Ulrich J. Krull

Localized surface plasmon resonance: Nanostructures, bioassays and biosensing—A review

Localized surface plasmon resonance (LSPR) is an optical phenomena generated by light when it interacts with conductive nanoparticles (NPs) that are smaller than the incident wavelength. As in surface plasmon resonance, the electric field of incident light can be deposited to collectively excite electrons of a conduction band, with the result being coherent localized plasmon oscillations with a resonant frequency that strongly depends on the composition, size, geometry, dielectric environment and separation distance of NPs. This review serves to describe the physical theory of LSPR formation at the surface of nanostructures, and the potential for this optical technology to serve as a basis for the development bioassays and biosensing of high sensitivity. The benefits and challenges associated with various experimental designs of nanoparticles and detection systems, as well as creative approaches that have been developed to improve sensitivity and limits of detection are highlighted using examples from the literature.

Beyond labels: A review of the application of quantum dots as integrated components of assays, bioprobes, and biosensors utilizing optical transduction

A comprehensive review of the development of assays, bioprobes, and biosensors using quantum dots (QDs) as integrated components is presented. In contrast to a QD that is selectively introduced as a label, an integrated QD is one that is present in a system throughout a bioanalysis, and simultaneously has a role in transduction and as a scaffold for biorecognition. Through a diverse array of coatings and bioconjugation strategies, it is possible to use QDs as a scaffold for biorecognition events. The modulation of QD luminescence provides the opportunity for the transduction of these events via fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), charge transfer quenching, and electrochemiluminescence (ECL). An overview of the basic concepts and principles underlying the use of QDs with each of these transduction methods is provided, along with many examples of their application in biological sensing. The latter include: the detection of small molecules using enzyme-linked methods, or using aptamers as affinity probes; the detection of proteins via immunoassays or aptamers; nucleic acid hybridization assays; and assays for protease or nuclease activity. Strategies for multiplexed detection are highlighted among these examples. Although the majority of developments to date have been in vitro, QD-based methods for ex vivo biological sensing are emerging. Some special attention is given to the development of solid-phase assays, which offer certain advantages over their solution-phase counterparts.

Real-Time PCR for Quantification of Giardia and Cryptosporidium in Environmental Water Samples and Sewage

ABSTRACT The protozoan pathogens Giardia lamblia and Cryptosporidium parvum are major causes of waterborne enteric disease throughout the world. Improved detection methods that are very sensitive and rapid are urgently needed. This is especially the case for analysis of environmental water samples in which the densities of Giardia and Cryptosporidium are very low. Primers and TaqMan probes based on the β-giardin gene of G. lamblia and the COWP gene of C. parvum were developed and used to detect DNA concentrations over a range of 7 orders of magnitude. It was possible to detect DNA to the equivalent of a single cyst of G. lamblia and one oocyst of C. parvum. A multiplex real-time PCR (qPCR) assay for simultaneous detection of G. lamblia and C. parvum resulted in comparable levels of detection. Comparison of DNA extraction methodologies to maximize DNA yield from cysts and oocysts determined that a combination of freeze-thaw, sonication, and purification using the DNeasy kit (Qiagen) provided a highly efficient method. Sampling of four environmental water bodies revealed variation in qPCR inhibitors in 2-liter concentrates. A methodology for dealing with qPCR inhibitors that involved the use of Chelex 100 and PVP 360 was developed. It was possible to detect and quantify G. lamblia in sewage using qPCR when applying the procedure for extraction of DNA from 1-liter sewage samples. Numbers obtained from the qPCR assay were comparable to those obtained with immunofluorescence microscopy. The qPCR analysis revealed both assemblage A and assemblage B genotypes of G. lamblia in the sewage. No Cryptosporidium was detected in these samples by either method.

Lanthanide upconversion nanoparticles and applications in bioassays and bioimaging: a review.

Through the process of photon upconversion, trivalent lanthanide doped nanocrystals convert long-wavelength excitation radiation in the infrared or near infrared region to higher energy emission radiation from ultraviolet to infrared. Such materials offer potential for numerous advantages in analytical applications in comparison to molecular fluorophores and quantum dots. The use of IR radiation as an excitation source reduces autofluorescence and scattering of excitation radiation, which leads to a reduction of background in optical experiments. The upconverting nanocrystals offer excellent photostability and are composed of materials that are not particularly toxic to biological organisms. Excitation at long wavelengths also minimizes damage to biological materials. In this review, the different mechanisms responsible for the upconversion process, and methods that are used to synthesize and decorate upconverting nanoparticles are presented to indicate how absorption and emission can be tuned. Examples of recent applications of upconverting nanoparticles in bioassays for the detection of proteins, nucleic acids, metabolites and metal ions offer indications of analytical advantages in the development of methods of analysis. Examples include multi-color and multi-modal imaging, and the use of upconverting nanoparticles in theranostics.

Modeling of DNA hybridization kinetics for spatially resolved biochips

The marriage of microfluidics with detection technologies that rely on highly selective nucleic acid hybridization will provide improvements in bioanalytical methods for purposes such as detection of pathogens or mutations and drug screening. The capability to deliver samples in a controlled manner across a two-dimensional hybridization detection platform represents a substantial technical challenge in the development of quantitative and reusable biochips. General theoretical and numerical models of heterogeneous hybridization kinetics are required in order to design and optimize such biochips and to develop a quantitative method for online interpretation of experimental results. In this work we propose a general kinetic model of heterogeneous hybridization and develop a technique for estimating the kinetic coefficients for the case of well-spaced, noninteracting surface-bound probes. The experimentally verified model is then incorporated into the BLOCS (biolab-on-a-chip simulation) 3D microfluidics finite element code and used to model the dynamic hybridization on a biochip surface in the presence of a temperature gradient. These simulations demonstrate how such a device can be used to discriminate between fully complementary and single-base-pair mismatched hybridization using fluorescence detection by interpretation of the unique spatially resolved intensity pattern. It is also shown how the dynamic transport of the targets is likely to affect the rate and location of hybridization as well as that, although nonspecific hybridization is present, the change in the concentration of hybridized targets over the sensor platform is sufficiently high to determine if a fully complementary match is present. Practical design information such as the optimum transport speed, target concentration, and channel height is presented. The results presented here will aid in the interpretation of results obtained with such a temperature-gradient biochip.

Fiber optic biosensor for fluorimetric detection of DNA hybridization

Abstract Single stranded deoxyribonucleic acid (ssDNA) thymidylic acid icosanucleotides (dT 20 ) were grown onto optical fibers. The fibers were first derivatized with γ-aminopropyltriethoxysilane (APTEs) onto which a spacer arm of 1,10 decanediol bis-succinate terminated with 5′-O-dimethoxytrityl-2′-deoxythymidine was covalently attached. The synthetic route used to grow the ssDNA was the well established solid-phase phosphoramidite methodology. The covalently immobilized oligormers were able to hybridize with available complementary ssDNA (cDNA) which was introduced into the local environmnet to forum double stranded DNA (dsDNA). This event was detected by the use of the fluorescent DNA stain ethidium bromide (EB). The sampling configuration utilized total internal reflection of optical radiation within the fiber, resulting in an intrinsic mode optical sensor. The non-optimized procedure used standard hybridization assay techniques to provide a detection limit of 86 ng ml −1 cDNA, a sensitivity of 83% fluorescence intensity increase per 100 ng ml −1 of cDNA initially present, with a hybridization analysis time of 46 min. The sensor has been observed to sustain activity after prolonged storage times (3 months) and harsh washing conditions (sonication).

Toward A Multiplexed Solid-Phase Nucleic Acid Hybridization Assay Using Quantum Dots as Donors in Fluorescence Resonance Energy Transfer

Solid-phase assays using immobilized quantum dots (QDs) as donors in fluorescence resonance energy transfer (FRET) have been developed for the selective detection of nucleic acids. QDs were immobilized on optical fibers and conjugated with probe oligonucleotides. Hybridization with acceptor labeled target oligonucleotides generated FRET-sensitized acceptor fluorescence that was used as the analytical signal. A sandwich assay was also introduced and avoided the need for target labeling. Green and red emitting CdSe/ZnS QDs were used as donors with Cy3 and Alexa Fluor 647 acceptors, respectively. Quantitative measurements were made via spectrofluorimetry or fluorescence microscopy. Detection limits as low as 1 nM were obtained, and the discrimination of single nucleotide polymorphisms (SNPs) with contrast ratios as high as 31:1 was possible. The assays retained their selectivity and at least 50% of their signal when tested in bovine serum and against a large background of noncomplementary genomic DNA. Mixed films of the two colors of QD and two probe oligonucleotide sequences were prepared for multiplexed solid-phase hybridization assays. It was possible to simultaneously detect two target sequences with retention of selectivity, including SNP discrimination. This research provides an important precedent and framework for the future development of QD-based bioassays and biosensors.

Paper-based solid-phase nucleic acid hybridization assay using immobilized quantum dots as donors in fluorescence resonance energy transfer.

A paper-based solid-phase assay is presented for transduction of nucleic acid hybridization using immobilized quantum dots (QDs) as donors in fluorescence resonance energy transfer (FRET). The surface of paper was modified with imidazole groups to immobilize QD-probe oligonucleotide conjugates that were assembled in solution. Green-emitting QDs (gQDs) were FRET-paired with Cy3 acceptor. Hybridization of Cy3-labeled oligonucleotide targets provided the proximity required for FRET-sensitized emission from Cy3, which served as an analytical signal. The assay exhibited rapid transduction of nucleic acid hybridization within minutes. Without any amplification steps, the limit of detection of the assay was found to be 300 fmol with the upper limit of the dynamic range at 5 pmol. The implementation of glutathione-coated QDs for the development of nucleic acid hybridization assay integrated on a paper-based platform exhibited excellent resistance to nonspecific adsorption of oligonucleotides and showed no reduction in the performance of the assay in the presence of large quantities of noncomplementary DNA. The selectivity of nucleic acid hybridization was demonstrated by single-nucleotide polymorphism (SNP) detection at a contrast ratio of 19 to 1. The reuse of paper over multiple cycles of hybridization and dehybridization was possible, with less than 20% reduction in the performance of the assay in five cycles. This work provides an important framework for the development of paper-based solid-phase QD-FRET nucleic acid hybridization assays that make use of a ratiometric approach for detection and analysis.

Biosensors Based on Thin Lipid Films and Liposomes

This article reports the theory and analytical applications of thin lipid films. Recent advances of electrochemical devices based on lipid membranes have lead to reports of construction of biosensors for environmental and food applications, and may provide opportunities for commercial fabrication. The methods of formation of lipid membranes on various supports including metals (silver, gold, stainless steel), agar, conducting polymers and ultrafiltration membranes have provided stabilization of lipid films with a diversity of analytical applications in real samples. Methods of immobilization and incorporation of various functional macromolecules are summarized. Several examples of the application of various methods for study of physical properties of supported bilayer lipid membranes are described. Applications of lipid-based biosensors in analytical chemistry for determination of compounds are demonstrated, including a diversity of chemical compounds such as environmental pollutants (ammonia and carbon dioxide, cyanide ions, etc.) and food toxins (aflatoxin M1and direct detection of toxin in real samples such as milk and milk preparations). Methods for application of liposomes as a sensing system are also summarized.

New opportunities in multiplexed optical bioanalyses using quantum dots and donor–acceptor interactions

AbstractThis review highlights recent trends in the development of multiplexed bioanalyses using quantum dot bioconjugates and donor–acceptor interactions. In these methods, multiple optical signals are generated in response to biorecognition through modulation of the photoluminescence of populations of quantum dots with different emission colors. The donor–acceptor interactions that have been used include fluorescence resonance energy transfer, bioluminescence resonance energy transfer, charge transfer quenching, and quenching via proximal gold nanoparticles. Assays for the simultaneous detection of between two and eight target analytes have been developed, where spectral deconvolution is an important tool. Target analytes have included small molecules, nucleic acid sequences, and proteases. The unique optical properties of quantum dots offer several potential advantages in multiplexed detection, and a large degree of versatility, for example, one pot multiplexing at the ensemble level, where only wavelength discrimination is required to differentiate between detection channels. These methods are not being developed to compete with array-based technologies in terms of overall multiplexing capacity, but rather to enable new formats for multiplexed bioanalyses. In particular, quantum dot bioprobes based on donor–acceptor interactions are anticipated to provide future opportunities for multiplexed biosensing within living cells. FigureMulticolor optical bioanalyses using donor-acceptor interactions between quantum dots and gold nanoparticles, fluorescent dyes, or redox active complexes.