RESUMO
E6 and E7 oncogenes are pivotal in the carcinogenic transformation in HPV infections and efficient diagnostic methods can ensure the detection and differentiation of HPV genotype. This study describes the development and validation of an electrochemical, label-free genosensor coupled with a microfluidic system for detecting the E6 and E7 oncogenes in cervical scraping samples. The nanostructuring employed was based on a cysteine and graphene quantum dots layer that provides functional groups, surface area, and interesting electrochemical properties. Biorecognition tests with cervical scraping samples showed differentiation in the voltammetric response. Low-risk HPV exhibited a lower biorecognition response, reflected in ΔI% values of 82.33 % ± 0.29 for HPV06 and 80.65 % ± 0.68 for HPV11 at a dilution of 1:100. Meanwhile, high-risk, HPV16 and HPV18, demonstrated ΔI% values of 96.65 % ± 1.27 and 93 % ± 0.026, respectively, at the same dilution. Therefore, the biorecognition intensity followed the order: HPV16 >HPV18 >HPV06 >HPV11. The limit of detection and the limit of quantification of E6E7 microfluidic LOC-Genosensor was 26 fM, and 79.6 fM. Consequently, the E6E7 biosensor is a valuable alternative for clinical HPV diagnosis, capable of detecting the potential for oncogenic progression even in the early stages of infection.
Assuntos
Técnicas Biossensoriais , Proteínas Oncogênicas Virais , Técnicas Biossensoriais/métodos , Humanos , Proteínas Oncogênicas Virais/genética , Feminino , Limite de Detecção , Proteínas E7 de Papillomavirus/genética , Colo do Útero/virologia , Grafite/química , Infecções por Papillomavirus/diagnóstico , Infecções por Papillomavirus/virologia , Técnicas Eletroquímicas/métodos , Proteínas Repressoras/genética , Técnicas Analíticas Microfluídicas/métodos , Técnicas Analíticas Microfluídicas/instrumentação , Pontos Quânticos/química , Dispositivos Lab-On-A-Chip , Papillomaviridae/genética , Papillomaviridae/isolamento & purificaçãoRESUMO
An innovative integrated paper-based microdevice was developed for protein separation by isoelectric focusing (IEF), allowing for robust design thanks to a 3D-printed holder integrating separation channel, reservoirs, and electrodes. To reach robustness and precision, the optimization focused on the holder geometry, the paper nature, the reservoir design, the IEF medium, and various focusing parameters. A well-established and stable pH gradient was obtained on a glass-fiber paper substrate with simple sponge reservoirs, and the integration of the electrodes in the holder led to a straightforward system. The separation medium composed of water/glycerol (85/15, v/v) allowed for reducing medium evaporation while being an efficient medium for most hydrophobic and hydrophilic proteins, compatible with mass spectrometry detection for further proteomics developments. To our knowledge, this is the first report of the use of glycerol solutions as a separation medium in a paper-based microdevice. Analytical performances regarding pH gradient generation, pI determination, separation efficiency, and resolution were estimated while varying the IEF experimental parameters. The overall process led to an efficient separation within 25 min. Then, this methodology was applied to a sample composed of saliva doped with proteins. A minimal matrix effect was evidenced, underscoring the practical viability of our platform. This low-cost, versatile and robust paper-based IEF microdevice opens the way to various applications, ranging from sample pre-treatment to integration in an overall proteomic-on-a-chip device.
Assuntos
Glicerol , Focalização Isoelétrica , Papel , Proteínas , Focalização Isoelétrica/instrumentação , Focalização Isoelétrica/métodos , Proteínas/análise , Proteínas/isolamento & purificação , Glicerol/química , Glicerol/análise , Concentração de Íons de Hidrogênio , Desenho de Equipamento , Humanos , Dispositivos Lab-On-A-Chip , Saliva/química , Técnicas Analíticas Microfluídicas/instrumentação , Proteômica/métodos , Interações Hidrofóbicas e HidrofílicasRESUMO
Leukocyte count is routinely performed for diagnostic purposes and is rapidly emerging as a significant biomarker for a wide array of diseases. Additionally, leukocytes have demonstrated considerable promise in novel cell-based immunotherapies. However, the direct retrieval of leukocytes from whole blood is a significant challenge due to their low abundance compared to erythrocytes. Here, we introduce a microfluidic-based platform that isolates and recovers leukocytes from diluted whole blood in a single step. Our platform utilizes a novel, sheathless method to initially sediment and focus blood cells into a dense stream while flowing through a tubing before entering the microfluidic device. A hexagonal-shaped structure, patterned at the device's inlet, directs all the blood cells against the channel's outer walls. The focused cells are then separated based on their size using the deterministic lateral displacement (DLD) microfluidic technique. We evaluated various parameters that could influence leukocyte separation, including different focusing structures (assessed both computationally and experimentally), the orientation of the tubing-chip interface, the effects of blood sample hematocrit (dilution), and flow rate. Our device demonstrated the ability to isolate leukocytes from diluted blood with a separation efficiency of 100%, a recovery rate of 76%, and a purity of 80%, while maintaining a cell viability of 98%. The device operates for over 30 min at a flow rate of 2 µL min-1. Furthermore, we developed a handheld pressure controller to drive fluid flow, enhancing the operability of our platform outside of central laboratories and enabling near-patient testing. Our platform can be integrated with downstream cell-based assays and analytical methods that require high leukocyte purity (80%), ranging from cell counting to diagnostics and cell culture applications.
Assuntos
Separação Celular , Leucócitos , Técnicas Analíticas Microfluídicas , Leucócitos/citologia , Humanos , Técnicas Analíticas Microfluídicas/instrumentação , Separação Celular/instrumentação , Desenho de Equipamento , Dispositivos Lab-On-A-ChipRESUMO
Microfluidic paper-based analytical devices (µPADs) allow user-friendly and portable chemical determinations, although they provide limited applicability due to insufficient sensitivity. Several approaches have been proposed to address poor sensitivity in µPADs, but they frequently require bulky equipment for power and/or read-outs. Universal serial buses (USB) are an attractive alternative to less portable power sources and are currently available in many common electronic devices. Here, USB-powered µPADs (USB µPADs) are proposed as a fusion of both technologies to improve performance without adding instrumental complexity. Two ITP USB µPADs were developed, both powered by a 5 V potential provided through standard USB ports. The first device was fabricated using the origami approach. Its operation was analyzed experimentally and numerically, yielding a two-order-of-magnitude sample focusing in 15 min. The second ITP USB µPAD is a novel design, which was numerically prototyped with the aim of handling larger sample volumes. The reservoirs were moved away from the ITP channel and capillary action was used to drive the sample and electrolytes to the separation zone, predicting 25-fold sample focusing in 10 min. USB µPADs are expected to be adopted by minimally-trained personnel in sensitive areas like resource-limited settings, the point-of-care and in emergencies.
Assuntos
Isotacoforese/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Papel , Fontes de Energia Elétrica , Eletrólitos/química , Desenho de EquipamentoRESUMO
We report a microfluidic immunosensor for the electrochemical determination of IgG antibodies anti-Toxocara canis (IgG anti-T. canis). In order to improve the selectivity and sensitivity of the sensor, core-shell gold-ferric oxide nanoparticles (AuNPs@Fe3O4), and ordered mesoporous carbon (CMK-8) in chitosan (CH) were used. IgG anti-T. canis antibodies detection was carried out using a non-competitive immunoassay, in which excretory secretory antigens from T. canis second-stage larvae (TES) were covalently immobilized on AuNPs@Fe3O4. CMK-8-CH and AuNPs@Fe3O4 were characterized by transmission electron microscopy, scanning electron microscopy, energy dispersive spectrometry, cyclic voltammetry, electrochemical impedance spectroscopy, and N2 adsorption-desorption isotherms. Antibodies present in serum samples immunologically reacted with TES, and then were quantified by using a second antibody labeled with horseradish peroxidase (HRP-anti-IgG). HRP catalyzes the reduction from H2O2 to H2O with the subsequent oxidation of catechol (H2Q) to p-benzoquinone (Q). The enzymatic product was detected electrochemically at _100â¯mV on a modified sputtered gold electrode. The detection limit was 0.10â¯ngâ¯mL-1, and the coefficients of intra- and inter-assay variation were less than 6%, with a total assay time of 20â¯min. As can be seen, the electrochemical immunosensor is a useful tool for in situ IgG antibodies anti-T. canis determination.
Assuntos
Anticorpos Anti-Helmínticos/imunologia , Ouro/química , Nanopartículas Metálicas/química , Técnicas Analíticas Microfluídicas/instrumentação , Toxocara canis/imunologia , Toxocaríase/imunologia , Animais , Anticorpos Anti-Helmínticos/sangue , Técnicas Biossensoriais/instrumentação , Carbono/química , Técnicas Eletroquímicas/instrumentação , Desenho de Equipamento , Óxido Ferroso-Férrico/química , Humanos , Imunoensaio/instrumentação , Limite de Detecção , Porosidade , Toxocaríase/sangueRESUMO
Exosomes are a specific subpopulation of extracellular vesicles that have gained interest because of their many potential biomedical applications. However, exosome isolation and characterization are the first steps toward designing novel applications. This work presents a direct current-insulator-based dielectrophoretic (DC-iDEP) approach to simultaneously capture and separate exosomes by size. To do so, a microdevice consisting of a channel with two electrically insulating post sections was designed. Each section was tailored to generate different nonuniform spatial distributions of the electric field and, therefore, different dielectrophoretic forces acting on exosomes suspended in solution. Side channels were placed adjacent to each section to allow sample recovery. By applying an electric potential difference of 2000 V across the length of the main channel, dielectrophoretic size-based separation of exosomes was observed in the device. Analysis of particle size in each recovered fraction served to assess exosome separation efficiency. These findings show that iDEP can represent a first step toward designing a high-throughput, fast, and robust microdevice capable of capturing and discriminating different subpopulations of exosomes based on their size.
Assuntos
Eletroforese/instrumentação , Exossomos , Técnicas Analíticas Microfluídicas/métodos , Eletroforese/métodos , Técnicas Analíticas Microfluídicas/instrumentação , Tamanho da PartículaRESUMO
Tristeza is a disease that affects citrus crops in general, caused by the Citrus tristeza virus (CTV). It is considered an economically important virus diseases in citrus, which is present in the main citrus producing regions all around the world. Early detection of CTV is crucial to avoid any epidemics and substantial economic losses for the citrus growers. Consequently, the development of rapid, accurate, and sensitive methods capable of detecting the virus in the early stages of the disease is highly desired. Based on that, a low-cost and rapid magneto-immunoassay methodology to detect the capsid protein from CTV (CP-CTV) was proposed. For this, magnetic beads were decorated with antibodies anti-CP-CTV and horseradish peroxidase enzyme (HRP) and applied for the capture and separation of CP-CTV from the sample solutions. The magnetically captured biomarker was detected using a simple disposable microfluidic electrochemical device (DµFED) constructed by rapid prototyping technique and composed by an array of immunosensors. In DµFED, the electrodes were modified with monoclonal antibody anti-CP-CTV and the detection was carried out using amperometry, based on the hydroquinone/H2O2 catalytic redox reaction due to the presence of HRP label in an immune-sandwich structure. The proposed immunoassay presented excellent linearity with a wide linear range of concentration of 1.95-10.0â¯×â¯103â¯fgâ¯mL-1 and ultralow detection limit of 0.3â¯fgâ¯mL-1. The disposable device was successfully applied for the detection of Citrus tristeza virus in healthy and infected plant samples, where it showed good agreements with the comparative method of enzyme-linked immunosorbent assay (ELISA). The developed immunoassay methodology showed a sensitive and selective way in the detection of CTV. Hence, it can be considered as a promising analytical alternative for rapid and low-cost diagnosis of Tristeza disease in citrus.
Assuntos
Closterovirus/isolamento & purificação , Dispositivos Lab-On-A-Chip , Animais , Anticorpos Imobilizados/imunologia , Anticorpos Monoclonais Murinos/imunologia , Proteínas do Capsídeo/análise , Proteínas do Capsídeo/imunologia , Citrus/virologia , Técnicas Eletroquímicas/instrumentação , Técnicas Eletroquímicas/métodos , Eletrodos , Ouro/química , Imunoensaio/métodos , Separação Imunomagnética/métodos , Limite de Detecção , Nanopartículas Metálicas/química , Camundongos , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Doenças das Plantas/virologia , Folhas de Planta/virologia , Reprodutibilidade dos TestesRESUMO
An electronic tongue (e-tongue) is a multisensory system employed in the analysis of liquid samples, transforming the raw data into specific recognition patterns through computational and statistical analysis. Distinct types of e-tongues have been reported in the literature, with a plethora of applications in several areas of research. Recently, e-tongues have been integrated into microfluidic devices, which offer advantages such as the use of continuous flow for faster and more accurate analysis, and reduction in size of the devices and volumes for sampling and discharge, which in turn reduces waste and cost. Here we describe the procedures and methodologies recently used in our research group in the development of a microfluidic e-tongue sensing system.
Assuntos
Nariz Eletrônico , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/instrumentação , Dimetilpolisiloxanos/química , Eletrodos , Ouro/química , Nanotecnologia/métodosRESUMO
This study describes for the first time the development of 3D printed microfluidic devices with integrated electrodes for label-free counting of E. coli cells incorporated inside droplets based on capacitively coupled contactless conductivity detection (C4D). Microfluidic devices were fully fabricated by 3D printing in the T-junction shape containing two channels for disperse and continuous phases and two sensing electrodes for C4D measurements. The disperse phase containing E. coli K12 cells and the continuous phase containing oil and 1% Span® 80 were pumped through flow rates fixed at 5 and 60 µLâ¯min-1, respectively. The droplets with incorporated cells were monitored in the C4D system applying a 500-kHz sinusoidal wave with 1 Vpp amplitude. The generated droplets exhibited a spherical shape with average diameter of 321⯱â¯9⯵m and presented volume of 17.3⯱â¯0.5â¯nL. The proposed approach demonstrated ability to detect E. coli cells in the concentration range between 86.5 and 8650â¯CFU droplet-1. The number of cells per droplet was quantified through the plate counting method and revealed a good agreement with the Poisson distribution. The limit of detection achieved for counting E. coli cells was 63.66 CFU droplet-1. The label-free counting method has offered instrumental simplicity, low cost, high sensitivity and compatibility to be integrated on single microfluidic platforms entirely fabricated by 3D printing, thus opening new possibilities of applications in microbiology.
Assuntos
Contagem de Células/métodos , Condutividade Elétrica , Técnicas Eletroquímicas/métodos , Escherichia coli K12/isolamento & purificação , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/métodos , Técnicas Eletroquímicas/instrumentação , Eletrodos , Limite de Detecção , Técnicas Analíticas Microfluídicas/instrumentação , Impressão TridimensionalRESUMO
Early diagnosis of cancer by biomarker detection has been widely studied since it can lead to an increase in patient survival rates. Magnetic nanoparticles (MNPs) play an important role in this field acting as a valuable tool in the biomarker immunocapture and detection. In this work, Co0.25Zn0.75Fe2O4 (CoZnFeONPs) nanoparticles were synthesized and applied as enzyme mimics of peroxidase-like catalysis in a disposable enzyme-free microfluidic immunoarray device (µID). The catalytic activity of CoZnFeONPs was evaluated by hydrogen peroxide detection using cyclic voltammetry and the apparent Michaelis-Menten constant was estimated by Lineweaver-Burk equation showing good Km values. In µID, the immunosensors were assembled with monoclonal antibody against CYFRA 21-1 covalently immobilized on graphene oxide previously deposited on the screen-printed carbon-based electrodes. Under optimized conditions, the method presented a good linear response for CYFRA 21-1 in the range of 3.9-1000â¯fgâ¯mL-1 achieving an ultralow limit of detection (LOD) of 0.19â¯fgâ¯mL-1. For comparison, Fe3O4 nanoparticles (FeONPs) was also synthetized and presented results slight inferior to that obtained with CoZnFeONPs. The methods developed using both MNPs exhibited countless advantages when compared with the immunosensors developed for CYFRA-21-1, previously reported in the literature. The methods were successful applied for the detection of CYFRA 21-1 in real serum samples of healthy and prostate cancer patients and showed good correlation with results obtained with the enzyme-linked immunosorbent assay (ELISA). The CoZnFeONPs associated with the disposable microfluidic immunoarray device provides a simple and effective method for biomarker detection that could satisfy the need for a low-cost and rapid test for early diagnosis of cancer.