Novel Magnetic Beads-based Instantaneous Derivatization For The Chemiluminescence Detection Of Sequence-specific DNA

Medical reports indicate that most of the cancers and genetic diseases are associated with genetic mutation. Furthermore, many infectious diseases are caused by virus and pathogens in the air. Therefore, it is very important and urgently needed to develop sequence-specific DNA biosensors and single nucleotide polymorphism detection techniques due to their significantadvantages in various fields such as genetic screening, forensic analysis, the early-stageidentification of genetic disorders, the diagnosis of infections and proteomic investigations. Fast and reliable DNA biosensors have grown tremendously over the past few years, most of which focus on the development of sensitive and selective techniques by the employment of various labels, such as enzyme, radioisotope and fluorescent dyes, quantum dots, electrochemical and CL tags. Given that the complicated procedure and much more time-consumption for labeling, currently, label-free alternatives attract increasing interest. Due to its simple optical system and low background nature, CL analysis has been widely applied in clinical, biological and environmental fields and is becoming one of the most powerful techniques for use in analytical chemistry.In 2003, our research group firstly reported a label-free CL technique (magnetic beads-based instantaneous derivatization) for DNA detection based on the reaction between a specific CL reagent-3, 4, 5-trimethoxyphenylglyoxal (TMPG) and guanine nucleotides. Herein, a series of label-free CL protocols for sequence-specific DNA assays have been developed in order to further improve its sensitivity and selectivity, including G₃₀ sequence, DNAzyme compound, G-rich sequence loaded polystyrene microspheres or surface-functionalized carbon nanotubes as the amplification platforms. Overall, simple, rapid and stable CL protocols coupled the high sensitivity of CL analysis with effective magnetic separation and good differentiation ability for unwanted constituents such as single-base mismatches, hence, offer great promise for DNA hybridization analysis. Description of research in my thesis is presented as follows: Chapter 1: This thesis first addresses the need to develop sequence-specific DNA and single nucleotide polymorphism detection techniques, and then presents current state of knowledge on DNA analysis, including fluorescent, electrochemical, SPR and QCM techniques, etc. The second phase of this chapter reviews current main CL systems such as luminol, peroxyoxalate, acridinium ester and alkane phosphatase-related CL reactions, and their recent applications in various fields, especially in immunoassay and DNA assay. Following that, objectives and significance of this research are summarized.Chapter 2: Poly T-modified magnetic bead-based instantaneous derivatization for CL detection of sequence-pecific DNA1．Label-free CL detection of telomere sequence-specific DNATelomere is the repeat units to the 3'-end of chromosomal DNA synthesized by a ribonucleoprotein-telomerase which provids cells with a mechanism to prevent gradual telomere erosion. Telomere and telomerase are thought to be responsible for the continuous and uncontrolled growth of cancer cells. In this work, based on magnetic instantaneous derivatization, a novel label-free CL detection protocol was presented as exemplified by the successful determination of sequence-specific DNA--telomere. This method was composed of three steps: (1) the dT₂₀ arm-modified in the magnetic beads was hybridized with the dA₂₀ -modified capture DNA; (2) the first step was followed by the second hybridization of the bead-captured probe with telomere; (3) the amount of telomere was determined by the guanine CL reaction with TMPG. The results showed that this simple and sensitive protocol was much suitable for the determination of sequence-specific DNA such as telomere with a good linear correlation in the concentration range of 5-100 nM (R²=0．9918) .2．Label-free CL approach for sequence-specific DNA associated with the anthrax lethal factorsAnthrax, as a world-wide biowarfare agent, has attracted great attention of public and military agencies. The practical and effective methods are desired to identify and monitor the biowarfare agent, and control its infections in public. In this section, we established the 锘縧abel-free CL approach to detect the 30-mer oligonucleotide related to the anthrax lethal factors. Furthermore, an amplified approach was investigated to improve the sensitivity. This amplified approach was composed of the following steps: (1) the dT₂₀ -modified arm in the magnetic beads was hybridized with the dA₂₀ -modified capture DNA; (2) the bead-captured probe hybridized the 30-mer anthrax target DNA; (3) the third hybridization proceeded between target DNA and reporter DNA containing a guanine-rich oligonucleotide (G₃₀ sequence); (4) the amount of the target DNA was determined by the guanine CL reaction with TMPG. The results showed that this simple and sensitive protocol was much suitable for the determination of sequence-specific DNA related to the anthrax lethal factors. Using nonamplifed method, a good linear correlation was obtained in the concentration range of 6-60 nM with the lowest detection concentration of 6 nM. In contrast, by using G₃₀ amplified method, a good linear correlation was achieved in the range of 0．45-6 nM with the lowest detection concentration of 0．45 nM, which was 10-fold improvement on the nonamplifed one. Moreover, this new CL protocol had a good discrimination for A-A single-base mismatch.鈽匔hapter 3: Carboxyl terminated magnetic bead-based instantaneous derivatization for the label-free CL detection of sequence-specific DNA鈽匟erein, an amplified CL approach was investigated employing G-rich sequence as the amplification unit. This novel method was composed of the following steps: (1) the carboxyl terminated magnetic beads were activated by EDC, and then coupled the amine-modified capture DNA; (2) the bead-captured probe hybridized the 30-mer target DNA; (3) the third hybridization proceeded between target DNA and reporter DNA (G-rich sequence); (4) guanine rich in target and reporter DNA reacted with TMPG to generate the CL signal. The results showed that this novel protocol was simple, stable and suitable for the determination of sequence-specific DNA related to the anthrax lethal factors. Both nonamplified and amplified approaches achieved a good linear correlation. The lowest detection concentration was 2 nM for nonamplified method and 50 pM for amplified one, which was 2．5-fold and 10-fold improvement on the previous work based on Poly T-modified magnetic beads, respectively. Moreover, this new CL protocol had a good discrimination of perfect complementary part from A-A single-base mismatch.Chapter 4: G-rich sequence-functionalized carboxyi terminated carbon nanotubes for the amplified CL detection of sequence-specific DNAWe developed a novel CL approach with high sensitivity and good selectivity, by taking advantage of magnetic beads as the preconcentration carrier and carboxyl modified carbon nanotubes (CNTs-COOH) as the amplification platform. Briefly, a "sandwich-type" detection strategy was employed in our design, which involved capture probe DNA immobilized on the surface of carboxyl terminated magnetic beads and multiple amine-modified reporter DNA self-assembled on the surface of CNTs-COOH. The reporter DNA included a G-rich sequence domain for the generation of light and an additional tethered nucleic acid domain complementary with target DNA. Four kinds of CNTs-COOH were prepared by a well controllable modification method, i.e., oxidized by a 9:1 concentrated sulphuric acid/30% hydrogen peroxide aqueous solution, their characteristics were investigated by TEM and zeta potential system etc., and then two of them were selected for the amplified detection of DNA. The CL signal was obtained via an instantaneous derivatization reaction between TMPG and guanines rich in target and reporter DNA. This new detection scheme eliminated the need to first release the hybridized target DNA prior to optical detection, as well as the need for any chemical manipulation of the nucleic acids. As a result, the entire assay could be completed within 3-4 hrs. By employing this strategy, we demonstrated that this DNA assay was reproducible, stable, easy to use, and could sensitively detect femtomolar target DNA related to the anthrax lethal factors with excellent differentiation ability for single-base mismatches. An excellent linearity was found within the range of 2-200 fmol (R²=0．992 for MW-1 and R²=0．998 for SW) with the lowest detection concentration of 10 pM (1 fmol). This was a 200-fold improvement on nonamplified measurement. Overall, this new CL protocol coupled the high sensitivity of CL analysis with effective magnetic separation for discriminating against unwanted constituents such as single-base mismatches, hence, offered great promise for DNA hybridization analysis. Chapter 5: Polystyrene microspheres-based amplified approach for highly sensitive CL detection of sequence-specific DNAThe commercial streptavidin-modified polystyrene microspheres (PS-SA) were employed as an amplification platform to develop a time-, labor-, and cost-effective CL approach. Compared with the CNTs-based amplified method, this new detection scheme eliminated the need to first preparation of the amplification carriers, shortened the assay time and improved the experimental reproducibility. Briefly, a "sandwich-type" detection strategy was employed in our design, which involved capture probe DNA immobilized on the surface of carboxyl terminated magnetic beads and multiple biotinylated reporter DNA self-assembled on the surface of PS-SA, both of which flanked target DNA. Prior to the binding of capture probe DNA, magnetic beads were activated by EDC. As a result, in the presence of target DNA, the capture probe brought target DNA, along with reporter DNA on the surface of PS-SA, proximal to the magnetic beads surface. The CL signal was obtained via the instantaneous derivatization reaction between TMPG and guanines rich in target and reporter DNA. As a result, the entire assay could be completed within 2-3 hrs. By employing this strategy, we demonstrated that this DNA assay was reproducible, stable and easy to use. An excellent linearity was found within the range of 1-100 fmol (R²=0．996) with the lowest detection concentration of 5 pM (0．5 fmol), which was 400-fold and 2-fold improvement on nonamplified and CNTs-based amplified methods, respectively. This novel approach provided great promise for DNA assay with good differentiation ability for various single-base mismatches.Chapter 6: DNAzyme-based novel instantaneous derivatization CL detection for sequence-specific DNAHerein, we wish to introduce a new CL method for the determination of the sequence-specific DNA by the coupling of catalytic nucleic acid label (DNAzyme) based CL detection route with an efficient magnetic isolation of the hybrid. The assay relied on (1) the immobilization of amine-modified capture DNA on the surface of carboxyl-terminated magnetic beads activated by EDC; (2) first hybridization event occurring between the bead-captured DNA probe and one 15-mer portion (5'-AAT ATT GAT AAG GAT-3') of the target sequence; (3) second hybridization happening between one part of the DNAzyme-modified reporter sequence and another 15-mer portion (5'-GAG GGA TTA TTG TTA-3') of the target sequence, and then direct detection of CL signal on the surface of the magnetic beads in a basic solution of luminol and H₂O₂．The influence of relevant experimental variables, including the effect of the amount of the magnetic beads, the duration of the hybridization and the parameters affecting CL signal, was examined and optimized. The results showed that this simple and sensitive protocol was suitable for the determination of sequence-specific DNA with a good linear correlation in the concentration range of 0．2-20 nM (R²=0．9987) and a detection limit of 0．1 nM. Overall, this new CL protocol coupled the high sensitivity of CL analysis with effective magnetic separation for discriminating against unwanted constituents such as mismatches and proteins, hence, offers great promise for DNA hybridization analysis.Chapter 7: Chemiluminescence method for the determination of surfactants based on its quenching effect on the Iuminoi-NaIO₄-cyclodextrin reactionThis was an early-stage work, which focused on developing a general flow injection CL assay to detect various types of surfactants, i.e., anionic, cationic and non-ionic surfactants. Surfactants are a class of compounds that are found in a multitude of domains, from industrial settings, to research laboratories, to household products as well as environmental pollutants. Due to their prevalence in so many domains, the need for analysis is paramount.All types of surfactants could be simply and sensitively determined, by directly quenching the CL signal between luminol and NaIO₄ in a basic solution containing one polyhydroxyl compound such as cyclodextrin (CD), glucose or glycerol. This specific quenching effect was attributed to the change of the microenvironment of the CL reaction, caused by the addition of various surfactants. Based on this fact, the potential use of this CL reaction was exemplified by the cationic surfactant CTMAB, anionic surfactant SDS and non-ionic surfactant Triton X-100．It was found that the measurable range of CTMAB, SDS and Triton X-100 were 4-400 fiM by using a basic CD-luminol-NaI04 CL reaction. With our simple setup, CTMAB, SDS and Triton X-100 were detectable at a concentration as low as 2μM. A series of seven repetitive measurements of 10μM surfactant were used for estimating the precision, yielded reproducible signals with a relative standard deviation of 3．0%. Overall, this new CL reaction is quite promising for the post-column determination of surfactant mixtures.