Method and apparatus for analyzing lipoprotein in blood sample

Abstract

To provide a method for analyzing lipoprotein in blood by an approach different from a conventional one.SOLUTION: The blood analyzer 1 includes a pore device 100, a measurement apparatus 200, and a data processing apparatus 300. The pore port 100 includes a first chamber 106 and a second chamber 108 partitioned by a pore, a first electrode-side E1 provided in the first chamber 106, and a second electrode-side E2 provided in the second chamber 108. The measuring device 200 measures a current flowing between the first and second electrodes E1 and E2. The data processing device 300 generates a particle size distribution of the lipoprotein 4 contained in the blood sample 2 based on the current measured by the measuring device 200.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates to the analysis of lipoproteins in blood samples. [Background technology]

[0002] LDL (Low Density Lipoprotein) cholesterol in the blood is known as "bad cholesterol," as it is a factor that promotes arteriosclerosis and myocardial infarction. In contrast, HDL (High Density Lipoprotein) is known as "good cholesterol," as it is a factor that prevents arteriosclerosis. LDL cholesterol in the blood is measured for the early diagnosis of coronary heart disease and other conditions.

[0003] Humans and animals absorb and digest dietary lipids, such as triglycerides and cholesterol, in the small intestine. However, these lipids cannot remain in the blood as they are; instead, they are stably present in the blood as lipoproteins, which are bound to apoproteins. Lipoproteins are a collective term for tiny lipid transporters of various particle sizes, and are called, in descending order of particle size, chylomicrons, very-low-density lipoproteins (VLDL), intermediate-density lipoproteins (IDL) or midband, low-density lipoproteins (LDL), small, dense LDL, and high-density lipoproteins (HDL). Lipoprotein particles are tiny particles, measuring 10 to 100 nm in diameter, approximately 1 / 70th the diameter of a red blood cell.

[0004] Commonly known methods for measuring total cholesterol (TC), LDL cholesterol (LDL-C), HDL cholesterol (HDL-C), and triglycerides (TG) involve breaking down particulate lipoproteins and chemically quantifying the cholesterol and triglycerides contained within, but do not directly measure the lipoprotein particles themselves.

[0005] However, since the metabolism of blood lipids in the body is centered on the breakdown and catabolism of lipoproteins, metabolic and therapeutic options cannot be determined without examining the lipoproteins themselves: VLDL, IDL, LDL, small, dense LDL, and HDL. Because lipoproteins themselves are extremely small and fragile, there is currently no way to directly measure them, and we have had to rely on quantitative measurements of cholesterol, etc. However, it is not true that myocardial infarction can be prevented by treating only the amounts of TC and TG, and the need for qualitative testing of lipoprotein properties remains unchanged.

[0006] Recently, in line with the term metabolic syndrome, the "bad" lipoproteins have been frequently discussed as a cause of arteriosclerosis and other conditions. These "bad" lipoproteins are IDL and small, dense LDL, and are recognized by medical specialists as risk factors for myocardial infarction. It is noteworthy that IDL and small, dense LDL can be eliminated with appropriate antihyperlipidemic medication, so it is said that detecting IDL and small, dense LDL is important.

[0007] Polyacrylamide gel disc electrophoresis (PAGE) (Patent Document 1) has been proposed as a method for measuring lipoproteins without destroying particulate lipoproteins. PAGE does not measure the particle size of lipoproteins, but rather separates and analyzes lipoproteins in order of particle size. In PAGE, lipoproteins in serum or plasma are stained with Sudan Black B, a known lipoprotein stain, before electrophoresis. The lipoproteins are then separated and analyzed in order of particle size by the molecular sieving effect of a concentrating gel and a uniform separating gel, resulting in separation into VLDL, IDL, LDL, small, dense LDL, and HDL. In addition to PAGE, there are also agarose gel and cellulose acetate membrane electrophoresis methods that utilize differences in the charge of lipoproteins for analysis.

[0008] This method involves first separating lipoproteins by charge, immobilizing them, and then staining them with lipids. The lipoproteins are then labeled beta (β), pre-beta (pre-β), and alpha (α) lipoproteins, starting from the cathode, according to their respective fractional locations in blood. If the dye used for this staining were changed to cholesterol dye, it would become a cholesterol fractionation measurement method; if triglyceride dye were used, it would become a triglyceride fractionation measurement method (Patent Documents 3 and 4). However, because agarose gel or cellulose acetate membrane electrophoresis analyzes lipoproteins in order of charge, it cannot be considered an analysis of particle size. Larger pre-β particles migrate faster than smaller β particles, and large particles tend to remain trapped in the mesh structure of the agarose gel, resulting in poor separation ability. Furthermore, because staining is performed after electrophoresis, quantitation is difficult due to variations in staining conditions.

[0009] Furthermore, density gradient-type gradient gel electrophoresis (GGE) is a method in which the gel concentration of the support gradually increases, and is said to be able to separate lipoproteins in order of particle size more accurately than a uniform gel. However, the gel used is difficult to prepare, and it has only been performed in certain laboratories (Non-Patent Document 1). Details of the gel shown in Non-Patent Document 1 have not been made public, making it extremely difficult to reproduce.

[0010] Furthermore, the method described in Non-Patent Document 2 identifies lipoprotein particle size by GGE using blood proteins such as thyroglobulin and ferritin as markers, but it was not possible to reproduce this method because ferritin was not consistently obtained and the gel plate itself was a product of a specific manufacturer and its manufacturing method was not publicly disclosed. Furthermore, protein staining was required after electrophoresis, making it not a method that anyone could easily use for confirmation testing.

[0011] High-performance liquid chromatography (HPLC) is also known as a method for analyzing lipoproteins in order of particle size (Patent Document 2). This method measures cholesterol and neutral fats after first separating lipoproteins in order of particle size, but does not measure the particle size of lipoproteins. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] Japanese Patent Application Laid-Open No. 2005-121619 [Patent Document 2] Japanese Patent Application Publication No. 8-320313 [Patent Document 3] Japanese Patent Application Publication No. 11-230937 [Patent Document 4] Japanese Patent Application Laid-Open No. 2000-356641 [Patent Document 5] Japanese Patent Application Laid-Open No. 2003-28779 [Patent Document 6] Japanese Patent Application Laid-Open No. 2004-258014 [Patent Document 7] Japanese Patent Application Laid-Open No. 2013-205411 [Non-patent literature]

[0013] [Non-Patent Document 1] CLIN. CHEM. 34 / 8 (B), B78-B83(1988) [Non-patent document 2] Biophysical Chemistry, Vol.44, pp303-307, 2000 Summary of the Invention [Problem to be solved by the invention]

[0014] Considering that lipoprotein metabolism is primarily the breakdown and catabolism of lipoprotein particles, it is important to know which lipoprotein particles there are, how many of them there are, and how they are metabolized. Knowing how many lipoproteins there are and what particle sizes they have may also help to clarify which lipoproteins are involved in the development of arteriosclerosis and myocardial infarction.

[0015] The present application has been made in this context, and an exemplary purpose of one embodiment thereof is to provide a method for analyzing lipoproteins in blood using an approach different from conventional methods. [Means for solving the problem]

[0016] An aspect of the present disclosure relates to a method for analyzing lipoproteins in a blood sample, in which the particle size distribution of lipoproteins contained in the blood sample is measured by an electrical detection zone method using a pore device.

[0017] Another aspect of the present disclosure is an apparatus for analyzing lipoproteins in a blood sample. The apparatus includes a pore device, a measuring device, and a processing device. The pore device has a first chamber and a second chamber separated by a pore, a first electrode provided in the first chamber, and a second electrode provided in the second chamber, and contains a solution containing blood. The measuring device measures the current flowing between the first electrode and the second electrode. The processing device generates a particle size distribution of lipoproteins contained in the blood sample based on the current measured by the measuring device.

[0018] Any combination of the above elements, or mutual substitution of elements or expressions between methods, devices, systems, etc., are also valid aspects of the present invention or the present disclosure. Furthermore, the description in this section (Means for Solving the Problems) does not explain all essential features of the present invention, and therefore, subcombinations of the described features may also constitute the present invention. [Effects of the Invention]

[0019] According to certain aspects of the present disclosure, measurement accuracy can be improved. [Brief explanation of the drawings]

[0020] [Figure 1] FIG. 1 is a block diagram of a blood analyzer according to an embodiment. [Figure 2] FIG. 10 is a waveform diagram of an exemplary minute current Is measured by a measuring device. [Figure 3] FIG. 1 is a perspective view of a pore tip according to an embodiment. [Figure 4] FIG. 4 is a cross-sectional view of the pore tip of FIG. 3. [Figure 5]FIG. 10 is a diagram showing the simulation results of the potential distribution of the pore chip according to the embodiment. [Figure 6] FIG. 1 shows the results of measuring the particle size distribution of lipoproteins in a blood sample. [Figure 7] FIG. 1 shows the results of measuring the particle size distribution of an LDL standard sample. [Figure 8] FIG. 1 shows an in-liquid transmission electron microscope (TEM) image of an LDL standard sample observed under water using a TEM. [Figure 9] FIG. 1 shows the results of measuring the particle size distribution of an LDL standard sample obtained from a liquid TEM image. [Figure 10] FIG. 1 is a cross-sectional view of a pore tip having a low aspect ratio. [Figure 11] FIG. 10 shows a histogram generated when measuring standard particles using a low aspect pore tip. [Figure 12] FIG. 1 is a schematic diagram illustrating the cause of histogram splitting in a low aspect ratio pore chip. [Figure 13] FIG. 1 shows the measurement results of particle size distribution of chylomicrons. DETAILED DESCRIPTION OF THE INVENTION

[0021] (Outline of the embodiment) A summary of some exemplary embodiments of the present disclosure is provided. This summary is intended to provide a simplified overview of some concepts of one or more embodiments in order to provide a basic understanding of the embodiments as a prelude to the more detailed description that follows. It is not intended to limit the scope of the invention or disclosure. This summary is not an exhaustive overview of all possible embodiments, and is not intended to identify key elements of all embodiments or to delineate the scope of some or all aspects. For convenience, the term "one embodiment" may refer to one embodiment (example or variant) or multiple embodiments (examples or variants) disclosed herein.

[0022] In one embodiment of the method for analyzing lipoproteins in a blood sample, the particle size distribution of lipoproteins contained in the blood sample is measured by an electrical detection zone method using a pore device.

[0023] According to this embodiment, lipoproteins can be measured without staining. This allows accurate measurement without altering the lipoproteins and maintaining their original diameter. This is an analytical method that faithfully reflects the lipoproteins in the blood of a living body. Furthermore, by simultaneously measuring apo B concentration and TC, the number of LDL particles (moles) in a certain blood volume and cholesterol in LDL can also be measured.

[0024] In one embodiment, the blood sample may contain serum. According to the above-described method, the diameter of lipoproteins in blood can be measured in a few seconds without staining, without requiring centrifugation, which requires several hours, and thus the test time can be significantly reduced compared to conventional methods.

[0025] In one embodiment, the blood sample may include blood after centrifugal separation. When it is desired to measure the particle size distribution of only a specific lipoprotein, it is effective to perform the measurement in combination with centrifugal separation.

[0026] In one embodiment, the pore diameter of the pore device is 80 nm to 120 nm, and the particle size distribution of LDL (Low Density Lipoprotein) may be measured.

[0027] In one embodiment, the pore diameter of the pore device is 2 μm to 3 μm, and the particle size distribution of chylomicrons may be measured.

[0028] A blood analysis device according to one embodiment includes a pore device having a first chamber and a second chamber separated by a pore, a first electrode provided in the first chamber, and a second electrode provided in the second chamber, and containing a blood sample; a measuring device that measures the current flowing between the first electrode and the second electrode; and a processing device that generates a particle size distribution of lipoproteins contained in the blood sample based on the current measured by the measuring device.

[0029] In one embodiment, the analyzer may measure blood in serum form.

[0030] In one embodiment, the analyzer may measure the blood in a state after centrifugation.

[0031] In one embodiment, the pore diameter is 80 nm to 120 nm, and the particle size distribution of LDL (Low Density Lipoprotein) may be measured.

[0032] (Embodiment) Preferred embodiments will be described below with reference to the drawings. The same or equivalent components, parts, and processes shown in each drawing will be given the same reference numerals, and redundant explanations will be omitted as appropriate. Furthermore, the embodiments are examples and do not limit the disclosure and invention, and all features and combinations thereof described in the embodiments are not necessarily essential to the disclosure and invention.

[0033] In addition, the dimensions (thickness, length, width, etc.) of each component shown in the drawings may be enlarged or reduced as appropriate for ease of understanding. Furthermore, the dimensions of multiple components do not necessarily represent their relative sizes, and even if a component A is depicted as being thicker than another component B in the drawings, it is possible that component A is thinner than component B.

[0034] In this specification, "a state in which component A is connected to component B" includes not only a case in which component A and component B are directly physically connected to each other, but also a case in which component A and component B are indirectly connected to each other via other components that do not substantially affect the electrical connection between them or that do not impair the function or effect achieved by their connection.

[0035] Similarly, "a state in which component C is connected (provided) between component A and component B" includes not only a case in which component A and component C, or component B and component C, are directly connected, but also a case in which they are indirectly connected via other components that do not substantially affect the electrical connection state between them or that do not impair the function or effect achieved by their combination.

[0036] In addition, in this specification, symbols attached to electrical signals such as voltage signals and current signals, or circuit elements such as resistors, capacitors, and inductors, represent the respective voltage values, current values, or circuit constants (resistance values, capacitance values, inductances) as necessary.

[0037] 1 is a block diagram of a blood analyzer 1 according to an embodiment. The blood analyzer 1 measures the particle size distribution of lipoproteins in a blood sample based on a particle size / particle size distribution measurement method known as the electrical sensing zone method (Coulter principle).

[0038] In this measurement method, an electrolyte solution containing particles is passed through a small pore called a nanopore. When the particle passes through the pore, the electrolyte in the pore decreases by an amount equivalent to the volume of the particle, increasing the electrical resistance of the pore. Therefore, by measuring the electrical resistance of the pore, the volume of the particle (i.e., particle size) can be measured.

[0039] The blood analyzer 1 includes a pore device 100, a measuring device 200, and a data processing device 300. The pore device 100 includes:

[0040] The interior of the pore device 100 is filled with a blood sample 2 containing lipoprotein 4, which is a particle to be measured. The interior of the pore device 100 is separated into a first chamber 106 and a second chamber 108 by a pore chip 102, and electrodes E1 and E2 are provided in the first chamber 106 and the second chamber 108. A pore 104 is provided in the pore chip 102. When a potential difference is generated between electrodes E1 and E2, an ionic current flows between the electrodes. Lipoprotein 4 moves between the first chamber 106 and the second chamber 108 via the pore 104 by electrophoresis or in response to the pressure generated by a pump (not shown).

[0041] The measurement device 200 generates a potential difference between the electrode pair E1 and E2 and acquires information correlated with the resistance value Rp between the electrode pair. The measurement device 200 includes a transimpedance amplifier 210, a voltage source 220, and a digitizer 230. The voltage source 220 generates a potential difference Vb between the electrode pair E1 and E2. This potential difference Vb serves as a driving source for electrophoresis and also as a bias signal for measuring the resistance value Rp.

[0042] A minute current Is that is inversely proportional to the resistance of the pore 104 flows between the pair of electrodes E1 and E2. Is=Vb / Rp …(1)

[0043] The transimpedance amplifier 210 converts the minute current Is into a voltage signal Vs. When the conversion gain is r, the following equation holds: Vs = -r × Is … (2) Substituting equation (1) into equation (2) gives equation (3). Vs = -Vb × r / Rp … (3) The digitizer 230 converts the voltage signal Vs into digital data Ds. In this manner, the measurement device 200 can obtain the voltage signal Vs that is inversely proportional to the resistance value Rp of the pore 104.

[0044] 2 is a waveform diagram of an exemplary minute current Is measured by the measurement device 200. Note that the vertical and horizontal axes of the waveform diagrams and time charts referred to in this specification have been appropriately enlarged or reduced for ease of understanding, and each waveform shown has been simplified, exaggerated, or emphasized for ease of understanding.

[0045] During the short period that lipoprotein 4 particles pass through the pore 104, the resistance value Rp of the pore 104 increases. Therefore, the current Is decreases in a pulsed manner each time a lipoprotein 4 passes through. The amplitude of each pulse current correlates with the particle size. The data processing device 300 processes the digital data Ds and analyzes the number and particle size distribution of lipoprotein 4 contained in the blood sample 2. Part of the data processing device 300 may be a server or a cloud.

[0046] FIG. 3 is a perspective view of a pore chip 400 according to an embodiment. The pore chip 400 is incorporated into the pore device 100 of FIG. 1. The pore chip 400 includes a membrane 410. The pore chip 400 may further include a support member (not shown) that supports the membrane 410. The membrane 410 has a pore (opening) 412 extending therethrough. d denotes the diameter of the pore 412, and t denotes the thickness of the membrane. The support member is an insulating substrate such as quartz (SiO2) glass, and the membrane 410 is a nitride film such as SiN or an oxide film such as SiO2. In a pore device, noise increases when the parasitic capacitance formed in the support member is large. Parasitic capacitance is proportional to the dielectric constant. For example, the dielectric constant of Si is 11.9, while the dielectric constant of SiO2 is 3.9, which significantly reduces the parasitic capacitance. In this embodiment, the pore chip 400 does not include a semiconductor, which reduces parasitic capacitance compared to technologies that use Si as a support member, thereby reducing the effects of noise and improving the signal-to-noise ratio.

[0047] 4 is a cross-sectional view of the pore chip 400 of FIG. 3. In this embodiment, the aspect ratio t / d of the pore 412 is 1≦t / d<2 The relationship is fulfilled.

[0048] 5 is a diagram showing the simulation results of the potential distribution of the pore chip 400 according to the embodiment. The gradation represents the potential, and the arrows represent the electric field vectors. d=300 nm, t=300 nm, and the aspect ratio t / d=1. SiN is used as the material for the membrane 410.

[0049] The pore diameter of the pore device suitable for measuring lipoproteins is explained below. The size of lipoprotein particles in blood is as follows: HDL 8nm LDL 25nm IDL 30nm VLDL 50nm~80nm Chylomicrons 200nm or larger Therefore, when pores of 80 nm or larger are used for measurement, lipoproteins other than chylomicrons (200 nm or larger), specifically VLDL (80 nm or smaller), IDL (30 nm), LDL (25 nm), and HDL (8 nm) can be measured.

[0050] Furthermore, the S / N ratio limit of the blood analyzer 1 according to this embodiment is approximately 20% of the pore diameter. Therefore, if the measurement target is LDL up to 25 nm and the S / N ratio limit is set to 24 nm, the upper limit of the pore diameter should be set to 24 nm / 20% = 120 nm.

[0051] By setting the upper limit of the pore diameter at 120 nm, chylomicrons will not pass through the pores and there will be no risk of the pores becoming clogged.

[0052] From this, it can be said that the pore diameter suitable for measuring LDL is 80 to 120 nm. By using a pore device with such a pore diameter, particle size distribution can be measured directly, easily, and quickly by simply diluting and suspending the sample in 1x PBS (phosphate buffered saline) without filtration or the need for reagents.

[0053] A pore device with d=100nm and t=100nm was fabricated, and the particle size distribution of LDL in blood was actually measured.

[0054] The procedure for preparing a blood sample is as follows: 1 μL of serum is mixed with 999 μL of 1x PBS and diluted 1000 times. The mixture is then suspended to create a blood sample. A predetermined amount (for example, 20 μL) is withdrawn from the blood sample, injected into a pore device, and measured. Lipoproteins may be altered in pure water, but diluting them with an isotonic solution of 1x PBS (0.15 M) allows for measurement without altering the lipoproteins.

[0055] 6 shows the results of measuring the particle size distribution of lipoproteins in a blood sample. The bias voltage Vb applied between the electrodes was 0.1 V.

[0056] The mean particle size across the entire distribution was 28.323 nm, which corresponds to the typical particle size of LDL cholesterol. The mode was 24.096 nm.

[0057] The overall median (D50) was 26.228 nm, the overall 10% value (D10) was 23.116 nm, and the overall 90% value (D90) was 35.802 nm. The difference between the D10 and D90 values ​​normalized by the median (D90-D10) / D50 was 0.484.

[0058] FIG. 6 shows a Gaussian fitting line, and the particle size (mean) at the peak was 25.161 nm, the standard deviation (SD) of the fitting line was 2.394 nm, and the coefficient of variation (CV), which is the standard deviation divided by the mean, was 9.52%.

[0059] 7 shows the results of measuring the particle size distribution of an LDL standard sample. The LDL standard sample used was manufactured by Athens Research and Technology. This standard sample was prepared by extracting LDL by ultracentrifugation.

[0060] The mean particle size across the entire distribution was 30.384 nm, and the mode was 26.343 nm.

[0061] The overall median (D50) was 28.806 nm, the 10% of the overall value (D10) was 25.561 nm, and the 90% of the overall value (D90) was 37.718 nm. The median normalized value (D90-D10) / D50, which shows how far the D10 and D90 values ​​are separated, was 0.422.

[0062] FIG. 8 shows an in-liquid transmission electron microscope (TEM) image of an LDL standard sample in a 1xPBS solution, observed underwater with a TEM.

[0063] Figure 9 shows the particle size distribution of an LDL standard sample obtained from a liquid TEM image. While a typical TEM image is obtained by observing a dry sample, the inventors performed TEM observation of an LDL standard sample that had not been altered by suspending it in an isotonic solution of 1x PBS (0.15 M), and the measurement results are equivalent to those in the state of the sample present in blood.

[0064] The measurement results based on the blood sample that was not subjected to ultracentrifugation (FIG. 6) and the measurement results of the LDL standard sample extracted by ultracentrifugation (FIGS. 7 and 9) are in good agreement. This experiment confirmed that the blood analyzer 1 according to the embodiment can accurately measure particle size using a blood sample simply prepared from serum that is not subjected to ultracentrifugation.

[0065] It should be noted that measuring the particle size distribution of LDL cholesterol using the electrical detection zone method should not be considered common technical knowledge. The lower limit of particle size measurable by commercially available devices is 40 nm according to specifications, and there have been no reports to date of accurate measurement of particle size distribution in the range below 40 nm. Furthermore, in reality, particle sizes below 100 nm cannot be measured with high accuracy. Therefore, it is a breakthrough that LDL cholesterol with a diameter of approximately 30 nm can be measured using the electrical detection zone method.

[0066] According to this embodiment, the particle size distribution of LDL to small dense LDL can be measured. A blood sample can be prepared simply by diluting it with 1x PBS (phosphate buffered saline) and suspending it, and no filtration, staining, or reagents are required. Therefore, particle size distribution can be measured more simply and quickly than with conventional techniques.

[0067] Furthermore, since no treatment that would alter the lipoproteins is performed during the blood sample preparation process or during measurement by the blood analyzer 1, the particle size distribution of lipoproteins can be accurately measured under the same conditions as those in blood.

[0068] We explain why equal aspect ratio pore devices are suitable for measuring lipoprotein particle size.

[0069] 10 is a cross-sectional view of a pore chip 800 having a low aspect ratio. The pore chip 800 includes a membrane 810, and a pore (opening) 812 is formed in the membrane 810. In conventional pore chips 800, the membrane 810 is made of SiN or SiO2, and the membrane 810 has a thickness t of several tens of nanometers. This is because (i) by reducing the thickness t, a highly crystalline film can be formed and the film formation time can be shortened, (ii) procurement is easy, i.e., thin films are less expensive and have a shorter delivery time, and (iii) the film processability is good, i.e., it is convenient for subsequent processing by dry etching or the like.

[0070] On the other hand, the membrane's support material is a semiconductor (Si), which makes it susceptible to noise.

[0071] When a membrane 810 having a thickness t of several tens of nanometers is used, the diameter d of the pore 812 (called the pore diameter) and the thickness t of the membrane 810 are as follows: d>t When the aspect ratio of the pore is defined as t / d, it can be said that the conventional pore chip 800 has a low aspect ratio.

[0072] FIG. 11 shows a histogram generated when measuring standard particles using a low-aspect pore chip 800. The pore chip 800 used in the experiment had a d of 3 μm and an aspect ratio of 0.017 (t of 0.050 μm). The diameter of the standard particle was 0.9 μm. The horizontal axis of the histogram represents the particle size estimated from the current measured when the particle passed through the pore, and the vertical axis represents the number of particles.

[0073] Ideally, the histogram should be unimodal because standard particles with the same diameter are being measured, but the measurement results show that the histogram splits into two, exhibiting a strong bimodal distribution. A bimodal histogram makes it difficult to estimate the particle diameter, reducing measurement accuracy.

[0074] The present inventors focused on the electric field strength within the pore 812 as the reason why bimodality occurred when the conventional pore chip 800 was used.

[0075] FIG. 12 is a schematic diagram illustrating the cause of histogram splitting in a low-aspect ratio pore chip. The pore chip 800 is housed in a case 900. The case 900 is divided into two chambers 902 and 904 by the pore chip 800. The chambers 902 and 904 are filled with a sample 2 containing particles. Particles 4 can take different paths when passing through a pore 812. Two paths (i) and (ii) are shown representatively in FIG. 12. If the electric field strength within the pore 812 is uniform, signals of the same intensity will be measured regardless of the path taken. However, if the electric field strength within the pore 812 is non-uniform, signals of different intensities will be measured when particles 4 of the same particle size pass through, depending on the path taken. This results in a split in the histogram.

[0076] The particle size of LDL in blood can change depending on factors such as the presence or absence of oxidative modification. Therefore, it is possible that the particle size distribution itself has multiple peaks. When measuring LDL with such a distribution using a pore device with low aspect ratio pores, it is unclear whether the multiple peaks are due to the presence or absence of oxidative modification.

[0077] By using a pore device with equal aspect ratio pores according to the embodiment, it is possible to accurately measure the particle size distribution of LDL with a resolution of 2 nm. This may enable not only the differentiation of LDL to small dense LDL, but also the differentiation of subtle differences in particle state, such as the presence or absence of oxidative modification, which is effective for the early diagnosis of coronary heart disease.

[0078] Next, we will explain the results of measuring chylomicrons. As mentioned above, the particle size of chylomicrons is 200 nm or more. Therefore, we measured a pore device with a pore diameter of 3 μm (3000 nm) and measured the particle size distribution of chylomicrons in serum.

[0079] 13 shows the measurement results of the particle size distribution of chylomicrons. The bias voltage Vb applied between the electrodes was 0.1 V.

[0080] The mean particle size across the distribution was 750.650 nm, and the mode was 740.536 nm.

[0081] The overall median (D50) was 736.895 nm, the 10% of the overall value (D10) was 689.753 nm, and the 90% of the overall value (D90) was 810.906 nm. The difference between the D10 and D90 values ​​normalized by the median (D90-D10) / D50 was 0.164.

[0082] FIG. 13 shows a Gaussian fitting line, with the particle size (mean) at the peak being 732.467 nm, the standard deviation (SD) of the fitting line being 44.497 nm, and the coefficient of variation (CV), which is the standard deviation divided by the mean, being 6.07%.

[0083] In this way, by increasing the pore diameter, the particle size distribution of chylomicrons can also be measured accurately.

[0084] Next, a modified example will be described.

[0085] (Variation 1) In the embodiment, a pore device of a type in which pores are formed in a membrane has been described, but the type of pore device is not limited to this. For example, a similar measurement can be performed using a channel-type pore device.

[0086] (Variation 2) In the embodiment, particle size distribution was measured without filtration, assuming a pore device with a pore diameter of 80 to 120 nm, but the present disclosure is not limited thereto. Furthermore, if the S / N ratio limit is improved, pores larger than 120 nm may be used as long as they are not affected by chylomicrons. When filtration is performed, a pore device with a pore diameter smaller than 80 nm may also be used.

[0087] For example, when measuring the particle size distribution of only LDL, a blood sample is prepared from which particles larger than LDL are removed using a filter, and measurement is then performed using a pore device with a pore diameter of approximately 50 nm to 70 nm. This allows for more accurate measurement of the particle size distribution of LDL while suppressing clogging.

[0088] Alternatively, when measuring the particle size distribution of HDL, a blood sample may be prepared from which large lipoproteins have been removed using a filter of about 40 nm, and then the sample may be measured using a pore device with a pore diameter of 10 nm to 30 nm.

[0089] (Variation 3) Similarly, when targeting chylomicrons, particle size distribution was measured without filtration, assuming a pore device with a pore diameter of 2000 to 3000 nm, but the present disclosure is not limited thereto. Furthermore, if the signal-to-noise ratio limit is improved, pores larger than 3000 nm may be used. When filtration is performed, a pore device with a smaller pore diameter that allows chylomicrons to pass through can be used.

[0090] (Variation 4) The blood sample preparation protocol is not limited to that described in the embodiment. Instead of 1x PBS, other solutions that are isotonic with blood may be used as the buffer solution. Furthermore, the dilution level is not limited to that described in the embodiment.

[0091] (Variation 5) The data processing device 300 may determine the particle size value from the particle size distribution (histogram) by statistical processing other than fitting using a normal distribution, or may determine the particle size value based on a model using machine learning.

[0092] The present invention has been described based on the embodiments, but the embodiments merely illustrate the principles and applications of the present invention, and many modifications and changes in arrangement are permitted to the embodiments as long as they do not deviate from the concept of the present invention defined in the claims. [Explanation of symbols]

[0093] 1 Blood analyzer 2. Sample 4. Lipoproteins 100 pore device 102 Pore Tip 104 pores 106 Room 1 108 Room 2 200 Measuring Equipment 210 Transimpedance Amplifier 220 Voltage Source 230 digitizer 300 Data processing device

Claims

1. 1. A method for analyzing lipoproteins in a blood sample, comprising: An analytical method comprising measuring the particle size distribution of lipoproteins contained in the blood sample by an electrical detection zone method using a pore device.

2. 2. The analytical method according to claim 1, wherein the blood sample contains serum.

3. 2. The analytical method according to claim 1, wherein the blood sample is prepared by suspending serum in phosphate-buffered saline.

4. 2. The analytical method according to claim 1, wherein the blood sample contains blood after centrifugal separation.

5. 5. The analytical method according to claim 1, wherein the pore diameter of the pore device is 80 nm to 120 nm, and the particle size distribution of LDL (Low Density Lipoprotein) is measured.

6. 5. The analytical method according to claim 1, wherein the pore diameter of the pore device is 2 μm to 3 μm, and the particle size distribution of chylomicrons is measured.

7. 1. An apparatus for analyzing a blood sample, comprising: a pore device for containing the blood sample, the pore device having a first chamber and a second chamber separated by a pore, a first electrode provided in the first chamber, and a second electrode provided in the second chamber; a measuring device for measuring a current flowing between the first electrode and the second electrode; a processing device that generates a particle size distribution of lipoproteins contained in the blood sample based on the current measured by the measuring device; An analytical device comprising:

8. 8. The analyzer according to claim 7, wherein the blood sample contains serum.

9. 8. The analyzer according to claim 7, wherein the blood sample is a suspension of serum in phosphate buffered saline.

10. 8. The analyzer according to claim 7, wherein the blood sample includes blood after centrifugal separation.

11. 11. The analyzer according to claim 7, wherein the pore diameter is 80 nm to 120 nm, and the analyzer measures the particle size distribution of LDL (Low Density Lipoprotein).

12. 11. The analytical device according to claim 7, wherein the diameter of the pores is 2 μm to 3 μm, and the particle size distribution of chylomicrons is measured.

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