Patent Publication Number: US-2021181216-A1

Title: Analysis method, analysis substrate, analysis kit, and analysis apparatus

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a Continuation Application of PCT Application No. PCT/IB2020/051714, filed Feb. 28, 2020 and based upon and claiming the benefit of priority from Japanese Patent Application No. 2019-164882, filed Sep. 10, 2019, the entire contents of all of which are incorporated herein by reference. 
    
    
     FIELD 
     Embodiments described herein relate generally to an analysis method, an analysis substrate, an analysis kit, and an analysis apparatus. 
     BACKGROUND 
     In the field of disease treatment or cosmetics, as a method for delivering an objective substance (such as drugs, nucleic acids and beauty components) to a cell, there is a method for preparing a lipid particle that contains the objective substance in a lipid membrane and contacting the lipid particle with the cell. 
     A lipid particle is manufactured, for example, by mixing a lipid serving as a material of a lipid membrane and an objective substance in a solution. A quality of a manufactured particle group is evaluated from a fill ratio of the objective substance. A fill ratio is obtained by measuring an amount of objective substances remaining in the solution without being contained in the lipid particle and by substituting the amount into the following formula: Fill ratio=((Total amount of objective substance input)−(Amount of objective substances remaining in solution))/(Total amount of objective substance input). 
     The higher the fill ratio, the higher the quality. The reason is that a higher fill ratio indicates that more objective substances are contained in a lipid particle. Furthermore, it is recommended to perform quality control to even out a fill ratio. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows a schematic view illustrating an example of a lipid particle group according to an embodiment. 
         FIG. 2  shows a flowchart of an example of an analysis method according to a first embodiment. 
         FIG. 3  shows a schematic view illustrating the example of the analysis method according to the first embodiment. 
         FIG. 4  shows a block diagram illustrating an example of an analysis apparatus according to the first embodiment. 
         FIG. 5  shows a plan view illustrating an example of an analysis substrate according to the first embodiment. 
         FIG. 6  shows a plan view illustrating a detector for the analysis substrate according to the first embodiment. 
         FIG. 7  shows a flowchart of another example of the analysis method according to the first embodiment. 
         FIG. 8  shows a block diagram illustrating another example of the analysis apparatus according to the first embodiment. 
         FIG. 9  shows a flowchart of an example of an analysis method according to a second embodiment. 
         FIG. 10  shows a graph showing an example of a relationship between a scattered light intensity and the number of lipid particles in the second embodiment. 
         FIG. 11  shows a graph showing experimental results of Example 1. 
         FIG. 12  shows a graph showing experimental results of Example 2. 
     
    
    
     DETAILED DESCRIPTION 
     In general, according to one embodiment, an analysis method is for determining an abundance ratio of first lipid particles containing an objective substance in a lipid particle group which contains a plurality of lipid particles. The analysis method comprising: irradiating step of irradiating a solution including the lipid particle group with light; measuring step of measuring at least two of the total number of the plurality of lipid particles, the number of the first lipid particles, and the number of second lipid particles not containing the objective substance in the solution by detecting scattered light obtained from the plurality of lipid particles in the lipid particle group; and calculating step of calculating, based on a result obtained from the measuring step, an abundance ratio of the first lipid particles by Formula (I): Abundance ratio of the first lipid particles=The number of the first lipid particles/The total number of the plurality of lipid particles in the lipid particle group. 
     Various embodiments will be described hereinafter with reference to the drawings. Each figure is a schematic view that promotes a better understanding about embodiments. Shapes, dimensions, ratios, and the like in each figure are different from actual ones and are designed appropriately with reference to the following description and known technology. 
     First Embodiment 
     (Analysis Method) 
     An analysis method of a first embodiment is to determine an abundance ratio of lipid particles containing an objective substance in a solution including a lipid particle group. 
     As illustrated in  FIG. 1 , a lipid particle group  1  includes a plurality of lipid particles  4 . The lipid particles  4  may include a lipid particle  4   a  that contains an objective substance  2  in a lipid membrane  3  (hereinafter also referred to as “encapsulated particle(s)” or “first lipid particle(s)”) and/or a lipid particle  4   b  that does not contain the objective substance  2  in the lipid membrane  3  (hereinafter also referred to as “non-encapsulated particle(s)” or “second lipid particle(s)”). The terms “particle” and “particles” as used herein may include both a particle (singular) and particles (plural, a plurality of particles). 
     The lipid membrane  3  is a substantially spherical hollow body of a lipid membrane formed by a plurality of non-covalently bonded lipid molecules. 
     Examples of the objective substance  2  include, but not limited to, nucleic acids, proteins, peptides, glycoproteins, low-molecular compounds, antibodies, or antigen-binding fragments. The objective substance  2  may also be, for example, drugs (such as therapeutic drugs or diagnostic drugs), cosmetic components, or genes to be delivery into cells. 
     Each of the plurality of lipid particles  4  included in the lipid particle group  1  desirably has a particle size, for example, less than 1 μm. A particle size of the lipid particles is measured, for example, by dynamic light scattering (DLS) or electrical sensing zone. 
     A solution including the lipid particle group  1  may be obtained by mixing a material of the lipid membrane  3  and the objective substance  2  (and other substances to be contained in the lipid membrane  3  as necessary) in a liquid. Alternatively, the solution may be obtained as follows: separate the lipid particle group  1  from the above liquid mixture; and add the separated lipid particle group  1  to another solvent. 
     The solution may be, for example, a reagent for delivery the objective substance  2  into a cell, a pharmaceutical product or a cosmetic product, which contains lipid particles. Alternatively, the solution may be a crude product a sample partially taken from a production lot in process of producing the reagent, the pharmaceutical product or the cosmetic product described above. 
     An analysis method according to an embodiment includes, for example, the following steps as illustrated in  FIG. 2 : 
     (S 1 ) a mixing step of mixing the solution including the lipid particle group  1  with a first substance that has lipid membrane permeability, binds to the objective substance  2  and contains a dye which generate fluorescence;
 
(S 2 ) an irradiating step of irradiating the resulting mixture with excitation light that generates fluorescence from the dye;
 
(S 3 ) a first measuring step of measuring the number of lipid particles  4  causing scattered light, that is, the total number of the plurality of lipid particles by detecting scattered light;
 
(S 4 ) a second measuring step of measuring the number of lipid particles  4  causing fluorescence, that is, the number of lipid particles containing the objective substance  2  (encapsulated particles  4   a ) by detecting fluorescence; and
 
(S 5 ) a calculating step of calculating an abundance ratio of the lipid particles containing the objective substance by Formula (I): (Abundance ratio of encapsulated particles  4   a =Number of encapsulated particles  4   a /Total number of the plurality of lipid particles in lipid particle group), based on a result obtained from the measuring step.
 
     Hereinafter, each step will be described in detail with reference to  FIG. 3 . 
     A first substance  5 , which is mixed with the solution including the lipid particle group  1  in the mixing step (S 1 ), includes, for example, a dye and a binding site that binds to the objective substance  2 . The first substance  5  may be a combination of two substances, that is, for example, a dye and a substance including a binding site. Alternatively, the first substance  5  may be a single substance that includes both a binding site and a dye. The first substance  5  has lipid membrane permeability as a whole. 
     The dye is a substance that does not generate fluorescence when being unbound to the objective substance  2  and generates fluorescence when being bound to the objective substance  2 . Alternatively, as will be described in detail, the dye may be a substance that generates fluorescence with different wavelengths depending on whether the dye is bound to the objective substance  2 . 
     Since the first substance  5  has lipid membrane permeability, the first substance  5  permeates and penetrates the lipid membrane  3  of each encapsulated particle  4   a  and non-encapsulated particle  4   b . In each encapsulated particle  4   a , the first substance  5  and the objective substance  2  are bound, which cause the dye to generate the fluorescence. 
     Next, in the irradiating step (S 2 ), the mixture is irradiated with excitation light that excites the dye to generate fluorescence. The light irradiated in the irradiating step (S 2 ) is excitation light that generates scattered light from the lipid particles  4  and excites the dye to generate fluorescence. 
     By the light irradiation, the light may fall on the lipid particles  4  in the lipid particle group  1  and may be scattered. 
     Accordingly, the total number of lipid particles is obtained by detecting the scattered light obtained from the lipid particles  4  and by counting the number of the plurality of lipid particles  4  causing scattered light  6   a  during the irradiating step (S 2 ) (first measuring step (S 3 )). 
     In addition, the light irradiation excites the dye in the first substance  5 . As a result, fluorescence  6   b  is obtained from the encapsulated particles  4   a  but not from the non-encapsulated particles  4   b.    
     Accordingly, the number of encapsulated particles  4   a  is obtained by detecting the fluorescence  6   b  and by measuring the number of encapsulated lipid particles  4   a  causing the fluorescence  6   b  during the irradiating step (S 2 ) (second measuring step (S 4 )). Next, in the calculating step (S 5 ), the total number of the plurality of lipid particles  4  and the number of encapsulated particles  4   a  are used to calculate an abundance ratio of the encapsulated particles  4   a , for example, by the following Formula (I): 
       Abundance ratio of encapsulated particles=Number of encapsulated particles 4 a /Total number of the plurality of lipid particles in lipid particle group  (I)
 
     In this manner, an abundance ratio of the encapsulated particles  4   a  is determined. An abundance ratio of the encapsulated particles  4   a  is a new index for evaluating a quality of the lipid particle group  1 . 
     In the related art, a fill ratio is used as an index for evaluating a quality of a lipid particle. A fill ratio is obtained by measuring an amount of residual objective substances which remains in a solution without being contained in a lipid particle. For example, in a case where the objective substance  2  is a nucleic acid, an amount of residual nucleic acid is estimated from a turbidity of a solution. In other words, this method is to evaluate a quality of a lipid particle group from estimation of a total amount of objective substances contained in lipid particles. 
     On the other hand, the analysis method of the embodiment enables direct measurement of the total number of lipid particles and the number of encapsulated particles  4   a . The present inventors have found that an abundance ratio of the encapsulated particles  4   a  according to the embodiment has a high correlation with the delivery efficiency of the objective substance  2  to a cell. On the other hand, a fill ratio according to the related art has a low correlation with the delivery efficiency of the objective substance  2 . This finding indicates that an abundance ratio of particles containing the objective substance  2  has a greater influence on the delivery efficiency of the objective substance  2  than an amount of the objective substances  2  contained in lipid particles. Therefore, an abundance ratio of the encapsulated particles  4   a  according to the embodiment may be a new index that enables more accurate evaluation of a quality of the lipid particle group  1 . 
     For the above reasons, in producing the lipid particle group  1 , when trying to uniform a quality based on a fill ratio in the related art, an abundance ratio of the encapsulated particles  4   a  is not always constant, which may vary the delivery efficiency to a cell. On the other hand, when a quality is controlled based on an abundance ratio of the encapsulated particles  4   a  by the method according to the embodiment, it is possible to produce the lipid particle group  1  with more stable introduction efficiency into a cell. In addition, the measurement of an amount of objective substances remaining in a solution as in the related art is not always accurate, depending on compositions of a solution. However, according to the method of the embodiment, it is possible to directly count the number of encapsulated particles  4   a , which makes it possible to obtain more accurate information than the method in the related art. 
     From the above facts, the analysis method of the embodiment provides a new index (an abundance ratio of the encapsulated particles  4   a ) for more accurate evaluation of a quality of the lipid particle group  1  and provides a simple method for determining the index. 
     Hereinafter described are details of each material and procedure used in the method of the embodiment. 
     A lipid molecule used as a material of the lipid membrane  3  is, for example, a main component of a biomembrane. The lipid molecule is a phospholipid cationic lipid, cholesterol, or sphingolipid, such as diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, kephalin, cerebroside, or the like or combinations thereof. 
     The lipid membrane  3  may further include other components. Examples of other components include a component that adjusts an acid dissociation constant of the lipid membrane  3 , a component that controls a particle size of the lipid membrane  3 , a component that increases an amount of the objective substances  2  contained, a component that facilitates fusion between the lipid membrane  3  and a cell, and/or a component that facilitates delivery of the objective substance  2  into a cell. 
     The lipid membrane  3  may be a lipid monomolecular membrane or a lipid bilayer membrane. Furthermore, the lipid particles may be a single-layer film or a multilayer film. The lipid membrane  3  may be particles including lipids called liposomes, vesicles or capsules. 
     The objective substance  2  may include one type of substance or a plurality type of substances. 
     The lipid particles  4  may contain a substance other than the objective substance  2  such as a drug that makes it easier for the lipid membrane  3  to contain the objective substance  2 , a drug that increases an amount of the objective substances  2  contained, and/or a drug that facilitates delivery of the objective substance  2  into a cell. 
     A type of the first substance  5  is selected according to a type of the objective substance  2 . The first substance  5  is not limited and may be any substance having a binding site that binds to the objective substance  2 , including a dye that generates fluorescence by the binding, and having lipid membrane permeability. 
     As the first substance  5 , a substance having or estimated to have the above properties may be used. Alternatively, a combination of those substances may be used. Alternatively, when it is unknown whether a candidate substance for the first substance  5  has a binding property to the objective substance  2  or has lipid membrane permeability, the first substance  5  may be determined by studying properties or permeability of the candidate substance. 
     A binding property to the objective substance  2  is studied by, for example, electrophoresis, column chromatography, dot blot, surface plasmon resonance measurement, or circular dichroism spectrum measurement. 
     Lipid membrane permeability is studied, for example, by parallel artificial membrane permeability assay in which a lipid as a material of the lipid membrane  3  is applied to an artificial membrane to assess permeability, or by a method in which a living cell is used to assess permeability into the cell. In the parallel artificial membrane permeability assay, a space in a container is separated by a porous sheet such as a membrane filter coated with lipid. One side of the space is filled with a solution including a candidate substance, and the other side of the space is filled with a solution not including a candidate molecule. The candidate substance is studied whether it transfers from one side to the other side of the space so as to study lipid membrane permeability. The lipid membrane used herein has preferably as few holes as possible, and the lipid is preferably the same as one used in the lipid membrane  3 . In the method using a living cell, an animal cell such as human colon cancer-derived cell line Coco-2 is brought into contact with a candidate substance and is monitored with a microscope. When fluorescence is observed in a cell membrane, the candidate substance is determined to have lipid membrane permeability. 
     The binding site of the first substance  5  is not limited in type. The first substance  5  may be a substance having a binding site that binds to the objective substance  2  by any one of hydrogen bonding, ionic bonding, van der Waals forces or hydrophobic bonding. 
     For example, when the objective substance  2  is a nucleic acid, the first substance  5  is a nucleic acid binding stain. Examples of the nucleic acid binding stain include PicoGreen, SYBR Green, Eva Green, and AccuBlue. Alternatively, a nucleic acid having a sequence that hybridizes with a nucleic acid objective substance  2  may be used as the binding site of the first substance  5 . This nucleic acid is, for example, modified to have lipid membrane permeability. 
     When the objective substance  2  is an antigen, the binding site of the first substance  5  may be an antigen-binding protein labeled with a fluorescent dye. The antigen-binding protein may be, for example, an antibody or a fragment thereof. The antigen-binding protein is modified and/or has a size adjusted to have lipid membrane permeability. 
     When the objective substance  2  is an enzyme substrate, the binding site of the first substance  5  may be a fragment having a correspondent enzyme or a binding site to the enzyme. 
     The binding site of the first substance  5  may be a receptor corresponding to the objective substance  2  or a fragment thereof having a binding site to the objective substance  2 . 
     Alternatively, the above combination of the objective substance  2  and the binding site of the first substance  5  may be inverted. For example, the objective substance  2  may be an antigen-binding protein and the binding site of the first substance may be an antigen. Other combinations are also available. 
     The dye is not limited in type. Any dye having a wavelength in a range detected by a standard fluorescence sensor may be used. 
     As the dye, it is also possible to use a substance that generates fluorescence having different wavelengths depending on whether the objective substance  2  and the first substance  5  are bound. For example, it is possible to employ a dye that causes fluorescence resonance energy transfer (FRET) and varies in fluorescence wavelength when binding to the objective substance  2 . In a case where the dye having a variable fluorescence wavelength is used, the excitation light irradiated which excites fluorescence after a wavelength varied by the binding is preferably used in the irradiating step (S 2 ). Accordingly, scattered light is obtained from the plurality of lipid particles  4  in the first measuring step (S 3 ), and fluorescence is obtained from the encapsulated particles  4   a  in the second measuring step (S 4 ). 
     The first measuring step (S 3 ) may be performed, for example, by capturing an image of the mixture obtained in the mixing step (S 1 ) with a detector (for example, a camera and a microscope) that detects scattered light during the irradiating step (S 2 ) and by counting spots where scattered light is obtained. Alternatively, the number of lipid particles may be reckoned by flowing the lipid particle group  1  through the flow channel. The method comprising; irradiating a specific region of the flow channel with the excitation light (the irradiating step (S 2 )); detecting scattered light in the region over time by an optical sensor; and counting the number of particles passed through the flow channel as the number of times of the scattered light is detected. 
     The second measuring step (S 4 ) is performed in a similar manner to the first measuring step (S 3 ) except that fluorescence is measured instead of scattered light. A detector to be used in this step may comprise a filter for detecting a desired fluorescence wavelength. 
     In the first measuring step (S 3 ) and the second measuring step (S 4 ), it is not always necessary to measure the number of lipid particles in the whole solution. The total number of the plurality of lipid particles  4  and the number of encapsulated particles  4   a  may be measured for part of the solution. Using the value as a representative value, an abundance ratio may be calculated and the resulting value may be used as an abundance ratio of the whole solution. For example, an image obtained by capturing part of the solution may also be used. In addition, an abundance ratio may be determined, for example, by calculating the total number of the plurality of lipid particles  4  and the encapsulated particles  4   a  per unit volume of the solution based on values of those numbers. Alternatively, an abundance ratio of part of the solution may be measured for a several times, and the average may be used as an abundance ratio of the solution. 
     The irradiating step (S 2 ), the first measuring step (S 3 ), and the second measuring step (S 4 ) may be performed at once using, for example, an apparatus that enables measurement of both scattered light and fluorescence. An example of such an apparatus includes an apparatus using a nanoparticle tracking analysis (NTA). 
     (Analysis Apparatus/Analysis Substrate) 
     The analysis method of the embodiment may be performed by an analysis apparatus. 
     As illustrated in  FIG. 4 , an analysis apparatus  10  includes, for example, an analysis substrate  100 , a storage  20 , a processor  30 , a liquid feed unit  40 , and a display  50 . 
     First, the analysis substrate  100  will be described with reference to  FIG. 5 . As illustrated in  FIG. 5 , the analysis substrate  100  includes a base plate  101 , a flow channel  102  that is disposed on the base plate  101  and has an upstream-side end divided into two branches, and a detector  103  disposed on a downstream-side end of the flow channel  102 . The analysis substrate  100  is divided into three sections according to functions. In other words, the analysis substrate  100  is provided with: a mixing unit  104  that includes the upstream-side end of the flow channel  102 ; a detecting unit  106  that includes the downstream-side end of the flow channel  102  and includes the detector  103 ; and a separating unit  105  disposed between the mixing unit  104  and the detecting unit  106 . 
     The mixing unit  104  includes a sample injection port  107  and a reagent injection port  108 . A flow channel  102   a  having the sample injection port  107  at the end thereof, and a flow channel  102   b  having the reagent injection port  108  at the end thereof are joined together and connect to the flow channel  102 . 
     The sample injection port  107  is an opening for injecting the solution including the lipid particle group  1  into the flow channel  102 . The reagent injection port  108  is an opening for injecting a solution including the first substance  5  into the flow channel  102 . 
     The flow channel  102  leads to the separating unit  105 . In the separating unit  105 , the flow channel  102  is filled with a separating agent (not illustrated). The separating agent is a material that changes moving speeds of the lipid particles  4  that pass through the flow channel  102  according to a particle size. Examples of the separating agent include nanopillars, gels, nanomeshes, and porous agents. The flow channel  102  further leads to the detecting unit  106 . 
     The detecting unit  106  includes the detector  103  disposed at a first measurement region R of the flow channel  102 . As illustrated in  FIG. 6 , the detector  103  includes a light source  109 , a scattered light detector  111 , and a fluorescence detector  112 . The light source  109  is configured to irradiate the lipid particle group  1  in at least the first measurement region R of the flow channel  102  with excitation light  110 . The scattered light detector  111  and the fluorescence detector  112  are disposed on side surfaces of the flow channel  102  in the first measurement region R. 
     The scattered light detector  111  includes a scattered light sensor (not illustrated) such as a CMOS image sensor which enables detection of scattered light from the lipid particle group  1  in the flow channel  102 . 
     The fluorescence detector  112  includes a fluorescence sensor (not illustrated) such as a CMOS image sensor which enables detection of scattered light from the lipid particle group  1  in the flow channel  102 . For example, the fluorescence sensor includes a filter  113  on the side closer to the flow channel  102 . 
     The scattered light detector  111  and the fluorescence detector  112  face each other across the flow channel  102  in  FIG. 6  but are not limited to such arrangements. The scattered light detector  111  and the fluorescence detector  112  may be disposed at any positions as long as each light is detected from the lipid particle group  1  in the flow channel  102 . For example, both detectors may be disposed on one side surface of the flow channel  102 , and one of the detectors may be disposed close to the upstream side, and the other close to the downstream side. 
     As illustrated in  FIG. 5 , the downstream-side end of the flow channel  102  has an outlet  114 . 
     The analysis substrate  100  does not necessarily include the separating unit  105 . However, the separating unit  105  may make it possible to obtain accurate information associated with a particle size of the lipid particles  4 . 
     The analysis substrate  100  is, for example, detachably attached to the analysis apparatus  10 . 
     Next, other units of the analysis apparatus  10  will be described with reference to  FIG. 4 . 
     The storage  20  is electrically connected to the analysis substrate  100  and includes, for example: scattered light measurement data  21  obtained from the scattered light detector  111  (for example, an image or video obtained by capturing scattered light obtained from the lipid particle group  1 , a scattered light intensity and/or the number of times the scattered light is obtained); fluorescence measurement data  22  obtained from the fluorescence detector  112  (for example, an image or video obtained by capturing fluorescence obtained from the lipid particle group  1 , a fluorescence intensity and/or the number of times the fluorescence is obtained); a number arithmetic expression  23  for measuring the total number of particles based on the scattered light measurement data  21  and measuring the total number of encapsulated particles  4   a  based on the fluorescence measurement data  22 ; total particle number data  24 ; encapsulated particle number data  25 ; an abundance ratio arithmetic expression  26  for calculating an abundance ratio of the encapsulated particles  4   a  based on each number data; an abundance ratio  27  of the encapsulated particles  4   a ; and/or a program P for controlling each unit. 
     The processor  30  is electrically connected to the storage  20  and includes, for example, a number arithmetic unit  31  and an abundance ratio arithmetic unit  32 . For example, using the number arithmetic expression  23 , the number arithmetic unit  31  measures the total number of particles from the scattered light measurement data  21  and measures the number of encapsulated particles  4   a  from the fluorescence measurement data  22 . Using the abundance ratio arithmetic expression  26 , the abundance ratio arithmetic unit  32  calculates, for example, an abundance ratio of the encapsulated particles  4   a  from the total particle number data  24  and the encapsulated particle number data  25 . 
     The display  50  is electrically connected to the processor  30  and the storage  20 , and may include, for example, a display or a printer. 
     The analysis apparatus  10  may further include an input unit (not illustrated) that enables an operator to start or stop each operation of the analysis apparatus  10 . The input unit includes, for example, a button and a keyboard electrically connected to the processor  30  and the storage  20 , and the processor  30  may send an instruction to each unit based on an input signal. The display  50  may also include a touch-screen input unit. 
     The storage  20 , the processor  30 , the display  50 , and the input unit may be configured to as a computer. 
     The liquid feed unit  40  includes, for example, a sample reservoir, a reagent reservoir, tubes, and pumps (not illustrated). For example, the sample reservoir is connected to the sample injection port  107  via the tube and reserves the solution including the lipid particle group  1 . The reagent reservoir is connected to the reagent injection port  108  via the other tube and reserves the solution including the first substance  5 . The pumps are disposed at any position in the tubes and are configured to deliver the solutions from the reservoirs to corresponding injection ports. 
     Hereinafter described is the analysis method using the analysis apparatus  10 . 
     First, the solution including the lipid particle group  1  which is to be analyzed is reserved in the sample reservoir. In addition, the solution including the first substance  5  is reserved in the reagent reservoir. The analysis substrate  100  is attached to the analysis apparatus  10 . The pumps of the liquid feed unit  40  are driven so that the solution including the lipid particle group  1  and the solution including the first substance  5  are delivered to the flow channel  102  from the sample reservoir and the reagent reservoir through to the sample injection port  107  and the reagent injection port  108 , respectively. Accordingly, the solution including the lipid particle group  1  and the solution including the first substance  5  are mixed. 
     Alternatively, the solution including the lipid particle group  1  may be mixed with the first substance  5  in advance, and the resulting mixture may be injected from the sample injection port  107  and/or the reagent injection port  108 . In that case, the flow channel  102  is not necessarily branched at the upstream side, and an analysis substrate with one injection port may be used. 
     Next, to feed the mixture to the detecting unit  106  via the separating unit  105 , another solution is injected into the flow channel  102  by the liquid feed unit  40  or the solution is drawn out of the outlet  114 . Lipid particles  4  that have passed through the separating unit  105  flow to the detecting unit  106  in ascending order (or descending order) of particle size. 
     Next, in the detector  103  of the detecting unit  106 , excitation light is irradiated from the light source  109 , and the scattered light detector  111  measures scattered light from the lipid particle group  1  in the flow channel  102  so as to obtain the scattered light measurement data  21 . In addition, the fluorescence detector  112  measures fluorescence to obtain the fluorescence measurement data  22 . 
     For example, the analysis substrate  100  includes pads (not illustrated) connected to each of the scattered light detector  111  and the fluorescence detector  112 . The scattered light measurement data  21  and the fluorescence measurement data  22  are taken from each pad, and these pieces of data are stored in the storage  20 . 
     Next, the number arithmetic unit  31  of the processor  30  takes the scattered light measurement data  21  and the number arithmetic expression  23  from the storage  20  and calculates the total number of lipid particles based on the number arithmetic expression  23 . The total particle number data  24  is stored in the storage  20 . Furthermore, the fluorescence measurement data  22  and the number arithmetic expression  23  are taken from the storage  20  and the number of encapsulated particles  4   a  is calculated based on the number arithmetic expression  23 . The encapsulated particle number data  25  is stored in the storage  20 . 
     Next, in the abundance ratio arithmetic unit  32  of the processor  30 , the total particle number data  24 , the encapsulated particle number data  25 , and the abundance ratio arithmetic expression  26  are taken from the storage  20  so as to calculate the abundance ratio  27  of the encapsulated particles  4   a . The abundance ratio  27  is stored in the storage  20 , and the abundance ratio  27  is displayed on the display  50 . 
     Each of the procedures may be automatically performed by the program P or may be performed manually by an operator using the input unit of the apparatus. 
     On completion of the analysis method, the analysis substrate  100  may be removed from the analysis apparatus  10  and discarded. When the analysis method is performed again, a new analysis substrate  100  may be used. 
     The analysis apparatus does not necessarily include the analysis substrate  100  and may be equipped with a sample reservoir (for example, a flow channel), a light source, a scattered light sensor and/or a fluorescence sensor. 
     (Analysis Kit) 
     According to the first embodiment, there is provided an analysis kit including the analysis substrate  100  and the first substance  5 . The first substance  5  is provided, for example, in a liquid state, being included in a desired solvent. The solvent is selected according to a type of the first substance  5 . Examples of the solvent include physiological saline or a buffer solution. Alternatively, the first substance  5  may be in a powder state. 
     (Analysis Method for Evaluating a Quality of a Lipid Particle Group) 
     In another embodiment, the analysis method may be a method for evaluating a quality of a lipid particle group. A quality of a lipid particle group is evaluated from an index, that is, an abundance ratio of the encapsulated particles  4   a  obtained by Formula (I). 
     As illustrated in  FIG. 7 , such an analysis method includes, for example, the above steps (S 1  to S 5 ) and an evaluating step (S 6 ). In the evaluating step (S 6 ), a quality of a lipid particle group is evaluated from an abundance ratio as an index obtained from a result of the calculating step (S 5 ). For example, in the evaluating step (S 6 ), a threshold of an abundance ratio is determined in advance. When an abundance ratio is over the threshold, a quality of the lipid particle group  1  is evaluated as good. When an abundance ratio is equal to or less than the threshold, a quality of the lipid particle group  1  is evaluated as poor. The threshold is not limited and may be determined according to the use of the lipid particle group  1 . 
     The analysis apparatus may be configured to further perform an evaluating step for the lipid particle group  1  as illustrated in  FIG. 7 . For example, as illustrated in  FIG. 8 , an analysis apparatus  11  is provided with a storage  20  that further includes an abundance ratio threshold  28 , and a processor  30  that further includes an evaluation unit  33 . The evaluation unit  33  compares an abundance ratio  27  with the abundance ratio threshold  28 . When the abundance ratio  27  is higher than the abundance ratio threshold  28 , the evaluation unit  33  displays on a display  50  that a quality of the lipid particle group  1  is good. When the abundance ratio  27  is lower than the abundance ratio threshold  28 , the evaluation unit  33  may display on the display  50  that a quality of the lipid particle group  1  is poor. 
     In another embodiment, the analysis apparatus  10  or the analysis apparatus  11  may be, for example, attached to a device that produces the lipid particle group  1 . In this case, the liquid feed unit  40  of the analysis apparatus  10  may include a sampling system that samples part of the solution including the lipid particle group  1  from a production lot and injects the solution into the sample injection port  107  of the analysis substrate  100 . In addition, the processor  30  may include a changing unit that changes production parameters based on an abundance ratio or a quality evaluation result obtained by the analysis apparatus  10  or the analysis apparatus  11 , and the changing unit may send instructions to the production device to change the parameters. Alternatively, in regard to a lipid particle group  1  evaluated as good based on an abundance ratio or a quality evaluation result, the processor  30  may instruct the production device to deliver the lipid particle group  1  as a final product to a line for shipping preparation. 
     The above analysis method and the analysis apparatus can provide a lipid particle group with a higher quality. In addition, immediately before using a lipid particle group, evaluating the lipid particle group by the analysis method or with the analysis apparatus of the embodiment enables prediction of the cell introduction efficiency according to results. Therefore, it is possible to select and use the lipid particle group  1  with a higher quality. 
     Second Embodiment 
     (Analysis Method) 
     In an analysis method according to a second embodiment, an abundance ratio of encapsulated particles  4   a  is determined by measuring scattered light without measuring fluorescence. As illustrated in  FIG. 9 , the analysis method includes the following steps: 
     (S 11 ) an irradiating step of irradiating a solution including a lipid particle group with light;
 
(S 12 ) a measuring step of measuring at least two of the total number of the plurality of lipid particles, the number of encapsulated particles  4   a , and the number of non-encapsulated particles  4   b  in the solution, by detecting scattered light obtained from each particle included in the lipid particle group; and
 
(S 13 ) a calculating step of calculating, based on a result of the measuring step, an abundance ratio of the encapsulated particles by Formula (I) (Abundance ratio of encapsulated particles  4   a =The number of encapsulated particles  4   a /The total number of the plurality of lipid particles in the lipid particle group).
 
     Hereinafter, each step will be described in detail. 
     The light to be irradiated in the irradiating step (S 11 ) is not limited as long as scattered light is obtained from each particle included in a lipid particle group  1 . 
     In the measuring step (S 12 ), for example, a scattered light intensity obtained from each lipid particle  4  is detected. The scattered light intensity may increase with an increase in density of the lipid particles  4 . Since the encapsulated particles  4   a  have a higher density than the non-encapsulated particles  4   b , the encapsulated particles  4   a  may have a higher scattered light intensity. 
     For example, when plotting the number of lipid particles  4  with respect to the scattered light intensity, two peaks may be obtained as illustrated in  FIG. 10 . A peak P 1  where the scattered light intensity may be large corresponds to the encapsulated particle  4   a . Accordingly, the number of particles at the peak P 1  is set as the number of encapsulated particles  4   a . A peak P 2  where the scattered light intensity may be small corresponds to the non-encapsulated particle  4   b . Accordingly, the number of particles at the peak P 2  is set as the number of non-encapsulated particles  4   b.    
     When, for example, a threshold of the scattered light intensity is determined using a reference lipid particle group with a known abundance ratio of the encapsulated particles  4   a , the number of particles in a range higher than the threshold is measured as the number of particles at the peak P 1 . On the other hand, particles in a range lower than the threshold are counted as the non-encapsulated particles  4   b  at the peak P 2 . 
     Regardless of the scattered light intensity, the number of lipid particles  4  causing scattered light represents the total number of lipid particles. 
     An abundance ratio of the encapsulated particles  4   a  is calculated from two of the following three values obtained as described above, that is, the total number of lipid particles, the number of encapsulated particles  4   a , and the number of non-encapsulated particles  4   b . Therefore, measuring at least two of the three numbers is enough. 
     The measuring step (S 12 ) may be performed by, for example, nanoparticle tracking analysis (NTA). For example, analysis by NTA may be performed by a commercially available measurement instrument such as NanoSight (trade name, manufactured by Malvern Panalytical). 
     The calculating step (S 13 ) may be performed in a similar manner to the calculating step (S 5 ) of the first embodiment. 
     According to this method, an abundance ratio of the encapsulated particles  4   a  is determined without detecting fluorescence. 
     The analysis method of the second embodiment may also include an evaluating step similar to the evaluating step (S 6 ). 
     According to another embodiment, scattered light may be detected over time in the measuring step (S 12 ), and furthermore, a Brownian momentum of the scattered light may be measured. The Brownian momentum of the scattered light increases with an increase in particle size. Since a particle size of the encapsulated particles  4   a  may be larger than a particle size of the non-encapsulated particles  4   b , particles with a Brownian momentum equal to or more than a threshold may be counted as the encapsulated particles  4   a , and particles with a Brownian momentum equal to or less than the threshold may be counted as the non-encapsulated particles  4   b.    
     According to another embodiment, a solution including the lipid particle group  1  and a first substance  5  may be mixed before the irradiating step (S 11 ). Such a condition is preferable in that the scattered light intensity to be detected is further increased so that the scattered light can be detected with higher sensitivity. 
     It is possible to perform the analysis method according to the second embodiment with an analysis substrate and an analysis apparatus. The analysis substrate used in the analysis method may not include a fluorescence detector  112 , and other configurations may be similar to those in the analysis substrate according to the first embodiment. For example, a number arithmetic unit  31  is configured to measure the number of encapsulated particles  4   a  and the number of non-encapsulated particles  4   b  from, for example, a scattered light intensity and a Brownian momentum of particles. 
     EXAMPLE 
     Hereinafter described are Examples in which an analysis method of the embodiment was used. 
     Example 1. Measure the Number of DNA-Containing Lipid Particles 
     DNA and a lipid were mixed to obtain a solution including a lipid particle group. A nucleic acid binding stain (PicoGreen) having lipid membrane permeability was added to the solution. Particles were irradiated with laser light (wavelength 488 nm) by an NTA device (NanoSight, manufactured by Malvern Pnanalytical). During the irradiation, scattered light and fluorescence from each lipid particle were measured to determine the number of lipid particles that generate each light. There was prepared a scatter diagram of the particle size and the number obtained from a scattered light or fluorescence intensity and a moving speed of the Brownian motion of the particles (diffusion coefficient D) ( FIG. 11 ). 
     Mainly, two peaks (A and B) were obtained in the axis of the scattered light or fluorescence intensity. Since the fluorescence intensity correlates with particle density, it is inferred that the peaks A and B represent DNA-encapsulated particles and DNA-non-encapsulated particles, respectively. Accordingly, it becomes clear that using a nucleic acid binding stain with lipid membrane permeability enables measurement of the number of DNA-encapsulated particles and DNA-non-encapsulated particles. 
     Example 2. Compare Method of Embodiment with Measurement of Fill Ratio 
     Three samples were collected from each of two lots (lot A and lot B) for producing lipid particles containing DNA including a reporter gene (the samples were referred to as samples A 1 , A 2 , A 3 , B 1 , B 2 , and B 3 ). Using the samples A 1  to A 3  and B 1  to B 3 , i) abundance ratio of nucleic acid-containing lipid particles, ii) fill ratio, and iii) DNA expression ratio were studied. 
     In regard to i), using a device similar to Example 1, an abundance ratio of DNA-encapsulated particles was calculated from Formula (I), referring the number of particles causing scattered light as the total number of lipid particles and referring the number of particles causing fluorescence as the number of DNA-encapsulated particles. 
     In regard to ii), an amount of residue nucleic acids remaining in a solution without being contained in a lipid particle was measured, and a fill ratio was measured from the following formula: Fill ratio=((Amount of nucleic acids input)−(Amount of residual nucleic acids))/(Amount of nucleic acids input). 
     In regard to iii), each sample was brought into contact with a cell (Jurkat cell) to introduce DNA into the cell, and a value of fluorescence from the reporter gene was measured. 
     Table 1 and  FIG. 12  show the results. 
     
       
         
           
               
               
               
               
               
               
             
               
                   
                 TABLE 1 
               
               
                   
                   
               
               
                   
                 Number of 
                 Number of 
                 Abundance 
                   
                 Fluorescence 
               
               
                   
                 particles 
                 particles 
                 ratio of 
                   
                 intensity 
               
               
                   
                 (scattered light 
                 (fluorescence 
                 encapsulated 
                 Fill 
                 (DNA expression 
               
               
                   
                 was detected) 
                 was detected) 
                 particles 
                 ratio 
                 ratio) 
               
               
                   
                 [Particles/ml] 
                 [Particles/ml] 
                 [%] 
                 [%] 
                 [lux] 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Lot A 
                 A1 
                 6.20E+12 
                 1.87E+12 
                 30.16 
                 68.5 
                 2735 
               
               
                   
                 A2 
                 8.54E+12 
                 2.26E+12 
                 26.46 
                 57 
                 1766 
               
               
                   
                 A3 
                 6.91E+12 
                 1.90E+12 
                 27.5 
                 61.5 
                 1659 
               
               
                   
                 Average 
                   
                   
                 28.04 
                 62.3 
                 2053.3 
               
               
                 Lot B 
                 B1 
                 8.19E+12 
                 2.82E+12 
                 34.43 
                 48 
                 8130 
               
               
                   
                 B2 
                 9.01E+12 
                 3.63E+12 
                 40.29 
                 62.45 
                 10661 
               
               
                   
                 B3 
                 8.59E+12 
                 5.34E+12 
                 62.17 
                 69.3 
                 7245 
               
               
                   
                 Average 
                   
                   
                 45.63 
                 60 
                 8678.7 
               
               
                   
               
            
           
         
       
     
     As shown in Table 1, values of iii) DNA expression ratio were 2053.3 in lot A and 8678.7 in lot B, indicating that lot B had a higher value than lot A. Values of ii) fill ratio in Lot A and lot B were 62.3 and 60.0, respectively. These are substantially equivalent values and had no correlation with the result of iii). On the other hand, according to the index of i) abundance ratio of encapsulated particles, values in lot A and lot B were 28.04 and 45.63, respectively. Lot B had a higher value than lot A and had correlation with the result of iii) DNA expression ratio. 
     These results show that ii) fill ratio, the index in the related art, cannot accurately evaluate a quality of lipid particles (introduction efficiency of DNA). On the other hand, the results clearly show that a quality of lipid particles is more accurately evaluated from the index of i) abundance ratio of encapsulated particles in the method according to the embodiment. 
     This fact suggests that a quality of lipid particles (introduction efficiency of an objective substance) is more influenced by a ratio of encapsulated particles and non-encapsulated particles than an amount of nucleic acids contained in a lipid particle. 
     Example 3. Study Dye Types 
     In a similar manner to in Example 1, an abundance ratio of encapsulated particles in a solution including lipid particles was determined. 
     In a similar manner to in Example 1, an abundance ratio of encapsulated particles was determined using a nucleic acid binding stain (Eva Green) instead of the nucleic acid binding stain (PicoGreen). 
     Table 2 shows the results. 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 2 
               
               
                   
                   
               
               
                   
                   
                 Number of 
                 Number of 
                 Abundance 
               
               
                   
                   
                 particles 
                 particles 
                 ratio of 
               
               
                   
                 Addition 
                 (scattered light 
                 (fluorescence 
                 encapsulated 
               
               
                   
                 amount 
                 was detected) 
                 was detected) 
                 particles 
               
               
                   
                 [μl] 
                 [Particles/ml] 
                 [Particles/ml] 
                 [%] 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 PicoGreen 
                 5 
                 2.83E+12 
                 8.96E+11 
                 32 
               
               
                 EvaGreen 
                 5 
                 2.84E+12 
                 5.79E+11 
                 20 
               
               
                   
               
            
           
         
       
     
     When Eva Green was used, the abundance ratio of the encapsulated particles decreased compared to a case where PicoGreen was used. This is because a filter in a fluorescence measurement device used in this experiment was not suitable for detecting a fluorescence wavelength of Eva Green. 
     Accordingly, when using a device that enables detection of a fluorescence wavelength of Eva Green with high sensitivity, the abundance seems to be equivalent to the result in the case where PicoGreen was used. 
     While certain embodiments have been described, these embodiments have been presented by way of example only, and are not intended to limit the scope of the inventions. Indeed, the novel embodiments described herein may be embodied in a variety of other forms; furthermore, various omissions, substitutions and changes in the form of the embodiments described herein may be made without departing from the spirit of the inventions. The accompanying claims and their equivalents are intended to cover such forms or modifications as would fall within the scope and spirit of the inventions.