Label-free Spectrophotometric Detection Of Small Biomolecules Based On DNA And Au Nanoparticles

In this dissertation, the work is focused on developing label-free spectrophotometric methods to detect small biomolecules based on DNA and Au nanoparticles system.Two main aspects were included in the dissertation:(1) A visual colorimetric method for the detection of neurogenin3(ngn3) synthetic peptide fragment (SKQRRSRRKKANDRERNRMH) is proposed on the basis of the color change caused by the aggregation of gold nanoparticles (AuNPs). Ngn3is a basic helix-loop-helix (bHLH) transcription factor involved in the differentiation of neural precursor cells. Under the experimental conditions, glutathione-modified AuNPs (GSH-AuNPs) with negative charge presented wine red color owing to the electrostatic repulsion between nanoparticles, which could prevent the GSH-AuNPs from aggregation. Upon the addition of ngn3peptide, the aggregation of GSH-AuNPs occured under acertain concentration of salt and the color of AuNPs solution changed from red to blue. Thus, a rapid detection method for ngn3peptide using GSH-AuNPs as colorimetric probe was established. Under the optimum conditions, the assay showed a linear response in the peptide concentration range of20-300ng/mL with a detection limit being8ng/mL and exhibited excellent selectivity for ngn3peptide.(2) Based on the pH induced conformational switch of i-motif DNA, a facile fluorescence biosensor for the detection of glucose is proposed for the first time. Under slightly acidic conditions, a cytosine-rich single-stranded DNA could form a four-stranded DNA structure called i-motif because the cytosine residues were partially protonated and formed a intramolecular noncanonical C-CH(+) base pairs. Glucose could be oxidized by oxygen (O2) in the presence of glucose oxidase (GOD), and the generated gluconic acid could decrease the pH value of the solution and then induced the fluorophore and quencher-labeled cytosine-rich single strand DNA to fold into a close-packed i-motif structure. As a result, the fluorescence quenching occured because of the energy transfer between fluorophore and quencher. Based on this working principle, the concentration of glucose could be detected by the decrease of fluorescence inensity. This glucose biosensor had been successfully applied to quantitative determination of glucose in real samples, suggesting that it may have great potential in the practical applicability.