Fully Automated Quantification of Insulin Concentration Using a Microfluidic-Based Chemiluminescence Immunoassay.

A fully automated microfluidic-based detection system for the rapid determination of insulin concentration through a chemiluminescence immunoassay has been developed. The microfluidic chip used in the system is a double-layered polydimethylsiloxane device embedded with interconnecting micropumps, microvalves, and a micromixer. At a high injection rate of the developing solution, the chemiluminescence signal can be excited and measured within a short period of time. The integral value of the chemiluminescence light signal is used to determine the insulin concentration of the samples, and the results indicate that the measurement is accurate in the range from 1.5 pM to 391 pM. The entire chemiluminescence assay can be completed in less than 10 min. The fully automated microfluidic-based insulin detection system provides a useful platform for rapid determination of insulin in clinical diagnostics for diabetes, which is expected to become increasingly important for future point-of-care applications.

Determination of base binding strength and base stacking interaction of DNA duplex using atomic force microscope.

As one of the most crucial properties of DNA, the structural stability and the mechanical strength are attracting a great attention. Here, we take advantage of high force resolution and high special resolution of Atom Force Microscope and investigate the mechanical force of DNA duplexes. To evaluate the base pair hydrogen bond strength and base stacking force in DNA strands, we designed two modes (unzipping and stretching) for the measurement rupture forces. Employing k-means clustering algorithm, the ruptured force are clustered and the mean values are estimated. We assessed the influence of experimental parameters and performed the force evaluation for DNA duplexes of pure dG/dC and dA/dT base pairs. The base binding strength of single dG/dC and single dA/dT were estimated to be 20.0 ± 0.2 pN and 14.0 ± 0.3 pN, respectively, and the base stacking interaction was estimated to be 2.0 ± 0.1 pN. Our results provide valuable information about the quantitative evaluation of the mechanical properties of the DNA duplexes.

Improving atomic force microscopy imaging by a direct inverse asymmetric PI hysteresis model.

A modified Prandtl-Ishlinskii (PI) model, referred to as a direct inverse asymmetric PI (DIAPI) model in this paper, was implemented to reduce the displacement error between a predicted model and the actual trajectory of a piezoelectric actuator which is commonly found in AFM systems. Due to the nonlinearity of the piezoelectric actuator, the standard symmetric PI model cannot precisely describe the asymmetric motion of the actuator. In order to improve the accuracy of AFM scans, two series of slope parameters were introduced in the PI model to describe both the voltage-increase-loop (trace) and voltage-decrease-loop (retrace). A feedforward controller based on the DIAPI model was implemented to compensate hysteresis. Performance of the DIAPI model and the feedforward controller were validated by scanning micro-lenses and standard silicon grating using a custom-built AFM.

Nanoscale depth reconstruction from defocus: within an optical diffraction model.

Depth from defocus (DFD) based on optical methods is an effective method for depth reconstruction from 2D optical images. However, due to optical diffraction, optical path deviation occurs, which results in blurring imaging. Blurring, in turn, results in inaccurate depth reconstructions using DFD. In this paper, a nanoscale depth reconstruction method using defocus with optical diffraction is proposed. A blurring model is proposed by considering optical diffraction, leading to a much higher accuracy in depth reconstruction. Firstly, Fresnel diffraction in an optical system is analyzed, and a relationship between intensity distribution and depth information is developed. Secondly, a blurring imaging model with relative blurring and heat diffusion is developed through curving fitting of a numerical model. In this way, a new DFD method with optical diffraction is proposed. Finally, experimental results show that this new algorithm is more effective for depth reconstruction on the nanoscale.

Rapid and label-free separation of Burkitt's lymphoma cells from red blood cells by optically-induced electrokinetics.

Early stage detection of lymphoma cells is invaluable for providing reliable prognosis to patients. However, the purity of lymphoma cells in extracted samples from human patients' marrow is typically low. To address this issue, we report here our work on using optically-induced dielectrophoresis (ODEP) force to rapidly purify Raji cells' (a type of Burkitt's lymphoma cell) sample from red blood cells (RBCs) with a label-free process. This method utilizes dynamically moving virtual electrodes to induce negative ODEP force of varying magnitudes on the Raji cells and RBCs in an optically-induced electrokinetics (OEK) chip. Polarization models for the two types of cells that reflect their discriminate electrical properties were established. Then, the cells' differential velocities caused by a specific ODEP force field were obtained by a finite element simulation model, thereby established the theoretical basis that the two types of cells could be separated using an ODEP force field. To ensure that the ODEP force dominated the separation process, a comparison of the ODEP force with other significant electrokinetics forces was conducted using numerical results. Furthermore, the performance of the ODEP-based approach for separating Raji cells from RBCs was experimentally investigated. The results showed that these two types of cells, with different concentration ratios, could be separated rapidly using externally-applied electrical field at a driven frequency of 50 kHz at 20 Vpp. In addition, we have found that in order to facilitate ODEP-based cell separation, Raji cells' adhesion to the OEK chip's substrate should be minimized. This paper also presents our experimental results of finding the appropriate bovine serum albumin concentration in an isotonic solution to reduce cell adhesion, while maintaining suitable medium conductivity for electrokinetics-based cell separation. In short, we have demonstrated that OEK technology could be a promising tool for efficient and effective purification of Raji cells from RBCs.

Atomic force microscopy imaging of live mammalian cells.

Atomic force microscopy (AFM) was used to examine the morphology of live mammalian adherent and suspended cells. Time-lapse AFM was used to record the locomotion dynamics of MCF-7 and Neuro-2a cells. When a MCF-7 cell retracted, many small sawtooth-like filopodia formed and reorganized, and the thickness of cellular lamellipodium increased as the retraction progressed. In elongated Neuro-2a cells, the cytoskeleton reorganized from an irregular to a parallel, linear morphology. Suspended mammalian cells were immobilized by method combining polydimethylsiloxane-fabricated wells with poly-L-lysine electrostatic adsorption. In this way, the morphology of a single live lymphoma cell was imaged by AFM. The experimental results can improve our understanding of cell locomotion and may lead to improved immobilization strategies.

Atomic force microscopy study of the antigen-antibody binding force on patient cancer cells based on ROR1 fluorescence recognition.

Knowledge of drug-target interaction is critical to our understanding of drug action and can help design better drugs. Due to the lack of adequate single-molecule techniques, the information of individual interactions between ligand-receptors is scarce until the advent of atomic force microscopy (AFM) that can be used to directly measure the individual ligand-receptor forces under near-physiological conditions by linking ligands onto the surface of the AFM tip and then obtaining force curves on cells. Most of the current AFM single-molecule force spectroscopy experiments were performed on cells grown in vitro (cell lines) that are quite different from the human cells in vivo. From the view of clinical practice, investigating the drug-target interactions directly on the patient cancer cells will bring more valuable knowledge that may potentially serve as an important parameter in personalized treatment. Here, we demonstrate the capability of AFM to measure the binding force between target (CD20) and drug (rituximab, an anti-CD20 monoclonal antibody targeted drug) directly on lymphoma patient cancer cells under the assistance of ROR1 fluorescence recognition. ROR1 is a receptor expressed on some B-cell lymphomas but not on normal cells. First, B-cell lymphoma Raji cells (a cell line) were used for ROR1 fluorescence labeling and subsequent measurement of CD20-rituximab binding force. The results showed that Raji cells expressed ROR1, and the labeling of ROR1 did not influence the measurement of CD20-rituximab binding force. Then the established experimental procedures were performed on the pathological samples prepared from the bone marrow of a follicular lymphoma patient. Cancer cells were recognized by ROR1 fluorescence. Under the guidance of fluorescence, with the use of a rituximab-conjugated tip, the cellular topography was visualized by using AFM imaging and the CD20-Rituximab binding force was measured by single-molecule force spectroscopy.

Nanoscale mapping and organization analysis of target proteins on cancer cells from B-cell lymphoma patients.

CD20, a membrane protein highly expressed on most B-cell lymphomas, is an effective target demonstrated in clinical practice for treating B-cell non-Hodgkin's lymphoma (NHL). Rituximab is a monoclonal antibody against CD20. In this work, we applied atomic force microscopy (AFM) to map the nanoscale distribution of CD20 molecules on the surface of cancer cells from clinical B-cell NHL patients under the assistance of ROR1 fluorescence recognition (ROR1 is a specific cell surface marker exclusively expressed on cancer cells). First, the ROR1 fluorescence labeling experiments showed that ROR1 was expressed on cancer cells from B-cell lymphoma patients, but not on normal cells from healthy volunteers. Next, under the guidance of ROR1 fluorescence, the rituximab-conjugated AFM tips were moved to cancer cells to image the cellular morphologies and detect the CD20-rituximab interactions on the cell surfaces. The distribution maps of CD20 on cancer cells were constructed by obtaining arrays of (16×16) force curves in local areas (500 × 500 nm(2)) on the cell surfaces. The experimental results provide a new approach to directly investigate the nanoscale distribution of target protein on single clinical cancer cells.

Imaging and measuring the molecular force of lymphoma pathological cells using atomic force microscopy.

Atomic force microscopy (AFM) provides a new technology to visualize the cellular topography and quantify the molecular interactions at nanometer spatial resolution. In this work, AFM was used to image the cellular topography and measure the molecular force of pathological cells from B-cell lymphoma patients. After the fluorescence staining, cancer cells were recognized by their special morphological features and then the detailed topography was visualized by AFM imaging. The AFM images showed that cancer cells were much rougher than healthy cells. CD20 is a surface marker of B cells and rituximab is a monoclonal antibody against CD20. To measure the CD20-rituximab interaction forces, the polyethylene glycol (PEG) linker was used to link rituximab onto the AFM tip and the verification experiments of the functionalized probe indicated that rituximab molecules were successfully linked onto the AFM tip. The CD20-rituximab interaction forces were measured on about 20 pathological cells and the force measurement results indicated the CD20-rituximab binding forces were mainly in the range of 110-120 pN and 130-140 pN. These results can improve our understanding of the topography and molecular force of lymphoma pathological cells.

Atomic force microscopy imaging and mechanical properties measurement of red blood cells and aggressive cancer cells.

Mechanical properties play an important role in regulating cellular activities and are critical for unlocking the mysteries of life. Atomic force microscopy (AFM) enables researchers to measure mechanical properties of single living cells under physiological conditions. Here, AFM was used to investigate the topography and mechanical properties of red blood cells (RBCs) and three types of aggressive cancer cells (Burkitt's lymphoma Raji, cutaneous lymphoma Hut, and chronic myeloid leukemia K562). The surface topography of the RBCs and the three cancer cells was mapped with a conventional AFM probe, while mechanical properties were investigated with a micro-sphere glued onto a tip-less cantilever. The diameters of RBCs are significantly smaller than those of the cancer cells, and mechanical measurements indicated that Young's modulus of RBCs is smaller than those of the cancer cells. Aggressive cancer cells have a lower Young's modulus than that of indolent cancer cells, which may improve our understanding of metastasis.

A fully automated system for measuring cellular mechanical properties.

As a novel effective label-free biomarker, the mechanical properties of cells have become increasingly important. However, the current methods of mapping cellular mechanical properties are mostly carried out manually, resulting in measurements that are time-consuming with low efficiency. In this article, a fully automated system of measuring the mechanical properties of cells based on atomic force microscopy (AFM) is proposed. In this system, the cells are recognized using an image-processing method and the relative position of the cell, and the AFM tip is accurately calibrated by the local scan method, meaning that the mechanical properties of cells can be measured sequentially without performing the step of AFM imaging. In addition, with the implementation of the automation, the high-throughput measurement of cellular mechanical properties can be performed rapidly. The capability of our system is validated on Raji cells, and the results indicate that the measurement rate of our system is 26 times faster than that of the traditional manual method, providing the technology for high-throughput measurement of cellular mechanical properties.

Imaging and measuring the rituximab-induced changes of mechanical properties in B-lymphoma cells using atomic force microscopy.

The topography and mechanical properties of single B-lymphoma cells have been investigated by atomic force microscopy (AFM). With the assistance of microfabricated patterned pillars, the surface topography and ultrastructure of single living B-lymphoma cell were visualized by AFM. The apoptosis of B-lymphoma cells induced by rituximab alone was observed by acridine orange/ethidium bromide (AO/EB) double fluorescent staining. The rituximab-induced changes of mechanical properties in B-lymphoma cells were measured dynamically and the results showed that B-lymphoma cells became dramatically softer after incubation with rituximab. These results can improve our understanding of rituximab'effect and will facilitate the further investigation of the underlying mechanisms.

Fabrication of Schottky barrier carbon nanotube field effect transistors using dielectrophoretic-based manipulation.

Nanoscale electronic devices made from carbon nanotubes (CNTs) such as transistors and sensors are much smaller and potentially more versatile than those built using conventional IC technology. In this paper, we present a method that uses dielectrophoretic (DEP) manipulation process for the fabrication of single-channel and multi-channel carbon nanotube field effect transistors (CNT-FETs). For a typical fabrication process, single-walled carbon nanotubes (SWCNTs) are first pre-aligned to micron-precision range between two microelectrodes using DEP technique. The typically applied alternating current (AC) voltage to generate the DEP force for manipulation has a frequency of 1 MHz and amplitude of 10 V. We first demonstrated single-channel or multi-channel structures of CNT-FETs. An AFM is then used to "clean" or "sweep away" unwanted particles or CNTs around the electrodes. Lastly, the fabricated FETs were covered in a polymethylmethacrylate (PMMA) thin film and treated with an annealing process. The PMMA covered devices show improved performances over the non-covered devices.

Drift compensation and faulty display correction in robotic nano manipulation.

Random drift and faulty visual display are the main problems in Atomic Force Microscopy (AFM) based robotic nanomanipulation. As far as we know, there are no effective methods available to solve these problems. In this paper, an On-line Sensing and Display (OSD) method is proposed to solve these problems. The OSD method mainly includes two subprocesses: Local-Scan-Before-Manipulation (LSBM) and Local-Scan-After-Manipulation (LSAM). During manipulation, LSBM and LSAM are on-line performed for random drift compensation and faulty visual display correction respectively. Through this way, the bad influence aroused from random drift and faulty visual display can be eliminated in real time. The visual feedback keeps consistent with the true environment changes during the process of manipulation, which makes several operations being finished without an image scan in between. Experiments show the increased effectiveness and efficiency of AFM based nanomanipulation.

Detecting CD20-rituximab specific interactions on lymphoma cells using atomic force microscopy.

Elucidating the underlying mechanisms of cell physiology is currently an important research topic in life sciences. Atomic force microscopy methods can be used to investigate these molecular mechanisms. In this study, single-molecule force spectroscopy was used to explore the specific recognition between the CD20 antigen and anti-CD20 antibody Rituximab on B lymphoma cells under near-physiological conditions. The CD20-Rituximab specific binding force was measured through tip functionalization. Distribution of CD20 on the B lymphoma cells was visualized three-dimensionally. In addition, the relationship between the intramolecular force and the molecular extension of the CD20-Rituximab complex was analyzed under an external force. These results facilitate further investigation of the mechanism of Rituximab's anti-cancer effect.

Carbon nanotube-sensor-integrated microfluidic platform for real-time chemical concentration detection.

This paper presents the development of a chemical sensor employing electronic-grade carbon nanotubes (EG-CNTs) as the active sensing element for sodium hypochlorite detection. The sensor, integrated in a PDMS-glass microfluidic chamber, was fabricated by bulk aligning of EG-CNTs between gold microelectrode pairs using dielectrophoretic technique. Upon exposure to sodium hypochlorite solution, the characteristics of the carbon nanotube chemical sensor were investigated at room temperature under constant current mode. The sensor exhibited responsivity, which fits a linear logarithmic dependence on concentration in the range of 1/32 to 8 ppm, a detection limit lower than 5 ppb, while saturating at 16 ppm. The typical response time of the sensor at room temperature is on the order of minutes and the recovery time is a few hours. In particular, the sensor showed an obvious sensitivity to the volume of detected solution. It was found that the activation power of the sensor was extremely low, i.e. in the range of nanowatts. These results indicate great potential of EG-CNT for advanced nanosensors with superior sensitivity, ultra-low power consumption, and less fabrication complexity.

AFM based MWCNT nanomanipulation with force and visual feedback.

To real-timely feel and see the manipulation process of multi-wall carbon nanotube (MWCNT) is required to better control its assembly based on atomic force microscope. Here real-time three-dimensional interactive forces between the probe and the sample can be fed back to the operator according to the proposed force model and position sensitive detector's signals, and MWCNT motion can be online displayed on the visual interface according to probe position and applied force based on the proposed MWCNT motion model and virtual reality technology. Based on force and visual feedback, the process and result of MWCNT manipulation can be online controlled, and MWCNT manipulation experiment will be performed to verify the effectiveness of the method.

Integrated Vortex Micropumps and Active Micromixers in Polymer Substrates for Automating Bio-fluidic Manipulation.

This paper presents a microfluidic mixing module array developed for bio-fluid/chemical delivery and mixing. Vortex micropumps, microchannels and pillared-surface diaphragm (PSD) active micromixers were successfully integrated into a single polymer-based microfluidic chip, consisting of three mixing modules. The pumping characteristics of the vortex micropump were investigated with both analytical and experimental results. Experiments were also conducted to examine the factors affecting the PSD-based mixing. The integrated mixing module further demonstrated the feasibility of chemical mixing and concentration control. An integrated fluidic system was shown successfully to deliver bio-fluid for real-time SPR based detection. A computer-controlled fluid manipulation system is also proposed for the real-time microfluidic operation control. The digitally controllability of the pumping and mixing operations could potentially improve the accuracy and efficiency, and hence, the functionality of the integrated microfluidic system.