Interferometric detection and enumeration of viral particles using Si-based microfluidics

Single-particle interferometric reflectance imaging sensor enables optical visualization and characterization of individual nanoparticles without any labels. Using this technique, we have shown end-point and real-time detection of viral particles using laminate-based active and passive cartridge configurations. Here, we present a new concept for low-cost microfluidic integration of the sensor chips into compact cartridges through utilization of readily available silicon fabrication technologies. This new cartridge configuration will allow simultaneous detection of individual virus binding events on a 9-spot microarray, and provide the needed simplicity and robustness for routine real-time operation for discrete detection of viral particles in a multiplex format.This work was supported in part by a research contract with the ASELSAN Research Center, Ankara, Turkey, and in part by the European Union's Horizon 2020 FET Open program under Grant 766466-INDEX. (ASELSAN Research Center, Ankara, Turkey; 766466-INDEX - European Union's Horizon 2020 FET Open program)First author draf

System Integration - A Major Step toward Lab on a Chip

Microfluidics holds great promise to revolutionize various areas of biological engineering, such as single cell analysis, environmental monitoring, regenerative medicine, and point-of-care diagnostics. Despite the fact that intensive efforts have been devoted into the field in the past decades, microfluidics has not yet been adopted widely. It is increasingly realized that an effective system integration strategy that is low cost and broadly applicable to various biological engineering situations is required to fully realize the potential of microfluidics. In this article, we review several promising system integration approaches for microfluidics and discuss their advantages, limitations, and applications. Future advancements of these microfluidic strategies will lead toward translational lab-on-a-chip systems for a wide spectrum of biological engineering applications

Electrochemical microfluidic multiplexed biosensor platform for point-of-care testing

Early and accurate diagnosis of a specific disease plays a decisive role for its effective treatment. However, in many cases the clinical findings, based on a single biomarker detection alone, are not sufficient for the appropriate diagnosis as well as monitoring of its treatment. Furthermore, it is highly desirable to screen multi-analytes (e.g. various diseases and drugs) at the same time enabling a low-cost, quick and reliable quantification. Thus, multiplexing, simultaneous detection of different analytes from a single sample, has become in recent years essential for diagnostics, especially for point-of-care testing (POCT). This thesis focuses on the scientific issue regarding the sensitivity enhancement of microfluidic biosensor platforms. Simulations, design studies and experiments are employed to investigate the interplay between the immobilization area and the resulting sensitivity. Thereby, a novel concept comprising design rules for microfluidic biosensors using the stop-flow technique has been introduced. In combination with different technical measures it allows the realization of an electrochemical lab-on-a-chip (LOC) platform for the fast, sensitive and simultaneous POCT in clinically relevant samples. This system employs a universally applicable, bioaffinity based biomolecule immobilization along with an amperometric readout. By means of the dry film photoresist technology, the fabrication of disposable microfluidic biosensors is enabled with high yield on wafer-level. The presented LOC platform offers three different biosensors with a microfluidic channel network of two, four or eight discrete immobilization sections, each with a volume of 680  nl. They can be actuated by individual channel inlets allowing a high flexibility in the assay design with respect to its format (e.g. competitive) and its technology (e.g. genomics). The feasibility for multiplexing is successfully demonstrated with DNA-based antibiotic assays for tetracycline and streptogramin, both important growth promoters in livestock breeding. The extensive usage of antibiotics is one of the major causes of the multi-drug-resistant bacteria and so, it has to be kept under surveillance. This platform allows the simultaneous POCT of different antibiotics from human plasma along with a limit of detection of less than 10  ng  ml⁻¹, a wide working range up to 1,600  ng ml⁻¹ and inter-assay precisions of about 10  %. Moreover, the microfluidic LOC system provides a low consumption of reagent and sample, reduces the total assay time drastically with a sample-to-result time of only 10  min. The shelf-life of the biosensors is proven to be at least 3 months at +4  °C. The introduced design concept with specific technical measures facilitates the implementation of microfluidic multiplexed biosensors in a low-cost, compact, and at the same time sensitive manner. This platform targets the POCT in the first place, yet, owing to its multiplexing approach it can be expanded for in vitro diagnostics

Nucleic Acid-based Detection of Bacterial Pathogens Using Integrated Microfluidic Platform Systems

The advent of nucleic acid-based pathogen detection methods offers increased sensitivity and specificity over traditional microbiological techniques, driving the development of portable, integrated biosensors. The miniaturization and automation of integrated detection systems presents a significant advantage for rapid, portable field-based testing. In this review, we highlight current developments and directions in nucleic acid-based micro total analysis systems for the detection of bacterial pathogens. Recent progress in the miniaturization of microfluidic processing steps for cell capture, DNA extraction and purification, polymerase chain reaction, and product detection are detailed. Discussions include strategies and challenges for implementation of an integrated portable platform

Punch Card Programmable Microfluidics

Small volume fluid handling in single and multiphase microfluidics provides a promising strategy for efficient bio-chemical assays, low-cost point-of-care diagnostics and new approaches to scientific discoveries. However multiple barriers exist towards low-cost field deployment of programmable microfluidics. Incorporating multiple pumps, mixers and discrete valve based control of nanoliter fluids and droplets in an integrated, programmable manner without additional required external components has remained elusive. Combining the idea of punch card programming with arbitrary fluid control, here we describe a self-contained, hand-crank powered, multiplex and robust programmable microfluidic platform. A paper tape encodes information as a series of punched holes. A mechanical reader/actuator reads these paper tapes and correspondingly executes a series of operations onto a microfluidic chip coupled to the platform in a plug-and-play fashion. Enabled by the complexity of codes that can be represented by a series of holes in punched paper tapes, we demonstrate independent control of fifteen on-chip pumps with enhanced mixing, on-off valves and a novel on-demand impact-based droplet generator. We demonstrate robustness of operation by encoding a string of characters representing the word "PUNCHCARD MICROFLUIDICS" using the droplet generator. Multiplexing is demonstrated by implementing an example water quality test utilizing colorimetric assays for pH, ammonia, nitrite and nitrate content in different water samples. With its portable and robust design, low cost and ease-of-use, we envision punch card programmable microfluidics will bring complex control of microfluidic chips into field-based applications in low-resource settings and in the hands of children around the world bringing microfluidics and low-Reynolds number hydrodynamics to everyday classrooms.Comment: 29 pages, 6 figure

Microfabricated Sampling Probes for Monitoring Brain Chemistry at High Spatial and Temporal Resolution

Monitoring neurochemical dynamics has played a crucial role in elucidating brain function and related disorders. An essential approach for monitoring neurochemicals is to couple sampling probes to analytical measurements; however, this approach is inherently limited by poor spatial and temporal resolution. In this work, we have developed miniaturized sampling probes and analytical technology to overcome these limitations. Conventional sampling probes were handmade and have several disadvantages, including large sizes (over 220 µm in diameter) and limited design flexibility. To address these disadvantages, we have used microfabrication to manufacture sampling probes. By bulk micromachining of Si, microchannels and small sampling regions can be fabricated within a probe, with an overall dimension of ~100 µm. For development of a dialysis probe, nanoporous anodic aluminum oxide was adapted for monolithically embedding a membrane. Coupling the probe to liquid chromatography-mass spectrometry, multiple neurochemicals were measured at basal conditions, including dopamine and acetylcholine. Comparing to conventional dialysis probes, the microfabricated dialysis probe provided at least 6-fold improvement in spatial resolution and potentially had lower tissue disruption. Furthermore, we have continued the development of a microfabricated push-pull probe. We enhanced functionality of the probe by integrating an additional channel into the probe for chemical delivery. Further, we demonstrated that the probe can feasibly be coupled to droplet microfluidic devices for improved temporal resolution. Nanospray ionization mass spectrometry was used for multiplexed measurements of neurochemicals in nanoliter droplet samples. Utility of the integrated system was demonstrated by monitoring in vivo dynamics during potassium stimulation of 4 neurochemicals, including glutamate and GABA. The probe provided unprecedented spatial resolution and temporal resolution as high as ~5 s. Additionally, we highlighted versatility of the method by coupling the probe to another high-throughput assay, i.e., droplet-based microchip capillary electrophoresis for rapid separation (less than 3 s) and measurement of multiple amino acid neurochemicals. This collection of work illustrates that development of the microfabricated sampling probes and their compatible microfluidic systems are highly beneficial for studying brain chemistry. The integrated miniaturized analytical technology can potentially be useful for solving other problems of biological significance.PHDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/144094/1/nonngern_1.pd

Configurable and Up-Scalable Microfluidic Life Science Platform for Cell Based Assays by Gravity Driven Sequential Perfusion and Diffusion

Microfluidics has the potential to significantly change the way modern biology is performed, but for this potential to be realized several on-chip integration and operation challenges have to be addressed. Critical issues are addressed in this work by first demonstrating an integrated microfluidic tmRNA purification and real time nucleic acid sequence based amplification (NASBA) device. The device is manufactured using soft lithography and a unique silica bead immobilization method for the nucleic acid micro purification column. The integrated device produced a pathogen-specific response in < 3 min from the chip-purified RNA. Further enhancements in the device design and operation that allow the on-chip integration of mammalian cell handling and culturing produced a novel integrated NASBA array. This system demonstrated for the first time that it is possible to combine on a single micro-device cell culture and real time NASBA. In order to expand the cell based assay capabilities of the integrated NASBA array and simplify the device operation novel hydrodynamics and cell sedimentation within trench structures and gravity driven sequential perfusion and diffusion mechanisms were developed. These mechanisms were characterized and implemented within an iCell array device. iCell array can completely integrate cell based assays with bio-analytical read-out. The device is highly scalable and can enable the configurable on-chip integration of procedures such as adherent and non-adherent cell-culture, cellstimulation, cell-lysis, cell-fixing, protein-immunoassays, bright field and fluorescent microscopic monitoring, and real time detection of nucleic acid amplification. The device uses on-board gravity driven flow control which makes it simple and economical to operate with dilute samples (down to 5 cells per reaction), low reagent volumes (50 nL per reaction), highly efficient cell capture (100% capture rates) and single cell protein and gene expression sensitivity. The key results from this work demonstrate a novel technology for versatile, fully integrated microfluidic array platforms. By multiplexing this integrated functionality, the device can be used from routine applications in a biology laboratory to high content screenings

Flexible Superwettable Tapes for On-Site Detection of Heavy Metals

Bioinspired superwettable micropatterns that combine superhydrophobicity and superhydrophilicity have been proved to exhibit outstanding capacity in controlling and patterning microdroplets and possessed new functionalities and possibilities in emerging sensing applications. Here, we introduce a flexible tape-based superhydrophilic–superhydrophobic tape toward on-site heavy metals monitoring. On such a superwettable tape, capillarity-assisted superhydrophilic microwells allow directly anchoring indicators in fixed locations and sampling into a test zone via simple dip-pull from an origin specimen solution. In contrast, the superhydrophobic substrate could confine the microdroplets in the superhydrophilic microwells for reducing the amount of analytical solution. The tape-based microchip also displays excellent flexibility against stretching, bending, and torquing for expanding wearable and portable sensing devices. Qualitative and quantitative colorimetric assessments of multiplex heavy metal analyses (chromium, copper, and nickel) by the naked eye are also achieved. The superwettable tape-based platforms with a facile operation mode and accessible signal read-out represent unrevealed potential for on-site environmental monitoring

Disposable cartridge based platform for real-time detection of single viruses in solution

Label-free imaging of viruses and nanoparticles directly in complex solutions is important for virology, vaccine research, and rapid diagnostics. These fields would all benefit from tools that allow for more rapid and sensitive characterization of viruses. Traditionally, light microscopy has been used in laboratories for detection of parasites, fungi, and bacteria for both research and clinical diagnosis because it is portable and simple to use. However, virus particles typically cannot be explored using light microscopy without the use of secondary labels due to their small size and low contrast. Characterization and detection of virus particles therefore rely on more complex approaches such as electron microscopy, ELISA, or plaque assay. These approaches require a significant level of expertise, purification of the virus from its natural environment, and often offer indirect verification of the virus presence. A successful virus visualization technique should be rapid, sensitive, and inexpensive, while needing minimal sample preparation or user expertise. We have developed a disposable cartridge based platform for real-time, sensitive, and label free visualization of viruses and nanoparticles directly in complex solutions such as serum. To create this platform we combined an interference reflectance imaging technique (SP-IRIS) with a sealable microfluidic cartridge. Through empirical testing and numeric modelling, the cartridge parameters were optimized and a flow rate of ~3 µL/min was established as optimal. A complex 2-dimensional paper based capillary pump was incorporated into the polymer cartridge to achieve a constant flow rate. Using this platform we were able to reliably show virus detection in a 20 minute experiment. We demonstrate sensitivity comparable to laboratory-based assays such as ELISA and plaque assay, and equal or better sensitivity compared to paper based rapid diagnostic tests. These results display a platform technology that is capable of rapid multiplexed detection and visualization of viruses and nanoparticles directly in solution. This disposable cartridge based platform represents a new approach for sample-to-answer label-free detection and visualization of viruses and nanoparticles. This technology has the potential to enable rapid and high-throughput investigation of virus particle morphology, as well as be used as a rapid point-of-care diagnostic tool where imaging viruses directly in biological samples would be valuable