Supporting Information â âLarge Core-Shell PMMA Colloidal Clusters ...
Supporting Information — “Large Core-Shell PMMA Colloidal Clusters: Synthesis, Characterization and Tracking” Mark T. Elsesser,∗ Andrew D. Hollingsworth, Kazem V. Edmond, and David J. Pine
E-mail: [email protected]
Characterization of Fluorescent Monomer The fluorescent dye was chosen such that its excitation maximum was close to that of a green laser line, e.g., argon-ion (λ0 = 514 nm), frequency doubled Nd:YAG (λ0 = 532 nm), or He-Ne laser (λ0 = 543 nm). Rhodamine B has a relatively broad absorption spectrum in the green range and exhibits good photostability. The dye monomer RAS was produced via the nucleophilic addition of RITC with 4-aminostyrene at room temperature. The reaction product, a substituted thiourea, contained one carbon-carbon double bond that could be detected with infrared spectroscopy. Unlike previously reported results, 1,2 we performed the conjugation reaction in DMF, an anhydrous, aprotic solvent that helped to suppress undesirable side reactions. Using FTIR, we determined that the use of alcoholic reaction media results in essentially no coupling between the functionalized dye and the primary aromatic amine. The infrared spectra of RITC before and after exposure to DMF and, separately, to ethanol was measured. As shown in Figure 1A, the broad absorption signal centered at 2074 cm−1 wavenumber indicates that the isothiocyanate group 3 was essentially unaffected by the solvent, while the peak’s absence after ∗ To
whom correspondence should be addressed
1
exposure to ethanol demonstrates the deleterious effect alcohols can have on this chemical group. Isothiocyanates are known to react readily with water and alcohols, significantly reducing the yield of desired product. 4,5 We therefore performed the RAS synthesis in DMF and used FTIR to both verify the presence of –N=C=S in RITC, and to confirm that we had obtained the desired reaction product. The results are shown in Figure 1B. The absence of an isothiocyanate peak in the RAS spectra indicates the formation of a covalent bond between RITC and the aromatic amine. The spectra also displayed a new absorption peak at 1680–1620 cm−1 that we identified as an alkenyl stretch. 3 This feature indicates the presence of a vinyl group necessary for the copolymerization of RAS, MMA and MA during the particle synthesis. We measured the fluorescence excitation and emission spectra of the RAS dye dissolved in methanol using a fluorescence spectrometer (Hitachi model F-2500). The normalized intensity data are presented in Figure 2, along with the emission spectra corresponding to the RAS-labeled PMMA particles. The particles were characterized using the spectral scanning confocal microscope. The RAS dye has peak excitation and emission wavelengths at 547 and 567 nm, respectively. The particle emission peak was shifted to longer wavelengths indicating that the incorporation of RAS into the particles modified the fluorescence slightly. We attempted to synthesize other dye monomers in order to produce a stronger fluorescent signal using the green laser line, and also to increase the reactivity of the unsaturated carboncarbon bond. For example, we coupled allylamine, the simplest unsaturated primary aliphatic amine, with RITC. Upon adding allylamine to a solution of RITC in DMF or ketones, we observed an immediate color change and the gradual attenuation of the fluorescent signal. Allylamine is more basic than 4-aminostyrene and may likely adjust RITC’s environment, causing it to undergo a molecular reconfiguration that results in decreased fluorescence. It was possible to restore the dye’s fluorescence by titrating 1 mM RITC (plus 5X molar excess allylamine) in ketones with oxalic acid. Rhodamine dyes display similar pH-dependent behavior in aqueous environments. 6 While the FTIR results indicated the formation of a covalent bond between the allylamine and RITC, there was no discernible absorbance peak at 1680–1620 cm−1 , suggesting that the vinyl
2
group was not preserved. We also tried to covalently attach the mixed isomer of carboxyrhodamine 6G, succinimidyl ester (CR6G) (λex = 525 nm) to allylamine and also to acrylamide via N-hydroxysuccinimide (NHS) ester chemistry, a technique commonly used to attach fluorescent labels to biomolecules. While we were successful in coupling the dye to the monomer, we determined that the dye-monomer was insoluble in the reaction medium and, therefore, was not suitable for use in our synthesis.
References (1) Bosma, G.; Pathmamanoharan, C.; de Hoog, E.; Kegel, W.; van Blaaderen, A.; Lekkerkerker, H. Journal of Colloid and Interface Science 2002, 245, 292–300 (2) Dullens, R.; Claesson, M.; Derks, D.; van Blaaderen, A.; Kegel, W. Langmuir 2003, 19, 5963– 5966 (3) Coates, J. Interpretation of Infrared Spectra, A Practical Approach. In Encyclopedia of Analytical Chemistry; John Wiley and Sons Ltd.: Chichester, UK, 2000 (4) Vinson, J. Analytical Chemistry 1969, 41, 1661–1662 (5) Siggia, S.; Hanna, J. Analytical Chemistry 1948, 20, 1084 (6) Adamczyk, M.; Grote, J. Bioorganic & Medicinal Chemistry Letters 2003, 13, 2327–2330
3
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80
-N=S=C 60
40
20
0 1000
1200
1400
1600
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2000
2200
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Wavenumber (cm-1)
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B
% Transmittance (a.u.)
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-N=S=C 60
40
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C=C-C 0 1000
1200
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1600
1800
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Wavenumber (cm-1)
Figure 1: (A) Comparison of FTIR spectra for RITC (dotted line) after exposure to ethanol (solid line) and DMF (dashed line). Absence of the ITC peak confirms the deleterious effect ethanol has on RITC. (B) FTIR spectra for RITC (dotted line) and the reaction product (solid line) when it is coupled to 4-aminostyrene.
4
Rel. absorption, emission intensity
Wavelength (nm)
Figure 2: Normalized fluorescence spectra of the pure RAS dye (≈ 0.5 µM in methanol): excitation (dotted line) and emission (solid line). The symbols represent the emission spectra of 1.30 µm diameter, RAS-labeled PMMA particles in cis-decalin.
5
E-mail: [email protected]
Characterization of Fluorescent Monomer The fluorescent dye was chosen such that its excitation maximum was close to that of a green laser line, e.g., argon-ion (λ0 = 514 nm), frequency doubled Nd:YAG (λ0 = 532 nm), or He-Ne laser (λ0 = 543 nm). Rhodamine B has a relatively broad absorption spectrum in the green range and exhibits good photostability. The dye monomer RAS was produced via the nucleophilic addition of RITC with 4-aminostyrene at room temperature. The reaction product, a substituted thiourea, contained one carbon-carbon double bond that could be detected with infrared spectroscopy. Unlike previously reported results, 1,2 we performed the conjugation reaction in DMF, an anhydrous, aprotic solvent that helped to suppress undesirable side reactions. Using FTIR, we determined that the use of alcoholic reaction media results in essentially no coupling between the functionalized dye and the primary aromatic amine. The infrared spectra of RITC before and after exposure to DMF and, separately, to ethanol was measured. As shown in Figure 1A, the broad absorption signal centered at 2074 cm−1 wavenumber indicates that the isothiocyanate group 3 was essentially unaffected by the solvent, while the peak’s absence after ∗ To
whom correspondence should be addressed
1
exposure to ethanol demonstrates the deleterious effect alcohols can have on this chemical group. Isothiocyanates are known to react readily with water and alcohols, significantly reducing the yield of desired product. 4,5 We therefore performed the RAS synthesis in DMF and used FTIR to both verify the presence of –N=C=S in RITC, and to confirm that we had obtained the desired reaction product. The results are shown in Figure 1B. The absence of an isothiocyanate peak in the RAS spectra indicates the formation of a covalent bond between RITC and the aromatic amine. The spectra also displayed a new absorption peak at 1680–1620 cm−1 that we identified as an alkenyl stretch. 3 This feature indicates the presence of a vinyl group necessary for the copolymerization of RAS, MMA and MA during the particle synthesis. We measured the fluorescence excitation and emission spectra of the RAS dye dissolved in methanol using a fluorescence spectrometer (Hitachi model F-2500). The normalized intensity data are presented in Figure 2, along with the emission spectra corresponding to the RAS-labeled PMMA particles. The particles were characterized using the spectral scanning confocal microscope. The RAS dye has peak excitation and emission wavelengths at 547 and 567 nm, respectively. The particle emission peak was shifted to longer wavelengths indicating that the incorporation of RAS into the particles modified the fluorescence slightly. We attempted to synthesize other dye monomers in order to produce a stronger fluorescent signal using the green laser line, and also to increase the reactivity of the unsaturated carboncarbon bond. For example, we coupled allylamine, the simplest unsaturated primary aliphatic amine, with RITC. Upon adding allylamine to a solution of RITC in DMF or ketones, we observed an immediate color change and the gradual attenuation of the fluorescent signal. Allylamine is more basic than 4-aminostyrene and may likely adjust RITC’s environment, causing it to undergo a molecular reconfiguration that results in decreased fluorescence. It was possible to restore the dye’s fluorescence by titrating 1 mM RITC (plus 5X molar excess allylamine) in ketones with oxalic acid. Rhodamine dyes display similar pH-dependent behavior in aqueous environments. 6 While the FTIR results indicated the formation of a covalent bond between the allylamine and RITC, there was no discernible absorbance peak at 1680–1620 cm−1 , suggesting that the vinyl
2
group was not preserved. We also tried to covalently attach the mixed isomer of carboxyrhodamine 6G, succinimidyl ester (CR6G) (λex = 525 nm) to allylamine and also to acrylamide via N-hydroxysuccinimide (NHS) ester chemistry, a technique commonly used to attach fluorescent labels to biomolecules. While we were successful in coupling the dye to the monomer, we determined that the dye-monomer was insoluble in the reaction medium and, therefore, was not suitable for use in our synthesis.
References (1) Bosma, G.; Pathmamanoharan, C.; de Hoog, E.; Kegel, W.; van Blaaderen, A.; Lekkerkerker, H. Journal of Colloid and Interface Science 2002, 245, 292–300 (2) Dullens, R.; Claesson, M.; Derks, D.; van Blaaderen, A.; Kegel, W. Langmuir 2003, 19, 5963– 5966 (3) Coates, J. Interpretation of Infrared Spectra, A Practical Approach. In Encyclopedia of Analytical Chemistry; John Wiley and Sons Ltd.: Chichester, UK, 2000 (4) Vinson, J. Analytical Chemistry 1969, 41, 1661–1662 (5) Siggia, S.; Hanna, J. Analytical Chemistry 1948, 20, 1084 (6) Adamczyk, M.; Grote, J. Bioorganic & Medicinal Chemistry Letters 2003, 13, 2327–2330
3
100
A
% Transmittance (a.u.)
80
-N=S=C 60
40
20
0 1000
1200
1400
1600
1800
2000
2200
2400
Wavenumber (cm-1)
100
B
% Transmittance (a.u.)
80
-N=S=C 60
40
20
C=C-C 0 1000
1200
1400
1600
1800
2000
2200
2400
Wavenumber (cm-1)
Figure 1: (A) Comparison of FTIR spectra for RITC (dotted line) after exposure to ethanol (solid line) and DMF (dashed line). Absence of the ITC peak confirms the deleterious effect ethanol has on RITC. (B) FTIR spectra for RITC (dotted line) and the reaction product (solid line) when it is coupled to 4-aminostyrene.
4
Rel. absorption, emission intensity
Wavelength (nm)
Figure 2: Normalized fluorescence spectra of the pure RAS dye (≈ 0.5 µM in methanol): excitation (dotted line) and emission (solid line). The symbols represent the emission spectra of 1.30 µm diameter, RAS-labeled PMMA particles in cis-decalin.
5
