To investigate simple fabrication approaches for multiple assay needs, we created a fluid-absorbing channel system made of cotton material. This device was used to establish a multiple detection platform, and solve contamination issues that commonly affect lateral flow-based biomedical devices, for clinical urinalysis of nitrite, total protein, and urobilinogen.
A robust, low-cost analytical device should be user-friendly, rapid, and affordable. Such devices should also be able to operate with scarce samples and provide information for follow-up treatment. Here, we demonstrate the development of a cotton-based urinalysis (i.e., nitrite, total protein, and urobilinogen assays) analytical device that employs a lateral flow-based format, and is inexpensive, easily fabricated, rapid, and can be used to conduct multiple tests without cross-contamination worries. Cotton is composed of cellulose fibers with natural absorptive properties that can be leveraged for flow-based analysis. The simple but elegant fabrication process of our cotton-based analytical device is described in this study. The arrangement of the cotton structure and test pad takes advantage of the hydrophobicity and absorptive strength of each material. Because of these physical characteristics, colorimetric results can persistently adhere to the test pad. This device enables physicians to receive clinical information in a timely manner and shows great potential as a tool for early intervention.
The development of point-of-care (POC) diagnostic devices that are affordable, robust, and easily used is imperative for improving global health1,2. In particular, devices composed of cellulose substrates (e.g., paper, thread, and cotton) provide promising analytical platforms for low-cost analysis because of their ubiquity, affordability, ease of use, robustness, and capacity to provide rapid results3-7.
Here, we unveil the development of a cotton-based analytical device that uses a lateral flow-based format for urinalysis. This cotton-based analytical device provides an alternative detection approach with several key advantages: i) fabrication with minimal human effort; ii) low cost; iii) the capacity to be used to conduct multiple, different assays without cross-contamination concerns; iv) device independence, i.e., the ability to be run without additional equipment and/or electricity; and, v) speed (colorimetric assays can be completed within 10 min).
The structure of this cotton-based analytical device can be divided into four parts: i) cotton that is naturally hydrophobic on its external hydrophobic layer; ii) cotton that is internally hydrophilic and serves as a transportation channel for liquid wicking; iii) lamination film that binds and compresses the cotton being used but contains drilled out holes for the placement of reaction/test pads; and, iv) chromatography paper test pads, which are coated/embedded with reactive reagents, placed on the exterior surface of the cotton (specifically, in the space drilled out of the lamination film) as reaction areas for colorimetric assays (i.e., nitrite, total protein, pH, and urobilinogen assays) and results display.
The underlying mechanism of the test is as follows. The cotton-based analytical device is scored with lines that penetrate half way through the depth of the cotton material to create a flow channel that allows sample fluid to reach the reactive pads being used. The absorptive edge of the analytical device is immersed into the target sample, whereupon the solution is wicked along the fluidic channel from the absorption end to the test pads (Figure 1). Because the absorptive strength of the test pad is greater than that of cotton, solutions absorbed by the test pads are firmly contained inside the test pad paper so that there is no reflow back into the fluidic channel, and the colorimetric results become subsequently fixed on the test pad material. At the end of the reaction, colorimetric results are recorded via a desktop scanner, and analyzed via image analyzing software.
CAUTION: Proper laboratory hygiene practice is required. Gloves and universal precautions are required when using this POC device. Contamination of results or infection may occur if adequate sterilization procedures are not carried out properly.
1. Prepare Test Strip Devices
2. Prepare Standards and Indicator Solutions for Urinalysis
3. Prepare Indicator Pads
4. Create Standard Curves for the Cotton-based Analytical Device
5. Determine Unknown Sample Concentrations Using Established Standard Curve Values
We successfully demonstrated the development of cotton-based analytical devices by using commercially available cleansing cotton characterized by hydrophilic (inner portion) and hydrophobic (exterior portion) properties (Figure 1A). Figure 3 shows the results of contact angle measurement. The hydrophobic interface of exterior cotton was 127.35° ± 4.73°. From a user-friendly perspective, colorimetric assays employed here could be directly observed by the naked eye, and further quantified through color intensity analyses (Figure 2F). Urine total protein in a normal individual should be less than 150 mg/dl (4 µM). The value of urine nitrite ions is associated with urinary tract infections or bacterial infections, so the analytical sensitivity of urine nitrite detection should be as low as possible when it comes to the development of analytical devices for a nitrite assay. Urobilinogen is excreted by the feces and recovered by the enterohepatic circulation, but some levels of urobilinogen, approximately 1 – 4 mg/daily, can be found in urine. Figure 4A–C illustrates the results from our efforts to create standard curves for each assay target. These standard curves were established by examining mean colorimetric intensity results and comparing them to the concentrations of standard concentrations that we established. This allowed us to evaluate the limit of detections (LODs) for each assay. The LODs for nitrite, BSA, and urobilinogen in buffer systems were, 0.147 mM, 3.672 µM, and 4.861 mg/L, respectively. This also provided us with a mathematical framework for determining the concentrations of nitrite, BSA, and urobilinogen in unknown solutions.
Figure 1. Schematic Diagram of Cotton-based Analytical Device. Top and cross-section view images display the dimensions of each test pad (0.5 cm diameter) and cotton-based device (1 cm width x 5.5 cm length). Please click here to view a larger version of this figure.
Figure 2. Fabrication Process of Cotton-based Analytical Device, Sampling, and Results Analysis. (A) A paper cutter was used to cut pieces of cleansing cotton to the specified size. (B) A pen was used to mark the locations for lamination film holes in the assembly. (C) A laminator was used to sandwich the assembly between two layers of laminating film by heating. (D) The laminated pieces of the completed device were separately cut. (E) The test pads were installed onto the cotton device. (F) Urobilinogen, BSA, and nitrite assays were performed with our cotton-based analytical device. (G) Images of the results were made and imported into a computer using a scanner. (H) Image analysis software was used to analyze the images by gray-scale contrast state analysis. (I) The obtained intensity results were fitted into a standard curve. Please click here to view a larger version of this figure.
Figure 3. Contact Angle Results for the Cleansing Cotton. DI water (1 µl) was dropped onto the cotton's external, hydrophobic layer to determine contact angle.
Figure 4. Analysis Results for Nitrite, Urobilinogen, and BSA on Cotton-based Analytical Device. The standard curves for (A) nitrite (0.156-2.5 mM), (B) BSA (1.875-30 µM), (C) urobilinogen (7.8 – 125 µM). For each concentration, tests were performed in triplicate and the R2 values for the fitting curve were nitrite: 0.99, BSA: 0.98, and urobilinogen: 0.95. Please click here to view a larger version of this figure.
Critical steps in this protocol included determining the appropriate combination of cotton material (with varying hydrophobicity/hydrophilicity) and filter paper (chromatography filter paper or quantitative filter paper). A well-planned and executed device design renders the best performance attributes for colorimetric assays. From our colorimetric assay results, the cotton-based analytical device presented herein demonstrates great potential as a platform for multiple disease detection.
Most current lateral flow-base products (e.g., pregnancy test) provide no more than a single assay functon14. While some dipstick tests perform multiple-biomarker assays15, their use runs the risk of collateral sample contamination because assay reagents directly contact test samples. Our device overcomes this obstacle and can be used to implement multiple colorimetric assays in a single lateral flow-based channel device.
This method is only limited by target analyte molecular size. The R 2 and LOD values of nitrite are much greater than those of the other assays (BSA and urobilinogen), suggesting that our devices are more suitable for low molecular analysis5 (Figure 4).
Some further optimization may still be required in order to enhance the practicality of this device for clinical practice. This optimization includes improving the reliability and stability of assay reagents when exposed to the outside environment for long periods of time. Nevertheless, we believe that this invention creates a decisive and valuable inroad into the development of low-cost analytical devices that can be reliably used in clinical practice, especially in regards to fulfilling needs for rapid and accurate analysis and suitable follow-up analysis of communicable and infectious diseases.
The authors have nothing to disclose.
This work was supported in part by grants from Taiwan's Ministry of Science and Technology (MOST 104-2628-E-007-001-MY3 (CMC)), and Taichung Veterans General Hospital (TCVGH-1056904C (MYH)).
bovine serum albumin | Sigma-Aldrich, US | No. 9048468 | ≥ 99% |
nitrite | Sigma-Aldrich, US | No. 7632000 | ≥ 99% |
urobilinogen | Santa Cruz Bio, US | No. SC-296690 | |
citrate | Sigma-Aldrich U.S | No. 6132043 | ≥ 99% |
tetrabromophenol blue | Sigma-Aldrich U.S | No. 4430255 | ≥ 99% |
sulfanilamide | Sigma-Aldrich U.S | No. 63741 | ≥ 99% |
citric acid | Sigma-Aldrich U.S | No. 77929 | ≥ 99.5% |
N-(1-naphthyl) ethylenediamine dihydrochloride | Sigma-Aldrich U.S | No. 1465254 | ≥ 98% |
4-(Dimethylamine)benzaldehyde | AlfaAesar, U.S | No. A11712 | ≥ 98% |
Methyl Red sodium salt | sigma, U.S | No. 114502 | ≥95% |
Bromothyle blue | sigma, U.S | No. 114413 | ≥95% |
Shiseido Cleansing Cotton | Shiseido, Japan | No. 79014 | |
chromatography paper | GE Healthcare Whatman, Springfield Mill, UK | No. 30306132 | |
plastic substrate | lamination film, MAS | A4 | 216 mm × 303 mm |
scanner | microtek scanmaker | i2400 | |
paper cutter | Life paper cutter | No.306 | |
laminator | AURORA | LM4231H | |
laminator film | UNI LAMI | 4A |