SYNTHESIS METHOD OF POLYMERIC NANOPARTICLE MOLECULARLY IMPRINTED BY MICROFLUIDIC SYSTEM

Technical Field

The present invention relates to a method for synthesizing molecularly imprinted polymeric nanoparticles which are used for detecting target molecule, bacteria, virus, protein, lipid, aptamer, carbohydrate, extracellular vesicle (exosome, microvesicle, apoptotic particles), nucleic acid or cells, by means of a microfluidic device.

Background of the Invention

Molecularly imprinted polymers are artificial receptors which can be used in many areas such as biosensors and purification and have recognition sites specifically prepared for target molecules. These structures which can be prepared in accordance with the molecules aimed to be detected, are more affordable, have high durability and reusability can be used as an alternative to antibodies. Whereas molecularly imprinted nanoparticles, which can be synthesized in nano sizes, attract attention due to their features such as high surface area-to-volume ratio, fast binding kinetics, and high compatibility with surface modification and various biomedical applications. Methods such as precipitation polymerization, emulsion polymerization, solid phase imprinting and core-shell polymerization are used for the bulk synthesis of molecularly imprinted polymers. However, problems such as requiring more equipment and long polymerization times, and also implementation difficulty of optimization of synthesis conditions and the fact that the quality of the nanomaterials acquired in each synthesis varies from batch to batch are included in these systems. In addition to providing solutions to these problems, continuous flow systems based on microfluidics with integrated fluid control elements offer advantages such as reusability, on-site traceability of reactions and rapid scanning of parameters.

Therefore, there is need for a method which enables to obtain a microfluidic device to be used for synthesizing molecularly imprinted polymeric nanoparticles and to synthesize molecularly imprinted polymeric nanoparticles by using this device.

The International patent document no. W02017001451, an application included in the state of the art, discloses a molecularly imprinted polymer and a preparation method thereof.

Summary of the Invention

An objective of the present invention is to realize a method which enables to synthesize molecularly imprinted polymeric nanoparticles by using a microfluidic device.

Another objective of the present invention is to realize a method which enables to obtain a microfluidic device so as to be used in synthesis of molecularly imprinted polymeric nanoparticles.

Another objective of the present invention is to realize a method which enables to require a shorter synthesis time in comparison to conventional synthesis methods of molecularly imprinted polymeric nanoparticles.

Another objective of the present invention is to realize a method which enables to make the nanoparticle synthesis more economical due to the fact that low volume of chemical use is required in a microfluidic system. Another objective of the present invention is to realize a nanoparticle synthesis method which enables to control synthesis conditions easily and to obtain high efficiency due to the fact that a microfluidic system can operate at high speeds.

Detailed Description of the Invention

The “Synthesis Method of Polymeric Nanoparticle Molecularly Imprinted by Microfluidic System” realized to fulfil the objectives of the present invention is shown in the figures attached, in which:

Figure l is a flow chart of the inventive method.

Figure 2 is a view related to the 3D CAD design of the microfluidic device used in the inventive method.

Figure 3 is (A) 30 minutes (B) 60 minutes (C) 90 minutes (D) 120 minutes (E) 150 minutes (F) 180 minutes graphs related to zeta size analysis of BSA (sample molecule) imprinted nanoparticles.

Figure 4 is (A) 30 minutes (B) 60 minutes (C) 90 minutes (D) 120 minutes (E) 150 minutes (F) 180 minutes graphs related to nanosight tracking analysis of BSA (sample molecule) imprinted nanoparticles.

Figure 5 is (A) 2D, (B) 3D AFM images of BSA (sample molecule) imprinted nanoparticles.

Figure 6 is XPS analysis of desorbed and non-desorbed nanoparticles. (A- B) Cis, (C-D) Nls, (E-F) Ols, spectra. The corresponding chemical groups and their binding energies are indicated in the graphs (A, C, E: non-desorbed; B, D, F: desorbed nanoparticles).

Figure 7 illustrates the wavelength shifts (A) 10 M, (B) 20 pM, (C) 30 pM, (D) 40 pM of different BSA concentrations against time by using BSA (sample molecule) imprinted nanoparticles.

The components illustrated in the figure are individually numbered, where the numbers refer to the following: 100. Method

The inventive method (100) for synthesizing molecularly imprinted polymeric nanoparticles which are used for detecting target molecule, bacteria, virus, protein, lipid, aptamer, carbohydrate, extracellular vesicle (exosome, microvesicle, apoptotic particles), nucleic acid or cells, by means of a microfluidic device comprises the steps of

- preparing the microfluidic device (101);

- preparing a pre-complex according to the selected target protein (102);

- preparing the first phase mixture (103);

- preparing the second phase mixture (104);

- carrying out homogenization by adding a co-monomer and a cross-linker to the second phase mixture (105);

- mixing the pre-complex and the first phase into the homogenized mixture and then taking the mixture into the first syringe (106);

- preparing a polymerization initiator mixture and then taking the mixture into the second syringe (107);

- connecting the first and second syringe to the microfluidic device (108); and

- obtaining molecularly imprinted polymeric nanoparticles of the target protein upon the bulk polymerization is realized by the mixtures received from the syringe in the microfluidic device (109).

In the step of performing preparing the microfluidic device (101) of the inventive method (100); negative molds for a microfluidic device with a spiral form are modelled in a 3D mechanical design program (Figure 2). In one embodiment of the invention, the design program used is Autodesk Inventor Professional. The negative molds modelled are printed by means of a desktop 3D printer by using stereolithography technique (STL). The negative molds are printed by using transparent (clear resin), elastic, flexible, hard (rigid), resistant to high temperatures, ceramic and surgical resin which can resist the temperatures at which polydimethylsiloxane (PDMS) is cured and the printing sensitivity is set to 25-100 pm. Upon the printing is completed, the raw (unprocessed) resin residue is immersed into isopropyl alcohol (99.9%) for 1-30 minutes according to the type of resin used as solvent treatment at first. Then, it is then cured by being exposed to UV-light treatment for 5-60 minutes. Following the curing process, the negative molds are covered with a tape made of copper, polyimide, polyester, kapton, vinyl, silver, polyethylene or teflon that is resistant to 0-500°C. PDMS containing a hardener, which is silicone elastomer, within the ratios of 5: 1; 6:1; 7: 1; 8: 1; 9: 1, 10: 1 is prepared and then poured onto 3D printed negative molds for processing by means of soft lithography technique. Thereafter, the negative molds are kept in an oven at 20-150°C for periods ranging from 10 minutes to 48 hours in order to cure the PDMS. At the end of 80°C, the cured PDMS is carefully removed from the molds. A channel inlet is created by drilling a hole of 1 mm on the PDMS molds and it is exposed to oxygen plasma in order to seal it with another PDMS mold with similar geometry. For this process, the plasma chamber is set to 15-30°C and the substrate temperature is set to 15-30°C. Oxygen flow is set as 10-50 cm ³ /min and plasma is administered for 10 seconds-5 minutes by using 10-300 W power. At the end of the process, two PDMS molds are quickly sealed in order to obtain a microfluidic device with spiral channels ready to be used in nanoparticle synthesis. In one embodiment of the invention, the total channel length, the channel width and the channel height of the microfluidic device are determined as 0.5-10 m, 0.1-1 mm and 0.1-1 mm, respectively.

In the step of preparing a pre-complex according to the selected target protein (102) of the inventive method (100); bovine serum albumin (BSA) protein as target protein and methacrylic acid as functional monomer are combined in a ratio of 4: 1 and mixed for 1-4 hours at room temperature at 10-50 rpm in order to form a pre-complex. In the step of preparing the first phase mixture (103) of the inventive method (100); polyvinyl alcohol and sodium dodecyl sulfate are combined in a ratio of 1 : 1.

In the step of preparing the second phase mixture (104) of the inventive method (100); polyvinyl alcohol, sodium dodecyl sulfate and sodium bicarbonate are combined in a ratio of 8: 1.2: 1.

In the step of carrying out homogenization by adding a co-monomer and a crosslinker to the second phase mixture (105) of the inventive method (100); after adding 2-hydroxyethylmethacrylate as co-monomer and ethylene glycol dimethacrylate as cross-linker into the second phase mixture, it is homogenized at 20000-50000 rpm for 15-30 minutes.

In the step of mixing the pre-complex and the first phase into the homogenized mixture and then taking the mixture into the first syringe (106) of the inventive method (100); a polymer mixture is obtained by adding the pre-complex and then the first phase into the homogenized mixture, and the polymer mixture is transferred into a syringe of 0.5-50 ml.

In the step of preparing a polymerization initiator mixture and then taking the mixture into the second syringe (107) of the inventive method (100); ammonium persulfate and sodium bisulfite are mixed in a ratio of 2: 1 and transferred into a syringe of 0.5-50 ml.

In the step of connecting the first and second syringe to the microfluidic device (108) of the inventive method (100); the first syringe comprising the polymer mixture and the second syringe comprising the polymerization initiator are connected to the syringe pump and the mixtures included in the syringe are delivered to the microfluidic device by means of capillary tubes. The speed is set to 10-500 pL/min for the polymer mixture and 0.2-10 pL/min for the polymerization initiator.

In the step of obtaining molecularly imprinted polymeric nanoparticles of the target protein upon the bulk polymerization is realized by the mixtures received from the syringe in the microfluidic device (109) of the inventive method (100); the; the microfluidic device is placed inside the oven at 30-50°C in order to create bulk polymerization environmental conditions and subjected to polymerization for 30-200 minutes. When the bulk polymerization is completed, BSA (sample molecule) protein-imprinted nanoparticles of 50-300 nm in size are obtained from the microfluidic device. Synthesis of BSA (sample molecule) protein-imprinted nanoparticles is carried out via miniemulsion polymerization method by using two different liquid phases.

The nanoparticle sampled obtained by means of the inventive method (100) nanoparticle samples were collected every half hour and separated for characterization. In addition, each experimental set was repeated for 3 times. The collected samples were centrifuged at 6000 rpm for 5 minutes before characterization. Following the centrifugation, the supernatants were used for characterization. The sizes of the synthesized nanoparticles were measured by using Nano Zetasizer. For size analysis, upon the prepared nanoparticle solution was placed into the sample housing, light scattering was measured with an incidence angle of 90°. Measurements were repeated three times and averaged. Zeta size analysis results of nanoparticles are given in the Figure 3. The sizes of the particles collected every 30 minutes are in the range of 86-103 nm. In order to analyze the concentration, diameter and size distribution of nanoparticles, the Nanosight Tracking Analysis instrument -which uses a laser-based optical technique that tracks the Brownian motion of individual particles included in solution- was used. After the sample is introduced into the device by means of a syringe, the size distribution and number of particles are measured upon determining the diameter of the particles by using the Stokes-Einstein equation. Before measurement analysis, the system is cleaned with distilled water and then values such as viscosity, temperature, and dilution rates are adjusted before the measurements. For each sample, three video recordings -each with a duration of 60 s- were performed. According to the results obtained, the concentration of the nanoparticle samples collected at 30-minute intervals was found to be 6-8 x 101 ² particles/mL, as shown in the Figure 4.

Atomic Force Microscope was used in order to perform topographic analysis of the surface. The surface analysis results of the BSA (sample molecule) imprinted nanoparticles attached to gold-coated plastic nanoperiodic surfaces are shown in the Figures 5 A and B. According to the results obtained, the surface depth was found to be 60.1 nm, whereas the surface roughness was found to be 14.16 nm.

XPS (K-Alpha XPS, Thermo Fisher Scientific, America) analysis was performed as well for the characterization of BSA (sample molecule) imprinted and nonimprinted nanoparticles. According to the results obtained, the general elemental analysis of the nanoparticles was obtained. The results are presented in the Figure 6. The element composition of both nanoparticles comprises three types of carbon atoms corresponding to C-C/C-H at 284 eV, C-C/C-H at 288 eV and C=O at 288 eV and two types of oxygen atoms corresponding to C-0 at 532 eV and C= at 532 eV. It O. (the Figures 6 E-F). When the BSA protein was removed from imprinted nanoparticles by being desorbed, the intensity of the peaks corresponding to the C=O bond reduced due to the reduction of carboxylic acid groups originating from the BSA.

In order to search the binding capacity of BSA (sample molecule) protein to BSA- imprinted nanoparticles, different concentrations of BSA protein (10-40 pM) were interacted with nanoparticles. According to the results obtained, it was observed that the amount of wavelength shifts increased together with the increasing protein concentration. (Figure 7) By means of the microfluidic device designed to be used in the synthesis of molecularly imprinted nanoparticles in the inventive method (100), the particle synthesis time is shortened in comparison to conventional methods and the particle synthesis becomes more economical with the use of lower volumes of chemicals. Besides, the fact that it is possible to control the synthesis conditions easily makes this system advantageous. Since these devices can be operated at higher speeds, efficiency increases and industrial production becomes possible. With the said method (100), protein imprinted nanoparticle production can be achieved in a short time such as 30 minutes.

Within these basic concepts; it is possible to develop various embodiments of the inventive “Synthesis Method (100) of Polymeric Nanoparticle Molecularly Imprinted by Microfluidic System”; the invention cannot be limited to examples disclosed herein and it is essentially according to claims.