| Water-soluble fluorescent conjugated polymers, have good performance of luminescence and biocompatibility, have made much progress in biological detection in recent years. Water-soluble polythiophene, as an important fluorescent conjugated polymer, was widely applied into biological fluorescent probe detection.In this paper, two intermediates and water-soluble polythiophene derivatives were synthesised:2-(3-thienyl)-p-toluenesulfonyl ethanol (Intermediate 1), (S)-3-[2-(3-thienyl)-ethoxy]-2-tert-butoxycarbonylamino-propionic acid (Intermediate2), poly (3-[(S)-5-amino-5-carboxyl-3-oxapentyl]-2,5-thiophenylene hydrochloride) (POWT) was synthesized. Molecular structures of intermediates and polymer were charactered by^-NMR, FT-IR, MALDI-TOF-MS, UV-Vis and PL.Photophysical properties of Intermediate 1, Intermediate 2, POWT were measured by UV-Vis absorption spectrum and fluorescence spectrum. Results showed that the maximum absorption wavelengths (λmax) of Intermediate 1, Intermediate 2 are located at 244nm and 248nm respectively, the maximum fluorescence emission wavelengths (kem) are located at 423nm and 419nm respectively. Solution of all the intermediates emit bule light. While the maximum absorption wavelength and the maximum fluorescence emission wavelength of POWT were 370nm and 560nm respectively. POWT emited bright orange yellow light. The relative fluorescence quantum yield of POWT were determined by "suction point excitation fluorescence correction integral area" method and the measured value were low.POWT was applied to BRCA1 and TB4 ssDNA detection. The following research were conducted through the fluorescence spectra. Firstly, buffer pH, concentration of NaCl and the component of buffer were optimized. When the pH was 8.4 and the concentration of NaCl was 0.2M, PL intensity of POWT(in 0.01M Tris-HCl buffer solution) were the highest. Tris, a buffer solute, could quench the PL intensity of POWT. Adding few Triton-X-100 surfactant into the solution, the PL intensity of POWT recover to its initial level, which will contribute to improve the sensitivity. Secondly, the response performance of POWT towards BRCA1 and TB4 ssDNA were investigated. Upon addition of BRCA1 or TB4 ssDNA, the PL intensity of POWT was quenched, accompanying with a bathochromic shift in PL spectra. The PL intensity was quenched to a larger extent as the concentration of ssDNA increased, the maximum △I/I values were 62.98% and 48.71%. When the concentration of ssDNA range from 1.0×10-11M to 1.0×10"9M, Stern-volmer equation could be used to fit the relationship between Io/I and the concentration of ssDNA. When the concentrations of ssDNA varied from 1.0×10-11M to 5.0×10-11M, two fine lines Stern-Volmer plots were obtained and their equations were I0/=1+8.254×109C and I0/I=1+6.924×109C (C:nM) respectively and the correlations were all 0.9999. The Ksv value were 8.254×109M-1 and 6.924×109M-1 respectively, and the LODs of these two ssDNA sequences were 2.2×10-12M and 2.7×10-12M respectively. Thirdly, the mismatched bases recognition performance of the POWT-ssDNA complex probe was investigated. Upon the addition of completely complentary ssDNA to the POWT-ssDNA solution, the PL intensity recover to its initial intensity after annealing. Whearas, sequences with 1-3 mismatched bases, the emission intensity would also increase to some extent, but less compared with the completely complementary strand. With the increase of mismatched bases, the recovery rates decreased, which was the strong demonstration of the selectivity of this probe. This method can be used into clinical diagnosis of the mutations of BRCA1 and TB4 gene. The interactive model between POWT and the two DNA was evaluated. The results show that the length of POWT was shorter than the two DNA, whereas, the molecular number of POWT was more than DNA, so one DNA molecule might interact with more POWT molecule. Lastly, it has been estimated that there are two factors affect the quenching efficiency. On the one hand, different numbers of bases. Generally speaking, the more bases ssDNA has, the stronger π-π stacking effect between ssDNA and POWT, so the A I/I value would become higher. On the other hand, for the same numbers of bases of different ssDNA, they have different numbers of purines and pyrimidines. The numbers of molecules which have five-membered rings and six-membered rings were not equal to the numbers of molecules which only have six-membered rings, the π-π stacking effect between ssDNA and POWT was not equal, so the quenching effect was different. Generally speaking, the more purines ssDNA had, the higher △ I/I value it will be. |
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