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Protease functional assay on membrane
Sensors & Actuators: B. Chemical 305 (2020) 127442
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Protease functional assay on membrane Garima Goyal a b c
a,b,c
, Alagappan Palaniappan
b,c,
T
*, Bo Liedberg
a,b,c,
*
Interdisciplinary Graduate School, Nanyang Technological University, 50 Nanyang Avenue, 639798, Singapore School of Materials Science and Engineering, Nanyang Technological University, 50 Nanyang Avenue, 639798, Singapore Center for Biomimetic Sensor Science, Nanyang Technological University, 50 Nanyang Drive, 637553, Singapore
A R T I C LE I N FO
A B S T R A C T
Keywords: Proteases Functional assay Paper based assay Matrilysin Colorimetric sensors
Targeting enzymes, proteases in particular, using various assay formats has attracted considerable interest for early disease diagnosis. While affinity-based assays that report the presence of proteases in samples have been widely explored, it is of utmost importance to develop functional assays that provide information on the activity of proteases for a more precise indication of the disease state and progression. Herein, we report a paper-based functional assay that yields naked eye observable and concentration dependent responses for the active form of proteases. The paper-based assay involves a facile single step fabrication process of depositing protease specific peptide functionalized gold nanoparticles on paper membranes. Matrilysin is used as a model protease to validate the proposed methodology. The proteolysis-driven aggregation of nanoparticles on the membrane yields a colorimetric response that has limit of detection that is ∼4 times lower (3.1 μg/mL) than for the same assay performed in homogeneous solution (12.5 μg/mL). To the best of our knowledge, this is the first report on heterogeneous protease assay on paper relying on aggregation of peptide-functionalized nanoparticles. The approach easily can be extended to assay other enzymes by functionalizing the gold nanoparticles using specific peptides.
1. Introduction
either the substrate or protease is immobilized on a surface/electrode of an assay platform [12]. Typical homogeneous functional assays are based on methodologies such as mass spectrometry, Förster Resonance Energy Transfer (FRET) etc. Homogeneous functional assays generally are very sensitive, but often requires sophisticated instrumentation or experience susceptibility to photo-bleaching and pH sensitivity [13]. On the other hand, heterogeneous assays could provide opportunities for the development of cost effective and easy-to-use assays, owing to their robustness and minimized liquid handling complexity. Till date, heterogeneous protease assays have been demonstrated by immobilizing substrates on glass/gold surface for SPR based detection or Raman spectroscopy or on conductive electrodes for electrochemical detection. However, most of these approaches are labor intensive, time consuming and require trained personnel [14–16]. Herein, we report a paper-based functional assay that yield naked eye observable and concentration dependent responses of proteases in buffer and synthetic urine. The proposed paper-based assay involves a facile single step fabrication process of depositing protease specific peptide functionalized gold nanoparticles (AuNPs) on paper membranes. AuNPs are chosen as reporters because of their high surface area and extinction coefficient [17,18]. Colorimetric sensing is realized by monitoring colloidal AuNPs aggregation on paper membranes induced
Monitoring differential expression of proteases for disease diagnosis has been an area of intense research over the past few decades. It has been reported that the up and downregulation of proteases can be associated with the state and progression of several diseases [1] including AIDS and hepatitis [2], cancer [3–5], arthritis [6], Alzheimer’s [7], diabetes [8] and inflammatory complications [9]. Therefore, attempts have been made to develop assays for in-vitro/in-vivo detection and for real time monitoring of proteases [4,10]. In general, proteases in diverse sample matrices have been detected by affinity (e.g., immunological) or functional (e.g., digestion) assays [11]. Affinity assays report the presence of proteases, regardless of its activity, based on binding between the proteases and specific recognition entities such as antibodies or aptamers. On the other hand, functional protease assays aim to detect only the active form of proteases, based on enzymatic activity of the target protease over a substrate. Therefore, functional protease assays generally are regarded more reliable for disease diagnosis and is preferred for protease assaying over the affinity assays. Functional protease assays can be classified as homogeneous or heterogeneous; where in the homogeneous assays both the substrate and protease are free to react in the aqueous phase, while in the latter, ⁎
Corresponding authors at: School of Materials Science and Engineering, Nanyang Technological University, 50 Nanyang Avenue, 639798, Singapore. E-mail addresses: [email protected] (A. Palaniappan), [email protected] (B. Liedberg).
https://doi.org/10.1016/j.snb.2019.127442 Received 11 October 2019; Received in revised form 11 November 2019; Accepted 18 November 2019 Available online 19 November 2019 0925-4005/ © 2019 Elsevier B.V. All rights reserved.
Sensors & Actuators: B. Chemical 305 (2020) 127442
G. Goyal, et al.
Scheme 1. Comparison of the size of the membrane with respect to a 50 cents coin. Schematics of the assay with peptide functionalized AuNPs on PVDF membrane, which yields a change in color from reddish to violet due to aggregation induced by MMP-7. These illustrative images were taken using Sony ILCE-7R.
functionalization replaces citrate on the AuNPs surface and modulates the net negative charge of the AuNPs (-5), which further stabilize the AuNPs and prevent them from aggregating in solution. Upon exposure to MMP-7, the peptide is hydrolyzed at two sites (A–L and AQ-L), leading to a decrease in net negative charge from -5 to -1 [25]. This reduction in net negative charge of the AuNPs reduces the colloidal stability inducing aggregation, which concomitantly causes a change in color of the suspension. Experimental observations clearly suggest that the addition of MMP-7 to the peptide functionalized AuNPs on the paper membrane also leads to an identical aggregation phenomenon and color change that enables rapid, sensitive and specific colorimetric detection of MMP-7. The proposed methodology involves a single step fabrication process of drop casting the peptide functionalized AuNPs on the membrane. The membranes are then rinsed to wash off the AuNPs that are not adsorbed on the membrane followed by air drying. Among the membranes tested, PVDF yields a homogeneous distribution of peptide functionalized AuNPs, as compared to cellulose acetate and nylon membranes (Fig. S2(a)). FESEM was used to independently characterize the distribution of AuNPs on different membranes tested. The AuNPs appear to be aggregated on cellulose acetate (Fig. S2(b)) and nylon membranes (Fig. S2(c)), whereas a well-dispersed distribution of AuNPs is evident on the PVDF membrane (Fig. 1 (top panel: a, b, c)). Also, the role of Tween 20 on the aggregation phenomenon on the paper membrane was studied and reported in Fig. S3. EDX analysis confirms the deposition of AuNPs on PVDF membrane. The two peaks, viz., carbon and fluorine are attributed to the PVDF membrane, while Au peaks ascertain the presence of AuNP on PVDF membranes before (Fig. S4(a)) and after (Fig. S4(b)) addition of MMP-7. Furthermore, the peak at 522 nm observed in the diffuse reflectance spectra of peptide functionalized AuNPs on the PVDF membrane also ascertains the deposition of AuNP on PVDF membrane (Fig. S5). The peak corresponding to AuNP broadens and shifts to approximately 550 nm upon addition of MMP-7, which is indicative of aggregation of AuNPs. The net negative charge of -5 is sufficient to maintain the AuNPs at a certain interparticle distance on the membrane such that they remain dispersed and hence appear reddish/brownish in color. Upon exposure of the membrane to MMP-7, hydrolysis of the immobilized peptide
by proteolysis. AuNPs aggregate on paper membrane because of increased electrostatic interparticle attraction upon proteolysis, consequently yielding a colorimetric response, as schematically represented in Scheme 1. This communication emphasizes on the feasibility of using peptide functionalized AuNPs on paper membranes, in the dry state, as an alternative to the typically adopted homogeneous solution state assays. Thus, we report a heterogeneous protease assay on paper membrane that monitors highly specific analyte-induced changes in the aggregation state of peptide functionalized AuNPs. Experimental results indicate that the colorimetric responses of AuNPs on membranes are more sensitive than the corresponding responses of a homogeneous solution state assay. The proposed approach would be ideal for applications in resource-limited settings, where small volume of samples, without tedious pre-treatment, can be added on the paper membrane containing AuNPs for assaying proteases via colorimetric responses that are observable by the naked eye or in combination with ubiquitous technologies like smartphones. Furthermore, the proposed methodology is a generic approach for detection of a wide range of proteases upon functionalizing AuNPs with specific peptides. 2. Result and discussion Matrilysin, one of the simplest proteases from a 24-member family of matrix metalloproteinases (MMPs), is used as a model protease to validate the proposed functional protease assay [19]. Matrilysin, from now on referred to as MMP-7, is known to be involved in the precise regulation of extracellular matrix degradation. An aberration in its expression can lead to various diseases such as pancreatic, colorectal and lung cancer [20–23]. The MMP-7 substrate used in this study is a synthetic peptide composed of 42 amino acids (NAADLEKAIEALEKHLEAKGPCDAAQLEKQLEQAFEAFERAG) with two digestion sites (labelled in bold) and a cysteine residue (underlined) for conjugation to AuNPs using gold-thiol chemistry [24]. The UV–vis absorbance spectrum of AuNPs shifts from 520 nm to 522 nm upon functionalization with peptide, and it further shifts to 550 nm upon aggregation induced by MMP-7 hydrolysis, as shown in Fig. S1 (a). Moreover, the TEM images shown in Fig. S1 (b) depict the dispersed and aggregated states of peptide functionalized AuNPs. Mechanistically, the peptide 2
Sensors & Actuators: B. Chemical 305 (2020) 127442
G. Goyal, et al.
Fig. 1. Field Emission Scanning Electron Microscope images of (top panel) peptide functionalized AuNPs on the PVDF membrane at (a) 2 K magnification (b) 15 K magnification (c) 60 K magnification; (bottom panel) MMP-7 induced aggregation peptide functionalized AuNPs on the PVDF membrane at (d) 2 K magnification (e) 15 K magnification (f) 60 K magnification. Red highlighted boxes show AuNPs aggregated after addition of MMP-7 (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.). Fig. 2. (a) Digital images of the peptide functionalized AuNPs immobilized on the PVDF membrane upon addition of 20 μL of MMP-7 in buffer at varying concentrations (0−25 μg/ mL) recorded after 30 min of incubation. (b) RED component histogram of the 200*200 pixels taken from the membrane showing the maximum and minimum value of the RED component of every pixel on the RGB scale of 0-255. (c) Calibration curve of the MMP-7 assay in buffer for averaged RED intensity vs MMP-7 concentration (y = –0.92 + 91, R2 = 0.94). Digital images were taken using Sony ILCE-7R (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.). Fig. 3. Digital images and their corresponding RED components of AuNPs deposited PVDF membranes upon addition of 20 μL of buffer (Blank), active MMP-7; heat-treated MMP-7, EDTA treated MMP-7 and inhibitor II treated MMP-7; ADAM-17, OmpT and trypsin at a concentration of 50 μg/mL recorded for illustrating the specificity of the assay. Digital images were taken using Sony ILCE-7R (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
decrease in repulsive forces between the AuNPs upon addition of MMP7. In order to ascertain that the peptide functionalized AuNPs remain fully functional when adsorbed on the membrane, a control experiment using zinc ions was performed. The peptide utilized in this study has been reported to fold and dimerize in the presence of zinc ions [26].The color of membrane with the peptide functionalized AuNPs changed instantly from red to violet upon addition of ZnCl2, whereas no such color change was observed upon addition of MilliQ water or EDTAchelated Zn2+ions (Fig. S6). Thus, the peptide functionalized AuNPs on
decreases the net negative surface charge of the remaining peptide fragments on AuNP to -1. Therefore, the repulsive forces decrease between the AuNPs, resulting in a reduction of the interparticle distance and hence causing aggregation of AuNPs on the membrane, which can be visualized by a change in color from reddish/brownish to violet. Moreover, FESEM images recorded after addition of MMP-7 to the peptide functionalized AuNPs on a PVDF membrane are shown in Fig. 1 (bottom panel: d, e, f). The AuNPs undoubtedly appear clustered/aggregated (highlighted in red boxes), which we again attribute to the 3
Sensors & Actuators: B. Chemical 305 (2020) 127442
G. Goyal, et al.
Fig. 4. (a) Digital images of the peptide functionalized AuNPs immobilized on the PVDF membrane upon addition of 20 μL of MMP-7 in synthetic urine for varying concentrations (0–25 μg/mL) recorded after 30 min of incubation. (b) RED component histogram of the 200*200 pixels taken from the membrane showing the maximum and minimum value of the RED component of every pixel on the RGB scale of 0-255 (c) Calibration curve of the MMP-7 assay in synthetic urine (y = –1.04x + 99.8, R2 equal to 0.91). Digital images were taken using Sony ILCE-7R (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
(Fig. 3). However, the response is not as significant as compared to that of MMP-7. Thus, in principle, any proteolytic enzyme with a specific peptide functionalized AuNPs immobilized on the membrane can be assayed using the proposed format. MMP-7 is present in numerous body fluids such as serum [22], plasma [20], saliva [28], and urine [29]. To test the proposed paperbased assay for applications in increasingly complex matrices, we have evaluated the colorimetric responses using synthetic urine. As observed from Fig. 4(a), colorimetric responses for assaying MMP-7 spiked in synthetic urine and the visual LOD of 3.125 μg/mL are comparable to that of the responses and visual LOD obtained in buffer. The red component, on the scale of 0-255, decreases with increasing MMP-7 concentrations as shown in Fig. 4(b). The linear concentration dependent response as shown in Fig. 4(c) yields an objective LOD of 3.5 μg/mL (185 nM). Furthermore, the standard deviation in red components (Fig. 4(c)) of less than 5 % (for all test concentrations, n = 3), illustrates that the developed assay possesses great potential for assaying MMP-7 in increasingly complex matrices without requiring tedious sample pretreatment protocols. It should be emphasized that it may not be appropriate to directly compare the LODs obtained using the proposed heterogeneous assay on membrane with previously reported homogeneous assays [25,30]. The MMP-7 utilized in these previous reports possessed a 10-fold higher enzymatic activity than the MMP-7 utilized in the current study, which is synthesized in house [31] with a controlled and stable activity. Thus, by taking this 10-fold higher activity into account, the LOD obtained for our membrane-based assay is almost on par with the visual LOD previously reported for a homogeneous MMP-7 assay using identical peptide functionalized AuNPs [30]. Furthermore, the proposed approach is specific for MMP-7 and enables detection of MMP-7 in synthetic urine, thereby providing a viable avenue towards onsite diagnosis. Our current research focuses on evaluating different clean-up strategies for detection of proteases in even more complex matrices including, for example, patient samples (urine).
membrane respond identically to external triggers like MMP-7 and Zn2+ ions as peptide functionalized AuNPs in homogeneous solution [25,26]. Concentration dependent responses are obtained (using the optimized concentration of AuNPs (Fig. S7)) by adding buffer solutions containing different amounts of MMP-7 on the membrane. No change in color of AuNPs on membrane is observed (after 30 min) upon addition of buffer in the absence of MMP-7. However, colorimetric responses are evident after 30 min of reaction time in presence of MMP-7. The color changes from reddish/brownish to violet with increasing concentrations of MMP-7 (Fig. 2(a)), and they can be seen by the naked eye for MMP-7 concentrations above 3.125 μg/mL (165 nM), which is considered as the visual limit of detection (LOD). RGB analysis was then carried out to objectively study the colorimetric responses and the red component was utilized to evaluate the colorimetric responses. Note, the variations in red components are significant as compared to the blue and green components, as shown in Fig. S8. The red component, on the scale of 0-255, decreases with increasing concentrations of MMP-7 (Fig. 2(b)), concurring with the visual observations. More importantly, the membrane configuration yields a better response as compared to the typically adopted homogenous assay with a visual LOD at 12.5 μg/mL (650 nM), as shown in Fig. S9. Thus, the proposed heterogeneous assay, on membrane, exhibits a LOD that is four times lower than that of the homogeneous assay. Moreover, the objective LOD obtained from the linear fit of averaged red intensity and MMP-7 concentration in Fig. 2(c) is calculated to be 3.3 μg/mL, which concurs with the visual LOD of the assay. In addition, stability studies performed over a period of 5 days suggests that the AuNP on membranes remain stable and functionally active when stored at 4 °C, as shown in Fig. S10. Colorimetric responses of peptide-functionalized AuNPs to inhibited MMP-7 were also obtained to evaluate the specificity of the developed assay. MMP-7 was partially inactivated by heating at 100 °C for 2 h. It was also fully inactivated by adding a chelating agent (EDTA, 250 mM) to remove Ca2+ and Zn2+ ions (metal ions that are essential for the activity of MMP-7) or by adding MMP inhibitor II (250 μM) that specifically bind to catalytic Zn2+ ions [27]. Partially inactivated MMP-7 leads to a moderate change in color as compared to the corresponding response seen upon addition of same concentration of active MMP-7. Fully inactivated MMP-7 (using EDTA or MMP inhibitor II) does not modulate the color. If fact, the color is comparable to that obtained in buffer without MMP-7 (Fig. 3). The values in parenthesis in Fig. 3 refer to the red component of the respective image. Moreover, proteases such as ADAM-17 (a disintegrin and metalloproteinase, belonging to the same family as MMP-7) and OmpT (Outer membrane protease) are then added at high concentrations (50 μg/mL) to the membranes to evaluate the specificity of the developed assay. As shown in Fig. 3, ADAM-17 and OmpT yield no significant colorimetric response, ensuring excellent specificity, respectively of the assay. Trypsin also was tested as it cleaves the peptide at the arginine and lysine residues towards the C terminal and a color change is apparent upon addition of Trypsin
3. Conclusion We have successfully demonstrated a functional protease assay on a paper membrane. The facile assay involves immobilization of peptide functionalized AuNPs on paper membranes, resulting in a rapid, sensitive and cost-effective heterogeneous assay as compared to the conventional solution-based homogenous assays. The rapid change in color of the membrane upon exposure to proteases offers an opportunity to evaluate the state of the disease and its progression. Experiments in synthetic urine suggest that the developed assay possesses the potential to yield colorimetric responses in complex matrices. Furthermore, this assay can be adapted for onsite applications as AuNPs easily can be patterned on the paper substrate to indicate presence or absence of proteases via facile and comprehensible readouts. Additionally, the proposed approach can be expanded into a multiplexed format for 4
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simultaneous detection of proteases using AuNPs functionalized with specific peptides.
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Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements The Institute for Nanomedicine jointly established between Northwestern University, U.S.A. and Nanyang Technological University, Singapore are acknowledged for financial support. We would like to thank Robert Selegård and Daniel Aili for the synthesis of the 42 mer peptide used in the assay. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.snb.2019.127442. References [1] K. Welser, R. Adsley, B.M. Moore, W.C. Chan, J.W. Aylott, Protease sensing with nanoparticle based platforms, Analyst 136 (2011) 29–41, https://doi.org/10.1039/ c0an00429d. [2] K. Lin, R.B. Perni, A.D. Kwong, C. Lin, VX-950, a novel hepatitis C virus (HCV) NS34A protease inhibitor, exhibits potent antiviral activities in HCv replicon cells, Antimicrob. Agents Chemother. 50 (2006) 1813–1822, https://doi.org/10.1128/ AAC.50.5.1813-1822.2006. [3] M.P. Look, J.A. Foekens, Clinical relevance of the urokinase plasminogen activator system in breast cancer, APMIS 107 (1999) 150–159, https://doi.org/10.1111/j. 1699-0463.1999.tb01538.x. [4] S.C. Dixon, K.B. Knopf, W.D. Figg, The control of prostate-specific antigen expression and gene regulation by pharmacological agents, Pharmacol. Rev. 53 (2001) 73–91 http://pharmrev.aspetjournals.org/content/53/1/73. [5] R. Roy, J. Yang, M.A. Moses, Matrix metalloproteinases as novel biomarkers and potential therapeutic targets in human cancer, J. Clin. Oncol. 27 (2009) 5287–5297, https://doi.org/10.1200/JCO.2009.23.5556. [6] W.-F.T. Lai, C.-H. Chang, Y. Tang, R. Bronson, C.-H. Tung, Early diagnosis of osteoarthritis using cathepsin B sensitive near-infrared fluorescent probes, Osteoarthr. Cartil. 12 (2004) 239–244, https://doi.org/10.1016/j.joca.2003.11.005. [7] K.-I. Saito, J.S. Elcet, J.E. Hamos, R.A. Nixon, Widespread activation of calciumactivated neutral proteinase (calpain) in the brain in Alzheimer disease: a potential molecular basis for neuronal degeneration, Proc. Natl. Acad. Sci. U.S.A. 90 (1993) 2628–2632, https://doi.org/10.1073/pnas.90.7.2628. [8] S.H. Havale, M. Pal, Medicinal chemistry approaches to the inhibition of dipeptidyl peptidase-4 for the treatment of type 2 diabetes, Bioorg. Med. Chem. 17 (2009) 1783–1802, https://doi.org/10.1016/j.bmc.2009.01.061. [9] J.H. Ryu, A. Lee, M.S. Huh, J. Chu, K. Kim, B.-S. Kim, K. Choi, I.C. Kwon, J.W. Park, I. Youn, Measurement of MMP activity in synovial fluid in cases of osteoarthritis and acute inflammatory conditions of the knee joints using a fluorogenic peptide probe-immobilized diagnostic kit, Theranostics 2 (2012) 198–206, https://doi.org/ 10.7150/thno.3477. [10] J. Cazzulo, V. Stoka, V. Turk, The major cysteine proteinase of trypanosoma cruzi: a valid target for chemotherapy of chagas disease, Curr. Pharm. Des. 7 (2001) 1143–1156, https://doi.org/10.2174/1381612013397528. [11] G. Zhang, Protease Assays, (n.d.). https://www.ncbi.nlm.nih.gov/books/ NBK92006/pdf/Bookshelf_NBK92006.pdf. [12] I.L.H. Ong, K.-L. Yang, Recent developments in protease activity assays and sensors, Analyst 142 (2017) 1867–1881, https://doi.org/10.1039/C6AN02647H.
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Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Protease functional assay on membrane Garima Goyal a b c
a,b,c
, Alagappan Palaniappan
b,c,
T
*, Bo Liedberg
a,b,c,
*
Interdisciplinary Graduate School, Nanyang Technological University, 50 Nanyang Avenue, 639798, Singapore School of Materials Science and Engineering, Nanyang Technological University, 50 Nanyang Avenue, 639798, Singapore Center for Biomimetic Sensor Science, Nanyang Technological University, 50 Nanyang Drive, 637553, Singapore
A R T I C LE I N FO
A B S T R A C T
Keywords: Proteases Functional assay Paper based assay Matrilysin Colorimetric sensors
Targeting enzymes, proteases in particular, using various assay formats has attracted considerable interest for early disease diagnosis. While affinity-based assays that report the presence of proteases in samples have been widely explored, it is of utmost importance to develop functional assays that provide information on the activity of proteases for a more precise indication of the disease state and progression. Herein, we report a paper-based functional assay that yields naked eye observable and concentration dependent responses for the active form of proteases. The paper-based assay involves a facile single step fabrication process of depositing protease specific peptide functionalized gold nanoparticles on paper membranes. Matrilysin is used as a model protease to validate the proposed methodology. The proteolysis-driven aggregation of nanoparticles on the membrane yields a colorimetric response that has limit of detection that is ∼4 times lower (3.1 μg/mL) than for the same assay performed in homogeneous solution (12.5 μg/mL). To the best of our knowledge, this is the first report on heterogeneous protease assay on paper relying on aggregation of peptide-functionalized nanoparticles. The approach easily can be extended to assay other enzymes by functionalizing the gold nanoparticles using specific peptides.
1. Introduction
either the substrate or protease is immobilized on a surface/electrode of an assay platform [12]. Typical homogeneous functional assays are based on methodologies such as mass spectrometry, Förster Resonance Energy Transfer (FRET) etc. Homogeneous functional assays generally are very sensitive, but often requires sophisticated instrumentation or experience susceptibility to photo-bleaching and pH sensitivity [13]. On the other hand, heterogeneous assays could provide opportunities for the development of cost effective and easy-to-use assays, owing to their robustness and minimized liquid handling complexity. Till date, heterogeneous protease assays have been demonstrated by immobilizing substrates on glass/gold surface for SPR based detection or Raman spectroscopy or on conductive electrodes for electrochemical detection. However, most of these approaches are labor intensive, time consuming and require trained personnel [14–16]. Herein, we report a paper-based functional assay that yield naked eye observable and concentration dependent responses of proteases in buffer and synthetic urine. The proposed paper-based assay involves a facile single step fabrication process of depositing protease specific peptide functionalized gold nanoparticles (AuNPs) on paper membranes. AuNPs are chosen as reporters because of their high surface area and extinction coefficient [17,18]. Colorimetric sensing is realized by monitoring colloidal AuNPs aggregation on paper membranes induced
Monitoring differential expression of proteases for disease diagnosis has been an area of intense research over the past few decades. It has been reported that the up and downregulation of proteases can be associated with the state and progression of several diseases [1] including AIDS and hepatitis [2], cancer [3–5], arthritis [6], Alzheimer’s [7], diabetes [8] and inflammatory complications [9]. Therefore, attempts have been made to develop assays for in-vitro/in-vivo detection and for real time monitoring of proteases [4,10]. In general, proteases in diverse sample matrices have been detected by affinity (e.g., immunological) or functional (e.g., digestion) assays [11]. Affinity assays report the presence of proteases, regardless of its activity, based on binding between the proteases and specific recognition entities such as antibodies or aptamers. On the other hand, functional protease assays aim to detect only the active form of proteases, based on enzymatic activity of the target protease over a substrate. Therefore, functional protease assays generally are regarded more reliable for disease diagnosis and is preferred for protease assaying over the affinity assays. Functional protease assays can be classified as homogeneous or heterogeneous; where in the homogeneous assays both the substrate and protease are free to react in the aqueous phase, while in the latter, ⁎
Corresponding authors at: School of Materials Science and Engineering, Nanyang Technological University, 50 Nanyang Avenue, 639798, Singapore. E-mail addresses: [email protected] (A. Palaniappan), [email protected] (B. Liedberg).
https://doi.org/10.1016/j.snb.2019.127442 Received 11 October 2019; Received in revised form 11 November 2019; Accepted 18 November 2019 Available online 19 November 2019 0925-4005/ © 2019 Elsevier B.V. All rights reserved.
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Scheme 1. Comparison of the size of the membrane with respect to a 50 cents coin. Schematics of the assay with peptide functionalized AuNPs on PVDF membrane, which yields a change in color from reddish to violet due to aggregation induced by MMP-7. These illustrative images were taken using Sony ILCE-7R.
functionalization replaces citrate on the AuNPs surface and modulates the net negative charge of the AuNPs (-5), which further stabilize the AuNPs and prevent them from aggregating in solution. Upon exposure to MMP-7, the peptide is hydrolyzed at two sites (A–L and AQ-L), leading to a decrease in net negative charge from -5 to -1 [25]. This reduction in net negative charge of the AuNPs reduces the colloidal stability inducing aggregation, which concomitantly causes a change in color of the suspension. Experimental observations clearly suggest that the addition of MMP-7 to the peptide functionalized AuNPs on the paper membrane also leads to an identical aggregation phenomenon and color change that enables rapid, sensitive and specific colorimetric detection of MMP-7. The proposed methodology involves a single step fabrication process of drop casting the peptide functionalized AuNPs on the membrane. The membranes are then rinsed to wash off the AuNPs that are not adsorbed on the membrane followed by air drying. Among the membranes tested, PVDF yields a homogeneous distribution of peptide functionalized AuNPs, as compared to cellulose acetate and nylon membranes (Fig. S2(a)). FESEM was used to independently characterize the distribution of AuNPs on different membranes tested. The AuNPs appear to be aggregated on cellulose acetate (Fig. S2(b)) and nylon membranes (Fig. S2(c)), whereas a well-dispersed distribution of AuNPs is evident on the PVDF membrane (Fig. 1 (top panel: a, b, c)). Also, the role of Tween 20 on the aggregation phenomenon on the paper membrane was studied and reported in Fig. S3. EDX analysis confirms the deposition of AuNPs on PVDF membrane. The two peaks, viz., carbon and fluorine are attributed to the PVDF membrane, while Au peaks ascertain the presence of AuNP on PVDF membranes before (Fig. S4(a)) and after (Fig. S4(b)) addition of MMP-7. Furthermore, the peak at 522 nm observed in the diffuse reflectance spectra of peptide functionalized AuNPs on the PVDF membrane also ascertains the deposition of AuNP on PVDF membrane (Fig. S5). The peak corresponding to AuNP broadens and shifts to approximately 550 nm upon addition of MMP-7, which is indicative of aggregation of AuNPs. The net negative charge of -5 is sufficient to maintain the AuNPs at a certain interparticle distance on the membrane such that they remain dispersed and hence appear reddish/brownish in color. Upon exposure of the membrane to MMP-7, hydrolysis of the immobilized peptide
by proteolysis. AuNPs aggregate on paper membrane because of increased electrostatic interparticle attraction upon proteolysis, consequently yielding a colorimetric response, as schematically represented in Scheme 1. This communication emphasizes on the feasibility of using peptide functionalized AuNPs on paper membranes, in the dry state, as an alternative to the typically adopted homogeneous solution state assays. Thus, we report a heterogeneous protease assay on paper membrane that monitors highly specific analyte-induced changes in the aggregation state of peptide functionalized AuNPs. Experimental results indicate that the colorimetric responses of AuNPs on membranes are more sensitive than the corresponding responses of a homogeneous solution state assay. The proposed approach would be ideal for applications in resource-limited settings, where small volume of samples, without tedious pre-treatment, can be added on the paper membrane containing AuNPs for assaying proteases via colorimetric responses that are observable by the naked eye or in combination with ubiquitous technologies like smartphones. Furthermore, the proposed methodology is a generic approach for detection of a wide range of proteases upon functionalizing AuNPs with specific peptides. 2. Result and discussion Matrilysin, one of the simplest proteases from a 24-member family of matrix metalloproteinases (MMPs), is used as a model protease to validate the proposed functional protease assay [19]. Matrilysin, from now on referred to as MMP-7, is known to be involved in the precise regulation of extracellular matrix degradation. An aberration in its expression can lead to various diseases such as pancreatic, colorectal and lung cancer [20–23]. The MMP-7 substrate used in this study is a synthetic peptide composed of 42 amino acids (NAADLEKAIEALEKHLEAKGPCDAAQLEKQLEQAFEAFERAG) with two digestion sites (labelled in bold) and a cysteine residue (underlined) for conjugation to AuNPs using gold-thiol chemistry [24]. The UV–vis absorbance spectrum of AuNPs shifts from 520 nm to 522 nm upon functionalization with peptide, and it further shifts to 550 nm upon aggregation induced by MMP-7 hydrolysis, as shown in Fig. S1 (a). Moreover, the TEM images shown in Fig. S1 (b) depict the dispersed and aggregated states of peptide functionalized AuNPs. Mechanistically, the peptide 2
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Fig. 1. Field Emission Scanning Electron Microscope images of (top panel) peptide functionalized AuNPs on the PVDF membrane at (a) 2 K magnification (b) 15 K magnification (c) 60 K magnification; (bottom panel) MMP-7 induced aggregation peptide functionalized AuNPs on the PVDF membrane at (d) 2 K magnification (e) 15 K magnification (f) 60 K magnification. Red highlighted boxes show AuNPs aggregated after addition of MMP-7 (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.). Fig. 2. (a) Digital images of the peptide functionalized AuNPs immobilized on the PVDF membrane upon addition of 20 μL of MMP-7 in buffer at varying concentrations (0−25 μg/ mL) recorded after 30 min of incubation. (b) RED component histogram of the 200*200 pixels taken from the membrane showing the maximum and minimum value of the RED component of every pixel on the RGB scale of 0-255. (c) Calibration curve of the MMP-7 assay in buffer for averaged RED intensity vs MMP-7 concentration (y = –0.92 + 91, R2 = 0.94). Digital images were taken using Sony ILCE-7R (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.). Fig. 3. Digital images and their corresponding RED components of AuNPs deposited PVDF membranes upon addition of 20 μL of buffer (Blank), active MMP-7; heat-treated MMP-7, EDTA treated MMP-7 and inhibitor II treated MMP-7; ADAM-17, OmpT and trypsin at a concentration of 50 μg/mL recorded for illustrating the specificity of the assay. Digital images were taken using Sony ILCE-7R (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
decrease in repulsive forces between the AuNPs upon addition of MMP7. In order to ascertain that the peptide functionalized AuNPs remain fully functional when adsorbed on the membrane, a control experiment using zinc ions was performed. The peptide utilized in this study has been reported to fold and dimerize in the presence of zinc ions [26].The color of membrane with the peptide functionalized AuNPs changed instantly from red to violet upon addition of ZnCl2, whereas no such color change was observed upon addition of MilliQ water or EDTAchelated Zn2+ions (Fig. S6). Thus, the peptide functionalized AuNPs on
decreases the net negative surface charge of the remaining peptide fragments on AuNP to -1. Therefore, the repulsive forces decrease between the AuNPs, resulting in a reduction of the interparticle distance and hence causing aggregation of AuNPs on the membrane, which can be visualized by a change in color from reddish/brownish to violet. Moreover, FESEM images recorded after addition of MMP-7 to the peptide functionalized AuNPs on a PVDF membrane are shown in Fig. 1 (bottom panel: d, e, f). The AuNPs undoubtedly appear clustered/aggregated (highlighted in red boxes), which we again attribute to the 3
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Fig. 4. (a) Digital images of the peptide functionalized AuNPs immobilized on the PVDF membrane upon addition of 20 μL of MMP-7 in synthetic urine for varying concentrations (0–25 μg/mL) recorded after 30 min of incubation. (b) RED component histogram of the 200*200 pixels taken from the membrane showing the maximum and minimum value of the RED component of every pixel on the RGB scale of 0-255 (c) Calibration curve of the MMP-7 assay in synthetic urine (y = –1.04x + 99.8, R2 equal to 0.91). Digital images were taken using Sony ILCE-7R (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
(Fig. 3). However, the response is not as significant as compared to that of MMP-7. Thus, in principle, any proteolytic enzyme with a specific peptide functionalized AuNPs immobilized on the membrane can be assayed using the proposed format. MMP-7 is present in numerous body fluids such as serum [22], plasma [20], saliva [28], and urine [29]. To test the proposed paperbased assay for applications in increasingly complex matrices, we have evaluated the colorimetric responses using synthetic urine. As observed from Fig. 4(a), colorimetric responses for assaying MMP-7 spiked in synthetic urine and the visual LOD of 3.125 μg/mL are comparable to that of the responses and visual LOD obtained in buffer. The red component, on the scale of 0-255, decreases with increasing MMP-7 concentrations as shown in Fig. 4(b). The linear concentration dependent response as shown in Fig. 4(c) yields an objective LOD of 3.5 μg/mL (185 nM). Furthermore, the standard deviation in red components (Fig. 4(c)) of less than 5 % (for all test concentrations, n = 3), illustrates that the developed assay possesses great potential for assaying MMP-7 in increasingly complex matrices without requiring tedious sample pretreatment protocols. It should be emphasized that it may not be appropriate to directly compare the LODs obtained using the proposed heterogeneous assay on membrane with previously reported homogeneous assays [25,30]. The MMP-7 utilized in these previous reports possessed a 10-fold higher enzymatic activity than the MMP-7 utilized in the current study, which is synthesized in house [31] with a controlled and stable activity. Thus, by taking this 10-fold higher activity into account, the LOD obtained for our membrane-based assay is almost on par with the visual LOD previously reported for a homogeneous MMP-7 assay using identical peptide functionalized AuNPs [30]. Furthermore, the proposed approach is specific for MMP-7 and enables detection of MMP-7 in synthetic urine, thereby providing a viable avenue towards onsite diagnosis. Our current research focuses on evaluating different clean-up strategies for detection of proteases in even more complex matrices including, for example, patient samples (urine).
membrane respond identically to external triggers like MMP-7 and Zn2+ ions as peptide functionalized AuNPs in homogeneous solution [25,26]. Concentration dependent responses are obtained (using the optimized concentration of AuNPs (Fig. S7)) by adding buffer solutions containing different amounts of MMP-7 on the membrane. No change in color of AuNPs on membrane is observed (after 30 min) upon addition of buffer in the absence of MMP-7. However, colorimetric responses are evident after 30 min of reaction time in presence of MMP-7. The color changes from reddish/brownish to violet with increasing concentrations of MMP-7 (Fig. 2(a)), and they can be seen by the naked eye for MMP-7 concentrations above 3.125 μg/mL (165 nM), which is considered as the visual limit of detection (LOD). RGB analysis was then carried out to objectively study the colorimetric responses and the red component was utilized to evaluate the colorimetric responses. Note, the variations in red components are significant as compared to the blue and green components, as shown in Fig. S8. The red component, on the scale of 0-255, decreases with increasing concentrations of MMP-7 (Fig. 2(b)), concurring with the visual observations. More importantly, the membrane configuration yields a better response as compared to the typically adopted homogenous assay with a visual LOD at 12.5 μg/mL (650 nM), as shown in Fig. S9. Thus, the proposed heterogeneous assay, on membrane, exhibits a LOD that is four times lower than that of the homogeneous assay. Moreover, the objective LOD obtained from the linear fit of averaged red intensity and MMP-7 concentration in Fig. 2(c) is calculated to be 3.3 μg/mL, which concurs with the visual LOD of the assay. In addition, stability studies performed over a period of 5 days suggests that the AuNP on membranes remain stable and functionally active when stored at 4 °C, as shown in Fig. S10. Colorimetric responses of peptide-functionalized AuNPs to inhibited MMP-7 were also obtained to evaluate the specificity of the developed assay. MMP-7 was partially inactivated by heating at 100 °C for 2 h. It was also fully inactivated by adding a chelating agent (EDTA, 250 mM) to remove Ca2+ and Zn2+ ions (metal ions that are essential for the activity of MMP-7) or by adding MMP inhibitor II (250 μM) that specifically bind to catalytic Zn2+ ions [27]. Partially inactivated MMP-7 leads to a moderate change in color as compared to the corresponding response seen upon addition of same concentration of active MMP-7. Fully inactivated MMP-7 (using EDTA or MMP inhibitor II) does not modulate the color. If fact, the color is comparable to that obtained in buffer without MMP-7 (Fig. 3). The values in parenthesis in Fig. 3 refer to the red component of the respective image. Moreover, proteases such as ADAM-17 (a disintegrin and metalloproteinase, belonging to the same family as MMP-7) and OmpT (Outer membrane protease) are then added at high concentrations (50 μg/mL) to the membranes to evaluate the specificity of the developed assay. As shown in Fig. 3, ADAM-17 and OmpT yield no significant colorimetric response, ensuring excellent specificity, respectively of the assay. Trypsin also was tested as it cleaves the peptide at the arginine and lysine residues towards the C terminal and a color change is apparent upon addition of Trypsin
3. Conclusion We have successfully demonstrated a functional protease assay on a paper membrane. The facile assay involves immobilization of peptide functionalized AuNPs on paper membranes, resulting in a rapid, sensitive and cost-effective heterogeneous assay as compared to the conventional solution-based homogenous assays. The rapid change in color of the membrane upon exposure to proteases offers an opportunity to evaluate the state of the disease and its progression. Experiments in synthetic urine suggest that the developed assay possesses the potential to yield colorimetric responses in complex matrices. Furthermore, this assay can be adapted for onsite applications as AuNPs easily can be patterned on the paper substrate to indicate presence or absence of proteases via facile and comprehensible readouts. Additionally, the proposed approach can be expanded into a multiplexed format for 4
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simultaneous detection of proteases using AuNPs functionalized with specific peptides.
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Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements The Institute for Nanomedicine jointly established between Northwestern University, U.S.A. and Nanyang Technological University, Singapore are acknowledged for financial support. We would like to thank Robert Selegård and Daniel Aili for the synthesis of the 42 mer peptide used in the assay. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.snb.2019.127442. References [1] K. Welser, R. Adsley, B.M. Moore, W.C. Chan, J.W. Aylott, Protease sensing with nanoparticle based platforms, Analyst 136 (2011) 29–41, https://doi.org/10.1039/ c0an00429d. [2] K. Lin, R.B. Perni, A.D. Kwong, C. Lin, VX-950, a novel hepatitis C virus (HCV) NS34A protease inhibitor, exhibits potent antiviral activities in HCv replicon cells, Antimicrob. Agents Chemother. 50 (2006) 1813–1822, https://doi.org/10.1128/ AAC.50.5.1813-1822.2006. [3] M.P. Look, J.A. Foekens, Clinical relevance of the urokinase plasminogen activator system in breast cancer, APMIS 107 (1999) 150–159, https://doi.org/10.1111/j. 1699-0463.1999.tb01538.x. [4] S.C. Dixon, K.B. Knopf, W.D. Figg, The control of prostate-specific antigen expression and gene regulation by pharmacological agents, Pharmacol. Rev. 53 (2001) 73–91 http://pharmrev.aspetjournals.org/content/53/1/73. [5] R. Roy, J. Yang, M.A. Moses, Matrix metalloproteinases as novel biomarkers and potential therapeutic targets in human cancer, J. Clin. Oncol. 27 (2009) 5287–5297, https://doi.org/10.1200/JCO.2009.23.5556. [6] W.-F.T. Lai, C.-H. Chang, Y. Tang, R. Bronson, C.-H. Tung, Early diagnosis of osteoarthritis using cathepsin B sensitive near-infrared fluorescent probes, Osteoarthr. Cartil. 12 (2004) 239–244, https://doi.org/10.1016/j.joca.2003.11.005. [7] K.-I. Saito, J.S. Elcet, J.E. Hamos, R.A. Nixon, Widespread activation of calciumactivated neutral proteinase (calpain) in the brain in Alzheimer disease: a potential molecular basis for neuronal degeneration, Proc. Natl. Acad. Sci. U.S.A. 90 (1993) 2628–2632, https://doi.org/10.1073/pnas.90.7.2628. [8] S.H. Havale, M. Pal, Medicinal chemistry approaches to the inhibition of dipeptidyl peptidase-4 for the treatment of type 2 diabetes, Bioorg. Med. Chem. 17 (2009) 1783–1802, https://doi.org/10.1016/j.bmc.2009.01.061. [9] J.H. Ryu, A. Lee, M.S. Huh, J. Chu, K. Kim, B.-S. Kim, K. Choi, I.C. Kwon, J.W. Park, I. Youn, Measurement of MMP activity in synovial fluid in cases of osteoarthritis and acute inflammatory conditions of the knee joints using a fluorogenic peptide probe-immobilized diagnostic kit, Theranostics 2 (2012) 198–206, https://doi.org/ 10.7150/thno.3477. [10] J. Cazzulo, V. Stoka, V. Turk, The major cysteine proteinase of trypanosoma cruzi: a valid target for chemotherapy of chagas disease, Curr. Pharm. Des. 7 (2001) 1143–1156, https://doi.org/10.2174/1381612013397528. [11] G. Zhang, Protease Assays, (n.d.). https://www.ncbi.nlm.nih.gov/books/ NBK92006/pdf/Bookshelf_NBK92006.pdf. [12] I.L.H. Ong, K.-L. Yang, Recent developments in protease activity assays and sensors, Analyst 142 (2017) 1867–1881, https://doi.org/10.1039/C6AN02647H.
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