A diffusion–reaction model for DNA microarray assays

Gadgil, Chetan; Yeckel, Andrew; Derby, Jeffrey J.; Hu, Wei Shou

doi:10.1016/j.jbiotec.2004.05.008

Cited by 45 publications

(43 citation statements)

References 46 publications

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“…At the probe surface a mass balance is applied to relate the flux of target from the aqueous phase with the forward and reverse rates of hybridization. Several approaches have been used to formulate this condition, including its treatment as a thin disk into which analyte diffuses before reacting (24)(25)(26), but in many studies the spot is treated as a planar surface on which solid-phase hybridization occurs (27)(28)(29). In its simplest form the hybridization reaction may be treated as a one-step, reversible reaction between target and probe with rate constants k 1 and k Ϫ1 (30), which is the approach used here.…”

Section: P-labeled Radiometric Assay Of Both Dna Surface Density Andmentioning

confidence: 99%

Array feature size influences nucleic acid surface capture in DNA microarrays

Dandy¹,

Grainger

2007

Proc. Natl. Acad. Sci. U.S.A.

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Analyte affinity capture by surface-immobilized diagnostic agents is a routinely used assay format for profiling numerous medically and technologically important target analytes. These assays suffer from numerous performance limitations, including sensitivity and rapidity. Assay miniaturization is advocated to improve surfacecapture performance, specifically exploiting the inverse relationship between analyte flux and capture feature size under mass transfer-limiting capture conditions that characterize many such assay formats. Reduced capture feature sizes, e.g., microarrays, are proposed to overcome mass transfer limitations, yet this is difficult to achieve across several size scales. This study validates certain advantages advocated for capture spot miniaturization using a rationale to understand surface capture miniaturization strategies. Experimentally derived immobilized ligand and target capture densities as a function of microspot size for DNA oligomers immobilized on model gold substrates are compared directly with theoretical analysis, validating the hypothesis that miniaturization yields many practical assay advantages. Specifically, results show that transitions from assay mass transfer limiting to kinetically limiting conditions as feature size decreases identify an optimal microspot size range for a specific bioassay system. Analytical advantages realized from such assay miniaturization are more uniform target-spot coverage and substantially increased rate of capture (hybridization), increasing assay signal and rapidity.bioassay ͉ mass transport ͉ miniaturization N ew strategies to improve bioanalytical methods, clinical assay designs, diagnostic devices, and rapid screening tools for disease biomarkers, biosecurity threats, and food pathogens have nearly universally emphasized miniaturization as a route to improve performance, cost, convenience, speed-to-answer, and portability. Reducing size scales for these applications has many practical implications to the measurement of biological analytes and such assay designs. Optimal device sizing is a key design feature for assays that commonly involve affinity binding of analytes to surfaces. Surface capture microassays employ diverse affinity reagents (e.g., antibodies, aptamers, and DNA) to capture broad varieties of analytes (e.g., small molecules, peptides, proteins, nucleic acids, and pathogens). Without active transport (e.g., stirring or field-induced), all current microassay platforms suffer from severe mass transfer limitations, that is, rates of analyte transport to the assay capture surface significantly lag rates of analyte binding. This problem is particularly important in producing rapid results in DNA microassays, where resulting DNA-DNA charge-charge interactions produce complications. A long-standing yet experimentally tentative assertion is that surface capture assays benefit significantly from reduced capture feature (i.e., microarray spot) size, specifically, that these assay systems capitalize on the inverse relationship between an...

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Section: P-labeled Radiometric Assay Of Both Dna Surface Density Andmentioning

confidence: 99%

Array feature size influences nucleic acid surface capture in DNA microarrays

Dandy¹,

Grainger

2007

Proc. Natl. Acad. Sci. U.S.A.

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“…5,11,21,38 While, some suggested that hybridization may be reaction limited, 11 others showed that the hybridization is reaction limited at early times when the target DNA concentration is high, but becomes diffusion limited at longer times as the target is consumed. 21,39 Experimental studies showed that continuous mixing or convective flow improved the rate and intensity of hybridization signals, suggesting that diffusion is indeed a limiting factor in surface hybridization. 5,40,55,56 In addition to translational diffusion, our model accounts for rotational diffusion and its effect on surface reaction and suggests that the relative contribution of these processes may depend on the size of target DNA.…”

Section: Discussionmentioning

confidence: 99%

“…38,40 A more recent study by Gadgil et al used a reaction-diffusion model that was solved for small sample volume and gap height (20 lL and 140 lm, respectively) and for large target DNA with average length of 2000 nucleotides. 21 Their results showed that only DNA close to the surface hybridized and that the bulk concentration at a distance of 250 lm or longer remained unchanged for as long as 12 h. Interestingly, the model also predicted that the intensity of hybridized targets best reflects their concentration in solution when the reaction is far from equilibrium.…”

Section: Introductionmentioning

confidence: 99%

An Integrated Reaction-Transport Model for DNA Surface Hybridization: Implications for DNA Microarrays

2008

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DNA microarrays have the potential to revolutionize medical diagnostics and development of individualized medical treatments. However, accurate quantification of scantily expressed genes and precise measurement of small differences between different treatments is not currently feasible. A major challenge remains the understanding of physicochemical processes and rate-limiting steps of hybridization of complex mixtures of DNA targets on immobilized DNA probes. To this end, we developed a mathematical model to describe the effects of molecular orientation and transport on the kinetics and efficiency of hybridization. First, we calculated the hybridization rate constant based on the distance between the complementary nucleotides of the target and probe DNA. The surface reaction rate was then integrated with translational and rotational transport of target DNA to the surface to calculate the kinetics of hybridization. Our model predicts that hybridization of short DNA targets is diffusion limited but long targets are kinetically limited. In addition, for DNA targets with wide size distribution, it may be difficult to distinguish between specific binding of long targets from nonspecific binding of short ones. Our model provides novel insight into the process of DNA hybridization and suggests operating conditions to improve the sensitivity and accuracy of microarray experiments.

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“…In passive assays, the overall hybridization rate is strongly limited by the slow diffusion of DNA, leading to long chemical equilibration times (Gadgil et al, 2004;Kim et al, 2006). Microfluidic, flow-over hybridization systems have been shown to increase the reaction rate.…”

Section: Introductionmentioning

confidence: 99%