ENZYME ASSEMBLY AND CATALYTIC
ACTIVITY IN A REUSABLE BIOMEMS
PLATFORM FOR METABOLIC ENGINEERING
Xiaolong Luo1,6, Angela Lewandowski2,3, Gregory Payne3, Reza Ghodssi4,6, William Bentley1,3 and Gary Rubloff5,6
1Fischell Department of Bioengineering, 2Department of Chemical and Biomolecular Engineering, 3University of Maryland Biotechnology Institute
(UMBI), 4Department of Electrical and Computer Engineering, 5Department of Materials Science and Engineering, 6Institute for Systems Research,
University of Maryland, College Park, MD 20742, USA
Abstract
We report a reversible biofunctionalization strategy for assembling a
catalytically-active enzyme in a reusable bioMEMS that supports
programmable bio-component assembly at selected sites. A control
system supervises the sequential assembly of various bio-components
onto specific electrodes in microfluidic channels. We demonstrate (a) the
assembly of a Pfs enzyme at a specific electrode address and (b) that the
enzyme is catalytically active in the bioMEMS. Enzymatic activity is
robust, remaining over days. In addition, the chitosan-mediated
biofunctionalization can be reversed, making a new type of biopolymerbased bioMEMS reusable for repeated assembly and catalytic activity.
Day 1
SAH
Enzymatic
reactions
Electric
signal
Day 3
Day 2
SAH
Day 2
Day 3
Enzyme assembly,
disassembly and
reassembly
In PBS
Day 4 ~ 7
SAH
SAH
Day 4
HPLC
analysis
HPLC
analysis
Day 8
HPLC
analysis
HPLC
analysis
Protein
% Conversion
chitosan film
chitosan film
(b) Tyrosinase conjugation of Pfs and chitosan
O
O
+
Tyrosinase
+
+
Pfs
Pfs-Chitosan
Conjugate
Chitosan
+
Active
OH
++
+
+
+ +
Pfs-chitosan
conjugate
(d) Enzymatic small molecule reaction
substrate
Product
8
10
In PBS (4 days)
Re-assembly
0
2
0
2
Product
4
6
8
10
0
2
4
6 (hours)
• Experiment: 46% conversion,
Control: 18% conversion at
3uL/min flow rate
HPLC analysis
• Electrode area is 0.2% of total
area in microchannel
Æ highly specific assembly
15
10
5
1
2
3
4 (hour)
40
• Estimated specific activity of
3.7 μmol SAH/min/mg Pfs is
within the range of reported
value (0.05~156) in literature
• Enhanced stability of assembled
enzyme over time
20
0
Conclusion and Future Work
(e) Mild acid wash
+
+
+
+ +
+
+ +
BioMEMS device and bioMEMS control system
(c)
(a)
6
20
0
Electric signal
5% HCl
4
60
+
+ +
+
+
2
0
analysis
Channel
side wall
Sealing layer
(b)
Pfs
Enzyme
0
After Pfs non-specific
assembly
Conjugation
(c) Biofunctionalization
(a) Prefabricated device
+
60
50
40
30
20
10
0
(a) SAH
+
Pfs
Biochemical
Activation
5
Negative control
Flow rate (μL/min)
Programmable enzyme assembly and reusable bioMEMS
¾Biochemical activation to prepare enzyme-chitosan conjugate
¾Electrical signal-guided enzyme assembly (spatial and temporal control)
¾Prefabricated device for biofunctionalization when needed
¾Reusable bioMEMS device and low average usage-cost
Inactive
10
0
Cell
% Conversion
electric
signal
15
In PBS (4 days)
Biomolecule assembly
20
Re-assembly
¾
25
Acid wash (10min)
Amine chemistry
Electrodeposition
chitosan
molecule
Electrode
PfsChitosan
Acid
Electric
signal
Acid wash (10min)
•
DNA
Wafer
PfsChitosan
Flow rate (μL/min)
Chitosan
pH responsive solubility
¾
Reversible enzyme assembly and catalytic activity
(a) Reproducible enzyme assembly and catalytic reactions
(b) Reproducible catalytic activity and stability of assembled enzyme over time
Method
•
Results
PC with
LabView
Electric
signal
Control
Flow
Control
Power supply
This work demonstrates:
¾Programmable enzyme assembly in bioMEMS (spatial and temporal)
¾Reversibility of biofunctionalization Æ Reusable bioMEMS
¾Retained catalytic activity and enhanced stability of assembled enzyme
Metabolic engineering in bioMEMS
Æ Multi-step cell-signaling process (autoinducer-2 production)
Æ BioMEMS platform for quorum sensing (QS) to study bacterial
pathogenicity.
AI-2 synthesis enzymes
waste
Fluid
flow
Valve
(b)
500μm
Electric I/O
Fluidic I/O
Micropump
Products
or waste
Water, PBS, Chitosan ……
Pfs
DPD
Signal
molecule
AI-2
LuxS
Acknowledgements
Valve
Channel
Electrode
SRH
SAH
Counter Chitosan
electrode
This work was supported in part by the Robert W. Deutsch Foundation,
the NSF IMI program, the Laboratory for Physical Sciences and the
Maryland NanoCenter.