Microwell assay plate and related methods
A microplate comprising: a plurality of wells arranged in a two-dimensional array, each of the wells comprising: a bottom surface, sidewalls extending from the bottom surface to form an open top; and at least two subwells in the bottom surface, the at least two subwells having sidewalls that extend below the bottom surface of the well, wherein each of the at least two subwells comprises a capture binding agent that is configured to bind to a target analyte, if present, in a sample. A sample is added to the well such that the sample fluidically contacts each of the at least two subwells such that a target analyte, if present, binds to the capture binding agent in one or more of the at least two subwells.
1. A method of detecting a target analyte and/or performing a spatial multiplexed assay, the method comprising:
providing a microplate comprising:
a plurality of wells arranged in a two-dimensional array, each of the wells comprising:
a bottom surface,
sidewalls extending from the bottom surface to form an open top;
at least two subwells in the bottom surface, the at least two subwells having sidewalls that extend below the bottom surface of the well, wherein each of the at least two subwells comprises a capture binding agent that is configured to bind to a target analyte, if present, in a sample;
adding a sample to the well such that the sample fluidically contacts each of the at least two subwells such that a target analyte comprising human chorionic gonadotropin (hCG) or placental growth factor (PIGF), if present, binds to the capture binding agent in one or more of the at least two subwells, wherein an upconverting nanoparticle (UCNP) labeled conjugate in the sample binds with the target analyte, if present; and
detecting a label of the upconverting nanoparticle (UCNP) labeled conjugate at one or more of the at least two subwells to thereby determine whether the target analyte is present in the sample.
2. The method of claim 1 , wherein the capture binding agent comprises at least a first capture binding agent and a second capture binding agent in different ones of the at least two subwells.
3. The method of claim 2 , wherein the UCNP labeled conjugate comprises a first UCNP labeled conjugate and a second UCNP labeled conjugate that are configured to bind to corresponding target analytes bound to the first and second capture binding agents, respectively, in the at least two subwells.
4. The method of claim 3 , wherein a UCNP of the first and second UCNP labeled conjugates is the same UCNP, and detecting a label of the labeled conjugate at one or more of the at least two subwells to thereby determine whether the target analyte is present in the sample comprises determining whether first and second target analytes are present based on which of the at least two subwells emit a signal from the UCNP labeled conjugate.
5. The method of claim 4 , wherein detecting a UCNP label of the UCNP labeled conjugate at one or more of the at least two subwells comprises impinging photons on the at least two subwells such that the UCNP labeled conjugate emits a signal.
6. The method of claim 5 , further comprising determining a concentration of the target analyte based on a signal strength of a signal from the UCNP labeled conjugate, wherein the signal has a signal strength that corresponds to a concentration of the target analyte, if present, in the sample.
7. The method of claim 6 , wherein at least one of the first and second capture binding agents comprises a control capture binding agent and other of the first and second capture binding agents comprises a test capture binding agent, wherein a signal strength of a UCNP labeled conjugate bound to the test capture binding agent relative to a signal strength of a UCNP labeled conjugate bound to the control capture binding agent corresponds to the concentration of the target analyte, if present, in the sample.
8. The method of claim 6 , wherein determining the concentration of the target analyte based on the signal strength of the signal from the UCNP labeled conjugate is based on an empirically-based model of actual experience.
9. The method of claim 1 , wherein the capture binding agent comprises a first half of a binding pair and the capture binding agent is bound to a surface of the at least two subwells via binding of the first half of the binding pair to a second half of the binding pair on the surface of the at least two subwells.
10. The method of claim 1 , wherein the upconverting nanoparticle (UCNP) labeled conjugate is present on a bottom and/or side of the well prior to adding the sample to the well.
11. The method of claim 1 , further comprising adding the upconverting nanoparticle (UCNP) labeled conjugate to the sample.
12. The method of claim 1 , wherein the target analyte is human chorionic gonadotropin (hCG) and the sample comprises a culture media in which an embryo is cultured.
13. The method of claim 1 , wherein the target analyte is placental growth factor (PIGF) and the sample comprises a culture media in which an embryo is cultured.
14. A method of detecting a target analyte and/or performing a spatial multiplexed assay, the method comprising:
providing a microplate comprising:
a plurality of wells arranged in a two-dimensional array, each of the wells comprising:
a bottom surface,
sidewalls extending from the bottom surface to form an open top;
at least two subwells in the bottom surface, the at least two subwells having sidewalls that extend below the bottom surface of the well, wherein each of the at least two subwells comprises a capture binding agent that is configured to bind to a target analyte, if present, in a sample, wherein the target analyte comprises human chorionic gonadotropin (hCG);
adding a sample to the well such that the sample fluidically contacts each of the at least two subwells such that a target analyte, if present, binds to the capture binding agent in one or more of the at least two subwells, wherein an upconverting nanoparticle (UCNP) labeled conjugate in the sample binds with the target analyte, if present, wherein the sample comprises a culture media in which an in vitro embryo is cultured;
detecting a label of the upconverting nanoparticle (UCNP) labeled conjugate at one or more of the at least two subwells to thereby detect the target analyte comprising hCG in the sample; and
evaluating the in vitro embryo based on the detection of the target analyte comprising hCG in the sample comprising the culture media in which the in vitro embryo is cultured.
15. The method of claim 14 , wherein the sample media comprises between 1 and 100 μL of culture media in which the embryo was cultured.
16. The method of claim 14 , wherein detecting the label of the upconverting nanoparticle (UCNP) labeled conjugate at one or more of the at least two subwells to thereby detect the target analyte comprising hCG comprises detecting a concentration of hCG at a limit of detection of less than 25 mIU/mL.
17. The method of claim 14 , wherein the target analyte comprises human chorionic gonadotropin (hCG) is a first target analyte in a first one of the at least two subwells, wherein a second target analyte in a second one of the at least two subwells comprises placental growth factor (“PIFG”), and evaluating the in vitro embryo further comprises evaluating the in vitro embryo based on the detection of the first target analyte comprising hCG in the sample and the second target analyte comprising PIFG comprising the culture media in which the in vitro embryo is cultured.
18. The method of claim 14 , wherein evaluating the in vitro embryo comprises evaluating samples from first and second culture media.
19. The method of claim 18 , wherein the first and second culture media are obtained from different days after fertilization of the embryo.
20. The method of claim 14 , wherein evaluating the in vitro embryo comprises comparing a signal strength of the label with an unknown concentration of target analyte with the signal strength(s) of the label in reference sample(s) with a known concentration of the target analyte.