US 11,738,312 B2

7

selectively controls transmission of energetic crosslinking
particles 207 through shadow mask 211 via apertures 230
disposed in shadow mask 211. Shadow mask 211 includes a
plurality of aperture 230 that transmits energetic crosslink-
ing particles 207. The transmission efficiency of aperture
230 can be partial or complete. Exemplary shadow masks
include metals, plastics, ceramic, and the like. Further,
aperture 230 can be disposed in shadow mask 211 ina select
pattern and can have an arbitrary cross-sectional shape (e.2..
polygonal, round) and size that can be used to limit a flux of
energetic crosslinking particles 207 or form a particular
cross-sectional shape of energetic crosslinking particles 207.

In an embodiment, with additional reference to FIG. 4,
multidimensional printer 200 further includes sample trans-
lation stage 213 disposed in sample chamber 206 and in
mechanical communication with solid crosslinked polymer
structure 210 produced from crosslinking polymers 202 by
energetic crosslinking particles 207, wherein sample trans-
lation stage 213 translates away from membrane 209,

toward membrane 209, or laterally with respect to mem- 2

brane 209 so that a two-dimensional or three-dimensional
size or shape of solid crosslinked polymer structure 210 can
be spatially controlled during formation of solid crosslinked
polymer structure 210 from polymers 202. Exemplary trans-
lation stages 213 includes a piezoelectric stage and the like.
Astep size of sample translation stage 213 can be from 1 nm
to 10* nm, specifically from 10 nm to 1000 nm, Further,
sample translation stage 213 can perform X, Y, Z indepen-
dent movements with aforementioned step sizes as well
rotation from and tilt from 0° to 360° with precision from
0.001 rad to 1 rad.

Sample chamber 206 receives energetic crosslinking par-
ticles 207 from membrane 209 and also receives liquid
composition 201 for interaction with energetic crosslinking
particles 207. Sample chamber 206 can provide a static
volume of liquid composition 201 or can include elements
for flowing liquid composition 201 through sample chamber
206. Accordingly, during formation of solid crosslinked
polymer structure 210, liquid composition 201 in sample
chamber 206 is a static liquid volume or is subject to fluid
flow through sample chamber 206. In an embodiment, with
reference to FIG. 4 and FIG. 5, sample chamber 206
includes flow chamber 214; flow channel 215 bounded by
wall 216 of flow chamber 214; inlet port 217 that receives
liquid composition 201 for fluid communication through
flow channel 215 in a direction of fluid flow 219; outlet port
218 for outflow of liquid composition 201 from sample
chamber 206; gasket 220 received in a gland embossed on
a surface of flow chamber 214 to seal flow chamber 214

against membrane support 221 that receives membrane 209: 5

and chamber lid 222 in mechanical communication with
membrane support 221 and flow chamber 214 and that is
fastened to flow chamber 214 to maintain force on mem-
brane support 221 and gasket 220. Elements of sample

chamber 206 can include a material compatible with liquid s

composition 201 and energetic crosslinking particles 207.
Exemplary materials include metals, plastics, glass, and the
like. A flow rate of liquid composition 201 through flow
channel 215 can be from 0 uL/sec to 100 mL/s, specifically
from 0 pL/sec to 100 uL/sec, and more specifically from 0
yL/sec to 10 L/sec. A volume of flow channel 215 proxi-
mate to membrane 209 can be from 0.01 wL to 1 L,
specifically from 0.1 0 UL to 1000 wl, and more specifically
from 1 0 pL to 10 wh. It is contemplated that a temperature
of liquid composition 201 in sample chamber 206 can be
controlled at an arbitrary temperature or allowed to obtain a
temperature of liquid composition 201 entering inlet port

&

&

8

217. Additionally, motion and position of solid crosslinked
polymer structure 210 formed in sample chamber 206 proxi-
mate to membrane 209 in flow channel 215 can be controlled.
by sample translation stage 213 disposed in sample chamber
206. Sample translation stage 213 can have electrical con-
nections to external control electronics that provide electri-
cal power for manipulation of the position of movement of
sample translation stage 213 relative to membrane 209.

In an embodiment, sample chamber 206, membrane 209
includes a silicon chip with nine SiN 50 nm thin membranes
disposed on the silicon chip and sealing the volume of flow
channel 215 filled with liquid composition 201 that includes
a PEGDA water solution. In this configuration, printing of
solid crosslinked polymer structure 210 from liquid com-
position 201 was performed inside a scanning electron
microscope (SEM) with beam energies of energetic cross-
linking particles 207 from 3 keV to 30 keV via rastering
focused electron beam with pre-programmed pitch size and
dwell time.

Liquid hydrogel composition 201 includes polymers 202
and can be a static liquid volume or fluidic (e.g., microflu-
idic, exchangeable). It is contemplated that a portion of
liquid composition 201 that is exposed to and receives
energetic crosslinking particles 207 can be part of a system
such as lab-on-a-chip or organ-on-a-chip with crosslinking
polymers 202 being a part of processing with such system.
Polymers 202 can be a single species or a plurality of
species. Some of the species include cross-linkable moieties
that can include a multiple bond such as a double bond or
triple bond. Polymers 202 can be selected based on an
application of solid crosslinked polymer structure 210. In an
embodiment, polymer 202 includes poly(ethylene glycol)
diacrylate that crosslinks via double bonds with initiation of
crosslinking by interaction with energetic crosslinking par-
ticles 207. Polymer 202 can include a block polymer,
copolymer, and the like. A molecular weight of polymer 202
can be from 100 Da to 10° Da, specifically from 100 Da to
10* Da, and more specifically from 100 Da to 10° Da.
Polymers 202 can be monodisperse or polydisperse.

In an embodiment, solvent 203 of liquid composition 201
ed to solvate polymers 202. Exemplary solvents 203
include water, alcohols, phenylenediamines, ethylene car-
bonate, sulfuric acid, benzene, halogenated hydrocarbons,
methyl ethyl ketone, hydrocarbons, esters, phenol, chloro-
phenol, phenol, dioxane, biphenyl, phenyl ether, xylene,
1,2-dichlorobenzene, toluene, chloroform, methanol, cyclo-
hexanone, ethylene carbonate, and the like or combination
thereof. In an embodiment, solvent 203 is water.

Solute 204 can be disposed in liquid composition 201 to
receive energetic crosslinking particles 207 and cause local-
ized crosslinking among polymers 202 proximate to solute
204. Exemplary solute 204 includes quantum dots, nanopar-
ticles, or a combination comprising at least one of the
foregoing solutes.

In according with the foregoing, a spatial resolution of
forming solid crosslinked polymer structure 210 depends on
a size of an interaction volume between polymers 202 and
energetic crosslinking particles 207 and energy of energetic
crosslinking particles 207. In this respect, the spatial reso-
lution is tunable and is from 10 nm to 90 jm, specifically
from 20 nm to 1000 nm, and more specifically from 50 nm
to 100 nm. To achieve a selected geometrical shape or size,
liquid composition 201 is subjected to relative rastering of
energetic crosslinking particles 207 in liquid composition
201 for two-dimensional patterning of polymers 202 to form
solid crosslinked polymer structure 210 having a selected
two-dimensional pattern. The two-dimensional pattern can



US 11,738,312 B2

Sheet 12 of 14

Aug. 29, 2023

U.S. Patent

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US 11,738,312 B2

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distances before reacting out completely. One of the possible
crosslinking agents particularly abundant in water under
aerated conditions is hydroperoxyl radical. FIG. 9E com-
pares the energy dose distribution upon water irradiation
with 3 keV focused beam (left panel) with the corresponding
HO,> concentration profile (right panel). As can be seen,
HO,~ crosslinking activator concentration remains high,
well beyond the electron dose gelation threshold and there-
fore may account for the systematically increased size of the
printed features.

With regard to printing controls, parameters tuned to
control the size, shape, and sharpness of features within the
individual printed layer were present. In raster scanning
mode, parameters include: electron beam energy (E), dwell
time (t,) at a pixel location, step-size (L) during the scan
and exposure dose D per pixel defined as D=I,,,n/L? where
I, is an electron (photon) beam current (intensity) and n is
number of scans.

FIG. 10A-G shows 3D structures printed using in-liquid
crosslinking by electrons (panels A-E) and soft X-rays
(panels F, G). The primary energy of electrons determines
the range of electrons in the solvent and therefore the height
profiles of the printed structure ranging from ca 100 nm to
ca 10 um while varying beam energy from 3 keV to 20 keV
(FIG. 10B, D). The dynamic range of heights exceeding 100
aspect ratio was achieved via beam energy variation, how-
ever, the sharpness of the features drops concomitantly with
energy. The comparable aspect ratios of 3D structures can be
obtained via varying only the dwell time. Different from the
beam energy Variations, which requires refocusing, the
facile and instant modulation of the dwell time and step-size
allows the fabrication of the high-aspect ratio microstruc-
tures, e.g. a flagella-like object in FIG. 10C in a single run
with a writing speed of ca. 5 mm/s. Note that similar
structures have been used for locomotion in liquids after
being functionalized with magnetic nanoparticles. The effect
of the beam intensity and writing sequence on the linear
feature size and morphology can be seen in the inset of FIG.
10H. Feature size increases with the beam intensity and then
saturates. This is a result of the increase and saturation of the
crosslink density inside the excitation volume similarly to
the radiation induced crosslinking in a dry state. Feature size
can be controlled effectively via printing with different
X-ray photon energies just below and above the element
specific absorption edge (e.g., Ols in FIG. 9B). The example
in the FIG. 10G shows that this can be performed even
through an already printed ture. Compared to printing
with electrons, the aspect ratio of the X-ray-induced struc-
tures can be appreciably larger similar to the deep X-ray

lithography results on solid films. In addition, the crosslink 5

density of X-ray printed hydrogel structures was noticeably
lower compared to their e-beam counterparts reflecting
significant surface rippling of the features upon drying. FIG.
10H-J quantifies the measured heights of the crosslinked

dried gel features as a function of each one of these s

parameters with others being unchanged. In addition to the
apparent ineré of the feature size with electron beam
energy (FIG. 10H), the height variation with exposure dose
has characteristic fast rise followed by saturation behavior
what was also commonly observed in dry films. Critical dose
threshold (on the order of 1 e~/nm?) provides through-
membrane crosslinking of a stable gel structure. While the
dose increase does not affect the dimensions of the interac-
tion volume in water, it expands the boundary at which the
critical concentration of crosslinking radicals can be main-
tained. The same is valid for the feature heights increase
with the dwell-time (FIG. 101). Step-size becomes a rather

20

&

16

important parameter when the beam is rastered across the
sample surface. By increasing the step-size and therefore the
pixel area, one can tune the overlap between the interaction
volumes (diffusion zones) of individual adjacent voxels,
thus reducing or increasing the effective thickness (and
width) of the printed feature. An important distinction
between the electron and soft X-ray beam writing used in
this study is the size of the probe: electron beam has a
diameter of ca. 5 nm, while for X-rays it is ca. 150 nm in our
setups. On the other hand, the effective diameter of the
interaction volume of a few keV electron beam is apprecia-
bly larger (see FIG. 9A) compared to one for soft X-rays.
‘Therefore, for X-rays, the formation of corrugated/discon-
tinuous patterns can be observed as soon as the step-size
becomes larger than 150 nm (see inset in FIG. 10J). On the
contrary, few keV electron beams generate continuous pat-
tems, for step-size values even larger than 100 nm.

For printing applications, dwell-time and step-size can be
independent irradiation parameters to control the size and
crosslink density of raster printed gel structures. Unlike the
beam energy and intensity, these two parameters are
decoupled in the SEM controls and can be tuned during the
scanning without refocusing. In the case of the e-beam
printing, the lateral and longitudinal resolution of the resul-

stant features are coupled and are proportional to each other

(FIG. 9A). As one intensifies any of the parameters: beam
energy, intensity, dwell time, both the width and height of
the pattern increase. Finally, the smallest feature size and
maximum resolution for SEM based gelation can be
achieved via lowering the energy of electrons that can
penetrate through the SiN window. We were able to rou-
tinely write ca. 150 nm thin and 100 nm wide gel lines
through a 50 nm SiN window using 3 keV electron beam
energy. Sub-100 nm features are attainable if thinner mem-
branes are used.

With regard to in-liquid direct writing for layer-by-layer
printing with electron beams, sequentially crosslinked fea-
tures | and 2 are shown in FIG. 11A that overlap and have
double-exposed region (OFR) as a second layer. OER can
have an increased thickness compared to a parent structure.
Since the
FIG. 10H), this height difference can vary from its maximum
value at very low doses to zero at doses above ca. 10?
e/nm. Moreover, the size and shape of the resultant
structure (including OER) may not depend on the writing
sequence (i.e. shape,,-shape,,). For in-liquid crosslinking,
layer-by-layer log-pile structures (FIG. 11B) were printed in
PEGDA solution and studied as a function of writing
sequence, electron energy, and dose.

SEM images in FIG. 11C show log-pile structures pat-
temed side-by-side with low (left) and high (right side)
doses. As expected, due to complete crosslinking along the
length of the structures, the high-dose structure does not
show any noticeable increase of the thickness of nods in
comparison to the thickness of the regular bar. The mor-
phology of the structure printed in liquid with a low dose,
however, exhibit a noticeable dependence on the writing
sequence and does not follow an expected commutative
exposure rule characteristic for solid resists (e.g.
shape,,shape,;). We assume that in-liquid electron beam
writing proceeds in a different way compared to solid resist
and the mechanical stress induced by densification of the
OER upon sequential writing leads to mechanical lifting and
eventually delamination of the prior-printed construct (FIG.
JIC, left panel).

Delamination of the printed layer from the optical win-
dows has been a speed limiting factor of the first bottom-up



U.S. Patent Aug. 29, 2023 Sheet 3 of 14 US 11,738,312 B2

YN y 2
Mt 22 40 244 206
FIG. 3A

200



US 11,738,312 B2

9

be arbitrary. Exemplary two-dimensional patterns include
linear interconnects, closed structures, arbitrary geometrical
shapes, and the like. It is contemplated that liquid compo-
sition 201, in contact with solid crosslinked polymer struc-
ture 210 having the selected two-dimensional pattern, can be 5
subjected to modulation of an energy or intensity of ener-
getic crosslinking particles 207 in liquid composition 201
such that addition of polymers 202 to solid crosslinked
polymer structure 210 provide a selected three-dimensional
geometry to solid crosslinked polymer structure 210 with the
two-dimensional pattern. The two-dimensional pattern can
be arbitrary. Exemplary two-dimensional patterns include
linear interconnects, circular structures, geometrical figures
of arbitrary shapes, and the like. In some embodiments,
liquid composition 201, in contact with solid crosslinked
polymer structure 210 having the selected two-dimensional
pattern, receives additional polymers for crosslinking in
presence of energetic crosslinking particles 207 such that
additional polymers 202 are crosslinked to solid crosslinked
polymer structure 210 to provide a selected three-dimen- 20
sional geometry to solid crosslinked polymer structure 210
with the two-dimensional pattern.

Multidimensional printer 200 can be made in various
ways. In an embodiment, a process for making multidimen-
sional printer 200 includes filling the sample chamber 206 25
with printing solution, disposing energetic crosslinking par-
ticle source 208 on vacuum chamber 205; optionally dis
posing shadow mask 211 in vacuum chamber 205; disposing
vacuum chamber 205 in communication with membrane
209; disposing sample chamber 206 in communication with
membrane 209; disposing liquid composition 201 in sample
chamber 206; and optionally disposing sample translation
stage 213 in sample chamber 206 and in communication
with sample chamber 206.

It will be appreciated that in making multidimensional 35
printer 200 elements thereof are arranged components to be
in communication so that energetic crosslinking particles
207 propagates from vacuum chamber 205 and received by
liquid composition 201 in sample chamber 206 with vapor
isolation of vacuum chamber 205 and sample chamber 206 4:
by membrane 209 and arranging electrical components to
communicate electrical signals amongst the various electri-
cal components. Fluid flow and stoppage of liquid compo-
sition 201 in sample chamber 206 can occur via a fluid pump
or like device. 4

The process for making multidimensional printer 200 also
can include filling chamber 206 with precursor solution with
or Without solute inclusions solute 204; printing the layer via
radiation induced crosslinking; activating delamination
agent (electrochemical, electrophoretic, mechanical, capil- 5
lary, thermal, photoinduced or their combination) to detach
the print from the membrane if working without free liquid-
gas interface; moving the stage and replenishing a liquid
interface; repeating the printing of the next layer; and
removing the sample from the solution upon completion

Multidimensional printer 200 has numerous advantageous
and unexpected benefits and u: In an embodiment, a
process for making a multidimensional structure from a
liquid composition with multidimensional printer 200
includes: disposing liquid composition 201 in sample cham-
ber 206; producing, by energetic crosslinking particle source
208, energetic crosslinking particles 207; receiving, by
vacuum chamber 205, energetic crosslinking particles 207,
from energetic crosslinking particle source 208; communi-
cating energetic crosslinking particles 207 from vacuum 6
chamber 205 to membrane 209; receiving, by membrane
209, energetic crosslinking particles 207 from vacuum

10

chamber 205; transmitting, by transmission layer 229 of
membrane 209, energetic crosslinking particles 207 for
communication to sample chamber 206; receiving, by liquid
composition 201 in sample chamber 206, energetic cross-
linking particles 207 from membrane 209; subjecting the
cross-linkable moieties of polymers 202 to energetic cross-
linking particles 207; crosslinking portions of polymers 202
proximate to the cross-linkable moieties subjected to ener-
getic crosslinking particles 207 in response to receiving
energetic crosslinking particles 207; and forming solid
crosslinked polymer structure 210 in response to cr
ing polymers 202 to make the multidimensional st

In an embodiment, the process for making a multidimen-
sional structure from a liquid composition with multidimen-
sional printer 200 further includes: receiving, by shadow
mask 211 interposed between energetic crosslinking particle
source 208 and membrane 209, energetic crosslinking par-
ticles 207 from energetic crosslinking particle source 208;
communicating, energetic crosslinking particles 207 to
membrane 209 from shadow mask 211; and selectively
controlling transmission, by shadow mask 211, energetic
crosslinking particles 207 through shadow mask 211 via
apertures 230 disposed in shadow mask 211. Shadow mask
211 can be moved relative to solid crosslinked polymer
structure 210 to control formation of crosslinks and resulting
shape or size of solid crosslinked polymer structure 210.

In an embodiment, the process for making a multidimen-
sional structure from a liquid composition with multidimen-
sional printer 200 further includes spatially controlling,
during formation of the solid crosslinked polymer structure
210 from the polymers 202, a two-dimensional or three-
dimensional size or shape of the solid crosslinked polymer
structure 210 by translating away from the membrane 209,
toward the membrane 209, or laterally with respect to the
membrane 209 the growing solid crosslinked polymer struc-
ture 210. Such motion can be provided by sample translation
stage 213.

In an embodiment, the process for making a multidimen-
sional structure from a liquid composition with multidimen-
sional printer 200 further includes two-dimensional pattern-
ing of polymers 202 by subjecting liquid composition 201 to
relative rastering of energetic crosslinking particles 207 in
liquid composition 201 to form solid crosslinked polymer
structure 210 having a selected two-dimensional pattern.
Relative rastering can occur by rastering the radiation beam,
shadow mask 211, sample stage 213 or their combination.

In an embodiment, the process for making a multidimen-
sional structure from a liquid composition with multidimen-
sional printer 200 further includes: subjecting liquid com-
position 201 in contact with solid crosslinked polymer
structure 210 having the selected two-dimensional pattern to
modulation of an energy or intensity of energetic crosslink-
ing particles 207 in liquid composition 201; adding poly-
mers 202 to solid crosslinked polymer structure 210 by
crosslinking in response to subjecting liquid composition
201 to the modulation; and providing a selected three-
dimensional geometry to solid crosslinked polymer structure
210 with the two-dimensional pattern in response to adding
polymers 202 to solid crosslinked polymer structure 210.

In an embodiment, the process for making a multidimen-
sional structure from a liquid composition with multidimen-
sional printer 200 further includes: receiving, by liquid
composition 201 in contact with solid crosslinked polymer
structure 210 having the selected two-dimensional pattem,
additional polymers; crosslinking the additional polymers to
solid crosslinked polymer structure 210 by energetic cross-
linking particles 207; and forming a selected three-dimen-



US 11,738,312 B2

21

wherein the shadow mask selectively controls transmis-
sion of the energetic crosslinking particles through the
shadow mask via apertures disposed in the shadow
mask.

2. The multidimensional printer of claim 1, further com-
prising a sample translation stage disposed in the sample
chamber and in mechanical communication with the solid
crosslinked polymer structure produced from crosslinking
the polymers by the energetic crosslinking particles,

wherein the sample translation stage translates away from

the membrane, toward the membrane, or laterally with
respect to the membrane so that a two-dimension or
three-dimensional size or shape of the solid crosslinked
polymer structure can be spatially controlled during
formation of the solid crosslinked polymer structure
from the polymers.

3. The multidimensional printer of claim 1, wherein the
energetic crosslinking particles comprise electrons.

4. The multidimensional printer of claim 1, wherein the 2

energetic crosslinking particles comprise X-rays.

5. The multidimensional printer of claim 1, wherein the
energetic crosslinking particles comprise poly(ethylene gly-
col) diacrylate.

6. The multidimensional printer of claim 1, wherein a
spatial resolution of forming the solid crosslinked polymer
strueture depends on a size of an interaction volume between
the polymers and the energetic crosslinking particles and
energy of the energetic crosslinking particles.

7. The multidimensional printer of claim 1, wherein the
spatial resolution is tunable and is from 10 nm to 90 jum.

8. The multidimensional printer of claim 1, wherein the
membrane comprises a barrier layer comprising silicon and
a transmission layer comprising silicon nitride disposed on
the barrier layer.

9. The multidimensional printer of claim 1, further com-
prising

the liquid composition that comprises the solvent and the
plurality of polymers disposed in the solvent, wherein
the polymers comprise the cross-linkable moiety.

3

&

22

10. The multidimensional printer of claim 1, further
comprising:

the multidimensional structure comprising the solid cross-

linked polymer structure disposed on the membrane,
the solid crosslinked polymer structure obtained from
the polymer in response to crosslinking the polymer via
the cross-linkable moiety.

11. The multidimensional printer of claim 9, wherein
solute is disposed in the liquid composition such that solute
receives energetic crosslinking particles and locally cross-
links the polymers proximate to the solute.

12. The multidimensional printer of claim 11, wherein the
solute comprises quantum dots, nanoparticles, or a combi-
nation comprising at least one of the foregoing solutes.

13. The multidimensional printer of claim 10, wherein,
during formation of the solid crosslinked polymer structure,
the liquid composition in the sample chamber is a static
liquid volume or is subject to fluid flow through the sample
chamber.

14. The multidimensional printer of claim 9, wherein the
liquid composition is subjected to relative rastering of the
energetic crosslinking particles in the liquid composition for
two-dimensional patterning of the polymers to form the
solid crosslinked polymer structure having a selected two-
dimensional pattern.

15. The multidimensional printer of claim 14, wherein the
liquid composition, in contact with the solid crosslinked
polymer structure having the selected two-dimensional pat-
tem, is subjected to modulation of an energy or intensity of
the energetic crosslinking particles in the liquid composition
such that addition of polymers to the solid crosslinked
polymer structure provide a selected three-dimensional
geometry to the solid crosslinked polymer structure with the
two-dimensional pattern.

16. The multidimensional printer of claim 14, wherein the
liquid composition, in contact with the solid crosslinked
polymer structure having the selected two-dimensional pat-
tem, receives additional polymers for crosslinking in pres-
ence of the energetic crosslinking particles such that addi-
tional polymers are crosslinked to the solid crosslinked
polymer structure to provide a selected three-dimensional
geometry to the solid crosslinked polymer structure with the
two-dimensional pattern.

+e ee


US 11,738,312 B2

13
Electrochemical delamination tests have been made using
PBS-based 20% w/v PEDGA solution.

With regard to composite hydrogels, gold nanoparticles
(50 nm in dia.) suspension in water was pre-concentrated by
centrifuging (2000 r/min, 5 min) and was subsequently
extracted and mixed with a 20% w/v PEGDA solution. The
prepared composite mixture was irradiated in the liquid
phase with an electron or soft X-ray beams through 50 nm
SiN membrane. The chip with the printed composite gel
structures was then developed in water and subsequently
analyzed as described in the article and Supporting material.

With regard to live cells interfacing and proliferation
printing. Caco-2 cells were thawed and cultured in DMEM
(Dulbecco’s Modification of Eagle’s medium) with 4.5 g/L
glucose and L-glutamine without sodium pyruvate for a few
days. These were subsequently washed in phosphate-buff-
ered saline solution (PBS) and DMEM. Lifting off process
was carried out using 2 ml of 0.05% (w/v) trypsin-EDTA and
left for 5 min to 10 min until the attached cells become

mobile on the slide. Neutralization of trypsin is done by 2

adding an equal volume of growth medium. The obtained
cell suspension is concentrated by centrifuge. The cell
concentrate was added to the PBS-based 20% w/v PEDGA
solution and cells were allowed to adhere to the SiN mem-
brane. For viability tests after irradiation, the crosslinked gel
with encapsulated cells was rinsed in the growth medium for
cell and exposed to calcein green dye in the growth medium
for 1 hr. Inside the live cells, the non-fluorescent i
converted into green-fluorescent calcein via de-esterification
of the acetoxymethy! group by the esterases only produced
by a live cell.

With regard to modeling, a stack of 50 nm SiN and
20-micron thick water layer was modeled with the electron
beam incident on the SiN membrane. The Monte Carlo
(MC) simulations (described in the SI section) generated the
trajectories and corresponding energy deposited (Gy). The
parameters used to generate FIG. 2 a) were as follows:
6.25-10° electrons for a 5 nm beam diameter for 3 keV, 5
keV, 10 keV, and 20 keV primary beam energy. The energy
deposition results in Gy for 6.25-10° electrons were scaled
depending on the current value to obtain the rate of ener;
deposition (Gy/s) and fed into the radiolysis kinetics model
(described in’ more details in the SI section). To generate
results shown in FIG. 2d), the CFD model was executed for
3 keV primary beam, and currents current values: 50 pA, 85
pA, 125 pA, 160 pA, 200 pA and 215 pA.

With regard to focused beam induced crosslinking pro-
cess in polymer aqueous solutions, upon impinging on the
liquid interface of the PEGDA aqueous solution, the elec-

trons experience a cascade of elastic and inelastic scattering 5

events, which slow down and broaden the beam thus creat-
ing a droplet-like highly excited interaction volume where
the crosslinking of the PEGDA polymer molecules into
insoluble gel matrix takes place (FIG. 9A, right facet).

Without wishing to be bound by theory, radiation-induced

crosslinking in polymer solutions occurs via two mecha-
nisms: directly, by activating the reactive groups in the
polymer solute with primary or secondary electrons or
indirectly, by electron beam-induced solvent radiolysis that
generates a variety of radicals promoting crosslinking. The
partitioning between these two reaction channels depends on
multiple parameters such as the solute concentration,
molecular weight of the polymer, beam energy, its intensity,
radical diffusion coefficient, and the like. This, in turn,
provides flexibility for highly penetrating ionizing radiation
with spatially homogenous excitation and diffusional pro-
files. Compared to broad beam irradiation conditions,

&

&

14

focused electron or X-ray beams generate highly localized,
spatially inhomogeneous radiolitic volumes where the radia-
tion chemistry and kinetics are less explored. The compara-
tive side-to-side -Raman analysis of irradiated and pristine
PEDGA regions (FIG. 9B) reveals quick quenching of the
1410 cmTM', 1640 cmTM! C=C stretching bands upon electron
irradiation, as is expected for complete crosslinking of
PEGDA via abstracting hydrogen atoms and opening the
carbon double bonds in its acrylate end groups predomi-
nantly by the radiolitic OH radicals.

When soft X-rays trigger crosslinking, the net effect is
similar, although it proceeds via different electronic excita-
tion/relaxation pathways. X-ray photon ionize/excites the
valence and core electrons of the solute and solution mol-
ecules. The relaxation of core hole proceeds primarily via
emission of Auger electrons After such a de-excitation, the
energy is effectively absorbed by the liquid via the same
inelastic electron scattering mechanism as described above
for electron beam induced crosslinking. The major differ-
ences between the electron and soft X-ray induced cross-
linking, therefore, are due to their ionization cross-sections
as can be seen from the liquid water example (FIG. 9C). The
values of liquid water photoionization cross-section for soft
X-rays (100 eV to 2000 eV) are on the average ca. 100 times

5 smaller compared to the few keV electrons. Therefore, such

X-rays can penetrate significantly deeper into the precursor
solution forming gel features with larger aspect ratios and
lower crosslinking densities. In addition, the electron ion-
ization cross-section of water is a smooth function of energy
(FIG. 9C), thus the range of electrons in water solution and
printed feature size always increase with energy. In the case
of soft X-rays however, the photoionization cross-section
sharply increases at the onset of the Ols core level excita-
tions (FIG. 9C). This chemical selectivity of the X-rays
range in the solution will be used to control the aspect ratio
of the printed features or chemically selective objects encap-
sulation.

Since the crosslinking in moderately concentrated
PEGDA solutions proceeds primarily via interaction with
radiolytic radicals, their diffusion beyond the electron-
sample interaction volume can be the feature size determin-
ing factor. To evaluate the effect of radical diffusion on
crosslinking we compared the experimental feature sizes the
e-beam printed in a 20% w/v PEGDA aqueous solution with
the modeled ones applying the same irradiation conditions.
Monte Carlo (MC) simulated spatial distributions of the
radiation dose deposited in aqueous solution through a 50
nm thick SiN membrane are shown in FIG. 9D for a few
different energies and a 5 nm wide electron beam. Assuming,
the critical energy dose for PEGDA gelation being within the
range 10° Gy to 10° Gy, the height of stable hydrated objects
can be from 200 nm for 3 kV to 8 micrometers for 20 keV.
electron beams (FIG. 9D).

Empirical data on thicknesses of hydrated features
obtained under the same conditions (dashed lines in FIG.
9D) are larger than the depths at the onset of gelation critical
density obtained from Monte Carlo simulations. Th«
indicates that indirect crosslinking via a runaway di
of radiolytic activators may indeed be responsible for the
observed size increase of the printed features. To support the
observation further, we adapted a kinetic radiolysis model to
our SEM conditions. The numerical simulations based on
this model predict a short lifetime and therefore a small
runaway diffusion length (<200 nm) for the most abundant
crosslinking OH radical outside the electron beam interac-
tion volume, thus implying that there can exist other radicals
with larger lifetimes that can, in principle, diffuse to longer



US 11,738,312 B2

3

a larger portion of the curve is from beam parking with a
longer dwell time. Panel D shows a dome-shaped structure
formed from four overlapping coaxial rings, shown in panel
E, by varying electron beam energy and dose for every ring.
Panel F shows letters printed with 536 eV and 150 nm wide
X-ray beam with 25 ms dwell time. Photon flux was about
2x10” photons/nm? s and 100 nm step size. Panel G shows
die printed with two photon energies for 13 um base squares
at 536 eV and 2.5 jum squares at 526 eV. Panels H to J show
heights measured by SEM of the dry rectangular and linear
structures as a function of exposure dose (panel H), dwell
time (panel I) and step size (panel J) for different energies of
electrons and X-rays beams. Insets show an SEM image of
structure written with variable beam current (panel H), dwell
time along its length using 536 eV X-rays (panel 1), and
step-size L using 536 eV X-rays and 3 keV electrons;
FIG. 11 shows layer-by-layer beam writing of overlap-
ping structures in liquid composition 201 that included
PEGDA. Panel A shows sequential writing of overlapping
structures. Panel B shows a model log-pile structure printed
with and without anodic potential on a SiN/Pt electrode.
Panel C shows morphology of the e-beam printed PEGDA
structure depended on the writing sequence 1-2-3-4 for
low-dose exposure and was independent for saturated cross-
linking density; Panel D shows electrochemical size and
adhesion control of in-liquid crosslinked structures for no
electrochemical bias (V=0) applied and for applied cathodic
potential V>1 volt and current density J-20 pA/mm?,
wherein reduction of feature size and footprint are shown as
result of presence of energetic crosslinking particles: and
FIG. 12 shows solid crosslinked polymer structure formed
for applications of a multidimensional printer with e-beam
and X-ray printing in liquid composition. Panel A shows
e-beam induced encapsulation and interconnection of micro-
objects and nanoobjects using in liquid cured gel. Step (1)
involves irradiation and in-liquid patterning of the nanopar-
ticles or cells in a hydrogel precursor suspension through a
SiN membrane followed by step (2) or step (3) for micro-
scopic and spectroscopic characterization in a dry or wet
stage. Step (4) involves hydrogel interfaced cells treated
with a fluorescent probe for viability tests. Step (5) involves
fluorescence microscopy of biological objects interfaced
with a gel. Panel B shows excitation and recording of LSPR
signal from e-beam printed Au NP/PEGDA composite
hydrogel plasmonic sensor as a function of ambient humid-
ity. Panel C shows normalized reflectance signal collected
from PEGDA incapsulated 50 nm Au assembly as a function
of the humidity. Insets show dark-field optical image of
-printed composite hydrogel pack and SEM images of Au

NP assembly where signal was collected from. Panel D 5

shows an optical microscopy image of hydrogel after elec-
tron beam irradiation of cell-laden PEGDA solution. Panel
E shows a fluorescence microscopy image of gel interfaced
cells stained with viability indicator such as alcein green
dye, wherein a dashed line indicates a necrotic cell.

DETAILED DESCRIPTION.

A detailed description of one or more embodiments is
presented herein by way of exemplification and not limita-
tion,

It has been discovered a multidimensional printer and
process for printing described herein provide patterning of
hydrogels in a native, liquid state by electrons or x-rays
respectively transmitted through an electron or x-ray trans-
parent membrane. The membrane can be molecularly imper-
meable or partially molecularly permeable and separates

20

&

&

s

4

electron optics or x-ray optics from a pressure of gas from
a liquid sample. Dependence of an attenuation length of
electrons and x-ray radiation in a condensed medium on,
energy controls crosslinking of a polymer in a liquid com-
position to make two-dimensional and three-dimensional gel
structures, referred to as a solid crosslinked polymer struc-
ture, with submicron spatial resolution. An advantage of
such compared to conventional dry gel patterning includes
micropatterning of the liquid composition in a liquid state
and additive fabrication of multi-compositional two-dimen-
sional and three-dimensional solid crosslinked polymer
structures as gel constructs, e.g., for tissue or soft robotics
engineering; addressable gel-encapsulation of an object,
e.g., a drug, biomolecule, functional nanoparticle, and the
like; or gel-coating or patterning of functional entities such
asa live biological object, composite, electrochemical elec-
trode, and the like.

Beneficially, the multidimensional printer herein provides
fabrication of two-dimensional and three-dimensional 3D
structures out of liquid biocompatible hydrogel solutions
(the liquid composition) for tissue engineering, soft robotics,
biosensing, drug delivery, wound treatment, or other bio-
medical research.

Conventional additive manufacturing from liquid gels is

5 based on photoinduced crosslinking of the polymer solution

and can be limited to micron-level spatial resolution by a
diffraction limit of photon optics. The multidimensional
printer overcomes this limitation and include:
X-rays as energetic crosslinking particles to
linking with smaller wavelength and mean free path in
condensed phase matter, wherein the energetic crosslinking
particles can be controlled by their energy. In an absence of
multidimensional printer 200 and just using e-beam lithog-
raphy or X-ray lithography, one obtains a two-dimensional
lithography process that requires vacuum conditions and dry
samples, ic., operation in an absence of a target (e.g., a
cross-linkable polymer) disposed in a liquid and lacks
three-dimensional printing in a continuous process. The
multidimensional printer 200 herein overcomes this techni-
cal limitation and includes the electron or X-ray transparent
membrane to isolate a vacuum of a radiation source chamber
source the liquid chamber.

In the multidimensional printer, the electron or X-ray
beam penetrates into the liquid after passing through the
membrane and crosslinks the polymers in the liquid com-
position within an interaction volume. The spatial resolution
of the multidimensional printer can depend on a size of the
interaction volume, which can be a function of electron or
X-ray energy that can be tuned, e.g., from 10 nm to tens of
microns. In addition, a structural size or degree of cross-
linking can be controlled by an irradiation dose (e.g. flux,
exposure time, dwell time, pitch size in case of scanning),
temperature (e.g... diffusion), or concentration of polymer, or
inclusion of a solute.

Multidimensional printer 200 makes a multidimensional
structure from a liquid composition. In an embodiment, with
reference to FIG. 1, FIG. 2, and FIG. 3, multidimensional
printer 200 includes: energetic crosslinking particle source
208 that produces energetic crosslinking particles 207;
vacuum chamber 205 in vacuum communication with ener-
getic crosslinking particle source 208 and that receives
energetic crosslinking particles 207 from energetic cross-
linking particle source 208 and communicates energetic
crosslinking particles 207 to membrane 209; membrane 209
in particle communication with vacuum chamber 205 and
including barrier layer 228 and transmission layer 229
disposed on barrier layer 228, wherein a portion of mem-



U.S. Patent Aug. 29, 2023 Sheet 8 of 14 US 11,738,312 B2

FIG. 8


U.S. Patent Aug. 29, 2023 Sheet 5 of 14 US 11,738,312 B2



US 11,738,312 B2

Sheet 4 of 14

Aug. 29, 2023

U.S. Patent

22
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FIG. 4


US 11,738,312 B2

5

brane 209 has some of transmission layer 229 in an absence
of barrier layer 228 for transmission of energetic crosslink-
ing particles 207 through transmission layer 229 without
being obstructed by barrier layer 228, such that membrane
209: receives energetic crosslinking particles 207 from
vacuum chamber 205; blocks, by barrier layer 228, energetic
crosslinking particles 207 from being further communicated
in multidimensional printer 200; and transmits, by transmis-
sion layer 229, energetic crosslinking particles 207 for
further communication in multidimensional printer 200; and
sample chamber 206 in communication with membrane 209
and that: receives liquid composition 201 that includes
solvent 203 and a plurality of polymers 202 disposed in
solvent 203, polymers 202 including a cross-linkable moi-
ety; receives energetic crosslinking particles 207 communi-
cated from transmission layer 229 of membrane 209; sub-
jects the cross-linkable moieties of polymers 202 to
energetic crosslinking particles 207 such that portions of
polymers 202 proximate to the cross-linkable moieties sub-

jected to energetic crosslinking particles 207 crosslink to. 2

form solid crosslinked polymer structure 210, wherein mem-
brane 209 isolates a vacuum of vacuum chamber 205 from
vapor of liquid composition 201 in sample chamber 206.

With reference to FIG. 3, multidimensional printer 200
can be configured in various ways, wherein membrane 209
seals sample chamber 206 that can, e.g., be disposed in
vacuum chamber 205 of an electron microscope as shown in
panel A of FIG. 3. An output optical element of the micro-
scope can be capped with membrane 209 and liquid com-
position 201 with free liquid surface being under ambient
conditions shown in panels B and C in FIG. 3. Lateral
resolution for crosslinking polymers 202 in liquid compo-
sition 201 by energetic crosslinking particles 207 can be
provided by electron optics or X-ray optics disposed in
energetic crosslinking particle source 208 or vacuum cham-
ber 205 to provide focused radiation as shown in panels A
and B in FIG. 3 or by placement of shadow mask 211 as
shown in panel C of FIG. 3 for unfocused radiation. In some
embodiments, zone plate 227 and order sorting aperture 226
can be interposed between energetic crosslinking particle
source 208 and membrane 209 for controlling a beam profile
of energetic crosslinking particles 207 received by liquid
composition 201 as shown in panel D of FIG. 3. Further,
two-dimensional patterning of solid crosslinked polymer
structure 210 can be achieved via rastering of the focused
beam of energetic crosslinking particles 207 or solid cross
linked polymer structure 210 via their relative motion or via
X-positioning or Y-positioning of shadow mask 211 relative
to liquid composition 201 for unfocused energetic crosslink-
ing particles 207.

Three-dimensional features in solid crosslinked polymer
structure 210 can be formed by aforementioned two-dimen-
sional positioning with either modulation of the energy and
intensity of energetic crosslinking particles 207 or additively

replenishing a liquid layer of liquid composition 201 in ;

contact with solid crosslinked polymer structure 210 such
that the polymers 202 in the replenished liquid layer is
subject to crosslinking added polymers to existing solid
crosslinked polymer structure 210.

Energetic crosslinking particle source 208 produces ener-
getic crosslinking particles 207. Energetic crosslinking par-
ticles 207 includes electrons or X-rays. Energetic crosslink-
ing particle source 208 can include elements that focus,
modulate, or energy-select energetic crosslinking particles
207 such that energetic crosslinking particles 207 can be
formed in a beam that is continuous or modulated. Further,
energetic crosslinking particles 207 can be focused (e.g., in

&

6

a focused beam of energetic crosslinking particles 207, or
diffuse, such as in a flood beam of energetic crosslinking
particles 207. Exemplary energetic crosslinking. particle
source 208 that provide electrons as energetic crosslinking
particles 207 includes an electron gun, filament, cathode,
electron microscope, and the like. Exemplary energetic
crosslinking particle source 208 that provide X-rays as
energetic crosslinking particles 207 includes an X-ray tube
and the like. An energy of energetic crosslinking particles
207 can be from 10 eV to 1000 keV, specifically from 1 keV
to 30 keV, and specifically from 1 keV to 10 keV. A type of
energetic crosslinking particles 207 can be electrons, hard
UV photons, X-ray photons or ions. In an embodiment,
energetic crosslinking particles 207 includes gamma rays.

Vacuum chamber 205 provides a vacuum environment for
energetic crosslinking particle source 208 and a propagation
medium through which energetic crosslinking particles 207
propagate from energetic crosslinking particle source 208 to
membrane 209. A pressure of vacuum chamber 205 can be
selected to provide a mean free path or energy filtering of
energetic crosslinking particles 207. It is contemplated that
the pressure of vacuum chamber 205 can be from 10-'° Pa
to 1 Pa, specifically from 10-* Pa to 107' Pa, and more
specifically from 10~> to 10°? Pa.

In an embodiment, with additional reference to FIG. 6,
membrane 209 includes barrier layer 228 and transmission
layer 229 disposed on barrier layer 228. Barrier layer 228
can include a material that attenuates an intensity of ener-
getic crosslinking particles 207 transmitted through barrier
layer 228. The attenuation efficiency can be partial or
complete, i.e., 100%. Attenuation includes absorption or
redirection of a trajectory of energetic crosslinking particles
207 such as refraction or reflection. Membrane 209 can be
made of any material such as a low-Z material with a
thickness that is less than electron or X-ray inelastic mean
free path in such material at an energy of printing and having
mechanical strength that withstands a pressure differential
between sample chamber 206 and vacuum chamber 205.
Exemplary barrier layers 228 include a material that attenu-
ate electron transmission such as silicon, material that
attenuates X-ray transmission such as silicon, or a combi-
nation thereof.

Transmission layer 229 can include a material that trans-
mits energetic crosslinking particles 207. The transmission
efficiency of transmission layer 229 can be partial or com-
plete, ic., 100%. Exemplary transmission layers 229 include
a material that transmits electrons such as silicon nitride,
material that transmits X-rays such as silicon nitride, or a
combination thereof. First thickness T1 of barrier layer 228
can be from 10 nm to 10* nm, specifically from 100 nm to
1000 nm. Second thickness T2 of transmission layer 229 can
be from 0.34 nm to 1000 nm, specifically from 5 nm to 100
nm, and more specifically from 10 nm to 30 nm. Further,
barrier layer 228 can include a plurality of apertures in a
select pattern over which or in which is disposed transmis-
sion layer 229. In this manner, membrane 209 isolates gas
from leaving sample chamber 206 and entering vacuum
chamber 205 and spatially selects energetic crosslinking
particles 207 from vacuum chamber 205 being transmitted
to liquid composition 201 through transmission layer 229.

In an embodiment, with additional reference to FIG. 7,
multidimensional printer 200 further includes shadow mask
211 interposed between energetic crosslinking particle
source 208 and membrane 209 and that receives energetic
crosslinking particles 207 from energetic crosslinking par-
ticle source 208 and communicates energetic crosslinking
particles 207 to membrane 209, wherein shadow mask 211



U.S. Patent Aug. 29, 2023 Sheet 7 of 14 US 11,738,312 B2

FIG. 7A



US 11,738,312 B2

17

3D laser printers. This has been resolved via implementation
of continuous liquid interface production technology which
employed quenching of the polymer crosslinking at the
liquid-window interface with a controlled through-the-win-
dow delivery of molecular oxygen inhibitor. Along these
lines, electrochemically driven pH variations in PEG solu-
tions have be used to reduce the crosslinking density of
hydrogel to improve their cellular adhesion and permeabil-
ity. The role of the electrochemically generated inhibitors on
the size and adhesion of the electron beam printed structures
nvestigated. The supporting SiN membrane was coated
few tens of nanometers thick Pt layer defined as the
‘ing electrode in a two-electrode electrochemical cell
filled with PEGDA/PBS aqueous electrolyte. The anodic
potential of the working electrode was adjusted to near the
‘onset of water splitting reaction (FIG. 1B). SEM images in
FIG. 1D show two log-pile constructs printed using the same
irradiation parameters but with (right panel) and without
(left pane) anodic potential applied to the conducting mem-
brane with respect to the counter electrode. The drastic
reduction of the size and adhesion of the printed construct
(right panel) can be observed, which might be explained by
the electrochemical increase of the near electrode concerta-
tion of the crosslinking inhibitors like O, that hamper beam

induced crosslinking process. Cathodic potential, on the 2

other hand, leads to electrochemical polymerization of the
PEGDA solution at the membrane electrode and over-
whelms the beam-induced prints. The electrochemical
quenching/promoting of the PEGDA crosslinking not only
provides an additional control of the e-beam printed feature
size but has a direct consequence for the additive fabrication
of overlying structures in a layer-by-layer manner.

With regard to composite hydrogels as plasmonic
microsensors, hydrogel applications has broadened via syn-
thesis of composite formulations for the rational engineering
of optical, electrical, mechanical, and magnetic properties
for numerous applications. Fabrication of composite organic
hydrogels can be classified as: (i) in situ ones where func-
tional inclusions (e.g. nanoparticles) or precursors are pre-
mixed in the prepolymer solution and become stabilized in
the hydrogel during the crosslinking process, whereas (ii) ex
situ techniques typically involve an inclusion impregnation
process, applied after the crosslinking of the host matrix. In
situ encapsulation offers the advantages of embedding
objects independently of their size, homogeneously across
the bulk, while the post-crosslinking impregnation depends
on surface-to-bulk diffusion of chemicals into the gel’s
matrix which is often hampered for the objects larger than
gel’s mesh size. FIG. 12A (panels land 2) shows in-liquid

entrapping of nanoparticles and via focused e-beam induced

cross-linking of nanoparticles suspension. Due to facile
coupling of the mechanical optical, chemical and electrical
properties of such composite gels to external stimuli they
become a common platform for numerous sensor designs.

FIG. 11B shows a plasmonic humidity microsensor e-beam 5

printed out of PEGDA/SO nm Au nanoparticles suspension.
The broadband light source excites the localized surface
plasmon resonances (LSPR) in the selected gel-imbedded
nanoparticle assembly (see inset in the FIG. 11C). The
scattered light is collected in the reflection dark field mode
and the shift of the LSPR maximum L is determined
spectroscopically as a function of the humidity level above
the sensor. The observed blue shifi of the 4,, with increase
of the humidity level (FIG. 11C) is presumably due to a
combination of two factors: reduction of the gel refractive
index with humidity and weakening of the dipole-dipole
coupling between the adjacent nanoparticles in the aggregate

20

&

&

18

(which scales as (D/d)*) upon gel matrix swelling, here D is
Au NP diameter and d is the interparticle distance. The size
of the e-beam gelated composite in this particular study was
ca. 100 jumx100 jumx5 um, however the plasmonic hot spots
were located inside Au NP assembly that is only ca. 500 nm
wide (see insets in FIG. 11C).

With regard to cellular interfacing with hydrogel patches,
computer-brain interfacing with neural probes or cardiac
cellular electrophysiology can involve development of soft
microelectrode arrays that match mechanical chemical and
electrical properties of the individual live cells. PEG-based
hydrogels are prospective soft interface electrode materials
due to their biocompatibility or adjustable elastic moduli and
ionic conductance. Sub-micron indexing of life cells with
PEGDA microelectrodes can be made. To compensate for
small polymerization cross-section and preserve sufficient
reaction rate inside the mesoscopic voxel, high concentra-
tions of cytotoxic photo-initiators can be use that may
compromise the biocompatibility of the electrodes. To over-
come these shortcomings, we employ in-liquid gel focused
electron (X-ray) beam crosslinking technique for cellular
interfacing. In addition to highest lateral resolution of the
membrane patching and flexible electrodes patterning, the
advantage such an approach lays in a nanometer depth
control of the electron range and crosslinking density across
the irradiated patch such as desired degree of electrical and
gentle mechanical contact can be achieved at the very
cellular membrane-gel electrode interface. PEGDA contact
formation can be administered on a live cell before its
membrane irreversibly degrades. The extent of radiation
damage of the biological objects (cells) during electron
beam-induced PEGDA crosslinking is not well known and
generated reactive radiolytic species like OH', O-, and H,O,
at high concentrations are known to be detrimental to the
cells. Critical dose values span from ca. 10-* e“/nm? to ca.
10° e“/nm? that are considered acceptable for live mamma-
lian cells, yeasts and other microorganisms. Such a margin
manifests a fundamental challenge of high-resolution elec-
tron microscopy of live cells as well as the variance in
live/dead criteria applied. PEGDA crosslinking threshold
dose is on the order of 1 e“/nm? in our setup and can be
reduced by at least an order of magnitude if higher molecular
weight PEG is used. Since the imaging during crosslinking
(which usually leads to high irradiation dose) is not a
requirement, the encapsulation of live microorganisms using
our in-liquid lithography can be feasible. The test process
flow is depicted in FIG. 12A, panels 1, 3, 4. Once a
prepolymer solution with premixed and proximal to SiN
membrane live cells was exposed to electrons, the cross-
linked gel with trapped/interfaced cells was tested with
standard calcein-AM cell viability assay. Live cells uptake
the non-fluorescent calcein-AM ester. Inside the living cell,
ester reacts with cytosolic esterases, which convert it into a
green-fluorochrome: calcein to which cellular membrane is
not permeable. Bright-field optical image in FIG. 12D shows
a SiN window containing cell-laden PEGDA solution after
exposure to 10 keV primary beam with an average exposure
dose of 8 e“/nm? (absorbed dose ca 3x10° Gy). Fluorescent
microscopy image of gel-immobilized cells in the FIG. 12.
complements FIG. 12D and indicates (i) that cells B-E do
produce fluorescent calcein after encapsulation; (ii) calcein
distribution remains confined within the cellular borders
indicating the integrity of cellular membrane and (iii) some
of the cells (cell A in the FIG. 12E) appear dark implying its
necrosis, while the rest of the cells apparently survive the
interfacing procedure. Although local absorption dose at the
point of beam incidence can be few orders of magnitude



United States Patent

383

12B2

USOI11

(12) (0) Patent No: US 11,738,312 B2
Kolmakov et al. (45) Date of Patent: Aug. 29, 2023
(54) MULTIDIMENSIONAL PRINTER (58) Field of Clas: n Search
CPC... BOID 69/125; BOID 67/0088
(71) Applicant: Government of the United States of See application file for complete search history.
America, as represented by the
Secretary of Commerce, Gaithersburg, (56) References Cited
MD (US)
U.S. PATENT DOCUMENTS
(72) Inventors: Andrei A. Kolmakov, Frederick, MD 5674346 A 10/1907 Kunde
(US); Glenn Emerson Holland, 2004/0046120 Al* 3/2004 Moses .... .. HOLI37/20
Rockville, MD (US); Tanya Gupta, 250/311
Gaithersburg, MD (US) (Continued)
(73) Assignee: GOVERNMENT OF THE UNITED FOREIGN PATENT DOCUMENTS
STATES OF AMERICA, AS
REPRESENTED BY THE oN 1974608 A 6/2007
SECRETARY OF COMMERCE,
Gaithersburg, MD (US) OTHER PUBLICATIONS
(*) Notice: — Subject to any disclaimer, the term of this Winkler, R., et al., “3D nanoprinting via focused electron beams”,
patent is extended or adjusted under 35 Journal of Applied Physics, 2019, p. 210901, vol. 125.
USC. 154(b) by 334 days. (Continued)
(21) Appl. No.: 16/944,571 Primary Examiner — Nicholas R Krasnow
(74) Attorney, Agent, or Firm — Office of Chief Counsel
(22) Filed: Jul. 31, 2020 for National Institute of Standards and ‘Technology
(65) Prior Publication Data (57) ABSTRACT
US 2021/0031150 Al Feb. 4, 2021 A multidimensional printer makes a multidimensional struc-
ture from a liquid composition and includes: an energetic
crosslinking particle source; a vacuum chamber that receives
Related U.S. Application Data energetic crosslinking particles from the energetic crosslink-
. . ing particle source; a membrane that transmits the energetic
(60) Provisional application No. 62/881,677, filedon Aug.  crogstinking particles; and a sample chamber that: receives
ul a liquid composition that includes a solvent and polymers,
(1) Int. CL the polymers including a cross-linkable moiety subjected to
B29C 64/00 (2017.01) the energetic crosslinking particles such that portions of the
BOLD 69/12 (2006.01) polymers proximate to the cross-linkable moieties subjected
= getic crosslinking particles crosslink to form a
_ (Continued) inked polymer structure, wherein the membrane
(52) U.S. Cl. isolates a vacuum of the vacuum chamber from vapor of the
CPC ...... BOLD 69/125 (2013.01); BOID 67/00045

(2022.08), BOID 67/0088 (2013.01);
(Continued)

200

liquid composition in the sample chamber.

16 Claims, 14 Drawing Sheets



U.S. Patent Aug. 29, 2023

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US 11,738,312 B2

11
sional geometry of solid crosslinked polymer structure 210
with two-dimensional pattern in response to crosslinking the
additional polymers to solid crosslinked polymer structure

Multidimensional printer 200 and processes disclosed
herein have numerous beneficial, including higher spatial
resolution, fast printing rate, and absence of chemical ini-
tiators. Advantageously, multidimensional printer 200 over-
comes limitations of technical deficiencies of conventional
compositions such as using crosslink initiators for two-
photon crosslinking. Further, fine features as small as few
tens of nanometers can be printed.

Conventional photo-induced three-dimensional printing
exhibits resolution of order of hundreds of micrometers due
to technical diffraction limitations of optical wavelength
manipulation. Multidimensional printer 200 overcomes such
technical limitations and provides higher resolution such as
hundreds of nanometers in lateral and vertical directions in
forming solid crosslinked polymer structure 210. Printing of
solid crosslinked polymer structure 210 directly from liquid
state of liquid composition 201 provides gel encapsulation
of objects that may not be delivered inside a gel structure
after fabrication with conventional methods and overcomes
technical barriers of non-uniform distribution in manufac-
turing processes, where encapsulation is done post-gelation.
Multidimensional printer 200 can encapsulate live cells for
tissue engineering. Further, multidimensional printer 200
provides flexibility in resolution, size, or shape of solid
crosslinked polymer structure 210 that can be made via
tuning parameters that include beam energy, current, dwell
time of energetic crosslinking particles 207 received by
liquid composition 201. Diverse materials can be subjected
to being printed to form solid crosslinked polymer structure
210 by multidimensional printer 200, wherein such material
can include compositional gel material that may not be
optically transparent and may not be printable with UV
printing.

Multidimensional printer 200 and processes herein pro-
vide electron and x-ray lithography to be conducted in liquid
state. The latter drastically increases the number of material,
composites and devices to be processed and fabricated with
unprecedented thickness and dimensional control.

The articles and processes herein are illustrated further by
the following Example, which is non-limiting.

Example

Electron and X-ray Focused Beam Induced Crosslinking
in Liquids

Conventional additive fabrication of three-dimensional 5

(3D) provides micron to centimeters size constructs made of
polymers and soft materials from photo-curable formula-
tions suitable for optical photolithography, holographic, and
stereolithography.

Polymerization and crosslink chemical reactions were s

initiated inside a liquid composition with focused electron
beams. The beam writing was done through the thin elec-
tron/X-rays transparent membranes separating high-vacuum
equipment from the volatile precursor solution. Using a
hydrogel as a model soft material, in-liquid direct writing
with sub 100 nm resolution was performed. Focused soft
X-rays with variable photon energies provide gel crosslink-
ing with chemical and spatial selectivity. Crosslinking in
hydrogel solutions takes place at low radiation doses that are
below a viability threshold for biological cells with high
spatial resolution for applications in tissue micro-engineer-
ing, drugs delivery, biosensing, bio-electronics, and the like.

20

&

4

12

To deliver focused electron or soft X-ray beams to
vacuum-incompatible liquid solutions and for patterning and
imaging in liquids, fluidic and closed chambers were
equipped with 30 nm to 50 nm thin silicon nitride (SiN)
membranes to isolate the liquid solution from the vacuum of
the microscope, wherein panel A of FIG. 3 shows a con-
figuration for electrons and panel D of FIG. 3 shows a
configuration for X-rays.

FIG. 4, FIG. §, and FIG. 8 show an SEM-compatible
chamber and results therefrom that were used with electron
focused beam-induced polymer crosslinking in volatile solu-
tions. The chamber for X-ray studies contained about 5
microliters of enclosed hydrogel precursor solution and was
smaller in size. Both chambers were equipped with a SiN/Si
chip pattemed with an array of nine 50 nm thick and 100
jmx100 jum wide SiN suspended membranes capable of
withstanding a 1 Bar (10° Pa) pressure differential. This
single-use exchangeable SiN/Si chip is vacuum-sealed
against the body of the fluidic (or enclosed) cell and the
interior of the chamber was filled with poly(ethylene glycol)
diacrylate (PEGDA) 20% w/v aqueous solution. Nine iden-
tical membrane windows were used for writing multiple
features and combinatorial data collection within a single

5 experiment as show in FIG. 8. Arrays of rectangular and fine

linear structures were printed on individual membrane win-
dows by varying only one of the parameters: beam energy,
irradiation intensity, step-size or dwell time while keeping
others locked. After rinsing off the uncured solution with
water, the dimensions of the cross-linked stable gel struc-
tures were inspected in the hydrated state using atomic force
microscopy (AFM) and optical profilometry and more pre-
cisely after drying by SEM. By comparing the height of the
same objects in their hydrated and dry state the gel’s average
vertical swelling ratio was estimated as a function of the
polymer molecular mass, concentration of the solution and
typical electron beam irradiation conditions. Such calibra-
tions were used to estimate the size of the dried-up hydrated
gel objects.

With regard to hydrogel synthesis and printing, printing
was performed with a stagnant liquid setup wher 5-10 pL
of 20% w/v PEGDA (average molar mass 0.7 kg/mol, no
initiator) aqueous solution was drop-casted on to Si chip
with an array of nine 50 nm thick SiN membranes. The chip
was bonded to supporting plate and sealed with vacuum-
tight (flow) chamber with silicone rubber gasket. Inside
SEM, liquid prepolymer solution was patterned through the
SiN window using focused electron beam with a known
amount of dose at every pixel. The experiment was repeated
in another part of the 100 mx100 pm window or at the
different SiN window using a different set of irradiation
parameters. After e-beam exposure, the chip was taken out
of the chamber and gently rinsed in DI water to remove the
uncured solution. This leaves an array of printed gel features
adhered to SiN membranes. The latter were inspected in a
hydrated state with AFM or optical microscopy/profilometry
and/or ina dry state using SEM, AFM, EDS, XPS, u-Raman
and other characterization tools. Printing with soft X-rays
was performed at ESCA microscopy beamline equipped
with zone plate optics capable to focus monochromatized
light to a spot 150 nm in diameter. The undulator and
monochromator have been set to operate either at 526 eV or
at 536 eV with the photon flux in the order of 10° photons/s
at 150 nm wide focal spot. The chamber equipped with the
same chip with nine SiN membranes array was filled with
PEGDA solution, sealed and scanned in front of the beam in
a pre-programmed path to generate a required pattern.



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US 11,738,312 B2

1
MULTIDIMENSIONAL PRINTER

CROSS REFERENCE TO REI
APPLICATIONS

PED

‘The application claims priority to U.S. Provisional Patent
Application Ser. No. 62/881,667, filed Aug. 1, 2019, the
disclosure of which is incorporated herein by reference in its
entirety.

STATEMENT REGARDING FEDERALLY,
SPONSORED RESEARCH,

This invention was made with United States Government
support from the National Institute of Standards and Tech-
nology (NIST), an agency of the United States Department
of Commerce and The Government has certain rights in the
invention. Licensing inquiries may be directed to the Tech-
nology Partnerships Office, NIST, Gaithersburg, Md.,
20899; voice (301) 301-975-2573; email tpo@nist.gov; ref-
erence NIST Docket Number 19-018US1.

BRIEF DESCRIPTION

Disclosed is a multidimensional printer for making a
multidimensional structure from a liquid composition, the
multidimensional printer comprising: an energetic crosslink-
ing particle source that produces energetic crosslinking
particles; a vacuum chamber in vacuum communication
with the energetic crosslinking particle source and that
receives the energetic crosslinking particles from the ener-
getic crosslinking particle source and communicates the
energetic crosslinking particles to a membrane: the mem-
brane in particle communication with the vacuum chamber
and comprising a barrier layer and a transmission layer
disposed on the barrier layer, wherein a portion of the
membrane has some of the transmission layer in an absence
of the barrier layer for transmission of the energetic cross-
linking particles through the transmission layer without
being obstructed by the barrier layer, such that the mem-
brane: receives the energetic crosslinking particles from the
vacuum chamber; blocks, by the barrier layer, the energetic
crosslinking particles from being further communicated in
the multidimensional printer; and transmits, by the trans-
mission layer, the energetic crosslinking particles for further
communication in the multidimensional printer; and a
sample chamber in communication with the membrane and
that: receives a liquid composition that comprises a solvent
and a plurality of polymers disposed in the solvent, the

polymers comprising a cross-linkable moiety; receives the 5

energetic crosslinking particles communicated from the
transmission layer of the membrane; subjects the cross-
linkable moieties of the polymers to the energetic crosslink-
ing particles such that portions of the polymers proximate to

the cross-linkable moieties subjected to the energetic cross- 5

linking particles crosslink to form a solid crosslinked poly-
mer structure, wherein the membrane isolates a vacuum of
the vacuum chamber from vapor of the liquid composition
in the sample chamber.

Disclosed is a process for making a multidimensional
structure from a liquid composition with the multidimen-
sional printer, the process comprising: disposing a liquid
composition in the sample chamber; producing, by the
energetic crosslinking particle source, the energetic cross-
linking particles; receiving, by the vacuum chamber, the
energetic crosslinking particles, from the energetic cross-
linking particle source; communicating the energetic cross-

20

&

&

2

linking particles from the vacuum chamber to the mem-
brane; receiving, by the membrane, the energetic
crosslinking particles from the vacuum chamber; transmit-
ting, by the transmission layer of the membrane, the ener-
getic crosslinking particles for communication to the sample
chamber; receiving, by the liquid composition in the sample
chamber, the energetic crosslinking particles from the mem-
brane; subjecting the cross-linkable moieties of the polymers
to the energetic crosslinking particles; crosslinking portions
of the polymers proximate to the cross-linkable moieties
subjected to the energetic crosslinking particles in response
to receiving the energetic crosslinking particles; forming the
solid crosslinked polymer structure in response to crosslink-
ing the polymers to make the multidimensional structure.

BRIEF DESCRIPTION OF THE DRAWINGS

The following description should not be considered lim-
iting in any way. With reference to the accompanying
drawings, like elements are numbered alike.

FIG. 1 shows a multidimensional printer for making a
solid crosslinked polymer structure:

FIG. 2 shows a multidimensional printer with crosslinks
in a solid crosslinked polymer structure;

FIG. 3 shows a plurality of configurations for a multidi-
mensional printer in panels A, B, C, and D;

FIG. 4 shows a cross-section of a sample chamber;

FIG. 5 shows an exploded view of the sample chamber
shown in FIG. 4;

FIG. 6 shows a plan view of a membrane in panel A and,
in panel B, a cross-section along line A-A of the membrane:

FIG. 7 shows a plan view of a shadow mask in panel A
and, in panel B, a cross-section along line A-A of the shadow

FIG. 8 shows a three-dimensional surface plot of an
optical image of solid crosslinked polymer structures that
were electron beam printed on a membrane;

FIG. 9 shows in panel A, top facet: electron beam gelation
in a liquid polymer solution through a 50 nm thick SiN
membrane. Lower energy electrons generate smaller exci-
tation volume and a finer feature size. The left facet of panel
‘A shows a spatial distribution of energy deposition by
electron beams in water, and the right facet of panel A shows
direct and indirect, through radiolytic radicals R, poly(eth-
ylene glycol) diacrylate (PEGDA) polymer crosslinking.
Panel B shows decreasing C=C bonds in PEGDA solution
upon irradiation with ca. 10° Gy dose for crosslinking of
precursor molecules. Panel C shows a comparison of energy
dependence of electron and soft X-ray ionization cross
sections of liquid water. Panel D shows absorbed energy
doses as a function of depth for electrons at 100 pA current
and 1 ms dwell time and for X-rays at 2x10” photons/s and
10 ms dwell time, wherein dashed vertical lines depict
empirical hydrated feature sizes, and error bars are standard
deviations from three separate experiments. Panel E shows
an energy deposited distribution for 3 keV 200 pA electron
beam in the left panel and distribution of hydroperoxyl
radical concentration in the right panel;

FIG. 10 shows SEM images of 3D features that were
patterned with focused electron beam in panel A to E and
patterned with soft X-rays in panels F and G. Numbers
indicated inside each panel are energy, beam currents as
photons intensities, dwell times, and step sizes used. In panel
A, NIST was formed with 3 keV electrons at 100 pA and 1
ms dwell time with 10 nm pixel size. Panel B shows an
annular structure formed by writing 1 pm wide ring with a
radius of 10 um. Panel C shows a curved structure, wherein



U.S. Patent Aug. 29, 2023 Sheet 1 of 14 US 11,738,312 B2

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US 11,738,312 B2

19

higher than the cell’s lethal dose limit, interfacing of the life
cells with PEGDA electrode may be due to radiation damage
that is mainly localized within a submicron gap of solution
between the cellular and SiN membranes (FIG. 12A). Cells
that are poorly adhered or floating in solution ca micron
away from the SiN membrane see a significantly diluted
load of radiolitic species. Moreover, the majority of the
reactive species produced by the beam becomes scavenged
during gel crosslinking process what, therefore effectively
reduce the concentration of toxic species seen by the cells.

Spatially controlled crosslinking inside liquid polymer
solutions employing scanning electron and soft X-ray
microscopy occurred. Gel interfacing of live cells, fabrica-
tion of a composite hydrogel plasmonic device, and 3D
printing of model hydrogel structures with a sub-micrometer
resolution can be made. Threshold dose required for electron
beam-induced PEGDA cross-linking in a liquid state can be
on the order of 1 e~/nm? and explored the effect of diffusion
of radiolitic species and other empirically adjustable and
tunable parameters such as electrons (or X-rays) energy,
beams intensity, exposure time, electrochemically driven pH
value and the like on the resolution and size of the features
formed. High spatial resolution printing of a large class of
hydrogels precursor solutions in the liquid state can also be
extended to gas phase polymerization and offers unique
advantages in shape, size and precision compared to tradi-
tional dry gel lithography and can complement the existing
multiphoton polymerization methods. The multidimensional
printer 200 can be implemented in a high vacuum, environ-
mental SEM, atmospheric SEM, synchrotrons or laboratory-
based X-ray microscope, and the like. The tunability of
‘X-ray energy at synchrotrons opens an additional opportu-
nity to conduct element specific 3D gel printing in solutions
relevant to biomedical, soft micro-robotics, electrochemical
and other applications. Moreover, the combination of our
method with the recently proposed implosive fabrication
technique can result in nanometer-scale 3D printing.

While one or more embodiments have been shown and
described, modifications and substitutions may be made
thereto without departing from the spirit and scope of the
invention. Accordingly, it is to be understood that the present
invention has been described by way of illustrations and not
limitation. Embodiments herein can be used independently
or can be combined.

All ranges disclosed herein are inclusive of the endpoints,
and the endpoints are independently combinable with each
other. The ranges are continuous and thus contain every
value and subset thereof in the range. Unless otherwise
stated or contextually inapplicable, all percentages, when

expressing a quantity, are weight percentages. The suflix 5

“(s)” as used herein is intended to include both the singular
and the plural of the term that it modifies, thereby including
at least one of that term (e.g., the colorant(s) includes at least
one colorants). “Optional” or “optionally” means that the

subsequently described event or circumstance can or cannot 5

occur, and that the description includes instances where the
event occurs and instances where it does not. As used herein,
“combination” is inclusive of blends, mixtures, alloys, rea
tion products, and the like.

As used herein, “a combination thereof” refers to a
combination comprising at least one of the named constitu-
ents, components, compounds, or elements, optionally
together with one or more of the same class of constituents,
components, compounds, or elements.

All references are incorporated herein by reference.

The use of the terms “a” and “an” and “the” and similar
referents in the context of describing the invention (espe-

20

0

&

0

45

at

0

65

20

cially in the context of the following claims) are to be
construed to cover both the singular and the plural, unless
otherwise indicated herein or clearly contradicted by con-
text. “Or” means “and/or.” It should further be noted that the
terms “first,” “second,” “primary,” “secondary,” and the like
herein do not denote any order, quantity, or importance, but
rather are used to distinguish one element from another. The
modifier “about” used in connection with a quantity is
inclusive of the stated value and has the meaning dictated by
the context (eg., it includes the degree of error associated
with measurement of the particular quantity). The conjunc-
tion “or” is used to link objects of a list or alternatives and
is not disjunctive; rather the elements can be used separately
or can be combined together under appropriate circum-
stances

What is claimed i:
1. A multidimensional printer for making a multidimen-
sional structure from a liquid composition, the multidimen-
sional printer comprising:
an energetic crosslinking particle source that produces
energetic crosslinking particles;
a vacuum chamber in vacuum communication with the
energetic crosslinking particle source and that receives
the energetic crosslinking particles from the energetic
crosslinking particle source and communicates the
energetic crosslinking particles to a membrane;
the membrane in particle communication with the vacuum
chamber and comprising a barrier layer and a trans-
mission layer disposed on the barrier layer, wherein a
portion of the membrane has some of the transmission
layer in an absence of the barrier layer for transmission
of the energetic crosslinking particles through the trans-
mission layer without being obstructed by the barrier
layer, such that the membrane:
receives the energetic crosslinking particles from the
vacuum chamber;

blocks, by the barrier layer, the energetic crosslinking
particles from being further communicated in the
multidimensional printer; and

transmits, by the transmission layer, the energetic
crosslinking particles for further communication in
the multidimensional printer; and
a sample chamber in communication with the membrane
and that:
receives a liquid composition that comprises a solvent
and a plurality of polymers disposed in the solvent,
the polymers comprising a cross-linkable moiety;

receives the energetic crosslinking particles communi-
cated from the transmission layer of the membrane;
and

subjects the cross-linkable moieties of the polymers to
the energetic crosslinking particles such that portions
of the polymers proximate to the cross-linkable
moieties subjected to the energetic crosslinking par-
ticles crosslink to form a solid crosslinked polymer
structure,

wherein the membrane isolates a vacuum of the vacuum
chamber from vapor of the liquid composition in the
sample chamber; and

a shadow mask interposed between the energetic cross-
linking particle source and the membrane and that
receives the energetic crosslinking particles from the
energetic crosslinking particle source and communi-
cates the energetic crosslinking particles to the mem-
brane,


US 11,738,312 B2

Page 2
(51) Int. Cl. 40/00 (2014.12); B33 40/10 (2020.01); B33¥
BOID 67/00 (2006.01) 50/00 (2014.12); B33¥ 50/02 (2014.12); B33¥
B29C 64/307 (2017.01) 70/00 (2014.12); B33¥ 99/00 (2014.12); GOIN
B29C 64/245 (2017.01) 23/2251 (2013.01): HOLJ 37/3244 (2013.01):
B33Y 40/00 (2020.01) BOID 2323/30 (2013.01); B33Y 10/00
B29C 65/82 (2006.01) (2014.12); B33Y 30/00 (2014.12); B33Y 40/20
B33Y 50/02 (2015.01) (2020.01); B33¥ 80/00 (2014.12): GOIN 33/50
ay wan ort (2013.01); GOIN 2021/95676 (2013.01); GOIN
3: (2015.01) 2223/418 (2013.01); GO2B 21/34 (2013.01):
B29C 64/255 (2017.01) 3G 7
B29C 6430 (2017.01) G03G 2215/2054 (2013.01); GOSB
BDIF 12/82 (2021.01) 2219/49023 (2013.01); GO6T 2207/10061
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B33Y 40/10 (2020.01) HOLJ 2237/2003 (2013.01); HOLJ 2237/2608
HOLS 3732 (2006.01) (2013.01) ¥108 148/143 2013.01) Y10T
B22F 12/00 (2021.01) 156/172 (2015.01); YIOT 156/1798 (2015.01)
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* cited by examiner


U.S. Patent Aug. 29, 2023 Sheet 11 of 14 US 11,738,312 B2

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US 11,738,312 B2

Sheet 14 of 14

Aug. 29, 2023

U.S. Patent

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