Micro Milling Process for the Rapid Prototyping of Microfluidic Devices

Micro milling process has become an attractive method for the rapid prototyping of micro devices. The process is based on subtractive manufacturing method in which materials from a sample are removed selectively. A comprehensive review on the fabrication of circular and rectangular cross-section channels of microfluidic devices using micro milling process is provided this review work. Process and machining parameters such as micro-tools selection, spindle speed, depth of cut, feed rate and strategy for process optimization will be reviewed. A case study on the rapid fabrication of a rectangular cross section channel of a microflow cytometer device with 200 um channel width and 50 um channel depth using CNC micro milling process is provided. The experimental work has produced a low surface roughness micro channel of 20 nm in roughness and demonstrated a microflow cytometer device that can produce hydrodynamic focusing with a focusing width of about 60 um.

A Portable Impedance Microflow Cytometer for Measuring Cellular Response to Hypoxia

This paper presents the development and testing of a low-cost (< $60), portable, electrical impedance based microflow cytometer for single cell analysis under controlled oxygen microenvironment. The system is based on an AD5933 impedance analyzer chip, a microfluidic chip, and an Arduino microcontroller operated by a custom Android application. A representative case study on human red blood cells (RBCs) affected by sickle cell disease is conducted to demonstrate the capability of the cytometry system. An equivalent circuit model of a suspended biological cell is used to interpret the electrical impedance of single flowing RBCs. RBCs exhibit decreased mean membrane capacitance by 24% upon hypoxia treatment while the mean cytoplasmic resistance remains consistent. RBCs affected by sickle cell disease exhibit decreased cytoplasmic resistance and increased membrane capacitance upon hypoxia treatment. Strong correlations are identified between the changes in the cells’ subcellular electrical components and the hypoxia-induced cell sickling process. The results reported in this paper suggest that the developed method of testing demonstrates the potential application for low-cost screening technique for sickle cell disease and other diseases in the field and low-resource settings. The developed system and methodology can be extended to analyze cellular response to hypoxia in other cell types.

Machine learning issues and opportunities in ultrafast particle classification for label-free microflow cytometry

Machine learning offers promising solutions for high-throughput single-particle analysis in label-free imaging microflow cytomtery. However, the throughput of online operations such as cell sorting is often limited by the large computational cost of the image analysis while offline operations may require the storage of an exceedingly large amount of data. Moreover, the training of machine learning systems can be easily biased by slight drifts of the measurement conditions, giving rise to a significant but difficult to detect degradation of the learned operations. We propose a simple and versatile machine learning approach to perform microparticle classification at an extremely low computational cost, showing good generalization over large variations in particle position. We present proof-of-principle classification of interference patterns projected by flowing transparent PMMA microbeads with diameters of $${15.2}\,\upmu \text {m}$$ 15.2 μ m and $${18.6}\,\upmu \text {m}$$ 18.6 μ m . To this end, a simple, cheap and compact label-free microflow cytometer is employed. We also discuss in detail the detection and prevention of machine learning bias in training and testing due to slight drifts of the measurement conditions. Moreover, we investigate the implications of modifying the projected particle pattern by means of a diffraction grating, in the context of optical extreme learning machine implementations.

A Portable Impedance Microflow Cytometer for Measuring Cellular Response to Hypoxia

This paper presents the development and testing of a low-cost, portable microflow cytometer based on electrical impedance sensing, for single cell analysis under controlled oxygen microenvironment. The cytometer system is based on an AD5933 impedance analyzer chip, a microfluidic chip, and an Arduino microcontroller operated by a custom Android application. A representative case study on human red blood cells (RBCs) affected by sickle cell disease is conducted to demonstrate the capability of the cytometry system. Equivalent circuit model of a suspending biological cell is used to interpret the electrical impedance of single flowing RBCs. In normal blood, cytoplasmic resistance and membrane capacitance do not change significantly with the change in oxygen tension. In contrast, RBCs affected by sickle cell disease show that upon hypoxia treatment, the cytoplasmic resistance decrease from 11.6 MΩ to 23.4 MΩ, and membrane capacitance decrease from 1.1 pF to 0.8 pF. Strong correlations are identified between the changes in these subcellular electrical components of single cells and the cell sickling process induced by hypoxia treatment. The representative results reported in this paper suggest that single cell electrical impedance can be used as a sensitive biophysical marker for quantifying cellular response to change in oxygen concentration. The developed flow cytometry system and the methodology can also be extended to analysis of cellular response to hypoxia in other cell types.

A Systematic Study on Transit Time and Its Impact on Accuracy of Concentration Measured by Microfluidic Devices

Gating or threshold selection is very important in analyzing data from a microflow cytometer, which is especially critical in analyzing weak signals from particles/cells with small sizes. It has been reported that using the amplitude gating alone may result in false positive events in analyzing data with a poor signal-to-noise ratio. Transit time (τ) can be set as a gating threshold along with side-scattered light or fluorescent light signals in the detection of particles/cells using a microflow cytometer. In this study, transit time of microspheres was studied systematically when the microspheres passed through a laser beam in a microflow cytometer and side-scattered light was detected. A clear linear relationship between the inverse of the average transit time and total flow rate was found. Transit time was used as another gate (other than the amplitude of side-scattering signals) to distinguish real scattering signals from noise. It was shown that the relative difference of the measured microsphere concentration can be reduced significantly from the range of 3.43%–8.77% to the range of 8.42%–111.76% by employing both amplitude and transit time as gates in analysis of collected scattering data. By using optimized transit time and amplitude gate thresholds, a good correlation with the traditional hemocytometer-based particle counting was achieved (R2 > 0.94). The obtained results suggest that the transit time could be used as another gate together with the amplitude gate to improve measurement accuracy of particle/cell concentration for microfluidic devices.

Development of a Low-Cost Electrical Impedance-Based Microflow Cytometer

Sickle Cell Disease (SCD) is a genetic condition caused by a mutated hemoglobin molecule (HbS) found in red blood cells (RBCs). HbS polymerizes in low oxygen environments and contribute to painful vaso-occlusion in patients. Laboratory diagnosis of SCD is typically made by detection of the presence of sickle cells by peripheral blood smear, and presence of HbS by electrophoresis and high-performance liquid chromatography. Recently, flow cytometry technique in companion with sickling assays has demonstrated the capability in quantitative measurements of sickle cells at single-cell level, using software algorithm for cell-imaging analysis (Van Beers et. al. American Journal of Hematology 2014), and electrical impedance (Liu et. al. Sensors and Actuators B: Chemical 2018). Here, we show a portable, cost-efficient electrical impedance-based sensor and its capability to be used in conjunction with microfluidics-based sickling assay for microflow cytometry of sickle cells. The impedance microflow cytometer is based on a commercially available integrated circuit (IC), the AD5933. Using a microcontroller and additional circuitry on a custom designed printed circuit board, we are able to produce sinusoidal signals of up to 100kHz in frequency and sample up to 200 data points per second, at a cost under $60 in materials to create. The impedance measurement range is optimized to work in companion with microfluidic chips in general. In order to measure sickle cells, the impedance microflow cytometer is used in companion with our unique Polydimethylsiloxane (PDMS) microfluidic cell sickling assay (Du et. al. PNAS 2014). Cells are suspended in phosphate buffered saline (PBS) medium and move in the microchannel using a pressure driven flow. Impedance measurement is achieved using two Ti/Au electrodes embedded in the microchannel as cells flow past the electrodes. Data is captured and made available for post processing using a customized MATLAB script. RBCs from healthy donors and SCD patients were used to demonstrate the capability of the developed system. The results showed that our system can separate between normal RBCs and sickle cells, as well as between sickled and unsickled cells. The performance in detection of sickle cells is comparable to a commercial impedance analyzer. This proof-of-concept design aims to minimize the physical space needed for cytometry as well as bring affordable and reliable cytometry results within its given limitations. Figure Disclosures Alvarez: Forma Therapeutics: Consultancy; Novartis: Consultancy.

Handheld Microflow Cytometer Based on a Motorized Smart Pipette, a Microfluidic Cell Concentrator, and a Miniaturized Fluorescence Microscope

Miniaturizing flow cytometry requires a comprehensive approach to redesigning the conventional fluidic and optical systems to have a small footprint and simple usage and to enable rapid cell analysis. Microfluidic methods have addressed some challenges in limiting the realization of microflow cytometry, but most microfluidics-based flow cytometry techniques still rely on bulky equipment (e.g., high-precision syringe pumps and bench-top microscopes). Here, we describe a comprehensive approach that achieves high-throughput white blood cell (WBC) counting in a portable and handheld manner, thereby allowing the complete miniaturization of flow cytometry. Our approach integrates three major components: a motorized smart pipette for accurate volume metering and controllable liquid pumping, a microfluidic cell concentrator for target cell enrichment, and a miniaturized fluorescence microscope for portable flow cytometric analysis. We first validated the capability of each component by precisely metering various fluid samples and controlling flow rates in a range from 219.5 to 840.5 μL/min, achieving high sample-volume reduction via on-chip WBC enrichment, and successfully counting single WBCs flowing through a region of interrogation. We synergistically combined the three major components to create a handheld, integrated microflow cytometer and operated it with a simple protocol of drawing up a blood sample via pipetting and injecting the sample into the microfluidic concentrator by powering the motorized smart pipette. We then demonstrated the utility of the microflow cytometer as a quality control means for leukoreduced blood products, quantitatively analyzing residual WBCs (rWBCs) in blood samples present at concentrations as low as 0.1 rWBCs/μL. These portable, controllable, high-throughput, and quantitative microflow cytometric technologies provide promising ways of miniaturizing flow cytometry.