Microfluidic Biosensors

Microfluidic Biosensors provides a comprehensive overview covering the most recent emerging technologies on the design, fabrication, and integration of microfluidics with transducers. These form various integrated microfluidic biosensors with device configurations ranging from 2D to 4D levels. Coverage also includes advanced printed microfluidic biosensors, flexible microfluidics for wearable biosensors, autonomous lab-on-a-chip biosensors, CMOS-base microanalysis systems, and microfluidic devices for mobile phone biosensing. The editors and contributors of this book represent both academia and industry, come from a varied range of backgrounds, and offer a global perspective. This book discusses the design and principle of microfluidic systems and uses them for biosensing applications.

The microfluidic fabrication technologies covered in this book provide an up-to-date view, allowing the community to think of new ways to overcome challenges faced in this field. The focus is on existing and emerging technologies not currently being analyzed extensively elsewhere, providing a unique perspective and much-needed content. The editors have crafted this book to be accessible to all levels of academics from graduate students, researchers, and professors working in the fields of biosensors, microfluidics design, material science, analytical chemistry, biomedical devices, and biomedical engineering. It can also be useful for industry professionals working for microfluidic device manufacturers, or in the industry of biosensors and biomedical devices.

• Presents an in-depth overview of microfluidic biosensors and associated emerging technologies such as printed microfluidics and novel transducers
• Addresses a range of microfluidic biosensors with device configurations ranging from 2D to 4D levels
• Includes the commercialization aspects of microfluidic biosensors that provide insights for scientists and engineers in research and development

• Language: English
• Publisher: Academic Press
• Release date: Nov 5, 2022
• ISBN: 9780128238479

Book preview

Chapter 1

Printed microfluidic biosensors and their biomedical applications

Jacky Fong Chuen Loo¹,², Aaron Ho Pui Ho¹ and Wing Cheung Mak¹,³,    ¹Department of Biomedical Engineering, The Chinese University of Hong Kong, Shatin, Hong Kong,    ²Department of Neuroscience and Biomedical Engineering, School of Science, Aalto University, Aalto, Espoo, Finland,    ³Division of Sensor and Actuator Systems, Department of Physics, Chemistry and Biology (IFM), Biosensors and Bioelectronics Centre, Linköping University, Linköping, Sweden

Abstract

In the last few decades, printed microfluidics exhibited attractive unique advantages, such as fast turnaround time and high flexibility in customization and fabrication, showing potentials in on-site production and downstream biosensing. The advancement in technical methods of fabrication, assembly, and integration has simplified the construction of these biosensors without sacrificing their biosensing performance. Since their development in the 1990s, microfluidic technologies have enabled the processing of fluid on micron scale, hence reducing the need for a large volume of the sample, from milliliter in conventional biosensing methods to pico-or micro-liter. It also supports automation in fluidic actuation to realize sample-to-answer biosensing, an essential feature for its use as a point-of-care and point-of-use test. This chapter first gives a brief overview of printed microfluidic biosensors, an introduction to the origins of microfluidics, and types of printed microfluidics compared to traditional microfluidics. It then covers each type of printed microfluidics (2D, pseudo-3D, 3D, and 4D) and its corresponding fabrication and assembly methods. Next, it discusses different approaches to conjugating the recognition elements, such as nucleic acid probes and antibodies, into the printed microfluidics and the integration with actuators and signal detecting units to make functional biosensors. Optical and electrochemical detection techniques are our main focus. The chapter then highlights in-depth researches on using these biosensors for two biomedical applications, i.e., disease screening and food safety. Lastly, we discuss the outlook on the development of printed microfluidics. This chapter aims to provide the reader with the updated knowledge on the advanced technologies for constructing printed microfluidic biosensors and increasing awareness of the importance of these advanced techniques and their practical real-life applications.

Keywords

Printed microfluidic biosensors; biomedical applications; microfluidic biosensors; printed microfluidics; pseudo-3D microfluidics; functional biosensors

1.1 Introduction

1.1.1 The emerging need of microfluidic biosensors

Global health issues remain major challenges across the world, and rapid disease diagnostics is one of the keys to disease control and surveillance. Take the 2014–2016 Western African Ebola virus epidemic as an example. It is essential to differentiate the Ebola virus from other viruses which share similar symptoms and exclude false detection of co-circulating diseases, such as malaria, to curb the spread of this highly contagious virus. This called for an effective on-site diagnostic for multiplexed detection of the Ebola virus and other viruses. Since medical centers, resources, and transportation are scarce in West Africa, sending samples to centralized laboratories for tests is not always feasible. This hampered large-scale diagnosis while addressing the epidemic. Even though conventional rapid test kits were available, e.g., ELISA kit, handling of biological fluid samples, and processing of the reaction posed a risk to healthcare workers administering the assay or monitoring the readout. Commercial rapid tests with paper-based lateral flow assays on other diseases, such as HIV, tuberculosis, influenza, and Escherichia coli (E. coli)O157:H7 infection, are designed to be simple test strips. Still, they only provide a yes/no answer and test for a single disease [1]. Therefore, a more reliable diagnostic method for multiple diseases is desired [2].

Biosensor is an integrated self-contained receptor–transducer system, which is capable of providing quantitative or semiquantitative analytical information. The main components include (1) a recognition element or bioreceptor, which is responsible for the specific targeting of the target analyte to support a high selectivity and specificity; (2) a transducer, which converts the interaction between the bioreceptor and the target into a signal, such as optical and electrochemical signals; and (3) electronics and display, which convert the signal obtained from the transducer into a processed, organized format to be read by human. Biosensors are often used in self-diagnosis and monitoring of disease progression. The earliest example of point-of-care testing (POCT) biosensors is the glucose biosensor that monitors the blood glucose level of diabetic patients. It is portable and generates results rapidly. To make the POC diagnostics with biosensors practical for use in the developing world, the World Health Organization issued criteria under the acronym ASSURED, i.e., Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free, Deliverable to end users [3]. In order to achieve ASSURED, biosensors are now designed to be inexpensive, portable, and perform as a standalone device. The desired way of performing the POC diagnostics is sample-to-answer biosensing. The user injects the sample into the inlet. The testing procedures, including reactions and signal acquisition, are automated, and the result readout show on the display without the need for further judgment or post-analysis. In order to perform automation inside a biosensor of limited space and weight, microfluidics handle fluid actuation and hence its reaction is the best option to be incorporated into the biosensor.

1.1.2 Printed microfluidics biosensors

Microfluidics is the process of fluid in micron scale, in the range of pico- to micro-liter. This process includes:

1. actuation of fluid of a particular volume to a specific location;

2. separation of the components in the fluid based on different properties, e.g., viscosity, density, etc.;

3. mixing of several kinds of fluid in a controlled temporal and spatial manner, causing bioreactions in a controlled manner.

Compared to conventional laboratory methods, it reduces the need for large volume of the sample, and labor-intensive works to process samples and reagents. Microfluidics is often applied in basic life science and applied science research [4]. In basic life science research, biomolecular and cell analysis, especially single-cell analysis, makes assay systems based on mammalian cells for developing drugs as individualized medicine [5–8]. In high-throughput screening in medical diagnosis requiring multiplex reactions, as microfluidic chambers are equivalent to multiple test tubes, microfluidics guides the sample through microchannels to simultaneously control fluidic flow to the sequential reaction sites and eventually to the sensing chambers [9].

Commercial printing technologies have been used for decades, with flexographic printing on a plastic sheet to fabricate printed sensing electrode or noncontact liquid line printing on paper to fabricate a lateral flow paper strip. Other printed components, such as printed circuit boards (PCBs) and 3D-printed machine exterior, have been used to construct biosensors [10]. It is, therefore, reasonable to extend the concept and advantages of printing into microfluidics fabrication and integration.

The COVID-19 pandemic showed us the importance of local production. Since transportation is limited or hauled due to restrictions, the import of medical supplies is insufficient to meet the skyrocketing rise in demand. The lack of medical supplies further impaired the already strained healthcare system. In this context, 3D printing of medical supplies, such as masks, face shields, and ventilators, was approved in many medical centers and became standard practice during emergency periods. Besides, visits to medical centers by patients with chronic illnesses and noncommunicable diseases were also disrupted due to risks of infection with the SARS-CoV-2 and limited medical resources. Similar to 3D printing of medical supplies, demand of POC diagnostics for the COVID-19 and other tests during the pandemic can be tackled by printing technology [11].

Printed microfluidics can be fabricated in 2D to 4D scale, depending on the complexity of the biosensing reaction. Table 1.1 summarizes some of the examples of sensing targets and the choices of microfluidic dimension. For example, immunoassay requires simple interaction between target molecules and recognition elements for visual readout and can therefore be performed in 2D microfluidics, such as lateral flow paper strip. For reactions requiring multiple procedures, e.g., target extraction and purification, washing, reagent mixing, before detecting the target, such as E. coli detection from food, 3D microfluidics are commonly employed. 4D or dynamic microfluidics possess reversible properties and respond to external stimuli, or responsive signals, such as light and heat, although there is a lack of example in biosensing target currently. More importantly, by leveraging the integration with sensor components, which can also be printed, the printed microfluidics can be translated into a printed biosensor to be practically used without compromising the advantages of how printing technology tackle the emergency [12].

Table 1.1

ABS, Acrylonitrile butadiene styrene; DLP, digital light processing; FDM, fused deposition modeling.

aKey: AFP: fetoprotein, CA153: carcinoma antigen 153, CA199: carcinoma antigen 199, CEA: carcinoembryonic antigen.

Fig. 1.1 illustrates how the printed microfluidic biosensors tackle the current and upcoming emergency needs around the globe. Printed microfluidics have exhibited attractive unique advantages, including fast turnaround time of production, flexibility in customization, and sustainability in fabrication, to fit this need. In conventional production methods (routes colored in red), the design of microfluidic biosensors is sent to the factory for mass production, stored in a warehouse, shipped to the destination, and transported to the target site. This kind of industrial production is less flexible in customizing the product design and the production schedule and is confined to factories due to various requirements of equipment and technicians. On the contrary, printed microfluidics can be printed anywhere, after obtaining the design in a digital format via the internet or cloud storage, anytime by anyone, as the printer is usually automated and printing can be easily controlled via software with the imported pre-optimized setting. They are also highly flexible in customization, and the design can be tailored or modified according to conditions and requirements. Printing microfluidic biosensors also promotes sustainability by reducing unnecessary waste from excessive production, extra storage, and power to maintain warehouses.

Figure 1.1 The illustration shows the logistic of fabricating printed microfluidic biosensors (blue route) and their advantages over conventional biosensing systems (red route).

1.2 Technologies in fabrication and assembly of printed microfluidics

This section describes printing technologies, especially advanced methods investigated and demonstrated in the past few years, for microfluidic fabrication and assembly. It also discusses the design, materials, and methods of printing and possible steps for post-processing of 2D, 3D, and 4D-printed microfluidics.

1.2.1 2D microfluidics

Cellulose-based paper has long been one of the most popular printing substrates. The advantages of paper are that it comes with a wide range of thickness and weight for commercial and industrial use at a low cost and that its ease of functionalization supports modifying the paper surface properties, e.g., hydrophilicity, permeability, piezoelectricity, and reactivity. Its high biocompatibility allows the printing of recognition elements on paper. Paper is also an environment friendly material which degrades quickly without polluting the environment and does not create toxins upon incineration. Paper-based microfluidics usually is more straightforward and more affordable. They rarely require an external power source, since fluid movement through the device can easily be facilitated by capillary forces. Cellulose fibers, chromatography paper, e.g., Whatman grade 1 chromatography paper, are commonly employed for printing, thanks to the detailed specification of well-defined properties in terms of pore size, water absorption property, hydrophobicity, etc. [23,24].

Nitrocellulose (NC) membrane is another popular printing substrate in 2D microfluidics. NC and polyvinylidene fluoride (PVDF) membranes have been extensively used in immunoblot for protein and nucleic acid analysis since the 1970s, thanks to their high protein- and nucleic acid-binding affinity as well as the availability of different pore sizes for capturing particular molecular size protein [25]. Although PVDF membrane is more popular given its better retention and less brittle nature than NC membrane, it is less desirable in POCT as it has to be pre-wetted with bipolar solvents, such as methanol or ethanol, prior to interaction with biomolecules in biosensing. Since NC membrane has better intrinsic protein and nucleic acid-binding properties than chromatography paper [13,26], it has been popularly used in conventional NC paper-based strip assay where antibodies can be immobilized on the NC surface to form a test line and control line to enable the end users to visualize the results more easily in comparison to color-change based assay. Similar to the chromatography paper, the well-defined properties of NC can define the flow rate in a 2D microfluidic, and geometric flow control, in terms of width, length, input angle, and output angle of the microchannel, in an NC membrane provides a new dimension of additional dynamic flow rate control of fluidic actuation [27,28].

To design 2D microfluidics, office software as simple as Microsoft (Ms) Paint, as long as they can generate the print layout to the printer, can be theoretically used for the drawing, although the accurate control of scaling is compromised. Therefore, professional computer-aided design (CAD) software are generally considered better at drawing the fine details of the micrometer resolution. Open-source software, e.g., Sketchup, Blender, and commercial software, such as AutoCAD or Solidwork, are commonly used for 2D-printed microfluidics design. 2D microfluidics contains microchannels of various lengths and widths and microchambers of various sizes and shapes, surrounded by the hydrophobic barrier such as wax. Therefore, after the dimensions and positions of the channels and chambers are determined in the drawing, the drawing is converted into the inverted image, i.e., the regions of channels and chambers are left blank while the surroundings are shown in black color, so that the printer head will move to the black region to print the wax materials according to the image.

Wax is the most common printing material. Printing wax channels on paper or NC membrane and then subjecting the paper to heat treatment causes wax penetration to form the wax barrier, of which the resolution and the channels can be as low as approximately 500 μm [24,29,30]. The percentage of wax in the printing material determines the hydrophobicity and fluidic properties, e.g., flow rate of the fluid. Commercial 2D inkjet printers commonly used in households can print 2D microfluidics with other materials instead of toner and ink. Fig. 1.2A demonstrates the use of a commercial printer (Xerox ColorQube color printers) for routinely printing paper-based microfluidics of different wax percentages with the addition of distinguishable color dyes, i.e., bright cyan (C), magenta (M), yellow (Y), black (K) that can produce a full range of colors, to various percentages of paraffin wax and resin ranging 50%–60% and 10%–20%, respectively. The color based on the combination of CMYK colors on the paper microfluidics are shown with the defined hydrophobicity, so that the behavior of fluid can be controlled conveniently [31].

Figure 1.2 2D-printed microfluidics. (A) The Xerox ColorQube 8570 model wax-based color printers for creating paper microfluidics of different color combinations of the wax ink, i.e., cyan, magenta, yellow, magenta + yellow, cyan + yellow, and cyan + magenta. (B) Fabrication and preparation of the self-calibrating microfluidic paper-based analytic device (left): its assembly, schematic diagram and flow of procedures, and (right) the image of the microfluidics for color intensity determination and plot fitting for detection in standard concentration solution (5 mM glucose and 5 mM lactate) and plasma samples [31,32]. Reprinted with permission from J. Potter, P. Brisk, W.H. Grover, Using printer ink color to control the behavior of paper microfluidics, Lab Chip 19 (2019) 2000–2008. https://doi.org/10.1039/C9LC00083F; S. Kim, D. Kim, S. Kim, Simultaneous quantification of multiple biomarkers on a self-calibrating microfluidic paper-based analytic device, Anal. Chim. Acta 1097 (2020) 120–126. https://doi.org/10.1016/j.aca.2019.10.068.

However, wax printing requires additional post-processing steps for melting wax into the paper to make the final product, e.g., heating at 120°C for 90 seconds and then cooling at 25°C. The heating and cooling need to be controlled accurately to guarantee the consistency of the end product. Prolonged heating might cause the melted wax to diffuse into the surrounding area. Due to its instability at high temperatures, storage and operation of the printed microfluidics should be kept at a controlled temperature. Also, printing quality depends on the smoothness of the paper or membrane surface. Inconsistent deposition of wax affects its hydrophobicity and hence its functionality.

2D-printed microfluidics can be inserted into a detector, such as potentiometric, fluorimetric, or colorimetric sensors, for visualization or signal acquisition [33]. Integration of 2D-printed microfluidics is relatively easy thanks to its properties such as thin layering, flexibility, and compatibility with daily-use adhesive materials. The microfluidics can be joined to any contact area of a detector at any orientation and geometry with simple adhesion such as adhesive tape and glue.

Another practical use of printed microfluidics in real-life applications is quantification accuracy. Calibration with standard concentrations is often used in the laboratory when determining the concentration to minimize the effect of environmental conditions (i.e., ambient light intensity, temperature, humidity, and pressure). However, on-site testing with calibration of printed biosensors can be challenging. Some 2D microfluidics introduces self-calibration and reserve areas for pre-loading and actuating calibrants. Fig. 1.2B shows an example of the microfluidic that supports automatic partition of the injected sample into different zones, pre-loaded with varying concentrations of the sensing elements such as glucose and lactate. The microfluidic is then imaged to determine the color intensity of each detection zone. Post-processing of the data by fitting the data plot with linear interpolation is used to simultaneously determine the glucose and lactate concentration [32].

1.2.2 Pseudo-3D microfluidics

Pseudo-3D microfluidics is defined as the 3D microfluidics in which fabrication of microfluidics is in a 2D manner, with extra procedures transforming multiple 2D microfluidics into 3D microfluidics. Stacking and folding are the two main methods to produce pseudo-3D microfluidics. Since most of the 2D microfluidics are printed on paper or membrane, its flexible feature enables folding without breaking the microfluidic bases.

Stacking multiple 2D microfluidics is popular because it extends the dimensional scale of microfluidics. It also reduces the size of the microfluidics while retaining a similar fluidic path distance. The difference in the diffusing speed of various molecules across layers of 2D microfluidic chambers, mostly with filter paper of defined pore size as a barrier, generates functions like molecule separation, and filtration, which is not conveniently done in conventional 2D microfluidics. Fig. 1.3A shows the schematics of pseudo-3D-printed well-based microfluidics based on stacking of multiple printed layers with laminate support and components to hold the assembled chip [15]. In detail, five layers of wax-printed chromatography paper were stacked together. Hydrophobic parafilm layers with hole-punch sites, creating a sample loading site, were then placed on both the top and the bottom of the stack layer. After proper alignment, all these were assembled manually with polyvinyl chloride enclosure and clips.

Figure 1.3 Pseudo-3D-printed microfluidics. (A) Stacking of multiple layers comprises five laminate sheet layers of wax-printed chromatography paper to form a paper wall. (B) Schematic of folding the 2D-printed microfluidic to generate a hydrophilic channel and function as separation of food colorants in the mixture [15,34]. Reprinted with permission from G.C. Ilacas, A. Basa, K.J. Nelms, J.D. Sosa, Y. Liu, F.A. Gomez, Paper-based microfluidic devices for glucose assays employing a metal-organic framework (MOF), Anal. Chim. Acta 1055 (2019) 74–80. https://doi.org/10.1016/j.aca.2019.01.009; F.M. Gharaghani, M. Akhond, B. Hemmateenejad, A three-dimensional origami microfluidic device for paper chromatography: application to quantification of Tartrazine and Indigo carmine in food samples, J. Chromatogr. A 1621 (2020) 461049. https://doi.org/10.1016/j.chroma.2020.461049.

On the other hand, folding and sliding action can join printed compartments of channels and chambers to build functional microfluidics. This simplifies the fabrication because printing is completed on a single surface instead of printing on multiple surfaces and then assembling them. The alignment by folding is more feasible than by stacking since the alignment can be well defined in the microfluidic design, while stacking requires external position markers, such as labels at particular corners, and positioning tools to facilitate the stacking process [16–18,34,35]. Fig. 1.3B shows origami-based three-dimensional paper chromatography (3D-PC) as a micro-chromatographic microfluidic platform. In this microfluidic, repeated mountain and valley folds are created during the folding and microchambers are formed. Chromatographic separation is possible only when the folding is completed to form a fluidic path across the printed layers. After the separation, the paper microfluidic is unfolded to reveal the color of all regions, and it is then followed by a colorimetric detection by determining the colors and their positions to identifying the target molecules [34].

However, the disadvantages of stacking and folding include the post-processing of stacking multiple printed materials to make it 3D, creating the folding lines that make the crease pattern to facilitate accurate folding upon usage, and the tedious process of folding during the assay. Their lack of robustness and automation hinders mass production and extensive use in large-scale screening tests. Therefore, researchers started moving to 3D-printed microfluidics in the last decade when the technology became widely available.

1.2.3 3D printed microfluidics

3D printing has recently become a popular area in the additive manufacturing process, as its technology has matured over decades and turned into commercial products widely available in the last decade. Its popularity can be observed from the availability of the 3D printing design and viewing software, education materials, and access to the equipment. In the 3D design, Microsoft changed its default drawing software from MS Paint to Paint 3D in the latest Windows operating system (Windows 10) on personal computers. It accelerates the education of basic 3D drawing. Open-source designs for 3D printing are free on online platforms under the Creative Commons – Attribution license, such as Thingiverse, Sketchfab, GrabCAD with digital design for physical objects and 3D printing services on demand, encouraging people to participate in the design and exploitation of 3D printing. Apart from the widely available commercial 3D printers that can produce high resolution, more and more open-source 3D printers are available for less than 1000 USD while restraining its printing resolution below submillimeter scale. Free online tutorials illustrate the procedure of building a 3D printer from scratch using another 3D printer, i.e., the replication of a 3D printer in-house, and it makes possible urgent repairing of a printer by replacing the damaged parts with 3D-printed parts. On the other hand, high-resolution 3D printing services on demand are now available at an affordable price. This section focuses on illustrating the practical procedure from design to production of 3D-printed microfluidics, particularly designing with CAD software to production and post-processing with various types of 3D