Studies On Sers Sensing Methods Based On Multiple Cycles Of Dna-Related Processes And The Application For Cancer Biomarker Detection

In this study, a series of SERS signal amplification methods were developed including autonomous DNA machine isothermal amplification, DNAzyme activated target recycling cascade amplification, bifunctional aptamer-based SERS assay using Primer self-generation etc. These methods were used to detect genes, Ramos cells, protein and small bioactive molecular. 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 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 from5.0×10-16M to10-12M, with the detection limits of DNA and Ramos cell as low as0.2fM and8cells 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 for biomedical research and cancer clinical early diagnosis.2. A simple and novel SERS method is demonstrated to detect L-histidine based on DNAzyme which can specifically recognize L-histidine. The unmodified DNAzyme as a molecular recognition module is firstly introduced into the ultrasensitive SERS biosensing applications based on the DNA amplification method. The system consists of target recycling-oriented strand displacement amplification and circular hairpin assisted exponential amplification reaction only involved one kind of DNAzyme-based probe, hairpin DNA, primer, SERS probe and polymerase. 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 of0.56nM (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 cancer biomarkers.3. A bifunctional SERS assay based on primer self-generation strand-displacement polymerization (PS-SDP) is developed to detect small molecule or protein in parallel. This contribution describes the first instance of the use of PS-SDP for protein-aptamer binding that is suitable for DNA recycling amplification, also accomplishing the first development of SERS assay for the parallel analysis of triphosphate (ATP) and lysozyme. The target binding-induced molecular structure-switching of the integrated aptamer probe allows transduction of the interaction between targets and signaling probes into the Raman signal. This method takes advantage of the dramatic increase in the intensity of the Raman signal upon target molecule binding that can trigger DNA recycling amplification. With the use of this SERS amplification system, ATP and lysozyme could be specifically detected at a concentration down to0.05nM and10fM, respectively. This method is validated to be an effective tool for recognizing target elements in a complicated environment, the human serum-containing sample. With its advantages of rapidity, sensitivity, and specificity, this described bifunctional SERS amplification method would provide a promising platform for SERS parallel detection of cancer biomarkers.4. A novel hairpin DNA aptamer-SERS system is designed with DNA multiple amplification for the detection of lysozyme. The haipin DNA aptamer as a molecular recognition module is firstly introduced into the ultrasensitive SERS biosensing applications based on the DNA amplification method. The system consists of target recycling-oriented strand displacement amplification and circular hairpin assisted exponential amplification reaction only involved o hairpin DNA aptamer, SERS probe, polymerase and nicking enzyme. This novel sensing system is simple in design and can be easily carried out by simple mixing and incubation. This flexible biosensing system exhibits not only high sensitivity and specificity with a wide dynamic range of3orders for DNA detection from5.0×10-15M to10-12M, with the detection limits of lysozyme as low as2fM. Due to its high sensitivity, excellent specificity and good performance in real flood sample, this hairpin DNA aptamer-based SERS detection system may offer a new approach to sensitively and selectively detect a wide spectrum of analytes.