CN114958748B - Nanometer magnetic affinity material for efficiently capturing and nondestructively releasing circulating tumor cells - Google Patents

Nanometer magnetic affinity material for efficiently capturing and nondestructively releasing circulating tumor cells Download PDF

Info

Publication number
CN114958748B
CN114958748B CN202210433043.3A CN202210433043A CN114958748B CN 114958748 B CN114958748 B CN 114958748B CN 202210433043 A CN202210433043 A CN 202210433043A CN 114958748 B CN114958748 B CN 114958748B
Authority
CN
China
Prior art keywords
bcn
avidin
boric acid
aptamer
nano
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202210433043.3A
Other languages
Chinese (zh)
Other versions
CN114958748A (en
Inventor
杨婷
王建华
王思怡
Original Assignee
东北大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 东北大学 filed Critical 东北大学
Priority to CN202210433043.3A priority Critical patent/CN114958748B/en
Publication of CN114958748A publication Critical patent/CN114958748A/en
Application granted granted Critical
Publication of CN114958748B publication Critical patent/CN114958748B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/405—Concentrating samples by adsorption or absorption
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
    • C12N2509/10—Mechanical dissociation

Landscapes

  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Nanotechnology (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • General Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Medical Informatics (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Oncology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Cell Biology (AREA)
  • Composite Materials (AREA)
  • Condensed Matter Physics & Semiconductors (AREA)
  • Materials Engineering (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The application belongs to the technical field of biological materials, and in particular relates to a nano magnetic affinity material for efficiently capturing and nondestructively releasing circulating tumor cells. The nano magnetic affinity material comprises a boric acid modified magnetic carbon nitride nano sheet M-BCN and an avidin-biotinylation aptamer Apt-Avi, wherein the avidin-biotinylation aptamer Apt-Avi is prepared by preassembling a biotinylation aptamer and avidin; the avidin-biotinylated aptamer is bound to the M-BCN by directed linkage of the avidin glycosyl to the boronic acid ligand in the M-BCN, and is loaded onto the M-BCN. By orderly arranging the aptamer on the surface of the material, the interaction between the aptamer and the CTC is promoted, so that the capturing efficiency of the CTC is improved. Meanwhile, after the CTC cells are captured, the material can release CTC in a non-destructive manner in a mild and reversible release manner, so that the activity of releasing CTC is improved, and the CTC can be subjected to subsequent analysis.

Description

Nanometer magnetic affinity material for efficiently capturing and nondestructively releasing circulating tumor cells
Technical Field
The application belongs to the technical field of biological materials, and in particular relates to a nano magnetic affinity material for efficiently capturing and nondestructively releasing circulating tumor cells.
Background
Circulating tumor cells (Circulating tumor cells, CTCs) are tumor cells shed from primary tumors into the blood circulation and are important factors that lead to tumor metastasis and recurrence. Circulating tumor cells not only carry the entire genome of tumor cells, but also are already present in the peripheral blood circulation at the early stage of cancer lesions, and thus, are used as an important subject for cancer research and an important marker for early diagnosis of cancer. The efficient and accurate separation and enrichment method is used for obtaining high-purity, nondestructive and active CTC cells from complex blood components, which is a precondition for downstream analysis. However, CTCs are very small in whole blood, making their isolation challenging. Thus, the preparation of affinity materials that meet the high efficiency of cell capture and non-destructive release is a hotspot of current research.
Aptamers (aptamers) are a class of nucleic acids with secondary structures, known as chemical substitutes for antibodies, which are often used to make cell capture affinity materials due to their programmable design, low cost, and ease of modification. In addition to capture, release of CTCs from the capture material remains a difficult challenge for downstream analysis. The captured CTCs often adhere to the matrix or become trapped in the microstructure, which prevents its reliable biochemical information from being extracted. Thus, a mild and controlled release of CTCs is a prerequisite for their in-depth analysis. In recent years, based on the corresponding CTC capture principle, a large number of researchers have been working on developing different cell release methods. The existing release principle of capturing CTC based on an aptamer mainly comprises the steps of simply changing the conformation of the aptamer to lose affinity and specificity, so that the purpose of releasing CTC is achieved, and the methods comprise thermal denaturation (Lab Chip 2012,12,3504), nuclease digestion (ACS appl. Mater. Interfaces 2015,7,24001), complementary sequence hybridization (Nano Res.2018,11,2592) and the like. However, these methods more or less damage the cells. In addition, the aptamer on the capture interface has low effectiveness and poor capture effect due to the improper modification method.
On the cell capture interface of the aptamer, direct binding tends to result in inactivation of the aptamer due to strong interactions between the aptamer and the cell capture interface; interfaces without orderly arranged aptamers also suffer from inter-strand entanglement and localized overcrowding, which greatly reduces the efficiency of the aptamers and thus does not allow efficient capture of CTCs.
For the common CTC release method, when CTCs are released by using a thermal deformation method, thermal denaturation of the aptamer occurs with an increase in temperature, fragile tumor cells may be damaged to some extent at the increased temperature, and some aptamers having a high melting point need a relatively high temperature to be thermally denatured, so that viable CTCs cannot be released. By release of the nuclease, the nuclease damages the cells to a certain extent, significantly reduces the cell activity, and the nuclease degradation is ineffective against chemically stable aptamers. Strategies based on complementary sequence hybridization provide a non-destructive method of releasing CTCs for downstream analysis. However, this method requires complex complementary sequence designs, careful selection of appropriate reaction conditions, and relatively long processing times. Therefore, there is an urgent need to open a new CTC capture affinity material to achieve controlled, mild, non-destructive release of CTCs.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a nano magnetic affinity material, which promotes the interaction between an aptamer and CTC by orderly arranging the aptamer on the surface of the material, thereby improving the capturing efficiency of CTC. Meanwhile, after the CTC cells are captured, the material can release CTC in a non-destructive manner in a mild and reversible release manner, so that the activity of releasing CTC is improved, and the CTC can be subjected to subsequent analysis.
The specific scheme of the invention is as follows:
a nano-magnetic affinity material for efficiently capturing and releasing circulating tumor cells in a nondestructive way comprises a boric acid-modified magnetic carbon nitride nano-sheet (M-BCN) and an avidin-biotinylation aptamer (Apt-Avi), wherein the avidin-biotinylation aptamer Apt-Avi is prepared by preassembling the biotinylation aptamer and the avidin.
The avidin-biotinylated aptamer is bound to the M-BCN by directed linkage of the avidin glycosyl group to the boronic acid ligand in the M-BCN, and is loaded onto the M-BCN.
According to the nano magnetic affinity material, based on the action between boric acid and glycosyl, the avidin-biotinylation aptamer is directionally connected to the magnetic carbon nitride nanosheet modified by boric acid, the biotinylation aptamer can achieve specific capture of CTCs, and through the directional coupling action of boric acid, the ordered arrangement of the aptamer on the surface of the material is effectively improved, and the capture capacity and capture efficiency of the material to CTCs are improved. Due to the action of B-N, the cyclic borate can be formed by the cis-dihydroxyl of the glycosyl in the boric acid ligand and the avidin under the physiological condition, the cyclic borate is dissociated in the weak acid medium, and under the condition that fructose exists, the dissociation can be further accelerated due to the competition effect, so that the nondestructive release of CTCs by combining the competition effect of fructose under the weak acid condition is realized.
Preferably, the pre-assembly of the biotinylated aptamer (Bio-Apt) with Avidin (Avidin) is carried out in a molar ratio of 4: 1.
The biotinylated aptamer (Bio-Apt) is an aptamer capable of targeting CTC cells or CTC biomarkers, such as TLS11a (sequence: 5-Bio-TTT TTA CAG CAT CCC CAT GTG AAC AAT CGC ATT GTG ATT GTT ACG GTT TCC GCC TCA TGG ACG TGC TG-3), or aptamer SYL3C, MUC1, and the like.
The boric acid modified magnetic carbon nitride nano sheet M-BCN uses magnetism g-C 3 N 4 The nano sheet is a matrix material, and boric acid ligand is modified on the surface of the nano sheet. The boric acid used for modification is a compound containing a carboxyl group and a boric acid group, preferably carboxyphenylboric acid, more preferably 4-carboxyphenylboric acid, whose carboxyl group in the molecular structure is bonded to graphite-phase carbon nitride (g-C 3 N 4 ) The amino groups on the surface condense into an amide bond and the boronic acid group acts as a ligand for binding to the sugar group in the avidin.
A preparation method of the material comprises the following steps:
s1: preparation of boric acid modified g-C 3 N 4 : dissolving carboxyphenylboronic acid in solvent under drying condition, adding amide condensing agent, and adding g-C 3 N 4 Reacting the powder; after the reaction is finished, centrifugal separation is carried out, sediment obtained by centrifugal separation is taken out, solvent and amide condensing agent are washed off, and boric acid modified g-C is obtained after freeze drying 3 N 4 。
The purpose of this step is to combine the carboxyl groups in carboxyphenylboronic acid with graphite-phase carbon nitride (g-C 3 N 4 ) The amino groups on the surface are condensed into amide bonds by reaction to realize g-C 3 N 4 Boric acid modification of carboxyphenylboronic acid with g-C 3 N 4 The mass ratio of the powder is preferably (3-4): 1. Specific reaction conditions may be determined depending on the kind of the amide condensing agent.
Preferably, the solvent is N, N-Dimethylformamide (DMF), the amide condensing agent is a condensing agent for promoting the amidation reaction, preferably N, N-Diisopropylethylamine (DIEA) and 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea Hexafluorophosphate (HATU) are used, in order to ensure that the reaction proceeds adequately, carboxyphenylboronic acid: DIEA: the molar ratio of HATU can be 1 (1-3) (1-1.5), the reaction temperature is about 40 ℃ and the reaction time is more than 15 hours.
The amide condensing agent may be any combination of HBTU (benzotriazole-N, N, N ', N' -tetramethyluronium hexafluorophosphate)/DIEA, EDC/NHS (carbodiimide condensing agent/hydroxysuccinimide), or the like, as long as it is capable of promoting the amidation reaction of amino groups and carboxyl groups, and specific reaction conditions for promoting the amidation may be as described in the prior art.
If the above DMF solvents and DIEA/HATU condensing agents are used, one method of washing away the precipitated surface solvents and amide condensing agents is: washing with DMF to remove HATU; washing with ethanol to remove DMF; then washing with water to remove ethanol; washing with dilute hydrochloric acid to remove DIEA; finally, the diluted hydrochloric acid and the salt are removed by washing with water.
S2: modification of g-C with boric acid 3 N 4 Peeling to obtain boric acid modified carbon nitride nano-sheet (BCN).
This step can be carried out using the g-C method known in the art 3 N 4 The method for stripping into nano-sheets preferably adopts an ultrasonic-assisted liquid stripping method, and the specific method is to modify g-C with boric acid 3 N 4 The powder is placed in water and treated by ultrasonic power of 600-800W for 16-20h. The suspension after sonication can collect the exfoliated BCN by:
firstly, carrying out centrifugal separation (4000-6000 rpm) at a lower rotating speed on the suspension after ultrasonic treatment, collecting supernatant, namely the separated BCN dispersion liquid, carrying out centrifugal separation (more than 15000 rpm) at a higher rotating speed on the BCN dispersion liquid, collecting precipitate, namely the separated BCN, and freeze-drying for later use.
S3: preparing boric acid modified carbon nitride nano-sheet BCN into boric acid modified magnetic carbon nitride nano-sheet (M-BCN).
To facilitate separation of nanomaterials after CTCs are captured, BCN is prepared as a magnetic material by methods known in the art for preparing magnetic carbon nitrides, such as hydrothermal reaction of ferric and ferrous salts to form Fe under alkaline conditions 3 O 4 The particles are supported on the carbon nitride surface.
The following provides a specific reference method for preparing BCN into magnetic material M-BCN: placing the BCN obtained in the step S2 into water for ultrasonic dispersion, and adding FeCl after the BCN is uniformly dispersed 3 ·6H 2 O and FeCl 2 ·4H 2 O, continuing ultrasonic dispersion, transferring the mixed solution into a reaction kettle, regulating the pH value to be that ferric ions and ferrous ions can generate coprecipitation (pH value is 9-10), reacting for 1-1.5 hours at the temperature of 80-120 ℃, cooling to room temperature, washing the separated solid, and drying for later use to obtain the prepared M-BCN; the BCN, feCl 3 ·6H 2 O and FeCl 2 ·4H 2 The mass ratio of O is 2 (2-2.5) to 0.8-1.
S4: aptamer modification: after incubation (0.5-2 h) of the biotinylated aptamer solution and the avidin solution, they are mixed with each other for pre-assembly, as described above, preferably at 4:1 in molar ratio.
Then adding M-BCN, reacting at room temperature (accelerating reaction by vibration and the like for 15-30 min), and obtaining the M-BCN (Apt-Avi-BCN) loaded with the avidin-biotinylation aptamer Apt-Avi, namely the nano magnetic affinity material to be prepared, and washing and re-suspending for later use.
The ratio of Apt-Avi to M-BCN in this step may be sufficient to achieve a sufficient loading of Apt-Avi, and specifically, the mass ratio of avidin-biotinylated aptamer Apt-Avi to M-BCN may be 1-1.5.
The nanomagnetic affinity material may be washed with a PBS-BSA solution, preferably 1% BSA. BSA may reduce non-specific adsorption of materials.
The application method of the nano magnetic affinity material comprises the following steps:
adding a body fluid sample containing CTC into the nano magnetic affinity material dispersion for incubation, and capturing CTC in the body fluid; and (3) performing magnetic separation on the mixed liquid after CTC capture, and removing supernatant after the magnetic separation to obtain nano magnetic affinity material sediment containing the CTC captured.
When it is desired to release CTCs, a slightly acidic (ph 6.6-6.8) PBS buffer with dissolved fructose is added to the pellet for incubation to release CTCs from the surface of the nanomagnetic affinity material, followed by magnetic separation to obtain CTCs from the supernatant.
Unlike traditional chemical conjugation method to fix the capture ligand randomly on the capture interface, the covalent binding between boric acid and avidin glycoprotein in the nano magnetic affinity material has site specificity and high orientation, so that the aptamer is ordered. Due to ordered arrangement of the aptamer, local multivalent effect and buffer effect of the avidin, the problems of entanglement and local overcrowding among aptamer chains are greatly avoided, and the affinity of the material to CTC is greatly improved. Meanwhile, since borate coupling is reversible, gentle cell release is easily achieved by competitive binding of fructose under acidic conditions.
The schematic diagram of the preparation process of the nano magnetic affinity material Apt-Avi-BCN and the principle of capturing and releasing CTC is shown in figure 1.
The invention has the beneficial effects that:
the nano magnetic affinity material uses boric acid as a bio-orthogonal probe, and affinity factors are orderly arranged on the surface of a boric acid modified magnetic carbon nitride nano sheet, so that the nano magnetic affinity material is used for directional orderly assembly of biotinylation aptamer. The ordered orientation of the aptamer greatly alleviates the problems of inter-strand entanglement and localized overcrowding. Meanwhile, the avidin plays a role in buffering, and inactivation caused by direct interaction between the aptamer and the interface is avoided. The local multivalent effect of avidin/biotin 1:4 assembly also enhances the cell capture capacity of the engineering interface. The CTC affinity of the cell capture material of the present invention is improved by about 100-fold compared to the free aptamer. In addition, reversible boronic acid coupling allows for gentle release of the captured CTCs under acidic fructose conditions and higher cell viability after release. The material interface of the invention has the capturing efficiency of 91.85 percent and the release efficiency of 92.41 percent on the CTC of a whole blood sample, can directly separate the CTC from the peripheral blood of a tumor patient, and has clinical potential in early diagnosis and treatment of cancers.
Drawings
Fig. 1: (A) Apt-Avi-BCN material preparation procedure. (B) Apt-Avi-BCN interface captures and releases CTC working principle schematic diagram. (C) Apt-Avi-BCN interface 3D structure diagram. Schematic of CTC binding and release profile to material interface.
Fig. 2: (A) Capture and release efficiency of Apt-Avi-BCN after addition of different amounts of HepG2 to whole blood. (B) Fluorescence images of HepG2 living cells released by Apt-Avi-BCN (green, AO staining) and dead cells (red, PI staining), where only two are dead cells and the rest are living cells, indicated by arrows. (C) Proliferation images of released HepG2 cells after 0-96h of culture. Scale bar 50 μm.
Fig. 3: (A) Representative immunostained images of isolated WBCs and CTCs in blood samples from liver cancer patients. Scale bar 20 μm. CTC 1: epcam negative; CTC 2 epcam positive; (B) Number of CTCs in blood samples from healthy volunteers and from patients with liver cancer of different stages.
Detailed Description
The nano-magnetic affinity materials of the present invention and methods for their preparation and use are described below in connection with specific embodiments, but are not intended to be limiting.
g-C in this example 3 N 4 The powder is synthesized by the following method: weighing 3g dicyandiamide in a 50mL alumina crucible, covering a cover, wrapping and sealing with aluminum foil paper, placing in a muffle furnace, and standing at 3deg.C for min -1 Is of the speed of (1)The temperature was raised to 600℃and maintained for 2h. Cooling the reactant to room temperature, taking out, and obtaining light yellow solid which is g-C 3 N 4 g-C was prepared by using an agate mortar 3 N 4 Grinding into uniform solid powder for later use.
The nano magnetic affinity material is prepared according to the following specific method:
s1: preparation of boric acid modified g-C 3 N 4 : under dry conditions, 166mg of 4-carboxyphenylboronic acid (4-CPBA) was weighed into a 50mL conical flask, 30mL of DMF was slowly added along the wall of the flask, after stirring and dissolving at 40℃0.4mL of DIEA and 456mg of HATU (the molar ratio of 4-CPBA, DIEA, HATU was about 1:2.3:1.2) were added and kept at 40℃for 10min, followed by 50mg of g-C prepared by grinding 3 N 4 The powder was reacted at 40℃with stirring for 16h.
After the reaction, the sample in the conical flask is centrifuged for 3min at 6000rpm, the supernatant is discarded, and the precipitate is washed: washing with DMF to remove HATU; washing with ethanol to remove DMF; then washing with water to remove ethanol; washing with dilute hydrochloric acid to remove DIEA; finally, washing with water to remove dilute hydrochloric acid and salt; freeze-drying after the cleaning is finished to obtain the boric acid modified g-C 3 N 4 。
S2: boric acid modification of g-C by ultrasonic assisted liquid stripping 3 N 4 Exfoliation to boric acid modified carbon nitride nanoplatelets (BCN): taking 100mg of boric acid modified g-C 3 N 4 In a 250mL Erlenmeyer flask, 100mL of secondary water was poured. Ultrasound was continued for 16h with an ultrasound power of 800W. And centrifuging the cream yellow suspension obtained by ultrasonic treatment at 5000rpm for 15min, and collecting the supernatant to obtain BCN dispersion. The resulting supernatant was centrifuged at 15000rpm for 10min and the pellet, BCN, was collected. And finally, freeze-drying the precipitate and weighing the precipitate for later use.
S3: synthesis of boric acid modified magnetic carbon nitride nanoplatelets (M-BCN): 0.1g of BCN is weighed and added into a conical flask, 19mL of a mixed solution of absolute ethyl alcohol and 19mL of water is added, and 800W of ultrasonic wave is carried out for 5 hours, so that the uniformly dispersed BCN is obtained. Further 0.12g FeCl was added 3 ·6H 2 O and 0.047g FeCl 2 ·4H 2 And O, performing ultrasonic dispersion for 30min. Transfer the mixture to 100mL of PolytetrafluoroethaneAdding 2mL of ammonia water into the liner of the alkene reaction kettle, uniformly mixing, adjusting the pH to about 10, and putting the reaction kettle into a baking oven to react for 1h at the temperature of 100 ℃. Naturally cooling to room temperature, washing the separated solid with absolute ethyl alcohol and deionized water, drying in a vacuum drying oven at 60 ℃ for 12h, and taking out for standby.
S4: aptamer modification: biotinylated aptamer TLS11a (Bio-Apt, sequence: 5-Bio-TTT TTA CAG CAT CCC CAT GTG AAC AAT CGC ATT GTG ATT GTT ACG GTT TCC GCC TCA TGG ACG TGC TG-3) was used for specific targeting. mu.L of Bio-Apt (13.3. Mu.M) was first combined with 250. Mu.L of avidin solution (200. Mu.g mL -1 ) The Bio-Apt was pre-assembled with avidin at a ratio of 4:1 after incubation for 1 hour at 37 ℃. 500 mu L M-BCN (250. Mu. GmL) was added -1 ) The resulting Bio-Apt loaded M-BCN (Apt-Avi-BCN) was resuspended by repeated washing in 500. Mu.L PBS (1% BSA to reduce non-specific adsorption) with shaking at room temperature for 30min.
The Apt-Avi-BCN nano magnetic affinity material prepared by the method can be used for capturing and releasing cells: and adding 500 mu L of Apt-Avi-BCN dispersion liquid into 500 mu L of a sample to be detected, incubating for 30min at 37 ℃, and finally magnetically separating to remove supernatant to obtain a precipitate containing the cell-capturing nano magnetic affinity material.
After cell capture, 1ml of PBS buffer (ph=6.8) containing 60mM fructose was added to the pellet and gently swirled at 37 ℃ for 30min. Magnetic separation was performed and the released cells were left in the supernatant after magnetic separation and counted under a microscope.
The prepared Apt-Avi-BCN nanomagnetic affinity material was tested for CTC cell capturing performance in blood using the following method: hepG2 cells pre-stained with DAPI were added to whole blood. Then, 500. Mu.L of blood was added to the 500. Mu.LApt-Avi-BCN dispersion and the mixture was incubated at 37℃for 30 minutes. The uncaptured cells in the supernatant were then separated by magnetic force and counted under a fluorescence microscope. The cell capture efficiency was calculated as follows:
capture efficiency = (number of cells added-number of cells not captured)/number of cells added x 100%
The prepared Apt-Avi-BCN nano magnetic affinity material is tested for release performance in blood by the following method: after cell capture, 1ml of PBS buffer containing 60mM fructose (ph=6.8) was added and incubated for 30 minutes with gentle shaking at 37 ℃. The released cells were magnetically separated from the capture material and counted. The cell release efficiency was calculated as follows:
release efficiency = number of released cells/number of captured cells x 100%
Cell viability assay and in vitro culture of released CTCs: cell viability of released HepG2 cells was assessed by live/dead staining method. Acridine Orange (AO) can penetrate living cells and fluoresce green, while dead cells are stained with Propidium Iodide (PI) and display red fluorescence. Released cells were co-stained with AO and PI and viability was calculated by counting live and dead cells as observed under fluorescence microscopy. Released HepG2 cells at 37℃and 5% CO 2 And DMEM (10% fbs and 1% antibiotics). Bright field images were taken at some time points with an inverted microscope.
FIG. 2 shows the results of the capture performance, release performance, cell viability assay and in vitro culture described above. As can be seen from FIG. 2, when the HepG2 cell number was between 50 and 1000, the capture efficiency was > 91.85%, the release efficiency was > 92.41% (FIG. 2A), and the DAPI/AO staining was examined, with 98% survival (FIG. 2B). Cells were collected, cultured in 96-well plates, stably attached for 24h, and cultured for 96h to become full of cells (fig. 2C).
The clinical application of the Apt-Avi-BCN material described above was further evaluated by analyzing blood samples of 18 liver cancer patients and 17 healthy volunteers without any pretreatment: without pretreatment, blood samples (0.5 mL) were added to 0.5mL of the Apt-Avi-BCN dispersion and incubated at 37℃for 30min, the supernatant was removed by magnetic separation, and the Apt-Avi-BCN was gently washed. Cells captured on Apt-Avi-BCN were released after incubation with PBS solution containing 60mM fructose (ph=6.8) at 37 ℃ for 30min with gentle shaking, and cells were obtained from the supernatant by magnetic separation.
Isolated cells were stained for immunofluorescence to identify CTCs: cells were fixed with 4% polyoxymethylene solution, blocked with 3% goat serum solution, and immunofluorescent staining was performed with DAPI, anti-CK and anti-CD 45. Finally, the cells were observed under a positive fluorescence microscope and photographed. Cells that were CD45 negative, DAPI and CK positive (DAPI+/CK+/CD 45-) were identified as CTCs, and cells that were CD45 and DAPI positive, CK negative (DAPI+/CK-/CD45+) were identified as WBC (FIG. 3A). CTCs were found and isolated in all liver cancer patients, with 5-43 CTCs per 1ml of blood, whereas no CTCs were found in healthy donor blood. At the same time, the number of CTCs captured was not only significantly different between healthy donor and patient, but also between patients in three phases (fig. 3B).

Claims (1)

1. The nano magnetic affinity material is characterized by comprising a boric acid modified magnetic carbon nitride nano sheet M-BCN and an avidin-biotinylation aptamer Apt-Avi, wherein the avidin-biotinylation aptamer Apt-Avi is prepared by pre-assembling a biotinylation aptamer and avidin;
the circulating tumor cells refer to HepG2 cells;
the biotinylated aptamer is TLS11a, sequence: 5-bio-TTT TTA CAG CAT CCC CAT GTG AAC AAT CGC ATT GTG ATT GTT ACG GTT TCC GCC TCA TGG ACG TGC TG-3;
the avidin-biotinylation aptamer Apt-Avi is combined with the oriented connection of the boric acid ligand in the boric acid-modified magnetic carbon nitride nanosheet M-BCN through the glycosyl of the avidin, and is loaded on the boric acid-modified magnetic carbon nitride nanosheet M-BCN;
in the avidin-biotinylated aptamer Apt-Avi, the biotinylated aptamer and avidin were present in a ratio of 4:1, pre-assembling the mixture in a molar ratio of 1;
in the boric acid modified magnetic carbon nitride nanosheet M-BCN, the matrix material is magnetic g-C 3 N 4 The nanometer sheet, the modified boric acid is carboxyphenylboric acid, the carboxyl in the molecular structure and the magnetism g-C 3 N 4 The amino groups on the surfaces of the nano-sheets are condensed into amide bonds;
adding a sample containing HepG2 cells into the nano magnetic affinity material dispersion for incubation, and capturing the HepG2 cells; performing magnetic separation on the mixed liquid after the acquisition of the HepG2 cells, removing supernatant after the magnetic separation, adding PBS buffer solution with pH of 6.6-6.8 and dissolved with fructose for incubation, so that the HepG2 cells are released from the surface of the nano magnetic affinity material, performing magnetic separation, and obtaining the HepG2 cells from the supernatant; when the number of HepG2 cells in whole blood is 50-1000 cells/mL, the capturing efficiency is more than 91.85%, the releasing efficiency is more than 92.41%, and 98% of the cells survive;
the preparation method of the nano magnetic affinity material for efficiently capturing and nondestructively releasing the circulating tumor cells comprises the following steps:
s1: preparation of boric acid modified g-C 3 N 4 : dissolving carboxyphenylboronic acid in N, N-dimethylformamide under drying condition, adding amide condensing agent, and adding g-C 3 N 4 Reacting the powder; the amide condensing agent is N, N-diisopropylethylamine and 2- (7-aza-benzotriazol) -N, N, N ', N' -tetramethyl urea hexafluorophosphate; carboxyphenylboronic acid: n, N-diisopropylethylamine: the molar ratio of the 2- (7-aza-benzotriazol) -N, N, N ', N' -tetramethyl urea hexafluorophosphate is 1 (1-3): 1-1.5; carboxyphenylboronic acid and g-C 3 N 4 The mass ratio of the powder is (3-4) 1;
after the reaction is finished, centrifugal separation is carried out, sediment obtained by centrifugal separation is taken out, solvent and amide condensing agent are washed off, and boric acid modified g-C is obtained after freeze drying 3 N 4 ；
S2: modification of g-C with boric acid 3 N 4 Stripping to obtain boric acid modified carbon nitride nano-sheet BCN; the stripping method adopts ultrasonic-assisted liquid stripping, the liquid adopts water, the ultrasonic power is 600-800W, and the ultrasonic time is 16-20 h;
s3: preparing boric acid modified carbon nitride nano-sheet BCN into boric acid modified magnetic carbon nitride nano-sheet M-BCN; placing the BCN obtained in the step S2 into water for ultrasonic dispersion, and adding FeCl after the BCN is uniformly dispersed 3 ·6H 2 O and FeCl 2 ·4H 2 O, continuing ultrasonic dispersion, transferring the mixed solution into a reaction kettle, adjusting the pH, reacting for 1-1.5h at the temperature of 80-120 ℃, cooling to room temperature, washing the separated solid, and drying for later use; the BCN, feCl 3 ·6H 2 O and FeCl 2 ·4H 2 The mass ratio of O is 2 (2-2.5) to 0.8-1;
s4: aptamer modification: and (3) incubating the biotinylated aptamer solution and the avidin solution, then pre-assembling, adding the boric acid modified magnetic carbon nitride nano-sheet M-BCN, reacting at room temperature to obtain the boric acid modified magnetic carbon nitride nano-sheet M-BCN loaded with the avidin-biotinylated aptamer Apt-Avi, namely the nano magnetic affinity material to be prepared, wherein the mass ratio of the avidin-biotinylated aptamer Apt-Avi to the boric acid modified magnetic carbon nitride nano-sheet M-BCN is (1-1.5): 1, washing by adopting the PBS-BSA solution, and then re-suspending for later use.
CN202210433043.3A 2022-04-24 2022-04-24 Nanometer magnetic affinity material for efficiently capturing and nondestructively releasing circulating tumor cells Active CN114958748B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210433043.3A CN114958748B (en) 2022-04-24 2022-04-24 Nanometer magnetic affinity material for efficiently capturing and nondestructively releasing circulating tumor cells

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210433043.3A CN114958748B (en) 2022-04-24 2022-04-24 Nanometer magnetic affinity material for efficiently capturing and nondestructively releasing circulating tumor cells

Publications (2)

Publication Number Publication Date
CN114958748A CN114958748A (en) 2022-08-30
CN114958748B true CN114958748B (en) 2024-02-13

Family

ID=82980013

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210433043.3A Active CN114958748B (en) 2022-04-24 2022-04-24 Nanometer magnetic affinity material for efficiently capturing and nondestructively releasing circulating tumor cells

Country Status (1)

Country Link
CN (1) CN114958748B (en)

Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003065042A1 (en) * 2001-02-16 2003-08-07 Immunivest Corporation Methods and reagents for the rapid and efficient isolation of circulating cancer cells
JP2004333232A (en) * 2003-05-02 2004-11-25 Canon Inc Separating method, detecting method, and screening method for target component
WO2005119255A2 (en) * 2004-05-12 2005-12-15 Applera Corporation Constrained cis-diol-borate bioconjugation system
WO2011112188A1 (en) * 2010-03-10 2011-09-15 Perfinity Biosciences, Inc. Method for recognition and quantification of multiple analytes in a single analysis
CN104983716A (en) * 2015-07-20 2015-10-21 广西医科大学 Tumor cell membrane/nuclear membrane double-targeting tumor nano-drug slow-release system and preparation and application thereof
CN105486865A (en) * 2014-09-15 2016-04-13 浙江大学 Micro-fluidic chip used for cell sorting and gathering and application of micro-fluidic chip
CN105717288A (en) * 2016-01-28 2016-06-29 上海交通大学 Surface modified magnetic nanoparticles and preparation method and application thereof
CN108254346A (en) * 2018-01-19 2018-07-06 苏州大学 Using DNA as the magnetic fluorescence copolymer nano probe of template and application
CN109517722A (en) * 2018-09-28 2019-03-26 张珝 A kind of device and its making and use method capturing specific few cells
CN111337328A (en) * 2020-04-14 2020-06-26 厦门大学 Method for non-injury rapid capture and release of circulating tumor cells in whole blood
CN111454907A (en) * 2020-04-14 2020-07-28 厦门大学 Circulating tumor cell rapid non-invasive capture, release and detection kit
CN112300994A (en) * 2019-08-02 2021-02-02 中国科学院苏州纳米技术与纳米仿生研究所 Nano magnetic bead for capturing circulating tumor cells and preparation method and application thereof
CN113189066A (en) * 2021-04-26 2021-07-30 东北大学 Bionic nano probe and preparation method and application thereof

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020172987A1 (en) * 1998-02-12 2002-11-21 Terstappen Leon W.M.M. Methods and reagents for the rapid and efficient isolation of circulating cancer cells
US10466243B2 (en) * 2013-11-20 2019-11-05 University Of Florida Research Foundation, Incorporated Antibody and aptamer ensemble for cell isolation and enrichment
WO2017180499A2 (en) * 2016-04-13 2017-10-19 President And Fellows Of Harvard College Methods for capturing, isolation, and targeting of circulating tumor cells and diagnostic and therapeutic applications thereof

Patent Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003065042A1 (en) * 2001-02-16 2003-08-07 Immunivest Corporation Methods and reagents for the rapid and efficient isolation of circulating cancer cells
JP2004333232A (en) * 2003-05-02 2004-11-25 Canon Inc Separating method, detecting method, and screening method for target component
WO2005119255A2 (en) * 2004-05-12 2005-12-15 Applera Corporation Constrained cis-diol-borate bioconjugation system
WO2011112188A1 (en) * 2010-03-10 2011-09-15 Perfinity Biosciences, Inc. Method for recognition and quantification of multiple analytes in a single analysis
CN105486865A (en) * 2014-09-15 2016-04-13 浙江大学 Micro-fluidic chip used for cell sorting and gathering and application of micro-fluidic chip
CN104983716A (en) * 2015-07-20 2015-10-21 广西医科大学 Tumor cell membrane/nuclear membrane double-targeting tumor nano-drug slow-release system and preparation and application thereof
CN105717288A (en) * 2016-01-28 2016-06-29 上海交通大学 Surface modified magnetic nanoparticles and preparation method and application thereof
CN108254346A (en) * 2018-01-19 2018-07-06 苏州大学 Using DNA as the magnetic fluorescence copolymer nano probe of template and application
CN109517722A (en) * 2018-09-28 2019-03-26 张珝 A kind of device and its making and use method capturing specific few cells
CN112300994A (en) * 2019-08-02 2021-02-02 中国科学院苏州纳米技术与纳米仿生研究所 Nano magnetic bead for capturing circulating tumor cells and preparation method and application thereof
CN111337328A (en) * 2020-04-14 2020-06-26 厦门大学 Method for non-injury rapid capture and release of circulating tumor cells in whole blood
CN111454907A (en) * 2020-04-14 2020-07-28 厦门大学 Circulating tumor cell rapid non-invasive capture, release and detection kit
CN113189066A (en) * 2021-04-26 2021-07-30 东北大学 Bionic nano probe and preparation method and application thereof

Non-Patent Citations (12)

* Cited by examiner, † Cited by third party
Title
A Rapid and Facile Separation–Detection Integrated Strategy for Exosome Profiling Based on Boronic Acid-Directed Coupling Immunoaffinity;Wang, Y等;《 Anal. Chem》;第93卷;摘要，第16060页右列倒数第1段-第16061页左列第1段 *
A rapid and simple method for efficient capture and accurate discrimination of circulating tumor cells using aptamer conjugated magnetic beads and surface-enhanced Raman scattering imaging;Changlong Sun等;《Analytical and Bioanalytical Chemistry》;第407卷;8883-8892 *
A Versatile Dynamic Mussel-Inspired Biointerface: From Specific Cell Behavior Modulation to Selective Cell Isolation;Lei Liu等;《Angew Chem Int Ed Engl》;第57卷(第26期);第8007页左列倒数第2段、右列第1段 *
Aptamer-functionalized nano/micro-materials for clinical diagnosis: isolation, release and bioanalysis of circulating tumor cells;Yaju Zhao等;《Integrative Biology》;第9卷(第3期);188-205 *
Discrimination of pathogenic bacteria with boronic acid modified protonated g-C3N4 nanosheets at various pHs;Yang, J.-Y等;《Sens. Actuators, B》;第340卷;全文 *
Dynamic Synthetic Biointerfaces: From Reversible Chemical Interactions to Tunable Biological Effects;Ma Y等;《Acc. Chem. Res》;第52卷;第1612页右列倒数第1段-1613页左列第1段、第1614页左列第2段-右列第1段，图3 *
Flower-like-flake Fe3O4/g-C3N4 nanocomposite: Facile synthesis, characterization, and enhanced photocatalytic performance;Dinglong Zhu等;《Colloids and Surfaces A: Physicochemical and Engineering Aspects》;第537卷;372-382 *
Reversible and Highly Ordered Biointerfaces for Efficient Capture and Nondestructive Release of Circulating Tumor Cells;Siyi Wang等;《Anal Chem》;第94卷;9450-9458 *
共沉淀法制备Fe3O4/石墨相氮化碳磁性复合材料及其移除有机废水的研究;张璐等;《化工新型材料》;第48卷(第7期);第120页右列第2段 *
基于核酸适配体的微流控芯片的构建及对循环肿瘤细胞筛选性能的测定;肖莉莉;刘晖;贾春锋;;福建医药杂志(第02期);全文 *
循环肿瘤细胞捕获微纳技术;刘洋;刘绍琴;;自然杂志(第03期);全文 *
核酸适配体修饰的铁纳米粒对EGFR阳性肿瘤细胞的靶向热疗效应;于连元等;《基础医学与临床》 *

Also Published As

Publication number Publication date
CN114958748A (en) 2022-08-30

Similar Documents

Publication Publication Date Title
Hens et al. Nanodiamond bioconjugate probes and their collection by electrophoresis
Fang et al. Aptamer-conjugated upconversion nanoprobes assisted by magnetic separation for effective isolation and sensitive detection of circulating tumor cells
Zhou et al. Multifunctional luminescent immuno-magnetic nanoparticles: toward fast, efficient, cell-friendly capture and recovery of circulating tumor cells
CN103630440A (en) Enriching method of circulating tumor cells
CN109312293A (en) For carrying out magnetic floating isolated composition and method
CN107858144B (en) A kind of magnetic material and preparation method thereof for efficient detection circulating tumor cell
Polito et al. One-step bioengineering of magnetic nanoparticles via a surface diazo transfer/azide–alkyne click reaction sequence
Peng et al. Fluorescent-magnetic-catalytic nanospheres for dual-modality detection of H9N2 avian influenza virus
CN106366196B (en) EpCAM antibody immunomagnetic beads and preparation method thereof
Chang et al. Biomimetic immunomagnetic gold hybrid nanoparticles coupled with inductively coupled plasma mass spectrometry for the detection of circulating tumor cells
CN106018806B (en) One kind is based on Au Fe3O4The preparation method and application for the interlayer type immunosensor that@Pd ZIF 8 are marked
CN112924695B (en) Composite magnetic nano material based on DNA tetrahedron, preparation and application
KR20110033575A (en) Mesoporous silica conjugate integrating magnetic nano particles having peroxidase activity and enzymes and method for manufacturing the same
CN111337328A (en) Method for non-injury rapid capture and release of circulating tumor cells in whole blood
CN104880456A (en) Preparation method and application of electrochemiluminescence immunosensor constructed on basis of GO/MWCNTs-COOH/Au @ CeO2
Elingarami et al. Surface-engineered magnetic nanoparticles for molecular detection of infectious agents and cancer
Bloemen et al. Antibody-modified iron oxide nanoparticles for efficient magnetic isolation and flow cytometric determination of L. pneumophila
JP2009531302A (en) Targeted iron oxide nanoparticles
Cheng et al. Hedgehog-inspired immunomagnetic beads for high-efficient capture and release of exosomes
CN114958748B (en) Nanometer magnetic affinity material for efficiently capturing and nondestructively releasing circulating tumor cells
CN112300994B (en) Nanometer magnetic bead for capturing circulating tumor cells and preparation method and application thereof
Rashid et al. Surface modification and bioconjugation of anti-CD4 monoclonal antibody to magnetic nanoparticles as a highly efficient affinity adsorbent for positive selection of peripheral blood T CD4+ lymphocytes
CN106366195B (en) PD-L1 antibody immunomagnetic beads and preparation method thereof
CN111701031A (en) Nano material for loading circRNA by targeted mitochondria and preparation method and application thereof
CN107522239A (en) A kind of method based on gallic acid metallic ion coordination chemical regulation nano ferriferrous oxide decentralization and particle diameter

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant