Nanopore single molecule analyte detection without electrodes

Abstract

An electrodeless system for identifying an analyte, based on optical measurements of ion flow through a nanopore driven by a chemical gradient, and a method for identifying an analyte by using the system. An electrodeless nanopore array for parallel identification of different analytes, a method for identifying different analytes by using the array, and a method for producing the array.

Description

Technical Field

[0001] This invention relates to a method for identifying analytes using protein nanopores. Background Technology

[0002] Natural transmembrane transport is facilitated by various membrane transport proteins [1]. The transported solutes, such as small ions [2], water [3], sugars [4], or even genetic material [5], are crucial for regulating various cellular activities. Although the detailed transport mechanisms vary [6], the fact that single-molecule identity can be reported during channel translocation forms the basis of nanopore sequencing as a biomimetic approach [7,8]. Nanopore sensing has been reported from planar lipid membranes [9], droplet interfacial bilayers (DIBs)

[10] , hydrogel interfacial bilayers

[11] , synthetic solid membranes

[12] , glass nanopipettes

[13] , or cell membranes

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

[0003] In electrophysiological processes, the Ag / AgCl electrode pair is used to apply transmembrane potential, thereby driving Cl... - And the continuous electromigration of charged analytes. It is also used to record ion current fluctuations for single-molecule recognition. Figure 1 a). In the absence of electrodes, although vibrational thermal diffusion of ions through nanopores exists in both directions, due to the law of electroneutrality, the net ion flux and electric field throughout the electrolyte-containing space are strictly zero. Figure 1 b)

[15] .

[0004] Electrophysiological measurements offer fairly good temporal (~10 μs) and amplitude resolution (<0.1 pA)

[16] , meeting the needs of single-channel recording-based applications, but have limitations in throughput

[17] . Despite the urgent need in nanopore sequencing and drug screening, it is not possible to achieve simultaneous reading of 1 million channels without sacrificing cost or device size[17,18]. Therefore, this urgent need prompts us to rethink a simplified high-throughput channel recording strategy, which may be further derived from bionics.

[0005] When the host cell remains intact, bacterial phage T4 injects its genomic DNA via channel proteins

[19] . Staphylococcus aureus α-hemolysin (α-HL) causes hemolysis of target cells due to the passive leakage of nutrients through the inserted channels

[20] . These spontaneous molecular transports, acquired from natural evolution, remind us that external electronic devices are not essential for molecular transport. The remaining challenge is how to acquire nanopore sensing signals without electrical connections.

[0006] Recent advances in optical single-channel recording (oSCR) [21-25] demonstrate an alternative strategy that optically monitors Ca 2+ Flow Figure 1 c) [21, 23]. Although oSCR has advantages in high-throughput measurements, it still utilizes a pair of electrodes to electrophoretically drive Ca 2+ through the nanopore

[23] . The electrodes are manually inserted into the aqueous droplet, which requires delicate micromanipulation skills and can cause a high risk of double-layer rupture

[23] , hindering its widespread application in academic research and industrial applications. SUMMARY

[0007] The present invention provides a system for identifying an analyte based on optical measurement of ion flow through a nanopore driven by a chemical gradient. The present invention also provides methods of identifying an analyte using the system, including methods of identifying small molecules or DNA (such as dsDNA or ssDNA).

[0008] In one aspect of the present invention, there is provided an electrodeless system for identifying an analyte, the system comprising:

[0009] (a) a first compartment having a first aqueous solution therein, wherein the first aqueous solution comprises a fluorescent reporter molecule capable of emitting fluorescence when bound to an ionic species;

[0010] (b) a second compartment having a second aqueous solution therein, wherein the second aqueous solution comprises an ionic species that specifically binds to the fluorescent reporter molecule; and

[0011] (c) a membrane separating the first compartment and the second compartment;

[0012] wherein the membrane between the first compartment and the second compartment has at least one inserted nanopore, such that the first compartment and the second compartment are connected through the nanopore;

[0013] wherein there is a chemical gradient of the ionic species between the first compartment and the second compartment that can drive diffusion of the ionic species through the nanopore from the second compartment to the first compartment.

[0014] In some embodiments, the membrane is a solid membrane.

[0015] In some embodiments, the membrane is a semi-permeable membrane.

[0016] In some embodiments, the second aqueous solution has a higher osmolarity than the first aqueous solution or a higher osmolality than the first aqueous solution; or the second aqueous solution has an equal osmolarity as the first aqueous solution or an equal osmolality as the first aqueous solution; or the second aqueous solution has a lower osmolarity than the first aqueous solution or a lower osmolality than the first aqueous solution.

[0017] In some embodiments, the semi-permeable membrane is composed of amphiphilic molecules; preferably, the amphiphilic molecules are lipids or triblock copolymers.

[0018] In some embodiments, the semi-permeable membrane is a bilayer composed of amphiphilic molecules

[0019] In some embodiments, the amphiphilic molecules are lipids.

[0020] In some embodiments, the lipids are one or more selected from the group consisting of fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenollipids, saccharolipids, polyketides, phospholipids, glycolipids, and cholesterols.

[0021] In some embodiments, the lipids are one or more selected from the group consisting of glyceryl monooleate; 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC); 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC); palmitoyloleoyl phosphatidylcholine (POPC); l-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE); l-palmitoyl-2-oleoyl-phosphatidylethanolamine; l-palmitoyl-2-oleoyl phosphatidylglycerol (POPE / POPG) mixture; and mixtures thereof.

[0022] In some embodiments, the first compartment is provided by an aqueous droplet.

[0023] 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.

[0024] In some embodiments, the nanopore is selected from the group consisting of a protein nanopore, a DNA nanopore, or a solid nanopore.

[0025] In some embodiments, the protein nanopore is one or more selected from the group consisting of: a-HL, ClyA, Phi29 connector protein, aerolysine, MspA, OmpF, OmpG, FraC, HlyA, SheA, sp1, and variants thereof; and an ion channel.

[0026] In some embodiments, the protein nanopore is ClyA-RR or a-HL.

[0027] In some embodiments, the ionic species is one or more selected from the group consisting of: 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+ , OH - , Pb 2+ , Pd 2+ , Pt 2+ , Pt 4+ , Ru 3 + , Sb 3+ , Sc 3+ , Sn 2+ , Sr 2+ , Tb 3+ , Tl + , and Zn 2+ .

[0028] 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 acids, BAPTA, SBFI, Asante NaTRIUM Green-1, Asante NaTRIUM Green-2, Thallos potassium ionophore 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.

[0029] In some embodiments, the second aqueous solution comprises calcium chloride and optionally a buffer.

[0030] In some embodiments, the concentration of calcium chloride in the second aqueous solution is 0.01-6.76 M.

[0031] In some embodiments, the first aqueous solution comprises a chelator and optionally a buffer, wherein the chelator is capable of binding to the ionic species; preferably, the chelator is EDTA, BAPTA, EGTA, CyDTA, DTPA, EDDP, HIDA, IDA, NTA, NTPO, TTHA, CA, TA, GA, HEDTA, or DEG.

[0032] 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, an LED, a halogen lamp, a xenon lamp; preferably, the light sensor is a CCD, a sCMOS sensor, a photodiode; more preferably, the light sensor is an EMCCD or an avalanche photodiode (APD).

[0033] In some embodiments, the system further comprises a device for total internal reflection fluorescence (TIRF), wide-field fluorescence microscopy, or confocal microscopy.

[0034] In some embodiments, the first aqueous solution or the second aqueous solution comprises the analyte.

[0035] In some embodiments, the analyte is selected from the group consisting of a small molecule, a macromolecule, and a biological macromolecule.

[0036] In some embodiments, the analyte is selected from the group consisting of a chemical 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.

[0037] In another aspect of the application, there is provided a method of identifying an analyte, the method comprising the steps of:

[0038] (a) providing any of the above systems, wherein the analyte is provided in the first compartment or the second compartment;

[0039] (b) applying light capable of exciting the fluorescent reporter molecule to a region of the first compartment adjacent to the nanopore;

[0040] (c) measuring a fluorescent signal from the fluorescent reporter molecule to identify the analyte.

[0041] In another aspect of the application, there is provided a method of producing an electrodeless system, comprising:

[0042] providing a first compartment having a first aqueous solution therein, wherein the first aqueous solution comprises a fluorescent reporter molecule capable of emitting fluorescence when bound to an ionic species;

[0043] providing a second compartment having a second aqueous solution therein, wherein the second aqueous solution comprises an ionic species that specifically binds to the fluorescent reporter molecule;

[0044] bringing the first compartment and the second compartment together in a hydrophobic medium containing an amphiphilic molecule, such that a semi-permeable membrane having an inserted nanopore is formed between the first compartment and the second compartment;

[0045] wherein a protein nanopore is provided in the first aqueous solution or the second aqueous solution.

[0046] In some embodiments, the second aqueous solution has a higher osmolality than the first aqueous solution or a higher osmolality than the first aqueous solution; or the second aqueous solution has an equal osmolality than the first aqueous solution or an equal osmolality than the first aqueous solution; or the second aqueous solution has a lower osmolality than the first aqueous solution or a lower osmolality than the first aqueous solution.

[0047] In some embodiments, the semi-permeable membrane is composed of amphiphilic molecules; preferably, the amphiphilic molecules are lipids or triblock copolymers.

[0048] In some embodiments, the semi-permeable membrane is a bilayer composed of amphiphilic molecules.

[0049] In some embodiments, the amphiphilic molecules are lipids.

[0050] In some embodiments, the lipids are one or more selected from the group consisting of fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, polyketides, phospholipids, glycolipids, and cholesterols.

[0051] In some embodiments, the lipids are one or more selected from the group consisting of glyceryl monooleate; 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC); 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC); palmitoyloleoyl phosphatidylcholine (POPC); l-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE); l-palmitoyl-2-oleoyl-phosphatidylethanolamine; l-palmitoyl-2-oleoyl phosphatidylglycerol (POPE / POPG) mixture; and mixtures thereof.

[0052] In some embodiments, the first compartment is provided by an aqueous droplet.

[0053] 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.

[0054] In some embodiments, the nanopore is selected from the group consisting of a protein nanopore, a DNA nanopore, or a solid nanopore

[0055] In some embodiments, the protein nanopore is one or more selected from the group consisting of: a-HL, ClyA, Phi29 connector protein, Aerolysin, MspA, OmpF, OmpG, FraC, HlyA, SheA, sp1, and variants thereof; and an ion channel.

[0056] In some embodiments, the protein nanopore is ClyA-RR or a-HL.

[0057] In some embodiments, the ionic species is one or more selected from the group consisting of: 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+ , OH - , Pb 2+ , Pd 2+ , Pt 2+ , Pt 4+ , Ru 3 + , Sb 3+ , Sc 3+ , Sn 2+ , Sr 2+ , Tb 3+ , Tl + , and Zn 2+ .

[0058] 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 acids, 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.

[0059] In some embodiments, the second aqueous solution comprises calcium chloride and optionally a buffer.

[0060] In some embodiments, the concentration of calcium chloride in the second aqueous solution is 0.01-6.76 M.

[0061] In some embodiments, the first aqueous solution comprises a chelator and optionally a buffer, wherein the chelator is capable of binding to the ionic species; preferably, the chelator is EDTA, BAPTA, EGTA, CyDTA, DTPA, EDDP, HIDA, IDA, NTA, NTPO, TTHA, CA, TA, GA, HEDTA, or DEG.

[0062] In some embodiments, the analyte is provided in the first aqueous solution or the second aqueous solution.

[0063] In some embodiments, the analyte is selected from the group consisting of small molecules, macromolecules, and biological macromolecules.

[0064] 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.

[0065] In another aspect of the application, there is provided an electrodeless nanopore array for identifying a plurality of analytes, the nanopore array comprising a plurality of systems in parallel and each system comprising:

[0066] (a) a first compartment having a first aqueous solution therein, wherein the first aqueous solution comprises a fluorescent reporter molecule capable of emitting fluorescence when bound to an ionic species;

[0067] (b) a second compartment having a second aqueous solution therein, wherein the second aqueous solution comprises an ionic species that specifically binds to the fluorescent reporter molecule; and

[0068] (c) a membrane separating the first compartment and the second compartment;

[0069] wherein in each system, the membrane between the first compartment and the second compartment has at least one inserted nanopore, such that the first compartment and the second compartment are connected through the nanopore in each system;

[0070] wherein in each system, there is a chemical gradient of the ionic species between the first compartment and the second compartment that can drive diffusion of the ionic species through the nanopore from the second compartment to the first compartment;

[0071] wherein the plurality of systems are configured such that the measured fluorescence of each system can be distinguished.

[0072] In some embodiments, in each system, the membrane between the first compartment and the second compartment system is a solid membrane.

[0073] In some embodiments, in each system, the membrane between the first compartment and the second compartment is a semi-permeable membrane.

[0074] In some embodiments, in each system, the second aqueous solution has a higher osmotic molarity by volume than the first aqueous solution or the second aqueous solution has a higher osmotic molarity by weight than the first aqueous solution; or the second aqueous solution has an osmotic molarity by volume equal to the first aqueous solution or the second aqueous solution has an osmotic molarity by weight equal to the first aqueous solution; or the second aqueous solution has a lower osmotic molarity by volume than the first aqueous solution or the second aqueous solution has a lower osmotic molarity by weight than the first aqueous solution.

[0075] In some embodiments, the semi-permeable membrane is composed of an amphiphilic molecule; preferably, the amphiphilic molecule is a lipid or a triblock copolymer.

[0076] In some embodiments, the semi-permeable membrane is a bilayer composed of an amphiphilic molecule.

[0077] In some embodiments, the amphiphilic molecule is a lipid.

[0078] In some embodiments, the lipid is one or more selected from the group consisting of a fatty acyl, a glycerolipid, a glycerophospholipid, a sphingolipid, a sterol lipid, an isoprenoid lipid, a saccharolipid, a polyketide, a phospholipid, a glycolipid, and a cholesterol.

[0079] In some embodiments, the lipid is one or more selected from the group consisting of glyceryl monooleate; 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC); 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC); palmitoyloleoyl phosphatidylcholine (POPC); l-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE); l-palmitoyl-2-oleoyl-phosphatidylethanolamine; l-palmitoyl-2-oleoyl phosphatidylglycerol (POPE / POPG) mixture; and mixtures thereof.

[0080] In some embodiments, the first compartment of the plurality of systems is separate from each other.

[0081] In some embodiments, the first compartment of each system is provided by an aqueous droplet.

[0082] In some embodiments, the second compartment of the plurality of systems is a single compartment.

[0083] 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.

[0084] In some embodiments, the second compartment of the plurality of systems is provided by a single hydrogel layer.

[0085] In some embodiments, in each system, the nanopore is selected from the group consisting of a protein nanopore, a DNA nanopore, or a solid nanopore.

[0086] In some embodiments, the protein nanopore is one or more selected from the group consisting of a-HL, ClyA, Phi29 connector protein, Aerolysin, MspA, OmpF, OmpG, FraC, HlyA, SheA, sp1, and variants thereof; and an ion channel.

[0087] In some embodiments, in each system, the protein nanopore is ClyA-RR or a-HL.

[0088] In some embodiments, in each system, wherein the ionic species is one or more selected from the group consisting of 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+ , OH - , Pb 2+ , Pd 2+ , Pt 2+ , Pt 4+ , Ru 3+ , Sb 3+ , Sc 3+ , Sn 2+ , Sr 2+ , Tb 3+ , Tl + , and Zn 2+ .

[0089] 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 acids, 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.

[0090] In some embodiments, in each system, the second aqueous solution comprises calcium chloride and optionally a buffer.

[0091] In some embodiments, in each system, the concentration of calcium chloride in the second aqueous solution is 0.01-6.76 M.

[0092] In some embodiments, in each system, the first aqueous solution comprises a chelator and optionally a buffer, wherein the chelator is capable of binding to the ionic species; preferably, the chelator is EDTA, BAPTA, EGTA, CyDTA, DTPA, EDDP, HIDA, IDA, NTA, NTPO, TTHA, CA, TA, GA, HEDTA, or DEG.

[0093] In some embodiments, the array further comprises a light source for illumination and a light sensor for detecting fluorescence; preferably, the light source is a laser, an LED, a halogen lamp, a xenon lamp; preferably, the light sensor is a CCD, a sCMOS sensor, a photodiode; more preferably, the light sensor is an EMCCD or an avalanche photodiode (APD).

[0094] In some embodiments, the array further comprises a device for total internal reflection fluorescence (TIRF), wide-field fluorescence microscopy, or confocal microscopy.

[0095] In some embodiments, in each system, the first aqueous solution or the second aqueous solution comprises the analyte.

[0096] In some embodiments, in each system, the analyte is selected from the group consisting of small molecules, large molecules, and biological macromolecules.

[0097] 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; more preferably, the RNA is miRNA, siRNA, or tRNA.

[0098] In some embodiments, different analytes are physically separated into various systems.

[0099] In some embodiments, the density of the systems in the nanopore array is 10-1000 per mm 2 ,

[0100] In some embodiments, the total area provided by the plurality of systems is 1-100 mm 2 .

[0101] In some embodiments, the number of the plurality of systems is 4-1,000,000; preferably, the number of the plurality of systems is 10-1000.

[0102] In another aspect of the application, there is provided a multiplex method for identifying a plurality of analytes, the method comprising:

[0103] (a) providing any one of the above nanopore arrays, wherein two or more analytes are provided in various systems of the nanopore array;

[0104] (b) applying a light signal capable of exciting the fluorescent reporter molecule contained in each of the first compartments to a region adjacent to the nanopore in a plurality of first compartments;

[0105] (c) measuring a plurality of fluorescent signals from the fluorescent reporter molecule contained in each system to identify a plurality of analytes.

[0106] In another aspect of the application, there is provided a method of producing an electrodeless nanopore array, the method comprising:

[0107] providing a plurality of aqueous droplets, wherein each of the aqueous droplets comprises a first aqueous solution comprising a protein nanopore, an analyte, and a fluorescent reporter molecule capable of emitting fluorescence when bound to an ionic species;

[0108] providing a hydrogel layer, wherein the hydrogel layer comprises the ionic species;

[0109] The plurality of aqueous droplets and the hydrogel layer are brought together in a hydrophobic medium comprising an amphiphilic molecule, such that a semipermeable membrane is formed between the aqueous droplets and the hydrogel layer.

[0110] In some embodiments, 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.

[0111] In some embodiments, the semipermeable membrane consists of an amphiphilic molecule; preferably, the amphiphilic molecule is a lipid or a triblock copolymer.

[0112] In some embodiments, the semipermeable membrane is a bilayer consisting of an amphiphilic molecule.

[0113] In some embodiments, the amphiphilic molecule is a lipid.

[0114] In some embodiments, the lipid is one or more selected from the group consisting of a fatty acyl, a glycerolipid, a glycerophospholipid, a sphingolipid, a sterol lipid, a prenol lipid, a saccharolipid, a polyketide, a phospholipid, a glycolipid, and a cholesterol.

[0115] In some embodiments, the lipid is one or more selected from the group consisting of monoglyceride oleate; 1,2-dioleoyl-sn-glycero-S-phosphocholine (DOPC); 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC); palmitoyloleoyl phosphatidylcholine (POPC); l-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE); l-palmitoyl-2-oleoyl-phosphatidylethanolamine; l-palmitoyl-2-oleoyl phosphatidylglycerol (POPE / POPG) mixture; and mixtures thereof.

[0116] In some embodiments, the hydrogel layer comprises 0.1-20% (w / v) agarose; preferably, the hydrogel layer comprises 2-5% (w / v) agarose.

[0117] In some embodiments, in each system, the nanopore is selected from the group consisting of a protein nanopore, a DNA nanopore, or a solid nanopore.

[0118] In some embodiments, the protein nanopore in each aqueous droplet is one or more selected from the group consisting of: a-HL, ClyA, Phi29 connector protein, Aerolysin, MspA, OmpF, OmpG, FraC, HlyA, SheA, sp1, and variants thereof; and an ion channel.

[0119] In some embodiments, the protein nanopore in each aqueous droplet is ClyA-RR or a-HL.

[0120] In some embodiments, the ionic species in the hydrogel layer is one or more selected from the group consisting of: 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+ , OH - , Pb 2+ , Pd 2+ , Pt 2+ , Pt 4+ , Ru 3+ , Sb 3+ , Sc 3+ , Sn 2+ , Sr 2+ , Tb 3+ , Tl + , and Zn 2+ .

[0121] 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 acids, 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.

[0122] In some embodiments, the hydrogel layer comprises calcium chloride and optionally a buffer.

[0123] In some embodiments, the concentration of calcium chloride in the hydrogel layer is 0.01-6.76 M.

[0124] In some embodiments, each aqueous droplet comprises a chelator and optionally a buffer, wherein the chelator is capable of binding to the ionic species; preferably, the chelator is EDTA, BAPTA, EGTA, CyDTA, DTPA, EDDP, HIDA, IDA, NTA, NTPO, TTHA, CA, TA, GA, HEDTA, or DEG.

[0125] In some embodiments, the analyte in each aqueous droplet is selected from the group consisting of small molecules, macromolecules, and biological macromolecules.

[0126] 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; more preferably, the RNA is miRNA, siRNA, or tRNA.

[0127] In some embodiments, different analytes are provided in various systems.

[0128] In some embodiments, the number of aqueous droplets is 4-1,000,000; preferably, the number of aqueous droplets is 10-1000.

[0129] In another aspect, the present application provides use of the above system for optical analyte analysis.

[0130] In another aspect, the present application provides use of the above nanopore array for optical analyte analysis.

[0131] In another aspect, the present application provides a kit for forming a nanopore array, the kit comprising:

[0132] A filled hydrogel comprising agarose, a buffer, and an ionic species capable of specifically binding to a fluorescent reporter molecule to cause it to emit fluorescence;

[0133] An aqueous solution comprising a chelating agent, the fluorescent reporter molecule capable of emitting fluorescence when bound to the ionic species, and a buffer; wherein the chelating agent is capable of binding to the ionic species;

[0134] A hydrophobic medium containing an amphiphilic molecule;

[0135] A solid support.

[0136] In some embodiments, the filled hydrogel has a higher osmolality than the aqueous solution or the filled hydrogel has a higher molality than the aqueous solution; or the filled hydrogel has an osmolality equal to the osmolality of the aqueous solution or the filled hydrogel has a molality equal to the molality of the aqueous solution; or the filled hydrogel has a lower osmolality than the aqueous solution or the filled hydrogel has a lower molality than the aqueous solution.

[0137] In some embodiments, the amphiphilic molecule is a lipid.

[0138] In some embodiments, the lipid is one or more selected from the group consisting of a fatty acyl, a glycerolipid, a glycerophospholipid, a sphingolipid, a sterol lipid, a prenol lipid, a saccharolipid, a polyketide, a phospholipid, a glycolipid, and a cholesterol.

[0139] In some embodiments, the lipid is one or more selected from the group consisting of: glycerol monooleate; 1,2-dioleoyl-sn-glycero-S-phosphocholine (DOPC); 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC); palmitoyloleoyl phosphatidylcholine (POPC); l-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE); l-palmitoyl-2-oleoyl-phosphatidylethanolamine; l-palmitoyl-2-oleoyl phosphatidylglycerol (POPE / POPG) mixture; and mixtures thereof.

[0140] In some embodiments, the aqueous solution further comprises a protein nanopore.

[0141] In some embodiments, the nanopore is selected from the group consisting of a protein nanopore, a DNA nanopore, or a solid nanopore.

[0142] In some embodiments, the protein nanopore is one or more selected from the group consisting of: a-HL, ClyA, Phi29 connector protein, Aerolysin, MspA, OmpF, OmpG, FraC, HlyA, SheA, sp1, and variants thereof; and an ion channel

[0143] In some embodiments, the protein nanopore is ClyA-RR or a-HL.

[0144] In some embodiments, the ionic species in the hydrogel layer is one or more selected from the group consisting of: 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+ OH - Pb 2+ Pd 2+ Pt 2+ Pt 4+ Ru 3+ Sb 3+ Sc 3+ Sn 2+ Sr 2+ Tb 3+ Tl + and Zn 2+ .

[0145] 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 acids, 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.

[0146] In some embodiments, the filled hydrogel comprises calcium chloride to provide Ca 2+ as the ionic species.

[0147] In some embodiments, the concentration of calcium chloride in the hydrogel layer is 0.01-6.76 M; preferably, the concentration of calcium chloride in the second aqueous solution is 0.15 M-6 M.

[0148] In some embodiments, the filled hydrogel comprises 2.5% agarose, 1.5 M CaCl2, and 10 mM HEPES, pH 7.0.

[0149] In some embodiments, the aqueous solution further comprises KCl.

[0150] In some embodiments, the aqueous solution can comprise 1.5 M KC1, 400 mM EDTA, 40 mM Fluo-8, 10 mM HEPES, pH 7.0.

[0151] In some embodiments, the hydrophobic medium containing an amphiphilic molecule can be a lipid oil comprising a dry film of 5 mg DPHPC lipids dissolved in a 2 mL mixture of hexadecane and silicone oil in a 1 : 1 volume ratio.

[0152] In some embodiments, the kit further comprises a coating hydrogel comprising agarose in water; preferably, the coating hydrogel can comprise 0.75% (w / v) agarose in water. BRIEF DESCRIPTION OF DRAWINGS

[0153] Figure 1 DiffusiOptoPhysiology and its application in TriM-β-CD sensing are shown. a-d, Schematic of ion transport through nanopores in different measurement platforms. a, During electrophysiological recordings, when a transmembrane potential is applied through a pair of Ag / AgCl electrodes, Cl - Electrophoretic movement through nanopores. b, In the absence of electrodes, although there are two directions of thermal movement of ions through the nanopore, there should be no net flow of ion transport according to the rules of electro-neutrality. c, During oSCRs, Ca 2+ ions are driven electrophoretically through the nanopore, building a steep Ca 2+ gradient. After binding to Fluo-8 on the cis side, the Fluo-8 / Ca 2+ complex near the pore emits strong fluorescence. d, During DiffusiOptoPhysiology, due to the thermal movement of ions, a shallow Ca 2+ gradient can be built near the pore. After binding to Fluo-8, the fluorescence emission is expected to be weaker than in c. e, Cross-sectional view of the spatial distribution of Fluo-8 / Ca 2+ complexes around the pore. Dotted box: magnified view of the immediate vicinity of the nanopore. f, Top left panel: corresponding image results from computer simulations. Top right panel: the simulated fluorescence intensity distribution follows a Gaussian profile. Bottom left panel: representative frame acquired from DOP recordings of a single WT a-HL nanopore. Bottom right panel: the corresponding fluorescence intensity distribution also follows a Gaussian profile. Scale bar, 4 pm. g, Single-molecule sensing of TriM-β-CD (75 mM) using a-HL nanopores during electrodeless oSCRs. Scale bar, 4 pm. h, Mean inter-event interval (1 / τ on ) and mean residence time (1 / τ off) versus TriM-β-CD concentration. Mean and standard deviation from three independent experiments (N=3) for each condition. i, τ obtained from electrophysiological recordings at DOP and +20 mV, respectively off and F P Statistics of the results. DOP recordings shown above were performed in conditions of 1.5 M KCl, 400 μΜ EDTA, 40 μΜ Fluo-8, 10 mM HEPES, pH 7.0 (cis side) and 0.75 M CaCl2, 10 mM HEPES, pH 7.0 (trans side). Electrophysiological recordings were performed in conditions of 1.5 M KCl, 10 mM HEPES, pH 7.0 on both sides of the membrane. TriM-β-CD was added at a final concentration of 75 mM on the cis side.

[0154] Figure 2 The geometry of the FEM model is shown. A sphere of 10 μιη in radius, filled with an electrolyte solution, is divided into two compartments (cis: upper, trans: lower) by a semi-permeable membrane of 10 nm in thickness. Only liquid, but not ions, is allowed to cross the membrane. In the center of the membrane, a single nanoscale cylindrical pore of varying diameter (2-8 nm) is placed, which is the only passage for liquid and ion transport between the two compartments. The boundary conditions on the cis side are set to have varying KCl concentrations (1-2.5 M), while the boundary conditions on the trans side are set to have varying CaCl2concentrations (0.5-1.5 M). All FEM simulations in this work were performed using this geometry.

[0155] Figure 3 A schematic of the setup is shown. a, Cross-sectional view of the electrodeless oSCR setup. When immersed in a lipid / oil environment (2.5 mg / ml DPHPC in a 1 : 1 volume ratio mixture of hexadecane / silicone oil), the aqueous droplet and the agarose matrix spontaneously form a droplet interface bilayer (DIB) when brought together

[21] . The aqueous droplet consists of 1-2.5 M KCl, 400 μΜ EDTA, 40 μΜ Fluo-8, 10 mM HEPES (pH 7.0) and has a biological nanopore. The agarose matrix 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, previously dissolved in the droplet, spontaneously inserts into the DIB and enables Ca2+transport from the trans to the cis side. 2+ thermodynamic diffusion into the droplet. Transported Ca2+ 2+ binds immediately to Fluo-8 in the droplet, which produces a fluorescent emission around the pore when imaged by total internal reflection fluorescence (TIRF) microscopy. b, Brightfield image of the DIB. The boundary of the DIB can be visually distinguished from the brightfield image.

[0156] Figure 4 Dextrans binding kinetics are shown. a-e, Representative current traces using applied potentials of +20, +40, +60, +80 and +100 mV, respectively. Trimethyl- -dextran (TriM- -CD) was added to the cis side to a final concentration of 4 mM. As the applied potential was increased, the frequency of event detection systematically decreased. This suggests that there can be an opposing electroosmotic flow in the nanopore that decreases the likelihood of TriM- -CD binding to the pore. f, 1 / τ on Plot as a function of applied voltage. 1 / τ on Statistics are based on three independent electrophysiological recordings (N = 3) with a duration of 90 s per condition. Electrophysiological recordings were performed across the membrane in 1.5 M KCl, 10 mM HEPES, pH 7.0. WT a-HL was added to the cis side.

[0157] Figure 5 Definition of signal and background during oSCR is shown. a, Representative image frame of a nanopore acquired directly from the electrodeless oSCR. b, 2D Gaussia fit of a Results. Parameters such as peak center (x c ,y c ), peak amplitude (A + z0) and full width at half maximum (FWHM) can be derived from the fit results (see Methods). c, Definition of signal and background according to FWHM. Briefly, the total pixel value within a circle of diameter 2 FWHM is defined as signal. The total pixel value within the outer ring between diameters 3 FWHM and 4 FWHM is defined as background. Image processing shown was performed automatically using MATLAB.

[0158] Figure 6 Demonstration of fluorescence trace normalization is shown. Due to the laser, power fluctuations, focal plane drift or nanopore motion, sometimes low-frequency fluctuations are observed in the raw fluorescence time traces. However, these fluctuations can be reduced by standard trace correction. a, Correction of fluorescence intensity. Sample traces corresponding to TriM- -CD sensing based on electrodeless oSCR were used as a demonstration Figure 1 ). See Figure 10 for definition of signal and background during electrodeless oSCR. Trace correction was performed according to the equation F cal = (F sig - F bkg ) / F bkg . F cal , F sig and F bkgRepresent corrected fluorescence intensity, raw fluorescence signal and raw fluorescence background, respectively. After normalization, low-frequency fluctuations observed in the raw fluorescence time trace are minimized. b, Normalized fluorescence time trace. For quantitative analysis, the fluorescence amplitude corresponding to the open-pore state is further normalized to 1.

[0159] Figure 7 Statistics of translocation events are shown. a, Representative fluorescence trace showing TriM-β-CD translocation through an a-HL pore. Event dwell times (t off ) and inter-event intervals (t on ) are defined with markers in the trace. b, Histogram of dwell times (t off ). The black line is a single-exponential fit to the histogram data. The time constant τ off is derived from the fit result. c, Histogram of blockage levels (%F b ). The peak x c is defined with the mean percentage blockage value F P . d, Histogram of inter-event intervals (t on ). The black line is a single-exponential fit to the histogram data. The time constant τ on is derived from the fit result.

[0160] Figure 8 The sensing performance through adjusting the osmotic enhancement during DOP recording is shown. a, Fluorescence traces obtained from DOP recording using different electrolyte combinations. DOP recording was performed using an a-HL nanopore. TriM-β-CD was added to the cis side at a final concentration of 15 mM. The inter-event interval τ on decreases when a larger osmotic gradient is established between the cis and trans sides. b, Osmotic pressure and 1 / τ onPlot of fluorescence intensity as a function of KCl concentration in the cis side. Mean and standard deviation from three independent experiments (N=3). c, SBR analysis of fluorescence imaging results at different KCl concentrations in the cis side. Top panel shows representative images acquired from DOP recordings. Images from left to right were obtained by DOP recordings with [KCl] of 1.0, 1.5, 2.0 and 2.5 M in the cis side. The value of SBR decreased as the KCl concentration in the cis side increased. Experiments in a-c were performed in the presence of 1 M-2.5 M KCl, 400 μΜ EDTA, 40 μΜ Fluo-8, 10 mM HEPES, pH 7.0 (in the cis side) and 0.75 M CaCl2, 10 mM HEPES, pH 7.0 (in the trans side). d, Plot of simulated fluorescence intensity as a function of KCl concentration in the cis side. Top panel shows the corresponding fluorescence intensity 2D profiles in the simulation, similar to the results obtained from the electrodeless oSCR in c. The simulation was performed in the presence of 1 M-2.5 M KCl (in the cis side) and 0.75 M CaCl2 (in the trans side). e, Cross-sectional plot of the spatial distribution of Fluo-8 in the simulation. The boundary conditions for the simulation were set to 1.0 M KCl (in the cis side) and 0.75 M CaCl2 (in the trans side). The continuous permeation flow from the cis side to the trans side resulted in an enriched distribution of Fluo-8 near the DIB.

[0161] Figure 9 Baseline comparison during TriM-β-CD sensing is shown. a, Representative fluorescence trace from DOP recordings, acquired in the presence of 2.5 M KCl, 400 μΜ EDTA, 40 μΜ Fluo-8, 15 mM TriM-β-CD, 10 mM HEPES, pH=7.0 (in the cis side) and 0.75 M CaCl2, 10 mM HEPES, pH=7.0 (in the trans side). b, Representative fluorescence trace from DOP recordings, acquired in the presence of 1 M KCl, 400 μΜ EDTA, 40 μΜ Fluo-8, 15 mM TriM-β-CD, 10 mM HEPES, pH=7.0 (in the cis side) and 0.75 M CaCl2, 10 mM HEPES, pH=7.0 (in the trans side). Both traces in a and b were recorded using a-HL nanopores. A reduction in thermal noise was observed when a permeation flow from the cis side to the trans side was present, which was a result of the enhanced DOP recording fluorescence intensity.

[0162] Figure 10FEM modeling showing Fluo-8 distribution. Fluo-8H placed in cis side is a cell impermeable molecule (AAT bioquest). The osmotic flow of water across the membrane leads to enrichment of Fluo-8 around the cis side of DIB. This phenomenon should help to enhance the SBR of nanopore fluorescence when the osmotic molarity of the electrolyte volume in the cis side is set lower than in the trans side. Fluo-8 concentration was simulated using FEM modeling (Methods). For all simulations, the concentrations of KCl, EDTA and Fluo-8 on the cis side boundary were set to 0.5-1.5 M, 400 μΜ and 40 μΜ, respectively. The CaCl2 concentration on the trans side boundary was kept constant at 0.75 M. These simulation parameters were set on the boundary of the simulation space, representing the steady state of the electrolyte buffer far from the nanopore. a, Cross-sectional view of the 3D distribution of Fluo-8 when the KCl concentration on the cis side boundary is 0.5 M. In this case, there is a strong osmotic flow from the cis to the trans side, leading to enrichment of Fluo-8 near the membrane. b, Cross-sectional view of the 3D distribution of Fluo-8 when the KCl concentration on the cis side boundary is 1 M. A decrease in the enrichment of Fluo-8 is observed. c, Cross-sectional view of the 3D distribution of Fluo-8 when the KCl concentration on the cis side boundary is 1.5 M. In this case, there is a weak osmotic flow from the trans to the cis side, leading to a decrease in the Fluo-8 concentration near the membrane.

[0163] Figure 11 SBR enhancement during DOP recording with increasing Ca 2+ flux. a, Imaging results (top) and corresponding 2D Gaussian fits (bottom) acquired from DOP recordings. The CaCl2 concentration in the trans side was increased while the KCl concentration in the cis side was adjusted accordingly, so that the osmotic molarity of the volume was kept iso-osmotic. The fluorescent spots correspond to Ca 2+ flux, which increased with increasing Ca 2+The increase in flow becomes brighter. Scale bar, 4 μm. b, FWHM and SBR of the fluorescence imaging signal under different electrolyte-osmolarity conditions (N = 12). c, Representative fluorescence traces show the translocation signal of PEG1500 through WT a-HL nanopores, as obtained by DOP recording. PEG1500 was added to the agarose matrix to a final concentration of 20 mM. A combination of 2.25 M KCl buffer in the cis side and 1.5 M CaCl2buffer in the trans side was used during DOP recording. d, Simulated total fluorescence intensity as a function of the weight-osmolarity, showing four different pore diameters of 2 nm, 4 nm, 6 nm, and 8 nm, respectively. The electrolyte concentration was kept iso-osmolar to avoid osmotic effects in this demonstration. e, Left panel: simultaneous DOP imaging of a-HL and ClyA-RR. ClyA-RR shows larger and brighter spots compared to WT a-HL in the same field of view due to larger channel conductance. Right panel: fluorescence intensity profiles along the vertical lines labeled by positions 1 and 2, respectively. The fluorescence intensity profiles fit a Gaussian distribution. Scale bar, 5 μm. f, FWHM and SBR of the fluorescence imaging signal of WT a-HL and ClyA-RR (N = 5). DOP recordings shown in e, f were performed under conditions of 1.5 M KCl, 400 μΜ EDTA, 40 μΜ Fluo-8, 10 mM HEPES, pH 7.0 (in the cis side) and 1.5 M CaCl2, 10 mM HEPES, pH 7.0 (in the trans side). g, Simulated osmotic flow through a virtual cylindrical pore with a diameter of 6 nm. The wider pore geometry results in a larger osmotic flow.

[0164] Figure 12Preparation and characterization of ClyA-RR is shown. a, Dodecameric ClyA-RR was characterized using blue native gel electrophoresis (4-15% polyacrylamide gradient gel). Lane M: precision plus protein standards (Bio-Rad); Lane 1 : ClyA-RR prepared using prokaryotic expression (Methods). Lane 2: ClyA-RR after addition of DDM to a final concentration of 0.25% (w / v). The gel shows that the monomers have self-assembled prior to addition of DDM, but we still use DDM for production to stabilize the dodecameric ClyA-RR. b, Continuous ClyA-RR membrane insertion observed during electrophysiological recordings. Measurements were made at a constant voltage of +50 mV. Dodecameric ClyA-RR nanopores were added on the cis side. c, Representative I-V curve of ClyA-RR nanopores. d, Histogram of open pore currents of ClyA-RR at +100 mV potential. Currents are centered at 1761.428 pA. Statistics of open pore currents are based on 20 independent electrophysiological recordings (N=20). All measurements (b-d) shown were performed under conditions of 1.5 M KCl, 10 mM HEPES, pH 7.0, on both sides of the membrane.

[0165] Figure 13 FEM modeling of osmotic flow in the pore is shown. Simulations were performed using different pore diameters under conditions of 1 M KCl, 400 μΜ EDTA, 40 μΜ Fluo-8 (in the cis side) and 1.5 M CaCl2(in the trans side). a, Plot of flow velocity at the center of the pore versus pore diameter. b-d, Cross-sectional views of simulated osmotic flow in different sized pores. From the simulations, it is clear that larger pore diameters result in a significant increase in flow velocity.

[0166] Figure 14shows dsDNA translocation through ClyA-RR nanopores. a, Schematic of dsDNA translocation through ClyA-RR during DOP recording. b, DOP imaging and corresponding fluorescence trace of ClyA-RR nanopores. No dsDNA was added in the droplet. c, DOP imaging and corresponding fluorescence trace of ClyA-RR nanopores when 2 mM dsDNA was added in the droplet. Continuous deep and long resident fluorescence blockades were clearly observed. Scale bar: 5 pm. d, dsDNA translocation through ClyA-RR nanopores was recorded electrophysiologically at +6 mV, +4 mV and +2 mV, respectively. Current blockades corresponding to dsDNA translocation events were still observed at a voltage as low as +2 mV. e, Histogram of dwell time of dsDNA translocation events. Fast dsDNA translocations could not be fully resolved due to the limited acquisition time of EM-CCD (30 ms). DOP recordings were performed in 1.5 M KCl, 400 mM EDTA, 40 mM Fluo-8, 10 mM HEPES, pH 7.0 (in the cis side) and 1.5 M CaCl2, 10 mM HEPES, pH 7.0 (in the trans side). Electrophysiological recordings were performed in 1.5 M KCl, 10 mM HEPES, pH 7.0 (in the cis side) and 1.5 M CaCl2, 10 mM HEPES, pH 7.0 (in the trans side). dsDNA was added in the cis side at a final concentration of 2 mM. Data from DOP recording (shown in olive) were acquired at a frame rate of 30 ms. Electrophysiological traces (shown in black) were recorded at a sampling rate of 25 kHz and a low-pass filter of 1 kHz.

[0167] Figure 15 shows statistics of dsDNA translocation through ClyA. a, Representative histogram of blockade level (%F b ) when dsDNA translocated through ClyA-RR pores. F P value was 0.610 ± 0.138 (center value ± FWHM). b-d, Representative histograms of blockade level (%I b ) when dsDNA translocated through ClyA-RR pores at +2 mV (b), +4 mV (c) and +6 mV (d). I P values were 0.786 ± 0.224, 0.605 ± 0.460 and 0.611 ± 0.357, respectively. The optical and electrical blockade levels were essentially the same. Both electrodeless oSCR and electrophysiological recordings were performed in 1.5 M KCl, 10 mM HEPES, pH 7.0 (in the cis side) and 1.5 M CaCl2, 10 mM HEPES, pH 7.0 (in the trans side). 2 mM 78 bp dsDNA was added in the cis side.

[0168] Figure 16 Multiplexed DOP recording in microchips is shown. a, Fingertip-sized device for DOP recording. b, Chip setup during DOP recording. The DIB in setup was excited with a 473 nm laser and imaged using total internal reflection fluorescence (TIRF) microscopy (Methods). c, Simultaneous imaging of WT a-HL and ClyA-RR nanopores in the same DIB. The two types of nanopores can be easily distinguished by the size and intensity of the fluorescent spots (yellow dashed circles: WT a-HL, red dashed circles: ClyA-RR). d, Schematic of a micro-DIB array for multiplexed electrodeless oSCR. e, Brightfield image of the mini-DIB array. f, Frames of a ClyA-RR nanopore inserted into a micro-DIB. The DIB is ~40 pm in diameter. The DIB was constructed in conditions of 1.5 M KCl, 400 mM EDTA, 40 mM Fluo-8, 10 mM HEPES, pH 7.0 (in the cis side) and 1.5 M KCl, 10 mM HEPES, pH 7.0 (in the trans side).

[0169] Figure 17 Electrodeless ssDNA sensing using a-HL nanopores is shown. (a) Representative DOP acquisition and derived fluorescence trace of a-HL nanopores. No ssDNA was added to the droplet. (b) Representative DOP acquisition and derived fluorescence trace of a-HL nanopores when oligo ssDNA was added to the droplet at a final concentration of 50 mM. Continuous short-residence fluorescence blockades were observed consistently and clearly. Scale bar in A, B: 5 pm. (c) Statistics of the blockade levels (%F b ) of the electrical resistance events. The mean value of %F was 0.289 ± 0.175 (center value ± FWHM). The DOP recording shown was performed in conditions of 3 M KCl, 400 mM EDTA, 40 mM Fluo-8, 10 mM HEPES, pH 7.0 (in the cis side) and 1.5 M CaCl2, 10 mM HEPES, pH 7.0 (in the trans side). The DNA sequence used in this assay: 5'-GATAGTGAGCCAAATTTAAA-3'.

[0170] Figure 18Electrodeless ssDNA sensing using ClyA-RR nanopores is shown. (a) DOP imaging of ClyA-RR nanopores and corresponding fluorescence trace. No ssDNA was added to the droplet. (b) DOP imaging of ClyA-RR nanopores and corresponding fluorescence trace when ssDNA-a (78 nt) was added to the droplet at a final concentration of 2 μΜ. A continuous deep and long residence fluorescence block was clearly observed. Scale bar in image inset: 5 μm. (c) DOP imaging of ClyA-RR nanopores and corresponding fluorescence trace when poly A 78 was added to the droplet at a final concentration of 50 μΜ. A continuous shallow and short residence fluorescence block was clearly observed. Scale bar: 5 μm. (d) Representative histogram of the block level (%F b ) when ssDNA-a interacted with ClyA-RR nanopores. The mean value of %F was 0.583 ± 0.014 (center value ± FWHM). (e) Representative histogram of the block level (%F 78 ) when poly A b interacted with ClyA-RR nanopores. The mean value of %F was 0.780 ± 0.006 (center value ± FWHM). DOP recordings were performed under conditions of 2.25 M KCl, 400 μΜ EDTA, 40 μΜ Fluo-8, 10 mM HEPES, pH 7.0 (in the cis side) and 1.5 M CaCl2, 10 mM HEPES, pH 7.0 (in the trans side).

[0171] Figure 19 A PMMA measurement device is shown. The PMMA device was fabricated as previously reported [ref]. (A) Bottom view of the DIB device used in the experiments. Scale bar: mm. (B) Front view of the DIB device used in the experiments. Scale bar: mm. The molten gel can be injected into the device along the green marked route. (C) 3D model of the DIB device. The gel inlet and outlet holes for the molten agarose are shown. The additional holes on the outlet side help to expel air bubbles during the filling process. Four independent droplet slots were designed for parallel measurements. Scale bar: 2 mm. (D) Photograph of the prepared device. The DIB setup uses 473 nm laser excitation and total internal reflection fluorescence (TIRF) imaging. Scale bar: 10 mm. DETAILED DESCRIPTION

[0172] The present invention is based on DiffusiOptoPhysiology (DOP), which simplifies the oSCR by dispensing with all electrical connections, optically monitors the diffusion of Ca 2+ and its indicator dye Fluo-8 through the nanopore sensor (DOP) and the fluorescence emission Figure 1d). Direct sensing of small molecules, macromolecules, and biological macromolecules was subsequently demonstrated by direct fluorescence readout. By dispensing with the need for electrode configuration, DOP enables parallel measurements from thousands of nanopores with highly accessible and biocompatible materials at a single-use cost of <$1. Thus, new concepts of clinical diagnostics can be developed using disposable chips equipped with nanotechnology sensors. Reduced technical barriers due to cost, facilities, and skills enable nanopore measurements by almost untrained researchers. Different research fields, such as high-throughput drug screening or basic research of ion channels, can thus benefit.

[0173] The present invention provides an electrodeless system for identifying an analyte, based on optical measurement of ion flow through a pore driven by a chemical gradient. The present invention also provides methods of using the system for identifying an analyte, including methods of identifying small molecules or DNA, such as dsDNA or ssDNA.

[0174] The electrodeless system includes two compartments separated by a membrane: a first compartment contains a first aqueous solution containing fluorescent reporter molecules, and a second compartment contains a second aqueous solution containing free ionic species. When bound to the ionic species, the fluorescent reporter molecules produce a specific fluorescence emission that can be distinguished by an optical sensor. The membrane between the first and second compartments has at least one nanopore, such that the first and second compartments are connected through the nanopore. The ionic species are driven by a chemical gradient to pass through the nanopore in the membrane from the second compartment into the first compartment, and bind to the fluorescent reporter molecules in the first compartment, causing the reporter molecules to emit fluorescence. Since the passage of ionic species is limited to the nanopore, this produces fluorescence in the region adjacent to the nanopore. The intensity of the fluorescence signal in the region adjacent to the pore depends on the rate of flux of ionic species into the first compartment. The fluorescence emission of the fluorescent reporter molecules can then be detected. When the nanopore is blocked or partially blocked by an analyte passing through the nanopore, the transport of ionic species through the nanopore is impeded, which is measured as a reduction in fluorescence adjacent to the nanopore compared to the fluorescence produced by unimpeded flow of ionic species through the pore. Depending on the size, shape, etc. of the analyte, different analytes impede the transport of ionic species through the nanopore to different extents, resulting in different degrees of fluorescence reduction. The reduction in fluorescence due to blocking or partial blocking of the pore is related to the degree to which the transport of ions through the pore is impeded, which in turn reflects information about the properties of the analyte. The magnitude of the reduction in fluorescence in the region adjacent to the nanopore can be used to identify the blocking analyte passing through the nanopore. The magnitude of the reduction in fluorescence can be characterized by event dwell time and percent block depth.

[0175] An analyte can be added to the first compartment or the second compartment. The analyte is driven across the nanopore, for example by a chemical gradient, from the first compartment into the second compartment or from the second compartment into the first compartment and blocks or partially blocks the nanopore, resulting in a decrease in fluorescence.

[0176] The method of identifying an analyte comprises:

[0177] (a) providing a system of the invention comprising an analyte in the first compartment or the second compartment;

[0178] (b) applying light capable of exciting the fluorescent reporter molecule to a region in the first compartment adjacent to the nanopore;

[0179] (c) measuring the fluorescent signal from the fluorescent reporter molecule to identify the analyte.

[0180] The ionic species binds to the fluorescent reporter molecule and can be any ionic species that causes a particular fluorescent emission of another molecule. Such ionic species are well known to those skilled in the art. In some embodiments, the ionic species includes, but is not limited to, 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+ , OH - , 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 can be one ionic species or a combination of two or more ionic species.

[0181] The term "fluorescent reporter molecule" can be any molecule that produces a specific fluorescent emission that can be distinguished by the optical sensor when bound to the ionic species as shown above. Such fluorescent reporter molecules are well known to those skilled in the art. In some embodiments, the fluorescent reporter molecule can be a calcium fluorescent probe, a sodium fluorescent probe, or a zinc fluorescent probe, which is a molecule, such as a small molecule, that can chelate calcium ions, sodium ions, or zinc ions, respectively. In some embodiments, the fluorescent reporter molecule includes, but is 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 acids, or BAPTA (1,2-bis(o- aminophenoxy)ethane-N,N,N',N'-tetraacetic acid). In some embodiments, the fluorescent reporter molecule includes, but is 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 can be one type of fluorescent reporter molecule or a combination of two or more types of fluorescent reporter molecules. The fluorescent reporter molecule can be membrane impermeable, such as bilayer impermeable.

[0182] In the systems and methods of the present application, the ionic species used should be capable of binding to the fluorescent reporter molecule used, such that the fluorescent reporter molecule can produce a specific fluorescent emission. 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.

[0183] A first aqueous solution can be present in the first compartment. A second aqueous solution can be present in the second compartment. The first compartment can be completely filled with the first aqueous solution. The first compartment can be incompletely filled with the first aqueous solution, and there can be some space or other matrix in addition to the first aqueous solution in the first compartment. At least the portion of the first compartment immediately adjacent to the membrane is filled with the first aqueous solution. The second compartment can be completely filled with the second aqueous solution. The second compartment can be incompletely filled with the second aqueous solution, and there can be some space or other matrix in addition to the second aqueous solution in the first compartment. At least the portion of the second compartment immediately adjacent to the membrane is filled with the second aqueous solution. The first and second compartments can independently be of any form or any shape. The form or shape of the first and second compartments can be the same or different. In some embodiments, the first and / or second compartments can or can not have a boundary layer. The boundary layer of the first and second compartments can each be fixed or variable. In some embodiments, the first compartment can be provided by an aqueous droplet. In some embodiments, the second compartment can be provided by a hydrogel layer.

[0184] The first aqueous solution includes a fluorescent reporter molecule. The second aqueous solution includes an ionic species. The second aqueous solution can include a salt to provide the ionic species, such as a calcium salt. The first and second aqueous solutions can independently include or not include other ingredients. The first aqueous solution can not include an ionic species capable of binding the fluorescent reporter molecule to cause it to emit fluorescence. The second aqueous solution can not include a fluorescent reporter molecule capable of binding the ionic species and emitting fluorescence.

[0185] The first aqueous solution can include a salt that is different from the salt in the second aqueous solution. The ions in the first aqueous solution should not bind to the fluorescent reporter molecule to cause it to emit fluorescence. The salt in the first aqueous solution can be any salt that does not bind to the fluorescent reporter molecule to cause it to emit fluorescence. In some embodiments, the first aqueous solution includes sodium chloride. In some embodiments, the first aqueous solution includes potassium chloride. In some embodiments, the second aqueous solution includes calcium chloride. The concentration of potassium chloride in the first aqueous solution can be about 0-3.4 M. In some embodiments, the concentration of potassium chloride is less than or equal to 3 M. In some embodiments, the concentration of potassium chloride is less than or equal to 0.75 M, less than or equal to 1.0 M, less than or equal to 1.5 M, less than or equal to 2.25 M, or less than or equal to 2.5 M. The concentration of calcium chloride in the second aqueous solution can be about 0.01-6.76 M. In some embodiments, the concentration of calcium chloride is greater than or equal to 0.15 M, greater than or equal to 0.5 M, greater than or equal to 0.75 M, greater than or equal to 1 M, or greater than or equal to 1.5 M, greater than or equal to 2 M, greater than or equal to 3 M, greater than or equal to 4 M, greater than or equal to 5 M, greater than or equal to 6 M. In some embodiments, the concentration of calcium chloride is less than or equal to 6 M,

[0186] The inventors found that increasing the concentration of the ionic species can improve the sensing signal by increasing the transmembrane chemical gradient of the ionic species, thereby causing more ionic species to flow 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 can further include a chelator for competitive binding of ions such as Ca 2+ ) whereby the fluorescence is attenuated as it moves away from the center of the nanopore due to the competitive binding. 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 can include a buffer to control the pH, for example, Bis-tris, Tris, Hepes, sodium phosphate, and / or potassium phosphate. In some embodiments, the first aqueous solution can include a potassium chloride buffer (e.g., 10 mM HEPES, pH 7.0 and KCL) and the second aqueous solution can include a calcium chloride buffer (e.g., 10 mM HEPES, pH 7.0 and CaCl2). In some embodiments, the first aqueous solution can include 1.0 M KCl, 400 mM EDTA, 10 mM HEPES, pH 7.0; 1.5 M KCl, 400 mM EDTA, 10 mM HEPES, pH 7.0; 2.25 M KCl, 400 mM EDTA, 10 mM HEPES, pH 7.0; or 2.5 M KCl, 400 mM EDTA, 10 mM HEPES, pH 7.0; or 2.5 M KCl, 400 mM EDTA, 10 mM HEPES, pH 7.0. In some embodiments, the second aqueous solution can include 0.5 M CaCl2, 10 mM HEPES, pH 7.0; 0.75 M CaCl2, 10 mM HEPES, pH 7.0; 1 M CaCl2, 10 mM HEPES, pH 7.0; or 1.5 M CaCl2, 10 mM HEPES, pH 7.0. Salts can be included in the first aqueous solution or the second aqueous solution for other reasons, for example, to stabilize proteins, control binding components, control osmolarity / tonicity, and / or activate fluorescent probes.

[0187] The first aqueous solution can include an analyte. The analyte is driven by a chemical gradient through a nanopore in the membrane from the first compartment into the second compartment and blocks or partially blocks the nanopore, resulting in a decrease in fluorescence from the fluorescent reporter molecule. The analyte can also be included in the second aqueous solution, where the analyte is driven by a chemical gradient through a nanopore in the membrane from the second compartment into the first compartment and blocks or partially blocks the nanopore, resulting in a decrease in fluorescence from the fluorescent reporter molecule.

[0188] In some cases, the first compartment can be provided by an aqueous droplet. The aqueous droplet can include or consist of the first aqueous solution. In some cases, the second compartment can be provided by a hydrogel layer, such as a hydrogel layer including an agarose matrix. The hydrogel can include the second aqueous solution. When the aqueous droplet and the hydrogel layer are brought together in a hydrophobic medium including amphiphilic molecules that are selectively permeable to water molecules, the two can spontaneously form a droplet interface bilayer (DIB) consisting of the amphiphilic molecules. The protein nanopore can be provided in the aqueous droplet or the hydrogel, such that the protein nanopore can spontaneously insert into the DIB when the DIB forms. The aqueous droplet can also include an analyte.

[0189] The matrix of the hydrogel layer can include or consist of a hydrophilic polymer. The matrix of the hydrogel layer can include or consist of a substantially transparent hydrophilic polymer. The matrix of the hydrogel layer can include or consist of agarose. Other hydrogel materials can be suitable, such as polyacrylamide, cross-linked polyethylene glycol, or nitrocellulose. The hydrogel layer can include 0.1-20% (w / v) agarose. In some embodiments, the hydrogel layer can include less than 5% (w / v) agarose, less than 4% (w / v) agarose, or about 3% (w / v) agarose. The hydrogel can include greater than 1% (w / v) agarose, greater than 2% (w / v) agarose. The hydrogel can include about 2% to about 4% agarose. The hydrogel can include about 2.5% (w / v) to about 3.5% (w / v) agarose. The hydrogel layer can include an analyte.

[0190] The membrane separating the first compartment and the second compartment can be any material capable of supporting a nanopore. The membrane can be a natural membrane, a synthetic membrane, or a man-made membrane. The membrane can be a solid membrane. The membrane can include or consist of a solid matrix, such as SiNx, glass, silicon dioxide, molybdenum disulfide, graphene, aluminum oxide, or CNT (carbon nanotube).

[0191] The membrane can be a semipermeable membrane. The semipermeable membrane can be selectively permeable to water molecules. Ionic species, fluorescent reporter molecules, analytes, and the like are impermeable to the semipermeable membrane, and thus, their passage is limited to the nanopore. Such semipermeable membranes and methods of their production are well known to those of skill in the art. The semipermeable membrane can include or consist of amphiphilic molecules that are selectively permeable to water molecules.

[0192] The semi-permeable membrane can be composed of amphiphilic molecules. The amphiphilic molecules can be lipids or polymers, such as block copolymers. The membrane can be a monolayer or a bilayer, for example, it can comprise or consist of amphiphilic molecules. Examples include a monolayer comprising or consisting of a polymer, such as a block copolymer, and a bilayer comprising or consisting of lipids. The bilayer can be a lipid bilayer. The bilayer can be artificial, for example, non-natural. The bilayer can not be a cell bilayer. The bilayer can not be a cell patch-clamp bilayer. The skilled person will appreciate that there are a number of methods of providing a bilayer. The bilayer can be provided by a droplet hydrogel bilayer (DHB) method, for example, as provided in WO2009024775, the contents of which are incorporated herein by reference.

[0193] In some embodiments, the semi-permeable membrane can be provided by a first compartment-second compartment interaction in a hydrophobic medium containing amphiphilic molecules, such as lipids or block copolymers. In some embodiments, the semi-permeable membrane can be provided by immersing the first and second compartments in a hydrophobic medium containing amphiphilic molecules, and bringing the first and second compartments together such that the amphiphilic molecules form a semi-permeable membrane. As a result, a semi-permeable membrane composed of amphiphilic molecules will spontaneously form between the first and second compartments. In some embodiments, a protein nanopore can be provided in the first or second compartment, which can spontaneously insert into the bilayer when the membrane composed of amphiphilic molecules spontaneously forms. In some embodiments, the semi-permeable membrane can be provided by immersing the first and second compartments, either of which has a protein nanopore in it, in a hydrophobic medium containing amphiphilic molecules. As a result, a semi-permeable membrane composed of amphiphilic molecules will spontaneously form between the first and second compartments, and the protein nanopore can spontaneously insert into the bilayer.

[0194] In some embodiments, a bilayer can be provided by contacting a first compartment having a monolayer of amphiphilic molecules with a second compartment having a monolayer of amphiphilic molecules to spontaneously form a bilayer. In some embodiments, a bilayer can be provided by immersing the first and second compartments, either of which has a protein nanopore in it, in a hydrophobic medium containing amphiphilic molecules, thereby forming a monolayer of amphiphilic molecules on the surface of the first and second compartments, and then bringing the first and second compartments together such that the monolayer of amphiphilic molecules forms a bilayer. As a result, a bilayer composed of amphiphilic molecules will spontaneously form between the first and second compartments, and the protein nanopore can spontaneously insert into the bilayer.

[0195] Thus, in one aspect of the application, there is provided a method of producing the system described above, comprising:

[0196] a first compartment having a first aqueous solution therein, wherein the first aqueous solution comprises a fluorescent reporter molecule capable of emitting fluorescence when bound to an ionic species;

[0197] a second compartment having a second aqueous solution therein, wherein the second aqueous solution comprises an ionic species that specifically binds to the fluorescent reporter molecule;

[0198] bringing the first compartment and the second compartment together in a hydrophobic medium containing an amphiphilic molecule, such that a semi-permeable membrane with an inserted nanopore is formed between the first compartment and the second compartment;

[0199] wherein a protein nanopore is provided in the first aqueous solution or the second aqueous solution

[0200] wherein a semi-permeable membrane consisting of amphiphilic molecules will spontaneously form between the first compartment and the second compartment, and a protein nanopore can spontaneously insert into the semi-permeable membrane;

[0201] The first compartment, the second compartment, the fluorescent reporter molecule, the ionic species, the protein nanopore, the semi-permeable membrane, the bilayer, the hydrophobic medium, the amphiphilic molecule, the analyte, and other features mentioned herein are as described in the context of the present specification.

[0202] In the present invention, an analyte can be provided in the first aqueous solution or the second aqueous solution prior to semi-permeable membrane formation. An analyte can also be added to the first aqueous solution or the second aqueous solution after semi-permeable membrane formation and prior to detection initiation.

[0203] In some embodiments, the amphiphilic molecule can selectively permeate water molecules. The amphiphilic molecule used in any method of the present invention can be a polymeric or lipid molecule, in particular, a surfactant molecule can be used. The lipid molecule can be selected from the group comprising fatty acyl, glycerolipid, glycerophospholipid, sphingolipid, sterol lipid, prenol lipid, glycolipid, polyketide, phospholipid, glycolipid, and cholesterol. The lipid is any one of the group comprising monoglyceride oleate; 1,2-dioleoyl-sn-glycero-S-phosphocholine (DOPC); 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC); palmitoyloleoyl phosphatidylcholine (POPC); l-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE); l-palmitoyl-2-oleoyl-phosphatidylethanolamine; l-palmitoyl-2-oleoyl phosphatidylglycerol (POPE / POPG) mixture; and mixtures thereof.

[0204] The polymer can be a block copolymer, such as a triblock copolymer, capable of forming a semi-permeable membrane, for example as provided in Discher, D. E. & Ahmed, F. Polymersomes. Annu. Rev. Biomed. Eng. 8, 323-341 (2006); Nardin, C, Winterhalter, M. & Meier, W. Giant free-standing ABA triblock copolymer membranes. Langmuir 16, 7708-7712 (2000); Meier, W., Nardin, C, & Winterhalter, M. Reconstitution of channel proteins in (polymerized) ABA triblock copolymer membranes. Angew. Chem. Int. Ed. 39, 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).

[0205] The hydrophobic medium can comprise an oil. In some embodiments, the hydrophobic medium can be an oil. The hydrophobic medium comprising an amphiphilic molecule can comprise or consist of a lipid-in-oil. The oil can be a hydrocarbon, which can be branched or unbranched, and which can be substituted or unsubstituted. For example, the hydrocarbon can 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 a n-alkane, such as a C10 to C17 n-alkane, for example n-hexadecane (C16). In some embodiments, the hydrophobic medium can be an oil, for example a mixture of hexadecane and silicone oil. In some embodiments, the oil can comprise a 1 : 1 (v:v) mixture of hexadecane and silicone oil AR20 (Sigma-Aldrich).

[0206] Alternatively, other bilayer formation methods can be used. For example, the bilayer can be provided by any of the following techniques known to those skilled in the art: patch-clamp, e.g. optical patch-clamp; black lipid membrane (BLM); also known as painted BLM; supported lipid bilayer (SLB); and tethered bilayer lipid membrane (t-BLM). The bilayer can be formed across a pore according to WO2008102121 (the contents of which are incorporated herein by reference). The bilayer can be formed at the interface of a droplet with a droplet according to WO2014064444 (the contents of which are incorporated herein by reference).

[0207] In the present invention, the term "nanopore" refers to a channel having an opening at its narrowest point, which has a diameter that allows passage of an analyte through the opening. The nanopore is narrow enough that blockage of the channel by the analyte can be detected by a change in a particular signal, such as a fluorescent signal.

[0208] The size of the nanopore in the membrane can vary depending on the intended application of the system, but must be large enough to allow passage of ions of the ionic species used in the present invention. Preferably, the nanopore is also small enough to prevent passage of the fluorescent reporter molecule. The nanopore can be large enough to allow passage of the analyte.

[0209] Whether the membrane is a solid membrane or a semi-permeable membrane, the nanopore can be a solid nanopore, a protein nanopore of a DNA nanopore. The nanopore can be natural, e.g. derived from a biological organism, or the nanopore can be synthetic. The nanopore can be produced recombinantly. The nanopore can be a biomolecule, such as a protein nanopore (which can also be referred to as a nanopore-forming protein). In some cases, the nanopore can be formed from a protein, which can be referred to as a protein nanopore or a nanopore-forming protein. The protein nanopore used in the present invention preferably does not have spontaneous gating activity and / or preferably remains open in the absence of the analyte. The protein nanopore used in the present invention can be any one. Examples of protein nanopores or nanopore-forming proteins include a-HL, ClyA, Phi29 connector protein, aerolysin, MspA, OmpF, OmpG, FraC, HlyA, SheA, sp1 or a variant thereof. In some embodiments, the one or more nanopores is ClyA-RR

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

[0210] In the present invention, the term "a-HL" can also be referred to as a-hemolysin, and can be selected from the group consisting of wild-type a-hemolysin, mutant a-hemolysin, wild-type a-hemolysin paralog or homolog hemolysin, and mutant a-hemolysin paralog or homolog hemolysin. In some embodiments, the a-hemolysin can be wild-type a-hemolysin. The a-hemolysin useful in the present invention should be capable of forming a nanopore. In some embodiments, the a-HL is a heptamer.

[0211] In the present invention, the term "ClyA" can be selected from the group consisting of wild-type ClyA, mutant ClyA, wild-type ClyA paralog or homolog ClyA, and mutant ClyA paralog or homolog ClyA. In some embodiments, the ClyA can be wild-type a-hemolysin. In some embodiments, the ClyA can be mutant ClyA. The preferred mutant ClyA is ClyA-RR. The ClyA useful in the present invention should be capable of forming a nanopore. In some embodiments, the ClyA or ClyA-RR is a dodecamer.

[0212] The sequences of protein nanopores (e.g. a-HL and ClyA or mutants thereof) are known to those skilled in the art. Methods of making protein nanopores (e.g. a-HL or ClyA or mutants thereof) are known to those skilled in the art, for example can be made by prokaryotic expression and are readily purified by gel electrophoresis or chromatography. Protein nanopores can be formed by self-assembly of several protein monomers, for example dodecameric ClyA, dodecameric ClyA-RR or heptameric a-HL. In some cases, protein nanopores can self-assemble into the semi-permeable membrane.

[0213] The nanopore can be a solid-state nanopore, for example comprising a synthetic material such as silicon nitride or graphene. Solid-state nanopores are typically nanoscale pores formed in a synthetic membrane such as SiNx or Si02. Solid-state nanopores can be produced by focused ion or electron beams, whereby the size of the pore can be adjusted. The nanopore can be a hybrid nanopore comprising a pore-forming protein formed in a synthetic material.

[0214] Methods of forming nanopores in a membrane are well known to those skilled in the art, for example by adding nanopore molecules to the semi-permeable membrane after formation of the semi-permeable membrane or during formation of the semi-permeable membrane. In some embodiments, the protein nanopore can be provided in the first compartment or the second compartment, and can spontaneously insert into the semi-permeable membrane when the membrane comprised of the amphiphilic molecules spontaneously forms.

[0215] The analyte is not limited to a particular molecule and can be any molecule that is capable of blocking or partially blocking the nanopore when passing through the nanopore. The analyte can include, but is not limited to, a small molecule, a large molecule, or a biological macromolecule. A small molecule refers to a molecule or ion that is low in molecular weight, small in size, much smaller than the pore size of the nanopore, and easily passes through the nanopore. The small molecule can include, but is not limited to, a compound, a drug, a sugar, an ion, a neurotransmitter, an amino acid, or a nucleotide. A large molecule refers to a very large molecule, usually composed of thousands or more atoms. The large molecule can include, but is not limited to, a biological polymer such as a nucleic acid, a protein, a carbohydrate, or a lipid; a large non-polymeric molecule such as a lipid or a macrocycle; or a synthetic macromolecule. An example of a large molecule is a biological macromolecule, which includes, but is not limited to, a polypeptide, a polysaccharide, or a polynucleotide, for example, DNA (including ssDNA or dsDNA) or RNA (including miRNA, siRNA, or tRNA). The length of the DNA (such as ssDNA or dsDNA) can be 10-1000 nt. The length of the DNA (such as ssDNA or dsDNA) can be greater than 15 nt, greater than 20 nt, greater than 30 nt, greater than 40 nt, greater than 50 nt, greater than 60 nt, greater than 70 nt, greater than 80 nt, greater than 90 nt, or greater than 100 nt. The length of the DNA (such as ssDNA or dsDNA) can be less than 500 nt, less than 4000 nt, less than 300 nt, less than 200 nt. The length of the RNA (such as miRNA, siRNA, or tRNA) can be greater than 15 nt, greater than 20 nt, greater than 30 nt, greater than 40 nt, greater than 50 nt, greater than 60 nt, greater than 70 nt, greater than 80 nt, greater than 90 nt, or greater than 100 nt. The length of the RNA (such as miRNA, siRNA, or tRNA) can be less than 500 nt, less than 4000 nt, less than 300 nt, less than 200 nt.

[0216] The analyte can be placed in the first aqueous solution or the second aqueous solution. The analyte can be included in the first aqueous solution or the second aqueous solution when the first aqueous solution or the second aqueous solution is prepared, i.e., the analyte can be prepared into the first aqueous solution or the second aqueous solution together with other intended ingredients. The analyte can be added to the first aqueous solution or the second aqueous solution after the system is ready and detection is started.

[0217] In the present disclosure, the term "identifying" includes detecting or analyzing identity, for example, the type of analyte or obtaining structural information of the analyte, for example, the structure of a polymer, or the structure of a polynucleotide or a polypeptide, such as the primary structure or the secondary structure of a polynucleotide.

[0218] Blocking analytes that pass through the nanopore can be identified by the magnitude of the reduction in fluorescence in the region adjacent to the nanopore. Based on the disclosure herein, one of skill in the art knows how to identify an analyte based on the magnitude of the reduction in fluorescence, e.g., which can be characterized by event dwell time and percent block depth. Event dwell time is the residence time of the analyte occupying the nanopore. Percent block depth is defined as b / I o where I b and I o represent the absolute blocked current and open pore current, respectively. For example, the fluorescence emission caused by the blockage of ion flow can be measured and compared to the fluorescence emission of a reference substance under the same detection conditions to determine whether the analyte and the reference are the same. The event dwell time and / or the percent block depth of the fluorescence emission can be the comparison term.

[0219] Fluorescence detection can include microscopy or spectroscopy of the membrane and membrane regions. Fluorescence detection can include the use of total internal reflection fluorescence (TIRF), wide-field fluorescence microscopy or confocal microscopy. Fluorescence detection can include the use of HiLo microscopy, for example as provided by Tokunaga et al (2008. Highly inclined thin illumination enables clear single-molecule imaging in cells. Nat Meth 5, 159-161). Fluorescence detection can include the use of other grazing incidence illumination techniques. Any suitable means of fluorescence detection can be used to detect fluorescence signals / emissions in the membrane and membrane regions immediately adjacent to the nanopore. Fluorescence detection can include the use of surface plasmon resonance. Fluorescence detection can include the use of super-resolution microscopy, for example deterministic super-resolution, including STED, GSD, RESOLFT or SSIM; or stochastical super-resolution, including SOFI, or single molecule localisation methods (SMLM) such as SPDM, SPDMphymod, PALM, FPALM, STORM or dSTORM. Fluorescence detection can include the use of epifluorescence microscopy, confocal laser scanning microscopy (LSM) or total internal reflection fluorescence (TIRF) microscopy. Fluorescence detection can include the use of fluorescence correlation spectroscopy (FCS). Image correlation spectroscopy (ICS) can be used to calculate the spatial correlation function of image fluorescence intensity fluctuations, which can be obtained by confocal or two-photon LSM or TIRF microscopy. Fluorescence detection techniques can be described in Ana J. Garcia-Saez, Petra Schwille. Surface analysis of membrane dynamics Biochimica et Biophysica Acta 1798 (2010) 766-776, the contents of which are incorporated by reference.

[0220] Detection of fluorescence emissions from the fluorescent reporter molecule can require a light source and a light sensor. The light source and the light sensor can be contained 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.

[0221] Accordingly, in some embodiments, the system comprises a light source capable of illuminating the membrane region adjacent to the nanopore. In some embodiments, the light source provides light in a specific wavelength range. In some embodiments, the light source can be a laser, an LED, a halogen lamp, or a xenon lamp. Those skilled in the art know how to use a light source to irradiate a fluorescent reporter molecule.

[0222] In some embodiments, the system comprises a light sensor capable of detecting the optical signal in the membrane region adjacent to the nanopore. The light sensor can be a light sensitive device sensitive to weak light (i.e. fluorescence), such as a charge-coupled device (CCD), an electron-multiplying CCD (EMCCD), a sCMOS sensor, or a photodiode, such as an avalanche photodiode (APD). Fast light sensitive devices are preferred. Preferably, the light sensor is an EMCCD or an avalanche photodiode (APD).

[0223] The light sensor can also be a microscopy imaging system, a photomultiplier tube, or a light sensor that can detect fluorescence using the above-mentioned fluorescence detection techniques. In some embodiments, a total internal reflection fluorescence (TIRF) imaging system, such as total internal reflection fluorescence microscopy (TIRFM), can be used to detect and / or record the optical signal in the membrane region adjacent to the nanopore. In some embodiments, a wide-field fluorescence imaging system or a confocal imaging system can be used to detect and / or record the optical signal in the membrane region adjacent to the nanopore. Those skilled in the art know how to use a light sensor to detect the fluorescence emission from a fluorescent reporter molecule.

[0224] In some embodiments, the light sensor and the light sensor can be a single device. Some fluorescence detection devices can also be used for illumination. For example, TIRFM can be used for illumination and imaging.

[0225] The inventors found that the osmotic flow between the first compartment and the second compartment is advantageous for further improving the fluorescence detection of the analyte by the nanopore. The difference in the osmotic molarity / weight osmotic molarity between the first compartment and the second compartment can drive the directional flow of water carrying ions and analytes through the biological nanopore inserted in the membrane. Therefore, by introducing this asymmetry, the translocation efficiency of the analyte should be improved. In addition, the fluorescence signal can be amplified by the osmotic flow between the first compartment and the second compartment. When the osmotic molarity (or weight osmotic molarity) of the second aqueous solution is higher than that of the first aqueous solution, due to the impermeability of the fluorescent reporter molecule to the semi-permeable membrane, the osmotic flow of water across the membrane leads to the enrichment of the fluorescent reporter molecule around the membrane in the first compartment, thus the fluorescence intensity is enhanced.

[0226] Thus, while in some embodiments the methods of the application can be practiced in an environment where the first aqueous solution and the second aqueous solution are maintained iso-osmotic or where the osmolality (or the onco-osmolarity) of the second aqueous solution is lower than the osmolality (or the onco-osmolarity) of the first aqueous solution, in some embodiments the osmolality (or the onco-osmolarity) of the second aqueous solution can be higher or lower than the osmolality (or the onco-osmolarity) of the first aqueous solution.

[0227] Both osmolality and onco-osmolarity are defined in terms of osmole. Osmole is a unit of measure that describes the number of moles of a compound that contribute to the osmotic pressure (i.e., the hydrostatic pressure that results from a concentration gradient across a surface on both sides, such as across a semipermeable membrane) of a chemical solution. Osmolality is defined as the number of osmole of solute per volume of solution. It is usually expressed in osmol / L. Onco-osmolarity is very similar, but it is defined as the number of osmole of solute per kilogram of pure solvent, and is usually expressed in osmol / kg. For example, a 1 mol / L NaCl solution corresponds to an osmolality of 2 osmol / L. The NaCl salt particles dissociate completely in water into two separate particles: Na + ions and Cl - ions. Thus, each mole of NaCl becomes two moles in solution, one mole of Na + and one mole of Cl - . Similarly, a 1 mol / L CaCl2solution produces a solution of 3 osmol / L (Ca 2+ and 2 Cl - ). Those skilled in the art are well aware of how to determine the osmolality or the onco-osmolarity of a solution.

[0228] The osmolarity of the second aqueous solution can be increased by increasing the concentration of the ionic species or by additionally adding other solutes that can increase the osmolarity / osmolality of the first aqueous solution. Preferably, the osmolarity / osmolality of the second aqueous solution is increased by increasing the concentration of the ionic species because increasing the concentration of the ionic species can improve the sensing signal. The osmolarity / osmolality of the first aqueous solution can be decreased by decreasing the concentration of solutes that can contribute to osmotic pressure or even by not containing salt in the first aqueous solution. In some embodiments, the osmolarity 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 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 greater than the osmolarity of the first aqueous solution.In some embodiments, the second aqueous solution has a weight osmolality that 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 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 than the weight osmolality of the first aqueous solution.

[0229] By measuring the fluorescence emitted from the fluorescent reporter molecule, the present application is able to record the flow through many parallel nanopores without the need for an expensive array of electrodes. The measured fluorescence can be separated into multiple fluorescence traces for each nanopore, which can be applied to situations requiring high-throughput screening, such as nanopore arrays.

[0230] In another aspect of the present application, an electrodeless nanopore array is provided. The nanopore array comprises a plurality of the present systems in parallel, each system being as described above. The nanopore array can be used to identify a plurality of analytes in parallel. The nanopore array of the present application can be used without electrodes, thereby reducing the size of the device and saving cost.

[0231] The plurality of systems are configured such that the measured fluorescence of each system can be distinguished. At least a portion of the plurality of systems are separated from one another such that each system can be used independently to detect an analyte therein and the measured fluorescence of each system can be distinguished. In some embodiments, at least a first compartment of the plurality of systems are separated from one another. In some embodiments, a second compartment of the plurality of systems are separated or not separated from one another. The plurality of systems can be the same or different.

[0232] The density of the systems in the array of nanopores can be up to 10 / mm 2 up to 50 / mm 2 up to 100 / mm 2 up to 200 / mm 2 up to 300 / mm 2 up to 400 / mm 2 up to 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 higher.

[0233] The total area provided by the plurality of systems can be up to 1 mm 2 up to 2 mm 2 up to 5 mm 2 up to 10 mm 2 up to 15 mm 2 up to 20 mm 2 up to 25 mm 2 up to 30 mm 2 up to 35 mm 2 up to 40 mm 2 up to 45 mm 2 up to 50 mm 2 up to 55 mm 2 up to 60 mm 2 up to 65 mm 2 up to 70 mm 2 up to 75 mm 2 up to 80 mm 2 up to 85 mm 2 up to 90 mm 2 up to 95 mm 2 up to 100 mm 2 or higher.

[0234] A plurality of analytes can be provided in two or more or each system of the array, thereby physically separating the plurality of analytes into the various systems. The plurality of analytes can be the same or different, or can be partially the same or partially different. In some embodiments, at least two of the plurality of analytes can be different. In some embodiments, the same analyte can be provided in two or more or each system of the array. In some embodiments, different analytes can be provided in two or more or each system of the array. In some embodiments, different analytes can be provided in different systems, respectively. The plurality of analytes pass in parallel through different nanopores, resulting in a reduction in fluorescence of the region adjacent to each nanopore, each nanopore can be separated into a plurality of fluorescence traces, thereby determining the identity of each analyte.

[0235] Such a nanopore array can be used in a multiplexed method of identifying a plurality of analytes, the method comprising:

[0236] (a) providing a nanopore array of the invention comprising a plurality of analytes, wherein two or more analytes are provided in various systems of the nanopore array;

[0237] (b) applying light capable of exciting a fluorescent reporter molecule contained in each first compartment to the region adjacent to the nanopore in the plurality of first compartments;

[0238] (c) measuring a plurality of fluorescence signals from the fluorescent reporter molecule contained in each first compartment to identify the plurality of analytes.

[0239] The first compartments, second compartments, fluorescent reporter molecules, ionic species, protein nanopores, membranes, bilayers, hydrophobic media, amphiphilic molecules, analytes, and other features mentioned herein are as described in the context of this specification.

[0240] In the nanopore array, different systems can comprise the same or different fluorescent reporter molecules and different ionic species. For ease of detection, preferably, different systems comprise the same fluorescent reporter molecule and different ionic species.

[0241] In the nanopore array, different systems can comprise the same or different nanopores. For ease of detection, preferably, different systems comprise the same nanopore.

[0242] In some embodiments, the first compartments of different systems are separated from each other and the second compartments of different systems are not separated from each other. In some embodiments, a nanopore array can be provided by placing a plurality of first compartments with the second compartments in a hydrophobic medium comprising amphiphilic molecules that are selectively permeable to water molecules, wherein at least a protein nanopore is included in each first compartment. A semipermeable membrane composed of amphiphilic molecules will spontaneously form between each first compartment and the second compartment, and the protein nanopore can spontaneously insert into the semipermeable membrane. In some embodiments, each first compartment includes a protein nanopore, a fluorescent reporter molecule, and optionally an analyte, wherein different analytes can be physically separated into various water-in-oil compartments.

[0243] In some embodiments, the first compartments of each system are provided by aqueous droplets, and the second compartments of each system are provided by a hydrogel layer (e.g., a hydrogel layer comprising an agarose matrix). In some embodiments, the second compartments of a plurality of systems can be provided by a single hydrogel layer. In some embodiments, a nanopore array can be provided by placing a plurality of aqueous droplets with the hydrogel layer in a hydrophobic medium comprising amphiphilic molecules that are selectively permeable to water molecules, wherein each aqueous droplet includes a protein nanopore, a fluorescent reporter molecule, and an analyte, wherein 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, and the protein nanopore can spontaneously insert into the semipermeable membrane.

[0244] According to another aspect of the application, there is provided a method of producing a nanopore array, comprising:

[0245] providing a plurality of aqueous droplets, wherein the aqueous droplets each include a protein nanopore, an analyte, and a fluorescent reporter molecule capable of emitting fluorescence when bound to an ionic species;

[0246] providing a hydrogel layer, wherein the hydrogel layer includes an ionic species;

[0247] placing the plurality of aqueous droplets and the hydrogel layer together in a hydrophobic medium containing amphiphilic molecules, thereby forming a semipermeable membrane between each aqueous droplet and the hydrogel layer.

[0248] The volume of each aqueous droplet can 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, e.g., about 30 pL. The density of the droplet-hydrogel array can be at least 10 droplets / mm 2 , at least 50 droplets / mm 2 , at least 100 droplets / mm 2 , at least 200 droplets / mm 2 , at least 300 droplets / mm2 at least 400 droplets / mm 2 at least 500 droplets / mm 2 at least 600 droplets / mm 2 at least 700 droplets / mm 2 at least 800 droplets / mm 2 at least 900 droplets / mm 2 at least 1000 droplets / mm 2 such as highly ordered droplet-hydrogel arrays that can be formed with the help of microfluidics technology.

[0249] In one nanopore array of the application, the number of aqueous droplets can be from 4 to 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 greater than 1000.

[0250] The light source, light sensor and recording device described above for the single system or method can be used in the nanopore array of the application. As known to the person skilled in the art, up to and more than 2500 pores can be recorded simultaneously using an electron multiplying CCD camera (ixon3, Andor).

[0251] The amplification of the fluorescent signal by the difference in osmolarity by volume / osmolarity by weight also applies to the case of the multiplex system and multiplex method of the application.

[0252] According to another aspect of the application, there is provided a kit for forming a nanopore array, the kit comprising:

[0253] a filling hydrogel comprising agarose, a buffer and an ionic species capable of specifically binding to a fluorescent reporter molecule to cause it to emit fluorescence;

[0254] an aqueous solution comprising a chelating agent, the fluorescent reporter molecule capable of emitting fluorescence when bound to the ionic species, and a buffer; wherein the chelating agent is capable of binding to the ionic species;

[0255] a hydrophobic medium containing an amphiphilic molecule;

[0256] a solid support.

[0257] The solid support can have any structure suitable for bringing together the hydrogel and aqueous solution droplets in the hydrophobic medium containing the amphiphilic molecule and thereby forming a semi-permeable membrane consisting of the amphiphilic molecule between the hydrogel and aqueous solution droplets. The solid support can be made of PMMA or glass. The solid support can be a microfluidic chip having, for example,Figure 19 PMMA measurement device. As shown Figure 3 and Figure 19 As shown, the PMMA measurement device can have four independent droplet channels in the central recessed area, a gel inlet for filling the filling hydrogel, an outlet for venting, and a coverslip for supporting the hydrogel. The coverslip can be an oxygen plasma treated coverslip. PMMA is known as polymethyl methacrylate methyl methacrylate.

[0258] The kit can also include a coating hydrogel, which includes agarose in water. To form a nanopore array, the coverslip can be spin-coated with molten coating hydrogel and adhered to the PMMA measurement device by filling the molten filling hydrogel through the gel inlet. The coverslip can then be immersed in a hydrophobic medium containing an amphiphilic molecule. An aqueous droplet with protein nanopores and analytes can be pipetted into the hydrophobic medium for incubation. The droplet and agarose hydrogel can be brought together in the hydrophobic medium, and a nanopore array in the form of DIBs can spontaneously form.

[0259] In some embodiments, the filling hydrogel has an osmolarity higher than or equal to the osmolarity of the aqueous solution or the filling hydrogel has an osmolality higher than or equal to the osmolality of the aqueous solution; or the filling hydrogel has an osmolarity lower than or equal to the osmolarity of the aqueous solution or the filling hydrogel has an osmolality lower than or equal to the osmolality of the aqueous solution.

[0260] In some embodiments, the fluorescent reporter molecule can be fluo-8 and the ionic species can be Ca 2+ .

[0261] The filling hydrogel or the aqueous solution can also include a protein nanopore.

[0262] The aqueous solution can also include a salt, such as KCl or NaCl.

[0263] The fluorescent reporter molecule, ionic species, protein nanopore, hydrophobic medium, amphiphilic molecule, chelator, analyte, and other features mentioned herein are as described in the context of this specification.

[0264] In some embodiments, the coating hydrogel can include 0.75% (w / v) agarose in water.

[0265] In some embodiments, the filling hydrogel can include 2.5% agarose, 1.5 M CaCl2, and 10 mM HEPES, pH 7.0.

[0266] In some embodiments, the aqueous solution can include 1.5 M KCl, 400 mM EDTA, 40 mM Fluo-8, 10 mM HEPES, pH 7.0.

[0267] In some embodiments, the hydrophobic medium containing amphiphilic molecules can be a lipid oil including a dried film of 5 mg DPHPC lipids dissolved in a 2 mL mixture of hexadecane and silicone oil in a 1 : 1 volume ratio. In another aspect, the present application provides a nanopore array formed by the above production method.

[0268] In another aspect, the present application provides use of the above system or the above nanopore array for optical analyte analysis.

[0269] Unless otherwise stated, most features of the system and method are the same in different systems and different methods, such as the features of the first and second compartments, the first and second aqueous solutions, the membrane, the nanopores, the fluorescent reporter molecules and / or the ionic species, and how they are formed, how they are used, etc. Unless otherwise stated or not possible, the features of the nanopore array, for example, the first and second compartments, the first and second aqueous solutions, the membrane, the nanopores, the fluorescent reporter molecules and / or the ionic species, and how they are formed, how they are used, etc. can be as described above in the single system.

[0270] In the present application, when referring to components in a solution, "in the first aqueous solution" and "in the first compartment" can be used interchangeably, and "in the second aqueous solution" and "in the second compartment" can be used interchangeably

[0271] The embodiments described herein can be understood more readily by reference to the following detailed description, examples, and claims, and their previous and later descriptions. It is to be understood that the embodiments described herein are not limited to a specific use, method, and / or product. It is also to be understood that the terminology used herein is for the purpose of describing specific aspects only and is not intended to be limiting.

[0272] Furthermore, the following description is provided as an enabling teaching of the various implementations. Those skilled in the relevant art will recognize that many changes can be made to the described implementations, while still falling within the scope of the present disclosure. It will also be apparent that some desired benefits of the present application can be obtained by selecting some of the features of the variations described, without using other features. Accordingly, those who work in the art will recognize that many modifications and adaptations to the implementations described are possible and can even be desirable in certain circumstances, and are part of the present disclosure. Thus, the following description is provided as illustrative of the principles of the implementations set forth by this document and is not meant to limit or otherwise in any way constrain the claims appended hereto.

[0273] In this application, the term "about" is used to indicate that a value includes the standard deviation or standard error of the system or method being used to determine the value. In any embodiment discussed in the context of a value used in conjunction with the term "about," it is specifically contemplated that the term "about" can be omitted.

[0274] Throughout this specification, it is to be understood that the singular includes the plural unless specifically stated to the contrary. Thus, for example, it is to be understood that the singular articles (e.g., English articles "a," "an," "the," etc.) include the plural unless specifically stated to the contrary.

[0275] 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. Thus, for example, it is to be understood that the use of the terms "including," "comprising," or "having" includes the presence of zero or more of the stated elements or process steps and any combinations thereof. Furthermore, it is to be understood that the use of the term "about" in describing the aspects is intended to convey that the description should be understood to include not only the precise

[0276] All patents and publications, including all sequences disclosed therein, disclosed herein are expressly incorporated herein by reference.

[0277] Example

[0278] Example 1

[0279] DOP monosensor of trimethyl-β-cyclodextrin: proof of concept demonstration

[0280] According to Figure 1 The basic configuration of the DOP recording includes asymmetric electrolyte buffers separated by a semi-permeable membrane with an inserted nanopore. The compartment filled with KCl, Fluo-8, and EDTA is defined as the cis side. While the compartment filled with CaCl2is defined as the trans side. The biological nanopore forms the only conductive path between the cis and trans sides, and the thermodynamic diffusion of the channel transport facilitates the Ca 2+and Fluo-8, driven by a chemical gradient. FluoCa is Ca 2+ and Fluo-8, around each nanopore to report the open state of the sensor.

[0281] In theory, finite element method (FEM) simulation was constructed, which was adapted from Poisson-Nernst-Planck-Stokes (PNPS) model

[26] (Methods, Figure 2 ). To simulate the experimental operation, simulation parameters could be adjusted by setting different boundary conditions, such as different combinations of reagent concentrations. Tentatively, the simulation was performed by setting the boundary condition on the cis side as 1.5 M KCl, 40 μΜ Fluo-8, 400 μΜ EDTA, the boundary condition on the trans side as 0.75 M CaCl2, and the cylindrical channel geometry with a diameter of 2 nm. According to the results, a concentration gradient of FluoCa was constructed right above the nanopore Figure 1 e). Due to the simultaneous emission of FluoCa, a strong fluorescence intensity contrast was expected at the top of the nanopore. The intensity profile generated as a simulation of TIRF imaging (Methods) followed a Gaussian distribution, with a full width at half maximum (FWHM) of 2.670 μm Figure 1 f top panel).

[0282] Experimentally, a droplet interface bilayer (DIB) was constructed between an aqueous droplet and a 100 nm thick hydrogel sheet Figure 3 ). The aqueous droplet was composed of 1.5 M KCl, 400 μΜ EDTA, 40 μΜ Fluo-8, 10 mM HEPES, pH 7.0. The hydrogel sheet was composed of 0.75 M CaCl2, 10 mM HEPES, pH 7.0, and 2.5% (v / w) low-melting-point agarose. WT α-HL (a-HL) placed in the aqueous droplet spontaneously inserted into the DIB and appeared as a bright fluorescent spot during TIRF imaging Figure 1 f bottom panel). The fluorescence intensity profile from a representative frame of TIRF imaging followed an approximate Gaussian distribution, with a FWHM of 2.583 μm Figure 1 f bottom panel), similar to that obtained from the simulation.

[0283] TriM-β-CD interacts with the restriction site of the WT a-HL nanopore, generating long residence and deep pore blockage events during electrophysiological recordings [27-29]. This easily observable property makes TriM-β-CD a representative small molecule analyte for the proof-of-concept of single-molecule sensing using DOPs. To maintain a stable analyte concentration during DOP recordings, TriM-β-CD was added to the cis side at a final concentration of 75 mM. TriM-β-CD binding from the cis side was also verified by corresponding electrophysiological measurements Figure 4 ). During DOP recordings, the random binding of TriM-β-CD to a-HL leads to a restricted flow of Ca 2+ through the channel, resulting in a highly distinguishable image contrast between the open (Fo) and blocked state (Fb) of the a-HL nanopore Figure 1 g). From the corresponding fluorescence traces Figure 1 g), consecutive pore blockages were observed, which were derived from continuously recorded image series (Methods, Figure 5 ). To allow quantitative comparisons between different experiments, all fluorescence traces were corrected and normalized prior to analysis Figure 6 ).

[0284] From the normalized fluorescence traces, the characteristics of single-molecule sensing events were characterized by the event residence time (t off ), the inter-event duration (t on ), and the fractional blockage depth (%F b ). Histograms of t off and t on showed exponential distributions, which could be fitted and characterized by their mean time constants τ off and τ on , respectively Figure 7 ). By varying the TriM-β-CD concentration in the cis side, the inverse of the residence time (1 / τ off ) remained constant, while the inverse of the inter-event interval (1 / τ on ) linearly correlated with the TriM-β-CD concentration in the cis side Figure 1 h, Table 1).

[0285] Table 1. 1 / τ on and 1 / τ off of TriM-β-CD using different [TriM-β-CD].

[0286]

[0287] DOP recordings were constructed in the presence of 1.5 M KCl, 400 mM EDTA, 40 mM Fluo-8, 10 mM HEPES, pH 7.0 (in the cis side) and 0.75 M CaCl2, 10 mM HEPES, pH 7.0 (in the trans side). TriM-β-CD was added to the cis side. Three independent measurements were performed to form statistical data.

[0288] From DOP recordings, the average τ off value was 0.347 ± 0.067 s and the average F P value was 0.078 ± 0.010. Here, F P is defined as the average blockage depth from the %F b value of each DOP recording experiment. Figure 7 ). Whereas the corresponding electrophysiological results obtained under a +20 mV potential bias produced a τ off value of 0.386 ± 0.392 s and an I P value of 0.065 ± 0.002. Here, I P is defined as the average blockage depth from each electrophysiological recording experiment. Three independent experiments were performed for each measurement condition to form statistical data. Thus, the similarity of this result confirms the feasibility of DOP single-molecule sensing Figure 1 i).

[0289] Although not proven, single-molecule sensing of other small molecules (such as sugars [30, 31], ions

[32] , nucleotides

[33] , neurotransmitters

[34] , amino acids

[35] , etc.) can in principle be similarly performed by DOP recordings with the added advantage of flux. Since 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 fluorescence emission intensity and analyte binding efficiency can be further improved to match electrophysiology.

[0290] Example 2

[0291] Enhancing DOP sensing by directed permeation

[0292] During conventional electrophysiological recordings, the applied electrochemical gradient is essential for driving the continuous flow of charged particles (such as ions and analytes). Intuitively, to drive the directed flow of analytes into the nanopore sensor without the use of electrodes, other forms of asymmetry must be introduced.

[0293] DIB is a self-assembled membrane consisting of 1,2-diphytanoyl-sn-glycero-3- phosphocholine (DphPC) lipids that selectively permeates water molecules but not ions [36, 37]. When a difference in molar concentration of volume (C solute = iM solute ) exists across the DIB, the osmotic pressure is constructed according to Δπ = (C solute,cis - C solute,trans )RT, where i is the dimensionless van't Hoff index representing the number of dissociated ions per solute molecule, M solute is the molar concentration of solute, R is the ideal gas constant, and T is the temperature in Kelvin. Here, the positive direction of osmotic pressure is defined as from the cis side to the trans side (this is an easy-to-understand definition, although it is possible that the osmotic pressure is higher on the cis side than on the trans side). This osmotic pressure then drives the directional flow of water, ions, and analytes through the biological nanopore inserted in the membrane

[22] . Thus, by introducing this asymmetry, it should be possible to improve the translocation efficiency of the analyte.

[0294] To experimentally verify this hypothesis, a series of DOP recordings Figure 3 were performed in DIBs with different KCl concentrations (1.0-2.5 M) in the cis side while the CaCl2concentration in the trans side was kept constant (0.75 M). α-HL and TriM-β-CD were again tentatively chosen as the model sensor and analyte, with the concentration of TriM-β-CD fixed at 15 mM in the cis side. From a representative DOP recording, an enhanced capture rate of TriM-β-CD was observed from the time-extended fluorescence trace when the KCl concentration in the cis side was reduced from 2.5 M to 1.0 M Figure 3 . By evaluating the 1 / τ on values from events in independent measurements, it was observed that 1 / τ on systematically decreased with the reduction in osmotic pressure Figure 8 , Table 2), which indicates that a higher event detection rate was observed with the help of the directional osmotic flow.

[0295] Table 2. 1 / τ on values of TriM-β-CD using different [KCl] in the cis side.

[0296]

[0297] DIBs were constructed in the presence of 1-2.5 M KCl, 400 μΜ EDTA, 40 μΜ Fluo-8, 10 mM HEPES, pH 7.0 (in the cis side) and 0.75 M CaCl2, 10 mM HEPES, pH 7.0 (in the trans side). 15 mM TriM-β-CD was added to the cis side. Three independent measurements were performed to form statistical data.

[0298] It was also found that when a permeation flow from the cis side to the trans side was present, a significantly improved contrast of the fluorescence image was observed from the DOP recordings. This phenomenon can be noticed from the reduction of thermal noise in the fluorescence trace under the measurement conditions of lower KCl concentration Figure 8 a, Figure 9 ). Here, the high thermal noise observed from the fluorescence trace is a result of the reduced photon count in the imaging process.

[0299] To further investigate the reason why the fluorescence intensity of DOP recordings can be adjusted by permeation Figure 8 c, Table 3), a set of different experiments were performed in the presence of 1-2.5 M KCl, 400 μΜ EDTA, 40 μΜ Fluo-8, 10 mM HEPES, pH 7.0 (in the cis side) and 0.75 M CaCl2, 10 mM HEPES, pH 7.0 (in the trans side). To avoid the interference of analyte binding, TriM-β-CD was omitted. To avoid the interference of inhomogeneous TIRF illumination or laser power fluctuations when evaluating the fluorescence brightness, a signal-to-background ratio (SBR) value was introduced to quantitatively compare different DOP recording experiments (Methods). From the representative image frames and the corresponding SBR values, it can be seen that the brightness of the fluorescent spots was significantly enhanced when a larger osmotic pressure from the cis side to the trans side was introduced. Each condition included five independent measurements to form statistical data Figure 8 c).

[0300] Table 3. FWHM and SBR using different [KCl]

[0301]

[0302] DIBs were constructed in the presence of 1-2.5 M KCl, 400 μΜ EDTA, 40 μΜ Fluo-8, 10 mM HEPES, pH 7.0 (in the cis side) and 0.75 M CaCl2, 10 mM HEPES, pH 7.0 (in the trans side). Five independent measurements were performed to form statistical data.

[0303] This phenomenon can also be observed from the corresponding FEM simulation, which is performed by setting the boundary conditions on the cis side as 1-2.5 M KCl, 40 μΜ Fluo-8, 400 μΜ EDTA and on the trans side as 0.75 M CaCl2( Figure 8 d) By plotting the Fluo-8 distribution within the simulation space, it can be clearly found that when there is a directional permeation flow from the cis side to the trans side, a concentrated Fluo-8 distribution is built near the cis side of the membrane. This is because Fluo-8 is impermeable to the lipid membrane, enriched by the permeation flow, thus the fluorescence intensity is enhanced Figure 10 ).

[0304] Example 3

[0305] Using amplified Ca 2+ Flow further optimizes SBR

[0306] However, the permeation enrichment of Fluo-8 should not occur in solid-state nanopore devices where the membrane does not have semi-permeable characteristics. Alternatively, more Ca 2+ flux can be introduced through the nanopore to improve the SBR recorded by DOP. A direct solution to follow this strategy is to increase [Ca 2+ ] in the trans side, which directly increases the chemical gradient of [Ca 2+ ] across the membrane. To verify this hypothesis, a series of DOP recordings were performed by gradually increasing the CaCl2concentration in the trans side. To avoid the interference of permeation, the KCl concentration in the cis side was adjusted accordingly, so that the osmotic pressure concentration of the cis and trans sides always remained isotonic.

[0307] Experimentally, DIB was built in the conditions of 0.75 M, 1.5 M or 2.25 M KCl, 400 μΜ EDTA, 40 μΜ Fluo-8, 10 mM HEPES, pH 7.0 (in the cis side) and 0.5 M, 1 M or 1.5 M CaCl2, 10 mM HEPES, pH 7.0 (in the trans side). Representative image frames show that the fluorescent dot size systematically increases when obtained using electrolyte buffer combinations with higher [CaCl2] in the trans side Figure 11 a) The fluorescence intensity obtained under these conditions was compared more directly according to the color coding of the fitted amplitudes Figure 11 a) The fluorescence intensity obtained under these conditions was compared more directly according to the color coding of the fitted amplitudes Figure 11b Quantitative measurements of SBR and FWHM in DOP recordings obtained using these electrolyte combinations are shown, whereby FWHM and SBR (Table 4) increase when the molar osmotic pressure concentration across the membrane is upregulated. Each condition includes 12 independent measurements to form statistical data.

[0308] Table 4. FWHM and SBR using different [CaCl2]

[0309]

[0310] DIBs were constructed in conditions of KCl (0.75 M, 1.5 M and 2.25 M), 400 mM EDTA, 40 mM Fluo-8, 10 mM HEPES, pH 7.0 (in the cis side) and CaCl2(0.5 M, 1 M and 1.5 M), 10 mM HEPES, pH 7.0 (in the trans side). 12 independent measurements were performed to form statistical data.

[0311] PEG is a macromolecule that is electrically neutral when dissolved in buffer at neutral pH and has been shown to translocate through a-HL nanopores during electrophysiological recordings

[38] . It has been reported that the capture rate improves and event dwell times are prolonged when measurements are performed using electrolyte buffers with higher salt concentrations

[39] . As a demonstration, PEG 1500 was chosen as a model analyte for DOP single-molecule detection of macromolecules.

[0312] Experimentally, DIBs were constructed in conditions of 2.25 M KCl, 10 mM HEPES, 400 mM EDTA, 40 mM Fluo-8, 20 mM PEG1500, 10 mM HEPES, pH 7.0 (in the cis side) and 1.5 M CaCl2, 10 mM HEPES, pH 7 (in the trans side). The addition of 20 mM PEG 1500 to the cis side immediately resulted in a large number of sharp peak translocation events from the resulting fluorescence trace Figure 11 c). The similarity of these pore translocation signatures to the reported electrophysiological data confirms that PEG1500 can be sensed by DOP recordings, similar to what was demonstrated for TriM- -CD.

[0313] However, the solubility of the analyte is typically reduced in electrolyte buffers with high salt concentrations

[39] . Furthermore, the salt concentration is also limited by the maximum solubility of the electrolyte in water (CaCl2: 6.767 M, KCl: 3.408 M, 20 °C). To bring more Ca 2+ Flow, without reaching this limit, can be introduced in the DOP recordings by incorporating nanopore sensors with larger pore diameters, which has been investigated by corresponding FEM studies (Methods, Figure 11d) was confirmed. According to the reported crystallographic results, the constriction of ClyA nanopore with a diameter of 3.8 nm is 2.7 times larger than the diameter of a-HL

[40] . ClyA and its variants have been exploited for sensing large biological macromolecules, such as dsDNA or small proteins, thanks to their large channel opening [40-44]. ClyA-RR was reported to be a charge-optimized mutant that can efficiently translocate dsDNA during electrophysiological recordings

[42] and was selected for DOP recordings (Methods, Figure 12 ) Although not demonstrated, phi29 connector protein

[45] or solid-state nanopores [24, 25] are also good candidates.

[0314] Attemptively, DIBs were constructed under conditions of 1.5 M KCl, 400 mM EDTA, 40 mM Fluo-8, 10 mM HEPES, pH 7.0 (in the cis side) and 1.5 M CaCl2, 10 mM HEPES, pH 7.0 (in the trans side). The electrolyte combination of 1.5 M KCl (cis) / 1.5 M CaCl2 (trans) was chosen considering the osmotic pressure ( Figure 8 ), CaCl2concentration ( Figure 11 ) and the pore permeability

[22] . To quantitatively compare the nanopores with different channel openings during DOP recordings, dodecameric ClyA-RR nanopores and heptameric a-HL nanopores were inserted into the droplets to be measured simultaneously from the same DIB.

[0315] Upon insertion, ClyA-RR nanopores showed a huge and bright fluorescent spot, while a-HL nanopores were smaller in size and dimmer in intensity ( Figure 11 e). As more Ca 2+ flow was introduced across the membrane, the FWHM and SBR derived from DOP recordings using ClyA-RR were significantly better than those derived from a-HL ( Figure 11 f, Table 5). Five independent measurements were performed to form statistical data.

[0316] Table 5. FWHM and SBR of aHL and ClyA-RR nanopores

[0317]

[0318] DIBs were constructed under conditions of 1.5 M KCl, 400 mM EDTA, 40 mM Fluo-8, 10 mM HEPES, pH 7.0 (in the cis side) and 1.5 M CaCl2, 10 mM HEPES, pH 7.0 (in the trans side). The pores were added to the cis side. Five independent measurements were performed to form statistical data.

[0319] In addition to improving SBR, the large pore size of ClyA also helps to enhance permeation flow, as predicted by FEM simulation, which can help to provide the driving force for DNA translocation Figure 11 g and Figure 13 ). Although much effort has been paid to counteract the electrophoretic force during DNA translocation

[46] , the electrophoretic force is still considered indispensable during DNA sensing process, which effectively unwinds the coiled DNA during translocation [9]. However, the long persistence length of dsDNA

[47] and the large opening of ClyA nanopore can lower the entropy barrier for dsDNA translocation

[48] . In addition, the large vestibule of ClyA can also accommodate dsDNA in a partially translocated form to report the sensing signal of dsDNA during DOP recording.

[0320] Example 4

[0321] Translocation of dsDNA and ssDNA through ClyA nanopore

[0322] Experimentally, DIB was constructed under the condition of 1.5 M KCl, 10 mM HEPES, 400 mM EDTA, 40 mM Fluo-8, 2 mM dsDNA (78 bp), 10 mM HEPES, pH 7.0 (in the cis side) and 1.5 M CaCl2, 10 mM HEPES, pH 7.0 (in the trans side) Figure 14 a). The molar osmotic pressure concentration on both sides of DIB aims to construct a persistent osmotic pressure from the cis side to the trans side. The dsDNA consists of 78 bp (Table 6), which is optionally dissolved in aqueous droplets with a final concentration of 2 mM.

[0323] Table 6. Nucleic acid abbreviations and sequences

[0324]

[0325] Note: To form dsDNA, complementary ssDNA (78 nt ssDNA-a and b) was dissolved in 1.5 M KCl buffer (1.5 M KCl, 10 mM HEPES, pH 7.0), heated to 95 °C on a PCR thermocycler (ABI 2720) and gradually cooled (-5 °C / min) to room temperature (25 °C).

[0326] Without the addition of any dsDNA in the droplet, representative fluorescence traces from ClyA nanopores exhibit stable opening, without spontaneous gating activity Figure 14 b). When dsDNA was added in the droplet, continuous fluorescence blockage spontaneously occurred during DOP recordingFigure 14 c) The observed blockades from DOP recordings show an average F P of 0.625 ± 0.014 and an average τ off of 2.538 ± 0.849 s (N = 3). These results indicate that the interaction of dsDNA with ClyA-RR occurs without the application of an electrochemical gradient. The longer dwell times can be due to the absence of electrophoretic forces during the measurement or can be due to the dsDNA being trapped in the large vestibule structure of ClyA.

[0327] To further validate this phenomenon by conventional electrophysiological recordings, planar lipid membranes were constructed under conditions of 1.5 M KCl, 10 mM HEPES, pH = 7.0 in the cis side and 1.5 M CaCl2, 10 mM HEPES, pH = 7.0 in the trans side. 78 bp dsDNA was added to the cis side at a final concentration of 2 μΜ. A very low voltage was applied as a mimic of DOP recordings with a strict zero transmembrane potential. Figure 14 d shows representative electrophysiological traces recorded at +6 mV, +4 mV or +2 mV transmembrane potential, respectively. Similar shifting events observed from DOP recordings were monitored, which confirmed our hypothesis that dsDNA can interact with ClyA and produce a detectable sensing signal when optically monitored without electrodes. I P The 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 are qualitatively consistent with the blockades observed from DOP recordings Figure 15 , Table 7). During the electrophysiological recordings, the dwell times were widely distributed from 1 to 10 5 ms, while due to the limited bandwidth of the acquisition of DOP recordings, fast events below 30 ms could not be detected optically Figure 14 d).

[0328] Table 7. Blockade levels of dsDNA shifting.

[0329]

[0330] DIB was constructed under conditions of 1.5 M KCl, 400 μΜ EDTA, 40 μΜ Fluo-8, 10 mM HEPES, pH 7.0 in the cis side and 1.5 M CaCl2, 10 mM HEPES, pH 7.0 in the trans side. 2 μΜ dsDNA was added to the cis side. Three independent measurements were performed to form statistical data.

[0331] To further validate ssDNA translocation through nanopores, electrodeless 20-nt ssDNA sensing was performed using a-HL nanopores. The results are shown in Figure 17 Figure 22B. a-HL WT nanopores were used, which only allow ssDNA but not dsDNA to pass through. Due to the relatively small size of this nanopore, a high concentration of 50 pmol / L ssDNA was used. The percentage block depth indicates that 20-nt ssDNA can pass through a-HL nanopores.

[0332] Another electrodeless 78-nt ssDNA sensing was performed using ClyA-RR, and the results are shown in Figure 18 Figure 23B. ClyA-RR has a larger pore size and allows both ssDNA and dsDNA to pass through. For ssDNA, the sequence and secondary structure of ssDNA significantly affect the fluorescence emission characteristics since ssDNA can not be perfectly straight when passing through the nanopore. 78-nt ssDNA-a and poly A 78 exhibit very different fluorescence emission characteristics. Poly A 78 has a uniform and shallow percentage block depth. 78-nt ssDNA-a has different percentage block depths and longer event dwell times. This indicates that ssDNA can be sensed by DOP.

[0333] Example 5

[0334] Multiplexed DOP recording using fingertip-sized devices and future prospects

[0335] By eliminating the need for electrode configurations, DOP enables a more compact device size while still retaining the advantages of low cost (<1$) and high throughput. This configuration is suitable for the production of disposable nanopore chips for clinical diagnostics where cross-contamination should be strictly prohibited. As a proof of concept, a miniaturized device (10 mm x 10 mm x 1 mm) was fabricated from bulk polymethyl methacrylate methyl acrylic acid (PMMA) Figure 16 a, Figure 2 ). DOP recording can be performed by placing the chip directly above a TIRF objective, which is used for both illumination and imaging. As a demonstration, DOP recording was performed from a-HL and ClyA using this miniaturized device, where both a-HL and ClyA can be visually monitored Figure 16 c).

[0336] However, DOP measurements from a single DIB are limited to one combination of pore and analyte. By eliminating the need to house electrodes, DOP is able to perform multiplexed recording from different DIBs with an extremely simple configuration and greatly reduced measurement volume, where different analytes can be physically separated into various water-in-oil compartments.

[0337] As a proof of concept, microdroplets (~30 pL) containing ClyA-RR nanopores were generated and pipetted into a measurement reservoir filled with a solution of lipid oil Figure 16 d) Although not monodisperse in size, many independent DIBs can spontaneously form to very easily enable subsequent DOP recordings Figure 16 e) In DIBs of ~40 pm in diameter, a single inserted ClyA nanopore was clearly observed as a bright fluorescent spot Figure 16 f) EDTA in the droplet persisted for ~10 minutes before being depleted by Ca 2+ binding. This corresponds to an effective measurement density of 10 3 independent DIBs / mm 2 which can be formed with the help of microfluidic devices

[49] . However, due to the complexity of electronic integration, oSCR or electrophysiology cannot easily achieve such high measurement densities.

[0338] Despite the advantages of being electrodeless, DiffusiOptoPhysiology is not without limitations. As a fluorescence imaging technique, the temporal resolution of DOP is typically limited to ~10 ms per frame when recorded in the full field of view (135 pm x 135 pm). Reading fluorescence from reduced image pixels can immediately improve the acquisition speed. With the addressability of highly ordered nanopore arrays improved, high-speed DOP recordings can be performed with spinning-disk confocal imaging

[50] . Without an electric field, the limit of detection (LOD) during DOP is typically higher than that of electrophysiology or oSCR (~pM) (as low as ~nM). However, since no electrodes need to be accommodated, the trade-off is the need for a smaller measurement volume (as low as ~30 pL), which in fact reduces the sample absolute cost.

[0339] Conclusion

[0340] In summary, we have demonstrated how to draw inspiration from natural passive channel transport to use DiffusiOptoPhysiology as a nanopore sensing platform. While the fluorescence emission during DOP recordings is due to passive diffusion as well as subsequent Ca 2+binding of Fluo-8, but as demonstrated, the fluorescence intensity is sufficient to meet the needs of various single-molecule sensing applications. After optimization of the binding electrolyte and channel size, this technology can achieve high-throughput nanopore measurements while the sensing performance can still be comparable to traditional electrophysiological recordings or oSCR. Although demonstrated using total internal reflection fluorescence (TIRF) microscopy, DOP can in principle be flexibly used with any fluorescence platform, such as confocal or epi-fluorescence microscopy. Without the need for space to accommodate electrodes, the measurement volume of DOP is further reduced to ~30 pL, the lowest record ever reported, which can be suitable for measuring analytes with extremely low abundance. Using microdroplet array to record DOP can also perform multiplexed measurements from independent compartments, which are simply constructed from water-in-oil separation. While having an integrated nanotechnology sensor as a chip, the omission of electronics significantly reduces the cost and size of the device. Therefore, this scheme can inspire future clinical applications using disposable nanopore chips in various applications.

[0341] Method

[0342] Materials

[0343] Hexadecane, silicone oil AR20, pentane, ethylenediaminetetraacetic acid (EDTA), Triton X-100, Genapol X-80 and PEG1500 were from Sigma-Aldrich. Potassium chloride, calcium chloride, magnesium chloride and sodium chloride were from Aladdin. Isopropyl-β-D-thiogalactopyranoside (IPTG) free of dioxane, dodecyl β-D-maltopyranoside (DDM), kanamycin sulfate, tris(hydroxymethyl)aminomethane (Tris) and imidazole were from Solarbio. Low-melt agarose and wide-range DNA ladder (20-500 bp) were from Takara. Precision plus protein marker and 4-15% polyacrylamide gel were from Bio-Rad. Ethanol and acetone were from Sinopharm. Fluo-8 H sodium salt (Fluo-8) was from AAT Bioquest. 1,2-Diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) was from Avanti Polar Lipids. 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) was purchased from Shanghai Yuanye Bio-Technology. Escherichia coli strain BL21(DE3) was from BioMed. Trimethyl-β-cyclodextrin was from Tokyo chemical industry (Shanghai). LB broth and LB agar were from Hopebio. All the above listed items were used as received.

[0344] KCl and CaCl2 buffers (1-2.5 M KCl, 10 mM HEPES, pH 7.0) were membrane filtered (0.2 pm cellulose acetate, Nalgene). For simplicity, 1-2.5 M KCl buffer represents 1-2.5 M KCl, 10 mM HEPES, pH 7.0 unless otherwise stated. 0.5-1.5 M CaCl2 buffer represents 0.5-1.5 M CaCl2, 10 mM HEPES, pH 7.0. KCl buffer was treated with Chelex 100 resin (Bio-Rad) overnight to remove contaminating divalent ions before use.

[0345] High-performance liquid chromatography (HPLC)-purified DNA (Table 6) was dissolved in DNase / RNase-free water before use. To form dsDNA, 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 and gradually cooled (-5 °C / min) to room temperature (25 °C) on a PCR thermocycler (ABI 2720).

[0346] Protein nanopores used in this work were a-HL WT and ClyA-RR Figure 7 ), which were expressed in E. coli and purified according to published protocols [22, 42].

[0347] ClyA-RR preparation

[0348] A gene encoding monomeric ClyA-RR (D64R / C87A / L99Q / E103G / S110R / F166Y / I203V / C285S / K294R / H307Y) protein was custom synthesized and constructed in a pET 30a(+) plasmid (Genescript, New Jersey). A hexahistidine 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, which were grown on LB agar plates containing 50 pg / mL kanamycin for 18 h. A single colony was inoculated into LB medium containing 50 pg / mL kanamycin and grown at 37 °C until OD600= 0.4. Protein expression was induced with 1 mM IPTG and the culture was grown at 37 °C for 4 h. Cells were harvested by centrifugation at 5000 x g for 20 min at 4 °C and the pellet was stored at -80 °C until use. 600A final OD600 of 4.0 was reached. Protein expression was induced by adding isopropyl β-D-thiogalactoside (IPTG) to the LB medium to a final concentration of 1 mM. The culture was further shaken (200 rpm) at 15 °C for 16 h. Cells were then harvested by centrifugation (4000 rpm, 4 °C, 20 min). The pellet was collected and resuspended in lysis buffer (150 mM NaCl, 50 mM Tris-HCl, 10% glycerol, pH 8.0), sonicated (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 eluted using 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; buffer D: 500 mM NaCl, 15 mM Tris-HCl, 10% glycerol, 20 mM imidazole, pH 8.0). The eluted fractions containing ClyA-RR monomers were determined using SDS-PAGE gel electrophoresis Figure 12 ) and stored at -80 °C in 270 mM NaCl, 50 mM Tris-HCl, 10% glycerol, 0.2% Triton 100, pH 8.0 buffer.

[0349] According to previous studies

[42] , 0.25% (w / v) β-dodecylmaltoside (DDM) was added to promote pore oligomerization. After incubation at 25 °C for 15 min, the results of pore oligomerization were characterized by blue native polyacrylamide gel electrophoresis (BN-PAGE, Bio-Rad) using 4-15% polyacrylamide gels Figure 12 ). The gel showed that monomers had self-assembled into oligomers before the addition of DDM. However, to strictly follow previous studies

[42] , the DDM-added ClyA-RR dodecamer was still used for subsequent measurements. Here, the band corresponding to the dodecameric 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 out of the gel was collected by centrifugation (20,000 g, 4 °C, 20 min). The collected dodecameric ClyA-RR protein was immediately used for subsequent experiments or stored at 4 °C for up to 14 days.

[0350] DIB formation

[0351] Detailed descriptions on how to create the droplet / hydrogel bilayer have been previously reported

[10] . Briefly, oxygen plasma treated coverslips (24 mm x 40 mm) were spin-coated (3000 rpm, 30 s) with 200 pL of molten agarose (0.75% w / v in Mili Q water). The coverslips were mounted on a PMMA device by filling the microfluidic channels within the device with molten agarose (2.5% w / v in CaCl2buffer)

[10] . A lipid / oil solution was prepared by dissolving a dry film of 5 mg DPHPC lipid in a 1:1 volume ratio mixture of 2 mL hexadecane and silicon oil. A lipid monolayer was formed on the agarose-coated glass coverslips when immersed in the lipid-oil solution. When preparing the aqueous droplets, protein nanopores and other analytes can be added to an aqueous buffer consisting of 1 M-2.5 M KCl, 400 pM EDTA, 40 pM Fluo-8, 10 mM HEPES, pH 7.0. Different volumes of the aqueous droplets were pipetted into the lipid / oil solution for incubation. After 5 min, a self-assembled lipid monolayer can be formed at the water-oil interface. A stable bilayer (DIB) can be spontaneously formed when this droplet and agarose matrix are brought together in the lipid / oil solution.

[0352] TIRF imaging and optical recordings

[0353] 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 (iXon 3897, Andor). Exposure times were set between 3-30 ms. The maximum field of view was 135 pm x 135 pm.

[0354] Electrical recordings

[0355] Electrophysiological recordings were performed as previously reported8. Electrophysiological traces were acquired at a 25 kHz sampling rate, 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).

[0356] Finite element modeling (FEM) simulation

[0357] Ca 2+ Binding with calcium indicator dye Fluo-8 results in fluorescent emission near the pore. Excess Ca 2+ binds with EDTA, resulting in a decrease in fluorescent background. These two full reactions can be described in equations (1, 2), where a and b represent the forward and reverse binding rates, respectively.

[0358]

[0359]

[0360] Optical single-channel recording (oSCR) can be simulated by FEM using the Poisson-Nernst-Planck-Stokes (PNPS) model [24, 26], where the Nernst-Planck-Stokes equation is described in equation (3).

[0361]

[0362] In the case of electrodeless oSCR, the potential V is set to be constant within the simulation space. Therefore, equation (3) is further simplified (equation (4)), where the motion of ions is only driven by passive diffusion, chemical reaction, and fluid flow.

[0363]

[0364] where [c i ] represents the concentration of different ionic species. R i represents the chemical reaction term, and u represents the fluid velocity. Free Ca 2+ can bind with Fluo-8 or EDTA, as described in equations (1, 2).

[0365] For different ions, equation (4) is further extended, where the identity of the ion is annotated by the corresponding superscript, as described in equations (5-10). Where, FluoCa and EDTACa represent the binding form of Fluo-8 and EDTA with Ca 2+ , respectively.

[0366]

[0367]

[0368]

[0369]

[0370]

[0371]

[0372] The electrostatic potential in the standard PNPS model is governed by the Poisson equation, as described by equation (11):

[0373]

[0374] However, in the electrodeless oSCR process, the potential V is constant within the simulation space, so the equation simplifies to equation (12):

[0375] z Ca [Ca 2+ ]+z k [K + ]+z Cl [Cl - ] = 0 (12)

[0376] The simulation parameters are mainly obtained from

[26] . Among them, D is the diffusion constant (D Fluo = D FluoCa = 15 pm 2 s -1 ,D k = D cl = D Ca = D EDTA = D EDTACa = 200 pm 2 s -1 ). z is the charge number (z Ca = +2, z k = +1, z cl = -1). F is the Faraday constant. k b is the Boltzmann constant. T is the temperature (300 k). V is the potential. a is the forward binding rate (a E = 5 pm -1 s -1 , a F = 150 s -1 ). b is the reverse binding rate (b E = 0.75 pm -1 s -1 , b F = 450 s -1 ). Footnotes E and FEDTA and Fluo-8, respectively. ε is the dielectric constant of water. The boundary conditions on the cis side were set to different KCl concentrations (0.5 M to 2.5 M), while the boundary conditions on the trans side were set to 0.75 M CaCl2.

[0377] Comsol 5.3a was used to numerically solve the steady-state distribution of ions under different simulation conditions. Briefly, an axisymmetric simulation geometry was defined as two hemispherical spaces separated by a semipermeable membrane, which only allowed the passage of liquid but not ions Figure 2 ) between the two hemispheres on the cis and trans sides, respectively, were connected through a cylindrical nanopore on the membrane, where the passage of liquid and ions was allowed freely.

[0378] When illuminated in TIRF mode, the excitation intensity decays exponentially in the z-direction. To simulate the fluorescence intensity in the projected x-y plane, Equation (13) was used, where γ is the evanescent wave decay constant in the z-direction:

[0379]

[0380] While the total fluorescence intensity was estimated according to Equation (14):

[0381] F Total = ∫∫ F(x, y) dx dy (14)

[0382] 2D Gaussian fit

[0383] The fluorescence intensity profile, shown as a bright spot, during the electrodeless oSCR process, was fitted to a 2D Gaussian distribution according to Equation (15):

[0384]

[0385] where f(x, y) represents the fitted fluorescence intensity in the x-y plane. z0represents the base level, “A” represents the fitted amplitude, x c and y c represent the fitted centroid. σ x and σ y represent the standard deviation of the distribution in the x and y directions, respectively.

[0386] This function allows the position of a tracked point with sub-pixel resolution to be located. The full width at half maximum (FWHM) of the 2D Gaussian function describes the width at half height, which can be used to evaluate the size of the light spot. The 2D Gaussian fitting was performed using the cftool module in MATLAB Figure 5 ). The FWHM was derived from Equation (16):

[0387]

[0388] We define pixels in a circle of diameter 2 FWHM as signal and pixels in the annulus between circles of diameter 3 FWHM and 4 FWHM as background. Figure 5 ).

[0389] Signal-to-background ratio (SBR) evaluation

[0390] The SBR value is introduced to quantitatively evaluate the performance of DOP recording from different DOP recording experiments. The SBR value is calculated as follows:

[0391]

[0392] where peak(sig) is the peak amplitude (A + z0) of the signal obtained from 2D Gaussian fitting Figure 5 ). mean(bkg) is the mean pixel intensity of the background (z0). std(bkg) is the standard deviation of the background pixel intensity. The definitions of signal and background are shown in Figure 5 .

[0393] References

[0394] 1. André, B. An overview of membrane transport proteins in Saccharomyces cerevisiae. Yeast 11, 1575-1611 (1995).

[0395] 2. Gouaux, E. & MacKinnon, R. Principles of selective ion transport in channels and pumps. Science 310, 1461-1465 (2005).

[0396] 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).

[0397] 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).

[0398] 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).

[0399] 6. Bressloff, P. C. & Newby, J. M. Stochastic models of intracellular transport. Rev Mod Phys 85, 135-196 (2013).

[0400] 7. Deamer, D., Akeson, M. & Branton, D. Three decades of nanopore sequencing. Nat Biotechnol 34, 518-524 (2016).

[0401] 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).

[0402] 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).

[0403] 10. Bayley, H. et al. Droplet interface bilayers. Mol Biosyst 4, 1191-1208 (2008).

[0404] 11. Sapra, K. T. & Bayley, H. Lipid-coated hydrogel shapes as components of electrical circuits and mechanical devices. Sci Rep-Uk 2, 848 (2012).

[0405] 12. Li, J. et al. Ion-beam sculpting at nanometre length scales. Nature 412, 166-169 (2001).

[0406] 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).

[0407] 14. Geng, J. et al. Stochastic transport through carbon nanotubes in lipid bilayers and live cell membranes. Nature 514, 612 (2014).

[0408] 15. Chou, L. Y., Ming, K. & Chan, W. C. Strategies for the intracellular delivery of nanoparticles. Chemical Society Reviews 40, 233-245 (2011).

[0409] 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).

[0410] 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).

[0411] 18. Jain, M. et al. Nanopore sequencing and assembly of a human genome with ultra-long reads. Nat Biotechnol 36, 338-+(2018).

[0412] 19. Letellier, L., L., Bonhivers, M. & Boulanger, P. Phage DNA transport across membranes. Research in microbiology 150, 499-505 (1999).

[0413] 20. Song, L. et al. Structure of staphylococcal a-hemolysin, a heptameric transmembrane pore. Science 274, 1859-1865 (1996).

[0414] 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).

[0415] 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).

[0416] 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).

[0417] 24. Anderson, B. N. et al. Probing solid-state nanopores with light for the detection of unlabeled analytes. Acs Nano 8, 11836-11845 (2014).

[0418] 25. Ivankin, A. et al. Label-Free Optical Detection of Biomolecular Translocation through Nanopore Arrays. Biophys J 108, 331a-331a (2015).

[0419] 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).

[0420] 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).

[0421] 28. Gu, L. Q., Cheley, S. & Bayley, H. Capture of a single molecule in an anocavity. Science 291, 636-640 (2001).

[0422] 29. Kang, X. F., Cheley, S., Guan, X. Y. & Bayley, H. Stochastic detection of enantiomers. J Am Chem Soc 128, 10684-10685 (2006).

[0423] 30. Bayley, H. & Cremer, P. S. Stochastic sensors inspired by biology. Nature 413, 226 (2001).

[0424] 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).

[0425] 32. Braha, O. et al. Simultaneous stochastic sensing of divalent metal ions. Nat Biotechnol 18, 1005 (2000).

[0426] 33. Clarke, J. et al. Continuous base identification for single-molecule nanopore DNA sequencing. Nat Nanotechnol 4, 265 (2009).

[0427] 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).

[0428] 35. Boersma, A. J. & Bayley, H. Continuous stochastic detection of amino acid enantiomers with a protein nanopore. Angewandte Chemie 124, 9744-9747 (2012).

[0429] 36. Sacerdote, M. & Szostak, J. Semipermeable lipid bilayers exhibit diastereoselectivity favoring ribose. Proceedings of the National Academy of Sciences 102, 6004-6008 (2005).

[0430] 37. Raghunathan, A. & Aluru, N. Molecular understanding of osmosis in semipermeable membranes. Phys Rev Lett 97, 024501 (2006).

[0431] 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).

[0432] 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).

[0433] 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).

[0434] 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).

[0435] 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).

[0436] 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).

[0437] 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).

[0438] 45. Wendell, D. et al. Translocation of double-stranded DNA through membrane- adapted phi29 motor protein nanopores. Nat Nanotechnol 4, 765 (2009).

[0439] 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).

[0440] 47. Tinland, B., Pluen, A., Sturm, J. & Weill, G. Persistence length of single- stranded DNA. Macromolecules 30, 5763-5765 (1997).

[0441] 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).

[0442] 49. Chen, Z. T. et al. Centrifugal micro-channel array droplet generation for highly parallel digital PCR. Lab Chip 17, 235-240 (2017).

[0443] 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. An electrodeless system for identifying an analyte, the system comprising: (a) a first compartment having a first aqueous solution therein, wherein the first aqueous solution comprises a fluorescent reporter molecule capable of emitting fluorescence when bound to an ionic species; (b) a second compartment having a second aqueous solution therein, wherein the second aqueous solution comprises the ionic species that specifically binds to the fluorescent reporter molecule; and (c) a membrane separating the first compartment and the second compartment; wherein the membrane between the first compartment and the second compartment has at least one inserted nanopore such that the first compartment and the second compartment are connected by the nanopore; wherein there is a chemical gradient of the ionic species between the first compartment and the second compartment that can drive diffusion of the ionic species through the nanopore from the second compartment to the first compartment; wherein an analyte passing through the nanopore is capable of blocking or partially blocking the nanopore and impeding transport of the ionic species through the nanopore, resulting in a decrease in fluorescence from the fluorescent reporter molecule, thereby identifying the blocking analyte that passed through the nanopore.

2. The system of claim 1, wherein the membrane is a solid membrane.

3. The system of claim 1, wherein the membrane is a semi-permeable membrane.

4. The system of claim 3, wherein the second aqueous solution has a higher osmolality than the first aqueous solution or the second aqueous solution has a higher molality than the first aqueous solution; or the second aqueous solution has an equal osmolality as the first aqueous solution or the second aqueous solution has an equal molality as the first aqueous solution; or the second aqueous solution has a lower osmolality than the first aqueous solution or the second aqueous solution has a lower molality than the first aqueous solution.

5. The system of claim 3, wherein the semi-permeable membrane is comprised of an amphiphilic molecule.

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

7. The system of claim 3, wherein the semi-permeable membrane is a bilayer comprised of amphiphilic molecules.

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

9. The system of claim 8, wherein the lipid is one or more selected from the group consisting of a fatty acyl, a glycerolipid, a glycerophospholipid, a sphingolipid, a sterol lipid, an isoprenoid lipid, a saccharolipid, a polyketide, a phospholipid, a glycolipid, and a cholesterol. ​ 10. The system of claim 8, wherein the lipid is one or more selected from the group consisting of: monolein; 1,2-dioleoyl-sn-glycero-S-phosphocholine (DOPC); 1,2-diphytanoyl-sn-glycero-3-phosphatidylcholine (DPhPC); palmitoyloleoyl phosphatidylcholine (POPC); l-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE); l-palmitoyl-2-oleoyl-phosphatidylethanolamine and l-palmitoyl-2-oleoyl phosphatidylglycerol (POPE / POPG) mixture; and mixtures thereof.

11. The system of claim 1, wherein the first compartment is provided by an aqueous droplet.

12. The system of claim 1, wherein the second compartment is provided by a hydrogel layer.

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

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

15. The system of claim 1, wherein the nanopore is selected from the group consisting of a protein nanopore, a DNA nanopore, or a solid nanopore.

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

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

18. The system of any one of claims 1-17, wherein the ionic species is one or more selected from the group consisting of 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+ , OH - , Pb 2+ , Pd 2+ , Pt 2+ , Pt 4+ , Ru 3+ , Sb 3+ , Sc 3+ , Sn 2+ , Sr 2+ , Tb 3+ , Tl + , and Zn 2+ .

19. The system of any one of claims 1-17, 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 acids, 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.

20. The system of any one of claims 1-17, wherein the second aqueous solution comprises calcium chloride and optionally a buffer.

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

22. The system of any one of claims 1-17, wherein the first aqueous solution comprises a chelator and optionally a buffer, wherein the chelator is capable of binding to the ionic species.

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

24. The system of any one of claims 1-17, wherein the system further comprises a light source for illumination and a light sensor for detecting fluorescence.

25. The system of claim 24, wherein the light source is a laser, LED, halogen lamp, xenon lamp.

26. The system of claim 24, wherein the light sensor is a CCD, sCMOS sensor, photodiode.

27. The system of claim 26, wherein the light sensor is an EMCCD or avalanche photodiode.

28. The system of any one of claims 1-17, wherein the system further comprises a device for total internal reflection fluorescence (TIRF), wide-field fluorescence microscopy, or confocal microscopy.

29. The system of any one of claims 1-17, wherein the first aqueous solution or the second aqueous solution comprises the analyte.

30. The system of claim 29, wherein the analyte is selected from the group consisting of small molecules, macromolecules, and biological macromolecules.

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

32. The system of claim 31, wherein the polynucleotide is DNA or RNA.

33. The system of claim 32, wherein the DNA is dsDNA or ssDNA.

34. The system of claim 32, wherein the RNA is miRNA, siRNA, or tRNA.

35. A method of identifying an analyte, the method comprising the steps of: (a) providing the system of any one of claims 1-34, wherein the analyte is provided in the first compartment or the second compartment; (b) applying light capable of exciting the fluorescent reporter molecule to a region in the first compartment adjacent to the nanopore; (c) measuring a fluorescent signal from the fluorescent reporter molecule to identify the analyte; the method being a method for non-diagnostic purposes and non-therapeutic purposes.

36. A method of producing an electrodeless system, comprising: (a) providing a first compartment having a first aqueous solution therein, wherein the first aqueous solution comprises a fluorescent reporter molecule capable of emitting fluorescence when bound to an ionic species; (b) providing a second compartment having a second aqueous solution therein, wherein the second aqueous solution comprises the ionic species that specifically binds to the fluorescent reporter molecule; (c) bringing the first compartment and the second compartment together in a hydrophobic medium comprising an amphiphilic molecule, such that a semi-permeable membrane having an inserted nanopore is formed between the first compartment and the second compartment; wherein a protein nanopore is provided in the first aqueous solution or the second aqueous solution; wherein the electrodeless system is used to identify an analyte that, when passing through the nanopore, is capable of blocking or partially blocking the nanopore and obstructing the transport of the ionic species through the nanopore, resulting in a decrease in fluorescence from the fluorescent reporter molecule, thereby identifying the blocking analyte that passed through the nanopore.

37. The method of claim 36, wherein the second aqueous solution has a higher osmolality than the first aqueous solution or the second aqueous solution has a higher osmolarity than the first aqueous solution; or the second aqueous solution has an osmolality that is equal to the osmolality of the first aqueous solution or the second aqueous solution has an osmolarity that is equal to the osmolarity of the first aqueous solution; or the second aqueous solution has a lower osmolality than the first aqueous solution or the second aqueous solution has a lower osmolarity than the first aqueous solution.

38. The method of claim 36, wherein the semi-permeable membrane is comprised of an amphiphilic molecule.

39. The method of claim 38, wherein the amphiphilic molecule is a lipid or a triblock copolymer.

40. The method of claim 39, wherein the lipid is one or more selected from the group consisting of a fatty acyl, a glycerolipid, a glycerophospholipid, a sphingolipid, a sterol lipid, a prenol lipid, a saccharolipid, a polyketide, a phospholipid, a glycolipid, and a cholesterol.

41. The method of claim 39, wherein the lipid is one or more selected from the group consisting of glycerol monooleate; 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC); 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC); palmitoyloleoyl phosphatidylcholine (POPC); l-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE); l-palmitoyl-2-oleoyl-phosphatidylethanolamine and l-palmitoyl-2-oleoyl phosphatidylglycerol (POPE / POPG) mixture; and mixtures thereof.

42. The method of claim 36, wherein the first compartment is provided by an aqueous droplet.

43. The method of claim 36, wherein the second compartment is provided by a hydrogel layer.

44. The method of claim 43, wherein the hydrogel layer comprises 0.1-20% (w / v) agarose.

45. The method of claim 44, wherein the hydrogel layer comprises 2-5% (w / v) agarose.

46. The method of claim 36, wherein the nanopore is selected from the group consisting of a protein nanopore, a DNA nanopore, or a solid nanopore.

47. The method of claim 46, wherein the protein nanopore is one or more selected from the group consisting of a-HL, ClyA, Phi29 connector protein, Aerolysin, MspA, OmpF, OmpG, FraC, HlyA, SheA, sp1, and variants thereof; and an ion channel.

48. The method of claim 47, wherein the protein nanopore is ClyA-RR or a-HL.

49. The method of any one of claims 36-48, wherein the ionic species is one or more selected from the group consisting of 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+ , OH - , Pb 2+ , Pd 2+ , Pt 2+ , Pt 4+ , Ru 3+ , Sb 3+ , Sc 3+ , Sn 2+ , Sr 2+ , Tb 3+ , Tl + , and Zn 2+ .

50. The method of any one of claims 36-48, 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 acids, 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.

51. The method of any one of claims 36-48, wherein the second aqueous solution comprises calcium chloride and optionally a buffer.

52. The method of claim 51, wherein the concentration of calcium chloride in the second aqueous solution is 0.01-6.76 M.

53. The method of any one of claims 36-48, wherein the first aqueous solution comprises a chelator and optionally a buffer, wherein the chelator is capable of binding to the ionic species.

54. The method of claim 53, wherein the chelator is EDTA, BAPTA, EGTA, CyDTA, DTPA, EDDP, HIDA, IDA, NTA, NTPO, TTHA, CA, TA, GA, HEDTA, or DEG.

55. An electrodeless nanopore array for identifying a plurality of analytes, the nanopore array comprising a plurality of systems in parallel and each system comprising: (a) a first compartment having a first aqueous solution therein, wherein the first aqueous solution comprises a fluorescent reporter molecule capable of emitting fluorescence when bound to an ionic species; (b) a second compartment having a second aqueous solution therein, wherein the second aqueous solution comprises the ionic species that specifically binds to the fluorescent reporter molecule; and (c) a membrane separating the first compartment and the second compartment; wherein in each system, the membrane between the first compartment and the second compartment has at least one inserted nanopore, such that the first compartment and the second compartment are connected through the nanopore in each system; wherein in each system, there is a chemical gradient of the ionic species between the first compartment and the second compartment that can drive diffusion of the ionic species through the nanopore from the second compartment to the first compartment; wherein the plurality of systems are configured such that the measured fluorescence of each system can be distinguished; and wherein an analyte passing through the nanopore is capable of blocking or partially blocking the nanopore and impeding the transport of the ionic species through the nanopore, resulting in a decrease in fluorescence from the fluorescent reporter molecule, thereby identifying the blocking analyte passing through the nanopore.

56. The nanopore array of claim 55, wherein in each system, the membrane between the first compartment and the second compartment system is a solid membrane.

57. The nanopore array of claim 55, wherein in each system, the membrane between the first compartment and the second compartment is a semi-permeable membrane.

58. The nanopore array of claim 57, wherein in each system, the second aqueous solution has a higher osmolality than the first aqueous solution or the second aqueous solution has a higher osmolarity than the first aqueous solution; or the second aqueous solution has an equal osmolality as the first aqueous solution or the second aqueous solution has an equal osmolarity as the first aqueous solution; or the second aqueous solution has a lower osmolality than the first aqueous solution or the second aqueous solution has a lower osmolarity than the first aqueous solution.

59. The nanopore array of claim 57, wherein the semi-permeable membrane is comprised of an amphiphilic molecule.

60. The nanopore array of claim 59, wherein the amphiphilic molecule is a lipid or a triblock copolymer.

61. The nanopore array of claim 57, wherein the semi-permeable membrane is a bilayer comprised of amphiphilic molecules.

62. The nanopore array of claim 61, wherein the amphiphilic molecule is a lipid.

63. The nanopore array of claim 62, wherein the lipid is one or more selected from the group consisting of a fatty acyl, a glycerolipid, a glycerophospholipid, a sphingolipid, a sterol lipid, a prenol lipid, a saccharolipid, a polyketide, a phospholipid, a glycolipid, and a cholesterol.

64. The nanopore array of claim 62, wherein the lipid is one or more selected from the group consisting of: monolein; 1,2-dioleoyl-sn-glycero-S-phosphocholine (DOPC); 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC); palmitoyloleoyl phosphatidylcholine (POPC); l-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE); l-palmitoyl-2-oleoyl-phosphatidylethanolamine and l-palmitoyl-2-oleoyl phosphatidylglycerol (POPE / POPG) mixture; and mixtures thereof.

65. The nanopore array of claim 55, wherein the first compartment of the plurality of systems are separate from one another.

66. The nanopore array of claim 65, wherein the first compartment of each system is provided by an aqueous droplet.

67. The nanopore array of claim 55, wherein the second compartment of the plurality of systems is a single compartment.

68. The nanopore array of claim 67, wherein the second compartment of each system is provided by a hydrogel layer.

69. The nanopore array of claim 68, wherein the hydrogel layer comprises 0.1-20% (w / v) agarose.

70. The nanopore array of claim 69, wherein the hydrogel layer comprises 2-5% (w / v) agarose.

71. The nanopore array of claim 68, wherein the second compartment of the plurality of systems is provided by a single hydrogel layer.

72. The nanopore array of claim 55, wherein in each system, the nanopore is selected from the group consisting of a protein nanopore, a DNA nanopore, or a solid nanopore.

73. The nanopore array of claim 72, wherein the protein nanopore is one or more selected from the group consisting of: a-HL, ClyA, Phi29 connector protein, aerolysin, MspA, OmpF, OmpG, FraC, HlyA, SheA, sp1, and variants thereof; and an ion channel.

74. The nanopore array of claim 73, wherein the protein nanopore is ClyA-RR or a-HL.

75. The nanopore array of any one of claims 55-74, wherein in each system the ionic species is one or more selected from the group consisting of 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+ , OH - , Pb 2+ , Pd 2+ , Pt 2+ , Pt 4+ , Ru 3+ , Sb 3+ , Sc 3+ , Sn 2+ , Sr 2+ , Tb 3+ , Tl + , and Zn 2+ .

76. The nanopore array of any one of claims 55-74, wherein 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 acids, 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 nanopore array of any one of claims 55-74, wherein in each system, the second aqueous solution comprises calcium chloride and optionally a buffer.

78. The nanopore array of claim 77, wherein in each system, the concentration of calcium chloride in the second aqueous solution is 0.01-6.76 M.

79. The nanopore array of any one of claims 55-74, wherein in each system, the first aqueous solution comprises a chelator and optionally a buffer, wherein the chelator is capable of binding to the ionic species.

80. The nanopore array of claim 79, wherein the chelator is EDTA, BAPTA, EGTA, CyDTA, DTPA, EDDP, HIDA, IDA, NTA, NTPO, TTHA, CA, TA, GA, HEDTA, or DEG.

81. The nanopore array of any one of claims 55-74, wherein the array further comprises a light source for illumination and a light sensor for detecting fluorescence.

82. The nanopore array of claim 81, wherein the light source is a laser, LED, halogen lamp, xenon lamp.

83. The nanopore array of claim 81, wherein the light sensor is a CCD, sCMOS sensor, photodiode.

84. The nanopore array of claim 83, wherein the light sensor is an EMCCD or avalanche photodiode.

85. The nanopore array of claim 81, wherein the array further comprises a device for total internal reflection fluorescence (TIRF), wide-field fluorescence microscopy, or confocal microscopy.

86. The nanopore array of any one of claims 55-74, wherein in each system, the first aqueous solution or the second aqueous solution comprises the analyte.

87. The nanopore array of claim 86, wherein in each system, the analyte is selected from the group consisting of a small molecule, a large molecule, and a biological macromolecule.

88. The nanopore array of claim 86, wherein in each system, the analyte is selected from the group consisting of a chemical compound, a drug, a sugar, an ion, a neurotransmitter, an amino acid, a nucleotide, a polymer, a polypeptide, a polysaccharide, and a polynucleotide.

89. The nanopore array of claim 88, wherein the polynucleotide is DNA or RNA.

90. The nanopore array of claim 89, wherein the DNA is dsDNA or ssDNA.

91. The nanopore array of claim 89, wherein the RNA is miRNA, siRNA, or tRNA.

92. The nanopore array of claim 86, wherein different analytes are physically separated into various systems.

93. The nanopore array of any one of claims 55-74, wherein the density of the system in the nanopore array is 10-1000 per mm 2 , 94. The nanopore array of any one of claims 55-74, wherein a total area provided by the plurality of systems is 1-100 mm 2 .

95. A multiplexed method for identifying a plurality of analytes, the method comprising: (a) providing the nanopore array of any one of claims 55-94, wherein two or more analytes are provided in various systems of the nanopore array; (b) applying a light signal capable of exciting the fluorescent reporter molecule contained in each of the first compartments to a region adjacent to the nanopore in a plurality of first compartments; (c) measuring a plurality of fluorescent signals from the fluorescent reporter molecule contained in each system to identify the plurality of analytes; the method being a method for non-diagnostic and non-therapeutic purposes.

96. A method of producing an electrodeless nanopore array, comprising: (a) providing a plurality of aqueous droplets, wherein each of the aqueous droplets comprises a first aqueous solution comprising a protein nanopore, an analyte, and a fluorescent reporter molecule capable of emitting fluorescence when bound to an ionic species; (b) providing a hydrogel layer, wherein the hydrogel layer comprises the ionic species; (c) bringing the plurality of aqueous droplets and the hydrogel layer together in a hydrophobic medium comprising an amphiphilic molecule, such that a semipermeable membrane is formed between each of the aqueous droplets and the hydrogel layer; wherein the electrodeless nanopore array is used to identify an analyte that, when passing through the nanopore, is capable of blocking or partially blocking the nanopore and obstructing the transport of the ionic species through the nanopore, resulting in a decrease in fluorescence from the fluorescent reporter molecule, thereby identifying the blocking analyte that passed through the nanopore.

97. The method of claim 96, wherein the hydrogel has a higher osmolality than the osmolality of each aqueous droplet or the hydrogel has a higher osmolality 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.

98. The method of claim 96, wherein the semi-permeable membrane consists of an amphiphilic molecule.

99. The method of claim 98, wherein the amphiphilic molecule is a lipid or a triblock copolymer.

100. The method of claim 99, wherein the lipid is one or more selected from the group consisting of a fatty acyl, a glycerolipid, a glycerophospholipid, a sphingolipid, a sterol lipid, a prenol lipid, a saccharolipid, a polyketide, a phospholipid, a glycolipid, and a cholesterol.

101. The method of claim 99, wherein the lipid is one or more selected from the group consisting of glycerol monooleate; 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC); 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC); palmitoyloleoyl phosphatidylcholine (POPC); l-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE); l-palmitoyl-2-oleoyl-phosphatidylethanolamine and l-palmitoyl-2-oleoyl phosphatidylglycerol (POPE / POPG) mixture; and mixtures thereof.

102. The method of claim 96, wherein the hydrogel layer comprises 0.1-20% (w / v) agarose.

103. The method of claim 102, wherein the hydrogel layer comprises 2-5% (w / v) agarose.

104. The method of claim 96, wherein the nanopore is selected from the group consisting of a protein nanopore, a DNA nanopore, or a solid nanopore.

105. The method of claim 104, wherein the protein nanopore in each aqueous droplet is one or more selected from the group consisting of a-HL, ClyA, Phi29 connector protein, aerolysin, MspA, OmpF, OmpG, FraC, HlyA, SheA, sp1, and variants thereof; and an ion channel.

106. The method of claim 105, wherein the protein nanopore in each aqueous droplet is ClyA-RR or a-HL.

107. The method of any one of claims 96-106, wherein the ionic species in the hydrogel layer is one or more selected from the group consisting of 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+ , OH - , Pb 2+ , Pd 2+ , Pt 2+ , Pt 4+ , Ru 3+ , Sb 3+ , Sc 3+ , Sn 2+ , Sr 2+ , Tb 3+ , Tl + , and Zn 2+ .

108. The method of any one of claims 96-106, 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 acids, 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.

109. The method of any one of claims 96-106, wherein the hydrogel layer comprises calcium chloride and optionally a buffer.

110. The method of claim 109, wherein the concentration of calcium chloride in the hydrogel layer is 0.01-6.76 M.

111. The method of any one of claims 96-106, wherein each aqueous droplet comprises a chelator and optionally a buffer, wherein the chelator is capable of binding to the ionic species.

112. The method of claim 111, wherein the chelator is EDTA, BAPTA, EGTA, CyDTA, DTPA, EDDP, HIDA, IDA, NTA, NTPO, TTHA, CA, TA, GA, HEDTA, or DEG.

113. The method of any one of claims 96-106, wherein the analyte in each aqueous droplet is selected from the group consisting of small molecules, macromolecules, and biological macromolecules.

114. The method of any one of claims 96-106, 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.

115. The method of claim 114, wherein the polynucleotide is DNA or RNA.

116. The method of claim 115, wherein the DNA is dsDNA or ssDNA.

117. The method of claim 115, wherein the RNA is miRNA, siRNA, or tRNA.

118. The method of any one of claims 96-106, wherein different analytes are provided in various systems.

119. The method of any one of claims 96-106, wherein the number of aqueous droplets is 4-1,000,000.

120. The method of claim 119, wherein the number of aqueous droplets is 10-1000.

121. Use of the system of any one of claims 1-34 for optical analyte analysis, the use being a non-diagnostic and non-therapeutic use.

122. Use of the nanopore array of any one of claims 55-94 for optical analyte analysis, the use being a non-diagnostic and non-therapeutic use.

123. The method of any one of claims 36-48, wherein the analyte is provided in the first aqueous solution or the second aqueous solution.

124. The method of claim 123, wherein the analyte is selected from the group consisting of small molecules, macromolecules, and biological macromolecules.

125. The method of claim 123, 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.

126. The method of claim 125, wherein the polynucleotide is DNA or RNA.

127. The method of claim 126, wherein the DNA is dsDNA or ssDNA.

128. The method of claim 126, wherein the RNA is miRNA, siRNA, or tRNA.

129. The nanopore array of any one of claims 55-74, wherein the number of the plurality of systems is 4-1,000,000.

130. The nanopore array of claim 129, wherein the number of the plurality of systems is 10-1000.

131. A kit for forming a nanopore array, the kit comprising: a filled hydrogel comprising agarose, a buffer, and an ionic species capable of specifically binding to a fluorescent reporter molecule to cause it to emit fluorescence; an aqueous solution comprising a chelator, the fluorescent reporter molecule capable of emitting fluorescence when bound to the ionic species, and a buffer; wherein the chelator is capable of binding to the ionic species; a hydrophobic medium containing an amphiphilic molecule; and a solid support; wherein the nanopore array is used to identify an analyte capable of blocking or partially blocking a nanopore when it passes through the nanopore and obstructing the transport of the ionic species through the nanopore, resulting in a decrease in fluorescence from the fluorescent reporter molecule, thereby identifying a blocking analyte that passes through the nanopore.

132. The kit of claim 131, wherein the volume osmolality of the stentable hydrogel is higher than the volume osmolality of the aqueous solution or the weight osmolality of the stentable hydrogel is higher than the weight osmolality of the aqueous solution; or the volume osmolality of the stentable hydrogel is equal to the volume osmolality of the aqueous solution or the weight osmolality of the stentable hydrogel is equal to the weight osmolality of the aqueous solution; or the volume osmolality of the stentable hydrogel is lower than the volume osmolality of the aqueous solution or the weight osmolality of the stentable hydrogel is lower than the weight osmolality of the aqueous solution.

133. The kit of claim 131, wherein the amphiphilic molecule is a lipid or a tri-block copolymer.

134. The kit of claim 133, wherein the lipid is one or more selected from the group consisting of a fatty acyl, a glycerolipid, a glycerophospholipid, a sphingolipid, a sterol lipid, a prenol lipid, a saccharolipid, a polyketide, a phospholipid, a glycolipid, and a cholesterol.

135. The kit of claim 133, wherein the lipid is one or more selected from the group consisting of glyceryl monooleate; 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC); 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC); palmitoyloleoyl phosphatidylcholine (POPC); l-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE); l-palmitoyl-2-oleoyl-phosphatidylethanolamine and l-palmitoyl-2-oleoyl phosphatidylglycerol (POPE / POPG) mixture; and mixtures thereof.

136. The kit of claim 131, wherein the aqueous solution further comprises a nanopore.

137. The kit of claim 136, wherein the nanopore is selected from the group consisting of a protein nanopore, a DNA nanopore, or a solid nanopore.

138. The kit of claim 137, wherein the protein nanopore is one or more selected from the group consisting of a-HL, ClyA, Phi29 connector protein, aerolysin, MspA, OmpF, OmpG, FraC, HlyA, SheA, sp1, and variants thereof; and an ion channel.

139. The kit of claim 138, wherein the protein nanopore is ClyA-RR or a-HL.

140. The kit of any one of claims 131-139, wherein the ionic species in the hydrogel layer is one or more selected from the group consisting of 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+ , OH - , Pb 2+ , Pd 2+ , Pt 2+ , Pt 4+ , Ru 3+ , Sb 3+ , Sc 3+ , Sn 2+ , Sr 2+ , Tb 3+ , Tl + , and Zn 2+ .

141. The kit of any one of claims 131-139, 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 acids, 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.

142. The kit of any one of claims 131-139, wherein the filling hydrogel comprises calcium chloride to provide Ca 2+ as the ionic species.

143. The kit of claim 142, wherein the concentration of calcium chloride in the hydrogel layer is 0.01-6.76 M.

144. The kit of any one of claims 131-139, wherein the filled hydrogel comprises 2.5% agarose, 1.5 M CaCl2, and 10 mM HEPES, pH 7.

0.

145. The kit of any one of claims 131-139, wherein the aqueous solution further comprises KCl.

146. The kit of claim 145, wherein the aqueous solution can comprise 1.5 M KCl, 400 μΜ EDTA, 40 μΜ Fluo-8, 10 mM HEPES, pH 7.

0.

147. The kit of any one of claims 131-139, wherein the hydrophobic medium containing an amphiphilic molecule can be a lipid oil comprising a dry film of 5 mg DPHPC lipids dissolved in a 2 mL mixture of hexadecane and silicone oil at a volume ratio of 1:

1.

148. The kit of any one of claims 131-139, wherein the kit further comprises a coating hydrogel comprising agarose in water.

149. The kit of claim 148, wherein the coating hydrogel can comprise 0.75% (w / v) agarose in water.

Images