For the Sensing of Viral DNA: An Integrated Polydimethylsiloxane Accurate CRISPR Detection (IMPACT) System

Infectious disease outbreaks have become more frequent and extreme in recent years, and as populations continue to grow and the world becomes more interconnected, they show no signs of stopping. The current COVID-19 pandemic affecting the world and grinding economies to a halt was known about months ago but could not be contained. One of the largest issues facing the containment of infectious disease is a lack of real-time, point-of-care detection devices which can accurately and effectively identify those who are infected so they can be treated and quarantined. Here, an Integrated Micropillar Polydimethylsiloxane Accurate CRISPR Detection (IMPACT) system is developed for detection of viral DNA. Single-stranded DNA reporter probes with fluorescent dyes are immobilized within the system, taking advantage of the increased surface area from the micropillar. A CRISPR-Cas12a and crRNA complex is then injected into the system, and if double-stranded target DNA is present, the CRISPR enzyme is activated and indiscriminately cleaves reporter probes, greatly increasing the fluorescent signal. The system can then be washed and the supernatant collected and measured, revealing accurate detection of the viral DNA target down to 0.1 nM concentration with no fluorescence background

Micro/Nano-Chip Electrokinetics

Micro/nanofluidic chips have found increasing applications in the analysis of chemical and biological samples over the past two decades. Electrokinetics has become the method of choice in these micro/nano-chips for transporting, manipulating and sensing ions, (bio)molecules, fluids and (bio)particles, etc., due to the high maneuverability, scalability, sensitivity, and integrability. The involved phenomena, which cover electroosmosis, electrophoresis, dielectrophoresis, electrohydrodynamics, electrothermal flow, diffusioosmosis, diffusiophoresis, streaming potential, current, etc., arise from either the inherent or the induced surface charge on the solid-liquid interface under DC and/or AC electric fields. To review the state-of-the-art of micro/nanochip electrokinetics, we welcome, in this Special Issue of Micromachines, all original research or review articles on the fundamentals and applications of the variety of electrokinetic phenomena in both microfluidic and nanofluidic devices

Magnetic tunable microstructured surfaces for thermal management and microfluidic applications

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2013.Cataloged from PDF version of thesis.Includes bibliographical references (p. 46-47).Micro and nanostructured surfaces have broad applications including heat transfer enhancement in phase-change systems and liquid manipulation in microfluidic devices. While significant efforts have focused on fabricating static micro/nanostructured arrays, uniform arrays that can be dynamically tuned have not yet been demonstrated. In this work, we present a novel fabrication process for magnetically tunable microstructured surfaces, where the tilt angle can be controlled upon application of an external magnetic field. We also demonstrated this platform for droplet manipulation in heat transfer applications. The tunable surfaces consist of ferromagnetic nickel (Ni) pillars on a soft PDMS substrate. The pillars have diameters of 23-35 [mu]m, pitches of 60-70 [mu]m, and heights of 70-80 [mi]m. We used vibrating sample magnetometry to obtain hysteresis loops of the Ni pillar arrays which match well the properties of bulk Ni. With a field strength of 0.5 tesla and a field angle of 600, a uniform 10.5± 1 tilt angle of the pillar arrays was observed. Furthermore, we developed a model to capture the tilt angle as a function of the magnetic field, and showed that by replacing nickel to cobalt, the tilt angle could be increased to 30' with the same field. Meanwhile, simulations show good agreement with the experiments. Future work will focus on using these surfaces to actively transport water droplets and spread the liquid film via pillar movement. This work promises tunable surface designs for important device platforms in microfluidics, biological and optical applications.by Yangying Zhu.S.M

Advances in Microfluidics and Lab-on-a-Chip Technologies

Advances in molecular biology are enabling rapid and efficient analyses for effective intervention in domains such as biology research, infectious disease management, food safety, and biodefense. The emergence of microfluidics and nanotechnologies has enabled both new capabilities and instrument sizes practical for point-of-care. It has also introduced new functionality, enhanced sensitivity, and reduced the time and cost involved in conventional molecular diagnostic techniques. This chapter reviews the application of microfluidics for molecular diagnostics methods such as nucleic acid amplification, next-generation sequencing, high resolution melting analysis, cytogenetics, protein detection and analysis, and cell sorting. We also review microfluidic sample preparation platforms applied to molecular diagnostics and targeted to sample-in, answer-out capabilities

The use of microfluidic technology for cancer applications and liquid biopsy

© 2018 by the authors. There is growing awareness for the need of early diagnostic tools to aid in point-of-care testing in cancer. Tumor biopsy remains the conventional means in which to sample a tumor and often presents with challenges and associated risks. Therefore, alternative sources of tumor biomarkers is needed. Liquid biopsy has gained attention due to its non-invasive sampling of tumor tissue and ability to serially assess disease via a simple blood draw over the course of treatment. Among the leading technologies developing liquid biopsy solutions, microfluidics has recently come to the fore. Microfluidic platforms offer cellular separation and analysis platforms that allow for high throughout, high sensitivity and specificity, low sample volumes and reagent costs and precise liquid controlling capabilities. These characteristics make microfluidic technology a promising tool in separating and analyzing circulating tumor biomarkers for diagnosis, prognosis and monitoring. In this review, the characteristics of three kinds of circulating tumor markers will be described in the context of cancer, circulating tumor cells (CTCs), exosomes, and circulating tumor DNA (ctDNA). The review will focus on how the introduction of microfluidic technologies has improved the separation and analysis of these circulating tumor markers

HIGH-THROUGHPUT APTAMER DISCOVERY AND APTAMER INTEGRATION INTO MICROFLUIDIC DEVICES FOR RARE CELL ANALYSIS

Precision medicine is the idea where diagnostics and therapeutics are catered to each individual patient to provide personalized care that is optimally effective. For this to be achieved, technologies must exist that extensively examine samples and provide a highly detailed diagnosis for each patient, and processes must exist that can produce personalized drugs that specifically target the patient’s illness. Aptamers are short single stranded nucleic acids that bind to their targets with high affinity and specificity. Aptamers could make a substantial impact toward the goal of precision medicine. However, one of the main challenges preventing aptamers from reaching their potential is the efficient discovery of new high-affinity aptamers. Currently, aptamer selections are very time consuming and expensive, and often do not result in the discovery of a high-quality aptamer. The ability to reliably select aptamers with high affinity and specificity is paramount to the widespread use of aptamers. Consequently, there is great interest in improving selection technology to obtain high-quality aptamers much more rapidly. Toward this effort, we have developed a Microplate-based Enrichment Device Used for the Selection of Aptamers (MEDUSA) that uses affinity microcolumn chromatography. Its versatile 96-well microplate-based design allows this device to be compatible with downstream plate-based processing in aptamer selections, and it lends itself to automation using existing microplate-based liquid-handling systems. MEDUSA is also reconfigurable and is able to operate in serial and/or parallel mode with up to 96 microcolumns. We have demonstrated its use in high-throughput aptamer selections, characterization and optimization of the aptamer selection process, and characterization of previously selected aptamers. More specifically, MEDUSA was used to perform 96 simultaneous tests that determined the optimal target loading on resin to maximize aptamer enrichment for three target proteins, GFP, HSF, and NELF-E. These tests also verified the specificity of aptamers to these three proteins, as well as the non-specific binding of two suspected background binding aptamers. MEDUSA was also used to performed novel RNA aptamer selections to 19 different targets simultaneously. For these selections, a new, more efficient selection strategy was tested that greatly reduced the selection time and reagent consumption. Through the use of MEDUSA, aptamer selections can be optimized and performed in a high-throughput manner, and the success rate of novel aptamer discovery can be drastically improved. In addition to the improvement of novel aptamer discovery, developing valuable applications that use aptamers is of equal importance. An area of study in which aptamers could be of great benefit is cancer. Cancer cells are extremely diverse and contain genetic mutations that allow them to escape the regulatory processes necessary for the healthy function of tissues and organs. Moreover, there are numerous mechanisms for malignancy each with different combinations of genetic mutations, and cancer cells are constantly evolving, which makes cancer treatment difficult with varying levels of efficacy. Aptamers can be selected that bind specifically to cancer cells, and this can be accomplished without any knowledge about the cancer cell surface composition. We have developed a diagnostic device that uses cancer cell-specific aptamers to capture and filter out cancer cells from complex samples, such as blood. Within this same device, the captured cancer cells are lysed, and their genomic DNA (gDNA) is isolated using a micropillar array. The long strands of gDNA are physically entangled within the array, and remain in the microchannel even under flow. With this device, we have demonstrated the successful capture of human cervical and ovarian cancer cells. In addition, we have developed a custom isothermal amplification technique within the microchannel that amplifies a specific gene of interest for subsequent sequencing and analysis to determine the presence of any genetic mutations. Following the capture of cervical and ovarian cancer cells, we successfully amplified the TP53 gene and sequenced a fragment from this gene. By comparing the sequencing results to the known human TP53 gene sequence, we successfully detected a point mutation in the ovarian cancer cells, whereas the cervical cancer cells contained the wildtype version of this gene fragment. This device can be used to amplify multiple genes consecutively, since the gDNA is retained, so many genes of interest in cancer can be tested in the same small population of cancer cells. Using our device to test patients’ cancer cells, a large amount of information can be provided to clinicians about each specific patient that will help them to prescribe the most effective treatment strategy. From the research presented here, aptamers could have a great impact in many areas, and technologies like MEDUSA would help move the field forward by enabling novel aptamers to be successfully discovered rapidly and efficiently. Moreover, aptamers that bind specifically to cancer cells in diagnostics like the one presented here would enable highly informed decisions to be made by clinicians about treatment options. Furthermore, aptamers could also be used in targeted drug delivery and therapies. Therefore, technologies such as these are examples of key steps toward truly personalized and precision medicine

Microparticle Array on Gel Microstructure Chip for Multiplexed Biochemical Assays

Ph.DDOCTOR OF PHILOSOPH

Negative Enrichment of Circulating Tumor Cells Via 3D Printed Microfluidic Device

Circulating tumor cells (CTCs) are cells that detach from the primary tumor inside the cancer patient’s body to circulate in the bloodstream and cause the metastasis of the cancer. Routine and reliable isolation of CTCs from peripheral blood would both allow effective monitoring of the disease burden and guide the development of personalized treatments for cancer patients. Negative enrichment of CTCs by depleting normal blood cells from patient samples not only ensures against a biased selection of a subpopulation but also allows the assay to be used on patients with different tumor types. In my doctoral thesis, I developed an additively manufactured microfluidic device that can negatively enrich viable CTCs from clinically-relevant volumes of unmanipulated whole blood samples. The microfluidic device depletes nucleated blood cells based on their surface antigens and the smaller anucleated cells based on their size contrast with the tumor cells. Also, enriched CTCs are made available off the device in a suspension making our technique compatible with standard immunocytochemical, molecular and functional assays. The densely micropatterned 3D device could deplete >99.5% of white blood cells from 10 mL of unmanipulated whole blood while recovering >90% of spiked tumor cells. I also demonstrated the clinical utility of the device by isolating CTCs from blood samples collected from patients with prostate and pancreatic cancers. This creates a universal CTC assay that can differentiate tumor cells from normal blood cells with the specificity of clinically established membrane antigens yet with no labels on the sample offers the potential to enable routine screening of blood samples for tumor load at the point-of-care.Ph.D