CellMap: An Automated Multielectrode Array Cell Culture Analysis System Based on Electrochemical Impedance Spectroscopy

The objective of this research is to develop fundamental understanding of cell-substrate (CS) and cell-cell (CC) interactions in the culture space for time evolving cell cultures. Space resolved CC and CS interactions are important indicators of cell-density distribution, localized cellular behavior, and multiple cell-layers which are differentiators of normal and abnormal cell behavior. In this research, CS and CC interactions and the variations therein due to a) Cell growth, 2) cell-drug interaction, and 3) effect of Cytotoxin were studied using multielectrode, multi-frequency Electrochemical Impedance Spectroscopy (EIS). Contemporary impedance based methods sense either CC or CS interaction as a space averaged macroscopic quantity. A major contribution of this research is that, both CC and CS interactions are recorded and analyzed with spatio-temporal resolution. This research led to the development of an automated cell culture monitoring system, namely, CellMap. A planar eight electrode sensor was fabricated on a glass substrate and interfaced with a switching circuit. The switching circuit sequentially selects consecutive electrodes upon input of a 5V trigger pulse which is generated by the frequency response analyzer at the end of each frequency scan, thereby facilitating automated switching and recording of multielectrode dataset. Calibration standards and protocols were developed to null the channel parasitics of individual channels. A set of eight impedance measurements for eight electrodes constitutes a frame . Frames are recorded at regular time intervals over the desired course of time. Impedance mapping of adhesion, spreading, motility and detachment of OvCa429 ovarian cancer cells was performed over a period of 70 hours. The cell-layer resistance, which indicates cell-cell contact, increased as a function of time until confluence, and decreased thereafter due to cell death and detachment. This was also confirmed by optical microscopy observations. Similarly, the cell layer Constant Phase Element (CPE) parameters, which were found to correlate well with cell density distribution, also increased as a function of time until confluence and decreased thereafter. Additionally, the cell-growth mapping revealed that the CellMap system is able to resolve non-uniform cell distributions in the culture space, which may be useful in differentiating between normal and pathological cells

Population and fertility by age and sex for 195 countries and territories, 1950–2017: a systematic analysis for the Global Burden of Disease Study 2017

Background: Population estimates underpin demographic and epidemiological research and are used to track progress on numerous international indicators of health and development. To date, internationally available estimates of population and fertility, although useful, have not been produced with transparent and replicable methods and do not use standardised estimates of mortality. We present single-calendar year and single-year of age estimates of fertility and population by sex with standardised and replicable methods. Methods: We estimated population in 195 locations by single year of age and single calendar year from 1950 to 2017 with standardised and replicable methods. We based the estimates on the demographic balancing equation, with inputs of fertility, mortality, population, and migration data. Fertility data came from 7817 location-years of vital registration data, 429 surveys reporting complete birth histories, and 977 surveys and censuses reporting summary birth histories. We estimated age-specific fertility rates (ASFRs; the annual number of livebirths to women of a specified age group per 1000 women in that age group) by use of spatiotemporal Gaussian process regression and used the ASFRs to estimate total fertility rates (TFRs; the average number of children a woman would bear if she survived through the end of the reproductive age span [age 10–54 years] and experienced at each age a particular set of ASFRs observed in the year of interest). Because of sparse data, fertility at ages 10–14 years and 50–54 years was estimated from data on fertility in women aged 15–19 years and 45–49 years, through use of linear regression. Age-specific mortality data came from the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2017 estimates. Data on population came from 1257 censuses and 761 population registry location-years and were adjusted for underenumeration and age misreporting with standard demographic methods. Migration was estimated with the GBD Bayesian demographic balancing model, after incorporating information about refugee migration into the model prior. Final population estimates used the cohort-component method of population projection, with inputs of fertility, mortality, and migration data. Population uncertainty was estimated by use of out-of-sample predictive validity testing. With these data, we estimated the trends in population by age and sex and in fertility by age between 1950 and 2017 in 195 countries and territories. Findings: From 1950 to 2017, TFRs decreased by 49\ub74% (95% uncertainty interval [UI] 46\ub74–52\ub70). The TFR decreased from 4\ub77 livebirths (4\ub75–4\ub79) to 2\ub74 livebirths (2\ub72–2\ub75), and the ASFR of mothers aged 10–19 years decreased from 37 livebirths (34–40) to 22 livebirths (19–24) per 1000 women. Despite reductions in the TFR, the global population has been increasing by an average of 83\ub78 million people per year since 1985. The global population increased by 197\ub72% (193\ub73–200\ub78) since 1950, from 2\ub76 billion (2\ub75–2\ub76) to 7\ub76 billion (7\ub74–7\ub79) people in 2017; much of this increase was in the proportion of the global population in south Asia and sub-Saharan Africa. The global annual rate of population growth increased between 1950 and 1964, when it peaked at 2\ub70%; this rate then remained nearly constant until 1970 and then decreased to 1\ub71% in 2017. Population growth rates in the southeast Asia, east Asia, and Oceania GBD super-region decreased from 2\ub75% in 1963 to 0\ub77% in 2017, whereas in sub-Saharan Africa, population growth rates were almost at the highest reported levels ever in 2017, when they were at 2\ub77%. The global average age increased from 26\ub76 years in 1950 to 32\ub71 years in 2017, and the proportion of the population that is of working age (age 15–64 years) increased from 59\ub79% to 65\ub73%. At the national level, the TFR decreased in all countries and territories between 1950 and 2017; in 2017, TFRs ranged from a low of 1\ub70 livebirths (95% UI 0\ub79–1\ub72) in Cyprus to a high of 7\ub71 livebirths (6\ub78–7\ub74) in Niger. The TFR under age 25 years (TFU25; number of livebirths expected by age 25 years for a hypothetical woman who survived the age group and was exposed to current ASFRs) in 2017 ranged from 0\ub708 livebirths (0\ub707–0\ub709) in South Korea to 2\ub74 livebirths (2\ub72–2\ub76) in Niger, and the TFR over age 30 years (TFO30; number of livebirths expected for a hypothetical woman ageing from 30 to 54 years who survived the age group and was exposed to current ASFRs) ranged from a low of 0\ub73 livebirths (0\ub73–0\ub74) in Puerto Rico to a high of 3\ub71 livebirths (3\ub70–3\ub72) in Niger. TFO30 was higher than TFU25 in 145 countries and territories in 2017. 33 countries had a negative population growth rate from 2010 to 2017, most of which were located in central, eastern, and western Europe, whereas population growth rates of more than 2\ub70% were seen in 33 of 46 countries in sub-Saharan Africa. In 2017, less than 65% of the national population was of working age in 12 of 34 high-income countries, and less than 50% of the national population was of working age in Mali, Chad, and Niger. Interpretation: Population trends create demographic dividends and headwinds (ie, economic benefits and detriments) that affect national economies and determine national planning needs. Although TFRs are decreasing, the global population continues to grow as mortality declines, with diverse patterns at the national level and across age groups. To our knowledge, this is the first study to provide transparent and replicable estimates of population and fertility, which can be used to inform decision making and to monitor progress. Funding: Bill & Melinda Gates Foundation

Impedance spectroscopy-based cellular analysis device

An electrical impedance sensing device which integrates multielectrode automated impedance spectroscopy capability with automatic parameter extraction and data analysis to create an automated cell behavior monitoring system. The device comprises radial electrodes and an out-of-plane counter electrode. Quantitative impedance data provided information on cell adhesion, spreading, proliferation and detachment due to cell cycle processes as well as cell-drug interaction, with spatio-temporal resolution. The resulting dataset is processed for impedance distribution and used to characterize cellular motion, morphology, electrochemical and dielectric properties. Also, a method is described for studying cell-cell and cell-matrix interactions, determining electrical characteristics of cell layers, and identifying specific impedance parameters for cancer screening, drug screening, bacterial growth monitoring, organ transplant compatibility, and cell-drug interaction among other applications

Wet etching process

The present invention illustrates a bulk silicon etching technique that yields straight sidewalls, through wafer structures in very short times using standard silicon wet etching techniques. The method of the present invention employs selective porous silicon formation and dissolution to create high aspect ratio structures with straight sidewalls for through wafer MEMS processing

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

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.ASTAR (Agency for Sci., Tech. and Research, S’pore

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

10.1002/smll.201401674Small118943-95

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.ASTAR (Agency for Sci., Tech. and Research, S’pore)Accepted Versio

CMOS high density electrical impedance biosensor array for tumor cell detection

We present a high-throughput label-free electrical-impedance spectroscopy (EIS) platform composed of a 96 × 96 microelectrode array for counting and analysis of breast tumor cells (MCF-7). These Auelectrodes are densely packed within a 3.5 mm × 3.5 mm interrogation region. Each Au-electrode has an edge-length of 25 μm designed to detect single tumor cell by electrical impedance spectroscopy. The detected signal is read out via an integrated circuit (IC) of addressable electrode-selection-switch array manufactured with 0.18μm CMOS technology. The independently addressable microelectrode array is integrated onto a printed circuit board (PCB) containing associated circuitry for electrode switching and readout. MCF-7 cells were dispensed onto the chip surface and EIS measurements were recorded before and after the cell dispensation. On an average 20% increase in impedance was found associated with cell occupancy on the electrode surface. A positive correlation was observed between cell detection with optical microscopy and with electrical scanning.Accepted versio

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