Multidisciplinary Microfluidic and Nanofluidic Lab-on-a-Chip: Principles and Applications

Multidisciplinary Microfluidic and Nanofluidic Lab-on-a-Chip: Principles and Applications provides chemists, biophysicists, engineers, life scientists, biotechnologists, and pharmaceutical scientists with the principles behind the design, manufacture, and testing of life sciences microfluidic systems. This book serves as a reference for technologies and applications in multidisciplinary areas, with an emphasis on quickly developing or new emerging areas, including digital microfluidics, nanofluidics, papers-based microfluidics, and cell biology. The book offers practical guidance on how to design, analyze, fabricate, and test microfluidic devices and systems for a wide variety of applications including separations, disease detection, cellular analysis, DNA analysis, proteomics, and drug delivery.

Calculations, solved problems, data tables, and design rules are provided to help researchers understand microfluidic basic theory and principles and apply this knowledge to their own unique designs. Recent advances in microfluidics and microsystems for life sciences are impacting chemistry, biophysics, molecular, cell biology, and medicine for applications that include DNA analysis, drug discovery, disease research, and biofluid and environmental monitoring.

• Provides calculations, solved problems, data tables and design rules to help understand microfluidic basic theory and principles
• Gives an applied understanding of the principles behind the design, manufacture, and testing of microfluidic systems
• Emphasizes on quickly developing and emerging areas, including digital microfluidics, nanofluidics, papers-based microfluidics, and cell biology

• Language: English
• Publisher: Elsevier Science
• Release date: Sep 19, 2021
• ISBN: 9780444594617

Book preview

Preface

XiuJun (James) Li¹, ², ³, Chaoyong Yang⁴ and Paul C.H. Li⁵, ¹Department of Chemistry and Biochemistry, University of Texas at El Paso, El Paso, TX, United States, ²Border Biomedical Research Center, Biomedical Engineering, University of Texas at El Paso, El Paso, TX, United States, ³Environmental Science and Engineering, University of Texas at El Paso, El Paso, TX, United States, ⁴Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, P.R.China; Institute of Molecular Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, P.R. China, ⁵Department of Chemistry, Simon Fraser University, Burnaby, BC, Canada

There are not too many research fields as multidisciplinary as microfluidic/nanofluidic lab-on-a-chip. In 1957 the photolithography technique was first developed by Jay Andrus et al. to create patterns for printed circuit boards and was further developed by Jack Kilby at Texas Instruments. Jack Kilby’s demonstration of the first integrated circuit using the technique provoked a revolution in microelectronics. This photolithography technique was widely used in the field of microelectro-mechanical systems (MEMS) and then was applied in the discipline of chemistry to demonstrate various miniaturized chemical analysis systems since the 1990s. This field of MEMS grew rapidly and expanded quickly from chemistry to other disciplines such as biology, biomedicine, physics, and bioengineering. Hence, we developed this book, Multidisciplinary Microfluidic and Nanofluidic Lab-on-a-Chip: Principles and Applications, to highlight various topics in several disciplines of microfluidics/nanofluidics from classic to emerging techniques and applications. This book aims to provide to a broad audience the comprehensive review of both fundamentals and the state of the art of microfluidics/nanofluidics.

The book consists of 16 chapters comprehensively covering fundamentals and applications of microfluidic/nanofluidic lab-on-a-chip. Classic techniques in micro/nanofluidics include micromachining methods (Chapter 1: Fabrication of Microfluidic Chips) and capillary electrophoresis chip techniques (Chapter 4: Microfluidic Capillary Electrophoresis Chip Techniques: Theory and Different Separation Modes). The next two chapters are related to fundamentals of microfluidic dynamics in physics and engineering, namely, microfluid–substrate interactions (Chapter 2: Fluid–Substrate Interactions) and magnetism (Chapter 3: Magnetism in Microfluidics: Computational Fluid Dynamics Simulations, Mixing, Transport, and Control of Fluids and Particles at Micro Scale). In addition, a number of applications, especially in biochemical and biomedical areas, are represented by cellular analysis (Chapter 5: Cell Manipulation and Cellular Analysis), organ-on-a-chip (Chapter 6: Organ-on-a-Chip), nucleic acid analysis (Chapter 7: Recent advances in nucleic acid analysis and Detection With Microfluidic and Nanofluidics), multiplexed nucleic acid tests (Chapter 8: Conventional and Unconventional Methodologies for Multiplex Nucleic Acid Tests), protein analysis (Chapter 9: Microfluidic Protein Analysis and Applications), drug discovery and delivery (Chapter 10: Microfluidics in Three Key Aspects of the Drug-Development Process: Biomarker Discovery, Preclinical Studies, and Drug Delivery Systems), and point-of-care analysis (Chapter 12: Rapid Disease Diagnosis Using Low-cost Paper and Paper-Hybrid Microfluidic Devices).

Furthermore, the last six chapters are dedicated to emerging techniques and their corresponding applications such as acoustics (Chapter 11: Fundamentals and Applications of Acoustics in Microfluidics), paper-hybrid microfluidic devices (Chapter 12: Rapid Disease Diagnosis Using Low-Cost Paper and Paper-Hybrid Microfluidic Devices), self-powered digital microfluidic devices (Chapter 13: Energy Harvesting and Self-Powered Devices in Droplet Microfluidics), three-dimensional printed microfluidics (Chapter 14: Integration of Three-Dimensional Printing and Microfluidics), nanofluidics (Chapter 15: Principles and Applications of the Nano-in-Nano Integration for Multidisciplinary Nanofluidics), and microfluidics and nanomaterials (Chapter 16: Microfluidics for Nanomaterial Synthesis). Different from other books on microfluidics, this book also includes nanofluidics and nanomaterials.

Each chapter in this book provides not only the state of the art of microfluidics/nanofluidics but also the future trends of each topic in the section of Perspectives at the end of each chapter. The Study Questions section featured in each chapter will be appealing to instructors that might consider this book as a textbook, making this book stand out from other microfluidics books.

This book provides chemists, biophysicists, engineers, life scientists, and members of the biotechnology and pharmaceutical sectors with a full understanding of the principles behind the design, manufacture, and applications of microfluidic/nanofluidic systems in life sciences. We hope this book will serve as a valuable source of information on these topics for beginners as wells as experts.

Part I

Microfluidic Techniques

Outline

Chapter 1 Fabrication of microfluidic chips

Chapter 2 Fluid–substrate interactions

Chapter 3 Magnetism in microfluidics: computational fluid dynamics simulations, mixing, transport, and control of fluids and particles at micro scale

Chapter 4 Microfluidic capillary electrophoresis chip techniques: theory and different separation modes

Chapter 1

Fabrication of microfluidic chips

Hui Chen, Bin Yang and Zhejun Yang,    Department of Chemistry, Fudan University, Shanghai, P.R. China

Abstract

The micro/nano-fluidic chips have played a more and more significant role in various fields including biomedical diagnosis, environmental monitoring, food safety, etc., in the past two decades. This chapter presents various fabrication technologies of micro/nano-fluidic chips from the traditional micro-electro-mechanical systems, soft photolithography, thermoplastic polymer, hydrogels and paper-based microfluidics, to the latest three-dimensional printing and modular microfluidics concept. The advantages and limitations of various fabrication methods are also discussed.

Keywords

Micro-electro-mechanical systems (MEMS); soft photolithography; thermoplastic polymer (hydrogels or paper)–based microfluidics; 3D printing; modular microfluidics; nanofluidics

1.1 Introduction

In recent thirty years, the microfluidic chips have been widely applied in the monitoring of environment, food safety and biomedical diagnosis areas due to its advantages such as low requirements of sample volume, easy operation and integration, portability, and short analysis time. The micro- or nano-scale channels with smooth inner surfaces are the key parts of chips, which must be made with specialized micromachining methods and technologies. The fabrication and micromachining of microfluidic chips are the fundamentals of research and application, which transfers specialized patterns into chips with high precision. Historically, the origin and blooming development of microfluidic and nanofluidic lab-on-a-chip depended strongly on the maturity of silicon-based micro-electro-mechanical system (MEMS) technology for semiconductor and integrated circuits industry. MEMS-based micromachining method paved the way of microfluidic chips and different types of microfabrication have also been developed in the past 30 years. The first microfluidic electrophoresis chips (micro total analysis systems, μ-TAS) was fabricated using the MEMS method in 1990s by Andreas Manz (Ciba-Geigy Corporation, Switzerland). MEMS technology has been also used to fabricate the highly integrated microfluidic chip with multifunctions such as separation, reaction, and detection in 2002.¹ To 2014, an organ-on-a-chip containing continuously perfused chambers inhabited by living cells has been fabricated on polydimethylsiloxane (PDMS) by a MEMS method to simulate tissue- and organ-level physiology, which advanced the study of tissue development, organ physiology, drug discovery, and disease etiology.² Moreover, in 2015, droplet chips fabricated by MEMS method have been firstly employed to generate genome—wide profile individual cells at highly parallel scale, which opened up the new era of single cell RNA sequence.³

However, the MEMS technology has high instruments requirement, complex design, and large variation of chips from piece to piece. Some more convenient, cost effective and reliable ways have been developed such as soft lithography,⁴ elastic PDMS molding,⁵ and thermo-pressing methods.⁶ PDMS-based chips are easy and inexpensive to manufacture. PDMS is also elastic, breathable, and compatible with cell culture. For the massive manufacture of microfluidic chips, polymethylmethacrylate (PMMA) or other polymers like polycarbonate (PC) are also applied in the field. The well-developed manufacture process of PC makes PC become one of the most optimal materials for massive production of disk-like microfluidic chips, integrated with separation, reaction, and detection functions. This kind disk chips have been massively manufactured for food safety monitoring and point-of-care test (POCT) with the low cost (US$1.00) and one-time use.⁷ Moreover, paper-based microfluidic chips are a promising aspect in some POCT or fast and portable detection situations due to the low cost, easy fabrication, and capillary effect.⁸ Paper strips and colorimetric detection mode play prominent roles in paper-based microfluidic chips. However, the precise control of fluids and channels in microscale still remains as the neck bottle of both PC- and paper-based microfluidic chips. With the developing of new technologies and new materials, three-dimensional (3D) printing and hydrogel have been also adapted in microfluidic chip micromachining for their simplicity and streamlines.⁹,⁷⁰ The designed structures can be quickly modified and updated, which reduces the time required for optimization and biological application. For the various application fields, the flexibility and the modular combination have attracted more and more attention, like Lego components.

In this chapter, the traditional micromachining methods such as MEMS and soft lithography will be reviewed and discussed. Furthermore, new technologies and materials such as 3D printing, hydrogel-based micromachining methods will be highlighted. Some micromachining methods with mature industrial arts such as paper- or PC-based will be emphasized for their potential massive manufacture. An up-to-date fabrication technique for micromachining micro and nano-fluidic devices will also be presented.

1.2 Microelectromechanical systems

The MEMS technology was developed on single-crystal silicon wafers for the manufacture of integrated circuits, which mainly includes standard photolithography and etching process. For the application of microfluidic chips, some extra processes (removing the photoresist film, bonding, etc.) have been supplemented and modified. Furthermore, glass and quartz are also often used for microfluidic chips manufactory with similar MEMS processes.¹⁰ The detailed processes are shown in the following.

1.2.1 Photomask

To get the specific designed channels of microfluidic chip and control the fluid precisely, the photomask with specialized pattern and high resolution is the perquisites of MEMS processes with high quality. The basic function of a photomask is forming the different photo transmitting effects on areas with and without patterns under ultraviolet (UV) light. There are two types of photomasks (as shown in Fig. 1.1). AutoCAD or other computer software can be used to design the patterns, which can be printed on transparent plastic films using high-resolution printers (1200 dpi or higher). These transparent plastic films are readily used as the photomask for standard photolithography with the requirement of 20-μm resolution of line width and line distance.

Figure 1.1 Two kinds of photomasks.

1.2.2 Cleaning of silicon substrate

To remove contaminants presented on the surface of silicon wafer and make sure the strong adhesion of photoresist onto the silicon wafer, several effective cleaning processes including degreasing, polishing, and acid or water washing must be conducted on the silicon wafer, followed by drying.

1.2.3 Thin-film deposition

Thin-film deposition is an additive process that adds a thin film on the silicon substrate surface to realize different functionalities (protection, conduction, insulation, etc.) after photolithography. The normally deposited films include photoresist, various conductive metals, silicon dioxide, silicon nitride, etc. The deposition methods depend on different materials.

Photoresist plays prominent role in photolithography, which is a solution of a light-sensitive polymer.¹¹ The photoresist normally includes two types, called positive and negative, where degradation reaction and crosslinking reaction happened, respectively. Positive resists have higher resolution than negative. Either positive or negative resists can be selected, depending on whether it is desirable to have the opaque regions of the photomask for the protection of the resist during UV exposure, or vice versa, as shown in Fig. 1.2. Spin coating method is normally used in the photoresist deposition on the spin-coating machine to get even, adhesive thin film with controllable thickness. Combined with two different photomasks (Fig. 1.1), four types of patterns can be formed on substrates.

Figure 1.2 The main steps of micro-electro-mechanical systems.

1.2.4 Baking

To remove and evaporate the solvent in photoresist film, baking process is performed in the oven at about 95, for 1 hour (depends on the thickness of photoresist film). The baking process can enhance the adhesion and polishing resistance between the photoresist film and the substrate.

1.2.5 Exposure

After baking, the photoresist is selectively exposed under UV light (normally 300–500 nm) for transferring the pattern on the photomask to the photoresist thin film. The photomask is placed between the UV light and silicon wafer with a photoresist film. Take the positive photoresist SU-8 as the example, crosslinking reaction happens in the area exposed with UV light and resulted in the changes of properties of the film and the transferring of patterns.¹² Therefore, the exposure process is the most important step in the whole MEMS technology. The mercury lamp is mostly used. The exposure mode includes contacting and noncontacting.

1.2.6 Development

The development process is to remove the undesirable areas of photoresist films and get the same (positive photoresist) or inverse (negative photoresist) patterns on mask using a developing solution (1-methoxy-2-propyl acetate as the main ingredient). The developing time depends on the different operation conditions and is normally 1–3 minutes.

1.2.7 Etching

The etching process is to remove the material of the silicon substrate and obtain predesigned pattern on the surface of substrate under the protection of a patterned photoresist film by physical or chemical methods. Generally, etching methods are divided into wet etching with chemical solutions ¹³ and dry etching with plasma methods ¹⁴ as shown in Fig. 1.3.

Figure 1.3 Two types of etching: (A) wet etching and (B) dry etching.

Wet etching uses chemical reactions between chemical etching solutions and silicon substrate to strip the substrate materials. The usually used etching solution is HF.¹⁴

Si(s) + 4HF(aq) = SiF4(aq) + 2H2(g)

SiF4(aq) + 2HF(aq) = H2SiF6(aq)

The wet etching method holds high selectivity and uniformity, no damage to silicon wafer, and is applicable to all metal, glass, and plastic substrates. However, the wet etching is isotropic and silicon wafer can be etched in all directions at nearly the same rate. So the fidelity of pattern is low and it is not easy to control the minimum line width.

Dry etching uses the reaction between the gas with high energy (plasma) and the silicon substrate.¹⁴ The most attractive advantage is that the etching rate at vertical direction is much higher than that at horizontal direction, which guarantees the fidelity of fine patterns and higher aspect ratio. But the instrument of dry etching is expensive and dry etching method is rarely applied in the microfluidic chip fabrications.

1.2.8 Removing the photoresist film

After etching, the photoresist has finished its mission and should be removed using organic solvent, oxidant, plasma, or strong UV lights. Finally, the substrate for microfluidic chip with microchannels has been obtained.

1.2.9 Bonding

To handle the fluid in closed microchannels, the above fabricated micro fluidic chips must be bonded with flat silicon or glass cover slide. To ensure the strong bonding, especially no fluid leakage at high pressure and temperature, the microfluidic chips and cover slides must be cleaned strictly and the remaining small particles, organic contaminations, and metal stuff must be removed completely. Some oxidation solvents such as piranha solution (H2SO4:H2O2=3:1) are normally used, followed by flushing with lots of deionized water. Oxygen or O3 plasma and UV are also often used to remove the organic contaminations on the glass or quartz surface and to make the surface hydrophilic as well. The flatness and roughness are critical to the successful bonding.

The bonding methods include the following three ways:

1.2.9.1 High-temperature annealing

High-temperature annealing is mostly used in glass–glass bonding, as shown in Fig. 1.4. The clean microfluidic chip side with microchannels and the cover slide are matched. All sets are put into a vacuum instrument and firstly vacuumed for 1 hour. A polished graphite board and a steel board were both pressed on the top of the cover slides and on the bottom of microfluidic chip substrate, respectively. All this sets are heated in programmed oven to certain temperature (300–800°C) and cooled down to room temperature.¹⁵

Figure 1.4 High-temperature bonding methods.

1.2.9.2 Anodic bonding

Anodic bonding is a simple, effective, and long-lasting technique to bond silicon–glass, as shown in Fig. 1.5. The clean silicon and glass slides are firstly matched. The silicon and glass slides are connected with anodic and cathode electrodes, respectively. A high voltage about 500–1000 V is conducted on the two electrodes at 300–500 oC. Na+ in the glass slide moves to the cathode and negative charges are formed on the glass slide and positive charges are formed on the silicon slide. The electrostatic interaction between silicon and glass slides promotes the bonding.¹⁶

Figure 1.5 Anodic bonding method.

1.2.9.3 Low-temperature bonding

HF is often used for the bonding of glass due to its reaction with Na2SiO3 in glass, as shown in Fig. 1.6. 1% HF is dropped into the gap between two glass slides and certain pressure is brought for several hours at room temperature. Epoxy and liquid PDMS can also be used as an adhesive.¹⁷

Figure 1.6 Adhesive bonding method.

1.3 Soft photolithography

Some of the existing work to fabricate microfluidics belongs to the field known as soft photolithography, which is named for the use of soft elastomer during the preparation procedures. The earliest soft photolithography used high-resolution elastomer stamps and possessed the ability to form self-assembled monolayer chemical inks on the substrates.⁴ This single layer can guide the deposition or removal of material from the substrate to create patterns from desired materials. The interest in this technology stems from the ability to use ordinary chemical laboratory equipment to form submicron-sized structures without expensive equipment. This technology has been widely used in academic and industrial laboratories around the world. The complexity of the chip structure and performance of soft photolithography patterning methods are rapidly developing. The key technologies in soft photolithography largely contain microcontact printing, replica molding, microtransfer molding, micro-molding in capillaries, and solvent-assisted micromolding.¹⁸ However, this section focuses on trends of soft photolithography method, including the innovation and transformation of these microfluidics from lab to off-the-shelf.

1.3.1 Fabrication of PDMS-based microfluidic chips

Compared to silica glass materials, elastomers are easier to obtain and cheaper. One of the most viable elastomers for microfabrication is PDMS. It is easy and inexpensive to manufacture. Another advantage of PDMS is its high elasticity. It is also breathable and compatible with cell culture, which is the most attractive merits in biology-related application such as organ-on-a-chip. Therefore, PDMS-based equipment is widely used in biological materials research, such as cell culture and screening, and biochemical testing. It also has limitations. The porous matrix of PDMS consists of a Si–O bonds, which is the main chain covered with alkyl groups. Therefore, it is incompatible with organic solvents.⁵ However, some limitations of PDMS chip lie in leakage, light absorption of base material, and low adhesion of biomolecules.

The soft photolithography process can be divided into two parts: the manufacture of elastomeric components and the use of these components to pattern geometric shapes defined by the relief structure of the components. PDMS is commonly used for this purpose. Through optimized materials and chemical composition, the manufacturing process has a high fidelity. As shown in Fig. 1.7, in general, a PDMS microfluidic chip is obtained by the following steps.¹⁹ First, a silicon master mold is fabricated according to the conventional microfluidic photolithography technique, successively by casting, exposure, and developing (Fig. 1.7A). Then, a PDMS precursor mixture at a defined weight ratio of base-to-curing agent (commonly 10:1) is poured carefully onto the master after surface hydrophobic treatment, which is stirred for 15 minutes and vacuumed for 15 minutes to eliminate bubbles and cured at 80°C for 4 hours (Fig. 1.7B). And, the cured PDMS replica is gently peeled off from the silicon master (Fig. 1.7C). Hole punchers and scalpels are manually used to drill the shaped inlets, outlets, and gas vents. The cured PDMS replica is then treated by plasma for bonding. Afterwards, another PDMS membrane without pattern is placed on its top (Fig. 1.7D). This blank PDMS membrane is used as a sealing membrane. Before biochemical assays, the PDMS microfluidic chip should be sterilized with 75% ethanol and washed with PBS three times. Finally, a sample solution is added into the layer via a flow springe-pump.

Figure 1.7 Fabrication process flow for traditional polydimethylsiloxane (PDMS) molding. (A) Mold definition via photolithography. (B) PDMS chip curing. (C) Peeling off of cured PDMS. (D) Bonding and package.

In fact, recent work has shown that replicas with nanometer depth and width can be accurately reproduced.²⁰ In the past decade, the soft lithography has developed rapidly, starting with the pioneering work of George M. Whitesides laboratory,²¹ and has now become a business involving dozens of independent companies. The practicality of these methods and the abundance of materials science, chemistry, and physics that control their operation may continue to arouse interest in the coming years.

1.3.2 Development of PDMS-based microfluidic chips

Whitesides’s group proposed the soft photolithography technique in 1998 to design PDMS devices, aiming to study the laminar flow characteristics in narrow, well-defined narrow channels. Elastic multiple PDMS layers and arrays were also used to construct the gas-controlled valve in the integrated microfluidic chips to control the fluid, as shown in Fig. 1.8.²² On the top layer, there are three linear control channels for pneumatically driving film formation, while on the bottom layer a T-joint channel for floating flow is integrated. Interestingly, by controlling the pressure in the top channel, the membrane is deformed to switch the fluid in bottom channel In/Out. Therefore, the three valves controlled the fluid flow in or out, forming a pump. The pumping rate was determined by controlling the distance between the adjacent two air channels on the top.

Figure 1.8 Gas controlled valve for on/off switch constructed by multiple polydimethylsiloxane layers.²² Unger, M.A.; Chou, H.P.; Thorsen, T.; Scherer, A.; Quake, S.R. Monolithic Microfabricated Valves and Pumps by Multilayer Soft Lithography. Science 2000, 288 (5463), 113–116.Reproduced with permission from 2000 AAAS.

Based on the above concept, Sim and colleagues introduced a double-layer PDMS substrate to prepare microparticles.²³ By controlling the pressure in the top channel, the membrane is deformed to limit the suspension to the designed shape, and then the particles obtained by UV irradiation and are restored to the original shape by release. Finally, the particles can be discharged through the bottom channel. Recently, PDMS-based soft lithography has been applied to the fabrication of organs-on-chips for 3D cell culture and organ mimic due to the bio-compatibility and flexible design of PDMS.²

Of course, the application of soft photolithography has some limitations, such as the shrinkage deformation of PDMS after curing is 1%, and the extension of the aspect ratio under the action of toluene and ethane. The elasticity and thermal expansion of PDMS make it difficult to obtain accuracy. Since the elastic film is too soft, a large aspect ratio cannot be obtained. The ratio of depth to width will cause deformation of the microstructure after deformation.²⁴

1.4 Thermoplastic polymer-based microfluidics

Thermoplastics are a class of synthetic polymers. Due to the long polymer backbone, it exhibits softening behavior when returns to their original chemical state after cooling.²⁵ Thermoplastic polymers differ from elastomers or thermosetting plastics in that it can be softened or completely melted and reshaped when heated, while still maintaining chemical and dimensional stability over a wide range of operating temperatures and pressures. Coupled with customizable chemical and physical properties, this feature makes thermoplastics very suitable for use as substrates for microfluidic applications. The manufacturing technology of thermoplastic polymer-based microfluidic chips is very different from that of glass chips. In this section, the manufacturing techniques include hot pressing, injection molding, and lithographie (LIGA).

1.4.1 Hot pressing

Hot pressing is a chip manufacturing technology that has been widely used to quickly replicate microstructures.²⁶ The thermoplastic polymer substrate and the mold are aligned and heated to apply a certain pressure to obtain a micro-structured chip. The mold used for the simple hot pressing method may be a wire material having a diameter of 50 μm or less or a positive film of a microchannel wafer having embossed protrusions. Simple microchannels use wire as a mold for intelligent manufacturing. And the intersection of the channels is pressed into an irregular shape on the same plane, which adversely affects the injection and separation. A microfluidic chip is manufactured by etching a microchannel silicon positive film to obtain complex microchannels, and the channel junction has a satisfactory structure.

1.4.2 Injection molding

Injection molding is a method in which raw materials are placed in an injection machine, heated to become a fluid into the mold with designed patterns and then cooled to release the mold.²⁷ The materials for patterned mold can be epoxy resin, SU-8 photoresist, AZ positive photoresist, and silicon material or glass. PC and PMMA are mostly used as the injecting materials. In the injection molding process, mold manufacturing is complicated and the cycle is long. The cost and technical requirements are high. Usually, one mold can produce 300,000–500,000 polymer chips, with good repeatability, short production cycle, and low cost. Therefore, injection molding is suitable for the mass production of molded chips.

The fabrication process of the injection molding is shown in Fig. 1.9. The design of microfluidic chip molds has several main considerations. First, the external dimensions of the chip are centimeters, and the width of microchannel is in the range of micrometers. So, the sizes of chips and microchannels should be calculated carefully and controlled effectively. Second, the microfluidic chip contains a micro-scale structure, which is sensitive to temperature changes or etching agent. Third, as shown in Fig. 1.9E, after carefully evaluating various microstructures, optimal polymer is selected to fill the thin-walled structure for the microfluidic chip.

Figure 1.9 Schematic of injection molding design for microfluidics. ²⁸ Viehrig, M.; Thilsted, A.H.; Matteucci, M.; Wu, K.; Catak, D.; Schmidt, M.S.; et al. Injection-Molded Microfluidic Device for SERS Sensing Using Embedded Au-Capped Polymer Nanocones. ACS Appl. Mater. Interface. 2018, 10 (43), 37417–37425.Reproduced with permission from 2018 American Chemical Society.

Fig. 1.10 shows the disk-like chips manufactured by injection molding from PC materials, which integrates multiple sample pretreatment, separation driven by centrifuge force, analysts reactions, and in situ fluorescent detection functions. This kind of disk-like chips have been commercialized for the food safety monitoring and clinical disease diagnosis.²⁹ Usually, one mold can produce 300,000–500,000 polymer chips, with good repeatability, short production cycle, and low cost.

Figure 1.10 (A) Image of the microfluidic chip; (B) design and functional partition of the microfluidic chip; and (C) stages in the fluid flow process in part of the microfluidic chip as viewed under a microscope (50× magnification).⁷ Ye, X.; Li, Y.; Fang, X.; Kong, J. Integrated Microfluidic Sample-to-Answer System for Direct Nucleic Acid-Based Detection of Group B Streptococci in Clinical Vaginal/Anal Swab Samples. ACS Sens. 2020, 5 (4), 1132–1139.Reproduced with permission from 2020 American Chemical Society.

1.4.3 LIGA

LIGA is the abbreviation of German lithography galanoformung abformung. It includes three parts: X-ray deep lithography, micro electroforming, and micro replication. It is mainly used to manufacture high amplitude microfluidic chips. The first step is synchrotron X-ray deep lithography. Using X-rays of synchrotron radiation source with good parallel performance and high radiation intensity, a few millimeters thick X-ray-sensitive photosensitive material is coated on the highly conductive metal film layer. The pattern on the film is usually transferred to the photoresist layer at a lithographic depth of several hundred microns. The second step is electroforming, that is, metal is deposited in the gap of the photoresist pattern after development, and electroplating can be used, while the underlying metal film of the photo-adhesive is used directly as an electrolytic electrode. In the third step, the electrophoresis chip is replicated by injection molding using thermoplastic polymer materials (e.g., PMMA, PC).

1.5 Hydrogels for the fabrication of microfluidic chips

Hydrogels are materials based on a network of hydrophilic polymer chains that are swelling in water but insoluble in water.³⁰ Natural hydrogels include polysaccharides which are extracted from cells (e.g., agarose, alginate, hyaluronic acid, and chitosan) and proteins (e.g., collagen, gelatin, and fibrin). For example, agarose is obtained from the cell wall of sterile seaweed. It is a neutral polysaccharide that forms a thermally reversible hydrogel below its gel temperature. Agarose is biologically inert, and it does not adhere to proteins and cells, so it has been widely used in biomedicine, such as cell culture and tissue engineering. Synthetic hydrogels (e.g., polyacrylic acid and polyethylene oxide) are also widely used. Hydrogels can be prepared from synthetic polymers (e.g., polyethylene glycol, polyacrylic acid, polyvinyl alcohol, polyacrylamide) and their derivatives. Hydrogel is generally made from monomers in aqueous solution, mixed with initiator and cross-linking agent, and then polymerized by various methods such as photopolymerization. Synthetic hydrogels have greater mechanical strength than natural hydrogels, but they may not be degraded by organisms.

In the past decade, hydrogels such as agarose, Matrigel, polyethylene glycol diacrylate (PEG-DA) sodium alginate, and chitosan have been frequently used to manufacture different microfluidic devices. Hydrogels in microfluidic systems have many advantages as the following ³¹:

1. Free diffusion of small molecules. Most cell nutrients and growth factors can be diffused in hydrogels, so hydrogel-based devices can easily create diffusion membranes for different applications.

2. Optical clarity. This characteristic of hydrogel makes it possible to observe the diffusion of fluorescent molecules and cell behavior within the gel structure under a microscope.

3. Easy to obtain. Most hydrogels, such as agarose, are commercially available and inexpensive.

4. Fine fabrication. Hydrogels have good ductility and can be designed into various shapes and precise dimensions.

5. Cell friendly. Most hydrogels are nontoxic and have Young’s modulus which are comparable to cells.

There are three main methods for hydrogel crosslinking: temperature, UV, and chemical chelating agents. Ionic or covalent crosslinking will combine hydrogel monomer, copolymer, or macromonomer to form an insoluble polymer matrix. Therefore, the physical properties, mechanical properties, and diffusion characteristics of hydrogels such as swelling, mechanics, rheology, and adhesion highly depend on the type of polymer chains, cross-linked molecules, and cross-link density.

1.5.1 Soft lithography using hydrogels

Soft lithography is the most commonly used method for microfluidic chip manufacturing. Hydrogels can also be used as soft elastomeric materials in the manufacturing process. For example, agarose is melt into liquid at a high temperature and solidify into solid at a low temperature. We can cast high-temperature liquid agarose onto a premade template. When the agarose cools down, it can naturally turn into solid and can be easily peeled off from the template. Using soft lithography, we can easily make hydrogel-based microfluidics repeatedly. Liu developed an agarose-glass hybrid microfluidic chip and a fork-shaped microchannel was fabricated between agarose and glass. The hybrid microfluidic chip successfully preconcentrated exosomes and detected exosomes at a low concentration.³²

1.5.2 Thermal controlled flow-solidification of hydrogels for subchannel construction

It is difficult to change the internal structure of the chip once the template is finished using soft lithography. The characteristic of hydrogels can help to partially modify the internal structure of the microfluidics.

Generally, the liquid in the chip behaves as a laminar flow, and adjacent liquids do not mix during they flow. Therefore, we can use laminar flow to construct hydrogel subchannels to modify the channels. We can inject the reaction solution, liquid hydrogel, and another reaction solution side by side into the channel. After the hydrogel cures, a subchannel forms, separating the solution on both sides. For example, Matrigel cures at 25°C–37°C. Wong et al. injected liquid Matrigel and PEG8000 (almost the same viscosity) into the main channel at 4°C, so that Matrigel formed hydrogel microblocks in the microchannel and separated the different flows from each other as shown in Fig. 1.11.³³

Figure 1.11 (A) Partitioning microchannels with slabs of hydrogel. (PE: polyethylene)³³ Wong Perez-Castillejos, R., Christopher Love, J., Whitesides, G.M.A.P. Partitioning Microfluidic Channels with Hydrogel to Construct Tunable 3-D Cellular Microenvironments. Biomaterials 2008, 29(12), 1853–1861. Reproduced with permission from 2008 Elsevier. (B) A diagram of the fabrication method and images demonstrating a variety of shapes that were polymerized within 35 seconds.³⁴ PDMS, Polydimethylsiloxane. Beebe, D.J.; Moore, J.S.; Bauer, J.M.; Yu, Q.; Liu, R.H.; Devadoss, C.; et al. Functional Hydrogel Structures for Autonomous Flow Control inside Microfluidic Channels. Nature 2000, 404 (6778), 588–590. Reproduced with permission from2000 Nature.

1.5.3 Photopolymerization of hydrogels for microstructures

Curing hydrogels through temperature changes is a very convenient method. However, this method cannot accurately control the pattern of the gel.

Hydrogel photopolymerization can make up for this deficiency. The photopolymerization has a high spatial resolution, and can easily integrate a more complicated gel structure in the microfluidics. Some examples are shown in Fig. 1.11B.³⁴ Gel materials that can be used for UV curing include PEG-DA, polyisopropylacrylamide (PNIPAAm), polyacrylamide, and many other polymers.

The light source used in the photopolymerization method is mostly UV light. Some studies are now using visible light for polymerization. The UV photopolymerization can integrate microstructures into microfluidic devices precisely, but the limitations of this method are also obvious. UV-cured polymers are expensive and cannot be used for cell mixing experiments.

Hydrogels can also form a series of hydrogel droplets in the channels of the microfluidic chip.³⁵ The hydrogel droplet formation is similar to the normal droplet formation. After the droplets are generated, they can be solidified into hydrogel droplets by the methods mentioned above. The porous structure of hydrogels can transport oxygen, nutrients, and growth factors, and the hydrogel droplets can simulate the extracellular environment as a cell culture medium. The curing of hydrogels can keep the reactants inside hydrogels without diffusion, and facilitate subsequent operations and long-term storage as shown in Fig. 1.12.³⁶ Hydrogel droplets mimic the extracellular environment and enable long-term single cell culture.

Figure 1.12 Design and operating principles for the rapid preparation of alginate hydrogel droplets.³⁶ Lee, D.H.; Bae, C.Y.; Han, J.I.; Park, J.K. In Situ Analysis of Heterogeneity in the Lipid Content of Single Green Microalgae in Alginate Hydrogel Microcapsules. Anal. Chem. 2013, 85 (18), 8749–8756. Reproduced with permission from 2013 American Chemical Society.

1.6 Three-dimensional printing

Three-dimensional printing, also known as additive manufacturing, is a process of continuous adding, stacking raw materials under computer control. According to the 3D model of the object, almost any shape and geometric feature can be printed.

Compared with PDMS, 3D printing fabrication is easy and can be modified quickly. It can save a lot of time to iterate to make a more desirable chip. The process is automatic and the cost of a single chip is much cheaper than PDMS.³⁷

In general, the main steps of 3D printing are ³⁸

1. Draw a 3D model on CAD and save it as a.stl file, which is a standard file format for data transfer between CAD and a 3D printer.

2. The 3D printer interprets the.stl file and divides the 3D model into continuous two-dimensional (2D) fault sections.

3. The 3D printer superimposes the materials in a layer-by-layer manner, and finally constructs objects with a 3D structure.

4. Some methods require the removal of supporting materials inside the structure.

Many materials are suitable for 3D printing, from polymer materials to various biological materials and even living cells. The materials and properties of different 3D printing methods are also different (Table 1.1). Here are three widely used methods shown in Fig. 1.13.

Table 1.1

Figure 1.13 Three-dimensional printing methods. (A) Stereolithography. (B) Inkjet printing. (C) Fused deposition modeling. ³⁹ Gross, B.; Lockwood, S.Y.; Spence, D.M. Recent Advances in Analytical Chemistry by 3D Printing. Anal. Chem. 2017, 89 (1), 57–70. Reproduced with permission from 2017 American Chemical Society.

1.6.1 Stereolithography

Stereolithography is to irradiate a laser to a specific site to cause the liquid resin material to polymerize and cure in situ. Afterwards, the vertical height of the object is extended by the lifting of the loading platform. In the process of superposition, the new resin material is smeared on the unprocessed section by the sliding blade, and then shaped by the laser, repeating the above process until the entire object is printed.⁴⁰

UV light was the earliest mainly used laser, but modern high-intensity lasers and focused LED light sources can be used in the visible wavelength range using appropriate types of photoinitiators.⁹ The construction resolution of stereolithography depends on the diameter of laser spot and the absorption characteristics of the photocurable resin. The printed material mainly uses the same epoxy or acrylate resin material. After printing is completed, the support material and the nonpolymerized resin need to be removed.

1.6.2 Inkjet printing

In an inkjet printing process, solid particles are flattened by a roller into a uniform thin layer. And then, glue, chemical adhesive, or UV is used to bond particles layer by layer in a predesigned 3D cross-sectional pattern. The ink is dispensed onto a solid support simultaneously with sacrificial support materials. Once printing is completed, sacrificial support materials can be removed. The printed material mainly uses Acrylonitrile, VisiJet crystal, ABS (acrylonitrile butadiene styrene), ThermoJet 2000,³⁷ and the sacrificial support materials mostly are water-soluble gel-like materials or meltable wax.⁴¹ Besides, multiple materials can be printed at the same time.

1.6.3 Fused deposition modeling

Fused deposition modeling (FDM) involves extruding heated thermoplastic materials from a possible nozzle, so this technique is also known as thermoplastic extrusion. FDM can print biocompatible and inexpensive polymers such as ABS, polylactic acid (PLA, biodegradable polymer), PC, polyamide and poly styrene, polyethylene terephthalate, PC, nylon, and thermoplastic elastomer. FDM materials have good compatibility and cost-effective which has greatly promoted the widespread popularity of 3D printing.

Kadimisetty and colleagues used FDM technology to print a low-cost, sensitive, supercapacitor-powered electrochemiluminescent protein immune array shown in Fig. 1.14. This immunosensor can detect three cancer biomarker proteins in serum within 35 minutes. The entire device costs only 1.2 euros. This device is printed by PLA, including three reagent reservoirs equipped along with inserts, wash reservoir module. The size of the base is 40 mm long × 30 mm wide, and the microfluidic channels are all filled with 160 microliters of liquid. By removing the 3D printed inserts, the liquid in the reservoir can be controlled to enter the detection channel in