SPR (SPR) has been demonstrated as a powerful technique for rapid, sensitive, and label-free genetic analysis [1-5]. When the sensor surface is coated with a single sensing (probe) DNA, SPR can be used for both affinity binding studies (i.e., kinetic measurements) and concentration detection of a target DNA. However, the concentration detection levels for SPR and SPR imaging are typically at low nanomolar (nM) [3, 4], which cannot rival fluorescence measurements. The remarkable sensitivity of SPR based on a position-sensitive device, on the other hand, enables researchers to measure oligonucleotide (ODN) and polynucleotide targets at sub-picomolar (sub-pM) [1]. Recently, low femtomolar level detections have even been achieved by utilizing ODN-capped gold nanoparticles [2] and enzyme-catalyzed reactions [4, 5]. This application note describes the use of a Biosensing Instrument (BI) SPR instrument, in conjunction with an enzyme-catalyzed precipitation reaction, to achieve highly sensitive and sequence-specific DNA assays. To measure the DNA targets at ultratrace levels, aminated ODN capture probes were affixed onto a carboxylated self-assembled monolayer preformed onto gold SPR sensor chips (available from Biosensing Instrument) via the well-known EDC/NHS (1-(3-dimethylaminopropyl)-3-ethyl-carbodiimidehydrochloride/N-hydroxysuccinimide) cross-linking reaction (FIG. 1). Upon hybridization with DNA targets of different concentrations or in various samples, the sensor surface was exposed to a biotinylated detection probe. This was followed by the attachment of the horseradish peroxydase-tagged streptavidin. For the amplified SPR gene detection shown in FIG. 1, a six-port, through-the-handle injection valve was used to inject hydrogen peroxide (H2O2) and 4-chloro-1-naphthol (CN) loaded in a 50-μL sample loop into the flow cell. As depicted in FIG. 1, the enzyme, horseradish peroxydase, anchored onto the sensor surface due to sequence-specific DNA hybridization, catalyzes the reduction of CN in the presence of H2O2.[5] A precipitate produced from this reduction deposits onto the sensor surface, resulting in a considerable change in the SPR signal. The concentration detection levels using the BI-SPR instrument in tandem with the signal amplification are quite noteworthy. The plot of SPR signal vs. ODN target concentration (not shown here) exhibits two regions. The lower curve (from 10 fM to 5 pM) has a slope that is much steeper than that of the upper section (from 5 pM to 100 pM). The variations in the slopes (sensitivities) between the lower and upper regions can be interpreted on the basis of the surface coverage of the duplexes formed at different target concentrations. Thus, ODN concentrations as low as a few femtomolar can be measured. Such remarkably low detection levels can be attributed to the combination of this highly sensitive SPR instrument and the signal amplification scheme.
