Spin characterization

CHAPTER 2. Design and characterization of diagnostic fidget spinner (Dx-FS)

2.3 Designing Diagnostic fidget spinner (Dx-FS)

2.3.5 Spin characterization

After identifying the design and components of the device we set to understand how the liquid will behave in motion inside the Dx-FS. First we set out to understand the spin speed profile of Dx-FS was quantified using the homemade setup shown in Figure 2.3a. In brief, a 532 nm laser (CPS532-C2;

Thorlabs, NJ, USA) illuminated a light sensor (LX1972; DFROBOT, Shanghai, China) configured with an Arduino controller (DFRduino UNO R3; DFROBOT, Shanghai, China). Dx-FS was installed in the light path and blocked the light during every spin. The light intensity was acquired at a rate of 6 kHz.

The laser intensity measurements were analyzed using custom-built software (Figure 2.3b) in MATLAB (MathWorks, MA, USA). We also captured the detailed spinning motion of Dx-FS using a high-speed camera (Phantom Miro 310; Vision Research, NJ, USA). Videos were recorded at 500 frames per second, and the frames are shown in Figure 2.3b. The spinning duration vs number of spin was plotted as shown in Figure 2.3c. The graph plotting revolution per minute (RPM) vs number of spins shows that the device goes to a maximum of 1200 RPM in a single spin without the liquid. Spin acceleration was measured by estimating derivative of the angular velocity. We confirm that the spinner accelerated to higher angular velocity in two seconds and decays over a time period of 1.4 minutes without the fluid. In case when it contains fluid of 1mL during the operation the resistance of the Nano porous membrane delays resist the flow and causes uneven flow condition on the membrane interface but with suitable changes in the condition we achieved uniform flow for the spin profile as shown in Figure 2.1e, f. Its important to notice that the maximum angular velocity can defer from subject to subject and can cause variation in the result. To make a rational of how the device can be used we performed the tests with 10 subjects (five men and five women) aged 19–45 years were carried out (Figure 2.4d, e). Condition of the fluid to mimic different density and various concentration of bacteria

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were tested for better understand the working range and practical utility of the device. The final design was modified, and the inlets and vent zone were positioned in a way that the liquid doesn’t leak out during the operation.

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Figure 2.2. Dimensions and assembly of Dx-Fidget Spinner (Dx-FS). (a) Dimensions of Dx-FS. The top view of the device with a length of 106 mm, a width of 50 mm, and a filtration chamber for installation of the filter with a diameter of 10 mm (upper panel). The side view of the filtration unit within the device with the sample loading area (yellow) of the NC membrane and the area for the backwater (blue) (lower panel). (b) Exploded view of the device. The top layer and main body were attached using an adhesive layer.

Figure 2.3. Spin characterization setup for Dx-FS. (a) Setup for spin characterization. Dx-FS was installed on an optical post and rotated by hand. The spinning motion of Dx-FS was captured using a high-speed camera and custom spin detector, as described in Methods. (b) Example spin measurements of Dx-FS using the setup in (a) shown as the normalized light intensity versus time. Here, Dx-FS blocks the laser twice per rotation, resulting in low optical density measurements from the light detector. (c) Measurements from (b) analyzed and plotted as the spinning duration (left panel) and rotational speed (right panel) for characterizing the rotational speed of Dx-FS. Spin acceleration was measured by estimating derivative of the angular velocity.

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Figure 2.4. Characterization of the fluid flow and operation. (a) Cross-sectional view of the device depicting sample bacterial enrichment through the filter membrane in the conventional (upper) and FAST (lower) setups. In FAST-based particle separation, the fluid flow caused by centrifugal force is in a direction perpendicular to the filtration flow through the membrane, similar to the tangential-flow filtration case. In addition, the drainage chamber underneath the membrane remains fully filled with the liquid during the entire filtration process (also refer Kim et al.30 for details). This is achieved by placing a buffer solution in the bottom chamber of the membrane prior to the spinning process, which ensures uniform filtration across the entire area of the membrane and significantly reduces the hydrodynamic resistance. (b) Images showing effective filtration during rotation and fluorescence images of bacterial cells enriched on the membrane for (1 and 3) conventional and (2 and 4) FAST filtration. Scale bar: 2 mm / 0.1 mm. (c) Flow volume of Dx-FS estimated at ω_max. (d) Number of spins by 10 independent subjects who spun Dx-FS. The angular velocities were measured, and the number of spins to elute 1 mL of liquid was estimated. (e) Measurements from (d) plotted for each subject. Red lines dashed red lines, and gray and red boxes over the measurements (gray dots) denote the mean, median, standard error of mean, and standard deviation. The gray areas indicate the number of spins in (d).

Figure 2.5. Bacterial cells retained on the surface of the membrane in Dx-FS. (a) An axial section of confocal images of the membrane containing GFP E. coli bacteria with a concentration of 10⁶ Bacteria/mL isolated using the Dx-FS. (b) SEM of the bacteria on the membrane.

48 2.4 Enrichment of bacteria with a few spins.

Dx-FS harnesses the kinetic energy imparted by hand when pushed and converts it into a centrifugal force (Figures 2.1e). Unlike motor-driven centrifugal devices, Dx-FS relies on the strength of each individual operator, which typically generates an angular rotational frequency ω smaller than 300 rad/s. Figure 2.1f shows the measurements of ω for Dx-FS over time (Figure 2.3). As the edge of the device is pushed by a finger, it shortly reaches a maximum angular rotational frequency ω max and then slows down over a minute.

2.4.1 Characterization of the filtration rate of Dx-FS.

To characterize the flow rate in Dx-FS-based filtration, we measured the liquid decanting time through the membrane at different angular velocities using our home-built spinning platform, as described previously.⁸⁶ Briefly, a servo motor (EDB2000-56V24/48-S; ERAETECH, South Korea) spun Dx-FS at the given rotational frequency. Images of the device were captured using a charge- coupled device (CCD) camera at five frames per second. The motor and camera were synchronized;

therefore, photographs were acquired at the same position of the device. The system was controlled using a custom-built LabVIEW software (National Instruments; TX, USA) program. The flow volume at a given angular rotational frequency can be estimated by integrating the flow rate over time. From the initial spin of Dx-FS, the angular rotational frequency reached its maximum and decreased over time (Figure 2.1f). To understand these conditions, spinning was simulated with a given initial angular rotational frequency with a specific angular acceleration. The flow volume was estimated by integrating the flow rate at the simulated angular rotational frequency (Figure 2.6). Similarly, the total flow volume from a single spin was estimated from the measured angular rotational frequency of Dx-FS (Figure 2.4c). All computations were performed using MATLAB software (MathWorks, MA, USA).

2.4.2 Introducing Fluid-assisted separation technology (FAST) in Dx-FS.

In early experiments when different concertation of bacteria and different density of liquids were filtered using Dx-FS, we notice that the device was unable to filter denser fluids in single spin. To understand the problem, we performed additional tests and found that the membrane interface was the bottle neck. Taking an electrical circuit analogy for Dx-FS, the flow at the membrane which in this case is nano porous (0.45 m, NC) adds additional resistance to the liquid – air interface when the fluid initial stating position (Figure 2.4 a). Practical use of Dx-FS becomes difficult with such level of resistance. The solutions to overcome this problem are by increase the number of spin or adapting an electrical motor as a driving force or reducing the membrane resistance. Despite the relatively low

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spinning speed with large variations, the filtration efficiency of Dx-FS is high because of the introduction of fluid-assisted separation technology (FAST)⁸² (Figure 2.4a). However, in conventional non-FAST case, where the space below the membrane is partially filled with air, a non-uniform pressure difference across the membrane is produced, which makes filtration possible only through the most vulnerable point (the highest-pressure difference). In such cases, the hand-powered spin speed is not sufficiently large, resulting in a significant volume of the sample remaining on the membrane (Figure 2.4a, Post-spin and Figure 2.4b-1, marked in red). However, no residual sample was observed in the FAST case (Figure 2.4b-2).

To further confirm the hypothesis of distribution we are filtering green fluorescent labeled bacteria cells of 10⁸ CFU/mL, on the membrane using both FAST and Non- FAST condition. The filters were imaged the membrane under high power fluorescent microscope. We observed a non-uniform distribution of bacteria in conventional filtration, which implies that the non-uniform fluid flow deposits concentrate the bacterial cells on the high-pressure area of the filter (Figure 2.4b1 and 3). When the same experiment was repeated with FAST condition, we could have observed a uniform spreading of bacterial cells on the porous filter, as it uses the entire membrane (Figures 2.4b2 and 4 and Figure 2.5).

We concluded that the FAST enable effective filtration by few hand spins while uniformly spreading bacteria on the membrane. Next to confirm the location of bacteria on the filter membrane and quantify the difference between both the approach the membranes were fixed and subjected to confocal imaging.

The Figure 2.5 clearly demonstrate that the E. Coli bacteria were stagnant on the membrane and didn’t enter the membrane while filtration. The intensity from the signal was measured and was found to be 11.6 times higher than the non-FAST method.

2.4.3 Comparing flowrates with FAST in Dx-FS

Owing to uniform filtration, the flow rate during filtration was also enhanced, as shown in Figure 2.4c and Figure 2.6.a & b. After understanding the difference in filtration using bacteria cells, we further tested liquid samples with various densities and levels of bacterial contamination and confirmed that such conditions do not affect the flow rate during the operation of Dx-FS (Figure 2.6 c

& d). However, both theoretically and experimentally it’s clear that the effect of fast shows close to 1.5 mL of liquid filtration at the same rotational frequencies, where non fast can only filter one third of the volume (Figure 2.4c ). Similarly, in the plot Figure 2.6a and b, in which the both the conditions were compared for flow rate to that of angular frequencies, the filtration was more than twice in FAST case and potentially filter about 1.5 mL/ min. To compare different density of fluids we first measured the density of water, Phosphate-buffer saline (PBS), PBS with bacteria 10⁵ &10⁶ bacteria / mL and also synthetic urine with similar number of bacteria (Figure 2.6c). The range of density was between 1.00

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g/cm³ to 1.10 g/cm³. Compared to non-FAST, FAST showed favorable filtration efficiency across 7 different solutions.

2.4.4 Understanding operator variation while using Dx-FS

Another challenge associated with hand-powered centrifugation is that each operator may use a different power to spin the device. To measure this difference, subjects across different age groups (19–45 years) were asked to perform the spinning operation. Figures 2.4d and e summarize the of ω max measurements of Dx-FS for 10 subjects with multiple trials. We measured ω max in the range of 90–300 rad/s from 62 measurements, and the average and standard deviation were 151 ± 48 rad/s (Figure 2.4d). There were large variations in the spin speed among subjects and during trials (Figure 2.4e). Here, we found that more than 12 manual spins were required to filter 1mL of the sample in conventional centrifugation. However, only one or two manual spins were sufficient to decant the same volume of the sample using FAST-enabled Dx-FS, which requires approximately 1–3 min. The completion of filtration process was confirmed by visual inspection of the empty filter chamber.

Similarly, in case of women we observed lower ω max, so we concluded that in some cases we would require additional spins until the entire volume is filtered. Overall, we confirmed that Dx-FS equipped with FAST-based filtration helps in achieving robust, fast, and efficient bacteria enrichment with minimal human effort.

2.4.5 Bacterial cell enrichment in Dx-FS

During the process of filtration, the bacteria from 1 mL of the sample, in this case being urine or water can be effectively enriched on a nitrocellulose membrane that the 10 m in diameter and 100 mm thick with 95% porosity. Figure 2.8 shows that mL of urine (left panel) containing bacterial cells (green) is processed using Dx-FS. All the urine is removed via hand-spinning and bacterial cells remain on the membrane (Figure 2.4b). The plot in Figure 2.8b shows that the Input liquid, i.e. 1 mL urine, is in 1,000 mm3 and the membrane is in 7.8540 mm3 in volume (left panel). Bacterial cells are with a 99.35%

recovery (Figure 3.1c), therefore, the Dx-FS makes the concentration 126.5 times higher than the input urine sample (right panel). In short, the membrane when filtered with 10⁵ bacteria /mL can be reduced to 10⁵ bacteria /10 L on the membrane.

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Figure 2.7. Inlet and Outlet sealing and FAST solution loading in Dx-FS. (a) Dx-FS equipped with rubber stoppers at the inlet and vent holes to avoid liquid overflow. The inlets (yellow) and vent holes (white) of the Dx-FS were incorporated with latex rings that prevent backflow or leakage during the operation of the device. (b) To realize the FAST method, water is loaded into the back chamber in Dx- FS using a syringe. The filter chamber is covered by a plastic cover with a hole, which is sealed by Para film layers. (c) Operation example. (1) The FAST solution (colored water in the example) was loaded using a syringe. (2) The sample solution was loaded into the sample chamber of Dx-FS. (3) The device is spun by hand. To visualize potential leakage, filter paper was taped over the hole before spinning. (d) Micrograph of the Para film layers in (c). The red inset shows a needle mark.

Figure 2.8. Bacterial cell enrichment in Dx-FS. (a) 1 mL of urine (left panel) containing bacterial cells (green) is processed using Dx-FS. All the urine is removed via hand-spinning and bacterial cells remain on the membrane (right panel, see Figure 2b). (b) Input liquid, i.e. 1 mL urine, is in 1,000 mm³ and the membrane is in 7.8540 mm³ in volume (left panel). Bacterial cells are with a 99.35% recovery (Figure 3.1c), therefore, the Dx-FS makes the concentration 126.5 times higher than the input urine sample (right panel)

a b

c d

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CHAPTER 3. Dx-FS for EPOCT/ POCT urinary tract infection diagnosis

3.1 Urinary tract infection diagnosis using Dx-FS.

From previous chapter (Figure 2.4, 2.5, 2.7 and 2.8) we know that bacteria can be effectively isolated using DX-FS, however to be used as IVD diagnostic device understanding of the working range of the Dx-FS and bacterial concentrating range need to be understood and how the level of bacteria in the samplecan be reported instrument free to the end user can dictate its practical adaptation in EPOCT settings. In this chapter we will focus on the isolation of bacteria from water and urine samples. Post isolation detection using three different methods were tested and ideal candidate will be further used in clinical testing of Dx-FS with UTI patients.

3.1.1 Spiked bacteria isolation using Dx-FS

An E. coli strain, MG1655, a kind gift from Prof. Sung-Kuk Lee, UNIST, was cultured as described previously.⁸⁷ Briefly, the E. coli strain was transformed with pGREEN, which encodes the constitutive expression of a green fluorescent protein.⁸⁸ Cells were streaked from their stocks on Luria–

Bertani agar plates. The plates were incubated at 30 °C for 24 h. Positive clones were selected using ampicillin. Bacterial cells were cultured overnight and then centrifuged for 15 min at 4000 rpm. The culturing medium was discarded, and the remaining bacterial pellet was re-suspended in phosphate buffered saline (1×PBS). Cells were counted using a hemocytometer. Water, synthetic urine (Biozoa Biological Supply, South Korea), or urine from a healthy individual was spiked with the desired number of cells for further experiments.

We first evaluated the dynamic range of the device using synthetic urine samples spiked with bacteria and the concentration ranged from 0 to 10⁶ E. coli/mL. The membranes were observed under a fluorescence microscope (Figure 3.1b). The quantified results from the fluorescence microscopy images confirmed that Dx-FS could filter 99.35% bacterial cells from a 1 mL sample in the concentration range of 10²–10⁶ E. coli/mL (Figure 3.1c). Here, the bacterial cells were enriched within the filter membrane, which eventually reduced the sample volume from 1 mL to <8 μL, resulting in a concentration more than 126.5 times higher than that of the input sample (Figure 2.8).

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Figure 3.1. Dx-FS as a versatile bacterial infection diagnostic platform. (a) Operation of the device preloaded with FAST solution (1) a sample is loaded into the sample chamber; (2) the sample flows through the membrane by spinning; (3) the detection solution is added, followed by incubation; and (4) bacterial cell contamination is visualized as an orange color. (b) Bacterial cells enriched using Dx-FS.

The membrane was imaged using microscopy. Scale bar: 20 μm. (c) Number of bacterial cells detected from the membrane shown in (b). (d) Colorimetric bacterial cell viability assay performed using Dx- FS. Water (upper panel) or a urine sample (lower panel) was spiked with bacterial cells. (e) Quantification of the orange color intensities in (d). The color intensities were measured and the averages (bars) and standard deviations (error bars) were plotted (n = 3). According to the UTI guidelines provided by the Centers for Disease Control and Prevention (CDC), the clinical threshold level for positive UTI diagnostics is a bacterial load level higher than 10⁵ CFU/mL (CFU/mL) in addition to the clinical symptoms. Further, the American Society of Microbiology and the European urinalysis guidelines29 states that UTI occurs when the bacterial load in urine reaches 10³ CFU/mL, when most UTI-related symptoms appear.

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3.1.2 Bacterial cell detection from fluorescence microscopy images.

The fluorescently labeled bacterial cells were isolated using Dx-FS and then imaged using fluorescence microscopy (Figures 2.1b, 3.1b and 2.5). A sample containing bacteria was processed using Dx-FS, as described earlier. The NC membrane from Dx-FS was transferred to a glass slide. The membrane was embedded in 50% glycerol in water and was sealed using a glass coverslip. The membranes were imaged using a standard inverted fluorescence microscope (Eclipse Ti-E; Nikon, Japan or IX 71; Olympus, Japan) equipped with a motorized stage (for large-area scanning, MS-2000;

ASI, OR, USA). Imaging was performed using a 10× dry objective lens. The bacterial cells in the microscopy images were counted using a wavelet-transform-based spot detection algorithm47. The fluorescence microscopy images were preprocessed by median filtering to remove the salt-and-pepper noise, followed by wavelet-transform-based spot detection with a scale factor of two. All computations were performed using MATLAB software (MathWorks, MA, USA).

3.1.3 Bacterial cell detection using colorimetric assay in Dx-FS.

Bacterial counting via the colorimetric assay in Dx-FS was carried out using a commercial kit (Microbial Viability Assay Kit-WST; Dojindo Molecular Technologies, Inc., Figure 3.2) according to the manufacturer’s recommended protocols. In brief, 100 μL of the detection solution (20 μL WST-8 diluted in 80 μL distilled water) was added to Dx-FS and incubated. Through a gentle spinning, the liquid was transported to the top of the membrane and incubated for 45 minutes at 37 °C. The solution turned orang depending on the bacterial cell count. To retain the same volume of the detection solution on top of the membrane, we removed the liquid from the back chamber before loading the detection solution. For the practical utility of Dx-FS as a POCT platform, we simplified the overall operation to three manual steps: raw sample injection, bacterial enrichment, and final detection (Figure 3.1a). First, 1 mL of a raw urine sample is introduced into the device preloaded with the FAST solution (Supplementary Figure 7) using a plastic pipette through the inlet hole (Figure 3a-1). The bacterial cells in the urine sample are enriched by spinning the device (Figure 3.1a-2). Spinning is repeated until the sample is completely filtered. Finally, the FAST solution is removed to retain the detection solution on top of the membrane, followed by addition of the detection solution (see Methods), which is then spun for short interval until the solution is localized to the membrane chamber (Figure 3.1a-3). The color change is measured after 45 minutes and translated into the bacterial load (Figure 3.1a-4).