Microfluid biosensor for detection of hpv in patient scraping samples: Determining E6 and E7 oncogenes.

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.

Trends and challenges in microfluidic methods for protein manipulation-A review.

Proteins are important molecules involved in an immensely large number of biological processes. Being capable of manipulating proteins is critical for developing reliable and affordable techniques to analyze and/or detect them. Such techniques would enable the production of therapeutic agents for the treatment of diseases or other biotechnological applications (e.g., bioreactors or biocatalysis). Microfluidic technology represents a potential solution to protein manipulation challenges because of the diverse phenomena that can be exploited to achieve micro- and nanoparticle manipulation. In this review, we discuss recent contributions made in the field of protein manipulation in microfluidic systems using different physicochemical principles and techniques, some of which are miniaturized versions of already established macro-scale techniques.

Comprehensive model of electromigrative transport in microfluidic paper based analytical devices.

A complete mathematical model for electromigration in paper-based analytical devices is derived, based on differential equations describing the motion of fluids by pressure sources and EOF, the transport of charged chemical species, and the electric potential distribution. The porous medium created by the cellulose fibers is considered like a network of tortuous capillaries and represented by macroscopic parameters following an effective medium approach. The equations are obtained starting from their open-channel counterparts, applying scaling laws and, where necessary, including additional terms. With this approach, effective parameters are derived, describing diffusion, mobility, and conductivity for porous media. While the foundations of these phenomena can be found in previous reports, here, all the contributions are analyzed systematically and provided in a comprehensive way. Moreover, a novel electrophoretically driven dispersive transport mechanism in porous materials is proposed. Results of the numerical implementation of the mathematical model are compared with experimental data, showing good agreement and supporting the validity of the proposed model. Finally, the model succeeds in simulating a challenging case of free-flow electrophoresis in paper, involving capillary flow and electrophoretic transport developed in a 2D geometry.

Droplet-Based Microfluidics Methods for Detecting Enzyme Inhibitors.

Sub-nanoliter droplets produced in microfluidic devices have gained an enormous importance for performing all kinds of biochemical assays. One of the main reasons is that the amounts of reagents employed can be reduced in approximately five orders of magnitude compared to conventional microplate assays. In this chapter, we describe how to carry out the design, fabrication, and operation of a microfluidic device that allows performing enzyme kinetics and enzyme inhibition assays in droplets. This procedure can be used effectively to screen a small size library of compounds. Then, we describe how to use this droplet microfluidic setup to screen for potential inhibitor compounds eluted from a coupled high-performance liquid chromatography (HPLC) system that separates crude natural extracts.

Electrokinetically Driven Exosome Separation and Concentration Using Dielectrophoretic-Enhanced PDMS-Based Microfluidics.

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.

Ultrasensitive immunoassay for detection of Citrus tristeza virus in citrus sample using disposable microfluidic electrochemical device.

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 1.95-10.0 × 103 fg mL-1 and ultralow detection limit of 0.3 fg 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.

Recent advances and challenges in the recovery and purification of cellular exosomes.

Exosomes are nanovesicles secreted by most cellular types that carry important biochemical compounds throughout the body with different purposes, playing a preponderant role in cellular communication. Because of their structure, physicochemical properties and stability, recent studies are focusing in their use as nanocarriers for different therapeutic compounds for the treatment of different diseases ranging from cancer to Parkinson's disease. However, current bioseparation protocols and methodologies are selected based on the final exosome application or intended use and present both advantages and disadvantages when compared among them. In this context, this review aims to present the most important technologies available for exosome isolation while discussing their advantages and disadvantages and the possibilities of being combined with other strategies. This is critical since the development of novel exosome-based therapeutic strategies will be constrained to the effectiveness and yield of the selected downstream purification methodologies for which a thorough understanding of the available technological resources is needed.

Label-free counting of Escherichia coli cells in nanoliter droplets using 3D printed microfluidic devices with integrated contactless conductivity detection.

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 µL 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 µm and presented volume of 17.3 ±â€¯0.5 nL. The proposed approach demonstrated ability to detect E. coli cells in the concentration range between 86.5 and 8650 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.

Novel enzyme-free immunomagnetic microfluidic device based on Co0.25Zn0.75Fe2O4 for cancer biomarker detection.

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 3.9-1000 fg mL-1 achieving an ultralow limit of detection (LOD) of 0.19 fg 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.

Milli-channel array for direct and quick reading of root elongation bioassays.

A novel platform to perform systematic analysis and direct reading of root elongation bioassays is presented. The device was designed to include multiplexed microenvironments for the germination and growth of individual seeds, which allows observation by the naked eye or by optical systems, notably cellphone cameras. Prototypes were fabricated by laser micromachining on a highly transparent material that is fully compatible with biological systems. The effectiveness of the milli-channel array was verified against the conventional system (Petri dish). Lactuca sativa was chosen as a model species and glyphosate as a typical toxic agent. All tests were run according to standardized procedures and data analysis was carried out through different statistical indicators such as the root elongation and germination indexes. Results attained in the milli-channel array were identical to those in Petri dish, with the remarkable benefit that several steps required in the conventional system were avoided, which enormously decreases the operation time and the possibility of experimental errors. Further advantages of the milli-channel array are also reported, such as the capability to achieve live imaging of plant organs growth through a simple experiment. The developed device has been proven to be effective, versatile, easy-to-use, and integrable to cellphones, which naturally provide facilities for data recording, analysis, and networking. These improvements open the route to novel applications of bioassays in the wide field of ecotoxicology and environmental studies.

Disposable Voltammetric Immunosensors Integrated with Microfluidic Platforms for Biomedical, Agricultural and Food Analyses: A Review.

Disposable immunosensors are analytical devices used for the quantification of a broad variety of analytes in different areas such as clinical, environmental, agricultural and food quality management. They detect the analytes by means of the strong interactions between antibodies and antigens, which provide concentration-dependent signals. For the herein highlighted voltammetric immunosensors, the analytical measurements are due to changes in the electrical signals on the surface of the transducers. The possibility of using disposable and miniaturized immunoassays is a very interesting alternative for voltammetric analyses, mainly, when associated with screen-printing technologies (screen-printed electrodes, SPEs), and microfluidic platforms. The aim of this paper is to discuss a carefully selected literature about different examples of SPEs-based immunosensors associated with microfluidic technologies for diseases, food, agricultural and environmental analysis. Technological aspects of the development of the voltammetric immunoassays such as the signal amplification, construction of paper-based microfluidic platforms and the utilization of microfluidic devices for point-of-care testing will be presented as well.

Effects of diffusion and mixing pattern on microfluidic-assisted synthesis of chitosan/ATP nanoparticles.

Chitosan (CHI) nanoparticles present promising applications in pharmaceutical and biomedical fields, including drug and gene delivery. Among different approaches, microfluidics emerges as a resourceful tool for nanoparticle production in low-cost, reproducible processes with predictable fluid dynamics. However, microfluidic-assisted synthesis of CHI nanoparticles has not been widely explored in the literature. In this context, we systematically investigated different process parameters that influence the synthesis of CHI/ATP nanoparticles. We highlight the effects and limitations of diffusion and distinct mixing patterns developed through the microchannels on the final physicochemical characteristics of CHI/ATP nanoparticles produced. To address these hurdles, here we describe a simple, feasible, and reproducible method for the production of CHI/ATP nanoparticles. This strategy enables the development of a continuous and homogeneous production process for CHI nanoparticles to be applied in the most varied fields of research.

Enhanced Performance of Colorimetric Biosensing on Paper Microfluidic Platforms Through Chemical Modification and Incorporation of Nanoparticles.

This chapter describes two different methodologies used to improve the analytical performance of colorimetric paper-based biosensors. Microfluidic paper-based analytical devices (µPADs) have been produced by a stamping process and CO2 laser ablation and modified, respectively, through an oxidation step and incorporation of silica nanoparticles on the paper structure. Both methods are employed in order to overcome the largest problem associated with colorimetric detection, the heterogeneity of the color distribution in the detection zones. The modification steps are necessary to improve the interaction between the paper surface and the selected enzymes. The enhanced performance has ensured reliability for quantitative analysis of clinically relevant compounds.

Dielectrophoretic behavior of PEGylated RNase A inside a microchannel with diamond-shaped insulating posts.

Ribonuclease A (RNase A) has proven potential as a therapeutic agent, especially in its PEGylated form. Grafting of PEG molecules to this protein yields mono-PEGylated (mono-PEG) and di-PEGylated (di-PEG) RNase A conjugates, and the unreacted protein. Mono-PEG RNase A is of great interest. The use of electrokinetic forces in microdevices represents a novel alternative to chromatographic methods to separate this specie. This work describes the dielectrophoretic behavior of the main protein products of the RNase A PEGylation inside a microchannel with insulators under direct current electric fields. This approach represents the first step in route to design micro-bioprocesses to separate PEGylated RNase A from unreacted native protein. The three proteins exhibited different dielectrophoretic behaviors. All of them experienced a marked streaming pattern at 3000 V consistent with positive dielectrophoresis. Native protein was not captured at any of the conditions tested, while mono-PEG RNase A and di-PEG RNase A were captured presumably due to positive dielectrophoresis at 4000 and 2500 V, respectively. Concentration of mono-PEG RNase A with a maximal enrichment efficiency of ≈9.6 times the feed concentration was achieved in few seconds. These findings open the possibility of designing novel devices for rapid separation, concentration, and recovery of PEGylated RNase A in a one-step operation.

Host-guest supramolecular chemistry in solid-state nanopores: potassium-driven modulation of ionic transport in nanofluidic diodes.

We describe the use of asymmetric nanopores decorated with crown ethers for constructing robust signal-responsive chemical devices. The modification of single conical nanopores with 18-crown-6 units led to a nanodevice whose electronic readout, derived from the transmembrane ion current, can be finely tuned over a wide range of K(+) concentrations. The electrostatic characteristics of the nanopore environment arising from host-guest ion-recognition processes taking place on the pore walls are responsible for tuning the transmembrane ionic transport and the rectification properties of the pore. This work illustrates the potential and versatility of host-guest chemistry, in combination with nanofluidic elements, as a key enabler to achieve addressable chemical nanodevices mimicking the ion transport properties and gating functions of specific biological channels.

A simple method for patterning poly(dimethylsiloxane) barriers in paper using contact-printing with low-cost rubber stamps.

This paper presents a simple and low-cost method for patterning poly(dimethylsiloxane) (PDMS) barriers in porous support such as paper for the construction of flexible microfluidic paper-based analytical devices (µPADs). The fabrication method consisted of contact-printing a solution of PDMS and hexane (10:1.5 w/w) onto chromatographic paper using custom-designed rubber stamps containing the patterns of µPADs. After penetrating the paper (∼30 s), the PDMS is cured to form hydrophobic barriers. Under optimized conditions, hydrophobic barriers and hydrophilic channels with dimensions down to 949±88 µm and 771±90 µm (n=5), respectively, were obtained. This resolution is well-suitable for most applications in analytical chemistry. Chemical compatibility studies revealed that the PDMS barriers were able to contain some organic solvents, including acetonitrile and methanol, and aqueous solutions of some surfactants. This find is particularly interesting given that acetonitrile and methanol are the most used solvents in chromatographic separations, non-aqueous capillary electrophoresis and electroanalysis, as well as aqueous solutions of surfactants are suitable mediums for cell lyses assays. The utility of the technique was evaluated in the fabrication of paper-based electrochemical devices (PEDs) with pencil-drawn electrodes for experiments in static cyclic voltammetry and flow injection analysis (FIA) with amperometric detection, in both aqueous and organic mediums.

Microfluidic Isolation of CD34-Positive Skin Cells Enables Regeneration of Hair and Sebaceous Glands In Vivo.

Skin stem cells resident in the bulge area of hair follicles and at the basal layer of the epidermis are multipotent and able to self-renew when transplanted into full-thickness defects in nude mice. Based on cell surface markers such as CD34 and the α6-integrin, skin stem cells can be extracted from tissue-derived cell suspensions for engraftment using the gold standard cell separation technique of fluorescence-activated cell sorting (FACS). This paper describes an alternative separation method using microfluidic devices coated with degradable antibody-functionalized hydrogels. The microfluidic method allows direct injection of tissue digestate (no preprocessing tagging of cells is needed), is fast (45 minutes from injected sample to purified cells), and scalable. This method is used in this study to isolate CD34-positive (CD34+) cells from murine skin tissue digestate, and the functional capability of these cells is demonstrated by transplantation into nude mice using protocols developed by other groups for FACS-sorted cells. Specifically, the transplantation of microfluidic isolated CD34+ cells along with dermal and epidermal cells was observed to generate significant levels of hair follicles and sebaceous glands consistent with those observed previously with FACS-sorted cells.

Continuous flow generation of magnetoliposomes in a low-cost portable microfluidic platform.

We present a low-cost, portable microfluidic platform that uses laminated polymethylmethacrylate chips, peristaltic micropumps and LEGO® Mindstorms components for the generation of magnetoliposomes that does not require extrusion steps. Mixtures of lipids reconstituted in ethanol and an aqueous phase were injected independently in order to generate a combination of laminar flows in such a way that we could effectively achieve four hydrodynamic focused nanovesicle generation streams. Monodisperse magnetoliposomes with characteristics comparable to those obtained by traditional methods have been obtained. The magnetoliposomes are responsive to external magnetic field gradients, a result that suggests that the nanovesicles can be used in research and applications in nanomedicine.

Hybrid microfluidic fuel cell based on Laccase/C and AuAg/C electrodes.

A hybrid glucose microfluidic fuel cell composed of an enzymatic cathode (Laccase/ABTS/C) and an inorganic anode (AuAg/C) was developed and tested. The enzymatic cathode was prepared by adsorption of 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and Laccase on Vulcan XC-72, which act as a redox mediator, enzymatic catalyst and support, respectively. The Laccase/ABTS/C composite was characterised by Fourier Transform Infrared (FTIR) Spectroscopy, streaming current measurements (Zeta potential) and cyclic voltammetry. The AuAg/C anode catalyst was characterised by Transmission electron microscopy (TEM) and cyclic voltammetry. The hybrid microfluidic fuel cell exhibited excellent performance with a maximum power density value (i.e., 0.45 mW cm(-2)) that is the highest reported to date. The cell also exhibited acceptable stability over the course of several days. In addition, a Mexican endemic Laccase was used as the biocathode electrode and evaluated in the hybrid microfluidic fuel cell generating 0.5 mW cm(-2) of maximum power density.

Recent advances in low-cost microfluidic platforms for diagnostic applications.

The use of inexpensive materials and cost-effective manufacturing processes for mass production of microfluidic devices is very attractive and has spurred a variety of approaches. Such devices are particularly suited for diagnostic applications in limited resource settings. This review describes the recent and remarkable advances in the use of low-cost substrates for the development of microfluidic devices for diagnostics and clinical assays. Thus, a plethora of new and improved fabrication methods, designs, capabilities, detections, and applications of microfluidic devices fabricated with paper, plastic, and threads are covered.