WO2022231447A2 - Flow deproteinizer and a method of deproteinization of liquid biological samples - Google Patents
Flow deproteinizer and a method of deproteinization of liquid biological samples Download PDFInfo
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- WO2022231447A2 WO2022231447A2 PCT/PL2022/000028 PL2022000028W WO2022231447A2 WO 2022231447 A2 WO2022231447 A2 WO 2022231447A2 PL 2022000028 W PL2022000028 W PL 2022000028W WO 2022231447 A2 WO2022231447 A2 WO 2022231447A2
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- deproteinization
- deproteinizer
- chamber
- channel
- protein
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- 238000000034 method Methods 0.000 title claims abstract description 96
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/14—Preparation by elimination of some components
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/34—Purifying; Cleaning
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D21/00—Separation of suspended solid particles from liquids by sedimentation
- B01D21/28—Mechanical auxiliary equipment for acceleration of sedimentation, e.g. by vibrators or the like
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D21/00—Separation of suspended solid particles from liquids by sedimentation
- B01D21/30—Control equipment
- B01D21/32—Density control of clear liquid or sediment, e.g. optical control ; Control of physical properties
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N1/4077—Concentrating samples by other techniques involving separation of suspended solids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N1/4077—Concentrating samples by other techniques involving separation of suspended solids
- G01N2001/4083—Concentrating samples by other techniques involving separation of suspended solids sedimentation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N1/4077—Concentrating samples by other techniques involving separation of suspended solids
- G01N2001/4088—Concentrating samples by other techniques involving separation of suspended solids filtration
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N2035/00465—Separating and mixing arrangements
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Sampling And Sample Adjustment (AREA)
Abstract
The flow deproteinizer, comprising the line for the liquid sample containing the protein, the deproteinization chamber and the line for discharging the liquid deproteinized sample, is characterized by the fact that it operates in a precipitation mode and is equipped with a vibration system ensuring the possibility of shaking the contents of the deproteinization chamber (2) which has a volume of 1.2-1.8 cm3, preferably 1.5 cm3, and the shape of a prism or inverted cartridge oriented in the vertical axis, the height of which is greater than the diagonal of the base, thanks to which it is possible to easily spontaneously separate the deproteinized fraction from the fraction containing the protein precipitate, whereby the deproteinizer is connected to the analytical system by means of flexible lines enabling it to vibrate, and it also includes a deproteinized sample drainage system, a protein-containing precipitate drainage system, a deproteinization chamber cleaning system and a venting system, whereby all inlet and outlet channels are equipped with electronically controlled valves and, preferably, an optical system for measuring the turbidity of the deproteinized sample. The method of deproteinizing liquid biological samples using a protein denaturation reaction with a precipitating agent is characterized in that the denaturation reaction of a protein contained in a biological sample is initiated by introducing a precipitation agent and carried out in the flow deproteinizer described above. The flow deproteinizer which enables the deproteinization of biological samples in the flow-through mode, allows to shorten the analysis time and simplify the procedure of sample preparation in relation to the deproteinization performed in the manual mode, as well as to reduce the costs of analysis and operation of the system in relation to the deproteinization in the flow-through mode with membranes.
Description
Flow deproteinizer and a method of deproteinization of liquid biological samples
The present invention relates to a deproteinizer for use in flow systems for clinical analysis of liquid biological samples, as well as a method for deproteinizing liquid biological samples used in clinical analyses.
Deproteinization of a biological sample (so-called deproteinization) is often a necessary step in clinical analysis, because proteins contained in biological fluids (e.g. serum, plasma) are interferents in many analytical methods, for example in the determination of creatinine by the Jaffe method in the non-kinetic variant [1], determination of iron by the photometric method with ferrozine orferene S [2], determination of copper with the photometric method based on the catalytic properties of copper [3], determination of nitrates by the Griess method [4] or determination of L-tyrosine by the fluorimetric method with tyrosinase [5]. In addition, due to their high viscosity, samples containing large amounts of proteins (e.g. blood plasma) can pose problems in flow analysis methods where equipment with small tubing diameter is used, usually not exceeding 1.0-2.0 mm, which involves the difficulty of flowing too viscous liquids.
One of the most common methods of deproteinization is protein precipitation with trichloroacetic acid (TCA), which requires mixing the sample with TCA-containing reagent and centrifuging the resulting thick precipitate containing the denatured protein [6]. This procedure allows the protein to be removed almost completely from biological samples, but has a number of disadvantages because it is performed outside the flow system. This complicates the analytical procedure and lengthens the entire analysis due to the need to transfer the sample between the analytical systems.
There are also other methods of precipitation deproteinization of biological samples, using as a precipitating agent, for example acetonitrile [7], metaphosphoric acid [7], sodium chlorate(VII) [7], zinc sulfate with NaOH addition [7], ethanol [7], methanol [7], ammonium sulfate [7) or tungsten acid [5]. However, these methods suffer from all the disadvantages of the precipitation method described above.
There are known methods of deproteinization used in flow systems that use the process of filtering biological samples on membranes with appropriate permeability parameters [8]. This type of procedure allows for quick and efficient deproteinization, but may require the use of a flow system equipped with a high-pressure system, allowing the sample to be pumped at a pressure of several bars, which generates additional costs of analysis. Moreover, the use of a membrane system has inconveniences related to the clogging of the membranes and the necessity of frequent cleaning and periodic replacement, which additionally increases the costs of analyses.
There is therefore an unmet need for a cheap and simple analytical system capable of deproteinizing a biological sample in a flow regime, showing compatibility with the systems used in clinical analysis.
The essence of the invention
The flow deproteinizer comprising the liquid sample supply line containing a protein, the deproteinization chamber and the line for discharging the liquid deproteinized sample, is characterized by the fact that it operates in a precipitation mode and is equipped with a vibration system ensuring the possibility of shaking the contents of the deproteinization chamber (2), which has a volume of 1, 2-1.8 cm3, preferably 1.5 cm3, and the shape of a prism
or inverted cartridge oriented in the vertical axis, the height of which is greater than the diagonal of the base, so that an easy spontaneous separation of the deproteinized fraction from the fraction containing the protein precipitate, wherein the deproteinizer is connected to the analytical system by flexible tubing enabling it to vibrate, and it also includes a deproteinized sample drainage system, a protein-containing precipitate drainage system, a deproteinization chamber cleaning system and a venting system, wherein all supply and drain channels are equipped with electronically controlled valves and, preferably, an optical system for measuring the turbidity of the deproteinized sample.
According to the invention, the deproteinizer has at least three inlet channels (A, B, C) to the deproteinization chamber, provided with flexible lines connecting the deproteinizer with the remaining elements of the analytical system, from which channel A supplies the biological sample, channel B supplies the precipitating reagent, and channel C supplies the cleaning solvent, wherein channel A and the line connected to it have a diameter of 0.5-0.6 mm, preferably 0, 56 mm, and the channels B and C and the lines connected thereto have a diameter of 1.0-1.2 mm, preferably 1.07 mm. Preferably, the deproteinizer has an additional fourth inlet channel (D) to the deproteinization chamber for supplying a second cleaning solvent, wherein channel D and the line connected to it has the same diameter as channels B and C. The inlets of channels A, B, and C are on the side walls in the middle of the deproteinization chamber (2), and the inlet of channel D is on the bottom wall of the deproteinization chamber (2) . The deproteinizer has at least two outlet channels (E, F) from the deproteinization chamber, equipped with flexible lines connecting the deproteinizer with other elements of the analytical system, from which the E channel leads the deproteinized supernatant, and the F channel leads the precipitate containing the denatured protein, wherein the E channel has diameter selected in such a way that a line connected to it, having a diameter equal to the diameter of channel A, passes through it and penetrates the interior of the deproteinization chamber (2), and channel F and line connected to it have the same diameter as conduits B, C, and D. The outlet of the E channel is located on the upper wall of the deproteinization chamber (2) and the line passing through it has an outlet in the central part of the deproteinization chamber in its vertical axis in the region of the optical system, preferably above the optical system, preferably at the level of 60% of the height of the deproteinization chamber (2), thanks to which after sedimentation of the precipitate it is possible to remove the deproteinized solution only from the upper part of the chamber, and the inlet of channel F is located on the bottom wall of the deproteinization chamber (2). Flexible lines are made of a chemically and biologically inert material, preferably a perfluorinated polymer material, most preferably PTFE or FEP. According to the invention, the vibrating system uses a laboratory shaker (12), preferably a microshaker, equipped with a holder (13) containing a pocket (14) closely engaging the lower part of the deproteinizer body (1).
According to the invention, the optical system comprises an optical axis that passes previously through the deproteinization chamber (2) in the area filled with the reaction solution. Preferably, the optical system comprises a sight glass in the form of a removable window (5) for visual assessment of the turbidity of a biological sample, wherein the deproteinizer body (1) has an opening on the front wall for access to the deproteinization chamber (2), a seat (3) around the opening in the form of a cavity or a rim on the wall of the deproteinizer body (1), as well as a gasket (4) and a window (5) placed in the seat (3) and a pressing element (6) ensuring their proper pressure against the deproteinizer body (1) which element (6) has an opening on the front wall of the same shape in the opening in the deproteinizer body (1), and on the rear wall
a set of screws (7) spreading the space between the element (6) and the deproteinizer body (1) ensuring the pressure of the window (5) and gasket (6) against the body (1). Alternatively, the optical system comprises a radiation source (9) positioned in the central part of the deproteinization chamber (2) and directed inside it in a plane perpendicular to its vertical axis, and a radiation detector (10) placed in the same plane at an angle of 180° to the optical emitter axis, allowing for turbidimetric monitoring of sample turbidity, or a radiation detector (11) placed in the same plane at an angle of 90° to the optical axis of the emitter, allowing nephelometric monitoring of sample turbidity.
According to the invention, the system draining the solutions formed after shaking generates an underpressure in the drainage channels thanks to the use of independent microsolenoid pumps in the lines connected to the channels E and F, which enables the deproteinized supernatant and denatured protein to be sucked off from the deproteinization chamber (2). The deproteinization chamber cleaning system (2) uses the supply channels C and D, a shaker (12) and the draining channel F. The deproteinization chamber venting system (2) is an open channel G, connecting the upper wall of the deproteinization chamber (2) with the surroundings.
According to the invention, the deproteinizer body (1) and the holder (13) are made of a chemically and biologically inert material with hydrophobic properties, preferably made of ABS, PEEK, Nylon, PP, PP-GF, PET or PETG, preferably by 3D printing technique, pressing element (6) is made of chemically inert material, preferably ABS or PLA, preferably by 3D printing technique, the window (5) is made of a transparent chemically and biologically inert material, preferably polystyrene, and the gasket (4) is made of a flexible chemically and biologically inert material, preferably silicone or rubber.
The method of deproteinizing liquid biological samples using a protein denaturation reaction with a precipitating agent is characterized in that the denaturation reaction of a protein contained in a biological sample is initiated by introducing a precipitation agent and carried out in the flow deproteinizer described above.
According to the invention, the deproteinization process comprises sequentially
- introducing a biological sample with a volume of 0.20-0.30 ml, preferably 0.24 ml, to the deproteinization chamber (2) through channel A,
- introducing 0.60-0.90 ml, preferably 0.72 ml, of the precipitating agent through channel B, preferably 5-20% (w/v) of trichloroacetic acid (TCA), preferably 10% TCA, 10% (w/v) trifluoroacetic acid (TFA), zinc sulphate, ethanol or methanol,
- 2-15 second shaking of the deproteinizer, preferably 5 seconds, preferably at least twice with a 20 second break, by means of a vibrating system, preferably by means of a laboratory shaker (12) or a laboratory microshaker (12) equipped with a handle (13) stabilizing the deproteinizer during shaking,
- leaving the deproteinizer and its contents for 5-10 minutes, preferably 8 minutes, until the denatured precipitate is completely sedimented,
- drawing the deproteinized fraction of the supernatant off with a line passing through the E channel, and subjecting to further analytical procedures,
- extraction of the protein precipitate through the channel F,
- cleaning the deproteinization chamber (2).
According to the invention, the cleaning process of the deproteinization chamber (2) is carried out manually by mechanical manipulations after disassembly of the window (5), or automatically using the cleaning system described above. Preferably, the automatic process of
cleaning the deproteinization chamber (2) is carried out by introducing 0.5-1.25 ml of concentrated alcohol through channel C, preferably 1 ml of at least 90% ethyl alcohol, shaking the deproteinizer for 2-15 seconds, preferably for 3 seconds, and withdrawing the solution through the channel F, followed by introducing 0.5-1.25 ml of concentrated urea through the channel D, preferably 1 ml of 8 M urea, shaking the deproteinizer for 2-15 seconds, preferably for 3 seconds, and draining the solution through channel F, after which the entire cleaning process is repeated at least four times, while it is also possible to introduce alcohol through channel D, and urea through the channel C, or both agents through the same C or D channel.
The flow deproteinizer which enables the deproteinization of biological samples in the flow mode allows to shorten the analysis time and simplify the procedure of sample preparation in relation to the deproteinization performed in the manual mode, as well as to reduce the costs of analysis and operation of the system in relation to the deproteinization in the flow mode with membranes.
The flow deproteinizer according to the invention is shown in the attached drawing in which:
Fig. 1 shows a visualization of the flow deproteinizer in a variant with a visual optics in a vertical section passing through the sight glasses on its front wall and the deproteinization chamber;
Fig. 2 shows a spatial visualization of the flow deproteinizer in the variant with the visual optics in a front view and a back view;
Fig. 3 shows the projections and a three-dimensional visualization of the body of the flow deproteinizer in the variant with the visual system;
Fig. 4 shows the projections and spatial visualization of the pressing element in the variant of the deproteinizer with the visual system;
Fig. 5 shows the projections of the window and the gasket and the spatial visualization of their mutual position with respect to the deproteinizer body in a variant with a visual optics; Fig. 6 shows a visualization of the flow deproteinizer in the variant with a three diode nephelometric and turbidimetric optics;
Fig. 7 shows cross-sections of the flow deproteinizer in the variant with a three diode nephelometric and turbidimetric optics;
Fig. 8 shows the projections of the side walls of the flow deproteinizer in the variant with three diode nephelometric and turbidimetric optics;
Fig. 9 shows the projections and spatial visualization of the socket of the laboratory shaker holder with the deproteinizer embedded in it in the variant with the visual optics;
Fig. 10 shows photographs of an exemplary prototype implementation of a deproteinizer in the variant with the visual optics mounted in a holder mounted on a laboratory shaker (left) and a close-up of the deproteinizer window after the deproteinization process is completed, where the separation of the supernatant layer from the precipitate layer is visible (right);
Fig. 11 shows a schematic diagram of a flow analytical system specifying a schematic of a flow deproteinizer in field B;
Fig. 12 shows a program for controlling the flow analytical system shown in Fig. 11;
Fig. 13 shows photographs of a flow deproteinizer placed in a holder on a laboratory shaker;
Fig. 13 shows the comparison of the signals obtained by the fluorimetric method for the determination of creatinine in the flow regime in a non-deproteinized sample (light green line) and in a deproteinized sample according to the invention (dark green line), where the test was performed with a creatinine solution with a concentration of 250 pmol-L 1 with the addition of 50 g/L of bovine serum albumin, and due to the deproteinization method, the quenching of the analytical response caused by the presence of the protein was reduced;
Fig. 14 shows a graph of the correlation between the results of creatinine determinations by the fluorimetric method in control sera deproteinized with the classic manual procedure, taking into account the centrifugation of the resulting protein precipitate, and the control sera deproteinized by the method according to the invention (left) and the Bland-Altman graph showing the compliance of the classical manual method with the method according to the invention.
Detailed description of the invention
The flow deproteinizer according to the invention enables the deproteinization of liquid biological samples in an automated flow system. Using the 3D printing technology, a deproteinizer was produced which was connected with flexible lines, equipped with microsolenoid pumps and microsolenoid valves, with reagent reservoirs and a further analytical system for determining the concentration of analytes in the tested biological samples. The schematic diagram of the entire analytical system is shown in Fig. 11, and its operation is shown in Fig. 12.
The method of deproteinizing liquid biological samples according to the invention uses the flow deproteinizer described above, and provides the possibility of deproteinizing the biological sample with a low-cost, flow-through precipitation method. The method uses the classic protein denaturation reaction induced by trichloroacetic acid or other precipitation agents with a similar effect. The precipitate of the precipitated protein is deposited at the bottom of the deproteinization chamber, leaving above the supernatant with a low protein content, about 10% of the initial content which allows subsequent analytical measurements without the risk of their disturbance due to protein interference.
Flow deproteinizer
The flow deproteinizer according to the invention comprises a deproteinization chamber (2) in which the process of protein precipitation from the tested sample takes place, a set of lines supplying appropriate reagents (A, B, C, D), a set of lines for draining the products of deproteinization (E, F), a vent line (G), a set of valves and pumps, preferably microsolenoid ones and a vibration system, a deproteinized sample drainage system, a protein-containing precipitate drainage system, a deproteinization chamber cleaning system, a venting system, and preferably an optical system for measuring the turbidity of the deproteinized sample.
The deproteinization chamber (2) has the shape of a prism oriented along the vertical axis of the deproteinizer. Preferably, the deproteinization chamber has a circular base. The lower plane of the deproteinization chamber (2) is spherically or elliptically deformed downwards, giving the chamber the shape of an inverted cartridge. The purpose of this distortion is to facilitate the gravitational outflow of the stream of contamination through the drainage channel (F) discharging contaminants after the deproteinization process, the inlet of which is located on the lower plane of the deproteinization chamber (2). It is essential that the
deproteinization chamber (2) has a shape with a vertical elongation, i.e. that its height is greater than the diagonal of the base, preferably 3-4 times greater. Thanks to this, it is possible to easily and spontaneously separate the deproteinized fraction from the fraction containing the precipitate due to sedimentation of the sediment at the bottom of the deproteinization chamber (2). Otherwise, the height of the liquid column would be too low to ensure that the deproteinized fraction could be safely sucked off without risk of sucking up protein precipitate. When determining the dimensions of the deproteinization chamber, it is important to design it in such a way that, during shaking, about 1/3 of its volume remains empty after mixing all the reagents, to ensure the correctness of this process. According to the invention, the deproteinization chamber (2) has a volumel.2-1.8 cm3 (diameter: 5-10 mm, height 30-40 mm), preferably 1.5 cm3 (diameter: 8.5 mm, height 33 mm), and is adapted to deproteinize a sample with a volume of 0,20-0.30 cm3, preferably 0.24 cm3, for a total protein content of about 50 g/L. Reagents used in the process of deproteinization have a volume of 0.60-0.90 cm3, preferably 0.72 cm3. The use of such a size deproteinization chamber (2) is related to the nature of the measurement being carried out which determines the availability of low-volume samples. In the case of smaller biological samples, it may even be justified to reduce the dimensions of the deproteinization chamber below 1 cm3, for example to a volume of 0.5 cm3 which will require a corresponding reduction in its dimensions.
The deproteinizer (1) is equipped with a vibration system that allows the contents of the deproteinization chamber to be shaken, which is crucial for ensuring a high degree of the protein denaturation reaction in the biological sample. It was found experimentally that shaking for a few seconds, preferably 5 seconds, carried out in 2 series, increases the effectiveness of deproteinization to about 90%. The vibrating system according to the invention comprises a shaker, for example a laboratory shaker, provided with a holder (9) capable of receiving the body of the deproteinizer (1). An exemplary vibratory system is shown in Fig. 9 and Fig. 10. However, it is possible to perform the deproteinization process without a mechanical shaking step, or by a manual shaking process or a mixing process, for example by means of a magnetic stirrer placed in the deproteinization chamber, preferably in a compartment ensuring its free flow movement and contact of the stirrer with the sample, but preventing it from falling out of the deproteinization chamber (2).
The deproteinizer is connected by flexible lines (tubing) with the analytical system for the determination of selected analytes in the deproteinized sample. The use of flexible lines allows the deproteinizer body to vibrate using a vibration system while ensuring that no vibrations are transmitted to the analytical system which may be shock sensitive. The lines are made of a chemically and thermally inert material, preferably perfluorinated one, most preferably PTFE or FEP. The wall thickness of the lines is 0.02-0.50 mm, preferably 0.0254 mm for the smaller diameter and 0.3048 mm for the larger diameter.
The deproteinization chamber (2) is fed by at least three inlet channels (A, B, C), preferably by four inlet channels (A, B, C, D) running inside the deproteinizer body (1). Channel A feeds the biological sample, channel B feeds the precipitant, preferably trichloroacetic acid, channel C feeds the cleaning solvent, preferably concentrated alcohol, most preferably more than 96% ethanol, and channel D feeds the second cleaning solvent, preferably urea solution, most preferably 8 M aqueous urea solution. In the three-channel variant, channel C supplies two different cleaning agents alternatively. Channel A and the line connected thereto have a diameter of 0.5-0.6 mm, preferably 0.56 mm, and lines B, C, and D and the lines connected thereto have a diameter of 1.0-1.2 mm, preferably 1.07 mm. The diameter of the channel A is
about twice smaller than the diameter of the channels B, C, and D in order to minimize the consumed volume of the biological sample. The inlets of channels A, B; and C are in the middle of the deproteinization chamber (2) and the inlet of channel D is in the bottom of the deproteinization chamber (2). The organization of the inlets of channels C and D in different areas of the deproteinization chamber (2) is aimed at increasing the washing capacity of the post-reaction precipitate. In the three-channel variant, the inlet of channel C is also located in the middle of the deproteinization chamber (2) to ensure the possibility of gravity flushing of the precipitate from the walls and spontaneous gravitation of the flushed precipitate to the outlet channel F.
The deproteinization chamber (2) has at least two outlet channels (E and F). The E channel discharges the deproteinized supernatant, and the F channel discharges a precipitate containing the denatured protein. Channel F and the line connected thereto have the same diameter as channels B, C, and D, and the inlet of channel F is at the bottom of the deproteinization chamber (2) to ensure the correct gravity discharge of precipitate. In turn, the E channel has a diameter selected in such a way that the line connected to it passes through it and penetrates the interior of the deproteinization chamber (2). The diameter of the line passing through the channel E is the same as the diameter of the channel A which means that the diameter of the channel E is enlarged relative to the diameter of the channel A by twice the wall thickness of the polymer lines used. The small diameter of the E channel is to minimize the consumed volume of the biological sample. The E channel inlet is located in the upper part of the deproteinization chamber (2), and the supernatant discharge hose passing through the channel E can take the supernatant from the centre of the deproteinization chamber (2), above the precipitate boundary, thus minimizing turbulence caused by suction of the supernatant which is desirable due to ensure the highest possible level of deproteinization of the sample. The location of the effective inlet to the line passing through the channel E in the middle of the deproteinization chamber (2) is to ensure the reproducibility of the supernatant collection process, because with constant volumes of the reagents used, the volume of supernatant available above the mouth of the channel E is reproducible. It has been experimentally determined that the most proteinized biological samples produce a precipitate which, after sedimentation, occupies 50-60% of the height of the liquid column filling the deproteinization chamber (2), which corresponds to 33-40% of the deproteinization chamber height, assuming 65-70% of it is filled with the reaction mixture. Therefore, placing the inlet of the line passing through the channel E in the area of approx. 60% of the height of the deproteinization chamber (2) ensures the collection of the supernatant free from the precipitated protein fraction which, after the end of denaturation, falls by gravity to the bottom of the deproteinization chamber, below the mouth of the channel E.
In a preferred embodiment, the deproteinizer is equipped with an optical system including an optical axis that crosses the deproteinization chamber (2) in the area filled with the reaction solution. The objective of the optical system is to enable the assessment of the turbidity of the solution after the end of the protein denaturation reaction in order to determine the shortest possible time needed for sedimentation of the precipitate, ensuring the possibility of sucking off the supernatant not contaminated with protein precipitate. It is also possible to perform a deproteinization without optical control of the sample turbidity. Then, a series of preliminary calibration measurements should be carried out to determine the maximum sedimentation times of the protein precipitate in biological samples ensuring the possibility of routine discharge of the supernatant not contaminated with protein precipitate.
The optical system may be in the form of a viewing window (5) that allows visual assessment of the turbidity of a biological sample. In this variant, the window is located in the central part of the deproteinization chamber in the area of the mouth of channel E. The window (5) is removable and it has an element (6) pressing it against the deproteinizer body (1) and a sealing member positioned (4) between the window and the deproteinizer body (1). Dismantling the window (5) allows direct access to the interior of the deproteinization chamber (1) for periodic mechanical cleaning.
In the variant with the visual optics, the deproteinizer body (1) has an opening in the front wall, giving direct access to the central part of the deproteinization chamber (2). Around this opening, on the outer wall, there is preferably a seat (3) for a gasket (4) and a window (5) closing the deproteinization chamber (2) externally, having a shape corresponding to said seat (3). Preferably, the seat (3) is in the form of a recess in the deproteinizer body (1), or a rim protruding beyond the deproteinizer body (1). The window (5) is pressed from the outside by means of a pressing element (6) in the shape of a prism with no lower and upper base, with a base shape corresponding to the shape of the base of the deproteinizer body (1), and with internal dimensions slightly larger than the outer contour of the deproteinizer body (1). The pressing element (6) has a cut-out on the front face of the shape and size corresponding to the hole in the front wall of the deproteinizer body (1). Around this opening, on the inside of the body of the pressing element (6), there is a socket for receiving the window (5) which is shaped according to the shape of the window (5), and the socket may be in the form of a recess or an rim. The set of both sockets ensures the stability of placing the window (5) in the place of the opening in the deproteinizer body (1), allowing for effective visual inspection of the interior of the deproteinization chamber (2) and assessment of the turbidity of the deproteinized sample. The pressing of the window (5) is realized by a pressing element (6) having a set of screws (7) on the opposite wall, preferably above three screws (7), tightening of which opens the space between the pressing element (6) and the rear wall of the deproteinizer body (1), pressing the window (5) against its front surface and at the same time squeezing the gasket (4) ensuring the tightness of the deproteinization chamber (2). On the rear wall of the deproteinizer body (1) there are round sockets (8) receiving the expanding screws (7) in the form of recesses or rims, additionally stabilizing the position of the pressing element (7) in relation to the opening in the front wall of the deproteinizer body (1).
Alternatively, the optical system may include a radiation source (9) positioned in the centre of the deproteinization chamber (2) and directed inwardly in a plane perpendicular to its vertical axis, and a radiation detector (10) positioned in the same plane at an angle of 180° to the optical axis of the emitter (9), allowing for turbidimetric monitoring of sample turbidity, or the radiation detector (11) placed in the same plane at an angle of 90° to the optical axis of the emitter (9), allowing nephelometric monitoring of sample turbidity. A system of this type is known from the literature and is perfect for assessing the turbidity of solutions by spectrophotometric measurement [9]. A deproteinizer equipped with an optical system of this type may additionally have a sight glass or other removable housing element that provides access to the interior of the deproteinization chamber (2).
The deproteinizer is equipped with a system for draining the shaking solution. This system generates an underpressure independently in the outlet channels E and F by connecting them to microsolenoid pumps, which allows the suction of the deproteinized supernatant and denatured protein. The outlet of the line passing through the channel E from the deproteinization chamber (2) is located in its central part, in the area of the optical system,
preferably above it, thanks to which, after the optically determined sedimentation of the precipitate, it is possible to remove the deproteinized solution from above the precipitate boundary, only from the upper part of the deproteinization chamber (2). In turn, the outlet of the channel F is located at the base of the deproteinization chamber (2) in order to increase the efficiency of removing the protein precipitate by gravity.
The deproteinizer is equipped with a deproteinization chamber cleaning system (2) using feed channels C and D, a shaker (12) and a discharge channel F. In the automatic cleaning process, the deproteinization chamber (2) is flooded with the cleaning solution, agitated in order to remove the protein precipitate as much as possible, and then emptied through the channel F. The cleaning process is repeated several times, preferably four times.
The deproteinizer is equipped with a venting system of the deproteinization chamber, in the form of an open channel G with a diameter of 1 mm, connecting the upper wall of the deproteinization chamber (2) with the environment outside the deproteinizer. The venting system regulates the pressure while filling the deproteinization chamber (2). In the absence of the vent channel G, filling the deproteinization chamber (2) was impossible due to the presence of air inside it and the tightness of the system.
The deproteinizer has the shape of a prism with external dimensions exceeding that of the deproteinization chamber (2) by at least 20 mm in each of the horizontal dimensions and by at least 30 mm in the vertical dimensions. The excess material of construction is necessary to ensure adequate stiffness of the deproteinizer body (1) which is subjected to shaking during routine use. An additional allowance of construction material in the vertical direction makes it possible to route the channels A-G appropriately and stably inside the body of the deproteinizer body (1) so that they can leave the body of the deproteinizer body (1) in a place convenient for the user. Preferably, in the variant with the spectrophotometric optical system (9, 10, 11) and in the version without the optical system, the deproteinizer body (1) has the shape of a prism with a regular octagon base, preferably 70-80 mm high and 25-30 mm wide, preferably 73.5 mm and 28 mm wide. The octagonal shape gives a very high stiffness while minimizing material consumption, and is also perfect for 3D printing. In turn, in the variant with the visual optics, the deproteinizer is preferably prism-shaped with a pentagonal base formed by subtracting from a regular octagon a trapezoid formed by three adjacent sides of the octagon, the window being placed on the middle of the short-sided walls. The deproteinizer body (1) in this variant preferably has a height of 70-80 mm, a width of 25-30 mm and a depth of 15-20 mm, preferably a height of 73.5 mm, a width of 28 mm, and a depth of 16.7 mm. Exemplary implementations of the deproteinizer body in various variants are shown in Figs. 1-3 and Figs. 6-8.
Due to the complicated internal shape of the deproteinization chamber (2) and the channels A - G, the production of the deproteinizer body (1) requires the use of specialized equipment. It is possible to produce it from many elements shaped by machining techniques. Alternatively, the deproteinizer body (1) is manufactured by 3D printing. The deproteinizer body (1) is made of chemically and biologically inert materials, preferably plastics, most preferably plastics that can be modelled by 3D printing. Among the available filaments for 3D printing, preferably the deproteinizer is made of filaments having hydrophobic properties, most preferably made of filaments such as ABS, PEEK, Nylon, PP, PP-GF, PET or PETG. It has been experimentally established that the protein precipitation occurs faster in a deproteinizer made of a material with hydrophobic properties, compared to the precipitate sedimentation rate in deproteinizers made of hydrophilic materials (for example, in a deproteinizer made of PLA, the precipitate stuck to the walls of the deproteinization chamber and did not fall effectively to its
bottom), probably due to electrostatic interactions between the protein denatured with trichloroacetic acid and the walls of the deproteinizer.
In the variant with the visual system, the pressing element (6) is made in the same technique as the deproteinizer body (1). Preferably, the pressing element is produced by 3D printing from a filament the same or different from the deproteinizer body (1), preferably from PLA or ABS, but most of the filaments can be used to manufacture this element. The window (5) is preferably made of polystyrene or other transparent material that is chemically and mechanically resistant, but glass windows can also be used. The gasket (4) is made of a chemically and biologically inert flexible material, preferably silicone or rubber.
A holder (13) for a shaker (12) with a seat (14) receiving the lower part of the deproteinizer body (1) is manufactured by techniques such as the deproteinizer body (1), preferably using the same materials of construction. The deproteinizer body (1) is placed in a seat (14) of the holder (13) which preferably tightly receives it and stabilizes it by a system of two or more elastic pressing elements that generate friction by pressure perpendicular to the outer planes of the lower part of the deproteinizer. There is no need to use additional locks and safeguards, especially since the routine shaking time does not exceed a total of 15 seconds. This solution is shown in Fig. 9, and an exemplary implementation is shown in Fig. 10. However, this does not limit the possibility of using other types of seats and holders, also those using fastening systems such as: a snap system, a magnetic system, etc.
The microsolenoid valves and pumps are located on channels A - F. Preferably, they are located in the areas of these channels outside the deproteinizer body (1) in order to provide them with better working conditions, not subjecting them to vibrations. According to the invention, however, it is possible to locate the respective pumps and valves within the deproteinizer body (1). Preferably, lines (hoses) made of inert perfluorinated materials, for example PTFE or FEP, are used routinely in analytical practice. The lines are connected to the channels A-F in a classic way, preferably by pressing on the hose on the projection protruding from the deproteinizer body (1) at the outlet of the channel (A-F) or by pressing into the cavity in the deproteinizer body (1) at the outlet the desired channel (A - F). Preferably microsolenoid type valves and pumps are used.
Deproteinization method
The prior art problem of necessity to analyze liquid biological samples in a multi-step procedure comprising a stationary process for removing interfering protein has not yet been solved. Classically, the protein is removed by the precipitation method, and then the obtained precipitate is centrifuged in laboratory centrifuges. It is a method leading to almost complete removal of protein from biological samples, but requires a centrifugation step that cannot be incorporated into the flow-through mode of analysis, preferred in analytical work due to time savings and high repeatability of measurements. An alternative to the classical methods of protein removal was to leave it in the sample and acceptance of interference resulting from its presence, with the possibility of introducing appropriate corrections of the obtained measurement results. From the point of view of the convenience and reliability of conducting analyses, none of the above solutions is satisfactory.
The present invention uses the classical protein denaturation reaction, but instead of the multi-step process using the step of centrifuging the resulting precipitate, it envisages the use of the deproteinizer described above which ensures that all manipulations related to the deproteinization of the sample are performed in one device in a flow regime. The method
according to the invention using the phenomenon of gravitational sedimentation of the precipitate, the formation of which is enhanced by the shaking process, and providing for the collection of the supernatant from the upper part of the deproteinization chamber (2), after the sedimentation process is completed, confirmed by optical evaluation of the solution turbidity. The deproteinization efficiency of the method according to the invention reaches 90% which is an acceptable level of deproteinization in many modern analytical methods (for example the creatinine determination procedure using the fluorimetric method with 3,5-dinitrobenzoic acid). The current process does not allow for complete deproteinization because a small fraction of denatured protein forms a fine light precipitate which would require the use of a centrifuge to remove. Nevertheless, the content of this light fraction usually does not exceed 10% of the original protein content which is an acceptable level of contamination in routine analytical measurements. The advantages of being able to use a flow-through mode of analysis covering the entire analytical process, including the sample deproteinization step, significantly outweigh the disadvantages associated with the presence of a small protein fraction in the deproteinized biological sample.
The method of deproteinizing biological samples uses the flow deproteinizer described above. The deproteinization process is carried out inside the deproteinization chamber (2) to which the tested biological sample is delivered, where the reaction of protein precipitation of the tested sample takes place. Due to its unique structure, the deproteinizer enables repeatable and reliable separation of the deproteinized fraction from the fraction containing the protein precipitate.
The deproteinization method according to the invention comprises the following steps, sequentially:
- introducing to a biological sample with a volume of 0.20-0.30 ml, preferably 0.24 ml, into the deproteinization chamber (2) via channel A,
- introducing 0.60-0.90 ml, preferably 0.72 ml, of the precipitating agent through channel B, preferably 10% (w/v) of trichloroacetic acid (TCA), 10% (w/v) of trifluroacetic acid (TFA), zinc sulphate, ethanol or methanol,
- 2-15 second shaking of the deproteinizer, preferably 5 seconds, preferably at least twice with a 20 second break, by means of a vibrating system, preferably by means of a laboratory shaker (12) or a laboratory microshaker (12) equipped with a handle (13) stabilizing the deproteinizer during shaking,
- leaving the deproteinizer and its contents for 5-10 minutes, preferably 8 minutes, until the denatured protein precipitate is completely sedimented,
- draining the deproteinized fraction of the supernatant through a line passing through the channel E and subjecting to further analytical processes,
- draining the protein precipitate through the channel F,
- cleaning the deproteinization chamber (2), manually or automatically by introducing 0.5-1.25 ml of concentrated alcohol through the channel C, preferably 1 ml of at least 90% ethyl alcohol, shaking the deproteinizer for 2-15 seconds, preferably for 3 seconds, and draining the solution through the channel F, followed by introducing 0.5-1.25 ml of concentrated urea through the channel D, preferably 1 ml of urea with a concentration of 8 , shaking the deproteinizer for 2-15 seconds, preferably for 3 seconds, and draining the solution through channel F, after which the whole cleaning process is repeated at least four times. It is also possible to introduce alcohol through the channel D and urea through the channel C, or both through the same channel C or D.
The deproteinization method of the invention, described above in various embodiments, was laboratory tested and provided effective deproteinization of biological and synthetic samples.
Deproteinization is performed with trichloroacetic acid due to the high efficiency of deproteinizing biological samples and due to the coarse-grained form of the formed precipitate which allows the denatured protein to fall spontaneously without the need to centrifuge the deprotein content. The concentration of TCA as a precipitating factor of 10% was determined on the basis of literature values [6]. However, it is possible to use the precipitation agent in a concentration of 5-20% with equally good results. However, it is possible to use other precipitation agents, for example trifluoroacetic acid, zinc sulphate, ethanol or methanol, but then it is necessary to experimentally determine the optimal sedimentation time or use a deproteinizer with an optical system to determine the optimal sedimentation time.
The volume ratio of the biological sample and the precipitating agent was experimentally set at 1:3 which allowed the protein precipitate to sediment faster, while not causing excessive dilution of the sample compared to other volume ratios. However, it is possible to carry out the deproteinization process using the volume ratio of the reagents in the range of 1:2-1:4, without a significant deterioration of the deproteinization results. However, the use of a 1:2 volume ratio is associated with a longer sedimentation time of the protein precipitate.
The presented optimal shaking times and the shaking interval times of the deproteinizer before sucking off the supernatant were determined on the basis of experimental observations of the precipitation of sediment from blood serum samples with the use of trichloroacetic acid as a precipitation agent. It is worth noting that for other types of samples and other precipitation agents, the settling times of the protein precipitate may be different which will require reference measurements or the use of a deproteinizer with an optical system to determine the optimal shaking and sedimentation times.
It is possible to use any cleaning agents and solutions to rinse the deproteinization chamber (2) after the deproteinization process is completed. However, literature reports suggest that the best results in dissolving denatured protein can be achieved using concentrated solutions of ethanol (96%) and urea (8 M). The method according to the invention provides for alternating washing of the deproteinization chamber with these solutions, with the additional application of shaking which ensures uniform access of these solutions to all parts of the deproteinization chamber (2). Portions of the solutions with a volume equal to approx. 2/3 of the volume of the deproteinization chamber (2) are used which ensures optimal shaking efficiency. It has been experimentally confirmed that shaking 1-5 seconds, preferably 3 seconds, with repetition of the cycle at least four times (a total of at least 8 shakes), gives the best results from the point of view of ensuring the optimal level of cleanliness of the deproteinization chamber (2) in the shortest possible time which is extremely important when working in the flow regime. The use of short shaking allows to perform more washings with fresh portions of the solution per unit time which has a positive effect on the level of washing away contaminants.
Summary
The flow deproteinizer according to the invention allows for efficient, cheap and effective deproteinization of a biological sample to the extent that allows minimizing protein interferences occurring in many analytical methods. The construction of the deproteinizer is simple which enables cheap and quick production using 3D printing technology. Ease of
production ensures virtually unlimited availability of the deproteinizer and allows its frequent replacement to ensure the optimal level of cleanliness of the tested samples and the safety of users.
The method of deproteinization of liquid biological samples is carried out with the use of the deproteinizer described above, in a mechanized way in the flow regime which significantly simplifies the analytical procedure of sample preparation for analysis, compared to the classical methods of deproteinization carried out in a manual manner. Moreover, the method according to the invention provides the possibility of lowering the costs of analysis and operation of the flow system compared to the costs related to the operation of systems equipped with flow membrane deproteinizers.
A flow deproteinizer and a deproteinization method using the deproteinizer of the invention are described below in the embodiments.
Example 1. The flow deproteinizer in the variant with a visual optical system was made by 3D printing using ABS filament for the production of the deproteinizer body (1) and the shaker holder (13), and the PLA filament for the production of the pressing element (6). The window (5) is made of polystyrene and the gasket (4) is made of silicone. The deproteinizer body had the shape of a prism, 73.5 mm high, 28.0 mm wide and 16.7 mm deep, with a pentagonal base as shown in Fig. 3. The deproteinization chamber had a volume of 1.5 ml and had a vertical cross- section in the shape of an inverted cartridge, 32.6 mm high and 8.5 mm radius, the deproteinization chamber (2) having an opening in the front wall of the body (1). The deproteinization chamber (2) was closed from the front by a window (5) and a rectangular gasket (4), 44.3 mm high and 20.5 mm wide which were pressed against the element (6) with the shape shown in Fig. 4 as shown in Fig. 5. The deproteinizer body had channels A-F equipped with PTFE lines connecting the deproteinization chamber (2) with the rest of the analytical system. Channel A and the line connected thereto had an internal diameter of 0.56 mm, and the channels B, C, D, F and the lines connected thereto had a diameter of 1.07 mm. Channel E had a diameter of 1.3 mm and a 0.56 mm diameter line passed through it which penetrated the deproteinization chamber (2) and had an outlet in the region of 60% of its height. Channels A, B, C had an outlet on the side walls of the deproteinization chamber (2) in the middle of its height, and channels D and F had an outlet on the lower wall of the deproteinization chamber (2). The body (1) had a vent channel G with a diameter of 1 mm, connecting the deproteinization chamber (2) with the surroundings and having an outlet on its upper wall. The handle (13) had a pentagonal shaped seat (14) capable of receiving the lower part of the deproteinizer body (1) and holding it on the shaker (12).
Example 2. The flow deproteinizer in the variant with the spectrophotometric optical system was made by 3D printing using ABS filament to produce the deproteinizer body (1) and the shaker handle (13). The deproteinizer body had the shape of a prism, 73.5 mm high, 28.0 mm wide, the base of which was a regular octagon, as shown in Figs. 6-8. The deproteinization chamber had a volume of 1.5 ml and had a vertical cross-section in the shape of an inverted cartridge with a height of 32.6 mm and a radius of 8.5 mm, whereby the deproteinization chamber (2). In the central part of the deproteinization chamber, in the middle of its height, there were elements of the optical system in the form of a diode constituting a radiation source with a wavelength of 630 nm directed towards the interior of the deproteinization chamber (2) in a plane perpendicular to its vertical axis, as well as a radiation detector (10) placed in the same plane at an angle of 180° to the optical axis of the emitter, allowing for turbidimetric monitoring
of the sample turbidity, and the radiation detector (11) placed in the same plane at an angle of 90° to the optical axis of the emitter, allowing nephelometric monitoring of the sample turbidity. The deproteinizer body had channels A-F equipped with PTFE lines connecting the deproteinization chamber (2) with other elements of the analytical system. Channel A and the line connected thereto had an internal diameter of 0.56 mm, and the channels B, C, D, F and the lines connected thereto had a diameter of 1.07 mm. Channel E had a diameter of 1.3 mm and a 0.56 mm diameter line passed through it which penetrated the deproteinization chamber (2) and had an outlet in the region of 60% of its height. Channels A, B, C had an outlet on the side walls of the deproteinization chamber (2) in the middle of its height, and channels D and F had an outlet on the lower wall of the deproteinization chamber (2). The body (1) had a vent channel G with a diameter of 1 m , connecting the deproteinization chamber (2) with the surroundings and having an outlet on its upper wall. The handle (13) had a pentagonal-shaped seat (14) capable of receiving the lower part of the deproteinizer body (1) and immobilizing it on a shaker (12). Example 3. Tests were carried out on the flow-through deproteinizers prepared in examples 1 and 2 in order to compare their effectiveness with the classical method of deproteinization, as well as to check the influence of the performed deproteinization on the analytical response in comparison with the measurements of non-deproteinized samples. Control sera by volume of0.24 ml with different creatinine and protein contents were deproteinized with the use of flow deproteinizers in the analytical system according to the scheme shown in Fig. 11 using the program shown in Fig. 12, using 0.72 ml of 10% trichloroacetic acid as a precipitating agent and the deproteinization procedure according to the invention, i.e. after mixing the reagents in the deproteinization chamber, two shakings for 5 seconds were performed, keeping a 20-second pause between shakings, then the solution was left for 8 minutes to sediment, and after finding a sufficiently low turbidity of the supernatant, it was withdrawn through the channel E and further analyzed, and the deproteinization chamber (2) was emptied through the channel F, then the chamber (2) was washed with 96% ethanol with 3 seconds shaking and drained, then washed with 8 M urea with 3 seconds shaking and drained, and the cleaning procedure was repeated four times. The deproteinization according to the classical manual procedure included precipitation of the precipitate and its 10-minute centrifugation. In order to maintain the same degree of sample dilution, the manual procedure used a 1:3 volume ratio of the sample to the precipitating agent (TCA), i.e. the same as in the flow deproteinizers. The supernatants of both deproteinizers according to the invention, as well as the classical deproteinization sample and the non-deproteinized sample, were tested for creatinine content using the fluorimetric method with the use of 3,5-dinitrobenzoic acid, according to the classical analytical procedure [10]. The results of the analyses of the samples deproteinized in the flow deproteinizers according to the invention were consistent. The intensity of the analytical response of the samples deproteinized in the flow deproteinizers according to the invention was significantly higher than the intensity recorded during the analysis of the non-proteinaceous sample (Fig. 13). The results of the analyses of the samples deproteinized with the method according to the invention correlated well with the results of the analysis of the classically deproteinized sample, and the results of the correlation analysis are shown in Fig. 14.
Example 4. The efficacy of the flow deproteinizer described in Example 1 was tested. For this purpose, an aqueous protein solution (bovine serum albumin) at a concentration of 50 g/L and an aqueous solution of the precipitating agent (trichloroacetic acid) at a concentration
of 10% (m/v) were pumped into it through separate channels. One minute was wated to allow the reaction to proceed and then the contents of the reactor was shaken twice for 5 seconds with an interval of 4 minutes. After the second shaking, a further 4 minutes was allowed for the resulting precipitate to sediment to the bottom of the reactor. The progress of the sedimentation process was visually monitored through the sight glass. Then, the solution was pumped out of the precipitate. Before subsequent use, the reactor was washed 4 times with a 96% ethanol solution.
The obtained supernatants were diluted 10-fold and their protein content was determined by the Bradford method: 50 mί of the supernatant was mixed with 2.5 mL of Bradford's reagent and after 5 minutes the absorbance of the mixture was measured at the wavelength l = 595 nm. Albumin concentration was determined on the basis of a previously prepared calibration curve with the formula: y = 6.87 10-4 [AU-L-mg'1] · x + 0.00258 [AU]
The results obtained are shown in Table 1 below. The average efficiency of the deproteinization process was determined at the level of 90±2%, in accordance with the following relationship:
Table 1. Absorbance values (A) for 10-fold diluted supernatants, calculated protein contents (ck - in diluted supernatant, CSN - in initial supernatant, cw - in initial sample) and deproteinization efficiency EdeP.
References
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[3] S. M. Ramasamy, H. A. Mottola; Flow injection (closed-loop configuration) catalytic determination of copper in human blood serum, Anal. Chim. Acta 127 (1981) 39-46.
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[8] N. Ali, H. Sofiah, A. Asmadi, A. Endut; Preparation and characterization of a polysulfone ultrafiltration membrane for bovine serum albumin separation: Effect of polymer concentration, Desalination Water Treat. 32 (2011) 248-255
[9] K. Strzelak, R. Koncki; Nephelometry and turbidimetry with paired emitter detector diodes and their application for determination of total urinary protein, Anal. Chim. Acta 788 (2013) 68-73.
[10] I. Lewinska, M. Michalec, t. Tymecki, From the bottom of an old jar: A fluorometric method for the determination of creatinine in human serum, Anal. Chim. Acta 1135 (2020) 116-122.
Claims
1. A flow deproteinizer, comprising a line for supplying the liquid sample containing the protein, a deproteinization chamber and a line for discharging the liquid deproteinized sample, characterized in that it operates in a precipitation mode and is equipped with a vibration system ensuring the possibility of shaking the contents of the deproteinization chamber (2) which has a volume of 1.2-1.8 cm3, preferably 1.5 cm3, and the shape of a prism or inverted cartridge oriented in the vertical axis, the height of which is greater than the diagonal of the base which allows easy spontaneous separation of the deproteinized fraction from the fraction containing the protein precipitate, while the deproteinizer is connected to the analytical system by flexible lines that enable it to vibrate, and it also includes a deproteinized sample drainage system, a protein-containing precipitate drainage system, a deproteinization chamber cleaning system and a venting system, whereby all inlet and outlet channels are equipped with electronically controlled valves and, preferably, an optical system for measuring the turbidity of the deproteinized sample.
2. The deproteinizer according to claim 1, characterized in that it has at least three inlet channels (A, B, C) to the deproteinization chamber, equipped with flexible lines connecting the deproteinizer with other elements of the analytical system, from which channel A supplies the biological sample, channel B supplies the precipitating agent, and channel C supplies the cleaning solvent, whereby channel A and the line connected to it have a diameter of 0.5-0.6 mm, preferably 0.56 mm, and lines B and C and lines connected thereto have a diameter of 1.0-1.2 mm, preferably 1.07 mm.
3. The deproteinizer according to claim 2, characterized in that it has an additional fourth inlet channel (D) to the deproteinization chamber for supplying a second cleaning solvent, whereby the channel D and the line connected thereto having the same diameter as the channels B and C.
4. The deproteinizer according to claim 2, characterized in that the inlets of channels A, B and C are located on the side walls in the central part of the deproteinization chamber (2), and the inlet of channel D is located on the bottom wall of the deproteinization chamber (2).
5. The deproteinizer according to claim 1, characterized in that it has at least two outlet channels (E, F) from the deproteinization chamber, equipped with flexible lines connecting the deproteinizer with other elements of the analytical system, from which the channel E leads the deproteinized supernatant, and the channel F leads the precipitate containing the denatured protein, whereby the channel E has a diameter selected in such a way that the line connected to it, having a diameter equal to the diameter of channel A, passes through it and penetrates the interior of the deproteinization chamber (2), and the channel F and the line connected to it have the same diameter as channels B, C and D.
6. The deproteinizer according to claim 5, characterized in that the outlet of the channel E is located on the upper wall of the deproteinization chamber (2) and the line passing through it has an outlet in the central part of the deproteinization chamber in its vertical axis in the region of the optical system, preferably above the optical system, preferably at the level of 60% of the height of the deproteinization chamber (2), thanks to which after sedimentation of the precipitate it is possible to withdraw the deproteinized solution only from the upper part of the chamber, and the channel F inlet is located on the lower wall of the deproteinization chamber (2).
7. The deproteinizer according to claims 2 and 5, characterized in that the flexible lines are made of a chemically and biologically inert material, preferably a perfluorinated polymer material, most preferably PTFE or FEP.
8. The deproteinizer according to claim 1, characterized in that the vibrating system uses a laboratory shaker (12), preferably a microshaker, provided with a holder (13) comprising a seat (14) closely engaging the lower part of the deproteinizer body (1),
9. The deproteinizer according to claim 1, characterized in that the optical system comprises an optical axis that passes formerly through the deproteinization chamber (2) in the area filled with the reaction solution.
10. The deproteinizer according to claim 9, characterized in that the optical system comprises a sight glass in the form of a removable window (5) enabling visual assessment of the turbidity of a biological sample, the deproteinizer body (1) having an opening on the front wall providing access to the deproteinization chamber (2), a seat (3) around this opening in the form of a recess or rim on the body deproteinizer (1) wall, as well as a gasket (4) and a window (5) placed in the socket (3) and a pressing element (6) ensuring their proper pressure against the deproteinizer body (1) which element (6) has an opening on the front wall with a shape identical to the opening in the deproteinizer body (1), and on the rear wall a set of screws (7) spreading the space between the element (6) and the deproteinizer body (1) ensuring the pressure of the window (5) and gasket (6) against the body (1).
11. The deproteinizer according to claim 9, characterized in that the optical system comprises a radiation source (9) placed in the central part of the deproteinization chamber (2) and directed inside it in a plane perpendicular to its vertical axis, as well as a radiation detector (10) placed in the same plane at an angle of 180° to the optical axis of the emitter, allowing for turbidimetric monitoring of sample turbidity, or a radiation detector (11) located in in the same plane at an angle of 90° to the optical axis of the emitter, allowing nephelometric monitoring of sample turbidity.
12. The deproteinizer according to claim 1, characterized in that the system draining the solutions formed after shaking generates an underpressure in the discharge channels thanks to the use of independent microsolenoid pumps in the lines connected to channels E and F which enables the ability to aspirate deproteinized supernatant and denatured protein from the deproteinization chamber (2).
13. The deproteinizer according to claim 1, characterized in that the deproteinization chamber cleaning system (2) uses supply channels C and D, a shaker (12) and a discharge channel F.
14. The deproteinizer according to claim 1, characterized in that the venting system of the deproteinization chamber (2) is an open channel G connecting the upper wall of the deproteinization chamber (2) with the environment.
15. A deproteinizer according to claim 1, characterized in that the deproteinizer body (1) and the holder (13) are made of a chemically and biologically inert material with hydrophobic properties, preferably of ABS, PEEK, Nylon, PP, PP-GF, PET or PETG, preferably by 3D printing, the pressing element (6) is made of a chemically inert material, preferably ABS or PLA, preferably by 3D printing, the window (5) is made of transparent chemically and biologically inert material, preferably polystyrene, and the gasket (4) is made of a flexible chemically and biologically inert material, preferably silicone or rubber.
16. A method of deproteinization of liquid biological samples using a protein denaturation reaction with a precipitating agent, characterized in that the denaturation reaction of a protein contained in a biological sample is initiated by introducing a precipitation agent and is carried out in a flow deproteinizer as described in claims 1-15.
17. The method according to claim 16, characterized in that the deproteinization process comprises sequentially
- introducing into a biological sample with a volume of 0.20-0.30 ml, preferably 0.24 ml, into the deproteinization chamber (2) through channel A,
- introducing 0.60-0.90 ml, preferably 0.72 ml, of the precipitating agent through channel B, preferably 5-20% (w/v) of trichloroacetic acid (TCA), preferably 10% TCA, 10% (w/v) trichloroacetic acid (TFA), zinc sulphate, ethanol or methanol,
- 2-15 second shaking of the deproteinizer, preferably 5 seconds, preferably at least twice with a 20 second break, by means of a vibrating system, preferably by means of a laboratory shaker (12) or a laboratory microshaker (12) equipped with a handle (13) stabilizing the deproteinizer during shaking,
- leaving the deproteinizer and its contents for 5-10 minutes, preferably 8 minutes, until the denatured protein precipitate is completely sedimented,
- draining the deproteinized fraction of the supernatant through a line passing through the channel E and subjecting to further analytical processes,
- draining of the protein precipitate through the channel F,
- cleaning the deproteinization chamber (2).
18. The method according to claim 17, characterized in that the process of cleaning the deproteinization chamber (2) is carried out manually by mechanical manipulations after disassembly of the window (5) or automatically using the cleaning system described in claim 13.
19. The method according to claim 17, characterized in that the automatic process of cleaning the deproteinization chamber (2) is carried out by introducing 0.5-1.25 ml of concentrated alcohol through the channel C, preferably 1 ml of at least 90% ethyl alcohol, shaking the deproteinizer for 2-15 seconds, preferably for 3 seconds, and draining the solution through the channel F, followed by introducing 0.5-1.25 ml of concentrated urea through the channel D, preferably 1 ml of 8 M urea, shaking the deproteinizer for 2-15 seconds, preferably for 3 seconds, and draining the solution through channel F, after which the whole cleaning process is repeated at least four times, whereby it is also possible to introduce alcohol through channel D and urea through channel C, or both through the same channel C or D.
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| PL437753A PL437753A1 (en) | 2021-04-29 | 2021-04-29 | Flow deproteinizer and method of deproteinization of liquid biological samples |
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