Set-Reset Flip-Flop
Memory Based on Enzyme Reactions:
Towards Memory Systems Controlled by Biochemical Pathways The enzyme-based set-reset flip-flop
memory system was designed with the core part composed of horseradish
peroxidase and diaphorase biocatalyzing oxidation and reduction of
redox species (2,6-dichloroindophenol or ferrocianide). The
biocatalytic redox transformations were activated by H2O2
and NADH produced in situ by
different enzymatic reactions allowing transformation of various
biochemical signals (glucose, lactate, D-glucose-6-phosphate, ethanol)
into reduced or oxidized states of the redox species. The current redox
state of the system, controlled by the set and reset signals, was read
out by optical and electrochemical means. The multi-well setup with the
flip-flop units separately activated by various set/reset signals
allowed encoding of complex information. For illustrative purposes, the
words “Clarkson” and then “University” were encoded using ASCII
character codes. The present flip-flop system will allow additional
functions of enzyme-based biocomputing systems, thus enhancing the
performance of multi-signal biosensors and actuators controlled by
logically processed biochemical signals. The integrated enzyme logic
systems and flip-flop memories associated with signal-responsive
chemical actuators are envisaged as basic elements of future
implantable biomedical devices controlled by immediate physiological
conditions.
M. Pita, G. Strack, K. MacVittie, J. Zhou, E. Katz, Set-reset flip-flop memory based on enzyme reactions: Towards memory systems controlled by biochemical pathways. J. Phys. Chem. B 2009, 113, 16071-16076. |
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(left)
The enzyme biocatalytic system mimicking set-reset flip-flop memory
operations using HRP and Diaph as the components of the core part and
GOx and AlcDH as terminal biocatalysts converting primary set-reset
signals (glucose and ethanol) to H2O2 and NADH inputs controlling the
switchable core part.
(above) Words encoded using ASCII character codes upon application of different set-reset signals (H2O2-NADH, respectively) to the multi-well flip-flop system composed of HRP and Diaph as the biocatalysts and K4[Fe(CN)6] as the redox species. Optical read out of the words Clarkson (A) and University (B) at 415 nm. Color photo of the multi-well reactor with the encoded word University (C) and the respective ASCII codes for the used characters (D). It should be noted that an undergraduate student Kevin MacVittie participated in the project working in a team of graduate students/postdocs. |
Enzyme-Based
Multiplexer and Demultiplexer
Digital 2-to-1 multiplexer and 1-to-2
demultiplexer were mimicked by biocatalytic reactions involving
concerted operation of several enzymes. Using glucose oxidase (GOx) and
laccase (Lac) as the data input signals and variable pH as the
addressing signal, ferrocyanide oxidation in the output channel was
selectively activated by one from two inputs, thus mimicking the
multiplexer operation. Demultiplexer based on the enzyme system
composed of GOx, glucose dehydrogenase (GDH) and horseradish peroxidase
(HRP) allowed selective activation of different output channels
(oxidation of ferrocyanide or reduction of NAD+) by the
glucose input. The selection of the output channel was controlled by
the addressing input of NAD+. The designed systems represent
important novel components of future branched enzyme networks
processing biochemical signals for biosensing and bioactuating.
M.A. Arugula, V. Bocharova, M. Pita, J. Halámek, E. Katz, Enzyme-based multiplexer and demultiplexer. J. Phys. Chem. B 2010, 114, 5222-5226. |
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(left)
The biocatalytic system mimicking the 2-to-1 multiplexer, where glucose
oxidase (GOx) and laccase (Lac) are the data input signals (Input1 and
Input2, respectively) and the pH change is the addressing signal
(Address). The same Output, K3[Fe(CN)6] is
produced in the both reacting pathways.
(above) The electronic equivalent circuitry of the 2-to-1 multiplexer based on the enzyme catalyzed reactions. |
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(left)
The
biocatalytic system mimicking the 1-to-2 demultiplexer, where glucose
is the data input signal (Input) and NAD+ is the addressing signal
(Address). Two different output channels (Output1 and Output1) are
represented by ABTSox and NADH, which production is triggered by the
same data input and selected by the addressing input.
(above) The electronic equivalent circuitry of the 1-to-2 demultiplexer based on the enzyme catalyzed reactions. |
Biochemical Filter with
Sigmoidal Response to pH Changes
We realize a biochemical filtering process
based on the introduction of a buffer in a biocatalytic
signal-transduction logic system based on the function of an enzyme,
esterase. The input, ethyl butyrate, is converted into butyric acid—the
output signal, which in turn is measured by the drop in the pH value.
The developed approach offers a versatile "network element" for
increasing the complexity of biochemical information processing
systems. Evaluation of an optimal regime for quality filtering is
accomplished in the framework of a kinetic rate-equation model.
(left)
Schematic presentation of the buffering-based pH-signal "logic filter."
The reaction biocatalyzed by an enzyme, here esterase, results in the
hydrolysis of ethyl butyrate (the logic Input) to yield butyric acid
which releases H+ ions upon dissociation. A limited quantity of a
buffer, here HEPES, if introduced, consumes most of the
biocatalytically produced H+ ions when the input is applied at a low
concentration. The pH change (the logic Output, measured by the pH
drop, as indicated by an arrow) sets in when the biocatalytically
produced H+ ions overwhelm the buffer. The biocatalytic process and
buffering combined, yield a sigmoidal dependence of the pH change as a
function of the input concentration. The inset illustrates the onset of
the sigmoidal response in our experimental system. The solid curves
show the output, y, vs. the input, x, properly redefined/rescaled to
vary in the "binary-logic ranges" from 0 to 1, as explained in the
text. Experimental data were fitted by using rate equations appropriate
for the processes involved, and the results are shown, here for the
reaction time 120 min, for increasing buffer (HEPES) concentrations.
The top (red) curve corresponds to [HEPES] = 0; middle (blue): [HEPES]
= 50 mM, bottom (green): [HEPES] = 100 mM. The dashed black curve does
not correspond to experimental data but rather illustrates a desirable,
"ideal" filter response with small slopes at both binary logic points 0
and 1, and with a steep, symmetrically positioned inflection region in
the middle.
M. Pita, V. Privman, M.A. Arugula, D. Melnikov, V. Bocharova, E. Katz, Towards biochemical filter with sigmoidal response to pH changes: Buffered biocatalytic signal transduction. PhysChemChemPhys 2011, 13, 4507-4513. |
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Measured pH values at the reaction time t = 120 min, shown vs. the initial substrate concentration, for different amounts of HEPES. Red (bottom) symbols/curve correspond to [HEPES] = 0, blue (middle): [HEPES] =50 mM, green (top): [HEPES] = 100 mM. The circular symbols are the actual pH values, whereas the solid curves are the theoretical model fits. (These curves were shown in the inset in the figure above, rescaled in terms of the logic-range variables). |
Top: Experimental dependence of pH on the
initial substrate concentration (ethyl butyrate) and reaction time, for
[HEPES] = 100 mM. Bottom: Numerically computed dependence for this
system, based on the kinetic model. Dr. Marcos Pita developed the system. Prof. Vladimir Privman suggested the filter concept and analyzed the experimental results. Mary Arugula performed most of the experiments. |
Biochemical
Filter with Sigmoidal Response: Increasing the Complexity of
Biomolecular Logic
The first realization of a designed,
rather than natural, biochemical filter process is reported and
analyzed as a promising network component for increasing the complexity
of biomolecular logic systems. Key challenge in biochemical logic
research has been achieving scalability for complex network designs.
Various logic gates have been realized, but a "toolbox" of analog
elements for interconnectivity and signal processing has remained
elusive. Filters are important as network elements that allow control
of noise in signal transmission and conversion. We report a versatile
biochemical filtering mechanism designed to have sigmoidal response in
combination with signal-conversion process. Horseradish
peroxidase-catalyzed oxidation of chromogenic electron donor by H2O2,
was altered by adding ascorbate, allowing to selectively suppress the
output signal, modifying the response from convex to sigmoidal. A
kinetic model was developed for evaluation of the quality of filtering.
The results offer improved capabilities for design of scalable
biomolecular information processing systems.
(left)
The convex and sigmoidal response for the "identity" logic gate mapping
0 to 0, and 1 to 1. The inset illustrates an "ideal" sigmoidal curve
passing through the two logic points, with a steep and symmetrically
positioned central inflection part, surrounded by broad small-slope
regions at the logic points, and with no measurable noise in the curve
itself (unlike in the actual experimental data). The extensions of the
curve indicate that the response could also be considered and measured
somewhat beyond the logic points, if physically relevant. The schematic
outlines the experimental system, "color-coded" to the plots. The Red
and Ox labels refer to the redox states of the chromogen, TMB; DHA
referes to dehydroascorbic acid — the product of irreversible oxidation
of ascorbate (Asc).
V. Privman, J. Halámek, M.A.
Arugula, D. Melnikov, V. Bocharova, E. Katz, Biochemical filter with sigmoidal
response: Increasing the complexity of biomolecular logic. J.
Phys. Chem. B 2010, 114, 14103-14109.
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Experimental dependence of the
concentration of the charge transfer species (the blue product),
measured by the absorbance, A, on the initial concentration of H2O2,
for varying reaction time, tg, with different initial amounts of
ascorbate, the concentration of which is shown above each plot.
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Theoreticl fit of the experimental data
shown in the figure at the left with the model rate equations.
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