Label-free methods of sensing

A method of sensing comprising:

• providing a sensor comprising:
• a first layer having at least one single nanohole structure or at least one dual nanohole structure, and
• a second layer having at least one nanopore,
• wherein the single nanohole structure comprises only one nanohole,
• wherein the dual nanohole structure comprises a first nanohole and a second nanohole connected by a gap, and
• wherein the one nanohole or the gap of the first layer is aligned with the nanopore of the second layer in a direction corresponding to a translocation direction across the first and second layers;
• providing a test sample comprising an analyte;
• contacting the test sample with the first layer of the sensor;
• irradiating the single nanohole structure or the dual nanohole structure of the first layer of the sensor with a beam of electromagnetic radiation;
• optically trapping the analyte in the single nanohole structure or in the dual nanohole structure and/or in the gap of the first layer of the sensor;
• applying a first electric field across the nanopore to draw one or more of the analytes into the nanopore, wherein the first electric field comprises a direct current (DC) electric field;
• applying a second electric field across the nanopore after applying the first electric field, wherein the second electric field comprises a pulsed, modulated, or alternating current (AC) electric field;
• and
• measuring one or more of:
• change in current and/or phase across the nanopore during application of the second electric field while the analyte is optically trapped and/or during one or more translocation events of the analyte through the nanopore;
• or
• at least one kinetic parameter of the analyte within the nanopore after removing or turning off the second electric field.