Cell-free assay device and methods of use
a cell-free assay and assay technology, applied in the field of cell-free assay devices, can solve the problems of muscle weakness, impaired respiratory function, and malfunction of the nervous system, and achieve the effect of enhancing an interaction
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example 1
Assay Device
[0161]An example of BoNT toxin is described, but should not be considered limiting in any way.
[0162]As depicted in FIG. 2, initially, BoNT toxin is introduced into the left chamber (first electrolyte volume). The rate of the HC insertions into the lipid bilayer and LC translations through the HCs, is monitored FIG. 2B. In this example, a DC bias is applied to measure the conductance through the HC. Unoccluded HCs, where the LC has translocated through and been released from the HC, can be differentiated from HC / LC complexes based on their respective current signature. After the LC has translocated into the middle chamber (second electrolyte volume), the rate of SNARE protein cleavage is monitored using a channel (e.g., by an endopeptidase assay) that is coated with a binding agent (e.g., an antibody) that specifically binds a cleaved form of the SNARE protein (e.g. the enzymatic target that has been acted upon by the toxin or protein) (FIG. 2C).
[0163]In certain embodimen...
example 2
Device Using an EIB or DIB
[0174]The general workflow of the cell-free assay device concept using an EIB and the example of BoNT, is depicted in FIG. 2.
[0175]A generic cell-free assay device utilizing a GNM to hold a PLB and an internal probe for monitoring the endopeptidase activity of a toxin that inserts itself into and / or translocates through the PLB is shown in FIG. 4. In this example, the protein or toxin's interaction, translocation, or binding to the bilayer is measured using a lipid bilayer that separates a first electrolyte volume from a second electrolyte volume. The second electrolyte volume comprises a component to measure the fluorescence of the enzymatic target after it has been acted upon by the toxin or protein.
[0176]In FIG. 5 a cell-free assay device is comprised of an encapsulated lipid bilayer, a first electrolyte volume (left side, FIG. 5), and a second electrolyte volume (right side, FIG. 5). The first electrolyte volume is constructed with a first electrode. Th...
example 3
Use of Pressure for Detecting a Modified Target
[0178]In order to demonstrate the basic feasibility of using an antibody coated pore to characterize the concentration of an antigen, an antibody-modified glass nanopore membrane (AMGNM) was developed. An AMGNM is sometimes referred to as “a site specific chemically modified nanopore” and is described in international patent application number PCT / US12 / 33142, which is incorporated herein by reference. In the work described herein, the antigen or modified target is a cleaved form of SNAP-25, cSNAP-25, which is the enzymatic target of botulinum toxin type A (BoNT / A). FIG. 7. Shows a portion of a fabrication process of an AMGNM, starting with a nanodisk electrode 103. Initially, the outside glass surface of a nanodisk electrode, whose Pt diameter matches that of the desired channel size, was chemically protected with cyano-silane 105 (3-cyanopropyldimethylchlorosilane) that limits non-specific binding (7C). A small amount of non-specific b...
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