Analyte detection with electrode-free nanopores

Abstract

The present invention provides an electrodeless system for identifying analytes based on optical measurement of ion flow through a nanopore driven by a chemical gradient, and methods for identifying analytes using such a system. The present invention also provides an electrodeless nanopore array for simultaneously identifying different analytes, methods for identifying different analytes using such an array, and methods for manufacturing such an array.

Description

[Technical Field]

[0001] The present invention relates to a method for identifying an analyte using a protein nanopore. [Background technology]

[0002] Transport across natural membranes is assisted by a variety of membrane transport proteins [1]. Transported solutes, such as small ions [2], water [3], sugars [4], and even genetic material [5], are crucial for regulating various cellular activities. While the detailed transport mechanisms vary [6], the ability to report the identity of single molecules as they translocate through the channel forms the foundation for nanopore sequencing as a biomimicry approach [7, 8]. Nanopore sensing has been reported from planar lipid membranes [9], droplet interface bilayers (DIBs)

[10] , hydrogel interface bilayers

[11] , synthetic solid membranes

[12] , glass nanopipettes

[13] , and cell membranes

[14] . However, the core mechanism, adopted from electrophysiology, has remained unchanged since its first appearance in 1996 [9].

[0003] In electrophysiological phenomena, the Ag / AgCl electrode pair applies a transmembrane potential and - The nanopore serves to drive the continuous electromigration of charged analytes and, for single-molecule identification, to record ionic current fluctuations (Figure 1a). In the absence of electrodes, although active thermal diffusion of ions across the nanopore exists in both directions, the law of electroneutrality dictates that the net ionic flow and the electric field across the electrolyte-containing space are exactly zero (Figure 1b)

[15] .

[0004] Electrophysiological measurements offer sufficient time resolution (approximately 10 μs) and amplitude resolution (<0.1 pA)

[16] to meet the needs of single-channel recording-based applications, but they suffer from throughput limitations

[17] . Nanopore sequencing and drug screening are urgently needed, but simultaneous readout from a million channels is not yet feasible without sacrificing device cost and size [17, 18]. This pressing need motivated us to rethink simplified strategies for high-throughput channel recording, which may be further gained from biomimicry.

[0005] The bacterial phage T4 injects its genomic DNA through a channel protein when interacting with a host cell

[19] . The α-hemolysin (α-HL) of Staphylococcus Aureus causes hemolysis of target cells by passive leakage of nutrients through an inserted channel

[20] . These spontaneous molecular transport events acquired through natural evolution suggest that external electronic components are not necessarily required for molecular transport. How to obtain nanopore sensing signals without electrical connections remains a challenging problem.

[0006] Recent developments in optical signal channel recording (oSCR) [21–25] offer an alternative strategy, which involves the transfer of Ca through individual nanopores embedded in the droplet interface bilayer (DIB). 2+ The oSCR is advantageous for high-throughput measurements, but continuous Ca flux through the nanopore is not sufficient. 2+ Electrode pairs were still used to electrophoretically drive the flow.

[23] Manual insertion of the electrodes into the aqueous droplets required delicate micromanipulation skills and potentially increased the risk of bilayer collapse.

[23] This hindered widespread use in academic research and industrial applications. Summary of the Invention

[0007] The present invention provides systems for identifying analytes based on optical measurement of ion flux through a nanopore driven by a chemical gradient. The present invention also provides methods of using such systems to identify analytes, including methods for identifying small molecules or DNA, such as dsDNA or ssDNA.

[0008] In one aspect of the invention, there is provided an electrodeless system for identifying an analyte, comprising: (a) a first compartment containing a first aqueous solution therein, said first aqueous solution comprising a fluorescent reporter molecule capable of fluorescing when bound to an ionic species; (b) a second compartment containing a second aqueous solution, said second aqueous solution containing said ionic species that specifically binds to said fluorescent reporter molecule; and (c) a membrane separating the first and second compartments; Including, at least one nanopore is inserted in the membrane between the first compartment and the second compartment such that the first compartment and the second compartment are connected by the nanopore; The ionic species can diffuse from the second compartment to the first compartment through the nanopore, and a chemical gradient of the ionic species exists between the first compartment and the second compartment. A system is provided.

[0009] In some embodiments, the membrane is a solid membrane.

[0010] In some embodiments, the membrane is a semi-permeable membrane.

[0011] In some embodiments, the osmolality of the second aqueous solution is higher than the osmolality of the first aqueous solution, or the osmolality of the second aqueous solution is higher than the osmolality of the first aqueous solution; or the osmolality of the second aqueous solution is equal to the osmolality of the first aqueous solution, or the osmolality of the second aqueous solution is equal to the osmolality of the first aqueous solution; or the osmolality of the second aqueous solution is lower than the osmolality of the first aqueous solution, or the osmolality of the second aqueous solution is lower than the osmolality of the first aqueous solution.

[0012] In some embodiments, the semi-permeable membrane is comprised of amphiphilic molecules; preferably, the amphiphilic molecules are lipids or triblock copolymers.

[0013] In some embodiments, the semipermeable membrane is a bilayer composed of amphiphilic molecules.

[0014] In some embodiments, the amphiphilic molecule is a lipid.

[0015] In some embodiments, the lipid is one or more selected from the group consisting of fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, polyketides, phospholipids, glycolipids, and cholesterol.

[0016] In some embodiments, the lipid is one or more selected from the group consisting of monoolein; 1,2-dioleoyl-sn glycero-S-phosphocholine (DOPC); 1,2-diphytanoyl-sn-glycero-3-phosphatidylcholine (DPhPC); palmitoyloleoylphosphatidylcholine (POPC); 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE); 1-palmitoyl-2-oleoyl-phosphatidylethanolamine; 1-palmitoyl-2-oleoylphosphatidylglycerol (POPE / POPG) mixture; and mixtures thereof.

[0017] In some embodiments, the first compartment is provided by an aqueous droplet.

[0018] In some embodiments, the second compartment is provided by a hydrogel layer; preferably, the hydrogel layer comprises 0.1-20% (w / v) agarose; more preferably, the hydrogel layer comprises 2-5% (w / v) agarose.

[0019] In some embodiments, the nanopore is selected from the group consisting of a protein nanopore, a DNA nanopore, or a solid-state nanopore.

[0020] In some embodiments, the protein nanopore is one or more selected from the group consisting of α-HL, ClyA, Phi29 connector protein, erolysin, MspA, OmpF, OmpG, FraC, HlyA, SheA, sp1, and variants thereof; and an ion channel.

[0021] In some embodiments, the protein nanopore is ClyA-RR or α-HL.

[0022] In some embodiments, the ionic species is Ag + , Ag 2+ , Al 3+ , As 3+ , Au + , Ba2+ , Bi 3+ , Ca 2+ , Cd 2+ , Ce 3+ , Ce 4+ , Cl - , Co 2+ , Cr 3+ , Cu + , Cu 2+ , Dy 3+ ,EU 3+ , Fe2 + , Fe 3+ , Ga 3+ , H + , Hg + , Hg 2+ , In 3+ , K. + , La 3+ , Mg 2+ , Mn 2+ , Mo 3+ , Na + , Ni 2+ , O.H. - , Pb 2+ , Pd 2+ , Pt 2+ , Pt 4+ , Ru 3+ , Sb 3+ ,Sc. 3+ , Sn 2+ , Sr 2+ , Tb 3+ , Tl + , and Zn 2+ and one or more selected from the group consisting of:

[0023] In some embodiments, the fluorescent reporter molecule is one or more selected from the group consisting of Fura-2, Indo-1, Fluo-2, Fluo-3, Fluo-4, Fluo-8, Calcium Green-1, DCFH, DHR, SNARF, Cal-520, calcium-specific aminopolycarboxylic acid, BAPTA, SBFI, Asante NaTRIUM Green-1, Asante NaTRIUM Green-2, Thallos Potassium Ion Channel Reagent, Asante Potassium Green-1, Asante Potassium Green-2, Asante Potassium Green-3, PBFI, Fluo-2 Mg, Fura-2 Mg, Indo-1 Mg, Asante Magnesium Green, SPQ, MQAE, TSQ, TFL-Zn, and ZINQUIN.

[0024] In some embodiments, the second aqueous solution comprises calcium chloride and, optionally, a buffering agent.

[0025] In some embodiments, the concentration of calcium chloride in the second aqueous solution is 0.01 to 6.76M.

[0026] In some embodiments, the first aqueous solution comprises a chelating agent and, optionally, a buffering agent, wherein the chelating agent is capable of binding to the ionic species; preferably, the chelating agent is EDTA, BAPTA, EGTA, CyDTA, DTPA, EDDP, HIDA, IDA, NTA, NTPO, TTHA, CA, TA, GA, HEDTA, or DEG.

[0027] In some embodiments, the system further comprises a light source for illumination and a light sensor for detecting fluorescence; preferably, the light source is a laser, LED, halogen light, xenon light; preferably, the light sensor is a CCD, sCMOS sensor, or photodiode; more preferably, the light sensor is an EMCCD or an avalanche photodiode (APD).

[0028] In some embodiments, the system further comprises a device for total internal reflection fluorescence (TIRF), wide-field fluorescence microscopy, or confocal microscopy.

[0029] In some embodiments, the first aqueous solution or the second aqueous solution comprises the analyte.

[0030] In some embodiments, the analyte is selected from the group consisting of a small molecule, a macromolecule, and a biopolymer.

[0031] In some embodiments, the analyte is selected from the group consisting of a compound, a drug, a sugar, an ion, a neurotransmitter, an amino acid, a nucleotide, a polymer, a polypeptide, a polysaccharide, and a polynucleotide; preferably, the polynucleotide is DNA or RNA; more preferably, the DNA is dsDNA or ssDNA; more preferably, the RNA is miRNA, siRNA, or tRNA.

[0032] In another aspect of the invention, there is provided a method for identifying an analyte, comprising the steps of: (a) providing any one of the systems above, wherein said analyte is provided in said first compartment or in said second compartment; (b) directing light capable of exciting the fluorescent reporter molecule at a region within the first compartment proximal to the nanopore; (c) measuring a fluorescent signal from the fluorescent reporter molecule to identify the analyte; A method is provided which includes:

[0033] In another aspect of the invention, there is provided a method of making an electrodeless system, comprising the steps of: providing a first compartment containing a first aqueous solution therein, said first aqueous solution including a fluorescent reporter molecule capable of fluorescing when bound to an ionic species; providing a second compartment containing a second aqueous solution therein, said second aqueous solution comprising said ionic species that specifically binds to said fluorescent reporter molecule; contacting the first compartment with the second compartment in a hydrophobic medium containing amphipathic molecules such that a semipermeable membrane having a nanopore inserted therein is formed between the first compartment and the second compartment; Including, A protein nanopore is provided in the first aqueous solution or the second aqueous solution; A method is provided.

[0034] In some embodiments, the osmolality of the second aqueous solution is higher than the osmolality of the first aqueous solution, or the osmolality of the second aqueous solution is higher than the osmolality of the first aqueous solution; or the osmolality of the second aqueous solution is equal to the osmolality of the first aqueous solution, or the osmolality of the second aqueous solution is equal to the osmolality of the first aqueous solution; or the osmolality of the second aqueous solution is lower than the osmolality of the first aqueous solution, or the osmolality of the second aqueous solution is lower than the osmolality of the first aqueous solution.

[0035] In some embodiments, the semi-permeable membrane is comprised of amphiphilic molecules; preferably, the amphiphilic molecules are lipids or triblock copolymers.

[0036] In some embodiments, the semipermeable membrane is a bilayer composed of amphiphilic molecules.

[0037] In some embodiments, the amphiphilic molecule is a lipid.

[0038] In some embodiments, the lipid is one or more selected from the group consisting of fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, polyketides, phospholipids, glycolipids, and cholesterol.

[0039] In some embodiments, the lipid is one or more selected from the group consisting of monoolein; 1,2-dioleoyl-sn glycero-S-phosphocholine (DOPC); 1,2-diphytanoyl-sn-glycero-3-phosphatidylcholine (DPhPC); palmitoyloleoylphosphatidylcholine (POPC); 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE); 1-palmitoyl-2-oleoyl-phosphatidylethanolamine; 1-palmitoyl-2-oleoylphosphatidylglycerol (POPE / POPG) mixture; and mixtures thereof.

[0040] In some embodiments, the first compartment is provided by an aqueous droplet.

[0041] In some embodiments, the second compartment is provided by a hydrogel layer; preferably, the hydrogel layer comprises 0.1-20% (w / v) agarose; more preferably, the hydrogel layer comprises 2-5% (w / v) agarose.

[0042] In some embodiments, the nanopore is selected from the group consisting of a protein nanopore, a DNA nanopore, or a solid-state nanopore.

[0043] In some embodiments, the protein nanopore is one or more selected from the group consisting of α-HL, ClyA, Phi29 connector protein, erolysin, MspA, OmpF, OmpG, FraC, HlyA, SheA, sp1, and variants thereof; and an ion channel.

[0044] In some embodiments, the protein nanopore is ClyA-RR or α-HL.

[0045] In some embodiments, the ionic species is Ag + , Ag 2+ , Al 3+ , As 3+ , Au + , Ba 2+ , Bi 3+ , Ca 2+ , Cd 2+ , Ce 3+ , Ce 4+ , Cl - , Co 2+ , Cr 3+ , Cu + , Cu 2+ , Dy 3+ ,EU 3+ , Fe2 + , Fe 3+ , Ga 3+ , H + , Hg + , Hg 2+ , In 3+ , K. + , La 3+ , Mg 2+ , Mn 2+ , Mo 3+ , Na + , Ni 2+ , O.H. - , Pb 2+ , Pd 2+ , Pt 2+ , Pt 4+ , Ru 3+ , Sb 3+ ,Sc. 3+ , Sn 2+ , Sr 2+ , Tb 3+ , Tl + , and Zn 2+ and one or more selected from the group consisting of:

[0046] In some embodiments, the fluorescent reporter molecule is one or more selected from the group consisting of Fura-2, Indo-1, Fluo-2, Fluo-3, Fluo-4, Fluo-8, Calcium Green-1, DCFH, DHR, SNARF, Cal-520, calcium-specific aminopolycarboxylic acid, BAPTA, SBFI, Asante NaTRIUM Green-1, Asante NaTRIUM Green-2, Thallos Potassium Ion Channel Reagent, Asante Potassium Green-1, Asante Potassium Green-2, Asante Potassium Green-3, PBFI, Fluo-2 Mg, Fura-2 Mg, Indo-1 Mg, Asante Magnesium Green, SPQ, MQAE, TSQ, TFL-Zn, and ZINQUIN.

[0047] In some embodiments, the second aqueous solution comprises calcium chloride and, optionally, a buffering agent.

[0048] In some embodiments, the concentration of calcium chloride in the second aqueous solution is 0.01 to 6.76M.

[0049] In some embodiments, the first aqueous solution comprises a chelating agent and, optionally, a buffering agent, wherein the chelating agent is capable of binding to the ionic species; preferably, the chelating agent is EDTA, BAPTA, EGTA, CyDTA, DTPA, EDDP, HIDA, IDA, NTA, NTPO, TTHA, CA, TA, GA, HEDTA, or DEG.

[0050] In some embodiments, an analyte is provided in the first aqueous solution or the second aqueous solution.

[0051] In some embodiments, the analyte is selected from the group consisting of a small molecule, a macromolecule, and a biopolymer.

[0052] In some embodiments, the analyte is selected from the group consisting of a compound, a drug, a sugar, an ion, a neurotransmitter, an amino acid, a nucleotide, a polymer, a polypeptide, a polysaccharide, and a polynucleotide; preferably, the polynucleotide is DNA or RNA; more preferably, the DNA is dsDNA or ssDNA; more preferably, the RNA is miRNA, siRNA, or tRNA.

[0053] In another aspect of the invention, there is provided an electrodeless nanopore array for identifying multiple analytes, comprising a plurality of systems in parallel, each system comprising: (a) a first compartment containing a first aqueous solution therein, said first aqueous solution comprising a fluorescent reporter molecule capable of fluorescing when bound to an ionic species; (b) a second compartment containing a second aqueous solution, said second aqueous solution containing said ionic species that specifically binds to said fluorescent reporter molecule; and (c) a membrane separating the first and second compartments; Including, In each system, at least one nanopore is inserted in the membrane between the first compartment and the second compartment, such that in each system the first compartment and the second compartment are connected by a nanopore; a chemical gradient of the ionic species exists between the first and second compartments of each system, allowing the ionic species to diffuse from the second compartment to the first compartment through the nanopore; the plurality of systems being arranged such that the measured fluorescence is distinguishable in each system; A nanopore array is provided.

[0054] In some embodiments, in each system, the membrane between the first compartment and the second compartment system is a solid membrane.

[0055] In some embodiments, in each system, the membrane between the first compartment and the second compartment is a semi-permeable membrane.

[0056] In some embodiments, in each system, the osmolality of the second aqueous solution is higher than the osmolality of the first aqueous solution, or the osmolality of the second aqueous solution is higher than the osmolality of the first aqueous solution; or the osmolality of the second aqueous solution is equal to the osmolality of the first aqueous solution, or the osmolality of the second aqueous solution is equal to the osmolality of the first aqueous solution; or the osmolality of the second aqueous solution is lower than the osmolality of the first aqueous solution, or the osmolality of the second aqueous solution is lower than the osmolality of the first aqueous solution.

[0057] In some embodiments, the semi-permeable membrane is comprised of amphiphilic molecules; preferably, the amphiphilic molecules are lipids or triblock copolymers.

[0058] In some embodiments, the semipermeable membrane is a bilayer composed of amphiphilic molecules.

[0059] In some embodiments, the amphiphilic molecule is a lipid.

[0060] In some embodiments, the lipid is one or more selected from the group consisting of fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, polyketides, phospholipids, glycolipids, and cholesterol.

[0061] In some embodiments, the lipid is one or more selected from the group consisting of monoolein; 1,2-dioleoyl-sn glycero-S-phosphocholine (DOPC); 1,2-diphytanoyl-sn-glycero-3-phosphatidylcholine (DPhPC); palmitoyloleoylphosphatidylcholine (POPC); 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE); 1-palmitoyl-2-oleoyl-phosphatidylethanolamine; 1-palmitoyl-2-oleoylphosphatidylglycerol (POPE / POPG) mixture; and mixtures thereof.

[0062] In some embodiments, each of the first compartments of the plurality of systems are separated from one another.

[0063] In some embodiments, the first compartment of each system is supplied by an aqueous droplet.

[0064] In some embodiments, the second compartment of the multiple systems is a single compartment.

[0065] In some embodiments, the second compartment of each system is provided by a hydrogel layer; preferably, the hydrogel layer comprises 0.1-20% (w / v) agarose; more preferably, the hydrogel layer comprises 2-5% (w / v) agarose.

[0066] In some embodiments, the second compartments of the multiple systems are provided by a single hydrogel layer.

[0067] In some embodiments, in each system, the nanopore is selected from the group consisting of a protein nanopore, a DNA nanopore, or a solid-state nanopore.

[0068] In some embodiments, the protein nanopore is one or more selected from the group consisting of α-HL, ClyA, Phi29 connector protein, erolysin, MspA, OmpF, OmpG, FraC, HlyA, SheA, sp1, and variants thereof; and an ion channel.

[0069] In some embodiments, in each system, the protein nanopore is ClyA-RR or α-HL.

[0070] In some embodiments, in each system, the ionic species is Ag + , Ag 2+ , Al 3+ , As 3+ , Au + , Ba 2+ , Bi 3+ , Ca 2+ , Cd 2+ , Ce 3+ , Ce 4+ , Cl - , Co 2+ , Cr 3+ , Cu + , Cu 2+ , Dy 3+ ,EU 3+ , Fe2 + , Fe 3+ , Ga 3+ , H + , Hg + , Hg 2+ , In 3+ , K. + , La 3+ , Mg 2+ , Mn 2+ , Mo 3+ , Na + , Ni 2+ , O.H. - , Pb 2+ , Pd 2+ , Pt 2+ , Pt 4+ , Ru 3+ , Sb 3+ ,Sc. 3+ , Sn 2+ , Sr 2+ , Tb 3+ , Tl + , and Zn2+ and one or more selected from the group consisting of:

[0071] In some embodiments, in each system, the fluorescent reporter molecule is one or more selected from the group consisting of Fura-2, Indo-1, Fluo-2, Fluo-3, Fluo-4, Fluo-8, Calcium Green-1, DCFH, DHR, SNARF, Cal-520, calcium-specific aminopolycarboxylic acid, BAPTA, SBFI, Asante NaTRIUM Green-1, Asante NaTRIUM Green-2, Thallos Potassium Ion Channel Reagent, Asante Potassium Green-1, Asante Potassium Green-2, Asante Potassium Green-3, PBFI, Fluo-2 Mg, Fura-2 Mg, Indo-1 Mg, Asante Magnesium Green, SPQ, MQAE, TSQ, TFL-Zn, and ZINQUIN.

[0072] In some embodiments, in each system, the second aqueous solution comprises calcium chloride and, optionally, a buffering agent.

[0073] In some embodiments, the concentration of calcium chloride in the second aqueous solution is 0.01 to 6.76M in each system.

[0074] In some embodiments, in each system, the first aqueous solution comprises a chelating agent and, optionally, a buffer, wherein the chelating agent is capable of binding the ionic species; preferably, the chelating agent is EDTA, BAPTA, EGTA, CyDTA, DTPA, EDDP, HIDA, IDA, NTA, NTPO, TTHA, CA, TA, GA, HEDTA, or DEG.

[0075] In some embodiments, the array further comprises a light source for illumination and a photosensor for detecting fluorescence; preferably, the light source is a laser, LED, halogen light, xenon light; preferably, the photosensor is a CCD, sCMOS sensor, or photodiode; more preferably, the photosensor is an EMCCD or an avalanche photodiode (APD).

[0076] In some embodiments, the array further comprises a device for total internal reflection fluorescence (TIRF), wide-field fluorescence microscopy, or confocal microscopy.

[0077] In some embodiments, in each system, the first aqueous solution or the second aqueous solution contains the analyte.

[0078] In some embodiments, in each system, the analyte is selected from the group consisting of small molecules, macromolecules, and biopolymers.

[0079] In some embodiments, in each system, the analyte is selected from the group consisting of a compound, a drug, a sugar, an ion, a neurotransmitter, an amino acid, a nucleotide, a polymer, a polypeptide, a polysaccharide, and a polynucleotide; preferably, the polynucleotide is DNA or RNA; more preferably, the DNA is dsDNA or ssDNA; and more preferably, the RNA is miRNA, siRNA, or tRNA.

[0080] In some embodiments, different analytes are physically separated into different systems.

[0081] In some embodiments, the density of the systems in the nanopore array is between 10 and 1000 systems / mm 2 is.

[0082] In some embodiments, the total area provided by the plurality of systems is between 1 and 100 mm 2 is.

[0083] In some embodiments, the number of systems in the plurality is between 4 and 1,000,000; preferably, the number of systems in the plurality is between 10 and 1000.

[0084] In another aspect of the invention there is provided a multiplex method for identifying multiple analytes, comprising the steps of: (a) providing any one of the nanopore arrays described above, wherein two or more analytes are provided to various systems of the nanopore array; (b) directing a light signal capable of exciting the fluorescent reporter molecule contained in each of the first compartments at a region within the plurality of first compartments proximal to the nanopore; (c) measuring a plurality of fluorescent signals from the fluorescent reporter molecules contained in each system to identify the plurality of analytes; A method is provided, comprising:

[0085] In another aspect of the invention, there is provided a method of fabricating an electrodeless nanopore array, comprising the steps of: providing a plurality of aqueous droplets, each containing a first aqueous solution including a protein nanopore, an analyte, and a fluorescent reporter molecule capable of fluorescing when bound to an ionic species; providing a hydrogel layer containing the ionic species; contacting the plurality of aqueous droplets with the hydrogel layer in a hydrophobic medium containing amphiphilic molecules such that a semipermeable membrane is formed between each of the aqueous droplets and the hydrogel layer; A method is provided which includes:

[0086] In some embodiments, the osmolality of the hydrogel is greater than the osmolality of each aqueous droplet; or the osmolality of the hydrogel is greater than the osmolality of each aqueous droplet; or the osmolality of the hydrogel is equal to the osmolality of each aqueous droplet; or the osmolality of the hydrogel is equal to the osmolality of each aqueous droplet; or the osmolality of the hydrogel is less than the osmolality of each aqueous droplet; or the osmolality of the hydrogel is less than the osmolality of each aqueous droplet.

[0087] In some embodiments, the semi-permeable membrane is comprised of amphiphilic molecules; preferably, the amphiphilic molecules are lipids or triblock copolymers.

[0088] In some embodiments, the semipermeable membrane is a bilayer composed of amphiphilic molecules.

[0089] In some embodiments, the amphiphilic molecule is a lipid.

[0090] In some embodiments, the lipid is one or more selected from the group consisting of fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, polyketides, phospholipids, glycolipids, and cholesterol.

[0091] In some embodiments, the lipid is one or more selected from the group consisting of monoolein; 1,2-dioleoyl-sn glycero-S-phosphocholine (DOPC); 1,2-diphytanoyl-sn-glycero-3-phosphatidylcholine (DPhPC); palmitoyloleoylphosphatidylcholine (POPC); 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE); 1-palmitoyl-2-oleoyl-phosphatidylethanolamine; 1-palmitoyl-2-oleoylphosphatidylglycerol (POPE / POPG) mixture; and mixtures thereof.

[0092] In some embodiments, the hydrogel layer comprises 0.1 to 20% (w / v) agarose; preferably, the hydrogel layer comprises 2 to 5% (w / v) agarose.

[0093] In some embodiments, in each system, the nanopore is selected from the group consisting of a protein nanopore, a DNA nanopore, or a solid-state nanopore.

[0094] In some embodiments, the protein nanopore in each aqueous droplet is one or more selected from the group consisting of α-HL, ClyA, Phi29 connector protein, erolysin, MspA, OmpF, OmpG, FraC, HlyA, SheA, sp1, and variants thereof; and an ion channel.

[0095] In some embodiments, the protein nanopore in each aqueous droplet is ClyA-RR or α-HL.

[0096] In some embodiments, the ionic species in the hydrogel layer is Ag + , Ag 2+ , Al 3+ , As 3+ , Au + , Ba 2+ , Bi 3+ , Ca 2+ , Cd 2+ , Ce 3+ , Ce4+ , Cl - , Co 2+ , Cr 3+ , Cu + , Cu 2+ , Dy 3+ ,EU 3+ , Fe2 + , Fe 3+ , Ga 3+ , H + , Hg + , Hg 2+ , In 3+ , K. + , La 3+ , Mg 2+ , Mn 2+ , Mo 3+ , Na + , Ni 2+ , O.H. - , Pb 2+ , Pd 2+ , Pt 2+ , Pt 4+ , Ru 3+ , Sb 3+ ,Sc. 3+ , Sn 2+ , Sr 2+ , Tb 3+ , Tl + , and Zn 2+ and one or more selected from the group consisting of:

[0097] In some embodiments, the fluorescent reporter molecule in each aqueous droplet is one or more selected from the group consisting of Fura-2, Indo-1, Fluo-2, Fluo-3, Fluo-4, Fluo-8, Calcium Green-1, DCFH, DHR, SNARF, Cal-520, calcium-specific aminopolycarboxylic acid, BAPTA, SBFI, Asante NaTRIUM Green-1, Asante NaTRIUM Green-2, Thallos Potassium Ion Channel Reagent, Asante Potassium Green-1, Asante Potassium Green-2, Asante Potassium Green-3, PBFI, Fluo-2 Mg, Fura-2 Mg, Indo-1 Mg, Asante Magnesium Green, SPQ, MQAE, TSQ, TFL-Zn, and ZINQUIN.

[0098] In some embodiments, the hydrogel layer comprises calcium chloride and optionally a buffering agent.

[0099] In some embodiments, the concentration of calcium chloride in the hydrogel layer is 0.01 to 6.76M.

[0100] In some embodiments, each aqueous droplet comprises a chelating agent and, optionally, a buffer, wherein the chelating agent is capable of binding to the ionic species; preferably, the chelating agent is EDTA, BAPTA, EGTA, CyDTA, DTPA, EDDP, HIDA, IDA, NTA, NTPO, TTHA, CA, TA, GA, HEDTA, or DEG.

[0101] In some embodiments, the analyte in each aqueous droplet is selected from the group consisting of small molecules, macromolecules, and biopolymers.

[0102] In some embodiments, the analyte in each aqueous droplet is selected from the group consisting of a compound, a drug, a sugar, an ion, a neurotransmitter, an amino acid, a nucleotide, a polymer, a polypeptide, a polysaccharide, and a polynucleotide; preferably, the polynucleotide is DNA or RNA; more preferably, the DNA is dsDNA or ssDNA; and more preferably, the RNA is miRNA, siRNA, or tRNA.

[0103] In some embodiments, different analytes are provided to the various systems.

[0104] In some embodiments, the number of aqueous droplets is 4 to 1,000,000; preferably, the number of aqueous droplets is 10 to 1,000.

[0105] In another aspect, the present invention provides the use of the above system for carrying out optical analyte analysis.

[0106] In another aspect, the present invention provides the use of the nanopore array described above for performing optical analyte analysis.

[0107] In another aspect, the present invention provides a kit for forming a nanopore array, comprising: a filling hydrogel comprising agarose, a buffer, and an ionic species capable of specifically binding to a fluorescent reporter molecule and causing it to emit fluorescence; an aqueous solution comprising a chelating agent, the fluorescent reporter molecule capable of fluorescing when bound to the ionic species, and a buffer; the chelating agent capable of binding to the ionic species; a hydrophobic medium containing amphiphilic molecules; solid support, A kit is provided, comprising:

[0108] In some embodiments, the osmolality of the filling hydrogel is greater than the osmolality of the aqueous solution, or the osmolality of the filling hydrogel is greater than the osmolality of the aqueous solution; or the osmolality of the filling hydrogel is equal to the osmolality of the aqueous solution, or the osmolality of the filling hydrogel is equal to the osmolality of the aqueous solution; or the osmolality of the filling hydrogel is less than the osmolality of the aqueous solution, or the osmolality of the filling hydrogel is less than the osmolality of the aqueous solution.

[0109] In some embodiments, the amphiphilic molecule is a lipid.

[0110] In some embodiments, the lipid is one or more selected from the group consisting of fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, polyketides, phospholipids, glycolipids, and cholesterol.

[0111] In some embodiments, the lipid is one or more selected from the group consisting of monoolein; 1,2-dioleoyl-sn glycero-S-phosphocholine (DOPC); 1,2-diphytanoyl-sn-glycero-3-phosphatidylcholine (DPhPC); palmitoyloleoylphosphatidylcholine (POPC); 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE); 1-palmitoyl-2-oleoyl-phosphatidylethanolamine; 1-palmitoyl-2-oleoylphosphatidylglycerol (POPE / POPG) mixture; and mixtures thereof.

[0112] In some embodiments, the aqueous solution also includes a protein nanopore.

[0113] In some embodiments, the nanopore is selected from the group consisting of a protein nanopore, a DNA nanopore, or a solid-state nanopore.

[0114] In some embodiments, the protein nanopore is one or more selected from the group consisting of α-HL, ClyA, Phi29 connector protein, erolysin, MspA, OmpF, OmpG, FraC, HlyA, SheA, sp1, and variants thereof; and an ion channel.

[0115] In some embodiments, the protein nanopore is ClyA-RR or α-HL.

[0116] In some embodiments, the ionic species in the hydrogel layer is Ag + , Ag 2+ , Al 3+ , As 3+ , Au + , Ba 2+ , Bi 3+ , Ca 2+ , Cd 2+ , Ce 3+ , Ce 4+ , Cl - , Co 2+ , Cr 3+ , Cu + , Cu 2+ , Dy 3+ ,EU 3+ , Fe2 + , Fe 3+ , Ga 3+ , H + , Hg + , Hg 2+ , In 3+ , K. + , La 3+ , Mg 2+ , Mn 2+ , Mo 3+ , Na + , Ni 2+ , O.H. - , Pb 2+ , Pd 2+ , Pt 2+ , Pt 4+ , Ru 3+ , Sb3+ ,Sc. 3+ , Sn 2+ , Sr 2+ , Tb 3+ , Tl + , and Zn 2+ and one or more selected from the group consisting of:

[0117] In some embodiments, the fluorescent reporter molecule in each aqueous droplet is one or more selected from the group consisting of Fura-2, Indo-1, Fluo-2, Fluo-3, Fluo-4, Fluo-8, Calcium Green-1, DCFH, DHR, SNARF, Cal-520, calcium-specific aminopolycarboxylic acid, BAPTA, SBFI, Asante NaTRIUM Green-1, Asante NaTRIUM Green-2, Thallos Potassium Ion Channel Reagent, Asante Potassium Green-1, Asante Potassium Green-2, Asante Potassium Green-3, PBFI, Fluo-2 Mg, Fura-2 Mg, Indo-1 Mg, Asante Magnesium Green, SPQ, MQAE, TSQ, TFL-Zn, and ZINQUIN.

[0118] In some embodiments, the filling hydrogel includes calcium chloride, thereby providing Ca as the ionic species. 2+ supply.

[0119] In some embodiments, the concentration of calcium chloride in the hydrogel layer is 0.01 to 6.76M; preferably, the concentration of calcium chloride in the second aqueous solution is 0.15M to 6M.

[0120] In some embodiments, the filling hydrogel comprises 2.5% agarose, 1.5 M CaCl2, and 10 mM HEPES (pH 7.0).

[0121] In some embodiments, the aqueous solution also contains KCl.

[0122] In some embodiments, the aqueous solution may contain 1.5 M KCl, 400 μM EDTA, 40 μM Fluo-8, 10 mM HEPES (pH 7.0).

[0123] In some embodiments, the hydrophobic medium containing amphiphilic molecules may be a lipid oil comprising 5 mg of dry DPHPC lipid membrane dissolved in 2 mL of a 1:1 volumetric mixture of hexadecane and silicone oil.

[0124] In some embodiments, the kit also includes a coating hydrogel, which may preferably comprise 0.75% (w / v) agarose in water. [Brief explanation of the drawings]

[0125] [Figure 1]Figure 1 illustrates DiffusiOptoPhysiology and its application in TriM-β-CD sensing. (a-d) Schematic diagrams of ion transport through a nanopore in various measurement platforms. (a) During electrophysiological recording, a membrane potential difference is applied via an Ag / AgCl electrode pair, and the electrophoretic behavior of Cl- is observed through the nanopore. (b) In the absence of electrodes, thermal ion motion across the nanopore exists in both directions, but according to the law of electroneutrality, no net ion transport flow should occur. (c) In oSCR, directional Ca2+ movement is electrophoretically driven through the nanopore, establishing a steep Ca2+ concentration gradient. Upon binding to Fluo-8 on the cis side, the Fluo-8 / Ca2+ complex near the pore emits strong fluorescence. (d) In DiffusiOptoPhysiology, a gentle Ca2+ concentration gradient can be established near the pore due to the thermal ion motion. Upon binding to Fluo-8, weaker fluorescence is expected to be emitted than in c. (e) Cross-section of the spatial distribution of Fluo-8 / Ca2+ complexes around the pore. The dotted box shows a magnified view of the immediate vicinity of the nanopore. (f) Top left: Corresponding image results obtained from computer simulation. Top right: The simulated fluorescence intensity profile follows a Gaussian distribution. Bottom left: Representative frames acquired from DOP recording of a single WT α-HL nanopore. Bottom right: The corresponding fluorescence intensity profile also follows a Gaussian distribution. Scale bar: 4 μm. (g) Single-molecule sensing of TriM-β-CD (75 mM) by the α-HL nanopore for electrodeless oSCR. Scale bar: 4 μm. (h) Plot of the reciprocal of the mean inter-event interval (1 / τon) and the reciprocal of the mean residence time (1 / τoff) versus TriM-β-CD concentration. Average values ​​and standard deviations are obtained from three independent experiments (N=3) for each condition. (i) Statistics of τ and FP results obtained from DOP and electrophysiological recordings at +20 mV, respectively. The DOP recordings described above were performed in cis with 1.5 M KCl, 400 μM EDTA, 40 μM Fluo-8, 10 mM HEPES (pH 7.0), and in trans with 0.75 M CaCl, 10 mM HEPES (pH 7.0).Electrophysiological recordings were performed using 1.5 M KCl, 10 mM HEPES (pH 7.0) on both sides of the membrane. TriM-β-CD was added to the cis side at a final concentration of 75 mM. [Figure 2] Figure 2 shows the geometry of the FEM model. A 10 μm radius sphere filled with electrolyte solution is divided into two chambers (cis: upper, trans: lower) by a 10 nm thick semipermeable membrane. Only liquid, not ions, is allowed to pass across the membrane. A single nanoscale cylindrical opening of various diameters (2 nm to 8 nm) is located in the center of the membrane and serves as the sole pathway for liquid and ion transport between the two chambers. The cis-side boundary conditions were set with varying KCl concentrations (1 M to 2.5 M), and the trans-side boundary conditions were set with varying CaCl concentrations (0.5 M to 1.5 M). All FEM simulations in this paper were performed using this geometry. [Figure 3] Figure 3 shows a schematic diagram of the setup. (a) Cross-section of the electrodeless oSCR setup. When immersed in a lipid / oil environment (2.5 mg / ml DPHPC in a 1:1 volumetric mixture of hexadecane and silicone oil), the aqueous droplet and agarose substrate spontaneously form a droplet interface bilayer (DIB) upon contact

[21] . The aqueous droplet consists of 1 M–2.5 M KCl, 400 μM EDTA, 40 μM Fluo-8, 10 mM HEPES (pH 7.0), and a biological nanopore. The agarose substrate consists of 0.5–1.5 M CaCl2, 10 mM HEPES (pH 7.0), and 2.5% (v / w) low-melting-point agarose. The biological nanopore, pre-dissolved in the droplet, spontaneously inserts into the DIB, allowing Ca2+ to thermodynamically diffuse into the droplet. The transported Ca2+ immediately binds to Fluo-8 in the droplet, emitting fluorescence near the pore when imaged by total internal reflection fluorescence (TIRF) microscopy. (b) Bright-field image of the DIB. The boundaries of the DIB are visually resolvable from the bright-field image. [Figure 4]Figure 4 shows the cyclodextrin binding kinetics. (a-e) Representative current traces for applied voltages of +20 mV, +40 mV, +60 mV, +80 mV, and +100 mV, respectively. Trimethyl-β-cyclodextrin (TriM-β-CD) was added in cis to a final concentration of 4 mM. As the applied voltage increased, the event detection frequency decreased regularly. This suggests that there may be a reverse electroosmotic flow within the nanopore, which reduces the probability of TriM-β-CD binding to the pore. (f) Plot of 1 / τ as a function of applied voltage. 1 / τ statistics were based on three independent electrophysiological recordings (N = 3), with a duration of 90 s for each condition. Electrophysiological recordings were performed with 1.5 M KCl, 10 mM HEPES (pH 7.0) on both sides of the membrane. WT α-HL nanopores were added in cis. [Figure 5] Figure 5 shows the definition of signal and background during oSCR. (a) Representative image frame obtained directly from electrodeless oSCR of the nanopore. (b) 2D Gaussian fitting of a.

number

[0126] The present invention is based on DiffusiOptoPhysiology (DOP), which is simplified from oSCR by removing all electrical connections, and uses nanopore sensors to measure Ca 2+ The fluorescence emission resulting from the diffusion-mediated binding of the nanopore with its indicator, Fluo-8 dye, was optically monitored (Figure 1d). Direct detection of small molecules, large molecules, and biopolymers was subsequently demonstrated through direct fluorescence readout. By eliminating the need for electrode placement, DOP enables parallel measurements from thousands of nanopores using accessible, biocompatible materials at a cost of less than $1 per use. This potentially opens up new possibilities for clinical diagnostics using disposable chips with nanotechnology sensors. The reduced technical barriers in terms of cost, equipment, and skills allow any researcher to easily perform nanopore measurements with little or no training. Various research fields, such as high-throughput drug screening and basic research on ion channels, could benefit from this approach.

[0127] The present invention provides an electrodeless system for identifying analytes based on optical measurement of ion flux through a pore driven by a chemical gradient. The present invention also provides methods of using such systems to identify analytes, including methods for identifying small molecules such as dsDNA or ssDNA, or DNA.

[0128] The electrodeless system includes two compartments separated by a membrane: the first compartment holds a first aqueous solution containing a fluorescent reporter molecule; the second compartment holds a second aqueous solution containing a free ionic species. Upon binding to the ionic species, the fluorescent reporter molecule emits a specific fluorescent light that can be detected by an optical sensor. The membrane between the first and second compartments has at least one nanopore, connecting the first and second compartments via the nanopore. Driven by a chemical gradient, the ionic species pass through the nanopore in the membrane from the second compartment to the first compartment, bind to the fluorescent reporter molecule in the first compartment, and emit fluorescence from the reporter molecule. Because the passage of the ionic species is confined to the nanopore, this results in fluorescence being present in the region proximal to the nanopore. The intensity of the fluorescent signal in the region proximal to the nanopore depends on the flux rate of the ionic species into the first compartment. The fluorescent emission of the fluorescent reporter molecule can then be detected. When the nanopore is blocked or partially blocked by an analyte passing through it, the translocation of ionic species through the nanopore is impeded, which is measured as a decrease in fluorescence near the nanopore and compared to the fluorescence resulting from the unimpeded flow of ionic species through the pore. Different analytes, depending on the size and shape of the analyte, impede the translocation of ionic species across the nanopore to different degrees, resulting in different degrees of fluorescence decrease. The decrease in fluorescence due to pore blockage or partial blockage correlates with the degree to which ionic translocation through the pore is impeded, which reflects information about the properties of the analyte. The magnitude of the fluorescence decrease in the nanopore-proximal region can be used to identify analytes that are causing blockage while passing through the nanopore. The magnitude of the fluorescence decrease can be characterized by the event dwell time and the percentage blockage depth.

[0129] An analyte can be added to either the first compartment or the second compartment, and the analyte passes through the nanopore, e.g., driven by a chemical gradient, from the first compartment to the second compartment or from the second compartment to the first compartment, blocking or partially blocking the nanopore and reducing fluorescence.

[0130] 1. A method for identifying an analyte, comprising: (a) providing a system of the invention comprising an analyte in the first compartment or the second compartment; (b) directing light capable of exciting the fluorescent reporter molecule at a region within the first compartment proximal to the nanopore; (c) measuring a fluorescent signal from the fluorescent reporter molecule to identify the analyte; A method comprising:

[0131] The ionic species can be any ionic species selected in conjunction with the fluorescent reporter molecule to cause a specific fluorescent emission of another molecule. Such ionic species are well known to those skilled in the art. In some embodiments, the ionic species includes Ag + , Ag 2+ , Al 3+ , As 3+ , Au + , Ba 2+ , Bi 3+ , Ca 2+ , Cd 2+ , Ce 3+ , Ce 4+ , Cl - , Co 2+ , Cr 3+ , Cu + , Cu 2+ , Dy 3+ ,EU 3+ , Fe2 + , Fe 3+ , Ga 3+ , H + , Hg + , Hg 2+ , In 3+ , K. + , La 3+ , Mg 2+ , Mn2+ , Mo 3+ , Na + , Ni 2+ , O.H. - , Pb 2+ , Pd 2+ , Pt 2+ , Pt 4+ , Ru 3+ , Sb 3+ ,Sc. 3+ , Sn 2+ , Sr 2+ , Tb 3+ , Tl + , and / or Zn 2+ The ionic species may be one type of ionic species or a combination of two or more types of ionic species.

[0132] The term "fluorescent reporter molecule" may refer to any molecule that, upon binding to an ionic species such as those listed above, produces a specific fluorescent emission distinguishable by an optical sensor. Such fluorescent reporter molecules are well known to those skilled in the art. In some embodiments, the fluorescent reporter molecule may be a calcium fluorescent probe, a sodium fluorescent probe, or a zinc fluorescent probe, which are molecules, such as small molecules, that can chelate calcium ions, sodium ions, or zinc fluorescence, respectively. In some embodiments, fluorescent reporter molecules include, but are not limited to, Fura-2, Indo-1, Fluo-2, Fluo-3, Fluo-4, Fluo-8, Calcium Green-1, DCFH, DHR, SNARF, Cal-520, calcium-specific aminopolycarboxylic acid, or BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid). In some embodiments, fluorescent reporter molecules include, but are not limited to, SBFI, Asante NaTRIUM Green-1, Asante NaTRIUM Green-2, Thallos Potassium Ion Channel Reagent, Asante Potassium Green-1, Asante Potassium Green-2, Asante Potassium Green-3, PBFI, Fluo-2 Mg, Fura-2 Mg, Indo-1 Mg, Asante Magnesium Green, SPQ, MQAE, TSQ, TFL-Zn, ZINQUIN, and the like. The ionic species may be a single fluorescent reporter molecule or a combination of two or more fluorescent reporter molecules. The fluorescent reporter molecules may be membrane-impermeable, such as bilayer-impermeable.

[0133] In the systems and methods of the present invention, the ionic species used should be capable of binding to the fluorescent reporter molecule used and of causing the fluorescent reporter molecule to emit a specific fluorescence. In some embodiments, the ionic species is Ca 2+and the fluorescent reporter molecule is a calcium fluorescent probe such as Fluo-8 or Cal-520.

[0134] The first compartment may contain a first aqueous solution therein. The second compartment may contain a second aqueous solution therein. The first compartment may be completely filled with the first aqueous solution. The first compartment may not be completely filled with the first aqueous solution, and some space or other material may be present in the first compartment in addition to the first aqueous solution. At least the portion of the first compartment immediately proximal to the membrane is filled with the first aqueous solution. The second compartment may be completely filled with the second aqueous solution. The second compartment may not be completely filled with the second aqueous solution, and some space or other material may be present in the first compartment in addition to the second aqueous solution. At least the portion of the second compartment immediately proximal to the membrane is filled with the second aqueous solution. The first and second compartments may independently be of any shape or form. The shape or form of the first and second compartments may be the same or different. In some embodiments, the first compartment and / or the second compartment may or may not have a boundary layer. The boundary layers of the first and second compartments may each be fixed or variable. In some embodiments, the first compartment may be provided by an aqueous droplet. In some embodiments, the second compartment may be provided by a hydrogel layer.

[0135] The first aqueous solution contains a fluorescent reporter molecule. The second aqueous solution contains an ionic species. The second aqueous solution may contain a salt that provides the ionic species, such as a calcium salt. The first and second aqueous solutions may or may not independently contain other components. The first aqueous solution may not contain an ionic species that can bind to a fluorescent reporter molecule and cause fluorescence emission. The second aqueous solution may not contain a fluorescent reporter molecule that can bind to an ionic species and emit fluorescence.

[0136] The first aqueous solution may contain a salt that may be different from the salt in the second aqueous solution. The ions in the first aqueous solution should not bind to the fluorescent reporter molecule and cause it to emit fluorescence. The salt in the first aqueous solution may be any salt that does not bind to the fluorescent reporter molecule and cause it to emit fluorescence. In some embodiments, the first aqueous solution contains sodium chloride. In some embodiments, the first aqueous solution contains potassium chloride. In some embodiments, the second aqueous solution contains calcium chloride. The potassium chloride concentration in the first aqueous solution may be about 0-3.4M. In some embodiments, the potassium chloride concentration is 3M or less. In some embodiments, the potassium chloride concentration is 0.75M or less, 1.0M or less, 1.5M or less, 2.25M or less, or 2.5M or less. The calcium chloride concentration in the second aqueous solution may be about 0.01-6.76M. In some embodiments, the concentration of calcium chloride is at least 0.15 M, at least 0.5 M, at least 0.75 M, at least 1 M, at least 1.5 M, at least 2 M, at least 3 M, at least 4 M, at least 5 M, or at least 6 M. In some embodiments, the concentration of calcium chloride is 6 M or less.

[0137] The inventors have discovered that increasing the concentration of ionic species can improve the sensing signal by increasing the chemical gradient of the ionic species across the membrane, resulting in greater flux of the ionic species through the nanopore. However, the salt concentration is also limited by the maximum solubility of the electrolyte in water (e.g., CaCl2: 6.767 M at 20°C). In some embodiments, the first aqueous solution contains the ions (Ca 2+The nanopore may further include a chelator for competitive binding of the fluorescent dye (e.g., EDTA, BAPTA, EGTA, CyDTA, DTPA, EDDP, HIDA, IDA, NTA, NTPO, TTHA, CA, TA, GA, HEDTA, or DEG), resulting in attenuation of fluorescence when the fluorescent dye moves away from the center of the nanopore. Examples of chelators include, but are not limited to, EDTA, BAPTA, EGTA, CyDTA, DTPA, EDDP, HIDA, IDA, NTA, NTPO, TTHA, CA, TA, GA, HEDTA, or DEG. In some embodiments, the first aqueous solution or the second aqueous solution may include a buffer for adjusting pH, such as Bis-tris, Tris, Hepes, sodium phosphate, and / or potassium phosphate. In some embodiments, the first aqueous solution may include a potassium chloride buffer (e.g., 10 mM HEPES (pH 7.0) and KCL), and the second aqueous solution may include a calcium chloride buffer (e.g., 10 mM HEPES (pH 7.0) and CaCl). In some embodiments, the first aqueous solution may include 1.0 M KCl, 400 μM EDTA, 10 mM HEPES, pH 7.0; 1.5 M KCl, 400 μM EDTA, 10 mM HEPES, pH 7.0; 2.25 M KCl, 400 μM EDTA, 10 mM HEPES, pH 7.0; or 2.5 M KCl, 400 μM EDTA, 10 mM HEPES, pH 7.0. In some embodiments, the second aqueous solution may include 0.5 M CaCl, 10 mM HEPES, pH 7.0; 0.75 M CaCl, 10 mM HEPES, pH 7.0; 1 M CaCl, 10 mM HEPES, pH 7.0; or 1.5 M CaCl, 10 mM HEPES, pH 7.0. Salts may be included in the first or second aqueous solutions for other reasons, e.g., to stabilize proteins, to adjust binding moieties, to adjust osmolality / osmolality, and / or to activate fluorescent probes.

[0138] The first aqueous solution may contain an analyte that migrates, driven by a chemical gradient, through a nanopore in the membrane from the first compartment to the second compartment, blocking or partially blocking the nanopore and reducing fluorescence from the fluorescent reporter molecule. The second aqueous solution may contain an analyte that migrates, driven by a chemical gradient, through a nanopore in the membrane from the second compartment to the first compartment, blocking or partially blocking the nanopore and reducing fluorescence from the fluorescent reporter molecule.

[0139] In some cases, the first compartment may be provided by an aqueous droplet. The aqueous droplet may comprise or consist of a first aqueous solution. In some cases, the second compartment may be provided by a hydrogel layer, such as a hydrogel layer comprising an agarose substrate. The hydrogel may comprise a second aqueous solution. When contacted in a hydrophobic medium containing amphiphilic molecules that are selectively permeable to water molecules, the aqueous droplet and hydrogel layer may spontaneously form a droplet interface bilayer (DIB) consisting of the amphiphilic molecules. The protein nanopore may be provided in the aqueous droplet or the hydrogel, allowing the protein nanopore to spontaneously insert into the DIB during DIB formation. The aqueous droplet may contain an analyte.

[0140] The substrate of the hydrogel layer may comprise or consist of a hydrophilic polymer. The substrate of the hydrogel layer may comprise or consist of a substantially transparent hydrophilic polymer. The substrate of the hydrogel layer may comprise or consist of agarose. Other hydrogel materials may be suitable, such as polyacrylamide, cross-linked polyethylene glycol, or nitrocellulose. The hydrogel layer may comprise 0.1 to 20% (w / v) agarose. In some embodiments, the hydrogel layer may comprise less than 5% (w / v) agarose, less than 4% (w / v) agarose, or about 3% (w / v) agarose. The hydrogel may comprise more than 1% (w / v) agarose, more than 2% (w / v) agarose, or about 2% to about 4% agarose. The hydrogel may comprise about 2.5% (w / v) to about 3.5% (w / v) agarose. The hydrogel layer may comprise an analyte.

[0141] The membrane separating the first and second compartments may be any material capable of supporting a nanopore. The membrane may be natural, synthetic, or artificial. The membrane may be a solid membrane. The membrane may include or consist of a solid substrate, such as SiNx, glass, silicon dioxide, molybdenum disulfide, graphene, aluminum oxide, or CNTs (carbon nanotubes).

[0142] The membrane may be a semipermeable membrane. A semipermeable membrane is selectively permeable to water molecules. Ionic species, fluorescent reporter molecules, analytes, etc. cannot pass through the semipermeable membrane and, as a result, their passage is restricted to the nanopore. Such semipermeable membranes and methods for their manufacture are well known to those skilled in the art. The semipermeable membrane may comprise or consist of amphipathic molecules that are selectively permeable to water molecules.

[0143] The semipermeable membrane may comprise or consist of amphiphilic molecules. The amphiphilic molecules may be lipids or polymers, such as block copolymers. The membrane may be a monolayer or a bilayer, e.g., a monolayer or a bilayer that may comprise or consist of amphiphilic molecules. Examples include monolayers comprising or consisting of polymers, such as block copolymers, and bilayers comprising or consisting of lipids. The bilayer may be a lipid bilayer. The bilayer may be an artificial bilayer, e.g., a non-natural bilayer. The bilayer may not be a cell bilayer. The bilayer may not be a patch-clamp cell bilayer. Those skilled in the art will appreciate that there are multiple methods for obtaining a bilayer. The bilayer may be provided by the droplet hydrogel bilayer (DHB) method, for example, as provided in International Publication No. WO2009024775, the contents of which are incorporated herein by reference.

[0144] In some embodiments, a semipermeable membrane may be obtained by interaction between the first and second compartments in a hydrophobic medium containing amphiphilic molecules, such as lipids or block copolymers. In some embodiments, a semipermeable membrane may be obtained by immersing the first and second compartments in a hydrophobic medium containing amphiphilic molecules and contacting the first and second compartments such that the amphiphilic molecules form a semipermeable membrane. As a result, a semipermeable membrane composed of the amphiphilic molecules spontaneously forms between the first and second compartments. In some embodiments, a protein nanopore may be provided in the first or second compartment, and the protein nanopore may spontaneously insert into the bilayer as the membrane composed of amphiphilic molecules spontaneously forms. In some embodiments, a semipermeable membrane may be obtained by immersing the first and second compartments, either of which has a protein nanopore, in a hydrophobic medium containing amphiphilic molecules. As a result, a semipermeable membrane of the amphiphilic molecules spontaneously forms between the first and second compartments, allowing the protein nanopore to spontaneously insert into the bilayer.

[0145] In some embodiments, the bilayer may be obtained by contacting a first compartment having a monolayer of amphipathic molecules with a second compartment having a monolayer of amphipathic molecules, thereby allowing the bilayer to spontaneously form. In some embodiments, the bilayer may be obtained by immersing the first compartment and the second compartment, one of which has a protein nanopore, in a hydrophobic medium containing amphipathic molecules, thereby allowing the monolayer of amphipathic molecules to form on the surfaces of the first compartment and the second compartment, and then contacting the first compartment and the second compartment such that the monolayer of amphipathic molecules forms a bilayer. As a result, a bilayer consisting of the amphipathic molecules spontaneously forms between the first compartment and the second compartment, allowing the protein nanopore to spontaneously insert into the bilayer.

[0146] Thus, one aspect of the invention is a method for producing the above system, comprising the steps of: providing a first compartment containing a first aqueous solution therein, the first aqueous solution including a fluorescent reporter molecule capable of fluorescing when bound to an ionic species; providing a second compartment containing a second aqueous solution therein, the second aqueous solution comprising said ionic species that specifically binds to said fluorescent reporter molecule; contacting the first compartment with the second compartment in a hydrophobic medium containing amphipathic molecules such that a semipermeable membrane having a nanopore inserted therein is formed between the first compartment and the second compartment; Including, a protein nanopore is provided in the first aqueous solution or the second aqueous solution; a semipermeable membrane composed of the amphiphilic molecule is spontaneously formed between the first compartment and the second compartment, and the protein nanopore can be spontaneously inserted into the semipermeable membrane; A method is provided.

[0147] The features described herein, such as the first compartment, the second compartment, the fluorescent reporter molecule, the ionic species, the protein nanopore, the semipermeable membrane, the bilayer, the hydrophobic medium, the amphipathic molecule, the analyte, etc., are as described in the context of this specification.

[0148] In the present invention, the analyte may be provided in the first or second aqueous solution before the semipermeable membrane is formed, or the analyte may be added to the first or second aqueous solution after the semipermeable membrane is formed and before detection begins.

[0149] In some embodiments, the amphiphilic molecule may be selectively permeable to water molecules. The amphiphilic molecule used in any of the methods of the present invention may be a polymer molecule or a lipid molecule, and in particular, a surfactant molecule. The lipid molecule may be selected from the group including fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, glycolipids, polyketides, phospholipids, glycolipids, and cholesterol. The lipid may include any of the group including monoolein; 1,2-dioleoyl-sn-glycero-S-phosphocholine (DOPC); 1,2-diphytanoyl-sn-glycero-3-phosphatidylcholine (DPhPC); palmitoyloleoylphosphatidylcholine (POPC); 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE); 1-palmitoyl-2-oleoyl-phosphatidylethanolamine; and 1-palmitoyl-2-oleoylphosphatidylglycerol (POPE / POPG) mixture; or a mixture thereof.

[0150] The polymer may be a block copolymer, e.g., a triblock copolymer, capable of forming a semipermeable membrane, such as those provided in Discher, DE and Ahmed, F., Polymersomes. Annu. Rev. Biomed. Eng., Vol. 8, pp. 323-341 (2006); Nardin, C., Winterhalter, M., and Meier, W., Giant free-standing ABA triblock copolymer membranes. Langmuir, Vol. 16, pp. 7708-7712 (2000); Meier, W., Nardin, C., and Winterhalter, M., Reconstitution of channel proteins in (polymerized) ABA triblock copolymer membranes. Angew. Chem. Int. Ed., Vol. 39, pp. 4599-4602 (2000), or CN104936682B, which are incorporated herein by reference. In one embodiment, the triblock copolymer is poly(2-methyloxazoline)-block-poly(dimethylsiloxane)-block-poly(2-methyloxazoline) (PMOXA-PDMS-PMOXA) or poly(2-methyloxazoline)-block-poly(ethylene)-block-poly(2-methyloxazoline) (PMOXA-PE-PMOXA).

[0151] The hydrophobic medium may comprise an oil. In some embodiments, the hydrophobic medium may be an oil. The hydrophobic medium containing the amphiphilic molecules may comprise or consist of a lipid-in-oil. The oil may be a hydrocarbon, which may be branched or unbranched, substituted or unsubstituted. For example, the hydrocarbon may have 5 to 20 carbon atoms, more preferably 10 to 17 carbon atoms. Suitable oils include alkanes or alkenes such as hexadecane, decane, pentane, or squalene, or fluorinated oils, or silicone-based oils, or carbon tetrachloride; or mixtures thereof. In some embodiments, the oil is an n-alkane, such as a C10 to C17 n-alkane, such as n-hexadecane (C16). In some embodiments, the hydrophobic medium may be an oil, such as a mixture of hexadecane and silicone oil. In some embodiments, the oil may comprise a 1:1 (v:v) mixture of hexadecane and silicone oil AR20 (Sigma-Aldrich).

[0152] Other bilayer formation methods can also be used instead. For example, bilayers can be obtained by any one of the following techniques known to those skilled in the art, including patch clamping, e.g., optical patch clamping; black lipid membranes (BLMs), also known as painted BLMs; supported lipid bilayers (SLBs); and tethered bilayer lipid membranes (t-BLMs). Bilayers can be formed at apertures according to International Publication No. 2008102121, the contents of which are incorporated herein by reference. Bilayers can also be formed at the interface between droplets according to International Publication No. 2014064444, the contents of which are incorporated herein by reference.

[0153] As used herein, the term "nanopore" refers to a channel having an opening at its narrowest point, the opening having a diameter that allows the passage of an analyte. The nanopore is sufficiently narrow that blockage of the channel by the analyte is detectable by a change in a particular signal, such as a fluorescent signal.

[0154] The membrane nanopore can vary in size depending on the intended use of the system, but must be large enough to allow ionic passage of the ionic species used in the present invention. Preferably, the nanopore is also small enough to prevent passage of fluorescent reporter molecules. The nanopore may also be large enough to allow passage of the analyte.

[0155] Regardless of whether the membrane is solid or semipermeable, the nanopore may be a solid-state nanopore, a DNA nanopore, or a protein nanopore. The nanopore may be natural, e.g., biologically derived, or alternatively, the nanopore may be synthetic. The nanopore may be recombinantly produced. The nanopore may be a biomolecule, such as a protein nanopore (sometimes referred to as a nanopore-forming protein). In some cases, the nanopore may be formed by a protein, sometimes referred to as a protein nanopore or nanopore-forming protein. Protein nanopores used in the present invention preferably do not have spontaneous gating activity and / or preferably remain open in the absence of an analyte. The protein nanopore used in the present invention may be any type. Examples of protein nanopores or nanopore-forming proteins include α-HL, ClyA, Phi29 connector protein, erolysin, MspA, OmpF, OmpG, FraC, HlyA, SheA, sp1, or variants thereof. In some embodiments, one or more nanopores are ClyA-RR

[42] . ClyA-RR is a mutant of ClyA (D64R / C87A / L99Q / E103G / S110R / F166Y / I203V / C285S / K294R / H307Y). Other examples of biomolecular nanopores include nanopores formed by DNA self-assembly. Nanopores that can be used in the present invention may also be ion channels, such as potassium channels or sodium channels.

[0156] In the present invention, the term "α-HL," which may also be referred to as α-hemolysin, may be selected from the group consisting of wild-type α-hemolysin, mutant α-hemolysin, a paralog or homolog hemolysin of wild-type α-hemolysin, and a paralog or homolog hemolysin of mutant α-hemolysin. In some embodiments, the α-hemolysin may be wild-type α-hemolysin. The α-hemolysin that may be used in the present invention should be capable of forming a nanopore. In some embodiments, the α-HL is a heptamer.

[0157] In the present invention, the term "ClyA" may be selected from the group consisting of wild-type ClyA, mutant ClyA, a paralog or homologue of wild-type ClyA, and a paralog or homologue of mutant ClyA. In some embodiments, the ClyA may be wild-type α-hemolysin. In some embodiments, the ClyA may be a mutant ClyA. A preferred mutant ClyA is ClyA-RR. The ClyA that may be used in the present invention should be capable of forming a nanopore. In some embodiments, the ClyA or ClyA-RR is a dodecamer.

[0158] The sequences of protein nanopores, such as the sequences of α-HL and ClyA or their variants, are known to those skilled in the art. Methods for producing protein nanopores, such as α-HL or ClyA or their variants, are known to those skilled in the art, and can be produced, for example, by prokaryotic expression and simple purification by gel electrophoresis or chromatography. Protein nanopores can be formed by the self-assembly of several protein monomers, such as dodecamer ClyA, dodecamer ClyA-RR, or heptameric α-HL. In some examples, protein nanopores can self-assemble into semipermeable membranes.

[0159] The nanopore may be a solid-state nanopore, such as one comprising a synthetic material such as silicon nitride or graphene. A solid-state nanopore is typically a nanometer-sized hole formed in a synthetic membrane (such as SiNx or SiO2). Solid-state nanopores can be fabricated with focused ion or electron beams, allowing for tunable pore size. The nanopore may also be a hybrid nanopore, comprising a set of pore-forming proteins in a synthetic material.

[0160] Methods for forming nanopores in membranes are well known to those of skill in the art, such as by adding nanopore molecules to the semipermeable membrane after or during its formation. In some embodiments, the protein nanopore may be provided in the first compartment or the second compartment and spontaneously inserted into the semipermeable membrane as the membrane of amphiphilic molecules spontaneously forms.

[0161] The analyte is not limited to a specific molecule and may be any molecule capable of blocking or partially blocking the nanopore when passing through it. Analytes may include, but are not limited to, small molecules, polymers, or biopolymers. A small molecule refers to a molecule or ion with a low molecular weight and size that is much smaller than the nanopore diameter and easily passes through the nanopore. Small molecules may include, but are not limited to, chemical compounds, drugs, sugars, ions, neurotransmitters, amino acids, or nucleotides. A polymer refers to a very large molecule, typically composed of thousands or more atoms. Polymers may include, but are not limited to, biopolymers such as nucleic acids, proteins, carbohydrates, or lipids; large non-polymeric molecules such as lipids or macrocycles; or synthetic polymers. Examples of polymers include biopolymers, such as, but not limited to, polypeptides, polysaccharides, or polynucleotides, such as DNA (including ssDNA or dsDNA) or RNA (including miRNA, siRNA, or tRNA). The length of DNA such as ssDNA or dsDNA may be 10 to 1000 nt. The length of DNA such as ssDNA or dsDNA may be more than 15 nt, more than 20 nt, more than 30 nt, more than 40 nt, more than 50 nt, more than 60 nt, more than 70 nt, more than 80 nt, more than 90 nt, or more than 100 nt. The length of DNA such as ssDNA or dsDNA may be less than 500 nt, less than 4000 nt, less than 300 nt, or less than 200 nt. The length of RNA such as miRNA, siRNA, or tRNA may be more than 15 nt, more than 20 nt, more than 30 nt, more than 40 nt, more than 50 nt, more than 60 nt, more than 70 nt, more than 80 nt, more than 90 nt, or more than 100 nt. The length of RNA such as miRNA, siRNA, or tRNA may be less than 500 nt, less than 4000 nt, less than 300 nt, or less than 200 nt.

[0162] The analyte may be placed in the first or second aqueous solution. The analyte may be included in the first or second aqueous solution at the time of preparation, i.e., the analyte may be formulated into the first or second aqueous solution along with other desired components. The analyte may be added to the first or second aqueous solution once the system is ready and the test is to be initiated.

[0163] In the present invention, the term "identification" includes obtaining identifying information, e.g., detection or analysis of the type of analyte, or structural information about the analyte, e.g., the structure of a polymer, or the structure of a polynucleotide or polypeptide, such as the primary or secondary structure of a polynucleotide.

[0164] Blocked analytes passing through the nanopore can be identified by the magnitude of the fluorescence decrease in the nanopore proximal region. With the present disclosure, those skilled in the art will know how to identify analytes based on the magnitude of the fluorescence decrease, which can be characterized, for example, by event residence time and percent blockage depth. Event residence time is the residence time that the analyte occupies the nanopore. Percent blockage depth is I b / I o where I b and I o and represent absolute blockage current and open pore current, respectively. For example, the fluorescence emission resulting from the obstruction of ion flow can be measured and compared to the fluorescence emission of a reference substance under the same test conditions to determine whether the analyte and the reference are identical. Comparisons can be made based on the event residence time of the fluorescence emission and / or the percent blockage depth.

[0165] Fluorescence detection may include microscopic or spectroscopic observation of the membrane and membrane region. Fluorescence detection may include the use of total internal reflection fluorescence (TIRF), wide-field fluorescence microscopy, or confocal microscopy. Fluorescence detection may include the use of thin-film oblique illumination (HiLo microscopy), for example, as provided by Tokunaga et al. (2008, Highly inclined thin illumination enables clear single-molecule imaging in cells. Nat Meth, vol. 5, pp. 159-161). Fluorescence detection may also include the use of other grazing incidence illumination methods. Any suitable fluorescence detection means may be used to detect the fluorescent signal / emission in the membrane and the membrane region immediately surrounding the nanopore therein. Fluorescence detection may also include the use of surface plasmon resonance. Fluorescence detection may involve the use of super-resolution microscopy, such as deterministic super-resolution microscopy, including STED, GSD, RESOLFT, or SSIM; or stochastic super-resolution, including SOFI; or single-molecule localization microscopy (SMLM), such as SPDM, SPDMphymod, PALM, FPALM, STORM, or dSTORM. Fluorescence detection may involve the use of epifluorescence microscopy, confocal laser scanning microscopy (LSM), or total internal reflection fluorescence (TIRF) microscopy. Fluorescence detection may involve the use of fluorescence correlation spectroscopy (FCS). Image correlation spectroscopy (ICS) may be used to calculate the spatial correlation function of fluctuations in fluorescence intensity in images acquired by confocal LSM or two-photon LSM, or using TIRF microscopy. Fluorescence detection methods may be as described in Ana J. Garcia-Saez, Petra Schwille., Surface analysis of membrane dynamics, Biochimica et Biophysica Acta, 1798 (2010), pp. 766-776, the contents of which are incorporated herein by reference.

[0166] Detection of fluorescent emission from the fluorescent reporter molecule may require a light source and a light sensor. The light source and light sensor may be in the same device or in separate devices. The light source should be capable of providing light at a wavelength or range of wavelengths capable of exciting the fluorescent reporter molecule in the presence of the ionic species, and the light sensor should be capable of detecting light at a wavelength or range of wavelengths emitted by the fluorescent reporter molecule in the presence of the ionic species.

[0167] Thus, in some embodiments, the system includes a light source capable of illuminating the membrane region proximal to the nanopore. In some embodiments, the light source provides light within a specific range of wavelengths. In some embodiments, the light source may be a laser, an LED, a halogen light, or a xenon light. Methods for illuminating fluorescent reporter molecules with a light source are known to those of skill in the art.

[0168] In some embodiments, the system includes an optical sensor capable of detecting an optical signal in the membrane region proximal to the nanopore. The optical sensor may be a light-sensitive device sensitive to low levels of light (i.e., fluorescence), such as a charge-coupled device (CCD), an electron-multiplying CCD (EMCCD), an sCMOS sensor, or a photodiode, such as an avalanche photodiode (APD). High-speed light-sensitive devices are preferred. Preferably, the optical sensor is an EMCCD or an avalanche photodiode (APD).

[0169] The optical sensor may also be a microscope imaging system, a photomultiplier tube, or an optical sensor capable of detecting fluorescence using the fluorescence detection techniques described above. In some embodiments, a total internal reflection fluorescence (TIRF) imaging system, such as a total internal reflection fluorescence microscope (TIRFM), may be used to detect and / or record the optical signal in the membrane region proximal to the nanopore. In some embodiments, a wide-field fluorescence imaging system or a confocal imaging system may be used to detect and / or record the optical signal in the membrane region proximal to the nanopore. Methods for detecting fluorescent emissions from fluorescent reporter molecules using optical sensors are known to those of skill in the art.

[0170] In some embodiments, the optical sensor and the optical detector may be a single device. Some fluorescence detection devices may be used for illumination. For example, TIRFM may be used for both illumination and imaging.

[0171] The inventors have discovered that osmotic flow between the first and second compartments is advantageous for further improving the fluorescent detection of analytes by nanopores. The osmolality / osmolality difference between the first and second compartments can drive directional water flow, carrying ions and analytes, through a biological nanopore inserted into the membrane. As a result, the introduction of this asymmetry should achieve enhanced analyte translocation efficiency. Furthermore, osmotic flow between the first and second compartments can amplify the fluorescent signal. Due to the fact that the fluorescent reporter molecule is impermeable to the semipermeable membrane when the osmolality (or osmolality) of the second aqueous solution is higher than that of the first aqueous solution, osmotic flow across the membrane concentrates the fluorescent reporter molecule around the membrane in the first compartment, resulting in enhanced fluorescence intensity.

[0172] Thus, in some embodiments, the methods of the invention can be carried out in an environment where the first and second aqueous solutions remain isotonic, or where the osmolality (or osmolality) of the second aqueous solution is lower than the osmolality (or osmolality) of the first aqueous solution, although in some embodiments the osmolality (or osmolality) of the second aqueous solution can be higher or lower than the osmolality (or osmolality) of the first aqueous solution.

[0173] Both osmolality and osmolality are defined in units of osmoles. Osmole is a unit of measure that describes the number of moles of a compound contributing to the osmotic pressure of a chemical solution, i.e., the hydrostatic pressure resulting from a concentration gradient across two sides, such as a concentration gradient across a semipermeable membrane. Osmolality is defined as the number of osmoles of solute per volume of solution. Osmolality is commonly expressed in osmol / L. Osmolality is very similar, but is defined as the number of osmoles of solute per kilogram of pure water solvent and is commonly expressed in osmol / kg. For example, a solution of 1 mol / L NaCl corresponds to an osmolality of 2 osmol / L. NaCl salt particles completely dissociate in water, forming two separate particles, Na + ions and Cl - ions. Therefore, each mole of NaCl becomes 1 mole of Na in solution. + and 1 mole of Cl - Similarly, a solution of 1 mol / L CaCl2 has a concentration of 3 osmol / L (Ca 2+ and 2Cl - Methods for determining the osmolality or osmolality of a solution are known to those skilled in the art.

[0174] The osmolality / osmolality of the second aqueous solution may be increased by increasing the concentration of ionic species or by further adding other solutes that can increase the osmolality / osmolality of the first aqueous solution. Since increasing the concentration of ionic species can improve the sensing signal, preferably, the osmolality / osmolality of the second aqueous solution is increased by increasing the concentration of ionic species. The osmolality / osmolality of the first aqueous solution may be decreased by decreasing the concentration of solutes that can contribute to osmotic pressure, or the first aqueous solution may not contain salt. In some embodiments, the osmolality of the second aqueous solution is at least 0.01 osmol / L, at least 0.05 osmol / L, at least 0.1 osmol / L, at least 0.2 osmol / L, at least 0.3 osmol / L, at least 0.4 osmol / L, at least 0.5 osmol / L, at least 0.6 osmol / L, at least 0.7 osmol / L, at least 0.8 osmol / L, at least 0.9 osmol / L, at least 1.0 osmol / L, at least 1.5 osmol / L, at least 2.0 osmol / L, at least 2.5 osmol / L, at least 3.0 osmol / L, at least 3.5 osmol / L, at least 4.0 osmol / L, at least 4.5 ... 0.5 osmol / L, at least 5.0 osmol / L, at least 5.5 osmol / L, at least 6.0 osmol / L, at least 6.5 osmol / L, at least 7.0 osmol / L, at least 7.5 osmol / L, at least 8.0 osmol / L, at least 8.5 osmol / L, at least 9.0 osmol / L, at least 9.5 osmol / L, at least 10 osmol / L, at least 11 osmol / L, at least 12 osmol / L, at least 13 osmol / L, at least 14 osmol / L, at least 15 osmol / L, at least 16 osmol / L, at least 17 osmol / L, at least 18 osmol / L, at least 19 osmol / L, or at least 20 osmol / L higher.In some embodiments, the osmolality of the second aqueous solution is at least 0.01 osmol / kg, at least 0.05 osmol / kg, at least 0.1 osmol / kg, at least 0.2 osmol / kg, at least 0.3 osmol / kg, at least 0.4 osmol / kg, at least 0.5 osmol / kg, at least 0.6 osmol / kg, at least 0.7 osmol / kg, at least 0.8 osmol / kg, at least 0.9 osmol / kg, at least 1.0 osmol / kg, at least 1.5 osmol / kg, at least 2.0 osmol / kg, at least 2.5 osmol / kg, at least 3.0 osmol / kg, at least 3.5 osmol / kg, at least 4.0 osmol / kg, at least 4.5 ... 0.5 osmol / kg, at least 5.0 osmol / kg, at least 5.5 osmol / kg, at least 6.0 osmol / kg, at least 6.5 osmol / kg, at least 7.0 osmol / kg, at least 7.5 osmol / kg, at least 8.0 osmol / kg, at least 8.5 osmol / kg, at least 9.0 osmol / kg, at least 9.5 osmol / kg, at least 10 osmol / kg, at least 11 osmol / kg, at least 12 osmol / kg, at least 13 osmol / kg, at least 14 osmol / kg, at least 15 osmol / kg, at least 16 osmol / kg, at least 17 osmol / kg, at least 18 osmol / kg, at least 19 osmol / kg, or at least 20 osmol / kg higher.

[0175] By measuring the fluorescence emitted from fluorescent reporter molecules, the present invention allows for the simultaneous recording of flow through many nanopores without the need for expensive electrode arrays. The measured fluorescence can be separated into multiple fluorescence traces for each nanopore, making it applicable to situations requiring high-throughput screening, such as nanopore arrays.

[0176] In another aspect of the present invention, an electrodeless nanopore array is provided. The nanopore array comprises a plurality of systems of the present invention in parallel, each system being as described above. The nanopore array can be used to simultaneously identify multiple analytes. The nanopore array of the present invention can be used without electrodes, thereby reducing the size and cost of the device.

[0177] The multiple systems are arranged such that the measured fluorescence is distinguishable in each system. At least some of the multiple systems are separated from one another so that each system can be used independently to detect an analyte therein and so that the measured fluorescence is distinguishable in each system. In some embodiments, at least the first compartments of the multiple systems are separated from one another. In some embodiments, the second compartments of the multiple systems are separated from one another or are not separated from one another. The multiple systems may be the same or different.

[0178] The density of the nanopore array is up to 10 / mm 2 , max. 50 / mm 2 , max. 100 / mm 2 , max. 200 / mm 2 , up to 300 / mm 2 , up to 400 / mm 2 , max. 500 / mm 2 , up to 600 / mm 2 , up to 700 / mm 2 , up to 800 / mm 2 , up to 900 / mm 2 , up to 1000 / mm 2 , or even more.

[0179] The total area provided by the above multiple systems is up to 1mm 2 , max. 2mm 2 , max. 5mm 2 , max. 10mm 2 , up to 15mm 2 , max. 20mm 2 , up to 25mm 2 , up to 30mm 2 , up to 35mm2 , up to 40mm 2 , up to 45mm 2 , max. 50mm 2 , up to 55mm 2 , up to 60mm 2 , up to 65mm 2 , up to 70mm 2 , up to 75mm 2 , up to 80mm 2 , up to 85mm 2 , up to 90mm 2 , up to 95mm 2 , or up to 100 mm 2 , or even more.

[0180] Multiple analytes can be provided to two or more systems of the array, or to each system, such that the analytes are physically separated into the various systems. The multiple analytes can be the same or different, or partially the same or partially different. In some embodiments, at least two of the multiple analytes can be different. In some embodiments, the same analyte can be provided to two or more systems of the array, or to each system. In some embodiments, different analytes can be provided to two or more systems of the array, or to each system. In some embodiments, different analytes can be provided to different systems. When multiple analytes pass simultaneously through different nanopores, the fluorescence in the region proximal to each nanopore decreases, allowing for separation into multiple fluorescence traces for each nanopore, thereby enabling identification of each analyte.

[0181] Such nanopore arrays provide a multiplex method for identifying multiple analytes, (a) providing a nanopore array of the invention comprising a plurality of analytes, wherein two or more analytes are provided to various systems of the nanopore array; (b) directing light capable of exciting a fluorescent reporter molecule contained in each of the first compartments at a region within the plurality of first compartments proximal to the nanopore; (c) measuring a plurality of fluorescent signals from the fluorescent reporter molecules contained in each of the first compartments to identify a plurality of analytes; It can be used in multiplexed methods including

[0182] The features described herein, such as the first compartment, the second compartment, the fluorescent reporter molecule, the ionic species, the protein nanopore, the membrane, the bilayer, the hydrophobic medium, the amphipathic molecule, the analyte, etc., are as described in the context of this specification.

[0183] In a nanopore array, the different systems may contain the same or different fluorescent reporter molecules and different ionic species. For convenience of detection, the different systems preferably contain the same fluorescent reporter molecules and different ionic species.

[0184] In a nanopore array, the different systems may contain the same or different nanopores. For convenience of detection, preferably, the different systems contain the same nanopores.

[0185] In some embodiments, the first compartments of different systems are separated from one another, and the second compartments of different systems are not separated from one another. In some embodiments, a nanopore array may be obtained by contacting a plurality of first and second compartments in a hydrophobic medium containing amphiphilic molecules that are selectively permeable to water molecules, where each first compartment contains at least a protein nanopore therein. A semipermeable membrane composed of amphiphilic molecules will spontaneously form between the first and second compartments, allowing the protein nanopore to spontaneously insert into the semipermeable membrane. In some embodiments, each first compartment contains a protein nanopore, a fluorescent reporter molecule, and optionally an analyte therein, and different analytes may be physically separated into various water-in-oil compartments.

[0186] In some embodiments, the first compartment of each system is provided by an aqueous droplet, and the second compartment of each system is provided by a hydrogel layer, such as a hydrogel layer comprising an agarose substrate. In some embodiments, the second compartments of multiple systems are provided by a single hydrogel layer. In some embodiments, a nanopore array may be obtained by contacting multiple aqueous droplets with a hydrogel layer in a hydrophobic medium containing amphiphilic molecules that are selectively permeable to water molecules, where each aqueous droplet contains a protein nanopore, a fluorescent reporter molecule, and an analyte, and different analytes can be physically separated into various water-in-oil droplets. A semipermeable membrane composed of amphiphilic molecules will spontaneously form between each aqueous droplet and the hydrogel layer, allowing the protein nanopores to spontaneously insert into the semipermeable membrane.

[0187] In another aspect of the invention, there is provided a method of fabricating a nanopore array, comprising the steps of: providing a plurality of aqueous droplets, each containing a protein nanopore, an analyte, and a fluorescent reporter molecule capable of fluorescing when bound to an ionic species; providing a hydrogel layer containing ionic species; contacting a plurality of aqueous droplets with a hydrogel layer in a hydrophobic medium containing amphiphilic molecules such that a semipermeable membrane is formed between each of the aqueous droplets and the hydrogel layer; A method is provided which includes:

[0188] The volume of each aqueous droplet may be less than 100 pL, less than 90 pL, less than 80 pL, less than 70 pL, less than 60 pL, less than 50 pL, less than 40 pL, for example, about 30 pL. The density of the droplet-hydrogel array is less than 1 mm 2 At least 10 droplets per 1mm 2 At least 50 droplets per 1 mm 2 At least 100 droplets per 1mm 2 At least 200 droplets per 1mm 2 At least 300 droplets per 1mm 2 At least 400 droplets per 1mm 2At least 500 droplets per 1mm 2 At least 600 droplets per 1mm 2 At least 700 droplets per 1mm 2 At least 800 droplets per 1mm 2 At least 900 droplets per 1mm 2 The number of droplets may be at least 1000 per well, such as highly ordered droplet-hydrogel arrays that can be formed with the assistance of microfluidics.

[0189] In one nanopore array of the present invention, the number of aqueous droplets may be between 4 and 1,000,000. In some embodiments, the number of aqueous droplets is greater than 10, greater than 100, or greater than 1000. In some embodiments, the number of aqueous droplets is less than 100,000, less than 10,000, or less than 1000.

[0190] The light sources, optical sensors, and recording devices described for the single systems or methods above can be used with the nanopore arrays of the invention. As known to those skilled in the art, over 2500 pores may be recorded simultaneously with an electron-multiplying CCD camera (ixon3, Andor).

[0191] The amplification of the fluorescent signal by the osmolality / osmolality difference is also applicable to the multiplex systems and methods of the present invention.

[0192] In another aspect of the invention, there is provided a kit for forming a nanopore array, comprising: a filling hydrogel comprising agarose, a buffer, and an ionic species capable of specifically binding to a fluorescent reporter molecule and causing it to emit fluorescence; an aqueous solution comprising a chelating agent, a fluorescent reporter molecule capable of fluorescing when bound to an ionic species, and a buffer; wherein the chelating agent is capable of binding to an ionic species; a hydrophobic medium containing amphiphilic molecules; solid support, A kit is provided comprising:

[0193] The solid support may have any structure suitable for contacting a hydrogel with a droplet of aqueous solution in a hydrophobic medium containing amphiphilic molecules to form a semipermeable membrane between the hydrogel and the droplet of aqueous solution. The solid support may be made of PMMA or glass. The solid support may be a PMMA measurement device having a structure as shown in Figure 19. As shown in Figures 3 and 19, the PMMA measurement device may have four independent droplet wells in a central recessed area, a gel inlet for filling the hydrogel, an outlet for discharging the hydrogel, and a cover glass for supporting the hydrogel. The cover glass may be an oxygen plasma-treated cover glass. PMMA is also known as polymethyl methacrylatemethacrylic acid.

[0194] The kit may also include a coating hydrogel containing agarose in water. To form a nanopore array, a cover glass may be spin-coated with the dissolved coating hydrogel and attached to a PMMA measurement device by filling the dissolved filling hydrogel through the gel inlet. The cover glass may then be immersed in a hydrophobic medium containing amphiphilic molecules. Aqueous droplets containing the protein nanopores and analytes may be pipetted into the hydrophobic medium and incubated. The droplets and agarose hydrogel may be brought into contact in the hydrophobic medium, and a nanopore array in the form of a DIB may spontaneously form.

[0195] In some embodiments, the osmolality of the filling hydrogel is greater than the osmolality of the aqueous solution, or the osmolality of the filling hydrogel is greater than the osmolality of the aqueous solution; alternatively, the osmolality of the filling hydrogel is equal to the osmolality of the aqueous solution, or the osmolality of the filling hydrogel is equal to the osmolality of the aqueous solution; alternatively, the osmolality of the filling hydrogel is less than the osmolality of the aqueous solution, or the osmolality of the filling hydrogel is less than the osmolality of the aqueous solution.

[0196] In some embodiments, the fluorescent reporter molecule may be Fluo-8 and the ionic species is Ca 2+ It may also be possible to use the following.

[0197] The filling hydrogel or aqueous solution may also contain protein nanopores.

[0198] The aqueous solution may contain a salt such as KCl or NaCl.

[0199] The features described herein, such as fluorescent reporter molecules, ionic species, protein nanopores, hydrophobic media, amphipathic molecules, chelators, analytes, etc., are as described in connection with the present specification.

[0200] In some embodiments, the hydrogel for application may be 0.75% (w / v) agarose in water.

[0201] In some embodiments, the filling hydrogel may include 2.5% agarose, 1.5 M CaCl2, and 10 mM HEPES (pH 7.0).

[0202] In some embodiments, the aqueous solution may contain 1.5 M KCl, 400 μM EDTA, 40 μM Fluo-8, 10 mM HEPES (pH 7.0).

[0203] In some embodiments, the hydrophobic medium containing the amphiphilic molecules may be a lipid oil comprising 5 mg of dry DPHPC lipid membrane dissolved in 2 mL of a 1:1 volumetric mixture of hexadecane and silicone oil. In another aspect, the present invention provides a nanopore array formed by the above-described manufacturing method.

[0204] In another aspect, the present invention provides the use of the above system or the above nanopore array for performing optical analyte analysis.

[0205] Unless otherwise specified, most of the features of the systems and methods are common to the different systems and methods, such as features of the first and second compartments, first and second aqueous solutions, membranes, nanopores, fluorescent reporter molecules, and / or ionic species, as well as methods of forming them, methods of using them, etc. For example, features of the first and second compartments, first and second aqueous solutions, membranes, nanopores, fluorescent reporter molecules, and / or ionic species in a nanopore array, as well as methods of forming them, methods of using them, etc. may be as described in the single system above, unless otherwise specified or possible.

[0206] In the present invention, when referring to components in solution, "in a first aqueous solution" and "in a first compartment" may be used interchangeably, and "in a second aqueous solution" and "in a second compartment" may be used interchangeably.

[0207] The embodiments described herein can be more readily understood by reference to the following detailed description, examples, and claims, as well as the accompanying text. It is to be understood that the embodiments described herein are not limited to particular uses, methods, and / or products. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.

[0208] Furthermore, the following description is provided as an enabling teaching of various embodiments of the best currently known aspects. Those skilled in the relevant art will recognize that many variations can be made to the described aspects while still obtaining beneficial results of the present disclosure. It will also be apparent that some of the desired advantages of the present invention can be obtained by selecting some of the features of the various embodiments without utilizing other features. Thus, those skilled in the art will recognize that many modifications and adaptations to the various embodiments described herein are possible and may even be desirable in certain circumstances and are part of the present disclosure. That is, the following description is provided by way of illustration of the principles of the embodiments described herein, not in limitation thereof.

[0209] Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error in the system or method being used to determine the value. In any embodiment discussed in connection with a numerical value used in conjunction with the term "about," it is specifically contemplated that the term "about" can be omitted.

[0210] Throughout this specification, singular terms should be understood to include the plural reference unless otherwise specified. Thus, for example, singular articles (such as "a," "an," and "the") should be understood to include the plural reference unless otherwise specified.

[0211] It should also be understood that terms used herein have the definitions commonly used in the art unless otherwise specified. All technical and scientific terms have the same meaning as commonly used by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification (including definitions) will control.

[0212] According to the present invention, the following inventions are provided. [1] An electrodeless system for identifying an analyte, comprising: (a) a first compartment containing a first aqueous solution therein, said first aqueous solution comprising a fluorescent reporter molecule capable of fluorescing when bound to an ionic species; (b) a second compartment containing a second aqueous solution, the second aqueous solution containing the ionic species that specifically binds to the fluorescent reporter molecule; and (c) a membrane separating the first and second compartments; Including, at least one nanopore is inserted in the membrane between the first compartment and the second compartment such that the first compartment and the second compartment are connected by the nanopore; the ionic species can diffuse from the second compartment to the first compartment through the nanopore; and a chemical gradient of the ionic species exists between the first compartment and the second compartment. system. [2] The system according to [1] above, wherein the membrane is a solid membrane. [3] The system described in [1] above, wherein the membrane is a semipermeable membrane. [4] The system according to [3] above, wherein the osmolality of the second aqueous solution is higher than that of the first aqueous solution, or the osmolality of the second aqueous solution is higher than that of the first aqueous solution; or the osmolality of the second aqueous solution is equal to that of the first aqueous solution, or the osmolality of the second aqueous solution is equal to that of the first aqueous solution; or the osmolality of the second aqueous solution is lower than that of the first aqueous solution, or the osmolality of the second aqueous solution is lower than that of the first aqueous solution. [5] The system according to [3] or [4] above, wherein the semipermeable membrane is made of amphiphilic molecules; preferably, the amphiphilic molecules are lipids or triblock copolymers. [6] The system according to [3] or [4] above, wherein the semipermeable membrane is a bilayer composed of amphiphilic molecules; preferably, the amphiphilic molecules are lipids. [7] The system described in [6] above, wherein the lipid is one or more selected from the group consisting of fatty acid acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, polyketides, phospholipids, glycolipids, and cholesterol. [8] The system described in [6] above, wherein the lipid is one or more selected from the group consisting of monoolein; 1,2-dioleoyl-sn-glycero-S-phosphocholine (DOPC); 1,2-diphytanoyl-sn-glycero-3-phosphatidylcholine (DPhPC); palmitoyloleoylphosphatidylcholine (POPC); 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE); 1-palmitoyl-2-oleoyl-phosphatidylethanolamine; 1-palmitoyl-2-oleoylphosphatidylglycerol (POPE / POPG) mixture; and mixtures thereof. [9] The system according to any one of [1] to [8] above, wherein the first compartment is provided by an aqueous droplet.

[10] The system according to any one of [1] to [9] above, wherein the second compartment is provided by a hydrogel layer; preferably, the hydrogel layer contains 0.1 to 20% (w / v) agarose; more preferably, the hydrogel layer contains 2 to 5% (w / v) agarose.

[11] The system according to any one of [1] to

[10] above, wherein the nanopore is selected from the group consisting of a protein nanopore, a DNA nanopore, or a solid-state nanopore.

[12] The system described in

[11] above, wherein the protein nanopore is one or more selected from the group consisting of α-HL, ClyA, Phi29 connector protein, erolysin, MspA, OmpF, OmpG, FraC, HlyA, SheA, sp1, and variants thereof; and ion channels; preferably, the protein nanopore is ClyA-RR or α-HL.

[13] The ion species is Ag + , Ag 2+ 、Al 3+ , As 3+ , Au + , Ba 2+ , Bi 3+ , Ca 2+ , Cd 2+ , Ce 3+ , Ce 4+ 、Cl - , Co 2+ 、Cr 3+ , Cu + , Cu 2+ , Dy 3+ ,EU 3+ , Fe2 + , Fe 3+ , Ga 3+ 、H + , Hg+ , Hg 2+ , In 3+ 、K + , La 3+ , Mg 2+ , Mn 2+ , Mo 3+ , Na + , Ni 2+ , O.H. - , Pb 2+ , Pd 2+ , Pt 2+ , Pt 4+ , Ru 3+ , Sb 3+ ,Sc. 3+ , Sn 2 + 、Sr 2+ , Tb 3+ 、Tl + , and Zn 2+ The system according to any one of [1] to

[12] above, which is one or more selected from the group consisting of:

[14] The system according to any one of [1] to

[13] above, wherein the fluorescent reporter molecule is one or more selected from the group consisting of Fura-2, Indo-1, Fluo-2, Fluo-3, Fluo-4, Fluo-8, Calcium Green-1, DCFH, DHR, SNARF, Cal-520, calcium-specific aminopolycarboxylic acid, BAPTA, SBFI, Asante NaTRIUM Green-1, Asante NaTRIUM Green-2, Thallos Potassium Ion Channel Reagent, Asante Potassium Green-1, Asante Potassium Green-2, Asante Potassium Green-3, PBFI, Fluo-2 Mg, Fura-2 Mg, Indo-1 Mg, Asante Magnesium Green, SPQ, MQAE, TSQ, TFL-Zn, and ZINQUIN.

[15] The system according to any one of the above [1] to

[14] , wherein the second aqueous solution contains calcium chloride and, optionally, a buffering agent.

[16] The system according to

[15] above, wherein the concentration of calcium chloride in the second aqueous solution is 0.01 to 6.76M.

[17] The system according to any one of [1] to

[16] above, wherein the first aqueous solution comprises a chelating agent and, optionally, a buffer, the chelating agent being capable of binding to the ionic species; preferably, the chelating agent is EDTA, BAPTA, EGTA, CyDTA, DTPA, EDDP, HIDA, IDA, NTA, NTPO, TTHA, CA, TA, GA, HEDTA, or DEG.

[18] The system according to any one of [1] to

[17] above, further comprising a light source for illumination and a photosensor for detecting fluorescence; preferably, the light source is a laser, an LED, a halogen light, or a xenon light; preferably, the photosensor is a CCD, an sCMOS sensor, or a photodiode; more preferably, the photosensor is an EMCCD or an avalanche photodiode.

[19] The system according to any one of [1] to

[17] above, further comprising a device for total internal reflection fluorescence (TIRF), wide-field fluorescence microscopy, or confocal microscopy.

[20] The system according to any one of [1] to

[19] above, wherein the first aqueous solution or the second aqueous solution contains the analyte.

[21] The system according to

[20] above, wherein the analyte is selected from the group consisting of small molecules, macromolecules, and biopolymers.

[22] The system of

[20] above, wherein the analyte is selected from the group consisting of a compound, a drug, a sugar, an ion, a neurotransmitter, an amino acid, a nucleotide, a polymer, a polypeptide, a polysaccharide, and a polynucleotide; preferably, the polynucleotide is DNA or RNA; more preferably, the DNA is dsDNA or ssDNA; and more preferably, the RNA is miRNA, siRNA, or tRNA.

[23] A method for identifying an analyte, comprising: (a) preparing the system according to any one of the above [1] to

[22] , in which the analyte is provided in the first compartment or the second compartment; (b) directing light capable of exciting the fluorescent reporter molecule at a region within the first compartment proximal to the nanopore; (c) measuring a fluorescent signal from the fluorescent reporter molecule to identify the analyte; A method comprising:

[24] A method for producing an electrodeless system, comprising: (a) providing a first compartment containing a first aqueous solution therein, the first aqueous solution including a fluorescent reporter molecule capable of fluorescing when bound to an ionic species; (b) providing a second compartment containing a second aqueous solution therein, said second aqueous solution comprising said ionic species that specifically binds to said fluorescent reporter molecule; (c) contacting the first compartment with the second compartment in a hydrophobic medium containing amphipathic molecules such that a semipermeable membrane having a nanopore inserted therein is formed between the first compartment and the second compartment; Including, A method wherein a protein nanopore is provided in the first aqueous solution or the second aqueous solution.

[25] The method according to

[24] above, wherein the osmolality of the second aqueous solution is higher than that of the first aqueous solution, or the osmolality of the second aqueous solution is higher than that of the first aqueous solution; or the osmolality of the second aqueous solution is equal to that of the first aqueous solution, or the osmolality of the second aqueous solution is equal to that of the first aqueous solution; or the osmolality of the second aqueous solution is lower than that of the first aqueous solution, or the osmolality of the second aqueous solution is lower than that of the first aqueous solution.

[26] The system according to

[24] or

[25] above, wherein the semipermeable membrane is made of amphiphilic molecules; preferably, the amphiphilic molecules are lipids or triblock copolymers.

[27] The method according to

[26] above, wherein the lipid is one or more selected from the group consisting of fatty acid acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, polyketides, phospholipids, glycolipids, and cholesterol.

[28] The method according to

[26] above, wherein the lipid is one or more selected from the group consisting of monoolein; 1,2-dioleoyl-sn-glycero-S-phosphocholine (DOPC); 1,2-diphytanoyl-sn-glycero-3-phosphatidylcholine (DPhPC); palmitoyloleoylphosphatidylcholine (POPC); 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE); 1-palmitoyl-2-oleoyl-phosphatidylethanolamine; 1-palmitoyl-2-oleoylphosphatidylglycerol (POPE / POPG) mixture; and mixtures thereof.

[29] The method according to any one of

[24] to

[28] above, wherein the first compartment is provided by an aqueous droplet.

[30] The method according to any one of [2] to

[29] above, wherein the second compartment is provided by a hydrogel layer; preferably, the hydrogel layer contains 0.1 to 20% (w / v) agarose; more preferably, the hydrogel layer contains 2 to 5% (w / v) agarose.

[31] The method according to any one of

[24] to

[30] above, wherein the nanopore is selected from the group consisting of a protein nanopore, a DNA nanopore, or a solid-state nanopore, and the protein nanopore is one or more selected from the group consisting of α-HL, ClyA, Phi29 connector protein, erolysin, MspA, OmpF, OmpG, FraC, HlyA, SheA, sp1, and variants thereof; and an ion channel.

[32] The method according to

[31] above, wherein the protein nanopore is one or more selected from the group consisting of α-HL, ClyA, Phi29 connector protein, erolysin, MspA, OmpF, OmpG, FraC, HlyA, SheA, sp1, and variants thereof; and ion channels; preferably, the protein nanopore is ClyA-RR or α-HL.

[33] The ion species is Ag + , Ag 2+ 、Al 3+ , As 3+ , Au + , Ba 2+ , Bi 3+ , Ca 2+ , Cd 2+ , Ce 3+ , Ce 4+ 、Cl - , Co 2+ 、Cr 3+ , Cu + , Cu 2+ , Dy 3+ ,EU 3+ , Fe2 + , Fe 3+ , Ga 3+ 、H + , Hg + , Hg 2+ , In3+ 、K + , La 3+ , Mg 2+ , Mn 2+ , Mo 3+ , Na + , Ni 2+ , O.H. - , Pb 2+ , Pd 2+ , Pt 2+ , Pt 4+ , Ru 3+ , Sb 3+ ,Sc. 3+ , Sn 2+ 、Sr 2+ , Tb 3+ 、Tl + , and Zn 2+ The method according to any one of

[24] to

[32] above, wherein the compound is one or more selected from the group consisting of:

[34] The method according to any one of

[24] to

[33] above, wherein the fluorescent reporter molecule is one or more selected from the group consisting of Fura-2, Indo-1, Fluo-2, Fluo-3, Fluo-4, Fluo-8, Calcium Green-1, DCFH, DHR, SNARF, Cal-520, calcium-specific aminopolycarboxylic acid, BAPTA, SBFI, Asante NaTRIUM Green-1, Asante NaTRIUM Green-2, Thallos Potassium Ion Channel Reagent, Asante Potassium Green-1, Asante Potassium Green-2, Asante Potassium Green-3, PBFI, Fluo-2 Mg, Fura-2 Mg, Indo-1 Mg, Asante Magnesium Green, SPQ, MQAE, TSQ, TFL-Zn, and ZINQUIN.

[35] The method according to any one of the above

[24] to

[34] , wherein the second aqueous solution contains calcium chloride and, optionally, a buffering agent.

[36] The method according to

[35] above, wherein the concentration of calcium chloride in the second aqueous solution is 0.01 to 6.76 M.

[37] The method according to any one of

[24] to

[36] above, wherein the first aqueous solution comprises a chelating agent and, optionally, a buffer, the chelating agent being capable of binding to the ionic species; preferably, the chelating agent is EDTA, BAPTA, EGTA, CyDTA, DTPA, EDDP, HIDA, IDA, NTA, NTPO, TTHA, CA, TA, GA, HEDTA, or DEG.

[38] An electrodeless nanopore array for identifying multiple analytes, comprising: It includes multiple systems in parallel, each system being: (a) a first compartment containing a first aqueous solution therein, said first aqueous solution comprising a fluorescent reporter molecule capable of fluorescing when bound to an ionic species; (b) a second compartment containing a second aqueous solution, the second aqueous solution containing the ionic species that specifically binds to the fluorescent reporter molecule; and (c) a membrane separating the first and second compartments; Including, in each system, at least one nanopore is inserted in the membrane between the first compartment and the second compartment, such that in each system, the first compartment and the second compartment are connected by a nanopore; a chemical gradient of the ionic species exists between the first and second compartments of each system, allowing the ionic species to diffuse from the second compartment to the first compartment through the nanopore; the plurality of systems are arranged such that the measured fluorescence is distinguishable in each system; Nanopore array.

[39] The nanopore array described in

[38] above, wherein in each system, the membrane between the first compartment and the second compartment system is a solid membrane.

[40] The nanopore array described in

[38] above, wherein in each system, the membrane between the first compartment and the second compartment is a semipermeable membrane.

[41] The nanopore array described in

[40] above, wherein in each system, the osmolality of the second aqueous solution is higher than the osmolality of the first aqueous solution, or the weight osmolality of the second aqueous solution is higher than the weight osmolality of the first aqueous solution; or the osmolality of the second aqueous solution is equal to the osmolality of the first aqueous solution, or the weight osmolality of the second aqueous solution is equal to the weight osmolality of the first aqueous solution; or the osmolality of the second aqueous solution is lower than the osmolality of the first aqueous solution, or the weight osmolality of the second aqueous solution is lower than the weight osmolality of the first aqueous solution.

[42] The nanopore array according to

[40] or

[41] above, wherein the semipermeable membrane is made of amphiphilic molecules; preferably, the amphiphilic molecules are lipids or triblock copolymers.

[43] A nanopore array according to

[40] or

[41] above, wherein the semipermeable membrane is a bilayer composed of amphiphilic molecules; preferably, the amphiphilic molecules are lipids.

[44] The nanopore array described in

[43] above, wherein the lipid is one or more selected from the group consisting of fatty acid acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, polyketides, phospholipids, glycolipids, and cholesterol.

[45] The nanopore array described in

[43] above, wherein the lipid is one or more selected from the group consisting of monoolein; 1,2-dioleoyl-sn-glycero-S-phosphocholine (DOPC); 1,2-diphytanoyl-sn-glycero-3-phosphatidylcholine (DPhPC); palmitoyloleoylphosphatidylcholine (POPC); 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE); 1-palmitoyl-2-oleoyl-phosphatidylethanolamine; 1-palmitoyl-2-oleoylphosphatidylglycerol (POPE / POPG) mixture; and mixtures thereof.

[46] A nanopore array according to any one of

[38] to

[45] above, wherein the first compartments of the multiple systems are separated from each other.

[47] A nanopore array according to

[46] above, wherein the first compartment of each system is supplied by an aqueous droplet.

[48] ​​A nanopore array according to any one of

[38] to

[46] above, wherein the second compartment of the multiple systems is a single compartment.

[49] A nanopore array as described in

[48] above, wherein the second compartment of each system is provided by a hydrogel layer; preferably, the hydrogel layer contains 0.1 to 20% (w / v) agarose; more preferably, the hydrogel layer contains 2 to 5% (w / v) agarose.

[50] A nanopore array according to

[49] above, wherein each of the second compartments of the multiple systems is provided by a single hydrogel layer.

[51] The nanopore array according to any one of

[38] to

[50] above, wherein in each system, the nanopore is selected from the group consisting of a protein nanopore, a DNA nanopore, or a solid-state nanopore.

[52] The nanopore array described in

[51] above, wherein the protein nanopore is one or more selected from the group consisting of α-HL, ClyA, Phi29 connector protein, erolysin, MspA, OmpF, OmpG, FraC, HlyA, SheA, sp1, and variants thereof; and ion channels; preferably, the protein nanopore is ClyA-RR or α-HL.

[53] In each system, the ionic species is Ag + , Ag 2+ 、Al 3+ , As 3+ , Au + , Ba 2+ , Bi 3+ , Ca 2+ , Cd 2+ , Ce 3+ , Ce 4+ 、Cl - , Co 2+ 、Cr 3+ , Cu + , Cu 2+ , Dy 3+ ,EU 3+ , Fe2 + , Fe 3+ , Ga 3+ 、H + , Hg + , Hg 2+ , In 3+ 、K + , La 3+ , Mg2+ , Mn 2+ , Mo 3+ , Na + , Ni 2+ , O.H. - , Pb 2+ , Pd 2+ , Pt 2+ , Pt 4+ , Ru 3+ , Sb 3+ ,Sc. 3+ , Sn 2+ 、Sr 2+ , Tb 3+ 、Tl + , and Zn 2+ The nanopore array according to any one of

[38] to

[52] above, wherein the nanopore array is one or more selected from the group consisting of:

[54] The nanopore array according to any one of

[38] to

[53] above, wherein the fluorescent reporter molecule is one or more selected from the group consisting of Fura-2, Indo-1, Fluo-2, Fluo-3, Fluo-4, Fluo-8, Calcium Green-1, DCFH, DHR, SNARF, Cal-520, calcium-specific aminopolycarboxylic acid, BAPTA, SBFI, Asante NaTRIUM Green-1, Asante NaTRIUM Green-2, Thallos Potassium Ion Channel Reagent, Asante Potassium Green-1, Asante Potassium Green-2, Asante Potassium Green-3, PBFI, Fluo-2 Mg, Fura-2 Mg, Indo-1 Mg, Asante Magnesium Green, SPQ, MQAE, TSQ, TFL-Zn, and ZINQUIN.

[55] The nanopore array according to any one of

[38] to

[54] above, wherein in each system, the second aqueous solution contains calcium chloride and, optionally, a buffering agent.

[56] The nanopore array according to

[55] above, wherein in each system, the concentration of calcium chloride in the second aqueous solution is 0.01 to 6.76 M.

[57] A nanopore array according to any one of

[38] to

[56] above, wherein in each system, the first aqueous solution comprises a chelating agent and, optionally, a buffer, the chelating agent being capable of binding to the ionic species; preferably, the chelating agent is EDTA, BAPTA, EGTA, CyDTA, DTPA, EDDP, HIDA, IDA, NTA, NTPO, TTHA, CA, TA, GA, HEDTA, or DEG.

[58] A nanopore array according to any one of

[38] to

[57] above, further comprising a light source for illumination and a light sensor for detecting fluorescence; preferably, the light source is a laser, LED, halogen light, or xenon light; preferably, the light sensor is a CCD, sCMOS sensor, or photodiode; more preferably, the light sensor is an EMCCD or an avalanche photodiode.

[59] The nanopore array of

[58] above, further comprising a device for total internal reflection fluorescence (TIRF), wide-field fluorescence microscopy, or confocal microscopy.

[60] The nanopore array according to any one of

[38] to

[59] above, wherein in each system, the first aqueous solution or the second aqueous solution contains the analyte.

[61] The nanopore array according to

[60] above, wherein in each system, the analyte is selected from the group consisting of small molecules, polymers, and biopolymers.

[62] The nanopore array of

[60] above, wherein in each system, the analyte is selected from the group consisting of a compound, a drug, a sugar, an ion, a neurotransmitter, an amino acid, a nucleotide, a polymer, a polypeptide, a polysaccharide, and a polynucleotide; preferably, the polynucleotide is DNA or RNA; more preferably, the DNA is dsDNA or ssDNA; and more preferably, the RNA is miRNA, siRNA, or tRNA.

[63] The nanopore array according to any one of

[60] to

[62] above, wherein different analytes are physically separated into various systems.

[64] The density of the system in the nanopore array is 10 to 1000 / mm 2 The nanopore array according to any one of

[38] to

[63] above,

[65] The total area provided by the plurality of systems is 1 to 100 mm 2 The nanopore array according to any one of

[38] to

[64] above,

[66] A multiplex method for identifying multiple analytes, comprising: (a) preparing a nanopore array according to any one of

[38] to

[65] above, in which two or more analytes are supplied to various systems of the nanopore array; (b) directing a light signal capable of exciting the fluorescent reporter molecule contained in each of the first compartments at a region within the plurality of first compartments proximal to the nanopore; (c) measuring a plurality of fluorescent signals from the fluorescent reporter molecules contained in each system to identify the plurality of analytes; A method comprising:

[67] A method for fabricating an electrodeless nanopore array, comprising: (a) providing a plurality of aqueous droplets, each containing a first aqueous solution that includes a protein nanopore, an analyte, and a fluorescent reporter molecule capable of fluorescing when bound to an ionic species; (b) providing a hydrogel layer containing said ionic species; (c) contacting the plurality of aqueous droplets with the hydrogel layer in a hydrophobic medium containing amphiphilic molecules such that a semipermeable membrane is formed between each of the aqueous droplets and the hydrogel layer; A method comprising:

[68] The method of

[67] above, wherein the osmolality of the hydrogel is higher than the osmolality of each aqueous droplet, or the osmolality of the hydrogel is higher than the osmolality of each aqueous droplet; or the osmolality of the hydrogel is equal to the osmolality of each aqueous droplet, or the osmolality of the hydrogel is equal to the osmolality of each aqueous droplet; or the osmolality of the hydrogel is lower than the osmolality of each aqueous droplet, or the osmolality of the hydrogel is lower than the osmolality of each aqueous droplet.

[69] The system according to

[67] or

[68] above, wherein the semipermeable membrane is composed of amphiphilic molecules; preferably, the amphiphilic molecules are lipids or triblock copolymers.

[70] The method according to

[69] above, wherein the lipid is one or more selected from the group consisting of fatty acid acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, polyketides, phospholipids, glycolipids, and cholesterol.

[71] The method according to

[69] above, wherein the lipid is one or more selected from the group consisting of monoolein; 1,2-dioleoyl-sn-glycero-S-phosphocholine (DOPC); 1,2-diphytanoyl-sn-glycero-3-phosphatidylcholine (DPhPC); palmitoyloleoylphosphatidylcholine (POPC); 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE); 1-palmitoyl-2-oleoyl-phosphatidylethanolamine; 1-palmitoyl-2-oleoylphosphatidylglycerol (POPE / POPG) mixture; and mixtures thereof.

[72] The method according to any one of

[67] to

[71] above, wherein the hydrogel layer contains 0.1 to 20% (w / v) agarose; preferably, the hydrogel layer contains 2 to 5% (w / v) agarose.

[73] The method according to any one of

[67] to

[72] above, wherein the nanopore is selected from the group consisting of a protein nanopore, a DNA nanopore, or a solid-state nanopore.

[74] The method according to

[73] above, wherein the protein nanopore in each aqueous droplet is one or more selected from the group consisting of α-HL, ClyA, Phi29 connector protein, erolysin, MspA, OmpF, OmpG, FraC, HlyA, SheA, sp1, and variants thereof; and ion channels; preferably, the protein nanopore in each aqueous droplet is ClyA-RR or α-HL.

[75] The ionic species in the hydrogel layer is Ag + , Ag 2+ 、Al 3+ , As 3+ , Au + , Ba 2+ , Bi 3+ , Ca 2+ , Cd 2+ , Ce 3+ , Ce 4+ 、Cl - , Co 2+ 、Cr 3+ , Cu + , Cu 2+ , Dy 3+ ,EU 3+ , Fe2 + , Fe 3+ , Ga 3+ 、H + , Hg + , Hg 2+ , In 3+ 、K + , La 3+ , Mg2+ , Mn 2+ , Mo 3+ , Na + , Ni 2+ , O.H. - , Pb 2+ , Pd 2+ , Pt 2+ , Pt 4+ , Ru 3+ , Sb 3+ ,Sc. 3+ , Sn 2+ 、Sr 2+ , Tb 3+ 、Tl + , and Zn 2+ The method according to any one of

[67] to

[74] above, wherein the method is one or more selected from the group consisting of:

[76] The method according to any one of

[67] to

[75] above, wherein the fluorescent reporter molecule in each aqueous droplet is one or more selected from the group consisting of Fura-2, Indo-1, Fluo-2, Fluo-3, Fluo-4, Fluo-8, Calcium Green-1, DCFH, DHR, SNARF, Cal-520, calcium-specific aminopolycarboxylic acid, BAPTA, SBFI, Asante NaTRIUM Green-1, Asante NaTRIUM Green-2, Thallos Potassium Ion Channel Reagent, Asante Potassium Green-1, Asante Potassium Green-2, Asante Potassium Green-3, PBFI, Fluo-2 Mg, Fura-2 Mg, Indo-1 Mg, Asante Magnesium Green, SPQ, MQAE, TSQ, TFL-Zn, and ZINQUIN.

[77] The method according to any one of the above

[67] to

[76] , wherein the hydrogel layer contains calcium chloride and, optionally, a buffering agent.

[78] The method according to

[77] above, wherein the concentration of calcium chloride in the hydrogel layer is 0.01 to 6.76M.

[79] The method according to any one of

[67] to

[78] above, wherein each aqueous droplet comprises a chelating agent and, optionally, a buffer, said chelating agent being capable of binding to said ionic species; preferably, said chelating agent is EDTA, BAPTA, EGTA, CyDTA, DTPA, EDDP, HIDA, IDA, NTA, NTPO, TTHA, CA, TA, GA, HEDTA, or DEG.

[80] The method according to any one of

[67] to

[79] above, wherein the analyte in each aqueous droplet is selected from the group consisting of small molecules, macromolecules, and biopolymers.

[81] The method according to any one of

[67] to

[79] above, wherein the analyte in each aqueous droplet is selected from the group consisting of a compound, a drug, a sugar, an ion, a neurotransmitter, an amino acid, a nucleotide, a polymer, a polypeptide, a polysaccharide, and a polynucleotide; preferably, the polynucleotide is DNA or RNA; more preferably, the DNA is dsDNA or ssDNA; and more preferably, the RNA is miRNA, siRNA, or tRNA.

[82] The method according to any one of

[67] to

[81] above, wherein different analytes are supplied to the various systems.

[83] The method according to any one of the above

[67] to

[82] , wherein the number of the aqueous droplets is 4 to 1,000,000; preferably, the number of the aqueous droplets is 10 to 1,000.

[84] Use of the system according to any one of [1] to

[22] above for carrying out optical analyte analysis.

[85] Use of a nanopore array as described in

[38] to

[65] above for optical analyte analysis.

[86] The method according to any one of the above

[24] to

[37] , wherein an analyte is provided in the first aqueous solution or the second aqueous solution.

[87] The method according to

[86] above, wherein the analyte is selected from the group consisting of small molecules, macromolecules, and biopolymers.

[88] The system of

[86] above, wherein the analyte is selected from the group consisting of a compound, a drug, a sugar, an ion, a neurotransmitter, an amino acid, a nucleotide, a polymer, a polypeptide, a polysaccharide, and a polynucleotide; preferably, the polynucleotide is DNA or RNA; more preferably, the DNA is dsDNA or ssDNA; and more preferably, the RNA is miRNA, siRNA, or tRNA.

[89] A nanopore array according to any one of

[38] to

[65] above, wherein the number of the plurality of systems is 4 to 1,000,000; preferably, the number of the plurality of systems is 10 to 1,000.

[90] A kit for forming a nanopore array, comprising: a filling hydrogel comprising agarose, a buffer, and an ionic species capable of specifically binding to a fluorescent reporter molecule and causing it to emit fluorescence; an aqueous solution comprising a chelating agent, said fluorescent reporter molecule capable of fluorescing when bound to said ionic species, and a buffer; wherein said chelating agent is capable of binding to said ionic species; a hydrophobic medium containing amphiphilic molecules; solid support, Includes a kit.

[91] The kit of

[90] above, wherein the osmolality of the filling hydrogel is higher than the osmolality of the aqueous solution, or the osmolality of the filling hydrogel is higher than the osmolality of the aqueous solution; or the osmolality of the filling hydrogel is equal to the osmolality of the aqueous solution, or the osmolality of the filling hydrogel is equal to the osmolality of the aqueous solution; or the osmolality of the filling hydrogel is lower than the osmolality of the aqueous solution, or the osmolality of the filling hydrogel is lower than the osmolality of the aqueous solution.

[92] The kit according to

[90] or

[91] above, wherein the amphiphilic molecule is a lipid or a triblock copolymer.

[93] The kit described in

[92] above, wherein the lipid is one or more selected from the group consisting of fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, polyketides, phospholipids, glycolipids, and cholesterol.

[94] The kit described in

[92] above, wherein the lipid is one or more selected from the group consisting of monoolein; 1,2-dioleoyl-sn-glycero-S-phosphocholine (DOPC); 1,2-diphytanoyl-sn-glycero-3-phosphatidylcholine (DPhPC); palmitoyloleoylphosphatidylcholine (POPC); 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE); 1-palmitoyl-2-oleoyl-phosphatidylethanolamine; 1-palmitoyl-2-oleoylphosphatidylglycerol (POPE / POPG) mixture; and mixtures thereof.

[95] The kit according to any one of

[90] to

[94] above, wherein the aqueous solution also contains a protein nanopore.

[96] The kit according to

[95] above, wherein the nanopore is selected from the group consisting of a protein nanopore, a DNA nanopore, or a solid-state nanopore.

[97] The kit described in

[96] above, wherein the protein nanopore is one or more selected from the group consisting of α-HL, ClyA, Phi29 connector protein, erolysin, MspA, OmpF, OmpG, FraC, HlyA, SheA, sp1, and variants thereof; and ion channels; preferably, the protein nanopore is ClyA-RR or α-HL.

[98] The ionic species in the hydrogel layer is Ag + , Ag 2+ 、Al 3+ , As 3+ , Au + , Ba 2+ , Bi 3+ , Ca 2+ , Cd 2+ , Ce 3+ , Ce 4+ 、Cl - , Co 2+ 、Cr 3+ , Cu + , Cu 2+ , Dy 3+ ,EU 3+ , Fe2 + , Fe 3+ , Ga 3+ 、H + , Hg + , Hg 2+ , In 3+ 、K + , La 3+ , Mg 2+ , Mn 2+ , Mo 3+ , Na + , Ni2+ , O.H. - , Pb 2+ , Pd 2+ , Pt 2+ , Pt 4+ , Ru 3+ , Sb 3+ ,Sc. 3+ , Sn 2+ 、Sr 2+ , Tb 3+ 、Tl + , and Zn 2+ The kit according to any one of

[90] to

[97] above, wherein the kit is one or more selected from the group consisting of:

[99] The kit according to any one of

[90] to

[98] above, wherein the fluorescent reporter molecule in each aqueous droplet is one or more selected from the group consisting of Fura-2, Indo-1, Fluo-2, Fluo-3, Fluo-4, Fluo-8, Calcium Green-1, DCFH, DHR, SNARF, Cal-520, calcium-specific aminopolycarboxylic acid, BAPTA, SBFI, Asante NaTRIUM Green-1, Asante NaTRIUM Green-2, Thallos Potassium Ion Channel Reagent, Asante Potassium Green-1, Asante Potassium Green-2, Asante Potassium Green-3, PBFI, Fluo-2 Mg, Fura-2 Mg, Indo-1 Mg, Asante Magnesium Green, SPQ, MQAE, TSQ, TFL-Zn, and ZINQUIN.

[0100] When the filling hydrogel contains calcium chloride, the ionic species is Ca 2+ The kit according to any one of

[90] to

[99] above, which supplies

[0101] The kit described in the above

[0100] , wherein the concentration of calcium chloride in the hydrogel layer is 0.01 to 6.76M.

[0102] The filling hydrogel is 2.5% agarose, 1.5M CaCl 2 and 10 mM HEPES (pH 7.0).

[0103] The kit according to any one of the above

[90] to

[0102] , wherein the aqueous solution also contains KCl.

[0104] The kit described in

[0103] above, wherein the aqueous solution may contain 1.5 M KCl, 400 μM EDTA, 40 μM Fluo-8, and 10 mM HEPES (pH 7.0).

[0105] A kit described in any of

[90] to

[0104] above, wherein the hydrophobic medium containing amphipathic molecules may be a lipid oil containing 5 mg of dried DPHPC lipid membrane dissolved in 2 mL of a 1:1 volumetric mixture of hexadecane and silicone oil.

[0106] A kit described in any of

[90] to

[0105] above, which also includes a hydrogel for application containing agarose in water; preferably, the hydrogel for application may contain 0.75% (w / v) agarose in water. All patents and publications referenced herein, including all sequences disclosed within such patents and publications, are expressly incorporated by reference. [Example]

[0213] Example 1: Single-molecule sensing of trimethyl-β-cyclodextrin with DOP: Proof-of-concept According to Figure 1d, the basic configuration for DOP recording involves an asymmetric electrolyte buffer solution separated by a semipermeable membrane into which a nanopore is inserted. The compartment filled with KCl, Fluo-8, and EDTA is defined as the cis side, whereas the compartment filled with CaCl2 is defined as the trans side. The biological nanopore forms the only conduction pathway between the cis and trans sides, allowing Ca2+ to enter the membrane by thermodynamic diffusion through a channel driven by a chemical gradient. 2+ FluoCa promotes the binding of Ca 2+ and Fluo-8 in their combined form, which emits fluorescence around each nanopore to report that the sensor is in an open state.

[0214] Theoretically, a finite element method (FEM) simulation was established by adapting the Poisson-Nernst-Planck-Stokes (PNPS) model

[26] (Methods, Figure 2). Simulation parameters, such as various combinations of reagent concentrations, could be adjusted by setting various boundary conditions to mimic experimental procedures. Simulations were performed using a cylindrical channel geometry with a diameter of 2 nm, with cis boundary conditions of 1.5 M KCl, 40 μM Fluo-8, and 400 μM EDTA, and trans boundary conditions of 0.75 M CaCl2. The results showed that a concentration gradient of FluoCa was established directly above the nanopore (Figure 1e). Due to the simultaneous release of FluoCa, a strong fluorescence intensity profile was expected at the top of the nanopore. The intensity profile was generated to mimic TIRF imaging (Methods) and followed a Gaussian distribution with a full width at half maximum (FWHM) of 2.670 μm (Figure 1f, top).

[0215] Experimentally, a droplet interface bilayer (DIB) was established between an aqueous droplet and a 100-nm-thick hydrogel sheet (Figure 3). The aqueous droplet consisted of 1.5 M KCl, 400 μM EDTA, 40 μM Fluo-8, and 10 mM HEPES (pH 7.0). The hydrogel sheet consisted of 0.75 M CaCl2, 10 mM HEPES (pH 7.0), and 2.5% (v / w) low-melting-point agarose. WT α-HL (α-HL) placed in the aqueous droplet spontaneously intercalated into the DIB, appearing as a bright fluorescent spot during TIRF imaging (Figure 1f, bottom). The fluorescence intensity profile of a representative frame obtained from TIRF imaging roughly followed a Gaussian distribution with a FWHM of 2.583 μm (Figure 1f, bottom), similar to the simulated results.

[0216] Trimethyl-β-cyclodextrin (TriM-β-CD) interacts with the WT α-HL nanopore confinements, resulting in long-lasting, deep pore-blocking events during electrophysiological recordings [27-29]. This observability allows TriM-β-CD to be used as a representative small-molecule analyte, enabling proof-of-concept single-molecule sensing using DOP. To maintain a stable analyte concentration during DOP recordings, TriM-β-CD was added in cis at a final concentration of 75 mM. TriM-β-CD binding from cis was also verified by corresponding electrophysiological measurements (Figure 4). During DOP recordings, stochastic binding of TriM-β-CD to α-HL facilitates Ca transport through the channel. 2+ Flow restriction resulted in highly discriminative image contrast between the open (Fo) and closed (Fb) states of the α-HL nanopore (Figure 1g). Corresponding fluorescence traces extracted from a series of continuously recorded images (Methods, Figure 5) confirmed sequential pore closure (Figure 1g). To enable quantitative comparisons between different trials, all fluorescence traces were calibrated and normalized before analysis (Figure 6).

[0217] From the normalized fluorescence traces, single-molecule sensing events are identified as events with dwell times (t off ), the inter-event period (t on), and percent blockage depth (%F b ) was characterized by t off and t on The histogram of shows an exponential distribution, and the mean time constant τ off and τ on The fitting and characterization were performed by (Figure 7). Varying the TriM-β-CD concentration in cis-phase changed the reciprocal of the residence time (1 / τ off ) is constant, but the inverse of the event interval (1 / τ on ) is linearly correlated with the TriM-β-CD concentration in cis (Fig. 1h, Table 1).

[0218] [Table 1]

[0219] DIBs were established in cis with 1.5 M KCl, 400 μM EDTA, 40 μM Fluo-8, and 10 mM HEPES (pH 7.0) and in trans with 0.75 M CaCl and 10 mM HEPES (pH 7.0). TriM-β-CD was added in cis. Three independent measurements were performed and statistics were compiled.

[0220] From the DOP recording, the average τ off Average F value 0.347±0.067 seconds P A value of 0.078 ± 0.010 was recorded, where F P is defined as the mean blockage depth, and %F from each trial of DOP recording b On the other hand, the corresponding electrophysiological results obtained with a +20 mV potential bias showed that τ off Values ​​0.386 ± 0.392 seconds and I P A value of 0.065±0.002 was obtained, where I P is defined as the average blockade depth from each trial of electrophysiological recording. Three independent trials were performed for each measurement condition and statistical analysis was performed. The similarity of the results confirmed the feasibility of single-molecule sensing by DOP (Figure 1i).

[0221] Although not demonstrated, single-molecule sensing of other small molecules, such as sugars [30, 31], ions

[32] , nucleotides

[33] , neurotransmitters

[34] , and amino acids

[35] , could in principle also be performed with DOP recordings, with the advantage of increased throughput. Because analyte capture is driven by a chemical gradient rather than an electrochemical gradient, the charge of the analyte is not important for DOP recordings. However, the intensity of the fluorescence emission and the analyte binding efficiency could be further improved to be comparable to those in electrophysiology.

[0222] Example 2: Enhanced DOP sensing through directional penetration During conventional electrophysiological recordings, applied electrochemical gradients are crucial for driving the continuous flow of charged particles, such as ions and analytes. Intuitively, directing the flow of analytes into a nanopore sensor without electrodes requires the introduction of other forms of asymmetry.

[0223] DIBs are self-assembled membranes composed of 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DphPC) lipids that selectively allow water molecules to pass through, but not ions [36, 37]. When a difference in osmolarity (C(solute) = iM(solute)) exists across the DIB, osmotic pressure is established according to Δπ = (C(solute, cis) = C(solute, trans))RT, where i is the dimensionless van't Hoff exponent representing the number of dissociated ions from each solute molecule, M(solute) is the molar concentration of the solute, R is the ideal gas constant, and T is the temperature in Kelvin. Here, the positive direction of osmotic pressure is defined as cis to trans (for ease of understanding; the osmotic pressure on the cis side may be higher than that on the trans side). This osmotic pressure then drives the directional flow of water, ions, and analytes through biological nanopores inserted into the membrane

[22] . As a result, the introduction of this asymmetry should result in an enhanced transfer efficiency of the analyte.

[0224] To experimentally verify this hypothesis, a series of DOP recordings were performed in DIB (Figure 3) by varying the cis KCl concentration (1.0-2.5 M) while keeping the trans CaCl concentration constant (0.75 M). For this experiment, α-HL and TriM-β-CD were again selected as the model sensor and analyte, respectively, and the cis TriM-β-CD concentration was fixed at 15 mM in cis. From representative DOP recordings, the enhancement of the capture rate of TriM-β-CD was confirmed by the time extension of the fluorescence traces when the cis KCl concentration was reduced from 2.5 M to 1.0 M (Figure 3). The independent measurements yielded a 1 / τ per event. on By evaluating the value of 1 / τ in response to the decrease in osmolality on A regular decrease in the event detection rate was observed (Fig. 8, Table 2), which means that a higher event detection rate was observed with the aid of directed osmotic flow.

[0225] [Table 2]

[0226] DIBs were established in cis with 1–2.5 M KCl, 400 μM EDTA, 40 μM Fluo-8, and 10 mM HEPES (pH 7.0), and in trans with 0.75 M CaCl2 and 10 mM HEPES (pH 7.0). 15 mM TriM-β-CD was added in cis. Three independent measurements were performed and statistical analysis was performed.

[0227] We also found that DOP recordings in the presence of cis-to-trans osmotic flow exhibited a significant improvement in fluorescence image contrast. This phenomenon was evident from the reduction in thermal noise in the fluorescence traces when using measurement conditions with lower KCl concentrations (Figures 8a and 9). Here, the high thermal noise observed in the fluorescence traces is the result of a reduction in the number of photons during imaging.

[0228] Furthermore, to investigate why the fluorescence intensity obtained from DOP recordings could be adjusted by osmotic pressure (Figure 8c, Table 3), we performed a series of experiments using 1–2.5 M KCl, 400 μM EDTA, 40 μM Fluo-8, and 10 mM HEPES (pH 7.0) in the cis phase and 0.75 M CaCl2 and 10 mM HEPES (pH 7.0) in the trans phase. TriM-β-CD was omitted to avoid interference from analyte binding. To avoid interference from uneven TIRF illumination or fluctuations in laser power when evaluating fluorescence brightness, we introduced a signal-to-background ratio (SBR) value to quantitatively compare different trials of DOP recordings (Methods). Representative image frames and corresponding SBR values ​​clearly showed that the brightness of the fluorescent spot was enhanced when greater osmotic pressure was introduced from cis to trans. Five independent measurements were performed for each condition, and statistics were compiled (Figure 8c).

[0229] [Table 3]

[0230] DIBs were established in cis with 1–2.5 M KCl, 400 μM EDTA, 40 μM Fluo-8, and 10 mM HEPES (pH 7.0), and in trans with 0.75 M CaCl2 and 10 mM HEPES (pH 7.0). Five independent measurements were performed and statistical analysis was performed.

[0231] This phenomenon was also confirmed by the corresponding FEM simulations performed with the cis boundary conditions set to 1–2.5 M KCl, 40 μM Fluo-8, and 400 μM EDTA, and the trans boundary condition set to 0.75 M CaCl2 (Figure 8d). By plotting the Fluo-8 distribution in the simulation space, we clearly see that a concentrated Fluo-8 distribution was established near the cis side of the membrane in the presence of directional osmotic flow from cis to trans. This was due to the concentration of Fluo-8, which is impermeable to the lipid membrane, via osmotic flow, resulting in an enhanced fluorescence intensity (Figure 10).

[0232] Example 3: Ca 2+ Further optimization of SBR by expanding flow However, Fluo-8 enrichment by permeation should not occur in solid-state nanopore devices where the membrane is not semipermeable. Instead, Ca 2+ Introducing more flow could improve the SBR from the DOP recordings. An immediate solution to this strategy is to increase the trans [CaCl2], which increases the [Ca 2+ This directly generates a chemical gradient of [C]. To test this hypothesis, a series of DOP recordings was performed by gradually increasing the trans CaCl concentration. To avoid interference from osmosis, the cis KCl concentration was adjusted accordingly to ensure that the cis and trans osmolarity remained isotonic.

[0233] Experimentally, DIBs were established using 0.75 M, 1.5 M, or 2.25 M KCl, 400 μM EDTA, 40 μM Fluo-8, and 10 mM HEPES (pH 7.0) in cis, and 0.5 M, 1 M, or 1.5 M CaCl2, 10 mM HEPES (pH 7.0) in trans. Representative image frames acquired using electrolyte buffer combinations with elevated [CaCl2] in trans demonstrate a regular expansion of the fluorescent spot size (Figure 11a). The corresponding 2D Gaussian fits (Figure 11a) are color-coded according to the fitting amplitude, allowing for easier comparison of the fluorescence intensities obtained under these conditions. SBR and FWHM were quantitatively measured from DOP recordings acquired using these electrolyte combinations and are shown in Figure 11b. From this figure, it can be seen that both FWHM and SBR increase when the osmolality on both sides of the membrane is increased (Table 4). Twelve independent measurements were performed for each condition and statistics were calculated.

[0234] [Table 4]

[0235] DIBs were established in cis with KCl (0.75 M, 1.5 M, and 2.25 M), 400 μM EDTA, 40 μM Fluo-8, and 10 mM HEPES (pH 7.0), and in trans with CaCl (0.5 M, 1 M, and 1.5 M), 10 mM HEPES (pH 7.0). Twelve independent measurements were performed and statistical analysis was performed.

[0236] PEG, an electrically neutral polymer dissolved in a neutral pH buffer, has been shown to translocate through α-HL nanopores during electrophysiological recordings.

[38] Higher salt concentrations in the electrolyte buffer during measurements have been reported to enhance capture rates and extend event residence times.

[39] As an illustration, PEG1500 was selected as a model analyte for single-molecule sensing of polymers using DOP.

[0237] Experimentally, DIBs were established using 2.25 M KCl, 10 mM HEPES, 400 μM EDTA, 40 μM Fluo-8, 20 mM PEG1500, and 10 mM HEPES (pH 7.0) in cis, and 1.5 M CaCl2, 10 mM HEPES (pH 7.0) in trans. Upon addition of 20 mM PEG1500 in cis, numerous nail-like translocation events immediately appeared in the extracted fluorescence trace (Figure 11c). These pore translocation features, along with the similarity to reported electrophysiological data, confirmed the detectability of PEG1500 in DOP recordings, as demonstrated with TriM-β-CD.

[0238] However, the solubility of analytes usually decreases in electrolyte buffers with high salt concentrations

[39] . Furthermore, the salt concentration is limited by the maximum solubility of the electrolytes in water (CaCl2: 6.767 M, KCl: 3.408 M at 20 °C). 2+To achieve this, nanopore sensors with larger openings could be introduced during DOP recordings, a confirmation of which is provided by corresponding FEM studies (Methods, Figure 11d). According to reported crystallographic results, the limiting diameter of the ClyA nanopore measured 3.8 nm, 2.7 times the diameter of α-HL

[40] . Given its large channel opening, ClyA and its variants have been developed to sense large biopolymers such as dsDNA and small proteins [40-44]. ClyA-RR, a charge-optimized variant reported to efficiently translocate dsDNA during electrophysiological recordings

[42] , was selected for DOP recordings (Methods, Figure 12). Although unproven, the phi29 connector protein

[45] and solid-state nanopores [24, 25] are also important candidates.

[0239] In our experiments, DIBs were established using 1.5 M KCl, 400 μM EDTA, 40 μM Fluo-8, and 10 mM HEPES (pH 7.0) in cis and 1.5 M CaCl2 and 10 mM HEPES (pH 7.0) in trans. The electrolyte combination of 1.5 M KCl (cis) / 1.5 M CaCl2 (trans) was chosen considering osmolality (Figure 8), CaCl2 concentration (Figure 11), and pore mobility

[22] . To quantitatively compare nanopores with different channel openings during DOP recordings, both the dodecamer ClyA-RR nanopore and the heptameric α-HL nanopore were placed in the droplet for simultaneous measurements from the same DIB.

[0240] Upon insertion, the ClyA-RR nanopore appears as a huge, dazzling fluorescent spot, whereas the α-HL nanopore appears smaller in size and dimmer in intensity (Figure 11e). The FWHM and SBR obtained from DOP recordings with ClyA-RR are clearly superior to those with α-HL, indicating that more Ca passes across the membrane. 2+ Flow is introduced (Fig. 11f, Table 5). Five independent measurements were performed and statistics were collected.

[0241] [Table 5]

[0242] DIBs were established in cis with 1.5 M KCl, 400 μM EDTA, 40 μM Fluo-8, and 10 mM HEPES (pH 7.0), and in trans with 1.5 M CaCl2 and 10 mM HEPES (pH 7.0). Pores were added in cis. Five independent measurements were performed and statistics were compiled.

[0243] In addition to improving SBR, as predicted by FEM simulations, the larger opening of ClyA may also contribute to enhanced osmotic flow, which may act as a driving force for DNA translocation (Figures 11g and 13). Although significant efforts have been made to counteract the electrophoretic force during DNA translocation

[46] , the electrophoretic force, which efficiently uncoils DNA during translocation [9], was considered essential for DNA sensing. However, the long-term persistence length of dsDNA

[47] and the large opening of the ClyA nanopore may reduce the entropic barrier for dsDNA translocation

[48] . Furthermore, the large entrance of ClyA may also play a role in accommodating dsDNA in a partially translocated form to report dsDNA sensing signals during DOP recording.

[0244] Example 4: Translocation of dsDNA and ssDNA through ClyA nanopores Experimentally, DIBs were established using 1.5 M KCl, 10 mM HEPES, 400 μM EDTA, 40 μM Fluo-8, 2 mM dsDNA (78 bp), and 10 mM HEPES (pH 7.0) in cis, and 1.5 M CaCl2, 10 mM HEPES (pH 7.0) in trans (Figure 14a). The osmolarity on both sides of the DIB was designed to establish sustained osmotic pressure from cis to trans. 78 bp dsDNA (Table 6) was dissolved in aqueous droplets at a final concentration of 2 μM, as desired.

[0245] [Table 6]

[0246] NOTE: To form dsDNA, complementary ssDNA (78 nt ssDNA-a and -b) (SEQ ID NOs: 1 and 2) were dissolved in 1.5 M KCl buffer (1.5 M KCl, 10 mM HEPES, pH 7.0) and heated to 95°C in a PCR thermal cycler (ABI2720) and gradually cooled (-5°C / min) to room temperature (25°C).

[0247] When no dsDNA was added to the droplet, a representative fluorescence trace from a ClyA nanopore appeared stably open and free of spontaneous gating activity (Figure 14b). Upon addition of dsDNA to the droplet, continuous fluorescent blockade spontaneously appeared during the DOP recording (Figure 14c). The blockade observed from the DOP recording had an average F of 0.625 ± 0.014. P and a mean τ of 2.538 ± 0.849. off (N = 3). These results suggest that dsDNA interaction with ClyA-RR occurred without applying an electrochemical gradient. The long residence time may be due to the removal of the electrophoretic force during the measurement or the trapping of dsDNA within the large entrance structure of ClyA.

[0248] To further verify this phenomenon using conventional electrophysiological recordings, planar lipid membranes were established using 1.5 M KCl and 10 mM HEPES (pH = 7.0) in cis and 1.5 M CaCl2 and 10 mM HEPES (pH = 7.0) in trans. 78 bp dsDNA was added to the cis membrane at a final concentration of 2 μM. To mimic DOP recordings, extremely low potentials were applied, resulting in a membrane potential difference of exactly zero. Figure 14d shows representative electrophysiological traces recorded at membrane potential differences of +6 mV, +4 mV, or +2 mV, respectively. Translocation events similar to those observed from DOP recordings were monitored, confirming the hypothesis that dsDNA can interact with ClyA and generate a detectable sensing signal when monitored optically without electrodes. PThe values ​​were 0.611 ± 0.357 at +6 mV, 0.605 ± 0.460 at +4 mV, and 0.786 ± 0.224 at +2 mV, which were qualitatively consistent with the blockade observed from DOP recordings (Figure 15, Table 7). During electrophysiological recordings, dwell times ranged from 1 ms to 10 ms. 5 Although the time distribution was wide, down to milliseconds, events faster than 30 milliseconds cannot be optically detected due to the limited bandwidth available from the DOP recording (Fig. 14d).

[0249] [Table 7]

[0250] DIBs were established in cis with 1.5 M KCl, 400 μM EDTA, 40 μM Fluo-8, and 10 mM HEPES (pH 7.0) and in trans with 1.5 M CaCl2 and 10 mM HEPES (pH 7.0). 2 μM dsDNA was added in cis. Three independent measurements were performed and statistics were compiled.

[0251] To further verify ssDNA translocation through the nanopore, we performed electrodeless 20nt ssDNA sensing using the α-HL nanopore. The results are shown in Figure 17. We used the αHL WT nanopore, which only allows ssDNA to pass through, but not dsDNA. Because this nanopore is relatively small, we used a high concentration of 50 μmol / L ssDNA. The percent blockage depth indicates that 20nt ssDNA can pass through the α-HL nanopore.

[0252] Electrodeless 78nt ssDNA sensing using ClyA-RR was also performed, and the results are shown in Figure 18. ClyA-RR has a larger opening, allowing both ssDNA and dsDNA to pass through. In the case of ssDNA, the ssDNA may not be perfectly linear when passing through the nanopore, so the fluorescence emission characteristics are greatly affected by the sequence and secondary structure of the ssDNA. 78nt ssDNA-α and polyA 78 showed very different fluorescence emission characteristics. 78have uniformly shallow percent blockage depths. 78nt ssDNA-a has distinct percent blockage depths and prolonged event dwell times. ssDNA is found to be detectable by DOP.

[0253] Example 5: Multiple DOP recording using a fingertip-sized device and future prospects By eliminating the need for electrode placement, DOP allows for much more compact device size while retaining the advantages of low cost (<$1) and high throughput. This configuration is suitable for fabricating disposable nanopore chips for clinical diagnostics, where cross-contamination must be strictly prevented. As a proof-of-concept, a miniaturized device (10 mm × 10 mm × 1 mm) was fabricated from bulky polymethyl methacrylatemethacrylic acid (PMMA) (Figure 16a, Figure 2). DOP recordings could be performed by placing the chip directly above the TIRF objective used for both illumination and imaging. As an illustration, DOP recordings from α-HL and ClyA were performed using this miniaturized device, and both α-HL and ClyA could be visually monitored (Figure 16c).

[0254] However, DOP measurements from a single DIB were limited to one pore and analyte combination. By eliminating the need for electrode placement, DOP enabled multiplexed recordings from different DIBs with a significantly simpler configuration and a much smaller measurement volume, where different analytes could be physically separated into various water-in-oil compartments.

[0255] As a proof-of-concept, microdroplets (approximately 30 pL) containing ClyA-RR nanopores were fabricated and pipetted into a measurement reservoir filled with lipid oil (Fig. 16d). Although not monodisperse in size, numerous independent DIBs spontaneously formed, which allowed for easy DOP recording (Fig. 16e). In a DIB with a diameter of approximately 40 μm, a single inserted ClyA nanopore was clearly visible as a bright fluorescent spot (Fig. 16f), indicating that EDTA in the droplets was converted to Ca. 2+ This continued for approximately 10 minutes until depletion by binding, which was achieved by 1 mm 2 10 per 3 This corresponds to an effective measurement density of 10 independent DIBs

[49] . However, this high measurement density is not easily achievable with oSCR or electrophysiology due to the complexity of incorporating electronics.

[0256] Despite its electrodeless advantages, DiffusiOptoPhysiology is not without limitations. As a fluorescence imaging technique, the temporal resolution of DOP is typically limited to approximately 10 ms / frame when recorded over a full field of view (135 μm × 135 μm). Acquisition speed could be significantly increased by reading fluorescence from a reduced number of image pixels. The improved addressability from highly ordered nanopore arrays enabled high-speed DOP recordings using spinning-disk confocal imaging

[50] . In the absence of an electric field, the limit of detection (LOD) during DOP was typically higher (~μM) than that from electrophysiology or oSCR (~nM). However, the elimination of electrodes is compensated by the much smaller required measurement volume (~30 pL), which actually reduces absolute sample costs.

[0257] conclusion In summary, we have demonstrated the use of DiffusiOptoPhysiolgy, inspired by natural passive channel transport, as a nanopore sensing platform. Fluorescence emission during DOP recordings indicates Ca2+ The fluorescence intensity, caused by the passive diffusion and subsequent binding of Fluo-8 to the nanopore, is strong enough to be useful for various single-molecule sensing applications, as demonstrated. By combining optimization of the electrolyte and channel size, this technique enables high-throughput nanopore measurements while maintaining sensing performance comparable to conventional electrophysiological recordings and oSCR. While demonstrated using total internal reflection fluorescence (TIRF) microscopy, DOP can, in principle, be flexibly applied to any fluorescence platform, including confocal and epifluorescence microscopy. Because it does not require space for electrode installation, the DOP measurement volume is further reduced to approximately 30 pL, the smallest volume reported to date, potentially suitable for measuring extremely low-abundance analytes. Furthermore, DOP recording using microdroplet arrays enables multiplexed measurements from independent compartments, a feasible approach based on simple water-in-oil separations. While incorporating nanotechnology sensors as chips, omitting the electronics significantly reduces the cost and size of the device. This method may provide a hint for future clinical applications of disposable nanopore chips for a variety of purposes.

[0258] method material Hexadecane, silicone oil AR20, pentane, ethylenediaminetetraacetic acid (EDTA), Triton X-100, Genapol X-80, and PEG 1500 were obtained from Sigma-Aldrich. Potassium chloride, calcium chloride, magnesium chloride, and sodium chloride were obtained from Aladdin. Dioxane-free isopropyl-β-D-thiogalactopyranoside (IPTG), dodecyl β-D-maltopyranoside (DDM), kanamycin sulfate, trimethylamine methane (Tris), and imidazole were obtained from Solarbio. Low-melting-point agarose and wide-range DNA ladders (20–500 bp) were obtained from Takara. Precision Plus protein markers and 4–15% polyacrylamide gels were obtained from BioRad. Ethanol and acetone were obtained from Sinopharm. Fluo-8H sodium salt (Fluo-8) was obtained from AAT Bioquest. 1,2-Diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) was obtained from Avanti Polar Lipids. 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) was obtained from Shanghai Yuanye Bio-Technology Co., Ltd. Escherichia coli strain BL21(DE3) was obtained from BioMed. Trimethyl-β-cyclodextrin was obtained from Tokyo Chemical Industry Co., Ltd. (Shanghai). LB broth and LB agar were obtained from Hopebio. All materials were used as received.

[0259] KCl buffer (1–2.5 M KCl, 10 mM HEPES, pH 7.0) and CaCl buffer (0.5–1.5 M CaCl, 10 mM HEPES, pH 7.0) were filtered through a membrane filter (0.2 μm cellulose acetate, Nalgene). For simplicity, unless otherwise noted, 1–2.5 M KCl buffer refers to 1–2.5 M KCl, 10 mM HEPES, pH 7.0, and 0.5–1.5 M CaCl buffer refers to 0.5–1.5 M CaCl, 10 mM HEPES, pH 7.0. The KCl buffer was treated overnight with Chelex 100 resin (Bio-Rad) to remove contaminating divalent ions.

[0260] DNA purified by high-performance liquid chromatography (HPLC) (Table 6) was dissolved in DNase-free / RNase-free water before use. To form dsDNA, the complementary ssDNA was further dissolved in 1.5 M KCl buffer (1.5 M KCl, 10 mM HEPES, pH 7.0), heated to 95°C in a PCR thermal cycler (ABI2720), and gradually cooled (-5°C / min) to room temperature (25°C).

[0261] The protein nanopores used herein were α-HL WT and ClyA-RR (Figure 7), which were expressed in E. coli and purified according to published protocols [22, 42].

[0262] ClyA-RR preparation The gene encoding the monomeric ClyA-RR (D64R / C87A / L99Q / E103G / S110R / F166Y / I203V / C285S / K294R / H307Y) protein was custom synthesized and constructed in the pET30a(+) plasmid (Genescript, New Jersey). A hexa-histidine tag was introduced at the C-terminus of the protein for subsequent chromatographic purification. The plasmid was transformed into E. coli BL21(DE3) competent cells and cultured on LB agar plates containing 50 μg / mL kanamycin for 18 hours. A single colony was plated on LB medium containing 50 μg / mL kanamycin and grown at OD . 600The medium was incubated at 37°C until the pH reached 4.0. Protein expression was induced by adding isopropyl β-D-thiogalactoside (IPTG) to the LB medium to a final concentration of 1 mM. The medium was then shaken (200 rpm) at 15°C for 16 hours. Cells were then harvested by centrifugation (4000 rpm, 4°C, 20 min). The pellet was collected, resuspended in lysis buffer (150 mM NaCl, 50 mM Tris·HCl, 10% glycerol, pH 8.0), lysed by sonication (15 min), and centrifuged (14,000 rpm, 4°C, 40 min) to remove intact cells. After syringe filtration, the supernatant was loaded onto a nickel affinity column (HisTrap™ HP, GE Healthcare). After washing the column with wash buffer A (150 mM NaCl, 50 mM Tris·HCl, 10% glycerol, 20 mM imidazole, pH 8.0), the target protein was sequentially eluted with three wash buffers (buffer B: 500 mM NaCl, 15 mM Tris·HCl, 10% glycerol, 300 mM imidazole, pH 8.0; buffer C: 500 mM NaCl, 15 mM Tris·HCl, 10% glycerol, 50 mM imidazole, pH 8.0; and buffer D: 500 mM NaCl, 15 mM Tris·HCl, 10% glycerol, 20 mM imidazole, pH 8.0). Eluted fractions containing ClyA-RR monomer were identified using SDS-PAGE gel electrophoresis (Figure 12) and stored at -80°C in a buffer containing 270 mM NaCl, 50 mM Tris-HCl, 10% glycerol, and 0.2% Triton 100, pH 8.0.

[0263] Following previous work

[42] , pore oligomerization was promoted by adding 0.25% (w / v) β-dodecyl maltoside (DDM). After 15 min of incubation at 25 °C, the pore oligomerization results were characterized by blue native polyacrylamide gel electrophoresis (BN-PAGE, BioRad) using a 4–15% polyacrylamide gel (Figure 12). The gel showed that the monomers had self-assembled into oligomers before the addition of DDM. However, to strictly follow previous work

[42] , we continued to use the ClyA-RR dodecamer prepared with DDM for follow-up measurements. Here, the band corresponding to the dodecamer ClyA-RR was excised from the gel and soaked in 150 mM NaCl, 15 mM Tris·HCl, pH 7.5, supplemented with 0.2% DDM and 10 mM EDTA for 3 h. The supernatant containing the dodecameric protein that had diffused from the gel was collected by centrifugation (20,000 g, 4°C, 20 min). The collected dodecameric ClyA-RR protein was either used immediately for subsequent experiments or stored at 4°C for up to 14 days.

[0264] DIB formation A detailed description of the droplet / hydrogel bilayer fabrication method has been reported previously

[10] . Briefly, oxygen-plasma-treated coverslips (24 mm × 40 mm) were spin-coated (3000 rpm, 30 s) with 200 μL of molten agarose (0.75% w / v in MiliQ water). The coverslip was attached to the PMMA device by filling the microfluidic channels within the device with molten agarose (2.5% w / v in CaCl2 buffer)

[10] . A lipid / oil solution was prepared by dissolving 5 mg of dried DPHPC lipid film in 2 mL of a 1:1 volumetric mixture of hexadecane and silicone oil. Upon immersion in the lipid-oil solution, a lipid monolayer was formed on the agarose-coated coverslip. To prepare aqueous droplets, protein nanopores and other analytes were added to a buffer solution consisting of 1M–2.5M KCl, 400 μM EDTA, 40 μM Fluo-8, and 10 mM HEPES, pH 7.0. Various volumes of aqueous droplets were pipetted into lipid / oil solutions and incubated. After 5 min, a self-assembled lipid monolayer was formed at the water-oil interface. When these droplets were contacted with an agarose substrate in the lipid / oil solution, a stable bilayer (DIB) spontaneously formed.

[0265] TIRF imaging and optical recording DIBs were imaged using an inverted microscope (Eclipse Ti-U, Nikon) equipped with a 60x oil-immersion TIRF objective (NA = 1.49, Plan Apo, Nikon). Fluorescence was excited with a 473 nm diode-pumped solid-state (DPSS) laser (100 mW, Changchun New Industries Optoelectronics Technology). Images were acquired using an electron-multiplying CCD camera (iXon3 897, Andor). Exposure times were set at 3–30 ms. The maximum field of view was 135 μm × 135 μm.

[0266] Electrical recording Electrophysiological recordings were performed as previously reported 8 Electrophysiological traces were acquired at a sampling rate of 25 kHz, low-pass filtered at 1 kHz (Axopatch 200B, Molecular Devices), digitized, and recorded using a Digidata 1550A digitizer (Molecular Devices). Subsequent data analysis was performed using Clampfit 10.7 (Molecular Devices).

[0267] Finite Element Modeling (FEM) Simulation Ca 2+ Binding of the calcium indicator Fluo-8 dye to the pore results in fluorescence emission near the pore. 2+ binds to EDTA, reducing the fluorescence background. These two complete reactions can be described as equations (1, 2), where α and β represent the forward and reverse binding rates, respectively.

[0268]

number

[0269] Optical single channel recording (oSCR) was simulated by FEM using the Poisson-Nernst-Planck-Stokes (PNPS) model [24, 26]. The Nernst-Planck-Stokes equation is given in Eq. (3).

[0270]

number

[0271] For the electrodeless oSCR, the potential V is set to be constant within the simulation space. Therefore, equation (3) simplifies to (equation (4)), and ion movement is driven only by passive diffusion, chemical reaction, and fluidic flow.

[0272]

number

[0273] where [c i ] represents the concentration of various ion species. i represents the chemical reaction term and u represents the liquid velocity. 2+ could be conjugated with Fluo-8 or EDTA as shown in formula (1, 2).

[0274] For various ions, we further expand Eq. (4) and annotate the ion identities with corresponding footnotes as described in Eqs. (5-10). Here, FluoCa and EDTACa are Fluo-8 and Ca. 2+ Binding form of EDTA and Ca 2+ represents the bonding form.

[0275]

number

[0276] The electrostatic potential of the standard PNPS model is controlled by the Poisson equation expressed as Eq. (11).

[0277]

number

[0278] However, in the electrodeless oSCR, the potential V is constant within the simulation space, so the above equation can be simplified to equation (12).

[0279]

number

[0280] The simulation parameters are mainly taken from the literature

[26] , where D is the diffusion coefficient (D Fluo =D FluoCa = 15 μm2 s -1 , D k =D cl =D Ca =D EDTA =D EDTACa = 200 μm 2 s -1 ). z is the number of charges (z Ca =+2, z k =+1, z cl =-1), where F is the Faraday constant. b is the Boltzmann constant. T is the voltage. α is the forward binding velocity (α E = 5 μM -1 s -1 , α F =150 s-1 ) and β is the reverse binding rate (β E =0.75μM -1 s -1 , β F =450s -1 ) E and F The σ and σ represent EDTA and Fluo-8, respectively. ε is the relative dielectric constant of water. The cis-side boundary conditions are set by varying the KCl concentration (0.5 M to 2.5 M), and the trans-side boundary conditions are set as 0.75 M CaCl2.

[0281] The steady-state distribution of ions under various simulation conditions is numerically solved using COMSOL5.3a. Briefly, the axisymmetric simulation geometry is defined as two hemispherical spaces separated by a semipermeable membrane that allows only the passage of liquid, but not ions (Figure 2). The two hemispheres, designated the cis and trans sides, are connected by a cylindrical nanopore on the membrane that allows the free passage of both liquid and ions.

[0282] When illuminated in TIRF mode, the excitation intensity decays exponentially in the z direction. To simulate the fluorescence intensity in the projected xy plane, we use equation (13), where γ is the decay constant of the evanescent light in the z direction.

[0283]

number

[0284] Meanwhile, the total fluorescence intensity is estimated according to equation (14).

[0285]

number

[0286] 2D Gaussian fitting In electrodeless oSCR, the fluorescence intensity profile, which appears as a bright spot, was fitted to a 2D Gaussian distribution according to equation (15).

[0287]

number

[0288] where f(x, y) represents the fitted fluorescence intensity in the xy plane, Z represents the reference level, A represents the fitting amplitude, and x c and y c represents the center of gravity of the fitting. σ x and σ y represent the standard deviations of the distribution in the x and y directions, respectively.

[0289] This function allows for sub-pixel resolution localization of the tracked spot. The full width half magnitude (FWHM) of the 2D Gaussian function represents the width at half height and can be used to estimate the spot size. Here, we perform a 2D Gaussian fitting using the cftool module of MATLAB (Figure 5). The FWHM is derived from Eq. (16):

[0290]

number

[0291] Pixels within a circle with a diameter of 2 FWHM were defined as signal, and pixels within the annulus between the circles with diameters of 3 FWHM and 4 FWHM were defined as background (Fig. 5).

[0292] Signal-to-background ratio (SBR) evaluation The SBR value was introduced to quantitatively evaluate the performance of DOP recording from different trials of DOP recording. The SBR value was calculated as follows:

[0293]

number

[0294] Here, peak (sig) is the peak amplitude (A + z0) of the signal obtained by 2D Gaussian fitting (Figure 5). bkg ) is the average pixel intensity of the background (z0). std( bkg ) is the standard deviation of the pixel intensity of the background. The definitions of signal and background are shown in Figure 5.

[0295] References 1. Andre, B. An overview of membrane transport proteins in Saccharomyces cerevisiae. Yeast 11, 1575-1611 (1995). 2. Gouaux, E. & MacKinnon, R. Principles of selective ion transport in channels and pumps. Science 310, 1461-1465 (2005). 3. Nielsen, S. et al. Specialized membrane domains for water transport in glial cells: high-resolution immunogold cytochemistry of aquaporin-4 in rat brain. Journal of Neuroscience 17, 171-180 (1997). 4. Wood, I.S. & Trayhurn, P. Glucose transporters (GLUT and SGLT): expanded families of sugar transport proteins. British Journal of Nutrition 89, 3-9 (2003). 5. Choi, K.H. et al. Insight into DNA and protein transport in double-stranded DNA viruses: the structure of bacteriophage N4. Journal of molecular biology 378, 726-736 (2008). 6. Bressloff, P.C. & Newby, J.M. Stochastic models of intracellular transport. Rev Mod Phys 85, 135-196 (2013). 7. Deamer, D., Akeson, M. & Branton, D. Three decades of nanopore sequencing. Nat Biotechnol 34, 518-524 (2016). 8. Huang, S. Nanopore-based sensing devices and applications to genome sequencing: a brief history and the missing pieces. Chin. Sci. Bull. 59, 4918-4928 (2014). 9. Kasianowicz, J.J., Brandin, E., Branton, D. & Deamer, D.W. Characterization of individual polynucleotide molecules using a membrane channel. P Natl Acad Sci USA 93, 13770-13773 (1996). 10. Bayley, H. et al. Droplet interface bilayers. Mol Biosyst 4, 1191-1208 (2008). 11. Sapra, K.T. & Bayley, H. Lipid-coated hydrogel shapes as components of electrical circuits and mechanical devices. Sci Rep-Uk 2, 848 (2012). 12. Li, J. et al. Ion-beam sculpting at nanometre length scales. Nature 412, 166-169 (2001). 13. Zhang, B. et al. Bench-top method for fabricating glass-sealed nanodisk electrodes, glass nanopore electrodes, and glass nanopore membranes of controlled size. Anal Chem 79, 4778-4787 (2007). 14. Geng, J. et al. Stochastic transport through carbon nanotubes in lipid bilayers and live cell membranes. Nature 514, 612 (2014). 15. Chou, L.Y., Ming, K. & Chan, W.C. Strategies for the intracellular delivery of nanoparticles. Chemical Society Reviews 40, 233-245 (2011). 16. Barry, R.J., Clarke, A.R. & Johnstone, S.J. A review of electrophysiology in attention-deficit / hyperactivity disorder: I. Qualitative and quantitative electroencephalography. Clinical neurophysiology 114, 171-183 (2003). 17. Dunlop, J., Bowlby, M., Peri, R., Vasilyev, D. & Arias, R. High-throughput electrophysiology: an emerging paradigm for ion-channel screening and physiology. Nat Rev Drug Discov 7, 358-368 (2008). 18. Jain, M. et al. Nanopore sequencing and assembly of a human genome with ultra-long reads. Nat Biotechnol 36, 338-+ (2018). 19. Letellier, L., Plancon, L., Bonhivers, M. & Boulanger, P. Phage DNA transport across membranes. Research in microbiology 150, 499-505 (1999). 20. Song, L. et al. Structure of staphylococcal α-hemolysin, a heptameric transmembrane pore. Science 274, 1859-1865 (1996). 21. Huang, S., Romero-Ruiz, M., Castell, O.K., Bayley, H. & Wallace, M.I. High-throughput optical sensing of nucleic acids in a nanopore array. Nat Nano 10, 986-U996 (2015). 22. Wang, Y.Q. et al. Osmosis-Driven Motion-Type Modulation of Biological Nanopores for Parallel Optical Nucleic Acid Sensing. Acs Appl Mater Inter 10, 7788-7797 (2018). 23. Heron, A.J., Thompson, J.R., Cronin, B., Bayley, H. & Wallace, M.I. Simultaneous measurement of ionic current and fluorescence from single protein pores. J. Am. Chem. Soc 131, 1652-1653 (2009). 24. Anderson, B.N. et al. Probing solid-state nanopores with light for the detection of unlabeled analytes. Acs Nano 8, 11836-11845 (2014). 25. Ivankin, A. et al. Label-Free Optical Detection of Biomolecular Translocation through Nanopore Arrays. Biophys J 108, 331a-331a (2015). 26. Shuai, J. & Parker, I. Optical single-channel recording by imaging Ca2+ flux through individual ion channels: theoretical considerations and limits to resolution. Cell Calcium 37, 283-299 (2005). 27. Gu, L.Q., Braha, O., Conlan, S., Cheley, S. & Bayley, H. Stochastic sensing of organic analytes by a pore-forming protein containing a molecular adapter. Nature 398, 686-690 (1999). 28. Gu, L.Q., Cheley, S. & Bayley, H. Capture of a single molecule in a nanocavity. Science 291, 636-640 (2001). 29. Kang, X.F., Cheley, S., Guan, X.Y. & Bayley, H. Stochastic detection of enantiomers. J Am Chem Soc 128, 10684-10685 (2006). 30. Bayley, H. & Cremer, P.S. Stochastic sensors inspired by biology. Nature 413, 226 (2001). 31. Fennouri, A. et al. Kinetics of enzymatic degradation of high molecular weight polysaccharides through a nanopore: experiments and data-modeling. Anal Chem 85, 8488-8492 (2013). 32. Braha, O. et al. Simultaneous stochastic sensing of divalent metal ions. Nat Biotechnol 18, 1005 (2000). 33. Clarke, J. et al. Continuous base identification for single-molecule nanopore DNA sequencing. Nat Nanotechnol 4, 265 (2009). 34. Boersma, A.J., Brain, K.L. & Bayley, H. Real-time stochastic detection of multiple neurotransmitters with a protein nanopore. Acs Nano 6, 5304-5308 (2012). 35. Boersma, A.J. & Bayley, H. Continuous stochastic detection of amino acid enantiomers with a protein nanopore. Angewandte Chemie 124, 9744-9747 (2012). 36. Sacerdote, M. & Szostak, J. Semipermeable lipid bilayers exhibit diastereoselectivity favoring ribose. Proceedings of the National Academy of Sciences 102, 6004-6008 (2005). 37. Raghunathan, A. & Aluru, N. Molecular understanding of osmosis in semipermeable membranes. Phys Rev Lett 97, 024501 (2006). 38. Robertson, J.W. et al. Single-molecule mass spectrometry in solution using a solitary nanopore. Proceedings of the National Academy of Sciences 104, 8207-8211 (2007). 39. Rodrigues, C.G., Machado, D.C., Chevtchenko, S.F. & Krasilnikov, O.V. Mechanism of KCl enhancement in detection of nonionic polymers by nanopore sensors. Biophys J 95, 5186-5192 (2008). 40. Soskine, M. et al. An engineered ClyA nanopore detects folded target proteins by selective external association and pore entry. Nano letters 12, 4895-4900 (2012). 41. Biesemans, A., Soskine, M. & Maglia, G. A Protein Rotaxane Controls the Translocation of Proteins Across a ClyA Nanopore. Nano letters 15, 6076-6081 (2015). 42. Franceschini, L., Brouns, T., Willems, K., Carlon, E. & Maglia, G. DNA Translocation through Nanopores at Physiological Ionic Strengths Requires Precise Nanoscale Engineering. Acs Nano 10, 8394-8402 (2016). 43. Franceschini, L., Soskine, M., Biesemans, A. & Maglia, G. A nanopore machine promotes the vectorial transport of DNA across membranes. Nature communications 4, 2415 (2013). 44. Soskine, M., Biesemans, A. & Maglia, G. Single-Molecule Analyte Recognition with ClyA Nanopores Equipped with Internal Protein Adaptors. Journal of the American Chemical Society 137, 5793-5797 (2015). 45. Wendell, D. et al. Translocation of double-stranded DNA through membrane-adapted phi29 motor protein nanopores. Nat Nanotechnol 4, 765 (2009). 46. Manrao, E.A. Reading DNA at single-nucleotide resolution with a mutant MspA nanopore and phi29 DNA polymerase. Nature Biotechnol. 30, 349-353 (2012). 47. Tinland, B., Pluen, A., Sturm, J. & Weill, G. Persistence length of single-stranded DNA. Macromolecules 30, 5763-5765 (1997). 48. Nomidis, S.K., Hooyberghs, J., Maglia, G. & Carlon, E. DNA capture into the ClyA nanopore: diffusion-limited versus reaction-limited processes. Journal of Physics: Condensed Matter (2018). 49. Chen, Z.T. et al. Centrifugal micro-channel array droplet generation for highly parallel digital PCR. Lab Chip 17, 235-240 (2017). 50. Nakano, A. Spinning-disk confocal microscopy-a cutting-edge tool for imaging of membrane traffic. Cell structure and function 27, 349-355 (2002).

Claims

1. 1. An electrodeless system for identifying an analyte, comprising: (a) a first compartment containing a first aqueous solution therein, said first aqueous solution comprising a fluorescent reporter molecule capable of fluorescing when bound to an ionic species; (b) a second compartment containing a second aqueous solution therein, said second aqueous solution comprising said ionic species that specifically binds to said fluorescent reporter molecule; and (c) a membrane separating the first compartment from the second compartment; Including, at least one nanopore is inserted in the membrane between the first compartment and the second compartment such that the first compartment and the second compartment are connected by a nanopore; a chemical gradient of the ionic species exists between the first compartment and the second compartment, allowing the ionic species to diffuse from the second compartment to the first compartment through the nanopore; A system wherein, upon passing through the nanopore, the analyte inhibits the translocation of the ionic species across the nanopore.

2. The system of claim 1 , wherein the membrane is a solid membrane or a semi-permeable membrane.

3. 3. The system of claim 2, wherein the osmolality of the second aqueous solution is higher than the osmolality of the first aqueous solution, or the osmolality of the second aqueous solution is higher than the osmolality of the first aqueous solution; or the osmolality of the second aqueous solution is equal to the osmolality of the first aqueous solution, or the osmolality of the second aqueous solution is equal to the osmolality of the first aqueous solution; or the osmolality of the second aqueous solution is lower than the osmolality of the first aqueous solution, or the osmolality of the second aqueous solution is lower than the osmolality of the first aqueous solution.

4. 4. The system of claim 2 or claim 3, wherein the semipermeable membrane is composed of amphiphilic molecules.

5. The system of claim 4 , wherein the amphiphilic molecule is a lipid or a triblock copolymer.

6. 4. The system of claim 2 or claim 3, wherein the semipermeable membrane is a bilayer composed of amphiphilic molecules.

7. The system of claim 6 , wherein the amphiphilic molecule is a lipid.

8. 8. The system of claim 7, wherein the lipid is one or more selected from the group consisting of fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, polyketides, phospholipids, glycolipids, and cholesterol.

9. 8. The system of claim 7, wherein the lipid is one or more selected from the group consisting of monoolein; 1,2-dioleoyl-sn glycero-S-phosphocholine (DOPC); 1,2-diphytanoyl-sn-glycero-3-phosphatidylcholine (DPhPC); palmitoyloleoylphosphatidylcholine (POPC); 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE); 1-palmitoyl-2-oleoyl-phosphatidylethanolamine; 1-palmitoyl-2-oleoylphosphatidylglycerol (POPE / POPG) mixture; and mixtures thereof.

10. The system according to any one of claims 1 to 9, wherein the first compartment is provided by an aqueous droplet.

11. The system according to any one of claims 1 to 10, wherein the second compartment is provided by a hydrogel layer.

12. The system of claim 11, wherein the hydrogel layer comprises 0.1 to 20% (w / v) agarose.

13. The system of claim 11, wherein the hydrogel layer comprises 2-5% (w / v) agarose.

14. The system of any one of claims 1 to 13, wherein the nanopore is selected from the group consisting of a protein nanopore, a DNA nanopore, or a solid-state nanopore.

15. 15. The system of claim 14, wherein the protein nanopore is one or more selected from the group consisting of α-HL, ClyA, Phi29 connector protein, erolysin, MspA, OmpF, OmpG, FraC, HlyA, SheA, sp1, and variants thereof; and an ion channel.

16. The system of claim 15, wherein the protein nanopore is ClyA-RR or α-HL.

17. The ion species is Ag ＋ , Ag ２＋ , Al ３＋ , As ３＋ , Au ＋ , Ba ２＋ , Bi ３＋ , Ca ２＋ , Cd ２＋ , Ce ３＋ , Ce ４＋ , Cl － , Co ２＋ , Cr ３＋ , Cu ＋ , Cu ２＋ , Dy ３＋ , Eu ３＋ , Fe2 ＋ , Fe ３＋ , Ga ３＋ , H ＋ , Hg ＋ , Hg ２＋ , In ３＋ , K. ＋ , La ３＋ , Mg ２＋ , Mn ２＋ , Mo ３＋ , Na ＋ , Ni ２＋ , O.H. － , Pb ２＋ , Pd ２＋ , Pt ２＋ , Pt ４＋ , Ru ３＋ , Sb ３＋ , Sc ３＋ , Sn ２＋ , Sr ２＋ , Tb ３＋ , Tl ＋ , and Zn ２＋ The system according to any one of claims 1 to 16, wherein the system is one or more selected from the group consisting of:

18. The fluorescent reporter molecule is Fura-2, Indo-1, Fluo-2, Fluo-3, Fluo-4, Fluo-8, Calcium Green-1, DCFH, DHR, SNARF, Cal-520, calcium-specific aminopolycarboxylic acid, BAPTA, SBFI, Asante NaTRIUM Green-1, Asante NaTRIUM Green-2, Thallos Potassium Ion Channel Reagent, Asante Potassium Green-1, Asante Potassium Green-2, Asante Potassium Green-3, PBFI, Fluo-2 18. The system of any one of claims 1 to 17, wherein the ZnO is one or more selected from the group consisting of Zn, Fura-2 Mg, Indo-1 Mg, Asante Magnesium Green, SPQ, MQAE, TSQ, TFL-Zn, and ZINQUIN.

19. A system according to any one of claims 1 to 18, wherein the second aqueous solution comprises calcium chloride and, optionally, a buffering agent.

20. 20. The system of claim 19, wherein the concentration of calcium chloride in the second aqueous solution is 0.01 to 6.76M.

21. 21. The system of any one of claims 1 to 20, wherein the first aqueous solution comprises a chelating agent and, optionally, a buffering agent, the chelating agent being capable of binding to the ionic species.

22. 22. The system of claim 21, wherein the chelating agent is EDTA, BAPTA, EGTA, CyDTA, DTPA, EDDP, HIDA, IDA, NTA, NTPO, TTHA, CA, TA, GA, HEDTA, or DEG.

23. The system of any one of claims 1 to 22, further comprising a light source for illumination and a light sensor for detecting fluorescence.

24. 24. The system of claim 23, wherein the light source is a laser, an LED, a halogen light, or a xenon light, and the light sensor is a CCD, an sCMOS sensor, or a photodiode.

25. 24. The system of claim 23, wherein the optical sensor is an EMCCD or an avalanche photodiode.

26. The system of any one of claims 1 to 22, further comprising a device for total internal reflection fluorescence (TIRF), wide-field fluorescence microscopy, or confocal microscopy.

27. The system of any one of claims 1 to 26, wherein the first aqueous solution or the second aqueous solution comprises the analyte.

28. 28. The system of claim 27, wherein the analyte is selected from the group consisting of small molecules, macromolecules, and biopolymers.

29. 28. The system of claim 27, wherein the analyte is selected from the group consisting of a compound, a drug, a sugar, an ion, a neurotransmitter, an amino acid, a nucleotide, a polymer, a polypeptide, a polysaccharide, and a polynucleotide.

30. 30. The system of claim 29, wherein the polynucleotide is DNA or RNA.

31. 31. The system of claim 30, wherein the DNA is dsDNA or ssDNA and the RNA is miRNA, siRNA or tRNA.

32. 1. A method for identifying an analyte, comprising: (a) providing a system according to any one of claims 1 to 31, wherein the analyte is provided in the first compartment or in the second compartment; (b) directing light capable of exciting the fluorescent reporter molecule at a region within the first compartment proximal to the nanopore; (c) measuring a fluorescent signal from the fluorescent reporter molecule to identify the analyte; A method comprising:

33. 1. An electrodeless nanopore array for identifying multiple analytes, comprising: a plurality of systems in parallel, each system being a system according to any one of claims 1 to 31; the plurality of systems are arranged such that the measured fluorescence is distinguishable in each system; Nanopore array.

34. 34. The nanopore array of claim 33, wherein each of the first compartments of the plurality of systems is separated from one another.

35. 35. The nanopore array of claim 33 or 34, wherein the second compartment of the plurality of systems is a single compartment.

36. The nanopore array of any one of claims 33 to 35, wherein different analytes are physically separated into different systems.

37. The density of the systems in the nanopore array is 10 to 1000 / mm ２ The nanopore array according to any one of claims 33 to 36, wherein

38. The total area provided by the plurality of systems is 1 to 100 mm ２ The nanopore array according to any one of claims 33 to 37,

39. The nanopore array of any one of claims 33 to 38, wherein the number of the plurality of systems is between 4 and 1,000,000.

40. The nanopore array of claim 39, wherein the number of the plurality of systems is between 10 and 1000.

Images