Song Fayi, Zhou Feimeng, Wang Jun, Tao Nongjian, Lin Jianqiao, Vellanoweth Robert L, Morquecho Yvonne, Wheeler-Laidman Janel
Department of Chemistry and Biochemistry, California State University, Los Angeles, 5151 State University Drive, Los Angeles, CA 90032, USA.
Nucleic Acids Res. 2002 Jul 15;30(14):e72. doi: 10.1093/nar/gnf072.
A flow-injection (FI) device is combined, through the use of a low-volume (4 microl) flow cell, with an ultrasensitive surface plasmon resonance (SPR) spectrometer equipped with a bi-cell photodiode detector. The application of this novel FI-SPR device for sequence-specific ultratrace analysis of oligodeoxynucleotides (ODNs) and polydeoxynucleotides was demonstrated. Self-assembled monolayers of ODN probes are tethered onto Au films with a mercaptohexyl group at the 3' ends. The FI-SPR provides a detection level (< or =54 fM) 2-3 orders of magnitude lower than other SPR devices and compares well with several ultrasensitive detection methods for labeled DNA targets (e.g. fluorophore-tagged and radiolabeled DNA samples). The technique is also highly selective, since a 47mer ODN target with a single-base mismatch yielded a much smaller SPR signal, and a specific interaction was detected when the complementary target was present at 0.001% of the total DNA. The FI-SPR was extended to the measurement of two individual genes in a cDNA mixture transcribed from an Arabidopsis thaliana leaf mRNA pool. The greatly enhanced sensitivity not only obviates the necessity of DNA labeling, but also significantly reduces sample consumption, allowing direct quantification of low abundance mRNAs in cellular samples without amplification.
流动注射（FI）装置通过使用小体积（4微升）流通池，与配备双电池光电二极管探测器的超灵敏表面等离子体共振（SPR）光谱仪相结合。展示了这种新型FI-SPR装置在寡脱氧核苷酸（ODN）和多脱氧核苷酸的序列特异性超痕量分析中的应用。ODN探针的自组装单分子层通过3'端带有巯基己基的方式连接到金膜上。FI-SPR提供的检测水平（≤54 fM）比其他SPR装置低2-3个数量级，与几种用于标记DNA靶标的超灵敏检测方法相当（例如荧光团标记和放射性标记的DNA样品）。该技术也具有高度选择性，因为具有单碱基错配的47聚体ODN靶标产生的SPR信号要小得多，并且当互补靶标占总DNA的0.001%时能检测到特异性相互作用。FI-SPR被扩展用于测量从拟南芥叶mRNA库转录的cDNA混合物中的两个个体基因。大大提高的灵敏度不仅消除了DNA标记的必要性，还显著减少了样品消耗，无需扩增即可直接定量细胞样品中的低丰度mRNA。
