A reusable digestion tube for amino acid assay

Abstract

The presented system introduces a reusable and user-friendly apparatus tailored for the digestion of samples for amino acid assay. This specialized device comprises a purpose built tube crafted from borosilicate glass, accompanied by a cap constructed from polytetrafluoroethylene. The tube is designed with a gas inlet for the introduction of nitrogen gas and a gas outlet to facilitate the expelling of air from the tube, preventing any spillage of the hydrochloric acid used in the sample digestion. A notable feature of the tube's design is its constriction, allowing the cap to establish an airtight seal, effectively sealing off both the gas inlet and gas outlet. By maintaining a nitrogen atmosphere within the tube throughout the digestion process, the risk of oxidation of amino acids is mitigated, ensuring accurate assessment of quantities of amino acids. The apparatus is characterized by its ease of use and reusability, providing a practical solution for digestion of samples for precise and efficient amino acid assay.

Description

A REUSABLE DIGESTION TUBE FOR AMINO ACID ASSAYTECHNICAL FIELD

[0001] The present invention relates to the field of biotechnology, and more specifically, systems and methods for amino acid assay.BACKGROUND

[0002] The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0003] Amino acid assay is a systematic analytical procedure employed to precisely quantify the amounts of various amino acids in a sample containing proteins. Several methods are available for conducting amino acid assay, with a common requirement being the preliminary digestion of the sample in 6N hydrochloric acid. This digestion process occurs at a high temperature, typically 110 degree Celsius, and spans a duration of 26 hours. Following digestion, the sample is subjected to analysis using an amino acid analyzer. A crucial aspect of this digestion process is the necessity for it to transpire in an inert atmosphere. The rationale behind this lies in the susceptibility of proteins to oxidation in the presence of oxygen during digestion. Such oxidation results in the detection of a reduced number and lower quantities of amino acids in the sample. To circumvent this issue, the digestion process is preferably conducted in an environment devoid of oxygen, commonly achieved by utilizing a nitrogen atmosphere.

[0004] US2288065 discloses a specialized digestion tube designed to prevent liquid spattering during boiling operations, even in cases of vigorous boiling or when dealing with viscous liquids. The tube retains the familiar straight tube design while incorporating a unique construction to eliminate the possibility of liquid or any portion thereof being expelled directly from the tube's mouth. This is achieved by laterally offsetting the upper and lower portions of the tube,with the closed end positioned significantly away from the axis of the open-ended upper portion. The connection between these segments involves a reversely curved intermediate portion. The tube's diameter and the design of the curved intermediate section are carefully calibrated to prevent any liquid droplets propelled upward during boiling from passing directly through the tube's mouth. Instead, such droplets are either intercepted by the curved intermediate section or the upper portion of the tube, ensuring that they flow downward and return to the boiling liquid, averting any unwanted spattering.

[0005] US4645746 discloses an enhanced method for quantifying nitrogen content or other elemental content in diverse materials representing a significant improvement over conventional approaches. This innovative process eliminates the need for traditional catalysts, offering a simpler and faster alternative with remarkably high accuracy. The key feature of this method is the continuous addition of hydrogen peroxide to the ongoing digestion process, conducted through boiling and refluxing with a fractionating column. Beyond facilitating the quantitative determination of nitrogen, this digestion process extends its capability to assess various elements with precision, showcasing its versatility and efficiency in elemental analysis.

[0006] US4882286 describes a microwave-based apparatus designed for the swift digestion of a sample through the Kjeldahl method. The apparatus comprises a reaction vessel enveloped by an insulator, such as a cup, to enhance the insulation process. Positioned within the internal chamber of a microwave system, the reaction vessel is surrounded by a wall featuring an aperture. A connector tube, capable of sliding movement within the aperture, connects one end to the mouth of the reaction vessel and the other end to an external suction device. External to the chamber, a biasing element is incorporated to aid in securely attaching the slidable connector tube to the vessel's mouth. This design allows for the easy removal of the vessel from the chamber without disengaging the biasing element, ensuring efficiency and convenience in the digestion process.

[0007] US4946797describes an expedited digestion technique related to Kjeldahl method utilizing microwave energy. The method involves applyingmicrowave energy to an acid / sample mixture at the initiation of digestion and continuously throughout the process. Following the cessation of microwave energy application, the digestate undergoes dilution through a combination of pulsed and continuous water addition. This gradual dilution method serves to prevent a sudden escalation in gas evolution, eliminating the necessity for an intervening cooling step and thereby reducing overall processing time. Additionally, the description includes a technique for separating fat from a protein sample containing fat during the digestion process.

[0008] While these devices may be effective in estimating protein levels, they prove unsuitable for accurately assessing amino acids due to the oxidation of amino acids within the samples when processed in these devices. Although heat sealing can be employed to render these devices airtight, it complicates the procedure and renders the apparatus non-reusable. Additionally, the use of such improvised devices also raises concerns about potential spills of hydrochloric acid. This underscores the necessity for a dedicated apparatus designed specifically for the digestion of samples in amino acid assay.

[0009] As a result, there exists a need for improvements over the prior art and more particularly for a more efficient way.SUMMARY OF THE INVENTION

[0010] The disclosed invention introduces a novel and efficient apparatus tailored for the digestion of samples in amino acid assays. At the core of this innovation is a purpose-built tube crafted from borosilicate glass, renowned for its exceptional resistance to chemical corrosion and its capacity to withstand high temperatures. This choice of material ensures the durability and suitability of the tube for the rigorous digestion process inherent in amino acid assays.

[0011] Complementing the robust tube design is the cap, meticulously constructed from polytetrafluoroethylene (PTFE). PTFE is chosen for its inert nature, providing resistance against chemical reactions with the sample components or the digestion process itself. The thoughtful selection of materialscontributes to the overall reliability and effectiveness of the apparatus. Critical to the functionality of the apparatus are the incorporated gas inlet and gas outlet features on the tube. The gas inlet facilitates the controlled injection of nitrogen gas into the apparatus, while the gas outlet allows for the gradual release of air. This design element serves a dual purpose, preventing spills of the hydrochloric acid used in the sample digestion and enhancing the overall safety of the process.

[0012] A distinctive feature in the tube's design is the presence of a constriction, strategically positioned to enable the cap to establish an airtight seal. This seal plays a crucial role in blocking both the gas inlet and gas outlet, effectively isolating the internal atmosphere. This airtight seal ensures that external air is excluded, allowing for precise control over the atmosphere within the tube. The controlled atmosphere within the tube is maintained by the injection of nitrogen gas, a process that serves as a safeguard against the oxidation of proteins in the sample during digestion. This crucial feature ensures the accuracy of amino acid assessments during the subsequent analytical phase, contributing to the reliability of the entire assay process. Furthermore, the apparatus is designed with user-friendliness in mind. The mechanism for injecting nitrogen and releasing air is straightforward, facilitating ease of use for laboratory technicians. An additional advantage lies in the reusability of the apparatus. Its design allows for easy cleaning and preparation for subsequent assays, aligning with sustainability goals and minimizing waste.

[0013] This specialized apparatus offers a comprehensive solution to the challenges associated with amino acid assays. Its robust materials, controlled atmosphere, and user-friendly features make it a valuable tool in the analytical toolkit, ensuring accurate and efficient determination of amino acid quantities in diverse samples.

[0014] The summary of the invention does not necessarily disclose all the features essential for defining the invention. The invention may reside in a subcombination of the disclosed features. The various combination and subcombination are fully described in the detailed description.BRIEF DESCRIPTION OF DRAWINGS

[0015] The accompanying drawings are included to provide a further understanding of the present disclosure, and are incorporated in and constitute a part of this specification. The drawings illustrate exemplary embodiments of the present disclosure and, together with the description, serve to explain the principles of the present disclosure.

[0016] The diagrams are for illustration only, which thus is not a limitation of the present disclosure, and wherein:

[0017] FIG. l is a longitudinal section view of the tube, in accordance with a preferred embodiment of the invention.

[0018] FIG. 2 is a longitudinal section view of the cap, in accordance with a preferred embodiment of the invention.

[0019] FIG. 3 is a longitudinal section view of the apparatus with the cap completely closing the tube, in accordance with a preferred embodiment of the invention.DETAILED DESCRIPTION OF DRAWINGS

[0020] Those skilled in the art will understand that when an element or part in the drawings is referred to as being “on” (or “connected” to or “coupled” to or “attached” to) another element, it can be directly on (or attached to) the other element or intervening elements may also be present. Furthermore, relative terms such as “inner”, “outer”, “upper”, “above”, “lower”, “beneath”, and “below”, and similar terms, may be used herein to describe a relationship of one element to another element. It is understood that these terms are intended to encompass different orientations of the device in addition to the orientation depicted in the figures.

[0021] The present invention describes a reusable apparatus designed for sample digestion for amino acid assay comprises a borosilicate glass tube and a polytetrafluoroethylene (PTFE) cap, each contributing to the precision and efficacy of the process. The borosilicate glass tube is chosen for its robustness inwithstanding chemical corrosion and high temperatures. Within this tube, a gas inlet and a gas outlet are strategically positioned at different levels and on diametrically opposite sides. These features allow for controlled gas flow during the digestive process. Additionally, a constriction is incorporated below both the gas inlet and the gas outlet, serving a critical role in the sealing mechanism. The PTFE cap complements the tube's design, featuring a shank extending from its top. This shank becomes instrumental in creating a secure seal when the cap is closed. The upper half of the shank is threaded with male threading, mirroring the corresponding female threading on the inner surface of the upper half of the tube's neck. The threaded engagement of these components facilitates a tight closure, ensuring the integrity of the apparatus. Upon the cap's complete closure, the shank comes into contact with the constriction in the tube. This interaction is pivotal, creating an airtight seal that effectively blocks both the gas inlet and the gas outlet. The cap's threading and the shank-constriction seal combine to establish a closed atmosphere within the tube, preventing the ingress of external air. In summary, design intricacies of the apparatus, such as the placement of gas inlet and gas outlet, the presence of a constriction, and the threaded sealing mechanism, collectively contribute to its ability to create a controlled and closed environment for the accurate and precise digestion of samples for amino acid assay.

[0022] FIG. l is a longitudinal section view of the tube, in accordance with a preferred embodiment of the invention. The tube is constructed from borosilicate glass with a thickness of 0.5 cm. It measures 20 cm in length and boasts an internal diameter of 2 cm. Positioned at the top, the mouth (101) of the tube leads through a cylindrical body to a rounded bottom (102). A neck region (103), extending 5 cm below the mouth, is a distinctive feature. The upper half of this neck (104) is internally threaded (105) with female threading, while the lower half (106) remains unthreaded. This lower section encompasses a gas inlet (107) and a gas outlet (108), with the gas inlet situated 4 cm below the mouth. The gas inlet, perpendicular to the tube, possesses an internal diameter of 0.5 cm, extending 7 cm in length. Similarly, the gas outlet, located 3 cm below the mouth, features an internal diameter of 0.5 cm and extends perpendicularly for a length of7 cm. Notably, the gas inlet and gas outlet are on diametrically opposite sides of the tube. A constriction (109) in the tube emerges just below the neck, precisely 5 cm beneath the mouth. This constriction spans 1 cm in length, reducing the internal diameter of the tube to 1 cm at this specific point. The detailed dimensions and features outlined contribute to the precise design and functionality of the tube in the proposed apparatus.

[0023] FIG. 2 is a longitudinal section view of the cap, in accordance with a preferred embodiment of the invention. The cap is constructed from polytetrafluoroethylene, commonly known as Teflon. Its primary function is to seal the mouth of the tube. The uppermost part of the cap (201) possesses a thickness of 2 cm and a diameter of 3 cm. Below this top section, a cylindrical shank (202) is integral to the cap, extending to a length of 5 cm. The shank is characterized by two distinct halves. The upper half (203) of the shank is threaded with male threading (204), while the lower half (205) remains unthreaded. This threading on the upper portion of the shank is designed to engage with the female threading (105) present in the upper half of the neck of the tube. This interaction allows for a secure closure of the tube when the cap is fully inserted. Upon complete insertion into the tube, the shank traverses the entire length of the neck, reaching down to the constriction in the tube. A critical aspect of the design is that, upon reaching the constriction, the bottom of the shank establishes an airtight seal. This airtight seal is crucial for maintaining the controlled atmosphere within the tube during the digestion process, ensuring the efficacy and precision of the amino acid assay.

[0024] FIG. 3 is a longitudinal section view of the apparatus with the cap completely closing the tube, in accordance with a preferred embodiment of the invention. The method of using the apparatus begins by holding the tube vertically, and the sample intended for amino acid assay is introduced through the mouth (101) of the tube. Subsequently, 6N hydrochloric acid (HPLC grade) is carefully poured into the tube through the same mouth. The volume of hydrochloric acid added is precisely determined, with 3 ml poured for every 1 gram of protein present in the sample. Following the addition of hydrochloricacid, the cap is positioned at the mouth of the tube. The top part (201) of the cap is then slowly rotated, facilitating the entry of the shank (202) into the tube until the shank is positioned just above the gas outlet (108). At this point, a cylinder of nitrogen gas is connected to the gas inlet (107), and nitrogen is injected into the tube at a pressure ranging from 1.1 to 1.3 atm. The injected nitrogen displaces the air within the tube, and the displaced air exits through the gas outlet (108). This nitrogen injection process, lasting 3 to 4 minutes, establishes an inert atmosphere within the tube. Upon completion of the nitrogen injection phase, the cap is rotated once again, gradually closing the tube entirely, and the supply of nitrogen is halted. The bottom of the shank (202) creates an airtight seal (301) precisely at the constriction (109) of the tube, effectively blocking both the gas inlet (107) and the gas outlet (108). With this seal in place, the sample is now exposed to hydrochloric acid within an inert atmosphere, a critical condition for accurate amino acid assays. The sealed tube can now be heated to 110 degree Celsius for a duration of 26 hours. Following this digestion period, the cap is opened, and nitrogen evaporation is carried out. Subsequently, the amino acid assay of the sample is conducted using standard methodologies in an amino acid analyzer. This comprehensive procedure ensures the controlled digestion of the sample in a nitrogen-rich environment, safeguarding the accuracy and reliability of the amino acid assay results.

[0025] The utilization of this specialized apparatus for amino acid assays presents several distinct advantages that contribute to its efficiency and practicality.

[0026] Reusability: One primary advantage lies in the reusability of the apparatus. After its initial use, the apparatus can be thoroughly cleaned and prepared for subsequent sample analyses. This feature not only makes the apparatus a sustainable choice but also significantly enhances its costeffectiveness, as it eliminates the need for frequent replacements.

[0027] User-Friendly Design: The apparatus excels in user-friendliness, as it eliminates the requirement for heat sealing. Unlike some other methods that necessitate complex sealing processes, this apparatus streamlines the procedure,making it accessible and straightforward for laboratory technicians. The absence of intricate sealing steps contributes to the overall ease of use, enhancing the efficiency of the amino acid assay process.

[0028] Enhanced Safety Measures: Safety is paramount in laboratory settings, and this apparatus incorporates safety features to mitigate potential risks. The inclusion of a gas inlet for injecting nitrogen gas and a separate gas outlet for the controlled release of air is a key safety measure. This design ensures that the hydrochloric acid used in the sample digestion is contained within the apparatus, minimizing the risk of spills. The controlled gas flow enhances the overall safety of the amino acid assay process, providing a secure environment for laboratory personnel.

[0029] While the subject invention is described and illustrated with respect to certain preferred and alternative embodiments, it should be understood that various modifications can be made to those embodiments without departing from the subject invention, the scope of which is defined in the following claims.

Claims

CLAIMS1. A reusable apparatus for controlled digestion of samples for amino acid assay comprising: a borosilicate glass tube and a polytetrafluoroethylene (PTFE) cap; wherein the borosilicate glass tube comprises a gas inlet (107) and a gas outlet (108) at different levels, positioned diametrically opposite to each other; a constriction (109) strategically located below the gas inlet (107) and gas outlet (108); wherein the PTFE cap incorporates a shank (202) with male threading (204) in its upper half (203); an upper half (104) of the tube's neck (103) is equipped with female threading (105) on its inner surface; the male threading (204) on the shank (202) and the female threading (105) on the tube's neck (103) engage seamlessly, ensuring a secure and tight closure; upon complete closure, the shank (202) of the cap makes contact with the constriction (109) in the tube; an airtight seal (301) effectively blocks both the gas inlet (107) and the gas outlet (108), creating a closed and controlled atmosphere inside the tube, facilitating digestion of samples for precise amino acid assay.

2. The apparatus of claim 1, wherein the gas inlet (107) and gas outlet (108) are strategically positioned to facilitate controlled gas flow, ensuring an inert atmosphere for digestion of samples for amino acid assay.

3. The apparatus of claim 1, wherein the constriction (109) below the gas inlet (107) and gas outlet (108) serves to enhance the sealing mechanism during closure, contributing to the creation of an inert atmosphere inside the tube.

4. The apparatus of claim 1, wherein the threaded engagement between the male threading (204) on the shank and the female threading (105) on the neck of the tube provides a secure and tight closure mechanism, optimizing the sealing.

5. A method for controlled digestion of samples for amino acid assay using the reusable apparatus comprising: placing a sample intended for amino acid assay into a borosilicate glass tube through its mouth (101); pouring 6N hydrochloric acid into the tube through its mouth (101); placing a polytetrafluoroethylene (PTFE) cap in the mouth (101) of the tube; slowly rotating the cap until the shank (202) of the cap reaches just above the gas outlet (108); injecting nitrogen gas into the tube through the gas inlet (107) at a pressure ranging from 1.1 to 1.3 atm for a duration of 3 to 4 minutes to displace the air inside the tube through the gas outlet (108);slowly rotating the cap again to completely close the tube, thereby blocking off the gas inlet (107) and the gas outlet (108); heating the tube to digest the sample.

6. The method of claim 5, wherein the sample intended for amino acid assay is introduced into the borosilicate glass tube through its mouth (101), providing a controlled and precise initiation of the digestion process.

7. The method of claim 5, wherein nitrogen gas is injected into the tube through the gas inlet (107), displacing the air present inside the tube through the gas outlet (108) to establish an inert atmosphere, ensuring optimal conditions for digestion of samples for amino acid assay.

8. The method of claim 5, wherein the cap is rotated slowly to completely close the tube, blocking off the gas inlet (107) and the gas outlet (108), facilitating the creation of an inert atmosphere essential for digestion of samples for precise amino acid assay.

9. The method of claim 5, wherein the tube is heated at 110 degree Celsius for a specified period of 26 hours, optimizing the digestion process for accurate amino acid assay results.

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