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A low-cost, handheld centrifugal microfluidic system for multiplexed visual detection based on recombinase polymerase amplification (RPA) was developed. A concise centrifugal microfluidic chip featuring four reaction units was developed to run multiplexed RPA amplification in parallel. Additionally, a significantly shrunk-size and cost-effective handheld companion device was developed, incorporating heating, optical, rotation, and sensing modules, to perform multiplexed amplification and visual detection. After one-time sample loading, the metered sample was equally distributed into four separate reactors with high-speed centrifugation. Non-contact heating was adopted for isothermal amplification. A tiny DC motor on top of the chip was used to drive steel beads inside reactors for active mixing. Another small DC motor, which was controlled by an elaborate locking strategy based on magnetic sensing, was adopted for centrifugation and positioning. Visual fluorescence detection was optimized from different sides, including material, surface properties, excitation light, and optical filters. With fluorescence intensity-based visual detection, the detection results could be directly observed through the eyes or with a smartphone. As a proof of concept, the handheld device could detect multiple targets, e.g., different genes of African swine fever virus (ASFV) with the comparable LOD (limit of detection) of 75 copies/test compared to the tube-based RPA.
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Técnicas de Amplificação de Ácido Nucleico , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Técnicas de Amplificação de Ácido Nucleico/métodos , Vírus da Febre Suína Africana/isolamento & purificação , Vírus da Febre Suína Africana/genética , Dispositivos Lab-On-A-Chip , Limite de Detecção , Centrifugação/instrumentação , Animais , Smartphone , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Técnicas Analíticas Microfluídicas/economiaRESUMO
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.
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Técnicas Biossensoriais , MicroRNAs , Ácidos Nucleicos , Ressonância de Plasmônio de Superfície , MicroRNAs/genética , DNA/genética , Limite de Detecção , Hibridização de Ácido NucleicoRESUMO
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.
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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 â 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.
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Técnicas Analíticas Microfluídicas , Microfluídica , Animais , Masculino , Ovinos , Microfluídica/métodos , Imunoensaio/métodos , Testes Imediatos , Medições Luminescentes/métodos , Análise de Sequência com Séries de Oligonucleotídeos , Técnicas Analíticas Microfluídicas/métodos , Sistemas Automatizados de Assistência Junto ao LeitoRESUMO
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.
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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.
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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.
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Infecções por HIV/urina , Imunoensaio/instrumentação , Testes Imediatos , Urinálise/instrumentação , Custos e Análise de Custo , Infecções por HIV/diagnóstico , Humanos , Imunoensaio/economia , Urinálise/classificação , Urinálise/economiaRESUMO
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, â¼130 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.
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Técnicas Analíticas Microfluídicas , Plasma , Centrifugação , Filtração , Sistemas Automatizados de Assistência Junto ao Leito , Testes Imediatos , PorosidadeRESUMO
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.
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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.
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An integrated microfluidic system has been developed for rapid enumeration of CD4 + T lymphocytes at point-of-care (POC) settings. A concise microfluidic chip, which consists of three separate chambers, respectively, for reaction, detection and waste storage, is developed to automate CD4 detection. To simplify CD4 + T lymphocyte enumeration, a single polycarbonate bead immobilized with CD4 antibody is adopted by the microfluidic chip to capture the CD4 antigen in the lysed testing sample. Desired performance is achieved by actuating the single bead for efficient mixing, as well as transferring it between different reaction chambers to reduce non-specific reaction. A controllable external magnetic field is applied to drive the single bead with a built-in ferrous core for different purposes. Chemiluminescence reaction is implemented in an independent chamber to reduce non-specific binding of enzyme. A simple flow control strategy is adopted to conveniently release the waste reagent into the waste storage chamber by just opening the vent hole without actively pumping. A sensitive CCD camera is used to collect the reaction signal by taking picture from the single bead, and then the signal intensity is further analyzed for CD4 + T lymphocyte enumeration. Experimental results show that rapid, convenient, accurate and low-cost CD4 + T lymphocyte enumeration can be obtained with the developed microfluidic system at POC test.
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Contagem de Linfócito CD4 , Dispositivos Lab-On-A-Chip , Medições Luminescentes/métodos , Antígenos CD4 , Linfócitos T CD4-Positivos/citologia , Desenho de Equipamento , Humanos , Processamento de Imagem Assistida por Computador , Imunoensaio/instrumentação , Imunoensaio/métodos , Medições Luminescentes/instrumentação , Testes Imediatos , SmartphoneRESUMO
Formaldehyde is one of the most dangerous air pollutants, which can cause sick building syndrome. Thus, it is very crucial to precisely determine formaldehyde with a low cost and simple operation. In this paper, a smartphone-based microfluidic colorimetric sensor is devised for gaseous formaldehyde determination with high sensitivity and selectivity. Specifically, a novel microfluidic chip is proposed based on the 4-aminohydrazine-5-mercapto-1,2,4-triazole (AHMT) method to determine formaldehyde; the chip consists of two reagent reservoirs, one reaction reservoir and a mixing column. In this design to prevent the fluid from flowing out while letting the gas molecule in, a hydrophobic porous poly tetra fluoroethylene (PTFE) membrane is put on the top of the reaction reservoir. Using the microfluidic chip sensor, a smartphone-based formaldehyde determination system is developed, which makes the measuring process automated and simple. As per the experiment results, the limit-of-detection (LOD) of the system is as low as 0.01 ppm, which is much lower than the maximum exposure concentration (0.08 ppm) recommended by the World Health Organization (WHO). Moreover, the sensor is hardly affected by acetaldehyde, volatile organic compounds (VOCs) or acidic-alkaline, which shows great selectivity. Finally, the performance of the proposed sensor is verified by using it for the determination of formaldehyde in a newly decorated house.
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In order to evaluate the thiolysis of NBD (7-nitro-1,2,3-benzoxadiazole) amines for development of H2S probes, herein we investigated the reactivity and selectivity of a series of NBD amines for the first time. The piperazinyl- and piperidyl-based NBD probes could react efficiently with micromolar H2S in buffer (pH 7.4), while such NBD(S) (nitrobenzothiadiazole) derivatives showed much slow thiolysis even in the presence of millimolar H2S. Low reactivity was also observed for thiolysis of these ethylamino-, ethanolamino- and anilino-based NBD probes. Therefore, almost all NBD amines used in bioimaging should be stable, in consideration of the presence of only micromolar endogenous H2S in vivo. Moreover, the piperazinyl-NBD derivatives could be efficient in the development of fluorescent H2S probes and for directly visualizing H2S by paper-based detection.
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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.
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Técnicas Biossensoriais , Ressonância de Plasmônio de Superfície , Ressonância de Plasmônio de Superfície/métodos , Fator de Crescimento Epidérmico , Gefitinibe , Imagem Óptica , Interações MedicamentosasRESUMO
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.
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Imageamento Hiperespectral , Microfluídica , Microfluídica/métodos , Preparações Farmacêuticas , Técnicas de Cocultura , Análise de Célula ÚnicaRESUMO
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.
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Técnicas Analíticas Microfluídicas , Microfluídica , Humanos , Imunoglobulina G , RimRESUMO
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.
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COVID-19 , Microfluídica , COVID-19/diagnóstico , Humanos , Técnicas de Amplificação de Ácido Nucleico , Reação em Cadeia da Polimerase , SARS-CoV-2/genéticaRESUMO
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.
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Vírus da Febre Suína Africana , Febre Suína Africana , Febre Suína Africana/diagnóstico , Animais , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico/métodos , Sensibilidade e Especificidade , Ressonância de Plasmônio de Superfície , SuínosRESUMO
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).
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Refratometria , Ressonância de Plasmônio de Superfície , HumanosRESUMO
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.