Development and Characterization of Microfluidic Devices and Systems for Magnetic Bead-Based Biochemical Detection

This paper presents the development and characterization of an integrated microfluidic biochemical detection system for fast and low-volume immunoassays using magnetic beads, which are used as both immobilization surfaces and bio-molecule carriers. Microfluidic components have been developed and integrated to construct a microfluidic biochemical detection system. Magnetic bead-based immunoassay, as a typical example of biochemical detection and analysis, has been successfully performed on the integrated microfluidic biochemical analysis system that includes a surface-mounted biofilter and electrochemical sensor on a glass microfluidic motherboard. Total time required for an immunoassay was less than 20 min including sample incubation time, and sample volume wasted was less than 50 microl during five repeated assays. Fast and low-volume biochemical analysis has been successfully achieved with the developed biofilter and immunosensor, which is integrated to the microfluidic system. Such a magnetic bead-based biochemical detection system, described in this paper, can be applied to protein analysis systems.

In this paper, recent research activities at the Microsystems and BioMEMS Laboratory at the University of Cincinnati (UC) will be presented, which includes handheld biochemical detection systems with sampling and analysis capabilities and plastic based disposable biochips for clinical diagnostics. In the past few years, numerous microfluidic prototype devices and systems have been developed, specifically for biochemical warfare detection systems and portable diagnostic applications. BioMEMS-research team at the University of Cincinnati has been working on the development of a remotely accessible generic microfluidic system for biochemical detection and biomedical analysis, based on the concepts of both surface-mountable microfluidic motherboards and electrochemical detection techniques. The goal of this work is to develop a generic MEMS-based microfluidic system and to apply the fluidic system to detect bio-molecules such as specific proteins and/or antigens in liquid samples.

The dominant physical transport processes are analyzed in a free-surface microfluidic and surface-enhanced Raman spectroscopy (SERS) chemical detection system. The analysis describes the characteristic fluid dynamics and mass transport effects occurring in a microfluidic detection system whose analyte absorption and concentration capability is designed to operate on principles inspired by canine olfaction. The detection system provides continuous, real-time monitoring of particular vapor-phase analytes at concentrations of 1 ppb. The system is designed with a large free-surface-to-volume ratio microfluidic channel which allows for polar or hydrophilic airborne analytes to readily be partitioned from the surrounding gas phase into the aqueous phase for detection. The microfluidic stream can concentrate certain molecules by up to 6 orders of magnitude, and SERS can enhance the Raman signal by 9-10 orders of magnitude for molecules residing in the so-called SERS "hot spots", providing extremely high detection sensitivity. The resulting vibrational spectra are sufficiently specific to identify the detected analyte unambiguously. Detection performance was demonstrated using a nominal 1 ppb, 2,4-dinitrotoluene (2,4-DNT) vapor stream entrained within N(2) gas. Applications to homeland security arise from the system's high sensitivity and its ability to provide highly reproducible, continuous chemical detection monitoring with minimal sampling requirements.

Single-cell biophysical properties play a crucial role in regulating cellular physiological states and functions, demonstrating significant potential in the fields of life sciences and clinical diagnostics. Therefore, over the last few decades, researchers have developed various detection tools to explore the relationship between the biophysical changes of biological cells and human diseases. With the rapid advancement of modern microfabrication technology, microfluidic devices have quickly emerged as a promising platform for single-cell analysis offering advantages including high-throughput, exceptional precision, and ease of manipulation. Consequently, this paper provides an overview of the recent advances in microfluidic analysis and detection systems for single-cell biophysical properties and their applications in the field of cancer. The working principles and latest research progress of single-cell biophysical property detection are first analyzed, highlighting the significance of electrical and mechanical properties. The development of data acquisition and processing methods for real-time, high-throughput, and practical applications are then discussed. Furthermore, the differences in biophysical properties between tumor and normal cells are outlined, illustrating the potential for utilizing single-cell biophysical properties for tumor cell identification, classification, and drug response assessment. Lastly, we summarize the limitations of existing microfluidic analysis and detection systems in single-cell biophysical properties, while also pointing out the prospects and future directions of their applications in cancer diagnosis and treatment.

A microfluidic concentration gradient colorimetric system for rapid detection of nitrite in surface water

This paper first prepared and characterized an all solid zinc ion selective electrode using electrode deposition, and further established a microfluidic chip system integrating zinc ion ASS-ISE and a self-made potential detection device. The stability of the self-made potential detection device was verified by comparing it with traditional electrochemical workstations. The accuracy of the microfluidic chip detection system was verified through comparative testing with traditional atomic absorption spectrophotometry (AAS). The experimental results show that the accuracy and stability of the microfluidic chip system integrated with ASS-ISE and self-made potential detection device meet the requirements of on-site rapid detection.

In this thesis, nanometer scale semiconductor lasers and micrometer scale polymer based dye lasers are our focus in bringing the miniaturized lasers to applications in data transmission; ultra-small chemical / biological sensors; and ultra-compact spectroscopic sources. Combining the advantage of electrically driven semiconductor lasers and the advantage of a broad emission spectrum of dye molecules would utilize the highly dense multi-functional lab-on-a-chip by integrating microfluidic PCR, microfluidic fluorescent detection system, and compact visible and NIR detectors which are commercially available. On the other hand, in the meantime of pushing the size limit of the laser cavities, new phenomena with the nanoscale lasers enable further exploration and understanding in fundamental physics. In the first part of this thesis, two sub-micron scale semiconductor lasers are presented. The smallest lasers utilizing the disk structures—with diameters of approximately 600 nm—were realized in the InGaP/InGaAlP quantum well material system at room temperature, featuring ultra-small mode volumes of approximately 0.03 cubic?microns, and exhibiting single-mode operation at low threshold powers. And the first visible photonic crystal ultra-small mode volume lasers, with cavity volumes of approximately 0.01 cubic?microns, are realized in the same material system. They are ideally suited for use as spectroscopic sources and both of them can be lithographically tuned from 650 – 690 nm. In the second part of this thesis, two sub-millimeter-scale polymer-based dye lasers—a poly(dimethylsiloxane) (PDMS)-based mechanically tunable DFB dye laser and a poly(methylmethacrylate) (PMMA)-based second-order circular grating distributed feedback dye laser—are presented. Both of them are compatible with microfluidic technology, which gives freedom in integrating the lasers with the microfluidic chips. Compared to the soft lithography used in the PDMS-based dye laser, the nanoimprint lithography used in the PMMA-based dye laser would be more useful for fabricating ultra-small dye lasers and enabling mass production in the near future. At the end of the thesis, a nano-linewidth metal grating mask pattern transferred transient grating (MPT-TG) technique is described as a potential technique using the ultra-small lasers for molecular-dynamics study in solutions.

On-chip micro-electromagnets for magnetic-based bio-molecules separation

This paper presents the development of a generic microfluidic system toward portable biochemical detection. Microfluidic and electrochemical detection devices such as microvalves, flow sensors, biofilters, and immunosensors have been successfully developed and characterized in this work. A magnetic bio-bead approach has been adopted for both sampling and manipulating target biological molecules. The microfluidic and electrochemical immunosensing experiment results obtained from this work have shown that the biochemical sensing capability of the complete microfluidic subsystem is suitable for portable biochemical detection of bio-molecules.

Early detection of pathogens in high-value agricultural species is crucial. Therefore, many methods which can detect agricultural pathogens to prevent economic loss have been developed. Among them, immunoassays, nucleic acid hybridization and polymerase chain reaction have been demonstrated to detect pathogens in Phalaenopsis orchid successfully with satisfactory sensitivity and specificity. However, above-mentioned methods all have some disadvantages including lengthy process or requiring specialized laboratory facilities and well-trained technicians. The current study therefore presents an integrated micro fluidic system for rapid and automatic detection of pathogens in agricultural species. The entire procedure, including pathogen-specific ribonucleic acid (RNA) purification, nucleic acid amplification using reverse transcription loop-mediated-isothermal-amplification (RT-LAMP) and optical detection, can be automatically performed on a single chip within 65 minutes. Moreover, the detection module can be interchanged between a fluorescent detector and a turbidity detector, depending on the need of the operator. Furthermore, the developed system can detect pathogens directly from fresh agriculture tissues such as leaves and flowers of the Phalaneopsis orchids. This is the first time that an interchangeable integrated micro fluidic system for the detection of Phalaneopsis orchids has been demonstrated. Some of the most prevalent Phalaenopsis orchid pathogens., such as Cymbidium mosaic virus (CymMV) and Tomato spotted wilt virus (TSWV) for Phalaneopsis orchids were used in the current study to demonstrate the capabilities of the developed system. It is concluded that this system with dual detection units can directly detect pathogens from crude agricultural materials successfully.

Microelectromechanical systems (MEMS) technology has matured to the point where practical biological and chemical applications are possible. One particularly active research area is in the development of lab-on-a-chip type systems. In order to create successful lab-on-a-chip and other microfluidic systems, it is necessary to have the capability of controlling and directing fluid flow. Such functionality can be found on the front end of a microfluidic system and is known as a fluid delivery or dosing subsystem. For a MEMS micro fluid dosing system to be realized, several components are necessary. The essential components include a fluid actuator, a fluidic control device, and micro plumbing. A prototype fluid delivery system is demonstrated here using a micropump as the fluid actuator, a thermal flow sensor as the fluidic control device, and micromachined couplers as plumbing. The technology to build these components has been developed and each of these components have been fabricated and tested. A prototype constructed of discrete components has also been demonstrated. A truly integrated, channel-based fluid dosing system can be achieved through device scaling.

Human papillomavirus (HPV) causes the prevalent sexually transmitted infection that accounts for the majority of cervical cancer incidences. Therefore, the development of a rapid, accurate, automatic and affordable nucleic acid detection strategy is urgently required for HPV tests, among which microfluidic chip is a promising diagnostic method. In this work, we developed a microfluidic detection system consisting of a microfluidic chip and the corresponding detection equipment to diagnose high-risk HPV. The proposed method integrates nucleic acid purification, isothermal amplification and real-time fluorescence detection into one device. Moreover, it demonstrates good detection performance such as high specificity of primer sets (100%) and exceptional stability (coefficient of variation <6%) among five HPV genotypes. Besides, the microfluidic loop-mediated isothermal amplification (LAMP) assay is accurate (specificity of 91.7% and sensitivity of 100%) and fast (average time threshold = 10.56 minutes) when considering the conventional qPCR assay as the gold standard. The integrated microfluidic detection system offers automated and rapid diagnosis within 40 minutes and shows broad potential to deliver point-of-care detection in resource-limited circumstances owing to its simplicity and affordability.

The usage of calorimeters is limited due to its long measurement time and large sample consumption, despite its many advantages including universal applicability and simple sample preparation. Miniaturization of calorimeters not only resolves these problems, it also enables high-throughput measurements with array operations. We have developed microfluidic chip calorimeters with high sensitivity and reliable microfluidics-based sample handling. Immense sensitivity improvements are attained through reduction of the thermal conductance via on-chip vacuum insulation. This is enabled by Parylene thin-film microfluidic systems. Polydimethylsiloxane microfluidic systems, combined with the Parylene microfluidic system, gives easy and accurate control of picoliter-scale sample volume in a manner that is easily scalable to large, complex systems. Two device classes have been realized. Heat conduction calorimeters for biochemical reactions with 3.5 nL sample volume were built and validated by measurements of the heat of mixing and of enzyme activity. The thermal conductance of these devices was 15.5 µW/K and their power sensitivity was 4.2 nW. These devices can be built as calorimetric arrays to enable high-throughput heat of reaction measurements upon libraries of biomolecular interactions. Flow calorimeters were designed for sensor applications and measurements of cellular metabolism. The thermal conductance of these devices was 4.7 µW/K and their power sensitivity was 1.5 nW. Further reduction of thermal conductance and optimal thermocouple materials will deliver sensitivity of order ~1 pW, which will enable real-time measurement of single cell metabolism.

Recently, micro-electro-mechanical-systems (MEMS) technology and micromachining techniques have enabled miniaturization of biomedical devices and systems. Not only do these techniques facilitate the development of miniaturized instrumentation for biomedical analysis, but they also open a new era for integration of microdevices for performing accurate and sensitive diagnostic assays. A so-called “micro-total-analysis-system”, which integrates sample pretreatment, transport, reaction, and detection on a small chip in an automatic format, can be realized by combining functional microfluidic components manufactured by specific MEMS technologies. Among the promising applications using microfluidic technologies, nucleic acid-based detection has shown considerable potential recently. For instance, micro-polymerase chain reaction chips for rapid DNA amplification have attracted considerable interest. In addition, microfluidic devices for rapid sample pretreatment prior to nucleic acid-based detection have also achieved significant progress in the recent years. In this review paper, microfluidic systems for sample preparation, nucleic acid amplification and detection for fast diagnosis will be reviewed. These microfluidic devices and systems have several advantages over their large-scale counterparts, including lower sample/reagent consumption, lower power consumption, compact size, faster analysis, and lower per unit cost. The development of these microfluidic devices and systems may provide a revolutionary platform technology for fast sample pretreatment and accurate, sensitive diagnosis.

The goal of this research was to develop enhanced signal detection mechanisms for immunosensing using carbon nanotubes (CNTs). The utilization of CNT labels for direct electrical measurement was implemented on lateral flow system and microfluidic integrated interdigitated array microelectrodes. These sensing mechanisms in simple and miniaturized system provided higher sensitivity and autonomous flow control for rapid detection aimed at point-of-care diagnostics. Specific functionalization protocols were carried out to chemically modify the surface of the CNTs for uniform dispersion and antibody conjugation in aqueous solution. Surfactant assisted dispersion of the CNTs was studied using PVP and PEG. Covalent conjugation of antibodies on the carboxyl groups of the CNTs was accomplished using EDC/Sulfo-NHS coupling chemistry. The adsorption of surfactant and antibodies were manipulated in order to optimize immunoassay detection capability based on electrical measurements. Following surface functionalization methods, CNTs as a sensing label were employed on a lateral flow system. Competitive and sandwich immunoassay formats were demonstrated based on antibody and antigen binding. The lateral flow system was used for immobilization of capture molecules and passive sample transport by capillary action. CNTs conjugated to antibodies formed conductive network at the capture zone providing a visual indication corresponding to the amount of binding. Most importantly, significant change in electrical conductance was measured for varying low antigen concentrations, detecting anti-human Immunoglobulin G concentration below 1 ng/ml. Research was also conducted to obtain on-chip immunoassay detection using CNT labels. An IDA microelectrode was used as a binding surface and integrated within a PDMS microfluidic system. The sample and reagents were delivered to the sensing area through a microchannel. The capture of target analyte was indicated by the conjugated CNTs that formed a conducting matrix across the IDA. The detection was based on the selective binding between HSA and anti-HSA, where the conductimetric signal of the binding reaction was monitored through the IDA. The developed miniaturized system provided simple and sensitive immunosensing with detection capability below 1 ng/ml concentration using only 5 ìl of sample volume. Simulation was performed in order to understand the influence of the parameters in the microfluidic detection system.