Molecularly imprinted polymer nanoparticles compatible with biological samples and preparation method thereof

Abstract

This invention provides molecularly imprinted polymer nanoparticles compatible with biological samples, and in particular pure biological samples and a preparation method thereof. Said molecularly imprinted polymer nanoparticles have a crosslinking degree exceeding 50%, a particle diameter of 10 to 500 nm, hydrophilic polymer brushes on its surfaces and can be prepared by introducing appropriate hydrophilic macromolecular chain transfer agents into reversible addition-fragmentation chain transfer (RAFT) precipitation polymerization systems through the one-pot synthesis. The preparation method is simple, features a broad range of application and yields a pure product. The obtained hydrophilic molecularly imprinted polymer nanoparticles have prospects for a wide range of application in biological sample analysis, medical clinical immune analysis, food and environmental monitoring, biomimetic sensors, etc.

Claims

1. A molecularly imprinted polymer nanoparticle with surface-grafted hydrophilic polymer brushes, wherein said nanoparticle has a core formed by the crosslinked polymer networks and hydrophilic polymer brushes on the surfaces of the core, said core has a crosslinking degree exceeding 50% and a particle diameter of 10-500 nm, and the core of said nanoparticle has molecularly imprinted cavities that are capable of recognizing the template molecules in biological samples.

2. The molecularly imprinted polymer nanoparticle according to claim 1, wherein said molecularly imprinted polymer nanoparticle is synthesized by a reversible addition-fragmentation chain transfer (RAFT) precipitation polymerization of monovinyl functional monomer and crosslinker mediated by hydrophilic macromolecular chain transfer agent and optional small molecule chain transfer agent in the presence of template molecule.

3. The molecularly imprinted polymer nanoparticle according to claim 2, wherein said reversible addition-fragmentation chain transfer (RAFT) precipitation polymerization is induced by the combined use of the hydrophilic macromolecular chain transfer agent and the small molecule chain transfer agent.

4. The molecularly imprinted polymer nanoparticle according to claim 2, wherein said template molecule is selected from the group consisting of amino acid, biological receptor, nucleic acid, steroid, immunosuppressant, hormone, heparin, antibiotics, vitamin, small pathological and disease biomarker, toxin, pesticide, herbicide, explosive, neurotoxin, endocrine disrupter, nucleotide, nucleoside, oligomeric nucleotide, metabolin, secondary metabolite, drug metabolite, drug, drug intermediate, and pharmic organic molecule.

5. The molecularly imprinted polymer nanoparticle according to claim 2, wherein said monovinyl functional monomer is selected from the group consisting of C.sub.2-C.sub.8 monoolefin, aromatic vinyl compounds, heterocyclic aromatic vinyl compounds, monoolefin type unsaturated carboxylic acid, carboxylate, ester and amide, vinyl alcohol and its ester, allyl alcohol and its ester, vinyl ether, vinyl lactam, vinyl halide, 1,1-dihaloethylene, (meth)acrylic acid and its ester, salt, amide, vinylpyridine compound, trifluoromethylacrylic acid and combinations thereof.

6. The molecularly imprinted polymer nanoparticle according to claim 2, wherein said crosslinker is a monomer with two or more nonconjugated unsaturated double bond.

7. The molecularly imprinted polymer nanoparticle according to claim 2, wherein said hydrophilic macromolecular chain transfer agent is hydrophilic polymers with a terminal dithioester or trithiocarbonate group.

8. The molecularly imprinted polymer nanoparticle according to claim 2, wherein said small molecule chain transfer agent has the following structure: ##STR00002## wherein R=alkyl, alkenyl, alkynyl, aryl, aralkyl, substituted alkyl, substituted aryl, carbocyclic or heterocyclic, alkyloxy or dialkylamine; Z=alkyl, aryl, aralkyl, substituted alkyl, substituted aryl, carbocyclic or heterocyclic, alkylthiol, arylthiol, alkoxycarbonyl, aralkoxycarbonyl, acyloxy, carbamoyl, cyano, N-alkyl, N-aryl-substituted amino, alkoxy, aryloxy, dialkyl or diaryl phosphate, dialkyl or diaryl phosphinic.

9. The molecularly imprinted polymer nanoparticle according to claim 2, wherein the molar ratio of the template molecule to monovinyl functional monomer to crosslinker is 1:1-10:4-80.

10. The molecularly imprinted polymer nanoparticle according to claim 2, wherein the molar ratio of the total amount of monovinyl functional monomer and crosslinker to that of the hydrophilic macromolecular chain transfer agent and optional small molecule chain transfer agent is 500:1-10:1.

11. The molecularly imprinted polymer nanoparticle according to claim 2, wherein the molar percentage of the small molecule chain transfer agent in the total amount of both small molecule chain transfer agent and hydrophilic macromolecular chain transfer agent is 0-95%.

12. The molecularly imprinted polymer nanoparticle according to claim 2, wherein the number-average molecular weight of said hydrophilic macromolecular chain transfer agent is in a range from of 500 to 50,000.

13. The molecularly imprinted polymer nanoparticle according to claim 1, wherein said molecularly imprinted polymer nanoparticle has a narrow particle size distribution, and the narrow particle size distribution indices of said nanoparticle determined by dynamic laser scattering according to ISO 13321 is less than or equal to 0.6.

14. The molecularly imprinted polymer nanoparticle according to claim 1, wherein said hydrophilic polymer brushes are formed by the macromolecular chain transfer agent.

15. A kit comprising the molecularly imprinted polymer nanoparticle with surface-grafted hydrophilic polymer brushes according to claim 1.

16. A sensor applicable for biological samples comprising the molecularly imprinted polymer nanoparticle with surface-grafted hydrophilic polymer brushes according to claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) In order to further clarify the above-mentioned and other advantages and characteristics of the present invention, a more detailed description of the present invention will be provided by referring to the specific embodiment described in the Figures. It should be noted that these Figures only describe the illustrative embodiment of the present invention, and they should not be considered to limit the scope of the invention. The present invention will be described and explained in a more specific and detailed way by using the following Figures, wherein:

(2) FIG. 1. Schematic illustration of the preparation of MIP nanoparticles with surface-grafted hydrophilic polymer brushes via hydrophilic macromolecular chain transfer agent-mediated RAFT precipitation polymerization.

(3) FIG. 2. Scanning electron microscope image of the MIP nanoparticles (with 2,4-D as the template) with surface-grafted hydrophilic poly(2-hydroxyethyl methacrylate) brushes (M.sub.n,NMR=4800) prepared via hydrophilic macromolecular chain transfer agent-mediated RAFT precipitation polymerization.

(4) FIG. 3. Scanning electron microscope image of the non-imprinted polymer nanoparticles (corresponding to the MIP in FIG. 2) with surface-grafted hydrophilic poly(2-hydroxyethyl methacrylate) brushes (M.sub.n,NMR=4800) prepared via hydrophilic macromolecular chain transfer agent-mediated RAFT precipitation polymerization.

(5) FIG. 4. Scanning electron microscope image of the MIP nanoparticles (with propranolol as the template) (propranolol-MIP-2) with surface-grafted hydrophilic poly(2-hydroxyethyl methacrylate) brushes (M.sub.n,NMR=4800) prepared via hydrophilic macromolecular chain transfer agent-mediated RAFT precipitation polymerization.

(6) FIG. 5. Scanning electron microscope image of the non-imprinted polymer nanoparticles (corresponding to the MIP in FIG. 4) (propranolol-CP-2) with surface-grafted hydrophilic poly(2-hydroxyethyl methacrylate) brushes (M.sub.n,NMR=4800) prepared via hydrophilic macromolecular chain transfer agent-mediated RAFT precipitation polymerization.

(7) FIG. 6. Equilibrium bindings of 2,4-D to the MIP (with 2,4-D as the template, filled symbols) and non-imprinted polymer (empty symbols) nanoparticles with surface-grafted hydrophilic poly(2-hydroxyethyl methacrylate) brushes (M.sub.n,NMR=4800) in pure milk (square) and pure bovine serum (diamond) (binding temperature: 25 C., the concentration of 2,4-D: 0.02 mM).

(8) FIG. 7. Selective bindings of the MIP (with 2,4-D as the template) and non-imprinted polymer (CP) nanoparticles with surface-grafted hydrophilic poly(2-hydroxyethyl methacrylate) brushes (M.sub.n,NMR=4800) toward a mixture of 2,4-D and its analogue phenoxyacetic acid (POAc) in pure milk and pure bovine serum (binding temperature: 25 C., both the concentrations of 2,4-D and POAc are 0.02 mM, and the concentrations of MIP and CP are 12 mg/mL).

(9) FIG. 8. Equilibrium bindings of propranolol to the MIP (propranolol-MIP-2, filled symbols) and non-imprinted polymer (propranolol-CP-2, empty symbols) nanoparticles with surface-grafted hydrophilic poly(2-hydroxyethyl methacrylate) brushes (M.sub.n,NMR=4800) in pure milk (square) and pure bovine serum (diamond) (binding temperature: 25 C., the concentration of propranolol: 0.05 mM).

(10) FIG. 9. Selective bindings of the MIP (propranolol-MIP-2) and non-imprinted polymer (CP) (propranolol-CP-2) nanoparticles with surface-grafted hydrophilic poly(2-hydroxyethyl methacrylate) brushes (M.sub.n,NMR=4800) toward a mixture of propranolol and its analogue atenolol in pure milk and pure bovine serum (binding temperature: 25 C., both the concentrations of propranolol and atenolol are 0.05 mM, the concentrations of propranolol-MIP-2 and propranolol-CP-2 are 2 mg/mL).

DETAILED DESCRIPTION

Definition

(11) As used in the present invention the term pure biological samples denotes the samples that are rich in biological matrices such as proteins (e.g., serum, milk, and urine, etc.).

(12) The term biological samples includes many types of samples obtained from the subjects and useful in the present invention. The biological samples may include, but not limited to, solid tissue samples, liquid tissue samples, biological fluids, aspirates, cells, and cell fragments. The specific examples of biological samples include, but not limited to, solid tissue samples, pathological samples, archived samples, biopsy samples, or tissue cultures obtained by remove operation or the cells and cell offsprings obtained from them as well as any section or smear prepared from the above sources. The non-restrictive examples of biological samples include those samples obtained from breast tissue, lymph nodes, and breast cancer. The biological samples also include any material obtained from the vertebrate body, include but not limited to blood, cerebrospinal fluid, serum, plasma, urine, nipple aspirate, fine needle aspiration, tissue lavage fluid such as ductal lavage fluid, saliva, phlegm, ascitic fluid, liver, kidney, breast, bone, marrow, orchis, brain, oarium, skin, lung, prostate, gland, pancreas, uterine neck, stomach, intestines, colon and rectum, bladder, nostril, uterus, semen, lymph, vaginal mixture, synovia, spinal fluid, head and neck, nasopharyngeal neoplasms, amniotic fluid, breast milk, pulmonary or pulmonary surfactant, urine, excrement and liquid samples of biological origin.

(13) As used in the present invention the term MIP nanoparticles denotes the polymers containing at least some fractions of cavities (or gaps) complementary with one or more templates with their diameters ranging from 10 to several hundreds of nanometers, wherein the template molecules are added into the monomer solutions containing the crosslinker prior to the polymerization. The resulting polymers have many cavities complementary with the template molecules.

(14) As used in the present invention the term RAFT precipitation polymerization is one type of controlled/living radical polymerizations. Dithioester or trithiocarbonate derivatives are normally added into a RAFT polymerization system to act as chain transfer agent, which can react with the propagating chain radical to form dormant intermediate during the polymerization process, thus suppressing the irreversible termination side reactions between the propagating chain radicals, and leading to well-controlled polymerization. Self-fragmentation of such dormant intermediate can give rise to a new radical from the corresponding sulfur atom, which is able to reinitiate the monomer to form propagating chain. Since the rates of both the addition or fragmentation processes are much faster than the chain propagation process, which allows the dithioester or trithiocarbonate derivative to quickly transfer between the reactive radicals and dormant species, thus resulting in narrow molecular-mass distribution and controlled/living polymerization.

(15) As used in the present invention the term particle size distribution denotes the percentages of particles with different diameters in a group of particles, also named as dispersion degree of the particles.

(16) As used in the present invention the term crosslinking degree denotes the ratio of the amount of the incorporated crosslinker in the polymer to that of the incorporated crosslinker and functional monomer. The crosslinking degrees of the MIP nanoparticles with surface-grafted hydrophilic polymer brushes obtained according to the present invention are determined by using ASTM D 2765. The crosslinking degree of the MIP obtained according to the present invention is larger than 50%, preferably larger than 60%, more preferably larger than 70%, and more preferably larger than 80%.

EXAMPLES

(17) The following examples are only used to illustrate the embodiments of the present invention, and they should not be interpreted as the limitation of the invention in any way.

Preparation Examples

Example 1

(18) 2,4-D (0.83 mmol) is added into a one-neck round-bottom flask (100 mL) containing a mixture of methanol and water (4:1 v/v, 60 mL), and a clear solution is obtained after magnetic stirring, and then 4-vinylpyridine (0.83 mmol) is added into the above solution. After their thorough mixing for 0.5 h, ethylene glycol dimethacrylate (2.50 mmol), small chain transfer agent cumyl dithiobenzoate (CDB) (0.055 mmol), poly(2-hydroxyethyl methacrylate) macromolecular chain transfer agent (M.sub.n,NMR=4800) (0.034 mmol), and azobisisobutyronitrile (AIBN) (0.028 mmol) are added successively. After being purged with argon for 30 min, the reaction mixture is sealed and immersed into a thermostatted oil bath at 60 C. The polymerization is allowed to take place for 10 h. The resulting polymer particles are collected by high speed centrifugation.

(19) The above product is purified by being washed with methanol/acetic acid (9:1 v/v) and methanol until no template is detectable in the centrifugated supernatant. After being dried at 40 C. under vacuum for 48 h, MIP nanoparticles with surface-grafted poly(2-hydroxyethyl methacrylate) brushes (M.sub.n,NMR=4800) are obtained. Said MIP nanoparticles have a diameter of 111 nm (DLS, with methanol as the solvent, the same below) and a crosslinking degree of 75%.

(20) The non-imprinted nanoparticles with surface-grafted poly(2-hydroxyethyl methacrylate) brushes (M.sub.n,NMR=4800) are synthesized and purified under the identical conditions except that the template is omitted.

Example 2

(21) 2,4-D (0.83 mmol) is added into a one-neck round-bottom flask (100 mL) containing a mixture of methanol and water (4:1 v/v, 60 mL), and a clear solution is obtained after magnetic stirring, and then 4-vinylpyridine (0.83 mmol) is added into the above solution. After their thorough mixing for 0.5 h, ethylene glycol dimethacrylate (2.50 mmol), small chain transfer agent cumyl dithiobenzoate (CDB) (0.055 mmol), poly(2-hydroxyethyl methacrylate) macromolecular chain transfer agent (M.sub.n,NMR=3250) (0.034 mmol), and azobisisobutyronitrile (AIBN) (0.028 mmol) are added successively. After being purged with argon for 30 min, the reaction mixture is sealed and immersed into a thermostatted oil bath at 60 C. The polymerization is allowed to take place for 10 h. The resulting polymer particles are collected by high speed centrifugation.

(22) The above product is purified by being washed with methanol/acetic acid (9:1 v/v) and methanol until no template is detectable in the centrifugated supernatant. After being dried at 40 C. under vacuum for 48 h, MIP nanoparticles with surface-grafted poly(2-hydroxyethyl methacrylate) brushes (M.sub.n,NMR=3250) are obtained. Said MIP nanoparticles have a diameter of 147 nm and a crosslinking degree of 75%.

(23) The non-imprinted nanoparticles with surface-grafted poly(2-hydroxyethyl methacrylate) brushes (M.sub.n,NMR=3250) are synthesized and purified under the identical conditions except that the template is omitted.

Example 3

(24) Propranolol (0.83 mmol) is added into a one-neck round-bottom flask (100 mL) containing a mixture of acetonitrile and methanol (1:1 v/v, 60 mL), and a clear solution is obtained after magnetic stirring, and then methacrylic acid (0.83 mmol) is added into the above solution. After their thorough mixing for 0.5 h, ethylene glycol dimethacrylate (2.50 mmol), small chain transfer agent cumyl dithiobenzoate (CDB) (0.055 mmol), poly(2-hydroxyethyl methacrylate) macromolecular chain transfer agent (M.sub.n,NMR=4800) (0.034 mmol), and azobisisobutyronitrile (AIBN) (0.028 mmol) are added successively. After being purged with argon for 30 min, the reaction mixture is sealed and immersed into a thermostatted oil bath at 60 C. The polymerization is allowed to take place for 24 h. The resulting polymer particles are collected by high speed centrifugation.

(25) The above product is purified by being washed with methanol/acetic acid (9:1 v/v) and methanol until no template is detectable in the centrifugated supernatant. After being dried at 40 C. under vacuum for 48 h, MIP nanoparticles with surface-grafted poly(2-hydroxyethyl methacrylate) brushes (M.sub.n,NMR=4800) (propranolol-MIP-1) are obtained. Said MIP nanoparticles have a diameter of 220 nm and a crosslinking degree of 75%.

(26) The non-imprinted nanoparticles with surface-grafted poly(2-hydroxyethyl methacrylate) brushes (M.sub.n,NMR=4800) (propranolol-CP-1) are synthesized and purified under the identical conditions except that the template is omitted.

Example 4

(27) Propranolol (0.83 mmol) is added into a one-neck round-bottom flask (100 mL) containing a mixture of acetonitrile and methanol (2:1 v/v, 60 mL), and a clear solution is obtained after magnetic stirring, and then methacrylic acid (0.83 mmol) is added into the above solution. After their thorough mixing for 0.5 h, ethylene glycol dimethacrylate (2.50 mmol), small chain transfer agent cumyl dithiobenzoate (CDB) (0.055 mmol), poly(2-hydroxyethyl methacrylate) macromolecular chain transfer agent (M.sub.n,NMR=4800) (0.034 mmol), and azobisisobutyronitrile (AIBN) (0.028 mmol) are added successively. After being purged with argon for 30 min, the reaction mixture is sealed and immersed into a thermostatted oil bath at 60 C. The polymerization is allowed to take place for 16 h. The resulting polymer particles are collected by high speed centrifugation.

(28) The above product is purified by being washed with methanol/acetic acid (9:1 v/v) and methanol until no template is detectable in the centrifugated supernatant. After being dried at 40 C. under vacuum for 48 h, MIP nanoparticles with surface-grafted poly(2-hydroxyethyl methacrylate) brushes (i.e., propranolol-MIP-2) are obtained. Said MIP nanoparticles have a diameter of 182 nm and a crosslinking degree of 75%.

(29) The non-imprinted nanoparticles with surface-grafted poly(2-hydroxyethyl methacrylate) brushes (propranolol-CP-2) are synthesized and purified under the identical conditions except that the template is omitted.

Example 5

(30) 2,4-D (0.83 mmol) is added into a one-neck round-bottom flask (100 mL) containing a mixture of methanol and water (4:1 v/v, 60 mL), and a clear solution is obtained after magnetic stirring, and then 4-vinylpyridine (0.83 mmol) is added into the above solution. After their thorough mixing for 0.5 h, ethylene glycol dimethacrylate (3.32 mmol), small chain transfer agent cumyl dithiobenzoate (CDB) (0.055 mmol), poly(2-hydroxyethyl methacrylate) macromolecular chain transfer agent (M.sub.n,NMR=4800) (0.034 mmol), and azobisisobutyronitrile (AIBN) (0.028 mmol) are added successively. After being purged with argon for 30 min, the reaction mixture is sealed and immersed into a thermostatted oil bath at 60 C. The polymerization is allowed to take place for 10 h. The resulting polymer particles are collected by high speed centrifugation.

(31) The above product is purified by being washed with methanol/acetic acid (9:1 v/v) and methanol until no template is detectable in the centrifugated supernatant. After being dried at 40 C. under vacuum for 48 h, MIP nanoparticles with surface-grafted poly(2-hydroxyethyl methacrylate) brushes (M.sub.n,NMR=4800) are obtained. Said MIP nanoparticles have a diameter of 180 nm and a crosslinking degree of 80%.

Example 6

(32) The MIP nanoparticles with surface-grafted hydrophilic polymer brushes according to the present invention are prepared in the same way as example 5 except that the molar ratio of the template to functional monomer is 1:10. Said MIP nanoparticles have a diameter of 215 nm and a crosslinking degree of 80%.

Example 7

(33) The MIP nanoparticles with surface-grafted hydrophilic polymer brushes according to the present invention are prepared in the same way as example 5 except that the molar ratio of the template to functional monomer is 1:5. Said MIP nanoparticles have a diameter of 195 nm and a crosslinking degree of 80%.

Example 8

(34) The MIP nanoparticles with surface-grafted hydrophilic polymer brushes according to the present invention are prepared in the same way as example 5 except that the molar ratio of the functional monomer to crosslinker is 1:80. Said MIP nanoparticles have a diameter of 850 nm and a crosslinking degree of 98.8%.

Example 9

(35) The MIP nanoparticles with surface-grafted hydrophilic polymer brushes according to the present invention are prepared in the same way as example 5 except that the molar ratio of the functional monomer to crosslinker is 1:40. Said MIP nanoparticles have a diameter of 480 nm and a crosslinking degree of 97.6%.

Example 10

(36) The MIP nanoparticles with surface-grafted hydrophilic polymer brushes according to the present invention are prepared in the same way as example 5 except that the molar ratio of the functional monomer to crosslinker is 1:20. Said MIP nanoparticles have a diameter of 384 nm and a crosslinking degree of 95.2%.

Example 11

(37) The MIP nanoparticles with surface-grafted hydrophilic polymer brushes according to the present invention are prepared in the same way as example 5 except is that the molar ratio of the functional monomer to crosslinker is 1:60. Said MIP nanoparticles have a diameter of 620 nm and a crosslinking degree of 98.4%.

Example 12

(38) The MIP nanoparticles with surface-grafted hydrophilic polymer brushes according to the present invention are prepared in the same way as example 5 except that the molar ratio of the functional monomer to crosslinker is 0.5:100. Said MIP nanoparticles have a diameter of 240 nm and a crosslinking degree of 80%.

Example 13

(39) The MIP nanoparticles with surface-grafted hydrophilic polymer brushes according to the present invention are prepared in the same way as example 5 except that the molar ratio of the initiator to the functional monomer and crosslinker is 5:100. Said MIP nanoparticles have a diameter of 130 nm and a crosslinking degree of 80%.

Example 14

(40) The MIP nanoparticles with surface-grafted hydrophilic polymer brushes according to the present invention are prepared in the same way as example 5 except that the molar ratio of the initiator to the functional monomer and crosslinker is 10:100. Said MIP nanoparticles have a diameter of 100 nm and a crosslinking degree of 80%.

Example 15

(41) The MIP nanoparticles with surface-grafted hydrophilic polymer brushes according to the present invention are prepared in the same way as example 5 except that only hydrophilic macromolecular chain transfer agent is used in the polymerization system. Said MIP nanoparticles have a diameter of 106 nm and a crosslinking degree of 80%.

Example 16

(42) The MIP nanoparticles with surface-grafted hydrophilic polymer brushes according to the present invention are prepared in the same way as example 5 except that the molar ratio of the initiator to chain transfer agents is 1:1. Said MIP nanoparticles have a diameter of 220 nm and a crosslinking degree of 80%.

Example 17

(43) The MIP nanoparticles with surface-grafted hydrophilic polymer brushes according to the present invention are prepared in the same way as example 5 except that the molar ratio of the initiator to chain transfer agents is 1:20. Said MIP nanoparticles have a diameter of 137 nm and a crosslinking degree of 80%.

Example 18

(44) The MIP nanoparticles with surface-grafted hydrophilic polymer brushes according to the present invention are prepared in the same way as example 5 except that the molar ratio of the initiator to chain transfer agents is 1:10. Said MIP nanoparticles have a diameter of 145 nm and a crosslinking degree of 80%.

Example 19

(45) The MIP nanoparticles with surface-grafted hydrophilic polymer brushes according to the present invention are prepared in the same way as example 5 except that the molar ratio of the initiator to chain transfer agents is 1:5. Said MIP nanoparticles have a diameter of 150 nm and a crosslinking degree of 80%.

Example 20

(46) The MIP nanoparticles with surface-grafted hydrophilic polymer brushes according to the present invention are prepared in the same way as example 5 except that the molar percentage of the small chain transfer agent in the total chain transfer agents is 95%. Said MIP nanoparticles have a diameter of 450 nm and a crosslinking degree of 80%.

Example 21

(47) The MIP nanoparticles with surface-grafted hydrophilic polymer brushes according to the present invention are prepared in the same way as example 5 except that the molar percentage of the small chain transfer agent in the total chain transfer agents is 50%. Said MIP nanoparticles have a diameter of 104 nm and a crosslinking degree of 80%.

Example 22

(48) The MIP nanoparticles with surface-grafted hydrophilic polymer brushes according to the present invention are prepared in the same way as example 5 except that the template is paraquat. Said MIP nanoparticles have a diameter of 169 nm and a crosslinking degree of 80%.

Example 23

(49) The MIP nanoparticles with surface-grafted hydrophilic polymer brushes according to the present invention are prepared in the same way as example 5 except that the functional monomer is itaconic acid. Said MIP nanoparticles have a diameter of 180 nm and a crosslinking degree of 80%.

Example 24

(50) The MIP nanoparticles with surface-grafted hydrophilic polymer brushes according to the present invention are prepared in the same way as example 5 except that the functional monomer is N-vinylpyrrolidone. Said MIP nanoparticles have a diameter of 120 nm and a crosslinking degree of 80%.

Example 25

(51) The MIP nanoparticles with surface-grafted hydrophilic polymer brushes according to the present invention are prepared in the same way as example 5 except that the crosslinker is trimethylolpropane trimethacrylate. Said MIP nanoparticles have a diameter of 208 nm and a crosslinking degree of 80%.

Example 26

(52) The MIP nanoparticles with surface-grafted hydrophilic polymer brushes according to the present invention are prepared in the same way as example 5 except that the crosslinker is divinylbenzene. Said MIP nanoparticles have a diameter of 174 nm and a crosslinking degree of 80%.

Example 27

(53) The MIP nanoparticles with surface-grafted hydrophilic polymer brushes according to the present invention are prepared in the same way as example 5 except that the hydrophilic macromolecular chain transfer agent is polyethylene glycol and the solvent is a mixture of N,N-dimethylformamide and acetonitrile (4:1 v/v). Said MIP nanoparticles have a diameter of 370 nm and a crosslinking degree of 80%.

Example 28

(54) The MIP nanoparticles with surface-grafted hydrophilic polymer brushes according to the present invention are prepared in the same way as example 5 except that the hydrophilic macromolecular chain transfer agent is poly(N-isopropylacrylamide) and the solvent is a mixture of N,N-dimethylformamide and acetonitrile (4:1 v/v). Said MIP nanoparticles have a diameter of 480 nm and a crosslinking degree of 80%.

Example 29

(55) The MIP nanoparticles with surface-grafted hydrophilic polymer brushes according to the present invention are prepared in the same way as example 5 except that the small chain transfer agent is O-ethyl S-(2-ethyloxycarbonyl-2-propyl) dithiocarbonate. Said MIP nanoparticles have a diameter of 500 nm and a crosslinking degree of 80%.

Example 30

(56) The MIP nanoparticles with surface-grafted hydrophilic polymer brushes according to the present invention are prepared in the same way as example 5 except that the solvent is butanone. Said MIP nanoparticles have a diameter of 450 nm and a crosslinking degree of 80%.

(57) As illustrated in FIGS. 2, 4, and 5, MIP nanoparticles with surface-grafted hydrophilic polymer brushes are obtained by using the method described in the present invention. Without being bound to any theory, the inventors believe that the active initiating center will transfer to the macromolecular chain transfer agent after the living RAFT polymerization is initiated because of the exixtence of the macromolecular chain transfer agent described in the present invention in the beginning of the polymerization, and then in the specific reaction condition, a number of active initiating centers from chain transfer agent will initiate the living crosslinking polymerization of the monovinyl functional monomer and crosslinker, leading to nanoparticles and forming hydrophilic polymer brushes outside the cores of nanoparticles by the grafting of the end of the macromolecular chain transfer agent. In addition, the obtained nanoparticles have uniform sizes, i.e., narrow size distribution, because of the living nature of RAFT polymerization, as shown in FIGS. 2, 4, and 5. After removal of the template molecules by washing, imprinted cavities of the template molecules will be created in the cores of the nanoparticles, the nanoparticles in the present invention are thus capable of specific bindings toward the template molecules in the samples, i.e., high selective bindings, as demonstrated by the following examples.

Measurement Examples

(58) FIG. 6, different amounts of MIP and non-imprinted polymer nanoparticles with surface-grafted poly(2-hydroxyethyl methacrylate) brushes (M.sub.n,NMR=4800) are added into a 2,4-D solution in pure milk or pure bovine serum (the concentration of 2,4-D: 0.02 mM), and the mixed solutions are incubated at ambient temperature for 16 h. After the solid particles are removed by centrifugation, 400 L of the supernatants are collected, to which 600 L of acetonitrile is added. After 5 mM of ultrasonic treatment of the above mixtures, the samples are centrifuged and all the supernatants are collected, to which some acetonitrile/water (3/2 v/v) is added to reach a final volume of 1 mL. The amounts of 2,4-D in the solutions are determined by HPLC, from which the template 2,4-D bound to the MIP and non-imprinted polymer nanoparticles with surface-grafted hydrophilic poly(2-hydroxyethyl methacrylate) brushes (M.sub.n,NMR=4800) can be derived.

(59) HPLC measurement condition: A mixture of methanol and 0.5% aqueous solution of acetic acid (3/1 v/v for milk sample and 4/1 v/v for bovine serum sample) is utilized as the mobile phase at a flow rate of 1 mL/min. The wavelength used for the determination is 284 nm.

(60) FIG. 7, a certain amount of MIP and non-imprinted polymer nanoparticles with surface-grafted poly(2-hydroxyethyl methacrylate) brushes (M.sub.n,NMR=4800) is added into a mixed solution of 2,4-D and POAc in pure milk and pure bovine serum (both the concentrations of 2,4-D and POAc are 0.02 mM), and the mixed solutions are incubated at ambient temperature for 16 h. After the solid particles are removed by centrifugation, 400 L of the supernatants are collected, to which 600 L of acetonitrile is added to precipitate all the proteins in the samples. After 5 min of ultrasonic treatment of the above mixtures, the samples are centrifuged and all the supernatants are collected, to which some acetonitrile/water (3/2 v/v) is added to reach a final volume of 1 mL. The amounts of 2,4-D and POAc in the solutions are determined by HPLC, from which the template 2,4-D and its analogue POAc bound to the MIP and non-imprinted polymer nanoparticles with surface-grafted hydrophilic poly(2-hydroxyethyl methacrylate) brushes (M.sub.n,NMR=4800) can be derived.

(61) HPLC measurement condition: A mixture of methanol and 0.5% aqueous solution of acetic acid (3/2 v/v) is utilized as the mobile phase at a flow rate of 1 mL/min, and the wavelength used for the determination is 272 nm.

(62) FIG. 8, different amounts of MIP (propranolol-MIP-2) and non-imprinted polymer (CP) nanoparticles with surface-grafted poly(2-hydroxyethyl methacrylate) brushes (M.sub.n,NMR=4800) are added into a propranolol solution in pure milk or pure bovine serum (the concentration of propranolol: 0.05 mM), and the mixed solutions are incubated at ambient temperature for 16 h. After the solid particles are removed by centrifugation, 400 L of the supernatants are collected, to which 600 L of acetonitrile is added. After 5 min of ultrasonic treatment of the above mixtures, the samples are centrifuged and all the supernatants are collected, to which some acetonitrile/water (3/2 v/v) is added to reach a final volume of 1 mL. The amounts of propranolol in the solutions are determined by HPLC, from which the template propranolol bound to the MIP (propranolol-MIP-2) and non-imprinted polymer (CP) nanoparticles with surface-grafted hydrophilic poly(2-hydroxyethyl methacrylate) brushes (M.sub.n,NMR=4800) can be derived.

(63) HPLC measurement condition: A mixture of acetonitrile and 0.4% aqueous solution of triethylamine (7/3 v/v) is utilized as the mobile phase at a flow rate of 1 mL/min, and the wavelength used for the determination is 293 nm.

(64) FIG. 9, a certain amount of MIP (propranolol-MIP-2) and their non-imprinted polymer (CP) nanoparticles with surface-grafted poly(2-hydroxyethyl methacrylate) brushes (M.sub.n,NMR=4800) is added into a mixed solution of propranolol and atenolol in pure milk or pure bovine serum (both the concentrations of propranolol and atenolol are 0.05 mM), and the mixed solutions are incubated at ambient temperature for 16 h. After the solid particles are removed by centrifugation, 400 L of the supernatants are collected, to which 600 L of acetonitrile is added. After 5 min of ultrasonic treatment of the above mixtures, the samples are centrifuged and all the supernatants are collected, to which some acetonitrile/water (3/2 v/v) is added to reach a final volume of 1 mL. The amounts of propranolol and atenolol in the solutions are determined by HPLC, from which the template propranolol and its analogue atenolol bound to the MIP (propranolol-MIP-2) and non-imprinted polymer (CP) nanoparticles with surface-grafted hydrophilic poly(2-hydroxyethyl methacrylate) brushes (M.sub.n,NMR=4800) can be derived.

(65) HPLC measurement condition: A mixture of acetonitrile and 0.4% aqueous solution of triethylamine (7/3 v/v) is utilized as the mobile phase at a flow rate of 1 mL/min, and the wavelength used for the determination is 275 nm.

(66) As demonstrated by the above measurement examples, the MIP nanoparticles obtained in the present invention can show specific template binding capability in aqueous samples as well as biological samples, and in particular pure biological samples, as good as what they show in the organic solvent (after considering the inevitable experimental errors). Without being bound to any theory, the applicants believe that the above results are attributed to generation of MIP nanoparticles with specific structures and properties by the method described in the present invention, and such MIP nanoparticles can efficiently recognize the template molecules in aqueous samples as well as biological samples, and in particular pure biological samples, and they are not interfered by the existence of components rich in biological samples such as proteins. This is unknown and unpredictable prior to the present invention. Therefore, the MIP nanoparticles obtained in the present invention can be efficiently used in aqueous samples and biological samples, and in particular pure biological samples, for specifically recognizing the targeted template molecules. For example, the MIP nanoparticles obtained according to the present invention can conveniently recognize the antibiotic and pesticide residues in milk, which is of great potential in food safety area.

(67) On the basis of their above-mentioned characteristics, the MIP nanoparticles obtained in the present invention can be used in analyses of biological samples, medical clinical immunoassays, foodstuff and enviromnental monitoring, and/or fabrication of biomimetic sensors. For example, the MIP nanoparticles obtained in the present invention can be used for the preparation of sensors applicable for biological samples.

(68) Although the present invention has been illustrated by using specific embodiments, many other alterations, modifications and other applications will become obvious to those skilled in the art. Therefore, preferably the present invention is not limited by the specific disclosed content of the above description, but is defined solely by the claims appended hereto.