Quantitative Analysis of Nucleic Acid Hybridization on Magnetic Particles and Quantum Dot-Based Probes

In the present study we describe sandwich design hybridization probes consisting of magnetic particles (MP) and quantum dots (QD) with target DNA, and their application in the detection of avian influenza virus (H5N1) sequences. Hybridization of 25-, 40-, and 100-mer target DNA with both probes was analyzed and quantified by flow cytometry and fluorescence microscopy on the scale of single particles. The following steps were used in the assay: (i) target selection by MP probes and (ii) target detection by QD probes. Hybridization efficiency between MP conjugated probes and target DNA hybrids was controlled by a fluorescent dye specific for nucleic acids. Fluorescence was detected by flow cytometry to distinguish differences in oligo sequences as short as 25-mer capturing in target DNA and by gel-electrophoresis in the case of QD probes. This report shows that effective manipulation and control of micro- and nanoparticles in hybridization assays is possible

Developments in nanoparticles for use in biosensors to assess food safety and quality

The following will provide an overview on how advances in nanoparticle technology have contributed towards developing biosensors to screen for safety and quality markers associated with foods. The novel properties of nanoparticles will be described and how such characteristics have been exploited in sensor design will be provided. All the biosensor formats were initially developed for the health care sector to meet the demand for point-of-care diagnostics. As a consequence, research has been directed towards miniaturization thereby reducing the sample volume to nanolitres. However, the needs of the food sector are very different which may ultimately limit commercial application of nanoparticle based nanosensors. © 2014 Elsevier Ltd

Development of nucleic acid-based nanoplatforms for applications in disease diagnostics and the study of nanomaterials-protein interactions

The impact of nucleic acids on scientific and medical progress has been enormous. DNA is the blueprint for structure and function from the level of individual cells up to whole organisms, and various forms of RNA are all involved in the regulation of genetic information. The identification of specific nucleic acid sequences and/or their level of expression provide key information for the molecular identity and organism functional state. This is very useful in areas like biomedicine. Nucleic acids are also versatile structural materials at the nanoscale. The precise recognition pattern of the Watson-Crick base pairs makes them not only successful as genetic materials, but also capable of directing the assembly of highly structured materials with unique nanoscale features. Nucleic acids, either alone or in combination with other materials, have been used to create a number of nanoscale structures and devices that perform actively in an engineered environment. In this thesis, research towards nucleic acids as a fundamental tool for both diagnostic purpose and structural applications is described. The thesis mainly consists of two studies. The first study is focused on nucleic acid-based biosensing using fluorescent quantum dots (QDs) as donors in a fluorescence resonance energy transfer (FRET) assay for addressing the analytical needs for DNA or RNA detection. The optimization of protocols for synthesizing QD-DNA constructs and their applications in biosensing assays are discussed. A highly sensitive and specific microRNA (miRNA) assay was then developed by the integration of the QD-DNA constructs and an isothermal enzyme-mediated target recycling step, with a detection limit of 42 fM and excellent selectivity for miR-148 versus base-mismatched sequences and other miRNAs. This proposed method was successfully employed for detection of miR-21 using an alternative FRET pair, which was compared to qRT-PCR for the quantitative analysis of miR-21 in biological samples. The second study is focused on construction and characterization of nucleic acid-based hierarchical porous nanostructures and presents a concept for exploiting such constructs as scaffolds for enzyme immobilization and activity studies. Based on rolling circle replication (RCR), DNA or RNA structures with flower-shaped morphologies were synthesized by interactions between inorganic magnesium pyrophosphate (Mg2PPi) crystals and DNA or RNA strands in a time-dependent manner. Focusing on RNA-based structures, various characterization techniques were applied to understand the composition and structure of the RNA particles, and different methods were taken for immobilizing protein or enzymes onto RNA particle. As a proof of principle study, β-galactosidase (β-gal) and horseradish peroxidase (HRP) enzymes were coupled to the RNA particles, and both exhibited enhanced enzymatic activity and improved stability in comparison to free enzymes. This RNA-based biomaterial provides a model to develop a wide range of biocatalysts and offers the promise of potent protein loading and delivery system for biomedical applications.Open Acces

A digital microarray using interferometric detection of plasmonic nanorod labels

DNA and protein microarrays are a high-throughput technology that allow the simultaneous quantification of tens of thousands of different biomolecular species. The mediocre sensitivity and dynamic range of traditional fluorescence microarrays compared to other techniques have been the technology's Achilles' Heel, and prevented their adoption for many biomedical and clinical diagnostic applications. Previous work to enhance the sensitivity of microarray readout to the single-molecule ('digital') regime have either required signal amplifying chemistry or sacrificed throughput, nixing the platform's primary advantages. Here, we report the development of a digital microarray which extends both the sensitivity and dynamic range of microarrays by about three orders of magnitude. This technique uses functionalized gold nanorods as single-molecule labels and an interferometric scanner which can rapidly enumerate individual nanorods by imaging them with a 10x objective lens. This approach does not require any chemical enhancement such as silver deposition, and scans arrays with a throughput similar to commercial fluorescence devices. By combining single-nanoparticle enumeration and ensemble measurements of spots when the particles are very dense, this system achieves a dynamic range of about one million directly from a single scan

Nanomaterial-Assisted Signal Enhancement of Hybridization for DNA Biosensors: A Review

Detection of DNA sequences has received broad attention due to its potential applications in a variety of fields. As sensitivity of DNA biosensors is determined by signal variation of hybridization events, the signal enhancement is of great significance for improving the sensitivity in DNA detection, which still remains a great challenge. Nanomaterials, which possess some unique chemical and physical properties caused by nanoscale effects, provide a new opportunity for developing novel nanomaterial-based signal-enhancers for DNA biosensors. In this review, recent progress concerning this field, including some newly-developed signal enhancement approaches using quantum-dots, carbon nanotubes and their composites reported by our group and other researchers are comprehensively summarized. Reports on signal enhancement of DNA biosensors by non-nanomaterials, such as enzymes and polymer reagents, are also reviewed for comparison. Furthermore, the prospects for developing DNA biosensors using nanomaterials as signal-enhancers in future are also indicated

Design and Development of Nanoconjugates for Nanotechnology

Nanotechnology builds devices from the bottom up with atomic accuracy. Among the basic nano-components to fabricate such devices, semiconductor nanoparticle quantum dots (QDs), metal nanocrystals, proteins, and nucleic acids have attracted most interests due to their potential in optical, biomedical, and electronic areas. The major objective of this research was to prepare nano-components in order to fabricate functional nano-scale devices. This research consisted of three projects. In the first two projects, we incorporated two desirable characteristics of QDs, which are their abilities to serve as donors in fluorescence energy transfer (FRET) and surface energy transfer (SET) as well as to do multiplexing, to engineer QD-based nanoconjugates for optical and biomedical applications. Immobilizing luminescent semiconductor CdSe/ZnS QDs to a solid platform for QD-based biosensors offers advantages over traditional solution-based assays. In the first project, we designed highly sensitive CdSe/ZnS QD SET-based probes using gold nanoparticles (AuNPs) as FRET acceptors on polystyrene (PS) microsphere surfaces. The emission of PS-QD was significantly quenched and restored when the AuNPs were attached to and then removed from the surface. The probes were sensitive enough to analyze signals from a single bead and for use in optical applications. The new PS-QD-AuNP SET platform opens possibilities to carry out both SET and FRET assays in microparticle-based platforms and in microarrays. In the second project, we applied the QD-encoded microspheres in FRET-based analysis for bio-applications. QDs and Alexa Fluor 660 (A660) fluorophores are used as donors and acceptors respectively via a hairpin single stranded DNA. FRET between QD and A660 on the surface of polystyrene microspheres resulted in quenching of QD luminescence and increased A660 emission. QD emission on polystyrene x microspheres was restored when the targeted complementary DNA hybridized the hairpin strand and displaced A660 away from QDs. The third project involved fabrication of different nanoconjugates via self-assembly of template-based metal nanowires and metal nanoparticles using oligonucleotides as linkers. These nanoconjugates can serve as building blocks in nano-electronic circuits. The template method restricted the oligonucleotides attachment to the tip of the nanowires. Nanowires tagged with hybridizable DNA could connect to complementary DNA-modified metal crystals in a position-specific manner

Digital microarrays: single-molecule readout with interferometric detection of plasmonic nanorod labels

DNA and protein microarrays are a high-throughput technology that allow the simultaneous quantification of tens of thousands of different biomolecular species. The mediocre sensitivity and limited dynamic range of traditional fluorescence microarrays compared to other detection techniques have been the technology’s Achilles’ heel and prevented their adoption for many biomedical and clinical diagnostic applications. Previous work to enhance the sensitivity of microarray readout to the single-molecule (“digital”) regime have either required signal amplifying chemistry or sacrificed throughput, nixing the platform’s primary advantages. Here, we report the development of a digital microarray which extends both the sensitivity and dynamic range of microarrays by about 3 orders of magnitude. This technique uses functionalized gold nanorods as single-molecule labels and an interferometric scanner which can rapidly enumerate individual nanorods by imaging them with a 10× objective lens. This approach does not require any chemical signal enhancement such as silver deposition and scans arrays with a throughput similar to commercial fluorescence scanners. By combining single-nanoparticle enumeration and ensemble measurements of spots when the particles are very dense, this system achieves a dynamic range of about 6 orders of magnitude directly from a single scan. As a proof-of-concept digital protein microarray assay, we demonstrated detection of hepatitis B virus surface antigen in buffer with a limit of detection of 3.2 pg/mL. More broadly, the technique’s simplicity and high-throughput nature make digital microarrays a flexible platform technology with a wide range of potential applications in biomedical research and clinical diagnostics.The authors wish to thank Oguzhan Avci and Jacob Trueb for thoughtful comments and suggestions regarding numerical optimization of the optical system. This work was funded in part by a research contract with ASELSAN, Inc. and the Wallace H. Coulter Foundation 2010 Coulter Translational Award. (ASELSAN, Inc.; Wallace H. Coulter Foundation Coulter Translational Award)Accepted manuscrip

Analytical Ancestry: “Firsts” in Fluorescent Labeling of Nucleosides, Nucleotides, and Nucleic Acids

Fluorescently labeled nucleosides, nucleotides, and nucleic acids are important types of reagents for biological assay methods and underpin current methods of chromosome analysis, gel staining, DNA sequencing and quantitative PCR. Although these methods use predominantly organic fluorophores, new types of particulate fluorophores in the form of nanoparticles, nanorods, and nanotubes may provide the basis of a new generation of fluorescent labels and nucleic acid detection methods

Hydrogel microparticles for biosensing

Due to their hydrophilic, biocompatible, and highly tunable nature, hydrogel materials have attracted strong interest in the recent years for numerous biotechnological applications. In particular, their solution-like environment and non-fouling nature in complex biological samples render hydrogels as ideal substrates for biosensing applications. Hydrogel coatings, and later, gel dot surface microarrays, were successfully used in sensitive nucleic acid assays and immunoassays. More recently, new microfabrication techniques for synthesizing encoded particles from hydrogel materials have enabled the development of hydrogel-based suspension arrays. Lithography processes and droplet-based microfluidic techniques enable generation of libraries of particles with unique spectral or graphical codes, for multiplexed sensing in biological samples. In this review, we discuss the key questions arising when designing hydrogel particles dedicated to biosensing. How can the hydrogel material be engineered in order to tune its properties and immobilize bioprobes inside? What are the strategies to fabricate and encode gel particles, and how can particles be processed and decoded after the assay? Finally, we review the bioassays reported so far in the literature that have used hydrogel particle arrays and give an outlook of further developments of the field. Keywords: Hydrogel; Biosensor; Microparticle; Multiplex assayNovartis Institutes of Biomedical Research (Presidential Fellowship)Novartis Institutes of Biomedical Research (Education Office)National Cancer Institute (U.S.) (Grant 5R21CA177393-02)National Science Foundation (U.S.) (Grant CMMI-1120724)Institute for Collaborative Biotechnologies (Grant W911NF-09-0001)United States. Army Research Offic

A digital microarray using interferometric detection of plasmonic nanorod labels

DNA and protein microarrays are a high-throughput technology that allow the simultaneous quantification of tens of thousands of different biomolecular species. The mediocre sensitivity and dynamic range of traditional fluorescence microarrays compared to other techniques have been the technology's Achilles' Heel, and prevented their adoption for many biomedical and clinical diagnostic applications. Previous work to enhance the sensitivity of microarray readout to the single-molecule ('digital') regime have either required signal amplifying chemistry or sacrificed throughput, nixing the platform's primary advantages. Here, we report the development of a digital microarray which extends both the sensitivity and dynamic range of microarrays by about three orders of magnitude. This technique uses functionalized gold nanorods as single-molecule labels and an interferometric scanner which can rapidly enumerate individual nanorods by imaging them with a 10x objective lens. This approach does not require any chemical enhancement such as silver deposition, and scans arrays with a throughput similar to commercial fluorescence devices. By combining single-nanoparticle enumeration and ensemble measurements of spots when the particles are very dense, this system achieves a dynamic range of about one million directly from a single scan.First author draf