The Research And Application Of Metal Nanoparticles And DNAzyme In Electrochemical And Colorimetric Sensor

Nanotechnology has given a new insight into the development and application of nanomaterials. In the modern science, combined with chemistry, biology, physics and medicine science, nanotechnology has become one of the most exciting forefront fields in developing the ultra-sensitive detection and imaging analysis. Owing to their small size (normally in the range of1-100nm), metal nanoparticles exhibit unique chemical, physical and electronic properties that are different from those of bulk materials, and can be used to construct novel and improved sensing devices; especially, electrochemical sensors and biosensors. We take advantage of the promotion of electronic transfer, catalytic performance and large specific surface area of the metal nanoparticles for electrochemical sensors; and with the large surface and the small mass, they can also be used as carriers fixed on other molecules, such as DNA, enzyme, and small molecule. In addition to the catalytic and fixed functions, some reports have focused on the optical properties of the metal nanoparticles. Using the metal nanoparticles can improve the sensitivity, lower the detection limit of the sensor, and perform the work that other materials can’t finish. In addition, when the distance between the nanoparticles is different, the color of the solution may also be different, thus it owns broad application prospects in colorimetric analysis.DNAzymes are catalytic DNA. To date, various DNAzymes have emerged through in vitro selection. Some DNAzymes are able to catalyze the incision or ligation of nucleic acids, and others have the catalytic activity of kinase or peroxidase. One interesting example of DNAzymes is the horseradish peroxidase mimicking DNAzyme, which has the catalytic activity of horseradish peroxidase and is more stable than horseradish peroxidase. Thus, this kind of DNAzyme can be used to replace protein enzymes in chemo/biosensing for signal amplification and sensitive detection. Furthermore, the catalytic activity of DNAzyme can be regulated through DNA, which is superior to protein enzymes. Cooperating with peroxide, DNAzyme can catalyze some substrates to generate color change for colorimetric assay. However, the catalytic activity of DNAzyme is inferior to that of protein enzymes; therefore, increasing the activity of DNAzyme through straightforward methods will be a significant and challenging work. Based on the above consideration and literatures reported previously, takeing the advantage of AuNPs and mimicking-HRP DNAzyme, we have developed a series of electrochemical and colorimetric chemo/biosensors. We also have studied the way to improve and inhibit the activity of DNAzyme. Additionally, we have developed logic gates based on specific metal ions binding peptides and AuNPs. The details are summarized as follows:1. Electrochemical sensor based on the metal nanoparticlesA strategy to construct H3PMo12O40(PMo12) and platinum nanoparticles-chitosan (Pt-CHIT) multilayer film ((PMo12/Pt-CHIT)n) has been developed in chapter2. Negatively charged PMo12and positively charged CHIT have been employed to fabricate stable ultrathin multilayer film on the multiwalled carbon nanotubes coated pyrolytic graphite (CPG) electrode using layer-by-layer self-assembly technique. The doping of Pt nanoparticles minimized the disadvantage of CHIT. Cyclic voltammetry was used to confirm the consecutive growth of the multilayer film and to investigate the electrochemical behavior of the resulting (PMo12/Pt-CHIT)4/CPG detailedly. As an IO3-sensor, the (PMo12/Pt-CHIT)4/CPG exhibits excellent characteristics, such as high sensitivity, wide linear range, lower detection limit and fast response time.A sensitive, label-free electrochemical aptasensor for small molecule detection has been developed in chapter3based on gold nanoparticles (AuNPs) amplification. This aptasensor was fabricated as a tertiary hybrid DNA-AuNPs system, which involved the anchored DNA (ADNA) immobilized on gold electrode, reporter DNA (RDNA) tethered with AuNPs and target-responsive DNA (TRDNA) linking ADNA and RDNA. Electrochemical signal is derived from chronocoulometric interrogation of [Ru(NH3)6]3+(RuHex) that quantitatively binds to surface-confined DNA via electrostatic interaction. Using adenosine triphosphate (ATP) as a model analyte and ATP-binding aptamer as a model molecular recognization element, the introduction of ATP triggers the structure switching of the TRDNA to form aptamer-ATP complex, which results in the dissociation of the RDNA capped AuNPs (RDNA-AuNPs) and the release of abundant RuHex molecules trapped by RDNA-AuNPs. The incorporation of AuNPs in this strategy significantly enhances the sensitivity because of the amplification of electrochemical signal by the RDNA-AuNPs/RuHex system. Under optimized conditions, a wide linear dynamic range of4orders of magnitude (1nM-10μM) was reached with the minimum detectable concentration at sub-nanomolar level (0.2nM). These results demonstrate that our nanoparticles-based amplification strategy is feasible for ATP assay and presents a potential universal method for other small molecule aptasensors.2. Colorimetric detection of the activities of ethyltransferase and restriction endonuclease based on DNAzyme and the reform of the DNAzyme activityDNA methylation catalyzed by methyltransferase (MTase) is a significant epigenetic process for modulating gene expression. Traditional methods to study MTase activity required laborious and costly DNA labeling process. In chapter4, we report a simple, colorimetric, and label-free methylation-responsive DNAzyme (MR-DNAzyme) strategy for MTase activity analysis. This new strategy relies on horseradish peroxidase (HRP) mimicking DNAzyme and the methylation-responsive sequence (MRS) of DNA which can be methylated and cleaved by MTase/endonuclease coupling reaction. Methylation-induced scission of MRS would activate the DNAzyme that can catalyze the generation of a color signal for the amplified detection of methylation events. Taking Dam MTase and DpnI endonuclease as examples, we have developed two colorimetric methods based on MR-DNAzyme strategy. The first method is to utilize an engineered hairpin-DNAzyme hybrid probe for facile turn-on detection of Dam MTase activity, with a wide linear range (6-100U/mL) and a low detection limit (6U/mL). Furthermore, this method could be easily expanded to profile the activity and inhibition of restriction endonuclease. The second method involves a methylation-triggered DNAzyme-based DNA machine, which achieves the ultra-high sensitive detection of Dam MTase activity (detection limit=0.25U/mL) by two-step signal amplification cascade.The activity of DNAzyme is much poorer than that of protein enzymes. Thus, it is significant to improve the activity of DNAzyme for expanding its application and improving the sensitivity. In chapter5, a new method to improve the activity of DNAzyme is introduced, called neighboring improved effect. We have found that if the DNAzyem sequence with an A base at3’terminal, its activity can be improved about5fold. This effect has terminal and base selectivity (A base at3’terminal). In addition, it also has the topology configuration selectivity that trends forming a G4parallel structure. The position of base A between hemin and G4(3’terminal) is a factor to affect the DNAzyme activity. In this chapter, we try to give a possible mechnism of the catalytic reaction and employ it to assay the activity of nuclease. Compared with other DNAzyme activity regulation method, neighboring improved effect is simple for that it can be realized only by adding an A base to the3’terminal of DNAzyme sequence. This method doesn’t need to introduce extra components or factors, and can be widely applied in analytical field. It also can help us to deepen our understanding of the DNAzyme catalytic mechanism.General method for the inhibition of the DNAzyme activity is based on the binding or release of partial DNAzyme sequence. In chapter6, a more simple and feasible method for inhibition of the DNAzyme activity is developed, called neighboring inhibited effect. We found that, if the first base at3’terminal of DNAzyme sequence is purine base, DNAzyme activity can be almost fully inhibited just by hybriding this purine base. The inhibition effect of this strategy is superior to general methods, and it doesn’t need to hybrid any base of DNAzyme sequence.3. Rapid colorimetric Zn2+assay and colorimetric logic gates based on bifunctional peptide and unmodified AuNPsIn chapter7, a new and simple mechanism for the label-free and rapid colorimetric Zn2+assay is developed based on bifunctional peptide probe and unmodified AuNPs. This bifunctional peptide probe contains specific Zn2+binding site and arginine that can lead to the color change of AuNPs. The combination of hydrosulfuryl in peptide with Au in AuNPs can make the arginine residual close to AuNPs surface, and the AuNPs will be unstable and aggregated. Once the-SHs in the side chain of peptide are all coordinated with Zn2+, the peptide probe can’t combine to the surface of AuNPs, therefore, the arginine residual cannot cause the aggregation of AuNPs. This colorimetric method for the detection of Zn2+exhibits excellent characteristics, such as rapid detection process (less than0.5min), high sensitivity, lower detection limit (10nM), and label-free. This strategy has been successfully used in Zn2+analysis of foods and drugs.Using the principle in chapter7, we have designed a series of colorimetric logic gates based on peptide and AuNPs in chapter8. Employing Zn2+and chymotrypsin as input, the color of the solution of AuNPs as output, it is easy to achieve the "YES"("NO"),"OR" and "AND" logic gates.