Chemical Strategies for Generating Protein Biochips

Jonkheijm, Pascal; Weinrich, Dirk; Schröder, Hendrik; Niemeyer, Christof M.; Waldmann, Herbert

doi:10.1002/anie.200801711

Cited by 557 publications

(474 citation statements)

References 364 publications

(497 reference statements)

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“…18,19 High specificity of the whole biochemical scheme determines the detection limit of the technique. In parallel to already well-studied antibodies, 18,20 the use of engineered receptor molecules, which are more stable and can be developed at low cost, i.e., aptamers, still need to be optimized for their wider implementation into biochemical assays.…”

Section: ■ Introductionmentioning

confidence: 99%

Patterned Biochemical Functionalization Improves Aptamer-Based Detection of Unlabeled Thrombin in a Sandwich Assay

Römhildt

Pahlke²,

Zörgiebel³

et al. 2013

ACS Appl. Mater. Interfaces

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Here we propose a platform for the detection of unlabeled human α-thrombin down to the picomolar range in a fluorescence-based aptamer assay. In this concept, thrombin is captured between two different thrombin binding aptamers, TBA1 (15mer) and TBA2 (29mer), each labeled with a specific fluorescent dye. One aptamer is attached to the surface, the second one is in solution and recognizes surface-captured thrombin. To improve the limit of detection and the comparability of measurements, we employed and compared two approaches to pattern the chip substrate−microcontact printing of organosilanes onto bare glass slides, and controlled printing of the capture aptamer TBA1 in arrays onto functionalized glass substrates using a nanoplotter device. The parallel presence of functionalized and control areas acts as an internal reference. We demonstrate that both techniques enable the detection of thrombin concentrations in a wide range from 0.02 to 200 nM with a detection limit at 20 pM. Finally, the developed method could be transferred to any substrate to probe different targets that have two distinct possible receptors without the need for direct target labeling.

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Section: ■ Introductionmentioning

confidence: 99%

Patterned Biochemical Functionalization Improves Aptamer-Based Detection of Unlabeled Thrombin in a Sandwich Assay

Römhildt

Pahlke²,

Zörgiebel³

et al. 2013

ACS Appl. Mater. Interfaces

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“…20,33,35,76 Such nonfouling surfaces, however, are limiting for numerous biomedical applications, for which selected interactions with the biological media are required. Alternatively, controlled derivatization of bioinert surfaces with small molecules, 15,41,49 polypeptides 9,23,51,63,79 or oligo and polysaccharides 60 yields interfaces that mediate biospecific interactions and suppress nonspecific interactions.…”

Section: Introductionmentioning

confidence: 99%

“…12,13,18,36,65,82 The expanding demands of biophotinic and bioelectronic engineering poses requirements for the development of bioactive coatings on materials such as silicate glasses and silicon. 33 Herein, we demonstrate a method for generation of protein-functionalized coatings directly anchored to the surfaces of glass and silicon (Scheme 1). All components of the coatings are covalently attached to each other and to the substrate.…”

Section: Introductionmentioning

confidence: 99%

Surface-Bound Proteins with Preserved Functionality

et al. 2009

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Abstract-Biocompatibility of materials strongly depends on their surface properties. Therefore, surface derivatization in a controllable manner provides means for achieving interfaces essential for a broad range of chemical, biological, and medical applications. Bioactive interfaces, while manifesting the activity for which they are designed, should suppress all nonspecific interaction between the supporting substrates and the surrounding media. This article describes a procedure for chemical derivatization of glass and silicon surfaces with polyethylene glycol (PEG) layers covalently functionalized with proteins. While the proteins introduce the functionality to the surfaces, the PEGs provide resistance against nonspecific interactions. For formation of aldehyde-functionalized surfaces, we coated the substrates with acetals (i.e., protected aldehydes). To avoid deterioration of the surfaces, we did not use strong mineral acids for the deprotection of the aldehydes. Instead, we used a relatively weak Lewis acid for conversion of the acetals into aldehydes. Introduction of a,x-bifunctional polymers into the PEG layers, bound to the aldehydes, allowed us to covalently attach green fluorescent protein and bovine carbonic anhydrase to the surfaces. Spectroscopic studies indicated that the surface-bound proteins preserve their functionalities. The surface concentrations of the proteins, however, did not manifest linear proportionality to the molar fractions of the bifunctional PEGs used for the coatings. This finding suggests that surface-loading ratios cannot be directly predicted from the compositions of the solutions of competing reagents used for chemical derivatization.

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“…18 Among the different immobilization strategies for generating protein biochips, LAMI is considered to be the one offering uniform orientation and covalent binding to the surface. 21 LAMI requires the presence of aromatic residues and disulphide bridges in the protein to be immobilized. The 3D structure of PSA shows that this protein has aromatic residues in close spatial proximity of disulphide bridges (Fig.…”

Section: Discussionmentioning

confidence: 99%

Arraying prostate specific antigen PSA and Fab anti‐PSA using light‐assisted molecular immobilization technology

et al. 2010

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We here report for the first time the creation of prostate specific antigen (PSA) and Fab anti-PSA biosensor arrays using UV light-assisted molecular immobilization (LAMI), aiming at the detection and quantification of PSA, a cancer marker. The technology involves formation of free, reactive thiol groups upon UV excitation of protein aromatic residues located in spatial proximity of disulphide bridges, a conserved structural feature in both PSA and Fab molecules. The created thiol groups bind onto thiol reactive surfaces leading to oriented covalent protein immobilization. Protein activity was confirmed carrying out immunoassays: immobilized PSA was recognized by Fab anti-PSA in solution and immobilized Fab anti-PSA cross-reacted with PSA in solution. LAMI technology proved successful in immobilizing biomedically relevant molecules while preserving their activity, highlighting that insight into how light interacts with biomolecules may lead to new biophotonic technologies. Our work focused on the application of our new engineering principles to the design, analysis, construction, and manipulation of biological systems, and on the discovery and application of new engineering principles inspired by the properties of biological systems.

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Chemical Strategies for Generating Protein Biochips

Cited by 557 publications

References 364 publications

Patterned Biochemical Functionalization Improves Aptamer-Based Detection of Unlabeled Thrombin in a Sandwich Assay

Patterned Biochemical Functionalization Improves Aptamer-Based Detection of Unlabeled Thrombin in a Sandwich Assay

Surface-Bound Proteins with Preserved Functionality

Arraying prostate specific antigen PSA and Fab anti‐PSA using light‐assisted molecular immobilization technology

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