CZ201140A3 - Two-dimensional method of isolation and analysis of substances from biological specimens - Google Patents

Abstract

The object of the solution is to isolate and analyze substances from biological samples using surface-modified magnetizable particles and chip capillary electrophoresis. The method is based on the manipulation of magnetized particles directly in the capillary electrophoretic reservoir electrophoresis reservoir, whereby the desired analyte is isolated by external magnetic field from the biological sample and subsequently separated, identified and quantified by chip capillary electrophoresis.

Description

The invention relates to a method for the selective isolation of biomolecules from complex biological samples by means of magnetizable particles in combination with rapid and simple analysis by capillary electrophoresis.

BACKGROUND OF THE INVENTION

The complexity of biological samples is a fundamental problem that makes it difficult to determine selected analytes. Each determination must be preceded by some form of extraction, separation or isolation process. Methods of detecting biologically important substances from very small sample volumes are generally very important.

Extraction or isolation of minor components of the complicated mixture leads to significant losses in the sample volume and this phenomenon represents a significant problem especially in samples whose acquisition in larger volumes may be complicated (cerebrospinal fluid, tumor effusions, etc.). The current trend leads to intense pressure on speed, simplicity and complexity of analytical methods. This is also related to the effort to miniaturize analytical instruments that would allow the portability of these devices and their use outside the laboratory, ie directly at the sampling site (eg in nature or at the patient's bed). In-situ analysis speeds up the entire analytical process and reduces overall analysis costs.

SUMMARY OF THE INVENTION

The above requirements are largely met by the method of isolation and analysis of the biologically important compounds of the invention.

The subject of the invention is a two-dimensional method of isolation and analysis of substances from biological samples, where the analyzed substances are isolated by surface modified magnetizable particles (one dimension) in the electrophoretic reservoir of the capillary electrophoresis chip before being analyzed by capillary electrophoresis.

T

The external magnetic field capillary electrophoresis chip separates from the modified magnetizable particles before the second separation and analysis of the substances is performed using standard capillary electrophoresis in a chip arrangement (second dimension).

Isolation by means of surface-modified magnetic particles is carried out directly in the electrophoretic reservoir of the capillary electrophoresis chip by the action of an external magnetic field, which is realized by means of a stationary magnet or electromagnet. In this method it is possible to use a very small volume of readily available sample, such as all types of body fluids (blood, blood serum, cerebrospinal fluid, sweat, urine, tumor effusions).

The method of the invention integrates the process of isolating substances by means of magnetizable particles with a surface selectively modified for the analyte under study and identifying the substances to be analyzed by capillary gel electrophoresis in a microfluidic arrangement. Magnetizable particles with a surface selectively modified for capture of proteins, nucleic acids or other analytes, placed in the reservoir of a commercial microfluidic chip specific for a particular analyte serve as a large surface extraction phase and thus also with a high extraction capacity. Capillary electrophoresis optimized for analysis of a specific analyte subsequently provides information not only on the concentration of the substance, but also on purity, size of molecules and other parameters.

In a preferred embodiment, a commercially available electrophoretic chip is utilized, in the reservoir of which the substance of interest (nucleic acid, protein) is isolated by means of modified magnetizable particles. An external magnetic field whose source is located below the electrophoretic chip is preferably used to manipulate the magnetizable particles. An alternative is to use a tubular magnet inserted into the electrophoretic chip reservoir. The magnetic field can be realized using either a stationary magnet of the desired size or an electromagnet. After the isolation washing processes have been completed, the "waste (or used) magnetic particles can be left in the reservoir because they do not migrate through the separation channel and thereby interfere with the assay, or they can be removed using the tubular magnet.

The term "surface-modified magnetizable particles" includes particles made of magnetic materials (eg Fe ₂ O ₃ , Fe ₃ O _4, etc.) having dimensions of the order of nano- or micrometers modified by a substance capable of interacting with an analyte (eg antibody, oligonucleotide complementary, etc.).

.X

The term "electrophoretic chip reservoir" refers to the space on the electrophoretic chip that is used to deposit solutions. It is a miniature container that is part of the plastic casing of the chip and defines the inputs to the individual channels in the chip.

The term "tubular magnet" refers to a rod-shaped magnet of dimensions suitable for insertion into the electrophoretic chip reservoir.

The term 'electromagnet' refers to a rod-shaped magnet of dimensions suitable for insertion into a reservoir of an electrophoretic chip whose magnetic properties are controlled by the passage of an electric current.

The basic procedure for the isolation of biomolecules using magnetizable particles is shown in Fig. In tube (a) there is a homogenate of the sample to which magnetizable particles have been added. In a process called hybridization (interaction of two strands of nucleic acids), conjugation (protein-protein interaction) or other physico-chemical interactions (adsorption, ionic interaction), specific biomolecules (DNA, RNA, proteins) are bound to a specifically modified surface of magnetizable particles (b) which is modified according to the kind of isolated molecules. For example, for nucleic acid isolation, the surface of the magnetizable particles is modified with a strand complementary to the sequence of interest or a repetitive sequence (oligo dT, oligo G, and the like). For protein isolation, modification with streptavidin, avidin, neutravidin, G, A proteins, polymeric compounds or, for example, antibodies (monoclonal, polyclonal) is used. The conjugated components in the hybridization are labeled for the final detection of the biological interaction; the most commonly used techniques of labeling include streptavidin-biotin or avidin-biotin, neutravidin-biotin, and the like. This is followed by washing of the magnetizable particles with the already bound molecules to remove other interfering substances (c). Separation of the isolated biomolecules from the magnetizable particles is achieved by increasing the temperature (d), changing the ionic strength, changing the elution solution composition, or other physicochemical changes in the hybridization environment (e). After separation of the isolated biomolecules, the magnetizable particles are attracted by a magnet and the target biomolecules are pipetted into a new tube (f). The biomolecules thus separated are ready for further analysis.

The object of the invention is to link the principle of biomolecule isolation by means of magnetizable particles with separation and subsequent analysis by chip capillary electrophoresis (Fig. 2). A schematic representation of a commercially available chip is shown in section (A). A magnet is attached to this chip, allowing the work with magnetizable particles directly in the chip (A), (B). Figure (C) shows the principle of separation by magnetizable particles, homogenate with target biomolecules, interaction of particles with target group of biomolecules, washing and removal

AND

- 'S.

denaturation and subsequent separation in conjunction with detection of target biomolecules by chip capillary electrophoresis.

Possibilities of manipulation of magnetic particles in the electrophoretic chip well are shown in Fig. 3. The particles can be held at the bottom of the well using a flame magnet placed under the chip (Fig. 3A), or the particles can be removed from the well using a thin tubular magnetic mechanism. 3C). Precise handling of the chip along the x and y axes, as well as handling the z-axis magnetic separator, is ideally ensured by a 3D micro manipulator. The realization of the magnetic separator is possible by means of a permanent magnet of suitable shape and dimensions or by means of an electromagnet.

Capillary electrophoresis using any chip provides information not only about the concentration of the substance of interest, but multiplies the separation potential of the present patent by the second dimension, i. isolated molecules by magnetic particles are further resolved according to size and determined by their concentration according to the determination of the given chip. The method according to the invention makes it possible to extend the possibilities of commercially available capillary electrophoresis chips to the efficient, easy and fast isolation and analysis of substances from very small volumes of biological samples outside the laboratory without the need for specialized separation channels with magnets.

BRIEF DESCRIPTION OF THE DRAWINGS

Giant. 1: The procedure for the isolation of biomolecules using magnetizable particles

Fig. 2: Isolation of biomolecules by magnetizable particles in connection with chip capillary electrophoresis

Fig. 3: Possibilities of manipulation with magnetic particles

The invention will be described in more detail by way of example and the accompanying drawings. This example is not limiting in terms of other possible embodiments within the scope of the claims.

• 5 “J

DETAILED DESCRIPTION OF THE INVENTION

Example 1:

Isolation of hepatitis B virus-specific oligonucleotides

Streptavidin-modified magnetic particles were conjugated to a biotin-labeled oligonucleotide (HBV) complementary to the hepatitis B virus-specific oligonucleotide of interest.

Preparation of magnetizable particles for out-of-chip hybridization in a microtube

The magnetizable particles in the native solution were collected in a volume of 110 μΐ, placed in a microtube, the microtube was applied to the magnet, and the native solution was aspirated. 200 μΐ of wash solution (50 mM HEPES) was added, the solution was mixed by pipette 5 times, applied to the magnet, and the wash solution was aspirated.

The biotin-labeled HBV oligonucleotide was dissolved in a 2 M NaCl solution to a final concentration of 5 µg / ml and added to the washed particles. The vial was placed in a thermostat and shaken for 30 min at 1100 rpm and 25 ° C. It was then transferred to a magnet, 2M NaCl solution with HBV oligonucleotide was aspirated and washed 3 times with 50 mM HEPES solution, after the last wash the solution was removed so that only HBV oligonucleotide-modified magnetizable particles remained in the microtube.

110 µ prom of hybridization solution (100 mM Na ₂ HPO ₄ , 100 mM NaH ₂ PO ₄ , 0.5 M NaCl and 0.15 M Tris-basjfc with adjusted pH = 7) was added to the washed modified magnetizable particles in the HBV oligonucleotide microtube, 5.

Plasma isolation and analysis of target molecules on the chip

6 μΐ of prepared hybridization particles were pipetted into each of the 11 sample wells per sample and a maximum of 5 μΐ of sample was pipetted into each well. The sample with the magnetizable particles was mixed by pipette 5 times.

The whole chip with pipetted samples was covered with parafilm and placed in a thermostat and shaken for 10 min at 600 rpm and 25 ° C. The chip was then placed on the magnet for 30-60 seconds (see Gjbr. 2A) until the magnetizable particles attracted to the magnet, gently aspirated the supernatant solution and added 10 μΐ wash solution (50 mM HEPES). The chip was then removed from the magnet and the sample with the wash solution in each well was mixed 5 times with a pipette. Washing was repeated three times, at the last wash the wash solution was aspirated and the chip was left on the magnet.

10 μΐ 50 mM HEPES was added to each well, the chip covered with paraffin, placed on a thermostat and shaken for 10 min at 600 rpm and 85 ° C. After releasing the sample from the magnetic particles (elution), the chip was immediately placed on the magnet and ice. Analysis of the samples by capillary chip electrophoresis was performed according to the instructions supplied with the actual chip and capillary chip electrophoresis, and analysis was started. Thus, the sought-after biotin-labeled HBV oligonucleotide was isolated by means of streptavidin-modified magnetic particles and subsequently analyzed by capillary electrophoresis. Using the software supplied with the chip, samples were evaluated to obtain information about the amount of hepatitis B virus-specific oligonucleotides, their purity and the size of the oligonucleotide fragment.

Industrial applicability

The two-dimensional method of isolation and analysis of substances in the chip arrangement of capillary electrophoresis enables simultaneous isolation of biologically important molecules from biological samples and analysis on a single device. This method of isolating the analyte with the surface-modified magnetizable particles does not result in the loss of the biological sample taken. The method according to the invention makes it possible to extend the possibilities of commercially available capillary electrophoresis chips to the efficient, easy and fast isolation and analysis of substances from very small volumes of biological samples outside the laboratory without the need for specialized separation channels with magnets.

6 ^Z

Λ'Κ- —tyCL ~ 7 7

Modified patent nations

Claims (3)

PATENT CLAIMS 1. A two-dimensional method for isolating and analyzing substances from biological samples, characterized in that the analyzed substances are isolated by surface modified magnetizable particles in the electrophoretic reservoir of the capillary electrophoresis chip prior to commencing capillary electrophoresis analysis, then subjected to external magnetic they separate the fields from the modified magnetizable particles before the second separation of the substances and analysis by standard capillary electrophoresis in a chip arrangement is performed. Method according to claim 1, characterized in that the magnetic field is realized by means of a stationary magnet or an electromagnet. Method according to claims 1 to 2, characterized in that the magnetizable particles are paramagnetic micro- or nanoparticles.