Genomic sequencing using modified protein pores and ionic liquids

A method of detecting the presence of one or more analytes in a liquid sample, comprising the steps of:

• contacting one or more analytes in the liquid sample with boromycin to form an analyte-boromycin mixture;
• incubating the analyte-boromycin mixture for at least 30 minutes at room temperature;
• placing the analyte-boromycin mixture in a trans compartment of a single-channel sensor;
• contacting the analyte-boromycin mixture with a pore assembly comprising a synthetic membrane or wild type or modified bacterial transmembrane protein covalently or non-covalently coupled with an agent that modifies ionic current;
• applying a potential to the chamber;
• determining a current across the applied potential;
• measuring one or more transient blockades in the ionic current;
• and
• comparing the transient blockades in the ionic current to one or more known transient current blockades to determine the identity of the one or more analytes.

An organic ion conducting solution composition comprising:

• a solvent comprising an organic ion conducting molecule, the molecule comprising:
• one or more heterocyclic rings comprising one or more heteroatoms;
• one or more side-chains attached to the one or more heteroatoms;
• and
• one or more negatively charged groups associated with the one or more of heteroatoms to form an ion conducting solution.

An organic ion conducting solution composition comprising:

• a solvent comprising an organic ion conducting molecule, the molecule comprising:
• one or more acyclic heteroatoms;
• one or more side-chains attached to the one or more heteroatoms;
• and
• one or more negatively charged groups are associated with the one or more of heteroatoms to form an organic ion conducting solution.

A method of synthesizing an organic ion conducting solution, comprising the steps of:

• heating a mixture comprising the acyclic or heterocyclic compound and the side-chain derivative with stirring at 60° C. or greater for at least 6 hours to form the organic ionic compound;
• dissolving the organic ionic compound in water;
• removing the excess acyclic or heterocyclic compound and the side-chain derivative by organic solvent extraction;
• repeating the solvent extraction process;
• and
• evaporating the water using a rotary evaporator to isolated the organic ionic liquid.

A method of synthesizing butylymethylimidazolium chloride, comprising the steps of:

• heating a mixture comprising 1-methylimidazole and 1-chlorobutane with stirring at 60° C. or greater for at least 6 hours to form the butylmethylimidazolium chloride;
• dissolving the butylymethylimidazolium chloride in water;
• removing the excess 1-methylimidazole and 1-chlorobutabe by solvent extraction with ethyl acetate;
• repeating the solvent extraction with ethyl-acetate;
• and
• evaporating the water using a rotary evaporator to isolated the butylymethylimidazolium chloride.

A method of slowing down translocation of one or more analytes in a nanopore sensor with a genetically modified bacterial transmembrane protein pore assembly by a technique comprising one or more of the following approaches:

• forming a weak non-covalent bond between the analytes and the protein pore assembly;
• employing an organic salt solution;
• changing the concentration of an ionic salt in the buffer;
• changing temperature of the nanopore sensor assembly;
• changing the pH of a buffer system;
• and
• varying a dielectric field.

A method for generating an oligonucleotide fingerprint comprising the steps of:

• dissolving one or more oligonucleotides in water or a buffer solution containing an ionic salt to form a solution;
• placing the oligonucleotide solution in a cis compartment of a single-channel sensor;
• contacting the solution with a pore assembly comprising a genetically modified bacterial transmembrane protein toxin;
• applying an electric potential to the sensor;
• determining an ionic current across the electric potential;
• measuring one or more transient blockades in the ionic current;
• and
• identifying one or more current modulations or sub-states in the ionic current.

A method of sequencing from an oligonucleotide fingerprint comprising the steps of:

• determining one or major current value states (I1 and I0) and an amplitude ΔI (=I1−I0) from an all-points histogram;
• determining one or more probability values of the major current value states (i.e., PI0 and PI1);
• and
• comparing the major current value states and the probability values with different oligonucleotides to identify molecules with identical base compositions with different sequences.

A single-channel, dual-chamber molecular analysis device comprising:

• a cis chamber;
• a trans chamber;
• a boundary layer comprising a lipid bi-layer or any natural or synthetic membrane on a Teflon septum separating the cis and trans chambers;
• a genetically modified bacterial transmembrane protein pore attached to the boundary layer;
• a conducting electrolyte in the chamber;
• and
• a terminus for establishing electrical connectivity between the cis and trans chambers.

A method for fabricating a single-channel, dual-chamber molecular analysis device, comprising the steps of:

• depositing a bilayer comprising two individual monolayers of a lipid molecule in an aperture of a Teflon septum;
• forming the bilayer at an air-water interface by hydrophobic apposition and the joining of the hydrocarbon chains of at least one individual monolayer;
• monitoring the bilayer formation using a function generator;
• adding a pore selected from a wild type bacterial transmembrane protein or a modified bacterial transmembrane protein to the bilayer or utilizing a porous synthetic membrane;
• and
• adding the conducting electrolyte to the chambers.