АКТУАЛЬНЫЕ НАПРАВЛЕНИЯ СОВРЕМЕННОЙ
НАУКИ:
УГЛЕРОДНЫЕ НАНОТРУБКИ КАК
ОСНОВА ДЛЯ СОЗДАНИЯ
БИОСЕНСОРОВ
wrapped-DNA SWNT
Д.ф.-м.н. Виктор Алексеевич Карачевцев
ОТДЕЛ МОЛЕКУЛЯРНОЙ БИОФИЗИКИ
Физико-технический институт
низких
температур НАН Украины
им. Б. И. Веркина, Харьков
.
karachevtsev@ilt.kharkov.ua
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 a1
and a2 and the roll-up vector Ch_na1_ma2
. 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:
Ch=na1+ ma2=(n,m)
Nanotube diameters: d=31/2ac-c(m2+mn+n2)1/2/π
Chiral angle:
θ=tan-131/2m(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
metallic
E11
E22
semiconducting
E11
Light absorption of SWNT in NIR region is caused by electronic transitions between pairs of van
Hove singularities. So, light absorption in the region 1800-1000 nm is assigned to electronic
transitions E11S in the semiconducting nanotube (band gap transition), absorption at the energy
900-500 nm results from the E22S transitions of these types of SWNTs and absorption at 500-300 nm
is assigned to the E11m 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
DNA-decorated carbon nanotubes for
chemical sensing
A. T. C Johnson et al, phys. stat.
sol. (b) 243, No. 13, 3252–3256
(2006)
We demonstrate a versatile class of
nanoscale chemical sensors based
on single-stranded DNA (ssDNA)for
chemical recognition and singlewalled 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.
Enzyme-Coated Carbon Nanotubes as SingleMolecule Biosensors
K.Besteman, J-O Lee, F.G. M. Wiertz, H.A. Heering, and C. Dekker
Nano Lett.,, 2003
Color Detection Using ChromophoreNanotube Hybrid Devices
A nanoscale color detector based on a singlewalled 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
X.Zhou et al. Nano Lett., 2009, 9 (3), pp 1028–1033
Sandia National Laboratories, Livermore,
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
Спектры комбинационного рассеяния
одностенных углеродных нанотрубок.
A1g
E1g
A1g
He-Ne лазер з λexc=632.8 нм (1,96 еВ)
SWNTs aggregate in bundles as a result of substantial
van der Waals attractions between tubes
bar 10nm
bar 500nm
R Smalley et al, J. Phys. Chem. 2001
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.
Биосенсоры на основе УОНТ
Биосовместимость нанотрубок
Присоединение к нанотрубке распознающей биомоле
Индивидуальные нанотрубки
Биосовместимость
нанотрубок
Нанотрубки в жгутах
Расщепление жгутов,
получение индивид. нанотрубок
и удержание их от слипания
Присоединение к нанотрубке
распознающей биомолекулы
Биосовместимость
нанотрубок
Присоединение к нанотрубке
распознающей биомолекулы
Создание функционального устройства
Использование жгутов
из нанотрубок
Улучшение
характеристик
созданного ранее
устройств
Изучены особенности адсорбции органических молекул на
поверхность одностенной углеродной нанотрубки
Расщепление жгутов
нанотрубок
10
B
Ультразву
к
8
SWNT
SWNT + pyrene
6
4
2
0
1500
1550
1600
Raman shift, cm
1650
-1
Molecular 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 πсопряженных молекул величина их взаимодействия с нанотрубкой в значительной степени определяется
соотношением площади молекулы и диаметром нанотрубки
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
Hydrophilic
Hydrophobic
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 selfstacking structures
From 30-nucleotides:
12 adenines were in   stacking with the
nanotube surface,
7 bases formed selfstacking (two dimers
and one trimer) and
other adenines were out
of stacking with SWNT
surface
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 H2O 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
MP2/6311++G(2d,p) level of theory).
Stepanian et al J. Phys. Chem. A, 2009, 113 (15), 3621
Многослойное навивание ДНК вокруг нанотрубки
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.
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.
Структура ДНК
Double-stranded DNA:SWNT hybrid
ds- DNA is a quite
rigid polymer
In this model the ds-DNA-SWNT hybrid
will be weak hydrophilic
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.
G. Gladchenko, M. Karachevtsev et. al. (2006) 104, 3193 Mol. Phys.
Carbon Nanotube DNA Sensor
Detection of DNA Hybridization Using the NearInfrared Band-Gap Fluorescence of SingleWalled Carbon Nanotubes
Nano Lett., Vol. 6, No. 8, 2006
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
900 C does not lead
to polymer release
from tube surface
Гібридизація двох полінуклеотидів на поверхні
вуглецевих нанотрубок є дефектною
Енергія утворення пари аденін
(rА)-урацил (rU) у воді ~ 19, а
енергія взаємодії поли(rА)-НТ ~
33 кДж/моль.
Адсорбція полімеру на
поверхні нанотрубки
перешкоджає утворенню
комплементарної пари з
водневими зв’язками.
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 зв’язків між азотними основами двох полімерів .
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
SWNT emission
(9,2)
(9,2)
(14,0)
(11,3)
(9,4)
(10,2)
(7,5)
(9,4)
(6,5)
(11,4)
. 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.
Emission bands are correspond to individual
semiconducting nanotubes of different diameters
Luminescence was excited by DPSS green laser at
λex=532 nm.
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 singlewalled carbon nanotubes
The enzyme was immobilized directly
on the nanotube.
M.S. Strano et al. Nature materials, 4 (2005), 86.
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 Rchymotrypsin 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) 025102).
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 singlestranded 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 19550 H2O 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 0.796 nm,
respectively.
Polynucleotide used in
the simulations were 15nucleotides 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.
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 10070
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.
DNA
interacts effectively with the enzyme shell due to Hbonding 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.
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