Карачевцев В.А. Углеродные нанотрубки

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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

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 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
-
1
3
1/2
m(m+2n)

metallic conduction in
(
n
,
m
)

carbon
nanotube is achieved when

2
n
+
m
=

3
q

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

1800
-
1000

nm

is

assigned

to

electronic

transitions

E
11
S

in

the

semiconducting

nanotube

(band

gap

transition),

absorption

at

the

energy

900
-
500

nm

results

from

the

E
22
S

transitions

of

these

types

of

SWNTs

and

absorption

at

500
-
300

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

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. 2001

bar 10nm

bar 500nm

Биосенсоры на основе УОНТ

Биосовместимость нанотрубок

Присоединение к нанотрубке распознающей биомолекулы

Индивидуальные нанотрубки

Биосовместимость


нанотрубок

Присоединение к нанотрубке

распознающей биомолекулы

Нанотрубки в жгутах

Расщепление жгутов,

получение индивид. нанотрубок

и удержание их от слипания

Биосовместимость

нанотрубок

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

P
hys
.

(
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 base
s formed self
-
stacking (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 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

MP
2
/
6
-
311
++G(
2
d,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
.

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
.

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 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

.

Енергія утворення пари аденін
(
r
А)
-
урацил (
rU
) у воді ~ 19, а
енергія взаємодії поли(
r
А)
-
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

E
11
(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
immobiliz
ed

directly
on the nanotube
.


T
he problem of enzyme immobilization on nanotube
and
of
keeping its native activity

I
t was shown
re
cently

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) 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

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

19550

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

0
.
796

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

NH
2

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

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
.


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