Biosensor, method for obtaining the biosensor, method for detecting pseudomonas aeruginosa and detection kit using the sensor for the rapid detection of p. aeruginosa

Abstract

The present invention relates to a biosensor, a method for obtaining same and the use thereof to detect Pseudomonas aeruginosa in clinical samples from patients, based on the use of molecular gates on porous organic–inorganic hybrid supports that allow specific and selective recognition of bacterial DNA. The molecular gate developed comprises an oligonucleotide sequence complementary to a specific region of the DNA of this bacterium. The external surface of the porous support is chemically modified to provide reactive groups capable of bonding with short-sequence oligonucleotides (O1) designed to act as an anchor and to subsequently bond oligonucleotides (O2) specific for the recognition of P. aeruginosa. In the presence of P. aeruginosa genomic DNA, the oligonucleotides (O2) are capable of specific recognition by complementarity and of detaching from the pore surface, leading to the release of a fluorescent indicator.

Description

[0001]

[0002] Biosensor, procedure for obtaining said biosensor, method for the detection of Pseudomonas aeruginosa and detection kit using said sensor for the rapid detection of P. aeruginosa.

[0003] TECHNICAL SECTOR

[0004] The present invention relates to a biosensor based on molecular gates for the early and accurate detection of Pseudomonas aeruginosa, the procedure for obtaining the same and its application in a detection or diagnostic kit, such as test strips.

[0005] Thus, the present invention offers a simple-to-use biosensor that allows for the rapid and effective detection of this pathogen in the clinical environment.

[0006] PRIOR ART

[0007] Pseudomonas aeruginosa is an opportunistic pathogen responsible for the most important bacterial infections associated with clinical settings. It is estimated that P. aeruginosa infections account for between 7.1 and 7.3% of healthcare-associated infections (Weiner LM, Webb AK, Limbago B, Dudeck MA, Patel J, Kallen AJ, et al. Antimicrobial-Resistant Pathogens Associated With Healthcare-Associated Infections: Summary of Data Reported to the National Healthcare Safety Network at the Centers for Disease Control and Prevention, 2011-2014. Infect Control Hosp Epidemiol, November 2016;37(11):1288-301). P. aeruginosa infection accounts for 23% of all infections acquired by patients in intensive care units (ICUs), this being the most common place where respiratory infections caused by this bacterium develop (Vincent JL, Sakr Y, Singer M, Martin-Loeches I, Machado FR, Marshall JC, et al.Prevalence and Outcomes of Infection Among Patients in Intensive Care Units in 2017. JAMA. April 21, 2020;323(15):1478-87). This opportunistic pathogen is capable of causing acute infections that frequently become chronic, exhibiting high resistance to antibiotics.

[0008] For these reasons, P. aeruginosa has been listed as a 'critical priority pathogen' by the World Health Organization (WHO) since 2017 (Tacconelli E, Carrara E, Savoldi A, Harbarth S, Mendelson M, Monnet DL, et al. Discovery, research, and development of new antibiotics: the WHO priority list of antibiotic-resistant bacteria and tuberculosis. Lancet Infect Dis. March 2018;18(3):318-27). P. aeruginosa infections primarily occur in hospitalized patients, especially those with neutropenia or who are debilitated or immunocompromised, including patients with severe burns, diabetes, cancer, organ transplants, or additional immunodeficiencies, being, as already mentioned, one of the main causes of acute nosocomial infections (Curran CS, Bolig T, Torabi-Parizi P. Mechanisms and Targeted Therapies for Pseudomonas aeruginosa Lung Infection. Am J Respir Crit Care Med. March 15, 2018;197(6):708-27).

[0009] P. aeruginosa can produce a wide variety of infections, particularly bacteremia, ventilator-associated pneumonia, respiratory infections in patients with cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), heart tract infections, urinary tract infections, and surgical infections. Beyond the prevalence of this bacterium as a nosocomial pathogen worldwide, the high mortality and morbidity associated with the numerous infections it is capable of causing are concerning, especially respiratory and bloodstream infections (Thaden JT, Park LP, Maskarinec SA, Ruffin F, Fowler VG, Van Duin D. Results from a 13-Year Prospective Cohort Study Show Increased Mortality Associated with Bloodstream Infections Caused by Pseudomonas aeruginosa Compared to Other Bacteria. Antimicrob Agents Chemother. June 2017;61(6):e0267116.,6), (Micek ST, Kollef MH, Torres A, Chen C, Relio J, Chastre J, et al.Pseudomonas aeruginosa Nosocomial Pneumonia: Impact of Pneumonia Classification. Infect Control Hosp Epidemiol. 2015 / 07 / 20 ed. 2015;36(10):1190).

[0010] Due to the clinical importance of P. aeruginosa, there are now several methods of bacterial identification available for clinical use. The gold standard detection technique in clinical samples is plate culture, based on the biological characteristics of the bacteria and / or the activities of its main bacterial molecules such as acetamidase, oxidase, arginine dihydrolase, or pyocyanin (Reyes EA, Bale MJ, Cannon WH, Matsen JM. Identification of Pseudomonas aeruginosa by pyocyanin production on Tech agar. J Clin Microbiol, March 1981;13(3):456-8), (Tang Y, Ali Z, Zou J, Jin G, Zhu J, Yang J, et al. Detection methods for Pseudomonas aeruginosa, history and future perspective. RSC Adv. 2017;7(82):51789-800), (Capatina D, Feier B, Hosu O, Tertis M, Cristea C. Analytical methods for the characterization and diagnosis of infection with Pseudomonas aeruginosa'. A critical review. Anal Chim Minutes. April 29, 2022; 1204:339696).This microbiological technique requires at least 24 hours for bacterial culture and additionally, phenotypic and biochemical assays of 24-48 hours in order to identify the bacteria, evaluate its sensitivity to antimicrobials and thus generate the diagnosis (Tang Y, Ali Z, Zou J, Jin G, Zhu J, Yang J, et al. Detection methods for Pseudomonas aeruginosa', history and future perspective. RSC Adv. 2017;7(82):51789-800), (Szita G, Biró G. A synthetic, selective culture medium for Pseudomonas aeruginosa. Acta Vet Hung. 1990;38(3):187 — 194). Furthermore, the results are sometimes inconsistent due to sample contamination during culture and erroneous detection of other species of the genus Pseudomonas that commonly generate false positives, such as Pseudomonas fluorescens (Wang C, Ye Q, Jiang A, Zhang J, Shang Y, Li F, et al.Pseudomonas aeruginosa Detection Using Conventional PCR and Quantitative Real-Time PCR Based on Species-Specific Novel Gene Targets Identified by Pangenome Analysis. Front Microbiol. May 4, 2022;13:820431). Due to this situation, other diagnostic methods for P. aeruginosa have been implemented in the clinical setting, such as molecular techniques, immunological techniques, and proteomic techniques (Tang Y, Ali Z, Zou J, Jin G, Zhu J, Yang J, et al. Detection methods for Pseudomonas aeruginosa: history and future perspective. RSC Adv. 2017;7(82):51789-800), (Capatina D, Feier B, Hosu O, Tertis M, Cristea C. Analytical methods for the characterization and diagnosis of infection with Pseudomonas aeruginosa: A critical review. Anal Chim Acta. April 29, 2022; 1204: 339696).While molecular and proteomic techniques offer greater sensitivity and specificity than plate culture, the samples require extensive preparation that is susceptible to contamination. Immunological techniques overcome these problems by allowing for on-site sample analysis, although they are less specific and sensitive than the former. Generally, all of these techniques still face long diagnostic turnaround times, complex and extensive sample preparation, and the need for highly trained personnel, which limits rapid diagnosis and their implementation in healthcare settings.

[0011] In recent years, the identification of quorum sensing biomarkers of P. aeruginosa or the development of biosensors based on the generation of optical, electrical, or piezoelectric signals have emerged as alternatives to conventional techniques. However, measurement in complex biological matrices remains challenging because, due to the presence of other interfering agents, nonspecific binding to the material surface leads to false, low-sensitivity, and irreproducible results (Reyes EA, Bale MJ, Cannon WH, Matsen JM. Identification of Pseudomonas aeruginosa by pyocyanin production on Tech agar. J Clin Microbiol, March 1981; 13(3):456-8). Research on these biosensors remains limited, and they cannot be fully implemented in clinical practice.

[0012] Given the urgent need to develop new sensors that address the challenges of current diagnostic techniques with greater specificity, sensitivity, and shorter response times, nanotechnology-based detection techniques have emerged. One attractive application of this technology is the development of materials equipped with molecular gates for the controlled release of a charge in the presence of a specific stimulus (Descalzo AB, Martínez-Máñez R, Sancenón F, Hoffmann K, Rurack K. The Supramolecular Chemistry of Organic-Inorganic Hybrid Materials. Angew Chem Int Ed. September 11, 2006;45(36):5924-48), (Sancenón F, Pascual L, Oroval M, Aznar E, Martínez-Máñez R. Gated Silica Mesoporous Materials in Sensing Applications. ChemistryOpen. August 2015;4(4):418-37). In this type of system, the pores of the porous hybrid material are loaded with therapeutic molecules, diagnostic markers, or an indicator molecule.Subsequently, the pores are blocked with molecules or supramolecular structures that act as a 'molecular gate', capable of being opened and releasing the cargo when a certain external stimulus is applied (Aznar E, Oroval M, Pascual L, Murguia JR, Martínez-Máñez R, Sancenón F. Gated Materials for OnCommand Release of Guest Molecules. Chem Rev. January 27, 2016;116(2):561-718).

[0013] Several sensors that apply this technology are known in the prior art. Patent document ES2767873A1 discloses a biosensor for the detection and quantification of Staphylococcus aureus based on a porous support that includes an indicator in its pores and an aptamer anchored to the surface of the support that specifically recognizes a S. aureus sequence. In the presence of the bacteria, the aptamer recognizes and binds to it, allowing the indicator to be released from the porous support, thus enabling the identification and quantification of the presence of S. aureus.

[0014] Patent documents EP3680345A1, ES2702999 A1, and WO2019048722 A1 disclose a porous material for the detection of Candida albicans. This material comprises an indicator species within its internal pores and has at least one DNA sequence complementary to a fragment of the C. albicans genome attached to its outer surface. In the presence of C. albicans, this DNA sequence binds to the pathogenic microorganism, allowing the indicator species to be released. Furthermore, patent documents ES2763043A1 and WO2021219910A1, as well as the article by Pía Luis et al., also disclose a porous material for the detection of Candida albicans.The article titled “Oligonucleotide-capped nanoporous anodic alumina biosensor as a diagnostic tool for rapid and accurate detection of Candida auris in clinical samples”, published in the journal Emerging Microbes & Infections on January 1, 2021, describes a porous support with an indicator inside the pores, and an oligonucleotide that specifically recognizes a Candida auris sequence, wherein the oligonucleotide is anchored to the surface of the porous support in a position such that it controls, in the absence / presence of C. auris DNA, the release of the indicator to the outside.

[0015] The annotated state-of-the-art documents disclose different sensors for the detection and quantification of different microorganisms such as S. aureus, C. albicans or C. auris, which comprise supports such as nanoporous anodic alumina that houses an indicator substance in its pores and has anchored on its surface complementary oligonucleotide sequences of a fragment of the genome of one of these microorganisms.

[0016] However, none of the cited background information is applicable for the detection and quantification of P. aeruginosa, which, as detailed above, is one of the main causes of acute nosocomial infections. This is because the development of each of the indicated biosensors is unique to the pathogen they recognize, and furthermore, none of the oligonucleotide sequences reported in the background information are specific to the DNA of this bacterium.

[0017] For all the above reasons, the applicant of the present invention detects the need to develop a new specific biosensor for the early, accurate and simple detection of P. aeruginosa in the clinical environment, as well as the procedure for preparing it and its application in test strips that solve the aforementioned problems, as described below.

[0018] DESCRIPTION OF THE INVENTION

[0019] The present invention provides and claims a biosensor for the detection of *P. aeruginosa*, as well as the process for obtaining it, an in vitro detection method, and its use in a detection kit, such as a test strip for the rapid and accurate in vitro detection of this bacterium in a sample, thus solving the problem described above. In this way, the biosensor of the invention allows the detection of *P. aeruginosa* DNA and therefore the diagnosis of an infection caused by it. To this end, the biosensor is based on a porous support that includes a molecular gate comprising an oligonucleotide sequence complementary to a specific region of the DNA of this bacterium, such that a specific molecular interaction occurs between the *P. aeruginosa* DNA and the complementary oligonucleotide sequence located on the porous system.Oligonucleotides have proven to be excellent candidates for blocking pores and acting as molecular gates, as they exhibit a high degree of specificity and sensitivity in infection detection systems, as demonstrated by numerous studies ((Sancenón F, Pascual L, Oroval M, Aznar E, Martínez-Máñez R. Gated Silica Mesoporous Materials in Sensing Applications. ChemistryOpen. August 2015;4(4):418-37), (Pascual L, Baroja I, Aznar E, Sancenón F, Marcos MD, Murguia JR, et al. Oligonucleotide-capped mesoporous silica nanoparticles as DNA-responsive dye delivery systems for genomic DNA detection. Chem Commun. 2015;51 (8):1414-6)).

[0020] To this end, the interior of the porous material is loaded, preferably by diffusion and under gentle agitation (preferably at 10–200 rpm, for 24 h, and at 25°C), with an indicator molecule such as rhodamine B. Subsequently, the external surface of the support is chemically modified to add an organic group, such as (3-isocyanatopropyl)thetoxysylane, under agitation (preferably 10–200 rpm, 5 h 30 min, 25°C) to provide reactive groups, such as isocyanate groups, capable of forming a chemical bond (e.g., a urea bond) with a first short-sequence binding oligonucleotide (hereafter abbreviated as O1). Oligonucleotide O1 acts as an anchor to which at least one second oligonucleotide designed to specifically recognize P. aeruginosa (hereafter abbreviated as O2) is attached.

[0021] The genome of the bacterium *P. aeruginosa* is approximately 5.2 to 7 Mbp in size, and its gene set varies from 10,000 to 40,000 genes among different strains of *P. aeruginosa*. Therefore, to design the oligonucleotide *O2*, which is capable of recognizing *P. aeruginosa* DNA, the first step was to identify suitable regions for specific and sensitive diagnosis of the bacterium. This required an in-depth search of *P. aeruginosa* databases for all clinical and potentially pathogenic strains for humans. The result was the high specificity of the designed sequence (molecular gateway) for all clinical strains of *P. aeruginosa*.The absence of molecular gates in P. aeruginosa and other common bacterial species in intensive care units has been experimentally demonstrated, as detailed below, offering highly advantageous results compared to other previously developed molecular gate-based techniques. Furthermore, to design the specific molecular gate targeting a particular region of the bacterial genome, a laborious study of secondary structure folding predictions was conducted to obtain the best spatial arrangement of the sequence in the biosensor, thereby enabling optimal recognition of the target sequence. Finally, to select from the different candidate sequences designed to recognize the specific region of the P. aeruginosa genome...aeruginosa, an in-depth study was carried out that included different concentration optimization processes, exemplified in the preferred embodiment section of this report and in figures 5 and 6, in order to evaluate the molecular recognition potential between base pairs, in order to ensure the displacement of the molecular gate from the surface of the porous material.

[0022] On the other hand, the final development of the biosensor requires a specific procedure to effectively anchor the molecular gate. For each type of nucleotide design of the molecular gates, a comprehensive study of the different synthesis steps of the sensor is necessary to fine-tune its operation. Specifically, the selection and design of the 01 / 02 recognition pair is critical. Finding the right conditions to achieve good recognition of P. aeruginosa DNA, and ensuring that this recognition is selective and functions in biological media, is not straightforward and requires considerable experimentation.

[0023] The present invention is defined according to the set of claims accompanying this specification. Thus, a first aspect of the present invention relates to a biosensor, a biosensor being understood as an analytical device that converts a biological response into an optical, electrical, or any other type of signal that provides qualitative or quantitative information. The biosensor of the invention comprises:

[0024] - a porous support surface-functionalized with a reactive organic group, preferably isocyanate groups, wherein the support is loaded with an indicator inside at least a plurality of its pores, and wherein said pores provide access to the exterior of the porous support; and

[0025] - at least one oligonucleotide 02 comprising the nucleotide sequence SEQ ID NO: 2, an oligonucleotide that specifically recognizes a region of the DNA of the bacterium P. aeruginosa.

[0026] As for the reactive group, this can be a neutral or cationic organic group. Preferably, the neutral organic group is selected from the group consisting of carboxylic acid (-COOH), alcohol (-OH), aldehyde (-CHO), C2-C30 alkenyl, C2-C30 alkynyl, amine (-NH2 or -NR'R"), amide (-C(O)NR'R"), azide (-N3), ketone (-O=O), ester (-COOR 1 ), ether (R'-OR” ), halogen-containing group, amine (RR'C=NR"), isocyanate (-N=C=O), isothiocyanate (-N=C=S), nitrile (-C=N), nitro (-NO2) and thiol (-SH), each R' and R” independently representing a hydrogen, a C2-C30 alkyl, a C2-C30 alkenyl, or a C2-C30 alkynyl, all of these groups can be linear or branched, and have substituents or not.

[0027] If the reactive group with which the porous support is functionalized is a cationic organic group, it is selected from the group consisting of amines (-NHs + ), guanidinium groups ([CHeNs]*), phosphonium (-PH4+ ) or quaternary ammonium (-NR4 + ), where R is independently selected from a linear or branched C1-C30 alkyl and a C3-C6 cycloalkyl.

[0028] The porous support has an internal nanoporous structure with pores ranging in diameter from 1 nm to 100 nm. Thus, the internal porous structure of the support can be macroporous (with pores of approximately 50 nm or greater in diameter, or distance between the internal or opposite walls of a pore), mesoporous (with pores between 2 nm and 50 nm in diameter), or microporous (with pores of approximately 2 nm or less in diameter). Preferably, the support of the invention has a mesoporous structure, with pores between 2 nm and 50 nm, and more preferably between 3 nm and 7 nm.

[0029] Thus, the porous support can be made up of any material that has an internal porous structure, where at least a plurality of these pores offer access to the outside, such as nanoporous anodic alumina, nanoporous silica, titanium oxide or graphene among other non-limiting alternatives of the present invention.

[0030] On the other hand, for the purposes of the present invention, an 'indicator' shall be understood to mean any compound or molecule capable of being detected, visualized, and / or quantified. Furthermore, the term 'indicator' may be interchanged with 'signal' or 'reporter' compound or molecule. Examples of indicators include, but are not limited to, dyes, fluorophores, electrochemiluminescent substances, redox-active substances, substances with plasmon resonance, or biologically active substances such as proteins, small biomolecules (smaller than 50 nm), enzymes, or nucleic acid fragments.In preferred embodiments, the indicator is selected from the group consisting of a colorimetric indicator (dye), a fluorimetric indicator (fluorophore), an electrochemiluminescent indicator or a mixture thereof, even more preferably a fluorimetric indicator, such as [Ru(bipi)s]Cl2, rhodamine 101, rhodamine 110, rhodamine B, safranin, fluorescein, sodium salt of sulforodamine B and / or PEG-Bodipi.

[0031] With regard to the term 'oligonucleotide', this refers to single-stranded DNA or RNA molecules of short sequence (up to 200 nucleotides), both natural and artificial, which can fold into a variety of structures, and where the nucleotides can be modified, for example, by 5' methylation. The oligonucleotides developed for the present invention (oligonucleotide 02 comprising the sequence SEQ ID NO: 2) are bifunctional, that is, on the one hand, they perform the function of specifically recognizing a region of P. aeruginosa DNA, binding to this target with an affinity in the micro-picomolar range, and, on the other hand, they block the pores of the porous support, preventing the exit of the indicator.

[0032] Thus, oligonucleotide 02, comprising the nucleotide sequence SEQ ID NO: 2, which recognizes P. aeruginosa DNA, is anchored to the surface of the porous support via a first binding oligonucleotide comprising the oligonucleotide sequence SEQ ID NO: 1. This binding oligonucleotide is linked to the support through reactive groups that functionalize its surface, such as isocyanate groups, forming a urea bond, such that the chemical bond / 01 / 02 assembly constitutes a molecular gate. The molecular gate is covalently anchored to the surface of the porous support in a position such that, in the absence of P. aeruginosa DNA, the molecular gate blocks access to the exterior through the support pores, preventing the indicator from exiting. In the presence of P. aeruginosa DNA, the oligonucleotide SEQ ID NO: 2 recognizes the DNA and binds to a specific region of the P. aeruginosa DNA.aeruginosa, moving from the surface of the porous support, which allows the unlocking of access to the pores of the porous support to the outside and allowing the indicator to exit.

[0033] According to a preferred embodiment of the invention, the nucleotide sequences employed are SEQ ID NO: 1 (NH2-(CH2)e-5'-AAA AAA CCC CCC-3'), which contains an amino group attached to a 6-carbon chain as a linker for the linking oligonucleotide 01, designed to link to the 5' terminal region of oligonucleotide 02 by means of a primary amino group thereof; and for the second oligonucleotide 02 it is SEQ ID NO: 2 (5- TTTTTGGGGGGACTCCCAGTCGGGGAAGCAGCGCTCGAGCCACTCGGCGAGCCGCCT GAGGCTCTCATGGCCCCGGAAGGGGGGTTTTT-3'), the second oligonucleotide 02 being capable of recognizing P. aeruginosa DNA.

[0034] Finally, the sequence SEQ ID NO: 3 (5'-

[0035] TTCCGGGGCCATGAGAGCCTCAGGCGGCTCGCCGAGTGGCTCGAGCGCTGCTTCCCC GACTGGGAGT-3') comprises a specific R region P hzA2 of the phzA2 gene of the bacterium P. aeruginosa, which preferentially recognizes SEQ ID NO: 2, as shown in Table 1, where R has been highlighted P hzA2.

[0036] Table 1. Location and genetic sequence of the target region of the phzA2 gene of P. aeruginosa, R P hzA2, (SEQ ID NO: 3) chosen for the design of oligonucleotide 02. In gray, the R region is highlighted P hzA2, complementary to the designed O2. The DNA and amino acid sequence of the phzA2 gene was obtained from the Pseudomonas and Uniprot genomic databases. of MREYQRLKGFTDNLELRRRNRATVEHYMRMKGAERLQRHSLFVEDGCAGNWTTESGEPLVFRG amino acid HESLRRLAEÍggggggglgggg||ggggggDPNHLWV£CDGRGKAbVPGYPQGYCENHYIHSF'E LENGRIKRNREFMNPÍÍQKLRALGIÁVPQI KRDGI PT

[0037] Advantageously, the SEQ ID NO: 2 sequence is specific to P. aeruginosa and does not exist in biosensors for other pathogens, guaranteeing the specificity of the biosensor of the invention for P. aeruginosa.

[0038] Uses of the biosensor

[0039] A second aspect of the present invention relates to the use of the biosensor for the detection of P. aeruginosa DNA, that is, to identify or report its presence in an isolated sample. A 'sample' is understood to be a portion, manipulated or not, representative of a whole that is subjected to analysis or experimentation, the analysis in this case being the presence or absence of P. aeruginosa DNA. Samples may be environmental or clinical. Environmental samples may be those that come from the surroundings or environment, such as wastewater samples, laboratory solutions, culture media, etc. Clinical samples are those isolated from a subject, such as a human being. Thus, clinical samples refer to blood or other biological fluids, solid tissue samples, cultured cells, cell supernatants, cell lysates, serum, plasma, or other biological fluids.Thus, non-limiting examples of clinical samples may include a sample of blood, urine, serum, sputum, pleural, peritoneal, synovial or cerebrospinal fluid.

[0040] In the context of the present invention, clinical samples are isolated from a subject, the 'subject' being understood to be any animal, preferably a mammal, and more preferably a human being.

[0041] A third aspect of the present invention relates to the use of the biosensor for in vitro diagnosis of P. aeruginosa infection in a subject. 'Diagnosis' for the purposes of the present invention refers to identifying a specific infection or disease, nosological entity, syndrome, or any health-disease condition by analyzing a series of clinical parameters or symptoms characteristic of that disease and distinguishing it from other diseases with similar clinical presentations. In the present invention, P. aeruginosa infection can refer to a wide variety of infections, particularly bacteremia, ventilator-associated pneumonia, urinary tract infections, respiratory infections in patients with cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD), heart tract infections (such as endocarditis), and surgical site infections.Pseudomonas aeruginosa also plays a significant role in causing medical device-associated infections, such as corneal infections related to contact lenses and urinary catheters. Specifically, the lungs are the most common site of P. aeruginosa infection, resulting in high morbidity and mortality, particularly in respiratory and bloodstream infections. In short, P. aeruginosa can cause a variety of infections in humans, depending on the affected body part, and is particularly problematic in individuals with weakened immune systems. In the lungs, it can cause pneumonia, which manifests with symptoms such as cough, fever, and shortness of breath. When it infects the skin and soft tissues, especially wounds and burns, it can cause pain, inflammation, and pus accumulation.In the ear, this bacterium is known to cause otitis externa, also called swimmer's ear, which presents with pain, redness, and discharge. If the infection affects the eyes, it can lead to keratitis, which causes redness, pain, and, in severe cases, vision loss. When the bacteria enter the bloodstream, it can cause bacteremia, an infection that can result in high fever, chills, and septic shock.

[0042] The detailed definitions of 'sample', 'subject', 'diagnose', 'infection' apply to all inventive aspects of this memory.

[0043] Detection and diagnostic methods

[0044] A fourth aspect of the invention relates to the in vitro method for the detection and / or quantification of P. aeruginosa in a sample comprising the steps of:

[0045] - to bring the biosensor of the invention described above into contact with a sample, and

[0046] - detect or measure the indicator in the medium, where the presence of the indicator molecule in the medium shows the presence of the DNA of the bacterium P. aeruginosa in the sample, according to the sample definition provided above and its realization examples.

[0047] Thus, the in vitro method for detecting P. aeruginosa of the invention involves, firstly, contacting the biosensor with a sample, which may be pretreated, if necessary, by contacting it with a buffer solution. Secondly, after waiting a period of time, for example 15 minutes, the indicator is measured in the sample medium by visual observation, fluorescence spectroscopy, or ultraviolet-visible spectrophotometry, among other non-limiting options. In a preferred embodiment, detection and quantification are performed by measuring the fluorescence intensity of the medium; for example, if rhodamine B is being used as the indicator, preferably an excitation wavelength between 500 and 550 nm and an emission wavelength between 550 and 600 nm are used.

[0048] Thus, the detection of the indicator in the medium allows us to conclude that P. aeruginosa DNA is present in the sample, since, upon contact of the biosensor with the sample, the oligonucleotide of SEQ ID NO: 2 will have recognized the P. aeruginosa DNA and will have hybridized, or bound, to it, thereby unblocking the pores of the porous support containing the indicator, releasing it into the medium and thus revealing the presence of the bacterial DNA. A fifth aspect of the present invention relates to an in vitro method for detecting an infection caused by the bacterium P. aeruginosa in a biological sample from a subject, for example, urine, a method comprising the steps of:

[0049] - to bring the biosensor into contact with an isolated biological sample from a subject, and

[0050] - detect the indicator in the medium, so that the presence of the indicator in the medium, detectable by the techniques discussed above, shows that the subject, such as a human being, from whom the biological sample comes, contains P. aeruginosa DNA.

[0051] Before putting the biological sample in contact with the biosensor, it is possible to pretreat it, for example, by putting it in contact with a buffer solution (for example, TRIS (trisaminomethane) or a lysis buffer).

[0052] As in the previous method, the presence of the indicator in the medium allows us to conclude that the subject is infected with P. aeruginosa, since, when the DNA of the same is present in the sample, the oligonucleotide of SEQ ID NO: 2 interacts with it, which produces the unblocking of the pores of the porous support that contain the indicator, releasing it into the medium and therefore evidencing the presence of the bacteria, and therefore, the infection caused by it.

[0053] In both the detection and diagnostic methods described, the sample can be either an environmental or a clinical sample. The clinical sample is preferably selected from the group consisting of blood, urine, serum, sputum, pleural, peritoneal, synovial, or cerebrospinal fluid, and the subject is preferably a human. On the other hand, the infection caused by P. aeruginosa is selected from the group consisting of bacteremia, pneumonia, respiratory infections in patients with cystic fibrosis or COPD, cardiac infections, surgical site infections, corneal infections, skin infections, soft tissue infections, otitis, keratitis, and sepsis.

[0054] Kit comprising the biosensor of the invention and uses thereof

[0055] A sixth aspect of the invention relates to a kit comprising the described biosensor, enabling the implementation of the diagnostic and detection uses and methods described above. Preferably, the kit is embodied in a test strip containing the described biosensor.

[0056] The kit may additionally include other elements that facilitate the object of the present invention, such as buffers, delivery vehicles, material supports, positive and / or negative control components, etc. Optionally, these controls include modifications of the biosensor for use as a control, such as, for example, oligonucleotides that can block the pores of the support but not recognize P. aeruginosa DNA.

[0057] In preferred embodiments, the test kit is a lateral flow assay (LFA) kit. In the context of the present invention, an LFA generally refers to an assay in which a sample flows by capillary action through a test strip comprising one or more fibrous membranes to enable the detection of at least one analyte, or to enable confirmation of the absence of at least one analyte. The LFA may be purely qualitative, providing a yes / no result, or it may be a quantitative assay to determine the total amount or concentration of an analyte. The LFA may be designed so that the presence and intensity of a signal are proportional to the concentration of an analyte. If the analyte is associated with a disease, the LFA may be used for the diagnosis of that disease.

[0058] In some embodiments, the lateral flow assay kit comprises a strip formed by at least one fibrous membrane on which the biosensor material is immobilized. In this configuration, the fibrous membrane preferably comprises: a sample zone for applying the sample to be analyzed; a deposit zone on which the biosensor material of the invention is immobilized; and a detection zone.

[0059] After application, the sample flows by capillary action through the fibrous membrane into the reservoir zone, where the system of the invention is located. If the sample contains the analyte of interest, the indicator contained within the biosensor material is released. Finally, the indicator is detected and / or quantified in the detection zone. In preferred embodiments, the test kit is a lateral flow test kit comprising a strip formed by at least one fibrous membrane onto which the biosensor material of the system of the invention is immobilized. In this configuration, the porous membrane preferably comprises: a sample zone for applying the sample to be analyzed; a reservoir zone onto which the biosensor material of the system of the invention is immobilized; and a detection zone.

[0060] In this case, if the sample contains the analyte of interest, it flows by capillary action through the fibrous membrane to the deposition zone, where the biosensor material is immobilized. The analyte then releases the indicator. Finally, the indicator is detected and / or quantified in the detection zone.

[0061] The membrane used in the lateral flow test kit of the present invention may be made of a variety of materials through which the sample to be examined can pass or move and which are known to a person skilled in the art. Examples of useful membrane materials include, but are not limited to, paper (cellulose), nitrocellulose, sintered glass ceramic, glass fiber (GF), polyvinylidene fluoride, nylon, filler-modified nylon, polyethersulfone, and Fusion 5™ (borosilicate glass fibers), or a combination thereof.

[0062] Typically, the membrane is mounted or adhered onto a backing surface that acts as a support and facilitates handling of the strip.

[0063] The lateral flow assay kit can be configured to function as a strip that is immersed in (or otherwise comes into contact with) the sample to be tested, or as a rigid plastic cassette provided with a well onto which the sample is applied.

[0064] The kit can measure the indicator in the sample medium, through visual observation, fluorescence or electrochemiluminescence, among other non-limiting options.

[0065] The kit can be supplemented with a portable fluorescence reader, which allows not only the detection of P. aeruginosa but also its quantification. In a preferred embodiment, detection and quantification are performed by measuring fluorescence intensity using a mobile phone.

[0066] Additionally, the kit may include instructions for its proper handling, either printed on a support that accompanies the kit, or through a QR code, among other non-limiting options.

[0067] A seventh aspect of the present invention relates to the use of the kit comprising the biosensor for the detection of DNA of the bacterium P. aeruginosa in an isolated sample.

[0068] An eighth aspect of the present invention relates to the use of the kit comprising the biosensor for diagnosing in vitro an infection caused by P. aeruginosa in a subject, such as a human being.

[0069] In both uses described, the sample can be an environmental sample or a clinical sample, which in the latter case can be a sample of blood, urine, serum, sputum, pleural, peritoneal, synovial or cerebrospinal fluid, among other non-limiting options, while the infection caused by P. aeruginosa can be: bacteremia, pneumonia, respiratory infections in patients with cystic fibrosis or COPD, heart tract infections, surgical infections, corneal infections, skin infections, soft tissue infections, otitis, keratitis and / or sepsis.

[0070] Procedure for preparing a biosensor

[0071] A ninth aspect of the present invention relates to the biosensor preparation process, the specific steps of which determine the correct functioning of the biosensor in accordance with the object of the invention. Thus, the biosensor preparation process comprises at least four steps: a) introducing an indicator into a porous support, where the porous support has an internal nanoporous porous structure with pores of diameter between 1 nm and 100 nm, preferably between 2 and 50 nm, such that a plurality of these pores provide access to the surface of the support to the outside, resulting in a loaded porous support; b) chemical modification of the surface of the loaded porous support by attaching reactive groups, such as isocyanate groups, to the outer surface of the loaded porous support, resulting in a loaded support functionalized with reactive groups on its outer surface;c) adding a first short-sequence binding oligonucleotide (up to 200 nucleotides) 01, comprising the nucleotide sequence SEQ ID NO: 1, to the charged and functionalized support, so as to obtain a support with anchoring elements by forming chemical bonds, such as urea bonds, between the reactive groups, such as isocyanate groups, and the amino group of oligonucleotide 01; d) adding a second oligonucleotide 02 by hybridization with the short sequence 01, oligonucleotide 02 recognizing the DNA of the bacterium P. aeruginosa 4, obtaining a biosensor;

[0072] Steps a), b), c), and d) are carried out under controlled agitation, temperature, and humidity conditions. Preferably, agitation is maintained between 10 and 200 rpm for a time between 1 and 32 hours at a temperature between 20 and 30°C. Humidity control is important because, in the presence of water, the anchoring of the reactive group and the formation of the bond between the organic groups and the O1 linking oligonucleotide could be compromised.

[0073] Advantages of the biosensor and its applicability

[0074] As explained, there is a significant challenge in the field of developing new technologies for the rapid and reliable detection of novel biomarkers that would allow for the early diagnosis of P. aeruginosa infections. However, even the most innovative technologies recently developed continue to present problems, mainly related to long diagnostic times, the need for highly qualified personnel, and high equipment costs, which hinder their implementation in hospitals with limited resources.

[0075] This new identification system based on nanoporous materials with molecular gates allows the detection of P. aeruginosa in a short time (less than 30 minutes in suspension and less than 5 minutes using the lateral flow assay on a reagent strip, compared to 3-4 days for conventional techniques) from a concentration of 0.153 ng pL -1 of genomic DNA in suspension and 0.92 ng pL-1 using lateral flow assay, which represents a significant diagnostic advance. It should be noted that the detection limit of the assays performed is 0.05 ng / L -1 Therefore, they were carried out above this limit.

[0076] Its high specificity at short time points allows for the discrimination of P. aeruginosa DNA against DNA from other bacteria and fungi that cause nosocomial infections, as well as other species of the genus Pseudomonas. Thanks to its high discriminatory power, the biosensor of the invention solves the significant problem of erroneously detecting other microbial species, especially in the case of P. fluorescens, one of the main problems with conventional P. aeruginosa detection techniques. Furthermore, the biosensor exhibits high sensitivity, superior to existing techniques, with very low bacterial detection limits (between 0.23 and 28.6 CFU mL⁻¹). 1), a fact that allows the detection of the presence of P. aeruginosa even at the beginning of the infection process, and not during late stages like most of the methods used.

[0077] The ability to diagnose at early stages, thereby preventing physicians from resorting to empirical treatments, translates into a better quality of life for patients, faster initiation of therapy, and more targeted use of antibiotics, thus avoiding contributing to the development of P. aeruginosa resistance. These excellent results have been made possible by the successful design of the molecular gate, which has proven to be a previously unknown target for the highly selective and sensitive detection of P. aeruginosa.

[0078] All these properties of the developed method have been successfully validated with real biological urine samples from patients of the Hospital Universitari i Politécnic La Fe with active infections by this microorganism, demonstrating its potential application in the clinical field.

[0079] The developed system can be used by healthcare professionals, such as those in Clinical Microbiology Departments, allowing them to tailor the antimicrobial treatment assigned to the patient and improve the clinical prognosis of P. aeruginosa infections thanks to the speed, sensitivity, and specificity of this technology. This will prevent the over-prescription of antibiotics, which is advantageous in terms of the economic sustainability of the healthcare system, the patient's quality of life, and the reduction of antimicrobial resistance in P. aeruginosa. Furthermore, the simplicity and low cost of this sensor system are noteworthy, as it can be obtained quickly and reproducibly on an industrial scale, making the method highly competitive.The rapid diagnostic time, the simplicity of the procedure, and its biocompatibility mean that this method does not require specialized technologists and significantly reduces implementation costs in the healthcare sector, especially in hospitals with fewer resources. Therefore, clinical analysis of the patient in the primary care and / or outpatient setting is considered highly feasible.

[0080] This proposed technology has not been previously used for the diagnosis of infectious diseases caused by P. aeruginosa and, therefore, it is a method of high inventive capacity resulting from a notable R&D process that would significantly differentiate the biosensor of the present invention from what is known in the State of the Art.

[0081] BRIEF DESCRIPTION OF THE DRAWINGS

[0082] To complement the description that follows and to aid in a better understanding of the characteristics of the invention, in accordance with the preferred examples of its practical embodiment detailed below, a set of drawings is included as an integral part of said description, in which, for illustrative and non-limiting purposes, the following has been represented:

[0083] Figure 1 shows a schematic representation of the biosensor synthesis procedure of the invention.

[0084] Figure 2 shows a schematic representation of the method for detecting and quantifying the presence of the bacterium P. aeruginosa in a sample, illustrating the process of controlled release of the indicator, the fluorescent molecule rhodamine B, according to an example embodiment, after the specific recognition of O2 with the R region PFigure 3A shows the predicted secondary structure of the phzA2 protein from P. aeruginosa. The isocyanate reactive groups are not shown in this figure for simplicity. Figure 3A shows the predicted secondary structure of the phzA2 protein. The secondary structure of the phzA2 gene from P. aeruginosa was estimated using the DNA secondary structure prediction programs VectorBuilder and RNAfoId (University of Vienna). The color scale indicates the probability of nucleotide hybridization (0-1), with 0 representing the minimum probability and 1 representing the maximum probability of self-linking. The arrows indicate the first and last nucleotides of a first candidate for the target region R. P hzA2, RL (which comprises the nucleotide sequence SEQ ID NO:3 ((5'— TTCCGGGGCCATGAGAGCCTCAGGCGGCTCGCCGAGTGGCTCGAGCGCTGCTTC CCCGACTGGGAGT-3')), as well as the first and last nucleotide of another candidate sequence to be the target R PhzA2, RS (which comprises the nucleotide sequence SEQ ID NO:5 (5'- GTTGTCGGTAAACCCTTTCAACCGTTGGTACTCTCGCAT-3'), which was ultimately discarded after experimental selectivity studies.

[0085] Figure 3.B shows the details of the candidate target regions RS and RL according to Figure 3.A.

[0086] Figure 4 shows the predicted tertiary structure of the phzA2 protein. The three-dimensional arrangement of the structure is shown in the lower left corner. The arrows indicate the location of the first and last amino acids encoded by the first and last triplets of the RS and RL target sequences. This tertiary structure prediction was performed using Alphafold, and the image was obtained and modified from Uniprot.

[0087] Figure 5 shows the results of experiments on the controlled release of rhodamine B from the pores of the S4(O2L) biosensor in TRIS buffer (pH 7.5) in the absence and presence of the purified RL pzhA2 gene target sequences to evaluate the concentration and optimal secondary structure of the pore block. Figure 5A shows an experiment in which the pores of the biosensor (S4) have been blocked with 2.5 pM of the oligonucleotide O2i_, (oligonucleotide O2 developed for the recognition of the candidate target region RL). Figure 5B shows an experiment in which the pores of the support (S4) have been blocked with 5 pM of the oligonucleotide O2L. Figure 5C shows an experiment in which the pores of the support S4 are blocked with 7.5 pM of the oligonucleotide O2L. Figure 5D shows an experiment in which the pores of the support (S4) have been blocked with 10 pM of the oligonucleotide O2L.The x-axis represents time in minutes, while the y-axis represents intensity in arbitrary units. Data series a) corresponds to measurements taken in the presence of the purified RL region of the P. aeruginosa phzA2 gene, while data series b) corresponds to measurements taken in the absence of said RL region.

[0088] Figure 6 shows the result of experiments on the controlled release of rhodamine B from the pores of the S4(O2s) material in TRIS buffer (pH 7.5) in the absence and presence of the purified RS pzhA2 gene target sequences to evaluate the optimal concentration and secondary structure for pore blocking.Figure 6A shows an experiment in which the pores of the S4 support have been blocked with 2.5 pM of the O2S oligonucleotide designed for recognition of the RS candidate region, such that the O2S oligonucleotide comprises the sequence SEQ ID NO:4 (5 - TTTTTGGGGGGGTTGTCGGTAAACCCTTTCAACCGTTGGTACTCTCGCATGGGGGGTTT TT-3'). Figure 6B shows an experiment in which the pores of the support (S4) have been blocked with 5 pM of the O2S oligonucleotide. Figure 6C shows an experiment in which the pores of the S4 support have been blocked with 8.33 pM of the O2S oligonucleotide. Figure 6D shows an experiment in which the pores of the S4 support have been blocked with 12.5 pM of the O2S oligonucleotide. The x-axis represents time in minutes, while the y-axis represents intensity in arbitrary units.Data series a) show the intensity of rhodamine B released in the presence of the purified RS region of the pzhA2 gene of P. aeruginosa, while data series b) show the intensity of rhodamine B released in the absence of the RS region of P. aeruginosa.

[0089] Figure 7 shows the results of controlled rhodamine B release experiments from the pores of S4 material in TRIS buffer (pH 7.5) in the absence and presence of the purified phzA2 gene target sequences RS and RL. Figures 7A, 7B, and 7C show experiments to verify the intensity of rhodamine B release in the presence of the purified RL region of the P. aeruginosa phzA2 gene on S4 supports blocked with 5 pM of the oligonucleotide O2L (SEQ ID NO: 2). Figure 7D shows the assays of rhodamine B release intensity on S4(O2S) supports in the presence of purified RS (n = 12 experiments), while Figure 7E shows the assays of rhodamine B release intensity on S4(O2L) supports in the presence of purified RL (n = 15 experiments). The x-axis represents time in minutes, while the y-axis represents intensity in arbitrary units.Data series a) show the intensity of rhodamine B released in the presence of the target region, while data series b) show the intensity of rhodamine B released in the absence of the target region.

[0090] Figure 8 shows the results of a controlled release study of the fluorophore from the porous interior of the material in the presence of P. aeruginosa ATCC® 27853™ in TRIS buffer (pH 7.5). Figure 8.A details the fluorescence intensity in the presence of 2 ng pL-1 of P. aeruginosa ATCC® 27853™ genomic DNA (data series a)) and in its absence (data series b)), while Figure 8.B details the fluorescence intensity in the presence of 48 CFU mL -1 of P. aeruginosa ATCC 2785 ATCC® 27853™ and in absence (data series b). The x-axis represents time in minutes, while the y-axis represents intensity in arbitrary units.

[0091] Figure 9 shows the results of a series of experiments designed to evaluate the biosensor's response to the presence of genomic DNA from other fungi and bacteria and its sensitivity. Figure 9.A shows the fluorescent intensity released from the pores in the presence of 0.2 ng pL. -1 Figure 9B shows the fluorescence intensity of P. aeruginosa, C. albicans, S. aureus, Pseudomonas putida, P. fluorescens, Escherichia coli, and Klebsiella pneumoniae at 10 minutes, with the different microorganisms represented on the x-axis and the intensity in arbitrary units on the y-axis. Figure 9B shows the fluorescence intensity with increasing concentrations of Colony Forming Units of P. aeruginosa, with the bacterial concentration in CFU / mL represented on the x-axis and the intensity in arbitrary units on the y-axis. Figure 9C shows the fluorescent intensity released from the pores in the presence of 0.2 ng pL-1 Figure 9.D shows the intensity of rhodamine B release in the presence of human urine samples after 30 minutes in TRIS buffer (pH 7.5), where data series a) corresponds to positive samples and data series b) corresponds to negative samples. Finally, Figure 9.E shows the ROC curve analysis, which indicates the fluorescence discrimination limit of the clinical samples, 1.92 fluorescence units, with specificity (in %) on the x-axis and sensitivity (in %) on the y-axis.

[0092] Figure 10 shows a schematic representation of the kit comprising the biosensor of the invention, materialized in a lateral flow assay kit that includes a test strip, as well as the scheme of the recognition of P. aeruginosa DNA using said test strip based on the S4-GF biosensor (acronym referring to the fact that the test strip comprises a glass fiber membrane).

[0093] Figure 11 shows a representation of the intensity of rhodamine B release in the presence of P. aeruginosa genomic DNA samples and human urine at 1 minute in TRIS buffer (pH 7.5), where the concentration of P. aeruginosa genomic DNA in ng uL is represented on the abscissa axis -1 and on the y-axis the intensity as (I- lo / lo- An image of the rhodamine B released as a function of P. aeruginosa using the developed kit is also included.

[0094] Figure 12 shows a representation of the intensity of rhodamine B release in the presence of human urine samples obtained using the S4-GF biosensor and recording the fluorescence using a mobile phone camera, where the sample type has been represented on the abscissa axis, with data series a) being positive samples and data series b) being negative samples, while the intensity has been represented on the ordinate axis as (l-lo / lo).

[0095] EXAMPLES

[0096] Materials and instrumentation

[0097] Chemical reagents

[0098] The following reagents and solvents were used to perform the corresponding studies on the prepared materials: (3-isocyanatopropyl)triethoxysilane, triethylamine (TEA), tris(hydroxymethyl)aminomethane (TRIS), potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, potassium chloride, hydrochloric acid, TRITON, ethylenediaminetetraacetic acid (EDTA), rhodamine B, RNase enzyme, ammonium acetate, sodium dodecyl sulfate (SDS), chloroform, phenol, isoamyl alcohol, and ethanol. All these reagents were obtained from Sigma-Aldrich Química (Madrid, Spain). The nanoporous anodic alumina supports were purchased from InRedox® (CO, USA). In all experiments, the hybridization buffer was based on a mixture of 20 Mm Tris and 37.5 mM MgCl2 adjusted by HCI to a pH of 7.5.

[0099] Microorganisms and growth conditions

[0100] All clinical microorganisms used in this study were isolated from patients from the Hospital Universitah i Politécnic La Fe and identified by the Department of Severe Infection of the La Fe Health Research Institute.

[0101] The culture media used for their growth were Thyptone Soy Broth (TSB), Lysogeny Broth (LB), and Yeast Extract Peptone Dextrose (YPD). P. aeruginosa sp., P. putida sp., P. fluorescens sp., S. aureus, and K. pneumoniae were grown in TSB medium; E. coli in LB medium; and C. albicans in YPD medium. All microorganisms were grown for 16–24 h at 37°C.

[0102] Example 1: Oligonucleotide Design 02

[0103] After obtaining a comprehensive list of potential P. aeruginosa-specific genes identified through Next Generation Sequencing (NGS) and pangenome studies, each gene was analyzed and its specificity and selectivity for P. aeruginosa against other Pseudomonas species and microorganisms was verified individually. This process was performed manually using the BLAST and Uniprot platforms. The BLAST search was optimized by selecting the 'highly similar sequences (megablast)' filter, and sequences with 98% or greater coverage were considered. All candidate sequences identified by Uniprot that showed a high probability of self-folding were excluded. Many of the candidate genes for P. aeruginosa were discarded because they shared a large portion of their nucleotide sequence with other bacteria of the genus Pseudomonas, such as P. fluorescens and P. putida.Next, short regions (between 30 and 70 nucleotides (nt)) of the finalist genes were manually selected to identify and identify the target region. By shortening the number of nucleotides compared to the complete genes, the final selectivity screening was performed, leaving only different potential target regions from the same gene. This was the final step in selecting the R region. P hzA2 and the corresponding molecular gate (O2) were the different studies of secondary structure prediction through VectorBuilder and RNAfoId until the optimal conformation to exert a correct pore block and R recognition P hzA2 of P. aeruginosa, where two candidate target regions were obtained: of 39 nucleotides (RS, comprising the sequence SEQ ID NO: 5 (5'-

[0104] GTTGTCGGTAAACCCTTTCAACCGTTGGGTACTCTCGCAT-3')) and 67 nucleotides (RL, comprising the sequence SEQ ID NO: 3 (5'-

[0105] TTCCGGGGCCATGAGAGCCTCAGGCGGCTCGCCGAGTGGCTCGAGCGCTGCTTCCCC GACTGGGAGT-3') ), as detailed in Figures 3 and 4. This last step was evaluated and confirmed experimentally, carrying out a series of controlled release experiments of rhodamine B from the pores of the biosensor (S4), experiments illustrated in Figures 5 to 7. In this way, the design procedure of the O2 followed to specifically recognize P. aeruginosa has made it possible to reach a target sequence of only 67 nucleotides (nt) compared to the 5.2 - 7 Mbp of the P. aeruginosa genome, being a process that has required a great inventive effort on the part of the research team of the present patent application.Thus, the oligonucleotide sequence 02 designed for the finally selected target region RL has 89 nts, of which 22 nts (11 at each end) have been designed at their ends to hybridize with 01, while the 67 central nts are used to close the pore and recognize its target sequence in P. aeruginosa.

[0106] Thus, Figure 5 shows a series of experiments performed in triplicate to analyze the controlled release of rhodamine B from the pores of the biosensor (S4) containing the oligonucleotide O2 for the candidate region of 67 nucleotides RL, S4(O2L) experiments in TRIS buffer (pH 7.5), using an O2L concentration of 2.5 pM, 5 pM, 7.5 pM and 10 pM.

[0107] On the other hand, Figure 6 shows a series of experiments performed in triplicate to analyze the controlled release of rhodamine B from the pores of the biosensor (S4) containing the oligonucleotide O2 for the candidate region of 39 nucleotides RS, S4(O2s) experiments in TRIS buffer (pH 7.5), using an O2s concentration of 2.5 pM, 5 pM, 8.33 pM and 12.5 pM.

[0108] Finally, Figure 7 shows a series of experiments to analyze the controlled release of rhodamine B from the pores of the S4 biosensor material in TRIS buffer (pH 7.5) in the absence and presence of the purified pzhA2 gene target sequences, RS and RL.

[0109] Following subsequent selectivity and sensitivity tests (illustrated in Figure 9), the RL sequence and its molecular gate O2L were chosen as the specific recognition sequences of the biosensor of the invention.

[0110] As shown in Figure 8, the detection system exhibits virtually no release in the absence of P. aeruginosa in the medium. However, in the presence of both purified bacterial genomic DNA (Fig. 8.1) and P. aeruginosa Colony Forming Units (CFU) (Fig. 8.2), the biosensor's molecular gates are able to be displaced by specific hybridization with bacterial DNA on the porous surface, thereby releasing the fluorescent indicator in a very short time.

[0111] Example 2: Biosensor Synthesis

[0112] The following details an example of the synthesis of the biosensor, according to a preferred embodiment of the invention. The first step of the procedure for obtaining the biosensor (4) consists of loading the interior of the porous support (1) by diffusion with the indicator (2), in this case, the fluorescent molecule rhodamine B.

[0113] For this purpose, 24 porous inorganic supports (1) of nanoporous anodic alumina (NAA) with a diameter of 2-5 mm, a thickness of 5 ± 0.1 pm, and an internal mesoporous structure of 2-50 nm in diameter were immersed in a solution of rhodamine B dissolved in acetonitrile (CH3CN, 1.57 mM, 6 mg, 8 mL) by diffusion of the previously prepared solution into the porous support (1) with stirring at 50 rpm, for 24 h at a temperature of 25°C. This yielded a porous support (S1) loaded with the indicator molecule.Following the loading process of the indicator molecule (2) into the porous interior, the external surface of the support is chemically modified by functionalizing the porous surface of the 24 loaded AAN supports (S1) by immersing them in (3-isocyanatopropyl)triethoxysilane (1000 pL, 1.32 mmol), with stirring at 50 rpm, for 5.3 h at a temperature of 25°C in order to provide isocyanate groups (3) on the external surface of the AAN support, obtaining a loaded and functionalized support (S2).

[0114] The loaded and functionalized supports (S2) were stored at 4-10°C overnight. The isocyanate groups (3) are capable of forming a urea bond with short-sequence binding oligonucleotides (O1). For this purpose, each loaded and functionalized AAN support (S2) was immersed in a solution of rhodamine B in acetonitrile (262.5 pg, 1.57 mM, 87.5 pL), the oligonucleotide that will allow the covalent bonding (O1) comprising the sequence SEQ ID NO: 1 (NH2-(CH2)6-5'-AAA AAA CCC CCC-3') (100 pM, 1.25 pL), and triethylamine (TEA, 0.5 pL), which acts as a catalyst for the urea bond. In this stage, in addition to (01), the solution in which the support (S2) is immersed contains rhodamine B to compensate for the pressure losses generated when the sensor is still open (it is not yet closed with the molecular gate).Therefore, when immersing the supports that are not yet sealed at this stage, just as the porous interior is charged by diffusion, some of the rhodamine B could escape through the same diffusion process into the surrounding medium. Adding the rhodamine B solution ensures that, even though diffusion occurs between the porous interior and the exterior when immersed in the hybridization solution, this chemical equilibrium is maintained, minimizing the loss of rhodamine B to the outside.

[0115] This step of adding the oligonucleotide (01) to the support (S2) was performed with stirring at 50rpm, at 25°C, and lasted 3 hours.

[0116] Thus, a support with anchoring elements (S3) is obtained by forming urea bonds between the isocyanate groups (3) and the amino groups incorporated into the phosphate groups of the 5' ends of the oligonucleotides (O1) ((NH2)-5'- AAA AAA CCC CCC -3').

[0117] The binding oligonucleotides (01) with sequence SEQ ID NO: 1 (NH2-(CH2)6-5'-AAA AAA CCC CCC-3') anchored on the porous surface through chemical bonding were designed to act as an anchor and subsequently covalently bind the oligonucleotides (02) comprising the sequence SEQ ID NO:2 (5'- TTTTTGGGGGGACTCCCAGTCGGGGAAGCAGCGCTCGAGCCACTCGGCGAGCCGCC TGAGGCTCTCATGGCCCCGGAAGGGGGGTTTTT-3') and which are added to the support (S3) for obtaining the biosensor (S4). Oligonucleotides (O2) are specific in the recognition of P. aeruginosa, as outlined in Figure 1. Specifically, the 3'-T TTT GGG GGG-5' sequence included in the bacterial recognition oligonucleotide O2 hybridizes specifically with the 5'-A AAA CCC CCC-3' region of O1 by means of the covalent bond described above.Thus, to form the molecular gate, a bond is first formed between the reactive group present on the surface of the porous support and oligonucleotide 01 via its amino group. Next, oligonucleotide 02, which recognizes the bacterial DNA, is added through specific molecular hybridization between oligonucleotide 01 and oligonucleotide 02 via a 22-nucleotide sequence (11 at each end of 02) designed to specifically recognize oligonucleotide 01. This step of adding oligonucleotide 02 to the support (S2) was performed with stirring at 50 rpm, at 25°C, and lasted 2 hours.

[0118] The molecular gate designed in this study that includes the sequence (5 1 — TTTTTGGGGGGACTCCCAGTCGGGGAAGCAGCGCTCGAGCCACTCGGCGAGCCG CCTGAGGCTCTCATGGCCCCGGAAGGGGGGTTTTT-3') is capable of recognizing a specific region of the phzA2 gene of P. aeruginosa (R PhzA2), as shown in Table 1. The aforementioned sequence included in oligonucleotide 02 could slightly increase or decrease its size by adding more or fewer recognition nucleotides, or by varying the number of nucleotides at both ends, which are involved in the recognition with the binding oligonucleotide 01.

[0119] For successful sensor synthesis, it is essential to perform each step at a controlled temperature of 20–30°C with agitation (10–200 rpm) to ensure homogenization, as well as to control humidity (maximum 45–10%). To achieve this, a humidity control material, such as silica gel beads, is preferably added at all stages of the biosensor preparation procedure. These beads absorb water molecules from the environment to prevent hydration during the synthesis process. The porous surface of the supports obtained in steps a), b), and c) must be in direct contact with the corresponding hybridization medium and then carefully dried at room temperature. In this way, only in the presence of P's genomic DNA will the sensor react.aeruginosa oligonucleotides O2 are able to specifically recognize by complementarity and move from the surface of the pores, leading to the release of the fluorescent indicator, as detailed in Figure 2. biosensor sensitivity

[0120] To evaluate the biosensor's detection capability, an assessment of the system's specificity and sensitivity was conducted. The system's response was monitored in the absence and presence of genomic DNA from six microorganisms that can interfere with the detection of P. aeruginosa: C. albicans, S. aureus, P. putida, P. fluorescens, E. coli, and K. pneumoniae (Figure 9A). These interfering microorganisms were chosen because they share an infection niche with P. aeruginosa and cause common nosocomial infections in patients with similar symptoms in many cases; they also pose a significant challenge in hospital settings due to their drug resistance (1617). Of particular concern are the false-positive diagnostic errors generated by P. fluorescens and P. putida in current P. aeruginosa diagnostic techniques.aeruginosa, and therefore the ability of the biosensor of the invention to specifically differentiate P. aeruginosa from these species of the genus Pseudomonas is noteworthy. The results verify the high selectivity of the biosensor for the specific detection of P. aeruginosa in a significantly perceptible manner from very short times compared to other microorganisms that cause common nosocomial infections and to other species of the genus Pseudomonas, as shown in Figure 9.A.

[0121] Similarly, the sensitivity of the biosensor was evaluated using increasing concentrations of colony-forming units (CFU) of the bacterium P. aeruginosa ATCC® 27853™. Compared to current diagnostic techniques, the developed system exhibits extremely high sensitivity with a detection limit of 0.2–28.6 CFU / mL. -1lower than that obtained by standard molecular techniques used in clinical practice (Figure 9.B). The detection capacity of the new system was also tested against 17 clinical strains of P. aeruginosa isolated from patients at the Hospital Universitari i Politécnic La Fe. The results demonstrated the sensor's high capacity to detect the presence of P. aeruginosa, regardless of the specific clinical strain of the bacteria (Figure 9.C). In this scenario, the biosensor's performance was evaluated as an alternative diagnostic tool to existing techniques for detecting P. aeruginosa in 62 clinical urine samples from infected patients at the Hospital Universitari i Politécnic La Fe (24 P. aeruginosa-positive samples, 38 negative samples) (Figure 9.D). To evaluate the predictive power of the biosensor (S4), a receiver operating characteristic (ROC) analysis was performed with an area under the curve (AUC) of 0.961 (AUC: 0.961 ± 0.025; **** p-value < 0.0001). Based on these results, at the detection threshold between positive and negative clinical samples of 1.920, the biosensor (S4) demonstrated a sensitivity of 91.67%, a specificity of 97.30% and a selectivity of 96%, obtaining 2.63% false positives and 0% false negatives (Figure 9.E).

[0122] The developed system was successfully applied to the bacterial identification of P. aeruginosa in complex clinical urine samples from infected patients, obtaining highly accurate data in less than 30 minutes for suspension assays of the material containing the biosensor in a sample, and less than 5 minutes (even times close to 1 minute) in assays with the FLA kit containing the biosensor, making the biosensor an ideal element for implementation in the health field.

[0123] Example 3: Incorporation of the biosensor into LFA test strips and tests

[0124] The biosensors (S4) were incorporated into a test strip in the form of a highly selective and sensitive lateral flow assay (LFA) (5) for the in situ detection of P. aeruginosa with a reading, which advantageously can be carried out by means of a portable fluorescence reader device, which can be achieved with the help of an adapted conventional smartphone.

[0125] According to a preferred embodiment of the invention illustrated in Figure 10, the test strip has two zones: zone A, where the sensor material (S4) is deposited at the bottom of the strip, and zone B, where the fluorescence signal of the released dye (2) migrating with the solvent flow is collected, as shown in Figure 10. The difference between the experiment performed with an uncontaminated sample (7) containing P. aeruginosa and a contaminated sample (8) containing the bacteria is evident. The function of the absorbent pad (6) at the top is to absorb the fluid through the membrane by capillary forces. In this way, the released dye (2) can be transported through the membrane, allowing spatial separation between the nanoprobes and the released dye.When the test strip is immersed in a solution that does not contain the target analyte, no signal (or a weak signal) is recorded in zone B, as the dye (2) trapped in the pores of the biosensor (S4) is not released. However, when the strip is immersed in a solution containing P. aeruginosa, the interaction of the analyte with the biosensor (S4) results in the release of the rhodamine B charge (see above). The released dye flows with the solvent to zone B, where the fluorescence emission can be measured using, for example, a smartphone mounted on a 3D-printed housing that includes a strip holder, thus ensuring defined lighting conditions. Specifically, for the experiments, the strips were immediately inserted into a custom-designed 3D-printed strip holder.

[0126] The fluorescence of the released dye was measured using a CMOS camera on a mobile phone (Samsung Galaxy™ S6), with an LED emitting at 520 nm, a band filter centered at 515 ± 10 nm to eliminate residual light from the LED, and a long-pass filter at 550 nm to efficiently capture the fluorescent emission of rhodamine (2) released along the membrane. Images collected from the strips were analyzed using ImageJ software, analyzing the integrated red channel fluorescence density of the samples while correcting for background fluorescence. Thus, the release of rhodamine B (RhB) can be correlated with the amount of P. aeruginosa present in the sample by comparison with a calibration curve.

[0127] To study the response of the reagent strip membranes based on the concentration of P. aeruginosa genomic DNA in hybridization buffer, 100 pL of different concentrations of purified genomic DNA (0.001 - 0.25 ng pL) were added. -1 ) in Zone A of the test strip membranes (0.5 x 2.5 cm). Hybridization buffer without the P. aeruginosa DNA genomic sample was used as a negative control. After 1 minute, the test strip membranes were inserted into the smartphone's integrated LED reader to measure the fluorescence of the RhB released in Zone B.

[0128] As shown in Figure 11, there is a significant increase in emitted fluorescence as the bacterial genomic concentration increases. This study allows the calculation of a detection limit of 0.05 ng / pL. -1 of P. aeruginosa genomic DNA in just 1 minute.

[0129] Finally, the test strips containing the biosensor of the invention were tested on clinical urine samples from infected and uninfected patients at the Hospital Universitah i Politécnic La Fe, diagnosed by the reference method. They demonstrated the ability to detect the presence of P. aeruginosa in clinical urine samples within 1 minute in a statistically significant manner and without the need for prior sample pretreatment (Figure 12). As in the evaluation of the biosensor (S4), the discriminative capacity of the S4-LFA membrane against positive and negative clinical samples from patients was evaluated using ROC analysis, obtaining an AUC value of 0.985 ± 0.024. Following this criterion, 11 samples were classified as positive (True positives: 11 and False positives: 0), while 6 samples were classified as negative (True negatives: 6 and False negatives: 0).The results demonstrate a sensitivity of 100% and a specificity of 90.91%.

Claims

CLAIMS 1 a Biosensor characterized in that it comprises: - a porous support (1) surface functionalized with reactive groups (3) and loaded with an indicator (2) inside at least a plurality of its pores, where said pores provide access to the exterior of the porous support (1); and - at least one oligonucleotide (O2) comprising the nucleotide sequence SEQ ID NO: 2, oligonucleotide (O2) specifically recognizing a region of the DNA of the bacterium P. aeruginosa, wherein - the porous support (1) has an internal nanoporous porous structure with pores ranging in diameter from 1 nm to 100 nm; - the oligonucleotide (O2) is anchored to the surface of the porous support (1) via a linking oligonucleotide O1 comprising the nucleotide sequence SEQ ID NO: 1, the linking oligonucleotide (O1) being linked to the support (1) via chemical bonds established with the reactive groups (3); such that the O1 / O2 / chemical bond assembly constitutes a molecular gate such that: - In the absence of DNA from the bacterium P. aeruginosa, the molecular gate blocks access to the pores of the support (1) to the outside, preventing the indicator from leaving, and - In the presence of DNA from the bacterium P. aeruginosa, the oligonucleotide (O2) recognizes and binds to a specific region of the DNA of the bacterium P. aeruginosa, unlocking access of the pores of the porous support (S4) to the outside and allowing the indicator to exit. 2 a Biosensor according to claim 1 a, characterized in that the DNA region is a specific region of the pzhA2 gene of the bacterium P. aeruginosa. 3 a Biosensor according to claim 1 a , characterized in that the reactive group (3) is a neutral or cationic organic group. 4 a Biosensor according to claim 3 a , characterized in that the neutral organic group is selected from the group consisting of carboxylic acid (-COOH), alcohol (-OH), aldehyde (-CHO), C2-C30 alkenyl, C2-C30 alkynyl, amine (-NH2 or -NR'R"), amide (-C(O)NR'R"), azide (-N3), ketone (-O=O), ester (-COOR 1 ), ether (R'-OR” ), halogen-containing group, amine (RR'C=NR"), isocyanate (-N=C=O), isothiocyanate (-N=C=S), nitrile (-C=N), nitro (-NO2) and thiol (-SH), each R' and R” independently representing a hydrogen, a C2-C30 alkyl, a C2-C30 alkenyl, or a C2-C30 alkynyl. 5 a Biosensor according to claim 3 a, characterized in that the cationic organic group is selected from the group consisting of amines (-NHs + ), guanidinium groups ([CHeNs]*), phosphonium (-PH4 + ) Or quaternary ammonium (-NR4 + ), where R is independently selected from a linear or branched C1-C30 alkyl and a C3-C6 cycloalkyl. 6 a Biosensor according to claim 1 a , characterized in that the porous support (1) is obtained from nanoporous anodic alumina, nanoporous silica, titanium oxide or graphene. 7 a Biosensor according to claim 1 a , characterized in that the porous support (1) is macroporous, mesoporous or microporous. 8 aBiosensor according to any of the preceding claims, characterized in that the indicator (2) is selected from a dye, a fluorophore, an electrochemiluminescent substance, a redox-active substance, a plasmonic resonance substance, or biologically active substances such as a protein, biomolecules smaller than 50 nm, enzymes, nucleic acid fragments, or a mixture thereof. 9 a Biosensor according to claim 8 a characterized in that the indicator (2) is selected from [Ru(bipi)3]Cl2, rhodamine 101, rhodamine 110, rhodamine B, safranin, fluorescein, sodium salt of sulforodamine B and / or PEG-Bodipis. 10 a Use of a biosensor according to any of the preceding claims for the detection of P. aeruginosa DNA in a sample. 11 a Use of a biosensor according to claim 10 awhere the sample is an environmental sample or a clinical sample, where the clinical sample is selected from the group consisting of blood, urine, serum, sputum, pleural, peritoneal, synovial, or cerebrospinal fluid. 12 a Use of a biosensor according to any of the claims in claim 1 a at 9 a to diagnose in vitro the infection caused by P. aeruginosa in a subject. 13 a Use of a biosensor according to claim 12 a where the infection caused by P. aeruginosa is selected from the group consisting of: bacteremia, pneumonia, respiratory infections in patients with cystic fibrosis or COPD, heart tract infections, urinary tract infections, surgical infections, corneal infections, skin infections, soft tissue infections, otitis, keratitis, and sepsis. 14 a Use of a biosensor according to claim 12 a or 13 awhere the subject is a human being. 15 a . In vitro method for detecting P. aeruginosa bacterium DNA in a sample comprising the following steps: - to put a biosensor into contact according to any of claim 1 a a 9 a with a sample, and - detect the indicator in the medium, where the presence of the indicator in the medium demonstrates the presence of the DNA of the bacterium P. aeruginosa in the sample. 16 a In vitro method for diagnosing P. aeruginosa infection in a subject comprising the following stages: - to put a biosensor into contact according to any of claim 1 a a 9 a with an isolated biological sample from a subject, and - detect the indicator in the medium, where the presence of the indicator in the medium shows that the subject suffers from an infection caused by the bacteria P. aeruginosa. 17 aMethod according to claim 16 a where the infection caused by P. aeruginosa is selected from the group consisting of: bacteremia, pneumonia, urinary tract infections, respiratory infections in patients with cystic fibrosis or COPD, heart tract infections, surgical infections, corneal infections, skin infections, soft tissue infections, otitis, keratitis, and sepsis. 18 a . Method according to any of the claims of 15 a at 5 PM a where the sample is an environmental sample or a clinical sample, where the clinical sample is selected from the group consisting of blood, urine, serum, sputum, pleural, peritoneal, synovial, or cerebrospinal fluid. 19 a . Method according to any of the claims of 15 a at 6 pm a where the sample is pretreated with a buffer solution. 20 a. Method according to any of the claims of 15 a at 19 a where the subject is a human being. 21 a A kit comprising a biosensor according to any of the claims in claim 1 a at 9 a . 22 a A kit according to claim 21 a characterized by the fact that it is materialized in a test strip. 23 a A kit according to claim 21 a characterized in that it is materialized in a lateral flow test kit (5). 24 a A kit according to claim 23 a characterized in that it comprises a strip formed by at least one fibrous membrane on which the biosensor material is immobilized, such that the fibrous membrane preferably comprises: a sample zone for applying the sample to be analyzed; a deposit zone on which the biosensor material is immobilized; and a detection zone. 25a Use of a kit according to any of the claims in claim 21 a at 24 a for the detection of P. aeruginosa bacterium DNA in a sample. 26 a Use of a kit according to any of the claims in claim 21 a at the 25th a for the in vitro diagnosis of infection caused by the bacterium P. aeruginosa in a subject. 27 a Use according to claim 25 a or 26 a where the sample is an environmental sample or a clinical sample, where the clinical sample is selected from the group consisting of blood, urine, serum, sputum, pleural, peritoneal, synovial, or cerebrospinal fluid. 28 a Use according to claim 26 awhere the infection caused by P. aeruginosa is selected from the group consisting of: bacteremia, pneumonia, urinary tract infections, respiratory infections in patients with cystic fibrosis or COPD, heart tract infections, surgical infections, corneal infections, skin infections, soft tissue infections, otitis, keratitis, and sepsis. 29 a Use according to claim 26 a or 27 a where the subject is a human being. 30 a . Method for preparing a biosensor according to any of the claims in claim 1 a a 9 acomprising the steps of: a) Introduction of an indicator (2) into a porous support (1), wherein the porous support (1) has an internal nanoporous porous structure with pores of diameter between 1 nm and 100 nm, such that a plurality of these pores gives access to the surface of the support (1) to the outside, obtaining a charged porous support (S1); b) chemical modification of the surface of the charged porous support (S1) by attaching reactive groups (3) to the outer surface of the charged porous support (S1), obtaining a charged and functionalized support (S2) with reactive groups (3) on its outer surface; c) addition of a first short-sequence binding oligonucleotide (O1), comprising the nucleotide sequence SEQ ID NO: 1, to the charged and functionalized support (S2), such that a support with anchoring elements (S3) is obtained by the formation of chemical bonds between the reactive groups (3) and the oligonucleotides (O1);d) addition of a second oligonucleotide (O2) by hybridization with the short sequence (O1), oligonucleotide (O2) that recognizes the DNA of the bacterium P. aeruginosa (4), obtaining a biosensor (S4), where steps a), b), c) and d) are carried out under controlled agitation, temperature and humidity conditions.; 31 a . Method for preparing a biosensor, according to claim 30 a , characterized in that step a) is carried out by diffusion, by introducing the support (1) into a solution containing the indicator (2) under controlled agitation and temperature conditions. 32 a . Method for preparing a biosensor, according to claim 30 a , characterized by the fact that the diffusion of stage a) is a passive diffusion. 33 a . Method for preparing a biosensor, according to claim 31 a or 32 a, characterized in that the diffusion of step a) is carried out under agitation conditions of between 10 and 200 rpm, at a temperature between 20°C and 30°C, and during a time interval between 1 and 32 hours. 34 a . Method for preparing a biosensor, according to claim 30 a , characterized in that step b) is carried out by introducing the loaded support (S1) into a solution of 3-isocyanatopropyltriethoxysilane under controlled stirring and temperature conditions. 35 a . Method for preparing a biosensor, according to claim 30 a characterized in that step b) is carried out under agitation conditions of between 10 and 200 rpm, at a temperature between 20°C and 30°C, and for a time interval between 1 and 32 hours. 36 a . Method for preparing a biosensor, according to claim 30 a, characterized in that step c) is carried out by immersing the loaded and functionalized support (52) in a solution containing the indicator (2) and the first oligonucleotide (01) under controlled stirring and temperature conditions. 37 a . Method for preparing a biosensor, according to claim 36 a , characterized in that step c) is carried out under stirring conditions of 10-200 rpm and a temperature of 25°C for 3 hours. 38 a . Method for preparing a biosensor, according to claim 30 a , characterized in that stage d) is carried out by immersing the support with anchoring elements (53) in a solution containing the second oligonucleotide (O2) capable of recognizing a specific region of the phzA2 gene of the DNA of the bacterium P. aeruginosa, under agitation and controlled temperature conditions. 39 a. Method for preparing a biosensor, according to claim 38 a , characterized in that step d) is carried out under stirring conditions of 10-200 rpm and a temperature of 25°C for 2 hours. 40 a . Method for preparing a biosensor, according to claim 30 a , characterized in that in stages a), b), c) and / od) a moisture control material is added. 41 a . Method for preparing a biosensor, according to claim 30 a , characterized in that the porous support (1) is obtained from nanoporous anodic alumina, nanoporous silica, titanium oxide or graphene. 42 a . Method for preparing a biosensor, according to claim 30 a , characterized in that the porous support (1) is macroporous, mesoporous or microporous. 43 a . Method for preparing a biosensor, according to claim 30 a, characterized in that the indicator (2) is selected from a dye, a fluorophore, an electrochemiluminescent substance, a redox-active substance, a plasmonic resonance substance, a biomolecule such as a protein, enzymes, nucleic acid fragments, or a mixture thereof. 44 a . Method for preparing a biosensor, according to claim 43 a , characterized in that the indicator (2) is selected from [Ru(bipi)s]Cl2, rhodamine 101, rhodamine 110, rhodamine B, safranin, fluorescein, sodium salt of sulforodamine B and / or PEG-Bodipis.

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