Method and apparatus for analyzing lipoprotein in a blood sample

Abstract

Provided is a method for analyzing lipoprotein in blood by an approach different from the conventional one. 【Solution means】The blood analyzer 1 includes a pore device 100, a measuring device 200, and a data processing device 300. The pore device 100 has a first chamber 106 and a second chamber 108 partitioned by pores, a first electrode E1 provided in the first chamber 106, and a second electrode E2 provided in the second chamber 108, and accommodates a blood sample 2. The measuring device 200 measures the current flowing between the first electrode E1 and the second electrode 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.

Description

Technical Field

[0001] The present disclosure relates to the analysis of lipoproteins in a blood sample.

Background Art

[0002] LDL (Low Density Lipoprotein) cholesterol in blood is regarded as a promoter of arteriosclerosis and myocardial infarction and is called bad cholesterol. In contrast, HDL (High Density Lipoprotein) is regarded as a factor that prevents arteriosclerosis and is called good cholesterol. LDL cholesterol in blood is measured for early diagnosis of coronary heart disease and the like.

[0003] Humans and animals absorb and digest lipids such as neutral fat and cholesterol in food in the small intestine, but they cannot exist in blood as they are, and can stably exist in blood as lipoproteins bound to apoproteins. The lipoproteins are a general term for fine lipid carriers with various particle sizes. Larger particle-sized ones are called chylomicrons, very-low-density lipoproteins (VLDL), intermediate-density lipoproteins (IDL) or mid-band, low-density lipoproteins (LDL), small, dense LDL, and high-density lipoproteins (HDL). The diameter of lipoprotein particles is a minute particle of about 1 / 70 of that of red blood cells and is about 10 nm to 100 nm.

[0004] Generally known methods for measuring total cholesterol (TC), LDL cholesterol (LDL-C), HDL cholesterol (HDL-C), and triglyceride (TG) chemically quantify cholesterol and triglyceride in the broken particulate lipoproteins and are not methods for directly measuring lipoprotein particles themselves.

[0005] However, in vivo, the metabolism of lipids in the blood is mainly centered around the degradation and catabolism of lipoproteins. Therefore, without considering the lipoproteins themselves, namely VLDL, IDL, LDL, small, dense LDL, and HDL, it is impossible to lead to the selection of metabolism and treatment methods. Since lipoproteins themselves are extremely tiny and very fragile, there is still no method for direct measurement currently, and we have no choice but to rely on the quantification of cholesterol and the like. However, it is not the case that simply treating by observing only the amounts of TC and TG can prevent myocardial infarction and the like. The fact that a qualitative examination of lipoproteins, that is, the so-called quality of lipoproteins, is still necessary remains unchanged.

[0006] Recently, in line with the term "metabolic syndrome", the extremely bad lipoproteins are often cited as the cause of arteriosclerosis and the like. These extremely bad lipoproteins refer to IDL and small, dense LDL, and they are recognized as risk factors for myocardial infarction even among professional doctors. And what should be noted is that these IDL and small, dense LDL can be eliminated by using appropriate hyperlipidemia treatment drugs, so the detection of IDL and small, dense LDL is said to be important.

[0007] Regarding the measurement method of lipoproteins, as a method for measuring without destroying particulate lipoproteins, polyacrylamide gel disc electrophoresis (PAGE method) (Patent Document 1) has been proposed. The PAGE method is not a measurement method for measuring the particle size of lipoproteins, but a method for separating and analyzing lipoproteins in the order of particle size. The PAGE method stains lipoproteins in serum or plasma with Sudan black B, which is said to be a lipoprotein staining agent, before electrophoresis, and then is separated and analyzed in the order of particle size by the molecular sieving effect of the concentrating gel and the uniform separation gel, and is separated into VLDL, IDL, LDL, small, dense LDL, and HDL. In addition to the PAGE method, there are agarose gel or cellulose acetate membrane electrophoresis methods that analyze by utilizing the difference in the charge of lipoproteins.

[0008] This method first separates lipoproteins by charge, fixes the lipoproteins, and then performs lipid staining. There is an analysis method that names β, pre-β, and α lipoproteins from the cathode side according to the fractionation position of blood proteins. If the dye used for this staining is changed to cholesterol staining, it becomes a cholesterol fraction measurement method, and if neutral fat is stained, it becomes a neutral fat fraction measurement method (Patent Documents 3 and 4). However, agarose gel or cellulose acetate membrane electrophoresis analyzes lipoproteins in the order of charge, so it cannot be said to be an analysis of particle size. There are also drawbacks such as the larger pre-β particles migrating faster than the smaller β particles and the larger particles remaining in the network structure of the agarose gel, resulting in poor separation ability. Furthermore, since staining is performed after electrophoresis, quantification cannot be performed due to unevenness in the concentration of the staining conditions.

[0009] Also, the density gradient type gradient gel electrophoresis (GGE) method is made in such a way that the gel concentration of the support gradually becomes denser, and it is said that lipoproteins can be separated more accurately in the order of particle size than a uniform gel. However, the preparation of the gel used is difficult and it has only been carried out in specific laboratories (Non-Patent Document 1). The details of the gel shown in Non-Patent Document 1 have not been published and it is extremely difficult to reproduce.

[0010] Also, the method shown in Non-Patent Document 2 identifies the particle size of lipoproteins by GGE using thyroglobulin or ferritin in blood proteins as markers. However, it was not possible to stably secure ferritin, the gel plate itself used was also a product of a specific manufacturer, and its preparation method etc. have not been published and cannot be reproduced. Furthermore, protein staining had to be performed after electrophoresis, so it was not in a state where anyone could easily conduct a confirmation test.

[0011] As a method for analyzing lipoproteins in the order of particle size, the high performance liquid chromatography (HPLC) method is also known (Patent Document 2). This method first separates lipoproteins in the order of particle size and then measures cholesterol and neutral fat, but it 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 Laid-Open No. Hei 8-320313 [Patent Document 3] Japanese Patent Application Laid-Open No. Hei 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 Document]

[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] [Problems to be Solved by the Invention]

[0014] Considering that the metabolism of lipoproteins mainly involves the decomposition and catabolism of lipoprotein particles, it is important to know which lipoprotein particles there are and how they are metabolized. By knowing the number of lipoprotein particles with a certain particle diameter, it is expected to clarify which lipoproteins are involved in the development of arteriosclerosis and myocardial infarction.

[0015] This application has been made in this situation, and an exemplary purpose of one of its aspects is to provide a method for analyzing lipoproteins in blood by a different approach from the conventional ones. [Means for Solving the Problems]

[0016] One aspect of the present disclosure relates to a method for analyzing lipoproteins in a blood sample. In this analysis method, 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. This analyzer includes a pore device, a measuring device, and a processing device. The pore device has a first chamber and a second chamber partitioned by pores, a first electrode provided in the first chamber, and a second electrode provided in the second chamber, and houses 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] In addition, combinations of the above components arbitrarily, and components and expressions mutually substituted between methods, apparatuses, systems, etc. are also valid as aspects of the present invention or the present disclosure. Furthermore, the description of this item (means for solving the problems) does not explain all the essential features of the present invention, and therefore, sub-combinations of these described features can also be the present invention.

Advantages of the Invention

[0019] According to one aspect of the present disclosure, the measurement accuracy can be improved.

Brief Description of the Drawings

[0020]

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Mode for Carrying Out the Invention

[0021] (Overview of the Embodiment) The overview of some exemplary embodiments of the present disclosure will be described. This overview is for the purpose of providing a basic understanding of the embodiments as a prelude to the detailed description to follow, and simplifies and explains some concepts of one or more embodiments. It does not limit the scope of the invention or the disclosure. This overview is not an all-inclusive overview of all possible embodiments, nor is it intended to identify the important elements of all embodiments or to demarcate the scope of some or all aspects. For convenience, "one embodiment" may be used to refer to one embodiment (example or variation) or a plurality of embodiments (examples or variations) disclosed in this specification.

[0022] The method for analyzing lipoproteins in a blood sample according to one embodiment measures the particle size distribution of lipoproteins contained in the blood sample by an electrical detection zone method using a pore device.

[0023] According to this aspect, it is possible to measure without staining lipoproteins. As a result, it is possible to accurately measure while maintaining the original diameter without altering the lipoproteins. This is an analysis method that faithfully reflects lipoproteins in the bleeding of a living body. Also, by simultaneously measuring the apoB concentration and TC, the number (moles) of LDL particles and the cholesterol in LDL in a certain blood volume can also be measured.

[0024] In one embodiment, the blood sample may contain serum. According to the above method, it is possible to measure the diameter of lipoproteins in blood in seconds without staining, without performing a centrifugation process that takes several hours, and the inspection time can be significantly shortened compared to the conventional method.

[0025] In one embodiment, the blood sample may contain blood after centrifugation. When it is desired to measure the particle size distribution focusing only on specific lipoproteins, it is effective to measure in combination with a centrifugation process.

[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] The blood analyzer according to one embodiment includes a first chamber and a second chamber partitioned through a pore, a first electrode provided in the first chamber and a second electrode provided in the second chamber, a pore device that houses a blood sample, a measurement device that measures the current flowing between the first electrode and the second electrode, and a processing device that generates the particle size distribution of lipoproteins contained in the blood sample based on the current measured by the measurement device.

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

[0030] In one embodiment, the analyzer may measure 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) Hereinafter, preferred embodiments will be described with reference to the drawings. The same or equivalent components, members, and processes shown in each drawing are denoted by the same reference numerals, and repeated descriptions will be omitted as appropriate. Also, the embodiments are illustrative rather than limiting the disclosure and the invention, and all features and combinations thereof described in the embodiments are not necessarily essential to the disclosure and the invention.

[0033] Also, the dimensions (thickness, length, width, etc.) of each member shown in the drawings may be enlarged or reduced as appropriate for ease of understanding. Furthermore, the dimensions of a plurality of members do not necessarily represent their size relationships. On the drawing, even if a member A is drawn thicker than another member B, member A may be thinner than member B.

[0034] In this specification, the phrase "member A is in a state of being connected to member B" includes not only the case where member A and member B are physically directly connected, but also the case where member A and member B are indirectly connected via other members without substantially affecting their electrical connection state or impairing the functions and effects achieved by their connection.

[0035] Similarly, the state where "member C is connected (provided) between member A and member B" includes not only the case where member A and member C, or member B and member C are directly connected, but also the case where they are indirectly connected via other members that do not substantially affect their electrical connection state or impair the functions and effects achieved by their combination.

[0036] Also, in this specification, the symbols attached to electrical signals such as voltage signals and current signals, or circuit elements such as resistors, capacitors, and inductors, shall represent their respective voltage values, current values, or circuit constants (resistance values, capacitance values, inductance) as required.

[0037] FIG. 1 is a block diagram of a blood analysis apparatus 1 according to an embodiment. The blood analysis apparatus 1 measures the particle size distribution of lipoproteins in a blood sample based on a particle size and particle size distribution measurement method called the electrical sensing zone method (Coulter principle).

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

[0039] The blood analysis apparatus 1 includes a pore device 100, a measurement device 200, and a data processing device 300. The pore device 100

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

[0041] The measuring device 200 generates a potential difference between the electrode pair E1, E2 and acquires information correlated with the resistance value Rp between the electrode pair. The measuring 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, E2. This potential difference Vb is a driving source for electrophoresis and also serves as a bias signal for measuring the resistance value Rp.

[0042] A minute current Is that is inversely proportional to the resistance of the pores 104 flows between the electrode pair E1, 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), Equation (3) is obtained. Vs = -Vb × r / Rp …(3) The digitizer 230 converts the voltage signal Vs into digital data Ds. In this way, the measuring device 200 can obtain a voltage signal Vs that is inversely proportional to the resistance value Rp of the pores 104.

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

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

[0046] FIG. 3 is a perspective view of the pore chip 400 according to the 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 is provided with a penetrating pore (opening) 412. d represents the diameter of the pore 412, and t represents 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 the pore device, if the parasitic capacitance formed in the support member is large, the noise becomes large. The 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, whereby the parasitic capacitance can be greatly reduced. In this embodiment, the pore chip 400 has a configuration that does not contain a semiconductor, and compared with the technology using Si as the support member, the parasitic capacitance is reduced, the influence of noise can be suppressed, and thus the SNR can be improved.

[0047] FIG. 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 satisfies the relationship.

[0048] FIG. 5 is a diagram showing the simulation result of the potential distribution of the pore chip 400 according to the embodiment. The shading gradation represents the potential, and the arrow represents the electric field vector. d = 300 nm and t = 300 nm, and the aspect ratio is t / d = 1. As the material of the membrane 410, SiN is used.

[0049] The pore diameter of the pore device suitable for the measurement of lipoproteins will be described. The sizes of lipoprotein particles in the blood are as follows. HDL 8 nm LDL 25 nm IDL 30 nm VLDL 50 nm - 80 nm Chylomicron 200 nm or more Therefore, when pores of 80 nm or more are used for measurement, lipoproteins other than chylomicrons (200 nm or more), specifically, VLDL (80 nm or less) - IDL (30 nm) - LDL (25 nm) - HDL (8 nm) can be measured.

[0050] Also, the SN ratio limit of the blood analyzer 1 according to the embodiment is about 20% of the pore diameter. Therefore, considering up to LDL of 25 nm as the measurement target, when the SN ratio limit is 24 nm, the upper limit of the pore diameter may be set to 24 nm / 20% = 120 nm.

[0051] By setting the upper limit of the pore diameter to 120 nm, chylomicrons do not pass through the pores, and there is no worry about clogging the pores.

[0052] From this, it can be said that the pore diameter suitable for the measurement of LDL is 80 - 120 nm. By using a pore device having such a pore diameter, after dilution with 1xPBS (phosphate buffered saline) and simply suspending, the particle size distribution can be measured directly, simply, and quickly without filtration and without the need for reagents.

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

[0054] The procedure for preparing the blood sample is as follows. Mix 1 μL of serum and 999 μL of 1×PBS and dilute 1000-fold. Then, perform suspension to obtain a blood sample. Withdraw a predetermined amount (for example, 20 μL) from the blood sample, inject it into the pore device, and measure. Since lipoproteins may be denatured in pure water, by diluting with an isotonic solution of 1×PBS (0.15 M), it is possible to measure without denaturing the lipoproteins.

[0055] Figure 6 is a diagram showing the measurement results of the particle size distribution of lipoproteins in the blood sample. The bias voltage Vb applied between the electrodes is 0.1 V.

[0056] The average value (Mean) of the particle size over the entire distribution is 28.323 nm, which is consistent with the typical particle size of LDL cholesterol. The mode was 24.096 nm.

[0057] Also, the median value (D50) of the whole was 26.228 nm, the value of 10% of the whole (D10) was 23.116 nm, and the value of 90% of the whole (D90) was 35.802 nm. The value (D90 - D10) / D50 obtained by normalizing the distance between the D10 and D90 values with the median value was 0.484.

[0058] In Figure 6, a Gaussian fitting line is shown. The particle size (average value Mean) at the peak of the fitting line is 25.161 nm, the standard deviation (S.D.) of the fitting line is 2.394 nm, and the coefficient of variation (C.V.), which is the standard deviation divided by the average value, is 9.52%.

[0059] Figure 7 is a diagram showing the measurement results of the particle size distribution of the LDL standard sample. The LDL standard sample used was manufactured by Athens Research and Technology. This standard sample is a sample from which LDL was extracted by ultracentrifugation.

[0060] The average value (Mean) of the particle size across the whole distribution was 30.384 nm, and the mode was 26.343 nm.

[0061] Also, the median value (D50) of the whole was 28.806 nm, the value of 10% of the whole (D10) was 25.561 nm, and the value of 90% of the whole (D90) was 37.718 nm. The value (D90 - D10) / D50 obtained by normalizing the distance between the D10 and D90 values with the median was 0.422.

[0062] Figure 8 is a diagram showing a liquid-phase TEM image obtained by observing an LDL standard sample in a 1xPBS solution with a transmission electron microscope (TEM) in water.

[0063] Figure 9 is a diagram showing the measurement results of the particle size distribution of the LDL standard sample obtained from the liquid-phase TEM image. A normal TEM image is one that observes a dried sample, but the inventors have observed a sample in which the lipoprotein has not been denatured by suspending the LDL standard sample in an isotonic solution of 1×PBS (0.15M) by TEM, and the measurement results are equivalent to the state existing in the blood.

[0064] The measurement results based on the blood sample without ultracentrifugation treatment (Figure 6) and the measurement results of the LDL standard sample extracted by ultracentrifugation treatment (Figures 7 and 9) are in good agreement. From this experiment, it was verified that the particle size can be accurately measured by the blood analysis device 1 according to the embodiment using a blood sample simply prepared from serum without performing ultracentrifugation.

[0065] Note that it should not be regarded as common general knowledge to measure the particle size distribution of LDL cholesterol by the electrical sensing zone method. The lower limit particle size measurable by the commercially available devices is 40 nm according to the specifications, and there has been no report that the particle size distribution could be accurately measured in the region of 40 nm or less. Also, the particle sizes of 100 nm or less have not been accurately measured in actual practice. Therefore, it is epoch-making that LDL cholesterol with a diameter of about 30 nm could be measured by the electrical sensing 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 1xPBS (phosphate buffered saline) and then suspending it, and filtration, staining, and reagents are not required. Therefore, the particle size distribution can be measured more simply and quickly compared with the prior art.

[0067] And, since there is no treatment that denatures lipoproteins during the process of preparing the blood sample or during the measurement by the blood analyzer 1, the particle size distribution of lipoproteins can be accurately measured under the same conditions as in the blood.

[0068] Explain the reason why a pore device with an aspect ratio is suitable for measuring the particle size of lipoproteins.

[0069] FIG. 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 pores (openings) 812 are formed in the membrane 810. In the conventional pore chip 800, the material of the membrane 810 is SiN or SiO2, and a membrane thickness t of several tens of nm has been used. This is because (i) by reducing the membrane thickness t, a highly crystalline membrane can be formed and the film formation time can be shortened, (ii) the availability is good, that is, a thin film is less costly and has a short delivery time, and (iii) the membrane processability is good, that is, it is convenient when processing later by dry etching or the like.

[0070] On the other hand, since the support member of the membrane is semiconductor (Si), there was a problem of being vulnerable to noise.

[0071] When using a membrane 810 with a film thickness t of several tens of nm, between the diameter of the pore 812 (referred to as the pore diameter) d and the film thickness t of the membrane 810, d > t The relationship will hold. When defining the aspect ratio of the pore as t / d, it can be said that the conventional pore chip 800 had a low aspect ratio.

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

[0073] Since standard particles having the same diameter are being measured, ideally the histogram should be unimodal, but in the measurement results, the histogram is split into two and shows strong bimodality. When the histogram has bimodality, it becomes difficult to estimate the diameter of the particles and the measurement accuracy decreases.

[0074] The inventors focused on the electric field strength in the pore 812 as the reason for the occurrence of bimodality when using the conventional pore chip 800.

[0075] FIG. 12 is a schematic diagram explaining 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 partitioned by the pore chip 800 into two chambers 902 and 904. The interiors of the chambers 902 and 904 are filled with the sample 2 containing particles. When the particles 4 pass through the pores 812, they can take different paths. In FIG. 12, two paths (i) and (ii) are typically shown. If the electric field strength within the pores 812 is uniform, signals of the same intensity will be measured regardless of the passing path. However, if the electric field strength within the pores 812 is non-uniform, when particles 4 having the same particle size pass through, signals of different intensities will be measured depending on the passing path. As a result, the histogram splits.

[0076] The particle size of LDL in blood can vary depending on the presence or absence of oxidative modification, etc. Therefore, it is also conceivable that the particle size distribution itself has multiple peaks. When measuring LDL having such a distribution using a pore device having pores with a low aspect ratio, it is not known whether multiple peaks appear depending on the presence or absence of oxidative modification.

[0077] By using the pore device having pores with an equal aspect ratio according to the embodiment, the particle size distribution of LDL can be accurately measured with a resolution of 2 nm. As a result, it may be possible to distinguish not only between LDL and small dense LDL, but also slight differences such as the presence or absence of oxidative modification of each particle, which is effective for early diagnosis of coronary heart disease and the like.

[0078] Subsequently, the results of measuring chylomicrons will be described. As described above, the particle size of chylomicrons is 200 nm or more. Therefore, a pore device with a pore diameter of 3 μm (3000 nm) was measured to measure the particle size distribution of chylomicrons in serum.

[0079] FIG. 13 is a diagram showing the measurement results of the particle size distribution of chylomicrons. The bias voltage Vb applied between the electrodes is 0.1 V.

[0080] The average value (Mean) of the particle size across the whole distribution was 750.650 nm, and the mode value was 740.536 nm.

[0081] Also, the median value (D50) of the whole was 736.895 nm, the value of 10% of the whole (D10) was 689.753 nm, and the value of 90% of the whole (D90) was 810.906 nm. The value (D90 - D10) / D50 obtained by normalizing the distance between the D10 and D90 values with the median value was 0.164.

[0082] In Fig. 13, a Gaussian fitting line is shown. The particle size (average value Mean) at its peak was 732.467 nm, the standard deviation (S.D.) of the fitting line was 44.497 nm, and the coefficient of variation (C.V), which is the standard deviation divided by the average value, was 6.07%.

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

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

[0085] (Modified Example 1) In the embodiment, a pore device of the type in which pores are formed in a membrane has been described, but the form of the pore device is not limited thereto. For example, similar measurements can also be performed using a flow channel type pore device.

[0086] (Modified Example 2) In the embodiment, assuming a pore device having a pore diameter of 80 to 120 nm, the measurement of the particle size distribution was performed without filtration, but the present disclosure is not limited thereto. Also, when the SN ratio limit is improved, pores larger than 120 nm may be used within the range not affected by kyro-microns. When filtration is assumed, a pore device with a pore diameter smaller than 80 nm can also be used.

[0087] For example, when measuring the particle size distribution only for LDL, a blood sample with particles larger than LDL removed by a filter is prepared, and the measurement is performed using a pore device having a pore diameter of about 50 nm to 70 nm, so that the particle size distribution of LDL can be measured more accurately while suppressing clogging.

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

[0089] (Modification 3) Similarly, when targeting chylomicrons, the particle size distribution was measured without filtration on the premise of a pore device having a pore diameter of 2000 to 3000 nm, but the present disclosure is not limited thereto. Also, when improving the signal-to-noise ratio limit, pores larger than 3000 nm may be used. When filtration is assumed, a pore device with a smaller pore diameter through which chylomicrons can pass can be used.

[0090] (Modification 4) The protocol for preparing the blood sample is not limited to that described in the embodiment. As the buffer solution, a solution other than 1×PBS that is isotonic with blood may be used. Also, the dilution ratio is not limited to that described in the embodiment.

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

[0092] Although the present invention has been described based on the embodiments, the embodiments merely show the principles and applications of the present invention, and many modifications and arrangement changes are allowed within the scope not departing from the idea of the present invention defined in the claims.

Explanation of Reference Numerals

[0093] 1 Blood analysis device 2 Sample 4 Lipoprotein 100 Pore device 102 Pore chip 104 Pore 106 First chamber 108 Second chamber 200 Measuring device 210 Transimpedance amplifier 220 Voltage source 230 Digitizer 300 Data processing device

Claims

1. A method for analyzing lipoproteins in a blood sample, by an electrical sensing zone method using a pore device having pores with a pore diameter d of 80 nm to 120 nm, measuring the particle size distribution of LDL (Low Density Lipoprotein) contained in the blood sample, when the aspect ratio of the pore is t / d using the membrane thickness t of the membrane of the pore device, 1 ≤ t / d < 2 The analysis method characterized by satisfying.

2. A method for analyzing lipoproteins in a blood sample, by an electrical sensing zone method using a pore device having pores with a pore diameter d of 2 μm to 3 μm, measuring the particle size distribution of chylomicrons contained in the blood sample, when the aspect ratio of the pore is t / d using the membrane thickness t of the membrane of the pore device, 1 ≤ t / d < 2 The analysis method characterized by satisfying.

3. The analysis method according to claim 1 or 2, wherein the blood sample contains serum.

4. The analysis method according to claim 1 or 2, characterized in that serum is suspended in phosphate buffered saline to prepare the blood sample.

5. The analysis method according to claim 1 or 2, wherein the blood sample contains blood after centrifugation.

6. An analyzer for a blood sample, comprising a first chamber and a second chamber partitioned through pores with a pore diameter d of 80 nm to 120 nm, a first electrode provided in the first chamber, and a second electrode provided in the second chamber, and a pore device for accommodating the blood sample; a measuring device for measuring the current flowing between the first electrode and the second electrode; a processing device for generating a particle size distribution of LDL (Low Density Lipoprotein) contained in the blood sample based on the current measured by the measuring device; equipped with, when the aspect ratio of the pore is t / d using the membrane thickness t of the membrane of the pore device, 1 ≤ t / d < 2 The analyzer characterized by satisfying.

7. An analyzer for a blood sample, comprising a first chamber and a second chamber partitioned through pores with a pore diameter d of 2 μm to 3 μm, a first electrode provided in the first chamber, and a second electrode provided in the second chamber, and a pore device for accommodating the blood sample; a measuring device for measuring the current flowing between the first electrode and the second electrode; A processing device that generates a particle size distribution of chylomicrons contained in the blood sample based on the current measured by the measurement device, comprising, When the aspect ratio of the pores is set to t / d using the membrane thickness t of the membrane of the pore device, 1 ≦ t / d < 2 An analyzer characterized by satisfying the above. **Claim 8** The analyzer according to claim 6 or 7, wherein the blood sample contains serum. **Claim 9** The analyzer according to claim 6 or 7, wherein the blood sample is obtained by suspending serum in phosphate buffered saline. **Claim 10** The analyzer according to claim 6 or 7, wherein the blood sample contains blood after centrifugation.

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