Final Project

1. The Bridge Probe (The Gravity Switch)This is the “A-B-C” chimeric probe that anchors the hydrophobic cholesterol to the biotinylated glass surface.Sequence: 5’-[Biotin-TEG] TTT TTT TTT TTT TTT rUrU rUrU rUrU rUrU rUrU rUrU TTT TTT TTT TTT TTT [Cholesterol-TEG]-3’Structure: Biotin—(dT)15—(rU)10—(dT)15—Cholesterol.Notes: Ensure you specify TEG (Triethylene Glycol) spacers for both the Biotin and Cholesterol modifications. This prevents steric hindrance, allowing the Cas13a to access the central RNA (rU10) cleavage site easily.Purification: HPLC purification is required for this dual-modified chimeric oligo.2. crRNA (The SARS-CoV-2 N-Gene Guide)This guide RNA targets a highly conserved region of the SARS-CoV-2 Nucleocapsid (N) gene (specifically the N2 region).Sequence: 5’-GAA UUU ACC CUU CGG GGU AGU CUA AAU GGU GAU GCU GCU CUU GCU UUG AGA G-3’Breakdown: * Direct Repeat (DR): GAAUUUACCCUUCGGGGUAGUCUAAAUSpacer: GGUGAUGCUGCUCUUGCUUUGAGAGNotes: This must be ordered as a single-stranded RNA (ssRNA).3. Fluorescent Reporter (Optional Confirmation)If you are using fluorescence for secondary verification alongside the visual liquid motion, this is the standard reporter.Sequence: 5’-[6-FAM] rU rU rU rU rU [BHQ-1]-3’Notes: A simple poly-rU pentamer labeled with FAM and BHQ-1.4. Positive Control (Target Activator)To test your reaction mix without a clinical sample, order this synthetic RNA fragment that matches the N-gene target.Sequence: 5’-CUC UCA AAG CAA GAG CAG CAU CAC C-3’Quick Ordering Summary TableComponentSequence (5’ to 3’)TypeModificationBridge ProbeBiotin-TEG-T15-rU10-T15-Cholesterol-TEGDNA/RNABiotin (5’), Cholesterol (3’)crRNAGAAUUUACCCUUCGGGGUAGUCUAAAU-GGUGAUGCUGCUCUUGCUUUGAGAGRNANoneReporterFAM-rUrUrUrUrU-BHQ1RNA6-FAM (5’), BHQ-1 (3’)Target RNACUCUCAAAGCAAGAGCAGCAUCACCRNANonePro-Tips for Your Build:Purification Matters: Chimeric oligos (DNA mixed with RNA) like the Bridge Probe are notoriously tricky. Ask your supplier (like IDT or GenScript) for HPLC or PAGE purification to ensure you don’t get truncated products that might fail to anchor.Glass Preparation: Since you’re using glass tubes, remember that the surface silanization (typically with APTES or FAS-17) is the most sensitive step. If the “blank” group liquid is moving too fast, your FAS-17 concentration is likely a bit too high.

Ultra-Sensitive Single-Tube Biosensor (USTB): Triple-Readout Protocol

This comprehensive protocol integrates mechanical Gravity readout with biochemical fluorescence and visual color change for maximum reliability. This “Triple-Readout” system ensures high diagnostic specificity for SARS‑CoV‑2 (N‑gene) detection.

I. Procurement Guide: What to Order

From Twist Bioscience (Custom Oligos)

Twist is the preferred source for the high‑purity, modified RNA/DNA tethers required for the “Hi‑to‑Ho” switch. Order the following three sequences:

From Thermo Fisher / Fisher Scientific

II. Step‑by‑Step Protocol

Part 1: Tube Functionalization (“Arming”)

Prepare these in advance; “Armed” tubes are stable for up to 30 days at 4 °C.

1. Dilute Probe – Reconstitute your Bridge Probe to 100 nM in 1× PBS.
2. Coat – Add 150 μL of probe solution to a Streptavidin‑Coated 1.5 mL tube.
3. Incubate – Let sit for 30 minutes at room temperature (RT).
4. Wash – Remove liquid and wash the tube 3 times with 200 μL of PBS‑T (1× PBS + 0.05% Tween‑20).
5. Dry – Air‑dry and store in a sealed bag with a silica desiccant.

Part 2: HUDSON Sample Processing

This “instrument‑free” method releases RNA directly from saliva or nasal swabs.

1. Mix – Combine 50 μL of sample with 50 μL of Lysis Buffer (100 mM TCEP‑HCl + 2 mM EDTA).
2. Heat – Incubate at 95 °C for 5 minutes (heat block or boiling water works).
3. Cool – Allow the lysate to reach RT before adding it to the CRISPR mix.

Part 3: Triple‑Readout CRISPR Reaction

Assemble the master mix on ice before adding the sample.

Master Mix Assembly (100 μL per test):

Note: Low buffer capacity is critical for the color shift.

1. Reaction – Add 11 μL of lysate to 100 μL of master mix inside your Armed Tube.
2. Incubation – Incubate at 37 °C for 15–20 minutes.

III. Interpretation of Results

IV. Featured Equipment & Reagents

Thermo Scientific Pierce TCEP‑HCl
A potent, odorless reducing agent. High‑purity TCEP ensures complete viral lysis and stable RNA for attomolar detection.

Thermo Scientific Pierce TCEP‑HCl, No‑Weigh Format – $186.00
Thermo Fisher Scientific

Fisher Scientific SUPERase·In RNase Inhibitor
Essential for maintaining CRISPR reaction activity even in “dirty” clinical samples. More stable across a wide temperature range than traditional inhibitors.

Fisher Scientific SUPERase·In RNase Inhibitor (20 U/μL) – $227.00
Invitrogen