Optical Biosensors

During the last three decades we have witnessed remarkable research and development activity aimed at the realization of optical sensors for the measurement of chemical and biological quantities. The first optical chemical sensors were based on the measurement of changes in the absorption spectrum and were developed for the measurement of CO. and O2 concentrations [6] . Since then, a large variety of optical methods have been used in chemical sensors and biosensors including ellip-sometry, spectroscopy (luminescence, phosphorescence, fluorescence, Raman), interferometry (white light interferometry, modal interferometry in optical waveguide structures), spectroscopy of guided modes in optical waveguide structures (grating coupler, resonant mirror), and surface plasmon resonance. In these sensors, a desired quantity is determined by measuring the refractive index, absorb-ance and fluorescence properties of analyte molecules or a chemo-optical transducing medium [7- 10] .

The most widely used and simple optical sensors making use of hybrid nanoparticles are based on the detection of analytes that either absorb or emit light and basically rely on the entrapment of biomolecules that contain chromophoric or fluorescent groups (i.e. flavoproteins, heme proteins, fluorescently-tagged proteins), or can be prepared by co-entrapping a biomolecule along with a chromo-phore or fluorophore that responds to the protein-mediated reaction (i.e. pH change, variation in O2 levels). Alternatively, bioluminescence can also be used as a signal to report on the presence of species that alter the function of luminescent proteins such as aequorin [11] . An interesting example mimicking cell function has been described for recognition of Listeriolysin O (LLO) [12] . LLO is a pore-forming hemolysin secreted by the foodborne pathogen Listeria monocytogenes and is required for bacterial virulence. Current detection methods for L. monocytogenes are time-consuming, labor-intensive, and expensive, which is impractical considering the limitations of food storage. To overcome these problems, Zhao et al. have developed a liposome-doped silica nanocomposite as a simple, inexpensive, and highly stable biosensor material that mimics existing whole-cell assays for LLO. Small unilamellar liposomes containing fluorescent dyes were immobilized within porous silica using alcohol-free sol-gel synthesis methods. The immobilized liposomes served as cellular surrogates for membrane insertion and pore formation by LLO. The integrity of liposomes in the solid-state sol-gel glass was investigated by fluorescence quenching and leaching assays. The materials were stable for at least 5 months in ambient conditions. Both free and immobilized liposomes responded to LLO at pH 6.0 with concentration-dependent kinetics. The pore formation of LLO in liposome-doped silica composites displayed similar kinetic curves as free liposomes but with slower rates. LLO insertion into the immobilized liposomes was pH dependent. No increase in membrane permeability was observed at pH 7.4 for the liposome-doped composites in the presence of LLO. Immobilized liposomes can detect LLO in ca 1.5 h using a steady state calibration and within 30 min using a kinetic calibration. These liposome silica composites potentially could be used for the detection of hemolysin-producing L. monocytogenes as well as many other bacteria that produce pore-forming toxins.

Attention has also been paid to the use of quantum dots for massively parallel and high-throughput analysis of a number of biological molecules. Han et al. reported the use of multicolor optical coding for biological assays by embedding different-sized quantum dots (zinc sulfide-capped cadmium selenide nanocrystals) into polymeric microbeads at precisely controlled ratios [13]. Their novel optical properties (e.g., size-tunable emission and simultaneous excitation) rendered these highly luminescent quantum dots ideal fluorophores for wavelength and intensity multiplexing. The use of ten intensity levels and six colors could theoretically code one million nucleic acid or protein sequences. Imaging and spectroscopic measurements indicated that the quantum dots-tagged beads are highly uniform and reproducible, yielding bead identification accuracies as high as 99.99% under favorable conditions. DNA hybridization studies demonstrated that the coding and target signals can be simultaneously read at the single-bead level. This spectral coding technology was expected to open new opportunities in gene expression studies, high-throughput screening, and medical diagnostics (Figs. 15.1 and 15.2)

The incorporation of fluorescence-quenching strategies into the sensing mechanism is of great interest because it allows extending the range of analytes to include nonabsorbing species that modulate decay properties of entrapped fluorescent species. Moreover, in the cases where fluorescent signaling is used, it allows enhancing the sensitivity limit up to single molecule detection limit. One recent new development taking advantage of such quenching strategies is a novel class of oligonucleotide

Fig. 15.1 (a) Schematic illustration of optical coding based on wavelength and intensity multiplexing. Large spheres represent polymer microbeads, in which small colored spheres (multicolor quantum dots) are embedded according to predetermined intensity ratios. Molecular probes (A-E) are attached to the bead surface for biological binding and recognition, such as DNA-DNA hybridization and antibody-antigen/ligand-receptor interactions. The number of colored spheres (red, green, and blue) does not represent individual quantum dots, but are used to illustrate the fluorescence intensity levels. Optical readout is accomplished by measuring the fluorescence spectra of single beads. Both absolute intensities and relative intensity ratios at different wavelengths are used for coding purposes; for example (1:1:1), (2:2:2), and (2:1:1) are distinguishable codes. (b) Ten distinguishable emission colors of ZnS-capped CdSe quantum dots excited with a near-UV lamp. From left to right (blue to red), the emission maxima are located at 443, 473, 481, 500, 518, 543, 565, 587, 610, and 655 nm. Fluorescence micrograph of a mixture of CdSe/ZnS quantum dots-tagged beads emitting single-color signals. The beads were spread and immobilized on a polylysine-coated glass slide, which caused a slight clustering effect. (Reprinted with permission from [13]; copyright Nature Publishing Group)

Fig. 15.1 (a) Schematic illustration of optical coding based on wavelength and intensity multiplexing. Large spheres represent polymer microbeads, in which small colored spheres (multicolor quantum dots) are embedded according to predetermined intensity ratios. Molecular probes (A-E) are attached to the bead surface for biological binding and recognition, such as DNA-DNA hybridization and antibody-antigen/ligand-receptor interactions. The number of colored spheres (red, green, and blue) does not represent individual quantum dots, but are used to illustrate the fluorescence intensity levels. Optical readout is accomplished by measuring the fluorescence spectra of single beads. Both absolute intensities and relative intensity ratios at different wavelengths are used for coding purposes; for example (1:1:1), (2:2:2), and (2:1:1) are distinguishable codes. (b) Ten distinguishable emission colors of ZnS-capped CdSe quantum dots excited with a near-UV lamp. From left to right (blue to red), the emission maxima are located at 443, 473, 481, 500, 518, 543, 565, 587, 610, and 655 nm. Fluorescence micrograph of a mixture of CdSe/ZnS quantum dots-tagged beads emitting single-color signals. The beads were spread and immobilized on a polylysine-coated glass slide, which caused a slight clustering effect. (Reprinted with permission from [13]; copyright Nature Publishing Group)

probes, called molecular beacons (MBs). MBs, first developed by Tyagi and Kramer, are single-stranded oligonucleotide probes that possess a stem-and-loop structure [14]. The loop portion of the molecule can report the presence of a specific complementary nucleic acid [14-19] , The five bases at the two ends of the MBs are complementary to each other, forming the stem. A fluorophore and a quencher are linked to the two ends of the stem as shown in Fig. 15.3. The stem keeps these two moieties in close proximity to each other, causing the fluorescence of the fluorophore to be quenched by energy transfer. When the probe encounters a target DNA molecule, it forms a hybrid that is longer and more stable than the stem, and its rigidity and length preclude the simultaneous existence of the stem hybrid. Thus the MB undergoes a spontaneous conformational reorganization that forces the stem apart and causes the fluorophore and the quencher to move away from each other, leading to the restoration of fluorescence. Therefore, at room temperature, the MBs emit an intense fluorescent signal only when hybridized to their

Fig. 15.2 Schematic illustration of DNA hybridization assays using quantum dots-tagged beads. Probe oligos (No. 1-4) were conjugated to the beads by crosslinking, and target oligos (No. 1-4) were detected with a blue fluorescent dye such as Cascade Blue. After hybridization, non-specific molecules and excess reagents were removed by washing. For multiplexed assays, the oligo lengths and sequences were optimized so that all probes had similar melting temperatures (Tm = 66°-99°C) and hybridization kinetics (30 min). (Reprinted with permission from [13]; copyright Nature Publishing Group)

Fig. 15.2 Schematic illustration of DNA hybridization assays using quantum dots-tagged beads. Probe oligos (No. 1-4) were conjugated to the beads by crosslinking, and target oligos (No. 1-4) were detected with a blue fluorescent dye such as Cascade Blue. After hybridization, non-specific molecules and excess reagents were removed by washing. For multiplexed assays, the oligo lengths and sequences were optimized so that all probes had similar melting temperatures (Tm = 66°-99°C) and hybridization kinetics (30 min). (Reprinted with permission from [13]; copyright Nature Publishing Group)

target molecules [14-18, 21-23]. The size of the loop and its content can be varied by designing different MBs. Also, the quencher and the fluorophores can be changed according to the problem studied. There have been a variety of applications of MBs, [14-18, 21-23] including the real-time monitoring of polymerase chain reactions, [ 14] and even the investigation of HIV-1 disease progression [ 17, 18] . MBs have extremely high selectivity with single-base pair mismatch identification capability. They hold great promise for studies in genetics, disease mechanisms, and molecular interactions, for applications in disease diagnostics, and in new drug development.

MBs and colloidal gold nanoparticles have been combined to develop a new class of nanobiosensors that was able to recognize and detect specific DNA sequences and single-base mutations in a homogeneous format [20]. At the core of this biosensor was a 2.5-nm gold nanoparticle that functions as both a nanoscaffold and a nanoquencher (efficient energy acceptor). Attached to this core were oligo-nucleotide molecules labeled with a thiol group at one end and a fluorophore at the other. This hybrid bio/inorganic construct was found to spontaneously assemble into a constrained arch-like conformation on the particle surface. Binding of target molecules resulted in a conformational change, which restores the fluorescence of the quenched fluorophore. Unlike conventional MBs with a stem-and-loop structure, the nanoparticle probes did not require a stem, and their background fluorescence increased little with temperature. In comparison with the organic quencher

Fig. 15.3 Nanoparticle-based probes and their operating principles. Two oligonucleotide molecules (oligos) are shown to self-assemble into a constrained conformation on each gold particle (2.5 nm diameter). A T6 spacer (six thymines) is inserted at both the 3'- and 5'-ends to reduce steric hindrance. Single-stranded DNA is represented by a single line and doublestranded DNA by a cross-linked double line. In the assembled (closed) state, the fluorophore is quenched by the nanoparticle. Upon target binding, the constrained conformation opens, the fluorophore leaves the surface because of the structural rigidity of the hybridized DNA (double-stranded), and fluorescence is restored. In the open state, the fluorophore is separated from the particle surface by about 10 nm. See text for detailed explanation. Au, gold particle; F, fluorophore; S, sulfur atom. (Reprinted with permission from [20]; copyright ACS publications)

Fig. 15.3 Nanoparticle-based probes and their operating principles. Two oligonucleotide molecules (oligos) are shown to self-assemble into a constrained conformation on each gold particle (2.5 nm diameter). A T6 spacer (six thymines) is inserted at both the 3'- and 5'-ends to reduce steric hindrance. Single-stranded DNA is represented by a single line and doublestranded DNA by a cross-linked double line. In the assembled (closed) state, the fluorophore is quenched by the nanoparticle. Upon target binding, the constrained conformation opens, the fluorophore leaves the surface because of the structural rigidity of the hybridized DNA (double-stranded), and fluorescence is restored. In the open state, the fluorophore is separated from the particle surface by about 10 nm. See text for detailed explanation. Au, gold particle; F, fluorophore; S, sulfur atom. (Reprinted with permission from [20]; copyright ACS publications)

Dabcyl (4,4'-dimethylaminophenyl azo benzoic acid), metal nanoparticles have unique structural and optical properties for new applications in biosensing and molecular engineering.

However, the field where these sensors based on fluorescent emission of organic dyes and functionalized quantum dot nanoparticles are of great help is in vivo applications. A recent and interesting work in the field of in vivo imaging is the development and characterization of a highly-selective magnesium fluorescent optical nanosensor, made possible by PEBBLE (probe encapsulated by biologically localized embedding) technology [24]. A ratiometric sensor has been developed by co-immobilizing a dye that is both sensitive to and highly selective for magnesium. The sensors were prepared via a microemulsion polymerization process which entraps the sensing components inside a polymer matrix. The resultant spherical sensors were ca 40 nm in diameter. The Coumarin 343 (C343) dye, which by itself does not enter the cell, when immobilized in a PEBBLE was used as the magnesium-selective agent that provides the high and necessary selectivity over other intra-cellular ions, such as Ca2+, Na+. and K+. The dynamic range of these sensors was 1-30 mM, with a linear range from 1 to 10 mM, with a response time of <4 s. In contrast to free dyes, these nano-optodes were not perturbed by proteins. They were fully reversible and exhibit minimal leaching and photobleaching over extended periods of time. In vitro intracellular changes in Mg2+ concentration were monitored in C6 glioma cells, which remained viable after PEBBLE delivery via gene gun injection. The selectivity for Mg2+ along with the biocompatibility of the matrix provides a new and reliable tool for intracellular magnesium measurements.

An interesting alternative for in vivo imaging is the use of quantum dots. Fluorescent semiconductor nanocrystals (quantum dots) have the potential to revolutionize biological imaging, given their much higher fluorescent quantum yields and lower photobleaching than organic dyes. However, their use has been limited by difficulties in obtaining nanocrystals that are biocompatible. The encapsulation of individual nanocrystals in phospholipid block-copolymer micelles is of great help to address this problem, as recently demonstrated both in vitro and in vivo imaging [25] (Fig. 15.4). When conjugated to DNA, the nanocrystal-micelles acted as in vitro fluorescent probes to hybridize to specific complementary sequences. Moreover, when injected into Xenopus embryos, the nanocrystal-micelles were stable, nontoxic (<5 x 109 nanocrystals per cell), cell autonomous, and slow to photobleach (Fig. 15.4). Nanocrystal fluorescence could be followed to the tadpole stage, allowing lineage-tracing experiments in embryogenesis.

A nice example for in vivo imaging has recently been reported. In particular, gold nanoparticle-oligonucleotide complexes were used as intracellular gene regulation agents for the control of protein expression in cells [26] . These oligonucle-otide-modified nanoparticles had affinity constants for complementary nucleic acids that were higher than their unmodified oligonucleotide counterparts, were less susceptible to degradation by nuclease activity, exhibited greater than 99% cellular uptake, could introduce oligonucleotides at a higher effective concentration than conventional transfection agents, and were nontoxic to the cells under the conditions studied. By chemically tailoring the density of DNA bound to the surface of gold nanoparticles, the authors demonstrated a tunable gene knockdown. The authors conclude that despite these systems have not yet been optimized for maximum efficacy, the ability to systematically control the oligonucleotide loading on the nanoparticle surface makes it possible to identify several features that make them attractive candidates for antisense studies and therapies (Fig. 15.5). The ability to modify the gold nanoparticle surface allows one to realize unusual cooperative properties that led to enhanced target binding and allows the introduction of a variety of functional groups that have proven to be informative in terms of studying how the structure works within a cell. Moreover, this platform will allow to add functionality [27, 28] that could direct the oligonucleotide-modified nanoparticle agents to specific cell types and different components within the cell, thus opening the door for new possibilities in the study of gene function and nanotherapies.

For in vitro applications, the potential of sensors based on surface plasmon resonance (SPR) and evanescent-field waveguide optics (EF) for characterization of thin films [29] and monitoring processes at interfaces [30] has been widely recognized. EF and SPR sensors work similarly; both types of waves are guided, the waveguide modes by total internal reflection at the waveguiding film interface and the surface plasmon by the metal dielectric interface, and both sensors respond

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