阅读说明：本技术 发光增强剂、酶促化学发光底物及制备方法与试剂盒 (Luminescence enhancer, enzymatic chemiluminescence substrate, preparation method and kit ) 是由 黄记有 任凯瑜 周峻 于 2019-11-13 设计创作，主要内容包括：本发明涉及生物检测领域,具体而言,提供了一种发光增强剂、酶促化学发光底物及制备方法与试剂盒。本发明提供的1,2-二氧杂丁烷化合物发光作用的发光增强剂包括荧光剂与改性PEG偶联的化合物,荧光剂与AMP偶联的化合物,或,荧光剂与Tris偶联的化合物中的至少一种。该发光增强剂不仅能提高1,2-二氧杂丁烷化合物底物液的稳定性,还可以显著改善底物液的发光效率。(The invention relates to the field of biological detection, and particularly provides a luminescence enhancer, an enzymatic chemiluminescence substrate, a preparation method and a kit. The luminescence enhancement agent for the luminescence effect of the 1, 2-dioxetane compound provided by the invention comprises at least one of a compound of a fluorescer coupled with modified PEG, a compound of a fluorescer coupled with AMP, or a compound of a fluorescer coupled with Tris. The luminescence enhancer not only can improve the stability of the 1, 2-dioxetane compound substrate solution, but also can obviously improve the luminous efficiency of the substrate solution.)

1. A luminescence enhancement agent for the luminescence of a 1, 2-dioxetane compound, wherein the luminescence enhancement agent comprises at least one of a fluorescer coupled to a modified PEG, a fluorescer coupled to AMP, or a fluorescer coupled to Tris.

2. The luminescence enhancement agent of claim 1, wherein the luminescence enhancement agent comprises a compound of fluorescer coupled to modified PEG, a compound of fluorescer coupled to AMP, and a compound of fluorescer coupled to Tris;

preferably, the ratio of the amount of fluorescer to modified PEG coupled compound, fluorescer to AMP coupled compound, and fluorescer to Tris coupled compound is (6-8): (1-3): 1.

3. the luminescence enhancement agent according to claim 1, wherein the fluorescent agent comprises a fluorescein-based fluorescent agent, a rhodamine-based fluorescent agent, a coumarin-based fluorescent agent, a naphthalimide-based fluorescent agent, or a benzimidazole-based fluorescent agent, preferably a fluorescein-based fluorescent agent;

preferably, the modified PEG includes PEG where the hydrophilic arm is bifunctional or PEG where the hydrophilic arm is monofunctional.

4. The luminescence enhancement agent of claim 1, wherein the structural formula of the luminescence enhancement agent comprises formula (i), formula (ii), formula (iii), formula (iv), or formula (v) as follows:

wherein m is an integer from 1 to 3, n is an integer from 1 to 12;

preferably, the structural formula of the luminescence enhancer includes the following formula (I-1):

5. the luminescence enhancement agent of claim 1, wherein the concentration of the luminescence enhancement agent is in the range of 0.0001mmol/L to 1mmol/L, preferably in the range of 0.1mmol/L to 0.5 mmol/L.

6. The luminescence enhancement agent of claim 1, further comprising at least one of magnesium ions, a buffer, a preservative, or a surfactant;

preferably, the buffer comprises AMP buffer or Tris buffer;

preferably, the concentration of the buffer solution is 20mmol/L-1000mmol/L, and the pH value is 8-11.5; preferably at a concentration of 50mmol/L to 100mmol/L, preferably at a pH of 8.5 to 9.5;

preferably, the salt containing the magnesium ion comprises magnesium chloride, magnesium sulfate or magnesium acetate;

preferably, the concentration of the magnesium ions is 0.0001-0.1 mol/L;

preferably, the preservative comprises sodium azide or Proclin 300;

preferably, the content of the sodium azide is 0.01-5% by mass;

preferably, the content of the Proclin300 is 0.01g/L-1.0 g/L;

preferably, the surfactant comprises at least one of CTAC, CTAB, Triton X-100, 3-sulfopropyl hexadecyl dimethyl betaine, 3-sulfopropyl tetradecyl dimethyl betaine, or Tween-20;

preferably, the concentration of the surfactant is 0.01mmol/L to 10 mmol/L.

7. A method for producing a luminescence enhancement agent for the luminescence of a 1, 2-dioxetane compound, characterized in that the components of the luminescence enhancement agent according to any one of claims 1 to 6 are dissolved in water.

8. A kit for enhancing the luminescence of a 1, 2-dioxetane compound, said kit comprising the luminescence-enhancing agent of any one of claims 1 to 6.

9. An enzymatic chemiluminescent substrate for alkaline phosphatase comprising a 1, 2-dioxetane compound and a luminescence enhancer of any one of claims 1-6;

preferably, the ratio of the amounts of the 1, 2-dioxetane compound and the fluorescent light-emission enhancer is 1:2 to 2: 1.

10. A kit for the enzymatic chemiluminescent substrate of alkaline phosphatase comprising the enzymatic chemiluminescent substrate of alkaline phosphatase as defined in claim 9.

Technical Field

The invention relates to the field of biological detection, and in particular relates to a luminescence enhancer, an enzymatic chemiluminescence substrate, a preparation method and a kit.

Background

With the development of medical technology and the continuous improvement of consciousness of residents in medical care and treatment before illness, the diagnosis requirement of the in vitro diagnosis industry on various diseases is higher, and how to detect the diseases more quickly, more efficiently, more sensitively and cheaper becomes an important subject of the industry. The detection technology in the in vitro diagnosis industry can be classified into biochemical diagnosis, immunodiagnosis, molecular diagnosis, microbial diagnosis, blood diagnosis, POCT and the like from a large methodological classification mode, wherein the immunodiagnosis accounts for the highest proportion, so the immunodiagnosis field still belongs to a diagnosis methodology with relatively high market share and a promising prospect.

Immunodiagnosis gradually develops from an initial radioimmunoassay and a time-resolved fluorescence method into the coexistence of multiple technologies such as enzyme-linked immunosorbent assay, colloidal gold, latex turbidimetry, fluorescence immunization, chemiluminescence and the like, and chemiluminescence develops rapidly due to the advantages of high efficiency, sensitivity, high adaptability to high flux and easiness in automation popularization, and keeps a strong market prospect in a certain time range in the future.

Chemiluminescence refers to the phenomenon that in a reaction system, an excited product is produced through a chemical reaction, and in the process of returning to a ground state, the released energy is converted into photons, so that luminescence is generated. Chemiluminescence can be divided into acridinium ester labeling chemiluminescence, horseradish peroxidase enzymatic chemiluminescence, alkaline phosphatase enzymatic chemiluminescence and the like according to a labeling mode. Commonly used luminescent substances include luminol, acridinium ester, AMPPD (1, 2-dioxane derivative), and the like. Among them, the alkaline phosphatase-AMPPD luminescence system is currently the most important and sensitive luminescence system, but the luminescence intensity is to be improved.

Currently, a class of AMPPD substrate solution optimization techniques mainly perform special functional group optimization at certain positions on the skeleton of AMPPD, so as to change the stability of molecules after phosphorus hydrolysis to obtain desired luminous efficiency, luminous time, quantum yield and the like. The main technical points comprise two aspects: firstly, a plurality of electrophilic groups or nucleophilic groups are added on the original AMPPD skeleton or the corresponding positions are changed, so that the electron distribution of the whole molecular structure is changed, and the structural stability, the luminous efficiency, the luminous time, the quantum yield and the like of the AMPPD are changed; on the other hand, the luminescence enhancement substance is connected to the skeleton molecules by a chemical technology, the technology makes up the solvent action, micelle delay effect and the like of the luminescence enhancement substance in the prior art, and greatly reduces the loss of a photon transfer process, thereby improving the luminous efficiency, shortening the platform period and the like.

The other AMPPD substrate liquid optimization technology is mainly characterized in that various special branched chains or special functional group combinations and surfactant of quaternary ammonium salt quaternary phosphonium salt combined with hydrophilic and hydrophobic structural units are added to form a substrate micelle environment which can be controlled in stability or light excitation, the substance can improve the light-emitting signal value, but the signal increase amplitude is limited compared with economic and easily-obtained CTAC or CTAB, and the substances are not easy to obtain and can be specially customized and are not beneficial to scale.

In addition, in the above optimization techniques, the stability and the luminous efficiency or the luminous rate or the quantum yield of the AMPPD substrate solution have a certain property of trade-off, and how to combine the two to achieve the optimal balance requires more experiments.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a luminescence enhancer for the luminescence effect of a 1, 2-dioxetane compound and a preparation method thereof, so as to solve the technical problems that the luminescence enhancer in the prior art is difficult to produce, high in cost and incapable of simultaneously considering stability and luminescence property.

The second object of the present invention is to provide a kit for enhancing the luminescence of a 1, 2-dioxetane compound.

A third object of the present invention is to provide an enzymatic chemiluminescent substrate for alkaline phosphatase and a kit.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

a luminescence enhancement agent for the luminescence of a 1, 2-dioxetane compound, said luminescence enhancement agent comprising at least one of a fluorescer coupled to a modified PEG, a fluorescer coupled to an AMP, or a fluorescer coupled to a Tris compound.

Further, the luminescence enhancement agent comprises a fluorescent agent coupled to the modified PEG, a fluorescent agent coupled to AMP, and a fluorescent agent coupled to Tris;

preferably, the ratio of the amount of fluorescer to modified PEG coupled compound, fluorescer to AMP coupled compound, and fluorescer to Tris coupled compound is (6-8): (1-3): 1.

further, the fluorescent agent includes a fluorescein fluorescent agent, a rhodamine fluorescent agent, a coumarin fluorescent agent, a naphthalimide fluorescent agent or a benzimidazole fluorescent agent, and preferably a fluorescein fluorescent agent.

Further, the modified PEG includes PEG in which the hydrophilic arm is bifunctional or PEG in which the hydrophilic arm is monofunctional.

Further, the structural formula of the luminescence enhancer includes the following formula (I), formula (II), formula (III), formula (IV) or formula (V):

wherein m is an integer from 1 to 3, n is an integer from 1 to 12;

preferably, the structural formula of the luminescence enhancer includes the following formula (I-1):

further, the concentration of the luminescence enhancer is 0.0001mmol/L-1mmol/L, preferably 0.1mmol/L-0.5 mmol/L.

Further, the luminescence enhancer further comprises at least one of magnesium ions, a buffer, a preservative or a surfactant;

preferably, the buffer comprises AMP buffer or Tris buffer;

preferably, the concentration of the buffer solution is 20mmol/L-1000mmol/L, and the pH value is 8-11.5; preferably at a concentration of 50mmol/L to 100mmol/L, preferably at a pH of 8.5 to 9.5;

preferably, the salt containing the magnesium ion comprises magnesium chloride, magnesium sulfate or magnesium acetate;

preferably, the concentration of the magnesium ions is 0.0001-0.1 mol/L;

preferably, the preservative comprises sodium azide or Proclin 300;

preferably, the content of the sodium azide is 0.01-5% by mass;

preferably, the content of the Proclin300 is 0.01g/L-1.0 g/L;

preferably, the surfactant comprises at least one of CTAC, CTAB, Triton X-100, 3-sulfopropyl hexadecyl dimethyl betaine, 3-sulfopropyl tetradecyl dimethyl betaine, or Tween-20;

preferably, the concentration of the surfactant is 0.01mmol/L to 10 mmol/L.

A method for preparing a luminescence enhancer for 1, 2-dioxetane compounds, comprises dissolving the above components in water.

A kit for enhancing the luminescence of a 1, 2-dioxetane compound, said kit comprising the above-described luminescence-enhancing agent.

An enzymatic chemiluminescent substrate for alkaline phosphatase comprising a 1, 2-dioxetane compound and a luminescence-enhancing agent as defined above;

preferably, the ratio of the amounts of the 1, 2-dioxetane compound and the substance of the luminescence-enhancing agent is from 1:2 to 2: 1.

A kit for an enzymatic chemiluminescent substrate for alkaline phosphatase comprising the enzymatic chemiluminescent substrate for alkaline phosphatase as described above.

Compared with the prior art, the invention has the beneficial effects that:

the luminescence enhancement agent for the luminescence effect of the 1, 2-dioxetane compound provided by the invention comprises at least one of a compound of a fluorescer coupled with modified PEG, a compound of a fluorescer coupled with AMP, or a compound of a fluorescer coupled with Tris. The inventor finds that the compound obtained by coupling the fluorescent agent with modified PEG, AMP or Tris can improve the luminous effect of the 1, 2-dioxetane compound, not only can improve the stability of the substrate solution of the 1, 2-dioxetane compound, but also can obviously improve the luminous efficiency of the substrate solution. On one hand, the luminescence enhancer avoids the high and complicated development process of improving the 1, 2-dioxetane compound substrate in the prior art and the production cost caused by the need of special instruments for large-scale production; on the other hand, compared with quaternary ammonium salt compounds or quaternary phosphonium salt compounds with complex structures, the components of the luminescence reinforcing agent are simple and easy to obtain, and can be obtained through simple chemical reaction, so that the production process is greatly simplified, and the production cost is reduced. The luminescence enhancer is applied to luminescence detection of the 1, 2-dioxetane compound, can remarkably improve the sensitivity and accuracy of detection, has good stability, and is suitable for being made into products such as kits for production and sale.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a graph showing the detection curves of chemiluminescent signals of different chemiluminescent liquids at different times in accordance with one embodiment of the present invention.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer.

Unless otherwise defined, technical and scientific terms used herein have the same meaning as is familiar to those skilled in the art. In addition, any methods or materials similar or equivalent to those described herein can also be used in the present invention.

A luminescence enhancement agent for the luminescence of a 1, 2-dioxetane compound, the luminescence enhancement agent comprising at least one of a fluorescer coupled to a modified PEG, a fluorescer coupled to AMP, or a fluorescer coupled to Tris.

The inventor finds that the compound obtained by coupling the fluorescent agent with modified PEG, AMP or Tris can improve the luminous effect of the 1, 2-dioxetane compound, not only can improve the stability of the substrate solution of the 1, 2-dioxetane compound, but also can obviously improve the luminous efficiency of the substrate solution. On one hand, the luminescence enhancer avoids the high and complicated development process of improving the 1, 2-dioxetane compound substrate in the prior art and the production cost caused by the need of special instruments for large-scale production; on the other hand, compared with quaternary ammonium salt compounds or quaternary phosphonium salt compounds with complex structures, the components of the luminescence reinforcing agent are simple and easy to obtain, and can be obtained through simple chemical reaction, so that the production process is greatly simplified, and the production cost is reduced. In addition, the inventor researches and discovers that when the compound in the luminescence enhancer provided by the invention is a combined type, a special inhomogeneous multi-level micelle can be formed, the compound can be easily cracked to emit photons in an enzymatic process, and the photons are transmitted to fluorescent groups with different energy range differences under different optical paths, so that the lossless enhanced luminescence is realized.

It should be noted that the 1, 2-dioxetane compound (AMPPD) specifically means: 3- (2-spiroadamantane) -4-methoxy-4- (3-phosphonooxy) -phenyl-1, 2-dioxetane. The fluorescent agent may be a fluorescent agent commonly used in the art, and may be, for example, but not limited to, a fluorescein-based fluorescent agent, a rhodamine-based fluorescent agent, a coumarin-based fluorescent agent, a naphthalimide-based fluorescent agent, or a benzimidazole-based fluorescent agent, and the like. AMP is 2-amino-2-methyl-1-propanol. Tris is Tris hydroxymethyl aminomethane. The modified PEG refers to PEG (polyethylene glycol) modified with functional groups or branched-chain type PEG at one end or two ends. The luminescence enhancement agent may include, for example, but is not limited to, a compound in which fluorescer is coupled to modified PEG and a compound in which fluorescer is coupled to AMP, a compound in which fluorescer is coupled to modified PEG and a compound in which fluorescer is coupled to Tris, a compound in which fluorescer is coupled to Tris and a compound in which fluorescer is coupled to AMP, a compound in which fluorescer is coupled to AMP and a compound in which fluorescer is coupled to Tris, a compound in which fluorescer is coupled to AMP, and the like.

In a preferred embodiment, the luminescence enhancement agent comprises a fluorescent agent coupled to the modified PEG, a fluorescent agent coupled to AMP, and a fluorescent agent coupled to Tris, preferably in a ratio of (6-8): (1-3): 1. the inventor researches and discovers that a compound of a fluorescent agent coupled with modified PEG, a compound of the fluorescent agent coupled with AMP and a compound of the fluorescent agent coupled with Tris can achieve the effects of improving the stability and the luminous performance, further, the combined use effect of the fluorescent agent and the compound of the Tris is better, and simultaneously, the inventor optimizes the better mixture ratio of the combined composition of the fluorescent agent, the fluorescent agent and the Tris to be (6-8): (1-3): 1. the ratio of the amount of fluorescent agent to the substance of the modified PEG-conjugated compound, fluorescent agent to AMP-conjugated compound, and fluorescent agent to Tris-conjugated compound may be, but is not limited to, 6:3:1, 8:1:1, or 7:2: 1.

In a preferred embodiment, the fluorescer comprises a fluorescein-based fluorescer, a rhodamine-based fluorescer, a coumarin-based fluorescer, a naphthalimide-based fluorescer or a benzimidazole-based fluorescer, preferably a fluorescein-based fluorescer.

In a preferred embodiment, the modified PEG comprises PEG where the hydrophilic arm is difunctional or PEG where the hydrophilic arm is monofunctional. The PEG with the hydrophilic arm as the bifunctional group means that both ends of the PEG are substituted by the functional group; the PEG with a monofunctional hydrophilic arm means that one end of the hydrophilic arm to which the PEG is coupled to the fluorescer is substituted with a functional group. The functional group is an atom or an atomic group that determines the chemical properties of the organic compound. Common functional groups include carbon-carbon double bonds, carbon-carbon triple bonds, hydroxyl groups, carboxyl groups, ether bonds, aldehyde groups, carbonyl groups, and the like. The organic chemical reaction mainly occurs on functional groups, the functional groups play a role in determining the properties of organic matters, and the functional groups are X, -OH, -CHO, -COOH, -NO2, -SO3H, -NH2 and RCO-, and the functional groups determine the chemical properties of halogenated hydrocarbon, alcohol or phenol, aldehyde, carboxylic acid, nitro compound or nitrite, sulfonic organic matters, amines and amides in the organic matters. Preferably, this refers primarily to PEG having an amino functional group at one end.

In preferred embodiments, the structural formula of the luminescence enhancer comprises formula (I), formula (II), formula (III), formula (IV) or formula (V) as follows:

wherein m is an integer from 1 to 3, n is an integer from 1 to 12;

preferably, the structural formula of the luminescence enhancer includes the following formula (I-1):

in a preferred embodiment, the concentration of the luminescence enhancing agent is in the range of 0.0001mmol/L to 1mmol/L, preferably in the range of 0.1mmol/L to 0.5 mmol/L. The concentration of the compound is typically, but not limited to, 0.0001mmol/L, 0.001mmol/L, 0.01mmol/L, 0.1mmol/L, 0.2mmol/L, 0.3mmol/L, 0.4mmol/L, 0.5mmol/L, 0.6mmol/L, 0.7mmol/L, 0.8mmol/L, 0.9mmol/L or 1 mmol/L.

In a preferred embodiment, the luminescence enhancer further comprises at least one of magnesium ions, a buffer, a preservative or a surfactant.

In a preferred embodiment, the buffer comprises AMP buffer or Tris buffer, preferably at a concentration of 20mmol/L to 1000mmol/L, at a pH of 8 to 11.5; the concentration is preferably 50mmol/L to 100mmol/L, and the pH is preferably 8.5 to 9.5. Buffer concentrations are typically, but not limited to, 20mmol/L, 50mmol/L, 100mmol/L, 200mmol/L, 300mmol/L, 400mmol/L, 500mmol/L, 600mmol/L, 700mmol/L, 800mmol/L, 900mmol/L or 1000 mmol/L; the pH is typically, but not limited to, 8, 9, 10, 11 or 11.5.

In a preferred embodiment, the salt containing magnesium ions includes magnesium chloride, magnesium sulfate or magnesium acetate, and the concentration of magnesium ions is preferably 0.0001 to 0.1 mol/L. The magnesium ion concentration is typically, but not limited to, 0.0001mol/L, 0.001mol/L, 0.01mol/L, or 0.1 mol/L.

In a preferred embodiment, the preservative comprises sodium azide or Proclin300, and when the preservative is sodium azide, the content is preferably 0.01-5% by mass; when the preservative is Proclin300, the content is preferably 0.01g/L to 1.0 g/L. The content of sodium azide is typically, but not limited to, 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, or 5%; proclin300 is typically, but not limited to, 0.01g/L, 0.1g/L, 0.2g/L, 0.3g/L, 0.4g/L, 0.5g/L, 0.6g/L, 0.7g/L, 0.8g/L, 0.9g/L, or 1 g/L.

In a preferred embodiment, the surfactant comprises at least one of CTAC, CTAB, Triton X-100, 3-sulfopropyl hexadecyl dimethyl betaine, 3-sulfopropyl tetradecyl dimethyl betaine, or Tween-20, and the concentration of the surfactant is preferably 0.01mmol/L to 10 mmol/L. Surfactants may be, for example, but are not limited to, CTAC and CTAB, Triton X-100 and 3-sulfopropylhexadecyldimethylbetaine, Tween-20, or CTAC, CTAB, Triton X-100, 3-sulfopropylhexadecyldimethylbetaine, 3-sulfopropyltetradecyldimethylbetaine, Tween-20, and the like.

A method for preparing a luminescence enhancer for the luminescence effect of a 1, 2-dioxetane compound is to dissolve the components of the luminescence enhancer provided by the invention in water.

A kit for enhancing the luminescence of a 1, 2-dioxetane compound, comprising a luminescence-enhancing agent provided by the present invention, wherein the components of the luminescence-enhancing agent are packaged individually or in any combination.

An enzymatic chemiluminescent substrate for alkaline phosphatase comprising a 1, 2-dioxetane compound and a luminescence enhancer provided by the present invention wherein the ratio of the amounts of the 1, 2-dioxetane compound and the luminescence enhancer is 1:2-2: 1. The enzymatic chemiluminescence substrate has the advantages of obviously improved stability and luminous efficiency, improved sensitivity and accuracy of the detection reagent, low cost, and suitability for batch production and wide application.

A kit of an enzymatic chemiluminescent substrate for alkaline phosphatase comprises the enzymatic chemiluminescent substrate for alkaline phosphatase provided by the invention, and the components in the enzymatic chemiluminescent substrate are packaged separately or in any mixture.

The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

Examples

1L of 100mol/L AMP (2-amino-2-methyl-1-propanol) buffer having a pH of 9.5 was prepared with ultrapure water, and then 150mg of AMPPD, 1.6mg of lucigenin, 250mg of CTAC, 40mg of fluorescein sodium, and 400mg of MgCl₂、500mg Proclin300、5mgZnCl₂The mixed solution is used as a basic luminous solution;

0.1mmol of the following substances (a) to (e) were added to the above base luminescent liquid, respectively:

(a) PEG-Fluorescein [ represented by the following formula (I-1) ];

(b) AMP-fluorochein [ formula (IV) below ];

(c) tris-fluorochein [ formula (V) below ];

(d) PEG-Fluorescein [ formula (I-1) below ], AMP-Fluorescein [ formula (IV) below ] and Tris-Fluorescein [ formula (V) below ] in a ratio of 7:2: 1;

(e)0.1mmol of sodium fluorescein [ the following formula (VI) ].

5 kinds of chemiluminescent liquid are obtained.

20 mul of 20ng/ml alkaline phosphatase solution is taken as a sample, 100 mul of the 5 kinds of chemiluminescent solutions are respectively added, chemiluminescent signals are measured on a chemiluminescent detector, specific detection results are shown in the following table, a time curve of luminescent reaction is made, and the results are shown in figure 1.

While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.