WO2015040633A1

WO2015040633A1 - Method for extraction of biomolecules by magnetic particles

Info

Publication number: WO2015040633A1
Application number: PCT/IN2014/000589
Authority: WO
Inventors: Rucha Pradipkumar DESAI; Aniruddha BHATI; Ramchand Nanappan Chaniyilparampu; Hilor Shubhash PATHAK; Ramesh Venkataramaiah UPADHYAY
Original assignee: The Registrar, Charotar University of Science & Technology (CHARUSAT)
Priority date: 2013-09-17
Filing date: 2014-09-12
Publication date: 2015-03-26

Abstract

The present invention provides a method for extraction of biomolecules preferably protein by magnetic particles. Magnetic particle is preferably uncoated /bared magnetic particle. The present invention also provides a process for the extraction of protein by steps of addition of magnetic particle into biological system, application of external magnetic field, collection of protein-magnetic particle pellet, resolubilization and finalization of collecting solublized protein and for drug extraction the step includes resuspendasion of the collected supernatant in mobile phase. The magnetic nanoparticle is synthetic analogues of any suitable magnetic material or combination of materials, such as magnetite, ulvospinel, hematite, ilmenite, maghemite, jacobsite, trevorite, magnesioferrite, pyrrhotite, greigite, troilite, goethite, lepidocrocite, feroxyhyte, iron, nickel, cobalt, awaruite, wairauite, or any combination thereof. It is also being made up of transition metal such as iron, manganese, nickel, cobalt, zinc, etc. The magnetic nanoparticles are various sizes and shapes.

Description

"METHOD FOR EXTRACTION OF BIOMOLECULES BY MAGNETIC PARTICLES"

Field of the invention:

The present invention provides method for extraction of biomolecules preferably proteins and drugs from crude mixture using magnetic particle without altering protein's structure.

Background of the invention:

Several methods for protein extraction are well known. Salting out, isoelectric point precipitation, precipitation by organic solvents, non-ionic hydrophilic polymers, etc. are known method for the extraction of protein. Method available in the art gives maximum yield of 60- 70%. Some of those techniques denature the protein of interest. Thus, protein extracted by these methods can not be used for further experimentation. Furthermore, these methods available in the art consume 6-8 hours for the extraction of protein of interest. Also, these processes include use of sophisticated instruments and costly chemicals. Thus, methods available in the art need expertise to handle and time consuming. So, there is need to develop the method for the extraction of biomolecules which could be proceed easier than existing methods. (Philip L.R. Bonner, Protein purification, Pub. Taylor & Francis Group, see page 19, 50, 71 , 75, 77, 2007 and references therein; Castellanos- Serra, L. R., Fernandez-Patron, C, Hardy, E., Santana, H., and Huerta, V. ( 1997) ,High yield elution of proteins from sodium dodecyl sulfate-polyacrylamide gels at the low-picomole level : application to N-term inal sequencing of a scarce protein and to in-solution biological activity analysis of on-gel renatured proteins.; J. Protein Chem. 16, 4 1 5-41 9, The Protein Protocols Handbook, ₂nd Edition, John M. Walker, 2002 Humana Press Inc. New Jersey, and references therein; Protein Purification Handbook, Amersham Pharmacia Biotech AB 1999, Sweden, and references therein) Uses of magnetic nanoparticles, i.e. paramagnetic and/or super paramagnetic for separation of biomolecules have been reported widely in literature (C. Xu et al. "Nitri lotriacetic acid-Modified magnetic nanoparticles as a general agent to bind Histidine-tagged proteins.' J.Am. Chem.Soc. 200, 26, 3392; Nam, j.-M. et al, "Bioactive Protein Nanoarrays on Niclkel Oxide Surfaces Formed by Dip-pen nanolithography." Angew. Chem. Int. Ed.2004,43, 1246 Zhu, H et al, "Global analysis of protein activities using proteome chips" Science 2001 , 293, 2101 ; Direct binding of protein to magnetic particles, RV Mehta et al, biotechnology techniques, 1 1(7), 1997, 493-496). The biomolecules attached with the magnetic particles, can easily isolated by means of external magnetic field. However, based on the availability of binding site present on the functional magnetic particles, appropriate protein/enzyme can be attached.

WO 2010/062586 relates to liquid purification using magnetic nanoparticles. These magnetic particles have surface functionalized by a moiety selected from moieties that are reactive to and combine with a predetermined target present in a liquid. The moiety is selected from the group consisting of dextran, polyethylene glycol, other modified polyethylene glycols, gold, azide, carboxyl groups, activated carbon, zeolites, am ines, and charged polymers.

US 201 10139719 provide magnetic particles immobilize the intended substance easily and efficiently. It discloses magnetic particle, comprising an aggregate which comprises a magnetic substance and a compound having an alkyl group, and a gel layer which covers the aggregate, in which the gel layer has a hydrophilic group.

WO 2008/010687 relates to a method for selective binding, separation or purification of proteins using magnetic nanoparticles. Furthermore, the present invention relates to a method for selective binding, separation, or purification of a specific protein using magnetic nanoparticle. US 4554088 relates to micron-sized magnetic particle coated with si lane polymer and coupling with diazotization, carbodimide and glutaraldehyde, etc.

WO 201 1/103539 describes use of fluoride/phosphate passivated metal oxide for isolation and storage of biomolecules from biological samples.

In prior art, coated or bounded or functional ized magnetic nanoparticle with any surface activating ligands were used for the extraction of biomolecules. In case of uncoated magnetic particle, difficulties of irreversible binding of biomolecule with magnetic particle and loss of activity of biomolecule after extraction were raised. In the present invention, inventors have developed a method by which biomolecules preferably total protein can be extracted using uncoated magnetic particle. Proteins extracted by the method described herein will be functionally active. Furthermore, a method for the extraction of total protein described herein involves lesser time for the extraction of protein as compare to existing conventional method for the extraction of protein. Present invention offers use of magnetic particle in various fields. Here, a method for the extraction of biomolecule preferably protein from the crude mixture is described. Extraction of protein from various biological systems can be done using method described herein. Furthermore, a method described herein avoids use of sophisticated instruments like ultracentrifuge. Thus, present invention provides cost effective method for the extraction of biomolecules preferably protein using uncoated magnetic particle.

Object of the invention

The main object of the present invention is to provide a method for extraction of protein from various biological systems such as blood, plant system, milk and/or bacteria by magnetic particles.

In further object, the present invention provides method for extraction of protein by magnetic particle in which protein will be functionally active after extraction. In yet another object, the present invention provides a process for extraction of total protein from the biological system. Process described herein does not involve use of sophisticated instruments. The process described herein provides functionally active protein after purification.

Still in another object, the present invention provides a method for extraction of biomolecules by magnetic particle preferably protein in which protein binds irreversibly with the magnetic particle. Thus, biomolecu!e is easily detached from the magnetic particle without applying specific process condition.

In further object, present invention provides total protein free of contaminants like nucleic acid and other small molecules e.g. dextran using magnetic particle.

In another object, the present invention provides a method to extract drugs using magnetic particles.

Still another object of the present invention is to demonstrate the method foi^¬ extraction of total protein (-100%) present in biological system.

Summary of the invention:

Detailed description of Figures:

Figure 1 shows flow chart for extraction of protein and/or drug.

Figure 2 shows Fourier Transform Infrared (FTIR) spectra of Bovine Serum Albumin (BSA), pellet of Bovine Serum Albumin-magnetic particles (BSA- P pellet) and magnetic particles (MP).

Figure 3 shows TGA data revealing weight loss (%) of BSA, BSA-M P and MNP. Figure 4 shows extraction efficiency and protein loading capacity of the magnetic particles.

Figure 5 shows Transmission Electron Microscopy (TEM) image of the whole blood protein- MP pellet.

Figure 6 shows TEM image of the magnetic particles after eluting the protein.

Figure 7 shows FTIR spectra of magnetic particles, blood plasma and pellet of plasma protein-magnetic particles.

Figure 8 shows TGA data of pellet of blood serum protein-magnetic particles.

Figure 9 shows SDS-PAGE image protein extracted from whole blood.

Figure 10 shows Agarose gel electrophoresis of whole blood and plasma before and after extracting the protein.

Figure 1 1 shows results of the glucose test performed on (a) plasma (before extraction), (b) protein eluted from plasma using the developed process, and (c) standard sample.

Figure 12 shows SDS-PAGE data of E-coli bacterial system.

Figure 13 shows influence of magnetic fluid on the extraction of calitriol drug.

Figure 14 shows extraction of calcitriol drug in terms of area under the curve for various concentrations of the drug. Figure 15 shows SDS-PAGE image of scaled up system.

Detailed description of the invention:

The present invention describes method for extraction of biomolecules preferably proteins from crude mixture using magnetic particle. Magnetic particle is preferably uncoated/bared magnetic particle. The magnetic nanopa ticle is synthetic analogues of any suitable magnetic material or combination of materials, such as magnetite, ulvospinel, hematite, ilmenite, maghemite, jacobsite, trevorite, magnesioferrite, pyrrhotite, greigite, troilite, goethite, lepidocrocite, feroxyhyte, iron, nickel, cobalt, awaruite, wairauite, or any combination thereof. It is also being made up of transition metal such as iron, manganese, nickel, cobalt, zinc, etc. The magnetic nanoparticles are various sizes and shapes.

A method described herein does not involve any sophisticated instruments. Further, it does not involve any process for detachment of biomolecule of i nterest from the magnetic particle.

In a preferred embodiment, the present invention provides a process for the extraction of protein involves steps shown as flow-chart in the Figure 1 and described below.

In one embodiment the 100 μΐ biological sample, 50 j_ig magnetic particles

(i.e. 5 μΐ magnetic fluid) and 300- 1000 μΐ pre-cooled binding buffer is added. In case of cell-based system one more step is need to lyses the cells using lysis buffer. The system is then cooled down to 0- 10°C for at least 0-20 minutes. The lysis buffer is any surfactant like sodium dodecyl sulfate, Triton X-100, etc., polymer like poly- ethylene glycol, ethylene glycol, etc. or mixture of surfactants. The binding buffer enables efficient binding of proteins on the magnetic nanoparticles. Binding buffer is surfactants/acids like CTAB, Tween 80, SDS, PEG 6000, urea, acids like 0.1N HC1 and/or 0.1 N H₂S0 , bases like sodium hydroxide, potassium hydroxide, salts/polymers like ammonium sulfate, sodium sulfate etc. and non-ionic polymers like dextrans, or solvents like acetone, ethanol, methanol, acetonitrile, propanol, etc.. Then the biological system is placed under the external magnetic field. This will form a pellet of protein-magnetic particles. The supernatant obtained is almost colorless. The supernatant and pellet consists of protein-magnetic particles is to be separated either by inverting the system or by pipetting.

From this step the process differs for the protein and/or drug extraction. For the protein and/or drug extraction pellet composed of protein-magnetic particles is processed further, as described in below process

In the protein-magnetic particles pellet elution buffer is added. The elution buffer facilitate desorption of protein from the protein-MNP pellet. Additionally it maintains the enzymatic action of the extracted protein. The elution buffers have pH from 5.4 to 8 depending on the biological system and its application. Mechanical or thermal energy is provided to desorb the protein from the MNP. Mechanical energy is in term of ultrasound in the frequency range 20-40 kHz. Alternatively, thermal energy in terms of water bath is provided to the protein-MNP pellet at the temperature range 40-70°C. In order to collect the magnetic particles and to separate protein, the system is kept under the magnet field as mentioned earlier, which will form pellet of magnetic particles (without protein). The supernatant thus obtained is collected. Optionally, this process is to be repeated. The supernatant contains total protein without contamination of nucleic acid, i.e. DNA and/or RNA and/or other small molecules.

The supernatant is either directly loaded for the estimation of the drug or dried, resuspended in mobile phase and after passing through anal chem. cartridge and finally loaded for the estimation of drug. In the present invention, LC-MS/MS instrument was used to estimate the drug concentration. The time required to extract the total protein is 30-80 minutes for the moderate size sample (< 300 ml). This time is reducing by increasing the strength of external magnetic field. Analytical methods used in the present invention

TEM: JEOL JEM 2100 200-kV transmission electron microscope (TEM) with a point resolution of 50X to 1.5MX has been used. Sample for TEM measurement have been prepared as follows: Magnetic particles or protein-magnetic particle clusters were suspended in volatile solvent like ethanol and homogeneous suspension was obtained by ultrasonicating the system. Immediately, a drop of this suspension was loaded on amorphous carbon coated copper grid and allowed to dry at the ambient conditions. TGA-DSC: A combine set of Therogravimetric analyzer (TGA) and Differential Scanning Calorimetry (DSC) (make-Mettler-Toledo, model TGA/DSC 1 ) have been used to study temperature dependent decomposition of protein attached with the magnetic particles. The temperature was varied from 300 to 6000 °C. STARe software was used to analyse weight loss in the system.

SDS-PAGE: A gel was prepared using standard Acrylamide - Bisacrylamide solution with β-merkeptoethanol and Tetramethylethylenediamine (TEMED) for gelling and cross linking. Protein was denaturized using SDS and heat, and loaded on gel. Midi- electrophoresis unit was used to run the gel. The gei was stained using silver staining procedure. The results were observed by a Gel documentation assembly (Bio-Rad). Thus, qualitative and quantitative determination of extracted proteins is carried out.

LC/MS-MS: It is the combination of sophisticated high-performance liquid chromatography and mass spectrometry and mainly used to quantitative analysis. The analysis was performed using the Standard Operating procedures (SOP) for the respective drugs. The samples were prepared using the procedure discussed in the example. The drug suspended in mobile phase was loaded in the LC-MS/MS (Waters and/ or AB-SCIEX). It gives peak for respective drug and the internal standard. The area under the curve represents the concentration of extracted drug. Folin-Lowry method: It is most widely used method to estimate the amount of proteins in the biological systems. In this, protein sample is treated with copper ion in alkali solution, incubated, and then Folin Reagent (make-Merck) was added. The system was incubated in dark, where aromatic amino acids in the treated sample reduce the phosphomolybdatephosphotungstic acid present in the Folin Reagent. The end product of this reaction has a blue color, indicating presence of protein. Before estimating the protein present in the sample, standard curve using Bovine Serum Albumin (BSA) was generated, and calibration constant was determined. The amount of proteins in the sample is to be estimated using spectrophotometer by reading the absorbance at 750 nm.

Bradford estimation: The procedure is based on the formation of a complex betwee the dye, Brilliant Blue G, and proteins in solution. The protein dye complex causes a shift in the absorption maximum of the dye from 465 to 595 nm. The sample was added into Bradford reagent (make-Sigma Aldrich). Using spectrophotometer absorbance was recorded at 595 nm, and protein value was estimated. In the beginning of each experiment, standard curve was generated following the procedure described by manufacturer of Bradford reagent.

SGPT and SGOT enzyme assay kit: Kit based Modified IFCC (The International Federation of Clinical Chemistry Working Group) method was used for enzyme activity assay. The experiment was done at room temperature by recording the change in absorbance of the system at 340 nm using spectrophotometer. The system was prepared by mixing kit-reagent and extracted protein sample. These kits are useful to calculate the activity of SGPT and SGOT enzymes.

Glucose estimation kit (GOD-POD): Glucose oxidase (GOD) converts glucose present in the sample into gluconate. The hydrogen peroxide (H₂0₂) produced in the reaction is degraded by peroxidases (POD). Development of pink color indicates presence of glucose. Sample provided with kit was used as standard. This method was used for the detection and estimation of glucose in the extracted protein to check its purity.

In order to optimize the concentrations of magnetic particles/magnetic fluid and also to determine the protein loading capacity of the magnetic particles, Bovine serum albumin fraction- V is used as test system.

The stock solutions of Bovine serum albumin fraction- V in the concentrations range 20-600 mg/ml were prepared. In the 100 μΐ BSA solution, 50 μg iron oxide superparamagnetic magnetic particles (i.e. 5 μΐ magnetic fluid) and 700 μΐ pre-cooled acetone was added. The system was then cooled down to 4°C for 10 minutes. The system was placed under magnetic field of 2500 Oe. This was form a pellet of protein-magnetic particles. In the presence of magnetic field, almost colorless supernatant obtained was separated either by inverting the system or by pipetting.

The confirmation of binding of BSA (protein) and magnetic particles was obtained using Fourier Transform Infrared (FTIR) spectroscopy and Thermogravimetric Analyzer (TGA) techniques.

Figure 2 shows FTIR spectra of BSA, pel let of BSA-magnetic particles (BSA- MP), and magnetic particles (MP). As shown in figure 2, in the spectrum of pure BSA, the peak near 1650 cm^{' 1} is the amide I band. It results from the C=0 stretching vibrations of the peptide bond. Similarly, the peaks near 1540 cm^"1 (N-H bending vibration/C-N stretching vibration) and 1240 cm^{' 1} (C-N stretching vibration/ -H bending vibration) are called the amide II band, and amide III band, respectively. The peak near 3300 cm^{" 1} is thought to be N-H bending vibration and the peak near 1400 cm^{' 1} to result from protein side-chain COO^". The peak near 3300 cm^"1 is thought to be N-H bending vibration and the peak near 1400 cm^"' to result from protein side-chain COO^". All the peaks observed in the BSA-MP pellet matches, within the experiment error (~2 cm^{" 1}) to the peak positions of pure BSA. The change/shift in peak position in the FTiR indicates either modification in the structure via breaking/bonding of chemical moieties. However, in the present case, no shift in peak position reveals that BSA structure does not get modified while formation of BSA-MP complex, and also suggests physic-adsorption of BSA molecules on the surface of MP. The FTIR spectrum of the magnetic nanoparticles reveals peak positions around 590 cm^"1 and 480 cm^"1 , both theses peaks are not observable in the spectrum of BSA-MP complex, which is due to low concentrations of MP compared to BSA.

Thermogravimetric data during the temperature scan gives two types of information: (i) thermally induced structural transition (e.g. phase transition), and (ii) dissociation of bounded molecules with reference to the thermal cycle.

Figure 3 shows the TGA data of. MP, BSA-MP and BSA. In case of MP nominal weight loss of 4% is observed. It is due to the fact that MP used in the present invention are bare/uncoated and hence observed 4% weight loss is due to moisture absorbed by the magnetic particles (MP) from the environment. Around 75% weight losses are observed in case of BSA, this indicates that in the experimental temperature range BSA completely deforms and/or undergoes structural transition. The residual weight is due to the ash produced during the temperature sweep. Similar weight loss (%) is also obtained for the BSA-MP. This confirms 100% binding of BSA molecules on the magnetic particles (MP). The first derivative of weight loss (or rate of change of weight dM/dT ) with temperature reveals that peak position remains unaltered. This confirms the structural integrity of BSA protein after binding with the magnetic particles.

On confirming the adsorption of protein on the magnetic particles (MP) completely, the next step is to desorb the protein from the magnetic particles (MP). For this, 500 μΐ elution buffer is added in the protein-magnetic particles pellet. Mechanical or thermal energy is provided to this system to desorb the protein from the MNP. Mechanical energy is to be in term of ultrasound in the frequency range 20- 40 kHz for 60 seconds. In other way, thermal energy in terms of water bath is provided to the protein-MNP pellet at the temperature range 40-50°C for 10 minutes. The system was kept under the magnet field as mentioned earlier. While doing this, magnetic nanoparticles pelletized, collect the supernatant. The supernatant collected is basically suspension of total protein, and estimated through protein assay or other suitable methods. In the present case, extracted protein is estimated using Bradford reagent.

Figure 4 shows the extraction efficiency and protein loading capacity of the magnetic nanoparticles for fixed 50 μg MNP, i.e. 5 μΐ magnetic fluid. Thus the extraction efficiency achieved varies from 100 - 68% for the protein concentration range 2-55 mg/100 μΐ BSA suspension. Figure 4 shows that at various places demonstrate that the size of the protein-magnetic particles pellet increases with protein concentrations.

The protein extraction efficiency in the range of 68-90% is obtained foi^¬ corresponding 55-28 mg/100 μΐ BSA suspensions at 50 μg magnetic nanoparticles. This means that 1 mg MNP is loading 1 100-560 g BSA, with the 68-90% extraction efficiency.

The protein extraction efficiency in the range of 90-95% is obtained for corresponding 28-22 mg/100 μΐ BSA suspensions at 50 μg magnetic nanoparticles. This means that 1 mg MNP is load 560-440 g BSA, with the 90-95% extraction efficiency.

The protein extraction efficiency in the range of 95- 100% is obtained for corresponding 22-2 mg/100 μΐ BSA suspensions at 50 μg magnetic nanoparticles. This means that 1 mg MNP is load 440-0.2 g BSA, with the 95-99.99% extraction efficiency. The lower limit of the protein concentration do not suggest minimum range, it is only used as reference. It is also possible to detect the protein even for low concentration than described here.

It is summarized from this experiment that the protein loading capacity of the magnetic particles (MP) ranges from 40- 1 100 g BSA per 1 g magnetic particles (MP). The obtained values are much higher than the earlier reported values of 348 mg BSA/l g of particles [Li et al, Chem Mater, 2006, 1 8, 6403]. It is to be noted that the loading capacity of the magnetic particles (MP) obtained using the present process is three orders of magnitude higher than the reported values.

Figure 9 depicts SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) of the protein present before and after performing the extraction process described in the invention. Figure 9 shows marker (lane 1), total protein in blood plasma (lane 2) (i.e. before extraction - control/reference), and total protein eluted using the developed process (lane 3-5). Lane 4 & 5 results into repetition of elusion step. Qualitative and quantitative analysis of electrophoresis SDS-PAGE data confirms presence of almost all proteins, ranging from 14 kDa to 206 kDa, before and after extraction procedure.

In another way, controlled sample is prepared by extracting the protein using conventional protocols. In this, the protein precipitation had been carried out either using salting out or by organic solvents. While using salting out it is to be salts l ike ammonium sulphate, sodium chloride and potassium chloride etc., or pre-chilled organic solvents like methanol, ethanol, acetone etc. The protein amount obtained using either of the process depending on the biological system which gives maximum yield is compared here with our developed process. In conventional process, as mentioned above, protein was precipitated by adding either saturated salt solutions (e.g. ammonium sulphate) or pre-chilled solvents (e.g. ethanol) while stirring. In case of organic solvents, system was incubated for 15 minutes at -20 °C, while system is not incubated in case of salt solutions. The sample is centrifuged at 10,000g for 15 minutes. The supernatant thus obtained is discarded and the pellet is resuspended using vortexing. When ammonium sulphate was used, salt impurities also eluted out along with the protein, which was removed by dialysis of the sample for overnight.

While using organic solvents the protein pellet resolubalization is very difficult which leads to loss of protein recovery and subsequent loss of its activity. Moreover processing time taken by both the processes is higher which made the protein more amenable for denaturation due to heat and mechanical stresses.

Protein extracted from both (conventional and process comprising use of magnetic particles) are shown in the form of purity and yield in Table- 1.

Table- 1 : Protein extraction from blood/plasma system (Bradford reagent, Sigma Aldrich (B6916)

Purity of extracted protein was analyzed by performing following tests:

(1 ) Agarose gel electrophoresis for the detection of nucleic acids, i.e DNA and/or RNA : Figure 10 shows agarose gel electrophoresis of the supernatant collected after elusion step from the whole blood and blood plasma (lanes 1 & 2) and lysed whole blood and lysed plasma (lanes 5 & 6). Lane 3 id DNA ladder. In the later case faint band corresponding to DNA/RNA is observed while protein extracted using the invented method do not contain signatures of DNA/RNA, i.e. nucleic acids.

(2) Glucose test using Cognet-Autospan Glucose test kit (Enzymatic GOD-POD method) - To analyze the presence of sugar as a contaminant glucose test was performed.

Figure 1 1 shows results of the glucose test performed on (a) plasma (before extraction), (b) protein eluted from plasma using the developed process, and (c) standard sample. In this test, light pink color indicates the presence of glucose. It is noted that in case of protein extracted using the developed process, solution remains colorless, which confirms that impurity of small molecules like glucose is to be avoided.

(3) Enzyme activity assay - Enzyme activity assay was performed by SGPT and SGOT-Corel Modified IFCC method, and Alkaline Phosphatase- Bio-in-vitro diagnostics PNPP based estimation kit. The results of the activity assay performed for certain enzymes of whole blood and plasma is shown in Table 2. It confirms that activity of the enzyme remains intact after extraction.

Table 2: Enzyme activity of various enzymes before and after the extraction using magnetic particles.

In another embodiment of the present invention drug present in blood/plasma is estimated. Protein binding efficiency and solubility of drug plays vital role in the drug extraction efficiency. The process invented is shown in the form of flow chart in the Figure 1. In order to reduce the data error,, sample quantity and all related parameters is to be increased. Calcitriol extraction: In the 300μ1 spiked plasma, magnetic particles (MP) /magnetic fluid followed by binding buffer (e.g. ethanol) is added. The magnetic fluid concentration was varied from 10 to 500 μΐ that corresponds to 100 to 5000 μg respectively.

Supernatant collected by magnetic decantation is dried, resuspended in mobile phase and after passing through anal chem. cartridge and finally loaded in liquid chromatography-tandem mass spectrometry (LC-MS/MS) (make: Waters and Ab- Sciex) for estimation. Figure 13 shows the area under the curve of calcitriol drug for various concentrations of magnetic fluids. The results showed that the addition of magnetic fluid/magnetic particles (MP) increases the extraction of drug as compared to the conventional method. Also an optimal concentration for extraction is elucidated.

Drug extraction efficiency of the magnetic particles/magnetic fluid is performing. The protein binding efficiency of Calcitriol is >99%. The Calcitriol drug concentration is varied by keeping the concentration of magnetic particle constant. As described above, similar process is carried out. The area under the curve obtained for this drug is plotted for varying concentration from 0 to 2100 pg/ml of Calcitriol drug.

Figure 14 shows the extraction of calcitriol drug in terms of area under the curve for various concentrations of the drug. Linearity in the graph confirms the reproducibility of the developed method. However, area under the curve without spiking calcitriol drug was also obtained. The estimated value of calcitriol based on the standard curve was ~ 40 pg/ml. This may be due to vitamin D3 present in the blood plasma. The results shown here were repeated at least 10 times and each time sample loaded in triplicate.

Similar process was followed to estimate drug concentration present in plasma. The values obtained were shown in Table 3. The values estimated by the process described herein are at least 7 to 18 % more than conventional methods. Extraction using Extraction without

Sr. Plasma protein

Name of the drug magnetic magnetic no. binding

nanoparticles nanoparticles

1 Calcitriol >99% 51 % 43 %

Omega acid

2 60 % 47.2 % 40%

(EPA)

Omega Acid

3 76 % 77.1 66.4 %

(DHA)

Hydroxy

4 84% 85 % 71 %

Bupropion

5 Pazopanib >99% 100 % 98 %

6 Retigabine 60 % 73.6 % 69.9 %

Table 3: Extraction of various drugs from plasma by magnetic particles

Scaling-up of total protein extraction process is performing. System is scaled up from initial 0.1 ml upto 300 ml by varying all the parameters linearly. Milk was chosen for the scaling-up. The quantitative results are calculated using Folin-Lowry protein estimation method and qualitative confirmation is done by gel electrophoresis. Table 4 illustrates the total protein extracted efficiency in milk.

Table 4: Comparing protein extraction efficiencies in scaled up system for Milk using Folin Lowry estimation method.

Figure 15 shows SDS-PAGE image of scaled up system; where lane 1 , 2 represents protein extracted from 0.1 ml system, Lane 3, 4 from 1 ml and lane 5, 6 from 100 ml systems; Lane 7 and 8 were total protein present in milk systems (stock). Loading was done in duplicate. The data confirms high extraction efficiency over wide range of sample sizes. This showed that on scaling up the system the extraction efficiency remains unchanged and there was no loss of protein in the procedure.

Similarly, this process was also applicable for the plasma fraction process, which was increase the yield and extraction efficiency. The wastage of precious human protein was to be reduced by the present process. The protein extracted was to be stored and further used for the experimentation.

Results and discussion:

The present invention provides method for extracting protein and/or drug from the biological system by magnetic particles. The method for the extraction of protein described herein above gives maximum yield of >99%. Further, activity of extracted protein or enzyme remains intact. Thus, the protein extracted by the method described herein above is to be stored and further used for the experimentation. The method described here involves 30-40 minutes. The extraction efficiency of smal l drug molecule is to be increased up to 75-90% which is 10-20% higher than other existing method for the extraction. One of the major advantages of the present invention is any sophisticated instrument like Cyro-centrifugation, Cryo-incubation are not required.

The method to use magnetic particles (MP) described here is appl ied in proteomic analysis and in diagnosis of disease using biomarker.

The obtained product at the end of the extraction is formulated by suitable techniques known in the art. Also, this process is useful for almost all biological system. The invention is illustrated more in detail in the following examples. The examples describe and demonstrate embodiments within the scope of the present invention. These examples are given solely for the purpose of illustration and are not to be construed as limitations of the present invention, as many variations thereof are possible without departing from the and scope.

Examples

Example 1: Extraction of total protein from whole blood

In the 100 μΐ human whole blood, 100 μΐ 1 % SDS lysis buffer was added, and the system was allowed to mix gently. 5 μΐ iron oxide superparamagnetic magnetic fluid was added in this system, and then 700 μΐ pre-cooled acetone binding buffer was added. The system was then cooled down to 4°C for at least 13 minutes.

The system was placed under magnetic field of 3000 Oe. This was form a pellet of protein-magnetic particles. In the presence of magnetic field, almost colorless supernatant obtained was separated either by inverting the system or by pipetting. The morphology of the pellet was observed by transmission electron microscopy (TEM) as shown in the Figure 5. The high resolution TEM image shown as an inset of figure 5 confirms the presence of magnetic particle. This was confirms that the cluster formed by the protein-magnetic particles remains in the nano-regime.

Further, the process for eluting the protein from the protein-MP pellet was followed. In this, 500 μΐ phosphate buffer was added in the protein-magnetic particles pellet. Mechanical or thermal energy is provided to this system to desorb the protein from the MNP. Mechanical energy was in term of ultrasound in the frequency range 40 kHz for 60 seconds. In other way, thermal energy in terms of water bath was provided to the protein-MNP pellet at the temperature range 40-50°C for 10 minutes. The system was kept under the magnet field as mentioned earlier. While doing this, magnetic nanoparticles pelletized, collect the supernatant. The total protein obtained by above mentioned process was collected and analyzed using Bradford protein assay and electrophoresis using SDS-PAGE. The extracted protein estimated using Bradford protein assay in case of whole blood system was shown in Table- 1.

Figure 6 shows TEM image of the magnetic particles (MP) after performing the elusion/extraction step. The morphological confirmation of extraction of total protein from the magnetic particles (MP) was confirmed using TEM image. It reveals bare/uncoated particles, i.e. without the presence of protein and/or protein-MP clusters. This support of finding that the total protein (>99%) extraction of total protein is possible using magnetic particles.

Example 2: Extraction of total protein from blood plasma

Similar process for extraction of total protein from blood plasma as fol lowed in Example 1 is performed. The FTIR spectra and TGA data of the blood plasma protein-MP pellet were shown in the Figures 7 and 8 respectively. Figure 7 shows FTIR spectra of (a) magnetic particles (before process), (b) blood plasma, and (c) pellet of protein-magnetic particles. The shift in the peak positions indicates interaction between protein and magnetic particles (MP).

Figure 8 shows TGA-DSC data of pellet of protein-magnetic particles. Only ~ 2.5% weight loss was observed up to 200° C temperatures. Maximum weight loss of ~ 87.5 % had been observed during the temperature range of 200° C, to 420°C. Further, in two steps ~ 7.15 % weight loss had been observed in temperature range of 420° C to 550°C. Therefore, total weight loss observed in the protein-magnetic particle pellet comes around ~ 97.2 % of the total weight. Remaining 2.8% weight loss corresponds to weight of magnetic particles, which do not decomposed in the given temperature range. The observed 97.2% weight loss was completely due to the protein adsorbed on the magnetic nanoparticles and hence confirms >99.9 % binding of protein-MP. Both the figures confirm adsorption of protein on the magnetic particles (MP) without altering the structural integrity. Example 3: Extraction of total protein from plant system

Tobacco and Bougainviliea plants were used as a plant system to cany out extraction of total protein. Procedure for the extraction had been followed as given in the Example 1. Total protein extracted from tobacco and bougainviliea plant was estimated by Bradford protein assay. Result of assay was shown in Table-5. It confirms that most of the protein gets extracted using the process described in the present invention.

Table 5: Extraction of protein from plant system

The extracted protein from plant leaves was tested for structural integrity using Peroxides enzyme assay. In this test, total sample volume taken was 1 ml,

Pyrogallol and hydrogen peroxide was kept at concentration of lOOmM in the system.

A control system was also tested which contained distilled water in place of extracted protein. The substrate used was Pyrogallol in presence of hydrogen peroxide. Table 6 shows the results of the enzyme assay. It was confirmed from the table that peroxides activity and hence structural integrity remains intact.

Extracted protein 0.1 16 ± 0.01

Control 0

Table 6: Peroxidase activity of extracted plant protein Example 4: Extraction of total protein from bacterial system

E.coli was used as a bacterial system to carry out extraction of total protein. Procedure for the extraction has been followed as given in the Example 2. Total proteins extracted from E-coli bacteria were shown in Table-7. Protein values were estimated by Folin-Lowry method (Folin-Ciocalteu's phenol reagent, Merck, 109001 ). Comparison of protein extracted with conventional method, depicts that large amount of protein was extracted using the process described herein.

Table 7: Extraction of protein from E. coli bacterial system.

SDS-PAGE of the extracted protein obtained by the process described in present invention and by the conventional process was shown in Figure 12. Where, lane 1 : total protein eluted using the invented process, lane 2: protein yield obtained by repetition of the process, lane 3&4: protein yield obtained followed by conventional method, lane 5: pure bacterial culture (control). For comparison purpose, pure culture following standard procedure for loading the sample was carried out The SDS-PAGE data. It confirms that almost all the bands of proteins were present in the sample of extracted protein obtained by the process of the present invention. Quantitative analysis suggest that amount of protein obtained using present process was higher than the conventional methods. Results are shown in Table 7.

While various embodiments of the present invention been described in detailed, it is apparent that modification and adaptation of those embodiments wi ll occur to those skilled in the art. It is expressly understood, however, that such modifications and adaptations are within the spirit and scope of the present invention as set forth in the following claims.

Claims

We Claim,

1. A method for extraction of biomolecules by magnetic particles comprises following process;

a) adding of magnetic particle into biological system;

b) adding binding buffer to allow formation of clusters of protein- magnetic particle;

c) applying the external magnetic field for magnetic decantation; d) separating supernatant and collecting magnetic particle-protein pellet; e) removing of external magnetic field from the protein-magnetic particle pellets;

f) adding buffer for resolubilization of protein;

g) applying external magnetic field for magnetic decantation; h) repeating steps (f) and (g);

i) collecting the supernatant containing soluble total protein; wherein, said separated supernatant of step (d) is resuspended in mobile phase to extract drug.

2. The method for extraction of biomolecules by magnetic particles as claimed in claim 1 (b), wherein 1 :3 to 1 : 10 binding buffer selected from CTAB, Tvveen 80, SDS, PEG 6000, urea, acids is selected from 0. 1 N HCL and/or 0.1 N H₂S0₄. bases is selected from sodium hydroxide and potassium hydroxide, salts and polymers is selected from ammonium sulfate, sodium sulfate and non-ionic polymers like dextrans, or solvents like acetone, ethanoi, methanol, acetonitrile and propanol is used.

3. The method for extraction of biomolecules by magnetic particles as claimed in claim 1 (c), wherein 400 Oe to 4000 Oe external magnetic field is applied for magnetic decantation.

4. The method for extraction of biomolecules by magnetic particles as claimed in claim 1 (f), wherein 1 :5 to 1 :6 buffer is added for resolubilization of protein.

5. The method for extraction of biomolecules by magnetic particles as claimed in claim 1 (a), wherein optionally 1 : 1 to 1 :2 lysing agent is used to lyse the cell.

6. The method for extraction of biomolecules by magnetic particles as claimed in claim 1 (b), wherein optionally cooling of system is done at 0 °C to 10 °C to allow formation of larger pellet of protein-magnetic particle.

7. The method for extraction of biomolecules by magnetic particles as claimed in claim 1 (e), wherein optionally 20 kHz to 40 kHz external energy is applied to expedite the solubilization process.

8. The method for extraction of biomolecules by magnetic particles as claimed in claim 1 , wherein preferably uncoated/bared magnetic particles are synthetic analogues of magnetic material or combination of materials selected from magnetite, ulvospinel, hematite, ilmenite, maghemite, jacobsite, trevorite, magnesioferrite, pyrrhotite, greigite, troilite, goethite, lepidocrocite, feroxyhyte, iron, nickel, cobalt, awaruite, wairauite, or any combination thereof; wherein optionally transition metal selected from iron, manganese, nickel, cobalt and zinc is used to form magnetic particles.

9. The method for extraction of biomolecules by magnetic particles as claimed in claim 1 , wherein the lysis buffer is selected from sodium dodecyl sulphate, triton X- 100, polymer like poly-ethylene glycol and ethylene glycol.

10. The method for extraction of biomolecules by magnetic particles as claimed in claim 1 , wherein the eluted protein is resokibiiized in bio-compatible buffer at 5 pH to 8 pH.