Part:BBa_K3714002

Biosensor gard-337 for lateral flow assay

The template of aptazyme gard-337 with two probes (binding BBa_K3714007 and BBa_K3714008) on both terminal for lateral flow assay (LFA). It could be used as a biosensor of gardiquimod to verify our design on a colloidal gold based detection platform (Figure.1).

Figure.1 | Design of lateral flow assay. T7 transcription products with or without certain molecule are used as input of LFA. When the certain molecule exists, positive signal could be seen on test line. T line, test line. C line, control line.

The probes was chosen manually before tested with RNA structure prediction model. We minimized the interaction between aptazyme domain and two probes. We also checked the possibility of aptazyme dimer.
Then we tested if the probes added would interrupt the activity of aptazyme. We use same method to determine its cleave fraction under a concentration gradient of gardiquimod (Figure.1). We found that the probes added do have a considerable but bearable influence in the sensibility of aptazyme (Figure.1, also see BBa_K3714000).

Figure.2 | Verification of cleavage property of aptazyme. T7 transcription products were loaded on a denatured PAGE to determine its cleave fraction.

Next, we questioned if this influence would damage its potential usage in LFA. The validation of LFA could be checked in BBa_K3714009. We used the same concentration gradient in LFA, and the outcome indicated that this aptazyme can response to gardiquimod of low concentration (Figure.3).

Figure.3 | Verification of lateral flow assay. T7 transcription product with a target concentration gradient were used as input of LFA.

Having tested the sensibility of aptazyme, we wondered if this aptazyme could be used in detection, where intensity should be a linear function of target concentration. We calculated the intensity of T lines on the strips, and make a linear regression to see if we can acquire a linear interval. The result indicated that our design could work well for rough quantification purpose when gardiquimod concentration falls between 0 and 10 μM.

Figure.4 | Performance of lateral flow assay. The strips in Figure.3 were analyzed by ImageJ, and we used a two-phase linear regression to fit the results. r = 0.982 in the first phase, and r = 0.976 in the second phase. Data are presented as mean±SD, n = 3.

These results have proved that the part K3714002 is capable of detecting and quantifying gardiquimod of low concentration in a short time (~50min, transcription included). We further made attempts (see BBa_K3714010 for more details) to control the cleavage activity by toehold mediated strand displacement (TMSD) reaction to allow the detection be finished within 5 minutes.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]