阅读说明：本技术 用于稳定活性生物材料的组合复合材料、其生产方法及用途 (Composite material for stabilizing active biological material, method for producing same and use thereof ) 是由 路易斯·古斯塔沃·戈迪尼奥·巴雷里亚 于 2018-07-20 设计创作，主要内容包括：本发明涉及合成溶胶-凝胶以固定活性或可活化生物材料的技术的用途。更具体地,本发明涉及一种用于将活性或可活化生物材料固定在溶胶-凝胶组合复合材料中的新方法,其中在保持固有生物活性的同时,减少了所述生物材料从所获得的组合复合材料中浸出而造成的损失。本发明还涉及通过该新方法获得的复合材料及其用途,即在生物催化和诊断方法中的用途。(The present invention relates to the use of the technique of synthesizing sol-gels to immobilize active or activatable biological materials. More specifically, the present invention relates to a new method for immobilizing active or activatable biomaterials in a sol-gel composite material, wherein the loss of said biomaterials by leaching out of the obtained composite material is reduced while maintaining the intrinsic biological activity. The invention also relates to the composite material obtained by the novel process and to the use thereof, namely in biocatalytic and diagnostic methods.)

1. A method for producing a composite material for stabilizing active or activatable biological material, comprising the steps of:

a) providing a simple composite material in the form of a sol-gel immobilizing one or more active or activatable biomaterials of different nature, said simple composite material being finely divided and having a low water content;

b) providing a second sol-gel immobilizing said simple composite of a) comprising:

i) preparing a suspension of said simple composite material of a) in a phosphate buffer solution of 100mM, pH 6.8 ± 0.2, at a mass/volume concentration of 0.83% to 3.34% with respect to the volume of the final reaction mixture;

ii) adding to the tetramethylorthosilicate a HCl solution in a concentration comprised between 4.50mM and 8.34mM in a proportion of 28% of the final volume of the mixture, the tetramethylorthosilicate being hydrolyzed by acid catalysis;

iii) adding the mixture obtained from ii) to an equal volume of the suspension obtained from i) at 25 ℃ to 30 ℃, allowing polymerization to occur until a composite material is obtained having a consistency such that it can be disintegrated;

iv) combining the composite obtained in iii)The material is broken into two types of size particles: equal to and less than about 2mm³And about 2mm³To 4mm³；

v) optionally, incubating the fragmented composite of iv) in 2.0% m/v Bovine Serum Albumin (BSA) dissolved in 10mM phosphate buffer pH 7.3 at a ratio of three volumes of incubation solution to one volume of the composite for 24 hours;

vi) by drying on the surface until the mass is reduced by about 70% and the available water content (a)_w) About 20ppm, maturing the disintegrated composite,

thereby, the composite material is obtained with the active or activatable biomaterial doubly immobilized in a sol-gel, wherein the losses due to leaching of the biomaterial are reduced, maintaining its activity or activation capacity.

2. The method of claim 1, wherein in the composite material, loss due to leaching of the dual immobilized biological material is reduced and its activity or activation capacity is maintained for at least 20 cycles of use.

3. The method according to claim 1 or 2, characterized in that the effective water content (a) of the simple composite material of a)_w) From 10ppm to 15 ppm.

4. The method according to any of the preceding claims, characterized in that the particle size of the simple composite material of a) is 100 μ ι η³To 110 μm³。

5. The process according to any of the preceding claims, characterized in that in step b) iii), the polymerization takes place in about 30 to 90 minutes.

6. The method of any one of the preceding claims, wherein the active or activatable biological material is from one or more of the following: biologically active proteins, such as enzymes, coenzymes; and immunologically active proteins, such as antigens, antibodies, hapten proteins; or any other member of a particular biological relationship.

7. The method according to claim 6, wherein the active or activatable biological material is one or more from an immunologically active protein such as an antigen, an antibody, a hapten protein and the method comprises step v).

8. Composite material for the double immobilization of active or activatable biomaterials in a sol-gel, obtainable by the method according to any one of claims 1 to 7.

9. The composite material for dual immobilization of active or activatable biomaterials in a sol-gel is characterized by reduced losses due to leaching of the immobilized biomaterials and by an activity that is maintained for at least 20 cycles of use.

10. The composite material for the dual immobilization of active or activatable biomaterial in a sol-gel according to any of claims 8 or 9, characterized in that the immobilized active or activatable biomaterial is one or more from the following: biologically active proteins, such as enzymes, coenzymes; and immunologically active proteins, such as antigens, antibodies, hapten proteins; or any other member of a particular biological relationship.

11. Composite material for the double immobilization of active or activatable biomaterials in sol-gel according to any of claims 8 to 10, characterized in that the particle size of the simple composite material, immobilized and matured and subdivided, is 100 μ ι η³To 110 μm³。

12. The composite material of any of claims 8 to 11, wherein the active or activatable biomaterial is one or more of a bioactive protein, such as an enzyme or a coenzyme, and the composite material is to be used in biocatalysis.

13. The composite active or activatable biomaterial dual immobilized in a sol-gel according to any of claims 8 to 11, characterized in that the active or activatable biomaterial is a specific binding immunoreactive protein and the composite is to be used for the detection of its respective specific ligand.

14. The composite material for dual immobilization of active or activatable biomaterial in sol-gel according to claim 13, characterized in that said specific binding immunocompetent protein is an antigen, an antibody, a hapten protein and said composite material is to be used for the diagnosis of diseases.

15. Use of a composite of active or activatable biomaterial doubly immobilized in a sol-gel according to any of claims 8 to 14 or produced by the method according to any of claims 1 to 7 in medical diagnostics.

16. A method of diagnosing a disease in a human or higher animal subject comprising the steps of:

a) contacting a biological fluid sample from a subject suspected to contain an analyte associated with a disease, in particular blood, urine, tissue-soaked supernatant or any other fluid obtained from the subject, with a composite material that double immobilizes a biological material in a sol-gel that specifically binds to the analyte, wherein the composite material is the composite material of any one of claims 8 to 14 or produced by the method of any one of claims 1 to 7; and

b) detecting specific binding between the analyte present in the sample and the biological material immobilized in the composite material, the presence or absence of the specific binding indicating a positive or negative diagnosis of the disease in the subject, respectively.

17. Diagnostic method according to claim 16, wherein said disease is any disease leading to the presence of antigens and/or antibodies in the fluids and/or tissues of a human or higher animal subject.

18. The diagnostic method of claim 16 or 17, wherein the analyte in the sample is an antigen or an antibody and the specific binding produces an immune complex.

19. The diagnostic method of claim 18, wherein said detection of specific binding comprises the steps of:

a) contacting the (first) immune complex with a second antibody labeled with a detectable tracer, which second antibody specifically binds to the analyte of the biological sample; and

b) detecting said tracer in said sample directly or by its interaction with a specific reagent.

20. The diagnostic method of claim 19, wherein said detectable tracer in said second antibody is: an enzyme, the detection of which is carried out by color development after contact with its chromogenic substrate; or a photosensitive molecule, the detection of which is performed by fluorescence emission after luminescence excitation of the corresponding wavelength.

21. The diagnostic method of any one of claims 16 to 20, further comprising: step c) recycling the composite material; and repeating steps a) to c) at least 20 times.

22. An analysis chamber for the detection of specific ligands of an immunologically active protein, the analysis chamber comprising as a filling a combinatorial composite of immobilized immunologically active proteins according to any one of claims 8 to 14 for use in medical diagnostics or a combinatorial composite of immobilized immunologically active proteins produced by a method according to any one of claims 1 to 7.

23. Apparatus for the detection of specific ligands of immunologically active proteins, said apparatus comprising up to four sets of two chambers configured in parallel, each said set consisting of one analysis chamber according to claim 22 and one negative control chamber filled with a composite material of immobilized immunologically inactive proteins for medical diagnosis.

24. The apparatus of claim 23, wherein each of the immunologically active proteins immobilized in each of the composite materials filling each of the analysis chambers is associated with a different specific disease.

25. The apparatus of claim 23 or 24, wherein the apparatus is reused at least 20 times.

Technical Field

The present invention relates to the immobilization of active or activatable biomaterials using sol-gel synthesis. More specifically, the present invention relates to a new method that envisages the immobilization of active or activatable biomaterials in a composite sol-gel, wherein the loss of said biomaterials by leaching from the composite is reduced while maintaining the biological activity of these materials.

Background

Sol-gel matrices are highly porous materials, mainly derived from silica, whose synthesis conditions are thought to be beneficial for the encapsulation of most biomolecules.

In a typical sol-gel synthesis scheme, the precursor is Si (OR)₄Alkoxides of the type or XSi (OR)₃Or XX' Si (OR)₂Alkoxysilanes of the type wherein X and X' mean that the organic group is directly linked to the silicon atom via a Si-C bridge in one head and presents several other functional groups on the other head. Fig. 1 shows an alkoxide, wherein the R group is a methyl group: tetramethyl orthosilicate (TMOS).

The first reaction of sol-gel synthesis is hydrolysis of the precursor [1], in which one OR ligand is substituted by an OH group:

(for example)

Condensation after hydrolysis:

(for example)

Those hydrolysis and condensation reactions are slow reactions, but the rate of hydrolysis can be increased by donating an acid (e.g., HCl) with a positive charge that can attack the oxygen of the alkoxy group (alcoxyde). A silica gel having a texture similar to that of the polymer gel is thus obtained. Proton acceptor media, on the other hand, such as alkaline solutions, accelerate the rate of condensation, resulting in the formation of denser colloidal particles. The ability to control these kinetics is important to adapt the encapsulation conditions to better respond to biomolecules.

Some additives also show a beneficial effect on the stability of biomolecules encapsulated in sol-gels: polyvinyl alcohol or glycerol can increase the activity of encapsulated glycolytic enzymes [2] [3 ]. Besides the additives, the amount of water used for hydrolysis of the precursor also affects the activity of the immobilized biomolecules, so that effective water must ensure the mobility of the biomolecules in the mature matrix: after addition of small amounts of water, the encapsulated beta-galactosidase was more active in the recently gelled composite than in the mature composite [4 ].

One disadvantage associated with encapsulation technology is the gradual decline in activity of the sol-gel matrix seen as an active composite: leaching of the encapsulated molecules results in a decrease in the activity of the matrix composite and thus limits its efficiency for long-term use.

Several strategies have been proposed to circumvent this obstacle to control the leaching of sol-gel encapsulated molecules. Some authors aimed to reduce leaching by adjusting sol-gel composition and gelation/maturation conditions to reduce pore size, as mentioned in t.m. butler et al [5], Lobnik, i.oehme et al [6] and g.e. badini et al [7 ]. However, the reduced diffusion of analyte within the matrix results in longer response reaction times.

Covalent attachment of biomolecules to condensation networks in sol-gel synthesis has also been proposed to reduce leaching in some way [8] [9] [10 ]. An alternative strategy is to increase the size of immobilized biomolecules by attachment to inert macromolecular transporters [11] [12] [13] or encapsulation within supramolecular constructs [14 ]. However, the activity of biomolecules requires some fluidity, and those methods decrease the desired activity.

The problem addressed by the present invention is therefore the stability of the active or activatable biomaterial in the sol-gel matrix, reducing its loss by leaching, while maintaining its activity. This problem is solved by a new method for immobilizing active or activatable biomaterials, wherein a composite is obtained: a sol-gel immobilized composite material, instead of previously produced sol-gel, in which active or activatable biomaterials are encapsulated with good physicochemical stability and reactivity.

Disclosure of Invention

The terms "immobilization", "encapsulation", "confinement" and corresponding deductions as used indifferently herein have the meaning of stabilizing the biological material within a sol-gel composite matrix.

By dual immobilization of active or activatable biomaterials within the sol-gel, it is desirable to reduce losses due to leaching. However, it is also desirable that the active sites of the immobilized biological material are less accessible, and contact with the active sites of the immobilized biological material results in a decrease in its activity.

There are practical difficulties in manufacturing such composite composites. By secondarily fixing another sol-gel (a simple composite) in the sol-gel, this produces a variety of unsuccessful results, since the gelling completely fails until the physicochemical properties of the final material are not suitable.

Without any prejudice, those that were not theoretically successful were due to the high hygroscopicity of the simple composite, which characteristic had caused mislevelness to occur due to water saturation under the reaction conditions, resulting in insufficient water required for the hydrolysis of the orthosilicate precursor.

As a result of intensive research, the present inventors have demonstrated that the method described herein results in a composite material, one sol-gel immobilizing another sol-gel that was previously obtained, encapsulating active or activatable biomaterials with good physicochemical stability, and finally formulated to reduce the loss of the biomaterials by leaching and retain the respective activities.

The inventors have surprisingly demonstrated that the double immobilization in a combined sol-gel composite of an active or activatable biomaterial obtained by the process of the invention not only significantly reduces the losses caused by leaching of the mentioned materials, but also retains its activity in the following manner: so that the immobilized biological material can respond for at least 20 cycles.

Thus, according to the present invention, there is provided a novel method for producing a composite material for stabilizing active or activatable biological material, the method comprising the steps of:

a) providing a simple composite material in the form of a sol-gel that immobilizes one or more active or activatable biomaterials of different properties, the simple composite material being finely ground and having a low effective water level;

b) providing a second sol-gel immobilizing a) the simple composite material comprising:

i) preparing a suspension of the simple composite of a) in a phosphate buffer solution of 100mM at a pH of 6.8 ± 0.2 at a concentration of 0.83% to 3.34% with respect to the volume of the final reaction mixture;

ii) hydrolysis of tetramethylorthosilicate by acid catalysis by adding 28% of the final volume of the mixture of HCl solution at a concentration of 4.50mM to 8.34 mM;

iii) adding the mixture of ii) to an equal volume of the suspension of i) at a temperature of 25 ℃ to 30 ℃ such that polymerization occurs until a composite material is obtained having a consistency capable of being disintegrated;

iv) fragmenting the combined composite obtained in iii) to a particle size of about and less than 2mm³And about 2mm³To 4mm³；

v) optionally, incubating the fragmented composite of iv) in a 2.0% m/v solution of Bovine Serum Albumin (BSA) dissolved in 10mM phosphate buffer pH 7.3 at a ratio of three volumes of incubation solution to one volume of composite for 24 hours;

vi) maturing the composite material by drying on the glass surface until the mass is reduced by about 70% and the effective water level is about 20 ppm;

thus a composite material is obtained which double immobilizes the active or activatable biomaterial in a sol-gel matrix, wherein the losses due to leaching of said material are reduced, yet retaining its activity or activation capacity.

In a preferred embodiment of the method of the invention, the obtained composite material with active or activatable biomaterial doubly immobilized in sol-gel has a reduced loss of said material and retains its activity for at least 20 cycles of use.

The active or activatable biomaterial that can be immobilized in the composite of the invention is not limited and allows to be one or more of any immobilizable molecule in a simple composite, preferably a member of a specific linkage, such as an enzyme-substrate, antibody-antigen or any other paired specific linkage, preferably a biologically active protein, more preferably an enzyme or coenzyme, or a specifically linked immunologically active protein, more preferably an antibody, an antigen or a hapten protein.

The simple composite material to be immobilized in the composite material is provided by any method known in the art, preferably by acid hydrolysis of tetramethyl orthosilicate to produce a hydrophilic composite material.

In a preferred embodiment of the invention, the simple composite material to be immobilized in the composite material and which in turn has immobilized active or activatable biological material, has a low grade of effective water of 10ppm to 15ppm and is ground to a particle size of 100 μm³To 110 μm³。

The formation of composite composites immobilizing previously prepared simple composites was achieved by acid hydrolysis of tetramethyl orthosilicate using HCl as catalyst at concentrations of 4.50mM to 8.34 mM. This concentration is determined by the amount of water displaced in the functional domain of the protein immobilized in the simple composite.

When the biological material immobilized in the composite material is one or more immunologically active proteins, such as antigens, antibodies, haptenic proteins, the method of the invention comprises the additional step v): the fragmented composite composites were incubated for 24 hours in 2.0% m/v bovine serum albumin dissolved in 10mM phosphate buffer pH 7.3 at a ratio of three volumes of incubation solution to one volume of composite. This additional step significantly increases the protein content of the mature composite material to 3.0 to 3.5 relative to the protein content of the simple composite material.

The invention also relates to a composite material obtained by the method of the invention, which doubly immobilizes an active or activatable biomaterial in a sol-gel. The composite is also referred to herein as a "doped" composite having a biomaterial immobilized therein.

The composite material obtained by the method of the invention, which doubly immobilizes an active or activatable biomaterial in a sol-gel, exhibits the robustness and improved retention capacity of said active or activatable immobilized biomaterial. At the same time, the composite material exhibits an internal structure or porosity that allows penetration until the active sites of those immobilized biological materials, analytes (ligands, substrates or activated molecules specific for active or activatable biological materials) are present in an aqueous sample that is contacted with the composite material. In other words, the composite material of the present invention not only exhibits improved physical retention of biological materials, but also retains the activity or activation capability of those retained biological materials. The robustness of the composite of the invention and its ability to retain the activity of the immobilized biomaterial enables the composite of the invention to be used for several consecutive use cycles at all times.

In a preferred embodiment, a composite according to the invention can be used for at least 20 cycles of use.

Each event of interaction/reaction between the biological material immobilized within the composite and the respective ligand or specific substrate or other specifically interacting (or analyte) molecule should be considered as a usage cycle of the doped composite by mixing said composite with a sample containing one analyte. For example, when the immobilized biological material is an enzyme or a coenzyme, one use cycle will be one cycle of an enzymatic reaction, and when the immobilized biological material is an antibody, an antigen, or a hapten protein, one use cycle will be one cycle of a reaction to form an immune complex.

One use cycle will preferably include the step of conditioning and washing the combined composite prior to the next cycle. It may also comprise other steps for detecting the occurrence of said interaction/reaction.

Thus, the composite material of the invention may be used in any application where an active or activatable biomaterial is to be used, more preferably, when a biomolecule is used in an aqueous medium, as for and without limitation to some, in the formation of immune complexes in medical diagnostics, or in the formation of products by biocatalysis of immobilized enzymes.

The advantages of biocatalysis over traditional catalysis have been widely recognized, resulting from enzyme immobilization and resulting in higher catalytic efficiency, higher enzymatic stability, higher substrate selectivity and lower cost of use due to lower thermal operating conditions requirements, and being more environmentally friendly [15 ].

In this way, in one embodiment of the composite of the invention, it can be used as a carrier for enzymes, thus having enzymatic activity and several cycles of reaction. Further, examples of alkaline phosphatase will be provided, but not limited thereto.

In a preferred embodiment of the composite material of the invention, it will act as a carrier for an immunologically active protein, such as an antibody, antigen or hapten protein, which is made active in a continuous cycle linked to the respective ligand. Thus, in each use cycle, it will be possible to detect the attachment of specific ligands of those immunologically active proteins immobilized within the composite material.

With respect to the above-mentioned immunologically active proteins or respective ligands immobilized in the composite material associated with a particular disease, the composite material can be used for diagnosing such a disease by detecting the presence of the specific ligand in the biological sample. Preferably, the biological sample will be from a human or higher animal, more preferably a biological fluid such as plasma/serum from blood, urine, supernatant from tissue maceration or any other aqueous fluid that does not have cells obtained from an individual with medical diagnostic advice. The disease to be diagnosed may be any disease that triggers the presence of antigens and/or antibodies in the fluids and/or tissues of a human or higher animal.

Thus, one use of the composite material of the present invention is in medical diagnostics, which constitutes a preferred use.

In more detail, but not exclusively, the use of the composite material of the invention in medical diagnostics follows the principle of immunodiagnosis, in that one biological sample suspected of containing therein an analyte of interest (e.g. an antibody, antigen or other analyte) is brought into contact with the respective specific ligand. These specific ligands will be antigens, antibodies or any other specific ligand immobilized in a composite material having a porosity that allows the aforementioned analytes present in the aqueous liquid sample to permeate until they come into contact with the specific immobilized ligand. These and other classes or both classes of analytes or respective specific ligands will be associated with disease.

Thus, by contacting the biological sample to be analyzed with a combinatorial complex material doped with a specific ligand for a specific analyte and the analyte being present in the aqueous sample, a specific complex analyte-ligand, e.g. an immune complex antigen-antibody, is formed, which can be further detected, thereby concluding the presence or absence of the analyte in the biological sample and thus making a positive or negative diagnosis of the relevant disease.

The detection may be by any suitable method, for example by staining the labelled antibody or reproducing fluorescence.

An example of this embodiment will be further described, in which an immune complex (first) formed after the biological sample is contacted with the combined composite material to which the antigen (or antibody) is immobilized is detected by a second labeled antibody (labeled with an enzyme, such as peroxidase), which is specifically linked to the antibody (or antigen) of the biological sample, thereby forming a second immune complex. Further addition of a chromogenic substrate degraded by the labeling enzyme of the second antibody provides a rich color appearance of the combined composite material, thereby completing detection of the first complex.

The detection can be verified by the absence of color after the same biological sample has been applied to the same composite material incorporating the non-immunogenic protein.

Recycling the composite material makes possible its use in a continuous cycle of use. As already described and further demonstrated, the composite material of the invention is capable of being reused at least 20 cycles of use.

In one aspect of this embodiment, the composite of the invention, double immobilized with an immunologically active protein, will be used to fill an analysis chamber designed to detect the presence of a specific ligand for the immunologically active protein (target-analyte) in a biological sample in which the composite will be contacted with the biological sample.

The fill profile of the analysis chamber with the composite material will be adjusted according to the target-analyte and/or immobilized biological material and will be determined by preliminary experiments for adjusting the operating rheology. In the same way, the usage scheme of the analysis chamber, the reagents and the dilution ratio of the biological sample will also be determined by routine preliminary tests in order to meet the sensitivity and specificity criteria of the target-analyte and/or immobilized biological material.

Thus, the minimum amount of simple composite material to be immobilized in the composite material, which will enable the detection of the presence of an analyte in a biological sample (sensitivity), and the maximum amount of simple composite material to be immobilized in the composite material, which will demonstrate no cross-reactivity (specificity) between different analytes, will be determined. At the same time, the most appropriate particle size gradient curve that will satisfy the analysis chamber will be determined.

Thus, a device is conceivable which will comprise a series of the above-mentioned analysis chambers, preferably up to 8 chambers arranged vertically in parallel, and which is provided on top of a common liquid collector where the liquid sample will be placed. Next to each analysis chamber dedicated to the detection of a unique analyte is a similar chamber dedicated to the negative control test that will be completely filled with a composite material having the same particle size gradient profile that immobilizes the same amount of simple composite material but incorporates a non-immunogenic protein.

Thus, the device will consist of pairs of chamber-analysis chambers and corresponding negative control chambers, each filled with a composite material doubly immobilizing a different active or activatable biomaterial as described above, and next to one negative control chamber filled with a composite material doubly immobilizing a non-immunoreactive biomaterial. Such a device would be able to detect different amounts, preferably up to 4, of target-analytes in a single unique biological sample and in each cycle of use.

A preferred embodiment of the invention is therefore a portable and easy-to-use device, which does not require an electrical energy supply and which will make possible a positive or implicit diagnosis in each biological sample, the number of analytes present preferably being (up to 4) as large as the number of analysis chambers constituting the construction. In particular, since each analyte in a unique sample of biological fluid from a human or higher animal suspected of having a particular disease is associated with a disease, the device will be able to differentially diagnose up to four diseases. Furthermore, the device can be reused for at least 20 consecutive analytical use (diagnostic test) cycles.

The device may be presented in kit form and will include, in addition to the device itself, instructions for use and all reagents required for the diagnostic test procedure.

Some examples of preferred ways of carrying out the invention will be described in the foregoing, and some mistakes and attempted trials of the invention will also be reported.

Drawings

FIG. 1: structural schematic of alkoxide-tetramethyl orthosilicate (TMOS);

FIG. 2: mass loss of simple composites as they mature while staying indoors;

FIG. 3: mass loss of the composite as it matures while staying indoors;

FIG. 4: production of p-nitrophenol catalyzed by alkaline phosphatase immobilized in a simple composite, correlated with the quality of the immobilized enzyme (hydrolysis of p-nitrophenol phosphate in 100mM phosphate buffer aqueous medium at pH 9.1);

FIG. 5: production of p-nitrophenol catalyzed by alkaline phosphatase immobilized in the composite, correlated with the quality of the immobilized enzyme (hydrolysis of p-nitrophenol phosphate in 100mM phosphate buffer aqueous medium at pH 9.1);

FIG. 6: dye concentration of samples collected every 3 minutes, eluted with 13 parts of 1.0ml of distilled water; the procedure started with elution with 1.0ml of 1.4mg/100ml Evan-blue (Evan-blue) aqueous solution and the upper layer (1.35G, particle size (G) > 1.0 mm) was infiltrated³；1.8g，1.0mm³＞G＞710μm³(ii) a The central layer was 2.70g, 710 μm³＞G＞212μm³；1.60g，1.0mm^3＞G＞710μm³) A particle size gradient of the composite material; the composite material is filled to 12cm³The reactor (2) has a size of 4cm_{Long and long}×3cm_{Width of}×1cm_{Height of}；

FIG. 7: dye concentration of samples collected every 30 seconds, eluted with 12 parts of 1.0ml of distilled water; the procedure started with elution with 1.0ml of 1.4mg/100ml Evans blue in water, permeating the upper layer (1.35G, particle size (G) > 1.0 mm)³；1.8g，1.0mm³＞G＞710μm³(ii) a The central layer was 2.70g, 710 μm³＞G＞300μm³；1.16g，1.0mm^3＞G＞710μm³) A particle size gradient of the composite material; the composite material is filled to 12cm³The reactor (2) has a size of 4cm_{Long and long}×3cm_{Width of}×1cm_{Height of}；

FIG. 8: the dye concentration of the sample collected every 10 seconds was eluted with 11 parts of 1.0ml of distilled water; the procedure started with elution with 1.0ml of 1.4mg/100ml Evans blue in water, permeating the upper layer (1.35G, particle size (G) > 1.0 mm)³；1.8g，1.0mm³＞G＞710μm³(ii) a The central layer was 2.70g, 710 μm³＞G＞500μm³；0.97g，1.0mm^3＞G＞710μm³) A particle size gradient of the composite material; the composite material is filled to 12cm³The reactor (2) has a size of 4cm_{Long and long}×3cm_{Width of}×1cm_{Height of}；

FIG. 9: dye concentration of samples collected every 90 seconds, eluted with 15 parts of 1.0ml of distilled water; the procedure started with elution with 1.0ml of 1.4mg/100ml Evan-blue (Evan-blue) aqueous solution, passing through the upper layer (1.35G, particle size (G) > 1.0 mm)³；1.8g，1.0mm³＞G＞710μm³(ii) a The central layer was 2.70g, 500 μm³＞G＞300μm³；1.90g，1.0mm^3＞G＞710μm³) The composite material with certain granularity gradient is infiltrated; the composite material is filled to 12cm³The reactor (2) has a size of 4cm_{Long and long}×3cm_{Width of}×1cm_{Height of}；

FIG. 10: so that the ratio of 1: 100 and 1: 1.0ml of human blood diluted 200 (in 10mM phosphate buffer pH 7.3) permeated the upper layer (1.35G, particle size (G) > 1.0 mM)³；1.8g，1.0mm³＞G＞710μm³) Center layer (2.70g, 710 μm)³＞G＞500μm³；0.97g，1.0mm³＞G＞710μm³) The hemoglobin concentration in the fraction of 1.0ml distilled water collected after the composite material of a certain particle size gradient; the composite material is filled to 12cm³The reactor (2) has a size of 4cm_{Long and long}×3cm_{Width of}×1cm_{Height of}；

FIG. 11: so that the ratio of 1: 100 dilutions (dilution in 10mM phosphate buffer pH 7.3) of 1.0ml human blood permeate the upper layer (1.35G, particle size (G) > 1.0 mM)³；1.8g，1.0mm³＞G＞710μm³) Center layer (2.70g, 710 μm)³＞G＞500μm³；0.97g，1.0mm³＞G＞710μm³) Hemoglobin concentration in the initial 10.0ml and continuous 1.0ml fractions of distilled water collected after the composite of a certain particle size gradient; the composite material is filled to 12cm³The reactor (2) has a size of 4cm_{Long and long}×3cm_{Width of}×1cm_{Height of}；

FIG. 12: the upper layer (1.35G, particle size (G) > 1.0 mM) was permeated with 1.0ml of human blood at various dilutions (diluted in 10mM phosphate buffer pH 7.3)³；1.8g，1.0mm³＞G＞710μm³) Center layer (2.70g, 710 μm)³＞G＞500μm³；0.97g，1.0mm³＞G＞710μm³) The hemoglobin concentration in distilled water of three 10.0ml fractions and a final 1.0ml fraction collected after combining the composite material in a certain particle size gradient; the composite material is filled to 12cm³The reactor (2) has a size of 4cm_{Long and long}×3cm_{Width of}×1cm_{Height of}；

FIG. 13: examples for calibrating the concentration of p-nitrophenol in the alkaline phosphatase reaction product;

FIG. 14: hydrolysis reaction yield of p-nitrophenol phosphate added in 4.0ml portions at a concentration of 0.75mM (in phosphate buffer 100mM pH 9, 1), which is permeated with a certain particle size gradient of the composite material doped with alkaline phosphatase at a concentration of 0.43% (m/m); the gradient of the upper layer is: 1.35G, particle size (G) > 1.0mm³；1.8g，1.0mm³＞G＞710μm³(ii) a The central layer is: 2.70g, 500 μm³＞G＞300μm³；1.90g，1.0mm^3＞G＞710μm³(ii) a The composite material is filled to 12cm³The reactor (2) has a size of 4cm_{Long and long}×3cm_{Width of}×1cm_{Height of}；

FIG. 15: photographs of two chambers containing the composite material; the left chamber is filled with the composite material doped with bovine serum albumin, and the right chamber is filled with the composite material doped with mucin; images were obtained after testing the supernatants of antibody-anti-mucin producing hybridoma cell cultures; chromogenic peroxidase substrate 3,3 ', 5, 5' -tetramethylbenzidine revealed the linkage of the second antibody;

FIG. 16: photograph of a prototype of the apparatus, in which all chambers are placed in parallel in a disposable configuration, below a common liquid collector and above a bottom collection tray.

Detailed Description

Example 1 composite formulation

Example 1.1. -simple composite Material

In preparing a precursor of a simple composite material with an indicative volume of 20ml, the protocol described by Alstein and co-workers [16] was followed, using 5.0ml of tetramethyl orthosilicate (TMOS) as orthosilicate precursor; 4.84ml hydrochloric acid (2.5 mM); 1.0ml polyethylene glycol (0.4 kDa). The mixture was stirred in a vortex until the liquid medium appeared clear. Homogenization is exothermic and the mixture is then sonicated for 30 minutes. 180mg to 500mg of lyophilized proteins (e.g. enzymes, antigens/antibodies) were dissolved in 10ml of HEPES buffer (50mM pH 7.5) beforehand.

10ml of the sonicated mixture was added to the same volume of HEPES buffer in which the proteins were dissolved. The mixture was homogenized while gelation occurred for about 6 minutes. The cured gel is then finely comminuted and spread on the glass surface to form a thin layer for exposure to dryness in a room. At maturity, about 84% of the volume mass is lost: approximately 3.0g of the mature complex was obtained from an initial liquid volume of 20.0ml of the precursor.

The simple composite was then ground to less than 106 μm³The particle size of (a). Water content of mature simple composite (a)_w) About 11 ppm. The protein concentration of the mature simple composite (protein mass/composite mass) is between 0.5% and 1.8%.

For immunoassays, a simple composite was also synthesized for a negative control immobilized with bovine serum albumin, whose antigen/antibody quality was identical to that described above.

Example 1.2. composite Material

1.2.1. Failure to fix composite materials

Attempts were made to synthesize a composite material, following conventional sol-gel preparation protocols, in an attempt to immobilize a previously synthesized simple composite material, rather than the free protein. Knowing the proton availability required for chemical attack of the orthosilicate structure, the following experiments were performed: 1.5ml of TMOS was hydrolyzed with 50 μ l of a solution of 100mM HCl in different volumes of water in a buffer medium at pH 7.0, in order to obtain a hydrophilic composite compatible with the simple composites described previously.

The water deficit in the reaction medium was found to be proportional to the mass of the simple composite, although linearity followed the initial ratio of the simple composite mass to the precursor volume. Thus, TMOS hydrolysis failed completely. Table 1 lists the compiled experience, illustrating the difficulty of fixing a simple composite in a second sol-gel.

Table 1: the experience of trying to fix simple composites in sol-gels.

1.2.2. Practical formula of composite material

Simple composites were synthesized at concentrations from 0.83% (m/v) up to 3.34% (m/v). Adding 25mg to 100mg of particles having a particle size of less than 106 μm relative to a unit precursor volume of 3.0ml³And water content (a)_w) About 11ppm of simple composite. The simple composite was suspended in 1.5ml Phosphate Buffer (PB) using 38mM Na₂HPO₄.2H₂O; KH of 62mM₂PO₄And (4) preparation. The PB was further adjusted to pH 6.8 ± 0.2 (1.0M NaOH aliquot was added) at 25 ℃.

An HCl solution was prepared, the concentration of which was dependent on the simple composite material of higrophilia and was adjusted by the respective immobilized proteins. For example, but not limited to, [ HCl ] 4.5mM for alkaline phosphatase; for mucin, [ HCl ] 8.3 mM.

540. mu.l of distilled water was mixed with 50. mu.l of HCl solution and then added to 1.5ml of TMOS. The mixture was homogenized for the first 120 seconds and after 3 minutes 1.5ml of this mixture was transferred to the same volume of PB suspension of simple composite material. Gelation occurs within 10 to 15 seconds during which the medium is homogenized to ensure uniform distribution of the simple composite particles.

The polymerization was carried out for 30 minutesFor 90 minutes, and then fragmenting the cured gel to an approximate volume size of less than 2mm³And 2mm³To 4mm³The particles of (1).

In immobilizing the antigen/antibody, the solidified gel was incubated in 2.0% (m/v) Bovine Serum Albumin (BSA) for 24 hours with orbital stirring at 400 rpm. The solvent was 7.6mM Na₂HPO₄.2H₂O；2.4mM KH₂PO₄(ii) a 137mM NaCl; 2.7mM KCl in 10mM phosphate buffer pH 7.3. + -. 0.3. Incubation volumes were given as a ratio of three volumes of solution to one volume of cured gel, which was transferred to a clean, dry glass surface after incubation. Starting on the glass surface, the glass was replaced three times: at the time of maturation for 24 hours, at the time of maturation for 48 hours and at the time of maturation for 72 hours.

Maturation of the composite (whether or not incubated in BSA) was carried out for 7 days by exposure to room conditions, after which the loss of volume mass was about 70%, i.e.the final water content a_W≈19ppm。

The protein concentration of the mature composite after BSA incubation was 3.0 to 3.5 times the protein concentration in the simple composite, and the protein concentration of the mature composite without BSA incubation was 0.2 to 0.4.

After maturation, particle size (G) separation was performed and classified into five categories:

example 2 maturation time

The mass loss of simple composites and composite composites with exposure to indoor conditions was investigated. Monitoring is carried out from the moment of gelation until the estimated mass value changes by less than 2.0%.

Samples of similar mass and two orders of magnitude mass were monitored for both composites to verify the dependence of maturation on precursor volume (initial mass): samples of 1.5ml and 3.0ml were studied. Fig. 2 and 3 show the collected data for the simple composite and the composite, respectively.

From the results obtained, it can be concluded that the stabilization times for the mass loss of the simple composite and the combined composite are 72 hours and 50 hours, respectively. Thus, the maturation interval was determined to be 7 days for both formulas, with the final moisture content of maturation:

1. simple composite materials a_w＝11ppm±0.2ppm；

2. Composite material a_w＝19ppm±0.6ppm。

Example 3 Leaching study

3.1. Protein quantification method for assessing protein concentration of composite materials and supernatants

The protein concentration was determined by a modified Lowry method [17 ]. However, the initial step does not guarantee a zero mass-balance between the proteins initially present in the composite sample (zero-mass-balance) and the proteins are transferred to the supernatant due to thermoalkaline digestion (composite samples immersed in 100M NaOH at 100 ℃, 10min, followed by ice incubation).

The method used herein was optimized to ensure that all composite samples were digested and the inherent protein content was transferred to NaOH solution: the composite material sample has a mass of not more than 5.0mg and a particle size of less than 106 μm³. From each digestion medium, 200. mu.l aliquots were taken for analysis。

Protein concentrations were calibrated using BSA standard solutions (100% purity) at concentrations of 20. mu.g/ml to 200. mu.g/ml: each concentration was calibrated using 200. mu.l aliquots. The lowry reagent (1.0ml) was added to the assay supernatant and BSA standard solution, and 200 μ l of Folin-ciancaut (Folin-Ciocalteau) reagent (diluted 1: 4) was added after 40 minutes. After 10min, when the recorded value is higher than 1.0, the absorbance of the culture broth sample (the sample has been diluted) at a wavelength of 750nm is read.

3.2. Protein loss from leaching

3.2.1. Simple composite material

First, three proteins were immobilized: samples of simple composites of Bovine Serum Albumin (BSA), alkaline phosphatase (ALP) and universal antibody (IgG) were tested for leaching.

The protein loading as defined herein is the mass of lyophilized reagent added relative to the total volume of precursor still in the sol-gel preparation. Two sets of composites were aimed at, with different protein-order concentrations (1.0% and 10.0%) of BSA and ALP, and one-third of the classes immobilized antibodies (IgG). Thus, the loss of protein from the composite was monitored in three trials for different protein concentrations and with different protein types.

The amount of protein retained in the composite was quantified before (matured and dried composite) and after the leaching test, where each test sample was dried at 40 ℃ for 24 hours and then exposed indoors for 10 days.

Considering that the protein leaching process is directly proportional to the area/volume ratio of the particles, two particle sizes were tested: 1mm³Above and 750 μm³The following.

First, experiments were performed using 30mg of mass sample and by incubating the matured disintegrated composite in distilled water under orbital stirring at 200rpm for 72 hours.

In 10ml vials, the volume of the incubation liquid was 1.0ml, at the end of each experiment the liquid medium was decanted and centrifuged at 11K rpm for 10 min. Three 200 μ Ι aliquots of the clear supernatant were analyzed in three separate quantification events to determine the protein concentration in the supernatant.

The step of determining the leached protein still remaining in the composite material follows the protocol described above for a sample mass of 5.0ml to ensure complete digestion of the sol-gel, thereby releasing the immobilized protein in large quantities into the supernatant to be analyzed. Three aliquots of 200. mu.l were again analyzed in three separate protein concentration quantification events.

From the data obtained, mass balances were performed to quantify the protein loss relative to the initial concentration determined accordingly. Based on this, the difference between the protein concentration of the composite material was calculated before and after the leaching experience. In addition, the amount of protein present in the total mass of the composite sample prior to leaching (using the previous data) was compared to the total amount of protein in 1.0ml incubation medium. The recorded data were averaged and the corresponding numbers are listed in table 2.

Table 2: protein concentrations obtained in leaching tests of simple composites doped with different types of proteins and different protein concentrations.

These results confirm that the smaller the particle size, the more closely the erosion of retained protein is to some extent related to it: the greater the particle size tested, the closer the concentration of the composite material recorded after leaching to the initial loading.

On the other hand, for the lower particle size samples, the difference between the initial loading of the composite material and the mass of dissolved protein in the supernatant was smaller, which is an indication of protein transfer to the leaching medium. The results of the different protein types retained in the simple composite, and the corresponding results of the different loadings, together demonstrate that the leaching phenomenon is not only proportional to the initial protein loading, but also proportional to the particle size.

The data recorded for IgG confirmed the results of the increased retention of antibodies in the composite material mentioned by Alstein and coworkers [16], where the difference between the two particle sizes before and after leaching was of the same order of magnitude.

In addition, 5mg and 60mg mass samples of simple composite incorporating ALP were also tested for leaching at maximum protein loading and smaller particle size under the same experimental conditions. The numbers obtained are listed in table 3, where they are compared with a 30mg mass sample.

Table 3: protein concentrations obtained in leaching tests of simple composites incorporating alkaline phosphatase at the same concentration but different sample masses.

From these figures it can be concluded that the loss of retained protein is influenced by the diffusion of particles in the liquid medium: for the same batch size, the lower the mass of the test sample, the higher the difference in the protein concentration retained in the composite before and after leaching.

On the other hand, for larger mass samples, it is clear that there is a large difference between the initial protein load of the composite material and the protein concentration in the leach supernatant, which means that there is less protein transfer into the broth than for smaller mass samples.

The effect of smaller protein attack and larger simple composite mass for the same reaction volume allows to conclude that the simpler the composite used in the same batch reactor volume, the less total loss of protein occurs and therefore the more stable the bioprocess activity of the doped simple composite.

3.2.2. Composite material

The same experimental procedure was performed on the combined composite immobilized with simple composites doped with the same protein, allowing for comparison with the quantitative protein loss recorded in the simple composite, which would further allow monitoring of the enzymatic (alkaline phosphatase) activity reaction.

The combined composite with the highest protein loading (43.11 μ g/mg) immobilized simple composite was synthesized to obtain two sets of samples with different protein concentrations: 8.81. mu.g/mg and 23.14. mu.g/mg. Similar to the previous experiment, a small particle size 30mg of a crushed sample was tested. The data processing and experimental procedures were the same as those monitored for simple composite leaching, and the results obtained are listed in table 4.

Table 4: protein concentrations obtained in leaching tests of composite composites prepared from simple composites doped with alkaline phosphatase of varying masses.

The difference between the combined composite with the highest initial concentration of protein before and after leaching was about 10 times lower (0.78. mu.g/mg vs 9.94. mu.g/mg) than that of the simple composite, and the protein concentration was about two times lower (23.14. mu.g/mg vs 43.11. mu.g/mg) than that of the simple composite.

Likewise, leaching tests were performed using 5mg and 60mg mass samples of the low particle size composite. The results obtained are listed in table 5.

Table 5: protein concentrations obtained in leaching tests of composite composites of different sample masses incorporating alkaline phosphatase at a concentration of 23.14 μ g/mg.

It is demonstrated that trace loss of protein is inversely proportional to the amount of composite material in the same liquid volume.

The results obtained finally demonstrate a smaller loss of protein in the combined composite compared to the simple composite results: the difference in protein retained in the composite before and after leaching. At the same time, a comparison of the protein content in the initial immobilized protein and the supernatant was made, and a greater difference was noted, which reflects a higher retention grade.

3.2.3. Saline medium exposed to too high a concentration

The simple composite samples and the combined composite samples with the highest protein concentration and smaller particle size were exposed to a high salt concentration solution of 2.0M NaCl to assess protein loss in high ion stress media.

For both composites, 60mg mass samples were tested by immersion for 72 hours under orbital agitation at 200rpm, and the leached composites and corresponding supernatants were subjected to quantitative protein analysis according to the same protocol as before. The data obtained are listed in table 6.

Table 6: protein concentrations obtained in leaching tests of simple composites and composite composites doped with alkaline phosphatase and immersed in 2.0M NaCl solution.

The values recorded for the difference in protein concentration retained in the composite before and after exposure to the salt solution were of the same order of magnitude as the values recorded by exposure to distilled water.

At the end of the experiment, the mass number of proteins in the initial composite sample was compared with the mass number of proteins in the supernatant, in which case there was clearly a smaller difference, probably due to the phenomenon of the higher ionic concentration of the instinctive reaction leading to a more intense transfer of proteins into the culture broth.

However, these figures must be considered as the fact that the leach solution is at a salt concentration that is too high compared to the expected physiological sample that can be used, and that the exposure time is much longer than that used in the preferred embodiment.

Example 4 catalytic test

The formulation of the composite material of the invention has previously been demonstrated to have the ability to immobilize/retain proteins, and then aims to verify whether a protein is still active as an enzyme. In this case, the activity of alkaline phosphatase (ALP; Sigma Aldrich Cat.N.times. 10567752001) was monitored. First as a free state and then as an immobilized enzyme in simple composites and composite composites.

Enzymatic assay spectrophotometric methods were repeated to quantify the concentration of p-nitrophenol (pNP) which is the product of a standard reaction product of the hydrolysis of p-nitrophenol phosphate (pNPP), a reaction known to have a 1:1 stoichiometric amount.

4.1. Enzymes immobilized in simple composites

Two samples of simple composite immobilized ALP were synthesized at precursor concentrations (m/v) of 8% and 10% according to conventional free enzyme assays. At the end of the 13-day maturation period, the concentrations were 4.8mg each_Protein/g_{Composite material}And 6.2mg_Protein/g_{Composite material}。

Breaking the immobilized mature composite sample to 1mm, with a view to reducing the external accessibility limit of the substrate molecules³To 2mm³Range of particle sizes. The test was performed using simple composite samples with a mass of 5.0g to 8.3g and the enzyme concentration of the batch was deduced from the protein concentration of the respective composite and the respective mass of the sample.

The enzymatic assay was performed in a 10ml effective volume reactor to which was added 3.6ml of the same buffer solution (phosphate buffer 100mM, pH 9.1) used in the free enzyme assay at room temperature. Kinetic studies began with the addition of 0.4ml of substrate solution (pNPP in pH 9.1 buffer), the reaction was carried out for 28 minutes, and 200 μ l of liquid medium was collected over a 1:30min interval. The initial substrate concentration at the total reaction volume was the same, about 3.0 mM. Data processing recorded that, over a period of 28 minutes, the collection of samples progressively reduced the volume of the reaction medium, but the mass of catalyzed composite was the same.

The values recorded make it possible to plot the kinetics as a function of the enzyme loading, as shown in FIG. 4.

The results obtained show that for an identical initial substrate concentration, the product concentration is directly proportional to the mass of composite material (containing immobilized enzyme).

4.2. Composite material having immobilized simple composite material doped with enzyme

A combined composite from the same formulation (protein concentration 23.14 μ g/ml) was studied for leaching tests and samples of mass 14mg to 20mg were tested.

The particle size of the samples, the experimental procedures and data processing of the enzymatic assays were exactly the same as those of the simple composite dynamics study. Similarly, the enzyme present in the reaction medium is quantified based on the predetermined protein concentration of the composite and the mass of the sample used for each test. The results obtained are shown in FIG. 5.

Comparison of the maximum yields of pNPP hydrolysate pNP catalyzed by the same enzyme immobilized in the two composite preparations made it clear that for the same mass of enzyme (11.65 mg)_{Simple composite material}；10.8mg_{Simple composite material}vs 12mg_{Composite material}；11mg_{Composite material}) Simple composite-generated pNP (40.57 mM; 26.76mM) compared to pNP produced from the composite (5.03 mM; 3.03mM) 8 to 9 times more.

Considering that the composite material of the invention is a coating of a simple composite material doped with an enzyme (encapsulated in a particle size range with robustness and external accessibility of the substrate), it is therefore understandable that fewer and fewer active sites of the enzyme are present, and therefore the yield of the enzyme metabolite is lower. However, as demonstrated earlier in leaching studies, lower catalytic efficiency was compensated by more durable retention of immobilized protein.

Example 5 emissions at different particle size profiles

A hydraulic test of the penetration of the particulate composite material with an aqueous dye solution and diluted blood will now be performed to obtain a residence time compatible with use in a medical diagnostic apparatus.

The composite materials tested herein are used as a packed bed of columns, also referred to as analysis chambers or reactors, for immunoassays or enzymatic tests, respectively. It is a rectangular box with a size of 4cm_{Long and long}×3cm_{Width of}×1cm_{Height of}Made of a clear acrylic material, the interior of which is visible. It is composed ofThe treatment of the contents is carried out by a drilled removable cap which, once mounted on the cartridge, allows the passage of the eluent through its interior. The surface of the opposite top is also drilled to allow liquid to flow out, the holes of both caps being 1.0mm in diameter.

The column is planned to be packed with the particulate composite material, with reference to an effective mass value of 9 grams (evaluated by weighing the entire column flooded with water (1 g/ml by volume mass)). The amount of this mass is excessive with respect to the application on the diagnostic device apparatus, and is estimated to be finally in the range of 3: 1 are reduced. The purpose of these studies was to test the discharge mechanism of liquid samples, and therefore, although particle size is the only conditioning variable, composite composites with the same maturity were therefore used.

The particle size gradient curve was initially programmed to completely fill the effective volume and the respective values were:

bottom layer: 15% (1.35g)Particle (G) > 1.0mm³；

Bottom intermediate layer: 20% (1.80g)1.0mm³＞G＞710μm³；

An intermediate layer: at 710 μm³Lower, 0% (2.70g)Variable particle size;

top intermediate layer: 20% (1.80g)1.0mm³＞G＞710μm³；

Top layer: 15% (1.35g)Particles > 1.0mm³。

5.1. Monitoring discharge of dye solution

After separation according to particle size, the two bottom layers were placed correspondingly, and the central layer ranged from 710 μm³To 212 μm³The size of the particles. An attempt was made to place the upper layer of the middle layer as programmed, but only 1.6g could be placed. After each layer was allowed to stand, it was washed with distilled water to increase the compactness of the particles and reduce the preferential run-off pattern, thus completing the filling step. The column was then exposed to a dry heat environment at 40 ℃ for 24 h.

The test started with measuring the amount of water that saturates the column contents-the effective liquid volume (ULV) (water was added to the dry granular composite contents until the first drop appeared at the bottom): it was found to be 3.0 ml. It is recommended to evaluate the volume of retained water under such a gradient curve. After saturation, it is clear that for every 1 ml addition, corresponding to 1 ml drained from the bottom of the column, it is evident that a quantity of liquid equal to ULV should be added to incubate the entire contents of the column.

In addition, the elapsed run-off time per ml of addition was monitored until at least 900 μ l was collected and this measurement was performed in 25 trials, where 3 minutes is the average recorded time. The column was then exposed to 40 ℃ dry heat for 24 h. The composite particle gradient was again brought to water saturation and a 1.0ml portion of evans blue (1.4mg/100ml) was eluted.

The evans blue solution was absorbance calibrated (λ 608nm) (starting from 1:1 ═ 1.426% ± 6%; until 1:10 ═ 0.134% ± 10%). The dye solution breakthrough was monitored by spectrophotometric reading of successive eluted/collected 1.0ml distilled water fractions, the respective values recorded are shown in figure 6.

After removing the wet particulate composite, a new dry particulate composite was placed in the column following the same procedure except that the center layer was 710 μm³To 300 μm³And the upper layer again consists only of 1.0mm³＞G＞710μm³But this time 1.16g of the particulate composite material was consumed. The elapsed time per ml of addition was monitored untilAt least 900 μ l was collected and this measurement was performed in 30 trials, where 25 seconds is the average recording time.

The column was then exposed to 40 ℃ dry heat for 24 h. The volume of residual liquid (1.9ml) was further evaluated and the test of dye solution discharge was repeated. The data collected is shown in figure 7.

After removing the wet particulate composite, a new dry particulate composite was placed in the column following the same procedure except that the center layer was 710 μm³To 500 μm³And the upper layer again consists only of 1.0mm³＞G＞710μm³But this time 0.98g of the particulate composite material was consumed. The elapsed run-off time per ml of addition was monitored until at least 900 μ l was collected and this monitoring was done for 25 trials, where 10 seconds is the average recorded time. The column was then exposed to 40 ℃ dry heat for 24 h. The volume of residual liquid (1.0ml) was further evaluated and the test of dye solution discharge was repeated. The data collected is shown in figure 8.

After removing the wet particulate composite, a new dry particulate composite was placed in the column following the same procedure except that the center layer was made from 500 μm³To 300 μm³And the upper layer again consists only of 1.0mm³＞G＞710μm³But this time 1.90g of the particulate composite was consumed. The elapsed run-off time per ml of addition was monitored until at least 900 μ l was collected and this monitoring was performed for 25 trials, where 1:30 minutes is the average recorded time.

The column was then exposed to 40 ℃ dry heat for 24 h. The volume of residual liquid (2.0ml) was further evaluated and the test of dye solution discharge was repeated. The data collected is shown in figure 9.

All these results show that the amount of residual liquid and the run-off time are limited by the smallest particle size layer for the same volume occupied by the composite and penetrated by water.

Table 7 shows these cases, where the particle size values and the corresponding quality of the bottom and bottom intermediate layers were maintained. At the same time, changing the particle size of the central layer but maintaining its mass content results in a change in the residence time of the permeate.

Table 7: the amount of drainage of the central layer (2.7g) of the combined composite of different particle sizes also affects the effective volume of residual liquid and the total mass held in the column.

In the center layer, for the same particle size interval, the discharge time of 1.0ml of dye solution:

1. and 10 seconds (710 μm)³＞G＞500μm³) In contrast, at about 200 μm³Within the range of (a): 90 seconds (500 μm)³＞G＞300μm³)；

2. And 30 seconds (710 μm)³＞G＞300μm³) In contrast, the larger range: 180 seconds (700 μm)³＞G＞212μm³)。

Emissions in the same particle size interval:

a. and 12 seconds (710 μm)³＞G＞500μm³) In contrast, at about 200 μm³Within the range of (a): 15ml (500 μm)³＞G＞300μm³)；

b. And 12ml (710 μm)³＞G＞300μm³) In contrast, the larger range: 13ml (710 μm)³＞G＞212μm³)。

In summary, at 710 μm³The penetration volume of the aqueous sample in the compacted layer of the particle-sized composite is directly proportional to the magnitude of the particle size interval and inversely proportional to the respective discharge time.

It is also evident that the total mass of composite material in the column is inversely proportional to the particle size. This finding is expected with respect to compaction (the smaller the particles the more pronounced) and thus the smaller inter-particle spaces that result in higher densities.

5.2. Monitoring blood discharge

For discharging diluted bloodTests were carried out and it was found from the particle size gradient that the flow of the dye solution was better (710 μm in the central layer)³＞G＞500μm³). Samples collected by the present inventors were anticoagulated with EDTA (50mg/ml) and diluted with phosphate buffer (10mM, pH 7.3). The 1.0ml of diluted sample was eluted, then the 1.0ml distilled water fraction was eluted, and the run off was evaluated by reading the collected fraction at an absorbance at a wavelength of 540nm, which targets the hemoglobin (Hb) molecule. Hb concentrations of 0.6mg/ml (absorbance 0.314% ± 7%) to 3.0mg/ml (absorbance 1.635% ± 4%) were calibrated. The Hb concentration of the sampled blood was 150mg/ml (+ -1.0%). The results recorded for three trials are shown in figure 10.

Prior to these results, it was evident that the diluted blood run-off occurred predominantly after 4.0ml of permeate water, consistent with previous data on dye solution discharge. It can therefore be concluded that the permeation of the blood sample in the dilutions mentioned herein is the same as the run-off recorded for the evans blue solution at a concentration of 1.4mg/100ml, and that they are two rheologically identical liquid media.

Therefore, it is inferred that 1: 200 and 1: it seems reasonable that the same drainage mechanism for the 100 blood sample on the other gradient particles of the composite material would be expected, which would predict a situation where the immobilized protein (antigen/antibody) would be well approached by a biological sample fluid of similar viscosity and density to water.

5.2.1. Monitoring blood flush discharge

The flush discharge of the combined composite gradient particle size was studied according to the recorded runoff curves of evans blue or blood solutions, first permeated with blood, then the first portion used 10.0ml of distilled water, followed by five successive elutions, each with 1.0ml of distilled water. The method comprises the following steps of 1: in a series of three experiments with 100 dilutions of blood, 96% ± 0.5% of the added Hb was collected directly in the first 10.0ml aliquot as shown in fig. 11.

Based on these results, the use of a less diluted blood sample (as low as 1:30) to wash the particulate composite was then investigated. In the protocol between each experimental event, the particulate contents of the column were washed thoroughly with distilled water and then dried at 35 ℃ for 24 hours. The particulate composite was saturated with 20.0ml of distilled water before adding a new blood sample.

The clearance of the collected fractions was monitored in elution with 3 parts of 10.0ml of distilled water and in elution with the last 1.0ml of distilled water, and the results are shown in FIG. 12.

It is evident from the collected data that even with the most concentrated blood samples, the discharge of 1.0ml of blood per unit volume of analysis occurs at most in the case of elution with 10.0ml of water, since the Hb concentration is lower than 1.0mg/ml in the third and fourth collected fractions.

Considering that these tests were performed with a total column packing mass of 6.82g, and the scaling ratio of these tests was also about 3: 1, then for the same gradient (relative proportion) the estimated mass of the combined composite will be between 1.5g and 3.0g at the scale of final use. In view of this, and the studies herein that linearly scale the particle size gradient, it is reasonable to estimate the effective wash volume to be 10 ml.

Example 6 composite materials in enzyme reactor

In this example, the enzymatic activity of at least 20 cycles of activity of the combined composite packed in the above acrylic column was studied, provided by a portion of the substrate solution that was pushed by the atmospheric pressure to penetrate the particulate composite under a vertical plug flow regime. The proposed protocol is to quantify the reaction yield of the conversion of p-nitrophenol phosphate (pNPP) to p-nitrophenol (pNP) catalyzed by alkaline phosphatase immobilized in a composite.

Using enzyme concentration thresholds for specific activity and enzyme loading: 0.4% (m/m) [18 ]][19]. The particle size gradient previously found was used as the optimal runoff for the enzymatic process: the residence time is at least 1.5 minutes (center layer particle size 500 μm)³＞G＞300μm³). The combined composite had a total mass of 7.75g and was compacted to minimize preferential run-off. The volume of the remaining liquid was 2.0 ml.

After packing the column, the run started at room temperature with the same eluent previously used for a similar catalytic run (phosphate buffer 100mm, pH 9.1) also used here as eluent to thoroughly wash the particulate gradient composite. To this was then added 4.0ml of substrate solution (dissolved at pH 9.1), and after 15 minutes of incubation, the particulate composite was infiltrated with three 2.0ml portions of eluent followed by eight 1.0ml portions of eluent. Once the respective elution fractions are added, one aliquot of the same volume, the enzymatic reaction product can be collected (in contrast to the batch process described previously). Quantitation of metabolite concentrations is derived directly from absorbance readings

Data processing begins with the comparison of 1.0: the absorbance values (λ 405nm) were calibrated for product (pNP) concentrations up to 56 μ M (see example figure 13).

Abs obtained from each of the 11 fractions collected₄₀₅The nm values were converted to pNP concentrations. Knowing the volume of each collected fraction, the number of moles present in each collected fraction was calculated. The collected moles of reaction product are added. In addition, knowing the substrate (pNPP) concentration, the respective moles initially provided were calculated based on the volume of solution fed to the reactor (see table 8).

Table 8: data processing of the first experimental results of p-nitrophenol phosphate hydrolysis in a bioreactor immobilized with a composite of alkaline phosphatase.

The reaction is known as 1:1 stoichiometric percentage, calculating the conversion: product moles 100/substrate moles. In the first run, the substrate concentration used was 318. mu.M, and a yield of 7.4% was recorded. The next experiment used an order of magnitude higher substrate solution: 3.03mM, yield 19.5%.

The third experiment maintained the substrate concentration and the incubation time was extended for 20 minutes. The yield was recorded to be 25.7%. From this result, it was concluded that a longer incubation time allowed for more deep hydrolysis of the added substrate. The fourth trial was a third repeat and a yield of 24.1% was recorded.

In the fifth run, the substrate concentration was reduced to 1/2, and a yield of 23.1% was recorded, and in the next run, under the same conditions, a value of 29.7% was recorded.

In the seventh experiment, the substrate concentration was reduced to 1/4: 750 μ M. The yield was recorded to be 36.4%. The final experiment was repeated twice, and the yields reported were: 38.9% and 35.6%.

In the next experimental event, three trials were performed, the last protocol was repeated, and the yields recorded were: 32.9 percent; 28.8 percent; and 28.2%. In a new series of five trials, the yields reported were: 25.4 percent; 24.9 percent; 27.5 percent; 27.8 percent; and 25.3%. In another experimental event of three trials, the yields recorded were: 16.6 percent; 19.0 percent; and 18.9%.

In the two subsequent experiments, the yields reported were: 21.7% and 21.1%. In the last experimental event, 9 trials were performed, and the yields reported were: 18.2 percent; 16.0 percent; 15.5 percent; 16.7 percent; 16.3 percent; 14.6 percent; 13.9 percent; 14.0 percent; and 19.0%.

The normalization procedure for these mentioned numbers after the 7 th experiment is compiled in fig. 14, where a substrate concentration of 750 μ M and an incubation time of 20 minutes is used. The figure shows the yield recorded, which is referred to the value obtained in the second experiment of the series (38.9%), keeping it at a maximum: 100 percent.

Before these results are reached, it can be reasonably concluded that the composite material is always suitable for the immobilization of enzymes (alkaline phosphatase) while retaining its activity. Considering also that the experimental standardization was achieved after a step adjustment of seven trials, it is reasonable to foresee the use of the composite formulation for a number of cycles greater than the 20 cycles originally indicated.

EXAMPLE 7 composite materials are preferably used in diagnostic devices

One preferred use of the composite material of the present invention will now be described, which is applied to a portable device configured as a vertical parallel operation unit for medical diagnosis. It accommodates up to 4 units, each unit comprising two chambers, an analysis chamber and a negative control chamber.

All chambers are filled with a particulate composite material, wherein the chamber for analysis is filled with a composite material in which an immune response protein, antigen or antibody relevant to diagnosis of human or higher animal diseases is immobilized, and the negative control chamber is filled with a composite material in which an immune non-response protein is immobilized.

The function of the device is based on the principle of immunodiagnosis, in which a biological sample is suspected of carrying an antibody (or antigen) associated with a disease, and by manipulating the device those molecules are linked to the corresponding specific ligands. The specific ligand will be an antigen (or antibody) immobilized in a composite material, the physical properties of the pore size of which make these liquid biological samples permeable. After antigen-antibody ligation, a primary immune complex will form and the composite must be washed to remove excess debris and unbound protein.

In the first case of this example, the response of a composite [20] immobilized with a simple composite incorporating mucin 1.8% (m/m) was preliminarily tested. The composite material is used to fill a test column that is coherent with one equipment unit. The test column was packed with a particulate composite material with a particle size gradient curve similar to the blood discharge test.

Immobilized antigen mucins are molecules with a glycosidic structure sialic acid homologous to a surface ligand of a tumor cell (e.g., breast cancer). Formation of the first complex was tested by antibody-reagent ligation as provided by the immunology groups of the scientific and Technology university of New Liiss (Science and Technology Faculty of university Nova de Lisboa). This antibody has previously shown high binding affinity for neoplastic tissues [21 ]. The biological sample in those tests is the supernatant from a culture of antibody-producing animal cells (hybridoma cells).

After incubation of the biological sample, the particulate composite is washedMaterial to remove excess unbound antibody. The immune complexes formed were detected by the addition of a secondary antibody labeled with peroxidase. Such an antibody has a specific affinity for the first antibody. Addition of a second antibody provides for the formation of a second immune complex: antigens_{Immobilization of}-antibodies_{Biological sample}-antibodies_Marking. The granular composite material is washed again to remove excess unattached protein.

The chromogenic substrate for peroxidase was then added: 3,3 ', 5, 5' -tetramethylbenzidine. The substrate is degraded by the second antibody and thus imparts a blue-green color to the composite, revealing the presence of the first antibody in the biological sample. This procedure was verified by a similar test performed on a homoparticulate composite material incorporating the non-immunogenic protein bovine serum albumin, as shown in figure 15, in which no colour was obtained.

The recovery of the particulate composite material makes it possible to reuse them separately, and this step is accomplished by elution with a chaotropic solution that disrupts the linkage of the first and second antibodies. The particulate composite material is then washed to remove any released protein.

7.1. Preliminary test protocol

When the composite material of the present invention is preferentially used in medical diagnosis, preliminary tests must be performed to test the criteria of sensitivity and specificity.

A sample of composite composites immobilized with the same simple composite having the same concentration of antigen/antibody/BSA will be tested. The protocol included 8 test bottles, each having a sample of 100mg of particulate composite material in the particle size range of 100 μm³To 300 μm³：

i) Wherein each of the four vials has 25mg immobilized thereon; 50 mg; 75 mg; 100mg of a sample of particulate composite of simple composite containing BSA. The mass of these simple composites corresponds to 3.0ml of precursor when synthesizing the composite.

ii) each of the other four vials had 25mg immobilized; 50 mg; 75 mg; 100mg of a sample of particulate composite material containing a simple antigen/antibody composite. When a composite material is to be synthesized, the mass of these simple composite materials corresponds to 3.0ml of precursor.

7.1.1. Reaction solution

Phosphate buffer 10mM, pH 7.3 ± 0.3(25 ℃) + 0.05% (m/v) tween-20 (PBS-T) was used:

diluting the biological sample;

dilution of the labeled secondary antibody;

washing the particulate composite.

Chromogenic peroxidase substrate: 3,3 ', 5, 5' -Tetramethylbenzidine (TMB) was used diluted with distilled water.

The recovery method comprises the following steps: restore^TMWestern Blot Stripping Buffer (Western Blot striping Buffer); thermo Scientific (Sb) was used diluted in distilled water.

7.1.2. Experimental procedure Using bottles

Step 1: and (4) adjusting the composite material.

The particulate composite material will be wetted with 2 × 2.5ml PBS-T: incubation time: and 5 min. The liquid medium is then poured off.

Step 2: the biological sample is eluted.

The biological fluid samples were first diluted 1:10 (in PBS-T) to 2.5ml volumes each. The aim is to obtain a result of a definite coloration of the composite doped with antigen (or antibody) and a clear elimination of the composite doped with BSA, so that the dilution scale of the biological sample will vary from 1:5 to 1: 13. Incubation time: and 20 min. The liquid medium is then poured off.

And step 3: first washed with 2X 2.5ml PBS-T. The liquid medium is then poured off.

And 4, step 4: the secondary antibody was eluted.

The secondary antibody labeled with peroxidase will first be labeled with 1: 3333 dilution (in PBS-T) to 2.5ml volumes each. The aim is to obtain a result of a definite coloration of the composite doped with antigen (or antibody) and a clear elimination of the composite doped with BSA, so that the dilution scale will be from 1: 2000 to 1: 5000. Incubation time: and 5 min. The liquid medium is then poured off.

And 5: a second wash was performed with 4X 2.5ml PBS-T. The liquid medium is then poured off.

Step 6: eluting the chromogenic substrate.

The peroxidase chromogenic substrate was first treated with distilled water at a rate of 1:4 to a respective volume of 2.5 ml. The TMB dilution scale will be optimized to 1:3 in order to obtain a clear staining of the antigen (or antibody) doped composites and a clear clearing of the BSA doped composites. Incubation time: 10min to 20 min. The liquid medium is then poured off.

As a result: the coloration of the composite material is the result of immune complex formation. The color intensity obtained in each tested composite sample will give an indication of the analytical standard of the method:

sensitivity-fewer simple composites (25 mg in time) are trapped in the combined composite, which will enable detection of the presence of antibodies (or antigens) in a biological sample;

specificity-confinement of the maximum mass of a simple composite (suitably 100mg) in the combined composite, without showing cross-reactions:

I. the negative control composite material is not colored;

the composite tested with biological samples containing different specific antibodies (or antigens) is free of staining.

And 7: and (6) recovering.

The stripping buffers were first diluted 1:16 with distilled water to a volume of 2.5ml each. The Sb dilution liquid was optimized from 1:10 to 1:32 depending on the clearance obtained. Incubation time: for 10 min. The liquid medium is then poured off.

And 8: a third wash was performed with 6X 2.5ml PBS-T. The liquid medium is then poured off.

7.1.3. Filling chamber

This embodiment relates to the operation of a diagnostic medical device that envisages a maximum containment of eight chambers as previously mentioned. The effective internal volume of each chamber was 9cm³And filled as described previously.

After 7 days of maturation, the particulate composite material was washed with distilled water and then dried at 37 ℃ for 3 hours. Particle size separation defines 5 particle size classes:

the filling of each chamber consists of 6 layers, with the following masses of particle size from top to bottom:

6 th layer:

layer 5:

layer 4:

layer 3:

layer 2:

layer 1:

the compaction of each layer was optimized by elution with 3.0ml to 5.0ml of distilled water, thereby reducing the formation of preferential run-off patterns. Once all layers have been deposited, the remaining free volume of the chamber is filled from the top (with glass spheres having a diameter of 0.8mm to 1.2 mm) up to 1.0 cm. This upper space is left so as to have a visible reflux window.

The entire 8 chambers were left at 37 ℃ for 48 hours, and then the volume of the remaining liquid was quantified by elution with 10ml of distilled water. The difference obtained by measurement after collection of water was 2.0 ml. + -. 0.4 ml.

7.1.4. Operation of the apparatus

The biological sample placed on the liquid collector drains directly into the analysis chamber placed below, as shown in fig. 16. The liquid discharge in all experimental steps did not fill the reflux window to avoid cross contamination from chamber to chamber. The experimental procedure described above involves a laboratory operation, the dilution scale having been previously determined in preliminary experiments.

Since each analysis chamber is run simultaneously with the corresponding negative control chamber, the minimum liquid volume used during operation of the device is twice the value shown below.

Step 1: and (4) adjusting the composite material.

The granular composite was wetted with 10.0ml PBS-T.

Step 2: the biological sample is eluted.

Biological fluid samples were diluted (in PBS-T) at 1:5 up to 1:13 to a respective volume of 4.0 ml. Incubation time: and 20 min.

And step 3: first, the cells were washed with 2X 10.0ml of PBS-T.

And 4, step 4: the secondary antibody was eluted.

Stock solutions of peroxidase-labeled secondary antibodies were mixed with PBS-T at 1: 2000 up to 1:5000 dilutions were made to a volume of 4.0ml each. Incubation time: and 5 min.

And 5: a second wash was performed with 2 portions of 10.0ml PBS-T.

Step 6: eluting the chromogenic substrate.

Peroxidase chromogenic substrate was diluted 1:4 to 1:3 with distilled water to 4.0ml volumes each. Incubation time: 10min to 20 min.

As a result: the analysis chamber filled with the granular composite doped with mucin achieved a blue-green color approximately proportional to the mucin concentration, whereas the analysis chamber filled with the granular composite doped with BSA exhibited no color change for the corresponding mucin concentration.

And 7: and (6) recovering.

The stripping buffer was diluted 1:16 to 1:10 with distilled water to a volume of 7.0ml each. Incubation time: for 10 min.

And 8: a third wash was performed with 3 portions of 10.0ml PBS-T.

For the entire 8-chamber operation, the liquid volume used is of the order of magnitude linearly related to the value of one chamber.

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[20]Mucin,Bovine Submaxillary Gland,Cat:4999643-500MG.

[21]Antibody produced by Glicoimunology Group,of Faculty of Science and Technology of Universidade NOVA de Lisboa and submitted to Portuguese patent request N.°110526,“ANTIBODY,FUNCTIONAL FRAGMENT OR PROBE THEREOF AGAINST TUMOUR ANTIGENS”.