Miniaturized PCR chips for nucleic acid amplification and analysis: latest advances and future trends

Zhang, Chunsun; Xing, Da

Fluorescence-based DNA detection method. Fluorescencebased real-time and end-point DNA detection is a powerful and important detection technique. The realtime detection kinetically observes the fluorescence signals from the interaction between the dye or probe and the increasing amount of double-stranded DNA molecules during PCR (10, 16, 20, 37, 39, 50, 54, (77) (78) (79) (80) ; the end-point detection measures the fluorescence only before and after PCR amplification, by which it can be established whether the DNA template is successfully amplified (17, 49, 71) . Although the end-point method is simple and can be used for single cell or molecule analysis (17, 49) , it is difficult to obtain reliable results if the DNA template concentrations are calculated only by the fluorescence signals. The realtime method has become popular. It uses an analysis procedure called threshold method. The threshold value (also called C T value) represents the number of cycles necessary to produce an exact amount of DNA. By using a series of different C T values, one can calculate the DNA template concentrations and obtain the PCR efficiency. If the C T value is further used as a criterion for the PCR efficiency, the dynamic parameters of the onchip PCR can be optimized (80) . However, since the sequence-nonspecific fluorescence dyes are often utilized for the on-chip real-time PCR systems, both specific and nonspecific products will produce the same fluorescence signals that are difficult to differentiate. In order to overcome this challenge, after on-chip PCR a MCV can be performed to determine the purity of the PCR and to identify the specific target products (10, 16, 20, 78, 79) . Other on-chip fluorescence-based detection technologies are also being developed. One such example was the DNA-'up-converting' phosphor particles (UPT)-lateral flow (LF) assay on a single chip proposed by Wang et al. (63) , in which the PCR products were labeled with the UPT reporter particles in an incubation chamber, applied to the LF strip, bound to pre-immobilized ligands and detected with an IR laser.