Dioxetane-Doped Silica Nanoparticles as Ultrasensitive Reagentless Thermochemiluminescent Labels for Bioanalytics

Abstract

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.

Synthesis of 1,2‐Dioxetanes as Thermochemiluminescent Labels for Ultrasensitive Bioassays: Rational Prediction of Olefin Photooxygenation Outcome by Using a Chemometric Approach

“…Removing ( 2 m , 4 m ) or elongating ( 2 n , 4 n ) the ester group present in the parent compounds ( 2 a , 4 a ) provided the expected enhancement of φ F and decrease in E a values (Table , entries 7 and 8, respectively). This finding further confirmed the stabilizing effect postulated for the acetate moiety, due to its electron‐withdrawing character . Then, we investigated N ‐aryl derivatives ( 2 o – q , 4 o – q ; Table , entries 9–11) and observed, in general, a lower thermal stability of the dioxetanes with respect to the reference compound 4 a , even if the decrease was more pronounced for the EDG‐substituted compound 4 p than that for the para ‐fluorophenyl derivative 4 q .…”

Thermochemiluminescent peroxide crystals

“…If these chemiluminophores are chemically modified to be stable at room temperature, their decomposition can be induced by heating or by application of mechanical force, and the respective phenomena are referred to as thermo- and mechanochemiluminescence. It was demonstrated recently that mechanochemiluminescent polymers can be prepared by incorporating mechanoluminophores in their polymer backbone 5,6 , and thermochemiluminescence was also accomplished by heating of deposited nanoparticles 7–9 and microcrystalline powder 10 . Chemiluminescence in the pure macroscopic solid state, however, was deemed unfeasible due to the very limited ability of molecules for diffusion required for their reaction and the anticipated low quantum yields.…”

Thermochemiluminescent semiconducting polymer dots as sensitive nanoprobes for reagentless immunoassay

“…In previous works regarding acridin-based 1,2-dioxetanes entrapped in silica NPs, 21,23,26 we noticed a strong dependence of both the activation energy and preexponential factor of the thermal decomposition reaction on the surrounding environment. Thus, we investigated the effect of the polymeric environment of Pdots on the thermal decomposition of the 1,2-dioxetane 1 .…”

“…This chemical luminescence phenomenon has been reinvestigated by our group and its applicability as detection principle for bioassays has been revitalized by combining new TCL molecules with silica-based nanoparticle (NP) technology in order to overcome the above reported limitations. 21-23 TCL-based methods might benefit from several advantages, namely the high signal-to-noise ratios typical of chemical luminescence-based detection and the reagentless nature of the TCL process itself, which would simplify analytical devices in comparison to CL/BL. Indeed, the detection step of an immunoassay employing TCL biospecific probes only requires heating of the sample to generate a light signal proportional to the amount of the TCL probe.…”

“…As a result, the intensity of the TCL emission originating from the decomposition of the 1,2-dioxetane derivative 1 is enhanced thanks to the higher fluorescence quantum yield of CN-PPV ( ϕ F =60%) 45 in comparison to ketone 2 ( ϕ F =11%). 21 After conjugation with Streptavidin (SA), the TCL-Pdots were tested as TCL labelling agent in a model non-competitive immunoassay for detection of Biotinylated immunoglobulin G (IgG).…”