KR101177643B1 - Diagnostic method of aging and aging related diseases using isoelectric focusing electrophoresis and biochip and diagnostic kit - Google Patents

Abstract

PURPOSE: A method and a kit for diagnosing aging and aging related diseases using isoelectric focusing and a biochip are provided to predict risks of cardiovascular diseases, cerebrovascular diseases and metabolic diseases by simply analyzing transformed HDL(High Density Lipoprotein). CONSTITUTION: HDL is separated from plasma and refined. The transformation of the HDL is analyzed using an isoelectric focusing analysis and a micro-channel biochip or a lab-on-a-chip analysis. The HDL is HDL2 or HDL3. The isoelectric focusing analyze the transformation of the HDL using a microfluidic channel or computer simulation. The microfluidic channel is composed of polydimethylsiloxane.

Description

Diagnostic method of aging and aging related diseases using isoelectric focusing electrophoresis and biochip and diagnostic kit

The present invention is directed to native polyacrylamide gel electrophoresis, SDS-PAGE or isoelectric point electrophoresis, which allows for easier analysis and visualization of modified HDL detection. Selected electrophoresis assays, in particular isoelectric point electrophoresis analysis, and microchannel biochip or lab-on-a-chip assays, are directed to methods of diagnosing aging and age-related diseases.

Aging is a chronic process following the outbreak of various physical and biochemical modifications that affect high-density lipoprotein (HDL) structure and function, including proinflammatory cytokines, acute phase reactants, and C-reactive proteins (C). Accompanied by increased circulation of Reactive Protein (CRP).

Recently, atherosclerosis that increases with aging is associated with failure to upregulate antioxidant genes, and oxidative stress is known to play an important role in the development of aging vasculature (Collins et al . Cire. Res. 104, e42-). e54, 2008). However, there are no reports on the changes in the functional and structural properties of apolipoprotein (apoA-I), which is a major component of HDL and HDL.

Coronary heart disease (CHD), such as atherosclerosis, is one of the leading causes of death in Western society and is more prevalent in adults over 50 because age is an important risk factor for CHD. Plasma levels of HDL-C are known to be inversely related to the risk of CHD, and a variety of factors contribute to the invention of CHD, such as obesity, hypertriglyceridemia, insulin resistance, smoking and lack of exercise.

In addition, modification of apolipoproteins such as protein cleavage, oxidation, nitriding and chloride can lead to the production of dysfunctional apolipoproteins and loss of high-density lipoprotein (HDL), which can accelerate atherosclerosis. HDL is known to have anti-inflammatory and antioxidant properties as well as prevention of coronary artery disease. Apolipoproteins exhibit antioxidant activity by removing lipid peroxides from LDL and anti-inflammatory activity by inhibiting the expression of adherent molecules.

Currently, although biomarkers used for aging are known about telomere length, there is still a need to develop a method for effectively diagnosing aging and aging-related diseases in a general and non-invasive manner, which is related to aging and aging. Very important for the treatment and prevention of diseases.

Accordingly, the present inventors have made efforts to develop a method for early diagnosis of aging and aging-related diseases. As a result, the modified HDL detection can be easily analyzed and visualized using isoelectric point electrophoresis and lab-on-a-chip analysis. Revealed to complete the present invention.

An object of the present invention is to provide a diagnostic method that can be easily visualized while analyzing a modified HDL detection known as an early diagnostic indicator of aging and aging-related diseases.

In order to achieve the above object, the present invention comprises the steps of isolating and purifying high-density lipoprotein (HDL) from blood isolated from the human body; And electrophoretic analysis selected from native polyacrylamide gel electrophoresis, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or isoelectric focusing (IEF), and microchannel biochips Or it provides a diagnostic method for aging and aging-related diseases comprising the step of analyzing the modification of the high-density lipoprotein (HDL) through lab-on-a-chip analysis.

The high-density lipoprotein (HDL) is preferably HDL ₂ or HDL ₃ . The HDL can be isolated and purified from human plasma using any one method selected from the group consisting of ultracentrifugation, column chromatography, and organic solvent extraction.

The isoelectric focusing (IEF) can analyze the deformation of high-density lipoprotein (HDL) through either microfluidic channel or computer simulation.

More specifically, the isoelectric focusing (IEF) can analyze the change in any one or two or more of the electrophoretic or isoelectric point of the HDL through any one of the microfluidic channel or computer simulation.

In particular, according to one embodiment of the present invention, HDL ₃ (E-HDL ₃ ) obtained from the elderly layer shows faster electrophoresis in the microfluidic channel or simulation model, unlike HDL ₃ (Y-HDL ₃ ) obtained from the young layer. , Higher isoelectric point, that is, basic isoelectric point approximately pI 8.1. These properties of E-HDL ₃ were similar to those of glycated apoA-I (gA-I).

The microfluidic channel may be made of polydimethysiloxane (PDMS).

The aging-related disease may be any one selected from the group consisting of cardiovascular diseases such as hypertension and arteriosclerosis, cerebrovascular diseases such as dementia, and metabolic diseases such as diabetes and obesity.

In addition, the present invention comprises a sample input unit for providing high-density lipoprotein (HDL) purified from the plasma separated from the human body; And it provides a kit for diagnosing aging and aging-related diseases comprising a microfluidic channel portion for analyzing the modification of the high-density lipoprotein (HDL) through isoelectric focusing (IEF).

According to the present invention, the modified HDL detection, which is known as an early diagnosis index of aging and aging-related diseases, can be easily analyzed and visualized. In addition to predicting or diagnosing the risk of occurrence of metabolic diseases such as and the like, a diagnostic kit for these diseases can be developed.

1 shows the results of fluorescence intensity analysis of the degree of glycation of HDL (A: glycosylation comparison between nA-I-rHDL and gA-I-rHDL, B: glycosylation comparison between Y-HDL ₃ and E-HDL ₃ ). ,
FIG. 2 shows electrophoretic patterns (A: 8-25% polyacrylamide) at nA-I-rHDL (N), gA-I-rHDL (G), Y-HDL ₃ (Y) and E-HDL ₃ (E). Amide concentration gradient gel electrophoresis, B: 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis),
Figure 3 Electrophoresis on IEF gels (pH 3-10) in nA-I-rHDL (N), gA-I-rHDL (G), Y-HDL ₃ (Y) and E-HDL ₃ (E). Is a pattern,
Figure 4 shows the normalized concentration behavior of amphoteric material in the IEF process (a: 50 seconds, b: 200 seconds, c: 400 seconds, d: 1000 seconds),
FIG. 5 shows changes in pH distribution under a potential of 50 V using 31 amphoteric materials during the amphoteric material-based IEF process.
FIG. 6 shows fluorescence images of shifted patterns of nA-I-rHDL (N) and gA-I-rHDL (G) in microfluidic channels in the presence of amphoteric substances (pH 3-10) (A : Moving pattern image at 5 minutes, B: moving pattern image at 25 minutes, C: moving distance determined from the simulation model, D: pI range determined from the simulation model),
FIG. 7 shows fluorescence images of shifted patterns of Y-HDL ₃ (Y) and E-HDL ₃ (E) in microfluidic channels in the presence of amphoteric substances (pH 3-10) (A: 5 Moving pattern image in minutes, B: moving pattern image at 25 minutes, C: moving distance determined from the simulation model, D: pI range determined from the simulation model).

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited by these examples.

≪ Example 1 >

1.Isolation of HDL from Elderly and Youth Serum

Density is adjusted by addition of NaCl and NaBr according to previously known methods ( Methods Enzymol. , 1986, 128, 223-246) and standard protocols ( Int. J. Mol. Med. , 2011 in press) by prior studies . VLDL (d <1.019 g / ml), LDL (1.019 <d <1.063 g / ml), HDL ₂ (1.063 <d <1.125 g / ml) and HDL ₃ (1.125 <d <1.225 g / ml) Using serial ultracentrifugation (Himac CP-1, Hitachi) for plasma of elderly men (mean age, 71 ± 4 years, n = 26) and young men (mean age, 22 ± 2 years, n = 18) From. Samples were centrifuged for 24 hours at 100,000 g, 10 ℃ using a centrifuge (Himac CP-90α). After dialysis against PBS, each lipoprotein fraction was diluted equally and collected.

2. Glycated apoA-I

Lipid-free apolipoprotein AI (10 mg / ml) previously purified from human plasma was treated with 250 mM D-fructose [in 200 mM potassium phosphate / 0.02% sodium azide buffer (pH) for 90 hours at 37 ° C. under air containing 5% CO _2. 7.4)]. The extent of the final glycosylation reaction was determined by measuring the fluorescence intensity of the resulting fluorescent material at 370 nm (excitation) and 440 nm (emission).

3. rHDL Preparation and Fluorescent Labeling

The preparation of recombinant HDL (rHDL) containing unglycosylated apoA-I (nA-I) or glycated apoprotein AI (gA-I) is described in our previous article ( Mol). 95: using phosphocholine (POP), cholesterol, apolipoprotein AI and sodium cholate according to the methods described in Cells. RHDL with phospholipid bilayer was synthesized via sodium cholate dialysis at a molar ratio of 5: 1: 150.

Fluorescent cholesterol derivative 22- (N-7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino-23,24-bisnor-5-collen-3-ol {22- (N -7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino-23,24-bisnor-5-cholen-3-ol [NBD-cholesterol, Molecular Probe N-1148, 70 g of NBDcholesterol / 1 mg of apoA-I]} was added to rHDL for fluorescent labeling.

4. Electrophoresis Analysis

Free using HDL ₃ (E-HDL ₃₎ and HDL ₃ (E-HDL ₃₎ the same amount the Pharmacia Phast System (AmershamPharmacia, Uppsala, Sweden) obtained from young people obtained from older people in the free lipid status, and lipid binding rHDL state Electrophoresis was performed on casted 8% to 25% polyacrylamide gradient gels (GE Healthcare, Uppsala, Sweden). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to the method published by Laemmli et al. (Nature, 1970, 227, 680-685). Isoelectric focusing (IEF) was performed on pre-cast gels with PhastGel IEF 3-9 (17-0543-01; GE Healthcare) using PhastSystem (GE Healthcare, Uppsala, Sweden). At this time, the protein band was visualized by staining PhastGel Blue R (17-0518-01, GE Healthcare).

5. Glycation degree analysis (fluorescence analysis)

In order to compare the degree of glycation between the experimental groups, the content of the advanced glycation end-product (AGE) in each lipoprotein was determined by measuring the fluorescence intensity at 370 nm (excitation) and 440 nm (emission). The LS55 fluorescence spectrometer (Perkin-Elmer, Norwalk, CT, USA) with WinLab software package 4.00 (Perkin-Elmer) and 1-cm path length suprasil quartz cuvettes (Fisher Scientific, Pittsburg, PA, USA) Used.

6. Fabrication of PDMS Microchips

The procedure used for the manufacture of PDMS microchips for IEF was based on soft lithography techniques which are widely reported in the literature (Proc. Natl. Acad. Sci. U.S.A., 1984, 2684-2688). That is, for pattern duplication, a negative photoresist was formed on a silicon wafer substrate using the SU-8 2000 series. The spin rate is controlled and programmed according to MICROCHEM's process guidelines for the desired channel thickness and coating quality. For PDMS channel preparation, PDMS pre-polymer and curing agent (Sylgard 184; Dow Corning Inc., Midland, MI, USA) were uniformly mixed in a ratio of 10: 1 and 0.001 Torr (to remove bubbles in the PDMS mixture) torr) for 2 hours. The PDMS mixture formed on the pattern replica was cured in an oven at 80 ° C. for 4-8 hours. After this curing process, the cured PDMS was carefully removed from the silicon wafer substrate. The PDMS channel has holes as reservoirs at the ends of the channels. PDMS with the channel pattern and slide glass for the bottom of the channel was treated with a plasma cleaner for 20-40 seconds. These microchannels are 20 cm long, 250 μm wide and 25 μm deep.

7. IEF model based on ampholyte

A mathematical model of the IEF has been presented in many prior documents (Electrophoresis, 2007, 28, 1138-1145; Electrophoresis, 2007, 28, 572-586). An IEF model based on ampholyte requires three simultaneous solutions to mass conservation equations, charge conservation equations, and electrical neutral equations for amphoteric molecules. The mass conservation equation without the velocity of the bulk flow for each amphoteric element is given by

&Quot; (1) &quot;

은 전하 보존 방정식에 의해 사용된다.Where C _i , D _i and <μ _i > represent the concentration, diffusion coefficient and effective mobility for component I. Electric field

Is used by the charge conservation equation.

&Quot; (2) &quot;

Where ω _i is the absolute mobility, N is the total number of components, including H ₃ 0 ⁺ (hydronium) and hydroxyl, and <z _i > is the effective valence. The concentration of hydronium ions (H ₃ 0 ⁺ ) can be obtained from the electrical neutral equation below.

&Quot; (3) &quot;

Where C _H is the concentration of hydronium, K _w is the reaction constant of water, N is the number of all components that are proteins as well as amphoteric, except hydronium (C _H ) and hydroxyl (C _OH ).

8. Assumptions of Simulation

Joule heating is ignored because a relatively small electric field is used for IEF separation, and the electrokinetic flow induced by the electric field because the channel surface is coated with methylcellulose to suppress the flow of electric penetration. Is not considered.

9. Boundary conditions

으로 다뤄지고, 정 전위 (constant potential)가 전극 (끝단) 웰 (wells)에서 유지되도록 다뤄진다. 이 연구에서, 양극 웰 (wells)에서의 전위 (electric potential)는 50V이고, 음극에서의 전위는 0 (φ_L = 0)으로 유지된다.When solving the mass conservation equation, the net flux through the channel wall and the end well (reservior) is set to zero. Electrical potential is an insulation boundary condition

It is treated to maintain a constant potential in the electrode (end) wells. In this study, the electrical potential at the anode wells is 50V and the potential at the cathode is kept at 0 (φ _L = 0).

10. numerical scheme

및

으로 설정되었다. 아래첨자 n 및 n-1은 현재 및 이전 단계를 나타낸다. 전기적 중성 방정식의 경우, 히드로늄 이온의 수렴값을 얻기 위해 Newton-Raphson 방법이 이용되었다. 마이크로채널(길이 20cm × 너비 250㎛ × 높이 25㎛)에서 3천개의 그리드 포인트들이 이용되었다. 이러한 시뮬레이션은 XP 기반의 삼성 컴퓨터(인텔(R) 코어(TM)2 쿼드 CPU 2.5GHz, 1.96G RAM)에서 수행되었다. 안정한 31개의 양쪽성 기반 pH 구배를 만드는데 걸린 계산 시간은 약 3일이었다.Analytical algebraic equations were obtained at each grid point for mass conservation and modified charge conservation equations. For simplicity and stabilization using a computer, structured grids were considered in this simulation. A tridiagonal matrix algorithm (TDMA) was used to solve the analytical algebraic equations along the grid lines, and line-by-line iterations were applied until the results converged in the computer domain. The convergence criteria for the mass conservation and charge conservation equations are

And

Was set. Subscripts n and n-1 represent the current and previous steps. In the case of the electrical neutral equation, the Newton-Raphson method was used to obtain the convergence value of the hydronium ions. Three thousand grid points were used in the microchannel (20 cm long x 250 m wide x 25 m high). These simulations were performed on an XP-based Samsung computer (Intel (R) Core (TM) 2 Quad CPU 2.5GHz, 1.96G RAM). The calculation time for making 31 stable amphoteric based pH gradients was about 3 days.

11. Experimental results

1) AGE Fluorescence

As shown in FIG. 1A, the fluorescence intensity of gA-I was increased by about 10 times compared to that of nA-I. And, as shown in Figure 1B, E-HDL ₃ was 15 times higher than the Y-HDL ₃ fluorescence intensity was confirmed that due to the aging caused a lot of saccharification.

2) Electric mobility analysis of HDL

nA-I-rHDL exhibited a particle size of 98 to 100 microns, while gA-I-rHDL exhibited approximately 93 microns. As shown in FIG. 2A, from PAGGE analysis, E-HDL ₃ formed more aggregates than Y-HDL ₃ , showing different mobility patterns. In addition, Y-HDL ₃ showed three major bands (91, 74 and 70 Hz), while E-HDL ₃ showed two major bands around 106 and 70 Hz. As shown in FIG. 2B, from SDS-PAGE analysis, gA-I had a multiplexing pattern and showed weak band intensity compared to nA-I. Similarly, E-HDL ₃ exhibited weak band strength and increased multimerized apoAI, while Y-HDL ₃ showed clear band strength. In addition, since E-HDL ₃ shows a higher band position, it suggests that the mobility of HDL ₃ changes with aging.

3) IEF on Polyacrylamide Gel

As shown in FIG. 3, gA-I-rHDL has a lot of base region shifted to the bottom on the IEF gel (pH 3-10) with increased faint intensity than nA-I-rHDL. In addition, nA-I-rHDL showed lower pI than gA-I-rHDL as it showed clearer band intensity and less movement to the bottom. E-HDL ₃ showed a wider range of isoelectric point values, with a broader band range than Y-HDL ₃ with weak band intensity. gA-I-rHDL and E-HDL ₃ migrated to the bottom of the gel with a larger diffusion area.

4) IEF on Microchannels and Simulations

To obtain a pH gradient in the straight channel, an amphoteric-root IEF simulation was performed. These mathematical models were created based on mass preservation and ion dissociation correlation of amphoteric macromolecules, charge preservation and electroneutralization conditions. Based on the 2-D model, 31 amphoterics were used in the pH range of 3.5-9.5 (ΔpI = 0.2, ΔpK = 1). The ion mobility of these amphoterics was 3 × 10 ⁻⁸ m ² / Vs, and normalized initial concentrations were used. An electrical potential of 50 V was applied to the negative electrode (right side), while zero electrical potential was maintained on the positive electrode (left side).

As shown in FIG. 4, normalized concentration behavior was plotted for the amphoteric material in the IEF process. Initially, the concentrations of all amphoterics were evenly distributed along the channel. These concentrations developed simultaneously at the cathode and anode. Two peaks of each concentration occurred and moved to the center of the channel (FIGS. 4A and 4B). 31 amphoterics stopped at the pi point and concentrated until steady state conditions were reached (FIG. 4C). At approximately 1000 seconds, a 40-fold concentration was made at steady state conditions (FIG. 4C). 5 shows the pH profile during the IEF process. Initially, pH was flat along the channel at the same concentration of amphoteric material. However, as the concentration of amphoteric material at the cathode and anode increased, the pH showed a sharp gradient at 50 seconds. 31 amphoterics are sufficient to make a linear pH gradient, while small amphoterics can make a staged purchase. A pH profile was created as the amphoteric material changed from 100 to 400 seconds. Near the steady state time (1000 seconds) the pH profile did not change significantly. From these results, a linear pH gradient was created for 31 amphoteric substances in the pH range of 3.5-9.5 (ΔpI = 0.2, ΔpK = 1), and the pi point of the protein was identified according to the channel length.

5) Electric mobility in microfluidic channel

After 25 minutes on electrophoresis, nA-I-NBD-rHDL (26 ng of apoAI and 0.8 g of NBD in the channel), as shown in FIGS. 6A and 6B, exhibits a concentrated band shift pattern with more pronounced green fluorescence , gA-I-rHDL showed a scattered band pattern with weaker fluorescence. Similarly, Y-HDL ₃ showed a more concentrated band pattern with stronger fluorescence intensity, whereas E-HDL ₃ showed much wider band shift with lower fluorescence intensity (FIGS. 7A and 7B).

From pI determination through simulation, nA-I-rHDL and gA-I-rHDL exhibited approximately 6.2 ± 0.2 and 7.0 ± 0.2, respectively (FIGS. 6C and 6D). The pi of Y-HDL ₃ and E-HDL ₃ showed approximately 6.9 ± 0.5 and 8.1 ± 0.5, respectively (FIGS. 7C and 7D). From these results, glycosylation results in an increase in pI with a broader range, which is consistent with the results obtained in IEF gels (FIG. 3).

gA-I-rHDL and E-HDL moved faster than nA-I-rHDL and Y-HDL in the channel. In addition, gA-I-rHDL showed a longer band scattering pattern with 0.4 ± 0.1 mm than nA-I-rHDL. Similarly, E-HDL ₃ showed a band scattering pattern with 1.7 ± 0.6 mm longer than Y-HDL ₃ (0.5 ± 0.4 mm). These results indicate that protein glycosylation in HDL alters the pI value and electrophoretic mobility in the microfluidic channel.

Thus, according to the present invention, E-HDL and glycated HDL may have several physical properties, such as increased glycated end product (AGE), more basic isoelectric point transition, multiplexing tendency of apoAI and IEF and microstructure. Share faster mobility in the fluid channel. gA-I-rHDL and E-HDL ₃ have similar properties, especially elevated levels of non-tryptophan Maillar fluorescence (FIG. 1), spontaneous multiplexing (FIG. 2), increased pi (FIG. 3), and faster electrophoresis Figures 5 to 7 are shown.

Therefore, the deformation monitoring of the HDL using the lab-on-a-chip according to the embodiment may be a rapid and reliable diagnosis method for the degree of aging and the onset of aging-related diseases.

As mentioned above, although this invention was demonstrated by the limited embodiment and drawing, this invention is not limited by this, The person of ordinary skill in the art to which this invention belongs, Of course, various modifications and variations are possible within the scope of equivalent claims.

Claims (7)

Isolating and purifying high-density lipoprotein (HDL) from plasma isolated from the human body; Aging and aging associated with isoelectric focusing (IEF) analysis and micro-channel biochip or lab-on-a-chip analysis to analyze the modification of the high-density lipoprotein (HDL) How to provide the information needed for the diagnosis of the disease. The method of claim 1, wherein the high-density lipoprotein (HDL) is HDL ₂ or HDL ₃ . The aging of claim 1 or 2, wherein the isoelectric focusing (IEF) analyzes the deformation of high-density lipoprotein (HDL) through either microfluidic channels or computer simulation. And providing information necessary for the diagnosis of aging-related diseases. The method of claim 3, wherein the isoelectric focusing (IEF) is a change in any one or more of the electrophoretic or isoelectric point of high-density lipoprotein (HDL) through either microfluidic channel or computer simulation. Method for providing information necessary for the diagnosis of aging and age-related diseases, characterized in that for analyzing. The method of claim 3, wherein the microfluidic channel is made of polydimethysiloxane (PDMS). The method according to claim 1 or 2, wherein the aging-related diseases provide information necessary for the diagnosis of aging and aging-related diseases, characterized in that any one selected from the group consisting of cardiovascular disease, cerebrovascular disease and metabolic disease. How to. delete

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