Back‐Scattering Interferometry: A Versatile Platform for the Study of Free‐Solution versus Surface‐Immobilized Hybridization

Pesciotta, Esther N.; Bornhop, Darryl J.; Flowers, Robert A.

doi:10.1002/asia.201000614

Cited by 9 publications

(19 citation statements)

References 18 publications

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“…Pesciotta et al studied the influence of mismatch position both in the bulk solution and on the surface. They also found that the dissociation constant K D for a DNA duplex is significantly larger on the surface than in the bulk solution . Bartlett et al used surface-enhanced Raman spectroscopy (SERS) to study the effect of electrochemical polarization upon thiol-linked dsDNA denaturation on gold electrodes .…”

Section: Introductionmentioning

confidence: 99%

Sequence and Temperature Influence on Kinetics of DNA Strand Displacement at Gold Electrode Surfaces

Biała

Sedova

Flechsig

2015

ACS Appl. Mater. Interfaces

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Understanding complex contributions of surface environment to tethered nucleic acid sensing experiments has proven challenging, yet it is important because it is essential for interpretation and calibration of indispensable methods, such as microarrays. We investigate the effects of DNA sequence and solution temperature gradients on the kinetics of strand displacement at heated gold wire electrodes, and at gold disc electrodes in a heated solution. Addition of a terminal double mismatch (toehold) provides a reduction in strand displacement energy barriers sufficient to probe the secondary mechanisms involved in the hybridization process. In four different DNA capture probe sequences (relevant for the identification of genetically modified maize MON810), all but one revealed a high activation energy up to 200 kJ/mol during hybridization, that we attribute to displacement of protective strands by capture probes. Protective strands contain 4 to 5 mismatches to ease their displacement by the surface-confined probes at the gold electrodes. A low activation energy (30 kJ/mol) was observed for the sequence whose protective strand contained a toehold and one central mismatch, its kinetic curves displayed significantly different shapes, and we observed a reduced maximum signal intensity as compared to other sequences. These findings point to potential sequence-related contributions to oligonucleotide diffusion influencing kinetics. Additionally, for all sequences studied with heated wire electrodes, we observed a 23 K lower optimal hybridization temperature in comparison with disc electrodes in heated solution, and greatly reduced voltammetric signals after taking into account electrode surface area. We propose that thermodiffusion due to temperature gradients may influence both hybridization and strand displacement kinetics at heated microelectrodes, an explanation supported by computational fluid dynamics. DNA assays with surface-confined capture probes and temperature gradients should not neglect potential influences of thermodiffusion as well as sequence-related effects. Furthermore, studies attempting to characterize surface-tethered environments should consider thermodiffusion if temperature gradients are involved.

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Section: Introductionmentioning

confidence: 99%

Sequence and Temperature Influence on Kinetics of DNA Strand Displacement at Gold Electrode Surfaces

Biała

Sedova

Flechsig

2015

ACS Appl. Mater. Interfaces

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“…25 This work highlights the unique capability of BSI to perform assays in both the hetero- and homogeneous format, allowing one to quantify how a surface attachment strategy or fluorophore will influence the performance of the methodology (Figure 7A). …”

Section: Backscattering Interferometrymentioning

confidence: 95%

“…24 Recently, we used BSI to begin to quantify some of the perturbations caused by adding a fluorophore to a DNA binding pair. 25 …”

Section: Applications Of Interferometrymentioning

confidence: 99%

Interferometric Methods for Label-Free Molecular Interaction Studies

2011

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Figure 7. (A) BSI shows the difference in binding affinities for free-solution and surface-immobilized DNA hybridization. 25 (B) BSI detects the kinetic interaction of calmodulin and trifluoperazine. From ref 106. Reprinted with permission from American Association for the Advancement of Science, Copyright 2007. (C) Shows that the magnitude of binding as detected by BSI for α-crystallin to different T4L lysozyme mutants. Reprinted from ref 109.

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“…26,28 The ability to measure these properties allows a wide array of interactions (antibodies to DNA to small molecules) to be quantified with a large range of affinities (μM to pM) regardless of the mass of the interacting species. 26,34,37−39 Because RI changes may arise from molecular interactions unrelated to target binding, suitable blanks and controls must be prepared to ensure that the measured changes reflect an actual binding event. In addition, temperature control is employed to constrain background noise arising from thermal fluctuations.…”

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confidence: 99%

Toward Rapid, High-Sensitivity, Volume-Constrained Biomarker Quantification and Validation using Backscattering Interferometry

et al. 2014

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Realizing personalized medicine, which promises to enable early disease detection, efficient diagnostic staging, and therapeutic efficacy monitoring, hinges on biomarker quantification in patient samples. Yet, the lack of a sensitive technology and assay methodology to rapidly validate biomarker candidates continues to be a bottleneck for clinical translation. In our first direct and quantitative comparison of backscattering interferometry (BSI) to fluorescence sensing by ELISA, we show that BSI could aid in overcoming this limitation. The analytical validation study was performed against ELISA for two biomarkers for lung cancer detection: Cyfra 21-1 and Galectin-7. Spiked serum was used for calibration and comparison of analytical figures of merit, followed by analysis of blinded patient samples. Using the ELISA antibody as the probe chemistry in a mix-and-read assay, BSI provided significantly lower detection limits for spiked serum samples with each of the biomarkers. The limit of quantification (LOQ) for Cyrfa-21-1 was measured to be 230 pg/mL for BSI versus 4000 pg/mL for ELISA, and for Galectin-7, it was 13 pg/mL versus 500 pg/mL. The coefficient of variation for 5 day, triplicate determinations was <15% for BSI and <10% for ELISA. The two techniques correlated well, ranging from 3–29% difference for Cyfra 21-1 in a blinded patient sample analysis. The label-free and free-solution operation of BSI allowed for a significant improvement in analysis speed, with greater ease, improved LOQ values, and excellent day-to-day reproducibility. In this unoptimized format, BSI required 5.5-fold less sample quantity needed for ELISA (a 10 point calibration curve measured in triplicate required 36 μL of serum for BSI vs 200 μL for ELISA). The results indicate that the BSI platform can enable rapid, sensitive analytical validation of serum biomarkers and should significantly impact the validation bottleneck of biomarkers.

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Back‐Scattering Interferometry: A Versatile Platform for the Study of Free‐Solution versus Surface‐Immobilized Hybridization

Cited by 9 publications

References 18 publications

Sequence and Temperature Influence on Kinetics of DNA Strand Displacement at Gold Electrode Surfaces

Sequence and Temperature Influence on Kinetics of DNA Strand Displacement at Gold Electrode Surfaces

Interferometric Methods for Label-Free Molecular Interaction Studies

Toward Rapid, High-Sensitivity, Volume-Constrained Biomarker Quantification and Validation using Backscattering Interferometry

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