ГКР СПЕКТРОСКОПИЯ МОЛЕКУЛ БАКТЕРИОРОДОПСИНА, АДСОРБИРОВАННЫХ НА СЕРЕБРЯНЫЕ НАНООСТРОВКОВЫЕ ПЛЕНКИ
SERS OF BACTERIORHODOPSIN WITH OUT-DIFFUSED SILVER NANOISLANDS
1 ФОТОНИКА И ОПТОИНФОРМАТИКА PHOTONICS AND OPTOINFORMATICS
UDC 543.424.2, 538.97
SERS OF BACTERIORHODOPSIN WITH OUT-DIFFUSED SILVER NANOISLANDS
Fabian Heislera,b, Ekaterina S. Piliuginac, Semen D. Chervinskiid,e, Anton K. Samusevb, Andrey A. Lipovskiic,d
a Friedrich-Schiller-Universität Jena, 07743, Jena, Germany, fabian.heisler@uni-jena.de
b ITMO University, 197101, Saint-Petersburg, Russia c Saint-Petersburg State Academic Univesity, 194021, Saint-Petersburg, Russia d Saint-Petersburg State Polytechic University, 195251, Saint-Petersburg, Russia e University of Eastern Finland, 80101, Joensuu, Finland
Abstract. We present the studies on surface-enhanced Raman spectroscopy (SERS) of bacteriorhodopsin in purple membranes using self-assembled silver nanoisland films for Raman signal enhancement. These metal island films were fabricated on soda-lime glass slides subjected to silver-sodium ion exchange in molten Ag0.05Na0.95NO3 at the temperature of 325°C for 20 minutes and subsequent treatment in hydrogen atmosphere at the temperature of 250°C for 10 minutes. The films typically consisted of 20–30 nm closely placed nanoislands. Being tested as SERS substrates for rhodamine 6G the nanoisland films gave the possibility to observe respective characteristic Raman lines from a dried drop of rhodamine 6G dissolved in water in the concentration of 10–6 M. Similarly fabricated substrates were used to obtain SERS spectra of bacteriorhodopsin in purple membranes dispersed in water, and Raman peaks at 1000–1020 cm–1, 1150–1220 cm–1 and 1530– 1570cm–1 were resolved. The substrates made it possible to register characteristic Raman peaks only for an order of magnitude lower concentration of bacteriorhodopsin in contrast to the virgin glass substrate, that is the enhancement of Raman signal was considerably less than for rhodomin 6G. This is supposed to be due to bacteriophodopsin molecules packing in patches, and it prevents bacteriophodopsin in purple membranes from penetration between the nanoislands where the local enhancement of the electric field of exciting light wave is maximal. Keywords: SERS, silver nanoisland films, bacteriorhodopsin, rhodamine 6G Acknowledgements. This work was financially supported by the Ministry of Education and Science of the Russian Federation (Projects 11.G34.31.0020 and 16.1233.2014/K) and the Government of the Russian Federation, Grant 074-U01. For providing the samples of purple membranes and assistance with SERS experiments the authors are grateful to Vitaly Polovinkin, Valentin Borshchevskiy and Valentin Gordeliy (Laboratory for Advanced Studies of Membrane Proteins, Moscow Institute of Physics and Technology, Dolgoprudny, Russia; Institut de Biologie Structurale J.-P. Ebel, Grenoble, France; Institute of Complex Systems (ICS), ICS-6: Structural Biochemistry, Research Centre Juelich, Juelich, Germany; Institute of Crystallography, University of Aachen (Rheinisch-Westfälische Technische Hochschule), Aachen, Germany.
УДК 543.424.2, 538.97 ГКР СПЕКТРОСКОПИЯ МОЛЕКУЛ БАКТЕРИОРОДОПСИНА,
АДСОРБИРОВАННЫХ НА СЕРЕБРЯНЫЕ НАНООСТРОВКОВЫЕ ПЛЕНКИ Ф. Хайслерa, b, Е.С. Пилюгинас, С.Д. Червинскийd,e, А.К. Самусевb, А.А. Липовскийс, d
a Йенский Университет имени Фридриха Шиллера, 07743, Йена, Германия, fabian.heisler@uni-jena.de b Университет ИТМО, 197101, Санкт-Петербург, Россия с Санкт-Петербургский Академический университет – научно-образовательный центр нанотехнологий РАН, 194021, Санкт-Петербург, Россия d Санкт-Петербургский государственный политехнический университет, 195251, Санкт-Петербург, Россия e Университет Восточной Финляндии, 80101, Йоэнсуу, Финляндия
Аннотация. Представлены результаты экспериментов по поверхностно-усиленной рамановской (ГКР) спектроскопии молекул бактериородопсина в пурпурных мембранах, адсорбированных на самоорганизованных серебряных наноостровковых пленках. Островки серебра формировались на поверхности натрий-кальциевого силикатного стекла в результате ионного обмена в расплаве смеси нитратов серебра и натрия, Ag0,05Na0,95NO3, при температуре 325°C в течение 20 минут и последующем отжиге в водородной атмосфере при температуре 250°C в течение 10 минут. Типичные пленки состояли из расположенных близко друг к другу островков размером 20–30 нм. В ходе тестирования в качестве подложек для ГКР спектроскопии островковые пленки позволили наблюдать соответствующие характерные линии комбинационного рассеяния от высушенной капли водного раствора родамина 6G с концентрацией 10–6 моль/л. Аналогичным образом изготовленные подложки были использованы для исследования ГКР спектров бактериородопсина в пурпурных мембранах, диспергированных в воде. Нами были зарегистрированы линии комбинационного рассеяния в областях 1000–1020 см–1, 1150–1220 см–1 и 1530–1570см–1. Подложки со наноостровковыми пленками серебра позволили зарегистрировать характерные линии комбинационного рассеяния бактериородопсина с концентрацией лишь на порядок меньшей, чем в случае стеклянной подложки. Таким образом, усиление сигнала комбинационного рассеяния оказалось существенно меньшим, чем в случае родамина 6G. Предположительно, это связано с тем, что молекулы бактериородопсина упакованы в белковый кристалл, что не позволяет им проникать в промежутки между островками серебра, где локальное усиление электрического поля падающей волны максимально. Ключевые слова: ГКР, серебряные наноостровковые пленки, бактериородопсин, родамин 6G Благодарности. Работа выполнена при финансовой поддержке Министерства образования и науки Российской Федерации (гранты №№ 11.G34.31.0020 и 16.1233.2014/K), а также государственной финансовой поддержке ведущих
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Научно-технический вестник информационных технологий, механики и оптики Scientific and Technical Journal of Information Technologies, Mechanics and Optics
2014, № 5 (93)
Fabian Heisler, Ekaterina S. Piliugina, Semen D. Chervinskii, Anton K. Samusev, Andrey A. Lipovskii
университетов Российской Федерации (субсидия 074-U01). За предоставление образцов пурпурных мембран авторы благодарны Виталию Половинкину, Валентину Борщевскому и Валентину Горделию (Лаборатория «Перспективные исследования мембранных белков» Московского физико-технического института, г. Долгопрудный, Россия; Институт структурной биологии, Гренобль, Франция; Институт сложных систем ICS-6: структурная биология, Исследовательский центр Юлиха, Юлих, Германия; Институт кристаллографии, Университет Аахена (Rheinisch-Westfälische Technische Hochschule), Аахен, Германия).
Introduction
Bacteriorhodopsin (BR) is a light-driven proton pump from Halobacterium salinarum: it captures light energy and uses it to move protons through the membranes out of biological cells [1]. This gives the possibility for the conversion of light into chemical energy. In nature BR exists in so-called purple membranes (PM) which have patches consisting of BR incorporated into lipids with an approximate mass ratio BR: lipids equal to 3:1 [1]. BR is well studied by various biophysical methods [1–4] and belongs to the class of membrane proteins, which are of great interest to the pharmaceutical industry [5, 6]. This paper is devoted to the observation of surface-enhanced Raman scattering (SERS) [7] from BR in PM. The SERS is assisted by self-assembled silver nanoisland films formed on the surface of an ion-exchanged glass.
Silver nanoisland films: fabrication and characterization
The technique used to fabricate the silver nanoisland film on a glass substrate includes two steps (Fig. 1): silver ion exchange into glass followed by thermal treatment of the glass in a hydrogen atmosphere [8]. We used commercially available Menzel microscope slides [9] as glass substrates. These soda-lime glass substrates are placed for 20 minutes in Ag0.05Na0.95NO3 ion exchange batch at the temperature of 325°C (Fig. 1, a). This temperature is sufficient to activate the diffusion of alkali ions out of the glass and their replacement with silver ions from the melted silver-sodium nitrate. This results in the enrichment of the subsurface layer of the glass with silver ions.
a bc
Figure 1: Silver nanoisland film fabrication: ion exchange between glass and molten salt (a); thermal treatment in hydrogen atmosphere (b); Typical AFM picture of the fabricated nanoisland film used for SERS measurements (c)
After the ion exchange the glass substrate is treated for 10 minutes in hydrogen atmosphere at the
temperature of 250°C (Fig. 1, b). Hydrogen diffuses into the subsurface glass layer, reducing silver ions via the
following reaction:
Si-O-Ag+
+
1 2
H2
Si-O-H+
+Ag0
.
Due to the low solubility of neutral silver in the glass the silver atoms coagulate and form nanoparticles. In this
soft mode of hydrogen treatment the nanoparticles are being formed on the glass surface rather than in the bulk
of the glass and the self-arrangement of the formed nanoparticles results in the formation of the silver nanoisland
film. A typical atomic force microscopy (AFM) image of the surface of the glass film with nanoislands is shown
in Fig. 1, c. The size of the nanoislands obtained in the regime described above is about 20–30 nm.
Silver nanoisland films as SERS substrates. For the measurements of the Raman spectra we used the
confocal Raman spectrometer Horiba LabRAM™ HR UV-VIS-NIR and a frequency-doubled continuous-wave
Nd:YAG laser with a wavelength of 532 nm as a light source. The laser beam was focused at the sample surface
with a Mitutoyo M Plan Apo 50x/0.55 objective lens. The beam waist was approximately 1.5 µm.
To perform the Raman experiments, we deposited drops of the analytes dissolved in water in different
concentrations on the surface of the prepared glasses and dried them at room temperature; the diameter of the so
formed spots of the analytes was about 7 mm. All the experiments were carried out with analytes deposited both
on the nanoisland film and virgin glass surface.
To verify the ability of the prepared silver nanoisland films to enhance the Raman signal, we used rhodamine 6G (R6G) dissolved in water in the concentration of 10–6 M. The spectrum shown in Fig. 2 was
Научно-технический вестник информационных технологий, механики и оптики Scientific and Technical Journal of Information Technologies, Mechanics and Optics
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2014, № 5 (93)
SERS OF BACTERIORHODOPSIN WITH OUT-DIFFUSED SILVER NANOISLANDS
acquired from R6G on silver nanoisland film at 30 s exposition and averaged over 20 measurements while using 20 nW exciting laser radiation power at the sample. The observed characteristic peaks at 612 cm–1, 772 cm–1, 1362 cm–1, 1510 cm–1 and 1647 cm–1 are in good agreement with literature data [10]. Similar BR SERS results were reported earlier in our paper related to silver nanoislands formed on the surface of a silver-containing phosphate glass using out-diffusion technique [11]. The reference spectra obtained from R6G deposited on a virgin SERS-inactive glass substrate demonstrated a strong luminescent background which gave no possibility to distinguish any Raman peaks and to estimate the degree of the enhancement provided by the nanoisland film.
Raman intensity, counts
250 225 200 175 150 125
250 500 750 1000 1250 1500 1750 2000 Raman shift, cm–1
Figure 2: SERS spectrum from R6G on nanoisland film acquired at laser power of 20 nW, 30 s exposition.
Raman scattering of bacteriorhodopsin. The main goal of this study was to register SERS spectra of BR in PM assisted by fabricated silver nanoisland film. The experiments have been carried out using BR in concentrations of 0.48 and 0.048 mg/ml, see Fig. 3. We used 200 µW exciting laser power and acquired the spectrum within 50 s averaging it over 2 measurements. For the sample with a concentration of 0.48 mg/ml (Fig. 3, a) the observed Raman peaks at 1000–1020 cm–1, 1150–1220 cm–1 and 1530–1570 cm–1 are in good correspondence to the literature [12]. Raman peaks were registered using both virgin glass and the nanoisland film. Fig. 3, a, clearly shows that the nanoisland film makes it possible to resolve more Raman peaks than the virgin glass.
Raman measurements were performed using BR in concentration of 0.048 mg/ml. We have demonstrated that the spectrum obtained using the non-SERS-active glass substrate contains only background without any distinguishable peaks (Fig. 3, b). At the same time the Raman spectrum of BR acquired using the nanoisland film shows vivid peaks around 1020 cm–1 and 1530 cm–1. It is important to mention that BR molecules are packed in PM patches consisting of approximately 1000 molecules [1]. The thickness of each patch is about 5 nm and corresponds to the size of BR molecules, whereas the lateral size of each patch is of the order of several hundreds of nanometers. So BR molecules in PM are not located between the nanoislands where the local enhancement of the electric field in the light wave is maximal. These results in a weaker Raman signal were compared to the ones obtained for R6G molecules.
Raman intensity, 103 counts
2,8 2,4 1,0
2,0 0,8
1,6 0,6
1,2 0,4 0,8 0,4 0,2
0 500 750 1000 1250 1500 1750 2000 Raman shift, cm–1
0 500 750 1000 1250 1500 1750 2000 Raman shift, cm–1
Glass substrate
Glass substrate
SERS substrate
SERS substrate
ab
Figure 3. Raman spectra of BR in the concentrations of (a) 0.48 and (b) 0.048 mg/ml. The black and red curves
correspond to reference Raman spectra of BR on virgin glass substrate and SERS spectra of BR deposited on
the nanoisland film respectively. The spectra were acquired at laser power of 200 W, with an exposition of 50 s
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Научно-технический вестник информационных технологий, механики и оптики Scientific and Technical Journal of Information Technologies, Mechanics and Optics
2014, № 5 (93)
Fabian Heisler, Ekaterina S. Piliugina, Semen D. Chervinskii, Anton K. Samusev, Andrey A. Lipovskii
Conclusion
Finally, we have fabricated silver nanoisland films by means of hydrogen/metal ions reactive diffusion in glasses followed by the transport of neutral metal to the glass surface. This gave the possibility to obtain uniform nanoislands with the size of the order of tens of nanometers.
We have performed SERS measurements of R6G in order to verify the capability of the fabricated silver
nanoisland films to deliver enhancement for Raman spectroscopy. The BR in PM spot formed after drying a 5 l drop with BR in PM in concentration of 0.48 mg/ml demonstrated selective enhancement of Raman peaks already observed in experiments without SERS substrate. For an order of magnitude with lower concentration of BR in PM the usage of the fabricated silver nanoisland film gives the possibility to resolve characteristic Raman peaks in contrast to the virgin glass substrate.
References
1. Lanyi J.K. Bacteriorhodopsin // Annual Review of Physiology. 2004. V. 66. P. 665–688. 2. Maeda A. Application of FTIR spectroscopy to the structural study on the function of bacteriorhodopsin //
Israel Journal of Chemistry. 1995. V. 35. N 3-4. P. 387–400. 3. Nabiev I.R., Efremov R.G., Chumanov G.D. The chromophore-binding site of bacteriorhodopsin. Reso-
nance Raman and surface-enhanced resonance Raman spectroscopy and quantum chemical study // Journal of Biosciences. 1985. V. 8. N 1-2. P. 363–374. 4. Mathies R.A., Lin S.W., Ames J.B., Pollard W.T. From femtoseconds to biology: mechanism of bacteriorhodopsin’s light-driven proton pump // Annual Review of Biophysics and Biophysical Chemistry. 1991. V. 20. P. 491–518. 5. Krogh A., Larsson B., Heijne v.G., Sonnhammer E.L.L. Predicting transmembrane protein topology with a hidden markov model: application to complete genomes // Journal of Molecular Biology. 2001. V. 305. N 3. P. 567–580. 6. Overington J.P., Al-Lazikani B., Hopkins A.L. How many drug targets are there? // Nature Reviews Drug Discovery. 2006. V. 5. N 12. P. 993–996. 7. Kneipp K., Moskowitz M., Kneipp H. Surface Enhanced Raman Scattering. Physics and Applications. NY: Springer, 2006. 460 p. 8. Chervinskii S., Sevriuk V., Reduto I., Lipovskii A. Formation and 2D-patterning of silver nanoisland film using thermal poling and out-diffusion from glass // Journal of Applied Physics. 2013. V. 114. N 22. Art. 224301. 9. Menzel-Glaser: Microscope slides [Электронный ресурс]. Режим доступа: http://www.menzel.de/Microscope-Slides.687.0.html?&L=1, свободный. Яз. англ. (дата обращения 16.06.2014) 10. Zhou Q., Li Z., Yang Y., Zhang Z. Arrays of aligned, single crystalline silver nanorods for trace amount detection // Journal of Physics D: Applied Physics. 2008. V. 41. N 15. Art. 152007. 11. Zhurikhina V.V., Brunkov P.N., Melehin V.G., Kaplas T., Svirko Yu., Rutckaia V.V., Lipovskii A.A. Selfassembled silver nanoislands formed on glass surface via out-diffusion for multiple usages in SERS applications // Nanoscale Research Letters. 2012. V. 7. N 1. P. 676.
12. Terner J., Campion A., El-Sayed M.A. Time-resolved resonance Raman spectroscopy of bacteriorhodopsin on the millisecond timescale // Proc. National Academy of Sciences of the United States of America. 1977. V. 74. N 12. P. 5212–5216.
Fabian Heisler Ekaterina S. Piliugina Semen D. Chervinskii Anton K. Samusev Andrey A. Lipovskii
– student, Friedrich-Schiller-Universitatሷ Jena, 07743, Jena, Germany;
Trainee researcher, ITMO University, 197101, Saint Petersburg, Russia, fabian.heisler@uni-jena.de
– student, Saint Petersburg State Academic University-Scientific
Educational Center of Nanotechnologies RAS, 194021, Saint Petersburg, Russia, piliugina.ekaterina@gmail.com
– engineer, Saint Petersburg State Polytechic University, 195251, Saint
Petersburg, Russia; early-stage researcher, University of Eastern Finland, 80101, Joensuu, Finland
– PhD, scientific researcher, ITMO University, 197101, Saint Petersburg,
Russia, a.samusev@phoi.ifmo.ru
– D.Sc., Professor, Professor, Saint Petersburg State Academic Univesity,
194021, Saint Petersburg, Russia; Saint Petersburg State Polytechic University, 195251, Saint Petersburg, Russia, lipovsky@spbau.ru
Хайслер Фабиан
– студент, Йенский Университет имени Фридриха Шиллера, 07743, Йена, Германия; стажёр-исследователь, Университет ИТМО,
197101, Санкт-Петербург, Россия; fabian.heisler@uni-jena.de
Научно-технический вестник информационных технологий, механики и оптики Scientific and Technical Journal of Information Technologies, Mechanics and Optics 2014, № 5 (93)
21
SERS OF BACTERIORHODOPSIN WITH OUT-DIFFUSED SILVER NANOISLANDS
Пилюгина Екатерина Сергеевна
– студент, Санкт-Петербургский Академический университет –
научно-образовательный центр нанотехнологий РАН, 194021,
Санкт-Петербург, Россия, piliugina.ekaterina@gmail.com
Червинский Семен Дмитриевич
– инженер, Санкт-Петербургский государственный политехнический
университет, 195251, Санкт-Петербург, Россия; младший
исследователь, Университет Восточной Финляндии, 80101, Йоэнсуу,
Финляндия; semen.chervinsky@gmail.com
Самусев Антон Кириллович
– кандидат физ.-мат. наук, научный сотрудник, Университет ИТМО,
197101, Санкт-Петербург, Россия, a.samusev@phoi.ifmo.ru
Липовский Андрей Александрович – доктор физико-математических наук, профессор, Санкт-
Петербургский Академический университет – научно-
образовательный центр нанотехнологий РАН, 194021, Санкт-
Петербург,
Россия;
профессор,
Санкт-Петербургский
государственный политехнический университет, 195251, Санкт-
Петербург, Россия; lipovsky@spbau.ru
Принято к печати 26.06.14 Accepted 26.06.14
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Научно-технический вестник информационных технологий, механики и оптики Scientific and Technical Journal of Information Technologies, Mechanics and Optics
2014, № 5 (93)
1 ФОТОНИКА И ОПТОИНФОРМАТИКА PHOTONICS AND OPTOINFORMATICS
UDC 543.424.2, 538.97
SERS OF BACTERIORHODOPSIN WITH OUT-DIFFUSED SILVER NANOISLANDS
Fabian Heislera,b, Ekaterina S. Piliuginac, Semen D. Chervinskiid,e, Anton K. Samusevb, Andrey A. Lipovskiic,d
a Friedrich-Schiller-Universität Jena, 07743, Jena, Germany, fabian.heisler@uni-jena.de
b ITMO University, 197101, Saint-Petersburg, Russia c Saint-Petersburg State Academic Univesity, 194021, Saint-Petersburg, Russia d Saint-Petersburg State Polytechic University, 195251, Saint-Petersburg, Russia e University of Eastern Finland, 80101, Joensuu, Finland
Abstract. We present the studies on surface-enhanced Raman spectroscopy (SERS) of bacteriorhodopsin in purple membranes using self-assembled silver nanoisland films for Raman signal enhancement. These metal island films were fabricated on soda-lime glass slides subjected to silver-sodium ion exchange in molten Ag0.05Na0.95NO3 at the temperature of 325°C for 20 minutes and subsequent treatment in hydrogen atmosphere at the temperature of 250°C for 10 minutes. The films typically consisted of 20–30 nm closely placed nanoislands. Being tested as SERS substrates for rhodamine 6G the nanoisland films gave the possibility to observe respective characteristic Raman lines from a dried drop of rhodamine 6G dissolved in water in the concentration of 10–6 M. Similarly fabricated substrates were used to obtain SERS spectra of bacteriorhodopsin in purple membranes dispersed in water, and Raman peaks at 1000–1020 cm–1, 1150–1220 cm–1 and 1530– 1570cm–1 were resolved. The substrates made it possible to register characteristic Raman peaks only for an order of magnitude lower concentration of bacteriorhodopsin in contrast to the virgin glass substrate, that is the enhancement of Raman signal was considerably less than for rhodomin 6G. This is supposed to be due to bacteriophodopsin molecules packing in patches, and it prevents bacteriophodopsin in purple membranes from penetration between the nanoislands where the local enhancement of the electric field of exciting light wave is maximal. Keywords: SERS, silver nanoisland films, bacteriorhodopsin, rhodamine 6G Acknowledgements. This work was financially supported by the Ministry of Education and Science of the Russian Federation (Projects 11.G34.31.0020 and 16.1233.2014/K) and the Government of the Russian Federation, Grant 074-U01. For providing the samples of purple membranes and assistance with SERS experiments the authors are grateful to Vitaly Polovinkin, Valentin Borshchevskiy and Valentin Gordeliy (Laboratory for Advanced Studies of Membrane Proteins, Moscow Institute of Physics and Technology, Dolgoprudny, Russia; Institut de Biologie Structurale J.-P. Ebel, Grenoble, France; Institute of Complex Systems (ICS), ICS-6: Structural Biochemistry, Research Centre Juelich, Juelich, Germany; Institute of Crystallography, University of Aachen (Rheinisch-Westfälische Technische Hochschule), Aachen, Germany.
УДК 543.424.2, 538.97 ГКР СПЕКТРОСКОПИЯ МОЛЕКУЛ БАКТЕРИОРОДОПСИНА,
АДСОРБИРОВАННЫХ НА СЕРЕБРЯНЫЕ НАНООСТРОВКОВЫЕ ПЛЕНКИ Ф. Хайслерa, b, Е.С. Пилюгинас, С.Д. Червинскийd,e, А.К. Самусевb, А.А. Липовскийс, d
a Йенский Университет имени Фридриха Шиллера, 07743, Йена, Германия, fabian.heisler@uni-jena.de b Университет ИТМО, 197101, Санкт-Петербург, Россия с Санкт-Петербургский Академический университет – научно-образовательный центр нанотехнологий РАН, 194021, Санкт-Петербург, Россия d Санкт-Петербургский государственный политехнический университет, 195251, Санкт-Петербург, Россия e Университет Восточной Финляндии, 80101, Йоэнсуу, Финляндия
Аннотация. Представлены результаты экспериментов по поверхностно-усиленной рамановской (ГКР) спектроскопии молекул бактериородопсина в пурпурных мембранах, адсорбированных на самоорганизованных серебряных наноостровковых пленках. Островки серебра формировались на поверхности натрий-кальциевого силикатного стекла в результате ионного обмена в расплаве смеси нитратов серебра и натрия, Ag0,05Na0,95NO3, при температуре 325°C в течение 20 минут и последующем отжиге в водородной атмосфере при температуре 250°C в течение 10 минут. Типичные пленки состояли из расположенных близко друг к другу островков размером 20–30 нм. В ходе тестирования в качестве подложек для ГКР спектроскопии островковые пленки позволили наблюдать соответствующие характерные линии комбинационного рассеяния от высушенной капли водного раствора родамина 6G с концентрацией 10–6 моль/л. Аналогичным образом изготовленные подложки были использованы для исследования ГКР спектров бактериородопсина в пурпурных мембранах, диспергированных в воде. Нами были зарегистрированы линии комбинационного рассеяния в областях 1000–1020 см–1, 1150–1220 см–1 и 1530–1570см–1. Подложки со наноостровковыми пленками серебра позволили зарегистрировать характерные линии комбинационного рассеяния бактериородопсина с концентрацией лишь на порядок меньшей, чем в случае стеклянной подложки. Таким образом, усиление сигнала комбинационного рассеяния оказалось существенно меньшим, чем в случае родамина 6G. Предположительно, это связано с тем, что молекулы бактериородопсина упакованы в белковый кристалл, что не позволяет им проникать в промежутки между островками серебра, где локальное усиление электрического поля падающей волны максимально. Ключевые слова: ГКР, серебряные наноостровковые пленки, бактериородопсин, родамин 6G Благодарности. Работа выполнена при финансовой поддержке Министерства образования и науки Российской Федерации (гранты №№ 11.G34.31.0020 и 16.1233.2014/K), а также государственной финансовой поддержке ведущих
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Научно-технический вестник информационных технологий, механики и оптики Scientific and Technical Journal of Information Technologies, Mechanics and Optics
2014, № 5 (93)
Fabian Heisler, Ekaterina S. Piliugina, Semen D. Chervinskii, Anton K. Samusev, Andrey A. Lipovskii
университетов Российской Федерации (субсидия 074-U01). За предоставление образцов пурпурных мембран авторы благодарны Виталию Половинкину, Валентину Борщевскому и Валентину Горделию (Лаборатория «Перспективные исследования мембранных белков» Московского физико-технического института, г. Долгопрудный, Россия; Институт структурной биологии, Гренобль, Франция; Институт сложных систем ICS-6: структурная биология, Исследовательский центр Юлиха, Юлих, Германия; Институт кристаллографии, Университет Аахена (Rheinisch-Westfälische Technische Hochschule), Аахен, Германия).
Introduction
Bacteriorhodopsin (BR) is a light-driven proton pump from Halobacterium salinarum: it captures light energy and uses it to move protons through the membranes out of biological cells [1]. This gives the possibility for the conversion of light into chemical energy. In nature BR exists in so-called purple membranes (PM) which have patches consisting of BR incorporated into lipids with an approximate mass ratio BR: lipids equal to 3:1 [1]. BR is well studied by various biophysical methods [1–4] and belongs to the class of membrane proteins, which are of great interest to the pharmaceutical industry [5, 6]. This paper is devoted to the observation of surface-enhanced Raman scattering (SERS) [7] from BR in PM. The SERS is assisted by self-assembled silver nanoisland films formed on the surface of an ion-exchanged glass.
Silver nanoisland films: fabrication and characterization
The technique used to fabricate the silver nanoisland film on a glass substrate includes two steps (Fig. 1): silver ion exchange into glass followed by thermal treatment of the glass in a hydrogen atmosphere [8]. We used commercially available Menzel microscope slides [9] as glass substrates. These soda-lime glass substrates are placed for 20 minutes in Ag0.05Na0.95NO3 ion exchange batch at the temperature of 325°C (Fig. 1, a). This temperature is sufficient to activate the diffusion of alkali ions out of the glass and their replacement with silver ions from the melted silver-sodium nitrate. This results in the enrichment of the subsurface layer of the glass with silver ions.
a bc
Figure 1: Silver nanoisland film fabrication: ion exchange between glass and molten salt (a); thermal treatment in hydrogen atmosphere (b); Typical AFM picture of the fabricated nanoisland film used for SERS measurements (c)
After the ion exchange the glass substrate is treated for 10 minutes in hydrogen atmosphere at the
temperature of 250°C (Fig. 1, b). Hydrogen diffuses into the subsurface glass layer, reducing silver ions via the
following reaction:
Si-O-Ag+
+
1 2
H2
Si-O-H+
+Ag0
.
Due to the low solubility of neutral silver in the glass the silver atoms coagulate and form nanoparticles. In this
soft mode of hydrogen treatment the nanoparticles are being formed on the glass surface rather than in the bulk
of the glass and the self-arrangement of the formed nanoparticles results in the formation of the silver nanoisland
film. A typical atomic force microscopy (AFM) image of the surface of the glass film with nanoislands is shown
in Fig. 1, c. The size of the nanoislands obtained in the regime described above is about 20–30 nm.
Silver nanoisland films as SERS substrates. For the measurements of the Raman spectra we used the
confocal Raman spectrometer Horiba LabRAM™ HR UV-VIS-NIR and a frequency-doubled continuous-wave
Nd:YAG laser with a wavelength of 532 nm as a light source. The laser beam was focused at the sample surface
with a Mitutoyo M Plan Apo 50x/0.55 objective lens. The beam waist was approximately 1.5 µm.
To perform the Raman experiments, we deposited drops of the analytes dissolved in water in different
concentrations on the surface of the prepared glasses and dried them at room temperature; the diameter of the so
formed spots of the analytes was about 7 mm. All the experiments were carried out with analytes deposited both
on the nanoisland film and virgin glass surface.
To verify the ability of the prepared silver nanoisland films to enhance the Raman signal, we used rhodamine 6G (R6G) dissolved in water in the concentration of 10–6 M. The spectrum shown in Fig. 2 was
Научно-технический вестник информационных технологий, механики и оптики Scientific and Technical Journal of Information Technologies, Mechanics and Optics
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2014, № 5 (93)
SERS OF BACTERIORHODOPSIN WITH OUT-DIFFUSED SILVER NANOISLANDS
acquired from R6G on silver nanoisland film at 30 s exposition and averaged over 20 measurements while using 20 nW exciting laser radiation power at the sample. The observed characteristic peaks at 612 cm–1, 772 cm–1, 1362 cm–1, 1510 cm–1 and 1647 cm–1 are in good agreement with literature data [10]. Similar BR SERS results were reported earlier in our paper related to silver nanoislands formed on the surface of a silver-containing phosphate glass using out-diffusion technique [11]. The reference spectra obtained from R6G deposited on a virgin SERS-inactive glass substrate demonstrated a strong luminescent background which gave no possibility to distinguish any Raman peaks and to estimate the degree of the enhancement provided by the nanoisland film.
Raman intensity, counts
250 225 200 175 150 125
250 500 750 1000 1250 1500 1750 2000 Raman shift, cm–1
Figure 2: SERS spectrum from R6G on nanoisland film acquired at laser power of 20 nW, 30 s exposition.
Raman scattering of bacteriorhodopsin. The main goal of this study was to register SERS spectra of BR in PM assisted by fabricated silver nanoisland film. The experiments have been carried out using BR in concentrations of 0.48 and 0.048 mg/ml, see Fig. 3. We used 200 µW exciting laser power and acquired the spectrum within 50 s averaging it over 2 measurements. For the sample with a concentration of 0.48 mg/ml (Fig. 3, a) the observed Raman peaks at 1000–1020 cm–1, 1150–1220 cm–1 and 1530–1570 cm–1 are in good correspondence to the literature [12]. Raman peaks were registered using both virgin glass and the nanoisland film. Fig. 3, a, clearly shows that the nanoisland film makes it possible to resolve more Raman peaks than the virgin glass.
Raman measurements were performed using BR in concentration of 0.048 mg/ml. We have demonstrated that the spectrum obtained using the non-SERS-active glass substrate contains only background without any distinguishable peaks (Fig. 3, b). At the same time the Raman spectrum of BR acquired using the nanoisland film shows vivid peaks around 1020 cm–1 and 1530 cm–1. It is important to mention that BR molecules are packed in PM patches consisting of approximately 1000 molecules [1]. The thickness of each patch is about 5 nm and corresponds to the size of BR molecules, whereas the lateral size of each patch is of the order of several hundreds of nanometers. So BR molecules in PM are not located between the nanoislands where the local enhancement of the electric field in the light wave is maximal. These results in a weaker Raman signal were compared to the ones obtained for R6G molecules.
Raman intensity, 103 counts
2,8 2,4 1,0
2,0 0,8
1,6 0,6
1,2 0,4 0,8 0,4 0,2
0 500 750 1000 1250 1500 1750 2000 Raman shift, cm–1
0 500 750 1000 1250 1500 1750 2000 Raman shift, cm–1
Glass substrate
Glass substrate
SERS substrate
SERS substrate
ab
Figure 3. Raman spectra of BR in the concentrations of (a) 0.48 and (b) 0.048 mg/ml. The black and red curves
correspond to reference Raman spectra of BR on virgin glass substrate and SERS spectra of BR deposited on
the nanoisland film respectively. The spectra were acquired at laser power of 200 W, with an exposition of 50 s
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Научно-технический вестник информационных технологий, механики и оптики Scientific and Technical Journal of Information Technologies, Mechanics and Optics
2014, № 5 (93)
Fabian Heisler, Ekaterina S. Piliugina, Semen D. Chervinskii, Anton K. Samusev, Andrey A. Lipovskii
Conclusion
Finally, we have fabricated silver nanoisland films by means of hydrogen/metal ions reactive diffusion in glasses followed by the transport of neutral metal to the glass surface. This gave the possibility to obtain uniform nanoislands with the size of the order of tens of nanometers.
We have performed SERS measurements of R6G in order to verify the capability of the fabricated silver
nanoisland films to deliver enhancement for Raman spectroscopy. The BR in PM spot formed after drying a 5 l drop with BR in PM in concentration of 0.48 mg/ml demonstrated selective enhancement of Raman peaks already observed in experiments without SERS substrate. For an order of magnitude with lower concentration of BR in PM the usage of the fabricated silver nanoisland film gives the possibility to resolve characteristic Raman peaks in contrast to the virgin glass substrate.
References
1. Lanyi J.K. Bacteriorhodopsin // Annual Review of Physiology. 2004. V. 66. P. 665–688. 2. Maeda A. Application of FTIR spectroscopy to the structural study on the function of bacteriorhodopsin //
Israel Journal of Chemistry. 1995. V. 35. N 3-4. P. 387–400. 3. Nabiev I.R., Efremov R.G., Chumanov G.D. The chromophore-binding site of bacteriorhodopsin. Reso-
nance Raman and surface-enhanced resonance Raman spectroscopy and quantum chemical study // Journal of Biosciences. 1985. V. 8. N 1-2. P. 363–374. 4. Mathies R.A., Lin S.W., Ames J.B., Pollard W.T. From femtoseconds to biology: mechanism of bacteriorhodopsin’s light-driven proton pump // Annual Review of Biophysics and Biophysical Chemistry. 1991. V. 20. P. 491–518. 5. Krogh A., Larsson B., Heijne v.G., Sonnhammer E.L.L. Predicting transmembrane protein topology with a hidden markov model: application to complete genomes // Journal of Molecular Biology. 2001. V. 305. N 3. P. 567–580. 6. Overington J.P., Al-Lazikani B., Hopkins A.L. How many drug targets are there? // Nature Reviews Drug Discovery. 2006. V. 5. N 12. P. 993–996. 7. Kneipp K., Moskowitz M., Kneipp H. Surface Enhanced Raman Scattering. Physics and Applications. NY: Springer, 2006. 460 p. 8. Chervinskii S., Sevriuk V., Reduto I., Lipovskii A. Formation and 2D-patterning of silver nanoisland film using thermal poling and out-diffusion from glass // Journal of Applied Physics. 2013. V. 114. N 22. Art. 224301. 9. Menzel-Glaser: Microscope slides [Электронный ресурс]. Режим доступа: http://www.menzel.de/Microscope-Slides.687.0.html?&L=1, свободный. Яз. англ. (дата обращения 16.06.2014) 10. Zhou Q., Li Z., Yang Y., Zhang Z. Arrays of aligned, single crystalline silver nanorods for trace amount detection // Journal of Physics D: Applied Physics. 2008. V. 41. N 15. Art. 152007. 11. Zhurikhina V.V., Brunkov P.N., Melehin V.G., Kaplas T., Svirko Yu., Rutckaia V.V., Lipovskii A.A. Selfassembled silver nanoislands formed on glass surface via out-diffusion for multiple usages in SERS applications // Nanoscale Research Letters. 2012. V. 7. N 1. P. 676.
12. Terner J., Campion A., El-Sayed M.A. Time-resolved resonance Raman spectroscopy of bacteriorhodopsin on the millisecond timescale // Proc. National Academy of Sciences of the United States of America. 1977. V. 74. N 12. P. 5212–5216.
Fabian Heisler Ekaterina S. Piliugina Semen D. Chervinskii Anton K. Samusev Andrey A. Lipovskii
– student, Friedrich-Schiller-Universitatሷ Jena, 07743, Jena, Germany;
Trainee researcher, ITMO University, 197101, Saint Petersburg, Russia, fabian.heisler@uni-jena.de
– student, Saint Petersburg State Academic University-Scientific
Educational Center of Nanotechnologies RAS, 194021, Saint Petersburg, Russia, piliugina.ekaterina@gmail.com
– engineer, Saint Petersburg State Polytechic University, 195251, Saint
Petersburg, Russia; early-stage researcher, University of Eastern Finland, 80101, Joensuu, Finland
– PhD, scientific researcher, ITMO University, 197101, Saint Petersburg,
Russia, a.samusev@phoi.ifmo.ru
– D.Sc., Professor, Professor, Saint Petersburg State Academic Univesity,
194021, Saint Petersburg, Russia; Saint Petersburg State Polytechic University, 195251, Saint Petersburg, Russia, lipovsky@spbau.ru
Хайслер Фабиан
– студент, Йенский Университет имени Фридриха Шиллера, 07743, Йена, Германия; стажёр-исследователь, Университет ИТМО,
197101, Санкт-Петербург, Россия; fabian.heisler@uni-jena.de
Научно-технический вестник информационных технологий, механики и оптики Scientific and Technical Journal of Information Technologies, Mechanics and Optics 2014, № 5 (93)
21
SERS OF BACTERIORHODOPSIN WITH OUT-DIFFUSED SILVER NANOISLANDS
Пилюгина Екатерина Сергеевна
– студент, Санкт-Петербургский Академический университет –
научно-образовательный центр нанотехнологий РАН, 194021,
Санкт-Петербург, Россия, piliugina.ekaterina@gmail.com
Червинский Семен Дмитриевич
– инженер, Санкт-Петербургский государственный политехнический
университет, 195251, Санкт-Петербург, Россия; младший
исследователь, Университет Восточной Финляндии, 80101, Йоэнсуу,
Финляндия; semen.chervinsky@gmail.com
Самусев Антон Кириллович
– кандидат физ.-мат. наук, научный сотрудник, Университет ИТМО,
197101, Санкт-Петербург, Россия, a.samusev@phoi.ifmo.ru
Липовский Андрей Александрович – доктор физико-математических наук, профессор, Санкт-
Петербургский Академический университет – научно-
образовательный центр нанотехнологий РАН, 194021, Санкт-
Петербург,
Россия;
профессор,
Санкт-Петербургский
государственный политехнический университет, 195251, Санкт-
Петербург, Россия; lipovsky@spbau.ru
Принято к печати 26.06.14 Accepted 26.06.14
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Научно-технический вестник информационных технологий, механики и оптики Scientific and Technical Journal of Information Technologies, Mechanics and Optics
2014, № 5 (93)