U.S. patent application number 12/487741 was filed with the patent office on 2013-05-16 for nanostructured composite material and biosensor comprising it.
This patent application is currently assigned to Achilleas Tsoukalis. The applicant listed for this patent is Nikolaos Chaniotakis, Maria Chatzimarinaki, Vassiliki Stavgianoudaki, Achilleas Tsoukalis, Vassiliki Vamvakaki. Invention is credited to Nikolaos Chaniotakis, Maria Chatzimarinaki, Vassiliki Stavgianoudaki, Achilleas Tsoukalis, Vassiliki Vamvakaki.
Application Number | 20130122531 12/487741 |
Document ID | / |
Family ID | 41031019 |
Filed Date | 2013-05-16 |
United States Patent
Application |
20130122531 |
Kind Code |
A1 |
Chaniotakis; Nikolaos ; et
al. |
May 16, 2013 |
NANOSTRUCTURED COMPOSITE MATERIAL AND BIOSENSOR COMPRISING IT
Abstract
A nanostructured composite is provided which comprises a matrix
comprising carbon nanofibers, and a biochemical receptor,
especially an enzyme, immobilized in the matrix, where the carbon
nanofibers have been subject to an oxidation treatment. In a
preferred embodiment, the nanostructured composite further
comprises a biomimetically synthesized silica matrix.
Inventors: |
Chaniotakis; Nikolaos;
(Crete, GR) ; Tsoukalis; Achilleas; (Attiki,
GR) ; Chatzimarinaki; Maria; (Crete, GR) ;
Vamvakaki; Vassiliki; (Crete, GR) ; Stavgianoudaki;
Vassiliki; (Crete, GR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Chaniotakis; Nikolaos
Tsoukalis; Achilleas
Chatzimarinaki; Maria
Vamvakaki; Vassiliki
Stavgianoudaki; Vassiliki |
Crete
Attiki
Crete
Crete
Crete |
|
GR
GR
GR
GR
GR |
|
|
Assignee: |
Achilleas Tsoukalis
Attiki
GR
|
Family ID: |
41031019 |
Appl. No.: |
12/487741 |
Filed: |
June 19, 2009 |
Current U.S.
Class: |
435/14 |
Current CPC
Class: |
G01N 33/551 20130101;
B82Y 15/00 20130101; B82Y 30/00 20130101; C01B 32/342 20170801;
C12N 11/02 20130101 |
Class at
Publication: |
435/14 |
International
Class: |
C12N 11/02 20060101
C12N011/02 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 20, 2008 |
GR |
20080100409 |
Claims
1. Nanostructured composite material, especially for application in
biosensors, comprising: a silica matrix precipitate on a scaffold
comprising a polymer or a peptide, carbon nanofibres; and a
biochemical receptor immobilized on said carbon nanofibers, wherein
said carbon nanofibers are activated.
2. Nanostructured composite material according to claim 1, wherein
the biochemical receptor is selected from the group consisting of
isolated enzymes, microorganisms, immunosystems, tissues,
organelles, whole cells, antibodies, nucleic acids and combinations
thereof.
3. Nanostructured composite material according to claim 1, wherein
the amount of acidic surface functional groups in the activated
carbon nanofibers is at least 6*10.sup.-5 mol/gram.
4. (canceled)
5. Nanostructured composite material according to claim 1, wherein
the scaffold material is selected from the group consisting of
poly-L-lysine, polyethylenimine, polyallylamine, cellulose, R5
peptide, poly-L-arginine, poly-L histidine, lysine oligomers,
poly(allylamine hydrochloride).
6. Nanostructured composite material according to claim 1, wherein
porin molecules are incorporated in the silica matrix.
7. Method for producing a nanostructured composite material
according to claim 1, comprising: activating carbon nanofibers by
treatment with an oxidizing agent; adjusting the pH value at the
surface of the carbon nanofibers to 7; immobilizing a biochemical
receptor on the activated carbon nanofibers; and optionally,
depositing a scaffold comprising a protein or polymer onto the
carbon nanofibers and precipitating a silica matrix onto the
scaffold.
8. Method according to claim 7, wherein activation of the carbon
nanofibers comprises: treating the carbon nanofibers with an
oxidizing agent selecting from the group consisting of hydrogen
peroxide, nitric acid, mixtures of sulfuric acid and nitric acid,
oxygen, potassium permanganate (KMnO.sub.4) and ruthenium tetroxide
(RuO.sub.4); treating the carbon nanofibers with irradiation;
supplying thermal energy; or a combination thereof.
9. A biosensor comprising a nanostructured composite material
according to claim 1.
10. A biosensor according to claim 9, wherein the biosensor is a
single needle biosensor for subcutaneous measurement.
11. A biosensor according to claim 9, further comprising a layer
comprising a biocompatible material on at least a part of the
surface of the biosensor or the multi-biosensor device.
12. A multi-biosensor device comprising a plurality of biosensors,
wherein at least one of the biosensors comprises a nanostructured
composite material according to claim 1.
13. A multi-biosensor device according to claim 12, wherein means
are provided to achieve individual activation of each
biosensor.
14. A multi-biosensor device according to claim 13, wherein the
biosensors are provided with a covering layer comprising an
electrically erodible polymer which is substantially impermeable to
fluids, and means are provided to achieve sequential activation of
the individual biosensors by electroerosion of said electrically
erodible polymer and electronic activation of the uncovered
sensor.
15. A multi-biosensor device according to claim 12, wherein the
multi-biosensor device can be implanted subcutaneously.
16. A multi-biosensor device according to claim 12, further
comprising a layer comprising a biocompatible material on at least
a part of the surface of the biosensor or the multi-biosensor
device.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of Greek Patent
Application 20080100409, filed on Jun. 20, 2008, which is hereby
incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] The present invention relates to nanostructured composite
materials which are suitable for the application in biosensors, and
to biosensors comprising those nanostructured composite
materials.
[0003] A nanostructured composite material (nanocomposite) is a
composite material in which at least one of the phases has at least
one dimension which is of the order of nanometers.
[0004] A biosensor is a device that uses specific biochemical
reactions mediated by a receptor selected from the group consisting
of isolated enzymes, microorganisms, immunosystems, tissues,
organelles, whole cells, antibodies, nucleic acids and combinations
thereof to detect chemical compounds (analytes) usually by means of
electrical, thermal or optical signals. Within the biosensor, the
receptor is coupled to a transducer by means of immobilization.
DESCRIBED OF THE PRIOR ART
[0005] It has been suggested to immobilize the biochemical receptor
on a matrix formed of a nanostructured carbon material like carbon
nanotubes or carbon nanofibers (See, e.g. Vamvakaki, V; Tsageraki,
K. and Chaniotakis, N: Anal. Chem. 2006, 78, 5538-5542). Within a
biosensor generating electrical signals, nanostructured carbon
materials can fulfill a dual function i.e. they provide a matrix
for the immobilization of the receptor and at the same time act as
a transducer for the electrical signal generated by the biochemical
reaction. Thus, immobilization of the biochemical receptor on a
nanostructured matrix formed of a carbon material allows for a
direct electron transfer from the receptor to the immobilization
surface.
[0006] The use of carbon nanotubes in biosensors is limited by the
fact that the tubular nanotubes have closed shells which do not
allow for a high degree of functionalization. This is because
adsorption or covalent immobilization of a receptor can be achieved
only at the functionalized ends of the opened nanotubes. For this
reason, the amount of biochemical receptors, e.g. enzymes that can
be immobilized on carbon nanotubes is limited, resulting in
receptor-matrix composites with sensitivities and stability
properties that are not fully optimized.
[0007] On the other hand, carbon nanofibers, due to their more open
surface structure, are expected to have a much larger
functionalizable surface area. Carbon nanofibers are cylindrical
nanostructures comprising graphene layers arranged as stacked
cones, cups, or plates. Thus, for carbon nanofibers a significantly
higher ratio of the number of surface-active groups to the volume
can be expected compared to that of the glassy-like surface of the
carbon nanotubes. Additionally, nanofibers have a higher
conductivity than nanotubes of prior art, thus giving larger
signals to the underlying electrode surface (e.g. a metal wire) or
semiconductor substrate (in case of an integrated multi bio-sensor
ChemFet plate), resulting in a better signal to noise ratio, i.e.
providing more accurate and faster filtered results. However,
carbon nanofibers exhibit a remarkable hydrophobicity which is
detrimental to the adsorption of biochemical receptors.
SUMMARY OF THE INVENTION
[0008] The present invention provides a nanostructured composite
material comprising a matrix comprising carbon nanofibers, and a
biochemical receptor, especially an enzyme, immobilized in the
matrix comprising carbon nanofibers wherein the carbon nanofibers
have an increased amount of functional surface groups, especially
acidic functional groups, resulting in a more hydrophilic
character, thus providing for a reliable and durable immobilization
of a large number of receptor units.
[0009] The amount of surface functional groups can be increased by
subjecting the carbon nanofibers to an activation process
comprising treatment of the carbon nanofibers with oxidizing agents
before immobilization of the biochemical receptor thereon.
[0010] Accordingly, the nanostructured composite material of the
present invention comprises a matrix comprising carbon nanofibers,
and a biochemical receptor, especially an enzyme, immobilized in
the matrix comprising carbon nanofibers, characterized in that the
carbon nanofibers are activated, i.e. have been subject to an
oxidation treatment. Carbon materials which have been subject to an
activation treatment are characterized by a developed surface area,
formation of shallow and tiny pores and an increased number of
oxygen-containing surface functional groups, especially acidic
functional groups. Accordingly the hydrophilic character of the
surface of the carbon nanofibers is increased. Thus activated
carbon nanofibers provide a very large number of adsorption sites,
and can thus immobilize an increased amount of biochemical receptor
units.
[0011] Accordingly, the nanostructured composite material of the
present invention is obtained by a method comprising the steps of
activation of carbon nanofibers by treatment with an oxidizing
agent, adjustment of the pH value at the surface of the carbon
nanofibers to 7 and immobilization of a biochemical receptor on the
activated carbon nanofibers.
BRIEF DESCRIPTION OF THE DRAWING
[0012] In the following, the present invention will be described in
greater detail with reference to the accompanying figures, in
which:
[0013] FIG. 1 shows a schematic representation of a nanostructured
composite material according to a specific embodiment of the
present invention;
[0014] FIG. 2 shows SEM images of A) carbon nanofibers, B) carbon
nanofibers with a biomimetically synthesized silica matrix
thereon;
[0015] FIG. 3 displays the stability of a nanostructured composite
material according to a specific embodiment of the present
invention under continuous polarization (+300 mV, 25.degree.
C.);
[0016] FIG. 4 shows the results of a stability study of free and
immobilized AChE after incubation at 50.degree. C.;
[0017] FIG. 5 shows the results of a stability study of free and
immobilized AChE after incubation in protease solution (10
mg/mL);
[0018] FIG. 6 displays the stability of a nanostructured composite
material according to another specific embodiment of the present
invention under continuous polarization (+700 mV, 25.degree.
C.).
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0019] In the nanostructured composite materials of the present
invention, grahitized as well as non-graphitized carbon nanofibers
or mixtures thereof can be used.
[0020] Activation is carried out by applying a suitable oxidizing
agent and optional supply of additional thermal energy and/or
irradiation, e.g. UV irradiation. In a preferred method,
concentrated hydrogen peroxide is used as the oxidation agent.
Alternatively, activation can be carried out by treating the carbon
nanofibres with concentrated nitric acid or a mixture of
concentrated nitric and sulphuric acid, preferably under
ultrasonification. Further alternatively, activation can be carried
out by exposing the carbon nanofibers to oxygen or any other
suitable oxidation agent, e.g. potassium permanganate (KMnO.sub.4)
or ruthenium tetroxide (RuO.sub.4), preferably at elevated
temperature.
[0021] It is important that after the activation treatment
neutralization of the pH at the surface of the activated carbon
nanofibers is performed. Neutralization of the pH value ensures
that the activity of the enzyme or other biochemical receptor which
is to be immobilized on the oxidized carbon nanofibers will remain
intact and will not be damaged due to very low or very high pH
values.
[0022] As a result of the activation procedure, the number of
surface functional groups, especially the number of acidic surface
functional groups, increases at least by a factor of three.
Preferably, the amount of acidic surface functional groups in the
activated carbon nanofibers is at least 6*10.sup.-5 mol/g, as
determined by direct titration with a solution of 0.01 mol/l sodium
hydroxide. Higher amounts of surface functional groups can be
achieved, depending on the selected oxidation agent and the
duration of the oxidation treatment. Typical acidic surface
functional groups are carboxyl, lactone, and phenol groups. When
hydrogen peroxide or a mixture of concentrated nitric and sulphuric
acid are used as the oxidation agent, the amount of carboxylic
groups as determined by titration with a 0.01 mol/l solution of
sodium hydrogen carbonate is at least about a fourth of the overall
number of acidic surface functional groups. However, the fractions
of the different types of acidic functional groups depend on the
selected oxidation agent(s) and the duration of the oxidation
treatment.
[0023] The fibrous structure of the carbon nanofibers is retained
after the oxidation treatment. The carbon nanofibers usually form
an entangled, spaghetti-like network. Typically, the oxidized
nanofibers have a mean diameter of 60 to 200 nm while their length
is in the order of tens of micrometers (usually >20 .mu.m).
[0024] The nanostructured composite material of the invention is
obtained by immobilization of a biochemical receptor in a matrix
comprising carbon nanofibers. Any biochemical receptor capable of
generating a signal which can be transduced by carbon nanofibers
can be applied. Preferably, the biochemical receptor is a receptor
which is involved into an electrochemical reaction (redox reaction)
with the analyte, thus generating an electrical signal which is
transduced by the activated carbon nanofibers. The biochemical
receptor is selected from the group consisting of isolated enzymes,
microorganisms, immunosystems, tissues, organelles, whole cells,
antibodies, nucleic acids and combinations thereof. Preferably,
enzymes are used as receptors because the reaction of an enzyme
with an analyte is highly specific.
[0025] Due to the activation treatment preceding the immobilization
of the receptor, both the porosity and the hydrophilicity of the
surface of the carbon nanofibers has increased. The more developed
surface might enable deeper penetration of a biochemical receptor
like an enzyme into the spaghetti like nanofiber structure, while
the presence of an increased amount of surface functional groups
facilitates adsorption of an increased amount of receptor units,
and stronger electrostatic adhesion thereof. Thus the amount of the
immobilized biochemical receptor units and the stability thereof is
increased as a result of the oxidation treatment of the carbon
nanofibers.
[0026] In a preferred embodiment, the receptor comprises the enzyme
glucose oxidase (GOx). A nanostructured composite comprising a
matrix comprising activated carbon nanofibers and a biochemical
receptor comprising the enzyme glucose oxidase immobilized in said
matrix is suitable for application in a biosensor for continuous
monitoring of glucose. Such devices are needed for diabetes
care.
[0027] Upon immobilization of the biochemical receptor in a matrix
comprising activated carbon nanofibers, deep penetration of the
receptor into the matrix structure is facilitated by ultrasonic
treatment.
[0028] Even though adsorption or covalent immobilization of the
biochemical receptor onto the surface of carbon nanofibers which
have been subject to an oxidation treatment provides a significant
stabilization effect, this is not sufficient for long-term
implantable biosensor systems. Since enzymes and other biochemical
receptors are not very stable outside their biological environment,
they are prone to denaturation or inactivation by even very small
changes in pH, temperature, or the presence of traces of organic
solvents and detergents usually employed for the construction of a
biosensor. Another very important factor drastically influencing
the biosensor stability is the presence of proteases, which are
found in virtually any analyte solution. Finally, leaching of the
receptor away from the matrix is another problem, causing signal
drift, and decreasing the biosensor sensitivity over time. All
these parameters have a detrimental effect on the analytical
characteristics of the biosensor, decreasing both the operational
and storage stabilities of biosensors, effects that are very
profound when these biosensors are applied in harsh environments,
or in-vivo.
[0029] Therefore, in a preferred embodiment of the present
invention, the nanostructured composite is further stabilized by
deposition of a biomimetically synthesized silica matrix onto the
activated carbon nanofibres with the receptor immobilized
thereon.
[0030] The term "biomimetic" refers to a laboratory procedure
designed to imitate a natural chemical process and to a compound
that mimics a biological material in its structure and/or
function.
[0031] In biomimetic synthesis, after immobilization of the
biochemical receptor, a scaffold comprising of a protein or a
polymer is deposited as an intermediate layer onto the surface of
the nanostructured composite, which directs growth of a matrix
comprising silica precipitated from a precursor solution. The
silica matrix is mainly formed of a network of fused silica
particles.
[0032] Methods for the biomimetic synthesis of silica are known in
the art, see e.g. Siddharth V. Patwardhan, Stephen J. Clarson and
Carole C. Perrya: On the role(s) of additives in bioinspired
silicification Chem. Commun., 2005, 1113-1121. In this document, a
plurality of suitable scaffold-forming proteins and polymers are
listed, which can be used in the method of production of a
nanostructured composite material according to the present
invention. Examples of suitable scaffold forming materials are e.g.
poly-L-lysine, polyethylenimine, polyallylamine, cellulose, R5
peptide, poly-L-arginine, poly-L histidine, lysine oligomers,
poly(allylamine hydrochloride).
[0033] Accordingly, a method for producing a nanostructured
composite material according to the preferred embodiment described
above comprises the steps of activation of the carbon nanofibers by
treatment with an oxidizing agent, adjustment of the pH value at
the surface of the carbon nanofibers to 7, immobilization of a
biochemical receptor on the activated carbon nanofibers, deposition
of a scaffold comprising a protein or polymer onto the carbon
nanofibers and precipitation of a silica matrix onto the
scaffold.
[0034] A schematic representation of a nanostructured composite
comprising oxidized carbon nanofibers, a biochemical receptor
immobilized thereon and a silica matrix precipitate on a scaffold
comprising a polymer or a peptide is shown in FIG. 1. Receptor
molecules are attached to the functional groups at the surface of a
carbon nanofiber strand. The composite formed of oxidized carbon
nanofibers and the biochemical receptor immobilized thereon is
encapsulated by a matrix comprising biomimetically synthesized
silica (represented by dashed lines) on a scaffold comprising a
polymer or a protein. The carbon nanofiber strand provides
electrical contact to the surface of a working electrode, which is
connected to electronics for signal conditioning and display. The
oxidized carbon nanofibers facilitate the immobilization and first
stabilization of the receptor, while the silica matrix acts as an
encapsulating protection layer. Thus the oxidized carbon nanofibers
in combination with the biomimetically synthesized silica matrix
provide ideal architectures for the design and development of
highly stable biosensor systems.
[0035] Furthermore, since the silica protective layer itself is
substantially free of receptor, the substrate (analyte) can freely
penetrate through the pores in the silica layer, eliminating the
diffusion of other redox interfering species, as well as cells and
large proteins. Due to the immobilization of the receptor directly
on the oxidized carbon nanofibres a direct electron transfer
between the receptor and the carbon nanofiber transducer is
achieved, mediating the signal very efficiently to the electronics.
Thus fast kinetics are obtained, free from interference and signal
drifts, with increased receptor stabilization over time, resulting
in a better performing biosensor with accuracy and stability over
an increased time and against chemical "noise" substances, so that
it can according to our measurements be used in-vivo and in a
closed loop infusion setup
[0036] The silica matrix must be thick enough for sufficient
protection, but thin enough in order to allow for the substrate
(glucose in the case of a receptor comprising Glucose Oxidase) to
reach the active center of the receptor. The pores must also be
small enough not to allow for any proteases to reach the receptor.
Based on TEM images, the thickness of this silica matrix ranges
between 0.5 and 100 nm. The porosity is controlled by the thickness
of the silica. The thicker the silica matrix the lower is the
porosity.
[0037] In a further preferred embodiment, porin molecules or
synthetic nanoporous materials are incorporated into the silica
matrix of the nanostructured composite material. Porins are beta
barrel proteins that cross a cellular membrane and act as a pore
through which molecules can diffuse. Unlike other membrane
transport proteins, porins are large enough to allow passive
diffusion--i.e., they act as channels that are specific to
different types of molecules. They are present in the outer
membrane of Gram-negative bacteria, the mitochondria, and the
chloroplast. Porins incorporated into the silica matrix behave like
selective channels since only molecules of small diameter can
freely diffuse into the interior of the composite and reach the
biochemical receptor immobilized on the oxidized carbon nanofibers
while large molecules like proteases cannot pass the channel so
that they do not reach the receptor.
[0038] The present invention is further directed to biosensors,
especially electrochemical biosensors, comprising the
nanostructured composite described herein above.
[0039] An electrochemical biosensor can be constructed either as a
two electrode sensor or as a three electrode sensor. A two
electrode sensor comprises a working electrode, preferably made of
platinum in contact with the nanostructured composite material
described herein above, and a reference electrode preferably a
Ag/AgCl electrode, in direct contact with analyte or living tissue.
In a three-electrode sensor a third electrode, preferably made of
platinum, too, is located in close proximity to the working
electrode. The nanostructured composite material may be deposited,
precipitated or coated onto the working electrode, thus forming a
layer on the surface of the working electrode. These methods are
known in the art and well established. For instance, the electrode
surface can be pretreated mechanically by a roughening treatment
(e.g. by grit-blasting) and/or chemically (e.g. by etching) in
order to facilitate adherence of the carbon nanofibres.
[0040] The related electronics and housing of biosensors is known
in the art and therefore does not need to be described here in
detail.
[0041] In a preferred embodiment, the biosensor is a single needle
biosensor which is implantable for a subcutaneous measurement
lasting several days as long as such needle can be accepted without
fear of contamination, usually a week.
[0042] A further preferred embodiment comprises a plurality of
biosensors (multi-biosensor device) with at least one of the
biosensors comprising the nanostructured composite described herein
above. For instance the device is formed of a plate comprising
several islands of biosensors. An exemplary application is an
implantable pump comprising a plurality of biosensors on the
surface thereof.
[0043] In a preferred multi-biosensor device, each biosensor is
adapted to be individually activated, i.e. means are provided to
achieve individual activation of each individual biosensor. This
activation can be achieved by removing a protective covering layer
from the individual sensor to be activated and electronic
activation of the uncovered sensor. Preferably, the removable
covering layer is substantially impermeable to fluid, since contact
with body fluids might degrade the biochemical receptor of the
sensor even when the sensor is not electronically activated yet.
For instance, activation of individual biosensors can be initiated
by a process triggered electrically (i.e. by application of an
electric current or potential), like electroerosion. Therefore, in
a preferred embodiment the biosensors are provided with a covering
layer comprising an electrically erodible polymer which is
substantially impermeable to fluids, and means are provided to
achieve sequential activation of the individual biosensors by
electroerosion of said electrically erodible polymer and electronic
activation of the uncovered sensor. Suitable removable polymers are
known in the art, see, e.g. Kwon et al., "Electrically erodible
polymer gel for controlled release of drugs", Nature, 354:291-93
(1991).
[0044] Sequential activation of the biosensors (one after the
other) allows for significant increase of the time the device is
capable of providing accurate measurement results, compared to a
device comprising a single biosensor only. Since the multi-sensor
device comprises a plurality of biosensors, in the case that the
active biosensor appears to cease working accurately, one of the
other biosensors can be activated and take over from the one being
activate so far.
[0045] Preferably the biosensor or the multi-biosensor device is
implantable into a being, especially a human being. Immobilization
of the receptor into the matrix comprising activated carbon
nanofibers, and the additional protection provided by the
biomimetically synthesized silica matrix and/or a removable
substantially impermeable covering layer as described above and/or
a further coating comprising a biocompatible material (see below)
ensure stability of the receptor over a long period of time even in
the implanted state. Thus the accuracy of the biosensor is not
deteriorated when implanted in a living being. Preferably, the
biosensors or multi-biosensor devices are implantable by means of
minimal invasive technique. In a preferred embodiment, the
multi-biosensor device can be implanted subcutaneously.
[0046] In a further preferred embodiment the biocompatibility of
the biosensor or the multi-biosensor device is improved by
providing a layer comprising a biocompatible material on at least a
part of the surface of the biosensor of the multi-biosensor device.
Preferably the layer comprises a biocompatible polymer or a
combination of different biocompatible polymers. A suitable
biocompatible polymer material is polyxylene. Other suitable
materials are sulfonated polytetrafluoroethylene,
polytetrafluoroethylene, preferably expanded
polytetrafluoroethylene, polyester, porous silicone, hydrophilic
polyvinylidene fluoride, mixed cellulose esters, polyvinyl
chloride, polypropylene, polyethylene, polysulfone,
polyethersulfone, cellulose acetate, nylon, polycarbonate, and
polymethylmethacrylate. The biocompatible layer facilitates
attachment of the device to the fibrous tissue build up in response
of the organism to the introduction of a foreign material. Thus the
biocompatible layer acts an angiogenic layer i.e. it promotes and
maintains the formation and healing of blood microcirculation
capillaries around the implanted device, thus maintaining the blood
circulation in the proximity of the implanted device and providing
realistic amounts of the analyte. The angiogenic layer is porous so
that the newly growing tissue penetrates into the angiogenic
material, thus interlocking with the implanted sensor device. In
the region proximate to the electrodes (e.g. around the tip of a
needle-type sensor), the biocompatible layer may be designed as a
composite angiogenic and filtration layer, i.e. as a
multifunctional layer. The filtration layer controls access to the
receptor, i.e. it allows passing of the analyte, but keeps out
proteases, tissue macrophages and any other species which could
destabilize or degrade the enzyme or other receptor immobilized on
the carbon nanofibers. Thus in addition to the silica matrix,
further protection of the receptor is achieved. Layers of different
materials can be combined to achieve the combination of multiple
functions like angiogenese and filtration. According to their
related functions in the multi-layer composite, the angiogenic
layer is oriented outwards from the sensor, while the semipermeable
filtration layer is oriented towards the interior of the sensor
device. Preferably, the semipermeable filtration layer has smaller
pores than the angiogenic layer. Suitable materials for the
filtration layer are polyurethane, polypropylene, polysulfone,
polytetrafluoroethylene and polyethylene terephtalate. The
filtration layer itself can be a combination of layers different in
composition and/or porosity.
[0047] Thus, in a preferred embodiment of an implantable sensor
device or multisensory device, e.g. an implantable pump comprising
a plurality of biosensors on the surface thereof, the outermost
(external) surface of the device comprises an angiogenic layer as
described above, optionally with removable impermeable coverings
provided on the individual sensor islands beneath the outer
angiogenic layer. Thus starting from the working electrode as the
innermost part, the sensor device might comprise the following
layer sequence: (i) metallic surface of the working electrode, (ii)
matrix comprising oxidized carbon nanofibers with the receptor
immobilized therein, (iii) optionally a biomimetically synthesized
silica matrix as described above, (iv) optionally a removable cover
layer which is non-permeable to fluids and which is removed when
the activation of the sensor is initiated as described above, (v)
optionally an angiogenic layer, which might also cover the whole or
parts of the surface of the housing.
[0048] In a preferred embodiment, the receptor of the biosensor
comprises the enzyme glucose oxidase (GOx). Thus the biosensor is
suitable as a continuous glucose monitoring device for diabetes
care. Existing sensing devices are not enough accurate and have a
drift in time, so that it was not possible so far to replace the
patient's centric manual adjustment of the insulin level by an
automatic algorithmic process (closed loop), although algorithms
were progressing. It is also a fact that the automatic process
needs a remote monitoring from professionals and large databases'
knowledge in an external secondary and more "knowledgeable"
algorithmic loop, and cannot be left alone in the local algorithmic
process or patient's knowledge.
[0049] The improved accuracy and stability of the biosensor of the
present invention permit the realization of closed loop infusion,
with local algorithmic loop. A web and GSM/GPRS based
telemonitoring system as described in the U.S. Pat. No. 7,161,484
B2 and in the European Patent Application EP 1 385 420 A0 (both
assigned to Micrel Medical Devices S.A.) can preferably make the
measurement results available for a distant physician or paramedics
to validate algorithmic insulin infusion control, and distant
closed loop algorithmic decisions based on large databases located
in distant servers. The local loop is a short term infusion control
(needed in case SGM or WiFi or WiMax transmission is impossible for
some time), while the web based loop is a longer term control and
validation of local loop control algorithmic parameters. An RFID
type of communication between biosensor and extracorporeal analyzer
is the preferred communication type.
[0050] It goes without saying that features of different preferred
embodiments described herein can be combined, if desired.
EXAMPLES
Activation Treatment of Carbon Nanofibers
[0051] 150 mg of carbon nanofibers (type HTE from Electrovac AG,
Austria) are dispersed in 150 ml of a solution containing 30 w/w %
of hydrogen peroxide. The dispersion is refluxed for two hours,
while irradiated with a UV-lamp. The oxidised CNFs are rinsed with
0.01 mol/l NaOH solution and deionized water until neutral pH. The
acid treated CNF are dried under vacuum at 100.degree. C.
overnight.
[0052] In another run, graphitized carbon nanofibers (type GFE from
Electrovac AG, Austria) were treated with a mixture of concentrated
nitric and sulphuric acid (volumic ratio 1:3) under
ultrasonification.
[0053] The amount of acidic surface functional groups is determined
by direct titration with a solution of 0.01 mol/l sodium hydroxide.
The amount of carboxylic groups is determined by titration with a
0.01 mol/l solution of sodium hydrogen carbonate. Data obtained
from carbon nanofibers with and without activation treatment are
compiled in table 1:
TABLE-US-00001 Carbon nanofibers type HTE Carbon nanofibers type
GFE oxidized with oxidized with original H.sub.2O.sub.2 original
H.sub.2SO.sub.4/HNO.sub.3 Acidic 16.8 .times. 10.sup.-6 62.4
.times. 10.sup.-6 26.2 .times. 10.sup.-6 345 .times. 10.sup.-6
groups (mol/g) COOH -- 17.9 .times. 10.sup.-6 -- 81.4 .times.
10.sup.-6 groups (mol/g)
[0054] As a consequence of the oxidative treatment the amount of
acidic surface groups has surface groups has substantially
increased.
[0055] Immobilization of Enzymes on Oxidized Carbon Nanofibers.
[0056] Glucose oxidase was immobilized on oxidized carbon
nanofibers (type HTE) which were prepared as described above. For
comparison, in a parallel experiment glucose oxidase was
immobilized on non-oxidized carbon nanofibers. Deep penetration of
glucose oxidase into the matrix structure is facilitated by
ultrasonic treatment.
[0057] The amount of the enzyme glucose oxidase adsorbed was found
to be 14 and 18 units/g for non-oxidized and oxidized HTE carbon
nanofibers respectively. Thus, due to the oxidation of the carbon
nanofibers, the amount of immobilized enzyme increased by more than
one third.
[0058] Preparation of Nanostructured Composites Comprising a
Biomimetically Silica Matrix.
[0059] To demonstrate the stabilizing effect of the combination of
an immobilizing matrix comprising oxidized carbon nanofibers and an
additional encapsulating matrix of biomimetically synthesized
silica, the inherently unstable enzyme acetylcholinesterase from
Drosophila melanogaster (Dm. AChE) is chosen as the receptor. For
comparison two different nanostructured composites were studied, a
first one comprising the enzyme AChE immobilized on carbon
nanofibers of the type HTE (Electrovac AG, Austria) which were
subjected to an oxidation treatment as described above (further
referred to as CNF/AChE); and a second one comprising AChE
immobilized on carbon nanofibers of the type HTE (Electrovac AG,
Austria) which were subjected to an oxidation treatment as
described above, and a biomimetically synthesized silica matrix
(further referred to as silica/CNF/PLL/AChE).
[0060] The precipitation mixture for silica/CNF/PLL/AChE consisted
of 100 .mu.L of poly-L-lysine solution (1.0 mM in nanopure
H.sub.2O), 500 .mu.L of immobilized Dm. AChE (immobilized on
oxidized carbon nanofibers) phosphate solution, 300 .mu.L of
nanopure water and 100 .mu.L of freshly prepared stock solution
obtained by hydrolysis of TMOS (tetramethylorthosilicate) at a
concentration of 1.0 M with 1.0 mM HCl solution. The mixture was
mixed in a vortex slowly, followed by centrifugation at 4,000 rpm.
The products obtained were washed twice with nanopure water.
Finally, the samples obtained were dried at room temperature
overnight.
[0061] SEM Studies
[0062] FIG. 2A shows the spaghetti-like network formed of carbon
nanofibers. As can be seen on FIG. 2B the precipitation of the
biomimetically synthesized silica matrix introduces silica
particles into the cavities of the spaghetti-like networks and
along the fiber axis of the carbon nanofibers. Thus, the carbon
nanofibers and the silica matrix are interlocked, resulting in a
strong attachment of the silica matrix to the carbon
nanofibers.
[0063] Stability Tests
[0064] The stabilizing effect of silica/carbon nanofiber
architecture to the enzyme AChE was examined by measuring the
sensitivity of the corresponding biosensor under continuous
polarization over time. It was found that successive additions of
acetylthiocholine lead to an increase of the anodic current, which
is attributed to the oxidation of the enzyme's hydrolysis product
thiocholine on the surface of the electrode. Thus the sensitivity
of the biosensor is directly related to the enzyme activity,
allowing the continuous monitoring of enzyme activity. The initial
sensitivity of the biosensor comprising the composite
silica/CNF/PLL/AChE is calculated to be 7.8 .mu.A/mM. The response
of biosensor is linear at acetylthiocholine concentrations from
0.04 mM to 0.3 mM. The response time of the biosensor is between 2
to 4 minutes and the sensor-to-sensor reproducibility is less than
10% RSD (n=3).
[0065] As shown in FIG. 3, the biosensor has a remaining activity
of 100% under continuous polarization for 13 days at 25.degree. C.,
biased at +300 mV vs. Ag/AgCl. The initial increase of the
biosensor sensitivity is a well-known phenomenon in biosensor
systems, attributed to changes of the enzyme's three-dimensional
structure inside the immobilization matrix upon rehydration. Of
considerable interest is the fact that the biosensor presented a
remaining activity of 70% after 3.5 months of continuous
polarization, while the free enzyme and the enzyme immobilized on
carbon nanofibers (CNF/AChE) showed a considerable decrease of
their activity to 50% after 4 and 25 days respectively (data not
shown). It is thus evident that the silica/nanofiber architecture
provides an environment within which relatively unstable enzymes
are highly stabilized.
[0066] The free enzyme and the nanostructured composites were also
examined for the stability of the enzyme activity under elevated
temperatures (50.degree. C.). FIG. 4 shows the stability of the
free enzyme, the enzyme immobilized on oxidized carbon nanofibers
and the enzyme in the composite material silica/CNF/PLL/AChE, as
calculated by the residual activity as a function of incubation
time. The stability is expressed as the time at which the enzymatic
activity is reduced to 50% (t.sub.50) compared to the initial
activity. The t.sub.50 of the free enzyme is measured to be only 4
min, while the immobilization of the enzyme on the oxidized carbon
nanofibers provides a 20fold increase in the stabilization as the
t.sub.50 increases to about 80 min. If the carbon nanofibers with
the immobilized AChE thereon are encapsulated by means of a
biomimetically synthesized silica matrix, then the t.sub.50 of the
system increases to 150 min. It is thus evident that enzyme
immobilization on oxidized carbon nanofibers with subsequent silica
precipitation provides superior enzyme stabilization against
thermal shock and protein denaturation.
[0067] Finally the stability of the free and immobilized enzyme
against external proteolysis was investigated. The resistance to
proteolysis is a direct proof of the physical protection provided
by the architecture of the nanostructured composite of the present
invention. Since proteases can be found in virtually any non
sterile solution, their effect on the biosensor element stability
is a very important study needed to be performed in order to prove
the ability of the system to be applied to continuous analysis of
real samples. As shown in FIG. 5 the free enzyme loses its activity
completely after only 1 h in protease (pronase) solution, while the
enzyme immobilized onto the oxidized carbon nanofibers retains 37%
of its initial activity after 2 h in the same solution. On the
other hand, when the AChE enzyme is additionally protected by the
silica matrix, its activity is virtually unaffected even after 12
hours of exposure to the protease.
[0068] According to the results shown in FIGS. 3 to 5,
immobilization on a nanostructured matrix of oxidized (activated)
carbon nanofibers provides a stabilization which is effective even
with an inherently unstable enzyme like AChE. The presence of a
biomimetically synthesized silica matrix further increases the
stability of the enzyme. This result might be attributed to a
synergy effect of both nanostructured matrix phases, i.e. the
oxidized carbon nanofibers and the biomimetically synthesized
silica matrix.
[0069] Stability Test of a Glucose Sensor
[0070] In the same manner as described above for the nanostructured
composite silica/CNF/PLL/AChE a nanostructured composite material
comprising glucose oxidase (GOx) immobilized on a matrix of
oxidized carbon nanofibers encapsulated in a biomimetically
synthesized silica matrix is obtained.
[0071] The sensitivity of that biosensor is directly related to the
activity of GOx, thus allowing the continuous monitoring of glucose
oxidase activity under continuous polarization as described above.
After three months an activity of 103% is found, and the experiment
is still going on. This result (see FIG. 6 showing a plot of the
remaining enzyme activity vs. time) shows that the nanostructured
composite is suitable for the application in a biosensor for
long-term glucose monitoring as it is necessary in diabetes
care.
* * * * *