Efficient microfluidic enrichment of nano-/submicroparticle in viscoelastic fluid.

The enrichment and focusing of the nano-/submicroparticle (e.g., 150-1000 nm microvesicle shed from the plasma membrane) in the viscoelastic fluid has great potentials in the biomedical and clinical applications such as the disease diagnosis and the prognostic test for liquid biopsy. However, due to the small size and the resulting weak hydrodynamic force, the efficient manipulation of the nano-/submicroparticle by the passive viscoelastic microfluidic technology remains a major challenge. For instance, a typically long channel length is often required to achieve the focusing or the separation of the nano-/submicroparticle, which makes it difficult to be integrated in small chip area. In this work, a microchannel with gradually contracted cross-section and high aspect ratio (the ratio of the height to the average width of channel) is utilized to enhance the hydrodynamic force and change the force direction, eventually leading to the efficient enrichment of nano-/submicroparticles (500 and 860 nm) in a short channel length (2 cm). The influence of the flow rate, the particle size, the solid concentration, and the channel geometry on the enrichment of the nano-/submicroparticles are investigated. With simple structure, small footprint, easy operation, and good performance, the present device would be a promising platform for various lab-chip microvesicle-related biomedical research and disease diagnosis.

Enhanced viscoelastic focusing of particle in microchannel.

A novel method is reported to enhance the focusing of microparticle in the viscoelastic fluid. Gradually contracted geometry is designed in microchannel, which changes the distribution of the elastic lift force on the cross section. Additionally, it induces the viscous drag force and the Saffman lift force in the lateral direction. Under the combined effect of these forces, microparticles fast migrate to the center of the channel. In comparison to the channel with constant cross section, the present channel significantly enhances the particle's lateral migration, leading to efficient viscoelastic particle focusing in a short channel length. The influence of flow rate, channel length, particle size and fluid property on the particle focusing is also investigated. With simple structure, small footprint and perfect particle focusing performance, the present device has great potential in the particle focusing processes in various lab-on-a-chip applications.

A passive microfluidic device for continuous microparticle enrichment.

A passive microfluidic device is reported for continuous microparticle enrichment. The microparticle is enriched based on the inertial effect in a microchannel with contracting-expanding structures on one side where microparticles/cells are subjected to the inertial lift force and the momentum-change-induced inertial force induced by highly curved streamlines. Under the combined effect of the two forces, yeast cells and microparticles of different sizes were continuously focused in the present device over a range of Reynolds numbers from 16.7 to 125. ∼68% of the particle-free liquid was separated from the sample at Re = 66.7, and ∼18 µL particle-free liquid was fast obtained within 10 s. Results also showed that the geometry of the contracting-expanding structure significantly influenced the lateral migration of the particle. Structures with a large angle induced strong inertial effect and weak disturbance effect of vortex on the particle, both of which enhanced the microparticle enrichment in microchannel. With simple structure, small footprint (18 × 0.35 mm), easy operation and cell-friendly property, the present device has great potential in biomedical applications, such as the enrichment of cells and the fast extraction of plasma from blood for disease diagnose and therapy.

A microfluidic device for noninvasive cell electrical stimulation and extracellular field potential analysis.

We developed a device that can quickly apply versatile electrical stimulation (ES) signals to cells suspended in microfluidic channels and measure extracellular field potential simultaneously. The device could trap cells onto the surface of measurement electrodes for ES and push them to the downstream channel after ES by increasing pressure for continuous measurement. Cardiomyocytes, major functional cells in heart, together with human fibroblast cells and human umbilical vein endothelial cells, were tested with the device. Extracellular field potential signals generated from the cells were recorded. We found that under electrical stimulation, cardiomyocytes were triggered to alter their field potential, while non-excitable cells were not triggered. Hence this device can noninvasively distinguish electrically excitable cells from electrically non-excitable cells. Results have also shown that increased cardiomyocyte cell number led to increased magnitude and occurrence of the cell responses. This relationship could be used to detect the viable cells in a cardiac tissue. Application of variable ES signals on different cardiomyocyte clusters has shown that the application of ES clearly boosted cardiomyocytes electrical activities according to the stimulation frequency. In addition, we confirmed that the device can apply ES onto and detect the electrical responses from a mixed cell cluster; the responses from the mixed cluster is dependent on the ratio of cardiomyocytes. These results demonstrated that our device could be used as a tool to optimize ES conditions to facilitate the functional engineered cardiac tissue development.

Microfluidic Magnetic Bead Assay for Cell Detection.

We present a novel cell detection device based on a magnetic bead cell assay and microfluidic Coulter counting technology. The device cannot only accurately measure cells size distribution and concentration but also detect specific target cells. The device consists of two identical micro Coulter counters separated by a fluid chamber where an external magnetic field is applied. Antibody-functionalized magnetic beads were bound to specific antigens expressed on the target cells. A high-gradient magnetic field was applied to the chamber closer to the second counter via an external cylindrical magnet. Because of the magnetic interaction between the magnetic beads and the magnetic field, target cells were retarded by the magnetic field; transit time of a target cell (bound with magnetic beads) passing through the second counter was longer than that through the first counter. In comparison, transit times of a nontarget cell remained nearly the same when it passed through both counters. Thus, from the transit time delay we can identify target cells and quantify their concentration in a cell suspension. The transit time and the size of each cell were accurately measured in terms of the width and amplitude of the resistive pulses generated from the two Coulter counters. Experiments demonstrated that for mixed cells with various target cell ratios, the transit time delay increased approximately linearly with the increasing target cell ratio. The limit of detection (LOD) of the assay was estimated to be 5.6% in terms of target cell ratio. Cell viability tests further demonstrated that most cells were viable after the detection. With the simple device configuration and easy sample preparation, this rapid and reliable method is expected to accurately detect target cells and could be applied to facilitate stem cell isolation and characterization.

pH-Sensitive Poly(histidine methacrylamide).

This research reports a synthetic amino acid based zwitterionic poly(histidine methacrylamide) (PHisMA), which possesses switchability among zwitterionic, anionic, and cationic states, pH-dependent antifouling properties, and chelation capability to multivalent metal ions. The PHisMA polymer brush surface shows good antifouling properties to resist protein adsorption and bacterial attachment in its zwitterionic state at pH 5. This study also demonstrates that the solution acidity significantly affects the mechanical properties of PHisMA hydrogels. PHisMA hydrogels show higher viscoelastic properties and lower swelling ratios in the zwitterionic state at pH 4 and pH 5, compared to higher or lower pH conditions. It was discovered that PHisMA can chelate multivalent metal ions, such as Ca(2+), Mg(2+), Cu(2+), Ni(2+) and Fe(3+). This study provides us a better understanding of structure-property relationships of switchable zwitterionic polymers. PHisMA can potentially be adapted for a broad range of applications including wound care, water treatment, bioseparation, coating, drug and gene delivery carriers, etc.

Structure-Function Relationships of a Tertiary Amine-Based Polycarboxybetaine.

Zwitterionic polycarboxybetaine (PCB) materials have attracted noticeable interest for biomedical applications, such as wound healing/tissue engineering, medical implants, and biosensors, due to their excellent antifouling properties and design flexibility. Antifouling materials with buffering capability are particularly useful for many biomedical applications. In this work, an integrated zwitterionic polymeric material, poly(2-((2-hydroxyethyl)(2-methacrylamidoethyl)ammonio)acetate) (PCBMAA-1T), was synthesized to carry desired properties (antifouling, switchability and buffering capability). A tertiary amine was used to replace quaternary ammonium as the cation to endow the materials with buffering capability under neutral pH. Through this study, a better understanding on the structure-property relationship of zwitterionic materials was obtained. The tertiary amine cation does not compromise antifouling properties of zwitterionic materials. The amount of adsorbed proteins on PCBMAA-1T polymer brushes is less than 0.8 ng/cm(2) for fibrinogen and 0.3 ng/cm(2) (detection limit of the surface plasmon resonance sensor) for both undiluted blood plasma and serum. It is found that the tertiary amine is favorable to obtain good lactone ring stability in switchable PCB materials. Titration study showed that PCBMAA-1T could resist pH changes under both acidic (pH 1-3) and neutral/basic (pH 7-9) conditions. To the best of our knowledge, such an all-in-one material has not been reported. We believe this material might be potentially used for a variety of applications, including tissue engineering, chronic wound healing and medical device coating.

Label-free biomarker assay in a microresistive pulse sensor via immunoaggregation.

We present a label-free biomarker detection method based on immunoaggregation and resistive pulse sensing technology. In this approach, target biomarkers and antibody (Ab)-functionalized microparticles are mixed to form biomarker-microparticle aggregates. A resistive pulse sensor is then used to measure the sizes and count the number of aggregates. The measured volume fraction of the aggregates represents the concentration of the targeted biomarker. In our tests, human ferritin, used as a biomarker, triggered the aggregation of antiferritin Ab-functionalized microparticles in phosphate-buffered saline (PBS). The volume fraction of aggregates increased with the increased ferritin concentration. We also demonstrated the detection of human ferritin in 10% fetal bovine serum (FBS) to mimic a real detection environment in complex media. The detection range from 0.1 to 208 ng/mL was achieved. In addition, we demonstrated that the detection range can be shifted to lower and higher concentrations by decreasing and increasing microparticle concentrations. This biomarker detection method is label-free, rapid, and able to quantitatively measure the concentration of any macromolecular biomarker as long as an antibody can be found, with simple measurement setup and sample preparations.

Rapid Surface Charge Mapping Based on a Liquid Crystal Microchip.

Rapid surface charge mapping of a solid surface remains a challenge. In this study, we present a novel microchip based on liquid crystals for assessing the surface charge distribution of a planar or soft surface. This chip enables rapid measurements of the local surface charge distribution of a charged surface. The chip consists of a micropillar array fabricated on a transparent indium tin oxide substrate, while the liquid crystal is used to fill in the gaps between the micropillar structures. When an object is placed on top of the chip, the local surface charge (or zeta potential) influences the orientation of the liquid crystal molecules, resulting in changes in the magnitude of transmitted light. By measuring the intensity of the transmitted light, the distribution of the surface charge can be accurately quantified. We calibrated the chip in a three-electrode configuration and demonstrated the validity of the chip for rapid surface charge mapping using a borosilicate glass slide. This chip offers noninvasive, rapid mapping of surface charges on charged surfaces, with no need for physical or chemical modifications, and has broad potential applications in biomedical research and advanced material design.

[Effect of early intervention of liver-smoothing and blood-activating decoction combined with acupuncture on patients with post-stroke depression].

To study the effect of early intervention of liver-soothing and Blood-activating decoction combined with acupuncture in improving neurological functions, depressive symptom and life quality of patients with post-stroke depression, and compare with fluoxetine hydrochloride. Specifically, 63 patients with post-stroke depression were randomly divided into the traditional Chinese medicine (TCM) acupuncture group (31 cases) and the western medicine group (32 cases). On the basis of the conventional treatment of the primary disease, the TCM acupuncture group was treated with liver-soothing and blood-activating decoction and acupuncture, while the western medicine group was treated with fluoxetine hydrochloride for four weeks. In the follow-up visit six months later, scores of HAMD, NIHSS and SS-QOL were observed. The scores of HAMD and NIHSS of both groups were significantly decreased (P < 0.01), while the scores of SS-QOL increased significantly, with a notable difference compared with that before the treatment (P < 0.01). Specifically, the TCM acupuncture group's was superior to the western medicine group (P < 0.05). The study suggests that the early intervention of liver-soothing and blood-activating decoction combined with acupuncture on patients with post-stroke depression has the effect in relieving depression symptom and improving neurological functions, thereby improving their quality of life and prognosis.

Rapid Detection of Microparticles Using a Microfluidic Resistive Pulse Sensor Based on Bipolar Pulse-Width Multiplexing.

Rapid and accurate analysis of micro/nano bio-objects (e.g., cells, biomolecules) is crucial in clinical diagnostics and drug discovery. While a traditional resistive pulse sensor can provide multiple kinds of information (size, count, surface charge, etc.) about analytes, it has low throughput. We present a unique bipolar pulse-width, multiplexing-based resistive pulse sensor for high-throughput analysis of microparticles. Signal multiplexing is enabled by exposing the central electrode at different locations inside the parallel sensing channels. Together with two common electrodes, the central electrode encodes the electrical signal from each sensing channel, generating specific bipolar template waveforms with different pulse widths. Only one DC source is needed as input, and only one combined electrical output is collected. The combined signal can be demodulated using correlation analysis and a unique iterative cancellation scheme. The accuracy of particle counting and sizing was validated using mixtures of various sized microparticles. Results showed errors of 2.6% and 6.1% in sizing and counting, respectively. We further demonstrated its accuracy for cell analysis using HeLa cells.

Cell Surface Charge Mapping Using a Microelectrode Array on ITO Substrate.

Many cellular functions are regulated by cell surface charges, such as intercellular signaling and metabolism. Noninvasive measurement of surface charge distribution of a single cell plays a vital role in understanding cellular functions via cell membranes. We report a method for cell surface charge mapping via photoelectric interactions. A cell is placed on an array of microelectrodes fabricated on a transparent ITO (indium tin oxide) surface. An incident light irradiates the ITO surface from the backside. Because of the influence of the cell surface charge (or zeta potential), the photocurrent and the absorption of the incident light are changed, inducing a magnitude change of the reflected light. Hence, the cell surface charge distribution can be quantified by analyzing the reflected light intensity. This method does not need physical or chemical modification of the cell surface. We validated this method using charged microparticles (MPs) and two types of cells, i.e., human dermal fibroblast cells (HDFs) and human mesenchymal stem cells (hMSC). The measured average zeta potentials were in good agreement with the standard electrophoresis light scattering method.

Recent Advances in Biomolecular Detection Based on Aptamers and Nanoparticles.

The fast, accurate detection of biomolecules, ranging from nucleic acids and small molecules to proteins and cellular secretions, plays an essential role in various biomedical applications. These include disease diagnostics and prognostics, environmental monitoring, public health, and food safety. Aptamer recognition (DNA or RNA) has gained extensive attention for biomolecular detection due to its high selectivity, affinity, reproducibility, and robustness. Concurrently, biosensing with nanoparticles has been widely used for its high carrier capacity, stability and feasibility of incorporating optical and catalytic activity, and enhanced diffusivity. Biosensors based on aptamers and nanoparticles utilize the combination of their advantages and have become a promising technology for detecting of a wide variety of biomolecules with high sensitivity, reliability, specificity, and detection speed. Via various sensing mechanisms, target biomolecules have been quantified in terms of optical (e.g., colorimetric and fluorometric), magnetic, and electrical signals. In this review, we summarize the recent advances in and compare different aptamer-nanoparticle-based biosensors by nanoparticle types and detection mechanisms. We also share our views on the highlights and challenges of the different nanoparticle-aptamer-based biosensors.

Sheathless size-based acoustic particle separation.

Particle separation is of great interest in many biological and biomedical applications. Flow-based methods have been used to sort particles and cells. However, the main challenge with flow based particle separation systems is the need for a sheath flow for successful operation. Existence of the sheath liquid dilutes the analyte, necessitates precise flow control between sample and sheath flow, requires a complicated design to create sheath flow and separation efficiency depends on the sheath liquid composition. In this paper, we present a microfluidic platform for sheathless particle separation using standing surface acoustic waves. In this platform, particles are first lined up at the center of the channel without introducing any external sheath flow. The particles are then entered into the second stage where particles are driven towards the off-center pressure nodes for size based separation. The larger particles are exposed to more lateral displacement in the channel due to the acoustic force differences. Consequently, different-size particles are separated into multiple collection outlets. The prominent feature of the present microfluidic platform is that the device does not require the use of the sheath flow for positioning and aligning of particles. Instead, the sheathless flow focusing and separation are integrated within a single microfluidic device and accomplished simultaneously. In this paper, we demonstrated two different particle size-resolution separations; (1) 3 µm and 10 µm and (2) 3 µm and 5 µm. Also, the effects of the input power, the flow rate, and particle concentration on the separation efficiency were investigated. These technologies have potential to impact broadly various areas including the essential microfluidic components for lab-on-a-chip system and integrated biological and biomedical applications.

Ultrasensitive detection of small biomolecules using aptamer-based molecular recognition and nanoparticle counting.

Detection of small biomolecules is critical for understanding molecular mechanisms in biological systems and performing in vitro diagnosis in clinics. Current antibody based detection methods face large challenges in detecting small biomolecules at low concentrations. We report a new method for detecting small biomolecules based on molecular recognition and nanoparticle (NP) counting. Aptamer-functionalized NPs are attached to complementary sequence (CS)-conjugated microparticle (MP) carriers. In the presence of target small biomolecules at ultra low concentrations, NPs would be released from the MP carriers. Coupled with a resistive pulse sensor (RPS) using a micropore that counts the released NPs, this method can measure the concentrations of target biomolecules at low concentrations with high sensitivity and high throughput. Adenosine was used as a model to demonstrate the feasibility of this method. It is demonstrated that this method can detect a wide range of adenosine concentrations with a low detection limit of 0.168 nM, which is 10 times lower than that of the ELISA kit. With its simple structure, high sensitivity, and high reproducibility, this detection method holds great potential for the ultrasensitive detection of low abundance small biomolecules.

Mapping Surface Charge Distribution of Single-Cell via Charged Nanoparticle.

Many bio-functions of cells can be regulated by their surface charge characteristics. Mapping surface charge density in a single cell's surface is vital to advance the understanding of cell behaviors. This article demonstrates a method of cell surface charge mapping via electrostatic cell-nanoparticle (NP) interactions. Fluorescent nanoparticles (NPs) were used as the marker to investigate single cells' surface charge distribution. The nanoparticles with opposite charges were electrostatically bonded to the cell surface; a stack of fluorescence distribution on a cell's surface at a series of vertical distances was imaged and analyzed. By establishing a relationship between fluorescent light intensity and number of nanoparticles, cells' surface charge distribution was quantified from the fluorescence distribution. Two types of cells, human umbilical vein endothelial cells (HUVECs) and HeLa cells, were tested. From the measured surface charge density of a group of single cells, the average zeta potentials of the two types of cells were obtained, which are in good agreement with the standard electrophoretic light scattering measurement. This method can be used for rapid surface charge mapping of single particles or cells, and can advance cell-surface-charge characterization applications in many biomedical fields.

A Microfluidic Sensor for Continuous, in Situ Surface Charge Measurement of Single Cells.

Cell surface charge has been recognized as an important cellular property. We developed a microfluidic sensor based on resistive pulse sensing to assess surface charge and sizes of single cells suspended in a continuous flow. The device consists of two consecutive resistive pulse sensors (RPSs) with identical dimensions. Opposite electric fields were applied on the two RPSs. A charged cell in the RPSs was accelerated or decelerated by the electric fields and thus exhibited different transit times passing through the two RPSs. The cell surface charge is measured with zeta potential that can be quantified with the transit time difference. The transit time of each cell can be accurately detected with the width of pulses generated by the RPS, while the cell size can be calculated with the pulse magnitude at the same time. This device has the ability to detect surface charges and sizes of individual cells with high tolerance in cell types and testing solutions compared with traditional electrophoretic light scattering methods. Three different types of cells including HeLa cancer cells, human dermal fibroblast cells, and human umbilical vein endothelial cells (HUVECs) were tested with the sensor. Results showed a significant difference of zeta potentials between HeLa cells and fibroblasts or HUVECs. In addition, when HeLa cells were treated with various concentrations of glutamine, the effects on cancer cell surface charge were detected. Our results demonstrated the great potential of using our sensor for cell type sorting, cancer cell detection, and cell status analysis.

Static and dynamic responses of polyelectrolyte brushes under external electric field.

The static and dynamic behaviors of partially charged and end-grafted polyelectrolyte brushes in response to electric fields were investigated by means of molecular dynamics simulation. The results show that the polymer brushes can be partially or fully stretched by applying an external electric field. Moreover, the brushes can switch reversibly from collapsed to stretched states, fully responding to the AC electric stimuli, and the gating response frequency can reach a few hundred MHz. The effects of the grafting density, the charge fraction of the brushes and the strength of the electric field on the average height of the polymer brushes were studied through the simulations.

Enabling single cell electrical stimulation and response recording via a microfluidic platform.

Electrical stimulation (ES) has been recognized to play important roles in regulating cell behaviors. A microfluidic device was developed for the electrical stimulation of single cells and simultaneous recording of extracellular field potential (EFP). Each single cell was trapped onto an electrode surface by a constriction channel for ES testing and was then driven to the outlet by the pressure afterward. This design allows the application of ES on and detection of EFP of single cells continuously in a microfluidic channel. Human cardiomyocytes and primary rat cortex neurons were tested with specific ES with the device. Each cell's EFP signal was detected and analyzed during the ES process. Results have shown that after applying specific ES on the excitable single cells, the cells evoked electrical responses. In addition, increased secretion of glutamic acid was detected from the stimulated neurons. Altogether, these results indicated that the developed device can be used to continuously apply ES on and accurately determine cell responses of single cells with shorter probing time. The throughput of the measurement can achieve 1 cell per minute, which is higher than the traditional ES methods that need culturing cells or manually positioning the cells onto the electrode surface. Before and after the application of ES, the cell viability had no significant change. Such a device can be used to study the biological process of various types of cells under electrical stimulation.

Lab-on-a-chip electrical multiplexing techniques for cellular and molecular biomarker detection.

Signal multiplexing is vital to develop lab-on-a-chip devices that can detect and quantify multiple cellular and molecular biomarkers with high throughput, short analysis time, and low cost. Electrical detection of biomarkers has been widely used in lab-on-a-chip devices because it requires less external equipment and simple signal processing and provides higher scalability. Various electrical multiplexing for lab-on-a-chip devices have been developed for comprehensive, high throughput, and rapid analysis of biomarkers. In this paper, we first briefly introduce the widely used electrochemical and electrical impedance sensing methods. Next, we focus on reviewing various electrical multiplexing techniques that had achieved certain successes on rapid cellular and molecular biomarker detection, including direct methods (spatial and time multiplexing), and emerging technologies (frequency, codes, particle-based multiplexing). Lastly, the future opportunities and challenges on electrical multiplexing techniques are also discussed.