U.S. patent application number 12/579084 was filed with the patent office on 2010-09-30 for sensor for detecting stem cell differentiation based on electrochemical methods.
This patent application is currently assigned to Industry-University Cooperation Foundation Sogang University. Invention is credited to Jeong-Woo Choi, Cheol-Heon Yea.
Application Number | 20100243479 12/579084 |
Document ID | / |
Family ID | 41462436 |
Filed Date | 2010-09-30 |
United States Patent
Application |
20100243479 |
Kind Code |
A1 |
Choi; Jeong-Woo ; et
al. |
September 30, 2010 |
SENSOR FOR DETECTING STEM CELL DIFFERENTIATION BASED ON
ELECTROCHEMICAL METHODS
Abstract
This invention relates to a sensor for detecting a stem cell
differentiation, including (a) an electrode; and (b) a substrate of
an alkaline phosphatase. The phosphorylation or dephosphorylation
of the substrate for an alkaline phosphatase as a stem cell
undifferentiation marker which dephosphorylates its substrate may
be measured using an electrical signal in the present sensor.
Therefore, the sensor of the present invention enables to
electrically detect a stem cell status in a high-throughput manner
and to determine the stem cell differentiation.
Inventors: |
Choi; Jeong-Woo; (Seoul,
KR) ; Yea; Cheol-Heon; (Seoul, KR) |
Correspondence
Address: |
EDWARDS ANGELL PALMER & DODGE LLP
P.O. BOX 55874
BOSTON
MA
02205
US
|
Assignee: |
Industry-University Cooperation
Foundation Sogang University
Seoul
KR
|
Family ID: |
41462436 |
Appl. No.: |
12/579084 |
Filed: |
October 14, 2009 |
Current U.S.
Class: |
205/777.5 ;
204/403.14 |
Current CPC
Class: |
C12N 5/0606 20130101;
C12Q 1/42 20130101; C12Q 1/005 20130101 |
Class at
Publication: |
205/777.5 ;
204/403.14 |
International
Class: |
C12Q 1/42 20060101
C12Q001/42; G01N 33/50 20060101 G01N033/50 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 27, 2009 |
KR |
10-2009-0026353 |
Claims
1. A sensor for detecting a stem cell differentiation, comprising:
(a) an electrode; and (b) a substrate for an alkaline
phosphatase.
2. The sensor according to claim 1, wherein the electrode is
selected from the group consisting of a gold, copper, glass carbon,
platinum and Ag/AgCl electrode.
3. The sensor according to claim 1, wherein the substrate is
selected from the group consisting of 1-naphthyl phosphate, calcium
phosphate, phenyl phosphate, 2-aminophenyl phosphate, 4-aminophenyl
phosphate, p-aminophenyl phosphate, 3-indoxyl phosphate,
5-bromo-4-chloro-3-indoxyl phosphate, 4-methylumbelliferyl
phosphate, 6-chloro-3-indoxyl phosphate and hydroquinone
diphosphate.
4. The sensor according to claim 1, wherein the stem cell is an
embryonic stem cell or a germ stem cell.
5. The sensor according to claim 1, wherein the stem cell comprises
an alkaline phosphatase where the stem cell is an undifferentiated
stem cell; and the stem cell comprises no alkaline phosphatase
where the stem cell is a differentiated stem cell.
6. The sensor according to claim 1, wherein the sensor measures the
phosphorylation or dephosphorylation of the substrate for alkaline
phosphatase on the stem cell through an electrical signal to
determine the differentiation of the stem cell.
7. The sensor according to claim 6, wherein the dephosphorylated
substrate is selected from the group consisting of 1-naphtol,
calcium ion, phenol, 2-aminophenol, 4-aminophenol, p-aminophenol,
3-indoxol, 5-bromo-4-chloro-3-indoxol, 4-methylumbelliferol,
6-chloro-3-indoxol and hydroquinone.
8. A method for determining a stem cell differentiation, comprising
the steps of: (a) preparing the sensor for detecting the stem cell
differentiation according to claim 1; (b) contacting cells of
interest to the sensor; and (c) measuring the phosphorylation or
dephosphorylation of a substrate for alkaline phosphatase on the
cells through an electrical signal.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims under 35 U.S.C. .sctn.119(a) the
benefit of Korean Application No. 10-2009-0026353 filed Mar. 27,
2009, the entire contents of which are incorporated herein by
reference.
BACKGROUND
[0002] 1. Technical Field
[0003] The present invention relates to a sensor for detecting a
stem cell differentiation, including: (a) an electrode; and (b) a
substrate for an alkaline phosphatase.
[0004] 2. Background Art
[0005] Cell chip technology is a promising tool for utilization in
cell based assays. There are two kinds of cell detection systems
for these chips. These systems are based on optical detection and
electrical (electrochemical) detection respectively. Optical
systems allow one to observe visual changes of the cells and have
high sensitivity and selectivity. Optical systems are limited by
size of the instrument and the process of transforming the optical
signal into an electrical signal [1-2].
[0006] While electrical cell detecting systems are relatively less
developed than optical systems, they have the ability to be
miniaturized and the signals are easily analyzed. There have been
some attempts to analyze living cells as electrochemically dynamic
systems by detecting electron generation and electron transfer at
the interface [3].
[0007] Living cells have been studied by many electrochemical
situations such as electron transfer at electro active centers in
cells, open circuit potential at the cell/sensor interface,
electric cell-substrate impedance sensing (ECIS), scanning
electrochemical microscopy (SECM) to obtain images of the
respiratory activity of collagen-embedded living cells,
electrochemical impedance spectroscopy (EIS), and activation of an
oxygen electrode [4-11].
[0008] However, there has been no attempt to detect the
differentiation of MES cells using electrical or electrochemical
systems.
[0009] The above information disclosed in this Background section
is only for enhancement of understanding of the background of the
invention and therefore it may contain information that does not
form the prior art that is already known in this country to a
person of ordinary skill in the art.
[0010] Throughout this application, various publications and
patents are referred and citations are provided in parentheses. The
disclosures of these publications and patents in their entities are
hereby incorporated by references into this application in order to
fully describe this invention and the state of the art to which
this invention pertains.
SUMMARY
[0011] The present inventors have intensive studies to develop a
novel sensor and method for detecting a stem cell differentiation
based on electrochemical methods. As results, we have discovered
that the phosphorylation or dephosphorylation of the substrate for
alkaline phosphatase as a stem cell undifferentiation marker which
dephosphorylates its substrate could be measured using an
electrical signal to determine a stem cell differentiation in an
electrochemical manner.
[0012] Accordingly, it is an object of the invention to provide a
sensor for detecting a stem cell differentiation.
[0013] It is another object of this invention to provide a method
for determining a stem cell differentiation.
[0014] Other objects and advantages of the present invention will
become apparent from the detailed description to follow taken in
conjugation with the appended claims and drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 schematically represents the electrochemical
detection system in which 1-naphthyl phosphate (NP) as a substrate
of an alkaline phosphatase (AP) involved in the sensor of the
present invention is changed into 1-naphtol by the alkaline
phosphatase of a mouse embryonic stem cell undifferentiated.
[0016] FIG. 2a represents a cyclic voltammogram of 1-NP used as a
substrate in the sensor of the present invention and FIG. 2b
represents a linear plot of reduction current peak as increasing
concentration of 1-NP.
[0017] FIG. 3a represents a cyclic voltammogram of mouse embryonic
stem cells according to addition of 1-NP and FIG. 3b represents a
linear plot of reduction current peak as increasing concentration
of MES cells. Data are presented as the mean.+-.standard deviation
of three different experiments. Arrow indicates increasing cell
number.
[0018] FIG. 4 is a linear plot of reduction current peak as
increasing concentration of (a) differentiated MES cells and (b)
undifferentiated MES cells. The scan rate was 100 mV/s. The
temperature was 37.+-.0.5.degree. C. and the CO.sub.2 was 5%. Data
are presented as the mean.+-.standard deviation of three different
experiments.
[0019] FIG. 5a represents a viability test according to 1 mM 1-NP
treatment. Data are presented as the mean of three different
experiments. FIG. 5b-5c represent AP staining assay of (b)
undifferentiated MES cells and (c) MES cells treated with 1-NP.
DETAILED DESCRIPTION
[0020] In one aspect of this invention, there is provided a sensor
for detecting a stem cell differentiation, including: (a) an
electrode; and (b) a substrate for an alkaline phosphatase.
[0021] The present inventors have intensive studies to develop a
novel sensor and method for detecting a stem cell differentiation
based on electrochemical methods. As results, we have discovered
that the phosphorylation or dephosphorylation of the substrate for
alkaline phosphatase as a stem cell undifferentiation marker which
dephosphorylates its substrate could be measured using an
electrical signal to determine a stem cell differentiation in an
electrochemical manner.
[0022] The sensor for detecting the stem cell differentiation of
the present invention is constituted of a conventional
three-electrode system known to those ordinarily skilled in the
art, which includes a working electrode, a counter electrode and a
reference electrode.
[0023] According to a preferable embodiment, the electrode involved
in the sensor of the present invention is selected from the group
consisting of a gold, copper, glass carbon, platinum and Ag/AgCl
electrode. More preferably, the electrode includes a gold, copper
or glass electrode as the working electrode, a platinum electrode
as the counter electrode, and a Ag/AgCl electrode as the reference
electrode, and most preferably a gold electrode as the working
electrode, a platinum electrode as the counter electrode, and a
Ag/AgCl electrode as the reference electrode.
[0024] The alkaline phosphatase used in the present invention
refers to a marker representing an undifferentiation status of stem
cells.
[0025] The substrate for alkaline phosphatase contained in the
sensor of the present invention may include various compounds
containing a phosphate group known to those ordinarily skilled in
the art, preferably 1-naphthyl phosphate, calcium phosphate, phenyl
phosphate, 2-aminophenyl phosphate, 4-aminophenyl phosphate,
p-aminophenyl phosphate, 3-indoxyl phosphate,
5-bromo-4-chloro-3-indoxyl phosphate, 4-methylumbelliferyl
phosphate, 6-chloro-3-indoxyl phosphate or hydroquinone
diphosphate, more preferably 1-naphthyl phosphate, phenyl
phosphate, 2-aminophenyl phosphate, 4-aminophenyl phosphate or
3-indoxyl phosphate, and most preferably 1-naphthyl phosphate.
[0026] In particular, 1-naphthyl phosphate as a substrate for
alkaline phosphatase is a compound in which the phosphate group is
linked to two benzene rings (FIG. 1), and is dephosphorylated by
alkaline phosphatase present in undifferentiated stem cells so as
to be changed into 1-naphtol. It is demonstrated that the
electrochemical characteristics of the modified substrate have
quite different to those of the pre-modified substrate (FIG.
2).
[0027] According to another preferable embodiment, the stem cell
used in the sensor of the present invention is an embryonic stem
cell or a germ stem cell, and more preferably an embryonic stem
cell.
[0028] According to still another preferable embodiment, the stem
cell used in the sensor of the present invention is a human or
mouse-derived stem cell, and more preferably a mouse-derived stem
cell.
[0029] According to further still another preferable embodiment,
the stem cell used in the sensor of the present invention includes
an alkaline phosphatase where the stem cell is an undifferentiated
stem cell; and the stem cell includes no alkaline phosphatase where
the stem cell is a differentiated stem cell.
[0030] According to another preferable embodiment, the sensor of
the present invention measures the phosphorylation or
dephosphorylation of alkaline phosphatase substrate using an
electrical signal to determine the differentiation of the stem
cell.
[0031] According to still another preferable embodiment, the
substrate dephosphorylated by alkaline phosphatase in the present
invention includes 1-naphtol, calcium ion, phenol, 2-aminophenol,
4-aminophenol, p-aminophenol, 3-indoxol,
5-bromo-4-chloro-3-indoxol, 4-methylumbelliferol,
6-chloro-3-indoxol or hydroquinone, more preferably 1-naphtol,
phenol, 2-aminophenol, 4-aminophenol or 3-indoxol, and most
preferably 1-naphtol.
[0032] In another aspect of this invention, there is provided a
method for determining a stem cell differentiation, comprising the
steps of: (a) preparing the sensor for detecting the stem cell
differentiation; (b) contacting cells of interest to the sensor;
and (c) measuring the phosphorylation or dephosphorylation of a
substrate for alkaline phosphatase on the cells through an
electrical signal.
[0033] To examine whether the stem cells are differentiated or not,
the method according to the step of this invention is minutely
explained as follows:
[0034] (a) The step preparing the sensor for detecting the stem
cell differentiation.
[0035] The sensor for detecting a stem cell differentiation
containing an electrode; and a substrate for an alkaline
phosphatase is prepared.
[0036] Since the electrode of the sensor and the substrate for the
alkaline phosphatase are described above, the common descriptions
between them are omitted in order to avoid undue redundancy leading
to the complexity of this specification.
[0037] (b) The step contacting cells of interest to the sensor.
[0038] And then, the stem cells of interest are contacted to the
sensor for verifying a differentiation status.
[0039] (c) The step measuring the phosphorylation or
dephosphorylation of the substrate for alkaline phosphatase on the
cells through an electrical signal.
[0040] The substrate is dephosphorylated by alkaline phosphatase in
undifferentiated stem cells, and the substrate dephosphorylated may
be measured using a cyclic voltammetry because the modified
substrate has quite different electrochemical properties compared
to the pre-modified substrate.
[0041] Data for undifferentiated stem cells may be quantitated by
tracing the electrochemical signal of the alkaline phosphatase
substrate using its electrochemical characteristics, and the signal
of the alkaline phosphatase substrate changed according to
differentiation may be detected using an electrochemical method to
determine the differentiation of the stem cell.
[0042] Likewise, the electrochemical signal of the alkaline
phosphatase substrate in the undifferentiated stem cells is
quantitated according to the amounts of stem cells, and the
electrochemical signal of the alkaline phosphatase substrate in the
differentiated stem cells is also quantitated according to the
amounts of stem cells. And then, these results are compared to
determine the extent of the stem cell differentiation.
[0043] In a view of identifying the differentiation of mouse
embryonic stem (MES) cells as a preferable embodiment,
differentiated MES cells show an independent relationship between
electrical signal and increasing cell number, while
undifferentiated MES cells represent a linear correlation between
electrical signal and increasing cell number. R.sup.2 value
representing the linearity of undifferentiated MES cells is
significantly calculated as 0.9432, indicating that the cell status
could be identified using this electrochemical method and the
detection limit is 50,000 undifferentiated MES cells (FIG. 4).
[0044] As described above in detail, the present invention provides
a sensor for detecting a stem cell differentiation, including (a)
an electrode; and (b) a substrate for an alkaline phosphatase. The
phosphorylation or dephosphorylation of the substrate for an
alkaline phosphatase as a stem cell undifferentiation marker which
dephosphorylates its substrate may be measured using an electrical
signal in the present sensor. Therefore, the sensor of the present
invention enables to electrically detect a stem cell status in a
high-throughput manner and to determine the stem cell
differentiation.
[0045] The present invention will now be described in further
detail by examples. It would be obvious to those skilled in the art
that these examples are intended to be more concretely illustrative
and the scope of the present invention as set forth in the appended
claims is not limited to or by the examples.
EXAMPLES
Experimental Materials and Methods
2.1 Materials
[0046] 1-Naphthyl phosphate and phosphate buffered saline (PBS) (pH
7.4, 10 mM) solution was purchased from Sigma-Aldrich. All other
chemicals that are used in this study were obtained commercially as
reagent grade.
2.2 Undifferentiated MES Cell Culture and Embryonic Body
Formation
[0047] J1 cells, mouse embryonic stem (ES) cells, were cultured in
Dulbecco's modified Eagle's medium (DMEM) supplemented with 15%
FBS, 1 mM sodium pyruvate, 10.sup.-4M 2-mercaptoethanol,
1.times.nonessential amino acids, and 1,000 U of leukemia
inhibitory factor (LIF) per ml at 37.degree. C. and 5% CO.sub.2. EB
formation was performed with J1 ES cells as previously described
[13]. ES cells (2.times.10.sup.6 cells) were seeded onto the
bacterial-grade Petri dishes in DMEM containing 10% FBS without
LIF. After 2 days of suspension culture, EBs were collected and
trypsinized. A total of 1.37.times.10.sup.6 cells were replated on
0.2% gelatin-coated 6-well plates containing the same medium. The
number of cells was counted after 3 days.
2.3 Fabrication of Electrochemical Cell
[0048] The culture chamber is a regular square chamber and each
side is 2 mm long. We used 1 the working gold electrode (CHI101),
the Ag/AgCl reference electrode (CHI111), and the platinum counter
electrode (CHI115). The J1 cells were transferred into the chamber
at a known cell density by infusion with fresh culture medium. The
number of cells was determined by a trypan blue dye method using a
hemacytometer.
2.4 Electrochemical Sensing of MES Cells
[0049] The cyclic voltammetry experiments were performed using a
CHI660A machine controlled by a general electrochemical system
software. A three-electrode system was designed which was comprised
of the gold as the working electrode, a platinum wire as the
counter electrode, and an Ag/AgCl cell as the reference electrode.
Measurements were carried out to study the electrical properties of
MES cells as well as detect the differentiation of the mouse
embryonic stem cells by the interaction between 1-NP and
differentiation marker alkaline phosphatase. PBS (10 mM, pH 7.4)
was used as an electrolyte at a scan rate of 0.1 V/s. Before the
experiment occurs, the culture media was removed and the 1-NP
containing PBS was injected into the chamber. The experiment was
started 15 min after buffer changing, and the measurement
conditions were maintained at 37.degree. C., 5% CO.sub.2.
2.5 Alkaline Phosphatase Staining Immunoassay
[0050] Alkaline Phosphatase (AP) staining immunoassay kit was
purchased from Sigma-Aldrich. First, the cells were fixed using a
citrate-acetone-formaldehyde fixative solution. After fixation, an
alkaline-dye mixture which is composed of sodium nitrate,
FRV-alkaline solution, and naphthol AS-BI alkaline solution was
added to the cells in a dark room and allowed to stain for 15 min.
Finally, the cells were counterstained using hematoxylin
solution.
[0051] 3. Results
3.1 Signal Detecting of 1-Naphtyhyl Phosphate
[0052] We quantified the 1-NP signal as follows. The concentration
of 1-NP was ranged from 1 .mu.M to 5 mM in an electrolyte buffer of
10 mM PBS (FIGS. 2a-2b). FIG. 2a describes the electrochemical
property of the 1-NP. The peak was observed around 0.1 V and the
potential slowly changed to negative voltage as the concentration
of 1-NP decreased. FIG. 2b shows the direct, linear, relationship
between 1-NP peak current and concentration of 1-NP. Linear
regression analysis was performed which proved the reliability of
the electrochemical quantifying method using cyclic voltammetry
(R.sup.2=0.9543). These results indicate that 1-NP can be
quantified by this cyclic voltammetry assay.
3.2 Electrochemical Response of Undifferentiated MES Cells
[0053] By incorporating 1-NP into the substrate of the MES cells,
alkaline phosphatase (an embryonic stem cell marker) can be tracked
by its dephosphorylating action on 1-NP. When the dissolved 1-NP
changes to 1-Naphthol there is a corresponding change in electrical
signal as 1-Napthol cannot be seen by the boundary conditions set
up to detect 1-NP. The detection was performed in 15 minutes after
the 1-NP was injected. FIG. 3a shows the electrochemical property
of MES cells after addition of 1-NP. The entire potential was
slightly changed to positive direction, but the reduction peak
current was decreased with an increasing concentration of MES
cells. The reason for this potential change could include the
media, because media has a lot of different materials. The linear
plot is showed in FIG. 3b and the linear regression analysis is
represented by R.sup.2 value, 0.9419. These results indicate good
correlation between cell number and CV reduction current peak which
is estimated to 1-NP.
3.3 Detecting Differentiation of MES Cells
[0054] In order to detect the differentiation of MES cells, we
compared electrochemical signals from undifferentiated MES cells
and differentiated MES cells. Each group of cells was seeded into a
culture chamber to a specific cell number. Because the
undifferentiated MES cells have better adhesion and growth than
differentiated MES cells, we seeded two times more differentiated
cells than the undifferentiated MES cells.
[0055] FIG. 4 shows the relationship between undifferentiated MES
cells and differentiated MES cells. Differentiated MES cells showed
an independent relationship between electrical signal and
increasing cell number, while undifferentiated MES cells showed a
linear relationship between signal and cell number. The linearity
of undifferentiated MES cells is represented by a high R.sup.2
value of 0.9432. The P-value for reliability between
undifferentiated MES cells and differentiated MES cells is 0.095,
0.001, 0.006 and 0.002 respectively for each point starting from
the second data point of the cell number. Other than the lowest two
cell numbers, all other points were significantly different at a 5%
confidence level. That means the third point of cell number is the
detection limit to identify the differentiation of MES cells. These
results indicate that we could identify the cell status using this
electrochemical method and the detection limit is 50,000
undifferentiated MES cells.
3.4 1-Naphthyl Phosphate Effect to Mouse MES Cells
[0056] To ensure that the presence of 1-NP is not affecting our
tests by altering MES cell viability or differentiation, an
Alkaline Phosphatase (AP) staining assay was performed. FIG. 5a
shows the result of the viability test after treating the cells
with 1 mM 1-NP. The cell viability was 99.65% as compared with
control. FIGS. 5b-5c show the results of AP staining before NP
treated stem cells and after NP treated stem cells. Each figure
shows well stained stem cells, which means that 1-NP does not
affect the differentiation of stem cells.
[0057] 4. Discussion
[0058] In this paper, we propose a cell based sensor that could
detect differentiation of MES cells based on electrochemical tools.
Electrochemical cell based sensors have been investigated before,
but most of them are used for detecting cell viability. These kinds
of cell based sensor have been applied to drug screening of cancer
cells or to environmental toxicant effect to cells [12].
[0059] Electrochemical cell based sensors can be divided into two
groups based on measuring techniques. One technique is based on
measuring impedance [14-16]. Cells are generally insulative. If
cells cover a large area of the electrode, there is a relatively
high level of impedance. In order for this technique to work the
cells must be able to attach to the electrode surface. This limits
this technique's usefulness to cells which are the type to be
adherent. This technique is based on the assumption that the
surface attached cells are alive and detached cells are dead.
[0060] Another type of electrochemical cell based sensor utilizes
an amperometric technique [6]. Amperometric technique involves
detecting electrical current with a fixed potential. Sometimes CV
technique is introduced as a means to determine the potential for
an amperometric technique. The amperometric technique measures the
amount of oxygen and the amount of dopamine around cells to
determine cell viability. Recently, the signal of HeLa cell was
quantified by CV and Potential Stripping Analysis (PSA) techniques
[12]. However, to date there has been no attempt to measure the
differentiation of stem cells using electrical or electrochemical
tools.
[0061] In summary, we report the successful demonstration of
identifying and quantifying ES cell differentiation utilizing a
novel technique base on 1-NP measured via an electrochemical
method. We have shown that measuring the 1-NP is a feasible means
to determine ES cell differentiation, and the presence of this
indicator did not affect the viability and differentiation of these
cells. For this experiment, we tested 1-NP as a substrate for
detecting the differentiation of MES cells. We have successfully
quantified the 1-NP by an electrochemical method, and this
technique was applied to MES cells to detect the differentiation
status of the cells. The peak current of 1-NP increases only with
undifferentiated MES cell number indicating that 1-NP only reacts
with undifferentiated MES cells. Finally, we compared the signal of
differentiated MES cells and undifferentiated MES cells, and we
determined the detection limit of undifferentiated MES cell number
to be .gtoreq.50,000. The proposed electrochemical measurement
system can be applied to electrical stem cell chip for diagnosis,
drug detection and on-site monitoring.
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[0078] Having described a preferred embodiment of the present
invention, it is to be understood that variants and modifications
thereof falling within the spirit of the invention may become
apparent to those skilled in this art, and the scope of this
invention is to be determined by appended claims and their
equivalents.
* * * * *