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A Novel Microfluidic Magnetic Bead Assay for Cell Detection Fan Liu, Pawan KC, Ge Zhang, and Jiang Zhe Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b02716 • Publication Date (Web): 04 Dec 2015 Downloaded from http://pubs.acs.org on December 15, 2015
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Analytical Chemistry
A Novel Microfluidic Magnetic Bead Assay for Cell Detection Fan Liu, † Pawan KC, § Ge Zhang, § * and Jiang Zhe† * † §
Department of Mechanical Engineering, University of Akron, Akron, Ohio 44325, United States Department of Biomedical Engineering, University of Akron, Akron, Ohio 44325, United States
ABSTRACT: We present a novel cell detection device based on a magnetic bead cell assay and microfluidic Coulter counting technology. The device can not 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 2nd counter via an external cylindrical magnet. Due to 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 2nd counter was longer than that through the 1st counter. In comparison, transit times of a non-target 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.
Cell detection is a vital task in cell biology to monitor environmental conditions1–3, diagnose diseases4–6, and study cell growth and differentiation7–12. Presenting at low concentrations in the total cell population, rare cells such as tumor cells often indicate crucial health status message13,14. Additionally under therapy or external stimuli, target cell population can change over a short time period8,15,16. Therefore it is important to be able to make accurate detection of cells and dynamically monitor target cell population changes within a short time frame, with portable, inexpensive and reliable devices. Cell’s genotype and surface characteristics are typically used to recognize and quantify target cells by various methods. Nucleic acid-based methods such as polymerase chain reaction (PCR) assay have been used in bacterial pathogen detection in terms of cell’s genotype17,18. As an example, qRT-PCR was used to detect specific cell types in terms of their unique mRNA transcripts obtained from PCR19,20. However, they required multi-step sample preparations including cell lysis and 5 - 24 hours long enrichment steps. In addition, typically no downstream analysis can be further performed after the PCR assay. Detection methods based on cell surface affinity, including enzyme-linked immunosorbent assay (ELISA)21,22, fluorescence-activated cell sorting (FACS)23,24 and surface plasmon resonance assays (SPR)25,26 have been widely used for cell properties measurement. Although these methods provide comprehensive information of cells, they require labeling cells with fluorescence or radioactive tags, which need lengthy sample preparation and skilled personnel. Furthermore, the instruments based on these methods are bulky, expensive and typically cannot perform onsite cell analysis.
Immunomagnetic separation has been widely used to isolate various cell populations based on the specific surface antigens. Target cells bind to magnetic particles conjugated with antibodies, and can be isolated from heterogeneous cell population by an external magnetic field. In the immunomagnetic separation, magnetic particle size and the external magnetic field gradient govern the target cell magnetic mobility and affect the separation efficiency27,28. Magnetic activated cell separation (MACS®) system uses small magnetic particles (~50 nm) for fast and stable binding. However, to compensate the low magnetic mobility of those small beads, a high gradient magnetic field (HGMS) column must be used together with an external magnet29,30. Instead of using the complex structure, column free systems employ simple permanent magnet and larger magnetic beads (i.e. Dynabeads), allowing direct cell separation in tubes31–33. In contrast to these methods relying on capturing the target cells, continuous magnetic separation methods were developed to separate cells by controlling the cells’ trajectory with a magnetic field34–36. Larger number and multiple types of cells have been separated with the continuous system37–39. Nevertheless, the above-mentioned magnetic separation systems focus on high recovery rate and high purity for separation; they are not able to detect cells in-situ and take in-situ measurement of cell ratio or concentration in a heterogeneous population. To overcome the limitations of current cell isolation and detection systems, here we present a novel microfluidic device based on a magnetic bead assay that can not only accurately detect target cells but also rapidly measure cell concentration and size distribution in a continuous flow. Cells are kept alive after detection and can be collected for further downstream applications.
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Analytical Chemistry
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EXPERIMENTAL SECTION
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Figure 1. Schematic of the magnetic bead cell assay in terms of delayed transit time through two micro Coulter counters. Magnetic functionalized CD31 antibodies were bound to antigens specifically expressed on the cell membrane of target HUVEC cells. Target cells (attached with magnetic beads via specific antibody/antigen binding) interact with an external magnetic field and travel through the 2nd counter with a lower speed, creating a delayed transit time (t2>t1); non-target cells travel through the two counters with same transit time (t2=t1).
Sensing Principle. Figure 1 shows the concept of the magnetic bead assay for target cell detection. The device identifies target cells via their transit time difference through the two Coulter counters induced by their magnetic interactions with the external magnetic field. Mixed cell suspension, including target cells (HUVECs bound with specific magnetic beads) and non-target cells, flow through the two-stage micro Coulter counter. When a cell passes through each Coulter counter, a resistive pulse is generated40–42. Each cell’s size and transit time are measured in terms of the pulse amplitude and pulse width, respectively43–46. A high-gradient magnetic field is generated closer to the 2nd counter via an external cylindrical magnet. Due to the magnetic interaction, transit time of a target cell (specifically bound with CD31+ magnetic beads) traveling through the 2nd counter is longer than that through the 1st counter (t2> t1). In comparison, transit time of a non-target cell remains the same for both counters (t2= t1). The average transit time delay of mixed cells is expected to be proportional to the target cell ratio; thus, we can quantify target cell ratio by measuring the transit time delay of mixed cells.
Figure 2. Pictures of the two stage cell detection device. Inserts: 1) the 1st counter, 2) the 2nd counter with a cylindrical magnet mounted externally close to it (below the channel bottom).
Device fabrication and testing procedures. Standard soft lithography process was used to fabricate the device. First, a SU8 mold, consisting patterns for two micro-channels, fluidic chamber, inlet and outlet reservoirs, was created using photolithography. Microchannels, fluidic chamber and the reservoirs were formed by pouring Polydimethylsiloxane (PDMS) on the SU8 layer, followed by degassing and curing. Next, the PDMS was bonded onto a glass substrate after an oxygen plasma treatment (200 mTorr, 50 W, 50 s). The nominal dimensions of the two identical sensing channels are 50 µm wide and 40 µm deep. The channel dimensions measured by a surface profilometer (Dektak 150, Veeco Instrument) are 51.8 ± 0.9 µm wide and 36.9 ± 0.2 µm deep. One pair of Ag/AgCl electrodes (1 mm in diameter) were inserted on each side of each Coulter counting channel via two punched 1-mm holes (see Figure 2) to finish the fabrication of the resistive pulse sensor. A N40 grade cylindrical rare earth magnet (R125A-DM, Amazing Magnet) was mounted below the glass substrate to create a high gradient magnetic field ( =67.62 T2/m) over the 2nd counter. For each cell detection test, a 100 µL mixed cell suspension with a specific target cell ratio was loaded into and driven through the device by a syringe pump (KDS Legato 270, KD Scientific) at a flow rate of 300 µL/hr. Resistive pulses from each counter were converted to measurable voltage pulses by applying a DC voltage at 2.5 V to the two pairs of the Ag/AgCl electrodes. The voltage pulses were then amplified by an external circuit and continuously recorded by a NI-DAQ board (BNC-2110, National Instruments) at a sampling rate of 300 KHz. Cell size and transit time through each counter can then be back calculated from the recorded pulse magnitude and width. Cell Culturing and Magnetic Conjugation. Human umbilical vein endothelial cells (HUVECs, Lonza, USA) were cultured in EBMTM-2 BulletKit™ media supplemented with 1% antibiotic-antimycotic solution at 37oC and 5% CO2. HUVECs between passages 6 and 10 were used for all the experiments. The magnetic particles we used were functionalized CD31+ MicroBead® (Miltenyi Biotec). The magnetic beads were pre-
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Analytical Chemistry
pared by coating a layer of dextran to the magnetic iron oxide core.47 The effective magnetic susceptibility of the magnetic particle is 1.3 from independent measurement. The mean diameter of the magnetic particles is 50 nm. The magnetic beads were bound to HUVECs following the optimized protocol. In brief, cells were reacted with the CD31 antibody magnetic beads solution at the ratio of 5000 cells per 1 µL solution at 4°C with gentle shaking for 30 minutes. After reaction, the cells were then washed extensively using 1X phosphate buffered saline (PBS, pH 7.4, Gibco, USA) to avoid non-specific binding. Specifically, the volume ratio of 10:1 (1X PBS vs. reaction solution) was used for each wash to rinse off the unconjugated magnetic beads. Each rinse procedure included 1 minute of gentle shaking after adding the 1X PBS and 5 minutes centrifugation at 300 g to separate the floating unconjugated magnetic beads from the reaction solution. For every conjugation reaction, 3 rinses were performed. Magnetic Beads Quantification. To quantify the amount of magnetic beads successfully conjugated to target cells, a ferrozine-based colorimetric assay measuring iron content was used as previously described48,49. Briefly, 105 cells conjugated with magnetic beads were resuspended in 100 µL of 50 mM sodium hydroxide (NaOH, Sigma Aldrich, USA) and incubated at 37°C for 2 hours. The iron releasing reagent containing 0.7 M hydrochloric acid (HCl, EMD Chemicals, USA) and 2.25% (w/v) potassium permanganate (KMnO4, Fisher Scientific, USA) were added to the mixture at the volume ratio of 1:3.3 and incubated at 60°C with shaking (100 rpm) for 2 hours. After the mixture was cooled down to room temperature, a freshly prepared iron detecting solution containing 6.5 mM ferrozine (Sigma Aldrich, USA), 6.5 mM neocuproine (Sigma Aldrich, USA), 2.5 M ammonium acetate (Sigma Aldrich, USA), and 1 M of ascorbic acid (Sigma Aldrich, USA) was added to the mixture in the volume ratio of 1:11. The final mixture was vortexed until no visible particles were present and then incubated at room temperature for 30 minutes. Afterwards, the absorbance of the mixture was measured at 550 nm using a Synergy H1 Hybrid microplate reader (BioTek, USA). A standard curve measuring iron standard solution with the concentration from 0 µg/ml to 13.2 µg/ml (GFS Chemicals, USA) was generated for each testing. The relative iron content from the magnetic conjugated cells was calculated by comparing absorbance with standard curve and using nonconjugating cells as control. Cell Viability Assay. Cell viability was assessed using LIVE/DEAD® Viability/Cytotoxicity Kit for mammalian cells (Life Technologies, USA). Briefly, 1.5 × 105 HUVECs were seeded on a well of 24-well plate after passing through the microfluidics device at various flow rates of 300, 1200 and 3000 µL/hr. After 1-hour incubation at 37°C with 5% CO2, cells were stained with 1 µM calcien AM and 4 µM EthD-1 for 30 minutes in cell culture incubator. Samples were imaged using a fluorescence microscope (Carl Zesis Observer A1) and quantified using ImageJ software (NIH free software). Cells without passing through the microfluidic device were used as controls. For each flow rate, 3 independent trials were conducted. Cell counts were calculated using 3~6 technical duplicates of each sample. The sample’s mean viability was calculated and normalized to the initial viability of the culture. Statistical significance was determined using the Student's t-test with p
