Figure 1.
The sandwich-DNA hybridization assay principle.
(A) Capture and detection probes are added to the solution of target DNA. (B) After the denaturation step (99°C, 10 minutes), the hybridization of the probes to the DNA target is achieved under gentle agitation (55°C, 3 hours). (C) Streptavidin-coated magnetic micro beads are added to the solution (gentle agitation for 30 minutes at room temperature). (D) Hybridized duplexes are captured by a magnet, unbound compounds are washed out. (E) Hybridized detection probes are eluted (95°C, 20 minutes) and are detected by SERRS in proportion of the initial concentration of target.
Figure 2.
(A) SERRS spectra of the probe labeled with a single rhodamine 6G (R6G). The peak area beneath the most intense peak at 1650 cm−1 (A1650) is the parameter chosen for quantification. The black spectrum is the Raman signal of the PMMA cuvette, appearing in all spectra. Acquisition times for 0 M, 5×10−9 M, 5×10−8 M and 5×10−7 M are 1×15 s, 1×30 s, 1×10 s and 1×10 s, respectively. (B) Dilution study achieved on solutions of R6G probe. Calibration curve of A1650 normalized to the acquisition time in second, as a function of the initial concentration of the R6G probe (CR6G). Gray line represent the 1σ deviation. (C) Calibration curve of A1650 normalized to the acquisition time in second, as a function of the initial concentration of target DNA Gray lines represent the 1σ deviation.
Figure 3.
Specificity and co-contamination assays.
All spectra were acquired in 1 acquisition of 30 s. (A) In black, negative control without target DNA. (B) In blue, C. hircus, negative control (5.10−8 M). (C) In green, signal from R. rupicapra (5.10−8 M). (D) In red, signal obtained from an equimolar mix of R. rupicapra and C. hircus DNA (5.10−8 M each).
Figure 4.
Probes and DNA characteristics.
(A) Probes and DNA sequences. (B) Alignment of Non target DNA (C. hircus) sequence with complement sequences of Capture probe and Detection probe. Numbers present non-effective bases in the Non target DNA sequence.