Real-Time Measurements of DNA Hybridization on Microparticles with Fluorescence Resonance Energy Transfer

Henry, Michael R.; Stevens, Priscilla Wilkins; Sun, Jason; Kelso, David M.

doi:10.1006/abio.1999.4344

Cited by 115 publications

(114 citation statements)

References 52 publications

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“…A variety of aids to enhance hybridization performance is used, including surfactants and blocking agents to control nonspecific adsorption, washing procedures to develop contrast between fully and partially complementary sequences, and variation of assay parameters such as ionic strength or temperature. Physical studies (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27) aim to distill down this complexity into scenarios where specific features of surface hybridization are brought out; for example, the impact of probe surface coverage on target hybridization (17,20,22,23,28).…”

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confidence: 99%

DNA surface hybridization regimes

Gong

Levicky²

2008

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

219

282

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Surface hybridization reactions, in which sequence-specific recognition occurs between immobilized and solution nucleic acids, are routinely carried out to quantify and interpret genomic information. Although hybridization is fairly well understood in bulk solution, the greater complexity of an interfacial environment presents new challenges to a fundamental understanding, and hence application, of these assays. At a surface, molecular interactions are amplified by the two-dimensional nature of the immobilized layer, which focuses the nucleic acid charge and concentration to levels not encountered in solution, and which impacts the hybridization behavior in unique ways. This study finds that, at low ionic strengths, an electrostatic balance between the concentration of immobilized oligonucleotide charge and solution ionic strength governs the onset of hybridization. As ionic strength increases, the importance of electrostatics diminishes and the hybridization behavior becomes more complex. Suppression of hybridization affinity constants relative to solution values, and their weakened dependence on the concentration of DNA counterions, indicate that the immobilized strands form complexes that compete with hybridization to analyte strands. Moreover, an unusual regime is observed in which the surface coverage of immobilized oligonucleotides does not significantly influence the hybridization behavior, despite physical closeness and hence compulsory interactions between sites. These results are interpreted and summarized in a diagram of hybridization regimes that maps specific behaviors to experimental ranges of ionic strength and probe coverage.biosensor ͉ ferrocene ͉ ionic strength ͉ microarray ͉ probe coverage S olid-phase or ''surface'' hybridization is the foundation of modern microarray and biosensing technologies widely used in applied genomics for genotyping, drug discovery, gene expression profiling, and related applications based on measurement of genomic information (1, 2). These tools function through detection of interactions between nucleic acids immobilized on a solid support, or ''probes,'' with analyte ''target'' nucleic acids present in solution. Binding, or hybridization, between probes and targets to form an immobilized duplex takes place based on the degree of complementarity between the probe and target base sequences. The tremendous growth in applications of surface hybridization has been mirrorred by increased emphasis on experimental and fundamental aspects of the assays that directly impact measurement and interpretation of data (3, 4).As summarized in several recent reviews (5-8), physical studies under simplified experimental conditions have begun to unravel the rich phenomenology that occurs in diagnostic assays. Applications typically operate away from equilibrium and are faced with a highly diverse pool of target sequences competing for the probe sites. A variety of aids to enhance hybridization performance is used, including surfactants and blocking agents to control nonspecific adsorp...

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mentioning

confidence: 99%

DNA surface hybridization regimes

Gong

Levicky²

2008

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

219

282

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“…Moreover, the reaction kinetics are relatively slow owing to the lengthy diffusion of molecules, because the reaction is carried out on a planar surface. (10,11) As an alternative technique, bead-based multiplexed immunoassays have been intensively researched to overcome the drawbacks of microarray technology. (12)(13)(14)(15) In the case of beadbased immunoassays, multiplexing is achieved by preparing multiple antibody-immobilized beads corresponding to each analyte, and thus multiplexed immunoreactions can be implemented in a single reaction chamber.…”

Section: Introductionmentioning

confidence: 99%

Development of a Bead-Based Multiplexed Immunoassay Using Image Cytometric Analysis

2017

Sensors and Materials

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Keywords: multiplexed immunoassay image cytometry, bead-based assayIn this paper, we present the demonstration of a size-encoded bead-based multiplexed immunoassay with image cytometric decoding and readout. Antibodies were immobilized on polystyrene microspheres by physical adsorption and employed for the demonstration of this duplexed immunoassay. First, we carried out a duplexed immunoassay with a mouse and rat IgG detection system and discussed the problem of cross-reactivity inherent to these immunoreaction systems. We then carried out a duplexed immunoassay with a mouse IgG and human albumin detection system, which was less cross-reactive. We subsequently demonstrated the application of size-encoded image cytometric decoding and the detection of mouse IgG and human albumin using this duplexed immunoassay system. BackgroundImmunoassays are one type of analytical method, which are widely applied for the detection and quantitation of proteins, owing to their simplicity and high sensitivity.(1-4) Recent progress in proteomics has revealed correlations among disease, bioactivity, and molecular information, and this has strongly motivated the clinical application of multiplexed protein assay methods.(5-7)Protein microarrays, which are based on specific antibodies, are the ideal tool for multiplexed immunoassays. (8,9) A spot of antibody is printed locally using microprinting technology for the application of conventional, but multiplexed, immunoreactions. Therefore, antibody printing is becoming the main technical difficulty in carrying out these assays. Moreover, the reaction kinetics are relatively slow owing to the lengthy diffusion of molecules, because the reaction is carried out on a planar surface. (10,11) As an alternative technique, bead-based multiplexed immunoassays have been intensively researched to overcome the drawbacks of microarray technology. (12)(13)(14)(15) In the case of beadbased immunoassays, multiplexing is achieved by preparing multiple antibody-immobilized beads corresponding to each analyte, and thus multiplexed immunoreactions can be implemented

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“…1. On the other hand, real-time biosensors are capable of taking multiple temporal samples of a binding process [8,9,10]. However, analyte estimation therein is typically performed using only the data collected in the equilibrium, and rarely relies on the kinetics [11].…”

Section: Introductionmentioning

confidence: 99%

Limits of performance of real-time DNA microarrays

Vikalo

Hassibi

2009

2009 47th Annual Allerton Conference on Communication, Control, and Computing (Allerton)

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DNA microarrays rely on chemical attraction between the nucleic acid sequences of interest (mRNA and DNA sequences, referred to as targets) and their molecular complements which serve as biological sensing elements (probes). The attraction between the complementary sequences leads to binding, in which probes capture target molecules. Molecular binding is a stochastic process and hence the number of captured analytes at any time is a random variable. Today, majority of DNA microarrays acquire only a single measurement of the binding process, essentially taking one sample from the steady-state distribution of the binding process. Real-time DNA microarrays provide much more: they can take multiple temporal measurements which not only allow more precise characterization of the steady-state but also enable faster detection based on the early kinetics of the binding process. In this paper, we derive the Cramer-Rao lower bound on the mean-square error of estimating the target amounts in real-time DNA microarrays, and compare it to that of conventional microarrays. The results suggest that a few temporal samples collected in the early phase of the binding process are often sufficient to enable significant performance improvement of the real-time microarrays over the conventional ones.

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Real-Time Measurements of DNA Hybridization on Microparticles with Fluorescence Resonance Energy Transfer

Cited by 115 publications

References 52 publications

DNA surface hybridization regimes

DNA surface hybridization regimes

Development of a Bead-Based Multiplexed Immunoassay Using Image Cytometric Analysis

Limits of performance of real-time DNA microarrays

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