A high-throughput, low-volume enzyme assay on solid support

Article abstract of DOI:10.1021/ac048611n

A high-throughput enzyme assay is described that uses 1 μL or less of enzyme solution for each test Enzyme solutions are deposited by robotic handling in a throughput of over 1000 tests/h on the surface of silica gel plates that have been preimpregnated with fluorogenic substrates. The reaction is quantitated by fluorescence. The method is compatible with water-insoluble substrates (lipases), water-soluble substrates (glycosidases), whole-protein substrates (proteases), and enzyme inhibition measurements. Hydrolytically labile umbelliferyl esters can be used to assay lipases in this format without background hydrolysis. High throughput and reproducibility were tested by fingerprint analysis of lipases and esterases against 37 different fluorogenic ester substrates. A set of eight fluorogenic unbelliferyl esters was selected for optimal activity screening of lipases and esterases on silica gel plates.

Full text of DOI:10.1021/ac048611n

Anal. Chem. 2005, 77, 373-377
A High-Throughput, Low-Volume Enzyme Assay on
Solid Support
Peter Babiak and Jean-Louis Reymond*
Department of Chemistry and Biochemistry, University of Berne, Freiestrasse 3, CH-3012 Berne, Switzerland
lipases, esterases, glycosidases, and proteases. High throughput
and reproducibility were tested by fingerprint analysis of lipases
and esterases with fluorogenic ester substrates. Data analysis led
to an optimized set of eight substrates for screening lipases and
esterases.
A high-throughput enzyme assay is described that uses 1
µL or less of enzyme solution for each test. Enzyme
solutions are deposited by robotic handling in a through-
put of over 1000 tests/h on the surface of silica gel plates
that have been preimpregnated with fluorogenic sub-
strates. The reaction is quantitated by fluorescence. The
method is compatible with water-insoluble substrates
(lipases), water-soluble substrates (glycosidases), whole-
protein substrates (proteases), and enzyme inhibition
measurements. Hydrolytically labile umbelliferyl esters
can be used to assay lipases in this format without
background hydrolysis. High throughput and reproduc-
ibility were tested by fingerprint analysis of lipases and
esterases against 37 different fluorogenic ester substrates.
A set of eight fluorogenic unbelliferyl esters was selected
for optimal activity screening of lipases and esterases on
silica gel plates.
RESULTS AND DISCUSSION
Assay Development for Lipases and Esterases (Water-
Insoluble Substrates). Lipases and esterases are interfacial
enzymes hydrolyzing fatty acid esters, in particular triglycerides.
High-throughput assays for these enzymes are mostly based on
chromogenic nitrophenyl esters or fluorogenic umbelliferyl esters
of fatty acids.1 However, these substrates are only poorly soluble
in water, requiring the addition of cosolvents such as DMF or
DMSO to react, and hydrolyze spontaneously or in the presence
of noncatalytic proteins.2 Chemically stable analogues of these
chromogenic and fluorogenic esters are known that are compatible
with extreme pH and temperature conditions and only react with
active enzymes.3,4 However, these chemically stable substrates also
show limited solubility in water and still require a cosolvent to
react with the enzymes. We reasoned that an assay format
providing a large contact surface area between the substrate and
the enzyme might enable an insoluble substrate to react with
interfacial enzymes such as lipases and esterases. Because
noncatalytic proteins might not be able to solubilize the substrates
at the interface, this assay format might show reduced background
hydrolysis even with reactive substrates.
High-throughput enzyme assays are essential tools in enzyme
engineering and drug discovery. Such assays are most often
spectroscopic assays based on chromogenic or fluorogenic
substrates or sensors.1 One problem encountered in high-
throughput screening is to reliably assay many different samples
at the lowest possible cost and with very good accuracy. This can
be realized by minimizing sample volume and optimizing liquid
handling, for example, by using microtiter plates (MTPs) and
multichannel pipets. Assays in MTPs have the drawback that two
different solutions must be combined and evenly mixed in each
well. Pipetting and mixing operations are difficult to carry out in
small volumes, and artifacts due to solvent evaporation and
substrate precipitation cannot be controlled easily. Herein we
report a method for screening enzymes using silica gel plates
impregnated with fluorogenic substrates. The method ensures
even distrubution of the substrate to each enzyme sample and
enables high throughput (>1000 tests/h) while using small
reaction volumes (1 µL/assay). The assay is demonstrated for
Interfacial assay conditions were investigated using substrates
adsorbed on porous surfaces such as cellulose-based filter paper,
silica gel, alumina, or reversed-phase-C18 TLC plates. We found
that lipase substrates 1a/b-20a/b (Chart 1), comprising esters
and acyloxymethyl ethers of umbelliferone and 4-methylumbel-
liferone,5 could be uniformly distributed on the surface of silica
gel TLC glass plates by simply soaking the plates in a dichlo-
(2) Beisson, F.; Tiss, A.; Rivie`re, C.; Verger, R. Eur. J. Lipid Sci. Technol. 2000,
133-153. (b) Wahler, D.; Reymond, J.-L. Angew. Chem., Int. Ed. 2002,
41, 1229. (c) Wahler, D.; Boujard, O.; Lefe`vre, F.; Reymond, J.-L. Tetrahedron
2004, 60, 703.
(3) Lagarde, D.; Nguyen, H.-K.; Ravot, G.; Wahler, D.; Reymond, J.-L.; Hills,
G.; Veit, T.; Lefevre, F. Org. Process. R. D. 2002, 6, 441. (b) Nyfeler, E.;
Grognux, J.; Wahler, D.; Reymond, J.-L. Helv. Chim. Acta 2003, 86, 2919.
(c) Gonzalez-Garcia, E. M.; Grognux, J.; Wahler, D.; Reymond, J.-L. Helv.
Chim. Acta 2003, 86, 2458.
(4) Badalassi, F.; Wahler, D.; Klein, G.; Crotti, P.; Reymond, J.-L. Angew. Chem.,
Int. Ed. 2000, 39, 4067. (b) Grognux, J.; Wahler, D.; Nyfeler, E.; Reymond,
J.-L. Tetrahedron Asymmetry 2004, 15, 2981.
* To whom correspondence should be addressed. Fax: + 41 31 631 80 57.
E-mail: reymond@ioc.unibe.ch.
(1) Enzyme Assays: A Practical Approach; Eisenthal, R., Danson, M., Eds.;
Oxford University Press: New York, 2002. (b) Gul, S.; Sreedharan, S. K.;
Brocklehurst, K. Enzyme Assays: Essential Data; John Wiley & Sons:
Chichester, U.K., 1998. (c) Reetz, M. T. Angew. Chem. 2001, 113, 292-
320; Angew. Chem., Int. Ed. 2001, 40, 284-310. (d) Wahler, D.; Reymond,
J.-L. Curr. Opin. Chem. Biol. 2001, 5, 152-158. (e) Wahler, D.; Reymond,
J.-L. Curr. Opin. Biotechnol. 2001, 12, 535-544. (f). Goddard, J.-P.;
Reymond, J.-L. Trends Biotechnol. 2004, 22, 363-370.
(5) Leroy, E.; Bensel, N.; Reymond, J.-L. Bioorg. Med. Chem. Lett. 2003, 13,
2105. (b) Leroy, E.; Bensel, N.; Reymond, J.-L. Adv. Synth. Catal. 2003,
345, 859.
10.1021/ac048611n CCC: $30.25 © 2005 American Chemical Society
Published on Web 12/17/2004
Analytical Chemistry, Vol. 77, No. 2, January 15, 2005 373

Chart 1. Fluorogenic Ester Substrates Used for
Solid-Supported Enzyme Assay
Figure 1. Principle of enzyme assay on silica gel plates.
romethane solution of the substrate and drying. The substrate-
loaded plates were stable for at least two weeks when stored in a
dry package at -20 °C. Addition of a small drop (1 µL) of enzyme
solution (lipase or esterase, 0.1 mg mL-1) readily induced a
reaction, which was detected after a few minutes by the appear-
ance of a bright blue fluorescence indicating the formation of
umbelliferone. By contrast, addition of buffer only or a solution
of a noncatalytic protein such as bovine serum albumin (BSA, 1
mg mL-1) did not produce a detectable reaction. Addition of
glycerol (30% v/v) retarded solvent evaporation, so that even
weakly reactive enzymes could be detected by extending incuba-
tion times up to 24 h. Under these conditions, there was minimal
(<10%) hydrolysis with either BSA or buffer alone. Enzyme
activities in the silica gel plate format were comparable to those
observed with the same substrates in a classical MTP setup,6
indicating that the enzymes were not deactivated by the silica gel.
Enzyme solutions were delivered to the substrate-loaded silica
gel plates using a liquid-handling HPLC-autosampler robot (Fig-
ures 1 and 4). Spotting of 1-µL aliquots formed regular spots of
∼3-mm diameter, allowing 144 assays for a 5 × 5 cm plate (Figure
2). Product formation was either judged qualitatively by visual
inspection in a TLC-UV chamber or quantitated using a fluores-
cence MTP reader adapted with a custom-made plate holder and
plate-reading routine. When the same enzyme solution was
dispensed repetitively onto the entire surface of a substrate-loaded
silica gel plate, there was ∼(10% fluctuation in the fluorescence
reading obtained in each assay across the entire plate. Variations
probably reflected slightly uneven substrate distribution and plate
surface inhomogeneity.7
Figure 2. Photo of an enzyme assay on silica gel plate. The plate
was soaked with substrate 19b (2 mM in CH2Cl2) and dried. The
following enzymes (1 µL/spot) were spotted (100/10/1 µg mL-1 in
rows A/B/C, D/E/F, G/H/I, J/K/L) Columns: 1, RNL; 2, PPL; 3, E1; 4,
E2; 5, PCL2; 6, MML; 7, L7; 8, L8; 9, L9; 10, L10. BSA (1 mg mL-1
)
was spotted in columns 11 and 12. Rows: ABC, PBS (10 mM
phosphate, 160 mM NaCl, pH 7.4); DEF, PBS + 30% v/v MeOH;
GHI, PBS + 10% v/v glycerol; JKL, PBS + 30% v/v glycerol. The
picture was taken after 120 min at 25 °C.
Glycosidase Assay (Water-Soluble Substrates). Although
initially designed for water-insoluble substrates, the assay also
proved suitable for water-soluble substrates, as shown for the case
of glycosidases. Silica gel plates impregnated with four different
4-methylumbelliferyl glycosides (R-glu, â-glu, R-gal, â-gal) were
prepared by soaking the plates in solutions of the substrates in
tetrahydrofuran. These plates were remarkably stable and could
be kept for several weeks at room temperature without noticeable
degradation of substrate. The glycosidase activity assays were
carried out by spotting the corresponding glycosidase enzymes
in aqueous HEPES buffer. Product formation was visible as a
bright fluorescent blue ring under UV after 5 min. The reaction
or each of the different glycoside substrates only took place with
the corresponding glycosidase, and there was no reaction with
(7) Fluorescence reading of an empty silica gel plate showed a background
fluorescence signal or up to 5% of the signal recorded when reading the
assays.
(6) % v/v DMSO was added as cosolvent in the reference solution assay, which
also showed a high level of nonspecific background hydrolysis.
374 Analytical Chemistry, Vol. 77, No. 2, January 15, 2005

Figure 3. â-Galactosidase activity and inhibition on silica gel plate.
The plate was soaked with a 1 mM solution of 4-methyl-umbelliferyl
â-D-galactoside in THF and dried. A solution of â-galactosidase from
Aspergillus oryzae (Sigma G5160, 255 000 units/mg) in 0.1 M HEPES
buffer pH 6.8 was spotted (1 µL/spot). The picture was taken after
10 min at 25 °C. Lane A 100 µg/mL enzyme; lane B, 10 µg/mL
enzyme. Column 1, no inhibitor, Inhibitor concentrations (µM): column
2, 100; 3, 10; 4, 1; 5, 0.1; 6, 0.01; 7, 0.001; 8A, 1 µg/mL enzyme, no
inhibitor; 8B, buffer control.
Figure 5. Reproducibility of activity patterns on substrate-impreg-
nated silica gel plates. The net fluorescence is reported as gray scale
(white 0, black maximum) after substraction of background (sample
B1 with BSA, ∼10% of maximum signal). Plate A: 1-µL drops (PBS,
30% v/v glycerol) were spotted. After 24 h at 25 °C, fluorescence
intensity was recorded using a fluorescence MTP reader (λex ) 360
nm, λem ) 460 nm). Plate B: the same experiment was repeated 2
weeks later. The following samples were spotted: A1, fluorescein
reference; B1, BSA 1 mg mL-1 ; A2-A12, C1-C12, E1-E12, G1-
G3, different lipases and esterases, 100 µg mL-1 ; B2-B12, D1-
D12, F1-F12, H1-H3, the same enzyme series at 10 µg‚mL-1 ; G5-
G12, H5-H12, recombinant lipases.
Protease Assay (Whole-Protein Substrate). The assay was
further extended to a protease assay to demonstrate the use of
whole-protein substrates. We used a fluorescence assay recently
developed in our group based on the indirect detection of free
amino acids using the copper-calcein complex.9 This assay can
be used to assay proteases using BSA as substrate. BSA-
impregnated silica gel plates were obtained by soaking the plates
for a few seconds in a 1 mg mL-1 solution of BSA in PBS buffer,
followed by drying under vaccuum. The assay was carried out by
spotting solutions of three different proteases (trypsin, elastase,
and chymotrypsin at 100 µg mL-1) in aqueous buffer with 30%
v/v glycerol containing the fluorescence quenched copper-calcein
complex, followed by overnight incubation. Fluorescence increase
indicative of proteolysis were not visible by the eye under UV
but could be detected using the MTP reader at the appropriate
wavelength (λex ) 470 nm, λem ) 509 nm). Proteases reproducibly
gave a 100 ( 30% fluorescence increase compared to the
fluorescence signal without protease or with a nonproteolytic
enzyme. This signal modulation was weaker than for the same
assay in solution (>800% increase) but would be sufficient in the
context of simple activity detection assays.
Figure 4. Autosampler with custom-made MTP/silica gel plate
holder. An HPLC needle is mounted on the XY arm at the left and is
connected to the washing pump (center). The needle takes 30 µL
from one of the MTP wells, distributes 1 µL onto each of the 22 silica
gel plates, and goes to a washing cycle before resuming distribution
with the next MTP well.
mismatched glycosidase or with BSA. The concentration of
enzyme necessary to induce the reaction was the same as for the
assay in MTPs.
Formation of fluorescent rings showed that the water-soluble
substrates underwent latteral diffusion upon spotting. The ring-
shaped fluorescence was suitable for qualitative reading but could
not be quantified automatically with the MTP reader. Homo-
geneous spots without lateral diffusion could be obtained by
spotting the enzyme solution manually in much smaller volumes
(∼100 nL) using a glass capillary. Unfortunately, this procedure
could not be automated with the available robot. The possibility
of tracking enzyme inhibition was tested using an aminocyclo-
pentitol-type inhibitor for â-galactosidases.8 A buffered solution
of â-galactosidase was treated with the inhibitor at various
concentrations and spotted on a TLC plate impregnated with
High-Throughput Measurements. High throughput and
reproducibility of the silica gel plate assay were tested by activity
profiling of fluorogenic ester substrates 1a/b-20a/b against a
series of lipases and esterases. A custom-made plate holder
allowed robotic pipetting from a 96-well plate into 22 different 5
4-methylumbelliferyl â-D-galactoside substrate. Indeed. there was
no measurable activity in the presence of the inhibitor, while the
control showed full activity (Figure 3).
(8) Leroy, E.; Reymond, J.-L. Org. Lett. 1999, 1, 775-777. (b) Gartenmann
Dickson, L.; Leroy, E.; Reymond, J.-L. Org. Biomol. Chem. 2004, 2, 1217-
1226.
(9) Klein, G.; Reymond, J.-L. Angew. Chem., Int. Ed. 2001, 40, 1771. (b) Dean,
K. E. S.; Klein, G.; Renaudet, O.; Reymond, J.-L. Bioorg. Med. Chem. Lett.
2003, 10, 1653.
Analytical Chemistry, Vol. 77, No. 2, January 15, 2005 375

Figure 6. Color-coded activity patterns of fluorogenic esters substrates against lipases and esterases using the silica gel plate assay. Enzyme
solutions (0.1 mg mL-1 in PBS + 30% v/v glycerol) were spotted (1 µL) on silica gel plates preimpregnated with each substrate (soaking in 2
mM substrate solution in CH2Cl2 and drying). The fluorescence signal (λex ) 360, λem ) 460 nm) was recorded after 24 h at 25 °C. (a) For
abbreviation for enzyme use, see Supporting Information. (b) Percentage of maximum observable fluorescence signal observed with the most
reactive substrate for each enzyme. The color-coding is calculated relative to this maximum for each column (color code at right). Substrates
are ordered by similarity analysis of their reactivity pattern against the different enzymes.
× 5 cm silica gel substrate-loaded silica gel plates (Figure 5). The
96-well plate was filled (200 µL/well) with 96 test solutions with
various enzymes and controls (Figure 6). The entire 96-well plate
was transferred onto 37 different substrate-loaded silica gel plates
in duplicate, amounting to 7104 spotting operations of 1 µL each
and 3404 different enzyme reactions. Dispensing of the 96-well
plate to 22 silica gel plates was completed in ∼3 h, so that the
entire double measurement was completed in ∼12 h of robot
operating time, consuming less than 0.1 mL of each enzyme test
solution and less than 0.2 mg of substrate per 5 × 5 cm silica gel
plate. Product formation in each spot was quantitated by measur-
ing fluorescence intensity using the fluorescence MTP reader (see
above).
Reproducibility was judged by comparing the activity patterns
produced by the 96 test samples onto two silica gel plates of the
same substrate (Figure 5). Similarity analysis of activity patterns
by data clustering showed that two activity measurements with
the same substrate were always more similar to one another than
to a different substrate. Visual inspection confirmed that the
reactivity pattern of each substrate across the 96 test samples was
indeed very well conserved between the two measurements.
Nevertheless, due to the significant activity fluctuations observed
in individual cases, duplicate measurements might be preferable
for high-throughput screening with this method.
Optimal Substrate Set for Lipases and Esterases. Cluster-
ing of enzyme activity patterns showed that several substrates
behaved identically across all enzymes tested. A subset of 15
functionally different acyl groups was selected, and the enzyme
reactivity data for the corresponding umbelliferyl ester and
4-methyl umbelliferyl ester was measured for 35 lipases and
esterases and computed as enzyme activity fingerprints (Figure
6).10 The fingerprint data showed that six substrates were sufficient
to cover most of the observed reactivity, namely, the C6, C12,
and C18,ester of umbelliferone (5a, 10a, 13a) and the cyclohex-
anecarboxylic, C8, and C16,ester of 4-methylumbelliferone (6b,
7b, 12b). In addition the isobutyryl ester of umbelliferone 2a
and cyclobutylcarboxylic ester of 4-methylumbelliferone 4b showed
singular reactivites with esterases. This subset of eight different
substrates (2a, 4b, 5a, 6b, 7b, 10a, 12b, 13a) might be
sufficient for a broad-based detection of interfacial lipase and
esterase activities using the silica gel plate assay.
CONCLUSION
The experiments above demonstrate a high-throughput en-
zyme assay based on fluorogenic substrates adsorbed on silica
gel plates. The reactivity of the enzymes at the silica gel surface
appears to be similar to that in solution. Evaporation of water from
the silica gel plate limits the reaction time to ∼15 min without
cosolvent and several hours in the presence of glycerol. The assay
is compatible with water-insoluble, water-soluble, whole-protein
substrates, and enzyme inhibition measurements. Water-insoluble
lipase substrates were found to react at the surface of silica gel
plates without addition of any cosolvent. Under these conditions,
the background hydrolysis of these rather labile umbelliferyl
esters was negligible. It should be noted that nitrophenyl esters
were not stable at the silica gel surface and also did not yield a
sufficiently strong color for product detection.
The silica gel plate assay presented here offers a practical
alternative to microtiter plate-based high-throughput screening and
allows miniaturization by ∼100-fold (100 to 1 µL assay) at
essentially no cost. The substrate conditioning method is broadly
applicable with fluorogenic substrates and sensors. This simple
method might be preferable to more sophisticated assays in
microarray format, which require complex substrate attachement
or dispensing procedures,11 and are based on microarrayers and
microarray scanners that are still relatively expensive instru-
ments.
(10) Wahler, D.; Badalassi, F.; Crotti, P.; Reymond, J.-L. Angew. Chem., Int. Ed.
2001, 40, 4457. (b) Wahler, D.; Badalassi, F.; Crotti, P.; Reymond, J.-L.
Chem. Eur. J. 2002, 8, 3211. (c) Reymond, J.-L.; Wahler, D. ChemBioChem
2002, 3, 701-708. (d) Grognux, J.; Reymond, J.-L. ChemBioChem 2004,
5, 826-831. (e) Goddard, J.-P.; Reymond, J.-L. J. Am. Chem. Soc. 2004,
126, 11116-11117.
376 Analytical Chemistry, Vol. 77, No. 2, January 15, 2005

EXPERIMENTAL SECTION
recorded after 24 h at 25 °C using a fluorescence MTP reader
(λex ) 470 nm, λem ) 509 nm). All plates prepared for assay were
used immediately or were stored in a drybox in a refrigerator at
-20 °C for later use. The maximum storage time tested was 2
weeks, after which there was no noticeable degradation.
Autosampler. Automatic sample delivery was carried out with
a Gilson 233XL on-line column switching and Gilson 402 syringe
pump with Gilson 735 sampler software v. 5.20.
Preparation of Substrate-Impregnated Silica Gel Plates
and Dispensing of Enzyme Samples. Lipases and Esterase
Assay. Silica gel TLC glass plates G-25 (Macherey-Nagel (silica
gel 40-63 µm, surface 550 m2/g, layer 0.25 mm) were soaked in
a solution of substrate (1a/b-20a/b, 2 mM in CH2Cl2) for 2 min
and dried. The 1-µL aliquots of enzyme samples (in PBS: 10 mM
phosphate, 160 mM NaCl, pH 7.4) with 30% v/v glycerol) were
spotted at 25 °C. Product formation was recorded after 2-24 h.
Glycosidase Assay. Silica gel TLC glass plates were soaked
in a solution of 4-methylumbelliferyl glycoside (2 mM in THF)
for ∼2 min and dried. Enzyme samples (∼100 nL) in 0.1 M HEPES
buffer pH 6.8 were spotted manually using a thin glass capillary.
Product formation was measured by digital photos under UV after
15 min.
ACKNOWLEDGMENT
This work was supported financially by the University of Berne,
the Swiss National Science Foundation, the Office Fe´de´ral Suisse
de l’Education et de la Science, the COST-program D16, and
Prote´us SA, N`ımes, France.
Protease Assay. Silica gel TLC glass plates were soaked in a
solution of BSA (0.1 mg mL-1 in PBS) for 10 s and dried under
vaccuum. The proteases (trypsin, chymotrypsin, elastase, ther-
molysin, each 100 µg mL-1) were conditioned in PBS + 30% v/v
glycerol containing 10 µM CuCl2, 1 µM calcein (F 21039) and
spotted as a 1-µL drop using the autosampler. Fluorescence was
SUPPORTING INFORMATION AVAILABLE
Synthesis of 1-20a/b; List of commercially available lipases
and esterases; spectral data, 1H NMR and 13C NMR, for
1-20a/b. This material is available free of charge via the Internet
at http://pubs.acs.org.
(11) Salisbury, C. M.; Maly, D. J.; Ellman, J. A. J. Am. Chem. Soc. 2002, 50,
14868-14870. (b) Zhu, Q.; Uttamchandani, M.; Li, D.; Lesaicherre, M. L.;
Yao, S. Q. Org. Lett. 2003, 8, 1257-1260. (c) Hauseman, B. T.; Huh, J. H.;
Kron, S. J.; Mrksich, M. Nat. Biotechnol. 2002, 20, 270-274. (d) Gosalia,
D. N.; Diamond, S. L. Proc. Natl. Acad. Sci. U.S.A. 2003, 15, 8721-8726.
Received for review September 20, 2004. Accepted
December 1, 2004.
AC048611N
Analytical Chemistry, Vol. 77, No. 2, January 15, 2005 377