A highly integrated digital PCR system with on-chip heating for accurate DNA quantitative analysis.

Digital polymerase chain reaction (dPCR) is extensively used for highly sensitive disease diagnosis due to its single-molecule detection ability. However, current dPCR systems require intricate DNA sample distribution, rely on cumbersome external heaters, and exhibit sluggish thermal cycling, hampering efficiency and speed of the dPCR process. Herein, we presented the development of a microwell array based dPCR system featuring an integrated self-heating dPCR chip. By utilizing hydrodynamic and electrothermal simulations, the chip's structure is optimized, resulting in improved partitioning within microwells and uniform thermal distribution. Through strategic hydrophilic/hydrophobic modifications on the chip's surface, we effectively secured the compartmentalization of sample within the microwells by employing an overlaying oil phase, which renders homogeneity and independence of samples in the microwells. To achieve precise, stable, uniform, and rapid self-heating of the chip, the ITO heating layer and the temperature control algorithm are deliberately designed. With a capacity of 22,500 microwells that can be easily expanded, the system successfully quantified EGFR plasmid solutions, exhibiting a dynamic linear range of 105 and a detection limit of 10 copies per reaction. To further validate its performance, we employed the dPCR platform for quantitative detection of BCR-ABL1 mutation gene fragments, where its performance was compared against the QuantStudio 3D, and the self-heating dPCR system demonstrated similar analytical accuracy to the commercial dPCR system. Notably, the individual chip is produced on a semiconductor manufacturing line, benefiting from mass production capabilities, so the chips are cost-effective and conducive to widespread adoption and accessibility.

High-Performance Plasma Biomarker Panel for Alzheimer's Disease Screening Using a Femtomolar-Level Label-Free Biosensing System.

Alzheimer's disease (AD) is the most common cause of dementia in older people. However, diagnosing AD through noncognitive methods, such as invasive cerebrospinal fluid sampling or radioactive positron emission tomography, has limited applications. Herein, the femtomolar levels of AD biomarkers amyloid ß 40 (Aß40), amyloid ß 42 (Aß42), phosphorylated tau 181 (P-tau181), phosphorylated tau 217 (P-tau217), and neurofilament light chain (NfL) were determined in human plasma in multicenter clinical cohorts using an ultrasensitive graphene field-effect transistor sensor. A machine-learning algorithm was also used to assemble these plasma biomarkers and optimize their performance in discriminating individual stages of Alzheimer's dementia progression. The "composite-info" biomarker panel, which combines these biomarkers and clinical information, considerably improved the staging performance in AD progression. It achieved an area under the curve of >0.94 in the receiver operator characteristic (ROC) curve. In addition, the panel demonstrated an advantage in the individual-based stage assessment compared with that of the Mini-Mental State Examination/Montreal Cognitive Assessment and nuclear magnetic resonance imaging. This study provides a composite biomarker panel for the screening and early diagnosis of AD using a rapid detection system.

Absolute Copy Numbers of ß-Actin Proteins Collected from 10,000 Single Cells.

Semi-quantitative studies have located varied expressions of ß-actin proteins at the population level, questioning their roles as internal controls in western blots, while the absolute copy numbers of ß-actins at the single-cell level are missing. In this study, a polymeric microfluidic flow cytometry was used for single-cell analysis, and the absolute copy numbers of single-cell ß-actin proteins were quantified as 9.9 ± 4.6 × 105, 6.8 ± 4.0 × 105 and 11.0 ± 5.5 × 105 per cell for A549 (ncell = 14,754), Hep G2 (ncell = 36,949), and HeLa (ncell = 24,383), respectively. High coefficients of variation (~50%) and high quartile coefficients of dispersion (~30%) were located, indicating significant variations of ß-actin proteins within the same cell type. Low p values (≪0.01) and high classification rates based on neural network (~70%) were quantified among A549, Hep G2 and HeLa cells, suggesting expression differences of ß-actin proteins among three cell types. In summary, the results reported here indicate significant variations of ß-actin proteins within the same cell type from cell to cell, and significant expression differences of ß-actin proteins among different cell types, strongly questioning the properties of using ß-actin proteins as internal controls in western blots.

Microfluidic Analyzer Enabling Quantitative Measurements of Specific Intracellular Proteins at the Single-Cell Level.

This paper presents a microfluidic instrument capable of quantifying single-cell specific intracellular proteins, which are composed of three functioning modules and two software platforms. Under the control of a LabVIEW platform, a pressure module flushed cells stained with fluorescent antibodies through a microfluidic module with fluorescent intensities quantified by a fluorescent module and translated into the numbers of specific intracellular proteins at the single-cell level using a MATLAB platform. Detection ranges and resolutions of the analyzer were characterized as 896.78⁻6.78 × 105 and 334.60 nM for Alexa 488, 314.60⁻2.11 × 105 and 153.98 nM for FITC, and 77.03⁻5.24 × 104 and 37.17 nM for FITC-labelled anti-beta-actin antibodies. As a demonstration, the numbers of single-cell beta-actins of two paired oral tumor cell types and two oral patient samples were quantified as: 1.12 ± 0.77 × 106/cell (salivary adenoid cystic carcinoma parental cell line (SACC-83), ncell = 13,689) vs. 0.90 ± 0.58 × 105/cell (salivary adenoid cystic carcinoma lung metastasis cell line (SACC-LM), ncell = 15,341); 0.89 ± 0.69 × 106/cell (oral carcinoma cell line (CAL 27), ncell = 7357) vs. 0.93 ± 0.69 × 106/cell (oral carcinoma lymphatic metastasis cell line (CAL 27-LN2), ncell = 6276); and 0.86 ± 0.52 × 106/cell (patient I) vs. 0.85 ± 0.58 × 106/cell (patient II). These results (1) validated the developed analyzer with a throughput of 10 cells/s and a processing capability of ~10,000 cells for each cell type, and (2) revealed that as an internal control in cell analysis, the expressions of beta-actins remained stable in oral tumors with different malignant levels.

A Microfluidic Fluorescent Flow Cytometry Capable of Quantifying Cell Sizes and Numbers of Specific Cytosolic Proteins.

This study presents a microfluidics based cytometry capable of characterizing cell sizes and counting numbers of specific cytosolic proteins where cells were first bound by antibodies labelled with fluorescence and then aspirated into a constriction microchannel in which fluorescent levels were measured. These raw fluorescent pulses were further divided into a rising domain, a stable domain and a declining domain. In addition, antibody solutions with labelled fluorescence were aspirated through the constriction microchannel, yielding curves to translate raw fluorescent levels to protein concentrations. By using key parameters of three domains as well as the calibration curves, cell diameters and the absolute number of ß-actins at the single-cell level were quantified as 14.2 ± 1.7 µm and 9.62 ± 4.29 × 105 (A549, ncell = 14 242), 13.0 ± 2.0 µm and 6.46 ± 3.34 × 105 (Hep G2, ncell = 35 932), 13.8 ± 1.9 µm and 1.58 ± 0.90 × 106 (MCF 10 A, ncell = 16 650), and 12.7 ± 1.5 µm and 1.09 ± 0.49 × 106 (HeLa, ncell = 26 246). This platform could be further adopted to measure numbers of various cytosolic proteins, providing key insights in proteomics at the single-cell level.

Mechanical property characterization of hundreds of single nuclei based on microfluidic constriction channel.

As label-free biomarkers, the mechanical properties of nuclei are widely treated as promising biomechanical markers for cell type classification and cellular status evaluation. However, previously reported mechanical parameters were derived from only around 10 nuclei, lacking statistical significances due to low sample numbers. To address this issue, nuclei were first isolated from SW620 and A549 cells, respectively, using a chemical treatment method. This was followed by aspirating them through two types of microfluidic constriction channels for mechanical property characterization. In this study, hundreds of nuclei were characterized, producing passage times of 0.5 ± 1.2 s for SW620 nuclei in type I constriction channel (n = 153), 0.045 ± 0.047 s for SW620 nuclei in type II constriction channel (n = 215) and 0.50 ± 0.86 s for A549 nuclei in type II constriction channel. In addition, neural network based pattern recognition was used to classify the nuclei isolated from SW620 and A549 cells, producing successful classification rates of 87.2% for diameters of nuclei, 85.5% for passage times of nuclei and 89.3% for both passage times and diameters of nuclei. These results indicate that the characterization of the mechanical properties of nuclei may contribute to the classification of different tumor cells.

Development of microfluidic impedance cytometry enabling the quantification of specific membrane capacitance and cytoplasm conductivity from 100,000 single cells.

This paper presents a new microfluidic impedance cytometry with crossing constriction microchannels, enabling the characterization of cellular electrical markers (e.g., specific membrane capacitance (Csm) and cytoplasm conductivity (σcy)) in large cell populations (~ 100,000 cells) at a rate greater than 100 cells/s. Single cells were aspirated continuously through the major constriction channel with a proper sealing of the side constriction channel. An equivalent circuit model was developed and the measured impedance values were translated to Csm and σcy. Neural network was used to classify different cell populations where classification success rates were calculated. To evaluate the developed technique, different tumour cell lines, and the effects of epithelial-mesenchymal transitions on tumour cells were examined. Significant differences in both Csm and σcy were found for H1299 and HeLa cell lines with a classification success rate of 90.9% in combination of the two parameters. Meanwhile, tumour cells A549 showed significant decreases in both Csm and σcy after epithelial-mesenchymal transitions with a classification success rate of 76.5%. As a high-throughput microfluidic impedance cytometry, this technique can add a new marker-free dimension to flow cytometry in single-cell analysis.

Single-Cell Protein Assays: A Review.

Quantification of single-cell proteomics provides key insights in the field of cellular heterogeneity. This chapter discusses the emerging techniques that are being used to measure the protein copy numbers at the single-cell level, which includes flow cytometry, mass cytometry, droplet cytometry, microengraving, and single-cell barcoding microchip. The advantages and limitations of each technique are compared, and future research opportunities are highlighted.

A microfabricated 96-well wound-healing assay.

This article presents a microfabricated 96-well wound-healing assay enabling high-throughput measurement of cellular migration capabilities. Within each well, the middle area is the wound region, made of microfabricated gold surface with self-assembled PEG repellent for cell seeding. After the formation of a cellular confluent monolayer around the wound region, collagen solution was applied to form three-dimensional matrix to cover the PEG surface, initiating the wound-healing process. By interpreting the numbers of migrated cells into the wound regions as a function of specific stimuli with different concentrations, EC50 (half-maximal effective concentration) was obtained. Using H1299 as a model, values of EC50 were quantified as 8% and 160 ng/ml for fetal bovine serum and CXCL12, respectively. In addition, the values of EC50 were demonstrated not to be affected by variations in compositions of extracellular matrix and geometries of wounds, which can thus be regarded as an intrinsic marker. Furthermore, the migration capabilities of a second cell type (HeLa) were characterized by the developed wound-healing assay, producing EC50 of 2% when fetal bovine serum was used as the stimuli. These results validated the proposed high-throughput wound-healing assay, which may function as an enabling tool in studying cellular capabilities of migration and invasion. © 2017 International Society for Advancement of Cytometry.

A microfluidic flow cytometer enabling absolute quantification of single-cell intracellular proteins.

Quantification of single-cell proteomics provides key insights into cellular heterogeneity while conventional flow cytometry cannot provide absolute quantification of intracellular proteins of single cells due to the lack of calibration approaches. This paper presents a constriction channel (with a cross sectional area smaller than cells) based microfluidic flow cytometer, capable of collecting copy numbers of specific intracellular proteins. In this platform, single cells stained with fluorescence labelled antibodies were forced to squeeze through the constriction channel with the fluorescence intensities quantified and since cells fully filled the constriction channel during the squeezing process, solutions with fluorescence labelled antibodies were flushed into the constriction channel to obtain calibration curves. By combining raw fluorescence data and calibration curves, absolute quantification of intracellular proteins was realized. As a demonstration, copy numbers of beta-actin of single tumour cells were quantified to be 0.90 ± 0.30 µM (A549, ncell = 14 228), 2.34 ± 0.70 µM (MCF 10A, ncell = 2455), and 0.98 ± 0.65 µM (Hep G2, ncell = 6945). The travelling time for individual cells was quantified to be roughly 10 ms and thus a throughput of 100 cells per s can be achieved. This microfluidic system can be used to quantify the copy numbers of intracellular proteins in a high-throughput manner, which may function as an enabling technique in the field of single-cell proteomics.

The Instrumentation of a Microfluidic Analyzer Enabling the Characterization of the Specific Membrane Capacitance, Cytoplasm Conductivity, and Instantaneous Young's Modulus of Single Cells.

This paper presents the instrumentation of a microfluidic analyzer enabling the characterization of single-cell biophysical properties, which includes seven key components: a microfluidic module, a pressure module, an imaging module, an impedance module, two LabVIEW platforms for instrument operation and raw data processing, respectively, and a Python code for data translation. Under the control of the LabVIEW platform for instrument operation, the pressure module flushes single cells into the microfluidic module with raw biophysical parameters sampled by the imaging and impedance modules and processed by the LabVIEW platform for raw data processing, which were further translated into intrinsic cellular biophysical parameters using the code developed in Python. Based on this system, specific membrane capacitance, cytoplasm conductivity, and instantaneous Young's modulus of three cell types were quantified as 2.76 ± 0.57 µF/cm², 1.00 ± 0.14 S/m, and 3.79 ± 1.11 kPa for A549 cells (ncell = 202); 1.88 ± 0.31 µF/cm², 1.05 ± 0.16 S/m, and 3.74 ± 0.75 kPa for 95D cells (ncell = 257); 2.11 ± 0.38 µF/cm², 0.87 ± 0.11 S/m, and 5.39 ± 0.89 kPa for H460 cells (ncell = 246). As a semi-automatic instrument with a throughput of roughly 1 cell per second, this prototype instrument can be potentially used for the characterization of cellular biophysical properties.

Specific membrane capacitance, cytoplasm conductivity and instantaneous Young's modulus of single tumour cells.

As label-free biomarkers, biophysical properties of cells are widely used for cell type classification. However, intrinsic biophysical markers, e.g., specific membrane capacitance (Cspecific membrane), cytoplasm conductivity (σconductivity) and instantaneous Young's modulus (Einstantaneous) measured for hundreds of single cells were not yet reported. In this study, single cells in suspension (adherent cells treated with trypsin) were aspirated through a microfluidic constriction channel at 25 °C, and the entry processes and impedance profiles were recorded and translated to Cspecific membrane, σconductivity and Einstantaneous. Cspecific membrane, σconductivity and Einstantaneous of five cell types were quantified as 2.10±0.38 µF cm-2, 0.91±0.15 S m-1 and 5.52±0.95 kPa for H460 cells (ncell=437); 2.52±0.54 µF cm-2, 0.83±0.12 S m-1 and 5.54±1.04 kPa for H446 cells (ncell=410); 2.45±0.57 µF cm-2, 0.99±0.18 S m-1 and 5.16±1.68 kPa for A549 cells (ncell=442); 1.86±0.31 µF cm-2, 1.07±0.18 S m-1 and 3.86±0.81 kPa for 95D cells (ncell=415); 2.03±0.35 µF cm-2, 0.99±0.16 S m-1 and 3.49±0.70 kPa for 95C cells (ncell=290). The database of Cspecific membrane, σconductivity and Einstantaneous may serve as a reference for future studies of cellular biophysical properties.

A Microfabricated 96-Well 3D Assay Enabling High-Throughput Quantification of Cellular Invasion Capabilities.

This paper presents a 96-well microfabricated assay to study three-dimensional (3D) invasion of tumor cells. A 3D cluster of tumor cells was first generated within each well by seeding cells onto a micro-patterned surface consisting of a central fibronectin-coated area that promotes cellular attachment, surrounded by a poly ethylene glycol (PEG) coated area that is resistant to cellular attachment. Following the formation of the 3D cell clusters, a 3D collagen extracellular matrix was formed in each well by thermal-triggered gelation. Invasion of the tumor cells into the extracellular matrix was subsequently initiated and monitored. Two modes of cellular infiltration were observed: A549 cells invaded into the extracellular matrix following the surfaces previously coated with PEG molecules in a pseudo-2D manner, while H1299 cells invaded into the extracellular matrix in a truly 3D manner including multiple directions. Based on the processing of 2D microscopic images, a key parameter, namely, equivalent invasion distance (the area of invaded cells divided by the circumference of the initial cell cluster) was obtained to quantify migration capabilities of these two cell types. These results validate the feasibility of the proposed platform, which may function as a high-throughput 3D cellular invasion assay.

Development of Droplet Microfluidics Enabling High-Throughput Single-Cell Analysis.

This article reviews recent developments in droplet microfluidics enabling high-throughput single-cell analysis. Five key aspects in this field are included in this review: (1) prototype demonstration of single-cell encapsulation in microfluidic droplets; (2) technical improvements of single-cell encapsulation in microfluidic droplets; (3) microfluidic droplets enabling single-cell proteomic analysis; (4) microfluidic droplets enabling single-cell genomic analysis; and (5) integrated microfluidic droplet systems enabling single-cell screening. We examine the advantages and limitations of each technique and discuss future research opportunities by focusing on key performances of throughput, multifunctionality, and absolute quantification.

Electrical Property Characterization of Neural Stem Cells in Differentiation.

Electrical property characterization of stem cells could be utilized as a potential label-free biophysical approach to evaluate the differentiation process. However, there has been a lack of technology or tools that can quantify the intrinsic cellular electrical markers (e.g., specific membrane capacitance (Cspecific membrane) and cytoplasm conductivity (σcytoplasm)) for a large amount of stem cells or differentiated cells. In this paper, a microfluidic platform enabling the high-throughput quantification of Cspecific membrane and σcytoplasm from hundreds of single neural stem cells undergoing differentiation was developed to explore the feasibility to characterize the neural stem cell differentiation process without biochemical staining. Experimental quantification using biochemical markers (e.g., Nestin, Tubulin and GFAP) of neural stem cells confirmed the initiation of the differentiation process featured with gradual loss in cellular stemness and increased cell markers for neurons and glial cells. The recorded electrical properties of neural stem cells undergoing differentiation showed distinctive and unique patterns: 1) in the suspension culture before inducing differentiation, a large distribution and difference in σcytoplasm among individual neural stem cells was noticed, which indicated heterogeneity that may result from the nature of suspension culture of neurospheres; and 2) during the differentiation in adhering monolayer culture, significant changes and a large difference in Cspecific membrane were located indicating different expressions of membrane proteins during the differentiation process, and a small distribution difference in σcytoplasm was less significant that indicated the relatively consistent properties of cytoplasm during the culture. In summary, significant differences in Cspecific membrane and σcytoplasm were observed during the neural stem cell differentiation process, which may potentially be used as label-free biophysical markers to monitor this process.

Development of Microfluidic Systems Enabling High-Throughput Single-Cell Protein Characterization.

This article reviews recent developments in microfluidic systems enabling high-throughput characterization of single-cell proteins. Four key perspectives of microfluidic platforms are included in this review: (1) microfluidic fluorescent flow cytometry; (2) droplet based microfluidic flow cytometry; (3) large-array micro wells (microengraving); and (4) large-array micro chambers (barcode microchips). We examine the advantages and limitations of each technique and discuss future research opportunities by focusing on three key performance parameters (absolute quantification, sensitivity, and throughput).

A simple multi-well stretching device to induce inflammatory responses of vascular endothelial cells.

We herein introduce a novel multi-well stretching device that is made of three polydimethylsiloxane layers, consisting of a top hole-punched layer, middle thin membrane, and bottom patterned layer. It is the first time that such a simple device has been used to supply axisymmetric and nonuniform strains to cells cultured on well bottoms that are stretchable. These mechanical stimuli can somewhat mimic the stretching at the bending sites of blood vessels, where the strains are complicated. In this device, nonuniform strain is given to cells through the deformation of a membrane from a flat surface to a spherical cap during the injection of a certain volume of water into the chamber between the middle membrane and bottom layer. EA.hy926 cells (a human umbilical vein endothelial cell line) were seeded on the well bottoms and exposed to axisymmetric strain under a 5, 10, 15, and 20% degree of deformation of the membrane. The cellular responses were characterized in terms of cell morphology, cell viability, and expression of inflammatory mRNAs and proteins. With increasing the degree of deformation, the cells exhibited an inclination toward detachment and apoptosis; meanwhile the expression of inflammatory mRNAs and proteins, such as MCP-1, IL-8, IL-6 and ICAM-1, showed a significant increment. The obtained results demonstrate that the inflammatory responses of EA.hy926 cells can be induced by increasing the magnitude of the strain. This simple device provides a useful tool for in vitro investigation of the inflammatory mechanisms related to vascular diseases.

Osteocyte culture in microfluidic devices.

This paper presents a microfluidic device (poly-dimethylsiloxane micro channels bonded with glass slides) enabling culture of MLO-Y4 osteocyte like cells. In this study, on-chip collagen coating, cell seeding and culture, as well as staining were demonstrated in a tubing-free manner where gravity was used as the driving force for liquid transportation. MLO-Y4 cells were cultured in microfluidic channels with and without collagen coating where cellular images in a time sequence were taken and analyzed, confirming the positive effect of collagen coating on phenotype maintaining of MLO-Y4 cells. The proliferating cell nuclear antigen based proliferation assay was used to study cellular proliferation, revealing a higher proliferation rate of MLO-Y4 cells seeded in microfluidic channels without collagen coating compared to the substrates coated with collagen. Furthermore, the effects of channel dimensions (variations in width and height) on the viability of MLO-Y4 cells were explored based on the Calcein-AM and propidium iodide based live/dead assay and the Hoechst 33258 based apoptosis assay, locating the correlation between the decrease in channel width or height and the decrease in cell viability. As a platform technology, this microfluidic device may function as a new cell culture model enabling studies of osteocytes.