Chemical, Gas, and Biosensors for Internet of Things and Related Applications

Chemical, Gas, and Biosensors for the Internet of Things and Related Applications brings together the fields of sensors and analytical chemistry, devices and machines, and network and information technology. This thorough resource enables researchers to effectively collaborate to advance this rapidly expanding, interdisciplinary area of study. As innovative developments in the Internet of Things (IoT) continue to open new possibilities for quality of life improvement, sensor technology must keep pace, Drs. Mitsubayashi, Niwa and Ueno have brought together the top minds in their respective fields to provide the latest information on the numerous uses of this technology.

Topics covered include life-assist systems, network monitoring with portable environmental sensors, wireless livestock health monitoring, point-of-care health monitoring, organic electronics and bio-batteries, and more.

• 2020 PROSE Awards - Winner: Category: Chemistry and Physics: Association of American Publishers
• Describes the latest advances and underlying principles of sensors used in biomedicine, healthcare, biotechnology, nanotechnology and food and environment safety
• Focuses on sensors’ methods of data communication, logging and analysis for IoT applications
• Explains the specific requirements of sensor design and performance improvement, helping researchers enhance sensitivity, selectivity, stability, reproducibility and response time

• Language: English
• Publisher: Elsevier Science
• Release date: Jun 14, 2019
• ISBN: 9780128154106

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Preface

The growing interest in medical and environmental sensors can be attributed to the increasing awareness that health is essential for enjoying and keeping a good quality of life. The advent of wearable and/or flexible sensors for healthcare represents such a leap. The demand for quality healthcare has strained the current healthcare system, which faces large workforce shortages and inadequate medical facilities. It is paramount to develop consumer-based devices to augment the current healthcare demands. One emerging technology is sensor networks connected to the Internet of Things (IoT), a human-oriented network of tiny human monitoring devices developed on the body and in the surrounding infrastructure.

Chemicals, gas, and biosensors will be required for preventive medicine and environmental assessment for healthcare demands in the near future. The reason for this stems from the fact that all branches of modern medicine, ranging from prevention to complex intervention, rely on early and accurate diagnosis followed by close monitoring of the results. The research field of gas and bio/chemical sensors has been developed by a group of truly interdisciplinary researchers involving chemists, biologists, physicists, material scientists, and computer engineers to create a range of novel configurations exploiting bio/chemical recognition systems allied with physiochemical transducers. Novel chemical, gas, and biosensors would further progress with the introduction of a range of optical, acoustic, magnetic, thermal, and electrical technologies, coupled with microelectronics and MEMS devices for the future medical and healthcare systems. This book is organized with the following parts and chapters. Part 1 introduces novel bio/chemical sensors with several recognition materials such as enzymes, antibodies, aptamers, receptors, and artificial materials for analytes in body fluids and gas-phase samples. In Part 2, flexible and mobile sensors and related techniques are explained with some wearable and cavitas (body cavity) sensors, and smell detectors. Information and network technologies for the sensors are illustrated with industrial applications in Part 3.

This book is intended for graduate students, academic researchers, and professors who work in the field of medical and environmental research, and also for industry professionals involved in development of devices and systems with IoT for human bio/chemical measurement, medical monitoring, and healthcare services with Internet technologies. We would like to sincerely express our appreciation to the distinguished authors of the chapters whose expertise has certainly contributed significantly to the book. We hope that this book can shed light on various technological aspects related to bio/chemical sensors with IoT and related applications in a healthcare context, and will stimulate further research in this field.

Kohji Mitsubayashi, Osamu Niwa and Yuko Ueno

Editors

Part I

Sensors and Devices for Internet of Things Applications

Outline

1 Portable urine glucose sensor

2 Design, application, and integration of paper-based sensors with the Internet of Things

3 Membrane-type Surface stress Sensor (MSS) for artificial olfactory system

4 Sensing technology based on olfactory receptors

5 Advanced surface modification technologies for biosensors

6 Development of portable immunoassay device for future Internet of Things applications

7 Sensitive and reusable surface acoustic wave immunosensor for monitoring of airborne mite allergens

8 Aptameric sensors utilizing its property as DNA

9 Electrochemical sensing techniques using carbon electrodes prepared by electrolysis toward environmental Internet of Things sensor

10 Chemical sensors for environmental pollutant determination

1

Portable urine glucose sensor

Narushi Ito¹, Mariko Miyashita² and Satoshi Ikeda²,    ¹PROVIGATE Inc., Tokyo, Japan,    ²TANITA Corporation, Tokyo, Japan

Abstract

This section describes the development of a portable urine glucose sensor for diabetic care in home use. Urine glucose monitoring is thought to be a quite useful monitor for the self-management of diabetes, as it can be done without pain, and it allows us to detect prediabetes by monitoring the postprandial urine glucose level.

Keywords

Urine glucose; enzyme sensor; noninvasive; postprandial hyperglycemia; clinical application

1.1 Introduction

Development of a noninvasive blood glucose monitoring system is based on measurement of near infrared light passing through fingers and arms [1,2], measurement of interstitial fluid collected from the skin surface with a micro glucose sensor [3,4], and measurement of contact lens type tears [5,6], etc. It has been done over the past 30 years and enormous amount of research and development has been published, however, there are still no practical products.

A series of products that have succeeded in development include a continuous blood glucose monitor and a flash glucose monitor that place small needles in the abdomen and the like. These are minimally invasive, and continuous monitoring for 2 weeks is possible. Meanwhile, as a noninvasive measurement, a portable urine glucose meter that makes it possible to quantitatively measure urine glucose levels correlated with blood glucose levels has been put to practical use.

In this section, we describe the principle and structure of the microplanar type urine glucose sensor and examples of application of commercialized urine glucose meter to healthcare.

1.2 Significance of urine glucose measurement

In the urine glucose tests, diabetes screening tests are being conducted to test positive (+) or negative (−) by measuring fasting urine such as common in medical examinations.

Positive (+) is based on 100 mg/dL as the urine glucose concentration. Medically urine glucose positivity is considered as a suspicious indicator of diabetes first. Then further inspections are necessary, because there are transient cases such as renal diabetes, stress, pregnancy, etc., in addition to other findings for diagnosis of diabetes.

Ultimately diabetes is confirmed by the 75 g oral glucose tolerance test. Urine glucose test is regarded as an auxiliary test and a large number of screening tests are still being carried out at present, due to its advantage of noninvasive measurement.

Changes in urine glucose concentration after meals are shown in Fig. 1.1. The postprandial urine glucose concentration is linked to blood glucose levels, and it is important that it does not overlook postprandial hyperglycemia occurring after meals. When the urine glucose concentration exceeds 50 mg/dL, it means that the blood glucose level exceeds the glucose excretion threshold of 160–180 mg/dL in the kidney.

Figure 1.1 Relationship between urine glucose concentration and blood glucose concentration after meal.

Even when blood glucose level rises with the diet, it decreases after 1 hour due to the action of insulin. In other words, the blood glucose level measured at 2 hours after a meal usually returns to the normal range. On the other hand, it is known that the urine glucose concentration 2 hours after a meal reflects the elevated blood glucose level with the meal. As well, it is demonstrated that urine glucose level correlates with the mean blood glucose level.

1.3 Operating principle of urine glucose sensor and laminated structure

1.3.1 Principle of operation

Urine glucose sensor is an enzyme electrode method combining glucose oxidase (GOX) and hydrogen peroxide electrode.

The electrode is fabricated by photolithography technology. Glucose is enzymatically converted to hydrogen peroxide (H2O2) by GOX, and the yielded H2O2 is electrochemically detected by the electrodes.

The enzymatic reaction (1) and the electrode reactions (2) are as follows:

1. Enzymatic reaction

GOX: Glucose+O2→gluconolactone+H2O2

2. Electrochemical reactions at electrodes

Working electrode: H2O2→2H++O2+2e−

Counter electrode: 2H+ +1/2O2+2e−→H2O

Entire electrode system: H2O2→H2O+1/2O2

A perspective view of the sensor is shown in Fig. 1.2. Three electrodes, a working electrode, a counter electrode, and a reference electrode, are formed in the hole of the cartridge. The working electrode and the counter electrode are Pt electrodes, and thin film Ag/AgCl electrodes are formed as reference electrode. The reference electrode has the role of stabilizing the potential after immersion in the solution. The outermost layer of the electrode is coated with a thin film of a fluorinated polymer to prevent contamination due to urine components while protecting the electrode system as a whole and stabilizing the operation of the electrodes for more than 1 year in solution.

Figure 1.2 Perspective view of the sensor.

1.3.2 Laminated structure of urine glucose sensor

To accurately measure postprandial urine, means to eliminate the influence of vitamin C (ascorbic acid) among substances released from foods are required. Ascorbic acid has a reaction that gives electrons to an electrode and another reaction decomposing hydrogen peroxide, and it is typical of an interfering substance of an amperometric sensor.

Furthermore, it is necessary to fabricate a membrane structure so that interfering substances other than ascorbic acid contained in the urine do not affect the measured values.

Fig. 1.3 shows a laminated structure of the urine glucose sensor. This sensor is composed of four layers: a restricted permeable layer, an enzyme immobilized layer, a cation-exchanging layer, and an adhesive layer.

1. The restricted permeable layer has a wide measurement range from 10 to 2000 mg/dL, limiting the diffusion of molecules larger than glucose. It has the role of preventing the influence of adhered substances in urine.

2. The enzyme immobilized layer is the one where enzyme (GOX) and bovine serum albumin are crosslinked and immobilized so as not to inactivate the enzyme; as a result, repetition of the sensor is possible.

3. The cation-exchanging layer has the role of permeating hydrogen peroxide and limiting the diffusion of molecules larger than hydrogen peroxide. Furthermore, it has the function of preventing permeation of ionized molecules.

4. The adhesive layer has the role of covalently bonding the selectively permeable film, which is an organic material, to the surface of the glass substrate or the electrode and stably adhering for a long time in water.

Figure 1.3 Laminated structure of the urine glucose sensor.

This sensor is formed of a thin film of four layers with a total thickness of 1 μm or less, effectively eliminating the influence of interfering substances in the urine and an early time response [7].

1.4 Development of portable urine glucose meter

1.4.1 Composition of urine glucose meter

This urine glucose meter consists of a body and a sensor. Portability is designed so that the sensor section is folded down to be compact, and at the time of measurement it is extended. Photo 1.1 shows a urine glucose meter in a stored state. Photo 1.2 shows the urine glucose meter extended at the time of measurement. The urine glucose meter sensor section is composed of a preservation solution bottle, which makes the sensor wet. The preservation solution is reserved to hold the sensor, which can cause optimal enzymatic reaction with pH buffer and physiological saline. After the sensor is taken out, it becomes possible to measure instantly.

Photo 1.1 Urine glucose meter folded (closed).

Photo 1.2 Urine glucose meter extended (opened).

Photo 1.2 shows a urine glucose meter at the time of measurement. When measured, total length 210 mm of the meter can be directly applied to urine with one hand. The urine glucose sensor at the tip is equipped with a thermistor for detecting the water temperature so that the output can be corrected. The sensor needs to be replaced either after 200 measurements or within 60 days due to the removable socket structure. The main body measures the minute current and controls the device. It also has a liquid crystal display that indicates urine glucose concentration, and switches for calibration and measurement value recall. It also operates for 8 months with one lithium battery.

1.4.2 Performance evaluation of urine glucose meter

The final test of the urine glucose meter requires performance evaluation by the urine of patients. The results of simultaneous measurement of patients’ urine specimens with urine glucose meter and clinical urine glucose analyzer (A&T GA03R) and correlation evaluation are shown in Fig. 1.4. The primary equation obtained by the method of least squares is urine glucose meter ① Y=0.925 X+53.3, R=0.987, urine glucose meter ② Y=0.939 X+62.9, R=0.987, urine glucose meter ③ Y=0.968 X+40.4, R=0.99, showing a high correlation with the conventional clinical urine glucose analyzer. Especially, the deviation of the measured values in the low concentration region is small, although the deviation is medically regarded as a problem. However, Fig. 1.4 demonstrates sufficient results for performance of a compact and simple measuring instrument. As well, those results showed that as a self-measuring tool at home, its portability is a plus, and it can measure urine glucose with high accuracy with a small size of 210 mm in total length [8].

Figure 1.4 Correlation between urine glucose meter and clinical urine glucose analyzer.

1.5 Clinical application of urine glucose meter

1.5.1 Relationship between the amount of boiled rice and urine glucose concentration in impaired glucose tolerance

Meal load test was conducted on subjects judged to have impaired glucose tolerance by 75 g oral glucose tolerance test. In the method, blood glucose level and urine glucose level up to 3 hours after starting a meal were measured for 320 kcal salad and meat, boiled rice with different amount of 100–300 g (145–435 kcal). The blood glucose level was measured using a self-monitoring blood glucose meter (GLUCOCARD: ARKRAY), and the urine glucose concentration was measured with a developed urine glucose meter.

Fig. 1.5 (A) shows changes in blood glucose concentration, and (B) shows changes in urine glucose concentration. The results confirmed an increase in urine glucose concentration with the rise in blood glucose concentration reflecting the difference in the amount of boiled rice. In particular, differences in urine glucose concentrations of 400 and 600 mg/dL are difficult to determine with conventional urine glucose test paper. The above results indicate that the quantitative measurement using the urine glucose meter can accurately capture postprandial hyperglycemia that changes depending on the carbohydrate intake [9].

Figure 1.5 Blood glucose concentration (BG) and urine glucose concentration (UG) differ by changing the volume of rice (In case of impaired glucose tolerance). (A) BG changes after meal; (B) UG changes after meal.

1.5.2 Results of urine glucose monitoring on impaired glucose tolerance case

The results of urine glucose measurement before and after a meal for 7 days are shown in Fig. 1.6. In this impaired glucose tolerance case, self-monitoring of urine glucose (SMUG) was carried out by instructing the user to pay attention to the relationship between urine glucose concentration after meals and meal contents. The meal content ingested was recorded at the same time. As a result, it was revealed that dietary control becomes possible by monitoring the urine glucose concentration after meals. Also, over the next 8 months, as a result of eating meals that did not raise the urine glucose concentration after meals, weight decreased from 63.9 to 59.0 kg, body fat decreased from 20.5% to 13.7%, and it was also effective to reduce body weight. In conclusion, postprandial hyperglycemia that occurs from early stage of diabetes can be controlled by urine glucose meter [9].

Figure 1.6 Results of urine glucose measurement before and after meal for 7 days (In case of impaired glucose tolerance).

1.5.3 Results of a case of self-monitoring of urine glucose in diabetes

SMUG was conducted for 6 months on a voluntary type 2 diabetes patient (woman aged 69 years, height 153.2 cm, weight 51.8 kg, BMI 22.1 kg/m²). The method was to measure urine glucose concentration 8 times a day (before morning, before breakfast, after breakfast, before lunch, after lunch, before dinner, after dinner, before going to bed). Also, meal contents were recorded at the same time. The doctor monitored the feedback of the relationship between the urine glucose concentration and meal contents after meals. Meanwhile, at a hospital every month, HbA1c and body weight were measured. The results of SMUG are shown in Fig. 1.7

Figure 1.7 Results of SMUG during 4 months.

The transition of measured values over 100 mg/dL during 4 months from the start of urine glucose measurement was 9 times in the first 2 weeks after SMUG began, 5 times in the next 2 weeks, 6 times in the next 2 weeks, then 3 times, 1 time, 0 times, 3 times, 0 times, and 3 times, all 2-week periods. HbA1c and body weight change are shown in Fig. 1.8. During the 5 months before SMUG, HbA1c had been in the 8% range, but it decreased from 8.7% (glycemic control status: unacceptable) to 5.8% (glycemic control status: excellent) in about 3 months after starting urine glucose measurement. Therefore, glycemic control improved. As a result, a decrease in HbA1c was observed 1 month after starting measurement of postprandial urine glucose concentration, and it was effective for blood glucose control of type 2 diabetic patients. The finding of the interview after use is that urine glucose measurement is easy to introduce and continue because it is noninvasive and the measured value changes dynamically in the range of 10–2000 mg/dL, so the results show it was easy to understand, and it was thought that the motivation for the patient’s blood glucose control was improved.

Figure 1.8 Results of HbA1c(JDS) and body weight change.

1.6 Conclusions

In conclusion, (1) urine glucose concentration at 2 hours after a meal is higher in proportion to the amount of ingested carbohydrate; and (2) SMUG is available for control of the meal contents, which suppresses postprandial hyperglycemia, indicating that HbA1c and body weight can be reduced. Recently, clinical trials comparing SMBG and SMUG levels of type 2 diabetes revealed that there is no difference in diet therapy effectiveness [10]. Postprandial hyperglycemia stimulates glucose spikes to vascular endothelial cells. As a result, it has been clarified that not only diabetes but also arteriosclerosis causing stroke and myocardial infarction can be incubated. As well, it is one of risk factors for dementia. Monitoring postprandial hyperglycemia with a urine glucose meter from the earliest stage of diabetes is recommended as a noninvasive healthcare tool that helps modify lifestyle of diet and exercise. Furthermore, a portable urine glucose meter integrating IoT and AI not only supports meals and exercise, but is thought to become an effective diabetes prevention tool customized to characteristics of individuals.

References

1. Heise HM, et al. Noninvasive blood glucose sensors based on near-infrared spectroscopy. Artif Organs. 1994;18:439.

2. US Patent 5,553,616, Determination of concentrations of biological substances using raman spectroscopy and artificial neural network discriminator.

3. US Patent 2005/0215872, Monitoring of physiological analytes.

4. Ito N, et al. Development of a transcutaneous blood-constituent monitoring method using a suction effusion fluid collection technique and an ion-sensitive field-effect transistor glucose sensor. Med Biol Comput. 1994;32:242.

5. March WF, et al. Ocular glucose sensor. Trans Am Soc Artif Intern Organs. 1982;28:232.

6. WO2014/113174, Encapsulated electronics.

7. Miyashita M, et al. Development of urine glucose meter based on micro-planer amperometric biosensor and its clinical application for self-monitoring of urine glucose. Biosensors Bioelectr. 2009;24:1336.

8. Yamaguchi I, et al. Performance evaluation of urine glucose meter: repeatability, effects of interferential substances, and comparison with clinical glucose analyzer. Rinsyoukensa. 2009;53:237.

9. Ohashi A, et al. Effect of food intake and its contents on postprandial urine glucose in diabetes candidates by digital urine glucose meter. Japan Soc Med Biol Eng. 2004;42:280.

10. Lu J, et al. Comparable efficacy of self-monitoring of quantitative urine glucose with seif-monitoring of blood glucose on glycemic control in non-insulin-treated type 2 diabetes. Diab Res Clin Pract. 2011;93:179.

2

Design, application, and integration of paper-based sensors with the Internet of Things

Jen-Hsuan Hsiao¹, Yu-Ting Tsao², Chung-Yao Yang³ and Chao-Min Cheng⁴,    ¹Department of Power Mechanical Engineering, National Tsing Hua University, Hsinchu, Taiwan,    ²School of Traditional Chinese Medicine, Chang Gung University College of Medicine, Taoyuan, Taiwan,    ³Hygeia Touch Inc., Taipei, Taiwan,    ⁴Institute of Biomedical Engineering, National Tsing Hua University, Hsinchu, Taiwan

Abstract

Paper-based analytical devices (PADs) are simple, convenient, and portable devices that can detect selected substances and quickly display results. PADs are being adopted at increasing rates for use in an expanding array of fields including biochemistry, drug test assaying, and environmental analysis. Compared with traditional analytical methodology, PADs provide several advantages including the following: (1) the capacity for easy mass production; (2) portability; (3) low cost; and (4) small sample volume requirements. These features position PADs as significantly useful tools in rural areas and developing countries, where sophisticated laboratories are limited. In addition to these advantages, PADs offer great potential for integration with the Internet and new artificial intelligence technology. Integrating what may be called lab-on-a-paper technology with the Internet of Things offers new opportunities for individuals, in the lab, in the field, and even at home, to carry out advanced examinations and analyses of a variety of things including point-of-care health diagnostics, health status, and environmental quality. In this way, PADs can significantly improve quality of life for a great number of people.

Keywords

Paper-based analytical devices; biochemistry; environment analysis; Internet of Things

2.1 Introduction

Paper-based analytical devices (PADs) have attracted significant and growing attention since their early development by the Whitesides research group [1]. Since that time, a number of fabrication methods, applications, and technological integrations, including integration with smartphones, have been investigated, demonstrated, and applied. Common fabrication methods for PADs now include photolithography [1], wax printing [2], wax dipping [3], screen printing [4–6], inkjet printing [7–10], plasma treatment [11–13], laser-based fabrication procedure [14], and the utilization of craft-cutting tools [15]. In addition to the significantly impactful development of two-dimensional PADs, the development of three-dimensional PADs has produced a crop of new tool platforms for the collection of rich data and valuable, health-altering diagnostics [16–19]. Paper-based sensors have proven themselves to be useful in a broad spectrum of fields. Multiple applications have been developed for both human diagnostics and environmental analysis. Among the diagnostic developments, a variety of tests examining urine, blood, and even very small amounts of aqueous humor have been successfully demonstrated. They have targeted a variety of diseases and disease states including paraquat poisoning, Alzheimer’s disease, age-related macular degeneration, glaucoma, corneal dystrophy, and chronic wound care to name but a few. Among the environmental analytical devices developed, these have successfully demonstrated the capacity to detect a host of target items including contaminants such as metals, nonmetals, organic molecules, pesticides, and microorganisms. All of these tools have the potential for integration with additional tools as well as, and perhaps most significantly, the Internet of Things.

2.2 Bioapplications of paper-based analytical devices

PAD bioapplications are continuing to be developed with a focus on health diagnostics for hastening and improving care via early diagnosis and disease state monitoring. Among these tools, an enzymatic assay for determining urinary creatinine was developed by Talalak et al. [20] that proved useful for investigating kidney function. When creatinine was present in the urine sample, the assays that had immobilized creatinine reagents would react and form pink-red quinoneimine dye. Color intensity was proportional to the creatinine concentration in the sample, and a linear range of 2.5–25, 2.0 mg/dL of detection limit was achieved (illustrated in Fig. 2.1A).

Figure 2.1 The design of PAD approaches for human usage. (A) The design and working principle of an ePAD for the detection of urine creatinine [20]. (B) The schematic of a distance-based PAD for ABO and Rh blood groups detection [21]. (C) The schematic illustration of the fabrication and sensing mechanism of an electrochemical-based ketamine detection PAD [22]. (D) Image of micropatterened liver function test (LFT) PADs assembled with a GX PSM and a Fusion 5 filter [23].

Accurate blood typing is essential for blood transfusion, tissue, and organ transplantation. In the research conducted by Al-Tamimi et al. [24], Kleenex paper was selected from a list of different paper substrates for its porous characteristic. Using this substrate, nonagglutinated red blood cells (RBCs) were eluted following application of 0.9% NaCl buffer. This paper-based assay was able to accurately detect the blood types of 100 samples within 1 minute, including 4 weak AB and 4 weak RhD samples. Another paper-based assay that could perform Rh and forward and reverse ABO blood typing was developed by Noiphung et al. [21]. They used a combination of wax printing and wax dipping methods to fabricate the PADs depicted in Fig. 2.1B. The advantage of this assay was that results could be maintained at room temperature for at least 7 days, a timespan that conventional slide or tube methods cannot achieve. Songjaroen and Laiwattanapaisal [25] described a PAD for simultaneous forward and reverse ABO blood group typing. The results were designed to be a barcode-like chart that could be read via comparison to a Tween-20 buffer wicking path.

Apart from blood typing applications, whole blood or serum samples are often used for various clinical diagnostics. Vella et al. [22] described a vertical flow paper-based assay that could detect alkaline phosphatase and aspartate aminotransferase, the two biomarkers of liver function, and total serum protein. This PAD comprised of a complete assay that could perform the functions of sample separation, distribution, and detection (illustrated in Fig. 2.1C). Test results could be captured using a smartphone and data could be sent to trained professionals for further analysis. Zhang et al. [26] developed a double-channel PAD that was coated with an enzyme–starch solution containing glucose oxidase, lactate oxidase, and horseradish peroxidase. To demonstrate the efficiency of detecting glucose and lactate, individual, mixed, and samples with RBCs were tested. Noiphung et al. [27] used an electrochemical detection method in their enzymatic PADs (ePADs) to measure the current of H2O2 at the optimum potential of −0.1 V (vs Ag/AgCl), which was proportional to whole blood glucose concentrations. This ePAD could also be used to measure other biochemical markers if H2O2 was a product of its enzymatic reaction.

In addition to general blood and serum test, PADs have also been developed to assist diagnosis in many medical fields such as infection, infertility, and rapid drug test. Tsai et al. [28] used unmodified gold nanoparticles and single-stranded detection oligonucleotides on a paper assay platform to detect target TB DNA sequences. These PADs could complete the diagnostic assay within 1 hour and the detection limit was 2.6 nM mycobacterium tuberculosis complex (MTBC) target sequences. They also introduced a model using a smartphone camera to take the diagnostic results and then transmit them for cloud computing. Matsuura et al. [29] used thiazine dye to stain sperm and evaluate sperm concentration in semen. They also used a tetrazolium-based colorimetric assay (MTT (3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay) to estimate human sperm motility in semen. The calculation of sperm concentration and motility by PADs helps men to evaluate their sperm quality in an easy way with increased privacy. Wang et al. [30] demonstrated a lateral flow immunoassay to detect dengue virus serotype-2 nonstructural protein-1 antigens in a buffer system. This lateral flow immunoassay could provide real-time diagnosis of dengue fever and create an opportunity for early remedial action. PADs have also been developed for isolating extracellular vesicles, which contain considerable data regarding intercellular communications. This approach may be further developed for nucleic acids analysis