This work was funded by the Dorothy J. Wingfield Phillips Chancellor Faculty Fellowship. Emily Kight was funded by NSF GRFP.

1. Fabricate and assemble the urinalysis device
<ol>
	<li>Fabricate the base plate (Figure 1A).
	<ol>
		<li>Use a computer-aided design (CAD) software to draw a rectangular area with dimensions 2.1641 in x 0.0547 in x 6.3828 in (W x H x L) using the polyline tool.</li>
		<li>Measure the test area (rectangular area encompassing the distance between the first and last pad and the width of the pads) on the dipstick. 
		NOTE: This information is needed to draw the through-holes that hold the dipstick in place and separate the liquid in between the pads (to prevent cross-contamination).</li>
		<li>Add through-holes that mimic the size and position of each test pad in the test area.</li>
		<li>Draw two raised side ledges that measure 2.1641 in x 0.6797 in (W x L).</li>
		<li>Draw a stop (0.1172 in by 0.2109 in (W x L)) using the polyline tool to facilitate alignment between the base plate and the slide. The stop should be perpendicular to the ledges and physically stops the slide from moving passed the urine dipstick pads.</li>
		<li>Select the lines for the stop and ledge to make one region using the Region command. Use the Extrude command to raise the region up to a height of 0.0703 in. Repeat this step on the other side of the device.</li>
		<li>Create a notch (0.1895 in by 0.3500 in (W x L)) on both ledges to facilitate alignment with the box. Position it 0.466 in from the bottom edge of the ledge. Use the Region command to create a rectangle and make the extrusion height 0.1250 in.</li>
		<li>Use the Solid Subtract command, select device, press Enter, select the notch region and press Enter. Repeat on the other side of the device. 
		NOTE: The shape will be removed from the device.</li>
		<li>Print the base plate on a 3D printer and sand the top face area between the ledges with sandpaper to roughen the surface. 
		NOTE: Sanding is important so that the hydrophobic coating can adhere to the base plate securely.</li>
		<li>Tape the ledges with adhesive tape (to avoid spraying the ledges) and spray the base plate with a hydrophobic spray. Apply several (4-8) coats of the basecoat to the base plate. Hold the can approximately 8-12 inches away from the base plate when spraying. The device should have a milky white appearance upon drying. 
		CAUTION: Follow manufacturer instructions for appropriate location and PPE for spraying.</li>
		<li>Wait 30 minutes before applying the topcoat several times (6-8x). Allow the base plate to dry for 12 hours before use. Remove the tape from the ledges.</li>
	</ol>
	</li>
	<li>Fabricate the top plate (Figure 1B).
	<ol>
		<li>Draw a rectangular area to measure 2.05 in x 5.470 in (W x L) in a CAD software using the polyline tool.</li>
		<li>Add a rectangular through-hole (the &#8220;viewing through-hole&#8221;) slightly larger than the size of the test area of the dipstick (e.g., 0.230 in x 3.147 in (W x L)). Place it 0.921 in from the top, 1.165 in from the left, and 1.165 in from the right edges of the top plate.</li>
		<li>Draw a second through-hole (the &#8220;Inlet-hole&#8221;) sized 0.075 in x 3.146 in (W x L). Place it 0.236 in from the bottom edge, 1.737 in from the top edge, and 1.162 in from the left and right edges of the top plate.</li>
		<li>Cut the top plate from a piece of clear acrylic with a laser cutter. Wipe off any remaining dust or debris.</li>
	</ol>
	</li>
	<li>Fabricate the inlet cover (Figure 1B).
	<ol>
		<li>Draw a rectangular area with dimensions 0.247 in x 3.3378 in (W x L) in a CAD software using the polyline tool. Add two circular through-holes with a diameter of 0.127 in approximately 0.073 in from the two edges of the inlet cover, one on either side.</li>
		<li>Cut the inlet cover from a piece of clear acrylic with a laser cutter.</li>
	</ol>
	</li>
	<li>Fabricate the slide (Figure 1C)
	<ol>
		<li>Draw a rectangular area in CAD software that measures measure 2.771 in x 0.0625 in x 5.000 in (W x H x L) using the polyline tool.</li>
		<li>Add through-holes that match the position of each test pad in the test area. Draw the first 0.105 in square through-hole to overlap with the placement of the first test pad: 1.096 in from the left and right edges of the slide, 0.960 in from the top edge, and 1.681 in from the bottom edge. Add more through-holes as needed (usually 10 total) for the selected dipstick brand of choice. Space each next through-hole by measuring the distance between test pads on the dipstick. 
		NOTE: The size of the through-holes is important in order to deposit the correct volume of liquid onto the dipstick pad. For our brand of dipstick, we created holes which deposit 15 ul onto each dipstick pad.</li>
		<li>Cut the slide from a piece of clear acrylic using a laser cutter. Wipe off any remaining dust or debris.</li>
		<li>Spray the front of the slide with a hydrophobic spray. Apply several coats (6-8x) of basecoat to the slide. Hold the can approximately 8 -12 in away from the slide when spraying.</li>
		<li>Wait 30 minutes before applying the topcoat several times (8-12x). Allow the slide to dry for 12 hours before use.</li>
		<li>Download a QR code from an online QR code generator and print the desired code on paper with sticky adhesive backing. Place the QR code 0.17 in from the right of the first through-hole along the same row as all the through-holes. 
		NOTE: As long as the QR code is adjacent to the through-holes, accurate placement is not important.</li>
		<li>Use clear tape to cover the QR code and secure it to the slide.</li>
	</ol>
	</li>
	<li>Assemble the inlet and plate sleeve (Figure 1D).
	<ol>
		<li>Fabricate the inlet by using acrylic cement to glue the inlet cover onto the top plate where the inlet-hole is located. Wait 24-48 hours to securely bond the pieces.</li>
		<li>Spray the back of the top plate with a hydrophobic spray once the inlet cover is securely bonded to the top plate. Place the top plate upside down. Apply the first basecoat several times (4-8x).</li>
		<li>Hold the spray 8-12 inches away from the top plate and wait 30 minutes for it to dry. Apply the topcoat several times (6-8x). Allow the top plate to dry for 12 hours before use.</li>
		<li>Assemble the plate sleeve (combined top plate and base plate) by gluing the completed top plate to the ledges of the base plate with acrylic cement. The two pieces are easy to align by visual inspection, as the bottom edge of the top plate will align with that of the base plate. Apply a clamp to the base plate ledges to secure it during drying and wait 24-48 hours before use, as per the manufacturer&#8217;s instructions.</li>
	</ol>
	</li>
	<li>Create the chart sticker.
	<ol>
		<li>Download the color chart for the brand of dipstick from the manufacturer&#8217;s website.</li>
		<li>Open the downloaded file in a graphics editor software.</li>
		<li>Open the digital file for the top plate template previously used for the laser cutter (Step 1.2 of this protocol) in a graphics editor software.</li>
		<li>Create the color boxes for the chart sticker by matching color boxes from the manufacturer color chart. Select the first block of color on the manufacturer&#8217;s chart with the dropper tool in the graphics editor software and then use the box shape tool to make a box shape in the same color on the top plate template, in the same row where the dipstick pad will be located. Repeat this for each color block corresponding to each pad row.</li>
		<li>Delete the layers associated with the top plate template.</li>
		<li>Print the chart sticker as a vinyl sticker with an online sticker print service. Place the chart sticker onto the plate sleeve and align it with each through-hole.</li>
	</ol>
	</li>
	<li>Fabricate the box (Figure 1E).
	<ol>
		<li>Draw the two long-sided box pieces (parts &#8220;a&#8221; and &#8220;b&#8221;) in the CAD software as rectangles with dimensions of 4.92 in x 6.63 in (W x L). Add a cut-out to part &#8220;a&#8221; centered on the bottom edge measuring 0.2 in x 6.11 in (W x L).</li>
		<li>Draw the two narrow-sided box pieces (parts &#8220;d&#8221; and &#8220;e&#8221;) in the CAD software as rectangles with dimensions measuring 1.805 in x 6.63 in (W x L).</li>
		<li>Draw the box top (part &#8220;c&#8221;) as a rectangle with dimensions 1.805 in x 6.63 (W x L). Draw the &#8220;imaging through-hole&#8221; on the top: 0.74 in x 0.910 in (W x L), positioned 3.17 in from the bottom, 2.53 in from the top, 0.65 in from the right edge, and 0.42 in from the left edge. 
		NOTE: The exact position of the imaging through-hole should be selected on the basis of the cell phones that will be used for the analysis.</li>
		<li>Draw each box piece to feature a pattern of interlocking edges that will allow all the box sides to snap together on each edge as described in Figure 1D. To make an interlock edge pattern, alternate an extrusion/intrusion pattern on the long edge with 0.135 in by 1.17 in (W x L) protrusions. Draw two extrusions on each long edge for every side of the box. Use the same extrusion/intrusion pattern for the short edge, but with intrusions measuring 0.135 in by 0.460 in (W x L).</li>
		<li>Cut the five pieces with a laser cutter or print them with a 3D printer. 
		NOTE: A laser-cut component using acrylic pieces will be cheap to manufacture and can be flattened for easy shipping. Use black acrylic as it is helpful to absorb scattered light during testing.</li>
		<li>Add black color construction paper to the box interior to prevent scatter from the flash during image analysis if the box material has a gloss finish.</li>
	</ol>
	</li>
</ol>
2. Prepare the test
<ol>
	<li>Download the UrineTest mobile application from GitHub (https://github.com/Iftak/UrineTestApp).&#160;</li>
	<li>Install the app onto a mobile phone.&#160; 
	NOTE: This step only has to be done once for all future uses of a given phone. If needed, enable developer status on the phone to do this.&#160;</li>
	<li>Launch the UrineTest application in the phone (Figure 2A).</li>
	<li>Read the instructions to change the analyte names and reading timings (Figure 2B) to match those for the dipstick of interest (based on the manufacturer&#8217;s specifications) and insert new input via the text holder window on the screen (Figure 2C). 
	NOTE: The necessary readout time for each dipstick pad will depend on the brand of the dipstick used.</li>
	<li>Assemble the various components together and insert the dipstick into the through-holes underneath the plate sleeve (Figure 1F).&#160;</li>
	<li>Place the plate sleeve inside the box so that its notch is aligned with the box gap.&#160;</li>
	<li>Place the slide inside the plate sleeve so that its through-holes align with the inlet.</li>
	<li>Place the phone on the top of the box with the back-camera lens facing the viewing through-hole to enable imaging. Ensure the camera visibility is not occluded by checking for the image on the phone screen prior to testing. The app will enable the flashlight on the phone automatically.</li>
	<li>Read the instruction for phone alignment (Figure 2D) and align the phone accordingly so that the dipstick coincides with the boundaries of the black rectangular overlay on screen (Figure 2E).</li>
	<li>Click the Start button on the app window to begin the test. 
	NOTE: This will open the phone camera to read the QR code once in view (Figure 2F).</li>
</ol>
3. Conduct the test
<ol>
	<li>Deposit urine into the inlet hole with a disposable polyethylene transfer pipet containing approximately 0.5 mL of urine (Figure 3). 
	NOTE: The exact amount of liquid is not important, but it should be at least 0.5 mL to ensure that all the through-holes receive sufficient urine. Upon adding the liquid, observe that it moves across inlet and is deposited in each through-hole of the slide.</li>
	<li>Initiate the test by pushing the slide into the plate sleeve until it is stopped by the base plate stop. 
	NOTE: Urine should make contact with dipstick pad when the QR code is in the field of view of the cell phone. After reading the QR code, the application will open a window to analyze the color changes (Figure 2G) and show the results automatically within the same window (Figure 2H).</li>
	<li>Discard the urine appropriately and clean the plate sleeve and slide with 10% bleach solution and rinse again with de-ionized water. Allow it to dry before additional use.</li>
</ol>

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The authors have nothing to disclose.

Traditional dipstick urinalysis is affordable and convenient but requires manual attention to detail to yield accurate results. Manual dipstick urinalysis is subject to variable lighting conditions, individual color perception differences and cross-contamination. Many clinics and hospitals already have instruments to automate urine dipstick analysis, but the instruments are usually bulky, expensive, and still rely on proper performance of the dip-wipe method. Additionally, these instruments require yearly calibration and...

Urine is a non-invasive source of multiple metabolic indicators of disease or health. Urinalysis, the physical and/or chemical analysis of urine, can be performed quickly to detect renal disease, urinary tract disease, liver disease, diabetes mellitus, and general hydration1. Urinalysis dipsticks are affordable, semi-quantitative diagnostic tools that rely on colorimetric changes to indicate approximate physiological levels. Each dipstick can perform a wide variety of assays including testing for pH, osmolality, hemoglobin/myoglobin, hematuria, leukocyte esterase, glucose, proteinuria, nitrite, ketone, and bilirubin2. The principle of dipstick urinalysis relies on the occurrence of a timed reaction through which a color change on the dipstick pad can be compared to a chart to determine analyte concentration3. Given their affordability and ease of use, dipsticks are one of the most common tools for urinalysis in healthcare.
Traditionally, dipstick urinalysis relies on a trained nurse or medical technician to manually insert the dipstick into a cup of urine sample, wipe off excess urine, and compare the color pads to chart colors at specific times. While the dip-and-wipe method is the gold standard for dipstick analysis, its reliance on human visual assessment limits the quantitative information that can be obtained. Moreover, the two manual steps of dipstick urinalysis &#8211; the dip-wipe step and colorimetric result comparison &#8211; require accurate technique, which limits the possibility of reliable testing in home settings by patients directly. Cross-contamination of the sample pads due to wiping can cause inaccurate color changes. Additionally, inconsistent volumes resulting from the lack of volume control during wiping can result in improper measurement of analyte concentrations. Importantly, the time between dipping the urine (i.e., the start of the assay) and comparison to a chart is critical for accurate analysis of the results and is a huge potential source of human error. The difficulty in manual colorimetric comparison is that many pads must be read at the same time, while some pads are read at different times. Even perfectly timed color comparisons still depend on the visual acuity of the human reader, who may suffer from color blindness or perceive different colors in different lighting environments4. These challenges underscore why clinicians can only rely on dipstick urinalysis performed by trained personnel. However, an automated urinalysis system could address all the aforementioned concerns by eliminating the need for manual dip-wipe steps, incorporating timing controls, and enabling simultaneous color comparisons with calibrated color references. This, in turn, would reduce user error, allowing for possible adoption in home settings.
In the last 20 years, automatic analyzers have been employed to read the results of dipstick urine tests with the same accuracy as or exceeding visual analysis5. Many clinics and doctor offices use such machines to rapidly analyze and print traditional dipstick results. Most urinalysis machines minimize visual inspection errors and ensure consistency in results6. They are easy to use and more efficient than manual inspection but still require the user to perform the dip-wipe method correctly. Hence, these machines have limited ability to be operated by untrained persons such as at-home users; moreover, they are extremely expensive.
Recently, cell phones have emerged as a resourceful tool for various biological colorimetric measurements7,8,9,10, including for urinalysis11,12,13. Given their remote sensing capabilities and high imaging resolution, cell phones have become effective healthcare analytical devices14,15. Indeed, the FDA has cleared several smartphone-based home urine tests16,17,18. Some of the new smartphone-based commercial products incorporate established urinalysis dipsticks, while others feature proprietary colorimetric pads. All such products feature proprietary methods to calibrate for different lighting conditions across different phones types. Still, a problem with these solutions is that the user must manually take a picture at the right time in addition to carrying out a proper manual dip-wipe method (i.e., without cross-contamination). Notably, none of these tests control the volume deposited onto the dipsticks, which we have found can affect the color change19 and interpreted physiological result. The present gaps and costs in the workflows of these devices suggest an additional need to enable a human-free, volume-controlled urine deposition procedure and hands-free dipstick photography.
We describe a protocol for volume-controlled, automated dipstick urinalysis without the need for a manual dip-wipe step. The key to the automated process is a device19 whose underlying principle is based on the SlipChip20 and that transfers liquid between different layers using surface chemistry effects. In brief, the hydrophobic coating on the transfer slide and surrounding plate sleeve force the liquid to move effortlessly through the device and to release onto the dipstick pad once the slide is in its final position, at which point the bottom hydrophobic barrier is replaced with air. Additionally, the coordinated light-blocking box standardizes the lighting conditions, camera angle of view, and the distance for camera focus to ensure accurate and repeatable results that are not influenced by ambient lighting conditions. An accompanying software app automates the capture of images and colorimetric analysis. Following description of the protocol, we provide representative results of the urinalysis test under different conditions. Comparisons with the standard dip-wipe method demonstrate reliability of the proposed method.

<table><tbody><tr><td>Black Cast Acrylic Sheet&lt;br/&gt; 12&quot; x 24&quot; x 1/8&quot;</td><td>McMaster Carr</td><td>8505K742</td><td>$14.27</td></tr><tr><td>Chart sticker</td><td>Stickeryou.com</td><td></td><td>$12.39</td></tr><tr><td>Clear Scratch- and UV-Resistant Cast Acrylic Sheet&lt;br/&gt; 12&quot; x 24&quot; x 1/16&quot;</td><td>McMaster Carr</td><td>8560K172</td><td>$9.52</td></tr><tr><td>disposable polyethylene transfer pipet</td><td>Fischer Brand</td><td>13-711-9AM</td><td>lot# : 14311021</td></tr><tr><td>Fortus ABS-M30</td><td>Stratasys</td><td>345-42207</td><td>lot# : 108078</td></tr><tr><td>Githut: https://github.com/Iftak/UrineTestApp</td><td></td><td></td><td></td></tr><tr><td>Innovating Science - Replacement Fluids for Urinalysis Diagnostic Test Kit (IS3008)</td><td>Amazon</td><td></td><td>$49</td></tr><tr><td>Nonwhitening Cement for Acrylic&lt;br/&gt; Scigrip 4, 4 oz. Can</td><td>MCM</td><td>7517A1</td><td>$9.22</td></tr><tr><td>Rust-Oleum 274232 Repelling treatment base coat-9 oz and top-coat 9-oz , Frosted Clear</td><td>Amazon</td><td>Color: Frosted Clear</td><td>$6.99</td></tr><tr><td>Urinalysis Reagent Strips 10 Panel (100 Tests) MISSION BRAND</td><td>Medimpex United, Inc</td><td>MUI-MS10</td><td>$10.59</td></tr></tbody></table>

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.

Figure 4 demonstrates how the urine is transferred to the dipstick during a urinalysis test. During a typical test, the transfer of urine is not observable because the box occludes the view. Once the sample is deposited in the inlet using a pipette (Step 3.1), it will fill the holes on the slide (Figure 4A). Figure 4B and Figure 4C, respectively, show the progressive movement of the urine across the pla...

Watch this Scientific Journal Video about Low-Cost, Volume-Controlled Dipstick Urinalysis for Home-Testing at JoVE.com

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

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 ...

low-cost-volume-controlled-dipstick-urinalysis-for-home-testing

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JoVE Journal

Bioengineering

5.4K Views.  Vanderbilt University. Dipstick urinalysis is a quick and affordable method of assessing one’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.

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

Integration of Miniaturized Solid Phase Extraction and LC-MS/MS Detection of 3-Nitrotyrosine in Human Urine for Clinical Applications

Free 3-nitrotyrosine (3-NT) has been extensively used as a possible biomarker for oxidative stress. Increased levels of 3-NT have been reported in a wide variety of pathological conditions. However, existing methods lack the sufficient sensitivity and/or specificity necessary to measure the low endogenous level of 3-NT reliably and are too cumbersome for clinical applications. Hence, analytical improvement is urgently needed to accurately quantify the levels of 3-NT and verify the role of 3-NT in pathological conditions. This protocol presents the development of a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) detection combined with a miniaturized solid phase extraction (SPE) for the rapid and accurate measurement of 3-NT in human urine as a non-invasive biomarker for oxidative stress. SPE using a 96-well plate markedly simplified the process by combining sample cleanup and analyte enrichment without tedious derivatization and evaporation steps, reducing solvent consumption, waste disposal, risk of contamination and overall processing time. The employment of 25 mM ammonium acetate (NH4OAc) at pH 9 as the SPE elution solution substantially enhanced the selectivity. Mass spectrometry signal response was improved through adjustment of the multiple reaction monitoring (MRM) transitions. Use of 0.01% HCOOH as additive on a pentafluorophenyl (PFP) column (150 mm x 2.1 mm, 3 &#181;m) improved signal response another 2.5-fold and shortened the overall run time to 7 min. A lower limit of quantitation (LLOQ) of 10 pg/mL (0.044 nM) was achieved, representing a significant sensitivity improvement over the reported assays. This simplified, rapid, selective and sensitive method allows two plates of urine samples (n = 192) to be processed in a 24 h time-period. Considering the markedly improved analytical performance, and non-invasive and inexpensive urine sampling, the proposed assay is beneficial for pre-clinical and clinical studies.

A selective and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method coupled with an efficient solid phase extraction on a mixed-mode cation-exchange (MCX) 96-well microplate was developed for the measurement of free 3-nitrotyrosine (3-NT) in human urine with high throughput, which is suitable for clinical applications.

Free 3-nitrotyrosine (3-NT) has been extensively used as a possible biomarker for oxidative stress. Increased levels of 3-NT have been reported in a wide ...

Chemistry

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 ...

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 ...

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

Real-Time Void Spot Assay

Normal voiding behavior is the result of the coordinated function of the bladder, the urethra, and the urethral sphincters under the proper control of the nervous system. To study voluntary voiding behavior in mouse models, researchers have developed the void spot assay (VSA), a method that measures the number and area of urine spots deposited on a filter paper lining the floor of an animal&#8217;s cage. Although technically simple and inexpensive, this assay has limitations when used as an end-point assay, including a lack of temporal resolution of voiding events and difficulties quantifying overlapping urine spots. To overcome these limitations, we developed a video-monitored VSA, which we call real-time VSA (RT-VSA), and which allows us to determine voiding frequency, assess voided volume and voiding patterns, and make measurements over 6 h time windows during both the dark and light phases of the day. The method described in this report can be applied to a wide variety of mouse-based studies that explore the physiological and neurobehavioral aspects of voluntary micturition in health and disease states.

This article describes a new method to study mouse voiding behavior by incorporating video monitoring in the conventional void spot assay. This approach provides temporal, spatial, and volumetric information on the voiding events and details of mouse behavior during the light and dark phases of the day.

Normal voiding behavior is the result of the coordinated function of the bladder, the urethra, and the urethral sphincters under the proper control of the ...

Behavior

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 ...

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 ...

A Decentralized (Ex Vivo) Murine Bladder Model with the Detrusor Muscle Removed for Direct Access to the Suburothelium during Bladder Filling

Previous studies have established the release of chemical substances from flat bladder mucosa sheets affixed in Ussing chambers and exposed to changes in hydrostatic pressure or mechanical stretch and from cultured urothelial cells upon hydrostatic pressure changes, stretch, cell swelling, or drag forces, and in bladder lumen at end of filling. Such findings led to the assumption that these mediators are also released in suburothelium (SubU)/lamina propria (LP) during bladder filling, where they affect cells deep in the bladder wall to ultimately regulate bladder excitability. There are at least two obvious limitations in such studies: 1) none of these approaches provide direct information about the presence of mediators in SubU/LP, and 2) the stimuli used are not physiological and do not recapitulate authentic filling of the bladder. Here, we discuss a procedure that enables direct access to the suburothelial surface of the bladder mucosa in the course of bladder filling. The murine detrusor-free preparation we created closely resembles filling of the intact bladder and allows pressure-volume studies to be performed on the bladder in the absence of confounding signaling from spinal reflexes and detrusor smooth muscle. Using the novel detrusor-free bladder model, we recently demonstrated that intravesical measurements of mediators cannot be used as a proxy to what has been released or present in the SubU/LP during bladder filling. The model enables examination of urothelium-derived signaling molecules that are released, generated by metabolism and/or transported into the SubU/LP during the course of bladder filling to transmit information to neurons and smooth muscle of the bladder and regulate its excitability during continence and micturition.

A Decentralized Ex Vivo Murine Bladder Model with the Detrusor Muscle Removed for Direct Access to the Suburothelium during Bladder Filling

The detrusor-free bladder model enables direct access to the suburothelium to study local mechanisms for regulation of biologically active mediator availability in suburothelium/lamina propria during storage and voiding of urine. The preparation closely resembles filling of an intact bladder and allows pressure-volume studies to be performed without systemic influences.

Previous studies have established the release of chemical substances from flat bladder mucosa sheets affixed in Ussing chambers and exposed to changes in ...

Medicine

A High-Throughput Electrochemiluminescence 7-Plex Assay Simultaneously Screening for Type 1 Diabetes and Multiple Autoimmune Diseases

Islet autoantibodies (IAbs) are widely used in type 1 diabetes (T1D) diagnosis and prediction. Four major IAbs to insulin (IAA), glutamate decarboxylase-65 (GADA), insulinoma antigen-2 (IA-2A), and zinc transporter-8 (ZnT8A) are equally important in disease prediction. Presently, up to 40% of patients diagnosed with T1D go on to develop other autoimmune disorders. Unfortunately, current screening methods using a single autoantibody for measurement are laborious and inefficient for large scale screening studies. We recently developed a simple multiplexed electrochemiluminescence (ECL) assay to address these current issues. The assay combines all 7 autoantibody tests into one well. Each well includes three IAbs (IAA, GADA, and IA-2A), autoantibodies to thyroid peroxidase (TPOA) and thyroid globulin (ThGA) to detect autoimmune thyroid disease, autoantibodies to tissue transglutaminase (TGA) for celiac disease, and autoantibodies to interferon alpha (IFN&#945;A) for autoimmune polyglandular syndrome-1 (APS-1); all of which screen for T1D and other relevant autoimmune diseases, simultaneously. The multiplex ECL assay is based on the single ECL assay platform, but instead uses the multiplex plate combining multiple autoantibody assays, up to 10, into a single well. The main difference from the single ECL assay is that each antibody-antigen complex formed in the fluid-phase is restrained onto a specific spot on each well through a linker system on the multiplex plate. The 7-Plex ECL assay, in the present study, is validated against standard radio-binding assays (RBA) and single ECL assays, using a large cohort of newly diagnosed T1D patients and age-matched healthy controls, resulting in excellent assay sensitivity and specificity.

We model a simple multiplexed ECL assay that combines 7 autoantibody assays together. The assay is capable of screening for T1D and multiple other autoimmune diseases, simultaneously, including celiac disease, autoimmune thyroid disease, and autoimmune polyglandular syndrome 1.

Islet autoantibodies (IAbs) are widely used in type 1 diabetes (T1D) diagnosis and prediction. Four major IAbs to insulin (IAA), glutamate ...

Collection, Processing, and Storage Consideration for Urinary Biomarker Research

There are several urinary proteins that show promise as novel markers of urinary tract infections. The identification of a novel biomarker that has greater predictive accuracy compared to current diagnostic methods has the potential to greatly improve the ability to manage patients with urinary tract infections. However, sample collection, processing, and storage can all potentially impact the results of biomarker research. Understanding the effects of each of these stages on biomarker studies is necessary to inform future, high-quality research in this area, as well as to critically review other studies in this area. Here, the study reviews the literature regarding the effects of each stage of urine sample processing and reports the effects of various conditions on urinary proteins. The protocol will focus on collection techniques, time and temperature of storage, processing techniques, use of reagents, and long-term freezing on biomarker stability. It will focus on proteins but will briefly discuss other materials that may be utilized in biomarker research. In doing so, this protocol will provide a guide to future researchers to assist in the design of urinary biomarkers studies.

This protocol aims to provide considerations for urine sample collection, processing, and storage for urine tract infection biomarker studies.

There are several urinary proteins that show promise as novel markers of urinary tract infections. The identification of a novel biomarker that has ...