RESUMO
Inflammatory bowel disease (IBD) is a term used to describe disorders that involve chronic inflammation in the gastrointestinal tract, affecting more than 6.8 million people worldwide. Biological therapy is used in the most severe cases of IBD where anti-tumour necrosis factor-alpha (TNF-α) antibodies are the first choice for a biological treatment. When administrated to patients, these antibodies interact with TNF-α, usually overexpressed in these diseases, neutralizing its biological activity. Because of the chronic nature of these diseases, a recurring administration of the therapeutic antibodies is required, thus making therapy monitorization essential for the correct management of these diseases. The aim of this work is the development of an enzyme-linked immunosorbent assay (ELISA) microfluidic biosensor to quantify the therapeutic antibodies in IBD patient plasma samples, where the commercial monoclonal antibody Infliximab (IFX) is used as a model target. By providing a faster and more accurate measurement of IFX, the proposed method leads to improved therapy scheduling and a reduced risk of endogenous anti-drug antibodies (ADAs) reducing the efficacy of the treatment. The time needed between sample insertion and result output for the microfluidic ELISA (mELISA) is 24 minutes, drastically shorter than the time required by the conventional ELISA (cELISA). The mELISA presented in this work has a LoD of 0.026 µg mL-1, while commercially available solutions provide a LoD of 0.15 µg mL-1. Results acquired by the mELISA are highly correlated with the results obtained from the cELISA (r = 0.998; R2 = 0.996; p < 0.0001), demonstrating the validity of the microfluidic approach for the quantification of IFX from patient plasma and its potential for use at the point-of-care (POC).
Assuntos
Doenças Inflamatórias Intestinais , Microfluídica , Anticorpos Monoclonais , Monitoramento de Medicamentos , Ensaio de Imunoadsorção Enzimática , Humanos , Doenças Inflamatórias Intestinais/diagnóstico , Doenças Inflamatórias Intestinais/tratamento farmacológico , Infliximab , Fator de Necrose Tumoral alfaRESUMO
The assurance of food and feed safety, including the identification and effective monitoring of multiple biological and chemical hazards, is a major societal challenge, given the increasing pace at which food commodities are demanded, produced and traded across the globe. Within this context, mycotoxins are globally widespread secondary fungal metabolites, which can contaminate crops either in the field or during storage and have serious human and animal health impacts such as carcinogenic, teratogenic and hepatotoxic effects. Therefore, their presence in a wide range of foods and feeds is strictly regulated, particularly in the European Union. In order to perform effective and routine monitoring of mycotoxin levels in the field prior to further processing, during transport or during processing, rapid, simple, portable and sensitive means of screening of regulated mycotoxins are in high demand. This review focuses on (1) discussing the relevance of mycotoxins and the standard approaches for their sampling and monitoring; and (2) compiling and discussing recent advances in miniaturized analytical tools for mycotoxin detection. This provides insights into current research efforts and opportunities to develop a truly integrated and fit-for-purpose analytical tool, suitable for use at critical points of the food, feed and raw material processing and distribution chains.
Assuntos
Ração Animal/análise , Contaminação de Alimentos/análise , Micotoxinas/análise , Animais , Microbiologia de Alimentos , Fungos , HumanosRESUMO
Multimodal ligands are synthetic molecules comprising multiple types of interactions that have been increasingly used for the capture of different biopharmaceutical compounds within complex biological mixtures. For monoclonal antibodies (mAbs) in particular, these ligands have shown the possibility of direct capture from cell culture supernatants in native conditions, as well as enhanced selectivity and affinity compared to traditional single-mode ligands. However, performing the capture of a target mAb using multimodal chromatography comes with the need for extensive optimization of the operating conditions, due to the multitude of interactions that can be promoted in parallel. In this work, a high-throughput microfluidic platform was developed for the optimization of chromatographic conditions regarding the capture of an anti-interleukin 8 mAb, using a multimodal ligand (2-benzamido-4-mercaptobutanoic acid), under a wide range of buffer pH and conductivities. The interaction of the ligand with the fluorescently labeled target mAb was also analyzed with respect to the individual contribution of the hydrophobic (phenyl) and electrostatic (carboxyl) moieties using fluorescence microscopy. The results were further validated at the macroscale using prepacked columns in standard chromatography assays, and recovery yield values of 94.6% ± 5.2% and 97.7% ± 1.5% were obtained under optimal conditions for the miniaturized and conventional approaches, respectively. In summary, this study highlights that a microfluidic-based approach is a powerful analytical tool to expedite the optimization process while using reduced reagent volumes (<50 µL), less resin (â¼70 nL), and delivering results in less than 1 min per assay condition.
Assuntos
Anticorpos Monoclonais/isolamento & purificação , Dimetilpolisiloxanos/química , Ensaios de Triagem em Larga Escala , Técnicas Analíticas Microfluídicas , Adsorção , Anticorpos Monoclonais/química , Ensaios de Triagem em Larga Escala/instrumentação , Ligantes , Técnicas Analíticas Microfluídicas/instrumentação , Estrutura Molecular , Tamanho da PartículaRESUMO
A hydrogenated amorphous silicon (a-Si:H) photosensor was explored for the quantitative detection of a HIV-1 virion infectivity factor (Vif) at a detection limit in the single nanomolar range. The a-Si:H photosensor was coupled with a microfluidic channel that was functionalized with a recombinant single chain variable fragment antibody. The biosensor selectively recognizes HIV-1 Vif from human cell extracts.
Assuntos
Infecções por HIV/diagnóstico , Infecções por HIV/virologia , HIV-1/isolamento & purificação , Técnicas Analíticas Microfluídicas/instrumentação , Silício/química , Produtos do Gene vif do Vírus da Imunodeficiência Humana/isolamento & purificação , Células HEK293 , Humanos , Óptica e Fotônica/instrumentaçãoRESUMO
The spatial and temporal control of biological species is essential in complex microfluidic biosystems. In addition, if the biological species is a cell, microfluidic handling must ensure that the cell's metabolic viability is maintained. The use of DEP for cell manipulation in microfluidics has many advantages because it is remote and fast, and the voltages required for cell trapping scale well with miniaturization. In this paper, the conditions for bacterial cell (Escherichia coli) trapping using a quadrupole electrode configuration in a PDMS microfluidic channel were developed both for stagnant and for in-flow fluidic situations. The effect of the electrical conductivity of the fluid, the applied electric field and frequency, and the fluid-flow velocity were studied. A dynamic exchange between captured and free-flowing cells during DEP trapping was demonstrated. The metabolic activity of trapped cells was confirmed by using E. coli cells genetically engineered to express green fluorescent protein under the control of an inducible promoter. Noninduced cells trapped by negative DEP and positive DEP were able to express green fluorescent protein minutes after the inducer was inserted in the microchannel system immediately after DEP trapping. Longer times of trapping prior to exposure to the inducer indicated first a degradation of the cell metabolic activity and finally cell death.
Assuntos
Sobrevivência Celular/fisiologia , Células Imobilizadas/metabolismo , Eletroforese/métodos , Escherichia coli/metabolismo , Técnicas Analíticas Microfluídicas/métodos , Células Imobilizadas/citologia , Simulação por Computador , Condutividade Elétrica , Escherichia coli/citologia , Proteínas de Fluorescência Verde/química , Proteínas Recombinantes/químicaRESUMO
The lack of biomimetic in vitro models capable of reproducing the complex architecture and the dynamic environment of the gastric mucosa, delay the development of diagnostic and therapeutic tools. Recent advances in microengineering made possible the fabrication of bioinspired microdevices capable of replicating the physiological properties of an organ, inside a microfluidics chip. Herein, a bioinspired stomach-on-a-chip (SoC) device is described, supporting peristalsis-like motion and reconstituting organ-level epithelial architecture and function. The device simulates the upper epithelial interface, representing the three innermost layers of the gastric mucosa, namely the epithelial barrier, the basement membrane and the lamina propria. The dynamic environment imparted by mechanical actuation of the flexible on-chip cell culture substrate, was the main driver in the development of epithelial polarization and differentiation traits characteristic of the native gastric mucosa, and allowed partial recapitulation of gastric barrier function. These traits were not affected by the addition of a mesenchymal population to the system, which was able to remodel the surrounding extracellular matrix, nor by the potential epithelial-mesenchymal cross-talk. The engineered platform highlights the importance of addressing the mechanical microenvironment of the native organ, to potentiate an organ-level response of the artificial tissue. The proposed SoC represents an appealing tool in personalized medicine, with bio-relevance for the study of gastric diseases and an alternative to current animal models.
Assuntos
Técnicas de Cultura de Células , Matriz Extracelular , Animais , Humanos , Matriz Extracelular/química , Microfluídica , Estômago , Dispositivos Lab-On-A-ChipRESUMO
In the last decade, there has been a growing interest in developing microfluidic systems as new scale-down models for accelerated and cost-effective biopharmaceutical process development. Nonetheless, the research in this field is still in its infancy and requires further investigation to simplify and accelerate the microfabrication process. In addition, integration of different label-free sensors into the microcolumn systems has utmost importance to minimize result discrepancies during the scale-up process. In this study, we developed a simple, low-cost integrated microcolumn (26 µl). Micromilling technology was employed to define the geometry and shape of microfluidic structures using poly(methylmethacrylate) (PMMA). The design of PMMA microstructure was transferred to polydimethylsiloxane (PDMS), and interdigitated planar microelectrodes (IDE) were integrated into the system. To evaluate the scalability of the developed microcolumn column, column performance was assessed and compared with a conventional 1-ml prepacked column. Computational Fluid Dynamics (CFD) studies were performed for both columns to understand the differences between theoretical and experimental results regarding retention time and peak broadening. Despite obtaining an acceptable asymmetric factor for the microcolumn (1.03 ± 0.02), the reduced plate height value was still higher than the recommended range with the value of 4.14 ± 0.18. Nevertheless, the consistency and significant improvement of microcolumn efficiency compared to previous studies provide the possibility of developing robust simulation tools for transferring acquired experimental data for larger-scale units.
Assuntos
Hidrodinâmica , Microtecnologia , Simulação por Computador , Impedância ElétricaRESUMO
One of the most significant challenges in capturing and detecting biomarkers is the choice of an appropriate biomolecular receptor. Recently, RNA G-quadruplexes emerged as plausible receptors due to their ability to recognize with high-affinity proteins. Herein, we have unveiled and characterized the capability of the precursor microRNA 149 to form a G-quadruplex structure and determined the role that some ligands may have in its folding and binding capacity to nucleolin. The G-quadruplex formation was induced by K+ ions and stabilized by ligands, as demonstrated by nuclear magnetic resonance and circular dichroism experiments. Surface plasmon resonance measurements showed a binding affinity of precursor microRNA 149 towards ligands in the micromolar range (10-5-10-6 M) and a strong binding affinity to nucleolin RNA-binding domains 1 and 2 (8.38 × 10-10 M). Even in the presence of the ligand PhenDC3, the binding remains almost identical and in the same order of magnitude (4.46 × 10-10 M). The molecular interactions of the RNA G-quadruplex motif found in precursor miRNA 149 (5'-GGGAGGGAGGGACGGG- 3') and nucleolin RNA-binding domains 1 and 2 were explored by means of molecular docking and molecular dynamics studies. The results showed that RNA G-quadruplex binds to a cavity between domains 1 and 2 of the protein. Then, complex formation was also evaluated through polyacrylamide gel electrophoresis. The results suggest that precursor microRNA 149/ligands and precursor microRNA 149/nucleolin RNA-binding domains 1 and 2 form stable molecular complexes. The in vitro co-localization of precursor microRNA 149 and nucleolin in PC3 cells was demonstrated using confocal microscopy. Finally, a rapid and straightforward microfluidic strategy was employed to check the ability of precursor microRNA 149 to capture nucleolin RNA-binding domains 1 and 2. The results revealed that precursor microRNA 149 can capture nucleolin RNA-binding domains 1 and 2 labeled with Fluorescein 5-isothiocyanate in a concentration-dependent manner, but PhenDC3 complexation seems to decrease the ability of precursor microRNA 149 to capture the protein. Overall, our results proved the formation of the G-quadruplex structure in the precursor microRNA 149 and the ability to recognize and detect nucleolin. This proof-of-concept study could open up a new framework for developing new strategies to design improved molecular receptors for capture and detection of nucleolin in complex biological samples.
Assuntos
Quadruplex G , MicroRNAs , Fosfoproteínas , Proteínas de Ligação a RNA , Linhagem Celular , Humanos , MicroRNAs/genética , Simulação de Acoplamento Molecular , Fosfoproteínas/genética , Proteínas de Ligação a RNA/genética , NucleolinaRESUMO
In recent years, there has been an increased interest in exploring the potential of micro-and mesoscale milling technologies for developing cost-effective microfluidic systems with high design flexibility and a rapid microfabrication process that does not require a cleanroom. Nevertheless, the number of current studies aiming to fully understand and establish the benefits of this technique in developing high-quality microsystems with simple integrability is still limited. In the first part of this study, we define a systematic and adaptable strategy for developing high-quality poly(methyl methacrylate) (PMMA)-based micromilled structures. A case study of the average surface roughness (Ra) minimization of a cuboid column is presented to better illustrate some of the developed strategies. In this example, the Ra of a cuboid column was reduced from 1.68 µm to 0.223 µm by implementing milling optimization and postprocessing steps. In the second part of this paper, new strategies for developing a 3D microsystem were introduced by using a specifically designed negative PMMA master mold for polydimethylsiloxane (PDMS) double-casting prototyping. The reported results in this study demonstrate the robustness of the proposed approach for developing microfluidic structures with high surface quality and structural integrability in a reasonable amount of time.
RESUMO
Organ-on-a-chip technology promises to revolutionize how pre-clinical human trials are conducted. Engineering an in vitro environment that mimics the functionality and architecture of human physiology is essential toward building better platforms for drug development and personalized medicine. However, the complex nature of these devices requires specialized, time consuming, and expensive fabrication methodologies. Alternatives that reduce design-to-prototype time are needed, in order to fulfill the potential of these devices. Here, a streamlined approach is proposed for the fabrication of organ-on-a-chip devices with incorporated microactuators, by using an adaptation of xurography. This method can generate multilayered, membrane-integrated biochips in a matter of hours, using low-cost benchtop equipment. These devices are capable of withstanding considerable pressure without delamination. Furthermore, this method is suitable for the integration of flexible membranes, required for organ-on-a-chip applications, such as mechanical actuation or the establishment of biological barrier function. The devices are compatible with cell culture applications and present no cytotoxic effects or observable alterations on cellular homeostasis. This fabrication method can rapidly generate organ-on-a-chip prototypes for a fraction of cost and time, in comparison to conventional soft lithography, constituting an interesting alternative to the current fabrication methods.
Assuntos
Técnicas de Cultura de Células/métodos , Elastômeros , Desenho de Equipamento/métodos , Dispositivos Lab-On-A-Chip , Linhagem Celular Tumoral , Humanos , VácuoRESUMO
Microfluidic strategies combined with transduction and electronic integration have the promise of enabling miniaturized, combinatorial assays at higher speeds and lower costs, while at the same time mimicking the local chemical concentrations and force fields of the cellular in vivo environment. In this chapter we introduce a microfluidic structure with hydrodynamic cell traps and a culture volume in the nanoliter range (50 nL), to quantitatively evaluate the transient calcium response of the endogenous Muscarinic type 1 receptor (M1) in HEK 293 T cells. The microfluidic fabrication protocol is described as well as a methodology to monitor the cell response in real time, after stimulation with M1 agonists (e.g., carbachol) and antagonists (e.g., Pirenzepine).
Assuntos
Cálcio/metabolismo , Carbacol/farmacologia , Microfluídica/métodos , Pirenzepina/farmacologia , Receptor Muscarínico M1/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Agonistas Colinérgicos/farmacologia , Células HEK293 , Humanos , Antagonistas Muscarínicos/farmacologia , Transdução de SinaisRESUMO
We have investigated the expression of nucleolin (NCL) in liquid biopsies of prostate cancer (PCa) patients and healthy controls to determine its correlation with tumor prognosis. To detect NCL we used a modified AS1411 aptamer designated by AS1411-N5. In presence of NCL, AS1411-N5 increases the fluorescence by assuming a G-quadruplex (G4) structure, while in the absence of NCL the fluorescence signal remains quenched. The structural characterization of AS1411-N5 was performed by biophysical studies, which demonstrated the formation of G4 parallel conformation in the presence of 100â¯mMâ¯K+ and the ability to recognize NCL with high affinity (KDâ¯=â¯138.1⯱â¯5.5â¯nM). Furthermore, the clinical relevance of NCL in PCa liquid biopsies was assessed by using an NCL-based ELISA assay. The protein was measured in the peripheral blood mononuclear cells (PBMCs) cell lysate of 158 individuals, including PCa patients and healthy individuals. The results depicted a remarkable increase of NCL levels in the PBMC's lysate of PCa patients (mean of 626.1â¯pg/mL whole blood) when compared to healthy individuals (mean of 198.5â¯pg/mL whole blood). The ELISA results also provided evidence for the usefulness of determining NCL levels in advanced PCa stages. Furthermore, a microfluidic assay showed the ability of AS1411-N5 in recognizing NCL in spiked human plasma samples.
Assuntos
Leucócitos Mononucleares , Fosfoproteínas/análise , Neoplasias da Próstata , Proteínas de Ligação a RNA/análise , Aptâmeros de Nucleotídeos , Humanos , Leucócitos Mononucleares/metabolismo , Masculino , Oligodesoxirribonucleotídeos , Neoplasias da Próstata/diagnóstico , NucleolinaRESUMO
Over the past decade significant progress has been found in the upstream production processes, shifting the main bottlenecks in current manufacturing platforms for biopharmaceuticals towards the downstream processing. Challenges in the purification process include reducing the production costs, developing robust and efficient purification processes as well as integrating both upstream and downstream processes. Microfluidic technologies have recently emerged as effective tools for expediting bioprocess design in a cost-effective manner, since a large number of variables can be evaluated in a small time frame, using reduced volumes and manpower. Their modularity also allows to integrate different unit operations into a single chip, and consequently to evaluate the effect of each stage on the overall process efficiency. This paper describes the development of a diffusion-based microfluidic device for the rapid screening of continuous chemical lysis conditions. The release of a recombinant green fluorescent protein (GFP) expressed in Escherichia coli (E. coli) was used as model system due to the simple evaluation of cell growth and product concentration by fluorescence. The concept can be further applied to any biopharmaceutical production platform. The microfluidic device was successfully used to test the lytic effect of both enzymatic and chemical lysis solutions, with lysis efficiency of about 60% and close to 100%, respectively, achieved. The microfluidic technology also demonstrated the ability to detect potential process issues, such as the increased viscosity related with the rapid release of genomic material, that can arise for specific lysis conditions and hinder the performance of a bioprocess. Finally, given the continuous operation of the lysis chip, the microfluidic technology has the potential to be integrated with other microfluidic modules in order to model a fully continuous biomanufacturing process on a chip.
Assuntos
Bactérias , Técnicas Analíticas Microfluídicas , Proteínas Recombinantes , Bactérias/química , Bactérias/citologia , Bactérias/metabolismo , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/metabolismo , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Proteínas Recombinantes/análise , Proteínas Recombinantes/metabolismoRESUMO
The optimization of chromatography ligands for the purification of biopharmaceuticals is highly demanded to meet the needs of the pharmaceutical industry. In the case of monoclonal antibodies (mAbs), synthetic ligands comprising multiple types of interactions (multimodal) provide process and economic advantages compared to protein-based affinity ligands. However, optimizing the operation window of these ligands requires the development of effective high-throughput screening platforms. Here, a novel microfluidics-based methodology to perform rapid and multiplexed screening of various multimodal ligands relative to their ability to bind different target molecules is demonstrated. The microfluidic structure comprises three individual chambers (≈8 nL each) packed with different types of chromatography beads in series with the feed flow. An artificial mixture composed of immunoglobulin G (IgG) and bovine serum albumin, labeled with different thiol-reactive neutral fluorescent dyes, is used as a model to quantitatively optimize the performance (yield and purity) of the separation. This approach can potentially be used as a predictive analytical tool in the context of mAb purification, allowing low consumption of molecules and providing results in <3 min. Furthermore, this versatile approach can potentially be extended not only with respect to the number of different resins and target molecules, but also for parallel analysis of multiple conditions.
Assuntos
Cromatografia/instrumentação , Imunoglobulina G/isolamento & purificação , Microfluídica/instrumentação , Corantes Fluorescentes/química , Ligantes , Soroalbumina Bovina/químicaRESUMO
The rapid and sensitive detection of specific nucleic acid sequences at the point-of-care (PoC) is becoming increasingly in demand for a variety of emergent biomedical applications ranging from infectious disease diagnostics to the screening of antimicrobial resistance. To meet such demand, considerable efforts have been invested towards the development of portable and integrated analytical devices combining microfluidics with miniaturized signal transducers. Here, we demonstrate the combination of rolling circle amplification (RCA)-based nucleic acid amplification with an on-chip size-selective trapping of amplicons on silica beads (~8 nL capture chamber) coupled with a thin-film photodiode (200â¯×â¯200⯵m area) fluorescence readout. Parameters such as the flow rate of the amplicon solution and trapping time were optimized as well as the photodiode measurement settings, providing minimum detection limits below 0.5 fM of targeted nucleic acids and requiring only 5⯵L of pre-amplified sample. Finally, we evaluated the analytical performance of our approach by benchmarking it against a commercial instrument for RCA product (RCP) quantification and further investigated the effect of the number of RCA cycles and elongation times (ranging from 10 to 120â¯min). Moreover, we provide a demonstration of the application for diagnostic purposes by detecting RNA from influenza and Ebola viruses, thus highlighting its suitability for integrated PoC systems.
Assuntos
Técnicas Biossensoriais , Ebolavirus/isolamento & purificação , RNA/isolamento & purificação , Sequência de Bases , Ebolavirus/química , Ebolavirus/genética , Fluorescência , Dispositivos Lab-On-A-Chip , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico , Sistemas Automatizados de Assistência Junto ao Leito , RNA/genética , Dióxido de Silício/químicaRESUMO
Colorectal cancer (CRC) is among the five most commonly diagnosed cancers worldwide, constituting 6% of all cancers and the third leading cause of cancer death. CRC is the third and second most frequent cancer in men and women worldwide, accounting for 14% and 13% of all cancer incidence rates, respectively. CRC incidence is decreasing in older populations, but it has been significantly rising worldwide in adolescents and adults younger than 50â¯years old. Significant advances in the screening methods and surgical procedures have been underlying the reduction of the CRC incidence rate in older populations. However, there is an urgent demand for the development of alternative effective therapeutic options to overcome advanced metastatic CRC, while preventing disease recurrence. This review addresses the immune and CRC biology, summarizing the recent advances on the immune and/or therapeutic regimens currently in clinical use. We will focus on the emerging role of nanotechnology in the development of combinational therapies targeting and thereby regulating the function of the major players in CRC progression and immune evasion.
Assuntos
Antineoplásicos/administração & dosagem , Neoplasias Colorretais/terapia , Imunoterapia , Nanotecnologia , Animais , Neoplasias Colorretais/imunologia , HumanosRESUMO
The field of microfluidics holds great promise for the development of simple and portable lab-on-a-chip systems. The use of capillarity as a means of fluidic manipulation in lab-on-a-chip systems can potentially reduce the complexity of the instrumentation and allow the development of user-friendly devices for point-of-need analyses. In this work, a PDMS microchannel-based, colorimetric, autonomous capillary chip provides a multiplexed and semi-quantitative immunodetection assay. Results are acquired using a standard smartphone camera and analyzed with a simple gray scale quantification procedure. The performance of this device was tested for the simultaneous detection of the mycotoxins ochratoxin A (OTA), aflatoxin B1 (AFB1) and deoxynivalenol (DON) which are strictly regulated food contaminants with severe detrimental effects on human and animal health. The multiplexed assay was performed approximately within 10min and the achieved sensitivities of<40, 0.1-0.2 and<10ng/mL for OTA, AFB1 and DON, respectively, fall within the majority of currently enforced regulatory and/or recommended limits. Furthermore, to assess the potential of the device to analyze real samples, the immunoassay was successfully validated for these 3 mycotoxins in a corn-based feed sample after a simple sample preparation procedure.
Assuntos
Aflatoxina B1/isolamento & purificação , Técnicas Biossensoriais , Ocratoxinas/isolamento & purificação , Tricotecenos/isolamento & purificação , Aflatoxina B1/química , Humanos , Imunoensaio/métodos , Dispositivos Lab-On-A-Chip , Ocratoxinas/química , Smartphone , Tricotecenos/químicaRESUMO
Portable, rapid, cost effective and simple analytical tools are in increasing demand to facilitate the routine monitoring of target chemical/biological compounds at the point-of-need. Such devices are highly relevant within the context of food safety, particularly concerning the screening of highly toxic and strictly regulated mycotoxins. To achieve ultrarapid detection of mycotoxins, namely aflatoxin B1, ochratoxin A and deoxynivalenol, at the point-of-need, a novel multiplexed bead-based microfluidic competitive immunosensor, coupled with an array of a-Si:H thin-film photodiodes for integrated fluorescence signal acquisition, is reported. Simultaneously measuring the initial binding rate for each analyte of the sample under analysis against an internal reference, this device provided limits of detection below 1 ng mL-1 for all mycotoxins in a single-step assay and within 1 minute after mixing the sample under analysis with a fluorescent conjugate. The compatibility of the device with the analysis of mycotoxins spiked in corn samples was further demonstrated after performing a sample preparation procedure based on aqueous two-phase extraction. The short times of analysis and sensitivities in the low ng mL-1 range make these devices potentially competitive with the lateral flow devices that are currently the standard for this application. Furthermore, this device architecture and concept is amenable of being expanded to other analytes in food safety, biomedical and other applications.
Assuntos
Imunofluorescência/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Micotoxinas/análise , Desenho de Equipamento , Imunofluorescência/métodos , Limite de Detecção , Modelos Lineares , Ligação Proteica , Reprodutibilidade dos Testes , Zea mays/química , Zea mays/microbiologiaRESUMO
The rapid detection of mycotoxins in feed samples is becoming an increasingly relevant challenge for the food production sector, in order to effectively enforce current regulations and assure food and feed safety. To achieve rapid mycotoxin detection, several biosensing strategies have been published, many reaching assay times of the order of a few minutes. However, the vast majority of these rely on sample preparation based on volatile organic solvents, often comprising complex multi-step procedures and devoid of clean-up and/or concentration effects. Here, a novel sample preparation methodology based on a green, non-toxic and inexpensive polyethylene glycol-sodium citrate aqueous two-phase system is reported, providing single-step extraction and concentration of three target mycotoxins within 20min: aflatoxin B1 (AFB1), ochratoxin A (OTA) and deoxynivalenol (DON). With point-of-need applications in mind, the extraction procedure was optimized and validated using a rapid multi-toxin microfluidic competitive immunoassay. The assay was successfully tested with spiked complex solid matrices including corn, soy, chickpea and sunflower-based feeds and limits of detection of 4.6ngg-1±15.8%, 24.1ngg-1±8.1% and 129.7ngg-1±53.1% (±CV) were obtained in corn for AFB1, OTA and DON, respectively. These sensitivities are fit-for-purpose at the required regulatory and recommended limits for animal feed, providing an effective and safe semi-quantitative mycotoxin analysis that can be performed in the field.
Assuntos
Aflatoxina B1/análise , Ração Animal/análise , Ocratoxinas/análise , Tricotecenos/análise , Aflatoxina B1/isolamento & purificação , Animais , Cicer/química , Citratos/química , Contaminação de Alimentos/análise , Imunoensaio , Técnicas Analíticas Microfluídicas , Ocratoxinas/isolamento & purificação , Polietilenoglicóis/química , Citrato de Sódio , Glycine max/química , Espectrofotometria , Tricotecenos/isolamento & purificação , Zea mays/químicaRESUMO
Antibodies and other protein products such as interferons and cytokines are biopharmaceuticals of critical importance which, in order to be safely administered, have to be thoroughly purified in a cost effective and efficient manner. The use of aqueous two-phase extraction (ATPE) is a viable option for this purification, but these systems are difficult to model and optimization procedures require lengthy and expensive screening processes. Here, a methodology for the rapid screening of antibody extraction conditions using a microfluidic channel-based toolbox is presented. A first microfluidic structure allows a simple negative-pressure driven rapid screening of up to 8 extraction conditions simultaneously, using less than 20µL of each phase-forming solution per experiment, while a second microfluidic structure allows the integration of multi-step extraction protocols based on the results obtained with the first device. In this paper, this microfluidic toolbox was used to demonstrate the potential of LYTAG fusion proteins used as affinity tags to optimize the partitioning of antibodies in ATPE processes, where a maximum partition coefficient (K) of 9.2 in a PEG 3350/phosphate system was obtained for the antibody extraction in the presence of the LYTAG-Z dual ligand. This represents an increase of approx. 3.7 fold when compared with the same conditions without the affinity molecule (K=2.5). Overall, this miniaturized and versatile approach allowed the rapid optimization of molecule partition followed by a proof-of-concept demonstration of an integrated back extraction procedure, both of which are critical procedures towards obtaining high purity biopharmaceuticals using ATPE.