DESCRIPTION

1. Object of the invention

[0001] The present invention refers to the utilization of sol- gel synthesis to immobilize active or activable biological materials. More specifically the present invention refers to a new method conceived to immobilize active or activable biological materials in a combined composite sol-gel in which the loss by- leaching of the mentioned biological materials from that combined composite is reduced while the biological activity of those materials is preserved.

2. State of ths Art

[0002] Sol-gel matrices are highly porous materials mainly derived from silica whose synthesis conditions are recognized as benign for encapsulation of most biological molecules.

[0003] In a typical protocol of synthesis of sol-cjel the precursors are alkoxides of the type Si (OR) 4 or alkoxysilanes of the type XSi (OR) ₃ or XX "Si (OR) a in which X and X refers to organic groups directly ligated to silica atom by a Si-C bridge in one top and presenting several other functional groups at the other top, Figure 1 illustrates one alkoxide where R-group is a methyl group: tetramethyi orthosilicate (TMOS) .

[0004] The first reaction of sol-gel synthesis is hydrolysis of precursors [1] in which one OR ligand is replaced by OH group:

Si (OR) 4 + H20 <h> Si (OR) 3 (OH) 4- ROH

(e.g. ) Si (OCH ₃ ) « + H ₂ 0 <0· Si(OCH ₃ ) ₃ (OH) + CH ₃ OH

[0005] Hydrolysis is then followed by condensation:

(e.g.) Si (OCRs) 3 (OH) + Si (OCH ₃ ) 3 (OH) (H ₃ CO) ₃ SiOSi (OCRs) 3 H ₂ 0 [0006] Those hydrolysis and condensation are slow reactions but hydrolysis rate can be increased by an acid (e.g HCl! which donate positive charges capable of attacking oxygen of the alcoxyde group. Thus it is obtained a gel-of-silica with texture similar to a polymeric-gel. On the other hand protons acceptor media, an alkaline solution for instance, accelerates condensation rate leading to the formation of denser colloidal particles. The ability of controlling those kinetics is important to adapt encapsulation conditions for a better response of biological molecules.

[0007] Some additives have also demonstrate :i beneficial effects on biomolecules stability encapsulated in so1 -ge.1 : po1yvi ny1 alcohol or glycerol can improve the activity of encapsulated lypases [2] [3] . Besides additives, the amount of water used to hydrolyse precursors can influence the activity of immobi1i zed biomolecules and thus the available-water must assu:re the mobility of biomolecul.es in the matured matrices: with a 1ow addition of water the activity of encapsulated jS-galactos idase was higher at a recently gelified composite than at the respective matured composite [41 ,

[0008] One disadvantage associated to encapsulation techniques is the progressi e loss of activity of the sol-gel matrix seen as an active-composite: the leach of encapsulated molecules result in decrease of activity of the matrix-composite and so limiting the efficiency on its long-standing use,

[0009] Several strategies have been come up to circumvent this hurdle in order to control the leaching of sol -gel encapsulated molecules. Some author aim to reduce leaching through decreasing pore-size by adjusting sol-gel composition and geiification/maturation conditions as referred by T.M, Butler et al. [5], Lobnik, I. Oehme et al. [6] and G.S. Badini efc al, [7] . However the diffusion of analytes within matrices is reduced which results in longer response reaction times. [0010] It was also proposed the coval.ent~l.ink of bioraolecul.es to a polycondensed net in the synthesis of sol -gel having somehow reduced the leach [8] [9] [10 _. ] ₌ An alternative strategy was to increase the size of the immobilized biomolecules by linking to an 1nert maeromo1 ecu.1 ar t ransporter [11] [12] [13] or yet an encapsulation within a supramolecular construct. [14] . However the activity of biomolecules requires some mobility and those approaches have reduced the expected activity.

[0011] Thus the problem this invention focused was the stabilization of an active or activable biological material in a sol-gel matrix reducing so its loss by leaching and at the same time retaining its activity. That problem was solved by a new method of immobilization of active or activable biological materials in -which a combined composite was obtained: one sol-gel immobilizing one other sol-gel previously produced within which active or activable biological mate ials were encapsulated in a good physical-chemical stability and response activity.

3. Description of the Invention

[00 2] The terms "immobi 11 zatlor/% "encapsulation", "imprisonment" and respective deductions here non-differentially used have the significance of stabilization of biological materials within a matrix of sol-gel composite.

[0013] With a double iromobi1ization within sol-gel of an active or activable biological materials, reduction of its loss by leaching is expected. However it should also be expected a lesser access to the active sites of immobilized biological materials and consequently a decrease of its activity.

[0014] There were some practical difficulties on manufacturing such combined composite. By effecting a second immobilization in sol-gel of one other sol-gel, the simple composite, it complied a -wide range of unsuccessful outcomes since the complete failure of gelification until inappropriate physical-chemical properties of the f.1.na1 ateria.ls .

[0015] Uncompromisingly to the theoretical basis, those non successes would come from the highly higrophil.ic character of the simple composite which in the reaction conditions have induced a dislocation of water for its saturation resulting in a deficit of water required for hydrolysis of the orthos i Iicate precursor.

[0016] As result of an intensive research, the inventor verified that the method herein described produce a combined composite, one sol-gel immobilizing another sol-gel previously obtained encapsulatincj an active or activable biological materials with good physical-chemical stability and in which final formulation the loss of ^‘ biological materials by leaching is reduced and respective activity is retained.

[0017] The present inventor surprisingly verified that double immobilization in a combined sol-gel composite of an active or activable biological materials obtained by the invented method not only remarkably reduced the loss by leaching of the mentioned materials but also preserved its activity in such a way that enabled at least 20 cycles of response from the immobilized biological material.

[0018] Thus according to the present invention it Is provided a new method of production of a combined composite for stabilization of an active or activable biological material, which coirtprises the following steps: a) to provide a simple composite under the shape of a sol -gel immobilizing an active or activable biological material of one or more different natures, finely grinded and at low available- water grade; b) to provide a second sol-gel immobilizing the simple composite of a ) inc1uding : i) preparing a suspension of the simple composite of a) in phosphate buffer solution 100 mM pH - 6,8 ± 0,2 at concentration between 0,83% and 3,34% in reference to the volume of the final reacting mixture; ii) effecting the hydrolysis of tetra ethyl orthosilicate by acid catalysis adding HC1 solution in a concentration of 4,50 m.M to 8,34 mM in a proportion of 28% of the final volume of this mixture; iii) adding the mixture ii) to equal volume of the suspension i) at 25°C to 30°C temperature allowing polymerization to occur until the combined composite obtained have consistency that enable to be fragmented; iv) fragmenting the combined composite obtained in iii) at granulometry approximately equal or less than 2 mm ³ and approximately between 2 ram ³ and 4 mm-h v) optionally incubating the fragmented combined composite of iv) during 24 hours in a solution of bovine serum albumin

(BSA) at 2,0% m/v solubilized in phosphate buffer saline

10 mM pH—7 , 3 in a proportion of three volumes of incubating solution to one volume of composite; vi) mat rating the combined composite by drying on a glass surface until a mass-decrease of circa 70% and available water grade of circa 20 ppm;

[0019] It is hence obtained a combined composite doubly immobilizing in a sol-gel matrix the active or activable biological material in which the loss by leaching of the mentioned material is reduced being additionally preserved its activity or activation eapacity .

[0020] In a preferred embodiment of the invented method, the obtained combined composite doubly immobilizing in sol-gel an active or activable biological material, has a reduced loss of the mentioned material and nreserved its activity for at least 20 cycles of utilization.

[0021] The active or activable biological materials that can be immobilized in the combined composite of the invention are not. limited, allowing to be one or more of any immobilizable molecule in the simple composite, preferably one member of the specific ligation such as enzyme-substrate, antibody-antigen or any other pair of specific ligation: preferably one biologically active protein, more preferably one enzyme or co-enzyme or one immunologxcally active protein of specific ligation, more preferably one antibody, one antigen or one hapten-protein.

[0022] The simple co p site which will be immobilized in the combined composite is provided by any methodology known at the state-of-the-art that produces a hydrophilic composite preferably by acid hydrolysis of tetramethyi orthosilicate.

[0023] In a preferred embodiment of the invention the simple composite that will be immobilized in the combined composite and that by its turn it has immobilized active or activable biological materials,, have a low grade of available-water between 10 and 15 ppm and is grinded to a granulometry between 100 and 110 p ³ .

[0024] The formation of the combined composite immobilizing the simple composite previously prepared is attained by acid hydrolysis of tetramethyi orthos ilicate using HC1 as catalyser in a concentration between 4,50 mM and 8,34 mM. That concentra ion is dictated by the water displacement in function of the protein immobilized in the simple composite.

[0025] When the bioiogical material immobilized in the combined composite is one or more of the immunologxcally active proteins such as an antigen, an antibody, a hapten-protein, the method of the invention include!S the additional stec v) of incubation of the fragmented combined composite during 24 hours in bovine serum albumin at 2, 0% m/v solubilized in phosphate buffer saline 10 raM pH—7 , 3 in a proportion of three volumes of the incubating solution to one volume of combined composite This additional step elevates significantly the protein content of the matured combined composite until 3, 0 to 3,5 relative to the protein content of the simple composite.

[0026] The present invention refers as well to a combined composite doubly immobilizing an active or activable biological material in sol-gel obtained by the method of the invention. This combined composite is herein also referred as "doped" composite with the biological material which is immobilized within it,

[0027] The combined composite obtained by the method of this invention doubly immobilizing in sol-gel an active or activable biological material, presents a robustness and improved retention capacity of the referred active or activable immobilized biological material. At the same time the referred combined composite presents an internal structure, or porosity, that allows the permeation until the active sites of those immobilized biological materials, of an analyte (ligand, substrate or activating molecule specific of the active or activable biological material) present in an aqueous sample placed in contact ith the referred combined composite, In other words the composite of this invention, presents not only an improved physical retention of the biological materials but also preserves the activity or the capacity of activation of those retained biological materials . The robustness of the combined composite of this invention together with its ability of maintaining the activity of the immobilized biological materials, enable the composite o this invention to be used along several successive utilization cycles.

[0028] In a preferred embodiment, one combined composite according to this invention can be used for at least 20 cycles of utilization , [0029] It ought to be intended as cycle of utilization of the doped composite each event of interaction/reaction among the biological material immobilized within the composite and the respective ligand or specific substrate, or other molecule of specific interaction (or analyte) , by contacting the referred combined composite with a sample containing one analyte. For example, one cycle of utilization will be one cycle of an enzymatic reaction when the immobilized material is an enzyme or co-enzyme, one cycle of reaction of formation of an immune-complex when the immobilized biological material is an antibody, an antigen or a hapten-protei.n -

[0030] One cycle of utilization will preferably include steps of conditioning and washing: of the combined composite before one next cycle. It might also include additional procedures for detection of the occurrence of the mentioned interact.ion/reaction .

[0031] The combined composite of the invention can hence be utilized in any application where will be utilized an active or activable biological material, more preferable when there will be utilized biomolecules in aqueous media, as for and without limited instance, the formation of immune-complexes in medical diagnosis, or the formation of products by biocatalysis with immobilized e zymes .

[0032] It has been widely recognized the vantages of biocatalysis relative to the conventional catalysis, that comes from the immobilization of enzymes and results in more catalytic efficiency, greater enzymatic stability, more selectivity of the substrates and minor costs of utilization by the less demanding thermal operational conditions and environmentally more friendly [15] .

[0033] By this manner, in an embodiment of the combined composite of the invention it can be utilized as support of enzymes, thus with enzymatic activity along with several reaction Further it 'will be presented an example, but not limitative, with alkaline phosphatase.

[003d ] In one of the preferred embodiment of the combined composite of this invention it will be utilized as support of immunologically active proteins, as for example antibodies, antigens or hapten-proteins, with activity in successive cycles of ligation to the respective ligands. So, in each cycle of utilization, it will be possible to detect the ligation of the specific ligands of those immunologically active proteins immobilized within the combined composite.

[0035] Being the, mentioned immunological1y active proteins immobilized in the combined composite, or the respective ligands, associated to specific pathologies, the combined composite can be utilized in diagnosis of such diseases through the detection of the specific ligands present in a biological sample- In preference the biological sample will be from a human being or superior animal, more preferably a biological fluid such as plasma/serum from blood, urine, supernatant from tissue macerate or any other aqueous fluid without cells obtained from an individual for medical diagnosis proposes. The disease to be diagnosed can be any pathology that trigger the presence of antigens and/or antibodies in fluids and/or tissues of a human being or superior animal.

[0036] So one utilization of this invented comhi.ned eompos i te is at medical diagnosis which constitutes one preferential utilization .

[0037] In more detail but without limitation, the utilization of invented combined composite in medical diagnosis follows the principals of immune-diagnosis in that one biological sample to which it is suspected to contain the analytes of interest, for example antibodies, antigens or other analytes, is provided to get in contact with respective specific ligands. These last ones will be antigens, antibodies or any other specific ligands immobilized in the combined composite whose porosity enable the permeation of 1015SPOT

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10

the mentioned analytes existent in the aqueous liquid samples, until they contact with the referred specific immobilized ligands. Ones and others, or both, the analyte or the respective specific ligand will be associated to a pathology.

[0038] So by contact of the biological sample to be analysed with the combined composite doped with a specific ligand for a certain analyte, and that analyte being present in the aqueous sample it will be formed the specific complex analyte-ligand, for example an immune-complex antigen-antibody, that can further be detected and so elucidating the conclusion of the presence or the absence of the analyte in the biological sample and consequently the affirmative or negative diagnostic of the associated disease.

[0039] The referred detection can be made by any adequate met od such as colouring or fluorescence recurring for instance labelled antibodies.

[0040] It will be further described an example of this embodiment in which the detection of the immune-complex (primary) formed after contact of a biological sample with the combined composite immobilizing an antigen (or antibody) is effected by a secondary labelled antibody (labelled with an enzyme e.g, peroxidase) that links specifically to the antibody (or antigen) of the biological sample consequently forming a secondary immune- complex. A further addition of a chromogenic substrate being degraded by the labelling enzyme of the secondary antibody provides a colourful appearance of the combined composite so that detection of the referred primary complex is accomplished,

[0041] The above mentioned detection can be validated by the absence of colour after applying the same biological sample at a same composite doped with a non- immunogenic protein.

[0042] Recycling; of the combined composite enables it to be used in successive cycles of utilization. The combined composite ll

of the invention is capable ot being reused at least for 20 cycles of utilization as already described and as farther demo: strated .

[0043] In one aspect of this embodiment the combined composite of the invention doubly immobilizing one immunologically active protein., will be used as filling an analytical-chamber designed for detection of a specific ligand of the referred immunologies1ly active protein (target-analyte) existent in a biological sample towhich it will be put in contact along in such analytical-chamber .

[0044] The filling profile of the analytical-chamber with comfoined composite ill be adjusted to tbe target-analyte and/or to the immobilized biological material, and. will be determined by preliminary essays for adjusting operational rheology. In the same way, the protocol of use of the analytical-chamber, dilution-rates of reagents and biological samples will also be determined by routine preliminary essays for meeting criteria of Sensibility and Specificity, in function of the target-analyte and/or immobilized biological material.

[0045] Thus it will be determined the least content of simple composite to be immobilized in the combined composite that will enable to detect the existence of the analyte in the biological sample (Sensibili y) and the greatest content; of simple composite to be immobilized in the combined composite that will evidence no cross response between different analytes (Specificity) . At the same time it will be determined the most adequate profile of granulometry gradient that will fulfil the analytical-chamber .

[0046] So it is conceivable a device that will include a series of the above described analytical-chambers, preferably until 8 chamber disposed vertically in parallel and supplied on top by a common liquid-collector in which the liquid samples will be placed. Each analytical-chamber dedicated to detect one distinct analyte will be sided by a similar chamber dedicated to negative-control test which will be filled exactly 'with the same profile of granulometry gradient of combined composite immobilizing the same content of simple composite but doped with a non-immunogenic protein .

[0047] The device will hence be constituted by pairs of chambers, analytical and respective negative control chambers, being each analytical-chamber filled with combined composite doubly immobilizing one differ*· t active or act.ivab.le biological material as described above, and sided by a negative-control chamber filled with combined composite doubly immobilising one non-Immunologle responsive biological material. Such device will enable to detect a number preferably until 4, different target- analytes in one unique biological sample and in each cycle of utilization .

[0048] Thus one preferred embodiment of this invention is a portable and easily usable device, with no requirements of electric energy supply and that will enable to diagnose affirmatively or negatively in each biological sample, the existence of a number of analytes preferably until 4 as much as the number of analytical chambers that will comprise the construct. In particular, being each of the mentioned analytes related to pathologies, from one unique sample of biological fluid of a human being or: superior animal to whom is suspected one specific pathology, this device will enable the differential diagnosis of until 4 pathologies. Additionally the device is reusable for at least 20 successive cycles of analytical utilization (diagnostic trials) .

[0049] This device can be presented in a kit format which beyond the device itself will include instructions of use and all the reagents required for the operation of diagnostic trials.

[0050] It will ahead be described some examples the preferred 'ways of putting in practice this invention and will a.lsc be reported several error-and-trial essays of the invention, 3.015 SPOT

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4. Description of the Figures [0051]

Figure 1: structural representation of an alkoxide, tetramethyl orthosilicate (TMOS) . igure 2: loss of mass at room exposition maturation of the simple composite .

Figure 3: loss of mass at room exposition maturation of the corabined composite .

Figure 4: production of p-nitrophenol catalysed by alkaline phosphatase immobilized in simple composite (hydrolysis of p~ nitrophenylphosphate in aqueous medium of phosphate buffer 100 mil pH-9,1) in relation to the mass of immobilized enzyme.

Figure 5: production of p-nitrophenol catalysed by alkaline phosphatase immobilized in combined composite (hydrolysis of p- nitrophenylphosphate in aqueous medium of phosphate buffer 100 raM pH=9, 1 ) in relation to the mass of immobilized enzyme.

Figure 6: dye concentration of samples collected at 3 minutes intervals by the elution of 13 fractions of 1,0 ml of distilled water. The experience started with the elution of 1,0 ml of Evan- blue aqueous solution at concentration of 1,4 mg/100 ml by permeating a granulometry gradient of combined composite on ascendant layers (1,35 g of granulometry (G) > 1,0 mm ³ ; 1,80 g of 1,0 mm ³ > G > 710 pm ³ ; central layer of 2, 70g 710 mki ³ > G > 212 pm ³ ; 1,60 g of 1,0 mm ³ > G > 710 pm ³ ). The combined composite fulfilled a 12 cm ³ reactor of 4 cmiengh *3 c midth *1 cm _heigh c dimens ions ,

Figure 7: dye concentration of samples collected at 30 seconds intervals by the elution of 12 fractions of 1,0 mi of distilled water, The experience started with the elution of 1,0 ml of Evan- blue aqueous solution at concentration of 1,4 mg/lGO ml by permeating a granulometry gradient of combined composite on ascendant layers (1,35 g of granulometry ( G) > 1,0 m ³ ; 1,80 g of 1,0 mm ³ > G > 710 pm ³ ; central layer of 2,70g 710 pm ³ > G > 300 um ^J ; 1,16 g of 1,0 mm ³ > G > 710 pm ³ ) . The combined composite fulfilled a 12 c ³ reactor ot 4 cmie _ngh ^x 3 crn _Width *1 citi _height dimensions ,

8: dye concentration of samples collected at 10 seconds intervals by the elution of 11 fractions of 1,0 ml of distilled water. The experience started with the elution of 1,0 ml of Evan- blue aqueous solution at concentration of 1,4 g/100 ml by permeating a granulometry gradient of combined composite on ascendant layers (1,35 g of granulometry ( G) > 1,0 mm ³ ; 1, 80 g of 1,0 Kira ³ > G > 710 pm ³ ; central layer of 2, 7Qg 710 p ³ > G > 500 pm ³ ; 0,97 g of 1,0 mm ³ > G > 710 pm ³ }, The combined composite fulfilled a 12 cm ³ reactor of 4 cmi _anah : 3 crrtwidth Cnihei ht dimensions , Figure dye concentration of samples collected at 90 seconds intervals by the elution of 15 fractions of 1,0 ml of distilled water. The experience started with the elution of 1,0 ml of Evan- blue aqueous solution at concentration of 1,4 mg/100 ml by- permeating a granulometry gradient of combined composite on ascendant layers (1,35 g of granulometry (G) > 1,0 mm ³ ; 1,80 g of 1,0 mm ³ > G > 710 pm ³ ; central layer of 2, 70g 500 pm ³ > G > 300 unn 1,90 a OJ 0 mm ³ > G > 710 pm ³ } , The combined composite fulfilled a 12 cm ³ reactor of 4 cmienqh *3 cr«idth *1 cmheight dimensions . Figure 10: haemoglobin concentration in fractions of 1,0 ml of distilled water collected after permeation of 1,0 ml human blood at dilution of 1:100 and 1:200 (diluted in phosphate buffer s line 10 mM pH ^™ 7 , 3 ) through a granulometry gradient of combined composite on ascendant layers of 1,35 g of granulometry (G) > 1,0 mm ³ ; 1,80 g of 1,0 man ³ > G > 710 pm ³ ; central layer 2, 70g of 710 pm ³ > G > 500 pm ³ ; 0,97 g of 1,0 mm ³ > G > 710 pm ³ . The combined composite fulfilled a 12 cm ³ reactor of 4 cmiengh *3 cm _Width *1 cniheig _ht dimensions . Figure 11: haemoglobin concentration in an initial fractions of 10,0 ml and sequent fractions of 1,0 mi of distilled water collected after permeation of 1,0 ml human blood at dilution of 1:100 (diluted in phosphate buffer saline 10 mM rί< 7 , 3 ) through a granulometry gradient of combined composite on ascendant layers of 1,35 g of granulometry (G) > 1,0 ram ³ ; 1,80 g of 1,0 mm ³ > G > 710 pm ³ ; central layer of 2, 70g 710 pm ³ > G > 500 p ³ ; 0,97 g of 1,0 ran ³ > G > 710 pm ³ . The combined composite fulfilled a 12 era ³ reactor of 4 emieogh *3 cruwi th *1 cruheight dimensions.

Figure 12: haemoglobin concentration in three fractions of 10,0 ml and one last fraction fractions of 1,0 ml of distilled water collected after permeatio of 1,0 ml human blood at different dilutions (diluted in phosphate buffer saline 10 raM pH=7 , 3 ) through a granulometry gradient of combined composite on ascendant layers of 1,35 g o granulometry (G) > 1,0 nan ³ ; 1,80 g of 1,0 ram ³ > G > 710 pm ³ ; central layer of 2, 7Qg 710 pm ³ > G > 500 pm ³ ; 0,97 g of 1,0 mm ³ > G > 710 pm ³ . The combined composite fulfilled a 12 cm ³ reactor of 4 crctienh *3 en ath *1 CTUheight dimensions.

Figure 13: example of calibration of concentrations of the alkaline phosphatase reaction product, p~n.ltrophenol .

Figure 14: reaction yields of hydrolysis of p-nitrophenylphosphate at concentration of 0,75 mM (i phosphate buffer 100 mid pH=9,l) added in fractions of 4,0 ml that permeated a granulometry gradient of combined composite doped with alkaline phosphatase at concentration of 0,43% (m/m). The gradient was posed in ascendant layers of 1,35 g of granulometry (G) > 1,0 mm ³ ; 1,80 g of 1,0 mm ³ > G > 710 pm ³ ; central layer 2,70g of 500 pm ³ > G > 300 pm ³ ; 1,90 g of 1,0 mm ³ > G > 710 pm ³ . The combined composite fulfilled a 12 cm ³ reactor of 4 cmie _ngh *3 c _Width *1 cmheight dimensions.

Figure 15: photograph of two chambers fulfilled with combined composite. The chamber on the left was filled with combined composite doped with albumin serum bovine and the chamber on the right was filled with combined composite doped with mucin. The image was obtained after an essay with supernatant of hybridoma- cells cu.11ure producing antibodies-ant imucin. Ligation of secondary antibodies was revealed by chromogenic pe oxidase substrate 3, 3 ^' , 5 , 5 ' -tetramethylben zidine .

Figure 16; photograph of the device prototype in which all the chamber are posed in parallel in a disposable construct, located underneath a common liquid-collector and above the bottom- collection-tray . xample 1 ~ Composite Formulations Exa ple 1.1, - Simple Composite

[0052] In the preparation of an indicative volume of 20 ml of precursors of simple composite _; it was followed the protocol described by A.1stein and co-workers [16] using as orthosilicate precursor 5,0 mi of tetramefchyl orthosilicate (TMOS) ; 4,84 ml of HC1 2,5 inM; 1,0 ml of polyethyleneglycol 0,4 KDa . The mixture was agitated in a vortex until liquid medium presented transparent. Homogenization was exothermic and the mixture was then sonicated for 30 minutes. It was previously solubilized 180 to 500 g of 1yophi1 ized protein, (e.g. enzyme, antigens / antibodies) in 10rn m1 HEPES buffer 50 mM pH ^::: 7,5.

[0053] It was added 10 ml of the sonicated mixture to the same volume of HEPES buffer solubilizing protein. This mixture was homogenized while gelification occurred for about 6 minutes. Solidified gel was then finely fragmented and spread onto a glass surface forming a thin layer for room exposure drying. At maturation there was a loss of about 84% of volumie mass; from the initial liquid volume of 20,0 ml of precursor it was approximately obtained 3,0 g of matured composite.

[0054] The simple composite was then grinded for a granulometry lesser than 106 gm ³ . The water content of the matured simple composite (a _w ) was around 11 ppm. The matured simple compos.it:e had a protein concentrat ion (mass of protein / mass of composite) among

[0055] For 1 une-essays it was also synthetized simple composite for negative control immobili ing bovine serum albumin with exactly the same mass o : antigens./antibodies as described above ,

1.2.1, Failures of immobilizing a composite

[0056] In tempt of sy.nthetiz.ing a combined composite it was followed the conventional protocol of sol-gel preparation trying to immobilize one simple composite previously synthetized instead of a free protein. Having in knowledge the proton availability required for chemical attack of the orthosilicate structure it 'was experimented a fraction of 50 mΐ of HC1 100 raM at pH-7,0 buffered media with different volumes of water for hydrolysis of 1,5 ml TMOS in order to obtain a hydrophilic composite compatible with the afore describe simple composite.

[0057] It was found a water deficit in the reacting medium proportional to the amount of mass of simple composite despite having linearly followed the initial proportion mass-of-simple- composite versus volume-of-precursors . Consequently there was a complete failure in TMOS hydrolysis. Table 1 presents a compilation of the experiences which elucidate the difficulties of immobilizing a simple composite in a second sol-gel. Table 1: experiences in tempting to immobilize a simple composite in sol-gel .

1.2.2, Practiced Formulation of the Combined Composite [0058] The synthesis was effected with concentration of simple composite since 0,83% until 3,34% (ra/v) . In reference to a unit precursors volume of 3,0 ml it was dosed 25 g to 100 mg of simple composite at granulometry lesser than 106 urn ³ and with water content (a _w ) of circa 11 ppm. That simple composite was suspended in 1,5 ml of phosphate buffer (PB) that was prepared with 38 M of N zHP04.2H ₂ 0; 62 mM of KH2PO4. PB was further adjusted pH === 6,8 ±

0,2 at 25 °C temperature (adding 1,0 M NaOH aliquots).

[0059] It was prepa ed an HC1 solution whose concentration depended on higrophilia of the simple composite and conditioned by l

respective immobilized protein. For instance but not limited, for alkaline phosphatase [HC1] = 4,5 mM ; for mucin [HC1] = 8,3 mM.

[0060] It was mixed 540 mΐ of distilled 'water with 50 mΐ of HC1 solution and then added tol, 5 ml of TM03, The mixture was homogenized during the initial 120 seconds and after 3 minutes 1,5 ml of that mixture was transferred to the same volume of PB suspension of simple composite. Gelification occurred in 10 to 15 seconds during which the media was homogenized in order to assure a homogeneous distribution of simple composite granules.

[0061] Polymerization went for 30 to 90 minutes after which the solidified gel was fragmented at granules of approximate volumetric dimensions less than 2 ram ³ and between 2 mm ³ and 4 mm ³ .

[0062] At immobilization of antigens/antibodies the solidified gel was incubated in 400 ^; rpm orbital acjitation during 24 hours in bovine serum albumin (BSA) at 2,0% (ra/v) . The solvent was phosphate buffer saline 10 mM pH=7, 3 1 0,3 prepared with 7,6 mM NazHPCb , 2¾0; 2,4 mM KH ₂ PO ₄ ; 137 mM NaCl ; 2,7 M KC1. The incubating volume was in a proportion of three volumes of the solution to one volume of solidified gel and after incubation it was transferred onto a clean-dry glass surface. From there on cjlass surfaces were changed for three times : at 24 hours, 48h and 72b of maturation.

[0063] The maturation of the combined composites either or not incubated in BSA, ran for seven days by exposure to room conditions after which there was a loss of volumetric mass of about 70% being the final water content a» ^s: 19 ppm.

: 0064 _.i The protein concentration of the combined composite matured after BSA incubation was arnona 3 , 0 to 5 times the concent ation found at the simple composite and without BSA incubation was in the range of 0,2 to 0,4.

[0065] After maturation the granule-size (G) separation was made and grouped in five classes:

Gi 96 [b) > 1 , 0 mm ³ ;

Example 2 Ti a of Maturation

[0066] It was studied the loss of mass of the composites, simple and combined, along the exposure to room conditions- That monitoring was made from the moment of gelification until the assessed mass values variated less than 2,0%,

[0067] For both composites, samples of similar masses and with two orders-of-magnitude of mass were monitored in order to verify the maturation dependence from the volume of precursors {initial mass) : they were studied samples of 1,5 and 3,0 ml, Figures 2 and 3 illustrate the collected data of simple and combined composites respectively .

[0068] From the obta i ned results it was inferred 72 and 50 hours the times of stabilizer ion of mass-loss respectively for simple and combined composites, Consequently it was established 7 days as maturation interval for both formulation at the end of which the ater contents ere :

1 Simple composite a _w = 11 ± 0,2 ppm;

2. Combined composite a _w -· 19 ± 0,6 ppm.

Example 3 Leaching Studies

3.1. Protein quantification method used to assess protein

[0069] Determination of protein concentration was made by modified Lowry method [17] However the initial procedures did not guaranty a zero-mass-balance among .initially existent protein in the composite samples and the protein transferred to the supernatant resulting from thermal-alkaline digestion (composite- sample submerged in 1,0 M NaOH during 10 min at 100 °C followed by ice incubation) .

[0070] Methodology used herein was optimised in order to assure that all composite-samples were digested and inherent protein content was transferred to NaOH solution: mass of composite samples not bigger than 5,0 mg with granulometry lesser than 106 gird. From each digestion medium 200 mΐ fractions were taken to be analysed.

[0071] Calibration of protein concentrations was made with BSA standard-solution {100% purity) at concentrations from 20 to 200 pg/mi: from each concentration 200 pi fractions were used to calibrate. Lowry-reagent was added (1,0 ml) to analysis- supernatant and BSA-standard solutions, and after 40 minutes 200 mΐ of Folin-Ciocaiteau reagent (diluted 1:4) was added. After 10 min. of incubation liquid samples were absorbance read at 750 nm wavelength having samples been diluted when recorded values were higher than 1,0.

[0072] It was firstly tested the leaching with samples of simple composite immobilizing three sorts of protein; bovine serum albumin (BSA) , alkaline phosphatase (ALP) and generic antibodies (IgG) .

[0073] The protein loads herein defined, were masses of lyophilized reagents added relatively to total volume of precursors still in sol-gel preparation. It was intended to obtain two groups of composites with different protein order-of- magnitudes of concentrations {1,0% and 10,0%) of BSA and ALP and one third typology with immobilized antibodies (IgG) . So composite losses of protein were monitored in t iple-essays in reference to different protein concentrations and using different proteins types , [0074] The quantifications of: protein contents retained in the composites were made before leaching (matured and dried composites) and after leaching essays at which the respective tested samples were 24h dried at 40 °C followed by 10 days room exposure .

[0075] Having in mind protein leaching process would be proportional to area/volume ratio of grains two granulometries were tested: above 1 mm ³ and under 750 mpi ³ ,

[0076] At first instance ¹ mass samples of 30 mg were used and experiences were performed by incubating matured fragmented composites in distilled water under orbital agitation of 200 rprn during 72 hours.

[0077] The incubation liquid volume was 1,0 ml in vials of 10 ml and at the end of each essay, the liquid media was decanted and centrifuged for 11 K rprn during 10 min. From the clarified supernatants three fraction of 200 mΐ were analysed in three separated quantification episodes to determine protein concentration in supernatants.

[0078] The procedures to determine after-leaching protein still retained in the composites followed the above described protocol of 5,0 ml sample-mass to ensure complete digestion of sol-gel and thus extensive release of immobilized protein to the analytical supernatant. Again three fraction of 200 mΐ were analysed in three separated protein concentration quantification episodes.

[0079] From the attained data mass-balance was made to quantify the losses of protein relative to the initial concentrations accordingly determined. On that view differentials 'were calculated among protein concentrations of the composites before arid after leaching experiences. Additionally it was compared the amount of existent protein in the total mass of composite-samples (using previously mentioned data) before leaching and the total amount of protein in the volume of 1,0 mi incubating medium, Recorded data were averaged and respective numbers are presented at table 2.

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

[0080] Those results confirmed a somehow erosion of retained protein being much relevant as smaller the grain size: concentrations recorded at composites after leaching are as closer to the initial loads as bigger the essayed grain-size.

[0081] On the other hand with samples of lower granulometry there was a smaller difference between initial loads of composites and mass of protein solubilized in supernatant which is symptomatic of more transference of protein to the leaching media. The conjunct of results for the different protein types retained in simple composite and respective different loads confirm the leaching phenomena is proportional not only to the initial protein loads but to the grain-si e as e11.

[0082] The recorded data regarding XgG confirm results mentioned by Alstein and co-workers [16] of an elevated retention of antibodies in this composite in which the difference before and after leaching is at the same order-of-magnitude for both granulometries .

[0083] Additionally it was tested the leachincj of 5 mg and 60 mg mass-samples of simple composite doped with ALP at the maximum protein loads and minor granulometry at the same experimental conditions. The obtained numbers are presented at table 3 in which they are comparable with 30 mg mass-samples .

Table 3: protein concentrations obtained in leaching test of simple composite doped with alkaline phosphatase at the same concentration _; but with samples of different mass.

[0084] The view of these figures permits to conclude the loss of retained protein was influenced by the spread-of-grain in the liquid media: for the same batch volume the differences of protein concentrations retained in the composite before and after leaching are as higher as the lower mass of the essayed samples,

[0085] On the other hand with greater-mass samples it is apparent a wider difference among initial protein loads of the composites and protein concentrations at leaching supernatants that means a lesser transference of protein into the incubation media comparing to the smaller-mass samples.

[0086] The effect of minor protein erosion with greater mass of simple composite for the same reaction-volume permits to deduce the more the simple composite -will be used in the same batch- reactor volume the less protein total-loss will occur and consequently more stable will be the bioprocess activity of the doped simp1e composite .

3.2.;

[0087] Having in mind the comparison with quantified protein loss recorded in simple composite the same experimental procedures were followed with combined composite immobilizing simple composite doped with the same protein which would further permit to monitor also the enzyme-activity response; alkaline- phosphatase .

[0088] It was synthesized combined composite immobilizing simple composite with the highest protein load (43,11 gg/'reg) to obtain two groups of samples with different protein concentrations: 8,81 gg/mg and 23,14 gg/mg. Fragmented samples of 30 mg were essayed at small grain-size similar to previous experiments . Data treatment and experimental procedures were the same as those followed with simple composite leaching monitoring and table 4 presents the obtained results.

Table 4: protein concentrations obtained in leaching test of combined composite prepared from different masses of simple composite doped with alkaline phosphatase .

[0089] The combined composite with the initial highest protein concentration presented differential values before and after leaching of circa ten times lower than the simple composite (0,78 pg/mg versus 3,94 ug/mg) having a protein concentration about twice lower than the simple composite (23/14 gcj/irtg versus 3 , 11 mg /mg } .

[0090] Likewise leaching essays using 5 mg and 60 mg mass- samples of combined composite were performed with low granulometry. Table 5 presents the obtained results. Table S: protein concentrations obtained in leaching test of combined composite doped with alkaline phosphatase at concentration of 23/14 gg/mg but with samples of different mass.

[0091] The view of the figures confirm that trace-loss of protein is inversely proportional to the quantity of combined composite in the same liquid volume.

[0092] The last obtained results compared with simple composite results turn out to be evident the smaller loss of protein in combined composite: differences at protein retained in the composite before and after leaching. At the same time wider differences were recorded at combined composite comparing initial 1Q156FCT

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immobilized protein and protein content in supernatants which ref1ects greater retent ion g rades .

[0093] Simple and combined composite samples with the highest protein concentrations and smaller granulometries were exposed to high saline concentration solution 2,0 M NaCl in order to assess the loss of protein at a high ionic stress medium.

[0094] For both composites 60 g mass-samples were tested by submergincj 72 hours at 200 rpm orbital agitation, and protein quantification of leached composites and respective supernatants followed the same previous protocols, Table 6 presents the obtained data .

Table 6: protein concentrations obtained in leaching test of simple and combined composites doped with alkaline phosphatase and submerged in a solution of 2,0 M NaCl,

[0095] The recorded values regarding the difference between retained protein concentrations in composites before and after exposure to saline solution are in the same order-of-magni tude of those recorded by exposure to distilled water. [0096] Comparing numbers of mass-protein within initial composites samples and mass-protein at supernatants at the end of the experiments a smaller difference is apparent at this case which could be symptomatic of a more intense transference of protein to the incubation media as reflex of the high ionic concentration.

[0097] These figures must however be seen as leaching solution was at an over-elevated saline concentration comparative to predicted usable physiological samples and time of exposure was considerable longer than it will be used at the preferable embodiment .

Example 4 ~ Catalytic Essays

[0098] Having previously been verified that the invented formulation of combined composite has the capacity to ircmobilize/retain proteins, it was then intended to verify if one protein being an enzyme, is still active, In that context the activity of alkaline phosphatase (ALP; Sigma Aldrich Gat. N° 10 567 752 001) was monitored firstly as free and then as immobilized enzyme within simple and combine composites.

[0099] Enzymatic essays recurred to spectrophotoirtetric method to quantify the concentration of p-n11rophenoi (pNP) as product of the standard-reaction of p-nitrophenyipnosphate (pNPP) hydrolysis having in knowledge the respective 1:1 stoichiometry.

4.1, Enzyme immobilized in the simple composite

[0100] After conventional free enzyme essays two samples of simple composite immobilizing ALP at precursors concentrations

(m/v) of 8% and 10% were synthetized. At the end of 13 maturation days concentrations were respectively 4,8 and 6,2 mgprotein /gcomposite.

[0101] Having in mind to reduce external access limitation of substrates molecules, the immobi1izing matured composite samples were fragmented to grain-size at the range of 1 to 2 mm ³ . The essays were performed with simple composite samples of mass from 5,0 to 8,3 g and enzyme concentrations in batch-volumes were deduced from protein concentration of respective composites and correspondent mass of the samples.

[01.02] Enzymatic essays were performed in 10 ml useful-volume reactors to which 3,6 ml of same buffer solution used in free- enzy e trials (phosphate buffer 100 mM pH=9, 1) , was added at room temperature. Kinetic studies started at the moment 0,4 ml of substrate solution (pNPP, solubilized at pH-9,1 buffer) was added and reactions ran for 28 minutes being collected 200 mΐ of liquid medium at 1:30 min, intervals, Initial substrate concentrations at the total reaction volumes were identical and around 3,0 mM. Data treatment had i account that along 28 min. time the reaction medium volume was progressively reduced by the collected samples but the mass of catalytic composite was the same.

[0103] The recorded values made possible to graph the kinetic profiles in function of enzyme loads as illustrated i Figure 4.

[0104] The obtained results turned evident that product concentrations were proportional to the mass of composite (with im obili zed enzyme) for one same initial substrate concentration.

4,2. Combined composite immobilizing simple composite doped with enzyme

[0105] It was studied combined composite from the same formulation used at leaching tests {protein concentration of 23,14 pg/ml) and samples of mass from 14 to 20 g were tested.

[0106] Grain-size of the samples, enzymatic essays experimental procedures and data treatment were exactly the same as those followed at simple composite kinetic studies. Similarly the quantification of existent enzyme in reaction media was made from the previously determined protein concentration of combined composite and the mass of samples used for each essay. Obtained resulted are presented in Figure 5.

30

[0107] Comparing maximum production of pNP, product of pNPP hydrolysis catalysed by the same enzyme immobilized in the two composite formulation it is evident that for identical masses of enzyme {li,65 riig.s-i/spie-cotBp site 10;8 Xti simple-coxposice ve sus 12 gcombined- composite?; 11 Ttxcji combined-composi te ) and at a similar time reaction (15 min.) it. was produced 8 to 9 times more of pNP with simple composite (40,57 mM; 26, 76 mM) than with combined composite (5, 03 M; 3,03 mM) ,

[0108] Having in mind that combined composite of this invention is a coating of an enzyme-doped simple composite (encapsulated in a grain-size of compromise with robustness and external access of substrates) it is comprehensive that enzyme active-sites are less available and _, consequently, there is a lower production of enzymatic metabolite. However the lower catalytic efficiency is compensated by a more sustained retention of immobilized protein as early demonstrated by leaching studies.

[0109] It will now be presented the hydraulic tests performed with an aqueous dye solution and diluted blood permeating grained combined composite, in order to obtain residence-times compatible with utilization in a medical diagnostic device,

[0110] The combined composite herein tested was used as a filling-bed of a column also named analysis-chamber or reactor accordingly used at immune-essays or enzymatic tests. It was a rectangular box with dimensions of A emiangch ^x 3 cirwidtft ^x 1 cmdepeh made of transparent acrylic material that allowed o visualize the interior. The handling of its content was made by a drilled removable top-cap that once set. at the box enabled elution of liquids through its interior. The surface of the opposite top was drilled as well to allow the exit of the liquids and the holes of both top-caps were 1,0 m diameter, [0111] Having in reference the useful mass value of 9 grams assessed by weighting the complete filling of water of the column ( oiumic-mass of Ig/ i) it was planned to fill the column with grained composite. Such amount of mass was over-dimensioned relative to the application on diacjnostic-device apparatus being estimated a final scale-down of 3:1. The aim of those studies was to test the drainage regime of liquid samples and thus grain-size was though as unique conditioning variable smd accordingly it was used combined composite with same maturation grade.

[0112] Granulometry gradient profile was initially programmed to completely fill the useful volume and respective values were:

• Bottom layer: 15% (1, 35g) <£> grain (G) > 1,0 mm ³ ;

• Bottom intermedium layer: 20% {1, 80g) <> 1,0 mm ³ > G > 710 mhi ³ ;

* Central layer: 3 _, 0% {2, /0g) <3 variable granulometry under 710 mth ³ ;

• Top intermedium layer: 20% (1, 80g) 1, 0 mm ³ > G > 710 urn ³ ?

• Top layer: 15% {1, 35g) <3 grain > 1,0 mm ³ .

5.1. Monitoring drainage of dye solution

[0113] After grain-size separation the two bottom layers were posed accordingly and the central layer was composed of grain-size ranged from 710 pm ³ to 212 am ³ . Immediate above layer was tried to be placed as programmed but only 1,6 g was able to be posed. The filling process was complied with washing with distilled water after posing each layer to improve grain compaction and reduce preferential run-off ways. Column was then left exposed 24h to 40 °C dry-heat,

[0114] The essay started by measuring the volume o1 : water saturating the column-content (adding water to the dry grained composite content until first drops came up at the bottom) useful liquid volume (ULV) : 3,0 mi was measured. The propose was tc> <¾ S SQss I0156FCT

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the volume of retained water at such gradient profile. After- saturation it was evident that at each added millilitre corresponded one millilitre drained at the bottom of the column, which proved that intending to incubate the whole content of the column, it should be added a liquid volume equal to ULV.

[0115] Additionally it was monitored the run-off time elapsed by each millilitre added until at least 900 u.l were collected and such measuring was made for 25 trials at which 3 minutes was the average recorded times. Column was then left 24h exposed to 40 °C dry-heat. Composite grain-gradient was again water saturated and one 1,0 mi fraction of Evans-blue (1,4 mg/100 ml) was eluted,

[0116] Absorbance calibration (l ^:::: 608 nm) of Evans-blue solution was made {since 1:1 = 1,426 ± 6%; until 1:10 ^::: 0,134 i 10% ). Drainage flow of dye-solution was monitored by spectrophotometric readings of successive eluted/collected fractions of 1,0 ml distilled water and respective recorded values are presented in Figure 6.

[0117] After removing wet grained composite new dry grained composite was placed in the column following the same procedures except that central layer was composed by grain ranged from 710 pm ³ to 300 pm ³ and the upper layer was once again exclusively composed by grain 1,0 mm ³ > G > 710 pm ³ but this time took 1,16 g of grained composite. It was monitored the run-off time elapsed by each millilitre added until at least. 500 pi was collected and such measuring was made for 30 trials at which 25 seconds was the average recorded time,

[0118] Column was then left 24h exposed to 40 °C dry-heat. It was further assessed the volume of retained liquid (1,9 ml) and replicated the essay of dye-solution drainage. Collected data are presented at Figure 7,

[0119] After removing wet grained composite new dry grained composite was placed in the column following the same procedures except that central layer was composed by grain ranged from 710 pm ³ to 500 pm ³ and the upper layer was once again exclusively composed by grained composite 1,0 ram ³ > G > 710 pm ³ but this time took 0,98 g. It was monitored the run-off time elapsed by each millilitre added until at least 900 pi was collected and such monitoring was made for 25 trials at which 10 seconds was the average recorded time. Column was then left 24h exposed to 40 °C dry-heat. It was further assessed the volume of retained liquid (1,0 ml) and replicated the essay of dye-solution drainage. Collected data are presented at Figure 8,

[0120] After removing wet grained composite new dry grained composite was placed in the column following the same procedures except that central ^: layer was composed by grain ranged from 500 pm ³ to 300 urn ³ and the upper layer was once again exclusively composed by grained composite 1,0 mxa ³ > G > 710 pm ³ but this time took 1,90 g. It 'was monitored the run-off time elapsed by each millilitre added until at least 900 pi was collected and such monitoring was made for 25 trials at which 1:30 minutes was the average recorded time ,

[0121] Coliimn was then left 24h exposed to 40 °C dry-heat was further assessed the volume of retained liquid (2,0 ml) and replicated the essay of dye-solution drainage , Collected data are presented at Figure 9,

[0122] All of these results indicate that for one same volume occupied by this composite and permeated by 'water, the amount of retained liquid and the run-off times were conditioned by the smailest grain-size laye .

[0123] Table 7 illustrate these instances where values of granulometry and respective masses of bottom and bottom- intermedium layers were maintained, At. same time variating granulometry of central layers but keeping its mass-content, resulted in variation of residence-time of permeating liquid. Table 7; water drainage values with different grain-sizes of 2, Iq central-layer of combined composite, which also affected the useful volume of retained liquid and total mass held in the column.

[0124] The drainage times of 1,0 ml of dye~solution for identical grandometry interva1s at the central layer :

1. in the range of about 200 pm ³ : 90 seconds (500 mih ³ > G > 300 pm ³ ) that compares with 10 seconds (710 p.m ³ > G > 500 pm ³ ) ; 2. for a wider range: 180 seconds (710 um ³ > G > 212 pm. ³ } that compares with 30 seconds (710 urn ³ > G > 300 pm ³ ) .

[0125] The drainage volumes for an identical granulometry intervals : a. in a range of about 200 pm ³ : 15 ml (500 pm ³ > G > 300 pm ³ } that compares with 12 ml (710 pm ³ > G > 500 pm ³ }; b. for a wider range: 13 ml (710 pm ³ > G > 212 pm ³ } that compares with 12 ml (710 pm ³ > G > 300 um ³ } ,

[0126] In summary in a compacted layer of combined composite of grain-size under 710 pm ³ the permeation volume of an aqueous sample is proportional to the magnitude of the interval of grain-sizes and inversely proportional to the respective drainage times. [0127] It was also evident that composite total mass within the column was inversely proportional to grain-size. Such finding ^• would be expectable in face of compaction (as bigger as the smaller the grain) and consequently less inter-particles space that enables higher densities.

[0128] Based on granulometry gradient found as better flowing recrime of dye-solution (central layer 710 mai ³ > G > 500 pm ³ ) it was tested drainage of diluted blood. Samples collected from the inventor were anticoagulated with EOTA (50 mg/ml) and diiuted with phosphate buffer saline 10 ¹ mM pH=7 , 3. Diluted samples of 1,0 ml were eluted followed by fractions of 1,0 ml of distilled water and run-offs were assessed by reading collected fraction at absorbance of 540 nm wavelength which targets haemoglobin (Jib) molecule.

Calibration was made for ¹ Jib concentrations from 0,6 mg ./ml (absorbance = 0,314 ± 7%) to 3,0 g/mi (absorbance == 1,635 ± 4%) . Sampled blood Ho concentration was 150 mg/ml (± 1,0%) . Results recorded from three trial are presented at Figure 10.

[0129] Before these results it is evident that diluted blood run-off occurred mainly after 4,0 ml of permeating water which agrees with previous data of dye-solution drainage. So it can be concluded that permeation of blood samples at dilutions herein referred was identical to run-offs recorded with Evans-blue solution concentrated at 1,4 mg/lOOml and those are two rheological identical liquid media.

[0130] Thus it seems reasonable to infer that drainage of 1:200 and 1:100 blood samples will have the same drainage regime at others gradient-grain of combined composite which enable to preview a scenario of good access to immobilized proteins (antigens/antibcdies) by biological-samp1e fIuids w11h v.1 scos ity and density similar to water. 5.2,1. Monitoring fch.© washin drainage of blood

[0131] According to the recorded run-off profiles either with Evans-blue or with blood solutions it was studied the washing drainage of the combined composite gradient-granulometries firstly permeated by blood and then using distilled water in a first fraction of 10,0 ml followed by five sequent elutions of 1,0 ml. At the series of three essays performed with 1:100 diluted blood it was collected 96 i 0,5% of added Mb right at the first fraction of 10,0 ml as illustrated in Figure 11.

[0132] Based on those results it was then studied the washing of the grained composite using lesser diluted samples of blood down to 1:30. The protocol in between every experimental episode, the grained content 6f the column was abundantly washed with distilled water and then 35 °C dried for 24 hours. Previous to addition of a new blood-sample the grained composite was saturated with 20,0 ml of disti .lied water .

[0133] It wats monitored the clearance of the collected fractions at elution of three fractions of 10,0 ml of distilled water and one last of 1,0 ml and the obtained results are presented at Figure 12.

[0134] From the collected data it is evident that even for the most concentrated blood-samples the drainage of 1,0 ml analytical blood-unit- olume occurred mostly with elution of 10,0 ml water being that in third and fourth collected fractions, the nb concentrations were lower than 1,0 mg/ml.

[0135] Having in mind these tests were performed with a total column-mass-filling of 6, 82 g and the scale of these tests was also approximately 3:1 then at the final utilization scale the estimated mass of combined composite for the same gradient (relative proportions} will range among 1,5 g to 3,0 g. At this view and in a linear scaling of granulometry gradien s herein studied, it is reasonable to estimate an effective washing volume of 10 ml.

Examples b " Combined Composite in z matic Reactor

[0136] At this example it was studied the enzymatic activity for at least 20 cycles of activity of the combined composite filling afore described acrylic column and supplied by fractions of substrate-solution that permeated the grained-composite pushed by atmospheric pressure in a vertical plug-flow regime . The propose was to quantify the reaction-yield of conversion of p- nitrophenylphosphate ^: (pNPP) into p-nitrophenol (pNP) catalysed by alkaline phosphatase immobilized in combined composite.

[0137] It was used the enzyme concentration threshold of correspondence among specific activity and load-of-enzyme : 0,4 % (m/m) [18] [19] . It was used the grain-size gradient previously found as the best run-off for enzymatic process: residence time of at least 1,5 minutes (central layers granulometry 500 pm ³ > G > 300 urn ³ ) . Total amount of combined composite mass was 7,75 g and its compacting was effected to minimize preferential run-off 'ways. The volume of retained liquid was 2,0 ml.

[0138] After filling the column the experience started with abundant wash of the grained gradient composite at room temperature with the same eluent previously used in similar catalytic experience (phosphate buffer 100 mm pH = 9, 1) and herein used as eluent too. It was then added 4,0 ml of substrate solution (solubilized in pH = 9,1) and after 15 minutes incubation the grained composite was permeated by three fractions of 2,0 ml of eluent followed by eight fractions of 1,0 ml. Quantification of metabolite concentration was direct from absorbance readings once the enzymatic reaction product was collected at one same volume added at respective elution fraction (contrarily to the batch process early described) . [0139] Data treatment started by calibration of concentrations of the product (pNP) for absorbance values (l~405 nra) under 1,0: up to 56 mM (see the example Figure 13),

[0140] AbS405 _nre values attained in each of the 11 collected fractions were converted to pNP concentration . Having in known the volume of each collected fraction it was computed the number of moles existent in each collected fraction. It was summed the number of collected moles of reaction product , Additionally knowing the substrate (pNPP) concentration it was computed the respective number of moles initially supplied based of the solution-volume fed to the reactor (see table 8) .

Table 8: data treatment of the first experiment results at bioreactor of _: combined · composite immobilizing alkaline phosphatase', on hydrolysis of p-nitrophenylphosphate .

[0141] Knowing this reaction is 1:1 stoichiometry percentage conversion yields were calculated: product number of moles * 100 / substrate number of moles. At the first trial it was used a substrate concentration of 313 pM and the recorded yield was 1,4 % . Next trial used a substrate solution one order-of-magnitude higher: 3, 03 M and the yield was 19,5 % .

[0142] The third trial kept substrate concentration and prolonged incubation period for 20 minutes. The recorded yield was 25,7 % . From that result it was inferred longer incubation time allowed a more extensive hydrolysis of the added substrate. Fourth trial was a replication of the third and the recorded yield was 24,1 % .

[0143] At the fifth trial substrate concentration wa; reduced to ½ a.nd the recorded yield _' was 23,1%, and at the next t ial with the sa.me cbndit.ions the value recorded 'was 29,7%,

[0144] At seventh trial substrate concentration was reduced to ¼ : 750 pM, The recorded yi.e1 d was 36,4 % . That last tra i1 was replicated for twice and : ecorded yields were: 38,9% and 35,6%.

[0145] At one next experimental episode three trial were performed replicating the last protocol and recorded yields were: 32,9% ; 28,8% and 28,2%. At one new series of five trials the recorded yie1ds were: 25,4% ; 21,9%; 27,5%; 27,8% and 25,3%. At one other experimental episode of three trial the recorded yields were: 16,6% ; 19,0% and 18,9%,

[0146] At the two following trails the recorded yields were: 21,7% and 21,1%, At one last experimental episode nine trial -were performed and the recorded yields were: 18,2% ; 16,0% ; 15,5% ; 16,7% ; 16,3% ; 14,6% ; 13,9% ; 14,0% and 19,0%.

[0147] Compilation of those mentioned figures is presented at Figure 14 for the normalized procedures established after the seventh trial in which it was used a 750 pM substrate concentration and 20 minutes incubation time. The. graphic shows recorded yields in reference to the value obtained at second experiment of that series (38,9%) that holds as maximum value: 100%.

[0148] Before these results it is reasonable to conclude that conibined composite is consistently applicable at immobilization of an enzyme, alkaline phosphatase, while retciining its activity, Having additionally in mind that exoeri enta 1 normalization was attained after seven essays of procedure adjustments it becomes reasonable t preview the tilisation of t is composite formulation f >r a wider number of cycles of more tha those 20 initially pointed.

Example 7 ~ Preferred Utilisation of Combined Composite at

Diagnostic Device

[0149] Now it will be described one preferential utilization of the invented combined composite applied to medical diagnosis onto a portable device constructed as a conjunct of vertical parallel operating units, it complies the maximum of 4 units and each unit includes two chambers, one analytical and one negative control chamber .

[0150] All the chamber are filled with grained combined composite in which the analytical one is filled with combined composite immobilizing an immunological1y responsive protein, an antigen or an antibody associated with the diagnosis of a human being or superior animal pathology and. the negative-control chamber is filled with combined composite immobilizing an ίmmanalogically non-responsive protein.

[0151] The functioning of the device is based on immune- diagnosis principles where a biological sample suspected to carry antibodies (or antigens) related to a pathology, by operating the device those molecules will ligate to respective specific ligands. The latter will be antigens (or antibodies) immobilized in the combined composites whose physical characteristics of pore-size enable the permeation of those liquid biological samples. After antigen-antibody ligation a primary immune-complex will be formed and the composite must be washed to remove the excess of debris and non-ligated proteins,

[0152] At the first instance of this example it was preliminary tested the response of the combined composite immobilizing a simple composite doped with mucin [201 1,8% (ra/m) . The combined composite was used to fulfil a test-column homologous to one device-unit. The test-column was filled with grained composite with granulometry gradient-profile analogous to blood drainage tests.

[0153 The immobilized antigen mucin is a molecule with giycidic structure of sialic-acids homologous to surface-ligands of tumour cells (e.g, breast cancer) . It was tested the formation of primary complex by ligation of an antibody-reagent kindly supplied by Glycoim nology-Group of Science and Technology Faculty of Universid.ade Nova de Lisboa, Such antibodies had previously demonstrated a high ligation affinity to neoplasia tissues [21] , The biologic samples at those tests were the supernatants from the culture of antibody-producer animal cells

( h bridoma-ce11s ) .

[G154 ] After incubation of biological sample, the grained composite was washed to remove the excess of non-ligated antibodies, The detection of the formed immune-complexes was made by addition of a secondary antibody labelled with peroxidase. Such antibody had specific affinity to the referred primary antibody. The addition of the secondary antibody provided the formation of a secondary immune-complex : antigen imtobiuzsd ~ antibody bi oiogi cai- i ne grainec composite was washed once again to remove the excess of non-ligated proteins .

[0155] It was then added the chromogenic substrate of peroxidase: .3 , 3 ' , 5 , 5 " -tetramethy1-benzidine , The substrate was degraded by the secondary antibody and consequently conferred blue-green colouring to the combined composite thus revealing the existence of the primary’- antibodies in the biological sample . The procedu e was validated by the similar test performed with homologous grained combined composite doped with the non- immunogenic : protein bovine serum albumin, at which the combined composite c iid not gain any colour as illustrated by Figure 15, [0156] Recycling the grained combined compos .te enabled the respective re-utilization and that procedure was made by elution of a chaotrcpic solution that disrupted the ligations of primary and secondary antibodies- Grained combined composites were then washed to remove all the released proteins. 7.1, Preliminary

[0157] At the preferential utilization of the invented combined composite in medical diagnosis preliminary essays must be performed in order to test Sensitivity and Specificity criteria.

[0158] Samples of combined composite immobilizing one same simple composite with a unique concentration of antigen / antibody / 8SA will be tested. Protocol includes 8 essay-via.Is having each vial a sample of 100 mg of grained composite with granulometry range of 100 pm ³ to 300 pm ³ : i) four essay-vials in which one of each vial will have a sample of grained combined composite immobilizing respectively 25; 50; 75; 100 mg of simple composite with BSA. Those masses of simple composite are relative to 3,0 ml of precursors when combined composite was synthetized. ii ) four essay-vials in which one of each vial will have a sample of grained combined composite immobilizing respectively 25; 50; 75; 100 g of simple composite with antigen / antibody. Those masses of simple composite are relative to 3,0 ml of precursors when combined composite will be synthetized. 7 1,1. Reaction solutions

[0159] Phosphate Buffer Saline 10 raM pH=7 , 3 ± 0,3 (25 °C ) +

0,05% (m/v) Tween-20 (PBS-T) used at:

® Dilution of the biological samples; « Dilution of the secondary labelled antibody;

• Washing the grained combined composite.

[0160] Chro ogenic peroxidase substrate: 3, 3 ^" , 5, 5 ^' -tetramethy benzidine (TMB) used under dilution with distilled water.

[0161] Recycling solution Restore ^® Western Blot Stripping Buffer; Thermo Scientific (Sb) used under dilution with distilled water.

Step 1: Conditioning of the composite.

Grained combined composite will be humidified with 2* 2,5 ml PBS-T: Incubation time: 5 min. Liquid medium will then be decanted.

Step 2: Elution of the biological sample.

Biological liquid sample will initially be diluted (in PBS- T) at 1:10 for the respective volume of 2,5 ml. It is intended to obtain a result of unequivocal colouring of the composite doped with antigen (or antibody) and unequivocal clearance of the composite doped with BSA and so dilution grade of biological samples will be optimized since 1 : 5 to 1:13. Incubation time : 20 min. Liquid medium will then be decanted. Step First wash with 2* 2,5 ml PBS-T. Liquid medium will then be decanted. Step 4: Elution of the secondary antibody.

Secondary antibody labelled with peroxidase will initially be diluted (in PBS-T) at 1:3333 for the respective volume of 2,5 ml. It is intended to obtain a result of unequivocal colouring of the composite doped with antigen (or antibody) and unequivocal clearance of the composite doped with BSA and so dilution grade will be optimized since 1:2000 to 1:5000. Incubation time: 5 min. Liquid medium will then be decanted,

Step 5: Second wash with 4* 2,5 mi PBS-T. Liquid medium will then be decanted,

Step 6 : Elution of the chromogenic substrate.

Peroxidase chromogenic’ substrate, will initially be diluted with distilled water at 1:4 for the respective volume of 2,5 ml. It is intended to obtain a result of unequivocal colouring of the composite doped with antigen (or antibody) and unequivocal clearance of the composite doped 'with BSA and so TMB dilution grade will be optimized to 1:3. Incubation time: 10 to 20 rain, Liquid medium will then be decanted.

[0163] Results: colouring of the combined composite is consequence of immune-complexes formation. Intensity of the attained colour in each of the essayed composites samples will give indication of analytical criteria of the method;

Sensitivity - the lesser mass of simple composite imprisoned at the combined composite {trendily 25 mg) that will enable to detect the existence of antibodies (or antigens) in biological samples;

Specificity --· the greatest mass amount of simple composite imprisoned at the combined composite (trendily 100 g) that will reveal no cross-reaction at:

I, no colouring of the nega ive-control contained composite; II, no colouring of combined composite tested with biological samples containing different specificity antibodies (or antigens) ,

Step 7: Recycling.

Striping buffer, will initially be diluted with distilled water at 1:16 for the respective volume of 2,5 ml . According to the obtained clearance Sb dilution 'will be optimized since 1:10 to 1:32. Incubation time: 10 min. Liquid mediu will then be decanted.

Step 8: Third wash with 6* 2,5 ml PBS-T. Liquid medium will then be decanted.

7.1.3. Filling of chambers

[0164] This example refers the operation of the diagnosis medical devide conceived ' for a maximum capacity of eight chamber as early mentioned. Each chamber had the useful internal volume of 9 cm ³ and was filled as forwardly described.

[0165] After seven days maturation the grained combined composite was washed with distilled water and then dried for 3 hours at 37 °C. Grain-size separation defined 5 granulometry classes :

Gi G > 1 , 0 mm ³ ;

G _IT 1,0 rmn ³ > G > 710 p ³

Gm G > 710 pm ³ > G > 500 pm ³

Giv 72 G > 500 pm ³ > G > 300 gm ³

Gv G > 300 pm ³ > G > 106 pm ³

[0166] The filling of each chamber was composed by 6 layers as referred below from top to bottom -with respective mass of grain- size

• Layer 6: Gi 200 mg;

• Layer 5; Gm <= 450 g; • Layer 4 _° c¾v 450 mg;

* Layer 3: Gy 120 mg;

® Layer 2 : Gn 210 mg;

® Layer 1; Gi 350 mg;

[0167] The compaction of each layer was optimized by eluting 3,0 to 5,0 ml of distilled water reducing so the formation of preferential run-off ways. Once deposited all the layers rest free-volume of the chamber was filled (with glass spheres of to 1,2 mm diameter) up to 1, 0 cm from the top. Timat upper space was left free in order to have a visible regurgitat:ion window.

[0163] The whole piece of 8 chamber was left 48 hours at 37 "C and after that retained liquid volumes were quantified by eluting 10 ml of distilled water. By measuring after the collected water the differentials were 2,0 ± 0,4 ml.

7.1.4. Operation of the device

[0169] Biological samples placed at liquids-collector drained directly to analytical-chambers posed underneath as presented in Figure 16 photograph. The drainage of liquids along all the experimental procedures did .not fulfil the regurgitation windows in order avoid lateral contamination between chambers. The forwardly described experimental procedures refer the operation of one chamber and the dilution grades were previously determined in preli inary essays .

[0170] The minimal liquid volumes utilised on operation of the device were twice the values indicated below as each analysis- chamber was operated simultaneously with respective negative- oontro1-chamber ,

[0171]

Step 1: Conditioning of the composite.

Grained combined composites were humidified with 10,0 ml

PBS-T . Step 2: Elution of the biological sample.

Biological liquid samples were diluted (in PBS-T) at 1:5 up to 1:13 for the respective volume of 4,0 mi. Incubation time: 20 min.

Step 3: First wash with 10,0 ml PBS-T.

Step 4: Elution of the secondary antibody.

Mother solution of secondary antibody labelled with peroxidase was PBS-T diluted at 1:2000 up to 1:5000 for the respective volume of 4,0 ml. Incubation time: 5 min.

Step 5: Second wash with 2 fractions of 10,0 ml PBS-T,

Step 6: Elution of the chromogenic substrate.

Peroxidase chromogenic substrate was diluted with distilled water at 1:4 up to 1 : 3 for the respective volume of 4,0 ml. Incubation time: 10 to 20 min.

[0172] Results: analytical-chambers filled with grained combined composites doped with mucin gained blue-green colour approximately proportional to mucin concentration and analytical- chambers filled with grained combined composites doped with BSA at mucin-concentration correspondence gained no colour.

Step 7: Recycling.

Striping buffer was diluted with distilled water at 1:16 up to 1:10 for the respective volume of 7,0 ml. Incubation time: 10 min.

Step 8: Third wash with 3 fractions of 10,0 ml PBS-T,

[0173] For the operation of the whole 8 chambers the liquid- volumes used were at the order of magnitude o linear correlation to the values referred to one chamber. 8. References [0174]

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[20] Mucin, Bovine Submaxillary Gland, Calbiochem ^® 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

110526, "ANTIBODY, FUNCTIONAL FRAGMENT PROBE THEREOF AGAINST TUMOUR ANTIGENS",