Nanobeads

Kohler J.M., Csaki A., Reichert J., Moller R., Straube W., Fritzsche W., Selective labeling of oligonucleotide monolayers by metallic nanobeads for fast optical readout of DNA-chips, Sens Act B 2001 76 166-172. 

Abstract Dye-doped polymeric micro- and nanobeads represent smart analytical tools that have become very popular recently. They enable noninvasive contactless sensing and imaging of various analytical parameters on a nanoscale and are also widely employed in composite sensing materials, in suspension arrays, and as labels. This contribution gives an overview of materials and techniques used for preparation of dye-doped polymeric beads. It also provides examples of bead materials and their applications for optical sensing and imaging. 

Micro- and nanobeads with magnetic properties have recently become popular since these tools can be manipulated, e.g., collected in the region of interest. Magnetite nanoparticles are introduced in order to render the polymeric beads magnetic. Preparation and application of magnetic beads will be discussed in more detail in Sect 5.5. 

As in the case of bulk optodes, plasticizers can be added to modify the properties of polymers (e.g., gas permeability). Plasticizers are mainly used to design ion-sensitive nanobeads. 

Polymeric beads obtained via emulsion polymerization, precipitation, etc. can be stained with dyes providing that both have functional groups available [7]. Covalent coupling is mostly preferred but the attachment based on strong electrostatic interactions is also feasible. This method is mostly used to design pH- and ion-sensitive micro- and nanobeads. The dynamic response of such systems can be... 

Precipitation was found to be a very useful method for preparation of nanobeads with magnetic properties [17] since not only indicators but also small lipophilic magnetite nanobeads (having diameter of a few nanometers) can be incorporated inside the polymeric beads. Such multifunctional magnetic beads can be guided to the region of interest, be collected and manipulated there. 

Dual lifetime referencing (DLR) is another powerful technique that enables referenced measurements in case of fluorescent indicators [23]. In this method, the analyte-dependent signal from an indicator is referenced against the signal from an inert luminophore. This can be realized in both the time domain [24] and in the frequency domain [25]. Often, a luminescent reference dye is embedded into gas blocking nanobeads to avoid oxygen quenching. Polymers with very low gas permeability such as poly(acrylonitrile) [24] or poly(vinylidene chloride-co-acry-lonitrile) [26] are the best choice here. 

It was demonstrated that poly(styrene-b/oc -vinylpyrrolidone) beads (0 220 nm) are suitable for preparation of pH nanosensors [12]. Various fluorescein derivatives were embedded and did not leach out of the beads due to functionalization with highly lipophilic octadecyl anchor. The pK., of the indicators inside the nanobeads varied from 5.8 to 7.7 making them suitable for various biotechnological, biological and marine applications. The beads based on a lipophilic l-hydroxypyrene-3,6, 8-trisulfonate (pKa 6.9) were also manufactured. 

Similarly to dyes, some fluorescent proteins can be incorporated into polymeric beads to be used as an alternative for ion sensing. For example, a reporter protein (composed of a phosphate-binding protein, a FRET donor (cyan fluorescent protein) and a FRET acceptor (yellow fluorescent protein)) was incorporated into polyacrylamide nanobeads by Sun et al. [46]. FRET was inhibited upon binding of phosphate. Kopelman and co-workers [47] used a similar approach to design a nanosensor for copper ions. They have found that fluorescence of red fluorescent protein DsRed (commonly used as a label) is reversibly quenched by Cu2+ and Cu+. Both DsRed and Alexa Fluor 488 (used as a reference) were entrapped into polyacrylamide nanobeads. Typically, up to 2 ppb of copper ions can be reliably measured. It should be mentioned, that in contrast to much more robust dyes, mild conditions upon polymerization and purification are very important for immobilization of the biomolecule to avoid degradation. 

Zenkl et al. [51] presented another approach to design saccharide-sensitive nanobeads. They prepared particles (0 380 nm) based on poly (/V- i sopropy I aery I am ide) cross-linked with phenylboronic acid moieties. In the presence of a saccharide (glucose or fructose) the particles reversibly swell due to the formation of negative charges. A FRET-indicator couple (fhiorescein/rhodamine) is used to monitor the... 

Borisov SM, Klimant I (2009) Luminescent nanobeads for optical sensing and imaging of dissolved oxygen. Microchim Acta 164 7-15... 

Borisov SM, Flerrod DL, Klimant I (2009) Fluorescent poly(styrene-block-vinylpyrrolidone) nanobeads for optical sensing of pH. Sens Actuators B 139 52-58... 

Reichert, J. (2000). Chip-based optical detection of dna hybridization by means of nanobead labeling, Anal. Chem., 72 (24), 6025-6029. 

More recently, Priego-Capote et al. reported on the production of MIP nanoparticles with monoclonal behaviour by miniemulsion polymerisation [63]. In the synthetic method that they employed, they devised to use a polymerisable surfactant that was also able to act as a functional monomer by interacting with the template (Fig. 4). The crosslinker content was optimised at 81% mol/mol (higher or lower contents leading to unstable emulsions). In this way, the authors were able not only to produce rather small particles (80-120 nm in the dry state) but also to locate the imprinted sites on the outer particle surface. The resulting MIP nanobeads were very effective as pseudostationary phases in the analysis of (/ ,S)-propranolol by CEC. 

The combination of different fluorescent metal indicators with inert luminescent reference beads consisting of poly(acrylonitrile) containing Ru(dpp)3 leads to a sensor array in a microwell plate format, suited for ratiomet-ric time-resolved imaging [95]. The data can be acquired with the help of the f-DLR method (for details see Sect. 2.3). A cross-reactive sensor array was arranged for the determination of mixtures of calcium(II), copper(II), nickel(II), cadmium(II), and zinc(II) ions by nine different commercially available fluorescent indicators (Table 3). For a successful application, it is mandatory that all luminophores can be excited at the same wavelength range between 400 and 500 nm, and that the excitation and emission spectra of all indicators overlap with those of the reference dye encapsulated in the nanobeads. 

Fig.26 Pseudo-color time-resolved image of polystyrene nanobeads containing Pt porphyrin and conjugated to streptavidin immobilized to a biotinylated microarray surface (black teflon coated 96-well glass slide, spot diameter 1 mm, Erie Scientific) in different concentrations (25, 15, 10, 5, Ong streptavidin per well) [167]...

Wang H, Abe T, Maruyama S, Iriyama Y, Ogumi Z, Yoshikawa K. Graphitized carbon nanobeads with an onion texture as a lithium-ion battery negative electrode for high-rate use. Adv Mater 2005 17 2857-2860. 

Spin-valve and other magnetoresistive devices detect the stray field from a magnetic micro- or nanobead, as illustrated by Fig. 2. Lithographically fabricated microcircuits [93-96] may be used to manipulate the magnetic particles. Detection limits in the 102 nM can be achieved, and detection of single particles is theoretically possible. 

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