This work was funded by an NIH Director&#39;s New Innovator Award (Award No. DP2HD091793). Q.D.M. is supported by the NSF Graduate Research Fellowships Program (Grant No. DGE-1650044). B.A.H is supported by the National Institutes of Health GT BioMAT Training Grant under Award Number 5T32EB006343 as well as the Georgia Tech President&#39;s Fellowship. G.A.K. holds a Career Award at the Scientific Interface from the Burroughs Welcome Fund. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Institutional approval from Institutional Animal Care and Use Committee (IACUC) at the researcher&#39;s institution is necessary to carry out the following animal experiments. Additionally, standard animal care facilities (e.g., housing chambers, sterile animal hoods, isofluorane chambers for anesthetization, and CO2 chambers for ethical endpoint euthanization) are necessary to properly carry out these experiments. Special training and assistance with these facilities can be provided by the Physiological Research Laboratory (PRL) at one&#39;s institution. All animal work was approved by IACUC at Georgia Tech (protocol: A14100).
1. Iron Oxide Nanoparticle (IONP) Synthesis
NOTE: Safety: The entire Iron Oxide Nanoparticle synthesis should be performed using personal protective equipment and underneath a chemical fume hood.
<ol>
	<li>In a 15 mL conical tube, chill 1 mL of 30% ammonium hydroxide in an ice bucket for 30&#160;min.</li>
	<li>Wash a 250 mL Erlenmeyer flask with deionized water and add 10 mL double distilled water (ddH2O). Submerge the outside of the flask in an ice bath resting on a stir/heat plate. Use a plastic pipette to flow N2 gas into the ddH2O in the flask. Bubble for 15 minutes with N2 to deoxygenate.</li>
	<li>Weigh 224 &#181;mol (4.5 grams) of dextran (Molecular Weight (MW) = 20 kDa) into a 50 mL conical tube. Add H2O such that the final volume of the mixture, including the dextran, is 20 mL. Vortex vigorously to dissolve the dextran.</li>
	<li>Weigh 290 &#181;mol (78.5 grams) of iron(III) chloride hexahydrate, add to dextran solution and vortex to dissolve.</li>
	<li>Filter the resulting solution through a 0.2 &#181;m filter.</li>
	<li>Transfer 1 mL chilled ammonium hydroxide into 9 mL of the deoxygenated water. Return to 4&#160;&#176;C.</li>
	<li>Weigh 367 &#181;mol (73.0 grams) of iron(II) chloride tetrahydrate, dissolve in 1 mL of oxygen-free ddH2O, and filter through a 0.2 &#181;m filter.</li>
	<li>Transfer the dextran-iron(III) solution into the 250 mL Erlenmeyer flask and deoxygenate with N2 for 15 minutes on ice. Mix with a magnetic stir bar spinning at 1600 rpm.</li>
	<li>To create a homogenous nitrogen atmosphere, cap the flask with a rubber septum. Puncture the septum with an 18 gauge needle to flow N2 into the flask. Insert a separate 18 gauge needle as a flow outlet.</li>
	<li>Add 467 &#181;L of the iron(II) solution to the dextran-iron(III) solution with a 1 mL syringe, stirring with a magnetic stir bar at 1600 rpm. This ratio of iron(II) to iron(III) results in a balanced reaction that will produce magnetite, or Fe3O4.</li>
	<li>Add the chilled dilute ammonium hydroxide dropwise into the Iron(II)-Iron(III)-dextran solution to initiate the nucleation process21. Make sure each droplet mixes well before dropping in another. Finish the reaction after the addition of 1-2 mL of ammonium hydroxide.</li>
	<li>Stop the flow of N2 and remove the rubber septum. Remove the ice bath and replace with a bath of warm water, while continuing to stir the solution. Ensure that the temperature of the solution reaches 75&#160;&#176;C and incubate for 75 minutes.</li>
	<li>Remove the flask from the stir/hot plate and doubly filter the solution through 0.2 &#181;m then 0.1 &#181;m filters to remove coarse particles.</li>
	<li>Buffer exchange the particles into ddH2O, using 100 kDa molecular weight cut off (m.w.c.o.) concentrators to remove excess dextran from the solution, which should be very viscous. Replace filters if the flow through remains dark even after 2 - 3 spins, which indicates that the filter may have broken.
	<ol>
		<li>To buffer exchange with spin filters, centrifuge at 4&#160;&#176;C at 4800 X g for 15 minutes, discard the flow-through, and add new buffer to the IONP solution. Repeat 3-5 times.</li>
	</ol>
	</li>
	<li>Determine the concentration of the nanoparticles using a spectrophotometer/plate reader. Take absorbance measurements at 400 nm and use the molar absorptivity of IONP at this wavelength (&#949; = 2.07 x 106 cm-1 M-1) to determine the concentration of IO particles.</li>
	<li>Resuspend the IONPs to 10 mg/mL in ddH2O (usually total volume is ~3 mL) and make sure to use polypropylene tubes for this step for chemical compatibility. Add 1.6 volumes of 5 M NaOH, then add 0.65 volumes of epichlorohydrin. Rigorously mix on a plate shaker at room temperature for 12 hours to crosslink dextran.</li>
	<li>Use a 20 mL syringe with an 18-gauge needle to transfer the IONP solution into the 50 kDa m.w.c.o. dialysis membrane. Place the dialysis membrane into 4 L ddH2O and replace H2O a few times (every 2 - 3 hours). Incubate overnight.</li>
	<li>Measure the concentration and bring IONPs to 5 mg/mL (see step 1.15). Add ammonium hydroxide to reach 20% (v/v) and shake at room temperature for &#62; 12 hours to aminate the surface of IONPs. Buffer exchange using 30 kDa m.w.c.o. filters (see 1.14).</li>
	<li>Adjust the volume down to 2.5 - 3.5 mL before final purification by Fast Protein Liquid Chromatography (FPLC; see Table of Materials for gel filtration column).</li>
	<li>Use dynamic light scattering (DLS) to determine the hydrodynamic radius of IONPs (expected range 10 - 100 nm, average size 40 - 50 nm). Do this by aliquoting 1 mL of 100X diluted IONP sample in a cuvette, place in the machine, and use the manufacturer&#39;s software to take the measurement. 
	NOTE: The total population of IONPs will be between 10 and 100 nm by DLS measurements, but the majority of the population is around the average diameter, which ranges from 40 - 50 nm. If one wants to limit the size range, one can further use size exclusion chromatography to isolate fractions with different diameters. IONPs should be stored at 4&#160;&#176;C.</li>
</ol>
2. Peptide Design, Conjugation to IONP, and In Vitro Validation
<ol>
	<li>Synthesize a peptide substrate (e.g., by a core facility or commercially) for a target protease with an N-terminal fluorescent reporter such as Fluorescein isothiocyanate (FITC) and a C terminal cysteine residue to allow thiol-mediated coupling. 
	NOTE: In the case of this study for MMP9, the peptide used was FITC-PLGVRGK-C, with kcat/Km ~ 2.0 x 105 M-1s-1.</li>
	<li>Aliquot 0.5 mg of IONP (at 1 mg/mL), and buffer exchange into coupling buffer (50 mM Sodium Borate with 1 mM EDTA, pH = 8.5) using a 10 kDa m.w.c.o. spin filter (see step 1.14).</li>
	<li>Dissolve Succinimidyl iodoacetate (SIA) in dimethylformamide (DMF) to reach a concentration of ~30 mg/mL, and add SIA to IONP at a mole ratio of 500 SIA:IONP. 
	NOTE: SIA is a heterobifunctional cross linker that mediates coupling of amines on IONPs to the sulfhydryl groups on the peptide substrate.</li>
	<li>Incubate for 1 - 2 hours at room temperature in the dark. If leaving overnight, incubate at 4&#160;&#176;C.</li>
	<li>Buffer exchange using a 10 kDa m.w.c.o. spin filter into coupling buffer to remove unreacted SIA (see step 1.14).</li>
	<li>Bring the final product solution to 1 mg/mL (0.5 mL solution). Mix the peptide of interest at a molar ratio of 90:1 (peptide:IONP) with 20 kDa thiol-terminated polyethylene glycol (PEG) at a molar ratio of 20:1 (PEG:IONP). Mix this peptide-PEG solution with IONP.</li>
	<li>Incubate overnight at room temperature on a plate shaker on highest speed, covering tubes in foil to prevent photobleaching of fluorescent molecules.</li>
	<li>Add L-cysteine at a molar ratio of 500:1 (C:IONP) to neutralize any unreacted SIA molecules. Incubate for 1 hour at 25&#160;&#176;C on a shaker at highest speed at room temperature.</li>
	<li>Purify via FPLC (see 1.1.19). Use a spectrophotometer to measure the absorbance of the sample solution and calculate the Peptide:IONP ratio according to the following. After purification, store product at 1 mg/mL at 4&#160;&#176;C in PBS.
	<ol>
		<li>Use a spectrophotometer to measure the absorbance of the sample solution at 400 nm (Asample,400) and the wavelength of maximum absorbance for the fluorophore used, which is 488 nm for FITC (Asample,488). Measure the absorbance of a stock amine IONP solution at the same two wavelengths (400 nm and 488 nm), which are respectively represented as ANP,400 and ANP,488.</li>
		<li>Use equations 1 and 2 to calculate the normalized A400 and A488 values. 
		<img alt="Equation 1" src="/files/ftp_upload/57937/57937eq1.jpg" />&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;Equation 1 
		<img alt="Equation 2" src="/files/ftp_upload/57937/57937eq2.jpg" />&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;Equation 2</li>
		<li>A400 and A488 represent the normalized values of the sample. Use these values to calculate the ratio of Peptide:IONP with equation 3. 
		<img alt="Equation 3" src="/files/ftp_upload/57937/57937eq3.jpg" />&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;Equation 3</li>
		<li>Where <img alt="Equation 4" src="/files/ftp_upload/57937/57937eq4.jpg" /> and&#160;<img alt="Equation 5" src="/files/ftp_upload/57937/57937eq5.jpg" /> are the molar absorptivities of the IONP and the fluorophore, respectively. For these particles <img alt="Equation 4" src="/files/ftp_upload/57937/57937eq4.jpg" /> = 2.06 x 106 M-1cm-1 and for FITC <img alt="Equation 5" src="/files/ftp_upload/57937/57937eq5.jpg" /> = 72,000 M-1cm-1, so the value of&#160; <img alt="Equation 6" src="/files/ftp_upload/57937/57937eq6.jpg" /> should be 28.75. Typical Peptide:IONP ratios should be in the range of 20:1 to 50:1.</li>
	</ol>
	</li>
	<li>Validate functionality of the probes by performing an in vitro cleavage assay.
	<ol>
		<li>With PBS (1% BSA), make an 18 &#181;L nanoparticle solution with a 200 nM concentration of peptide. Mix with 2 &#181;L of protease of interest (MMP9; 0.1 - 1 mg/mL).</li>
		<li>Incubate in a plate reader at 37&#160;&#176;C for 1 hour, taking fluorescence measurements (using the appropriate excitation and emission wavelengths, which are 485 nm and 528 nm for FITC, respectively) every 1 - 2 minutes to monitor cleavage.</li>
	</ol>
	</li>
</ol>
3. Administration of Nanosensors and Urine Detection of Cancer
NOTE: For more details on the example model, see our previous report13.
<ol>
	<li>Create a metabolic cage for urine collection by securing a cylindrical sleeve to the top of a 96 well plate. During urine collection, place a mouse inside the sleeve and cover with a Petri dish cover to prevent escape of the animal.</li>
	<li>Prepare an injection solution (200 &#181;L maximum volume) containing nanosensors at a concentration of 3000 mg/kg (~50 &#181;mol) by peptide in sterile saline.</li>
	<li>In female, 8-week old, nude mice bearing xenograft LS174T colorectal tumors at a burden of ~100-300 mm3, which should occur on approximately day 10, administer nanosensor solution via tail vein injection. Immediately after injection, place mice into metabolic cages and note the time of injection for each mouse.</li>
	<li>At 60 - 90 minutes after injection, remove cylindrical sleeve from 96-well plate. Restrain mouse and apply slight pressure on the bladder to induce mice to void any remaining urine onto the plate. Collect all urine (200 - 500 &#181;L).</li>
	<li>Analyze urine samples by immunoprecipitation to purify FITC from the urine and increase sensitivity. Use magnetic beads coupled with anti-FITC antibodies.
	<ol>
		<li>First, wash 25 mg of the magnetic beads 3 times with coating buffer, and bring the final volume to 225 &#181;L. 
		NOTE: It is important to make the coating buffer (0.1 M sodium borate, pH 9.5), blocking buffer (PBS, 0.5% BSA, 0.05% Tween-20, pH 7.4), and washing buffer (PBS, 0.1% BSA, 0.05% Tween-20, pH 7.4) fresh each time, and approximately 20 mL of each is necessary.</li>
		<li>Add 200 &#181;L of anti-FITC (5 mg/mL) and 200 &#181;L of ammonium sulfate (3 M). Incubate for 16-24 hours at 37 C on a rotator.</li>
		<li>Add the solution to a magnetic separator, remove the supernatant, replace with 625 <img alt="Equation 7" src="/files/ftp_upload/57937/57937eq7.jpg" />&#923; blocking buffer, and incubate at 37 C overnight. Then wash 3 times with wash buffer and store in 1.25 mL wash buffer.</li>
		<li>Incubate 2 &#181;L of urine with 5 &#181;L of magnetic beads (20 mg/mL) and bring to a total volume to 50 &#181;L with PBS (0.01% Tween 20). Incubate for 60&#160;min.</li>
		<li>Wash twice with 50 &#181;L PBS (0.01% Tween 20) using a magnetic separator to collect magnetic beads after each wash.</li>
		<li>Elute twice with 32.5 &#181;L of 5% glacial Acetic Acid. Neutralize pooled elution (70 &#181;L) with 35 &#181;L of 2 M Tris to achieve a final pH of ~7.</li>
		<li>Read on a plate reader (see Table of Materials) at appropriate excitation and emission wavelengths to quantify urine fluorescence. Calculate the concentration of fluorescent reporter against a ladder of known concentrations of free fluorophore.</li>
	</ol>
	</li>
</ol>

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Dr. Kwong is co-founder and serves as consultant to Glympse Bio, which is developing products related to the research described in this paper. This study could affect his personal financial status. The terms of this arrangement have been reviewed and approved by Georgia Tech in accordance with its conflict of interest policies

This method describes the development of activity-based nanosensors consisting of protease substrates conjugated to a nanoparticle core. The event of proteolytic cleavage is dubbed the &#34;pharmacokinetic switch&#34;, because cleaved peptide products are smaller than the renal size filtration limit of 5 nm23&#160;and filter into urine to produce a noninvasive signal. Therefore, it is important to use nanoparticles or carriers with a hydrodynamic radius that is larger than 5 nm, as anything smalle...

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and have significant control over many biological processes, including homeostasis, allostasis, and disease1. An altered state of protease activity has been correlated to a variety of diseases, including cancer and cardiovascular disease, making proteases attractive candidates for development into clinical biomarkers2,3. Moreover, protease activity is functionally linked to distinct pathogeneses, patient outcomes, and prognosis of disease4. Broadly, biosensors have been developed to detect various biological phenomena and diseases, such as cancer, neurodegenerative disease, and electron transfer processes5,6,7,8,9. More specifically, substrate-based protease sensors have been developed to detect protease activity, and include fluorogenic probes for diagnostic imaging10 and isotopically labeled peptide substrates for in vitro detection by mass spectrometry11. In addition, activity-based probes have been developed, which contain substrate-like regions that bind or modify the target protease12. With this method, the target protease is irreversibly inhibited when the active site is modified, and analysis requires harvesting of tissue, which limits in vivo applications. However, it is important to sense protease activity in vivo, because regulation of protease activity is heavily dependent on the context of other biological activities such as the presence of endogenous inhibitors.
The goal of this work is to describe the formulation of activity-based nanosensors that detect protease activity in vivo by producing a measurable signal in urine. This platform is used as a noninvasive diagnostic to discriminate complex diseases such as cancer by using dysregulated protease activity as a functional biomarker. Our nanosensor platform consists of iron oxide nanoparticles (IONP) conjugated to protease substrates. These substrates are terminated by a fluorescent reporter which is released when proteases cleave the substrate. These IONPs circulate in vivo, localize to disease sites, and expose substrates to active disease-associated proteases. After cleavage, fluorescent reporters are released and, due to their small size, are filtered into urine, while uncleaved substrates on the IONP remain in the body. Therefore, an increase in protease activities in vivo will result in higher concentrations of reporter in urine (Figure 1). Since our platform is a urine test, no imaging platform is required and diagnostic signals are enriched in urine.
This platform can be engineered to detect a variety of diseases including cancer, fibrosis, and thrombosis13,14. Here we describe the design of nanosensors to detect elevations in Matrix metallopeptidase 9 (MMP9) activity as a biomarker of colorectal cancer. Colorectal cancer is the second leading cause of cancer death in the United States, with an estimated 136,800 new cases and 50,300 deaths in 2014 alone15. Colorectal tumor cells produce MMP9, which has been shown to drive malignant progression , matrix degradation, as well as metastasis16. Additionally, we identified a suitable peptide substrate (PLGVRGK) for MMP9 from the literature17. This platform may be used for early cancer detection and low-cost point-of-care diagnostics13,14,18,19,20,21.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57937/57937fig1.jpg" /> 
Figure 1: Schematic of Nanosensor Activity In vivo. Nanosensors circulate through the body and localize to sites of disease. Then, disease-related proteases cleave peptide substrates presented by IONPs. The size of cleaved fragments allows for renal clearance, causing them to localize in the urine. After the animal urinates, these peptide fragments can be analyzed by their reporter molecule. <a href="https://www.jove.com/files/ftp_upload/57937/57937fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>0.2 &amp;micro;m syringe filters</td><td>VWR</td><td>4652</td><td></td></tr><tr><td>18G needle</td><td>VWR</td><td>89134-024</td><td></td></tr><tr><td>15 mL conicals</td><td>VWR</td><td>89039-670</td><td></td></tr><tr><td>250 mL Erlenmeyer flask</td><td>VWR</td><td>89000-362</td><td></td></tr><tr><td>Stir bar</td><td>VWR</td><td>58949-006</td><td></td></tr><tr><td>Hot Plate/Magnetic Stirrer</td><td>VWR</td><td>97042-634</td><td></td></tr><tr><td>Glacial acetic acid</td><td>VWR</td><td>97064-482</td><td></td></tr><tr><td>Albumin from Bovine Serum (BSA)</td><td>Thermo Fisher</td><td>A13100</td><td></td></tr><tr><td>Iron (III) chloride hexahydrate</td><td>Sigma</td><td>236489</td><td></td></tr><tr><td>Iron (II) chloride tetrahydrate</td><td>Sigma</td><td>44939</td><td></td></tr><tr><td>Epichlorohydrin</td><td>Sigma</td><td>45340-500ML-F</td><td></td></tr><tr><td>DMF</td><td>Sigma</td><td>D4551</td><td></td></tr><tr><td>Ammonium Hydroxide</td><td>Sigma</td><td>320145-500ML</td><td></td></tr><tr><td>Sodium Hydroxide pellets</td><td>Sigma</td><td>221465-500G</td><td></td></tr><tr><td>EDTA</td><td>Sigma</td><td>E9884</td><td></td></tr><tr><td>Sodium Borate</td><td>Sigma</td><td>B9876</td><td></td></tr><tr><td>L-Cysteine</td><td>Sigma</td><td>168149-100G</td><td></td></tr><tr><td>Tris-HCl</td><td>Sigma</td><td>T5941</td><td></td></tr><tr><td>Tris base</td><td>Sigma</td><td>T6066</td><td></td></tr><tr><td>PBS tablets</td><td>Sigma</td><td>P4417</td><td></td></tr><tr><td>Dextran</td><td>Pharmacosmos</td><td>5510 0020 9006</td><td></td></tr><tr><td>Amicon 15 mL 10k filters, 24 pk</td><td>Millipore</td><td>UFC901024</td><td></td></tr><tr><td>Amicon 15 mL 30k filters, 24 pk</td><td>Millipore</td><td>UFC903024</td><td></td></tr><tr><td>Amicon 15 mL 100k filters, 24 pk</td><td>Millipore</td><td>UFC910024</td><td></td></tr><tr><td>Zetasizer Nano ZS</td><td>Malvern Panalytical</td><td>NanoZS</td><td></td></tr><tr><td>Slide-A-Lyzer Dialysis Cassette</td><td>LifeTech</td><td>66130</td><td></td></tr><tr><td>Dynabeads MyOne Tosylactivated</td><td>LifeTech</td><td>65501</td><td></td></tr><tr><td>SIA</td><td>Life Tech</td><td>22349</td><td></td></tr><tr><td>PEG 20k</td><td>Laysan Bio</td><td>MPEG-SH-20K-1g</td><td></td></tr><tr><td>Fluorescein Antibody [2A3]</td><td>GeneTex</td><td>GTX10257</td><td></td></tr><tr><td>Hiload 16/600 superdex 200</td><td>GE Healthcare</td><td>45-002-490</td><td></td></tr><tr><td>Plate Reader</td><td>Fisher</td><td>BTCYT5M</td><td></td></tr><tr><td>BD Insulin Syringes</td><td>Fisher</td><td>NC0872854</td><td></td></tr></tbody></table>

nanosensors to detect protease activity in vivo for noninvasive diagnostics

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and allostasis. Moreover, dysregulated protease activity drives pathogenesis and is a functional biomarker of diseases such as cancer; therefore, the ability to detect protease activity in vivo may provide clinically relevant information for biomedical diagnostics. The goal of this protocol is to create nanosensors that probe for protease activity in vivo by producing a quantifiable signal in urine. These protease nanosensors consist of two components: a nanoparticle and substrate. The nanoparticle functions to increase circulation half-life and substrate delivery to target disease sites. The substrate is a short peptide sequence (6-8 AA), which is designed to be specific to a target protease or group of proteases. The substrate is conjugated to the surface of the nanoparticle and is terminated by a reporter, such as a fluorescent marker, for detection. As dysregulated proteases cleave the peptide substrate, the reporter is filtered into urine for quantification as a biomarker of protease activity. Herein we describe construction of a nanosensor for matrix metalloproteinase 9 (MMP9), which is associated with tumor progression and metastasis, for detection of colorectal cancer in a mouse model.

The majority of the population of the IONPs is around the average diameter, which ranges from 40 - 50 nm. After pegylation, this size range has a circulation half-life of approximately 6 hours13&#160;in vivo (see Figure 2a). If one wants to select for a particular size range, one can use size exclusion chromatography to isolate IONP fractions with different diameters. The nanoparticles by TEM will appear as individual spherica...

Watch this Scientific Journal Video about Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics at JoVE.com

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are tightly regulated enzymes involved in fundamental biological processes, and dysregulated protease activity drives progression of complex diseases such as cancer. This method&#39;s goal is to create nanosensors that measure protease activity in vivo by producing a cleavage signal that is detectable from host urine and discriminates disease.

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and ...

nanosensors-to-detect-protease-activity-vivo-for-noninvasive

Research

JoVE Journal

Bioengineering

16.2K Views.  Georgia Tech College of Engineering and Emory School of Medicine. Proteasen zijn strak gereguleerde enzymen die betrokken zijn bij de fundamentele biologische processen en dysregulated protease-activiteit stations progressie van complexe ziekten zoals kanker. Doel van deze methode is het creëren van nanosensors die door het produceren van een decollete-signaal dat is aantoonbaar uit host urine en discrimineert ziekte protease activiteit in vivo meten.

Video: Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Detection of Protease Activity by Fluorescent Peptide Zymography

The purpose of this method is to measure the proteolytic activity of complex biological samples. The samples are separated by molecular weight using electrophoresis through a resolving gel embedded with a degradable substrate. This method differs from traditional gel zymography in that a quenched fluorogenic peptide is covalently incorporated into the resolving gel instead of full length proteins, such as gelatin or casein. Use of the fluorogenic peptides enables direct detection of proteolytic activity without additional staining steps. Enzymes within the biological samples cleave the quenched fluorogenic peptide, resulting in an increase in fluorescence. The fluorescent signal in the gels is then imaged with a standard fluorescent gel scanner and quantified using densitometry. The use of peptides as the degradable substrate greatly expands the possible proteases detectable with zymographic techniques.

Here, we present a detailed protocol for a modified zymographic technique in which fluorescent peptides are used as the degradable substrate in place of native proteins. Electrophoresis of biological samples in fluorescent peptide zymograms enables detection of a wider range of proteases than previous zymographic techniques.

The purpose of this method is to measure the proteolytic activity of complex biological samples. The samples are separated by molecular weight using ...

Cancer Research

Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, they are important drug targets. We have developed a magnetic-agarose-bead-based assay platform for the investigation of proteolytic activity, which is based on the use of recombinant fusion protein substrates. In order to demonstrate the use of this assay system, a protocol is presented on the example of human immunodeficiency virus type 1 (HIV-1) protease. The introduced assay platform can be utilized efficiently in the biochemical characterization of proteases, including enzyme activity measurements in mutagenesis, kinetic, inhibition, or specificity studies, and it may be suitable for high-throughput substrate screening or may be adapted to other proteolytic enzymes.
In this assay system, the applied substrates contain N-terminal hexahistidine (His6) and maltose-binding protein (MBP) tags, cleavage sites for tobacco etch virus (TEV) and HIV-1 proteases, and a C-terminal fluorescent protein. The substrates can be efficiently produced in Escherichia coli cells and easily purified using nickel (Ni)-chelate-coated beads. During the assay, the proteolytic cleavage of bead-attached substrates leads to the release of fluorescent cleavage fragments, which can be measured by fluorimetry. Additionally, cleavage reactions can be analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A protocol for the in-gel renaturation of assay components is also described, as partial renaturation of fluorescent proteins enables their detection based on molecular weight and fluorescence.

Here, we present the detailed procedure of a recently developed protease assay platform utilizing N-terminal hexahistidine/maltose-binding protein and fluorescent protein-fused recombinant substrates attached to the surface of nickel-nitrilotriacetic acid magnetic agarose beads. A subsequent in-gel analysis of the assay samples separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is also presented.

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, ...

Biochemistry

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. Nucleases display a variety of vital physiological roles in prokaryotic and eukaryotic organisms, ranging from maintaining genome stability to providing protection against pathogens. Altered nuclease activity has been associated with several pathological conditions including bacterial infections and cancer. To this end, nuclease activity has shown great potential to be exploited as a specific biomarker. However, a robust and reproducible screening method based on this activity remains highly desirable.
Herein, we introduce a method that enables screening for nuclease activity using nucleic acid probes as substrates, with the scope of differentiating between pathological and healthy conditions. This method offers the possibility of designing new probe libraries, with increasing specificity, in an iterative manner. Thus, multiple rounds of screening are necessary to refine the probes&#39; design with enhanced features, taking advantage of the availability of chemically modified nucleic acids. The considerable potential of the proposed technology lies in its flexibility, high reproducibility, and versatility for the screening of nuclease activity associated with disease conditions. It is expected that this technology will allow the development of promising diagnostic tools with a great potential in the clinic.

Altered nuclease activity has been associated with different human conditions, underlying its potential as a biomarker. The modular and easy to implement screening methodology presented in this paper allows the selection of specific nucleic acid probes for harnessing nuclease activity as a biomarker of disease.

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. ...

Use of the Protease Fluorescent Detection Kit to Determine Protease Activity

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect protease activity uses casein labeled with fluorescein isothiocyanate (FITC) as the substrate.
Protease activity results in the cleavage of the FITC-labeled casein substrate into smaller fragments, which do not precipitate under acidic conditions. After incubation of the protease sample and substrate, the reaction is acidified with the addition of trichloroacetic acid (TCA). The mixture is then centrifuged with the undigested substrate forming a pellet and the smaller, acid soluble fragments remaining in solution. The supernatant is neutralized and the fluorescence of the FITC-labeled fragments is measured.
The described kit procedure detects the trypsin protease control at a concentration of approximately 0.5 &mu;g/ml (5 ng of trypsin added to the assay). This sensitivity can be increased with a longer incubation time, up to 24 hours. The assay is performed in microcentrifuge tubes and procedures are provided for fluorescence detection using either cuvettes or multiwell plates.

The Protease Fluorescent Detection Kit is designed for the measurement of protease activity using fluorometry. It is also suitable for detection of trace amounts of protease contamination. The method is based on the proteolytic hydroysis of a proprietary formulation of a FITC-labeled casein substrate.

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect ...

Biology

Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform for reduced sample volume and assay time as well as full automation are required for potential use in point-of-care-diagnostics. Recently, we have demonstrated ultrasensitive detection of serum proteins, C-reactive protein (CRP) and cardiac troponin I (cTnI), utilizing a lab-on-a-disc composed of TiO2 nanofibrous (NF) mats. It showed a large dynamic range with femto molar (fM) detection sensitivity, from a small volume of whole blood in 30 min. The device consists of several components for blood separation, metering, mixing, and washing that are automated for improved sensitivity from low sample volumes. Here, in the video demonstration, we show the experimental protocols and know-how for the fabrication of NFs as well as the disc, their integration and the operation in the following order: processes for preparing TiO2 NF mat; transfer-printing of TiO2 NF mat onto the disc; surface modification for immune-reactions, disc assembly and operation; on-disc detection and representative results for immunoassay. Use of this device enables multiplexed analysis with minimal consumption of samples and reagents. Given the advantages, the device should find use in a wide variety of applications, and prove beneficial in facilitating the analysis of low abundant proteins.

This protocol demonstrates how to achieve femto molar detection sensitivity of proteins in 10 &#181;L of whole blood within 30 min. This can be achieved by using electrospun nanofibrous mats integrated in a lab-on-a-disc, which offers high surface area as well as effective mixing and washing for enhanced signal-to-noise ratio.

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform ...

Time-Resolved In Vivo Measurement of Neuropeptide Dynamics by Capacitive Immunoprobe in Porcine Heart

The ability to measure biomarkers in vivo relevant to the assessment of disease progression is of great interest to the scientific and medical communities. The resolution of results obtained from current methods of measuring certain biomarkers can take several days or weeks to obtain, as they can be limited in resolution both spatially and temporally (e.g., fluid compartment microdialysis of interstitial fluid analyzed by enzyme-linked immunosorbent assay [ELISA], high-performance liquid chromatography [HPLC], or mass spectrometry); thus, their guidance of timely diagnosis and treatment is disrupted. In the present study, a unique technique for detecting and measuring peptide transmitters in vivo through the use of a capacitive immunoprobe biosensor (CI probe) is reported. The fabrication protocol and in vitro characterization of these probes are described. Measurements of sympathetic stimulation-evoked neuropeptide Y (NPY) release in vivo are provided. NPY release is correlated to the sympathetic release of norepinephrine for reference. The data demonstrate an approach for the fast and localized measurement of neuropeptides in vivo. Future applications include intraoperative real-time assessment of disease progression and minimally invasive catheter-based deployment of these probes.

Time-Resolved In Vivo Measurement of Neuropeptide Dynamics by Capacitive Immunoprobe in Porcine Heart

Established immunochemical methods to measure peptide transmitters in vivo rely on microdialysis or bulk fluid draw to obtain the sample for offline analysis. However, these suffer from spatiotemporal limitations. The present protocol describes the fabrication and application of a capacitive immunoprobe biosensor that overcomes the limitations of the existing techniques.

The ability to measure biomarkers in vivo relevant to the assessment of disease progression is of great interest to the scientific and medical ...

Monitoring Neutrophil Elastase and Cathepsin G Activity in Human Sputum Samples

Proteases are regulators of countless physiological processes and the precise investigation of their activities remains an intriguing biomedical challenge. Among the ~600 proteases encoded by the human genome, neutrophil serine proteases (NSPs) are thoroughly investigated for their involvement in the onset and progression of inflammatory conditions including respiratory diseases. Uniquely, secreted NSPs not only diffuse within extracellular fluids but also localize to plasma membranes. During neutrophil extracellular trap (NETs) formation, NSPs become an integral part of the secreted chromatin. Such complex behavior renders the understanding of NSPs pathophysiology a challenging task. Here, detailed protocols are shown to visualize, quantify and discriminate free and membrane-bound neutrophil elastase (NE) and cathepsin G (CG) activities in sputum samples. NE and CG are NSPs whose activities have pleiotropic roles in the pathogenesis of cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD): they promote tissue remodeling, regulate downstream immune responses and correlate with lung disease severity. The protocols show how to separate fluid and cellular fraction, as well as the isolation of neutrophils from human sputum for enzymatic activity quantification via small-molecule F&#246;rster resonance energy transfer-based (FRET) reporters. To gather specific insights into the relative role of NE and CG activities, a FRET readout can be measured by different technologies: i) in vitro plate reader measurements allow for high-throughput and bulk detection of protease activity; ii) confocal microscopy spatiotemporally resolves membrane-bound activity at the cell surface; iii) small-molecule FRET flow cytometry enables for the rapid evaluation of anti-inflammatory treatments via single-cell protease activity quantification and phenotyping. The implementation of such methods opens the doors to explore NSPs pathobiology and their potential as biomarkers of disease severity for CF and COPD. Given their standardization potential, their robust readout and simplicity of transfer, the described techniques are immediately shareable for implementation across research and diagnostic laboratories.

The protocols herein described provide a guide to visualize and quantify the activity of neutrophil proteases in human sputum. The applications of such analysis span from the evaluation of anti-inflammatory treatments, to biomarker validation, drug screening and large cohort clinical studies.

Proteases are regulators of countless physiological processes and the precise investigation of their activities remains an intriguing biomedical ...

Immunology and Infection

CRISPR-Cas Synthetic Urine Biomarker Test for Cancer Diagnostics

CRISPR-Cas-mediated Multianalyte Synthetic Urine Biomarker Test for Portable Diagnostics

Creating synthetic biomarkers for the development of precision diagnostics has enabled detection of disease through pathways beyond those used for traditional biofluid measurements. Synthetic biomarkers generally make use of reporters that provide readable signals in the biofluid to reflect the biochemical alterations in the local disease microenvironment during disease incidence and progression. The pharmacokinetic concentration of the reporters and biochemical amplification of the disease signal are paramount to achieving high sensitivity and specificity in a diagnostic test. Here, a cancer diagnostic platform is built using one format of synthetic biomarkers: activity-based nanosensors carrying chemically stabilized DNA reporters that can be liberated by aberrant proteolytic signatures in the tumor microenvironment. Synthetic DNA as a disease reporter affords multiplexing capability through its use as a barcode, allowing for the readout of multiple proteolytic signatures at once. DNA reporters released into the urine are detected using CRISPR nucleases via hybridization with CRISPR RNAs, which in turn produce a fluorescent or colorimetric signal upon enzyme activation. In this protocol, DNA-barcoded, activity-based nanosensors are constructed and their application is exemplified in a preclinical mouse model of metastatic colorectal cancer. This system is highly modifiable according to disease biology and generates multiple disease signals simultaneously, affording a comprehensive understanding of the disease characteristics through a minimally invasive process requiring only nanosensor administration, urine collection, and a paper test which enables point-of-care diagnostics.

Author Spotlight: Engineering Molecular Tools for Disease Detection and Imaging

This protocol describes a CRISPR-Cas-mediated, multianalyte synthetic urine biomarker test that enables point-of-care cancer diagnostics through the ex vivo analysis of tumor-associated protease activities.

Creating synthetic biomarkers for the development of precision diagnostics has enabled detection of disease through pathways beyond those used for ...

Use of Enzymatic Biosensors to Quantify Endogenous ATP or H2O2 in the Kidney

Enzymatic microelectrode biosensors have been widely used to measure extracellular signaling in real-time. Most of their use has been limited to brain slices and neuronal cell cultures. Recently, this technology has been applied to the whole organs. Advances in sensor design have made &#160;possible the measuring of&#160;cell signaling in blood-perfused in vivo kidneys. The present protocols list the steps needed to measure ATP and H2O2 signaling in the rat kidney interstitium. Two separate sensor designs are used for the ex vivo and in vivo protocols. Both types of sensor&#160;are coated with&#160;a thin enzymatic biolayer on top of a permselectivity layer to give fast responding, sensitive and selective biosensors. The permselectivity layer protects the signal from the interferents in biological tissue, and the enzymatic layer utilizes&#160;the sequential catalytic reaction of glycerol kinase and glycerol-3-phosphate oxidase in the presence of ATP to produce H2O2. The set of sensors used for the ex vivo studies further detected analyte by oxidation of H2O2 on a platinum/iridium (Pt-Ir) wire electrode. The sensors for the&#160;in vivo studies are instead based on the reduction of H2O2 on a mediator coated gold electrode designed for blood-perfused tissue. Final concentration changes&#160;are detected by real-time amperometry followed by calibration to known concentrations of analyte. Additionally, the specificity of the amperometric signal can be confirmed by the addition of enzymes such as catalase and apyrase that break down H2O2 and ATP correspondingly. These sensors also rely heavily on accurate calibrations before and after each experiment. The following two protocols establish the study of real-time detection of ATP and H2O2 in kidney tissues, and can be further modified to extend the described method for use in other biological preparations or whole organs.

Use of Enzymatic Biosensors to Quantify Endogenous ATP or H2O2 in the Kidney

Enzymatic microelectrode biosensors enable real-time measurements of extracellular cell signaling in biologically-relevant concentrations. The following protocols extend the applications of biosensors to the ex vivo and in vivo detection of ATP and H2O2 in the kidney.

Enzymatic microelectrode biosensors have been widely used to measure extracellular signaling in real-time. Most of their use has been limited to brain ...

A Fluorogenic Peptide Cleavage Assay to Screen the Proteolytic Activity of Proteases

Source: Jaimes, J. A., et al. <a href="https://app.jove.com/v/58892">A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation.</a> J. Vis. Exp. (2019).
This video demonstrates an assay to screen for the proteolytic activity of proteases using fluorogenic peptides. The protease recognizes its cleavage site on the peptide, cleaving it and separating the quencher from the fluorophore, enabling its fluorescence emission. The fluorescence signal is detected and analyzed to check for the cleavage efficiency of different peptide variants.

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

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