| In this study, a series of signal amplification methods were developed including aptamer-based assay using Primer self-generation, circular hairpin assisted exponential amplification reaction, autonomous DNA machine isothermal amplification etc. These methods were used to detect small bioactive molecular, protein, genes, and Ramos cells. Because of the sensitivity, simplicity, rapidity and low-cost, these methods could be easily used for biological sample analysis. The experimental protocols could be summarized as follows:1. A novel Electrogenerated Chemiluminescence (ECL) assay based on primer self-generation strand-displacement polymerization (PS-SDP) is developed to detect triphosphate (ATP). This contribution describes the first instance of the use of PS-SDP for protein-aptamer binding that is suitable for DNA recycling amplification,. The target binding-induced molecular structure-switching of the integrated aptamer probe allows transduction of the interaction between targets and signaling probes into the ECL signal. This method takes advantage of the dramatic increase in the intensity of the ECL signal upon target molecule binding that can trigger DNA recycling amplification. With the use of this ECL amplification system, ATP could be specifically detected at a concentration down to3.1nM..With its advantages of rapidity, sensitivity, and specificity, this described ECL amplification method would provide a promising platform for electrochemical of small bioactive molecular.2. A simple and novel SERS method is demonstrated to detect lysozyme based on aptamer which can specifically recognize lysozyme. DNA recycling amplification is firstly introduced into the ultrasensitive SERS biosensing applications based on aptamer DNA recongnition, also accomplishing the first development of SERS amplification assay for the analysis of lysozyme. The circular hairpin assisted exponential amplification reaction is accomplished only involved one kind of aptamer probe, trigger DNA1, hairpin DNA, and SERS probe. This novel sensing system is simple in design and can be easily carried out by simple mixing and incubation. The SERS signals are significantly enhanced with a detection limit of5fM(3σ). Due to its high sensitivity, excellent specificity and good performance in cellular homogenate, this system could be expected to be developed into a promising practical assay for other proteins.3. A novel surface-enhanced Raman scattering (SERS) detection system is designed to detect a specific DNA sequence and cell based on the autonomous DNA machine, the autonomous scission-replication-displacement-rolling circle amplification (RCA) process is triggered by the recognition of target DNA1including two cycles and RCA amplification. In addition, the two cycles are independent and happen simultaneously as an effective amplified replication system. The problem of high background induced by excess bio-barcodes is circumvented using magnetic bead (MB) as the carrier of RCA products. Overall, with multiple amplification steps and one magnetic separation procedure, this flexible biosensing system exhibits not only high sensitivity and specificity with a wide dynamic range of4orders for DNA detection from5.0×10-15M to5.0×10-12M, with the detection limits of DNA and Ramos cell as low as1.0fM and10cells respectively. Given the unique and attractive characteristics, the ultrasensitive SERS biosensing method provides a universal method for quantitative analysis of gene, cells and also protein, etc, and supplies valuable information forbiomedical research and cancer clinical early diagnosis. |
