Study of cell and drug interactions based on dual-mode detection using SPR and fluorescence imaging.

The investigation of the interactions between cells and drugs forms a crucial aspect of biological and clinical medical studies. Generally, single-cell or local-cellular studies require a microscopic imaging system with high magnifications, which suffers from low detection throughputs and poor time responses. The study presented in this paper combined SPR and fluorescence to achieve cell localization, real-time monitoring of cell images and quantitative analysis of drugs. In order to obtain more comprehensive, accurate and real-time data, a dual-mode system based on surface plasmon resonance (SPR) and fluorescence was constructed based on a 4× magnification lens. This enables simultaneous studies of an entire cell and a specific region of the cell membrane. An adaptive adjustment algorithm was established for distorted SPR images, achieving temporal and spatial matching of the dual-mode detection. The combination of SPR and fluorescence not only achieved micro-detection but also complemented the qualitative or quantitative limitations of SPR or fluorescence method alone. In system characterization, the response signal of SPR was noticed to increase with the increasing concentration of EGF in stimulated cells. It indicated that this platform could be employed for quantitative detection of the cell membrane region. Upon addition of EGF, a peak in the SPR curve was observed, and the cells in the corresponding SPR image turned whiter. This indicated that the platform can simultaneously monitor the SPR response signal and image changes. The response time of fluorescence in EGF testing was several seconds earlier than SPR, revealing that signal transduction first occurred in the whole cell and then propagated to the cell membrane region. The inhibitory ability of Gefitinib on cells was verified in a fast and real-time manner within 20 min. The results indicated that the detection limit of this method was 20 IU/mL for EGF and 10 µg/mL for Gefitinib. In conclusion, this study demonstrates the advantages of SPR and fluorescence dual-mode techniques in the analysis of cell-drug interactions, as well as their strong potential in drug screening.

Analysis of cellular response to drugs with a microfluidic single-cell platform based on hyperspectral imaging.

BACKGROUND: Cellular response to pharmacological action of drugs is significant for drug development. Traditional detection method for cellular response to drugs normally rely on cell proliferation assay and metabolomics examination. In principle, these analytical methods often required cell labeling, invasion analysis, and hours of co-culture with drugs, which are relatively complex and time-consuming. Moreover, these methods can only indicate the drug effectiveness on cell colony rather than single cells. Thus, to meet the requirements of personal precision medicine, the development of drug response analysis on the high resolution of single cell is demanded. RESULTS: To provide precise result for drug response on single-cell level, a microfluidic platform coupled with the label-free hyperspectral imaging was developed. With the help of horizontal single-cell trapping sieves, hundreds of single cells were trapped independently in microfluidic channels for the purposes of real-time drug delivery and single-cell hyperspectral image recording. To significantly identify the cellular hyperspectral change after drug stimulation, the differenced single-cell spectrum was proposed. Compared with the deep learning classification method based on hyperspectral images, an optimal performance can be achieved by the classification strategy based on differenced spectra. And the cellular response to different reagents, for example, K+, Epidermal Growth Factor (EGF), and Gefitinib at different concentrations can be accurately characterized by the differenced single-cell spectra analysis. SIGNIFICANCE AND NOVELTY: The high-throughput, rapid analysis of cellular response to drugs at the single-cell level can be accurately performed by our platform. After systematically analyzing the materials and the structures of the single-cell microfluidic chip, the optimal single-cell trapping method was proposed to contribute to the further application of hyperspectral imaging on microfluidic single-cell analysis. And the hyperspectral characterization of single-cell with cancer drug stimulation proved the application potential of our method in personal cancer medication.

Multi-parameter surface plasmon resonance instrument for multiple nucleic acid quantitative detection.

Multiplex nucleic acid assays can simultaneously detect the characteristics of different target nucleic acids in complex mixtures and are used in disease diagnosis, environmental monitoring, and food safety. However, traditional nucleic acid amplification assays have limitations such as complicated operation, long detection time, unstable fluorescent labeling, and mutual interference of multiplex nucleic acids. We developed a real-time, rapid, and label-free surface plasmon resonance (SPR) instrument for multiplex nucleic acid detection. The multiparametric optical system based on total internal reflection solves the multiplex detection problem by cooperating with linear light source, prism, photodetector, and mechanical transmission system. An adaptive threshold consistency correction algorithm is proposed to solve the problem of inconsistent responsiveness of different detection channels and the inability of quantitative comparison. The instrument achieves label-free and amplification-free rapid detection of these biomarkers for miRNA-21 and miRNA-141, which are widely expressed in breast cancer and prostate cancer. The multiplex nucleic acid detection takes 30 min and the biosensor has good repeatability and specificity. The instrument has a limit of detection (LODs) of 50 nM for target oligonucleotides, and the smallest absolute amount of sample that can be detected is about 4 pmol. It provides a simple and efficient point-of-care testing (POCT) detection platform for small molecules such as DNA and miRNA.

[Detection of IgG protein in human urine based on vertical flow paper microfluidic chip].

The kidney is the body's most important organ and the protein components in urine could be detected for diagnosing certain diseases. The amount of IgG protein in urine could be used to determine the degree of kidney function damage. IgG protein in human urine was detected by vertical flow paper-based microfluidic chip, double-antibody sandwich immunoreaction, and cell phone image processing. The results showed that using an IgG antibody concentration of 500 µg/mL and a gold standard antibody concentration of 100 µg/mL, the image signal showed a good linear relationship in the range of IgG concentration of 0.2-3.2 µg/mL, with R2=0.973 3 achieved. A complete set of detection devices were designed and the detection method showed good non-specificity.

A fully-enclosed prototype 'pen' for rapid detection of SARS-CoV-2 based on RT-RPA with dipstick assay at point-of-care testing.

A fully-enclosed prototype 'pen' for rapid detection of SARS-CoV-2 based on reverse transcriptase isothermal recombinase polymerase amplification (RT-RPA) with dipstick assay was developed. The integrated handheld device, consisting of amplification, detection and sealing modules, was developed to perform rapid nucleic acid amplification and detection under a fully enclosed condition. After RT-RPA amplification with a metal bath or a normal PCR instrument, the amplicons were mixed with dilution buffer prior to being detected on a lateral flow strip. To avoid aerosol contamination causing false-positive, from amplification to final detection, the detection 'pen' had been enclosed to isolate from the environment. With colloidal gold strip-based detection, the detection results could be directly observed by eyes. By cooperating with other inexpensive and rapid methods for POC nucleic acid extraction, the developed 'pen' could detect COVID-19 or other infectious diseases in a convenient, simple and reliable way.

An automated microfluidic system with one-dimensional beads array for multiplexed torch detection at point-of-care testing.

An automated microfluidic system with functionalized beads has been developed for multiplexed TORCH detection at point-of-care testing. A concise microfluidic chip consisting of a one-dimensional beads array is developed to simultaneously detect TOX, RUB, CMV, HSV-I and HSV-II respectively with five functionalized beads. A compact liquid handling module has been developed to automate the sandwiched chemiluminescence immunoassay within the one-dimensional beads array of the microfluidic chip. A precise ram pump is adopted to not only add reagent into the microfluidic chip from outside, but also facilitate elaborate fluid control inside the microfluidic chip for improved performance. A large-size waste chamber with a liquid-absorbing sponge holds the waste reagent within the microfluidic chip to prevent backflow. The one-dimensional beads array is heated from double-sides at 37 ℃ for sensitive detection with reduced time. A sensitive CMOS camera is adopted to take chemiluminescence image from the one-dimensional beads array, and a custom processing algorithm is adopted to analyze the image. For each serum sample, five different infections can be simultaneously detected with the automated microfluidic system. Experimental results show that efficient, sensitive, and accurate multiplexed TORCH detection can be conveniently achieved with the integrated microfluidic system.

A microfluidic system for rapid nucleic acid analysis based on real-time convective PCR at point-of-care testing.

A microfluidic system for rapid nucleic acid analysis based on real-time convective PCR is developed. To perform 'sample-in, answer-out' nucleic acid analysis, a microfluidic chip is developed to efficiently extract nucleic acid, and meanwhile convective PCR (CPCR) is applied for rapid nucleic acid amplification. With an integrated microfluidic chip consisting of reagent pre-storage chambers, a lysis & wash chamber, an elution chamber and a waste chamber, nucleic acid extraction based on magnetic beads can be automatically performed for a large size of test sample within a limited time. Based on an easy-to-operate strategy, different pre-stored reagents can be conveniently released for consecutive reaction at different steps. To achieve efficient mixing, a portable companion device is developed to introduce properly controlled 3-D actuation to magnetic beads in nucleic acid extraction. In CPCR amplification, PCR reagent can be spontaneously and repeatedly circulated between hot and cool zones of the reactor for space-domain thermal cycling based on pseudo-isothermal heating. A handheld real-time CPCR device is developed to perform nucleic acid amplification and in-situ detection. To extend the detection throughput, multiple handheld real-time CPCR devices can be grouped together by a common control system. It is demonstrated that influenza A (H1N1) viruses with the reasonable concentration down to 1.0 TCID50/ml can be successfully detected with the microfluidic system.

Integration of a multichannel surface plasmon resonance sensor chip and refractive index matching film array for protein detection in human urine.

Most prism-based surface plasmon resonance (SPR) experiments use matching fluid with a similar refractive index to mount the chip on the optical prism. The fluidity of the matching fluid easily affects the transmission of the optical signal. In this paper, an integrated SPR sensor chip comprises a three-layer structure of flow layer, metal layer and refractive index matching layer is demonstrated to address the problems related to consistency and uniformity. The Young's modulus, array spacing, shape and other parameters of the matching film were calculated and optimized. The chip can self-adhere to the optical prism, and effectively avoids the generation of air bubbles. The refractive index detection sensitivity of the integrated SPR sensor chip was 3.4359 × 10-6 RIU (refractive index unit), and the chip stabilization time has been effectively shortened. The integrated SPR sensor chip was also used to detect kappa light chain protein and human serum albumin (HSA) in urine samples. The detection limit of kappa light chain protein was 0.06 µg/mL compared with 18.5 µg/mL by conventional immunoturbidimetry. The integrated SPR sensor chip based on refractive index matching film array has great potential in biomedical detection and other fields, including point-of-care testing (POCT).

Real-Time Detection of LAMP Products of African Swine Fever Virus Using Fluorescence and Surface Plasmon Resonance Method.

African swine fever (ASF) is a swine disease with a very high fatality rate caused by a complex double-stranded DNA virus. The fluorescence PCR detection method is widely used for virus nucleic acid detection. Surface plasmon resonance (SPR) is a label-free and real-time detection method, unlike the fluorescence PCR detection method. In this research, we detected the loop-mediated isothermal amplification (LAMP) products of the African swine fever virus by using the SPR and fluorescence methods separately and simultaneously. By comparing the positive and negative control results, we found that the SPR response unit is completely different before and after the LAMP process. In addition, the fluorescence results on a chip showed that with an increase in the concentration of the sample, the cycle threshold (CT) value decreased, which is consistent with commercial instruments. Both the decline rate of the SPR response unit and the CT value of the fluorescence realized were used to distinguish the positive control from the negative control and water, which indicates that the SPR method can be combined with fluorescence to detect LAMP products. This research provides a label-free and simple method for detecting LAMP products.

[Isothermal amplification technology based on microfluidic chip].

Polymerase chain reaction (PCR) is the gold standard for nucleic acid amplification in molecular diagnostics. The PCR includes multiple reaction stages (denaturation, annealing, and extension), and a complicated thermalcycler is required to repetitively provide different temperatures for different stages for 30-40 cycles within at least 1-2 hours. Due to the complicated devices and the long amplification time, it is difficult to adopt conventional PCR in point-of-care testing (POCT). Comparing to conventional PCR, isothermal amplification is able to provide a much faster and more convenient nucleic acid detection because of highly efficient amplification at a constant reaction temperature provided by a simple heating device. When isothermal amplification is combined with microfluidics, a more competent platform for POCT can be established. For example, various diagnosis devices based on isothermal amplification have been used to rapidly and conveniently detect SARS-CoV-2 viruses. This review summarized the recent development and applications of the microfluidics-based isothermal amplification. First, different typical isothermal amplification methods and related detection methods have been introduced. Subsequently, different types of microfluidic systems with isothermal amplification were discussed based on their characteristics, for example, functionality, system structure, flow control, and operation principles. Furthermore, detection of pathogens (e.g. SARS-CoV-2 viruses) based on isothermal amplification was introduced. Finally, the combination of isothermal amplification with other new technologies, e.g. CRISPR, has been introduced as well.

Methods and platforms for analysis of nucleic acids from single-cell based on microfluidics.

Single-cell nucleic acid analysis aims at discovering the genetic differences between individual cells which is well known as the cellular heterogeneity. This technology facilitates cancer diagnosis, stem cell research, immune system analysis, and other life science applications. The conventional platforms for single-cell nucleic acid analysis more rely on manual operation or bulky devices. Recently, the emerging microfluidic technology has provided a perfect platform for single-cell nucleic acid analysis with the characteristic of accurate and automatic single-cell manipulation. In this review, we briefly summarized the procedure of single-cell nucleic acid analysis including single-cell isolation, single-cell lysis, nucleic acid amplification, and genetic analysis. And then, three representative microfluidic platforms for single-cell nucleic acid analysis are concluded as valve-, microwell-, and droplet-based platforms. Furthermore, we described the state-of-the-art integrated single-cell nucleic acid analysis systems based on the three platforms. Finally, the future development and challenges of microfluidics-based single-cell nucleic acid analysis are discussed as well.

Rapid PCR powered by microfluidics: A quick review under the background of COVID-19 pandemic.

PCR has been widely used in different fields including molecular biology, pathogen detection, medical diagnosis, food detection and etc. However, the difficulty of promoting PCR in on-site point-of-care testing reflects on challenges relative to its speed, convenience, complexity, and even cost. With the emerging state-of-art of microfluidics, rapid PCR can be achieved with more flexible ways in micro-reactors. PCR plays a critical role in the detection of SARS-CoV-2. Under this special background of COVID-19 pandemic, this review focuses on the latest rapid microfluidic PCR. Rapid PCR is concluded in two main features, including the reactor (type, size, material) and the implementation of thermal cycling. Especially, the compromise between speed and sensitivity with microfluidic PCR is explored based on the system ratio of (thermal cycling time)/(reactor size). Representative applications about the detection of pathogens and SARS-CoV-2 viruses based on rapid PCR or other isothermal amplification are discussed as well.

Seepage Time Soft Sensor Model of Nonwoven Fabric Based on the Extreme Learning Machine Integrating Monte Carlo.

Nonwoven fiber materials are materials with multifunctional purposes, and are widely used to make masks for preventing the new Coronavirus Disease 2019. Because of the complexity and particularity of their structure, it becomes difficult to model the penetration and flow characteristics of liquid in nonwoven fiber materials. In this paper, a novel seepage time soft sensor model of nonwoven fabric, based on Monte Carlo (MC), integrating extreme learning machine (ELM) (MCELM) is proposed. The Monte Carlo method is used to expand data samples. Then, an ELM method is used to establish the prediction model of the dyeing time of the nonwoven fiber material overlaps with the porous medium, as well as the insertion degree and height of the different quantity of hides. Compared with the back propagation (BP) neural network and radial basis function (RBF) neural network, the results show that the prediction model based on the MCELM method has significant power in terms of accuracy and prediction speed, which is conducive to the precise and rapid manufacture of nonwoven fiber materials in practical applications between liquid seepage characteristics and structural characteristics of porous media. Furthermore, the relationship between the proposed models has certain value for predicting the behavior and use of nonwoven fiber materials with different structural characteristics and related research processes.

A plasma separator with a multifunctional deformable chamber equipped with a porous membrane for point-of-care diagnostics.

Traditionally, plasma is extracted from whole blood using centrifuges in clinical laboratories, which is unsuitable for on-site testing. For point-of-care diagnostics, for example in HIV tests, to ensure the detection sensitivity for low-abundance analytical targets, a relatively large volume of plasma needs to be extracted from milliliters of blood with a simpler and easier-to-operate method than centrifugation. We report the development of a membrane-assisted, sedimentation-facilitated plasma separator with a multifunctional deformable chamber, which is able to perform plasma separation from undiluted whole blood in a short time. Multiple steps related to plasma separation, including cell sedimentation, cell filtration, and plasma driving and discharging, are all performed in or through the multifunctional deformable chamber equipped with a top-layer porous membrane, which significantly reduces the device complexity. Assisted by a simple jig or even hands, plasma separation can be conveniently performed upon mechanical actuation of the deformable chamber. Within 8 min, ∼130 µL of plasma can be conveniently extracted with the described device from 2.3 mL of whole blood. It has been demonstrated that HIV antibodies or virus spiked in whole blood can be successfully detected with reasonable sensitivity from the extracted plasma with the described pump-free device.

An immunoassay cassette with a handheld reader for HIV urine testing in point-of-care diagnostics.

Currently, most HIV tests are performed with blood samples, or alternatively saliva samples are used for HIV testing. Simple HIV tests need to be performed in hospitals or other medical agencies instead of more invasive HIV blood tests. To enable point-of-care (POC) HIV diagnostics, based on a recently developed lateral flow strip for HIV urine testing, a microfluidic immunoassay cassette with a handheld optical reader is developed. Based on lateral flow strip with gold colloid reporter, the integrated immunoassay cassette can perform sample introduction, metering, discharging, applying and detection which simplifies HIV testing. An indicator is incorporated into the cassette to guide sample introduction based on color change, and further, the excess test sample is stored inside the sealed cassette to avoid any contamination. The low-cost handheld optical reader can provide a test result within a few seconds, which is useful for simple, sensitive and affordable HIV onsite detection. Instead of using normal white LEDs, a customized back light module embedded with green LEDs is adopted to illuminate the lateral flow strip with an appropriate working current to achieve optimal performance. Compared to the standard lateral flow strips using a benchtop reader, with the disposable immunoassay cassette assisted by the handheld optical reader, more convenient, easier-to-operate, and more affordable HIV urine testing can be achieved in POC diagnostics.

Development of a Portable SPR Sensor for Nucleic Acid Detection.

Nucleic acid detection is of great significance in clinical diagnosis, environmental monitoring and food safety. Compared with the traditional nucleic acid amplification detection method, surface plasmon resonance (SPR) sensing technology has the advantages of being label-free, having simple operation, and providing real-time detection. However, the angle scanning system in many SPR angle modulation detection applications usually requires a high-resolution stepper motor and complex mechanical structure to adjust the angle. In this paper, a portable multi-angle scanning SPR sensor was designed. The sensor only uses one stepping motor to rotate a belt, and the belt pulls the mechanical linkages of incident light and reflected light to move in opposite directions for achieving the SPR angle scanning mode that keeps the incident angle and reflected angle equal. The sensor has an angle scanning accuracy of 0.002°, response sensitivity of 3.72 × 10-6 RIU (refractive index unit), and an angle scanning range of 30°-74°. The overall size of the system is only 480 mm × 150 mm × 180 mm. The portable SPR sensor was used to detect nucleic acid hybridization on a gold film chip modified with bovine serum albumin (BSA). The result revealed that the sensor had high sensitivity and fast response, and could successfully accomplish the hybridization detection of target DNA solution of 0.01 µmol/mL.

Free convective PCR: From principle study to commercial applications-A critical review.

Polymerase chain reaction (PCR) is an extremely important tool for molecular diagnosis, as it can specifically amplify nucleic acid templates for sensitive detection. As another division of PCR, free convective PCR was invented in 2001, which can be performed in a capillary tube pseudo-isothermally within a significantly short time. Convective PCR thermal cycling is implemented by inducing thermal convection inside the capillary tube, which stratifies the reaction into spatially separate and stable melting, annealing, and extension zones created by the temperature gradient. Convective PCR is a promising tool that can be used for nucleic acid diagnosis as a point-of-care test (POCT) due to the significantly simplified heating strategy, reduced cost, and shortened detection time without sacrificing sensitivity and accuracy. Here, we review the history of free convective PCR from its invention to development and its commercial applications.

Pressure Signal Enhancement of Slowly Increasing Leaks Using Digital Compensator Based on Acoustic Sensor.

Pipeline leak detection technologies are critical for the safety protection of pipeline transportation. However, they are insensitive to slowly increasing leaks. Therefore, this study proposes an enhancement method for slowly increasing leak signals. By analyzing the characteristics of pressure signals of slowly increasing leaks, a digital compensator is developed to overcome the disadvantages of pressure signals and enhance the pressure signals. According to the frequency response analysis of the digital compensator, the enhancement principle is the parameter adjustment of the digital compensator. Therefore, this paper further proposes an adaptive adjustment method of the parameter to enhance different degrees of leak signals online in real-time, and the proposed method is evaluated using two field pipelines. The experimental results demonstrate that this method is suitable not only for enhancing slowly increasing leaks but also for enhancing abrupt leaks.

Flexible capacitive pressure sensor with sensitivity and linear measuring range enhanced based on porous composite of carbon conductive paste and polydimethylsiloxane.

In recent years, the development of electronic skin and smart wearable body sensors has put forward high requirements for flexible pressure sensors with high sensitivity and large linear measuring range. However, it turns out to be difficult to increase both of them simultaneously. In this paper, a flexible capacitive pressure sensor based on a porous carbon conductive paste-polydimethylsiloxane composite is reported, the sensitivity and the linear measuring range of which were developed using multiple methods including adjusting the stiffness of the dielectric layer material, fabricating a microstructure and increasing the dielectric permittivity of the dielectric layer. The capacitive pressure sensor reported here has a relatively high sensitivity of 1.1 kPa-1 and a large linear measuring range of 10 kPa, making the product of the sensitivity and linear measuring range 11, which is higher than that of the most reported capacitive pressure sensors to our best knowledge. The sensor has a detection of limit of 4 Pa, response time of 60 ms and great stability. Some potential applications of the sensor were demonstrated, such as arterial pulse wave measuring and breath measuring, which shows it as a promising candidate for wearable biomedical devices. In addition, a pressure sensor array based on the material was also fabricated and it could identify objects in the shape of different letters clearly, which shows promising application in future electronic skins.

Development of a Surface Plasmon Resonance and Fluorescence Imaging System for Biochemical Sensing.

Surface plasmon resonance (SPR) biosensors are an extremely sensitive optical technique used to detect the changes in refractive index occurring at the sensor interface. Fluorescence involves the emission of light by a substance that has absorbed light or other electromagnetic radiation, and the parameters of the absorbed and emitted radiation are used to identify the presence and the amount of specific molecules in a specimen. SPR biosensors and fluorescence analysis are both effective methods for real-time detection. The combination of these technologies would improve the quantitative detection sensitivity of fluorescence analysis and the specificity of SPR detection. We designed and developed an SPR and fluorescence synchronous detection system. The SPR module was based on two kinds of modulation methods, and the fluorescence module was capable of switching between four wavelengths. The fluorescence microspheres and A549 cells of different concentration were both detected by the SPR and fluorescence method synchronously in real time. The fluorescent signal and the optical signal of the SPR were shown to correlate. The correlation coefficient for fluorescent microspheres detection reached up to 0.9866. The system could be used in cell analysis and molecule diagnosis in the future.