Colorimetric Detection of a Bacterial Biomarker Using a Modified Litmus Test

Start with an RNA-cleaving DNAzyme, a catalytic DNA that, on binding to a specific bacterial biomarker, gets activated.

The DNAzyme is linked to a streptavidin-coated magnetic bead via biotin at its 5' end. At the 3' end, it is bound to an oligonucleotide-linked urease, forming a functional magnetic bead.

To a tube containing functional magnetic beads, add an E. coli lysate.

A specific E. coli biomarker from the lysate, activates the DNAzyme, triggering cleavage of the RNA linkage, causing the detachment of the DNAzyme-urease conjugate.

Use a magnetic stand to immobilize the beads and remove the bead-bound DNAzyme-urease conjugate.

Transfer the released urease-containing supernatant to another tube.

Add a pH indicator dye — phenol red, and a urea solution.

The urease hydrolyzes urea into ammonia and carbon dioxide.

Ammonia, a weak base, raises the pH of the solution, causing a color change, that indicates the presence of the E. coli biomarker.

After preparing E. coli cells according to the text protocol, pre-wash a 1.5-milliliter microfuge tube by adding and vortexing 100 microliters of RB. Then, discard the buffer.

Add 15 microliters of assembled EC1 to the washed tube, then, plac