Enzymes
immobilized on a nano-structured surface were used to switch
the activity of one enzyme by a local pH change produced by another
enzyme. Immobilized amyloglucosidase (AMG) and trypsin were studied
as examples of the pH-dependent switchable “target enzymes.”
The reactions catalyzed by co-immobilized urease or esterase were
increasing or decreasing the local pH, respectively, thus operating
as “actuator enzymes.” Both kinds of the enzymes, producing
local pH changes and changing biocatalytic activity with the pH variation,
were orthogonal in terms of the biocatalytic reactions; however, their
operation was coupled with the local pH produced near the surface
with the immobilized enzymes. The “target enzymes” (AMG
and trypsin) were changed reversibly between the active and inactive
states by applying input signals (urea or ester, substrates for the
urease or esterase operating as the “actuator enzymes”)
and washing them out with a new portion of the background solution.
The developed approach can potentially lead to switchable operation
of several enzymes, while some of them are inhibited when the others
are activated upon receiving external signals processed by the “actuator
enzymes.” More complex systems with branched biocatalytic cascades
can be controlled by orthogonal biocatalytic reactions activating
selected pathways and changing the final output.
Advances
in protein engineering resulted in increased efforts to
create protein biosensors that can replace instrumentation-heavy analytical
and diagnostic methods. Sensitivity, amenability to multiplexing,
and manufacturability remain to be among the key issues preventing
broad utilization of protein biosensors. Here, we attempt to address
these by constructing arrays utilizing protein biosensors based on
the artificial allosteric variant of PQQ-glucose dehydrogenase (GDH).
We demonstrated that the silica nanoparticle-immobilized GDH protein
could be deposited on fiberglass sheets without loss of activity.
The particle-associated GDH activity could be monitored using changes
in the fluorescence of the commonly used electron mediator phenazine
methosulfate. The constructed biosensor arrays of macrocyclic immunosuppressant
drugs cyclosporine A and FK-506 displayed very low background and
a remarkable dynamic range exceeding 300-fold that resulted in a limit
of detection of 2 pM for both analytes. This enabled us to quantify
both drugs in human blood, serum, urine, and saliva. The arrays could
be stored in dry form and quantitatively imaged using a smartphone
camera, demonstrating the method’s suitability for field and
point-of-care applications. The developed approach provides a generalizable
platform for biosensor array development that is compatible with inexpensive
and potentially scalable manufacturing.
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