Development of a Microfluidic Device for Exosome Isolation in Point-of-Care Settings.

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.

Exosomal miRNA Biomarker Panel for Pancreatic Ductal Adenocarcinoma Detection in Patient Plasma: A Pilot Study.

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.

Selection of healthy sperm based on positive rheotaxis using a microfluidic device.

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.

Nano-engineered screen-printed electrodes: A dynamic tool for detection of viruses.

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.

Development of a Point-of-Care Assay for HIV-1 Viral Load Using Higher Refractive Index Antibody-Coated Microbeads.

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.

Design, Development, and Performance Comparison of Wide Field Lensless and Lens-Based Optical Systems for Point-of-Care Biological Applications.

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.

Advances in HIV diagnosis and monitoring.

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.

Cell phone based colorimetric analysis for point-of-care settings.

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.

Point-of-care Colorimetric Analysis through Smartphone Video.

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.

Strategies in Ebola virus disease (EVD) diagnostics at the point of care.

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.

Advances in Candida detection platforms for clinical and point-of-care applications.

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.

Engineering cancer microenvironments for in vitro 3-D tumor models.

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.

Development of a Diagnostic Microfluidic Chip for SARS-CoV-2 Detection in Saliva and Nasopharyngeal Samples.

The novel coronavirus SARS-CoV-2 was first isolated in late 2019; it has spread to all continents, infected over 700 million people, and caused over 7 million deaths worldwide to date. The high transmissibility of the virus and the emergence of novel strains with altered pathogenicity and potential resistance to therapeutics and vaccines are major challenges in the study and treatment of the virus. Ongoing screening efforts aim to identify new cases to monitor the spread of the virus and help determine the danger connected to the emergence of new variants. Given its sensitivity and specificity, nucleic acid amplification tests (NAATs) such as RT-qPCR are the gold standard for SARS-CoV-2 detection. However, due to high costs, complexity, and unavailability in low-resource and point-of-care (POC) settings, the available RT-qPCR assays cannot match global testing demands. An alternative NAAT, RT-LAMP-based SARS-CoV-2 detection offers scalable, low-cost, and rapid testing capabilities. We have developed an automated RT-LAMP-based microfluidic chip that combines the RNA isolation, purification, and amplification steps on the same device and enables the visual detection of SARS-CoV-2 within 40 min from saliva and nasopharyngeal samples. The entire assay is executed inside a uniquely designed, inexpensive disposable microfluidic chip, where assay components and reagents have been optimized to provide precise and qualitative results and can be effectively deployed in POC settings. Furthermore, this technology could be easily adapted for other novel emerging viruses.

Development of a LAMP-Based Diagnostic for the Detection of Multiple HIV-1 Strains.

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.

A wearable telehealth system for the monitoring of parameters related to heart failure.

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.

Biosensors for the Isolation and Detection of Circulating Tumor Cells (CTCs) in Point-of-Care Settings.

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.

The Molecular Basis and Clinical Consequences of Chronic Inflammation in Prostatic Diseases: Prostatitis, Benign Prostatic Hyperplasia, and Prostate Cancer.

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.

Synthesis of nano-textured biocompatible scaffolds from chicken eggshells.

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.

Electrical detection of cancer biomarker using aptamers with nanogap break-junctions.

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.

RT-LAMP-Based Molecular Diagnostic Set-Up for Rapid Hepatitis C Virus Testing.

Hepatitis C virus (HCV) infections occur in approximately 3% of the world population. The development of an enhanced and extensive-scale screening is required to accomplish the World Health Organization's (WHO) goal of eliminating HCV as a public health problem by 2030. However, standard testing methods are time-consuming, expensive, and challenging to deploy in remote and underdeveloped areas. Therefore, a cost-effective, rapid, and accurate point-of-care (POC) diagnostic test is needed to properly manage the disease and reduce the economic burden caused by high case numbers. Herein, we present a fully automated reverse-transcription loop-mediated isothermal amplification (RT-LAMP)-based molecular diagnostic set-up for rapid HCV detection. The set-up consists of an automated disposable microfluidic chip, a small surface heater, and a reusable magnetic actuation platform. The microfluidic chip contains multiple chambers in which the plasma sample is processed. The system utilizes SYBR green dye to detect the amplification product with the naked eye. The efficiency of the microfluidic chip was tested with human plasma samples spiked with HCV virions, and the limit of detection observed was 500 virions/mL within 45 min. The entire virus detection process was executed inside a uniquely designed, inexpensive, disposable, and self-driven microfluidic chip with high sensitivity and specificity.