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Article history: Abnormal protease activities are associated with cancers, vascular diseases and Alzheimer diseases.
Received 6 October 2017 Therefore, detection of protease activities has become increasingly important in recent years. Herein,
Received in revised form 2 March 2018 we report a semi-quantitative, liquid crystal (LC)-based protease assay for naked-eye detection of pro-
Accepted 22 April 2018
tease activity. In this assay, casein molecules are cleaved by proteases into small peptide fragments, which
Available online 24 April 2018
can be quantified either by using Lowry’s method or using LC. In the latter, peptide fragments adsorb on a
solid surface and disrupt LC to produce a bright spot for naked-eye detection. The bright spot is observed
Keywords:
only when the surface-adsorbed peptide density exceeds a critical value. In the assay, a major challenge
Continuous assay
Protease
is how to separate undigested casein and remaining protease from peptide fragments to prevent their
Liquid crystal interferences with LC. To overcome this issue, trichloroacetic acid (TCA) is added to precipitate casein
Trichloro acetic acid and proteases. By using the assay, we are able to detect 10 ng/mL of protease (activity 7.23 U/mg pro-
Syringe pump tease, R2 = 0.991) or 6.5 g/mL of peptide fragments. Finally, a continuous protease assay is developed to
minimize manual sampling and reduce errors in kinetic studies.
© 2018 Elsevier B.V. All rights reserved.
1. Introduction Recently, many simple and portable protease assays have been
developed [14–20]. These assays can be classified as specific and
Proteases are enzymes which are able to catalyze the hydroly- non-specific assays depending on the substrates used. For specific
sis of peptide bonds. They play important roles in food digestion, protease assays, custom-synthesized peptides with a well-defined
healing of wounds and blood coagulation [1–3]. Proteases attract sequence are used as a substrate for a particular protease. On
much attention in recent years because they are associated with the other hands, non-specific protease assays use low-cost pro-
cancers [4,5], vascular diseases [6] and Alzheimer diseases [7]. teins such as casein as a substrate, which can be digested by
Therefore, development of facile yet sensitive protease assays for proteases into peptide fragments [21]. After precipitation of undi-
the detection of protease activities is highly important. Tradi- gested casein and remaining proteases, the peptide fragments can
tionally, protease activities can be determined after incubation be quantified by using Lowry’s method with Folin–Ciocalteu (FC)
of proteases with their substrates, such as proteins or peptides. reagent, which reacts with tyrosine residue in the peptide frag-
After the incubation period, peptide fragments released from ments and gives a blue color. Although this method is simple and
the substrates (due to protease activity) are detected by using quantitative, the limit of detection (LOD) is only 0.1 U/mL [22].
analytical instrumentation such as high performance liquid chro- To address this issue, many researchers used labeled caseins as
matography coupled with mass spectroscopy (HPLC–MS), liquid substrates to lower LODs. For example, Manicourt and Lefebvre
nitrogen assisted spray ionization mass spectrometry (LNASI-MS), used [3 H] and [125 I]-labeled casein to detect matrix metallopro-
matrix-assisted laser desorption/ionization time-of-flight mass teinases (MMPs) and plasmin with LODs of 2.5 ng/mL and 5 g/mL,
spectrometry (MALDI-TOF-MS) and surface assisted laser desorp- respectively [23,24]. However, labeling of casein with radioisotopes
tion/ionization time-of-flight mass spectrometry (SALDI-TOF-MS) is tedious, time-consuming, expensive and requires special han-
[8–11]. Based on peptide mass fingerprints and peak heights, one dling of radioactive materials. Alternatively, casein can be labeled
can determine protease cleavage sites and protease activities. This with fluorescent dyes for fluorescence detection. For example,
method is accurate, label-free and highly specific, but it relies on Schade et al. and Mancini et al. employed 4,4-difluoro-4-bora-
expensive instrumentation and is time-consuming [12,13]. 3a,4a-diaza-s-indacene (BODIPY) and fluorescein-labeled caseins
to detect protease activities. They were able to achieve LODs of
50 ng/mL and 170 nM, respectively [25–27]. Although fluorescence
technique is highly sensitive, fluorescein becomes non-fluorescent
∗ Corresponding author.
in acidic solutions. Alternatively, some researchers derivatized
E-mail address: cheyk@nus.edu.sg (K.-L. Yang).
https://doi.org/10.1016/j.snb.2018.04.125
0925-4005/© 2018 Elsevier B.V. All rights reserved.
M. Jannat, K.-L. Yang / Sensors and Actuators B 269 (2018) 8–14 9
Fig. 3. Precipitation of casein (6.5 mg/mL) by using a TCA solution (110 mM) at dif-
ferent volume ratios. Numbers indicate TCA to casein ratios. (A) Visual appearance
of mixtures containing both TCA and casein after the mixing. (B–D) LC was used
to detect remaining casein in the supernatant after the precipitation of casein by
using TCA. The initial casein concentration was (B) 6.5 mg/mL, (C) 0.65 mg/mL and Fig. 4. Protease assays for determining protease activities. Released peptide frag-
(D) 65 g/mL, respectively. ments were quantified (as tyrosine equivalent) either by using (A) Lowry’s method
or (B) LC method. Number above each spot indicates the protease concentration
(g/mL).
ratio reached at 4:1, the supernatant was completely clear. At this
point, probably all casein molecules have precipitated. However,
it is difficult to determine the exact amount of TCA needed to assay, the amount of TCA required to precipitate protease is much
precipitate all casein molecules from the solution based on this less and did not need to be taken into account. In the following
experiment. To find the exact amount of TCA required, we repeated experiments, TCA to casein ratio was fixed at 1.5:1 to make sure all
the same experiment and used LC to detect residual casein in the undigested casein and remaining protease have been precipitated
supernatant. LC was used because we have shown that 20 g/mL from the reaction mixture.
of casein in the solution leads to a bright LC spot (Fig. 2A). Thus,
if LC gives a dark spot, then it can be concluded that the casein 3.3. Measurement of protease activity
concentration is less than 20 g/mL. As shown in Fig. 3B, when
the TCA to casein ratio was 0.25:1, the corresponding LC spot was After the precipitation of casein and protease, the remaining
bright. The LC result suggests that the remaining casein concentra- peptide fragments in the reaction mixture can be quantified to
tion in the supernatant was higher than 20 g/mL. Thus, it can be obtain protease activity. Firstly, we used Lowry’s method to deter-
concluded that the amount of TCA was not enough to precipitate mine the amount of peptide fragments (as tyrosine equivalent)
casein. Next, when the TCA to casein ratio was 1:1, a bright LC spot after incubation of casein with different concentrations of protease
was still observed. This result suggests that the amount of TCA was for 10 min [22]. Fig. 4A shows the amount of peptide fragments
still insufficient to precipitate all casein molecules. Finally, when in the supernatant as a function of the protease concentration. It
the TCA to casein ratio was 1.5:1, a dark LC spot appeared. At this is apparent from the figure that the amount of peptide fragments
mixing ratio, it seemed that all casein molecules (6.5 mg/mL) were increased linearly with increasing protease concentration. From
precipitated by TCA. Subsequently, similar TCA precipitation exper- the slope of the regression line, protease activity was found to be
iments were conducted by using two lower casein concentrations 7.23 U/mg protease (R2 = 0.991). This activity is comparable to man-
at 0.65 mg/mL and 65 g/mL, respectively. Fig. 3C shows that when ufacturer’s data (≥5 U/mg protease). From Fig. 4A, it can also be
the casein concentration was 0.65 mg/mL, a TCA to casein ratio of seen that 5 g/mL (or 0.04 U/mL) of protease can be detected by
0.67:1 was sufficient to precipitate all casein molecules. Similarly, using Lowry’s method. At this protease concentration, 3.26 mg/mL
Fig. 3D shows that when the casein concentration was 65 g/mL, a of peptide fragments were released after 10 min of protease reac-
TCA to casein ratio at 0.43:1 was sufficient to precipitate all casein tion. In addition, it is also found that this activity is not affected by
molecules. These results, when combined, suggest that the TCA to the pH of protease solution within 3–11.5 range (Fig. S3)
casein ratio for complete precipitation of 65 mg/mL, 0.65 mg/mL In addition to Lowry’s method, peptide fragments were also
and 65 g/mL of casein were 1.5:1, 0.67:1 and 0.43:1, respectively. detected by using the LC method. In this case, reaction mixtures
A similar experiment was also repeated for protease to determine from the protease assays were first mixed with TCA and then
how much TCA is required to precipitate protease (Fig. S2). Since the centrifuged to remove casein and protease. Subsequently, clear
casein concentration was much higher than that of protease in the supernatant was spotted on glass slides and LC cells were fabri-
12 M. Jannat, K.-L. Yang / Sensors and Actuators B 269 (2018) 8–14
Fig. 5. Protease kinetic study using a batch process. Protease concentrations were
100 g/mL (䊏), 30 g/mL (䊉), 5 g/mL () and 1 g/mL (), respectively. Original
data of this kinetic study is provided at supplementary document (Table S1).
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An electrophoretic method for the detection of chymotrypsin and trypsin structured intermediate, Protein Sci. 18 (2009) 980–993.
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722–731. Biographies
[34] C.-H. Jang, M.L. Tingey, N.L. Korpi, G.J. Wiepz, J.H. Schiller, P.J. Bertics, et al.,
Using liquid crystals to report membrane proteins captured by affinity
microcontact printing from cell lysates and membrane extracts, J. Am. Chem. Mahbuba Jannat is now pursuing her phD degree in the department of Chemical
Soc. 127 (2005) 8912–8913. and Biomolecular Engineering at the National University of Singapore (NUS). Before
[35] D. Hartono, S.L. Lai, K.-L. Yang, L.-Y.L. Yung, A liquid crystal-based sensor for joining NUS, she received her B.S degree in Chemical Engineering from Bangladesh
real-time and label-free identification of phospholipase-like toxins and their University of Engineering and Technology (BUET) in 2013. Her current research
inhibitors, Biosens. Bioelectron. 24 (2009) 2289–2293. interest is liquid crystal based biosensor development.
[36] Q. Zhu, K.-L. Yang, Microfluidic immunoassay with plug-in liquid crystal for
Dr. Yang Kun-Lin is an associate professor in the Department of Chemical and
optical detection of antibody, Anal. Chim. Acta 853 (2015) 696–701.
Biomolecular Engineering at the National University of Singapore (NUS). Before
[37] C.-H. Chen, K.-L. Yang, Functional protease assay using liquid crystals as a
joining NUS, he was a post-doctoral researcher in the Chemical and Biomolecu-
signal reporter, Biosens. Bioelectron. 35 (2012) 174–179.
lar Department at the University of Wisconsin −Madison working on liquid crystal
[38] L.H. Ong, X. Ding, K.-L. Yang, Mechanistic study for immobilization of
sensors. He received his BS and MS degrees in Chemical Engineering from National
cysteine-labeled oligopeptides on UV-activated surfaces, Colloids Surf. B 122
Taiwan University and PhD degree from Georgia Tech. He is the recipient of the
(2014) 166–174.
Defense Innovation Research Program Award and the A*Star Research Grant Award
[39] C.-Y. Xue, K.-L. Yang, Dark-to-bright optical responses of liquid crystals
for his work on liquid crystal sensors. His present research interests include biosen-
supported on solid surfaces decorated with proteins, Langmuir 24 (2008)
sors, liquid crystals and microfluidics.
563–567.