Individual Final Project: SARS-CoV-2 USTB
Ultra-Sensitive Single-Tube Biosensor (USTB)
🔬 Project Abstract
The USTB is an instrument-free diagnostic platform that converts molecular detection into a macroscopic visual signal: liquid motion. By leveraging the collateral cleavage activity of CRISPR-Cas13a, the sensor triggers a transition in surface wettability from Hydrophilic (Hi) to Hydrophobic (Ho). This project demonstrates a platform capable of achieving $\le 1$ aM sensitivity with a visual readout time of 1 minute.
🧬 The “Hi-to-Ho” Sensing Principle
The sensor utilizes a three-segment probe immobilized on the glass surface. This molecular assembly acts as a “trap door” for the sample liquid:
- Hi Group (Hydrophilic): An anchored 40T DNA segment that holds the liquid at the bottom of the tube.
- Res Group (Responsive): A 6U RNA bridge that serves as the cleavage site for activated Cas13a.
- Ho Group (Hydrophobic): A terminal Dodecane anchor that remains on the glass wall.
When the target viral RNA is present, the Cas13a enzyme is activated and shreds the Res Group. The hydrophilic “mask” is detached, leaving only the hydrophobic base. Gravity then overcomes the reduced surface tension, and the liquid falls.
🛠 Fabrication & Zone Architecture
The glass tube is engineered into three distinct chemical regions (ABC zones) to ensure high sensitivity and zero false-positives.
- Region A (Bottom): The active sensing zone. Functionalized with the 3-segment probe using APTES and Glutaraldehyde cross-linking.
- Region B (Middle): The hydrophobic threshold. Coated with 0.1% FAS-17 (Heptafluorodecyl trimethoxy silane) to create a “non-stick” band that prevents accidental liquid fall.
- Region C (Top): The target zone. Treated with a plasma pen to ensure the liquid spreads instantly upon contact, providing a permanent visual result.
🧪 Technical Specifications & Ordering
For the SARS-CoV-2 N-gene target, the following components are utilized:
| Component | Sequence (5’ to 3') | Modifications | Vendor Note |
|---|---|---|---|
| Bridge Probe | NH2-(T)40-(U)6-(CH2)12 | 5’: Amine / 3’: Dodecane | Order as individual tube |
| crRNA | GAAUUUACCCUUCGGGGUAGUCUAAA... | 53nt Full Guide | Pure RNA |
| Reporter (Opt.) | rUrUrUrUrUrU | 5’: 6-FAM / 3’: BHQ-1 | HPLC Purified |
📋 Experimental Workflow
1. Sample Processing (HUDSON)
- Mix the sample 1:1 with 100 mM TCEP-HCl / 2 mM EDTA.
- Heat to 96°C for 5 minutes. This inactivates endogenous RNases and releases the target RNA.
2. CRISPR Reaction & Readout
- Activation: Add 11 µL of lysate to 100 µL of Master Mix (Cas13a, crRNA).
- Incubation: 15–20 minutes at 37°C.
- Readout: Invert the tube. If the target is present, the liquid falls within 1 minute.
🟢 Optional: Dual-Accuracy Fluorescence
For verification, a FAM-rU6-BHQ reporter is included in the mix. Upon target detection, the tube will emit a bright green signal under 470nm blue light, providing an orthogonal confirmation to the gravity-based “flip.”
📈 Significance & Future Outlook
This USTB platform achieves $0.10-per-test affordability while maintaining the sensitivity of commercial RT-PCR kits (Ct values up to 36). Its modularity allows it to be rapidly adapted for environmental monitoring or agricultural pathogens by simply swapping the crRNA sequence.
Biosafety: All work is conducted using synthetic, non-infectious RNA fragments in a BSL-2 environment.