Multiplexed Detection Of Biomolecules Research Articles

Gold-nanorod-enhanced Raman spectroscopy encoded micro-quartz pieces for the multiplex detection of biomolecules.

The rapid analysis and detection of biomolecules has become increasingly important in biological research. Hence, here we propose a novel suspension array method that is based on gold nanorod (AuNR)-enhanced Raman spectroscopy and uses micro-quartz pieces (MQPs) as microcarriers. AuNRs and Raman reporter molecules are coupled together by Au-S bonds to obtain surface-enhanced Raman scattering labels (SERS labels). The SERS labels are then assembled on the surfaces of the MQPs via electrostatic interactions, yielding encoded MQPs. Experimental results showed that the encoded MQPs could be decoded using a Raman spectrometer. A multiplex immunoassay experiment demonstrated the validity and specificity of these encoded MQPs when they were used for bioanalysis. In concentration gradient experiments, the proposed method was found to give a linear concentration response to the target biomolecule at target concentrations of 0.46875-30nM, and the detection limit was calculated to be 1.78nM. The proposed method utilizes MQPs as carriers rather than conventional microbeads, which allows the interference caused by the background fluorescence of microbeads to be eliminated. The fluorescence of the encoded MQPs can be simply, rapidly, and inexpensively quantified using fluorescence microscopy. By dividing the quantitative and qualitative detection of biomolecules into two independent channels, crosstalk between the encoded signal and the labeled signal is averted and high decoding accuracy and detection sensitivity are guaranteed. Graphical abstract.

Multiplexed Nucleic Acid Sensing with Single-Molecule FRET.

Multiplex detection of biomolecules is important in bionanotechnology and clinical diagnostics. Multiplexing using engineered solutions such as microarrays, synthetic nanopores, and DNA barcodes is promising, but they require sophisticated design/engineering and typically yield semiquantitative information. Single-molecule fluorescence resonance energy transfer (smFRET) is an attractive tool in this regard as it enables both sensitive and quantitative detection. However, multiplexing with smFRET remains a great challenge as it requires either multiple excitation sources, an antenna system created by multiple FRET pairs, or multiple acceptors of the donor fluorophore, which complicates not only the labeling schemes but also data analysis, due to overlapping of FRET efficiencies ( EFRET). Here, we address these currently outstanding issues by designing interconvertible hairpin-based sensors (iHabSs) with nonoverlapping EFRET utilizing a single donor/acceptor pair and demonstrate a high-confidence multiplex detection of unlabeled nucleic acid sequences. We validated the reliability of our approach by systematically omitting one target at a time. Further, we demonstrate that these iHabSs are fully recyclable, sensitive with a limit of detection of ∼200 pM, and able to discriminate against single base mismatches. The multiplexed approach developed here has the potential to benefit the fields of biosensing and diagnostics by allowing simultaneous and quantitative detection of unlabeled nucleic acid biomarkers.

Multiplex electrical detection of avian influenza and human immunodeficiency virus with an underlap-embedded silicon nanowire field-effect transistor

The label-free electrical detection of the binding of antibodies and antigens of avian influenza (AI) and human immunodeficiency (HIV) viruses is demonstrated through an underlap-embedded silicon (Si) nanowire field-effect transistor. The proposed sensor was fabricated on a silicon bulk wafer by a top-down process. Specifically, a Si nanowire was fabricated by a combined isotropic and anisotropic patterning technique, which is one route plasma etching process. The sensor was fabricated by a self-aligned process to the gate with tilted implantation, and it allows precise control of the underlap region. This was problematic in earlier underlap field-effect transistors fabricated by a conventional gate-last process. As a sensing metric to detect the binding of a targeted antibody, the transfer characteristic change was traced. Before and after differences between the antibody binding results were caused by changes in the channel potential on the underlap region due to the charge effect arising from the biomolecules; this is also supported by a simulation. Furthermore, the multiplex detection of AI and HIV is demonstrated, showing distinctive selectivity in each case. Thus, the proposed device has inherent benefits for the label-free, electrical, and multiplex detection of biomolecules. Moreover, its processes are compatible with commercialized technology presently used to fabricate semiconductor devices. This advantage is attractive for those involved in the construction of a point-of-care testing (POCT) system on a chip involving simple, low-cost and low-risk fabrication processes of novel structures and materials.

Multifunctional Encoded Self-Assembling Protein Nanofibrils as Platform for High-Throughput and Multiplexed Detection of Biomolecules

A one-dimensional nanofibrillar array formed by the co-assembly of native and biotin-functionalized beta-amyloid (Aβ) peptide was developed for biomolecule sensing. With the presence of biotin moiety, a variety of biomolecular probes can be conjugated onto the nanofibrils, thus converting the protein assembly into a miniature biosensor. In this work, DNA probes were immobilized onto the fibril for the detection of cDNA sequences. The as-developed "DNA-nanoarray" achieved a detection limit at subattomole level (183 fM in 10 μL). This highly sensitive, yet simple, assay requires a trace amount of sample consumption (<10 μL) and is pretreatment-free. In addition, we reported the preparation of alternate-segmented amyloid nanofibrils with multifunctionality. The fibrils hereby serve as an encoded template that can be visualized with various fluorescence labeling dyes for barcode recognition purpose, and, hence, multiplex detection of biomolecules was achieved. Regarding that each protein nanofibril represents a single detection platform, a large number of single fibrils simultaneously are monitored with the dual-color TIRFM in a high-throughput manner.

Surface-Enhanced Raman Scattering-Active Nanostructures and Strategies for Bioassays

Surface-enhanced Raman scattering (SERS) techniques offer a number of advantages in molecular detection and analysis, particularly in terms of the multiplex detection of biomolecules. So far, many new SERS-based substrates and analytical techniques have been reported. For easy understanding, various SERS techniques are classified into the following four categories: adsorption-mediated direct detection; antibody- or ligand-mediated direct detection; binding catalyzed indirect detection; and tag-based indirect detection. Among these, recent successes of SERS tagging/encoding (nano/micro) materials and detection methods are highlighted, including our recent works. Some novel SERS-based strategies for the detection of several biological molecules are also introduced.

Fluorescence Dye Adsorption Assay to Quantify Carboxyl Groups on the Surface of Poly(methyl methacrylate) Microbeads

Microbead-based assays have evolved into powerful tools for the multiplex detection of biomolecules. Analytes are captured by DNA or protein capture molecules which are coupled on microbead surfaces. A homogeneous carboxylation of microbeads is essential for the optimal and reproducible coupling of capture molecules and thus a prerequisite for an optimal multiplex microbead-based assay performance. We developed a simple fluorescence dye adsorption assay for the description of microbead carboxylation and for the prediction of coupling successes of capture molecules. Using the fluorescence dye SYTO-62 it is possible to quantify the degree of carboxylation of poly(methyl methacrylate) (PMMA) microbeads within 1 h in a multiplex format by fluorescence microscopy or flow cytometry. Compared to conventional bulk assays which only provide an average degree of carboxylation the main advantage of the SYTO-62 assay is the single microbead analysis and therefore the description of the qualitative distribution of carboxylation in microbead populations. The SYTO-62 assay is sensitive enough to even determine weak carboxylation. Also, the quality of microbeads can be evaluated. To our knowledge this is the first report which applies a reversible noncovalent fluorescent dye adsorption assay to quantify the degree of carboxylation on surfaces.

Multiplex Label‐Free Detection of Biomolecules with an Imprinted Suspension Array

Appearing in print: Polymer beads with a photonic crystal structure can be prepared that display the molecular imprint of a label-free biomolecule to be analyzed. A series of beads corresponding to a series of analytes could be used for multiplex detection of biomolecules with remarkable sensitivity and specificity (see picture).