds, 300 K, circles, 310 K, squares. Note that the largest fluctuations in the measured points occur at the two highest frequencies. There is relatively more noise in the imaginary than the real measurements because the signals are an order of magnitude smaller. All 30 constant T data sets were fit in this manner; only this representative sample is shown.
  • Fig. 9. Transmission electron microscopy of Ocean nanoparticles with nominal diameters of 20, 25, 30, and 35 nanometers. The SHP-20 particles show a generally uniform intensity in TEM. In the images of the larger particles, an increasing number of particles exhibit intensity variations (light and dark bands), which we attribute to polycrystallinity.
  • Fig. 10 (A) The amplitude of the out-of-phase component of the AC susceptibility ⁇ " vs. frequency for the SHP particles. (B) The moment/kg observed by SQUID relaxometry (gray bars) correlates well with ⁇ " at 1.0 Hz (black bars).
  • Fig. 11 Normalized magnetization vs. field obtained by DC susceptometry (solid symbols) compared to the Langevin function computed for 25 nm (dashed line) and 8 nm (dotted line) particles. The fits to the data (solid lines) are described in the text.
  • Fig. 13 Normalized magnetization vs. field data (solid symbols) obtained by relaxometry. The solid line was computed using the Langevin function, assuming 27.4 nm particles. The dashed lines were computed using the Moment Superposition Model, as described in the text.
  • B In photomicrographs of slides prepared after the 60 min. incubation, the Jurkat cells are stained pink, while the nanoparticles are stained blue. Untreated cells (left) express high levels of the CD3 receptor and show greater nanoparticle binding, while the trypsin-treated cells (right) are stripped of cell surface antigens and show significantly reduced binding. The observed SQUID relaxometry signal appears to be attributable to binding of nanoparticles to cells and nanoparticle aggregation.
  • Fig. 17 is an illustration of an example Superconducting Quantum Interference Device (SQUID) sensor system for relaxometry.
  • SQUID Superconducting Quantum Interference Device
  • Fig. 18 is an illustration of results with particular cells.
  • Fig. 21 is an illustration of the application to a magnetic biopsy needle.
  • Fig. 22 is an illustration of issues associated with production and characterization of nanoparticles.
  • Fig. 23 is an illustration of susceptometry measurements of nanoparticle properties.
  • Fig. 24 is an illustration f properties before and after addition of PEG.
  • Fig. 26 is an illustration of SQUID detection of specific Nanoparticle Binding.
  • U937 cells 1.0 x 107) were incubated with varying numbers of nanoparticles (A and B) or varying number of cells were incubated with nanoparticles (C and D).
  • the curves shown are nonlinear fits of the data. Data represents results from three separate experiments.
  • Fig. 27 is a schematic representation of magnetic needle and sampling of spiked sample. Shown is the schematic of the magnetic needle covered with a polyimide sheath prepared for insertion into a bone marrow sample (A), as well as the sheath-covered needle itself (C) and the magnetic needle inserted into an experimental sample (B). Sample tubes are placed into a sample holder on a platform under the SQUID sensors (D).
  • Fig. 28 is an illustration of the percentage of lymphoblasts in pre-needle and needle harvest samples of three leukemia patients.
  • the patient samples were diluted 1:1024 in donor blood, and the lymphoblast percentage was calculated both before and after needle harvest. Since neutrophils were present primarily due to dilution of sample with blood so that the percentage of blasts was low, the percentage of lymphoblasts was calculated with neutrophils included in the WBC count (A) and excluded from the WBC count (B).
  • Fig. 29 is a schematic illustration of the production and operation of targeting agents according to the present invention.
  • Fig. 31 is a schematic illustration of an example preparation of a sample for measurement according to the present invention.
  • Fig. 33 is a schematic illustration of measurements from the process described in connection with Fig. 32.
  • Fig. 34 is a schematic illustration of an apparatus suitable for use in the present invention.
  • the present invention provides methods and apparatuses using magnetic nanoparticles that have been conjugated with targeting agents so that they are preferentially bound to cells of one or predetermined types. After the targeted nanoparticles have bound to such cells, those cells can be separated from the rest of a sample with a magnetic device. Also, the magnetic properties of the targeted nanoparticles can be used to measure the number of target cells in the sample using magnetic relaxation, as an alternative to or in connection with separation using a magnetic device.
  • Fig. 29 is a schematic illustration of the production and operation of targeting agents according to the present invention.
  • the illustrations in the figure are highly simplified and intended for ease of explanation only, and are not intended to represent the actual shapes, sizes, proportions, or complexities of the actual materials involved.
  • a sample 81 e.g., a cell culture, a blood or tissue sample, or in vivo blood or tissue, comprises some cells of the type of interest (shown in the figure as circles with "V" shaped structures around the periphery) and some cells of other types (shown in the figure as ovals with rectangular structures around the periphery).
  • a plurality of magnetic nanoparticles 82 is provided, shown in the figure as small circles.
  • a plurality of targeting agents 83 is also provided, shown in the figure as small triangles. The nanoparticles and targeting agent are combined (or conjugated), forming targeted nanoparticles 84.
  • the targeted nanoparticles can then be introduced into the sample 85.
  • Cells of the type of interest have binding sites or other affinities for the targeting molecule, illustrated in the figure by "V" shaped structures around the periphery of such cells.
  • the number of binding sites can vary, but some types of cancer cells have been found to have many thousands of binding sites for specific antibodies, so the 8 sites shown on each cell in the figure is a very significant underrepresentation of the density of nanoparticles that can attach to such cells.
  • the targeting agents attach to the cells of the type of interest, illustrated in the figure by the triangular targeting molecules situated within the "V" shaped structures. Generally, each cell will have a large number of such binding or affinity sites.
  • Cells of other types do not have such binding sites or affinities, illustrated in the figure by ovals with no targeted nanoparticles attached. Targeted nanoparticles that do not bind to cells are left free in the body, illustrated in the figure by small circles with attached triangles that are not connected with any specific cell (both inside and outside the organ or other structure represented by the irregular curve).
  • Fig. 30 is a schematic illustration of a sample 85 such as that in Fig. 29 after introduction of a magnetic device 86.
  • the magnetic device 86 has attracted the nanoparticles in the sample 85, with the strongest attraction for those cells 88 that have many nanoparticles attached by the targeting agent.
  • Cells without nanoparticles 87, i.e., those that did not bind to the targeting agent, are not attracted to the magnetic device 86.
  • the magnetic device 86 can be removed from the sample 85 and examined to determine whether any cells of the targeted type are present.
  • the targeting agent Since substantially all cells of the targeted type present in the sample will have nanoparticles bound to them by the targeting agent, if there are any cells of the targeted type present in the sample originally there will be cells of the targeted type on the magnetic device.
  • the figure shows roughly equal number of targeted cells and non-targeted cells; in practice, there can be many times as many non-targeted cells as targeted cells, and the operation of the targeted nanoparticles and magnetic device will serve to pull only targeted cells from the sample. Accordingly, the presence of targeted cells in the sample can be determined even in the presence of very low targeted cell concentrations.
  • EXAMPLE MAGNETIC DEVICE EMBODIMENT AND APPLICATION Acute leukemias are bone marrow-derived malignancies for which the development of sensitive detection methods is crucial to improving clinical detection and outcomes.
  • technologies used to examine bone marrow samples may fail to detect the presence of leukemia cells below 1% to 5% of total leukocytes, i.e., minimal residual disease.
  • a major difficulty in detecting minimal residual disease using bone marrow aspiration is that random sampling can neglect areas of focal disease. As a result, opportunities opportunities to intensify therapy may be overlooked, leading to relapsed disease. In these cases, the ability to reliably detect residual leukemia cells, when present below 5%, to monitor the efficacy of therapy is critical to improving care.
  • Magnetic nanoparticles have become an increasingly important tool in both targeting and detecting cancer cells.
  • Cellular targeting may be achieved through attachment of receptor-specific ligands, including antibodies.
  • receptor-specific ligands expressed on tumor cells, such as CD34 on acute leukemia cells, could allow for targeting nanoparticles labeled with antibodies.
  • receptor-specific binding of nanoparticles leads to internalization in vitro and in vivo, thus increasing the potential number of nanoparticles associated with each cell target.
  • SPIONs superparamagnetic nanoparticles composed of iron oxide (SPIONs), conjugated to anti-CD34 antibodies, we hypothesized that we could create magnetically charged leukemia cells that could be preferentially collected using a magnetic source during standard bone marrow sampling procedures.
  • SPIONs magnetorelaxometry, whereby nanoparticles are briefly magnetized by a pulsed field, and the SQUIDs detect the nanoparticle magnetization as it relaxes back to equilibrium.
  • SPIONs have three specific properties that make them highly compatible for SQUID relaxometry detection: (a) they are superparamagnetic, (b) the individual magnetic moments of these particles align with a magnetic field, so that cells labeled with sufficient numbers of bound single particles with magnetic moments of ⁇ 4 x 10-18 A-m2 are detectable by SQUIDs, and (c) unbound single particles, even when present in large numbers, do not generate detectable SQUID signals.
  • Magnetic moments measured by SQUID relaxometry provide additional information regarding cellular binding and a secondary confirmation of microscopy results from magnetic needle collections.
  • the present invention provides for the enhancement of leukemia cell sampling using a magnetic device and nanoparticles.
  • the sensitivity and ability of the SQUID to quantify cell sampling is also described.
  • the invention can provide enhanced technologies for marrow sampling and for extraction of leukemia cells from bone marrow biopsy samples, which can improve clinical decision making and patient outcomes.
  • U937, Jurkat, and GA-10 cells were purchased commercially from American Type Culture Collection and cultured in PMI supplemented with 10% fetal bovine serum (FBS; v/v; HyClone), 1% penicillin streptomycin (v/v; Life Technologies-Bethesda Research Laboratories), and 4 ⁇ g/mL ciprofloxacin (Bayer). Cells were cultured in an incubator at 37°C with 5% C02 and maintained at a cell concentration between 1 x 10 s and 1 x 10 6 viable cells/mL.
  • U937, GA-10, and Jurkat represent myeloid, B-cell, and T-cell lineage leukemia cell lines. Each cell line expresses CD34.
  • Peripheral blood and bone marrow collection Peripheral whole blood was obtained from donors through venous puncture and was anticoagulated in 10 U/mL of heparin (Becton Dickinson). Bone marrow aspirations were performed in patients with acute leukemia who required a bone marrow evaluation as a part of their routine clinical care. Human subjects provided consent in accordance with local and federal guidelines. The patients were placed in the supine position, and the sacral area was draped in a sterile fashion. Local anesthesia was achieved with 1% Xylocaine (Abraxis Pharmaceutical) administered s.c, and periosteally. A Jamshidi needle (Baxter Healthcare Corporation) was inserted into the cortex of the posterior superior iliac spine.
  • a Quantum Simply Cellular kit (Bangs) was used for quantitation of cellular antigen expression in antibody binding capacity units as per manufacturer's instructions and described briefly here.
  • the Quantum Simply Cellular bead populations provided a means for constructing a QuickCal calibration curve (antibody binding capacity values versus fluorescence intensity). Cells were compared with antibody-labeled beads, and cell surface antigen expression was quantified in antibody binding capacity units. Approximately 1 x 10 s cells and Quantum Simply Cellular beads were incubated with mouse FITC-labeled anti- human anti-CD34 (Caltag).
  • Nanopartlcles were brought to pH 8.0 with 50 mmol/L NaHC03 (Sigma-Aldrich), 50 ⁇ g of anti-CD34 antibody (BD Biosciences) was added, and the mixture was incubated at room temperature on a Lab- Quake shaker for 2 h.
  • the antibody-nanoparticle mixture was centrifuged at 7,500 relative centrifugal force for 30 min at 4°C.
  • the magnetic needle is composed of a 17-cm-long, 1-mm-diameter stainless steel rod, at the end of which are two cylindrical NdFeB magnets 2 mm in length and separated by a stainless steel spacer 2 mm in length.
  • the components of the magnetic needle are contained within a tight-fitting polyimide tube and the needle assembly is then inserted into a removable polyimide sheath. Magnetically labeled cells are attracted to the both poles on both magnets and stick to the outside of the sheath. After extracting magnetic material from the marrow, the sheath is removed from the needle and inserted into media where the cells are liberated from the sheath.
  • Samples in 1.5 mL microcentrifuge tubes were centered under the seven channel SQUID sensor array (BTi 2004, 4D-Neuroimaging) and placed at a distance of 3.4 cm below the center sensor.
  • a uniform magnetic field of 38 Gauss was produced at the location of the sample for 0.3 s using a square Helmholtz array.
  • the decaying magnetization at each sensor was then sampled at a rate of 1 kHz and digitized using a NI-PXI8336 16-channel digitizer and LabVIEW 8.5.1 acquisition software (National Instruments). The pulsing sequence was repeated 10 times and the results averaged to increase the signal to noise ratio.
  • unconjugated nanoparticles also produced a detectable signal, indicating that low amounts of cellular nanoparticle uptake can be detected by SQUID, but is not easily visualized by light microscopy and special staining, or that the nanoparticles are agglomerated so they intrinsically produce a small magnetic moment.
  • neutrophils from the blood used for dilution were seen in addition to the lymphoblasts. Neutrophils were only present in these samples due to the use of donor blood to dilute the bone marrow and should not present a significant factor in collection of lymphoblasts pure bone marrow specimens.
  • a second lymphoblast percentage without the neutrophil component was calculated. As neutrophils have a very different morphology than lymphoblasts, neutrophils were easily indentified visually and excluded when examining needle collection slides microscopically (Fig. 28). Excluding the neutrophils, virtually 100% of the cells identified were lymphoblasts. Since neutrophils are primarily present in these samples because peripheral blood was used to dilute the number of blasts to determine the detection limits of this technique, we would not anticipate the same number of neutrophils in undiluted specimens.
  • the present example provides improvements to cellular targeting using superparamagnetic particles that provide new opportunities for its use as a clinical tool.
  • nanoparticles several techniques, including antibody-bound, small-molecule modification, and aptamer-based technologies have been studied. Since the accurate detection of residual disease has previously been shown to lead to response-based improvements in leukemia-free survival, we investigated the binding of ligand-bearing nanoparticles to CD34+ cells to enhance collection of lymphoblast cells using a magnetic needle as an initial step toward the development of a tool to increase the sensitivity of detection of residual disease in acute leukemia patients. We found the binding of nanoparticles to CD34+ cell lines was dependant on cell line receptor expression and the presence CD34 antibody ligand on the nanoparticle surface.
  • the limit of nanoparticle binding to a cell may be a result of steric considerations, either based on cell size or access to the receptor, or incomplete coupling of the nanoparticles with antibodies directed against CD34. Since U937 has a reported diameter of between 10 and 20 ⁇ , and the nanoparticles used in this study have a diameter of 140 nm, and a rough calculation indicates that ⁇ 1.6 x 10 4 to 6.5 x 10 4 nanoparticles can bind to the surface of a cell. Our observed limit seems to be in close agreement with this theoretical calculation.
  • the future clinical use of this device may show even higher collection enhancement than we observed. Nonetheless, the number of lymphoblasts collected on the needle remained fairly constant regardless of dilution factor, and at the lowest dilutions, the number of lymphoblasts in the pre-needle sample approaches the number of lymphoblasts recovered on the needle (data not shown).
  • Nanoparticles in the absence or presence of cells but without a targeting ligand, also showed a measurable background magnetic relaxometry signal. This background is derived from two sources: nanoparticle agglomeration visible by light microscopy in sample without cells as well as nonspecific uptake and binding of naked nanoparticles to cells. Agglomeration of the nanoparticles causes a signal similar to cell-nanoparticle binding and was visible by SQUID relaxation
  • the magnetic needle when combined with microscopy, proved to enhance the collection and identification of CD34-expressing cells.
  • the present invention can provide for ex vivo extraction of leukemia cells from patient bone marrow samples. In addition, this sampling modality has the capacity to identify minimal residual disease earlier, which may improve survival and reduce therapy-related patient toxicity.
  • An advantage of the nanoparticle used in this study is its potential for in vivo use for the detection and possible treatment of leukemias.
  • the present invention can also provide an ability to directly inject the antibody-labeled nanoparticles into the bone marrow and then specifically harvest or kill CD34-expressing cells.
  • a level of 0.2 ng/ml of PSA in the blood would indicate a tumor containing ten to one hundred million cells which would be shedding some cells into the blood.
  • a more sensitive determination of metastatic cells is important to determine that treatment for this reoccurrence of cancer should begin at an earlier stage. Indications of increased numbers of markers or detection of metastatic cells in the blood can be followed by searches throughout the body for the tumors using magnetic relaxometry sensor arrays that are targeted towards the type of cancer identified by the markers or detected cells in the serum.
  • These sensor arrays use the same type of ultra-sensitive sensors, such as SQUIDs, that are used to detect the cells in the blood but are arranged in an array of sensors to permit localization of the metastatic tumor.
  • the patient can be placed under this array which then scans the body, after injection of magnetic nanoparticles with antibodies specific to the markers or cell cancer type identified in the serum assay. This method increases the specificity for finding cancer while also increasing the detection sensitivity since the cancer type has been identified.
  • This method of metastatic cell detection using magnetic relaxometry or sensitive marker detection can be followed by localization of the cancer site responsible for shedding these cells into the serum and can be used following any type of cancer treatment whether it is surgery, chemotherapy or ablation. Detection of presence of these cells or markers can then indicate further therapy is needed such as continuation of the chemotherapy or radiation or targeted therapy when the source of the cells or markers is located.
  • Detection of cells in the body's fluids can also be used to indicate that further imaging for cancer is desirable.
  • Magnetic relaxometry is a very sensitive method for determining the presence of cancer clusters resulting from metastasis and can be used to examine the entire body for growths of cancer. In this method, the treatment can then be focused on the specific areas where cancer cell clusters have been detected. This can result in a considerable decrease in side effects as compared with a general therapy applied to the entire individual. Therapy can also be more effective when there is minimal spread of the disease, made possible by detection before significant spread. The result is more effective treatment, a general cost savings in medical care, and increased quality of life because the body's normal cells are not subjected to therapeutical agents that harm normal cells.
  • Fig. 31 is a schematic illustration of an example preparation of a sample for measurement according to the present invention.
  • the illustrations in the figure are highly simplified and intended for ease of explanation only, and are not intended to represent the actual shapes, sizes, proportions, or complexities of the actual materials involved.
  • a sample 11, e.g., blood or serum or tissue comprises some cells of the type of interest (shown in the figure as circles with "V" shaped structures around the periphery) and some cells of other types (shown in the figure as ovals with rectangular structures around the periphery).
  • a plurality of magnetic nanoparticles 12 is provided, shown in the figure as small circles.
  • a plurality of targeting molecules 13 is also provided, shown in the figure as small triangles. The nanoparticles and targeting molecules are combined (or conjugated), forming targeted nanoparticles 14.
  • the targeted nanoparticles can then be combined with the sample 15.
  • Cells of the type of interest have binding sites or other affinities for the targeting molecule, illustrated in the figure by "V" shaped structures around the periphery of such cells.
  • the targeting molecules attach to the cells of the type of interest, illustrated in the figure by the triangular targeting molecules situated within the "V" shaped structures.
  • each cell will have a large number of such binding or affinity sites.
  • Cells of other types do not have such binding sites or affinities, illustrated in the figure by ovals with no targeted nanoparticles attached.
  • Targeted nanoparticles that do not bind to cells are left free in the prepared sample, illustrated in the figure by small circles with attached triangles that are not connected with any specific cell.
  • FIG. 32(a,b,c,d) provide a schematic illustration of an example measurement in accord with the present invention.
  • the prepared sample is as in Fig. 31, with the addition of arrows near each nanoparticle.
  • the arrows are representative of the magnetization of each nanoparticle, and indicate that the magnetization of the nanoparticles in the prepared sample is random (in the figure, the arrows are shown in one of four directions for ease of illustration only; in practice the magnetization can have any direction).
  • Fig. 32c illustrates the sample a short time after the magnetic field is removed.
  • the nanoparticles not bound to cells are free to move by Brownian motion, and their magnetization rapidly returns to random, represented in the figure by the magnetization arrows of the unbound nanoparticles pointing in various directions.
  • the nanoparticles bound to cells are inhibited from such physical motion and hence their magnetization remains substantially the same as when in the presence of the applied magnetic field.
  • Fig. 32d illustrates the prepared sample a longer time after removal of the applied magnetic field. The magnetization of the bound nanoparticles has by now also returned to random.
  • the magnetization can then be measured as the bound nanoparticles transition from uniform to random magnetization, corresponding to the state of Fig. 32d.
  • the characteristics of the measurement magnetization from the state of Fig. 32c to that of Fig. 32d are related to the number of bound nanparticles in the sample, and hence to the number of cells of the type of interest in the sample.
  • Fig. 34 is a schematic illustration of an apparatus suitable for use in the present invention.
  • a sample holder 41 is configured to contain a sample such as those described elsewhere herein.
  • a magnetizing system 42 for example Helmholtz coils, mounts relative to the sample holder so that the magnetizing system can apply a magnetic field to the sample.
  • a magnetic sensor system 43 counts relative to the sample so that it can sense the small magnetic fields associated with the magnetized nanoparticles. The system is controlled and the sensor data analyzed by a control and analysis system 44; for example by a computer with appropriate programming.
  • the present invention provides methods and apparatuses that can detect metastatic cancer cells in the blood, bone marrow, urine or other body fluids and that can identify the tumor source of these cells.
  • the present invention also provides methods and apparatuses that can identify the tumor source of cancer-indicating markers that can be found in the body serum.
  • the present invention uses biomagnetic sensors and targeted superparamagnetic nanoparticles.
  • the biomagnetic sensors detect magnetic nanoparticles bound by antibodies or other specific agents to cells of interest such as cancer cells.
  • body serum such as blood or bone marrow is extracted and has magnetic nanoparticles with specific targeting molecules attached, such as antibodies, mixed into it.
  • Any cells targeted by the targeting molecules e.g., cancer cells targeted by specific antibodies
  • the sample is placed under a magnetic field sensor system using magnetic relaxometry to magnetize any nanoparticles in the sample and observe their magnetic field decay.
  • the magnetic relaxometry system allows the specific detection of nanoparticles bound to targeted cells.
  • a sensor array consisting of these same or similar sensors can be used to search for tumors in the body of the same type of cells identified in the serum, again using magnetic relaxometry.
  • the particular type of cell identified by the body fluid survey identifies the specific targeting molecule for these cells.
  • Magnetic nanoparticles conjugated with these targeting molecules are injected into the body, providing a sensitive and specific test for finding the tumor or metastasis that has produced the metastatic cancer cells in the serum. This method is capable of detecting several hundred cells that might be localized in a growing tumor.
  • the presence of certain markers in the serum indicating that there is a metastatic tumor growing somewhere in the body can be used to perform a search for this tumor using a magnetic relaxometry sensor array.
  • markers in the serum indicating metastasis can be followed by a search for the metastatic tumor of the type identified by the marker.
  • Knowledge of the tumor type indicates the type of antibody or other targeting molecule that will be attached to the magnetic nanoparticle injected into the body to target the responsible tumor. This substantially increases the specificity and sensitivity of the sensor search for the metastatic tumor as compared with previous methods.
  • Magnetic relaxation detection of suitable magnetic nanoparticles in serum can be performed in 5 minutes or less.
  • the source contrast of the magnetic relaxation superparamagnetic nanoparticle method of the present invention is many orders of magnitude greater than other detection methods because only bound or hindered particles are observed; the measurement is not impaired by the presence of unbound nanoparticles.
  • the magnetic relaxation sensor method can compete with all prior methods for detecting metastatic cancer cells and offers a new method for then locating the tumor source of these cells in the body for therapeutical applications.
  • An example embodiment of the present invention using SQUID sensors and conventional shielding can detect roughly 200 cells or more in a typical blood or tissue sample.
  • An example embodiment using SQUID sensors and conventional shielding can detect about lOng or more of targeted cells in a typical blood or tissue sample.
  • NANOPARTICLE PRODUCTION AND MAGNETIC PROPERTIES. In order to label biological structures magnetically for detection by magnetic relaxometry, the nanoparticles with their coatings of specific antibodies should exhibit different behavior when attached to targets than when not attached to target objects. As an example, when the particles of interest are free to reorient in their suspending fluid, they have relaxation times much shorter than the observation time; however, if rotation of the nanocrystal is hindered by its attachment through antibody-antigen interactions with the target structure, the relaxation time can be comparable to the magnetic relaxometry observation time, typically of the order of a second.
  • the relaxation time for rotation in a fluid is governed by the well-known Brownian formula, linearly dependent on the viscosity and the particle's hydrodynamic volume and inversely on the temperature of the suspending fluid.
  • the magnetic moment of the single domain nanocrystal typically of the order of half a million Bohr magnetons in magnetite, reorients with characteristic relaxation times first discussed by Neel.
  • the Neel time is important because its exponential dependence on particle diameter can limit the sizes of the particles that are suitable for SQUID relaxometry.
  • the " magnetic relaxometry window” the material-dependent size range for which magnetic relaxometry can readily sense the decaying magnetism from relaxing nanoparticles, can be about 2 nm wide centered on a diameter of 25 nm at room temperature, for the magnetite particles discussed as examples here.
  • the nanoparticles used as examples in some of the description herein were obtained from Ocean NanoTech (Springdale, A , USA), designated Ocean SHP 30 lot DE4G. These magnetite particles were suspended in water with an iron content measured to be 28.8 mg [Fe] per ml.
  • the Feret diameter is the largest caliper measurement that could be made on the TEM image of the particle.
  • a caliper measurement is the distance between two parallel planes each just touching the surface of the object. The distribution peaked at 25 nm with a full width at half maximum (FWHM) of 4 nm.
  • the Neel relaxation time is also affected by the strength of the applied magnetic field.
  • the Langevin function The equilibrium polar angle ⁇ alignment of a classical dipole with an applied field is determined by a balance between the torque exerted on the dipole by the field and the disorienting effect of thermal fluctuations that increases with T. When these two effects are the only agents present, the equilibrium average value of the cosine of the polar angle is determined by the well-known Langevin function L, the classical limit of the quantum-mechanical Brillouin function.
  • L the classical limit of the quantum-mechanical Brillouin function.
  • the anisotropy should not be neglected; the anisotropy can hinder alignment with the external field and we can modify the Langevin function as described below.
  • the weighting factor in the determination of the average value of cos9 should include, in addition to the term coupling the moment to the field, a term due to the anisotropy energy that also depends on To compute this function we apply the Gibbs distribution: j e - uw, kT cos 0sin 6H0
  • This "Modified Langevin function", L(x, y) is odd in x and even in y.
  • the result of a numerical computation of eq. 4 is shown Fig. la for various values of y.
  • Fig. lb displays the ratio of the Modified Langevin to the Langevin as a function of particle diameter, taking the particles to be uniform spheres, and using bulk values of the parameters for magnetite at 300 K.
  • MMM Superposition Model
  • the particles are assumed to be spherical and homogeneous.
  • the observed induced magnetic moment and its subsequent decay arises from the mechanisms represented below.
  • a pulse of strength B is applied for a time tpulse, and the magnetic moment M(t) of the sample is given at a time t after the pulse is turned off.
  • n is the number of particles in the sample
  • ⁇ is the magnetic moment of a particle of diameter D .
  • ⁇ ⁇
  • L is the modified Langevin function discussed above.
  • w(D) is the diameter probability distribution of the sample.
  • neel ⁇ tpulse (1 - exp(-tpulse I N ))
  • M (t) is the magnetization (dipole moment per unit volume as a function of time) in an alternating magnetic field
  • H(t) H 0 cos(O)t)
  • M 0 (t) % 0 H 0 cos(OX) is the "equilibrium magnetization" in the applied field. That is, at any given instant the magnetization is relaxing toward a value it would have were the relaxation time zero. Ignoring frequency dependence of the equilibrium volume susceptibility ⁇ ⁇ , and using the dc expression :
  • N is the number of monodisperse magnetic dipoles with moment ⁇ divided by the total volume, however we wish to define it. This is the same as volume susceptibility of a single nanoparticle as described by Worm when we replace N by 1 / .
  • ⁇ 0 is the dc volume susceptibility of an ensemble of noninteracting monodisperse nanoparticles immobilized in a magnetically inert medium.
  • ⁇ 0 is the permeability of free space.
  • Fig. 6 presents the resulting estimates for the three parameters used in our model. In all three cases the parameters determined from the real and imaginary susceptibilities are much more consistent with each other for temperatures above 300 K than for those below. The likelihood this discrepancy arises from a deficiency in the lower wing of the measured size distribution is demonstrated in these figures, by showing the consequences of adding a small cluster of particles to the lower portion of the size distribution.
  • the size range of nanoparticles should correspond to a relaxation time of about 1 second. In that case log i ⁇ 0 , allowing us via Eq.l to find the effective particle diameter,
  • the relative sensitivities of the real and imaginary parts of the ac susceptibility at different frequencies depend on the size distribution and temperature. In our case there is indication from the real part of the susceptibility, which at low temperatures is very sensitive to the smallest sizes, that the measured size distribution is deficient in smaller particles. A possible, but not unique, explanation for this apparent deficit could be that a fraction of the largest particles have broken up into multiple domains. A small change in the number density of large particles could result in a large change in the number density of small particles. This possibility is supported by the observation that the larger particles tend to appear less round than the smaller ones.
  • the pulsed fields rise abruptly to a constant amplitude and are terminated abruptly after a fixed duration, with a decay time of a few ms, such that the lingering effects of the applied pulsed field and associated transients are essentially undetectable, beyond 50 ms past the switch-off, by a SQUID array above the sample.
  • the sample and the SQUID array lie along the axis of a Helmholtz pair, with the sample centered between the two coils.
  • Magnetic relaxometry window as the size range of a particular lot of nanoparticles for which the time constants will allow the SQUI Ds to pick up a measurable signal from the relaxation of the particles after the alignment pulse is switched off (Fig. 7b).
  • the amplitude for the maximum signal we can expect with the above- described apparatus is proportional to
  • the total magnetic moment of the sample is the total magnetic moment of the sample.
  • Fig. 8 presents the SQUID measurements of a) relaxation and b) excitation of the sample of magnetite nanoparticles for three different pulse durations compared with the model results, using parameters determined for 300 K.
  • the model predictions were normalized for Fig. 8a by one fitted number.
  • Fig. 8b in addition to an overall normalization, the exponent was optimized to 0.89. From the Fig. 8b normalization constant the number of particles involved may be extracted and compared with the number expected, as explained elsewhere herein. [001811 Determination of the number of particles.
  • the model moment prediction for the single nanoparticle, y Ms ⁇ [ fsw(D) ⁇ dD , produces a model data set, y ; , for a set of parameters.
  • the measured data set corresponding to the model set is d ; .
  • the predicted value for d ; is ny t , where n is the number of particles.
  • a least squares fit yields
  • n 1.5xl0 13 .
  • this sample a cotton tip, on which fluid containing the suspended particles had been allowed to evaporate, was estimated to contain 9.38xl0 12 particles.
  • SQUID relaxometry Detection of nanoparticles by relaxometry was performed using a seven-channel low-temperature SQUID array (BTi 2004, 4D-Neuroimaging, San Diego, CA) originally designed for magnetoencephalography. Second order gradiometers with a baseline of 4 cm are used to reject background magnetic fields due to distant sources, allowing the measurements to be performed in an unshielded environment. Due to RF interference, the sensitivity of the system is currently limited to ⁇ 10 12 T/VHZ.
  • the seven gradiometer coils are located at the bottom of the liquid He dewar, 1.9 cm from the outer dewar surface, arranged with six in a circle of 2.15 cm radius and one at the center.
  • the sample is located at a distance z ⁇ 2.8-3.5 cm below the bottom of the center coil.
  • the decaying magnetization is sampled at a rate of 1 kHz (beginning 50 ms after switching off the magnetizing pulse) and digitized using a National Instruments PXI8336 16-channel digitizer and LabVIEW 8.5.1 acquisition software (National Instruments, Austin, TX).
  • a multiple dipole model may be used to determine the spatial coordinates and moments for multiple discrete sources using n different sample positions - equivalent to a sensor array with 7n elements.
  • the particles should be immobilized.
  • the antibody-conjugated nanoparticles are immobilized by the binding of the antibodies to receptors on the cell surface.
  • 10-20 ⁇ of stock nanoparticle solution is applied to a Q-tips cotton swab (Unilever, Trumball, CT) and allowed to dry in air.
  • Nanoparticles were brought to pH 8.0 with 50 mM NaHC0 3 (Sigma-Aldrich, St. Louis, MO), 50 ⁇ g of antibody (BD Biosciences, San Jose, CA) was added, and the mixture was incubated at room temperature on a LabQuake shaker for 2 hours.
  • the antibody-nanoparticle mixture was centrifuged at 7,500 RCF for 30 minutes at 4°C. The supernatant was removed and 10 ml of double distilled water was added to the pelleted NPs.
  • Fig. 9A shows TEM images of each nanoparticle sample.
  • the SHP-20 particles show the most uniform intensity in TEM, whereas the number of particles exhibiting dark and light banding becomes increasingly prevalent in the SHP- 25, -30, and -35 particles.
  • the particle size distributions (feret diameter), determined by using the program Image J by analyzing 2500 particles from each sample using multiple TEM fields, are shown in Fig. 9B.
  • the average particle diameter determined for each set of particles is in good agreement with the nominal diameters specified by the manufacturer (see Table 2).
  • the narrowest size distribution is obtained for the SHP-20 particles, and the standard deviation in particle diameter is observed to increase with average diameter.
  • Fig. 10A shows the imaginary component of the AC susceptibility at room temperature as a function of frequency. While the SHP-20 particles show a clear peak at ⁇ 300 Hz and the SHP-25 sample appears to be peaking at ⁇ 0.1 Hz, there is only a weak maximum in the AC loss for the SHP- 30 sample, and no peak is evident for the SHP-35 sample.
  • the measurement timescale of the SQUID- relaxometry technique (50 ms to ⁇ 2 s) corresponds to frequencies of approximately 0.1-3 Hz.
  • the SHP-25 particles give rise to the largest detectable moment/kg, roughly 4 times greater than the moment/kg of the SHP-30 particles, and nearly an order of magnitude greater than the moment/kg of multi-core particles we have characterized previously. Therefore, we have achieved a significant improvement in detection sensitivity by switching from multi-core particles to single-core particles of relatively uniform diameter.
  • the improvement is not as great as anticipated based on the narrow size distributions of the SHP particles.
  • a relaxometry signal is detected from all four sets of particles, an examination of the size distributions in Fig. 9B suggests that the optimal particle diameter is roughly 26 nm (the diameter at which all of the distributions overlap). If this is the ideal diameter, then theoretically, particles in the 26 ⁇ 1 nm size range should have the appropriate relaxation time to contribute to the detected moment/kg. In that case, we can estimate from the size distributions in Fig. 9B that the detected moment/kg should be approximately 30 J/T/kg for the SHP-25 particles and 40 J/T/kg for the SHP-30 particles.
  • Fig. 11 shows magnetization curves measured by DC susceptometry near the blocking temperature (see Table 3) of each sample. The data is plotted as M/M s (dimensionless) vs. H to compare the shapes of the curves. For all samples, the magnetization does not rise nearly as sharply with field as expected for magnetite particles with diameters in the 20-40 nm range. The theoretical curves for 25 nm and 8 nm diameter particles, calculated using the Langevin function, are plotted in Fig. 11 (dashed and dotted lines, respectively) for comparison.
  • K would have to be extremely large (10-40 times the bulk value) to yield relaxation times of order 1 s from crystallites with diameters in the 7 - 10 nm range, suggesting that the small crystallites within the polycrystalline particles in the ensemble do not contribute to the observed SQUID relaxometry signal. This suggests that the lower than expected moment/kg observed by relaxometry can be explained by assuming that only monocrystalline particles of the correct diameter contribute to the observed relaxometry signal.
  • the SHP-25 magnetization curve has slightly negative curvature, and can also be fit by the Langevin function, the resulting value of ⁇ is 1.2x10 18 J/T, corresponds to a particle diameter of only 17.6 nm and is not at all consistent with the observed particle size distribution. Further, this diameter implies that K must be approximately 3.5 x 10 4 J/m 3 , about 3 times the bulk value, in order to obtain a 1 s relaxation time at room temperature. Because the SHP-35 magnetization curve has positive curvature, it cannot be fit by the Langevin function.
  • K obtained from the MSM analysis (9175 J/m 3 ) implies that a particle with a relaxation time of 1 s at 300 K is 27.6 nm in diameter, in reasonable agreement with our crude estimate of 26 nm (the diameter where the 4 size distributions in Fig. 9 overlap). Further experimental studies of nanoparticles exhibiting much lower polydispersity will be required to more precisely determine the size and magnetic moment of the "ideal" particle for detection by relaxometry.
  • the SQUID relaxometry technique is theoretically insensitive to unbound nanoparticles in solution due to their rapid Brownian relaxation; however, if the hydrodynamic diameter of nanoparticle aggregates is sufficiently large, the Brownian relaxation time of the aggregated particles may fall within the 50 ms - 2 s time scale of the SQUID relaxometry measurement, in which case the signal is detected (even though the particles are not bound to cells). For even larger aggregates, the Brownian time constant may exceed 2 s, in which case, the observed relaxation will be dominantly due to the Neel process, and the detected signal will be difficult to distinguish from that of cell-bound nanoparticles.
  • SiMAG Lots 1903/08 and 1803/08 showed increasing levels of aggregation by DLS (431 nm and 1760 nm average hydrodynamic diameters, respectively) that correlated with increasingly less negative zeta potentials (-18.8 mV and -8.5 mV, respectively) and an increasing tendency to aggregate after conjugation.
  • DLS 431 nm and 1760 nm average hydrodynamic diameters, respectively
  • zeta potentials 18.8 mV and -8.5 mV, respectively
  • SHP-30 nanoparticles were conjugated to a monoclonal antibody that targets the CD3 cell-surface antigen, whose expression is correlated with acute transplant rejection.
  • the CD3-conjugated nanoparticles were then incubated with 10 7 Jurkat cells, which express high levels of the CD3 antigen on the cell surface.
  • the same CD3-conjugated nanoparticles were also incubated with 10 7 Jurkat cells pre-treated with trypsin, an enzyme that hydrolyses proteins, resulting in the digestion of cell surface antigens.
  • trypsin an enzyme that hydrolyses proteins
  • nanoparticle aggregates some of which are cell-sized, are visible in both the untreated and trypsinized cell experiments, and these aggregates appear to be responsible for a significant fraction of the signal observed from the trypsinized cells.
  • a cell ⁇ 10 micron diameter
  • a single antibody-conjugated nanoparticle ⁇ 70 nm diameter.
  • single nanoparticles are not visible at the magnification shown, and visible aggregates in the photomicrographs can be assumed to be of order 500 nm or larger, large enough to give rise to detectable relaxometry signals.
  • the number of nanoparticles that will theoretically bind to a single cell is limited by steric hindrance to roughly 150,000, assuming a single random-close-packed layer of antibody-conjugated particles (modeled as spheres of diameter 70 nm covering a 15 micron diameter cell).
  • the observed signal due to CD3-specific binding is 1.9 x 10 5 pJ/T for 10 7 cells after 60 minutes. Given that the observable moment/kg is 0.83 J/T/kg[Fe 3 0 4 ], this indicates specific binding of 23 pg of magnetite per cell, which is equivalent to 550,000 nanoparticles per cell.
  • the number of specifically bound nanoparticles therefore exceeds both the steric limit for monolayer coverage and the number of CD3 receptors per cell ( ⁇ 100,000) determined by flow cytometry.
  • the higher than expected number of nanoparticles per cell is certainly beneficial from a detection sensitivity standpoint, but the exact mechanism is not yet understood.
  • the additional binding may be the result of internalization of the nanoparticles by the cells or the tendency of clusters of antigen-bound nanoparticles to attract additional nanoparticles to the cell surface. Further work may identify the cause of both the apparently enhanced antigen-specific binding and the significant non-specific signal, particularly the development of biocompatible surface coatings that minimize nanoparticle aggregation.
  • Example embodiments and applications - Magnetic Nanoparticles for In-Vivo Detection and Localization of Disease can be used in ultra-sensitive biomagnetic methods for: (1) early detection and localization of disease, (2) image-guided therapy to treat disease, (3) monitoring and controlling treatment.
  • Fig. 16 is an illustration of magnetic relaxometry for in-vivo detection of disease. Note, in relation to Fig.
  • Fig. 17 is an illustration of an example Superconducting Quantum Interference Device (SQUID) sensor system for relaxometry. Note, in relation to Fig. 17, a liquid helium dewar for cooling low temperature SQUIDs, seven 2 nd order gradiometers for sensors, coils for magnetizing fields, 3-D nonmetallic stage for holding cells and animals, larger coils for human subjects.
  • SQUID Superconducting Quantum Interference Device
  • Fig. 18 is an illustration of results with particular cells. Note, in relation to Fig. 18, that results for breast, ovarian, T-cells, and leukemia have been obtained; there is a large number of nanoparticles per cell, with the number dependent on the number of ligands per cell; unbound particles give no moment; some phagocytosis occurs; magnetic relaxometry gives moment/cell, sites/cell, number of cells in the sample; antibody sites per line: (a) MCF7 breast 11 x 10 6 , (b) SK- OV-3 ovarian, 6.39 x 10 6 , (c) BT-474 breast, 2.75 x 10 6 , (d) MCF7 breast 0.18 x 10 6 , (e) MDA-MB-231 0.11 x 10 6 , (f) non-breast/ovarian ⁇ 4000.
  • Fig. 19 is an illustration of biomagnetic sensitivity compared to other methods.
  • Fig. 20 is an illustration of results with animal models. Note, in relation to Fig. 20, (1) Human cancer cells injected into SCID mice and tumors allowed to grow; (2) Nanoparticles injected into Mice; (3) Mice imaged by SQUID sensor system as a function of time; (4) Tumor locations obtained and verified; 1 - 2 mm accuracy; (5) Cell numbers obtained and verified by histology.
  • Fig. 21 is an illustration of the application to a magnetic biopsy needle. Note, in relation to Fig. 21, (1) Magnetic Nanoparticles - CD34 labeled to target Leukemia Cells; (2) Added to bone marrow; (3) Needle with small magnets inserted; (4) After 2 min needle extracted; (5) Cells collected placed under SQUID; (6) M D determined. [00221] Fig. 22 is an illustration of issues associated with production and characterization of nanoparticles. Note, in relation to Fig. 22, (1) Size must be ⁇ 24 nm; (2) Ideal particles are monodispersed; (3) Maximum moment/mg (Fe); (4) Commercial products unreliable; (5) Particles must have biocompatible coatings.
  • Fig. 23 is an illustration of susceptometry measurements of nanoparticle properties.
  • Fig. 24 is an illustration of properties before and after addition of PEG.
  • Flynn ER Bryant, HC, Bergemann C, Larson RS, Lovato D, Sergatskov DA, Use of a SQUID array to detect T-cells with magnetic nanoparticles in determining transplant rejection, JMMM, 311 (2007) 429-435, PMID 18084633; Flynn ER, Detection and Treatment Possibilities of Disease with Magnetic Nanoparticles, 6th International Conference on the Scientific and Clinical Applications of Magnetic Carriers, Mayl7-20, 2006, Krems, Austria, Invited Talk;

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Abstract

L'invention concerne un ensemble embout de pistolet à pulvérisation à multiples embouts de pulvérisation réversible, qui comprend un support d'embout (20) et un barillet (22). Dans un mode de réalisation, le support d'embout (20) comprend une ouverture de barillet (64) et un passage de pulvérisation de fluide (66) qui est en communication avec l'ouverture de barillet (64). Le barillet (64) comprend un canon (24) comprenant une pluralité d'embouts de pulvérisation (32, 34) espacés les uns des autres et une poignée (26) pour faire tourner le canon (24) dans l'ouverture de barillet (64). Le support d'embout (20) ou le barillet (22) comprend une ou plusieurs projections (30) tandis que l'autre comprend une surface de came (48) dans laquelle s'insèrent par coulissement la ou les projections (30), de telle sorte que la rotation du barillet (22) par rapport au support d'embout (20) provoque la translation du barillet (22) par rapport au support d'embout (20) et l'alignement de chaque embout de pulvérisation (32, 34) avec le passage de pulvérisation de fluide (64) en position de pulvérisation et une position de fonctionnement.
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WO2014160844A2 (fr) * 2013-03-27 2014-10-02 Imra America, Inc. Nanoparticules magnétiques utiles pour une détection de capteur magnétique en particulier dans des applications de biocapteur
WO2014160844A3 (fr) * 2013-03-27 2014-12-04 Imra America, Inc. Nanoparticules magnétiques utiles pour une détection de capteur magnétique en particulier dans des applications de biocapteur
US10106623B2 (en) 2014-02-12 2018-10-23 Michael Uhlin Bispecific antibodies for use in stem cell transplantation
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TWI624661B (zh) * 2016-09-05 2018-05-21 財團法人工業技術研究院 生物分子磁感測器
WO2021134136A1 (fr) 2019-12-31 2021-07-08 Universidad De Santiago De Chile Magnétomètre portable à champ appliqué externe fixe pour la détection de signaux magnétiques à partir d'échantillons et l'évaluation de la quantité de matériau magnétique dans ledit échantillon
WO2021237283A1 (fr) * 2020-05-25 2021-12-02 King Paul Jeremy Utilisation de nanoparticules magnétiques pour la détection et la quantification d'un ou de plusieurs analytes
US11604187B2 (en) 2020-05-25 2023-03-14 Quantum Ip Holdings Pty Limited Use of magnetic nanoparticles for the detection and quantitation of analyte(s)
CN117434141A (zh) * 2023-10-13 2024-01-23 中国计量科学研究院 样品检测方法、装置、计算机设备和存储介质

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