# US 11,112,408 B2 · Biosensor for detecting the presence of bacteria > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,112,408 B2 - Document ID: US11112408B2 - Status: Granted - Kind: B2 - Application number: 16/317,149 - Filed: 2017-07-12 - Published: 2019-08-22 - Granted: 2021-09-07 - Earliest claimed: 2016-07-12 - Assignees: Centre National de la Recherche Scientifique (CNRS); Universite De Montpellier; AXLR, SATT du Languedoc Roussillon (SATT AXLR) - Applicants: Centre National de la Recherche Scientifique (CNRS); Universite de Montpellier; AXLR, SATT du Languedoc Roussillon (SATT AXLR) - Inventors: Tatiana Rabe Ralam; Arnaud Vena; Mamadou Balde; Brice Sorli; Morgane Nedelec; Héla Bahloul - Web page: https://patentgrep.com/patent/US11112408B2 ## CPC - G01N 33/56911: {Bacteria} - C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids} - C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)} - C12N 15/115: Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith {; Nucleic acids binding to non-nucleic acids, e.g. aptamers} - C12Q 1/02: involving viable microorganisms - G01N 31/22: using chemical indicators (G01N31/02 takes precedence) - G01N 31/229: {for investigating time/temperature history} - G01N 33/02: Food - G01N 33/58: involving labelled substances (G01N33/53 takes precedence) - C12N 2310/16: Aptamers - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES ## Abstract The present invention relates to a biosensor for detecting the presence and/or measuring the concentration of at least one biological agent, comprising a support on which a barcode is disposed, at least one area of which is functionalised by at least one aptamer capable of selectively immobilising said biological agent, said aptamer per se being directly or indirectly bonded to a pigment molecule capable either of producing colour, or of changing colour following the immobilisation of the biological agent by said aptamer. ## Claims ### Claim 1 A biosensor for detecting the presence and/or measuring the concentration of at least one biological agent, comprising a support on which a barcode is placed, at least one area of which is functionalized by at least one aptamer capable of selectively immobilizing said biological agent, said aptamer itself being linked, directly or indirectly, to a coloring molecule, wherein: said coloring molecule is linked via a labile bond to the aptamer and is able to produce color following an enzyme attack causing a break of said labile bond; or, alternatively, said coloring molecule is an antibiotic covalently bonded to the aptamer and is able to produce color or to change color following the immobilization of the biological agent by said aptamer. ### Claim 2 (depends on 1) The biosensor as claimed in claim 1, wherein the support is a flexible or rigid support on which the barcode is placed. ### Claim 3 (depends on 1) The biosensor as claimed in claim 1, wherein the support is made of glass, paper or plastic. ### Claim 4 (depends on 3) The biosensor as claimed in claim 3, wherein a layer of alumina is placed on the support. ### Claim 5 (depends on 1) The biosensor as claimed in claim 1, wherein the barcode is a one-dimensional barcode composed of an assembly of parallel black stripes and white stripes in which at least one area of at least one white stripe or black stripe is functionalized by at least one aptamer. ### Claim 6 (depends on 1) The biosensor as claimed in claim 1, wherein the barcode is a 2-dimensional barcode composed of an assembly of black pixels and white pixels, in which at least one white pixel or at least one black pixel is functionalized by at least one aptamer. ### Claim 7 (depends on 1) The biosensor as claimed in claim 1, wherein the aptamer capable of selectively immobilizing said biological agent is an RNA strand, a DNA strand or a peptide, the 3′ end of which is modified to bear a —COOH function, or else the 5′ end of which is modified to bear an —NH2 function. ### Claim 8 (depends on 1) The biosensor as claimed in claim 1, wherein the aptamer is covalently bonded to the barcode area via a first spacer. ### Claim 9 (depends on 1) The biosensor as claimed in claim 1, wherein the biological agent is selected from the group consisting of bacteria, prions and viruses, mycotoxins and peptides. ### Claim 10 (depends on 1) The biosensor as claimed in claim 1, wherein the biological agent is a bacterium and the aptamer is capable of selectively binding at least one membrane protein of said bacterium. ### Claim 11 (depends on 1) The biosensor as claimed in claim 1, wherein the biological agent is a bacterium selected from the group consisting of Escherichia coli, Pseudomonas aeroginosa, Acinetobacter baumannii, Staphylococcus aureus, Clostridium difficile, Clostridium botulinum, Streptococcus pneumoniae, Treponema pallidum, Salmonella gastroenteretis, Listeria monocytogenes and Legionella spp. ### Claim 12 (depends on 1) The biosensor as claimed in claim 1, wherein said coloring molecule is non-luminescent and may be selected from a chromogenic antibiotic or is luminescent, and may be selected from 5-FAM, 6-FAM, ATTO type dyes, especially ATTO 532 or ATTO 550, ATTO 647, DY-510XL, DY-530, HEX, CAL Fluor Orange 560. ### Claim 13 (depends on 1) The biosensor as claimed in claim 1, wherein said coloring molecule is non-luminescent in its form linked to the aptamer and luminescent, in its form not linked to the aptamer. ### Claim 14 (depends on 13) The biosensor as claimed in claim 13, wherein the coloring molecule has a light absorption band of from 600 nm to 700 nm, and a light emission band of from 600 nm to 700 nm or a minimum colorimetric shift for detection. ### Claim 15 (depends on 8) The biosensor as claimed in claim 8, wherein the coloring molecule is covalently bonded to the aptamer via a second spacer, said second spacer being linked to the coloring molecule by a labile chemical bond which is capable of breaking following immobilization of the biological agent by the aptamer. ### Claim 16 (depends on 15) The biosensor as claimed in claim 15, wherein the second spacer is a divalent radical comprising a β-lactam unit. ### Claim 17 (depends on 1) A process for analyzing contamination by at least one biological agent in an environment, comprising at least the steps consisting in: making available a biosensor as claimed in claim 1, bringing the biosensor into the presence of the environment to be analyzed, under conditions conducive to the immobilization of the biological agent(s) which may be present in said environment by the aptamers of the biosensor, reading the biosensor thus coated, so as to acquire the information encoded by the barcode of the biosensor thus coated, and generating, from the acquired information, information regarding the possible presence of at least one biological agent in the environment. ### Claim 18 (depends on 1) A method for analyzing contamination by at least one biological agent in an environment comprising using a biosensor as claimed in claim 1. ### Claim 19 (depends on 1) A device for analyzing contamination by at least one biological agent in an environment, comprising at least one biosensor as claimed in claim 1. ### Claim 20 (depends on 8) The biosensor as claimed in claim 8, wherein the first spacer is a spacer of formula —S—(CH2)n—C(O)—where n is an integer from 2 to 6 and wherein the sulfur atom is covalently bonded to the support and the carbon atom of the C(O) group is covalently bonded to a nitrogen atom of the aptamer or is bonded by a silane bond (OH-Silane-NH2), by a heterobifunctional linker (heterobifunctional SH-linker-NH2), or else by UV. ### Claim 21 (depends on 1) The biosensor as claimed in claim 1, wherein the support is made of microporous polyethylene or polypropylene. ### Claim 22 (depends on 1) The biosensor as claimed in claim 1, wherein the biological agent is chosen from lipopolysaccharides (LPS) and peptidoglycans (PPG). ### Claim 23 (depends on 8) The biosensor as claimed in claim 8, wherein the coloring molecule is covalently bonded to the aptamer via a second spacer, said second spacer being linked to the coloring molecule by a labile chemical bond capable of breaking in the presence of a β-lactamase. ### Claim 24 (depends on 15) The biosensor as claimed in claim 15, wherein the second spacer is a divalent radical comprising a cephalosporin unit. ### Claim 25 (depends on 1) The biosensor as claimed in claim 1, wherein the coloring molecule is an antibiotic covalently bonded to the aptamer, said coloring molecule comprising a chromogenic antibiotic. ### Claim 26 (depends on 1) The biosensor as claimed in claim 1, wherein the coloring molecule is an antibiotic covalently bonded to the aptamer, said coloring molecule being selected from cephalosporins and penicillins. ### Claim 27 (depends on 1) The biosensor as claimed in claim 1, wherein said coloring molecule is ATTO.