Review
奈米粉體報告
主題:奈米多孔膜生物傳感應用
組員: 奈米三乙 49814114 黃柏凱
奈米三乙 49814035 郭南億
摘要
在眾多的生物傳感應用，如血糖檢測，核酸檢測，細菌檢測和基於細胞
的檢測，已被用於合成奈米多孔膜。親和力的增加表面面積和增強輸出傳
感信號，使越來越多的多孔膜作為生物傳感平台的吸引力。表面改性技術
可用於提高變現 bioanalyte 固定，共軛和檢測的表面特性。聯合的變現檢
測技術，如電化學和光學檢測方法，多孔膜？為基礎的生物傳感器具有優
勢，包括快速響應，靈敏度高，成本低。在本文中，多孔膜生物傳感應用
的概述。包括聚合物膜，無機膜，採用光刻奈米孔的膜，和基於碳奈米管
膜的多孔膜的類型進行了介紹。多孔膜的製備技術進行了討論。多孔膜生
物傳感應用的關鍵要求，包括表面 bioanalyte 固定，生物相容性，機械和
化學穩定性，抗生物污染能力的功能。多孔膜的最新進展和發展基礎的生
物傳感器進行了討論，尤其是傳感機制和表面功能化戰略。最後，多孔膜
生物傳感應用的挑戰和未來發展進行了討論。
介紹
一個理想的膜接口，允許在奈米選擇性分子運輸是極大的興趣。在自然界中，
有許多生物奈米級膜，具有各種功能。例如，細胞膜選擇性地調節通過奈米通
道蛋白嵌入脂質雙層毛孔離子和小分子的運輸。已經花了很多的努力，設計和
製作合成的多孔膜，模仿自然生物細胞膜的功能。
多孔膜是一種多孔奈米材料，通常有 1 至 500nm 的高孔隙的孔徑。很高的
表面積，體積比可能是多孔材料最重要的屬性。
增加孔隙表面能夠與生物分子吸附到通過材料的毛孔或流動相互作用。與其
他高寬比如奈米管和奈米線，奈米材料或奈米結構，奈米多孔膜有能力來區分
大小，形狀為基礎的生物分子，具有奈米孔壁的相互作用，並允許選擇性的生
物分子交換，已取得了很大的興趣在生物分子分離，免疫隔離，並進行 DNA 測
序。
在生物醫學應用，奈米多孔膜必須具有的功能和結構的穩定性和抗生物污染
1230003-1
行為維持 在各種環境條件，如溫度，pH 值，存在一定的生物或化學化合物。
當前奈米製造技術，已使我們能夠精確地控制奈米孔的大小，分佈，形態，以
及物理和化學性質。尤其是表面作案¯離子技術可以用來實現孔洞，如粘接性能，
生物相容性，表面潤濕性的首選的表面特性。或孔洞內的不同功能組別的分佈，
可用於控制生物分子吸附，調節細胞/組織的相互作用，也逗號分開生物樣品。
所有這些優勢包括許多生物分子分離純化¯陽離子，免疫隔離，並擴散控制給藥
應用非常流行的多孔膜。除了上述的應用，基於多孔膜的生物傳感器已獲得近
年來相當大的興趣，因為他們增強的電子轉移速率和增加面積較平坦的基板表
面反應/親和力。
這次檢討的目的是介紹多孔膜的最新進展和發展生物傳感應用。包括聚合物
和無機多孔膜，多孔膜，不同類型的介紹。動機和多孔膜的生物醫學應用的最
新發展。多孔膜生物傳感的重要原則和要求進行了討論，其中包括表面功能，
生物相容性和抗生物污染。
心得
透過這次的報告，我對奈米的特性及相關的應用產品更了解，還對奈米
那麼微小的東西感到敬佩。隨著奈米不同形式的應用，所呈現的性質非常
不同，再針對此形式作更多的運用，奈米在物理、化學、生物以及機械工
程和電子工程、材料科學和生物醫學.....等等領域上，都有相關的應用，是
相當多元化的，在它各種特殊性下還有很多分支，奈米有多特色，如透氣
性、熱膨脹、耐化學腐蝕、高導熱性等特性，透過了解這些特性，可以將
之應用奈米結晶材料、奈米粉體、奈米纜線、奈米孔隙材料的產品就此應
運而生。
奈米孔隙材料此類材料指孔隙尺寸小於 100 奈米之多孔隙材料，包括
自然界中早已存在之生物膜與沸石，其高表面積（通常高達~102m2/g），
使之具高催化及吸附效應。奈米孔隙材料可由溶膠－凝膠法、微影蝕刻、
離子束等方法製得；奈米孔隙薄膜經鍍膜處理，可得奈米細管結構。
奈米孔隙材料可用開發改良催化劑，應用於石化工業等。利用孔隙結構，
在薄膜過濾系統純化／分離、藥物輸送植入裝置、及基因定序、醫學檢測
等，奈米孔隙材料均有相當大之應用潛能。氣膠為質輕之良好絕熱材料；
奈米孔隙薄膜可作為半導體業中之低介電材料；奈米多孔矽特殊的發光性
質，可作為固態雷射之材料；奈米多孔碳則具高電容特性，可應用於如手
提電腦、行動電話，乃至電動車等電池之開發。
1230003-2
Nano LIFE
Vol. 2, No. 1 (2012) 1230003 (13 pages)
© World Scienti¯c Publishing Company
DOI: 10.1142/S1793984411000323
NANOPOROUS MEMBRANE
FOR BIOSENSING APPLICATIONS
YANG MO* and TAN FEI
Department of Health Technology and Informatics
Biomedical Engineering Programme
The Hong Kong Polytechnic University, Hung Hom, Hong Kong
*htmems@polyu.edu.hk
Received 27 June 2011
Accepted 24 October 2011
Published 13 April 2012
Synthetic nanoporous membranes have been used in numerous biosensing applications, such as
glucose detection, nucleic acid detection, bacteria detection, and cell-based sensing. The increased
surface a±nity area and enhanced output sensing signals make the nanoporous membranes
increasingly attractive as biosensing platforms. Surface modi¯cation techniques can be used to
improve surface properties for realizable bioanalyte immobilization, conjugation, and detection.
Combined with realizable detection techniques such as electrochemical and optical detection
methods, nanoporous membrane—based biosensors have advantages, including rapid response,
high sensitivity, and low cost. In this paper, an overview of nanoporous membranes for biosensing
application is given. Types of nanoporous membranes including polymer membranes, inorganic
membranes, membranes with nanopores fabricated using nanolithography, and nanotube-based
membranes are introduced. The fabrication techniques of nanoporous membranes are also discussed. The key requirements of nanoporous membranes for biosensing applications include
surface functionality for bioanalyte immobilization, biocompatibility, mechanical and chemical
stability, and anti-biofouling capability. The recent advances and development of nanoporous
membrane—based biosensors are discussed, especially for the sensing mechanism and surface
functionalization strategies. Finally, the challenges and future development of nanoporous
membrane for biosensing applications are discussed.
Keywords: Nanoporous membrane; biosensing; surface modi¯cation.
1. Introduction
In the past few decades, great advances have been
made in the development of well-de¯ned, controllable and biocompatible interfaces for biomedical and clinical applications.1,2 Especially, an ideal
membrane interface that allows selective molecular
transport at nanoscale is of great interest. In nature,
there are many biological membranes at nanoscale
level, with various functions. For example, cell
membranes selectively regulate ions and small
molecules transport through
nanoscale channel
protein pores embedded in the lipid bilayers. A lot of
e®orts have been spent to design and fabricate synthetic nanoporous membranes to mimic the functions of natural biological membranes.
Nanoporous membrane is a type of porous
nanomaterial, which usually has pore diameters
between 1 ‘- 500 nm with a high porosity.3 The very
high surface-area-to-volume ratio is probably the
most important property of nanoporous materials.
1230003-3
Y. Mo & T. Fei
The increased pore surface is able to interact with
biomolecules adsorbed into the pores or °owing
through the materials. Compared with other highaspect-ratio nanomaterials or nanostructures, such
as nanotubes and nanowires, nanoporous membranes have the capability to discriminate biomolecules based on size, shape, and interactions with
nanopore walls and permit selective biomolecule
exchange, which has gained much interest in biomolecule separation,11 immunoisolation,35 and DNA
sequencing.27
In biomedical applications, nanoporous membrane must exhibit functional and structural stability and manintain anti-biofouling behaviors under
a variety of environmental conditions such as temperature, pH, and presence of certain biological or
chemical compounds.4,5 The current nano-manufacturing technique has made it possible to precisely
control the nanopore size, distribution, morphology,
as well as physical and chemical properties.6 Especially, the surface modi¯cation technique can be
used to achieve preferred surface properties of
nanopores such as adhesion properties, biocompatibility, and surface wettability. The distribution of
di®erent functional groups on or inside nanopores
can be used to control biomolecules adsorption,
modulate cells/tissue interactions, and also seperate
biological samples. All of these advantages have
made nanoporous membranes very popular for
many biological applications including molecule
separation and puri¯cation,7—9 immunoisolation,10
and di®usion-controlled drug delivery.11 In addition
to the above-mentioned applications, nanoporous
membrane—based biosensors have gained considerable interest in recent years because of their enhanced
electron transfer rate and increased surface reaction/
a±nity area compared with °at substrates.
The purpose of this review is to present the
recent advances and development of nanoporous
Table 1.
Pore Size
Surface area/Porosity
Permeability
Strength
Thermal stability
Chemical stability
Costs
Life
Meso-macro
Low > 0:6
Low—Medium
Medium
Low
Low-Medium
Low
Short
membrane for biosensing applications. In Sec. 2,
the di®erent types of nanoporous
membranes,
including polymeric and inorganic nanoporous
membranes, are introduced. The motivation and
recent development of nanoporous membrane for
biomedical applications is also given. In Sec. 3, important principles and requirements of nanoporous
membranes for biosensing are discussed, including
surface functionalization, biocompatibility, and
anti-biofouling.
In Sec. 4, the various biosensing applications
of nanoporous membrane are discussed. Finally,
challenges and future directions are discussed in
the conclusion section.
2. Nanoporous Membrane
Nanoporous materials can be classi¯ed on the basis
of their pore size, material constituents, and fabrication methods. Table 1 shows the available nanoporous membranes materials according to the
chemical compositions and technical characteristics.
For pore size, following the International Union of
Pure and Applied Chemistry (IUPAC) classi¯cation, mesopores are pores with 2 nm to 50 nm diameter and macropores are those with 50 nm to
100 nm diameter. According to types of materials,
nanoporous membranes include organic polymeric
membranes and inorganic membranes. The typical
organic polymeric nanoporous membrane materials
include polycarbonate (PC)
and polyethylene
terephthalate (PET). The common fabrication
methods for organic polymer nanoporous membrane
are ion-track etching12 and phase separation techniques.13 The typical pore size for organic nanoporous membrane is from 10 nm to 10 1um. Recently,
inorganic nanoporous membrane materials, including aluminum anodic oxide (AAO) and mesoporous
silica, have gained a lot of interest due to the
Classi¯cation of nanoporous materials.
Micro-meso
High 0.3—0.6
Low—medium
Low
High
High
High
Long
Glass
Alumina-silicate
Oxides
Metal
Meso-macro
Low 0.3—0.6
High
Strong
Good
High
High
Long
Micro-meso
High 0.3—0.7
Low
Weak
Medium—High
High
Low—Medium
Medium—Long
Micro-meso
Medium 0.3—0.6
Low—Medium
Weak—Medium
Medium—High
Very high
Medium
Long
Meso-macro
Low 0.1—0.7
High
Strong
High
High
Medium
Long
1230003-4
Nanoporous Membrane for Biosensing Applications
well-ordered pore structure and availability for
massive production. The pore size from 20 nm to
200 nm can be achieved for AAO via anodization
etching process. The pore size from 2 nm to 20 nm
can be achieved for mesoporous silica via the sol-gel
process.14
2.1. Nanoporous polymeric membrane
The organic polymeric nanoporous membranes have
been used for many biological applications, especially for ¯ltration applications. Many nanoporous ¯lters are made of organic polymers, including
PC and PET. The most common fabrication
method for organic polymeric nanoporous membrane is the ion-etching method.12,15 Generally, a
thin polymeric substrate is under irradiation of
accelerated heavy ions. The well-de¯ned small ion
tracks are then generated on the polymeric substrate surface and can be subsequently enlarged by
the chemical etching method. Cylindrical or conical
pores in the range from 10 nm to 10 1um are generated during the fabrication process.
2.2. Alumina nanoporous anodized
membrane
Among the nanoporous materials that are widely
used in biological science and other research ¯elds,
nanoporous alumina membrane, also known as
anodic aluminum oxide (AAO), has received great
attention due to its excellent biocompatibility and
well-established fabrication process. Nanoporous
alumina membrane is generally prepared by the
two-step anodization technique in acid electrolyte
solutions such as oxalic, sulfuric, chromic and
phosphoric acids.16,17 Figure 1 shows the SEM
images of anodized nanoporous alumina membranes. The two-step anodization process is considered to be the most successful technique to make
a nanoporous surface with a thin aluminum template. It has gained increasing importance due to
particular characteristics such as controllable pore
diameter, periodicity of patterns on surface, and
extremely narrow distribution of pore size, which
o®ers a promising route to synthesize a large surface
area and ordered nanostructure with high aspect
ratio. The resulting membrane has closely packed
hexagonal pore structure of 20—200 nm. The twostep anodization electrochemical method was ¯rst
introduced by Masuda and Fukuda to prepare
alumina with highly ordered nanopore arrays.6
During this fabrication process, the nanoporous
layer was stripped out by an acid etching solution
after the ¯rst anodization was performed. Then the
second anodization produced highly ordered, hexagonal close-packed pore array. The nanopores had
a minimum pore diameter around 70 nm, high pore
density in the range of 10 9 — 10 11 cm —2 , and an
aspect ratio of more than 100.
The pore size, geometry, and distribution can be
controlled by conditions during anodization process
such as anodization voltage and types of acid solutions. Anodization voltage is one of the most important factors to control the sizes and patterns of
nanopores. The e®ect of the anodization voltage can
be correlated with a voltage dependence of the
volume expansion of the aluminum during oxidation
and also the current e±ciency for oxide formation.
It a®ected the growth rate of nanopores and the
(a)
Fig. 1.
(b)
Nanoporous alumina membrane (a) top view, (b) cross-section view. (Adapted from Ref. 78, with permission).
1230003-5
Y. Mo & T. Fei
interpore distance. Except for anodized alumina,
various other inorganic nanoporous membranes
such as silica,14 titania,18 and zirconia19 have also
been developed.
2.3. Membranes with nanopores
fabricated by nanolithography
Recently, inorganic membranes with nanopores
have been fabricated using nanolithography techniques. These processes can precisely control nanopore size and easily integrate with microdevices.
Nanopores were drilled using techniques of focused
ion beam (FIB) and electron beam lithography
(EBL) on substrates, including silicon,20 silicon
nitride,21 and graphene.22,23 Among them, graphene
has been of intense interest for researchers due to its
excellent mechanical, electrical and optical properties. Graphene is a two-dimensional thin sheet of
carbon atoms packed in the form of a honeycomb
lattice. Generally, ultrathin graphene layers are
generated by mechanical exfoliation from graphite
on SiO2 or by synthesis. Especially, the robust
mechanical property makes it possible to generate
nanopores on free-standing ultrathin graphene
membranes using nanolithography technology such
as EBL. Nanopores on graphene can be new analytical platforms as nanopore sensor, especially for
DNA translocation and sequencing.24—27
2.4. Carbon nanotube membrane
During the nanoporous membrane design, the hollow inner pores ranging from around 1 nm of singlewalled nanotubes to around 10 nm of multi-walled
nanotubes render carbon nanotubes to be a good
candidate for biomolecule separation and drug
delivery. Nanotube membrane can be a ¯lm composed of open-ended carbon nanotubes perpendicularly aligned with the supporting substrate.28,29
The carbon nanotube membranes can be fabricated
by a template method using nanoporous alumina
membrane to generate aligned CNT membrane30 or
using extreme magnetic ¯eld.31 The transport
properties through the nanotube porous membrane
have been tested, and it was demonstrated that the
°ow of water through carbon nanotubes could be
controlled through electric current.32,33 Nanotube
membranes have many potential applications, such
as in the water desalination process.
2.5. Biological applications
of nanoporous membranes
Nanoporous membranes have been used for many
biological applications. Biomolecular separation in
nanopores has recently been explored for various
applications. The nanoporous membrane can act as
a blood ¯lter that retains serum proteins, which
allows smaller waste substances out.34 Immunoisolation needs encapsulation of implanted cells or drug
release system and protects them from immune
reaction.10 Small molecules such as oxygen, glucose,
and insulin can pass the small pores of nanoporous
semipermeable membrane, but the passage of much
larger immune system molecules such as immunoglobulin can be impeded by the nanopores.
Nanoporous silicon membrane interfaces have been
explored to treat diabetes in implantable arti¯cial
pancreas.35 Nanoporous membranes have also been
used for controlled drug delivery application.11 The
nanopore size, porosity, permeance, depth, and
thickness can be well controlled for many nanoporous materials, especially nanoporous membranes. It
provides a promising method for making capsules
that may be used for giving controlled release of
pharmacologic agents.36 Due to its excellent biocompatibility, nanoporous alumina membrane is a
good sca®old for cell culture and tissue engineering.37,38 It has been extensively used as a substrate
for tissue constructs.39 It is known that the surface
chemistry, including surface topography, is one of
the important factors to in°uence the cellular response. The impact of the nanoscale pores on cell
response is an important issue that can be investigated by evaluating cell adhesion, cell growth,
morphology changes, and extracellular matrix production via di®erent methods.
3. Requirements of Nanoporous
Membrane for Biosensing
In the past decades, great e®orts have been expended for the development of practical biosensors
to o®er new analytical platforms for applications
in pharmaceutical industry, environmental diagnostics, and food safety. A biosensing system is
typically composed of a biological component and a
physiochemical signal detection component to
detect biological species such as nucleic acids, proteins, cells, virus, and tissues. The biological component could be microorganisms, enzymes, cells,
1230003-6
Nanoporous Membrane for Biosensing Applications
antibodies, DNA, or a biomimic, while the transducers may be optical, piezoelectric, or electrochemical.40 The biosensor could be classi¯ed based
on the employed transducer, which plays a signi¯cant role in procedure of bacteria detection. The
transduction methods such as optical, piezoelectric,
and electrochemical methods are the most common
methods used in today's research for bacteria
detection.41 The working mechanism involves the
binding of bioanalyte targets to speci¯c receptors on
the surface. Then, the transducer converts the
physiochemical signal into an electrical signal for
detection. Due to the nanoscale dimension, the
nanoporous membranes have enhanced electrolyte
transfer rate and increased surface reaction and
a±nity area compared with °at substrates, which
can dramatically increase the output sensing signals. With the capabilities of integrating electrochemical or optical detection methods, nanoporous
membranes have been widely used for various biosensing applications. The nanoporous membrane
based biosensors have advantages such as high
sensitivity, rapid response, and low limit of
detection (LOD).
methods. The physical adsorption method is the
easiest way without complex chemical reaction. The
bonding e±ciency is mainly dependent on the surface properties of nanoporous membrane such as
hydrophobicity and roughness. However, this
physical bonding is generally weak, with low stability. For covalent bonding method, biomolecules
are linked with nanoporous membrane via strong
covalent
bonding.
Self-assembled monolayers
(SAMs) of silanes are commonly used to attach
functional groups on the nanoporous membrane
surface. For example, the immobilization of bacteria
antibody on nanoporous membrane via self assembly monolayer of epoxy silane is shown in Fig. 2.42
The nanoporous alumina membranes were ¯rst
treated with hydrogen oxide (H2O2) to generate
reactive hydroxyl group on the surface. GPMS
silane molecules were then self-assembled onto the
membrane surface. Finally, speci¯c antibodies were
grafted via silane monolayer. It has been demonstrated that nanoporous membrane—based biosensor
with immobilized antibody can detect Escherichia
coli O157:H7 or Staphylococcus aureus. The disadvantage of covalent immobilization is the possible
denaturation of antibodies during the covalent
bonding process.
3.1. Nanopore surface functionality
in biosensors
3.2. Anti-biofouling on nanoporous
membrane in biosensors
Nanoporous membranes with controllable pore size,
geometry, and morphology composed of various
materials have been used for biosensing applications. The key challenge is to realize speci¯c
capturing biomolecules on the membrane—liquid
interface. To achieve this, the biological sensing
element should be stably immobilized on the membrane. The immobilization process can be realized
by either physical adsorption or covalent bonding
When nanoporous membrane—based biosensor
contacts biological environments, nucleic acids,
proteins, and cells may accumulate on the membrane
biosensor surfaces. This biofouling process is caused
by the adsorption or adhesion interactions between
biological species and membrane surfaces. The
adsorbed proteins may decrease the di®usion
antibody
NH
O
O
O
HO
O
O
OH OH OH
Si
O
O
O
O
NH
O
O
O
O
GPMS
O
Si
Si
Si
O
O
O
O
antibody
O
O
nanoporous alumina membrane
NH
CH2
CH2
CH2
HO
HO
CH2 CH2
CH2
4 °C overnight
nanoporous alumina membrane -GPMS
Si
Si
Si
O
O
O
O
SAMs
nanoporous alumina membrane -antibody
Fig. 2. Schematic drawing of antibody immobilization on nanoporous alumina membrane. (Adapted from Ref. 42, with
permission).
1230003-7
Y. Mo & T. Fei
capabilities of small molecules through nanopores,
block active sensing area, and decrease sensing signals,
which may ¯nally lead to the failure of biosensors.
Many materials have been used to modify nanoporous membrane biosensor surface to prevent biofouling. These materials
include hydrogels,
phospholipids, and surfactants, which may prevent
nonspeci¯c protein adsorption and keep the small
molecules di®usion through the nanopores.43 Moreover, the application of these materials should not
decrease the sensitivity and respond rapidly to the
bioanalyte concentration change in the environment. Recently, poly (ethylene glycol) (PEG)
hydrogel has been successfully used for modi¯cation
of nanoporous membrane to increase the e±ciency
of preventing nonspeci¯c protein adsorption. The
hydrophilic and uncharged properties of PEG can
result in the formation of highly hydrated polymer
coils on the biosensor surface, which avoids further
protein adhesion. Due to the thermodynamic
mechanism, hydrated PEG coil on the biosensor
surface makes the process of protein adsorption
extremely unfavorable. This mechanism can be used
to create inert surfaces with further modi¯cation
with appropriate biomolecules such as peptides or
proteins to achieve the speci¯c interactions.
In Sang's research group, PEG monolayers were
grafted to nanoporous alumina membranes using
covalent silane grafting or physical adsorption
methods to form hybrid organic—inorganic membranes. The e®ect of PEG modi¯cation on the gas,
liquid, and protein permeabilities of the membranes
were demonstrated. It suggested that hybrid membrane could provide signi¯cantly improved functional behavior over existing organic or inorganic
membranes.44 Yu et al. has used generated PEG
micropattern covalently grafted on the nanoporous
alumina membrane to generate microarrays for
bacteria micro-patterning and detection.45
3.3. Biocompatibility of nanoporous
membrane in biosensors
It is important to design a biocompatible nanoporous membrane with multiple functions with longterm physical and chemical stability for biosensing
applications. Especially, e®orts have focused on
tuning surface chemistry to regulate cellular and
tissue responses for implantable nanoporous membrane biosensors. For example, the biocompatibility
of nanoporous alumina membrane was proven to be
Fig. 3. SEM images of osteoblast cultured on nanoporous
alumina showing processes extending into nanopores (Adapted
from Ref. 46, with permission).
an excellent biocompatible material for many biological applications. Tejal's research group studied
osteoblast cells response on nanoporous alumina
membranes. The impact of the nanoscale pores on
osteoblast response was studied by evaluating cell
adhesion, morphology, and matrix production. The
advantages of using nanoporous alumina membrane
with proven biocompatibility for improvement
of
46
cell response have been demonstrated. The longterm osteoblast cellular response to the nanoporous
alumina membranes was compared with those cultured on amorphous membranes, Anopore TM , glass,
latex, and aluminum substrates. Figure 3 showed
the Scanning Electron Microscope image of osteoblast cells cultured on nanoporous alumina surface.
The results indicated osteoblast cells adherent
on the nanoporous alumina membranes produced
more extracellular matrix protein compared to cells
adhered on other surfaces.39 Moreover, the cellular
1230003-8
Nanoporous Membrane for Biosensing Applications
response of adhesive cells can be regulated by
modi¯cation of the surface chemistry of the substrate. Substrate surface modi¯cation with polymer,
proteins, or peptide could in°uence cell adhesion
and growth on the substrate. Methods of surface
modi¯cation include physical adsorption, chemical
conjugation, mechanical methods, covalent attachment, and biological methods. Physically adsorbed
extracellular matrix proteins (ECM) such as vitronectin or covalently immobilized small functional
biological groups such as RGD peptides could be
used to enhance adhesions of osteoblasts.47
deposited onto platinum (Pt)-coated glass plate for
immobilization of glucose oxidase.52 Amperometric
measurement showed the linearity of glucose concentration in the range of 25 mg dL —1 to 300 mg dL —1 .
Joo et al. developed a nonenzymatic glucose sensing
system based on nanoporous platinum electrode
embedded in a micro°uidic chip. This system was
composed of micro°uidic channel network and a
miniaturized electrochemical cell. The nanoporous
Pt electrode was utilized for nonenzymatic glucose
detection by direct oxidation of glucose.53
4.2. Cholesterol detection
4. Applications of Nanoporous
Membrane in Biosensors
Biosensor technology is currently drawing a lot of
researchers' interests for its advantages of easy
operation, time saving, portability, with the added
bene¯ts of low cost and repeated reliable results. It
has overcome the limitations of traditional technology for detection. In the past decade, the nanoporous membrane has attracted great interest as a
popular platform for biosensing due to its high
surface-area-to-volume ratio, enhanced sensitivity,
biocompatibility, and easy surface functionalization. Various applications of nanoporous membrane
for biosensing are introduced as follows.
4.1. Glucose detection
Blood glucose monitoring is important for the
treatment of diabetes mellitus. Many glucose
membrane sensors have been developed with the
advantage of preventing the direct electrooxidation
of chemical agents in the physiological environment.
Moussy et al. developed a Na¯on-based membrane
biosensor for in vitro and in vivo detection of glucose
concentrations in dogs.48 However, cracking and
mineralization were exhibited, which a®ected the
permeability of this membrane sensor.49 Li et al.
used the porous nanocrystalline TiO2 ¯lm immobilized with glucose oxidase for amperometric detection of glucose due to its excellent biocompatibility
and high adsorption ability.50 Yang et al. developed
a surface-treated nanoporous ZrO2/chitosan composite matrix for glucose detection. Glucose oxidase
immobilized in the material kept the activity well.
This device had a rapid response of less than 10 s. The
linear range was from 1:25 x 10 —5 to 9:5 x 10 —3 M,
with a detection limit of 1:0 x 10 —5 M.51 Saha et al.
used the nanoporous cerium oxide (CeO2) thin ¯lm
Currently, cardiovascular diseases have become one
of the major causes for death of humans. High concentration of cholesterol in blood is one of the most
important reasons. Hence, it is essential to measure
cholesterol concentration in blood. Li et al. developed
a cholesterol biosensor based on a layer of porous
silica sol-gel matrix immobilized with cholesterol
oxidase (ChOx ). This biosensor had a half-life time
about 35 days. The detection cholesterol concentration range was from 1 x 10 —6 to 8 x 10 —5 mol/L,
with a detection limit of 1:2 x 10 —7 mol/L. This
sensor also had a high speci¯city, wherein interfering agents such as ascorbic acid and uric acid did
not a®ect the results.54 Singh et al. developed a
cholesterol biosensor based on zinc oxide (ZnO)
nanoporous thin ¯lms grown on gold surface.
The ChOx enzyme was immobilized onto ZnO
nanoporous ¯lm surface by physical adsorption
technique. The high isoelectric point of ZnO made it
possible to adsorb an enzyme with a low isoelectric
point e±ciently without any surface functionalization. The optical and cyclic voltammetric measurements demonstrated the detection
range of
cholesterol is within 25—400 mg/dl.55
4.3. Single molecule analysis
A lipid bilayer is an arti¯cial membrane composed
of lipid molecules (usually phospholipids). It is an
essential component of all biological membranes,
including mammalian cell membranes. And it is
important for the permeability properties of cell
membranes. The functional coupling of lipid bilayer
with inorganic solids became a very attractive topic
in the past 20 years.56 Many e®orts were spent
focusing on fabricating arti¯cial lipid membrane
structures attached to a solid surface to allow for the
1230003-9
Y. Mo & T. Fei
Fig. 4. Schematic drawing of the Te°on cell used for electrochemical impedance analysis and single-channel recordings.
(Adapted from Ref. 57, with permission).
insertion of functional transmembrane peptides for
detection of transport activity, which is the prerequisite of a lipid membrane based biosensor.57
When biomolecules go through the embedded protein nanopores within the lipid membrane, the
amplitude and current blockage duration are changed for the ion current.58 Information about the size,
structure, and sequence of small biomolecules can be
derived by ion current analysis. The in vitro lipid
membrane systems can be integrated with electrochemical or clamp signal measurements for ion
transport study. Figure 4 showed the schematic
setup for a nanoporous membrane based lipid layer
biosensor for single molecule analysis. The nanoporous membrane can be a good insulating platform
to support proteinembedded lipid layer for single
Fig. 5.
molecule analysis. The nanopore size, pore surface
chemistry, and nanoporous membrane topography
could be controlled in nanoscale during the fabrication process of many materials. It makes it possible to regulate analyte—surface interactions, which
has the potential to support the engineered pores for
single-molecule detection and analysis.59
Steinem et al. reported the application of using
nanoporous alumina membrane as support substrates for lipid bilayer to make arti¯cial membrane
based biosensor.57 The nanoporous alumina membranes with di®erent pore diameters were produced
by an anodization process. One side of the nanoporous membrane was covered with a thin gold layer
followed by chemisorption of a hydrophobic thiol
compound, which was a prerequisite for the formation
of suspending membranes with the name of nanoblack lipid membranes (nano-BLMs). The bilayer
formation process and long-term mechanical stability
of the nano-BLMs were tested by electrical impedance
spectroscopy. The membrane exhibited high membrane resistances, which were suited for single-channel
recordings. Gramicidin and alamethicin were successfully inserted into the arti¯cial membranes to
demonstrate the functionality of the nano-BLMs.57
This nano-BLM system was also used to detect the
photocurrents generated by bacteriorhodopsin upon
continuous light illumination. The system showed a
high long-term stability with stationary recorded
currents.60 The similar lipid membrane system was
also realized on porous silicon substrate for rupture
process and lateral motility analysis.61
4.4. DNA hybridization detection
Nanoporous membranes can be fabricated with a regularly arranged hexagonal pattern of nanopores with
controlled diameters. Such nanoporous membranes
Schematic drawing of nanoporous membrane based °uorescence based biosensor. (Adapted from Ref. 63, with permission).
1230003-10
Nanoporous Membrane for Biosensing Applications
Fig. 6. Schematic drawing of nanoporous membrane for DNA
hybridization detection via electrical measurement. (Adapted
from Ref. 65, with permission).
have high surface area, which allows binding relatively
large amounts of target molecules. In addition, due to
the advantages of low auto-°uorescence, high porosity,
which allows for high °ow rates through the membrane, and good transparency, as well as the small pore
diameter which is comparable to the nucleic acid
length, the nanoporous alumina membrane is widely
used in the application of DNA and RNA detection
and sensing.
Due to the property of optical transparency in
UV and IR regions, the nanoporous alumina membrane can be allowed to directly detect DNA molecules by method of optical adsorption. Vlassiouk
et al. used nanoporous alumina membrane to capture DNA molecules on amino silane—modi¯ed
surface Combined with the advantage of high surface area, the nanoporous alumina membrane was
successfully used to detect and separate DNA molecules e±ciently by optical and IR adsorption
methods.62 For °uorescence detection, the nonporous oxide membranes can a®ord a substantial
increase in the °uorescence signal intensity compared with °at substrates. It can be used as a convenient sensing platform for various biological
species, such as protein, DNA, or RNA, with
immunolabeling techniques. Smirnov et al. demonstrated the application of nanoporous alumina
membranes as substrates for °uorescence detection
of labeled biomolecules (Fig. 5).63 They used
aminosilane—succinimide chemistry to covalently
bind biotin monolayer on the nanopore surface to
detect streptavidin labeled with Alexa Flour 488 dye.
The °uorescence intensity in nanoporous alumina
membranes increased by a factor of ¯ve compared to
°at aluminum and enhancement as high as seven
times is observed compared to °at glass surface.
These membrane-based °uorescence sensors o®ered
signi¯cant advantages, such as increased density of
binding sites for target molecules, and enhanced °uorescence collection e±ciency.63 Yang et al. developed
a novel biofunctionalized three-dimensional ordered
nanoporous SiO2 device for chemiluminescent sensing. The functionalization of surface was achieved
by using 3-glycidoxypropyltrimethoxysilane as a
linker. The high e±ciency of chemiluminescence
signal collection was demonstrated for streptavidin to
biotin-labeled antibody recognition.64
The current nano-manufacturing technique can
fabricate nanopores with comparable size with small
biomolecules such as short DNA and RNA. It makes
it possible to detect DNA molecules using nanoporous membrane by monitoring ionic conductivity
change in the nanopores. Vlassiouk et al. used
nanoporous alumina membrane to monitor DNA
hybridization process via electrical measurements
(Fig. 6). Single-stranded DNA (ss-DNA) was ¯rst
immobilized on the inside walls of nanopores. The
ionic current was blocked through the nanopores
once the target DNA was captured after DNA hybridization. The ion current change can then be
monitored by cyclic voltammetry and impedance
spectroscopy. The electrical detection method provides the opportunity for inexpensive detection. The
theoretical sensitivity limit was as low as femtomolar for nanoporous membrane with 5 1um x 5 1um
area, and 0.5 1um thickness with 20 nm pore size.65
Takmakov et al. further used hydrothermally shrunk
alumina nanopores for DNA hybridization detection. After the hydrothermal treatment, the 60 nm
nanopores could shrink to a neck of less than 10 nm,
which signi¯cantly increase electrolyte resistance
through the small pores. The dominance of pore
resistance compared with electrolyte resistance
made it possible to develop this sensing platform in
a microarray format.66 Wang et al. used surface
charge e®ect to modulate ionic conductance for
label-free DNA sensing. A mixture of neutral silanes
and morpholinos (neutral analogs of DNA) was
optimized to modify nanopore surface. Upon DNA
binding, a strong e®ect will be generated for ion
conductance change.67 A capacitance sensor based
on a nanoporous alumina structure was fabricated
for DNA hybridization sensing.68 The membrane
served as a template, and the gold nanowires made
by depositing gold ¯lm on surface of membrane were
used as the working and counter electrodes respectively. The capacitance of the sensor decreased
greatly when the complementary DNA molecules
were captured.
1230003-11
Y. Mo & T. Fei
4.5. Cancer biomarker detection
There is always a strong demand for rapid screening of blood samples. A nanoporous
alumina
membrane—based platform was developed capable
of ¯ltering and detection of the cancer biomarker
CA15-3 in the blood samples without any sample
preparation. The nanoporous membrane was ¯rst
functionalized with 3-aminopropyltrimethoxysilane
(APS) and then immobilized with primary antiCA15-3 antibodies. The antibody immobilized
nanoporous membrane could capture immunoglobulins conjugated with gold nanoparticles in the
blood samples. The ion current through the nanopores was then blocked by this immunoreactions,
and the blocking e®ect was further enhanced by
silver deposition. By monitoring the impedance
spectrum signal change before and after capturing,
the low concentrations of immunoglobulin could be
detected. This developed nanoporous membrane
sensor achieved a detection limit of 52 U mL —1 of
CA15-3. This nanoporous membrane based immunoassay was promising for cancer diagnostics.69
4.6. Bacteria detection
Pathogenic bacteria detection is one of the most
important methods for food safety and public
health. To avoid diseases caused by pathogenic
bacteria, the process of detection and identi¯cation
is the ¯rst control point. Therefore, it is signi¯cantly
necessary to control these bacteria content in food
and water supply by e®ective detection and
inspection approaches. Wang et al. developed a
biosensor based on ssDNA probe—functionalized
AAO nanopore membranes for E. coli O157:H7
detection. They proposed a dynamic polymeraseextending (PE) DNA hybridization procedure,
where hybridization happened in the existence of
Taq DNA polymerase and dNTPs under controlled
reaction temperature. The probe of target DNA was
extended to increase the capability to block the
ionic °ow in the nanopores. Cyclic voltammetry and
impedance spectroscopy were used to measure the
ionic conductivity change during the DNA hybridization. This AAO membrane—based biosensor
provides a sensitivity limit of 0.5 nM for complementary target DNA by PE method for E. coli
O157:H7 pathogen detection.70
Yu et al. developed a micro°uidic chip with
nanoporous alumina membranes immobilized with
speci¯c antibody for E. coli O157:H7 detection.
PEG hydrogel layer with 20 x 20 microarray pattern was covalently bonded on silane-modi¯ed
nanoporous alumina surface for bacteria capture.45
The antibodies were immobilized on the hydrophobic silane-modi¯ed nanoporous membrane surface. The target
E. coli O157:H7 were then
successfully patterned and captured by the antibodies on the membrane inside the microwells. The
antigen—antibody immunocomplex continued to
react with °uorescence-labeled antibody to form
\sandwich" structure on the membrane. Figure 7
shows the sensing mechanism of this microchip,
which is based on nanopore blocking. Once bacteria
is captured by speci¯c antibody on silane-modi¯ed
nanoporous alumina membrane, the electrolyte
current will be blocked, which causes the impedance
amplitude increase. Impedance spectroscopy was
used to measure the impedance amplitude change
during the bacteria capturing process. A linear
concentration relationship was observed in the
range from 10 2 to 10 6 CFU/mL, with a detection
limit around 102 CFU/mL. Cheng et al. used a
nanoporous alumina membrane—modi¯ed platinum
electrode for speci¯c quantitative label-free detection of E. coli cells. Cyclic voltammetry was used to
detect the bacteria binding process. This biosensor
gave a low detection limit of 22 cfu mL —1 over a
wide linear working range of 10 to 106 cfu mL —1 .71
4.7. Cell-based biosensor
In the last decade, cell-based biosensors are of particular interest for cell monitoring methods due to
their simplicity, sensitivity, and low cost. The cellbased biosensor use the whole cell as sensing element
to detect agents with physiological e®ects to the
cells.72,73 For this purpose, it is of great interest to
develop an ideal interface to control the cell
Fluorescence labeled Antibody for E.coli
Impedance
Analyzer
Nanopores
Antibody for E.coli
Fig. 7. Mechanism for nanoporous membrane-based bacteria
impedance sensing. (Adapted from Ref. 76, with permission).
1230003-12
Nanoporous Membrane for Biosensing Applications
surrounding microenvironment for the study of cell
response to various agents. One popular method is
planar gold microelectrodes—based electric cellsubstrate impedance sensing (ECIS) technique for
cell proliferation, morphology, and motility monitoring.74,75 However, the metal electrodes have
polarization problems in the electrolyte solutions,
especially when the size of metal electrode decreases.
It will be hard to derive the impedance signal
component related to cells from the total impedance
signals. Using nonconductive nanoporous membrane materials is one promising method to solve
this problem.
Yu et al. used nanoporous alumina membrane for
the study of anticancer drug e®ect of retinoic acid
(RA) on human esophageal squamous epithelial
KYSE30 cancer cells with impedance spectroscopy.76 The sensing mechanism was based on
nanopore blocking e®ect by adherent cells cultured
on membrane
surfaces.
During
impedance
measurement, the applied electric ¯eld generated
ion current through the insulated membrane, but
without polarization e®ects. This device was successfully used to monitor cancer cell adhesion, proliferation, and anticancer drug—induced change by
impedance spectroscopy. Liu et al. further developed a PEG cell-based microarray on nanoporous
alumina membrane with cell micropatterning and
controlled drug delivery to cytotoxic e®ects of cisplatin.77 Jiang et al. developed a micro°uidic device
Table 2.
integrated with polycarbonate mesoporous membrane with pore size of 30 nm for detection of Hela
cells adhesion process.79
5. Challenges and Future Development
Table 2 summarizes the main nanoporous
membrane—based biosensor types. When these
nanoporous membrane—based biosensors are used
in vivo physiological environment for long-term
monitoring, several issues have to be addressed,
such as the stability of surface functionality, biofouling of nanopores, and long-term biocompatibility. These critical challenges will inspire the next
generation of nanoporous membrane—based biosensors, such as stimuli-responsive membranes,
which can regulate the interactions with physiological environment in a controlled way. These
smart nanoporous membranes can change their
surface and di®usion properties according to the
physiological environment change. Moreover, there
will be advances and development for cost-e®ective
manufacturing techniques of nanoporous membranes, which will lead to mass production of
cheaper nanoporous membrane sensing platforms.
Finally, the integration of the nanoporous membrane with micro°uidic devices with the requirement of packaging and functionalization will be
another trend for future development of nanoporous
membrane—based sensing platforms.
Biosensor types based on nanoporous membrane.
Biosensing applications
Nanoporous membrane types
Detection methods
Detection limit
Glucose detection
Na¯on,48,49 Titanium oxide50
ZrO2 /Chitosan composite51
Cerium oxide52
Silicic sol-gel matrix53 Zinc
oxide54
Lipid membrane56—61
Alumina62,63,65—68 Silicon
oxide64
Amperometric
10 —4 —10 —5 M
Cholesterol detection
Single molecule analysis
DNA hybridization detection
Cancer biomarker detection
Bacteria detection
Alumina69
Alumina71,72
Cell based biosensor
Alumina,76,77 Polycarbonate79
Amperometric Electrochemical
1:2 x 10 —7 M 6:5 x 10 —6 M
Amperometric or photo current
Optical and IR adsorption,62
Flurescence63
Chemiluminescence,64
Amperometric and
impedance
spectroscopy,65—67
Capacitance68
Impedance spectroscopy
Impedance spectroscopy and
cyclic voltammetry
Impedance spectroscopy
Single molecule
1—10 nM
1230003-13
52 U/mL
10 1 —10 2 CFU/mL
N/A
Y. Mo & T. Fei
6. Conclusion
Nanoporous membranes have been widely used for
many biosensing applications, such as DNA hybridization, cancer marker detection, single-molecule analysis, bacteria detection, and cellular
sensing, realizably, sensitively, and in real-time. The
key properties of nanoporous membranes for biosensing include narrow pore size distribution, biocompatibility, mechanical and chemical stability,
anti-biofouling capabilities, and reliable surface
immobilization. The recent advancement of the
nano-manufacturing technique can precisely control
nanopore size, geometry, and distribution for controlling protein, ion, and drug molecules transport.
Surface modi¯cation methods such as surface
treatment and self-assembled polymer grafting can
be used to achieve preferred surface properties. The
future nanoporous membrane for biosensing should
have multiple functions, be stimuli responsive, and
have programmable external control.
References
1. R. F. Turner, D. J. Harrison and R. V. Rajotte,
Matrix Comput. 12, 361 (1991).
2. J. E. Babensee, J. M. Anderson, L. V. McIntire and
A. G. Mikos, Adv. Drug Deliv. Rev. 33, 111 (1998).
3. G. Q. Liu and X. S. Zhao, Nanoporous Materials
Science and Engineering (Imperial College Press,
London, 2003).
4. J. Y. Han, J. P. Fu and R. B. Schoch, Lab. Chip 8,
23 (2007).
5. S. P. Adiga, C. M. Jin, L. A. Curtiss, N. A. Monteiro-Riviere and R. J. Narayan, Wiley Interdiscipl.
Rev. Nanomed. Nanobiotechnol. 1, 568 (2009).
6. H. Masuda, H. Yamada, M. Satoh, H. Asoh, M.
Nakao and T. Tamamura, Appl. Phys. Lett. 71,
2770 (1997).
7. H. D. Tong, H. V. Jansen, V. J. Gadgil, C. G.
Bostan, E. Berenschot, C. J. M. Van Rijn and M.
Elwenspoek, Nano Lett. 4, 283 (2004).
8. H. U. Osmanbeyoglu, T. B. Hurb and H. K. Him, J.
Membr. Sci. 343, 1 (2009).
9. C. C. Striemer, T. R. Gaborski, J. L. McGrath and
P. M. Fauche, Nature 445, 749 (2007).
10. L. Leoni, A. Boiarski and T. A. Desai, Biomed.
Microdev. 4, 131 (2002).
11. D. A. LaVan, T. McGuire and R. Langer, Nat.
Biotechnol. 21, 1184 (2003).
12. C. Trautmann, W. Brüchle, R. Sphor, J. Vetter and
N. Angert, Nucl. Instr. Meth. B 111, 70 (1996).
13. M. Ulbricht, Polymer 47, 2217 (2006).
14. W. Tanglumlert, S. Wongkasemjit and T. Imae, J.
Nanosci. Nanotechnol. 9, 1844 (2009).
15. S. Metz, C. Trautmann, A. Bertsch and P. Renaud,
J. Micromech. Microeng. 14, 324 (2004).
16. K. Itaya, S. Sugawara, K. Arai and S. Saito, J.
Chem. Eng. Jpn. 17, 514 (1984).
17. Y. Xu, G. E. Thompson and G. C. Wood, Trans.
Inst. Met. Finish. 63, 3 (1985).
18. V. Vega, V. Prida, M. Hern‘andez-V‘elez, E. Manova,
P. Aranda, E. Ruiz-Hitzky and M. V‘azquez,
Nanoscale Res. Lett. 2, 355, (2007).
19. T. Tsuru, M. Narita, R. Shinagawa and T. Yoshioka, Desalination 233, 1 (2008)
20. C. C. Striemer, T. R. Gaborski, J. L. McGrath and
P. M Fauchet, Nature 445, 749 (2007).
21. H. D. Tong, H. V. Jansen, V. J. Gadgil, C. G.
Bostan, E. Berenschot, C. J. M. van Rijn and
M. Elwenspoek, Nano Lett. 4, 283 (2004).
22. C. Dekker, Nature Nanotech. 2, 209 (2007).
23. K. S. Novoselov, A. K. Geim, S. V. Morozov, D.
Jiang, Y. Zhang, S. V. Gubonos, I. V. Grigorieva
and A. A. Firsov, Science, 306, 666 (2004).
24. S. Bae, H. Kim, Y. Lee, X. F. Xu, J. S. Park, Y. Zheng,
J. Balakrishnan, T. Lei, H. R. Kim, Y. I. Song, Y. J.
Kim, K. S. Kim, B. Ozyilmaz, J. H. Ahn, B. H. Hong
and S. Iijima, Nature Nanotech. 5, 574 (2010).
25. M. D. Fischbein and M. Drndić, Appl. Phys. Lett.
93, 113107 (2008).
26. S. Garaj, W. Hubbard, A. Reina, J. Kong, D. Branton
and J. A. Golovchenko, Nature 467, 190 (2010).
27. G. F. Schneider, S. W. Kowalczyk, V. E. Calado, G.
Pandraud, H. W. Zandbergen, L. M. K. Vandersypen and C. Dekker, Nano Lett. 10, 3163 (2010).
28. B. J. Hinds, N. Chopra, T. Rantell, R. Andrews,
V. Gavalas and L. G. Bachas, Science 303, 62
(2004).
29. L. Sun and R. M. Crooks, J. Am. Chem. Soc. 122,
12340 (2000).
30. C. L. Cheung, A. Kurtz, H. Park and C. Lieber, J.
Phys.Chem. B 106, 2429 (2002).
31. M. J. Casavant, D. A. Walters, J. J. Schmidt and R.
E. Smalley, J. Appl. Phys. 93, 2153 (2003).
32. Z. K. Wang, L. J. Ci, L. Chen, S. Nayak, P. M.
Ajayan and N. Koratkar, Nano lett. 7, 697 (2007).
33. M. A. Yu, H. H. Funke, J. L. Falconer and R. D.
Noble, J. Am. Chem. Soc. 32, 8285 (2010).
34. W. H. Fissell, H. D. Humes, A. J. Fleischman and
S. Roy, Blood Puri¯cation, 25, 12 (2007).
35. I. Tsujino, J. Ako, Y. Honda and P. J. Fitzgera,
Expert Opin. Drug. Deliv. 4, 287 (2007).
36. D. Gong, V. Yadavalli, M. Paulose, M. Pishko and
C. A. Grimes, Biomed. Microdev. 5, 75 (2003).
37. K. E. La Flamme, K. C. Popat, L. Leoni, E. Markiewicz, T. J. La Tempa, B. B. Roman, C. A. Grimes
and T. A. Desai, Biomaterials 28, 2638 (2007).
1230003-14
Nanoporous Membrane for Biosensing Applications
38. K. C. Popat, K. I. Chatvanichkul, G. L. Barnes,
T. J. Latempa, C. A. Grimes and T. A. Desai,
J. Biomed.Mater. Res. A 80, 955 (2007).
39. K. C. Popat, E. E. L. Swan, V. Mukhatyar, K. I.
Chatvanichkul, G. K. Mor, C. A. Grimes and T. A.
Desai, Biomaterials 26, 4516 (2005).
40. O. Lazcka, F. J. Del Campo and F. X. Munoz,
Biosensor Bioelectron. 22, 1205 (2007).
41. V. Velusamy, K. Arshak, O. Korostynska, K. Oliwa
and C. Adley, Biotechnol. Adv. 28, 232 (2010).
42. F. Tan, P. H. M. Leung, Z. B. Liu, Y. Zhang, L. D.
Xiao, W. W. Ye, X. Zhang, L. Yi and M. Yang,
Sens. Actuators B 159, 328 (2011).
43. R. J. Narayan et al., J. Nanosci. Nanotechnol. 7,
1486 (2007).
44. S. W. Lee, H. Shang, R. T. Haasch, V. Petrova and
G. U. Lee, Nanotechnology 16, 1335 (2005).
45. J. J. Yu, Z. B. Liu, Q. J. Liu, K. T. Yuen, A. F. T.
Mak, M. Yang and P. H. M. Leung, Sens. Actuators
A 154, 288 (2009).
46. E. E. L. Swan, K. C. Popat, C. A. Grimes and T. A.
Desai, J. Biomed. Mater. Res. A 72, 288 (2005).
47. E. E. L. Swan, K. C. Popat and T. A. Desai, Biomaterials 26, 1969 (2005).
48. F. Moussy, D. J. Harrison and R. V. Rajotte, Int. J.
Artif. Organ. 17, 95 (1994).
49. R. C. Mercado and F. Moussy, Biosens. Bioelectron.
13, 133 (1998).
50. Q. W. Li, G. A. Luo, J. Feng, Q. Zhou, L. Zhang and
Y. F. Zhu, Electroanalysis 13, 413 (2001).
51. Y. H. Yang, H. F. Yang, M. H. Yang, Y. L. Liu, G.
L. Shen and R. Q. Yu, Analytica Chimica Acta 525,
213 (2004).
52. S. Saha, S. K. Arya, S. P. Singh, K. Sreenivas, B. D.
Malhotra and V. Gupta, Biosensor Bioelectron. 24,
2040 (2009).
53. S. Joo, S. Park, T. D. Chung and H. C. Kim, Anal.
Sci. 23, 277 (2007).
54. J. P. Li, T. Z. Peng and Y. Q. Peng, Electroanalysis
15, 1031 (2003).
55. S. P. Singh, S. K. Arya 1, P. Pandey, B. D. Malhotra, S. Saha, K. Sreenivas and V. Gupta, Appl.
Phys. Lett. 91, 063901 (2007).
56. E. Sackmann, Science 271, 43 (1996).
57. W. Romer and C. Steinem, Biophys. J. 86, 955
(2004).
58. M. Akeson, D. Branton, J. J. Kasianowicz, E.
Brandin and D. W. Deamer, Biophys. J. 77, 3227
(1999).
59. H. Bayley and P. S. Cremer, Nature 413, 226 (2001).
60. C. Horn and C. Steinem, Biophys J. 89, 1046 (2005).
61. D. Weiskopf, E. K. Schmitt, M. H. Klühr, S. K. Dertinger and C. Steinem, Langmuir 28, 9134 (2007).
62. I. Vlassiouk, A. Krasnoslobodtsev, S. Smirnov and
M. Germann, Langmuir 20, 9913 (2004).
63. P. Takmakov, I. Vlassiouk and S. Smirnov, Anal.
Bioanal. Chem. 385, 954 (2006).
64. Z. J. Yang, Z. Y. Xie, H. L. Liu, F. Yan and H. X. Ju,
Adv. Funct. Mater. 18, 3991 (2008).
65. I. Vlassiouk, P. Takmakov and S. Smirnov, Langmuir 21, 4776 (2005).
66. P. Takmakov, I. Vlassiouk and S. Smirnov, Analyst
131, 1248 (2006).
67. X. Wang and S. Smirnov, ACS Nano 3, 1004 (2009).
68. B. Kang, U. Yeo and K. H. Yoo, Biosens. Bioelectron. 25, 1592 (2010).
69. A. D. L. Escosura-Muñiz and A. Merkoçi, Small
7, 675 (2011).
70. L. J. Wang, Q. J. Liu, Z. Y. Hu, Y. F. Zhang, C. S.
Wu, M. Yang and P. Wang, Talanta 78, 647 (2009).
71. M. S. Cheng, S. H. Lau, V. T. Chow and C. S. Toh,
Environ. Sci. Technol. 45, 6453 (2011).
72. P. Wang, G. Xu, Q. Liu, Y. Xu, Y. Li and R. Li,
Sens. Actuators B: Chem. 108, 576 (2005).
73. A. Rasooly and J. Jacobson, Biosen. Bioelectron.
21, 1851 (2006).
74. I. Giaever and C. R. Keese, Nature 366, 591 (1993).
75. C. R. Keese, J. Wegener, S. R. Walker and L. Giaever,
Proc. Natl. Acad. Sci. USA 101, 1554 (2004).
76. J. J. Yu, Z. B. Liu, A. F. T. Mak and M. Yang,
Talanta 80, 189 (2009).
77. Z. B. Liu, Y. Zhang, J. J. Yu, A. F. T. Mak, Y. Li
and M. Yang, Sens. Actuators B 143, 776 (2010).
78. E. Gultepe, D. Nagesha and L. Menon, Appl. Phys.
Lett. 90, 163119 (2007).
79. L. M. Jiang, J. M. Liu, J. Shi, X. Li, H. Li, J. Liu, J.
N. Ye and Y. Chen, Microelectron. Eng. 88, 1722
(2011).
1230003-15