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A modularized system is designed to replicate cellular environment and sequentially harvest extracellular vesicles released from cells. The system allows long-term culture under interstitial flow and simple metabolite collection via modularization.

Rapid detection of contaminants for the purpose of sensitive and quantitative monitoring of environmental hazards is an essential first step in realizing the avoidance of human health risks. In this regard, we present a fast and simple electrochemical method of detecting di

Extracellular vesicles (EV) have been emerging as potential biomarkers for disease monitoring. In particular, tumor‐derived EV (TDE) are known to carry oncogenic miRNA, so they can be used for diagnosis of early cancer by analyzing the expression levels of EV‐miRNA circulating in the blood. Here, using our novel microfluidic device, we rapidly and selectively isolate cancerous EV expressing breast cancer‐derived surface markers CD49f and EpCAM within 2 minutes. Based on seven candidates of miRNA nominated from The Cancer Genome Atlas (TCGA) database, the expression levels of miRNA in TDE were validated in a total of 82 individuals, including 62 breast cancer patients and 20 healthy controls. Among seven candidates, four miRNAs (miR‐9, miR‐16, miR‐21, and miR‐429) from the EV were highly elevated in early‐stage breast cancer patients compared with healthy donors. The combination of significant miRNAs from specific EV has high sensitivities of 0.90, 0.86, 0.88, and 0.84 of the area under the receiver operating characteristic curve (AUC) in each subtype (luminal A, luminal B, HER‐2, and triple‐negative) of early‐stage breast cancer. Our results suggest that the combination of four miRNA signatures of specific EV could serve as a sensitive and specific biomarker and enable early diagnosis of breast cancer using liquid biopsy., A new combination of miRNA from cancer‐specific extracellular vesicles was proposed for the highly sensitive and specific diagnosis of early‐stage breast cancer in association with a microfluidic‐based immuno‐isolation technique. These findings suggest that miRNA in EV could be used as important biomarkers for the early detection of breast cancer and identification of cancer subtype.

Coil spring-powered device for circulating biomarker isolation.

The contamination of food and drinking water by bacteria that cause food poisoning is a critical public health issue. In general, the procedures for testing the presence of pathogenic bacteria in public is not very effective, because it is time consuming and does not facilitate real-time monitoring in a field setting. Therefore, this study introduced a fully automated platform that would allow non-experts to easily examine the major food poisoning bacteria in a field setting, from pretreatment samples to final detection. Two enterobacteria species, of four different bacteria species (i.e., Escherichia coli and Salmonella typhimurium) were successfully immobilized on the surface of magnetic beads by exploiting the specific binding force with mannose. Subsequently, magneto-electrochemical impedance measurement technology allowed bacterial detection with a high sensitivity. E. coli, typical enterobacteria, was detected in 25 min with a detection limit of 100 CFU/mL. Moreover, we demonstrated that the automatic performance and the experimental consistency was greatly improved in comparison with those of manually conducted experiments.

In disease diagnostics and health surveillance, the use of saliva has potential because its collection is convenient and noninvasive. Over the past two decades, the development of salivary utilization for the early detection of cancer, especially oral cavity and oropharynx cancer has gained the interest of the researcher and clinician. Until recently, the oral cavity and oropharynx cancers are still having a five-year survival rate of 62%, one of the lowest in all major human cancers. More than 90% of oral cancers are oral squamous cell carcinoma (OSCC). Despite the ease of accessing the oral cavity in clinical examination, most OSCC lesions are not diagnosed in the early stage, which is suggested to be the main cause of the low survival rate. Many studies have been performed and reported more than 100 potential saliva biomarkers for OSCC. However, there are still obstacles in figuring out the reliable OSCC salivary biomarkers and the clinical application of the early diagnosis protocol. The current review article discusses the emerging issues and is hoped to raise awareness of this topic in both researchers and clinicians. We also suggested the potential salivary biomarkers that are reliable, specific, and sensitive for the early detection of OSCC.

Inflammation is an essential contributing factor in the development of thrombosis. Using a microfluidic flow chamber, we investigated how the proinflammatory cytokine interleukin 6 (IL-6) affects the cleavage of platelet-bearing ultra-large VWF (ULVWF) by plasma ADAMTS13. We found that IL-6-treated platelets perfused at arteriolar shear stress significantly enhanced the ULVWF-platelet complex formation on activated endothelial cells and suppressed their clearance by ADAMTS13 under flow conditions. We also detected the phosphorylation of the serine/threonine kinase Akt and extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in platelets treated with IL-6. Treatment of IL-6-primed platelets with either the phosphoinositol-3 kinase (PI3K) inhibitor LY294002 or the mitogen-activated protein kinase kinase (MEK) inhibitor U0126 reduced the ULVWF-platelet complex formation and restored the clearance of the complex by plasma ADAMTS13, compared to IL-6-primed platelets. Furthermore, IL-6 enhanced the phosphorylation of the intracellular adaptor molecule 14-3-3ζ, which regulates VWF binding to the glycoprotein (GP) Ib-IX complex. The 14-3-3 antagonist R18 significantly increased ADAMTS-13 cleavage of ULVWF strings with adherent IL-6-treated platelets. These findings indicate that IL-6 related intracellular signals of platelet is involved in regulating ULVWF-platelet binding and ULVWF cleavage by ADAMTS13.

Circulating tumor cell (CTC)-neutrophil clusters are highly potent precursors of cancer metastasis. However, their rarity in patients' blood has restricted research thus far, and moreover, studies on

We have developed a high-throughput pretreatment microfluidic chip for enrichment of microorganisms in food using magnetic particles and extracting DNAs using photothermal effects of magnetic particles. Magnetic particles modified with Concanavalin A can capture a variety of pathogens in the sample. As magnetic particles and bacteria injected into the microfluidic chip at a high flow rate, they bound actively in the mixing channel. After passing through the mixing channel, the combined bacteria and magnetic particles complexes were captured and enriched by magnetic force at the chambers which rectangular neodymium magnets were assembled in the form of dozens of arrays. After the magnet arrays were removed, the elution buffer was injected at a slightly lower flow rate and the eluted particles were captured in a small lysis chamber. The laser with a wavelength of 532 nm was irradiated at the lysis chamber to dissolve the captured bacteria as strong heat generated by the photothermal effects of the magnetic particles. Finally, the extracted DNAs were detected by real-time PCR.

Numerous studies reported that psychological fatigue is one of the main reasons leading fatal road crashes. In order to quantify fatigue level of each subject, we measured a concentration of salivary cortisol from 4 subjects (20-40 years of age) using the Smart Fatigue Phone, which consists of a lateral flow immunosensor and a smartphone-linked fluorescence signal reader, during 50-min driving session. Since the salivary cortisol needs to be measured below 1 ng/mL to distinguish the subjects from awaken-drivers, we have employed the fluorescence detection module (Limit of detection: 0.1 ng/mL). To validate correlation between fatigue status and salivary cortisol concentration measured by the Smart Fatigue Phone, the electroencephalogram (EEG) signal was simultaneously obtained from the participants. As a result, alpha wave and concentration of cortisol over time was highly correlated, reflecting that quantification of salivary cortisol can be used for real-time monitoring of driver fatigue (p 0.05). The Smart Fatigue Phone is expected to be a useful tool for drivers to recognize their fatigue status and subsequently to make a decision for driving a car. Thus, we assume that this fatigue detection system will consequently minimize road crashes by quantifying salivary cortisol in real time in the near future.

The estimation of bloodstain age is an important factor in forensic analysis. Previously, we have reported a smartphone-based colorimetric system for age estimation of bloodstain, in which Whole blood and EDTA whole blood were dropped on 4 different materials (700 μL) and captured using a smartphone for 72 h. In order to enhance sensitivity and accuracy of the previous system, the current work is dedicated towards the application of pattern recognition and classification of bloodstain images based on a smartphone. Three detection methods (blood pool, crack ratio, and colorimetric analysis) in terms of 6 steps of drying process of the bloodstain (coagulation, gelation, edge desiccation, center desiccation, crack propagation, and final desiccation) were applied to estimate age of the bloodstain accurately. Three parameters from the bloodstain images were then classified as comparing to those of stored reference images with similar trends in database. The bloodstain age was successfully determined by 9 h, 18 h, and 48 h with respect to the three detection methods mentioned above, respectively. The differences in bloodstain images were clearly distinguished every hour by using smartphone-based pattern recognition analysis. Therefore, our system is expected to shed a light on the field of forensic science by estimating bloodstain age in real time.

The outbreak of the COVID-19 pandemic has led to millions of fatalities worldwide. For preventing epidemic transmission, rapid and accurate virus detection methods to early identify infected people are urgently needed in the current situation. Therefore, an electrochemical biosensor based on the trans-cleavage activity of CRISPR/Cas13a was developed in this study for rapid, sensitive, and nucleic-acid-amplification-free detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Herein, a redox probe conjugated with ssRNA is immobilized on the electrode surface modified with a nanocomposite (NC) and gold nanoflower (AuNF) for enhancing the sensing performance. The SARS-CoV-2 RNA is captured by the Cas13a–crRNA complex, which triggers the RNase function of Cas13a. The enzymatically activated Cas13a–crRNA complex is subsequently introduced to the reRNA-conjugated electrochemical sensor, and consequently cleaves the reRNA. A change in current occurs due to the release of the redox molecule labeled on the reRNA, which is trans-cleaved from the Cas13a–crRNA complex. The biosensor can detect as low as 4.4 × 10−2 fg/mL and 8.1 × 10−2 fg/mL of ORF and S genes, respectively, over a wide dynamic range (1.0 × 10−1 to 1.0 × 105 fg/mL). Moreover, the biosensor was evaluated by measuring SARS-CoV-2 RNA spiked in artificial saliva. The recovery of the developed sensor was found to be in an agreeable range of 96.54–101.21%. The designed biosensor lays the groundwork for pre-amplification-free detection of ultra-low concentrations of SARS-CoV-2 RNA and on-site and rapid diagnostic testing for COVID-19.

Significance Skin-interfaced, wireless devices for clinical-grade monitoring of physiological parameters are of growing interest for uses that range from healthcare to sports performance. This paper introduces a multifunctional skin-mounted microfluidic platform for capture and biomarker analysis of microliter volumes of sweat, a biofluid that can be collected noninvasively, with potential relevance in biophysical sensing. The focus is on colorimetric and digital assessments of a collection of parameters related to stress, including concentrations of vitamin C, cortisol, and glucose, along with quantitative measurements of sweat rate and galvanic skin response. The results represent important additions to a portfolio of emerging capabilities in skin-interfaced technologies for physiological monitoring, with particular relevance to conditions that follow from unhealthy levels of physical and mental stress., Soft microfluidic systems that capture, store, and perform biomarker analysis of microliter volumes of sweat, in situ, as it emerges from the surface of the skin, represent an emerging class of wearable technology with powerful capabilities that complement those of traditional biophysical sensing devices. Recent work establishes applications in the real-time characterization of sweat dynamics and sweat chemistry in the context of sports performance and healthcare diagnostics. This paper presents a collection of advances in biochemical sensors and microfluidic designs that support multimodal operation in the monitoring of physiological signatures directly correlated to physical and mental stresses. These wireless, battery-free, skin-interfaced devices combine lateral flow immunoassays for cortisol, fluorometric assays for glucose and ascorbic acid (vitamin C), and digital tracking of skin galvanic responses. Systematic benchtop evaluations and field studies on human subjects highlight the key features of this platform for the continuous, noninvasive monitoring of biochemical and biophysical correlates of the stress state.

Through the development of organic synthetic skill, chemicals that mimic signaling mediators such as steroid hormones have been exposed to the environment. Recently, it has become apparent that this circumstance should be further studied in the field of physiology. Estrogenic action of chronic low-dose nonylphenol (NP) and di-(2-ethylhexyl) phthalate (DEHP) in mouse uterus was assessed in this study. Ten to twelve-week-old female mice (CD-1) were fed drinking water containing NP (50 or 500 μg/L) or DEHP (133 or 1,330 μg/L) for 10 weeks. Uterine diameter, the thickness of myometrium and endometrium, and the height of luminal epithelial cells were measured and the number of glands were counted. The expression levels of the known 17β-estradiol (E2)-regulated genes were evaluated with real-time RT-PCR methodology. The ration of uterine weight to body weight increased in 133 μg/L DEHP. Endometrial and myometrial thickness increased in 133 and 1,330 μg/L DEHP treated groups, and in 50, 500 μg/L NP and 133 μg/L DEHP, respectively. The height of luminal epithelial cell decreased in NP groups. The numbers of luminal epithelial gland were decreased in NP groups but increased in 50 μg/L DEHP group. The histological characters of glands were not different between groups. The mRNA expression profiles of the known 17β-estradiol (E2) downstream genes, Esr1, Esr2, Pgr, Lox, and Muc1, were also different between NP and DEHP groups. The expression levels dramatically increased in some genes by the NP or DEHP. Based on these results, it is suggested that the chronic low-dose NP or DEHP works as estrogen-like messengers in uterus with their own specific gene expression-regulation patterns.

Endocrine disruptors have been concerned in toxicology but now challenged as physiological point especially concerned with exposing dose and period. In this study the low-dose chronic administration of di(2-ethylhexyl) phthaltae (DEHP) during reproductive period was examined to evaluate the possible roles. Adult male and female CD-1 mice were exposed to DEHP with drinking water containing 133 1g/L and 1,330 /g/L DEHP in water according to OECD 433 guide line and sacrificed just after weaning. The weights of uterus and ovary were decreased by drinking of 1,330 /g/L DEHP water. There was not adverse effects on either accumulated mating rate and mating rate depend on estrus stage, pregnancy duration, and sex ration at birth. However, the accumulated rate of successful delivery and litter size were significantly high at 1,330 dg/L DEHP water. The number of epididymal sperm was significantly increased by drinking of 1,330 g/L DEHP water. In addition, the number of follicles (primary, secondary, tertiary) were more many than control at 1,330 /g/L DEHP water drunk mother. Though further studies are needed to identify what are the mechanism of DEHP in folliculogenesis and spermatogenesis. From this study we firstly report the effect of low-dose chronic administration of DEHP with drinking could change the ovarian follicle population size and spermatogenesis rate. Put together, those finding is different from previous high-dose effects and suggest the physiological role of DEHP in gonads and uterus.

Primary hyperparathyroidism (PHPT) is the leading cause of secondary osteoporosis. Although bone mineral density (BMD) tends to recover after parathyroidectomy in PHPT patients, the degree of recovery varies. Circulating microRNAs (miRNAs) profiles are known to be correlated with osteoporosis and fracture. We aimed to investigate whether osteoporotic fracture-related miRNAs are associated with postoperative BMD recovery in PHPT. Here, 16 previously identified osteoporotic fracture-related miRNAs were selected. We analyzed the association between the preoperative level of each miRNA and total hip (TH) BMD change. All 12 patients (among the 18 patients enrolled) were cured of PHPT after parathyroidectomy as parathyroid hormone (PTH) and calcium levels were restored to the normal range. Preoperative miR-19b-3p, miR-122-5p, and miR-375 showed a negative association with the percent changes in TH BMD from baseline. The association remained robust for miR-122-5p and miR-375 even after adjusting for sex, age, PTH, and procollagen type 1 N-terminal propeptide levels in a multivariable model. In conclusion, preoperative circulating miR-122-5p and miR-375 levels were negatively associated with TH BMD changes after parathyroidectomy in PHPT patients. miRNAs have the potential to serve as predictive biomarkers of treatment response in PHPT patients, which merits further investigation.

The rapid increase in the concentration of pathogenic airborne bacteria is a serious issue because of their infectivity. Due to the abundance of these infectious pathogens in the environment, researchers have developed devices to detect airborne pathogens using microfluidic technologies. Such devices represent a significant improvement over conventional techniques that rely on colony counting or biochemical assays, which are time as well as labor consuming. In addition to these detection methods, researchers have also shown that the photothermal effects of gold nanoparticles can be used to kill pathogenic bacteria. Thus, in this study, we combined a microfluidic device with a photothermal system to capture, lyse, and detect airborne bacteria. Specifically, we synthesized gold nanoparticles in poly(dimethylsiloxane) (PDMS) microfluidic chips to enrich and extract DNA from the pathogenic bacteria in air samples. The geometrically activated surface interaction (GASI) chip was employed, which was previously designed by our group and basically modified herringbone structure, to capture bacteria and increase the frequency of bacterial contact with the gold-nanoparticle-embedded PDMS. We design a microfluidic herringbone chip to capture bacteria and increase the frequency of bacterial contact with the gold-nanoparticle-embedded PDMS. Finally, the bacteria-captured PDMS microchips are irradiated at 532 nm using a 300-mW laser for 10 min, and the destruction of bacteria is verified by fluorescence microscopy and scanning electron microscopy (SEM).

In this study, our objective was to develop a photothermal biosensor (PTB) for measurement of C-reactive protein (CRP) concentrations in human saliva. The resulting PTB that we developed is rapid, simple, and minimally invasive, and can be used to measure the concentration of CRP in human saliva (7-μL sample volume). The CRP (0.1–100 ng/mL) is captured by the sandwich immunoassay on the sensor, and gold nanoparticles (GNPs) conjugated with antibodies cause a photothermal effect when irradiated with a laser (90 s, wavelength = 532 nm). The temperature changes caused by this effect correlate with the concentrations of CRP. Therefore, the sensor shows a higher correlation with the concentrations of CRP than commercial detection kits. Its detection limit is 0.1 ng/mL CRP.

4-Nonylphenol (NP) is a surfactant that is a well-known and widespread estrogenic endocrine disrupting chemical (EDC). Although it has been known that the affinity of NP to ERs is low, it has been suggested that low-dose NP has toxicity. In the present study, the endocrine disrupting effects on reproduction, and the weight of gonads, epididymis, and uterus were evaluated with the chronic lower-dose NP exposing. This study was designed by following the OECD test guideline 443 and subjected to a complete necropsy. In male, NP had an effect on the weight of the testis and epididymis in both F0 and F1. In females, NP decreased the weight of ovary and uterus in F0 but not in pre-pubertal F1 pubs. Fertility of male and female in F0 or F1 was no related with NP administration. The number of caudal-epididymal sperm by body weight (BW) was not different between groups in both F0 and F1. Besides, the difference of the sperm number between generations was not detected. The number of ovulated oocytes was similar between groups in F0, but significantly decreased in NP 50 group of F1. The litter size and sex ratios of offspring in F1 and F2 were not different. The accumulated mating rate and gestation period were not affected by the NP administration. Those results shows that chronic lower-dose NP administration has an effect of endocrine disruptor on the weight of gonads and epididymis of F0 and F1 but not in reproduction. Based on the results, it is suggested that chronic lower-dose NP exposing causes endocrine disruption in the weight of gonad and epididymis but not in the reproductive ability of next generations.

Estimating the age of a bloodstain is important for tracking down the true perpetrator of a crime from among the suspects. The current methods available for this estimation suffer from several shortcomings, including the time required and low accuracy. A mobile detection system, named Smart Forensic Phone, developed in this study enabled us to rapidly and precisely estimate the age of a bloodstain. We placed blood on five different materials (wallpaper, fabric, glass, wood, and A4 paper), monitored the RGB (Red, Green, and Blue) values per pixel of the bloodstain picture at 6-h intervals, and estimated the approximate age of the bloodstain using a smartphone application. The RGB values were converted into the V value of HSV (hue, saturation, and brightness). A rapid decline in RGB and V values was observed over the first 42 h and thereafter remained relatively unchanged. The age of the bloodstain could be calculated from the plot of V vs. time. This method enables the estimation of the age of the bloodstain if it is less than 42 h old. We will be continuing to work toward optimizing the method, so that the age of the bloodstain older than 42 h can be determined using the Smart Forensic Phone . Our detection system offers a novel method to both criminal investigators and researchers for predicting the time elapsed after the victim’s death, which is also called postmortem interval (PMI) or the approximate time when the crime was committed.

There is a growing interest in the area of affective disorder (i.e. psychological stress and depression) among advanced countries. Since the real-time diagnosis of human stress has been of great importance, we have recently reported the Smartphone Linked Stress Measurement (SLSM); the smartphone holder yet has two negative points, which are too excessive weight to carry and expensive cost to manufacture. In this paper, we have further developed the SLSM for the psychological stress in order to overcome the drawbacks mentioned above. We call the newly developed system as the Smartphone Diagnostics Unit (SDU). The SDU is composed of an origami smartphone holder, three cotton swabs, three strip biosensors, and three bottles of buffer solution. We demonstrated a light-weighted and economical origami smartphone holder to fulfill an easy-to-carry unit and low cost of fabrication, respectively. To validate the SDU, two kinds of stress-related biomarkers in human saliva, such as cortisol and C-reactive protein (CRP), were measured. The successful measurement of the concentration of salivary biomarkers enables the SDU to be useful for the real-time detection of human psychological stress in the future.

Salivary cortisol is frequently used as a biomarker of stress. However, no study has used salivary cortisol to evaluate stress levels related to personal characteristics with a paper-based lateral flow assay (LFA) strip and a smartphone holder, and a smartphone at the same time during simulated speech communication. In this paper, we investigated variations in levels of salivary cortisol, communication apprehension, and the effects of self-efficacy via the Smartphone Linked Stress Measurement (SLSM) that we have recently reported. Saliva samples were collected by 48 participants (30 male and 18 female) at three moments: immediately before public speaking (basal measurement), immediately after public speaking (speech measurement), and forty minutes after taking a rest (relaxed measurement). Results from the questionnaire showed that salivary cortisol levels, communication apprehension, and self-efficacy were significantly correlated. Also, anxiety about speaking can raise their stress levels during their speeches, as reflected in their increased cortisol levels. Therefore, this study establishes the effects of self-efficacy on communication apprehension and salivary cortisol.

The presence of circulating tumor cells (CTCs) in blood of cancer patients is thought to be an important indicator of disease progression and survival. Due to the rarity of CTCs in the bloodstream, CTCs must be enriched for molecular and cellular analyses for clinical use. Current CTC detection methods mostly rely on epithelial cell adhesion molecule (EpCAM); however, these methods may underestimate CTC number and potentially miss a critical subpopulation because of large heterogeneity among CTCs. We describe an integrated microfluidic chip, termed the μ-MixMACS chip, that can negatively exclude background cells from a CTC population. It comprises a micromixer that generates multivortexing flows to enhance mixing, which increasing binding between white blood cells and magnetic nanoparticles coated CD45 antibody, and a magnetic sorter to remove magnet-coated cells and minimize interference from white blood cells during subsequent CTC analysis. As all processes were carried out continuous manner, any CTC loss during isolation was minimized. Blood samples from 10 patients with adjuvant breast cancer were analyzed. CTCs were sorted as unfixed cells in solution. CTCs were isolated from 5 of 10 blood samples, 1–3 CTCs were found in each of these 5 samples. The proposed μ-MixMACS chip is expected to be useful for isolating CTCs from a variety of tumor types without any changes.

Medical disorders caused by second-hand smoke are a major public health concern worldwide. To estimate the level of second-hand smoke exposure, salivary diagnostics for cotinine analysis is a compelling alternative in conventional diagnostics using bio-fluids, such as blood and urine, owing to its simple and non-invasive collection method. However, there are several critical issues, such as tedious multisteps, demand for expertise, and field unavailability to collect and transport the purified saliva for further analysis. Here, an all-in-one platform is presented to simply collect real human saliva and directly deliver it onto the biosensing surface. The platform consists of a commercial cotton-swab-type collector, 3D-printed housing, and microfluidic channel integrated with an electrochemical competitive immunosensor to evaluate the level of salivary cotinine. The immunosensor is based on a competitive binding assay between cotinine-conjugated horseradish peroxidase (C-HRP) and cotinine for anti-cotinine binding sites. The current responses obtained from the HRP–thionine–H2O2 system decreased proportionally to the cotinine concentration. This immunosensor successfully detected its target over a range of 1 × 10−1 to 1 × 104 pg ml−1 with a low limit of detection of 6 × 10−2 pg ml−1 and a limit of quantification of 1 × 10−1 pg ml−1. In addition, the platform is applicable to various commercial cotton-swab-type saliva collectors and can successfully transfer the saliva in wide flow rates ranging from 0.1 to 30 ml min−1 without leakage or damage to the sensing surface. Furthermore, the practicality of the proposed platform was evaluated by measuring cotinine in real human saliva from eight non-smokers. The concentration of cotinine was from 45.7 to 890.8 pg ml−1, which was in good agreement with that measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The introduced all-in-one platform represented a reliable performance delivering simple and practical steps in salivary diagnostics.

Bloodstream infection by microorganisms is a major public health concern worldwide. Millions of people per year suffer from microbial infections, and current blood culture-based diagnostic methods are time-consuming because of the low concentration of infectious microorganisms in the bloodstream. In this study, we introduce an efficient automated microfluidic system for the continuous isolation of rare infectious bacteria (Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa) from blood. Bacteria received a balanced force between a fluidic drag force and a periodically controlled dielectrophoretic (DEP) force from tilted electrodes to minimize cell adhesion to the electrodes, which prevented the loss of rare infectious bacteria. Target bacteria were efficiently segregated from the undesired blood cells to ensure that only the bacteria received the DEP force under the hypotonic condition, while the blood cells received no DEP force and exited the channel via a laminar flow. Thus, the bacteria were successfully extracted from the blood with a high recovery yield of 91.3%, and the limit of the bacteria concentration for isolation was 100 cfu/ml. We also developed an automated system that performed every step from blood-sample loading to application of electricity to the microfluidic chip for bacteria separation. It reduced the standard deviation of the bacteria recovery yield from 6.16 to 2.77 compared with the conventional batch process, providing stable bacteria-extraction performance and minimizing errors and bacteria loss caused by user mistakes. © 2019 International Society for Advancement of Cytometry.

Photothermal sensors represent a novel type of probe having potential in the field of clinical diagnostics particularly due to the lack of a tedious sample pre-treatment, a sophisticated equipment and a skilled manpower. Herein, we describe the fabrication of a microfluidic nickel-resistive temperature detector (micro-Ni-RTD) for the selective monitoring of diabetes using a boronate-based sialic acid (SA) receptor. This work represents an improvement made to our previous photothermal sensor designs by reducing the cost by about 100-fold and significantly improving the sensitivity of the device, enabling the detection of sialic acid contents as low as 0.06 μmol/mL, a 4-fold improvement compared to the sensor described our previous study. The average temperature difference between healthy subjects and diabetes patients was found to be 0.181 °C dL/g with micro-Ni-RTD in comparison with 0.043 °C dL/g with our previous platinum-based resistive temperature detector (Pt-RTD) reported previously The enhancement in the discriminatory ability of micro-Ni-RTD for the diabetic clinical samples compared with healthy control can be attributed to the modified surface modification strategy and the incorporation of the microfluidic channels. Thus, micro-Ni-RTD represents a convenient, rapid, reliable, and low-cost biosensor for the clinical monitoring of diabetes patients.

We demonstrate the use of a parallel detection system with a nanograting-based microarray to accomplish high-throughput analysis of bio-molecular interactions in a label-free manner. Well-type label-free microarrays were fabricated to eliminate the risk of cross-contamination and to minimize sample volumes. Parallel analysis without the use of spectrometer arrays or a moving platform was accomplished by using scanning multiple optical probes generated by a spatial light modulator and microlens array. Additionally, multiple optical probe spots focused by the microlens array reduced detection errors while enhancing the signal-to-noise ratio within a high-density microarray. Finally, we verified the feasibility of the parallel detection system by analyzing the peak wavelength value (PWV) shift of human salivary cortisol and anti-cortisol in a competitive binding experiment.

The dissemination of circulating tumor cells (CTCs) requires the Epithelial-to-Mesenchymal transition (EMT), in which cells lose their epithelial characteristics and acquire more mesenchymal-like phenotypes. Current isolation of CTCs relies on affinity-based approaches reliant on the expression of Epithelial Cell Adhesion Molecule (EpCAM). Here we show EMT-induced breast cancer cells maintained in prolonged mammosphere culture conditions possess increased EMT markers and cancer stem cell markers, as well as reduced cell mass and size by quantitative phase microscopy; however, EpCAM expression is dramatically decreased in these cells. Moreover, CTCs isolated from breast cancer patients using a label-free microfluidic flow fractionation device had differing expression patterns of EpCAM, indicating that affinity approaches reliant on EpCAM expression may underestimate CTC number and potentially miss critical subpopulations. Further characterization of CTCs, including low-EpCAM populations, using this technology may improve detection techniques and cancer diagnosis, ultimately improving cancer treatment.

Much research has been performed over the past several decades in an attempt to conquer cancer. Tissue biopsy is the conventional method for gathering biological materials to analyze cancer and has contributed greatly to the understanding of cancer. However, this method is limited because it is time-consuming (requires tissue sectioning, staining, and pathological analysis), costly, provides scarce starting materials for multiple tests, and is painful. A liquid biopsy, which analyzes cancer-derived materials from various body fluids using a minimally invasive procedure, is more practical for real-time monitoring of disease progression than tissue biopsy. Biomarkers analyzable through liquid biopsy include circulating tumor cells (CTCs), exosomes, circulating cell-free DNA (cfDNA), miRNA, and proteins. Research on CTCs has been actively conducted because CTCs provide information on the whole cell, unlike the other biomarkers mentioned above. However, owing to the rarity and heterogeneity of CTCs, CTC research faces many critical concerns. Although exosomes and cfDNA have some technical challenges, they are being highlighted as new target materials. That is because they also have genetic information on cancers. Even though the number of exosomes and cfDNA from early stage cancer patients are similar to healthy individuals, they are present in high concentrations after metastasis. In this article, we review several technologies for material analyses of cancer, discuss the critical concerns based on hands-on experience, and describe future directions for cancer screening, detection, and diagnostics.

The quantification of cancer-derived exosomes has a strong potential for minimally invasive diagnosis of cancer during its initial stage. As cancerous exosomes form a small fraction of all the exosomes present in blood, ultra-sensitive detection is a prerequisite for the development of exosome-based cancer diagnostics. Herein, a detachable microfluidic device implemented with an electrochemical aptasensor (DeMEA) is introduced for highly sensitive and in-situ quantification of cancerous exosomes. To fabricate the aptasensor, a nanocomposite was applied on the electrode surface followed by electroplating of gold nanostructures. Subsequently, an aptamer against an epithelial cell adhesion molecule is immobilized on the electrode surface to specifically detect cancer-specific exosomes. A microfluidic vortexer is then constructed and implemented in the sensing system to increase the collision between the exosomes and sensing surface using hydrodynamically generated transverse flow. The microfluidic vortexer was integrated with the aptasensor via a 3D printed magnetic housing. The detachable clamping of the two different devices provides an opportunity to subsequently harvest the exosomes for downstream analysis. The DeMEA has high sensitivity and specificity with an ultra-low limit of detection of 17 exosomes/μL over a wide dynamic range (1 × 102 to 1 × 109) exosomes/μL in a short period. As proof of the concept, the aptasensor can be separated from the 3D printed housing to harvest and analyze the exosomes by real-time polymerase chain reaction. Moreover, the DeMEA quantifies the exosomes from plasma samples of patients with breast cancer at different stages of the disease. The DeMEA provides a bright horizon for the application of microfluidic integrated biosensors for the early detection of cancerous biomarkers.

Microbial forensics, exploiting bacteria, archaea, and eukaryotes, has been considered as one of the primary fields to trace the postmortem interval from the decaying cadavers. On the other hand, there remain several challenges of laboratory-based analysis for prediction of postmortem interval, including long-time measurement, complicated measuring procedure, and bacterial growth while carrying samples from the scene. Herein, we introduce the Smart Forensic Kit, which consists of a highly sensitive colorimetric gas sensor, a quality control algorithm, and a smartphone-based analysis method, to quantify the bacterial-derived ammonia gas in real-time. As a result, the estimation system of the postmortem interval has a superior selectivity to the ammonia gas with a detection limit of 38.7 ppb, response linearity to the target bacteria (Escherichia coli, Pseudomonas aeruginosa, and Pseudomonas putida), and short measuring time (10 min) with the maximum predicted postmortem interval from the mouse carcass (168 h). Furthermore, thanks to measuring the postmortem interval within 10 min, the negligible increase rate of bacterial concentration was observed. Consequently, the results reflected a high correlation between the ammonia gas emitted from bacteria and the postmortem interval so that we believe the Smart Forensic Kit will be applied for tracing down the decomposition of the cadavers in the near future.

Liquid biopsies are easier to acquire patient derived samples than conventional tissue biopsies, and their use enables real-time monitoring of the disease through continuous sampling after initial diagnosis, resulting in a paradigm shift to customized treatment according to the patient’s prognosis. Among the various liquid biopsy samples, saliva is easily obtained by spitting or swab sucking without needing an expert for sample collection. In addition, it is known that disease related biomarkers that exist in the blood and have undergone extensive research exist in saliva even at a lower concentration than the blood. Thus, interest in the use of saliva as a liquid biopsy has increased. In this review, we focused on the salivary exosome and cell-free DNA (cfDNA) among the various biomarkers in saliva. Since the exosome and cfDNA in saliva are present at lower concentrations than the biomarkers in blood, it is important to separate and concentrate them before conducting down-stream analyses such as exosome cargo analysis, quantitative polymerase chain reaction (qPCR), and sequencing. However, saliva is difficult to apply directly to microfluidics-based systems for separation because of its high viscosity and the presence of various foreign substances. Therefore, we reviewed the microfluidics-based saliva pretreatment method and then compared the commercially available kit and the microfluidic chip for isolation and enrichment of the exosome and cfDNA in saliva.

Paper-based lateral-flow assay (LFA) is a simple and inexpensive point-of-care device that has become commonplace in medicine, environmental monitoring, and over-the-counter personal use. Some LFAs have demonstrated comparable analytical performance with laboratory-based methods, but the detection limit or sensitivity of most LFAs is significantly inferior to other molecular techniques by 10-100 × . Consequently, LFAs are not viable for the early detection of disease-relevant biomarkers that are present in extremely small amounts in clinical specimens. Herein, we present a simple, cost-effective, and highly sensitive LFA sensor based on photothermal laser speckle imaging (PT-LSI). Under the illumination of a photothermal excitation light, gold nanoparticles (AuNPs), a common signal transduction medium in LFAs, absorb the light energy to produce heat, which subsequently induces modulation of the optical property and thermal deformation of the membrane. We measured these fluctuations through laser speckle imaging to quantify the concentration of AuNP-biomarker complexes. We experimentally demonstrate that the detection limit of our technique is superior to that of colorimetric detector by 68-125 × . The capability of our sensor for highly sensitive detection of disease biomarkers is validated by using U.S. FDA-approved LFA kits for cryptococcal antigens (CrAg).

Bisphenol A (BPA) is an organic monomer used to make common consumer goods such as plastic containers, sports equipment, and cosmetics which are heavily produced worldwide. A growing interest has been drawn to general public as BPA is one of the major endocrine disrupting chemicals threating human health. To date, numerous BPA sensors have been attempted to be developed but important challenges still remained such as limited linearity range, easy to use, and long term response time. To address the present issues, a microfluidic channel should be integrated into an electrochemical aptasensor and it is called Geometrically Activated Surface Interaction (GASI) chip. The vigorous generation of the micro-vortex in the GASI fluidic chamber provides the high collision chances between BPA and anti-BPA aptamer (BPAPT) and consequently more BPA molecules can be captured on the aptasensor surface, which finally results in high sensitivity of the aptasensor. To construct the integrated aptasensor, a miniaturized gold electrode is fabricated using shadow mask and e-beam evaporation process. Afterward, BPAPT is immobilized on a nanostructured gold electrode via thiol chemistry, and other terminus of the aptamer is labeled with a ferrocene (Fc) redox probe. Then, the microfluidic channel is mounted over the miniaturized gold electrode to introduce and enrich BPA to the aptasensor. Upon the specific interaction between BPA and its aptamer, configuration of aptamer is changed so that Fc tag approaches to the electrode surface and direct oxidation signal of Fc and BPA are followed as analytical signals. The unique microfluidic integrated electrochemical aptasensor delivers a wide linear dynamic range over 5 × 10–12 to 1 × 10−9 M, with a limit of detection 2 × 10–13 M. This aptasensor provides a precise platform for simple, selective and more importantly rapid detection of BPA. Such kind of sensing platforms can serve as a fertile ground for designing miniaturized portable sensors.

The emergence of a wide range of applications of smartphones along with advances in ‘liquid biopsy’ has significantly propelled medical research particularly in the field of in vitro diagnostics (IVD). Herein, we have presented a detailed analysis of IVD, its associated critical concerns and probable solutions. It also demonstrates the transition in terms of analytes from minimally invasive (blood) to non-invasive (urine, saliva and sweat) and depicts how the different features of a smartphone can be integrated for specific diagnostic purposes. This review basically highlights recent advances in the applications of smartphone-based biosensors in IVD taking into account the following factors: accuracy and portability; quantitative and qualitative analysis; and centralization and decentralization tests. Furthermore, the critical concerns and future direction of diagnostics based on smartphones are also discussed.

This is a one-step POC biosensor that guarantees rapid assay times, low-cost analysis, simple handling, stability, and is easy to mass product. However, it has several drawbacks such as limited sample volume and relatively low sensitivity. To overcome these disadvantages, we have designed and fabricated the µ-PAD to confine fluid flow by creating a hydrophobic channel in the paper to improve the sensitivity. The channel pattern was drawn by a computer-aided design program and directly printed by a commercially available wax printer which generates the hydrophobic channel pattern. While maintaining a constant sample, absorption, and conjugation pad, the width of the detection pad was reduced from 5 mm to 2 mm at intervals of 1 mm. The intensity of bands from the single- and double-orifice patterns increased identically compared with the device with no orifice. The relative intensity of the signal bands increased from 141 to 158. Also, numerical simulation was performed to validate the experimental result by using lattice Boltzmann method. We observed that the sensitivity was enhanced at a specific detection pad width (3 mm). Therefore, our simple structural change of the channel led to improve the colorimetric intensity. Cortisol, which is a known stress biomarker, was used to validation the device, an enhanced signal was obtained indicating that our device can be used for the detection psychological stress in humans.

Lateral flow assay is a simple and rapid diagnostic tool and consequently has been widely used in both academia and industry. Although the fabrication procedure for test strips is easy and simple, an expensive dispenser is necessary. Here we propose and develop a cheap and user-friendly dispenser using a fountain pen and antibody-conjugated gold nanoparticle ink to create test lines on a lateral flow immunosensor strip. The advantages of the pen type dispenser are that (1) depending on the size of the pen nib, various widths of lines can be drawn and (2) applying a small amount of the sample solution into the reservoir can conserve expensive antibody–gold colloidal solution in a small scale-experiment or feasibility test. In order to validate the newly developed lateral flow immunosensor, alpha-amylase and cortisol present in saliva were chosen as biomarkers representing the degree of human emotional stress. The biomarkers were successfully detected through our system and the results clearly show a correlation between the amount of analytes and signals developed after specific reactions.