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 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.

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.

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.

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.

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.

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.

Ultra-sensitive and selective detection of mercury ion (Hg2+) using free-standing silicon nanowire sensors.

In this paper, ultra-sensitive and highly selective Hg2+ detection in aqueous solutions was studied by free-standing silicon nanowire (SiNW) sensors. The all-around surface of SiNW arrays was functionalized with (3-Mercaptopropyl)trimethoxysilane serving as Hg2+ sensitive layer. Due to effective electrostatic control provided by the free-standing structure, a detection limit as low as 1 ppt was obtained. A linear relationship (R 2 = 0.9838) between log(CHg2+ ) and a device current change from 1 ppt to 5 ppm was observed. Furthermore, the developed SiNW sensor exhibited great selectivity for Hg2+ over other heavy metal ions, including Cd2+. Given the extraordinary ability for real-time Hg2+ detection, the small size and low cost of the SiNW device, it is expected to be a potential candidate in field detection of environmentally toxic mercury.

High-Performance Limiting Current Oxygen Sensor Comprised of Highly Active La0.75Sr0.25Cr0.5Mn0.5O3 Electrode.

Zirconia-based limiting current oxygen sensor gains considerable attention, due to its high-performance in improving the combustion efficiency of fossil fuels and reducing the emission of exhaust gases. Nevertheless, the Pt electrode is frequently used in the oxygen sensor, therefore, it restrains the broader application due to the high cost. Quite recently, La0.75Sr0.25Cr0.5Mn0.5O3 (LSCM) has been reported to be highly active to catalyze oxygen reduction. Herein, with the intention of replacing the frequently used Pt, we studied the practicability of adapting the LSCM to zirconia-based limiting current oxygen sensor. Through comparing the electrocatalytic activity of LSCM and Pt, it is confirmed that LSCM gave analogous oxygen reactivity with that of the Pt. Then, limiting the current oxygen sensors comprised of LSCM or Pt are fabricated and their sensing behavior to oxygen in the range of 2⁻25% is evaluated. Conclusively, quick response/recovery rate (within 7s), linear relationship, and high selectivity (against 5% CO2 and H2O) in sensing oxygen are observed for the sensors, regardless of the sensing materials (LSCM or Pt) that are used in the sensor. Particularly, identical sensing characteristics are observed for the sensors consisting of LSCM or Pt, indicating the practicability of replacing the Pt electrode by adapting the LSCM electrode to future zirconia-based oxygen sensors.

Investigation of Controllable Nanoscale Heat-Denatured Bovine Serum Albumin Films on Graphene.

Two-dimensional graphene devices are widely used for biomolecule detection. Nevertheless, the surface modification of graphene is critical to achieve the high sensitivity and specificity required for biological detection. Herein, native bovine serum albumin (BSA) in inorganic solution is denatured on the graphene surface by heating, leading to the formation of nanoscale BSA protein films adsorbed on the graphene substrate via π-stacking interactions. This technique yields a controllable, scalable, uniform, and high-coverage method for graphene biosensors. Further, the application of such nanoscale heat-denatured BSA films on graphene as a universal graphene biosensor platform is explored. The thickness of heat-denatured BSA films increased with heating time and BSA concentration but decreased with solvent concentration as confirmed by atomic force microscopy. The noncovalent interaction between denatured BSA films and graphene was investigated by Raman spectroscopy. BSA can act as a p-type and n-type dopant by modulating pH-dependent net charges on the layered BSA-graphene surface, as assessed by current-voltage measurements. Chemical groups of denatured BSA films, including amino and carboxyl groups, were verified by X-ray photoelectron microscopy, attenuated total reflectance-Fourier transform infrared spectra, and fluorescent labeling. The tailoring of the BSA-graphene surfaces through chemical modification, controlled thickness, and doping type via noncovalent interactions provides a controllable, multifunctional biosensor platform for molecular diagnosis without the possibility of nonspecific adsorption on graphene.

Facile Preparation of Bifunctional Monodisperse Nanospheres with Tunable Size and Luminescence.

Nanotechnology has found wide use in biomedical applications and the food and bioprocessing industry. In this light, we demonstrate a facile strategy to prepare bifunctional monodisperse silica nanospheres encapsulating chitosan-coated magnetic nanoparticles and CdTe quantum dots. The size of these composite spheres can be adjusted from 90 nm to 500 nm by varying the concentration of ammonia, water, tetraethyl orthosilicate, and the ratio of the chitosan-coated magnetic nanoparticles and CdTe quantum dots. The composite spheres are characterized using scanning electron microscope analyses, transmission electron microscope analyses, energy-dispersed spectrum studies, Malvern Zetasizer, vibrating sample magnetometer, and fluorescence microscopy. The spheres exhibit good monodispersion and favorable superparamagnetic and fluorescent properties. The luminescence of the spheres can be varied by using different types of coated quantum dots. Such composite spheres with tunable characteristics allow for external manipulation of research systems by magnetic fields together with the real-time fluorescent monitoring of multiple samples. The abovementioned properties can potentially be exploited for application in the biomedical and biosensing fields.

Graphene microelectrode arrays for neural activity detection.

We demonstrate a method to fabricate graphene microelectrode arrays (MEAs) using a simple and inexpensive method to solve the problem of opaque electrode positions in traditional MEAs, while keeping good biocompatibility. To study the interface differences between graphene-electrolyte and gold-electrolyte, graphene and gold electrodes with a large area were fabricated. According to the simulation results of electrochemical impedances, the gold-electrolyte interface can be described as a classical double-layer structure, while the graphene-electrolyte interface can be explained by a modified double-layer theory. Furthermore, using graphene MEAs, we detected the neural activities of neurons dissociated from Wistar rats (embryonic day 18). The signal-to-noise ratio of the detected signal was 10.31 ± 1.2, which is comparable to those of MEAs made with other materials. The long-term stability of the MEAs is demonstrated by comparing differences in Bode diagrams taken before and after cell culturing.

Microchip-based human serum atherogenic lipoprotein profile analysis.

Owing to the mounting evidence of serum lipid changes in atherosclerosis, there has been increasing interest in developing new methods for analyzing atherogenic lipoprotein profiles. The separation of lipoprotein and lipoprotein subclasses has been demonstrated using a microchip capillary electrophoresis (CE) system [Chromatographia 74 (2011) 799-805]. In contrast to this previous study, the current report demonstrates that sdLDL peak efficiencies can be improved dramatically by adding gold nanoparticles (AuNPs) to the sample. Moreover, NBD C6-ceramide was identified as a satisfactory dye for specific labeling and quantitation of individual serum lipoproteins. The accuracy of the method was evaluated by comparison with ultracentrifuge separated small, dense, low-density lipoprotein (sdLDL). A high correlation was observed between these two methods for sdLDL cholesterol. Lipid levels were investigated between atherosclerotic patients and healthy controls. The variation of serum atherogenic lipoprotein profiles for atherosclerotic patients pre- and post-treatment was assessed by microchip CE. This method has potential for the rapid and sensitive detection of different lipoprotein classes as well as their subclasses and, therefore, is suitable for routine clinical applications. Microchip-based atherogenic lipoprotein profile assays will greatly improve the analysis of risk factors in atherosclerosis and will provide useful information for monitoring the effect of therapies on atherosclerotic disease.

Rolling circle amplification immunoassay combined with gold nanoparticle aggregates for colorimetric detection of protein.

A highly sensitive and novel colorimetric rolling circle amplification (RCA) immunoassay for detecting C-reactive protein (CRP) has been developed. In the assay, a CRP capture antibody was immobilized on magnetic beads and a CRP detection antibody was conjugated with single-stranded DNA (ssDNA) using N-[ε-maleimidocaproyloxy] sulfosuccinimide ester. Along with the addition of CRP, a "sandwich" structure was formed. Subsequently, the ssDNA was used as a primer to initiate the RCA reaction in the presence of the circular template, phi29 DNA polymerase and deoxynucleotide triphosphates. The RCA product obtained by magnetic separation, and long tandem repeated sequences mediated the aggregation of gold nanoparticles (AuNPs), which could be observed by the naked eye or quantified using absorption spectra with a detection limit of 30 fg mL(-1) and a linear response range from 10 ng mL(-1) to 1 pg mL(-1). This assay offers the advantages of isothermal conditions, low cost and label-free quantification that could be hopeful for ultrasensitive and robust visual protein detection.

Simplified microchip electrophoresis for rapid separation of urine proteins.

BACKGROUND: Urine protein test has been widely used in clinics, but to determine the type of proteinuria is usually difficult due to technical limitations. METHODS: In the current study, a rapid and simple method to separate and determine urine proteins by a microchip electrophoresis (ME) system has been developed in which only 4 min are required. RESULTS: Optimal separation conditions have been established by using 15 s injection time at 500 and 1,500 V separation voltage in 75 mmol/l borate buffer containing 0.8 mmol/l calcium lactate and 1% ϕ ethylamine (pH 10.55). Relative standard deviation (RSD) of migration time with purified human albumin and human transferring was 2.68% and 2.24%, and RSD of the peak area was 5.85% and 4.96%, respectively. The linear detection range was 1.0-15.0 g/l for purified human albumin and 1.0-10.0 g/l for human transferrin, with the same detection limit (S/N = 3) of 0.4 g/l. Finally, comparing to conventional agarose gel electrophoresis, the same results were obtained by using ME by testing clinical samples including 60 selective proteinuria, 105 nonselective proteinuria, and 6 overflow proteinuria. CONCLUSION: This newly established ME could have broad applications to determine the type of proteinuria in clinics.

Activation of multiple signaling pathways during the differentiation of mesenchymal stem cells cultured in a silicon nanowire microenvironment.

Mesenchymal stem cells (MSC) offer an optimal source for bone tissue engineering due to their capability of undergoing multilineage differentiation, where the mechanical properties of the microenvironment of MSCs are vital for osteochondral formation. However, the mechanisms of how mechanical and microenvironmental cues control osteogenesis and chondrogenesis are yet to be elucidated. In this study, we investigated the effects of vertically aligned silicon nanowire (SiNW) array on the differentiation of MSCs and the associated molecular mechanisms involved in osteogenesis and chandrogenesis. The results showed that the microenvironment of SiNW array activated a number of mechanosensitive pathways (including Integrin, TGF-ß/BMP, Akt, MAPK, Insulin, and Wnt pathways) in MSCs, which converged to stimulate the osteogenesis and chondrogenesis via the Ras-Raf-MEK-ERK cascade. FROM THE CLINICAL EDITOR: This study reports on the mechanisms and microenvironmental influence of osteogenesis and chondrogenesis by mesenchymal stem cells interacting with vertically aligned silicon nanowire scaffolds.

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.

A colorimetric method for H1N1 DNA detection using rolling circle amplification.

A highly sensitive and specific colorimetry-based rolling circle amplification (RCA) assay has been successfully developed as a method for the effective detection of H1N1 DNA. Specific oligonucleotide and reporter primer probes were designed together with a circular template, and the oligonucleotide probes were attached to the surfaces of magnetic beads (MBs) to form functional MB-DNA conjugates as capture probes for the target H1N1 DNA molecules. Together with the addition of DNA targets and reporter primer probes to the MB-DNA conjugates, sandwiched hybrids were formed. The initiation of RCA amplification using the circular template in the presence of phi29 polymerase allowed for the amplification of a large number of repeat sequences of the single-stranded (ss)-DNA product. This RCA product accumulated gold nanoparticles (AuNPs), resulting in a colorimetric change that could be viewed by the naked eye or detected using UV-vis spectroscopy. According to this method, H1N1 DNA could be detected at the 1 pmol L(-1) level. This platform exhibited design convenience, simplicity, and cost-effectiveness, and could be used to provide a new diagnostic assay for H1N1, and other infectious diseases.

The advantage of circulating tumor cells over serum carcinoembryonic antigen for predicting treatment responses in rectal cancer.

AIM: The objective of this study was to investigate the clinical significance of circulating tumor cells (CTCs) on the evaluation and prediction of treatment responses in rectal cancer patients compared with serum carcinoembryonic antigen (CEA). MATERIALS & METHODS: Both CTCs and CEA levels of 103 rectal cancer patients (66 with stage II-III and 37 with recurrence or metastasis) were analyzed before and after chemoradiotherapy. CTCs were detected using EpCAM magnetic bead-based enrichment combined with cytometric identification. RESULTS: CTCs were detected in all patients while no tumor cells were found in healthy controls. CTC levels in metastatic patients were significantly higher than those with recurrence or stage II-III rectal cancer. There is a close relationship between CTC levels and treatment outcomes but serum CEA did not have any correlation. CONCLUSION: CTCs are promising markers for the evaluation and prediction of treatment responses in rectal cancer patients, superior to the conventional tumor marker CEA.