Organically modified silica nanoparticles doped with new acridine-1,2-dioxetane analogues as thermochemiluminescence reagentless labels for ultrasensitive immunoassays (original) (raw)
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2012
Thermochemiluminescence (TCL; the light emission originating by the thermally triggered decomposition of a molecule) was proposed in the late 1980s as a detection technique for immunoassays. However, after little pioneering work, this technique was abandoned because of the high temperatures required and the poor detectability in comparison to other labels. Here we describe for the first time a thermochemiluminescent acridine-based 1,2-dioxetane with a remarkably low (i.e., below 100°C) emission-triggering temperature, which made it possible to obtain light emission even in an aqueous environment, as well as amino-functionalized silica nanoparticles loaded with this compound and the fluorescent energy acceptor dipyridamole. Thanks to the signal amplification due to the large number of 1,2-dioxetane molecules in each nanoparticle (about 10 4) and the increased emission efficiency due to energy transfer to the fluorescent acceptor, the doped nanoparticles could be revealed with a detectability close to that of chemiluminescent enzyme labels (the limit of detection of doped nanoparticles by TCL imaging was 1 × 10 −16 mol mm −2 , thus approaching the value of 5 × 10 −17 mol mm −2 obtained for the enzyme label horseradish peroxidase with chemiluminescence detection). They could thus be used as highly detectable labels in the development of sensitive TCL-based immunoassays and nucleic acid hybridization assays, in which the detection step does not require any additional chemical reagent. We believe that these doped silica nanoparticles could pave the way for the revival of TCL detection in bioanalytics, taking advantage of the reagentless detection and the high signal/noise ratio in comparison with conventional luminescence detection techniques.
Ultrasensitive detection of biomolecules with fluorescent dye-doped nanoparticles
Analytical Biochemistry, 2004
Fluorescent-labeled molecules have been used extensively for a wide range of applications in biological detection and diagnosis. A new form of highly luminescent and photostable nanoparticles was generated by doping the fluorescent dye tris(2′2-bipyridyl)dichlororuthenium(II)hexahydrate (Rubpy) inside silica material. Because thousands of fluorescent dye molecules are encapsulated in the silica matrix that also serves to protect Rubpy dye from photodamaging oxidation, the Rubpy-dye-doped nanoparticles are extremely bright and photostable. We have used these nanoparticles successfully in various fluorescence labeling techniques, including fluorescent-linked immunosorbent assay, immunocytochemistry, immunohistochemistry, DNA microarray, and protein microarray. By combining the high-intensity luminescent nanoparticles with the specificity of antibody-mediated recognition, ultrasensitive target detection has been achieved. In all cases, assay results clearly demonstrated the superiority of the nanoparticles over organic fluorescent dye molecules and quantum dots in probe labeling for sensitive target detection. These results demonstrate the potential to apply these newly developed fluorescent nanoparticles in various biodetection systems.
2018
Purification of TCL-Pdots-SA was carried out through filtration over a 400 µm cutoff filter and centrifugation, using a 100 K molecular weight cutoff centrifugal membrane (Amicon Ultra-4, Ultracel-100K). Then, nanoparticles were further purified by size exclusion chromatography, using Sephacryl S-300 HR resin. Spectroscopic properties, in terms of absorption and emission spectra were recorded using a UV-Vis spectrophotometer (Varian Cary 50) and a Uv-Vis spectrofluorimeter (Carian Cary Eclipse). The 1 H and 13 C-NMR spectra were recorded on 400 NMR instrument with a 5 mm probe. All chemical shifts have been quoted relative to deuterated solvent signals. Dynamic light scattering (DLS) experiments were conducted using a Malvern Zetasizer NanoZS while TE images were acquired using a Philips CM100 transmission electron microscope (Philips/FEI Corp., Eindhoven, Holland). Fluorescence images of TCL-Pdots-SA conjugated with Biotin-IgG, before and after the eluition step, were obtained using an epifluorescence microscope with a 100x, 1.3 oil immersion objective and with a 532 nm diode laser, while the efficiency of FRET mechanism was calculated by Fluorescence Lifetime measurements, using a FluoTime 100 spectrometer (PicoQuant, PicoHarp 300). TCL signal was acquired using a portable batteryoperated CCD camera (model MZ-2PRO, MagZero, Pordenone, Italy) equipped with a thermoelectrically cooled monochrome CCD image sensor and an objective (low distortion wide angle lenses 1/3 in. 1.28 mm, f 1.8) obtained from Edmund Optics (Barrington, NJ). TCL images were analyzed using an open Electronic Supplementary Material (ESI) for Nanoscale.
Nanoscale, 2018
Thermochemiluminescence (TCL) is a potentially simple and sensitive detection principle, as the light emission is simply elicited by thermally-triggered decomposition of a molecule to produce a singlet excited-state product. Here we report about TCL semiconductive polymer dots (TCL-Pdots) obtained by doping fluorescent cyano-polyphenylene vinylene (CN-PPV) Pdots with an acridine 1,2-dioxetane derivative. The TCL-Pdots showed remarkable stability over time and minimum leaching of the thermo-responsive species. Furthermore, detectability of TCL-Pdots was improved by taking advantage of both the high number of 1,2-dioxetanes entrapped in each nanoparticle (about 20 molecules per Pdot) and the 5-fold enhancement of TCL emission due to energy transfer from 1,2-dioxetane to the polymer matrix, which itself acted as an energy acceptor. Indeed, upon heating the TCL-Pdots to 110 °C, 1,2-dioxetane decomposes generating an acridanone product in its electronically excited state. The latter tran...
A highly sensitive fluorescent immunoassay based on avidin-labeled nanocrystals
Analytical and Bioanalytical Chemistry, 2006
Nanocrystals of the fluorogenic precursor fluorescein diacetate (FDA) were applied as labels to enhance assay sensitivity in our previous studies. Each FDA nanocrystal can be converted into ~2.6 x 10 6 fluorescein molecules, which is useful for improving sensitivity and limits of detection of immunoassays. NeutrAvidin was simply adsorbed on the surface of the FDA nanocrystals which was coated with distearoylglycerophosphoethanolamine (DSPE) modified with amino(poly(ethylene glycol))(PEG(2000)-Amine) as an interface for coupling biomolecules. It can be applied to detect different kinds of analytes which are captured by corresponding biotinylated biomolecules in different bioanalytical applications. The applicability of the NeutrAvidin-labeled nanocrystals was demonstrated in an immunoassay using the labeled avidin-biotin technique. Biotinylated antibody and FDA-labeled avidin were applied to the assay sequentially. The performance was compared with the traditional sandwich type assay for mouse immunoglobulin G detection. Following the immunoreaction, the nanocrystals were released by hydrolysis and dissolution instigated by adding a large volume of organic solvent/sodium hydroxide mixture. The limit of detection was lowered by a factor of 2.5-21, and the sensitivity was 3.5-30-fold higher compared with the immunoassay using commercial labeling systems, i.e. FITC and peroxidase. This study shows that the fluorescent nanocrystals combined with the avidin-biotin technique can enhance the assay sensitivity and achieve a lower limit of detection without requiring long incubation times as in enzyme-based labels.
Silole nanocrystals as novel biolabels
Journal of Immunological Methods, 2004
A novel class of biofunctional silole nanocrystals with the potential to create highly sensitive immunoassay was firstly demonstrated. Biolabels were constructed by encapsulating nanocrystalline hexaphenylsilole [Ph 2 Si(CPh) 4 ; HPS] within ultrathin polyelectrolyte layers via the layer-by-layer (LbL) technique that provided an binterfaceQ for the attachment of antibodies. A high ratio of fluorescent dyes to biomolecules (F/P ratio; 2.4Â10 3 ) was achieved without self-quenching problem. The aggregation-induced emission (AIE) feature offered silole biolabels the sensitivity 40-to 140-fold higher than that of a start-of-the-art immunoassay using directly fluorescent-labeled antibodies. D
Development of novel dye-doped silica nanoparticles for biomarker application
Journal of Biomedical Optics, 2001
We report the development of novel luminescent nanoparticles composed of inorganic luminescent dye, Tris(2,2Ј-bipyridyl) dichlororuthenium (II) hexahydrate, doped inside a silica network. These dye doped silica (DDS) nanoparticles have been synthesized using a water-in-oil microemulsion technique in which controlled hydrolysis of the tetraethyl orthosilicate leads to the formation of monodispersed nanoparticles. They are prepared with a variety of sizes: small (5Ϯ1 nm), medium (63Ϯ4 nm), and large (400Ϯ10 nm), which shows the efficiency of the microemulsion technique for the synthesis of uniform nanoparticles. All these nanoparticles are suitable for biomarker application since they are much smaller than cellular dimension. These nanoparticles are highly photostable in comparison to most commonly used organic dyes. These nanoparticles have been characterized by various microscopic and spectroscopic techniques. The amount of dye content in these nanoparticles has been optimized to eliminate self-quenching. It has been observed that maximum luminescence intensity is achieved when the dye content is around 20 wt%. Silica surface of DDS nanoparticles is available for surface modification and bioconjunction. For demonstration as a biomarker, the DDS nanoparticle's surface has been biochemically modified to attach membrane-anchoring groups and applied successfully to stain human leukemia cells.
Nanocrystal Biolabels with Releasable Fluorophores for Immunoassays
Analytical Chemistry, 2004
A novel signal amplification technology based on a new class of biofunctional fluorescent nanocrystals holds promise to improve the sensitivity and the limits of detection of immunoassays. A two-step approach without layerby-layer techniques is described to encapsulate the fluorogenic precursor fluorescein diacetate (FDA) nanocrystals (107-nm average size) followed by conjugation of the antibody. Distearoylphosphatidylethanolamine (DSPE) modified with amino(poly(ethylene glycol)) (PEG(2000)-Amine) is coated on the surface of the FDA nanocrystals to provide a interface for the antibody coupling. Antimouse antibodies are attached to the nanocrystalline FDA biolabels by adsorption. A high molar ratio of fluorescent molecules to biomolecules (2.8 × 10 4 ) is achieved in this nanocrystal biolabel system. The analytical performance of the nanocrystal-based label system is evaluated in a model sandwich immunoassay for the detection of mouse IgG. After separation of the nonbound antibody nanocrystal labels, fluorophores are released by hydrolysis and dissolution of the nanocrystalline FDA. Due to the release of the fluorophores (fluoresceins) into a large volume of organic solvent/sodium hydroxide mixture, self-quenching is suppressed. The FDA[DSPE-PEG(2000)Amine]modified biolabels have a highly stable colloidal suspension with minimized nonspecific interactions. The limit of detection was lowered by a factor of 5-28, and the sensitivity was 400-2700-fold higher compared with a state-of-the-art immunoassay using directly fluorescentlabeled antibodies. Our approach provides high sensitivity and low limits of detection without the need for long incubation times, making it an interesting alternative in biolabel technology.
Nanomaterials in fluorescence-based biosensing
Analytical and Bioanalytical Chemistry, 2009
Fluorescence-based detection is the most common method utilized in biosensing because of its high sensitivity, simplicity, and diversity. In the era of nanotechnology, nanomaterials are starting to replace traditional organic dyes as detection labels because they offer superior optical properties, such as brighter fluorescence, wider selections of excitation and emission wavelengths, higher photostability, etc. Their size-or shape-controllable optical characteristics also facilitate the selection of diverse probes for higher assay throughput. Furthermore, the nanostructure can provide a solid support for sensing assays with multiple probe molecules attached to each nanostructure, simplifying assay design and increasing the labeling ratio for higher sensitivity. The current review summarizes the applications of nanomaterialsincluding quantum dots, metal nanoparticles, and silica nanoparticles-in biosensing using detection techniques such as fluorescence, fluorescence resonance energy transfer (FRET), fluorescence lifetime measurement, and multiphoton microscopy. The advantages nanomaterials bring to the field of biosensing are discussed. The review also points out the importance of analytical separations in the preparation of nanomaterials with fine optical and physical properties for biosensing. In conclusion, nanotechnology provides a great opportunity to analytical chemists to develop better sensing strategies, but also relies on modern analytical techniques to pave its way to practical applications.