Colorimetric Detection of a Bacterial Biomarker Using a Modified Litmus Test

Overview

This video demonstrates a colorimetric E. coli detection method, involving an E. coli biomarker activating a DNAzyme, leading to cleavage of RNA linkage and detachment of a DNAzyme-urease conjugate. A subsequent colorimetric assay detects the E. coli biomarker through ammonia-induced pH changes.

Protocol

1. Preparing E. coli Cells for Testing

1. For a desired cell suspension, transfer 1 ml of cultured stock to a 1.5 ml microfuge tube.
2. Centrifuge the cells at 6,000 x g for 10 min at 4 °C. Carefully remove the supernatant without disturbing the cell pellet.
3. Add 10 µl of reaction buffer to the cell pellet and resuspend the cells. Sonicate the cell suspension for 5 min. Transfer the cell suspension to an ice box for 5 min.
4. Sonicate the cell suspension for another 5 min.
5. Centrifuge the cell suspension at 13,000 x g for 10 min at 4 °C. Use the supernatant for testing (10 µl).

2. Litmus Test

1. In a 1.5 ml microfuge tube, prewash the tube by adding and vortexing 100 µl of reaction buffer (RB) in the microfuge tube and discarding the buffer.
2. Transfer 15 µl of assembled EC1 to the washed microfuge tube.
3. Wash the magnetic beads by placing the microfuge tube on a magnetic rack. Remove the supernatant by pipetting. Remove the microfuge tube from the rack, add 100 µl of RB, and carefully resuspend the magnetic beads.
4. Wash the MB two more times by repeating step 2.3.
5. Place the microfuge tube back on the magnetic rack, remove the supernatant, and add the 10 µl E. coli sample prepared from step 1.3.
6. Mix the sample and magnetic beads carefully by gently tapping on the microfuge tube.
7. Incubate the reaction at room temperature for 1 hr.
8. To the reaction, add 90 µl of ddH₂O and place the microfuge tube onto a magnetic rack.
9. After approximately 3 min of magnetic separation, carefully transfer 85 µl of the supernatant to a 0.5 ml microfuge tube. Withdraw the supernatant slowly to avoid collecting any magnetic beads.
10. To the above microfuge tube add 15 µl of 0.04% phenol red and 100 µl of substrate solution.
11. Take a photograph at specific time intervals to record color change.
    NOTE: The change in pH can also be monitored using a pH meter with a microelectrode. The starting pH should be approximately 5.2-5.5 (the solution is yellow). If not, the solution can be adjusted by the addition of 1 mM Acetate Buffer pH 5.0).

Materials

Name	Company	Catalog Number	Comments
Ethylenediaminetetraacetic acid (EDTA)	VWR AMRESCO	105
Sodium Hydroxide (NaOH) pellets	BIO BASIC CANADA INC	SB6789
Tris-base	VWR AMRESCO	497
Urea	VWR AMRESCO	M123
Xylenecyanol FF	SIGMA-ALDRICH	X-4126
0.04% Phenol red	SIGMA-ALDRICH	P3532
10x T4 polynucleotide kinase reaction buffer	Lucigen	30061-1
E. coli K12 (MG1655)	ATCC	ATCC700926
Magnetic Bead (BioMag)	Bangs Laboratories Inc
Magnetic Seperation Rack	New England BioLabs	S1506S
Microfuge tubes	Sarstedt	72.69
Cell culture incubator	Eppendorf Scientific	M13520000
Branson Ultrasonic cleaner	Branson	N/A