Dielectrophoresis: An Approach to Increase Sensitivity, Reduce Response Time and to Suppress Nonspecific Binding in Biosensors?

The performance of receptor-based biosensors is often limited by either diffusion of the analyte causing unreasonable long assay times or a lack of specificity limiting the sensitivity due to the noise of nonspecific binding. Alternating current (AC) electrokinetics and its effect on biosensing is an increasing field of research dedicated to address this issue and can improve mass transfer of the analyte by electrothermal effects, electroosmosis, or dielectrophoresis (DEP). Accordingly, several works have shown improved sensitivity and lowered assay times by order of magnitude thanks to the improved mass transfer with these techniques. To realize high sensitivity in real samples with realistic sample matrix avoiding nonspecific binding is critical and the improved mass transfer should ideally be specific to the target analyte. In this paper we cover recent approaches to combine biosensors with DEP, which is the AC kinetic approach with the highest selectivity. We conclude that while associated with many challenges, for several applications the approach could be beneficial, especially if more work is dedicated to minimizing nonspecific bindings, for which DEP offers interesting perspectives

Microfluidic Selection of Aptamers towards Applications in Precision Medicine

Precision medicine represents a shift in medicine where large datasets are gathered for massive patient groups to draw correlations between disease cohorts. An individual patient can then be compared to these large datasets to determine the best treatment strategy. While electronic health records and next generation sequencing techniques have enabled much of the early applications for precision medicine, the human genome only represents a fraction of the information present and important to a person’s health. A person’s proteome (peptides and proteins) and glycome (glycans and glycosylation patterns) contain biomarkers that indicate health and disease; however, tools to detect and analyze such biomarkers remain scarce. Thus, precision medicine databases are lacking a major source of phenotypic data due to the absence of available methods to explore these domains, despite the potential of such data to allow further stratification of patients and individualized therapeutic strategies. Available methods to detect non-nucleic acid biomarkers are currently not well suited to address the needs of precision medicine. Mass spectrometry techniques, while capable of generating high throughput data, lack standardization, require extensive preparative steps, and have many sources of errors. Immunoassays rely on antibodies which are time consuming and expensive to produce for newly discovered biomarkers. Aptamers, analogous to antibodies but composed of nucleotides and isolated through in vitro methods, have potential to identify non-nucleic acid biomarkers but methods to isolate aptamers remain labor and resource intensive and time consuming. Recently, microfluidic technology has been applied to the aptamer discovery process to reduce the aptamer development time, while consuming smaller amounts of reagents. Methods have been demonstrated that employ capillary electrophoresis, magnetic mixers, and integrated functional chambers to select aptamers. However, these methods are not yet able to fully integrate the entire aptamer discovery process on a single chip and must rely on off-chip processes to identify aptamers. In this thesis, new approaches for aptamer selection are developed that aim to integrate the entire process for aptamer discovery on a single chip. These approaches are capable of performing efficient aptamer selection and polymerase chain reaction based amplification while utilizing highly efficient bead-based reactions. The approaches use pressure driven flow, electrokinetic flow or a combination of both to transfer aptamer candidates through multiple rounds of affinity selection and PCR amplification within a single microfluidic device. As such, the approaches are capable of isolating aptamer candidates within a day while consuming <500 µg of a target molecule. The utility of the aptamer discovery approach is then demonstrated with examples in precision medicine over a broad spectrum (small molecule to protein) of molecular targets. Seeking to demonstrate the potential of the device to generate probes capable of accessing the human glycome (an emerging source of precision medicine biomarkers), aptamers are isolated against gangliosides GM1, GM3, and GD3, and a glycosylated peptide. Finally, personalized, patient specific aptamers are isolated against a multiple myeloma patient serum sample. The aptamers have high affinity only for the patient derived antibody

Minute-Level Speed Identification and Assessment of Bacteria/Cells Using Electrokinetic Assistance

Conventional techniques for detection and analysis of cells/bacteria use Western blot and ELISA kits that are high cost and long time consuming. An ideal advanced biosensor (molecular or whole cells detections) unit must have several important features: rapid detection time (&lt;15 minutes), high sensitivity (102 cells/ml for whole cell detection or sub-nM concentration for molecular detection), high specificity, small, and inexpensive instrumentation/configuration. Two novel platforms will be introduced here, including an optofluidic system for the rapid on-chip detection of bacterial infection and a cell-based biochip for the label-free assessment of drug susceptibility on cancer cells. Rapid identification of rare pathogen from a very dense human blood sample is realized through combining the hybrid electrokinetic concentration with on-chip surface-enhanced Raman spectroscopy (SERS) identification of bacteria based on their detected SERS spectra. Compared to the current method in the hospital, this simple and rapid platform accelerated the detection time from 2 days to a few minutes. The cell-based biochip uses a novel, rapid, and label-free approach- AC electric field induced electro-rotation (eROT) to evaluate the drug susceptibility of cancer cells. The isolated lung cancer cells were successfully analyzed using eROT approach for the rapid and label-free assessment of the drug susceptibility of cancer cells. eROT spectra for different drug-treated cancer cells was successfully determined to the drug resistance and susceptibilities through their frequency-dependent rotation speeds. The relationship and trend between eROT method and conventional method are very agreement

Label-free cell separation and sorting in microfluidic systems

Cell separation and sorting are essential steps in cell biology research and in many diagnostic and therapeutic methods. Recently, there has been interest in methods which avoid the use of biochemical labels; numerous intrinsic biomarkers have been explored to identify cells including size, electrical polarizability, and hydrodynamic properties. This review highlights microfluidic techniques used for label-free discrimination and fractionation of cell populations. Microfluidic systems have been adopted to precisely handle single cells and interface with other tools for biochemical analysis. We analyzed many of these techniques, detailing their mode of separation, while concentrating on recent developments and evaluating their prospects for application. Furthermore, this was done from a perspective where inertial effects are considered important and general performance metrics were proposed which would ease comparison of reported technologies. Lastly, we assess the current state of these technologies and suggest directions which may make them more accessible

Electrokinetic Mixing and Separation in Microfluidic Systems

Electrokinetics involves the study of liquid or particle motion under the action of an electric field; it includes electroosmosis, electrophoresis, dielectrophoresis, and electrowetting, etc. The applications of electrokinetics in the development of microfluidic devices have been widely attractive in the past decade. Electrokinetic devices generally require no external mechanical moving parts and can be made portable by replacing the power supply by small battery. Therefore, electrokinetic based microfluidic systems can serve as a viable tool in creating a lab-on-a-chip (LOC) for use in biological and chemical assays. Here we present our works of electrokenitic based mixing and separation in microfluidics systems. Firstly, we present a novel fast micromixer of quasi T-channel with electrically conductive sidewalls and some newly observed phenomena on mixing process. The sidewalls of the microchannel can be either parallel or non-parallel with an angle. The mixing behaviors in the micromixer with different angles between the two electrodes located at the sidewalls are studied in terms of velocity and scalar concentration distributions. It is found that mixing can be enhanced rapidly at a small angle about 5° between the two electrode sidewalls even at low AC voltage, compared with that in parallel sidewalls. The effectiveness of several parameters were explored for the further enhancement of the fluid mixing, including conductivity gradient, AC electric flied frequency, applied voltage, AC signal phase shift between the electrodes, etc. The results reveal that the mixing is the stronger under high conductivity gradient, low frequency, high voltage and 180º signal phase shift between the two electrodes. Fast mixing under high AC frequency can be achieved in this quasi T-channel micromixer as well. The most important observation is that for the first time turbulence can be achieved under AC electrokinetic forcing at low Reynolds number in the order of 1 in this novel design. Thus, turbulent mixing can also be generated in micofluidics to cause rapid mixing. The turbulent flow is also measured with laser induced fluorescence photobleaching anemometer. Secondly, we have successfully manipulated and isolated cancer cells from other cells and bio-particles, by dielectrophoresis (DEP) in a microfluidic platform in a continuous operation. In this cell sorter, the cancer cells were treated as target cells and were deflected to a side channel from a main channel as they experienced a negative DEP force, when an AC electric field at the cross-over frequency of the cancer cells was supplied. This motion consequently led to the separation of the cancer cells from other cells and bio-particles. Colorectal cancer cells (HCT116) were firstly separated from human Embryonic Kidney 293 cells (HEK 293) and Escherichia coli (E. coli) bacterium. Then prostate cancer cells (LNCaP) were separated from HCT116 cells. Furthermore, we developed a cascade configuration sorter to increase purity of the isolated target cells, and a staggered sorter with two side channels in opposite side walls to increase sample throughput without compromising enrichment factor. Comparing to a single side channel DEP cell sorter, the isolation purity was improved from 80% to 96% by single cascade sorter and the sample throughput was increased from 0.2 µL/min to 0.65 µL/min by a single staggered side channel sorter. Here we report the theory and method, experimental setup, results and discussion. The future work and direction will be proposed as well

Ultrafast Microfluidic Immunoassays Towards Real-time Intervention of Cytokine Storms

Biomarker-guided precision medicine holds great promise to provide personalized therapy with a good understanding of the molecular or cellular data of an individual patient. However, implementing this approach in critical care uniquely faces enormous challenges as it requires obtaining “real-time” data with high sensitivity, reliability, and multiplex capacity near the patient’s bedside in the quickly evolving illness. Current immunodiagnostic platforms generally compromise assay sensitivity and specificity for speed or face significantly increased complexity and cost for highly multiplexed detection with low sample volume. This thesis introduces two novel ultrafast immunoassay platforms: one is a machine learning-based digital molecular counting assay, and the other is a label-free nano-plasmonic sensor integrated with an electrokinetic mixer. Both of them incorporate microfluidic approaches to pave the way for near-real-time interventions of cytokine storms. In the first part of the thesis, we present an innovative concept and the theoretical study that enables ultrafast measurement of multiple protein biomarkers (<1 min assay incubation) with comparable sensitivity to the gold standard ELISA method. The approach, which we term “pre-equilibrium digital enzyme-linked immunosorbent assay” (PEdELISA) incorporates the single-molecular counting of proteins at the early, pre-equilibrium state to achieve the combination of high speed and sensitivity. We experimentally demonstrated the assay’s application in near-real-time monitoring of patients receiving chimeric antigen receptor (CAR) T-cell therapy and for longitudinal serum cytokine measurements in a mouse sepsis model. In the second part, we report the further development of a machine learning-based PEdELISA microarray data analysis approach with a significantly extended multiplex capacity using the spatial-spectral microfluidic encoding technique. This unique approach, together with a convolutional neural network-based image analysis algorithm, remarkably reduced errors faced by the highly multiplexed digital immunoassay at low analyte concentrations. As a result, we demonstrated the longitudinal data collection of 14 serum cytokines in human patients receiving CAR-T cell therapy at concentrations < 10pg/mL with a sample volume < 10 µL and 5-min assay incubation. In the third part, we demonstrate the clinical application of a machine learning-based digital protein microarray platform for rapid multiplex quantification of cytokines from critically ill COVID-19 patients admitted to the intensive care unit. The platform comprises two low-cost modules: (i) a semi-automated fluidic dispensing module that can be operated inside a biosafety cabinet to minimize the exposure of technician to the virus infection and (ii) a compact fluorescence optical scanner for the potential near-bedside readout. The automated system has achieved high interassay precision (~10% CV) with high sensitivity (<0.4pg/mL). Our data revealed large subject-to-subject variability in patient responses to anti-inflammatory treatment for COVID-19, reaffirming the need for a personalized strategy guided by rapid cytokine assays. Lastly, an AC electroosmosis-enhanced localized surface plasmon resonance (ACE-LSPR) biosensing device was presented for rapid analysis of cytokine IL-1β among sepsis patients. The ACE-LSPR device is constructed using both bottom-up and top-down sensor fabrication methods, allowing the seamless integration of antibody-conjugated gold nanorod (AuNR) biosensor arrays with microelectrodes on the same microfluidic platform. Applying an AC voltage to microelectrodes while scanning the scattering light intensity variation of the AuNR biosensors results in significantly enhanced biosensing performance. The technologies developed have enabled new capabilities with broad application to advance precision medicine of life-threatening acute illnesses in critical care, which potentially will allow the clinical team to make individualized treatment decisions based on a set of time-resolved biomarker signatures.PHDMechanical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/163129/1/yujing_1.pd

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

Fluidic Dielectrophoresis: Electrokinetic Polarization and Manipulation of Electrical Liquid Interfaces for Biological and Sensing Applications

Development of rapid, sensitive and portable detection systems are important for effective detection of diseases in developing countries, biowarfare/anti-terrorism applications, environmental monitoring, and for basic biological research. One of the most specific and popular sensing platform is the enzyme-linked immunosorbent assay (ELISA) platform, which identifies the presence of a substance, specifically an antigen, in a liquid sample. Biosensing assays, like ELISA, offer sensitivity and selectivity, however, its assay time is long due to immobilization and detection through a secondary antibody. The assay also requires periodic rinsing steps to avoid non-specific binding and to remove excess proteins. Finally, the fluorescent detection instrumentation is required, and is still too bulky and costly for widespread daily laboratory, clinical and point-of-care use. The main challenge for producing low cost, portable, and easy to operate biosensing systems is then to miniaturize the sensing platform without any sophisticated instrumentation and complicated reagent protocols. In this work, we first explore a well-known electrokinetic phenomenon called dielectrophoresis (DEP), which traditionally has been studied in cells and particles, but here at a liquid-liquid interface, which we call fluidic dielectrophoresis (fDEP). The liquid-liquid interface with disparaging electrical properties - conductivity and permittivity - is shown to move when subjected to an alternating current (ac) electric field and the direction and magnitude is studied at varying applied frequencies and voltages. We found that when a biomolecular reaction occurs at the liquid-liquid interface it alters the electrical properties, which is transduced by interfacial displacement; we call this novel transduction method interfacial electrokinetic transduction (IET). We began with a model biomolecular reaction between biotin and avidin to validate our detection scheme. We then performed detection of hCG in human serum. Finally, we implemented impedance spectroscopy to non-optical monitor the position of the interface. Coupled with IET, the system non-optically monitored the position of the electrical interface in the presence of a biomolecular reaction. Collectively, we successfully developed the first, in solution, label-free non-optical biosensor. This novel biosensor was shown to detect biomarkers down to femtomolar concentration in human serum within minutes

ELECTROKINETICS-ASSISTED ELECTRICAL SENSORS FOR RAPID DETECTION OF BACTERIA

Department of Mechanical Enginering (Mechanical Engineering)An array of microfabricated interdigitated electrodes (IDEs) is the most commonly used form of electrode geometry for dielectrophoretic manipulation of biological particles in microfluidic biochips owing to simplicity of fabrication and ease of analysis. However, the dielectrophoretic force dramatically reduces as the distance from the electrode surface increasestherefore, the effective region is usually close to the electrode surface for a given electric potential difference. Here, I present a novel two-dimensional computational method for generating planar electrode patterns with enhanced volumetric electric fields, which I call the ???microelectrode discretization (MED)??? method. It involves discretization and reconstruction of planar electrodes followed by selection of the electrode pattern that maximizes a newly defined objective function, factor S, which is determined by the electric potentials on the electrode surface alone. In this study, IDEs were used as test planar electrodes. Two arrays of IDEs and respective MED-optimized electrodes were implemented in microfluidic devices for the selective capture of Escherichia coli against 1-??m-diameter polystyrene beads, and I experimentally observed that 1.4 to 35.8 times more bacteria were captured using the MED-optimized electrodes than the IDEs (p < 0.0016), with a bacterial purity against the beads of more than 99.8%. This simple design method offered simplicity of fabrication, highly enhanced electric field, and uniformity of particle capture, and can be used for many dielectrophoresis-based sensors and microfluidic systems. Dielectrophoresis (DEP) is usually effective close to the electrode surface. Several techniques have been developed to overcome its drawbacks and to enhance dielectrophoretic particle capture. Here a simple technique was presented of superimposing alternating current DEP (high-frequency signals) and electroosmosis (EOlow-frequency signals) between two coplanar electrodes (gap: 25 ??m) using a lab-made voltage adder for rapid and selective concentration of bacteria, viruses, and proteins, where the voltages and frequencies of DEP and EO were controlled separately. This signal superimposition technique enhanced bacterial capture (Escherichia coli K-12 against 1-??m-diameter polystyrene beads) more selectively (>99 %) and rapidly (~30 s) at lower DEP (5 Vpp) and EO (1.2 Vpp) potentials than those used in the conventional DEP capture studies. Nanometer-sized MS2 viruses and troponin I antibody proteins were also concentrated using the superimposed signals, and significantly more MS2 and cTnI-Ab were captured using the superimposed signals than the DEP (10 Vpp) or EO (2 Vpp) signals alone (p < 0.035) between the two coplanar electrodes and at a short exposure time (1 min). This technique has several advantages, such as simplicity and low cost of electrode fabrication, rapid and large collection without electrolysis. Electrokinetic technologies such as AC electro-osmosis (EO) and dielectrophoresis (DEP) have been used for effective manipulation of bacteria to enhance the sensitivity of an assay, and many previously reported electrokinetics-enhanced biosensors are based on stagnant fluids. An effective region for positive DEP for particle capture is usually too close to the electrode for the flowing particles to move toward the detection zone of a biosensor against the flow directionthis poses a technical challenge for electrokinetics-assisted biosensors implemented within pressure-driven flows, especially if the particles flow with high speed and if the detection zone is small. Here, a microfluidic single-walled carbon nanotubes (SWCNTs)-based field-effect transistor immunosensor was presented with electrohydrodynamic (EHD) focusing and DEP concentration for continuous and label-free detection of flowing Staphylococcus aureus in a 0.01?? phosphate buffered saline (PBS) solution. The EHD focusing involved AC EO and negative DEP to align the flowing particles along lines close to the bottom surface of a microfluidic channel for facilitating particle capture downstream in the detection zone. For feasibility, 380-nm-diameter fluorescence beads suspended in 0.001?? PBS were tested, and 14.6 times more beads were observed to be concentrated on the detection area with EHD focusing. Moreover, label-free, continuous, and selective measurement of S. aureus in 0.01?? PBS was demonstrated, showing good linearity between the relative changes in electrical conductance of the SWCNTs and logarithmic S. aureus concentrations, a capture/detection time of 35 min, and limit of detection of 150 CFU/mL, as well as high specificity through electrical manipulation and biological interaction.ope