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Ionescu, R.E., Marks, R.S., Gheber,
L.A. (2003). Nanolithography Using Protease Etching
of Protein Surfaces. Nano Letters 3,
1639.
Ionescu, R. E.; Marks, R. S.; Gheber,
L. A., (2005) Manufacturing of nano-channels with
controlled dimensions, using protease
nano-lithography. Nano Letters 5, 821.
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A novel approach to
“direct-write” negative nano-lithography, based on
biological specificity, where a proteolytic enzyme is used
to etch grooves in a hardened protein layer. We are using
trypsin to
engrave features in a BSA layer, while flowing the trypsin
solution via a nano-pipette (controlled by a
SPM) to the
surface. Trypsin is a
proteolytic enzyme that cleaves very specifically on the
carboxyl side of lysine
and arginine
residues.
We deliver trypsin
solution via a nano-pipette, and control its position with a
Scanning Probe Microscope (SPM).
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181 ankyngvfqe ccqaedkgac llpkietmre kvltssarqr lrcasiqkfg eralkawsva
241 rlsqkfpkae fvevtklvtd ltkvhkecch gdllecaddr adlakyicdn qdtissklke
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421 lgeygfqnal ivrytrkvpq vstptlvevs rslgkvgtrc ctkpesermp ctedylslil
481 nrlcvlhekt pvsekvtkcc teslvnrrpc fsaltpdety vpkafdeklf tfhadictlp
541 dtekqikkqt alvellkhkp kateeqlktv menfvafvdk ccaaddkeac favegpklvv
601 stqtala
k-lysine
r-arginine
Amino acid sequence of Bovine Serum Albumin (BSA). |
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Trypsin on BSA, holding the pipette in one place for various
time durations |
Trypsin on BSA,
while
moving the sample relative to the pipette |
Water on BSA, holding the pipette in one place for various time
durations
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Water on BSA, while moving the sample relative to the pipette |
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(a)
Swelling of BSA, water only.
(b)
Etching of wells, trypsin solution
(c)
Deduced etching rate of trypsin (difference between (b) and (a))
Swelling starts upon contact, BSA swells and exposes lysine and
arginine residues to trypsin, which starts etching some 6.8
seconds later (see V=0 for (c)). Since etching rate is faster
than swelling rate (0.0188 > 0.0066), trypsin “wins”, and goes
below the original surface after ~8.5 seconds (see V=0 for (b)).
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100 nm pipette, 200 ms delay (1.5 µm/s)
Channel dimensions are influenced by pipette aperture
cross-section and writing velocity
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Etching is additive
since the depth of the depression at the intersection is the sum
of depths of the channels.
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Volume of trypsin delivered is proportional to channel
cross-section. The proportion changes with pipette aperture
diameter, thus the same volume delivered with a smaller diameter
pipette (at a slower writing speed) etches a larger volume than
when delivered with a larger diameter pipette. |

A network of channels |

Fluorescently labeled Avidin flows in BSA channel |

Avidin deposited with a nanopipette on a channel. |
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