Surface plasmon resonance (SPR) is a popular technique for label-free detection of biomolecular interactions at a surface. SPR yields quantitative kinetic association and dissociation constants of surface interactions such as the binding of two molecular species, one present in the liquid phase and the other immobilized at the surface. Current state-of-the-art SPR systems extract kinetic constants from measurements of the step response of the interaction versus time. The step response measurement is subject to the influence of noise and drift disturbances that limit its minimum-detectable mass changes. This paper presents a new SPR technique that measures the biomolecular interaction not in time but over a very narrow frequency range under periodic excitation. The measured response is, thus, locked to a very specific narrow band signal. This narrow band spectral sensing scheme has a very high degree of rejection to uncorrelated spurious signals. The signal-locked SPR technique was implemented using a chemical modulator chip connected to a set of functionalized Au sensing sites downstream. Binding experiments for a model system of carbonic anhydrase-II (CA-II) analyte and immobilized 4-(2-aminoethyl)benzenesulfonamide (ABS) ligand display a 100-fold (20 dB) improvement in the measured signal-to-noise ratio (SNR) when using the new technique compared to the SNR achieved using the conventional step response method.
Label-free techniques such as surface plasmon resonance (SPR) have used a step-response excitation method to characterize the binding of two biochemical entities. A major drawback of the step response technique is its high susceptibility to thermal drifts and noise which directly determine the minimum detectable binding mass. In this paper we present a new frequency-domain method based on the use of multisine chemical excitation that is much less sensitive to these disturbances. The multisine method was implemented in a PDMS microfluidic chip using a dual channel, dual multiplug chemical signal generator connected to functionalized and reference SPR binding spots. Kinetic constants for the reaction are extracted from the characteristics of the sense spot response versus frequency. The feasibility of the technique was tested using a model system of Carbonic Anhydrase-II analyte and amino-benzenesulfonamide ligand. The experimental signal to noise ratio (SNR) for the multisine measurement is about 32 dB; 7 dB higher than that observed with the single step-response method, while the overall measurement time is twice as long as the step method.
We demonstrate a new dual slope SPR technique that is ten-fold faster than the conventional step-response method. The new scheme utilizes rapid slope-based measurements followed by rapid reset, and it separates association and dissociation half reaction measurements at two separate sites inside a dual-chamber PDMS microfluidic chip. For a model CAII-ABS test system, the association and dissociation slopes were measured in 30 seconds compared to 5 minutes for step-response. The values of k(a) and k(d) calculated from the slope method are 3.66 ± 0.19 × 10(3) M(-1) s(-1) and 4.83 ± 0.17 × 10(-2) s(-1), respectively, matching well with step-response values while facilitating ~10 to 15 fold faster detection and quantification.
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