This study was supported in part by a Koch Institute Support Grant number P30-CA14051 from the National Cancer Institute (Swanson Biotechnology Center), a Core Center Grant P30-ES002109 from the National Institute of Environmental Health Sciences, the Koch Institute&#39;s Marble Center for Cancer Nanomedicine, the Koch Institute Frontier Research Program via the Kathy and Curt Marble Cancer Research Fund, and the Virginia and D. K. Ludwig Fund for Cancer Research. A.E.V.H. is supported by an NIH-funded predoctoral training fellowship (T32GM130546). S.N.B. is a Howard Hughes Medical Institute Investigator. L.H. is supported by a K99/R00 Pathway to Independence Award from the National Cancer Institute and the startup funding from Boston University.

All animal studies are approved by the Institutional Animal Care and Use Committee (IACUC) at the authors&#39; institution. Standard animal care facilities including housing chambers, sterile hoods, anesthetization, and ethical endpoint euthanization are required to properly carry out these experiments. All experiments are conducted in compliance with institutional and national guidelines and supervised by the veterinarian staff at the institution. Female BALB/c mice, used for the experiments, are obtained from a commercial source (see Table of Materials) and ranged in age from 6 to 8 weeks at the start of the study. Sequences for custom-synthesized DNA, crRNA, FRET-based peptide substrate probes, and sensor peptides are provided in Supplementary Table 1.
1. Protease-activated peptide substrate selection
<ol>
	<li>Collect and prepare tissue samples from healthy lung or lung tissue with tumor nodules following a previously published report13.
	<ol>
		<li>Homogenize tissues in pre-chilled phosphate buffered saline (PBS, pH 7.4) (200 mg tissue/mL PBS) with tissue dissociation tubes for the automated dissociation of tissues into single-cell suspensions.</li>
		<li>Centrifuge tissue homogenates at 6,000 x g for 5 min at 4 &#176;C. Retain the supernatant in a new tube.</li>
		<li>Centrifuge the supernatant at 14,000 x g for 25 min at 4 &#176;C.</li>
		<li>Measure the protein concentration using bicinchoninic acid (BCA) protein assay kit (see Table of Materials).</li>
		<li>Add PBS to the sample to prepare a 0.33 mg/mL solution.</li>
	</ol>
	</li>
	<li>Assess the proteolytic activity following the steps below.
	<ol>
		<li>In a 384-well plate, add 5 &#181;L, 6 &#181;M FRET-based peptide substrate probes to each well. For each probe, perform the reaction in triplicate. 
		NOTE: FRET-based peptide substrate probe contains a short peptide sequence (6-8 amino acids, designed to be specific to a target protease or group of proteases) terminated with a fluorophore and quencher pair. Two FRET probes are described in Supplementary Table 1 as an example. The full library of probes can be found in Hao et al.13.</li>
		<li>Centrifuge the well plate for 10 s at room temperature at 180 x g to ensure the probes are at the bottom of the plate.</li>
		<li>Add 25 &#181;L 0.33 mg/mL tissue sample or 40 &#181;M recombinant protease13 to each well. 
		NOTE: The final concentration of the tissue sample or recombinant protease may need to be optimized according to their intrinsic proteolytic activity. For instance, highly proteolytic tissues such as intestines may require higher dilution factors to allow for the monitoring of initial enzymatic cleavage.</li>
		<li>Centrifuge the well plate briefly at 180 x g (at room temperature) to mix the probes and tissue lysates.</li>
		<li>Immediately begin detecting probe cleavage by measuring fluorescence with the plate reader at 37 &#176;C every 2 min for 1 h (&#955;ex: 485 nm; &#955;em: 535 nm) to monitor the cleavage. 
		NOTE: Use the appropriate excitation and emission wavelengths for specific fluorophore and quencher pairs. In the case of this study, parameters (&#955;ex: 485 nm and &#955;em: 535 nm) are set for the FAM fluorophore.</li>
		<li>To analyze the fluorescent measurement data, utilize the Python (see Table of Materials) package for enzyme kinetics analysis available at https://github.com/nharzallah/NNanotech-Kinetic. This script calculates the initial reaction velocity (V0) using the slope of the linear fit of the first 8-10 initial time points.</li>
	</ol>
	</li>
</ol>
2. Sensor formulation and characterization
<ol>
	<li>Synthesize the conjugate of DNA and protease-activated peptide (PAP).
	<ol>
		<li>Incubate 20-mer phosphorothioated DNA reporters with 3&#39;-DBCO group (1.1 eq., see Table of Materials) with azide-terminated PAPs with C-terminus cysteine end in 100 mM phosphate buffer (pH 7.0) at room temperature for &#62;4 h. If leaving overnight, incubate at 4 &#176;C.</li>
		<li>Purify the product on a high-performance liquid chromatography (HPLC) system equipped with a column ideal for biomolecules (see Table of Materials). Set the HPLC gradient to start from 5% A buffer (0.05% TFA in H2O), keep isocratic for 20 min, and reach 80% B buffer (0.05% TFA, 99.95% acetonitrile) at 65 min with a flow rate of 0.3 mL min-1, and collect the conjugate product with retention time at ~31 min.</li>
		<li>Validate the purified conjugates by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry using &#945;-cyano-4-hydroxycinnamic acid as matrix14(see Table of Materials).</li>
	</ol>
	</li>
	<li>Synthesize the DNA-encoded activity-based nanosensors.
	<ol>
		<li>Dissolve 2 mg of multivalent PEG (40 kDa, 8-arm, see Table of Materials) with maleimide-reactive group in 1 mL of 100 mM phosphate buffer (pH 7.0) and filter (cutoff: 0.2 &#181;m).</li>
		<li>Add the cysteine-terminated DNA-peptide conjugates (2 eq., see Table of Materials) to the PEG and react at room temperature for &#62;4 h. If leaving overnight, incubate at 4 &#176;C.</li>
		<li>Remove the unconjugated materials using size exclusion chromatography with a commercially available dextran-agarose composite matrix column on a fast protein liquid chromatography (FPLC) (see Table of Materials). Run samples in PBS and monitor absorbance at 260 nm for DNA and 280 nm for peptide.</li>
		<li>Concentrate the synthesized nanosensors with centrifugal filter tubes (MWCO = 10 kDa) according to the manufacturer&#39;s recommended speed (see Table of Materials).</li>
		<li>Quantify the concentration of DNA using ssDNA assay kit (see Table of Materials) and a plate reader at &#955;ex: 485 nm and &#955;em: 535 nm. Store the nanosensors at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Characterize the DNA-encoded activity-based nanosensors.
	<ol>
		<li>Measure the hydrodynamic particle size of DNA-encoded nanosensors by dynamic light scattering (DLS). 
		NOTE: The expected size range of nanosensors is 15-50 nm, with an average size of 20-30 nm. If a limited size range is desired, FPLC (in step 2.3) can be used to isolate narrower fractions with different molecular weights.</li>
		<li>Concentrate the nanosensors to 0.5 mg/mL (by DNA concentration) with centrifugal filter tubes (MWCO = 10 kDa) and load the samples onto a carbon film-coated copper grid mounted on a cryoholder. Observe the morphology using cryogenic transmission electron microscopy (200 kV, magnification of 10,000-60,000)14.</li>
	</ol>
	</li>
</ol>
3. Sensor injection and urine collection
<ol>
	<li>Collect urine for baseline measurement.
	<ol>
		<li>Place the mouse into a custom housing chamber (see Supplementary Figure 1) with a 96-well plate as the base.</li>
		<li>Restrain the mouse and apply gentle pressure on the bladder to void any remaining urine onto the plate.</li>
		<li>Pipette the collected urine (~100-200 &#181;L) from the 96-well plate into a 1.5 mL tube after replacing the mouse to the normal housing.</li>
	</ol>
	</li>
	<li>Establish preclinical murine tumor model.
	<ol>
		<li>Inoculate 6 to 8-week-old BALB/c female mice by intravenous injection with luciferase-expressing MC26-Fluc cell line (100k cells/mouse) (see Table of Materials). Monitor tumor progression weekly using an in vivo fluorescence imaging system. 
		NOTE: Visible tumor burden indicated by luminescent signal occurs approximately in week 2 of injection of this particular cell line. Carefully check on tumor-bearing animals during the tumor progression on a regular basis.</li>
	</ol>
	</li>
	<li>Inject nanosensors at different time points after tumor implantation.
	<ol>
		<li>Prepare an injection solution (200 &#181;L maximum volume) containing nanosensors at a concentration of 1 nmol by DNA barcode in sterile PBS.</li>
		<li>Inject 200 &#181;L sensor solution in PBS into each experimental mouse intravenously.</li>
	</ol>
	</li>
	<li>Collect urine samples from healthy control and tumor-bearing mice at 1 h after sensor injection as described in steps 1.1-1.3. 
	&#8203;NOTE: Fresh urine samples can be processed to DNA barcode analysis directly or frozen immediately on ice.</li>
</ol>
4. CRISPR detection of DNA barcodes: fluorescence-based
<ol>
	<li>Use fresh urine samples or defrost&#160;frozen samples on ice. Centrifuge urine samples at 800 x g for 5 min at room temperature.</li>
	<li>Combine the reagents in Supplementary Table 2, add the Cas12a enzyme (see Table of Materials) last and gently mix the reaction by pipetting up and down. Incubate the reaction at 37 &#176;C for 30 min.</li>
	<li>Run the reporter reaction, as shown in Supplementary Table 3, in triplicate using the product of step 2. Add the reaction from Step 2 last and quickly bring to the plate reader.</li>
	<li>Detect LbaCas12a activation by measuring fluorescence with a plate reader at 37 &#176;C every 2 min for 3 h (&#955;ex: 485 nm and &#955;em: 535 nm) to monitor the cleavage kinetics of the DNA reporter.</li>
	<li>To analyze the fluorescent measurement data, utilize the Python package for enzyme kinetics analysis available at https://github.com/nharzallah/NNanotech-Kinetic. This script calculates the initial reaction velocity (V0) using the slope of the linear fit of the first 8-10 initial time points.</li>
</ol>
5. CRISPR detection of DNA barcodes: paper-based
<ol>
	<li>Centrifuge urine samples at 800 x g for 5 min at room temperature. 
	NOTE: Run the urine samples for fluorescence-based and paper-based CRISPR detection in parallel.</li>
	<li>Combine the reagents in Supplementary Table 2. Incubate at 37 &#176;C for 30 min. 
	NOTE: This incubation step is identical to that for the fluorescence-based CRISPR detection.</li>
	<li>Run the reporter reaction using FAM-biotin labeled DNA reporter for lateral flow assay on a paper strip (see Table of Materials). Combine the reagents in Supplementary Table 4 in a 96-well plate using the product of step 2. Cover with aluminum foil and incubate at 37 &#176;C for 1 h. 
	NOTE: Select the optimal incubation time according to the real-time kinetics monitoring in the fluorescence-based CRISPR detection assay described above.</li>
	<li>To a fresh well of a 96-well plate, add 80 &#181;L of PBS. Add 20 &#181;L of sample from step 3 to this well.</li>
	<li>Place one lateral flow paper strip to each well and wait until the liquid reaches the top of the strip (&#60;5 min). Look for the appearance of the control and/or sample band(s) on the paper strip.</li>
	<li>Take a picture of the lateral flow strip and quantify band intensity using ImageJ.</li>
</ol>

CRISPR-Cas Synthetic Urine Biomarker Test for Cancer Diagnostics

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	<li>Soleimany, A. P., Bhatia, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-based+diagnostics:+an+emerging+paradigm+for+disease+detection+and+monitoring.">Activity-based diagnostics: an emerging paradigm for disease detection and monitoring.</a> Trends Mol Med. 26 (5), 450-468 (2020).</li><li>Muir, R. K., Guerra, M., Bogyo, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-based+diagnostics:+recent+advances+in+the+development+of+probes+for+use+with+diverse+detection+modalities.">Activity-based diagnostics: recent advances in the development of probes for use with diverse detection modalities.</a> ACS Chem Biol. 17 (2), 281-291 (2022).</li><li>Heitzer, E., Haque, I. S., Roberts, C. E. S., Speicher, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+and+future+perspectives+of+liquid+biopsies+in+genomics-driven+oncology.">Current and future perspectives of liquid biopsies in genomics-driven oncology.</a> Nat Rev Genet. 20 (2), 71-88 (2019).</li><li>Dudani, J. S., Warren, A. D., Bhatia, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+protease+activity+to+improve+cancer+care.">Harnessing protease activity to improve cancer care.</a> Annu Rev Canc Biol. 2, 353-376 (2018).</li><li>Hanahan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallmarks+of+cancer:+new+dimensions.">Hallmarks of cancer: new dimensions.</a> Cancer Discov. 12 (1), 31-46 (2022).</li><li>Quail, D. F., Joyce, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microenvironmental+regulation+of+tumor+progression+and+metastasis.">Microenvironmental regulation of tumor progression and metastasis.</a> Nat Med. 19 (11), 1423-1437 (2013).</li><li>Kwong, G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+biomarkers:+a+twenty-first+century+path+to+early+cancer+detection.">Synthetic biomarkers: a twenty-first century path to early cancer detection.</a> Nat Rev Cancer. 21 (10), 655-668 (2021).</li><li>Kirkpatrick, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urinary+detection+of+lung+cancer+in+mice+via+noninvasive+pulmonary+protease+profiling.">Urinary detection of lung cancer in mice via noninvasive pulmonary protease profiling.</a> Sci Transl Med. 12 (537), eaaw0262 (2020).</li><li>Warren, A. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disease+detection+by+ultrasensitive+quantification+of+microdosed+synthetic+urinary+biomarkers.">Disease detection by ultrasensitive quantification of microdosed synthetic urinary biomarkers.</a> J Am Chem Soc. 136 (39), 13709-13714 (2014).</li><li>Kwong, G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass-encoded+synthetic+biomarkers+for+multiplexed+urinary+monitoring+of+disease.">Mass-encoded synthetic biomarkers for multiplexed urinary monitoring of disease.</a> Nat Biotechnol. 31 (1), 63-70 (2013).</li><li>Chen, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-Cas12a+target+binding+unleashes+indiscriminate+single-stranded+DNase+activity.">CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity.</a> Science. 360 (6387), 436-439 (2018).</li><li>Choi, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Renal+clearance+of+quantum+dots.">Renal clearance of quantum dots.</a> Nat Biotechnol. 25 (10), 1165-1170 (2007).</li><li>Hao, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-Cas-amplified+urinary+biomarkers+for+multiplexed+and+portable+cancer+diagnostics.">CRISPR-Cas-amplified urinary biomarkers for multiplexed and portable cancer diagnostics.</a> Nat Nanotechnol. 18 (7), 798-807 (2023).</li><li>Hao, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microenvironment-triggered+multimodal+precision+diagnostics.">Microenvironment-triggered multimodal precision diagnostics.</a> Nat Mater. 20 (10), 1440-1448 (2021).</li><li>Ghanta, K. 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S.N.B., L.H., and R.T.Z. are listed as inventors on a patent application related to the content of this work. S.N.B. holds equity in Glympse Bio, Satellite Bio, Lisata Therapeutics, Port Therapeutics, Intergalactic Therapeutics, Matrisome Bio, and is a director at Vertex; consults for Moderna, and receives sponsored research funding from Johnson &amp; Johnson, Revitope, and Owlstone.

Presented here is a highly customizable platform for multiplexed cancer detection with a portable urine test that assesses disease-associated proteolytic activity using a minimally invasive injected sensor. When activated by tumor proteases, peptide substrate cleavage is amplified via DNA barcode release into the urine. The synthetic DNA reporters in a urine sample can be read out by a secondary CRISPR-Cas-mediated enzymatic amplification using fluorometric detection or a simple paper-based test. DNA barcoding i...

Despite the significant effort to identify tumor biomarkers such as shed proteins and DNA, the cancer diagnostic field has been strained by their low abundance or rapid degradation in circulation1. As a complementary strategy, bioengineered synthetic biomarkers that selectively respond to disease features to generate amplified signals represent new avenues towards accurate and accessible diagnostics2,3. To aid detection, these synthetic biomarkers harness tumor-dependent activation mechanisms such as enzymatic amplification to produce analytes with improved signal-to-noise ratio4. Herein, a class of cancer-associated enzymes, proteases, are leveraged to activate injectable nanoscale sensors to release disease reporters detectable from the biofluids such as blood or urine5,6. In light of tumor heterogeneity, developing a panel of protease-activated sensors allows for multianalyte tests that combine different protease cleavage events into a &#39;disease signature&#39; to assess cancer incidence and progression in a more specific, multiplexed manner.
Protease-activated synthetic biomarkers have been developed that comprise peptide substrates conjugated to the surface of an inert carrier7. When injected in vivo, these peptides are carried to the tumor whereupon enzymatic cleavage by tumor proteases release reporters into blood or urine for detection. Multiplexed detection with protease-activated synthetic biomarkers requires each synthetic biomarker within a cocktail to be labeled with a unique molecular barcode. To this end, various approaches have been developed, including mass barcodes and ligand-encoded reporters8,9,10. As opposed to these methods of multiplexing which may be limited to a few different signal possibilities, DNA barcoding affords many more combinations in accordance with the high complexity and heterogeneity of human disease states. To expand the multiplexity of synthetic biomarkers, the sensors are barcoded by labeling each reporter with a unique DNA sequence for detection via CRISPR-Cas nuclease to amplify the biofluidic signal ex vivo. These single-stranded DNA (ssDNA) barcodes are designed to bind to complementary CRISPR guide RNAs (crRNAs), activating the target-triggered collateral nuclease activity of CRISPR-Cas12a11. This nuclease activity can be employed to cleave a reporter DNA strand detected through fluorescence kinetics or using paper strips.
In addition to molecular amplification via proteases (in vivo) and CRISPR-Cas (ex vivo), another key design feature of protease-activated synthetic biomarkers involves harnessing nanomaterial pharmacokinetics to increase diagnostic signal concentration in biofluids10. One approach is the use of a nanoparticle carrier to increase the circulation time of surface-conjugated peptide substrates. A polyethylene glycol (PEG) dendrimer is selected as a nanocarrier with relatively small hydrodynamic diameter and multivalency to increase delivery to tumors. While small enough to promote tumor delivery, the size of the PEG carrier is larger than the ~5 nm size cut-off of the kidney glomerular filtration barrier so that only cleaved peptide substrates can be cleared into the urine, taking advantage of size filtration by the kidneys12. In this protocol, the multiple-step workflow is outlined for the synthesis and application of DNA-barcoded activity-based nanosensors in a preclinical murine model, highlighting the setup of the CRISPR-Cas-mediated, multianalyte synthetic urine biomarker test, which has been employed by this group to classify disease status in murine models of multiple cancer types13. Owing to the versatile design principle, all three functional components of the nanosensor - the nanocarrier (PEG polymer), the stimuli-responsive module (protease-activated substrate), and the biofluidic reporter (DNA barcode) - can be precisely interchanged according to application-specific needs, allowing for modularity by tailoring the target and release specificities.

<table><tbody><tr><td>10x NEB Buffer 2.1</td><td>New England Biolabs</td><td>B6002SVIAL</td><td></td></tr><tr><td>20-mer phosphorothioated DNA reporters with 3&amp;rsquo;-DBCO group</td><td>IDT</td><td></td><td>Custom DNA</td></tr><tr><td>Agilent 1100 High Performance Liquid Chromatography system with Vydac 214TP510 C4 column</td><td>&amp;nbsp;Agilent</td><td></td><td>HPLC</td></tr><tr><td>&amp;Auml;KTA fast protein liquid chromatography (FPLC)</td><td>GE Healthcare</td><td></td><td>FPLC</td></tr><tr><td>Amicon ultracentrifuge tubes (MWCO = 10 kDa)</td><td>EMD millipore</td><td></td><td>Various volumes available</td></tr><tr><td>Azide-terminated PAPs with C-terminus cysteine</td><td>CPC Scientific</td><td></td><td>Custom peptide</td></tr><tr><td>crRNAs&amp;nbsp;</td><td>IDT</td><td></td><td>See Supplementary Table 1</td></tr><tr><td>Cryogenic transmission electron microscopy</td><td>JEM-2100F</td><td>JEOL</td><td>cyroTEM</td></tr><tr><td>Cysteine terminated DNA-peptide conjugates</td><td>CPC Scientific</td><td></td><td>Custom peptide</td></tr><tr><td>Dynamic light scattering (DLS)</td><td></td><td></td><td>DLS</td></tr><tr><td>EnGen &lt;em&gt;Lba&lt;/em&gt;Cas12a (Cpf1), 100 &amp;micro;M</td><td>New England Biolabs</td><td>M0653T</td><td></td></tr><tr><td>Experimental animals</td><td>Taconic Biosciences</td><td>BALB/cAnNTac</td><td>6&amp;ndash;8&amp;thinsp;weeks of age</td></tr><tr><td>gentleMACS C tubes</td><td>Miltenyi Biotec</td><td>130-093-237</td><td>tissue homogenization</td></tr><tr><td>HybriDetect Universal Lateral Flow Assay Kit</td><td>Miltenyi Biotec</td><td>MGHD 1</td><td></td></tr><tr><td>Matrix-assisted laser desorption/ionization&amp;ndash;time of flight (MALDI&amp;ndash;TOF) mass spectrometry&amp;nbsp;</td><td>Bruker</td><td></td><td>Microflex MALDI&amp;ndash;TOF</td></tr><tr><td>MC26-Fluc cell line</td><td></td><td></td><td>Kenneth K. Tanabe Laboratory, Massachusetts General Hospital</td></tr><tr><td>multivalent PEG (40 kDA, 8-arm) with maleimide-reactive group</td><td>JenKem</td><td>A10020-1 / 8ARM(TP)-MAL-40K,1 g</td><td></td></tr><tr><td>Python, Version 3.9</td><td></td><td></td><td>https://www.python.org/</td></tr><tr><td>Quant-iT OliGreen ssDNA Assay Kit and Quant-iT OliGreen ssDNA Reagent</td><td>Invitrogen</td><td>O11492</td><td>ssDNA assay kit</td></tr><tr><td>ssDNA FAM-T10-Quencher and&amp;nbsp; FAM-T10-Biotin reporter substrates</td><td>IDT</td><td></td><td>Custom DNA</td></tr><tr><td>Superdex 200 Increase 10/300 GL column</td><td>GE Healthcare</td><td>GE28-9909-44</td><td>For FPLC</td></tr><tr><td>Tecan Infinite Pro M200 plate reader</td><td>Tecan</td><td></td><td></td></tr><tr><td>ThermoFisher Pierce BCA Protein Assay Kit</td><td>ThermoFisher Scientific</td><td>23225</td><td></td></tr></tbody></table>

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.

Nominating protease-activated peptide substrates 
To design sensors which will reflect changes in the proteolytic activity of the tissue, protease activity in the tissue is first characterized using a library of peptide probes13 (Figure 1). Fresh and frozen tissue samples can provide substantial information about the proteolytic activity of the tumor microenvironment by combining tissue samples with FRET probes designed to detect substrate cleava...

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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.

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 ...

crispr-cas-mediated-multianalyte-synthetic-urine-biomarker-test-for

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Developing Custom Chinese Hamster Ovary-host Cell Protein Assays using Acoustic Membrane Microparticle Technology

Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to the complexity of development on alternate platforms. BioScale's proprietary Acoustic Membrane MicroParticle (AMMP) technology allows sandwich immunoassays to be easily developed for use on the ViBE platform, providing better sensitivity, reproducibility, and automated operation. Provided as an example, this protocol outlines the procedure for developing a custom Chinese Hamster Ovary- Host Cell Protein (CHO-HCP) assay. The general principles outlined here can be followed for the development of a wide variety of immunoassays.
An AMMP assay measures antigen concentration by measuring changes in oscillation frequency caused by the binding of microparticles to the sensor surface to calculate. It consists of four major components: (1) a cartridge that contains a functionalized eight sensor chip (2) antibody labeled magnetic microparticles, (3) hapten tagged antibody that binds to the surface of the functionalized chip (4) samples containing the antigen of interest. BioScale's biosensor is a resonant device that contains eight individual membranes with separate fluidic paths. The membranes change oscillation frequency in response to mass accumulating on the surface and this frequency change is used to quantitate the amount of added mass.
To facilitate use in a wide variety of immunoassays the sensor is functionalized with an anti-hapten antibody. Assay specific antibodies are modified through the covalent conjugation of a hapten tag to one antibody and biotin to the other. The biotin label is used to bind the antibody to streptavidin coupled magnetic beads which, in combination with the hapten-tagged antibody, are used to capture the analyte in a sandwich. The complex binds to the chip through the anti-hapten/hapten interaction. At the end of each assay run the sensors are cleaned with a dilute acid enabling the sequential analysis of columns from a 96-well plate.
Here, we present the method for developing a custom CHO-HCP AMMP assay for bioprocess development. Developing AMMP assays or modifying existing assays into AMMP assays can provide better performance (reproducibility, sensitivity) in complex samples and reduced operator time. The protocol shows the steps for development and the discussion section reviews representative results. For a more in-depth explanation of assay optimization and customization parameters contact BioScale. This kit offers generic bioprocess development assays such as Residual Protein A, Product titer, and CHO-HCP.

Development of custom assays on the ViBE platform for more sensitive, reproducible, automated results in complex matrices is described. The universal cartridge allows assays to be easily adapted for use with custom assays. This versatility enables rapid development and validation of novel assays or automated versions of existing manual assays, exemplified in this video.

Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to ...

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

High-throughput and Comprehensive Drug Surveillance Using Multisegment Injection-Capillary Electrophoresis-Mass Spectrometry

New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug crisis in public health. Conventional urine drug testing based on a two-tier immunoassay screen followed by a gas chromatography-tandem mass spectrometry (GC-MS/MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS) method are expensive and prone to bias while being limited to targeted panels of known drugs of abuse (DoA). Herein, we outline an improved method for drug surveillance that allows for the resolution and detection of an expanded panel of DoA and their metabolites when using multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS). Multiplexed separations of ten urine samples with a quality control by CE (&lt; 3 min/sample) in conjunction with full-scan data acquisition using a time-of-flight mass spectrometer (TOF-MS) under positive ion mode detection allows for the identification and quantification of DoA above recommended cut-off levels. An excellent resolution of drug isomers and isobars, including background interferences, are achieved when using MSI-CE-MS with an electrokinetic spacer between sample segments, where accurate mass/molecular formula together with the comigration of a matching deuterated internal standard and the detection of one or more bio-transformed metabolites facilitate DoA identification over a wider detection window. Additionally, urine samples can be analyzed directly without enzyme deconjugation for the rapid screening without complicated sample workup. MSI-CE-MS enables the surveillance of a broad spectrum of DoA that is required for the treatment monitoring of high-risk patients, including confirming prescribed drug adherence, revealing illicit drug use/substitution, and evaluating optimal dosage regimes as required for new advances in precision medicine.

Here we describe a high-throughput method for comprehensive drug surveillance that allows for the improved resolution and detection of large panels of drugs of abuse and their metabolites, with quality control based on multisegment injection-capillary electrophoresis-mass spectrometry.

New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug ...

Chemistry

Development of a Lateral Flow Immunochromatographic Strip for Rapid and Quantitative Detection of Small Molecule Compounds

Membrane-based lateral flow immunochromatographic strips (ICSs) are useful tools for low-cost self-diagnosis and have been efficiently applied to toxin, physiological index and clinical biomarker detection. In this protocol, we provide a detailed description of the steps to develop a rapid, sensitive and quantitative lateral-flow immunoassay (using AuNPs as a marker and mAbs as a probe). The procedure describes the preparation and characterization of colloidal gold, synthesis of the AuNP-mAb conjugate, assembly of the immunochromatographic strip, and methodological investigation of the assay. The results showed that the final strips can be further utilized for the rapid and convenient self-diagnosis of a small molecule, which may provide an alternative tool in the rapid and precise analysis of physiological and biological indices.

Membrane-based lateral flow immunochromatographic strips (ICSs) are useful tools for low-cost self-diagnosis and have been efficiently applied to toxin, ...

Biochemistry

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

Low-Cost, Volume-Controlled Dipstick Urinalysis for Home-Testing

Dipstick urinalysis provides quick and affordable estimations of multiple physiological conditions but requires good technique and training to use accurately. Manual performance of dipstick urinalysis relies on good human color vision, proper lighting control, and error-prone, time-sensitive comparisons to chart colors. By automating the key steps in the dipstick urinalysis test, potential sources of error can be eliminated, allowing self-testing at home. We describe the steps necessary to create a customizable device to perform automated urinalysis testing in any environment. The device is cheap to manufacture and simple to assemble. We describe the key steps involved in customizing it for the dipstick of choice and for customizing a mobile phone app to analyze the results. We demonstrate its use to perform urinalysis and discuss the critical measurements and fabrication steps necessary to ensure robust operation. We then compare the proposed method to the dip-and-wipe method, the gold standard technique for dipstick urinalysis.

Dipstick urinalysis is a quick and affordable method of assessing one&#8217;s personal state of health. We present a method to perform accurate, low-cost dipstick urinalysis that removes the primary sources of error associated with traditional dip-and-wipe protocols and is simple enough to be performed by lay users at home.

Dipstick urinalysis provides quick and affordable estimations of multiple physiological conditions but requires good technique and training to use ...

Design to Implementation Study for Development and Patient Validation of Paper-Based Toehold Switch Diagnostics

Access to low-burden molecular diagnostics that can be deployed into the community for testing is increasingly important and has meaningful wider implications for the well-being of societies and economic stability. Recent years have seen several new isothermal diagnostic modalities emerge to meet the need for rapid, low-cost molecular diagnostics. We have contributed to this effort through the development and patient validation of toehold switch-based diagnostics, including diagnostics for the mosquito-borne Zika and chikungunya viruses, which provided performance comparable to gold-standard reverse transcription-quantitative polymerase chain reaction (RT-qPCR) based assays. These diagnostics are inexpensive to develop and manufacture, and they have the potential to provide diagnostic capacity to low-resource environments. Here the protocol provides all the steps necessary for the development of a switch-based assay for Zika virus detection. The article takes readers through the stepwise diagnostic development process. First, genomic sequences of Zika virus serve as inputs for the computational design of candidate switches using open-source software. Next, the assembly of the sensors for empirical screening with synthetic RNA sequences and optimization of diagnostic sensitivity is shown. Once complete, validation is performed with patient samples in parallel with RT-qPCR, and a purpose-built optical reader, PLUM. This work provides a technical roadmap to researchers for the development of low-cost toehold switch-based sensors for applications in human health, agriculture, and environmental monitoring.

Access to decentralized, low-cost, and high-capacity diagnostics that can be deployed into the community for decentralized testing is critical for combating global health crises. This manuscript describes how to build paper-based diagnostics for viral RNA sequences that can be detected with a portable optical reader.

Access to low-burden molecular diagnostics that can be deployed into the community for testing is increasingly important and has meaningful wider ...

Optimized CRISPR-Based Molecular Diagnostics Protocol for Point-of-Care Use

Point-of-care CRISPR-based Diagnostics with Premixed and Freeze-dried Reagents

Molecular diagnostics by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based detection have high diagnostic accuracy and attributes that are suitable for use at point-of-care settings such as fast turnaround times for results, convenient simple readouts, and no requirement of complicated instruments. However, the reactions can be cumbersome to perform at the point of care due to their many components and manual handling steps. Herein, we provide a step-by-step, optimized protocol for the robust detection of disease pathogens and genetic markers with recombinase-based isothermal amplification and CRISPR-based reagents, which are premixed and then freeze-dried in easily stored and ready-to-use formats. Premixed, freeze-dried reagents can be rehydrated for immediate use and retain high amplification and detection efficiencies. We also provide a troubleshooting guide for commonly found problems upon preparing and using premixed, freeze-dried reagents for CRISPR-based diagnostics, to make the detection platform more accessible to the wider diagnostic/genetic testing communities.

Author Spotlight: Development of Simplified CRISPR-Based Tests for Rapid Detection of Infectious Diseases

Here, we present the step-by-step preparation of premixed, lyophilized recombinase-based isothermal amplification and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based reactions, which can be used for the detection of nucleic acid biomarkers of infectious disease pathogens or other genetic markers of interest.

Molecular diagnostics by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based detection have high diagnostic accuracy and attributes ...

Field-Deployable Candidatus Liberibacter asiaticus Detection Using Recombinase Polymerase Amplification Combined with CRISPR-Cas12a

The early detection of Candidatus Liberibacter asiaticus (CLas) by citrus growers facilitates early intervention and prevents the spread of disease. A simple method for rapid and portable Huanglongbing (HLB) diagnosis is presented here that combines recombinase polymerase amplification and a fluorescent reporter utilizing the nuclease activity of the clustered regularly interspaced short palindromic repeats/CRISPR-associated 12a (CRISPR-Cas12a) system. The sensitivity of this technique is much higher than PCR. Furthermore, this method showed similar results to qPCR when leaf samples were used. Compared with conventional CLas detection methods, the detection method presented here can be completed in 90 min and works in an isothermal condition that does not require the use of PCR machines. In addition, the results can be visualized through a handheld fluorescent detection device in the field.

Field-Deployable Candidatus Liberibacter asiaticus Detection Using Recombinase Polymerase Amplification Combined with CRISPR-Cas12a

In this work, a rapid, sensitive, and portable detection method for Candidatus Liberibacter asiaticus based on recombinase polymerase amplification combined with CRISPR-Cas12a was developed.

The early detection of Candidatus Liberibacter asiaticus (CLas) by citrus growers facilitates early intervention and prevents the spread of disease. A ...

Biology

DNA Virus Detection System Based on RPA-CRISPR/Cas12a-SPM and Deep Learning

We report a fast, easy-to-implement, highly sensitive, sequence-specific, and point-of-care (POC) DNA virus detection system, which combines recombinase polymerase amplification (RPA) and CRISPR/Cas12a system for trace detection of DNA viruses. Target DNA is amplified and recognized by RPA and CRISPR/Cas12a separately, which triggers the collateral cleavage activity of Cas12a that cleaves a fluorophore-quencher labeled DNA reporter and generalizes fluorescence. For POC detection, portable smartphone microscopy is built to take fluorescent images. Besides, deep learning models for binary classification of positive or negative samples, achieving high accuracy, are deployed within the system. Frog virus 3 (FV3, genera Ranavirus, family Iridoviridae) was tested as an example for this DNA virus POC detection system, and the limits of detection (LoD) can achieve 10 aM within 40 min. Without skilled operators and bulky instruments, the portable and miniature RPA-CRISPR/Cas12a-SPM with artificial intelligence (AI) assisted classification shows great potential for POC DNA virus detection and can help prevent the spread of such viruses.

We present a protocol that combines recombinase polymerase amplification with a CRISPR/Cas12a system for trace detection of DNA viruses and builds portable smartphone microscopy with an artificial intelligence-assisted classification for point-of-care DNA virus detection.

We report a fast, easy-to-implement, highly sensitive, sequence-specific, and point-of-care (POC) DNA virus detection system, which combines recombinase ...

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

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