Study On Novel Methods For DNA Detection Based On Biological Amplification Technologies

The accomplishment of the Human Genome Project and the progress in research of the functional genomics make gene diagnoses a hot point in the areas of molecular biology and biomedicine. Based on the hybridization of bases, DNA biosensors that can continuously, fast, sensitively detect the specific gene sequence have been quickly developed in recent years. DNA analysis is the key technologies for its biological function study, disease diagnosis, and drug development. This thesis focuses on exploring new methods for highly sensitive detection of nucleic acids and related molecules.1. Label-free surface-enhanced Raman spectroscopy for sensitive DNA detection by DNA-mediated silver nanoparticle growthThis work designed a label-free strategy for surface-enhanced Raman spectroscopic (SERS) detection of target DNA on a peptide nucleic acid (PNA) modified glass slide. Upon hybridization of the PNA with target DNA, the surface became negatively charged and allowed the absorption of silver ions on the DNA skeleton. After chemical reduction by hydroquinone, the formed silver nanoparticles could be further grown with a silver enhancement step to amplify the detectable SERS signal by absorbing rhodamine 6G as SERS reporter on silver nanoparticle surface. The growth of silver nanoparticles was characterized with X-ray photoelectron spectroscopy and scanning electron microscopic image. The label-free SERS method achieved the detection of DNA with a liner range from 1.0×10-10 to 1.0×10-6 M and a detection limit of 45 pM. Through introducing hybridization chain reaction to increase the DNA length, the Raman signal was further amplified, leading to a detection limit of 3.4 pM. This approach could discriminate perfectly matched target DNA from single-base mismatch DNA. This strategy possessed good capacity to integrate other amplification techniques for sensitive high-throughput detection of genes.2. Raman spectroscopic detection of sub-picomolar DNA by coupling silver catalyzed silver deposition with circular strand-replacement polymerization on magnetic nanoparticles.A highly sensitive surface-enhanced Raman spectroscopic (SERS) method was developed for detection of target DNA. This method combined circular strand-displacement polymerization (CSRP) with silver enhancement on magnetic nanoparticles (MNPs) to achieve dual signal amplification. The MNPs were used as immobilization support of the molecular beacon (MB) as recognition probe, which led to convenient separation of the CSRP product from the reaction mixture containing the primer and Raman dye assembled silver nanoparticles (reporter AgNPs). After the MB hybridized with target DNA, the reporter AgNPs was attached to MNPs by hybridization between primer and stem part of the MB to initiate a polymerization of DNA strand, which led to the release target and another polymerization cycle. Thus the CSRP produced the multiplication of target-related reporter AgNPs on MNPs surface. Silver enhancement was then performed on the surface to increase the SERS signal of Raman dye. This signal could discriminate perfect matched target DNA from 1-base mismatch DNA. The dynamic range of the sequence-specific DNA detection was from 10-13 to 10-8 mol L-1 with a detection limit down to sub-picomolar DNA. This proposed method exhibited an efficient amplification performance, and would open new opportunities for sensitive detection of other biorecognition events.3. Sub-femtomolar electrochemical detection of DNA using surface circular strand-replacement polymerization and gold nanoparticle catalyzed silver deposition for signal amplification.A highly sensitive method was developed for detection of target DNA. This method combined circular strand-displacement polymerization (CSRP) with silver enhancement to achieve dual signal amplification. After molecular beacon (MB) hybridized with target DNA, the reporter gold nanoparticle (Au NPs) was attached to electrode surface by hybridization between Au NP labeled primer and stem part of the MB to initiate a polymerization of DNA strand, which led to the release of target and another polymerization cycle. Thus the CSRP produced the multiplication of target-related reporter Au NPs on the surface. The Au NPs then catalyzed silver deposition for subsequent stripping analysis of silver. The dual signal amplification offered a dramatic enhancement of the stripping response. This signal could discriminate perfect matched target DNA from 1-base mismatch DNA. The dynamic range of the sequence-specific DNA detection was from 10-16 to 10-12 M with a detection limit down to sub-femtomolar level. This proposed method exhibited an efficient amplification performance, and would open new opportunities for sensitive detection of other biorecognition events.4. Sensitive fluorescence detection of DNA using isothermal exponential amplification coupled quantum dots coated silica nanosphere as labelA new strategy to combine isothermal exponential amplification (IEA) with CdTe quantum dots (QDs) functionalized silica nanosphere label was designed for highly sensitive fluorescent detection of target DNA. A well plate was used as support to immobilize molecular beacon (MB) as recognition probe and perform the IEA procedure. After the MB recognized target DNA and opened its cycle, the stem part could hybridize with a primer to initiate the polymerization of DNA strand, which led to the release of target to open another MB molecule and start next cycle of strand-replacement polymerization. Meantime, the formed double-strand DNA was recognized by nicking endonuclease, leading to an endonuclease-based strand-replacement polymerization, which produced DNA trigger to open more MB. The opened MB molecules were finally bound to the label by biotin-streptavidin coupling. Upon a dissolving process, the released cadmium cation could sensitize the fluorescent emission of Rhod-5N to achieve cascade signal amplification. The proposed method could detect target DNA ranging from 10-17 to 10-11 M with a detection limit down to～50 copies. It also showed high selectivity. This highly sensitive and specific assay had potential to become a promising DNA quantification method in biomedical research and clinical diagnosis.5. Assistant DNA recycling with nicking endonuclease and molecular beacon for signal amplification using a target-complementary arched structureA simple and universal method for ultrasensitive "signal on" detection of DNA was developed with a target-complementary arched structure to release assistant DNA, which was recycled with nicking endonuclease to amplify the detectable fluorescent signal of molecular beacon. The target-complementary arched structure was successfully designed to trigger the A-DNA recycling with NEase and MB for signal amplification, which led to a highly sensitive method fluorescent detection of target DNA. The entire detection time was less than 2 h. Based on the A-DNA recycling, the strategy showed a detection limit at sub-picomolar level, which was about 3 orders of magnitude lower than that of the conventional hybridization without NEase-based amplification. Moreover, since the A-DNA recycling replaced the target DNA recycling, the proposed strategy provided a universal strategy in DNA detection without requirement of NEase-specific recognition sequence in target DNA. The novel concept of A-DNA recycling strategy can be expected to design the integrated, portable and low cost device for DNA detection based on the arched structure.6. Ultrasensitive fluorescence detection of bleomycin via exonuclease Ⅲ-aided DNA recycling amplificationA simple and universal method for ultrasensitive "signal on" detection of bleomycin was developed based on bleomycin (BLM)-Fe(Ⅱ) mediated molecular beacon scission to release R-DNA, which was recycled with exonuclease Ⅲ to amplify the detectable fluorescent signal. The entire detection time was less than 1 h. Based on the exonuclease Ⅲ (Exo Ⅲ) aid DNA recycling, the strategy showed a detection limit at sub-picomolar level, which was about 2 orders of magnitude lower than that of the conventional hybridization without Exo Ⅲ-based amplification. Importantly, the proposed sensor also exhibited high selectivity for BLM among the four important antitumor antibiotics and satisfactory performance in trace BLM determination in serum samples. On the basis of the findings and results, we believe that this method shows distinct advantages over conventional methods in terms of high specificity, low detection limit, and simple-to-implement procedure and it could offer an interesting alternative approach for the determination of trace amounts of BLM in clinical samples.