This article describes a method for detecting specific E. coli biomarkers using an RNA-cleaving DNAzyme linked to a urease. The detection process involves colorimetric changes in response to the presence of the biomarker.
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, place the tube on the magnetic rack, and aspirate the supernatant. Remove the tube from the rack, add 100 microliters of RB, and carefully resuspend the magnetic beads.
After using RB to wash the beads two more times and removing the RB, add 10 microliters of the E. coli sample to the tube, and gently mix the contents by tapping on the tube. Then, incubate the reaction at room temperature for one hour.
After the incubation, add 90 microliters of doubly-distilled water, and place the tube on the magnetic rack. Following approximately three minutes of magnetic separation, carefully transfer 85 microliters of the supernatant to a 0.5-milliliter microfuge tube.
It is very important to pipette the solution carefully without taking up any of the magnetic beads. These beads are loaded with urease and will generate a false positive signal.
Add 15 microliters of 0.04% phenol red and 100 microliters of substrate solution. Then, take photographs at specific time intervals to record a color change.
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