АКТУАЛЬНЫЕ НАПРАВЛЕНИЯ СОВРЕМЕННОЙ НАУКИ: УГЛЕРОДНЫЕ НАНОТРУБКИ КАК ОСНОВА ДЛЯ СОЗДАНИЯ БИОСЕНСОРОВ ОТДЕЛ МОЛЕКУЛЯРНОЙ БИОФИЗИКИ Физико-технический институт низких температур НАН Украины им. Б. И. Веркина, Харьков. wrapped-DNA SWNT Д.ф.-м.н. Виктор Алексеевич Карачевцев

СОДЕРЖАНИЕ СОДЕРЖАНИЕ Введение: углеродные нанотрубки – материал 21 века Электронная структура углеродных одностенных нанотрубок (УОНТ). Спектроскопия ОУНТ: Рамановская спектроскопия, спектроскопия поглощения, люминесценция. Экспериментальное определение хиральности нанотрубок. Достоинства УОНТ при создании биосенсоров. Нековалентная фунционализация ОУНТ с помощью органических молекул Индивидуальные нанотрубки в водной суспензии – способ их приготовления: а) за счет образования мицелл б) благодаря воднорастворимым полимерам. Биосовместимость УОНТ посредством ДНК (однонитевой и двунитевой полимер). Наногибрид УОНТ:ДНК:фермент в качестве элемента люминесцентного сенсора для определения уровня глюкозы Выводы

Nature, 2006

Carbon single-walled nanotube Schematic representation of a 2D graphite layer with the lattice vectors a 1 and a 2 and the roll-up vector C h _n a1 _m a2. Achiral tubes exhibit roll-up vectors derived from (n,0) (zigzag) or (n,n) (armchair). The translation vector T is parallel to the tube axis and defines the 1D unit cell. The rectangle represents an unrolled unit cell, defined by T and Ch. In this example, (n,m) =(4,2) Chiral vector: C h =na 1 + ma 2 =(n,m) Nanotube diameters: d=3 1/2 a c-c (m 2 +mn+n 2 ) 1/2 /π Chiral angle: θ=tan /2 m(m+2n) metallic conduction in (n,m) carbon nanotube is achieved when 2n+m = 3q where q is an integer. 1/3 of the nanotubes are metallic and 2/3 are semiconducting

SWNTs band structure

UV-visable-NIR light absorption of SWNTs E 11 semiconducting metallic E 22 E 11 Light absorption of SWNT in NIR region is caused by electronic transitions between pairs of van Hove singularities. So, light absorption in the region nm is assigned to electronic transitions E 11 S in the semiconducting nanotube (band gap transition), absorption at the energy nm results from the E 22 S transitions of these types of SWNTs and absorption at nm is assigned to the E 11 m transition in the metallic SWNTs.

Достоинства углеродных нанотрубок при построении сенсоров: а) химическая стойкость к воздействию различных агрессивных сред, а также в слабом влиянии на среду, в которой происходит диагностика, что особенно важно для биологических систем; б) термическая стойкость нанотрубок, которая может быть использована при регенерации сенсора; с) металлическая или полупроводниковая проводимость нанотрубок, которая значительно облегчает сам процесс создания сенсора на основе нанотрубок, поскольку она сама уже может являться элементом электронного устройства; д) размеры сенсора: одностенная нанотрубка имеет диаметр 0,5-2 нм и длину до нескольких мкм; е) разветленная поверхность (высокий показатель площади поверхности на единицу веса) - одностенная нанотрубка представляет собой углеродный монослой завернутый в цилиндр; ж) сверхпрочность.

Network Field-Effect Transistors Schematic illustration of layouts for bottom-gate (a) and top-gate (b) SWNT TFT devices

flexible electronic

We demonstrate a versatile class of nanoscale chemical sensors based on single-stranded DNA (ssDNA)for chemical recognition and single- walled carbon nanotube field effect transistors (SWNT FETs) for electronic read-out. SWNT FETs with a nanoscale coating of ssDNA respond to vapors that cause no detectable conductivity change in bare devices. Sensor responses differ in sign and magnitude for different gases and can be tuned by choice of the ssDNA base sequence. Sensors respond and recover rapidly (seconds), and the sensor surface is self-regenerating. Preliminary results of all-atom molecular dynamics simulations agree with experiment. A. T. C Johnson et al, phys. stat. sol. (b) 243, No. 13, 3252–3256 (2006) DNA-decorated carbon nanotubes for chemical sensing

Enzyme-Coated Carbon Nanotubes as Single- Molecule Biosensors K.Besteman, J-O Lee, F.G. M. Wiertz, H.A. Heering, and C. Dekker Nano Lett.,, 2003

Color Detection Using Chromophore- Nanotube Hybrid Devices X.Zhou et al. Nano Lett., 2009, 9 (3), pp 1028 – 1033 Sandia National Laboratories, Livermore, A nanoscale color detector based on a single- walled carbon nanotube functionalized with azobenzene chromophores, where the chromophores serve as photoabsorbers and the nanotube as the electronic read-out. By synthesizing chromophores with specific absorption windows in the visible spectrum and anchoring them to the nanotube surface, it was demonstrated the controlled detection of visible light of low intensity in narrow ranges of wavelengths. These measurements are suggested that upon photoabsorption, the chromophores isomerize from the ground state trans configuration to the excited state cis configuration, accompanied by a large change in dipole moment, changing the electrostatic environment of the nanotube.

Dependence of energy separation (between van Hoff singularities) from SWNTs diameters. For the each laser excitation energy the two lowest energy transitions are correspond to semiconducting nanotubes, the next higher energy transition is for metallic NT and so on. So, in Raman measurements the resonance condition will be occurred for nanotubes certain diameters.

Спектры комбинационного рассеяния одностенных углеродных нанотрубок. He-Ne лазер з λexc=632.8 нм (1,96 еВ) A 1g E 1g

SWNTs aggregate in bundles as a result of substantial van der Waals attractions between tubes Unfortunately, nanotubes are poorly soluble in the most of organic solvents and are insoluble in water. This is because NTs aggregate in bundles as a result of substantial van der Waals attractions between tubes. Solution nanotubes in water is realized if a surfactant or polymer is added into aqueous solution. Sonication of an aqueous SWNT solution containing a polymer can lead to debundled isolated SWNTs. Polymer wrapped around tubes can preclude its aggregation in bundles. R Smalley et al, J. Phys. Chem bar 10nm bar 500nm

Биосенсоры на основе УОНТ Биосовместимость нанотрубок Присоединение к нанотрубке распознающей биомолекулы Индивидуальные нанотрубки Биосовместимость нанотрубок нанотрубок Присоединение к нанотрубке распознающей биомолекулы Нанотрубки в жгутах Расщепление жгутов, получение индивид. нанотрубок и удержание их от слипания Биосовместимостьнанотрубок Присоединение к нанотрубке распознающей биомолекулы Использование жгутов из нанотрубок Улучшение характеристик созданного ранее устройств Создание функционального устройства

Изучены особенности адсорбции органических молекул на поверхность одностенной углеродной нанотрубки M olecular Phys. (2003) Изучено взаимодействие между молекулами пирен, нафталин и углеродной нанотрубкой в пленках. Определены структуры этих гибридов и рассчитана энергия взаимодействия. Показано, что для плоских π- сопряженных молекул величина их взаимодействия с нанотрубкой в значительной степени определяется соотношением площади молекулы и диаметром нанотрубки Ультразву к Расщепление жгутов нанотрубок

Aqueous solution of SWNT with surfactant ultrasonication micelle

Biofunctionalization of carbon nanotube by DNA Single-stranded biopolymer DNA wraping, R. Smalley et al. CPL, 2001 Zheng et al. Nature, 2003

Computer sumulation of SWNT:ssDNA hybrid Hydrophobic Hydrophilic The single-stranded DNA chain forms a helical wrapping around the nanotube at which the nitrogen bases are extended from the backbone and stack onto the nanotube while the hydrophilic sugar-phosphate backbone turns to water.

Структура однонитевой ДНК

Computer sumulation of SWNT:polyA hybrid Molecular dynamic simulation demonstrates that more than half of adenines is out of stacking with the tube surface and some of them can form self- stacking structures From 30-nucleotides: 12 adenines were in - stacking with the nanotube surface, 7 bases formed self- stacking (two dimers and one trimer) and other adenines were out of stacking with SWNT surface For For simulation the program package NAMD was employed with Charmm27 force field parameter set. During simulation a box was applied, being of 55×55×135 Å dimensions in which poly(rA)-wrapped SWNT was embedded in water (more than 9000 H 2 O molecules). For modeling, the periodical boundary conditions were provided. SWNT was selected as a zigzag (16.0) carbon nanotube. Its length and diameter were 11.0 nm and 1.27 nm, respectively. The 30-nucleotides length of poly(rA) was selected for our simulation. V.A. Karachevtsev, et al ( Chemical Physics &Physical Chemistry, 9, 2010 (2008 )

Calculation stacking energy interaction A-A, A-SWNT Ab initio calculated stacking energy of adenines dimer is not more than -37 kJ/mol. J. Sponer, J. Leszczynski, P. Hobza, Biopolymers 2002, 61, 3–31. The interaction energy in the complex formed by adenine with a zigzag(10,0) SWCNT is -57 kJ/mol (calculated at the MP2/ G(2d,p) level of theory). Stepanian et al J. Phys. Chem. A, 2009, 113 (15), 3621

Многослойное навивание ДНК вокруг нанотрубки Показано, что сравнительно длинная однонитевая ДНК может навиваться в несколько слоев вокруг нанотрубки по типу веретена. Предложена физическая модель такого гибрида, выполнены оценки энергии взаимодействия между отдельными фрагментами полимера. Karachevtsev et al. J. Nanoscience and Nanotechnology (2008) Компютерное моделирование показало, что такие гибриды стабильны и удержание верхних слоев ДНК над нижними осуществляется за счет образования водородных связей между азотистыми основаниями из разных нитей и фосфатным остовом. Компютерное моделирование показало, что такие гибриды стабильны и удержание верхних слоев ДНК над нижними осуществляется за счет образования водородных связей между азотистыми основаниями из разных нитей и фосфатным остовом.

Структура ДНК

Double-stranded DNA:SWNT hybrid In this model the ds-DNA-SWNT hybrid will be weak hydrophilic ds- DNA is a quite rigid polymer

Biofunctionalization of carbon nanotube by double-stranded DNA Will double-stranded biopolymer form a hybrid with nanotube by wrapping and form stable aqueous suspension? In our model of the formation of fds-DNA:SWNT hybrid we assume that at the beginning the denatured regions of the fragmented polymer wrap around the nanotube under sonication. The ss-polymer parts are highly flexible can wrap around nanotube surface.These polymer tails serve as anchor holding the ds-parts of DNA close to the nanotube. The ds-regions coming into the interaction with the SWNT surface further stabilize the complex holding the DNA nearest to the nanotube surface.In our model of the formation of fds-DNA:SWNT hybrid we assume that at the beginning the denatured regions of the fragmented polymer wrap around the nanotube under sonication. The ss-polymer parts are highly flexible can wrap around nanotube surface. These polymer tails serve as anchor holding the ds-parts of DNA close to the nanotube. The ds-regions coming into the interaction with the SWNT surface further stabilize the complex holding the DNA nearest to the nanotube surface. G. Gladchenko, M. Karachevtsev et. al. (2006) 104, 3193 Mol. Phys.

Carbon Nanotube DNA Sensor Nano Lett., Vol. 6, No. 8, 2006 Detection of DNA Hybridization Using the Near- Infrared Band-Gap Fluorescence of Single- Walled Carbon Nanotubes Nano Lett. 2006, 6, 371–375

UV-spectroscopy DNA: melting curve The conformational state of the polymer adsorbed on carbon nanotube surface can be also controlled by UV absorption spectroscopy.

Heat cycling of the SWNT:DNA aqueous solution The SWNT:DNA aqueous solution heat cycling up to 90 0 C does not lead to polymer release from tube surface

Гібридизація двох полінуклеотидів на поверхні вуглецевих нанотрубок є дефектною Адсорбція полімеру на поверхні нанотрубки перешкоджає утворенню комплементарної пари з водневими звязками. Цей результат слід враховувати при створені геносенсора на НТ. Доведено, що гібридизація ДНК на поверхні одностінної вуглецевої нанотрубки є дефектною. Це пояснюється більшою енергією адсорбції полімеру на поверхні нанотрубки у порівнянні з енергією утворення водневих звязків між азотними основами двох полімерів. Енергія утворення пари аденін (rА)-урацил (rU) у воді ~ 19, а енергія взаємодії поли(rА)-НТ ~ 33 кДж/моль. В.О. Карачевцев, Г.О. Гладченко, М.В. Карачевцев, В.С. Леонтьєв, В.О. Валєєв, О.C. Литвин ( Chemical Physics &Physical Chemistry, 9, 2010 (2008))

Polymer hybridization occurs with defects poly(rU) hybridization with poly(rA) NT occurs with defects along the whole polymer length because of π-π stacking between nitrogen bases and the nanotube surface, which hinders the usual hybridization process Karachevtsev et al. ChemPhysChem 2008, 9, 2010 – 2018

(9,2) (9,4) (6,5) (7,5) (10,2) (9,4) (9,2) (11,3) (14,0) (11,4) SWNT emission Emission bands are correspond to individual semiconducting nanotubes of different diameters Luminescence was excited by DPSS green laser at λex=532 nm.. For the semiconducting nanotubes their bandgap energy Eg is equal to the energy difference E11(dt) between the two van Hove singularities, the first subband edges of the valence and conduction bands. Quantum yield is 10 -3

Glucose sensor based on the following biochemical reactions: Detection of the influence of the electron (product of reaction) on the nanotubes luminescence intensity is the main idea of the sensor.

Near-infrared optical sensors based on single- walled carbon nanotubes M.S. Strano et al. Nature materials, 4 (2005), 86. The enzyme was immobilized directly on the nanotube.

The problem of enzyme immobilization on nanotube and of keeping its native activity It was shown recently that the activity of two enzymes: R-chymotrypsin and soybean peroxidase decreased significantly after their adsorption onto the surface of single-walled carbon nanotubes. Soybean peroxidase retained up to 30% of its native activity upon adsorption while the adsorbed R- chymotrypsin retained only 1% of its native activity. [S.S. Karajanagi, A.A. Vertegel, R.S. Kane, and J.S. Dordick. Langmuir; 20(26); 2004, p.11594]. DNA polymerase decreases activity after adsorption onto carbon nanotubes surface (Changqing Yi et al. Nanotechnology 18 (2007) ). Thus, the problem of enzyme immobilization on the nanotube and of keeping its native activity is the key feature Thus, the problem of enzyme immobilization on the nanotube and of keeping its native activity is the key feature that should be investigated and understood before the work on the development of biosensors is carried out. Our approach: indirect immobilization of enzyme (GOX) on SWNTs with preservation of its activity which implies using the polymer as an interlayer between them. The DNA wrapped around the nanotube is an interlayer/interface between the enzyme and SWNT surface. Karachevtsev et. al. CPL 435, 104 (2007).

AFM image of SWNT:DNA:GOX bionanohybrids The wire-like structure is associated with individual nanotubes around which single- stranded DNA was wrapped, and the distinct globular structures on the SWNT represent GOX molecules. AFM image cross-section analysis along the nanotube or enzyme revealed that the height of globular structures above SWNTs ranges from 4.7 to 5.4 nm and also indicates that enzymes are adsorbed on DNA wrapped around the tube.

BIONANIHYBRID SWNT:DNA:GOX – molecular dynamic modeling NAMD program Box: 94.77×80.66×95.54 Å H 2 O molecules For modeling the periodical boundary conditions were provided. In all the cases SWNT was selected as a zigzag (10.0) carbon nanotube. Its length and diameter were 5.1 and nm, respectively. Polynucleotide used in the simulations were 15- nucleotides polyC. Karachevtsev et. al. (2007) SPIE Proceed.

BIONANIHYBRID SWNT:DNA:GOX – molecular dynamic modeling Analysis of GOX and DNA interaction showed the availability of some molecular contacts, including H-bonds between some groups of amino acids that form the enzyme shell and DNA components. For example, we observed the formation of H-bond between oxygen of DNA phosphate group and OH group of tyrosine (left insert), between NH2 group of cytosine and CO group of glutamine (right insert) or asparagines.

High energy part of NIR luminescence spectra of carbon nanotubes in aqueous solution with DNA and DNA:GOX In the nanotube emission spectrum after GOX adding a small spectral blue shift (not above 28 cm -1 ) was observed after GOX immobilization close to the tube. A blue shift in the spectrum of nanotube emission is observed after expanding the tube surface covering with the polymer due to decreasing an area of the nanotube hydrophobic contact surface with water Luminescence was excited by DPSS green laser at λex=532 nm. Luminescence was excited by DPSS green laser at λex=532 nm.

Influence of glucose on intensity of nanotubes emission Figure demonstrates the glucose injections influence on the nanotube luminescence intensity. We controlled the integral intensity of the most intensive band in the emission spectrum at cm -1, corresponding to the (6,5) nanotube index chirality. Two first glucose injections (1 mM each portion) decrease significantly the luminescence intensity but after each adding the percent of diminution of the intensity. Karachevtsev et. al. (2007) SPIE Proceed.

Conclusions We have demonstrated a new approach for the indirect immobilization of the enzyme on SWNTs with keeping the enzyme activity. DNA wrapped around the nanotube is acting as interlayer/interface between the enzyme and nanotube surface. We have demonstrated a new approach for the indirect immobilization of the enzyme on SWNTs with keeping the enzyme activity. DNA wrapped around the nanotube is acting as interlayer/interface between the enzyme and nanotube surface. DNA interacts effectively with the enzyme shell due to H- bonding between enzyme components and serves as an anchorage for GOX. DNA interacts effectively with the enzyme shell due to H- bonding between enzyme components and serves as an anchorage for GOX. Such a method for enzyme immobilization onto a nanotube surface can be used in designing biosensors for glucose, lactate and others. Such a method for enzyme immobilization onto a nanotube surface can be used in designing biosensors for glucose, lactate and others.

ACKNOWLEDGMENTS Dr. U. Dettlaff-Weglikowska, Stuttgart, Germany, for SWNTs purification Dr. O.S. Lytvyn – Atom Force Microscopy study (ISP, Kyiv, Ukraine) ILTPE, Kharkov, Ukraine: Dr. G. Gladchenko – UV absorption spectroscopy A. Yu. Glamazda –luminescence study Dr. S. Stepanian and M.V. Karachevtsev – computer sumulation V.S. Leontiev – biochemistry samples manipulations