Novel Chemiluminescence Technologies For The Detection Of Biomolecules

The detection of biomolecules such as DNA、RNA and protein has gained considerable interest in clinical, environmental, and biodefense applications, etc. Many different types of techniques, such as electrochemical, optical, fluorescent, radiochemical, chemiluminescence (CL) methods have been applied for protein and DNA analysis. Each technique has its own pros and cons. Radiochemical method which is one of the commonly used methods suffers from the tedious labeling protocol, environmental pollution and the harmful effect of human health. Hence, the nonradioactive technology including electrochemistry, enzymic method, and fluorescent method have been developing quickly in recent years.CL has been exploited within a wide range of applications in various fields, due to their extremely high sensitivity along with their extra advantages such as simple instrumentation, wide calibration ranges, and suitability for miniaturization in analytical chemistry. By using gold nanoparticle amplification, template recognition and aptameric system, a series of CL biomolecules assays have been developed. Description of research in my thesis is presented as follows:Chapter1:This thesis first addresses different techniques with and without label, and then presents current state of knowledge, including nanoparticle, enzyme as label, etc. The second phase of this chapter reviews current main amplification methods and their applications in biosensor fields, and discusses the development in this field. Following that, objectives and significance of this research are summarized. Chapter2:New Chemiluminescence Techniques for the Detection DNA and Hg2+Ion based on Gold Nanoparticles.Combining the advantage of gold nanoparticle as biological tag and the high sensitivity of CL detection of gold through luminol-AgNO3reaction, gold nanoparticle is employed as a label in the CL assay. While NH2OH is thermodynamically capable of reducing Au3+ions to bulk metal, the reaction is dramatically accelerated by Au NPs and Hg2+ions. As a result, the added Au3+ions go into the production of larger Au particles or Hg2+-Au particles. According to this phenomenon, we report two new CL methods for the detection of DNA and Hg2+ions. For DNA detection, reporter DNA-functionalized gold nanoparticles are used. After HAUCl4-NH2OH amplification, the detection limit of this sandwich detection could be improved to be300aM. For the Hg2+detection, we use capture probe containing20T bases to capture Hg2+ions, and then add HAUCl4-NH2OH to form gold nanoparticles. The thus-formed Au NPs trigger the reaction between luminol and AgNO3, producing CL emission. This novel CL technique has several advantages including high sensitivity (10ppt) and selectivity over a spectrum of interfering metal ions. In view of these advantages, as well as the cost-effective, minimized working steps and portable features of the CL techniques, we expect that this CL sensor will be a promising candidate for the field detection of toxic Hg2+ions in environment, water, and food samples.Chapter3:DNA and Protein Detection via Template RecognitionTemplate recognition is employed in an entirely new type of sensing platform for the simple and robust detection of DNA and protein. Only in the presence of target molecules, the aptamer DNA can hybridize stably with the capture DNA to form a stem-loop structure due to the enhancement of base stacking. This leads to a strong chemiluminescence emission for simple protein detection. First, we develop a magnetic bead-based sensing platform for simple detection of short-length DNA, on the basis of a template dependent surface-hybridization assay. The method required the recognition of the target by a reporter probe to promote their efficient annealing with magnetic bead-attached capture DNA. The data show that this new biosensor offers the possibility of highly selective and sensitive detection of the short-length target DNA. A readily achieved detection limit was found to be0.01fmol. Second, we report an innovative protein detection technology to improve the simplicity and robustness of the aptameric system based on the formation of a stem-loop structure and enhancement of base stacking. Our new technique encompasses a design strategy whereby capture and aptamer probes that do not hybridize to each other are made to anneal to each other in the presence of a target protein via the formation of a protein-aptamer complex, leading to the formation of a stem-loop structure and enhancement of base stacking. The detection limit was estimated to be10fM at a signal-to-noise ratio of3, with a large dynamic range that spanned approximately4orders of magnitude (50fMto500pM), This technique provides a novel method for simple, fast, and convenient point-of-care diagnostics in the field monitoring of proteins and also toxic metal ion, etc.Chapter4:Label-free CL Technique for the Detection of miRNA Based on the Conformational SwitchHerein we developed an aMBs based sensing platform for simple detection of miRNA. The method required the opening of the aMBs by target miRNAs to activate its binding affinity to SA-MPs. First, we combined it with the hybridization chain reaction. The sensitivity of detection was improved through designing aMBs sequences which contained a sequence as a HCR trigger for signal amplification. Second, we used stranddisplacement amplification catalysed by DNA polymerase and restrictionendonuclease. These simple and rapid detections for miRNA let-7a do not require any modified DNA probes and had only two steps. In view of these advantages, the label-free aMBs assay, as a new CL strategy, might create a universal technology for developing simple biosensors in sensitive and selective detection of miRNA.