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Development of a Sampling Collection Device with Diagnostic Procedures Jhih-Yan Cheng, Mow-Jung Feng, Chia-Chi Wu, Jane Wang, Ting-Chang Chang, and Chao-Min Cheng Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b01269 • Publication Date (Web): 24 Jun 2016 Downloaded from http://pubs.acs.org on July 3, 2016

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Analytical Chemistry

Figure 1. (a) Prototype of a body fluid collector. (b) The schematic diagram of design and operation of a body fluid collector. (c) Lactate concentration of specimen collecting by collector (cotton swabs coated with nonwoven material) and cotton swabs (cotton swabs not coated with nonwoven material) 132x124mm (96 x 96 DPI)

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Figure 2. Comparison between glycogen assay and lactate assay (Sample number N=13) 132x119mm (96 x 96 DPI)


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Figure 3. ELISA analysis result for IL-6 in clinical sample. (Sample number for IL-6 assay N=39) 132x140mm (96 x 96 DPI)



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Figure 4. The results of IZON qNano analysis of (a) mean size and mode size for the MVs in the clinical sample. (b) MV concentration in the clinical sample. (Sample number N=20) 140x243mm (96 x 96 DPI)


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Cheng Page 1

Development of a Sampling Collection Device with Diagnostic Procedures Jhih-Yan Cheng1, Mow-Jung Feng2, Chia-Chi Wu2, Jane Wang1, Ting-Chang Chang2*, ChaoMin Cheng3* 1

Department of Chemical Engineering, National Tsing Hua University, Hsinchu 300, Taiwan

2

Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital Linkou Branch,

Taoyuan 333, Taiwan 3

Institute of Biomedical Engineering, National Tsing Hua University, Hsinchu 300, Taiwan

Corresponding author: Chao-Min Cheng, Institute of Biomedical Engineering, National Tsing Hua University, Hsinchu 300, Taiwan, e-mail: [email protected] Ting-Chang Chang, Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital Linkou Branch, Taoyuan 333, Taiwan, e-mail: [email protected]

Keywords: cervicovaginal fluid, female genital disease, diagnostic device

Development of a Sampling Collection Device with Diagnostic Procedures



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Cheng Page 2 Abstract Cervicovaginal fluid plays an important role in the detection of many female genital diseases, but the lack of suitable collection devices in the market severely challenges test success rate. Appropriate clinical sampling devices for cervicovaginal fluid collection would help physicians detect diseases and disease states more rapidly, efficiently, and accurately. The objective of this study was to develop a readily used sampling collection device that would eliminate macromolecular interference and accurately provide specimens for further studies. This study was designed to develop an effective device to collect cervicovaginal fluid from women with symptoms of endometrial lesions, women appearing in the clinic for a routine Papanicolaou smear, and/or women seeking a routine gynecologic checkup. Paper-based assay, ELISA, and qNano were used to pro-vide accurate diagnoses. A total of 103 patients successfully used the developed device to collect cervicovaginal fluid. Some of the collected specimens were used to detect glycogen, lactate, and pH for determining pathogen infection. Other specimen samples were tested for the presence of female genital cancer by comparing interleukin 6 concentration and microvesicle concentration. We proposed a non-invasive screening test for the diagnosis of female genital diseases using a dual-material collection device. The outer, nonwoven fabric portion of this device was designed to filter macromolecules, and the inner cotton portion was designed to absorb cervicovaginal fluid. Introduction The use of body fluid for research in the field of diagnostics is very common, and has been proven efficient. Dr. Watelet demonstrated that nasal secretion could be used to analyze immune response.

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Well-developed medical devices, i.e., sinus packs, were used to obtain sufficient

amounts of nasal secretion for standardized immunological analysis. Body fluids can also be Development of a Sampling Collection Device with Diagnostic Procedures


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Cheng Page 3 used for detecting and quantifying bacterial infection. Notably, secretion source can even be distinguished via the presence of specific, characteristic bacteria.

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Dr. Fleming found that

Lactobacillus crispatus and Lactobacillus gasseri existed in female genital tract secretions but not in other body fluids. 3 From a molecular perspective, the use of histatin mRNA and statherin peptide as subject biomarkers, can be useful in identifying body fluid that is secreted from different body parts. 4 The detection of uterine inflammation or lesions by secretion analysis may be considered a future trend. Glands in the cervix and endometrium produce fluid every day. This normal secretion gathers at the intersection of the cervix and the vagina, the vaginal hole, before passing to the outside. Cervicovaginal fluid plays an important role in the detection of many female genital diseases, but the lack of suitable collection devices in the market severely challenges the success rate of the tests. Appropriate clinical sampling devices for cervicovaginal fluid collection would help physicians detect diseases and disease states more rap-idly, efficiently, and accurately. The common sample collecting methods include the following: (1) using a sterile cotton swab to clean vaginal mucous;

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(2) using a pipette inserted into the vaginal hole to collect vaginal

secretion;5 (3) vaginal lavage;6 (4) using a vaginal swab;7 and, (5) using a cotton ball6. In addition, multiple commercial vaginal discharge collection devices are available (Table 1). All of these products are suitable for self-collection, and some are more suitable for collection by physicians in a clinic. In previous studies of gynecological diseases, cotton swabs were widely used to collect cervicovaginal fluid. There are two other recently patented cervicovaginal fluid collection methods available in the US that have been commercialized. The first one uses underwear as the sampling device. Using this approach, even small secretion volumes were usable because the highly absorptive material could still receive and hold sample. However, Development of a Sampling Collection Device with Diagnostic Procedures



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Cheng Page 4 sometimes the secretion would remain on the nearby skin rather than the underwear material6.The other patent employs a collection device formed by an elastomeric rim and a flexible film reservoir. The rim has a generally rectangular cross section and forms a collection space for collecting vaginal secretion. The reservoir may be collapsible so as to be substantially enclosed within the rim when the device is being used8. The disadvantage with this approach is that collection often fails from insufficient discharge volume. Although fluid from cotton swabs can be smeared onto glass slides, the swabs are not ideal because they may not release enough fluid for accurate reading. Swabs are also easily contaminated. In an article published in Nature Medicine on how pH paper was better than expensive kits when measuring acidity, Dr. Sullivan claimed that "If the swab stayed in the air too long, if it was put on a surface or dropped, that would affect the reading." 9 Up until now, invasive or mini-invasive procedures such as dilatation of the endocervical canal and curettage of endometrial tissue have been required to rule out the presence of genital diseases in women with abnormal vaginal bleeding or other symptoms. Additionally, small amounts of fragmented endometrial tissue taken via endometrial brushes, such as a Tao-brush, often result in inconclusive diagnostics. Most of the diagnostic technologies used today require laboratories, expensive equipment, and professional personnel. One of the main challenges for industry is to develop fast, quantitative, and low-cost devices. As the demand becomes apparent, some technologies that fulfill requirements mentioned above are emerging. Cellulose-based diagnostic platforms are one of the methods proposed in the past decade, and such platforms have become popular largely because of their simplicity. Among different kinds of cellulose, the most commonly used is paper, which was first used diagnostically by Whitesides and colleagues.10-13



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Cheng Page 5 The advantages of using microfluidic paper-based analytical devices (µPADs) include high throughput, low sample volumes, low cost, and robustness.12,14 For these reasons, µPADs are well established as a valuable tool in food, medical, environmental, veterinary, agricultural, and industrial diagnostics. A number of fabrication techniques have been established for µPAD production, including photolithography, inkjet printing, stamping, cutting, screen-printing, and wax printing.13,15-19 Wax printing is the fastest and simplest fabrication method to date. In this process, a wax printer employs melted wax in the same way that an ink jet printer uses ink cartridges. A hydrophobic barrier is created with the wax, enabling spatial control over fluid transport caused by the capillary flow in the paper. Devices that are non-invasive and easy-tooperate will greatly reduce discomfort and widen acceptance. Additionally, a device capable of repeat, long-term testing can effectively prevent the occurrence, or reoccurrence, of infections. If we can collect cervicovaginal fluid to detect physiological cycle-related or disease-related molecules, it may provide a useful means of screening for uterine diseases. Our device employs a cotton swab surrounded by a porous filter mesh to prevent macromolecules from contaminating the cotton swab. After sample collection, we focused on the use of paper as a microfluidic substrate employing an eco-friendly wax-printing technology to establish testing well regions. Three distinct colorimetric assays were completed using our wax-printed paper: 1) a lactate assay; 2) a pH assay; and, 3) a glycogen assay. We believe that our device can be used for a wide range of applications in analytical chemistry, analytical biochemistry, biomaterials, microfluidics and nanofluidics, and, ultimately, in obstetrics and gynecology. Materials and Methods Clinical Sample Collection. Clinical samples were collected from 200 adult patients (1) with known endometrial lesions, including any type of endometrial hyperplasia and any stage of Development of a Sampling Collection Device with Diagnostic Procedures



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Cheng Page 6 endometrioid carcinoma of the endometrium, and (2) with symptoms/signs of endometrial hyperplasia or endometrial cancer, including abnormal uterine bleeding with thick endometrial stripe or medically uncontrollable uterine bleeding. The control group was made up of another 100 adult patients who visited the clinic for routine Papanicolaou smears or routine gynecologic checkups and had no suspected endometrial disease, or who visited for uterine myoma or benign ovarian tumors. The institutional review board approved the collection and use of these samples and information for research purposes (102-3655A3). Collection Sample Recovery. After collecting cervicovaginal fluid with our collection device, the centrifuge method was used to remove specimens from the collector itself as shown in Figure SI-1. A centrifuge tube with separate, removable, upper and lower tubes was used. The upper tube contained a filter bottom, so that, when both tubes were combined, the filter acted to remove macromolecules under centrifugal force. The cotton swab was placed in the upper tube, and the cotton and stick were separated easily via an incision at the junction of cotton and stick. Samples were placed into a centrifuge (5000 g, 10 min) to collect the specimen in the lower tube, which contained buffer. After discarding the upper tubes, the liquid in the lower tube was kept as the specimen for further diagnosis or storage. Quantitative Detection of Analytes Using µPADs. Patterning a piece of paper into hydrophilic channels demarcated by hydrophobic barriers requires that these barriers extend through the entire thickness of the paper. Here, a wax printer (Xerox Phaser 8560) was used to print 96-well patterned designs in solid wax on paper (Whatman No.1 chromatography paper). Next, the paper was heated to re-melt the wax slightly so that it penetrated the paper to generate complete hydrophobic barriers. After patterning paper, the final step in the fabrication of µPADs was the addition of assay reagents to the µPAD reaction zones. Paper patterned in this manner can be Development of a Sampling Collection Device with Diagnostic Procedures


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Cheng Page 7 used for a variety of biological assays by adding appropriate reagents to the test areas. We demonstrated this concept by detecting lactate, pH, and glycogen. For our lactate assay, 5U lactate oxidase (2 µL) was spotted onto the test area by pipetman, followed by 3U HRP solution. For our pH assay, 4*10-4 N NaOH solution (25 mL) that contained 0.01g bromothymol blue and 5g resazurin was spotted onto the test area. For our glycogen assay, we catalyzed the glucose in 50 µL specimen samples with glycogen phosphorylase (2 µL) for 30 minutes. Then, glucose indicator (2 µL) that contained 15U HRP (3 µL), 75U glucose oxidase (3.75 µL), and the solvent ABTS (93.25 µL) were spotted onto the test zone. The spotted reagents were allowed to air dry at room temperature and the catalyzed sample (2 µL) was then added to each microzone. The fluid filled the entire pattern within approximately one second, but the assay required 3 minutes for the paper to dry and for the color to fully develop. The reflected light was captured by a digital camera. By measuring the intensity of the color in a digital image of the test zone, we could calculate analyte concentration by comparing its intensity to values on a calibration curve. Biomarker Detection by ELISA Sandwich Assay. The reagents for IL-6 were provided by a commercial ELISA kit (Invitrogen), and the manufacturer’s protocol was followed. Two antibodies were used to bind to different sites on the antigen or ligand. Anti-IL-6 antibody (capture antibody), which is highly specific for the antigen, was applied and allowed to attach to the solid substrate surface. The antigen was added, and then the second antibody, biotynilated anti-IL-6, was added to act as the detection antibody. The detection antibody binds the antigen at a different epitope than the capture antibody. As a result, the antigen is ‘sandwiched’ between the two antibodies. The antibody binding affinity for the antigen is usually the main determinant of immunoassay sensitivity. As the antigen concentration increases, the amount of detection antibody increases, leading to a higher measured response. The standard curve of a sandwichDevelopment of a Sampling Collection Device with Diagnostic Procedures



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Cheng Page 8 binding assay has a positive slope. To measure antigen, streptavidin-HRP (enzyme) was then attached to the biotinalyted anti-IL-6 antibody and stabilized chromogen (substrate) was added to elicit a colorimetric response that could be read by a spectrophotometric plate reader. The signal generated was proportional to the amount of target antigen present in the sample. Microvesicle Measurement. The instrument we used for detecting microvesicles was an IZON qNano. Fluid cells were washed with de-ionized water and then dried with a clean cloth. Once the lower fluid cell was in place, 70 µL of PBS solution was carefully placed into the center channel to ensure that no bubbles were introduced. The nanopore was primed with a drop of buffer on both the cis and the trans side before being placed onto the mechanical stretching jaws. The upper fluid cell was then set into place, and 40 µL of PBS was added to the upper fluid cell. Pore width was controlled by adjusting jaw width, pressure was controlled with an external pump connected to the upper fluid cell, and voltage was controlled by the connected computer. At constant voltage (nominally 0.3 V), the pore was known to be open when a direct correlation could be observed between ionic current and adjustment of jaw width - once this was achieved, particles were added into the upper fluid cell. The nanopore and cell were cleaned with deionized water and the cell reloaded with sample between runs. Particle suspensions were made by diluting a stock solution of particles in standard electrolyte. These particles were diluted in standard electrolyte and ultrasonicated for at least 5 minutes prior to use. Carboxylated polystyrene particles with nominal average sizes of and 220 nm (Thermo Scientific) was received at concentrations of and 1.7×1012 particles/mL. The clinical sample was eluted and extruded through a 200 nm polycarbonate filter (Sartorius Stedim) to eliminate the bulk contaminating components of complex biological samples including free lipids, lipoproteins, cell debris, and small molecules. After calibrating by standard solution, 40 µL samples were added to Development of a Sampling Collection Device with Diagnostic Procedures


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Cheng Page 9 the upper fluid cell. Through calibration, blockade magnitude was converted to particle diameter and concentration was calculated from the rate of particles passing through the pore over time. Results and Discussion Sampling Collection Device Prototyping. Prototypes were made for the collection of body secretion, here, cervicovaginal fluid. The cotton swab was wrapped up with a layer of porous film that acted as a filter to eliminate macromolecule interference (Figure 1b). Various porous membranes were tested for this purpose. We developed the collection device to that the nonwoven layer, called spunlace beauty facial gauze could not only exclude macromolecules (e.g., mucus) in cervicovaginal fluid but could be opened and removed easily with one hand. The sampled body fluid could be collected and the filter with macromolecules discarded. The design of this sampling collection device is patent pending. For clinical use, the collection device was made of materials approved by US FDA and is sterilized and well packed before use. To confirm that the nonwoven material covering the cotton swab did not interfere with specimen collection, we compared absorbed lactate concentrations from swabs covered with material to those not covered with material and established a calibration line between mean intensity and lactate concentration as shown in Figure SI-2. Both types of swabs were placed in the same lactate solution. Subsequent centrifugation and concentration examination showed that recovered lactate was comparable for both types as shown in Figure 1c. Thus, the nonwoven material did not affect detection results. When developing the collection device, different device sizes were used for patients. However, the small collection device was less comfortable for patients than the larger one. As a result, the large collection device shown in Figure 1a was chosen for our experiments. Note, before collecting the sample, the device was soaked in sodium chloride (NaCl) solution. Therefore, after Development of a Sampling Collection Device with Diagnostic Procedures



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Cheng Page 10 using the centrifuge method, the absorbed sample was directly spun down with NaCl solution. We tested solution volumes of 5 mL, 4 mL, and 3 mL as shown in Table 2-4, and found that our device could not fully absorb 5 mL of sodium chloride solution and still adequately collect and transfer secretion samples. Following examinations of samples using each different sodium chloride soaking volume, we found that soaking in the 4 mL sodium chloride solution was the most suitable. Subsequent testing was performed accordingly. Quantitative Detection of Analytes by µPADs. Vaginal epithelial cells normally proliferate in the presence of estrogen. Vaginal surface cells are rich in glycogens, which helps lactobacillus grow and simultaneously inhibits other pathogens, forming a balanced vaginal environment. When estrogen level drops, epithelial cells atrophy and glycogen concentration decreases. Such an environment becomes less ideal for lactobacillus growth, leading to the growth of other pathogens. The cervicovaginal fluid of healthy women contains higher concentrations of lactate and lower pH. In cases where lactate concentration lowers and pH rises, vaginal infection by pathogens may be suspected. The µPAD assays employed here were based on enzymatic reactions or small-molecule dyes. Solutions of the reagents for the assays were spotted by hand onto the test zones and dried. Quantitative colorimetric detection of analytes using µPADs was possible by using reflectance detection because color intensity developed in the test zones was a function of analyte concentration. Reflectance detection is based on the measurement of the light reflected off of the surface of the test zone. This reflected light can be captured by a digital camera. By measuring the intensity of the color in a digital image of the test zone, it is possible to calculate the concentration of the analyte by comparing its intensity to values on a calibration curve.



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Cheng Page 11 After establishing a standard curve (shown in Figure SI-3.), we could quantify our clinical samples for lactate concentration, glycogen concentration, and pH value as shown in Table 5. Comparisons demonstrated that high cervicovaginal glycogen correlated well with increased lactate concentration. (Figure 2) Quantitative Detection of Endometrial Cancer Potential Biomarker. Endometrial cancer is a cancer that arises from the inside lining of the uterus. There are roughly 142,000 women suffering from endometrial cancer and 42,000 women die each year worldwide.2 Between 2002 and 2007, the occurrence of endometrial cancer in Taiwan increased by 30.8%, while cervical cancer decreased by 26% as the popularity of the Papanicolaou test grew. Note, the occurrence of endometrial cancer, exceeded that of cervical cancer in 2010 and 2011. Based on the morphology of an individual cell or a small amount of cells, cytopathologists can diagnose the presence of squamous intraepithelial lesions after Papanicolaou staining. However, morphological aberrations in glandular cells are frequently subtle, and glandular lesions, including endometrial tumors, are not easy to diagnose through cellular morphology. Screening of endometrial cancer and its precursors is a process that has yet to be developed, and the demand for this type of screening has become more urgent within the last decade. One of the potential novel protein biomarkers for endometrial cancer screening is interleukin-6 (IL-6), which is a multifunctional cytokine that plays a central role in host defense due to its immune and hematopoietic activities and its potent ability to induce the acute phase response.20 We used a commercial ELISA kit to analyze the IL-6 concentration in cervicovaginal fluid. As shown in Figure SI-4., after we established a calibration curve for IL-6 in a standard solution, we could calculate IL-6 concentration in clinical samples based on this curve. IL-6 concentration




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Cheng Page 12 from 27 healthy women controls (mean 10.28 pg/mL) and 12 patients with endometrial carcinoma (mean 534.45pg/mL) showed significant difference (P

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