Cell surface receptors: rapid quantification of live cell receptors using bioluminescence in a flow-based microfluidic device (small 8/2015).

C. T. Lim and co-workers describe a rapid and sensitive bioluminescence-based microfluidic method for quantifying receptor numbers on live cells. On page 943, this integrated, lens-free optical platform allows the determination of signals from the cell surface with high sensitivity. Compared to conventional approaches, the combined use of bioluminescence and microfluidics makes it safe to use, reduces background noise, improves sensitivity, requires smaller sample volumes, and allows high-throughput sampling over thousands of cells.

Rapid quantification of live cell receptors using bioluminescence in a flow-based microfluidic device.

The number of receptors expressed by cells plays an important role in controlling cell signaling events, thus determining its behaviour, state and fate. Current methods of quantifying receptors on cells are either laborious or do not maintain the cells in their native form. Here, a method integrating highly sensitive bioluminescence, high precision microfluidics and small footprint of lensfree optics is developed to quantify cell surface receptors. This method is safe to use, less laborious, and faster than the conventional radiolabelling and near field scanning methods. It is also more sensitive than fluorescence based assays and is ideal for high throughput screening. In quantifying ß(1) adrenergic receptors expressed on the surface of H9c2 cardiomyocytes, this method yields receptor numbers from 3.12 × 10(5) to 9.36 × 10(5) receptors/cell which are comparable with current methods. This can serve as a very good platform for rapid quantification of receptor numbers in ligand/drug binding and receptor characterization studies, which is an important part of pharmaceutical and biological research.

Microfluidic platform for negative enrichment of circulating tumor cells.

Negative enrichment is the preferred approach for tumor cell isolation as it does not rely on biomarker expression. However, size-based negative enrichment methods suffer from well-known recovery/purity trade-off. Non-size based methods have a number of processing steps that lead to compounded cell loss due to extensive sample processing and handling which result in a low recovery efficiency. We present a method that performs negative enrichment in two steps from 2 ml of whole blood in a total assay processing time of 60 min. This negative enrichment method employs upstream immunomagnetic depletion to deplete CD45-positive WBCs followed by a microfabricated filter membrane to perform chemical-free RBC depletion and target cells isolation. Experiments of spiking two cell lines, MCF-7 and NCI-H1975, in the whole blood show an average of >90 % cell recovery over a range of spiked cell numbers. We also successfully recovered circulating tumor cells from 15 cancer patient samples.

3D numerical simulation of a Coulter counter array with analysis of electrokinetic forces.

Coulter counters have played an important role in biological cell assays since their introduction decades ago. Several types of high throughput micro-Coulter counters based on lab-on-chip devices have been commercialized recently. In this paper, we propose a highly integrated micro-Coulter counter array working under low DC voltage. The real-time electrical current change, including the pulse amplitude and width, of the micro-Coulter counter with novel structure is systematically investigated numerically. The major types of forces exerted on the particle in the micro-Coulter counter, including hydrodynamic force and electrokinetic force are quantitatively analyzed. The simulation in this study shows the pulse profile, such as width and amplitude, is affected by both particle size and the flow condition. The special cases of multiple particle aggregation and cross-talk between neighboring channels are also considered for their effects on the electric current pulses. This simulation provides critical insight and guidance for developing next new generations of micro-Coulter counter.

Towards an optimal and unbiased approach for tumor cell isolation.

Our current understanding of clinical significance or the lack thereof of circulating tumor cells (CTCs) is biased by the technology used to isolate these rare cells. Despite the presence of a vast number of academic and commercial technologies, the lack of a standardized and optimized platform has been widely noted. We present a negative enrichment approach, integrating WBC depletion and chemical-free RBC depletion in the same setup without the need for centrifugation, washing or multiple sample handling steps. This approach achieves an average of >90 % recovery of spiked tumor cells and >99 % total WBC depletion in whole blood across multiple cell lines, in a simple and easy-to-use assay. The results presented herein and ongoing improvements aim to fulfill the need for a highly reliable, unbiased, standardized, and optimized CTC isolation platform, using component technologies that are validated for cell isolation.

Effects of the electrode size and modification protocol on a label-free electrochemical biosensor.

In the present work, the effect of a surface modification protocol along with the electrode size has been investigated for developing an efficient, label-free electrochemical biosensing method for diagnosis of traumatic brain injury (TBI) biomarkers. A microdisk electrode array (MDEA) and a macroelectrode with a comb structure (MECS) were modified with an anti-GFAP (GFAP = glial fibrillary acidic protein) antibody using two protocols for optimum and label-free detection of GFAP, a promising acute-phase TBI biomarker. For the MDEA, an array of six microdisks with a 100 µm diameter and, for the MECS, a 3.2 mm × 5.5 mm electrode 5 µm wide with 10 µm spaced comb fingers were modified using an optimized protocol for dithiobis(succinimidyl propionate) (DSP) self-assembled monolayer formation. Anti-GFAP was covalently bound, and the remaining free DSP groups were blocked using ethanolamine (Ea). Sensors were exposed to solutions with different GFAP concentrations, and a label-free electrochemical impedance spectroscopy (EIS) technique was used to determine the concentration. EIS results confirmed that both types of Ea/anti-GFAP/DSP/Au electrodes modified with an optimized DSP-based protocol can accurately detect GFAP in the range of 1 pg mL(-1) to 100 ng mL(-1) with a detection limit of 1 pg mL(-1). However, the cross-use of the MDEA protocol on the MECS and vice versa resulted in very low sensitivity or poor signal resolution, underscoring the importance of proper matching of the electrode size and type and the surface modification protocol.

Design considerations in the development and application of microdisc electrode arrays (MDEAs) for implantable biosensors.

The use of microlithographically fabricated Microdisc Electrode Arrays (MDEAs) in the development of implantable voltammetric biosensors necessitates design criteria that balances the overall footprint of the device with the advantages to be derived from large separation distances between non-interacting microdisc elements. Using the dynamic electroanalytical techniques of Multiple Scan Rate Cyclic Voltammetry (MSRCV) experiments with finite element simulations and Electrochemical Impedance Spectroscopy with equivalent circuit modeling, three unique MDEA designs; MDEA 050 (r = 25 microm, 5,184 discs), MDEA 100 (r = 50 microm, 1,296 discs) and MDEA 250 (r = 125 microm, 207 discs) of constant critical dimensions (center-to-center d/r = 4) and area (A = 0.1 cm(2)) were studied in 1.0 mM ferrocene monocarboxylic acid (FcCO(2)H) solution (in 0.1 M Tris/0.1 M KCl buffer, pH = 7.2). The critical disc-to-disc spacing (d/r) required to archive 67% of maximal current response was defined as optimal. Based on the predictive model, new MDEA designs; MDEA 001 (r = 0.5 microm, 127,324 discs), MDEA 002.5 (r = 1.25 microm, 20,372 discs), MDEA 005 (r = 2.5 microm, 5,093 discs), MDEA 010 (r = 5 microm, 1,273 discs), MDEA 015 (r = 7.5 microm, 566 discs), MDEA 020 (r = 10 microm, 318 discs) were simulated at 10 and 100 mV/s. The final disc count of each MDEA was dictated by the need to maintain a comparable electroactive area between the MDEAs, which was chosen to be 0.001 cm(2), which in turn was dictated by the need to generate sufficient electrochemical current to be comfortably measured by common electrochemical detectors.

Enrichment, detection and clinical significance of circulating tumor cells.

Circulating Tumor Cells (CTCs) are shed from primary or secondary tumors into blood circulation. Accessing and analyzing these cells provides a non-invasive alternative to tissue biopsy. CTCs are estimated to be as few as 1 cell among a few million WBCs and few billion RBCs in 1 ml of patient blood and are rarely found in healthy individuals. CTCs are FDA approved for prognosis of the major cancers, namely, Breast, Colon and Prostate. Currently, more than 400 clinical trials are ongoing to establish their clinical significance beyond prognosis, such as, therapy selection and companion diagnostics. Understanding the clinical relevance of CTCs typically involves isolation, detection and molecular characterization of cells, ideally at single cell level. The need for highly reliable, standardized and robust methodologies for isolating and analyzing CTCs has been widely expressed by clinical thought leaders. In the last decade, numerous academic and commercial technology platforms for isolation and analysis of CTCs have been reported. A recent market report highlighted the presence of more than 100 companies offering products and services related to CTCs. This review aims to capture the state of the art and examines the technical merits and limitations of contemporary technologies for clinical use.

Electrokinetic Analysis of Cell Translocation in Low-Cost Microfluidic Cytometry for Tumor Cell Detection and Enumeration.

Conventional Coulter counters have been introduced as an important tool in biological cell assays since several decades ago. Recently, the emerging portable Coulter counter has demonstrated its merits in point of care diagnostics, such as on chip detection and enumeration of circulating tumor cells (CTC). The working principle is based on the cell translocation time and amplitude of electrical current change that the cell induces. In this paper, we provide an analysis of a Coulter counter that evaluates the hydrodynamic and electrokinetic properties of polystyrene microparticles in a microfluidic channel. The hydrodynamic force and electrokinetic force are concurrently analyzed to determine the translocation time and the electrical current pulses induced by the particles. Finally, we characterize the chip performance for CTC detection. The experimental results validate the numerical analysis of the microfluidic chip. The presented model can provide critical insight and guidance for developing micro-Coulter counter for point of care prognosis.

Size based sorting and patterning of microbeads by evaporation driven flow in a 3D micro-traps array.

We present a three-dimensional (3D) micro-traps array for size selective sorting and patterning of microbeads via evaporation-driven capillary flow. The interconnected micro-traps array was manufactured by silicon micromachining. Microliters of aqueous solution containing particle mixtures of different sized (0.2 to 20 µm diameter) beads were dispensed onto the micro-traps substrate. The smaller particles spontaneously wicked towards the periphery of the chip, while the larger beads were orderly docked within the micro-traps array.

Anti-EpCAM modified LC-SPDP monolayer on gold microelectrode based electrochemical biosensor for MCF-7 cells detection.

A succinimidyl 6-(3-[2-pyridyldithio]-propionamido) hexanoate (LC-SPDP) self-assembled monolayer (SAM) prepared onto a 500 µm (diameter) gold microelectrode (Au) surface has been utilized for covalent immobilization of anti-EpCAM antibody. Amino group on anti-EpCAM antibody was covalently bound with succinimidyl group on SAM via amide bond and unreacted active groups of LC-SPDP were blocked using 1% ethanol amine (EA). These anti-EpCAM/LC-SPDP/Au electrodes were characterized using cyclic voltammetric (CV) and fluorescence techniques, respectively. The anti-EpCAM/LC-SPDP/Au electrodes were exposed to solutions with different MCF-7 cell concentrations and CV technique was used to determine the cell concentration. Further, CV studies on blank 500 and 50 µm (diameter) gold microelectrodes were used to identify cell via molecular profiling using ferrocene amidopentyl carboxylic acid based redox tagging and magnetic beads based enhancement. CV results confirm that the anti-EpCAM/LC-SPDP/Au based biosensor could detect MCF-7 cells in the range of 1×10(5)-1×10(8) with correlation coefficient of 0.999 and detection limit of 1×10(5) cells ml(-1) i.e. 100 cells in solution used for incubation (1 µl). Molecular profiling studies suggest that smaller size microelectrode (50 µm; diameter) with magnetic beads based enhancement can be employed to identify cell type. This work establishes the feasibility of using microelectrode based platform for breast cancer specific MCF-7 cell concentration estimation and their molecular profiling.

Breast tumor cell detection at single cell resolution using an electrochemical impedance technique.

Gold micro-electrodes with various diameters (25, 50, 75, 100 and 250 µm) were manufactured using standard micro-fabrication techniques and optimized for counting of MCF-7 cells (breast tumor cells) with single cell resolution. For specific cell capture, anti-EpCAM was immobilized on 11-mercaptoundecanoic acid (11-MUA)-3-mercaptopropionic acid (3-MPA) mixed self-assembled monolayer (SAM) modified gold surface of micro-electrodes. Electrodes were characterized using optical, cyclic voltammetry and electrochemical impedance spectroscopic (EIS) techniques. Cell capture response recorded using EIS suggested that optimum electrode dimensions should be analogous to desired cell size. For MCF-7 cells with an average diameter of 18 ± 2 µm, an electrode with 25 µm diameter was established as the optimum electrode size for precise single cell recognition and enumeration. In EIS investigation, the 25 µm electrode exhibited an impedance change of ~2.2 × 10(7) Ω in response to a single tumor cell captured on its surface. On the other hand other electrodes (250, 100, 75 and 50 µm) showed much less response for a single tumor cell. In future, the use of high density arrays of such electrodes with surface modifications will result in miniaturized lab on a chip devices for precise counting of MCF-7 cells with single cell resolution.

Design rule for optimization of microelectrodes used in electric cell-substrate impedance sensing (ECIS).

This paper presents an experimentally derived design rule for optimization of microelectrodes used in electric cell-substrate impedance sensing (ECIS) up to 10MHz. The effect of change in electrode design (through electrode sensor area, lead trace widths, and passivation coating thickness) on electrode characteristics was experimentally evaluated using electrochemical impedance spectroscopy (EIS) measurements and analyzed using equivalent circuit models. A parasitic passivation coating impedance was successfully minimized by designing electrodes with either a thicker passivation layer or a smaller lead trace area. It was observed that the passivated lead trace area to passivation coating thickness ratio has a critical value of 5.5, under which the impedance contribution of the coating is minimized. The optimized design of ECIS-based microelectrode devices reported in this work will make it possible to probe the entire beta dispersion region of adherent biological cell layers by reducing measurement artifacts and improving the quality of data across the beta-dispersion region. The new design will enable the use of the commonly used ECIS technique to measure real-time cellular properties in high frequency ranges (beta dispersion) that was not possible thus far.

A detailed model for high-frequency impedance characterization of ovarian cancer epithelial cell layer using ECIS electrodes.

We report on the electrical impedance spectroscopy characterization of OVCA429 ovarian cancer cells. A commercially available eight-well cell culture impedance array (ECIS-8W1E), commonly used in electrical cell-substrate impedance sensing (ECIS), was used for OVCA429 characterization. Impedances of ECIS-8W1E array were recorded with cell culture medium (without cells) and with OVCA429 cell layer in the culture medium between 100 Hz and 10 MHz frequency. Physiological and interfacial components of experimental impedance data were modeled by equivalent circuit fitting, using a newly developed model. Impedance measurements with cell culture medium show only two semicircles in the admittance plane, which are identified as: 1) low-frequency semicircle due to impedance of gold electrode in contact with the electrolyte and 2) high-frequency semicircle due to impedance of polymer-coated region of the gold electrode in contact with the electrolyte. In the presence of OVCA429 cell layer, three convolved semicircles are observed in the Cole-Cole plane, which are identified as the interfacial impedance in series with the cell layer impedance. The average resistance and capacitance of the OVCA429 cell layer was found to be 152+/-59 ohm x cm(2) and 8.5+/-2.4 microF/cm(2), respectively.