JP4260975B2 - Lipid analysis method and lipid analyzer - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a technique for efficiently analyzing lipid concentration in a liquid.
[0002]
[Prior art]
Lipid is a general term for intracellular organic substances composed of two parts: a nonpolar group consisting of a relatively long hydrocarbon chain (hydrophobic chain) and a polar group (hydrophilic group) such as an ester group, hydroxyl group, and phosphate group. It is.
[0003]
Conventionally, when performing qualitative analysis or quantitative analysis of lipids, a chromatograph using liquid or gas has been used.
[0004]
A chromatograph is a method in which various solids or liquids are used as a stationary phase and a gas or liquid is used as a developing agent (mobile phase) to move the sample and separate using the difference in the adsorptivity or distribution coefficient of each component in the sample. It is.
[0005]
[Problems to be solved by the invention]
However, the analysis method using the chromatograph has a problem that it is skilled in operation and requires several hours for analysis per sample.
[0006]
In addition, most of the lipids with long hydrophobic chains have unknown measurement conditions when using a chromatograph, and it takes a lot of time and labor to investigate the measurement conditions. Such lipids have been difficult to detect with conventional analytical instruments such as chromatographs.
[0007]
An object of the present invention is to solve this problem and to provide a lipid analysis method and a lipid analysis apparatus capable of efficiently analyzing the lipid concentration in a liquid.
[0008]
[Means for Solving the Problems]
In order to achieve the object, the lipid analysis method of claim 1 of the present invention comprises:
The molecular film of the amphiphilic substance was immersed in the analyte solution, the lipids of the analyte solution with the opposite polarity charge to the molecule layer is adsorbed on the molecular film, of the molecular film by adsorption lipid It is characterized by analyzing lipids in a liquid to be analyzed based on a potential change.
[0009]
The lipid analysis method according to claim 2 of the present invention is the lipid analysis method according to claim 1,
The lipid concentration in the liquid to be analyzed is measured based on the potential change of the molecular film due to the adsorption of the lipid.
[0010]
Moreover, the lipid analyzer of claim 3 of the present invention is:
A reference electrode (23), the molecular film of amphiphilic substances with opposite polarity to the charge of the lipid analyte and (25) and the probe (22) having,
Voltage detection means (35) for detecting a potential difference between a reference electrode of the probe and a molecular film;
Storage means (37a) in which data indicating the relationship between the lipid concentration to be analyzed and the output voltage of the probe is stored in advance;
A calculation means (37) for calculating the lipid concentration in the analysis target liquid from the output voltage when the analysis target liquid is measured by the probe and the data stored in the storage means;
Output means (38) for outputting the calculation result of the calculation means.
[0011]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, embodiments of the present invention will be described with reference to the drawings.
FIG. 1 shows a configuration of a lipid analyzer 20 according to the embodiment.
[0012]
In FIG. 1, the lipid analyzer 20 includes a container 21 for containing a reference solution and a liquid to be analyzed, a probe 22, a voltage detector 35 for detecting a potential difference between the probes 22, and an output of the voltage detector 35 as a digital value. An A / D converter 36 that converts to an A / D converter, an arithmetic device 37 that performs arithmetic processing on the output of the A / D converter 36, and an output device 38 that outputs the arithmetic result of the arithmetic device 37.
[0013]
Probe 22 is intended to be used immersed in a liquid placed in a container 21, a reference electrode 23 for outputting the reference voltage on the measurement, and a molecular film 26 of the amphiphilic substance.
[0014]
The surface of the reference electrode 23 is covered with a buffer layer 24 in which 100 mMole / L potassium chloride is fixed with agar so as not to react with lipid in the liquid, and a lead wire 22a is connected thereto.
[0015]
The molecular film 25 is fixed in a state where one surface is exposed on the surface of a base material 26 such as acrylic, and a buffer layer equivalent to the buffer layer 24 of the reference electrode 23 is provided on the opposite surface of the molecular film 25. 27 is provided with an electrode 28, and a lead wire 22 b is connected to the electrode 28.
[0016]
Molecular film 25, amphipathic substances and a part requiring hydrophilicity moieties and polar with a hydrophobic nonpolar is integrally so as to form a film in a state in which the hydrophilic portion on the surface side When immersed in a liquid, the potential of the membrane changes according to the components in the liquid.
[0017]
A schematic structure of the molecular film 25 is shown in FIG. In FIG. 2, each molecule 31 is composed of a spherical hydrophilic group 31a and a hydrocarbon chain 31b whose atomic arrangement extends from the hydrophilic group 31a, and each of these molecular groups is arranged such that the hydrophilic group 31a side is aligned on the surface side. In addition, a part of the matrix 33 (surface structure, micro structure having a planar extension) on the surface of the membrane member 32 (made of a polymer and a plasticizer described later) is partially dissolved in the matrix (for example, Is contained in the molecule 31 ').
[0018]
Here, the polarity of the hydrophilic group 31a of each molecule 31 of the molecular film 25 must be opposite to the polarity of the hydrophilic group of the lipid to be analyzed.
[0019]
For example, when analyzing the lipid positive charge is dioctyl phosphate _(2C 8 POOH) has a negative charge, oleic acid _(C 10 COOH), diphenyl phosphoric acid, with a lipid content children, such as digital alcohol. Conversely, when analyzing a negatively charged lipid, a lipid molecule such as trioctylmethyl ammonium chloride (TOMA) or oleylamine having a positive charge is used.
[0020]
The molecular film 25, the above lipid component element, in which the the underlying polymer and a plasticizer were prepared by mixing at a predetermined ratio. For example, using a polyvinyl chloride (PVC) is a polymer, mixed with the lipid component child using dioctyl phthalate (DOP), dioctyl phenyl phosphonate (DOPP) or tricresyl phosphate (TCP) as a plasticizer 800 mg of the product was dissolved in 10 cc of THF, transferred to a flat-bottomed container (for example, a 85 mmφ petri dish), and kept at about 30 ° C. for 2 hours on a uniform heated plate to evaporate the THF. A molecular film of about 200 μm can be obtained.
[0021]
When the probe 22 is immersed in a liquid, the distance between the reference electrode 23 and the molecular film 25 is made constant so that the measurement conditions do not change, but the support electrode 29 keeps the reference electrode 23 and the base material 26 constant. You may make it support by the space | interval of.
[0022]
Lead wires 22 a and 22 b of the probe 22 having the molecular film 25 are connected to a voltage detector 35.
[0023]
The voltage detector 35 is configured by, for example, a differential amplifier, detects a difference (voltage) between the potential of the reference electrode 23 and the potential of the molecular film 25 and outputs the difference to the A / D converter 36.
[0024]
The A / D converter 36 converts the output voltage of the voltage detector 35 into a digital value and outputs it to the arithmetic unit 37.
[0025]
The arithmetic unit 37 is constituted by a microcomputer, and stores the output value of the A / D converter 36 in the memory 37a when receiving a storage instruction M from an operation unit (not shown) or the like, and stores the output value of the memory 37a when receiving the arithmetic command C. Based on the output value of the A / D converter 36, the calculation for lipid concentration analysis in the liquid is performed, and the calculation result is output to the output device 38.
[0026]
The output device 38 is configured by a display device, a printer, a communication device with another device, or the like, and transmits the calculation result of the calculation device 37 to the display output, print output, or other device.
[0027]
Here, although the case where there is one molecular film 25 will be described, lipid analysis can also be performed using multiple regression analysis of multivariate analysis based on measured values of a plurality of molecular films having different characteristics. .
[0028]
Next, a method for analyzing lipid concentration in a liquid using the lipid analyzer 20 will be described based on the flowchart of FIG.
[0029]
First, as a previous step, a reference solution that has components close to the analysis target liquid and does not contain the lipid to be analyzed, and a plurality of (for example, three types) concentration known liquids that contain different amounts of analysis target lipids. The same amount of the reference solution and each concentration known solution are put in each container 21.
[0030]
First, the probe 22 is immersed in the reference solution, and the output voltage V0 is stored in the memory 37a of the arithmetic unit 32 (S1).
[0031]
Next, the probe 22 is immersed in the first known concentration solution having the lowest concentration A1 for a certain time (S2).
[0032]
At this time, the molecular film 25 of the probe 22 adsorbs lipid molecules 40 in the first known solution having a polarity opposite to that of the molecular film 25 as shown in FIG. The electrical characteristics of 25 itself change.
[0033]
After a predetermined time has elapsed, the probe 22 is returned to the reference solution, and the output voltage V1 is stored in the memory 37a (S3).
[0034]
Next, the probe 22 is immersed in a second known concentration solution having the second lowest concentration A2 from the reference solution for a certain period of time, and then returned to the reference solution to store the output voltage V2 in the memory 36a. 22 is immersed in the third known concentration solution having the highest concentration A3 from the reference solution for a certain period of time and then returned to the reference solution, and the output voltage V3 is stored in the memory 37a (S4 to S7).
[0035]
As described above, when the voltages V0 to V3 measured in order from the lowest concentration are stored in the memory 37a of the arithmetic device 36, and the first arithmetic command Ca is input to the arithmetic device 36, the arithmetic device 36 then An arithmetic operation is performed to obtain relative voltage values Va, Vb, and Vc of each concentration known solution with respect to the reference solution (S8).
[0036]
Va = V1-V0
Vb = V2-V0
Vc = V3-V0
[0037]
Then, using the relative voltage values Va, Vb, and Vc and the concentrations A1, A2, and A3 of the known concentrations, the following calculations are performed to obtain the coefficients Ka, Kb, and Kc for the concentrations A1, A2, and A3. Calculate (S9).
[0038]
Ka = A1 / Va
Kb = A2 / Vb
Kc = A3 / Vc
[0039]
Next, the arithmetic unit 36 obtains an average value K of Ka to Kc, and stores this coefficient K in the memory 37a as calibration data indicating the relationship between the concentration of the lipid to be analyzed and the voltage (S10).
[0040]
Here, the data when the concentration of lipid TOMA (quaternary ammonium salt) having a positive charge is measured in ppb unit using the molecular film 25 having a negative charge (molecular film made of 2C10 lipid) is actually shown in FIG. Shown in
[0041]
This data shows the measurement results when the lipid TOMA concentration is 1 ppb (a), 3 ppb (b), 10 ppb (c), 30 ppb (d), and 100 ppb (e), and the relationship between the lipid TOMA concentration and the voltage is a solid line. As shown by Q, it changes almost linearly (especially in the range of 3 ppb or more).
[0042]
That is, the proportional coefficients Ka to Kc at the respective concentrations are substantially equal. Therefore, as described above, the average value K of Ka to Kc (corresponding to the slope of the solid line Q in FIG. 5) can be used as calibration data indicating the relationship between the lipid concentration to be analyzed and the voltage. By storing the coefficient K in the memory 37a in advance, the lipid concentration of a liquid whose concentration is unknown can be obtained by calculation.
[0043]
In addition, as described above, it is also possible to measure a liquid having a known lipid concentration using a plurality of molecular films having different characteristics as described above, and to perform a multiple regression analysis using each measured value as a multivariate. It is possible to obtain the relationship (calibration data) between the concentration of lysine and the measured value of each molecular membrane, and from this relationship and the measured value of each molecular membrane for the analyte solution, it is also possible to obtain the lipid concentration of the analyte solution, etc. is there. However, even in this case, each molecular film having a polarity opposite to that of the lipid to be analyzed is used.
[0044]
When the concentration is actually measured, the probe used in order to obtain the relationship between the concentration and the voltage 6 and the used probe 22 is washed to remove the lipid adsorbed on the molecular film 25 (S11).
[0045]
In this case, the probe 22 is first immersed in the above-described reference solution and the output voltage V0 ′ is stored in the memory 37a. The probe 22 is immersed in the analysis target solution for a certain period of time, and as shown in FIG. After the lipid in the analysis target liquid is adsorbed on the molecular film 25, the lipid is returned to the reference liquid and the output voltage V is stored in the memory 37a (S12 to S14).
[0046]
Here, when the second calculation command Cb is input, the calculation device 36 performs the following calculation:
Vx = V−V0 ′
To calculate the relative voltage Vx of the liquid to be analyzed with respect to the reference liquid, and using the relative voltage Vx and the coefficient K, the following calculation is performed:
Ax = K ・ Vx
To determine the lipid concentration A in the analysis target liquid (S15, S16).
[0047]
The calculated density A is displayed, printed, or transmitted to another device by the output device 37 so that the analyst can confirm (S17).
[0048]
In the above description, the concentration is calculated using one coefficient K with the relationship between the concentration and the voltage being a complete proportional relationship. However, as indicated by the broken line R in FIG. A characteristic obtained by linear interpolation (or curve interpolation) is stored in the memory 37a, and the lipid concentration of the analysis target solution may be obtained from the intersection of the voltage when the analysis target solution is measured and the broken line R. . In this case, interpolation calculation or the like is necessary, but the accuracy of the calculated density is higher.
[0049]
In the above description, the probe 22 is immersed in a liquid containing lipid for a certain period of time, the lipid in the liquid is adsorbed to the molecular film 25 of the probe, and a change in the output voltage of the probe due to this adsorption is detected in the reference solution. However, it is also possible to determine the rate of change per unit time of the output voltage of the probe 22 in a state in which the probe 22 is immersed in a lipid-containing liquid and detect the concentration.
[0050]
In this case, for example, as shown in FIG. 6, after the probe 22 is immersed in a known concentration solution of concentration A1, the output voltage V1a of the probe 22 at a certain timing t1 is obtained, and the output voltage V1b of the probe 22 at a different timing t2 And the change rate ΔV1 of the output voltage per unit time is expressed as ΔV1 = (V1b−V1a) / (t2−t1)
Ask for.
[0051]
Similarly, for the solution having a known concentration A2, the probe 22 is immersed, the output voltage V2a of the probe 22 at a certain timing t1 is obtained, the output voltage V2b at the timing t2 is obtained, and the output voltage per unit time is obtained. Change rate ΔV2 of ΔV2 = (V2b−V2a) / (t2−t1)
After the probe 22 is immersed in a known concentration solution of concentration A3, the output voltage V3a of the probe 22 at a certain timing t1 is obtained, the output voltage V3b at the timing t2 is obtained, and the output voltage per unit time is obtained. Change rate ΔV3 of
ΔV3 = (V3b−V3a) / (t2−t1)
Ask for.
[0052]
However, the timings (t1, t2) are not necessarily the same for each concentration. In this case, the probe 22 is washed every time the known concentration solution to be measured is changed.
[0053]
Here, the number of lipids adsorbed to the molecular film 25 per unit time is proportional to the lipid concentration in the liquid, and the output voltage of the probe 22 changes in proportion to the number of lipids adsorbed to the molecular film 25. The change rates ΔV1 to ΔV3 are proportional to the lipid concentration in the liquid.
[0054]
Therefore, the ratio of the concentration to the rate of change in voltage due to lipid is theoretically constant, but there is variation in measurement.
[0055]
Therefore, in the arithmetic unit 36, the ratios Ja to Jc for each concentration are similarly calculated as described above.
Ja = A1 / ΔV1
Jb = A2 / ΔV2
Jc = A3 / ΔV3
And the average value J is calculated.
[0056]
Then, after immersing the probe 22 in the analysis target liquid, the output voltage Va at a certain timing t1 and the output voltage Vb at a different timing t2 are obtained, and the voltage change rate ΔVx per unit time is obtained as follows.
ΔVx = (Vb−Va) / (t2−t1)
And the lipid concentration A in this analysis target solution is
A = J · ΔVx
And may be output to the output device 37.
[0057]
In this case as well, the ratio of the concentration to the rate of change in voltage is not completely constant, but the rate of change obtained at each concentration is interpolated with a straight line or curve, and this interpolation data is stored in the memory 37a. The interpolation data corresponding to the rate of change obtained by measuring the analysis target solution may be stored as the lipid concentration A of the analysis target solution.
[0058]
In the above description, the number of molecules per unit volume of the liquid is determined in units of ppb (1/1000 of ppm) as the lipid concentration, but the weight per unit volume is expressed in g / cm ³ units as the lipid concentration. Or may be determined by molar concentration.
[0059]
The change in the output voltage of the probe 22 is caused by the adsorption of one hydrophilic group of the lipid in the liquid corresponding to one hydrophilic group of the molecular film 25 of the probe 23. The unit may be the charge density of the lipid in the liquid.
[0060]
In the above description, the lipid to be analyzed is known in advance and data indicating the relationship between the concentration and the voltage is obtained in advance, and the lipid concentration in the analysis solution is obtained using this data. The change in the output voltage of the probe 22 is caused by the adsorption of one hydrophilic group of the lipid in the liquid corresponding to one hydrophilic group of the molecular film 25 of the probe 23. Even if the lipid itself is unknown, the charge density can be determined.
[0061]
For example, the charge density of a liquid containing known lipids at a known concentration is proportional to the concentration, so it can be calculated, and the relationship of voltage to concentration described above is replaced with the relationship of voltage to charge density. The data can be stored in advance in the memory, and the lipid charge density of the analysis target liquid can be obtained from the data and the voltage obtained for the analysis target liquid.
[0062]
If the number of charges per molecule of the lipid to be analyzed is known, the molar concentration can also be obtained. If the molecular weight of the lipid to be analyzed is known, the number of molecules per unit volume (ppm) is obtained. be able to.
[0063]
【The invention's effect】
As described above, the present invention is to immerse the molecular film of the amphiphilic substance to be analyzed liquid, the lipid analyte solution with opposite polarity charge as the molecular film adsorbed on the molecular film, Based on the potential change of the molecular film due to the adsorption of the lipid, the lipid concentration in the analysis target liquid is analyzed.
[0064]
For this reason, it is possible to analyze lipids in a short time without requiring skill in operation unlike conventional analytical instruments. In addition, even for lipids whose measurement conditions are not known, concentration analysis can be performed as long as the charge polarity of the hydrophilic group is known, and quaternary ammonium salts and the like that have been difficult with conventional analytical instruments can be easily obtained. Can be analyzed.
[Brief description of the drawings]
FIG. 1 is a diagram showing a configuration of a lipid analyzer according to an embodiment of the present invention. FIG. 2 is a diagram showing a schematic structure of a molecular membrane. FIG. 3 is a flowchart showing a procedure of an analysis method according to an embodiment. Fig. 5 is a diagram for explaining the movement of lipid in the analysis target liquid with respect to the molecular membrane. Fig. 5 is a diagram showing the relationship between the lipid concentration and the measured value. Fig. 6 is a diagram for explaining another concentration analysis method. Explanation of]
20 Lipid analyzer 22 Probe 22a, 22b Lead wire 23 Reference electrode 24 Buffer layer 25 Molecular film 26 Base material 27 Buffer layer 28 Electrode 29 Support material 31 Molecule 31a Hydrophilic group 31b Hydrophobic chain 35 Voltage detector 36 A / D converter 37 Arithmetic device 37a Memory 38 Output device

Claims (3)

The molecular film of the amphiphilic substance was immersed in the analyte solution, the lipids of the analyte solution with the opposite polarity charge to the molecule layer is adsorbed on the molecular film, of the molecular film by adsorption lipid A lipid analysis method comprising analyzing lipids in a liquid to be analyzed based on a potential change.   The lipid analysis method according to claim 1, wherein the lipid concentration in the analysis target liquid is measured based on a potential change of the molecular film due to the adsorption of the lipid. A reference electrode (23), the molecular film of amphiphilic substances with opposite polarity to the charge of the lipid analyte and (25) and the probe (22) having,
Voltage detection means (35) for detecting a potential difference between a reference electrode of the probe and a molecular film;
Storage means (37a) in which data indicating the relationship between the lipid concentration to be analyzed and the output voltage of the probe is stored in advance;
A calculation means (37) for calculating the lipid concentration in the analysis target liquid from the output voltage when the analysis target liquid is measured by the probe and the data stored in the storage means;
A lipid analyzer comprising output means (38) for outputting the calculation result of the calculation means.

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