Chapter 9
Formulation of Reactive Nanostructured Adhesive Microbial Ink-Jet Inks for Miniature Biosensors and Biocatalysis 1,3,4
1
M . C.Flickinger1,2,7,*,Ο. K. Lyngberg , E . A. Freeman , C. R. Anderson , and M . C. Laudon Downloaded by CORNELL UNIV on October 5, 2013 | http://pubs.acs.org Publication Date: June 12, 2009 | doi: 10.1021/bk-2009-1008.ch009
1,5
1,6
1BioTechnology Institute, University of Minnesota, St. Paul, MN 55108 2Departmentof Biochemistry, Molecular Biology and Biophysics, University of Minnesota, St. Paul, MN 55108 Current address: Bristol-Myers Squibb, New Brunswick, NJ 08903 4Departmentof Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, MN 55455 Current address: Grain Value LLC, St. Paul, MN 55118 Current address: Department of Plant Biology, University of Minnesota, St. Paul, MN 55108 Current address: Department of Microbiology; Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695 *Corresponding author:
[email protected] 3
5
6
7
Reactive adhesive microbial inks can be formulated from aqueous latex emulsions for drop on demand (DOD) piezoelectric deposition. Ink-jet printing of a high density of living microorganisms many be useful to generate microstructures for micro-biosensors, as biocatalytic coatings in micro-fluidic devices, or micro-channel bioreactors. Microbial inks are viable wet cell paste mixed with aqueous (organic solvent-free) emulsions of adhesive polymer particles
NOTE: Portions of this work were presented at the Bio-Printing and Bio -Patterning Workshop, Manchester Conference Centre, UMIST, the University of Manchester, Manchester, UK, September 27-28, 2004 and the European Coating Conference "Power of Ink Jet Materials III", Berlin, Germany, December 1-2, 2005. 156
© 2009 American Chemical Society
In Nanotechnology Applications in Coatings; Fernando, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2009.
157 that dry rapidly with arrested coalescence as thin, adhesive, nanoporous coatings. Latex ink viscosity (~1.5 to 3 cP) is semi-shear rate dependent. Nanoporosity is essential for the embedded microorganisms to retain viability and reactivity. The nanoporous latex inks in this study contain glycerol and sucrose added to a low T acrylate/vinyl acetate latex emulsion (particle dia. ~280nm, T ~10°C). The glycerol and sucrose arrest polymer particle coalescence during film formation (porogens) and also act as osmoprotectants. Viability and reactivity is measured by bioluminescence following deposition, drying, and rehydration in nitrogen-limited (nongrowth) media. As a model system, the reactivity of Escherichia coli (50% RH using the nano-plotter enclosed in a humidified chamber.
E. coli Bioluminescence in Ink-Jet Printed Patches, Micro-Wells, and Dot Arrays Bioluminescence Response ofInk-Jet Printed Patches +2
The Hg -inducible reactivity of ink-jet deposited latex inks containing E. coli pRB28 was initially demonstrated by duplicating the previously reported mercury-inducible bioluminescence in 12.7 mm diameter patches of 30 μπι layers of the same E. coli strain prepared in SF091 latex and coated onto polyester using a manual Mayer rod drawdown coating method (P). In order to duplicate these results using an office printer, a 9 χ 11 array of 12.7 mm diameter and 2 μπι thick patches spaced 17 mm on center was printed on an 21.6 cm χ 27.9 cm sheet of polyester with ink formulation I (Table III) using the Epson 640 printer (Figure 2). Total printed patch volume was 240 nl. Figure 3 shows the induction kinetics for these patches which is 1.5 logs less than the bioluminescence of the same diameter but much thicker 30μπι thick, -6 μΐ SF091 latex patches previously reported (P). The maximum luciferase induction kinetics was similar to that previously reported, but bioluminescence was maintained for a shorter period of time (5 hours versus 24 hours) because of the reduced volume printed. The minimum concentration of Hg detected was 10η M . At 1,000 nM Hg the maximum induction was less than at 100 nM Hg indicating mercury is toxic at >100 nM as previously reported (P). This demonstrated that E. coli remained viable and reactive (inducible) following ink-jet deposition in a SF091 latex ink formulation deposited through a 26 μπι diameter ink-jet aperture. +2
+2
Bioluminescence Response ofLatex Micro- Wells Filled with Nanoporous Latex Ink using Reservoir Dispense Deposition Latex emulsion inks can be formulated to retain nanopores when dried by the addition of glycerol or sucrose to arrest polymer particle coalescence (70,
In Nanotechnology Applications in Coatings; Fernando, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2009.
172
Downloaded by CORNELL UNIV on October 5, 2013 | http://pubs.acs.org Publication Date: June 12, 2009 | doi: 10.1021/bk-2009-1008.ch009
Figure 2. 12.7 mm diameter, 2 μm thick Ε. coli latex ink patches printed on polyester sheet. Epson 640printer, 26μm aperture (44). (See page 3 of color insert.)
|«*
=Φ ^
^ 4 è 10*
οto
3
8 io i
t
~^
V
-V- ^
3fc
TT
^ -~ ^ 10
^ y 15
^ 20
10° 10' 10° 10 10' Mercury concentration (nM) 1
Time (h)
1
(B)
(A)
Figure 3. Bioluminescence kinetics of 12.7 mm diameter, -2 μm thick patches ofE. coli pRB28 printed using latex ink Formulation I (Table III.) on polyester using an Epson 640printer. A. HgCl concentration: A, 1,000 nM; O, 100 nM; Ύ, 10 nM; O, 1 nM; +, 0.1 nM; X, 0.01 nM. Β. Ύ, Maximum lux expression (sensitivity, dynamic range) (44). 2
41-43) (formulations I-IV, Table III). Without these additives, SF091 latex polymer particles completely coalesce during ink drying to create very low permeability polymer coatings. These nonporous coatings can be used for printing walls, ceilings, and channels creating micro-fluidic structures containing whole-cell microbial reaction zones (44, 46). In order to demonstrate this concept, the Canon JP 1080A printer was used to print SF091 latex (formulation V Table III) to form the walls of square microwells of decreasing volume on polyester sheet. Two rows of droplets were deposited around a center of 4 χ 3 droplet spaces wide. Each row or column of droplets was 0.3 mm wide. The ink-jet printer head moved in the y direction (02000 pm) delivering a line of droplets and then advanced one row in the χ y
In Nanotechnology Applications in Coatings; Fernando, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2009.
Downloaded by CORNELL UNIV on October 5, 2013 | http://pubs.acs.org Publication Date: June 12, 2009 | doi: 10.1021/bk-2009-1008.ch009
173 direction (0-3000 μπι) and delivered a second row of drops. This was repeated until all 7 rows were deposited. The entire well was created by printing 10 consecutive layers (10 LBL) with a 60 s to 120 s drying step between deposition of each layer. Figure 4A shows a profilometer image of an empty micro-well and 4B a well filled by printing E. coli in ink formulation 77. Individual droplet deposits are visible as craters. The micro-well wall height was composed of two lines of droplets (Figure 4A). The average wall height at the ridges was -10 μπι. In Β the well is filled with formulation II (Table III) and the height of the center is lower than the wall material because of the higher water content of the ink containing E. coli compared to the walls (formulation V) formed by only SF091 latex. The minimum micro-well that could be constructed using the Canon 1080A printer and SF091 latex was 1 row of droplets deposited 10 LBL. The Hg -inducible bioluminescence of E. coli pRB28 printed in micro-wells was investigated for square micro-well sizes of the following number of droplets (10 LBL) on each side: 28 χ 28, 14 χ 14,4 χ 3, and 2 χ 2 (generating 8.4 mm χ 8.4 mm, 4.2 mm χ 4.2 mm, 1.2 mm χ 0.9 mm and 0.6 mm χ 0.6 mm micro-wells respectively) which corresponded to micro-well volumes of 141 nl, 35 nl, 2.2 nl, and 0.72 nl well arrays. The E. coli filled micro-wells were induced with 100 nM Hg and the luciferase activity measured by excising individual microwells and placing them in a liquid scintillation vial. Figure 5A shows the induction kinetics for different sizes of micro-wells. Wells of 28 χ 28, 14 χ 14 and 4 x 3 droplets were induced by H g within 2 hours and maintained activity for 12 hours or longer. Micro-wells of 2 χ 2 droplets (
