Method of determining target nucleic acid

A method of determining the presence and/or amount of single-stranded RNA sequences, the method comprising steps of:

• a) delivering a plurality of probes into the cells, wherein:
• each of the plurality of probes is capable of binding with the single-stranded RNA sequences present in the cells to form a double-stranded sequence, each of the plurality of probes has an overhang sequence at an end, and each of the single-stranded RNA sequences has a known sequence and is composed of 15 to 30 nucleotides;
• b) inserting an array of functionalized nanoneedles into the cells to bind with the double-stranded sequence, wherein the functionalized nanoneedles comprise a RNA binding protein for binding with the double-stranded sequence, wherein the RNA binding protein does not bind with any double-stranded sequence having less than 15 nucleotides or more than 30 nucleotides or any single stranded sequence;
• c) removing the array of functionalized nanoneedles with the bound double-stranded sequence from the cells;
• d) hybridizing the bound double-stranded sequence starting at the overhang sequence with an initiating agent, a first DNA sequence and a second DNA sequence to produce a hybridized product, the initiating agent being a single-stranded nucleotide sequence, the first DNA sequence being partially complementary to the initiating agent and partially complementary to the second DNA sequence, and the second DNA sequence being partially complementary to the first DNA sequence and partially identical to the initiating agent for binding with another first DNA sequence to extend the bound double-stranded sequence, at least one of the first and second DNA sequence comprising a fluorophore molecule, the fluorophore molecule being 1,1′-bis(3-hydroxypropyl)-3,3,3′,3′-tetramethylindocarbocyanine;
• e) washing the functionalized nanoneedles bound with the double-stranded sequence to remove impurities from the functionalized nanoneedles;
• and
• f) determining the presence and/or amount of the single-stranded RNA sequences in cells, which have a complementary sequence to the probes, based on signals of the fluorophore molecule,
• wherein the detection limit of the method is 10−16 M.