The authors thank all study participants and the UAB CCTS Bionutrition Core and UAB High Resolution Imaging Service Center for their contributions. This work was supported by NIH grants DK106284 and DK123542 (TM), and UL1TR003096 (National Center for Advancing Translational Sciences).

All experiments outlined in this work were approved by the University of Alabama at Birmingham (UAB) Institutional Review Board. Healthy adults (33.6 &#177; 3.3 years old; n=10) were enrolled in the study if they had a normal blood comprehensive metabolic panel, non-tobacco users, non-pregnant, a BMI between 20-30 kg/m2, and free of chronic medical conditions or acute illnesses. Healthy participants signed a written informed consent form prior to the start of the study.
1. Clinical protocol and urine collection
<ol>
	<li>Have participants consume a low oxalate diet prepared by the UAB Center for Clinical and Translational Sciences Bionutrition Core for 3 days and fast overnight before collecting their urine (24-hour sample).</li>
	<li>The following day, have participants return their 24-hour urine sample (pre-oxalate) before consuming an oxalate load (smoothie containing fruits and vegetables, ~8 mM oxalate). Have participants subsequently collect their urine for 24 hours (post-oxalate sample) and return their urine the following day.</li>
	<li>Maintain all urine samples at room temperature (RT) prior to processing as described below and shown in Figure 1.</li>
</ol>
2. Urine Processing
NOTE: All materials and equipment used are listed in Table of Materials. 
CAUTION: Wear personal protective equipment at all times while handling clinical samples and reagents. Specifically, gloves, face and eye shields, respiratory protection, and protective clothing.
<ol>
	<li>Measure and record urine pH and volume. Mix thoroughly prior to adding 50 mL of urine into a labeled sterile 50 mL conical tube.</li>
	<li>Centrifuge sample at 1200 x g for 10 min at RT using a benchtop centrifuge. 
	NOTE: Keep the sample at RT to prevent further crystal formation as cooler temperatures can promote crystallization.</li>
	<li>Discard the supernatant and wash and resuspend the pellet again with 5 mL of 100% ethanol. Centrifuge the sample at 1200 x g for 10 minutes at RT using a benchtop centrifuge.</li>
	<li>Discard the supernatant and resuspend the pellet in 1 mL of 100% ethanol. Store the sample at -20 &#176;C for later processing OR stain the sample as described below. 
	&#8203;NOTE: There is no significant difference in data points (i.e., particle size/concentration) between stored or freshly stained samples.</li>
</ol>
3. Nanoparticle Tracking Analysis (NTA)
<ol>
	<li>Sample Preparation
	<ol>
		<li>Gold nanoparticles: Use gold nanoparticles to optimize settings on the instrument. Dilute 100 nm sized gold nanoparticles 1:1000 in ultrapure water.</li>
		<li>Human Urine: Dilute urine samples 20 times in water prior to staining with 5 mM Fluo-4 AM (a calcium fluorescence dye) for 30 min in the dark. Analyze the samples using NTA.</li>
		<li>Prepare Calcium Oxalate (CaOx) crystals as previously described29. Dilute 10 mM stock solution (14.6 mg in 10 mL of water) to 50 &#181;M in water and stain the diluted samples using 5 mM Fluo-4 AM for 30 min in dark prior to analysis.</li>
		<li>Calcium Phosphate (CaP) crystals: Dilute 10 mM stock solution (50.4 mg in 10 mL of water) to 50 &#181;M in water and stain the diluted samples using 5 mM Fluo-4 AM for 30 min in dark prior to NTA analysis.</li>
	</ol>
	</li>
	<li>Instrument Set-up, Camera Settings, and Data Collection 
	NOTE: The computer and instrument setup used for this method are shown in Figure 2.
	<ol>
		<li>Turn on the computer and then the instrument. Open the software and turn on the camera.</li>
		<li>Once the software window is open, click the capture icon in the top left corner on the window to start the capture mode. The camera initialization takes a few seconds.</li>
		<li>Clean the platform by first pumping air into it using a 1 mL syringe until the platform appears clean. Gently add water to the apparatus 2-3 times using another 1 mL syringe to remove any air bubbles. 
		&#8203;NOTE: Look for any air bubbles in the platform as well as in the tubing. It is important to not have bubbles throughout the apparatus prior to and while running samples. If bubbles are present, clean the platform again with air and water.</li>
		<li>Once the platform is clean, add water to check for any contamination on the surface by viewing the camera. Next, add gold nanoparticles&#160;as a control to the sample loading pump injector to set up the instrument.</li>
		<li>Adjust the camera level on the screen or on the knob to the right side of the instrument until the image starts to display colored pixels and then reduce the camera level.</li>
		<li>Then adjust the screen to optimize the image. Left-click the mouse button on the video image. Hold the left mouse button and drag image up and down to get the entire view. 
		&#8203;NOTE: The normal camera lens and filter is used to assess gold nanoparticles and unstained samples.</li>
		<li>Set up the infusion speed and focus the camera so that the gold nanoparticles are visible on the camera screen. Set the infusion speed to high (i.e., 500 &#181;L/min) for initial set up to ensure the gold nanoparticles are detected. Once detected, reduce the speed to 50 &#181;L/min.</li>
		<li>Adjust the camera level to visualize the particles. For unstained samples, adjust the screen gain at level 5 to achieve the camera focus, and set the camera level at 8. Once the focus is set, record the sample (i.e., 1 measurement for 60 seconds only). 
		&#8203;NOTE: The focus and continuous flow speed are important for obtaining clear and sharp images of the particles for counting.</li>
		<li>After optimization, clean the apparatus again with water before assessing samples. View the camera to ensure that the tubing is clean and particles are not present. 
		&#8203;NOTE: Wash the chamber between each sample until no particles are detected by the camera.</li>
		<li>To analyze stained samples, adjust the camera to the filter position containing the suitable fluorescent filter. Load diluted and stained samples onto the sample loading pump injector and reduce the speed to 20 &#181;L/min for analysis of the sample.</li>
		<li>Next adjust the screen gain and camera level as these are important parameters. For stained (fluorescent) samples, set the screen gain to 5 and the camera level at level 13. 
		&#8203;NOTE: These parameters will vary based on the sample type and every sample will need to be optimized to gain focus.</li>
		<li>Use standard measurement to measure the samples for 5 captures per sample where one capture-duration is for 60 seconds.</li>
		<li>Save and store data after each measurement. The software will save image and video files for each measurement. The software provides output data (e.g., crystal size: 10 nM - 1000 nM and concentration) in both excel and pdf formats.</li>
		<li>Calculate the average number of nanoparticles for all 5 readings for each individual sample. Analyze the data using standard deviation or standard error of the mean and use t-tests for paired analysis.</li>
	</ol>
	</li>
</ol>

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The authors declare no conflicts of interest.

NTA has been used in the present study to assess nanocrystals in human urine using a calcium binding probe, Fluo-4 AM. There is no standard method available to detect nanocrystals in the urine. Some research groups have detected nanocrystals in the urine and relied on the use of extensive protocols or methods that are limited in their ability to quantify the samples27,28. This study shows a specific and sensitive method for detecting calcium containing nanocrysta...

Urinary crystals form when urine becomes supersaturated with minerals. This can occur in healthy individuals but is more common in individuals with kidney stones1. The presence and accumulation of urinary crystals can increase one&#39;s risk of developing a kidney stone. Specifically, this occurs when crystals bind to Randall&#39;s plaque, nucleate, accumulate, and grow over time2,3,4. Crystalluria precedes kidney stone formation and assessment of crystalluria may have predictive value in kidney stone formers3,5. Specifically, crystalluria has been suggested to be useful to predict the risk of stone recurrence in patients with a history of calcium oxalate containing stones6,7.
Crystals have been reported to negatively impact renal epithelial and circulating immune cell function8,9,10,11,12,13. It has been previously reported that circulating monocytes from calcium oxalate (CaOx) kidney stone formers have suppressed cellular bioenergetics compared to healthy individuals14. In addition, CaOx crystals reduce cellular bioenergetics and disrupt redox homeostasis in monocytes8. Consumption of meals rich in oxalate may cause crystalluria which could lead to renal tubule damage and alter the production and function of urinary macromolecules that are protective against kidney stone formation15,16. Several studies have demonstrated that urinary crystals can vary in shape and size depending on the pH and temperature of the urine17,18,19. Further, urinary proteins have been shown to modulate crystal behavior20. Daudon et al.19, proposed that crystalluria analysis could be helpful in the management of patients with kidney stone disease and in assessing their response to therapies. A few conventional methods currently available to evaluate the presence of crystals include polarized microscopy21,22, electron microscopy23, particle counters3, urine filtration24, evaporation3,5 or centrifugation21. These studies have provided valuable insight to the kidney stone field regarding crystalluria. However, a limitation of these methods has been the inability to visualize and quantify crystals less than 1 &#181;m in size. Crystals of this size may influence the growth of CaOx stones by attaching to Randall&#39;s plaque.
Nanocrystals have been shown to cause extensive injury to renal cells compared to larger microcrystals25. The presence of nanocrystals has been reported in urine using a nanoparticle analyzer26,27. Recent studies have used fluorescently labeled bisphosphate probes (alendronate-fluorescein/alendronate-Cy5) to examine nanocrystals using nanoscale flow cytometry28. The limitation of this dye is that it is not specific and will bind to almost all types of stones except cysteine. Thus, accurately assessing the presence of nanocrystals in individuals may be an effective tool to diagnose crystalluria and/or predict stone risk. The purpose of this study was to detect and quantify calcium containing nanocrystals (&#60;1 &#181;m in size) using nanoparticle tracking analysis (NTA). To achieve this, NTA technology was used in combination with a calcium binding fluorophore, Fluo-4 AM to detect and quantify calcium containing nanocrystals in the urine of healthy adults.

<table><tbody><tr><td>Benchtop Centrifuge</td><td>Jouan Centrifuge</td><td>CR3-12</td><td></td></tr><tr><td>Calcium Oxalate monohydrate</td><td>Synthesized in the lab as previously described&lt;sup&gt;29&lt;/sup&gt;.</td><td> </td><td>Store at RT; Stock 10 mM</td></tr><tr><td>Calcium Phosphate crystals (hydroxyapatite nanopowder)</td><td>Sigma</td><td>677418</td><td>Store at RT; Stock 10 mM</td></tr><tr><td>Ethanol</td><td>Fischer Scientific</td><td>AC615095000</td><td>Store at RT; Stock 100%</td></tr><tr><td>Fluo-4 AM*</td><td>AAT Bioquest, Inc.</td><td>20550</td><td>Store at Freezer (-20&amp;deg;C); Stock 5 mM</td></tr><tr><td>Gold Nanoparticles</td><td>Sigma</td><td>742031</td><td>Store at 2-8&amp;deg;C</td></tr><tr><td>NanoSight Instrument</td><td>Malvern Instruments, UK</td><td>NS300</td><td></td></tr><tr><td>Syringe pump</td><td>Harvard Apparatus</td><td>98-4730</td><td></td></tr><tr><td>Virkon Disinfectant</td><td>LanXESS Energizing Company, Germany</td><td>LSP</td><td></td></tr><tr><td>*Fluorescence dyes are light sensitive; stock and aliquots should be stored in the dark at -20&amp;deg;C.</td><td></td><td></td><td></td></tr></tbody></table>

estimation of urinary nanocrystals in humans using calcium fluorophore labeling and nanoparticle tracking analysis

Kidney stones are becoming more prevalent worldwide in adults and children. The most common type of kidney stone is comprised of calcium oxalate (CaOx) crystals. Crystalluria occurs when urine becomes supersaturated with minerals (e.g., calcium, oxalate, phosphate) and precedes kidney stone formation. Standard methods to assess crystalluria in stone formers include microscopy, filtration, and centrifugation. However, these methods primarily detect microcrystals and not nanocrystals. Nanocrystals have been suggested to be more harmful to kidney epithelial cells than microcrystals in vitro. Here, we describe the ability of Nanoparticle Tracking analysis (NTA) to detect human urinary nanocrystals. Healthy adults were fed a controlled oxalate diet prior to drinking an oxalate load to stimulate urinary nanocrystals. Urine was collected for 24 hours before and after the oxalate load. Samples were processed and washed with ethanol to purify samples. Urinary nanocrystals were stained with the calcium binding fluorophore, Fluo-4 AM. After staining, the size and count of nanocrystals were determined using NTA. The findings from this study show NTA can efficiently detect nanocrystalluria in healthy adults. These findings suggest NTA could be a valuable early detection method of nanocrystalluria in patients with kidney stone disease.

The findings from this study show NTA can efficiently detect the mean size and concentration of calcium containing urinary nanocrystals in human urine. This was achieved by using the fluorophore, Fluo-4 AM, and nanoparticle tracking analysis. Fluo-4 AM was able to bind to both CaOx and CaP crystals. As shown in Figure 3A, CaOx crystals were determined to be between 50-270 nm in size and have a mean concentration of 1.26 x 109 particles/mL. CaP crystals were between 30-225 nm in si...

Watch this Scientific Journal Video about Estimation of Urinary Nanocrystals in Humans using Calcium Fluorophore Labeling and Nanoparticle Tracking Analysis at JoVE.com

Estimation of Urinary Nanocrystals in Humans using Calcium Fluorophore Labeling and Nanoparticle Tracking Analysis

The objective of this study was to determine whether nanoparticle tracking analysis (NTA) could detect and quantify urinary calcium containing nanocrystals from healthy adults. The findings from the current study suggest NTA could be a potential tool to estimate urinary nanocrystals during kidney stone disease.

Kidney stones are becoming more prevalent worldwide in adults and children. The most common type of kidney stone is comprised of calcium oxalate (CaOx) ...

estimation-urinary-nanocrystals-humans-using-calcium-fluorophore

Research

JoVE Journal

Medicine

3.6K Views.  University of Alabama at Birmingham. The objective of this study was to determine whether nanoparticle tracking analysis (NTA) could detect and quantify urinary calcium containing nanocrystals from healthy adults. The findings from the current study suggest NTA could be a potential tool to estimate urinary nanocrystals during kidney stone disease. 

Video: Estimation of Urinary Nanocrystals in Humans using Calcium Fluorophore Labeling and Nanoparticle Tracking Analysis

Fluorescent Nanoparticles for the Measurement of Ion Concentration in Biological Systems

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion fluxes at the cellular level are required for initiating action potentials in excitable cells.2 Sodium regulation plays an important role in both of these cases; however, no method currently exists for continuously monitoring sodium levels in vivo 3 and intracellular sodium probes 4 do not provide similar detailed results as calcium probes. In an effort to fill both of these voids, fluorescent nanosensors have been developed that can monitor sodium concentrations in vitro and in vivo.5,6 These sensors are based on ion-selective optode technology and consist of plasticized polymeric particles in which sodium specific recognition elements, pH-sensitive fluorophores, and additives are embedded.7-9 Mechanistically, the sodium recognition element extracts sodium into the sensor. 10 This extraction causes the pH-sensitive fluorophore to release a hydrogen ion to maintain charge neutrality within the sensor which causes a change in fluorescence. The sodium sensors are reversible and selective for sodium over potassium even at high intracellular concentrations.6 They are approximately 120 nm in diameter and are coated with polyethylene glycol to impart biocompatibility. Using microinjection techniques, the sensors can be delivered into the cytoplasm of cells where they have been shown to monitor the temporal and spatial sodium dynamics of beating cardiac myocytes.11 Additionally, they have also tracked real-time changes in sodium concentrations in vivo when injected subcutaneously into mice.3 Herein, we explain in detail and demonstrate the methodology for fabricating fluorescent sodium nanosensors and briefly demonstrate the biological applications our lab uses the nanosensors for: the microinjection of the sensors into cells; and the subcutaneous injection of the sensors into mice.

Fluorescent nanoparticles produced in our lab are used for imaging ion concentrations and ion fluxes in biological systems such as cells during signaling and interstitial fluid during physiological homeostasis.

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion ...

Bioengineering

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

Localization and Relative Quantification of Carbon Nanotubes in Cells with Multispectral Imaging Flow Cytometry

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich cell structures and de facto there is still no quantitative method to assess their distribution at cell and tissue levels. What we propose here is an innovative method allowing the detection and quantification of CNTs in cells using a multispectral imaging flow cytometer (ImageStream, Amnis). This newly developed device integrates both a high-throughput of cells and high resolution imaging, providing thus images for each cell directly in flow and therefore statistically relevant image analysis. Each cell image is acquired on bright-field (BF), dark-field (DF), and fluorescent channels, giving access respectively to the level and the distribution of light absorption, light scattered and fluorescence for each cell. The analysis consists then in a pixel-by-pixel comparison of each image, of the 7,000-10,000 cells acquired for each condition of the experiment. Localization and quantification of CNTs is made possible thanks to some particular intrinsic properties of CNTs: strong light absorbance and scattering; indeed CNTs appear as strongly absorbed dark spots on BF and bright spots on DF with a precise colocalization.
This methodology could have a considerable impact on studies about interactions between nanomaterials and cells given that this protocol is applicable for a large range of nanomaterials, insofar as they are capable of absorbing (and/or scattering) strongly enough the light.

In this study, the main purpose was to monitor the cellular uptake and eventual exocytosis of carbon nanotubes (CNTs). From this perspective, we proposed here a unique method using multispectral imaging flow cytometry allowing a quantification and localization of CNTs in a statistically relevant number of cells.

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich ...

Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers that exist in biological fluids cannot be easily detected with mass spectrometry or immunoassays because they are present in very low concentration, are labile, and are often masked by high-abundance proteins such as albumin or immunoglobulin. Bait containing poly(N-isopropylacrylamide) (NIPAm) based nanoparticles are able to overcome these physiological barriers. In one step they are able to capture, concentrate and preserve biomarkers from body fluids. Low-molecular weight analytes enter the core of the nanoparticle and are captured by different organic chemical dyes, which act as high affinity protein baits. The nanoparticles are able to concentrate the proteins of interest by several orders of magnitude. This concentration factor is sufficient to increase the protein level such that the proteins are within the detection limit of current mass spectrometers, western blotting, and immunoassays. Nanoparticles can be incubated with a plethora of biological fluids and they are able to greatly enrich the concentration of low-molecular weight proteins and peptides while excluding albumin and other high-molecular weight proteins. Our data show that a 10,000 fold amplification in the concentration of a particular analyte can be achieved, enabling mass spectrometry and immunoassays to detect previously undetectable biomarkers.

Several pathological biomarkers cannot be easily detected by current techniques because of their low concentration in biological fluids, the presence of degrading enzymes, and large amounts of high molecular weight proteins. Chemically functionalized hydrogel nanoparticles can harvest, preserve and concentrate low abundance proteins enabling the detection of previously undetectable biomarkers.

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers ...

Nanoparticle Tracking Analysis of Gold Nanoparticles in Aqueous Media through an Inter-Laboratory Comparison

In the field of nanotechnology, analytical characterization plays a vital role in understanding the behavior and toxicity of nanomaterials (NMs). Characterization needs to be thorough and the technique chosen should be well-suited to the property to be determined, the material being analyzed and the medium in which it is present. Furthermore, the instrument operation and methodology need to be well-developed and clearly understood by the user to avoid data collection errors. Any discrepancies in the applied method or procedure can lead to differences and poor reproducibility of obtained data. This paper aims to clarify the method to measure the hydrodynamic diameter of gold nanoparticles by means of Nanoparticle Tracking Analysis (NTA). This study was carried out as an inter-laboratory comparison (ILC) amongst seven different laboratories to validate the standard operating procedure&#8217;s performance and reproducibility. The results obtained from this ILC study reveal the importance and benefits of detailed standard operating procedures (SOPs), best practice updates, user knowledge, and measurement automation.

The protocol described here aims to measure the hydrodynamic diameter of spherical nanoparticles, more specifically gold nanoparticles, in aqueous media by means of Nanoparticle Tracking Analysis (NTA). The latter involves tracking the movement of particles due to Brownian motion and implementing the Stokes-Einstein equation to obtain the hydrodynamic diameter.

In the field of nanotechnology, analytical characterization plays a vital role in understanding the behavior and toxicity of nanomaterials (NMs). ...

Chemistry

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.

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.

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

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 ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

Biology

Nanoparticle Tracking Analysis for the Quantification and Size Determination of Extracellular Vesicles

The physiological and pathophysiological roles of extracellular vesicles (EVs) have become increasingly recognized, making the EV field a quickly evolving area of research. There are many different methods for EV isolation, each with distinct advantages and disadvantages that affect the downstream yield and purity of EVs. Thus, characterizing the EV prep isolated from a given source by a chosen method is important for interpretation of downstream results and comparison of results across laboratories. Various methods exist for determining the size and quantity of EVs, which can be altered by disease states or in response to external conditions. Nanoparticle tracking analysis (NTA) is one of the prominent technologies used for high-throughput analysis of individual EVs. Here, we present a detailed protocol for quantification and size determination of EVs isolated from mouse perigonadal adipose tissue and human plasma using a breakthrough technology for NTA representing major advances in the field. The results demonstrate that this method can deliver reproducible and valid total particle concentration and size distribution data for EVs isolated from different sources using different methods, as confirmed by transmission electron microscopy. The adaptation of this instrument for NTA will address the need for standardization in NTA methods to increase rigor and reproducibility in EV research.

We demonstrate how to use a novel nanoparticle tracking analysis instrument to estimate the size distribution and total particle concentration of extracellular vesicles isolated from mouse perigonadal adipose tissue and human plasma.

The physiological and pathophysiological roles of extracellular vesicles (EVs) have become increasingly recognized, making the EV field a quickly evolving ...

Longitudinal Quantification of Cardiovascular Calcification Using PET/CT Imaging

Novel Quantification Protocol for Cardiovascular Calcification Progression Using Longitudinal MicroPET/MicroCT Images

Micro positron emission tomography (PET) and micro computed tomography (CT) imaging are powerful, ideal research tools for following the progression of cardiovascular calcification. Due to their non-invasive nature, small research animals can be imaged at multiple time points. The challenge lies in the accurate quantification of cardiovascular calcification. Here, we provide a protocol, using images from the later disease stages as a template, to accurately quantify the progression of cardiovascular calcification in longitudinal studies. The protocol involves 1) the alignment of the chest area in multiple images from the same animal during a longitudinal study as the first step, 2) the definition of a region of interest (ROI) situated within the heart and the aorta at the site of larger calcium deposits that become apparent in later images, and 3) simultaneous segmentation and quantification of calcium deposits across all images acquired during the longitudinal study. This streamlined method enhances the accuracy of image analysis in following the progression of cardiovascular calcification by improving the precision of ROI definition and reducing the variability associated with earlier techniques that analyze individual scans independently.

Author Spotlight: Enhanced Quantification of Cardiovascular Calcification Progression for Longitudinal Micro PET/CT Studies in Small Research Animals

This novel protocol entails the quantification of cardiovascular calcification progression from serial micro positron emission tomography (PET)/micro computed tomography (CT) images in small research animals.

Micro positron emission tomography (PET) and micro computed tomography (CT) imaging are powerful, ideal research tools for following the progression of ...

Estimation of Urinary Nanocrystals in Humans using Calcium Fluorophore Labeling and Nanoparticle Tracking Analysis

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Fluorescent Nanoparticles for the Measurement of Ion Concentration in Biological Systems

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Localization and Relative Quantification of Carbon Nanotubes in Cells with Multispectral Imaging Flow Cytometry

Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Nanoparticle Tracking Analysis of Gold Nanoparticles in Aqueous Media through an Inter-Laboratory Comparison

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

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

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

Nanoparticle Tracking Analysis for the Quantification and Size Determination of Extracellular Vesicles

Novel Quantification Protocol for Cardiovascular Calcification Progression Using Longitudinal MicroPET/MicroCT Images