Systems and methods for enhanced detection and quantification of analytes
Devices, systems, and methods for detecting molecules of interest within a collected sample are described herein. In certain embodiments, self-contained sample analysis systems are disclosed, which include a reusable reader component, a disposable cartridge component, and a disposable sample collection component. The reader component may communicate with a remote computing device for the digital transmission of test protocols and test results. In various disclosed embodiments, the systems, components, and methods are configured to identify the presence, absence, and/or quantity of particular nucleic acids, proteins, or other analytes of interest, for example, in order to test for the presence of one or more pathogens or contaminants in a sample.
1. A method for multiplexing first and second types of target analytes in a sample to determine at least one of a presence, absence, or quantity of the first and second types of target analytes in the sample, the method comprising:
introducing the sample comprising the first and second types of target analytes into fluid in a sample preparation reservoir of a sample analysis cartridge;
directly or indirectly binding the first type of target analyte to a first signaling agent in the fluid in the sample preparation reservoir, the first signaling agent having a first detectable potential for the first type of target analyte;
directly or indirectly binding the second type of target analyte to a second signaling agent in the fluid in the sample preparation reservoir, the second signaling agent having a second detectable potential, different from the first detectable potential, for the second type of target analyte;
releasing the fluid from the sample preparation reservoir into an analysis channel comprising a sensor such that the first type of target analyte directly or indirectly bound to the first signaling agent and the second type of target analyte directly or indirectly bound to the second signaling agent;
releasing a substrate solution from a substrate reservoir into the analysis channel, the substrate solution comprising a substrate;
reacting the substrate with the first signaling agent and the second signaling agent localized over the sensor;
electrically stimulating the reacted substrate-first signaling agent complex, and the reacted substrate-second signaling agent complex to generate a first signal and a second signal, which are detected by the sensor, wherein the signal for each substrate-signaling agent complex is based on the respective potential of the signaling agent for the analyte, and the sensor simultaneously detects the first and second signals;
and
processing the first and second signals to determine at least one of the presence, absence, or quantity of the first type of target analyte and the second type of target analyte.
2. The method of claim 1 , wherein the first type of target analyte directly or indirectly bound to the first signaling agent is a first amplicon complex in the sample preparation reservoir and the second type of target analyte directly or indirectly bound to the second signaling agent is a second amplicon complex in the sample preparation reservoir.
3. The method of claim 2 , wherein the first and second amplicons are amplified in the sample preparation reservoir with polymerase and primers.
4. The method of claim 1 , wherein the first and second signaling agents are redox dye and the substrate is ferricyanide or phosphate buffering saline (PBS).
5. The method of claim 1 , wherein the electrically stimulating is conducted using cyclic voltammetry, square wave voltammetry, or amperometry.
6. The method of claim 1 , wherein the first type of target analyte directly or indirectly bound to the first signaling agent and the second type of target analyte directly or indirectly bound to the second signaling agent are each directly or indirectly bound to a magnetic particle that permits localization over the sensor in the analysis channel via magnetic attraction to one or more magnetic generators.
7. The method of claim 1 , wherein the substrate is a first type of substrate and the substrate solution further comprises a second type of substrate, the method further comprising:
reacting the second type of substrate with the first signaling agent;
electrically stimulating the reacted second type of substrate-first signaling agent complex to generate a third signal;
reacting the second type of substrate with the second signaling agent;
electrically stimulating the reacted second type of substrate-second signaling agent complex to generate a fourth signal, wherein the signal for each second type of substrate-signaling agent complex is based on the respective potential of the signaling agent for the analyte, and the sensor simultaneously detects the third and fourth signals; and
processing the third and fourth signals to determine at least one of the presence, absence, or quantity of a third type of target analyte and a fourth type of target analyte.
8. A method for multiplexing first and second types of target analytes in a sample to determine at least one of a presence, absence, or quantity of the first and second types of target analytes in the sample, the method comprising:
introducing the sample comprising the first and second types of target analytes into fluid in a sample preparation reservoir of a sample analysis cartridge;
directly or indirectly binding the first type of target analyte to a signaling agent in the fluid in the sample preparation reservoir;
directly or indirectly binding the second type of target analyte to a signaling agent in the fluid in the sample preparation reservoir;
releasing the fluid from the sample preparation reservoir into an analysis channel comprising a sensor such that the first type of target analyte directly or indirectly bound to the signaling agent and the second type of target analyte directly or indirectly bound to the signaling agent;
releasing a substrate solution from a substrate reservoir into the analysis channel, the substrate solution comprising a first type of substrate having a first detectable potential for the first type of target analyte and a second type of substrate having a second detectable potential, different from the first detectable potential, for the second type of target analyte;
reacting the first type of substrate with the signaling agent;
electrically stimulating the reacted first type of substrate-signaling agent complex to generate a first signal;
reacting the second type of substrate with the signaling agent;
electrically stimulating the reacted second type of substrate-signaling agent complex to generate a second signal, wherein the signal for each substrate-signaling agent complex is based on the respective potential of the substrate, and wherein the sensor simultaneously detects the first and second signals; and
processing the first and second signals to determine at least one of the presence, absence, or quantity of the first type of target analyte and the second type of target analyte.
9. The method of claim 8 , wherein the first type of target analyte directly or indirectly bound to the signaling agent is a first amplicon complex in the sample preparation reservoir and the second type of target analyte directly or indirectly bound to the signaling agent is a second amplicon complex in the sample preparation reservoir.
10. The method of claim 8 , wherein the first amplicon complex and the second amplicon complex are amplified in the sample preparation reservoir with polymerase and primers.
11. The method of claim 8 , wherein the signaling agents are horseradish peroxidase (HRP) and redox dye and the first type of the substrate is Tetramethylbenzidine (TMB) and the second type of substrate is ferricyanide.
12. The method of claim 8 , wherein the electrically stimulating is conducted using cyclic voltammetry, square wave voltammetry, or amperometry.
13. The method of claim 8 , wherein the first type of target analyte directly or indirectly bound to the signaling agent and the second type of target analyte directly or indirectly bound to the signaling agent are each directly or indirectly bound to a magnetic particle that permits localization over the sensor in the analysis channel via magnetic attraction to one or more magnetic generators.
14. The method of claim 8 , wherein the signaling agent is a first signaling agent and the fluid in the sample preparation reservoir further comprises a second signaling agent, the method further comprising:
reacting the first type of substrate with the second signaling agent;
electrically stimulating the reacted first type of substrate-second signaling agent complex to generate a third signal;
reacting the second type of substrate with the second signaling agent;
electrically stimulating the reacted second type of substrate-second signaling agent complex to generate a fourth signal, wherein the signal for each substrate-signaling agent complex is based on the respective potential of the substrate, and wherein the sensor simultaneously detects the first and second signals; and
processing the third and fourth signals to determine at least one of the presence, absence, or quantity of a third type of target analyte and a fourth type of target analyte.
15. The method of claim 1 , further comprising amplifying the first type of target analyte and the second type of target analyte, the amplifying comprising isothermal amplification.
16. The method of claim 15 , wherein the isothermal amplification comprises one or more of rolling circle amplification, loop-mediated isothermal amplification, strand displacement amplification, recombinase polymerase amplification, helicase dependent amplification, polymerase spiral reaction, and nicking enzyme amplification reaction.
17. The method of claim 8 , further comprising amplifying the first type of target analyte and the second type of target analyte, the amplifying comprising isothermal amplification.
18. The method of claim 17 , wherein the isothermal amplification comprises one or more of rolling circle amplification, loop-mediated isothermal amplification, strand displacement amplification, recombinase polymerase amplification, helicase dependent amplification, polymerase spiral reaction, and nicking enzyme amplification reaction.