Molecular commonality detection using an artificial enzyme membrane for in situ one-stop biosurveillance

Article abstract of DOI:10.1021/ac0708902

Biodetection and biosensing have been developed based on the concept of sensitivity toward specific molecules. However, current demand may require more levelheaded or far-sighted methods, especially in the field of biological safety and security. In the fields of hygiene, public safety, and security including fighting bioterrorism, the detection of biological contaminants, e.g., microorganisms, spores, and viruses, is a constant challenge. However, there is as yet no sophisticated method of detecting such contaminants in situ without oversight. The authors focused their attention on diphosphoric acid anhydride, which is a structure common to all biological phosphoric substances. Interestingly, biological phosphoric substances are peculiar substances present in all living things and include many different substances, e.g., ATP, ADP, dNTP, pyrophosphate, and so forth, all of which have a diphosphoric acid anhydride structure. The authors took this common structure as the basis of their development of an artificial enzyme membrane with selectivity for the structure common to all biological phosphoric substances and studied the possibility of its application to in situ biosurveillance sensors. The artificial enzyme membrane-based amperometric biosensor developed by the authors can detect various biological phosphoric substances, because it has a comprehensive molecular selectivity for the structure of these biological phosphoric substances. This in situ detection method of the common diphosphoric acid anhydride structure brings a unique advantage to the fabrication of in situ biosurveillance sensors for monitoring biological contaminants, e.g., microorganism, spores, and viruses, without an oversight, even if they were transformed.

Full text of DOI:10.1021/ac0708902

Anal. Chem. 2007, 79, 5540-5546
Molecular Commonality Detection Using an
Artificial Enzyme Membrane for in Situ One-Stop
Biosurveillance
Shinya Ikeno,†,‡ Hitoshi Asakawa, and Tetsuya Haruyama*
†
,†,‡
Department of Biological Functions and Engineering, Kyushu Institute of Technology, Kitakyushu Science and
Research Park, Fukuoka, 808-0196, Japan, and CREST, Japan Science and Technology Agency, Kawaguchi,
Saitama, 332-0012, Japan
viruses, is important for maintaining safety and security.1-3 Many
biosurveillance techniques have been developed thus far. Sensor
technology will further contribute to the implementation of
effective biosurveillance. Hygienic applications are expanding
explosively, for example, in food production and hazard analysis
Biodetection and biosensing have been developed based
on the concept of sensitivity toward specific molecules.
However, current demand may require more levelheaded
or far-sighted methods, especially in the field of biological
safety and security. In the fields of hygiene, public safety,
and security including fighting bioterrorism, the detection
of biological contaminants, e.g., microorganisms, spores,
and viruses, is a constant challenge. However, there is
as yet no sophisticated method of detecting such contami-
nants in situ without oversight. The authors focused their
attention on diphosphoric acid anhydride, which is a
structure common to all biological phosphoric substances.
Interestingly, biological phosphoric substances are pecu-
liar substances present in all living things and include
many different substances, e.g., ATP, ADP, dNTP, pyro-
phosphate, and so forth, all of which have a diphosphoric
acid anhydride structure. The authors took this common
structure as the basis of their development of an artificial
enzyme membrane with selectivity for the structure com-
mon to all biological phosphoric substances and studied
the possibility of its application to in situ biosurveillance
sensors. The artificial enzyme membrane-based ampero-
metric biosensor developed by the authors can detect
various biological phosphoric substances, because it has
a comprehensive molecular selectivity for the structure
of these biological phosphoric substances. This in situ
detection method of the common diphosphoric acid
anhydride structure brings a unique advantage to the
fabrication of in situ biosurveillance sensors for monitor-
ing biological contaminants, e.g., microorganism, spores,
and viruses, without an oversight, even if they were
transformed.
4,5
critical control point (HACCP), clinical activities, clinical medi-
cine,6 good manufacturing practice (GMP),8,9 and biological
hazard security, including fighting bioterrorism. In conventional
biosurveillance, the plate culture method has been employed. This
method is appropriate for the detection of living microorganisms,
but it is time-consuming. In other cases of biosurveillance
methods, specific molecular markers are traced by mean of time-
consuming ex situ methods. Specific proteins located on the
surface of microorganisms, spores and viruses are a good markers
that show up readily in affinity-based assays allowing the recogni-
,7
1,10
tion of the species of contaminant.
However, affinity-based
assays can detect only the single contaminant species for which
they are specifically designed. They cannot detect all contaminant
comprehensively. At the frontline of biosurveillance, an in situ
monitoring method of detecting biological contaminants without
oversight is very much required. Conventional methods target
specific marker molecules. This type of strategy can be used to
determine specific species of biological contaminants, but it cannot
be used to perform comprehensive in situ surveys for a strict and
continuous surveillance. In order to perform comprehensive
biosurveillance and keep a strict watch on all biological contami-
nants without any oversight, the modification of the base concept
is probably required.
(
1) Koopmans, M.; Duizer, E. Int. J. Food Microbiol. 2004, 90, 23-41.
(2) Rueckert, A.; Ronimus, R. S.; Morgan, H. W. Food Microbiol. 2006, 23,
20-230.
2
(
3) Booth, T. F.; Kournikakis, B.; Bastien, N.; Ho, J.; Kobasa, D.; Stadnyk, L.;
Li, Y.; Spence, M.; Paton, S.; Henry, B.; Mederski, B.; White, D.; Low, D.
E.; McGeer, A.; Simor, A.; Vearncombe, M.; Downey, J.; Jamieson, F. B.;
Tang, P.; Plummer, F. J. Infect. Dis. 2005, 191, 1472-1477.
4) da Cruz, A. G.; Cenci, S. A.; Maia, M. C. A. Trends Food Sci. Technol. 2006,
17, 406-411.
The social demand for biological safety and security is
currently heightened in many different fields. In the fields of
hygiene, public safety including fighting bioterrorism, the detec-
tion of biological contaminant, e.g., microorganisms, spores, and
(
(
(
(
5) Stecchini, M. L.; Del Torre, M. Vet. Res. Commun. 2005, 29, 117-121.
6) Raspor, P. Acta Biochim. Pol. 2005, 52, 659-664.
7) Cassens, U.; Ahlke, C.; Garritsen, H.; Krakowitzky, P.; Wullenweber,
J.; Fischer, R. J.; Peters, G.; Sibrowski, W. Transfusion 2002, 42,
10-17.
*
To whom correspondence should be addressed, E-mail: haruyama@
life.kyutech.ac.jp, Phone and Fax: +81-(0)93-695-6065.
(8) Plumb, K. Chem. Eng. Res. Des. 2005, 83, 730-738.
(9) Velagaleti, R.; Burns, P. K.; Gill, M. Drug Inf. J. 2003, 37, 407-438.
(10) Chen, F. C.; Godwin, S. L. J. Food Prot. 2006, 69, 2534-2538.
†
Kyushu Institute of Technology.
‡
CREST.
5540 Analytical Chemistry, Vol. 79, No. 15, August 1, 2007
10.1021/ac0708902 CCC: $37.00 © 2007 American Chemical Society
Published on Web 07/07/2007

Figure 1. Hypothetical illustration of the concept of comprehensive contamination detection in biosurveillance.
The authors focused their attention on diphosphoric acid
anhydride, which is a structure common to all biological phos-
phoric acids. Interestingly, biological nucleic acids are peculiar
to all living things and include many different substances, e.g.,
ATP, ADP, dNTP, pyrophosphate, and so forth, all of which have
a diphosphoric acid anhydride structure. In this study, substances
that have diphosphoric acid anhydride structure are described
as biological phosphoric substances. ATP has been employed as
Those early studies were oriented toward the design of both
molecular selectivity and catalytic activity, but not toward specific
practical tasks. There have been notable facts that still determine
the design of artificial enzyme functions. For instance, Barr et al.
employed Cu2 -cyclodextrin complexes to induce the catalytic
properties of metal and the binding characteristics of cyclodex-
trins.25 These complexes catalyze the hydrolysis of phosphate
triesters, and the rate of reaction has been improved spectacularly.
Breslow et al. mimicked ribonuclease A in order to attach two
+
11,12
a marker in detection of living microorganisms.
However, any
1
6
practical biosurveillance method must be able to detect all
contaminants, e.g., living microorganisms (rich in ATP), dried
microorganisms and spores (rich in ADP), and viruses (containing
dNTP and diphosphoric acid), as detection target.
imidazole rings to the primary face of â-cyclodextrin. This
mimetic enzyme specifically catalyzes the hydrolysis of a catechol
cyclic phosphate carrying a 4-tert-butyl group. The key guideline
for the design of the catalytic site of artificial enzymes has been
set by these pioneering achievements.
The authors took this common structure as the basis of their
concept of comprehensive detection of biological contaminants
in biosurveillance as illustrated hypothetically in Figure 1. To
perform the comprehensive detection of contaminants, the authors
have developed an artificial enzyme membrane with molecular
selectivity for the structure common to all biological phosphoric
substances comprehensively and studied the possibility of its
application in in situ sensors for primary biosurveillance. This is
the key concept of the present research, as selectivity for a
structure common to many type of substances cannot be provided
by natural enzymes.
In this study, we designed an artificial enzyme membrane with
the intention of both providing selectivity for the structure
common to all biological phosphoric substances and applying it
2+
in a biosensor. In our molecular design strategy, the Cu complex
cavity acts as the catalytic active site, which runs the catalytic
hydrolysis reaction at the diphosphoric acid anhydride part of the
substrate. The active center cavities are accumulated in the
(
14) Chiang, L. C.; Konishi, K.; Aida, T.; Inoue, S. J. Chem. Soc., Chem. Commun.
992, 254-256.
(15) Breslow, R.; Fang, Z. L. Tetrahedron Lett. 2002, 43, 5197-5200.
1
(
(
16) Breslow, R.; Schmuck, C. J. Am. Chem. Soc. 1996, 118, 6601-6605.
17) Rousseau, C.; Nielsen, N.; Bols, M. Tetrahedron Lett. 2004, 45, 8709-
In the present research, we attempted the design and synthesis
of an artificial enzyme membrane that has selectivity for the
diphosphoric acid anhydride structure common to molecules
found in all biological organisms.
8711.
(18) Kofoed, J.; Reymond, J. L. Curr. Opin. Chem. Biol. 2005, 9, 656-664.
(
(
(
19) Ojida, A.; Inoue, M.; Mito-oka, Y.; Tsutsumi, H.; Sada, K.; Hamachi, I. J.
Am. Chem. Soc. 2006, 128, 2052-2058.
20) Suzuki, I.; Obata, K.; Anzai, J.; Ikeda, H.; Ueno, A. J. Chem. Soc. Perkin
Trans. 2 2000, 1705-1710.
Pioneering work on the molecular design of artificial enzymes
has focused on mimesis based on accurate organic synthesis.1
3-25
21) Seneque, O.; Campion, M.; Giorgi, M.; Le Mest, Y.; Reinaud, O. Eur. J.
(
11) Harvey, S. D.; Mong, G. M.; Ozanich, R. M.; Mclean, J. S.; Goodwin, S. M.;
Inorg. Chem. 2004, 1817-1826.
Valentine, N. B.; Fredrickson, J. K. Anal. Bioanal. Chem. 2006, 386, 211-
(22) Emgenbroich, M.; Wulff, G. Chem.-Eur. J. 2003, 9, 4106-4117.
(23) Hegg, E. L.; Burstyn, J. N. Coord. Chem. Rev. 1998, 173, 133-165.
(24) Motherwell, W. B.; Bingham, M. J.; Six, Y. Tetrahedron 2001, 57, 4663-
4686.
2
19.
(
12) Satoh, T.; Kato, J.; Takiguchi, N.; Ohtake, H.; Kuroda, A. Biosci. Biotechnol.
Biochem. 2004, 68, 1216-1220.
(
13) Breslow, R.; Huang, Y.; Zhang, X. J.; Yang, J. Proc. Natl. Acad. Sci. U.S.A.
(25) Barr, L.; Easton, C. J.; Lee, K.; Lincoln, S. F.; Simpson, J. S. Tetrahedron
Lett. 2002, 43, 7797-7800.
1997, 94, 11156-11158.
Analytical Chemistry, Vol. 79, No. 15, August 1, 2007 5541

Figure 2. Schematic of the synthesis procedure of the artificial enzyme membrane through coordinative self-assembly.
membrane to ensure suitable catalytic activity in every unit section
of the membrane matrix covering the electrode surface. The
membrane is designed and synthesized through coordinative self-
assembly among a metal coordinative cation polymer, Cu(II) ion,
and a counteranion polymer. In this study, the potential charac-
teristics of the artificial enzyme membrane were investigated
kinetically and were used to construct a molecular commonality
sensor for in situ biosurveillance with selectivity for molecule
structure common to all biological phosphoric substances.
dissolved into 10 mL of HEPES buffer (10 mM, pH 7.4). Then,
Cu(II) ions were added to the solution to a final concentration of
10 mM and allowed to react for 1 h to form polyhistidine-Cu
2
+
complexes. After coordination, 20 µmol (monomer units) of poly-
(sodium-4-styrenesulfonate) was added to the coordinative mixture
solution and was allowed to form polyion complexes with the
2+
shrunken polyhistidine-Cu complexes. The synthesized artificial
enzyme was collected by centrifugation and was repeatedly
washed with HEPES buffer (10 mM, pH 7.4) in order to remove
the free Cu(II) ions. Finally, the collected artificial enzyme was
dissolved into 1.0 mL of HEPES buffer (10 mM, pH 7.4).
Spectral Analysis. The amount of Cu(II) ion in the artificial
enzyme membrane was determined using ICP optical emission
spectroscopy (Perkin-Elmer, Optima 4300DV Cyclon, Wellesley,
MA). An organic elementary analyzer (Yanaco MT-5, Anatech
Yanaco Corp., Kyoto, Japan) was employed to determine the
amount of C, N, and H in the artificial enzyme membrane. The
ESR spectra were recorded on an ESR spectrometer (Jasco,
Tokyo, Japan).
EXPERIMENTAL SECTION
Reagents. The water used in the present experiment was first
deionized and passed through a Milli-Q water purification system
from Millipore Co. (Bedford, MA). Poly(
(
(
L
-histidine) hydrochloride
molecular weight 6700) was purchased from Sigma Chemical Co.
St. Louis, MO). Poly(sodium-4-styrenesulfonate) (molecular
weight 70 000) was purchased from Aldrich Chemical Corp.
Milwaukee, WI). ATP, ADP, AMP, GTP, CTP, TTP, sodium
(
tripolyphosphate, sodium pyrophosphate decahydrate, â-nicoti-
namide adenine dinucleotide disodium salt reduced form (NADH),
flavin adenine dinucleotide disodium salt (FAD), Cu(II) chloride
dihydrate, sodium acetate, ammonium dihydrogenphosphate,
tetra-n-butylammonium hydroxide, and methanol (HPLC grade)
were obtained from Wako Pure Chemical Industries, Ltd. (Osaka,
Japan). Sodium tetrapolyphosphate was obtained from Nacalai
Tesque (Kyoto, Japan). HEPES, MES, and CHES for the buffer
solutions were purchased from Dojindo (Kumamoto, Japan). Cu-
The Raman spectra were acquired at an excitation of 514.5 nm
under radiation from an argon ion laser (GLG2162, NEC) and were
recorded on an NRS-2000 Raman spectrometer with a liquid
nitrogen-cooled CCD detector (Jasco). The wave numbers of the
Raman bands were calibrated with the spectrum of a silicon wafer
-
1
and were reproducible to within 1.0 cm . All spectra were
recorded at room temperature (25 °C).
HPLC Analysis. The product of dephosphorylation of biologi-
cal phosphoric substances effected by the artificial enzyme was
analyzed by means of HPLC (Hitachi) on a Wakosil-II 3C18 RS
column (10 cm × 4.6 mm, i.d. 3 µm; Wako, Osaka, Japan). The
reaction mixture was prepared by adding the artificial enzyme
and an adenosine phosphate (ATP, ADP, or AMP) to the buffer
solution, and the reaction was initiated at that time. After the
reaction, the sample was centrifuged, and the supernatant was
(
II) mesotetra(4-sulfonatophenyl)porphine was purchased from
Frontier Scientific, Inc. (Logan, UT).
Synthesis of the Artificial Enzyme Membrane. The artificial
enzyme membrane is a kind of polymer metal complex, but the
molecular design is our original invention. It consists of two
polymers and one metal ion.
For the present molecular design of the artificial enzyme, 20
µmol (monomer units) of poly(
L-histidine) hydrochloride was
5542 Analytical Chemistry, Vol. 79, No. 15, August 1, 2007

analyzed on the HPLC system. The mobile phase was composed
of 20% methanol in water containing 20 mM ammonium dihydro-
genphosphate and 5 mM tetra-n-butylammonium hydroxide. This
-1
mobile phase was pumped at a rate of 1.0 mL min . The separated
sample was detected with a UV detector at 260 nm. The reaction
and separation were performed at room temperature (25 °C).
Electrochemical Evaluation. The electrochemical evaluation
of the biological phosphoric substances was performed using
three-electrode systems with an Ag/AgCl reference electrode, a
Pt counter electrode, and a sensor device. The sensor device was
fabricated by coating the glassy carbon electrode surface (3 mm
in diameter, BAS, Tokyo, Japan) with an artificial enzyme. The
electrochemical potential application and current recording were
operated by a computerized electrochemical analyzer (HZV-100,
Hokutodenko, Tokyo, Japan). For the electrochemical analysis,
HEPES buffer (0.1 M, pH 7.4, containing 0.1 M KCl) was used as
the electrolyte solution. Cyclic voltammograms of the artificial
enzyme membrane-coated electrode were performed at a potential
-
1
sweep speed of 10 mV s . Sensor response was obtained and
recorded at a constant applied potential of -250 mV versus Ag/
AgCl.
RESULTS AND DISCUSSION
Design and Synthesis of the Artificial Enzyme Membrane.
In order to obtain selectivity of molecular commonality for the
common diphosphoric acid anhydride structure, the authors
2
6-30
focused on the catalytic activity of Cu(II) complexes.
Cu(II)
complexes were obtained through our peculiar process of coor-
dinative self-assembly. The coordinatively self-assembled structure
is stabilized by the formation of a polyion-polyion complex with
a countercharged polymer, as illustrated in the hypothetical
synthesis scheme of Figure 2.
Figure 3. Spectrum investigation of the artificial enzyme structure.
A) Raman spectrum of the artificial enzyme. (a-c) show the Raman
spectra of Cu(II) mesotetra(4-sulfonatophenyl)porphine, deoxyglobin,
and the artificial enzyme, respectively. (B) ESR spectrum of the
artificial enzyme.
(
Structural Information on the Artificial Enzyme Mem-
brane. The synthesized artificial enzyme membrane was inves-
tigated by conventional means: element analysis and spectrum
analysis through ESR and Raman spectroscopy. The Raman
spectrogram indicated that the artificial enzyme was formed
through coordinative self-assembly from a coordinative cation
polymer (poly(L-histidine)) and Cu(II) ion. The Raman spectra of
the artificial enzyme and two Cu(II) complexes, mesotetra(4-
sulfonatophenyl) orphine, and deoxyglobin, are shown in Figure
3
1
A
// ) 17.0 mT. Following Peisach and Blumberg, the coordina-
2+
tion position of the Cu complex cavity was investigated. It is
clear from the ESR spectrum that it is a type II Cu complex. It
was verified that the coordinative position of the Cu complex
cavity was the square planar complexes of 4N or 2N2O, and
structural distortion was hardly observed. The composition of the
2
+
structural substances was estimated to be poly(L-histidine):Cu:
poly(styrenesulfonate) ) 5.1:1.0:4.3 by organic elementary ana-
lyzer and inductively coupled plasma analysis. The result strongly
suggests that the artificial enzyme membrane had densely
arranged Cu2 complex cavities in its membrane matrix structure,
which probably contributed to the prominent catalytic activity of
each unit membrane area. This structure is shown in Figure 5A,
which was modeled based on data from the spectrum investigation
and element analysis.
Recognition of Molecular Commonality through Catalytic
Dephosphorylation Activity on Biological Phosphoric Sub-
stances with the Artificial Enzyme Membrane. In order to
recognize the common molecular structure, we designed the
artificial enzyme membrane in such a way that it possesses a
specific catalytic activity (not molecule selectivity). The catalytic
activity of biological phosphoric substance dephosphorylation was
investigated kinetically. We investigated the initial rate of ATP
dephosphorylation by the artificial enzyme at pH 7.4 and 25 °C
3
A.
The formation of a coordinative bond between the imidazole
residue and the Cu(II) ion can be verified by comparing the three
+
-
1
spectra. A peak of N-copper stretching was observed at 220 cm
,
2
+
which is the characteristic Raman peak of the Cu -histidine
complex. Further structural information on the complex was
obtained from the ESR spectrum, as shown in Figure 3B. The
specific ESR parameters obtained were g// ) 2.26, g ) 2.06, and
(
26) Jakubiak, A.; Kolarz, B. N.; Jezierska, J. Macromol. Symp. 2006, 235, 127-
35.
1
(
27) Mendoza, A.; Aguilar, J.; Basallote, M. G.; Gil, L.; Hernandez, J. C.; Manez,
M. A.; Garcia-Espana, E.; Ruiz-Ramirez, L.; Soriano, C.; Verdejo, B. Chem.
Commun. 2003, 3032-3033.
(
(
(
28) Scarpellini, M.; Neves, A.; Horner, R.; Bortoluzzi, A. J.; Szpoganics, B.; Zucco,
C.; Silva, R. A. N.; Drago, V.; Mangrich, A. S.; Ortiz, W. A.; Passos, W. A.
C.; de Oliveira, M. C. B.; Terenzi, H. Inorg. Chem. 2003, 42, 8353-8365.
29) Maheswari, P. U.; Roy, S.; den Dulk, H.; Barends, S.; van Wezel, G.;
Kozlevcar, B.; Gamez, P.; Reedijk, J. J. Am. Chem. Soc. 2006, 128, 710-
7
11.
(31) Peisach, J.; Blumberg, W. E. Arch. Biochem. Biophys. 1974, 165, 691-
30) Liu, S. H.; Hamilton, A. D. Chem. Commun. 1999, 587-588.
708.
Analytical Chemistry, Vol. 79, No. 15, August 1, 2007 5543

Figure 5. Structure and sensor responses of the artificial enzyme
membrane-based sensor device. (A) Photograph of the artificial
enzyme membrane-coated GC electrode (sensor device) and the
structure model of the artificial enzyme membrane. The structure was
modeled by a molecular structure modeling software (Molecular
Operating Environment, MOE; Chemical Computing Group, Canada)
based on data from the spectrum invest,igations and element analysis.
(B) Typically, cyclic voltammograms of the sensor device in solutions
with (solid line) or without (broken line) ATP. (C) Sensor response
curves for adenosine phosphates (ATP, ADP, and AMP). The
concentration of each phosphate is 100 µM. The sensor response
was recorded at a constant potential of -250 mV vs Ag/AgCl.
Figure 4. Catalytic dephosphorylation activity of the artificial enzyme
membrane. (A) Initial velocity of ATP dephosphorylation by the
artificial enzyme. (B) The pH dependence of the initial velocity of
dephosphorylation of 100 µM ATP by the artificial enzyme. The
reaction buffer was 0.1 M acetate buffer (pH 5.0), MES buffer (pH
6.0), HEPES buffer (pH 7.0, pH 7.4, pH 8.0), and CHES buffer (pH
.0), respectively. (C) Initial velocity of dephosphorylation of each
9
adenosine phosphate (100 µM; ATP, ADP, AMP) by the artificial
enzyme. These initial velocities were calculated based on the HPLC
analysis data.
complex, followed by the dephosphorylation of the phosphate
terminal of ATP. The kinetic parameters were estimated to be
-
4
-1
V
max ) 2.5 × 10 mM min , K
m
) 1.4 mM, and Kcat ) 1.24 ×
-
5
-1
by means of HPLC analysis (Figure 4A). It is clear that the artificial
10
s
(pH 7.4, 25 °C).
enzyme has a Michaelis-Menten-type catalytic activity in ATP
The pH of the reaction mixture has several possible effects
(
diphosphoric acid anhydride) hydrolysis. These kinetics can be
on the structure and activity of natural enzymes. Therefore, the
effect of the pH on the activity of the artificial enzyme was
investigated by means of HPLC analysis. Figure 4B shows the
described as the binding of the substrate ATP to the metal center
in the nanocavity, to form an intermediate polymer-Cu2 -ATP
+
5544 Analytical Chemistry, Vol. 79, No. 15, August 1, 2007

Table 1. Sensor Responses of the Artificial Enzyme-Based Sensor to Biological Phosphoric Substances
a
Concentration of each phosphoric substance is 100 µM (Final concentration). R1, adenosine; R2, guanosine; R3, cytidine; R4. thymidine; R5,
b
2+
nicotinamide ribose; R6, riboflavin. Sensing by using polymer membrane(artificial enzyme without Cu coordination) coated sensor device.
c
AMP can be measured by present sensor but in case of high concentration level.
initial velocity of ATP dephosphorylation catalyzed by the artificial
enzyme membrane for each buffer pH. The pH was changed from
the nanocavity structure of the artificial enzyme membrane was
deformed and the catalytic activity decreased. In neutral solutions,
the artificial enzyme showed a catalytic activity as high as that of
a native enzyme. The artificial enzyme membrane in a pH 6
5
.0 to 9.0 using 0.1 M acetate buffer, MES buffer, HEPES buffer,
or CHES buffer. In general, hydrolysis is the predominant
pathway, followed by OH- as the nucleophile at high pH levels.
Native enzymes have a pH optimum for their catalytic activity;
this artificial enzyme showed its maximum catalytic activity at pH
a
solution, which is the pK of the imidazole residue of poly(L-
histidine), showed the highest catalytic ATP dephosphorylation
activity. The nanocavity structure of the artificial enzyme mem-
brane in the pH 6 solution was stable and the Cu(II) ion was able
6
.0, just like a native enzyme.
-
Changes in the pH level may affect the shape of the artificial
to coordinate with the OH in the nanocavity, which enabled the
enzyme membrane or the charge properties of the substrate, so
that either the substrate cannot bind to the active site or it cannot
undergo dephosphorylation. The nanocavities of the artificial
enzyme membrane were formed as a result of the electrostatic
interaction between the cationic and the anionic polymer. There-
fore, the polyion complex formed a tight structure at low-pH levels,
and the intrananocavity acquired an increased hydrophobicity due
to the functional polymer residue. The nanocavities were also
reduced in low-pH solutions, because the coordination of the Cu-
artificial enzyme membrane to show robust catalytic activity.
The artificial enzyme was designed and synthesized for the
purpose of applying it to biosurveillance biosensing. Its molecular
target is the structure common to all biological phosphoric
substances. In the experiment, three different biological phos-
phoric substances were used in order to investigate the molecular
selectivity of the artificial enzyme membrane for the biological
phosphoric substances as well as its catalytic activity. The initial
velocity of ATP, ADP, and AMP dephosphorylation catalyzed by
the artificial enzyme membrane is shown in Figure 4C. The results
show that the high-velocity catalytic activity was faster in the case
of compounds that have more phosphate residues. This is due to
the easy penetration of the phosphate terminal into the nanocavity
(the active center of the artificial enzyme) in the case of longer
phosphate residues. As indicated by the kinetic analysis, the
artificial enzyme can catalyze the dephosphorylation of three
(II) ion with poly(L-histidine) was reduced by the protonated
imidazole ligand. Therefore, the substrates had difficulty penetrat-
ing into the nanocavities, and the phosphate terminals were not
-
easily dephosphorylated by the OH in the nanocavities in low-
pH solutions. In high-pH solutions, the electrostatic interactions
between the anion and cation polymer were weakened by the
nonprotonated imidazole residue in poly(L-histidine). As a result,
Analytical Chemistry, Vol. 79, No. 15, August 1, 2007 5545

different biological phosphoric substances almost equally. This
observation clearly indicates that the artificial enzyme membrane
can be employed as the molecular recognition layer/molecular
transduction layer of biosensors and that biosurveillance with a
molecular commonality sensing for biological phosphoric sub-
stances can be performed as intended.
based assay is unique, in that it cannot be performed using natural
enzymes. Such comprehensive molecular selectivity is not a typical
for nature enzymes but this does not mean that the molecular
selectivity of our artificial enzyme is too wide. The membrane form
of the artificial enzyme reduces the risk of diffusion of interfer-
ences into the membrane. The molecular selectivity works within
the limits of the common phosphoric acid anhydride structure.
Molecular Commonality Sensor Based on the Recognition
of Biological Phosphoric Substances by the Artificial Enzyme
Membrane. Based on the artificial enzyme membrane, an
amperometric biosensor was fabricated by way of trial, as
schematically illustrated in Figure 5A. The artificial enzyme
membrane in jelly form can be mounted easily on a glassy carbon
electrode surface to form a molecular recognition layer/molecular
transduction layer. The sensor device was preliminarily examined
through electrochemical experimentation. It is very important to
CONCLUSION
Detection can be done in thick solutions such as serum-
containing culture media. Another important point is the working
potential. In the case of the artificial enzyme-based sensor, -250
mV versus Ag/AgCl is the working potential, and it is low enough
to preclude most potential interferences. Both the membrane-
based selectivity and the lower working potential ensure compre-
hensive selective sensing without disturbances.
3
-
point out that the product of the catalytic reaction is PO
4
, which
This molecular commonality assay, which focuses on the
structure common to all target molecules, represents a novel
analytical concept. Especially in the case of biological safety and
security applications, risk factors have to be recognized without
exception. The present artificial enzyme membrane sensor can
recognize living microorganisms (rich in ATP), dried microorgan-
isms and spores (rich in ADP), and viruses (containing dNTP and
diphosphoric acid), because it can detect most (such or these)
biological phosphoric substances. In the trial application, the
artificial enzyme-based sensor could detect microorganisms (E.
coli). Then, we are trying detection of the contents from both
spores and viruses by the sensor device. Other advantages of this
artificial enzyme-based sensor are very stable for long storage (for
over 3 months in dried form). The preserved sensor devise shows
excellent coefficient of variation of within 3%. Therefore, the sensor
matrix is very stable in its functions allowing a high level of
reproducibility.
Taking advantage of the molecular commonality sensing using
the artificial enzyme membrane, practical sensor devices can be
developed for one-stop biosurveillance in the fields of food
production, HACCP, clinical service, pharmaceutical development,
GMP, and biological hazard security including fighting bioterror-
ism. Biological safety and security are crucial and fundamental
matters for humanity.
hardly exists in neutral-pH aqueous solutions because it is
equilibrated immediately between the orthophosphate ion form
of H PO and HPO . The two forms of orthophosphate ion are
2 4 4
-
2-
electrochemically stable and do not undergo a redox reaction
under electrode potential. However, in the case of the artificial
3
-
enzyme membrane-based biosensor, the reaction product, PO
4
,
may accumulate between the artificial enzyme membrane layer
and the electrode and can be reduced by applying electrode
potential. This is clearly seen in the cyclic voltammograms of the
sensor device (Figure 5B). Remarkably, a catalytic reduction
current was observed in the cyclic voltammograms in the 1 mM
ATP solution (Figure 5B, solid line). In contrast, no cathodic
current response was observed in the absence of ATP (Figure
5B, broken line). The data indicate that the product of the artificial
enzyme membrane was reduced electrochemically. With ADP, the
current peak height reached two-thirds of that for ATP, because
the ADP molecule has only diphosphates. It was clearly shown
that the catalytic reaction of the artificial enzyme can be success-
fully coupled with the electrochemical reaction. In the case of the
artificial enzyme-based amperometric biosurveillance sensor, sen-
sor response can be obtained by applying a constant potential of
-250 mV versus Ag/AgCl. Typical sensor responses show that
the reduction current increases with the addition of adenosine
phosphate (100 µM: AMP, ADP, ATP) as typical biological
phosphoric substances (Figure 5C). The sensor can detect both
ATP and ADP because the molecular selectivity for biological
phosphoric substances, all of which have a diphosphoric acid
anhydride, is comprehensive. The sensor responds to all biological
phosphoric substances, but not to NADH and FAD that do not
contain the phosphoric acid hydride structures with terminal
group (Table 1). The amount of sensor response varies according
to the number of phosphates and other structural factors. In the
case of ATP, the biosurveillance sensor can determine the
concentration within the nanomolar to millimolar range.
ACKNOWLEDGMENT
This research was supported by the “Creation of Biodevices
and Biosystems with Chemical and Biological Molecules for
Medical Use” CREST, from the Japan Science and Technology
Agency. We thank Dr. Toshiaki Kamachi for the spectral mea-
surement of the artificial enzyme membrane using the ESR
spectrometer. We also thank Mr. Shigeya Suzuki (Kikkoman
Corp.) and his colleagues for the valuable information on hygiene
investigation precedents.
As shown in Table 1, the artificial enzyme membrane sensor
provides molecular commonality sensing for all molecules that
contain phosphoric acid hydride structures. This artificial enzyme-
Received for review May 1, 2007. Accepted June 3, 2007.
AC0708902
5546 Analytical Chemistry, Vol. 79, No. 15, August 1, 2007