WO2013044240A1 - Capture et libération sélectives de cellules par des aptamères - Google Patents

Capture et libération sélectives de cellules par des aptamères Download PDF

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Publication number
WO2013044240A1
WO2013044240A1 PCT/US2012/056926 US2012056926W WO2013044240A1 WO 2013044240 A1 WO2013044240 A1 WO 2013044240A1 US 2012056926 W US2012056926 W US 2012056926W WO 2013044240 A1 WO2013044240 A1 WO 2013044240A1
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Prior art keywords
cells
temperature
aptamer
microchamber
cell
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PCT/US2012/056926
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English (en)
Inventor
Qiao Lin
Jing Zhu
Milan N. Stojanovic
Renjun Pei
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The Trustees Of Columbia University In The City Of New York
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Publication of WO2013044240A1 publication Critical patent/WO2013044240A1/fr
Priority to US14/223,767 priority Critical patent/US20140296095A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/04—Cell isolation or sorting
    • 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
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells

Definitions

  • Isolation of cells from biological samples from heterogeneous mixtures such as blood or other body fluids can be used in cell biology research and clinical diagnostics.
  • the ability to detect and characterize cancer cells from blood or other body fluids can be helpful to detect cancer in early stages and understand cancer development and progression mechanisms, such as metastasis, thus can improve survival rates.
  • studies of phenotypically pure subpopulations of human lymphocytes can provide information concerning immune responses to injury and disease.
  • target cells can be selectively captured, and in some instances, such as tissue engineering and cell-based therapeutics, retrieved nondestructively without mechanical or biochemical damage.
  • Isolation of cells can be based on the size or volume, density, electrical properties, or surface characteristics, using methods such as filtration, centrifugation, dielectrophoresis, or affinity binding.
  • affinity binding recognizes cells by binding of ligands to receptors on cell membranes, and hence has high specificity to target cells.
  • Ligands for affinity binding can include antibodies, which can be generated in vivo against target antigens found on cell membranes.
  • Antibody-based cell isolation techniques can be implemented using methods such as magnetic activated cell sorting (MACS) and fluorescence- activated cell sorting (FACS).
  • aptamers can be used as affinity ligands for isolation of cells.
  • Aptamers are oligonucleotides or peptides that can bind with target molecules, such as proteins, peptides, as well as cellular targets. They can be obtained or prepared by a procedure called systematic evolution of ligands by exponential enrichment (SELEX), which selects aptamers from a randomized oligonucleotide or peptide library via an iterative process.
  • SELEX systematic evolution of ligands by exponential enrichment
  • Aptamers can be explored in microfluidic systems as affinity ligands for cell isolation.
  • surface-immobilized aptamers targeting prostate-specific membrane antigen (PSMA) and aptamers targeting protein tyrosine kinase 7 (PTK7) can be used to separate LNCaP cells and CC F-CEM cells, respectively, from heterogeneous cell mixtures. Release of captured cells can be achieved using tryptic digestion of target proteins, degradation of aptamers, and hydrodynamic shear by infused air bubbles.
  • PSMA prostate-specific membrane antigen
  • PTK7 protein tyrosine kinase 7
  • release of captured cells can be achieved using tryptic digestion of target proteins, degradation of aptamers, and hydrodynamic shear by infused air bubbles.
  • these methods can result in damage in cells such the destruction or impairment of the cell recognition surfaces.
  • the disclosed subject matter provides techniques for capturing and releasing target ceils.
  • the techniques can be based on a microdevice having a microchamber including an aptamer capable of binding with the target cells.
  • the aptamer can be immobilized on an inner surface of the microchamber, for example, by using streptavidin-biotin binding.
  • Heater and temperature sensor can be included in the microdevice to provide thermal regulation of the microchamber.
  • a sample is introduced into the microchamber, so that the target sells bind to the aptamer at an initial temperature, and are released at a second, different temperature.
  • the sample can include impurities other than the target cells, such as non-target cells, small molecules, and proteins.
  • the microchamber Before releasing the aptamer-bound target cells, the microchamber can be washed to remove cells or other substances not bound to the aptamer. After the washing, a second sample including the target cells can be introduced into the microchamber to increase the amount of the target cells to bind with the aptamer.
  • the target cells have membrane protein(s) and the binding between the target cells and the aptamer are via the interaction between the aptamer and the membrane protein(s).
  • the aptamer can be selected or developed to specifically bind with the membrane protein.
  • the membrane protein is PTK7.
  • the initial temperature can be about 20°C to 30°C, e.g., at about room temperature. In alternative embodiments, the initial temperature can be at physiological temperature (about 37°C). In some embodiments, the second temperature for the release of the target cells can be about from 35°C to about 55°C, e.g., at about 48°C. In alternative embodiments, the second temperature can be lower than the initial temperature, e.g., the second temperature can be from about 4°C to about 20°C.
  • Figures 1 a- 1 c are schematic diagrams illustrating cell capture and temperature-mediated release according to some embodiments of the disclosed subject matter.
  • Figure 2a is a schematic diagram of a microdevice for selective capture and release of target cells according to some embodiments of the disclosed subject matter. All dimensions are in microns.
  • Figures 2b-2g are schematic diagrams of an example fabrication procedure of the microdevice depicted in Figure 2a.
  • Figure 2h is an image of an example microdevice according to some embodiments of the disclosed subject matter.
  • Figure 2i is a close-up image of a portion of the image depicted in
  • Figure 2j is an example setup for operating a microdevice for capturing and temperature-mediated release of target cells according to some embodiments of the disclosed subject matter.
  • Figures 3a-3d present results of certain tests performed according to some embodiments of the disclosed subject matter.
  • Figures 3a and 3b are images of the microchamber of a microdevice after the introduction of a sample, and after the introduction of 10 samples and buffer washing, respectively.
  • Figure 3c is a plot depicting the time response of the amount of captured cells versus incubation time.
  • Figure 3d is a plot depicting the concentration response of cell capture.
  • Figure 4a-4e present results of certain tests performed according to some embodiments of the disclosed subject matter.
  • Figure 4a is a plot depicting percentage of captured cells remaining on the substrate as a function of time while rinsing at constant temperature (48 °C and room temperature) and flow rate (5 uJL/min);
  • Figure 4b is a plot depicting captured cell density versus the number of cell suspension samples introduced while the temperature was maintained at either 48 °C or room temperature;
  • Figure 4c is a plot showing the effect of temperature on cell release efficiency while rinsing at 5 ⁇ 7 ⁇ ;
  • Figure 4d is a plot showing the effect of flow rate on cell release efficiency while the microchamber temperature was maintained at 48°C;
  • Figure 4e is a bar graph showing cell capture and re-capture on the regenerated aptamer-functionalized surface: the normalized percentage of remaining cells after the first, second and third capture and regeneration cycle.
  • Figures 5a-5c illustrate the viability of cells subjected to capture and release according to some embodiments of the disclosed subject matter.
  • Figures 5a and 5b are image of PI stained cells (in 5a) and JC-1 stained cells (in 5b) following cell capture and release, generated by a combination of phase contrast and fluorescent micrographs.
  • Figure 5c is a bar graph showing concentrations of normal cells and heat-treated cells as a function of culture duration.
  • a device incorporating a microchamber can be provided, including an aptamer capable of binding with the target cells.
  • a sample including target cells can be introduced to the microchamber so that the target sells bind to the aptamer at an initial temperature, and are released at a second, different temperature.
  • the techniques utilize a microdevice 100 including a microchamber (or chamber) 110 which is functionalized on its inner surface with aptamers 120 that bind with the target cells 112, e.g., via certain membrane proteins of the target cells.
  • the dimensions noted on Figure 1 are only for purpose of illustration.
  • the overall length and width of the microdevice can be a few millimeters.
  • the length and width of the microchamber can be in the order of millimeters, and the depth of the microchamber can be from a few microns to a tens of microns to allow transportation of the target cells while retaining reasonable encounter probability between cells and aptamer.
  • the depth of the chamber can be from about 10 to about 100 microns.
  • the aptamers When a sample including the target cells is introduced to the microchamber, the aptamers bind with the target cells at a first temperature, e.g., room temperature ( Figure la). Thereafter, the microchamber can be washed to remove impurities in the sample, e.g., non-target cells, small molecules, proteins, or the like, that are not bound with the aptamers ( Figure lb). For certain target cells, the cell capture procedure can also be conducted at physiological condition (about 37°C).
  • the aptamer can be immobilized on an inner surface of the
  • the inner surface of the microchamber can be functionalized by certain proteins, e.g., streptavidin, which can bind an aptamer tagged with biotin.
  • the inner surface of the microchamber can be functionalized by certain proteins, e.g., streptavidin, which can bind an aptamer tagged with biotin.
  • the inner surface of the microchamber can be functionalized by certain proteins, e.g., streptavidin, which can bind an aptamer tagged with biotin.
  • microchamber can be modified with functional groups, e.g., a thiol group.
  • the thiol group can then be connected by crosslinker, e.g. N-gamma-Maleimidobutyryl- oxysuccinimide ester, together with the streptavidin.
  • another sample including the target cells can be introduced into the microchamber to allow increased amount of target cells to bind with the aptamers.
  • the temperature of the microchamber can be raised, e.g., via integrated resistive heaters 156 on the microchip, to a second, higher temperature to disrupt the binding between the aptamer and the target cells while maintaining the structural integrity and viability of the cells ( Figure lc).
  • the released cells can be collected for further analysis or detection.
  • the microdevice and the aptamers can be reused for processing further samples, hi alternative embodiments, the cell release can be achieved at a temperature lower than the initial temperature, e.g., by cooling the microchamber to disrupt the interactions between the aptamer and the bound target cells.
  • Such cooling can be thermoelectric cooling, e.g., by using a Peltier element incorporated as a part of the microdevice.
  • a suitable aptamer for MUC cells can capture MUC1 cells at about physiological condition (about 37°C) and release the cells at a lower temperature, e.g., about 4°C, or at a temperature than 37°C.
  • the microchamber 110 can be formed between a cavity or void between two PDMS layers 154, the bottom layer positioned atop a passivation layer 152 that covers embedded heaters 156, which are deposited on glass substrate 150.
  • the target cells can be any cells that have surface membrane proteins to which an aptamer can be selected or developed to specifically bind.
  • the cells can include CCRF-CEM, MCF7, LNCaP, Hs578T, and the corresponding membrane proteins can include PTK7, MUC 1 , PSMA, and PDGF, respectively.
  • the aptamers can be selected based on the membrane proteins of the target cells, or developed using SELEX procedure based on membrane proteins of the target cells. Particular aptamers can be generated which bind with specific equilibrium constants, kinetic parameters, and at specific temperatures. For example, for CCRF-CEM cells, a suitable aptamer can be sgc8c. For MCF-7 cells, a suitable aptamer can be MUC1-5TR-1. Aptamers for PSMA (on LNCaP cells) and PDGF (on Hs578T cell line) can be xPSM-A9 and PDGF-aptamer-36t, respectively. PDGF- aptamer-36t has a sequence of: 5'-CAC AGG CTA CGG CAC GTA GAG CAT CAC CAT GAT CCT GTG-3' (SEQ ID NO: 1).
  • the first temperature at which the aptamer binds with the target cells depend on the choice of aptamer-membrane protein of the target cells.
  • the first temperature can be about from 20°C to about 30°C, e.g. about 25°C. In other example embodiments, the first temperature can be about 37°C.
  • the second temperature at which the captured cells are released from the aptamer can also depend on the choice of aptamer-membrane protein of the target cells.
  • the second temperature can be about from 30°C to about 55°C, e.g., about 48°C. In alternative embodiments, the second temperature can be from about 4°C to about 20°C.
  • the duration of heating or cooling at the second temperature can be brief, e.g., between 1 to 5 minutes, e.g., about 2 minutes.
  • Example 1 Further details of device structure, fabrication, and operation procedures of the above-described embodiments can be found in the following Example, which is provided for illustration purpose only and not for limitation.
  • Example 1 Example 1
  • CCRF-CEM cells are a human ALL cell line. ALL is a common cancer for children younger than 14 years old, representing one third of all malignancies in that age group. CCRF-CEM cells can be recognized by the DNA aptamer sgc8c. Toledo cells, a human diffuse large-cell lymphoma cell line not recognized by sgc8c, were used as a control (non-target cells).
  • the microfluidic device used for cell capture and temperature-mediated cell release includes a micro chamber 210 situated on a temperature control chip 230.
  • the tapered chamber (2 mm in length, 1 mm in width and 20 ⁇ in height), whose surfaces are functionalized with aptamers specific to a target cell type, is connected to two inlets 215 (3.5 mm in length, 0.7 mm in width and 600 ⁇ in height) respectively for introduction of sample and washing buffer, and an outlet 218 for collection of released cells or waste fluids.
  • the microfluidic channels connecting these fiuidic ports and the chamber are 0.5 mm in width and 20 ⁇ in height.
  • Integrated on the temperature control chip 230 are a serpentine-shaped temperature sensor 252 (Imewidth: 25 ⁇ ) beneath the center of the chamber, and two serpentine-shaped heaters 256 (linewidth: 300 ⁇ ) on each side of the temperature sensor.
  • the chamber temperature can be controlled in closed loop using these integrated temperature sensor and heaters.
  • the temperature control chip 230 was fabricated using standard microfabrication techniques. A glass slide (Fisher HealthCare, Houston, TX) was cleaned by piranha. Chrome (10 nm) and gold (100 ran) thin films 256 were deposited by thermal evaporation and patterned by wet etching to generate the temperature sensor and heaters which were then passivated by 1 ⁇ of silicon dioxide that was deposited using plasma-enhanced chemical vapor deposition (PECVD). Finally, contact regions for electrical connections to the sensor and heaters were opened by etching the oxide layer using hydrofluoric acid (Figure 2b).
  • PECVD plasma-enhanced chemical vapor deposition
  • microchamber 210 was fabricated from
  • PDMS polydimethylsiloxane 259
  • Sylgard 184 Dow Corning Inc. Midland, MI
  • Layers of SU-8 photoresist 258 (MicroChem Corp., Newton, MA) were spin-coated on a silicon wafer 257 (Silicon Quest International, Inc., San Jose, CA), exposed to ultraviolet light through photomasks, baked, and developed to form a mold defining the microfluidic features.
  • a PDMS prepolymer solution base and curing agent mixed in a 10: 1 ratio
  • the resultmg sheet bearing the microfluidic features was then peeled off the mold ( Figure 2d).
  • the surface of the temperature control chip was treated with chlorotrimethylsilane 261 , and a PDMS layer 262 (approximately 100 ⁇ ) was spin-coated onto the chip (Figure 2e).
  • the PDMS sheet 259 was bonded to the PDMS layer 262 after treatment of the bonding interfaces with oxygen plasma for 15 seconds ( Figure 2f).
  • the PDMS sheet 250 can be easily removed from the temperature control chip, allowing the temperature control chip to be reused for the next test.
  • a fabricated and packaged device is shown in Figures 2h, and a close-up image of a selected portion of the device is shown in Figure 2i.
  • Chlorotrimethylsilane, (3-mercaptopropyl) trimethoxysilane (3-MPTS), 4- maleimidobutyric acid Nhydroxysuccinimide ester (GMBS), streptavidin and bovine serum albumin (BSA) were obtained from Sigma-Aldrich (St. Louis, MO).
  • JC-1 5,5',6,6'- tetrachloro-l, ,3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1), propidium iodide (PI), RPMI-1640 media, fetal bovine serum (FBS), penicillinstreptomycin (P/S, penicillin 10,000 unit/mL, streptomycin 10,000 g/mL), Dulbecco's
  • D-PBS phosphatebuffered saline
  • VybrantcS VybrantcS multicolor cell-labeling kit
  • ATCC American Type Culture Collection
  • the biotinylated sgc8c aptamer was functionalized in a freshly fabricated microdevice.
  • the microchamber was first treated with 4% (v/v) 3-MTPS in ethanol for 30 min at room temperature, followed by an ethanol wash. 2 mM GMBS in ethanol was then introduced and incubated for 20 min at room temperature, followed by an ethanol wash and drying by nitrogen.
  • the chamber was incubated overnight with 100 ⁇ g/mL streptavidin in D-PBS at 4 °C, followed by a D-PBS wash. Finally, 10 ⁇ of biotinylated sgc8c aptamer in D-PBS was introduced into the chamber and incubated at room temperature for 20 min.
  • a D-PBS wash was used to remove free aptamer molecules, leaving immobilized aptamer molecules on the surface.
  • the chamber Prior to cell introduction, the chamber was incubated with 1 mg/mL BSA solution in D-PBS at room temperature for at least 30 min to minimize nonspecific adsorption of cells.
  • Both CCRF-CEM and Toledo cells were incubated with RPMI-1 40 media supplemented with 10% FBS and 1% P/S, and were kept at 37 °C in a humidified incubator containing 5% C0 2 . Each cell type was collected through centrifugation, resuspended at 1 x 10 s cells/mL in D-PBS supplemented with 1 mg mL BSA, and then kept on ice. Cells were mixed or diluted to different concentrations prior to introduction into the microdevice.
  • microchamber of the microdevice 291 was achieved using the integrated temperature sensor and heaters (not shown) with a proportional-integral-derivative (PID) algorithm implemented in a Lab VIEW (National Instruments Corp., TX) program on a computer 292.
  • the resistance of the sensor was measured by a digital multimeter (3442 OA, Agilent Technologies Inc., CA), and the heaters were connected to a DC power supply 294 (E3631, Agilent Technologies Inc., CA).
  • the inlets of the microdevice were connected to two syringes that respectively contained cell mixture and D-PBS, and was each driven by a syringe pump 296 (KD210P, KD Scientific Inc., MA).
  • the outlet was connected to a microcentrifuge tube 295 for collection of released cells or waste. Unless indicated otherwise, all phase contrast images and fluorescent images of the chamber were taken using an inverted epifluorescence microscope (Diaphot 300, Nikon Instruments Inc., NY) with a CCD camera (Model 190CU, Micrometrics, NH).
  • CCRF-CEM cells During cell capture, a batch of CCRF-CEM cells was introduced into the chamber and incubated without any fluid flow for 1 min. This was repeated several times, followed by a wash with D-PBS at 5 ⁇ / ⁇ for approximately 1 min. An image of the cell-laden chamber was taken and used to manually count the number of captured cells, which was used to compute the captured cell density on the surface.
  • CCRF-CEM and Toledo cells were labeled with the fluorescent dyes DiO and Dil, respectively, and fluorescent images were taken after the first introduction of the cell mixture as well as after D-PBS washing.
  • the chamber was heated using the integrated heaters via closed loop temperature control to a desired temperature for 2 min, and flows of D-PBS at various rates were used to rinse the chamber. Images of the chamber were taken every 2 seconds, and used to manually count the cells that remained on the aptamer-immobilized surface.
  • the retrieved cells were kept in D-PBS with 10% FBS containing PI (2 ⁇ ) and JC-1 (10 g mL) at 37 °C for 1 hour, and then phase contrast and fluorescent images were taken with an inverted microscope (DMI6000B, Leica Microsystems Inc., IL) equipped with a digital camera (Retiga 2000R,
  • CCRF-CEM cells target cell type, 3.5* 10 6 cells/mL
  • Toledo cells non-target cell type, 5.0 l0 6 cells/mL
  • all non- specifically adsorbed Toledo cells were removed, leaving only specifically captured CCRF-CEM cells.
  • CCRF-CEM cell suspensions with concentrations of 5.0 ⁇ 10 6 cells/mL were introduced into the aptamer-functionalized chamber and allowed to incubate for varying lengths of time. After incubation, D-PBS was used to remove unbound cells. The fraction of captured cells in each introduction was calculated by ⁇ * N a /N b , where N a is the number of captured cells, i.e., cells that remained on the microfluidic aptamer-functionalized chamber surface after washing, and ⁇ 3 ⁇ 4 is the maximum number of cells that can be captured due to geometric limitations.
  • ⁇ 3 ⁇ 4 is also equal to the number of cells observed in the chamber before washing.
  • the effects of the cell suspension concentration on the surface density of captured cells were also determined.
  • the linear equation fitted the test data (R 2 > 0.99), resulting in a value of A equal to 0.3874 mL/mm 2 .
  • the cell-capture surface can be regenerated after the release of the captured cells.
  • three cycles were performed in the same device, with each cycle including first introducing a dilute cell solution to the microchamber at room temperature, then releasing cells at 48 °C and 5 ⁇ ,/ ⁇ for 2 min, and finally regenerating the aptamer-functionalized surface
  • PI red-fluorescent nuclear stain that is not permeant to live cells.
  • JC-1 accumulates in healthy mitochondria as indicated by red fluorescence, the intensity of which decreases along with mitochondrial depolarization occurring in the early stage of apoptosis. The results showed that the PI stained cells did not emit any red fluorescence ( Figure 5a), and the JC-1 stained cells exhibited bright red fluorescence ( Figure 5b), indicating that the collected cells were viable.

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Abstract

La présente invention concerne des procédés et des systèmes pour capturer et libérer des cellules cibles. Le système comprend un microdispositif ayant une microchambre comprenant un aptamère capable de se lier aux cellules cibles. Un échantillon comprenant des cellules cibles est introduit dans la microchambre, où les cellules cibles se lient à l'aptamère à une première température. Les cellules cibles capturées peuvent être libérées lorsque la température de la microchambre est changée à une deuxième température.
PCT/US2012/056926 2011-09-23 2012-09-24 Capture et libération sélectives de cellules par des aptamères WO2013044240A1 (fr)

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US9250169B2 (en) 2007-03-27 2016-02-02 The Trustees Of Columbia University In The City Of New York Selective capture and release of analytes
US9090663B2 (en) 2009-04-21 2015-07-28 The Trustees Of Columbia University In The City Of New York Systems and methods for the capture and separation of microparticles
US10058276B2 (en) 2011-07-29 2018-08-28 The Trustees Of Columbia University In The City Of New York MEMS affinity sensor for continuous monitoring of analytes
WO2014078521A1 (fr) * 2012-11-14 2014-05-22 The Trustees Of Columbia University In The City Of New York Isolement et enrichissement d'acides nucléiques sur micropuce
WO2015077441A3 (fr) * 2013-11-20 2015-11-12 University Of Florida Research Foundation, Incorporated Ensemble anticorps et aptamère pour l'isolement de cellules et l'enrichissement en cellules
US10466243B2 (en) 2013-11-20 2019-11-05 University Of Florida Research Foundation, Incorporated Antibody and aptamer ensemble for cell isolation and enrichment
US10294471B2 (en) 2014-08-05 2019-05-21 The Trustees Of Columbia University In The City Of New York Method of isolating aptamers for minimal residual disease detection

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