Detection of oligonucleotide hybridization at femtomolar level and sequence-speci®c gene analysis of the Arabidopsis thaliana leaf extract with an ultrasensitive surface plasmon resonance spectrometer

A ̄ow-injection (FI) device is combined, through the use of a low-volume (4 ml) ̄ow 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-speci®c ultratrace analysis of oligodeoxynucleotides (ODNs) and polydeoxynucleotides was demonstrated. Self-assembled monolayers of ODN probes are tethered onto Au ®lms with a mercaptohexyl group at the 3¢ ends. The FI±SPR provides a detection level (£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. ̄uorophore-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 speci®c 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 signi®cantly reduces sample consumption, allowing direct quanti®cation of low abundance mRNAs in cellular samples without ampli®cation.