Protein Quantification Using Controlled DNA Melting Transitions in Bivalent Probe Assemblies

Abstract

Homogenous protein assays, despite the potential for mix-and-read workflows, have eluded widespread acceptance due to interferences in biological matrices and limited multiplexability. Here, we employ standard qPCR instrumentation for thermofluorimetric analysis of bivalent probe (TFAB) assemblies, allowing protein levels to be quantitatively translated into multiplexable DNA melting transitions within 30 min. As protein-bound bivalent probes are thermodynamically more stable than unbound probes, differential thermal analysis can remove background analytically, without physical separation. Using either antibody-oligonucleotides or aptamers as probes, TFAB is validated for protein quantification in buffer, human serum, and human plasma and for assaying hormone secretions from endocrine cells. The direct optical method exhibits superior scalability, allowing detection of only 1 amol of protein in microfluidic channels of 100 pL volume. Overall, we demonstrate TFAB as a robust and generalizable homogeneous protein assay with superior performance in biological matrices.

Homogeneous Assays of Second Messenger Signaling and Hormone Secretion Using Thermofluorimetric Methods That Minimize Calibration Burden

“…We have previously shown that this approach allows separation of signal and background (two peaks for each trace in Figure 1B) and effectively negates matrix-based interferences. 10,13 …”

“…Interestingly, the quality of this separation was comparable to our prior results using thrombin aptamer pairs yet was significantly improved compared to our in-house synthesized antibody-oligos. 10 This enhancement could be a result of either the improved purification of the probes or the simplification of the proximity assembly in this version of the assay, which does not require connector oligos. Leveraging the blank (zero insulin) subtraction method approach presented in our prior work, 13 the insulin-dependent portions of the TFA curves could then be easily visualized (Figure 2B) and applied for the quantitative analysis presented below.…”

“…14,15 The recognition sequence of DNA is thus thermodynamically stabilized by the presence of cAMP, and a FRET pair can be used for readout. Again, we hypothesized that adaptation of our TFA method ology 10,13 to these probes would result in the concomitant improvements in performance.…”

“…In the future, it should be straightforward to adapt the antibody-oligonucleotide version of TFA to any protein or target with two antibodies (or aptamers), and the development of additional DNA-driven assays for small molecules and second messengers would expand the catalogue of TFA analytes. Multiplexing through spectrally resolved fluorophores and thermally resolved melting transitions has already been shown to be feasible, 10 thus, further work in this area should permit small-volume, mix-and-read assays on a variety of analytes to be carried out in a single tube. Finally, due to its nonamplified, direct-readout nature, adaptation of TFA to the microfluidic platform 10 is an obvious yet important field to explore in the future.…”

“…Multiplexing through spectrally resolved fluorophores and thermally resolved melting transitions has already been shown to be feasible, 10 thus, further work in this area should permit small-volume, mix-and-read assays on a variety of analytes to be carried out in a single tube. Finally, due to its nonamplified, direct-readout nature, adaptation of TFA to the microfluidic platform 10 is an obvious yet important field to explore in the future.…”