Chemiluminescence Technology For Homogeneous And Multiplexed DNA Assays

Simultaneous detection of multiple proteins has gained considerable interest in clinical study, environmental research, and biophylaxis applications, etc. At the same time, simultaneous multi-component quantitative detection has became a hot pot in protein, gene sequence analysis and other fields, especially in the clinical diagnosis, environmental monitoring. Simultaneous detection of multianalyte assays have focused more and more research workers' eyes, due to their unique advantages, such as less sample, reduced analysis time, minimized repetitions of tedious procedures, and lower cost compared with parallel single-analyte assays.Although the traditional high-density porous plate is still the main technique of biological analysis, with study on the simplicity and multifunction of microsphere in a deep-going way, the technique, including microcarrier and labeling, has begun to show a huge market potential. The current coding microspheres screening method which can reduce equipment costs and testing fees, faces how to improve the number of coding/decoding process in order to simplify and improve detection speed and accuracy, lower costs, and their resolving ways waits for a new coding material, encoding / decoding strategy/ invention, or computer-aided drug screening.This paper includes three parts: firstly, review of the current multi-component detection technology progress; secondly, construction of a new protocol for carrier-resolvedtechnology for homogeneous and multiplexed DNA assays; lastly, the parallel analyticalmethod for simultaneous detection of three different DNA sequences on the based oflabel-resolved technology. In the second part, we use thermo-sensitivepoly-N-isopropylacrylamide (PNIP), polystyrene beads, and magnetic beads for threecarriers and peroxidase-streptavidin polymer for chemiluminescence detection enzyme,which allowed for the simultaneous determination of three oligonucleotide sequences (60bases in length) associated with the breast cancer gene (BRCA1) and showed highselectivity and attomolar-femtomolar sensitivity. As the three carriers have differentphysical and chemical characteristics, such as PNIP, known to aggregate and precipitate outof water when the temperature is raised above the lower critical solution temperature(LCST), magnetic beads, separated by magnetic force, polystyrene beads precipitated bycentrifugation. The mixture of three different DNAs could react in a single vessel withoutany washing and cross interference, leading to the development of a "one-pot reactionsystem". Only one washing step prior to detection in our protocol is required, which standsout our whole procedure being simple and efficient. The results show the hybridizationresponse to sample mixtures containing increasing levels of each target which proportionalto the amount of corresponding DNA targets, indicating minimal cross interferences. In thethird part, we describe the development of a rapid and cost-effective chemiluminescence(CL) method for simultaneous detection of three DNA targets using magnetic beads as asingle carrier and three CL labels as different hybridization codes for label-resolvedtechnology for homogeneous and multiplexed DNA assays. The combination of goldnanoparticles with two enzyme CL labels offers great promise for multiple DNAhybridization analysis in point-of-care diagnostics and field analysis, since there is no crossreaction within simultaneous detection of horseradish peroxidase (HRP), alkalinephosphatase (ALP) and gold nanoparticles. Above all, three capture probe DNAs wereimmobilized on the magnetic beads. Then the mixture of three different capture probeconjugates first hybridized with three corresponding target sequences, sandwiched withthree label DNAs, after that react with streptavidin colloidal gold, anti-fluoresceinhorseradish peroxidase and anti-digoxin alkaline phosphatase conjugate, afterwards detecting the CL signals of magnetic beads, respectively. This CL technique was developed in this research due to its high sensitivity for the determination of enzyme and gold nanoparticles, wide calibration ranges, and suitability for miniaturization in analytical chemistry. There is no cross reaction within the simultaneous detection of the above-mentioned two enzymes, plus gold nanoparticles. The work presented here validates for the determination of three DNA targets with varied quantity range and provides the foundation for the development of highly sensitive techniques with increased multianalyte capability.To sum up, this paper established two kinds of new methods for the simultaneous determination of three different DNA and successfully made homogeneous and multiplexed DNA assays with carrier-resolved and label-resolved technologies. Integration of the two detection techniques is expected to determine more targets. In this paper, by the innovative construction of the multi-component CL technology, we demonstrate a valuable option for protein and DNA analysis method with simplicity and and high sensitivity.