Multiplexed assessment of the surface density of DNA probes on DNA microarrays by surface plasmon resonance imaging

Abstract

In terms of hybridization assays surface plasmon resonance imaging (SPRi) offers high throughput, label-free and real-time monitoring of the binding kinetics. This requires DNA microarrays on bare or modified gold SPRi chips, which are generally premade by an off-line microspotting procedure.Therefore, the surface density of the immobilized probes is not known although it is an essential quality control parameter, especially, when it can vary in a very broad range as in case of selfassembled thiol-labeled DNAs on gold surface. Here we show that the small molecular weight ruthenium(III) hexamine complex (RuHex) introduced earlier for electrochemical quantitation of DNA coverage on gold electrodes can be used also in SPRi to assess the surface density of DNA probes in DNA microarrays. A single injection of RuHex solution allows the simultaneous visualization and quantification of the surface density of DNA probes (ranging in this study from 4×10 11 to 1.7×10 13 molecules cm -2 ) on all spots of a DNA microarray made by microspotting thiol labeled short DNA probes both in prehybridized and single-stranded form on a gold SPRi chip. The methodology was applied to determine the effect of the surface density of DNA probes on the hybridization efficiency and kinetics of complementary microRNAs, using hsa-miR-208a-3p as model. Single mismatch duplexes were found to be more effectively destabilized than fully complementary duplexes by steric hindrance at large surface densities of the DNA probes, which offers an effective mean to increase single mismatch selectivity.Keywords: DNA surface density, microspotting, surface plasmon resonance imaging, DNA microarray, microRNA DNA probes the critical experimental parameters to be considered for proper hybridization efficiency are well known, e.g., surface density of probes [14-17], probe design [18], the type and length of spacers [19], the nanostructure of the surface [20,21], ionic strength [15]. However, the optimal deployment of nucleic acid probes is also dependent on the type of detection used in the assay that limits the interlope of optimized immobilization methods between the various platforms. In channelmultiplexed SPR systems [22] the immobilization is less complex as requires solely the injection of the relevant coupling agents and probes into the channels. However, also the level of multiplexing is significantly smaller compared to photolithography or methods based on the local delivery and

Finding the Optimal Surface Density of Aptamer Monolayers by SPR Imaging Detection‐based Aptamer Microarrays

“…The chips were inserted in the SPRi instrument and the surface density of aptamer probes was determined for all the different aptamer spots (see layout on Figure B) from a single injection of 50 μM of RuHex solution. Interestingly, we found earlier full reversibility of the RuHex binding for the short, 18‐mer, DNA hybridization probes, i. e. the baseline was recovered after switching back from the RuHex solution to the working buffer . However, in case of the ca.…”

“…This surface density is considerably smaller than the ca. 6×10 12 molecules cm −2 that we determined previously for hybridization assays with an 18‐mer DNA probe . It is also considerably smaller than reported for thrombin aptamers immobilized through streptavidin/biotin interaction where the highest bound target/immobilized aptamer ratio was found for an aptamer surface density of ca.…”

“…Since probably the most convenient methods for the preparation of nucleic acid microarrays with SPRi detection involves an off line methodology, e. g. various forms of microspotting, the surface concentration of aptamers in the different spots is unfortunately not assessable directly (during immobilization) by SPR measurements. Therefore, we developed recently a simple method based on detecting a small molecular weight marker cation, [Ru(NH 3 ) 6 ] 3+ (RuHex), that was shown earlier by chronocoulometry to bind by electrostatic interaction to the surface confined nucleic acid strands and in appropriate conditions to exactly compensate their negative charge. Establishing such conditions enables based on the stoichiometry of RuHex‐DNA binding to assess the surface concentration of surface confined nucleic acid strands, which unlike the chronocoulometric method, can be performed simultaneously on all spots without the need of individual electrical addressing, and use of short spacers.…”

“…Therefore, we developed recently a simple method based on detecting a small molecular weight marker cation, [Ru(NH 3 ) 6 ] 3+ (RuHex), that was shown earlier by chronocoulometry to bind by electrostatic interaction to the surface confined nucleic acid strands and in appropriate conditions to exactly compensate their negative charge. Establishing such conditions enables based on the stoichiometry of RuHex‐DNA binding to assess the surface concentration of surface confined nucleic acid strands, which unlike the chronocoulometric method, can be performed simultaneously on all spots without the need of individual electrical addressing, and use of short spacers. The highly reliable microspotting modification of the SPRi chips and the assessment of the surface concentration of DNA probes helped already to understand the appropriate conditions for highly efficient and selective hybridization assays .…”

Stochastic electrochemistry at ultralow concentrations: the case for digital sensors

“…31,32 This complicates sensor design because, besides the intrinsic kinetic properties of the recognition elements, their density is also an important factor (especially for highly charged capture probes such as DNA). 33,34 Hence, in addition to diffusional mass transport, the recognition event can also become the rate-limiting step.…”