A novel microfluidic chip integrated with Pt micro-thermometer for temperature measurement at the single-cell level.

Noninvasive and sensitive thermometry of a single cell during the normal physiological process is crucial for analyzing fundamental cellular metabolism and applications to cancer treatment. However, current thermometers generally sense the average temperature variation for many cells, thereby failing to obtain real-time and continuous data of an individual cell. In this study, we employed platinum (Pt) electrodes to construct an integrated microfluidic chip as a single-cell thermometer. The single-cell isolation unit in the microchip consisted of a main channel, which was connected to the inlet and outlet of a single-cell capture funnel. A single cell can be trapped in the funnel and the remaining cells can bypass and flow along the main channel to the outlet. The best capture ratio of a single MCF7 cell at a single-cell isolation unit was 90 % under optimal condition. The thermometer in the micro-chip had a temperature resolution of 0.007 °C and showed a good linear relationship in the range of 20-40 °C (R2 = 0.9999). Slight temperature increment of different single tumor cell (MCF7 cell, H1975 cell, and HepG2 cell) cultured on the chip was continuously recorded under normal physiological condition. In addition, the temperature variation of single MCF7 cell in-situ after exposure to a stimulus (4 % paraformaldehyde treatment) was also monitored, showing an amplitude of temperature fluctuations gradually decreased over time. Taken together, this integrated microchip is a practical tool for detecting the change in the temperature of a single cell in real-time, thereby offering valuable information for the drug screening, diagnosis, and treatment of cancer.

Rapid and sensitive electrochemiluminescence detection using easily fabricated sensor with an integrated two-electrode system.

The electrochemiluminescence (ECL) behavior of a tri(2,2'-bipyridyl)ruthenium(ii) (Ru(bpy)32+)/tripropylamine (TPrA) system was investigated in sensor chips with two kinds of integrated two-electrode systems, which included screen-printed electrodes (SPE) and physical vapor deposition (PVD) electrodes. Firstly, under excitation with an optimal transient potential (TP) within 100 ms, the ECL assay could be carried out on the microchips using an Au & Au electrode system, emitting strong and stable light signal. Secondly, on the PVD chip, the ECL intensity initiated by optimal TP was eight times stronger than the peak light signal emitted by the linear sweep voltammetry model. Finally, the logarithmic ECL intensities exhibited a linear increase with the logarithmic concentrations of Ru(bpy)32+ in both the SPE and PVD chips without any reference electrode (RE). Typically, the integration of an interdigital two-electrode system in the microchip significantly enhanced the ECL sensitivity of Ru(bpy)32+ because the large relative area between the working electrode (WE) and counter electrode (CE) achieved a highly efficient mass transfer. This improvement enabled the establishment of a reliable linear relationship across a wide concentration range, spanning from 1 pM to 1 µM (R2 = 0.998). Therefore, the exceptional ECL response of the Ru(bpy)32+/TPrA system on microfluidic chips using a two-electrode system and the TP excitation model has been demonstrated. This suggests that ECL chips without a RE have broad potential for the rapid and sensitive detection of multiple targets.

Detection of EGFR gene with a droplet digital PCR chip integrating a double-layer glass reservoir.

The lack of reliable and practical method for detecting rare hot mutation of epidermal growth factor receptor (EGFR) in circulating tumor DNA (ctDNA) for lung cancer has remained a challenge for general clinical application due to excess wild type DNA in clinical samples. In this study, we developed a droplet digital PCR (ddPCR) platform, integrating a PDMS chip and double-layer glass reservoir. The duplex T-junction droplet generators in PDMS chip can produce about one million uniform droplets of 4.187 pL within ∼10 min, which were then stored in the glass reservoir. The double-layer glass reservoir can protect droplets from evaporation and breaking, solving the problem of instability during thermal-cycling. The quantitative capabilities of the ddPCR chip were evaluated by testing EGFR exon gene 21, with a good linear correlation in the wide range of 101 to 106 copies/µL (R2 = 0.9998). We then demonstrated that the proposed ddPCR device can recognize rare EGFR L858R mutation under a background of 106 copies/µL wild-type DNA at a sensitivity of 0.0001%. Finally, we demonstrated this ddPCR platform could identify low amount of EGFR L858R mutation in ctDNA and CTCs of patients with lung cancer.

A batch microfabrication of a self-cleaning, ultradurable electrochemical sensor employing a BDD film for the online monitoring of free chlorine in tap water.

Free chlorine is one of the key water quality parameters in tap water. However, a free chlorine sensor with the characteristics of batch processing, durability, antibiofouling/antiorganic passivation and in situ monitoring of free chlorine in tap water continues to be a challenging issue. In this paper, a novel silicon-based electrochemical sensor for free chlorine that can self-clean and be mass produced via microfabrication technique/MEMS (Micro-Electro-Mechanical System) is proposed. A liquid-conjugated Ag/AgCl reference electrode is fabricated, and electrochemically stable BDD/Pt is employed as the working/counter electrode to verify the effectiveness of the as-fabricated sensor for free chlorine detection. The sensor demonstrates an acceptable limit of detection (0.056 mg/L) and desirable linearity (R 2 = 0.998). Particularly, at a potential of +2.5 V, hydroxyl radicals are generated on the BBD electrode by electrolyzing water, which then remove the organic matter attached to the surface of the sensor though an electrochemical digestion process. The performance of the fouled sensor recovers from 50.2 to 94.1% compared with the initial state after self-cleaning for 30 min. In addition, by employing the MEMS technique, favorable response consistency and high reproducibility (RSD < 4.05%) are observed, offering the opportunity to mass produce the proposed sensor in the future. A desirable linear dependency between the pH, temperature, and flow rate and the detection of free chlorine is observed, ensuring the accuracy of the sensor with any hydrologic parameter. The interesting sensing and self-cleaning behavior of the as-proposed sensor indicate that this study of the mass production of free chlorine sensors by MEMS is successful in developing a competitive device for the online monitoring of free chlorine in tap water.

Wet-Etched Microchamber Array Digital PCR Chip for SARS-CoV-2 Virus and Ultra-Early Stage Lung Cancer Quantitative Detection.

We report a novel design of chamber-based digital polymerase chain reaction (cdPCR) chip structure. Using a wet etching process and silicon-glass bonding, the chamber size can be adjusted independently of the process and more feasibly in a normal lab. In addition, the structure of the chip is optimized through hydrodynamic computer simulations to eliminate dead space when the sample is injected into the chip. The samples will be distributed to each separated microchambers for an isolated reaction based on Poisson distribution. Due to the difference in expansion coefficients, isolation of the sample in the microchambers by the oil phase on top ensures homogeneity and independence of the sample in the microchambers. The prepared microarray cdPCR chip enables high-throughput and high-sensitivity quantitative measurement of the SARS-CoV-2 virus gene and the mutant lung cancer gene. We applied the chip for the detection of different concentrations of the mix containing the open reading frame 1ab (ORF1ab) gene, the most specific and conservative gene region of the SARS-CoV-2 virus. In addition to this, we also successfully detected the fluorescence of the epidermal growth factor receptor (EGFR) mutant gene in independent microchambers. At a throughput of 46 200 microchambers, solution mixtures containing both genes were successfully tested quantitatively, with a detection limit of 10 copies/µL. Importantly, the chips are individually inexpensive and easy to industrialize. In addition, the microarray can provide a unified solution for other viral sequences, cancer marker assay development, and point-of-care testing (POCT).

Low-cost portable bioluminescence detector based on silicon photomultiplier for on-site colony detection.

A low-cost, portable bioluminescence detector based on a silicon photomultiplier (SiPM) was developed for on-site colony detection, the main components of which are a low-noise photoelectric signal detection and processing circuit, power management module, and high-performance embedded microcontroller subsystem with peripheral circuits. Balanced chopper modulation and lock-in amplification techniques were adopted to improve the signal-to-noise ratio, and a zero-adjustment technique was used to eliminate the dark current of the SiPM to expand the dynamic range. Using this bioluminescence detector, adenosine triphosphate could be determined in the range of 3.6 × 10-6 to 3.6 × 10-11 mol/L, and bacterial colonies could be determined in the range of 1.0 × 103 to 1.0 × 109 CFU/mL, with a limit of quantitation of 1.0 × 103 CFU/mL. Satisfactory recoveries and precision were obtained. Actual samples were accurately tested and the data were verified by comparison with those from the national standard method. The manufacturing cost of the bioluminescence detector was only $30, which is only approximately 1% of the price of current commercial instruments. This study provides a tool for rapid on-site detection of bacterial colonies, as well as a new concept for the development of low-cost portable detection equipment.

Recent developments in sensors for wearable device applications.

Wearable devices are a new means of human-computer interaction with different functions, underlying principles, and forms. They have been widely used in the medical and health fields, in applications including physiological signal monitoring; sports; and environmental detection, while subtly affecting people's lives and work. Wearable sensors as functional components of wearable devices have become a research focus. In this review, we systematically summarize recent progress in the development of wearable sensors and related devices. Wearable sensors in medical health applications, according to the principle of measurement, are divided into physical and chemical quantity detection. These sensors can monitor and measure specific parameters, thereby enabling continuously improvements in the quality and feasibility of medical treatment. Through the detection of human movement, such as breathing, heartbeat, or bending, wearable sensors can evaluate body movement and monitor an individual's physical performance and health status. Wearable devices detecting aspects of the environment while maintaining high adaptability to the human body can be used to evaluate environmental quality and obtain more accurate environmental information. The ultimate goal of this review is to provide new insights and directions for the future development and broader application of wearable devices in various fields.Graphical abstract.

Microfluidic sensor integrated with nanochannel liquid conjunct Ag/AgCl reference electrode for trace Pb(II) measurement.

Pollution due to heavy metals is becoming increasingly hazardous; therefore, demand for the large-scale deployment of sensor nodes for water quality monitoring has increased. The development of integrated and miniaturised sensors for detecting heavy metals is necessary. Herein, an integrated microfluidic sensor based on a "glass-silicon-glass" sandwich structure is proposed for Pb2+ detection. This micro-sensor consists of a nanochannel liquid conjunct Ag/AgCl reference electrode(RE), a working electrode with a three-dimensional Au micropillar array, and a detection chamber for sample measurement. The potential fluctuation of the RE in this sensor was only 0.62% over seven days, remaining relatively stable. Under optimal conditions, the limit of detection and sensitivity for lead were 0.13 µg L-1 (S/N = 3) and 52.30 nA (µg L-1)-1, respectively. The linearity of the sensor for detecting lead was good in the concentration range of 0.50-150 µg L-1 (R2 = 0.9989). Moreover, the proposed microsensor showed high selectivity for Pb2+ and achieved sensitive detection of trace Pb2+ in different water samples. Therefore, this integrated and miniaturised sensor is a practical tool for trace lead detection, allowing the development of large scale sensor network for water monitoring.

A high-precision thermometry microfluidic chip for real-time monitoring of the physiological process of live tumour cells.

Temperature changes in cells are generally accompanied by physiological processes. Cellular temperature measurements can provide important information to fully understand cellular mechanisms. However, temperature measurements with conventional methods, such as fluorescent polymeric thermometers and thermocouples, have limitations of low sensitivity or cell state disturbance. We developed a microfluidic chip integrating a high-precision platinum (Pt) thermo-sensor that can culture cells and monitor the cellular temperature in situ. During detection, a constant temperature system with a stability of 0.015 °C was applied. The temperature coefficient of resistance of the Pt thermo-sensor was 2090 ppm/°C, giving a temperature resolution of the sensor of less than 0.008 °C. This microchip showed a good linear correlation between the temperature and resistance of the Pt sensor at 20-40 °C (R2 = 0.999). Lung and liver cancer cells on the microchip grew normally and continuously. The maximum temperature fluctuation of H1975 (0.924 °C) was larger than that of HepG2 (0.250 °C). However, the temperature of adherent HepG2 cells changed over time, showing susceptibility to the environment most of the time compared to H1975. Moreover, the temperature increment of non-cancerous cells, such as hepatic stellate cells, was monitored in response to the stimulus of paraformaldehyde, showing the process of cell death. Therefore, this thermometric microchip integrated with cell culture could be a non-disposable and label-free tool for monitoring cellular temperature applied to the study of physiology and pathology.

Single-cell genetic analysis of lung tumor cells based on self-driving micro-cavity array chip.

Lung cancer is one of the common malignant tumors with a high incidence and mortality rate. Targeted therapies are efficient on lung cancer patients with specific gene mutations. Circulating tumor cells (CTCs) are used for liquid biopsy, providing genetic information for lung cancer treatment selection and prognosis. We developed a less costly self-driving micro-cavity array for simple molecular analysis at a single cell level to examine the genetic make-up of CTCs. This chip integrated sample detection structure and vacuum driving system to achieve cell loading, lysing, isothermal amplification (LAMP), and signal read-out on one chip. We used the "film-polydimethylsiloxane (PDMS) chip-film" structure and oil sealing method during amplification reaction to minimize water loss. We then conducted a LAMP assay using the self-driving device to detect epidermal growth factor receptor (EGFR) L858R mutation and identified an excellent linear in the range between 101-104 copies/µL (R2 = 0.997). We finally assessed the EGFR L858R gene expression of lung tumor cells (H1975 cells) as putative CTCs using the proposed detection platform. We discovered its ability to perform genetic analysis at the single-cell level. The EGFR L858R mutational gene expression levels were different in H1975 cells. In conclusion, the self-driving micro-cavity array is a less costly and simple tool for mutational gene profiling of single lung CTC. Besides, it can be used in personalized therapy and efficacy monitoring.

Strategies for the detection of target analytes using microfluidic paper-based analytical devices.

Microfluidic paper-based analytical devices (µPADs) have developed rapidly in recent years, because of their advantages, such as small sample volume, rapid detection rates, low cost, and portability. Due to these characteristics, they can be used for in vitro diagnostics in the laboratory, or in the field, for a variety of applications, including food evaluation, disease screening, environmental monitoring, and drug testing. This review will present various detection methods employed by µPADs and their respective applications for the detection of target analytes. These include colorimetry, electrochemistry, chemiluminescence (CL), electrochemiluminescence (ECL), and fluorescence-based methodologies. At the same time, the choice of labeling material and the design of microfluidic channels are also important for detection results. The construction of novel nanocomponents and different smart structures of paper-based devices have improved the performance of µPADs and we will also highlight some of these in this manuscript. Additionally, some key challenges and future prospects for the use of µPADs are briefly discussed.

Rapid developments in lateral flow immunoassay for nucleic acid detection.

Recently, lateral flow assay (LFA) for nucleic acid detection has drawn increasing attention in the point-of-care testing fields. Due to its rapidity, easy implementation, and low equipment requirement, it is well suited for use in rapid diagnosis, food authentication, and environmental monitoring under source-limited conditions. This review will discuss two main research directions of lateral flow nucleic acid tests. The first one is the incorporation of isothermal amplification methods with LFA, which ensures an ultra-high testing sensitivity under non-laboratory conditions. The two most commonly used methodologies will be discussed, namely Loop-mediated Isothermal Amplification (LAMP) and Recombinase Polymerase Amplification (RPA), and some novel methods with special properties will also be introduced. The second research direction is the development of novel labeling materials. It endeavors to increase the sensitivity and quantifiability of LFA testing, where signals can be read and analyzed by portable devices. These methods are compared in terms of limits of detection, detection times, and quantifiabilities. It is anticipated that future research on lateral flow nucleic acid tests will focus on the integration of the whole testing process into a microfluidic system and the combination with molecular diagnostic tools such as clustered regularly interspaced short palindromic repeats to facilitate a rapid and accurate test.

A batch microfabrication of a microfluidic electrochemical sensor for rapid chemical oxygen demand measurement.

Chemical oxygen demand (COD) is one of the key water quality parameters in environmental monitoring. However, fabricating a COD sensor with the characteristic of batch-processing and rapid measurement is always a challenging issue. This paper reports a microfluidic electrochemical sensor for the organic matter measurement based on advanced oxidization within a fixed microvolume detection chamber by a microfabrication technique/MEMS. By fabricating a silicon-based Ag/AgCl reference electrode and employing PbO2 as the working electrode with Pt as the counter electrode, we verified the superiority of the as-fabricated sensor by continuous potassium acid phthalate detection; an acceptable limit of detection (4.17 mg L-1-200 mg L-1), a low limit of detection (2.05 mg L-1), a desirable linearity (R2 = 0.982) and relative stability at different pH values and Cl- concentrations was witnessed. Particularly, a shorter detection time (2 s) was witnessed for the as-proposed sensor compared with traditional organic matter measurement methods. Each sensing process takes only 2 seconds for sensing because a micro-cavity with a volume of 2.5 µL was fabricated and used as a detection pool. Moreover, as the sensor was fabricated by a mass-production technique, potential response consistency of multiple sensors was expected and was verified via a series of parallel experiments. In this paper, a miniaturized (8 mm × 10 mm), low-cost and reliable COD sensor was designed and fabricated by MEMS, and it provided a core sensor component for construction of an online water environment monitoring network to meet the substantial demand for COD sensors in the Internet of Things (IOT) era.

Deep Learning on chromatographic data for Segmentation and Sensitive Analysis.

Lateral flow immunoassay (LFIA) is one of the most common methods in point-of-care testing, which is widely applied in some conditions for various applications. Image segmentation is an increasingly popular experimental paradigm to efficiently test the target area in LFIA. However, due to process pollution, and problems related to the experimental operation and irregular structure of the background of the reaction, currently available tools cannot be used to extract correct signals from these images, which affects the accuracy of detection. Machine learning has significantly improved modern biochemical analysis by pushing the limits of traditional techniques for the recognition and processing of images. In this paper, the U-Net, a variant of the convolutional neural network (CNN) is used for the quantitative analysis of LFIA images for the accurate quantification of single- and multi-target images. By graying, binarizing, and labeling different concentrations of test strips, the target area of LFIA images containing the T-/C-lines is extracted and obtained. Then it provides updated trends and directions for the development of LFIA technology. Several indicators are introduced to evaluate the proposed U-Net structure to verify the feasibility and effectiveness of its image processing capability. When the trained U-Net model was used to process images, the peak signal-to-noise ratio was 22.4 dB, significantly higher than prevalent methods in the area that have reported only a 4 dB improvement in the quality of the graphics. The intersection-over-union between samples also increased to above 93%. Our results show that the proposed method has significant potential for detecting a segmented target in an LFIA area, especially weak positive signals and multichannel detection. With other modifications, this deep learning method can be applied as a powerful tool to study rapid detection devices, systems, and biological images.

Smartphone-imaged multilayered paper-based analytical device for colorimetric analysis of carcinoembryonic antigen.

Paper-based immunoassays are effective methods that employ microfluidic paper-based analytical devices (µPADs) for the rapid, simple, and accurate quantification of analytes in point-of-care diagnosis. In this study, we developed a wax-printed multilayered µPAD for the colorimetric detection of carcinoembryonic antigen (CEA), where the device contained a movable and rotatable detection layer to allow the µPAD to switch the state of the sample solutions, i.e., flowing or storing in the sensing zones. A smartphone with a custom-developed program served as an automated colorimetric reader to capture and analyze images from the µPAD, before calculating and displaying the test results. After optimizing the crucial conditions for the assay, the proposed method exhibited a wide linear dynamic range from 0.5 to 70 ng/mL, with a low CEA detection limit of 0.015 ng/mL. The clinical performance of this method was successfully validated using 50 positive and 40 negative human serum samples, thereby demonstrating the high sensitivity of 98.0% and specificity of 97.5% in the detection of CEA. The proposed method is greatly simplified compared with the cumbersome steps required for traditional immunoassays, but without any loss of accuracy and stability, as well as reducing the time needed to detect CEA. Complex and bulky instruments are replaced with a smartphone. The proposed detection platform could potentially be applied in point-of-care testing. Graphical abstract.

Single-cell impedance analysis of osteogenic differentiation by droplet-based microfluidics.

Single-cell analysis is critical to understanding its heterogeneity and biological processes, such as stem cell differentiation, and elucidating the underlying mechanisms of cellular metabolism. New tools to promote intercellular variability studies help elucidate cellular regulation mechanisms. Here an impedance measurement and analysis system was built to monitor the osteogenic differentiation of single bone marrow mesenchymal stem cells (BM-MSCs) in droplets. The biochip including a microelectrode array was designed based on droplet microfluidics and fabricated. A novel theoretical electrical model was proposed to simulate the electrical properties of cells in the droplets. Impedance measurements showed that single cells are substantially heterogeneous during osteoblast differentiation at different stages (days 0, 7, 14 and 21) and different cell passages (passages 6, 7 and 11). This result was consistent with the appearance of two biomarkers (alkaline phosphatase and calcium nodules), which are the gold standard biomarkers of osteoblastogenesis and differentiation. The device enabled highly efficient single-cell trapping, accurate positioning, and sensitive, label-free and noninvasive impedance measurements of individual cells with multiple channels. This system provides a strategy for exploring the processes of osteoblastogenesis and differentiation at the single-cell level and has substantial potential for applications in the biomedical field.

Algorithms for immunochromatographic assay: review and impact on future application.

Lateral flow immunoassay (LFIA) is a critical choice for applications of point-of-care testing (POCT) in clinical and laboratory environments because of its excellent features and versatility. To obtain authentic values of analyte concentrations and reliable detection results, the relevant research has featured the application of a diversity of methods of mathematical analysis to technical analysis to allow for use with a small quantity of data. Accordingly, a number of signal and image processing strategies have also emerged for the application of gold immunochromatographic and fluorescent strips to improve sensitivity and overcome the limitations of correlative hardware systems. Instead of traditional methods to solve the problem, researchers nowadays are interested in machine learning and its more powerful variant, deep learning technology, for LFIA detection. This review emphasizes different models for the POCT of accurate labels as well as signal processing strategies that use artificial intelligence and machine learning. We focus on the analytical mechanism, procedural flow, and the results of the assay, and conclude by summarizing the advantages and limitations of each algorithm. We also discuss the potential for application of and directions of future research on LFIA technology when combined with Artificial Intelligence and deep learning.

Compact Yttria-Stabilized Zirconia Based Total NOx Sensor with a Dual Functional Co3O4/NiO Sensing Electrode.

Yttria-stabilized zirconia (YSZ) based potentiometric gas sensors have been widely utilized for detecting NOx (NO and NO2). Nevertheless, it is still remains challenging issue for YSZ-based sensors to sense total NOx due to the opposite response signals to NO and NO2. Herein, we report an efficient strategy to sense total NOx at high temperature (above 300 °C) by designing a dual functional sensing electrode (SE); namely, the SE will simultaneously convert NO (in NOx mixture) to NO2 and electrocatalyze all of the obtained NO2 to generate the response signal of total NOx. In comparison with those previously reported total NOx sensors, the proposed total NOx sensor will be featured with a simplified sensor configuration and desirable long-term stability. To confirm the practicability of the proposed strategy, the NO conversion rate of several metal oxides and their composites have been measured and it turns out that the Co3O4/NiO shows relatively high NO conversion rate. Further study indicates a YSZ-based sensor consisting of (Co3O4 + 20 wt % NiO)-SE and Mn-based RE demonstrates satisfactory performance in detecting total NOx. For instance, analogous response magnitude to NO and NO2 as well as the mixture of NO/NO2 (within 35 ppm) is witnessed for the sensor; particularly, the sensor gives acceptable stability and response/recovery rate at the operating temperature of 500 °C within the examined period. In summary, the use of dual functional SE (e.g., Co3O4/NiO composite SE) indeed addressed those issues of concern in monitoring the level of total NOx and has provided a promising alternative way for designing future high-performance total NOx sensor.

Detection platforms for point-of-care testing based on colorimetric, luminescent and magnetic assays: A review.

Along with the considerable potential and increasing demand of the point-of-care testing (POCT), corresponding detection platforms have attracted great interest in both academic and practical fields. The first few generations of conventional detection devices tend to be costly, complicated to operate and hard to move on account of early limitations in the level of technological development and relatively high requirement of performance. Owing to the requirements for rapidity, simplicity, accuracy and cost controlling in the POCT, reader systems are urgently needed to be developed, upgraded and modified constantly, realizing on-site testing and healthcare management without a specific place or cumbersome operation. Accordingly, numerous rapid detection platforms with diverse size and performance have emerged such as bench-top apparatuses, handheld devices and intelligent detection devices. This review discusses various devices developed mainly for the detection of lateral flow test strips (LFTSs) or microfluidic strips in the POCT and summarizes these devices by size and portability. Furthermore, on the basis of various detection methods and diverse probes usually containing specific nanoparticles composites, three most common aspects of detection rationale in the POCT are selected to elaborate each kind of detection platforms in this paper: colorimetric assay, luminescent detection and magnetic signal detection. Herein, we focus on their structures, detection mechanisms and assay results, accompany with discussions and comments on the performances, costs and potential application, as well as advantages and limitations of each technique. In addition, perspectives on the future advances of detection platforms and some conclusions are proposed.

EGFR point mutation detection of single circulating tumor cells for lung cancer using a micro-well array.

In view of their critical function in metastasis, characterization of single circulating tumor cells (CTCs) can provide important clinical information to monitor tumor progression and guide personal therapy. Single-cell genetic analysis methods based on microfluidics have some inherent shortcomings such as complicated operation, low throughput, and expensive equipment requirements. To overcome these barriers, we developed a simple and open micro-well array containing 26,208 units for either nuclear acids or single-cell genetic analysis. Through modification of the polydimethylsiloxane surface and optimization of chip packaging, we addressed protein adsorption and solution evaporation for PCR amplification on a chip. In the detection of epidermal growth factor receptor (EGFR) exon gene 21, this micro-well array demonstrated good linear correlation at a DNA concentration from 1 × 101 to 1 × 105 copies/µL (R2 = 0.9877). We then successfully integrated cell capture, lysis, PCR amplification, and signal read-out on the micro-well array, enabling the rapid and simple genetic analysis of single cells. This device was used to detect duplex EGFR mutation genes of lung cancer cell lines (H1975 and A549 cells) and normal leukocytes, demonstrating the ability to perform high-throughput, massively parallel duplex gene analysis at the single-cell level. Different types of point mutations (EGFR-L858R mutation or EGFR-T790M mutation) were detected in single H1975 cells, further validating the significance of single-cell level gene detection. In addition, this method showed a good performance in the heterogeneity detection of individual CTCs from lung cancer patients, required for micro-invasive cancer monitoring and treatment selection.