We present a robust, sensitive, fluorescent or radio label-free self-assembled optical diffraction biosensor that utilizes rolling circle amplification (RCA) and magnetic microbeads as a signal enhancement method. An aptamer-based sandwich assay was performed on microcontact-printed streptavidin arranged in 15-μm-wide alternating lines, and could specifically capture and detect platelet-derived growth factor B-chain (PDGF-BB). An aptamer served as a template for the ligation of a padlock probe and the circularized probe could in turn be used as a template for RCA. The concatameric RCA product hybridized to biotinylated oligonuclotides which then captured streptavidin-labeled magnetic beads. In consequence, the signal from the captured PDGF-BB was amplified via the concatameric RCA product, and the diffraction gratings on the printed areas produced varying intensities of diffraction modes. The detected diffraction intensity and the density of the microbeads on the surface varied as a function of PDGF-BB concentration. Our results demonstrate a robust biosensing platform that is easy to construct and use, and devoid of fluorescence microscopy. The self-assembled bead patterns allow both a visual analysis of the molecular binding events under an ordinary bright-field microscope and serve as a diffraction grating biosensor.Detection of proteins in a sensitive and rapid manner plays an essential role in clinical applications. Numerous studies have been reported on using antibody-based immunoassay systems as recognition elements for detecting proteins. 1-3 Antibodies, however, are generally produced in vivo, which generates difficulties in engineering their properties. In contrast, aptamers, generated by an in vitro selection process, are single-stranded oligonucleotides (DNA or RNA) that can specifically bind and recognize a variety of analytes ranging from small organic molecules to proteins, even to whole cells. As a result, aptamers have been usedTo whom correspondence should be addressed. Phone: +1 (765) as recognition elements in a number of biosensing platforms. 4-7 However, even though aptamers uniquely transduce the recognition of analytes into the generation of readily observable signals, analytes in small quantities are still difficult to detect with aptamers alone, pointing to a need for novel signal enhancement schemes.Among many other biosensing platforms, the effectiveness of optical diffraction based biosensors has been demonstrated for recognizing binding events of various biomolecules, which operate based on changes in effective height or refractive index on periodically patterned gratings. [8][9][10][11][12] In many studies, in order to detect small amount of biomolecules, additional signal enhancement was necessary. 13 The enhancement was accomplished either by microfabrication of solid diffraction gratings or by in situ assembled diffraction gratings that are self-fabricated by nano or micro-size particles. Compared to the microfabrication of diffraction gratings which increases cost, tim...

Plants are resistant to most microbial species due to nonhost resistance (NHR), providing broad-spectrum and durable immunity. However, the molecular components contributing to NHR are poorly characterised. We address the question of whether failure of pathogen effectors to manipulate nonhost plants plays a critical role in NHR. RxLR (Arg-any amino acid-Leu-Arg) effectors from two oomycete pathogens, Phytophthora infestans and Hyaloperonospora arabidopsidis , enhanced pathogen infection when expressed in host plants ( Nicotiana benthamiana and Arabidopsis, respectively) but the same effectors performed poorly in distantly related nonhost pathosystems. Putative target proteins in the host plant potato were identified for 64 P . infestans RxLR effectors using yeast 2-hybrid (Y2H) screens. Candidate orthologues of these target proteins in the distantly related non-host plant Arabidopsis were identified and screened using matrix Y2H for interaction with RxLR effectors from both P . infestans and H . arabidopsidis . Few P . infestans effector-target protein interactions were conserved from potato to candidate Arabidopsis target orthologues (cAtOrths). However, there was an enrichment of H . arabidopsidis RxLR effectors interacting with cAtOrths. We expressed the cAtOrth AtPUB33, which unlike its potato orthologue did not interact with P . infestans effector PiSFI3, in potato and Nicotiana benthamiana. Expression of AtPUB33 significantly reduced P . infestans colonization in both host plants. Our results provide evidence that failure of pathogen effectors to interact with and/or correctly manipulate target proteins in distantly related non-host plants contributes to NHR. Moreover, exploiting this breakdown in effector-nonhost target interaction, transferring effector target orthologues from non-host to host plants is a strategy to reduce disease.

Analytical assays that involve aptamers and nuleic acid amplification technologies can be used to detect sensitively target proteins, often in the nanomolar or picomolar range. However, in many cases there are no obvious advantages to amplification assays relative to other assay formats. In all likelihood all formats are limited by the dissociation constants of the aptamers themselves. The one exception to this is the proximity ligation assay, where sequence amplification allows the detection of extremely small quantities of ligands relative to background.