Reaserch Of Signal Amplification Methods Of Nucleic Acid Molecular Optical Probes In Biochemical Analysis

In recent years, nucleic acid molecule probes have become a class of important bioanalytical tools for wide application in the field of bioanalysis and biomedicine. Nucleic acid molecule probes are comprised of nucleic acid sequences to recognize targets by base pairing, electrostatic adsorption and other non-covalent interactions and produce optical, electrical or magnetic signal changes. In the different signal output-modes of detection methods, the optical probe nucleic acid molecules have get the depth and extensive research, because of its high sensitivity, wide range of targets, sundry measure methods, simple experimental operation, and so on. However, since the optical detection signal mode of nucleic acid molecule probes to the target is generally achieved through 1:1 transmission, so that detection sensitivity is limited within a certain range. In order to improve the detection sensitivity, the scientists have developed a variety of signal amplification methods such as enzyme-assisted signal amplification, nucleic acid hybridization-based signal amplification, nanomaterials-based signal amplification and fluorescent conjugated polymer-based signal amplification and so on. In this dissertation, we combined the nucleic acid molecule optical probe with some new signal amplification technology to detect nucleic acid molecules and enzymes. The main researches of this dissertation are summarized as follows.1. We developed homogeneous fluorescence DNA detection based on a novel competitive host-guest interaction mechanism between P-cyclodextrin polymer (poly P-CD) and pyrene-labeled probes. Pyrene labeling with oligonucleotide strands can be recruited and resided in lipophilic cavities of poly β-CD. This altered lipophilic microenvironment provides favored polarity for enhanced quantum efficiencies and extraordinarily increases the luminescence intensity of pyrene. However, with addition of complementary DNA, the pyrene-labeled probe formed double-strand DNA to hinder pyrene from entering the cavities of poly β-CD. The releasing of pyrene from poly β-CD, which are followed by fluorescence extinguishing, will provide clear signal turn-off in presence of target DNA. We also introduced Exodeoxyribonuclease I to improve the sensitivity of this system, and the following product of cleavage reaction, pyrene-nucleotide, could more easily host-guest interact with polyβ-CD and emit stronger fluorescence than pyrene-labeled probe.2. We synthesized a new cationic β-cyclodextrin polymer (cationic poly β-CD), and developed a sensitive enhanced fluorescence signal assay of DNA based on the competitive host-guest interaction mechanism between cationic poly β-CD and single-pyrene-labeled probes. Because of the branched amino-group, the cationic poly β-CD characterized positive electrical charge at pH 7 aqueous solution. Electrostatic interactions between cationic conjugated polymers and negatively charged DNA can assist the pyrene labeling with oligonucleotide to reside in lipophilic cavities of β-CD and emit strong fluorescence. However, with addition of complementary DNA, the pyrene-labeled probe formed double-strand DNA to hinder pyrene from entering the cavities of poly β-CD, and caused the fluorescence extinguishment. Ralative to the previous assay, this method only need single-pyrene-labeled probe, and avoid the introducation of enzyme. The proposed detection of DNA exhibits a dynamic range from 1.25-5.0 nmol/L and a detection limit of 0.3 nmol/L, present good selectivity to mismatch DNA. In addition, the successful detection of adenosine and mercury ion are also demonstrated by using the similar sensor scheme.3. We have demonstrated a new, label-free and highly sensitive strategy for gold nanoparticle (AuNP)-based colorimetric detection of sequence specific DNA via hybridization chain reaction (HCR) amplification. Two hairpin auxiliary probes were designed, which exposed sticky end, and stabilized AuNPs effectively against salt-induced aggregation. The target DNA hybridized with the hairpin auxiliary probes and triggered the formation of extended dsDNA polymers through HCR. In this state, the formed dsDNA polymers provide little stabilization, and AuNPs undergo aggregation when salt concentration is increased. As a result, a red-to-blue color variation is observed in the colloid solution. The system is simple in design and convenient in operation. The novel strategy eliminated the need of enzymatic reactions, separation processes, chemical modifications, and sophisticated instrumentations. The detection and discrimination process can be seen with the naked eye. The detection limit of this method is lower than or at least comparable to previous AuNP-based methods. This method offered ultrahigh detection sensitivity and provided a colorimetric detection limit of 50 pmol/L for instrument detection and 100 pmol/L for naked eye detectionImportantly, the protocol offers high selectivity for the determination between perfectly matched target oligonucleotides and targets with single base-pair mismatches.4. This work develops a fluorescence approach for sensitive detection of DNA methyltransferase activity based on endonuclease and rolling circle amplification (RCA) technique. In the presence of DNA adenine methylation (Dam) MTase, the methylation-responsive sequence of hairpin probe is methylated and cleaved by the methylation-sensitive restriction endonuclease Dpn I. The products cleaved by restriction endonuclease Dpn I then function as a signal primer to initiate RCA reaction by hybridizing with the circular DNA template. Each RCA product containing thousands of repeated sequences might hybridize with a large number of molecular beacons (detection probes), resulting in an enhanced fluorescence signal. In the absence of Dam MTase, neither methylation/cleavage nor RCA reaction can be initiated and no fluorescence signal is observed. The proposed method exhibits a dynamic range from 0.5 U/mL to 30 U/mL and a detection limit of 0.18 U/mL. This method can be used for the screening of antimicrobial drugs and has a great potential to be further applied in early clinical diagnosis.5. We have developed a new approach to detect the activity of alanine aminotransferase (ALT) by using molecular beacons (MBs). ALT catalyzes the transfer of an amino group from alanine to α-ketoglutarate, producing pyruvate and glutamate. Subsequently, lactate dehydrogenase mediates the oxidation of pyruvate, yielding lactate acid and NAD+. The E. coli DNA ligase would catalyze the ligation of two short oligonucleotides that complement with an MBs only in the presence of NAD+, resulting in the opening of the MBs and the restoration of fluorescent signal. As a result, the activity of ALT can be determined by measuring the fluorescence of MBs. The proposed method exhibits a dynamic range up to 3.13 U/L with a detection limit of 0.25 U/L.