Microfluidic Bioassays Research Articles

Flow-Through Carbon Nanofiber-Based Transducer for Inline Electrochemical Detection in Paper-Based Analytical Devices.

Point-of-care (POC) devices are rapid, simple, portable, inexpensive, and convenient, but typically they only deliver qualitative results when used in the form of a lateral flow assay (LFA). Electrochemical detection could improve their sensitivity and ensure quantitative detection; however, a breakthrough in material-based technology is needed. We demonstrate a new concept in which electrodes are directly embedded within the lateral flow, enabling flow-through and hence interaction with the entire sample. This is accomplished through laser-induced carbon nanofibers (LCNFs) made by electrospinning Matrimid into nanofiber mats with subsequent pyrolyzing of electrode structures through a CO2 laser. Their highly porous 3D structure and superior graphene-like electrochemical properties are ideally suited for flow-through electrochemical LFA (EC-LFA), where the LCNFs are simply added in line with the other membranes. After optimization of the setup, biological binding assays typical for LFA diagnostics were successfully implemented, enabling the highly sensitive and quantitative detection of 137 pM DNA target sequences of a pathogenic organism that rivals the performance of pump-controlled microfluidic bioassays. This demonstrates that LCNF-based transducers can transform paper-based diagnostic tests to enable precise, quantitative analysis without reliance on cost-intensive read-out systems.

Digital microfluidics for biological analysis and applications.

Digital microfluidics (DMF) is an emerging liquid-handling technology based on arrays of microelectrodes for the precise manipulation of discrete droplets. DMF offers the benefits of automation, addressability, integration and dynamic configuration ability, and provides enclosed picoliter-to-microliter reaction space, making it suitable for lab-on-a-chip biological analysis and applications that require high integration and intricate processes. A review of DMF bioassays with a special emphasis on those actuated by electrowetting on dielectric (EWOD) force is presented here. Firstly, a brief introduction is presented on both the theory of EWOD actuation and the types of droplet motion. Subsequently, a comprehensive overview of DMF-based biological analysis and applications, including nucleic acid, protein, immunoreaction and cell assays, is provided. Finally, a discussion on the strengths, challenges, and potential applications and perspectives in this field is presented.

Affordable mobile microfluidic diagnostics: minimum requirements for smartphones and digital imaging for colorimetric and fluorometric anti-dengue and anti-SARS-CoV-2 antibody detection

Background: Miniaturised bioassays permit diagnostic testing near the patient, and the results can be recorded digitally using inexpensive cameras including smartphone and mobile phone cameras. Although digital cameras are now inexpensive and portable, the minimum performance required for microfluidic diagnostic bioassays has not been defined. We present a systematic comparison of a wide range of different digital cameras for capturing and measuring results of microfluidic bioassays and describe a framework to specify performance requirements to quantify immunoassays. Methods: A set of 200 µm diameter microchannels was filled with a range of concentrations of dyes used in colorimetric and fluorometric enzyme immunoassays. These were imaged in parallel using cameras of varying cost and performance ranging from <£30 to >£500. Results: Higher resolution imaging allowed larger numbers of microdevices to be resolved and analysed in a single image. In contrast, low quality cameras were still able to quantify results but for fewer samples. In some cases, an additional macro lens was added to focus closely. If image resolution was sufficient to identify individual microfluidic channels as separate lines, all cameras were able to quantify a similar range of concentrations of both colorimetric and fluorometric dyes. However, the mid-range cameras performed better, with the lowest cost cameras only allowing one or two samples to be quantified per image. Consistent with these findings, we demonstrate that quantitation (to determine endpoint titre) of antibodies against dengue and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viruses is possible using a wide range of digital imaging devices including the mid-range smartphone iPhone 6S and a budget Android smartphone costing <£50. Conclusions: In conclusion, while more expensive and higher quality cameras allow larger numbers of devices to be simultaneously imaged, even the lowest resolution and cheapest cameras were sufficient to record and quantify immunoassay results.

Facile fabrication of plasmonic wettability contrast paper surface for droplet array-based SERS sensing

A facile and cost-effective method was developed to create superhydrophobic surfaces onto a superhydrophilic commercial paper for the application of droplet splitting of microliter droplets into nano and sub-nano-liter droplets. The high wettability contrast of nearly 180° between the superhydrophobic region created via candle soot coated onto the patterned poly (dimethylsiloxane) and the paper causes the self-partitioning of a 1 µl droplet while sliding through the surface with a speed ranging from 0.001 to 7.3 mm/s. Additionally, the splitting of the droplet demonstrated for µl droplets of surface tension ranging from 72 mN/m to 38.5 mN/m. The plasmonic droplet assay for surface-enhanced Raman spectroscopy was fabricated via in-situ reduction of Ag nanoparticles onto the superhydrophilic region. The Raman scattering studies of methyl orange using the prepared substrates exhibit a limit of detection of 670 pM. Further, by using methyl orange as a probe molecule, selective detection of Hg2+ ions up to a concentration of 100 pM in nanoliter droplet volume is demonstrated. It is envisaged that these cost-effective plasmonic nanoliter droplet assays created via patterned superhydrophilic-superhydrophobic can find applications in high throughput screening, microfluidic bioassays, medical diagnosis.

Affordable mobile microfluidic diagnostics: minimum requirements for smartphones and digital imaging for colorimetric and fluorometric anti-dengue and anti-SARS-CoV-2 antibody detection.

Background: Miniaturised bioassays permit diagnostic testing near the patient, and the results can be recorded digitally using inexpensive cameras including smartphone and mobile phone cameras. Although digital cameras are now inexpensive and portable, the minimum performance required for microfluidic diagnostic bioassays has not been defined. We present a systematic comparison of a wide range of different digital cameras for capturing and measuring results of microfluidic bioassays and describe a framework to specify performance requirements to quantify immunoassays. Methods: A set of 200 µm diameter microchannels was filled with a range of concentrations of dyes used in colorimetric and fluorometric enzyme immunoassays. These were imaged in parallel using cameras of varying cost and performance ranging from <£30 to >£500. Results: Higher resolution imaging allowed larger numbers of microdevices to be resolved and analysed in a single image. In contrast, low quality cameras were still able to quantify results but for fewer samples. In some cases, an additional macro lens was added to focus closely. If image resolution was sufficient to identify individual microfluidic channels as separate lines, all cameras were able to quantify a similar range of concentrations of both colorimetric and fluorometric dyes. However, the mid-range cameras performed better, with the lowest cost cameras only allowing one or two samples to be quantified per image. Consistent with these findings, we demonstrate that quantitation (to determine endpoint titre) of antibodies against dengue and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viruses is possible using a wide range of digital imaging devices including the mid-range smartphone iPhone 6S and a budget Android smartphone costing <£50. Conclusions: In conclusion, while more expensive and higher quality cameras allow larger numbers of devices to be simultaneously imaged, even the lowest resolution and cheapest cameras were sufficient to record and quantify immunoassay results.

Microfluidic and Microscale Assays to Examine Regenerative Strategies in the Neuro Retina.

Bioengineering systems have transformed scientific knowledge of cellular behaviors in the nervous system (NS) and pioneered innovative, regenerative therapies to treat adult neural disorders. Microscale systems with characteristic lengths of single to hundreds of microns have examined the development and specialized behaviors of numerous neuromuscular and neurosensory components of the NS. The visual system is comprised of the eye sensory organ and its connecting pathways to the visual cortex. Significant vision loss arises from dysfunction in the retina, the photosensitive tissue at the eye posterior that achieves phototransduction of light to form images in the brain. Retinal regenerative medicine has embraced microfluidic technologies to manipulate stem-like cells for transplantation therapies, where de/differentiated cells are introduced within adult tissue to replace dysfunctional or damaged neurons. Microfluidic systems coupled with stem cell biology and biomaterials have produced exciting advances to restore vision. The current article reviews contemporary microfluidic technologies and microfluidics-enhanced bioassays, developed to interrogate cellular responses to adult retinal cues. The focus is on applications of microfluidics and microscale assays within mammalian sensory retina, or neuro retina, comprised of five types of retinal neurons (photoreceptors, horizontal, bipolar, amacrine, retinal ganglion) and one neuroglia (Müller), but excludes the non-sensory, retinal pigmented epithelium.

Rapid EC50 determination of hydrophobic toxicants in continuous droplet biomicrofluidics

Abstract Droplet-based bioassays are attractive for increasing throughput while minimizing reagent consumption. However, the choice of continuous oil phase within the bioassay can substantially alter assay outcomes when hydrophobic reagents and analytes dynamically partition between the oil and aqueous phases. Fluorinated continuous phases have been recognized as a better alternative to hydrocarbons to prevent leakage from droplets but variations among them has not been quantitatively addressed and analysis is complicated by the presence of surfactants. Herein, surface modification strategies to avoid surfactants are demonstrated with fluorocarbon oils for the first time. Perfluorocarbons, hydrofluoroether and hexadecane are quantitatively compared based on their aqueous distribution coefficient with a model industrial toxicant, pentachlorophenol (PCP). PCP partition in the oil phase skews cytotoxicity evaluation, wherein a bacterial inhibition assay displays a wide range of EC50 values for the same toxicant. Prevention of PCP extraction in a real-time viability assay reveals a 40-fold difference in LD50 toxicity vs. hexadecane control. Theoretical and empirical approaches to limit bioassay partitioning are established for expansion outside cell viability analyses employed herein. Structure activity relationships of surface modification and oil-water partition are related to Hamaker constants and Hansen solubility parameters towards translation on disposable plastics beside poly(methyl acrylate). The results provide a theoretical basis towards design and selection of continuous phase oils and microfluidic substrates to achieve a range of aqueous/oil partitioning for droplet microfluidic bioassays.

Laser Inscription of Microfluidic Devices for Biological Assays.

A rapid and direct CO2 laser ablation method was developed to create superhydrophilic surfaces and arrays of hydrophobic-superhydrophilic patterns for application in bioassays. Here, a combination of superhydrophilic and hemiwicking wetting characteristics was exploited to create microfluidic slides that were used as biological assays that prevented cell aggregation. This feature allowed microscopic analyses to be carried out at the individual cell level. This bioassay enabled control of cell population in localized areas (15 cells cm-2). The device had 84% transparency, allowing direct fluorescence microscopy measurements in transmission mode. High adhesion of aqueous fluids on superhydrophilic areas surrounded by superhydrophobic boundaries provided selective retention and confinement. The adhered droplets maintained retention under 180° substrate tilt. These architectures provided rapid self-partitioning of the liquid into an array of droplets. The hydrophobic-superhydrophilic patterned arrays may have applications in microfluidic bioassays, high-throughput screening, and medical diagnostics.

Anti-myeloma activity and molecular logic operation by Natural Killer cells in microfluidic droplets

Immune-targeted therapies that activate effector lymphocytes such as Natural Killer (NK) cells are currently being investigated for the treatment of Multiple myeloma (MM), the second most common form of hematological cancer. However, individual NK cells are highly heterogeneous in their cytolytic potential, making it difficult to detect, quantify and correlate the outcome of dynamic effector-target cell interactions at single cell resolution. Here, we present a microfluidic bioassay platform capable of activity-based screening of cellular and molecular immunotherapies. We identified distinct functional signatures associated with NK-MM cell interaction. The addition of immunomodulatory drug lenalidomide altered responses of NK-susceptible MM cells but not that of NK-tolerant MM cells. Antitumor cytotoxicity was significantly increased by the blockade of PD1/PDL1 axis as well as the clinically relevant cell line NK92, which were used to construct molecular logic functions (AND and NOT gates). A predictive agent-based mathematical model was developed to simulate progressive disease states and drug efficacy. The findings of the current study validate the applicability of this microfluidic cytotoxicity assay for immunotherapy screening, biocomputation and for future employment in detection of patient-specific cell response for precision medicine.

Development of a Microfluidic System Comprising Dialysis and Secretion Components for a Bioassay of Renal Clearance.

We have developed a microfluidic bioassay system that mimics glomerular filtration and tubular secretion in the kidney. The system consists of a peristaltic micropump (heart), a dialysis component (renal corpuscle), and a secretion component (renal proximal tubule). Analytes were separated by size using a dialysis membrane in the dialysis component. Model cells were cultured on a membrane in the secretion component, and active transport mediated by P-glycoprotein (P-gp) was confirmed using the P-gp substrate rhodamine 123 with or without the P-gp inhibitor quinidine sulfate. The system achieved both size separation and selective transport by P-gp on a single microchip. This proof-of-concept model may find applications in drug excretion assays, including studies of drug-drug interactions during tubular secretion.

Disposable luciferase-based microfluidic chip for rapid assay of water pollution.

In the present study, we demonstrate the use of a disposable luciferase-based microfluidic bioassay chip for environmental monitoring and methods for fabrication. The designed microfluidic system includes a chamber with immobilized enzymes of bioluminescent bacteria Photobacterium leiognathi and Vibrio fischeri and their substrates, which dissolve after the introduction of the water sample and thus activate bioluminescent reactions. Limits of detection for copper (II) sulfate, 1,3-dihydroxybenzene and 1,4-benzoquinone for the proposed microfluidic biosensor measured 3μM, 15mM, and 2μM respectively, and these values are higher or close to the level of conventional environmental biosensors based on lyophilized bacteria. Approaches for entrapment of enzymes on poly(methyl methacrylate) (PMMA) plates using a gelatin scaffold and solvent bonding of PMMA chip plates under room temperature were suggested. The proposed microfluidic system may be used with some available luminometers and future portable luminescence readers.

Paper-Based Biochip Assays and Recent Developments: A Review

Biomolecules in human body serve as biomarker for diagnosis of diseases, and paper-based biochips have become of much interest for development of biomolecule sensing. Cellulose micro-/nanofiber matrices consisting of paper allow not only capillary-driven flow without external pumping owing to abundant micro-/nanopores but also three-dimensional (3D) hierarchical micro-/nanostructures as 3D templates for microfluidic bioassays. Besides, colloidal and thermal evaporated metal nanoparticles on cellulose fibers offer huge opportunities in nanoplasmonic biosensing. Here, we review paper-based biochips including microfluidic paper-based assays and nanoplasmonic biosensors and further discuss micro-/nanofabrication of paper-based biochips and their applications in biomolecule detection.

Real-Time Continuous Identification of Greenhouse Plant Pathogens Based on Recyclable Microfluidic Bioassay System.

The development of a real-time continuous analytical platform for the pathogen detection is of great scientific importance for achieving better disease control and prevention. In this work, we report a rapid and recyclable microfluidic bioassay system constructed from oligonucleotide arrays for selective and sensitive continuous identification of DNA targets of fungal pathogens. We employ the thermal denaturation method to effectively regenerate the oligonucleotide arrays for multiple sample detection, which could considerably reduce the screening effort and costs. The combination of thermal denaturation and laser-induced fluorescence detection technique enables real-time continuous identification of multiple samples (<10 min per sample). As a proof of concept, we have demonstrated that two DNA targets of fungal pathogens (Botrytis cinerea and Didymella bryoniae) can be sequentially analyzed using our rapid microfluidic bioassay system, which provides a new paradigm in the design of microfluidic bioassay system and will be valuable for chemical and biomedical analysis.

Double Sweeping: Highly Effective Sample Preconcentration Using Cationic and Anionic Micelles and Its Application to a Multiple Enzyme Activity Assay.

In conventional microfluidic bioassays, short optical path length and extremely small quantities of analytes sometimes can significantly reduce detectability. Various sample preconcentration techniques have been reported for improving the detectability of bioassays. In the present study, we developed a novel preconcentration technique, "double sweeping", utilizing cationic and anionic micelles simultaneously. Microscopic observations demonstrated that double sweeping enabled a sample solution, which initially filled a capillary, to be focused into an extremely narrow band. Initially, the sample molecules are swept from cathode to anode, or anode to cathode, based on conventional sweeping with an anionic or cationic micellar solution, respectively. Then, the fronts of the swept bands collide with each other in the capillary and halt at the interface between the bands. The sample molecules in the micellar solutions continue to move toward the interface because of the electrophoretic migration of the micelles, which results in further focusing and suppression of the band broadening due to molecular diffusion. We demonstrated that higher preconcentration efficiency was achieved using double sweeping than using conventional sweeping. In addition, double sweeping was successfully incorporated into a multiple enzyme activity assay using an arrayed reagent-release capillary, resulting in simple, rapid, simultaneous, and highly sensitive assays of caspase-3, alkaline phosphatase, and trypsin.

An electrochemical-sensor system for real-time flow measurements in porous materials.

Flow monitoring in porous materials is critical for the engineering of paper-based microfluidic bioassays. Here, we present an electrochemical-sensor system that monitors the liquid flow in porous materials without affecting the real flow in paper-strip samples. The developed microfluidic sensor records an amperometric signal created by the solution movement mediated by paper wicking. This approach allows the in situ monitoring of the different hydrodynamic conditions of a specific paper geometry or composition. In addition, the method proposed in this work was employed to characterise the fluid flow of different nitrocellulose paper strips after oxygen-plasma treatment or dextran coating. The dextran fluid-flow modifiers were further used on the paper strip-based assays as means of signal enhancement. The proposed electrochemical-sensing method offers a valuable alternative to existing optical-based monitoring techniques for flow measurement in paper-based microfluidic systems.

User-friendly 3D bioassays with cell-containing hydrogel modules: narrowing the gap between microfluidic bioassays and clinical end-users' needs

Cell-containing hydrogel modules as cell-hydrogel microunits for creating a physiologically relevant 3D in vivo-like microenvironment with multiple cell types and unique extracellular matrix (ECM) compositions facilitate long-term cell maintenance and bioassays. To date, there have been many important advances in microfluidic bioassays, which incorporate hydrogel scaffolds into surface-accessible microchambers, driven by the strong demand for the application of spatiotemporally defined biochemical stimuli to construct in vivo-like conditions and perform real-time imaging of cell-matrix interactions. In keeping with the trend of fostering collaborations among biologists, clinicians, and microfluidic engineers, it is essential to create a simpler approach for coupling cell-containing hydrogel modules and an automated bioassay platform in a user-friendly format. In this article, we review recent progress in hydrogel-incorporated microfluidics for long-term cell maintenance and discuss some of the simpler and user-friendly 3D bioassay techniques combined with cell-containing hydrogel modules that can be applied to mutually beneficial collaborations with non-engineers. We anticipate that this modular and user-friendly format interfaced with existing laboratory infrastructure will help address several clinical questions in ways that extend well beyond the current 2D cell-culture systems.

Femtosecond laser fabrication of monolithically integrated microfluidic sensors in glass.

Femtosecond lasers have revolutionized the processing of materials, since their ultrashort pulse width and extremely high peak intensity allows high-quality micro- and nanofabrication of three-dimensional (3D) structures. This unique capability opens up a new route for fabrication of microfluidic sensors for biochemical applications. The present paper presents a comprehensive review of recent advancements in femtosecond laser processing of glass for a variety of microfluidic sensor applications. These include 3D integration of micro-/nanofluidic, optofluidic, electrofluidic, surface-enhanced Raman-scattering devices, in addition to fabrication of devices for microfluidic bioassays and lab-on-fiber sensors. This paper describes the unique characteristics of femtosecond laser processing and the basic concepts involved in femtosecond laser direct writing. Advanced spatiotemporal beam shaping methods are also discussed. Typical examples of microfluidic sensors fabricated using femtosecond lasers are then highlighted, and their applications in chemical and biological sensing are described. Finally, a summary of the technology is given and the outlook for further developments in this field is considered.

Complex magnetic fields breathe life into fluids.

The vast majority of materials research exploits equilibrium or quasi-equilibrium processes to produce inert materials. In contrast, living systems depend on far-from-equilibrium kinetic processes that require a continuous flux of energy to persist and perform useful tasks. The Greek god Hephaestus forged metal automatons that he miraculously animated to perform the tasks of living creatures. Is something like this actually possible? Here we show that subjecting magnetic fluids suspended in an immiscible liquid to uniform, multidimensional, time-dependent magnetic fields, generates a variety of life-like collective dynamics, including various forms of locomotion, swarming and feeding, that are sustained by the continuous injection of energy via the applied field. These leaderless emergent behaviors occur autonomously, without human guidance, and are quite surprising. Such self-healing, remotely-powered fluid automatons could be used as an extraction/separation technology to efficiently purify water by scavenging toxic chemicals and microorganisms, or alternatively enable the controlled release of chemicals. Other possible applications include vigorous fluid mixing and even microdroplet manipulation for microfluidic bioassays.

Poly(oligoethylene glycol methacrylate) dip-coating: turning cellulose paper into a protein-repellent platform for biosensors.

The passivation of nonspecific protein adsorption to paper is a major barrier to the use of paper as a platform for microfluidic bioassays. Herein we describe a simple, scalable protocol based on adsorption and cross-linking of poly(oligoethylene glycol methacrylate) (POEGMA) derivatives that reduces nonspecific adsorption of a range of proteins to filter paper by at least 1 order of magnitude without significantly changing the fiber morphology or paper macroporosity. A lateral-flow test strip coated with POEGMA facilitates effective protein transport while also confining the colorimetric reporting signal for easier detection, giving improved performance relative to bovine serum albumin (BSA)-blocked paper. Enzyme-linked immunosorbent assays based on POEGMA-coated paper also achieve lower blank values, higher sensitivities, and lower detection limits relative to ones based on paper blocked with BSA or skim milk. We anticipate that POEGMA-coated paper can function as a platform for the design of portable, disposable, and low-cost paper-based biosensors.

Microflow injection potassium bioassay based on G-quadruplex DNAzyme-enhanced chemiluminescence.

By taking advantage of microflow injection chemiluminescence analysis, we developed a distinctive microfluidic bioassay method based on G-Quadruplex DNAzyme-enhanced chemiluminescence for the determination of K(+) in human serum. AGRO100, the G-rich oligonucleotide with high hemin binding affinity was primarily selected as a K(+) recognition element. In the presence of K(+), AGRO100 folded into G-quadruplex and bound hemin to form DNAzyme, which catalyzed the oxidation of luminol by H2 O2 to produce chemiluminescence. The intensity of chemiluminescence increased with the K(+) concentration. In the study, the DNAzyme showed both long-term stability and high catalytic activity; other common cations at their physiological concentration did not cause notable interference. With only 6.7 × 10(-13) mol of AGRO100 consumption per sample, a linear response of K(+) ranged from 1 to 300 µmol/L, the concentration detection limit 0.69 µmol/L (S/N = 3) and the absolute detection limit 1.38 × 10(-12) mol were obtained. The precision of 10 replicate measurements of 60 µmol/L K(+) was found to be 1.72% (relative standard deviation). The accuracy of the method was demonstrated by analyzing real human serum samples.