Pb2 Detection With Fluorescently Modified DNAzyme

Label the 5'-end of the substrate with a fluorophore such as 6-carboxytetramethylrhodamine (TAMRA), and the 3'-end of the enzyme strand with a fluorescence quencher, such as 4-(4'-dimethylaminophenylazo) benzoic acid (dabcyl). The TAMRA-labeled substrate is named Rh-17DS, and the dabcyl-labeled enzyme is named 17E-Dy (see Table 1). The structure of the fluorophore and quencher, and their linkage to the DNA are shown in Fig. 3B.

After hybridizing to 17E-Dy, the fluorescence of Rh-17DS is suppressed, because the fluorophore and the quencher are close to each other. Upon addition of Pb2+, the enzyme is activated and the substrate strand is cleaved into two pieces. Compared with the uncleaved substrate, the piece containing fluorophore hybridizes to the enzyme strand with lower stability and is released from the enzyme strand under the experimental conditions. Therefore, the fluorescence of TAMRA is unmasked in the presence of Pb2+. The activity of the DNAzyme is much lower in the presence of other metal ions, and very little fluorescence increase is observed. Thus, the presence of Pb2+ can be detected by the increase of fluorescence. The detection process is summarized in Fig. 3A. The detailed protocols for performing the assays for Pb2+ using the Pb2+-dependent DNAzyme are presented next.

1. DNA handling: DNA samples received from commercial sources are normally in a powdered form, requiring careful handling. Spin the received DNA container on a bench top centrifuge for 1 min before opening the cap. According to the amount received, dissolve the DNA sample in a dilution buffer (e.g., 5 mM of Tris-acetate, pH 7.2-8.2) to prepare the DNA stock solution, so that the concentration of DNA is 100 |M to 1 mM. Confirm the concentration of DNA by measuring the absorption at 260 nm. Aliquot the DNA stock solution into several microcentrifuge tubes to minimize loss from contamination. Wear gloves while handling DNA or RNA.

Fig. 3. (A) Concept and design of metal ion biosensors, using lead sensors as an example (4). (B) Structures of fluorescence tag C (6-carboxytetramethylrhodamine) and quencher (as 4-[4'-dimethylaminophenylazo] benzoic acid ) with linkage to DNA. (C) Selec- § tivity of the DNAzyme sensor. The bar plot shows the quantification of the initial rate of fluorescence increase. Inset: the fluores- c cence increase in the presence of Pb2+ and other divalent metal ions. c

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Fig. 3. (A) Concept and design of metal ion biosensors, using lead sensors as an example (4). (B) Structures of fluorescence tag C (6-carboxytetramethylrhodamine) and quencher (as 4-[4'-dimethylaminophenylazo] benzoic acid ) with linkage to DNA. (C) Selec- § tivity of the DNAzyme sensor. The bar plot shows the quantification of the initial rate of fluorescence increase. Inset: the fluores- c cence increase in the presence of Pb2+ and other divalent metal ions. c

2. Sensor sample preparation: dissolve Rh-17DS and 17E-Dy to final concentrations of 50 nM each in 50 mM of HEPES, pH 7.5, and 50 mM of NaCl. Each assay uses 600 |L of sample. The volume of sample prepared can vary depending on how many assays are needed.

3. DNA annealing: anneal the substrate and enzyme strand from the sensor sample preparation using the following steps. First, incubate the sample in a 90°C water bath (containing 500 mL of water) for 2 min. Turn off the water bath and allow the sample to naturally cool in the water bath to room temperature. It takes approx 2 h. Finally, place the sample with the water bath in a 4°C refrigerator for an additional hour (see Notes 2 and 3). A high annealing efficiency is very important to ensure low background fluorescence.

4. Pb2+ detection: transfer 600 |L of annealed sample into a quartz cuvet of 0.5-cm path length on each side. Place the cuvet into the sample holder of the fluorom-eter (cooled to 4°C). Mix the sample by a stirrer from the top of the cuvet. The position of the stirrer should be above the light path system of the fluorometer. Insert a length of micro tubing into the cuvet so that one end of the tubing is in the sensor solution next to the stirrer. Connect the needle of a 10-| L syringe to the other end of the tubing. Inject 1-2 |L of concentrated metal ion solution into the cuvet using the 10-||L syringe to initiate the cleavage reaction (see Note 4).

5. Fluorometer settings: set the fluorometer to monitor steady-state fluorescence. Monitor the TAMRA emission at 580 nm at 2-s intervals by exciting at 560 nm. Initiate the reading of the fluorometer at the moment of injection of metal ions.

6. Kinetics data fitting: an example of the time-dependent fluorescence increase in the presence of different metal ions is shown in the inset of Fig. 3C. The observed kinetics data can be fit to the equation: Ft = F0 + F (l — e kt), where Ft is the fluorescence at time t; F0 is the initial fluorescence; F is the final fluorescence after complete cleavage; and k is the apparent rate constant reflecting the overall rate of cleavage and release of the cleaved fragment.

7. Measuring the initial fluorescence increase rate: the kinetics curves have an initial linear increase of fluorescence with time (see Fig. 3C, Inset). For biosensor applications, a convenient method of quantification is to calculate the fluorescence increase rate in the initial linear region. A bar plot of comparison of the initial fluorescence increase for the fluorescently labeled "8-17" DNAzyme is presented in Fig. 3C.

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