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Exosomes have gained recognition in cancer diagnostics and therapeutics. However, most exosome isolation methods are time-consuming, costly, and require bulky equipment, rendering them unsuitable for point-of-care (POC) settings. Microfluidics can be the key to solving these challenges. Here, we present a double filtration microfluidic device that can rapidly isolate exosomes via size-exclusion principles in POC settings. The device can efficiently isolate exosomes from 50-100 µL of plasma within 50 min. The device was compared against an already established exosome isolation method, polyethylene glycol (PEG)-based precipitation. The findings showed that both methods yield comparable exosome sizes and purity; however, exosomes isolated from the device exhibited an earlier miRNA detection compared to exosomes obtained from the PEG-based isolation. A comparative analysis of exosomes collected from membrane filters with 15 nm and 30 nm pore sizes showed a similarity in exosome size and miRNA detection, with significantly increased sample purity. Finally, TEM images were taken to analyze how the developed devices and PEG-based isolation alter exosome morphology and to analyze exosome sizes. This developed microfluidic device is cost-efficient and time-efficient. Thus, it is ideal for use in low-resourced and POC settings to aid in cancer and disease diagnostics and therapeutics.
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Exosomas , MicroARNs , Neoplasias , Humanos , Sistemas de Atención de Punto , MicrofluídicaRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is rapidly becoming one of the leading causes of cancer-related deaths in the United States, and with its high mortality rate, there is a pressing need to develop sensitive and robust methods for detection. Exosomal biomarker panels provide a promising avenue for PDAC screening since exosomes are highly stable and easily harvested from body fluids. PDAC-associated miRNAs packaged within these exosomes could be used as diagnostic markers. We analyzed a series of 18 candidate miRNAs via RT-qPCR to identify the differentially expressed miRNAs (p < 0.05, t-test) between plasma exosomes harvested from PDAC patients and control patients. From this analysis, we propose a four-marker panel consisting of miR-93-5p, miR-339-3p, miR-425-5p, and miR-425-3p with an area under the curve (AUC) of the receiver operator characteristic curve (ROC) of 0.885 with a sensitivity of 80% and a specificity of 94.7%, which is comparable to the CA19-9 standard PDAC marker diagnostic.
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Carcinoma Ductal Pancreático , Exosomas , MicroARNs , Neoplasias Pancreáticas , Humanos , MicroARNs/genética , Proyectos Piloto , Biomarcadores de Tumor/genética , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/genética , Carcinoma Ductal Pancreático/diagnóstico , Carcinoma Ductal Pancreático/genética , Exosomas/genética , Curva ROC , Neoplasias PancreáticasRESUMEN
For conception, sperm cells travel towards the oocyte. This journey is accomplished by only a few sperm cells, following various guidance mechanisms. Of these mechanisms, rheotaxis plays a significant role in guiding the sperm over a long distance. By taking advantage of this natural rheotaxis behavior of sperm, we have developed a microfluidic chip that isolates healthy sperm cells. The developed chip consists of different chambers separated by microchannels that facilitate separation of motile sperm cells from unprocessed semen samples with the help of fluid flow. The sperm cells are subjected to different velocities in different parts of the chip that direct functional sperm towards the collection chamber utilizing positive rheotaxis. The results from the developed microfluidic chip (with 0.5 µL min-1 flow rate) have shown almost 100% motility, a significantly higher percentage of morphologically normal sperm cells with lesser sperm DNA fragmentation than the control (no-flow) and raw semen sample. This chip satisfies the need of a clinical setting as it is low-cost, easy to operate and uses a small semen volume for sperm sorting.
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Dispositivos Laboratorio en un Chip , Motilidad Espermática , Animales , Fragmentación del ADN , Masculino , Análisis de Semen , EspermatozoidesRESUMEN
There is a growing interest in the development of portable, cost-effective, and easy-to-use biosensors for the rapid detection of diseases caused by infectious viruses: COVID-19 pandemic has highlighted the central role of diagnostics in response to global outbreaks. Among all the existing technologies, screen-printed electrodes (SPEs) represent a valuable technology for the detection of various viral pathogens. During the last five years, various nanomaterials have been utilized to modify SPEs to achieve convincing effects on the analytical performances of portable SPE-based diagnostics. Herein we would like to provide the readers a comprehensive investigation about the recent combination of SPEs and various nanomaterials for detecting viral pathogens. Manufacturing methods and features advances are critically discussed in the context of early-stage detection of diseases caused by HIV-1, HBV, HCV, Zika, Dengue, and Sars-CoV-2. A detailed table is reported to easily guide readers toward the "right" choice depending on the virus of interest.
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The detection of viruses using imaging techniques is challenging because of the weak scattering of light generated by the targets of sizes in the nanometer range. The system we have developed overcomes the light scattering problems by utilizing antibody-coated microbeads of higher index of refraction that can specifically bind with viruses and increase the acceptance angle. Using the new technology, we have developed a portable, cost-effective, and field-deployable platform for the rapid quantification of HIV-1 viral load for point-of-care (POC) settings. The system combines microfluidics with a wide field of view lensless imaging technology. Highly specific antibodies are functionalized to a glass slide inside a microchip to capture HIV-1 virions. The captured virions are then bound by antibody-conjugated microbeads, which have a higher refraction index. The microbeads-HIV-1 virions complexes generate diffraction patterns that are detected with a custom-built imaging setup and rapidly and accurately quantified by computational analysis. This platform technology enables fast nanoscale virus imaging and quantification from biological samples and thus can play a significant role in the detection and management of viral diseases.
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VIH-1 , Microesferas , Sistemas de Atención de Punto , Refractometría , Carga ViralRESUMEN
Lensless biological imaging systems are an emerging alternative to conventional microscopic systems because they enable a wide field of view imaging. While most microscopic systems sacrifice the field of view for magnification, lensless systems have taken advantage of small imaging pixel size, projection, digital magnification, and post-processing to compensate for diffracted images. A new lens-based system is designed to have the exact same wide field of view as that of a basic lensless setup. A new compound lens system design is utilized to achieve an explicit aim to have the same fields of view as the lensless setup. Then the characteristics of these two optical imaging setups (lensless and lens-based setups) are compared at this level of complexity to see what the minimal systems principles are needed to achieve the biological imaging goals for simplified and less expensive future designs. For both imaging systems, images of biological entities are recorded with the help of the same CMOS imaging device and computer software. The main contribution of this work is an exhaustive comparison between the performance characteristics of both systems using optical standards and biological images.
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Although highly active antiretroviral therapy (HAART) has been introduced over twenty years ago to treat Human Immunodeficiency Virus (HIV) positive patients, acquired immunodeficiency syndrome (AIDS) is still one of the deadliest diseases found worldwide. AIDS prevalence and mortality rates are usually more pronounced in resource-constrained countries than in the developed world. The lack of trained medical technicians, sophisticated diagnostic equipment, and the overall scarcity of medical infrastructures have severely impacted HIV/AIDS diagnostics, which hinders the initiation and periodic monitoring of antiretroviral therapy (ART). Currently, available HIV viral load assays are not well-suited for resource-limited settings due to their high cost and a requirement for medical/technical infrastructures. In this paper, we review current and emerging diagnostic assays for HIV detection, with a focus on point-of-care (POC) based immunoassays for viral load measurement, drug resistance, and HIV recurrence. We also discuss the limitations of the available HIV assays and highlight the technological advancements in cellphone, paper, and flexible material-based assays which have the potential to improve HIV diagnosis and monitoring, thus assisting with the management of the disease.
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Técnicas de Laboratorio Clínico/métodos , Infecciones por VIH/diagnóstico , Infecciones por VIH/tratamiento farmacológico , Fármacos Anti-VIH/uso terapéutico , Farmacorresistencia Viral , Infecciones por VIH/virología , VIH-1/aislamiento & purificación , Humanos , Sistemas de Atención de Punto , Carga Viral , Latencia del VirusRESUMEN
Cell phones show considerable promise for point-of-care (POC) diagnostic procedures because they are accessible, connected, and computationally powerful. Cell phone image processing methods are being developed for the detection and quantification of a wide range of targets, employing methods from microscopy to fluorescence techniques. However, most of the lab-based biological and biochemical assays still lack a robust and repeatable cell phone analogue. Existing solutions require external smartphone hardware to obtain quantitative results, imposing a design tradeoff between accessibility and accuracy. Here, we develop a cell phone imaging algorithm that enables analysis of assays that would typically be evaluated via spectroscopy. The developed technique uses the saturation parameter of hue-saturation-value color space to enable POC diagnosis. Through the analysis of over 10 000 images, we show that the saturation method consistently outperforms existing algorithms under a wide range of operating field conditions. The performance improvement is also proven analytically via the mathematic relationship between the saturation method and existing techniques. The method presented here is a step forward towards the development of POC diagnostics by reducing the required equipment, improving the limit of detection (LOD), and increasing the precision of quantitative results.
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Algoritmos , Teléfono Celular , Colorimetría/métodos , Proteína p24 del Núcleo del VIH/inmunología , Infecciones por VIH/diagnóstico , Procesamiento de Imagen Asistido por Computador/instrumentación , Sistemas de Atención de Punto , VIH/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , Humanos , Límite de Detección , Imagen Óptica/métodosRESUMEN
Point-of-care (POC) tests often rely on smartphone image methods for colorimetric analysis, but the results of such methods are frequently difficult to reproduce or standardize. The problem is aggravated by unpredictable image capture conditions, which pose a significant challenge when low limits of detection (LOD) are needed. Application-specific smartphone attachments are often used to standardize imaging conditions, but there has recently been an interest in equipment-free POC colorimetric analysis. Improved output metrics and preprocessing methods have been developed, but equipment-free imaging often has a high LOD and is inappropriate for quantitative tasks. Additional work is necessary to replace external smartphone attachments with algorithms. Towards this end, we have developed a video processing method that synthesizes many images into a single output metric. We use image features to select the best inputs from a large set of video frames and demonstrate that the resulting output values have a stronger correlation with laboratory methods and a lower standard error. The developed algorithm only requires 20 seconds of video and can easily be integrated with existing processing methods. We apply our algorithm to the NS1-based sandwich ELISA for Zika detection and show that the LOD is two times lower when our video-based method is used.
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Ebola virus disease (EVD) is a devastating, highly infectious illness with a high mortality rate. The disease is endemic to regions of Central and West Africa, where there is limited laboratory infrastructure and trained staff. The recent 2014 West African EVD outbreak has been unprecedented in case numbers and fatalities, and has proven that such regional outbreaks can become a potential threat to global public health, as it became the source for the subsequent transmission events in Spain and the USA. The urgent need for rapid and affordable means of detecting Ebola is crucial to control the spread of EVD and prevent devastating fatalities. Current diagnostic techniques include molecular diagnostics and other serological and antigen detection assays; which can be time-consuming, laboratory-based, often require trained personnel and specialized equipment. In this review, we discuss the various Ebola detection techniques currently in use, and highlight the potential future directions pertinent to the development and adoption of novel point-of-care diagnostic tools. Finally, a case is made for the need to develop novel microfluidic technologies and versatile rapid detection platforms for early detection of EVD.
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Ebolavirus/genética , Ebolavirus/aislamiento & purificación , Fiebre Hemorrágica Ebola/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , África Occidental/epidemiología , Técnicas Biosensibles/métodos , Brotes de Enfermedades , Progresión de la Enfermedad , Genoma Viral/genética , Fiebre Hemorrágica Ebola/epidemiología , Fiebre Hemorrágica Ebola/virología , Humanos , Sistemas de Atención de Punto , Reacción en Cadena en Tiempo Real de la Polimerasa , Resonancia por Plasmón de Superficie/métodosRESUMEN
Invasive candidiasis remains one of the most serious community and healthcare-acquired infections worldwide. Conventional Candida detection methods based on blood and plate culture are time-consuming and require at least 2-4 days to identify various Candida species. Despite considerable advances for candidiasis detection, the development of simple, compact and portable point-of-care diagnostics for rapid and precise testing that automatically performs cell lysis, nucleic acid extraction, purification and detection still remains a challenge. Here, we systematically review most prominent conventional and nonconventional techniques for the detection of various Candida species, including Candida staining, blood culture, serological testing and nucleic acid-based analysis. We also discuss the most advanced lab on a chip devices for candida detection.
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Candida albicans/aislamiento & purificación , Candidiasis/diagnóstico , Infección Hospitalaria/diagnóstico , Pruebas en el Punto de Atención/tendencias , Candida albicans/patogenicidad , Candidiasis/microbiología , Infección Hospitalaria/microbiología , Humanos , Dispositivos Laboratorio en un Chip , Sistemas de Atención de PuntoRESUMEN
The natural microenvironment of tumors is composed of extracellular matrix (ECM), blood vasculature, and supporting stromal cells. The physical characteristics of ECM as well as the cellular components play a vital role in controlling cancer cell proliferation, apoptosis, metabolism, and differentiation. To mimic the tumor microenvironment outside the human body for drug testing, two-dimensional (2-D) and murine tumor models are routinely used. Although these conventional approaches are employed in preclinical studies, they still present challenges. For example, murine tumor models are expensive and difficult to adopt for routine drug screening. On the other hand, 2-D in vitro models are simple to perform, but they do not recapitulate natural tumor microenvironment, because they do not capture important three-dimensional (3-D) cell-cell, cell-matrix signaling pathways, and multi-cellular heterogeneous components of the tumor microenvironment such as stromal and immune cells. The three-dimensional (3-D) in vitro tumor models aim to closely mimic cancer microenvironments and have emerged as an alternative to routinely used methods for drug screening. Herein, we review recent advances in 3-D tumor model generation and highlight directions for future applications in drug testing.
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Since its first appearance in 1981, HIV-1 has remained a global concern. Current methods for diagnosing HIV-1, while effective, are mostly specific to a given subtype of HIV-1 and often require expensive equipment and highly trained individuals to collect and process the sample. It is necessary to develop a sensitive diagnostic method that can be administered with minimal equipment to provide better care in low-resource settings. Loop-mediated isothermal amplification is a rapid and sensitive method for detecting the presence of specific nucleic acid sequences. Herein we report the development and comparison of two different HIV LAMP assays, integrase and VPR, as well as the comparison between TRIZol and magnetic beads RNA extraction methods for each assay. Our analysis shows that the integrase assay was able to detect the virus from multiple subtypes in under 30 min with a variable limit of detection (LOD) that was dependent on the HIV-1 subtype.
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Infecciones por VIH , VIH-1 , Técnicas de Amplificación de Ácido Nucleico , VIH-1/aislamiento & purificación , VIH-1/genética , Técnicas de Amplificación de Ácido Nucleico/métodos , Humanos , Infecciones por VIH/diagnóstico , Infecciones por VIH/virología , Técnicas de Diagnóstico Molecular/métodos , Límite de Detección , ARN Viral/análisisRESUMEN
Heart failure is a cardiovascular disease in which heart fails to pump sufficient blood required by the body. Significant signs of worsening heart failure include decreased thoracic impedance, increased heart rate, irregular electrocardiogram (ECG), and lack of motion activity of the patient. Heart failure can be better managed if monitored continuously and in real-time. The existing solutions for continuous monitoring of these parameters are invasive and hence are not only expensive but can also cause serious health risks. This paper discusses the development of a telehealth system that consists of an Internet of Things including a wearable device connected to a cloud-based database and a mobile application using Wi-Fi. The wearable device is a noninvasive monitor that consists of different sensors embedded with a microcontroller and can be a potential solution for better management of heart failure. It continuously monitors the above-mentioned parameters and sends data to the mobile application using a cloud-based system. The mobile application has separate portals for patients and doctors where doctor can monitor a specific patient enrolled under his profile. The performance of the developed device is validated in 10 healthy individuals.
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Circulating tumor cells (CTCs) are cells that have been shed from tumors and circulate in the bloodstream. These cells can also be responsible for further metastases and the spread of cancer. Taking a closer look and analyzing CTCs through what has come to be known as "liquid biopsy" has immense potential to further researchers' understanding of cancer biology. However, CTCs are very sparse and are therefore difficult to detect and capture. To combat this issue, researchers have attempted to create devices, assays, and further techniques to successfully isolate CTCs for analysis. In this work, new and existing biosensing techniques for CTC isolation, detection, and release/detachment are discussed and compared to evaluate their efficacy, specificity, and cost. Here, we specifically aim to evaluate and identify the potential success of these techniques and devices in point-of-care (POC) settings.
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Chronic inflammation is now recognized as one of the major risk factors and molecular hallmarks of chronic prostatitis, benign prostatic hyperplasia (BPH), and prostate tumorigenesis. However, the molecular mechanisms by which chronic inflammation signaling contributes to the pathogenesis of these prostate diseases are poorly understood. Previous efforts to therapeutically target the upstream (e.g., TLRs and IL1-Rs) and downstream (e.g., NF-κB subunits and cytokines) inflammatory signaling molecules in people with these conditions have been clinically ambiguous and unsatisfactory, hence fostering the recent paradigm shift towards unraveling and understanding the functional roles and clinical significance of the novel and relatively underexplored inflammatory molecules and pathways that could become potential therapeutic targets in managing prostatic diseases. In this review article, we exclusively discuss the causal and molecular drivers of prostatitis, BPH, and prostate tumorigenesis, as well as the potential impacts of microbiome dysbiosis and chronic inflammation in promoting prostate pathologies. We specifically focus on the importance of some of the underexplored druggable inflammatory molecules, by discussing how their aberrant signaling could promote prostate cancer (PCa) stemness, neuroendocrine differentiation, castration resistance, metabolic reprogramming, and immunosuppression. The potential contribution of the IL1R-TLR-IRAK-NF-κBs signaling molecules and NLR/inflammasomes in prostate pathologies, as well as the prospective benefits of selectively targeting the midstream molecules in the various inflammatory cascades, are also discussed. Though this review concentrates more on PCa, we envision that the information could be applied to other prostate diseases. In conclusion, we have underlined the molecular mechanisms and signaling pathways that may need to be targeted and/or further investigated to better understand the association between chronic inflammation and prostate diseases.
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Cell adhesion, morphology and growth are influenced by surface topography at nano and micrometer scales. Nano-textured surfaces are prepared using photolithography, plasma etching and long polymer chemical etching which are cost prohibitive and require specialized equipment. This article demonstrates a simple approach to synthesize nano-textured scaffolds from chicken eggshells. Varieties of pattern are made on the eggshells like micro-needle forests and nanopores, giving very uniform nano-textures to the surfaces. The surfaces are characterized for chemical composition and crystal phase. The novel patterns are transferred to PDMS surfaces and the nano-textured PDMS surfaces are used to study the effect of texturing on human fibroblast cell growth and attachment. The effects of surface topographies, along with laminin coating on cell cultures, are also studied. We find an exciting phenomenon that the initial seeding density of the fibroblast cells affects the influence of the nano-texturing on cell growth. These nano-textured surfaces give 16 times more fibroblast growth when compared to flat PDMS surfaces. The novel nano-textured patterns also double the laminin adsorption on PDMS.
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Materiales Biocompatibles/química , Cáscara de Huevo/química , Nanoestructuras/química , Andamios del Tejido/química , Animales , Proliferación Celular , Forma de la Célula , Células Cultivadas , Pollos , Dimetilpolisiloxanos/química , Fibroblastos/citología , Humanos , Nanoestructuras/ultraestructuraRESUMEN
Epidermal growth factor receptor (EGFR) is a cell surface protein overexpressed in cancerous cells. It is known to be the most common oncogene. EGFR concentration also increases in the serum of cancer patients. The detection of small changes in the concentration of EGFR can be critical for early diagnosis, resulting in better treatment and improved survival rate of cancer patients. This article reports an RNA aptamer based approach to selectively capture EGFR protein and an electrical scheme for its detection. Pairs of gold electrodes with nanometer separation were made through confluence of focused ion beam scratching and electromigration. The aptamer was hybridized to a single stranded DNA molecule, which in turn was immobilized on the SiO(2) surface between the gold nanoelectrodes. The selectivity of the aptamer was demonstrated by using control chips with mutated non-selective aptamer and with no aptamer. Surface functionalization was characterized by optical detection and two orders of magnitude increase in direct current (DC) was measured when selective capture of EGFR occurred. This represents an electronic biosensor for the detection of proteins of interest for medical applications.
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Aptámeros de Nucleótidos/química , Biomarcadores de Tumor/análisis , Técnicas Biosensibles/instrumentación , Conductometría/instrumentación , Receptores ErbB/análisis , Microelectrodos , Nanotecnología/instrumentación , Técnicas Biosensibles/métodos , Diseño de Equipo , Análisis de Falla de EquipoRESUMEN
Human immunodeficiency virus (HIV) is a global epidemic; however, many individuals are able to obtain treatment and manage their condition. Progression to acquired immunodeficiency syndrome (AIDS) occurs during late-stage HIV infection, which compromises the immune system, making it susceptible to infections. While there is no cure, antiretroviral therapy can be used provided that detection occurs, preferably during the early phase. However, the detection of HIV is expensive and resource-intensive when tested with conventional methods, such as flow cytometry, polymerase chain reaction (PCR), or enzyme-linked immunosorbent assays (ELISA). Improving disease detection in resource-constrained areas requires equipment that is affordable, portable, and can deliver rapid results. Microfluidic devices have transformed many benchtop techniques to on-chip detection for portable and rapid point-of-care (POC) testing. These devices are cost-effective, sensitive, and rapid and can be used in areas lacking resources. Moreover, their functionality can rival their benchtop counterparts, making them efficient for disease detection. In this review, we discuss the limitations of currently used conventional HIV diagnostic assays and provide an overview of potential microfluidic technologies that can improve HIV testing in POC settings.
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Infecciones por VIH , Dispositivos Laboratorio en un Chip , Humanos , Sistemas de Atención de Punto , Infecciones por VIH/diagnóstico , Pruebas en el Punto de Atención , Citometría de FlujoRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) remains a difficult tumor to diagnose and treat. To date, PDAC lacks routine screening with no markers available for early detection. Exosomes are 40-150 nm-sized extracellular vesicles that contain DNA, RNA, and proteins. These exosomes are released by all cell types into circulation and thus can be harvested from patient body fluids, thereby facilitating a non-invasive method for PDAC detection. A bioinformatics analysis was conducted utilizing publicly available miRNA pancreatic cancer expression and genome databases. Through this analysis, we identified 18 miRNA with strong potential for PDAC detection. From this analysis, 10 (MIR31, MIR93, MIR133A1, MIR210, MIR330, MIR339, MIR425, MIR429, MIR1208, and MIR3620) were chosen due to high copy number variation as well as their potential to differentiate patients with chronic pancreatitis, neoplasms, and PDAC. These 10 were examined for their mature miRNA expression patterns, giving rise to 18 mature miRs for further analysis. Exosomal RNA from cell culture media was analyzed via RTqPCR and seven mature miRs exhibited statistical significance (miR-31-5p, miR-31-3p, miR-210-3p, miR-339-5p, miR-425-5p, miR-425-3p, and miR-429). These identified biomarkers can potentially be used for early detection of PDAC.