2D polymer based targets for serial X-ray crystallography
View Patent ↗Systems and methods for producing and using polymer based fixed targets for hydrated, room temperature, high-throughput serial protein crystallography with minimal background and minimal sample consumption are provided. The fixed targets can also be used with non-protein samples such as nanoparticle inorganic crystals. The targets maintain full crystal hydration that allows for on-chip crystallization as well as maintaining long term stability of the sample within the chip. The target chips are optimized to minimize background by controlling the sample thickness in the beam to match crystal size, enabling fast raster scanning at lower fidelity and high hit rates that facilitate dynamic measurements. The targets provide an inexpensive, flexible substrate for on-chip, micro-batch or vapor diffusion crystallization facilitated by polymer brushes for sample concentration and surface initiated crystallization thereby eliminating damaging crystal sample transfer.
1 . A fixed target apparatus for serial x-ray crystallography, the apparatus comprising:
(a) a rigid base frame;
(b) a base polymer sheet mounted to the base frame, said sheet patterned with at least one window;
(c) a bottom low water permeability barrier film;
(d) a top low water permeability barrier film;
(e) spacers separating said bottom low permeability barrier film from said top low permeability barrier film, said spacers and barrier films forming at least one chamber;
(f) an upper polymer sheet patterned with at least one window; and
(g) an upper rigid frame;
(h) said at least one chamber further comprising barrier film surface functionalization for surface-initiated sample crystallization and in situ diffraction.
2 . The apparatus of claim 1 , further comprising:
a target mounting bracket coupled with said rigid base frame, said mounting bracket configured to fit in an X-ray or XFEL diffractometer.
3 . The apparatus of claim 1 , further comprising:
one or more input channels fluidly coupled to at least one input port in said top ridged frame and to at least one chamber; and
one or more output channels fluidly coupled to at least one chamber and to at least one output port in said top ridged frame;
wherein fluids can be introduced to at least one chamber through the input port; and
wherein fluids or gases can be withdrawn from at least one chamber through the output port.
4 . The apparatus of claim 3 , wherein said input channels, output channels and said at least one chamber further comprise surface treatments producing a hydrophilic surface for improved sample loading of the apparatus.
5 . The apparatus of claim 4 wherein said surface treatment comprises an oxygen-plasma treatment.
6 . The apparatus of claim 1 , wherein said pattern of windows of said patterned upper and lower polymer sheets comprises parallel rows of elongate windows configured to allow controlled exposure of a chamber interion to an orthogonal beam through the windows.
7 . The apparatus of claim 1 , wherein said spacers comprise a planar spacer layer with an open center pattern circumscribing one or more chambers, said spacer layer having a thickness that defines a volume of said one or more chambers.
8 . The apparatus of claim 7 , wherein said spacer layer has a chamber pattern selected from the group of patterns consisting of a plurality of parallel channels, a single central chamber, and a chamber with radiating channels.
9 . The apparatus of claim 1 , wherein said barrier film surface functionalization comprises:
ultraviolet light photografting of poly acrylic acid brushes onto film surface regions; and
wherein target proteins are concentrated and crystallized at said surface regions.
10 . A fixed target apparatus for serial x-ray crystallography, the apparatus comprising:
(a) a top polymer frame with at least one inlet port and at least one outlet port;
(b) an upper polymer sheet mounted to the top frame, said sheet patterned with a plurality of windows;
(c) at least one microfluidic chamber, each chamber comprising:
(i) an upper barrier film;
(ii) a lower barrier film;
(iii) a spacer layer separating said upper barrier film from said lower barrier film, said spacer having open center patterns circumscribing said chamber, said spacer layer having a thickness that defines a volume of said chamber;
(iv) an inlet channel fluidly coupled to at least one microfluidic chamber and said inlet port; and
(v) an outlet channel fluidly coupled to said outlet port and said at least one microfluidic chamber;
(d) a bottom polymer sheet patterned with a plurality of windows, said sheet mounted to the lower barrier film of said microfluidic chamber; and
(e) a bottom polymer frame mounted to the patterned bottom polymer sheet;
(f) wherein said spacer layer thickness enables tuning of chamber volume to match target crystal dimensions and crystallization conditions while minimizing background.
11 . A fixed target apparatus for serial x-ray crystallography, the apparatus comprising:
(a) a top polymer frame with at least one inlet port and at least one outlet port;
(b) an upper polymer sheet mounted to the top frame, said sheet patterned with a plurality of windows;
(c) at least one microfluidic chamber, each chamber comprising:
(i) an upper barrier film;
(ii) a lower barrier film;
(iii) a spacer layer separating said upper barrier film from said lower barrier film, said spacer having open center patterns circumscribing said chamber, said spacer layer having a thickness that defines a volume of said chamber;
(iv) an inlet channel fluidly coupled to at least one microfluidic chamber and said inlet port; and
(v) an outlet channel fluidly coupled to said outlet port and said at least one microfluidic chamber;
(d) a bottom polymer sheet patterned with a plurality of windows, said sheet mounted to the lower barrier film of said microfluidic chamber; and
(e) a bottom polymer frame mounted to the patterned bottom polymer sheet;
(f said at least one chamber further comprising barrier film surface functionalization for surface-initiated sample crystallization and in situ diffraction.