Technical Field of the Invention

The invention relates to a method developed to selectively detect biomarkers in the presence of a magnetic field using a solution containing polymer-encapsulated superparamagnetic iron oxide micelle nanoparticles. The present invention has the potential for rapid and easy diagnosis of many different diseases in biological analysis applications and the biomedical sector.

State of the Art (Prior Art)

In magnetic levitation platforms, cell groups and materials with different densities can be aligned at their specific levitation heights using paramagnetic solutions. In one of these methods, different types of cancer cells and blood cells were collected at characteristic levitation heights using Gadavist (Gd) as the paramagnetic solution without using any biomarking method. In yet another application, spherical or non-spherical materials with similar densities in a manganese (II) chloride (MnCh) solution were collected at various heights using a magnetic levitation platform that imparts inclination based on gravity force. Magnetic levitation platforms, which do not require additional equipment, are also important in rapidly diagnosing biomarker proteins in various diseases. In another method called immunosandwich assay, the experiment was performed using magnetic and polymer microspheres in the presence of Gd solution, and mouse immunoglobulin G (IgG) protein in serum was detected with a detection limit of >10 ng/ml.

Another solution used in magnetic levitation systems is superparamagnetic (SPM) solutions. SPM solutions enable faster analysis thanks to their higher magnetic sensitivity compared to their paramagnetic counterparts. In a study, the densities of plasma proteins were determined using superparamagnetic iron oxide nanoparticles as the solution in the magnetic levitation technique. In this study, the densities of plasma proteins, which had been reported in the literature to have a density of 1.35 g/cm ³ , were determined as 1.03 ± 0.02 g/cm ³ using microspheres levitated in a magnetic field. In this particular study, although the magnetic levitation method was employed, the platform used in the magnetic levitation setup was consisting of two magnets having dimensions of 25.4 mm x 25.4 mm x 50.8 mm and a plastic cuvette with a volume of 4 mL. Since this system requires high volumes of solutions, it increases the cost. Additionally, the separation of the microspheres at different densities required a waiting time of 180 minutes, leading to an increase in analysis time. Moreover, selective biomarker detection cannot be performed in this method. The proteins in the sample are collected in a specific band, allowing only the total protein quantity to be detected.

In another study, the presence of opioid peptides in human plasma samples was detected by a magnetic levitation system. In this study, it was observed that the individuals with opioids in their plasma samples took longer to reach equilibrium compared to plasma samples from healthy individuals in the presence of a magnetic field.

The patent document numbered W02020/023443A1, describes systems and methods for separating a large number of molecular entities with different densities. The system comprises: a pair of magnetic poles of similar polarity for the generation of the magnetic field and a container holding a large number of molecular entities in a liquid medium containing nanoparticles that significantly alter the magnetic sensitivity of the liquid medium, thereby effectively producing sufficient gradients in the liquid medium inside the container when the container is placed in the magnetic field. To elevate multiple molecular entities to the relevant layers in the container, each layer corresponds to a specific density. The nanoparticles can be superparamagnetic iron oxide nanoparticles.

The study reported by Ali Akbar Ashkarran et al. introduces the capabilities of magnetic levitation (Maglev) to detect diseases (Opioid Use Disorder being used as a model disease) using levitation of human plasma proteins. The proof-of-concept findings reveal that optically imaged plasma proteins subjected to magnetic levitation carry significant information about the health spectrum of plasma donors. Furthermore, the liquid chromatography-mass spectrometry analysis of magnetically levitated plasma proteins has shown notable differences between healthy individuals' plasma and patients with Opioid Use Disorder. Generally, the presented method provides diagnostic value for disease detection by using optically imaged evolving magnetically levitated plasma proteins and/or proteomic information. The document also demonstrates a levitation model of human plasma proteins in a superparamagnetic iron oxide nanoparticle solution functionalized with a commercially available product called ferumoxytol. In this study, biomarkers were not detected selectively. The changes in plasma protein density are examined concerning opioid use.

Heterogeneous methods require a surface for detection, while homogeneous methods can perform detection within the liquid without the need for a surface. Therefore, homogeneous methods do not require surface chemistry processes or washing steps. This allows homogeneous methods to be more easily and cost-effectively implemented.

Heterogenous analysis methods used for biomarker detection can considerably extend the time needed for diagnosis since it involves an attachment step of biomarkers onto a surface, additional modifications of the surface to facilitate this attachment, and a series of distinct and time-consuming steps to accurately identify the biomarkers. Although it has been previously shown that biomarkers can be selectively detected specifically on microparticles, the magnetic levitation techniques used in other researches do not allow a uniform analysis approach that eliminates processing difficulties and does not require any surface for biomarker identification.

Brief Description and Objectives of the Invention

The main object of the present invention is to develop a method for selectively detecting and quantifying biomarkers used in diagnosing and monitoring diseases in the presence of a magnetic field without the need for any surface. These biomarkers can be proteins, molecules, DNA, and RNA. Specifically, particular biomarkers like Immunoglobulin G (IgG), Prostate Specific Antigen (PSA), C-Reactive Protein (CRP), p24, troponin, or creatinine can also be used. In the present invention, IgG is selected as the preferred biomarker for detection.

The object of the present invention is to develop a cheap and easy-to-use method that allows for the quantitative determination of biomarkers used in the diagnosis and monitoring of diseases. The method of the present invention offers a simple and cost-effective detection without requiring surface chemistry and washing procedures.

The object of the present invention is to achieve a fast analysis method by using significantly reduced volumes of solution, thereby reducing costs and saving time with a 15-minute analysis time in the magnetic levitation platform. The term solution here refers to the solutions used throughout the detection process. The total volume of solution used for a single experiment is between 10-100 pL, preferably 30 pL. Moreover, the selective detection of the desired biomarker is possible. Using this platform, the IgG as a sample biomarker can be detected under a magnetic field within a short period of 15 minutes, achieving limit of detection (LOD) of <1 pg/mL in phosphate-buffered saline (PBS) and <10 pg/mL in fetal bovine serum (FBS).

In the present invention, biomarkers are detected using the magnetic levitation technique in a superparamagnetic solution directly from the liquid sample (e.g., serum, urine, saliva) with a homogeneous analysis method without the need for any detection surface. Since this method does not require multistep pre-preparation surface modification, it dramatically decreases the procedure time observed in the heterogeneous analysis method. In the present invention, it takes advantage of the naturally occurring biological binding process between the biomarker in the sample liquid and its specific conjugate (such as protein, antibody, DNA, aptamer, or polymer), any surface is not required for biomarker or conjugate binding unlike heterogeneous methods. Moreover, the detection steps are reduced by mixing the sample with the conjugates compared to heterogeneous methods. This enables fast detection. Moreover, the use of small volumes of biological and chemical materials for diagnosis is considered another advantage. In the present invention, the biomarker to be analyzed is directly mixed with a superparamagnetic solution and a specific fluorescent conjugate for the biomarker's detection at specific concentrations. Therefore, it is reported that rapid and easy -only depending on changes in fluorescence intensity- detection of biomarkers will light the way for early diagnosis of diseases and the creation of personalized diagnostic kits.

Definitions of Figures Explaining the Invention

Figure 1: The schematic representation for the used magnetic levitation platform. Here, it shows g gravity acceleration.

Figure 2: Inside the capillary loaded with superparamagnetic iron oxide micelle nanoparticle (SPION), cases od (A) without antigen and (B) with antigen. The fluorescence intensity changes are shown (C) without antigen and (D) with antigen at 0 ^th and 15 ^th min. The fluorescence intensity changes are shown for different concentrations of antigen (mouse immunoglobulin G, IgG) inside (E) phosphate buffer saline (PBS) and (F) fetal bovine serum (FBS). Definitions of Elements/Parts/Pieces Forming the Invention

In order to better explain the magnetic levitation platform developed with the present invention, the parts/pieces/elements in the figure prepared for the process given as an example are individually numbered, and the explanation of each number is given below.

1. Magnet

2. Mirror

3. Glass Capillary Channel

4. Florescent Light Source

5. Objective and filter

6. Camera

7. Antibody

8. Antigen

9. Fluorescent Signal Distribution of without Antigen at the 0 ^th minute

10. Fluorescent Signal Distribution of without Antigen at 15 ^th minutes

11. Fluorescent Signal Distribution of with Antigen at 0 ^th minute

12. Fluorescent Signal Distribution of with Antigen at 15 ^th minutes

13. Decrease in Fluorescence Intensity at 0 mg/mL Antigen Concentration Incubated in Phosphate Buffered Salt Solution

14. Decrease in Fluorescence Intensity at 0.001 mg/mL Antigen Concentration Incubated in Phosphate Buffered Salt Solution

15. Decrease in Fluorescence Intensity at 0.01 mg/mL Antigen Concentration Incubated in Phosphate Buffered Salt Solution

16. Decrease in Fluorescence Intensity at 0 mg/mL Antigen Concentration Incubated in Fetal Bovine Serum

17. Decrease in Fluorescence Intensity at 0.001 mg/mL Antigen Concentration Incubated in Fetal Bovine Serum

18. Decrease in Fluorescence Intensity at 0.01 mg/mL Antigen Concentration Incubated in Fetal Bovine Serum Detailed Description of the Invention

In the present invention, targeted biomarkers interact selectively with antibody, and then the biomarker and the antibody form a large complex structure. Superparamagnetic iron oxide nanoparticles (SPIONs) in the magnetic levitation platform environment move towards the magnets over time, so the magnetic field strength in the channel decreases with time. When the biomarker and antibody complex is placed in the channel, it collects in an area near the middle of the channel, while the complex begins to disperse over time at the midpoint of the channel as the SPIONs move towards the magnets. Since fluorescent-labeled antibodies are used in the present invention, their distribution can be clearly observed under the fluorescent microscope. In the absence of an antibody-selective biomarker, antibodies do not form complexes with the biomarker. In this structure, the distribution profile of the antibodies differs from the case where the selective biomarkers are present. Therefore, biomarker determination can be detected by the fluorescence intensity change over time.

This method, which was developed for the detection of the biomarkers in the presence of a magnetic field using a micellar superparamagnetic iron oxide nanoparticles (SPION) solution, comprises the following steps;

• Forming the biomarker-fluorescent conjugate complex by incubating the sample, which will be used in the biomarker analysis with its specific fluorescent conjugate

• Adding a solution containing superparamagnetic iron oxide micellar nanoparticles encapsulated with poly (ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (PEG-PPG-PEG) polymer to the complex,

• Loading this mixture into the capillary channel (3), and then placing the same on the magnetic levitation platform containing at least two magnets (1) whose poles are facing each other, and at least one mirror (2) so as to obtain the image.

• Analyzing the time-dependent change in fluorescence intensity of the images by observing the mixture under a fluorescent microscope and determining the amount of the biomarker in the sample by analyzing the fluorescence change.

The magnetic levitation platform used consists of two N52-NdFeB magnets (1), at least one mirror (2) placed at an angle of 45° so as to obtain the image, and parts produced with a three- dimensional printer, which are the carriers of all these. The magnetic levitation platform is placed on the fluorescent microscope so as to obtain the channel image. The microscope image is provided by at least one mirror (2) placed at an angle of 45° (Figure 1). With this mirror (2), the radiation provided by the fluorescent light source (4) on the fluorescent microscope passes through the objective and filter elements (5) and illuminates the capillary channel (3), it can be captured by the camera (6) by passing through the elements (5). Fluorescent images of the channel are taken immediately (0 minutes) and 15 minutes after it is placed on the magnetic levitation platform. The presence or absence of the targeted biomarker in the channel (Fig. 2A, B) changes the time-dependent fluorescence in the channel. In order to measure these changes, fluorescent signal distribution (9-12) changing from the lower magnet to the upper magnet (Figure 2C, D; 0 pm distance shows the lower magnet position) is detected in the middle of the channel by analyzing the fluorescent channel images. The presence of biomarker is detected by measuring the decreased percentage of the maximum value of this fluorescent signal relative to Oth minute to the 15 ^th minute (13-18) (Figure 2E-F).

Moreover, the micellar iron oxide nanoparticles used in the levitation system were synthesized in the laboratory in two steps. In summary, firstly, approximately 7 nm cubic SPIONs having homogeneous size distribution were obtained in hexane using the thermal decomposition method of the iron-oleate complex. In this synthesis, oleic acid was used as a surfactant capping agent, and 1 -octadecene (ODE) was used as the solvent. The synthesis was heated up to 320 °C at a constant temperature increase rate (5.5 °C/min) and kept at this temperature for half an hour. After the cleaning procedure, characterizations of the material were performed.

Then, in order to obtain micellar iron oxide nanoparticles in the form of supraparticles, first, iron oxide nanoparticles dispersed in the cyclohexane and the polymer solution in water with a concentration of 2% (w/v) were mixed with a vortex. Following this, using the ultraprobe sonication technique, SPIONs were encapsulated with Poly(ethylene glycol)-block- poly(propylene glycol)-block-poly(ethylene glycol) (PEG-PPG-PEG) in order to transfer the SPIONs in the organic phase to the water phase and obtain the micellar iron oxide nanoparticles. The cap of the vial, including the organic solvent-water mixture, was left open for a few days to allow the organic solvent to evaporate. After the iron oxide micellar nanoparticles remaining in the water were characterized, they were stored at +4 °C to be used in the magnetic levitation system. The polymer used in this procedure is an amphiphilic polymer called as a nonionic triblock copolymer, and its main chain is hydrophobic polyoxypropylene. Two hydrophilic polyoxyethylene chains follow both sides of this hydrophobic chain. If you imagine this amphiphilic polymer as a three-armed structure, the hydrophobic chain remains inside, while the other two hydrophilic arms are directed towards the outer hydrophilic solution when the micelle is formed. In this way, hydrophobic iron oxide nanoparticles located in the center of the micelle structure, while the PEG-based hydrophilic arms are directed towards the water.

Because iron oxide nanoparticles tend to stick to each other, the water phase transition of these materials is a complicated process. In the present invention, PEG-PPG-PEG polymer was preferred to form iron oxide nanoparticle micelles in water because it has an amphiphilic structure. Thanks to this method, SPIONs can be transferred to the water phase with an easy technique and can maintain their stability for a long time. Moreover, the fact that the triblock copolymer is based on PEG is another reason why it is preferred because PEG can be used safely in many biological studies thanks to its nontoxic structure and biocompatibility.

Instead of this triblock polymer, coating the iron oxide nanoparticles with only PEG could also be an alternative technique. However, in this case, a single SPION would be covered with PEG, and more than one iron oxide nanoparticle structure would not be formed in a single micelle. In addition, the process would be more complex as the surface chemistry would be changed, and the SPIONs would be more likely to aggregate at the end of the process.

Although iron oxide materials gain a superparamagnetic structure as their size decreases, magnetic force decreasing with the decreasing size is a challenging problem in the literature. With the micellar SPION production, it is aimed to overcome this problem. Thanks to the encapsulation of more than one iron oxide nanoparticles in the micelle structure, the superparamagnetic characteristics are preserved while the magnetic field of the nanoparticles is strengthened. The invention also has an innovative approach in terms of the use of micellar iron oxide nanoparticles in the diagnosis of biomarkers in magnetic levitation systems.

Prior to the loading with the sample (PBS, serum, etc.), the glass capillary channel (3) that will be used in the biomarker analysis was exposed to air plasma for 1.5 minutes under 100 Watt 0.5 bar pressure. Then, the sample was incubated with fluorescent antibodies for 15 minutes and the prepared sample was loaded into the glass capillary channel with a pipette without an air gap. Following this, it was placed through two magnets (1) located on the platform and observed under a fluorescent microscope. Fluorescent images were taken at 0 and 15 minutes and their time-dependent fluorescence intensity variation were analyzed using these images. The biomarker concentration in the sample was quantified by comparing the time-dependent variation of the fluorescence intensity in different times.

For the identification of biomarkers, first, the fluorescently labeled antibody was incubated with the sample for 1-30 minutes, ideally 15 minutes. Then, superparamagnetic iron oxide nanoparticles in micellar structure with a concentration of 0.01-0.5 mg/mL, preferably 0.153 mg/mL, were added into this solution. The micelle structure was important for the organic to water phase transition of the iron oxide nanoparticles. Various techniques such as ligand exchange, encapsulation with silica or a polymer can also be used for this phase transition. However, micelles were preferred in the present invention because of their superior magnetic advantages. When a micelle was considered as a huge polymer particle involving a plurality of iron oxide nanoparticle, using this technique, the obtained nanostructures will hold both superparamagnetic characteristics and will be magnetically stronger due to the coexistence of a plurality of iron oxide close to each other.

Then, this mixture with micelle SPION was loaded into the capillary channel (3) and the channel was placed on the magnetic levitation platform. Imaging was performed under fluorescent light for 0-30 minutes. At the end of the imaging times, the time-dependent percentage changes are calculated by measuring the maximum fluorescence value changes within the channel. By considering these changes, the amount of the target biomarker/antigen present in the sample was determined (Figure 2). Throughout the analysis, the magnetic forces within the glass capillary decrease with time due to the attraction of the micelle SPIONs towards the lower magnet. Accordingly, there was a difference in the fluorescence intensity of the solution. When the samples with antigen were compared with the samples without antigen, it was observed that there was a greater variation in fluorescence intensity of samples with antigen. Moreover, it was observed that the fluorescence difference of the sample increases with the increasing antigen concentration. Therefore, based on this intensity difference, the limit of detection (LOD) values for the used detection method were calculated as <1 pg/ml in phosphate buffered solution (PBS) and <10 pg/ml in fetal bovine serum (FBS). In this technique, since the detection was performed by using the freely circulating antigen and antibodies in the liquid sample and the platform does not require a particular surface for the detection, the present invention allows to a homogenous detection assay. Since the antigen was the desired biomarker to be detected here, it was used to observe whether there was any detection. The group without antigen was used as the control group.