Harnessing Contact-Quenched, Profluorescent Chemical Probes for Sensitive Determination and High-Throughput Measurements of Enzyme Activity
Bibliographic record
Abstract
High Resolution Image Download MS PowerPoint Slide Dual-labeled, profluorescent chemical probes have been developed to quantify and visualize a specific enzyme’s activity in complex biological media. The intact chemical probes often exhibit minimal fluorescence due to fluorescence quenching, but their intrinsic fluorescence can be released by a favorable enzyme-catalyzed reaction. Contact quenching represents one of several fluorescence quenching mechanisms. However, it is seldom intentionally implemented in the synthesis of dual-labeled, profluorescent chemical probes, because the structure of a contact quenching construct requires the formation of a ground-state fluorophore-quencher/-fluorophore complex. The ability of such dual-labeled molecular probes to act as intramolecular dimers cannot be predicted. We previously revealed that a mono exo -bicyclo[6.1.0]nonyne ( exo -BCN)-derivatized cystamine framework was critical to synthesizing sensitive, dual-labeled, profluorescent chemical probes capable of contact quenching. Here, we exploited the nonsymmetrical mono - exo -BCN-cystamine backbone by sequentially coupling it with two different carboxyfluorescein (FAM) derivatives and subsequently synthesizing four nonsymmetrical bis -FAM chemical probes. The fluorescence turn-on properties of the bis -FAM chemical probe with the lowest background FAM fluorescence were characterized by kinetic studies. Moreover, the release of two FAM equivalents from the fluorescence turn-on chemical probe during reactions was utilized to develop sensitive assays for measuring the activity and inhibition of two serum biomarkers, butyrylcholinesterase (BChE) and paraoxonase 1 (PON1) lactonase. We also developed an efficient, high-throughput assay for detecting BChE activity based on the chemical probe. Finally, the fluorescence assays successfully quantified the activities of BChE and PON1 lactonase in human serum.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.000 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.001 | 0.001 |
| Open science | 0.001 | 0.000 |
| Research integrity | 0.001 | 0.001 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".