Simultaneous evaluation of multiple microarray surface chemistries through real-time interferometric imaging

Chiodi, Elisa; Solá, Laura; Brambilla, Dario; Cretich, Marina; Marn, Allison M.; Ünlü, M. Selim; Chiari, Marcella

doi:10.1007/s00216-019-02276-1

Cited by 12 publications

(9 citation statements)

References 20 publications

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“…The physical-chemical properties of the surface environment can affect the interaction between the probes and the targets in solution. In the case of protein-protein interaction, e.g., antibody-antigen, in which the binding occurs via a localized recognition site of large-structured molecules, surface properties mainly affects signal amplitudes, but have small effects on the interaction parameters [34]. In contrast, the details of surface immobilization strongly affect nucleic acids hybridization, in which the binding involves the conformational locking of flexible chains, and even weak spurious interactions with a single monomer can reduce the hybridization affinity [31].…”

Section: Discussionmentioning

confidence: 99%

Copolymer Coatings for DNA Biosensors: Effect of Charges and Immobilization Chemistries on Yield, Strength and Kinetics of Hybridization

et al. 2021

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The physical–chemical properties of the surface of DNA microarrays and biosensors play a fundamental role in their performance, affecting the signal’s amplitude and the strength and kinetics of binding. We studied how the interaction parameters vary for hybridization of complementary 23-mer DNA, when the probe strands are immobilized on different copolymers, which coat the surface of an optical, label-free biosensor. Copolymers of N, N-dimethylacrylamide bringing either a different type or density of sites for covalent immobilization of DNA probes, or different backbone charges, were used to functionalize the surface of a Reflective Phantom Interface multispot biosensor made of a glass prism with a silicon dioxide antireflective layer. By analyzing the kinetic hybridization curves at different probe surface densities and target concentrations in solution, we found that all the tested coatings displayed a common association kinetics of about 9 × 104 M−1·s−1 at small probe density, decreasing by one order of magnitude close to the surface saturation of probes. In contrast, both the yield of hybridization and the dissociation kinetics, and hence the equilibrium constant, depend on the type of copolymer coating. Nearly doubled signal amplitudes, although equilibrium dissociation constant was as large as 4 nM, were obtained by immobilizing the probe via click chemistry, whereas amine-based immobilization combined with passivation with diamine carrying positive charges granted much slower dissociation kinetics, yielding an equilibrium dissociation constant as low as 0.5 nM. These results offer quantitative criteria for an optimal selection of surface copolymer coatings, depending on the application.

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Section: Discussionmentioning

confidence: 99%

Copolymer Coatings for DNA Biosensors: Effect of Charges and Immobilization Chemistries on Yield, Strength and Kinetics of Hybridization

et al. 2021

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“…Similar to epoxysilane, the coating process is rapid and does not require any particular laboratory equipment or expertise. Moreover, the polymer structure is customizable, and from MCP-2 a whole family of polymers was developed, ranging from click-chemistry polymers based on NHS esters, to fluoropolymers [44]. Moreover, it can be utilized to coat a variety of materials, including paper [45].…”

Section: Monolayer Polymeric Coatings: the Sweet Spotmentioning

confidence: 99%

The Effects of Three-Dimensional Ligand Immobilization on Kinetic Measurements in Biosensors

et al. 2022

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The field of biosensing is in constant evolution, propelled by the need for sensitive, reliable platforms that provide consistent results, especially in the drug development industry, where small molecule characterization is of uttermost relevance. Kinetic characterization of small biochemicals is particularly challenging, and has required sensor developers to find solutions to compensate for the lack of sensitivity of their instruments. In this regard, surface chemistry plays a crucial role. The ligands need to be efficiently immobilized on the sensor surface, and probe distribution, maintenance of their native structure and efficient diffusion of the analyte to the surface need to be optimized. In order to enhance the signal generated by low molecular weight targets, surface plasmon resonance sensors utilize a high density of probes on the surface by employing a thick dextran matrix, resulting in a three-dimensional, multilayer distribution of molecules. Despite increasing the binding signal, this method can generate artifacts, due to the diffusion dependence of surface binding, affecting the accuracy of measured affinity constants. On the other hand, when working with planar surface chemistries, an incredibly high sensitivity is required for low molecular weight analytes, and furthermore the standard method for immobilizing single layers of molecules based on self-assembled monolayers (SAM) of epoxysilane has been demonstrated to promote protein denaturation, thus being far from ideal. Here, we will give a concise overview of the impact of tridimensional immobilization of ligands on label-free biosensors, mostly focusing on the effect of diffusion on binding affinity constants measurements. We will comment on how multilayering of probes is certainly useful in terms of increasing the sensitivity of the sensor, but can cause steric hindrance, mass transport and other diffusion effects. On the other hand, probe monolayers on epoxysilane chemistries do not undergo diffusion effect but rather other artifacts can occur due to probe distortion. Finally, a combination of tridimensional polymeric chemistry and probe monolayer is presented and reviewed, showing advantages and disadvantages over the other two approaches.

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“…Copoly Azide 10% is an MCP-4 derivative where the activated ester of NAS is replaced by an azide moiety that allows azide-alkyne cycloaddition reactions, also known as "click" chemistry reactions [19]. Copoly Azide 10% was synthesized as described in [20,21]. The structure or the polymers is reported in Figure 1.…”

Section: Surface Functionalizationmentioning

confidence: 99%

A Reliable, Label Free Quality Control Method for the Production of DNA Microarrays with Clinical Applications

Chiodi

Damin²,

Solá³

et al. 2021

Polymers

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The manufacture of a very high-quality microarray support is essential for the adoption of this assay format in clinical routine. In fact, poorly surface-bound probes can affect the diagnostic sensitivity or, in worst cases, lead to false negative results. Here we report on a reliable and easy quality control method for the evaluation of spotted probe properties in a microarray test, based on the Interferometric Reflectance Imaging Sensor (IRIS) system, a high-resolution label free technique able to evaluate the variation of the mass bound to a surface. In particular, we demonstrated that the IRIS analysis of microarray chips immediately after probe immobilization can detect the absence of probes, which recognizably causes a lack of signal when performing a test, with clinical relevance, using fluorescence detection. Moreover, the use of the IRIS technique allowed also to determine the optimal concentration of the probe, that has to be immobilized on the surface, to maximize the target recognition, thus the signal, but to avoid crowding effects. Finally, through this preliminary quality inspection it is possible to highlight differences in the immobilization chemistries. In particular, we have compared NHS ester versus click chemistry reactions using two different surface coatings, demonstrating that, in the diagnostic case used as an example (colorectal cancer) a higher probe density does not reflect a higher binding signal, probably because of a crowding effect.

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Simultaneous evaluation of multiple microarray surface chemistries through real-time interferometric imaging

Abstract: OpenBU http://open.bu.edu Electrical and Computer Engineering BU Open Access Articles 2020-01-04 Simultaneous evaluation of multiple microarray surface chemistries through real-t...

Cited by 12 publications

References 20 publications

Copolymer Coatings for DNA Biosensors: Effect of Charges and Immobilization Chemistries on Yield, Strength and Kinetics of Hybridization

Copolymer Coatings for DNA Biosensors: Effect of Charges and Immobilization Chemistries on Yield, Strength and Kinetics of Hybridization

The Effects of Three-Dimensional Ligand Immobilization on Kinetic Measurements in Biosensors

A Reliable, Label Free Quality Control Method for the Production of DNA Microarrays with Clinical Applications

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