2.1. Introduction
A microfluidic system is a small portable system that can complete sample pretreatment, separation, dilution, mixing, chemical reaction, detection, and product extraction. These systems can increase analysis speed and efficiency as well as reduce the consumption of samples or reagents. Moreover, the process of analysis can be completely automated, eliminating human interference, preventing pollution, and allowing for efficient repeating of experiments. The microfluidic chips used in laboratory medicine are representative of this technology. Microfluidic devices have been widely used in chemistry, biology, physics, engineering, and biomedical sciences. Microfluidic devices are considered mobile devices because a sample analysis can be performed entirely within the small, portable device [
15]. The reduced reagent use of microfluidics is also beneficial in areas with scarce resources or challenging terrain. Miniaturization and a low reagent consumption reduce the cost of each experiment, facilitating detection activities in underdeveloped areas [
16].
A POCT is a portable device used for analysis and detection outside a conventional laboratory [
17]. POCTs can provide early and rapid diagnoses for patients with the disease. The advantages of POCTs are their portability and detection speed. POCT technology can also be used for animal disease detection. Pascual-Garrigos et al., improved the loop mediated isothermal amplification (LAMP) assay to realize the early POC detection of bovine respiratory diseases (BRD), so as to reduce the risk of morbidity and mortality of dairy cows on the farm and reduce the economic burden of farmers. It can be seen that POCT technologies can be widely used in different occasions [
18].
Microfluidic systems have had a substantial influence on the applications of POCTs in medical diagnosis. Microfluidic systems have a high sensitivity and can obtain results rapidly; thus, microfluidic systems combined with POCTs are the most portable and least expensive devices for rapid detection [
19].
2.2. Advantages of Microfluidic POC Devices in Medical Laboratory
The detection of biomarkers is key in medical detection. The conventional detection method for protein biomarkers is an enzyme-linked immunosorbent assay (ELISA). ELISA is an ensemble test that requires enough target particles to generate measurable signals as readings. ELISA is primarily a plate-based assay. The method uses a variety of enzyme substances, such as alkaline enzyme, horse radish oxidase and β-galactosidase. Specific substrates, such as ortho-phenyldiamine dihydrochloride, are used to hydrolyze through the top of the enzyme to produce colored results [
20]. ELISA has a high sensitivity in detecting protein content, therefore it is the gold standard method for identifying or quantifying proteins [
21].
ELISA can also detect and analyze infectious pathogens such as viruses [
22]. ELISA has been successfully used to detect the dengue virus [
23], Zika virus [
24], influenza virus [
25], and numerous other viruses, including the SARS-CoV-2 virus [
26,
27].
Conventional ELISA must be improved for it to obtain the high detection sensitivity necessary for protein detection [
28]. Rissin et al., reported a method capable of detecting ultra-low protein concentrations. Each protein molecule was captured by microbeads to form a sandwich complex structure, which was deposited in the reaction chamber array. Enzyme labeling was used to detect which microbeads captured a single protein and which beads did not. Single-molecule ELISA (digital ELISA) based on the singulation of enzyme labels can detect proteins with concentrations
in serum. Digital ELISA uses the same reagents developed for standard ELISA, reducing the development costs.
Figure 1 shows the process of digital ELISA [
29].
On the basis of digital ELISA, Chang et al., proposed a theoretical model that generates signals as a function of time, concentration, and binding constant [
30]. Kan et al., improved the efficiency of the bead reading by improving the efficiency of the bead hole falling and developed a digital ELISA method that can detect IL-17A with 100% efficiency [
31]. In addition, later studies have revealed that capillary force and magnetic force or ionic concentration-polarization (CP)-based biomolecule preconcentration and dielectrophoresis (DEP) can improve the efficiency of bead pore falling, improving the sensitivity of digital ELISA [
31,
32]. For single-molecule protein detection, Rissan et al., developed multiple single-molecule immunoassays that can simultaneously detect multiple protein molecules that were called multiplexed digital ELISA. In this method, the microbeads are divided into different types, and each type of microbead has different specific antibodies that combine with different protein molecules. Each type of microbead also has different fluorescence characteristics, presenting different fluorescence images to distinguish the different protein molecules, which is shown in
Figure 2 [
33]. The most common method for multiple molecular detections is flow cytometry [
34]. xMAP-based technologies are also a recent research pathway for multiple molecular detection technologies. xMAP-based technologies can simultaneously detect, for example, viruses, bacteria, and proteins and are widely used in pharmaceutical, clinical, and research laboratories [
35].
Although digital ELISA can detect multiple protein markers simultaneously and can achieve a higher sensitivity compared with conventional ELISA, these detection methods often require nonportable instruments and are unsuitable for rapid detection, especially in underdeveloped regions. These detection methods must become more widespread, and a method to achieve this goal is microfluidic technologies. Current microfluidic technologies can be combined with other medical detection technologies to achieve better detection results.
Mass spectrometry (MS) based proteomics has become an indispensable technology to interpret the coding information in the genome. Mass spectrometry can identify and accurately quantify thousands of proteins from complex samples [
36]. A combination of MS and a microfluidic chip (μchip–MS) can improve the tools currently used by clinicians. It can provide new methods for early detection, diagnosis, monitoring, and the treatment of noncommunicable diseases (such as heart disease, stroke, cancer, and diabetes) [
37,
38,
39]. The emergence of this platform constitutes a new application of microfluidic chips and compared with conventional immunoassay technologies, μchip–MS has a higher sensitivity and specificity in proteomic analysis and requires less labor and time. Surface plasmon resonance (SPR) is a useful method, which can provide optical and label free detection of target analytes. The SPR detection method has many advantages, such as a low production cost, high detection accuracy and sensitivity, and has been successfully applied to cancer biomarkers and virus detection [
40,
41]. In 2019, Liu et al., used nanoparticle and microfluidic technologies to realize the SPR detection of the target protein. The experimental results were better than those of the single SPR detection method [
42], thus, the inclusion of microfluidic technologies in existing technologies can increase the sensitivity of conventional tests.
Numerous conventional medical detection technologies have incorporated microfluidic POC devices. These devices can detect biomarkers in blood and can obtain excellent detection results [
43,
44]; these characteristics indicate the suitability of microfluidics for disease detection in underdeveloped areas.
2.3. Classification of Microfluidic POC Equipment Types
The first microfluidic device was a micro gas analysis system based on gas chromatography developed by Stanford University in the 1970s. The main components were made from silicon and glass by using photolithography and chemical etching technology [
45]. Microfluidic chips based on polydimethylsiloxane (PDMS) materials have matured, and current commercial microfluidic devices are made of a PDMS polymer by using soft lithography [
46].
Recently, cheap paper and three-dimensional (3D) printing technology have been used for microfluidic chip fabrication, reducing the cost of manufacturing [
47,
48]. These devices can be used for POC testing in underdeveloped areas. Mobile sensors based on integrated microfluidic devices and smartphones are a successful example of combining microfluidics with POC technologies [
49]. Handheld centrifugal microfluidic devices and microfluidic POC devices using DEP technology also constitute special research topics, extending the research into microfluidic POC devices [
50].
Microfluidic equipment based on PDMS, paper, and 3D printing and systems integrating microfluidics and smartphones is described in the following sections. Subsequently, and finally, handheld centrifugal microfluidic devices and microfluidic POC deceives using DEP technology are introduced.
2.3.1. Microfluidic Equipment Made of PDMS
PDMS and thermoplastic molding methods are typically used in microfluidic manufacturing [
51,
52]. The most common commercial microfluidic devices are made of a PDMS polymer. The biggest advantage of the PDMS polymer is its low price, which enables the large-scale production of microfluidic systems. Soft lithography is a non-lithographic method used to copy patterns, simplifying the carving process. It is particularly suitable for fabricating channels in bulk polymers [
53]. PDMS and soft lithography technology are critical for the inexpensive production of microfluidic devices [
54]. Microfluidics require little infrastructure, are inexpensive, and are easy to manufacture, thus, people can learn to use them at low cost, promoting the continued development of PDMS microfluidic devices.
We needed only to make the template of a PDMS microfluidic chip in a dust-free room to carry out continuous replication and production, which really makes it easier for us to design and manufacture the chip in the early stage of the experimental project; however, PDMS-based microfluidic chips still have some shortcomings. First, utilizing PDMS to produce chips is not suitable due to a great demand for the output of chips in large-scale production. In comparison, the method of making microchannels with thermoplastic elastomers is more convenient and can be applied to large-scale microfluidic chip production. Unlike PDMS, thermoplastic elastomers can be processed using many industrial polymer manufacturing technologies, making them cheaper and easier to be mass-produced. Therefore, in the actual application, the manufacturing of microfluidic chips with thermoplastic elastomers is more widely used [
55]. Moreover, PDMS microfluidic chips have a two-dimensional structure. Compared with a three-dimensional structure, the two-dimensional structure is unable to simulate the flow of fluid in the stereoscopic space. Thus, PDMS microfluidic chips require continual improvement [
56].
2.3.2. Paper-Based Microfluidic Devices
Paper-based microfluidic systems have become popular in practice due to their low cost, ease of manufacture, and portability. The first paper-based microfluidic device was designed by Whitesides et al., in 2007 [
57]. Paper-based microfluidic devices are widely used in POC applications such as public health and environmental monitoring [
58].
Because paper is cheap, it is particularly popular for applications requiring a low cost, easy operation, and rapid analysis, such as for disease diagnosis in low and medium resource areas [
59]. In addition, patterning technology is the key factor that renders paper-based microfluidic devices usable in the field of POC. The drawing of microchannels on paper-based microfluidics by wax printing can yield control over liquid flow that is comparable with that of standard microfluidics; this has resulted in paper-based microfluidic technology emerging as a new field of microfluidic device-related research [
60].
Paper-based microfluidic chips can be applied to analyze various analytes in the human body, including urine, saliva, blood, tears, and other bodily or exocrine fluids. These microfluidic chips can also be used for POC detection of diseases [
61], for example, the analysis of tear fluid components can be used for the early diagnosis of dry eye disease. Yetisen et al., designed a portable microfluidic control system that can quantitatively analyze the electrolytes in tear fluid. The control system consisted of a paper-based microfluidic device, a portable readout device and a smartphone for data acquisition. In the monitoring process, the operator only needs to put the tear sample in the paper-based microfluidic device and wait for the capillary in the fluorescent probes to absorb the sample. Finally, after putting the fluorescent probes into the portable readout device, the measurement results can be observed with the smartphone. Experiments on samples with different ion concentrations revealed that the system could be used for early POC diagnosis of dry eye disease, which is shown in
Figure 3 [
62]. For the early detection of acute myocardial infarction (AMI) after the onset of chest pain symptoms, Lim et al., developed a highly sensitive microfluidic paper-based device. The device could simultaneously detect multiple cardiac biomarkers and was effective for both the early and late diagnosis of AMI [
43].
The low cost of a paper-based microfluidic device increases its applicability for POC detection in underdeveloped areas; however, if only paper is used as the main detection material, the microfluidic system often cannot compare with the conventional laboratory detection methods for disease detection.
The paper-based microfluidic device may not be able to detect complex samples accurately, but because paper-based microfluidic devices often use a colorimetric method as the basis for detection, it is convenient to be integrated with smartphones as the detection result can be obtained by analyzing the color of the picture. There is no doubt that due to the very low cost of paper, paper-based microfluidic devices would be the key to many fast and real-time detection applications for a period of time to come.
2.3.3. 3D-Printed Microfluidic Devices
In general, 3D printing technologies have influenced the development of microfluidics [
63]; these technologies are automated, eliminating the human resources required for manufacturing conventional PDMS microfluidics [
64], and have not only reduced costs but also achieved a good resolution and throughput, increasing their recognition by the scientific community [
65,
66]. High latitude means that the real flow of fluid can be simulated more accurately; thus, microfluidic devices with more dimensions are currently being developed [
67]. Furthermore, 3D printing methods have substantial commercial potential and can rapidly produce prototype products, increasing the frequency and efficiency of experiments, and enabling the rapid commercialization of experimental technologies [
68,
69].
In fact, microfluidic POCT devices based on 3D printing technology have been applied in practical applications. Song et al., developed a structured platform with a built-in microfluidic detection box. The platform uses reverse-transcription loop-mediated isothermal amplification (RT-LAMP) technology to realize the rapid detection of the Zika virus. The detection box is manufactured by 3D printing technology and it does not need to be heated by an external power supply, but through chemical heating to meet the conditions required for the experiment. Only saliva samples were needed to detect the Zika virus [
70]. Additionally, Kadimisetty et al., developed a microfluidic POCT device with a simple operation and low cost for nucleic acid amplification tests to diagnose infectious diseases [
71].
Three-dimensional printing technology has an unprecedented processing capability, providing new opportunities in microfluidic POCT by accelerating experiments and the commercialization of the results. Although 3D microfluidic chips are still less precise relative to traditional PDMS microfluidic chips, 3D printing technology is useful for producing microfluidic POCTs. Due to the rapid manufacturing capacity of 3D printing technology, large-scale 3D-printed microfluidic equipment production has become possible. In this approach, 3D-printed microfluidic POC devices can play a significant role in underdeveloped areas.
2.3.4. Mobile Sensors Based on Integrated Microfluidic Devices and Smart Phones
The continual development of microelectronics since the beginning of the 21st century has resulted in smartphones; these handheld devices now have a processing power equivalent to that of a small computer and can smoothly complete simple data processing tasks. Smartphones can replace conventional computers for data processing, computing, and data collection in underdeveloped areas [
72,
73,
74]. In particular, combining smartphones with microfluidic devices is a comprehensive solution for the new generation of mobile sensing applications, known as MS
2. Portable mobile sensors based on microfluidic devices integrated with smartphones are useful for disease detection in remote areas.
The Zika virus is transmitted by infected mosquitoes and can cause fever, headache, rash, and joint muscle pain, especially for newborns, making timely detection necessary. Kaarj et al., developed a wax paper microfluidic chip using reverse transcription loop-mediated isothermal amplification (RT-LAMP). The color change induced by ZIKV RNA in the microfluidic detection area is observable within 15 min, and virus detection can be completed with a smartphone camera [
75]. Urine also contains many markers that can be used to detect disease. Jalal et al., developed a microfluidic device combining reagent paper and polycarbonate (PC) plastic materials. A smartphone camera can capture colorimetric changes after the reaction of the analyte, and algorithms were designed to detect the chemical components in urine. Thus, diagnosis can be achieved based on urine composition [
76].
MS2 applications are easy to use, enabling untrained users to obtain reliable test results and we believe that MS2 is a valuable and promising technology; however, many difficulties remain in this emerging field. More complex detection experiments require the introduction of experimental samples into existing MS2 systems as well as additional interface equipment for the detection. Therefore, applications of MS2 still require improvement in MS2 devices and development in the relevant industries.
2.3.5. Handheld Centrifugal Microfluidic Devices
Human blood analysis yields important health information. The analysis of serum biomarkers is an essential tool for modern medical diagnosis and bio information extraction [
77]. To prepare blood samples, centrifugation is the first critical step and because an excess of red blood cells affects the background fluorescence and interferes with such detection [
78], the plasma, serum, and parasites must first be separated [
79]. Commercial centrifuges are expensive, bulky, require supporting equipment, such as power systems, and require trained personnel to carry out regular maintenance and inspections. Thus, conventional centrifuges are unsuitable for POC diagnosis in remote areas, and portable centrifuging equipment that can be used in resource-limited environments must, therefore, be developed [
80].
Bhamla et al., developed an ultra-low-cost manual centrifuge called “paperfuge”. The cost of each centrifuge is no more than USD 0.20, and the device is very small with a weight of only 2 g. It can reach speeds of 125,000 rpm for 30,000×
g of centrifugal force; thus, it is can easily separate whole blood. Centrifugal microfluidic devices can be developed by using 3D printing technology, PDMS, and plastic.
Figure 4 represents the classifications of handheld microfluidic centrifuge [
81]. These devices can be quickly put to use in resource-poor environments, providing a new direction of development for microfluidic POCTs.
The paperfuge has some shortcomings. For example, it can centrifuge whole blood but it cannot effectively collect relevant samples. Li et al., improved the system and designed a paper-based integrated diagnosis system. The system effectively integrates the paperfuge and an immunoassay unit that can analyze biomarkers in blood while centrifuging. ELISA-level analysis of carcinoembryonic antigens (CEA) and alpha fetoproteins (AFP) in human serum was achieved. The system is called the ‘Fully Integrated hand-powered Centrifuge and Analysis paper-based microfluidic devices’ (FICA-μPADs). The operation of this system is also very simple. It only needs to settle the blood sample of the patient’s finger in the capillary tube of the hand-powered centrifuge for centrifugation, and then complete the corresponding ELISA operation through the immunoassay unit, before finally reading the corresponding signal through the portable inter-reader to obtain the experimental results. Crucially, in the absence of any process optimization, the manufacturing cost of a single unit was less than USD 0.50, which is suitable for POC detection.
Figure 5 depicts the structure and operation of the FICA-μPADs [
82].
The development of 3D printing technology has resulted in new methods of rapid POC detection [
83]. Byagathvalli et al., designed the 3D-Fuge using 3D printing technology, and it could process samples up to 2 mL, overcoming the limitation of traditional handheld centrifuges can only process 20 μL samples each time. Up to four samples could be stored and centrifuged simultaneously to achieve nucleotide extraction. The production cost of these devices is less than USD 1.00, and they can be produced with any type of 3D printing equipment. Thus, this design is suitable for large-scale production [
84].
Figure 6 represents the production of the 3D-Fuge.
Since Bhamla et al., invented an ultra-low-cost manual centrifuge, the development of similar devices has accelerated. PDMS, plastic, and 3D-printed microfluidic centrifuges have been developed, broadening the potential applications of microfluidic POCTs.
However, these handheld centrifuges still have the inherent drawbacks of small-capacity systems. Additionally, many people doubt whether small, low-cost equipment can perform effectively. The market for and profit margin of handheld centrifuges is also low, limiting the development; however, these devices could be used in areas with limited resources and become more popular with time [
85].
2.3.6. Microfluidic POC Devices Using DEP Technology
Dielectrophoresis (DEP) is a label free, noninvasive, independent, rapid and sensitive particle manipulation and characterization technology. In the past decades, the progress of MEMS technology has made the biomedical application of DEP possible. The system developed through these technologies can use a small number of biological samples and reagents for analysis and detection. Lab on chip (LOC) equipment capable of processing micro/nano samples has promoted the development of biotechnology and chemistry.
DEP is a major electrical phenomenon in electrical technology. DEP can deal with charged particles and dielectric particles at the same time, such as cells and bacteria. DEP is a phenomenon of relative motion between suspension and medium under a non-uniform electric field. The force leading to this motion is called a dielectrophoretic force [
86]. Designing and analyzing new and more advanced LOC equipment require accurate modeling and simulation of sample/particle dynamics in such devices. Through dielectrophoretic force, we can realize the control of small particles, so as to realize more possibilities [
87].
Blood is mainly composed of plasma and cells, and there are a variety of cell types in blood. Therefore, blood contains significant information about systemic function. Moreover, the dielectric properties of cells can be separated by Dep. Han et al., designed a laterally driven continuous dielectric electrophoresis (DEP) differentiator, which can effectively separate red blood cells and white blood cells in whole blood samples [
88]. In addition, DEP has many applications, including the separation of fetal cells from maternal blood, microbial separation, and stem cell and cancer cell separation [
89].
POC detection equipment has been widely used in clinical laboratories and in patients’ disease detection. The development of POC equipment can provide a faster and low-cost method for disease diagnosis. With the development of micro manufacturing technology, cell separation and concentration based on DEP have become possible in practice and because DEP is simple to operate, does not need skilled technicians, has low voltage requirements and does not need high use requirements, DEP has the potential to be used in portable POC medical devices.
Chen et al., designed an integrated microfluidic platform that uses optically induced dielectrophoresis to separate and recover extracellular vesicles, which has high potential in cancer-related exosomal protein and miRNA analysis applications [
90]. Sahin et al., proposed a microfluidic device which can use the DEP method to separate red blood cells and bacterial cells [
91].
Although DEP technology and microfluidic technology have many applications, there are still many challenges to realize a POC application. Firstly, the accurate measurement of the dielectric performance of the battery is an important problem in the design of microfluidic POC equipment based on DEP. Secondly, the differences of the cell molecules and physiological states will also lead to changes of the dielectric properties, which leaves many applications in the experimental stage only and which cannot be put into use. Although there are still some problems to be solved, DEP technology can undoubtedly play an important role in microfluidic POC equipment, so that the microfluidic POC equipment has a wider range of applications. As these problems are overcome in the future, DEP technology can be used to manufacture many microfluidic POC devices for the early diagnosis and prognosis of diseases.
Table 1 describes the overview of the microfluidic POC equipment types.
2.4. Advantages over Non-Microfluidic POC Devices
POCT can provide early and rapid diagnosis results for patients who need to detect related diseases. The advantages of POCT lies in its portability and rapid detection ability. POC equipment has formed a large commercial scale and has been applied in different disease detection fields [
93].
It can be seen that there are many similarities between the microfluidic system and POCT. In fact, the microfluidic system has changed many applications of POCT in medical diagnosis. A microfluidic system has a high sensitivity in detection and can quickly obtain detection results, therefore, the combination of a microfluidic system and POCT could be used to design more portable and low-cost devices for rapid detection. The integration of microfluidics in medical point detection has significantly changed disease diagnosis and pathogen detection. Being easy to use, having no need for skilled personnel or heavy equipment, requiring a low sample size and delivering fast results makes POCT equipment an indispensable part of the healthcare industry.
There are many on-site detection and diagnosis POC devices in the market, including but not limited to glucose detection and infectious disease monitoring; however, among the POC detection devices, microfluidic POC devices with POC integrated microfluidic technology occupy the vast majority of the share, because microfluidic POC devices can allow fluid operation and detection to be carried out in the same device, which is more integrated than non-microfluidic POC devices. At the same time, many mature application technologies in the laboratory, such as ELISA and lamp, have been successfully applied to microfluidic devices, which greatly increases the disease detection ability of microfluidic POC devices. At the same time, there are many kinds of microfluidic POC devices, which can adapt to different detection environments, thus, it is easier to commercialize.
Table 2 summarizes the advantages of microfluidic POC devices over non-microfluidic POC devices.