Article pubs.acs.org/ac
Dioxetane-Doped Silica Nanoparticles as Ultrasensitive Reagentless Thermochemiluminescent Labels for Bioanalytics Aldo Roda,* Massimo Di Fusco, Arianna Quintavalla,* Massimo Guardigli, Mara Mirasoli, Marco Lombardo, and Claudio Trombini Department of Chemistry “G. Ciamician”, Alma Mater Studiorum, University of Bologna, Via Selmi 2, 40126 Bologna, Italy 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 104) 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. (a fluorescent energy acceptor). Decomposition of 1,2dioxetane upon heating produced adamantanone in the first singlet and triplet electronically excited states, which led to light emission via energy transfer to the energy acceptor. However, after little pioneering work, TCL detection was abandoned because of methodological problems, such as the high temperatures required to trigger the TCL emission (typically in the 200−250 °C range) and the poorer detectability in comparison with fluorescent and enzyme labels. No applications of TCL detection in other bioassays, such as nucleic acid hybridization assays, as well as in luminescence imaging have been reported. Nevertheless, TCL still offers interesting and largely unexplored analytical opportunities. As in BL/CL and ECL, there is no nonspecific signal due to the matrix components and the main background signal is determined by instrumental noise; thus, TCL emission could be detected with high sensitivity using the photodiodes7 or charge-coupled-device (CCD) imaging sensors8 already developed and optimized for BL/CL measurements. In addition, being TCL emission simply triggered by heat, this technique would allow for reagentless
L
uminescence-based detection techniques are particularly attractive for bioanalytical applications and biosensors because they combine high detectability with simple instrumentation. Photoluminescence is by far the most employed luminescence detection technique. However, despite a potentially intense light signal, it suffers from problems of nonspecific signals due to sample matrix autofluorescence and difficulties in obtaining reliable quantitative data.1 Superior analytical performance can be achieved using other luminescence detection techniques, such as biochemiluminescence (BL/CL) and electrogenerated chemiluminescence (ECL), which offer higher detectability because of the absence of a background signal and easier implementation in analytical devices because no excitation source nor wavelength selection systems are required. Owing to these advantages, BL/CL and ECL are nowadays extensively used in ultrasensitive biosensors and miniaturized analytical devices.2,3 Thermochemiluminescence (TCL), i.e., the light emission originating from the thermolysis of a suitable molecule, was proposed in the late 1980s as a further luminescence-based detection technique for immunoassays.4−6 In the so-called fluorescence-amplified thermochemiluminescence immunoassay (FATIMA), antibodies were labeled with “light bulbs” constituted by a dual conjugate of bovine serum albumin with adamantylidene adamantane 1,2-dioxetane (1,2-dioxetanes are still today the main TCL species) and 9,10-diphenylanthracene © 2012 American Chemical Society
Received: August 10, 2012 Accepted: November 2, 2012 Published: November 2, 2012 9913
dx.doi.org/10.1021/ac302306u | Anal. Chem. 2012, 84, 9913−9919
Analytical Chemistry
Article
1 (0.25 g, 0.90 mmol) and adamantanone (0.13 g, 0.90 mmol) in dry tetrahydrofuran (THF; 12 mL) was added dropwise to a suspension of TiCl3·3THF (1.0 g, 2.70 mmol) and zinc powder (0.18 g, 2.70 mmol) in dry THF (3.0 mL) over a period of 30 min. The reaction mixture was heated at reflux for 4 h, cooled to room temperature, and then filtered over silica gel. Silica was washed with ethyl acetate (50 mL), and the filtrate was evaporated in vacuum. The crude product was purified by flash chromatography on silica gel using 1:1 (v/v) dichloromethane/ hexane as the eluent. Compound 2 was obtained as a white solid (0.31 g, 0.78 mmol, yield 87%). Mp: 209−212 °C. 1H NMR (400 MHz, CDCl3): δ 1.29 (t, 3H, J = 7.2 Hz), 1.31− 2.19 (m, 12H), 3.46 (s, 2H), 4.28 (q, 2H, J = 7.2 Hz), 4.66 (s, 2H), 6.79 (d, 2H, J = 8.4 Hz), 7.01 (t, 2H, J = 7.6 Hz), 7.15− 7.19 (m, 2H), 7.24−7.27 (m, 2H). 13C NMR (100 MHz, CDCl3): δ 14.2, 25.0, 32.1, 37.1, 48.7, 61.3, 112.3, 120.0, 120.5, 126.1, 126.2, 127.4, 143.1, 144.7, 169.8. IR (neat): ν 2907, 2847, 1751, 1684, 1653, 1592, 1457 cm−1. MS: m/z 400 ([M + H]+). Synthesis of Compound 3. Compound 3 was synthesized by adapting previously reported procedures.13,14 Compound 2 (0.1 g, 0.25 mmol) was dissolved in CH2Cl2 (10 mL), then 0.05 g of polymer-bound Bengal Rose were added, and the suspension was cooled to −78 °C in an acetone/dry ice bath. The solution was bubbled with oxygen and irradiated for about 8 h under stirring using a 400 W halogen lamp equipped with an UV cutoff filter (0.5% transmission at 400 nm). The reaction product was purified by flash chromatography on silica gel using dichloromethane as the eluent. Compound 3 was obtained as a yellow solid (0.07 g, 0.16 mmol, yield 64%). Mp: 124−127 °C. 1H NMR (400 MHz, CDCl3): δ 0.64 (d, 2H, J = 12.8 Hz), 1.17 (d, 2H, J = 11.6 Hz), 1.28 (t, 3H, J = 7.2 Hz), 1.39−2.11 (m, 8H), 2.29 (s, 2H), 4.29 (q, 2H, J = 7.2 Hz), 4.66 (s, 2H), 6.83 (d, 2H, J = 8.4 Hz), 7.21 (t, 2H, J = 7.6 Hz), 7.36−7.40 (m, 2H), 8.22−8.24 (m, 2H). 13C NMR (100 MHz, CDCl3): δ 14.2, 17.4, 25.50, 25.7, 26.9, 31.7, 32.9, 36.1, 39.3, 48.4, 61.6, 86.8, 97.8, 111.8, 121.0, 121.6, 128.4, 129.1, 139.3, 169.3. IR (neat): ν 2919, 2855, 1749, 1635, 1598, 1467, 1374 cm−1. MS: m/z 432 ([M + H]+). Synthesis of Doped SiNPs. Doped amino-functionalized SiNPs were prepared according to a published procedure15 via the hydrolysis of triethoxyvinylsilane and (3-aminopropyl)triethoxysilane in the nonpolar core of Aerosol OT/1-butanol/ water micelles. For the synthesis of 3-doped SiNPs, the micelles were obtained by dissolving 0.44 g of the surfactant Aerosol OT and 800 μL (0.56 g) of the cosurfactant 1-butanol in 20 mL of deionized water by vigorous magnetic stirring. A total of 30 μL of a 30 mmol L−1 solution of 3 in DMF was added to the solution, followed by 200 μL (0.18 g) of neat triethoxyvinylsilane. The resulting solution was stirred for about 1 h, then 10 μL (9.5 mg) of neat (3-aminopropyl)triethoxysilane was added, and the solution was again stirred for about 20 h. The entire reaction was carried out at room temperature. The nanoparticles were collected by centrifugation (15000g, 15 min), washed several times with deionized water, and stored suspended in water at −20 °C. The synthesis of 3/DP-doped SiNPs was carried out using the same procedure, except that 30 μL of a DMF solution containing 30 mmol L−1 3 and 60 mmol L−1 DP was added. Transmission Electron Microscopy (TEM) Imaging. Bright-field TEM images were acquired using a Philips CM100 transmission electron microscope (Philips/FEI Corp., Eindhoven, Holland). Samples were suspended in doubly distilled
luminescence-based detection. However, the successful implementation of TCL detection in bioassays requires TCL labels with lower emission-triggering temperatures and higher detectability. Indeed, the availability of TCL molecules with low triggering temperatures would allow one to perform TCL detection directly in an aqueous environment using the miniaturized heaters already developed for on-chip polymerase chain reactions.9 Moreover, TCL labels that decompose producing efficient fluorophores in the singlet excited state would eliminate the need of a fluorescent energy acceptor, and a significant improvement in label detectability could be achieved by employing multiple labeling strategies. Herein we report for the first time the TCL properties of 1,2dioxetane containing an N-ethyl acetate acridine moiety and of amino-functionalized silica nanoparticles (SiNPs) incorporating the dioxetane, either alone or together with the fluorescent energy acceptor dipyridamole (DP), to be used as TCL labels in bioassays. The TCL emission of the SiNPs can be triggered at low temperatures (i.e., below 100 °C) and, thanks to signal amplification due to high loading of 1,2-dioxetane, the detectability of SiNPs is close to that of common CL enzyme labels. Thus, bioassays based on biospecific reagents (e.g., antibodies) labeled with the doped SiNPs could, in principle, achieve a performance similar to those of enzyme-based assays.
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EXPERIMENTAL SECTION Materials. All of the chemicals were purchased from SigmaAldrich Co. (St. Louis, MO) and used as received. Deionized water (>18 MΩ·cm) obtained by a Milli-Q Synthesis A10 system (Merck Millipore, Billerica, MA) was used in the preparation of buffer solutions. Characterization of Compounds. 1H and 13C NMR spectra were recorded on a Varian Inova 400 NMR instrument (Varian Inc., Palo Alto, CA). IR spectra were recorded using a Nicolet 205 FT-IR spectrometer (Thermo Fisher Scientific, Waltham, MA). Samples were measured as films between NaCl plates. Mass spectrometry was performed using a MSD 1100 single-quadrupole mass spectrometer (Agilent Technologies, Palo Alto, CA) equipped with an electrospray ionization source. Melting points were determined using a Büchi apparatus and are uncorrected. Synthesis of Compound 1. Compound 1 was synthesized according to a published procedure.10 A solution of 9(10H)acridanone (1.0 g, 5.12 mmol) in N,N-dimethylformamide (DMF; 20 mL) was added to a suspension of NaH (0.15 g, 6.06 mmol) (97%) in dry DMF (10 mL). The mixture was stirred for 30 min at room temperature and then, after cooling to 0 °C, ethyl 2-bromoacetate (1.28 g, 7.66 mmol) and tetrabutylammonium iodide (20 mg, 0.054 mmol) were added. The solution was stirred for a further 24 h at room temperature and then poured into 25 mL of cold water. The precipitate was collected by filtration, dried under vacuum, and purified by flash chromatography on silica gel using 9:1 (v/v) dichloromethane/ethyl acetate as the eluent. Compound 1 was obtained as a pale-yellow solid (0.87 g, 3.1 mmol, yield 60%). Mp: 182−185 °C. 1H NMR (400 MHz, CDCl3): δ 1.31 (t, 3H, J = 7.2 Hz), 4.33 (q, 2H, J = 7.2 Hz), 5.08 (s, 2H), 7.32−7.37 (m, 4H), 7.72−7.76 (m, 2H), 8.58−8.60 (m, 2H). 13C NMR (100 MHz, CDCl3): δ 14.1, 48.4, 62.1, 114.2, 121.8, 122.6, 127.9, 134.0, 142.2, 168.3, 178.1. IR (neat): ν 2926, 1748, 1628, 1599, 1496 cm−1. MS: m/z 282 ([M + H]+). Synthesis of Compound 2. Compound 2 was synthesized by adapting a previously reported procedure.11,12 A solution of 9914
dx.doi.org/10.1021/ac302306u | Anal. Chem. 2012, 84, 9913−9919
Analytical Chemistry
Article
a fluorescent energy acceptor, we investigated 1,2-dioxetane derivatives with the general formula shown in Figure 1. In fact,
water and sonicated for 2 min to disperse the SiNPs without any additional treatment. A drop of the suspension was transferred onto porous carbon foils supported on conventional copper microgrids. The SiNP size distribution was obtained by measuring about 100 nanoparticles for each sample using MetaMorph image analysis software (Molecular Devices, LLC, Sunnyvale, CA). TCL Measurements. TCL and fluorescence spectra were recorded using a Varian Eclipse spectrofluorimeter (Varian Inc., Palo Alto, CA). TCL imaging experiments were performed using a LB-980 Night Owl low-light luminograph (Berthold Technologies, Bad Wildbad, Germany). Image integration times varied from 5 s (for evaluation of the TCL decay kinetics) to 5 min (for assessment of the detectability by TCL imaging). TCL images were acquired and analyzed to measure the TCL signals using the image analysis software (WinLight 32) provided with the instrument. For TCL imaging measurements, microscope glass slides glued to a flat heating element or high-resistivity (70−100 Ω) indium−tin oxide (ITO)-coated microscope glass slides (SPI Supplies/Structure Probe Inc., West Chester, PA) were used as solid supports. In the latter case, heating was provided by a suitable electrical current flowing through the ITO coating. The temperature of the support was controlled by varying the applied current and monitored by a copper/constantan thermocouple. Samples were deposited on the supports as acetonitrile solutions (3) or water suspensions (3-doped and 3/ DP-doped SiNPs) either with a micropipet or using a manual microarrayer (Glass Slide Microarrayer, V&P Scientific Inc., San Diego, CA). The manual microarrayer deposited arrays of spots of about 10 nL with diameters in the range of 500−800 μm depending on the nature of the surface. The spots were left to dry before the TCL measurement. For covalent immobilization of doped SiNPs, the glass slides were functionalized with (glycidoxypropyl)trimethoxysilane (GOPS) to introduce amino-reactive epoxy groups. Glass slides were cleaned by soaking in a Piranha solution for 30 min, thoroughly washed in ethanol and water, and then dried in an oven at 80 °C for 2 h. Afterward, the slides were treated with a 10% (v/v) solution of GOPS in toluene for 2 h, washed in toluene, and finally dried in an oven at 80 °C for 20 min. To immobilize doped SiNPs onto the epoxy-functionalized slides, a water suspension of SiNPs was spotted onto the slide. After an overnight incubation, the slides were thoroughly washed to remove unbound SiNPs and dried, and the TCL signals were measured.
Figure 1. General structure of the 1,2-dioxetane derivatives containing the N-substituted acridine moiety investigated in this work.
thermal decomposition of these compounds should lead to Nsubstituted acridanones in the singlet excited state, which have fluorescence quantum yields16 higher than that of adamantanone (ΦF = 0.01517). Indeed, 1,2-dioxetanes containing an acridine moiety have been postulated as intermediates in the CL reaction of acridinium salts and acridane esters.18 According to data reported in the literature,19 the N-methylsubstituted derivative (Figure 1, R = −CH3) is thermochemiluminescent but also poorly stable at room temperature in the solid state. Therefore, we selected the N-ethyl acetatesubstituted derivative 3 because of its much higher stability (the estimated half-life in nonpolar, aprotic solvents was 27.0 months at 25 °C), which has been attributed to the electronwithdrawing character of the ethyl acetate moiety.13 Even though TCL of compound 3 was not studied, a relatively intense emission with ΦCL = 4.1 × 10−4 (ΦCL represents the efficiency of a CL reaction, being defined as the ratio of the number of photons emitted to the number of molecules reacted) was obtained when decomposition of the molecule was triggered by alkaline hydrolysis of the ester moiety. This emission has been attributed to a singlet-excited-state acridone produced by a chemically initiated electron exchange luminescence process.13 This supported the hypothesis that the thermally induced decomposition of this compound could also lead to light emission. In addition, the ethyl acetate group could be easily modified to permit chemical conjugation to biospecific probes (e.g., the introduction of an N-hydroxysuccinimide group could allow binding to primary amino groups of antibodies) or to scaffolds for multiple labeling of bioprobes. Suitable substituents in the acridine moiety could also be used to control the emission properties (e.g., the emission wavelength) or to influence the thermal stability. Compound 3 was synthesized as shown in Figure 2 by adapting previously reported procedures. Briefly, commercially available 9(10H)-acridanone was reacted with ethyl bromoacetate to obtain 10-ethyl acetate-9-acridanone (1). Then, 1 was coupled with adamantanone using a McMurry-like reaction to give 9-adamantylidene-10-ethyl acetate-9-acridane (2). Finally, Bengal Rose-mediated photooxygenation of 2 led to the 1,2dioxetane derivative 3 in a reasonable yield. Upon heating of 3, a weak TCL emission was observed starting from 60 °C, both in the solid state and in solution. With increasing temperature, the TCL reaction became faster and the emission intensity increased. In the temperature interval between 70 and 100 °C, the TCL emission showed a first-order decay and reasonably fast reaction kinetics (i.e., t1/2
