Patent Description:
The device of the invention is useful for detecting and quantifying peptides or proteins of interest (POI) present in a given biological sample by visual comparison of colour change providing an independent read-out method, which is equipment-free, accurate, simple, low cost, having a long shelf-life able to detect very low amounts of peptides or proteins in a sample that can be used for tracking any (bio)molecule of interest, such as the used in health, environment and food safety industries.

The present invention is in the area of biosensors, biochemistry applied to protein/peptide detection.

Colorimetric or optical biosensors have been extensively studied in sensing biomolecules, metal ions, and other compounds. These biosensors have shown great advantages over conventional assays, particularly concerning their visible response signals, since being possible to observe the results directly by naked eye, there is no need of special equipment for readout.

Several colorimetric assays have been reported in the literature for proteins detection in point-of-care. These includes the well-known lateral flow assays (LFAs). These assays show advantage in terms of portability, free - readout and equipment free. However, this approach is complex, because it uses a porous membrane with specific antibodies or proteins immobilized in lines. Also, LFAs show other concerns, as it is based in a "one-step" assay, in which the biosensor surface is not easily washed and, subsequently, may suffer from interference from sample components that pre-block the strips. Besides that, these assays demonstrate a qualitative and semi-quantitative nature. In addition, sometimes it is needed to label the antibody for increasing the sensitivity becoming a more complicated and expansive assay, especially accounting the needs of a very selective antibody.

A similar tool to LFA using cellulose as a support material is the dipstick-based sensing system. When the dipstick gets in touch with the sample for example of urine or of other physiological fluid, a colour change in the stick is generated.

These methods are a simple but of offer a response of semi-quantitative nature, thereby limiting the accuracy of the analytical data generated.

In addition, the methods reported in the literature use as a biorecognition element enzymes or antibodies, becoming the assay expansive and with a limiting lifetime.

Recently, protein immobilization on oxidized paper enhancement was described being further quantified by colorimetric assay with Ponceau dye. The modified cellulose paper is applied as a sensor for protein quantification in urine, a test able to detect levels of proteinuria and even microalbuminuria. However, this method did not present a specific biorecongiton layer, limiting its selectivity (Imamura et al.

Another interesting disclosure refers to an aptamer-based biochip for protein detection and quantitation which combines the recent biochip technology and the conventional staining methods. This assay shows very promising features in terms of simplicity. However, being the assay based in an aptamer as a biorecognition element and glass as a support material, it becomes a very expensive assay (Lee and Hah <NUM>).

Natural receptors have been widely used in different areas. However, the stability of such biomolecules is limited, and they can generally not be used under harsh conditions.

Artificial receptors offer promising alternative to natural recognition elements for biosensors since they provide several advantages in terms of long-term storage stability, resistance to high temperatures, potential re-usability, robustness, and ease of preparation.

There is a growing interest to design and develop simple, reliable, rapid, and inexpensive biosensors for monitoring and detecting proteins as biomarkers for several chronic diseases as cancer and neurodegenerative conditions.

Plastic antibodies rely on molecular imprinted polymer technology (MIP technology) wherein a target molecule is imprinted over the surface, thereby generating artificial binding positions for these molecules.

Document X. Wang et al. (<NUM>) discloses a paper-based platform for colorimetric detection of thyroglobulin which is based on the use of a MIP layer for protein recognition and the color change produced upon bringing into contact the platform with a staining solution (i. a solution containing tetramethylbenzidin which acts as colorimetric indicator). The paper-based platform of said document is made of a Whatman filter paper (according to the manufacturer it comprises more than <NUM>% alpha-cellulose). In said document, the paper filter is pretreated with hemin/graphene nanosheets to form a composite before coating the paper with a MIP layer, and the MIP layer is specific for thyroglobulin.

Documents M. Pereira et al. (<NUM>) and N. Ferreira et al. (<NUM>) discloses a cellulose paper-based platform for electrochemical detection of peptides/proteins based on the use of a MIP as biorecognition layer. In these documents, the MIP is formed by electropolymerization, which implies that the MIP is built on top of an electrode (graphite) layer deposited on top of the cellulose paper support.

Document G. Ertürk & B. Mattiason (<NUM>) discloses a protocol for grafting a layer of MIP on a surface bearing hydroxilic groups via an intermediate amine-functionalized layer further derivatized with glutardialdehyde. The substrate is a magnetic Fe<NUM><NUM><NUM> particle and the target analyte is not defined.

Document F. Chevalier et a. (<NUM>) discloses some dye compounds commonly used for protein staining. It is silent about MIPs and paper-based sensing platforms.

Document <CIT> discloses computerized techniques for processing results data from colorimetric assays carried out with cellulose paper based test stripes with a MIP as recognition layer. Said document is silent about and intermediate amine layer.

Document Q. Kong et al. (<NUM>) discloses a paper-based microfluidic platform for colorimetric detection of bisphenol A comprising a Whatman cellulose paper filter coated with a MIP layer. The MIP layer is formed by bulk polymerization "in situ" of suitable monomers around a template with a crosslinker and an initiator. In analogy to document X. Wang et al. (<NUM>), the MIP is built on top of a layer of magnetic ferrite nanoparticles used to coat the underlying paper substrate.

Document Y. Peng et al. (<NUM>) discloses a paper-based colorimetric biosensing device, wherein said device comprises a substrate made of cellulose filter paper and a biorecognition layer, wherein the cellulose surface is functionalized with an intermediate layer with surface-active amine groups which are further derivatized with a dialdehyde compound, and with a top (biorecognition) layer comprising antibodies. Said document is silent about molecularly imprinted polymers; i.e. the antibodies are not "plastic antibodies".

The present invention provides a paper-strip device that allows to detect and quantify proteins/peptides of interest (POI) in a very sensitive way, simple and low-cost by coupling the biorecognition function of a plastic antibody imprinted by MIP on the paper strip with colour changes properties thus enabling to detect and quantify said POI without the need of specific equipment, enzymes or antibodies as the biosensors of the prior art above-mentioned, even in the point-of care.

After addition of GLU into APTES/CP, the colour of the solution turned into brick-red instantly. When APTES was mixed with GLU, the amino groups on APTES reacted with GLU to form Schiff bases and led to the formation of APTES dimer, which displays brick-red colour.

<FIG> presents the FTIR spectra of different immobilization steps of the CP based sensor, wherein:.

From <FIG> it is possible to observe that absorbances at <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM>-<NUM> are associated with to CP. The peak at <NUM>-<NUM> corresponds to intramolecular OH stretching, including hydrogen bonds and at <NUM>-<NUM> due to CH and CH2 stretching. Other vibrational peaks at <NUM>-<NUM> correspond to OH from absorbed water, <NUM>-<NUM> due to CH2 symmetric bending, <NUM>-<NUM> due to CH bending, <NUM>-<NUM> due to C-O-C asymmetric stretching <NUM>-<NUM> due to C-O/C-C stretching and <NUM>-<NUM> due to the asymmetric out-of-plane stretching vibrations.

Further, it is possible to observe that, after GLU immobilization onto CP modified with APTES an additional band appears at <NUM>-<NUM> and is representative of C=N stretching. One of the aldehyde group of GLU (-CHO) reacted with the amine previously immobilized on the cellulose paper by means of a nucleophilic addition mechanism forming an imine (-C=N).

After polymerization, a broad band is visible at <NUM>-<NUM> in MIP/GLU/APTES/CP, being this peak representative of simple mono-substituted C=C stretch.

An additional small band is present at <NUM>-<NUM> representative of amide group from the polymeric matrix. <FIG> presents the gradient of colour after test-strips incubation with different concentrations of Aβ-<NUM> POI with <NUM>, <NUM>,<NUM> and <NUM> ng/mL respectively.

The present invention, as defined in the claims, relates to a method and to a biosensing device for detecting and quantifying selected biomolecules such as peptides or proteins of interest (POI) present in a biological sample.

The biosensing device is based on a cellulose paper colour test-strip platform built-in by assembling a molecular imprinting polymer treated with said POI on the paper strip, acting as the biorecognition element of the device.

Because of this, it is possible to detect and quantify the POI by direct contact of the biosensor with the sample to be analysed. The target peptide/protein (POI) can be visually detected by conventional protein staining methods and quantified by colour graduation after promoting the contact of the biosensor device with a biological sample having said POI therein.

The biosensing device, as defined on the claims, is based on a cellulose paper (CP) test-strip support comprising a plastic antibody acting as the biorecognition element of the device, which is specific for a given POI.

The biosensing device further comprises adequate staining compounds able to induce chemical changes upon the modified cellulose substrate (CP) with the biorecognition element, attributing it the ability to react with a given peptide or protein present in a biological sample and to produce a colour change therein, when in presence of a target peptide (POI).

These staining compounds can be provided apart of the biosensor device, for example in the format of a kit, thus comprising the cellulose paper (CP) test-strip support and the specific plastic antibody as a first element of the kit, and the staining solution as a second element of the kit.

The kit for detecting and quantifying a selected peptide or protein of interest (POI), as defined in the claims, is provided with a third element, which is a washing solution for removing the excess of staining solution.

Therefore, by using the above-mentioned kit, the method of the invention provides an easy, reliable, and accurate way to detect and quantify a given POI present in a biological sample in the point-of care.

In the scope of the present invention a "plastic antibody" means a biorecognition layer built by molecular imprinting polymer (MIP) technology.

MIP is the <NUM>-D or <NUM>-D imprint of a certain molecule in a rigid polymeric matrix build with synthetic materials of organic (vinyl functional derivatives) or inorganic (silica derivatives) nature.

Biomimetic materials are mostly prepared by MIP technology, where the target molecule is imprinted over the surface, thereby generating artificial binding positions for these molecules.

In the scope of the present invention, the imprinted molecule is a peptide of interest (POI), and therefore, the biosensing device of the invention is able to recognize said POI after having contacted a biological sample comprising it even in very low concentrations.

The recognition of a given POI produces a change of colour and the intensity of the colour generated gives an accurate quantification of said POI in the analysed biological sample, when compared by a standard with a colour gradient.

The cellulose material that can be used as support material of the biosensing device is of the type qualitative filter paper, circles, diam. <NUM>, such as the one provided by Whatman.

Other cellulose paper (CP) that can be used in the invention are Whatman® cellulose chromatography papers, Whatman® qualitative filter paper, Grade <NUM>, nitrocellulose film slides, Whatman Grade <NUM>(qualitative filter paper circles), GE healthcare Whatman™ cellulose filter paper, and cellulose and polyester fibre (cleanroom paper wipers).

The cellulose paper can be cut into strips of approx. <NUM>×<NUM><NUM> slides.

Strips of cellulose paper (CP) are treated with a compound (<NUM>) capable of providing an amine group (-NH<NUM> ) for the formation of the amine layer onto the test-strips as described above.

As defined in the claims, compounds capable of providing an amine group for the formation of the amine layer can be selected from the group preferably comprising <NUM>-aminopropyltriethoxysilane (APTES), ethylenediamine, phenylenediamine, poly(diallyl dimethyl ammonium chloride, poly(vinylamine) hydrochloride, poly(L-lysine hydrobromide), polyethyleneimine hydrochloride, poly(allylamine hydrochloride), poly(<NUM>-aminostyrene), poly(allylamine), poly (ethylene glycol) bis (<NUM>-aminoethyl), chitosan, poly(I-lysine hydrobromide).

The CP strips with an amine-layer (<NUM>) prepared as above is further incubated with a solution comprising a dialdehyde compound (<NUM>) capable of providing two terminal aldehyde functional groups (-CHO) to form an aldehyde-layer.

The two terminal aldehyde groups of compound (<NUM>) react with the amine group of the amine-layer (<NUM>) by means of a nucleophilic addition mechanism forming an imine (-C=N) group thus resulting in an aldehyde-layer (<NUM>).

Dialdehyde compounds adequate for the invention include glutaraldehyde (GLU), benzene-<NUM>,<NUM>-dialdehyde, and succindialdehyde.

The biorecognition element of the device is produced by molecularly imprinting of a POI (<NUM>) onto a treated CP-strip having an amine-layer modified with an aldehyde-layer.

In the scope of the present invention, any protein or peptide of interest (POI) suitable for the invention include but are not limited to peptide Aβ-<NUM>, cardiac myoglobin, troponin T, CK-MB isoenzyme, cancer MUC-<NUM> protein, carcinoembryonic antigen and carbohydrate antigen (CA <NUM>-<NUM>), neurodegenerative biomarkers such as amyloid β42, Tau protein, interleukin, antibodies such as SARS-CoV-<NUM> (<NUM>-nCoV), antibodies for malaria, zika and dengue virus and respective antigens, viral proteins SARS-CoV-<NUM> (<NUM>-nCoV) antigen), bacterial proteins such as protein A, serum human albumin amongst others.

Compound (<NUM>) comprises two free aldehydes groups, one reacting with the amine group of CP-strip as described above, and the other reacting with the amine groups of a POI (<NUM>) thus, also forming an imine group. Consequently, the protein/peptide is attached to the previously treated CP-strip as described above.

The biorecognition layer is made by assembling a molecular imprinting polymer (MIP) with a selected POI (<NUM>) on the paper strip. The imprinted sites (<NUM>) are obtained once the peptide/protein (<NUM>) is removed.

The biosensing device based on a colorimetric test-strip for detecting and quantifying selected proteins or peptides (POI) thus comprises a modified support of cellulose filter paper (CP) having a layer formed by amine groups (<NUM>) linked to aldehyde groups (<NUM>), a and biorecognition layer built by molecular imprinting polymer technology comprising imprinted sites (<NUM>) for a given POI (<NUM>) able to capture said POI (<NUM>) when contacting a biological sample comprising it.

The biosensing device of the invention is able detect and quantify proteins/peptides of interest (POI) present in a biological sample by visual comparison of colour change providing an independent read-out method, which is equipment-free, accurate, simple, low cost, having a long shelf-life able to detect very low amounts of peptides or proteins after staining with conventional staining compositions and methods.

Detection and quantitation of the peptide (POI) bound to the biosensor can be assessed by staining the test-strip with conventional protein dyes and staining methods.

Suitable dyes for the invention include but are not limited to Coomassie brilliant blue R-<NUM>, bromophenol, methylene blue, silver nitrate and zinc nitrate, fluorescent dyes as eosin, rhodamine, and Nile red and brilliant cresyl blue oxazone.

After staining the biosensing device of the invention for the detection and quantification of a POI the excess of dye can be removed by washing with an inert solution such as acetate, citrate, phosphate, ethanol, methanol and water.

The biosensing device can be provided as a kit comprising the test-strip having imprinted sites for a given POI, a container with a staining solution and another container with a washing solution. Preferably, the kit comprises several test-strips and each container comprise an amount of the respective solution for staining and washing the correspondent number of test-strips included in the package of the kit. Each test-strip is enough for performing the method of detecting and quantifying the specific POI for which the plastic antibody of the biosensor is designed.

The biosensing device of the invention, as described in the previous section, is prepared according to the following steps, and as shown in <FIG> and defined in the claims:.

Cellulose material as described in the previous section is cut into strips of approx. <NUM>×<NUM><NUM> slides.

Cellulose paper-strips can be advantageously pre-treated by incubation in ethanol absolute during <NUM> to remove some organic compounds on its surface.

The amine-layer (<NUM>) is formed by incubating the CP-strips in a solution <NUM>% of a compound (<NUM>), able to provide an amine (-NH<NUM> ) functional group, preferably <NUM>-aminopropyltriethoxysilane (APTES), in <NUM>% of an OH-solution, preferably an ethanolic solution. The monolayer (<NUM>) thus obtained has a standing-up composition with the amine groups (-NH<NUM> ) exposed to the external surface of the PC.

The CP-strips having an amine layer (<NUM>) are further incubated with a solution comprising a dialdehyde compound (<NUM>) able to provide two terminal aldehyde functional groups (-CHO), preferably glutaraldehyde (GLU), to form an aldehyde + amine-layer (<NUM>+<NUM>). This step can be performed at room temperature (<NUM>-<NUM>), for <NUM> minutes at a pH value of approx. After incubation, the thus obtained CP-strips are repeatedly washed with distilled water for removal of undesired excess of residues.

A peptide of interest (<NUM>) is immobilized onto the cellulose paper-strips (PC) treated as explained above by incubation of a solution of said POI (<NUM>) in an adequate buffer, such as PBS buffer at a pH value of approx. <NUM>, for around <NUM> at a low temperature of <NUM> to <NUM>, preferably of <NUM> to <NUM>, more preferably at <NUM>.

The CP-strips with immobilized POI (<NUM>) as above described can be washed to remove non-bound POI with an adequate solution such as deionised water and PBS buffer.

The plastic antibody of the biosensor is produced by imprinting a selected POI according to any MIP known method, such as the one described by <NPL> ).

In the scope of the present invention, molecular imprinting starts by self-organizing the monomeric structures of a polymer monomers (MON) around a template (<NUM>) followed by polymerizing the MON with a cross-linker (CL). The polymerization starts with addition of a radicalar initiator (<NUM>). The imprinted sites (<NUM>) are obtained once the peptide/protein (<NUM>) is removed.

Monomers (MON) adequate for the invention include, but are not limited to, acrylamide monomer (AAM), vinyl benzene boric acid, <NUM>-vinyl benzaldehyde, <NUM>-vinyl aniline, tert-butyl p-vinylphenylcarbonate, acrylic acid, methacrylic acid, trifluoromethyl acrylic acid methyl methacrylate, p-vinylbenzoic acid, itaconic acid, <NUM>-ethylstyrene, styrene, <NUM>-vinylpyridine,vinylpyridine, <NUM>-vinylimidazole, acrylamide, <NUM>-acrylamido-<NUM>-methyl-<NUM>-propane sulfonic acid, <NUM>-hydroxyethyl methacrylate, trans-<NUM>-(<NUM>-pyridyl)-acrylic acid.

Suitable crosslinkers (CL) for the invention include but not limited to N,N'-methylenebis(acrylamide) (N,N-BISAA), bis-(<NUM>-(tert-butylperoxy)-<NUM>-methylethyl)-benzene, ethylene glycoldimethacrylate, N,N;-methylenediacrylamide, divinylbenzene, <NUM>,<NUM>-diisopropenyl benzene, N,N' -<NUM>,<NUM>-phenylenediacrylamine, <NUM>,<NUM>-bisacryloylamidopyridine, <NUM>,<NUM>-bis(acryloylamido)benzoicacid, tetramethylene dimethacrylate, trimethylpropane trimethacrylate.

CP-strips treated as described previously can be imprinted with a given POI by incubating a monomer (MON) with a cross-linker (<NUM>), for approx. <NUM> at room temperature at a pH of approx.

After incubation, a solution of an initiator is added to start the polymerization. The polymerization is carried out at room temperature (<NUM>-<NUM>), for approx. <NUM>, after which the sensor is thoroughly washed with deionised water.

Suitable initiators (<NUM>) for the invention include but are not limited to ammonium persulphate (APS), tetramethylethylenediamine (TEMED), azobisisobutyronitrile azobisdimethylvaleronitrile, <NUM>,<NUM>-azo(<NUM>-cyanovaleric acid), benzoylperoxide , dimethylacetal of benzyl.

Finally, the previously attached POI (<NUM>) is removed to empty the imprinted sites (<NUM>), according to described by <NPL> ) to empty the imprinted sites. This step takes approx. The imprinted material is then washed and conditioned in PBS buffer, pH <NUM>, in order to increase the pH value and to remove the amino acid fractions produced by the treatment.

Suitable chemicals for the template removal from the polymeric matrix include, but are not limited to, oxalic acid, acetic acid, sulphuric acid, methanol and enzymes (trypsin and proteinase K).

The staining solutions of the invention can be prepared by any of known methods for this purpose. It can be prepared by dissolving a suitable dye for peptide-staining in a buffer to achieve a staining solution such as bromophenol (blue), methylene blue (blue), silver nitrate (grey) and zinc nitrate (grey), fluorescent dyes as eosin, rhodamine, and Nile blue oxazone.

Washing solutions comprise solvents or buffer solutions to remove the excess of staining solution of the test-strips. Typically, they are prepared by mixing prepared in acidic medium as acetate, citrate, phosphate or organic solvents as ethanol, methanol or in water.

Detection and quantification of a given peptide (POI) present in a biological sample can be performed by contacting the biosensor of the invention with a biological sample.

Biological samples, in the scope of the present invention are for example, urine, serum and saliva.

Detection and quantitation of the peptide (POI) bound to the biosensor can be assessed by staining the test-strip of the invention with conventional protein dyes.

The results can be assessed either by visual comparison of the colour or by the different colour intensity in the standard curve made with IMAGEJ software with RGB coordinates.

The method of detecting and quantifying a given POI present in a biological sample comprises the following steps:.

Suitable peptide staining compositions for the invention include but are not limited to Coomassie brilliant blue R-<NUM> (CB), bromophenol, methylene blue, silver nitrate and zinc nitrate, fluorescent dyes as eosin, rhodamine, and Nile blue oxazone, as indicated before.

The test-strips are incubated with a peptide staining composition for approx. <NUM> minutes to promote the development of the colour of the correspondent staining composition. Preferably, at least half of a test-strip is dipped into the biological sample.

Biological samples as plasma or serum can be pre-treated. Serum is the liquid fraction of whole blood and thus it can be collected after blood clotting. The clot is removed by centrifugation and the resulting supernatant, designated serum, is carefully removed using a Pasteur pipette. Plasma is produced when whole blood is collected in tubes treated with an anticoagulant (e.g., EDTA-treated or citratetreated). The blood does not clot in the plasma tube. The cells are removed by centrifugation. The supernatant, designated plasma, is carefully removed from the cell pellet using a Pasteur pipette.

Liquid samples such as urine, serum, plasma, saliva, sweat, can be put directly in contact with the test-strips of the invention.

Different colours are generated by different staining compositions. As an example, the following dyes are presented followed by the corresponding colour generated: Bromophenol (blue), methylene blue (blue), silver nitrate (grey) and zinc nitrate (grey); fluorescent dyes such as eosin, rhodamine, and Nile blue oxazone can also be used.

Cellulose paper-strips (CP) were subjected to a multi-step treatment to obtain the cellulose test-strip based biosensor with a MIP as a biorecognition element.

Cellulose paper Whatman® qualitative filter paper was commercially acquired, and several strips <NUM>×<NUM><NUM> slides were obtained by cutting the cellulose paper with a scissor.

Cellulose paper-strips were incubated by immersion in different solutions in a container (<NUM> per paper slide) under constant stirring (lateral motion) at room-temperature (<NUM>-<NUM>), in a chamber under protective light conditions, ensuring that the paper-slides were completely submerged in the solution.

Twenty cellulose strips were used to prepare a biosensor device based on a paper colour test-strip platform.

Twenty cellulose strips were pre-treated by incubation in <NUM> ethanol solution <NUM>% of during <NUM>, to remove residual organic compounds on its surface. Incubation was conducted under constant stirring (lateral motion), usually at room-temperature (<NUM>-<NUM>), unless specified otherwise, in a chamber protected from light and ensuring that the paper was completely submerged in the solution.

An amine layer was obtained on the pre-treated paper strips by immersing the strips in a <NUM>% solution of <NUM>-aminopropyltriethoxysilane (APTES) in ethanol <NUM>%, during <NUM>, with continuous stirring (<NUM> slides were placed into <NUM> flask containing <NUM> of the ethanolic solution).

The previously aminated test strips with APTES were modified by reaction with glutaraldehyde (GLU). The reaction took place with a <NUM>% solution of GLU, at room temperature, for <NUM> minutes (<NUM> strips were placed into <NUM> flask containing <NUM> of the MES buffer pH <NUM>). The test-strips were then repeatedly washed with distilled water (each strip has been in contact with distilled water for <NUM> seconds).

The paper-strips previously modified with GLU were further incubated with <NUM>µL of <NUM>µg/mL of a peptide Aβ-<NUM> (POI) solution (pH <NUM>, in phosphate buffered solution PBS buffer), for <NUM> at <NUM> in order to form a covalent imine group, for <NUM> at <NUM>. After peptide Aβ-<NUM> immobilization, cellulose strips were washed with deionised water and PBS buffer by immersing in a <NUM> of each solution during ~<NUM> seconds.

Paper-strips comprising the peptide Aβ-<NUM> therein attach were washed with deionised water and PBS buffer followed by incubation with <NUM> of <NUM> mmol L-<NUM> , acrylamide monomer (AAM) and <NUM> mmol L<NUM> of crosslinker N,N'-methylene-bis(acrylamide) (N,N-BISAA), in PBS solution (pH <NUM>).

Next, <NUM>µlL of <NUM> mmol L<NUM> of ammonium persulfate, (APS) and (<NUM>% of tetramethylethylenediamine (TEMED) solution prepared in PBS buffer pH <NUM> was added to the previous solution in order to start the polymerization.

The polymerization was carried out at room temperature for <NUM>, after which each test-strip was thoroughly washed with deionised water and PBS buffer by immersing each slide in a <NUM> of each solution during ~<NUM> seconds.

Finally, the attached peptide was removed by reaction with <NUM> mol L-<NUM>, oxalic acid (Oac) to form the imprinted sites, being each strip immersed in <NUM> of Oac solution. This step was carried out at room temperature for <NUM>. The imprinted material was washed and conditioned in PBS buffer, pH <NUM>, to increase the pH and remove the amino acids fractions produced by Oac treatment (see <FIG>).

The blank material (NIP) was synthetized in parallel excluding from the procedure the peptide attach step, for comparison purposes.

The binding assay of Aβ-<NUM> to the test-strip based biosensor was verified against incubation with <NUM>µL of different concentrations of Aβ-<NUM> prepared in PBS buffer pH <NUM>, during <NUM> minutes at room temperature. For this purpose, different test -strips were used:.

Thereafter, test-strips were washed with PBS buffer (<NUM> PBS solution/ slide) and let dry at room temperature for <NUM> minutes.

Detection and quantitation of the POI bound to the biosensor was confirmed by conventional Coomassie brilliant blue R-<NUM> (CB) staining method.

For this purpose, the modified paper strips were incubated with CB staining solution for <NUM> minutes.

Each strip was incubated with CB solution <NUM>% (W/V) prepared in (ethanol- phosphoric acid -water) (EtOH - H<NUM>PO<NUM> - H<NUM>O; <NUM>:<NUM>:<NUM>) solution during <NUM> minutes in order to promote the development of the Blue/green colour.

Then, strips were washed with (EtOH- H<NUM>PO<NUM> - H<NUM>O; <NUM>:<NUM>:<NUM>) solution, for <NUM> minutes, and with PBS buffer at pH <NUM> in order remove the unreacted CB staining solution.

After taking pictures, the obtained signals were evaluated with Image J program of Windows by measuring the colour coordinates of each image acquired by the RGB colour system (red, green and blue).

Claim 1:
A biosensing device for detecting and quantifying a selected peptide or protein of interest (POI) (<NUM>) present in a biological sample characterized by comprising a cellulose paper (CP) test-strip platform comprising an amine-layer (<NUM>), and a biorecognition layer for said POI (<NUM>) which is built by molecular imprinting polymer (MIP) technology and is capable of generating a change of colour after being stained with an adequate peptide staining solution;
wherein said cellulose paper test-strip platform is a qualitative filter type paper; wherein said amine layer is obtainable by treating the cellulose paper (CP) test-strip platform with a compound selected from a list consisting of: <NUM>-aminopropyltriethoxysilane (APTES), ethylenediamine, phenylenediamine, poly(diallyldimethyl ammonium chloride, poly(vinylamine) hydrochloride, poly(L-lysine hydrobromide), polyethyleneimine hydrochloride, poly(allylamine hydrochloride), poly(<NUM>-aminostyrene), poly(allylamine), polyethylene glycol)bis(<NUM>-aminoethyl), chitosan, poly(I-lysine hydrobromide);
wherein said amine-layer is further incubated with a solution comprising a dialdehyde compound capable of providing two terminal aldehyde functional groups, thus forming an amine-aldehyde layer (<NUM>),
wherein a molecularly imprinted polymer (MIP) layer able to recognize said POI (<NUM>) is built on top of said amine-aldehyde layer; and
wherein said POI (<NUM>) is selected from a list consisting of: peptide Aβ-<NUM>, cardiac myoglobin, troponin T, CK-MB isoenzyme, cancer MUC-<NUM> protein, carcinoembryonic antigen and carbohydrate antigen (CA <NUM>-<NUM>), neurodegenerative biomarkers such as amyloid β42, Tau protein, interleukin, antibodies such as SARS-CoV-<NUM> (<NUM>-nCoV), antibodies for malaria, zika and dengue virus and respective antigens, viral proteins SARS-CoV-<NUM> (<NUM>-nCoV) antigen), bacterial proteins such as protein A, and serum human albumin.