The document describes the development of an improved fluorescent protease assay. The original PDQ protease assay had limitations including a short shelf-life and detection limits between 100 ng to 10 μg. The new fluorescent assay uses a fluorescently labeled protease substrate immobilized on metal-enhanced nanoparticles. This allows for a single-step assay with lower detection limits over 100-fold lower than the original assay. The fluorescent assay is also adaptable to high-throughput systems and has a longer minimum shelf-life of one year, making it more cost-effective to manufacture.

1. PDQ™ Protease Assay
Dave Samaroo, Rosalind Ramsey, and Sheldon E. Broedel, Jr.
Athena Environmental Sciences, Inc. 1450 South Rolling Road, Baltimore, MD 21227
Introduction
Importance:
Proteases are well known commercial enzymes that are exploited in the
detergent, food, pharmaceutical, diagnostics, and fine chemical industries.
Their use as detergent additives is the largest application of industrial enzymes
in terms of volume and value. Interest in proteases has increased with the
realization that they play a critical role in a variety of diseases. Modifications in
proteolytic systems may trigger multiple pathological conditions such as
cancer, neurodegenerative disorders, and cardiovascular diseases. In industrial
settings, the formation of biofilms is an economic problem and proteases are
often used to remove and clean these biofilms from surfaces. Further,
researchers and manufacturers of high valued proteins are concerned with
undesirable degradation by contaminating proteases. All of these applications
require the use of a quantitative measure of protease activity.
Background of PDQ™ Protease Assay:
The PDQ™ Protease Assay is a colorimetric assay that was designed to
simplify the measurement of protease activity. The assay format eliminates the
separation steps often used in historic protease assays thereby allowing for a
single step assay. The assay is a vial-based system consisting of cross-linked
sol-gel composed of albumin, azoalbumin, and gelatin. This substrate matrix is
susceptible to a wide range of enzymes commonly used in industry and
research. However, the manufacture of the assay is labor intensive and the
product suffers from a short shelf-life. Further, the detection limit is between
100 ng and 10 µg depending on the enzyme.
Objective of new format:
To develop an assay that has a lower detection limit, is adaptable to high
throughput systems and is more cost effective to manufacture, we evaluated the
use of fluorescence tagged protease substrate. The new format consists of a 96-
well plate using metal-enhanced nano-particles to increase the fluorescent
signal. The fluorescently labeled substrate is immobilized on the surface of the
metal nano-particles and protease activity is quantified by measuring the
amount of residual fluorescence remaining in the well or the amount of
fluorescence released into the buffer. This allows for a one step procedure with
reaction conditions that are matrix-independent including the use of opaque
samples.
References:
Kirk, O., Borchert, T., & Fuglsang, C. (2002). Industrial enzyme applications. Current Opinion
In Biotechnology, 13(4), 345-351. doi:10.1016/s0958-1669(02)00328-2.
Lopez-Otin, C., & Bond, J. (2008). Proteases: Multifunctional Enzymes in Life and
Disease.Journal Of Biological Chemistry, 283(45), 30433-30437. doi:10.1074/jbc.r800035200.
Selan, L., Berlutti, F., Passariello, C., Comodi-Ballanti, M., & Thaller, M. (1993). Proteolytic
enzymes: a new treatment strategy for prosthetic infections?. Antimicrobial Agents And
Chemotherapy, 37(12), 2618-2621. doi:10.1128/aac.37.12.2618
Royer, G. P. and Broedel, Jr., S. E. (1996). One Step Protease Assay Technical Brief. Retrieved 8
January 2015, from http://www.athenaes.com/tech_brief_protease.php (accessed 8 Jan 2015).
Aslan, K., Gryczynski, I., Malicka, J., Matveeva, E., Lakowicz, J., & Geddes, C. (2005). Metal-
enhanced fluorescence: an emerging tool in biotechnology. Current Opinion In
Biotechnology, 16(1), 55-62. doi:10.1016/j.copbio.2005.01.001.
Methods: QuantaWell™ strip wells were coated as in Fig. 3 and stored at 50ºC. At
the times indicated, a duplicate set of strips were reacted with 0.1 ml trypsin and the
amount of residual fluorescence measured as in Fig. 3.
Methods: QuantaWells™ were coated as in Fig. 3 and duplicate wells incubated at
37ºC with 0.1 ml of seven different proteases at concentrations ranging from 100
µg/ml to 10 pg/ml. After 1 hour, the amount of residual fluorescence was determined.
Summary and Conclusions:
The Fluorescent PDQ™ Protease Assay using metal nano-particle coated
QuantaWell™ microtiter plates exhibits:
• Lower detection limits by two or more orders of magnitude compared to
the original assay.
• Ability to measure a broad spectrum of proteases.
• Lower manufacturing costs.
• Increased product shelf life.
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
0.0 200.0 400.0 600.0 800.0 1000.0
RFU
Coating Concentration (µg/ml)
Released Substrate using 40 BAEE/ml Trypsin
Abstract
The colorimetric PDQ™ Protease Assay was first introduced in 1996. It
has since become a routinely used assay for measuring protease activity in
process samples or for certifying reagents as protease-free. However, while
the sol-gel substrate matrix allows for a one-step assay with nanogram
detection limits, the substrate matrix only has a three month shelf-life.
Alternative matrix formats, including the use of fluorescent substrates, have
not yielded a more robust assay. Here we describe development of a protease
assay using a fluorescent protease substrate in combination with metal nano-
particle coated microtiter wells. The metal-enhanced fluorescent signal
permitted the development of an assay with a detection limit more than 100-
fold lower than the original assay with detection limits below 10 pg for
certain proteases. An accelerate stability study suggested that the shelf-life
of the substrate is at least one year at 4ºC. Further, the assay is adaptable to
high-throughput formats.
Figure 1. Metal nano-particle coated plates increase the fluorescence
signal yielding a more sensitive assay.
Method: The wells of a Maxisorb™ (Nunc) and QuantaWell™ (Athena) plate were
coated with 100 µg of fluorescently labeled BSA. Duplicate wells were incubated with
0.1 ml of trypsin at different concentrations ranging from 50 to 0.05 BAEE Units/ml
and the residual level of fluorescence measured after 1 hour incubation at 37ºC.
Figure 2. The signal is proportional to the amount of substrate coated on
the well.
Methods: A QuantaWell™ plate was coated with 50 µg fluorescently labeled BSA.
Duplicate wells were incubated with 0.1 ml of 100 to 0.001 BAEE units/ml trypsin and
the residual fluorescence measured after 1 hour at 37ºC. The resulting calibrator
curve (right panel) was compared to the calibrator curve generated by the colorimetric
assay (left panel).
Figure 3. Enhanced fluorescence assay exhibited a 100-fold lower
detection limit compared to the standard colorimetric assay.
Method: A QuantaWell™ plate was coated with different concentrations of
fluorescently labeled BSA. Duplicate wells were incubated with 0.1 ml of 40 BAEE
units/ml trypsin for 1 hour at 37ºC and the amount of residual (not shown) and
released fluorescence measured. The selected coating concentration for further
development was 500 µg/ml (50 µg per well).
Released Residual
Figure 4. Measuring the residual fluorescence permitted detection of
lower amounts of protease activity.
Method: QuantaWells™ (Athena) were coated as in Fig. 3. Duplicate wells were
incubated with 0.1ml of trypsin at concentrations ranging from 102 to 10-3 BAEE
Units/ml and the amount of fluorescence released (left panel) and residual (right
panel) measured after 1 hour incubation at 37ºC.
LOD ~ 0.01 Unit/mlLOD ~ 1 Unit/ml
Trypsin Conc. (Units/ml)
Figure 6. The activity of a range of different type of proteases can be
measured using the fluorescent PDQ™ Protease Assay.
Figure 5. The substrate matrix coated on QuantaWells™ was stable at
elevated temperature for 48 days. This indicates a product shelf-life of 1
year at 4ºC.