An improved urine-testing system for people suffering from kidney stones inspired by nature and proposed by researchers from Penn State and Stanford University may enable patients to receive results within 30 minutes instead of the current turnaround time of a week or more.
Kidney stones occur due to buildup of certain salts and minerals that form crystals, which in turn stick together and enlarge to form a hard mass in the kidneys. The stones move into the urinary tract and can cause blood in the urine, considerable pain and blockages in the urinary system.
Metabolic testing of a kidney stone patient’s urine to identify metabolites such as minerals and solutes that cause stones to form is key for preventing future ones. This testing is currently done by requiring the patient to collect their urine over a 24-hour period in a large container. The container is then sent to a lab for analysis and the results normally come back in 7 to 10 days.
“The lengthy process, cumbersome collection procedure and delay in obtaining the results render 24-hour urine testing to be underutilized in clinical practice despite guideline recommendations,” said Pak Kin Wong, professor of biomedical engineering and mechanical engineering and principal investigator on the study. The research was published today in Science Advances.
Wong said that expensive special equipment is required to detect urinary solutes and minerals for a test result. The urine sample, therefore, has to be shipped to a commercial diagnostic lab for testing. To solve this, the research team developed a biomimetic detection system called slippery liquid-infused porous surface (SLIPS)-LAB.
SLIPS is a dynamic, extremely low-friction smooth surface created by locking lubricating liquids in micro/nanostructured substrates. This is inspired by nepenthes pitcher plants, which are carnivorous plants that have unique leaves shaped like pitchers and are filled with digestive liquid. The plants have evolved extremely slippery liquid-infused micro-textured rims that cause insects to fall into the “pitcher.”
“There are many aspects we can learn from nature and our environment, and our research is an example how biomedical engineers can make good use of it,” Wong said.
SLIPS-LAB works by enabling reagent and urine droplets to easily move over the slick surface of the testing device’s fluid addition channel and not get stuck. The droplet is driven by a Laplace pressure difference, a small pressure force due to surface tension, induced by the geometry of the device. This enables the reactants to combine with the urine at the necessary timed rate for reaction.
“We demonstrated that SLIPS-LAB enables the reagent and sample to move themselves and perform the reactions for us,” Wong said. “It means the technology doesn’t require a technician to run any test machinery, so it is possible to do the test in non-traditional settings, like a physician’s office or even the patient’s home.”
The test results can then be read using a scanner or a cell phone, and the scanned image can then be analyzed using a computer algorithm. All these steps, according to Wong, would take approximately 30 minutes in a physician’s office. An added benefit, Wong said, is that SLIPS-LAB is more cost-effective than regular, 24-hour testing.
“The low cost, rapidity and simplicity of SLIPS-LAB would reduce the barrier for the clinician and patient to undergo stone risk metabolite analysis,” Wong said. “This would improve the management of patients with urinary stone disease and open new possibilities for stone patients to test their urine samples in mobile health settings.”
The lead author of the study, Hui Li, graduate student in biomedical engineering, said another promising result of their research was demonstrating that the test also works as a spot test, which means a patient can monitor certain levels in their urine without 24-hour collection.
“SLIPS-LAB may open new opportunities in on-demand monitoring of urinary analytes and may potentially transform metabolic evaluation and clinical management of urinary stone disease,” Li said.
SLIPS-LAB liquid handling
We first demonstrated SLIPS-LAB for liquid handling of various viscous fluids and biological fluids (Fig. 1C and fig. S2). This capability is important because reagents and samples may have diverse viscosities and properties (A. Keiser, P. Baumli, D. Vollmer, D. Quéré, Universality of friction laws on liquid-infused materials. Phys. Rev. Fluids 5, 014005 (2020).
The viscous fluids included water, milk, juice, glycerol, syrup, and honey. These fluids cover a large range of viscosity (1 to ~5000 centipoise) and various liquid handling applications, such as drug screening, environmental monitoring, and food safety (table S1).
The biological fluids included urine, saliva, tracheal aspirate, plasma, and whole blood. The fluids were loaded in the bottom inlets (Fig. 1C, t = 0 s), and the device was placed horizontally. The droplet transportation process was initialized when the air hole was unsealed. Droplet movement was induced by the channel geometry without external pump, power, or control (movie S1).
Droplets were transported from the bottom inlets toward the reaction chambers (Fig. 1C, t = 6 s). Urine samples required less than 10 s to reach the reaction chamber. The samples were subsequently mixed in the chambers (Fig. 1C, t = 670 or 92 s). Droplet transportation was robust, and there were no visible residues or traces on the SLIPS-coated channels for all fluids.
Working principles of SLIPS-LAB
We investigated the working principles of SLIPS-LAB for performing important liquid handling procedures including volume metering, droplet transportation, reaction time control, and mixing for biochemical analysis (Fig. 2A and movie S2).
We first established two mechanisms of liquid sampling with a large range of volume (Fig. 2A, step 1). To sample a small volume, capillary force was sufficient to trap the liquid in a punched hole of the device when the sample passed through the hole (Fig. 2B and movie S3).
The punched holes, which served as the top inlets, controlled the sample volume (1 to 15 μl) by the hole diameter and height (Fig. 2G and fig. S3). This volume range represents fluid samples with a dimension smaller than the capillary length, lc = (γLV/ρg)0.5, where γLV is the interfacial tension of the liquid-vapor interface, ρ is the liquid density, and g is the gravitational acceleration.
The capillary length is approximately 2.7 mm for water (the cubic root of 15 μl or mm3 is ~2.5 mm). To sample a large volume (i.e., dimensions larger than lc), we designed a mechanism by drawing liquid from the reservoir (Fig. 2C and fig. S4). The device was dipped vertically into the reservoir with the air hole opened (P.-G. de Gennes, F. Brochard-Wyart, D. Quere, Capillarity and Wetting Phenomena: Drops, Bubbles, Pearls, Waves (Springer, 2004)).
The air hole was then sealed, and liquid droplets were trapped in the channel by air pressure when the channel was removed from the liquid reservoir. By engineering the dipping height and channel thickness, this approach was demonstrated for handling fluids in the range of 10 to 50 μl (Fig. 2H).
These ranges (both small volume and large volume) were sufficient for implementing all biochemical assays in this study. The sampling and volume metering processes were repeatable and were insensitive to the converging angle (0° to 20°) of the loading channels (fig. S5).
Autonomous droplet transportation was demonstrated in SLIPS-LAB (Fig. 2A, step 2, and fig. S6). We designed a converging channel geometry to create droplet motion (Fig. 2D). The geometry of the channel modulated the Laplace pressure and the projected area of the droplet.
In the converging channel, the droplet experienced a differential force between the sides, and a nonzero net force was created to drive the droplet toward the narrow side of the channel (Fig. 2E).
This transportation mechanism, however, does not work with a typical PDMS microchannel (e.g., the same channel without the lubricant) because of the large contact angle hysteresis (H. Mishra, A. M. Schrader, D. W. Lee, A. Gallo Jr., S. Y. Chen, Y. Kaufman, S. Das, J. N. Israelachvili, Time-dependent wetting behavior of PDMS surfaces with bioinspired, hierarchical structures. ACS Appl. Mater. Interfaces 8, 8168–8174 (2016)). Contact angle hysteresis, which is the difference between receding and advancing contact angles, represents a retention force against the droplet motion. In contrast, the contact angle hysteresis is markedly reduced to as few as 3° with SLIPS coatings (N. Vogel, R. A. Belisle, B. Hatton, T. S. Wong, J. Aizenberg, Transparency and damage tolerance of patternable omniphobic lubricated surfaces based on inverse colloidal monolayers. Nat. Commun. 4, 2167 (2013).), enabling autonomous droplet transportation. In our experiment, the loading speed was controlled by the converging angle and channel thickness (Fig. 2I and movie S4). The sample remained stationary in the straight channel (i.e., 0° converging angle) in the experiment.
Reaction sequence and delay time control for multistep biochemical reactions were realized by regulating the loading time in between droplets (Fig. 2A, step 3). Because the droplet loading speed depends on the channel geometry, the reaction delay time can be preprogrammed in the design of SLIPS-LAB. For instance, the converging angle and the channel thickness can be adjusted for reaction time control.
By preprogramming the converging angle between 5° and 20° and the thickness of the channel between 0.75 and 3.0 mm, the loading time was tuned from 3 s to over 4 min (Fig. 2I). As a demonstration, a SLIPS-LAB device with two inlets connected to a reaction chamber was designed with a converging angle of 20° on the left channel and a converging angle of 5° on the right channel (Fig. 2A).
In addition to water, reaction time control was demonstrated using human whole-blood samples (movie S5). Additional reaction time control, e.g., 30 min, could be implemented by resealing the air hole, which stopped the motion of the second droplet and opening the air hole again after the desired delay time (movie S6).
Fluid droplets generally stop at the end of the converging channel without entering the chamber (see movie S4 for an example). To address this issue, a 3D reaction chamber design was developed to guide the droplet from the channel to the reaction chamber for mixing and detection (Fig. 2A, step 4, and fig. S7A).
The 3D reaction chamber, which has a reduction in height and a curved chamber boundary, facilitates wetting of the fluid on the reaction chamber surface and loading of the droplet. The design was demonstrated in different configurations experimentally (Fig. 2A and fig. S7B).
Multiple air vents were incorporated in the design to avoid trapping of air between the droplets. Once the droplets are in contact, mixing of fluids was governed by coalescence kinetics (25, 26).
For instance, the sample in the top inlet was first mixed with the reagent loaded from the left inlet (Fig. 2A, t = 10 s, and Fig. 2F, step 1). A multistep reaction can be achieved with the second reagent loaded from the right inlet (Fig. 2A, t = 36 s, and Fig. 2F, steps 2 to 4). The liquid handling processes are repeatable, and the droplet transportation can be performed in the same device multiple times (figure S8) (M. J. Kreder, D. Daniel, A. Tetreault, Z. Cao, B. Lemaire, J. V. I. Timonen, J. Aizenberg, Film dynamics and lubricant depletion by droplets moving on lubricated surfaces. Phys. Rev. X 8, 031053 (2018).).
SLIPS-LAB for multiplex detection of urinary stone–associated analytes
We designed a six-plex SLIPS-LAB device for metabolic evaluation of urinary stone disease (Fig. 3, A to D, and fig. S9, A and B).
The device conducts colorimetric and enzymatic assays in parallel for detecting calcium, citrate, uric acid, oxalate, and pH, which are among the most clinically relevant urinary analytes to assess stone risk and treatment response (Fig. 3E).
The assay procedure for each analyte was designed according to the manufacturer’s instructions. For instance, the calcium assay requires mixing of the calcium detectors with o-cresolphthalein complexone and 8-quinolinol, whereas the citrate assay requires only a single master solution containing citrate lyase, horseradish peroxidase, and 10-acetyl-3,7-dihydroxyphenoxazine.
Uric acid and oxalate assays involve multiple steps with or without the requirement of a delay between the reactions. For example, the oxalate assay requires a minimum of 3 min between the first and second reactions (oxalate converter and enzyme mix).
Furthermore, the assays require handling of fluid volumes that are over 30-fold apart (from 1.4 to 47 μl). Additional descriptions of the assays are described in figure S9C and table S2. These reactions result in products that can be detected colorimetrically.
We engineered the thickness of the channels (bottom inlets) to sample liquids of desired volumes. Two channel thickness values (1.5 and 3.0 mm) were included in the design, and the device sampled fluids from a laser-machined reservoir array that has a height of 3 mm (190 μl per well).
The sample volumes drawn were 30 and 47 μl based on our calibration (Fig. 2H). The converging angle was adjusted between 5° and 20° to control the droplet loading time. For the calcium, citrate, and uric acid assays (3 mm thickness), the droplets reached the reaction chambers in ~3 s.
For the oxalate, pH, and control assays (1.5 mm thickness), the droplets reached the reaction chambers in ~6 s. An exception is that the master solution for oxalate detection was loaded in ~220 s by controlling the converging angle to implement the 3-min delay time required for the reaction (Fig. 2I).
In addition, the volume of the top inlet was controlled by the diameter of the punch hole (1.5 or 2.0 mm) and the thickness of the top PDMS layer (3.0 and 2.0 mm). The diameter, height, and location of the top inlets were optimized to sample liquids (Fig. 3C and fig. S9B).
Liquid handling with the six-plex SLIPS-LAB device was demonstrated (Fig. 3D and movie S7). Droplet transportation was initiated when the air holes were unsealed. The droplets were transported according to the design (Fig. 3D, boxes 1 and 3).
For the oxalate assay (Fig. 3D, box 2), as an example, the sample in the right inlet was first loaded and mixed with the reagent in the top inlet. The top inlet was positioned to the right to facilitate the merging of the urine and the converter. Agitation by gently shaking the device was performed to speed up the reactions. The volume and inlet position for each reagent are shown in Fig. 3E.
The colorimetric assays were measured using a desktop scanner to demonstrate SLIPS-LAB for point-of-care diagnosis without bulky supporting equipment and a centralized laboratory. These biochemical assays were calibrated using SLIPS-LAB (Fig. 3F).
The changes of the RGB (red, green, and blue) intensity values and the most sensitive color element or the combination of the sensitive color elements were determined for each assay (fig. S10). The calcium, uric acid, and citrate assays were represented by the green element, and the oxalate assay was represented by the blue element.
These color elements were consistent with the recommended wavelengths for absorbance measurement. Regression analysis was performed to determine a calibration curve for each assay (fig. S11). The pH value was determined by a combination of red and green elements.
More information: “SLIPS-LAB—A bioinspired bioanalysis system for metabolic evaluation of urinary stone disease” Science Advances (2020). advances.sciencemag.org/content/6/21/eaba8535