Sequential lateral flow device
The present disclosure provides methods and lateral flow devices for detecting a plurality of target analytes in a liquid sample. In some implementations, the disclosed lateral flow device comprises a housing unit, a capillary flow bed, a sample-receiving zone, a buffer-receiving zone, and a capture zone. The device is configured to control the flow of the sample and reagent buffer in a sequential manner with minimal mixing. In some implementations, the disclosed method is capable of detecting a plurality of target analytes in an assay by applying the binding agents and the signaling agents in separate or sequential steps.
1. A lateral flow device for detecting a plurality of target analytes in a liquid sample, the device comprising:
a housing unit comprising an inner surface that defines a cavity in the housing unit;
a capillary flow bed residing in the cavity, wherein the capillary flow bed transports the liquid sample from a proximal region of the capillary flow bed to a distal region of the capillary flow bed;
a buffer-receiving zone comprising a buffer-receiving pad (P B ) and a conjugate pad (P C ), the conjugate pad comprising a signaling agent for providing a detectable signal;
a capture zone (C) comprising a plurality of immobilized capture agents;
a sample-receiving zone disposed between the buffer-receiving zone and the capture zone, the sample-receiving zone comprising a sample-receiving pad (P S ), a transfer pad (P T ), and a substantially impermeable backing disposed between the sample-receiving zone and the capillary flow bed and extending at least partially underneath the sample-receiving pad and the transfer pad, the transfer pad comprising a plurality of binding agents that specifically bind to a plurality target analytes;
a bibulous reservoir pad (R) disposed in the distal region of the capillary flow bed;
a first inlet (I 1 ) in said housing for introducing the liquid sample into the sample-receiving pad, wherein the impermeable backing inhibits contact between the liquid sample and the capillary flow bed in the sample-receiving zone, thereby reducing backflow of the sample in a proximal direction;
a second inlet (I 2 ) in said housing for introducing a buffer into the buffer-receiving pad, wherein the buffer mobilizes the signaling agent to obtain a mobilized signaling agent; and
a reading window defined in the housing unit over the capture zone for observing a detectable signal produced by an interaction of the binding agent and the signaling agent in the presence of the target analyte;
wherein the bibulous reservoir pad draws the liquid sample and the mobilized signaling agent in the distal flow direction, and wherein the liquid sample flows along the capillary flow bed into the capture zone before the mobilized signaling agent flows into the capture zone.
2. The lateral flow device of claim 1 , wherein the inner surface of the housing unit comprises a series of ribs which retain excess fluid, thereby inhibiting the excess fluid from overflowing the sample-receiving pad or the buffer-receiving pad.
3. The lateral flow device of claim 2 , wherein the series of ribs are disposed over the sample-receiving pad on the upper inner surface of the housing unit.
4. The lateral flow device of claim 2 , wherein the series of ribs are disposed over the buffer-receiving pad on the upper inner surface of the housing unit.
5. The lateral flow device of claim 1 , wherein the inner surface of the housing unit comprises at least one pinch point pressing the buffer-receiving pad into the conjugate pad to create a flow path for the buffer to flow from the buffer-receiving pad to the conjugate-pad.
6. The lateral flow device of claim 1 , wherein the inner surface of the housing unit comprises at least one pinch point pressing the sample-receiving pad into the transfer pad to create a flow path for the liquid sample to flow from the sample-receiving pad to the transfer-pad.
7. The lateral flow device of claim 1 , wherein the inner surface of the housing unit comprises at least one pinch point pressing the transfer pad into a flow path bed to create a flow path for the liquid sample from the transfer-pad to the capillary flow bed.
8. The lateral flow device of claim 1 , wherein the capillary flow bed is slightly bent to promote capillary action and minimize flooding.
9. The lateral flow device of claim 1 , wherein the plurality of capture agents in the capture zone comprise one or more types of capture agents each adapted to a common antigen of at least a subset of target analytes in the liquid sample.
10. The lateral flow device of claim 1 , wherein the plurality of the capture agents bind a Gram-positive and/or Gram-negative bacterial antigen.
11. The lateral flow device of claim 1 , wherein the plurality of the capture agents comprise antibodies.
12. The lateral flow device of claim 1 , wherein the plurality of capture agents comprise one or more capture agent groupings on the capillary flow bed, the groupings spatially separated from each other, each grouping comprising an antibody that binds to a different target or analyte.
13. The lateral flow device of claim 1 , wherein the plurality of binding agents bind a Gram-positive and/or Gram-negative bacterial antigen.
14. The lateral flow device of claim 1 , wherein the plurality of binding agents comprise antibodies.
15. The lateral flow device of claim 1 , wherein each of the plurality of binding agents is labeled with a first member of a conjugate pair and the signaling agent is labeled with a second member of the conjugate pair.
16. The lateral flow device of claim 15 , wherein the conjugate pair comprises biotin and a biotin-binding protein, and either the binding agent or the signaling agent is labeled with biotin.
17. The lateral flow device of claim 1 , wherein the capillary flow bed comprises nitrocellulose.
18. The lateral flow device of claim 1 , wherein the sample-receiving pad, the buffer-receiving pad, the bibulous reservoir pad, and the conjugate pad each comprises a bibulous material.