Patent Publication Number: US-2013240180-A1

Title: System and method for superheating and/or supercooling of liquids and use of the system and/or method

Description:
The present invention relates to a system and method for superheating and/or supercooling of liquids and use of the system and/or method. 
     Superheating means: Bringing the temperature of a liquid above the boiling temperature at a defined pressure (here: the ambient pressure) without vaporisation of the liquid. Supercooling means: Bringing the temperature of a liquid below the freezing temperature at a defined pressure by keeping the liquid in the liquid phase without change of the phase. 
     Fast, reliable and inexpensive methods for e.g. protein identification and analysis of posttranslational modifications are important as proteins are being used as tools, targets and therapeutics. 
     For e.g. protein identification mass spectrometry (MS) of proteins is the state-of-art technique. However, for mass spectrometry (MS)-based identification of proteins a pre-treatment step to break the proteins into peptides is required for peptide mass fingerprinting (PMF) or peptide fragment fingerprinting. 
     Typically an enzymatic reaction based on proteases such as trypsin is employed. Besides the enzymatic reaction, researchers also use chemical degradation methods. Both processes are time consuming and expensive. 
     A more general approach than enzyme-based target molecule hydrolysation can be performed by energy-based destruction of peptide bonds within the proteins. 
     As known from MS fragmentation reactions, increasing amounts of energy lead to more unspecific fragmentation patterns and finally to complete atomization of the original molecule. 
     The rate of protein fragmentation is highly energy-dependent thus increasing temperature will dramatically increase the reaction rate. However, maximum achievable temperature is typically limited by the solvent boiling point. In case water is used as solvent, the solvent boiling point is 100° C. at ambient pressure, if nucleation centres inside the reaction vessel are abundant. 
     Nevertheless, if no nucleation centres are present, water temperature can be increased above the boiling point of water at ambient pressure. This water, which is not boiling but has a temperature above its boiling point, is called superheated water. 
     It would therefore be of great benefit if there were a system, which is able to superheat or supercool liquids so that energy-based dissociation of molecules in liquids can be achieved. 
     It is the object of the invention to provide a system which allows superheating and or supercooling of liquids in capillary tubes. 
    
    
     With reference to claim  1  the object is met by an open system for superheating and/or supercooling of liquids, wherein the liquid is within a capillary tube, wherein there is at least one heating and/or cooling means for heating the liquid above boiling point of the liquid at ambient pressure or cooling the liquid below freezing point of the liquid at ambient pressure, wherein the at least one heating and/or cooling means is in thermal contact with the capillary tube in an area, in which there is liquid inside the capillary tube, and wherein the capillary tube is at least nearly free of nucleation centers at its inner surface. 
     The meaning of “at least nearly free of nucleation centers” means that the inner surface is at least nearly free of scratches, impurities or other imperfections on the inner surface. 
     Open system means that the surface of the liquid, which is not in contact with the surface of the capillary tube, is in contact with ambient pressure. 
     Although the main point of application of the superheating and/or supercooling is described with reference to the treatment of proteins, peptides and biomolecules it has to be understood that the invention for superheating and/or supercooling of liquids can also be used in other applications. 
     Capillary tube means preferentially a cylindrical body wherein the liquid is inside this body. However, also capillary tubes with other inner geometric shape are imaginable. It is of importance that the inner surface of the capillary tube is free of nucleation centers (e.g. scratches or impurities). 
     The heating or cooling means are in thermal contact with the outer surface of the capillary tube. Thereby the inner volume of the capillary tube can be heated or cooled. Therefore it is advantageous if the material of the capillary tube has a high thermal conductivity. 
     The capillary tube can be made of glass, thermostable plastics or metal. The inner surface of this tube shall be at least nearly free of nucleation centers. This means that the inner surface should be scratch-free and also free of impurities. The glass can be treated as it is known for example in the field of advanced optic systems to get the surface at least nearly free of nucleation centers. It is also envisioned that the glass capillary can be coated (chemically modified) on its inner surface with e.g. silanes, silane-based surface modifications, surfactants or polymers such as e.g. polyelectrolytes. The inner surface of the metal capillary tube can be high polished or perhaps be coated, as described above for glass. The capillary tube can be made of thermostable plastics such as, but not limited to, PFA, PSA, PTFE, PE, PP. It can also be possible to bring another liquid into the tube, whereby this other liquid is phobic with respect to the liquid to be heated. This means that the two liquids keep separated and have a sharp boarder between the two liquids. The other liquid builds a thin film over the inner surface of the capillary tube. This leads to a “polished” inner surface, which it at least nearly free of nucleation centers because of the surface tension of the other liquid. 
     It is belonging to the invention that inner diameter of the capillary tube is in the range between 1 μm to 250 μm. However, so far capillary tubes with an inner diameter of 5, 50, 100 and 150 μm have been tested positively for use of the invented system. 
     With respect to claim  2  it is within the scope of the invented system that the area of the with liquid filled capillary tube extends on both sides beyond the area of the capillary tube where the heating and/or cooling means are in thermal contact with the capillary tube. 
     This is advantageous, because there is a temperature gradient in the liquid beyond the area in which the heating or cooling means are placed. Therefore there is no change of phase inside the liquid as it might happen if the outer surface of the liquid would be in the area of the capillary tube, which is directly heated. 
     Hence, according to claim  3 , it is further advantageous that the open system is operated in a flow-through mode. 
     It is beneficial to operate the system in flow-through mode for samples (liquids), for which only a short exposure period / time is required. It is also of importance that, by operating the system in flow-through mode, no manual sample handling is required. By avoidance of manual sample handling, the contamination risk of the sample can be reduced. However, it is within the scope of the invention, that the system can 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 accomplished by the operation of at least one feeder screw or pump for conveying the liquid through the area of the capillary tube where the at least one heating and/or cooling means are in thermal contact with the capillary tube. 
     It is within the scope of the invention that the feeder screw or pump can be operated with a defined speed, therefore conveying an accurate volume of liquid with a precise flow rate through the area of the capillary tube where the at least one heating and/or cooling means are in thermal contact with the capillary tube. The pump can be a syringe pump or a capillary pump or any other pump which allows conveyance of liquids. 
     It is hereby also envisaged that the operation of the feeder screw or pump can be controlled by a computer or such, which allows programming the feeder screw or pump with a defined sequence of slow and fast flow rates. The control can be of the kind of an open-loop control or a closed-loop control. It is possible to have the liquid for a defined time in the area, where the liquid is heated. Thereby it is possible to bring a defined amount of heat into the liquid. 
     As described in claim  5 , it is also within the scope of the invention that the open system is operated in a batch mode. 
     This is favorable in case extraordinary stable molecules require long exposure times for dissociation. For these, it might not be possible to realize the long exposure times in a flow-through mode, even at very low flow rates. 
     Claim  6  refers to a preferred use of one of the above mentioned systems or methods. It is therefore a preferred embodiment of the invention that the liquid is water or an organic solvent. 
     Many substances dissolve in water and it is commonly referred to as the universal solvent. 
     As described in claim  7 , it is feasible within the scope of the invention that the liquid contains biomolecules, wherein said biomolecules are dissociated when superheated or supercooled and exposed for an exposure period. 
     Dissociation means: Any kind of destruction of biomolecules, including energy-based hydrolysis, fragmentation, degradation but also treatment with chemicals, enzymes or metals for fragmentation. 
     Therefore the invention is of a very important relevance for the treatment of biomolecules for scientific applications. 
     The exposure period for dissociation of biomolecules is strongly influenced by the character of the molecule (e.g. amino acid sequence for proteins and peptides). However, it is favorable for the use of the invention that the exposure period is in the range of seconds to a few minutes. 
     As described in claim  8 , it is envisaged that the biomolecules are biopolymers selected from the group consisting of proteins, nucleic acids, carbohydrates and lipids. 
     As described in claim  9 , it is a preferred embodiment of the invention that the biomolecules are proteins or peptides. 
     Superheating-induced dissociation of proteins and peptides can be monitored by mass spectrometry. Optimizing of superheating conditions such as temperature and exposure time could lead to partial hydrolysis of sample molecules. 
     Simple samples such as di- and tri-peptides were used to demonstrate feasibility of the method. Subsequently, a set of short oligo-peptides can be fragmented under superheated conditions. Employing small molecules such as di- and tri-peptides can build a basis to calculate the masses of all possible reaction products. These products could result from original peptide fragmentation, adduct formation or elimination. 
     Partial hydrolysis could build a basis to identify sequence-specific weak points in peptide backbones. 
     In an ideal case, the invention can be used to increase the knowledge and experience in the mechanism of superheat-induced molecule decay. 
     Furthermore it is possible to predict hydrolysation products. 
     Finally, it may be possible to identify superheat-destroyed proteins in a PMF-like measurement by the peptide formation patterns. 
     Superheat-induced hydrolysation products could easily be detected by product mass and fragmentation behaviour in tandem mass spectrometry analysis. Therefore, the available MS systems such as electrospray ionization (ESI), quadrupole-ion trap/matrix assisted laser desorption ionization-tandem time of flight (MALDI-TOF 2 ) can help in mass analysis and structure elucidation (de novo sequencing) of superheat-resulting fragments. 
     The superheating technique could replace tryptic digestion of proteins. It would dramatically shorten reaction time currently required be enzymatic methods from typical few hours to seconds. Subsequently, the peptide analysis by mass spectrometry would then give the same level of information (PMF) in the “microfluidics meets proteomics”-approach as compared to much slower and far more expensive proteolytic digests. 
     Beside the identification of proteins other important factors must be analysed to determine e.g. activity, subcellular location and destiny of the molecule. Many different posttranslational protein-modifications (PTMs) are known to have influence on these parameters. Some of these groups are difficult to analyse, because of impossibility of specific manipulation. In case of glycosylations, mass difference before and after enzymatic cleave-off of glycans can be used to calculate chemical formula of the sugar molecules. In comparison to high energy transfer to such molecules is other systems (e.g. in from MSMS reaction), also superheating should provide random fragmentation patterns in amino acid side chains, protein- and peptide- modifications. Today, many hydrolysing or manipulating enzymes for PTMs are unknown. Superheating could close this gap to identify different steps of degradation processes in protein-modifying groups, finally for detailed analysis of such structures. Superheat may provide a method to treat the multitude of PTMs. 
     Superheating can replace a number of hydrolysation enzymes currently used in daily laboratory processes, including the digestion of DNA, fatty acids, isoprenoids and polyketides. In particular, lantibiotics are of special interest due to non-existence of hydrolysing enzymes to a majority of them. 
     Hence, it is within the scope of the invention that the biomolecules are selected from the group consisting of secondary metabolites, sugars, polycyclic aromatic carbohydrates, phospholipids, glycolipids, sterols, glycerolipids, vitamins, hormones, neurotransmitters, fatty acids, isoprenoids, lantibiotics and polyketides. 
     With respect to claim  11 , it is also within the scope of the invention that the liquid contains prokaryotes, eukaryotes or viruses. 
     Hereby it is envisioned that the system can be used for e.g. fast DNA or RNA extraction without addition of extraction solutions. The invented system allows a fast, simple, and inexpensive method for preparing e.g. genomic DNA for PCR amplification, genotyping, genetic studies, human identity testing, viral/microbial screening or other uses. 
     Nevertheless, it is also envisaged that the system can be used to serve as a unique tool for fragmentation of water insoluble molecules.