*3.8. Comparison of the Cleavage Activity of EC1T in the Presence of CIM-EC Obtained from E. coli Grown in Various Growth Media*

100 ȝL of 2500 CFU/mL glycerol stock of *E. coli* was inoculated into 2 mL of LB, LBM, SOB, SOC, TB, TH, or TSB. Following 7 h incubation at 37 °C, 1 mL of each culture was taken and centrifuged at 11,000 g for 5 min at RT. The cell pellet was re-suspended in 200 ȝL of 1× RB. Cleavage reactions were then conducted by mixing 41 ȝL of 1× RB, 5 ȝL of each CIM-EC, 1 ȝL of 2.5 ȝM FS1, 1 ȝL of 2.5 ȝM EC1T and 2 ȝL of 2× RB. Each reaction mixture was incubated at RT for 60 min, followed by 10% dPAGE analysis as described above.

#### *3.9. Comparison of the Cleavage Activity of EC1T in the Presence of Different Divalent Metals*

First, stocks of 2× RBc (100 mM HEPES, 300 mM NaCl, pH 7.5) and 150 mM MCl2 (M = Cd, Co, Mg, Mn, Ni, Cu, Zn and Ca) were prepared. The CIM-EC was also prepared from *E. coli* grown in SOB in the same way as described immediately above except for the use of 1× RBc instead of 1× RB. The cleavage reactions as shown in Figure 3A were set up by mixing 15.5 ȝL of water, 22.5 ȝL of 2× RBc, 5 ȝL of a relevant MCl<sup>2</sup> stock, 1 ȝL of 2.5 ȝM FS1, 1 ȝL of 2.5 ȝM EC1T, and 5 ȝL of the CIM-EC. The cleavage reactions as shown in Figure 3B were set up similarly except that the volume of water and 150 mM BaCl2 were co-varied to achieve a final [BaCl2] of 0, 1, 5, 7.5, 10, 15, 20, 25 and 50 mM. Each reaction mixture was incubated at RT for 60 min, followed by 10% dPAGE analysis as described above.

#### *3.10. Comparison of the Cleavage Activity of EC1T at Different Reaction Temperature*

A 2× RBBa stock (100 mM HEPES, 300 mM NaCl, 30 mM BaCl2, pH 7.5) was first prepared. Five cleavage reaction mixtures were then set up by mixing 19.5 ȝL of water, 22.5 ȝL of 2× RBBa, 1 ȝL of 2.5 ȝM FS1, 1 ȝL of 2.5 ȝM EC1T, and 5 ȝL of the CIM-EC prepared with 1× RBBa. These mixtures were incubated, respectively, at 4, 15, 23, 37 and 50 °C for 60 min, followed by 10% dPAGE analysis as described above.

#### *3.11. Comparison of the Cleavage Activity of EC1T at Different pH*

A series of 2× RBBac stock (300 mM NaCl, 30 mM BaCl2, along with a chosen buffering agent at 100 mM) were first prepared with pH being varied from 5.0 to 9.0 at an increasing interval of 0.5 units. MES was used for pH 5.0, 5.5 and 6.0; HEPES was used for pH 6.5, 7.0, 7.5 and 8.0; Tris was used for pH 8.5 and 9.0. The cleavage reactions were then conducted in a similar fashion as described in the section immediately above. Note that the CIM-EC for a given pH was prepared with a relevant 1× RBBac.

#### *3.12. Comparison of the Cleavage Activity of EC1T at Varying FS1/EC1T Ratios*

Stocks of EC1T at 2.5, 5, 12.5, 25, 62.5, 125, and 250 ȝM were first prepared. Cleavage reactions were then conducted by mixing 19.5 ȝL of water, 22.5 ȝL of 2× RBBa, 1 ȝL of 2.5 ȝM FS1, 1 ȝL of a given EC1T stock, and 5 ȝL of the CIM-EC prepared with 1× RBBa. Each reaction mixture was incubated at RT for 60 min, followed by 10% dPAGE analysis as described above.

#### *3.13. Specificity Test*

Five Gram-negative bacteria (*P. peli*, *Y. rukeri*, *H. alvei*, *A. xylosoxidans* and *E. coli*) and four Gram-positive bacteria (*L. mesenteroides*, *L. planturum*, *P. acidilactici* and *B. subtilis*) were tested in this experiment. Each bacterium was cultured in SOB for a different period of time until the OD600 reached ~1. The CIM was then prepared with 1× RBBa and tested with EC1T/FS1 under the optimal reaction condition (50 mM HEPES, pH 7.5, 150 mM NaCl and 15 mM BaCl2, room temperature, EC1T/FS1 = 50/1). Each reaction mixture was incubated at RT for 60 min, followed by 10% dPAGE analysis as described above.

#### *3.14. Detection Sensitivity*

First, a single colony of *E. coli* from an agar plate was taken, inoculated into 2 mL of SOB and grown for 14 h at 37 °C with shaking at 250 rpm. 10-fold serial dilution was then carried out as follows: 100 ȝL of the 14-h culture was mixed with 900 ȝL of fresh SOB. 100 ȝL of the diluted culture was again taken and mixed with 900 ȝL of fresh SOB. This process was repeated 7 times. 100 L of the final dilution were plated onto a TSB agar plate (done in triplicate), which was incubated at 37 °C for 15 h. Colonies in each plate were counted; the average number of colonies from the three plates was taken as the number of cells for this final dilution. This number was then used to calculate the number of cells for the other dilutions. 500 ȝL of each dilution was taken and centrifuged at 11,000 g for 5 min at RT. The cell pellet was re-suspended in 100 ȝL of 1× RBBa and used as the CEM-EC for this experiment (done in triplicate).

Cleavage reactions concerning EC1T/FS1 were set up and monitored as follows: 19.5 ȝL of water, 22.5 ȝL of 2× RBBa, 1 ȝL of 2.5 ȝM FS1, 1 ȝL of 125 ȝM EC1T were mixed in a quartz crystal cuvette, which was placed in a fluorimeter (Cary Eclipse Fluorescence Spectrophotometer; excitation wavelength = 488 nm and emission wavelength = 520 nm) set at RT. Fluorescence intensity was recorded every minute for 5 min; 5 ȝL of a relevant CIM-EC was then added into the cuvette and the solution was quickly mixed by pipetting the mixture up and down a few times. Following this step, the fluorescence intensity of the solution was recorded for 55 more minutes. All the reactions were conducted in 3 replicates and the average data are shown in Figure 6A. The final reaction mixture was also taken and analyzed by 10% dPAGE and data are shown in Figure 6B.

Cleavage reactions concerning RFD-EC1 were set up and monitored similarly: 20.5 ȝL of water, 22.5 ȝL of 2× RBBa, 1 ȝL of 2.5 ȝM RFD-EC1 was mixed in a cuvette. After reading fluorescence intensity for 5 min, 5 ȝL of a relevant CIM-EC was then added, followed by fluorescence intensity reading for 55 more minutes (Figure 6C). The final reaction mixture was also analyzed by 10% dPAGE (Figure 6D).

#### *3.15. Single Cell Detection via Culturing*

For isolating a single cell we followed our previously reported protocol [12]. Briefly, a glycerol stock containing 2 CFU/mL of *E. coli* was prepared. 100 ȝL of this stock was distributed to 10 culture tubes each with 2 mL of SOB. Since the concentration of the stock was 2 CFU/mL, only 2 out of the 10 tubes contained a single seeding cell (2 CFU/mL × 0.1 mL = 2). All the tubes were incubated at 37 °C with shaking at 250 rpm. At 2, 4, 6, 8 and 10 h, 200 ȝL of culture was harvested from each culture tube and CIMs were prepared (40 ȝL of 1× RBBa was used to dissolve the cell pellet). All the tubes were further incubated for 20 h to identify the two tubes containing *E. coli* cell (the culture in these tubes turned turbid while that in other 8 tubes stayed clear). Each CIM from *E. coli*-containing tubes was used to initiate the cleavage reaction by mixing 19.5 ȝL of water, 22.5 ȝL of 2× RBBa, 1 ȝL of 2.5 ȝM FS1, 1 ȝL of 125 ȝM EC1T, and 5 ȝL of a relevant CIM. The reaction and dPAGE analysis procedures were same as described above.

#### **4. Conclusions**

We recently described an RNA-cleaving fluorogenic DNAzyme, named RFD-EC1, which is active in the presence of the crude extracellular mixture (CEM) of the model Gram-negative bacterium *E. coli* [12–14]. RFD-EC1 was found to be highly active with CEM of *E. coli* but inactive with CEMs from a host of other Gram-negative and Gram-positive bacteria, and thus, RFD-EC1 can be used to develop a simple, "mix-and-read" fluorescence assay to achieve selective detection of *E. coli*. However, several parameters that are particularly relevant to the performance of this assay remained to be investigated. In this study we sought to establish a *trans*-acting DNA catalyst that cleaves an external substrate, optimize the reaction conditions that best support the catalytic activity of the DNAzyme, and determine the culturing conditions that enable the quickest detection of a single live bacterial cell.

The *trans*-acting DNAzyme was successfully established by segregating the substrate sequence domain from the sequence of the original DNA library. Also the two fixed sequence domains flanking the random-sequence domain could be removed without affecting the catalytic performance of the DNAzyme. The shortened, *trans*-acting DNAzyme, named EC1T, now contains 70 nucleotides.

Originally, the DNAzyme was isolated to cleave in the presence of the crude extracellular mixture (CEM) of *E. coli* and it has been determined that the target that activates the DNAzyme is a protein molecule based on the observation that the treatment of the CEM with proteases abolishes the DNAzyme activity [12]. Although the identity of this target is yet to be determined, we found that the target protein is much more abundant intracellularly and could be retrieved with a simple heating step (50 °C; 15 min). This led us to the use of the crude intracellular mixture (CIM) as the target of detection, translating into a better assay sensitivity.

Our results revealed that the nutritional factors in culture media played a vital role in growing the cells in faster rate (varying by as much as ~25-fold) with super Optimal Broth (SOB) which can substantially reduce the time required for single cell detection.

In order to establish an optimal reaction condition for EC1T, we examined the following reaction parameters: choice of divalent metal ions, reaction temperature and pH as well as the ratio between the substrate and the DNAzyme. Although EC1T was found to be active in the absence of any divalent ion, it exhibited much stronger activity in the presence of Ba2+, Ca2+, Mn2+ or Mg2+. We chose Ba2+ as the divalent metal ion cofactor because this metal ion does not impose any fluorescence quenching effect. The DNAzyme was originally derived at room temperature (~23 °C) and a solution pH of 7.5 and therefore it was not surprising that EC1T exhibited the strongest activity at 23 °C and pH 7.5. We further found that when the concentration of FS1 was kept at 50 nM, 2.5 ȝM EC1T was required to reach the optimal cleavage activity. All the above optimization experiments led to the establishment of the optimal reaction condition for EC1T: 50 mM HEPES, 150 mM NaCl, 15 mM BaCl2, pH 7.5, DNAzyme: substrate ratio = 50:1.

Under the above optimal reaction condition, the *trans*-acting system was able to detect 105 cells when the reaction was monitored in a fluorimeter. If the reaction mixture was analyzed by dPAGE (which separates the reaction product from the substrate), the system can detect 10<sup>4</sup> cells. When the original RFD-EC1 was used for the assay, the detection sensitivity was further improved: the fluorimeter method was able to detect 10<sup>4</sup> cells while the dPAGE method was able to detect as low as 103 cells. Importantly, the optimized assay did not compromise the specificity.

With a culturing step, the optimized assay is able to achieve the detection of *E. coli* from a single colony forming unit in 4–6 h (dependent on the method of choice), which represents a significant deduction in time (12 h) required by the same probe under unoptimized conditions. Overall, we have significantly improved the performance of our DNAzyme probe and demonstrate the utility of such probes as simple biosensors to achieve sensitive and speedy detection of bacterial pathogens.

#### **Acknowledgments**

This work was supported by research grants from the Natural Sciences and Research Council of Canada (NSERC) and Sentinel Bioactive Paper Network. We would like to thank Gerard Wright, Brian Coombes and Russell Bishop for providing various bacterial cells.

#### **Conflict of Interest**

The authors declare no conflict of interest.

#### **References**

