Apparatus for analyzing and detecting interactions and reactions of molecules
View Patent ↗An apparatus for label-free analysis of molecules, including interactions and reactions of the molecules, is disclosed. The apparatus is based on detecting molecule movement under the influence of an external electric field. The apparatus is able to achieve sensitive detection of molecular binding to proteins or other molecules, and conformational changes of proteins or other molecules and biochemical reactions of the proteins or other molecules. Applications of the apparatus include screening of drug molecules, kinetic analysis of posttranslational modification of proteins, and small molecule-protein interactions.
1. An apparatus for analyzing and detecting interactions and reactions of proteins, comprising:
an electrically conductive surface coated with streptavidin;
a molecular bridge binding a protein to said surface, wherein the molecular bridge comprises polyethylene glycol having a number average molar mass of 1.6 kD to 160 kD and a biotin group and a N-hydroxysuccinimide group on either end of the polyethylene glycol, and wherein the biotin group conjugates the streptavidin on the surface;
at least one electrode forming an alternating current (AC) electric field comprising a frequency between the at least one electrode and the surface and generating a response of the protein, wherein the response of the protein is periodic movement in response to the AC electric field;
a solution, wherein the surface, the molecular bridge, the protein, and at least a portion of the electrode are submerged in the solution and the electrode is disposed in the solution so that a direction of the electric field generated is substantially orthogonal to the surface;
a light emitting device exciting surface plasmons in the surface;
a sensor measuring a change in a distance between the protein and the surface; and
a filter which detects the movement of the protein in the electric field thereby selectively detecting the response of the protein at the frequency.
2. The apparatus of claim 1 , wherein the protein is directly bound to the molecular bridge.
3. The apparatus of claim 1 , wherein the solution has a pH of about 5.3.
4. The apparatus of claim 1 , wherein the surface comprises a glass substrate coated with a metal film.
5. The apparatus of claim 4 , wherein the metal film has a thickness of about 47 nm and comprises gold.
6. The apparatus of claim 1 , wherein the surface comprises a glass substrate coated with indium tin oxide.
7. The apparatus of claim 1 , wherein the surface comprises a glass substrate coated with graphene.
8. The apparatus of claim 1 , wherein the surface comprises a glass substrate coated with a thin carbon film.
9. The apparatus of claim 1 , wherein the molecular bridge has a length from about 10 nm to about 999 nm.
10. The apparatus of claim 1 , wherein a voltage of less than 10 volts is used to generate the electric field.
11. The apparatus of claim 1 , wherein the frequency is from about 0.1 Hz to 10,000 Hz.
12. The apparatus of claim 1 , further comprising a reference electrode.
13. The apparatus of claim 1 , wherein the light emitting device is selected from the group consisting of a laser, a light emitting diode, and a superluminescent light emitting diode.
14. The apparatus of claim 1 , wherein the sensor is selected from the group consisting of a surface plasmon resonance (SPR) sensor, a total internal reflection microscope, and a transmitted optical microscope.
15. The apparatus of claim 1 , wherein the filter is a fast Fourier transform (FFT) filter.
16. The apparatus of claim 1 , wherein the response is associated with a change in a charge of the protein.
17. An apparatus for analyzing and detecting interactions and reactions of proteins, comprising:
an electrically conductive surface coated with streptavidin;
a molecular bridge for binding a protein to the surface, wherein the molecular bridge comprises polyethylene glycol having a number average molar mass of 1.6 kD to 160 kD and a biotin group and a N-hydroxysuccinimide group on either end of the polyethylene glycol, and wherein the biotin group conjugates the streptavidin on the surface;
at least one electrode forming an electric field between the at least one electrode and the surface; and
a solution, wherein the surface, the molecular bridge, and at least a portion of the electrode are submerged in the solution.