KR20130105476A - System and method for superheating and/or supercooling of liquids and use of the system and/or method - Google Patents

Abstract

PURPOSE: A system for superheating and/or supercooling of a liquid is provided to quickly isolate DNA or RNA without an isolation solution. CONSTITUTION: An open system for overheating and/or supercooling of a liquid has: a capillary tube containing a liquid; and at least one heating and/or cooling means for heating the liquid above boiling point of the liquid at an ambient pressure or cooling the liquid below freezing point of the liquid at an ambient pressure. At least one heating and/or cooling means is (are) in thermal contact with the capillary tube in an area. The capillary tube is at least nearly free of nucleation centers at inner surface thereof. The open system is operated in a flow-through mode.

Description

FIELD AND METHOD FOR SUPERHEATING AND / OR SUPERCOOLING OF LIQUIDS AND USE OF THE SYSTEM AND / OR METHOD}

The present invention relates to systems and methods for overheating and / or subcooling of liquids and to the use of such systems and / or methods.

Overheating means raising the temperature of the liquid above its boiling point without vaporizing the liquid at a given pressure (here, atmospheric pressure). Subcooling means lowering the temperature of the liquid below its freezing point while maintaining the liquid in the liquid phase without changing phase at a given pressure.

Because proteins are being used as tools, targets and therapies, fast, reliable and inexpensive methods, for example for protein identification and analysis of post-translational modifications, are important.

For example, protein protein mass spectrometry (MS) is a state-of-the-art technology. However, for MS-based protein identification, peptide mass fingerprinting (PMF) or peptide fragment fingerprinting requires a pretreatment step to break down the protein into peptides.

In general, enzymatic reactions based on proteases such as trypsin are used. In addition to enzymatic reactions, the researchers also use chemical degradation methods. Both courses are time consuming and expensive.

More general approaches than hydrolysis of enzyme-based target molecules can be performed by energy-based disruption of peptide bonds in proteins.

As is known by the MS fragmentation reaction, an increase in the amount of energy results in more nonspecific fragmentation patterns, resulting in the complete atomization of the original molecule.

The rate of protein fragmentation is energy dependent and thus increasing temperature dramatically increases the rate of reaction. However, the maximum achievable temperature is generally limited by the boiling point of the solvent. If water is used as the solvent, the boiling point of the solvent is 100 ° C at atmospheric pressure if the nucleation center is abundant in the reaction vessel.

Nevertheless, if there are no nucleation centers, the water temperature can rise above the boiling point of water at atmospheric pressure. This water, which has a temperature higher than its boiling point but does not boil, is called superheated water.

This is a great help if you have a system that can superheat or supercool a liquid to achieve energy-based molecular dissociation in the liquid.

It is an object of the present invention to provide a system capable of overheating and / or supercooling a liquid in a capillary.

It is contemplated herein that the system can be used for rapid DNA or RNA extraction, for example without the addition of extracts. The system of the present invention enables fast, simple and inexpensive methods, for example, for the preparation of genomic DNA for PCR amplification, genotyping, genetic research, human identity screening, virus / microbial screening or other uses.

Nevertheless, it is also envisioned that the system can be used as a unique tool for fragmentation of water insoluble molecules.

With reference to claim 1, the object of the present invention is met by an open system for superheating and / or supercooling a liquid, wherein the liquid is in a capillary, where it is heated at atmospheric pressure or above the boiling point of the liquid or at atmospheric pressure. There is at least one heating and / or cooling means for cooling the liquid below the freezing point of the liquid, wherein the at least one heating and / or cooling means is in thermal contact with the capillary in the region where the liquid is present in the capillary, where the capillary is At least there is little nucleation center on its inner surface.

"At least there are few nucleation centers" means that there is at least little scratches, impurities or other defects on the inner side.

Open system means that the surface of the liquid that is not in contact with the surface of the capillary tube is in contact with atmospheric pressure.

Although the point of overheating and / or supercooling applications is described with regard to the treatment of proteins, peptides and biomolecules, it is to be understood that the present invention for superheating and / or supercooling liquids can also be used for other applications.

Capillary tube means first of all a cylindrical body of liquid in the body. However, one can also think of capillaries with different geometric shapes inside. It is important that the inner surface of the capillary is free of nucleation centers (eg, scratches or impurities).

The heating or cooling means is in thermal contact with the outer side of the capillary. By doing so, the inner space of the capillary can be heated or cooled. Therefore, it is advantageous if the material of the capillary has high thermal conductivity.

The capillary can be composed of glass, thermostable plastic or metal. The inner surface of this tube should have at least few nucleation centers. This means that there is no scratch on the inner surface and no impurities. The glass can be treated, for example, as known in the art of advanced optical systems, to obtain a surface with at least few nucleation centers. Glass capillary can also be expected that its inner surface can be coated (chemically modified) with a polymer such as, for example, silane, silane-based surface modification, surfactant or polyelectrolyte. The inner surface of the metal capillary may be highly polished or perhaps coated, such as the glass described above. The capillary can be composed of, but is not limited to, thermostable plastics such as PFA, PSA, PTFE, PE, PP. It is also possible to put another liquid in the tube, where the other liquid is incompatible with the liquid to be heated. This means that the two liquids remain separate and have a distinct boundary between the two liquids. The other liquid forms a thin film on the inner surface of the capillary. Because of the surface tension of other liquids, this inner surface becomes a "smooth" inner surface that is at least almost free of nucleation centers.

The inner diameter of the capillary tube of the present invention is in the range of 1 μm to 250 μm. To date, however, capillaries with internal diameters of 5, 50, 100 and 150 μm have been tested positive for use of the present system.

In connection with claim 2, it is within the system scope of the present invention that the region of the capillary filled with liquid extends both beyond the region of the capillary in thermal contact with the heating and / or cooling means.

This is advantageous because there is a temperature gradient in the liquid beyond the area where the heating or cooling means is located. Therefore, there is no phase change inside the liquid that can occur if the outer surface of the liquid is in the region of the directly heated capillary.

Therefore, according to claim 3, it is more advantageous for the open system to be operated in a flow-through mode.

It is useful to operate the system in flow through mode for samples (liquid) requiring only a short exposure period / hour. It is also important that manual sample handling is not required by operating the system in flow through mode. Prevention of manual sample processing can reduce the risk of sample contamination. However, within the scope of the present invention, the system may be connected to other flow through processing systems such as, for example, high performance liquid chromatography (HPLC).

As described in claim 4, the flow through mode can advantageously be achieved with the operation of one or more screws or pump feeders for conveying liquid through the region of the capillary in which one or more heating and / or cooling means are in thermal contact with the capillary. have.

Within the scope of the present invention, the screw or pump feeder is operated at a fixed rate, whereby one or more heating and / or cooling means may deliver the correct volume of liquid at the correct flow rate through the region of the capillary tube in thermal contact with the capillary tube. Can be. The pump may be an inhaler pump or a capillary pump, or any other pump that allows the liquid to move.

It is also contemplated herein that the operation of the screw or pump feeder may be controlled by a computer or the like capable of programming a predetermined sequence of slow and fast flow rates of the screw or pump feeder. The control may be such as open-loop control or closed-loop control. This makes it possible to remain in the liquid for a defined time in the area where the liquid is heated. By doing so it is possible to transfer a fixed amount of heat into the liquid.

As described in claim 5, within the scope of the present invention the open system is operated in batch mode.

This is appropriate for rare stable molecules that require long exposure times for dissociation. For these molecules, it would not have been possible to realize long exposure times in flow through mode even at very low flow rates.

Claim 6 represents a preferred use of one of the aforementioned systems or methods. Therefore, a preferred embodiment of the present invention is that the liquid is water or an organic solvent.

Many substances are dissolved in water and water is usually called a common solvent.

As described in claim 7, it is feasible within the scope of the present invention that the liquid comprises biomolecules, wherein the biomolecules are overheated or supercooled and exposed and dissociated during the exposure time.

Dissociation refers to the destruction of any type of biomolecule, including energy-based hydrolysis, fragmentation, degradation as well as treatment with chemicals, enzymes or metals for fragmentation.

Therefore, the present invention has a very important relevance for biomolecule processing for scientific applications.

Exposure time for dissociation of biomolecules is strongly influenced by the nature of the molecule (eg amino acid sequences of proteins and peptides). However, it is suitable for the use of the present invention that the exposure time is in the range of several seconds to several minutes.

As described in claim 8, the biomolecule is expected to be a biopolymer selected from the group consisting of proteins, nucleic acids, carbohydrates and lipids.

As described in claim 9, a preferred embodiment of the present invention is that said biomolecule is a protein or peptide.

Overheat-induced dissociation of proteins and peptides can be observed by mass spectrometry. Optimization of superheat conditions such as temperature and exposure time can result in partial hydrolysis of sample molecules.

Simple samples such as dipeptides and tripeptides are used to demonstrate the feasibility of the method. As a result, a set of short oligo-peptides can fragment under superheated conditions. Using small molecules, such as dipeptides and tripeptides, can generate criteria for calculating the mass of all possible reaction products. Such products may arise from peptide fragmentation, adduct formation or removal inherently.

Partial hydrolysis can create criteria for identifying sequence-specific weaknesses in the peptide backbone.

Ideally, the present invention can be used to increase knowledge and experience about the mechanism of overheat-induced molecular disruption.

Furthermore, it is possible to predict hydrolysis products.

Eventually, it will be possible to identify superheat-breaking proteins by peptide formation patterns in PMF-like assays.

Superheat-induced hydrolysis products can easily be detected by product mass and fragmentation behavior in tandem mass spectrometry analysis. Therefore, electrospray ionization (ESI), quadrupole-ion traps / matrix assisted laser desorption ionization-tandem time of flight (MALDI-TOF ² ) Available MS systems such as can assist in mass spectrometry and structural characterization (de novo sequencing) of overheat-result fragments.

Overheating techniques can replace trypsin digestion of proteins. This can easily be reduced to a few seconds, which normally requires several hours in enzymatic methods. As a result, peptide analysis by mass spectrometry gives the same level of information (PMF) in the "microfluidics meets proteomics" -approach compared to the much slower and much more expensive proteolytic digestion. do.

Other important factors besides the identification of proteins should be analyzed to determine, for example, activity, intracellular distribution and molecular fate. Many other posttranslational protein-modifications (PTM) are known to affect these factors. Some of these groups are difficult to analyze because of the impossibility of specific manipulations. For glycosylation, the difference in mass before and after enzymatic cleavage of glycans can be used to calculate the chemical formula of the sugar molecule. Compared to the high energy transfer to the molecule in other systems (eg in MSMS reactions), the superheat will also provide a random fragmentation pattern in the side chains, protein modifications, and peptide modifications of amino acids. Today, many hydrolytic or engineered enzymes for PTM are not known. Finally, for a detailed analysis of the structure, the superheat can narrow these gaps to identify different stages of degradation in the protein modification group. Superheat will provide a number of PTM treatment methods.

Superheat can replace a number of hydrolytic enzymes currently used in the course of daily experiments, including digestion of DNA, fatty acids, isoprenoids and polyketides. In particular, lantibiotics are of particular importance because of the absence of hydrolase in most of them.

Thus, within the scope of the present invention, the biomolecules are secondary metabolites, sugars, polycyclic aromatic carbohydrates, phospholipids, glycolipids, sterols, glycerol lipids, vitamins, hormones, neurotransmitters, fatty acids, isoprenoids, lantibiotics and poly It is selected from the group consisting of ketides.

With regard to claim 11, it is also within the scope of the present invention that the liquid comprises prokaryotes, eukaryotes or viruses.

Claims (11)

The liquid is in the capillary and there is at least one heating and / or cooling means for heating the liquid above the boiling point of the liquid at atmospheric pressure or for cooling the liquid below the freezing point of the liquid at atmospheric pressure, and the at least one heating and / or cooling means may be An open system for overheating and / or supercooling of a liquid, wherein the capillary is in thermal contact with the capillary in the region where the liquid is present, and the capillary has at least its nucleation center at least at its inner surface. 2. An open system according to claim 1, wherein the region of the capillary filled with liquid extends both beyond the region of the capillary in thermal contact with the heating and / or cooling means. Method for superheating and / or supercooling a liquid under use of the open system, characterized in that the open system according to claim 1 or 2 is operated in a flow-through mode. 4. The flow-through mode of claim 3, wherein the flow-through mode is achieved by operation of at least one screw or pump feeder for conveying liquid through the capillary region in which at least one heating and / or cooling means is in thermal contact with the capillary. Way. Method for superheating and / or supercooling a liquid under use of the open system, characterized in that the open system according to claim 1 is operated in a batch mode. Use of the open system and / or method according to any one of claims 1 to 5, characterized in that the liquid is water or an organic solvent. Use according to any one of claims 1 to 6, characterized in that the liquid comprises a biomolecule and the biomolecule is overheated or supercooled and exposed for dissociation during the exposure time. 8. The use according to claim 7, wherein the biomolecule is a biopolymer selected from the group consisting of proteins, nucleic acids, carbohydrates and lipids. 8. Use according to claim 7, wherein the biomolecule is a protein or a peptide. The method of claim 7, wherein the biomolecules are secondary metabolites, sugars, polycyclic aromatic carbohydrates, phospholipids, glycolipids, sterols, glycerol lipids, vitamins, hormones, neurotransmitters, fatty acids, isoprenoids, lantibiotic and polyke Use, characterized in that selected from the group consisting of tide. Use according to any one of claims 1 to 6, characterized in that the liquid comprises prokaryotes, eukaryotes or viruses.