Generally, for high sensitivity disease screening, 3 steps are essential: Sampling, Signal amplification and signal detection. Our PaDetector goes beyond traditional detection methods in every aspect.

This year, the goal of our project is to develop a household HPV screening device with high sensitivity, low cost and extreme convenience. Here are what we have done.

Cas12a biosensing system

With Cas12a biosensing system, we are able to detect HPV16 with high sensitivity and great specificity:

Figure 1. Result of Fluorophore quencher (FQ)-labelled reporter assay. If the reporter was cut, FAM would be able to emit green florescence, otherwise the florescence would stay red (from TAMRA)

FnCas12a is capable of detecting 1pM of HPV16 genome in room temperature. Although time is a little bit long (about 60min), it does not really matter because you can do it in your own house.

The result from quantitative FQ-labelled reporter assay is also consist with it.

Figure 2. Quantitative comparison between FnCas12a and LbCas12a.

The portable detection device

Based on Cas12a biosensing system, we designed an in vitro portable detection device.

Figure 3. The structure of the portable detection device.

With an UV flashlight, it can show the test result to users with high convenience and great clarity.

Figure 4. Result of the detection. (L:negative R:positive)

HCR biosensing system

With HCR biosensing system, we are able to detect and distinguish different target we synthesis according to HPV L1, but the specificity of our probe and sensitivity of the test paper we ordered from a company should be improved.

Preprocessing of HPV genome

HPV16 L1 digested by EcoRI, HPV16 L1 digested by EcoRI and PstI, HPV18 L1 digested by BcuI and PstI is chosen to produce S1.

Figure 5. Use two kinds of endonucleases combine with ExoIII to digest L1 region of HPV genome.

ExoIII assisted signal amplification-Version 1.0

Compared with lane 4, when S10 exists, the band of P2 undertook a significant migration, which indicates P20-S10 is degraded by ExoIII and release S2.

Figure 6. ExoIII assisted signal amplification-Version 1.0.

Hybridization chain reaction

Without the initiator (S2), the hybridization chain reaction cannot occur. When initiator (S2) exists, the hybridization chain reaction occurs, and the length nicked double helix varies with the concentration of initiator (S2).

Figure 7. Characterization of the HCR of pure hairpin DNA strands by 8% and 5% native PAGE.

Lateral flow nucleic acid biosensor

Compared with FITC/Cy3 test paper, Cy3/FITC test paper shows a better result, so we choose the Cy3/FITC test paper for further detection.

Figure 8. Detection of HCR production on test paper.

Team:ZJU-China/Demonstrate