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EPA Project Mercury Research
Mercury is a very toxic element that is widely spread in the atmosphere, lithosphere, and surface water. It can cause severe health problems even at a very low concentration (the maximum allowable level of mercury in drinking water is 2 ppb). Current approaches to detect Hg2+ include AAS/AES, ICP-MS, etc. But most of them are costly, time-consuming, complex, and nonportable. Therefore, it is desirable to develop a highly sensitive, facile, and practical sensor to monitor mercury pollution.
Our novel design of the Hg2+ sensor is mainly based on two biological approaches–rolling circle amplification (RCA) and thymine-mercury-thymine (T-Hg2+-T) mismatch, which can dramatically lower the detection limit.
RCA is a simple enzymatic process that can generate very long single-strand DNA (ssDNA) with tandem repeats.
A primer DNA first anneals to a circular DNA template.
The added DNA polymerase extends the primer continuously around the circular DNA generating a long DNA product that consists of many repeated copies of the circle.
T-Hg 2+ -T Mismatch
The normal Watson-Crick base pairs (upper) and the Hg(II)-mediated T-T mismatch (lower).
The binding strength in T-Hg2+-T is at least as stable as normal Watson-Crick base pairs.
1. Ligation: circular template synthesis
2. Rolling circle amplification
3. RCA product detection
Real Time Detection
Real time mercury detection. Hg2+(36.8 nM) was added at t = 875 s and continuously scanning for 2000 s . The sample were excited at λ ex = 490 nm and the fluorescence signal was collected at λ em = 525 nm.
Different concentrations of Hg2+detected by the RCA-based mercury biosensor. The sample was excited at λ ex= 490 nm, and scanned from 500 nm to 600 nm. Slit: 2 nm.
- A novel mercury biosensor has been developed based on RCA process and T-Hg2+-T mismatch phenomenon.
- The preliminary results prove the feasibility of the designed mercury sensor. However, the relatively high background signal limits its detection sensitivity.
- To lower the background signal and improve the sensitivity, molecular beacon is chosen as the detection signal instead of Eva Green. The experiment is in progress.
- Sensitivity Detection.Different concentrations of Hg2+will be detected and obtain the detection limit and linear range.
- Selectivity Detection.Other possible interference ions will be detected as well to prove the high selectivity of this method.
- Real Time Detection.Monitor the whole process of RCA by real time fluorescence detection.
- Real Sample Detection.Various water sources, such as river, lake, and tap water, will be detected.