Also, the control experiments for cantilever assay as well as measurement of frequency shift in dry air due to proteolysis are provided.
For measurement of resonant frequency shift in dry air due to enzymatic cleavage, we have to dry up the cantilever which was utilized for in situ bioassay.
Then the resonance of such a cantilever, on which the peptide chains are likely to be cleft by CTSB, is measured, and consequently, so is the resonant frequency shift in dry air due to proteolysis driven by CTSB.
From these analyses we conclude that 1) the moisture diffusivity scales with viscosity, which is the main parameter in shift in drying behavior.
The mutualistic design can be applied to prevent or even suppress the dynamics of catastrophic shifts in dry land ecosystems.
In other words, hydrodynamic loading change during the enzymatic cleavage can be also estimated from the resonant frequency shift measured in liquid, since the total amount of cleft peptides is directly evaluated from the resonant frequency shift measured in dry air.
As stated earlier in Methods and Materials, we have considered the resonant frequency shift measured in dry air due to protease in order to quantify the amount of cleft peptides.
On the other hand, such detection based on resonant frequency shift measured in dry air or vacuum restricts our understanding of biochemical reactions in fluid with a real-time, indicative for kinetics of biochemical reactions [20], [27].
The relation between frequency shift (measured in dry air), ΔωP*, due to proteolysis (protease) and total mass of cleft peptides, ΔmP, is given by ΔωP* = (ωI/2)[ ΔmP/ Mc + ΔmI ], where ωI is the resonant frequency of a cantilever, which was functionalized by peptides, in dry air, and Mc is the mass of a bare cantilever.
Since the total mass of cleft peptides can be measured from the resonant frequency shift measured in dry air due to proteolysis, the change of hydrodynamic loading due to proteolysis can be also estimated from the resonant frequency shift evaluated in buffer solution due to proteolysis (see Eqs. (3) and (4) and also Ref. [19]).
Table 1 shows the resonant frequencies of four bare cantilevers (before peptide immobilization), the resonant frequency shifts measured in dry air due to peptide immobilization, and the resonant frequency shifts evaluated in dry air due to proteolysis driven by protease with different protease concentrations (i.e. 0.28 µM, 1.12 µM, 1.53 µM, and 1.61 µM).
