Biosensor Based on Capture Molecules with Different Affinities

A method for biosensing a target substance, the method comprising:

• bringing a matrix containing the target substance into contact with a sensor device having a surface, a collection of particles tethered to the surface by tether molecules, a collection of target-analogues, and a collection of capture molecules,
• wherein the collection of capture molecules are attached to the particles or to the surface, and wherein the collection of target-analogues is attached to the particles or to the surface,
• wherein the capture molecules are selected to bind to the target substance and have a target dissociation rate, and the target-analogues are selected to bind to the capture molecules and have a target-analogue dissociation rate that differs from the target dissociation rate by at least a factor of three;
• detecting a time series of a spatial coordinate parameter of the particles, wherein the spatial coordinate parameter is measured relative to the surface,
• identifying within the detected time series a time sequence of individual association/dissociation events of the target substance with the capture molecules,
• wherein each of the events corresponds to a change between binding states of a capture molecule to a target substance or to a target-analogue;
• determining a concentration of the target substance in the matrix from the identified time sequence of individual association/dissociation events.

A biosensing device for detecting a target substance, the device comprising:

• a cell comprising on its interior a biosensing surface;
• a light source that emits light into the cell;
• a light detector that senses light from the cell;
• a processor that determines concentration of the target substance in the cell from sensed light from the cell;
• wherein the biosensing surface has a surface, a collection of particles tethered to the surface by tether molecules, and a collection of capture molecules,
• wherein the collection of capture molecules comprises first capture molecules bound to the particles or to the tether molecules, and second capture molecules bound to the surface or to the tether molecules,
• wherein the first capture molecules are selected to bind to the target substance and have a first target dissociation rate, and the second capture molecules are selected to bind to the target substance and have a second target dissociation rate that differs from the first target dissociation rate by at least a factor of three;
• wherein the processor detects a time series of a spatial coordinate parameter of the particles, and identifies within the detected time series a time sequence of individual association/dissociation events of the target substance with the capture molecules, and determines the concentration from the identified time sequence of individual association/dissociation events,
• wherein each of the events corresponds to a change between binding states of a first capture molecule and a second capture molecule to a target substance.