The Application Of Organic Small Molecule As Spectral Probes In Copper Ion And DNA Analysis

The studies are started with the aim of improving the selectivity of resonance light scattering (RLS) technique and establishing several methods for the target detection with simply and speed properties, which base on the technique of the resonance light scattering, the fluoroscope, and the investigation of many literatures. To exploit the potential of organic small molecule probes, and artificially synthesize a new organic small molecule probe with application potential, we have established several methods for detection of DNA and copper ion. To obtain the theoretic instruction for designing and synthesizing other new organic small molecule probes, we have studied the relationship between the structure and the properties of organic small molecule probes. The main investigations include in two sections.Section one mainly introduces the RLS quantity determination methods using spectral probes of organic small molecule, the main content as follows:1. Light scattering (LS) signals have been applied for analytical detections, but the selectivity is poor. In order to improve the selectivity, pre-separation or new machines are generally considered. Different from those technique methods, we synthesized a new oxamide ligand, N, N'-bis (2-aminophenyl) oxamide (NAPO), which was found that the LS signals of NAPO, measured with a common spectrofluorometer, could be selectively enhanced by copper ion in neutral medium. Thus, a new highly selective method for copper ion detection could be developed over the range of 0.9-31.0μmol/L with the limit of determination of 97.6 nmol/L (3σ). Foreign ions including Cd(II), Al(III) could be allowed even if present at the level of 7-fold more than that of Cu²⁺, avoiding pre-separation procedures from complicated samples such as in real wastewater samples. Mechanism studies have been showed that some kinds of clusters form from the reaction of copper ion and OPAME, which has the enhanced LS signals.2. The interaction of Amido black 10B (AB) with DNA in basic medium was studied in the presence of cetyltrimethylammonium bromide (CTMAB) based on the measurements of resonance light scattering (RLS), UV-vis, CD spectra, and RLS imaging. The interaction has been proved to give a ternary complex of CTMAB-DNA-AB in Britton-Robinson buffer of pH 11.55, which exhibits strong negative Cotton effect at 233.3 nm and 642.8 nm, and strong RLS signals characterized at 469 nm. Experiments showed that the enhanced RLS intensities (ΔI_rls) against the mixture of AB and CTMAB are proportional to the concentration of fish sperm DNA (fsDNA) and calf thymus DNA (ctDNA) respectively over the range of 0.03-1.0 and 0.05-1.5μg/mL, with the limits of determination (3σ) of 7.3 ng/mL for fsDNA and 7.0 ng/mL for ctDNA.3. A novel dual-wavelength resonance lighting scattering ratiometry (DRLS) is proposed for determination of Deoxyribonucleic acid (DNA) based on the interaction of Amaranth (AH) with DNA at present of cetyltrimethylammonium bromide (CTMAB). The interaction mechanism among DNA, AH, and CTMAB has been investigated based on the measurements of resonance light scattering (RLS), UV-vis, and CD spectra. The results show that a ternary complex of DNA-CTMAB-AH could be formed in Britton-Robinson buffer of pH 5.72, which displays strong RLS signals characterized at 570.0 nm, and clearly negative Cotton effect near 256.0 nm and 528.0 nm. By comparing the RLS signals of 570.0 nm (/570) and the RLS intensities ratio of 608 nm to that of 260 nm (I₆₀₈/I₂₆₀), respectively, we find that the detection range of DNA is 0.01-1.5μg/mL for RLS and DRLS with the limit (3σ) of 6.5 ng/mL for RLS and 1.0 ng/mL for DRLS. Thus, the DRLS method is obviously superior to the ordinary RLS method.Section two mainly introduces the application of OSMs as spectral probes in Single nucleotide polymorphisms and DNA hybridization detection, the main content as follows:1. The interactions of organic small molecules (OSMs) quinone-imine dyes including Acridine Yellow (AY), Neutral Red (NR), Acridine Orange (AO), Brilliant Cresyl Blue (BCB), Thionin (TN), Azur A (AA), Azur B (AB), and Methylene Blue (MB) respectively with double strand DNA (dsDNA) and single strand DNA (ssDNA) were investigated based on the resonance light scattering (RLS) and TEM measurements. Mechanism investigations have showed that the groove binding occurs between dsDNA and OSMs, which depends on G-C sequences of dsDNA and the volume of OSMs. With the amplified RLS signals, a new technique has been proposed to detection of the hybridization and mismatch of DNA labeling neither the target nor the probe DNA. The results show that the extent of the amplified RLS signals of dsDNA by AY is the maximum among these eight quinone-imine dyes, and therefore, it was selected as a typical model system for further discussions.2. Investigations have demonstrated that the amino of dGMP, dAMP, and cytosine could react easily with OPAME as the consequences of thio-subtituted isoindole compounds. Whereas, similar results could not be found as the reaction between thymine and OPAME, for it only has a secondary amino-group, which could not efficiently form thio-subtituted isoindole compounds. The investigation also shows hat the amino-groups of ssDNA can react easily with OPAME and form strong fluorescence products for ssDNA can uncoil sufficiently with the random structure and the exposed amino-group of bases. Whereas, if ssDNA binds with the target firstly, forming double-stranded DNA (dsDNA) structure, then the amino-groups of bases are screened in the interior of the double-stranded structure, preventing the amino-groups from reacting with OPAME. As a result, the amino-groups of bases display inert chemical activity with OPAME, yielding weak fluorescence emission. With that, a novel simple label-free sequence detection method for complementary and single-base mismatched ssDNA in the hybridization of DNA could be constructed.3. DNA sequences detection is significant to pathogen determination, biomedical research, and drug discovery in the post-genomic and proteomic era, thus, it is significant to establish simple and speedy detections for specific oligonucleotide sequences and single-base mismatches. In this contribution, we have proposed a novel simple and speedy assay for specific oligonucleotide sequences and single-base mismatch detection based on the different quench effects of silver ion on Texas Red tagged single and double stand DNA. Experiments have showed that silver ion can quench the fluorescent of Texas Red tagged oligonucleotide effectively. However, the quench efficiency of silver ion decreased when Texas Red tagged oligonucleotide (P) hybridized firstly with its complete complement ssDNA (T), thus, the fluorescence signals of the system were enhanced. With that, complementary and single-base mismatched oligonucleotide sequence could be identified in the hybridization of DNA. Comparison with the recent developed molecular beacons (MBs) technique, it dose not need so called the "stem-loop" structures. So, the hybridization process between P and T is easier than that between MBs and its target, as a result of enhancing the detection efficiency.The studies enlarge the content of investigation and the application area for organic small molecule as spectral probes, and it can provide the experimental and theoretic instruction in exploring the application of other organic small molecule probes in biochemical analysis.