Diffractometric Detection of Proteins Using Microbead-Based Rolling Circle Amplification

Abstract

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...

Detection of Proteins Using Nano Magnetic Particle Accumulation-Based Signal Amplification

“…After the separation step, there are various techniques to read out signal from magnetic particles; electrochemical detection [10], magnetometer [11], magnetic remanence [12], magnetoresistance [13,14], hall sensors [15], optical methods such as laser diffraction [16], optical light microscope [17], and fluorescent detection [18]. Common approaches to amplifying the signal from magnetic particle-based biosensors are the use of additional magnetic [16] or nonmagnetic particles, such as labels coated with biomolecules to either bind to a target molecule [19] or bind to magnetic beads [18,20].…”

“…Common approaches to amplifying the signal from magnetic particle-based biosensors are the use of additional magnetic [16] or nonmagnetic particles, such as labels coated with biomolecules to either bind to a target molecule [19] or bind to magnetic beads [18,20]. In a companion paper [21] we introduced an alternative signal amplification method based on magnetic bead accumulation.…”

Aptamer and rolling circle amplification-involved sandwich assay for platelet-derived growth factor-BB with absorbance analysis

“…23,24 RCA based assays for PDGF-BB using aptamers have been reported in different formats with electrochemical, uorescence, chemiluminescence, or other detection techniques. [24][25][26][27][28][29][30] In the sandwich formats, a pair of affinity ligands (e.g. two aptamers, or one aptamer and one antibody) are needed, while the strategy relying on a bindinginduced aptamer switch needs only one aptamer.…”

Recent developments in nucleic acid identification using solid-phase enzymatic assays

“…Isothermal RCA has also been very popular as a signal amplification method for cell and protein arrays [66,68,72,73]. The strategy for RCA detection of cells or proteins is very similar.…”

“…As an alternative to antibodies the use of DNA/RNA aptamers has also been reported (Fig. 7) [66,73]. In this method RCA starts in the presence of circular DNA and other reaction components.…”