| This thesis is composed of two parts. One part is DNAzyme sensors in the detection of metal ions, and the other part is the synthesis and application of upconversion nanocrystals in biosensing.I DNAzyme sensors in the detection of metal ions.DNAzymes, obtained by in vitro screening technique, are single-stranded DNAs with enzymatic activities. Compared to natural enzymes, DNAzymes can be superior in terms of stability, cost-effectiveness, ease of synthesis, modification and storage. As for some DNAzymes, they need specific metal ions as cofactors, and their enzymatic activities are highly dependent on the concentrations of the metal ions. As a result, their applications in the detection of metal ions have attracted more and more attention in these years.(1) A single-stranded DNAzyme-based Pb2+fluorescent sensor was designed by combining the substrate sequence and the enzyme sequence into one oligonucleotide strand. The intramolecular duplex structure of this single-stranded DNAzyme kept the fluorophore and the quencher, labeled at its two ends, in close proximity; thus the background fluorescence was significantly suppressed. Using this fluorescent sensor, Pb2+quantitation can be achieved with high sensitivity and high selectivity. In addition, the extraordinary stability of the intramolecular duplex structure could assure a low background fluorescence at high temperature, even if the number of complementary base pairs between the substrate sequence and the enzyme sequence was reduced, allowing the sensor to work well over a wide temperature range. Similar performances of the fluorescent sensor at4,25and37℃suggested that this sensor has a good ability to resist temperature fluctuations.(2) We reconstructed a Cu2+-dependent DNAzyme/substrate complex reported earlier. The reconstructed complex makes the use of intramolecular stem-loop structure possible, thus providing a good choice for the design of Cu2+sensors. To demonstrate this, a fluorescent sensor was designed on the basis of the reconstructed complex. In this sensor, the fluorophore/quencher pair was caged tightly in an intramolecular double-helix structure; thus, the background signal was greatly suppressed. Cu2+-dependent cleavage of the complex could cause the release of the fluorophore, leading to restoration of the fluorescence signal. High quenching efficiency provided the sensor with high sensitivity, it allowed specific detection of aqueous Cu2+down to a limit of0.6nM and within a linear range of5-500nM. The Cu2+sensor based on the reconstructed complex showed high tolerance of variations of temperature and ionic strength, making it more suitable for practical applications. To further demonstrate the tolerance of changes of temperature, the fluorescencere sponse of the sensor was measured as a function of Cu2+concentration at four temperatures:4℃,20℃,25℃and37℃. The four curves were almost overlapped and the linear regression equations obtained at these four temperatures were very similar, which indicated that the performance of the sensor was independent of temperature in the range of4-40℃. F/Fo was nearly constantat concentrations of NaCl in the range0.8-3.0M, indicating that the Cu2+sensor can work well in an ionic strength independent manner during this range.(3) Label-free metal ion detection methods were developed. To achieve these, a reconstructed Cu2+-specific DNA-cleaving DNAzyme (Cu2+-specific DNAzyme) with an intramolecular stem-loop structure was used. G-quadruplex-forming G-rich sequence(s), linked at the ends of double-helix stem of an intramolecular stem-loop structure, was partly caged in an intramolecular duplex or formed a split G-quadruplex. Cu2+-triggered DNA cleavage at a specific site decreased the stability of the double-helix stem, resulting in the formation or destruction of G-quadruplex DNAzyme that can effectively catalyze the2,2’-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS)-H2O2reaction. Based on these, two label-free, cost-effective and simple Cu2+sensors were designed. These two sensors followed different detection modes:’Turn-on’ and ’Turn-off’. As for the ’Turn-on’ sensor, the intramolecular stem-loop structure ensured a low background signal, and the co-amplification of detection signal by dual DNAzymes (Cu2+-specific DNAzyme and G-quadruplex DNAzyme) provided a high sensitivity. This sensor enabled the selective detection of aqueous Cu2+with the linear range is8-200nM and detection limit of3.9nM. Visual detection was possible. Although the ’Turn-off’sensor gave lower detection sensitivity than the ’Turn-on’ one, the characteristics of cost-effectiveness and ease of operation made it an important implement to reduce the possibility of pseudo-positive or pseudo-negative results. Combining the ability of Hg2+ion to stabilize T-T base mismatch, above dual DNAzymes-based strategy was further used for Hg2+sensor design. The proposed sensor allowed the specific detection of Hg2+ion with a detection of4.8nM. Visual detection was also possible.II The synthesis and application of upconversion nanocrystals in biosensing.Upconversion nanocrystals which emit high-energy photons under excitation by near-infrared low-energy light have been widely explored in recent years. They have lots of advantages, such as good chemical stability, low toxicity, no background fluorescence, high signal to noise ratio, deep light-penetration under NIR excitation, minimal photodamage to living organisms and nearly-zero background auto-fluorescence, which show great potential in bioanalytical chemistry and bioimaging.(4) In this chapter, we report a facile one-step hydrothermal method to synthesize carboxyl-functionalized fluorescence upconversion nanocrystals by using a small-molecule malonic acid as capping agent. The synthesized NaLuF4:Yb3+/Er3+products with hydrophilic carboxyl-functionalized surface offer efficient upconversion luminescent performance. Furthermore, the biomolecules can be linked towards the carboxyl-functionalized surfaces of the upconversion nanocrystals, thus indicating the potential bioapplications of these kinds of materials. Based on strong affinities between Graphene oxide (GO) and single stranded DNA, we conjuged the DNA strand on to the surface of the synthesized nanocrystals through the carboxyl functional groups, and then GO can efficiently quench the fluorescence of upconversion nanocrystal completely due to the π-π stacking with DNA. Thus the complex of GO and upconversion nanocrystals can offer a highly sensitive biosensing platform based on Fluorescence Resonance Energy Transfer (FRET) for detection excision enzyme I, and the detection limit is0.02U·mL-1. |
PDF Full Text Request