High sensitivity and label-free oligonucleotides detection using photonic bandgap sensing structures biofunctionalized with molecular beacon probes (original) (raw)
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Molecular Beacons for DNA Biosensors with Micrometer to Submicrometer Dimensions
Analytical Biochemistry, 2000
Ultrasensitive molecular beacon (MB) DNA biosensors, with micrometer to submicrometer sizes, have been developed for DNA/RNA analysis. The fluorescence-based biosensors have been applied in DNA/ RNA detection without the need for a dye-labeled target molecule or an intercalation reagent in the testing solution. Molecular beacons are hairpin-shaped oligonucleotides that report the presence of specific nucleic acids. We have designed a surface-immobilizable biotinylated ssDNA molecular beacon for DNA hybridization at a liquid-solid interface. The MBs have been immobilized onto ultrasmall optical fiber probes through avidin-biotin binding. The MB DNA biosensor has been used directly to detect, in real time, its target DNA molecules without the need for a competitive assay. The biosensor is stable and reproducible. The MB DNA biosensor has selectivity with single basepair mismatch identification capability. The concentration detection limits and mass detection limits are 0.3 nM and 15 amol for a 105-m biosensor, and 10 nM and 0.27 amol for a submicrometer biosensor, respectively. We have also prepared molecular beacon DNA biosensor arrays for simultaneous analysis of multiple DNA sequences in the same solution. The newly developed DNA biosensors have been used for the precise quantification of a specific rat ␥-actin mRNA sequence amplified by the polymerase chain reaction.
An elegant biosensor molecular beacon probe: challenges and recent solutions
Scientifica, 2012
Molecular beacon (MB) probes are fluorophore- and quencher-labeled short synthetic DNAs folded in a stem-loop shape. Since the first report by Tyagi and Kramer, it has become a widely accepted tool for nucleic acid analysis and triggered a cascade of related developments in the field of molecular sensing. The unprecedented success of MB probes stems from their ability to detect specific DNA or RNA sequences immediately after hybridization with no need to wash out the unbound probe (instantaneous format). Importantly, the hairpin structure of the probe is responsible for both the low fluorescent background and improved selectivity. Furthermore, the signal is generated in a reversible manner; thus, if the analyte is removed, the signal is reduced to the background. This paper highlights the advantages of MB probes and discusses the approaches that address the challenges in MB probe design. Variations of MB-based assays tackle the problem of stem invasion, improve SNP genotyping and si...
Label-free luminescent oligonucleotide-based probes
Chemical Society Reviews, 2013
Breakthrough advances in chemistry and biology over the last two decades have vastly expanded the repertoire of nucleic acid structure and function with potential application in multiple areas of science and technology, including sensing and analytical applications. DNA oligonucleotides represent popular tools for the development of sensing platforms due to their low cost, rich structural polymorphism, and their ability to bind to cognate ligands with sensitivity and specificity rivaling those for protein enzymes and antibodies. In this Review, we give an overview of the "label-free" approach that has been a particular focus of our group and others for the construction of luminescent DNA-based sensing platforms. The label-free strategy aims to overcome some of the drawbacks associated with the use of covalently-labeled oligonucleotides prevalent in electrochemical and optical platforms. Label-free DNA-based probes harness the selective interaction between luminescent dyes and functional oligonucleotides that exhibit a "structure-switching" response upon binding to analytes. Based on the numerous examples of label-free luminescent DNA-based probes reported recently, we envisage that this field would continue to thrive and mature in the years to come.
Journal of biophotonics, 2018
An experimental study of the influence of the conformational change suffered by molecular beacon (MB) probes-upon the biorecognition of nucleic acid target oligonucleotides over evanescent wave photonic sensors-is reported. To this end, high sensitivity photonic sensors based on silicon photonic bandgap (PBG) structures were used, where the MB probes were immobilized via their 5' termination. Those MBs incorporate a biotin moiety close to their 3' termination in order to selectively bind a streptavidin molecule to them. The different photonic sensing responses obtained toward the target oligonucleotide detection, when the streptavidin molecule was bound to the MB probes or not, demonstrate the conformational change suffered by the MB upon hybridization, which promotes the displacement of the streptavidin molecule away from the surface of the photonic sensing structure.
Materials Science and …, 1994
Hybridized pairs of oligonucleotides, one biotinylated and the other FITC-labeled, were captured on streptavidincoated surfaces via biotin-streptavidin binding. Detection of the fluoresceinated probe was achieved (1) potentiometrically by light-addressable potentiometric sensing (LAPS) using urease-conjugated anti-fluorescein antibodies and (2) by evanescent wave spectroscopy (EWS) using light internally reflected along a quartz fiber on which the hybrid duplexes were captured facilitating excitation of the FITC-labeled probe in the evanescent wave zone.
Biosensor for label-free DNA quantification based on Functionalized LPGs
Biosensors and Bioelectronics, 2015
A label-free fiber optic biosensor based on a long period grating (LPG) and a basic optical interrogation scheme using off the shelf components is used for the detection of in-situ DNA hybridization. A new methodology is proposed for the determination of the spectral position of the LPG mode resonance. The experimental limit of detection obtained for the DNA was 62 72 nM and the limit of quantification was 209 77 nM. The sample specificity was experimentally demonstrated using DNA targets with different base mismatches relatively to the probe and was found that the system has a single base mismatch selectivity.
Proceedings of 5th International Electronic Conference on Sensors and Applications
A label-free biosensor based on silicon-on-insulator (SOI) photonic bandgap (PBG) structures is performed for specific protein detection. First, the SOI sensing surface is functionalized using triethoxyvinylsilane (TEVS) organosilane. Then, a UV light photocatalyzed immobilization of polyclonal half anti-bovine serum albumin (haBSA) antibodies is performed. Finally, a direct detection of target BSA antigen is carried out. Both the immobilization and the detection steps are monitored by making a continuous tracking of the PBG edge shift. In order to confirm the recognition of the antigen by the immobilized antibody, a fluorophore-labelled secondary antibody was flowed at the end of the experiment in order to perform a confirmation fluorescence test after the photonic detection.
Modified Oligonucleotides for Biosensing Applications
Sensor Letters, 2009
There is a large interest in the use of the self assembling properties of biomolecules to fabricate new types of biosensors. In this work we describe the synthesis of a base analogue of cytosine bearing a thiol group. This group may be used to attach molecules of interest at internal positions of an oligonucleotide sequence and also to attach oligonucleotides to gold surfaces. Moreover we described the synthesis of an oligonucleotide with a photolabile linker at the loop of an intramolecular hairpin and an amino group at the 3 -end. The hairpin photolabile oligonucleotide when immobilized on a glass slide acts as a form of photo-resist: photolysis of an area results in the formation of single-stranded DNA that can direct the hybridization of a labeled complementary oligonucleotide.
Label-free detection of DNA hybridization using nanopillar arrays based optical biosensor
2014
In this study we fabricated nanopillar arrays (NPLAs) of silicon, through a process involving very-largescale integration and reactive ion etching, for use as two-dimensional periodic relief gratings on silicon surfaces. Oligonucleotides were successively immobilized on the pillar surface, allowing the system to be used as an optical detector specific for the targeted single-stranded DNAs (ssDNAs). The surfaces of the oligonucleotides-modified NPLAs underwent insignificant structural changes, but upon hybridizing with target ssDNA, the NPLAs underwent dramatic changes in terms of their pillar scale. Binding of the oligonucleotides to the NPLA occurred in a way that allowed them to retain their function and selectively bind the target ssDNA. We evaluated the performance of the sensor by capturing the target ssDNA on the NPLA and measuring the effective refractive index (n eff). The binding of the target ssDNA species to the NPLA resulted in a color change from pure blue to red, observable by the naked eye at an angle of 15 •. Moreover, we used effective medium theory to calculate the filling factors inside the NPLA and, thereby, examine the values of n eff during the structural changes of the NPLA. Accordingly, these new films have potential applications as label-free optical biosensors.
Sensors, 2009
SiO 2-TiO 2 thin films for use as fiber optic guiding layers of optical DNA biosensors were fabricated by the sol-gel dip coating technique. The chemical structure and the surface morphology of the films were characterized before immobilization. Single probe DNA strands were immobilized on the surface and the porosity of the films before the hybridization process was measured. Refractive index values of the films were measured using a Metricon 2010 prism coupler. On the surface of each film, 12 different spots were taken for measurement and calculation of the mean refractive index values with their standard deviations. The increased refractive index values after the immobilization of single DNA strands indicated that immobilization was successfully achieved. A further refractive index increase after the hybridization with target single DNA strands showed the possibility of detection of the E. coli O157:H7 EDL933 species using strands of 20-mers (5'-TAATATCGGTTGCGGAGGTG-3') sequence.