Clickable cellulosic surfaces for peptide-based bioassays

Odinolfi, Maria Teresa; Romanato, Alessandro; Bergamaschi, Greta; Strada, Alessandro; Solá, Laura; Girella, Alessandro; Milanese, Chiara; Chiari, Marcella; Gori, Alessandro; Cretich, Marina

doi:10.1016/j.talanta.2019.120152

Cited by 9 publications

(3 citation statements)

References 23 publications

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“…All these results suggest a great dependency on the spatial availability of BP for a more efficient EVs binding, which is improved when the distance between BP and agarose surface is increased. These results are in line with previous data on the relevance for peptide probes of strict control of surface exposure to complex biological samples ( Gori et al, 2016 ; Odinolfi et al, 2019 ). Taking it into account, further improvement of the EVs binding capacity of BP could be obtained using an agarose with an even longer arm between cobalt and the bead surface.…”

Section: Discussionsupporting

confidence: 92%

Proof of concept of using a membrane-sensing peptide for sEVs affinity-based isolation

Benayas,

Morales,

Gori

et al. 2023

Front. Bioeng. Biotechnol.

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Section: Discussionsupporting

confidence: 92%

Proof of concept of using a membrane-sensing peptide for sEVs affinity-based isolation

Benayas,

Morales,

Gori

et al. 2023

Front. Bioeng. Biotechnol.

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“…All these results suggest a great dependency on the spatial availability of BP for a more e cient EVs binding, which is improved when the distance between BP and agarose surface is increased. These results are in line with previous data on the relevance for peptide probes of strict control of surface exposure to complex biological samples (Gori et al, 2016) (Odinol et al, 2019). Taking it into account, further improvement of the EVs binding capacity of BP could be obtained using an agarose with an even longer arm between cobalt and the bead surface.…”

Section: Discussionsupporting

confidence: 91%

Proof of concept of using a membrane-sensing peptide for sEVs affinity-based isolation

Benayas,

Morales,

Gori

et al. 2023

Preprint

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Background: One main limitation in biomarker studies using EVs is the lack of a suitable isolation method rendering high yield and purity samples in a quick and easily standardized procedure. Here we report an affinity isolation method with a membrane-sensing peptide (MSP) derived from bradykinin. Results: We designed a protocol based on agarose beads carrying cation chelates to specifically bind to the 6His-tagged membrane-sensing peptide. This approach presents several advantages: i) cation-carrying agaroses are widely used and standardized for His-tagged protein isolation, ii) the affinity protocol can be performed in small volumes, feasible and manageable for clinical routine and iii) elution with imidazole or EDTA allows a gentle and easy recovery without EV damage, which allows subsequent characterization and functional analysis of EVs. We optimized all steps of the protocol to enhance peptide exposure on the beads leading to a final procedure that incubates 0.5mg of peptide for 10 minutes with 10µl of Long-arm Cobalt agarose before and overnight incubation with concentrated cell conditioned medium. EV downstream analyses can be performed on the agarose beads by simple adding lysis or nucleic-acid extraction buffers. Alternatively, EVs can be gently eluted by competition with imidazole, rendering a fully competent EV preparation. Conclusions: This new isolation methodology is based on the recognition of general membrane characteristics of EVs and thus can be a good option for a total isolation of EVs without a bias based on the surface markers. It can be used in any species EV sample, enabling this approach to samples from animal or plant species against which no suitable antibodies exist. Being an affinity method, the sample handling protocol is very simple, and less time-consuming than traditional methods, does not require specialized equipment and can be easily introduced in a clinical automated routine. We demonstrated the high purity and yield of the method in comparison with other commercially available kits. This method can also be scale up or down according to operator needs, with the possibility of analyzing very low amounts of sample. Finally, it is compatible with any downstream analyses thanks to the gentle elution procedure.

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“…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]. Our group has successfully shown the ability of MCP-2 to immobilize both hard and soft proteins [44], forming a layer of single probes which extend into the solution when the polymer is hydrated.…”

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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Clickable cellulosic surfaces for peptide-based bioassays

Cited by 9 publications

References 23 publications

Proof of concept of using a membrane-sensing peptide for sEVs affinity-based isolation

Proof of concept of using a membrane-sensing peptide for sEVs affinity-based isolation

Proof of concept of using a membrane-sensing peptide for sEVs affinity-based isolation

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

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