WO2025164876A1 - Method for extracting parameters of dielectrophoresis using dielectric spectroscopy, method for analyzing protein by dielectrophoresis, and biosensor - Google Patents

Abstract

The present invention relates to a method for obtaining parameters of dielectrophoresis (DEP) using dielectric spectroscopy and, more specifically, to a method for extracting parameters of dielectrophoresis (DEP) using complex permittivity spectra extracted from dielectric spectroscopy (DS). The non-labeled method of the present invention does not require a separate antibody or a complex pretreatment process, and can implement a dielectrophoresis method using the complex permittivity spectra of dielectric spectroscopy, such that the present invention may be utilized in various fields, such as analyzing intracellular biomaterials, and in particular, may be utilized to monitor the differentiation process of stem cells.

Description

A method for extracting parameters of dielectrophoresis from genomic spectroscopy, a method for analyzing proteins using dielectrophoresis, and a biosensor

The present invention relates to a method for obtaining parameters of dielectrophoresis using dielectric spectroscopy, and more specifically, to a method for extracting parameters of dielectrophoresis (DEP) using a complex permittivity spectra extracted from dielectric spectroscopy (DS).

The non-labeled method of the present invention does not require a separate antibody or a complex pre-treatment process, and can implement a dielectrophoresis method using the complex permittivity spectrum of dielectric spectroscopy, so it can be used in various fields such as analyzing intracellular biomaterials, and in particular, can be used to monitor the differentiation process of stem cells.

The self-renewal capacity of stem cells makes them useful for regenerating body tissues damaged by disease or accidents. However, undifferentiated stem cells have the potential to differentiate into cancer cells or other unwanted cells, making technology for monitoring stem cell differentiation crucial.

Various methods have been used to monitor the expression of specific proteins in cells or to characterize cell differentiation, such as immunostaining, Western blotting, flow cytometry, microarrays, immunocytochemistry, quantitative polymerase chain reaction (qPCR), and reverse transcription-polymerase chain reaction (RT-PCR). The most widely used technique is immunohistochemistry (IHC). IHC is a labeling method. It stains cells with antibodies that bind to specific antigens and uses the antigen-antibody reaction to determine the presence of a specific antigen from a fluorescent image. This reaction typically occurs between two large protein molecules. Glycoprotein antibodies bind to glycoproteins, lipoproteins, or protein antigens. The chemical composition of the antibody determines its specific binding to a single antigen.

These IHC assays have limitations. One of the major limitations of stem cell testing is the lack of all the markers necessary to differentiate cells from other cells. IHC is a multistep process, and the processing of multiple steps is time-consuming. IHC can be performed on fixed cells or tissues, which have been processed to allow the incorporation of antibodies. Secondary antibodies are then conjugated to the primary antibody for detection. The secondary antibody is fluorophore-labeled and observed under a fluorescence microscope. Several parameters are considered to prevent nonspecific antibody binding in the sample, including antibody design, concentration, and processing time; buffer composition and pH; and temperature. Furthermore, fixation methods can interfere with the binding of antigens to the primary antibody target, potentially targeting the epitope. To overcome this problem, an antigen retrieval step is often performed, pre-treating the tissue to recover antigens masked by fixation, making them more accessible for antibody binding. Effective antibody design is essential, as nonspecific antibodies can interfere with accurate data acquisition by generating more background noise through aberrant binding.

In other words, the biggest drawback of IHC is the lack of individual antibodies that respond to various antigens. This requires the development of antibodies specific to specific antigens, the development of fluorescent staining techniques for these antibodies, and the interpretation of fluorescent images of the samples produced by the antigen-antibody reaction, all of which are extremely complex processes for detecting specific antigens. Here, fluorescently tagged secondary antibodies are used to detect primary cells bound to the target protein, thereby identifying cell characteristics. Furthermore, the technique relies on qualitative assessment rather than quantitative analysis of the degree of cell differentiation. This technique has limited resolution and is not suitable for the highly sensitive characterization of stem cells.

Recently, in addition to the aforementioned immunohistochemical methods, several electrical sensing technologies have been reported for the detection of stem cell differentiation. Using electrode-based chips and impedance spectroscopy, the effect of chlorpyrifos (an insecticide) on adipocyte differentiation of hMSCs was investigated, and it was demonstrated that neural differentiation of hMSCs can be monitored using electrical cell-substrate impedance sensing.

However, methods for analyzing intracellular proteins, particularly during stem cell and cell differentiation, have not been presented. Furthermore, no label-free method has been proposed to provide differentiation-level numerical data. Therefore, a new technology capable of measuring the degree of protein differentiation across different stages of differentiation is needed.

In order to solve the problems of the above-mentioned prior art, the present invention aims to provide an effective method for analyzing intracellular biomaterials, especially proteins, without a labeling substance, and to provide a new method for monitoring the differentiation stage of stem cells by extracting parameters of the dielectrophoresis method.

In order to achieve the above object, the present invention provides a method for extracting parameters of dielectrophoresis (DEP) using complex permittivity spectra extracted from dielectric spectroscopy (DS).

In addition, the present invention provides a method for analyzing intracellular biomaterials using parameters of dielectrophoresis (DEP) extracted using complex permittivity spectra extracted from dielectric spectroscopy (DS).

In addition, the present invention provides a biosensor for analyzing intracellular biomaterials by extracting parameters of dielectrophoresis (DEP) using complex permittivity spectra extracted from dielectric spectroscopy (DS).

In addition, the present invention provides a method for monitoring a stage of differentiation from stem cells by analyzing stem cells or proteins differentiated from stem cells using a method of extracting parameters of dielectrophoresis (DEP) using complex permittivity spectra extracted from dielectric spectroscopy (DS).

The analytical method of the present invention utilizes dielectrophoresis to analyze intracellular biomaterials. It extracts parameters of dielectrophoresis using complex permittivity spectra from dielectric spectroscopy, thereby elucidating the relationship between dielectrophoresis and dielectric spectroscopy. By analyzing intracellular biomaterials, particularly proteins, using dielectrophoresis, it overcomes the limitations of existing labeling methods and enables real-time measurement, which is expected to find wide application in various fields.

Figure 1(a) shows an open-ended coaxial probe, and (b) is a setup for RF/microwave band measurements of aqueous biological materials using the open-ended coaxial probe.

Figure 2 is a measurement configuration diagram for the material-under-test (MUT).

Figures 3(a) and (b) show the real part permittivity spectra (a) and imaginary part permittivity spectra (b) of protein suspensions extracted from hMSCs and Saos-2 cells, respectively.

Figure 4 shows the imaginary part of the complex permittivity spectrum measured over the microwave frequency range.

Figure 5 compares (a) the real part and (b) the imaginary part of the dielectric spectra of the curve-fitted and measured results of protein suspensions extracted from hMSCs and Saos-2 cells.

Figure 6 is a photograph showing immunofluorescence staining for (a) CD 90; (b) CD 7; and (c) osteocalcin in hMSCs and Saos-2 cells. The scale bar represents 200 μm. Green staining indicates CD 90, CD 73, or osteocalcin, and blue staining indicates cell nuclei stained with 4',6-diamidino-2-phenylindole (DAPI).

Hereinafter, the present invention will be described in more detail through specific examples. The following examples illustrate preferred embodiments of the present invention, and the scope of the present invention is not limited to the matters described in the following examples.

In the present invention, “biomaterials” are biomolecules that exhibit a specific substrate, and can be interpreted as having the same meaning as target molecules or analytes. The biomolecules may be proteins, cells, viruses, nucleic acids, organic molecules, or inorganic molecules. In the case of the proteins, any biomaterials such as antigens, antibodies, substrate proteins, enzymes, and coenzymes may be used. In the case of the nucleic acids, they may be DNA (gDNA and cDNA), RNA, PNA, LNA, or a combination thereof. Nucleotides, which are the basic structural units of nucleic acid molecules, include not only natural nucleotides but also analogues in which sugar or base moieties are modified. The biomaterials may preferably include bacteria, viruses, molds, fungi, or a combination thereof.

In the present invention, “stem cells” are stem cells that have pluripotency to differentiate into all cells existing in a living body and also have proliferation ability, and include, but are not particularly limited to, embryonic stem (ES) cells, embryonic stem (ntES) cells derived from cloned embryos obtained by nuclear transfer, sperm stem cells (“GS cells”), embryonic germ cells (“EG cells”), artificially induced pluripotent stem (iPS) cells, and pluripotent cells (Muse cells) derived from cultured fibroblasts or bone marrow stem cells. Preferably, the pluripotent stem cells are ES cells, ntES cells, and iPS cells.

The present invention relates to a method for extracting parameters of dielectrophoresis (DEP) using a complex permittivity spectra extracted from dielectric spectroscopy (DS). In one aspect, the extracted complex permittivity spectra may be a method in which a numerical value is measured in the real part (ε′ _γ (ω)) or the imaginary part (ε″ _γ (ω)).

In one aspect, the extracted complex permittivity spectra may be a numerical value of Δε _β obtained from β-dispersion or a numerical value of a relaxation time (τ _β ) in β-dispersion.

In one aspect, the numerical value of Δε _β or the numerical value of the relaxation time (τ _β ) may be obtained by the following mathematical expressions 1, 2, 4 to 6.

[Mathematical Formula 1]

In the above mathematical expression 1, τ represents relaxation time, ω = 2π f , and f represents frequency.

[Equation 2]

In the above mathematical expression 2, ε′ _γ (ω) represents the relative electric energy storage capacity of the material, and ε″ _γ (ω) represents the attenuation when an electromagnetic wave passes through the material.

[Equation 4]

In the above mathematical expression 4, the unknown variables ε _γ , _∞ , Δε _β , Δε _γ , τ _β , and σ _dc are determined using a complex nonlinear least squares fit.

[Equation 5]

In the above mathematical expression 5, ε″ _rd (ω) and ε″ _rσ (ω) represent dielectric loss and conductor loss, respectively, ε″ _rσ (ω) is expressed as the absolute value of the last term of mathematical expression 4, and ε″ _r (ω) in the β-dispersion region is determined using the mathematical expression 6 below.

[Equation 6]

In one aspect, the parameter of the above dielectrophoresis (DEP) method may be a DEP cross-over frequency f _xo .

In addition, the present invention relates to a method for analyzing intracellular biomaterials using parameters of dielectrophoresis (DEP) extracted using complex permittivity spectra extracted from dielectric spectroscopy (DS).

In one aspect, the parameters of the dielectrophoresis (DEP) method may be at least one selected from the group consisting of a DEP cross-over frequency f _xo , a radius R of the biomaterial, d is a thickness of a shell of “bound water,” ε _bw is a relative permittivity of the “bound water,” and C _m is a capacitance of the membrane.

In one aspect, the above parameters may be obtained by the following mathematical equations 7 to 12.

[Equation 7]

In the above mathematical expression 7, σ _m is the electrical conductivity of the cell suspension medium.

[Equation 8]

In the above mathematical expression 8, C _m is the capacitance of the cell membrane, R is the radius of the spherical biomaterial, ε _eff is the effective permittivity of the spherical biomaterial, δ is the thickness of the cell membrane, ε _m is the average relative permittivity of the material forming the cell membrane structure, and φ _m is the membrane-folding factor of the cell membrane.

[Equation 9]

In the above mathematical expression 9, R represents the radius of the spherical biomaterial, η represents the viscosity of the suspension medium of the intracellular biomaterial, k _B represents the Boltzmann constant, T represents the absolute temperature, and τ _β represents the beta relaxation time in the beta-dispersion of the spherical biomaterial.

[Equation 10]

In the above mathematical expression 10, ε _bw represents the relative permittivity of the “bound water”, ε _e represents the effective permittivity of the spherical hydrated biomaterial, d represents the thickness of the shell of “bound water”, and ε _p represents the assumed relative permittivity of the spherical biomaterial.

[Equation 11]

In the above mathematical expression 11, ε _sus represents the relative permittivity of the suspension of intracellular biomaterial, ε _med represents the relative permittivity of the medium of the suspension, and p represents the volume fraction of the aqueous biomaterial.

[Equation 12]

In the above mathematical expression 12, d represents the thickness of the “bound water” shell, and R represents the radius of the spherical biomaterial.

In one aspect, the method may be one in which the cell is a stem cell or a cell differentiated from a stem cell.

In one aspect, the biomaterial may be at least one selected from the group consisting of proteins, cells, viruses, nucleic acids, organic molecules, and inorganic molecules, and is preferably a protein extracted from a cell. The method may be a protein extracted from a cell.

In addition, the present invention relates to a biosensor for analyzing intracellular proteins by a method of extracting parameters of dielectrophoresis (DEP) using complex permittivity spectra extracted from dielectric spectroscopy (DS).

In addition, the present invention relates to a method for monitoring a stage of differentiation from stem cells by analyzing stem cells or proteins differentiated from stem cells using a method of extracting parameters of dielectrophoresis (DEP) using complex permittivity spectra extracted from dielectric spectroscopy (DS).

Dielectric spectroscopy (DS) characterizes the rotation and relaxation of dipole molecules in solid, liquid, or gaseous states when an external electric field is applied to the material. By studying the motion of dipoles within the material-under-test (MUT), DS provides complex permittivity spectra of the material-under-test (MUT) over a broad frequency range. Since the foundational work of H. P. Schwan, who demonstrated electrical properties of tissue and cell dispersions over the frequency range of 10 Hz to 35 GHz, DS has been a primary technique for investigating the dielectric properties of biological materials. There are three main types of dispersion: α, β, and γ. α dispersion is difficult to measure due to electrode polarization but is typically found below 10 kHz. β dispersion is primarily due to interfacial polarization and is thought to be caused by the presence of cell plasma membranes. γ dispersion is due to the relaxation of water molecules. β- and γ-dispersion occur in the radio frequency (RF) band (MHz range) and microwave frequency band (GHz range). The dielectric properties of many human tissues, including blood, bone, brain, fat, and heart, were previously characterized by Gabriel et al. The tissues studied were measured over the frequency range of 10 Hz to 20 GHz. An open-ended coaxial probe, an impedance analyzer (10 Hz to 10 MHz), and two network analyzers (300 kHz to 3 GHz and 130 MHz to 20 GHz) were used to measure the human tissues.

Subsequently, DS was used to investigate genomic properties at the cellular level. Asami et al. simulated and measured mouse erythrocytes, lymphocytes, and plant protocytes at frequencies ranging from 10 kHz to 250 MHz. Dispersion was observed at approximately 5 MHz in mouse erythrocytes, while large dispersion was observed at approximately 1 MHz and small dispersion was observed at approximately 6 MHz in mouse lymphocytes.

The noninvasive, label-free, and real-time detection characteristics of DS technology make it ideal for clinical use. Genomic characteristics of normal and diseased breast tissue have been measured using open-ended coaxial probes in the frequency range of 1–20 GHz. However, the application of this DS technology to detect or analyze proteins extracted from cells has not been reported.

Dielectrophoresis (DEP) is a technology that separates or distinguishes heterogeneous cells or proteins by applying a non-uniform electric field to a heterogeneous biomaterial containing bioparticles such as cells, bacteria, viruses, proteins, or nucleic acids. Dielectrophoresis is caused by the interaction between a non-uniform electric field and the polarizability of the particles, and utilizes the force (DEP force) that spherical particles experience due to dielectrophoresis. Positive DEP refers to a force applied toward the stronger electric field, and negative DEP refers to a force that repels from the stronger field. Particles can be separated based on their electrical properties.

DS can provide complex permittivity spectra across a wide frequency range, but is limited below the MHz frequency band. Another label-free technique, dielectrophoresis (DEP), can be used to identify different cells at different stages of differentiation, but it has struggled to analyze the genetic properties of intracellular biomaterials.

The complex permittivity spectra of protein suspensions can be presented over a specific frequency range. The complex permittivity spectra clearly exhibit β- and γ-dispersion, and also exhibit three unique features in the real and imaginary parts, along with relaxation frequencies in the β-dispersion. These features can be used to distinguish hMSCs from Saos-2 cells. It has been demonstrated that this technique can replace immunohistochemistry, one of the most popular techniques.

In particular, the present invention can obtain dielectrophoretic parameters calculated from the complex permittivity spectra of protein suspensions extracted through dielectric spectroscopy, and calculate dielectrophoretic crossover frequencies. These calculations demonstrate that dielectrophoretic crossover frequencies of different proteins differ, demonstrating the utility of using dielectrophoresis to distinguish proteins.

Several methods for characterizing cell differentiation, including immunohistochemistry, Western blotting, flow cytometry, microarrays, immunocytochemistry, quantitative polymerase chain reaction (qPCR), and reverse transcription-polymerase chain reaction (RT-PCR), have been used to characterize cell differentiation. In particular, immunocytochemistry is a commonly used fluorescence method, where a fluorescently tagged secondary antibody detects primary cells bound to the target protein, thereby identifying cell characteristics. However, the resolution of these techniques is limited and unsuitable for highly sensitive characterization of stem cells.

In the present invention, we demonstrate that dielectrophoretic parameters calculated from complex permittivity spectra extracted from DS can be utilized to distinguish hMSCs from Saos-2 cells. The complex permittivity spectra ε _γ (ω) of protein suspensions extracted from hMSCs and Saos-2 cells provided by DS showed two unique dispersions, called β- and γ-dispersions. β-dispersion is caused by interfacial polarization between proteins and distilled water (DW). Interfacial polarization occurs between heterogeneous substances, and is characterized by a greater interfacial polarization as the difference in dielectric properties between the two substances increases.

The present invention is a method for analyzing proteins by implementing dielectrophoresis (DEP) using complex permittivity spectra extracted from dielectric spectroscopy (DS), thereby providing key parameters of dielectrophoresis. The present invention demonstrates that proteins from hMSCs and Saos-2 cells can be distinguished by DEP using the β-dispersion parameter of protein suspensions from DS.

A method for extracting a complex permittivity spectrum of a stem cell protein involves projecting a broadband (10 MHz to 43.5 GHz) electromagnetic wave onto a protein suspension containing proteins extracted from undifferentiated stem cells or stem cells at a specific differentiation stage to be analyzed and mixed with distilled water using an open-ended coaxial probe and a vector network analyzer (VNA), and extracting a complex permittivity spectrum from the measured reflection coefficient.

Typically, due to the limited bandwidth of VNAs, it is very difficult to extract the dispersion characteristics of dielectrics down to the kHz band. However, using mathematical models of cell or protein suspensions and curve-fitting techniques, the dispersion characteristics of dielectrics can be determined from the kHz band to the THz band. The mathematical model for the complex permittivity spectra of protein suspensions extracted from stem cells using a single-shell model is as follows.

DS provides the complex permittivity spectrum over the frequency band of interest. This spectrum can be expressed as a function of frequency as follows:

[Equation 2]

Here, ω = 2π f and f is frequency. ε′(ω) of a material represents the material's relative electrical energy storage capacity compared to air. ε″(ω) of a material represents the attenuation of electromagnetic waves as they pass through the material.

In this study, a comprehensive understanding of dielectric dissipation characteristics, particularly in the kHz range, is difficult due to the limited bandwidth of the VNA at low frequencies (10 MHz). However, mathematical models and curve-fitting techniques for cell or protein suspensions can be used to obtain a comprehensive picture of dielectric dissipation characteristics.

The genetic properties of cell suspensions can be analyzed using a "multi-stratified shell" model. The "multi-stratified shell" model can be expressed as follows:

[Equation 3]

Here, ε _γ , _∞ is the dielectric constant at f = ∞, Δε _k is the dielectric drop at the kth "unit" dispersion, and τ _k is the relaxation time at the kth "unit" dispersion. σ _dc and ε ₀ are the DC conductivity of the suspension medium and the electric permittivity of air, respectively. According to the "multilayer shell" model, the number of interfaces between dielectrics corresponds to the number of dielectric "unit" dispersions in the suspension.

When a protein suspension is mixed with water, one or two layers of water molecules (hydration shells) strongly associated with the protein surface are formed. This is called "bound water." As described above, β-dispersion and γ-dispersion were detected in the protein suspensions of hMSCs and Saos-2 cells. β-dispersion occurs because the "bound water" acts as a "single shell" for the protein. Therefore, with n = 1 in Equation 3 (referred to as the "single shell" model in the "multilayer" model), the dielectric properties of the protein solutions of hMSCs and Saos-2 cells can be modeled as follows:

[Equation 4]

The first variance (k = 1) and the second variance (k = 2) in Equation 3 correspond to the β-variance and the γ-variance in Equation 4, respectively. A complex nonlinear least squares fit can be used to determine the unknown parameters ε _γ , _∞ , Δε _β , Δε _γ , τ _β , and σ _dc in Equation 4. In this study, a commercial software tool (OriginPro 2021b, OriginLab Corporation, Northampton, MA, USA) using the Levenberg-Marquardt algorithm was used to determine the unknown parameters.

In the present invention, a dielectrophoresis method is implemented based on a complex permittivity spectrum extracted from the above-mentioned dielectric spectroscopy, and the dielectrophoresis cross-over frequency f _xo is a key parameter of DEP. For a spherical cell with a radius R , f _xo can be modeled by the following mathematical equation.

[Equation 7]

In the above mathematical expression 7, σ _m is the electrical conductivity of the suspension medium.

[Equation 8]

,

In the above mathematical expression 8, C _m is the capacitance of the cell membrane and is related to the effective permittivity ε _eff of a spherical cell with a radius R. δ is the thickness of the cell membrane, ε _m is the average relative permittivity of the materials forming the cell membrane structure, and φδ _m is the membrane-folding factor of the cell membrane, which is φδ _m = 1 for a perfectly smooth spherical cell.

While dielectrophoresis is typically applied to cell suspensions, modeling proteins as spherical particles allows dielectrophoresis to be applied to protein suspensions. The beta relaxation time (τ _β ) in the beta-dispersion of globular proteins can be derived from the mathematical model for the complex permittivity spectrum of protein suspensions in Equation 4. This study elucidates the correlation between the complex permittivity spectra of protein suspensions extracted using dielectric spectroscopy.

[Equation 9]

In the above mathematical expression 9, R represents the radius of the globular protein, η represents the viscosity of the suspension medium, k _B represents the Boltzmann constant, and T represents the absolute temperature. The radius of the globular protein can be obtained from mathematical expression 9 using τ _β obtained from dielectric spectroscopy.

[Equation 10]

Using the above mathematical equation (10), the relative permittivity of the “bound water” expressed as ε _bw can be obtained. ε _e is the effective permittivity of the spherical hydrated protein, d is the thickness of the shell of “bound water,” and ε _p is the assumed relative permittivity of the protein.

[Equation 11]

ε _e in the above mathematical expression 10 can be obtained from mathematical expression 11. ε _sus in mathematical expression 11 is the relative permittivity of the protein suspension, ε _med is the relative permittivity of the medium of the suspension, and p is the volume fraction of the aqueous protein.

ε _m and δ in Equation 8 are equivalent to ε _bw and d in Equation 10, respectively. It is known that the volume of hydrated protein is generally about 50% larger than that of unhydrated protein. Therefore, Equation 11 can be obtained.

[Equation 12]

The relative permittivity of the protein suspension, ε _sus , is extracted from dielectric spectroscopy, and ε _med is the relative permittivity of the medium of the suspension. In the embodiment of the present invention, since it is purified water, ε _med is extracted from dielectric spectroscopy, and together with this, the effective permittivity of the aqueous protein, ε _e , can be calculated using mathematical equation 11.

As shown in the examples below, dielectrophoretic parameters can be calculated from the complex permittivity spectra of protein suspensions extracted by dielectric spectroscopy, and dielectrophoretic crossover frequencies can be calculated. These calculations demonstrate that dielectrophoretic crossover frequencies of different proteins differ, making it possible to distinguish proteins using dielectrophoresis.

Additionally, different cell types were evaluated by immunohistochemistry using representative antibodies (a type of protein) including CD 90, CD 73, and osteocalcin. Altered genetic profiles using DS detection technology were attributed to the presence of other proteins, including CD 90 and CD 73.

Dielectrophoretic studies on proteins from hMSCs and Saos-2 cells demonstrated that the protein dielectrophoretic response could be obtained using the β-dispersion parameter from dielectric spectroscopy (DS) measurements, confirming that protein dielectrophoresis could distinguish proteins from hMSCs and Saos-2 cells.

In the present invention, stem cells, hMSCs, were used. hMSCs are multipotent cells capable of differentiating into various specialized tissue cells, including osteoblasts, chondrocytes, and adipocytes. Quality control of stem cell differentiation is crucial for clinical treatments that require precise cell surface markers and molecular expression. Stem cell markers such as CD 90, CD 73, and CD 105 are commonly used to isolate and identify stem cells. Currently, industry requires the characterization of hMSCs using CD 73, CD 90, CD 105, and CD 44. Positive hMSCs have been used for tissue regeneration. Cultured hMSCs express CD 105, CD 73, and CD 90, but do not express CD 31, CD 14, or other tissue-specific cell maturation markers. Saos-2 cells are widely used in studies of osteocyte differentiation, proliferation, and metabolism, and are known to be capable of osteogenic differentiation. They exhibit the most mature osteoblastic phenotype and are positive for alkaline phosphatase, osteocalcin, and collagens I and III.

The following examples illustrate the present invention without limiting its scope.

[Example 1]

<Preparation of samples>

Human mesenchymal stem cells (hMSCs; ATCC, Manassas, VA, USA) and human osteogenic sarcoma cells (Saos-2 cells; Korean Cell Line Bank, Seoul, Korea) were maintained in Dulbecco's modified Eagle's medium/Ham's F-12 50/50 (DMEM/F12; Gibco, New York, USA) supplemented with 10% fetal bovine serum (FBS; Gibco), 100 U/mL penicillin, and 100 μL streptomycin (Gibco) at 37°C in a humidified incubator with 5% CO2. The medium was changed periodically.

<Protein Separation and Measurement>

Sample Preparation for Dielectric Spectroscopy (DS) Cells were detached from the cell culture vessel by trypsinization, and the cell suspension was centrifuged at 1,500 rpm for 3 minutes. The supernatant was removed, and the cells were homogenized in distilled water (DW) using a homogenizer. To remove cell debris, the sample was transferred to a microtube and centrifuged at 10,000 rpm for 10 minutes. After centrifugation, the protein suspension of hMSCs or Saos-2 cells was collected in a new microtube. The total protein of the protein suspension was measured using the SMART BCA Protein Assay Reagent Kit (iNtRon Biotechnology, Inc., Seongnam, South Korea).

Measurement of DS

A 200 mm long open-ended coaxial probe is assembled with a 2.4 mm female coaxial connector and is shown in Figure 1(a). One end of the probe is connected to the VNA using a coaxial cable. The other end is shown immersed in aqueous biological material within a 2.0 mL microtube (see Figure 1(b)).

The measurement setup is shown in Figure 2. A coaxial cable assembled with 2.4 mm female coaxial adapters at both ends was connected to an open-ended coaxial probe and a VNA. The open-ended coaxial probe is a "Slim form probe" (Keysight Technologies, Santa Rosa, CA, USA).

The VNA (N5224B PNA Microwave Network Analyzer, Keysight Technologies) covers the frequency band from 10 MHz to 43.5 GHz.

After connecting the probe and VNA, the reference plane for the measurement was moved from the VNA test port to the tip of the probe via VNA calibration. This movement of the reference plane eliminated the interfaces between the probe and the coaxial cable, and between the coaxial cable and the 200 mm-long probe, from the measured one-port S-parameter (S11). The VNA calibration was performed in three steps. First, the probe was suspended freely in the middle, referred to as an "open circuit." Second, the probe was connected to a "slim form short" provided by the probe manufacturer, referred to as a "short circuit." Third, the probe was immersed in a 25 mL glass vial at room temperature, referred to as a "load." During the calibration process and the measurement of the material under test (MUT), the entire VNA frequency band (10 MHz to 43.5 GHz) was swept to measure S11 at 401 frequency data points on a logarithmic scale. S11 was averaged over 16 measurements.

The complex permittivity spectra were extracted from the measured S11 using software (Keysight N1500A Materials Measurement Suits-Coaxial probe method, pre-installed on the VNA). After the calibration process was completed, the spectra of distilled water (DW) from a second 25 mL vial were extracted to verify the calibration process. The complex permittivity spectra of distilled water (DW) are presented in the results below.

<Results of extracting complex permittivity spectra from DS>

The real (ε′ _γ ) and imaginary (ε″ _γ ) parts of the extracted complex permittivity spectra are plotted in Figs. 3(a) and 3(b), respectively. ε′ _γ of distilled water (DW) remained around 77–78, decreasing above 3 GHz due to γ-dispersion, as expected. The measured value was close to the known static permittivity of water, 78.35 ± 0.05. The maximum value of ε″ _γ was also observed at 19.6 GHz, which is the relaxation frequency (f _c , _γ ) of distilled water in γ-dispersion. In general, f _c of a dielectric can be converted to the relaxation time (τ ) using the relationship given in Equation 1:

[Mathematical Formula 1]

The τ _γ value of distilled water (DW) in γ-dispersion was 8.12 ps. This value is close to the previously reported value of 8.27 ± 0.02 ps. The ε′ _γ and ε″ _γ characteristics of distilled water (DW) in the frequency band from 10 MHz to 43.5 GHz confirmed that the experimental setup (Fig. 2) is effective in measuring and characterizing the complex permittivity spectra of aqueous biological materials.

The ε′ _γ spectra of the protein suspensions of hMSCs and Saos-2 cells were compared with each other and with the ε′ _γ of distilled water (DW) (Fig. 3(a)). Above 300 MHz, the ε′ _γ spectra of the three solutions were quite similar and difficult to distinguish. However, differences appeared below 300 MHz. The ε′ _γ values of the two types of proteins increased significantly compared to that of distilled water (DW) as the frequency decreased. At 30 MHz, the ε′ _γ of the protein suspension of hMSCs was 94.4, which was 7.4 and 15.7 higher than those of the protein suspension of Saos-2 cells and distilled water (DW), respectively. The increase in the ε′ _γ spectra of protein suspensions of hMSCs and Saos-2 cells was attributed to interfacial polarization, i.e., the Maxwell-Wagner effect. Interfacial polarization occurs when dielectric particles are placed in an ionic solution or electrolyte and an electric field is applied to the solution. This phenomenon, also known as β-dispersion, is commonly observed in biological materials in the RF range, typically below 1 GHz.

The ε″ _γ spectra of protein suspensions extracted from hMSCs and Saos-2 cells were compared with each other and with distilled water (DW) (Fig. 3(b)). Above 1 GHz, the ε″ _γ spectra of the three samples were similar and indistinguishable. However, below 1 GHz, the samples could be distinguished. The ε″ _γ values of the protein suspensions increased significantly with decreasing frequency compared to that of distilled water (DW). At 30 MHz, the ε″ _γ of the protein suspension from hMSCs was 28.7, which was 10.8 and 24.4 higher than those of the protein suspension from Saos-2 cells and distilled water (DW), respectively. The increase in the ε″ _γ spectra of the protein suspensions of hMSCs and Saos-2 cells was mainly due to the conductivity of the solutions, and the protein suspension of hMSCs had a higher conductivity than that of Saos-2 cells.

The ε′ _γ and ε″ _γ values of the three samples were compared at specific frequencies (Table 1). The protein suspensions of hMSCs and Saos-2 cells showed different ε′ _γ and ε″ _γ below 300 MHz and below 1 GHz, respectively, confirming the possibility of differentiating stem cells based on complex permittivity spectra. Table 1 below shows the ε′ _γ and ε″ _γ measurements of the three materials at specific frequencies.

In addition to β-dispersion, γ-dispersion was observed among the three samples. γ-dispersion is derived from the directional polarization of water molecules in the target material and was measured in the microwave frequency range. Directional polarization occurs when an electric field is applied to a dielectric material consisting of permanent dipoles, such as water. The ε″ _γ spectra of the three samples (Figure 3(b)) were focused in the microwave frequency range (Figure 4). The relaxation frequencies of the protein suspensions of hMSCs and Saos-2 cells were different from those of distilled water (DW) (Table 2). Table 2 below shows the relaxation frequencies (f _c , _γ) and relaxation times in γ-dispersion among the three materials. In the presence of stem cell proteins, the f _c and _γ of distilled water in γ-dispersion were slightly affected, shifting to lower frequencies by 2.3 and 1.9 GHz in the protein suspensions of hMSCs and Saos-2 cells, respectively. In γ-dispersion, the f _c , _γ values of stem cell protein solutions can be distinguished from those of distilled water (DW), but the f _c , _γ values of protein suspensions of hMSCs and Saos-2 cells are similar. Consequently, the f _c , _γ values of stem cells in γ-dispersion cannot be used to distinguish stem cells, whereas the f _c , _β values in β-dispersion can be used to distinguish stem cells.

DS provides the complex permittivity spectrum over the frequency band of interest. This spectrum can be expressed as a function of frequency as follows:

[Equation 2]

Here, ω = 2π f and f is frequency. ε′(ω) of a material represents the material's relative electrical energy storage capacity compared to air. ε″(ω) of a material represents the attenuation of electromagnetic waves as they pass through the material.

In this study, a comprehensive understanding of dielectric dissipation characteristics, particularly in the kHz range, is difficult due to the limited bandwidth of the VNA at low frequencies (10 MHz). However, mathematical models and curve-fitting techniques for cell or protein suspensions can be used to obtain a comprehensive picture of dielectric dissipation characteristics.

The genetic properties of cell suspensions can be analyzed using a "multi-stratified shell" model. The "multi-stratified shell" model can be expressed as follows:

[Equation 3]

Here, ε _γ , _∞ is the dielectric constant at f = ∞, Δε _k is the dielectric drop at the kth "unit" dispersion, and τ _k is the relaxation time at the kth "unit" dispersion. σ _dc and ε ₀ are the DC conductivity of the suspension and the electric permittivity of air, respectively. According to the "multilayer shell" model, the number of interfaces between dielectrics corresponds to the number of dielectric "unit" dispersions in the suspension.

When a protein suspension is mixed with water, one or two layers of water molecules (hydration shells) strongly associated with the protein surface are formed. This is called "bound water." As described above, β-dispersion and γ-dispersion were detected in the protein suspensions of hMSCs and Saos-2 cells. β-dispersion occurs because the "bound water" acts as a "single shell" for the protein. Therefore, with n = 1 in Equation (3) (referred to as the "single shell" model in the "multilayer" model), the dielectric properties of the protein solutions of hMSCs and Saos-2 cells can be modeled as follows:

[Equation 4]

The first variance (k = 1) and the second variance (k = 2) in Equation 3 correspond to the β-variance and the γ-variance in Equation 4, respectively. A complex nonlinear least squares fit can be used to determine the unknown parameters ε _γ , _∞ , Δε _β , Δε _γ , τ _β , and σ _dc in Equation 4. In this study, a commercial software tool (OriginPro 2021b, OriginLab Corporation, Northampton, MA, USA) using the Levenberg-Marquardt algorithm was used to determine the unknown parameters.

The "single-shell" model parameters were compared between protein solutions of hMSCs and Saos-2 cells (Table 3). The curve-fitted complex permittivity spectra (ε ^* _γ (ω)) were compared with the measured complex permittivity spectra (ε _γ (ω)) over an extended frequency range from 100 kHz to 1 THz (Fig. 5). It is possible to comprehensively represent the dielectric dispersion of protein suspensions of hMSCs and Saos-2 cells over an extended frequency range (100 kHz to 1 THz).

The curve-fitted ε′ _γ spectra of ε ^* _γ (ω) were in good agreement with the measured ε′ _γ (ω) (Fig. 5(a)). The "single-shell" model (Eq. 4) predicts the difference between the protein solutions of hMSCs and Saos-2 cells. For example, at 100 kHz, the difference is 57.134. However, when f = ∞, the difference between ε _γ , _∞ is negligible (Table 3).

As mentioned earlier, ε″(ω) represents the attenuation of electromagnetic waves passing through a material. The greater the attenuation of a material, the greater the loss in that material. In fact, the ε″(ω) spectrum can be expressed as follows:

[Equation 5]

In the above mathematical expression 5, ε″ _rd (ω) and ε″ _rσ (ω) represent dielectric loss and conductor loss, respectively. ε″ _rσ (ω) can be expressed as the absolute value of the last term of mathematical expression 4. ε″ _r (ω) in the β-dispersion region is mainly expressed as the last term of mathematical expression 5, Since it is dominated by , the influence of that term in ε″(ω) must be removed to expose the actual β-dispersion characteristics. For this, we use Equation 6:

[Equation 6]

Using Equation 6, the curve-fitted and measured ε″ _rσ (ω) values were calculated and compared with each other (Fig. 5(b)). The σ _dc values of the protein solutions of hMSCs and Saos-2 cells listed in Table 3 were used for these calculations. Although there was a slight deviation between the curve-fitted and measured ε″ _rd (ω) below 100 MHz, the curve-fitted ε″ _rd (ω) tended to follow the measured ε″ _rd (ω). There was a difference in the ε″ rd (ω) values at f _c , _β of the protein solutions of hMSCs and Saos-2 cells (Table 3). The difference in the ε″ _rd (ω) values at f _{c ,} β of the protein solutions of hMSCs and Saos-2 cells was 28.16. The difference in f _{c , β} between the protein solutions of hMSCs and Saos-2 cells was 723 kHz. However, in the γ-dispersion region, the ε″ _rd (ω) spectrum was hardly influenced by .

<Results of extracting DEP parameters from complex permittivity spectra extracted from DS>

The dielectrophoretic crossover frequency, f _xo , is a key parameter of DEP. For spherical cells with a radius R , f _xo can be modeled by the following mathematical equation:

[Equation 7]

In the above mathematical expression 7, σ _m is the electrical conductivity of the suspension medium.

[Equation 8]

In the above mathematical expression 8, C _m is the capacitance of the cell membrane and is related to the effective permittivity ε _eff of a spherical cell with a radius R. δ is the thickness of the cell membrane, ε _m is the average relative permittivity of the materials forming the cell membrane structure, and φδ _m is the membrane-folding factor of the cell membrane, which is φδ _m = 1 for a perfectly smooth spherical cell.

While dielectrophoresis is typically applied to cell suspensions, modeling proteins as spherical particles allows dielectrophoresis to be applied to protein suspensions. The beta relaxation time (τ _β ) of spherical proteins in the beta-dispersion can be derived from the mathematical model for the complex permittivity spectrum of protein suspensions in Equation 4:

[Equation 9]

In the above mathematical expression 9, R represents the radius of the globular protein, η represents the viscosity of the suspension medium, k _B represents the Boltzmann constant, and T represents the absolute temperature. The radius of the globular protein can be obtained from mathematical expression 9 using τ _β obtained from dielectric spectroscopy.

[Equation 10]

Using the above mathematical equation (10), the relative permittivity of the “bound water” expressed as ε _bw can be obtained. ε _e is the effective permittivity of the spherical hydrated protein, d is the thickness of the shell of “bound water,” and ε _p is the assumed relative permittivity of the protein.

[Equation 11]

ε _e in the above mathematical expression 10 can be obtained from mathematical expression 11. ε _sus in mathematical expression 11 is the relative permittivity of the protein suspension, ε _med is the relative permittivity of the medium of the suspension, and p is the volume fraction of the aqueous protein.

ε _m and δ in Equation 8 are equivalent to ε _bw and d in Equation 10, respectively. Typically, the volume of a hydrated protein is approximately 50% larger than that of an unhydrated protein. Therefore, Equation 12 is as follows.

[Equation 12]

The relative permittivity of the protein suspension, ε _sus , is extracted from dielectric spectroscopy, and ε _med is the relative permittivity of the medium of the suspension. In the embodiment of the present invention, since it is purified water, ε _med is extracted from dielectric spectroscopy, and together with this, the effective permittivity of the aqueous protein, ε _e , can be calculated using mathematical equation 11.

The dielectrophoretic crossover frequency can be calculated using Equations 7 to 12. As an example, the dielectrophoretic crossover frequency was calculated in a protein suspension containing proteins extracted from hMSCs and Saos-2 cells and purified water. In this calculation process, the electrical conductivity of purified water, σ _m = 0.032 S/m, and viscosity,  η = 0.9X10 ^-3 Pa s, were used, and the calculation was performed at room temperature ( T = 298 K). In Equation 11, p = 0.411 was used, which is the volume fraction of aqueous protein calculated from 0.3 g of water per 1 g of protein. In Equation 10, ε _p represents the assumed relative permittivity of the protein, and was assumed as (ε _e -ε _sus ) in this calculation process. The calculation results are shown in Table 4. Table 4 compares the dielectrophoresis parameters for each protein extracted from hMSCs and Saos-2 cells.

As shown in Table 4 above, dielectrophoretic parameters can be obtained from the complex permittivity spectra of protein suspensions extracted from dielectric spectroscopy, and dielectrophoretic crossover frequencies can be calculated. These calculations demonstrate that dielectrophoretic crossover frequencies of different proteins differ, demonstrating the potential for protein differentiation using dielectrophoresis.

[Comparative example]

To compare with the above DS measurement method, we intend to measure immunohistochemistry.

For immunofluorescence, cells were cultured at room temperature on cell culture slides (SPL Life Sciences Co., Ltd., Pocheon-si, Gyeonggi-do, Republic of Korea) after fixation with 4% (by volume) paraformaldehyde. After fixation, cells were stained using primary antibodies against CD 90 (Santa Cruz Biotechnology, Dallas, TX, USA), CD 73 (Santa Cruz Biotechnology), or osteocalsin (Santa Cruz Biotechnology). CD 90 and CD 73 are expressed in hMSCs and are used as phenotypic markers of mesenchymal stem cells. Osteocalcin is expressed in Saos-2 cells and is used as a marker of osteoblasts and bone. Cells were stained using fluorescein 5-isothiocyanate (FITC)-conjugated anti-mouse IgG secondary antibody (Jackson ImmunoResearch Laboratories Inc., West Baltimore Pike, PA, USA). Vectashield® mounting medium containing 4',6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Newark, CA, USA) was used to stain cell nuclei. Stained samples were analyzed by fluorescence microscopy.

<Measurement Results>

Immunohistochemistry (IHC) detects antigens or proteins in cells or tissue sections through the binding of specific antibodies to specific antigens. IHC is widely used in research and clinical laboratories to visualize the distribution and location of specific cellular components, such as proteins and other large molecules, within cells and tissues. Different cells express different proteins that can be identified through antigen-antibody interactions using IHC. In this study, hMSCs and Saos-2 cells were characterized by immunofluorescence using CD 90, CD 73, and osteocalcin (Figures 6(a) and (b)). hMSCs expressed CD 90 and CD 73, markers of mesenchymal stem cells. However, Saos-2 cells did not express any markers. Merging the marker and DNA images revealed that most hMSCs were positive for CD 90 and CD 73, confirming that hMSCs possessed the expected stem cell properties. In contrast, Saos-2 cells expressed osteocalcin, a marker specific for osteoblasts (Fig. 6(c)). Merging the marker and DNA images revealed that all Saos-2 cells were stained with osteocalcin, indicating that their osteoblastic characteristics were maintained.

Immunofluorescence staining for osteocalcin. Scale bar represents 200 μm. Green staining indicates CD 90, CD 73, or osteocalcin, and blue staining indicates cell nuclei by 4',6-diamidino-2-phenylindole 4 (DAPI) staining.

The above description is merely an example of the technical idea of the present invention, and those skilled in the art will appreciate that various modifications and variations can be made without departing from the essential characteristics of the present invention.

Accordingly, the embodiments disclosed in the present invention are intended to illustrate, rather than limit, the technical concept of the present invention, and the scope of the technical concept of the present invention is not limited by these embodiments. The scope of protection of the present invention should be interpreted by the following claims, and all technical concepts within the scope equivalent thereto should be construed as being included within the scope of the present invention.

Claims (10)

A method for extracting parameters of dielectrophoresis (DEP) using complex permittivity spectra extracted from dielectric spectroscopy (DS). In the first paragraph, the extracted complex permittivity spectra is a method in which the numerical value is measured in the real part (ε′ _γ (ω)) or the imaginary part (ε″ _γ (ω)). In the second paragraph, the extracted complex permittivity spectra is a numerical value of Δε _β obtained from β-dispersion or a numerical value of relaxation time (τ _β ) in β-dispersion, and the numerical value of Δε _β or the numerical value of relaxation time (τ _β ) is obtained by the following mathematical expressions 1, 2, 4 to 6: [Mathematical Formula 1] In the above mathematical expression 1, τ represents relaxation time, ω = 2π f , and f represents frequency. [Equation 2] In the above mathematical expression 2, ε′ _γ (ω) represents the relative electric energy storage capacity of the material, and ε″ _γ (ω) represents the attenuation when an electromagnetic wave passes through the material. [Equation 4] In the above mathematical expression 4, the unknown variables ε _γ , _∞ , Δε _β , Δε _γ , τ _β , and σ _dc are determined using a complex nonlinear least squares fit. [Equation 5] In the above mathematical expression 5, ε″ _rd (ω) and ε″ _rσ (ω) represent dielectric loss and conductor loss, respectively, ε″ _rσ (ω) is expressed as the absolute value of the last term of mathematical expression 4, and ε″ _r (ω) in the β-dispersion region is determined using the mathematical expression 6 below. [Equation 6] In the first paragraph, the parameter of the dielectrophoresis (DEP) method is a dielectrophoresis cross-over frequency (DEP cross-over frequency) f _xo . A method for analyzing intracellular biomaterials using parameters of dielectrophoresis (DEP) extracted from complex permittivity spectra extracted from dielectric spectroscopy (DS). In the fifth paragraph, the parameters of the dielectrophoresis (DEP) are at least one selected from the group consisting of a DEP cross-over frequency f _xo , a radius R of the biomaterial, d is a thickness of a shell of “bound water,” ε _bw is a relative permittivity of the “bound water,” and C _m is a capacitance of a cell membrane, and the parameters are obtained by the following mathematical equations 7 to 12: [Equation 7] In the above mathematical expression 7, σ _m is the electrical conductivity of the cell suspension medium. [Equation 8] In the above mathematical expression 8, C _m is the capacitance of the cell membrane, R is the radius of the spherical biomaterial, ε _eff is the effective permittivity of the spherical biomaterial, δ is the thickness of the cell membrane, ε _m is the average relative permittivity of the material forming the cell membrane structure, and φδ _m is the membrane-folding factor of the cell membrane. [Equation 9] In the above mathematical expression 9, R represents the radius of the spherical biomaterial, η represents the viscosity of the suspension medium of the intracellular biomaterial, k _B represents the Boltzmann constant, T represents the absolute temperature, and τ _β represents the beta relaxation time in the beta-dispersion of the spherical biomaterial. [Equation 10] In the above mathematical expression 10, ε _bw represents the relative permittivity of the “bound water”, ε _e represents the effective permittivity of the spherical hydrated biomaterial, d represents the thickness of the shell of “bound water”, and ε _p represents the assumed relative permittivity of the spherical biomaterial. [Equation 11] In the above mathematical expression 11, ε _sus represents the relative permittivity of the suspension of intracellular biomaterial, ε _med represents the relative permittivity of the medium of the suspension, and p represents the volume fraction of the aqueous biomaterial. [Equation 12] In the above mathematical expression 12, d represents the thickness of the “bound water” shell, and R represents the radius of the spherical biomaterial. A method according to claim 5, wherein the cell is a stem cell or a cell differentiated from a stem cell. A method according to claim 5, wherein the biomaterial is a protein extracted from a cell. A biosensor for analyzing intracellular proteins by a method according to any one of claims 5 to 8. A method for monitoring a stage of differentiation from stem cells by analyzing stem cells or proteins differentiated from stem cells by a method according to any one of claims 5 to 8.