Abstract:
A system and method for analyzing a sample of liquid having an NMR signal in response to a magnetic field for the presence of an analyte. Included is an NMR device having a testing section that is adapted to contain a liquid and apply a magnetic field to the liquid. A complex comprised of a conjugate having a field gradient bound to the analyte that is of sufficient magnitude to quench the NMR signal of the liquid when in the test section whereby the presence of the complex is determined by the absence of the NMR signal.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims the benefit U.S. Provisional Application No. 61/785,508, filed Mar. 14, 2013 and herein incorporated by reference. 
    
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
     Not Applicable. 
     INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC 
     Not Applicable. 
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     There is a need for efficient, inexpensive diagnostic tools that may be used in the detection of diseases in patients. A cost-effective diagnostic system that delivers timely results would enhance point of care analyses and ultimately save lives. 
     2. Brief Summary of the Invention 
     The present invention avoids the general drawbacks of the prior art by using nuclear magnetic resonance (NMR) spectroscopy that has the sensitivity to detect single magnetic nanoparticles in an aqueous solution. In one embodiment, the present invention provides a novel NMR microcoil spectroscopic flow cytometer (a Magnecytometer), which performs ultra-sensitive cell detection and isolation. The invention uses the technique of binding of antibody-conjugated, super-paramagnetic iron oxide nanoparticles (SPIONs) to tumor cells or other cells of interest and flow NMR spectroscopy of water in the surrounding buffer solution. The invention utilizes the SPION-induced alteration in the NMR relaxation of the water in an NMR microcoil as a detection mechanism. The invention has the sensitivity to detect a single cell obtained from a small volume of liquid. The invention may also be applied to the detection and capture of almost any type of cell, virus or macromolecule. 
    
    
     
       BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS 
         FIG. 1  is a schematic of an embodiment of the invention. 
         FIG. 2  illustrates a biological complex that may be used with the invention. 
         FIG. 3  is a schematic of another embodiment of the invention. 
         FIG. 4  is a flowchart of an embodiment of the invention. 
         FIG. 5  is a graph showing volume dependent loading and washing profiles of His-tagged BSA-SPION complex. The first D1 peak shows the amount of protein entering the column. The first D2 peak shows protein exiting the column. The second peak of D2 gives the washing profile. 
         FIGS. 6A and 6B  illustrate how SPIONs quench a magnetic signal of a liquid. 
         FIG. 7  is a graph showing the NMR continuous spectra of BSA complex. Approximately 250 beads were detected. 
         FIG. 8  is a graph showing the continuous NMR spectrum of complete complex using BSA.1.6E6 molecules detected. 
         FIG. 9  is a graph showing the continuous NMR spectrum of a complex with C4-2 cells. Approximately 5 cells were detected. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     This description is not to be taken a limiting sense, but is made merely for the purpose of illustrating the general principles of the invention. The scope of the invention is defined by the appended claims. In a preferred embodiment, the present invention provides a device and method for analyzing a sample of liquid having an NMR signal in response to a magnetic field for the presence of an analyte. As shown in  FIG. 1 , an analyte  110  such as a single cell, a cancer cell or even a single cancer cell, is located in a nuclear magnetic resonance device  100  having a sample testing section  102 , which may be 1 microliter or less in volume, in which a magnetic field is applied by coil  104  that surrounds tube  106 . Analyte  110  may be conjugated with a conjugate, which may be paramagnetic or superparamagnetic, having a field gradient that quenches all or sonic of the NMR signal of the liquid in the sample testing section  102 . Testing is performed by applying a magnetic field when a liquid containing a potential analyte is located in sample testing section  102  to determine the presence of the analyte  110  based upon the NMR signal  120  of the liquid being quenched by the conjugate. 
     For detecting a single bead in the device shown in  FIG. 1 , a bead was placed in agarose under high temperature conditions. The temperature was then lowered to fix the bead in place inside capillary tube. A coil was then wrapped around the capillary and NMR was performed. 
     To test a small sample size of liquid, another embodiment of the invention prepares the analyte for analysis by creating a biological complex  200  as show in  FIG. 2 . The biological complex is comprised of a recognition ligand  212  bound to the analyte  210 . The recognition ligand  212  has an affinity to the analyte and an affinity to an affinity column resulting in the attachment of the biological complex to the affinity column. This permits the conjugate  220  to bind with the analyte and also creates an elution solution for analysis when the biological complex is removed from the column. A biological marker  240  may also be attached to the analyte  210  for testing of the presence of the analyte prior to attaching the analyte to an affinity column. Moreover, to concentrate the analyte, the presence of the biological marker is also tested as the analyte passes through the affinity column with the process continuing until no analyte is detected passing through the column. After testing, a magnet may be used to remove the biological complexes from the elution solution. 
     For a complex formed in the column using a prostate cancer cell, the Nickel agarose bead non-covalently binds the His-Tagged antibody that is attached to a chromophore. The Antibody is then attached to the prostate cancer cell via the receptor PSMA. SPIONs are then attached to the cell via a streptavidin-biotinylated anti-PSMA  270  and  271 . 
     The above-described embodiment uses a nuclear magnetic resonance (NMR) based flow cytometer (a Magnecytometer) to provide a rapid, specific detection of small numbers of biological objects, such as cells, proteins, and viruses in native biological fluids without the need for preprocessing or separations. Since biochemistry is chemistry which takes place in water, the invention uses the NMR spectroscopic signal from the abundant (˜111 Molar) solvent water protons to produce a large NMR signal. The detection scheme relies on the modification of a water signal in the presence of a conjugate that acts as a signal modifier, such as super-paramagnetic iron oxide nanoparticles (SPIONs). SPIONs possess no intrinsic magnetic field, but when placed into the static field of an NMR magnet, they become magnetically-saturated and produce magnetic field gradients which extend radially up to 50 micrometers. Water protons diffusing in these strong gradients experience decreased transverse relaxation times with subsequent line broadening. Using this property of SPIONs it can be shown that a single 1 micrometer SPION would perturb the NMR signal from ˜0.5 nL of the surrounding water containing an easily-detectable ˜10 17  protons. 
     Accordingly, by matching the volume of liquid in test section  102  with a conjugate having a field gradient that quenches the NMR signal of the liquid in the test section  102  a single analyte may be detected. In a specific embodiment, matching an 80 micrometer diameter NMR microcoil having a testing section containing this volume of water with a single SPION conjugate will quench the NMR signal from the water in this volume. SPIONs can also be conjugated to molecules, such as antibodies, that recognize other biological molecules. Therefore, attaching a conjugated SPIONs to biological analytes, and separate analyte-bound SPIONs from those lacking bound analytes, the microcoil NMR  100  may be used to detect conjugates, such as SPIONs, acting as surrogates, or signal amplifiers, for cells, viruses or molecules that are associated with a variety of diseases. Signal modifiers such as SPIONs may be attached as conjugates to antibodies directed against cell receptors and then may be used for single cell detection. 
     Another aspect of the invention is a method for ensuring that only those conjugates that have bound to an analyte pass through the NMR microcoil for counting. Another aspect of the invention addresses the fact that biological objects of interest are often dilute, so that large fluid volumes would need to be processed or examined. The passage of large volumes (1-100 mL) of fluid through an NMR microcoil would require inconveniently-large amounts of time. 
     The present invention addresses both these considerations with the aid of affinity column chromatography as shown in  FIG. 3  which allows a biological complex  300 , as described above, to be assembled in a step-wise fashion on column  301 , by washing off the unbound, and non-specific material  310 , and only eluting the conjugate or SPION-bound complex  300  that is passed through the NMR device  100 . 
     During the assembly of the complex, the analyte may be concentrated by processing arbitrary amounts of fluid. The analyte may also be recovered with a magnet after it has passed through the NMR microcoil for later use because the analyte would be now attached to magnetic beads  220 . In this manner it is possible to use antibodies  212  as recognition ligands for single cell detection even in large amounts of biological fluids. 
     Possible uses for the invention include, but are not limited to, measuring circulating tumor cells in blood. The large number of unwanted erythrocytes and other blood cells would constitute a non-interfering background. It is also important to note than magnecytometry is minimally invasive, requiring only a small amount of blood of approximately 30 microliters. Thus, the invention may improve the way doctors diagnose diseases such as prostate cancer which often metastasizes and therefore circulating tumor cells can be found in the blood, even in early stages of the disease. 
     In one preferred embodiment, the affinity column  301  may be packed with nickel (Ni)-agarose  303 , then a His-tagged antibody  212  is run through the system and recycled to fully saturate the nickel agarose column. The next step is to run a sample through the column and wash with a 2.5 mM imidazole (Sigma™, St. Louis, Mo.) wash buffer, this ensures there are no containments other than the analyte  210  of interest attached to the column that will decrease the chance of a false positive running through the NMR. 
     As shown in  FIG. 4 , two spectrophotometers  402  and  406  on either side of the column may also be used. The first detector  402  detects proteins going into column  301 . The second detector  406  detects for a biological marker such as a chromophore  240  that is attached to the His-tagged antibody  212 . The His-tagged antibody attaches to the Ni-agarose column for the direct quantification of His-Tagged antibody that elutes from the column that is then released by the imidazole competitively binding to the Ni-agarose. The conjugates  220 , which may be SPIONs, are then run through the column which bind via a streptavidin  250  conjugated SPIONs and a biotinylated antibody  252  that then attaches to cells of interest completing biological complex  200  as shown in  FIG. 2 . 
     Once bound, the sample is eluted with a 200 mM imidazole elution buffer to cleave the His-tagged antibodies off the column that then run through the NMR. The properties of SPION may then be used to detect for the analytes or cells of interest. Samples from the NMR may also be gathered in a fraction collector for further analysis such as determination of the protein concentration, and iron assays by using a magnet. 
     The NMR was Bruker™ (Madison, Wis.) MiniSpec and the solenoid coil was developed using 50 gauge copper wire wound around the outer diameter of a glass capillary tube  106  (O.D. 170 μm; I.D. 100 μm). The π/2 pulse length for this coil is 80 μs. The resonance frequency is 40.015 MHz, and contains a 100 cm permanent magnet (MRT Inc. Tsukuba Japan), all data was collected using a Hewitt Packard Windows XP™ Running Magritek (Welington, New Zealand) Prospa™, and WinDaq™ (collects optical data from detectors) on one hard drive. Data was collected via a Magritek Kea and then stored to the console. A continuous pulse repeat macro was written with the following stipulations: The macro repeats a RF pulse every 0.861 seconds for a continuous reading on each coil volume. A macro was also written to integrate the FID data for every RF pulse and generate a plot. 
     The nickel agarose was obtained from Thermo Scientific™ and contains 6% beaded agarose with a binding capacity of 10 mg/mL. Anti-PSMA, clone J591 antibody was purchased from Neil H. Bander, MD (Cornell College of Medicine, USA). Proteins were His-Tagged using the Solulink™ procedure and reagents. The J-591 were biotinylated using the Lightning Link™ Biotin conjugation kit Type A from Innova Bioscience. The SPIONs are manufactured by MagSense™ and are streptavidin modified. The were conjugated with biotinylated anti-PSMA antibody J591 at a ratio of 75×10 3  beads per ng antibody; Typically 200 ng J591 antibody were incubated in 0.5 mL PBS (Phosphate Buffered Saline) containing 15×10 6  beads by gentle end-over-end rotation at room temperature. Antibody-functionalized SPIONs were then combined with prostate tumor cells in PBS at a ratio of 300 beads per cell. 
     For an embodiment in which prostrate cancer cells were detected using the invention, the PSMA receptor  260  was selected as a preferential target for SPION labeling and attaching the His-tags. The antibody for PSMA and the protein complex BSA had a His-tag placed upon them along with the hydrazonechromophore following the manufacturer&#39;s (Solulink™) procedure: The protein must first be desalted using Zeba (Thermo Scientific™, Rockford, Ill.) desalt columns. Protein concentration determination was also performed using Pierce™ Micro HCA Kit. S-4FB (linker containing chromophore) was subsequently added, using 2 mole equivalents of S-4FB per protein. The protein then was desalted using Zeba desalting columns, and protein concentration was then determined. The molar substitution ratio was subsequently determined based on the amount of protein. Protein conjugated to S-4FB was then conjugated to 6× His-Tag and was incubated at room temperature for 16 hours. Removal of excess His-tags was performed using MicroconUltracel YM-3 spin column with a 3000 molecular weight cut-off. The biological complex was then washed twice with PBS and concentration of protein complex determined, and labeling was determined using absorbance at 360 nm. The antibodies were also conjugated to biotin, using the Lightning Link™ Biotin conjugation Kit from Innova Biosciences. Protein concentration determination was done prior to biotinylation, then 1 μL of LL-modifier reagent was added for every 10 μL of antibody used. This mixture was then added to Lightning Link™ mix and resuspended resulting in a mixture that was then incubated for 3 hours at room temperature and 1 μL of LL-quencher was added for every 10 μL of antibody used. Protein concentration was again determined. 
     The C4-2 prostate cancer cell line that was grown in vented cell culture flasks (BD Biosciences, San Jose, Calif.) in Dulbecco&#39;s modified Eagle&#39;s media (DMEM) containing: 4.5 g/L glucose and 2 mM L-glutamine (Sigma™, St. Louis, Mo.), and supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan Utah) and penicillin/streptomycin at 100 U (Sigma™). The cells were grown at 37° C. in a humidified 95% O 2 /5% CO 2  atmosphere to passage numbers typically not exceeding 35 to avoid genotypic drifts; Cell detachment was in 0.5% trypsin containing 0.02% EDTA (Sigma™) for 30 seconds upon reaching 60-80% confluence; Trypsin action was stopped by adding medium and cells were harvested by centrifugation at 150×g for 10 minutes at room temperature, then resuspended in phosphate buffered saline (PBS; Sigma™). Cell numbers were counted using a hemocytometer. 
       FIG. 4  illustrates an exemplary process flow of an embodiment of the invention. As shown, sample  400  is injected and data is acquired by detector  402 , which may be set at 280 nm or any other suitable wavelength, and stored by a processor. The sample then flows through affinity column  404  where a desired protein binds to the His-tagged antibody specific for the cell or protein of interest and the remainder of the sample flows past detector  406  and can be recycled through the column to ensure maximum binding. Then the wash buffer containing 2.5 mM imidazole flows through the system releasing excess particles and molecules not bound to column. Elution buffer containing a high concentration (200 mM) of imidazole then releases His-Tagged antibodies bound to the column, also releasing cells/proteins of interest bound to SPIONs. 
     Detector  406  may be set at 360 nm, or any other suitable wavelength, detects the chromophore  240  attached to His-Tagged antibodies  212  that run through the NMR. Small quantities of SPIONs  220  can therefore be detected. The embodiment may be used to process small quantities of cells labeled with magnetic beads  220  (˜5 cells) for detection. 
     Continuing to pass analytes past detector  406  until no reading or a sufficiently low reading is obtained. This reduces false positives from occurring as shown in  FIG. 5 . A spectrophotometer functions as a detector was placed before the affinity column to detects the amounts of protein prior to entering the column as described above. For an example used in accordance with the present invention,  FIG. 5  shows that 368.11 mg/mL of protein entered the column as shown by peak  510  for detector  402  and 56.458 mg/mL of His-Tagged protein exited the column as demonstrated by peak  512  for detector  406 . Peak  510  shows protein entering the column. Peak  512  shows protein exiting the column. Peak  514  shows the wash buffer releasing unbound SPIONs and gives the washing profile. 
     As further shown in  FIG. 1 , a single biological complex  200  identified as number  110  was placed in test chamber or section  102  of device  100 . NMR was then performed showing the quenched water proton signal  120  indicating the presence of the analyte of interest. The detection of a single bead in the test chamber may then be used to count the number of analytes present as an elution solution flows through the device. 
       FIGS. 6A and 6B  illustrate the effect on water protons when stimulated by SPIONs at varying concentration of magnetic beads.  FIG. 6A  shows cells that do not express PSMA and therefore are not magnetically labeled with SPIONs.  FIG. 6B  shows cells that overexpress PSMA and bind SPIONs. 
       FIG. 7  illustrates the continuous NMR spectrum of the water protons being quenched by the BSA complex. An after iron assay was performed and it was determined that 3.6E6 beads and 1.8E6 BSA molecules (BSA binds two beads per molecule) were bound and eluted from the column.  FIGS. 8 and 9  are the NMR continuous spectra of C4-2 prostate cancer cells, which over express PSMA. The data shows that the cells were successfully bound to the column and detected using NMR. It was determined that approximately 5 cells were detected and characterized. Portion  800  shows the His-Tagged attached to the protein of interest. Portion  802  shows the wash removing the unbound SPIONs. Portion  902  shows C4-2 cells. Portion  900  shows the wash removing the unbound SPIONs.