Toward the Development of Nucleic Acid Assays Using Fluorescence Resonance Energy Transfer (FRET) and a Novel Label Free Molecular Switching Construct
Bibliographic record
Abstract
The research presented in this thesis introduces design criteria for development of a new type of self-contained optical biosensor. The study begins with evaluation of a dual label, fluorescence resonance energy transfer (FRET) bioassay format, and then goes on to demonstrate a signalling platform that uses an immobilized fluorescent intercalating dye so as to avoid labelling of both the target and probe strands. An extensive survey of FRET pairs that can be used to monitor hybridization events in solution and at solid interfaces was conducted in solution to provide a set of calculated Förster distances for the extrinsic labels Cyanine 3 (Cy3), Cyanine 5 (Cy5), Carboxytetramethylrhodamine (TAMRA), Iowa Black Fluorescence Quencher (IabFQ) and Iowa Black RQ (IabRQ). FRET parameters using thiazole orange (TO) intercalating dye as a FRET donor for various acceptor dye-labelled DNA conjugates in solution were determined. Limitations associated with quenching mechanisms other than those mediated by FRET motivated the development of a molecular switch that contained intercalating dye. The four binding sites associated with Neutravidin served for assembly of the switch using biotin interactions. One binding site was used to immobilize an unlabelled oligonucleotide probe. The adjacent site was used to immobilize a novel biotinylated TO derivative that could physically reach the probe. On hybridization of the probe with target, the intercalating dye was captured by the hybrid, leading to a change of fluorescence. This reversible signalling mechanism offers a method without nucleic acid labelling to detect nucleic acid association at an interface. A SNP discrimination strategy involving TO and formamide was investigated, and SNP discrimination without the requirement of thermal denaturation was achieved for multiple target lengths, including a 141-base pair PCR amplicon in solution. It was determined that formamide could also provide improvements of signal-to-noise when using thiazole orange to detect hybridization.
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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 | 0.002 | 0.001 |
| Meta-epidemiology (narrow) | 0.001 | 0.001 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.001 | 0.000 |
| Science and technology studies | 0.000 | 0.001 |
| Scholarly communication | 0.002 | 0.001 |
| Open science | 0.002 | 0.001 |
| Research integrity | 0.001 | 0.002 |
| Insufficient payload (model declined to judge) | 0.001 | 0.001 |
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".