ABSTRACT
We propose a spectral contrast method to map the transmission images of surface plasmon resonance (SPR) in metallic nanostructures. Comparing the intensities between two neighboring wavelength bands near the SPR wavelength, the signal-to-noise ratio for biosensing applications obtained using the proposed method is found to be ten times higher than that obtained by conventional intensity analysis and 1.6 times better than that obtained by peak-wavelength fitting. The dynamic range and linearity of the refractive index are comparable to the peak-wavelength shift measurement. Based on the detection method, a spectral modulation system for the optical microscope is developed, combined with a gold-capped nanowire array, to measure the biointeractions in microfluidic devices. The experimental results show that the proposed method obtained multiple detections with a detection limit of 1.04â¯×â¯10-5 refractive index units. Two types of analysis methods for SPR images are used to study the protein-antibody interactions. The region-of-interest analysis supports multiplexing detections in a compact microfluidic sensor. The effective pixel analysis eliminates low-response pixels and enhances the signal-to-noise ratios for sensitive label-free detection.
Subject(s)
Gold/chemistry , Nanostructures/chemistry , Optical Imaging/methods , Surface Plasmon Resonance/methods , Equipment Design , Glucose/analysis , Nanowires/chemistry , Optical Imaging/instrumentation , Refractometry , Surface Plasmon Resonance/instrumentationABSTRACT
In this work, we investigated aptamer selection against alpha-methylacyl-CoA racemase (AMACR) with three DNA libraries bearing different primers. Because of increased selection diversity, aptamers with varied structural properties, such as pre-folded, induced-folding, high and low primer-dependent conformations, were discovered. From the selection results, a dimeric aptamer was constructed and capable of detecting over-expression of AMACR in prostate cancer cell lines. In summary, this study demonstrates a library-based approach to obtain aptamers with different binding properties and provides distinct aptamers for flexible design of AMACR detection.
ABSTRACT
The controlled coverage of immobilized biomolecules is introduced, illustrating a concept for designing biomaterial surfaces such that the extent of manipulation employed to elicit biological responses is controlled according to density changes in the underlying chemical motifs and the density of immobilized biomolecules.
Subject(s)
Biocompatible Materials/chemistry , Cells, Immobilized/chemistry , Biological Phenomena , Cells, Immobilized/metabolism , Humans , ImmobilizationABSTRACT
A peroxidase-mimic DNAzyme is a G-quadruplex (G4) DNA-hemin complex, in which the G4-DNA resembles an apoenzyme, and hemin is the cofactor for hydrogen peroxide (H2O2) catalysis. Twenty-one-mer CatG4 is a well-proven G4-DNA as well as a hemin-binding aptamer for constituting a DNAzyme. This work studied if a multivalent DNAzyme with accelerated catalysis could be constructed using a multimeric CatG4 with hemin. We compared CatG4 monomer, dimer, trimer, and tetramer, which were prepared by custom oligo synthesis, for G4 structure formation. According to circular dichroism (CD) analysis, we found that a CatG4 multimer exhibited more active G4 conformation than the sum effect of equal-number CatG4 monomers. However, the DNAzyme kinetics was not improved monotonically along with the subunit number of a multimeric CatG4. It was the trivalent DNAzyme, trimeric CatG4:hemin, resulting in the rapidest H2O2 catalysis instead of a tetravalent one. We discovered that the trivalent DNAzyme's highest catalytic rate was correlated to its most stable hemin-binding G4 structure, evidenced by CD melting temperature analysis. Finally, a trivalent DNAzyme-based colorimetric glucose assay with a detection limit as low as 10 µM was demonstrated, and this assay did not need adenosine 5'-tri-phosphate disodium salt hydrate (ATP) as a DNAzyme boosting agent.
Subject(s)
Biocatalysis , Biosensing Techniques/methods , DNA, Catalytic/metabolism , G-Quadruplexes , Glucose/analysis , Hydrogen Peroxide/metabolism , Adenosine Triphosphate/metabolism , Colorimetry , Dimerization , Glucose/chemistry , Hemin/metabolism , Kinetics , Models, Molecular , Oxidation-Reduction , Polymerization , Transition TemperatureABSTRACT
This paper first reports DNA aptamers and a fluorescent enzyme-linked aptamer assay (ELAA) targeting alpha-methylacyl-CoA racemase (AMACR), an emerging prostate cancer biomarker. The aptamers were in vitro selected using a new single-bead SELEX approach, which was rapid and consumed only ca. 45 ng AMACR. Before SELEX, silane chemistry was used to prepare epoxide-functionalized glass microbeads (EGBs, 500 µm in size and manipulated by tweezers) for AMACR coating. Recombinant AMACR was also prepared. During SELEX, the ligand evolution was assured by a differential real-time quantitative PCR assay. After SELEX, the aptamers were identified by the alignment analysis and 2nd structure prediction from the selected, cloned sequences. The circular dichroism (CD) analysis revealed that the aptamers formed stable B-form, stem-loop conformations. The fluorescent ELAA method confirmed the nM-level affinity and high specificity of the aptamers against AMACR. Finally, an aptamer-based fluorescent AMACR assay was demonstrated. The assay featured a wide dynamic range (from 10(-1) to 10(3) nM of AMACR), a low detection limit of 0.44 nM (19.5 ng/mL), and high AMACR specificity and is promising for clinical AMACR diagnostics.
Subject(s)
Aptamers, Nucleotide/chemistry , Biosensing Techniques/methods , Racemases and Epimerases/analysis , SELEX Aptamer Technique/methods , Base Sequence , Biomarkers, Tumor/analysis , Humans , Limit of Detection , Male , Molecular Sequence Data , Prostatic Neoplasms/enzymologyABSTRACT
A new SELEX scheme is proposed for the selection of aptamers targeting a specific epitope of a native protein. Anti-sialic acid receptor (SAR) aptamers that inhibit H1 hemagglutination at a low picomole dose are selected accordingly.
Subject(s)
Aptamers, Nucleotide/genetics , Epitopes/drug effects , Hemagglutinins/drug effects , Receptors, Cell Surface/drug effects , SELEX Aptamer Technique , Antiviral Agents/chemical synthesis , Antiviral Agents/pharmacology , DNA, Single-Stranded/chemistry , Hemagglutination/drug effects , Humans , Influenza A Virus, H1N1 Subtype/drug effects , Structure-Activity RelationshipABSTRACT
This study describes a simple and sensitive method for determining the alkylphenolic compounds, 4-tert-octylphenol (4-t-OP), 4-nonylphenol isomers (4-NPs), and their monoethoxylates (4-t-OP1EO and 4-NP1EOs), in fresh fruits and vegetables. The method involves extracting a sample by a modified Nielson-Kryger steam distillation extraction using n-hexane for 1 h. The alkylphenolic compounds were identified and quantitated by gas chromatography-mass spectrometry (GC-MS) in selected ion monitoring (SIM) mode. Various pH values and amounts of NaCl added to the sample solution were evaluated as extraction conditions. The quantitation limit of this method was less than 0.2 ng/g in 10 g (fresh weight) of sample. Recovery of alkylphenolic compounds in spiked samples exceeded 64% while R.S.D. ranged from 1.0 to 9.8%. Alkylphenolic residues were detected in fresh fruits and vegetables at concentrations of 4-NPs and 4-t-OP from n.d. to 16 ng/g and from n.d. to 4.8 ng/g (fresh weight), respectively. NP1EO and OP1EO were always below the quantitation limit.
