High-Resolution Epifluorescence and Time-of-Flight Secondary Ion Mass Spectrometry Chemical Imaging Comparisons of Single DNA Microarray Spots

Abstract

DNA microarray assay performance is commonly compromised by spot-spot probe and signal variations as well as heterogeneity within printed microspots. Accurate metrics for captured DNA target signal rely upon uniform spot distribution of both probe and target DNA to yield reliable hybridized signal. While often presumed, this is neither easily achieved nor often proven experimentally. High resolution imaging techniques were used to determine spot heterogeneity in identical DNA array microspots comprising varied ratios of unlabeled and dye-labeled DNA probes contact printed onto commercial arraying surfaces. Epifluorescence imaging data for individual array microspots were correlated with time-of-flight secondary ion mass spectrometry (TOF-SIMS) chemical state imaging of the same spots. Epifluorescence imaging intensity distinguished varying DNA density distributed both within a given spot and from spot-to-spot. TOF-SIMS chemical analysis confirmed these heterogeneous printed DNA distributions by tracking bound Cy3 dye-, DNA base, and phosphate- specific ion fragments often correlating to fluorescence patterns within identical spots. TOF-SIMS ion fragments originating from probe DNA and Cy3 dye are enriched in microspot centers, correlating with high fluorescence intensity regions. Both TOF-SIMS and epifluorescence supports Marangoni flow effects on spot drying, with high density DNA-Cy3 located in spot centers and non-homogeneous DNA distribution within printed spots. Microspot image dimensional analysis results for DNA droplet spreading show differing DNA densities across printed spots. The study directly supports different DNA probe chemical and spatial microenvironments within spots that yield spot-spot signal variations known to affect DNA target hybridization efficiencies and kinetics. These variations critically affect probe-target duplex formation and DNA array signal generation.

Real-Time Fluorescent Image Analysis of DNA Spot Hybridization Kinetics To Assess Microarray Spot Heterogeneity

“…55 Printed dried probe spots often exhibit increased DNA density at spot centers compared to peripheries. This probe heterogeneity has implications for target uptake and resulting spot duplex heterogeneity as a consequence due to local differences in interfacial steric and coulombic properties that affect target-probe pairing efficiencies and kinetics.…”

“…Target hybridization therefore occurs from spot edges radially inwards as indicated by increasing Cy5 emission spot intensity profiles (Figure 3). Both non-uniform radial probe density from spot drying 55 and target radial transport issues 15 produce non-homogeneous target hybridization with time. Target mass transport depends on array format (static vs. flow), target concentration, and its diffusion coefficient.…”

Biophysical properties of nucleic acids at surfaces relevant to microarray performance

“…Routine conventional fluorescence scanner images provide information for integrated spot pixel intensity, shape and morphology, but lack important details for incorporating intra-spot ssDNA immobilized heterogeneity and intra-spot structural issues known to affect both target capture signals and duplex hybridization kinetics critical to this assay’s answer development and diagnostic reliability. [34] Additionally, signal quantitation is affected by numerous, known surface issues and background noise sources that confound reliable, quantitative correlations of spot-to-spot fluorescent assay signals and experiment-to-experiment comparisons. [17, 79] Significantly, integration of spot fluorescent intensity to report assay signal with commonly used fluorescence scanners demonstrates that the typical fluorescence scanner cannot discern microspot optical heterogeneity and fluorescence intensity variations within single printed spots to yield a reliable metric.…”

“…Inaccuracy in this fluorescent metric is attributed to fluorescent dye-dye and dye-surface interactions, leading to quenching of surface fluorescence signal. [34]…”

Optimization of Cyanine Dye Stability and Analysis of FRET Interaction on DNA Microarrays

“…Next to biological and biochemical sources, bias originates from photochemical processes and depends on the choice of labeling agent as well as the selected imaging procedure and environment. In earlier works, it was shown that the ubiquitous application of cyanine dye labeling causes significant bias due to the dyes’ disparate susceptibility to photobleaching and possible FRET interaction [19,20,26,28,29]. …”