Quantitative analysis of double-stranded DNA amplified by a polymerase chain reaction employing surface-enhanced Raman spectroscopy

Abstract

Surface-enhanced Raman spectroscopy (SERS) was utilized for the quantitative analysis of double-stranded (ds) DNA amplified by a polymerase chain reaction (PCR). 4?, 6-Diamidino-2-phenylindole dihydrochloride (DAPI), which intercalates into ds-DNA but does not form a complex with single-stranded (ss) DNA, was added to a DNA solution after amplification by PCR. When the solution was mixed, including ds-DNA-DAPI complexes and free DAPI with silver colloid sol, only free DAPI was adsorbed on the colloid surface. The dye on the colloid gave very intense SERS signals with excitation at 514.5 nm, whereas DAPI engaging in the intercalation with ds-DNA did not show any SERS signal. The SERS spectrum of DAPI on the colloid showed a strong band at 1610 cm(-1) due to the C?N stretching mode, and a linear relationship was observed between the peak intensity of the C?N stretching band and the concentration of free DAPI. Therefore one can determine the concentration of free DAPI by the SERS measurement. The more ds-DNA there is in the solution, the less free DAPI there is. Thus it is possible to quantitatively analyze the ds-DNA amplified by PCR indirectly by using SERS. The correlation coefficient between the peak intensity of the C?N stretching band and the concentration of ds-DNA amplified by PCR was calculated to be 0.988 for a concentration range from 0.1 to 1.3 mg/ml.

Detection of 4′,6-diamidino-2-phenylindole within silver-doped silica sol- and aerogels using surface-enhanced Raman spectroscopy

“…2a). The spectrum of DAPI has several notable features, however the most easily distinguished is the strong peak at approximately 1615 cm − 1 assigned to the C = N stretching mode [6,31]. Other notable peaks include four of approximately equal intensity in the 1450-1600 cm − 1 range due to the 19b, 19a, 8b and W2 vibrations and two peaks near 1150 cm −1 due to the W12 and W11 vibrations [32,33].…”

“…Raman spectroscopy is an important sensing technique that can be used in aqueous environments within biological media, while yielding a spectrum that is unique for a compound of interest. Because the Raman signal from water is weak, it contributes little to the background during spectral acquisition [5,6]. Normal Raman spectroscopy produces a weak signal.…”

“…However, the Raman signal can be enhanced by factors between 10 3 and 10 14 when target molecules are adsorbed to suitable metal surfaces resulting in surface enhanced Raman spectroscopy (SERS). Bioanalytical applications using SERS include analysis of amino acids, nucleotides, biological pigments and double stranded DNA [6][7][8]. The optimal size of the metal particles is 20 to 80 nm which can be achieved by roughening a metal electrode or by using nanoparticles of appropriate sizes [9,10].…”

“…1) [27][28][29]. DAPI is also Raman active with a distinct functional group that can adsorb to silver, and is thus an excellent SERS probe candidate [6]. In this study, we have used sol-gel chemistry to make Raman active substrates that contain silver nanoparticles and the biologically useful fluorescent probe, DAPI, forming both sol-gels and aerogels materials.…”

Direct visual evidence of end-on adsorption geometry of pyridine on silver surface investigated by surface enhanced Raman scattering and density functional theory calculations

“…SERS is a sensitive molecular technique for detection of DNA fragment http://dx.doi.org/10.1016/j.saa.2015.07.019 1386-1425/Ó 2015 Elsevier B.V. All rights reserved. [1][2][3][4][5], quantitative analysis of presence of low concentration in the samples [1,6], detection of change in pH [7][8][9][10] and determination of molecular structure of single-molecule junction by tip-enhanced Raman spectroscopy [10][11][12]. The SERS has drawn a lot of attention to the scientific community due to the large enhancement effect up to order of 10 6-9 [13].…”