Optofluidic multiplex detection of single SARS-CoV-2 and influenza A antigens using a novel bright fluorescent probe assay.

The urgency for the development of a sensitive, specific, and rapid point-of-care diagnostic test has deepened during the ongoing COVID-19 pandemic. Here, we introduce an ultrasensitive chip-based antigen test with single protein biomarker sensitivity for the differentiated detection of both severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A antigens in nasopharyngeal swab samples at diagnostically relevant concentrations. The single-antigen assay is enabled by synthesizing a brightly fluorescent reporter probe, which is incorporated into a bead-based solid-phase extraction assay centered on an antibody sandwich protocol for the capture of target antigens. After optimization of the probe release for detection using ultraviolet light, the full assay is validated with both SARS-CoV-2 and influenza A antigens from clinical nasopharyngeal swab samples (PCR-negative spiked with target antigens). Spectrally multiplexed detection of both targets is implemented by multispot excitation on a multimode interference waveguide platform, and detection at 30 ng/mL with single-antigen sensitivity is reported.

Optofluidic control of rodent learning using cloaked caged glutamate.

Glutamate is the major excitatory neurotransmitter in the brain, and photochemical release of glutamate (or uncaging) is a chemical technique widely used by biologists to interrogate its physiology. A basic prerequisite of these optical probes is bio-inertness before photolysis. However, all caged glutamates are known to have strong antagonism toward receptors of γ-aminobutyric acid, the major inhibitory transmitter. We have developed a caged glutamate probe that is inert toward these receptors at concentrations that are effective for photolysis with violet light. Pharmacological tests in vitro revealed that attachment of a fifth-generation (G5) dendrimer (i.e., cloaking) to the widely used 4-methoxy-7-nitro-indolinyl(MNI)-Glu probe prevented such off-target effects while not changing the photochemical properties of MNI-Glu significantly. G5-MNI-Glu was used with optofluidic delivery to stimulate dopamine neurons of the ventral tegmental area of freely moving mice in a conditioned place-preference protocol so as to mediate Pavlovian conditioning.

Optofluidic Sensor Based on Polymer Optical Microresonators for the Specific, Sensitive and Fast Detection of Chemical and Biochemical Species.

The accurate, rapid, and specific detection of DNA strands in solution is becoming increasingly important, especially in biomedical applications such as the trace detection of COVID-19 or cancer diagnosis. In this work we present the design, elaboration and characterization of an optofluidic sensor based on a polymer-based microresonator which shows a quick response time, a low detection limit and good sensitivity. The device is composed of a micro-racetrack waveguide vertically coupled to a bus waveguide and embedded within a microfluidic circuit. The spectral response of the microresonator, in air or immersed in deionised water, shows quality factors up to 72,900 and contrasts up to 0.9. The concentration of DNA strands in water is related to the spectral shift of the microresonator transmission function, as measured at the inflection points of resonance peaks in order to optimize the signal-over-noise ratio. After functionalization by a DNA probe strand on the surface of the microresonator, a specific and real time measurement of the complementary DNA strands in the solution is realized. Additionally, we have inferred the dissociation constant value of the binding equilibrium of the two complementary DNA strands and evidenced a sensitivity of 16.0 pm/µM and a detection limit of 121 nM.

In situ Detection of Cobaloxime Intermediates During Photocatalysis Using Hollow-Core Photonic Crystal Fiber Microreactors.

Hollow-core photonic crystal fibers (HC-PCFs) provide a novel approach for in situ UV/Vis spectroscopy with enhanced detection sensitivity. Here, we demonstrate that longer optical path lengths than afforded by conventional cuvette-based UV/Vis spectroscopy can be used to detect and identify the CoI and CoII states in hydrogen-evolving cobaloxime catalysts, with spectral identification aided by comparison with DFT-simulated spectra. Our findings show that there are two types of signals observed for these molecular catalysts; a transient signal and a steady-state signal, with the former being assigned to the CoI state and the latter being assigned to the CoII state. These observations lend support to a unimolecular pathway, rather than a bimolecular pathway, for hydrogen evolution. This study highlights the utility of fiber-based microreactors for understanding these and a much wider range of homogeneous photocatalytic systems in the future.

Real Time Water-In-Oil Emulsion Size Measurement in Optofluidic Channels.

In this work, we investigated a platform for real-time emulsion droplet detection and size measurement in optofluidic platforms. An 8.2 µm core diameter input optical fiber and a multi-mode Gradient Refractive Index (GRIN) output fiber were integrated into an acrylic microfluidic channel platform consisting of three layers. Water-in-oil emulsions were investigated, since relevant applications have emerged in the recent past for these types of emulsions, such as drug encapsulation as well as droplet-based Polymerase Chain Reaction (PCR) amplification of DNA, among others. The main contribution of this work is in understanding the main physical phenomena (i.e., total internal reflection, refraction, and interference) behind the complex transmittance pattern obtained for these droplets. For this purpose, a frequency domain electromagnetic wave propagation modelling of the structure using the Finite Element Method (FEM) was used along with experimental measurements.

Real-Time Measurement of Refractive Index Using 3D-Printed Optofluidic Fiber Sensor.

This work describes a 3D-printed optofluidic fiber sensor to measure refractive index in real time, combining a microfluidic system with an optical fiber extrinsic Fabry-Perot interferometer. The microfluidic chip platform was developed for this purpose through 3D printing. The Fabry-Perot cavity was incorporated in the microfluidic chip perpendicularly to the sample flow, which was of approximately 3.7 µL/s. The optofluidic fiber sensor platform coupled with a low-cost optical power meter detector was characterized using different concentrations of glucose solutions. In the linear regression analysis, the optical power shift was correlated with the refractive index and a sensitivity of -86.6 dB/RIU (r2 = 0.996) was obtained. Good results were obtained in terms of stability with a maximum standard deviation of 0.03 dB and a sensor resolution of 5.2 × 10-4 RIU. The feasibility of the optofluidic fiber sensor for dynamic analyses of refractive index with low sample usage was confirmed through real-time measurements.

A Novel Approach for the Creation of Electrically Controlled LC:PDMS Microstructures.

This work presents research on unique optofluidic systems in the form of air channels fabricated in PDMS and infiltrated with liquid crystalline material. The proposed LC:PDMS structures represent an innovative solution due to the use of microchannel electrodes filled with a liquid metal alloy. The latter allows for the easy and dynamic reconfiguration of the system and eliminates technological issues experienced by other research groups. The paper discusses the design, fabrication, and testing methods for tunable LC:PDMS structures. Particular emphasis was placed on determining their properties after applying an external electric field, depending on the geometrical parameters of the system. The conclusions of the performed investigations may contribute to the definition of guidelines for both LC:PDMS devices and a new class of potential sensing elements utilizing polymers and liquid crystals in their structures.

Optical Identification of Parenteral Nutrition Solutions Exploiting Refractive Index Sensing.

Parenteral artificial nutrition (PAN) is a lifesaving treatment for a large population of patients affected by different diseases, and it consists of intravenous injection of nutritive fluids by means of infusion pumps. Wrong PAN solutions are, unfortunately, often administered, thus threatening the patients' well-being. Here, we report an optofluidic label-free sensor that can distinguish PAN solutions on the basis of their volumetric refractive index (RI). In our system, a monochromatic light beam, generated by a laser diode, travels obliquely through a transparent, square-section polystyrene channel, is then back-reflected by a mirror, and finally exits the channel in a position that depends on the filling fluid RI. The displacement of the output light spot ΔXexperim is easily detected with a linear, 1-D position sensitive detector (PSD). We initially calibrated the sensor with water-glucose solutions demonstrating a sensitivity S = ΔXexperim/Δn = 13,960 µm/RIU. We then clearly distinguished six commercial PAN solutions, commonly administered to patients. To the best of our knowledge, this is the first reported healthcare sensing platform for remote contactless recognition of PAN fluids, which could be inserted into infusion pumps to improve treatment safety, by checking the compliance to the prescription of the fluid actually delivered to the patient.

Switching between laser-induced thermophoresis and thermal convection of liquid suspension in a microgap with variable dimension.

Phoretic motion of particles along a temperature gradient formed in a fluid, known as thermophoresis, often takes place under the influence of bulk motion caused by thermal convection. In this paper, using a laser heating method, the significance of two competing effects, that is, thermophoresis and thermal convection, for the particle transport in a liquid phase confined in a microgap is investigated experimentally by changing the gap size as a control parameter. It is found that there is a threshold of the gap size, above which the particles tend to accumulate around the heated spot, forming a ring-like particle distribution. On the contrary, if the gap size is below the threshold, the particles are depleted from the heated spot. Switching between these accumulation and depletion modes is expected to develop novel manipulation techniques.

Optofluidic Flow-Through Biosensor Sensitivity - Model and Experiment.

We present a model and simulation for predicting the detected signal of a fluorescence-based optical biosensor built from optofluidic waveguides. Typical applications include flow experiments to determine pathogen concentrations in a biological sample after tagging relevant DNA or RNA sequences. An overview of the biosensor geometry and fabrication processes is presented. The basis for the predictive model is also outlined. The model is then compared to experimental results for three different biosensor designs. The model is shown to have similar signal statistics as physical tests, illustrating utility as a pre-fabrication design tool and as a predictor of detection sensitivity.

Optofluidic Amplification-free Multiplex Detection of Viral Hemorrhagic Fevers.

Infectious disease outbreaks such as Ebola and other Viral Hemorrhagic Fevers (VHF) require low-complexity, specific, and differentiated diagnostics as illustrated by the recent outbreak in the Democratic Republic of Congo. Here, we describe amplification-free spectrally multiplex detection of four different VHF total RNA samples using multi-spot excitation on a multimode interference waveguide platform along with combinatorial fluorescence labeling of target nucleic acids. In these experiments, we observed an average of 8-fold greater fluorescence signal amplitudes for the Ebola total RNA sample compared to three other total RNA samples: Lake Victoria Marburg Virus, Ravn Marburg Virus, and Crimean-Congo Hemorrhagic Fever. We have attributed this amplitude amplification to an increased amount of RNA during synthesis of soluble glycoprotein in infection. This hypothesis is confirmed by single molecule detection of the total RNA sample after heat-activated release from the carrier microbeads. From these experiments, we observed at least a 5.3x higher RNA mass loading on the Ebola carrier microbeads compared to the Lake Victoria Marburg carrier microbeads, which is consistent with the known production of soluble glycoprotein during infection.

Greatly Enhanced Single Particle Fluorescence Detection Using High Refractive Index Liquid-Core Waveguides.

High sensitivity and easy integration with microfabrication techniques has made silicon photonics one of the leading technologies used to build biosensors for diagnostic applications. Here we introduce a new silicon dioxide based optofluidic platform having a planar solid-core (SC) waveguide orthogonally intersecting a liquid-core (LC) waveguide with high refractive index ZnI2 salt solution as core. This enables both more uniform collection of particle fluorescence by the core mode and its propagation to an off-chip detector. This approach results in ultra-high sensitivity performance, demonstrated by achieving 8X enhancement in signal-to-noise ratio, a 45x increase in detection efficiency, and a 100x lower detection limit of 80 aM of fluorescent nanobeads. This represents a key step towards an ultrasensitive biosensor system for analyzing pathogens at clinical concentrations.

Free-Space Excitation of Optofluidic Devices for Pattern-Based Single Particle Detection.

Optofluidic sensors have enabled single molecule sensing using planar, waveguide dependent multi-spot fluorescence excitation. Here, we demonstrate a new approach to single-particle fluorescence sensing using free-space, top-down illumination of liquid-core antiresonant reflecting optical waveguide (ARROW) devices using two different multi-spot excitation techniques. First, the liquid core ARROW waveguide is excited with a focused beam through a slit pattern milled into an opaque aluminum film, showing comparable performance for single bead fluorescence detection as in-plane, multi-mode interference waveguide based excitation. The second top-down illumination technique images the spot pattern from a Y-splitter SiO2 waveguide chip directly onto the detection device for efficient power utilization and circumventing the need for an opaque cover, producing a further 2.7x improvement in signal-to-noise ratio. The two top-down approaches open up new possibilities for chip-based optical particle sensing with relaxed alignment tolerances.

A New Mechanism of Light-Induced Bubble Growth to Propel Microbubble Piston Engine.

Radiation pressure refers to the momentum transfer of photons during light "particles" impacting a surface. The force is too small to drive microengines. Different from the classical radiation pressure, the indirect radiation pressure (Fm ) is introduced, coming from the momentum change of light-induced bubble expansion. Fm is shown to obey Fm ∼ (I·rb )2 , behaving faster growth of indirect radiation pressure versus light intensity I and bubble radius rb . An effective bubble size range is identified for Fm to suppress other forces for bubble in liquid. The top laser irradiation on nanofluid is used in this experiment. A well-defined bubble pulsating flow, being a new principle of bubble piston engine, is demonstrated. During pulse on (≈ns scale), Fm exceeding other forces generates an extremely large acceleration, which is three to four orders larger than the gravity acceleration, propelling the bubble traveling downward. During pulse off, the bubble is floating upward due to the nonexistence of Fm . In such a way, the piston engine sustains the oscillating ranges of 38-347 µm for bubble diameters and 2.7-457.9 µm for traveling distances of piston. This work is useful to manipulate bubble dynamics in solar energy systems, and can find various applications in optofluidics.

Hyperboloid-Drum Microdisk Laser Biosensors for Ultrasensitive Detection of Human IgG.

Whispering gallery mode (WGM) microresonators have been used as optical sensors in fundamental research and practical applications. The majority of WGM sensors are passive resonators that require complex systems, thereby limiting their practicality. Active resonators enable the remote excitation and collection of WGM-modulated fluorescence spectra, without requiring complex systems, and can be used as alternatives to passive microresonators. This paper demonstrates an active microresonator, which is a microdisk laser in a hyperboloid-drum (HD) shape. The HD microdisk lasers are a combination of a rhodamine B-doped photoresist and a silica microdisk. These HD microdisk lasers can be utilized for the detection of label-free biomolecules. The biomolecule concentration can be as low as 1 ag mL-1 , whereas the theoretical detection limit of the biosensor for human IgG in phosphate buffer saline is 9 ag mL-1 (0.06 aM ). Additionally, the biosensors are able to detect biomolecules in an artificial serum, with a theoretical detection limit of 9 ag mL-1 (0.06 aM ). These results are approximately four orders of magnitude more sensitive than those for the typical active WGM biosensors. The proposed HD microdisk laser biosensors show enormous detection potential for biomarkers in protein secretions or body fluids.

Droplet-based optofluidic systems for measuring enzyme kinetics.

The study of enzyme kinetics is of high significance in understanding metabolic networks in living cells and using enzymes in industrial applications. To gain insight into the catalytic mechanisms of enzymes, it is necessary to screen an enormous number of reaction conditions, a process that is typically laborious, time-consuming, and costly when using conventional measurement techniques. In recent times, droplet-based microfluidic systems have proved themselves to be of great utility in large-scale biological experimentation, since they consume a minimal sample, operate at high analytical throughput, are characterized by efficient mass and heat transfer, and offer high levels of integration and automation. The primary goal of this review is the introduction of novel microfluidic tools and detection methods for use in high-throughput and sensitive analysis of enzyme kinetics. The first part of this review focuses on introducing basic concepts of enzyme kinetics and describing most common microfluidic approaches, with a particular focus on segmented flow. Herein, the key advantages include accurate control over the flow behavior, efficient mass and heat transfer, multiplexing, and high-level integration with detection modalities. The second part describes the current state-of-the-art platforms for high-throughput and sensitive analysis of enzyme kinetics. In addition to our categorization of recent advances in measuring enzyme kinetics, we have endeavored to critically assess the limitations of each of these detection approaches and propose strategies to improve measurements in droplet-based microfluidics. Graphical abstract.

Enhanced Detection of Single Viruses On-Chip via Hydrodynamic Focusing.

Planar optofluidics provide a powerful tool for facilitating chip-scale light-matter interactions. Silicon-based liquid core waveguides have been shown to offer single molecule sensitivity for efficient detection of bioparticles. Recently, a PDMS based planar optofluidic platform was introduced that opens the way to rapid development and prototyping of unique structures, taking advantage of the positive attributes of silicon dioxide-based optofluidics and PDMS based microfluidics. Here, hydrodynamic focusing is integrated into a PDMS based optofluidic chip to enhance the detection of single H1N1 viruses on-chip. Chip-plane focusing is provided by a system of microfluidic channels to force the particles towards a region of high optical collection efficiency. Focusing is demonstrated and enhanced detection is quantified using fluorescent polystyrene beads where the coefficient of variation is found to decrease by a factor of 4 with the addition of hydrodynamic focusing. The mean signal amplitude of fluorescently tagged single H1N1 viruses is found to increase with the addition of focusing by a factor of 1.64.

Controlling the Trajectories of Nano/Micro Particles Using Light-Actuated Marangoni Flow.

The ability to manipulate small objects and to produce patterns on the nano- and microscale is of great importance, both with respect to fundamentals and technological applications. The manipulation of particles with diameters of the order of 100 nm or below is a challenge because of their Brownian motion but also because of the scaling behavior of methods such as optical trapping. The unification of optical and hydrodynamic forces is a potential route toward the manipulation of tiny objects. Herein we demonstrate the trapping and manipulation of nano- and microparticles based on interfacial flows controlled by visible light, a method we denote as "Light-Actuated Marangoni Tweezer (LAMT)". We experimentally study the manipulation of particles having diameters ranging from 20 nm to 10 µm, including quantum dots and polystyrene nano/microparticles. The particles can be manipulated by scanning a light beam along a liquid surface. In this way, we are able to define almost arbitrary particle trajectories, for example, in the form of letters. In addition, we are able to handle a number of particles in parallel by creating an optical "landscape" consisting of a multitude of laser spots. The inherent advantages of LAMTs are the linear scaling of the trapping force with the particle diameter and the fact that the force is less dependent on particle properties than in the case of conventional methods.

Creating Multifunctional Optofluidic Potential Wells for Nanoparticle Manipulation.

Optical forces have enabled various nanomanipulation in microfluidics such as optical trapping, sorting, and transporting of nanoparticles (NPs), but the manipulation is usually specific with a certain optical field. Tightly focused Gaussian beams can trap NPs but not sort them; moderately focused Gaussian beams allow sorting microparticles in a flow but not NPs; quasi-Bessel beams can sort NPs in a flow but cannot control their positions due to low trapping stiffness. All these methods rely on the axial variation of laser intensity. Here we show that multifunctional and tunable optofluidic potential wells can be created for nanomanipulation by synchronizing optical phase gradient force with fluid drag force. We demonstrate controlled trapping and transporting of 150 nm Ag NPs over 10 µm and sorting of 80 and 100 nm Au NPs using optical line traps with tunable phase gradients in experiments. Our simulations further predict that simultaneous sorting and trapping of sub-50 nm Au NPs can be achieved with a sorting resolution of 1 nm using optimized optical fields. Our method provides great freedom and flexibility for nanomanipulation in optofluidics with potential applications in nanophotonics and biomedicine.

Designable 3D Microshapes Fabricated at the Intersection of Structured Flow and Optical Fields.

3D structures with complex geometric features at the microscale, such as microparticles and microfibers, have promising applications in biomedical engineering, self-assembly, and photonics. Fabrication of complex 3D microshapes at scale poses a unique challenge; high-resolution methods such as two-photon-polymerization have print speeds too low for high-throughput production, while top-down approaches for bulk processing using microfabricated template molds have limited control of microstructure geometries over multiple axes. Here, a method for microshape fabrication is presented that combines a thermally drawn transparent fiber template with a masked UV-photopolymerization approach to enable biaxial control of microshape fabrication. Using this approach, high-resolution production of complex microshapes not producible using alternative methods is demonstrated, such as octahedrons, dreidels, and axially asymmetric fibers, at throughputs as high as 825 structures/minute. Finally, the fiber template is functionalized with conductive electrodes to enable hierarchical subparticle localization using dielectrophoretic forces.