JPH04177169A - Apparatus for automatically analyzing metal in body fluid - Google Patents

Abstract

PURPOSE:To accurately and rapidly quantity the metal element in a body fluid by using an extremely small amount of a sample by mounting the first reaction part releasing protein in the sample and a membrane separation part removing released protein. CONSTITUTION:A flow path is formed in an apparatus by a fine tube 10 and a liquid supply pump 11. As the fine tube 10, for example, a Teflon tube with an inner diameter of about 1mm or less is used. The flow passage in the fine tube 10 is connected to the front part of a sample introducing part 5 and a reaction part 6 is provided in succession to the introducing part 5 and a membrane separation part 7 is provided in succession to the reaction part 6. In the dissociation of protein, for example, a trichloroacetic acid-acidic reagent is used. When a solubilizing reagent is not used, a molecular sieve membrane is used as a separation membrane to remove protein. When solubilization is performed, an org. phase is separated by a phase separation membrane. In order to detect an element to be analyzed after the removal of protein, necessary pretreatment is performed. A sample solution is introduced into a spectropho tometer having a flow cell to detect the signal from the element to be analyzed and the obtained signal is compared with that obtained using a control solution to calculate the conc. of the element in the sample.

Description

[Detailed description of the invention] [Industrial application field] The present invention relates to an automatic metal analyzer in biological fluids.

[Prior art and its problems]

Quantitative analysis of metal elements in biological fluids has become increasingly important in the field of clinical medicine or clinical testing in recent years. For example, zinc is normally present in serum at 0.9-1
, lppm, but it decreases with diseases such as liver cirrhosis, acute hepatitis, leukemia, and myocardial infarction, so quantifying the amount present in serum is useful for early detection of these diseases. Although it is desirable that such an analysis be carried out promptly at the medical institution where the biological fluid was collected, it is generally performed by bringing the biological fluid sample to an institution specializing in analysis, for reasons such as the need for skill in processing.

For each biological fluid sample, specialized analytical laboratories typically add an acidic solution of trichloroacetic acid to release the proteins.
This is centrifuged to remove proteins, and if necessary, pretreatment is performed depending on the element to be analyzed, and the sample is introduced into an inspection device (such as an atomic absorption spectrometer) for analysis.

However, the above method has the following problems.

(1) The amount of sample required for analysis is large (0.2-0
．． 5m1 or more).

(2) Since protein removal, pretreatment, and detection are performed in sequential batches, it takes a long time from sample collection to obtaining test results.

(3) Since the analysis operation is performed in an open system, there is a risk that the sample will be contaminated during the operation.

(4) Since the analytical operations are complex, a skilled analytical engineer is required.

(5) Since the analysis is done manually, the measurement error is large.

On the other hand, the so-called "flow injection method" is known as an analysis method that suppresses the amount of sample required and enables continuous, simple, and highly accurate analysis. The flow injection method uses an appropriate liquid delivery system (such as a non-pulsating flow metering pump) to send a reagent solution into a thin tube such as a Teflon tube with an inner diameter of about 0.5 to 1 m +++. A fixed amount of sample solution is injected into the flow, the reagent solution and sample solution are mixed and reacted in a thin tube, and the reaction solution is introduced into the detection device for analysis.The analysis speed is fast. All chemical reactions, mixing, dilution, addition of reagents, etc. are performed within the tube, so the risk of contamination of the sample during the operation is extremely small. In addition, it is possible to control the dispersion of the sample by adjusting the geometrical and hydrodynamic conditions of the system, making it possible to use advanced analysis methods that analyze time changes in the detection signal by changing the physical and chemical conditions. Also corresponds. Furthermore, the device can be manufactured at low cost and the running cost is low.

However, for the determination of metal elements in biological fluids, it is essential to remove proteins in order to avoid interference during analysis, and analysis of metals in biological fluids using the flow injection method has not been proposed.

[Problem to be solved by the invention]

An object of the present invention is to provide an inexpensive automatic analysis system that accurately and quickly quantifies metal elements in biological fluids using a very small amount of sample (on the order of tens of microliters).

[Knowledge that leads to problem solving]

As a result of studying methods to solve the above problems, the present inventors applied a flow injection method to first release proteins from body fluids within a tubular system, and then separated the free proteins with a membrane within the tubular system. They discovered that by subsequently introducing analytical reagents into the system and conducting the analysis, it was possible to analyze metal elements in biological fluids all at once with high precision.

[Structure of the invention]

The present invention provides an automatic metal analyzer in a biological fluid using a flow injection method, which is characterized by having a first reaction section that releases proteins in a sample and a membrane separation section that removes the released proteins.

The basic parts of the apparatus of the present invention can be constructed using materials, parts, etc. commonly used in flow injection analysis methods. For the tubing system, it is preferable to use a Teflon tube with an inner diameter of 1 ml or less. The tube diameter may be partially varied to control sample dispersion. The reaction section can be formed by winding the above tube into a coil. It is desirable to use connectors that can be tightly attached to each part of the pipe and to connect the pipe to other parts, and to make each part replaceable if necessary. A pulseless metering pump such as a double plunger pump is used as the liquid pump.

Reagents used for protein release include trichloroacetic acid-acidic solution. Free protein may be solubilized using an organic solvent or an aqueous surfactant solution as a reagent solution along with a trichloroacetic acid solution. When a solubilizing reagent is not used, proteins are removed by fractionation based on molecular weight using a molecular sieve membrane as a separation membrane. When solubilization is performed, an organic phase (protein-containing phase) and an aqueous phase are separated using a phase separation membrane. These molecular sieve membranes and phase separation membranes may be commercially available products. Phase separation itself is a known technique in the flow injection method, and a ready-made phase splitter may be used. The flow injection method provides highly reproducible dispersion and residence time, so the sample solution diffuses into the reagent solution in the tube at a constant rate with good reproducibility, so the chemical reaction always proceeds at a constant rate. Therefore, even if protein removal has not progressed 100%, as long as protein removal has progressed at a certain rate over a certain period of time, there will be no problem in subsequent measurements.

In order to detect the target element after protein removal, necessary pretreatment is performed after protein removal. When the analysis means is inductively coupled plasma emission spectrometry, the sample solution is diluted in the second reaction section. When the analysis means is spectrophotometry or fluorescence photometry, reagents necessary for detection are added as pretreatment reagents.

It is not necessary that the pretreatment reagent or the second reaction part be a single one, and a plurality of pretreatments can be performed as necessary.

As the analytical means, spectrophotometry is simple and preferred.

A sample solution is introduced into a spectrophotometer with a flow cell to detect signals from the target element. The obtained signal is appropriately processed by a computer and compared with the signal obtained from a standard solution of the element to be analyzed, thereby calculating and displaying the concentration in the sample. A series of analysis systems can also be controlled by a computer.

[Specific disclosure of the invention]

The present invention will be explained below using the analysis of total zinc in human serum as an example.

Boiled for 1M The following experiment confirmed protein release and separation of the released protein.

0.5M trichloroacetic acid in 0.5+ml of human serum sample
A 0.1M hydrochloric acid solution was added. The solution became cloudy and it was confirmed that the protein was liberated. This solution was mixed with a pore size of 0.
When the serum was passed through a 45μ PTFE membrane, clean protein-free serum was obtained. In addition, as a membrane, the pore size is 0.2
0μm cellulose acetate membrane, 0.45μm cellulose nitrate membrane and molecular weight cutoff 20.000
Similar clean protein-depleted serum was obtained with the aromatic polyamide membrane.

Loss of M. Total zinc was analyzed using the apparatus of the present invention using human serum as a sample. The equipment used is shown in the figure.

(Device Configuration) A flow path is formed in the device by a thin tube 10 and a liquid feeding pump 11. A polytetrafluoroethylene (PTFE) tube with an inner diameter of 0.5 to 1.0 mm was used as the thin tube.
A double plunger type pump was used as the liquid pump. In the embodiment shown in the figure, a plurality of reagent introduction sections 1 and 2 are provided, each of which is connected to a capillary tube, and the channels within the capillary tubes are connected at a portion before reaching the sample introduction section 5. The sample injection section 5 uses an automatic six-way ceramic valve having a sample loop with a capacity of 100 μL, into which the sample is introduced by a peristaltic pump. A reaction section 6 is provided following the sample introduction section. The reaction section 6 is a PTFE tube wound into a coil with a length of 2 m and an inner diameter of 1.0 mm. A membrane separation section 7 is provided following the reaction section 6. A detection reagent is added to the separated aqueous phase through the reagent introduction section 3.

The reaction between the sample and the reagent takes place in the second reaction section 8.

The second reaction part 8 is a PRFE with a length of 2 m and an inner diameter of 0.5 mm.
It is a tube wound into a coil. A spectrophotometer, which is a detector, is connected to the reaction section. As a spectrophotometer, a Hitachi ratio beam spectrophotometer U-1000 equipped with a Z-type flow cell with an optical path length of 10+a was used to measure the absorbance at 560 nm.

These pumps, spectrophotometers, and automatic six-way valves are all controlled by a computer.

All digital signals from the spectrophotometer are processed by a computer.

(Analysis) 0.5M trichloroacetic acid-0.1M from reagent introduction section 1
Water or diluted hydrochloric acid is introduced into the hydrochloric acid solution as a carrier through the reagent introduction section 2, and human serum sample 1 is introduced through the sample introduction section 5.
00 μL was introduced. As a detection reagent, a zinc coloring reagent 2-(5-bromo-2-pyridylazo)-5(N-propyl-N-sulfopropylamino)phenol sodium salt (5-Br-PAPS) was used. The absorbance at 560 nm due to the formed zinc-5-Br-PAPS complex was measured, and the zinc concentration in the sample solution was calculated based on the relational expression between the zinc concentration and absorbance obtained for the standard solution. The results are shown in the table below along with the analysis results using the conventional method.

Table 0.89 0.920.95
0.931.2
1.21.0
1.1× For each measurement, the results obtained are in good agreement with the results obtained by the conventional centrifugal single atomic absorption method, as shown in the table of average values of three measurements. In addition, the measurement accuracy was 1.5% (C, V) when measuring the same sample five times.

%), and it was possible to measure 60 samples in 1 hour.

〔Effect of the invention〕

In the apparatus of the present invention, in order to analyze a sample, it is only necessary to introduce certain reagents and a sample into the apparatus, so that the analysis operation is simple. Furthermore, since protein removal and pretreatment for analysis are performed in a closed system, there is less contamination of the sample and highly accurate results can be obtained. Furthermore, since analysis operations can be automated, it is possible to analyze a large number of samples in a short period of time, and the reproducibility of analysis results is also high.

[Brief explanation of drawings]

The figure is a conceptual diagram showing a partial configuration of an apparatus according to the present invention. 1.2.3...Reagent introduction part, 5...Sample introduction part,
6.8... Reaction section, 7... Membrane separator, 9...
Detector, 10... Thin tube, 11... Liquid pump.

Claims (1)

[Claims] 1. An automatic metal analyzer in a biological fluid using a flow injection method, characterized by having a reaction section that releases proteins in a sample and a membrane separation section that removes the released proteins.