Recent advances in the design and development of microfluidics (MFs) devices have made it possible to miniaturize conventional biochemical laboratory protocols into a microchannel networking system, which has emerged as an efficient and cost-effective tool. Biomedical microdevices include integrated structures consisting of numerous micro- and nano-sized integrated devices, where many processes from particle manipulation to sensing take place in the platform. Although different types of microfluidic devices can perform similar tasks in biomedical applications, passive microfluidic systems are mainly used for particle manipulation ^{[1][2][3][4][5]}[1,2,3,4,5] and mixing liquids ^[4][6][4,6], while active types contribute more to particle trapping ^{[7][8][9][10][11][12][13][14][15][16][17][18][19]}[7,8,9,10,11,12,13,14,15,16,17,18,19] and sensing ^{[20][21][22][23][24]}[20,21,22,23,24]. Passive devices are governed by diffusion, inertial forces, secondary flows, and geometry-induced turbulence and particle manipulation; active microfluidic devices generate streams depending on external energy to disturb particles or fluids inside microfluidic devices. Depending on the geometric design, mixing ratios of fluids could be relatively high, and separation of particles, which have different sizes and densities under the influence of internal forces, can reach high-efficiency values in microfluidic channels. External forces due to acoustic pressure fields, electric fields, magnetic fields, thermal fields, pressure fields, and optical fields could manipulate biological or chemical particles and mix fluids in biomedical applications. In addition, some active manipulation techniques with functional surfaces coated on the transduction area can also sense some unique biological structures, such as DNAs and biomarkers. Briefly, passive MFs, where internal forces are effective, and active MFs, which perform operations under the influence of external forces, are two categories regarding microfluidic devices. There are several biomedical applications in microfluidics devices. One of the most promising applications of microfluidics in biomedical sciences is the diagnosis of diseases, including cancer diagnosis and infectious diseases. However, further development of microfabrication permits employment of microfluidics devices in disease modeling, tissue engineering, and organ-on-a-chip. Moreover, microfluidics–biosensing technology has become popular for applications such as point-of-care testing, biosensors, and cell manipulations [25].

2. Microfluidics in Diagnosis

2.1. Cancer Detection

Cancer, which can occur in any tissue, is one of the most common and deadly diseases in the world ^[26][176]. The importance of timely diagnosis of cancer is indisputable. Today, methods such as positron emission tomography, magnetic resonance imaging, and computed tomography are used for diagnosing and staging cancer masses ^[27][177]. There is certainly a need for novel approaches in the diagnosis and treatment of cancer, as these methods rely on patients’ exposure to high doses of radiation or chemotherapeutics ^[28][178]. Microfluidics, which involve miniaturized devices and precision analysis techniques, are promising for biomedical applications such as cell culture, drug delivery, DNA amplification, and point-of-care (POC). Chemotherapeutics used in cancer treatment often cause many side effects. By attaching any imaging or locating agent to nanoparticles, both treatment and diagnosis can be achieved. Microfluidic systems used for this purpose in this way include theranostic nanoparticles ^[29][179]. Theranostic nanoparticles can be used to monitor drug delivery, drug release, efficacy, the determination of cancer stage, and the mediation of drug delivery at the appropriate dose ^[30][180]. Nanocarriers loaded with chemotherapeutics cause the least systemic toxicity while delivering the drug to the target tissue. For instance, fluorescent 5-aminolevulinic chitosan nanoparticles, combined with alginate and conjugated with folic acid, were designed for endoscopic detection of colorectal cancer cells. These nanoparticles entered tumor cells via the folate receptor, accumulated protoporphyrin IX in the cell with the 5-aminolevulinic acid released from the lysosome, and, thus, were proved to be an ideal vector for photodynamic detection ^[31][181]. Ryu et al. ^[32][182] demonstrated that cathepsin B-sensitive fluorogenic peptide probes conjugated to the surface of glycol chitosan nanoparticles could filter metastatic cells from healthy ones in three mouse models. Another research effort includes the case of the use of hyaluronic acid, iron oxide, and homocamptothecin nanoparticles in human squamous cell carcinoma, both in in vitro and in in vivo studies ^[33][183]. Baghbani et al. ^[34][184] showed that ultrasound-mediated treatment of doxorubicin-loaded alginate-stabilized perfluorohexane nanodroplets caused tumor regression in mice with breast cancer. A study performed a photodynamic therapy system with near infrared/magnetic resonance imaging by loading Fe3O4 nanoparticles onto redox sensitive chlorine-e6 conjugated dextran nanoparticles to identify breast cancer cells ^[35][185]. Quantum dots are avant-garde in vivo imaging tools. For example, Shi et al. ^[36][186] developed luminescent magnetic graphene oxide quantum dot nanoplatforms to identify HEPG2 hepatocellular carcinoma from infected blood samples. In another study, quantum dots and anti-cancer drugs were loaded together on lipid carriers to feel and treat H22 cancer cells ^[37][187]. Microfluidic systems provide models for examining and eliminating essential mechanisms such as apoptosis, drug resistance, invasion, and metastasis in cancer. As an example, Han et al. ^[38][188] developed a redox and pH-sensitive system with mesoporous silica nanoparticles loaded with doxorubicin to overcome drug resistance in breast cancer. In another study, a paclitaxel and lonidamine loaded EGFR targeted polymer nanoparticle drug delivery system was developed for the combined treatment of drug-resistant cells in breast cancer ^[39][189]. In order to increase apoptosis and reduce drug resistance in lung cancer, an inhalation system containing siRNAs, targeting MRP1 and BCL2 and mesoporous nanoparticles loaded with doxorubicin and cisplatin, was designed ^[40][190]. Furthermore, microfluidics also automated tumor cell culture, enabling the creation of multicellular co-cultures and mimicry of cancer tissue with organoids ^[41][191]. For example, a multi-organ microfluidic chip mimicking lung cancer is physiologically suitable for recapitulating the metastasis process ^[42][192]. Nguyen et al. developed electrical impedance through a three-dimensional matrix microfluidic system to define single cancer cell migration ^[43][193]. Apart from these, research efforts examining tumor cell extravasation ^[44][194], invasion ^[45][195], and blood-tumor barrier models ^[46][196], with microfluidic platforms, have also been conducted. Microfluidic systems are also employed to specify cancer biomarkers such as CTCs, ctDNA, exosomes, ncRNA, and various cellular metabolites or proteins ^[41][191]. In addition, routine measurement of biomarkers in small amounts of fluid samples from cancer patients contribute to personalized medicine. As CTCs mostly express epithelial cell adhesion molecules, antibodies on CTC chips were used for their selection from blood ^[47][197]. In the following stages, debulking, inertial focusing, and magnetic separation steps were added to this system, which was named as CTC-iChip ^[48][198]. Ganesh et al. designed another microfluidic chip based on a ZnO electrode and pH sensors for the isolation of CTCs ^[49][199]. The rm chip combined two approaches based on cell size or immunoaffinity with Rhipsalis (Cactaceae)-like hierarchical structures ^[50][200]. However, the monolithic CTC-iChip is also noteworthy, which distinguishes CTCs using epitopes such as cytokeratin, HER2, and prostate-specific antigen ^[51][201]. In Western blotting with microfluidics, expression in patient-derived CTCs was profiled with an eight-plexed protein panel ^[52][202]. Aptamer nanovectors, used in CTC membrane protein profiling, identified different breast cancer subpopulations by multispectral orthogonal surface enhanced Raman spectroscopy analysis ^[53][203]. Microfluidic technologies such as acoustic waves ^[54][204], oscillating flow ^[55][205], Dean vortex flow ^[56][206], and cluster-chip ^[57][207] that can separate CTCs from blood in a label-free manner are also worth mentioning. Moreover, the immunoaffinity ^[58][59][60][208,209,210], nanomembrane filter ^[61][211], dielectrophoretic system ^[62][212], lateral displacement, and acoustic fluid ^[63][213] techniques were used to isolate exosomes. In addition, techniques for detecting exosomes include the fluorescence electrochemical technique ^[64][214] and mass spectrometry ^[65][215]. Among these techniques, the ExoPCD-chip, which combines the isolation and electrochemical analysis of exosomes, and the herringbone chip (HB chip), stand out due to their superior performance ^[66][67][216,217]. The liberated ctDNAs from tumor cells reflect mutation degree and progression of cancer. The microfluidic solid phase extraction (μSPE) device produced by Compos et al. includes the immobilization, extraction, and replication of cfDNA, and it can also be produced as a low cost platform ^[68][218]. In order to isolate cfDNA from serum while minimizing degradation, a rapid and automated microfluidic has developed that combines all three process of plasma separation, residual protein lysis, and cfDNA elution ^[69][219]. However, the ncRNAs play a regulatory role in tumor progression at the transcriptional and translational level. In this regard, an oil-saturated PDMS microfluidic system with droplet digital PCR was developed for lung cancer miRNA quantification ^[70][220]. By the multiplex qRT-PCR method developed on a microfluidic chip, 384 miRNAs, which are important in the diagnosis and prognosis of prostate cancer, could be purified ^[71][221]. Protein-structured substances such as growth factors, cytokines, and hormones secreted by cancerous cells, as a result of the increase in proliferation, are ideal diagnostic tools. Researchers designed a microfluidic integrated microarray in a single platform to identify PSA, TNF-α, IL-1β, and IL-6 proteins in serum samples from prostate cancer patients ^[72][222]. Fan et al. reported a blood barcode chip integrated microfluidic system that can rapidly measure a wide panel of proteins from blood ^[73][223].

2.2. Cardiovascular Disease Detection

Cardiovascular diseases (CVDs), such as stroke, coronary artery, and hypertension, arise from dysfunctionality of the heart and its relevant blood vessels. CVDs are a major cause of premature death worldwide. Social, environmental, cardiometabolic, and behavioral risk factors are some leading determinants of CVD ^[74][75][224,225]. However, aging is the essential factor of CVDs due to the induction of oxidative stress, which results in variations in biological reactions and reactive oxygen species (ROS) ^[76][226]. Diagnosis of CVD is crucially important to decrease the mortality rates, and several detection techniques depending upon biomarkers or molecular imaging (MOI) are currently applied in clinics. Nevertheless, improvements in the accuracy, sensitivity, and specificity of the current diagnostics for early-stage detections of CVDs are necessary to establish effective diagnostic systems ^[77][227]. Microfluidic diagnostic platforms present favorable features such as portability, fast-responsive analysis, and low reagent use to detect CVD biomarkers. For this purpose, microchannels were modified by particular antigens to determine CVD-associated biomarkers, and several studies have been performed ^{[78][79][80][81][82][83]}[228,229,230,231,232,233]. Plenty of blood-borne biomarkers such as cardiac troponin I (cTnI), fibrinogen, and C-reactive protein (CRP) are associated with CVDs. However, currently used assays for diagnosis are costly, time-inefficient, and susceptible to batch-to-batch changes. Sinha et al. built a portable microfluidic device with the integration of aptamer probes and field-effect transistor (FET) based sensor arrays ^[84][234]. The proposed device can identify four CVDs related biomarkers such as CRP, cTnI, fibrinogen, and N-terminal pro-b-type natriuretic peptide (Nt-proBNP) in only five minutes from small volumes of clinical samples and present favorable results for novel POCT of CVDs. Heart failure (HF) is a common CVD, and the changes in the level of NT-proBNP in the blood are related to the diagnosis of HF. However, current clinical CVD detection methods are not precise enough to evaluate severity and progression of HF according to one single cut-off value of the NT-proBNP biomarker, whereas a rising pattern for long time periods could be a signal for HF. Therefore, POC monitoring of NT-proBNP is vital to prevent HF. As an example, Beck et.al. developed a microfluidic biosensorchip to determine changes in the level of NT-proBNP by modification of silver nanoparticles (AgNPs) as a label ^[85][235]. For this purpose, laminar flow assay (LFA) and electrochemical analysis were combined by flow injection analysis (FIA) while detecting of antibody modified AgNPs. The developed biosensor allows for precise detection of NT-proBNP from a finger prick sample volume at home with simple use. Acute myocardial infarction (AMI) is an extensively encountered CVD disease that is life-threatening and sometimes challenging to diagnose since the symptoms could be confused with other diseases. For this reason, Yin et al. demonstrated a snail-shaped microfluidic platform to detect myoglobin (Myo), cTnI, and creatine kinase-MB (CK-MB) biomarkers for diagnosis of AMI. They designed a microfluidic chip by utilizing a chemiluminescence (CL) detector and coating the middle of the chip, which has reaction layer based on particular antibodies. Thus, they obtained a POCT candidate which is able to diagnose three AMI-related biomarkers with higher sensitivity and within a short time of period ^[86][236].

2.3. Respiratory Infection Detection (SARS-CoV-2)

A novel coronavirus disease (COVID-19) was first reported in late 2019 and resulted in the infection of over 66 million individuals approximately within a year after its discovery ^[87][88][237,238]. SARS-CoV-2 is a RNA virus that could quickly spread among individuals in intimate interaction through respiration and develops in certain regions such as the nasal cavity, pharynx, and lower respiratory tract ^[89][239]. One of the essential stages in controlling the spread of SARS-CoV-2 is early diagnosis ^[90][240]. As a result, researchers have been working to have a rapid, inexpensive, portable, and sensitive alternatives for detection. Microfluidic-based detection strategies have been widely developed for point-of-care COVID-19 disease detection throughout the pandemic. These strategies could be classified according to detection mechanism in microfluidic devices: antigen detection, anti-SARS-CoV-2 antibody detection, and nucleic acid detection ^[91][241]. In another investigation, Ho et al. ^[92][242] designed a disposable point-of-care digital microfluidic cartridge to detect the N gene in SARS-CoV-2 by utilizing real-time quantitative polymerase chain reaction (qPCR). According to the study, the DMF cartridge demonstrated uniform droplet formation, homogeneous temperature control, and a suitable fluorescence readout, enabling qPCR POC testing. Recently, paper-based microfluidic devices have been also emerging. Akarapipad et al. ^[93][243] utilized a paper based-microfluidic device for comfortable and facilitated detection of SARS-CoV-2 from saliva samples. Evaluation of the flow profile allowed for assessing infection status. The change in surface tension and capillary flow velocity resulted within particle-target immunoagglutination through the channel, which was consequently determined using a smartphone. Similarly, Kim et al. ^[94][244] introduced airborne droplets that could be trapped directly on a paper microfluidic device without additional apparatus in less than 30 mins, including capture-to-assay time. The working principle was based on the 10% human saliva samples with SARS-CoV-2 sprayed into the air to produce liquid droplets and aerosols. Subsequently, an antibody-conjugated particle was introduced to the paper channel, and the immunoagglutinated particles on the paper microchip were quantified using a smartphone-based fluorescence microscope. Therefore, SARS-CoV-2 could be identified directly from the air with a portable and low-cost approach. Furthermore, the detection of SARS-CoV-2 N protein utilizing a paper channel was reported, which demonstrated a paper-based enzyme-linked immunosorbent assay on the chip and visual detection and sensitivity of the N protein ^[95][245].

3. Drug Discovery and Delivery

Patients usually ingest drugs for treatment. In the traditional methods, high doses of drugs, high toxicity, and often side effects occur. Drug delivery systems aim to minimize cytotoxicity by increasing bioavailability and specificity. Microfluidic devices can also be platforms for drug delivery that are easy to control, scale, and replicate ^[96][247]. Nanotechnological developments facilitate the controlled release and targeted delivery of drugs by encapsulating them ^[97][248]. While experimental studies demonstrate the potential of drug delivery systems, it takes a long term to develop the clinical trial with the efficacy and safety standards that patients can use. Microfluidic systems allow people to control the effectiveness of drug delivery systems. Carriers are formed by encapsulating drugs with organic, inorganic and hybrid molecules. Dendrimers, micelles, liposomes, various polymers, and metallic nanostructures are frequently used in drug delivery systems ^[98][249]. Drugs that are less soluble in water become more soluble by the conjugation of drug and polymer complexes. However, the effectiveness of nanocarriers varies depending on their size, shape, and physical/chemical properties ^[29][179]. These targeted carriers must be biodegradable and compatible, as well as responsive to stimuli ^[97][248]. Doxil is the first successful PEGylated liposomal carrier approved by the FDA in 1995 and has fewer side effects and more toxic to tumor cells than doxorubicin ^[29][179]. InFed is an iron complex containing dextran and has been used to treat iron deficiency ^[99][250]. Another drug–polymer complex is Abraxane, nanoparticles of paclitaxel coated with human albumin ^[100][251]. PEGylated lipid nanoprticles also had success in the delivery of RNA therapeutics, such as Onpattro and Comirnaty ^[101][102][252,253]. In addition, various agents can be attached to the polymer backbone or functional side groups that facilitate targeting and imaging of nanoparticles. Spark microfluidic systems used in the fabrication of particles are divided into single, mixed, and fully aqueous emulsion templates. While traditional methods such as emulsion, dispersion polymerization, and spray drying are less effective in particle production, technologies such as droplet and flow lithography, electrohydrodynamic co-spraying, photolithography, soft lithography-based printing, and micro molding are considered to be more innovative ^[103][254]. Each phase in these systems ensures the production of particles in the appropriate size and shape, as well as is the desired physical, chemical, and biological properties. In this way, thy can be used to develop particles with complex structures such as core-shell, multi-core-shell, janus, and porous ones ^[104][255]. The formation of particles from monodisperse droplets occurs using various methods such as polymerization, ionic crosslinking, and solvent evaporation ^[103][254]. The chemical structure of drug targets must be among already characterized macromolecules such as nucleic acids, enzymes, proteins, and lipids. Thus, microfluidic systems can also be used for new drug discovery ^[105][256]. Microfluidic systems not only improve the drug delivery with precise fluid control, but they also provide benefits for testing drugs before clinical use ^[96][247]. For the clinical analysis of the drug delivery systems produced in experimental studies, the animal models should be primarily studied. The first limitations in directing drug delivery systems to the desired target are the barriers in the body at the systemic, microenvironmental, and cellular levels ^[106][257]. For example, ellipsoids, discoid-shaped nanoparticles, and nanorods adhere better to blood vessels than spheres ^[107][258]. Inhalation of nanoparticles allows rapid passage into lung tissue to avoid extravasation ^[108][259]. However, mucus barriers can pass smaller particles while larger ones are filtered out. Methods such as receptor-mediated transcytosis and glucose transporters can be used to cross the blood-brain barrier ^[109][260]. Oral administration of polymeric nanoparticles was found to be more active with the gastrointestinal tract than normal drugs ^[110][261]. Platforms have also been developed that allow the drug to be released only under certain pH and temperature conditions ^[111][262]. Enhanced permeability and retention effects in cancer tissues are also utilized for the accumulation of drugs. In addition, negatively charged particles are more difficult to adhere to the cell membrane, while positive ones can cause cytotoxicity in the cell ^[112][263]. Preclinical testing of drugs can be achieved by recapitulating the barriers in the body with microfluidic systems such as organ-on-chip and body-on-chip platforms. Microfluidics allows drug specificity and adjustable doses or combined drug strategies for personalized therapy ^[106][257]. Microfluidic devices can be used in disease remodeling and to increase drug accessibility to cancer cells ^[113][264]. It is also possible to evaluate them with their biomarker roles ^[114][115][265,266]. For example, graphene oxide nanoflakes have been used in the detection of pancreatic cancer due to their capacity to bind albumin in plasma ^[116][267]. The use of magnetically guided or Au nanoparticles is common. The use of photothermal CAR-T cells in solid tumors is also of interest ^[117][268]. Microfluidic systems have been also used for approaches such as gene therapy and gene editing ^[118][269].

4. Disease Modeling

Human diseases are controlled by sophisticated mechanisms that are intrinsically difficult to understand since there is a limitation in direct observation of interference of biological molecules. Hence, methods for disease modeling are of considerable interest for understanding disease pathophysiology and the development of advanced therapeutic strategies. The two-dimensional cell culture method has some drawbacks, including the possibility of cell morphology and polarity changes, which might cave to interruption in cellular-extracellular communication ^[119][270]. Moreover, the monolayer structure of two-dimensional cell culture leads to unrestricted availability to reach to the optimum medium, oxygen, and signal molecules. Significantly, the accessibility of the nutrients, oxygen, or/and signal molecules for cancer cells in a living organism could be changeable due to the inherent structure of the tumor ^[120][271]. On the other hand, three-dimensional cell culture platforms offer an opportunity to investigate complicated interactions by emulating a physiological environment that approximates the in vivo environment observed in patients ^[121][272]. The reason for the similarities between responses of animal models and tumor spheroids against drugs could be the increase in cellular interaction via adhesion ^[122][273]. Disease-on-chip models, however, attract widespread interest due to their potential emulation of the disease microenvironment, regulatory factors, and physiological circumstances surrounding organs. The shear force applied by the environment, cell patterning, cell–cell communication, and other factors can be controlled for mimicking the organ and relevant diseases ^[123][274]. Furthermore, these platforms offer multi-omic analysis and investigation of the primary biophysical and chemical reasons for cancer formation and cellular-extracellular conditional growth microenvironment ^[124][275].

4.1. Cancer Modeling

The studies on microfluidic cell culture technology in the literature pointed out different aspects of cancer modeling, including cancer cell invasion ^{[125][126][127][128][129][130][131][132]}[276,277,278,279,280,281,282,283], intravasation ^[133][134][284,285], extravasation ^{[135][136][137]}[286,287,288], and tumor microenvironment modeling ^[138][139][289,290]. Cell invasion refers to cell motility, including attachment, proteolysis, and relocation of cancer cells, which may result in cancer metastasis ^[140][291]. Conventional laboratory strategies, mainly two-dimensional approaches, are limited to providing adequate quantitative data, including multifactors for the determination of cell–matrix interaction, cell–cell communication, and cell invasion ^{[141][142][143]}[292,293,294]. Since multiple factors exist in tumor invasion, finding and distinguishing the function of such environmental factors are required to comprehend the intercellular dynamics of the tumor invasion. One of the significant factors is the interaction between the tumor environmental niche and human immune system. As an example, Surendran et al. ^[144][295] emulated the tumor-immune microenvironment (TIME) as a three-dimensional platform, which represented the role of neutrophils along with chemotaxis and neutrophil extracellular traps (NETosis) in the invasion of ovarian tumor cells. In another study on the cancer invasion, Samandari et al. ^[145][296] engineered a stand-alone microfluidic gradient generator to characterize transmission of the chemotactic factors over the hydrogel region, which utilized the hydrogel barriers to isolate the cell culture chamber from the signal channels. Moreover, the proposed detachable PDMS microfluidic chip enabled pump free activity and low-pressure operation, thereby preventing potential leakage. Besides, Amirabadi et al. ^[146][297] produced a two-layered three-dimensional environment, serving for invasion of different types of breast cancer cells consisting of wild type, mutated, and promoter hypermethylated E-cadherin containing cells. According to the results, MDA-MB-231 cells, as single cells, invaded the matrix more than MCF-7 and CAMA-1, while CAMA-1 cells unitedly invaded less than MCF-7. The cancer metastasis process could be defined as an intravasation, where transportation of the cancer cell through the blood vessel occurs. Cancer cells tend to intravasate at locations where the shear stress is lower through the vessel ^[147][298] and, therefore, trigger formation of angiogenesis-caused capillary branches ^[148][299]. Yankaskas et al. ^[149][300] displayed shear stress responses of normal and tumor cells throughout the migration to intravasation. Therefore, they utilized a particular molecule, which behaved as fluid shear sensor of the cells. The microfluidic platform modeled the transition from migration to intravasation, where the cells moving through longitudinal channels moved into an orthogonal channel with induced shear stress. However, recapitulating invasion and intravasation at the same time in cancer modeling is compelling due to the complex tumor microenvironment. Nevertheless, Nagaraju and Truonginvasion et al. ^[150][301] designed a microfluidic tumor-vascular model, including a three-dimensional tumor, stroma, and vasculogenesis to investigate invasion and intravasation in a single device. Besides, there have been various studies on emulated tumor microenvironments, such as tumor-on-a-chip or cancer-on-a-chip platforms ^[151][152][302,303]. For instance, Chi et al. ^[153][304] introduced a three-layered L-TumorChip platform, combining tumor stroma and microvasculature and investigated the effect of different stromal cells on cancer cell development and the stromal effects on drug responses. In another study, Strelez et al. ^[138][289] presented the colorectal cancer (CRC) on-a-chip platforms with the facets of CRC, stromal cross-talk, and mechanical force. Moreover, Fridman et al. ^[154][305] mimicked the breast tumor microenvironment, where tumor cells, immune cells, and fibroblasts were encapsulated into different hydrogel scaffolds within a microfluidic platform. Similarly, Haque et al. ^[139][290] used patient-derived organoids and mimicked pancreatic ductal adenocarcinoma by exhibiting epithelium–stroma communication and controlled the microenvironment-modulating agents in a lab-on-a-chip model.

4.2. Neurological Disease Modeling

Microfluidic modeling platforms have been rapidly developed over the past decade, allowing the advancement of in vitro human nervous system modeling and associated disease models. Central nervous system (CNS) modeling involves handling axons, synapses, and neuronal networks, as well as conditional growth in cell culture for mimicking neural diseases such as Parkinson’s disease (PD), Alzheimer’s disease (AD), and multiple sclerosis (MS) ^[155][306]. As an example, Virlogeux et al. ^[156][307] established a microfluidic model to ascertain Huntington’s disease (HD) corticostriatal network to understand the uncertain role of pre-and postsynaptic neurons during the first stage of the HD development. Since the overall process is unclear, it is formidable to come up with auspicious treatment strategies. Hence, Hyung et al. ^[157][309] established a microfluidic platform exhibiting the overall mechanism of myelination, demyelination, and remyelination under the favor of cocultured motor neurons and primary Schwann cells. Significantly, the emulated microenvironment enabled the preservation of long-term coculturing over 40 days. Similarly, Dittlau et al. ^[158][310] studied the effects of ALS-causing mutations in an in vitro microfluidic model. The results demonstrated that FUS mutations caused by ALS consequently led to poor neurite regeneration over axotomy and neurite outgrowth. They concluded that a selective HDAC6 inhibitor-enhanced neurite outgrowth and regeneration was at play and, therefore, that HDAC6 inhibition could be used to treat ALS.

4.3. Pulmonary/Lung Disease Modeling

Lung-on-a-chip platforms seek to model the evaluation of drug toxicity under physiological conditions and to provide technical assistance for drug screening and personalized diagnosis and therapy ^[159][311]. Furthermore, various research studies focused on developing lung disease models, such as lung inflammation, injury, and other pulmonary diseases due to the complexity of the lung anatomy and physiology, which involves airway transportation by small units such as bronchi, bronchioles, and alveoli ^[159][311], were developed. For instance, Huh et al. ^[160][312] investigated the human pulmonary edema on a microfluidic platform, which exhibited the alveolar–capillary interface of the lung. This system consisted of microchannels surrounded by tight layers of human endothelial cells and pulmonary epithelium subjected to air, fluid flow, and cyclic mechanical strain to simulate breathing activity. The other study showed that the pulmonary artery (PA)-on-a-chip platform allowed researchers to investigate pulmonary arterial hypertension (PAH) regarding molecular and functional alterations in pulmonary vascular endothelial and smooth muscle cells against drugs and disease impellers ^[161][313]. COPD (chronic obstructive pulmonary disease) is a serious lung illness caused by restricted airways, leading to breathing complications. Although COPD is associated with neutrophil outflow into the airways through chemotactic migration, there is plenty room to improve knowledge about the utilization of neutrophil chemotaxis for the diagnosis of COPD. As an example, Wu et al. ^[162][314] constructed a microfluidic system to quantify the neutrophil chemotaxis in sputum samples from COPD patients.

4.4. Liver Disease Modeling

Since liver diseases manifest and develop silently, it is vital to immediately take action following a diagnosis ^[163][315]. In vitro studies of the pathogenesis of liver disorders benefit from microfluidic disease-on-chip technologies ^[163][315]. Numerous liver disease-on-a-chip systems have been introduced, particularly for investigating fatty liver disease. Non-alcoholic fatty liver disease (NAFLD) emerges from lipid deposition in hepatocytes, which could ultimately lead to hepatic carcinoma. Lasli et al. ^[164][316] established a NAFLD-on-a-chip model to investigate steatosis, which was composed of spheroids formed inside inverted pyramid-shaped microwells. Moreover, spheroids were formed by coculturing human hepatocellular carcinoma (HepG2) cells and umbilical vein endothelial cells (HUVECs) in microwells. Steatosis progression might lead to inflammation, which is known as steatohepatitis ^[165][317]. Wang et al. ^[166][318] designed a NAFLD model as a liver-on-a-chip platform using human-induced pluripotent stem cells (hiPSC) cultured within spheroids. The essential pathogenic characteristics of liver organoids were linked to NAFLD that was investigated on-a-chip after induction by free fatty acid. In addition to NAFLD, scientists examined alcoholic liver disease (ALD) by mimicking physiology or anatomy of the liver. For instance, Lee et al. ^[167][319] established an ALD model on a chip, which consisted of mono- and co-cultured spheroids. Ethanol-exposed spheroids exhibited different levels of alcoholic injury. Subsequently, the viability, morphology, cytochrome P450 (CYP450) activity, and hepatic functions of spheroids were investigated.

5. Tissue Engineering

5.1. Replication of the Cellular Microenvironment

A cellular microenvironment can be formed by dynamic interactions of cells, interstitial fluid, and ECM that vitally affect the cellular process and functioning through physical, biochemical, and physicochemical mechanisms ^[168][169][330,331]. Therefore, it is crucially important to replicate the dynamics of the cellular microenvironment to analyze the phenotypes for disease modelling and therapeutics. Herein, microfluidic platforms are favorable to construct complex biofidelic cell microenvironments by precisely altering the distribution of oxygen and signaling molecules, controlling the mechanotransduction, and presenting a way to combine them with elements to induce the cells electrically, chemically, or mechanically ^[170][329]. The cellular behavior depends on the flow. Therefore, one of the particular flow processes observed in the cell microenvironment is the interstitial flow ^[171][332]. Interstitial flow (IF) is a one-way transport of fluid through ECM and a signal from the tumors which vitally affects cancer metastasis. Besides, it delivers proteins and soluble reagents through tumor stroma. In a recent study, a microfluidic platform was fabricated that allowed the investigation of activation and differentiation of cancer cells by mimicking the IF of tumor cells and transport of the soluble factors through tumor stroma from donor cells ^[172][333]. Biochemical factors also have a major role in the regulation of cell functioning. Zhang et al. investigated the effect of Ca⁺² and Sr⁺² metal ions on osteogenic differentiation of mesenchymal stem cells (MSCs) by employing a microfluidic platform ^[173][334]. In that study, the Ca⁺² and Sr⁺² crosslinked alginate microgels were produced and processed for encapsulation of single MSCs using a microfluidic system to mimic the three-dimensional stem cell microenvironment. In conclusion, they indicated that Ca⁺² crosslinked alginate hydrogels triggered the osteogenic differentiation by increasing the matrix mineralization. The stiffness of the microenvironment is another essential factor that alters the cell fate and functioning and tissue development ^[174][175][335,336]. In a recent study, the separation of nasopharyngeal carcinoma 43 (NPC43) cells and nasopharyngeal epithelial 460 (NP460) cells were performed by altering the stiffness, number of layers, and dimensions of the cell microenvironment ^[176][337]. These alterations caused changes in the migration of both cell types according to separation by a microfluidic platform.

5.2. Fabrication of Biomaterials

Biomaterials are the building blocks of tissue engineering that improve the replication of native ECM by inducing the required cellular functioning in injured tissues by utilization of an artificial framework ^[177][178][338,339]. Several techniques have been used to construct engineered biomaterials including particulate-leaching, freeze-drying, electrospinning, rapid prototyping, solvent casting, and microfluidics ^[179][340]. Among these techniques, microfluidics has become an advantageous approach for the fabrication of biomaterials as this approach is cost-effective, safe, and manageable ^[180][341]. Moreover, employing both fluid dynamics and shaped microchannels would lead to the fabrication of distinctive biomaterial carriers such as nanoparticles, microfibers, and microspheres ^[181][342]. In a recent study, Lei et al. have developed a microfluidic platform to prepare magnetic chitosan microspheres (MCMs) to trigger angiogenesis and epithelization for wound healing with antibacterial activity ^[182][343]. As a result, the developed microfluidic platform enhanced the efficient fabrication of MCMs with uniform size and shape. In another investigation by Utoh et. al, the microfluidic system was used to fabricate collagen microfibers by fragmentation phenomenon with continuous flow and altered shear stress ^[183][344]. Calcium phosphate (CaP) biomaterial is a commonly used material to promote bone regeneration and repair, and as Galván-Chacón et al. demonstrated, a microfluidic system could alter the physical and chemical properties of CaP for superior efficiency ^[184][345]. Furthermore, monodispersed CaP microparticles were synthesized in different sizes using droplet microfluidics, which could directly lead to monitoring of the responsive kinetics. The vascularization is one of the obstacles in tissue engineering, and it is considered an essential process to equally distribute the nutrients and oxygen successfully in engineered tissues ^[185][346]. In this regard, microfluidic platforms play a crucial role in intensifying the inherent laminar flow and perfusion flow through cells for vasculogenesis and represent a unique system for microvessels perfusion ^[170][186][329,347]. In a recent study, Wang et al. developed a microfluidics-based technique for the fabrication of the endothelized biomimetic microvessels (BMVs) by alginate–collagen composites ^[187][348]. The constructed BMVs exhibited a significant perfusion effect that was also able to induce osteogenic differentiation by releasing BMP-2 and PDGF-BB.

6. Organ-on-a Chip

6.1. Gut-on-a-Chip

The essential responsibility of a gut system is nutrient digestion and the restricting of transmission of undesired substances and pathogens for the protection of the body by barrier functioning ability. Nevertheless, the gut is not only vital for the digestive system, but also crucial for the desirable functionality of other organs. Thus, the improper functioning of the gut triggers several diseases ^[188][373]. To understand the physiology of the human gut system, animal models and static in vitro models were developed. However, the animal models are not sufficient to mimic the physiology of the human gut, and the static model is not efficient to replicate fluid flow, peristaltic movements, and the villi structures of intestines ^[189][374]. Hence, three-dimensional models are required to mimic the gut microenvironment dynamically and to investigate its physiology and pathology properly. Gut-on-a-chip (GoC) platforms are favorable to replicate gut dynamics by consistently perfused microchannels and the utilization of several intestinal cell types to mimic the in vivo morphology of the gut ^[190][375]. Maurer et al. developed an intestine-on-a-chip platform to understand microbial interactions in the gut microbiota by replicating the immune tolerance of the intestinal lumen with characteristics of mucosal macrophages and dendritic cells ^[191][376]. Hence, efficient investigation of microbial pathogenicity mechanisms under the immunocompetent intestine microenvironment was studied, which can be utilized to explore pathogenic diseases. Jeon et al. designed a gut-on-a-chip platform to study epithelial cell differentiation in vitro ^[192][377]. In addition, intestinal epithelial barrier functioning was analyzed with co-culturing of the damaged epithelial layer, and probiotics that consequently promoted healing of barrier functioning were recorded with the assistance of the human microbiome without bacterial overgrowth. In the case of intestinal drug absorption, tissue explants are favorable for drug screening. However, it is not possible to keep the explant tissue alive for a long period in a static environment. Amirabadi and his colleagues developed an intestinal explant barrier chip (IEBC) to analyze intestinal permeability ex vivo ^[193][378]. The novel platform incorporated human and porcine intestinal colon tissue explants in separate microchannels to study the intestinal absorption of therapeutics in a dynamic microenvironment with small non-specific binding of therapeutic molecules. The mentioned microfluidic device could be modified for the use in drug screening in other organs such as liver or skin.

6.2. Bone-on-a-Chip

The regulatory effects of the sympathetic nervous system (SNS) on breast cancer bone metastasis were exemplified recently, and Conceição et al. presented a fully humanized metastasis-on-a-chip platform to reproduce the influence of sympathetic stimulus on the cellular interaction between breast cancer cells and bone cells ^[194][379]. Three different cell types of osteoclasts, breast cancer cell variants, and sympathetic neurons were cultured in separate chambers, which allowed the dynamic paracrine signaling between the cells. According to the results, the aggression of breast cancer cells increased with the release of paracrine signaling from osteoclasts and sympathetic neurons. The essential role of bone marrow is to establish the hematopoiesis through its endosteal and perivascular niches. Glaser and his team developed a novel microfluidic platform that consisted of two niches of bone marrow separated by vascular network formation ^[195][380]. The CD34 + hematopoietic stem cells (HSPC) were cultured and differentiated into mature neutrophils. Thus, the platform allowed one to analyze the stem cell niche and could be utilized for drug screening and modeling of haematological diseases. Multiple myeloma (MM) is an incurable disease which is caused by the accumulation of monoclonal abnormal plasma cells and growth of osteolytic lesions. Nelson et al. ^[196][363] demonstrated a similar study based on human bone marrow on chip. However, Sui et al. presented a microfluidic device that replicated the stroma, sinusoidal circulation, and endothelium of the bone marrow microenvironment ^[197][381]. Subsequently, the effect of CXCL12- mediated MM cells on the barrier function of endothelial cells was observed, and the device could be used to investigate the spatiotemporal association of cancer cells in the bone marrow sinusoidal microenvironment.

6.3. Liver-on-a-Chip

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease, and its mechanism of progression is still complicated. Du et al. developed a microfluidic-based liver lobule chip (LC), which provided a platform for the co-culturing of hepatic cells and was recruited to investigate NAFLD accurately ^[198][360]. In vivo-like liver microtissue was obtained in the LC platform by a dual blood supply from hepatic artery (HA) and hepatic portal vein (PV). The NAFLD was modeled under exposure of nutrient supplies with changes in the lipid zonation for early-stage progression of NAFLD. Obesity is a metabolic disease that emerges with an excessive amount of lipid accumulation, together with escalated inflammation and forms hypertrophic adipocytes. A team led by Leung developed a novel adipose-on-chip (AOC) disease model to reproduce adipose tissue hypertrophy and inflammation under high concentrations of free fatty acid (FFA) ^[199][382]. The disease model was replicated by employing oleic acid (OA) and palmitic acid (PA) to initiate inflammation in adipocytes using hypertrophic lipid droplets. The developed model offered a new methodology to investigate obesity-associated metabolic diseases. A study conducted by Lee et al. ^[200][383] led to the development of a gut-liver chip to recapitulate hepatic steatosis. In another investigation, the OOC platforms were tested to investigate the toxicological pattern of the therapeutic agent and its metabolites in drug discovery. Soltantabar et al. designed a pumpless heart/liver-on-a-chip (HLC) microfluidic device to explore cardiotoxicity evaluation of doxorubicin (DOX) ^[201][384]. The presented HLC platform explained high viability of H9c2 rat cardiomyocytes and HepG2 hepatocellular carcinoma cells. This device was particularly suitable to monitor the damage on heart cells more efficiently compared to three-dimensional static culture. Thus, the developed HLC platform could be a promising tool to investigate cardiotoxicity in the heart.

6.4. Brain-on-a-Chip

Epilepsy is a complex neurologic disease that occurs due to recurrent epileptic seizures. Pelkonen et al. presented a microfluidic platform for epilepsy modeling consisting of a microelectrode array (MEA) that enables one to discriminate seizure-like activity ^[202][372]. Human pluripotent stem cells (hPSCs) and differentiated neurons were utilized to form functional neuronal networks, and seizure-like activity was mimicked by kainic acid (KA) treatment on neuronal networks. Several investigations indicated that neurocognitive facilities of the brain can be influenced by the gut environment, and exosomes could also moderate the signaling in the gut–brain axis (GBA). Kim et al. developed a GBA-on chip to investigate gut and brain communication ^[203][352]. This microchip was composed of the blood–brain barrier (BBB) and gut barrier, which emulated the co-culture of brain endothelial and gut endothelial cells. The barrier integrity was tested by trans-endothelial/epithelial electrical resistance (TEER), and changes were demonstrated in barriers after lipopolysaccharide (LPS) or butyrate treatment, which eventually induced an inflammatory response in the gut–brain axis and influenced permeability of BBB, respectively.

6.5. Heart-on-a-Chip

Human-induced pluripotent stem cell (hiPSCs) differentiated cardiomyocytes (CMs) (hiPSCs-CMs) are the key elements to build heart-on-chip platforms. Nevertheless, the immaturity of hiPSCs-CMs, according to the adult myocardium, cause a difficulty in the exact replication of heart physiology and disease. The team led by Zhang et al. developed a novel heart-on-chip platform to overcome the immaturity of hiPSCs-CMs ^[204][386]. That microfluidic platform offered a long-term dynamic culture of hiPSCs-CMs, while the real-time recording of hiPSC-CMs under applied electrical stimulation provided the maturation of CMs to replicate native cardiac tissue. The developed heart-on-chip demonstrated favorable results in drug tests and could be proposed as a platform to evaluate drug efficiency and cardiotoxicity.

6.6. Kidney-on-a-Chip

The glomerulus is the essential element of a kidney, which carries out regular filtration of blood using a capillary network and particular cells known as podocytes. Therefore, mimicking the glomerulus is crucial to investigate kidney physiology and diseases. Roye et al. proposed and demonstrated a personalized glomerulus chip to reproduce glomerulus barrier function utilizing hiPSCs-differentiated nephron progenitor cells and vascular endothelial cells (ECs) from a single patient to obtain a genetically matched tissue profile ^[205][387]. In another example, the kidney-organoid-on-a-chip platform was introduced by Lee et al. to investigate the biochemical effect on in vitro development of human pluripotent stem cell (hPSCs)-derived human kidney organoids. In addition, a disease model was also developed to investigate the glomerulus injury, and the results indicated that the glomerulus chip established promising outcomes to replicate a functional glomerulus and glomerulus-related diseases.

6.7. Lung-on-a-Chip

Reproducing the air–blood barrier in lung-on-a chip platforms is complicated, and the alveoli network is crucial to mimic physiological properties of the lung in vitro. Zamprogno et al. fabricated a lung-on a chip platform that enabled one to replicate an array of alveoli. This system has an advantage of biodegradability and elasticity in the biological membrane due to the presence of collagen, elastin, and proteins of lung ECM ^[206][389]. The platform exhibited an excellent model demonstration for prolonged air–blood barrier functioning using primary human lung endothelial and alveolar epithelial cells and proposed a novel technique to replicate biological barriers of the organs. Furthermore, the lung-on-a-chip platform studied by Zhu et al. ^[207][390] exhibited a favourable biomimetic breathing human lung with microphysiological breathing monitoring.

7. Microfluidics Biosensors

7.1. Enzyme-Based Microfluidic Biosensors

Enzymes are proteins in nature and are known to enhance the rate of efficiency of a reaction ranging from 105 to 1017 in comparison with non-catalyzers reactions. The biosensor-embedded enzyme generally belongs to the redox enzyme class, which catalyzes oxidation–reduction reactions. Enzymes are perfect biosensors because electrochemical monitoring is typically used to detect their turnover ^[208][391]. Since the first enzymatic biosensor was introduced in 1962 by Clark and Lyon et al. ^[209][392], enzymes have been utilized in a diversity of biosensing applications due to their intrinsic functional properties such as high selectivity, biocatalytic activity, and precise enzyme–substrate interactions ^[210][393]. By taking advantage of these features, enzyme-based biosensors constitute continuous monitoring and rapid, accurate analysis of several biomarkers ^[211][394]. They are usually coupled with microfluidic platforms due to automation, small and stable sensing area, and multiplexed functions ^[212][213][395,396]. In these platforms, reliability, long-term stability, and reusability of enzymes are the main concerns ^[214][397]. Enzyme immobilization is one of the most crucial techniques to address these challenges. Immobilization strategies of enzymes onto a surface of a transducer, as well as on the microchannels, have been widely reviewed in the literature ^{[215][216][217][218][219]}[398,399,400,401,402]. Most of the immobilization approaches rely on adsorption, covalent bonding, cross-linking, and entrapment of enzymes. Adsorption is forthright. Physical interactions such as ionic, hydrogen bonding, and Van der Waals forces are responsible for immobilization without disrupting the essential structure of the enzyme ^[220][403]. However, covalent bonding is more complicated. It requires strong interaction between surface groups of the enzyme and the surface ^[221][404]. Immobilization can be conducted via forming strong covalent bonds between enzymes as well. Cross-linking methods form three-dimensional enzyme complexes by utilizing cross-linking agents for immobilization ^[218][401]. Lastly, enzymes can be encapsulated within organic or inorganic polymer matrices to sustain the structural stability of the enzyme and diminish leakage ^{[218][219][220][221][222]}[401,402,403,404,405]. Glucose level measurement is mostly performed by this type of microfluidic biosensor, which were extensively studied in the literature due to the huge demand for diabetes management ^[223][406]. In these systems, the widely utilized enzyme is glucose oxidase (GOx) due to its high specificity, low cost, and durability against pH and temperature ^[223][224][406,407]. These advantages make GOx a potent enzyme for microfluidic biosensors to monitor glucose levels in the blood and noninvasive fluids such as saliva, tears, and sweat ^{[225][226][227][228][229]}[408,409,410,411,412]. Recently, Sun et al. ^[230][413] developed a microfluidic biosensor for glucose level monitoring from a single drop of any of these noninvasive fluids for the first time. This fully integrated nanoelectronic system was composed of a pump-free, flexible microfluidic enzymatic system (called iez Slice), coupled with a customized reusable potentiostat (called iezBar) for signal acquisition and wireless transference. In this microfluidic platform, to achieve glucose measurement in various raw biofluids including tear, saliva, and sweat, three-dimensional carbonaceous nanosphere network aerogels with hierarchical architectures (3D-CNAs) were used as glucose oxidase electrode substrates due to their higher-level electro-catalytic ability. Moreover, utilization of a microchannel made of high-concentration buffer powder-loaded Kimwipes (HBP-KWs) provided a distinctive stable glucose measurement from tears, sweat, and saliva. Because of the nature of HBP, this microchannel compels biofluids to maintain the same pH and high ionic strength as they do when they flow into it. They noted that, together with the HBP-KWs microchannel, this enzyme-based microfluidic biosensor accurately analyzed glucose from a 0.30 μL sample of raw noninvasive biofluids with a much higher r-value (≥0.96). Monitoring of glucose byproduct lactate level is prominent for athletes and high-performance workers. Additionally, the lactate level is expressed as the best marker of tissue hypoxia ^[231][414]. Recently, Shitanda et al. ^[232][415] developed a microfluidic sensing system for sweat lactate level tracking. They immobilized the lactate oxidase (LOx) enzyme by a covalent bonding method onto a MgO-templated carbon screen-printed electrode with the aid of an alkene of glycidyl methacrylate (GMA). This electrode configuration supplied a high surface area to acquire a high response readout. Then, this electrode was integrated into a microfluidic platform with eight sweat collecting channels to avoid turbulence and air trapping, and a chamber with a 10 mm radius was introduced. Monitoring the level of another biomarker, urea, provides valuable information about kidney and liver health ^[233][416]. Hence, numerous microfluidic platforms, based on the principle of urea hydrolysis by the urease enzyme, exist in the literature ^[233][234][416,417]. Additionally, monitoring creatinine levels for kidney health is among the top applications of enzyme-based biosensors ^[235][418]. Tzianni et al. ^[236][419] developed a smartphone-coupled paper-based sim card type biosensor for urinary creatinine measurement. Creatinine deiminase enzyme was immobilized onto pH-responsive copolymers PMMA-co-PMAA and demonstrated three conductive electrode configurations. This system was successfully mounted in a sim cardholder. Based on the same principle, various enzyme microfluidic paper-based analytic devices revolutionized the field of microfluidics in biomedical applications. Such an improvement has introduced further sensitivity and selectivity in analytical properties on paper-bound enzyme microfluidics systems ^[237][420]. Additionally, enzyme-immobilized papers demonstrate further mechanical and chemical stability due to their collegial effects. Nonetheless, such bindings increase the life span and enzyme stability ^[238][239][421,422]. An enzyme-based paper microfluidics system, μPADs, offers a cellulose matrix which is highly flexible, thin, and cost effective ^[240][423]. In addition, the paper has implicit capillary function with high surface-to-volume ratio, enabling the user to load various enzymes and markers. Moreover, the surface of microfluidic cellulose papers is conveniently adjustable for microfluidics channels via two zones attributed to sample and detection ^[241][137]. During the last decade, multiplexed analysis of simultaneous biomarkers in invasive and noninvasive biofluids has accelerated by leveraging microfluidics ^[242][425]. For instance, an enzymatic low volume microfluidic platform that simultaneously analyzes a lifestyle biomarker trio—alcohol, glucose, and lactate levels in a low volume of sweat (1–5 μL)—was developed by Bhide et al. ^[213][396]. Monitoring free amino acids in the blood serum could give prominent information about the state of several diseases, including cancers ^[243][426]. More recently, Kugimiya et al. ^[244][427] developed a laminated paper-based analytical device (LPAD), exploiting an aminoacyl-tRNA synthetase (aaRS) analysis system, to measure histidine, tryptophan, glycine, and lysine levels. It included a sample spot connected to four enzymatic reaction areas, each containing a specific tRNA synthetase for one amino acid type and four detection areas. Properties and dimensions of channels between detection and reaction areas specify the incubation time for the reaction mixture. In the detection zone, the colorimetric signal due to the molybdenum blue reaction was quantified using an image scanner. In another study, ammonia and ethanol concentration in sweat were measured, relying on an enzyme-based colorimetric readout in a multi-layer microfluidic platform. In this platform, super absorbent polymer (SAP) pumps and capillary burst valves were integrated for mixing purposes, as well as to increase the reaction kinetics control ^[245][428]. Apart from monitoring the health status of patients, these types of microfluidic sensors are also used to monitor and characterize microfluidic cell culture systems ^[246][429]. In an interesting study, Cedillo-Alcantar et al. ^[247][424] developed an automated microfluidic platform, relying on droplet generation technology, for this purpose. To emphasize an application, they performed a simultaneous analysis of glucose, bile acid, and lactate dehydrogenase (LDH) of a microfluidic cell culture platform comprising hepatocyte spheroids. To circumvent the problem of decreasing enzyme activities when immobilized, they injected the required enzymes throughout the measurement. With the aid of automated pneumatic valves, first enzymes were mixed with the substrates and then encapsulated in water-in-oil droplets. In each tiny droplet (<0.8 nL), a discrete enzymatic assay took place, and the results were quantified based on colorimetric and fluorescent readouts.

7.2. Nanozymes-Based Microfluidic Biosensors

Nanozymes are artificial nanomaterial enzymes that not only mimic the activity of enzymes, but also offer advantages over natural enzymes due to their impressive properties ^[248][430]. Compared to natural enzymes, they are easy and inexpensive to produce on a large scale, have a long storage time, and withstand harsh conditions such as high pH and temperatures ^{[248][249][250]}[430,431,432]. Since they were first introduced in 2007, the use of nanozymes in biosensors instead of enzymes has gained momentum. That year, the interesting enzyme mimetic property of magnetite (Fe₃O₄), similar to natural peroxidases, was discovered by Gao et al. ^[251][433]. They proposed a new immunoassay in which they used H₂O₂Fe₃O₄ instead of horseradish peroxidase (HRP) to catalyze the oxidation of various peroxidase substrates. In the following years, other nanomaterials with peroxidase-like properties, such as metal and metal oxides ^[252][434], metal–organic frameworks (MOFs), and carbon-based nanomaterials ^[253][435] were discovered ^[249][431]. Moreover, it was reported that numerous nanomaterials could mimic the activities of other enzymes, including oxidase ^[254][436], catalase ^[255][437], and superoxide dismutase (SOD) ^[256][257][438,439]. Together with the discovery of enzyme mimetic nanomaterials, their utilization in microfluidic biosensors to perform the function of enzymes has been accelerated. Considering the prominent research area of microfluidic glucose biosensors, nanozymes were effectively used for colorimetric, electrochemical, and fluorescence glucose measurement ^{[258][259][260]}[440,441,442]. Gomez et al. ^[259][441] used a supported metal–organic framework (MOF) to mimic the activity of peroxidase for the first time in a microfluidic paper-based analytical device (μPAD) and measured glucose levels using a small sample volume (10μl) of urine and serum. Another μPAD was designed for simultaneous detection of uric acid and glucose based on peroxidase mimicking platinum nanoparticles (Pt NPs) ^[261][443]. Hydrogen peroxide is an important analyte itself and a prominent product or substrate of catalyzed oxidation reactions ^[262][444]. Hence, various microfluidic biosensors utilizing nanozymes including Au@PtNP/GO ^[263][445], graphene oxide-gold ^[264][446], and cerium oxide nanosheets (NSs) ^[262][265][444,447] were developed for H202 measurement. Nanozymes are exploited for point-of-care (POC) cancer diagnosis as well ^{[260][266][267]}[442,448,449]. More recently Liu et al. ^[260][442] developed an electrochemical/visual microfluidic platform for sensitive detection of pheochromocytoma circulating cells (PCC-CTCs) based on the peroxidase mimicking activity of covalent–organic framework-based nanozymes (COF@Pt).

7.3. Microfluidics in Antibody Based Biosensing

Antibody-based microfluidic biosensors are based on the immobilization of various monoclonal antibodies as a rapid detection mechanism ^[268][269][450,451]. Using antibodies as biosensors has the advantage that the immunogen could be detected with no prior purification steps ^[270][452]. However, recent developments in recombinant technology made it possible to generate the fab fragment as the main antigen binding site for various general antibodies ^[271][453]. The limitation of traditional immunoassay techniques has been overcome in combination with microfluidics devices. In the recent pandemic, various portable microfluidics-based immunoassay strips presented accurate, quick, and convenient approaches for the detection of SARS CoV 2 IgG/IgM/antigen using pharyngeal swabs ^[272][454]. In this case, the microfluidic immunoassay relies on the interaction of viral protein with immobilized anti-SARS-CoV-2 antibodies on an electrode of sensor with the capacity to detect the change of the electric current. The detectors are generally coated with various materials, such as fluorine-doped tin oxide electrode (FTO) or screen-printed carbon electrode (SPE) or gold nanoparticles AuNPs) or graphene, which consequently act as an indicator for change in conductivity upon antigen/antibody interaction ^{[273][274][275]}[455,456,457]. The DNA-based microfluidic biosensors employ amplification of targeted DNA fragments followed by DNA hybridization of obtained sequences of the immobilized complementary target sequence in a single platform. The technique that generally requires separate amplification and base pairing on a gel substrate is integrated into a single tool with a convenient detection method by receiving the relevant change in physiochemical signals ^[276][464]. Currently, there are three main microfluidics biosensors developed to detect nucleic acid: PCR, (CRISPR)/clustered regularly interspaced short palindromic repeats, and isothermal amplification ^[277][465]. The microfluidics-integrated qPCR method that was reported by several research groups has the advantage of high throughput scheme processing, allowing massive quantitative analysis, including preparation and detection in a single chip platform ^[278][279][466,467]. A recent PCR-based microfluidic system introduced by Cojocaru et al. was a disposable chip functionalized with lyophilized probes and primers, which offered a rapid result (less than 30 mins) for as much as 1.2 μL volume per reaction ^[280][468]. In isothermal amplification, the thermocycler, the fundamental part of PCR, is replaced by incubator or water bath-integrated microfluidics devices. Several types of microfluidic-based isothermal amplifications developed, including rolling circle amplification (RCA) ^[281][469], recombinase polymerase amplification (RPA) ^[282][470], and loop-mediated isothermal amplification (LAMP) ^[283][471], which were remarkably efficient and accurate. Ramachandran et al. designed a microfluidics-based CRISPR where the CRISPR–Cas12 enzyme and a guide RNA were introduced to the device to bind to the selected target DNA and cleave it. The active compound then randomly chopped the probed single-stranded DNA labeled fluorophore−quencher. In this method, the CRISPR assay was analyzed by governing the gradients obtained from the change in the electric field and, subsequently, navigated target DNA, reporters, and Cas12–gRNA within the microfluidic device. This technique is known as isotachophoresis (ITP) enhanced microfluidic based CRISPR assay, which can detect the target nucleic acid, RNA, within less than 35 mins ^[284][472].

8. Artificial Cells

The microfluidics systems are also used to provide activated artificial cells via mechanical forces. In this system, stable double emulsion droplets (aqueous/oil/aqueous) are utilized to model mechanosensitive artificial cells. The microfluidics device is designed to trap such mixed drops in the form of the developed chamber, which implies pressure and target simultaneously. Such pressure is accompanied by a temporary raise and perpetual drops in the oil thickness. Therefore, the group observed consequent calcium ion influx due to activated artificial cell activity which was caused by diluted oil drops ^[285][473].

9. Microfluidics and Cryopreservation

Cryopreservation is a banking technique that enhances the store of bio-engineered materials under gradual/rapid cooling and dehydration by adding cryoprotective agents (CPA) ^[286][474]. The technique is significant in maintaining particular genetic characteristics of the culture at a certain point for the applications in clinics and tissue engineering ^[287][288][475,476]. In the cryopreservation technique, the biological function of living cells is quenched at low temperatures, which procures their long-term preservation ^[289][477]. The cryopreservation technique is very prone to induce cell damage due to dehydration, osmotic shock, and formation of ice crystals, thereby decreasing the cell viability ^[290][478]. Microfluidic CPA-integrated devices introduced a great advantage of cryopreservation in a single platform. These procedures, such as automated freeze-thawing cycles with potentially adjustable cell concentration in addition to low-CPA vitrification, were demonstrated with a series of functional microtubes in form of microfluidics channels ^[291][479]. In the case of fertility preservation, oocyte cryopreservation is a significant process and, as Guo et al. demonstrated, a microfluidic device could be utilized to minimize the osmotic stress injury (OSI) on porcine oocytes during CPA loading and unloading ^[292][480]. They indicated that their invented microfluidic system was able to decrease the potential osmotic damage drastically through the sequencing of loading and unloading CPAs and, thus, promotes the developmental capability of oocytes. Embryo vitrification is another vital process in fertility preservation. Tirgar et. al. designed an automatic standalone microfluidic-based cryopreservation system for mouse blastocyte vitrification ^[293][481]. This device enhanced, controlled, and offered a continuous CPA loading without damaging the spherical morphology of blastocytes and minimized the shrinkage rate. Moreover, in a recent study, Özsoylu et al. developed a method to preserve the cell-based biosensor chip, which allowed one to preserve adherent cells on sensor surfaces and, therefore, this method was called on-sensor cryopreservation (OSC) ^[294][482]. Here, the biosensor surface was modified by polyethylene vinyl acetate (PEVA) electrospun fibers to make the sensor durable for high temperature changes. Then, the microfluidic platform was integrated through the sensor to ensure fast thawing and decreasing thermal mass. The results indicated that the OSC method is an ideal technique for cryopreservation of adherent cells using sensors and, therefore, it is recommended for “ready-to-use on-site” applications.