Bioconjugate Chem. 2004, 15, 409−412
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Silicon Nanoparticles as a Luminescent Label to DNA L. Wang,* V. Reipa, and J. Blasic Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899-8312. Received October 28, 2003; Revised Manuscript Received December 26, 2003
We successfully conjugated 1-2 nm diameter silicon nanoparticles to a 5′-amino-modified oligonucleotide (60mer) that contains a C6 linker between amide and phosphate groups. The conjugation was implemented via two photoinduced reactions followed by a DNA labeling step through formation of a carboxamide bond. Photoluminescence of the conjugates is dominated by two blue bands (400 and 450 nm maximal) under 340 nm excitation. The quantum yield of oligonucleotide-conjugated nanoparticles was determined to be 0.08 as measured against quinine sulfate in 0.1 M HClO4 as a reference standard. We report a conjugation process that allows labeling of Si nanoparticles to an oligonucleotide in aqueous solutions. Ways to further optimize the procedure in order to achieve narrower and brighter photoluminescence are discussed.
INTRODUCTION
MATERIALS AND METHODS
The rapid development of pharmacogenomic research and drug discovery, diagnostics of infectious and genetic disease, and methodologies for forensic and genetic identification has stimulated the search for novel fluorophores. Recently, binary semiconductor quantum dots have been demonstrated to possess certain advantages over organic fluorophores (1). However, their inherent toxicity and chemical instability require polymer encapsulation before the bioconjugation step (2). Si nanoparticles (3) have been shown to have similar roomtemperature photoluminescence quantum yields, but higher chemical stability, thus making them attractive for bioconjugation. The surface chemistry of silicon is still a very active field despite the ubiquitous presence of silicon in microelectronics since the 1960s (4). The growing interest is partly attributed to novel applications of silicon chips, which require fine controls over the interfacial characteristics. Recently, silicon nanoparticles with discrete sizes and distinct emission in the blue, green, and red were produced by using anodic etching in hydrofluoric acid (3). Similar to binary semiconductor nanocrystals (5), Si nanoparticles display a relatively narrow emission and high quantum efficiency. Several studies have been published, aiming at functionalizing bulk Si surfaces for biomolecule attachment (6-9). Wagner and co-workers (6) produced bioreactive self-assembled monolayers on hydrogen-passivated Si(111) as atomically flat substrates for biological scanning probe microscopy. Wojtyk et al. (9) also reported the formation of activated ester monolayers on porous silicon surfaces that could be readily attached to biomolecules. The present study is intended for conjugating individual Si nanoparticles suspended in an organic medium to an oligonucleotide in water through chemical derivatization of the nanoparticle outer layer. We have characterized the nanoparticle-labeled nucleotide in terms of the overall size, absorbance, and luminescence quantum yield.
Si nanoparticles were produced using lateral electrochemical anodization (10). (100) Si wafer (p-type, B doped, resistance R ) 2 to 10 Ω, Virginia Semiconductor, VA)1 was cut into 5 cm × 5 cm pieces and etched in (vol) 20% HF, 60% H2O2, 20% ethanol bath at room temperature. A constant current of 50 mA was maintained by a galvanostat while the etching solution was slowly pumped into the cell. Electrical contact to the substrate was provided by InGa eutectic, and Pt mesh served as a cathode. A 100 W halogen lamp illuminated both wafer sides in the procedure. Total etching time per wafer was 30 min. Following etching, wafers were flushed with ethanol and dried in the Ar stream. They were immediately placed in Ar purged 1-octene (or 1-hexene) and sonicated for 1 h in an ultrasound bath (model SC507H, Sonicor Inc., Copiaque, NY). After centrifugation to remove aggregates, the nanoparticles in supernatant were illuminated by 236 nm light (Spectroline, ENF240C) in a 10 mL quartz cell for 2 h while maintaining a vigorous Ar purge. The residual solvent was evaporated under the Ar flow, resulting in dry 1-octene (or 1-hexene) monolayer-coated Si particles. Activation was carried out by adding 0.2 mL of a 15 mM solution of 4′-[3-(trifluoromethyl-3H-diazirin-3-yl)]benzoic acid, N-hydroxysuccinimide ester (TDBA-OSu) [Photoprobes, Industrie Nord, Switzerland] in anhydrous carbon tetrachloride to dried silicon particles and immediately illuminating with a 365 nm lamp (Spectroline, ENF-240C) for 15 min under vigorous magnetic stirring to ensure uniform illumination. After the reaction, carbon tetrachloride was evaporated using an Ar stream. The dried, activated silicon nanoparticles were dissolved in 15 µL of anhydrous DMSO and slowly added to 0.5 mL of the 5′-amino-modified oligonucleotide solution (2 µg/ mL) in water. The 5′ amino-modified oligonucleotide (60mer) was obtained from Qiagen (Valencia, CA, se-
* Corresponding author. Phone: (301) 975-2447; e-mail:
[email protected].
1 Certain commercial equipment, instruments, and materials are identified in this paper to adequately specify the experimental procedure. In no case does such identification imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment are necessarily the best available for the purpose.
10.1021/bc030047k Not subject to U.S. Copyright. Published 2004 by American Chemical Society Published on Web 02/28/2004
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with TBE (90 mM Tris, 90 mM boric acid, 2 mM EDTA) as the running buffer and the voltage set at 115 V for 40 min. A 10 bp DNA ladder from Invitrogen Corporation (Carlsbad, CA) was used to examine the equivalence in the size of Si nanoparticle-labeled oligonucleotide relative to double-stranded DNA. The labeled nucleotide was recovered from polyacrylamide gel using Ultrafree-MC centrifugal filter devices (Durapore 0.22 µm, Millipore Corporation Bedford, MA). The hybridization between the gel-purified nanoparticle-labeled nucleotide and the complementary strand was performed in 100 mM TrisHCl, 1 mM EDTA, pH 7.5, using the following protocol: 95 °C for 30 s, then decreasing the temperature to 60 °C at the rate of 0.2 °C/s, incubation for 30 s at 60 °C and then lowering the temperature to 4 °C. The complementary strand was also obtained from Qiagen with the sequence, 5′-GCTGATGGAGAGGCTCTCTGTCGACTACGGAAAGAAGTCCAAGCTGGAGTTCTCTTTTAA-3′. RESULTS AND DISCUSSION
Figure 1. A reaction diagram displaying the three steps used to produce oligonucleotide-conjugated Si nanoparticles by using single-surface Si-H bond and 1-octene as an example. In this case, carbene addition at the terminal methyl group is the main outcome of the reaction between the 1-octene-coated nanoparticle and the cross-linker, TDBA-OSu.
quence: 5′-TTAAAAGAGAACTCCAGCTTGGACTTCTTTCCGTAGTCGACAGAGAGCCTCTCCATCAGC-3′) and contained a C6 linker between amide and phosphate groups at the 5′ end. This sequence is part of the rat tubulin gene. The reaction was carried out in the dark for 1 h with magnetic stirring. The total volume was then decreased to ∼20 µL by vacuum centrifugation before purification by electrophoresis in 18% polyacrylamide gel
The multiple chemical steps for obtaining oligonucleotide-conjugated Si nanoparticles are illustrated in Figure 1. Under 236 nm illumination in 1-octene (or 1-hexene), hydrogen-passivated silicon particles attained from electrochemical etching are coated with a layer of 1-octene (or 1-hexene). The reaction is driven by formation of free radicals that interact with 1-octene to generate Si-C bonds (11-13). In the following step, TDBA-OSu, a photoactivatable aryldiazirine cross-linker, is inserted into the C-H bonds of the CH2 groups within the hydrocarbon chain through a highly reactive carbene intermediate under 365 nm illumination in dry carbon tetrachloride (14, 15). The report by Nassal (14) has demonstrated that under the radiation of high pressure mercury lamp the singlet carbene can react even with cyclohexane that is considered as an inert solvent. The author carefully characterized the photolysis products with cyclohexane in the presence and absence of oxygen. While purging argon prior to photolysis, the relative yield of carbene addition product is as high as 73% in addition to byproducts from the reaction of triplet carbene with residual oxygen. On the other hand, Cicero and co-
Figure 2. (a) Polyacrylamide gel electrophoresis of the products of the labeling reaction (Figure 1) in the presence (lane 2) and absence (lane 3) of TDBA-OSu as well as the 10 bp DNA ladder (lane 1). The gel portion with the DNA ladder was stained with ethidium bromide (5 µg/mL in TBE). The brightly luminescent band (the first band) in lane 2 is from nanoparticle-labeled oligonucleotide (the main product), which was cut and extracted for the spectral characterization and hybridization with the complementary strand. No luminescent product was detected for the labeling reaction in the absence of TDBA-OSu. (b) Polyacrylamide gel electrophoresis of gel-purified silicon nanoparticle-labeled oligonucleotide (lane 2) and its hybridization product with the complementary strand (lane 3) as well as the 10 bp DNA ladder (lane 1). The gel was stained with ethidium bromide in order to examine the positions of Si nanoparticle-labeled oligonucleotide and the hybridization product relative to double-stranded DNA. The band of the labeled nucleotide is below that of 10 bp DNA, and the band of the hybridization product is located near the 60mer, double-stranded DNA.
Silicon Nanoparticles as a Luminescent Label to DNA
workers (13) demonstrated that in the absence of oxygen, 1-octene could form a compact monolayer on the surface of hydrogen-terminated silicon(111) just alike 1-octadecene. In our case, since C6 hydrocarbon chains surrounding the nanoparticles might not form an ordered monolayer like C8 hydrocarbon chains the carbene addition could take place at multiple CH2 sites within the C6 carbon chain. However, we anticipate the insertion yields at sites closer to the surface of the particle are much lower than those at sites away from the surface of the particle because of the spatial hindrance of the sites near particle surface. As suggested by Nassal (14), the reactions by triplet carbene will not involve the silicon particles. The CH insertion at the terminal methyl group will likely be the main product, particularly in the case of C8 hydrocarbon chain-coated particles shown in Figure 1 as an example of the outcomes by the reactive singlet carbene. Additional control experiments were carried out to compare the results in the presence and absence of the photoactivatable aryldiazirine cross-linker, TDBAOSu. Figure 2a shows the products of the labeling reactions with TDBA-OSu (lane 2) and without the reagent (lane 3) as well as a 10 bp DNA ladder (lane 1). The bright, intrinsic luminescent band in lane 2 (the first band) is from silicon nanoparticle-labeled oligonucleotide. In the absence of the cross-linker, the labeling reaction was not successful (lane 3). Only the gel piece with DNA ladder was stained with ethidium bromide (EtBr). The control experiment clearly shows the importance of the photoactivatable cross-linker. The photoinduced reaction generates amino-reactive N-succinimidyl groups on the outer layer of the particles, which are used to immobilize the amino-modified oligonucleotide in the final step. Note that it is critically important to dissolve TDBA-OSuactivated particles in dry DMSO prior to the immobilization of the nucleotide. Without such step we were unable to produce nucleotide-conjugated nanoparticles. Figure 2b shows polyacrylamide gel electrophoresis of purified silicon nanoparticle-labeled 60mer oligonucleotides (lane 2) and their hybridization products with the complementary strand (lane 3) as well as the 10 bp DNA ladder (lane 1). The band of the labeled nucleotides and the hybridization products can be visualized under a UV lamp. The gel was stained with ethidium bromide (EtBr), and a transilluminator equipped with a UV lamp centered at 302 nm and a band-pass filter for EtBr detection was used to image the gel. This allows visualizing the DNA ladder and more importantly, confirming the existence of the hybridization products. The single-stranded 60mer oligonucleotide as a control runs out the gel in the time setting for the electrophoresis (40 min). Considering the diameter of B-form double-stranded DNA is about 2 nm, it is reasonable to estimate the diameter of Si nanoparticle conjugated to the 60mer oligonucleotide to be smaller than 2 nm. The diameter and mass of Si-nanoparticle resulted in the running location of labeled nucleotide in the polyacrylamide gel. The band of the hybridization products is somewhat spread out. This observation clearly shows that Si nanoparticles are conjugated to the 60mer oligonucleotide. The band at the same running position (lane 3) as that of Si nanoparticles (lane 2) is mostly due to the unhybridized particles. An additional experimental control in the absence of the 5′amino-modified oligonucleotide in the final immobilization step (Figure 1) did not produce a detectable band at this position. The absorption spectrum of the purified nucleotide is shown in Figure 3a. Absorbance above 300 nm is due to Si nanoparticles, while below 290 nm it can be attributed
Bioconjugate Chem., Vol. 15, No. 2, 2004 411
Figure 3. Spectroscopic characterization of purified oligonucleotide-conjugated Si nanoparticles: (a) absorbance; (b) excitation spectrum with luminescence monitored at 400 nm (dash line) and emission spectrum under 300 nm excitation (solid line), 1-hexene used in the first step; (c) excitation spectrum with luminescence monitored at 450 nm (dash line) and emission spectrum under 330 nm excitation (solid line), 1-octene used in the first step (see text).
to both DNA and nanoparticles. Figure 3b shows the excitation and photoluminescence spectra of the labeled nucleotides coated with a layer of 1-hexene. The excitation profile was recorded with photoluminescence fixed at 400 nm, and the emission spectrum was obtained with the excitation wavelength set at 300 nm. The excitation spectrum is peaked at 300 nm and luminescence spectrum contains a peak at 400 nm with a shoulder above 440 nm. When 1-octene is used instead of 1-hexene, both excitation and luminescence peaks shift to 332 and 450 nm, respectively (Figure 3c). Interestingly, the 400 nm emission peak is clearly resolved from the dominant peak at 450 nm. It may be that Si nanoparticles with two discrete sizes are present. According to Belomoin and coworkers (16), the emission at 400 and 450 nm is characteristic to ∼1 nm and ∼1.2 nm Si particles based on high-resolution transmission electron microscopy measurements. It is worthy to mention that the diluted nucleotide-conjugated Si nanoparticles are stable in aqueous solutions at least a week without deterioration in the luminescence. We determined the photoluminescence quantum yield of nucleotide-conjugated Si nanoparticles, using quinine sulfate in 0.1 M HClO4 as a reference standard (17). The quantum yields are 0.08 ( 0.002 (standard deviation) and 0.05 ( 0.003 for 1-octene- and 1-hexene-coated nanoparticles, respectively. Since a homogeneous nanoparticle
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preparation would be expected to have about 20 nm to 30 nm photoluminescence bandwidth (5), our conjugates suggest moderate size inhomogeneity; therefore, size exclusion chromatography separation would be justified in addition to ultracentrifugation prior to 1-alkene monolayer binding. Moreover, the use of a longer carbon chain would be expected to provide a better-ordered selfassembled monolayer (SAM), thus lowering nonradiative recombination channel concentration and increasing photoluminescence quantum yield. CONCLUSIONS
A key requirement for the effective use of Si nanoparticles in biological applications is aqueous solubility. In the present study, we successfully conjugated 1-2 nm diameter silicon nanoparticles to a 5′-amino-modified oligonucleotide through a three-step procedure. Under UV excitation, photoluminescence of the conjugates is dominated by two blue bands (400 and 450 nm maximal). The quantum yield of oligonucleotide-conjugated nanoparticles that were coated with a layer of 1-octene was determined to be 0.08 as measured against quinine sulfate in 0.1 M HClO4 as a reference standard. We have demonstrated the feasibility of the present coupling chemistry to obtain Si nanoparticle-labeled nucleotides. Ways to reduce size inhomogeneity and surface defects of Si nanoparticles in order to achieve narrower and brighter photoluminescence will be addressed in further studies. LITERATURE CITED (1) Ouellete, J. (2003) Quantum Dots for Sale. Ind. Phys. Feb/ March, 14-17. (2) Ding, Z., Quinn, M. B., Haram, A. K., Pell, L. E., Korgel, B. A., and Bard, A. J. (2002) Electrochemistry and Electrogenerated Chemiluminescence from Silicon Nanocrystal Quantum Dots. Science 296, 1293-1297. (3) Belomoin, G., Therrien, J., Smith, A., Rao, S., Twesten, R., Chaieb, S., Nayfeh, M. H., Wagner, L., and Mitas, L. (2002) Observation of a Magic Discrete family of Ultrabright Si Nanoparticles. Appl. Phys. Lett. 80, 841-843. (4) Buriak, J. M. (2002) Organometallic Chemistry on Silicon and Germanium Surfaces. Chem. Rev. 102, 1271-1308.
Wang et al. (5) Alivisatos, A. P. (1996) Perspectives on the Physical Chemistry of Semiconductor Nanoparticles. J. Phys. Chem. B 100, 13226-13239. (6) Wagner, P., Nock, S., Spudich, J. A., Volkmuth, W. D., Chu, S., Cicero, R. L., Wade, C. P., Linford, M. R., and Chidsey, C. E. D. (1997) Bioreactive Self-Assembled Monolayers on Hydrogen-Passivated Si(111) as a New Class of Atomically Flat Substrates for Biological Scanning Probe Microscopy. J. Struct. Biol. 119, 189-201. (7) Cicero, R. L., Wagner, P., Linford, M. R., Hawker, C. J., Waymouth, R. M., and Chidsey, C. E. D. (1997) Functionalization of Alkyl Monolayers on Surfaces with Diverse Amines: Photochemical Chlorosulfonation Followed by Sulfonamide Formation. Polym. Prepr. 38, 904-905. (8) Boukherroub, R., and Wayner, D. D. M. (1999) Controlled Functionalization and Multistep Chemical Manipulation of Covalently Modified Si(111) Surfaces. J. Am. Chem. Soc. 121, 11513-11515. (9) Wojtyk, J. T. C., Morin, K. A., Boukherroub, R., and Wayner, D. D. M. (2002) Modification of Porous Silicon Surfaces with Activated Ester Monolayers. Langmuir 18, 6081-6087. (10) Jung, K. H., Shih, S., Hsieh, T. Y., Kwong, D. L., and Lin, T. L. (1991) Intense Photoluminescence from Laterally Anodized Porous Si. Appl. Phys. Lett. 59, 3264-3266. (11) Linford, M. R., Fenter, P., Eisenberger, P. M., and Chidsey, C. E. D. (1995) Alkyl Monolayers on Silicon Prepared from 1-Alkenes and Hydrogen-Terminated Silicon. J. Am. Chem. Soc. 117, 3145-3155. (12) Boukherroub, R., Morin, S., Bensebaa, F., and Wayner, D. D. M. (1999) New Synthetic Routes to Alkyl Monolayers on the Si(111) Surface. Langmuir 15, 3831-3835. (13) Cicero, R. L., Linford, M. R., and Chidsey, C. E. D. (2000) Photoreactivity of Unsaturated Compounds with HydrogenTerminated Silicon(111). Langmuir 16, 5688-5695. (14) Nassal, M. (1984) 4′-(1-Azi-2,2,2-trifluoroethyl)phenylalanine, a Photolabile Carbene-generating Analogue of Phenylalanine. J. Am. Chem. Soc. 106, 7540-7545. (15) Brunner, J. (1993) New Photolabeling and Cross-linking Methods. Annu. Rev. Biochem. 62, 483-514. (16) Belomoin, G., Therrien, J., and Nayfeh, M. (2000) Oxide and Hydrogen Capped Ultrasmall Blue Luminescent Si Nanoparticles. Appl. Phys. Lett. 77, 779-781. (17) Velapoldi, R. A., and Mielenz, K. D. (1980) Standard Reference Materials: A Fluorescence SRM: Quinine Sulfate Dihydrate (SRM 936). NBS Spec. Publ. 260-264.
BC030047K
