Coupling stimuli-responsive magnetic nanoparticles with antibody-antigen detection in immunoassays.

Because current homogeneous immunoassays show some limitations, particularly low sensitivity, we developed a new immunoassay to overcome these limitations. The approach was based on magnetic nanoparticles with a thermoresponsive polymer layer, a negatively charged polymer, and streptavidin-biotin-based antibody-antigen detection and yielded higher sensitivity than commonly used heterogeneous immunoassays. Because no special equipment is needed, it can be applied to currently available absorbance-based systems for high-throughput assays.

DNA detection on a power-free microchip with laminar flow-assisted dendritic amplification.

In this paper, we describe DNA detection experiments using our two original technologies, power-free microchip and laminar flow-assisted dendritic amplification (LFDA), which were previously applied to immunoassays. A microchip was fabricated by combining a poly(dimethylsiloxane) (PDMS) part having microchannel patterns and a glass plate modified with probe DNA. We carried out two kinds of experiments: the detection of 21-base biotinylated target DNA and the detection of single-nucleotide polymorphism (SNP) in 56-base unlabeled target DNA by sandwich hybridization with biotinylated probe DNA. For both of the experiments, the necessary solutions were injected into microchannels not by an external power source, but by air dissolution into the PDMS part. After a hybridization reaction, the LFDA was started by injecting FITC-labeled streptavidin and biotinylated anti-streptavidin antibody onto the reaction site. With a detection time of 20 min, the limit of detection (LOD) for the biotinylated target was 2.2 pM, and the LOD for the SNP was 10-30 pM, depending on the SNP type.

Detection of non-cross-linking interaction between DNA-modified gold nanoparticles and a DNA-modified flat gold surface using surface plasmon resonance imaging on a microchip.

Gold nanoparticles (GNPs) with fully matched DNA duplexes on their surfaces aggregate together without molecular cross-linking at high salt concentrations. The mechanism of this non-cross-linking (NCL) interaction has been elusive. In this paper, NCL interaction between duplex-modified GNPs and a duplex-modified flat gold surface is presented for the first time. This new experimental platform has enabled us to study the NCL interaction between duplexes with different sequences. We immobilized 15-base single-stranded (ss) DNA onto the surfaces of GNPs with a diameter of 40nm and onto a flat gold substrate. The GNPs were hybridized with 15-base ssDNA at a low salt concentration. A microfluidic device was used for simultaneous delivery of the following three components onto the gold substrate: the duplex-modified GNPs, 15-base ssDNA to be hybridized onto the substrate, and NaCl at a high concentration. Adsorption of the GNPs onto the substrate was monitored using surface plasmon resonance imaging. When the GNPs and the substrate had an identical sequence, the adsorption behavior was analogous to the aggregation behavior of GNPs in test tubes. Furthermore, we investigated 12 cases in which the GNPs and the substrate had completely different sequences, and obtained results suggesting that the NCL attraction force primarily depends on the terminal base pairs of the duplexes. This means that the main mechanism of the NCL interaction is likely to be inter-duplex base stacking rather than formation of Holliday junctions.

Surface plasmon resonance imaging on a microchip for detection of DNA-modified gold nanoparticles deposited onto the surface in a non-cross-linking configuration.

Recently we reported that gold nanoparticles (GNPs) with fully matched duplexes on their surfaces are selectively deposited onto walls of poly(dimethylsiloxane) (PDMS) microchannels at high salt concentrations. In this study, the surface plasmon resonance (SPR) imaging technique was applied to monitor this phenomenon for improvement of detection sensitivity and elucidation of the phenomenon. The microchip was fabricated by bonding a surface-patterned PDMS plate and a gold thin film-deposited glass substrate. Probe oligonucleotide-modified GNPs were hybridized with target oligonucleotides to make fully matched or single-base-mismatched duplexes. The hybridized GNP solution was mixed with an NaCl solution in a Y-shaped microchannel. The deposition of the GNPs onto the gold sensor surface was detected by SPR imaging. Discrimination of the targets was possible with limit of detection of 32 nM (19 fmol) without temperature control in 5 min. Detailed analysis indicated that a seed layer of GNPs was initially adsorbed onto the sensor surface regardless of the target sequence. Therefore, in combination with a portable SPR device, the proposed method is promising for point-of-care testing of single-nucleotide polymorphsims.

Point mutation detection with the sandwich method employing hydrogel nanospheres by the surface plasmon resonance imaging technique.

We propose a surface modification procedure to construct DNA arrays for use in surface plasmon resonance (SPR) imaging studies for the highly sensitive detection of a K-ras point mutation, enhanced with hydrogel nanospheres. A homobifunctional alkane dithiol was adsorbed on Au film to obtain the thiol surface, and ethyleneglycol diglycidylether (EGDE) was reacted to insert the ethyleneglycol moiety, which can suppress nonspecific adsorption during SPR analysis. Then streptavidin (SA) was immobilized on EGDE using tosyl chloride activation. Biotinylated DNA ligands were bound to the SA surface via biotin-SA interaction to fabricate DNA arrays. In SPR analysis, the DNA analyte was exposed on the DNA array and hybridized with the immobilized DNA probes. Subsequently, the hydrogel nanospheres conjugated with DNA probes were bound to the DNA analytes in a sandwich configuration. The DNA-carrying nanospheres led to SPR signal enhancement and enabled us to discriminate a K-ras point mutation in the SPR difference image. The application of DNA-carrying hydrogel nanospheres for SPR imaging assays was a promising technique for high throughput and precise detection of point mutations.

Flow-stress-induced discrimination of a K-ras point mutation by sandwiched polymer microsphere-enhanced surface plasmon resonance.

The highly sensitive detection of a K-ras point mutation with the aid of DNA-carrying microspheres as a flow-stress receptor is proposed at the surface of a surface plasmon resonance (SPR) biosensor. Single-stranded DNAs were immobilized onto epoxy-group-derivatized gold surfaces and the hybridization of DNA targets was monitored. The subsequent interaction with DNA-carrying micospheres enhanced the SPR response. The increase of flow rate during the event of dissociation changed the amount of detachment of the DNA-carrying microspheres for the mismatched pair. In addition, the viscosity was changed by addition of glycerol to the buffer. The increase of shear stress from the flow resulted in detachment of DNA-carrying microspheres hybridized with the mismatched sequence and increased the ability to discriminate a point mutation. This is a new method which not only increases the lower detection limit of evanescent wave-based biosensors, but also the ability to discriminate a point mutation which is a critical factor for ultrasensitive DNA detection in flow devices.

Hydrogel-microsphere-enhanced surface plasmon resonance for the detection of a K-ras point mutation employing peptide nucleic acid.

Highly-sensitive detection of a K-ras point mutation in codon 12, frequently found in pancreatic cancer, based on DNA-carrying hydrogel microspheres as a response enhancer for surface plasmon resonance (SPR), is described. Acrylamide-based microspheres with carboxyl groups were conjugated with DNA probes. Use of the DNA-carrying microsphere in the sandwich method, that is, binding of the microspheres with target DNAs at the sensor surface, enhanced the SPR response as a combined result of increased dielectric constant by the DNA-carrying microspheres. Microspheres lead to response enhancement, as shown by a 100-fold increase in sensitivity compared to that of non-amplified DNA target hybridization. In addition, the advantage of peptide nucleic acid (PNA) in the detection of a K-ras point mutation at the sensor surface by increasing temperature and flow rate is discussed. Results illustrate that the sandwich method through DNA-carrying microspheres for a SPR sensor is a promising approach for ultrasensitive DNA detection.