Functional DNA Nanostructures And Its Applications In Diagnostics Of Biomarker

Early diagnosis and early treatment is the key to improve the prognosis of patients with lung cancer and to reduce the mortality, tumor markers play an important role in it. In addition to the traditional tumor markers, we have found many new and effective molecular biomarkers, including methylation, a variety of gene mutations, cirDNA and micro RNA in blood. Many research showed that the sensitivity and specificity of these markers for lung cancer diagnosis and prognosis estimation, even higher than the traditional tumor markers. Therefore, these markers may be widely used in clinical early prediction and diagnosis of lung cancer. DNA nanostructures can be readily functionalized with a variety of molecules and nanoparticles with nanometer-scale addressability.Since DNA nanostructures are essentially biomolecules, it is intuitive to explore their biological applications in diagnostics and therapeutics. In this paper, we focus on 'the construction and functional modification of DNA nanostructures and their applications in cancer diagnostics. DNA nanostructures are used to load more signal molecules as signal amplification probes or to be functionalized with variety of biomolecules as carrier for sensitive/simultaneous detection of multi-tumor markers for cancer.1. Evaluation of DNA methyltransferase activity and inhibition via chiroplasmonic assemblies of gold nanoparticlesRecently, chiral superstructures assembled by plasmonic nanoparticles (NPs) have gained significant attention due to their strong and intriguing chiroptical activity. Herein, circular dichroism(CD) spectroscopy has been explored for detection of methyltransferase (MTase) activity and inhibition based on DNA-induced chiroplasmonic assemblies of gold nanoparticles (AuNPs) and endonuclease Hpa?. DNA-modified AuNPs are assembled into AuNPs dimers by linker DNA,which display strong chiroptical activity and are used as signal probes. Endonuclease Hpa? can specifically recognize and cleave 5'-CCGG-3, sequence in the linker DNA,which destroys the AuNPs dimers and results in low CD signal. On the contrary, Hpa? doesn't work when 5'-CCGG-3'is methylated to 5'-CCmGG-3f by M.SssI. As a result, the intact AuNPs dimers remain strong chirality. Thus, a simple CD method for evaluation of MTase activity and inhibition is conducted.The CD intensity is in linear correlation with M.SssI concentration ranging from 0.5 to 150 U/mL,with a correlation coefficient of 0.9816 and a detection limit of 0.27 U/mL (S/N = 3). The inhibition of 5-Aza or procaine on M.SssI activity is also evaluated. Good accuracy, precision and sensitivity are obtained when the method is applied in complex matrix such as human serum samples, which is significant for clinical diagnosis and drug development.2. Chiroplasmonic assemblies of gold nanoparticles for ultrasensitive detection of 8-hydroxy-2'-deoxyguanosine in human serum sampleGold nanoparticles (AuNPs) has been extensively explored to be used in analytical methods such as electrochemical, colorimetric methods, and so on. However, only a few methods has been reported by using chirality of AuNPs although their chiral assembly has been studied extensively and circular dichroism (CD) spectroscopy is also a simple and sensitive analytical method. In this paper, sensitive CD spectroscopy method has been explored for detection of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a well-known biomarker for oxidative DNA damage,based on DNA-induced chiroplasmonic assemblies of AuNPs. First, 8-OHdG aptamer hybridized with its complementary sequence that modified with AuNPs based on precision matched bases.DNA-modified AuNPs were assembled into AuNPs dimers by 8-OHdG aptamer, which displayed strong chiroptical activity. Subsequently, in the presence of 8-OHdG, the high specific recognition and affinity constants of aptamer and 8-OHdG destroyed the hybrid of aptamer and its complementary sequence, as a result, AuNPs dimers were destroyed and showed low CD signal.The CD intensity was in log-linear correlation with the concentration of 8-OHdG ranging from 0.05 to 2 nM, with a correlation coefficient of 0.9951 and a detection limit of 33 pM (S/N = 3). The method has been successfully applied in complex matrix such as human serum samples. The recoveries were from 92.5% to 107% and the relative standard derivations were in the range of 4.89%?7.27%, indicating that the method had good accuracy and high precision. Therefore,these results indicated that the proposed CD method was simple and reliable, which held great potential for clinical examinations.3. Label-free ultrasensitive detection of telomerase activity in urine via multiple telomeric hemin/G-quadruplexes triggered polyaniline deposition and DNA tetrahedron-structure regulated signalIn this paper, a novel strategy for label-free ultrasensitive detection of telomerase activity in urine was proposed based on multiple telomeric hemin/G-quadruplexes triggered polyaniline deposition and DNA tetrahedron-structure regulated signal. Briefly, DTS-primers were skillfully designed and immobilized on the surface of gold electrode. Under the action of telomerase,telomeric repeats (TTAGGG)X elongated to the 3' end of primer and formed multiple telomeric hemin/G-quadruplexes in the presence of hemin and potassium ions, which have high catalytic activity to guide PANI deposition not only on themselves but also on the DNA tetrahedron-structure(DTS). Then, telomerase activity could be determined by using PANI as electrochemical signal molecules. Interestingly, the nanospacing between primers could be easily controlled with the regulation of DTS size. In the presence of DTS(37), the nanospacing between primers is closest to the telomerase size, which greatly improved the telomerase accessibility, elongation efficiency and detection sensitivity. With this label-free strategy, telomerase activity in cancer cells, such as MCF-7, A549, MDA-MB-231, and He La could be evaluated. The lowest detection limit was found to be 1 HeLa cell with a broad detection range from 5 cells to 5 000 cells. Compared with single-strand telomerase substrate primer, the sensitivity for telomerase detection was enhanced ?20 times by using DTS(37). Importantly, the application of this method for determination of the telomerase activity in bladder cancer samples was in good agreement with clinical diagnosis. In a word, the proposed method is label-free, simple, reliable, and has great potential to be developed as a noninvasive and ultrasensitive method for practical clinical diagnosis and evaluation of cancer development.4. Application of spectral crosstalk correction for improving multiplexed microRNA detection using a single excitation wavelengthMicroRNAs (miRNAs) play crucial roles in the regulation of cellular activities and are next-generation biomarkers for early cancer detection. Simultaneous monitoring of multiplexed miRNA is very important for enhancing the accuracy of cancer diagnostics. Traditional fluorescence methods for multicomponent analysis were usually operated under multiple excitation wavelengths, because spectral crosstalk is very detrimental to detecting accuracy for multicomponent analysis. Herein, we present a fluorescence strategy for multi-miRNAs detection in plasma under a single excitation wavelength. Nucleic acid stain TOTO-1 and three labeled fluorescence dyes Cy3, Cy3.5, and Cy5 emit no fluorescence in their free state. Target miRNA hybridized the auxiliary and probe oligonucleotides into duplex nucleic acid. Intercalation interaction localized TOTO-1 and labeled dyes into the duplex nucleic acid. As a result, TOTO-1 emitted strong fluorescence and efficient Forster resonance energy transfer (FRET) happened.MicroRNAs miRNA-155, miRNA-182, and miRNA-197, which are significant for the early diagnosis of lung cancer, were simultaneously detected as models. Deviations from spectral crosstalk in the presence of other miRNAs were corrected by mathematical methods. Results demonstrated that, after spectra crosstalk corrections, every miRNA at high or low concentration in plasma was determined accurately in the presence of either high or low concentrations of the other two miRNAs. This new multiplexed assay for miRNAs is promising for clinical diagnosis,prognosis, and therapeutic monitoring of early-stage lung cancer.5. Colorimetric detection of influenza A virus using antibody-functionalized gold nanoparticlesEarly and accurate diagnosis is considered the key issues to prevent the further spread of viruses and facilitate influenza therapy. Herein, we report a colorimetric immunosensor for influenza A virus (IAV) based on gold nanoparticles (AuNPs) modified with monoclonal anti-hemagglutinin antibody (mAb). The immunosensor allows for a fast, simple,and selective detection of IAV. In this assay, influenza-specific antibodies are conjugated to AuNPs to create mAb-AuNPs probes. Since IAV has multiple recognition sites for probes on the surface, the mAb-AuNPs probes can be specifically arranged on the virus surface due to their very specific antigen recognition. In this case, this aggregation of the mAb-AuNPs probes produces a red shift in the absorption spectrum due to plasmon coupling between adjacent AuNPs, and it can be detected with the naked eye as a color change from red to purple and quantified with the absorption spectral measurements. The aggregate formation is also confirmed with transmission electron microscopy(TEM) imaging and dynamic light scattering (DLS). Under the optimal conditions, the present immunoassay can sensitively measure H3N2 IAV (A/Brisbane/10/2007) with a detection limit of 7.8 hemagglutination units (HAU). This proposed immunosensor revealed high specificity,accuracy, and good stability. Notably, it is a single-step detection using AuNPs probes and UV-vis spectrophotometer for readout, and no additional amplification, e.g., enzymatic, is needed to read the result. This assay depends on an ordered AuNPs' structure covering the virus surface and could be applied to any virus pathogen by incorporating the appropriate pathogen-specific antibody.