JPH07239291A - Analysis of body fluid sample - Google Patents

Abstract

PURPOSE:To achieve highly sensitive analysis without performing special pretreatment or drying treatment by dripping a body fluid mixed with a mineral acid on a base plate to dry the same and irradiating the dried sample with X-rays to subject the same to total reflection fluorescent X-ray analysis. CONSTITUTION:When fluoric acid is used as a mineral acid, the purity thereof is pref. enhanced and the concn. thereof is 0.1-40%, more pref., 10-25%. The addition amt. of fluoric acid to a body fluid sample is pref. 0.1-2 times per unit volume. The sample soln. and fluoric acid are preliminarily mixed before dripping and the resulting mixture is pref. dripped immediately after mixing and the dripping amt. of the mixture is about 1-10mul usually and, in this case, the spreading of the mixture is affected by the viscosity thereof after mixing but the spreading diameter thereof on the base plate is about 1.5-10mm. The sample soln. spreads flatly and becomes pasty within about 0.5-1min by the reaction of the org. matter such as protein in the sample with fluoric acid. The pasty sample soln. is dried naturally or dried for 1-10min at 40-120 deg.C. The sample spreads on the surface of the base plate and the contact area with the base plate is wide and the close adhesiveness therewith is good and cracking is prevented by org. matter and the irregular reflection of X-rays is prevented.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for analyzing a biological fluid sample. More specifically, the present invention relates to a method of quantifying an element present in a minute amount in a biological fluid by a fluorescent X-ray analysis method.

[0002]

Quantification of trace elements such as iron, copper, zinc and manganese in biological fluids is an indispensable technique for evaluating the degree of heavy metal contamination and the effect on the living body. In addition, since the content of trace elements in biological fluids also changes due to the influence of drugs and metabolic disorders, quantitative analysis of such elements can be carried out by pathological examination of deficiency or hyperplasia (toxicosis) of specific elements. It plays an important role in the evaluation of drug side effects and the study of metabolic mechanisms. Conventionally, such trace elements have been quantified by atomic absorption method or ICP emission spectroscopy. But,
In the atomic absorption method, it takes a long time for measurement, and it is particularly necessary to change the wavelength of the measurement light for each element to be quantified. Therefore, it is not suitable for quantifying many elements. Further, the quantification accuracy is low in the ICP emission spectroscopy.
Therefore, recently, analysis of a biological fluid sample using a fluorescent X-ray analysis method has been proposed. For example, it has been reported that quantification of a trace amount of Se in blood and tissue can be performed at a ppb level by using an energy dispersive spectroscopy system (B. Ska et al., Radiochem. Radioanal. Lett., 31 , No. .
3, pp.165-170 (1977)). In order to reduce the background due to scattered radiation in such a method, it has been proposed to perform quantification by total internal reflection X-ray using a smooth sample substrate surface (P. Wobrauschek et al., X-ray Spectroscopy).
m., 8 , No. 2, pp. 57-62 (1979)).

In the total reflection X-ray fluorescence analysis, the sample is dropped on a smooth substrate, dried and solidified, and the secondary X-ray generated when the sample surface is irradiated with X-rays determines the contained element in the sample. This is a method, and it is necessary to use a substrate whose surface is as smooth as possible so as not to cause irregular reflection of irradiated X-rays, and a glass plate or the like has been conventionally used. However, in order to improve the detection accuracy of trace elements in a sample, a high-purity glass plate is required, and an ordinary glass plate has a high background, which makes accurate analysis impossible. Therefore, as a material having a high purity of substrate components and excellent smoothness, Si wafers and Ga
-It is known to use As wafers as substrates. When these wafers are used as substrates, the wafer surface is washed with hydrofluoric acid in advance and the sample solution is dropped after removing the dirt on the surface. However, if the wafer surface is washed with hydrofluoric acid, The native oxide film is removed, leaving an active hydrophobic surface. For this reason, when the biological fluid is dropped, the sample is repelled and becomes almost spherical, and the sample scatters due to slight vibrations and shocks, which is inconvenient to handle and unstable and easily peeled off even when dried. Further, since the sample is dried and solidified in a swollen state, in the case of a biological fluid sample, there is a problem that the influence of wrinkles on the surface caused by the coagulation of proteins becomes large and the accuracy of analysis is lowered.

[0004]

DISCLOSURE OF THE INVENTION The present invention provides an analysis method which solves the above problems in conventional total reflection X-ray fluorescence analysis, and provides a fluorescent X-ray analysis method capable of simultaneously quantifying a plurality of elements. When applied to a biological fluid sample, it achieves an analysis method that does not require special pretreatment or drying treatment and has high measurement sensitivity.

[0005]

According to the present invention, there is provided a method for analyzing a biological fluid sample having the following constitution. (1) A method for analyzing an element contained in a biological fluid sample by a fluorescent X-ray analysis method, which comprises mixing a biological fluid sample with a mineral acid, dropping the mixture on a substrate, and allowing it to dry, and then irradiating it with X-rays. A method for analyzing a biological fluid sample, which comprises performing a reflected fluorescent X-ray analysis. (2) The analysis method according to (1) above, wherein the mineral acid mixed with the biological fluid sample is hydrofluoric acid, and the substrate onto which the sample is dropped is a Si wafer. (3) The analysis method according to (1) above, wherein the mineral acid mixed with the biological fluid sample is hydrochloric acid, sulfuric acid or nitric acid, and the substrate onto which the sample is dropped is a Ga-As wafer. (4) The sample is a serum sample, and 0.1 to 2 times 0.1 to 40% mineral acid is mixed per unit volume of the dropped sample
The analysis method according to any one of (1) to (3). (5) The analysis method according to any one of (1) to (4) above, wherein the standard solution is added to the biological fluid sample.

The present invention performs quantitative analysis of elements contained in biological fluid by total reflection X-ray fluorescence analysis. Total reflection fluorescence X
In the line analysis, as shown in FIG. 1, the sample 2 is dropped on the surface of the substrate 1, the primary X-ray is irradiated to the sample with a small incident angle φ, and the fluorescent X-ray (secondary Characteristic X-ray) r
Is detected and counted by the detector 3, and the amount of element in the sample is measured by this intensity. The incident angle may be in a range satisfying the condition of total reflection, and specifically, may be 0.01 to 0.1 degree. As the detector 3, a dispersive crystal is mainly used in the wavelength dispersive system, and a semiconductor detector (SSD) is generally used in the energy dispersive system.
According to the total reflection X-ray fluorescence analysis, there is an advantage that the contained elements can be quantified at the same time without the need for pretreatment for removing proteins and the like in the liquid.

The substrate on which the sample is dropped is preferably a Si wafer, a Gs-As wafer, or the like having a high purity and a smooth surface so as to have a semiconductor grade purity. When quantifying an element having a relatively small atomic number such as Al or P having a characteristic X-ray peak close to Si, it is preferable to use a Ga-As wafer so that the peaks do not overlap. The surface roughness of these wafers is approximately 4 nm or less, and the scattering that occurs during X-ray irradiation is small, which is advantageous in improving the accuracy and sensitivity of quantitative determination. Since a natural oxide film is formed on the surface of the substrate, the surface may be washed with hydrofluoric acid to remove the natural oxide film during analysis. The hydrofluoric acid used for cleaning may be one commonly used in acid cleaning of wafers,
The concentration is approximately 0.1 to 5%.

[0008] The biological fluid sample to be analyzed includes various liquids collected from a living body. Specifically, circulating fluid such as blood and lymph fluid, secretory fluid such as digestive fluid, cerebrospinal fluid, amniotic fluid, exudate ,
Examples include liquids such as cell liquids or extracts from solid biological samples and culture media. For blood, a whole blood sample or serum is used. If necessary, these biological fluids may be diluted with pure water, an organic solvent or the like to adjust the viscosity and the protein or salt concentration in the sample, or various additives having known components may be added. The amount of sample required for one measurement may be such that it is dropped on the substrate, and is usually 1 to 10 μl (0.001 to 0.01).
ml) is enough.

In the present invention, when the biological fluid sample is dropped on the substrate, it is essentially important to mix the sample with a mineral acid. The mineral acid is selected according to the type of the substrate, and hydrofluoric acid is suitable when using a Si wafer as the substrate.
When using an As wafer, hydrochloric acid, sulfuric acid, nitric acid or the like is suitable. Hereinafter, a case where a Si wafer is used as a substrate and hydrofluoric acid is mixed with a biological fluid sample will be mainly described.
The same applies when a hydrochloric acid, sulfuric acid, nitric acid or the like is mixed with a biological fluid sample using an As wafer. The purity of hydrofluoric acid to be mixed with the sample is preferably ultra-high purity for semiconductors (99.9999%), and the concentration is suitably 0.1 to 40%.
25% is more preferable. When the concentration of hydrofluoric acid is less than 0.1%, the protein contained in the biological fluid sample is hardly denatured and the adherence between the substrate and the sample solution is low, which is not preferable.
On the other hand, when the concentration of hydrofluoric acid exceeds 40%, the denaturation of the protein in the biological fluid sample becomes remarkable, and the smoothness of the sample surface is significantly impaired. Further, the addition amount of hydrofluoric acid is preferably 0.1 to 2 times per unit volume of the sample. If the addition amount is less than 0.1 times per unit volume, the effect of improving the adherence of the sample cannot be seen. Further, when the amount of hydrofluoric acid added exceeds twice the unit volume of the sample, the error due to denatured protein becomes large. As for the mixing of the sample solution and hydrofluoric acid, it is preferable to mix the biological fluid sample and hydrofluoric acid in advance before dropping, and drop the mixture on the substrate. Immediately after dropping the hydrofluoric acid, the sample solution is dropped. Alternatively, if hydrofluoric acid is dropped immediately after dropping the sample, the smoothness of the sample surface and the adherence to the substrate are deteriorated. If the biological fluid and hydrofluoric acid are left to stand for a long time, the protein in the fluid will coagulate and it will be difficult to add drops, so it is preferable to add drops immediately after mixing.

The amount of the biological fluid dropped is usually about 1 to 10 μl, and in this case, the diameter spreads to about 1.5 to 10 mm on the substrate surface depending on the viscosity after mixing with hydrofluoric acid. When the sample concentration is not sufficient, the dropping may be repeated multiple times. After the dropping, the sample solution spreads flat on a horizontal substrate, and organic substances such as proteins in the biological fluid sample react with hydrofluoric acid to give 0.5
It becomes pasty in about 1 minute. The sample solution is naturally dried after dropping, or is allowed to stand for 1 to 10 minutes together with the substrate at a temperature of 40 to 120 ° C. to be dried to volatilize hydrofluoric acid and water to be solidified.

As shown in FIG. 2, the mixed sample solution 21 of the biological fluid and hydrofluoric acid of the present invention spreads flat on the substrate unlike the conventional sample 22 which bulges into a spherical shape on the wafer surface 23. Therefore, even if the sample liquid dries to cause small wrinkles on the surface, the influence of diffused reflection of irradiated X-rays is reduced. Since the conventional sample 22 dries in a state of bulging on the wafer surface,
Since the wrinkles formed on the surface of the sample are large and irregular, the irregular reflection of the irradiated X-rays has a large effect. Also,
Since the sample according to the present invention spreads on the surface of the substrate, it has a larger contact area with the substrate and better adhesion to the substrate than the conventional dropped sample. Further, the mixed hydrofluoric acid causes proteins and the like in the biological fluid to be denatured to form a paste, which enhances the adhesive strength with the substrate, thereby further improving the adhesiveness with the substrate. further,
Cracks on the surface of the sample are prevented by the organic substance that has been transformed into a paste, so that diffused reflection of irradiated X-rays is suppressed. In addition to this, by mixing hydrofluoric acid into the biological fluid, concentration segregation when the sample is dried and solidified can be prevented, and reliability detection accuracy can be obtained. Usually, in a high salt concentration sample such as serum, the salt concentration is likely to be non-uniform when the sample is dried, and the concentration is higher toward the center of the dropping solution. However, since the concentration segregation is prevented by adding hydrofluoric acid to the serum, the detection accuracy is high.

The elements that can be quantified according to the present invention are all elements that can be analyzed by fluorescent X-ray analysis, and are substantially all elements. For example, iron, copper, lead, zinc, chromium, manganese, heavy metals such as cobalt, nickel and mercury, alkali metals or alkaline earth metals such as calcium and magnesium, other metal elements such as aluminum, selenium, arsenic and phosphorus. Non-metal elements such as are subject to analysis. Quantitative analysis uses, for example, a calibration curve prepared in advance using a sample of known concentration or a calibration curve obtained by adding a standard solution to measure the intensity of the secondary X-ray obtained for the measurement sample to be quantified. This is done by matching the results with a calibration curve.
Since the logarithmic value of the number of counts per second (integrated intensity) usually has a linear relationship with the concentration, collation can be performed relatively easily. Since it is possible to measure the integrated intensity of the peak corresponding to each element by one measurement, it is possible to quantify a plurality of elements simultaneously. The quantifiable concentration is 100
It is about ppb to 0.1% and has a measurement accuracy of about 50 ppb. When a large amount of a specific element is contained, a known method such as removal of the element may be used in order to minimize the influence on the quantification of other elements.

A standard addition method can be used for the quantitative analysis of the present invention. In advance, various amounts of standard solution were added to the biological fluid sample, and 25% hydrofluoric acid was mixed with the biological fluid in the same amount to obtain Si.
Drop on the surface of the wafer, let it air dry, and fluoresce X on the surface of the sample.
Irradiate a line and measure its reflection intensity. For human serum samples, it is advisable to use the following standard solutions containing elements with known concentrations. Addition amount of standard solution is 1 ml of serum sample solution
On the other hand, about 10 to 100 μl is suitable. Ca: 1000 to 10000 ppb, Cu: 50 to 400 ppb,
Fe: 50-400 ppb, Zn: 25-200 ppb, P
t: 25 to 200 ppb

The type and concentration of the element contained in the standard solution are appropriately determined according to the type of biological fluid to be measured. For example, for urine, the following standard solution may be used. The addition amount of the standard solution is about 10 to 100 μl per 1 ml of the urine sample. Al: 0.5-5 mg / l, Co: 10-20 mg / l, C
u: 0.2-1 mg / l, Fe: 3-30 mg / l, Hg:
0.1 to 20 mg / l, Mn: 10 to 100 mg / l, Zn: 40
~ 80 mg / l

[0015]

EXAMPLES AND COMPARATIVE EXAMPLES Examples and comparative examples of the present invention are shown below. Example 1 A chip of a high-purity single crystal Si wafer for a semiconductor device (50 mm square, thickness 2 mm, surface roughness 4 nm) was dropped onto a surface thereof with 25% hydrofluoric acid (purity 99.9999%) to clean the chip surface. did. Next, 500 μl of human serum was added with a predetermined amount (0 μl, 10 μl, 20 μl) of the standard solution containing the element at the concentration shown in Table 2, and further added with 25% hydrofluoric acid (purity 99.99).
99%) was mixed in an amount of 500 μl, and 10 μl of this was dropped on the surface of the chip. After natural drying, the sample surface is irradiated with fluorescent X-rays (irradiation conditions: Mo target, voltage 40 Kv, current 5 m
A, irradiation angle 0.03 °), and the concentration of elements contained in the serum sample was measured by the secondary X-ray generation intensity. The results are shown in Table 1. In order to confirm the accuracy of this analysis method, the same biological fluid sample was analyzed for elemental concentration in the sample by a conventional method (high-frequency inductively coupled plasma, emission spectroscopy). The results are also shown in Table 1.

Comparative Example 1 A serum containing a predetermined amount (0 μl, 20 μl, 40 μl) of the standard solution shown in Table 2 was used, and 25 was added to the surface of the Si wafer chip of Example 1.
After the concentration of hydrofluoric acid was dropped to remove the natural oxide film on the surface, the serum sample solution was dropped, and the concentrations of the elements contained in the serum sample were measured under the same conditions as in Example 1. The results are summarized in Table 1.

[0017]

[Table 1]

[0018]

[Table 2]

As shown in Table 1, according to the analysis method of the present invention, the adherence between the sample and the substrate is excellent, and the flatness of the sample surface can be secured, so that the detection sensitivity of reflected X-rays is high. In addition, the concentration of trace elements such as Pt, which cannot be detected by the method of the comparative example, can be detected. Further, as is clear from the comparison with the measured value by the conventional IPC, etc., the detected accuracy is higher than that of the comparative example. high.

[0020]

According to the analysis method of the present invention, the adhesion between the biological fluid sample and the substrate is good, the handling is easy, and
Since the sample adheres evenly to the surface of the substrate, it is less affected by wrinkles on the dried sample surface, has good detection sensitivity, and has high measurement accuracy. Further, since the method of the present invention is total reflection X-ray fluorescence analysis, the contained elements in the sample biological fluid can be quantified at the same time, and there is no need for a pretreatment for removing organic matter in the liquid as in the conventional case.

[Brief description of drawings]

FIG. 1 is a cross-sectional view showing the outline of total reflection X-ray fluorescence analysis of the present invention.

FIG. 2 is a schematic diagram showing a state in which a dropped sample of the present invention and a conventional dropped sample are compared.

[Explanation of symbols]

1-Substrate, 2-Sample, 3-Detector, 4-B
e window, 5-collimator, 6-anode 21-sample solution of the present invention, 22-conventional sample solution,
23-Substrate surface

Claims (5)

[Claims] 1. A method for analyzing an element contained in a biological fluid sample by a fluorescent X-ray analysis method, which comprises mixing a biological fluid sample with a mineral acid, dropping the mixture on a substrate, and drying the mixture, and then irradiating with X-rays. A method for analyzing a biological fluid sample, which comprises performing a total reflection X-ray fluorescence analysis. 2. The analysis method according to claim 1, wherein the mineral acid mixed with the biological fluid sample is hydrofluoric acid, and the substrate onto which the sample is dropped is a Si wafer. 3. The analysis method according to claim 1, wherein the mineral acid mixed with the biological fluid sample is hydrochloric acid, sulfuric acid or nitric acid, and the substrate onto which the sample is dropped is a Ga-As wafer. 4. The analysis method according to claim 1, wherein the sample is a serum sample, and 0.1 to 2 times 0.1 to 40% of mineral acid is mixed per unit volume of the dropped sample. 5. The standard solution is added to the biological fluid sample.
Analysis method according to any one of 4 to 4.