(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization I International Bureau (10) International Publication Number (43) International Publication Date WO 2013/121201 Al 22 August 2013 (22.08.2013) P O P C T (51) International Patent Classification: Avenue, Oxford Science Park, Oxford Oxfordshire OX4 G01N 33/487 (2006.01) G01N 33/543 (2006.01) 4GA (GB). CLARKE, James Anthony; Oxford Nanopore G01N 33/53 (2006.01) Technologies Limited, Edmund Cartwright House, 4 Robert Robinson Avenue, Oxford Science Park, Oxford (21) International Application Number: Oxfordshire OX4 4GA (GB). WHITE, James; Oxford PCT/GB20 13/050348 Nanopore Technologies Limited, Edmund Cartwright (22) International Filing Date: House, 4 Robert Robinson Avenue, Oxford Science Park, 14 February 2013 (14.02.2013) Oxford Oxfordshire OX4 4GA (GB). (25) Filing Language: English (74) Agent: CHAPMAN, Lee Phillip; J A Kemp, 14 South Square, Gray's Inn, London Greater London WC1R 5JJ (26) Publication Language: English (GB). (30) Priority Data: (81) Designated States (unless otherwise indicated, for every 61/599,240 15 February 2012 (15.02.2012) US kind of national protection available): AE, AG, AL, AM, (71) Applicant: OXFORD NANOPORE TECHNOLOGIES AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, LIMITED [GB/GB]; Edmund Cartwright House, 4 Robert BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, Robinson Avenue, Oxford Science Park, Oxford Oxford DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, shire OX4 4GA (GB). HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, (72) Inventors: TURNER, Daniel John; Oxford Nanopore ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, Technologies Limited, Edmund Cartwright House, 4 NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, Robert Robinson Avenue, Oxford Science Park, Oxford RW, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, Oxfordshire OX4 4GA (GB). FORDHAM, Daniel TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, George; Oxford Nanopore Technologies Limited, Edmund ZM, ZW. Cartwright House, 4 Robert Robinson Avenue, Oxford Sci ence Park, Oxford Oxfordshire OX4 4GA (GB). GILL, (84) Designated States (unless otherwise indicated, for every Roger Charles; Oxford Nanopore Technologies Limited, kind of regional protection available): ARIPO (BW, GH, Edmund Cartwright House, 4 Robert Robinson Avenue, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ, Oxford Science Park, Oxford Oxfordshire OX4 4GA (GB). UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, BROWN, Clive Gavin; Oxford Nanopore Technologies EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, Limited, Edmund Cartwright House, 4 Robert Robinson MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, Avenue, Oxford Science Park, Oxford Oxfordshire OX4 TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, 4GA (GB). RED), Stuart; Oxford Nanopore Technologies ML, MR, NE, SN, TD, TG). Limited, Edmund Cartwright House, 4 Robert Robinson [Continued on nextpage] (54) Title: APTAMER METHOD d) o Fig 1 (57) Abstract: The invention relates to a new method of determining in a sample the presence or absence of one or more analyte o members of a group of two or more analytes. The invention therefore relates to a multiplex assay for determining the presence or ab - sence of each analyte in a group of multiple analytes. The assay uses aptamers and transmembrane pores. w o 2013/121201 A i llll II II 11III III I II II II I III IIII II I II Published: — with sequence listing part of description (Rule 5.2(a)) — with international search report (Art. 21(3)) — before the expiration of the time limit for amending the claims and to be republished in the event of receipt of amendments (Rule 48.2(h)) APTAMER METHOD Field of the invention The invention relates to a new method of determining in a sample the presence or absence of one or more analyte members of a group of two or more analytes. The invention therefore relates to a multiplex assay for determining the presence or absence of each analyte in a group of multiple analytes. The assay uses aptamers and transmembrane pores. Background of the invention Transmembrane pores (nanopores) have great potential as direct, electrical biosensors for a variety of analytes, such as polymers and small molecules. When a potential is applied across a nanopore, there is a change in the current flow when a molecule, such as a nucleotide or a polynucleotide, resides transiently in the barrel or channel of the nanopore for a certain period of time. Specific molecules, such as specific nucleotides and specific polynucleotides, give current changes of known signature and duration. Such current changes can be used to identify the nucleotide or polynucleotide present in the pore. Summary of the invention The inventors have demonstrated that a transmembrane pore can be used in a multiplex assay to determine the presence or absence of each analyte in a group of two or more analytes. More specifically, the inventors have demonstrated that the presence or absence of analytes can be determined using a transmembrane pore and panel of aptamer-containing probes. Each of the analytes of interest is recognised by at least one probe in the panel. Each probe also includes a tail that is capable of entering the pore and affecting the current flowing through the pore. Each tail affects the current flowing through the pore in different and distinctive ways depending on whether or not the probe is bound to one of the analytes of interest. The effect each probe in the panel has on the current flowing through the pore is also distinctive so that the identity of each probe can be detected. This combination of aptamers and tails is such that it is surprisingly possible to identify the binding of one or more of, or even all of, the probes in the panel to the analytes of interest by analyzing the effect of the probe tails on the current flowing through the pore. Accordingly, the invention provides a method of determining in a sample the presence or absence of one or more analyte members of a group of two or more analytes, the method comprising: (a) contacting the sample with a transmembrane pore and a panel of two or more probes, wherein each probe recognises one or more of the analyte members and comprises (i) an aptamer that binds to one or more of the analyte members and (ii) a tail which is capable of entering the pore and has different effects on the current flowing through the pore depending on whether or not the aptamer in the probe is bound to one of the analyte members, wherein each probe affects the current flowing through the pore in a distinctive manner, and wherein each analyte member in the group of two or more analytes is recognised by at least one probe in the panel; and (b) measuring the current flowing through the pore to determine which probes in the panel, if any, have bound to an analyte member and thereby determining the presence or absence of one or more analyte members in the sample. The invention also provides: a method of determining in a sample the concentration of one or more analyte members of a group of two or more analytes, the method comprising: (i) carrying out a method of the invention; and (ii) for one or more analyte members shown to be present in the sample comparing the current flowing through the pore in step (b) with control or reference data for each analyte member and thereby determining the concentration of the one or more analyte members in the sample; - a panel of probes for determining in a sample the presence, concentration or absence of one or more analyte members of a group of two or more analytes, the panel comprising two or more probes, wherein each probe recognises one or more of the analyte members and comprises (i) an aptamer that binds to one or more of the analyte members and (ii) a tail which is capable of entering a transmembrane pore and has different effects on the current flowing through the pore depending on whether or not the aptamer in the probe is bound to one of the analyte members, wherein each probe affects the current flowing through the pore in a distinctive manner, and wherein each analyte member in the group of two or more analytes is recognised by at least one probe in the panel; a kit for determining in a sample the presence, concentration or absence of one or more analyte members of a group of two or more analytes, comprising (a) a panel of probes of the invention and (b) a transmembrane pore; and an analysis apparatus for determining in a sample the presence, concentration or absence of one or more analyte members of a group of two or more analytes, comprising a plurality of pores and a panel of probes of the invention. Description of the Figures Figure 1 shows a cartoon representation of the probe configuration within the nanopore in the absence (a) and presence (b) of a bound analyte. The expected event traces corresponding to the absence (c) and presence (d) of a bound analyte are shown below (the y-axis = current and the x-axis = time for (c) and (d)). The dashed line represents a poly(dA) region and the cross region represents an abasic region.