Series: Getting Started with Microfluidics and Becoming Expert in 3 Hours (Ep14)
Episode 14: Introduction to Optofluidics and Applications - 1
Hi. Welcome back to my article for microfluidics. This is the Episode 14 in the series "Getting Started with Microfluidics and Becoming Expert in 3 Hours." In this and the next episode, I will introduce the impressive research works using optofluidics. Please enjoy it.
The article series "Getting Started with Microfluidics and Becoming Expert in 3 Hours" is to popularize the knowlegde of microfluidics and the applications to people who is interesting in microfluidics but don't even know what it is and people who tries to expend their horizon with useful information and hope to developing a microfluidic platform. All articles focus more on the phenomena explanation and application introduction instead of formula calculation; therefore, the content is friendly to everyone. Don't hesitate to read them and feel no pressure. You will definitely like them. Here is the links to another articles in this series.
================Another articles in the series================
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Applied external fields: Optic field - 1
Optofluidics is a category of microfluidics that operated with light (or genarally EM waves), including absorption, transmission, emittion, excitation, refraction, reflection, fluorescence, scattering, interference, and other photo effects on molecules in fluids and/or the devices themselves.
*Most microfluidic chips require optical microscopes to observe, which might make the divide between optofluidics and other microfluidics ambiguous and hard to define. Hence, in this and the next article, I specially selected the microfluidic platforms which directly incorporated with optical elements or the platforms which designed to perform specific optical effects instead of just observation.
What can an "optofluidic chip" do?
In previous articles, I have introduced research works using chemical and/or enzymatic colorimetry methods to detect specific compounds. They measured the concentrations of the chemicals based on the color change, which can be quantified by spectrometer or a smartphone camera. Comparatively, this paper shows a device which was directly integrated with optical fibers for absorption/transmission measurement, as shown in the figure below. The figure is reproduced with permission from Li et al. (2020). Micromachines, 11(1), 59. Copyright © 1996-2021 MDPI (Basel, Switzerland). CC BY 4.0. This microfluidic chip was designed to monitor the total phosphorus in water, including 4 typical phosphorus compounds, including sodium glycerophosphate, disodium guanosine 5' -monophosphate (Na2GMP), tetrasodium pyrophosphate, and sodium tripolyphosphate. This chip consisted of 3 parts: the spiral on-chip digestion unit, the serial convergent-divergent mixing unit, and the Z-shaped optical detecting unit. A microheater was placed below the spiral channel to maintain the high temperature for digestive reaction. The sample, after digestive reaction, was then mixed with chromogenic compound. Afterward, the mixture flow through the Z-shaped flow cell, and the optical properties was measured with 2 optical fiber collimators (OFCs). The absorbance measurement is governed by the Beer–Lambert law. Here, the optical length was designed to be 10 mm. The optofluidic chip, integrated with on-chip pretreatment and real-time measurement, can conduct the analysis within 10-20 min.
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As the result of the Beer-Lambert equation, the absorbance measurement and the sensitivity is directly affected by the optical length. However, it is not easy to make a good use of space when tests are parallelized into a tiny planar microfluidic chip with the Z-shaped channel. To address it, researcher has tried to integrated Fabry−Pérot cavities to detect the absorbance signal. Fabry−Pérot interferometer is a optical cell with 2 parallel surfaces at high reflecting rate at both sides of the cell. The distance between 2 reflecting surfaces is precisely controlled, which is usually in the µm ranges. Light can pass through it only if its wavelength allows it to resonate in the cell.
The following figure shows a parallel detection of phosphates using gradient generating microchannels with a Fabry−Pérot interferometer array (FPA). The figure is reproduced with permission from Zhu et al. (2020). ACS sensors, 5(5), 1381-1388. Copyright © 2020 American Chemical Society. This sensing platform was divided into 3 parts: the gradient FPA chip, the circuit broads with LEDs and photodiodes, and the smartphone user interface. The sample to detect and the reagent were firstly loaded into the gradient generating channels. After the fluids were well mixed at individual ratio, the optical signals were measured by the LEDs and photodiodes, which were strictly aligned above and below the detecting areas. The signals were then transmitted to smartphone via a bluetooth module. According to the color changes in the mixture at different ratio, the original concentration can be calculated. This miniature platform required only 80 s to detect a sample.
In the following work, the researchers has developed a waveguide-based droplet determination method. The figure is reproduced with permission from Bettella et al. (2019). Sensors and Actuators B: Chemical, 282, 391-398. Copyright © 2018 Bettella et al. CC BY-NC-ND 4.0. The working principle is straight forward. The waveguide channels were designed perpendicular to the fluid channel, allowing the waveguide to illuminate the fluid and objects. Droplets interacted with the monochrome light (He-Ne laser) from a waveguide channel and the transmitted light was collected by another one. The output waveguide was then analyzed by near field technique. The transmittance of the continuous phase and the dispersed phase of the light is different because of the refraction index. The signal from the beginning and the end of the droplet can be clearly distinguished. Accordingly, the sizes and the shapes of droplets can be analyzed. Compared with using high speed camera, the on-chip waveguide method to count droplets showed 50% higher capability. Besides, the post-processes were not required.
Fluorescence is a common technique to label or detect targets in biotech labs. Here, a group of researchers have developed a microfluidic device incorporated with an array of microlenses, which was able to enhance 8-fold fluorescent signal. The figure is reproduced with permission from Lim et al. (2013). Lab on a Chip, 13(8), 1472-1475. Copyright © 2013 The Royal Society of Chemistry. CC BY 3.0. The microlenses were made by photolithogrphy and thermal annealing. The photoresist (AZ9260) was fabricated into cylinders on a glass substrate with masked UV exposure. After that, the lens array was heated to 150℃, allowing the photoresist to reshape into spherical cap due to surface tension. The lens array was then bonded to the microchannels whose channel walls were pre-coated with gold and silver as mirror. Once a droplet flowed through the place corresponding to a lens, the focused exciting light lighted up the droplet. The fluorescent signal emitted in all directions was then focused again by the lens, as shown in the following figure.
Evanescent wave (EW) is a near field effect of total reflection. According to the Snell's law, there is a upper limit of incident angle of waves transmitted from medium of higher refractive index to another of lower ones. When the incident angle is higher than the limit, no refraction will be observed in a far field scale. In the microscopic view (close to the wavelength), the transmitted energy decays rapidly as the distance from the interface increases. This allows researchers to develop biochemical assays based on evanescent waves due to the length scale of surface reactions.
An EW-based fluorescent detection microfluidic chips is shown in the following figure, which is reproduced with permission from Wang et al. (2016). Sensing and bio-sensing research, 7, 7-11. Copyright © 2015 Wang et al. CC BY-NC-ND 4.0. In this paper, the researchers demonstrated a fluorescent sensor chip which emitted and was excited by EW. The excitation light source and the detection apparatus were at the same end of the chip. The fused silica capillaries were served as the material which allows EW to propagate. There were 3 capillaries embedded in 1 PDMS slab, allowing parallel detection of different targets. In order to prevent the excitation light directly illuminate the capillaries, which was not in the form of EW, the detection end of the chip except the capillaries was blocked with carbon paste. The operating processes are as follows. The antibody-immobilized capillaries were incubated with target and fluorophore-labelled antibodies sequentially. The high aspect ratio (viewed from the end of capillaries) and the nature of the EW can improve the sensitivity because the design can collect signal from the whole capillary without loss.
Protein characterization is a series of measurement to determine the characteristics of proteins, including but not limited to the sizes, charges, concentrations, sequences, structures, functionalities, diffusivities, and interactions between other proteins and/or biomolecules. Microfluidics allow to perform experiments in technically precise and high-throughput manners. Optical setups provide versatile and convenient approaches to detect with high sensitivity in real time. Here I am going to show you examples for protein characterization using fluorescence and chemiluminescence signals, respectively.
It is important to distinguish the fluorescence and chemiluminescence. Fluorescence is a light emission due to light excitation, which means it always require 2 optical instruments: a light source for excitation and a photo detector. It only happens when electrons in substances or molecules are excited by absorbing a photon (or photons). Whilst the chemiluminescence emits light when the specific reaction happens. One of the products of the reaction is in the excited form. The light is emitted because the excited electrons return to their ground states. Chemiluminescence doesn't require the excitation part since the energy is from a reaction.
A microfluidic chip to analyze the size distribution and rheological properties of proteins has been demonstrated. The biophysical properties are important for pharmaceutical industries. The figure is reproduced with permission from Kopp et al. (2018). Industrial & Engineering Chemistry Research, 57(21), 7112-7120. Copyright © 2018 American Chemical Society. In this paper, colloidal solutions of proteins labelled with fluorescent dyes at high concentrations were studied. The protein solution was flow-focused with 2 sheath flows in the channel. The collides in the channel diffused perpendicular to the channel due to the Brownian motion. The distributions of collides along the channel were recorded using fluorescence. It was also applicable with UV absorption of proteins and/or instinct fluorescence. The size distribution of collides can be calculated by fitting diffusivities. They firstly verified the feasibility of the platform by introducing bovine serum albumin (BSA, known to form oligomers in concentrated solutions) and observed the strong self-interactions by measuring diffusion constant. After that, antibodies were studied. According to their finding, the protein-protein interaction between antibodies was not observed even at high concentration.
The following figure is reproduced with permission from Chiu et al. (2020). ChemRxiv. (This content is a preprint and has not been peer-reviewed.) Copyright © 2015 Chiu et al. CC BY-NC-ND 4.0. The protein of interest was labelled with benzoisoindole, a fluorogenic structure, and dissolved at desired concentrations in the hydrogen peroxide solution before being introduced into the chip. Bis-(2,4,6-trichlorophenyl) oxalate (TCPO) in acetonitrile (ACN) were injected to the microfluidic chip from another 2 inlets (i). The chemicals in the lanimar flow were quickly mixed in the zig-zag channel and reacted to each other. A series of reaction allowed the benzoisoindole to relax to its ground state, and at the same time to released a photon. By capturing the photons, the protein concentration was calculated. This method can be performed regardless of the protein used. Besides, comparing with UV absorbance, fluorescence-based methods, and traditional lab approaches, such as BCA and bradford, it provided a much higher sensitivity.
Apart from the properties of proteins mentioned above, the thermal stability of folded protein using was also characterized by capillary microfluidics. The figure is reproduced with permission from Sagar et al. (2013). Scientific reports, 3(1), 1-6. Copyright © 2021 Springer Nature Limited. CC BY 3.0. The thermal gradient in the capillary with or without flow was generated by applying infrared (IR) laser (pointed by the arrow) perpendicular to the capillary, which allowed more precise control than using Peltier or resistive heating elements. The temperature profile and the protein stability (here green-fluorescent protein GFP used) were measured using a 2-laser system with a temperature-sensitive dye and the fluorescent signal of GFP at the same time, respectively. This setup minimized the required solution volume and experiment time (at the scales of nL and ms to min) compared with traditional water bath methods (sub-mL and hours) and provided accurate thermodynamic parameters, including time and space-dependent thermal profiles of its folding states.
Finally here. Time for a break. In the following article, other optical techniques for other applications will be introduced continuously. I hope you like this episode and have found some materials that draw your attention. See you in the next episode.